Study On The Physiological And Molecular Mechanisms Of Pitaya In Response To Drought Stres

Pitaya（Hylocereus polyrhizus L.）,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 transcriptomic and microarray technologies to detect drought-related expressed sequence tags.To date,however,proteomic data on pitaya have been very limited.Moreover,combination of physiological,transcriptomic and proteomic analysis to better understand the response mechanism of pitaya to drought stress have not been reported so far.The objective of this study was to decipher the response mechanism of pitaya to drought.The stems of pitaya plants regarding their morphological,anatomical and physiological characteristics,transcript levels,and protein abundance changes in response to drought stress simulated using polyethylene glycol（PEG）6000 were analyzed.The results of this study provide insights into the drought-tolerance mechanisms of pitaya.The main results showed as follows:1)Morphological and anatomical response of pitaya to drought stressThe stems of pitaya wilted,its color deepened and its edge became thinner under drought stress.Drought stress significantly increased the root length and root volume,and decreased the the mean root diameter and branch number.Drought stress also obviously decreased the cell diameter of the fleshy stems and the distance between fiber bundles.The ultrastructure of chloroplast was destroyed and plastid globules appeared.2)Drought stress enhanced the osmolyte accumulation and antioxidant enzyme activitiesDrought stress enhanced the soluble sugar,soluble protein,free proline,malondialdehyde（MDA）,superoxide anion(O₂^·-),and hydrogen peroxide（H₂O₂）contents as well as the superoxide dismutase（SOD）,and ascorbic peroxidase（APX）activities of pitaya.However,glutathione reductase（GR）activity was sensitive to drought stress.Overall,drought stress enhanced osmolyte accumulation,lipid peroxidation,and antioxidant enzyme activities.3)Differentially expressed genes and enriched pathways under drought stress in pitayaTranscriptomic data showed that 48,342 unigenes were obtained from 12 c DNA libraries,and24,810 unigenes were annotated and enriched in GO,KOG,KEGG,Swiss-Prot,Pfam,and NR databases,respectively.After unigenes screening,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（12co-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 47down-regulated）,and the number of genes in the OS6H vs NS6H comparison group was more abundant.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,c GMP-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.4)Differentially accumulated proteins and enriched pathways under drought stress in pitayaFor 4D label-free proteomics analysis,a total of 3,107,303 spectra were generated,including371,016 spectra corresponding to known spectra,43,489 peptides,41,084 unique peptides,5,929identified proteins,and 5,269 quantified proteins.To clarify the functions of the 5,269 quantified proteins,they were annotated according to GO terms,predicted functional domains,KEGG pathways,and KOG functional classifications.A total of 686 differentially accumulated proteins（DAPs）were identified in these two comparisons.Of these DAPs,58（32 upregulated and 26 downregulated）were common to both comparisons,285（133 upregulated and 152 downregulated）were specific to the OS6H vs.NS6H comparison,and 343 proteins（154 upregulated and 189 downregulated）were exclusive to the OS3D vs.NS3D comparison.Among the DAPs in the OS6H vs.NS6H comparison,the KEGG pathway enrichment analysis showed that the four most enriched pathways were carbon metabolism,starch and sucrose metabolism,carbon fixation in photosynthetic organisms,and porphyrin and chlorophyll metabolism.Of the DAPs in the OS3D vs.NS3D comparison,the three most enriched pathways were starch and sucrose metabolism,pyruvate metabolism,and alanine,aspartate,and glutamate metabolism.The enriched KEGG pathways were further classified into 5functional categories for analysis:carbohydrate metabolism（starch and sucrose metabolism,galactose metabolism,and TCA cycle）,energy metabolism（photosynthesis,oxidative phosphorylation,and carbon fixation in photosynthetic organisms,）,amino acid metabolism（aspartate,glutamate,arginine,proline and phenylalanine）,glutathione and ascorbate metabolism,and secondary metabolism（flavonoid,anthocyanin,phenylpropanoid and carotenoid biosynthesis）.These enhanced the osmotic regulation ability,enzyme-induced and non-enzyme-induced antioxidant ability of pitaya.5)Overexpression of the pitaya HpPEAMT gene enhanced drought stress in tobaccoPhosphoethanolamine N-methyltransferase（PEAMTase）plays an important role in the abiotic stress response.Although the PEAMT genes has been isolated from many species other than pitaya,its role in the drought stress response has not yet been fully elucidated.In the present study,we isolated a1,485 bp c DNA fragment of HpPEAMT from pitaya（Hylocereus polyrhizus）,which was differentially expressed at both transcription and protein levels in pitaya under drought stress,and a protein band with a predicted molecular mass around 60 k Da was obtained when the gene was expressed in E.coli BL21（DE3）.Phylogenetic analysis showed that,during its evolution,HpPEAMT has shown a high degree of amino acid sequence similarity with the orthologous genes in Chenopodiaceae species.To further investigate the function of HpPEAMT,we generated transgenic tobacco plants overexpressing HpPEAMT,and the transgenic plants accumulated significantly more glycine betaine（GB）than did the wild type（WT）.Drought tolerance trials indicated that,compared with those of the wild-type（WT）plants,the roots of the transgenic plants showed higher drought tolerance ability and exhibited improved drought tolerance.Further analysis revealed that overexpression of HpPEAM in Nicotiana tabacum resulted in upregulation of transcript levels of GB biosynthesis-related genes（Ni BADH,Ni CMO and Ni SDC）in the leaves.Furthermore,compared with the wild-type plants,the transgenic tobacco plants displayed a significantly lower malondialdehyde（MDA）accumulation and higher activities of the superoxide dismutase（SOD）and peroxidase（POD）antioxidant enzymes under drought stress.Taken together,our results suggested that HpPEAMT enhanced the drought tolerance of transgenic tobacco.6)Physiological and molecular responses of pitaya to drought stressIn combination with physiological,transcriptome and proteome studies,drought stress activated a series of signal transduction pathways（such as plant hormones,c GMP-PKG,Ras,phosphatidylinositol,Wnt,etc.）that regulated downstream gene expression.Carbohydrate degradation and conversion（sucrose and starch,galactose metabolism and pyruvate metabolism,etc.）and amino acid metabolism（such as alanine,glutamate,aspartate,proline,and tyrosine,etc.）improved the osmotic regulation ability,ascorbic acid and glutathione metabolism and secondary metabolism（such as flavonoid,carotenoid,and phenylpropanoid biosynthesis,etc.）improved the detoxification and antioxidant capacity,enhanced energy metabolism（oxidative phosphorylation,and carbon fixation in photosynthetic organisms）and partial carbohydrate metabolism（glycolysis and TCA cycle）might provide necessary energy for the activation of these metabolic pathways.The high drought resistance of pitaya is attributed to its osmotic regulation,detoxification and antioxidant capacity,thus avoiding significant oxidative damage.In the present study,the major morphological,physiological and molecular adaptation mechanisms of pitaya response to drought stress were preliminarily clarified,and the key genes,proteins and metabolic pathways were identified,which are helpful for the better understanding of mechanism underlying the high tolerance of pitaya to drought stress.