CbMYB108 integrates the regulation of diterpene biosynthesis and trichome development in Conyza blinii against UV-B.

Plants synthesize abundant terpenes through glandular trichomes (GTs), thereby protecting themselves from environmental stresses and increasing the economic value in some medicinal plants. However, the potential mechanisms for simultaneously regulating terpenes synthesis and GTs development remain unclear. Here, we showed that terpenes in Conyza blinii could be synthesized through capitate GTs. By treating with appropriate intensity of UV-B, the density of capitate GTs and diterpene content can be increased. Through analyzing corresponding transcriptome, we identified a MYB transcription factor CbMYB108 as a positive regulator of both diterpene synthesis and capitate GT density. Transiently overexpressing/silencing CbMYB108 on C. blinii leaves could increase diterpene synthesis and capitate GT density. Further verification showed that CbMYB108 upregulated CbDXS and CbGGPPS expression in diterpene synthesis pathway. Moreover, CbMYB108 could also upregulated the expression of CbTTG1, key WD40 protein confirmed in this study to promote GT development, rather than through interaction between CbMYB108 and CbTTG1 proteins. Thus, results showed that the UV-B-induced CbMYB108 owned dual-function of simultaneously improving diterpene synthesis and GT development. Our research lays a theoretical foundation for cultivating C. blinii with high terpene content, and broadens the understanding of the integrated mechanism on terpene synthesis and GT development in plants.

The Blinin Accumulation Promoted by CbMYB32 Involved in Conyza blinii Resistance to Nocturnal Low Temperature.

Blinin, a unique terpenoid from Conyza blinii (C. blinii), benefits our health even though this is not its primary function. Physiological and ecological studies have found that the great secondary metabolites participate in important biological processes and relate to species evolution, environmental adaptation, and so on. Moreover, our previous studies have shown that the metabolism and accumulation of blinin has a close correspondence with nocturnal low temperature (NLT). To find out the transcriptional regulation linker in the crosstalk between blinin and NLT, RNA-seq, comparative analysis, and co-expression network were performed. The results indicated that CbMYB32 is located in a nucleus without independent transcriptional activation activity and is probably involved in the metabolism of blinin. Furthermore, we compared the silence and overexpression of CbMYB32 with wild C. blinii. Compared with the overexpression and the wildtype, the CbMYB32 silence line lost more than half of the blinin and detected more peroxide under NLT. Finally, as a characteristic secret of C. blinii, it is reasonable to infer that blinin participates in the NLT adaptation mechanism and has contributed to the systematic evolution of C. blinii.

ABA and SA Participate in the Regulation of Terpenoid Metabolic Flux Induced by Low-Temperature within Conyza blinii.

Under dry-hot valley climates, Conyza blinii (also known as Jin Long Dan Cao), suffers from nocturnal low-temperature stress (LTS) during winter. Here, to investigate the biological significance of terpenoid metabolism during LTS adaptation, the growth state and terpenoid content of C. blinii under different LTS were detected, and analyzed with the changes in phytohormone. When subjected to LTS, the results demonstrated that the growth activity of C. blinii was severely suppressed, while the metabolism activity was smoothly stimulated. Meanwhile, the fluctuation in phytohormone content exhibited three different physiological stages, which are considered the stress response, signal amplification, and stress adaptation. Furthermore, drastic changes occurred in the distribution and accumulation of terpenoids, such as blinin (diterpenoids from MEP) accumulating specifically in leaves and oleanolic acid (triterpenoids from MVA) accumulating evenly and globally. The gene expression of MEP and MVA signal transduction pathways also changes under LTS. In addition, a pharmacological study showed that it may be the ABA-SA crosstalk driven by the LTS signal, that balances the metabolic flux in the MVA and MEP pathways in an individual manner. In summary, this study reveals the different standpoints of ABA and SA, and provides a research foundation for the optimization of the regulation of terpenoid metabolic flux within C. blinii.

Hemin-induced increase in saponin content contributes to the alleviation of osmotic and cold stress damage to Conyza blinii in a heme oxygenase 1-dependent manner.

