SlMYC1 Regulates Type VI Glandular Trichome Formation and Terpene Biosynthesis in Tomato Glandular Cells.

Tomato (Solanum lycopersicum) glandular trichomes function as biochemical factories that synthesize a diverse array of specialized metabolites. Terpenoids are the most diverse class of plant specialized metabolites, with volatile mono- and sesquiterpenes playing important roles in plant defense. Although the biosynthetic pathways of volatile terpenes in tomato glandular trichomes have been well described, little is known about their regulation. Here, we demonstrate that SlMYC1, a basic helix-loop-helix transcription factor, differentially regulates mono- and sesquiterpene biosynthesis in the type VI glandular trichomes of tomato leaves and stems. SlMYC1 functions as a positive regulator of monoterpene biosynthesis in both leaf and stem trichomes but as a negative regulator of sesquiterpene biosynthesis in stem trichomes. SlMYC1 is also essential for type VI glandular trichome development, as knocking down SlMYC1 led to the production of smaller type VI glandular trichomes at lower densities, and knocking out this gene led to their absence. Our findings reveal a role for SlMYC1 not only in type VI glandular trichome development but also in the regulation of terpene biosynthesis in tomato.

Comparison of root transcriptomes and expressions of genes involved in main medicinal secondary metabolites from Bupleurum chinense and Bupleurum scorzonerifolium, the two Chinese official Radix bupleuri source species.

Radix bupleuri, roots of Bupleurum species, is a widely used traditional Chinese medicine. Here, we compared the root transcriptomes of both Bupleurum chinense DC. and Bupleurum scorzonerifolium Willd. A total of 313 483 and 342 263 high quality expressed sequence tags were obtained, respectively. In addition, 17 117 (59.2%) and 19 416 (62.8%) unigenes for B. chinense and B. scorzonerifolium had homologous genes in the opposite dataset. For B. chinense, Kyoto Encyclopedia of Genes and Genomes database (KEGG) annotation identified carbohydrate metabolism, energy metabolism and amino acid metabolism as the three highest groups in the metabolism category. For B. scorzonerifolium, the lipid metabolism group had the most unigenes. Genes that may participate in the biosynthesis of terpenoid, triterpenoid, sterol, lignan and flavonoids were identified according to their annotations. (Tri)terpenoid-related genes were predominantly found in B. chinense. The expressions of certain genes were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in the roots of the two species. A total of 558 putative transcription factors (TFs) and 137 transcriptional regulators (TRs) among 1364 TFs and 327 TRs, and 610 TFs and 129 TRs among 1600 TFs and 323 TRs were specific for B. chinense and B. scorzonerifolium, respectively. Our transcriptome comparison reflects the different types and proportions of metabolites synthesized by the two species. The data, especially, those genes involved in the biosynthesis of particular types of metabolites, will provide the basis for further investigations of the secondary metabolite repertoire of the two Bupleurum species, as well as other species from the genus of Bupleurum.

[Expression analyses of BcUGT3 and BcUGT6, and their in vitro expression in Escherichia coli].

The tissue-specific and MeJA-induced transcriptional levels of BcUGT3 and BcUGT6 in Bupleurum chinense were analyzed in the present study. The transcriptional levels of BcUGT3 in root, leaf, flower and fruit were similar and they all were higher than those in stem. The transcriptional level of BcUGT6 was the highest in leaf and the lowest in flower among in all tested tissues. With non-treated adventitious roots as control, BcUGT6's transcriptional levels were elevated to nearly 2 folds for 2 h, 8 h, 24 h, 2 d and 4 d in MeJA-treated adventitious roots of B. chinense. It showed that the transcriptional level of BcUGT6 was slightly affected by MeJA. While, BcUGT3's transcriptional levels were gradually elevated, and till 4 d after MeJA treatment, the expression level was about 7 folds than that of non-treated control. Using pET-28a (+), the expressions of two genes was investigated. Induced by IPTG, the target proteins were expressed in E. coli and then purified. All the results obtained in the present study will be helpful for follow-up bio-function analysis of BcUGT3 and BcUGT6.

[Induction of hairy roots and plantlet regeneration of Bupleurum chinense DC].

In this study, the induction of hairy roots of Bupleurum chinense DC. was explored and established after experiments at different conditions: A. rhizogenes A4 was used to infect the leaves bases of B. chinense tube seedlings. The explants were co-cultured on Phytagel-solidified media for 3 days and then, were turned into solid media, similar with the co-culture media except that bacteriostat was added. After 10 days, rootlets began to appear and after 4 to 5 weeks, rootlets can be converted into liquid shaking culture stage. Plants regeneration from hairy root was useful for the research of new germplasm production and the variety improvement breeding. In the present study, the regenerated plants were obtained. One approach was to continuously culture under light conditions the seedlings which parting off spontaneously from the hairy roots during liquid shaking culture. The other approach was to culture the callus-like tissues produced by hairy roots with the optimized regeneration media for the induction of regenerated plants. The results of present study provide a technique to induce hairy roots and plantlet regeneration of B. chinense and this technique is helpful for the researches on metabolism, especially on the Agrobacterium-mediated genetic transformation of B. chinense.

[Expression analysis of glycosyltransferase BcUGT1 from Bupleurum chinense DC. and its expression in E. coli and the target protein purification].

