Skip to main content
NCBI home page
Journal of Experimental Botany logoLink to Journal of Experimental Botany
. 2013 Feb 11;64(6):1755–1767. doi: 10.1093/jxb/ert040

Lipid transfer protein 3 as a target of MYB96 mediates freezing and drought stress in Arabidopsis

Lin Guo 1, Haibian Yang 1, Xiaoyan Zhang 1, Shuhua Yang 1,*
PMCID: PMC3617838  PMID: 23404903

Abstract

Several lipid-transfer proteins were reported to modulate the plant response to biotic stress; however, whether lipid-transfer proteins are also involved in abiotic stress remains unknown. This study characterized the function of a lipid-transfer protein, LTP3, during freezing and drought stress. LTP3 was expressed ubiquitously and the LTP3 protein was localized to the cytoplasm. A biochemical study showed that LTP3 was able to bind to lipids. Overexpression of LTP3 resulted in constitutively enhanced freezing tolerance without affecting the expression of CBFs and their target COR genes. Further analyses showed that LTP3 was positively regulated by MYB96 via the direct binding to the LTP3 promoter; consistently, transgenic plants overexpressing MYB96 exhibited enhanced freezing tolerance. This study also found that the loss-of-function mutant ltp3 was sensitive to drought stress, whereas overexpressing plants were drought tolerant, phenotypes reminiscent of myb96 mutant plants and MYB96-overexpressing plants. Taken together, these results demonstrate that LTP3 acts as a target of MYB96 to be involved in plant tolerance to freezing and drought stress.

Key words: Arabidopsis, drought stress, freezing tolerance, gene regulation, lipid-transfer protein 3, MYB96.

Introduction

Low temperature is the environmental factor that limits plant growth, development, and distribution. Most temperate plants have evolved mechanisms to adapt to the seasonal variations in temperature through cold acclimation in which a prior exposure to low non-freezing temperatures increases their ability to survive under subsequent freezing temperatures. Cold acclimation involves transcriptional regulation that induces cold-regulated (COR) genes, some of which encode proteins and enzymes that mediate lipid and sugar metabolism to protect plant cells from freezing injury. In Arabidopsis, most COR genes are directly regulated by the CBF/DREB (C-repeat-binding factor/dehydration-response element-binding protein) family of transcription factors (Thomashow, 1999; Yamaguchi-Shinozaki and Shinozaki, 2006). Overexpression of CBF1-CBF3 in Arabidopsis upregulates COR gene expression and concomitantly enhances freezing tolerance (Jaglo-Ottosen et al., 1998; Liu et al., 1998; Kasuga et al., 1999; Gilmour et al., 2004), and the constitutive expression of COR genes also enhances freezing tolerance (Artus et al., 1996; Steponkus et al., 1998). CBF expression is transcriptionally regulated by the transcription factors ICE1 (inducer of CBF expression 1), CAMTA3 (calmodulin-binding transcription activator 3), and MYB15 (Chinnusamy et al., 2003; Agarwal et al., 2006; Doherty et al., 2009). ICE1 is negatively regulated by an E3 ubiquitin ligase, HOS1 (high expression of osmotically responsive genes 1) and positively regulated by a SUMO E3 ligase, SIZ1 (SAP and Miz), at the post-translational level (Dong et al., 2006a; Miura et al., 2007). In addition, several other components have been identified that modulate freezing tolerance without affecting CBF expression (Xin and Browse, 1998; Gong et al., 2005; Dong et al., 2006b; Lee et al., 2006; Knight et al., 2008; Zhu et al., 2008).

Plant non-specific lipid-transfer proteins (LTPs) are small, basic, secreted proteins (Kader, 1996). The conserved features of plant LTPs include four disulphide bonds of eight cysteine residues and two pentapeptide consensus motifs (Maldonado et al., 2002). These proteins are able to transfer several different phospholipids and can bind fatty acids. There are approximately 100 LTPs or putative LTPs in Arabidopsis, and four major groups are classified based on phylogenetic analysis (Chae et al., 2009). Among them, group I includes 13 SCA (stigma/style Cys-rich adhesin)-like LTPs which are related to conventional LTPs. Several group I LTPs have been characterized. The overexpression of barley LTP4 in tobacco (Nicotiana tabacum) enhances the plant’s resistance to a bacterial pathogen (Molina and Garcia-Olmedo, 1997), and tobacco LTP2 has been shown to mediate cell-wall loosening in vitro (Nieuwland et al., 2005). Arabidopsis LTP5 is implicated in pollen tube tip growth and fertilization (Chae et al., 2009). Groups II and III are mostly as LTP-like proteins. They include DIR1 and AZI1 which function in plant defence responses (Jung et al., 2009; Maldonado et al., 2002). LTPG in group III is required for the export of lipids to the plant surface (Debono et al., 2009). However, whether LTPs function in cold stress in plants remains unclear.

LTP3 from group I is induced by abiotic stress (Seo et al., 2011). This study investigated the biological function of LTP3 by altering its expression. The overexpression of LTP3 was constitutively enhanced the freezing tolerance of Arabidopsis. Further investigation indicated that the expression of LTP3 is positively regulated by MYB96, and in vitro and in vivo assays demonstrated that MYB96 could directly bind to the promoter of LTP3. Consistently, MYB96-overexpressing transgenic plants exhibited enhanced freezing tolerance that was similar to that of LTP3-overexpressing plants. In addition, similar to myb96, ltp3 was more sensitive to drought stress than wild-type plants. These results demonstrate that LTP3 is positively regulated by MYB96 and plays important roles in the response of Arabidopsis plants to abiotic stress.

Materials and methods

Plant materials and growth conditions

Arabidopsis thaliana ecotype Col-0 plants were used in this study. The plants were grown at 20±1 °C under a 16/8h light/dark cycle at 100 µmol m–2 s–1, with 50–70% relative humidity in soil or on MS medium (Sigma) containing 2% sucrose and 0.8% agar. The ltp3-1 (SALK_095248) and myb96-2 (SAIL_815_G17) mutants were obtained from The Arabidopsis Information Resource (TAIR, http://arabidopsis.org).

For the drought-tolerance assay, water was withheld from plants that had grown in soil under normal conditions in a growth room for 2 weeks, after which, the plants were watered and the number of surviving plants was counted after 1 week. For water loss of detached leaves, leaves were detached from 3-week-old plants, placed on a Petri dish for 1, 2, 3, 4, 5, and 6h, and weighed (Hua et al., 2012). Three independent experiments were performed, with 30 plants for each line in each experiment.

