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23 pages, 6558 KiB  
Article
Unravelling Different Water Management Strategies in Three Olive Cultivars: The Role of Osmoprotectants, Proteins, and Wood Properties
by Sara Parri, Claudia Faleri, Marco Romi, José C. del Río, Jorge Rencoret, Maria Celeste Pereira Dias, Sara Anichini, Claudio Cantini and Giampiero Cai
Int. J. Mol. Sci. 2024, 25(20), 11059; https://doi.org/10.3390/ijms252011059 - 15 Oct 2024
Viewed by 632
Abstract
Understanding the responses of olive trees to drought stress is crucial for improving cultivation and developing drought-tolerant varieties. Water transport and storage within the plant is a key factor in drought-tolerance strategies. Water management can be based on a variety of factors such [...] Read more.
Understanding the responses of olive trees to drought stress is crucial for improving cultivation and developing drought-tolerant varieties. Water transport and storage within the plant is a key factor in drought-tolerance strategies. Water management can be based on a variety of factors such as stomatal control, osmoprotectant molecules, proteins and wood properties. The aim of the study was to evaluate the water management strategy under drought stress from an anatomical and biochemical point of view in three young Italian olive cultivars (Giarraffa, Leccino and Maurino) previously distinguished for their physiological and metabolomic responses. For each cultivar, 15 individuals in pots were exposed or not to 28 days of water withholding. Every 7 days, the content of sugars (including mannitol), proline, aquaporins, osmotins, and dehydrins, in leaves and stems, as well as the chemical and anatomical characteristics of the wood of the three cultivars, were analyzed. ‘Giarraffa’ reduced glucose levels and increased mannitol production, while ‘Leccino’ accumulated more proline. Both ‘Leccino’ and ‘Maurino’ increased sucrose and aquaporin levels, possibly due to their ability to remove embolisms. ‘Maurino’ and ‘Leccino’ accumulated more dehydrins and osmotins. While neither genotype nor stress affected wood chemistry, ‘Maurino’ had a higher vessel-to-xylem area ratio and a larger hydraulic diameter, which allows it to maintain a high transpiration rate but may make it more susceptible to cavitation. The results emphasized the need for an integrated approach, highlighting the importance of the relative timing and sequence of each parameter analyzed, allowing, overall, to define a “strategy” rather than a “response” to drought of each cultivar. Full article
(This article belongs to the Special Issue Molecular Advances in Olive and Its Derivatives)
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Figure 1

Figure 1
<p>Sugar levels identified by HPLC in Giarraffa (GIA), Leccino (LEC) and Maurino (MAU) under control (CTRL, black) and drought stress (DS, orange). (<b>A</b>) Glucose in leaf; (<b>B</b>) glucose in stem; (<b>C</b>) fructose in leaf; (<b>D</b>) fructose in stem; (<b>E</b>) sucrose in leaf; (<b>F</b>) sucrose in stem; (<b>G</b>) mannitol in leaf; (<b>H</b>) mannitol in stem, all expressed in mg g<sup>−1</sup> tissue dry weight (DW). Data in each column are presented as mean ± standard error. Within each time point, different letters denote statistical significance (<span class="html-italic">p</span>-value &lt; 0.05) according to Tukey’s multiple post hoc tests.</p>
Full article ">Figure 1 Cont.
<p>Sugar levels identified by HPLC in Giarraffa (GIA), Leccino (LEC) and Maurino (MAU) under control (CTRL, black) and drought stress (DS, orange). (<b>A</b>) Glucose in leaf; (<b>B</b>) glucose in stem; (<b>C</b>) fructose in leaf; (<b>D</b>) fructose in stem; (<b>E</b>) sucrose in leaf; (<b>F</b>) sucrose in stem; (<b>G</b>) mannitol in leaf; (<b>H</b>) mannitol in stem, all expressed in mg g<sup>−1</sup> tissue dry weight (DW). Data in each column are presented as mean ± standard error. Within each time point, different letters denote statistical significance (<span class="html-italic">p</span>-value &lt; 0.05) according to Tukey’s multiple post hoc tests.</p>
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<p>Proline content in leaves (<b>A</b>) and stems (<b>B</b>) of Giarraffa (GIA), Leccino (LEC), and Maurino (MAU) cultivars under control (CTRL, black) and drought stress (DS, orange). Contents are expressed as μg g<sup>−1</sup> tissue dry weight (DW). Values in each column are presented as mean ± standard error. Within each time point, different letters denote statistical significance (<span class="html-italic">p</span>-value &lt; 0.05) according to Tukey’s multiple post hoc tests.</p>
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<p>PIP1 aquaporin levels in stems of Giarraffa (GIA), Leccino (LEC) and Maurino (MAU) cultivars under control (CTRL) and drought-stress (DS) conditions, at the beginning of stress (t0), two weeks later (t2) and four weeks later (t4). (<b>A</b>) Membranes immunoblotted with anti-aquaporin antibodies from the above experimental groups; (<b>B</b>) relative blot quantification expressed as integrated density (i.d.).</p>
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<p>Dehydrin levels in leaves of Giarraffa (GIA), Leccino (LEC) and Maurino (MAU) cultivars after two (t2) and four (t4) weeks of stress. (<b>A</b>) Membranes immunoblotted with anti-dehydrin antibodies from the above experimental groups; (<b>B</b>) relative quantification of the blots expressed as integrated density (i.d.).</p>
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<p>Dehydrin levels in stems of Giarraffa (GIA), Leccino (LEC) and Maurino (MAU) cultivars under control (CTRL) and drought-stress (DS) conditions, at the beginning of stress (t0), two weeks later (t2) and four weeks later (t4). (<b>A</b>) Membranes immunoblotted with anti-dehydrin antibodies from the above experimental groups; (<b>B</b>) relative blotting quantification expressed as integrated density (i.d.).</p>
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<p>Osmotin levels in leaves of Giarraffa (GIA), Leccino (LEC) and Maurino (MAU) cultivars after two (t2) and four (t4) weeks of stress. (<b>A</b>) Membranes immunoblotted with anti-osmotin antibodies from the above experimental groups; (<b>B</b>) relative quantification of blotting expressed as integrated density (i.d.).</p>
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<p>Osmotin levels in stems of Giarraffa (GIA), Leccino (LEC) and Maurino (MAU) cultivars under control (CTRL) and drought-stress (DS) conditions, at the beginning of stress (t0), two weeks (t2) and four weeks (t4). (<b>A</b>) Membranes immunoblotted with anti-osmotin antibodies from the above experimental groups; (<b>B</b>) relative blotting quantification expressed as integrated density (i.d.).</p>
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<p>2D-HSQC NMR spectra of stems from three olive cultivars (Giarraffa, Leccino, and Maurino) subjected to drought stress (DS) (bottom) and their corresponding stem controls (top). The primary lignin structures identified are also shown. A: β-<span class="html-italic">O</span>-4′ alkyl-aryl ethers; B: β-5′ phenylcoumarans; C: β-β′ resinols; F: β-1′-spirodienones Cinnamyl alcohol end-groups (I), cinnamaldehyde end-groups (J), <span class="html-italic">p</span>-hydroxyphenyl units (H), guaiacyl units (G), syringyl units (S), and Cα-oxidized syringyl units (Sʹ). The yellow boxes reflect semi-quantitative estimates of lignin units and compounds. Composition is expressed in molar percent (H + G + S = 100%), and end-groups are expressed as a fraction of the total lignin inter-unit linkage types A–F.</p>
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<p>Stem sections of <span class="html-italic">Olea europaea</span> cultivars Giarraffa (<b>A</b>), Leccino (<b>B</b>), and Maurino (<b>C</b>). ph: phloem, x: xylem vessels; cz: cambial zone; r: parenchyma ray; f: fibers; p: paratracheal parenchyma; bar corresponds to 20 µm. (<b>D</b>) Frequency distributions (number of vessels by 5 µm diameter) of vessel lumen diameters in the three olive cultivars.</p>
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24 pages, 9066 KiB  
Article
Genome-Wide Identification and Expression Analyses of the Thaumatin-Like Protein Gene Family in Tetragonia tetragonoides (Pall.) Kuntze Reveal Their Functions in Abiotic Stress Responses
by Zengwang Huang, Qianqian Ding, Zhengfeng Wang, Shuguang Jian and Mei Zhang
Plants 2024, 13(17), 2355; https://doi.org/10.3390/plants13172355 - 23 Aug 2024
Viewed by 796
Abstract
Thaumatin-like proteins (TLPs), including osmotins, are multifunctional proteins related to plant biotic and abiotic stress responses. TLPs are often present as large multigene families. Tetragonia tetragonoides (Pall.) Kuntze (Aizoaceae, 2n = 2x = 32), a vegetable used in both food and [...] Read more.
Thaumatin-like proteins (TLPs), including osmotins, are multifunctional proteins related to plant biotic and abiotic stress responses. TLPs are often present as large multigene families. Tetragonia tetragonoides (Pall.) Kuntze (Aizoaceae, 2n = 2x = 32), a vegetable used in both food and medicine, is a halophyte that is widely distributed in the coastal areas of the tropics and subtropics. Saline–alkaline soils and drought are two major abiotic stress factors significantly affecting the distribution of tropical coastal plants. The expression of stress resistance genes would help to alleviate the cellular damage caused by abiotic stress factors such as high temperature, salinity–alkalinity, and drought. This study aimed to better understand the functions of TLPs in the natural ecological adaptability of T. tetragonoides to harsh habitats. In the present study, we used bioinformatics approaches to identify 37 TtTLP genes as gene family members in the T. tetragonoides genome, with the purpose of understanding their roles in different developmental processes and the adaptation to harsh growth conditions in tropical coral regions. All of the TtTLPs were irregularly distributed across 32 chromosomes, and these gene family members were examined for conserved motifs of their coding proteins and gene structure. Expression analysis based on RNA sequencing and subsequent qRT-PCR showed that the transcripts of some TtTLPs were decreased or accumulated with tissue specificity, and under environmental stress challenges, multiple TtTLPs exhibited changeable expression patterns at short (2 h), long (48 h), or both stages. The expression pattern changes in TtTLPs provided a more comprehensive overview of this gene family being involved in multiple abiotic stress responses. Furthermore, several TtTLP genes were cloned and functionally identified using the yeast expression system. These findings not only increase our understanding of the role that TLPs play in mediating halophyte adaptation to extreme environments but also improve our knowledge of plant TLP evolution. This study also provides a basis and reference for future research on the roles of plant TLPs in stress tolerance and ecological environment suitability. Full article
(This article belongs to the Section Plant Molecular Biology)
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Figure 1
<p>Locations of the 37 <span class="html-italic">TtTLP</span>s on 16 chromosomes in <span class="html-italic">T. tetragonoides</span>.</p>
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<p>(<b>A</b>) Phylogenetic relationships of the 37 TtTLPs from <span class="html-italic">T. tetragonoides</span>. (<b>B</b>) The conserved motifs of each group of TtTLPs identified using the MEME web server. Different motifs are represented by different colored boxes, and the motif sequences are provided at the bottom.</p>
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<p>Exon–intron organization of the <span class="html-italic">TtTLP</span> genes constructed using GSDS 2.0.</p>
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<p>Phylogenetic relationships of the 37 TtTLPs from <span class="html-italic">T. tetragonoides</span>, 27 AtTLPs from <span class="html-italic">Arabidopsis thaliana</span>, and 30 OsTLPs from <span class="html-italic">Oryza sativa</span>. The amino acid sequences of these 94 TLPs from three plant species were compared with ClustalW alignment, and the phylogenetic tree was constructed in MEGA X using the neighbor-joining method, with 1000 bootstrap repetitions. The different branch colors represent different subgroups.</p>
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<p>Statistics for predicted <span class="html-italic">cis</span>-acting elements in the <span class="html-italic">TtTLP</span> promoters (ATG_upstream 2000 bp). (<b>A</b>) Summaries of the 13 <span class="html-italic">cis</span>-acting elements in the promoter regions of 37 <span class="html-italic">TtTLP</span>s. The scale bar represents 200 bp. (<b>B</b>) Distribution of the 13 <span class="html-italic">cis</span>-acting elements in the 37 <span class="html-italic">TtTLP</span> promoter regions. The elements are represented by different symbols. Information on these elements is listed in <a href="#app1-plants-13-02355" class="html-app">Table S3</a>.</p>
Full article ">Figure 5 Cont.
