Overexpression VaPYL9 improves cold tolerance in tomato by regulating key genes in hormone signaling and antioxidant enzyme

Abstract

Background: Abscisic acid (ABA) has been reported in controlling plant growth and development, and particularly dominates a role in resistance to abiotic stress. The Pyrabactin Resistance1/PYR1-Like /Regulatory Components of ABA receptors (PYR1/PYL/RCAR) gene family, of which the PYL9 is a positive regulator related to stress response in ABA signaling transduction. Although the family has been identified in grape, detailed VaPYL9 function in cold stress remains unknown.

Results: In order to explore the cold tolerance mechanism in grape, VaPYL9 was cloned from Vitis amurensis. The subcellular localization showed that VaPYL9 was mainly expressed in the plasma membrane. Yeast two-hybrid (Y2H) showed VaPCMT might be a potential interaction protein of VaPYL9. Through the overexpression of VaPYL9 in tomatoes, results indicated transgenic plants had higher antioxidant enzyme activities and proline content, lower malondialdehyde (MDA) and H₂O₂ content, and improving the ability to scavenge reactive oxygen species than wild-type (WT). Additionally, ABA content and the ratio of ABA/IAA kept a higher level than WT. Quantitative real-time PCR (qRT-PCR) showed that VaPYL9, SlNCED3, SlABI5, and antioxidant enzyme genes (POD, SOD, CAT) were up-regulated in transgenic tomatoes. Transcriptome sequencing (RNA-seq) found that VaPYL9 overexpression caused the upregulation of key genes PYR/PYL, PYL4, MAPK17/18, and WRKY in transgenic tomatoes under cold stress.

Conclusion: Overexpression VaPYL9 enhances cold resistance of transgenic tomatoes mediated by improving antioxidant enzymes activity, reducing membrane damages, and regulating key genes in plant hormones signaling and antioxidant enzymes.

Keywords: Abscisic acid; Cold stress; Expression analysis; Overexpression VaPYL9; RNA-seq.

Fig. 1

Evolution analysis of PYL genes from grape, Arabidopsis thaliana, and tomato. The phylogenetic tree was constructed using PYL sequences of grape, tomato, and A. thaliana in MEGA X, which was clustered into three subgroups. Different background colors represent different subgroups. VaPYL9 marked with red ellipse was in the III class. The green circle, blue circle, and pink circle represent grapes, A. thaliana, and tomato PYL genes, respectively

Fig. 2

(a). Subcellular localization of VaPYL9. Four-weekend tobaccos were infected with bacterial liquid. After 24 h, infected leaves were observed and photoed in cell membrane and cytoplasm. (b). Yeast two -hybrid (Y2H) assay. Combinations of pGADT7-T with pGBKT7-53 and pGBKT7-Lam were served as positive and negative controls, respectively. VaPYL9 and VaPCMT were commonly transformed into Y2H gold. The result indicated that there was an interaction relationship between VaPYL9 and VaPCMT Proteins

Fig. 3

Phenotype investigation and degree of membrane damage between WT and OE. Six-week-old tomatoes were used to detect physiological indexes related to cold stress. (a). Expression level of VaPYL9 in grape under 4 ℃ stress for 0 h, 6 h, 12 h, 24 h, 48 h. (b). Expression level of VaPYL9 in WT and transgenic tomatoes under cold stress and nonstress. (c). Tomatoes phenotype was observed in WT and OE under 4 ℃ for 0 h, 48 h. (d). The determination of relative electrolyte leakage in WT and OE plants under 4 ℃ for 0 h, 6 h, 12 h, 24 h, 48 h. (e). The DAB and NBT staining of WT and OE plants under 4 ℃ for 0 h, 12 h, 48 h. (f). The content of H₂O₂ was determined in WT and OE plants under 4 ℃ for 0 h, 6 h, 12 h, 24 h, 48 h. (g). The content of MDA was determined in WT and OE plants under 4 ℃ for 0 h, 6 h, 12 h, 24 h, 48 h. (h). The content of proline was determined in WT and OE plants under 4 ℃ for 0 h, 6 h, 12 h, 24 h, 48 h. The bar represents the value of standard error (p < 0.01). Lowercase letters represent the significance level. Means and SE values were calculated from at least three independent experiments

Fig.4

The changing of antioxidant enzyme activities and endogenous hormones in WT and OE plants. Six-weekend tomatoes were used as materials to detect enzyme activities and endogenous hormones. (a). POD, SOD, and CAT enzymes activities were determined in WT and OE plants under 4 ℃ for 0 h, 6 h, 12 h, 24 h, 48 h. b. (b). The content of IAA and ABA was determined, and ABA/IAA was calculated in WT and OE plants under 4 ℃ for 0 h, 6 h, 12 h, 24 h, 48 h. The bar represents the value of standard error (p < 0.01). Lowercase letters represent the significance level. Means and SE values were calculated from at least three independent experiments

Fig. 5

qRT-PCR analysis. (a). Expression level detection of key enzyme genes related to POD, SOD, and CAT genes under 4 ℃ for 0 h, 48 h. (b). Expression level detection of NCED3 and ABI5 genes related to ABA path under 4 ℃ for 0 h, 48 h. (c). Expression level detection of MAPK17/18, GsSRK, MYC2, PR5, PYL4, PYR/PYL, WRKY key genes in RNA-seq. The bar represents the value of standard error (p < 0.01). Lowercase letters represent significance level. Means and SE values were calculated from at least three independent experiments

Fig. 6

The analysis of transcriptome sequencing in WT and OE plants under 4 ℃ for 48 h. (a) The heatmap of total differential expression genes. Rectangles in red and blue represent up and down-regulated expression. (b) The number of DEGs was shown by the volcano plot. Dots in red, green and red represent up, down and normal regulated genes, respectively. The number of the dots indicates the number of DEG. (c) The number of DEGs in main GO enrichments. (d) The expression level of key differential expression genes in MAPK path. Rectangles in red and blue represent up and down-regulated expression. (e) Expression level of differential expression genes in plant hormone signaling path. Rectangles in red and blue represent up and down-regulated expression. (f) Key differential expression genes expression in transcription factors activity. Rectangles in red and blue represent up and down-regulated expression. (g) Key differential expression genes expression in signaling receptor activity. Rectangles in red and blue represent up and down-regulated expression. (h) VaPYL9 overexpression participated in MAPK and plant hormone signaling KEGG path, and improved the activity of transcription factors and signaling receptors

Fig. 7

A systematic model of the role of VaPYL9 in the response of tomatoes under 25 ℃, 4 ℃. Green and red arrows represent down and up-regulated expression levels in WT vs OE, respectively. The size of the red areas represents the degree of leaves damage