Plants were treated with abscisic acid (ABA) by soaking 2-week-old seedlings in an aqueous 10 µM solution for 0, 1, 3, 6, and 12h. Similarly, in treatment with methyl viologen (MV), an oxidative stress inducer (Dodge, 1971), plants were soaked in an aqueous 10 µM solution of MV for 0, 3, 6, and 12h. There were three replications, each replicate contained 50 seedlings, and the study was repeated three times. For all studies involving RNA extraction, seedlings were then rinsed in sterile distilled water, frozen in liquid nitrogen, and stored at –80 °C.

Plasmid construction and plant transformation

MYB96 genomic DNA was amplified using primers MYB96-1 and MYB96-2 and cloned into pBI121 to generate pSuper::MYB96. MYB96 cDNA was amplified using primers MYB96-3 and MYB96-2 and cloned into the binary vector pER8 (Zuo et al., 2000) to generate pER8::MYB96. MYB96 cDNA was amplified using primers MYB96-3 and MYB96-2 and cloned into pQE-80L (Qiagen) to generate MYB96-His-expressing vector. To construct pSuper::MYB96-Myc, MYB96 cDNA was amplified using the primers MYB96-3 and MYB96-4 and subsequently cloned into the pSuper1300 vector (Yang et al., 2010) containing a 6 × Myc tag.

LTP3 cDNA was amplified using the primers LTP3-1 and LTP3-2 and cloned into pSuper1300 to generate pSuper::LTP3. LTP3 cDNA was amplified using the primers LTP3-1 and LTP3-8 and cloned into pGEX-4T-1 (Chen et al., 2009) to generate the construct LTP3-GST. LTP3 cDNA was amplified using primers LTP3-1 and LTP3-4 and fused in-frame with GFP in pSuper1300::GFP (Shi et al., 2012) to generate pSuper::LTP3-GFP. For the molecular complementation assay, a 2.0-kb genomic fragment comprising the LTP3 promoter and the coding region was cloned into pCAMBIA1300 (Cambia) to generate the pLTP3::LTP3 construct. All of the primers used are listed in Supplementary Table S1 (available at JXB online).

Agrobacterium tumefaciens GV3101 was transformed with the different constructs and used to transform wild-type, myb96-2, and ltp3-1 plants via the floral dip method (Clough and Bent, 1998).

GUS activity

A 1.7-kb genomic fragment upstream of the LTP3 ATG start codon was amplified by PCR using the LTP3-p1 and LTP3-p2 primers. The putative MYB96 binding sites were named X, Y, and Z, so the XYZ, YZ, and Z binding site regions were amplified using the primers LTP3-P11 and LTP3-P2, LTP3-P13 and LTP3-P2, and LTP3-P15 and LTP3-P2, respectively. These fragments were cloned into the plasmid pPZPGUS2 (Diener et al., 2000) to generate different pLTP3::GUS constructs.

For transient GUS assay, pLTP3::GUS vectors and pSuper::MYB96 along with 35S::LUC were coinfiltrated into Nicotiana benthamiana leaves. The cotransformation of pZPGUS2 and pSuper::MYB96 along with 35S::LUC was used as the control. The GUS activity was assayed using methylumbelliferyl glucoronide (Sigma Aldrich), and the LUC activity was used as an internal control. The GUS/LUC ratio was used to determine the promoter activity (Shi et al., 2012).

Freezing tolerance, ion leakage, and proline content assays

The freezing-tolerance assay was performed as described (Shi et al., 2012). The electrolyte leakage was measured as previously described (Lee et al., 2002). The proline content was measured as described (Bates et al., 1972).

Lipid-binding assay

Fluorescent binding assays were performed as described (Buhot et al., 2004). The purified recombinant LTP3-GST protein was mixed with various concentrations of 2-p-toluidinonaphthalene-6-sulphonate (TNS) at 25 °C. This mixture was excited at 320nm and the emission at 437nm was recorded using a spectrofluorometer (Hitachi F-7000). Purified GST was used as a control. Kd value was calculated using Sigma Plot software.

RNA extraction and real-time PCR

Total RNA was extracted from 14-day-old seedlings grown on MS plates using TRIzol (Invitrogen), followed by treatment with DNase I (Takara). The treated RNA samples were subjected to first-strand cDNA synthesis using M-MLV reverse transcriptase (Promega). Real-time PCR was performed using SYBR Green PCR Master Mix (Takara), SYBR Premix Ex Taq (Takara), and an Applied Biosystems 7500 real-time PCR system, as previously described (Huang et al., 2010).

Recombinant protein purification and electrophoretic mobility shift assay

The MYB96-His vector was expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified using Ni-agarose affinity. An electrophoretic mobility shift assay (EMSA) was performed as described (Shi et al., 2012). The XYZ, XY regions, and XY mutant region of the LTP3 promoter were amplified using primers LTP3-P3 and LTP3-P4, LTP3-P3 and LTP3-P8, and LTP3-P5 and LTP3-P6, respectively. The biotin-labelled DNA fragments listed in Supplementary Table S1 were synthesized and used as probes, and the biotin-unlabelled DNA fragments of the same sequences were used as the competitors in this assay.

Chromatin immunoprecipitation assay

A chromatin immunoprecipitation (ChIP) assay was performed as previously described (Gendrel et al., 2005) using MYB96-MYC transgenic plants. Two-week-old MYB96-MYC seedlings were fixed in 1% formaldehyde for 15min and neutralized with 0.125M glycine for an additional 5min under vacuum. After being washed twice with cold sterilized water, the tissues were homogenized, and the chromatin was isolated and sonicated. The MYB96 protein was immunoprecipitated using an anti-Myc antibody (Sigma Aldrich). The enrichment of the DNA fragments was determined using quantitative real-time PCR (qRT-PCR) with the primers listed in Supplementary Table S1. The experiments were repeated three times with similar results.

Confocal laser microscopy

Protoplasts were prepared from leaves of 4-week-old plant under a 12/12h lightdark cycle as described (Yoo et al., 2007). The fluorescence of GFP in the transformed Arabidopsis protoplasts was visualized using a confocal laser-scanning microscope (LSM510, Carl Zeiss) at 2–4 days after infiltration.

Results

Expression pattern of LTP3 and localization of LTP3

LTP3 is induced by abiotic stress (Seo et al., 2011), implying this gene might be involved in the plant responses to abiotic stress. To study the biological function of LTP3, this study first examined the tissue-specific LTP3 expression in Arabidopsis. Total RNA was extracted from various tissues, including the roots, stems, leaves, flowers, and siliques, and the transcript levels were analysed by qRT-PCR. LTP3 was strongly expressed in the cauline leaves, flowers, and siliques (Fig. 1A), a result that was verified by the GUS staining of the transgenic plants that expressed GUS driven by the LTP3 promoter. The GUS activity was high in the leaves, flowers and siliques but was low in the roots, stems, and seeds (Fig. 1B).

