大豆籽粒发育过程中异黄酮合成相关酶基因的表达模式与异黄酮积累的相关分析

利用高效液相色谱法和实时定量PCR方法,分别测定了2个异黄酮含量显著差异的大豆品种鲁黑豆2号(LHD2)和南汇早黑豆(NHZ)在子粒发育过程中的异黄酮含量变化以及异黄酮合成相关酶基因的表达模式变化,试图分析异黄酮积累与各基因表达量变化的相关关系。结果表明在大豆子粒发育过程中,异黄酮含量逐渐升高,而不同异黄酮合成相关酶基因的表达趋势不同,CHS7、CHS8、CHR、CHI1A和IFS2的表达趋势与异黄酮积累模式基本一致,而IFS1和CHI1B1的表达趋势与异黄酮积累模式相反。IFR的表达模式在2个大豆品种中存在相反的趋势,在LHD2中与异黄酮组分积累趋势相反,而在NHZ中与异黄酮组分积累趋势相同。结果还表明,同一基因家族中不同基因在子粒发育过程中的表达量也存在差异。查尔酮合酶基因家族中CHS7和CHS8以及查尔酮异构酶基因家族的CHI1A的表达水平相对其他成员较高,异黄酮合酶基因家族中IFS2的表达量显著高于IFS1的表达量,预示这些基因家族在大豆子粒异黄酮积累过程中存在功能分化。此外,各基因表达模式与异黄酮积累的相关分析结果表明,不同基因表达模式与异黄酮积累的相关性在2个品种中也不尽相同。LHD2中CHS7、CHS8和IFS2在子粒发育过程中的表达量变化与不同异黄酮组分呈显著正相关,CHI1B1基因的表达量变化与不同异黄酮组分呈显著负相关。而在NHZ中,IFR在子粒发育过程中的表达量变化与多个异黄酮组分呈显著正相关。这预示了不同大豆品种异黄酮含量差异的潜在遗传基础。各异黄酮合成相关酶基因表达量变化的相关分析表明,在2个品种中,苯丙氨酸水解酶PAL1与4CL,4CL与CHS2以及CHS1与IFS2基因的表达量均呈现显著正相关。表明这些基因可能通过协同作用共同调控异黄酮的合成与积累。这些结果为今后利用基因工程提高大豆异黄酮含量奠定了基础。     

Interactions within a network of phytochrome, cryptochrome and UV-B phototransduction pathways regulate chalcone synthase gene expression in Arabidopsis leaf tissue

The Arabidopsis gene encoding the key flavonoid biosynthesis enzyme chalcone synthase (CHS) is regulated by several environmental and endogenous stimuli. Here we dissect the network of light signalling pathways that control CHS expression in mature leaves using cryptochrome (cry) and phytochrome (phy) deficient mutants. The UV-A/blue light induction of CHS is mediated principally by cry1, but neither cry1 nor cry2 is involved in UV-B induction or in the UV-A and blue light signalling pathways that interact synergistically with the UV-B pathway to enhance CHS expression. Moreover, these synergistic responses do not require phyA or phyB. Phytochrome is a positive regulator of the cry1 inductive pathway, mediating distinct potentiation and coaction effects. A red light pretreatment enhances subsequent cry1-mediated CHS induction. This potentiation is unaltered in phyA and phyB mutants but much reduced in a phyA phyB double mutant, indicating that it requires principally phyA or phyB. In contrast, the cry1-mediated induction of CHS, without pretreatment, is much reduced in phyB but not phyA mutants, indicating coaction between cry1 and phyB. Further experiments with phy-deficient mutants demonstrate that phyB is a negative regulator of the UV-B inductive pathway. We further show that phyB acts upstream of the points of interaction of the UV-A and blue synergism pathways with the UV-B pathway. We propose that phyB functions to balance flux through the cry1 and UV-B signalling pathways.     

