Medicago truncatula ß-glucosidase 17 contributes to drought and salt tolerance through antioxidant flavonoid accumulation.

Flavonoids are usually present in forms of glucosides in plants, which could be catabolized by ß-glucosidase (BGLU) to form their corresponding flavonoid aglycones. In this study, we isolated three abiotic-responsive BGLU genes (MtBGLU17, MtBGLU21 and MtBGLU22) from Medicago truncatula, and found only the recombinant MtBGLU17 protein could catalyse the hydrolysis of flavonoid glycosides. The recombinant MtBGLU17 protein is active towards a variety of flavonoid glucosides, including glucosides of flavones (apigenin and luteolin), flavonols (kaempferol and quercetin), isoflavones (genistein and daidzein) and flavanone (naringenin). In particular, the recombinant MtBGLU17 protein preferentially hydrolyses flavonoid-7-O-glucosides over their corresponding 3-O-glucosides. The content of luteoin-7-O-glucoside was reduced in the MtBGLU17 overexpression plants but increased in the Tnt-1 insertional mutant lines, whereas luteoin content was increased in the MtBGLU17 overexpression plants but reduced in the Tnt-1 insertional mutant lines. Under drought and salt (NaCl) treatment, the MtBGLU17 overexpression lines showed relatively higher DPPH content, and higher CAT and SOD activity than the wild type control. These results indicated that overexpression lines of MtBGLU17 possess higher antioxidant activity and thus confer drought and salt tolerance, implying MtBGLU17 could be potentially used as a candidate gene to improve plant abiotic stress tolerance.

Genome-wide characterization and expression analysis of the HAK gene family in response to abiotic stresses in Medicago.

The high-affinity K+ transporter (HAK) family plays a vital role in K+ uptake and transport as well as in salt and drought stress responses. In the present study, we identified 22 HAK genes in each Medicago truncatula and Medicago sativa genome. Phylogenetic analysis suggested that these HAK proteins could be divided into four clades, and the members of the same subgroup share similar gene structure and conserved motifs. Many cis-acting elements related with defense and stress were found in their promoter region. In addition, gene expression profiles analyzed with genechip and transcriptome data showed that these HAK genes exhibited distinct expression pattern in different tissues, and in response to salt and drought treatments. Furthermore, co-expression analysis showed that 6 homologous HAK hub gene pairs involved in direct network interactions. RT-qPCR verified that the expression level of six HAK gene pairs was induced by NaCl and mannitol treatment to different extents. In particular, MtHK2/7/12 from M. truncatula and MsHAK2/6/7 from M. sativa were highly induced. The expression level of MsHAK1/2/11 determined by RT-qPCR showed significantly positive correlation with transcriptome data. In conclusion, our study shows that HAK genes play a key role in response to various abiotic stresses in Medicago, and the highly inducible candidate HAK genes could be used for further functional studies and molecular breeding in Medicago.

ARF2 positively regulates flavonols and proanthocyanidins biosynthesis in Arabidopsis thaliana.

MAIN CONCLUSION: Auxin response factor 2 acts as a positive regulator to fine-tune the spatial and temporal accumulation of flavonoid compounds, mainly flavonols and proanthocyanidins in Arabidopsis. Auxin response factor (ARF) proteins are reported to involve in auxin-mediated regulation of flavonoid biosynthesis. However, the detailed regulation mechanism of ARF remains still unknown. Here, we provide genetic and molecular evidence that one of the twenty-three ARF members-ARF2-positively regulates flavonoid biosynthesis at multi-level in tissue-specific manner in Arabidopsis thaliana. Loss-of-function mutation of ARF2 led to significant reduction in flavonoid content (e.g., flavonols and proanthocyanidins) in the seedlings and seeds of the Arabidopsis arf2 mutants. Over-expression of ARF2 increased flavonols and proanthocyanidins content in Arabidopsis. Additionally, the changes of flavonoid content correlate well with the transcript abundance of several regulatory genes (e.g., MYB11, MYB12, MYB111, TT2, and GL3), and key biosynthetic genes (e.g., CHS, F3'H, FLS, ANS, ANR, TT12, TT19, and TT15), in the arf2 mutant and ARF2 over-expression lines. Transient transactivation assays with site-directed mutagenesis confirmed that ARF2 directly regulates the expression of MYB12 and FLS genes in the flavonol pathway and ANR in the proanthocyanidin pathway, and indirectly regulates MYB11 and MYB111 genes in the flavonol pathway, and ANS, TT12, TT19 and TT15 genes in the proanthocyanidin pathway. Further genetic results indicated that ARF2 acts upstream of MYB12 to regulate flavonol accumulation, and of TT2 to regulate proanthocyanidins accumulation. In particular, yeast two-hybrid assays revealed that ARF2 physically interacts with TT2, a master regulator of proanthocyanidins biosynthesis. Combined together, these results indicated that ARF2 functions as a positive regulator for the fine-tuned spatial and temporal regulation of flavonoids (mainly flavonols and proanthocyanidins) accumulation in Arabidopsis.