Fig. 1.

Fig. 1.

Open in a new tab

Expression patterns of LTP3. (A) LTP3 expression in various organs of Arabidopsis plants by qRT-PCR. Total RNA was isolated from roots (RT), four-leaf stage seedlings (SE), rosette leaves (RL), cauline leaves (CL), stems (ST), flowers (FL), mature siliques (SL), and seeds (SD). The data represent the means of three replicates ± SD. (B) GUS expression in seedlings at the two-leaf (i) and four-leaf (ii) stages, stem and cauline leaves (iii), inflorescence and flowers (iv), and mature siliques (v) of LTP3::GUS transgenic plants. (C–F) Expression of LTP3 under cold (C), drought (D), ABA (E), and MV treatment (F) by qRT-PCR. Total RNA was extracted from plants treated with cold (4 °C), drought, abscisic acid (ABA, 10 µM), or methyl viologen (MV, 10 µM) for the indicated times. The transcription levels were determined by qRT-PCR. The data represent the means of three replicates ± SD.

Next, the expression patterns of LTP3 were analysed by qRT-PCR under stress conditions. LTP3 was found to be slightly upregulated by cold stress after 3–6h treatment but was reduced thereafter (Fig. 1C). The transcript level of LTP3 was dramatically increased by dehydration and ABA treatment (Fig. 1D and E).

To determine the subcellular localization of LTP3, The pSuper::LTP3-GFP construct was generated and transformed into Arabidopsis protoplasts. The GFP signal was examined using confocal microscopy. The green fluorescence of the protoplasts harbouring pSuper::LTP3-GFP was observed throughout the cytoplasm; the control protoplasts containing pSuper::GFP were localized ubiquitously (Fig. 2A).

Fig. 2.

Fig. 2.

Open in a new tab

Subcellular localization of LTP3 in Arabidopsis protoplasts and the lipid-binding activity of LTP3. (A) pSuper::LTP3-GFP and pSuper::GFP plasmids transformed into Arabidopsis protoplasts and signals detected using a confocal laser-scanning microscope. From left to right, green fluorescence signals, chlorophyll red autofluorescence, an overlay of the green and red signals, and bright-field images. Bar: 20 µm. (B) Purified recombinant LTP3-GST binds the TNS lipophilic probe. Recombinant LTP3-GST was expressed in E. coli, purified using GST beads, and incubated with increasing concentrations of TNS. Black triangles indicate recombinant LTP3 proteins (5 µM), white circles indicate GST only, and black circles indicate the protein buffer control.

LTP3 possesses lipid-binding activity

As LTP3 is a putative lipid-transfer protein, this study examined whether LTP3 exhibited lipid-binding activity using recombinant LTP3 and a fluorescent lipid substrate. The LTP3-GST protein was expressed in E. coli, purified using GST beads, and incubated with the fluorescent probe TNS, which emits an intense fluorescence when bound in a hydrophobic condition (Debono et al., 2009). The fluorescence increased intensely with increasing concentrations of TNS, with a binding Kd of 9.8±4.7 µM (Fig. 2B). However, the fluorescence intensity did not increase with the TNS concentrations when a protein buffer or only GST was used. These results suggest that LTP3 protein has a lipid-binding activity in vitro.

Overexpression of LTP3 confers enhanced freezing tolerance

To dissect the biological function of LTP3 further, a T-DNA insertion line of LTP3 (SALK_095248), named ltp3-1, was obtained (Fig. 3A); qRT-PCR analysis showed that ltp3-1 was a loss-of-function mutant (Fig. 3B). This study also generated transgenic plants that overexpressed LTP3 driven by a Super promoter (LTP3-OE). A total of 20 independent transgenic plants were generated, and the T3 homozygous LTP3-OE3 and LTP3-OE4 plants were found to overexpress LTP3 (Fig. 3B) and were chosen for further studies. Under normal growth conditions, the ltp3-1 mutant plants and LTP3-OE plants were indistinguishable from the wild-type plants (Fig. 3C, Supplementary Fig. S1A). These results indicate that altered LTP3 expression does not significantly affect plant growth and development under normal growth conditions.

Fig. 3.

Fig. 3.

Open in a new tab

Freezing-tolerance assay of LTP3-OE plants. (A) The genomic structure of LTP3. Exons, untranscribed regions, and intron, are indicated by grey boxes, white boxes, and lines, respectively. The T-DNA position in the ltp3 mutant is shown. (B) LTP3 expression in 2-week-old ltp3-1 mutant and LTP3-overexpressing seedlings by qRT-PCR. The data represent the means of three replicates ± SD. (C) Freezing phenotypes of LTP3-OE transgenic plants. Two-week-old seedlings were treated at –5 °C for 1h (non-acclimated, NA) or –7 °C for 1h after 4 °C treatment for 4 d (acclimated, CA), and the pictures were taken after a 2-d recovery at 22 °C. (D) Survival rate of the seedlings in (C) after freezing. The surviving seedlings that could regrow were scored after a 2-d recovery. The data represent the mean values of three replicates ± SD; *P < 0.01 (Student’s t-test). At least three independent experiments were performed with similar results. (E) Ion leakage assay of the seedlings in (C) at the indicated freezing temperatures. The data are the mean values of three replicates ± SD; *P < 0.05, **P < 0.01 (Student’s t-test).

To investigate the possible role of LTP3 under stress conditions, this study first examined the freezing tolerance of the wild-type, ltp3-1, and LTP3-OE plants. Two-week-old LTP3-OE and wild-type seedlings grown on agar plates at 22 °C were subjected to a freezing treatment with or without cold acclimation, transferred to 4 °C for 12h, and then returned to the normal growth conditions for 4 d and assayed. The survival rates of the non-acclimated LTP3-OE seedlings were much higher than the wild-type seedlings after treatment at –5 and –6 °C (Fig. 3C and D). Consistently, following a cold acclimation at 4 °C for 4 days, the LTP3-OE seedlings were more tolerant to freezing than wild type after treatments at –7 and –8 °C (Fig. 3C and D). However, the ltp3-1 mutant resembled the wild-type plants with regard to freezing tolerance (Supplementary Fig. S1). Considering LTP3 shares high sequence similarity with several other LTP genes (Supplementary Fig. S2A), including LTP4, LTP8, and LTP12, this study checked whether their expression is affected in ltp3-1 mutant. Without cold treatment, expression of LTP4 and LTP12 was comparable to LTP3 in the wild type and ltp3-1 mutant. After cold treatment, the expression of LTP4 and LTP8 in ltp3-1 was higher than that in the wild type, whereas LTP12 expression in ltp3-1 was lower than the wild type (Supplementary Fig. S2B). These results suggest that the normal response of ltp3-1 mutant to freezing stress may due to the redundancy of LTP genes.