Hemin can improve the stress resistance of plants through the heme oxygenase system. Additionally, substances contained in plants, such as secondary metabolites, can improve stress resistance. However, few studies have explored the effects of hemin on secondary metabolite content. Therefore, the effects of hemin on saponin synthesis and the mechanism of plant injury relief by hemin in Conyza blinii were investigated in this study. Hemin treatment promoted plant growth and increased the antioxidant enzyme activity and saponin content of C. blinii under osmotic stress and cold stress. Further study showed that hemin could provide sufficient precursors for saponin synthesis by improving the photosynthetic capacity of C. blinii and increasing the gene expression of key enzymes in the saponin synthesis pathway, thus increasing the saponin content. Moreover, the promotion effect of hemin on saponin synthesis is dependent on heme oxygenase-1 and can be reversed by the inhibitor Zn-protoporphyrin-IX (ZnPPIX). This study revealed that hemin can increase the saponin content of C. blinii and alleviate the damage caused by abiotic stress, and it also broadened the understanding of the relationship between hemin and secondary metabolites in plant abiotic stress relief.

Involvement of several putative transporters of different families in ß-cyclocitral-induced alleviation of cadmium toxicity in quinoa (Chenopodium quinoa) seedlings.

Cadmium (Cd) has a serious negative impact on crop growth and human food security. This study investigated the alleviating effect of ß-cyclocitral, a potential heavy metal barrier, on Cd stress in quinoa seedlings and the associated mechanisms. Our results showed that ß-cyclocitral alleviated Cd stress-induced growth inhibition in quinoa seedlings and promoted quinoa seedling root development under Cd stress. Moreover, it maintained the antioxidant system of quinoa seedlings, including the enzymatic, i.e., superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and nonenzymatic, i.e., reduced glutathione (GSH) and ascorbic acid (ASA), antioxidants, which eliminate the damage from excessive reactive oxygen species (ROS). Our results showed that ß-cyclocitral could reduce the amount of Cd absorbed by roots. Furthermore, we systematically identified five transporter families from the quinoa genome, and the RT-qPCR results showed that ZIP, Nramp and YSL gene families were downregulated by ß-cyclocitral to reduce Cd uptake by roots. Thus, ß-cyclocitral promoted the growth, photosynthetic capacity and antioxidant capacity of the aboveground parts of quinoa seedlings. Taken together, these results suggested that the ß-cyclocitral-induced decrease in Cd uptake may be caused by the downregulation of several selected transporter genes.

Enhanced antioxidant capacity and upregulated transporter genes contribute to the UV-B-induced increase in blinin in Conyza blinii.

Conyza blinii (C. blinii) is a traditional Chinese medicinal plant mainly grown in Sichuan, China. C. blinii is suitable for studying the mechanism of plant tolerance to UV-B due to its living conditions, characterized by a high altitude and exposure to strong ultraviolet radiation. Our results showed that the growth and photosynthetic activity of C. blinii were improved under a specific intensity of UV-B, rather than being significantly inhibited. Although UV-B increased the content of reactive oxygen species (ROS) in C. blinii, the activities of antioxidative enzymes were elevated, including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), which contributed to the elimination of ROS. Additionally, the content of blinin, the characteristic diterpene in C. blinii, was markedly increased by UV-B. Furthermore, RNA sequencing analyses were used to explore the molecular mechanism of UV-B tolerance in C. blinii. According to the results, most of the key enzyme genes in the blinin synthesis pathway were upregulated by UV-B. In addition, 23 upregulated terpene transporter genes were identified, and these genes might participate in blinin transport during the response to UV-B. Taken together, these results implied that enhanced antioxidant capacity and upregulated transporter genes contributed to increased synthesis of blinin in response to UV-B in C. blinii.

Ferrous iron-induced increases in capitate glandular trichome density and upregulation of CbHO-1 contributes to increases in blinin content in Conyza blinii.

