Functional characterization of a UDP-glucose:flavonoid 3-O-glucosyltransferase from the seed coat of black soybean (Glycine max (L.) Merr.)

The seed coats of black soybean (Glycine max (L.) Merr.) accumulate red (cyanidin-), blue (delphinidin-), purple (petunidin-), and orange (pelargonidin-based) anthocyanins almost exclusively as 3-O-glucosides; however, the responsible enzyme has not been identified. In this study, the full-length cDNA which encodes the enzyme that catalyzes the final step in anthocyanin biosynthesis, namely UDP-glucose:flavonoid 3-O-glucosyltransferase (UGT78K1), was isolated from the seed coat tissue of black soybean using rapid amplification of cDNA ends (RACE). Of the 28 flavonoid substrates tested, the purified recombinant protein glucosylated only anthocyanidins and flavonols, and demonstrated strict 3-OH regiospecificity. Galactose could also be transferred with relatively low activity to the 3-position of cyanidin or delphinidin in vitro. These findings are consistent with previous reports of mainly 3-O-glucosylated and minor amounts of 3-O-galactosylated anthocyanins in the seed coat of black soybean. The recombinant enzyme exhibited pronounced substrate inhibition by cyanidin at 100 μM acceptor concentration. Transfer of UGT78K1 into the Arabidopsis T-DNA mutant (ugt78d2) deficient in anthocyanidin and flavonol 3-O-glucosyltransferase activity, restored the accumulation of anthocyanins and flavonols, suggesting the in vivo function of the enzyme as a flavonoid 3-O-glucosyltransferase. Genomic and phylogenetic analyses suggest the existence of three additional soybean sequences with high similarity to UGT78K1. RT-PCR confirmed the co-expression of one of these genes (Glyma08g07130) with UGT78K1 in the seed coat of black soybean, suggesting possible functional redundancies in anthocyanin biosynthesis in this tissue.     

Molecular genetic analysis of tandemly located glycosyltransferase genes,UGT73B1,UGT73B2, andUGT73B3, inArabidopsis thaliana

In theArabidopsis genome, approximately 120 UDP-glycosyltransferases (UGTs) have been annotated. They generally catalyze the transfer of sugars to various acceptor molecules, including flavonoids. To better understand their physiological roles, we analyzed a tandemly located putative flavonoid UGT cluster comprisingUGT73B1, UGT73B2, andUGT73B3 on Chromosome IV. We then isolated four loss-of-function mutations —ugt73b1- 1, ugt73b2- 1, ugt73b3- 1, andugt73b3- 2. In our expression analysis, the closely related UCTs exhibited tissue-specific patterns of expression that were severely altered in their respective mutant plants. For example,UGT73B2 was up-regulated inugt73b1- 1, whereasUGT73B7 was highly expressed inugt73b2- 1, ugt73b3- t, andugt73b3- 2. Interestingly, each recessive mutant was resistant to methyl viologen (paraquat), an herbicide thought to cause oxidative stress. Our results suggest thatUGTs play an important role in the glycosylation pathways when responding to oxidative stress.     

Molecular analysis of the<Emphasis Type="Italic"> CRINKLY4</Emphasis> gene family in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

The maize (Zea mays L.) CRINKLY4 (CR4) gene encodes a serine/threonine receptor-like kinase that controls an array of developmental processes in the plant and endosperm. The Arabidopsis thaliana (L.) Heynh. genome encodes an ortholog of CR4, ACR4, and four CRINKLY4-RELATED (CRR) proteins: AtCRR1, AtCRR2, AtCRR3 and AtCRK1. The available genome sequence of rice (Oryza sativa L.) encodes a CR4 ortholog, OsCR4, and four CRR proteins: OsCRR1, OsCRR2, OsCRR3 and OsCRR4, not necessarily orthologous to the Arabidopsis CRRs. A phylogenetic study showed that AtCRR1 and AtCRR2 form a clade closest to the CR4 group while all the other CRRs form a separate cluster. The five Arabidopsis genes are differentially expressed in various tissues. A construct formed by fusion of the ACR4 promoter and the GUS reporter, ACR4::GUS, is expressed primarily in developing tissues of the shoot. The ACR4 cytoplasmic domain functions in vitro as a serine/threonine kinase, while the AtCRR1 and AtCRR2 kinases are not active. The ability of ACR4 to phosphorylate AtCRR2 suggests that they might function in the same signal transduction pathway. T-DNA insertions were obtained in ACR4, AtCRR1, AtCRR2, AtCRR3 and AtCRK1. Mutations in acr4 show a phenotype restricted to the integuments and seed coat, suggesting that Arabidopsis might contain a redundant function that is lacking in maize. The lack of obvious mutant phenotypes in the crr mutants indicates they are not required for the hypothetical redundant function.     

Genetic regulation and photocontrol of anthocyanin accumulation in maize seedlings.