<p>Statistics for predicted <span class="html-italic">cis</span>-acting elements in the <span class="html-italic">TtTLP</span> promoters (ATG_upstream 2000 bp). (<b>A</b>) Summaries of the 13 <span class="html-italic">cis</span>-acting elements in the promoter regions of 37 <span class="html-italic">TtTLP</span>s. The scale bar represents 200 bp. (<b>B</b>) Distribution of the 13 <span class="html-italic">cis</span>-acting elements in the 37 <span class="html-italic">TtTLP</span> promoter regions. The elements are represented by different symbols. Information on these elements is listed in <a href="#app1-plants-13-02355" class="html-app">Table S3</a>.</p>
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<p>(<b>A</b>) Heatmaps showing the expression levels of the <span class="html-italic">TtTLP</span>s in the roots, stems, leaves, flower buds, and young fruit of <span class="html-italic">T. tetragonoides</span> plants. (<b>B</b>) Heatmaps showing the expression levels of the <span class="html-italic">TtTLP</span>s in <span class="html-italic">T. tetragonoides</span> seedlings under heat treatment (45 °C for 2 h). “-C” represents “control”. The RNA-seq data of the <span class="html-italic">TtTLP</span>s were listed in <a href="#app1-plants-13-02355" class="html-app">Table S4</a>. The heat map was constructed from log2-transformed FPKM (+1) values, and normalized treatments were carried out based on rows. The RNA-seq data of the <span class="html-italic">TtTLP</span>s are listed in <a href="#app1-plants-13-02355" class="html-app">Table S4</a>.</p>
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<p>Heatmaps showing the expression levels of the <span class="html-italic">TtTLP</span>s under salt, alkalinity, and high osmotic treatment in <span class="html-italic">T. tetragonoides</span> seedling roots (<b>A</b>), stems (<b>B</b>), and leaves (<b>C</b>). “-C” “-2 h”, and “-2 d” each represent “control”, “stress treatment for two hours”, and “stress treatment for two days (48 h)”.The heat map was constructed from log2-transformed FPKM (+1) values, and normalized treatments were carried out based on rows. The RNA-seq data of the <span class="html-italic">TtTLP</span>s were listed in <a href="#app1-plants-13-02355" class="html-app">Table S4</a>.</p>
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<p>Quantitative RT-PCR detection of the expression levels of the six candidate <span class="html-italic">TtTLP</span>s in response to heat stress in <span class="html-italic">T. tetragonoides</span> seedlings. The relative expression levels in the root, stem, and leaf samples under heat stress treatment (45 °C; 0 and 2 h) were calculated using the 2<sup>−ΔCt</sup> method, with the housekeeping gene <span class="html-italic">TtACT</span> as a reference gene. Bars show the mean values ± SD of <span class="html-italic">n</span> = 3–4 technical replicates. Asterisks indicate significant differences from the CK (control check, without heat stress, Student’s <span class="html-italic">t</span>-test, *** <span class="html-italic">p</span> &lt; 0.001, and **** <span class="html-italic">p</span> &lt; 0.0001).</p>
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<p>Relative expression levels of six candidate <span class="html-italic">TtTLP</span>s checked by qRT-PCR in root, stem, and leaf samples of <span class="html-italic">T. tetragonoides</span> seedlings under high salinity, high alkalinity, and high osmotic treatments (0, 2, 8, 24, and 48 h). Relative expression values were calculated using the 2<sup>−ΔCt</sup> method, with the housekeeping gene <span class="html-italic">TtACT</span> as a reference gene. Bars show the mean values ± SD of <span class="html-italic">n</span> = 3–4 technical replicates. Asterisks indicate significant differences from the CK (control check, without abiotic stresses, Student’s <span class="html-italic">t</span>-test, * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, and **** <span class="html-italic">p</span> &lt; 0.0001).</p>
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<p>Functional identification related to abiotic stress of six candidate <span class="html-italic">TtTLP</span>s in yeast using a heterologous expression assay. The yeast wild-type (WT) strain BY4741 was transformed with the empty vector pYES2 or six recombinant vectors, namely TtTLP6-pYES2, TtTLP10-pYES2, TtTLP11-pYES2, TtTLP12-pYES2, TtTLP22-pYES2, and TtTLP19-pYES2. Yeast cultures were adjusted to OD600 = 2, and 2 μL serial dilutions (10-fold, from left to right in each panel) were spotted on SDG-Ura medium plates supplemented with different NaCl concentrations (0, 0.8, 1, and 1.2 M) (<b>A</b>); NaHCO<sub>3</sub> concentrations (0, 9, 10, and 11 mM, pH 8.2) (<b>B</b>); mannitol concentrations (0, 0.8, 1, and 1.2 M) (<b>C</b>); heat challenges (52 °C for 20, 30, and 40 min, yeast strains without heat stress as control) (<b>D</b>); and freeze–thawing challenges (one, two, and three times, yeast strains without freeze-thawing as control) (<b>E</b>). The WT strain transformed with pYES2 was used as a positive control, and the yeast spots growing on the SDG-Ura medium plate without any challenge were the CK (check) control. The plates were incubated for 2–5 d at 30 °C.</p>
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<p>Functional identification related to hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) tolerance of six <span class="html-italic">TtTLP</span>s in yeast using heterologous expression assays. The yeast wild-type (WT) and two H<sub>2</sub>O<sub>2</sub>-sensitive mutant strains, <span class="html-italic">skn7∆</span> and <span class="html-italic">yap1∆</span>, were transformed with the empty vector pYES2 or six recombinant vectors, namely TtTLP6-pYES2, TtTLP10-pYES2, TtTLP11-pYES2, TtTLP12-pYES2, TtTLP22-pYES2, and TtTLP19-pYES2. (<b>A</b>) H<sub>2</sub>O<sub>2</sub> (0, 0.4, 0.5, and 0.6 mM) tolerance in <span class="html-italic">skn7∆</span>; (<b>B</b>) H<sub>2</sub>O<sub>2</sub> (0, 0.4, 0.5, and 0.6 mM) tolerance in <span class="html-italic">yap1∆</span>. The yeast strains were cultured and spotted, as described above.</p>
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<p>Functional identification related to heavy metal (HM) tolerance of six <span class="html-italic">TtTLP</span>s in yeast using heterologous expression assays. The yeast wild-type (WT) and a series of metal-sensitive mutant strains were transformed with the empty vector pYES2 or six recombinant vectors, namely TtTLP6-pYES2, TtTLP10-pYES2, TtTLP11-pYES2, TtTLP12-pYES2, TtTLP22-pYES2, and TtTLP19-pYES2. (<b>A</b>) Cadmium (Cd, 0, 30, 40, and 50 μM) tolerance in <span class="html-italic">ycf1∆</span>; (<b>B</b>) zinc (Zn, 0, 0.2, 0.3, and 0.4 mM) tolerance in <span class="html-italic">zrc1∆cot1∆</span>; (<b>C</b>) cobalt (Co, 0, 0.1, 0.15, and 0.2 mM) tolerance in <span class="html-italic">cot1∆</span>; (<b>D</b>) nickel (Ni, 0, 0.25, 0.5, and 1 mM) tolerance in <span class="html-italic">smf1∆</span>; and (<b>E</b>) manganese (Mn, 0, 0.5, 1, and 2 mM) tolerance in <span class="html-italic">smf1∆</span>. The yeast strains were cultured and spotted, as described previously.</p>
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17 pages, 371 KiB  
Review
Cognitive Impairment in Parkinson’s Disease: An Updated Overview Focusing on Emerging Pharmaceutical Treatment Approaches
by Yildiz Degirmenci, Efthalia Angelopoulou, Vasiliki Epameinondas Georgakopoulou and Anastasia Bougea
Medicina 2023, 59(10), 1756; https://doi.org/10.3390/medicina59101756 - 1 Oct 2023
Cited by 6 | Viewed by 4549
Abstract
Cognitive impairment in patients with Parkinson’s disease (PD) is one of the commonest and most disabling non-motor manifestations during the course of the disease. The clinical spectrum of PD-related cognitive impairment includes subjective cognitive decline (SCD), mild cognitive impairment (MCI) and PD dementia [...] Read more.
Cognitive impairment in patients with Parkinson’s disease (PD) is one of the commonest and most disabling non-motor manifestations during the course of the disease. The clinical spectrum of PD-related cognitive impairment includes subjective cognitive decline (SCD), mild cognitive impairment (MCI) and PD dementia (PDD). As the disease progresses, cognitive decline creates a significant burden for the family members and/or caregivers of patients with PD, and has a great impact on quality of life. Current pharmacological treatments have demonstrated partial efficacy and failed to halt disease progression, and novel, effective, and safe therapeutic strategies are required. Accumulating preclinical and clinical evidence shows that several agents may provide beneficial effects on patients with PD and cognitive impairment, including ceftriaxone, ambroxol, intranasal insulin, nilotinib, atomoxetine, mevidalen, blarcamesine, prasinezumab, SYN120, ENT-01, NYX-458, GRF6021, fosgonimeton, INT-777, Neuropeptide S, silibinin, osmotin, cordycepin, huperzine A, fibroblast growth factor 21, Poloxamer 188, ginsenoside Rb1, thioredoxin-1, tangeretin, istradefylline and Eugenia uniflora. Potential underlying mechanisms include the inhibition of a-synuclein aggregation, the improvement of mitochondrial function, the regulation of synaptic plasticity, an impact on the gut–brain axis, the modulation of neuroinflammation and the upregulation of neurotrophic factors, as well as cholinergic, dopaminergic, serotoninergic and norepinephrine neurotransmission. In this updated overview, we aim to cover the clinical aspects of the spectrum of PD-related cognitive impairment and discuss recent evidence on emerging treatment approaches that are under investigation at a preclinical and clinical level. Finally, we aim to provide additional insights and propose new ideas for investigation that may be feasible and effective for the spectrum of PD-related cognitive impairment. Full article
(This article belongs to the Section Neurology)
17 pages, 4526 KiB  
Article
Differential Regulations of Antioxidant Metabolism and Cold-Responsive Genes in Three Bermudagrass Genotypes under Chilling and Freezing Stress
by Zhou Li, Cheng Huang and Liebao Han
Int. J. Mol. Sci. 2023, 24(18), 14070; https://doi.org/10.3390/ijms241814070 - 14 Sep 2023
Cited by 2 | Viewed by 1089
Abstract
As a typical warm-season grass, bermudagrass growth and turf quality begin to decrease when the environmental temperature drops below 20 °C. The current study investigated the differential responses of three bermudagrass genotypes to chilling stress (8/4 °C) for 15 days and then freezing [...] Read more.