An allelic series for the chalcone synthase locus in Arabidopsis

Five new alleles of the Arabidopsis chalcone synthase (CHS) locus, tt4, have been characterized at the gene, protein, and end product levels as a genetic approach to understanding structure-function relationships in a key enzyme of plant secondary metabolism. Together with two previously described mutants, these tt4 lines represent one of the first allelic series for a central enzyme of the flavonoid pathway and include both null alleles and alleles with leaky, apparently temperature-sensitive, phenotypes. A variety of effects on accumulation of CHS protein and flavonoid glycosides were observed among these lines, including alterations in the apparent stability and activity of the enzyme. Assembly of the CHS homodimer also appeared to be impacted in several cases. A three-dimensional model of the Arabidopsis CHS protein, based on the recently determined structure for alfalfa CHS, predicts significant effects on protein structure or folding for several of the mutations. This allelic series should provide a useful genetic resource for ongoing studies of flavonoid enzyme structure, function, and subcellular organization.     

Chalcone synthase homologues from Humulus lupulus: some enzymatic properties and expression

The enzymatic properties of four chalcone synthase homologues CHS_H1, VPS, CHS 2 and CHS 4 from Humulus lupulus L. were investigated after heterologous expression in Escherichia coli. It was found that both VPS and CHS_H1 can utilize isovaleryl-CoA and isobutyryl-CoA as substrates producing compounds with positions in thin layer chromatography characteristic for phloroisovalerophenone and phloroisobutyrophenone. These reactions are accompanied by the formation of associated byproducts. The formation of naringenin chalcone can be catalyzed primarily by CHS_H1. Comparatively the ability of VPS to perform chalcone synthase reaction is very limited. Since only CHS_H1 has true chalcone synthase activity, this enzyme can be considered a key enzyme in prenylflavonoid biosynthesis. Both CHS 2 and CHS 4 utilize isovaleryl-CoA and isobutyryl-CoA as substrates, but the reactions were prematurely terminated. In comparison with VPS and CHS_H1, the optimum pH of CHS 2 was shifted to lower value. High expression of chalcone synthase-like genes were found in maturating hop cones of cultivars with high bitter acid content (Agnus, Magnum, Target) by Northern and Western blotting using probes specific for vps, chs_H1, chs 4 and polyspecific serum risen against recombinant protein CHS4, respectively. It was also found that these cultivars maintained expression of CHS homologues for a longer period of time during cone development in contrast to time-limited expression of CHS homologues in cultivars with low bitter acids content.     

Compute simulation to characterize structure and function of chalcone synthase from <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> georgi

Prediction and analysis of molecular structure and biochemical function are of theoretical guiding significance for gene discovery and application, and considered as one of the central problem of computational biology. Here, some characteristic features of chalcone synthase (CHS) family from Scutellaria baicalensis were described via bioinformatic analysis, and showed as following: the nucleic acid sequences and amino acid sequences of three chs member genes shared high similarity in the molecular structures and physicochemical properties; SbCHS proteins were localized to the cytosol, and possessed a good hydrophobic nature, with lacking any transmembrane topological structure. The phylogram analysis suggested that they were a group genes with significant functional association and genetic conservation. The secondary structures of the SbCHSs were mainly composed of α-helixes and random coils, and the tertiary structures contained malonyl CoA linkers, besides, each of CHS-A and CHS-B with N-glycosylation motif included. Taken together, these results demonstrate that CHS family from S. baicalensis has the typical molecular structure and function of chalcone synthase, compared with the experimental data for Medicago sativa CHS protein.     

