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

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.

Fig. 1.

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.

Fig. 2.

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.

Fig. 3.

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).

Fig. 4.

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).

Fig. 5.

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.

Fig. 6.

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.

Fig. 7.

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.