As a typical warm-season grass, bermudagrass growth and turf quality begin to decrease when the environmental temperature drops below 20 °C. The current study investigated the differential responses of three bermudagrass genotypes to chilling stress (8/4 °C) for 15 days and then freezing stress (2/−2 °C) for 2 days. The three genotypes exhibited significant variation in chilling and freezing tolerance, and Chuannong-3, common bermudagrass 001, and Tifdwarf were ranked as cold-tolerant, -intermediate, and -sensitive genotypes based on evaluations of chlorophyll content, the photochemical efficiency of photosystem II, oxidative damage, and cell membrane stability, respectively. Chuannong-3 achieved better tolerance through enhancing the antioxidant defense system to stabilize cell membrane and reactive oxygen species homeostasis after being subjected to chilling and freezing stresses. Chuannong-3 also downregulated the ethylene signaling pathway by improving CdCTR1 expression and suppressing the transcript levels of CdEIN3-1 and CdEIN3-2; however, it upregulated the hydrogen sulfide signaling pathway via an increase in CdISCS expression under cold stress. In addition, the molecular basis of cold tolerance could be associated with the mediation of key genes in the heat shock pathway (CdHSFA-2b, CdHSBP-1, CdHSP22, and CdHSP40) and the CdOSMOTIN in Chuannong-3 because the accumulation of stress-defensive proteins, including heat shock proteins and osmotin, plays a positive role in osmoprotection, osmotic adjustment, or the repair of denatured proteins as molecular chaperones under cold stress. The current findings give an insight into the physiological and molecular mechanisms of cold tolerance in the new cultivar Chuannong-3, which provides valuable information for turfgrass breeders and practitioners. Full article
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Figure 1
<p>Changes in (<b>A</b>) total Chl content, (<b>B</b>) Chl a content, (<b>C</b>) Chl b content (OP), and (<b>D</b>) the ratio of Chl a to Chl b among three bermudagrass genotypes (Tifdwarf, common bermudagrass, and Chuannong-3) under optimal, chilling, and freezing conditions. Vertical bars indicate positive standard error of mean (<span class="html-italic">n</span> = 4). Different letters above columns indicate significant differences under optimal, chilling, or freezing condition based on least significant difference (<span class="html-italic">p</span> &lt; 0.05), respectively.</p>
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<p>Changes in (<b>A</b>) Fv/Fm, and (<b>B</b>) PI<sub>ABS</sub> among three bermudagrass genotypes (Tifdwarf, common bermudagrass, and Chuannong-3) under optimal, chilling, and freezing conditions. Vertical bars indicate positive standard error of mean (<span class="html-italic">n</span> = 4). Different letters above columns indicate significant differences under optimal, chilling, or freezing condition based on least significant difference (<span class="html-italic">p</span> &lt; 0.05), respectively.</p>
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<p>Changes in (<b>A</b>) O<sub>2</sub><sup>−</sup> content, (<b>B</b>) H<sub>2</sub>O<sub>2</sub> content, (<b>C</b>) MDA content, and (<b>D</b>) EL among three bermudagrass genotypes (Tifdwarf, common bermudagrass, and Chuannong-3) under optimal, chilling, and freezing conditions. Vertical bars indicate positive standard error of mean (<span class="html-italic">n</span> = 4). Different letters above columns indicate significant differences under optimal, chilling, or freezing condition based on least significant difference (<span class="html-italic">p</span> &lt; 0.05), respectively.</p>
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<p>Changes in (<b>A</b>) SOD activity, (<b>B</b>) POD activity, and (<b>C</b>) CAT activity among three bermudagrass genotypes (Tifdwarf, common bermudagrass, and Chuannong-3) under optimal, chilling, and freezing conditions. Vertical bars indicate positive standard error of mean (<span class="html-italic">n</span> = 4). Different letters above columns indicate significant differences under optimal, chilling, or freezing condition based on least significant difference (<span class="html-italic">p</span> &lt; 0.05), respectively.</p>
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<p>Changes in (<b>A</b>) APX activity, (<b>B</b>) MDHAR activity, (<b>C</b>) DHAR activity, (<b>D</b>) GR activity, (<b>E</b>) GST, and (<b>F</b>) GPX among three bermudagrass genotypes (Tifdwarf, common bermudagrass, and Chuannong-3) under optimal, chilling, and freezing conditions. Vertical bars indicate positive standard error of mean (<span class="html-italic">n</span> = 4). Different letters above columns indicate significant differences under optimal, chilling, or freezing condition based on least significant difference (<span class="html-italic">p</span> &lt; 0.05), respectively.</p>
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<p>Changes in (<b>A</b>) ASA content, (<b>B</b>) DHA content, (<b>C</b>) GSH content, (<b>D</b>) GSSG, (<b>E</b>) The ratio of ASA to DHA, and (<b>F</b>) the ratio of GSH to GSSG among three bermudagrass genotypes (Tifdwarf, common bermudagrass, and Chuannong-3) under optimal, chilling, and freezing conditions. Vertical bars indicate positive standard error of mean (<span class="html-italic">n</span> = 4). Different letters above columns indicate significant differences under optimal, chilling, or freezing condition based on least significant difference (<span class="html-italic">p</span> &lt; 0.05), respectively.</p>
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<p>Changes in expression levels of (<b>A</b>) <span class="html-italic">CdEIN3-1</span>, (<b>B</b>) <span class="html-italic">CdEIN3-2</span>, and (<b>C</b>) <span class="html-italic">CdCTR1</span> among three bermudagrass genotypes (Tifdwarf, common bermudagrass, and Chuannong-3) under optimal, chilling, and freezing conditions. Vertical bars indicate positive standard error of mean (<span class="html-italic">n</span> = 4). Different letters above columns indicate significant differences under optimal, chilling, or freezing condition based on least significant difference (<span class="html-italic">p</span> &lt; 0.05), respectively.</p>
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<p>Changes in expression levels of (<b>A</b>) <span class="html-italic">CdHSBP-1</span>, (<b>B</b>) <span class="html-italic">CdHSFA-2b</span>, (<b>C</b>) <span class="html-italic">CdHSP22</span>, and (<b>D</b>) <span class="html-italic">CdHSP40</span> among three bermudagrass genotypes (Tifdwarf, common bermudagrass, and Chuannong-3) under optimal, chilling, and freezing conditions. Vertical bars indicate positive standard error of mean (<span class="html-italic">n</span> = 4). Different letters above columns indicate significant differences under optimal, chilling, or freezing condition based on least significant difference (<span class="html-italic">p</span> &lt; 0.05), respectively.</p>
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<p>Changes in expression levels of (<b>A</b>) <span class="html-italic">CdOSMOTIN</span>, and (<b>B</b>) <span class="html-italic">CdISCS</span> among three bermudagrass genotypes (Tifdwarf, common bermudagrass, and Chuannong-3) under optimal, chilling, and freezing conditions. Vertical bars indicate positive standard error of mean (<span class="html-italic">n</span> = 4). Different letters above columns indicate significant differences under optimal, chilling, or freezing condition based on least significant difference (<span class="html-italic">p</span> &lt; 0.05), respectively.</p>
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<p>Integrative pathways involved in antioxidant metabolism and cold-responsive genes in three bermudagrass genotypes (Tifdwarf, common bermudagrass, and Chuannong-3) under chilling and freezing conditions.</p>
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15 pages, 3303 KiB  
Article
Proteomic Analysis Reveals Salt-Tolerant Mechanism in Soybean Applied with Plant-Derived Smoke Solution
by Setsuko Komatsu, Taiki Kimura, Shafiq Ur Rehman, Hisateru Yamaguchi, Keisuke Hitachi and Kunihiro Tsuchida
Int. J. Mol. Sci. 2023, 24(18), 13734; https://doi.org/10.3390/ijms241813734 - 6 Sep 2023
Cited by 2 | Viewed by 1706
Abstract
Salt stress of soybean is a serious problem because it reduces plant growth and seed yield. To investigate the salt-tolerant mechanism of soybean, a plant-derived smoke (PDS) solution was used. Three-day-old soybeans were subjected to PDS solution under 100 mM NaCl for 2 [...] Read more.
Salt stress of soybean is a serious problem because it reduces plant growth and seed yield. To investigate the salt-tolerant mechanism of soybean, a plant-derived smoke (PDS) solution was used. Three-day-old soybeans were subjected to PDS solution under 100 mM NaCl for 2 days, resulting in PDS solution improving soybean root growth, even under salt stress. Under the same condition, proteins were analyzed using the proteomic technique. Differential abundance proteins were associated with transport/formaldehyde catabolic process/sucrose metabolism/glutathione metabolism/cell wall organization in the biological process and membrane/Golgi in the cellular component with or without PDS solution under salt stress. Immuno-blot analysis confirmed that osmotin, alcohol dehydrogenase, and sucrose synthase increased with salt stress and decreased with additional PDS solution; however, H+ATPase showed opposite effects. Cellulose synthase and xyloglucan endotransglucosylase/hydrolase increased with salt and decreased with additional PDS solution. Furthermore, glycoproteins decreased with salt stress and recovered with additional treatment. As mitochondrion-related events, the contents of ATP and gamma-aminobutyric acid increased with salt stress and recovered with additional treatment. These results suggest that PDS solution improves the soybean growth by alleviating salt stress. Additionally, the regulation of energy metabolism, protein glycosylation, and cell wall construction might be an important factor for the acquisition of salt tolerance in soybean. Full article
(This article belongs to the Collection Feature Papers in Molecular Plant Sciences)
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<p>Experimental design to investigate the effects of PDS solution on soybean under salt stress. Seeds were sown and treated with or without 2000 ppm PDS solution. After 3 days of sowing, soybean seedlings were treated with 100 mM NaCl for 2 days. Soybean seedlings were analyzed with morphological and proteomic methods before being confirmed. For confirmatory experiments, immuno-blot and enzymatic analyses were used. All experiments were performed using 3 independent biological replicates. GABA means gamma-aminobutyric acid.</p>
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<p>Morphological analysis of soybean treated with PDS solution under salt stress. Soybean seeds were sown and treated with or without 2000 ppm PDS solution. Three-day-old soybeans were treated for 2 days with or without salt stress. Four treatments were performed: control (blue), salt (orange), smoke (gray), and salt + smoke (yellow). Before morphological analysis, a photograph was taken (<b>A</b>). The bar in the picture indicates 1 cm. As morphological parameters, hypocotyl length (<b>B</b>), hypocotyl-fresh weight (<b>C</b>), main-root length (<b>D</b>), and total-root fresh weight (<b>E</b>) were analyzed at 5 days after sowing. The data are presented as mean ± SD from 5 independent biological replicates. Mean values in each point with different letters are significant according to one-way ANOVA followed by Tukey’s multiple comparisons (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Functional categories of proteins with differential abundance in soybean root with PDS solution under salt stress. Four kinds of treatments (control, salt, smoke, salt + smoke) were performed. Proteins extracted from soybean root after treatment were enriched, reduced, alkylated, and digested. After analysis by LC combined with MS/MS, the relative abundance of proteins from without (<a href="#app1-ijms-24-13734" class="html-app">Table S2</a>) or with (<a href="#app1-ijms-24-13734" class="html-app">Table S3</a>) PDS solution under salt stress was compared to that of the control. Functional categories of changed proteins were determined using gene-ontology analysis. Red and blue columns indicate protein increases and decreases, respectively.</p>
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<p>Immuno-blot analysis of the proteins involved in soybean treated with PDS solution under salt stress. Four treatments were performed: control (blue), salt (orange), smoke (gray), and salt+smoke (yellow). Proteins extracted from soybean root were separated on SDS-polyacrylamide gel by electrophoresis and stained with Coomassie-brilliant blue (<b>A</b>). A staining pattern with Coomassie-brilliant blue was used as a loading control. Proteins were transferred onto membranes. The membranes were cross-reacted with anti-osmotin (<b>B</b>), H<sup>+</sup>ATPase (<b>C</b>), alcohol dehydrogenase (<b>D</b>), sucrose synthase (<b>E</b>), and glutathione reductase (<b>F</b>) antibodies. The integrated densities of the bands were calculated using ImageJ software (version 1.8). The data are presented as mean ± SD from 3 independent biological replicates (<a href="#app1-ijms-24-13734" class="html-app">Figures S2–S7</a>). Statistical analysis is the same as in <a href="#ijms-24-13734-f002" class="html-fig">Figure 2</a>.</p>
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<p>Immuno-blot analysis of the proteins involved in soybean treated with PDS solution under salt stress. Proteins blotted on the membrane were cross-reacted with anti-xyloglucan endotransglucosylase/hydrolase (<b>A</b>) and cellulose synthase (<b>B</b>) antibodies. A staining pattern with Coomassie-brilliant blue was used as a loading control (<a href="#app1-ijms-24-13734" class="html-app">Figure S2</a>). Three independent experiments were performed as biological replicates (<a href="#app1-ijms-24-13734" class="html-app">Figures S8 and S9</a>). Data analysis is the same as in <a href="#ijms-24-13734-f004" class="html-fig">Figure 4</a>. Statistical analysis is the same as in <a href="#ijms-24-13734-f002" class="html-fig">Figure 2</a>.</p>
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<p>Lectin blot analysis of the proteins involved in soybean treated with PDS solution under salt stress. Proteins blotted on the membrane were cross-reacted with peroxidase-Concanavalin A (<b>A</b>). A staining pattern with Coomassie-brilliant blue was used as a loading control (<a href="#app1-ijms-24-13734" class="html-app">Figure S2</a>). The integrated densities of lectin blot were calculated using ImageJ software (version 1.8) (<b>B</b>). Data analysis is the same as in <a href="#ijms-24-13734-f004" class="html-fig">Figure 4</a>. Statistical analysis is the same as in <a href="#ijms-24-13734-f002" class="html-fig">Figure 2</a>.</p>
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<p>The contents of ATP and gamma-aminobutyric acid in soybean treated with PDS solution under salt stress. Soybean seeds were sown and treated with or without 2000 ppm PDS solution. Three-day-old soybeans were treated with or without salt stress for 2 days. Metabolites were extracted from the root. The ATP (<b>A</b>) and gamma-aminobutyric acid (<b>B</b>) contents were measured for each sample. Data analysis is the same as in <a href="#ijms-24-13734-f004" class="html-fig">Figure 4</a>. Statistical analysis is the same as in <a href="#ijms-24-13734-f002" class="html-fig">Figure 2</a>. GABA stands for gamma-aminobutyric acid.</p>
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21 pages, 14331 KiB  
Review
Tomato Biodiversity and Drought Tolerance: A Multilevel Review
by Veronica Conti, Luigi Parrotta, Marco Romi, Stefano Del Duca and Giampiero Cai
Int. J. Mol. Sci. 2023, 24(12), 10044; https://doi.org/10.3390/ijms241210044 - 12 Jun 2023
Cited by 12 | Viewed by 5529
Abstract
Ongoing global climate change suggests that crops will be exposed to environmental stresses that may affect their productivity, leading to possible global food shortages. Among these stresses, drought is the most important contributor to yield loss in global agriculture. Drought stress negatively affects [...] Read more.
Ongoing global climate change suggests that crops will be exposed to environmental stresses that may affect their productivity, leading to possible global food shortages. Among these stresses, drought is the most important contributor to yield loss in global agriculture. Drought stress negatively affects various physiological, genetic, biochemical, and morphological characteristics of plants. Drought also causes pollen sterility and affects flower development, resulting in reduced seed production and fruit quality. Tomato (Solanum lycopersicum L.) is one of the most economically important crops in different parts of the world, including the Mediterranean region, and it is known that drought limits crop productivity, with economic consequences. Many different tomato cultivars are currently cultivated, and they differ in terms of genetic, biochemical, and physiological traits; as such, they represent a reservoir of potential candidates for coping with drought stress. This review aims to summarize the contribution of specific physio-molecular traits to drought tolerance and how they vary among tomato cultivars. At the genetic and proteomic level, genes encoding osmotins, dehydrins, aquaporins, and MAP kinases seem to improve the drought tolerance of tomato varieties. Genes encoding ROS-scavenging enzymes and chaperone proteins are also critical. In addition, proteins involved in sucrose and CO2 metabolism may increase tolerance. At the physiological level, plants improve drought tolerance by adjusting photosynthesis, modulating ABA, and pigment levels, and altering sugar metabolism. As a result, we underline that drought tolerance depends on the interaction of several mechanisms operating at different levels. Therefore, the selection of drought-tolerant cultivars must consider all these characteristics. In addition, we underline that cultivars may exhibit distinct, albeit overlapping, multilevel responses that allow differentiation of individual cultivars. Consequently, this review highlights the importance of tomato biodiversity for an efficient response to drought and for preserving fruit quality levels. Full article
(This article belongs to the Special Issue Environmental Stress and Plants 2.0)
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<p>Scheme of the main gene activities that allow some tomato cultivars to be drought tolerant. Among the main changes in gene expression, the diagram shows those that are better characterized, such as OLP, TAS14, SlMAPK, SlJUB1, EPF2, DELLA, and SlPIP, and the function of the proteins encoded by these genes. For more information, please refer to the text. Image was created with the tools of BioRender (<a href="https://app.biorender.com/" target="_blank">https://app.biorender.com/</a> (accessed on 5 June 2023)).</p>
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<p>Summary of the main biochemical mechanisms used by tomato plants to cope with drought. In general, the protein-based mechanisms can be divided into two groups according to the time of response. Phase 1 response involves changes in cyclophilin, HSP70, aquaporins, SuSy, and SOD, in addition to changes in RuBisCO isoforms. Phase 2 includes changes in osmotin, dehydrin, and catalase content and activity. Image was created with the tools of BioRender (<a href="https://app.biorender.com/" target="_blank">https://app.biorender.com/</a> (accessed on 30 May 2023)).</p>
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<p>Summary of the main physiological mechanisms that allow tomato plants to escape drought. Mechanisms are those that are reported to vary between tomato cultivars and that may allow one cultivar to be more tolerant than another. Image was created with the tools of BioRender (<a href="https://app.biorender.com/" target="_blank">https://app.biorender.com/</a> (accessed on 28 May 2023)).</p>
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<p>Summary of the effects of drought stress on tomato fruit quality. ROS production following drought stress is known to induce early fruit development compared to irrigated plants, although yield is not affected. In addition, drought stress is reported to increase the content of bioactive molecules, such as vitamin C and lycopene. Image was created with the tools of BioRender (<a href="https://app.biorender.com/" target="_blank">https://app.biorender.com/</a> (accessed on 5 June 2023)).</p>
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29 pages, 1441 KiB  
Article
Proteomic Analysis of Proteins Related to Defense Responses in Arabidopsis Plants Transformed with the rolB Oncogene
by Yulia V. Vereshchagina, Anastasiya A. Mironova, Dmitry V. Bulgakov and Victor P. Bulgakov
Int. J. Mol. Sci. 2023, 24(3), 1880; https://doi.org/10.3390/ijms24031880 - 18 Jan 2023
Viewed by 2199
Abstract
During Agrobacterium rhizogenes–plant interaction, the rolB gene is transferred into the plant genome and is stably inherited in the plant’s offspring. Among the numerous effects of rolB on plant metabolism, including the activation of secondary metabolism, its effect on plant defense systems [...] Read more.
