Molecular Basis for Chemical Evolution of Flavones to Flavonols and Anthocyanins in Land Plants.

During the course of evolution of land plants, different classes of flavonoids, including flavonols and anthocyanins, sequentially emerged, facilitating adaptation to the harsh terrestrial environment. Flavanone 3ß-hydroxylase (F3H), an enzyme functioning in flavonol and anthocyanin biosynthesis and a member of the 2-oxoglutarate-dependent dioxygenase (2-ODD) family, catalyzes the hydroxylation of (2S)-flavanones to dihydroflavonols, but its origin and evolution remain elusive. Here, we demonstrate that functional flavone synthase Is (FNS Is) are widely distributed in the primitive land plants liverworts and evolutionarily connected to seed plant F3Hs. We identified and characterized a set of 2-ODD enzymes from several liverwort species and plants in various evolutionary clades of the plant kingdom. The bifunctional enzyme FNS I/F2H emerged in liverworts, and FNS I/F3H evolved in Physcomitrium (Physcomitrella) patens and Selaginella moellendorffii, suggesting that they represent the functional transition forms between canonical FNS Is and F3Hs. The functional transition from FNS Is to F3Hs provides a molecular basis for the chemical evolution of flavones to flavonols and anthocyanins, which contributes to the acquisition of a broader spectrum of flavonoids in seed plants and facilitates their adaptation to the terrestrial ecosystem.

Expression of Root Genes in Arabidopsis Seedlings Grown by Standard and Improved Growing Methods.

Roots of Arabidopsis thaliana seedlings grown in the laboratory using the traditional plant-growing culture system (TPG) were covered to maintain them in darkness. This new method is based on a dark chamber and is named the improved plant-growing method (IPG). We measured the light conditions in dark chambers, and found that the highest light intensity was dramatically reduced deeper in the dark chamber. In the bottom and side parts of dark chambers, roots were almost completely shaded. Using the high-throughput RNA sequencing method on the whole RNA extraction from roots, we compared the global gene expression levels in roots of seedlings from these two conditions and identified 141 differently expressed genes (DEGs) between them. According to the KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment, the flavone and flavonol biosynthesis and flavonoid biosynthesis pathways were most affected among all annotated pathways. Surprisingly, no genes of known plant photoreceptors were identified as DEGs by this method. Considering that the light intensity was decreased in the IPG system, we collected four sections (1.5 cm for each) of Arabidopsis roots grown in TPG and IPG conditions, and the spatial-related differential gene expression levels of plant photoreceptors and polar auxin transporters, including CRY1, CRY2, PHYA, PHYB, PHOT1, PHOT2, and UVR8 were analyzed by qRT-PCR. Using these results, we generated a map of the spatial-related expression patterns of these genes under IPG and TPG conditions. The expression levels of light-related genes in roots is highly sensitive to illumination and it provides a background reference for selecting an improved culture method for laboratory-maintained Arabidopsis seedlings.

Genome-wide analysis of DNA methylation in photoperiod- and thermo-sensitive male sterile rice Peiai 64S.

BACKGROUND: Epigenetic modifications play important roles in the regulation of plant development. DNA methylation is an important epigenetic modification that dynamically regulates gene expression during developmental processes. However, little studies have been reported about the methylation profiles of photoperiod- and thermo-sensitive genic male sterile (PTGMS) rice during the fertility transition. RESULTS: In this study, using methylated DNA immunoprecipitation sequencing (MeDIP-seq), the global DNA methylation patterns were compared in the rice PTGMS line PA64S under two different environments (different temperatures and day lengths). The profiling of the DNA methylation under two different phenotypes (sterility and fertility) revealed that hypermethylation was observed in PA64S (sterility), and 1258 differentially methylated regions (DMRs) were found between PA64S (sterility) and PA64S (fertility). Twenty differentially methylated genes of them were further validated through bisulfite sequencing, and four of these genes were analyzed by qRT-PCR. Especially, a differentially methylated gene (LOC_Os08g38210), which encoded transcription factor BIM2, is a component of brassinosteroid signaling in rice. The hypermethylated BIM2 gene may suppress some downstream genes in brassinosteroid signaling pathway, and thus affect the male fertility in PA64S. CONCLUSIONS: The results presented here indicated that hypermethylation was observed in PA64S (sterility). Gene Ontology (GO) analysis and KEGG analysis revealed that flavone and flavonol biosynthrsis, circadian rhythm, photosynthesis and oxidative phosphorylation pathways were involved in sterility-fertility transition of PA64S.

