Sugar Tech

 
DOI 10.1007/s12355-016-0431-4

RESEARCH ARTICLE

A Novel Non-specific Lipid Transfer Protein Gene

from Sugarcane (NsLTPs), Obviously Responded to Abiotic
Stresses and Signaling Molecules of SA and MeJA
Yun Chen1 • Jingjing Ma1 • Xu Zhang1 • Yuting Yang1 • Dinggang Zhou1 •

Qing Yu1 • Youxiong Que1 • Liping Xu1 • Jinlong Guo1

Received: 11 October 2015 / Accepted: 25 January 2016

Ó Society for Sugar Research & Promotion 2016

Abstract Non-specific lipid transfer proteins (NsLTPs) Keywords Sugarcane (Saccharum hybrid complex)

are soluble, small, basic proteins in plant, which have been Lipid transfer proteins (LTPs)
Gene expression

reported to be involved in plant physiological functions qRT-PCR
such as the catalyzing transfer of phospholipids and play an
important role in plant defense and stress responses. In this
study, a member of NsLTP family gene (ScNsLTP, Acc. Introduction
No. KR259657) was isolated from a full-length cDNA
library of sugarcane stalk. The cDNA of ScNsLTP was 671 Plant lipid transfer proteins, also known as plant LTPs or
bp long and contained a 312 bp open reading frame (ORF), PLTPs, were a group of highly conserved proteins of about
which can encode a protein of 103 amino acid residues 7–10 kDa found in higher plant tissues (Asero et al. 2000).
with molecular weight of 10.66 kDa. The ScNsLTP tran- LTPs were responsible for the shuttling of phospholipids
script levels in sugarcane seedlings decreased in response and other fatty acid groups between cell membranes and
to SA, whereas it increased under MeJA treatment, sug- were also able to bind acyl groups (Kader 1996). Lipid
gesting an antagonistic regulatory mechanism between the transfer proteins were found in a wide variety of organisms
signaling molecules of SA and MeJA. The transcript levels including animals, plants and bacteria, and they existed in
of ScNsLTP were obviously up-regulated under chilling different tissues with various functions and sizes (Kader
and PEG stresses, implying that the ScNsLTP gene was 1996; Wang et al. 2012).
involved in response to abiotic stresses and playing a Plant non-specific lipid transfer proteins (NsLTPs),
positive role in adaption to low temperature and drought named for their character of reversibly bind to various
stresses. These results provide important information for phospholipids and non-specific binding to different lipids
further functional studies of plant NsLTPs gene. (Ostergaard et al. 1993), were a group of small (about
6.5–10.5 kDa), soluble basic proteins (Wang et al. 2012). It
contained a highly conserved cysteine residues motif which
arranged as C-Xn-C-Xn-CC-Xn-CXC-Xn-C-Xn-C and
Electronic supplementary material The online version of this formed four conserved defined disulfide bonds (Samuel
article (doi:10.1007/s12355-016-0431-4) contains supplementary et al. 2002). According to their primary structure, NsLTPs
material, which is available to authorized users.
could be classified into three types (Boutrot et al. 2005).
& Liping Xu NsLTPs played an important role in plants for facili-
xlpmail@126.com tating the transfer of phospholipids, glycolipids, fatty acids
& Jinlong Guo and steroids between membranes (Seedorf et al. 1994;
jl.guo@163.com Wilmanns et al. 2006). Nowadays, more NsLTPs are
1
reported in plants. DIR1 encoded a non-specific lipid
Key Laboratory of Sugarcane Biology and Genetic Breeding,
transfer protein in Arabidopsis, which played an important
Ministry of Agriculture, Fujian Agriculture and Forestry
University, Shangxiadian Rd 15, Cangshan, Jinshan, Fuzhou role in plant defense against pathogens (Maldonado et al.
350002, Fujian, People’s Republic of China 2002; Lascombe et al. 2008). Besides, transgenic tobacco

