Combined metabolome and transcriptome analyses reveal that growing under Red shade affects secondary metabolite content in Huangjinya green tea.

Shading treatments impact the tea (Camellia sinensis L.) quality. The sunlight sensitive varieties can be grown under shading nets for better growth and secondary metabolite content. Here, we studied the responses of a sunlight sensitive green tea variety "Huangjinya" by growing under colored shading nets (red, yellow, blue, and black (75% and 95%) shading rates) to find out the most suitable color of the shading net. Red shading was the most promising treatment as it positively affected the weight and length of 100 one-bud-three leaves and reduced the degree and rate of new shoots burn compared to control (natural sunlight). We then explored the comparative metabolomic changes in response to red shading by using UPLC-ESI-MS/MS system. The amino acids and derivatives, flavonoids, and alkaloids were downaccumulated whereas lipids, organic acids, and lignans were upaccumulated in Red shade grown tea samples. The red shading nets caused a decreased catechin, epicatechin, dopamine, and L-tyramine contents but increased caffeine content. We then employed transcriptome sequencing to find key changes in expressions of related genes and pathways. Notably, key genes associated with the phenylpropanoid and flavonoid biosynthesis pathways exhibited complex regulation. These expression changes suggested a potential trend of polymerization or condensation of simple molecules like catechin or pelargonidin into larger molecules like glucoside or proanthocyanidins. Here, Red shading net triggered higher expression of genes enriched in lipid biosynthesis and jasmonic acid biosynthesis, suggesting an interplay of fatty acids and JA in improving tea performance. These findings contribute to the metabolic responses of Huangjinya tea to red shading nets which might have implications for flavor and health benefits. Our data provide a foundation for further exploration and optimization of cultivation practices for this unique tea variety.

OsbZIP18 Is a Positive Regulator of Phenylpropanoid and Flavonoid Biosynthesis under UV-B Radiation in Rice.

In plants exposed to ultraviolet B radiation (UV-B; 280-315 nm), metabolic responses are activated, which reduce the damage caused by UV-B. Although several metabolites responding to UV-B stress have been identified in plants, the accumulation of these metabolites at different time points under UV-B stress remains largely unclear, and the transcription factors regulating these metabolites have not been well characterized. Here, we explored the changes in metabolites in rice after UV-B treatment for 0 h, 6 h, 12 h, and 24 h and identified six patterns of metabolic change. We show that the rice transcription factor OsbZIP18 plays an important role in regulating phenylpropanoid and flavonoid biosynthesis under UV-B stress in rice. Metabolic profiling revealed that the contents of phenylpropanoid and flavonoid were significantly reduced in osbzip18 mutants compared with the wild-type plants (WT) under UV-B stress. Further analysis showed that the expression of many genes involved in the phenylpropanoid and flavonoid biosynthesis pathways was lower in osbzip18 mutants than in WT plants, including OsPAL5, OsC4H, Os4CL, OsCHS, OsCHIL2, and OsF3H. Electrophoretic mobility shift assays (EMSA) revealed that OsbZIP18 bind to the promoters of these genes, suggesting that OsbZIP18 function is an important positive regulator of phenylpropanoid and flavonoid biosynthesis under UV-B stress. In conclusion, our findings revealed that OsbZIP18 is an essential regulator for phenylpropanoid and flavonoid biosynthesis and plays a crucial role in regulating UV-B stress responses in rice.

NtERF4 promotes the biosynthesis of chlorogenic acid and flavonoids by targeting PAL genes in Nicotiana tabacum.