The electrolyte leakage as an indicator of membrane injury was measured in the non-acclimated and acclimated seedlings. The electrolyte leakage of the LTP3-OE seedlings was much lower than that of the wild-type seedlings in both non-acclimated and acclimated plants (Fig. 3E). These results indicate that the overexpression of LTP3 results in a constitutive freezing tolerance of plants.

Expression of stress-responsive genes in LTP3-overexpressing plants

The CBF pathway is known to play an important role in cold stress signalling (Thomashow, 1999). This current study next determined whether the enhanced freezing tolerance of LTP3-OE was caused by altered expression of cold-responsive genes in the CBF pathway. qRT-PCR analysis showed that CBF1, CBF2, CBF3, and their target genes, such as RD29A and COR47, were induced by cold in both wild-type and LTP3-OE plants; however, no obvious differences in the expression levels were observed between the wild-type and LTP3-OE plants before and after cold treatment (Supplementary Fig. S3). Therefore, LTP3 appears to be involved in freezing stress independently of the CBF pathway.

Overexpression of LTP3 increases soluble sugar accumulation

Because soluble sugar and proline are important osmolytes that protect plants against cold stress (Nanjo et al., 1999; Wanner and Junttila, 1999), the soluble sugar and proline contents were measured in wild-type and LTP3-OE plants. The LTP3-OE plants accumulated more soluble sugar than the wild-type plants at 22 °C (Supplementary Fig. S4A). Cold stress induced the accumulation of soluble sugar in the wild-type plants, and the increase in the soluble sugar content in the LTP3-OE plants was higher compared to the wild type after cold treatment (Supplementary Fig. S4A). In contrast, no notable differences in the proline levels were detected between the LTP3-OE and wild-type plants with or without cold treatment (Supplementary Fig. S4B).

Alteration in LTP3 expression affects plant responses to drought and oxidative stress

To investigate the role of LTP3 in other abiotic stress, this study next examined the effect of LTP3 on drought and oxidative stress. Two-week-old seedlings of ltp3-1, LTP3-OE, and wild type were exposed to dehydration by withholding water for 2 weeks. The plants were then watered and cultured continually for 1 week and the plant survival rates were measured. The ltp3-1 mutant was more sensitive to drought than the wild type, whereas the LTP3-OE plants exhibited an enhanced tolerance to drought (Fig. 4A and B). Moreover, ltp3-1 lost more water, whereas LTP3-OE lost less water than the wild type in the water loss assay of detached leaves (Fig. 4C). To determine whether the drought sensitivity of ltp3-1 was caused by the disruption of LTP3, a 2.0-kb wild-type genomic fragment containing LTP3 under the control of its own promoter was transformed into the ltp3-1 mutant. The transgene fully restored the drought-sensitive phenotype of the ltp3-1 mutant (Fig. 4A and C), indicating that the ltp3 mutation is responsible for the drought sensitivity. Taken together, these results demonstrate that LTP3 positively regulates the plant response to drought stress.

Fig. 4.

Fig. 4.

Open in a new tab

Phenotypes of ltp3-1 and LTP3-OE plants under drought and oxidative stress. (A) Drought-tolerance assay of the wild type, ltp3-1, LTP3-OE, and ltp3-1/LTP3 complemented plants. The plants were grown in soil, deprived of water for 2 weeks, and then watered. Similar results were observed in three independent experiments. (B) Survival rate of the plants in (A) under drought stress. The data presented are the means ± SD (n = 30); *P < 0.05 (Student’s t-test). Similar results were observed in three independent experiments. (C) Water loss of detached leaves of the wild type, ltp3-1, LTP3-OE, and ltp3-1/LTP3 complemented plants. The data presented are the means ± SD of three replicates (n = 30 for each experiment); *P < 0.05 (Student’s t-test). (D, E) Phenotype of ltp3-1 plants under methyl viologen treatment. The wild-type Col and ltp3-1 seeds were germinated at 22 °C on MS medium containing 0–0.5 µM methyl viologen. The photographs in D were taken on day 7 after stratification. The germination rates (E) are the means ± SD of three experiments (n = 30 for each experiment); *P < 0.05 (Student’s t-test).

The ltp3-1 mutant exhibited a hypersensitive phenotype when germinated on MS media containing different concentrations of MV, an oxidative stress inducer (Fig. 4D), and the germination rates of ltp3-1 were much lower than the wild type on MV-containing media (Fig. 4E). These observations, together with the strong induction of LTP3 by MV (Fig. 1F), suggest that LTP3 plays a positive role in the plant responses to oxidative stress.

MYB96 directly regulates LTP3 expression

To identify direct upstream regulators of LTP3, this study searched the publicly available data and found that LTP3 expression is decreased in a myb96-1 mutant and increased in a myb96-1D mutant that overexpresses MYB96 (Seo et al., 2011). This study obtained a loss-of-function mutant allele, named myb96-2, from TAIR and generated MYB96-overexpressing (MYB96-OE3 and MYB96-OE4) lines by transforming the pSuper::MYB96 plasmid into the Col-0 background (Fig. 5A). The expression of LTP3 was dramatically downregulated in the myb96-2 mutant and upregulated in the MYB96-OE plants compared to the wild type (Fig. 5A). Further MYB96-overexpressing lines were generated, in which MYB96 was driven by an inducible promoter (Zuo et al., 2000). MYB96 was dramatically upregulated after treatment with β-oestradiol, and LTP3 was induced accordingly (Fig. 5B). These results imply that LTP3 might be a direct target of the MYB96 protein.

Fig. 5.

Fig. 5.

Open in a new tab

LTP3 is a direct target of MYB96. (A) Expression of LTP3 and MYB96 in 2-week-old wild-type Col, myb96-2, and MYB96-OE plants by qRT-PCR. The data are the means of three replicates ± SD. (B) Expression of LTP3 and MYB96 in the transgenic plants expressing MYB96 under an oestradiol-inducible promoter with or without 5 µM β-oestradiol treatment for 6h. (C) Schematic diagrams showing the promoter structure of LTP3. The 1.5-kb upstream sequence is shown. X, Y, and Z show the putative MYB96 binding sites, and the translation start site (ATG) is shown at position +1. (D) ChIP assay of MYB96-MYC binding to the LTP3 promoter. The MYB96-binding sites are indicated in (C) and the primers used for the ChIP-qRT-PCR are listed in detail in Supplementary Table S1. A coding region of LTP3 was amplified as a control. The data are the means of three replicates ± SD from one experiment. At least three independent experiments were performed with similar results. (E) Transient expression of different pLTP3::GUS vectors with pSuper::MYB96 in Nicotiana benthamiana leaves. The cotransformation of pZPGUS2 with the pSuper::MYB96 vector was used as the control. The data are the means of three replicates ± SD. (F) EMSA assay for MYB96 binding to the promoters of LTP3. Each biotin-labelled DNA fragment was incubated with the MYB96-His protein. A competition assay for the labelled promoter sequences was performed by adding an excess of unlabelled wild-type or mutated probes.

