Effects of exogenous sorbitol spray on gene expression networks of peach leaves

Abstract:【Objective】In many fruit trees of the Rosaceae family, sorbitol serves as the end products of photosynthesis and transporter carbohydrates, as well as an important signal substance in tissue development. Some previous studies had indicated that the accumulation of sorbitol might have a positive influence on soluble carbohydrates content and growth development. To understand how exogenous sorbitol affect metabolic pathways in source and sink leaf tissues, we investigated the transcriptional regulation of the gene expression networks in metabolic pathways after spraying the sorbitol on mature leaves in peach using RNAseq analysis.【Methods】The 6-month-old peach seedlings were sprayed by exogenous sorbitol solution (100 mg·^L-1) , and the clean water treatment was employed as a control. Before spraying, the young leaves on the top of seedling were temporarily covered with a plastic bag, then removed2 hours later. 48 hours after the spray, total RNA was extracted using Tritol reagent from at least 3 pieces of leaves mixture in each peach seedling groups respectively. The transcriptome sequencing library (300-400 bp DNA insertion) was constructed using NEB approaches, then the sequencing was performed on Illuminar Hiseq 2500 platform. The final sequencing reads were processed to trim off adapters sequence and low-quality bases for following analysis. Finally all the filtered clean reads were aligned to the peach reference genome (Ppersica＿298＿v2.1, Phytozome) using Top Hat2 and statistical analyses for differential gene expression were performed by cufflink script to analyze the changes of gene expression in mature leaves (with sorbitol spray) and young leaves (without sorbitol spray) .【Re-sults】The reads were mapped to the peach genome. The percentages of unique reads pairs showed a perfect match from 74.8% to 80.7% in the young and the mature leaves under different spraying treatment.The results of transcriptome sequencing showed that the number of DEGs (Differentially Expressed Genes) in the mature leaves treated with sorbitol was more than that of the young leaves without sorbitol spray. A total of 665 DEGs were identified in the mature leaves and control samples. Among these DEGs, 408 (61.4%) genes were up-regulated, and 257 (38.6%) were down-regulated, while the number of that in the young leaves without sorbitol spraying were 253 (in total) , 90 (up-regulated) and 163 (down-regulated) respectively. 5 genes responding to environmental stress remarkably increased at transcription level in both the mature leaves and the young leaves. These genes encode a Ribulose bisphosphate carboxylase (Pp.1 G311400) , Homeodomain-like superfamily protein (Pp.2 G200400) , ferretin (Pp.6 G283700) , TIR-NBS-LRR (Pp.8 G023900) and TIR-NBS-LRR (Pp.8 G027100) respectively. Meanwhile, 3 genes related to ionic equilibrium (oligopeptide transporter, Pp.4 G004600; Aldolase-type TIM barrel protein, Pp.4 G129800; natural resistance-associated macrophage protein, Pp.6 G225900) and 2 genes involved in intracellular lipid metabolism (lipoxygenase, Pp.2 G005500; terpene synthase, Pp.3 G222200) were inhibited. Especially in the mature leaves, more stress genes identified and related secondary metabolites pathways were affected. Geneontology enrichment analysis showed that ATPase family (GO:0042626, 12 genes of DEGs, coupling to transmembrane movement of substances) , hydrolase family (GO:0016820, 12 genes of DEGs, catalyzing transmembrane movement of substances) , calcium ion transmembrane transporter family (GO:0015085, 7 genes of DEGs, stimulation-induced cellular transmembrane signal transduction) and primary active transmembrane transporter family (GO:0015399, 12 genes of DEGs) were crucial for osmolytes accumulation, transmembrane transportation and ion uptake under stress condition in the mature leaves after sorbitol spraying. Using Kyoto Encyclopedia of Genes and Genomes database, we annotated 52 differentially expressed genes participated in the biosynthesis of plant secondary metabolites regulating response to exogenous sorbitol, which resulted in the detected changes in gene expression of Linoleic acid metabolism, diterpenoid biosynthesis, propanoate metabolism, phenylpropanoid biosynthesis and amino sugar and nucleotide sugar metabolism. Unlike in the mature leaves, the expression of genes associated with carbohydrates metabolism was primarily modulated in the young leaves. It was noted that hydrolase family (GO:0004553, 7 genes of DEG) which hydrolyze O-glycosyl compounds and the special transcription factor (GO:0001071, GO:0003700, 6 genes of DEGs) played an acute role in response to endogenous stimulus from mature leaves after sorbitol spraying. The transcriptome evidence confirmed that the spraying treatment indirectly influenced carbohydrates metabolism in sink tissue, such as starch and sucrose metabolism, galactose metabolism, pentose phosphate metabolism, fructose and mannose metabolism, amino sugar and nucleotide sugar metabolism.【Conclusion】After the application of sorbitol, the stress-response mechanisms in the mature leaves of peach initiated and affected secondary product metabolism. When the corresponding stress signals were transmitted to the young leaves, carbohydrates metabolism showed unblance accordingly, responding to the relevant stimulus.