Analysis Of Betaine And Raffinose Metabolism In Response And Adaptation To Drought Stress In Jatropha Curcas Based On High-throughput Sequencing

Jatropha curcas has attracted wide attention as a potential biofuel crop owing to its hardiness,high oil content,and suitability of seed oil for biofuel conversion.Drought is one of the major environmental impacts,limiting the distribution,plantation and productivity of the plant.Thereafter,it is becoming a most urgent task now,to design genetic breeding strategies aimed at improving traits that underlie adaptation to drought stress.In-depth analysis emphasizes the complexity underlying genetic network responses to drought.In the present study,comparative genome-wide transcriptome analysis on Jatropha seedlings,treated with drought hardening（10% PEG）,followed by air drought stress,was performed by RNA-Seq technology.Expression patterns of drought-responsive genes involved in sugar metabolism,plant hormone signal transduction,carotenoid secondary metabolism,were discussed.Then two candidates encoding raffinose synthase（RS）were isolated from Jatropha.O ur investigations establish a solid foundation for future comprehensive analysis on molecular mechanism underlying the formation of dro ught-resistance and genetic improvement of Jatropha.The main results are shown as follows:1.Jatropha seedlings were treated with 10% PEG for drought hardening（6 h,12 h,24 h,48 h）,followed by air drought stress（24 h,48 h,72 h）.Then the leaves were collected for RNA-Seq analysis using Illumina Hiseq2000 platform.The low-quality reads were removed,resulting in the average number of clean reads at 11,933,126 for each sample.The alignment analysis with reference genome resulted in 26,979 genes with annotation.2.The NOISeq method was used to detect differentially expressed genes（DEGs）between two selected samples.Compared with CK（0 h）,altogether 306 DEGs were characterized during drought hardening,including 166 up-regulated and 186 down-regulated genes.Functional analyses including Gene Ontology（GO）and KEGG were performed and we found,drought hardening induced a bulk of transcription initiation factors,transcription factors（EREBP,MYC-like）,together with a repression on protein degradation;multiple pathways involving sugar metabolism were affected,for instance,an activation on raffinose and glucan biosynthesis,a reduction on polygalacturonic acid degradation.3.During the air drought stress treatment（for 24 h,48 h,72 h）,a number of 1 618 DEGs were identified,of which,671,742,696 were repressed,while 411,409,379 were induced.Most of the down-regulated genes related to degradation of biomolecules,such as pectinesterase,polygalacturonase,fatty acid hydrolase.Meanwhile,considerable genes encoding transporters were highly repressed,indicating the impact of drought stress on substance transportation.O n the contrary,most candidates involved in carotenoid and abscisic acid（ABA）biosynthesis performed a constant up-regulation during drought treatment.Besides,genes for key responders of plant hormone signal transduction,including PP2C（ABA）,GID1（Gibberellin）,and JAZ（jasmonic acid）,were mostly induced,implying that various plant hormones played vital roles in drought stress response in Jatropha.4.Betaine acts as a nontoxic protective osmolyte,its biosynthesis needs two key enzymes: betaine aldehyde dehydrogenase（BADH）and choline monooxygenase（CMO）.According to our RN A-seq data,two candidates for BADH and four for CMO were identified.Expression profiles revealed that,the main transcript for BADH and three CMO transcripts were all up-regulated to some extent,when exposed to drought treatment.Accordingly,the content of betaine in plant leaves also increased to approx 1.36 and 1.44 fold of the relative control,after drought hardening for 48 h and air drought stress for 72 h.All these data demonstrated the considerable function of betaine accumulation in drought response and adaptation of Jatropha.5.Accumulation of raffinose family oligosaccharides（RFOs）performs potential roles in stress amelioration in higher plants.Raffinose synthase（RS）is one of the key enzymes that channels sucrose into the RFOs biosynthetic pathway.Previous RNA-seq analysis indicated that,two transcripts for RS（XM₀12230751.1,XM₀12230752.1）kept an up-regulated expression profile during both drought hardening and air drought treatment,while another transcript（XM₀12225806.1）were induced only by air drought stress.These findings implied the RS family members should not be a simple functional redundancy.Then,we isolated and cloned the coding sequence of both candidates（XM₀12230752.1 and XM₀12225806.1）into pGEM-T vector.The total length of their CDS was 2280 bp and 2331 bp,respectively,both containing the conserved domain of raffinose synthase,while they were clustered into different clades when exposed to the phylogenetic classification.All these data present a theory foundation for further functional validation of RS candidates in future.