During Agrobacterium rhizogenes–plant interaction, the rolB gene is transferred into the plant genome and is stably inherited in the plant’s offspring. Among the numerous effects of rolB on plant metabolism, including the activation of secondary metabolism, its effect on plant defense systems has not been sufficiently studied. In this work, we performed a proteomic analysis of rolB-expressing Arabidopsis thaliana plants with particular focus on defense proteins. We found a total of 77 overexpressed proteins and 64 underexpressed proteins in rolB-transformed plants using two-dimensional gel electrophoresis and MALDI mass spectrometry. In the rolB-transformed plants, we found a reduced amount of scaffold proteins RACK1A, RACK1B, and RACK1C, which are known as receptors for activated C-kinase 1. The proteomic analysis showed that rolB could suppress the plant immune system by suppressing the RNA-binding proteins GRP7, CP29B, and CP31B, which action are similar to the action of type-III bacterial effectors. At the same time, rolB plants induce the massive biosynthesis of protective proteins VSP1 and VSP2, as well as pathogenesis-related protein PR-4, which are markers of the activated jasmonate pathway. The increased contents of glutathione-S-transferases F6, F2, F10, U19, and DHAR1 and the osmotin-like defense protein OSM34 were found. The defense-associated protein PCaP1, which is required for oligogalacturonide-induced priming and immunity, was upregulated. Moreover, rolB-transformed plants showed the activation of all components of the PYK10 defense complex that is involved in the metabolism of glucosinolates. We hypothesized that various defense systems activated by rolB protect the host plant from competing phytopathogens and created an effective ecological niche for A. rhizogenes. A RolB → RACK1A signaling module was proposed that might exert most of the rolB-mediated effects on plant physiology. Our proteomics data are available via ProteomeXchange with identifier PXD037959. Full article
(This article belongs to the Special Issue Advances and New Perspectives in Plant-Microbe Interactions 2.0)
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<p>Induction of defense proteins by the <span class="html-italic">rolB</span> gene. Fragments of two-dimensional gels of protein fractions from the control (At) and <span class="html-italic">rolB-</span>transformed AtB-1 (AtB) plants obtained in three separate experiments are presented. Gels were scanned using a VersaDoc MP 4000 system with PDQuest 8.0.1 Advanced software as described in <a href="#sec4dot3-ijms-24-01880" class="html-sec">Section 4.3</a>. Protein expression is presented as the average total spot intensity in a group of three repeated gels. (<b>A</b>) hevein-like preproprotein PR-4/HEL, marked with boxes (this protein was divided on gels into two isoforms by isoelectric mobility and by mass); (<b>B</b>) vegetative storage protein 1 (VSP1, boxes) and vegetative storage protein 2 (VSP2, circles).</p>
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<p>Downregulation of RACK1 proteins in <span class="html-italic">rolB</span>-expressing <span class="html-italic">Arabidopsis</span> plants. Gels were scanned using a VersaDoc MP 4000 system with PDQuest 8.0.1 Advanced software as described in <a href="#sec4dot3-ijms-24-01880" class="html-sec">Section 4.3</a>. Protein expression is presented as the average total spot intensity in a group of three repeated gels. Fragments of 2-D gels of protein fractions from the control (At) and <span class="html-italic">rolB-</span>transformed AtB-1 line (AtB) are presented in triplicate: RACK1A, marked with polygons; RACK1B (boxes) and RACK1C (circles).</p>
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<p>qPCR analysis of selected genes in <span class="html-italic">Arabidopsis</span> normal (At) and <span class="html-italic">rolB</span>-transformed AtB-1 line (AtB). (<b>A</b>) expression of <span class="html-italic">RACK1</span> genes; (<b>B</b>) expression of genes involved in defense reactions and plant immunity; (<b>C</b>) CERK1-associated genes; (<b>D</b>,<b>E</b>) chaperone-type genes. Data are presented as mean relative expression fold ± standard error across three different experiments with three technical replicates. Asterisks above the bars indicate significantly different mean values (* <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01), Fisher’s LSD.</p>
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19 pages, 4447 KiB  
Article
Applied Selenium as a Powerful Antioxidant to Mitigate the Harmful Effects of Salinity Stress in Snap Bean Seedlings
by Hoda A. S. Farag, Mohamed F. M. Ibrahim, Ahmed Abou El-Yazied, Hossam S. El-Beltagi, Hany G. Abd El-Gawad, Mohammed Alqurashi, Tarek A. Shalaby, Abdallah Tageldein Mansour, Abdulmalik A. Alkhateeb and Reham Farag
Agronomy 2022, 12(12), 3215; https://doi.org/10.3390/agronomy12123215 - 18 Dec 2022
Cited by 11 | Viewed by 2540
Abstract
Selenium (Se) plays several significant roles in regulating growth, development and plant responses to various abiotic stresses. However, its influence on sulfate transporters (SULTRS) and achieving the harmony with other salt-tolerance features is still limited in the previous literatures. This [...] Read more.
Selenium (Se) plays several significant roles in regulating growth, development and plant responses to various abiotic stresses. However, its influence on sulfate transporters (SULTRS) and achieving the harmony with other salt-tolerance features is still limited in the previous literatures. This study elucidated the effect of Se supplementation (5, 10 and 20 µM) on salt-stressed (50 mM NaCl) snap bean seedlings. Generally, the results indicated that Se had dual effects on the salt stressed seedlings according to its concentration. At a low level (5 µM), plants demonstrated a significant improvement in shoot (13.8%) and root (22.8%) fresh weight, chlorophyll a (7.4%), chlorophyll b (14.7%), carotenoids (23.2%), leaf relative water content (RWC; 8.5%), proline (17.2%), total soluble sugars (34.3%), free amino acids (FAA; 18.4%), K (36.7%), Ca (33.4%), K/Na ratio (77.9%), superoxide dismutase (SOD; 18%), ascorbate peroxidase (APX;12.8%) and guaiacol peroxidase (G-POX; 27.1%) compared to the untreated plants. Meanwhile, most of these responses as well as sulfur (S), Se and catalase (CAT) were obviously decreased in parallel with increasing the applied Se up to 20 µM. The molecular study revealed that three membrane sulfate transporters (SULTR1, SULTR2 and SULTR 3) in the root and leaves and salinity responsive genes (SOS1, NHX1 and Osmotin) in leaves displayed different expression patterns under various Se treatments. Conclusively, Se at low doses can be beneficial in mitigating salinity-mediated damage and achieving the functioning homeostasis to tolerance features. Full article
(This article belongs to the Section Farming Sustainability)
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Figure 1
<p>Effect of Se supplementation at 5, 10 and 20 µM on growth performance (<b>A</b>), shoot fresh weight (<b>B</b>), shoot fresh weight (<b>C</b>), shoot dry weight (<b>D</b>) and root dry weight (<b>E</b>) of non-stressed and salt-stressed (50 mM NaCl) snap bean seedlings (28 days after sowing). The results are expressed as mean values of three measurements ± SE using Duncan’s multiple range test (<span class="html-italic">p</span> = 0.05). Different letters indicate significant differences among the treatments.</p>
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<p>Effect of Se supplementation at 5, 10 and 20 µM on chlorophyll a (<b>A</b>), chlorophyll b (<b>B</b>), total chlorophyll (<b>C</b>) and carotenoids (<b>D</b>) of non-stressed and salt-stressed (50 mM NaCl) snap bean seedlings (28 days after sowing). The results are expressed as mean values of three measurements ± SE using Duncan’s multiple range test (<span class="html-italic">p</span> = 0.05). Different letters indicate significant differences among the treatments.</p>
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<p>Effect of Se supplementation at 5, 10 and 20 µM on relative water content; RWC (<b>A</b>), proline (<b>B</b>), total soluble sugars (<b>C</b>) or free amino acids; FAA (<b>D</b>) of non-stressed and salt-stressed (50 mM NaCl) snap bean seedlings (28 days after sowing). The results are expressed as mean values of three measurements ± SE using Duncan’s multiple range test (<span class="html-italic">p</span> = 0.05). Different letters indicate significant differences among the treatments.</p>
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<p>Effect of Se supplementation at 5, 10 and 20 µM on relative water content; malondialdehyde; MDA (<b>A</b>) and hydrogen peroxide; H<sub>2</sub>O<sub>2</sub> (<b>B</b>) in the leaves of non-stressed and salt-stressed (50 mM NaCl) snap bean seedlings (28 days after sowing). The results are expressed as mean values of three measurements ± SE using Duncan’s multiple range test (<span class="html-italic">p</span> = 0.05). Different letters indicate significant differences among the treatments.</p>
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<p>Effect of Se supplementation at 5, 10 and 20 µM on the activity of superoxide dismutase; SOD (<b>A</b>), ascorbate peroxidase; APX (<b>B</b>), guiacol peroxidase; G-POx (<b>C</b>) and catalase; CAT (<b>D</b>) of non-stressed and salt-stressed (50 mM NaCl) snap bean seedlings (28 days after sowing). The results are expressed as mean values of three measurements ± SE using Duncan’s multiple range test (<span class="html-italic">p</span> = 0.05). Different letters indicate significant differences among the treatments.</p>
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<p>Effect of Se supplementation at 5, 10 and 20 µM on the leaf content of K (<b>A</b>), Na (<b>B</b>), Ca (<b>C</b>), K/Na ratio (<b>D</b>), S (<b>E</b>) and Se (<b>F</b>) of non-stressed and salt-stressed (50 mM NaCl) snap bean seedlings (28 days after sowing). The results are expressed as mean values of three measurements ± SE using Duncan’s multiple range test (<span class="html-italic">p</span> = 0.05). Different letters indicate significant differences among the treatments.</p>
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<p>Effect of Se supplementation at 5, 10 and 20 µM on the relative expression of sulfate transporter genes. Root-<span class="html-italic">PvSULTR1</span> (<b>A</b>), Root-<span class="html-italic">PvSULTR2</span> (<b>B</b>), Root-<span class="html-italic">PvSULTR3</span> (<b>C</b>), Shoot-<span class="html-italic">PvSULTR1</span> (<b>D</b>) Shoot-<span class="html-italic">PvSULTR2</span> (<b>E</b>) and Shoot-<span class="html-italic">PvSULTR3</span> (<b>F</b>) of non-stressed and salt-stressed (50 mM NaCl) snap bean seedlings (28 days after sowing). The results are expressed as mean values of three measurements ± SE using Duncan’s multiple range test (<span class="html-italic">p</span> = 0.05). Different letters indicate significant differences among the treatments.</p>
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<p>Effect of Se supplementation at 5, 10 and 20 µM on relative expression of salt stress responsive genes. <span class="html-italic">SOS1</span> (<b>A</b>), <span class="html-italic">NHX1</span> (<b>B</b>) and <span class="html-italic">Osmotin</span> (<b>C</b>) of non-stressed and salt-stressed (50 mM NaCl) snap bean seedlings (28 days after sowing). The results are expressed as mean values of three measurements ± SE using Duncan’s multiple range test (<span class="html-italic">p</span> = 0.05). Different letters indicate significant differences among the treatments.</p>
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<p>Simplified model for the suggested effects of applied Se on snap bean plants grown under salinity stress. RWC, relative water content; MDA, malondialdehyde; FAA, free amino acids; G-POX, Guaiacol peroxidases; CAT, catalase; APX; ascorbate peroxidase; SOD, superoxide dismutase; blue solid lines indicate the positive effects of applied Se at low concentration; red dotted lines indicate the negative effects of applied Se at high level; green up arrow, increase; red down arrow.</p>
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15 pages, 3401 KiB  
Article
Identification and Validation of Quantitative Trait Loci for Grain Size in Bread Wheat (Triticum aestivum L.)
by Wenjing Hu, Sen Liao, Die Zhao, Jizeng Jia, Weigang Xu and Shunhe Cheng
Agriculture 2022, 12(6), 822; https://doi.org/10.3390/agriculture12060822 - 8 Jun 2022
Cited by 8 | Viewed by 2563
Abstract
Grain width (GW) and grain length (GL) are crucial components affecting grain weight. Dissection of their genetic control is essential for improving yield potential in wheat breeding. Yangmai 12 (YM12) and Yanzhan 1 (YZ1) are two elite cultivars released in the Middle and [...] Read more.
