A novel bHLH gene responsive to low nitrogen positively regulates the biosynthesis of medicinal tropane alkaloids in Atropa belladonna.

Medicinal tropane alkaloids (TAs), including hyoscyamine, anisodamine and scopolamine, are essential anticholinergic drugs specifically produced in several solanaceous plants. Atropa belladonna is one of the most important medicinal plants that produces TAs. Therefore, it is necessary to cultivate new A. belladonna germplasm with the high content of TAs. Here, we found that the levels of TAs were elevated under low nitrogen (LN) condition, and identified a LN-responsive bHLH transcription factor (TF) of A. belladonna (named LNIR) regulating the biosynthesis of TAs. The expression level of LNIR was highest in secondary roots where TAs are synthesized specifically, and was significantly induced by LN. Further research revealed that LNIR directly activated the transcription of hyoscyamine 6ß-hydroxylase gene (H6H) by binding to its promoter, which converts hyoscyamine into anisodamine and subsequently epoxidizes anisodamine to form scopolamine. Overexpression of LNIR upregulated the expression levels of TA biosynthesis genes and consequently led to the increased production of TAs. In summary, we functionally identified a LN-responsive bHLH gene that facilitated the development of A. belladonna with high-yield TAs under the decreased usage of nitrogen fertilizer.

BHLH IRIDOID SYNTHESIS 3 is a member of a bHLH gene cluster regulating terpenoid indole alkaloid biosynthesis in Catharanthus roseus.

Basic helix-loop-helix (bHLH) transcription factors (TFs) are key regulators of plant specialized metabolites, including terpenoid indole alkaloids (TIAs) in Catharanthus roseus. Two previously characterized subgroup-IVa bHLH TFs, BIS1 (bHLH Iridoid Synthesis 1) and BIS2 regulate iridoid biosynthesis in the TIA pathway. We reanalyzed the recently updated C. roseus genome sequence and discovered that BIS1 and BIS2 are clustered on the same genomic scaffold with a previously uncharacterized bHLH gene, designated as BIS3. Only a few bHLH gene clusters have been studied to date. Comparative analysis of 49 genome sequences from different plant lineages revealed the presence of analogous bHLH clusters in core angiosperms, including the medicinal plants Calotropis gigantea (giant milkweed) and Gelsemium sempervirens (yellow jessamine), but not in the analyzed basal angiosperm and lower plants. Similar to the iridoid pathway genes, BIS3 is highly expressed in roots and induced by methyl jasmonate. BIS3 activates the promoters of iridoid branch genes, geraniol synthase (GES), geraniol 10-hydroxylase (G10H), 8-hydroxygeraniol oxidoreductase (8HGO), iridoid synthase (IS), 7-deoxyloganetic acid glucosyl transferase (7-DLGT), and 7-deoxyloganic acid hydroxylase (7DLH), but not iridoid oxidase (IO). Transactivation of the promoters was abolished when BIS3 is converted to a dominant repressor by fusing with the ERF-associated amphiphilic repression (EAR) sequence. In addition, BIS3 acts synergistically with BIS1 and BIS2 to activate the G10H promoter in tobacco cells. Mutation of the known bHLH TF binding motif, G-box (CACGTG) in the G10H promoter significantly reduced but did not abolish the transactivation by BIS3. Promoter deletion analysis of G10H suggests that the sequences adjacent to the G-box are also involved in the regulation by BIS3. Overexpression of BIS3 in C. roseus flower petals significantly upregulated the expression of iridoid biosynthetic genes and increased loganic acid accumulation. BIS2 expression was significantly induced by BIS3 although BIS3 did not directly activate the BIS2 promoter. Our results advance our understanding of the regulation of plant specialized metabolites by bHLH TF clusters.

The bHLH gene family and its response to saline stress in Jilin ginseng, Panax ginseng C.A. Meyer.

Basic helix-loop-helix (bHLH) gene family is a gene family of transcription factors that plays essential roles in plant growth and development, secondary metabolism and response to biotic and abiotic stresses. Therefore, a comprehensive knowledge of the bHLH gene family is paramount to understand the molecular mechanisms underlying these processes and develop advanced technologies to manipulate the processes efficiently. Ginseng, Panax ginseng C.A. Meyer, is a well-known medicinal herb; however, little is known  about the bHLH genes (PgbHLH) in the species. Here, we identified 137 PgbHLH genes from Jilin ginseng cultivar, Damaya, widely cultivated in Jilin, China, of which 50 are newly identified by pan-genome analysis. These 137 PgbHLH genes were phylogenetically classified into 26 subfamilies, suggesting their sequence diversification. They are alternatively spliced into 366 transcripts in a 4-year-old plant and involved in 11 functional subcategories of the gene ontology, indicating their functional differentiation in ginseng. The expressions of the PgbHLH genes dramatically vary spatio-temporally and across 42 genotypes, but they are still somehow functionally correlated. Moreover, the PgbHLH gene family, at least some of its genes, is shown to have roles in plant response to the abiotic stress of saline. These results provide a new insight into the evolution and functional differentiation of the bHLH gene family in plants, new bHLH genes to the PgbHLH gene family, and saline stress-responsive genes for genetic improvement in ginseng and other plant species.

[Cloning and expression analysis of CtbHLH transcription factor gene from Carthamus tinctorius and construction of plant expression vector].

To clone bHLH( basic helix-loop-helix) gene from Carthamus tinctorius,analyze the expression level in different plant tissues and construct the plant expression vector. The bHLH1 gene was cloned by RT-PCR techniques,and the protein characteristics were analyzed by bioinformatics,and phylogenetic tree was constructed. The expression of bHLH1 gene in different tissues and the roots after inoculated by Fusarium oxysporum were analyzed using real time-PCR,and the plant expression vector p BASTA-bHLH1 was constructed. The obtained ORF sequence of bHLH1 gene was 897 bp,encoded a protein of 298 amino acids. Sequence alignment and phylogenetic tree analyses showed that C. tinctorius bHLH1 had a certain homology with other species of amino acids,and was the most similar to the amino acid sequence of tobacco. Real-time PCR results showed significant differences,CtbHLH1 gene in red flower petals in different tissues and different flowering period had remarkable difference in expression level,its high amount expressed in petals,flowers third day after blossom expressed the highest quantity,at the end of the flowering the expression quantity is low. In addition,it is expressed in the root,and the expression in the stem and leaves is extremely low. The bHLH1 gene of C. tinctorius is successfully cloned,and the expression is analyzed. The plant expression vector p BASTA-bHLH is constructed.