Project description:Transcriptomics and metabolomics assisted molecular characterization of novel ERF transcription factors involved in the regulation of specialized metabolism in Ophiorrhiza pumila
Project description:In this study, the genes that encode AP2/ERF transcription factors, namely OpERF1 to OpERF5, were isolated from HR of O. pumila. Phylogenetic analysis of AP2/ERF protein sequences suggested the close evolutionary relationship of OpERF1 with stress-responsive ERF factors in Arabidopsis and of OpERF2 with ERF factors reported to regulate alkaloid production, such as ORCA3 in Catharanthus roseus, NIC2-locus ERFs in tobacco, and JRE4 in tomato. We generated the HR lines of O. pumila, ERF1i and ERF2i, in which the expression of OpERF1 and OpERF2, respectively, was suppressed using RNA interference technique. The transcriptome and metabolome of these suppressed HR were analyzed for functional characterization of OpERF1 and OpERF2.
Project description:MicroRNAs (miRNAs) are a class of endogenous small RNAs that play important roles in growth, development, and environmental stress response processes in plants. Ulmus pumila is a typical deciduous broadleaved tree species of north temperate, and is widely distributed in central and northern Asia, which has important economic and ecological value. With the spread and aggravate of soil salinisation, salt stress has become a major abiotic stress that highly affects the normal growth and development of U. pumila. However, to date, no investigation into the influence of salt stress on U. pumila miRNAs has been reported. To identify miRNAs and predict their target mRNA genes under salt stress, three small RNA libraries were generated and sequenced from CK (without salt stress), LSS (light salt stress for a short time) and MSL (medium-heavy salt stress for a long time) roots of U. pumila seedlings. Through integrative analysis, 245 conserved miRNAs representing 30 families and 64 novel miRNAs were identified, of which 89 exhibited altered expression level under salt stress, and 232 potential targets for the miRNAs were predicted and annotated in U. pumila. The expressions of six differentially expressed miRNAs were validated by qRT-PCR. These salt responsive miRNAs may play crucial roles in U. pumila defense against salt stress, and our miRNA data provides valuable information regarding further functional analysis of miRNAs involved in salt tolerance of U. pumila and other forest tree species.
Project description:BackgroundThe anticancer drug camptothecin (CPT), first isolated from Camptotheca acuminata, was subsequently discovered in unrelated plants, including Ophiorrhiza pumila. Unlike known monoterpene indole alkaloids, CPT in C. acuminata is biosynthesized via the key intermediate strictosidinic acid, but how O. pumila synthesizes CPT has not been determined.ResultsIn this study, we used nontargeted metabolite profiling to show that 3α-(S)-strictosidine and 3-(S), 21-(S)-strictosidinic acid coexist in O. pumila. After identifying the enzymes OpLAMT, OpSLS, and OpSTR as participants in CPT biosynthesis, we compared these enzymes to their homologues from two other representative CPT-producing plants, C. acuminata and Nothapodytes nimmoniana, to elucidate their phylogenetic relationship. Finally, using labelled intermediates to resolve the CPT biosynthesis pathway in O. pumila, we showed that 3α-(S)-strictosidine, not 3-(S), 21-(S)-strictosidinic acid, is the exclusive intermediate in CPT biosynthesis.ConclusionsIn our study, we found that O. pumila, another representative CPT-producing plant, exhibits metabolite diversity in its central intermediates consisting of both 3-(S), 21-(S)-strictosidinic acid and 3α-(S)-strictosidine and utilizes 3α-(S)-strictosidine as the exclusive intermediate in the CPT biosynthetic pathway, which differs from C. acuminata. Our results show that enzymes likely to be involved in CPT biosynthesis in O. pumila, C. acuminata, and N. nimmoniana have evolved divergently. Overall, our new data regarding CPT biosynthesis in O. pumila suggest evolutionary divergence in CPT-producing plants. These results shed new light on CPT biosynthesis and pave the way towards its industrial production through enzymatic or metabolic engineering approaches.