Dissection of genotype-phenotype associations in rice grains using metabolome quantitative trait loci analysis

A comprehensive and large‐scale metabolome quantitative trait loci (mQTL) analysis was performed to investigate the genetic backgrounds associated with metabolic phenotypes in rice grains. The metabolome dataset consisted of 759 metabolite signals obtained from the grains of 85 lines of rice (Oryza sativa, Sasanishiki × Habataki back‐crossed inbred lines). Metabolome analysis was performed using four mass spectrometry pipelines to enhance detection of different classes of metabolites. This mQTL analysis of a wide range of metabolites highlighted an uneven distribution of 802 mQTLs on the rice genome, as well as different modes of metabolic trait (m‐trait) control among various types of metabolites. The levels of most metabolites within rice grains were highly sensitive to environmental factors, but only weakly associated with mQTLs. Coordinated control was observed for several groups of metabolites, such as amino acids linked to the mQTL hotspot on chromosome 3. For flavonoids, m‐trait variation among the experimental lines was tightly governed by genetic factors that alter the glycosylation of flavones. Many loci affecting levels of metabolites were detected by QTL analysis, and plausible gene candidates were evaluated by in silico analysis. Several mQTLs profoundly influenced metabolite levels, providing insight into the control of rice metabolism. The genomic region and genes potentially responsible for the biosynthesis of apigenin‐6,8‐di‐C‐α‐l‐ arabinoside are presented as an example of a critical mQTL identified by the analysis.     

Partial reconstruction of flavonoid and isoflavonoid biosynthesis in yeast using soybean type I and type II chalcone isomerases

Flavonoids and isoflavonoids are major plant secondary metabolites that mediate diverse biological functions and exert significant ecological impacts. These compounds play important roles in many essential physiological processes. In addition, flavonoids and isoflavonoids have direct but complex effects on human health, ranging from reducing cholesterol levels and preventing certain cancers to improving women's health. In this study, we cloned and functionally characterized five soybean (Glycine max) chalcone isomerases (CHIs), key enzymes in the phenylpropanoid pathway that produces flavonoids and isoflavonoids. Gene expression and kinetics analysis suggest that the soybean type I CHI, which uses naringenin chalcone as substrate, is coordinately regulated with other flavonoid-specific genes, while the type II CHIs, which use a variety of chalcone substrates, are coordinately regulated with an isoflavonoid-specific gene and specifically activated by nodulation signals. Furthermore, we found that some of the newly identified soybean CHIs do not require the 4′-hydroxy moiety on the substrate for high enzyme activity. We then engineered yeast (Saccharomyces cerevisiae) to produce flavonoid and isoflavonoid compounds. When one of the type II CHIs was coexpressed with an isoflavone synthase, the enzyme catalyzing the first committed step of isoflavonoid biosynthesis, various chalcone substrates added to the culture media were converted to an assortment of isoflavanones and isoflavones. We also reconstructed the flavonoid pathway by coexpressing CHI with either flavanone 3β-hydroxylase or flavone synthase II. The in vivo reconstruction of the flavonoid and isoflavonoid pathways in yeast provides a unique platform to study enzyme interactions and metabolic flux.     

Integrated multiple population analysis of leaf architecture traits in maize (Zea mays L.)

Leaf morphology in maize is regulated by developmental patterning along three axes: proximodistal, mediolateral, and adaxial-abaxial. Maize contains homologues of many genes identified as regulators of leaf development in other species, but their relationship to the natural variation of leaf shape remains unknown. In this study, quantitative trait loci (QTLs) for leaf angle, leaf orientation value, leaf length, and leaf width were mapped by a total of 256 F(2:3) families evaluated in three environments. Meta-analysis was used to integrate genetic maps and detect QTLs across several independent QTL studies, on the basis of the previously reported experimental results for leaf architecture traits. Candidate gene sequences for leaf architecture were mapped in the integrated consensus genetic map. In total, 21 QTLs and 17 meta-QTLs (mQTLs) were detected. Among these QTLs, qLA1-1 and qLA2 were consistently detected in five and three populations respectively, and six of seven QTLs with contributions (R(2)) >10% were integrated in mQTLs. Six key mQTLs (mQTL1-1, mQTL2-1, mQTL3-3, mQTL5-1, mQTL7-2, and mQTL8-1) with R(2) of some initial QTLs >10% included 4-6 initial QTLs associated with 2-4 traits. Therefore, the chromosome regions for six mQTLs with high QTL co-localization might be hot spots of the important QTLs for the associated traits. Fifteen key candidate genes controlling leaf architecture traits coincided with 11 corresponding mQTLs, namely DWARF4, KAN3, liguleless1, TAC1, ROT3, AS2/liguleless2, PFL2, yabby9/SE/LIC/yabby15, mwp1, CYCD3;2, and CYCB1. In particular, DWARF4, liguleless1, AS2/liguleless2, yabby9/SE/LIC/yabby15, and CYCD3;2 were mapped within the important mQTL1-1, mQTL2-1, mQTL3-3, mQTL5-1, and mQTL7-2 intervals, respectively. Fine mapping or construction of single chromosome segment lines for genetic regions of these five mQTLs is worth further study and could be put to use in marker-assisted breeding. In conclusion, the results provide useful information for further research and help to reveal the molecular mechanisms with regard to leaf architecture traits.     

