Four-Coumarate:CoA ligase gene (4CL) plays multiple important roles in plant growth and development by catalyzing the formation of CoA ester. 4CL belongs to the plant phenylpropane derivative, which is related to the synthesis of flavonoids and lignin and is a key enzyme in the biosynthetic pathway. In this study, 12 4CL genes of Fraxinus mandschurica were identified and named Fm4CL1-Fm4CL12, respectively. The analysis of the expression pattern of Fm4CL genes indicate that Fm4CL-like 1 gene may play a role in the lignin synthesis pathway. Our study indicate that overexpression of Fm4CL-like 1 increases the lignin content of transgenic tobacco by 39.5% compared to WT, and the S/G ratio of transgenic tobacco increased by 19.7% compared with WT. The xylem cell layer of transgenic line is increased by 40% compared to WT, the xylem cell wall thickness increased by 21.6% compared to the WT. Under mannitol-simulated drought stress, the root length of transgenic tobacco is 64% longer than WT, and the seed germination rate of the transgenic lines is 47% higher than that of WT. In addition, the H2O2 content in the transgenic tobacco was 22% lower than that of WT, while the POD and SOD content was higher than WT by 30 and 24% respectively, which showed Fm4CL-like 1 affect the accumulation of reactive oxygen species (ROS). The MDA content and relative conductivity was 25 and 15% lower than WT, respectively. The water loss rate is 16.7% lower than that of WT. The relative expression levels of stress-related genes NtHAK, NtAPX, NtCAT, NtABF2, and NtZFP were higher than those of WT under stress treatment. The stomatal apertures of OE (Overexpression) were 30% smaller than those of WT, and the photosynthetic rate of OE was 48% higher than that of WT. These results showed that the overexpression line exhibited stronger adaptability to osmotic stress than WT. Our results indicate that Fm4CL-like 1 is involved in secondary cell wall development and lignin synthesis. Fm4CL-like 1 play an important role in osmotic stress by affecting cell wall and stomatal development.

Background: Triterpenoids from birch (Betula platyphylla Suk.) exert antitumor and anti-HIV activities. Due to the complexity of plant secondary metabolic pathways, triterpene compounds in plants is not always determined by a single gene; they may be controlled by polygene quantitative traits. Secondary metabolism related to terpenoids involves tissue specificity and localisation of key biosynthetic enzymes. Terpene synthesis is influenced by light, hormones and other signals, as well as upstream transcription factor regulation. Results: Anchor Herein, we identified and characterised two birch MYB transcription factors (TFs) that regulate triterpenoid biosynthesis. BpMYB21 and BpMYB61 are R2R3 TFs that positively and negatively regulate responses to methyl-jasmonate (MeJA) and salicyclic acid (SA), respectively. Expression of BpMYB21 and BpMYB61 was elevated in leaves and stems more than roots during July/August in Harbin, China. BpMYB21 expression was increased by abscisic acid (ABA), MeJA, SA and gibberellins (GAs). BpMYB61 expression in leaves and BpMYB21 expression in stems was reduced by ABA, MeJA and SA, while GAs, ethylene, and injury increased BpMYB61 expression. BpMYB21 was localised in nuclei, while BpMYB61 was detected in cell membranes and nuclei. Promoters for both BpMYB21 (1302 bp) and BpMYB61 (850 bp) were active. BpMYB21 and BpMYB61 were ligated into pYES3, introduced into AnchorINVScl (yeast strain without exogenous genes), INVScl-pYES2-SSAnchorAnchor (transgenic yeast strain harbouring the SS gene from birch), and INVScl-pYES2-SE (transgenic yeast strain harbouring the SE gene from birch), and the squalene content was highest in AnchorINVScl-pYES-MYB21-SS (transgenic yeast strain harbouring SS and MYB21 genes) and INVScl-pYES3-MYB61 (transgenic yeast strain harbouring the MYB61 gene). In BpMYB21 transgenic birch key triterpenoid synthesis genes were up-regulated, and in BpMYB61 transgenic birch AnchorFPS (farnesyl pyrophosphate synthase) and SS (squalene synthase) were up-regulated, but HMGR (3-hydroxy-3-methylglutaryl coenzyme a reductase), BPWAnchor (lupeol synthase), SE (squalene epoxidase) and BPY (b-amyrin synthase) were down-regulated. Both BpMYB21 and BpMYB61 specifically activate SE and BPX (cycloartenol synthase synthesis) promoters. Conclusions: These findings support further functional characterisation of R2R3-MYB genes, and illuminate the regulatory role of BpMYB21 and BpMYB61 in the synthesis of birch triterpenoids.