Genome-Wide Identification and Characterization of Growth Regulatory Factor Family Genes in Medicago.

Growth Regulatory Factors (GRF) are plant-specific transcription factors that play critical roles in plant growth and development as well as plant tolerance against stress. In this study, a total of 16 GRF genes were identified from the genomes of Medicago truncatula and Medicago sativa. Multiple sequence alignment analysis showed that all these members contain conserved QLQ and WRC domains. Phylogenetic analysis suggested that these GRF proteins could be classified into five clusters. The GRF genes showed similar exon-intron organizations and similar architectures in their conserved motifs. Many stress-related cis-acting elements were found in their promoter region, and most of them were related to drought and defense response. In addition, analyses on microarray and transcriptome data indicated that these GRF genes exhibited distinct expression patterns in various tissues or in response to drought and salt treatments. In particular, qPCR results showed that the expression levels of gene pairs MtGRF2-MsGRF2 and MtGRF6-MsGRF6 were significantly increased under NaCl and mannitol treatments, indicating that they are most likely involved in salt and drought stress tolerance. Collectively, our study is valuable for further investigation on the function of GRF genes in Medicago and for the exploration of GRF genes in the molecular breeding of highly resistant M. sativa.

Identification and Characterization of Abiotic Stress-Responsive NF-YB Family Genes in Medicago.

Nuclear factor YB (NF-YB) are plant-specific transcription factors that play a critical regulatory role in plant growth and development as well as in plant resistance against various stresses. In this study, a total of 49 NF-YB genes were identified from the genomes of Medicago truncatula and Medicago sativa. Multiple sequence alignment analysis showed that all of these NF-YB members contain DNA binding domain, NF-YA interaction domain and NF-YC interaction domain. Phylogenetic analysis suggested that these NF-YB proteins could be classified into five distinct clusters. We also analyzed the exon-intron organizations and conserved motifs of these NF-YB genes and their deduced proteins. We also found many stress-related cis-acting elements in their promoter region. In addition, analyses on genechip for M. truncatula and transcriptome data for M. sativa indicated that these NF-YB genes exhibited a distinct expression pattern in various tissues; many of these could be induced by drought and/or salt treatments. In particular, RT-qPCR analysis revealed that the expression levels of gene pairs MsNF-YB27/MtNF-YB15 and MsNF-YB28/MtNF-YB16 were significantly up-regulated under NaCl and mannitol treatments, indicating that they are most likely involved in salt and drought stress response. Taken together, our study on NF-YB family genes in Medicago is valuable for their functional characterization, as well as for the application of NF-YB genes in genetic breeding for high-yield and high-resistance alfalfa.

Identification and Characterization of Salt- and Drought-Responsive AQP Family Genes in Medicagosativa L.