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over expressing of TaLTP3F1 gene showed bacterial, 24 h, respectively. The other two groups of plantlets were
fungal and aphid resistance (Choi et al. 2012). AZI1, a lipid grown in 1/2 Hoagland nutrient solution containing
transfer protein of Arabidopsis, played a vital role in salt 250 mM NaCl (Damaj et al. 2010; Que et al. 2009) and
tolerance (Pitzschke et al. 2014). 25 % polyethylene glycol (PEG) for 0, 12, 24 h, respec-
As for the sugarcane NsLTPs, one study, based on tively. Last group was subjected to low temperature (4 °C)
microarray technology, indicated that three LTPs homol- for 0, 12 and 24 h. All of the treatments were carried out in
ogous ESTs from sugarcane leaf libraries were all three replicates, three plantlets for each replicate. All col-
increased during water deficit, and one of them lected samples were immediately frozen in liquid nitrogen
(SCBGLR1120F01.g) was further confirmed by RT-PCR and stored at -80 °C until RNA extraction.
(Rodrigues et al. 2011). The other sugarcane LTP was Two-bud setts from Yacheng05-179 (10 months old)
reported as Puccinia melanocephala infection response were used for biotic treatments. The buds were inoculated
gene, which expression was increased in incompatible with 0.5 lL/each bud suspension containing 5 9 106
sugarcane genotype compared to healthy plants revealing Sporisorium scitamineum spores mL-1 in 0.01 % (v/v)
by quantitative expression analysis (Oloriz et al. 2012). Tween-20, while controls were mock-inoculated with
Though there were two earlier reports referring to LTP in 0.01 % (v/v) Tween-20 in sterile distilled water instead of
sugarcane, the categories and characteristics of sugarcane S. scitamineum spores (Moosawi-Jorf and Mahin 2007; Su
NsLTPs remained unclear. et al. 2013). Then, the inoculated setts grew at 28 °C under
In this study, a gene designated as ScNsLTP was isolated 60 % humid condition for 12 h light/12 h dark. Five buds
from full-length cDNA library of sugarcane stem. The for each replicate were collected at each of the time point
category and characteristics of ScNsLTP gene and its of 0 h, 2 days, 5 days and 7 days. Samples collected were
deduced protein were determined based on bioinformatics frozen in liquid nitrogen and stored at -80 °C until RNA
analysis. Furthermore, tissue-specific expression pattern of extraction.
ScNsLTP gene and its expression profiles under different
stress conditions in sugarcane were investigated by qRT- RNA Extraction
PCR. Our study aimed to lay the foundation for further
research on the function of ScNsLTP gene in sugarcane. Total RNA of Yacheng05-179 was extracted with TRIzol
reagent (Invitrogen, USA) according to the manufacture’s
protocol. The qualities of total RNAs were monitored by
Materials and Methods measuring the absorbance at 260, 280 nm (NanoVue plus,
GE, USA) and by electrophoresis on 1.5 % agarose gel.
Plant Materials and Treatments DNase I (Promega, USA) was used to remove any DNA
contamination. The first-strand cDNA synthesis was
Sugarcane materials of cultivar Yacheng05-179 were pro- obtained by the Prime-ScriptTM RT Reagent Kit (TaKaRa,
vided by the Key Laboratory of Sugarcane Biology and Dalian).
Genetic Breeding, Ministry of Agriculture, Fuzhou, China.
For tissue-specific expression analysis, six healthy and Sequence Analysis and Prediction of Protein
consistent growing plants of Yacheng05-179 of 10 months Property
age were randomly selected, which contained more than 25
internodes and had sufficient buds for sampling. For each Open reading frame (ORF) finder was performed to analyze
plant, the skin and the basic tissue (all part inside the skin), the ORF of the sequence and predict the amino acid
all the buds, the youngest fully expanded leaf viz ?1 leaf sequence. Multiple sequence alignments to determine pro-
with a visible dewlap (the collar between the leaf blade and tein similarities were carried out with DNAMAN 5.0. The
sheath) were collected. All collected samples were frozen physicochemical properties such as pI (isoelectric poin-
in liquid nitrogen and stored at -80 °C until RNA t),and Mw (molecular weight) predictions were performed
extraction. using the ProtParam (http://web.expasy.org/protparam/).
During abiotic treatments, uniform tissue culture plant- The ProtScale (http://web.expasy.org/cgi-bin/protscale/prot
lets of Yacheng05-179 were grown in 1/2 Hoagland scale.pl?1) was used to analyze protein hydrophobicity. Other
nutrient solution for 1 week and then treated by the fol- on-line tools such as PSORT (http://psort.hgc.jp/form.html)
lowing five different treatments at 28 °C with 16 h light/ and SignalP (http://www.cbs.dtu.dk/services/SignalP/) were
8 h dark. Two groups of plantlets (overground part) were used to predict intracellular localization and signal peptide,
sprayed with 5.0 mM salicylic acid (SA) solution and respectively (Su et al. 2013). Homology modeling of the 3D
100 lM methyl jasmonate (MeJA) in 0.1 % (v/v) ethanol structure of ScNsLTP was obtained using the SWISS-
and 0.05 % (v/v) Tween-20 (Li et al. 2010) for 0, 6, 12 and MODEL server (http://swissmodel.expasy.org/) (Arnold