Birch (Betula platyphylla Suk.) contains triterpenoids with anti-HIV and anti-tumor pharmacological activities. However, the natural abundance of these triterpenoids is low, and their chemical synthesis is costly. Transcription factors have the ability to regulate the metabolite pathways of triterpenoids via multi-gene control, thereby improving metabolite yield. Thus, transcription factors have the potential to facilitate the production of birch triterpenoids. Plant bHLH (basic helix-loop-helix) transcription factors play important roles in stress response and secondary metabolism. In this study, we cloned two genes, BpMYC4 and BpbHLH9, that encode bHLH transcription factors in Betula platyphylla Suk. The open reading frame (ORF) of BpMYC4 was 1452 bp and encoded 483 amino acids, while the ORF of BpbHLH9 was 1140 bp and encoded 379 amino acids. The proteins of BpMYC4 and BpbHLH9 were localized in the cell membrane and nucleus. The tissue-specific expression patterns revealed that BpMYC4 expression in leaves was similar to that in the stem and higher than in the roots. The expression of BpbHLH9 was higher in the leaves than in the root and stem. The expressions of BpMYC4 and BpbHLH9 increased after treatment with abscisic acid, methyl jasmonate, and gibberellin and decreased after treatment with ethephon. The promoters of BpMYC4 and BpbHLH9 were isolated using a genome walking approach, and 900-bp and 1064-bp promoter sequences were obtained for BpMYC4 and BpbHLH9, respectively. The ORF of BpbHLH9 was ligated into yeast expression plasmid pYES3 and introduced into INVScl and INVScl1-pYES2-SS yeast strains. The squalene and total triterpenoid contents in the different INVScl1 transformants decreased in the following order INVScl1-pYES-SS-bHLH9 > INVScl1-pYES3-bHLH9 > INVScl1-pYES2- BpSS > INVScl-pYES2. In BpbHLH9 transgenic birch, the relative expression of the genes that encodes for enzymes critical for triterpenoid synthesis showed a different level of up-regulation compair with wild birch(control), and the contents of betulinic acid, oleanolic acid and betulin in bHLH9–8 transgenic birch were increased by 11.35%, 88.34% and 23.02% compared to in wild birch, respectively. Our results showed that the modulation of BpbHLH9 by different hormones affected triterpenoid synthesis and triterpenoid contents. This is the first report of the cloning of BpbHLH9, and the findings are important for understanding the regulatory role of BpbHLH9 in the synthesis of birch triterpenoids.

The basic helix-loop-helix (bHLH) transcription factors (TFs) are involved in plant morphogenesis and various abiotic and biotic stress responses. However, further exploration is required of drought-responsive bHLH family members and their detailed regulatory mechanisms in Populus. Two bHLH TF genes, PxbHLH01/02, were identified in Populus simonii × P. nigra and cloned. The aim of this study was to examine the role of bHLH TFs in drought tolerance in P. simonii × P. nigra. The results showed that the amino acid sequences of the two genes were homologous to Arabidopsis thaliana UPBEAT1 (AtUPB1) and overexpression of PxbHLH01/02 restored normal root length in the AtUPB1 insertional mutant (upb1-1). The PxbHLH01/02 gene promoter activity analysis suggested that they were involved in stress responses and hormone signaling. Furthermore, Arabidopsis transgenic lines overexpressing PxbHLH01/02 exhibited higher stress tolerance compared with the wild-type. Populus simonii × P. nigra overexpressing PxbHLH02 increased drought tolerance and exhibited higher superoxide dismutase and peroxidase activities, lower H₂O₂ and malondialdehyde content, and lower relative conductivity. The results of transcriptome sequencing (RNA-seq) and quantitative real-time PCR suggested that the response of PxbHLH02 to drought stress was related to abscisic acid (ABA) signal transduction. Overall, the findings of this study suggest that PxbHLH02 from P. simonii × P. nigra functions as a positive regulator of drought stress responses by regulating stomatal aperture and promoting ABA signal transduction.