The Transcriptome And Physiology Analysis Of Cynanchum Thesioides Under Drought Stress

Drought is a worldwide problem that seriously limits plant growth and crop yield.Cynanchum thesioides?Freyn?K.Schum.and its variety Cynanchum thesioides?Freyn?K.Schum.var.australe?Maxim.?Tsiang et P.T.Li.belong to Cynanchum Linn.of Asclepiadaceae,they are upright,xerophytic shrub and kind of plants that are eaten as fruits but also used in drugs,animal feed,as industrial raw materials,and as pasture for soil and water conservation.At present,the amount of wild C.thesioides decreased year by year because of seasonal drought.Therefore,its drought resistance was comprehensively evaluated and explored from physiological and molecular aspects,so as to provide theoretical basis and excellent reserve genes for further study on the regulatory genes and molecular breeding of drought tolerance in Asclepiadaceae,and to improve desertification in China.In our study,water treatments of C.thesioides were set up by pot-culture methods in greenhouse.The effects of different drought treatments on seedlings of growth indicators,osmotic substances,antioxidant system in C.thesioides were studied and compared their drought resistance.The effects of different drought treatments on seedlings of growth indicators,osmotic substances,antioxidant system and secondary metabolite-succinic acid in C.thesioides were studied,the molecular mechanism of the response to drought stress was investigated combined with transcriptomics,and the candidate DEGs were verified and analyzed by qRT-PCR.The main results showed that:1.As drought stress increased,the plant height,root width,root length,root fresh weight and,fresh weight of aerial part of C.thesioides seedling gradually decreased.Soluble sugar?SS?and proline?Pro?content in roots of C.thesioides.,soluble protein?SP?content in leaves and roots of C.thesioides?Freyn?K.Schum.,and Pro content in leaves of C.thesioides?Freyn?K.Schum.var.australe?Maxim.?Tsiang et P.T.Li.gradually increased,while SS content in leaves of C.thesioides?Freyn?K.Schum.var.australe?Maxim.?Tsiang et P.T.Li.,and SP in roots and Pro in leaves of C.thesioides?Freyn?K.Schum.firstly increased and then decreased gradually.Superoxide anion free radical?O2·-?generation rate,peroxide hydrogen?H2O2?and malondialdehyde?MDA?content in leaves of C.thesioides increased gradually.The catalase?CAT?activity of C.thesioides firstly increased and then decreased,while the superoxide dismutases?SOD?activity of C.thesioides?Freyn?K.Schum.var.australe?Maxim.?Tsiang et P.T.Li.and ascorbate peroxidase?APX?activity of C.thesioides?Freyn?K.Schum.gradually increased.Stomatal width and area were decreased under drought stress,and stomatal density increased significantly with the increase of drought degree.The thickness of leaf,upper and lower epidermis cuticle,upper and lower epidermis,palisade tissue and sponge tissue decreased under the condition of mild drought,when the drought reached a certain level,C.thesioides could enhanced water holding capacity of leaves by increasing their thickness.Cluster analysis and correlation index showed that Pt/St,leaf thickness,cutical thickness of upper epidermis and lower epidermis thickness were closely related to drought resistance of C.thesioides.Through analysis of average membership functional values,the drought tolerance of C.thesioides?Freyn?K.Schum.var.australe?Maxim.?Tsiang et P.T.Li.was better than C.thesioides?Freyn?K.Schum.2.A total of 207.58 Gb clean data and 55,268 genes were obtained from leaves of C.thesioides seedling under drought stress.18,708 and 14,480 genes were annotated in GO and KEGG database,respectively.In compared with CK,mild,moderate and severe drought in C.thesioides?Freyn?K.Schum.contained 75,2768,and 6077 up-regulated and 83,3605,and 5383 down-regulated genes,respectively.In