Chalcone synthase-like gene in the liverwort, <Emphasis Type="Italic">Marchantia paleacea</Emphasis> var. <Emphasis Type="Italic">diptera</Emphasis>

A chalcone synthase (CHS)-like gene, MpCHSLK1, was isolated from liverwort, Marchantia paleacea var. diptera. Phylogenetic analysis revealed that MpCHSLK1 is closely related to stilbene synthase of the whisk fern, Psilotum nudum. Southern blot analysis using an MpCHSLK1 probe revealed that the gene belongs to a small gene family. Northern blot analysis indicated that CHS-like genes were expressed in either the mother plants or photoautotrophic cells. In photoautotrophic cells, the CHS-like genes were expressed light-dependently, and this expression was completely inhibited by the photosynthetic electron transport inhibitor, DCMU.Abbreviations CHS Chalcone synthase - DCMU 3-(3,4-Dichlorophenyl)-1-1-dimethylurea - POR Protochlorophyllide oxidoreductase - STS Stilbene synthase     

石笔木CHS基因家族成员的分析

用PCR方法从石笔木（Tutcheria spectabilis Dunn）的总DNA中扩增CHS基因外显子2的部分序列以代表该基因进行研究,经克隆后测序,得到长约740－780bp的序列共12个。以EMBL数据库中得到的紫花苜蓿和欧洲赤松各一个序列作为参照,进行排序和系统树的构建分析。结果显示,所有被测定的序列同源性均高于70％,为CHS基因家庭的成员,且这些序列由3大类共5种不同的基因拷贝组成：第一类家族成员因碱基的插入和缺失改变了读码框,推测已失去了CHS基因的功能,成为假基因,其中个别拷贝存在较大缺失,这在以前的研究中未见报道;第二类家族成员因活性位点的氨基酸发生突变,可能具有新的基因功能;第三类家族成员则具有原CHS基因的功能。综上结果,可预测,山茶科CHS基因家族较大,且有着复杂的进化式样。     

A chalcone synthase/stilbene synthase DNA probe for conifers

A probe for chalcone synthase (CHS) was generated by PCR using chalcone synthase conserved sequences. The cloned PCR product has high similarity to both chalcone synthase and stilbene synthase sequences. The probe was used to examine the organization of chalcone synthase and stilbene synthase genes in Abies procera, Pinus lambertiana, P. monticola, Picea glauca, P. sitchensis, Pseudostuga menziesii, Taxus brevifolia, and Thuja plicata. A large number of hybridizing bands were found in all species except T. plicata which did not cross hybridize. The hybridization patterns are highly polymorphic between the species and are also polymorphic within several of them.     

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.     

山茶属CHS基因家族的组成和分子进化初探

用PCR方法从4种山茶属(Camellia)(山茶科)(Theaceae)植物的总DNA中分别扩增到CHS基因外显子2的部分序列,经克隆、测序得到16个该基因的序列,这些序列与来自GenBank的该属另一种植物的3个序列及作为外类群的大豆(Glycine max (L.) Merr.)的2个序列一起进行分析.研究表明,山茶属CHS基因家族在进化过程中已分化为A、B、C三个亚家族,包括A1、A2、A3、B1、B2、C 等6类不同的基因成员;其中只有A2类成员为全部被研究的5种植物所共有,而其他5类成员只在部分被研究的植物中发现;所有这些CHS成员具有很高的同源性:在核苷酸水平上同一亚家族内基本上高于90%,不同亚家族间也在78%以上.从推测的氨基酸组成看,山茶属内CHS基因的功能已发生了分化,各类成员的碱基替代率有较大差异; 从分子系统发育树和可能的氨基酸组成分析,山茶属具有新功能的基因成员是在经过基因重复后,或是由少数几个位点的突变而成,或是由逐渐积累的突变而形成的.进一步分析认为,该属CHS基因的分化直到近期还在活跃地进行,并且不同种的进化式样有一定的差别,这种不同的进化式样可能是物种形成后受不同环境因素影响而形成的.     