MAIN CONCLUSION: Ferrous iron can promote the development of glandular trichomes and increase the content of blinin, which depends on CbHO-1 expression. Conyza blinii (C. blinii) is a unique Chinese herbal medicine that grows in Sichuan Province, China. Because the habitat of C. blinii is an iron ore mining area with abundant iron content, this species can be used as one of the best materials to study the mechanism of plant tolerance to iron. In this study, C. blinii was treated with ferrous-EDTA solutions at different concentrations, and it was found that the tolerance value of C. blinii to iron was 200 µM. Under this concentration, the plant height, root length, biomass, and iron content of C. blinii increased to the maximum values, and the effect was dependent on the upregulated expression of CbHO-1. At the same time, under ferrous iron, the photosynthetic capacity and capitate glandular trichome density of C. blinii also significantly increased, providing precursors and sites for the synthesis of blinin, thus significantly increasing the content of blinin. These processes were also dependent on the high expression of CbHO-1. Correlation analysis showed that there were strong positive correlations between iron content, capitate glandular trichome density, CbHO-1 gene expression, and blinin content. This study explored the effects of ferrous iron on the physiology and biochemistry of C. blinii, greatly improving our understanding of the mechanism of iron tolerance in C. blinii.

Purification, characterization and antioxidant activities in vitro of polysaccharides from Amaranthus hybridus L.

BACKGROUND: Amaranthus hybridus L. is an annual, erect or less commonly ascending herb that is a member of the Amaranthaceae family. Polysaccharides extracted from traditional Chinese medicines may be effective substances with antioxidant activity. METHODS: In this study, we isolated crude polysaccharides from A. hybridus (AHP-M) using microwave-assisted extraction. Then, the AHP-M was purified by chromatography with DEAE-32 cellulose, and two fractions, AHP-M-1 and AHP-M-2, were obtained. The structural characteristics of AHP-M-1 and AHP-M-2 were investigated, and their antioxidant activities were analyzed in vitro. RESULTS: We found that the monosaccharide composition of AHP-M-1 was different from that of AHP-M-2. The molecular weights of AHP-M-1 and AHP-M-2 were 77.625 kDa and 93.325 kDa, respectively. The results showed that the antioxidant activity of AHP-M-2 was better than that of AHP-M-1. For AHP-M-2, the DPPH radical scavenging rate at a concentration of 2 mg/mL was 78.87%, the hydroxyl radical scavenging rate was 39.34%, the superoxide anion radical scavenging rate was 80.2%, and the reduction ability of Fe3+ was approximately 0.90. The total antioxidant capacity per milligram of AHP-M-2 was 6.42, which was higher than that of Vitamin C (Vc). CONCLUSION: The in vitro test indicated that AHP-M-1 and AHP-M-2 have good antioxidant activity, demonstrating that A. hybridus L. polysaccharide has immense potential as a natural antioxidants.

Genome-wide investigation of the ZF-HD gene family in Tartary buckwheat (Fagopyrum tataricum).

BACKGROUND: ZF-HD is a family of genes that play an important role in plant growth, development, some studies have found that after overexpression AtZHD1 in Arabidopsis thaliana, florescence advance, the seeds get bigger and the life span of seeds is prolonged, moreover, ZF-HD genes are also participate in responding to adversity stress. The whole genome of the ZF-HD gene family has been studied in several model plants, such as Arabidopsis thaliana and rice. However, there has been little research on the ZF-HD genes in Tartary buckwheat (Fagopyrum tataricum), which is an important edible and medicinal crop. The recently published whole genome sequence of Tartary buckwheat allows us to study the tissue and expression profiles of the ZF-HD gene family in Tartary buckwheat on a genome-wide basis. RESULTS: In this study, the whole genome and expression profile of the ZF-HD gene family were analyzed for the first time in Tartary buckwheat. We identified 20 FtZF-HD genes and divided them into MIF and ZHD subfamilies according to phylogeny. The ZHD genes were divided into 5 subfamilies. Twenty FtZF-HD genes were distributed on 7 chromosomes, and almost all the genes had no introns. We detected seven pairs of chromosomes with fragment repeats, but no tandem repeats were detected. In different tissues and at different fruit development stages, the FtZF-HD genes obtained by a real-time quantitative PCR analysis showed obvious expression patterns. CONCLUSIONS: In this study, 20 FtZF-HD genes were identified in Tartary buckwheat, and the structures, evolution and expression patterns of the proteins were studied. Our findings provide a valuable basis for further analysis of the biological function of the ZF-HD gene family. Our study also laid a foundation for the improvement of Tartary buckwheat crops.