Localization of flavonoid enzymes in Arabidopsis roots

Immunofluorescence and immuno-electron microscopy have been used to test the hypothesis that flavonoid metabolism is organized as a membrane-associated enzyme complex. The cellular and subcellular locations of chalcone synthase (CHS) and chalcone isomerase (CHI), the first two enzymes of this pathway, were examined in Arabidopsis roots. High levels of both enzymes were found in the epidermal and cortex cells of the elongation zone and the root tip, consistent with the accumulation of flavonoid endproducts at these sites. Co-localization of CHS and CHI was observed at the endoplasmic reticulum and tonoplast in these cells, and also in electron-dense regions that are, as yet, unidentified. In addition, a striking asymmetric distribution was observed for these enzymes in cortex cells of the elongation zone, which may provide clues about the physiological function of flavonoids in roots. The accumulation of CHS and CHI was also examined in tt7(88), a mutant in the gene for flavonoid 3'-hydroxylase (F3'H), which has been postulated to serve as a membrane anchor for the flavonoid enzyme complex. CHS and CHI accumulated to lower levels in cortex cells and higher levels in epidermal cells in the roots of this mutant as compared with wild-type plants. Moreover, the electron-dense regions containing these two enzymes were not observed. However, localization of CHS and CHI to the ER and tonoplast did not appear to be affected, suggesting that other proteins may function in recruiting the "soluble" flavonoid enzymes to membranes. Staining of flavonoid endproducts with DPBA was consistent with expression of CHS and CHI in these seedlings.     

Biphenyl synthase,a novel type III polyketide synthase

Biphenyls and dibenzofurans are the phytoalexins of the Maloideae, a subfamily of the economically important Rosaceae. The carbon skeleton of the two classes of antimicrobial secondary metabolites is formed by biphenyl synthase (BIS). A cDNA encoding this key enzyme was cloned from yeast-extract-treated cell cultures of Sorbus aucuparia. BIS is a novel type III polyketide synthase (PKS) that shares about 60% amino acid sequence identity with other members of the enzyme superfamily. Its preferred starter substrate is benzoyl-CoA that undergoes iterative condensation with three molecules of malonyl-CoA to give 3,5-dihydroxybiphenyl via intramolecular aldol condensation. BIS did not accept CoA-linked cinnamic acids such as 4-coumaroyl-CoA. This substrate, however, was the preferential starter molecule for chalcone synthase (CHS) that was also cloned from S. aucuparia cell cultures. While BIS expression was rapidly, strongly and transiently induced by yeast extract treatment, CHS expression was not. In a phylogenetic tree, BIS grouped together closely with benzophenone synthase (BPS) that also uses benzoyl-CoA as starter molecule but cyclizes the common intermediate via intramolecular Claisen condensation. The molecular characterization of BIS thus contributes to the understanding of the functional diversity and evolution of type III PKSs.     

An Arabidopsis Mutant Tolerant to Lethal Ultraviolet-B Levels Shows Constitutively Elevated Accumulation of Flavonoids and Other Phenolics

The isolation and characterization of mutants hypersensitive to ultraviolet (UV) radiation has been a powerful tool to learn about the mechanisms that protect plants against UV-induced damage. To increase our understanding of the various mechanisms of defense against UVB radiation, we searched for mutations that would increase the level of tolerance of Arabidopsis plants to UV radiation. We describe a single gene dominant mutation (uvt1) that leads to a remarkable tolerance to UVB radiation conditions that would kill wild-type plants. Pigment analyses show a constitutive increase in accumulation of UV-absorbing compounds in uvt1 that increases the capacity of the leaves to block UVB radiation and therefore is likely to be responsible for the elevated resistance of this mutant to UVB radiation. These increases in absorption in the UV region are due, at least in part, to increases in flavonoid and sinapate accumulation. Expression of chalcone synthase (CHS) mRNA was shown to be constitutively elevated in uvt1 plants, suggesting that the increases in absorption may be a consequence of changes in gene expression. Expression of CHS in uvt1 was shown to be still inducible by UV, indicating that the uvt1 lesion may not affect the UV-mediated regulation of CHS gene expression. Our data support an important role for UV screens in the overall protection of plants to UVB radiation. The uvt1 mutant could prove to be an important tool to elucidate further the exact role of UV-absorbing pigments in UV protection as well as the relative contribution of other mechanisms to the overall tolerance of plants to UV radiation.     