Aquaporins (AQP) are distributed ubiquitously in plants, and they play important roles in multiple aspects of plant growth and development, as well as in plant resistance to various environmental stresses. In this study, 43 MsAQP genes were identified in the forage crop Medicago sativa. All the MsAQP proteins were clustered into four subfamilies based on sequence similarity and phylogenetic relationship, including 17 TIPs, 14 NIPs, 9 PIPs and 3 SIPs. Analyses of gene structure and conserved domains indicated that the majority of the deduced MsAQP proteins contained the signature transmembrane domains and the NPA motifs. Analyses on cis-acting elements in the promoter region of MsAQP genes revealed the presence of multiple and diverse stress-responsive and hormone-responsive cis-acting elements. In addition, by analyzing the available and comprehensive gene expression data of M. truncatula, we screened ten representative MtAQP genes that were responsive to NaCl or drought stress. By analyzing the sequence similarity and phylogenetic relationship, we finally identified the corresponding ten salt- or drought-responsive AQP genes in M. sativa, including three MsTIPs, three MsPIPs and four MsNIPs. The qPCRs showed that the relative expression levels of these ten selected MsAQP genes responded differently to NaCl or drought treatment in M. sativa. Gene expression patterns showed that most MsAQP genes were preferentially expressed in roots or in leaves, which may reflect their tissue-specific functions associated with development. Our results lay an important foundation for the future characterization of the functions of MsAQP genes, and provide candidate genes for stress resistance improvement through genetic breeding in M. sativa.

A Novel 3-O-rhamnoside: 2″-O-xylosyltransferase Responsible for Terminal Modification of Prenylflavonol Glycosides in Epimedium pubescens Maxim.

Prenylated flavonol glycosides in Epimedium plants, as key medicinal components, are known to have great pharmaceutical activities for human health. Among the main prenylated flavonol glycosides, the modification mechanism of different sugar moieties is still not well understood. In the current study, a novel prenylated flavonol rhamnoside xylosyltransferase gene (EpF3R2″XylT) was cloned from E. pubescens, and the enzymatic activity of its decoding proteins was examined in vitro with different prenylated flavonol rhamnoside substrates and different 3-O-monosaccharide moieties. Furthermore, the functional and structural domains of EpF3R2″XylT were analyzed by bioinformatic approaches and 3-D protein structure remodeling. In summary, EpF3R2″XylT was shown to cluster with GGT (glycosyltransferase that glycosylates sugar moieties of glycosides) through phylogenetic analysis. In enzymatic analysis, EpF3R2″XylT was proven to transfer xylose moiety from UDP-xylose to prenylated flavonol rhamnoside at the 2″-OH position of rhamnose. The analysis of enzymatic kinetics showed that EpF3R2″XylT had the highest substrate affinity toward icariin with the lowest Km value of 75.96 ± 11.91 mM. Transient expression of EpF3R2″XylT in tobacco leaf showed functional production of EpF3R2″XylT proteins in planta. EpF3R2″XylT was preferably expressed in the leaves of E. pubescens, which is consistent with the accumulation levels of major prenylflavonol 3-O-triglycoside. The discovery of EpF3R2″XylT will provide an economical and efficient alternative way to produce prenylated flavonol trisaccharides through the biosynthetic approach.

Genome-Wide Identification and Analyses of Drought/Salt-Responsive Cytochrome P450 Genes in Medicago truncatula.

Cytochrome P450 monooxygenases (P450s) catalyze a great number of biochemical reactions and play vital roles in plant growth, development and secondary metabolism. As yet, the genome-scale investigation on P450s is still lacking in the model legume Medicago truncatula. In particular, whether and how many MtP450s are involved in drought and salt stresses for Medicago growth, development and yield remain unclear. In this study, a total of 346 MtP450 genes were identified and classified into 10 clans containing 48 families. Among them, sixty-one MtP450 genes pairs are tandem duplication events and 10 MtP450 genes are segmental duplication events. MtP450 genes within one family exhibit high conservation and specificity in intron-exon structure. Meanwhile, many Mt450 genes displayed tissue-specific expression pattern in various tissues. Specifically, the expression pattern of 204 Mt450 genes under drought/NaCl treatments were analyzed by using the weighted correlation network analysis (WGCNA). Among them, eight genes (CYP72A59v1, CYP74B4, CYP71AU56, CYP81E9, CYP71A31, CYP704G6, CYP76Y14, and CYP78A126), and six genes (CYP83D3, CYP76F70, CYP72A66, CYP76E1, CYP74C12, and CYP94A52) were found to be hub genes under drought/NaCl treatments, respectively. The expression levels of these selected hub genes could be induced, respectively, by drought/NaCl treatments, as validated by qPCR analyses, and most of these genes are involved in the secondary metabolism and fatty acid pathways. The genome-wide identification and co-expression analyses of M. truncatulaP450 superfamily genes established a gene atlas for a deep and systematic investigation of P450 genes in M. truncatula, and the selected drought-/salt-responsive genes could be utilized for further functional characterization and molecular breeding for resistance in legume crops.