山茶属CHS基因家族的组成和分子进化初探

用PCR方法从4种山茶属(Camellia)(山茶科)(Tlaeaceae)植物的总DNA中分别扩增到CHS基因外显子2的部分序列,经克隆、测序得到16个该基因的序列,这些序列与来自GenBank的该属另一种植物的3个序列及作为外类群的大豆(Glycine max (L)Merr)的2个序列一起进行分析。研究表明,山茶属CHS基因家族在进化过程中已分化为A、B、c三个亚家族,包括A1、A2、A3、B1、B2、C等6类不同的基因成员;其中只有A2类成员为全部被研究的5种植物所共有,而其他5类成员只在部分被研究的植物中发现;所有这些CHS成员具有很高的同源性：在核苷酸水平上同一亚家族内基本上高于90％,不同亚家族间也在78％以上。从推测的氨基酸组成看,山茶属内CHS基因的功能已发生了分化,各类成员的碱基替代率有较大差异;从分子系统发育树和可能的氨基酸组成分析,山茶属具有新功能的基因成员是在经过基因重复后,或是由少数几个位点的突变而成,或是由逐渐积累的突变而形成的。进一步分析认为,该属CHS基因的分化直到近期还在活跃地进行,并且不同种的进化式样有一定的差别,这种不同的进化式样可能是物种形成后受不同环境因素影响而形成的。     

Molecular genetic analysis of chalcone synthase in Lycopersicon esculentum and an anthocyanin-deficient mutant

Summary Twelve loci have previously been identified in tomato (Lycopersicon esculentum) that control the intensity and distribution of anthocyanin pigmentation; these are useful genetic markers because they encode phenotypes that are readily visualized in the hypocotyls of emerging seedlings. In order to obtain molecular probes for tomato anthocyanin biosynthesis genes, we isolated two cDNAs which encode chalcone synthase (CHS), one of the key enzymes in anthocyanin biosynthesis, from a tomato hypocotyl cDNA library. By comparing their nucleic acid sequences, we determined that the two CHS cDNAs have an overall similarity of 76% at the nucleotide level and 88% at the amino acid level. We identified hybridization conditions that would distinguish the two clones and by Northern analysis showed that 1.5 kb mRNA species corresponding to each cDNA were expressed in cotyledons, hypocotyls and leaves of wild-type seedlings. Hybridization of the cDNAs at low stringency to genomic blots indicated that in tomato, CHS genes comprise a family of at least three individual members. The two genes that encode the CHS cDNAs were then placed onto the tomato genetic map at unique loci by restriction fragment length polymorphism mapping. We also assayed the activity of CHS and another enzyme in the anthocyanin pathway, flavone 3-hydroxylase, in hypocotyl extracts of wild-type tomato and a number of anthocyanin-deficient mutants. Five mutants had reduced CHS activity when compared to the wildtype controls. Of these, three were also reduce in flavone 3-hydroxylase activity, suggesting a regulatory role for these loci. The other two mutants were preferentially reduced in CHS activity, suggesting a more specific role for these loci in CHS expression.     

The chalcone synthase multigene family of Petunia hybrida (V30): differential,light-regulated expression during flower development and UV light induction

We have analysed the expression of the 8–10 members of the gene family encoding the flavonoid biosynthetic enzyme chalcone synthase (CHS) from Petunia hybrida. During normal plant development only two members of the gene family (CHS-A and CHS-J) are expressed. Their expression is restricted to floral tissues mainly. About 90% of the total CHS mRNA pool is transcribed from CHS-A, wheares CHS-J delivers about 10% in flower corolla, tube and anthers. Expression of CHS-A and CHS-J during flower development is coordinated and (red) light-dependent. In young seedlings and cell suspension cultures expression of CHS-A and CHS-J can be induced with UV light. In addition to CHS-A and CHS-J, expression of another two CHS genes (CHS-B and CHS-G) is induced in young seedlings by UV light, albeit at a low level. In contrast to CHS genes from Leguminoseae, Petunia CHS genes are not inducible by phytopathogen-derived elicitors. Expression of CHS-A and CHS-J is reduced to a similar extent in a regulatory CHS mutant, Petunia hybrida Red Star, suggesting that both genes are regulated by the same trans-acting factors. Comparison of the promoter sequences of CHS-A and CHS-J reveals some striking homologies, which might represent cis-acting regulatory sequences.     