植物查尔酮合成酶超基因家族的分子进化

查尔酮合成酶(CHS)超基因家族又称为植物类型III聚酮合酶超基因家族, 其编码酶通过催化和合成一系列结构多样及生理活性各异的次生代谢物, 在植物生长发育和适应环境的过程中扮演着重要角色。为全面了解CHS超基因家族在植物中的进化规律, 重建其进化历史, 该研究利用14种具有全基因组数据的代表植物, 通过生物信息学手段, 深入挖掘和分析了不同植物类群基因组中查尔酮合成酶超基因家族的成员构成, 推测了其可能的扩增机制和功能分歧, 并探讨了该超基因家族在植物中的总体进化趋势。结果共识别144条具有表达信息的同源序列, 它们全部来自9种陆生植物的基因组, 藻类植物基因组中没有发现相关序列。系统发育和进化分析表明, CHS超基因家族的起源古老, 它们可能为适应复杂的生态环境而出现在早期的陆生植物中, 之后在长期的进化过程中不断发生谱系的特异扩张和拷贝丢失, 最后通过功能分歧的形式在不同植物类群中被分别固定。此外, 进化检验也显示, 尽管CHS超基因家族内部发生了多样的遗传改变, 但整个超基因家族仍处于强烈的纯化选择之下, 并且个体基因中也无任何单氨基酸位点受到正向选择的影响。     

Flavonoid-related regulation of auxin accumulation in<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis>-induced plant tumors

Agrobacterium tumefaciens-induced plant tumors accumulate considerable concentrations of free auxin. To determine possible mechanisms by which high auxin concentrations are maintained, we examined the pattern of auxin and flavonoid distribution in plant tumors. Tumors were induced in transformants of Trifolium repens (L.), containing the beta-glucuronidase ( GUS)-fused auxin-responsive promoter ( GH3) or chalcone synthase ( CHS2) genes, and in transformants of Arabidopsis thaliana (L.) Heynh., containing the GUS-fused synthetic auxin response element DR5. Expression of GH3::GUS and DR5::GUS was strong in proliferating metabolically active tumors, thus suggesting high free-auxin concentrations. Immunolocalization of total auxin with indole-3-acetic acid antibodies was consistent with GH3::GUS expression indicating the highest auxin concentration in the tumor periphery. By in situ staining with diphenylboric acid 2-aminoethyl ester, by thin-layer chromatography, reverse-phase high-performance liquid chromatography, and two-photon laser-scanning microscopy spectrometry, tumor-specific flavones, isoflavones and pterocarpans were detected, namely 7,4'-dihydroxyflavone (DHF), formononetin, and medicarpin. DHF was the dominant flavone in high free-auxin-accumulating stipules of Arabidopsis leaf primordia. Flavonoids were localized at the sites of strongest auxin-inducible CHS2::GUS expression in the tumor that was differentially modulated by auxin in the vascular tissue. CHS mRNA expression changes corresponded to the previously analyzed auxin concentration profile in tumors and roots of tumorized Ricinus plants. Application of DHF to stems, apically pretreated with alpha-naphthaleneacetic acid, inhibited GH3::GUS expression in a fashion similar to 1-N-naphthyl-phthalamic acid. Tumor, root and shoot growth was poor in inoculated tt4(85) flavonoid-deficient CHS mutants of Arabidopsis. It is concluded that CHS-dependent flavonoid aglycones are possibly endogenous regulators of the basipetal auxin flux, thereby leading to free-auxin accumulation in A. tumefaciens-induced tumors. This, in turn, triggers vigorous proliferation and vascularization of the tumor tissues and suppresses their further differentiation.     