Identification of a bifunctional maize C- and O-glucosyltransferase.

Flavonoids accumulate in plant vacuoles usually as O-glycosylated derivatives, but several species can also synthesize flavonoid C-glycosides. Recently, we demonstrated that a flavanone 2-hydroxylase (ZmF2H1, CYP93G5) converts flavanones to the corresponding 2-hydroxy derivatives, which are expected to serve as substrates for C-glycosylation. Here, we isolated a cDNA encoding a UDP-dependent glycosyltransferase (UGT708A6), and its activity was characterized by in vitro and in vivo bioconversion assays. In vitro assays using 2-hydroxyflavanones as substrates and in vivo activity assays in yeast co-expressing ZmF2H1 and UGT708A6 show the formation of the flavones C-glycosides. UGT708A6 can also O-glycosylate flavanones in bioconversion assays in Escherichia coli as well as by in vitro assays with the purified recombinant protein. Thus, UGT708A6 is a bifunctional glycosyltransferase that can produce both C- and O-glycosidated flavonoids, a property not previously described for any other glycosyltransferase.

Microbial transformations of 3-methoxyflavone by strains of Aspergillus niger.

Microbial transformation of 3-methoxyflavone into 3'-hydroxyflavon-3-yloxymethyl myristate was presented. Six filamentous fungi were used as biocatalysts: a wild strain of Aspergillus niger KB, its four UV mutants (A. niger MB, SBP, SBJ, 13/5) and the strain of Penicillium chermesinum 113. The highest yields were observed for the strains of A. niger KB and A. niger SBP (69.8% and 63.1%, respectively).

Metabolic engineering of the flavone-C-glycoside pathway using polyprotein technology.

C-Glycosylated flavonoids are biologically active plant natural products linked to dietary health benefits. We have used polyprotein expression technology to reconstruct part of the respective biosynthetic pathway in tobacco and yeast, such that dihydrochalcone and flavanone precursors are directly converted to C-glycosides. The polyprotein system developed facilitated the simple and efficient co-expression of pathway enzymes requiring different sub-cellular localization in both plants and yeast. The pathway to flavone-C-glucosides comprised a flavanone 2-hydroxylase (F2H), co-expressed with a C-glucosyltransferase (CGT). While pathway engineering in tobacco resulted in only minor C-glycoside formation, when fed with the flavanone naringenin, yeast transformed with the F2H-CGT polyprotein construct produced high concentrations of 2-hydroxynaringenin-C-glucoside in the medium. These fermentation products could then be readily chemically converted to the respective flavone-C-glucosides. The efficiency of the biosynthesis was optimal when both the F2H and CGT were obtained from the same species (rice). These results confirm the coupled roles of the F2H and CGT in producing C-glucosides in vivo, with the use of the polyprotein expression system in yeast offering a useful system to optimize the synthesis of these natural products in quantities suitable for dietary studies.

Uncovering nutritional metabolites and candidate genes involved in flavonoid metabolism in Houttuynia cordata through combined metabolomic and transcriptomic analyses.