MYB96 can bind to the LTP3 promoter in vivo

MYB proteins can recognize the consensus CAGTTN motif in the promoters of their target genes (Urao et al., 1993). A sequence analysis revealed that the promoter region of LTP3 includes three CAGTTA elements, named the X, Y, and Z sites, respectively, within a 1.1-kb region upstream of the translation start site (Fig. 5C). ChIP was performed to determine whether MYB96 was able to bind directly to the LTP3 promoter in vivo. This study generated transgenic plants that expressed the MYB96-Myc fusion protein under the Super promoter, and the protein-DNA complexes were purified using an anti-Myc antibody. qRT-PCR was then used to detect whether the MYB96 protein can bind to the LTP3 promoter. As shown in Fig. 5D, the MYB96 protein strongly interacted with the fragment that contained the Y binding site but not the fragments that contained the X or Z binding sites or the exon region, implying that the Y binding site is important for the binding of MYB96 to the LTP3 promoter.

Then the tobacco transient cotransformation system that expressed the pSuper::MYB96 and pLTP3::GUS constructs was used to detect whether MYB96 can regulate LTP3 expression. pLTP3::GUS constructs driven by a series of LTP3 promoter fragments that contained different MYB96 binding sites were generated. In the presence of MYB96, the GUS activity increased significantly when the LTP3 promoter included the YZ or XYZ binding sites. However, a low GUS activity was detected in the LTP3 promoters that only contained the Z site or that did not contain an MYB binding site (Fig. 5E). Together, these results demonstrate that the MYB96 protein can specifically bind to the LTP3 promoter in vivo.

MYB96 can bind the LTP3 promoter in vitro

An EMSA was performed to test whether the MYB96 protein binds directly to the LTP3 promoter in vitro. Recombinant MYB96-His protein from E. coli was purified and incubated with biotin-labelled probes. A shifted DNA-binding band was detected with the addition of MYB96-His and labelled DNA probes containing XYZ or XY sites, but no band was detected in the absence of the MYB96-His protein. The DNA binding was abolished with increasing amounts of unlabelled probe (Fig. 5F). To determine the binding specificity, the mutated XY-containing probe in which the CAGTTA sequence was changed to ACGTAT was used; no DNA binding was detected after incubation with the MYB96-His protein (Fig. 5F). These results indicate that MYB96 specifically binds in vitro to the LTP3 promoter containing the CAGTTA element.

MYB96-OE plants show LTP3-OE-like enhanced freezing tolerance

MYB96 is induced by different stress conditions, including ABA, drought, and salt stress (Seo et al., 2009). Therefore, this study addressed whether MYB96 was induced by cold stress. qRT-PCR analysis showed that MYB96 could be slightly induced by cold conditions: the expression after 3-h cold treatment increased 2-fold over the expression at 0h, and the expression decreased thereafter, returning to the basal level after 12h under cold conditions (Fig. 6A).

Fig. 6.

Fig. 6.

Open in a new tab

Freezing tolerance is enhanced by the overexpression of MYB96. (A) Expression of MYB96 under cold by qRT-PCR. Total RNA was extracted from plants treated at 4 °C for the indicated times. The data represent the means of three replicates ± SD. (B) Freezing phenotypes of MYB96-OE transgenic plants. Two-week-old light-grown seedlings were treated at –5 °C for 1h (NA) or –7 °C for 1h after 4 °C treatment for 4 d (CA), and the surviving seedlings were scored after a 2-d recovery. (C) Survival rate of the seedlings in (B) after freezing. The data represent the means of three replicates ± SD; *P < 0.01 (Student’s t-test). At least three independent experiments were performed with similar results. (D) Ion leakage assay of the seedlings in (B) at the indicated freezing temperatures. The data are means of three replicates ± SD; *P < 0.01 (Student’s t-test). (E) Expression of CBF genes in MYB96-OE plants by qRT-PCR. Two-week-old seedlings grown at 22 °C were treated at 4 °C for 3h. The data represent the means of three replicates ± SD.

To explore the role of MYB96 in freezing tolerance, the survival rates of the wild-type and MYB96-OE seedlings after freezing treatment were determined. The non-acclimated MYB96-OE lines were more tolerant to freezing than the wild type after treatment at –5 °C (Fig. 6B). The survival rates for the MYB96-OE3 and MYB96-OE4 seedlings were 61 and 59%, respectively, whereas only 27% of the wild-type plants survived (Fig. 6C). A similar freezing-tolerant phenotype was observed in the acclimated MYB96-OE plants after being subjected to a freezing treatment (Fig. 6B and C). The electrolyte leakage was dramatically reduced in the acclimated and non-acclimated MYB96-OE plants versus the wild-type plants after the freezing treatment (Fig. 6D).

Furthermore, the expression patterns of CBF1-CBF3 in the MYB96-OE plants were not substantially altered compared to the wild type (Fig. 6E). Thus, MYB96 is involved in the plant response to freezing stress and is largely independent of the CBF genes.

Overexpression of LTP3 restores sensitivity to drought stress in the myb96 mutant

To investigate the genetic interaction of LTP3 and MYB96 further, this study generated myb96-2 LTP3-OE plants by transforming the pSuper::LTP3 plasmid into myb96-2 plants. The expression of LTP3 in the myb96-2 LTP3-OE mutant was higher than that in the myb96-2 plants, as determined by qRT-PCR (Fig. 7A). Notably, the constitutive expression of LTP3 fully restored the drought-sensitive phenotype of myb96-2 into the drought-tolerant phenotype of the wild-type plants (Fig. 7B and C). These results indicate that MYB96 acts upstream of LTP3 to function in drought tolerance.

Fig. 7.

Fig. 7.

Open in a new tab

Phenotypes of myb96-2 LTP3-OE plants under drought stress. (A) LTP3 expression in 2-week-old wild-type Col, LTP3-OE3, myb96-2, and myb96-2 LTP3-OE plants, as determined by semi-quantitative RT-PCR. (B) Drought-tolerance assay of the wild-type Col, LTP3-OE3, myb96-2, and myb96-2 LTP3-OE plants. The plants were grown in soil, deprived of water for 2 weeks, and then watered. Similar results were observed in three independent experiments. (C) Survival rate of the plants in (B) under drought stress. The data presented are the means ± SD (n = 30); *P < 0.05 (Student’s t-test). Similar results were observed in three independent experiments.

