International Journal of Molecular Sciences

Editorial

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9 pages, 257 KiB

Open AccessEditorial

Environmental Stress and Plants

by Lavinia Mareri, Luigi Parrotta and Giampiero Cai

Int. J. Mol. Sci. 2022, 23(10), 5416; https://doi.org/10.3390/ijms23105416 - 12 May 2022

Cited by 77 | Viewed by 7321

Abstract

Land plants are constantly subjected to multiple unfavorable or even adverse environmental conditions. Among them, abiotic stresses (such as salt, drought, heat, cold, heavy metals, ozone, UV radiation, and nutrient deficiencies) have detrimental effects on plant growth and productivity and are increasingly important [...] Read more.

Land plants are constantly subjected to multiple unfavorable or even adverse environmental conditions. Among them, abiotic stresses (such as salt, drought, heat, cold, heavy metals, ozone, UV radiation, and nutrient deficiencies) have detrimental effects on plant growth and productivity and are increasingly important considering the direct or indirect effects of climate change. Plants respond in many ways to abiotic stresses, from gene expression to physiology, from plant architecture to primary, and secondary metabolism. These complex changes allow plants to tolerate and/or adapt to adverse conditions. The complexity of plant response can be further influenced by the duration and intensity of stress, the plant genotype, the combination of different stresses, the exposed tissue and cell type, and the developmental stage at which plants perceive the stress. It is therefore important to understand more about how plants perceive stress conditions and how they respond and adapt (both in natural and anthropogenic environments). These concepts were the basis of the Special Issue that International Journal of Molecular Sciences expressly addressed to the relationship between environmental stresses and plants and that resulted in the publication of 5 reviews and 38 original research articles. The large participation of several authors and the good number of contributions testifies to the considerable interest that the topic currently receives in the plant science community, especially in the light of the foreseeable climate changes. Here, we briefly summarize the contributions included in the Special Issue, both original articles categorized by stress type and reviews that discuss more comprehensive responses to various stresses. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

Research

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14 pages, 1894 KiB

Open AccessArticle

Salicylic Acid Enhances Adventitious Root and Aerenchyma Formation in Wheat under Waterlogged Conditions

by Murali Krishna Koramutla, Pham Anh Tuan and Belay T. Ayele

Int. J. Mol. Sci. 2022, 23(3), 1243; https://doi.org/10.3390/ijms23031243 - 23 Jan 2022

Cited by 27 | Viewed by 3830

Abstract

The present study investigated the role of salicylic acid (SA) in regulating morpho-anatomical adaptive responses of a wheat plant to waterlogging. Our pharmacological study showed that treatment of waterlogged wheat plants with exogenous SA promotes the formation axile roots and surface adventitious roots [...] Read more.

The present study investigated the role of salicylic acid (SA) in regulating morpho-anatomical adaptive responses of a wheat plant to waterlogging. Our pharmacological study showed that treatment of waterlogged wheat plants with exogenous SA promotes the formation axile roots and surface adventitious roots that originate from basal stem nodes, but inhibits their elongation, leading to the formation of a shallow root system. The treatment also enhanced axile root formation in non-waterlogged plants but with only slight reductions in their length and branch root formation. Exogenous SA enhanced the formation of root aerenchyma, a key anatomical adaptive response of plants to waterlogging. Consistent with these results, waterlogging enhanced SA content in the root via expression of specific isochorismate synthase (ICS; ICS1 and ICS2) and phenylalanine ammonia lyase (PAL; PAL4, PAL5 and PAL6) genes and in the stem nodes via expression of specific PAL (PAL5 and PAL6) genes. Although not to the same level observed in waterlogged plants, exogenous SA also induced aerenchyma formation in non-waterlogged plants. The findings of this study furthermore indicated that inhibition of ethylene synthesis in SA treated non-waterlogged and waterlogged plants does not have any effect on SA-induced emergence of axile and/or surface adventitious roots but represses SA-mediated induction of aerenchyma formation. These results highlight that the role of SA in promoting the development of axile and surface adventitious roots in waterlogged wheat plants is ethylene independent while the induction of aerenchyma formation by SA requires the presence of ethylene. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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Figure 1

Figure 1
Root and shoot phenotype of non-waterlogged plants. Shoot growth (A) and tiller number (B), and total number of axile roots per plant (C), average axile root length (D), and number of branch roots per nodal axile root (E) in non-waterlogged plants treated with or without hormone and/or inhibitor. Data are means of three independent biological replicates (shoots and roots of at least three individual plants per replicate) ± SE. Different letters between any two samples show significant difference (one-way ANOVA; p < 0.05, Fisher’s LSD test). CTRL, non-waterlogged untreated; SA, salicylic acid; ABT, 1-aminobenzotriazole (SA biosynthesis inhibitor); AVG, aminoethoxyvinylglycine (ethylene biosynthesis inhibitor). Full article ">Figure 2
Root and shoot phenotype of waterlogged plants. Shoot growth (A) and tiller number (B), and total number of axile roots per plant (C), average axile root length (D), and number of branch roots per nodal axile root (E) in control non-waterlogged plants, and those waterlogged for 14 days and treated with or without hormone and/or inhibitor. Data are means of three independent biological replicates (shoots and roots of at least three individual plants per replicate) ± SE. Different letters between any two samples show significant difference (two-way ANOVA; p < 0.05, Fisher’s LSD test). CTRL, non-waterlogged untreated; WL, waterlogged untreated; SA, salicylic acid; ABT, 1-aminobenzotriazole (SA biosynthesis inhibitor); AVG, aminoethoxyvinylglycine (ethylene biosynthesis inhibitor). Full article ">Figure 3
Surface adventitious root and aerenchyma formation. Development of surface adventitious roots (A–D,I–L), transverse sections of nodal roots (4 cm from the root apex) (E–H,M–P), number of surface adventitious roots (Q) and percentage of aerenchyma in root cross-sections (R,S) in non-waterlogged and 14-day waterlogged plants treated with or without hormone and/or inhibitor. Data are means of three independent biological replicates ± SE. Different letters between any two samples show significant difference (two-way ANOVA; p < 0.05, Fisher’s LSD test). Arrows indicate surface adventitious roots emergence from stem nodes. CTRL, non-waterlogged untreated; WL, waterlogged untreated; SA, salicylic acid; ABT, 1-aminobenzotriazole (SA biosynthesis inhibitor); AVG, aminoethoxyvinylglycine (ethylene biosynthesis inhibitor); nd, not detected. Hormone and/or inhibitor treatments of non-waterlogged control plants did not induce surface adventitious roots. Full article ">Figure 4
Expression of salicylic acid biosynthesis genes and salicylic acid level in roots. Relative transcript levels of the TaICSs (A,B) and TaPALs (C–E), and salicylic acid level (F) in roots at 1, 7 and 14 days after waterlogging (DAWL). Transcript levels of the TaICSs and TaPALs were determined using Ta18SrRNA as the reference gene and expressed relative to the transcript levels of TaICS1 and TaPAL4 in control roots at 1 DAWL, respectively, which were arbitrarily set a value of 1. Data are means of three independent biological replicates ± SE. Asterisks show significant difference in transcript levels between the control (non-waterlogged) and waterlogged samples within waterlogging duration (one-way ANOVA; p < 0.05, Fisher’s LSD test). Full article ">Figure 5
Expression of salicylic acid biosynthesis genes and salicylic acid level in stem nodes. Relative transcript levels of the TaICSs (A,B) and TaPALs (C–E), and salicylic acid level (F) in stem nodes at 7 and 14 days after waterlogging (DAWL). Transcript levels of the TaICSs and TaPALs were determined using Ta18SrRNA as the reference gene and expressed relative to the transcript levels of TaICS1 and TaPAL4 in control stem nodes at 7 DAWL, respectively, which were arbitrarily set a value of 1. Data are means of three independent biological replicates ± SE. Asterisks show significant difference in transcript levels between the control (non-waterlogged) and waterlogged samples within waterlogging duration (one-way ANOVA; p < 0.05, Fisher’s LSD test). Full article ">Figure 6
Schematic representation of the role of salicylic acid in inducing aerenchyma formation and development of adventitious roots in wheat under waterlogging condition. Full article ">

24 pages, 5596 KiB

Open AccessArticle

Insights into the Mechanisms of Heat Priming and Thermotolerance in Tobacco Pollen

by Lavinia Mareri, Claudia Faleri, Iris Aloisi, Luigi Parrotta, Stefano Del Duca and Giampiero Cai

Int. J. Mol. Sci. 2021, 22(16), 8535; https://doi.org/10.3390/ijms22168535 - 8 Aug 2021

Cited by 5 | Viewed by 3314

Abstract

Global warming leads to a progressive rise in environmental temperature. Plants, as sessile organisms, are threatened by these changes; the male gametophyte is extremely sensitive to high temperature and its ability to preserve its physiological status under heat stress is known as acquired [...] Read more.

Global warming leads to a progressive rise in environmental temperature. Plants, as sessile organisms, are threatened by these changes; the male gametophyte is extremely sensitive to high temperature and its ability to preserve its physiological status under heat stress is known as acquired thermotolerance. This latter can be achieved by exposing plant to a sub-lethal temperature (priming) or to a progressive increase in temperature. The present research aims to investigate the effects of heat priming on the functioning of tobacco pollen grains. In addition to evaluating basic physiological parameters (e.g., pollen viability, germination and pollen tube length), several aspects related to a correct pollen functioning were considered. Calcium (Ca²⁺) level, reactive oxygen species (ROS) and related antioxidant systems were investigated, also to the organization of actin filaments and cytoskeletal protein such as tubulin (including tyrosinated and acetylated isoforms) and actin. We also focused on sucrose synthase (Sus), a key metabolic enzyme and on the content of main soluble sugars, including UDP-glucose. Results here obtained showed that a pre-exposure to sub-lethal temperatures can positively enhance pollen performance by altering its metabolism. This can have a considerable impact, especially from the point of view of breeding strategies aimed at improving crop species. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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15 pages, 4301 KiB

Open AccessArticle

Complex Analysis of Antioxidant Activity, Abscisic Acid Level, and Accumulation of Osmotica in Apple and Cherry In Vitro Cultures under Osmotic Stress

by Petra Jiroutova, Zuzana Kovalikova, Jakub Toman, Dominika Dobrovolna and Rudolf Andrys

Int. J. Mol. Sci. 2021, 22(15), 7922; https://doi.org/10.3390/ijms22157922 - 25 Jul 2021

Cited by 11 | Viewed by 2880

Abstract

Plant response to osmotic stress is a complex issue and includes a wide range of physiological and biochemical processes. Extensive studies of known cultivars and their reaction to drought or salinity stress are very important for future breeding of new and tolerant cultivars. [...] Read more.

Plant response to osmotic stress is a complex issue and includes a wide range of physiological and biochemical processes. Extensive studies of known cultivars and their reaction to drought or salinity stress are very important for future breeding of new and tolerant cultivars. Our study focused on the antioxidant activity, accumulations of osmotica, and the content of abscisic acid in apple (cv. “Malinové holovouské”, “Fragrance”, “Rubinstep”, “Idared”, “Car Alexander”) and cherry (cv. “Regina”, “Napoleonova”, “Kaštánka”, “Sunburst”, “P-HL-C”) cultivated in vitro on media containing different levels of polyethylene glycol PEG-6000. Our results indicated that the studied genotypes responded differently to osmotic stress manifested as reduction in the leaf relative water content (RWC) and increment in the activities of antioxidant enzymes, proline, sugars, and abscisic acid content. Overall, cherry cultivars showed a smaller decrease in percentage RWC and enzymatic activities, but enhanced proline content compared to the apple plants cultivars. Cultivars “Rubinstep”, “Napoleonova”, and “Kaštánka” exhibited higher antioxidant capacity and accumulation of osmoprotectants like proline and sorbitol that can be associated with the drought-tolerance system. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Effect of different concentration of polyethylene glycol (PEG-6000) on the growth and appearance of the in vitro culture of apple cultivar “Rubinstep” (A) and cherry cultivar “Kaštánka” (B); PEG concentration in g L−1 increasing from left to right (PEG0, PEG5, PEG10, PEG25, PEG50). Full article ">Figure 2
Effect of PEG-6000 concentration on the relative water content (%) for in vitro culture of apple (left) and cherry (right) cultivars. Error bars represent standard deviation (SD). Values within column, followed by the same letter(s), are not significantly different according to Tukey’s test (p < 0.05). Full article ">Figure 3
Effect of PEG-6000 concentration on proline content (µg g−1 FW) for in vitro culture of apple (left) and cherry (right) cultivars. Error bars represent standard deviation (SD). Values within column, followed by the same letter(s), are not significantly different according to Tukey’s test (p < 0.05). Full article ">Figure 4
Effect of PEG-6000 concentration on sugars content (mg g−1 FW) for in vitro culture of apple (A) and cherry (B) cultivars. All values were recalculated relative to the compound content in untreated samples taken as 100%. GLU, glucose; FRU, fructose; GLY, glycerol; SOR, sorbitol; SUC, sucrose. The statistical data are shown in <a href="#app1-ijms-22-07922" class="html-app">Supplementary Material Tables S1 and S2</a>. Full article ">Figure 5
Effect of PEG-6000 concentration on scavenging activity of DPPH radical (%) for in vitro culture of apple (left) and cherry (right) cultivars. Error bars represent standard deviation (SD). Values within column, followed by the same letter(s), are not significantly different according to Tukey’s test (p < 0.05). Full article ">Figure 6
Effect of PEG-6000 concentration on catalase activity CAT (µmol H2O2 g−1 FW), ascorbate peroxidase activity APX (nmol H2O2 g−1 FW), and superoxide dismutase activity SOD (%) for in vitro culture of apple (left) and cherry (right) cultivars. Error bars represent standard deviation (SD). Values within column, followed by the same letter(s), are not significantly different according to Tukey’s test (p < 0.05). Full article ">Figure 7
Effect of PEG-6000 concentration on abscisic acid content ABA (ng g−1 FW) for in vitro culture of apple (left) and cherry (right) cultivars. Error bars represent standard deviation (SD). Values within column, followed by the same letter(s), are not significantly different according to Tukey’s test (p < 0.05). Full article ">

13 pages, 1265 KiB

Open AccessArticle

Response of Wheat DREB Transcription Factor to Osmotic Stress Based on DNA Methylation

by Huihui Wang, Yanqiu Zhu, Ping Yuan, Shanglin Song, Tianyu Dong, Peilei Chen, Zhikun Duan, Lina Jiang, Longdou Lu and Hongying Duan

Int. J. Mol. Sci. 2021, 22(14), 7670; https://doi.org/10.3390/ijms22147670 - 18 Jul 2021

Cited by 12 | Viewed by 3190

Abstract

Dehydration-responsive element-binding protein (DREB) plays an important role in response to osmotic stress. In this study, DREB2, DREB6 and Wdreb2 are isolated from wheat AK58, yet they belong to different types of DREB transcription factors. Under osmotic stress, the transcript expression of [...] Read more.

Dehydration-responsive element-binding protein (DREB) plays an important role in response to osmotic stress. In this study, DREB2, DREB6 and Wdreb2 are isolated from wheat AK58, yet they belong to different types of DREB transcription factors. Under osmotic stress, the transcript expression of DREB2, DREB6 and Wdreb2 has tissue specificity and is generally higher in leaves, but their expression trends are different along with the increase of osmotic stress. Furthermore, some elements related to stresses are found in their promoters, promoters of DREB2 and Wdreb2 are slightly methylated, but DREB6’s promoter is moderately methylated. Compared with the control, the level of promoter methylation in Wdreb2 is significantly lower under osmotic stress and is also lower at CG site in DREB2, yet is significantly higher at CHG and CHH sites in DREB2, which is also found at a CHG site in DREB6. The status of promoter methylation in DREB2, DREB6 and Wdreb2 also undergoes significant changes under osmotic stress; further analysis showed that promoter methylation of Wdreb2 is negatively correlated with their expression. Therefore, the results of this research suggest the different functions of DREB2, DREB6 and Wdreb2 in response to osmotic stress and demonstrate the effects of promoter methylation on the expression regulation of Wdreb2. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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Figure 1
The expression analysis of wheat DREB genes under osmotic stress. Quantitative real-time PCR (qRT-PCR) analysis of DREB2, DREB6 and Wdreb2 expression are shown in (a–c); the transcript abundances of three DREBs are analyzed in wheat seedlings treated with 15% PEG6000 solution. The error bar is the standard error of the mean. * and ** represent significant difference relative to the control conditions at the levels of 0.05 and 0.01, respectively. Full article ">Figure 2
Methylation profiles in the promoter regions of wheat DREB genes under osmotic stress. (a–c) represent cytosine methylation maps for the promoter sequences of DREB2, DREB6 and Wdreb2 in wheat leaves stressed with 15% PEG6000 solution for 12 h. Red, blue and green circles represent CG, CHG or CHH. Filled and hollow circles denote methylated and unmethylated cytosine, respectively. Each row represents the sequencing result of one positive clone. Full article ">Figure 3
Effect of osmotic stress on methylation level of DREB promoters in wheat. (a–c) represent methylation rates for the promoter regions of DREB2, DREB6 and Wdreb2 in wheat leaves stressed with 15% PEG6000 solution for 12 h. The error bar is the standard error of the mean. * and ** represent significant difference relative to the control conditions at the levels of 0.05 and 0.01, respectively. Full article ">

30 pages, 6504 KiB

Open AccessArticle

Genome-Wide Identification and Characterization of PIN-FORMED (PIN) Gene Family Reveals Role in Developmental and Various Stress Conditions in Triticum aestivum L.

by Manu Kumar, Bhagwat Singh Kherawat, Prajjal Dey, Debanjana Saha, Anupama Singh, Shashi Kant Bhatia, Gajanan Sampatrao Ghodake, Avinash Ashok Kadam, Hyun-Uk Kim, Manorama, Sang-Min Chung and Mahipal Singh Kesawat

Int. J. Mol. Sci. 2021, 22(14), 7396; https://doi.org/10.3390/ijms22147396 - 9 Jul 2021

Cited by 49 | Viewed by 6380

Abstract

PIN-FORMED (PIN) genes play a crucial role in regulating polar auxin distribution in diverse developmental processes, including tropic responses, embryogenesis, tissue differentiation, and organogenesis. However, the role of PIN-mediated auxin transport in various plant species is poorly understood. Currently, no information is available [...] Read more.

PIN-FORMED (PIN) genes play a crucial role in regulating polar auxin distribution in diverse developmental processes, including tropic responses, embryogenesis, tissue differentiation, and organogenesis. However, the role of PIN-mediated auxin transport in various plant species is poorly understood. Currently, no information is available about this gene family in wheat (Triticum aestivum L.). In the present investigation, we identified the PIN gene family in wheat to understand the evolution of PIN-mediated auxin transport and its role in various developmental processes and under different biotic and abiotic stress conditions. In this study, we performed genome-wide analysis of the PIN gene family in common wheat and identified 44 TaPIN genes through a homology search, further characterizing them to understand their structure, function, and distribution across various tissues. Phylogenetic analyses led to the classification of TaPIN genes into seven different groups, providing evidence of an evolutionary relationship with Arabidopsis thaliana and Oryza sativa. A gene exon/intron structure analysis showed a distinct evolutionary path and predicted the possible gene duplication events. Further, the physical and biochemical properties, conserved motifs, chromosomal, subcellular localization, transmembrane domains, and three-dimensional (3D) structure were also examined using various computational approaches. Cis-elements analysis of TaPIN genes showed that TaPIN promoters consist of phytohormone, plant growth and development, and stress-related cis-elements. In addition, expression profile analysis also revealed that the expression patterns of the TaPIN genes were different in different tissues and developmental stages. Several members of the TaPIN family were induced during biotic and abiotic stress. Moreover, the expression patterns of TaPIN genes were verified by qRT-PCR. The qRT-PCR results also show a similar expression with slight variation. Therefore, the outcome of this study provides basic genomic information on the expression of the TaPIN gene family and will pave the way for dissecting the precise role of TaPINs in plant developmental processes and different stress conditions. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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31 pages, 7018 KiB

Open AccessArticle

Transcriptome Analysis of Tolerant and Susceptible Maize Genotypes Reveals Novel Insights about the Molecular Mechanisms Underlying Drought Responses in Leaves

by Joram Kiriga Waititu, Xingen Zhang, Tianci Chen, Chunyi Zhang, Yang Zhao and Huan Wang

Int. J. Mol. Sci. 2021, 22(13), 6980; https://doi.org/10.3390/ijms22136980 - 29 Jun 2021

Cited by 49 | Viewed by 4823

Abstract

Maize (Zea mays L.) is the most essential food crop in the world. However, maize is highly susceptible to drought stress, especially at the seedling stage, and the molecular mechanisms underlying drought tolerance remain elusive. In this study, we conducted comparative transcriptome [...] Read more.

Maize (Zea mays L.) is the most essential food crop in the world. However, maize is highly susceptible to drought stress, especially at the seedling stage, and the molecular mechanisms underlying drought tolerance remain elusive. In this study, we conducted comparative transcriptome and physiological analyses of drought-tolerant (CML69) and susceptible (LX9801) inbred lines subjected to drought treatment at the seedling stage for three and five days. The tolerant line had significantly higher relative water content in the leaves, as well as lower electrolyte leakage and malondialdehyde levels, than the susceptible line. Using an RNA-seq-based approach, we identified 10,084 differentially expressed genes (DEGs) with 6906 and 3178 DEGs been annotated and unannotated, respectively. Two critical sets of drought-responsive DEGs, including 4687 genotype-specific and 2219 common drought-responsive genes, were mined out of the annotated DEGs. The tolerant-line DEGs were predominantly associated with the cytoskeleton, cell wall modification, glycolysis/gluconeogenesis, transport, osmotic regulation, drought avoidance, ROS scavengers, defense, and transcriptional factors. For the susceptible line, the DEGs were highly enriched in the photosynthesis, histone, and carbon fixation pathways. The unannotated DEGs were implicated in lncRNAs, including 428 previously reported and 22% putative TE-lncRNAs. There was consensus on both the physiological response and RNA-seq outcomes. Collectively, our findings will provide a comprehensive basis of the molecular networks mediating drought stress tolerance of maize at the seedling stage. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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13 pages, 2204 KiB

Open AccessArticle

Effect of Salt Stress on the Expression and Promoter Methylation of the Genes Encoding the Mitochondrial and Cytosolic Forms of Aconitase and Fumarase in Maize

by Alexander T. Eprintsev, Dmitry N. Fedorin, Mikhail V. Cherkasskikh and Abir U. Igamberdiev

Int. J. Mol. Sci. 2021, 22(11), 6012; https://doi.org/10.3390/ijms22116012 - 2 Jun 2021

Cited by 20 | Viewed by 2975

Abstract

The influence of salt stress on gene expression, promoter methylation, and enzymatic activity of the mitochondrial and cytosolic forms of aconitase and fumarase has been investigated in maize (Zea mays L.) seedlings. The incubation of maize seedlings in 150-mM NaCl solution resulted [...] Read more.