Chlorogenic acid (CGA) and flavonoids are important secondary metabolites, which modulate plant growth and development, and contribute to plant resistance to various environmental stresses. ERF4 has been shown to be a repressor of anthocyanin accumulation in grape, but its full roles in regulating the biosynthesis of other phenylpropanoid compounds still needs to be further studied. In the present study, two NtERF4 genes were identified from N. tabacum genome. The expression level of NtERF4a was higher than that of NtERF4b in all the tobacco tissues examined. Over-expression of NtERF4a significantly promoted the accumulation of CGA and flavonoids in tobacco leaves, while silencing of NtERF4a significantly repressed the biosynthesis of CGA and flavonoids. RNA-seq analysis of NtERF4a-OE and WT plants revealed 8 phenylpropanoids-related differentially expressed genes (DEGs), including 4 NtPAL genes that encode key enzymes in the phenylpropanoid pathway. Activation of NtERF4a-GR fusion protein in tobacco significantly induced the transcription of NtPAL1 and NtPAL2 in the presence of protein synthesis inhibitor. Chromatin immunoprecipitation and Dual-Luc assays further indicated that NtERF4a could bind to the GCC box presented in the promoters of NtPAL1 and NtPAL2, thereby activating their transcription. Moreover, ectopic expression of NtERF4a induced the transcription of NtGSK1, NtMYC2, and NtJAZ3 genes, and enhanced the resistance of tobacco seedlings to salt and drought stresses, indicating multiple roles of NtERF4a in plants. Our findings revealed new roles of NtERF4a in modulating the accumulation of phenylpropanoid compounds in tobacco, and provided a putative target for improving phenylpropanoids synthesis and stress resistance in plants.

Integrated transcriptome and microRNA sequencing analyses reveal gene responses in poplar leaves infected by the novel pathogen bean common mosaic virus (BCMV).

Recently, a novel poplar mosaic disease caused by bean common mosaic virus (BCMV) was investigated in Populus alba var. pyramidalis in China. Symptom characteristics, physiological performance of the host, histopathology, genome sequences and vectors, and gene regulation at the transcriptional and posttranscriptional levels were analyzed and RT-qPCR (quantitative reverse transcription PCR) validation of expression was performed in our experiments. In this work, the mechanisms by which the BCMV pathogen impacts physiological performance and the molecular mechanisms of the poplar response to viral infection were reported. The results showed that BCMV infection decreased the chlorophyll content, inhibited the net photosynthesis rate (Pn) and stomatal conductance (Gs), and significantly changed chlorophyll fluorescence parameters in diseased leaves. Transcriptome analysis revealed that the expression of the majority of DEGs (differentially expressed genes) involved in the flavonoid biosynthesis pathway was promoted, but the expression of all or almost all DEGs associated with photosynthesis-antenna proteins and the photosynthesis pathway was inhibited in poplar leaves, suggesting that BCMV infection increased the accumulation of flavonoids but decreased photosynthesis in hosts. Gene set enrichment analysis (GSEA) illustrated that viral infection promoted the expression of genes involved in the defense response or plant-pathogen interaction. MicroRNA-seq analysis illustrated that 10 miRNA families were upregulated while 6 families were downregulated in diseased poplar leaves; moreover, miR156, the largest family with the most miRNA members and target genes, was only differentially upregulated in long-period disease (LD) poplar leaves. Integrated transcriptome and miRNA-seq analyses revealed 29 and 145 candidate miRNA-target gene pairs; however, only 17 and 76 pairs, accounting for 2.2% and 3.2% of all DEGs, were authentically negatively regulated in short-period disease (SD) and LD leaves, respectively. Interestingly, 4 miR156/SPL (squamosa promoter-binding-like protein) miRNA-target gene pairs were identified in LD leaves: the miR156 molecules were upregulated, but SPL genes were downregulated. In conclusion, BCMV infection significantly changed transcriptional and posttranscriptional gene expression in poplar leaves, inhibited photosynthesis, increased the accumulation of flavonoids, induced systematic mosaic symptoms, and decreased physiological performance in diseased poplar leaves. This study elucidated the fine-tuned regulation of poplar gene expression by BCMV; moreover, the results also suggested that miR156/SPL modules played important roles in the virus response and development of viral systematic symptoms in plant virus disease.

Combined analysis of metabolome and transcriptome of wheat kernels reveals constitutive defense mechanism against maize weevils.

Sitophilus zeamais (maize weevil) is one of the most destructive pests that seriously affects the quantity and quality of wheat (Triticum aestivum L.). However, little is known about the constitutive defense mechanism of wheat kernels against maize weevils. In this study, we obtained a highly resistant variety RIL-116 and a highly susceptible variety after two years of screening. The morphological observations and germination rates of wheat kernels after feeding ad libitum showed that the degree of infection in RIL-116 was far less than that in RIL-72. The combined analysis of metabolome and transcriptome of RIL-116 and RIL-72 wheat kernels revealed differentially accumulated metabolites were mainly enriched in flavonoids biosynthesis-related pathway, followed by glyoxylate and dicarboxylate metabolism, and benzoxazinoid biosynthesis. Several flavonoids metabolites were significantly up-accumulated in resistant variety RIL-116. In addition, the expression of structural genes and transcription factors (TFs) related to flavonoids biosynthesis were up-regulated to varying degrees in RIL-116 than RIL-72. Taken together, these results indicated that the biosynthesis and accumulation of flavonoids contributes the most to wheat kernels defense against maize weevils. This study not only provides insights into the constitutive defense mechanism of wheat kernels against maize weevils, but may also play an important role in the breeding of resistant varieties.