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et al. 2006). The phylogenetic tree was constructed using Protein Physicochemical Properties
neighbor-joining analysis of MEGA 5.0. Thirty-four unique
amino acid sequences from 26 species which referred to The physical and chemical properties of the ScNsLTP
Arondel et al. (2000), Boutrot et al. (2005), Liu et al. (2009) deduced protein are given in Table 1. The molecular for-
and Jin et al. (2015)were analyzed. The accession numbers mula of the ScNsLTP was C456H743N127O138S14, with a
of the chosen proteins are given in Table S1. theoretical isoelectric point (pI) of 6.68 and a calculated
molecular mass of 10.66 kDa (Table 1). ScNsLTP was
Real-Time Quantitative PCR Analysis predicted as an unstable hydrophobic protein based on the
instability index (II), the grand average of hydropathicity
The real-time quantitative PCR (qRT-PCR) was followed (GRAVY) and aliphatic index indicated that ScNsLTP was
the manufacture’s protocol of the SYBR Green Master a hydrophobic protein (Table 1, Fig. S1).
(ROX) (Roche, USA) on ABI PRISM7500 real-time PCR
system (Applied Biosystems, USA). The qRT-PCR reac- Prediction of the Intracellular Localization
tion procedure was as follows: 50 °C, 2 min; 95 °C, of Sugarcane ScNsLTP
10 min; 40 cycles of 94 °C, 15 s and 60 °C, 60 s. The
qRT-PCR results were analyzed by 2-DDCt method (Livak The intracellular localization of the ScNsLTP protein was
and Schmittgen, 2001). The primers for ScNsLTP (forward: predicted by PSORT software. The results elucidated that
ATTAGATTCAATGGCGAAAGCAC, reverse: CTGTC the ScNsLTP protein located in the vacuole with highest
CGTCGGGTCATTCAC) and glyceraldehyde-3-phosphate probability (90.0 %), and the lowest probability (10.0 %)
dehydrogenase (GAPDH) (forward: CACGGCCACTGGA was on membrane and lumen of endoplasmic reticulum.
AGCA, reverse: TCCTCAGGGTTCCTGATGCC) were
synthesized by Shanghai Sangon Biotechnology Company Analysis of Signal Peptide of ScNsLTP Protein
(Shanghai, China).
The SignalP server predicted the presence of an N-terminal
signal peptide in the protein (Fig. S2). As shown in Fig. 3,
the 14th valine residue (V) with the highest signal peptide
Results score (S) of 0.967, the isoleucine residue (I) at 26 had the
highest combined cleavage site score (Y) of 0.809 and the
Analysis of Gene Sequence highest raw cleavage site score (C) of 0.782.

A full-length cDNA of ScNsLTP gene was 671 bp with a Structural Analyses of ScNsLTP Protein
312 bp ORF, encoding a deduced protein with 103 amino
acid residues (Fig. 1). As predicted by conserved domain The secondary structure prediction indicated that 57 amino
database (CDD) in NCBI, ScNsLTP belonged to nsLTP- acid residues of the ScNsLTP formed by random coil with
like subfamily (Fig. 2). percentage of 55.34 %, while 20 amino acid residues for

Fig. 1 Nucleotide acid

sequence and deduced amino
acid sequence of ScNsLTP gene.
Note The C showed the
conservative cysteine residual
contained in the domains of
ScNsLTP

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Fig. 2 Prediction of conserved domain in sugarcane ScNsLTP protein

