The WRKY gene family in the halophyte Limonium bicolor: identification, expression analysis, and regulation of salt stress tolerance.

KEY MESSAGE: 63 L. bicolor WRKY genes were identified and their informatics was analyzed. The results suggested that the LbWRKY genes involved in the development and salt secretion of salt glands in L. bicolor. Salt stress, as a universal abiotic stress, severely inhibits the growth and development of plants. WRKY transcription factors play a vital role in plant growth and development, as well as in response to various stresses. Nevertheless, little is known of systematic genome-wide analysis of the WRKY genes in Limonium bicolor, a model recretohalophyte. In this study, 63 L. bicolor WRKY genes were identified (LbWRKY1-63), which were unevenly distributed across seven chromosomes and one scaffold. Based on the structural and phylogenetic characteristics, 63 LbWRKYs are divided into three main groups. Cis-elements in the LbWRKY promoters were related to growth and development, phytohormone responses, and stress responses. Colinearity analysis showed strong colinearity between LbWRKYs and GmWRKYs from soybean (Glycine max). Therefore, LbWRKY genes maybe have similar functions to GmWRKY genes. Expression analysis showed that 28 LbWRKY genes are highly expressed in roots, 9 in stems, 26 in leaves, and 12 in flowers and most LbWRKY genes responded to NaCl, ABA, and PEG6000. Silencing LbWRKY10 reduced salt gland density and salt secretion ability of leaves, and the salt tolerance of the species. Consistent with this, genes associated with salt gland development were markedly down-regulated in the LbWRKY10-silenced lines. Our findings suggested that the LbWRKY genes involved in the development and salt secretion of salt glands in L. bicolor. Our research provides new insights into the functions of the WRKY family in halophytes.

Promoting effect of low concentration strontium on photosynthetic performance of Chinese cabbage seedlings: Combined leaf characteristics, photosynthetic carbon assimilation and chlorophyll fluorescence.

Low concentration strontium (LC-Sr) can promote the growth of plants. In order to explore its promoting mechanism from the aspect of photosynthesis, the leaf characteristics, CO2 assimilation and chlorophyll (Chl) a fluorescence kinetics were investigated with hydroponically LC-Sr-treated Chinese cabbage seedlings. After a 28-d treatment to SrCl2 at different concentrations (0.1, 0.2, 0.5, and 1.0 mmol L-1), we observed an increase in the specific leaf weight (SLW) of Chinese cabbage compared with the control group. Notably, as the strontium concentration increased, a more pronounced improvement trend in the contents of Chl and protein in the leaves was observed, contributing to the enhancement of photosynthesis. However, the statistical differences in Pn among various LC-Sr treatments were not significant. Nevertheless, the leaf starch content exhibited a significant increase after LC-Sr treatments. Additionally, Chl a fluorescence transient has been used as a sensitive indicator of the promotional effect of LC-Sr on photosynthesis. The results of fluorescence parameters showed that LC-Sr treatments accelerated the light reaction speed of leaves (Tfm, dV/dto, dVG/dto), improved the energy utilization efficiency of photosystem (PSI and PSII) (ETo/CSo, ψET,ψRE, δRo, φRo), and ultimately enhanced the photosynthetic performance of leaves (PIabs, SFIabs, DFabs). The increased RCs/CSo and Sm contributed to the enhancement of the light reaction activity of strontium-treated leaves. The LC-Sr treatments had no interference with the calcium absorption, and notably enhanced the photosynthetic capacity of Chinese cabbage, shedding light on potential benefits of LC-Sr for crop cultivation.

[Effects of tHMGR from three different plant sources on mevalonate (MVA) pathway flux in Saccharomyces cerevisiae].