The ORF sequence of glycosyltransferase gene BcUGT1 cloned from Bupleurum chinense DC. was analyzed and its three dimentional structure was predicted. Using qRT-PCR method, the expression characteristics of BcUGT1 after methyl jasmonate (MeJA) induction and in different plant tissues were investigated. The results showed that BcUGT1 may be involved in saikosaponin biosynthesis in B. chinense. Thereafter, the recombinant vectors of BcUGT1 were constructed for its expression in E. coli. The target protein was successfully expressed and purified. In the present study, three vectors, pRSET-A, pET-28a (+) and pET-30a (+), and three isolates of E. coli, BL21 (DE3) plysS, BL21A1 and BL21-CodonPlus (DE3)-RIPL were used under different induction conditions, such as different concentrations and during times of inducers (L-arabinose and IPTG) and different inducing temperatures. The results showed that in the condition of 0.5 or 1 mmol x L(-1) IPTG, 16 degrees C, 20 h, target protein expressed in BL21-CodonPlus (DE3)-RIPL with pET-28a (+) or pET-30a (+) as vector. Using PrepEase His-tagged protein purification kit, the target protein was purified. The present work will be helpful for follow-up bio-function analysis of BcUGT1.

[Studies on high efficient plant regeneration system from anther callus of Bupleurum chinense].

The callus of Bupleurum chinense with anthers at the stage of uninucleate was induced. After several subcultures, anther calli of B. chinense were cultured at 20 MS culture mediums with different plant hormones to differentiate into plantlets. Differentiation of callus was detected after 21 and 49 days to select the most effective medium. There were 19 culture mediums in which anther callus could differentiate into plantlets with differentiation rate range from 3% to 60% , and most less than 20%. MS + KT 0.5 mg x L(-1) + sucrose 30 g c L(-1) + phytagel 5 g x L(-1) was the best differentiation medium with the differentiation rate of 60%, followed by MS + ZT 1.0 mg x L(-1) + sucrose 30 g x L(-1) + phytagel 5 g x L(-1) with the differentiation rate of 58%. Then plantlets were transferred to rooting medium to obtain whole plant. All plantlets could root in the rooting medium of MS + sucrose 30 g x L(-1) + phytagel 5 g x L(-1) and 1/2 MS + NAA 0.5 mg x L(-1) + sucrose 30 g x L(-1) + phytagel of 5 g L(-1) with the rooting rate of 100%. As a result, the high efficient and stable plant regeneration system was established from anther callus of B. chinense.

[Cloning of UGT gene of Bupleurum chinense and construction of over expressing and RNAi transgenic vectors].

OBJECTIVE: To clone the full-length cDNA of a uridine diphosphate glycosyltransferase (UGT) gene which may be involved in saikosaponin biosynthesis of Bupleurum chinense, and construct the transgenic vectors for over expression and RNAi of the cloned UGT. These works will provide foundation for further its function analysis by transgene study. METHOD: RAGE and LD-PCR were used to clone the full-length cDNA of the UGT, on the basis of its partial cDNA sequence obtained from our previous 454-sequenced dataset. The ORF and partial sequences of the UGT were PCR cloned using primers with corresponding restriction enzymes cutting sites. The PCR products were digested with corresponding restriction enzymes and then were inserted into pCAMBIA-SUPER 1 300 and pHANNIBAL. The recombinant pHANNIBAL were digested with Not I and then were inserted into a binary vector, pART27. Finally, the transgenic vectors for over expression and RNAi of the cloned UGT were constructed. RESULT: The full-length cDNA of a UGT were cloned from B. chinense. The recombinant vectors for over expression and RNAi of the UGT were obtained. CONCLUSION: Our works on full-length cDNA cloning and transgenic vectors construction provide a substantial foundation for follow-up biofunction analysis of the UGT through transgenic research.

Transcriptome analysis of Bupleurum chinense focusing on genes involved in the biosynthesis of saikosaponins.

BACKGROUND: Bupleurum chinense DC. is a widely used traditional Chinese medicinal plant. Saikosaponins are the major bioactive constituents of B. chinense, but relatively little is known about saikosaponin biosynthesis. The 454 pyrosequencing technology provides a promising opportunity for finding novel genes that participate in plant metabolism. Consequently, this technology may help to identify the candidate genes involved in the saikosaponin biosynthetic pathway. RESULTS: One-quarter of the 454 pyrosequencing runs produced a total of 195, 088 high-quality reads, with an average read length of 356 bases (NCBI SRA accession SRA039388). A de novo assembly generated 24, 037 unique sequences (22, 748 contigs and 1, 289 singletons), 12, 649 (52.6%) of which were annotated against three public protein databases using a basic local alignment search tool (E-value ≤1e-10). All unique sequences were compared with NCBI expressed sequence tags (ESTs) (237) and encoding sequences (44) from the Bupleurum genus, and with a Sanger-sequenced EST dataset (3, 111). The 23, 173 (96.4%) unique sequences obtained in the present study represent novel Bupleurum genes. The ESTs of genes related to saikosaponin biosynthesis were found to encode known enzymes that catalyze the formation of the saikosaponin backbone; 246 cytochrome P450 (P450s) and 102 glycosyltransferases (GTs) unique sequences were also found in the 454 dataset. Full length cDNAs of 7 P450s and 7 uridine diphosphate GTs (UGTs) were verified by reverse transcriptase polymerase chain reaction or by cloning using 5' and/or 3' rapid amplification of cDNA ends. Two P450s and three UGTs were identified as the most likely candidates involved in saikosaponin biosynthesis. This finding was based on the coordinate up-regulation of their expression with ß-AS in methyl jasmonate-treated adventitious roots and on their similar expression patterns with ß-AS in various B. chinense tissues. CONCLUSIONS: A collection of high-quality ESTs for B. chinense obtained by 454 pyrosequencing is provided here for the first time. These data should aid further research on the functional genomics of B. chinense and other Bupleurum species. The candidate genes for enzymes involved in saikosaponin biosynthesis, especially the P450s and UGTs, that were revealed provide a substantial foundation for follow-up research on the metabolism and regulation of the saikosaponins.