The perennial herb Houttuynia cordata has long been cultivated and used as medicinal and edible plant in Asia. Nowadays, increasing attention is attracted due to its numerous health benefits. Flavonoids are the main chemical constituents exerting pharmacological activities. In the present study, we investigated both metabolome and transcriptome of two H. cordata accessions (6# and 7#) with distinct flavonoids contents. In total 397 metabolites, i.e., 220 flavonoids, 92 amino acids and derivatives, 20 vitamins, and 65 saccharides were abundant in aboveground part. Cyanidin-3-O-rutinoside and quercetin-3-O-galactoside were the most abundant flavonoids, which can be categorized into seven classes, namely anthocyanidins, chalcones, flavanols, flavanones, flavanonols, flavones, and flavonols. Flavonols was the most abundant group. Contents of 112 flavonoids differed significantly between the two accessions, with catechin-(7,8-bc)-4α-(3,4-dihydroxyphenyl)-dihydro-2-(3H)-one, cinchonain Id, and cinchonain Ic being the dominant flavonoid metabolites among them. Pinocembrin-7-O-neohesperidoside, pinocembrin-7-O-rutinoside, and kaempferol-3-O-galactoside-4'-O-glucoside were uniquely abundant in accession 7. Transcriptome data revealed a total of 110 different expressed genes related to flavonoid metabolism, with more highly expressed genes observed in 7#. We annotated a total of 19 differential flavonoid metabolites and 34 differentially expressed genes that are associated with the flavonoid metabolic network. Based on the transcriptome and qPCR data a total of 8 key candidate genes involved in flavonoid metabolism were identified. The ANS gene were found to play an important role in the synthesis of cyanidin-3-O-glucoside, while the CHI, F3'H and FLS genes were mainly responsible for controlling the levels of flavanones, flavones, and flavonols, respectively. Collectively, the present study provides important insights into the molecular mechanism underlying flavonoid metabolism in H. cordata.

4-Coumaroyl-CoA ligases in the biosynthesis of the anti-diabetic metabolite montbretin A.

BACKGROUND: Montbretins are rare specialized metabolites found in montbretia (Crocosmia x crocosmiiflora) corms. Montbretin A (MbA) is of particular interest as a novel therapeutic for type-2 diabetes and obesity. There is no scalable production system for this complex acylated flavonol glycoside. MbA biosynthesis has been reconstructed in Nicotiana benthamiana using montbretia genes for the assembly of MbA from its various different building blocks. However, in addition to smaller amounts of MbA, the therapeutically inactive montbretin B (MbB) was the major product of this metabolic engineering effort. MbA and MbB differ in a single hydroxyl group of their acyl side chains, which are derived from caffeoyl-CoA and coumaroyl-CoA, respectively. Biosynthesis of both MbA and MbB also require coumaroyl-CoA for the formation of the myricetin core. Caffeoyl-CoA and coumaroyl-CoA are formed in the central phenylpropanoid pathway by acyl activating enzymes (AAEs) known as 4-coumaroyl-CoA ligases (4CLs). Here we investigated a small family of montbretia AAEs and 4CLs, and their possible contribution to montbretin biosynthesis. RESULTS: Transcriptome analysis for gene expression patterns related to montbretin biosynthesis identified eight different montbretia AAEs belonging to four different clades. Enzyme characterization identified 4CL activity for two clade IV members, Cc4CL1 and Cc4CL2, converting different hydroxycinnamic acids into the corresponding CoA thioesters. Both enzymes preferred coumaric acid over caffeic acid as a substrate in vitro. While expression of montbretia AAEs did not enhance MbA biosynthesis in N. benthamiana, we demonstrated that both Cc4CLs can be used to activate coumaric and caffeic acid towards flavanone biosynthesis in yeast (Saccharomyces cerevisiae). CONCLUSIONS: Montbretia expresses two functional 4CLs, but neither of them is specific for the formation of caffeoyl-CoA. Based on differential expression analysis and phylogeny Cc4CL1 is most likely involved in MbA biosynthesis, while Cc4CL2 may contribute to lignin biosynthesis. Both Cc4CLs can be used for flavanone production to support metabolic engineering of MbA in yeast.

