Chalcone synthase-like genes active during corolla development are differentially expressed and encode enzymes with different catalytic properties in Gerbera hybrida (Asteraceae)

Recent studies on chalcone synthase (CHS) and the related stilbene synthase (STS) suggest that the structure of chs-like genes in plants has evolved into different forms, whose members have both different regulation and capacity to code for different but related enzymatic activities. We have studied the diversity of chs-like genes by analysing the structure, expression patterns and catalytic properties of the corresponding enzymes of three genes that are active during corolla development in Gerbera hybrida. The expression patterns demonstrate that chs-like genes are representatives of three distinct genetic programmes that are active during organ differentiation in gerbera. Gchs1 and gchs3 code for typical CHS enzymes, and their gene expression pattern temporally correlates with flavonol (gchs1, gchs3) and anthocyanin (gchs1) synthesis during corolla development. Gchs2 is different. The expression pattern does not correlate with the pigmentation pattern, the amino acid sequence deviates considerably from the consensus of typical CHSs, and the catalytic properties are different. The data indicate that it represents a new member in the large superfamily of chs and chs-related genes.     

Organization of soybean chalcone synthase gene clusters and characterization of a new member of the family

Chalcone synthase (CHS; EC 2.3.1.74), the first committed enzyme of the multibranched pathway of flavonoid/isoflavonoid biosynthesis is encoded by a multigene family in soybean, (Glycine max L. Merrill). Our results suggest that this gene family comprises at least seven members, some of which are clustered. We have identified four chs clusters in the allo-tetraploid G. max genome and chs5, a newly characterized member of the chs gene family is present in two of them. We describe the complete nucleotide sequence of chs5, the identification of its immediate neighbors and the organization of the four hitherto identified chs clusters in the Gm genome.     

Chalcone synthase in rice (Oryza sativa L.): Detection of the CHS protein in seedlings and molecular mapping of the chs locus

The chalcone synthase is a key enzyme that catalyses the first dedicated reaction of the flavonoid pathway in higher plants. The chs gene and its protein product in rice has been investigated. The presence of a chalcone synthase (CHS) protein in rice seedlings and its developmental stage-specific expression has been demonstrated by western analysis. The chalcone synthase of rice was found to be immunologically similar to that of maize. A rice cDNA clone, Os-chs cDNA, encoding chalcone synthase, isolated from a leaf cDNA library of an indica rice variety Purpleputtu has been mapped to the centromeric region of chromosome 11 of rice. It was mapped between RFLP markers RG2 and RG103. RG2 is the nearest RFLP marker located at a genetic distance of 3.3 cM. Some segments of chromosome 11 of rice including chs locus are conserved on chromosome 4 of maize. The markers, including chs locus on chromosome 11 of rice are located, though not in the same order, on chromosome 4 of maize. Genetic analysis of purple pigmentation in two rice lines, Abhaya and Shyamala, used in the present mapping studies, indicated the involvement of three genes, one of which has been identified as a dominant inhibitor of leaf pigmentation. The Os-chs cDNA shows extensive sequence homology, both for DNA and protein (deduced), to that of maize, barley and also to different monocots and dicots.     

Cloning and analysis of two genes for chalcone synthase from Petunia hybrida

Summary With the help of a cDNA probe for a chalcone synthase gene of Petroselinum a cDNA clone for a chalcone synthase gene of Petunia hybrida could be identified. The homologous cDNA allowed the cloning of two genomic EcoRI fragments from Petunia hybrida containing complete chalcone synthase genes. It could be demonstrated that the genes on the two fragments are different and are not allelic but members of a gene family. The two genes are found in a variety of different Petunia lines including in the two conditional mutants affected in chalcone synthase expression in floral buds, White Joy and Red Star. The structure of the two chs genes from Petunia is compared to the chs gene from Antirrhinum majus.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Molecular evolution and functional specialization of chalcone synthase superfamily from Phalaenopsis Orchid