Discussion

This study found that the overexpression of LTP3 could constitutively enhance the tolerance to freezing stress. Under cold treatment, there are numerous physiological and biochemical changes in plants, including the expression of CBF genes, CBF regulons, a transient increase in the ABA level, changes in the membrane lipid composition, the accumulation of compatible osmolytes such as soluble sugars and proline, and increased levels of antioxidants (Thomashow, 1999; Xin and Browse, 2000). The expression of CBF1–CBF3 and their regulons was not notably affected in the LTP3-OE plants; however, the accumulation of soluble sugars in the LTP3-OE plants was dramatically increased compared to the wild-type plants. Therefore, the LTP3-mediated freezing tolerance appears to be at least partially attributed to the accumulation of soluble sugars but is uncoupled from the CBF pathway.

Little is known about how plants perceive changes in temperature or transfer a temperature signal. One hypothesis is that the fluidity of the cell membranes plays a key role in the perception of cold temperatures and the subsequent signal transduction (Murata and Los, 1997). The unsaturation of the lipids in thylakoid membranes has been shown to protect the photosynthetic machinery from photoinhibition at low temperatures (Nishida and Murata, 1996). The PLDα protein is the key enzyme involved in the biosynthesis of lipids. PLDα-deficient mutants, with lower levels of phosphatidylcholine and higher levels of phosphatidic acid than the wild type, display a destabilization of the membrane bilayer structure and improved tolerance to freezing stress (Welti et al., 2002). Because cellular membranes are thought to be the primary sites of damage due to cold stress, changes in the membrane behaviour under cold conditions must be critical to the development of freezing tolerance. Several plant LTPs are known to interact with lipids and fatty acids in vitro without ligand specificity (Zachowski et al., 1998; Guerbette et al., 1999; Hamilton, 2004). They may function as lipid carriers between intracellular organelles (Kader et al., 1984), and play a role in the delivery of wax components during the assembly of the cuticle (Sterk et al., 1991; Yeats and Rose, 2008; Debono et al., 2009). This study found that the overexpression of LTP3 results in a constitutively enhanced tolerance to freezing stress. As LTP3 is able to bind to lipids in an in vitro assay and it is localized in cytosol, it is hypothesized that LTP3 might act as a co-signal of lipids and bind and transfer these lipids from cytosol to the cell membrane or cell wall to form cuticular wax, thus protecting the plants against adverse environmental conditions, including freezing and drought stress. It is also possible that certain changes in the content and species of lipids might occur when overexpressed LTP3 gene in Arabidopsis, thereby affecting the responses of Arabidopsis plants to environmental stimuli. The type of lipids to which LTP3 binds and transfers remains to be investigated.

Several MYB proteins are reported to be involved in abiotic stress in Arabidopsis. For instance, MYB15 is reported to regulate CBF expression, thereby modulating freezing tolerance (Agarwal et al., 2006). MYB44 plays a role in an ABA-mediated signalling pathway that confers enhanced abiotic stress tolerance via the enhancement of stomatal closure (Jung et al., 2008), and MYB60 acts to regulate stomatal movement (Cominelli et al., 2005). MYB96 is involved in multiple physiological processes, including stomatal aperture, root development, pathogen infection, and cuticular wax accumulation. MYB96 is also suggested to act in the ABA-signalling pathway to regulate stomatal movement, thus regulating plant drought tolerance (Seo et al., 2009). It has also been shown that MYB96 participates in disease resistance through the salicylic acid-signalling pathway (Seo and Park, 2010). A recent study showed that MYB96 regulates cuticular wax biosynthesis by directly binding to the promoters of genes that encode very-long-chain fatty acid-condensing enzymes (Seo et al., 2011).

This study provides several lines of evidence showing that LTP3 is the direct target of the MYB96 protein in the positive regulation of the plant responses to freezing and drought stress. LTP3 expression was upregulated in the MYB96-OE plants and downregulated in the myb96 mutant. Further study showed that MYB96 directly bound to the promoter of LTP3 in vitro and in vivo. Consistent with this, the overexpression of LTP3 and MYB96 displayed similar enhanced freezing tolerance and drought tolerance, whereas the ltp3-1 mutant showed myb96-like drought-sensitive phenotypes. Furthermore, the overexpression of LTP3 in the myb96 mutant compromised the drought-sensitive phenotype of the myb96 mutant. These results add more evidence to support the previous finding that MYB96 transcription activator confers drought tolerance by modulating cuticular wax biosysnthesis (Seo et al., 2011). It is noteworthy that overexpression of MYB96 causes severe morphological defects such as dwarfed stature with altered leaf morphology, whereas overexpression of LTP3 resembles the wild type in normal conditions. This difference might be because that LTP3 is just one of MYB96 targets. The accumulation of cuticular wax in MYB96-overexpressing plants might also account for its enhanced freezing tolerance; however, this study did not observe obvious changes in accumulation of cuticular wax on the leaf surface of LTP3-overexpressing plants. Further investigation of molecular mechanism of LTP3 will shed more light on its function in environmental stress responses.

Supplementary material

Supplementary data are available at JXB online.

Supplementary Fig. S1. Response of ltp3-1 mutant to cold stress.

Supplementary Fig. S2. The effect of LTP3 on expression of its homologues.

Supplementary Fig. S3. Expression of cold-responsive genes in LTP3-OE plants.

Supplementary Fig. S4. Soluble sugar content and proline contents in LTP3-OE plants.

Supplementary Table S1. Primer sequences used in this study.

Supplementary Data
supp_64_6_1755__index.html (971B, html)

Acknowledgements

We thank Nam-Hai Chua (The Rockefeller University) for providing the pER8 vector, and Jian Hu (China Agricultural University) for sharing the ltp3-1 seeds. This work was supported by China National Funds for Distinguished Young Scientists (31225003), the National Basic Research Program of China (2009CB119100), the National Natural Science Foundation of China (31121002), and the Ministry of Agriculture of China for transgenic research (2011ZX08009–003–002).