Identification and Characterization of Abiotic Stress Responsive CBL-CIPK Family Genes in Medicago.

The calcineurin B-like protein (CBL) and CBL-interacting protein kinase (CIPK) play important roles in plant signal transduction and response to abiotic stress. Plants of Medicago genus contain many important forages, and their growth is often affected by a variety of abiotic stresses. However, studies on the CBL and CIPK family member and their function are rare in Medicago. In this study, a total of 23 CBL and 58 CIPK genes were identified from the genome of Medicago sativa as an important forage crop, and Medicaog truncatula as the model plant. Phylogenetic analysis suggested that these CBL and CIPK genes could be classified into five and seven groups, respectively. Moreover, these genes/proteins showed diverse exon-intron organizations, architectures of conserved protein motifs. Many stress-related cis-acting elements were found in their promoter region. In addition, transcriptional analyses showed that these CBL and CIPK genes exhibited distinct expression patterns in various tissues, and in response to drought, salt, and abscisic acid treatments. In particular, the expression levels of MtCIPK2 (MsCIPK3), MtCIPK17 (MsCIPK11), and MtCIPK18 (MsCIPK12) were significantly increased under PEG, NaCl, and ABA treatments. Collectively, our study suggested that CBL and CIPK genes play crucial roles in response to various abiotic stresses in Medicago.

Over-expression of a γ-tocopherol methyltransferase gene in vitamin E pathway confers PEG-simulated drought tolerance in alfalfa.

BACKGROUND: α-Tocopherol is one of the most important vitamin E components present in plant. α-Tocopherol is a potent antioxidant, which can deactivate photoproduced reactive oxygen species (ROS) and prevent lipids from oxidation when plants suffer drought stress. γ-Tocopherol methyltransferase (γ-TMT) catalyzes the formation of α-tocopherol in the tocopherol biosynthetic pathway. Our previous studies showed that over-expression of γ-TMT gene can increase the accumulation of α-tocopherol in alfalfa (Medicago sativa). However, whether these transgenic plants confer increased drought tolerance and the underlying mechanism are still unknown. RESULTS: In the present study, we further evaluate transgenic alfalfa lines, and found that over-expression of MsTMT led to an increase in α-tocopherol and total tocopherol level in the transgenic lines compared with the control plant. It was revealed that drought tolerance of the transgenic alfalfa was remarkably increased, with alleviated oxidative damage and accumulation of more osmolytic substances. The stomatal development in transgenic plants was significantly inhibited on both sides of leaves, which may be resulted from the repression of MsSPCHLESS (MsSPCH) gene. The reduced stomatal density of transgenic plants contributes to a lower stomatal conductance and higher water use efficiency (WUE). Moreover, both RNA-seq and qRT-PCR analyses indicate that regulatory mechanism of MsTMT in drought involved in both ABA-dependent and ABA-independent pathways. CONCLUSION: Our results suggest that MsTMT gene plays a positive role in regulating alfalfa response to PEG-simulated drought stress, which might involve complex mechanisms, including ROS scavenging system, stomatal development and multiple phytohormone signaling pathways. This study will broaden our view on the function of γ-TMT gene and provide new strategy for genetic engineering in alfalfa breeding.

GbMYBR1 from Ginkgo biloba represses phenylpropanoid biosynthesis and trichome development in Arabidopsis.