The influence of salt stress on gene expression, promoter methylation, and enzymatic activity of the mitochondrial and cytosolic forms of aconitase and fumarase has been investigated in maize (Zea mays L.) seedlings. The incubation of maize seedlings in 150-mM NaCl solution resulted in a several-fold increase of the mitochondrial activities of aconitase and fumarase that peaked at 6 h of NaCl treatment, while the cytosolic activity of aconitase and fumarase decreased. This corresponded to the decrease in promoter methylation of the genes Aco1 and Fum1 encoding the mitochondrial forms of these enzymes and the increase in promoter methylation of the genes Aco2 and Fum2 encoding the cytosolic forms. The pattern of expression of the genes encoding the mitochondrial forms of aconitase and fumarase corresponded to the profile of the increase of the stress marker gene ZmCOI6.1. It is concluded that the mitochondrial and cytosolic forms of aconitase and fumarase are regulated via the epigenetic mechanism of promoter methylation of their genes in the opposite ways in response to salt stress. The role of the mitochondrial isoforms of aconitase and fumarase in the elevation of respiration under salt stress is discussed. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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Figure 1
Analysis of CG dinucleotides in the promoters of the Fum1 and Fum2 genes from maize. The position of CG nucleotides is indicated by vertical lines. Full article ">Figure 2
Change in activity of aconitase in the mitochondrial and cytosolic fractions after the transfer of maize seedlings to 150-mM NaCl. The data represent the means of three biological repeats ± SD. The control (untreated) samples of activities did not exhibit statistically significant changes in the variation level during the experiment. Full article ">Figure 3
Relative level of transcripts of the genes Aco1 and Aco2 and the rate of methylation of their promoters in maize leaves in the course of incubation of maize seedlings in 150-mM NaCl. The data represent the means of three biological repeats ± SD. The control (untreated) samples of gene expression values did not exhibit statistically significant changes in the variation level. Full article ">Figure 4
Change in the activity of fumarase in the mitochondrial and cytosolic fractions after the transfer of maize seedlings in 150-mM NaCl. The data represent the means of three biological repeats ± SD. The control (untreated) samples of the values of activity did not exhibit statistically significant changes in the variation level. Full article ">Figure 5
Relative level of transcripts of the genes Fum1 and Fum2 and the rate of methylation of their promoters in maize leaves in the course of incubation of maize seedlings in 150-mM NaCl. The data represent the means of three biological repeats ± SD. The control (untreated) samples of gene expression values did not exhibit statistically significant changes in the variation level. Full article ">Figure 6
Relative level of transcripts of the gene ZmCOI6.1 (A) and of the genes Pif5 and Pif6 (B) in maize leaves in the course of incubation of maize seedlings in 150-mM NaCl. The data represent the means of three biological repeats ± SD. The control (untreated) samples of gene expression values did not exhibit statistically significant changes in the variation level. Full article ">

23 pages, 4254 KiB

Open AccessArticle

Heat Priming of Lentil (Lens culinaris Medik.) Seeds and Foliar Treatment with γ-Aminobutyric Acid (GABA), Confers Protection to Reproductive Function and Yield Traits under High-Temperature Stress Environments

by Anjali Bhardwaj, Kumari Sita, Akanksha Sehgal, Kalpna Bhandari, Shiv Kumar, P. V. Vara Prasad, Uday Jha, Jitendra Kumar, Kadambot H. M. Siddique and Harsh Nayyar

Int. J. Mol. Sci. 2021, 22(11), 5825; https://doi.org/10.3390/ijms22115825 - 29 May 2021

Cited by 16 | Viewed by 4788

Abstract

Gradually increasing temperatures at global and local scales are causing heat stress for cool and summer-season food legumes, such as lentil (Lens culinaris Medik.), which is highly susceptible to heat stress, especially during its reproductive stages of development. Hence, suitable strategies are [...] Read more.

Gradually increasing temperatures at global and local scales are causing heat stress for cool and summer-season food legumes, such as lentil (Lens culinaris Medik.), which is highly susceptible to heat stress, especially during its reproductive stages of development. Hence, suitable strategies are needed to develop heat tolerance in this legume. In the present study, we tested the effectiveness of heat priming (HPr; 6 h at 35 °C) the lentil seeds and a foliar treatment of γ-aminobutyric acid (GABA; 1 mM; applied twice at different times), singly or in combination (HPr+GABA), under heat stress (32/20 °C) in two heat-tolerant (HT; IG2507, IG3263) and two heat-sensitive (HS; IG2821, IG2849) genotypes to mitigate heat stress. The three treatments significantly reduced heat injury to leaves and flowers, particularly when applied in combination, including leaf damage assessed as membrane injury, cellular oxidizing ability, leaf water status, and stomatal conductance. The combined HPr+GABA treatment significantly improved the photosynthetic function, measured as photosynthetic efficiency, chlorophyll concentration, and sucrose synthesis; and significantly reduced the oxidative damage, which was associated with a marked up-regulation in the activities of enzymatic antioxidants. The combined treatment also facilitated the synthesis of osmolytes, such as proline and glycine betaine, by upregulating the expression of their biosynthesizing enzymes (pyrroline-5-carboxylate synthase; betaine aldehyde dehydrogenase) under heat stress. The HPr+GABA treatment caused a considerable enhancement in endogenous levels of GABA in leaves, more so in the two heat-sensitive genotypes. The reproductive function, measured as germination and viability of pollen grains, receptivity of stigma, and viability of ovules, was significantly improved with combined treatment, resulting in enhanced pod number (21–23% in HT and 35–38% in HS genotypes, compared to heat stress alone) and seed yield per plant (22–24% in HT and 37–40% in HS genotypes, in comparison to heat stress alone). The combined treatment (HPr+GABA) was more effective and pronounced in heat-sensitive than heat-tolerant genotypes for all the traits tested. This study offers a potential solution for tackling and protecting heat stress injury in lentil plants. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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21 pages, 4282 KiB

Open AccessArticle

Morphological and Metabolite Responses of Potatoes under Various Phosphorus Levels and Their Amelioration by Plant Growth-Promoting Rhizobacteria

by Leangsrun Chea, Birgit Pfeiffer, Dominik Schneider, Rolf Daniel, Elke Pawelzik and Marcel Naumann

Int. J. Mol. Sci. 2021, 22(10), 5162; https://doi.org/10.3390/ijms22105162 - 13 May 2021

Cited by 24 | Viewed by 4544

Abstract

Low phosphorus (P) availability is a major limiting factor for potatoes. P fertilizer is applied to enhance P availability; however, it may become toxic when plants accumulate at high concentrations. Therefore, it is necessary to gain more knowledge of the morphological and biochemical [...] Read more.

Low phosphorus (P) availability is a major limiting factor for potatoes. P fertilizer is applied to enhance P availability; however, it may become toxic when plants accumulate at high concentrations. Therefore, it is necessary to gain more knowledge of the morphological and biochemical processes associated with P deficiency and toxicity for potatoes, as well as to explore an alternative approach to ameliorate the P deficiency condition. A comprehensive study was conducted (I) to assess plant morphology, mineral allocation, and metabolites of potatoes in response to P deficiency and toxicity; and (II) to evaluate the potency of plant growth-promoting rhizobacteria (PGPR) in improving plant biomass, P uptake, and metabolites at low P levels. The results revealed a reduction in plant height and biomass by 60–80% under P deficiency compared to P optimum. P deficiency and toxicity conditions also altered the mineral concentration and allocation in plants due to nutrient imbalance. The stress induced by both P deficiency and toxicity was evident from an accumulation of proline and total free amino acids in young leaves and roots. Furthermore, root metabolite profiling revealed that P deficiency reduced sugars by 50–80% and organic acids by 20–90%, but increased amino acids by 1.5–14.8 times. However, the effect of P toxicity on metabolic changes in roots was less pronounced. Under P deficiency, PGPR significantly improved the root and shoot biomass, total root length, and root surface area by 32–45%. This finding suggests the potency of PGPR inoculation to increase potato plant tolerance under P deficiency. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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15 pages, 3161 KiB

Open AccessArticle

Thermal Analysis of Stomatal Response under Salinity and High Light

by Aleksandra Orzechowska, Martin Trtílek, Krzysztof Michał Tokarz, Renata Szymańska, Ewa Niewiadomska, Piotr Rozpądek and Katarzyna Wątor

Int. J. Mol. Sci. 2021, 22(9), 4663; https://doi.org/10.3390/ijms22094663 - 28 Apr 2021

Cited by 21 | Viewed by 3365

Abstract

A non-destructive thermal imaging method was used to study the stomatal response of salt-treated Arabidopsis thaliana plants to excessive light. The plants were exposed to different levels of salt concentrations (0, 75, 150, and 220 mM NaCl). Time-dependent thermograms showed the changes in [...] Read more.

A non-destructive thermal imaging method was used to study the stomatal response of salt-treated Arabidopsis thaliana plants to excessive light. The plants were exposed to different levels of salt concentrations (0, 75, 150, and 220 mM NaCl). Time-dependent thermograms showed the changes in the temperature distribution over the lamina and provided new insights into the acute light-induced temporary response of Arabidopsis under short-term salinity. The initial response of plants, which was associated with stomatal aperture, revealed an exponential growth in temperature kinetics. Using a single-exponential function, we estimated the time constants of thermal courses of plants exposed to acute high light. The saline-induced impairment in stomatal movement caused the reduced stomatal conductance and transpiration rate. Limited transpiration of NaCl-treated plants resulted in an increased rosette temperature and decreased thermal time constants as compared to the controls. The net CO₂ assimilation rate decreased for plants exposed to 220 mM NaCl; in the case of 75 mM NaCl treatment, an increase was observed. A significant decline in the maximal quantum yield of photosystem II under excessive light was noticeable for the control and NaCl-treated plants. This study provides evidence that thermal imaging as a highly sensitive technique may be useful for analyzing the stomatal aperture and movement under dynamic environmental conditions. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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Figure 1
Water content of four-old-week rosettes of non- and salt-treated A. thaliana plants. Results are presented as box-and-whisker plots, showing mean and standard error at the 95% confidence level (a). Concentration of sodium ions accumulated in A. thaliana leaf rosettes after one-time treatment with 0, 75, 150, and 220 mM NaCl (b). Asterisks (*) indicate significant differences in Na+ content between non- and salt-treated plants (ANOVA, p ≤ 0.05). Full article ">Figure 2
Total chlorophyll (a) and carotenoid (b) content in rosette leaves of four-week old A. thaliana plants, three days after exposure to salinity. Tests of statistical significance in Chl and Car content between the salt-treated plants compared to the controls were performed using ANOVA (p ≤ 0.05). Full article ">Figure 3
Stomatal conductance (a), transpiration rate (b), and the net CO2 assimilation rate (c) measured three days after inducing salt stress in 4-week old A. thaliana rosettes. Maximal PSII efficiency (FV/FM) in A. thaliana plants exposed to salinity under LL (empty box charts) and an acute HL (pattern-filled box charts) is shown in (d). Results are presented as box-and-whisker plots, showing mean, standard error, and outliers at the 95% confidence level. An asterisk (*) indicates significant differences (p ≤ 0.05, the Mann-Whitney U test) between control and salt-treated plants (a–c), and plants before (LL) and after an acute HL treatment (d). Full article ">Figure 4
Infrared thermograms of LL-growing (a) and HL-treated (b) A. thaliana plants acquired three days after exposure to salinity (upper panel), and the average temperature distribution over the leaf rosettes of control and salt-treated plants (lower panel). Data are presented as box-and-whisker plots showing mean, standard error, and the outliers at the 95% confidence level. An asterisk (*) indicates significant differences (p ≤ 0.05, ANOVA test) between the non- and salt-treated plants before (LL) and after an acute HL treatment. Full article ">Figure 5
Changes in the temperature of rosette leaves induced by an acute HL treatment measured in A. thaliana plants exposed to salinity. Each point on the graph represents the mean value of at least three randomly chosen spots on the thermal image for control (a), 75 (b), 150 (c), and 220 mM (d) NaCl treatments. The thermal kinetics were fitted using a mono-exponential function, y = y0 + ys·[1 − exp(−t/ts)]. Theoretically determined courses approximate the initial responses of plants to excess light three days after exposure to salinity. Each graph (a–d) shows the fitted thermal data along with 95% prediction (light red surfaces) and confidence (dark red surfaces) bands. The light intensity was 2000 µmol·m−2·s−1. Full article ">Figure 6
An exponential decay of thermal time constant estimated for control and salt-treated plants as a result of an acute HL treatment. Data are shown along with 95% prediction (light red surfaces) and confidence (dark red surfaces) bands. Full article ">

14 pages, 1740 KiB

Open AccessArticle

Functional Characterization of a Sugar Beet BvbHLH93 Transcription Factor in Salt Stress Tolerance

by Yuguang Wang, Shuang Wang, Ye Tian, Qiuhong Wang, Sixue Chen, Hongli Li, Chunquan Ma and Haiying Li

Int. J. Mol. Sci. 2021, 22(7), 3669; https://doi.org/10.3390/ijms22073669 - 1 Apr 2021

Cited by 37 | Viewed by 3328

Abstract

The basic/helix–loop–helix (bHLH) transcription factor (TF) plays an important role for plant growth, development, and stress responses. Previously, proteomics of NaCl treated sugar beet leaves revealed that a bHLH TF, BvbHLH93, was significantly increased under salt stress. The BvbHLH93 protein localized in [...] Read more.

The basic/helix–loop–helix (bHLH) transcription factor (TF) plays an important role for plant growth, development, and stress responses. Previously, proteomics of NaCl treated sugar beet leaves revealed that a bHLH TF, BvbHLH93, was significantly increased under salt stress. The BvbHLH93 protein localized in the nucleus and exhibited activation activity. The expression of BvbHLH93 was significantly up-regulated in roots and leaves by salt stress, and the highest expression level in roots and leaves was 24 and 48 h after salt stress, respectively. Furthermore, constitutive expression of BvbHLH93 conferred enhanced salt tolerance in Arabidopsis, as indicated by longer roots and higher content of chlorophyll than wild type. Additionally, the ectopic expression lines accumulated less Na⁺ and MDA, but more K⁺ than the WT. Overexpression of the BvBHLH93 enhanced the activities of antioxidant enzymes by positively regulating the expression of antioxidant genes SOD and POD. Compared to WT, the overexpression plants also had low expression levels of RbohD and RbohF, which are involved in reactive oxygen species (ROS) production. These results suggest that BvbHLH93 plays a key role in enhancing salt stress tolerance by enhancing antioxidant enzymes and decreasing ROS generation. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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23 pages, 4133 KiB

Open AccessArticle

Transcriptome Analysis Unravels Key Factors Involved in Response to Potassium Deficiency and Feedback Regulation of K⁺ Uptake in Cotton Roots

by Doudou Yang, Fangjun Li, Fei Yi, A. Egrinya Eneji, Xiaoli Tian and Zhaohu Li

Int. J. Mol. Sci. 2021, 22(6), 3133; https://doi.org/10.3390/ijms22063133 - 19 Mar 2021

Cited by 20 | Viewed by 3384

Abstract

To properly understand cotton responses to potassium (K⁺) deficiency and how its shoot feedback regulates K⁺ uptake and root growth, we analyzed the changes in root transcriptome induced by low K⁺ (0.03 mM K⁺, lasting three days) [...] Read more.

To properly understand cotton responses to potassium (K⁺) deficiency and how its shoot feedback regulates K⁺ uptake and root growth, we analyzed the changes in root transcriptome induced by low K⁺ (0.03 mM K⁺, lasting three days) in self-grafts of a K⁺ inefficient cotton variety (CCRI41/CCRI41, scion/rootstock) and its reciprocal grafts with a K⁺ efficient variety (SCRC22/CCRI41). Compared with CCRI41/CCRI41, the SCRC22 scion enhanced the K⁺ uptake and root growth of CCRI41 rootstock. A total of 1968 and 2539 differently expressed genes (DEGs) were identified in the roots of CCRI41/CCRI41 and SCRC22/CCRI41 in response to K⁺ deficiency, respectively. The overlapped and similarly (both up- or both down-) regulated DEGs in the two grafts were considered the basic response to K⁺ deficiency in cotton roots, whereas the DEGs only found in SCRC22/CCRI41 (1954) and those oppositely (one up- and the other down-) regulated in the two grafts might be the key factors involved in the feedback regulation of K⁺ uptake and root growth. The expression level of four putative K⁺ transporter genes (three GhHAK5s and one GhKUP3) increased in both grafts under low K⁺, which could enable plants to cope with K⁺ deficiency. In addition, two ethylene response factors (ERFs), GhERF15 and GhESE3, both down-regulated in the roots of CCRI41/CCRI41 and SCRC22/CCRI41, may negatively regulate K⁺ uptake in cotton roots due to higher net K⁺ uptake rate in their virus-induced gene silencing (VIGS) plants. In terms of feedback regulation of K⁺ uptake and root growth, several up-regulated DEGs related to Ca²⁺ binding and CIPK (CBL-interacting protein kinases), one up-regulated GhKUP3 and several up-regulated GhNRT2.1s probably play important roles. In conclusion, these results provide a deeper insight into the molecular mechanisms involved in basic response to low K⁺ stress in cotton roots and feedback regulation of K⁺ uptake, and present several low K⁺ tolerance-associated genes that need to be further identified and characterized. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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Figure 1
Effects of potassium (K+) deficiency on CCRI41 (a K+ inefficient cotton variety) self-grafts (CCRI41/CCRI41, scion/rootstock) and its reciprocal grafts (SCRC22/CCRI41) with SCRC22 (a K+ efficient variety). Grafting was performed hypocotyl-to-hypocotyl (a) when the cotyledons of rootstock just fully expanded. Grafts were subjected to low K+ (LK, 0.03 mM K+) at the three-leaf stage, with 2.5 mM K+ as control (CK). After 16 days, all leaves were photographed (b), and the chlorophyll content in the third (3rd L), fourth (4th L) and fifth (5th L) leaves was measured (c). The dry weight of roots, stem and leaves was recorded (d), and the root-shoot ratio was calculated (e). K+ concentration (f) and K+ accumulation (g) in roots, stem and leaves were determined or calculated. CK: Control; LK: Low K+ treatment. The data are shown as means ± SD of three replicates (n = 3); * and ** indicate significant differences at 5% and 1% level, respectively. Full article ">Figure 2
Transcriptome relationships among the roots of CCRI41 self-grafts (CCRI41/CCRI41, scion/rootstock) and reciprocal grafts (SCRC22/CCRI41) under potassium (K+) deficiency. (a) Cluster dendrogram of the root transcriptomes. (b) Number of differentially expressed genes (DEGs) in the roots of CCRI41/CCRI41 and SCRC22/CCRI41. (c) Venn diagram showing shared and unique DEGs in both grafts. CR and LR: Roots from CK and LK-treated plants; 41/41: CCRI41/CCRI41; 22/41: SCRC22/CCRI41; 1, 2, and 3: Number of replicates. R common: Common DEGs in the roots of both grafts. R22/41 unique: The DEGs specifically identified in the roots of SCRC22/CCRI41. Full article ">Figure 3
The differentially expressed genes (DEGs) involved in Ca2+ (calcium) signaling in R common (a) and R22/41 unique (b,c) group. R common: Common DEGs in roots of CCRI41/CCRI41 (scion/rootstock) and SCRC22/CCRI41 under potassium (K+) deficiency. R22/41 unique: DEGs specifically identified in roots of SCRC22/CCRI41. Full article ">Figure 4
The differently expressed genes (DEGs) involved in ROS (reactive oxygen species) signaling in R common (a) and R22/41 unique (b,c) group. R common: Common DEGs in roots of CCRI41/CCRI41 (scion/rootstock) and SCRC22/CCRI41 under potassium (K+) deficiency. R22/41 unique: DEGs specifically identified in roots of SCRC22/CCRI41. Full article ">Figure 5
The differentially expressed genes (DEGs) involved in phytohormone signaling in R common (a) and R22/41 unique (b) group. R common: Common DEGs in roots of CCRI41/CCRI41 (scion/rootstock) and SCRC22/CCRI41 under potassium (K+) deficiency. R22/41 unique: DEGs specifically identified in roots of SCRC22/CCRI41. Red and purple boxes contain genes that were up- and down-regulated, respectively. The red and blue colored boxes contain genes that were oppositely regulated in both grafts, the left and right boxes were assigned to CCRI41/CCRI41 and SCRC22/CCRI41, respectively. The “DNA” in diagram represents undefined genes. Full article ">Figure 6
The differentially expressed genes (DEGs) related to transporters in R common (a) and R22/41 unique (b,c) group. R common: Common DEGs in roots of CCRI41/CCRI41 (scion/rootstock) and SCRC22/CCRI41 under potassium (K+) deficiency. R22/41 unique: The DEGs specifically identified in roots of SCRC22/CCRI41. Full article ">Figure 7
The predicted transcription factors (TFs) that have binding sites in the 2-kb promotor region of the K+ transporter gene GhHAK5 (Gh_D01G1760) in R common (a) and R22/41 unique (b) group. R common: Common DEGs in roots of CCRI41/CCRI41 (scion/rootstock) and SCRC22/CCRI41 under potassium (K+) deficiency. R22/41 unique: DEGs specifically identified in roots of SCRC22/CCRI41. Full article ">Figure 8
Two members of ethylene response factor (ERF) TFs, GhERF15 and GhESE3, negatively regulate potassium (K+) uptake in cotton roots. GhERF15 and GhESE3 were silenced in the variety SCRC22 using agrobacterium-mediated virus-induced gene silencing (VIGS) at the cotyledonary stage. The relative expression of GhERF15 (a) and GhESE3 (b) indicates that they were silenced in both A and D subgenome. The seedlings at three-leaf stage were moved into K+-starvation solutions for 48 h, then transferred to measuring solution with 0.08 mM K+ to determine the net K+ uptake rate (c,d). * and ** indicate significant differences at 5% and 1% level, respectively. Full article ">Figure 9
A model of transcription regulation involved in response to potassium (K+) deficiency and feedback regulation of K+ uptake and root growth in cotton. Genes within the light grey box show the same responses to low K+ stress in CCRI41 (a K+ inefficient cotton variety) self-grafts (CCRI41/CCRI41, scion/rootstock) and its reciprocal grafts (SCRC22/CCRI41) with SCRC22 (a K+ efficient variety); while genes within the white box represent the components involved in feedback regulation of K+ uptake and root growth under K+ deficiency, they were specifically identified in the roots of SCRC22/CCRI41 or oppositely regulated by low K+ in CCRI41/CCRI41 and SCRC22/CCRI41. The red and blue highlighted genes were up- and down-regulated by low K+ respectively. Full article ">

17 pages, 4246 KiB

Open AccessArticle

Quantitative Analysis of UV-B Radiation Interception in 3D Plant Structures and Intraindividual Distribution of Phenolic Contents

by Hyo In Yoon, Hyun Young Kim, Jaewoo Kim, Myung-Min Oh and Jung Eek Son

Int. J. Mol. Sci. 2021, 22(5), 2701; https://doi.org/10.3390/ijms22052701 - 7 Mar 2021

Cited by 13 | Viewed by 2862

Abstract

Ultraviolet-B (UV-B) acts as a regulatory stimulus, inducing the dose-dependent biosynthesis of phenolic compounds such as flavonoids at the leaf level. However, the heterogeneity of biosynthesis activation generated within a whole plant is not fully understood until now and cannot be interpreted without [...] Read more.

Ultraviolet-B (UV-B) acts as a regulatory stimulus, inducing the dose-dependent biosynthesis of phenolic compounds such as flavonoids at the leaf level. However, the heterogeneity of biosynthesis activation generated within a whole plant is not fully understood until now and cannot be interpreted without quantification of UV-B radiation interception. In this study, we analyzed the spatial UV-B radiation interception of kales (Brassica oleracea L. var. Acephala) grown under supplemental UV-B LED using ray-tracing simulation with 3-dimension-scanned models and leaf optical properties. The UV-B-induced phenolic compounds and flavonoids accumulated more, with higher UV-B interception and younger leaves. To distinguish the effects of UV-B energy and leaf developmental age, the contents were regressed separately and simultaneously. The effect of intercepted UV-B on flavonoid content was 4.9-fold that of leaf age, but the effects on phenolic compound biosynthesis were similar. This study confirmed the feasibility and relevance of UV-B radiation interception analysis and paves the way to explore the physical and physiological base determining the intraindividual distribution of phenolic compound in controlled environments. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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11 pages, 4669 KiB

Open AccessArticle

Functional Characteristics Analysis of Dehydrins in Larix kaempferi under Osmotic Stress

by Xuechun Wang, Meng Zhang, Baohui Xie, Xiangning Jiang and Ying Gai

Int. J. Mol. Sci. 2021, 22(4), 1715; https://doi.org/10.3390/ijms22041715 - 9 Feb 2021

Cited by 12 | Viewed by 2724

Abstract

Dehydrins (DHN) belong to the late embryogenesis abundant II family and have been found to enhance plant tolerance to abiotic stress. In the present study, we reported four DHNs in Larix kaempferi (LkDHN) which were identified from the published transcriptome. Alignment analysis showed [...] Read more.