The flavonoid pathway leading to anthocyanin biosynthesis in maize is controlled by multiple regulatory genes and induced by various developmental and environmental factors. We have investigated the effect of the regulatory loci R, B, and Pl on anthocyanin accumulation and on the expression of four genes (C2, A1, Bz1, and Bz2) in the biosynthetic pathway during an inductive light treatment. The results show that light-mediated anthocyanin biosynthesis is regulated solely by R; the contributions of B and Pl are negligible in young seedlings. Induction of the A1 and Bz2 genes by high fluence-rate white light requires the expression of a dominant R allele, whereas accumulation of C2 and Bz1 mRNA occurs with either a dominant or recessive allele at R. A1 and Bz2 mRNA accumulate only in response to high fluence-rate white light, but Bz1 is fully expressed in dim red light. Some C2 mRNA is induced by dim red light, but accumulation is far greater in high fluence-rate white light. Furthermore, expression from both dominant and recessive alleles of the regulatory gene R is enhanced by high fluence-rate white light. Seedlings with a recessive allele at R produce functional chalcone synthase protein (the C2 gene product) but accumulate no anthocyanins, suggesting that, in contrast to the R-mediated coordinate regulation of C2 and Bz1 observed in the aleurone, C2 expression in seedlings is independent of R and appears to be regulated by a different light-sensitive pathway.     

Biochemical characterization of anArabidopsis glucosyltransferase with high activity toward Jasmonic acid

Biochemical characterization of the recombinant gene products from theArabidopsis glucosyltransferase multigene family has identified one enzyme with high activity toward the plant cellular regulator jasmonic acid (JA). The protein, AtJGT1 (UDP-glucose:JA glucosyltransferase), also has significant activities with other substrates, such as dihydrojasmonicacid, indole-3-acetic acid (IAA), indole-3-propionic acid, and indole-3-butyric acid. TheK _M values of AtJGT1 for JA or IAA are similar to those of anArabidopsis IAA glucosyltransferase UGT84B1 previously reported. Northern blot analysis showed thatAtJGTI is highly expressed in the leaves, but only slightly detectable in the roots, stems, and inflorescences. This study describes the first biochemical analysis of a recombinant glucosyltransferase with JA activity, and provides the foundation for future genetic approaches to understanding the role of JA-glucose inArabidopsis.     

Structural basis for acceptor‐substrate recognition of UDP‐glucose: anthocyanidin 3‐O‐glucosyltransferase from Clitoria ternatea

UDP‐glucose: anthocyanidin 3‐O‐glucosyltransferase (UGT78K6) from Clitoria ternatea catalyzes the transfer of glucose from UDP‐glucose to anthocyanidins such as delphinidin. After the acylation of the 3‐O‐glucosyl residue, the 3′‐ and 5′‐hydroxyl groups of the product are further glucosylated by a glucosyltransferase in the biosynthesis of ternatins, which are anthocyanin pigments. To understand the acceptor‐recognition scheme of UGT78K6, the crystal structure of UGT78K6 and its complex forms with anthocyanidin delphinidin and petunidin, and flavonol kaempferol were determined to resolutions of 1.85 Å, 2.55 Å, 2.70 Å, and 1.75 Å, respectively. The enzyme recognition of unstable anthocyanidin aglycones was initially observed in this structural determination. The anthocyanidin‐ and flavonol‐acceptor binding details are almost identical in each complex structure, although the glucosylation activities against each acceptor were significantly different. The 3‐hydroxyl groups of the acceptor substrates were located at hydrogen‐bonding distances to the Nε2 atom of the His17 catalytic residue, supporting a role for glucosyl transfer to the 3‐hydroxyl groups of anthocyanidins and flavonols. However, the molecular orientations of these three acceptors are different from those of the known flavonoid glycosyltransferases, VvGT1 and UGT78G1. The acceptor substrates in UGT78K6 are reversely bound to its binding site by a 180° rotation about the O1–O3 axis of the flavonoid backbones observed in VvGT1 and UGT78G1; consequently, the 5‐ and 7‐hydroxyl groups are protected from glucosylation. These substrate recognition schemes are useful to understand the unique reaction mechanism of UGT78K6 for the ternatin biosynthesis, and suggest the potential for controlled synthesis of natural pigments.     

The role of UDP-glucose:hydroxycinnamate glucosyltransferases in phenylpropanoid metabolism and the response to UV-B radiation in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Arabidopsis harbors four UDP-glycosyltransferases that convert hydroxycinnamates (HCAs) to 1-O-β-glucose esters, UGT84A1 (encoded by At4g15480), UGT84A2 (At3g21560), UGT84A3 (At4g15490), and UGT84A4 (At4g15500). To elucidate the role of the individual UGT84A enzymes in planta we analyzed gene expression, UGT activities and accumulation of phenylpropanoids in Arabidopsis wild type plants, ugt mutants and overexpressing lines. Individual ugt84A null alleles did not significantly reduce the gross metabolic flux to the accumulating compounds sinapoylcholine (sinapine) in seeds and sinapoylmalate in leaves. For the ugt84A2 mutant, LC/MS analysis revealed minor qualitative and quantitative changes of several HCA choline esters and of disinapoylspermidine in seeds. Overexpression of individual UGT84A genes caused increased enzyme activities but failed to produce significant changes in the pattern of accumulating HCA esters. For UGT84A3, our data tentatively suggest an impact on cell wall-associated 4-coumarate. Exposure of plants to enhanced UV-B radiation induced the UGT84A-encoding genes and led to a transient increase in sinapoylglucose and sinapoylmalate concentrations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