In this study, we used bioinformatic tools to analyze the 3-hydroxy-3-methylglutaryl-CoA reductase(HMGR) genes from Glycyrrhiza uralensis, Artemisia annua, and Arabidopsis thaliana. The results indicated that GuHMGR and AaHMGR contained two transmembrane regions while AtHMGR had three transmembrane regions. GuHMGR, AaHMGR, and AtHMGR all had the active center for catalysis. Three truncated HMGR genes(tHMGRs) of G. uralensi, A. annua, and A. thaliana were respectively ligated to pYES3 vector to construct the recombinant plasmids pYES3-tGuHMGR,pYES3-tAaHMGR,and pYES3-tAtHMGR. Afterwards, the control plasmid pYES3 and the three plasmids and were respectively introduced into Saccharomyces cerevisiae Cen.pk2-1 D, which yielded strains Y0, Y1, Y2, and Y3, respectively. The content of squalene, lanosterol, and ergosterol in these strains was measured by GC-MS. The relative expression of tGuHMGR, tAaHMGR, and tAtHMGR in strains Y1, Y2, and Y3 was determined by quantitative real-time PCR. The results showed that the strain overexpressing tAaHMGR had the highest yield of squalene and the highest total yield of squalene, ergosterol, and lanosterol. The quantitative real-time PCR showed higher relative expression of tAaHMGR than tGuHMGR, consistent with the strain fermentation result. We selected a superior tHMGR by comparing the effects of different tHMGRs on the mevalonate(MVA) pathway flux in S. cerevisiae. The findings can provide a reference for the construction of S. cerevisiae strains with high yields of squalene and terpenoid precursors.

[Regulation of ß-mercuryl alcohol metabolic flow in Saccharomyces cerevisiae cells].

In this study, citrate synthase gene(CIT2), and malate synthase gene(MLS1) were successfully knocked out in ß-amyrin-producing yeast cells by using CRISPR/CAS9. The promoter of phosphoglucose isomerase gene(PGI1) was replaced by that of cytochrome c oxidase subunit Ⅶa(Cox9)to weaken its expression, aiming to channel more carbon flux into the NADPH-producing pathway. The fermentation results showed that CIT2 deletion had no effect on the ß-amyrin production. Compared with the control strain, the production of ß-amyrin was increased by 1.85 times after deleting MLS1, reaching into 3.3 mg·L~(-1). By replacing the promoter of PGI1, the ß-amyrin yield was 3.75 times higher than that of the control strain, reaching up to 6.7 mg·L~(-1). This study successfully knocked out the CITT2 and MLS1 genes and weakened the PGI1 gene by using CRISPR/CAS9, which directly influenced the production of ß-amyrin and provided some reference for the the metabolic engineering of triterpernoid producing strain.

[Study of heterologous efficient synthesis of ß-amyrin and high-density fermentation].

In this study, the synthetic pathway of ß-amyrin was constructed in the pre-constructed Saccharomyces cerevisiae chassis strain Y0 by introducing ß-amyrin synthase from Glycyrrhiza uralensis, resulting strain Y1-C20-6, which successfully produced ß-amyrin up to 5.97 mg·L~(-1). Then, the mevalonate pyrophosphate decarboxylase gene(ERG19), mevalonate kinase gene(ERG12), 3-hydroxy-3-methylglutaryl-CoA synthase gene(ERG13), phosphomevalonate kinase gene(ERG8) and IPP isomerase gene(IDI1)were overexpressed to promoted the metabolic fluxto the direction of ß-amyrin synthesis for further improving ß-amyrin production, resulting the strain Y2-C2-4 which produced ß-amyrin of 10.3 mg·L~(-1)under the shake flask fermentation condition. This is 100% higher than that of strain Y1-C20-6, illustrating the positive effect of the metabolic engineering strategy applied in this study. The titer of ß-amyrin was further improved up to 157.4 mg·L~(-1) in the fed-batch fermentation, which was almost 26 fold of that produced by strain Y1-C20-6. This study not only laid the foundation for the biosynthesis of ß-amyrin but also provided a favorable chassis strain for elucidation of cytochrome oxidases and glycosyltransferases of ß-amyrin-based triterpenoids.

Overexpression of PagLOL1b improves drought tolerance through increasing water use efficiency and reactive oxygen species scavenging in transgenic poplar.

LESION SIMULATING DISEASE1 (LSD) family genes play a key role in plant response to abiotic and biotic stress. However, their functions in the resistance of tree to drought stress are still largely not clear. Here, five LSD family genes in poplar genome were identified. Phylogenetic and collinear relationship analysis showed that they belonged to LSD, LSD-one-like 1 (LOL1) and LSD-one-like 2 (LOL2) subfamilies, and experienced two segmental duplication events. PagLSDs were highly conserved in gene structure, and all PagLSDs contained at least two LSD domains. Expression pattern and cis-acting element analyses showed that PagLSDs were widely expressed in different organs, significantly induced by polyethylene glycol, and possessed a great number of plant growth, development, plant hormones, and biotic and abiotic stress elements in their promoter regions. Further physiological experiments with transgenic poplar plants revealed that overexpression of PagLOL1b significantly enhanced the drought tolerance of transgenic plants. The improved drought tolerance was closely associated with the significant increase in stomatal closure, water use efficiency, antioxidant enzyme gene expression and antioxidant enzyme activity in transgenic plants. The results in our study imply that PagLOL1b has great potential in the engineering of new tree varieties resistant to drought stress.

