Evolution of chalcone isomerase from a noncatalytic ancestor.

The emergence of catalysis in a noncatalytic protein scaffold is a rare, unexplored event. Chalcone isomerase (CHI), a key enzyme in plant flavonoid biosynthesis, is presumed to have evolved from a nonenzymatic ancestor related to the widely distributed fatty-acid binding proteins (FAPs) and a plant protein family with no isomerase activity (CHILs). Ancestral inference supported the evolution of CHI from a protein lacking isomerase activity. Further, we identified four alternative founder mutations, i.e., mutations that individually instated activity, including a mutation that is not phylogenetically traceable. Despite strong epistasis in other cases of protein evolution, CHI's laboratory reconstructed mutational trajectory shows weak epistasis. Thus, enantioselective CHI activity could readily emerge despite a catalytically inactive starting point. Accordingly, X-ray crystallography, NMR, and molecular dynamics simulations reveal reshaping of the active site toward a productive substrate-binding mode and repositioning of the catalytic arginine that was inherited from the ancestral fatty-acid binding proteins.

Construction of a chalcone reductase expression vector and transformation of soybean plants.

The present study aimed to clone the soybean chalcone reductase 3 (CHR3) and create a recombinant expression vector pCAMBIA3300­CHR3 containing Bar resistance gene as a selection marker, and then obtain transgenic soybean plants using Agrobacterium infection. The plant expression vector pCAMBIA3300­CHR3 was transferred into soybean receptor plants, Jinong 17 and Jilin 30. Polymerase chain reaction (PCR) and Southern blotting were used to confirm the positive transgenic plants. Additionally, reverse transcription­quantitative PCR (RT­qPCR) was used to detect CHR3 expression and isoliquiritigenin content was measured using high­performance liquid chromatography (HPLC) in the transgenic offspring. Soybean CHR3 (932 bp fragment) was successfully cloned into the plant expression vector pCAMBIA3300­CHR3, which was subsequently transferred into soybean receptor plants. In the T1 generation positive plants were validated by PCR analysis, including eight Jinong 17 and five Jilin 30 transgenic plants; Southern blotting demonstrated that the functional components of the pCAMBIA3300­CHR3 vector had been integrated into the soybean genome; RT­qPCR results demonstrated that the expression of CHR3 mRNA was increased by 2 to 20­fold in the transgenic plants compared with the non­transgenic soybean plants. Furthermore, the isoliquiritigenin content was increased by 8.56% in the transgenic Jinong 17, compared with control plants, as detected by HPLC. The CHR3 gene can produce isoliquiritigenin, a precursor of daidzein, which in turn can improve the ability of soybean to resist phytophthora root rot.

Transcriptome profiling shows gene regulation patterns in a flavonoid pathway in response to exogenous phenylalanine in Boesenbergia rotunda cell culture.

BACKGROUND: Panduratin A extracted from Boesenbergia rotunda is a flavonoid reported to possess a range of medicinal indications which include anti-dengue, anti-HIV, anti-cancer, antioxidant and anti-inflammatory properties. Boesenbergia rotunda is a plant from the Zingiberaceae family commonly used as a food ingredient and traditional medicine in Southeast Asia and China. Reports on the health benefits of secondary metabolites extracted from Boesenbergia rotunda over the last few years has resulted in rising demands for panduratin A. However large scale extraction has been hindered by the naturally low abundance of the compound and limited knowledge of its biosynthetic pathway. RESULTS: Transcriptome sequencing and digital gene expression (DGE) analysis of native and phenylalanine treated Boesenbergia rotunda cell suspension cultures were carried out to elucidate the key genes differentially expressed in the panduratin A biosynthetic pathway. Based on experiments that show increase in panduratin A production after 14 days post treatment with exogenous phenylalanine, an aromatic amino acid derived from the shikimic acid pathway, total RNA of untreated and 14 days post-phenylalanine treated cell suspension cultures were extracted and sequenced using next generation sequencing technology employing an Illumina-Solexa platform. The transcriptome data generated 101, 043 unigenes with 50, 932 (50.41%) successfully annotated in the public protein databases; including 49.93% (50, 447) in the non-redundant (NR) database, 34.63% (34, 989) in Swiss-Prot, 24,07% (24, 316) in Kyoto Encyclopedia of Genes and Genomes (KEGG) and 16.26% (16, 426) in Clusters of Orthologous Groups (COG). Through DGE analysis, we found that 14, 644 unigenes were up-regulated and 14, 379 unigenes down-regulated in response to exogenous phenylalanine treatment. In the phenylpropanoid pathway leading to the proposed panduratin A production, 2 up-regulated phenylalanine ammonia-lyase (PAL), 3 up-regulated 4-coumaroyl:coenzyme A ligase (4CL) and 1 up-regulated chalcone synthase (CHS) were found. CONCLUSIONS: This is the first report of Boesenbergia rotunda de novo transcriptome data that could serve as a reference for gene or enzyme functional studies in the Zingiberaceae family. Although enzymes that are directly involved in the panduratin A biosynthetic pathway were not completely elucidated, the data provides an overall picture of gene regulation patterns leading to panduratin A production.

