Identification and expression analysis of PYL gene families in grape

Abstract:【Objective】The purpose of this study was to clarify the response of PYL gene of grape (Vitis vinifera L.) to abiotic stress and enrich the function of PYR/PYL/RCAR (Pyrabactin Resistance/Pyr1-Like/Regulatory Components of ABA Receptor) in ABA signaling and initiating signal transduction.【Methods】The candidate PYL gene were screened by using the full-length of amino acid sequence of PYR/PYL/RCAR in Arabidopsis (Arabidopsis thaliana) , rice (Oryza sativa) and maize (Zea mays) from12 X V. vinifera‘Pinot Noir'website genome (quasi-homozygous line PN40024) and removing redundancy. The chromosomal location, gene structure, phylogeny, physical and chemical properties of the gene family were comprehensively analyzed by bioinformatics methods. The plantlets propagated in vitro of‘Red Globe'were cultured in artificial climate chamber and were treated with 400 mmol · L-1 Na Cl (T1) , 10% PEG (T2) , 50 μmol · L-1 ABA (T3) and 100 μmol · L-1 ABA (T4) for 0, 2, 6 and 24 h, respectively. The treatment of 0 h was used as control. The total RNA was extracted from stems and leaves of the plantlets with different treatments, and the expression level of the gene family under abiotic stresses was analyzed systematically.【Results】Six PYL genes were identified from grape genome, named as Vv PYL1-Vv PYL6, respectively. There are two PYL genes located on the chromosome 2, other members of the gene family are randomly distributed on different chromosomes without preference.Vv PYL1, Vv PYL4 and Vv PYL5 are distributed in the beginning of their chromosomes, and Vv PYL6 are located in the middle of their chromosomes, while Vv PYL2 and Vv PYL3 are distributed in the ends of their chromosomes. Vv PYL5 has no intron structure, and the other members contain at least three exons.Both the 5'and 3'ends of Vv PYL4 and Vv PYL5 do not contain noncoding regions. The two exons ofVv PYL1, Vv PYL2 and Vv PYL3, which are located in the 3'end, were highly conservative, and are 216 bp and 219 bp in length, respectively. The sum of the 3 exons of Vv PYL3 is 580 bp, which is the same as that of Vv PYL5. The average number of amino acids in the gene family is 201, and the number of amino acids in Vv PYL1-Vv PYL5 is small, with an average of 184, while the amino acid number of Vv PYL6 is 286, which is significantly different from that of other members of the family. In addition to Vv PYL4, this gene family is rich in acidic amino acids, and Vv PYL2 and Vv PYL5 are stable proteins.The secondary structure is dominated by alpha helix, extended chain structure and irregular coiling. Subcellular localization prediction found that Vv PYL5 and Vv PYL6 are localized only in the cytoplasm, Vv PYL4 are located on the cell membrane and in the nucleus, Vv PYL1, Vv PYL2 and Vv PYL3 exit in chloroplast, cytoplasm and nucleus, Vv PYL1 would also be located in the mitochondria. Phylogenetic analysis showed that the Vv PYL gene family could be divided into three sub groups, which is consistent with the classification results of Arabidopsis. There are several conserved sites in the PYL gene family of grape, which contain 4-8 motif. Among them, Vv PYL2 had the least number of motif (4) , and Vv PYL6 had the largest number of motif (8) . q RT-PCR (quantitative real-time PCR) analysis showed that after 400 mmol · L-1 Na Cl treatment, the expression level of Vv PYL1 was the lowest at 2 h, and the expression level gradually increased with the increase of stress time. The expression level of the gene24 h after the treatment was almost the same as that of the control. The expression level of Vv PYL2 2 h after the treatment with 400 mmol · L-1 Na Cl was significantly lower than that of the control, while the gene were significantly up-regulated 6 h and 24 h after the treatment, which were 1.3 and 2.2 times as high as that of the control, respectively. The expression levels of Vv PYL3, Vv PYL4 and Vv PYL5 were down regulated after 400 mmol · L-1 Na Cl treatment, and the expression level of Vv PYL6 24 h after treatment with 400 mmol · L-1 Na Cl was 1.6 times as high as that of the control. The response of Vv PYL1 to10% PEG treatment was basically the same as the Vv PYL2, and the expression level of the gene 2 h after treatment with 10% PEG was significantly lower than that of the control, and the expression levels of the gene 6 h and 24 h after treatment with 10% PEG were up-regulated. Among them, the expression level of the two genes 6 h after treatment with 10% PEG was 2.2 times and 2.1 times as high as that of the control, respectively. The expression levels of Vv PYL3, Vv PYL4, Vv PYL5 and Vv PYL6 were down regulated when the plantlets treated with 10% PEG compared with the control. The expression level of Vv PYL1 was 1.2, 1.5 and 1.7 times as high as that of the control group 2 h, 6 h and 24 h after 50 mol · L-1 ABA treatments, respectively. The expression level of Vv PYL2 6 h after the treatment with 50 mol · L-1 ABA was down regulated significantly, which was 0.2 times as high as that of the control. The expression levels of Vv PYL3 and Vv PYL4 were decreased to 1.6 and 1.4 times 24 h after the treatment with 50 mol·L-1 ABA. The expression levels of Vv PYL5 and Vv PYL6 24 h after treatment with 50 mol · L-1 ABA were the same as those of the control. The response of Vv PYL1 to 100 μmol·L-1 ABA was almost the same as Vv PYL2, and the expression levels of Vv PYL1 and Vv PYL2 6 h after treatment with 100 μmol·L-1 ABA was 1.8 and 1.3 times as high as that of the control, and they were 2 and 1.6 times as high as that of the control, respectively 24 h after the treatment with 100 μmol·L-1 ABA. The expression levels of Vv PYL3 and Vv PYL5 2 h and 6 h after treatment with 100 mol · L-1 ABA were significantly lower than the that of the control at 24 h. In addition, the expression level of Vv PYL4 was lower than that of the control under different stress times, and the expression level of Vv PYL6 was higher than that of the control at 2 h.【Conclusion】In this study, 6 grape PYL genes were cloned. The PYL gene family was highly conservative and could be divided into 3 sub groups, which was consistent with the classification results of Arabidopsis. Most members of the gene family could be induced by abiotic stresses. PYL family members of grape may have different biological functions and affect the downstream signal transduction process.This study provides a basis for functional study of PYL gene of grape in stress response.