Multifaceted roles of WRKY transcription factors in abiotic stress and flavonoid biosynthesis.

Increasing biotic and abiotic stresses are seriously impeding the growth and yield of staple crops and threatening global food security. As one of the largest classes of regulators in vascular plants, WRKY transcription factors play critical roles governing flavonoid biosynthesis during stress responses. By binding major W-box cis-elements (TGACCA/T) in target promoters, WRKYs modulate diverse signaling pathways. In this review, we optimized existing WRKY phylogenetic trees by incorporating additional plant species with WRKY proteins implicated in stress tolerance and flavonoid regulation. Based on the improved frameworks and documented results, we aim to deduce unifying themes of distinct WRKY subfamilies governing specific stress responses and flavonoid metabolism. These analyses will generate experimentally testable hypotheses regarding the putative functions of uncharacterized WRKY homologs in tuning flavonoid accumulation to enhance stress resilience.

A Novel R2R3-MYB Transcription Factor SbMYB12 Positively Regulates Baicalin Biosynthesis in Scutellaria baicalensis Georgi.

Scutellaria baicalensis Georgi is an annual herb from the Scutellaria genus that has been extensively used as a traditional medicine for over 2000 years in China. Baicalin and other flavonoids have been identified as the principal bioactive ingredients. The biosynthetic pathway of baicalin in S. baicalensis has been elucidated; however, the specific functions of R2R3-MYB TF, which regulates baicalin synthesis, has not been well characterized in S. baicalensis to date. Here, a S20 R2R3-MYB TF (SbMYB12), which encodes 263 amino acids with a length of 792 bp, was expressed in all tested tissues (mainly in leaves) and responded to exogenous hormone methyl jasmonate (MeJA) treatment. The overexpression of SbMYB12 significantly promoted the accumulation of flavonoids such as baicalin and wogonoside in S. baicalensis hairy roots. Furthermore, biochemical experiments revealed that SbMYB12 is a nuclear-localized transcription activator that binds to the SbCCL7-4, SbCHI-2, and SbF6H-1 promoters to activate their expression. These results illustrate that SbMYB12 positively regulates the generation of baicalin and wogonoside. In summary, this work revealed a novel S20 R2R3-MYB regulator and enhances our understanding of the transcriptional and regulatory mechanisms of baicalin biosynthesis, as well as sheds new light on metabolic engineering in S. baicalensis.

A Comparison of the Flavonoid Biosynthesis Mechanisms of Dendrobium Species by Analyzing the Transcriptome and Metabolome.

Dendrobium huoshanense, Dendrobium officinale, and Dendrobium moniliforme, as precious Chinese medicinal materials, have a variety of medicinal properties. Flavonoids are important medicinal components of Dendrobium, but their accumulation rules and biosynthesis mechanisms remain unclear. To explore the similarities and differences of flavonoid accumulation and biosynthesis in these three Dendrobium species, we performed flavonoid content determination, widely-targeted metabolomics and transcriptome sequencing on 1-4 years old Dendrobium species. The results showed that in different growth years, D. huoshanense stems had the highest flavonoid content in the second year of growth, while D. officinale and D. moniliforme stems had the highest flavonoid content in the third year of growth. A total of 644 differentially accumulated metabolites (DAMs) and 10,426 differentially expressed genes (DEGs) were identified by metabolomic and transcriptomic analysis. It was found that DAMs and DEGs were not only enriched in the general pathway of "flavonoid biosynthesis", but also in multiple sub-pathways such as "Flavone biosynthesis", and "Flavonol biosynthesis" and "Isoflavonoid biosynthesis". According to a combined transcriptome and metabolome analysis, the expression levels of the F3'H gene (LOC110096779) and two F3'5'H genes (LOC110101765 and LOC110103762) may be the main genes responsible for the differences in flavonoid accumulation. As a result of this study, we have not only determined the optimal harvesting period for three Dendrobium plants, but also identified the key genes involved in flavonoid biosynthesis and provided a basis for further study of the molecular mechanism of flavonoid synthesis.