Intra- and interspecific variations in vacuolar flavonoids among Ficus species from the Budongo Forest,Uganda

Leaves of 14 species of Ficus growing in the Budongo Forest, Uganda, were analysed for vacuolar flavonoids. Three to six accessions were studied for each species to see whether there was intraspecific chemical variation. Thirty-nine phenolic compounds were identified or characterised, including 14 flavonol O-glycosides, six flavone O-glycosides and 15 flavone C-glycosides. In some species the flavonoid glycosides were acylated. Ficus thonningii contained in addition four stilbenes including glycosides. Most of the species could be distinguished from each other on the basis of their flavonoid profiles, apart from Ficus sansibarica and Ficus saussureana, which showed a very strong intraspecific variation. However, on the whole flavonoid profiles were sufficiently distinct to help in future identifications.     

Investigation of Two Distinct Flavone Synthases for Plant-Specific Flavone Biosynthesis in Saccharomyces cerevisiae

Flavones are plant secondary metabolites that have wide pharmaceutical and nutraceutical applications. We previously constructed a recombinant flavanone pathway by expressing in Saccharomyces cerevisiae a four-step recombinant pathway that consists of cinnamate-4 hydroxylase, 4-coumaroyl:coenzyme A ligase, chalcone synthase, and chalcone isomerase. In the present work, the biosynthesis of flavones by two distinct flavone synthases was evaluated by introducing a soluble flavone synthase I (FSI) and a membrane-bound flavone synthase II (FSII) into the flavanone-producing recombinant yeast strain. The resulting recombinant strains were able to convert various phenylpropanoid acid precursors into the flavone molecules chrysin, apigenin, and luteolin, and the intermediate flavanones pinocembrin, naringenin, and eriodictyol accumulated in the medium. Improvement of flavone biosynthesis was achieved by overexpressing the yeast P450 reductase CPR1 in the FSII-expressing recombinant strain and by using acetate rather than glucose or raffinose as the carbon source. Overall, the FSI-expressing recombinant strain produced 50% more apigenin and six times less naringenin than the FSII-expressing recombinant strain when p-coumaric acid was used as a precursor phenylpropanoid acid. Further experiments indicated that unlike luteolin, the 5,7,4′-trihydroxyflavone apigenin inhibits flavanone biosynthesis in vivo in a nonlinear, dose-dependent manner.     

Uniformity and distinctness of phyllocladus as evidenced by flavonoid accumulation

The conifer genus Phyllocladus is shown by comparative flavonoid chemistry to be remarkably homogeneous and quite distinct from other studied genera in the Podocarpaceae. It is characterized by the accumulation (in the foliage) of a predominance of flavone O-glycosides, and in particular, luteolin 7- and 3′-O-glycosides. Lower levels of flavonol O-glycosides are also evident. Two flavone glycosides are reported for the first time, luteolin 3′-O-α-L-rhamnopyranoside and luteolin 7-O-α-L-rhamnoside.     

Identification of Gene-Specific Polymorphisms and Association with Capsaicin Pathway Metabolites in Capsicum annuum L. Collections

Pepper (Capsicum annuum L.) is an economically important crop with added nutritional value. Production of capsaicin is an important quantitative trait with high environmental variance, so the development of markers regulating capsaicinoid accumulation is important for pepper breeding programs. In this study, we performed association mapping at the gene level to identify single nucleotide polymorphisms (SNPs) associated with capsaicin pathway metabolites in a diverse Capsicum annuum collection during two seasons. The genes Pun1, CCR, KAS and HCT were sequenced and matched with the whole-genome sequence draft of pepper to identify SNP locations and for further characterization. The identified SNPs for each gene underwent candidate gene association mapping. Association mapping results revealed Pun1 as a key regulator of major metabolites in the capsaicin pathway mainly affecting capsaicinoids and precursors for acyl moieties of capsaicinoids. Six different SNPs in the promoter sequence of Pun1 were found associated with capsaicin in plants from both seasons. Our results support that CCR is an important control point for the flux of p-coumaric acid to specific biosynthesis pathways. KAS was found to regulate the major precursors for acyl moieties of capsaicinoids and may play a key role in capsaicinoid production. Candidate gene association mapping of Pun1 suggested that the accumulation of capsaicinoids depends on the expression of Pun1, as revealed by the most important associated SNPs found in the promoter region of Pun1.     