Grain width (GW) and grain length (GL) are crucial components affecting grain weight. Dissection of their genetic control is essential for improving yield potential in wheat breeding. Yangmai 12 (YM12) and Yanzhan 1 (YZ1) are two elite cultivars released in the Middle and Lower Yangtze Valleys Wheat Zone (MLYVWZ) and the Yellow-Huai River Valleys Wheat Zone (YRVWZ), respectively. One biparental population derived from YM12/YZ1 cross was employed to perform QTL mapping based on the data from four environments over two years to detect quantitative trait loci (QTL) for GW and GL. A total of eight QTL were identified on chromosomes 1B, 2D, 3B, 4B, 5A, and 6B. Notably, QGW.yz.2D was co-located with QGL.yz.2D, and QGW.yz.4B was co-located with QGL.yz.4B, respectively. QGW.yz.2D and QGL.yz.2D, with the increasing GW/GL allele from YZ1, explained 12.36–18.27% and 13.69–26.53% of the phenotypic variations for GW and GL, respectively. QGW.yz.4B and QGL.yz.4B, with the increasing GW/GL allele from YM12, explained 10.34–11.95% and 10.35–16.04% of the phenotypic variation for GW and GL, respectively. QGL.yz.5A, with the increasing GL allele from YM12, explained 10.04–12.48% of the phenotypic variation for GL. Moreover, the positive alleles of these three QTL regions could significantly increase thousand-grain weight, and QGW.yz.4B/QGL.yz.4B and QGL.yz.5A did not show significant negative effects on grain number per spike. QGL.yz.2D, QGW.yz.4B/QGL.yz.4B, and QGL.yz.5A have not been reported. These three QTL regions were then further validated using Kompetitive Allele-Specific PCR (KASP) markers in 159 wheat cultivars/lines from MLYVWZ and YRVWZ. Combining the positive alleles of the major QTL significantly increased GW and GL. Eleven candidate genes associated with encoding ethylene-responsive transcription factor, oleosin, osmotin protein, and thaumatin protein were identified. Three major QTL and KASP markers reported here will be helpful in developing new wheat cultivars with high and stable yields. Full article
(This article belongs to the Special Issue Molecular Markers and Marker-Assisted Breeding in Wheat)
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Figure 1
<p>(<b>a</b>) Grain morphology of two parents in Sihong Experimental Station of 2020. The scale bar represents 3 mm; (<b>b</b>) Frequency distribution of the YM12/YZ1 population for grain width and grain length of the <span class="html-italic">BLUE</span> datasets in the YM12/YZ1 population. <span class="html-italic">BLUE</span> represents the best linear unbiased estimator. YM12 represents Yangmai 12, YZ1 represents Yanzhan 1, and YM12/YZ1 represents Yangmai 12/Yanzhan 1 population.</p>
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<p>Allelic effects (<b>a</b>), genetic maps (<b>b</b>), physical locations (<b>c</b>), and expression patterns of genes (<b>d</b>) in the physical interval of major quantitative trait loci (QTL) <span class="html-italic">QGW.yz.2D/QGL.yz.2D</span>. In allelic effects (<b>a</b>), A and B indicated the lines with the alleles from YM12 and YZ1, respectively; *** represents significance at <span class="html-italic">p</span> &lt; 0.001; ns represents non-significance. In the genetic maps (<b>b</b>), the names of the markers are listed on the right side of the corresponding linkage group, and their genetic positions and QTL names are shown on the left (cM). The red rectangles at the chromosomes represent QTL regions. Blue blocks represent QTLs for grain width, and green blocks represent QTLs for grain length. In the expression patterns of genes (<b>d</b>), the red arrows represent the genes that were more highly expressed in grain or both in grain and whole endosperm than in root, leaf, stem, and spike. GW, grain width; GL, grain length; GNS, grain number per spike; TGW, thousand-grain weight. YM12, Yangmai 12; YZ1, Yangzhan 1.</p>
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<p>Allelic effects (<b>a</b>), genetic maps (<b>b</b>), physical locations (<b>c</b>), and expression patterns of genes (<b>d</b>) in the physical interval of major quantitative trait loci (QTL) <span class="html-italic">QGW.yz.4B/QGL.yz.4B</span>. In allelic effects (<b>a</b>), A and B indicated the lines with the alleles from YM12 and YZ1, respectively; *** represents significance at <span class="html-italic">P</span> &lt; 0.001; ns represents non-significance. In the genetic maps (<b>b</b>), the names of the markers are listed on the right side of the corresponding linkage group, and their genetic positions and QTL names are shown on the left (cM). The red rectangles at the chromosomes represent QTL regions. Blue blocks represent QTLs for grain width, and green blocks represent QTLs for grain length. In the expression patterns of genes (<b>d</b>), the red arrows represent the genes that were more highly expressed in grain or both in grain and whole endosperm than in root, leaf, stem, and spike. GW, grain width; GL, grain length; GNS, grain number per spike; TGW, thousand-grain weight. YM12, Yangmai 12; YZ1, Yangzhan 1.</p>
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<p>Allelic effects (<b>a</b>), genetic maps (<b>b</b>), physical locations (<b>c</b>), and expression patterns of genes (<b>d</b>) in the physical interval of major quantitative trait loci (QTL) <span class="html-italic">QGL.yz.5A</span>. In allelic effects (<b>a</b>), A and B indicated the lines with the alleles from YM12 and YZ1, respectively; * represents significance at <span class="html-italic">p</span> &lt; 0.05; ns represents non-significance. In the genetic maps (<b>b</b>), the names of the markers are listed on the right side of the corresponding linkage group, and their genetic positions and QTL names are shown on the left (cM). The red rectangles at the chromosomes represent QTL regions. Blue blocks represent QTLs for grain width, and green blocks represent QTLs for grain length. In the expression patterns of genes (<b>d</b>), the red arrows represent the genes that were more highly expressed in grain or both in grain and whole endosperm than in root, leaf, stem, and spike. GW, grain width; GL, grain length; GNS, grain number per spike; TGW, thousand-grain weight. YM12, Yangmai 12; YZ1, Yangzhan 1.</p>
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<p>Additive effects of <span class="html-italic">QGL.yz.2D</span> and <span class="html-italic">QGL.yz.4B</span> on grain width (<b>a</b>) and additive effects of <span class="html-italic">QGL.yz.2D</span>, <span class="html-italic">QGL.yz.4B</span>, and <span class="html-italic">QGL.yz.5A</span> on grain length (<b>b</b>) based on the <span class="html-italic">BLUE</span> datasets in the YM12/YZ1 population. A and B indicated the lines with the alleles from YM12 and YZ1, respectively. The different letters are significant at <span class="html-italic">p</span> &lt; 0.05. GW, grain width; GL, grain length; GNS, grain number per spike; TGW, thousand-grain weight. YM12, Yangmai 12; YZ1, Yangzhan 1.</p>
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<p>Kompetitive Allele-Specific PCR array and additive effects of the major QTL in the validation population. (<b>a</b>) KASP_2D; (<b>b</b>) KASP_4B; (<b>c</b>) KASP_5A. Blue dots represent the YM12-like genotypes, red dots represent the YZ1-like genotypes, and black dots in (<b>a</b>–<b>c</b>) are water controls. YM12 represents Yangmai 12, YZ1 represents Yanzhan 1. (<b>d</b>) Additive effects of <span class="html-italic">QGW.yz.2D/QGL.yz.2D</span> and <span class="html-italic">QGW.yz.4B/QGL.yz.4B</span> on grain width; (<b>e</b>) Additive effects of <span class="html-italic">QGW.yz.2D/QGL.yz.2D</span>, <span class="html-italic">QGW.yz.4B/QGL.yz.4B</span>, and <span class="html-italic">QGL.yz.5A</span> on grain length. A and B indicated the lines with the alleles from YM12 and YZ1, respectively. The different letters are significant at <span class="html-italic">p</span> &lt; 0.05. GW, grain width; GL, grain length; GNS, grain number per spike; TGW, thousand-grain weight. YM12, Yangmai 12; YZ1, Yangzhan 1.</p>
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16 pages, 3221 KiB  
Article
Folic Acid Confers Tolerance against Salt Stress-Induced Oxidative Damages in Snap Beans through Regulation Growth, Metabolites, Antioxidant Machinery and Gene Expression
by Hameed Alsamadany, Hassan Mansour, Amr Elkelish and Mohamed F. M. Ibrahim
Plants 2022, 11(11), 1459; https://doi.org/10.3390/plants11111459 - 30 May 2022
Cited by 22 | Viewed by 6674
Abstract
Although the effect of folic acid (FA) and its derivatives (folates) have been extensively studied in humans and animals, their effects are still unclear in most plant species, specifically under various abiotic stress conditions. Here, the impact of FA as a foliar application [...] Read more.
Although the effect of folic acid (FA) and its derivatives (folates) have been extensively studied in humans and animals, their effects are still unclear in most plant species, specifically under various abiotic stress conditions. Here, the impact of FA as a foliar application at 0, 0.1, and 0.2 mM was studied on snap bean seedlings grown under non-saline and salinity stress (50 mM NaCl) conditions. The results indicated that under salinity stress, FA-treated plants revealed a significant (p ≤ 0.05) increase in growth parameters (fresh and dry weight of shoot and root). A similar trend was observed in chlorophyll (Chl b), total chlorophyll, carotenoids, leaf relative water content (RWC), proline, free amino acids (FAA), soluble sugars, cell membrane stability index (CMSI), and K, Ca, and K/Na ratio compared to the untreated plants. In contrast, a significant decrease was observed in Na and salinity-induced oxidative damage as indicated by reduced H2O2 production (using biochemical and histochemical detection methods) and rate of lipid peroxidation (malondialdehyde; MDA). This enhancement was correlated by increasing the activities of antioxidant enzymes, i.e., superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (G-POX), and ascorbate peroxidase (APX). Gene expression analyses conducted using qRT-PCR demonstrated that genes coding for the Na+/H+ antiporter protein Salt Overly Sensitive 1 (SOS1), the tonoplast-localized Na+/H+ antiporter protein (NHX1), and the multifunctional osmotic protective protein (Osmotin) were significantly up-regulated in the FA-treated plants under both saline and non-saline treatments. Generally, treatment with 0.2 mM FA was more potent than 0.1 mM and can be recommended to improve snap bean tolerance to salinity stress. Full article
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<p>Effect of exogenous folic acid (FA; 0, 0.1, and 0.2 mM) on vegetative growth (<b>A</b>), shoot fresh weight (<b>B</b>), shoot dry weight (<b>C</b>), root fresh weigh (<b>D</b>) and root dry weight (<b>E</b>), of snap bean plants grown under non-stressed and saline-stressed (50 mM, NaCl) conditions. Values are the averages of three replicates ± SD. Different letters indicate significant differences according to Duncan’s multiple range tests (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Effect of exogenous folic acid (FA; 0, 0.1, and 0.2 mM) on photosynthetic pigments including Chl a (<b>A</b>), Chl b (<b>B</b>), total chlorophyll (<b>C</b>), and carotenoids (<b>D</b>) of snap bean plants grown under non-stressed and saline-stressed (50 mM, NaCl) conditions. Values are the averages of three replicates ± SD. Different letters indicate significant differences according to Duncan’s multiple range tests (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Effect of exogenous folic acid (FA; 0, 0.1, and 0.2 mM) on the histochemical detection of H<sub>2</sub>O<sub>2</sub> using diaminobenzidine (DAB) staining method (<b>A</b>), the concentration of H<sub>2</sub>O<sub>2</sub> (<b>B</b>), lipid peroxidation as indicated by malondialdehyde, MDA (<b>C</b>), and cell membrane stability index, CMSI (<b>D</b>), of snap bean plants grown under non-stressed and saline-stressed (50 mM, NaCl) conditions. Values are the averages of three replicates ± SD. Different letters indicate significant differences according to Duncan’s multiple range tests (<span class="html-italic">p</span> &lt; 0.05). Dark-brown DAB staining color indicates increase the accumulation of H<sub>2</sub>O<sub>2</sub>.</p>
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<p>Effect of exogenous folic acid (FA; 0, 0.1, and 0.2 mM) on the activities of antioxidant enzymes including superoxide dismutase, SOD (<b>A</b>), catalase, CAT (<b>B</b>), guaiacol peroxidase, G-POX (<b>C</b>), and ascorbate peroxidase, APX (<b>D</b>), of snap bean plants grown under non-stressed and saline-stressed (50 mM, NaCl) conditions. Values are the averages of three replicates ± SD. Different letters indicate significant differences according to Duncan’s multiple range tests (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Effect of exogenous folic acid (FA; 0, 0.1, and 0.2 mM) on leaf relative water content; RWC (<b>A</b>), proline (<b>B</b>), free amino acids, FAA (<b>C</b>), and soluble sugars (<b>D</b>) of snap bean plants grown under non-stressed and saline-stressed (50 mM, NaCl) conditions. Values are the averages of three replicates ± SD. Different letters indicate significant differences according to Duncan’s multiple range tests (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Effect of exogenous folic acid (FA; 0, 0.1, and 0.2 mM) on K (<b>A</b>), Na (<b>B</b>), Ca (<b>C</b>), and K/Na ratio (<b>D</b>) in the leaves of snap bean plants grown under non-stressed and saline-stressed (50 mM, NaCl) conditions. Bars represent standard deviation (SD) of the means (n = 3). Values are the averages of three replicates ± SD. Different letters indicate significant differences according to Duncan’s multiple range tests (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Effect of exogenous folic acid (FA; 0, 0.1, and 0.2 mM) on the relative expression of plasma membrane Na<sup>+</sup>/H<sup>+</sup> antiporter protein of salt overly sensitive gene (<span class="html-italic">SOS1</span>) (<b>A</b>), vacuolar-localized Na<sup>+</sup>/H<sup>+</sup> antiporter protein (<span class="html-italic">NHX1</span>) (<b>B</b>), and the multifunctional osmotic protective protein (<span class="html-italic">Osmotin</span>) (<b>C</b>) of snap bean plants grown under non-stressed and saline-stressed (50 mM, NaCl) conditions. Values are the averages of three replicates ± SD. Different letters indicate significant differences according to Duncan’s multiple range tests (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Simplified model for the suggested effect of exogenous folic acid as a foliar spray on snap bean plants grown under salinity stress. RWC, relative water content; CMSI, cell membrane stability index; SAM, S-adenosylmethionine; MDA, malondialdehyde; FAA, free amino acids; POX, peroxidase; CAT, catalase; APX, ascorbate peroxidase; SOD, superoxide dismutase; ASA-GSH, ascorbate glutathione cycle; green upward arrow, increase; red downward arrow, decrease.</p>
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23 pages, 3330 KiB  
Article
Distinct Tomato Cultivars Are Characterized by a Differential Pattern of Biochemical Responses to Drought Stress
by Veronica Conti, Claudio Cantini, Marco Romi, Maria Michela Cesare, Luigi Parrotta, Stefano Del Duca and Giampiero Cai
Int. J. Mol. Sci. 2022, 23(10), 5412; https://doi.org/10.3390/ijms23105412 - 12 May 2022
Cited by 6 | Viewed by 2565
Abstract
Future climate scenarios suggest that crop plants will experience environmental changes capable of affecting their productivity. Among the most harmful environmental stresses is drought, defined as a total or partial lack of water availability. It is essential to study and understand both the [...] Read more.