MAIN CONCLUSION: GbMYBR1, a new type of R2R3-MYB repressor from Ginkgo biloba, displayed pleiotropic effects on plant growth, phenylpropanoid accumulation, by regulating multiple related genes at different levels. Ginkgo biloba is a typical gymnosperm that has been thriving on earth for millions of years. MYB transcription factors (TFs) play important roles in diverse processes in plants. However, the role of MYBs remains largely unknown in Ginkgo. Here, an MYB TF gene from Ginkgo, designated as GbMYBR1, was found to act as a repressor in multiple processes. GbMYBR1 was mainly expressed in the leaves of Ginkgo. Over-expression of GbMYBR1 in Arabidopsis thaliana led to growth retardation, decreases in lignin content, reduced trichome density, and remarkable reduction in anthocyanin and flavonol contents in leaves. Proanthocyanidin content was decreased in the seeds of transgenic Arabidopsis, which led to light-brown seed color. Both qPCR and transcriptome sequencing analyses demonstrated that the transcript levels of multiple genes related to phenylpropanoid biosynthesis, trichome formation, and pathogen resistance were down-regulated in the transgenic Arabidopsis. In particular, we found that GbMYBR1 directly interacts with the bHLH cofactor GL3 as revealed by yeast two-hybrid assays. Our work indicated that GbMYBR1 has pleiotropic effects on plant growth, phenylpropanoid accumulation, and trichome development, mediated by interaction with GL3 or direct suppression of key pathway genes. Thus, GbMYBR1 represents a novel type of R2R3 MYB repressor.

Genistein-Specific G6DT Gene for the Inducible Production of Wighteone in Lotus japonicus.

Prenylated isoflavonoids have been found in several legume plants, and they possess various biological activities that play important roles in both plant defense and human health. However, it is still unknown whether prenylated isoflavonoids are present in the model legume plant Lotus japonicus. In the present study, we found that the prenylated isoflavonoid wighteone was produced in L. japonicus when leaf was supplemented with genistein. Furthermore, a novel prenyltransferase gene, LjG6DT, was identified, which shared high similarity with and was closely related to several known prenyltransferase genes involved in isoflavonoid biosynthesis. The recombinant LjG6DT protein expressed in yeast exhibited prenylation activity toward genistein as an exclusive substrate, which produced wighteone, a prenylated genistein at the C-6 position that occurs normally in legume plants. The LjG6DT-green fluorescent protein (GFP) fusion protein is targeted to plastids. The transcript level of LjG6DT is induced by glutathione, methyl jasmonate and salicylic acid, implying that LjG6DT is involved in stress response. Overexpression of LjG6DT in L. japonicus hairy roots led to increased accumulation of wighteone when genistein was supplied, indicating that LjG6DT is functional in vivo. Feeding assays with the upstream intermediate naringenin revealed that accumulation of wighteone in L. japonicus was dependent on genistein supplementation, and accumulation of wighteone is competed by genistein methylation. This study demonstrated that phytoalexin wighteone is inducibly produced in L. japonicus, and it provides new insight into the biosynthesis and accumulation of prenylated isoflavonoids in legume plants.

Genome-Wide Identification and Functional Characterization of UDP-Glucosyltransferase Genes Involved in Flavonoid Biosynthesis in Glycine max.