Dehydrins (DHN) belong to the late embryogenesis abundant II family and have been found to enhance plant tolerance to abiotic stress. In the present study, we reported four DHNs in Larix kaempferi (LkDHN) which were identified from the published transcriptome. Alignment analysis showed that these four LkDHNs shared close relationships and belonged to SK₃-type DHNs. The electrophoretic mobility shift assay indicated that these four LkDHNs all possess sequence-independent binding capacity for double-strands DNAs. The subcellular localizations of the four LkDHNs were in both the nucleus and cytoplasm, indicating that these LkDHNs enter the nucleus to exert the ability to bind DNA. The preparation of tobacco protoplasts with different concentrations of mannitol showed that LkDHNs enhanced the tolerance of plant cells under osmotic stress. The overexpression of LkDHNs in yeasts enhanced their tolerance to osmotic stress and helped the yeasts to survive severe stress. In addition, LkDHNs in the nucleus of salt treated tobacco increased. All of these results indicated that the four LkDHNs help plants survive from heavy stress by participating in DNA protection. These four LKDHNs played similar roles in the response to osmotic stress and assisted in the adaptation of L. kaempferi to the arid and cold winter of northern China. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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Figure 1
Alignment of LkDHNs (Dehydrin in Larix kaempferi) and DHNs from other plants. Six verified DHNs including AtDHN (Arabidopsis thaliana), MnDHN (Musa nana), OsDHN (Oryza sativa), PgDHN (Picea glauca), PmDHN (Pinus massonia), SpDHN (Stipa purpurea) and LkDHNs homologous sequence were aligned by DNAman 8.0. The S segment was indicated by blue box; K segments were indicated by red boxes; nuclear localization signals (NLS) were indicated by the purple line; phosphorylation sites were indicated by black dot. Full article ">Figure 2
Subcellular localization of LkDHNs in lower epidermis cells of the transformed tobacco leaves. (a–f) are wild type, 35S-GFP, 35S-LkDHN1-GFP, 35S-LkDHN2-GFP, 35S-LkDHN3-GFP, and 35S-LkDHN4-GFP at the wavelength of GFP, respectively; (g–l) are wild type, 35S-GFP, 35S-LkDHN1-GFP, 35S-LkDHN2-GFP, 35S-LkDHN3-GFP, and 35S-LkDHN4-GFP at the wavelength of DAPI, respectively; (m–r) are the merge of wild type, 35S-GFP, 35S-LkDHN1-GFP, 35S-LkDHN2-GFP, 35S-LkDHN3-GFP, and 35S-LkDHN4-GFP, respectively. The nucleuses were indicated by black arrow; the cytoplasms were indicated by white arrow. The scale bar is 25 μm. Full article ">Figure 3
Stress treatment enhanced the signal of LkDHNs in nucleus. (A) The effect of salt stress on the nuclear localization of LkDHNs was observed by a Leica SP8 laser confocal microscope. a and b are GFP with 0 and 200 mM NaCl, respectively; c and d are LkDHN1 with 0 and 200 mM NaCl, respectively; e and f are LkDHN2 with 0 and 200 mM NaCl, respectively; g and h are LkDHN3 with 0 and 200 mM NaCl, respectively; i and j are LkDHN4 with 0 and 200 mM NaCl, respectively. (B) Analysis of GFP fluorescence signal in nucleus under salt stress. a: GFP; b: LkDHN1; c: LkDHN2; d: LkDHN3; e: LkDHN4. The purple line represents the fluorescence after 30 min treatment with 200 mM NaCl, and the blue line represents the fluorescence under 0 mM NaCl. The nucleuses were indicated by red arrow. Full article ">Figure 4
The binding ability of LkDHNs to different DNA probes was analyzed by electrophoretic mobility shift assay (EMSA). Lane 1: free-probe; Lane 2: CSE-probe with LkDHN1; Lane 3: CSE-probe with LkDHN2; Lane 4: CSE-probe with LkDHN3; Lane 5: CSE-probe with LkDHN4; Lane 6: pET28a-probe with LkDHN1; Lane 7: pET28a-probe with LkDHN2; Lane 8: pET28a-probe with LkDHN3; Lane 9: pET28a-probe with LkDHN4. Full article ">Figure 5
LkDHNs increased the tolerance of tobacco protoplasts and yeasts to osmotic stress. (A) The tolerance of tobacco protoplasts with LkDHNs to osmotic stress was enhanced. a and b are the enzymatic hydrolysis of GFP at 0.4 and 0.6 M mannitol, respectively; c and d are the enzymatic hydrolysis of LkDHN1 at 0.4 and 0.6 M mannitol, respectively; e and f are the enzymatic hydrolysis of LkDHN2 at 0.4 and 0.6 M mannitol, respectively; g and h are the enzymatic hydrolysis of LkDHN3 at 0.4 and 0.6 M mannitol, respectively; i and j are the enzymatic hydrolysis of LkDHN4 at 0.4 and 0.6 M mannitol, respectively. The intact protoplasts were indicated by red arrow; the broken protoplasts were indicated by blue arrow. The scale bar is 500 μm. (B) Enhanced tolerance of yeast overexpressing LkDHNs to osmotic stress. a: spot assay of pPIC9 and LkDHNs on YPD medium; b: spot assay of pPIC9 and LkDHNs on YPD medium with 2 M sorbitol; c: spot assay of pPIC9 and LkDHNs on YPD medium with 1.5 M NaCl. Full article ">

23 pages, 4820 KiB

Open AccessArticle

Effect of RIP Overexpression on Abiotic Stress Tolerance and Development of Rice

by Pieter Wytynck, Jeroen Lambin, Simin Chen, Sinem Demirel Asci, Isabel Verbeke, Jeroen De Zaeytijd, Kondeti Subramanyam and Els J.M. Van Damme

Int. J. Mol. Sci. 2021, 22(3), 1434; https://doi.org/10.3390/ijms22031434 - 1 Feb 2021

Cited by 9 | Viewed by 3172

Abstract

Ribosome-inactivating proteins (RIPs) are a class of cytotoxic enzymes that can inhibit protein translation by depurinating rRNA. Most plant RIPs are synthesized with a leader sequence that sequesters the proteins to a cell compartment away from the host ribosomes. However, several rice RIPs [...] Read more.

Ribosome-inactivating proteins (RIPs) are a class of cytotoxic enzymes that can inhibit protein translation by depurinating rRNA. Most plant RIPs are synthesized with a leader sequence that sequesters the proteins to a cell compartment away from the host ribosomes. However, several rice RIPs lack these signal peptides suggesting they reside in the cytosol in close proximity to the plant ribosomes. This paper aims to elucidate the physiological function of two nucleocytoplasmic RIPs from rice, in particular, the type 1 RIP referred to as OsRIP1 and a presumed type 3 RIP called nuRIP. Transgenic rice lines overexpressing these RIPs were constructed and studied for developmental effects resulting from this overexpression under greenhouse conditions. In addition, the performance of transgenic seedlings in response to drought, salt, abscisic acid and methyl jasmonate treatment was investigated. Results suggest that both RIPs can affect methyl jasmonate mediated stress responses. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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24 pages, 3895 KiB

Open AccessArticle

Transcriptomics Reveals Fast Changes in Salicylate and Jasmonate Signaling Pathways in Shoots of Carbonate-Tolerant Arabidopsis thaliana under Bicarbonate Exposure

by Laura Pérez-Martín, Silvia Busoms, Roser Tolrà and Charlotte Poschenrieder

Int. J. Mol. Sci. 2021, 22(3), 1226; https://doi.org/10.3390/ijms22031226 - 27 Jan 2021

Cited by 17 | Viewed by 5000

Abstract

High bicarbonate concentrations of calcareous soils with high pH can affect crop performance due to different constraints. Among these, Fe deficiency has mostly been studied. The ability to mobilize sparingly soluble Fe is a key factor for tolerance. Here, a comparative transcriptomic analysis [...] Read more.

High bicarbonate concentrations of calcareous soils with high pH can affect crop performance due to different constraints. Among these, Fe deficiency has mostly been studied. The ability to mobilize sparingly soluble Fe is a key factor for tolerance. Here, a comparative transcriptomic analysis was performed with two naturally selected Arabidopsis thaliana demes, the carbonate-tolerant A1_(c+) and the sensitive T6_(c−). Analyses of plants exposed to either pH stress alone (pH 5.9 vs. pH 8.3) or to alkalinity caused by 10 mM NaHCO₃ (pH 8.3) confirmed better growth and nutrient homeostasis of A1_(c+) under alkaline conditions. RNA-sequencing (RNA-seq) revealed that bicarbonate quickly (3 h) induced Fe deficiency-related genes in T6_(c−) leaves. Contrastingly, in A1_(c+), initial changes concerned receptor-like proteins (RLP), jasmonate (JA) and salicylate (SA) pathways, methionine-derived glucosinolates (GS), sulfur starvation, starch degradation, and cell cycle. Our results suggest that leaves of carbonate-tolerant plants do not sense iron deficiency as fast as sensitive ones. This is in line with a more efficient Fe translocation to aerial parts. In A1_(c+) leaves, the activation of other genes related to stress perception, signal transduction, GS, sulfur acquisition, and cell cycle precedes the induction of iron homeostasis mechanisms yielding an efficient response to bicarbonate stress. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Location, soil parameters, physiological characterization, and plant nutrition. (A) Location of Arabidopsis thaliana natural populations A1(c+) and T6(c−) in a soil map of Catalonia. Orange areas represent calcareous soils and white areas non-calcareous soils. (B) Soil carbonate and pH levels (means ± Standard Error; n = 9) in the natural habitat of A1(c+) and T6(c−) populations. (C) Picture of 30-day-old Col-0, A1(c+), and T6(c−) plants under different alkaline treatments. Germination rate (D), rosette diameter (E), and silique number (F) of A1(c+), T6(c−), and Col-0 plants submitted to control (pH 5.9, grey bars), high pH (pH 8.3, light green bars), or bic (pH 8.3 with 10 mM of NaHCO3, dark green bars) in plates or hydroponic culture. Values are means ± SE; n = 45, n = 12, n = 9, respectively; letters indicate significant differences (p < 0.05, Tukey’s honestly significant difference HSD) per deme. (G–I) Standardized leaf mineral content of four plants per deme grown under pH 5.9 (grey lines), pH 8.3 (light green lines), or bic (dark green lines) for 2 weeks. Full article ">Figure 2
Compared transcriptomic profiles under different alkalinity treatments at two time points. Differential expressed genes (DEGs) bar plot (A), heatmap profile (B), and Venn diagram at 3 h (C) and 48 h (D) of the pairwise comparations pH 8.3 vs. pH 5.9 and bic vs. pH 5.9 in A1(c+) and T6 (c−) demes. DEGs were filtered at log fold change (LFC > 1, LFC < −1), and adjusted p-value < 0.05. Yellow areas indicate upregulated genes and blue areas indicate downregulated genes. Full article ">Figure 3
Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of bic vs. pH 5.9 treatments DEGs at two time points. Venn diagram of up- and downregulated DEGs in A1(c+) and T6(c−) comparing bic vs. pH 5.9 treatments after 3 h (A) and 48 h (B). Bubble plots indicating significant GO analysis of differentially expressed genes in bic vs. pH 5.9 comparison between A1(c+) and T6(c−) at 3 h (C) and 48 h (D). GO were filtered to adjusted p-value < 0.05. Scale colors indicate number of DEGs while bubble size indicates -log of adjusted p-value. GO terms were separated into biological function, cellular component, and molecular function. Arrows indicate up or downregulated genes. Heatmaps of KEGG pathway analysis from DEGs in bic 8.3 vs. pH 5.9 comparison between A1(c+) and T6(c−) at 3 h (E) and 48 h (F). KEGG pathway terms were filtered by p-value < 0.05. Scale colors indicate pathway fold enrichment. BP, biological process; CC, cellular component; MF, molecular function. Full article ">Figure 4
Differential expression of signal perception, transduction, and transcription factor (TF) genes involved in bicarbonate short responses. Heatmaps of DEGs from the receptor-like kinase gene family (A), and peroxidase, glutathione, and calmodulin gene families (B) in A1(c+) and T6(c+), comparing bic vs. pH 5.9 exposure for 3 h. Yellow color represents upregulated genes while blue color represents downregulated genes. (C) Bar plot indicating the total number of DEGs upregulated (right) or downregulated (left) within each transcription factor family. Tolerant deme A1(c+) is marked in grey, while sensitive deme T6(c−) is visualized in black color. Full article ">Figure 5
Protein–protein interaction network functional enrichment analysis of specific genes derived from the response to bicarbonate of A1 (c+). Gene protein interaction network of A1(c+) exclusive DEGs from bic vs. pH 5.9 comparison after 3 h exposure. Each sphere corresponds to one gene and nodes represent protein interactions. Gene pathways are shown in different colors. Arrows indicate up- or downregulation of the genes. Full article ">Figure 6
Protein–protein interaction network functional enrichment analysis of specific genes derived from the response to bicarbonate of T6 (c−). Gene protein interaction network of T6(c−) exclusive DEGs from bic vs. pH 5.9 comparison after 3 h exposure. Each sphere corresponds to one gene and nodes represent protein interactions. Gene pathways are shown in different colors. Arrows indicate up or downregulation of the genes. Full article ">

23 pages, 3575 KiB

Open AccessArticle

Whole-Transcriptome RNA Sequencing Reveals the Global Molecular Responses and CeRNA Regulatory Network of mRNAs, lncRNAs, miRNAs and circRNAs in Response to Salt Stress in Sugar Beet (Beta vulgaris)

by Junliang Li, Jie Cui, Cuihong Dai, Tianjiao Liu, Dayou Cheng and Chengfei Luo

Int. J. Mol. Sci. 2021, 22(1), 289; https://doi.org/10.3390/ijms22010289 - 30 Dec 2020

Cited by 40 | Viewed by 5296

Abstract

Sugar beet is an important sugar-yielding crop with some tolerance to salt, but the mechanistic basis of this tolerance is not known. In the present study, we have used whole-transcriptome RNA-seq and degradome sequencing in response to salt stress to uncover differentially expressed [...] Read more.

Sugar beet is an important sugar-yielding crop with some tolerance to salt, but the mechanistic basis of this tolerance is not known. In the present study, we have used whole-transcriptome RNA-seq and degradome sequencing in response to salt stress to uncover differentially expressed (DE) mRNAs, microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) in both leaves and roots. A competitive endogenous RNA (ceRNA) network was constructed with the predicted DE pairs, which revealed regulatory roles under salt stress. A functional analysis suggests that ceRNAs are implicated in copper redistribution, plasma membrane permeability, glycometabolism and energy metabolism, NAC transcription factor and the phosphoinositol signaling system. Overall, we conducted for the first time a full transcriptomic analysis of sugar beet under salt stress that involves a potential ceRNA network, thus providing a basis to study the potential functions of lncRNAs/circRNAs. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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Figure 1

Figure 1
Identification and analysis of differentially expressed mRNAs (DEmRNAs) under salt stress. (a) Violin Plot of gene expression patterns for each sample, with origin representing the median; ck represents the control group and ST represents the treatment group. (b) Volcano Plot of log2 FC(ST/CK) of leaves and roots mRNAs. (c) Visualization of DEmRNAs in each sample, where the intersection size represents those common to all samples, intersection size stands for the common DEmRNAs among different samples. (d–e) gene ontology (GO) annotation in leaves (d) and roots (e). (f,g) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment in leaves (f) and roots (g). (h,i) Mapman analysis of metabolism in leaves (h) and in roots (i), where each square represents mapping to the metabolic pathway and color represents up-regulation (red) or down-regulation (blue). Full article ">Figure 2
Identification and analysis of DElncRNAs under salt stress. (a–d) Comparison of transcript length, exon number, fragments per kilobase of transcript per million mapped reads (FPKM) value, numbers and ORF length in lncRNA versus mRNA. (e) Venn diagram of DElncRNAs in leaves and roots. (f,g) Heat map of DElncRNAs in leaves (g) and in roots. (h) GO enrichment of targets of DElncRNAs in roots; (i) KEGG enrichment of targets of roots DElncRNAs. Full article ">Figure 3
Identification and analysis of DEcircRNAs under salt stress. (a,b) All identified circRNAs chromosome distribution (a) and type distribution (b). (c) Venn diagram of DEcircRNAs in leaves and roots. (d) Number of DEcircRNAs in leaf and root. (e,f) Heat map of DEcircRNAs in leaves (e) and in roots (f). Full article ">Figure 4
Identification and analysis of DEmiRNAs under salt stress. (a,b) Identified miRNAs length distribution (a) and 5′ nucleotide composition (b). (c) Venn diagram of identified miRNAs in each sample. (d,e) Heat map of DEmiRNAs in leaves (d) and in roots (e). (f,g) GO annotation of targets of DEmiRNAs in leaves (f) and in roots (g). Full article ">Figure 5
Salt stress response ceRNA network constructed with DEmRNAs, DElncRNAs and DEmiRNAs in leaves (a) and roots (b). The color represents up-regulation (red) or down-regulation (green). Red solid lines represent regulation verified by degradome sequencing. Full article ">Figure 6
T-plots and alignments of miRNA–mRNA pairs validated by degradome sequencing. (a) bv1_023200_jmkt.t1 cleaved by gma-miR408a-3p_L-1R+5; (b) bv6_144730_qgsa.t1 cleaved by mtr-miR408-3p_L-1R+1; (c) bv1_023200_jmkt.t1 cleaved by mtr-miR408-3p_L-1R+1; (d) bv1_023200_jmkt.t1 cleaved by mtr-MIR408-p3_2ss18GT19GA; (e) bv6_144730_qgsa.t1 cleaved by mtr-MIR408-p3_2ss18GT19GA; (f) bv5_114390_pjnp.t1 cleaved by mtr-miR164d; (g) bv6_136680_juie.t1 cleaved by PC-3p-154_19269; (h) bv6_136690_fscn.t1 cleaved by PC-3p-154_19269; (i) bv_009820_mrec.t1 cleaved by mtr-miR395k_1ss1TC. The ‘lightning’ shape indicates the position of the cleavage site. Full article ">Figure 7
qRT-PCR analysis of all 50 components of the ceRNA network under salt stress in (a) leaves and (b) roots. FC(ST/CK) represents fold changes of relative expression levels between ST and CK, log2FC(ST/CK) is from the mean of three replicates, bars stand for ±SD, asterisk (*) indicates that the gene is not detected in ST and its value is artificially set to −4 instead of calculating log2(0). Full article ">Figure 8
Proposed model of ceRNA network that regulates response to salt stress in sugar beet. SS: sucrose synthase, PFK: fructose phosphate kinase, HK: hexokinase, PK: pyruvate kinase, PDH: pyruvate dehydrogenase, MDH: malic dehydrogenase. Red filling represents up-regulation, whereas green filling represents down-regulation. Full article ">

19 pages, 3402 KiB

Open AccessArticle

Compensation Mechanism of the Photosynthetic Apparatus in Arabidopsis thaliana ch1 Mutants

by Joanna Wójtowicz, Adam K. Jagielski, Agnieszka Mostowska and Katarzyna B. Gieczewska

Int. J. Mol. Sci. 2021, 22(1), 221; https://doi.org/10.3390/ijms22010221 - 28 Dec 2020

Cited by 8 | Viewed by 3026

Abstract

The origin of chlorophyll b deficiency is a mutation (ch1) in chlorophyllide a oxygenase (CAO), the enzyme responsible for Chl b synthesis. Regulation of Chl b synthesis is essential for understanding the mechanism of plant acclimation to various conditions. Therefore, the [...] Read more.

The origin of chlorophyll b deficiency is a mutation (ch1) in chlorophyllide a oxygenase (CAO), the enzyme responsible for Chl b synthesis. Regulation of Chl b synthesis is essential for understanding the mechanism of plant acclimation to various conditions. Therefore, the main aim of this study was to find the strategy in plants for compensation of low chlorophyll content by characterizing and comparing the performance and spectral properties of the photosynthetic apparatus related to the lipid and protein composition in four selected Arabidopsis ch1 mutants and two Arabidopsis ecotypes. Mutation in different loci of the CAO gene, viz., NW41, ch1.1, ch1.2 and ch1.3, manifested itself in a distinct chlorina phenotype, pigment and photosynthetic protein composition. Changes in the CAO mRNA levels and chlorophyllide a (Chlide a) content in ecotypes and ch1 mutants indicated their significant role in the adjustment mechanism of the photosynthetic apparatus to low-light conditions. Exposure of mutants with a lower chlorophyll b content to short-term (1LL) and long-term low-light stress (10LL) enabled showing a shift in the structure of the PSI and PSII complexes via spectral analysis and the thylakoid composition studies. We demonstrated that both ecotypes, Col-1 and Ler-0, reacted to high-light (HL) conditions in a way remarkably resembling the response of ch1 mutants to normal (NL) conditions. We also presented possible ways of regulating the conversion of chlorophyll a to b depending on the type of light stress conditions. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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Figure 1

Figure 1
Characterization of Arabidopsis plant phenotypes and analysis of the composition and arrangement of chlorophyll–protein (CP) complexes: (A)—morphological phenotypes of 8-week-old mutants ch1.1, ch1.2, ch1.3 and NW41 and Arabidopsis ecotypes: Ler-0, Col-1, in normal light (NL) conditions; (B)—native PAGE separation of CP complexes from thylakoids of plants grown in NL, low-light (LL) and high-light (HL) conditions; 1LL, 10LL—after 1st and 10th day of treatment; the picture shows true colors; a total of 8.3 µg of total chlorophyll was loaded into each well; interpretation of bands indicated in the figure. NW41 analysis was impossible due to a lack of material with acceptable quality. Full article ">Figure 2
Relative optical density of Lhcb1-6 proteins isolated from NW41, ch1.1, ch1.2, ch1.3, Ler-0 and Col-1 Arabidopsis plants in LL (low-light) and HL (high-light) conditions; in reference to NL (normal light) conditions; 1LL, 10LL—after 1st and 10th day of treatment. The data were obtained from immunoblot analysis of SDS-PAGE gels with 2 µg of total chlorophyll per well. Presented data are mean values ± SD from 3 independent experiments; pairs of results marked with an asterisk differ significantly at p = 0.05 (one-way ANOVA with the post hoc Tukey test). Full article ">Figure 3
Spectroscopic analysis of thylakoids isolated from NW41, ch1.1, ch1.2, ch1.3, Ler-0 and Col-1 Arabidopsis plants in NL (normal light), LL (low-light) and HL (high-light) conditions; 1LL, 10LL—after 1st and 10th day of treatment. Fluorescence emission spectra at 77K, excited at 470 nm, Chl concentration of 10 μg/mL in 20 mM HEPES-NaOH buffer (pH 7.5) containing 15 mM NaCl, 4 mM MgCl2 and 80% (v/v) glycerol. The spectra were normalized to the area of 100 under the spectrum. The presented spectra are representative of three separate experiments. Fluorescence emission at 685 and 695 nm corresponds to the PSII core and inner antenna, at 730 and 695 nm to the PSI core and inner antenna, at 681 to the LHCII trimers (outer PSII antennae) and at 700 nm to LHCII macroaggregates. Full article ">Figure 4
Relative mRNA levels of genes corresponding to antenna and core proteins of PSI and PSII: (A)—in reference to the corresponding ecotype in NL (normal light) conditions; (B)—in reference to normal growth conditions (NL) of every analyzed Arabidopsis plant in different stress conditions (1LL, 10LL, HL) for PSII genes. LL—low-light conditions (1, 10—after 1st and 10th day of treatment), HL—high-light conditions. The data are mean values ± SD from 3–4 independent experiments; results marked with an asterisk differ significantly at p = 0.05 from the corresponding ecotype/plant in NL conditions. Full article ">Figure 5
Relative mRNA levels of the CAO gene expression: (A)—in reference to the corresponding ecotype in NL (normal light) conditions, (B)—in reference to NL conditions for each plant; NL—normal light, LL—low-light conditions (1, 10—after 1st and 10th day of treatment), HL—high-light conditions. The data are mean values ± SD from 3 independent experiments; results marked with an asterisk differ significantly at p = 0.05 from the corresponding ecotype/plant in NL conditions. Full article ">Figure 6
Characterization of chlorophyllide a content in thylakoid extracts of analyzed plants: (A)—in proportion to the corresponding ecotype in NL conditions; (B)—in proportion to normal growth conditions (NL) of every analyzed Arabidopsis plant in different stress conditions (1LL, 10LL, HL) separately. NL—normal light, LL—low-light conditions (1, 10—after 1st and 10th day of treatment), HL—high-light conditions. The data were obtained by HPLC analysis. The data are mean values ± SD from 3 independent experiments; results marked with an asterisk differ significantly at p = 0.05 from the corresponding ecotype/plant in NL conditions. Full article ">Figure 7
Changes in the Chla/b ratio in thylakoids isolated from all analyzed plants in NL (normal light), LL (low-light) and HL (high-light) conditions. The data were obtained by HPLC analysis. The data are mean values ± SD from 3 independent experiments; results marked with an asterisk differ significantly at p = 0.05 from each plant in NL conditions. Full article ">Figure 8
Routes of chlorophyll a to b conversion. Two possible routes of chlorophyll a to b conversion were previously described (Tanaka and Tanaka, 2019). In the first route (*), chlorophyll a is dephytilated by chlorophyllase and then converted to chlorophyll b by chlorophyllide a oxygenase (CAO) and chlorophyll synthase. In the second route (**), chlorophyll a is directly converted to chlorophyll b by CAO activity. Both of the routes were presented separately for all investigated plants in the applied light conditions: normal light (NL), low-light (LL), high-light (HL). (A)—for ch1 mutants: NW41, ch1.1, ch1.2, ch1.3, in LL; (B)—for ch1 mutants: NW41, ch1.1, ch1.2, ch1.3, in NL; (C)—control plants: Ler-0 and Col-1, in LL; (D)—control plants: Ler-0 and Col-1, in HL. Arrows correspond to the increase/decrease in a specified chlorophyllide/chlorophyll amount and level of CAO mRNA in response to the applied light conditions. Full article ">

18 pages, 24323 KiB

Open AccessArticle

Reducing Flower Competition for Assimilates by Half Results in Higher Yield of Fagopyrum esculentum

by Marta Hornyák, Aneta Słomka, Klaudia Sychta, Michał Dziurka, Przemysław Kopeć, Jakub Pastuszak, Anna Szczerba and Agnieszka Płażek

Int. J. Mol. Sci. 2020, 21(23), 8953; https://doi.org/10.3390/ijms21238953 - 25 Nov 2020

Cited by 7 | Viewed by 2758

Abstract

Despite abundant flowering throughout the season, common buckwheat develops a very low number of kernels probably due to competition for assimilates. We hypothesized that plants with a shorter flowering period may give a higher seed yield. To verify the hypothesis, we studied nutrient [...] Read more.