Table 1 Physical and chemical properties of the ScNsLTP was constructed based on the putative amino acid sequences.
Figure 5 shows that ScNsLTP was categorized to type 2
Characteristics Predictive values
NsLTPs. The hypothetical three-dimensional structure of
Number of amino acids 103 ScNsLTP was compact and globular, comprising a-helix and
Theoretical pI 6.68 random coil (Fig. 6b). Compared ScNsLTP with the NsLTP
Molecular weight/Da 10,661.5 proteins from NtNsLTP2 (BAJ25798) and ZmNsLTP
Total number of negatively charged residues 8 (ACG30536), it was found that they showed similarity in the
(Asp ? Glu) three-dimensional structure (Fig. 6). These observations sug-
Total number of positively charged residues 8 gested that ScNsLTP has typical features of plant NsLTPs.
(Arg ? Lys)
Formula C456H743N127O138S14 Expression of the ScNsLTP Gene in Various Tissues
Instability index (II) 43.14
Grand average of hydropathicity (GRAVY) 0.365 qRT-PCR analysis revealed that ScNsLTP showed tissue-
Aliphatic index 85.53 specific expression (leaf and bud). Results of Fig. 7 indicated
that the expression level of ScNsLTP in bud and in leaf was 7.5
extended strand with percentage of 19.42 % and the other times and 4.5 times higher than that in the skin, respectively, and
26 for alpha helix with percentage of 25.24 % (Fig. S3). 1.5 times and 3.0 times more in the basic tissue, respectively.

Phylogenetic Analysis of ScNsLTP Amino Acid Effect of Abiotic and Biotic Treatments on ScNsLTP
Sequence Expression

ScNsLTP contained a highly conserved cysteine residues qRT-PCR was used to investigate the expression profile of
motif backbone with other plants (Fig. 4). A phylogenetic tree ScNsLTP in response to various exogenous stresses

Fig. 3 Prediction of signal

peptide of ScNsLTP

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of sugarcane smut pathogen S. scitamineum, no apparent

change in ScNsLTP transcript was observed in all the
samples of 0 h to 7 days (Fig. 8c).

Discussion

Plant NsLTPs were ubiquitous and low molecular mass

active proteins that had been demonstrated to play an
important role in plant resistance and defense against a
variety of biotic and abiotic stresses (Boutrot et al. 2005).
To date, NsLTPs had been identified in a variety of plant
species, such as wheat (Monnet et al. 2001; Jang et al.
2003; Boutrot et al. 2007), rice (Boutrot et al. 2008), corn
(Wei and Zhong 2014) and arabidopsis (Arondel et al.
Fig. 4 Alignment of the deduced ScLTP sequence with other plant 2000). As to sugarcane, although nsLTP-like genes had
NsLTP sequences
been observed in two former studies (Rodrigues et al. 2011;
Oloriz et al. 2012), the category and characteristics of
NsLTPs, together with its function in sugarcane under the
stresses of different abiotic and biotic factors, remained
unclear. In this study, a novel nsLTP-like gene was isolated
from sugarcane, which was categorized to be type 2 in
nsLTP-like family based on the phylogenetic tree using 34
nsLTP-like gene sequences from various plant species.
Besides, its expression profiles under different stresses
were investigated.
ScNsLTP deduced protein contained an N-terminal
signal peptide and was predicted as an extracellularly
located protein with a hydrophobic cavity structure
(Fig. 6b), and the ScNsLTP protein might be a secreted
protein with transport function (Fig. 3) which suggested
that it could be involved in the secretory pathway with the
functions in the synthesis of cutin (Sterk et al. 1991; Car-
valho and Gomes 2007). Plant NsLTPs were mainly clas-
sified into three types (type 1, type 2 and type 3) based on
the primary structures (Boutrot et al. 2005; Carvalho and
Gomes 2007; Yeats and Rose 2008; Arondel et al. 2000).
In the present study, ScNsLTP was categorized to plant
Fig. 5 Phylogenetic tree of ScNsLTP amino acid sequence and
type 2 NsLTPs and closely related to NtNsLTP2 from N.
homologous NsLTPs from other plants. Note HvLTP2 proteins have tabacum, which was similar to Jin et al. (2015). There was
been (7)- and (9)-indexed, with respect to their mature molecular another classification method which categorized NsLTPs
mass, because they share the same name but are different proteins from flowering plants in different types according to their
sequence similarity, intron position, as well as spacing
(Fig. 8). The expression of ScNsLTP were sharply induced between the cysteine residues (Boutrot et al. 2008; Edstam
by low temperature and PEG, which were about 3.2 times et al. 2011; Wei and Zhong 2014), which might provide a
and 7.3 times higher than that of the control under treat- different perspective but without involving in our current
ment for 24 h, while ScNsLTP transcriptional level fluc- studies.
tuated slightly under NaCl stress (Fig. 8a). The expression Previous studies showed that plant NsLTPs were widely
scenarios were complicated for the treatment of signal distributed in different types of tissues. In this study,
hormones (Fig. 8b) a slightly down-regulation in response ScNsLTP showed much higher expression level in leaf and
to exogenous hormone of SA, sharply induced by MeJA at bud (Fig. 7), though it was originally separated from stem
the latter stage of stress (24 h), while a down-regulation at cDNA library. The NsLTP from Salvia miltiorrhiza, how-
the earlier stage (6 and 12 h). Besides, under the challenge ever, was expressed at high levels in stem (Liu et al. 2011),