Genome-wide investigation of the AP2/ERF gene family in tartary buckwheat (Fagopyum Tataricum).

BACKGROUND: AP2/ERF transcription factors perform indispensable functions in various biological processes, such as plant growth, development, biotic and abiotic stresses responses. The AP2/ERF transcription factor family has been identified in many plants, and several AP2/ERF transcription factors from Arabidopsis thaliana (A. thaliana) have been functionally characterized. However, little research has been conducted on the AP2/ERF genes of tartary buckwheat (Fagopyum tataricum), which is an important edible and medicinal crop. The recently published whole genome sequence of tartary buckwheat allowed us to study the tissue and expression profiles of AP2/ERF genes in tartary buckwheat on a genome-wide basis. RESULTS: In this study, 134 AP2/ERF genes of tartary buckwheat (FtAP2/ERF) were identified and renamed according to the chromosomal distribution of the FtAP2/ERF genes. According to the number conserved domains and gene structure, the AP2/ERF genes were divided into three subfamilies by phylogenetic tree analysis, namely, AP2 (15 members), ERF (116 members) and RAV (3 members). A total of 10 motifs were detected in tartary buckwheat AP2/ERF genes, and some of the unique motifs were found to be important for the function of AP2/ERF genes. CONCLUSION: A comprehensive analysis of AP2/ERF gene expression patterns in different tissues and fruit development stages by quantitative real-time PCR (qRT-PCR) showed that they played an important role in the growth and development of tartary buckwheat, and genes that might regulate flower and fruit development were preliminarily identified. This systematic analysis establishes a foundation for further studies of the functional characteristics of FtAP2/ERF genes and improvement of tartary buckwheat crops.

The jasmonate-responsive transcription factor CbWRKY24 regulates terpenoid biosynthetic genes to promote saponin biosynthesis in Conyza blinii H. Lév.

Conyza blinii H. Lév., the most effective component is saponin, is a biennial medicinal material that needs to be overwintered. WRKY transcription factors family is a large protein superfamily that plays a predominant role in plant secondary metabolism, but their characteristics and functions have not been identified in C. blinii. The CbWRKY24 sequence was selectedfrom the transcriptome database of the C. blinii leaves constructed in our laboratory. Phylogenetic tree analysis revealed that it was associated with AaWRKY1 which can regulate artemisinin synthesis in Artemisia annua. Expression analysis in C. blinii revealed that CbWRKY24 was mainly induced by methyl jasmonate (MeJA) and cold treatments. Transcriptional activity assay showed that it had an independent biological activity. Overexpression of CbWRKY24 in transient transformed C. blinii resulted in improved totalsaponins content, which was attributed to upregulate the expression level of keys genes from mevalonate (MVA) pathway in transient transformed plants compared to wild type (WT) plants. Meanwhile, overexpression the CbWRKY24 in transient transformed tomato fruits showed that the transcript level of related genes in lycopene pathway decreased significantly when compared to WT tomatofruits. Additionally, the MeJA-response-element was found in the promoter regions of CbWRKY24 and the histochemical staining experiments showed that promoter had GUS activity in transiently transformed tobacco leaves. In summary, our results indicated that we may have found a transcription factor that can regulate the biosynthesis of terpenoids in C. blinii.

Genome-Wide Investigation of the Auxin Response Factor Gene Family in Tartary Buckwheat (Fagopyrum tataricum).