Despite abundant flowering throughout the season, common buckwheat develops a very low number of kernels probably due to competition for assimilates. We hypothesized that plants with a shorter flowering period may give a higher seed yield. To verify the hypothesis, we studied nutrient stress in vitro and in planta and analyzed different embryological and yield parameters, including hormone profile in the flowers. In vitro cultivated flowers on media with strongly reduced nutrient content demonstrated a drastic increase in degenerated embryo sacs. In in planta experiments, where 50% or 75% of flowers or all lateral ramifications were removed, the reduction of the flower competition by half turned out to be the most promising treatment for improving yield. This treatment increased the frequency of properly developed embryo sacs, the average number of mature seeds per plant, and their mass. Strong seed compensation under 50% inflorescence removal could result from increased production of salicylic and jasmonic acid that both favor more effective pollinator attraction. Plants in single-shoot cultivation finished their vegetation earlier, and they demonstrated greater single seed mass per plant than in control. This result suggests that plants of common buckwheat with shorter blooming period could deliver higher seed yield. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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Figure 1
Fagopyrum esculentum (cv. ‘Korona’) floral buds and their internal ovule and embryo sac development impairment cultured in vitro on media with different content of sugar, vitamins, and macro- and microelements. The same features were observed in cv. ‘Panda’: (a) bud laid out on Medium 1 with full content of nutrients at the time 0; (b) bud after 10 days of culture on Medium 1 with 30% content of nutrients; (c) bud after 10 days of culture on Medium 1 with full content of nutrients; (d,e) degeneration of the cells of embryo sacs (arrows); and (e) shrunken embryo sac (arrow). Bars: (d,e) 20 µm; and (f) 200 µm. For media content, see <a href="#sec4-ijms-21-08953" class="html-sec">Section 4</a>. Full article ">Figure 2
In planta degeneration of the embryo sacs cells and ovules after removal of 75% of flowers in Fagopyrum esculentum (cv. ‘Korona’). The same features were observed in cv. ‘Panda’: (a) 1-nucleate embryo sac (arrow); (b,c) egg apparatus of seven-cell embryo sacs (arrows); and (d) the whole ovule (arrow). Bars (a–c) 20 µm; and (d) 100 µm. Full article ">Figure 3
Normal pollen, female gametophyte and embryo development in Fagopyrum esculentum (cv. ‘Korona’) after removal of 50% of flowers. The same features were observed in cv. ‘Panda’: (a) tetrads of microspores in blue callose sheath (arrow); (b) microspores released from the callose sheath; (c) vacuolated microspores with thick sporodermis (arrow); (d) 1-nucleate pollen grains, nuclei visible (arrow); (e) degenerated (D) and normal (N) pollen grains stained with Alexander dye; (f,g) 2-nucleate embryo sac-successive stages, nuclei marked with arrows; (h–k) cells of two seven-cell embryo sacs (antipodal cells not shown) with secondary nucleus (arrow), egg cell (stars), and synergids (triangles); (i–k) successive stages of the same embryo sac; (l) pollen tube penetrating one of the two synergids (arrow); and (m) globular proembryo. Bars: (a,f–m) 20 µm; (b,c) 50 µm; and (d) 100 µm. Full article ">Figure 4
Macroscopic images of flower production (a,b) and seed setting (c–f) in cv. ‘Panda’ (a–d) and cv. ‘Korona’ (e,f) of Fagopyrum esculentum. Please compare control plants in bloom with plants from which 50% of flowers were removed:(a) vs. (b) and control plants in fruiting with plants with single shoot plants: (c) vs. (d,e) vs. (f). Full article ">Figure 5
Inflorescences and lateral stem removal performed during in planta experiments on Fagopyrum esculentum in 2019 and 2020. C, control plant with all lateral ramifications and with all inflorescences; 50%, plant with half of the spike of spikelets (every second removed); 75%, plant with only 25% of spike of spikelets (every second, third, and fourth removed); 1S, single shoot plant (all lateral ramifications removed). Full article ">

18 pages, 4843 KiB

Open AccessArticle

Wild Soybean Oxalyl-CoA Synthetase Degrades Oxalate and Affects the Tolerance to Cadmium and Aluminum Stresses

by Peiqi Xian, Zhandong Cai, Yanbo Cheng, Rongbin Lin, Tengxiang Lian, Qibin Ma and Hai Nian

Int. J. Mol. Sci. 2020, 21(22), 8869; https://doi.org/10.3390/ijms21228869 - 23 Nov 2020

Cited by 31 | Viewed by 3641

Abstract

Acyl activating enzyme 3 (AAE3) was identified as being involved in the acetylation pathway of oxalate degradation, which regulates the responses to biotic and abiotic stresses in various higher plants. Here, we investigated the role of Glycine sojaAAE3 (GsAAE3) in [...] Read more.

Acyl activating enzyme 3 (AAE3) was identified as being involved in the acetylation pathway of oxalate degradation, which regulates the responses to biotic and abiotic stresses in various higher plants. Here, we investigated the role of Glycine sojaAAE3 (GsAAE3) in Cadmium (Cd) and Aluminum (Al) tolerances. The recombinant GsAAE3 protein showed high activity toward oxalate, with a K_m of 105.10 ± 12.30 μM and V_max of 12.64 ± 0.34 μmol min⁻¹ mg⁻¹ protein, suggesting that it functions as an oxalyl–CoA synthetase. The expression of a GsAAE3–green fluorescent protein (GFP) fusion protein in tobacco leaves did not reveal a specific subcellular localization pattern of GsAAE3. An analysis of the GsAAE3 expression pattern revealed an increase in GsAAE3 expression in response to Cd and Al stresses, and it is mainly expressed in root tips. Furthermore, oxalate accumulation induced by Cd and Al contributes to the inhibition of root growth in wild soybean. Importantly, GsAAE3 overexpression increases Cd and Al tolerances in A. thaliana and soybean hairy roots, which is associated with a decrease in oxalate accumulation. Taken together, our data provide evidence that the GsAAE3-encoded protein plays an important role in coping with Cd and Al stresses. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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Figure 1

Figure 1
The effect of Cd and Al stresses and exogenous oxalate on wild soybean (BW69) root tips. (A) Cd and Al stresses induced oxalate accumulation. The seedlings were exposed to nutrient solution containing 0 or 30 μM CdCl2 or AlCl3 for 4 or 8 h. (B) The effect of exogenous oxalate on wild soybean root elongation and oxalate content. The seedlings were exposed to 0, 0.5, 1.0 mM sodium oxalate for 24 h. Root elongation was measured with a ruler before and after treatment (n = 16). After treatment, the root tips (0–2 cm) were ground into fine powder with liquid nitrogen and then extracted with distilled water for oxalate content analysis (n = 3). The lowercase letters mean statistical significance of comparisons of root elongation data, and the uppercase letters mean statistical significance of comparisons of oxalate content data. (C) Correlation between oxalate content and GsAAE3 expression (n = 3). The expression of GsAAE3 was determined by qRT-PCR. All data are presented as means ± SD. Different letters indicate statistically significant difference, using one-way ANOVA and Duncan’s test (p ≤ 0.05). Full article ">Figure 2
Phylogenetic analysis and multiple sequence alignment. (A) Phylogenetic trees of AAE3 proteins in representative species of major lineage of plants (sequences of representative species can be found in <a href="https://db.cngb.org/onekp/" target="_blank">https://db.cngb.org/onekp/</a>, accessed on 18 November 2020). Clades are indicated by different colors. (B) Phylogenetic analysis of AAE3 proteins in higher plants. All the available amino acid sequences and the accession numbers of AAE3 proteins were obtained from the NCBI databases (<a href="https://www.ncbi.nlm.nih.gov/" target="_blank">https://www.ncbi.nlm.nih.gov/</a>, accessed on 20 October 2020). The AAE3 proteins including Capsicum annuum (CaAAE3; NP_001311686), Solanum lycopersicum (SlAAE3; XP_004234395), Vitis vinifera (VvAAE3; XP_002267459.1), Amaranthus hypochondriacus (AhAAE3), Populus trichocarpa (PtAAE3; XP_002322473), Arabidopsis thaliana (AtAAE3; AT3G48990), Phaseolus vulgaris (PvAAE3; XP_007143422), Medicago truncatula (MtAAE3; XP_003599555), Vigna umbellate (VuAAE3; KX354978), Glycine max (GmAAE3-1; XP_003534000.1, GmAAE3-2; XP_014619651.1), Glycine soja (GsAAE3; BW679327.1), Fagopyrum esculentum (FeAAE3-1 and FeAAE3-2), Ricinus communis (RcAAE3; XP_0002509782), Zea mays (ZmAAE3; AEY64280), Sorghum bicolor (SbAAE3; KXG27467), Setaria italic (SiAAE3; XP_004960018), Oryza sativa (OsAAE3; Os04g0683700), Hordeum vulgare (HvAAE3; BAK00674), and Brachypodium distachyon (BdAAE3; XP_003579506). (C) Multiple sequence alignment of AAE3 proteins from Glycine soja (GsAAE3), Vigna umbellate (VuAAE3), Arabidopsis thaliana (AtAAE3), Medicago truncatula (MtAAE3), Amaranthus hypochondriacus (AhAAE3), and Fagopyrum esculentum (FeAAE3-1 and FeAAE3-2). The conserved AMP binding domain and acetyl-CoA synthetase domain are indicated. Full article ">Figure 3
Expression pattern analysis of GsAAE3. (A) Tissue expression pattern of GsAAE3. The samples of whole roots, stems, leaves, apex, flowers, pods, and seeds were collected when the plants were already in seed-filling stage. (B) (β-glucuronidase) GUS staining in GsAAE3 promoter-GUS transgenic hairy roots. (C) Dose-dependent expression of GsAAE3 in wild soybean root tips (0–2 cm). The seedlings were cultured in the nutrient solution added with 0, 7.5, 15, 30, or 50 μM CdCl2 or AlCl3 for 4 h. (D) Time-dependent expression of GsAAE3 in wild soybean root tips (0–2 cm). The seedlings were cultured in the nutrient solution added with 30 μM CdCl2 or AlCl3 for 0, 1, 2, 4, 8, 12, or 24 h. The expression of GsAAE3 was determined by qRT-PCR. Three independent biological replicates were performed, and the data are presented as the means ± SD. Different letters indicate statistically significant difference, using one-way ANOVA and Duncan’s test (p ≤ 0.05). Full article ">Figure 4
Biochemical analysis of GsAAE3. (A) SDS-PAGE gel of Ni-NTA purified recombinant protein (right), unpurified recombinant (middle), and molecular weight markers (left). (B) Kinetic analysis of GsAAE3 using a range of oxalate concentrations. Km and Vmax were determined from non-linear regression to the Michaelis–Menten kinetics for concentrations up to 1500 μM oxalate. Data are means ± SD of three independent biological replicates. Full article ">Figure 5
Subcellular localization of GsAAE3. (A,B) Green fluorescent protein (GFP) fluorescence images of tobacco leaves lower epidermal cells transiently expressing 35S::GFP and 35S::GsAAE3-GFP. DAPI (4′, 6-Diamidino-2-Phenylindole), a cell nucleus-specific fluorescence dye, was used to stain cell nucleus. Full article ">Figure 6
Overexpression of GsAAE3 in A. thaliana. (A,D) Cd and Al tolerances phenotypes of overexpressing GsAAE3 in transgenic A. thaliana lines. (B,E) Statistical analysis of relative root elongation. (C,F) The effect of Cd and Al stresses on oxalate content in wild-type and transgenic A. thaliana lines. Four days after vernalization, the T3 transgenic seeds (OE3 and OE8) and the wild-type (columbia-0) were cultivated on the Cd concentration gradients agar medium added with 0, 25, and 50 μM CdCl2 or Al concentration gradients agar medium added with 0, 25, and 50 μM AlCl3. After 7 days in culture, the images of the phenotypes of the GsAAE3 transgenic lines were recorded for statistical analysis. The root elongation was measured using Image J software (n = 3). After treatment, the roots of A. thaliana were ground into fine powder with liquid nitrogen and then extracted with distilled water for oxalate content analysis (n = 3). All data are presented as means ± SD. Different letters indicate statistically significant difference, using one-way ANOVA and Duncan’s test (p ≤ 0.05). Full article ">Figure 7
Performance of GsAAE3 transgenic hairy roots under Cd and Al stresses. (A) Cd and Al tolerance phenotypes of overexpressing GsAAE3 in soybean hairy roots. (B) Relative expression of GsAAE3 in transgenic and control hairy roots under Cd and Al stresses and blank. (C) Relative fresh weight of hairy roots in transgenic and control plants under Cd and Al stresses. (D,E) The effect of Cd and Al stresses on Cd and Al concentrations in control and transgenic soybean hairy roots. (F,G) The effect of Cd and Al stresses on oxalate content in control and transgenic soybean hairy roots. The expression of GsAAE3 was determined by qRT-PCR. The consistent growth positive transgenic and control hairy roots were chosen to transfer into nutrient solution supplemented with 23 μM CdCl2 or 25 μM AlCl3 or blank for 4 days. After treatment, the images of the phenotypes were recorded. The fresh weight and the concentrations of Cd and Al in transgenic and control hairy roots were measured. In addition, the soybean hairy roots were ground into fine powder with liquid nitrogen and then extracted with distilled water for oxalate content analysis. Three independent biological replicates were performed, and the data are presented as the means ± SD. Different letters indicate statistically significant difference, using one-way ANOVA and Duncan’s test (p ≤ 0.05). Full article ">

18 pages, 1034 KiB

Open AccessArticle

Metal Homeostasis and Gas Exchange Dynamics in Pisum sativum L. Exposed to Cerium Oxide Nanoparticles

by Elżbieta Skiba, Monika Pietrzak, Magdalena Gapińska and Wojciech M. Wolf

Int. J. Mol. Sci. 2020, 21(22), 8497; https://doi.org/10.3390/ijms21228497 - 11 Nov 2020

Cited by 17 | Viewed by 3339

Abstract

Cerium dioxide nanoparticles are pollutants of emerging concern. They are rarely immobilized in the environment. This study extends our work on Pisum sativum L. as a model plant, cultivated worldwide, and is well suited for investigating additive interactions induced by nanoceria. Hydroponic cultivation, [...] Read more.

Cerium dioxide nanoparticles are pollutants of emerging concern. They are rarely immobilized in the environment. This study extends our work on Pisum sativum L. as a model plant, cultivated worldwide, and is well suited for investigating additive interactions induced by nanoceria. Hydroponic cultivation, which prompts accurate plant growth control and three levels of CeO₂ supplementation, were applied, namely, 100, 200, and 500 mg (Ce)/L. Phytotoxicity was estimated by fresh weights and photosynthesis parameters. Additionally, Ce, Cu, Zn, Mn, Fe, Ca, and Mg contents were analyzed by high-resolution continuum source atomic absorption and inductively coupled plasma optical emission techniques. Analysis of variance has proved that CeO₂ nanoparticles affected metals uptake. In the roots, it decreased for Cu, Zn, Mn, Fe, and Mg, while a reversed process was observed for Ca. The latter is absorbed more intensively, but translocation to above-ground parts is hampered. At the same time, nanoparticulate CeO₂ reduced Cu, Zn, Mn, Fe, and Ca accumulation in pea shoots. The lowest Ce concentration boosted the photosynthesis rate, while the remaining treatments did not induce significant changes. Plant growth stimulation was observed only for the 100 mg/L. To our knowledge, this is the first study that demonstrates the effect of nanoceria on photosynthesis-related parameters in peas. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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31 pages, 10428 KiB

Open AccessArticle

Growth and Photosynthetic Activity of Selected Spelt Varieties (Triticum aestivum ssp. spelta L.) Cultivated under Drought Conditions with Different Endophytic Core Microbiomes

by Karolina Ratajczak, Hanna Sulewska, Lidia Błaszczyk, Aneta Basińska-Barczak, Katarzyna Mikołajczak, Sylwia Salamon, Grażyna Szymańska and Leszek Dryjański

Int. J. Mol. Sci. 2020, 21(21), 7987; https://doi.org/10.3390/ijms21217987 - 27 Oct 2020

Cited by 8 | Viewed by 3058

Abstract

The role of the microbiome in the root zone is critically important for plants. However, the mechanism by which plants can adapt to environmental constraints, especially water deficit, has not been fully investigated to date, while the endophytic core microbiome of the roots [...] Read more.

The role of the microbiome in the root zone is critically important for plants. However, the mechanism by which plants can adapt to environmental constraints, especially water deficit, has not been fully investigated to date, while the endophytic core microbiome of the roots of spelt (Triticum aestivum ssp. spelta L.) grown under drought conditions has received little attention. In this study, we hypothesize that differences in the endophytic core of spelt and common wheat root microbiomes can explain the variations in the growth and photosynthetic activity of those plants, especially under drought conditions. Our greenhouse experimental design was completely randomized in a 2 × 4 × 3 factorial scheme: two water regime levels (well-watered and drought), three spelt varieties (T. aestivum ssp. spelta L.: ‘Badenstern’, ‘Badenkrone’ and ‘Zollernspelz’ and one wheat variety: T. aestivum ssp. vulgare L: ‘Dakotana’) and three mycorrhizal levels (autoclaved soil inoculation with Rhizophagus irregularis, control (autoclaved soil) and natural inoculation (non-autoclaved soil—microorganisms from the field). During the imposed stress period, relative water content (RWC), leaf chlorophyll fluorescence, gas exchange and water use efficiency (WUE) were measured. Microscopic observations of the root surface through fungi isolation and identification were conducted. Our results indicate that ‘Badenstern’ was the most drought tolerant variety, followed by ‘Zollernspelz’ and ‘Badenkrone,’ while the common wheat variety ‘Dakotana’ was the most drought sensitive. Inoculation of ‘Badenstern’ with the mycorrhizal fungi R. irregularis contributed to better growth performance as evidenced by increased whole plant and stalk dry matter accumulation, as well as greater root length and volume. Inoculation of ‘Zollernspelz’ with arbuscular mycorrhizal fungi (AMF) enhanced the photochemical efficiency of Photosystem II and significantly improved root growth under drought conditions, which was confirmed by enhanced aboveground biomass, root dry weight and length. This study provides evidence that AMF have the potential to be beneficial for plant growth and dry matter accumulation in spelt varieties grown under drought conditions. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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19 pages, 3232 KiB

Open AccessArticle

The Grapevine Calmodulin-Like Protein Gene CML21 Is Regulated by Alternative Splicing and Involved in Abiotic Stress Response

by Olga A. Aleynova, Konstantin V. Kiselev, Zlata V. Ogneva and Alexandra S. Dubrovina

Int. J. Mol. Sci. 2020, 21(21), 7939; https://doi.org/10.3390/ijms21217939 - 26 Oct 2020

Cited by 38 | Viewed by 3267

Abstract

Calmodulin-like proteins (CMLs) represent a large family of plant calcium sensor proteins involved in the regulation of plant responses to environmental cues and developmental processes. In the present work, we identified four alternatively spliced mRNA forms of the grapevine CML21 gene that encoded [...] Read more.