References

  1. Agarwal M, Hao Y, Kapoor A, Dong CH, Fujii H, Zheng X, Zhu JK. 2006. A R2R3 type MYB transcription factor is involved in the cold regulation of CBF genes and in acquired freezing tolerance. Journal of Biological Chemistry 281,37636–37645 [DOI] [PubMed] [Google Scholar]
  2. Artus NN, Uemura M, Steponkus PL, Gilmour SJ, Lin C, Thomashow MF. 1996. Constitutive expression of the cold-regulated Arabidopsis thaliana COR15a gene affects both chloroplast and protoplast freezing tolerance. Proceedings of the National Academy of Sciences, USA 93,13404–13409 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bates LS, Waldren RP, Teare ID. 1972. Rapid determination of free proline for water-stress studies. Plant and Soil 39,205–207 [Google Scholar]
  4. Buhot N, Gomes E, Milat ML, Ponchet M, Marion D, Lequeu J, Delrot S, Coutos-Thevenot P, Blein JP. 2004. Modulation of the biological activity of a tobacco LTP1 by lipid complexation. Molecular Biology of the Cell 15, 5047–5052 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chae K, Kieslich CA, Morikis D, Kim SC, Lord EM. 2009. A gain-of-function mutation of Arabidopsis lipid transfer protein 5 disturbs pollen tube tip growth and fertilization. The Plant Cell 21, 3902–3914 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen H, Xue L, Chintamanani S, et al. 2009. ETHYLENE INSENSITIVE3 and ETHYLENE INSENSITIVE3-LIKE1 repress SALICYLIC ACID INDUCTION DEFICIENT2 expression to negatively regulate plant innate immunity in Arabidopsis. The Plant Cell 21, 2527–2540 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chinnusamy V, Ohta M, Kanrar S, Lee BH, Hong X, Agarwal M, Zhu JK. 2003. ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis. Genes and Development 17, 1043–1054 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clough SJ, Bent AF. 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal 16, 735–743 [DOI] [PubMed] [Google Scholar]
  9. Cominelli E, Galbiati M, Vavasseur A, Conti L, Sala T, Vuylsteke M, Leonhardt N, Dellaporta SL, Tonelli C. 2005. A guard-cell-specific MYB transcription factor regulates stomatal movements and plant drought tolerance. Current Biology 15, 1196–1200 [DOI] [PubMed] [Google Scholar]
  10. Debono A, Yeats TH, Rose JK, Bird D, Jetter R, Kunst L, Samuels L. 2009. Arabidopsis LTPG is a glycosylphosphatidylinositol-anchored lipid transfer protein required for export of lipids to the plant surface. The Plant Cell 21, 1230–1238 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Diener AC, Li H, Zhou W, Whoriskey WJ, Nes WD, Fink GR. 2000. Sterol methyltransferase 1 controls the level of cholesterol in plants. The Plant Cell 12, 853–870 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dodge AD. 1971. The mode of action of the bipyridylium herbicides, paraquat and diquat. Endeavor 30, 130–135 [DOI] [PubMed] [Google Scholar]
  13. Doherty CJ, Van, Buskirk HA, Myers SJ, Thomashow MF. 2009. Roles for Arabidopsis CAMTA transcription factors in cold-regulated gene expression and freezing tolerance. The Plant Cell 21, 972–984 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dong CH, Agarwal M, Zhang Y, Xie Q, Zhu JK. 2006a. The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1. Proceedings of the National Academy of Sciences, USA 103, 8281–8286 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dong CH, Hu X, Tang W, Zheng X, Kim YS, Lee BH, Zhu JK. 2006b. A putative Arabidopsis nucleoporin, AtNUP160, is critical for RNA export and required for plant tolerance to cold stress. Molecular and Cellular Biology 26, 9533–9543 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gendrel AV, Lippman Z, Martienssen R, Colot V. 2005. Profiling histone modification patterns in plants using genomic tiling microarrays. Nature Methods 2, 213–218 [DOI] [PubMed] [Google Scholar]
  17. Gilmour SJ, Fowler SG, Thomashow MF. 2004. Arabidopsis transcriptional activators CBF1, CBF2, and CBF3 have matching functional activities. Plant Molecular Biology 54, 767–781 [DOI] [PubMed] [Google Scholar]
  18. Gong Z, Dong CH, Lee H, Zhu J, Xiong L, Gong D, Stevenson B, Zhu JK. 2005. A DEAD box RNA helicase is essential for mRNA export and important for development and stress responses in Arabidopsis. The Plant Cell 17, 256–267 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Guerbette F, Grosbois M, Jolliot-Croquin A, Kader JC, Zachowski A. 1999. Lipid-transfer proteins from plants: structure and binding properties. Molecular and Cellular Biochemistry 192, 157–161 [PubMed] [Google Scholar]
  20. Hamilton JA. 2004. Fatty acid interactions with proteins: what X-ray crystal and NMR solution structures tell us. Progress in Lipid Research 43, 177–199 [DOI] [PubMed] [Google Scholar]
  21. Hua D, Wang C, He J, Liao H, Duan Y, Zhu Z, Guo Y, Chen Z, Gong Z. 2012. A plasma membrane receptor kinase, GHR1, mediates abscisic acid- and hydrogen peroxide-regulated stomatal movement in Arabidopsis. The Plant Cell 24, 2546–2561 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Huang X, Li J, Bao F, Zhang X, Yang S. 2010. A gain-of-function mutation in the Arabidopsis disease resistance gene RPP4 confers sensitivity to low temperature. Plant Physiology 154, 796–809 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jaglo-Ottosen KR, Gilmour SJ, Zarka DG, Schabenberger O, Thomashow MF. 1998. Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance. Science 280, 104–106 [DOI] [PubMed] [Google Scholar]
  24. Jung C, Seo JS, Han SW, Koo YJ, Kim CH, Song SI, Nahm BH, Choi YD, Cheong JJ. 2008. Overexpression of AtMYB44 enhances stomatal closure to confer abiotic stress tolerance in transgenic Arabidopsis. Plant Physiology 146, 623–635 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Jung HW, Tschaplinski TJ, Wang L, Glazebrook J, Greenberg JT. 2009. Priming in systemic plant immunity. Science 324, 89–91 [DOI] [PubMed] [Google Scholar]
  26. Kader JC. 1996. Lipid-transfer proteins in plants. Annual Review of Plant Physiology and Plant Molecular Biology 47, 627–654 [DOI] [PubMed] [Google Scholar]
  27. Kader JC, Julienne M, Vergnolle C. 1984. Purification and characterization of a spinach-leaf protein capable of transferring phospholipids from liposomes to mitochondria or chloroplasts. European Journal of Biochemistry 139, 411–416 [DOI] [PubMed] [Google Scholar]
  28. Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K. 1999. Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nature Biotechnology 17, 287–291 [DOI] [PubMed] [Google Scholar]
  29. Knight H, Mugford SG, Ulker B, Gao D, Thorlby G, Knight MR. 2008. Identification of SFR6, a key component in cold acclimation acting post-translationally on CBF function. The Plant Journal 58, 97–108 [DOI] [PubMed] [Google Scholar]