Expression of chalcone synthase and chalcone isomerase proteins in Arabidopsis seedlings

Antibodies have been developed against the first two enzymes of flavonoid biosynthesis in Arabidopsis thaliana. Chalcone synthase (CHS) and chalcone isomerase (CHI) were overexpressed and purified from Escherichia coli as fusion proteins with glutathione S-transferase from Schistosoma japonicum. The recombinant proteins were then used to immunize chickens and the resulting IgY fraction was purified from egg yolks. Immunoblots of crude protein extracts from Arabidopsis seedlings carrying wild-type and null alleles for CHS and CHI showed that the resulting antibody preparations provide useful tools for characterizing expression of the flavonoid pathway at the protein level. An initial analysis of expression patterns in seedlings shows that CHS and CHI proteins are present at high levels during a brief period of early seedling germination that just precedes the transient accumulation of flavonoid end-products.     

定点突变提高虎杖聚酮合酶的苯亚甲基丙酮合酶活性北大核心CSCD

虎杖(Polygonum cuspidatum)聚酮合酶(polyketide synthase 1,PcPKS1)同时具有查尔酮合酶(chalcone synthase,CHS)及苯亚甲基丙酮合酶(benzylidene acetone synthase,BAS)催化活性,能够催化生成聚酮类化合物柚皮素查尔酮和苯亚甲基丙酮,进而催化合成黄酮类或覆盆子酮等具有多种生物学活性的化合物。本研究通过分析虎杖PcPKS1与掌叶大黄(Rheum palmatum)BAS、拟南芥(Arabidopsis thaliana)CHS等家族成员的序列以及酶催化位点的构象,确定可能影响酶功能的3个氨基酸位点:Thr133、Ser134、Ser339。采用定点突变对PcPKS1进行分子修饰,成功获得2个突变体并进行相关体外酶促反应,高效液相色谱(high performance liquid chromatography,HPLC)产物分析结果表明,在pH 7.0和pH 9.0的体外酶促条件下,突变体T133LS134A和S339V维持BAS和CHS双功能活性,且BAS活性显著高于原PcPKS1。本研究为利用PcPKS1进行基因工程调节黄酮类和覆盆子酮化合物的生物合成提供理论依据。     

Structure function analysis of benzalacetone synthase from Rheum palmatum

Benzalacetone synthase (BAS) is a plant-specific chalcone synthase (CHS) superfamily type III polyketide synthase (PKS) that catalyzes a one-step decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA. The diketide forming activity of Rheum palmatum BAS is attributed to the characteristic substitution of the conserved active-site Phe215 with Leu (numbering in Medicago sativa CHS). To further understand the structure and function of R. palmatum BAS, four site-directed mutants (C197T, C197G, G256L, and S338V) were newly constructed. All the mutants did not change the product pattern, however, the activity was 2-fold increased in S338V, while reduced to half in G256L mutant. On the other hand, the C197 mutants were functionally almost identical to wild-type BAS, excluding the possibility that the second active-site Cys is involved in the enzyme reaction. Instead, homology modeling suggested a possibility that, unlike the case of CHS, BAS utilizes an alternative pocket to lock the coumaroyl moiety for the diketide formation reaction.     

Involvement of plasma membrane redox activity and calcium homeostasis in the UV-B and UV-A/blue light induction of gene expression in Arabidopsis.

UV and blue light are important regulators of plant gene expression and development. We investigated the signal transduction processes involved in the induction of chalcone synthase (CHS) and phenylalanine ammonia-lyase (PAL) gene expression by UV-B and UV-A/blue light in an Arabidopsis cell suspension culture. Experiments with electron transport inhibitors indicated that plasma membrane redox activity is involved in both signal transduction pathways. Calcium ionophore treatment stimulated expression of the TOUCH3 gene, and this induction was strongly antagonized by UV-A/blue and UV-B light, suggesting that both light qualities may promote calcium efflux from the cytosol. Consistent with this hypothesis, experiments with specific inhibitors indicated that UV-B and UV-A/blue light regulate calcium levels in a cytosolic pool in part via the action of specific Ca2+-ATPases. On the basis of these and previous findings, we propose that plasma membrane redox activity, initiated by photoreception, is coupled to the regulation of calcium release from an intracellular store, generating a calcium signal that is required to induce CHS expression.