Calmodulin-like proteins (CMLs) represent a large family of plant calcium sensor proteins involved in the regulation of plant responses to environmental cues and developmental processes. In the present work, we identified four alternatively spliced mRNA forms of the grapevine CML21 gene that encoded proteins with distinct N-terminal regions. We studied the transcript abundance of CML21v1, CML21v2, CML21v3, and CML21v4 in wild-growing grapevine Vitis amurensis Rupr. in response to desiccation, heat, cold, high salinity, and high mannitol stress using quantitative real-time RT-PCR. The levels of all four splice variants of VaCML21 were highly induced in response to cold stress. In addition, VaCML21v1 and VaCML21v2 forms were highly modulated by all other abiotic stress treatments. Constitutive expression of VaCML21v2 and VaCML21v4 improved biomass accumulation of V. amurensis callus cell cultures under prolonged low temperature stress. Heterologous expression of the grapevine CML21v2 and VaCML21v4 splice variants in Arabidopsis improved survival rates of the transgenic plants after freezing. The VaCML21v2 overexpression enhanced activation of the cold stress-responsive marker genes AtDREB1A and AtDREB2A, while VaCML21v4 overexpression—AtCOR47, AtRD29A, AtRD29B, and AtKIN1 genes after freezing stress in the transgenic Arabidopsis. The results indicate that the grapevine CML21 gene acts as a positive regulator in the plant response to cold stress. The detected variety of CML21 transcripts and their distinct transcriptional responses suggested that this expansion of mRNA variants could contribute to the diversity of grapevine adaptive reactions. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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Figure 1
The structure representation and protein sequence analysis of CML21 mRNA splice variants in grapevine. (a) Alternatively spliced CML21 transcript variants of Vitis amurensis identified in this work (VaCML21v2, VaCML21v3, VaCML21v4) and CML21 transcript variants of Vitis vinifera (VviCML21v1, VviCML21v2, VviCML21v3, VviCML21v4) predicted by genome sequence analysis and retrieved from the databases. Exons and introns are shown using boxes and lines, respectively, with white dashed boxes representing untranslated regions (UTRs). (b) Alignment and conserved motif analysis of CML21 deduced proteins in V. amurensis and V. vinifera. The EF hands were predicted by PROSITE scan [<a href="#B31-ijms-21-07939" class="html-bibr">31</a>,<a href="#B32-ijms-21-07939" class="html-bibr">32</a>] and the lipid modification sites were predicted using GPS-Lipid [<a href="#B33-ijms-21-07939" class="html-bibr">33</a>,<a href="#B34-ijms-21-07939" class="html-bibr">34</a>]. Multiple sequence alignment was conducted using BioEdit software [<a href="#B35-ijms-21-07939" class="html-bibr">35</a>]. Full article ">Figure 2
Expression analysis of VaCML21v1 (a), VaCML21v2 (b), VaCML21v3 (c), and VaCML21v4 (d) splice variants 6 h, 12 h, and 24 h post-treatment in the leaves of V. amurensis exposed to control (Control, filtered water, +25 °C), water-deficit (WD, cuttings laid on a paper towel, +25 °C), high salt (0.4 M NaCl, +25 °C), osmoticum (0.4 M mannitol, +25 °C), low temperature (+10 °C and +4 °C), and high temperature (+37 °C) abiotic stress conditions. The expression of the CMLs was profiled by quantitative real-time RT-PCR. The data are presented as the mean ± SE (three independent experiments). *, **—significantly different from the Control for each time point at p ≤ 0.05 and 0.01 according to Student’s t-test. Full article ">Figure 3
Expression levels of the CML21v1, CML21v2, and CML21v3 splice variants in Vitis vinifera in the most-damaged cultivar “Cabernet Franc” and the least-damaged cultivar “Sangiovese” cultivated under low-temperature stress conditions. The RNAseq libraries were obtained by Londo et al. [<a href="#B37-ijms-21-07939" class="html-bibr">37</a>] and analyzed in the present study for VviCML21 abundance. The V. vinifera plants were exposed to control treatment, freezing (gradual freezing to −3 °C for 45 min), acclimation or chilling (+4 °C for 48 h), and acclimated freezing (+4 °C for 48 h and −3 °C for 45 min). RPM—reads per million mapped reads. The data are presented as the mean ± SE. Means followed by the same letter were not different using one-way analysis of variance (ANOVA), followed by the Tukey HSD multiple comparison test. A value of p ≤ 0.05 was considered significant. Full article ">Figure 4
Characterization of the grapevine callus cell cultures transformed with the VaCML21v2 and VaCMLv4 splice variants. (a,b) Quantification of the VaCML21v2 and VaCML21v4 transgene mRNAs; (c,d) Quantification of the endogenous VaCML21v1/v2 and VaCML21v3/v4 mRNAs. (e,f) Fresh biomass accumulation in the VaCML21v2-transgenic and VaCML21v4-transgenic cell cultures after 30-day cultivation. v2-1, v2-2, v2-3—VaCML21v2-transformed calli; v4-1, v4-2, v4-3—VaCML21v4-transformed calli. KA-0—control cell culture transformed with the “empty” vector. The data are presented as the mean ± SE (two independent experiments). * p ≤ 0.05; ** p ≤ 0.01 versus values of VaCML21 expression in the empty vector-transformed KA-0 cell culture according to Student’s t-test. Full article ">Figure 5
Characterization and responses to freezing of vector control (KA-0), VaCML21v2- (v2L1, v2L2, v2L3) and VaCML21v4-transgenic (v4L1, v4L2, v4L3) Arabidopsis. (a,b) Quantification of the VaCML21v2 and VaCML21v4 mRNAs in A. thaliana performed by qRT-PCR. (c,d) 4-week-old KA-0 and VaCML21-transgenic plants were stressed at −10 °C for 45 min and then cultured at +4 °C for 1 h for recovery. Photographs of representative seedlings were taken after 7 d of recovery. (e) Survival rates determined as the number of visibly green plants 7 d after freezing. Values are the mean ± SE. Six plants of each line were used in each of ten experiments. Means followed by the same letter were not different using one-way analysis of variance (ANOVA), followed by the Tukey HSD multiple comparison test. A value of p ≤ 0.05 was considered as significant. Full article ">Figure 6
Influence of temperature (a,b), salt (c,d) and osmotic (e,f) stresses on the fresh biomass accumulation in the transgenic grapevine callus cell lines overexpressing the VaCML21v2 and VaCML21v4 splice variants. The average growth rates were assessed after 30 days of cultivation in the dark under the control (24 °C), cold stress (+16 °C), heat stress (+37 °C), salt stress (NaCl 50 and 100 mM), and high-mannitol (200 and 300 mM) conditions. v2-1, v2-2, v2-3—VaCML21v2-transformed calli; v4-1, v4-2, v4-3—VaCML21v4-transformed calli. KA-0—control cell culture transformed with the “empty” vector. The data are presented as the mean ± SE (two independent experiments with ten replicates each). Means followed by the same letter were not different using one-way analysis of variance (ANOVA), followed by the Tukey HSD multiple comparison test. A value of p ≤ 0.05 was considered as significant. Full article ">Figure 7
The expression of stress-responsive genes AtCOR47 (a), AtDREB1A (b), AtDREB2A (c), AtKIN1 (d), AtRD29A (e), and AtRD29B (f) in transgenic Arabidopsis transformed with the VaCML21v2 and VaCML21v4 splice variants in response to freezing stress. The 4-week-old control KA-0, 35S::VaCML21v2 (lines v2L1, v2L2, and v2L3), and 35S::VaCML21v4 (lines v4L1, v4L2, and v4L3) plants were exposed to control conditions (at 22 °C) or freezing (at −10 °C for 45 min and then cultured at +4 °C for 1 h for recovery). Total RNA was extracted from plants just before freezing (white bars) and 1 h after freezing (grey bars). The data are presented as the mean ± SE (two independent experiments). Means followed by the same letter were not different using one-way analysis of variance (ANOVA), followed by the Tukey HSD multiple comparison test. A value of p ≤ 0.05 was considered significant. Full article ">

17 pages, 8310 KiB

Open AccessArticle

Molecular Mechanisms Underlying Sugarcane Response to Aluminum Stress by RNA-Seq

by Thiago Mateus Rosa-Santos, Renan Gonçalves da Silva, Poornasree Kumar, Pratibha Kottapalli, Chiquito Crasto, Kameswara Rao Kottapalli, Suzelei Castro França and Sonia Marli Zingaretti

Int. J. Mol. Sci. 2020, 21(21), 7934; https://doi.org/10.3390/ijms21217934 - 26 Oct 2020

Cited by 17 | Viewed by 3567

Abstract

Some metals are beneficial to plants and contribute to critical physiological processes. Some metals, however, are not. The presence of aluminum ions (Al³⁺) can be very toxic, especially in acidic soils. Considerable parts of the world’s arable land are acidic in [...] Read more.

Some metals are beneficial to plants and contribute to critical physiological processes. Some metals, however, are not. The presence of aluminum ions (Al³⁺) can be very toxic, especially in acidic soils. Considerable parts of the world’s arable land are acidic in nature; mechanistically elucidating a plant’s response to aluminum stress is critical to mitigating this stress and improving the quality of plants. To identify the genes involved in sugarcane response to aluminum stress, we generated 372 million paired-end RNA sequencing reads from the roots of CTC-2 and RB855453, which are two contrasting cultivars. Data normalization resulted in 162,161 contigs (contiguous sequences) and 97,335 genes from a de novo transcriptome assembly (trinity genes). A total of 4858 and 1307 differently expressed genes (DEGs) for treatment versus control were identified for the CTC-2 and RB855453 cultivars, respectively. The DEGs were annotated into 34 functional categories. The majority of the genes were upregulated in the CTC-2 (tolerant cultivar) and downregulated in RB855453 (sensitive cultivar). Here, we present the first root transcriptome of sugarcane under aluminum stress. The results and conclusions of this study are a crucial launch pad for future genetic and genomic studies of sugarcane. The transcriptome analysis shows that sugarcane tolerance to aluminum may be explained by an efficient detoxification mechanism combined with lateral root formation and activation of redox enzymes. We also present a hypothetical model for aluminum tolerance in the CTC-2 cultivar. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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29 pages, 6350 KiB

Open AccessArticle

Precise Editing of the OsPYL9 Gene by RNA-Guided Cas9 Nuclease Confers Enhanced Drought Tolerance and Grain Yield in Rice (Oryza sativa L.) by Regulating Circadian Rhythm and Abiotic Stress Responsive Proteins

by Babar Usman, Gul Nawaz, Neng Zhao, Shanyue Liao, Yaoguang Liu and Rongbai Li

Int. J. Mol. Sci. 2020, 21(21), 7854; https://doi.org/10.3390/ijms21217854 - 23 Oct 2020

Cited by 85 | Viewed by 6927

Abstract

Abscisic acid (ABA) is involved in regulating drought tolerance, and pyrabactin resistance-like (PYL) proteins are known as ABA receptors. To elucidate the role of one of the ABA receptors in rice, OsPYL9 was mutagenized through CRISPR/Cas9 in rice. Homozygous and heterozygous mutant plants [...] Read more.

Abscisic acid (ABA) is involved in regulating drought tolerance, and pyrabactin resistance-like (PYL) proteins are known as ABA receptors. To elucidate the role of one of the ABA receptors in rice, OsPYL9 was mutagenized through CRISPR/Cas9 in rice. Homozygous and heterozygous mutant plants lacking any off-targets and T-DNA were screened based on site-specific sequencing and used for morpho-physiological, molecular, and proteomic analysis. Mutant lines appear to accumulate higher ABA, antioxidant activities, chlorophyll content, leaf cuticular wax, and survival rate, whereas a lower malondialdehyde level, stomatal conductance, transpiration rate, and vascular bundles occur under stress conditions. Proteomic analysis found a total of 324 differentially expressed proteins (DEPs), out of which 184 and 140 were up and downregulated, respectively. The OsPYL9 mutants showed an increase in grain yield under both drought and well watered field conditions. Most of the DEPs related to circadian clock rhythm, drought response, and reactive oxygen species were upregulated in the mutant plants. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that DEPs were only involved in circadian rhythm and Gene Ontology (GO) analysis showed that most of the DEPs were involved in response to abiotic stimulus, and abscisic acid-activated signaling pathways. Protein GIGANTEA, Adagio-like, and Pseudo-response regulator proteins showed higher interaction in protein–protein interaction (PPI) network. Thus, the overall results showed that CRISPR/Cas9-generated OsPYL9 mutants have potential to improve both drought tolerance and the yield of rice. Furthermore, global proteome analysis provides new potential biomarkers and understandings of the molecular mechanism of rice drought tolerance. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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21 pages, 4183 KiB

Open AccessArticle

Genome-Wide Identification of the Gossypium hirsutum NHX Genes Reveals That the Endosomal-Type GhNHX4A Is Critical for the Salt Tolerance of Cotton

by Wenyu Ma, Zhongying Ren, Yang Zhou, Junjie Zhao, Fei Zhang, Junping Feng, Wei Liu and Xiongfeng Ma

Int. J. Mol. Sci. 2020, 21(20), 7712; https://doi.org/10.3390/ijms21207712 - 18 Oct 2020

Cited by 23 | Viewed by 3701

Abstract

Soil salinization, which is primarily due to excessive Na⁺ levels, is a major abiotic stress adversely affecting plant growth and development. The Na⁺/H⁺ antiporter (NHX) is a transmembrane protein mediating the transport of Na⁺ or K⁺ and [...] Read more.

Soil salinization, which is primarily due to excessive Na⁺ levels, is a major abiotic stress adversely affecting plant growth and development. The Na⁺/H⁺ antiporter (NHX) is a transmembrane protein mediating the transport of Na⁺ or K⁺ and H⁺ across the membrane to modulate the ionic balance of plants in response to salt stress. Research regarding NHXs has mainly focused on the vacuolar-type NHX family members. However, the biological functions of the endosomal-type NHXs remain relatively uncharacterized. In this study, 22 NHX family members were identified in Gossypium hirsutum. A phylogenetic analysis divided the GhNHX genes into two categories, with 18 and 4 in the vacuolar and endosomal groups, respectively. The chromosomal distribution of the NHX genes revealed the significant impact of genome-wide duplication during the polyploidization process on the number of GhNHX genes. Analyses of gene structures and conserved motifs indicated that GhNHX genes in the same phylogenetic cluster are conserved. Additionally, the salt-induced expression patterns confirmed that the expression levels of most of the GhNHX genes are affected by salinity. Specifically, in the endosomal group, GhNHX4A expression was substantially up-regulated by salt stress. A yeast functional complementation test proved that GhNHX4A can partially restore the salt tolerance of the salt-sensitive yeast mutant AXT3. Silencing GhNHX4A expression decreased the resistance of cotton to salt stress because of an increase in the accumulation of Na⁺ in stems and a decrease in the accumulation of K⁺ in roots. The results of this study may provide the basis for an in-depth characterization of the regulatory functions of NHX genes related to cotton salt tolerance, especially the endosomal-type GhNHX4A. Furthermore, the presented data may be useful for selecting appropriate candidate genes for the breeding of new salt-tolerant cotton varieties. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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Figure 1
Phylogenetic analysis of the Na+/H+ antiporters (NHXs). The phylogenetic tree was generated with the neighbor-joining method of MEGA X (1000 bootstrap replicates) using 22 NHXs from G. hirsutum (Gh) (<a href="#app1-ijms-21-07712" class="html-app">Table S2</a>) and 49 NHXs from G. arboreum (Ga), G. raimondii (Gr), A. thaliana (At), P. trichocarpa (Pt), O. sativa (Os), and Z. mays (Zm) (<a href="#app1-ijms-21-07712" class="html-app">Tables S4 and S5</a>). The NHXs from G. hirsutum are presented by purple backgrounds, and the NHXs from A. thaliana are indicated by red stars. All NHXs are classified into one of two classes (Class 1: vacuolar-type, Class 2: endosomal-type), which are differentiated by different color arcs. Numbers on the branches are bootstrap values. Full article ">Figure 2
Chromosomal distribution and homologous relationships of the GhNHX genes. G. hirsutum chromosomes are presented in a circular form. The A and D subgenomes of G. hirsutum were labeled by blue and red fonts, respectively. Approximate positions of GhNHX genes are indicated with short gray lines on the circle. Homologous GhNHX genes between the A and D subgenomes are connected by yellow curves. The red and yellow circles in the middle represent the gene density on each chromosome, where red indicates high density and yellow indicates low density. Full article ">Figure 3
Phylogenetic relationships, gene structures, and motif architectures of GhNHX genes. (A) Phylogenetic relationships among GhNHX genes. The phylogenetic tree was constructed with the neighbor-joining method of MEGA X (1000 bootstrap replicates). All NHXs are classified into two classes, which are differentiated by color. The vacuolar-type members are indicated by orange branches, and the endosomal-type members are indicated by blue branches. Numbers on the branches are bootstrap values. (B) Exon-intron structures of GhNHX genes. Gene structure maps were drawn with the Gene Structure Display Server. The exons and introns are indicated by pink boxes and gray lines, respectively. (C) Motif compositions in proteins encoded by the GhNHX genes. Conserved motifs were predicted with the MEME software (<a href="http://meme-suite.org/" target="_blank">http://meme-suite.org/</a>). Motif lengths are presented proportionally. Full article ">Figure 4
Expression patterns of GhNHX genes in leaves after the 200 mM NaCl treatment. (A) Cluster analysis of expression profiles of GhNHX genes under the NaCl treatment. The cluster analysis was developed using the K-means method on the expression profiles of G. hirsutum NHX genes under the NaCl treatment. The expression pattern of each gene is indicated by a gray line, and the representative expression pattern of each cluster is represented by a purple line. The x-axis presents the examined time-points after the salt treatment, whereas the y-axis presents the scale of the relative expression levels. (B) Expression profiles of GhNHX genes under the NaCl treatment. The x-axis presents the examined time-points after the salt treatment, whereas the y-axis presents the relative expression levels. Error bars indicate the standard deviation of three biological replicates (* p < 0.05, ** p < 0.01; t-test). Full article ">Figure 5
Functional complementation of GhNHX4A in yeast mutants. W303: Wild-type strain W303; GhNHX4A: Mutant strain AXT3 transformed with pYES2-GhNHX4A; pYES2: Mutant strain AXT3 transformed with empty pYES2. Full article ">Figure 6
Subcellular localization of GhNHX4A in tobacco leaves. (A–D) 35S:GhNHX4A-eGFP, eGFP-tagged GhNHX4A; (E–H) 35S:eGFP, control; (A,E) Green fluorescence image; (B,F) Red fluorescence stained by FM4-64; (C,G) Red fluorescence, green fluorescence and bright field images are merged; (D,H) Bright field image. Full article ">Figure 7
Functional analysis of GhNHX4A in cotton response to salt stress. (A) Gene silencing efficiency of TRV:00 and TRV:GhNHX4A. TRV:CLA was used as the positive control, and GhHIS3 served as the internal reference gene. Error bars indicate the standard deviation of at least three biological replicates (** p < 0.01; t-test). (B) Representative images of TRV:00 and TRV:GhNHX4A plants treated with water (mock) or 200 mM NaCl. (C) Na+ and K+ contents and Na+/K+ ratios of the TRV:00 and TRV:GhNHX4A plants treated with water (mock) or NaCl. Red, blue, and purple refer to the roots, stems, and leaves, respectively. Error bars indicate the standard deviation of at least three biological replicates (** p < 0.01; t-test). Full article ">

21 pages, 1646 KiB

Open AccessArticle

The Anaerobic Product Ethanol Promotes Autophagy-Dependent Submergence Tolerance in Arabidopsis

by Li-Bing Yuan, Liang Chen, Ning Zhai, Ying Zhou, Shan-Shan Zhao, Li-Li Shi, Shi Xiao, Lu-Jun Yu and Li-Juan Xie

Int. J. Mol. Sci. 2020, 21(19), 7361; https://doi.org/10.3390/ijms21197361 - 5 Oct 2020

Cited by 15 | Viewed by 4042

Abstract

In response to hypoxia under submergence, plants switch from aerobic respiration to anaerobic fermentation, which leads to the accumulation of the end product, ethanol. We previously reported that Arabidopsis thaliana autophagy-deficient mutants show increased sensitivity to ethanol treatment, indicating that ethanol is likely [...] Read more.

In response to hypoxia under submergence, plants switch from aerobic respiration to anaerobic fermentation, which leads to the accumulation of the end product, ethanol. We previously reported that Arabidopsis thaliana autophagy-deficient mutants show increased sensitivity to ethanol treatment, indicating that ethanol is likely involved in regulating the autophagy-mediated hypoxia response. Here, using a transcriptomic analysis, we identified 3909 genes in Arabidopsis seedlings that were differentially expressed in response to ethanol treatment, including 2487 upregulated and 1422 downregulated genes. Ethanol treatment significantly upregulated genes involved in autophagy and the detoxification of reactive oxygen species. Using transgenic lines expressing AUTOPHAGY-RELATED PROTEIN 8e fused to green fluorescent protein (GFP-ATG8e), we confirmed that exogenous ethanol treatment promotes autophagosome formation in vivo. Phenotypic analysis showed that deletions in the alcohol dehydrogenase gene in adh1 mutants result in attenuated submergence tolerance, decreased accumulation of ATG proteins, and diminished submergence-induced autophagosome formation. Compared to the submergence-tolerant Arabidopsis accession Columbia (Col-0), the submergence-intolerant accession Landsberg erecta (Ler) displayed hypersensitivity to ethanol treatment; we linked these phenotypes to differences in the functions of ADH1 and the autophagy machinery between these accessions. Thus, ethanol promotes autophagy-mediated submergence tolerance in Arabidopsis. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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Figure 1
Differentially expressed genes in response to ethanol treatment. (A) Functional annotation of 2487 upregulated and 1422 downregulated genes after ethanol treatment. (B) Hierarchical clustering of differentially expressed ethanol-responsive genes from the AP2/ERF transcription factor subfamily. (C,E) Hierarchical clustering of differentially expressed ethanol-responsive genes from the ethylene (ET, C), jasmonic acid (JA, D), and salicylic acid (SA, E) biosynthesis and signaling pathways. (F,H) Hierarchical clustering of differentially expressed ethanol-responsive genes from the glutathione S-transferase (GST, F) gene family, reactive oxygen species (ROS, G)- and autophagy (ATG, H)-related genes. The log2 fold change values of the transcriptional profiles were calculated using the R program heatmap 2.0. Red and blue represent up- and downregulated genes, respectively. Differentially expressed genes were identified based on the criteria FDR < 0.005 and FC ≥ 1.5 or FC ≤ 0.67. Full article ">Figure 2
Induction of autophagy by ethanol treatment. (A) Confocal analysis of changes in transgenic lines expressing autophagy-related protein 8e fused to green fluorescent protein (GFP-ATG8e) in leaf cells in response to ethanol treatment. Leaves of 4-week-old GFP-ATG8e transformants were detached and immersed in 100 mM ethanol or water for 6 h, and GFP-ATG8e was visualized by fluorescence confocal microscopy. Bar, 50 µm. (B) Immunoblot analysis showing the free GFP generated from GFP-ATG8e fusion protein upon ethanol treatment. Detached leaves from 4-week-old GFP-ATG8e plants were immersed in 100 mM ethanol or water, and leaves were collected at 0, 3, 6, 12, and 24 h after treatment. Anti-GFP antibodies were used for immunoblotting. Coomassie blue-stained total proteins are shown at the bottom to indicate the amount of protein loaded per lane. Numbers below the protein bands indicate relative gray values of the bands. The numbers on the left indicate the molecular mass (kD) of the size markers. hpt, hours post-treatment. Full article ">Figure 3
Loss of function of ADH1 attenuates submergence tolerance and autophagosome formation. (A) Phenotypes of 4-week-old wild-type (WT), adh1-4, adh1-8, and adh1-16 plants before treatment (Air) and following 6 d of recovery after 7 d of light (normal light/dark cycle) submergence (LS) treatment (LS+R6). (B) ATG7 and ATG8a protein levels in 4-week-old WT, adh1-4, adh1-8, and adh1-16 plants before treatment (Light) and after 24 h of LS treatment. Coomassie blue-stained total proteins are shown below the blots to indicate the amount of protein loaded per lane. (C) Monodansylcadaverine (MDC) staining of mature root cells from one-week-old WT, adh1-4, adh1-8, and adh1-16 seedlings under normal light/dark (Light) conditions or following 24 h of LS treatment. Red arrows indicate labeled autophagosomes. Bars = 50 µm. (D) Number of puncta per root section in mature root cells from the WT, adh1-4, adh1-8, and adh1-16 seedlings shown in (C). Data are average values ± SD of three biological replicates. For each experiment, 15 sections were analyzed per genotype. Asterisks represent significant differences between WT and adh1 samples, as determined by Student’s t-test (* p < 0.05 and ** p < 0.01). Full article ">Figure 4
Two Arabidopsis accessions with different levels of submergence tolerance show different responses to ethanol. (A) Phenotypes of 4-week-old Columbia (Col-0) and Landsberg erecta (Ler) plants before submergence (Air) and after 60 h of dark submergence (DS), followed by recovery for 6 d (R6). The experiments were repeated three times with similar results. (B) and (C) survival rate (B) and dry weights (C) of Col-0 and Ler plants after DS treatment followed by recovery for 6 d. Data are means (± SD) of three biological replicates. For each biological repetition, 15 plants were used per accession. Asterisks indicate significant differences between Col-0 and Ler, as determined by Student’s t-test (** P < 0.01). (D) Protein abundance of ADH1 and PDC1 in 4-week-old Col-0 and Ler plants treated with dark submergence (DS) at various time points. Numbers below the protein bands indicate relative gray values of the bands. Coomassie blue-stained Rubisco is shown as a loading control. The experiments were repeated three times with similar results. (E) Measurement of alcohol dehydrogenase (ADH) activity in 2-week-old Col-0 and Ler plants after dark submergence (DS) treatment for 0, 3, 6, and 12 h. The experiments were biologically repeated three times with similar results. Error bars represent SD (n = 3 technical replicates). Asterisks indicate significant difference between Col-0 and Ler, as determined by Student’s t-test (* p < 0.05 and ** p < 0.01). (F) Col-0 and Ler seedlings grown on MS medium supplemented with different concentrations of ethanol. Images were taken 10 d after germination. The experiments were repeated three times with similar results. (G) Seedling growth index in (F). The colors in the columns correspond to seedlings with true leaves (dark green), seedlings with green (light green) or brown (yellow) cotyledons, and etiolated seedlings (pink). Data are means (± SD) of three biological replicates. C, Col-0; L, Ler. Full article ">Figure 5
Autophagosome formation in two Arabidopsis accessions in response to submergence. (A) Protein abundance of ATG7 and ATG8a in one-week-old Col-0 and Ler seedlings treated with light submergence (LS) at various time points. Numbers below the protein bands indicate relative gray values of the bands. Actin bands are shown below the blots to indicate the amount of protein loaded per lane. The experiments were repeated three times with similar results. (B) MDC staining of root cells from one-week-old Col-0 and Ler seedlings treated with light submergence (LS) at various time points. Red arrows indicate labeled autophagosomes. The experiments were repeated three times with similar results. Bars = 50 µm. (C) Number of puncta per root section in mature root cells of one-week-old Col-0 and Ler seedlings following LS treatment for 12 h in (B). Data are average values ± SD of three biological replicates. For each experiment, 15 sections were analyzed per accession. Asterisks indicate significant differences between Col-0 and Ler, as determined by Student’s t-test (** p < 0.01). Full article ">Figure 6
Proposed model for the role of ethanol-induced autophagy in regulating plant responses to submergence. Submergence causes hypoxia, which leads to deficiencies in cellular energy and carbohydrate shortages in plants. To survive hypoxic stress, plant cells switch from aerobic respiration to anaerobic fermentation, especially ethanolic fermentation. In this context, ADH1 (alcohol dehydrogenase1) activity increases rapidly in plant cells, resulting in the accumulation of ethanol. The increased ethanol levels promote autophagosome formation, which modulates hypoxia responses and facilitates plant survival by regulating ROS homeostasis, although the precise mechanism remains to be elucidated. By contrast, anaerobic fermentation produces much less ATP than aerobic respiration. The TOR (target of rapamycin) pathway and SnRK1 (Snf1-related protein kinase 1) play opposite roles in regulating autophagy in response to energy limitation. The energy sensor SnRK1 is activated under hypoxia stress and acts as a positive regulator of autophagy, whereas the negative effect of the TOR pathway on autophagy is repressed, ultimately improving plant survival following submergence. Full article ">

15 pages, 3365 KiB

Open AccessArticle

Identification of the Cytosolic Glucose-6-Phosphate Dehydrogenase Gene from Strawberry Involved in Cold Stress Response

by Yunting Zhang, Mengwen Luo, Lijuan Cheng, Yuanxiu Lin, Qing Chen, Bo Sun, Xianjie Gu, Yan Wang, Mengyao Li, Ya Luo, Xiaorong Wang, Yong Zhang and Haoru Tang

Int. J. Mol. Sci. 2020, 21(19), 7322; https://doi.org/10.3390/ijms21197322 - 3 Oct 2020

Cited by 26 | Viewed by 3014

Abstract

Glucose-6-phosphate dehydrogenase (G6PDH) plays an important role in plant stress responses. Here, five FaG6PDH sequences were obtained in strawberry, designated as FaG6PDH-CY, FaG6PDH-P1, FaG6PDH-P1.1, FaG6PDH-P2 and FaG6PDH-P0, which were divided into cytosolic (CY) and plastidic (P) isoforms based on [...] Read more.