Auxin signaling plays an important role in plant growth and development. It responds to various developmental and environmental events, such as embryogenesis, organogenesis, shoot elongation, tropical growth, lateral root formation, flower and fruit development, tissue and organ architecture, and vascular differentiation. However, there has been little research on the Auxin Response Factor (ARF) genes of tartary buckwheat (Fagopyrum tataricum), an important edible and medicinal crop. The recent publication of the whole-genome sequence of tartary buckwheat enables us to study the tissue and expression profile of the FtARF gene on a genome-wide basis. In this study, 20 ARF (FtARF) genes were identified and renamed according to the chromosomal distribution of the FtARF genes. The results showed that the FtARF genes belonged to the related sister pair, and the chromosomal map showed that the duplication of FtARFs was related to the duplication of the chromosome blocks. The duplication of some FtARF genes shows conserved intron/exon structure, which is different from other genes, suggesting that the function of these genes may be diverse. Real-time quantitative PCR analysis exhibited distinct expression patterns of FtARF genes in various tissues and in response to exogenous auxin during fruit development. In this study, 20 FtARF genes were identified, and the structure, evolution, and expression patterns of the proteins were studied. This systematic analysis laid a foundation for the further study of the functional characteristics of the ARF genes and for the improvement of tartary buckwheat crops.

The Potential Role of Auxin and Abscisic Acid Balance and FtARF2 in the Final Size Determination of Tartary Buckwheat Fruit.

Tartary buckwheat is a type of cultivated medicinal and edible crop with good economic and nutritional value. Knowledge of the final fruit size of buckwheat is critical to its yield increase. In this study, the fruit development of two species of Tartary buckwheat in the Polygonaceae was analyzed. During fruit development, the size/weight, the contents of auxin (AUX)/abscisic acid (ABA), the number of cells, and the changes of embryo were measured and observed; and the two fruit materials were compared to determine the related mechanisms that affected fruit size and the potential factors that regulated the final fruit size. The early events during embryogenesis greatly influenced the final fruit size, and the difference in fruit growth was primarily due to the difference in the number of cells, implicating the effect of cell division rate. Based on our observations and recent reports, the balance of AUX and ABA might be the key factor that regulated the cell division rate. They induced the response of auxin response factor 2 (FtARF2) and downstream small auxin upstream RNA (FtSAURs) through hormone signaling pathway to regulate the fruit size of Tartary buckwheat. Further, through the induction of fruit expansion by exogenous auxin, FtARF2b was significantly downregulated. The FtARF2b is a potential target for molecular breeding or gene editing.

Insights into the correlation between Physiological changes in and seed development of tartary buckwheat (Fagopyrum tataricum Gaertn.).

BACKGROUND: Tartary buckwheat (Fagopyrum tataricum Gaertn.) is a widely cultivated medicinal and edible crop with excellent economic and nutritional value. The development of tartary buckwheat seeds is a very complex process involving many expression-dependent physiological changes and regulation of a large number of genes and phytohormones. In recent years, the gene regulatory network governing the physiological changes occurring during seed development have received little attention. RESULTS: Here, we characterized the seed development of tartary buckwheat using light and electron microscopy and measured phytohormone and nutrient accumulation by using high performance liquid chromatography (HPLC) and by profiling the expression of key genes using RNA sequencing with the support of the tartary buckwheat genome. We first divided the development of tartary buckwheat seed into five stages that include complex changes in development, morphology, physiology and phytohormone levels. At the same time, the contents of phytohormones (gibberellin, indole-3-acetic acid, abscisic acid, and zeatin) and nutrients (rutin, starch, total proteins and soluble sugars) at five stages were determined, and their accumulation patterns in the development of tartary buckwheat seeds were analyzed. Second, gene expression patterns of tartary buckwheat samples were compared during three seed developmental stages (13, 19, and 25 days postanthesis, DPA), and 9 765 differentially expressed genes (DEGs) were identified. We analyzed the overlapping DEGs in different sample combinations and measured 665 DEGs in the three samples. Furthermore, expression patterns of DEGs related to phytohormones, flavonoids, starch, and storage proteins were analyzed. Third, we noted the correlation between the trait (physiological changes, nutrient changes) and metabolites during seed development, and discussed the key genes that might be involved in the synthesis and degradation of each of them. CONCLUSION: We provided abundant genomic resources for tartary buckwheat and Polygonaceae communities and revealed novel molecular insights into the correlations between the physiological changes and seed development of tartary buckwheat.

Research on homology modeling, molecular docking of the cellulase and highly expression of the key enzyme (Bgl) in Pichia pastoris.