Flavonoids, natural products abundant in the model legume Glycine max, confer benefits to plants and to animal health. Flavonoids are present in soybean mainly as glycoconjugates. However, the mechanisms of biosynthesis of flavonoid glycosides are largely unknown in G. max. In the present study, 212 putative UDP-glycosyltransferase (UGT) genes were identified in G. max by genome-wide searching. The GmUGT genes were distributed differentially among the 20 chromosomes, and they were expressed in various tissues with distinct expression profiles. We further analyzed the enzymatic activities of 11 GmUGTs that are potentially involved in flavonoid glycosylation, and found that six of them (UGT72X4, UGT72Z3, UGT73C20, UGT88A13, UGT88E19 and UGT92G4) exhibited activity toward flavonol, isoflavone, flavone and flavanol aglycones with different kinetic properties. Among them, UGT72X4, UGT72Z3 and UGT92G4 are flavonol-specific UGTs, and UGT73C20 and UGT88E19 exhibited activity toward both flavonol and isoflavone aglycones. In particular, UGT88A13 exhibited activity toward epicatechin, but not for the flavonol aglycones kaempferol and quercetin. Overexpression of these six GmUGT genes significantly increased the contents of isoflavone and flavonol glucosides in soybean hairy roots. In addition, overexpression of these six GmUGT genes also affected flavonol glycoside contents differently in seedlings and seeds of transgenic Arabidopsis thaliana. We provide valuable information on the identification of all UGT genes in soybean, and candidate GmUGT genes for potential metabolic engineering of flavonoid compounds in both Escherichia coli and plants.

Involvement of three putative glucosyltransferases from the UGT72 family in flavonol glucoside/rhamnoside biosynthesis in Lotus japonicus seeds.

Flavonols are one of the largest groups of flavonoids that confer benefits for the health of plants and animals. Flavonol glycosides are the predominant flavonoids present in the model legume Lotus japonicus. The molecular mechanisms underlying the biosynthesis of flavonol glycosides as yet remain unknown in L. japonicus. In the present study, we identified a total of 188 UDP-glycosyltransferases (UGTs) in L. japonicus by genome-wide searching. Notably, 12 UGTs from the UGT72 family were distributed widely among L. japonicus chromosomes, expressed in all tissues, and showed different docking scores in an in silico bioinformatics docking analysis. Further enzymatic assays showed that five recombinant UGTs (UGT72AD1, UGT72AF1, UGT72AH1, UGT72V3, and UGT72Z2) exhibit activity toward flavonol, flavone, and isoflavone aglycones. In particular, UGT72AD1, UGT72AH1, and UGT72Z2 are flavonol-specific UGTs with different kinetic properties. In addition, the overexpression of UGT72AD1 and UGT72Z2 led to increased accumulation of flavonol rhamnosides in L. japonicus and Arabidopsis thaliana. Moreover, the increase of kaempferol 3-O-rhamnoside-7-O-rhamnoside in transgenic A. thaliana inhibited root growth as compared with the wild-type control. These results highlight the significance of the UGT72 family in flavonol glycosylation and the role of flavonol rhamnosides in plant growth.

GmMYB58 and GmMYB205 are seed-specific activators for isoflavonoid biosynthesis in Glycine max.

KEY MESSAGE: GmMYB58 and GmMYB205 are key positive regulators that are involved in isoflavonoid biosynthesis in seeds of Glycine max, and they activate the expression of several structural genes in the isoflavonoid pathway. MYB transcription factors (TFs) are major regulators involved in flavonoid/isoflavonoid biosynthesis in many plant species. However, functions of most MYB TFs remain unknown in flavonoid/isoflavonoid pathway in Glycine max. In this study, we identified 321 MYB TFs by genome-wide searching, and further isolated and functionally characterized two MYB TFs, GmMYB58 and GmMYB205. The deduced GmMYB58 and GmMYB205 proteins contain highly conserved R2R3 repeat domain at the N-terminal region that is the signature motif of R2R3-type MYB TFs. GmMYB58 and GmMYB205 were highly expressed in early seed development stages than in the other tested organs. GmMYB58 and GmMYB205 GFP fusion proteins were found to be localized in the nucleus when they were transiently expressed in Arabidopsis thaliana mesophyll protoplast. Both GmMYB58 and GmMYB205 can activate the promoter activities of GmCHS, GmIFS2, and GmHID in the transient trans-activation assays, and the activation of GmHID by both GmMYB58 and GmMYB205 was further confirmed by yeast one-hybrid assay. In addition, over-expression of GmMYB58 and GmMYB205 resulted in significant increases in expression levels of several pathway genes in soybean hairy roots, in particular, IFS2 by more than fivefolds in GmMYB205-over-expressing lines. Moreover, isoflavonoid contents were remarkably enhanced in the GmMYB58 and GmMYB205 over-expressing hairy roots than in the control. Our results suggest that GmMYB58 and GmMYB205 are seed-specific TFs, and they can enhance isoflavonoid biosynthesis mainly through the regulation of GmIFS2 and GmHID in G. max.

