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Basic helix-loop-helix (bHLH) proteins are the second largest family of transcription factors (TFs) involved in developmental and physiological processes in plants. In this study, 205 putative bHLH TF genes were identified in the genome of Artemisia annua and expression of 122 of these was determined from transcriptomes used to construct the genetic map of A. annua. Analysis of gene expression association allowed division of the 122 bHLH TFs into five groups. Group V, containing 15 members, was tightly associated with artemisinin biosynthesis genes. Phylogenetic analysis indicated that two bHLH TFs, AabHLH106 and AabHLH112, were clustered with Arabidopsis ICE proteins. AabHLH112 was induced by low temperature, while AabHLH106 was not. We therefore chose AabHLH112 for further examination. AabHLH112 was highly expressed in glandular secretory trichomes, flower buds, and leaves. Dual-luciferase assays demonstrated that AabHLH112 enhanced the promoter activity of artemisinin biosynthesis genes and AaERF1 , an AP2/ERF TF that directly and positively regulates artemisinin biosynthesis genes. Yeast one-hybrid assays indicated that AabHLH112 could bind to the AaERF1 promoter, but not to the promoters of artemisinin biosynthesis genes. Overexpression of AabHLH112 significantly up-regulated the expression levels of AaERF1 and artemisinin biosynthesis genes and consequently promoted artemisinin production. [ABSTRACT FROM AUTHOR]

Rhodiola crenulata is a Tibetan native herbal plant belonging to the family of Crassulaceae, which produces the pharmaceutical icariside D2 with the activities of inhibiting angiotensin-converting enzyme and killing leukemia cancer cells. In this study, we functionally characterized a novel UDP-glycosyltransferase (RcUGT1) that converted tyrosol to specifically produce icariside D2 from R. crenulata at molecular and biochemical levels. RcUGT1 was highly expressed in flowers and roots, while the icariside D2 content was much higher in stems than that in other organs, suggesting the potential translocation of icariside D2 from flowers and roots to stems. The high production of icariside D2 in stems provided a reasonable suggestion to farmers to harvest stems instead of roots for icariside D2 production. Enzymatic assays of recombinant RcUGT1 indicated that it converted tyrosol to specifically form icariside D2, with the values of Km 0.97±0.10 mM, Vmax 286±8.26 pKat/mg, Kcat 0.01552 s−1, and Kcat/Km 159.55 s−1 M−1. Functional identification of RcUGT1 facilitated the icariside D2 production through metabolic engineering in plants or synthetic biology in microbes. [ABSTRACT FROM AUTHOR]

In recent years, although several related genes had been cloned and characterized, the role of aldehyde dehydrogenase 1 ( ALDH1), the newly cloned gene involved in artemisinin biosynthesis pathway, is still not clear. In this study, a 2,100-bp ALDH1 promoter region fused with GUS reporter gene was stably transferred into Arabidopsis thaliana. Histochemical staining showed the methyl jasmonate (MeJA) and wounding treatment induced the GUS gene expression specifically in the trichomes of transgenic A. thaliana, consistent with the results that the expression level of ALDH1 gene was increased in the A. annua under MeJA and wounding treatments. Two RAA motifs (AP2/ERF binding site) but no W box (WRKY binding site) motif were identified in the ALDH1 promoter by the analysis through PLACE and plantCARE. Through the dual luciferase reporter assay, we revealed that both AaORA and AaERF2, rather than AaWRKY1, could activate the expression of ALDH1 promoter. Our study shed light on the in-depth understanding of the role of ALDH1 in artemisinin biosynthesis. [ABSTRACT FROM AUTHOR]

Tyrosine decarboxylase initializes salidroside biosynthesis. Metabolic characterization of tyrosine decarboxylase gene from Rhodiola crenulata (RcTYDC) revealed that it played an important role in salidroside biosynthesis. Recombinant 53 kDa RcTYDC converted tyrosine into tyramine. RcTYDC gene expression was induced coordinately with the expression of RcUDPGT (the last gene involved in salidroside biosynthesis) in SA/MeJA treatment; the expression of RcTYDC and RcUDPGT was dramatically upregulated by SA, respectively 49 folds and 36 folds compared with control. MeJA also significantly increased the expression of RcTYDC and RcUDPGT in hairy root cultures. The tissue profile of RcTYDC and RcUDPGT was highly similar: highest expression levels found in stems, higher expression levels in leaves than in flowers and roots. The gene expressing levels were consistent with the salidroside accumulation levels. This strongly suggested that RcTYDC played an important role in salidroside biosynthesis in R. crenulata. Finally, RcTYDC was used to engineering salidroside biosynthetic pathway in R. crenulata hairy roots via metabolic engineering strategy of overexpression. All the transgenic lines showed much higher expression levels of RcTYDC than non-transgenic one. The transgenic lines produced tyramine, tyrosol and salidroside at higher levels, which were respectively 3.21–6.84, 1.50–2.19 and 1.27–3.47 folds compared with the corresponding compound in non-transgenic lines. In conclusion, RcTYDC overexpression promoted tyramine biosynthesis that facilitated more metabolic flux flowing toward the downstream pathway and as a result, the intermediate tyrosol was accumulated more that led to the increased production of the end-product salidroside. [ABSTRACT FROM AUTHOR]

Putrescine, produced via the arginine decarboxylase (ADC)/ornithine decarboxylase (ODC)-mediated pathway, is an initial precursor for polyamines metabolism and the root-specific biosynthesis of medicinal tropane alkaloids (TAs). These alkaloids are widely used as muscarinic acetylcholine antagonists in clinics. Although the functions of ODC in biosynthesis of polyamines and TAs have been well investigated, the role of ADC is still poorly understood. In this study, enzyme inhibitor treatment showed that ADC was involved in the biosynthesis of putrescine-derived metabolites and root growth in Atropa belladonna. Further analysis found that there were six ADC unigenes in the A. belladonna transcriptome, with two of them, AbADC1 and AbADC2 , exhibiting high expression in the roots. To investigate their roles in TAs/polyamines metabolism and root growth, RNA interference (RNAi) was used to suppress either AbADC1 or AbADC2 expression in A. belladonna hairy roots. Suppression of the AbADC1 expression resulted in a significant reduction in the putrescine content and hairy root biomass. However, it had no noticeable effect on the levels of N -methylputrescine and the TAs hyoscyamine, anisodamine, and scopolamine. On the other hand, suppression of AbADC2 expression markedly reduced the levels of putrescine, N -methylputrescine, and TAs, but had no significant effect on hairy root biomass. According to β -glucuronidase (GUS) staining assays, AbADC1 was mainly expressed in the root elongation and division region while AbADC2 was mainly expressed in the cylinder of the root maturation region. These differences in expression led to functional divergence, with AbADC1 primarily regulating root growth and AbADC2 contributing to TA biosynthesis. • RNAi reduced putrescine biosynthesis by suppressing AbADC1 or AbADC2 in A. belladonna hairy roots. • Knocking down AbADC1 reduced hairy root biomass, while knocking down AbADC2 did not. • Knocking down AbADC2 suppressed the biosynthesis of N -methylputrescine and TAs. • AbADC1 exhibited high expression in the root elongation and division region. • AbADC2 was highly expressed in the cylinder of the root maturation region. [ABSTRACT FROM AUTHOR]