Genetics of flavonoid, carotenoid, and chlorophyll pigments in melon fruit rinds.

External color has profound effects on acceptability of agricultural products by consumers. Carotenoids and chlorophylls are known to be the major pigments of melon (Cucumis melo L.) rinds. Flavonoids (especially chalcones and anthocyanins) are also prominent in other fruits but have not been reported to occur in melons fruit. We analyzed the pigments accumulating in rinds of different melon genotypes during fruit development. We found that melon rind color is based on different combinations of chlorophyll, carotenoids, and flavonoids according to the cultivar tested and their ratios changed during fruit maturation. Moreover, in "canary yellow" type melons, naringenin chalcone, a yellow flavonoid pigment previously unknown to occur in melons, has been identified as the major fruit colorant in mature rinds. Naringenin chalcone is also prominent in other melon types, occurring together with carotenoids (mainly ß-carotene) and chlorophyll. Both chlorophyll and carotenoid pigments segregate jointly in an F(2) population originating from a cross between a yellow canary line and a line with green rind. In contrast, the content of naringenin chalcone segregates as a monogenic trait independently to carotenoids and chlorophyll. Transcription patterns of key structural phenylpropanoid and flavonoid biosynthetic pathway genes were monitored in attempts to explain naringenin chalcone accumulation in melon rinds. The transcript levels of CHI were low in both parental lines, but C4H, C4L, and CHS transcripts were upregulated in "Noy Amid", the parental line that accumulates naringenin chalcone. Our results indicate that naringenin chalcone accumulates independently from carotenoids and chlorophyll pigments in melon rinds and gives an insight into the molecular mechanism for the accumulation of naringenin chalcone in melon rinds.

Biochemical and molecular analysis of pink tomatoes: deregulated expression of the gene encoding transcription factor SlMYB12 leads to pink tomato fruit color.

The color of tomato fruit is mainly determined by carotenoids and flavonoids. Phenotypic analysis of an introgression line (IL) population derived from a cross between Solanum lycopersicum 'Moneyberg' and the wild species Solanum chmielewskii revealed three ILs with a pink fruit color. These lines had a homozygous S. chmielewskii introgression on the short arm of chromosome 1, consistent with the position of the y (yellow) mutation known to result in colorless epidermis, and hence pink-colored fruit, when combined with a red flesh. Metabolic analysis showed that pink fruit lack the ripening-dependent accumulation of the yellow-colored flavonoid naringenin chalcone in the fruit peel, while carotenoid levels are not affected. The expression of all genes encoding biosynthetic enzymes involved in the production of the flavonol rutin from naringenin chalcone was down-regulated in pink fruit, suggesting that the candidate gene underlying the pink phenotype encodes a regulatory protein such as a transcription factor rather than a biosynthetic enzyme. Of 26 MYB and basic helix-loop-helix transcription factors putatively involved in regulating transcription of genes in the phenylpropanoid and/or flavonoid pathway, only the expression level of the MYB12 gene correlated well with the decrease in the expression of structural flavonoid genes in peel samples of pink- and red-fruited genotypes during ripening. Genetic mapping and segregation analysis showed that MYB12 is located on chromosome 1 and segregates perfectly with the characteristic pink fruit color. Virus-induced gene silencing of SlMYB12 resulted in a decrease in the accumulation of naringenin chalcone, a phenotype consistent with the pink-colored tomato fruit of IL1b. In conclusion, biochemical and molecular data, gene mapping, segregation analysis, and virus-induced gene silencing experiments demonstrate that the MYB12 transcription factor plays an important role in regulating the flavonoid pathway in tomato fruit and suggest strongly that SlMYB12 is a likely candidate for the y mutation.