Identification of UDP-glycosyltransferases involved in the biosynthesis of astringent taste compounds in tea (Camellia sinensis).

Galloylated catechins and flavonol 3-O-glycosides are characteristic astringent taste compounds in tea (Camellia sinensis). The mechanism involved in the formation of these metabolites remains unknown in tea plants. In this paper, 178 UGT genes (CsUGTs) were identified inC. sinensis based on an analysis of tea transcriptome data. Phylogenetic analysis revealed that 132 of these genes were clustered into 15 previously established phylogenetic groups (A to M, O and P) and a newly identified group R. Three of the 11 recombinant UGT proteins tested were found to be involved in the in vitro biosynthesis of ß-glucogallin and glycosylated flavonols. CsUGT84A22 exhibited catalytic activity toward phenolic acids, in particular gallic acid, to produce ß-glucogallin, which is the immediate precursor of galloylated catechin biosynthesis in tea plants. CsUGT78A14 and CsUGT78A15 were found to be responsible for the biosynthesis of flavonol 3-O-glucosides and flavonol 3-O-galactosides, respectively. Site-directed mutagenesis of the Q373H substitution for CsUGT78A14 indicated that the Q (Gln) residue played a catalytically crucial role for flavonoid 3-O-glucosyltransferase activity. The expression profiles of the CsUGT84A22, CsUGT78A14, and CsUGT78A15 genes were correlated with the accumulation patterns of ß-glucogallin and the glycosylated flavonols which indicated that these three CsUGT genes were involved in the biosynthesis of astringent compounds inC. sinensis.

Role of a chalcone isomerase-like protein in flavonoid biosynthesis in Arabidopsis thaliana.

Flavonoids are important natural products for plant defence and human health. Although almost all the flavonoid pathway genes have been well-documented by biochemical and/or genetic approaches, the role of the Arabidopsis chalcone isomerase-like (CHIL) gene remains unclear. Two chil mutants with a seed colour similar to that of wild-type Arabidopsis have been identified here, but in sharp contrast to the characteristic transparent testa seed phenotype associated with other known flavonoid pathway genes. CHIL loss-of-function mutations led to a strong reduction in the proanthocyanidin and flavonol levels in seeds, but not in the anthocyanin levels in leaves. CHIL over-expression could partially recover the mutant phenotype of the chil mutant and increased both proanthocyanidin and flavonol accumulation in wild-type Arabidopsis. However, the CHIL gene could not rescue the mutant phenotype of TT5 that encodes the intrinsic chalcone isomerase in Arabidopsis. Parallel phenotypical and metabolic analyses of the chil, tt5, chs, and f3h mutants revealed that, genetically, CHIL functions at the same step as TT5. Moreover, it is demonstrated that CHIL co-expresses, co-localizes, and interacts with TT5 in Arabidopsis for flavonoid production. Based on these genetic and metabolic studies, it is concluded that CHIL functions with TT5 to promote flavonoid production, which is a unique enhancer in the flavonoid pathway.

Functional characterization of proanthocyanidin pathway enzymes from tea and their application for metabolic engineering.