Plant genomes appear to exploit the process of gene duplication as a primary means of acquiring biochemical and developmental flexibility. The best example is the gene encoding chalcone synthase (CHS, EC2.3.1.74), the first committed step in flavonoid biosynthesis. In this study, we examined the molecular evolution of three CHS family members of Phalaenopsis including a novel chs gene (phchs5), which is slowly evolved. The inferred phylogeny of the chs genes of Phalaenopsis with other two orchid plants, Bromoheadia finlaysoniana and Dendrobium hybrid, suggested that gene duplication and divergence have occurred before divergence of these three genera. Relatively quantitative RT-PCR analysis identified expression patterns of these three chs genes in different floral tissues at different developmental stages. Phchs5 was the most abundantly expressed chs gene in floral organs and it was specifically transcribed in petal and lip at the stages when anthocyanin accumulated (stage1–4). Phchs3 and phchs4 were expressed at much lower levels than phchs5. Phchs3 was expressed in pigmented tissue (including lip, petal and sepal) at middle stages (stages 2–4) and in colorless reproductive tissue at late stage (stage 5). Phchs4 was only expressed in petal at earlier stages (stage 1–3) and in lip at middle stage (stage 4). These results present new data on differentiation of gene expression among duplicate copies of chs genes in Phalaenopsis.     

Tissue-specific expression of the chalcone synthase multigene family in Phaseolus vulgaris: development of a RT-PCR method for the expression profiling of the chs isogenes

Chalcone synthase is a key metabolic control point in the biosynthesis of a large number of flavonoids and isoflavonoid metabolites. Chs genes in bean comprise a multigene family, of which certain individual members can be differentially induced with respect to kinetics and extent of accumulation. A RT-PCR technique, based on primers designed complementary to a common conserved region and divergent 3′ sequences of the bean chs family, was developed to detect the expression of individual members of the chs family. The semi-quantitative technique is based on the amplification of short, overlapping sequences differing in size. The method was found to be sensitive, rapid, and capable of distinguishing among the individual chs members (chs 1, 4, 14, and 17). The tissue-specific expression of chs isogenes in bean seedlings, flowers and callus, as well as the effect of light on chs expression in etiolated tissue was documented.     

Promoter analysis of the chalcone synthase (chsA) gene of Petunia hybrida: a 67 bp promoter region directs flower-specific expression

In order to scan the 5 flanking region of the chalcone synthase (chs A) gene for regulatory sequences involved in directing flower-specific and UV-inducible expression, a chimaeric gene was constructed containing the chs A promoter of Petunia hybrida (V30), the chloramphenicol acetyl transferase (cat) structural sequence as a reporter gene and the chs A terminator region of Petunia hybrida (V30). This chimaeric gene and 5 end deletions thereof were introduced into Petunia plants with the help of Ti plasmid-derived plant vectors and CAT activity was measured. A 220 bp chs A promoter fragment contains cis-acting elements conferring flower-specific and UV-inducible expression. A promoter fragment from –67 to +1, although at a low level, was still able to direct flower-specific expression but could not drive UV-inducible expression in transgenic Petunia seedlings. Molecular analysis of binding of flower nuclear proteins to chs A promoter fragments by gel retardation assays showed strong specific binding to the sequences from –142 to +81. Promoter sequence comparison of chs genes from other plant species, combined with the deletion analysis and gel retardation assays, strongly suggests the involvement of the TACPyAT repeats (–59 and –52) in the regulation of organ-specificity of the chs A gene in Petunia hybrida. We also describe an in vitro organ-specific transient expression system, in which flower or purple callus protoplasts are used, that enables us to pre-screen organ-specific expression of a chimaeric reporter gene.     

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 characterization of a novel chalcone synthase gene from Phalaenopsis hybrida orchid flowers

Chalcone synthase (CHS, EC 2.3.1.74) is the key enzyme involved in flavonoid and anthocyanin biosynthesis. A complete DNA sequence of chalcone synthase gene designated Pchs1 was isolated by means of usual and then inverse polymerase chain reactions from genomic DNA of an orchid, Phalaenopsis hybrida, cv. Formosa rose. Nucleotide sequence analysis based on alignment with published Phalaenopsis chs cDNA revealed that Pchs1 contained an intact open reading frame of 1173-bp with one 109-bp intron at the conserved site. The deduced polypeptide (PCHS1) from Pchs1 comprised 390 amino acids with a predicted mol wt of 42.5 kD. PCHS1 showed 61–65% identities with CHS from other plants and retained most of the conserved residues. Some putative cis-regulatory elements were present at the 5′ and 3′ flanking regions of Pchs1. Southern blot analysis predicted at least four chs-like genes, thus indicating the presence of a small multigene chs family in P. hybrida. Relative quantitative RT-PCR showed that Pchs1 is expressed in petals at early flower development as well as in lip tissue when the flower has just opened. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 2, pp. 250–258. The text was submitted by the authors in English.     