Comparison of buckwheat genomes reveals the genetic basis of metabolomic divergence and ecotype differentiation.

Golden buckwheat (Fagopyrum dibotrys or Fagopyrum cymosum) and Tartary buckwheat (Fagopyrum tataricum) belong to the Polygonaceae and the Fagopyrum genus is rich in flavonoids. Golden buckwheat is a wild relative of Tartary buckwheat, yet golden buckwheat is a traditional Chinese herbal medicine and Tartary buckwheat is a food crop. The genetic basis of adaptive divergence between these two buckwheats is poorly understood. Here, we assembled a high-quality chromosome-level genome of golden buckwheat and found a one-to-one syntenic relationship with the chromosomes of Tartary buckwheat. Two large inversions were identified that differentiate golden buckwheat and Tartary buckwheat. Metabolomic and genetic comparisons of golden buckwheat and Tartary buckwheat indicate an amplified copy number of FdCHI, FdF3H, FdDFR, and FdLAR gene families in golden buckwheat, and a parallel increase in medicinal flavonoid content. Resequencing of 34 wild golden buckwheat accessions across the two morphologically distinct ecotypes identified candidate genes, including FdMYB44 and FdCRF4, putatively involved in flavonoid accumulation and differentiation of plant architecture, respectively. Our comparative genomic study provides abundant genomic resources of genomic divergent variation to improve buckwheat with excellent nutritional and medicinal value.

Hybrid Assembly and Annotation of the Genome of the Indian Punica granatum, a Superfood.

The wonder fruit pomegranate (Punica granatum, family Lythraceae) is one of India's economically important fruit crops that can grow in different agro-climatic conditions ranging from tropical to temperate regions. This study reports high-quality de novo draft hybrid genome assembly of diploid Punica cultivar "Bhagwa" and identifies its genomic features. This cultivar is most common among the farmers due to its high sustainability, glossy red color, soft seed, and nutraceutical properties with high market value. The draft genome assembly is about 361.76 Mb (N50 = 40 Mb), ∼9.0 Mb more than the genome size estimated by flow cytometry. The genome is 90.9% complete, and only 26.68% of the genome is occupied by transposable elements and has a relative abundance of 369.93 SSRs/Mb of the genome. A total of 30,803 proteins and their putative functions were predicted. Comparative whole-genome analysis revealed Eucalyptus grandis as the nearest neighbor. KEGG-KASS annotations indicated an abundance of genes involved in the biosynthesis of flavonoids, phenylpropanoids, and secondary metabolites, which are responsible for various medicinal properties of pomegranate, including anticancer, antihyperglycemic, antioxidant, and anti-inflammatory activities. The genome and gene annotations provide new insights into the pharmacological properties of the secondary metabolites synthesized in pomegranate. They will also serve as a valuable resource in mining biosynthetic pathways for key metabolites, novel genes, and variations associated with disease resistance, which can facilitate the breeding of new varieties with high yield and superior quality.

Regulation mechanisms of flavonoids biosynthesis of Hancheng Dahongpao peels (Zanthoxylum bungeanum Maxim) at different development stages by integrated metabolomics and transcriptomics analysis.

BACKGROUND: Flavonoids have strong free radical scavenging and antioxidant capacity. The high abundance of flavonoids in Chinese prickly ash peels have many benefits to human health. In this study, 'Hancheng Dahongpao', a main cultivar, was taken as materials to investigate the flavonoids biosynthesis mechanism of Zanthoxylum bungeanum Maxim at three key development stages by integration of metabolomics and transcriptomics analysis. RESULTS: A total of 19 differentially accumulated metabolites were identified, the key flavonoids compounds were kaempferol, quercetin and their glycoside derivatives, and two major anthocyanins (peonidin O-hexoside and peonidin 3-O-glucoside). 5 gene networks/modules including 15 important candidate genes were identified, which was highly correlated with flavonoids. Among these genes, ZM-163828 and ZM-184209 were strongly correlated with kaempferol and quercetin, and ZM-125833 and ZM-97481 were controlled the anthocyanins biosynthesis. Moreover, it was shown that MYB-ZM1, MYB-ZM3, MYB-ZM5, MYB-ZM6 and MYB-ZM7 coordinately controlled flavonoids accumulation through regulating the structural genes. CONCLUSIONS: Generally, this study systematically revealed the flavonoids metabolic pathways and candidate genes involved in flavonoids biosynthesis and laid a foundation for the potential targets for the breeding of new valuable Chinese prickly ash cultivars.