Tea (Camellia sinensis) is rich in specialized metabolites, especially polyphenolic proanthocyanidins (PAs) and their precursors. To better understand the PA pathway in tea, we generated a complementary DNA library from leaf tissue of the blister blight-resistant tea cultivar TRI2043 and functionally characterized key enzymes responsible for the biosynthesis of PA precursors. Structural genes encoding enzymes involved in the general phenylpropanoid/flavonoid pathway and the PA-specific branch pathway were well represented in the library. Recombinant tea leucoanthocyanidin reductase (CsLAR) expressed in Escherichia coli was active with leucocyanidin as substrate to produce the 2R,3S-trans-flavan-ol (+)-catechin in vitro. Two genes encoding anthocyanidin reductase, CsANR1 and CsANR2, were also expressed in E. coli, and the recombinant proteins exhibited similar kinetic properties. Both converted cyanidin to a mixture of (+)-epicatechin and (-)-catechin, although in different proportions, indicating that both enzymes possess epimerase activity. These epimers were unexpected based on the belief that tea PAs are made from (-)-epicatechin and (+)-catechin. Ectopic expression of CsANR2 or CsLAR led to the accumulation of low levels of PA precursors and their conjugates in Medicago truncatula hairy roots and anthocyanin-overproducing tobacco (Nicotiana tabacum), but levels of oligomeric PAs were very low. Surprisingly, the expression of CsLAR in tobacco overproducing anthocyanin led to the accumulation of higher levels of epicatechin and its glucoside than of catechin, again highlighting the potential importance of epimerization in flavan-3-ol biosynthesis. These data provide a resource for understanding tea PA biosynthesis and tools for the bioengineering of flavanols.

Over-expression of Medicago Acyl-CoA-binding 2 genes enhance salt and drought tolerance in Arabidopsis.

Acyl-CoA-binding proteins (ACBPs) are mainly involved in acyl-CoA ester binding and trafficking in eukaryotic cells, and they function in lipid metabolism, membrane biosynthesis, cellular signaling, stress response, disease resistance, and other biological activities in plants. However, the roles of ACBP family members in Medicago remain unclear. In this study, a total of eight ACBP genes were identified in the genome of Medicago truncatula and Medicago sativa, and they were clustered into four sub-families (Class I-IV). Many cis-acting elements related to abiotic response were identified in the promoter region of these ACBP genes, in particular light-responsive elements. These ACBP genes exhibited distinct expression pattern in various tissues, and the expression level of MtACBP1/MsACBP1 and MtACBP2/MsACBP2 gene pairs were significantly increased under NaCl treatment. Subcellular localization analysis showed that MtACBP1/MsACBP1 and MtACBP2/MsACBP2 were localized in the endoplasmic reticulum of tobacco epidermal cells. Arabidopsis seedlings over-expressing MtACBP2/MsACBP2 displayed increased root length than the wild type under short light, Cu2+, ABA, PEG, and NaCl treatments. Over-expression of MtACBP2/MsACBP2 also significantly enhanced Arabidopsis tolerance under NaCl and PEG treatments in mature plants. Collectively, our study identified salt and drought responsive ACBP genes in Medicago and verified their functions in increasing resistance against salt and drought stresses.

The APETALA2-MYBL2 module represses proanthocyanidin biosynthesis by affecting formation of the MBW complex in seeds of Arabidopsis thaliana.

Proanthocyanidins (PAs) are the second most abundant plant phenolic natural products. PA biosynthesis is regulated by the well-documented MYB/bHLH/WD40 (MBW) complex, but how this complex itself is regulated remains ill defined. Here, in situ hybridization and ß-glucuronidase staining show that APETALA2 (AP2), a well-defined regulator of flower and seed development, is strongly expressed in the seed coat endothelium, where PAs accumulate. AP2 negatively regulates PA content and expression levels of key PA pathway genes. AP2 activates MYBL2 transcription and interacts with MYBL2, a key suppressor of the PA pathway. AP2 exerts its function by directly binding to the AT-rich motifs near the promoter region of MYBL2. Molecular and biochemical analyses revealed that AP2 forms AP2-MYBL2-TT8/EGL3 complexes, disrupting the MBW complex and thereby repressing expression of ANR, TT12, TT19, and AHA10. Genetic analyses revealed that AP2 functions upstream of MYBL2, TT2, and TT8 in PA regulation. Our work reveals a new role of AP2 as a key regulator of PA biosynthesis in Arabidopsis. Overall, this study sheds new light on the comprehensive regulation network of PA biosynthesis as well as the dual regulatory roles of AP2 in seed development and accumulation of major secondary metabolites in Arabidopsis.