Molecular Evolution of SNAREs in Vitis vinifera and Expression Analysis under Phytohormones and Abiotic Stress

Abstract

SNARE proteins (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) play a key role in mediating a variety of plant biological processes. Currently, the function of the SNARE gene family in phytohormonal and abiotic stress treatments in grapevine is currently unknown, making it worthwhile to characterize and analyze the function and expression of this family in grapevine. In the present study, 52 VvSNARE genes were identified and predominantly distributed on 18 chromosomes. Secondary structures showed that the VvSNARE genes family irregular random coils and α-helices. The promoter regions of the VvSNARE genes were enriched for light-, abiotic-stress-, and hormone-responsive elements. Intraspecific collinearity analysis identified 10 pairs collinear genes within the VvSNARE family and unveiled a greater number of collinear genes between grapevine and apple, as well as Arabidopsis thaliana, but less associations with Oryza sativa. Quantitative real-time PCR (qRT-PCR) analyses showed that the VvSNARE genes have response to treatments with ABA, NaCl, PEG, and 4 °C. Notably, VvSNARE2, VvSNARE14, VvSNARE15, and VvSNARE17 showed up-regulation in response to ABA treatment. VvSNARE2, VvSNARE15, VvSNARE18, VvSNARE19, VvSNARE20, VvSNARE24, VvSNARE25, and VvSNARE29 exhibited significant up-regulation when exposed to NaCl treatment. The PEG treatment led to significant down-regulation of VvSNARE1, VvSNARE8, VvSNARE23, VvSNARE25, VvSNARE26, VvSNARE31, and VvSNARE49 gene expression. The expression levels of VvSNARE37, VvSNARE44, and VvSNARE46 were significantly enhanced after exposure to 4 °C treatment. Furthermore, subcellular localization assays certified that VvSNARE37, VvSNARE44, and VvSNARE46 were specifically localized at the cell membrane. Overall, this study showed the critical role of the VvSNARE genes family in the abiotic stress response of grapevines, thereby providing novel candidate genes such as VvSNARE37, VvSNARE44, and VvSNARE46 for further exploration in grapevine stress tolerance research.

Keywords: SNARE gene family; abiotic stress; grapevine; qRT-PCR; subcellular localization.

Figure 1

Phylogenetic analysis of the SNARE gene family in grapevine. Phylogenetic trees were constructed using the SNARE protein sequences. I–IV represent different subgroups, respectively. The NJ method was used, and the bootstrap value was set to 1000.

Figure 2

Gene structure analysis of SNARE gene family in grapevine. The exon–intron structure of VvSNARE genes. Exon CDS, upstream/downstream, and intron are indicated by the green boxes, pink boxes, and black lines, respectively. The scale bar represents 1 kb (right).

Figure 3

Cis-regulatory element analysis of the VvSNARE genes. Different colors on the right represent different elements. the numbers in the figure represent the number of cis acting elements.

Figure 4

(A,B) Analysis of the collinear of the SNARE gene family in grapevines. (A) Collinearity analysis of VvSNARE. The gray lines represent all collinear blocks in the grape genome, and the red lines represent gene pairs between the VvSNARE genes. (B) Collinearity analysis of SNARE genes in grapevines and three representative plants. The gray lines in the background show collinearity between the V. vinifera and A. thaliana, M. domestica, and O. sativa genomes. The red lines show collinearity between A. thaliana and V. vinifera, M. domestica and V. vinifera, and Oryza sativa and V. vinifera for VvSNARE genes.

Figure 5

(A) Codon parameter analysis of the SNARE genes in grapevines. “A3s, G3s, C3s and T3s” refers to the synonymous codon corresponding base frequency on the third. “CAI” refers to the codon adaptation index, “CBI” refers to the codon bias index, “FOP” refers to the frequency of optimal codons, “ENc” refers to the effective number of codons, “GC3s” refers to the amount of the third codon (G + C), and “GC” refers to the count of genes (G + C). (B) Tissue differential expression of VvSNARE. Heatmap experiments were performed with GeneChip microarrays obtained from the grape tissue expression database. Red or blue shading represents the expression level—significant or insignificant, respectively. The scale indicates the relative expression level.

Figure 6

(A,B) Relative expression levels of SNARE genes in grape tissue under ABA, NaCl, 4 °C, and PEG treatments. Grape plantlets grown for 30 days were treated with 400 mmol·L⁻¹ NaCl, 10% PEG and 0.2 mmol·L⁻¹ ABA, respectively. The treatment time was 24 h. Distilled water was used as the control. Three biological and technical replicates were set up for this experiment. Gene expression was normalized to the unstressed control expression level, which was given the value of 1. Error bars represent the mean ± SE from three biological replicates. Different letters indicate significant differences, while the same lowercase letters indicate no statistical difference (p < 0.05).

Figure 6

(A,B) Relative expression levels of SNARE genes in grape tissue under ABA, NaCl, 4 °C, and PEG treatments. Grape plantlets grown for 30 days were treated with 400 mmol·L⁻¹ NaCl, 10% PEG and 0.2 mmol·L⁻¹ ABA, respectively. The treatment time was 24 h. Distilled water was used as the control. Three biological and technical replicates were set up for this experiment. Gene expression was normalized to the unstressed control expression level, which was given the value of 1. Error bars represent the mean ± SE from three biological replicates. Different letters indicate significant differences, while the same lowercase letters indicate no statistical difference (p < 0.05).

Figure 7

Subcellular localization of VvSNAREs in the lower epidermal cells of Nicotiana benthamiana. The 35S::VvSNARE37-EGFP, 35S::VvSNARE44-EGFP, and 35S::VvSNARE46-EGFP constructs are expressed in Nicotiana benthamiana leaves. The top row shows the positive control (35S::EGFP), and the bottom row shows the 35S::VvSNARE-EGFP construct. The EGFP fluorescence image, bright field (BF) image, and the merged EGFP and BF images are shown. Scale bars correspond to 10 µm.