JA-induced FtBPM3 accumulation promotes FtERF-EAR3 degradation and rutin biosynthesis in Tartary buckwheat.

Buckwheat accumulates abundant flavonoids, which exhibit excellent health-promoting value. Flavonoids biosynthesis is mediated by a variety of phytohormones, among which jasmonates (JAs) induce numerous transcription factors, taking part in regulation of flavonoids biosynthesis genes. However, some transcriptional repressors appeared also induced by JAs. How these transcriptional repressors coordinately participate in JA signaling remains unclear. Here, we found that the disruption of the GCC-box in FtF3H promoter was associated with flavonoids accumulation in Tartary buckwheat. Further, our study illustrated that the nucleus-localized FtERF-EAR3 could inhibit FtF3H expression and flavonoids biosynthesis through binding the GCC-box in the promoter of FtF3H. The JA induced FtERF-EAR3 gene expression while facilitating FtERF-EAR3 protein degradation via the FtBPM3-dependent 26S proteasome pathway. Overall, these results illustrate a precise modulation mechanism of JA-responsive transcription suppressor participating in flavonoid biosynthesis, and will further help to improve the efficiency of flavonoids biosynthesis in Tartary buckwheat.

Both Two CtACO3 Transcripts Promoting the Accumulation of the Flavonoid Profiles in Overexpressed Transgenic Safflower.

The unique flavonoids, quinochalcones, such as hydroxysafflor yellow A (HSYA) and carthamin, in the floret of safflower showed an excellent pharmacological effect in treating cardiocerebral vascular disease, yet the regulating mechanisms governing the flavonoid biosynthesis are largely unknown. In this study, CtACO3, the key enzyme genes required for the ethylene signaling pathway, were found positively related to the flavonoid biosynthesis at different floret development periods in safflower and has two CtACO3 transcripts, CtACO3-1 and CtACO3-2, and the latter was a splice variant of CtACO3 that lacked 5' coding sequences. The functions and underlying probable mechanisms of the two transcripts have been explored. The quantitative PCR data showed that CtACO3-1 and CtACO3-2 were predominantly expressed in the floret and increased with floret development. Subcellular localization results indicated that CtACO3-1 was localized in the cytoplasm, whereas CtACO3-2 was localized in the cytoplasm and nucleus. Furthermore, the overexpression of CtACO3-1 or CtACO3-2 in transgenic safflower lines significantly increased the accumulation of quinochalcones and flavonols. The expression of the flavonoid pathway genes showed an upward trend, with CtCHS1, CtF3H1, CtFLS1, and CtDFR1 was considerably induced in the overexpression of CtACO3-1 or CtACO3-2 lines. An interesting phenomenon for CtACO3-2 protein suppressing the transcription of CtACO3-1 might be related to the nucleus location of CtACO3-2. Yeast two-hybrid (Y2H), glutathione S-transferase (GST) pull-down, and BiFC experiments revealed that CtACO3-2 interacted with CtCSN5a. In addition, the interactions between CtCSN5a and CtCOI1, CtCOI1 and CtJAZ1, CtJAZ1 and CtbHLH3 were observed by Y2H and GST pull-down methods, respectively. The above results suggested that the CtACO3-2 promoting flavonoid accumulation might be attributed to the transcriptional activation of flavonoid biosynthesis genes by CtbHLH3, whereas the CtbHLH3 might be regulated through CtCSN5-CtCOI1-CtJAZ1 signal molecules. Our study provided a novel insight of CtACO3 affected the flavonoid biosynthesis in safflower.

Functional and Structural Investigation of Chalcone Synthases Based on Integrated Metabolomics and Transcriptome Analysis on Flavonoids and Anthocyanins Biosynthesis of the Fern Cyclosorus parasiticus.