Transcription factor PdMYB118 in poplar regulates lignin deposition and xylem differentiation in addition to anthocyanin synthesis through suppressing the expression of PagKNAT2/6b gene.

R2R3-MYB transcription factors function as the master regulators of the phenylpropanoid pathway in which both lignin and anthocyanin are produced. In poplar, R2R3-MYB transcription factor PdMYB118 positively regulates anthocyanin production to change leaf color. However, the molecular mechanism by which it controls different branches of the phenylpropanoid pathway still remains poorly understood. Here, we reported that in addition to anthocyanin synthesis, lignin deposition and xylem differentiation were regulated by PdMYB118 through inhibiting PagKNAT2/6b gene expression. The transgenic poplar plants overexpressing PdMYB118 accumulated more xylem, lignin and anthocyanin. Transcriptome and reverse transcription quantitative PCR analyses revealed that the expression of PagKNAT2/6b gene which inhibited lignin deposition and xylem differentiation was significantly down-regulated in transgenic poplar plants. Subsequent dual-luciferase reporter and yeast-one-hybrid assays demonstrated that PdMYB118 directly inhibited the transcription of PagKNAT2/6b by binding to the AC elements in its promoter region. Further experiments with transgenic poplar plants overexpressing PagKNAT2/6b demonstrated that overexpression of PagKNAT2/6b in the PdMYB118 overexpression background rescued lignin accumulation and xylem width to the same level of wild type plants. The findings in this work suggest that PdMYB118 is involved in the lignin deposition and xylem differentiation via modulating the expression of PagKNAT2/6b, and the PdMYB118- PagKNAT2/6b model can be used for the genetic breeding of new woody tree with high lignin production.

PagDA1a and PagDA1b expression improves salt and drought resistance in transgenic poplar through regulating ion homeostasis and reactive oxygen species scavenging.

DA1/DAR proteins play a crucial role in plant biomass production. However, their functions in woody plants in response to abiotic stress are still unknown. In this study, a total number of six PagDA1/DAR family genes were identified in the poplar genome, and the biological functions of PagDA1a and PagDA1b in the resistance to salt and drought stresses were investigated in transgenic poplar. PagDA1a and PagDA1b were ubiquitously expressed in roots, stems, and leaves, with predominant expression in roots, and were significantly induced by abiotic stress and ABA. Transgenic poplar overexpressing either PagDA1a or PagDA1b showed restrained growth but improved resistance to salt and drought stresses. Further ion content and antioxidant enzyme expression analyses exhibited that transgenic poplar accumulated less sodium (Na+), hydrogen peroxide (H2O2) and malondialdehyde (MDA) in the leaves, accompanied with increased activity of superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT), and up-regulated transcription of SOD1, APX1, and CAT2. Our observations demonstrate that PagDA1a and PagDA1b improve salt and drought tolerance through ion homeostasis optimization and ROS scavenging ability enhancement in transgenic poplar, and both can be used for the future genetic breeding of new salt and drought tolerant tree species.

Production of 11-Oxo-ß-Amyrin in Saccharomyces cerevisiae at High Efficiency by Fine-Tuning the Expression Ratio of CYP450:CPR.

The production of glycyrrhetinic acid (GA) and 11-oxo-ß-amyrin, the major bioactive components in liquorice, was typically inhibited by P450 oxidation in Saccharomyces cerevisiae. This study focused on optimizing CYP88D6 oxidation by balancing its expression with cytochrome P450 oxidoreductase (CPR) for the efficient production of 11-oxo-ß-amyrin in yeast. Results indicated that a high CPR:CYP88D6 expression ratio could decrease both 11-oxo-ß-amyrin concentration and turnover ratio of ß-amyrin to 11-oxo-ß-amyrin, whereas a high CYP88D6:CPR expression ratio is beneficial for improving the catalytic activity of CYP88D6 and 11-oxo-ß-amyrin production. Under such a scenario, 91.2% of ß-amyrin was converted into 11-oxo-ß-amyrin in the resulting S. cerevisiae Y321, and 11-oxo-ß-amyrin production was further improved to 810.6 mg/L in fed-batch fermentation. Our study provides new insights into the expression of cytochrome P450 and CPR in maximizing the catalytic activity of P450s, which could guide the construction of cell factories in producing natural products.