Glucose-6-phosphate dehydrogenase (G6PDH) plays an important role in plant stress responses. Here, five FaG6PDH sequences were obtained in strawberry, designated as FaG6PDH-CY, FaG6PDH-P1, FaG6PDH-P1.1, FaG6PDH-P2 and FaG6PDH-P0, which were divided into cytosolic (CY) and plastidic (P) isoforms based on the bioinformatic analysis. The respective FaG6PDH genes had distinct expression patterns in all tissues and at different stages of fruit development. Notably, FaG6PDH-CY was the most highly expressed gene among five FaG6PDH members, indicating it encoded the major G6PDH isoform throughout the plant. FaG6PDH positively regulated cold tolerance in strawberry. Inhibition of its activity gave rise to greater cold-induced injury in plant. The FaG6PDH-CY transcript had a significant increase under cold stress, similar to the G6PDH enzyme activity, suggesting a principal participant in response to cold stress. Further study showed that the low-temperature responsiveness (LTR) element in FaG6PDH-CY promoter can promote the gene expression when plant encountered cold stimuli. Besides, FaG6PDH-CY was involved in regulating cold-induced activation of antioxidant enzyme genes (FaSOD, FaCAT, FaAPX and FaGR) and RBOH-dependent ROS generation. The elevated FaG6PDH-CY enhanced ROS-scavenging capability of antioxidant enzymes to suppress ROS excessive accumulation and relieved the oxidative damage, eventually improving the strawberry resistance to cold stress. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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17 pages, 3817 KiB

Open AccessArticle

Production of Antioxidant Molecules in Polygonum aviculare (L.) and Senecio vulgaris (L.) under Metal Stress: A Possible Tool in the Evaluation of Plant Metal Tolerance

by Mirko Salinitro, Sara Hoogerwerf, Sonia Casolari, Alessandro Zappi, Dora Melucci and Annalisa Tassoni

Int. J. Mol. Sci. 2020, 21(19), 7317; https://doi.org/10.3390/ijms21197317 - 3 Oct 2020

Cited by 16 | Viewed by 2675

Abstract

Plants growing on heavy metal (HM)-polluted soils show toxicity symptoms, such as chlorosis and growth reduction, and undergo oxidative stress due to the formation of reactive oxygen species (ROS). Plants overcome oxidative stress by producing a wide range of antioxidant molecules, such as [...] Read more.

Plants growing on heavy metal (HM)-polluted soils show toxicity symptoms, such as chlorosis and growth reduction, and undergo oxidative stress due to the formation of reactive oxygen species (ROS). Plants overcome oxidative stress by producing a wide range of antioxidant molecules, such as polyphenols and flavonoids. The aim of the present work was to study the accumulation of these molecules in response to increasing concentrations of Cd, Cr, Cu, Ni, Pb and Zn and to assess whether they can be used as a tool in assessing metal-related stress in Polygonum aviculare and Senecio vulgaris. On average, P. aviculare shoots accumulated lower amounts of metals than S. vulgaris shoots. The uptake of all six elements was correlated and proportional to their concentration in the nutrient solution (ρ > 0.9), with the bioaccumulation factor (BAF) being >1 for most of them. The present research demonstrated that 82% of the samples showed a good correlation (|ρ| > 0.5) between the level of polyphenols, flavonoids and antioxidant activity and the metal concentration in plant shoots, confirming that the metal stress level and production of phenolic compounds having antioxidant activity were strictly connected. Nonetheless, the mere quantification of these molecules cannot identify the type of metal that caused the oxidative stress, neither determine the concentration of the stressors. The five tested populations of each species did not show any specific adaptation to the environment of origin. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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Figure 1
Metal contents in shoots of P. aviculare grown in hydroponics at increasing metal concentrations (from control to D, see Methods section). (A) Cd; (B) Cr; (C) Cu; (D) Ni; (E) Pb; (F) Zn. Bars represent the average of data coming from five populations analyzed in three biological replicates (n = 15). Different small letters (from v to z) above the bars indicate a statistically significant difference among the treatments (p < 0.05) calculated with Dunn.test using R software. Full article ">Figure 2
Metal contents in shoots of S. vulgaris grown in hydroponics at increasing metal concentrations (from control to D, see Methods section). (A) Cd; (B) Cr; (C) Cu; (D) Ni; (E) Pb; (F) Zn. Bars represent the average of data coming from five populations analyzed in three biological replicates (n = 15). Different small letters (from v to z) above the bars indicate a statistically significant difference among the treatments (p < 0.05) calculated with Dunn.test using R software. Full article ">Figure 3
PCA analysis showing the grouping for P. aviculare data according to different metal treatments. The complete dataset reported in <a href="#app1-ijms-21-07317" class="html-app">Supplementary Table S1</a> was used to perform the analysis by means of the prcomp function of the R package. Treatments: control, no metal; A, urban metal concentration; B, medium metal concentration, C, high metal concentration, D, maximum metal concentration allowing plant survival. Full article ">Figure 4
PCA analysis showing the grouping for P. aviculare data according to different metal treatments. The complete dataset reported in <a href="#app1-ijms-21-07317" class="html-app">Supplementary Table S1</a> was used to perform the analysis by means of the prcomp function of the R package. Treatments: control, no metal; A, urban metal concentration; B, medium metal concentration, C, high metal concentration, D, maximum metal concentration allowing plant survival. Full article ">Figure 5
Shoot metal concentration in different P. aviculare populations. (A) Cd; (B) Cr; (C) Cu; (D) Ni; (E) Pb; (F) Zn. Populations: B, Bologna urban; M, Milan urban; N, Bologna woodland; T, Milan woodland; P, serpentine. Data represent the mean value of three biological replicates (n = 3). Different small letters (from x to z) above the bars indicate a statistically significant difference among the treatments (p < 0.05) calculated with Dunn.test using R software. Full article ">Figure 6
Shoot metal concentration in different S. vulgaris populations. (A) Cd; (B) Cr; (C) Cu; (D) Ni; (E) Pb; (F) Zn. Populations: B, Bologna urban; M, Milan urban; N, Bologna woodland; T, Milan woodland; P, Mt. Prinzera serpentine. Data represent the mean value of three biological replicates (n = 3). Different small letters (from x to z) above the bars indicate a statistically significant difference among the treatments (p < 0.05) calculated with Dunn.test using R software. Full article ">Figure 7
(A) Polygonum aviculare (L.), and (B) Senecio vulgaris (L.). Photo by Mirko Salinitro. Full article ">

16 pages, 5358 KiB

Open AccessArticle

Overexpression of UGT74E2, an Arabidopsis IBA Glycosyltransferase, Enhances Seed Germination and Modulates Stress Tolerance via ABA Signaling in Rice

by Ting Wang, Pan Li, Tianjiao Mu, Guangrui Dong, Chengchao Zheng, Shanghui Jin, Tingting Chen, Bingkai Hou and Yanjie Li

Int. J. Mol. Sci. 2020, 21(19), 7239; https://doi.org/10.3390/ijms21197239 - 30 Sep 2020

Cited by 29 | Viewed by 4545

Abstract

UDP-glycosyltransferases (UGTs) play key roles in modulating plant development and responses to environmental challenges. Previous research reported that the Arabidopsis UDP-glucosyltransferase 74E2 (AtUGT74E2), which transfers glucose to indole-3-butyric acid (IBA), is involved in regulating plant architecture and stress responses. Here, we [...] Read more.

UDP-glycosyltransferases (UGTs) play key roles in modulating plant development and responses to environmental challenges. Previous research reported that the Arabidopsis UDP-glucosyltransferase 74E2 (AtUGT74E2), which transfers glucose to indole-3-butyric acid (IBA), is involved in regulating plant architecture and stress responses. Here, we show novel and distinct roles of UGT74E2 in rice. We found that overexpression of AtUGT74E2 in rice could enhance seed germination. This effect was also observed in the presence of IBA and abscisic acid (ABA), as well as salt and drought stresses. Further investigation indicated that the overexpression lines had lower levels of free IBA and ABA compared to wild-type plants. Auxin signaling pathway gene expression such as for OsARF and OsGH3 genes, as well as ABA signaling pathway genes OsABI3 and OsABI5, was substantially downregulated in germinating seeds of UGT74E2 overexpression lines. Consistently, due to reduced IBA and ABA levels, the established seedlings were less tolerant to drought and salt stresses. The regulation of rice seed germination and stress tolerance could be attributed to IBA and ABA level alterations, as well as modulation of the auxin/ABA signaling pathways by UGT74E2. The distinct roles of UGT74E2 in rice implied that complex and different molecular regulation networks exist between Arabidopsis and rice. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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Figure 1
UGT74E2 catalyzes indole-3-butyric acid (IBA) glycosylation in rice. (A) RT-PCR analysis of the mRNA levels of UGT74E2 in transgenic rice plants. (B) HPLC profiling of IBA glucose conjugates (IBA-Glc) extracted from seven-day-old wild type (WT) and UGT74E2-overexpressed lines after 0 μM IBA or 100 μM IBA treatment for 12 h. (C) Relative IBA-Glc contents in UGT74E2 transgenic rice plants (* p < 0.05). (D) LC-MS confirmation of IBA-glucose ester under positive ion mode. Full article ">Figure 2
UGT74E2 affected the seed germination and postgermination growth of the transgenic plants. (A) Seed germination and postgermination growth of UGT74E2 transgenic plants. Here, at least 30 seeds were observed for each line (bars = 3 cm). (B) Shoot and root lengths of UGT74E2 overexpression lines for 2, 4, and 7 days. Bars represent standard deviation of at least 10 seedlings. (C) Expression of auxin signaling pathway genes in seven-day-old UGT74E2-overexpressed rice plants compared to WT. Transcript levels were normalized to the mRNA levels of OsActin. Data shown are means ± SD. Student’s t-test was performed (* p < 0.05, ** p < 0.01). Experiments were conducted for three biological replicates. (D) Location of GH3, IAA, and ARF genes in the auxin biosynthesis and signaling pathway. Full article ">Figure 3
UGT74E2-overexpressed plants are not sensitive to IBA and abscisic acid (ABA) treatment. (A) Seed germination of WT and UGT74E2 transgenic rice under IBA and ABA treatments for eight days. Here, at least 30 seeds were observed for each line (bars = 3 cm). (B) Time to 50% germination and four-day germination rates were calculated in the presence of IBA and ABA. (C) Shoot and root lengths of UGT74E2-overexpressed lines in the presence of IBA and ABA (scale bars = 3 cm). Experiments were conducted for three biological replicates. Bars represent standard deviation of at least 10 seedlings. Student’s t-test was performed (* p < 0.05, ** p < 0.01). Full article ">Figure 4
UGT74E2 altered IBA and ABA contents in germinating rice seeds. (A) The contents of IBA in germinating WT and UGT74E2 transgenic rice seeds after being imbibed for 24 h. (B) The contents of ABA in germinating WT and UGT74E2 transgenic rice seeds after being imbibed for 24 h. (C) Expression of OsABI3 and OsABI5 in germinating seeds in WT and UGT74E2 transgenic rice. Gene expression was normalized to the reference gene OsActin. For each experiment, three replicates were done; data are presented as means ± SD. Student’s t-test for each genotype was performed (* p < 0.05, ** p < 0.01). (D) Role of ABI3 and ABI5 genes in the ABA signaling pathway during seed germination. Full article ">Figure 5
UGT74E2 transgenic lines are not sensitive to salt and drought stresses during the germination and postgermination stagea. (A) Germination rates of WT and UGT74E2-overexpressed lines under NaCl, polyethylene glycol (PEG), and mannitol treatment. (B) Shoot and root lengths of UGT74E2-overexpressed lines under PEG and NaCl treatment (scale bars = 3 cm). (C) IBA and (D) ABA contents in seven-day-old rice after 200 mM NaCl and 15% PEG treatment for 12 h. Data are means ± SD. For each experiment, three biological replicates were done. Student’s t-test was performed (* p < 0.05, ** p < 0.01). Full article ">Figure 6
UGT74E2-overexpressed lines are less tolerant to polyethylene glycol (PEG) and NaCl during the seedling stage. The growth (A) and survival rates (B) of UGT74E2 transgenic rice plants upon exposure to 200 mM NaCl and 15% PEG for seven days (scale bars = 10 cm). For each experiment, three biological replicates were performed. Student’s t-test was performed (** p < 0.01). Full article ">Figure 7
Accumulation of ROS under NaCl and PEG treatments. (A) Two-week-old seedlings were subjected to nitroblue tetrazoliun (NBT) and diaminobenzidine (DAB) staining after treatment with NaCl and polyethylene glycol (PEG) for 24 h. (scale bars = 1 cm) (B) Genes encoding antioxidant enzymes are downregulated upon UGT74E2 overexpression under abiotic stress. For RNA extraction, seven-day-old rice plants were subjected to 200 mM NaCl and 15% PEG treatment for 12 h. Gene expression was normalized to the reference gene OsActin. For each experiment, three replicates were done; data are presented as means ± SD. Student’s t-test for each genotype was performed (** p < 0.01). Full article ">Figure 8
Expression of stress-responsive genes is downregulated upon UGT74E2 overexpression under abiotic stress. For RNA extraction, seven-day-old rice plants were subjected to 200 mM NaCl and 15% PEG (polyethylene glycol) treatment for 12 h. Gene expression was normalized to the reference gene OsActin. For each experiment, three replicates were done; data are presented as means ± SD. Student’s t-test for each genotype was performed (* p < 0.05, ** p < 0.01) Full article ">

20 pages, 5855 KiB

Open AccessArticle

The Interplay between Toxic and Essential Metals for Their Uptake and Translocation Is Likely Governed by DNA Methylation and Histone Deacetylation in Maize

by Sarfraz Shafiq, Asim Ali, Yasar Sajjad, Qudsia Zeb, Muhammad Shahzad, Abdul Rehman Khan, Rashid Nazir and Emilie Widemann

Int. J. Mol. Sci. 2020, 21(18), 6959; https://doi.org/10.3390/ijms21186959 - 22 Sep 2020

Cited by 25 | Viewed by 4170

Abstract

The persistent nature of lead (Pb) and cadmium (Cd) in the environment severely affects plant growth and yield. Conversely, plants acquire zinc (Zn) from the soil for their vital physiological and biochemical functions. However, the interplay and coordination between essential and toxic metals [...] Read more.

The persistent nature of lead (Pb) and cadmium (Cd) in the environment severely affects plant growth and yield. Conversely, plants acquire zinc (Zn) from the soil for their vital physiological and biochemical functions. However, the interplay and coordination between essential and toxic metals for their uptake and translocation and the putative underlying epigenetic mechanisms have not yet been investigated in maize. Here, we report that the presence of Zn facilitates the accumulation and transport of Pb and Cd in the aerial parts of the maize plants. Moreover, the Zn, Pb, and Cd interplay specifically interferes with the uptake and translocation of other divalent metals, such as calcium and magnesium. Zn, Pb, and Cd, individually and in combinations, differentially regulate the expression of DNA methyltransferases, thus alter the DNA methylation levels at the promoter of Zinc-regulated transporters, Iron-regulated transporter-like Protein (ZIP) genes to regulate their expression. Furthermore, the expression of histone deacetylases (HDACs) varies greatly in response to individual and combined metals, and HDACs expression showed a negative correlation with ZIP transporters. Our study highlights the implication of DNA methylation and histone acetylation in regulating the metal stress tolerance dynamics through Zn transporters and warns against the excessive use of Zn fertilizers in metal contaminated soils. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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Figure 1
Mobility index (%) of Pb, Cd, and Zn from roots to shoots and roots to leaves in response to Pb/Cd/Zn applied alone and in combinations. The values marked with different letters are statistically different (p ≤ 0.05), while the values marked with the same letters do not differ significantly. Full article ">Figure 2
The activities of Peroxidase (POD), Superoxide dismutase (SOD), and Catalase (CAT) do not differ much between Pb/Cd/Zn applied alone and in combinations. The results shown are the average of three biological replicates. Bars represent mean ± SD. The values marked with different letters are statistically different (p ≤ 0.05), while the values marked with the same letters do not differ significantly. Full article ">Figure 3
The expression of ZIP transporters in response to Pb/Cd/Zn applied alone and in combinations. (A) The expression of ZIP transporters was normalized with ZmUBQ5. The data presented are the averages of three biological replicates. Bars represent mean ± SD. The values marked with different letters are statistically different (p ≤ 0.05), while the values marked with the same letters do not differ significantly. (B) Multiple linear regression (MLR) equations for uptake of each heavy metal and expression of ZIP transporters. (C) Cumulative percentage contribution (CPC) for the expression of each ZIP transporter in response to the uptake of heavy metals. Full article ">Figure 4
The expression of histone deacetylases in response to Pb/Cd/Zn applied alone and in combinations. (A) The expression of histone deacetylases was normalized with ZmUBQ5. The data presented are the averages of three biological replicates. Bars represent mean ± SD. The values marked with different letters are statistically different (p ≤ 0.05), while the values marked with the same letters do not differ significantly. (B) Multiple linear regression (MLR) equations for uptake of each heavy metal and expression of HDACs. (C) Cumulative percentage contribution (CPC) for expression of each HDAC transporter in response to the uptake of heavy metals. Full article ">Figure 5
The expression of DNA methyltransferases in response to Pb/Cd/Zn applied alone and in combinations. (A) The expression of DNA methyltransferases was normalized with ZmUBQ5. The data presented are the averages of three biological replicates. Bars represent mean ± SD. The values marked with different letters are statistically different (p ≤ 0.05), while the values marked with the same letters do not differ significantly. (B) Multiple linear regression (MLR) equations for uptake of each heavy metal and expression of DNA methyltransferases. (C) Cumulative percentage contribution (CPC) for expression of each DNA methyltransferase in response to the uptake of heavy metals. Full article ">Figure 6
DNA methylation levels at the promoter of selected ZIP transporters in response to Pb/Cd/Zn applied alone and in combinations. DNA was digested with McrBc and equal amounts of digested or undigested DNA were used as template for PCR. McrBC digests the methylated DNA; therefore, lighter band intensity reflects higher DNA methylation level. Full article ">Figure 7
Pearson correlation between the expression of ZIP transporters and the expression of DNA methyltransferases (A) and HDACs (B). Red color represents the negative correlation (−1), green color represents the positive correlation (+1), and yellow represents the absence of correlation (0). * significant correlation at p ≤ 0.05, ** significant correlation at p ≤ 0.01. Full article ">Figure 8
Proposed model for the uptake and translocation interplay of Pb, Cd, and Zn through ZIP transporters. Heavy metals may alter the DNA methylation and histone acetylation levels through the activity of DNA methyltransferases and histone deacetylases, respectively. The resulting chromatin landscape may control the expression of certain ZIP transporters that may carry Pb and Cd along with Zn into the cell. Consequently, other metal transporters may facilitate the loading of toxic metals to xylem, and subsequent transport to shoots that result in the increased concentration of toxic metals in aerial parts of the plants. Moreover, the enrichment of toxic metals into the cell disturbs the uptake and translocation of other essential divalent metals, e.g., Ca/Mg. The gradient represents the metal concentration, and cylindrical structures represent the metal transporters that can load the metals to the xylem. Full article ">

14 pages, 3798 KiB

Open AccessArticle

Drought Disrupts Auxin Localization in Abscission Zone and Modifies Cell Wall Structure Leading to Flower Separation in Yellow Lupine

by Aleksandra Bogumiła Florkiewicz, Agata Kućko, Małgorzata Kapusta, Sebastian Burchardt, Tomasz Przywieczerski, Grażyna Czeszewska-Rosiak and Emilia Wilmowicz

Int. J. Mol. Sci. 2020, 21(18), 6848; https://doi.org/10.3390/ijms21186848 - 18 Sep 2020

Cited by 13 | Viewed by 3801

Abstract

Drought causes the excessive abscission of flowers in yellow lupine, leading to yield loss and serious economic consequences in agriculture. The structure that determines the time of flower shedding is the abscission zone (AZ). Its functioning depends on the undisturbed auxin movement from [...] Read more.