Cellulose is the most abundant and renewable biological resource on earth. As nonrenewable resources are becoming scarce, cellulose is expected to become a major raw material for food, energy, fuel and other products. 1,4-ß-glucosidase (Bgl), as a kind of cellulose, can be degraded cellulose into industrial available glucose. In this study, we constructed mutants of Bgl with enhanced activity based on homology modeling, molecular docking, and the site-directed mutagenesis of target residues to modify spatial positions, steric hindrances, or hydrophilicity/hydrophobicity. On the basis of the high-activity mutations were got (N347S and G235 M) by using site-directed mutagenesis and screening methods and introduced in the Pichia pastoris expression system, the enzymatic properties of mutant enzymes were analysed. Assays of the activity of the purified Bgl revealed that the two mutants exhibited increased activity. The pPICZαA-G235 M and pPICZαA-N347S mutants exhibited a >33.4% and 44.8% increase in specific activity respectively, with similar pH, temperature and metal ion requirements, compared to wild-type Bgl. These findings would be good foundation for improving production properties of Bgl in the future.

ß-Amyrin synthase from Conyza blinii expressed in Saccharomyces cerevisiae.

Conyza blinii H.Lév. is a widely used medicinal herb in southwestern China. The main pharmacological components of C. blinii are a class of oleanane-type pentacyclic triterpene glycosides known as conyzasaponins, which are thought to be synthesized from ß-amyrin. However, no genes involved in the conyzasaponin pathway have previously been identified. Here, we identify an oxidosqualene cyclase (OSC), a ß-amyrin synthase, which mediates cyclization of 2,3-oxidosqualene to yield ß-amyrin. Ten OSC sequences were isolated from C. blinii transcript tags. Phylogenetic analysis was used to select the tag Cb18076 as the putative ß-amyrin synthase, named CbßAS. The open reading frame of CbßAS is 2286 bp and encodes 761 amino acids. Its mature protein contains the highly conserved motifs (QXXXGXW/DCTAE) of OSCs and (MWCYCR) of ß-amyrin synthases. Transcription of CbßAS was upregulated 4-24 h after treatment of the seedlings of the C. blinii cultivar with methyl jasmonate. Furthermore, expression of CbßAS in Saccharomyces cerevisiae successfully yielded ß-amyrin. The chemical structures and concentrations of ß-amyrin were confirmed by GC-MS/MS. The target yeast ultimately produced 4.432 mg·L-1 ß-amyrin. Thus, CbßAS is an OSC involved in conyzasaponin biosynthesis.

The complete chloroplast genome sequence of medicinal plant Conyza blinii H.Lév.

Conyza blinii is a widely used medicinal plant in the southwest of Sichuan with great economic value. Its chloroplast (cp) genome was 152,503 bp in length with the typical quadripartite structures of the large (LSC, 84,210 bp) and small (SSC, 18,257 bp) single-copy regions, separated by a pair of inverted repeats (IRs, 25,018 bp). The genome contains 114 encoding genes, including 80 protein-coding genes, 30 tRNAs and 4 rRNAs. The overall GC content of the whole cp genome is 37.4% which is similar to the other reported asteridae cp genomes.

The complete chloroplast genome sequence of Tartary Buckwheat Cultivar Miqiao 1(Fagopyrum tataricum Gaertn.).

Chloroplasts (cp) are indispensable organelles in plant cells that perform photosynthesis amongst other functions, including producing pigments, synthesizing sugars and certain amino acids. The complete chloroplast genome of Tartary Buckwheat Cultivar Miqiao 1 is sequenced in this study. Miqiao 1 is currently the only Tartary Buckwheat variety that can produce the whole nutritive Tartary Buckwheat Rice by hulling. The Miqiao 1 cp genome size is 159,272 bp in length, including 79 protein-coding genes, 30 tRNA genes and 4 rRNA genes. A pair of inverted repeats (IRs) of 30,817 bp were disconnected by a large single copy (LSC) of 84,397 bp and a small single copy (SSC) of 13,241 bp. The cp genome with 37.9% GC content contained 113 annotated genes.