  30. Lee BH, Kapoor A, Zhu J, Zhu JK. 2006. STABILIZED1, a stress-upregulated nuclear protein, is required for pre-mRNA splicing, mRNA turnover, and stress tolerance in Arabidopsis. The Plant Cell 18, 1736–1749 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lee H, Guo Y, Ohta M, Xiong L, Stevenson B, Zhu JK. 2002. LOS2, a genetic locus required for cold-responsive gene transcription encodes a bi-functional enolase. EMBO Journal 21, 2692–2702 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki K, Shinozaki K. 1998. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. The Plant Cell 10, 1391–1406 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Maldonado AM, Doerner P, Dixon RA, Lamb CJ, Cameron RK. 2002. A putative lipid transfer protein involved in systemic resistance signalling in Arabidopsis. Nature 419, 399–403 [DOI] [PubMed] [Google Scholar]
  34. Miura K, Jin JB, Lee J, Yoo CY, Stirm V, Miura T, Ashworth EN, Bressan RA, Yun DJ, Hasegawa PM. 2007. SIZ1-mediated sumoylation of ICE1 controls CBF3/DREB1A expression and freezing tolerance in Arabidopsis. The Plant Cell 19, 1403–1414 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Molina A, Garcia-Olmedo F. 1997. Enhanced tolerance to bacterial pathogens caused by the transgenic expression of barley lipid transfer protein LTP2. The Plant Journal 12, 669–675 [DOI] [PubMed] [Google Scholar]
  36. Murata N, Los DA. 1997. Membrane fluidity and temperature perception. Plant Physiology 115, 875–879 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nanjo T, Kobayashi M, Yoshiba Y, Sanada Y, Wada K, Tsukaya H, Kakubari Y, Yamaguchi-Shinozaki K, Shinozaki K. 1999. Biological functions of proline in morphogenesis and osmotolerance revealed in antisense transgenic Arabidopsis thaliana. The Plant Journal 18, 185–193 [DOI] [PubMed] [Google Scholar]
  38. Nieuwland J, Feron R, Huisman BA, Fasolino A, Hilbers CW, Derksen J, Mariani C. 2005. Lipid transfer proteins enhance cell wall extension in tobacco. The Plant Cell 17, 2009–2019 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nishida I, Murata N. 1996. Chilling sensitivity in plants and cyanobacteria: The crucial contribution of membrane lipids. Annual Review of Plant Physiology and Plant Molecular Biology 47, 541–568 [DOI] [PubMed] [Google Scholar]
  40. Seo PJ, Lee SB, Suh MC, Park MJ, Go YS, Park CM. 2011. The MYB96 transcription factor regulates cuticular wax biosynthesis under drought conditions in Arabidopsis. The Plant Cell 23, 1138–1152 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Seo PJ, Park CM. 2010. MYB96-mediated abscisic acid signals induce pathogen resistance response by promoting salicylic acid biosynthesis in Arabidopsis. New Phytologist 186, 471–483 [DOI] [PubMed] [Google Scholar]
  42. Seo PJ, Xiang F, Qiao M, Park JY, Lee YN, Kim SG, Lee YH, Park WJ, Park CM. 2009. The MYB96 transcription factor mediates abscisic acid signaling during drought stress response in Arabidopsis. Plant Physiology 151, 275–289 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Shi Y, Tian S, Hou L, Huang X, Zhang X, Guo H, Yang S. 2012. Ethylene signaling negatively regulates freezing tolerance by repressing expression of CBF and type-A ARR genes in Arabidopsis. The Plant Cell 24, 2578–2595 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Steponkus PL, Uemura M, Joseph RA, Gilmour SJ, Thomashow MF. 1998. Mode of action of the COR15a gene on the freezing tolerance of Arabidopsis thaliana. Proceedings of the National Academy of Sciences, USA 95, 14570–14575 [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sterk P, Booij H, Schellekens GA, Van, Kammen A, De Vries SC. 1991. Cell-specific expression of the carrot EP2 lipid transfer protein gene. The Plant Cell 3, 907–921 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Thomashow MF. 1999. PLANT COLD ACCLIMATION: Freezing tolerance genes and regulatory mechanisms. Annual Review of Plant Physiology and Plant Molecular Biology 50, 571–599 [DOI] [PubMed] [Google Scholar]
  47. Urao T, Yamaguchi-Shinozaki K, Urao S, Shinozaki K. 1993. An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence. The Plant Cell 5, 1529–1539 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wanner LA, Junttila O. 1999. Cold-induced freezing tolerance in Arabidopsis. Plant Physiology 120, 391–400 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Welti R, Li W, Li M, Sang Y, Biesiada H, Zhou HE, Rajashekar CB, Williams TD, Wang X. 2002. Profiling membrane lipids in plant stress responses. Role of phospholipase D alpha in freezing-induced lipid changes in Arabidopsis. Journal of Biological Chemistry 277, 31994–32002 [DOI] [PubMed] [Google Scholar]
  50. Xin Z, Browse J. 1998. Eskimo1 mutants of Arabidopsis are constitutively freezing-tolerant. Proceedings of the National Academy of Sciences, USA 95, 7799–7804 [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Xin Z, Browse J. 2000. Cold comfort farm: the acclimation of plants to freezing temperatures. Plant, Cell and Environment 23, 893–902 [Google Scholar]
  52. Yamaguchi-Shinozaki K, Shinozaki K. 2006. Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annual Review of Plant Biology 57, 781–803 [DOI] [PubMed] [Google Scholar]
  53. Yang H, Shi Y, Liu J, Guo L, Zhang X, Yang S. 2010. A mutant CHS3 protein with TIR-NB-LRR-LIM domains modulates growth, cell death and freezing tolerance in a temperature-dependent manner in Arabidopsis. The Plant Journal 63, 283–296 [DOI] [PubMed] [Google Scholar]
  54. Yeats TH, Rose JK. 2008. The biochemistry and biology of extracellular plant lipid-transfer proteins (LTPs). Protein Science 17, 191–198 [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Yoo SD, Cho YH, Sheen J. 2007. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocols 2, 1565–1572 [DOI] [PubMed] [Google Scholar]
  56. Zachowski A, Guerbette F, Grosbois M, Jolliot-Croquin A, Kader JC. 1998. Characterisation of acyl binding by a plant lipid-transfer protein. European Journal of Biochemistry 257, 443–448 [DOI] [PubMed] [Google Scholar]
  57. Zhu J, Jeong JC, Zhu Y, et al. 2008. Involvement of Arabidopsis HOS15 in histone deacetylation and cold tolerance. Proceedings of the National Academy of Sciences, USA 105, 4945–4950 [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Zuo J, Niu QW, Chua NH. 2000. An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. The Plant Journal 24, 265–273 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Data
supp_64_6_1755__index.html (971B, html)
supp_ert040_jexbot090571_file001.pdf (269.1KB, pdf)

Articles from Journal of Experimental Botany are provided here courtesy of Oxford University Press

RESOURCES