Drought causes the excessive abscission of flowers in yellow lupine, leading to yield loss and serious economic consequences in agriculture. The structure that determines the time of flower shedding is the abscission zone (AZ). Its functioning depends on the undisturbed auxin movement from the flower to the stem. However, little is known about the mechanism guiding cell–cell adhesion directly in an AZ under water deficit. Therefore, here, we seek a fuller understanding of drought-dependent reactions and check the hypothesis that water limitation in soil disturbs the natural auxin balance within the AZ and, in this way, modifies the cell wall structure, leading to flower separation. Our strategy combined microscopic, biochemical, and chromatography approaches. We show that drought affects indole-3-acetic acid (IAA) distribution and evokes cellular changes, indicating AZ activation and flower abortion. Drought action was manifested by the accumulation of proline in the AZ. Moreover, cell wall-related modifications in response to drought are associated with reorganization of methylated homogalacturonans (HG) in the AZ, and upregulation of pectin methylesterase (PME) and polygalacturonase (PG)—enzymes responsible for pectin remodeling. Another symptom of stress action is the accumulation of hemicelluloses. Our data provide new insights into cell wall remodeling events during drought-induced flower abscission, which is relevant to control plant production. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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Figure 1

Figure 1
Soil drought stress increases proline content in flower abscission zone (AZ) of yellow lupine. Lupines were cultivated under water deficit conditions (25% WHC), while control plants were grown in the soil of optimal moisture (70% WHC). For analysis, sections of the abscission zone (AZ) were collected on the 48th day of cultivation. Data are presented as averages ± SE (n = 3). Significant differences in stressed plants in comparison to control are ** p < 0.01 (Student’s t-test). Full article ">Figure 2
Drought stress affects indole-3-acetic acid (IAA) localization in abscission zone (AZ) cells. Immunolocalization of IAA in the floral AZ of yellow lupine exposed to drought-stress (25% WHC, water deficit conditions) (B,E,F) and AZ from control plants growing in the soil of optimal moisture (70% WHC) (A,B). Green fluorescence indicates IAA presence, whereas blue labeling corresponds to nuclei stained with DAPI. AZ area is marked by a white dotted line (A,B). Image (D) presents the magnified control AZ area presented in (A). IAA is found in nuclei (C insert) and cells located near the vascular bundles (VB) (E,F). Bars are given in each image. Endogenous content of IAA in control and drought-treated AZs (G). For all analysis, sections of AZs were collected on the 48th day of cultivation. Data are presented as averages ± SE (n = 3). Significant differences in the stressed plants in comparison to control plants are ** p < 0.01 (Student’s t-test). Full article ">Figure 3
Drought causes changes in pectin distribution in floral abscission zone (AZ) of yellow lupine. Lupines were cultivated under water deficit conditions (25% WHC), while control plants were grown in the soil of optimal moisture (70% WHC). Sections of floral AZs were collected on the 48th day of cultivation. The total content of pectin based on pectin absorbance (A). Data are presented as averages ± SE (n = 3). Significant differences in the stressed plant in comparison to control plants are ** p < 0.01 (Student’s t-test). De-esterified pectin staining using Ruthenium red in the control AZ (B–D) and drought-stressed (E–G). AZ area is marked by the black dotted line (B,E). Arrowheads indicate the place of de-esterified pectin accumulation. Abbreviation: VB—vascular bundles. Bars are given in each image. Full article ">Figure 4
Drought leads to redistribution of low- and high-methylated pectin in the abscission zone (AZ) area. Immunolocalization of pectin in the flower AZ of yellow lupine cultivated under soil drought conditions (25% WHC, water deficit conditions) (E–J) and AZ of control lupines growing in the soil of optimal moisture (70% WHC) (A–D). For analysis, sections of AZs (marked by the white dotted line) were collected on the 48th day of cultivation. Low-methylated and un-methylated homogalacturonans (HGs) were detected by JIM5-Ab, while JIM7-Ab was used to localize high-methylated HGs. Green fluorescence indicates the pectin presence, while the blue signal corresponds to nuclei stained with DAPI. Images (B,D) are magnifications of AZ areas presented in (A,C), respectively. Images (F,G) are magnified regions presented in (E). Images (I,J) are magnified regions presented in (H). Abbreviations: DIST—distal region, flower pedicel fragment above the AZ; PROX—proximal region, stem fragment below the AZ. Bars are given on each image. Full article ">Figure 5
Drought influences the presence of pectin remodeling enzymes in the abscission zone (AZ) area. The impact of soil drought stress (25% WHC, water deficit conditions) on the localization of pectin-methylesterase (PME) (B,C) and polygalacturonase (PG) (E,F) was analyzed in the flower AZ of yellow lupine. Control was AZs excised from plants growing in the soil of optimal humidity (70% WHC) (A,D). For analysis, sections of AZs were collected on the 48th day of cultivation. Fixed material was dissected and incubated with monoclonal anti-PME-Ab (A–C) and anti-PG-Ab (D–F). Green fluorescence indicates the presence of the enzymes. Nuclei were stained with DAPI (blue fluorescence). AZ area is marked by white dotted lines (A,B,D,E). Abbreviations: DIST—a distal region of AZ, flower pedicel fragment above the AZ, PROX—a proximal region of AZ, stem fragment below the AZ, VB—vascular bundles. Bars are given in images. Full article ">Figure 6
Hemicellulose content is negatively regulated by soil water deficit. The effect of drought stress (25% WHC, water deficit conditions) on the absorbance of hemicelluloses in the flower abscission zone (AZ) of yellow lupine was analyzed. Control AZ fragments were collected from plants growing in the soil of optimal conditions (70% WHC). For analysis, sections of AZs were harvested on the 48th day of cultivation. Data are presented as averages ± SE (n = 3). Significant differences in the stressed plants in comparison to control plants are a p < 0.05 (Student’s t-test). Full article ">

21 pages, 2992 KiB

Open AccessArticle

Genome-Wide Analysis Reveals Dynamic Epigenomic Differences in Soybean Response to Low-Phosphorus Stress

by Shanshan Chu, Xiangqian Zhang, Kaiye Yu, Lingling Lv, Chongyuan Sun, Xiaoqian Liu, Jinyu Zhang, Yongqing Jiao and Dan Zhang

Int. J. Mol. Sci. 2020, 21(18), 6817; https://doi.org/10.3390/ijms21186817 - 17 Sep 2020

Cited by 20 | Viewed by 3279

Abstract

Low-phosphorus (low-P) stress has a significant limiting effect on crop yield and quality. Although the molecular mechanisms of the transcriptional level responsible for the low-P stress response have been studied in detail, the underlying epigenetic mechanisms in gene regulation remain largely unknown. In [...] Read more.

Low-phosphorus (low-P) stress has a significant limiting effect on crop yield and quality. Although the molecular mechanisms of the transcriptional level responsible for the low-P stress response have been studied in detail, the underlying epigenetic mechanisms in gene regulation remain largely unknown. In this study, we evaluated the changes in DNA methylation, gene expression and small interfering RNAs (siRNAs) abundance genome-wide in response to low-P stress in two representative soybean genotypes with different P-efficiencies. The DNA methylation levels were slightly higher under low-P stress in both genotypes. Integrative methylation and transcription analysis suggested a complex regulatory relationship between DNA methylation and gene expression that may be associated with the type, region, and extent of methylation. Association analysis of low-P-induced differential methylation and gene expression showed that transcriptional alterations of a small part of genes were associated with methylation changes. Dynamic methylation alterations in transposable element (TE) regions in the CHH methylation context correspond with changes in the amount of siRNA under low-P conditions, indicating an important role of siRNAs in modulating TE activity by guiding CHH methylation in TE regions. Together, these results could help to elucidate the epigenetic regulation mechanisms governing the responses of plants to abiotic stresses. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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Figure 1
DNA methylome features in soybean. (a) Relative proportions of mCs in three sequence contexts (CG, CHG, and CHH); (b) A circos plot of gene and transposon density and mCG, mCHG, and mCHH location in soybean. NN_HP represents ‘Nan-nong94-156′ under control conditions; NN_LP represents ‘Nan-nong94-156′ under low-P conditions; BG_HP represents ‘Bogao’ under control conditions, and BG_LP represents ‘Bogao’ under low-P stress. Full article ">Figure 2
Genomic DNA methylation profiles in soybean. (a) DNA methylation patterns across genes; (b) DNA methylation patterns across TEs; the vertical dashed line represents the boundaries of the gene body or transposon (TE) body. Full article ">Figure 3
Relationship between DNA methylation and gene expression. (a) Distributions of methylation levels within gene bodies partitioned by different expression levels: 1st_quintile is the lowest and 4th_quintile is the highest; genes with FPKM value < 0.1 were considered non-expressed (none); (b) expression profiles of methylated genes compared with unmethylated genes. Methylated genes were further divided into quintiles based on promoter and gene body region methylation levels: 1st_quintile is the lowest and 5th_quintile is the highest. Full article ">Figure 4
Differential methylome analysis under low-P stress. (a) Venn diagram of hyper/hypomethylated genes among ‘Nan-nong94-156′ and ‘Bogao’ under low-P stress; (b) KEGG pathway enrichment of hypermethylated and hypomethylated genes in two cultivars under low-P conditions. The size of the circle represents gene numbers, and the color represents the q-value. NN_LP vs. NN_HP, ‘NN’ low-P versus high-P; BG_LP vs BG_HP, ‘BG’ low-P versus high-P. Full article ">Figure 5
Assignment of differentially methylated genes among ‘Nan-nong94-156′ (a) and ‘Bogao’ (b) under low-P stress in Mapman bins. The red and blue squares indicate the hyper- and hypomethylated genes, respectively. Other different shaped graphics with different colors refer to some sensors and transcription factors (TFs) responding to low-P stress. (c) Venn map of differentially methylated transcriptional factors; (d) IGV software depicts the hypermethylation of GmSCL9 gene body region induced by low-P stress in ‘Nan-nong94-156′. NN_LP vs NN_HP, ‘NN’ low-P versus high-P; BG_LP vs BG_HP, ‘BG’ low-P versus high-P. Full article ">Figure 6
Differentially methylated TEs. (a) Numbers of differentially methylated TEs in ‘Nan-nong94-156′ or ‘Bogao’ under low-P stress; (b) heat maps of differentially methylated TEs. NN_LP vs NN_HP, ‘NN’ low-P versus high-P; BG_LP vs BG_HP, ‘BG’ low-P versus high-P. Full article ">Figure 7
The effect of methylation changes on transcriptional alterations. (a) Differentially expressed genes (DEGs) in ‘Nan-nong94-156′ or ‘Bogao’ in response to low-P stress. Each dot represents one gene. The red dots represent upregulated genes and the green dots represent downregulated genes. The black dots represent genes without differential expression. The X-axis is the log2 value of fold change and the Y-axis is the log10 value of false discovery rate (FDR); (b) heat maps of DEGs. NN_HP, ‘Nan-nong94-156′ under control conditions; NN_LP, ‘Nan-nong94-156′ under low-P conditions; BG_HP, ‘Bogao’ under control conditions; BG_LP, ‘Bogao’ under low-P conditions. Venn diagram of DMGs (differentially methylated genes) and DEGs in NN_LP vs NN_HP (c) and BG_LP vs BG_HP (d); (e) differential expression levels of all genes (red box), hypermethylated genes (green box), and hypomethylated genes (blue box) among NN_LP vs NN_HP and BG_LP vs BG_HP in three sequence contexts (CG, CHG, and CHH) are displayed as boxplots (boxes represent the quartiles; Wilcoxon P values are reported). NN_LP vs NN_HP, ‘NN’ low-P versus high-P; BG_LP vs BG_HP, ‘BG’ low-P versus high-P. Full article ">Figure 8
Number distribution of siRNAs in the TE and flanking 2-kb regions. NN_HP represents ‘Nan-nong94-156′ under control conditions; NN_LP represents ‘Nan-nong94-156′ under low-P conditions; BG_HP represents ‘Bogao’ under control conditions, and BG_LP represents ‘Bogao’ under low-P conditions. Full article ">

14 pages, 5610 KiB

Open AccessArticle

Functional Characterization of a Putative RNA Demethylase ALKBH6 in Arabidopsis Growth and Abiotic Stress Responses

by Trinh Thi Huong, Le Nguyen Tieu Ngoc and Hunseung Kang

Int. J. Mol. Sci. 2020, 21(18), 6707; https://doi.org/10.3390/ijms21186707 - 13 Sep 2020

Cited by 63 | Viewed by 4913

Abstract

RNA methylation and demethylation, which is mediated by RNA methyltransferases (referred to as “writers”) and demethylases (referred to as “erasers”), respectively, are emerging as a key regulatory process in plant development and stress responses. Although several studies have shown that AlkB homolog (ALKBH) [...] Read more.

RNA methylation and demethylation, which is mediated by RNA methyltransferases (referred to as “writers”) and demethylases (referred to as “erasers”), respectively, are emerging as a key regulatory process in plant development and stress responses. Although several studies have shown that AlkB homolog (ALKBH) proteins are potential RNA demethylases, the function of most ALKBHs is yet to be determined. The Arabidopsis thaliana genome contains thirteen genes encoding ALKBH proteins, the functions of which are largely unknown. In this study, we characterized the function of a potential eraser protein, ALKBH6 (At4g20350), during seed germination and seedling growth in Arabidopsis under abiotic stresses. The seeds of T-DNA insertion alkbh6 knockdown mutants germinated faster than the wild-type seeds under cold, salt, or abscisic acid (ABA) treatment conditions but not under dehydration stress conditions. Although no differences in seedling and root growth were observed between the alkbh6 mutant and wild-type under normal conditions, the alkbh6 mutant showed a much lower survival rate than the wild-type under salt, drought, or heat stress. Cotyledon greening of the alkbh6 mutants was much higher than that of the wild-type upon ABA application. Moreover, the transcript levels of ABA signaling-related genes, including ABI3 and ABI4, were down-regulated in the alkbh6 mutant compared to wild-type plants. Importantly, the ALKBH6 protein had an ability to bind to both m⁶A-labeled and m⁵C-labeled RNAs. Collectively, these results indicate that the potential eraser ALKBH6 plays important roles in seed germination, seedling growth, and survival of Arabidopsis under abiotic stresses. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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Figure 1
Subcellular localization and stress-responsive expression patterns of ALKBH6. (A) Schematic representation of the domain structure of ALKBH6. The conserved 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase (Oxy) domain is shown. aa, amino acid. (B) The ALKBH6-green fluorescence protein (GFP) fusion protein was transiently expressed in tobacco leaves, and the fluorescence signals were detected using a confocal microscope. 4′,6-diamidino-2-phenylindole (DAPI) was used to stain the nucleus, and the red signals indicate chloroplast auto-fluorescence. Bar = 10 μm. (C) Two-week-old Arabidopsis seedlings were subjected to drought, heat (42 °C), cold (10 °C), high salinity (300 mM NaCl), or abscisic acid (ABA) (100 μM) treatment, and the transcript levels of ALKBH6 were determined via real-time RT-PCR. Values are the mean ± SE obtained from three independent experiments, and the asterisks above columns indicate significant differences (* p ≤ 0.05). Full article ">Figure 2
Seedling and root growth of the alkbh6 mutants under drought stress. (A) The wild-type (WT) and alkbh6 mutants were grown on the (Murashige and Skoog) MS medium supplemented with 200 or 400 mM mannitol, and the fresh weight and root length of the seedlings were measured at the indicated days after germination (DAG). (B) Twenty-day-old seedlings grown in soil were subjected to drought stress for 13 days, and survival rates were measured 3 days after recovery. Values are the mean ± SE obtained from three independent experiments. Full article ">Figure 3
Seedling and root growth of the alkbh6 mutants under salt stress. (A) Wild-type Arabidopsis (WT) and alkbh6 mutants were grown on solid MS medium supplemented with 150 mM NaCl, and photographs for seedlings and roots were taken at the indicated days after germination (DAG). (B) Survival rate and fresh weight were measured 21 DAG, and root lengths were measured 14 DAG. Values are the mean ± SE obtained from three independent experiments, and the asterisks above columns indicate significant differences (* p ≤ 0.05). Full article ">Figure 4
Seedling and root growth of the alkbh6 mutants under cold or heat stress. (A) The wild-type (WT) and alkbh6 mutants were grown at 10 °C, and root lengths were measured four weeks after germination. (B) Three-day-old seedlings were treated at 37 °C for 1 h and then subjected to heat stress at 45 °C for 4 h. Survival rate and fresh weight of the plants were measured seven days after heat treatment. Values are the mean ± SE obtained from three independent experiments, and the asterisks above columns indicate significant differences (* p ≤ 0.05). Full article ">Figure 5
Effects of ABA on the seedling and root growth of the alkbh6 mutants. (A) Growth of wild-type (WT) plants and alkbh6 mutants was observed on solid MS medium supplemented with 2 or 4 μM ABA. (B) Cotyledon greening and fresh weight of the assessed plant lines were measured two and three weeks after germination, respectively. The transcript levels of (C) ABA biosynthesis-genes and (D) ABA signaling-related genes in 3-week-old seedlings were determined via qRT-PCR. Values are the mean ± SE obtained from three independent experiments, and the asterisks above columns indicate significant differences (* p ≤ 0.05). Full article ">Figure 6
RNA-binding ability of the ALKBH6 protein. Electrophoretic mobility shift assay showing the binding between the ALKBH6 protein (3, 5, and 7 μM) and the synthetic (A) RNA-A, (B) RNA-B, or (C) RNA-C. The modified adenine and cytosine bases are indicated in red. The 5′-fluorescein-labeled RNA substrates were mixed with the purified protein, and the reaction mixtures were separated on a 6% native acrylamide gel. The binding products were detected using an image analyzer. Full article ">

16 pages, 1570 KiB

Open AccessArticle

The Metabolic Reprogramming Induced by Sub-Optimal Nutritional and Light Inputs in Soilless Cultivated Green and Red Butterhead Lettuce

by Begoña Miras-Moreno, Giandomenico Corrado, Leilei Zhang, Biancamaria Senizza, Laura Righetti, Renato Bruni, Christophe El-Nakhel, Maria Isabella Sifola, Antonio Pannico, Stefania De Pascale, Youssef Rouphael and Luigi Lucini

Int. J. Mol. Sci. 2020, 21(17), 6381; https://doi.org/10.3390/ijms21176381 - 2 Sep 2020

Cited by 20 | Viewed by 3260

Abstract

Sub-optimal growing conditions have a major effect on plants; therefore, large efforts are devoted to maximizing the availability of agricultural inputs to crops. To increase the sustainable use of non-renewable inputs, attention is currently given to the study of plants under non-optimal conditions. [...] Read more.

Sub-optimal growing conditions have a major effect on plants; therefore, large efforts are devoted to maximizing the availability of agricultural inputs to crops. To increase the sustainable use of non-renewable inputs, attention is currently given to the study of plants under non-optimal conditions. In this work, we investigated the impact of sub-optimal macrocations availability and light intensity in two lettuce varieties that differ for the accumulation of secondary metabolites (i.e., ‘Red Salanova’ and ‘Green Salanova’). Photosynthesis-related measurements and untargeted metabolomics were used to identify responses and pathways involved in stress resilience. The pigmented (‘Red’) and the non-pigmented (‘Green Salanova’) lettuce exhibited distinctive responses to sub-optimal conditions. The cultivar specific metabolomic signatures comprised a broad modulation of metabolism, including secondary metabolites, phytohormones, and membrane lipids signaling cascade. Several stress-related metabolites were altered by either treatment, including polyamines (and other nitrogen-containing compounds), phenylpropanoids, and lipids. The metabolomics and physiological response to macrocations availability and light intensity also implies that the effects of low-input sustainable farming systems should be evaluated considering a range of cultivar-specific positive and disadvantageous metabolic effects in addition to yield and other socio-economic parameters. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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Score plot of orthogonal projection to latent structures discriminant analysis (OPLS-DA) supervised modeling carried out on untargeted metabolomics profiles of ‘Red’ (A) (R2Y = 0.99, Q2Y = 0.91) and ‘Green Salanova’ (B) (R2Y = 0.97, Q2Y = 0.69) subjected to different nutrient solutions. Full article ">Figure 2
Processes affected by the macrocation concentration in ‘Red’ (A) and ‘Green Salanova’ (B). Differential metabolites and their fold-change (FC) values were elaborated using the Omic Viewer Dashboard of the PlantCyc pathway Tool software (<a href="http://www.pmn.plantcyc.com" target="_blank">www.pmn.plantcyc.com</a>). In each class, the large dot represents the average (mean) logFC of the metabolites. Small dots represent the individual logFC for each metabolite. The abbreviated subcategory names on the x-axis correspond to: Nucleo: nucleosides and nucleotides; FA/Lipids: fatty acids and lipids; Amines: amines and polyamines; Carbohyd: carbohydrates; Cofactors: cofactors, prosthetic groups, electron carriers, and vitamins. Full article ">Figure 3
Score plot of OPLS-DA supervised modeling carried out on untargeted metabolomics profiles of ‘Red’ (A) (R2Y = 0.99, Q2Y = 0.92) and ‘Green’ (B) (R2Y = 1, Q2Y = 0.92) Salanova under optimal and sub-optimal light conditions. Full article ">Figure 4
Processes affected by the light intensity in ‘Red’ (A) and ‘Green Salanova’ (B). Differential metabolites and their fold-change (FC) values were elaborated using the Omic Viewer Dashboard of the PlantCyc pathway Tool software (<a href="http://www.pmn.plantcyc.com" target="_blank">www.pmn.plantcyc.com</a>). In each class, the large dot represents the average (mean) logFC of the metabolites. Small dots represent the individual logFC for each metabolite. The abbreviated subcategory names reported on the x-axis correspond to: Nucleo: nucleosides and nucleotides; FA/Lipids: fatty acids and lipids; Amines: amines and polyamines; Carbohyd: carbohydrates; Cofactors: cofactors, prosthetic groups, electron carriers and vitamins. Full article ">

14 pages, 3445 KiB

Open AccessArticle

Characterization of the Role of SPL9 in Drought Stress Tolerance in Medicago sativa

by Alexandria Hanly, Jim Karagiannis, Qing Shi Mimmie Lu, Lining Tian and Abdelali Hannoufa

Int. J. Mol. Sci. 2020, 21(17), 6003; https://doi.org/10.3390/ijms21176003 - 20 Aug 2020

Cited by 30 | Viewed by 3799

Abstract

Extreme environmental conditions, such as drought, are expected to increase in frequency and severity due to climate change, leading to substantial deficiencies in crop yield and quality. Medicago sativa (alfalfa) is an important crop that is relied upon as a staple source of [...] Read more.

Extreme environmental conditions, such as drought, are expected to increase in frequency and severity due to climate change, leading to substantial deficiencies in crop yield and quality. Medicago sativa (alfalfa) is an important crop that is relied upon as a staple source of forage in ruminant feed. Despite its economic importance, alfalfa production is constrained by abiotic stress, including drought. In this report, we investigate the role of Squamosa Promoter Binding Protein-Like 9 (SPL9), a target of miR156, in drought tolerance. Transgenic alfalfa plants with RNAi-silenced MsSPL9 (SPL9-RNAi) were compared to wild-type (WT) alfalfa for phenotypic changes and drought tolerance indicators. In SPL9-RNAi plants, both stem thickness and plant height were reduced in two- and six-month-old alfalfa, respectively; however, yield was unaffected. SPL9-RNAi plants showed less leaf senescence and had augmented relative water content under drought conditions, indicating that SPL9-RNAi plants had greater drought tolerance potential than WT plants. Interestingly, SPL9-RNAi plants accumulated more stress-alleviating anthocyanin compared to WT under both drought and well-watered control conditions, suggesting that MsSPL9 may contribute to drought tolerance in alfalfa, at least in part, by regulating anthocyanin biosynthesis. The results suggest that targeting MsSPL9 is a suitable means for improving alfalfa resilience towards drought conditions. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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17 pages, 3366 KiB

Open AccessArticle

Identification of a Novel Gene, Osbht, in Response to High Temperature Tolerance at Booting Stage in Rice

by Jae-Ryoung Park, Won-Tae Yang, Doh-Hoon Kim and Kyung-Min Kim

Int. J. Mol. Sci. 2020, 21(16), 5862; https://doi.org/10.3390/ijms21165862 - 15 Aug 2020

Cited by 16 | Viewed by 3400

Abstract

Rice is one of the world’s leading food crops, and over 90% of the world’s rice production stems from Asia. In particular, an increase of 1 °C in the minimum temperature reduces the quantity of rice by 10%. Therefore, the development of rice [...] Read more.