Nucleotide polymorphism in the chalcone synthase-A locus and evolution of the chalcone synthase multigene family of common morning glory Ipomoea purpurea

Chalcone synthase (CHS) is a small multigene family with at least four members (CHS-A, B, C and PS) in common morning glory Ipomoea purpurea ROTH. The chalcone synthase enzyme performs the initial condensation reaction that results in the 15-carbon three-ring structure that is the backbone of flavonoid biosynthesis. The biochemical pathway that commences with CHS is important in plant disease defence, pigment biosynthesis and UV protection. Accordingly, it is of substantial interest to characterize levels and patterns of molecular diversity for genes that encode this important enzyme. We report the sequence of 19 CHS-A alleles from Mexican and American populations of common morning glory. American populations of this annual self-compatible vine are believed to have been introduced from Mexico, where the species is native. Individual plants were sampled from populations of common morning glory throughout Mexico and the south-eastern USA. Four American alleles were sequenced and these, together with one allele from Mexico City, were identical in primary nucleotide sequence. These data suggest a restricted origin for the American population, probably as a consequence of selection for domestication by pre-Columbian peoples. Additionally the Mitontic (Chiapas, Mexico) population is significantly more homogeneous than expected by chance indicating that this population may also have experienced a recent population bottleneck. Estimates of nucleotide diversity from the Mexican CHS-A alleles were high. We present evidence that these estimates may, in part, result from low to moderate levels of interlocus recombination/gene conversion. We also present evidence that the ancient duplication of the CHS gene family, preceding the origin of the genus Ipomoea, was associated with heterogeneity in the rate of substitution between the resulting gene family members. The group of gene family members whose sequences possess a signature amino acid of the closely related Stilbene synthase exhibit a significantly faster proportional rate of nonsynonymous substitution.     

Likelihood Analysis of the Chalcone Synthase Genes Suggests the Role of Positive Selection in Morning Glories (<Emphasis Type="Italic">Ipomoea</Emphasis>)

Chalcone synthase (CHS) is a key enzyme in the biosynthesis of flavonoides, which are important for the pigmentation of flowers and act as attractants to pollinators. Genes encoding CHS constitute a multigene family in which the copy number varies among plant species and functional divergence appears to have occurred repeatedly. In morning glories (Ipomoea), five functional CHS genes (A–E) have been described. Phylogenetic analysis of the Ipomoea CHS gene family revealed that CHS A, B, and C experienced accelerated rates of amino acid substitution relative to CHS D and E. To examine whether the CHS genes of the morning glories underwent adaptive evolution, maximum-likelihood models of codon substitution were used to analyze the functional sequences in the Ipomoea CHS gene family. These models used the nonsynonymous/synonymous rate ratio ( = d_N/d_S) as an indicator of selective pressure and allowed the ratio to vary among lineages or sites. Likelihood ratio test suggested significant variation in selection pressure among amino acid sites, with a small proportion of them detected to be under positive selection along the branches ancestral to CHS A, B, and C. Positive Darwinian selection appears to have promoted the divergence of subfamily ABC and subfamily DE and is at least partially responsible for a rate increase following gene duplication.     