UDP-Glucuronosyltransferase activity is correlated to saponin production in Gypsophila paniculata root in vitro cultures

UDP-Glucuronosyltransferase (UGT, EC 2.4.1.17) activity was detected in excised root cultures of Gypsophila paniculata. The UGT activity (up to 0.8 nkat mg^–1 protein) correlated to the total saponin content (2 mg g^–1 dw) during the exponential phase of the batch culture. This gives rise to the hypothesis of a direct relationship in the biosynthetic pathway regulation between the enzyme activity and the saponin biosynthesis.     

ASK1 physically interacts with COI1 and is required for male fertility inArabidopsis

Jasmonates are a new class of plant hormones that play important roles in plant development and plant defense. TheCOI1 gene was previously shown to be required for jasmonate-regulated plant fertility and defense. We demonstrated for the first time that COI1 interacts with theArabidopsis SKP1-LIKE1 (ASK1) to form a complex that is required for jasmonate action inplanta. Functional analysis by antisense strategy showed thatASK1 is involved in male fertility.     

Characterisation of a barley (Hordeum vulgare L.) homologue of the Arabidopsis flowering time regulator GIGANTEA

Barley cDNA and genomic clones homologous to the Arabidopsis flowering time regulator GIGANTEA were isolated. Genetic mapping showed that GIGANTEA is present as a single copy gene in barley (3HS) and rice (1S), while two copies are present in maize (3S and 8S) at locations consistent with previous comparative mapping studies. Comparison of the barley peptide with rice and Arabidopsis gave 94% and 79% similarity, respectively. Northern and semi-quantitative RT-PCR analysis of the barley gene (HvGI) showed the presence of a single mRNA species, with a peak of expression between 6 h and 9 h after dawn in short days (8 h light) and a peak 15 h after dawn in long days (16 h light). This behaviour is similar to that seen in Arabidopsis and rice, showing that sequence and expression pattern were well conserved. A lack of correspondence with the map positions of QTL affecting flowering time (heading date) suggests that variation at HvGI does not provide a major source of adaptive variation in photoperiod response.     

The flowering-time geneFT and regulation of flowering inArabidopsis

Transition from vegetative to reproductive development (flowering) is one of the most important decisions during the post-embryonic development of flowering plants. More than twenty loci are known to regulate this process inArabidopsis. Some of these flowering-time genes may act at the shoot apical meristem to regulate its competence to respond to floral inductive signals and floral evocation. Genetic and phenotypic analyses of mutants suggest that the late-flowering geneFT may be a good candidate for such genes. To test this, we have cloned theFT gene using aFT-deficiency line associated with a T-DNA insertion. Cloned genes and loss-of-function mutants in hand, it is now possible to analyse the role ofFT and other genes in flowering at the biochemical and cellular levels as well as at the genetic level. The deduced FT protein has homology with TFL1 and CEN proteins believed to be involved in regulation of inflorescence meristem identity. Phylogenetic analysis suggests that theFT group and theTFL1/CEN group of genes diverged before the diversification of major angiosperm clades. This raises the interesting question of the evolutionary relationship between the regulation of vegetative/reproductive switching in the shoot apical meristem and the regulation of inflorescence architecture in angiosperms. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Fronitier of Plant Biology”     

<Emphasis Type="Italic">Gemini pollen 2</Emphasis>, a male and female gametophytic cytokinesis defective mutation

Gametophytic cytokinesis is essential for the development and function of the male and female gametophytes. We have previously described the isolation and characterisation of gemini pollen 1 (gem1) that acts gametophytically to disturb asymmetric division and cytokinesis at pollen mitosis I (PMI) in Arabidopsis. Here we describe the genetic and cytological analysis of an independent gametophytic mutant, gem2, with similar characteristics to gem1, but which maps to a different genetic locus. gem2 shows reduced genetic transmission through both male and female gametes and leads to the production of divided or twin-celled pollen. Developmental analysis revealed that gem2 does not affect karyokinesis at PMI, but leads to repositioning of the cell plate, and partial or complete failure of cytokinesis, resulting in symmetrical divisions or binucleate pollen grains, respectively. Symmetrical divisions lead to altered pollen cell fate with both sister cells displaying vegetative cell fate. Moreover, we demonstrate that the predominant female defect in gem2 is a lack of cellularisation of the embryo sac during megagametogenesis. GEM2 therefore defines an independent genetic locus that is involved in the correct specification of both male and female gametophytic cytokinesis.