Future climate scenarios suggest that crop plants will experience environmental changes capable of affecting their productivity. Among the most harmful environmental stresses is drought, defined as a total or partial lack of water availability. It is essential to study and understand both the damage caused by drought on crop plants and the mechanisms implemented to tolerate the stress. In this study, we focused on four cultivars of tomato, an economically important crop in the Mediterranean basin. We investigated the biochemical mechanisms of plant defense against drought by focusing on proteins specifically involved in this stress, such as osmotin, dehydrin, and aquaporin, and on proteins involved in the general stress response, such as HSP70 and cyclophilins. Since sugars are also known to act as osmoprotectants in plant cells, proteins involved in sugar metabolism (such as RuBisCO and sucrose synthase) were also analyzed. The results show crucial differences in biochemical behavior among the selected cultivars and highlight that the most tolerant tomato cultivars adopt quite specific biochemical strategies such as different accumulations of aquaporins and osmotins. The data set also suggests that RuBisCO isoforms and aquaporins can be used as markers of tolerance/susceptibility to drought stress and be used to select tomato cultivars within breeding programs. Full article
(This article belongs to the Section Molecular Plant Sciences)
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<p>Content of HSP70 in leaves of the four tomato cultivars from both control (CTRL) and stressed (DS) samples. (<b>A</b>) Immunoblotting with anti-HSP70 antibody. The arrow indicates the band with molecular weight between 70–75 kDa. Lane 1, Perina CTRL; lane 2, Perina DS; lane 3 Fragola CTRL; lane 4, Fragola DS; lane 5, Quarantino CTRL; lane 6 Quarantino DS; lane 7, Pisanello CTRL; lane 8, Pisanello DS. Here and in all subsequent gels an equal amount of protein (40 µg) was loaded into all lanes. (<b>B</b>) Quantitation of the relative content of individual bands in different samples. Green bars indicate control samples, red bars those that are drought stressed.</p>
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<p>Content of the cyclophilin family in the four tomato cultivars in both control (CTRL) and drought-stressed (DS) samples. (<b>A</b>) Immunoblot analysis of cyclophilin with the three bands identified at 25, 23, and 15 kDa. Lane 1, Perina CTRL; lane 2, Perina DS; lane 3 Fragola CTRL; lane 4, Fragola DS; lane 5, Quarantino CTRL; lane 6 Quarantino DS; lane 7, Pisanello CTRL; lane 8, Pisanello DS. (<b>B</b>) Quantitative analysis of the relative content of the three cyclophilin bands in the different samples. In green the control samples, in red the stressed samples.</p>
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<p>Content of dehydrins in both control and stressed plants of the four tomato cultivars. (<b>A</b>) Immunoblotting in leaves of the four tomato varieties analyzed. Lane 1, Perina CTRL; lane 2, Perina DS; lane 3 Fragola CTRL; lane 4, Fragola DS; lane 5, Quarantino CTRL; lane 6 Quarantino DS; lane 7, Pisanello CTRL; lane 8, Pisanello DS. The major dehydrins identified have molecular weights of 33.7, 30, 22, 17.8, and 15 kDa. (<b>B</b>) Relative content of dehydrins in the four tomato cultivars in both control (CTRL, in green) and drought-stressed (DS, in red) samples. Please note that in this graph the green bars of control samples are superimposed on the red bars of stressed samples.</p>
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<p>Content of osmotin in the leaves of the four tomato cultivars. (<b>A</b>) Immunoblotting with the anti-osmotin antibody. Lane 1, Perina CTRL; lane 2, Perina DS; lane 3 Fragola CTRL; lane 4, Fragola DS; lane 5, Quarantino CTRL; lane 6 Quarantino DS; lane 7, Pisanello CTRL; lane 8, Pisanello DS. The arrow indicates the position of the only immunoreactive band. (<b>B</b>) Relative quantization of the immunoblotting signal in controls (CTRL, green bar) and stressed samples (DS, red bar).</p>
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<p>Aquaporin content in leaves of the four tomato cultivars under both control and stressed conditions. (<b>A</b>) Immunoblotting with anti-aquaporin antibodies in the four cultivars, control samples (CTRL), and stressed samples (DS). On the right, molecular weights of the three main bands identified. (<b>B</b>) Relative quantization of blotting expressed as integrated density (y-axis). Green bars indicate control samples, red bars indicate drought-stressed samples.</p>
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<p>Content and isoform composition of RuBisCO in control and stressed plants of the four tomato cultivars. (<b>A</b>) 1D immunoblotting of RuBisCO in the four cultivars analyzed. Lane 1, Perina CTRL; lane 2, Perina DS; lane 3, Fragola CTRL; lane 4, Fragola DS; lane 5, Quarantino CTRL; lane 6 Quarantino DS; lane 7, Pisanello CTRL; lane 8, Pisanello DS. (<b>B</b>) Relative quantitative analysis of 1D immunoblotting in both control (green bar) and stressed samples (red bar). (<b>C</b>) Master (virtual) blot of RuBisCO isoforms after 2D electrophoresis. Each sample contained 300 μg of protein. The blot contains all the spots detected by the anti-RuBisCO antibody, which are numbered automatically by the PDQuest software. Relative percentage content of RuBisCO isoforms in both control and stressed samples of Perina (<b>D</b>), Fragola (<b>E</b>), Quarantino (<b>F</b>) and Pisanello (<b>G</b>) cultivars. Again, green bars represent control samples and red bars indicate stressed samples.</p>
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<p>Content of sucrose synthase (SuSy) in the leaves of the four cultivars, both in control (CTRL) and in stressed samples (DS). (<b>A</b>) Immunoblotting; the arrow indicates the position of the cross-reactive SuSy. Lane 1, Perina CTRL; lane 2, Perina DS; lane 3 Fragola CTRL; lane 4, Fragola DS; lane 5, Quarantino CTRL; lane 6 Quarantino DS; lane 7, Pisanello CTRL; lane 8, Pisanello DS. (<b>B</b>) Quantitative analysis of immunoblotting to SuSy. It should be noted that the level of SuSy in the control samples of Perina, Fragola and Quarantino was extremely low, almost indistinguishable from the background.</p>
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<p>Content in mg per g of sucrose (<b>A</b>), glucose (<b>B</b>), fructose (<b>C</b>) and water-soluble pectins (<b>D</b>) in leaves sampled from control (green bars) and drought stressed (red bars) plants belonging to four Tuscan tomato cultivars. Asterisk indicates significant difference between control and stressed plants with <span class="html-italic">p</span> ≤ 0.05 (*) or <span class="html-italic">p</span> ≤ 0.01 (**).</p>
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21 pages, 11280 KiB  
Article
LDHB Deficiency Promotes Mitochondrial Dysfunction Mediated Oxidative Stress and Neurodegeneration in Adult Mouse Brain
by Jun Sung Park, Kamran Saeed, Myeung Hoon Jo, Min Woo Kim, Hyeon Jin Lee, Chan-Bae Park, Gwang Lee and Myeong Ok Kim
Antioxidants 2022, 11(2), 261; https://doi.org/10.3390/antiox11020261 - 28 Jan 2022
Cited by 16 | Viewed by 4546
Abstract
Age-related decline in mitochondrial function and oxidative stress plays a critical role in neurodegeneration. Lactate dehydrogenase-B (LDHB) is a glycolytic enzyme that catalyzes the conversion of lactate, an important brain energy substrate, into pyruvate. It has been reported that the LDHB pattern changes [...] Read more.
