Transcriptome analysis of pitaya response to PEG simulated drought stress

Abstract:【Objective】Pitaya (Hylocereus spp.), also known as dragon fruit, is a member of the family Cactaceae. The pitaya cultivation area is expanding rapidly in many tropical and subtropical areas worldwide because it produces a nutritionally valuable fruit with an exotic appearance, striking colors, and health-promoting properties. Moreover, pitaya is a highly drought-tolerant plant, making it an excellent species for mining plant drought- tolerance genes. Previous studies on pitaya plant responses to drought stress mostly involved physiological and biochemical analyses, with some applying microarray technologies to detect drought- related expressed sequence tags. To date, however, transcriptomic data on pitaya have been very limited. Moreover, the combination of physiological and transcriptomic analysis to better understand the response mechanism of pitaya to drought stress has not been reported so far. The objective of this study was to decipher the response mechanism of pitaya to drought and provide the theoretical basis for breeding new drought-resistant germplasm.【Methods】The pitaya stems regarding their physiological characteristics and transcript levels between the control and drought stress simulated using polyethylene glycol (PEG)6000(-4.9 MPa) were compared. Seedlings not subjected to drought stress (0 MPa) were used as the control. At specific post-treatment time-points (0, 6, 12, and 18 h as well as 1, 3, 5, and 7 days), six pitaya stems of each time-point from stressed and the control were collected, immediately frozen in liquid nitrogen, and stored at −80 °C prior to analyzing their malondialdehyde (MDA) content, catalase (CAT) and peroxidase (POD) activities. Based on the physiological responses, 6 h and 3 days were selected as the optimal sampling time for the transcriptome assay. Therefore, pitaya seedlings exposed to drought stress for 6 h and 3 days were designated as OS6H and OS3D, respectively, with the corresponding controls designated as NS6H and NS3D, respectively. |Fold Change| ≥ 2 and FDR < 0.01 was used to screen differentially expressed genes (DEGs), which were then annotated and enriched in Gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), Eukaryotic orthologous groups (KOG), Swiss-Prot protein database (Swiss-Prot), Protein families (Pfam) and NCBI non-redundant protein database (NR) databases, respectively. Besides, To verify the accuracy and reliability of transcriptome data, 12 DEGs were randomly selected and analyzed by real- time fluorescence quantitative PCR (qRT- PCR).【Results】A total of 432 differentially expressed genes (DEGs) were identified from OS6H vs NS6H (ratio of 6-h drought stress to control) and OS3D vs NS3D (ratio of 3-d drought stress to control). There were 18 co-expressed DEGs in the two comparison groups (12 co-upregulated,4 co-downregulated, and 2 in reverse expression pattern), 288 DEGs expressed exclusively in OS6H vs NS6H comparison group (88 up- regulated, 200 down- regulated), 126 DEGs expressed exclusively in the OS3D vs NS3D comparison group (79 up- regulated and 47 downregulated), and the number of genes in the OS6H vs NS6H comparison group was more abundant. GO enrichment divided DEGs into biological processes (mainly metabolic process and cellular process), cell components (mainly membrane and membrane part) and molecular functions (mainly catalytic activity and binding). KEGG pathway enrichment analysis showed that the four most enriched pathways in the OS6H vs NS6H comparison group were starch and sucrose metabolism, photosynthesis-antenna protein, phenylpropanoid biosynthesis, and cyanoamino acid metabolism. Of the DEGs in the OS3D vs NS3D comparison, the four most enriched pathways were alanine, aspartate, and glutamate metabolism, starch and sucrose metabolism, cyanoamino acid metabolism and phenylpropanoid biosynthesis. The enriched KEGG pathways were further classified into 6 functional categories for analysis: signal transduction (such as plant hormones, cGMP- PKG, Ras, phosphatidylinositol, Wnt, etc.), carbohydrate metabolism (sucrose and starch, pyruvate metabolism and glycolysis, etc.), amino acid metabolism (e.g. alanine, glutamate, tyrosine, cysteine, and glutathione, etc.), transcription and transport (RNA degradation, ribosomes and endocytosis, etc.), secondary metabolism(e.g. flavonoids, phenylpropanoid, etc.) and lipid metabolism (а-linolenic acid metabolism, glycerophospholipid metabolism, cutin, suberine and wax biosynthesis). These enhanced the osmotic regulation, detoxification and antioxidant capacity of pitaya. Moreover, some DEGs identified in this study, including alanine-glyoxylate aminotransferase 2 homolog 3 (At3g08860), phenylcoumaran benzylic ether reductase (PT7), probable choline kinase 1 (CK1), salicylate carboxymethyltransferase (SAMT), scarecrow-like protein 28 (SCL28), putative disease resistance protein (RGA1) and exodium-like1 (EXL1), have rarely been reported as responsive to drought stress. The possible functions of these proteins influencing drought resistance need to be experimentally verified.【Conclusion】The molecular adaptation mechanism of pitaya seedlings to drought stress was preliminarily clarified. Drought stress activated a series of signal transduction pathways that regulate downstream gene expression. Through the degradation and conversion of carbohydrates, amino acid metabolism and secondary metabolism, the osmotic regulation, detoxification and antioxidant capacity of pitaya are enhanced, thus avoiding significant oxidative damage. The results of this study provide insights into the drought-tolerance mechanisms of pitaya.