Expression and functional analysis of the nobiletin biosynthesis-related gene CitOMT in citrus fruit.

Nobiletin, a polymethoxy flavone (PMF), is specific to citrus and has been reported to exhibit important health-supporting properties. Nobiletin has six methoxy groups at the 3',4',5,6,7,8-positions, which are catalyzed by O-methyltransferases (OMTs). To date, researches on OMTs in citrus fruit are still limited. In the present study, a novel OMT gene (CitOMT) was isolated from two citrus varieties Satsuma mandarin (Citrus unshiu Marc.) and Ponkan mandarin (Citrus reticulata Blanco), and its function was characterized in vitro. The results showed that the expression of CitOMT in the flavedo of Ponkan mandarin was much higher than that of Satsuma mandarin during maturation, which was consistent with the higher accumulation of nobiletin in Ponkan mandarin. In addition, functional analysis showed that the recombinant protein of CitOMT had methylation activity to transfer a methyl group to 3'-hydroxy group of flavones in vitro. Because methylation at the 3'-position of flavones is vital for the nobiletin biosynthesis, CitOMT may be a key gene responsible for nobiletin biosynthesis in citrus fruit. The results presented in this study will provide new strategies to enhance nobiletin accumulation and improve the nutritional qualities of citrus fruit.

Molecular Basis of Overdominance at a Flower Color Locus.

Single-gene overdominance is one of the major mechanisms proposed to explain heterosis (i.e., hybrid vigor), the phenomenon that hybrid offspring between two inbred lines or varieties show superior phenotypes to both parents. Although sporadic examples of single-gene overdominance have been reported over the decades, the molecular nature of this phenomenon remains poorly understood and it is unclear whether any generalizable principle underlies the various cases. Through bulk segregant analysis, chemical profiling, and transgenic experiments, we show that loss-of-function alleles of the FLAVONE SYNTHASE (FNS) gene cause overdominance in anthocyanin-based flower color intensity in the monkeyflower species Mimulus lewisii FNS negatively affects flower color intensity by competing with the anthocyanin biosynthetic enzymes for the same substrates, yet positively affects flower color intensity by producing flavones, the colorless copigments required for anthocyanin stabilization, leading to enhanced pigmentation in the heterozyote (FNS/fns) relative to both homozygotes (FNS/FNS and fns/fns). We suggest that this type of antagonistic pleiotropy (i.e., alleles with opposing effects on different components of the phenotypic output) might be a general principle underlying single-gene overdominance.

Differentially evolved glucosyltransferases determine natural variation of rice flavone accumulation and UV-tolerance.

Decoration of phytochemicals contributes to the majority of metabolic diversity in nature, whereas how this process alters the biological functions of their precursor molecules remains to be investigated. Flavones, an important yet overlooked subclass of flavonoids, are most commonly conjugated with sugar moieties by UDP-dependent glycosyltransferases (UGTs). Here, we report that the natural variation of rice flavones is mainly determined by OsUGT706D1 (flavone 7-O-glucosyltransferase) and OsUGT707A2 (flavone 5-O-glucosyltransferase). UV-B exposure and transgenic evaluation demonstrate that their allelic variation contributes to UV-B tolerance in nature. Biochemical characterization of over 40 flavonoid UGTs reveals their differential evolution in angiosperms. These combined data provide biochemical insight and genetic regulation into flavone biosynthesis and additionally suggest that adoption of the positive alleles of these genes into breeding programs will likely represent a potential strategy aimed at producing stress-tolerant plants.

The Influence of Cultivars and Phenological Phases on the Accumulation of Nevadensin and Salvigenin in Basil (Ocimum basilicum).