Age-related decline in mitochondrial function and oxidative stress plays a critical role in neurodegeneration. Lactate dehydrogenase-B (LDHB) is a glycolytic enzyme that catalyzes the conversion of lactate, an important brain energy substrate, into pyruvate. It has been reported that the LDHB pattern changes in the brain during ageing. Yet very little is known about the effect of LDHB deficiency on brain pathology. Here, we have used Ldhb knockout (Ldhb−/−) mice to test the hypothesis that LDHB deficiency plays an important role in oxidative stress-mediated neuroinflammation and neurodegeneration. LDHB knockout (Ldhb−/−) mice were generated by the ablation of the Ldhb gene using the Cre/loxP-recombination system in the C57BL/6 genetic background. The Ldhb−/− mice were treated with either osmotin (15 μg/g of the body; intraperitoneally) or vehicle twice a week for 5-weeks. After behavior assessments, the mice were sacrificed, and the cortical and hippocampal brain regions were analyzed through biochemical and morphological analysis. Ldhb−/− mice displayed enhanced reactive oxygen species (ROS) and lipid peroxidation (LPO) production, and they revealed depleted stores of cellular ATP, GSH:GSSG enzyme ratio, and downregulated expression of Nrf2 and HO-1 proteins, when compared to WT littermates. Importantly, the Ldhb−/− mice showed upregulated expression of apoptosis mediators (Bax, Cytochrome C, and caspase-3), and revealed impaired p-AMPK/SIRT1/PGC-1alpha signaling. Moreover, LDHB deficiency-induced gliosis increased the production of inflammatory mediators (TNF-α, Nf-ĸB, and NOS2), and revealed cognitive deficits. Treatment with osmotin, an adipoR1 natural agonist, significantly increased cellular ATP production by increasing mitochondrial function and attenuated oxidative stress, neuroinflammation, and neuronal apoptosis, probably, by upregulating p-AMPK/SIRT1/PGC-1alpha signaling in Ldhb−/− mice. In brief, LDHB deficiency may lead to brain oxidative stress-mediated progression of neurodegeneration via regulating p-AMPK/SIRT1/PGC-1alpha signaling, while osmotin could improve mitochondrial functions, abrogate oxidative stress and alleviate neuroinflammation and neurodegeneration in adult Ldhb−/− mice. Full article
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<p>Schematic diagram showing the generation of <span class="html-italic">Ldhb</span><sup>−/−</sup> mouse, experimental design, and verification of <span class="html-italic">Ldhb</span><sup>−/−</sup> model. (<b>A</b>) Schematic diagram illustrating the Cre-loxP and Flp-FRT system using pBS-<span class="html-italic">Ldhb</span> KO vectors. <span class="html-italic">Ldhb</span><sup>−/−</sup> mice were generated through gene knockout at the exon 3 loci. (<b>B</b>) Schematic diagram of experimental design, showing the period of osmotin treatment in <span class="html-italic">Ldhb</span><sup>−/−</sup> mice and behavior test. (<b>C</b>,<b>D</b>) The Western blot analysis showing the expression of LDHB in the cortex, hippocampus, kidney, and liver of <span class="html-italic">Ldhb</span><sup>−/−</sup> and WT mice. β-actin was used as a loading control. The bands were quantified using ImageJ software, and the differences are represented by histograms. (<b>E</b>) Confocal microscopic images showing the expression of LDHB in the cortex and hippocampus of <span class="html-italic">Ldhb</span><sup>−/−</sup>, WT mice. (<b>F</b>) Immunohistochemistry staining for LDHB in the cortex and hippocampus of <span class="html-italic">Ldhb</span><sup>−/−</sup>, WT mice.</p>
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<p>LDHB deficiency-induced oxidative stress in adult mouse brain. (<b>A</b>,<b>B</b>) Analysis of the generation of ROS and production of LPO in the cortex and hippocampus regions of <span class="html-italic">Ldhb</span><sup>−/−</sup> and WT mice. (<b>C</b>,<b>D</b>) The graphs represent the levels of glutathione (GSH) and GSH/GSSG ratio in the hippocampi of adult <span class="html-italic">Ldhb</span><sup>−/−</sup> and WT mice. (<b>E</b>) Quantitative analysis of cellular ATP levels in the cortical and hippocampal brain tissue lysate in the experimental groups. (<b>F</b>) Western blot analysis of Nrf2 and HO-1 in the cortex and hippocampus of adult mouse brains. The bands were quantified using ImageJ software, and the differences are represented by histograms. β-actin was used as a loading control. (<b>G</b>) Confocal immunofluorescence photomicrographs of Nrf-2 reactivity in the cortex and hippocampus of the experimental groups. The density values are relative to the WT group and expressed in arbitrary units (A.U.). The data are presented as the mean ± SEM of 4 mice per group, * <span class="html-italic">p</span> ≤ 0.05, ** <span class="html-italic">p</span> ≤ 0.01.</p>
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<p>LDHB deficiency-induced apoptotic cell death in the cortex and hippocampus of the experimental groups. (<b>A</b>) Western blot analysis of Bax, Bcl-2, Cytochrome C and Caspase-3 in the cortex and hippocampus of <span class="html-italic">Ldhb</span><sup>−/−</sup> and WT mice. The bands were quantified using ImageJ software, and the differences are represented by histograms. β-actin was used as a loading control. (<b>B</b>) Confocal immunofluorescence photomicrographs of Caspase-3 reactivity in the cortex and hippocampus of the experimental groups. The density values are relative to the WT group and expressed in arbitrary units (A.U.). The data are presented as the mean ± SEM of 4 mice per group, ** <span class="html-italic">p</span> ≤ 0.01. (<b>C</b>) Photomicrograph of LDHB Nissl staining in the cortex and hippocampus region of the mouse brain.</p>
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<p>Treatment with osmotin upregulated p-AMPK/SIRT1/PGC-1alpha signaling, reduced by LDHB deficiency. (<b>A</b>) Western blot analysis of AdipoR1, p-AMPK, AMPK, SIRT1, and PGC-1alpha in the cortex and hippocampus of the experimental groups. The bands were quantified using ImageJ software, and the differences are represented by histograms. β-actin was used as a loading control. (<b>B</b>) Confocal immunofluorescence photomicrographs of PGC-1alpha immunoreactivity in the cortex and hippocampus of the experimental groups. The data is presented as the mean ± SEM of 4 mice per group and expressed in arbitrary units (A.U.). ** <span class="html-italic">p</span> ≤ 0.01, <sup>#</sup> <span class="html-italic">p</span> ≤ 0.05, n.s = not significant.</p>
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<p>Osmotin alleviates oxidative stress and apoptosis, induced by LDHB deficiency in the cortex and hippocampus. (<b>A</b>,<b>B</b>) Analysis of the ROS generation and LPO production in the cortex and hippocampus of <span class="html-italic">Ldhb</span><sup>−/−</sup> and osmotin treated <span class="html-italic">Ldhb</span><sup>−/−</sup> mice. (<b>C</b>–<b>E</b>) The representative histograms show the glutathione (GSH) levels, GSH/glutathione disulfide (GSH/GSSG) ratio, and ATP levels in the brain tissue lysate of the adult mice. (<b>F</b>) Western blot analysis of Nrf2 and HO-1 in the cortex and hippocampus of adult mouse brains. (<b>G</b>) Confocal immunofluorescence photomicrographs of Nrf-2 reactivity in the cortex and hippocampus of <span class="html-italic">Ldhb</span><sup>−/−</sup> and Osmotin-treated groups. (<b>H</b>) Western blot analysis of Bax, Bcl-2, Caspase-3, and Cytochrome C in the cortex and hippocampus of adult mouse brains. (<b>I</b>) Confocal immunofluorescence photomicrographs of Caspase-3 reactivity in the cortex and hippocampus of <span class="html-italic">Ldhb</span><sup>−/−</sup> and Osmotin-treated groups. Western blot bands were quantified using ImageJ software, and the differences are presented by histograms. β-actin was used as a loading control. The density values are relative to the KO group and expressed in arbitrary units (A.U.). The data are presented as the mean ± SEM of 4 mice per group, * <span class="html-italic">p</span> ≤ 0.05, ** <span class="html-italic">p</span> ≤ 0.01.</p>
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<p>Osmotin alleviates immune response induced by LDHB deficiency in the cortex and hippocampus. (<b>A</b>,<b>C</b>) Western blot analysis of GFAP, Iba-1, TNF-α, Nf-ĸB, and NOS-2 in the cortex and hippocampus of adult mouse brains. The bands were quantified using ImageJ software, and the differences are represented by histograms. β-actin was used as a loading control. (<b>B</b>,<b>D</b>) Confocal immunofluorescence photomicrographs of GFAP and IL-1β reactivity, respectively, in the cortex and hippocampus of the experimental groups. The density values are relative to the WT group and expressed in arbitrary units (A.U.). The data are presented as the mean ± SEM of 4 mice per group, ** <span class="html-italic">p</span> ≤ 0.01, <sup>#</sup> <span class="html-italic">p</span> ≤ 0.05.</p>
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<p>Effect of osmotin on LDHB deficiency-induced cognitive and learning dysfunction. The behavioral studies were performed through the Y-maze test and the Morris water maze (MWM). (<b>A</b>,<b>B</b>) The graphs represent the % of spontaneous alternation behaviors and the total number of entries in the Y-maze test. The <span class="html-italic">Ldhb</span><sup>−/−</sup> mice revealed significantly reduced % spontaneous alternation, with a marked difference in the total number of arm entries compared to the WT group. Treatment with osmotin enhanced % spontaneous alternation, signifying improvement in short-term memory and working memory, as well as reduced total number of arm entries in <span class="html-italic">Ldhb</span><sup>−/−</sup> mice. (<b>C</b>) Represents trajectories in the Y-maze test. (<b>D</b>) Average escape latency time is taken by mice to reach the hidden platform (from training day 1 to 4 days) in the MWM test. The training period reveals that mice were successful in learning the maze. (<b>E</b>,<b>F</b>) In the probe test, <span class="html-italic">Ldhb</span><sup>−/−</sup> mice, compared to WT, illustrated a decline in time spent and an average number of crossing in the arbitrary quadrant in MWM. Osmotin treatment of <span class="html-italic">Ldhb</span><sup>−/−</sup> increased the total time spent and the number of crossings, implying an improvement in spatial reference and working memory (<b>G</b>) Diagram reveals representative trajectories in the MWM test. The data are presented as the mean ± SEM of 4 mice per group, ** <span class="html-italic">p</span> ≤ 0.01, <sup>#</sup> <span class="html-italic">p</span> ≤ 0.05.</p>
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<p>Schematic illustration of proposed detrimental consequences of LDHB deficiency on brain mitochondrial function and neurodegeneration. The schematic diagram shows that the deficiency of LDHB in the brain can induce neurodegeneration through mitochondrial dysfunction. Using osmotin improved mitochondrial bioenergetics and suppressed ROS induced oxidative stress, neuroinflammation, and neurodegeneration, probably by upregulating the p-AMPK/Sirt1/PGC-1α signaling pathway.</p>
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17 pages, 2934 KiB  
Article
Leaf Apoplast of Field-Grown Potato Analyzed by Quantitative Proteomics and Activity-Based Protein Profiling
by Kibrom B. Abreha, Erik Alexandersson, Svante Resjö, Åsa Lankinen, Daniela Sueldo, Farnusch Kaschani, Markus Kaiser, Renier A. L. van der Hoorn, Fredrik Levander and Erik Andreasson
Int. J. Mol. Sci. 2021, 22(21), 12033; https://doi.org/10.3390/ijms222112033 - 6 Nov 2021
Cited by 4 | Viewed by 3049
Abstract
Multiple biotic and abiotic stresses challenge plants growing in agricultural fields. Most molecular studies have aimed to understand plant responses to challenges under controlled conditions. However, studies on field-grown plants are scarce, limiting application of the findings in agricultural conditions. In this study, [...] Read more.
Multiple biotic and abiotic stresses challenge plants growing in agricultural fields. Most molecular studies have aimed to understand plant responses to challenges under controlled conditions. However, studies on field-grown plants are scarce, limiting application of the findings in agricultural conditions. In this study, we investigated the composition of apoplastic proteomes of potato cultivar Bintje grown under field conditions, i.e., two field sites in June–August across two years and fungicide treated and untreated, using quantitative proteomics, as well as its activity using activity-based protein profiling (ABPP). Samples were clustered and some proteins showed significant intensity and activity differences, based on their field site and sampling time (June–August), indicating differential regulation of certain proteins in response to environmental or developmental factors. Peroxidases, class II chitinases, pectinesterases, and osmotins were among the proteins more abundant later in the growing season (July–August) as compared to early in the season (June). We did not detect significant differences between fungicide Shirlan treated and untreated field samples in two growing seasons. Using ABPP, we showed differential activity of serine hydrolases and β-glycosidases under greenhouse and field conditions and across a growing season. Furthermore, the activity of serine hydrolases and β-glycosidases, including proteins related to biotic stress tolerance, decreased as the season progressed. The generated proteomics data would facilitate further studies aiming at understanding mechanisms of molecular plant physiology in agricultural fields and help applying effective strategies to mitigate biotic and abiotic stresses. Full article
(This article belongs to the Special Issue Plant Proteomic Research 4.0)
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Figure 1

Figure 1
<p>Word-cloud representation of the leaf apoplastic proteome from potato cultivar Bintje. Identified proteins were classified into families using Pfam analysis. Scale of the fonts and colors in the word cloud represents relative abundance of the protein family in the apoplast samples from field-grown plants.</p>
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<p>Principal components and heat map analyses of apoplastic proteome samples isolated from potato cultivar Bintje grown at two experimental sites (Borgeby and Mosslunda) and in two different growing seasons (2011 and 2012) in Mosslunda. (<b>A</b>) Unsupervised principal component analysis plot of the samples in Mosslunda and Borgeby; (<b>B</b>) Unsupervised principal component analysis plot of the samples in Mosslunda in 2011 and 2012; (<b>C</b>) Abundance of peptides from Borgeby respectively compared to those in Mosslunda; (<b>D</b>) Abundance of peptides collected in 2011 respectively compared to those collected in 2012 in Mosslunda. Two-group comparisons (<span class="html-italic">t</span>-test) were performed in Qlucore with false discovery rate using Benjamini−Hochberg correction (<span class="html-italic">q</span> &lt; 0.001). Heat maps are sorted using hierarchal clustering and red represents higher abundance (Fold change, log2).</p>
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<p>Quantitative analysis of apoplastic proteome samples isolated from potato cultivar Bintje grown in Mosslunda in 2012. (<b>A</b>) Unsupervised principal component analysis plot of all the samples collected in June (Mo_Jun_12), July (Mo_Jul_12), and August (Mo_Jul_12). Each circle represents one biological replicate. (<b>B</b>) Heat maps and the number of peptides up- and down-regulated in plants grown under field conditions in Mosslunda. We performed a multi-group comparison with false discovery rate &lt; 0.001 (according to the Benjamini−Hochberg procedure for determining <span class="html-italic">q</span>). Heat map of the differentially regulated peptides (<span class="html-italic">q</span> &lt; 0.001) was sorted using hierarchal clustering and red represents higher abundance (Fold change, log2). (<b>C</b>) STEM clustering analysis of apoplastic peptides in June, July, August of 2012 in Mosslunda. Proteins that were significantly (<span class="html-italic">q</span> ≤ 0.001) increased or decreased in at least one of the months across the growing season were used for the STEM clustering analysis. Top left of each box is the profile number and bottom left of each box indicates the number of peptides that fit the defined abundance pattern in June, July, and August. The STEM analysis identified 16 profiles, of which profiles 11 and 12 contains statistically significant number of proteins (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Quantitative analysis of apoplastic proteome samples isolated from potato cultivar Bintje grown under greenhouse and field conditions. (<b>A</b>) Unsupervised principal component analysis plot of all the samples. Each circle represents one biological replicate; (<b>B</b>) heat maps and numbers of peptides up- and down-regulated under greenhouse and field conditions according to a two-group comparison in Qlucore with a false discovery rate &lt; 0.001 (according to the Benjamini−Hochberg procedure for determining q). The heat map of the differentially regulated peptides (<span class="html-italic">q</span> &lt; 0.001) was sorted using hierarchal clustering and red represents higher abundance (Fold change, log2).</p>
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<p>Serine hydrolase and β-glycosidase activity profiling of potato cultivar Bintje grown under greenhouse and field conditions in Mosslunda in June, July, and August 2012. Apoplastic proteins were labeled by 2 µM probe for (<b>A</b>) serine hydrolase and (<b>B</b>) β-glycosidase. The probe-labelled proteins were separated on 12% sodium dodecyl sulfate-polyacrylamide electrophoresis gels and detected using a fluorescence scanner.</p>
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<p>Identification of serine hydrolases and β-glycosidases proteins that were captured by activity-based probes. Leaf apoplastic proteome of the potato sample was co-labelled by 5 μM biotinylated probes for β-glucosidase (JJB111) and serine hydrolases (FP-biotin). Biotinylated proteins were then affinity-purified with streptavidin beads and separated on 12% sodium dodecyl sulfate-polyacrylamide electrophoresis gel. The gel was stained by SYPRO Ruby staining.</p>
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10 pages, 13085 KiB  
Article
Arabidopsis OSMOTIN 34 Functions in the ABA Signaling Pathway and Is Regulated by Proteolysis
by Eun-Joo Park and Tae-Houn Kim
Int. J. Mol. Sci. 2021, 22(15), 7915; https://doi.org/10.3390/ijms22157915 - 24 Jul 2021
Cited by 16 | Viewed by 3243
Abstract
Plants have evolutionarily established resistance responses to a variety of abiotic stress conditions, in which ABA mediates the integrated regulation of these stress responses. Numerous proteins function at the transcription level or at the protein level when contributing to controls of the ABA [...] Read more.