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Fig. 6 Structure of ScNsLTP

and homologous NsLTPs from
other plants. Note 1
a NtNsLTP2; b ScNsLTP;
c ZmNsLTP. Note 2 Schematic
representation of the cysteine
residues in red

increased compared with the control (Fig. 8a), while

FxaNsLTP a type 1 NsLTP gene in the mature strawberry
was significantly decreased under the same condition
(Mar et al. 2002). Different with CpLTP I.1, the NsLTP
gene from Chimonanthus praecox activated by NaCl (Liu
et al. 2009), ScNsLTP showed mildly change at the
expression level when exposing to the stress of NaCl
(Fig. 8a), while the expression profile of ScNsLTP under
PEG stress was similar to CpLTP I.1 though both belong
to an osmotic stress (Fig. 8a). It was revealed that
ScNsLTP played a positive role in sugarcane responded to
drought and chilling stresses, while it played a minor role
in response to salinity stress. NsLTPs were responsive to
many plant hormones, such as abscisic acid (ABA), SA
and MeJA (Wang et al. 2009; Jung et al. 2006; Guan et al.
Fig. 7 ScNsLTP expression in sugarcane different tissues. Note 1, 2,
3, 4 represent the skin of stem, the basic tissue of stem, leaf and bud, 2013). Within those hormones, SA and MeJA were con-
respectively sidered to be signaling molecules in exogenous stress
response, and they modulated signaling pathways inter-
which was inconsistent with our results. In conclusion, the acting in an opposite manner (Niki et al. 1998; Kachroo
expression of NsLTP genes at different transcriptional level et al. 2001). Similarly, ScNsLTP expression was strongly
was observed in a wide variety of tissues and at diverse induced by MeJA treatment and slightly suppressed by
stages of development together with different plant physi- exogenous SA in this study (Fig. 8b), suggesting that the
ological conditions, which revealed that a variety of regulation of ScNsLTP gene in response to abiotic stress,
diverse roles for NsLTPs in plant (Carvalho and Gomes such as drought and chilling stresses, might be mediated
2007). via the signaling molecule of MeJA. Interestingly, the
Although plant NsLTPs previously thought to be transcripts of ScNsLTP were diversity in responsive to
involved in lipid shuttling between lipid bilayers and had different exogenous stresses, implying their multitudinous
been implicated in plant defense (Garcia-Olmedo et al. functional roles. Collective results suggest that ScNsLTP
1995), whose biological function in vivo remained elusive may play an important role in plant stress resistance and
(Pagnussat et al. 2009; Wei and Zhong 2014). To date, defense to the environment.
only a tiny fraction of plant NsLTPs had been functionally In conclusion, this study reports a plant type 2 non-
identified, and even fewer had been identified in sugar- specific lipid transfer protein (ScNsLTP) gene from sug-
cane. Plant NsLTPs were used to be demonstrated as arcane, which is distributed in different types of tissues and
potent inhibitors of bacterial and fungal plant pathogens shows leaf-specific and bud-specific expression pattern. In
(Molina et al. 1993; Park et al. 2002); however, the addition, the increased expression of ScNsLTP in sugarcane
transcriptional level of ScNsLTP in this study did not suggests that the signaling pathway of MeJA-induced may
exhibit a conspicuous change in buds challenged by fun- mediate the ScNsLTP gene in response to abiotic stresses of
gal smut pathogen (Fig. 8c). It seemed that ScNsLTP was drought and chilling. The results will help to understand
more sensitive to abiotic stress in this study. The the NsLTPs functions in the stress-resistant mechanisms in
expression of ScNsLTP under chilling stress was sharply plant.

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Fig. 8 Expression patterns of ScNsLTP under different stress treatments in sugarcane

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