The biosynthesis of flavonoids and anthocyanidins has been exclusively investigated in angiosperms but largely unknown in ferns. This study integrated metabolomics and transcriptome to analyze the fronds from different development stages (S1 without spores and S2 with brown spores) of Cyclosorus parasiticus. About 221 flavonoid and anthocyanin metabolites were identified between S1 and S2. Transcriptome analysis revealed several genes encoding the key enzymes involved in the biosynthesis of flavonoids, and anthocyanins were upregulated in S2, which were validated by qRT-PCR. Functional characterization of two chalcone synthases (CpCHS1 and CpCHS2) indicated that CpCHS1 can catalyze the formation of pinocembrin, naringenin, and eriodictyol, respectively; however, CpCHS2 was inactive. The crystallization investigation of CpCHS1 indicated that it has a highly similar conformation and shares a similar general catalytic mechanism to other plants CHSs. And by site-directed mutagenesis, we found seven residues, especially Leu199 and Thr203 that are critical to the catalytic activity for CpCHS1.

Transcriptome-based metabolic profiling of flavonoids in Agave lechuguilla waste biomass.

Agave lechuguilla is one of the most abundant species in arid and semiarid regions of Mexico, and is used to extract fiber. However, 85 % of the harvested plant material is discarded. Previous bioprospecting studies of the waste biomass suggest the presence of bioactive compounds, although the extraction process limited metabolite characterization. This work achieved flavonoid profiling of A. lechuguilla in both processed and non-processed leaf tissues using transcriptomic analysis. Functional annotation of the first de novo transcriptome of A. lechuguilla (255.7 Mbp) allowed identifying genes coding for 33 enzymes and 8 transcription factors involved in flavonoid biosynthesis. The flavonoid metabolic pathway was mostly elucidated by HPLC-MS/MS screening of alcoholic extracts. Key genes of flavonoid synthesis were higher expressed in processed leaf tissues than in non-processed leaves, suggesting a high content of flavonoids and glycoside derivatives in the waste biomass. Targeted HPLC-UV-MS analyses confirmed the concentration of isorhamnetin (1251.96 µg), flavanone (291.51 µg), hesperidin (34.23 µg), delphinidin (24.23 µg), quercetin (15.57 µg), kaempferol (13.71 µg), cyanidin (12.32 µg), apigenin (9.70 µg) and catechin (7.91 µg) per gram of dry residue. Transcriptomic and biochemical profiling concur in the potential of lechuguilla by-products with a wide range of applications in agriculture, feed, food, cosmetics, and pharmaceutical industries.

Temporospatial Flavonoids Metabolism Variation in Ginkgo biloba Leaves.

Ginkgo (Ginkgo biloba L.) is a high-value medicinal tree species characterized by its flavonoids beneficial effects that are abundant in leaves. We performed a temporospatial comprehensive transcriptome and metabolome dynamics analyses of clonally propagated Ginkgo plants at four developmental stages (time: May to August) across three different environments (space) to unravel leaves flavonoids biosynthesis variation. Principal component analysis revealed clear gene expression separation across samples from different environments and leaf-developmental stages. We found that flavonoid-related metabolism was more active in the early stage of leaf development, and the content of total flavonoid glycosides and the expression of some genes in flavonoid biosynthesis pathway peaked in May. We also constructed a co-expression regulation network and identified eight GbMYBs and combining with other TF genes (3 GbERFs, 1 GbbHLH, and 1 GbTrihelix) positively regulated the expression of multiple structural genes in the flavonoid biosynthesis pathway. We found that part of these GbTFs (Gb_11316, Gb_32143, and Gb_00128) expressions was negatively correlated with mean minimum temperature and mean relative humidity, while positively correlated with sunshine duration. This study increased our understanding of the molecular mechanisms of flavonoids biosynthesis in Ginkgo leaves and provided insight into the proper production and management of Ginkgo commercial plantations.

Fulvic acid ameliorates drought stress-induced damage in tea plants by regulating the ascorbate metabolism and flavonoids biosynthesis.