Plants have evolutionarily established resistance responses to a variety of abiotic stress conditions, in which ABA mediates the integrated regulation of these stress responses. Numerous proteins function at the transcription level or at the protein level when contributing to controls of the ABA signaling process. Although osmotin is identified as a salt-inducible protein, its function in the abiotic stress response is yet to be elucidated. To examine the role of Arabidopsis OSMOTIN 34 (OSM34) in the ABA signaling pathway, a deletion mutant osm34 generated by a CRISPR/Cas9 system and the double mutant osm34 osml (osmotin 34-like) were analyzed for various ABA responses. Both osm34 and osm34 osml showed reduced levels of ABA responses in seeds and leaves. Moreover, proline level and expression of the proline biosynthesis gene P5CS1 was significantly reduced in osm34 osml. Interestingly, OSM34 binds to SKP2A, an F-Box protein whose transcription is induced by ABA. The protein stability of OSM34 was determined to be under the control of the 26S proteasome. In conclusion, our data suggest that OSM34 functions as a positive regulator in the generation of ABA responses and is under post-translational control. Full article
(This article belongs to the Section Molecular Plant Sciences)
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Graphical abstract

Graphical abstract
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<p>Generation of the deletion mutant <span class="html-italic">osm34</span> by a CRISPR/Cas9 approach (<b>A</b>) Two guide RNAs targeting first exon region of <span class="html-italic">OSM34</span> were designed (red line with arrow heads, top), and a deletion mutant <span class="html-italic">osm34</span> was isolated (bottom). (<b>B</b>) PCR genotyping confirmed a large deletion in <span class="html-italic">osm34</span> and the removal of Cas9 by crossing into wild-type (Col-0). By crossing Cas9-free <span class="html-italic">osm34</span> into <span class="html-italic">osml</span> (<span class="html-italic">osmotin 34-like</span>), the double mutant <span class="html-italic">osm34 osml</span> was generated. (<b>C</b>) Sequence analyses of <span class="html-italic">osm34</span> revealed location of the deletion when compared to the wild-type sequences. The red color bar indicates the deleted sequences in <span class="html-italic">osm34</span>.</p>
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<p>The mutant <span class="html-italic">osm34</span> and <span class="html-italic">osm34 osml</span> produce defects in the generation of ABA responses (<b>A</b>) Seed germination assay using <span class="html-italic">osm34</span> and <span class="html-italic">osm34 osml</span> under 0.5 μM ABA. a/b/bc/c indicate statistically different groups based on one-way ANOVA Duncan post-hoc test (<span class="html-italic">p</span> &lt; 0.05). (<b>B</b>) Quantification of proline level using 1.25% ninhydrin solution. Three-week-old rosette leaves of wild-type (Col-0) and <span class="html-italic">osm34 osml</span> were sprayed with 30 μM ABA for two days. a/ab/bc/c indicate different groups based on one-way ANOVA Duncan post-hoc test (<span class="html-italic">p</span> &lt; 0.05). (<b>C</b>) The ABA-induced stomatal closure was analyzed using four-week-old wild-type (Col-0), <span class="html-italic">osm34</span>, and <span class="html-italic">osm34 osml</span> after 2 h of exposure to 10 μM ABA. a/ab/bc/c groups based on one-way ANOVA Duncan post-hoc test (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>The mutants <span class="html-italic">osm34</span> and <span class="html-italic">osm34 osml</span> exhibit reduced ABA induction of gene expression Six-day-old seedlings were treated with 50 μM ABA for 24 h and used for quantitative PCR analyses. Clathrin was used as an internal control. a/b/ab indicate different groups according to one-way ANOVA Duncan post-hoc test (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>OSM34 interacts with SKP2A and is destabilized via 26S proteasome-dependent pathway (<b>A</b>) Yeast two-hybrid assay showed direct interaction between OSM34 (42-244) and SKP2A (1-360). Co-transformed yeast colonies were grown on either DDO media (-Leu/-Trp) or QDO media (-Ade/-His/-Leu/-Trp) containing 10 mM 3-AT. (<b>B</b>) The Arabidopsis transgenic lines expressing either <span class="html-italic">OSM34-GFP</span> or <span class="html-italic">SKP2A-HA</span> were used for co-immunoprecipitation (co-IP) analysis. Input was collected before co-IP analysis. After immunoprecipitation with anti-GFP-sepharose beads, Western blot analysis was performed on the co-IP sample using anti-HA antibody. (<b>C</b>) Cell-free OSM34 degradation assay using tobacco leaves with transiently expressing either SKP2A-FLAG/GST-HA or OSM34-GFP/GST-HA. GST-HA was co-expressed as an internal control for each transient expression. Total proteins were incubated for 3 h with 20 μM MG132 or DMSO and subsequently analyzed by Western blot. * indicates the target GFP band. (<b>D</b>) In vivo stability of OSM34 was analyzed by Western blot assays using the transgenic lines co-expressing <span class="html-italic">OSM34-GFP</span> and <span class="html-italic">SKP2A-HA</span> or expressing <span class="html-italic">OSM34-GFP</span> alone.</p>
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19 pages, 5767 KiB  
Article
ROS Dependent Antifungal and Anticancer Modulations of Piper colubrinum Osmotin
by Rajeswari Gopal Geetha, Sivakumar Krishnankutty Nair Chandrika, Gayathri G. Saraswathy, Asha Nair Sivakumari and Manjula Sakuntala
Molecules 2021, 26(8), 2239; https://doi.org/10.3390/molecules26082239 - 13 Apr 2021
Cited by 4 | Viewed by 3409
Abstract
Osmotin, a plant defense protein, has functional similarity to adiponectin, an insulin sensitizingsensitising hormone secreted by adipocytes. We speculated that Piper colubrinum Osmotin (PcOSM) could have functional roles in obesity-related cancers, especially breast cancer. Immunofluorescence assays, flow cytometry, cell cycle analysis and a [...] Read more.
Osmotin, a plant defense protein, has functional similarity to adiponectin, an insulin sensitizingsensitising hormone secreted by adipocytes. We speculated that Piper colubrinum Osmotin (PcOSM) could have functional roles in obesity-related cancers, especially breast cancer. Immunofluorescence assays, flow cytometry, cell cycle analysis and a senescence assay were employed to delineate the activity in MDAMB231 breast cancer cell line. PcOSM pre-treated P. nigrum leaves showed significant reduction in disease symptoms correlated with high ROS production. In silico analysis predicted that PcOSM has higher binding efficiency with adiponectin receptor compared to adiponectin. PcOSM was effectively taken up by MDAMB231 cancer cells which resulted in marked increase in intracellular ROS levels leading to senescence and cell cycle arrest in G2/M stage. This study provides evidence on the ROS mediated direct inhibitory activity of the plant derived osmotin protein on the phytopathogen Phytophthora capsici, and the additional functional roles of this plant defense protein on cancer cells through inducing ROS associated senescence. The strong leads produced from this study could be pursued further to obtain more insights into the therapeutic potential of osmotin in human cancers. Full article
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Figure 1
<p>Purification of His<sub>6</sub> tagged recombinant <span class="html-italic">Piper colubrinum</span> Osmotin in <span class="html-italic">E. coli</span>: (<b>A</b>) SDS-PAGE analysis of the expression of 6xHis tagged PcOSM in <span class="html-italic">E. coli</span> cultures expressed at 37 °C Lane 1. Molecular mass marker (NEB), (2–4)-Total protein from non-induced, induced by 1mM IPTG for 6h and 8 h. (<b>B</b>) SDS PAGE of crude protein and partially purified recombinant PcOSM protein, (<b>C</b>) Western blot analysis of His tagged PcOSM, probed with Anti-polyhistidine antibody, (<b>D</b>) and (<b>E</b>) AKTA chromatogram obtained from purification optimization. (<b>F</b>) SDS-PAGE analysis of purified elute fractions obtained from purification optimization -Lane 1: Molecular marker (NEB), lane 2-9-elute, (<b>G</b>) LC-MS/MS analysis of osmotin showing mass spectrum of osmotin and the unique peptides identified were highlighted in different colours.</p>
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<p>Inhibitory role of PcOSM on <span class="html-italic">Phytophtora capsici.</span> Control <span class="html-italic">Piper nigrum</span> leaf disc infected with <span class="html-italic">P. capsici</span>. In vivo hyphal lysis of PcOSM- <span class="html-italic">Piper nigrum</span> leaf infiltrated with PcOSM 100µg/mL and 20o µg/mL and infected with <span class="html-italic">P. capsici</span> 1 h after infiltration. The leaf discs showed significant hyphal breakage of <span class="html-italic">P. capsici</span> followed by inhibition of mycelial growth.</p>
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<p>PcOSM induces ROS production in <span class="html-italic">Piper nigrum</span> leaves: Scale bar = 100 µm. (<b>A</b>) Fluorescence microscopic images (20×) of leaf disc stained with DCFDA to visualize ROS accumulation. (<b>B</b>) Mean values of integrated density of DCFDA staining. The asterisk indicates a significant difference (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Molecular docking studies: (<b>A</b>) The multiple alignments of amino acid sequences of ADIPOQ, PcOSM, NtOSM compiled with PRALINE. Coloured blocks indicate conserved regions. (<b>B</b>) The helixes of ADIPOR1 are embedded in lipid bilayer membrane subjected to supervised docking with OSM (purple) and ADIPOQ (green) restricting their docking grid confined to intra-cellular loops of ADIPOR1. The lowest energy docking solutions were submitted to MD simulation for structural refinement. (<b>C</b>) Representative snapshot for free energy minima of ADIPOR1/OSM and (<b>D</b>) ADIPOR1/ADIPOQ complexes from simulations showing functional residues contributing highly towards effective binding at the interaction interface.</p>
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<p>Cytotoxicity effect on MDA MB231 cells treated with PcOSM for 72 h measured by MTT assay. Results are reported as percentage of cell death. The bar shows mean ± Standard deviation of triplicates. Statistical significance of the treated and untreated cells were analyzed using one way ANNOVA. PcOSM treated cells were significantly different from control cells (<span class="html-italic">p</span> &lt; 0.001).</p>
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<p>Immunofluorescence assays: (<b>A</b>) MDA MB231 cells stained with FITC labelled secondary antibody for ADIPOR1 (green colour) and Hoechst 33342 to stain the nuclei (blue colour). (<b>B</b>) Cells were stained with Phycoerythrin labelled secondary antibody for histidine tagged PcOSM. (<b>C</b>) C0-immunofluorescence assay for co-localization of PCOSM and ADIPOR1, cells were co stained with secondary antibodies for ADIPOR1 and PcOSM. (<b>D</b>) Intensity profile of co-localization of ADIPO-R1 and PcOSM of an area indicated in the image analysed by NIS Elements imaging software.</p>
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<p>Phalloidin staining for cytoskeleton: (<b>A</b>) Untreated MDA MB231 cells stained with rhodamine- Phalloidin showing rigid cytoskeleton. (<b>B</b>) PcOSM treated MDAMB-231 cells showing cytoskeletal changes.</p>
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<p>PcOSM induced intracellular reactive oxygen species accumulation: (<b>A</b>) Flow cytometry analysis of reactive oxygen species (ROS) production in control cells, positive control and PcOSM treated MDAMB231 cells for 24, 48 and 72 h. (<b>B</b>) Representative histogram of DCFDA staining of cells treated with PcOSM. Columns indicate mean ± SD of three experiments. * <span class="html-italic">p</span> &lt; 0.05 vs. Control. (<b>C</b>) Confocal microscopy images (20X) showing ROS production in positive control and PcOSM after 72 h of treatment.</p>
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<p>PcOSM induced cellular senescence in MDAMB231 cells: Senescence associated beta galactosidase assay (SA-β-Gal) staining of cells indicated concentration of PcOSM after 72 h. Cells were fixed and stained with fresh SA-β-Gal. PcOSM treated cells exhibited blue colour indicating senescent cells.</p>
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<p>PcOSM induced cell cycle arrest: MDA MB231 cells were treated with PcOSM (200 µg/mL) for 72 h. Cell cycle analysis was performed using PI staining. Percentage of cell cycle distributions are shown. The results indicate the mean ± SD of three independent experiments.</p>
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