Cloning and functional analysis of heat shock transcription factor PpHSF18 in peach (Prunus persica)

Abstract:【Objective】Peach (Prunus persica L.) is an important economical fruit and is popular with consumers. In peach, the branch angle, as an important agronomic trait of tree architecture, affects fruit yield and quality, adaptive capacity to environment, and competitive capacity. The pillar peach, as one of the special germplasm resources, shows smaller branch angle and fewer secondary branches than the standard peach, which is an ideal tree architecture for implementing easy pruning and mechanized harvesting strategies. Heat shock factors (HSFs), especially for class A, play a vital role in not only biotic or abiotic stress but also normal plant growth and development, especially in plant tree architecture. However, PpHSFAs have not been studied in peach, especially the role in regulating branch angle. The objectives of this study aimed to screen and validate the candidate PpHSFAs function that might participate in formation of branch angle.【Methods】A pillar peach (Sahonglongzhu, S) and a standard peach (Okubo, O) with one- year old seedlings were selected for measuring branch angle, respectively. The shoot- tip of these two cultivars was taken for RNA- seq. The expression pattern of 11 PpHSFAs, PpTAC1 and PpLAZY1 were detected and the heatmap was plotted by TBtools software. The homologyof PpHSFAs with OsHSFA2D was detected using Blastp search and the gene with the highest expected value was selected as the candidate gene. PpHSF18 was selected and might be involved in the formation of branch angle, which showed a high similarity with OsHSFA2D. Then, PpHSF18 was cloned by PCR and transformed in pSAK277 vector, and promoted by 35S promoter, using the restriction enzymes Hind Ⅲ and Xba Ⅰ. The recombinant vector was transformed into GV3101 strain. The pSAK277-35S::PpHSF18 was transformed into Arabidopsis thaliana using floral dip method by Agrobacterium-mediated transformation. The positive plants were identified by PCR and the relative expression levels of PpHSF18 in transgenic lines were detected by quantitative real-time PCR (qRT- PCR). The Arabidopsis thaliana plants, transformed with the pSAK277-35S::PpHSF18 and WT, were photographed and the branch angles were measured and calculated in SPSS using ANOVA at significance level of p＜0.05.【Results】The branch angle of Okubo increased gradually with the development stage, ranging from 62° to 76°. The increased angle resulted in lager crown width. The branch angle of Sahonglongzhu showed no significant difference in the development stage, ranging from 31° to 34°. It was easy to observe that the branch angle of Okubo was significantly larger than that of Sahonglongzhu at all stages. Transcriptome analysis of shoot-tip with these two cultivars showed that the expression pattern of PpTAC1 and PpLAZY1, which belonged to IGT gene family, showed opposite pattern in Okubo and Sahonglongzhu. The expression level of PpTAC1 was higher in Okubo than that in Sahonglongzhu, but PpLAZY1 showed the opposite expression pattern. Except PpHSF16 with no expression in these two peach cultivars, the expression profiles of remaining 10 PpHSFAs were classed into two groups. PpHSF2, PpHSF3, PpHSF10, PpHSF11 and PpHSF14 showed co- expression with PpTAC1, and PpHSF1, PpHSF4, PpHSF7, PpHSF9 and PpHSF18 displayed co-expression with PpLAZY1. PpHSF18 showed the highest expected value with OsHSFA2D which was a key transcription factor in regulating rice tiller angle among all the PpHSFAs. PpHSF18 contained 1080 bp of ORF (open reading frame) and encoded 359 amino acids, which contained a 21 bp insertion between HR-A and HR-B region, as the characteristic of class A of HSF gene subfamily. PpHSF18 gene was successfully cloned using leaves of Okubo as the template. The pSAK277-35S::PpHSF18 overexpression vector was constructed and stably transformed into Arabidopsis thaliana. Four transgenic PpHSF18-overexpression T0 lines were obtained by PCR with a purpose band. All the transgenic lines showed higher PpHSF18 transcript levels than WT. The Line 1, Line 2 and Line 4, showing higher expression level among transgenic lines, were selected for further analysis. Compared to WT plants, PpHSF18 overexpression significantly decreased the branch angle in all three transgenic lines. The branch angle of three transgenic Arabidopsis lines was about 40°, while the branch angle of WT was about 60°. The branch angle of three transgenic lines was very significantly smaller than that of WT using ANOVA analysis. According to the above results of phenotype and qRT-PCR analysis of different transgenic lines, it was suggested that PpHSF18 as a key transcription factor could indeed repress plant branching angle.【Conclusion】A homology of OsHSFA2D denoted as PpHSF18 was cloned in standard peach cultivar Okubo. PpHSF18 encoded a class A HSF transcription factor with a 21 bp insertion between HR- A and HR-B region. PpHSF18 was expressed higher in pillar cultivar Sahonglongzhu than that in standard peach cultivar Okubo, showing a co-expression with PpLAZY1. Overexpression of PpHSF18 resulted in a highly significant decrease of branch angle in transgenic A. thaliana. These results indicated that PpHSF18 was involved in regulating branch angle and provided the theoretical basis for regulating branch angles using the molecular technique in peach.