Rice is one of the world’s leading food crops, and over 90% of the world’s rice production stems from Asia. In particular, an increase of 1 °C in the minimum temperature reduces the quantity of rice by 10%. Therefore, the development of rice varieties that can stably maintain the yield and quality of the rice even under these rapid climate changes is indispensable. In this study, we performed quantitative trait loci (QTL) mapping after treatment with heat stress during the booting stage in rice. We performed a QTL analysis using the Cheongcheong/Nagdong double haploid (CNDH) line and identified 19 QTLs during the 2 year analysis. Of these QTL regions, the 2.2 cM region of RM3709–RM11694 on chromosome 1 was shared among the six traits (heading date; culm length; panicle length; number of tiller; 1000 grain weight; and content of chlorophyll) examined. Rice Microsatellite (RM) 3709–RM11694 contained 27 high-temperature-tolerance candidate genes. Among the candidate genes, OsBHT showed a different gene expression level between CNDH75, which is a high-temperature tolerant line, and CNDH11 which is a susceptible line. Although some existing high-temperature-tolerant genes have been reported, OsBHT can be used more effectively for the development of heat tolerance in rice. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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Figure 1
Comparison of phenotypes after high-temperature treatment in rice at the booting stage. The Cheongcheong/Nagdong double haploid (CNDH) 120 line was treated with a high temperature during the booting stage. The phenotype that appeared after the booting stage was assessed. (A) Nagdong is a variety that is tolerant to high temperatures; (B) Cheongcheong is a variety that is susceptible to high temperatures. Culm length decreased and no heading was observed; (C) CNDH11 exhibited a decrease in tiller number after the high-temperature treatment and did not show heading; (D) CNDH109 exhibited a great decrease in tiller number after treatment with high temperature. Full article ">Figure 2
The frequency distribution of heading date, culm length, panicle length, number of tillers, 1000 grain weight, and content of chlorophyll in the CNDH population. As all traits showed a normal distribution, the investigated traits were considered to be quantitative traits. Cheongcheong is a high-temperature resistant variety, and Nagdong is a high-temperature sensitive variety. A, Cheongcheong; B, Nagdong. Full article ">Figure 3
Candidate genes between RM212 and RM3411 and changes in chlorophyll content during high-temperature treatment. (A) Forty-eight candidate genes were distributed by function. Among the candidate genes, the gene responsible for cell function was detected most frequently, and heat shock protein accounted for 8.3%. (B) Changes in chlorophyll content when high temperature was the treatment in rice at the booting stage. a, Cheongcheong; b, Nagdong. Full article ">Figure 4
Analysis of relative expression levels of candidate genes in high-temperature susceptible and tolerance lines. Expression analysis of candidate genes in rice at the booting stage exposed to 42 °C heat stress in a growth chamber, with rice leaf sampled at different time points at 0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 16 h, 24 h, 28 h, and 72 h. Data are presented as the mean ± SD (n = 3), Student’s t-test. CNDH11 is the high-temperature susceptible line, and CNDH75 is the high-temperature tolerance line. * Significant at the 0.05 level; ** significant at the 0.01 level. Full article ">Figure 5
Sequence analysis of Oryza sativa Booting stage Heat Tolerance (OsBHT). (A) Analysis of the relationship between the OsBHT gene and homology gene by phylogenetic tree. The phylogenetic tree was constructed by the parsimony method with 1000 bootstrap replicates. (B) As a result of comparing the protein sequences of the homologous genes of OsBHT, a very high similarity was shown in Oryza sativa, Arabidopsis thaliana, Zea mays, and Glycine soja. Full article ">Figure 6
OsBHT protein interaction prediction. OsBHT interact with the heat shock protein. Signaled by the guanosine triphosphate (GTP)-binding protein when plants are exposed to high temperatures, Hsp70 and Hsp90 work together with multiple co-chaperones to prevent protein denaturation. Full article ">

12 pages, 2132 KiB

Open AccessArticle

The Alleviation of Photosynthetic Damage in Tomato under Drought and Cold Stress by High CO₂ and Melatonin

by Rong Zhou, Hongjian Wan, Fangling Jiang, Xiangnan Li, Xiaqing Yu, Eva Rosenqvist and Carl-Otto Ottosen

Int. J. Mol. Sci. 2020, 21(15), 5587; https://doi.org/10.3390/ijms21155587 - 4 Aug 2020

Cited by 21 | Viewed by 4240

Abstract

The atmospheric CO₂ concentration (a[CO₂]) is increasing at an unprecedented pace. Exogenous melatonin plays positive roles in the response of plants to abiotic stresses, including drought and cold. The effect of elevated CO₂ concentration (e[CO₂]) accompanied by [...] Read more.

The atmospheric CO₂ concentration (a[CO₂]) is increasing at an unprecedented pace. Exogenous melatonin plays positive roles in the response of plants to abiotic stresses, including drought and cold. The effect of elevated CO₂ concentration (e[CO₂]) accompanied by exogenous melatonin on plants under drought and cold stresses remains unknown. Here, tomato plants were grown under a[CO₂] and e[CO₂], with half of the plants pre-treated with melatonin. The plants were subsequently treated with drought stress followed by cold stress. The results showed that a decreased net photosynthetic rate (P_N) was aggravated by a prolonged water deficit. The P_N was partially restored after recovery from drought but stayed low under a successive cold stress. Starch content was downregulated by drought but upregulated by cold. The e[CO₂] enhanced P_N of the plants under non-stressed conditions, and moderate drought and recovery but not severe drought. Stomatal conductance (g_s) and the transpiration rate (E) was less inhibited by drought under e[CO₂] than under a[CO₂]. Tomato grown under e[CO₂] had better leaf cooling than under a[CO₂] when subjected to drought. Moreover, melatonin enhanced P_N during recovery from drought and cold stress, and enhanced biomass accumulation in tomato under e[CO₂]. The chlorophyll a content in plants treated with melatonin was higher than in non-treated plants under e[CO₂] during cold stress. Our findings will improve the knowledge on plant responses to abiotic stresses in a future [CO₂]-rich environment accompanied by exogenous melatonin. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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Figure 1
Treatment flow of the experiments. “a[CO2]” and “e[CO2]” indicate 400 and 800 ppm CO2 concentration, respectively. The batch marked with “melatonin” indicates the plants were treated by seven times of 1 mM melatonin. “DS”, drought stress, 25/20 °C + no irrigation; “CS”, cold stress, 12/12 °C + irrigation; “R1” and “R2”, recovery, 25/20 °C + irrigation. Full article ">Figure 2
(A) Net photosynthetic rate (PN), (B) stomatal conductance (gs), (C) transpiration rate (E), (D) intracellular CO2 concentration (Ci), and (E) leaf temperature in the first fully expanded leaves of tomato from the top during different treatments. “a[CO2]” and “a[CO2] + M” indicates 400 ppm CO2 concentration without and with melatonin application. “e[CO2]” and “e[CO2] + M” indicates 800 ppm CO2 concentration without and with melatonin application. “Control”, 25/20 °C + irrigation; “DS”, drought stress, 25/20 °C + no irrigation, “CS”, cold stress, 12/12 °C + irrigation; “R1” and “R2”, recovery, 25/20 °C + irrigation. The data represent average values ± SD (n = 3). The ANOVA was conducted within all the treatments at different stages. Different small letters showed significant differences (p < 0.05). Full article ">Figure 3
(A) Chlorophyll a, (B) chlorophyll b, (C) carotenoid, and (D) chlorophyll a/b in the first fully expanded leaves of tomato from the top during different treatments. “a[CO2]” and “a[CO2] + M” indicates 400 ppm CO2 concentration without and with melatonin application. “e[CO2]” and “e[CO2] + M” indicates 800 ppm CO2 concentration without and with melatonin application. “Control”, 25/20 °C + irrigation; “Drought”, 25/20 °C + no irrigation for 30 h; “Cold”, 12/12 °C + irrigation for 60 h. The data represent average values ± SD (n = 3). The ANOVA was conducted within all the treatments. Different small letters showed significant differences (p < 0.05). Full article ">Figure 4
(A) Glucose, (B) fructose, (C) sucrose, and (D) starch in the first fully expanded leaves of tomato from the top during different treatments. Treatments are the same as in <a href="#ijms-21-05587-f003" class="html-fig">Figure 3</a>. The data represent average values ± SE (n = 3). The ANOVA was conducted within all the treatments. Different small letters showed significant differences (p < 0.05). Full article ">Figure 5
(A) Plant height, (B) leaf area, (C) leaf FW and (D) leaf DW, (E) stem FW, and (F) stem DW of tomato during the treatments. FW and DW are the abbreviations of fresh and dry weight, respectively. Treatments are the same as in <a href="#ijms-21-05587-f003" class="html-fig">Figure 3</a>. The data represent average values ± SD (n = 3). Different small letters showed significant differences (p < 0.05). Full article ">Figure 6
Plant morphology of tomato under different treatments. “a[CO2]” and “a[CO2] + M” indicates 400 ppm CO2 concentration without and with melatonin application. “e[CO2]” and “e[CO2] + M” indicates 800 ppm CO2 concentration without and with melatonin application. “Control”, 25/20 °C + irrigation; “Drought”, 25/20 °C + no irrigation for 32 h; “Cold”, 12/12 °C + irrigation for 68 h; “Recover”, 25/20 °C + irrigation for 24 h after cold stress. Full article ">Figure 7
Schematic diagram of the main findings. Full article ">

Review

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19 pages, 1279 KiB

Open AccessReview

Adjustment of K⁺ Fluxes and Grapevine Defense in the Face of Climate Change

by Houssein Monder, Morgan Maillard, Isabelle Chérel, Sabine Dagmar Zimmermann, Nadine Paris, Teresa Cuéllar and Isabelle Gaillard

Int. J. Mol. Sci. 2021, 22(19), 10398; https://doi.org/10.3390/ijms221910398 - 27 Sep 2021

Cited by 15 | Viewed by 2906

Abstract

Grapevine is one of the most economically important fruit crops due to the high value of its fruit and its importance in winemaking. The current decrease in grape berry quality and production can be seen as the consequence of various abiotic constraints imposed [...] Read more.

Grapevine is one of the most economically important fruit crops due to the high value of its fruit and its importance in winemaking. The current decrease in grape berry quality and production can be seen as the consequence of various abiotic constraints imposed by climate changes. Specifically, produced wines have become too sweet, with a stronger impression of alcohol and fewer aromatic qualities. Potassium is known to play a major role in grapevine growth, as well as grape composition and wine quality. Importantly, potassium ions (K⁺) are involved in the initiation and maintenance of the berry loading process during ripening. Moreover, K⁺ has also been implicated in various defense mechanisms against abiotic stress. The first part of this review discusses the main negative consequences of the current climate, how they disturb the quality of grape berries at harvest and thus ultimately compromise the potential to obtain a great wine. In the second part, the essential electrical and osmotic functions of K⁺, which are intimately dependent on K⁺ transport systems, membrane energization, and cell K⁺ homeostasis, are presented. This knowledge will help to select crops that are better adapted to adverse environmental conditions. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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14 pages, 1289 KiB

Open AccessReview

Ozone Induced Stomatal Regulations, MAPK and Phytohormone Signaling in Plants

by Md. Mahadi Hasan, Md. Atikur Rahman, Milan Skalicky, Nadiyah M. Alabdallah, Muhammad Waseem, Mohammad Shah Jahan, Golam Jalal Ahammed, Mohamed M. El-Mogy, Ahmed Abou El-Yazied, Mohamed F. M. Ibrahim and Xiang-Wen Fang

Int. J. Mol. Sci. 2021, 22(12), 6304; https://doi.org/10.3390/ijms22126304 - 11 Jun 2021

Cited by 66 | Viewed by 5822

Abstract

Ozone (O₃) is a gaseous environmental pollutant that can enter leaves through stomatal pores and cause damage to foliage. It can induce oxidative stress through the generation of reactive oxygen species (ROS) like hydrogen peroxide (H₂O₂) that [...] Read more.

Ozone (O₃) is a gaseous environmental pollutant that can enter leaves through stomatal pores and cause damage to foliage. It can induce oxidative stress through the generation of reactive oxygen species (ROS) like hydrogen peroxide (H₂O₂) that can actively participate in stomatal closing or opening in plants. A number of phytohormones, including abscisic acid (ABA), ethylene (ET), salicylic acid (SA), and jasmonic acid (JA) are involved in stomatal regulation in plants. The effects of ozone on these phytohormones’ ability to regulate the guard cells of stomata have been little studied, however, and the goal of this paper is to explore and understand the effects of ozone on stomatal regulation through guard cell signaling by phytohormones. In this review, we updated the existing knowledge by considering several physiological mechanisms related to stomatal regulation after response to ozone. The collected information should deepen our understanding of the molecular pathways associated with response to ozone stress, in particular, how it influences stomatal regulation, mitogen-activated protein kinase (MAPK) activity, and phytohormone signaling. After summarizing the findings and noting the gaps in the literature, we present some ideas for future research on ozone stress in plants Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
A proposed model associated with signaling in guard cells in response to ozone stress. Ozone leads to the formation of free radicals (ROS) in the apoplast and a rise in Ca2+ level in guard cells (a). Several apoplastic antioxidants including ascorbic acid (AsA) scavenge ROS and inhibit their generation (b). The mechanism of ROS defense triggered by high ozone concentrations involves two key antioxidants—peroxidases (PRX) and NADPH oxidases (RbohF) (c). RbohF in the signal transduction process is activated by second messengers including Ca2+, open stomata 1 (OST1), and calcium-dependent protein kinases (CPKs) (d). Apoplastic ROS may influence plasma membrane sensor proteins and receptor-like kinases, (RLKs) such that ROS (e.g., H2O2) can be transferred through aquaporin (AQP) channels (e). Cytosolic Ca2+ is sensed in chloroplasts by the calcium-sensing receptor (CAS). Apoplastic oxidized dehydroascorbate (DHA) and AsA move across the PM into the cytosol and regulate redox homeostasis (e), which is also sensed by redox-sensitive transcription factors (TFs). Triggering of mitogen-activated protein kinases 3/6 (MPK3/6) (f). Activation of MPK3/6 and translocation to the nucleus leads to phosphorylation and activates transcription factors (TFs) (g). Solid and broken lines indicate direct and indirect interactions, respectively. Full article ">Figure 2
A schematic model of phytohormone-induced guard-cell signaling associated with stomatal regulation in response to ozone (O3), adapted from Wilkinson and Davies (2010) [<a href="#B31-ijms-22-06304" class="html-bibr">31</a>]. (a) Ozone and ethylene (ET) can substitute within the abscisic acid (ABA) signal transduction branch leading to stomatal closure through H2O2 in the absence of ABA. (b) A higher level of H2O2 and NO might prevent stomatal closure in the presence of ABA, O3, and ET. (c) ET acts via the ETR1 receptor to inhibit response to ABA-induced H2O2 and prevent stomatal closure in the presence of ABA. Full article ">Figure 3
Programmed cell death (PCD) cycle induced by ozone (O3) associated with Phytohormones, adapted from Kangasjarvi et al. (2005) [<a href="#B67-ijms-22-06304" class="html-bibr">67</a>]. Reactive oxygen species (ROS) accumulation is caused by ozone stress and results in the accumulation of salicylic acid (SA) and PCD. Cell death activates ethylene (ET) production required for ROS generation resulting in PCD. Jasmonic acid (JA) antagonizes cell cycle progression by inducing cell death and promoting SA and ET function. Abscisic acid (ABA) antagonizes the function of ET, which may have an important role in ozone-induced PCD. Full article ">

22 pages, 2761 KiB

Open AccessReview

Plant Mitogen-Activated Protein Kinase Cascades in Environmental Stresses

by Li Lin, Jian Wu, Mingyi Jiang and Youping Wang

Int. J. Mol. Sci. 2021, 22(4), 1543; https://doi.org/10.3390/ijms22041543 - 3 Feb 2021

Cited by 84 | Viewed by 5917

Abstract

Due to global warming and population growth, plants need to rescue themselves, especially in unfavorable environments, to fulfill food requirements because they are sessile organisms. Stress signal sensing is a crucial step that determines the appropriate response which, ultimately, determines the survival of [...] Read more.

Due to global warming and population growth, plants need to rescue themselves, especially in unfavorable environments, to fulfill food requirements because they are sessile organisms. Stress signal sensing is a crucial step that determines the appropriate response which, ultimately, determines the survival of plants. As important signaling modules in eukaryotes, plant mitogen-activated protein kinase (MAPK) cascades play a key role in regulating responses to the following four major environmental stresses: high salinity, drought, extreme temperature and insect and pathogen infections. MAPK cascades are involved in responses to these environmental stresses by regulating the expression of related genes, plant hormone production and crosstalk with other environmental stresses. In this review, we describe recent major studies investigating MAPK-mediated environmental stress responses. We also highlight the diverse function of MAPK cascades in environmental stress. These findings help us understand the regulatory network of MAPKs under environmental stress and provide another strategy to improve stress resistance in crops to ensure food security. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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Figure 1

Figure 1
MAPK cascade in salt and drought stress. (a) The following three MAPK cascades can regulate salt stress in Arabidopsis: the AtMEKK1-AtMKK2-AtMPK4 cascade, the AtMEKK1-AtMKK5-AtMPK6 cascade and the AtMKK4-AtMPK3 cascade. The substrate of the AtMEKK1-AtMKK2-AtMPK4 and AtMKK4-AtMPK3 cascades is still unknown (marked as ?). The AtMEKK1-AtMKK5-AtMPK6 cascade confers tolerance to salt stress by regulating AtFSD2/3 expression. AtFSD2/3 are two major FSD-encoding genes in Arabidopsis. (b) The AtMAPKKK18-AtMKK3-AtMPK1/2/7/14 cascade can be activated by ABA after drought stress. The AtMAPKKK18-AtMKK3-AtMPK1/2/7/14 cascade positively regulates drought stress in an ABA-dependent manner. The substrate of AtMPK1/2/7/14 is unknown (marked as ?). (c) Two MAPK cascades are involved in drought stress in cotton. The GhMKK3-GhMPK7 cascade enhances drought tolerance in an ABA-dependent manner. Whether GhPIP1 is the substrate of GhMPK7 requires more experimental evidence (marked as ?). GhMAP3K15-GhMKK4-GhMPK6 positively regulates drought stress in an ABA-independent manner. The substrate of this cascade is GhWRKY59. GhWRKY59 can regulate GhDREB2 expression by directly binding the W-box of GhDREB2 promoters. This figure was created using BioRender (<a href="http://biorender.com/" target="_blank">http://biorender.com/</a>; accessed on 14 December 2020). Full article ">Figure 2
MAPK cascade regulates cold stress in Arabidopsis and rice. (a,b) In Arabidopsis, two pathways can regulate cold stress. AtMEKK1-AtMKK2-AtMPK4 positively regulates cold stress, whereas AtMKK4/5-AtMPK3/6 negatively regulates cold stress. (a) When cold stress occurs, AtMPK3/6 can phosphorylate AtICE1 and AtMYB15, which induces AtICE1 fast degradation and represses AtMYB15’s binding affinity, which, in turn, attenuates AtCBF3 transcription. The upstream targets of AtMKK4/5 are unknown (marked as ?). AtMEKK1-AtMKK2-AtMPK4 suppresses AtMPK3/6 activity. The substrate of AtMPK4 is still unknown (named TFs). (b) In the absence of cold stress, AtMPK3/6 cannot phosphorylate AtICE1 and AtMYB15. AtICE1 can be degraded by 26S proteasome, and AtMYB15 can bind the promoter of AtCBF3 to suppress AtCBF3 expression. (c,d) The OsMPK3-OsICE1 cascade regulates cold stress in rice. (c) Upon cold stress treatment, OsMPK3 phosphorylates OsICE1, which represses the interaction between OsICE1 and OsHOS1 and eventually induces OsTPP1 expression and trehalose production. OsMKK6 and other MAPKKs (Mitogen-activated protein kinase kinase) (marked as ?) are shown as upstream positive or negative regulators of this MAPK cascade. OsPP2C72 can dephosphorylate OsMPK3 and OsICE1 which represses the function of OsMPK3 and OsICE1 under cold stress. (d) Under warm temperature, OsMPK3 cannot phosphorylate OsICE1, which can be degraded by OsHOS1. This figure was created using BioRender (<a href="http://biorender.com/" target="_blank">http://biorender.com/</a>; accessed on 14 December 2020). Full article ">Figure 3
MAPK cascade function in biotic stress. (a) The MAPK cascade is involved in camalexin biosynthesis after Botrytis cinerea infection. (b) The AtMEKK1-AtMKK4/5-AtMPK3/6 cascade regulates ethylene (ET) production in two dependent ways. In one approach, AtMPK3/6 phosphorylates AtWRKY33, which can bind the promoters of AtACS2 and AtACS6 and activate AtACS2 and AtACS6 expression. In the other approach, AtMPK3/6 can directly phosphorylate AtACS2 and AtACS6, eventually promoting AtACS2 and AtACS6 stability. (c) AtMEKK1-AtMKK1/2-AtMPK4 negatively regulates salicylic acid (SA) production by negatively regulating AtPAD4 and AtEDS1 activities, whereas OsMKK10.2-OsMPK6 positively regulates SA production. (d) AtMAPKKK14-AtMKK3-AtMPK1/2/7/1 can be activated by jasmonic acid (JA) after insect feeding. The red arrows mean content increase. This figure was created using BioRender (<a href="http://biorender.com/" target="_blank">http://biorender.com/</a>; accessed on 14 December 2020). Full article ">

24 pages, 4834 KiB

Open AccessReview

Plant Morphological, Physiological and Anatomical Adaption to Flooding Stress and the Underlying Molecular Mechanisms

by Weitao Jia, Maohua Ma, Jilong Chen and Shengjun Wu

Int. J. Mol. Sci. 2021, 22(3), 1088; https://doi.org/10.3390/ijms22031088 - 22 Jan 2021

Cited by 89 | Viewed by 15292

Abstract

Globally, flooding is a major threat causing substantial yield decline of cereal crops, and is expected to be even more serious in many parts of the world due to climatic anomaly in the future. Understanding the mechanisms of plants coping with unanticipated flooding [...] Read more.

Globally, flooding is a major threat causing substantial yield decline of cereal crops, and is expected to be even more serious in many parts of the world due to climatic anomaly in the future. Understanding the mechanisms of plants coping with unanticipated flooding will be crucial for developing new flooding-tolerance crop varieties. Here we describe survival strategies of plants adaptation to flooding stress at the morphological, physiological and anatomical scale systemically, such as the formation of adventitious roots (ARs), aerenchyma and radial O₂ loss (ROL) barriers. Then molecular mechanisms underlying the adaptive strategies are summarized, and more than thirty identified functional genes or proteins associated with flooding-tolerance are searched out and expounded. Moreover, we elaborated the regulatory roles of phytohormones in plant against flooding stress, especially ethylene and its relevant transcription factors from the group VII Ethylene Response Factor (ERF-VII) family. ERF-VIIs of main crops and several reported ERF-VIIs involving plant tolerance to flooding stress were collected and analyzed according to sequence similarity, which can provide references for screening flooding-tolerant genes more precisely. Finally, the potential research directions in the future were summarized and discussed. Through this review, we aim to provide references for the studies of plant acclimation to flooding stress and breeding new flooding-resistant crops in the future. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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42 pages, 2405 KiB

Open AccessReview

Regulation of ROS Metabolism in Plants under Environmental Stress: A Review of Recent Experimental Evidence

by Mirza Hasanuzzaman, M. H. M. Borhannuddin Bhuyan, Khursheda Parvin, Tasnim Farha Bhuiyan, Taufika Islam Anee, Kamrun Nahar, Md. Shahadat Hossen, Faisal Zulfiqar, Md. Mahabub Alam and Masayuki Fujita

Int. J. Mol. Sci. 2020, 21(22), 8695; https://doi.org/10.3390/ijms21228695 - 18 Nov 2020

Cited by 313 | Viewed by 14768

Abstract

Various environmental stresses singly or in combination generate excess amounts of reactive oxygen species (ROS), leading to oxidative stress and impaired redox homeostasis. Generation of ROS is the obvious outcome of abiotic stresses and is gaining importance not only for their ubiquitous generation [...] Read more.

Various environmental stresses singly or in combination generate excess amounts of reactive oxygen species (ROS), leading to oxidative stress and impaired redox homeostasis. Generation of ROS is the obvious outcome of abiotic stresses and is gaining importance not only for their ubiquitous generation and subsequent damaging effects in plants but also for their diversified roles in signaling cascade, affecting other biomolecules, hormones concerning growth, development, or regulation of stress tolerance. Therefore, a good balance between ROS generation and the antioxidant defense system protects photosynthetic machinery, maintains membrane integrity, and prevents damage to nucleic acids and proteins. Notably, the antioxidant defense system not only scavenges ROS but also regulates the ROS titer for signaling. A glut of studies have been executed over the last few decades to discover the pattern of ROS generation and ROS scavenging. Reports suggested a sharp threshold level of ROS for being beneficial or toxic, depending on the plant species, their growth stages, types of abiotic stresses, stress intensity, and duration. Approaches towards enhancing the antioxidant defense in plants is one of the vital areas of research for plant biologists. Therefore, in this review, we accumulated and discussed the physicochemical basis of ROS production, cellular compartment-specific ROS generation pathways, and their possible distressing effects. Moreover, the function of the antioxidant defense system for detoxification and homeostasis of ROS for maximizing defense is also discussed in light of the latest research endeavors and experimental evidence. Full article

(This article belongs to the Special Issue Environmental Stress and Plants)

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