According to the earlier literature the optimum harvest time for basil is at the full flowering stage if accumulation of essential oil is taken into account. In this research we have investigated our gene-bank stored basil accessions to determine whether the harvest timing is variety specific or not considering their flavonoid accumulation pattern. In our work we have determined by HPLC the content of two main flavonoid compounds, salvigenin and nevadensin, of eight different gene bank accessions from 2013 of Ocimum basilicum L. Data were analysed with the nonparametric Kruskal-Wallis test. Multiple pairwise comparisons were made using the Conover-Iman procedure where the significance level was 5%. We have observed that the optimum harvest time is at the full flowering stage in the case of accessions 'Genovese' and 'Piros', but this was not verified for the others. The result of our experiment has shown that the maximum salvigenin and nevadensin content was detected both at the full- and early flowering period. Almost in all phenological phases the accession 'M. Grünes' accumulated the highest level of nevadensin, while accession 'Lengyel' produced the lowest results in all phenological phases. Generally it could be observed that compared with nevadensin more salvigenin is accumulated, and it is independent of the phenological phases. In the case of salvigenin, 'M. Grünes' accession produced the largest quantity and accession 'Dark Opal' showed the lowest values. Our analyses demonstrated that harvest at different phenological phases may result in different amounts of active agents according to the cultivar.

Functional characterization of cis-acting elements mediating flavone-inducible expression of CYP321A1.

How plant allelochemicals elicit herbivore counterdefense genes remains largely unknown. To define the cis-acting elements for flavone inducibility of the allelochemical-metabolizing CYP321A1 from Helicoverpa zea, functions of varying length of CYP321A1 promoter are examined in H. zea fatbody cells. Progressive 3' deletions reveal presence of positive elements in the 5' untranslated region (UTR). Progressive 5' deletions map out regions of one essential element, four enhancers, and two silencers. Further progressive 5'deletions localize the essential element to a 36-bp region from -109 to -74. This essential element, designated as xenobiotic response element to flavone (XRE-Fla), contains a 5' AT-only TAAT inverted repeat, a GCT mirror repeat and a 3' antioxidant response element-like element. Internal deletions and substitution mutations show that the TAAT repeat is only necessary for the maximal flavone inducibility, whereas the other two components are necessary for the basal and flavone-induced expression of CYP321A1. Electrophoresis mobility shift assays demonstrate that XRE-Fla specifically binds to H. zea fatbody cell nuclear extracts and flavone treatment increases the nuclear concentrations of the yet-to-be characterized transcription factors binding to XRE-Fla. Taken together, CYP321A1 expression is regulated primarily by XRE-Fla and secondarily by other cis elements scattered in its promoter and 5' UTR.

Robustness of QTLs across germplasm pools using a model quantitative trait.

Knowledge of the inheritance of C-glycosyl flavone synthesis in maize (Zea mays L.) silk tissues has been acquired through detailed genetic studies involving primarily germplasm from the Corn Belt Dent race. To test the robustness of this genetic knowledge, we examined C-glycosyl flavone synthesis in a genetically distinct germplasm pool, popcorn. C-glycosyl flavone profiles and levels and the involvement of three specific genes/quantitative trait loci (p, pr1, and sm1) in C-glycosyl flavone synthesis were examined in popcorn germplasm representing at least two races and various diverse geographic regions. Twenty-four inbred lines and 23 hybrids involving these inbred lines and inbred line R17 were characterized for their flavone profiles and levels in silk tissues. Two F2 mapping populations were constructed to examine the involvement of p, pr1, and sm1 on C-glycosyl flavone synthesis. C-glycosyl flavone levels threefold higher than previously reported in Corn Dent Belt materials and a novel class of compounds were discovered. The gene action of sm1 was different, the functional p allele was not always dominant, and pr1 did not affect mays in synthesis. Based on this rather simplistic "model" quantitative trait, it appears that caution should be exercised when attempting to apply quantitative trait locus knowledge accumulated in one germplasm base to a germplasm base that is known to be distinctly unique.