C.thesioides?Freyn?K.Schum.var.australe?Maxim.?Tsiang et P.T.Li.,there were 269,2103,and 2763 up-regulated and 375,2399,and 4061 down-regulated genes,respectively.Differentially expressed genes?DEGs?were significantly enriched in carbon metabolism,starch and sucrose metabolism,biosynthesis of amino acids,phenylpropanoid biosynthesis and plant hormone signal transduction pathways.3.Gene co-expression network of C.thesioides under drought stress was constructed by WGCNA.In C.thesioides?Freyn?K.Schum.,grey60 module was significantly correlated with mild drought stress,greenyellow,brown and orange module were significantly correlated with moderate drought stress,and orange,turquoise and violet module were significantly correlated with severe drought stress.In C.thesioides?Freyn?K.Schum.var.australe?Maxim.?Tsiang et P.T.Li.,lightgreen and saddlebrown module were significantly correlated with mild drought stress,lightcyan module was significantly correlated with moderate drought stress,darkorange and darkturquoise module were significantly correlated with severe drought stress.Modules significantly associated with severe drought were mainly enriched in plant hormone signaling pathways.The family of C3H,NF-X1,ERF,NAC,WRKY,CO-like,MYB_related,C2H2,G2-like and HD-ZIP were identified from C.thesioides.4.The succinic acid?SA?content in leaves,stems and roots of C.thesioides?Freyn?K.Schum.was 558.6,268.67 and 427.44 ?g·g-1,respectively.And those of C.thesioides?Freyn?K.Schum.var.australe?Maxim.?Tsiang et P.T.Li.was 593.46,368.69 and 141.08 ?g·g-1,respectively.With the increase of drought stress,the content of succinic acid in C.thesioides leaves and roots first increased and then decreased,and the content was the highest under moderate drought stress.The activity of ?-ketoglutaric dehydrogenase?OGDH?,succinyl-CoA synthetase?SUCLA?,succinate dehydrogenase?SDH?,glutamate decarboxylase?GAD?and y-aminobutyric acid?GABA?contents in C.thesioides leaves were the highest under severe drought stress.The correlation analysis showed that there was a negative correlation between succinic acid and GABA,SDH in leaves.146 and 172 genes involved in the succinic acid biosynthesis were detected in leaves of C.thesioides?Freyn?K.Schum.and C.thesioides?Freyn?K.Schum.var.australe?Maxim.?Tsiang et P.T.Li.,respectively.Succinic acid content was positively correlated with MDH?BMK_Unigene₀33366?,but negatively correlated with GAD?BMK_Unigene₀96180 and BMK_Unigene₀30468?,SDH?BMK_Unigene₁42841?.Eight differentially expressed genes involved in succinic acid synthesis were validated by qRT-PCR,the results were consistent with transcriptome data of C.thesioides.The relative expression trend of qRT-PCR was basically consistent with that of transcriptome sequencing?FPKM?,which further verified the reliability of transcriptome data of C.thesioides.5.Based on physiological and biochemical and transcriptome data,the molecular mechanism of response to drought was preliminarily analyzed.Drought transduced further via Ca2+,MAPK,ABA signaling transduction pathway and protein kinases?PP2C,MAPK,CDPKs,SnRKs,LRRs,CRKs,WAKs,SnFKs?resulting in the activation of downstream transcription factors?TFs?.The activation of TFs triggers the expression of a cascade of downstream drought-responsive genes,whose transcription further regulates the homeostasis of cellular metabolism,such as amino acid metabolism?P5CS,THAL?,starch and sucrose metabolism?AGPase,SPS,FBPase?,biosynthesis of phenylpropanes?PAL,C4H,F5H?,flavonoids?CHS,F3H?,and succinic acid?MDH,SDH,GAD?.Furthermore,excess ROS can be scavenged via antioxidant enzyme?SOD,CAT,APX,POD,GST,GPX?,which further affects ROS signaling.The single or synergistic effects of the above processes further regulate the dynamic balance of cell metabolism and improve the drought resistance of C.thesioides.