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.     

Characterization and structural features of a chalcone synthase mutation in a white-flowering line of Matthiola incanaR. Br. (Brassicaceae)

For Matthiola incana (Brassicaceae), used as a model system to study biochemical and genetical aspects of anthocyanin biosynthesis, several nearly isogenic colored wild type lines and white-flowering mutant lines are available, each with a specific defect in the genes responsible for anthocyanin production (genes e, f, and g). For gene f supposed to code for chalcone synthase (CHS; EC 2.3.1.74), the key enzyme of the flavonoid/anthocyanin biosynthesis pathway belonging to the group of type III polyketide synthases (PKS), the wild type genomic sequence of M. incana line 04 was determined in comparison to the white-flowering CHS mutant line 18. The type of mutation in the chs gene was characterized as a single nucleotide substitution in a triplet AGG coding for an evolutionary conserved arginine into AGT coding for serine (R72S). Northern blots and RT-PCR demonstrated that the mutated gene is expressed in flower petals. Heterologous expression of the wild type and mutated CHS cDNA in E. Scherichia coli, verified by Western blotting and enzyme assays with various starter molecules, revealed that the mutant protein had no detectable activity, indicating that the strictly conserved arginine residue is essential for the enzymatic reaction. This mutation, which previously was not detected by mutagenic screening, is discussed in the light of structural and functional information on alfalfa CHS and related type III PKS enzymes.     

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。     

Gerbera hybrida (Asteraceae) imposes regulation at several anatomical levels during inflorescence development on the gene for dihydroflavonol-4-reductase

In the ornamental cut flower plant Gerbera hybrida the spatial distribution of regulatory molecules characteristic of differentiation of the composite inflorescence is visualized as the various patterns of anthocyanin pigmentation of different varieties. In order to identify genes that the plant can regulate according to these anatomical patterns, we have analysed gene expression affecting two enzymatic steps, chalcone synthase (CHS) and dihydroflavonol-4-reductase (DFR), in five gerbera varieties with spatially restricted anthocyanin pigmentation patterns. The dfr expression profiles vary at the levels of floral organ, flower type and region within corolla during inflorescence development according to the anthocyanin pigmentation of the cultivars. In contrast, chs expression, although regulated in a tissue-specific manner during inflorescence development, varies only occasionally. The variation in the dfr expression profiles between the varieties reveals spatially specific gene regulation that senses the differentiation events characteristic of the composite inflorescence.     

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.     

Flavonols and fertilization in Petunia hybrida: localization and mode of action during pollen tube growth

Flavonols form an important class of flavonoids which serve an essential function during plant reproduction. Flavonoid biosynthesis is initiated by the enzyme chalcone synthase (CHS). A high abundance of flavonols and chs mRNA was demonstrated in male and female reproductive organs of Petunia hybrida. Detailed analyses revealed precise spatial and temporal regulation of the chs promoter and flavonol synthesis in the stigma, style and ovules. Transgenic plants were generated with a complete block of flavonol biosynthesis as the result of anti-sense inhibition of chs gene activity. The absence of flavonols by this dominant mutation rendered these plants self-sterile. Pollination experiments with wild-type and mutant plants revealed that the production of flavonols in either the anthers or the pistils was required for pollen tube growth and seed set. Mutant pollen without flavonols in their exine germinated normally. However, after a short period of in vitro pollen tube growth the tips of these tubes disrupted and the protoplasm was disloaded leading to the death of the pollen grain. Addition of flavonol aglycones but not other flavonoids complemented this phenotype. Confocal laser scanning microscopy revealed the localization of high levels of flavonols throughout the wild-type pollen tube. These compounds were not detected in the exine or cell wall of growing tubes. In addition, it was observed that the flavone apigenin could completely inhibit pollen tube growth. Taken together, it is shown that flavonols play an important role in the growth of the pollen tube and their mode of action is discussed.