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。     

小桐子低温诱导查耳酮合酶基因的克隆及其表达分析

为了解查耳酮合酶在小桐子(Jatropha curcas)抗冷性形成中的作用, 基于小桐子低温锻炼转录组和数字基因表达谱数据, 克隆了低温新诱导表达的小桐子查耳酮合酶基因(JcCHS), 并分析了该基因的表达特性和功能。结果表明, JcCHS 基因的cDNA全长为1386 bp, 包含完整开放阅读框(ORF) 1170 bp, 编码389个氨基酸, JcCHS的理论分子量为42.2 kDa、等电点为6.53, 与蓖麻CHS 蛋白序列的相似性高达93.6%, 具有III 型聚酮合酶家族保守的查耳酮合酶/ 对苯乙烯合酶结构域。半定量RTPCR分析表明, JcCHS 在小桐子各组织中都有表达, 其中根的表达量较高。JcCHS 基因的表达能在一定程度上提高重组酵母菌的低温抵抗能力, 这说明JcCHS 基因可能参与了小桐子的抗低温响应。     

Evidence that stilbene synthases have developed from chalcone synthases several times in the course of evolution

Chalcone (CHS) and stilbene (STS) synthases are related plant-specific polyketide synthases that are key enzymes in the biosynthesis of flavonoids and of stilbene phytoalexins, respectively. A phylogenetic tree constructed from 34 CHS and four STS sequences revealed that the STS formed no separate cluster but grouped with CHS from the same or related plants. This suggested that STS evolved from CHS several times independently. We attempted to simulate this by site-directed mutagenesis of an interfamily CHS/STS hybrid, which contained 107 amino acids of a CHS from Sinapis alba (N-terminal) and 287 amino acids of a STS from Arachis hypogaea. The hybrid had no enzyme activity. Three amino acid exchanges in the CHS part (Gln-100 to Glu, Val-103 to Met, Val-105 to Arg) were sufficient to obtain low STS activity, and one additional exchange (Gly-23 to Thr) resulted in 20–25% of the parent STS activity. A kinetic analysis indicated (1) that the hybrids had the same K_m for the substrate 4-coumaroyl-CoA but a lower V_max than the parent STS, and (2) that they had a different substrate preference than the parent STS and CHS. Most of the other mutations and their combinations led to enzymatically inactive protein aggregates, suggesting that the subunit folding and/or the dimerization was disturbed. We propose that STS evolved from CHS by a limited number of amino acid exchanges, and that the advantage gained by this enzyme function favored the selection of plants with improved STS activity.Abbreviations AA amino acid - CHS chalcone synthase - STS stilbene synthase Correspondence to: J. Schröder 0592The data are discussed on the level of the presently available CHS and STS sequences although many were published after beginning the experiments several years ago. The new information changed the CHS consensus in some details but otherwise confirmed the deductions on the potential significance of amino acid differences between CHS and STS     

Molecular analysis of chalcone and dihydropinosylvin synthase from Scots pine (Pinus sylvestris), and differential regulation of these and related enzyme activities in stressed plants

Chalcone synthase (CHS) and stilbene synthase (STS) are closely related polyketide synthases which are key enzymes in the biosynthesis of flavonoids and stilbenes. Scots pine (Pinus sylvestris) is an interesting plant for a direct comparison of the enzymes. It not only contains the usual flavonoids, but also an unusual chalcone derivative (pinocembrin), and it synthesizes stilbenes of the pinosylvin type. We analysed a CHS and a STS by molecular cloning and functional expression in Escherichia coli. The CHS was active not only with 4-coumaroyl-CoA (to naringenin chalcone), but also with cinnamoyl-CoA (leading to pinocembrin). The STS was identified as dihydropinosylvin synthase, because it preferred dihydrocinnamoyl-CoA to cinnamoyl-CoA. The protein deviated in 47 positions from the CHS consensus. It had 73.2% identity with the CHS from P. sylvestris and only 65.3% with a STS from peanut (Arachis hypogaea). We also investigated the regulation of both enzyme types in P. sylvestris plantlets exposed to stress. CHS was present in non-stressed plantlets, and induction led to a transient increase with a peak after 16 h. STS¹ type activities were regulated differently and were absent in non-stressed plantlets. Increases were observed after a lag period of at least 6 h, and highest activities were obtained after 30 h. The analysis of the reactions in the plant extracts and the substrate specificity of the cloned STS indicate that the plants contain at least two different types of STS: the cloned dihydropinosylvin synthase and a pinosylvin synthase which preferentially utilizes cinnamoyl-CoA as substrate.     