BACKGROUND: Fulvic acid (FA) is a kind of plant growth regulator, which can promote plant growth, play an important role in fighting against drought, improve plant stress resistance, increase production and improve quality. However, the function of FA in tea plants during drought stress remain largely unknown. RESULTS: Here, we examined the effects of 0.1 g/L FA on genes and metabolites in tea plants at different periods of drought stress using transcriptomics and metabolomics profiles. Totally, 30,702 genes and 892 metabolites were identified. Compared with controlled groups, 604 and 3331 differentially expressed metabolite genes (DEGs) were found in FA-treated tea plants at 4 days and 8 days under drought stress, respectively; 54 and 125 differentially expressed metabolites (DEMs) were also found at two time points, respectively. Bioinformatics analysis showed that DEGs and DEMs participated in diverse biological processes such as ascorbate metabolism (GME, AO, ALDH and L-ascorbate), glutathione metabolism (GST, G6PDH, glutathione reduced form and CYS-GYL), and flavonoids biosynthesis (C4H, CHS, F3'5'H, F3H, kaempferol, quercetin and myricetin). Moreover, the results of co-expression analysis showed that the interactions of identified DEGs and DEMs diversely involved in ascorbate metabolism, glutathione metabolism, and flavonoids biosynthesis, indicating that FA may be involved in the regulation of these processes during drought stress. CONCLUSION: The results indicated that FA enhanced the drought tolerance of tea plants by (i) enhancement of the ascorbate metabolism, (ii) improvement of the glutathione metabolism, as well as (iii) promotion of the flavonoids biosynthesis that significantly improved the antioxidant defense of tea plants during drought stress. This study not only confirmed the main strategies of FA to protect tea plants from drought stress, but also deepened the understanding of the complex molecular mechanism of FA to deal with tea plants to better avoid drought damage.

[Cloning,expression and characterization of chalcone isomerase from medicinal plant Chinese sumac (Rhus chinensis)].

Flavonoids are a group of secondary metabolites found in plants. They have many pharmacological functions and play an important role in Chinese sumac( Rhus chinensis),which is a well-known traditional Chinese medicinal plant. Chalcone isomerase( CHI,EC 5. 5. 1. 6) is one of the key enzymes in the flavonoids biosynthesis pathway. In this paper,the full-length c DNA sequence encoding the chalcone isomerase from R. chinensis( designated as Rc CHI) was cloned by RT-PCR and rapid-amplification of c DNA Ends( RACE). The Rc CHI c DNA sequence was 1 058 bp and the open reading frame( ORF) was 738 bp. The ORF predicted to encode a 245-amino acid polypeptide. Rc CHI gene contained an intron and two exons. The sequence alignments revealed Rc CHI shared47. 1%-71. 6% identity with the homologues in other plants. Real-time PCR analysis showed that the total flavonoid levels were positively correlated with tissue-specific expressions of Rc CHI mRNA in different tissues. The recombinant protein was successfully expressed in an Escherichia coli strain with the p GEX-6 P-1 vector. In this paper,the CHI gene was cloned and characterized in the family of Anacardiaceae and will help us to obtain better knowledge of the flavonoids biosynthesis of the flavonoid compounds in R. chinensis.

Molecular cloning and functional characterization of chalcone isomerase from Carthamus tinctorius.

Flavonoid is one of the widespread groups of plant secondary metabolites that provide several health benefits. However, the explicit mechanism of flavonoid biosynthesis in plants largely remains unclear. Chalcone isomerase an important class of enzyme presents crucial role during flavonoid metabolism in many plants. Here, we isolated the full-length cDNA (1161 bp) of a novel Chalcone Isomerase from safflower encoding 217 amino acid polypeptide using oligos from 5' and 3' ends. The result of Sanger sequencing and phylogenetic analysis revealed that CtCHI is highly homologous to other plants, including typical polyadenylation signals AATAA and Poly A tail. The transient expression in tobacco mesophyll cells using Green Fluorescent Protein tagging determined the subcellular localization of CtCHI in cell membrane and nucleus. The CtCHI ectopic expression in different safflower varieties at different flowering stages showed that CtCHI were found in abundance at the bud stage of Jihong No. 1. Further correlation analysis between CtCHI expression and flavonoid accumulation at various flowering phases suggested that CtCHI might play a potential role during flavonoid biosynthesis in safflower. In addition, the overexpression of pBASTA-CtCHI in transgenic Arabidopsis infiltrated with floral dip transformation showed relatively higher expression level and increased flavonoid accumulation than wild type. Moreover, the in vitro enzymatic activity and HPLC analysis of transgenic Arabidopsis confirmed the de novo biosynthesis of Rutin. Taken together, our findings laid the foundation of identifying an important gene that might influence flavonoid metabolism in safflower.