Duplication and sequence divergence of rice chalcone synthase genes

Chalcone synthase (CHS, EC 2.3.1.74) is a key enzyme in the biosynthesis of flavonoids, which plays an important role in flower pigmentation and protection against UV, plant-microbe interactions, and plant fertility. In many plants, genes encoding CHS constitute a multigene family, wherein sequence and functional divergence occurred repeatedly. Since the genome of rice (Oryza sativa) has been completely sequenced, many genes possessing typical CHS domains were assumed to be chs genes, although the sequence and functional divergence of this large gene family has not as yet been investigated. In this study, all putative CHS members from O. sativa were analyzed by the phylogenetic methods. Our results indicate that the members of rice CHS superfamily probably diverged into four branches. Members of each branch may perform specific functions. Two conserved chs genes clustered with chs genes from other monocotyledon and dicotyledon species are believed to encode true CHSs responsible for the biosynthesis of flavonoids and anthocyanins. Two chs genes in one distant branch might play some functions in fertility. Several other putative chs genes were clustered together, and the function of this branch could not be predicted. Many tentative chs genes were clustered together with fatty acid synthase (FAS) genes. These genes may belong to the fas gene family. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 3, pp. 460–465. This text was submitted by the authors in English.     

Cloning and molecular analysis of structural genes involved in flavonoid and stilbene biosynthesis in grape (Vitis vinifera L.)

Genes involved in flavonoid and stilbene biosynthesis were isolated from grape (Vitis vinifera L.). Clones coding for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydoxylase (F3H), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX) and UDP glucose:flavonoid 3-O-glucosyl transferase (UFGT), were isolated by screening a cDNA library, obtained from mRNA from seedlings grown in light for 48 h using snapdragon (Antirrhinum majus) and maize heterologous probes. A cDNA clone coding for stilbene synthase (StSy) was isolated by probing the library with a specific oligonucleotide. These clones were sequenced and when the putative products were compared to the published amino acid sequence for corresponding enzymes, the percentages of similarity ranged from 65% (UFGT) to 90% (CHS and PAL). The analysis of the genomic organization and expression of these genes in response to light shows that PAL and StSy genes belong to large multigene families, while the others are present in one to four copies per haploid genome. The steady-state level of mRNAs encoded by the flavonoid biosynthetic genes as determined in young seedlings is coordinately induced by light, except for PAL and StSy, which appear to be constitutively expressed.     

Isolation and sequence analysis of a chalcone synthase cDNA ofMatthiola incana R. Br. (Brassicaceae)

A cDNA clone (pcM12) of the chalcone synthase (CHS) ofMatthiola incana R. Br. (Brassicacease) was isolated from a cDNA library, sequenced and analysed. It comprises the complete coding sequence for the CHS and 5 and 3 untranslated regions. The deduced amino acid sequence shows that theMatthiola incana CHS consists of 394 amino acid residues. Comparison with CHS amino acid sequences of other plants indicates more than 82% homology.     

Two sequences encoding chalcone synthase in yellow lupin (Lupinus luteus l.) may have evolved by gene duplication

Two full copy cDNA sequences encoding chalcone synthase (CHS) were selected from a yellow lupin (Lupinus luteus L.) root and nodule cDNA library, and sequenced. Analysis of their open reading frames gave evidence that both encode the functional enzyme. Sequence alignment and phylogenetic studies on the DNA and protein level of these clones compared to the sequences of chalcone synthases from 54 other plant species reveal the possibility that lupin chalcone synthase is encoded by a multigene family consisting of at least two distinct genes that probably diverged by gene duplication. The duplication event is estimated to have taken place about 16 million years ago.