The influence of intracellular pools of phenylalanine derivatives upon the synthesis of capsaicin by immobilized cell cultures of the chilli pepper,Capsicum frutescens

In an attempt to determine the potential factors controlling the biosynthesis of the secondary metabolite capsaicin by immobilized cell cultures of the chilli pepper, Capsicum frutescens Mill, labelling techniques using the radioactive precursor [¹⁴C]phenylalanine have been employed. Following preincubation treatments with either capsaicin (the end-product of the pathway) or sinapic acid, [¹⁴C]phenylalanine was applied and the movement of the label through the pathway and its eventual fate was followed. Results have shown that capsaicin, through a feedback-inhibition mechanism, negatively influences its own synthesis. Furthermore, capsaicin synthesis in these cells is not controlled via the activity of the enzymes phenylalanine ammonia-lyase and cinnamate 4-hydroxylase which may determine the rate of entry of metabolites into the phenylpropanoid pathway. The importance of other sinks for phenylalanine derivatives, which may compete for capsaicin precursors, has also been investigated. Surprisingly, protein proved to be only a relatively minor sink for phenylalanine with the great majority of the label rapidly ending up in covalently bound phenolics in the cell wall. Attempts to prevent this by applying sinapic acid were only partially successful. The importance of these results in relation to the possible control mechanisms which operate to control secondary metabolite synthesis in vitro is discussed.     

Flavonoids as taxonomic markers in old world Lupinus species

In a leaf survey of 54 specimens of 11 Old World Lupinus species three classes of flavonoids were detected: flavones (in 82%), flavonols (in 36%) and flavone C-glycosides (in 55%). The rough-seeded species were clearly distinguished from the smooth-seeded taxa by the presence of a novel 2′-hydroxyflavone, luteolin and flavone C-glycosides as major leaf constituents and by the absence of flavonols. Within the smooth-seeded species, there are three flavonoid patterns: (a) flavonols only, L. albus; (b) flavones and flavonols, L. luteus, L. hispanicus and L. angustifolius; and (c) flavones only, L. micranthus. L. angustifolius further differed in uniquely producing diosmetin as a major leaf constituent. These divisions coincide exactly with previous groupings based on alkaloidal and morphological data. Amongst the 12 samples of L. angustifolius three chemical races were distinguished and a number of diosmetin glucoside malate esters detected. The flower flavonoid aglycone patterns of the nine Old World species surveyed differed markedly from the corresponding leaf profiles by the presence of flavones: luteolin and apigenin in eight and chrysoeriol in seven species as major constituents, while flavone C-glycosides were found only in trace amount in three species. In a leaf flavonoid survey of 13 representative New World Lupinus taxa, glycoflavones were major leaf components, a variety of methylated flavones were identified and flavonols were absent. The presence of the novel 2′-hydroxyflavone in five New World species may indicate some evolutionary link with the rough seeded taxa of the Old World.     

Quantitative trait loci analysis of seed‐specialized metabolites reveals seed‐specific flavonols and differential regulation of glycoalkaloid content in tomato

Given the potential health benefits (and adverse effects), of polyphenolic and steroidal glycoalkaloids in the diet there is a growing interest in fully elucidating the genetic control of their levels in foodstuffs. Here we carried out profiling of the specialized metabolites in the seeds of the Solanum pennellii introgression lines identifying 338 putative metabolite quantitative trait loci (mQTL) for flavonoids, steroidal glycoalkaloids and further specialized metabolites. Two putative mQTL for flavonols and one for steroidal glycoalkaloids were cross‐validated by evaluation of the metabolite content of recombinants harboring smaller introgression in the corresponding QTL interval or by analysis of lines from an independently derived backcross inbred line population. The steroidal glycoalkaloid mQTL was localized to a chromosomal region spanning 14 genes, including a previously defined steroidal glycoalkaloid gene cluster. The flavonoid mQTL was further validated via the use of transient and stable overexpression of the Solyc12g098600 and Solyc12g096870 genes, which encode seed‐specific uridine 5′‐diphosphate‐glycosyltransferases. The results are discussed in the context of our understanding of the accumulation of polyphenols and steroidal glycoalkaloids, and how this knowledge may be incorporated into breeding strategies aimed at improving nutritional aspects of plants as well as in fortifying them against abiotic stress.     

Apigenin and amentoflavone glycosides in the psilotaceae and their phylogenetic significance

Documentation of amentoflavone O-glucosides as the predominant flavonoid glycosides in both genera of the Psilotaceae clearly distinguishes this family from all other families of vascular plants. Psilotum and Tmesipteris also possess apigenin C- and O-glycosides as common flavonoid types. Apigenin 7-O-rhamnoglucoside occurs in both genera and the previously undocumented apigenin 7-O-rhamnoglucoside-4′-O-glucoside, although identified only in Tmesipteris, may also be present in Psilotum. The existence of flavone C-glycosides in both genera may provide a phytochemical relationship between the Psilotaceae and some ferns. The phylogenetic significance of these results is discussed.     

First bacterial chalcone isomerase isolated from Eubacterium ramulus

The human fecal anaerobe Eubacterium ramulus is capable of degrading various flavonoids, including the flavone naringenin. The first step in the proposed degradation pathway is the isomerization of naringenin to the corresponding chalcone. Cell-free extracts of E. ramulus displayed chalcone isomerase activity. The enzyme from E. ramulus was purified to homogeneity. Its apparent molecular mass was estimated to be 136 and 129 kDa according to gel filtration and native polyacrylamide gel electrophoresis, respectively. Chalcone isomerase is composed of one type of subunit of 30 kDa. The purified enzyme catalyzed the isomerization of naringenin chalcone, isoliquiritigenin, and butein, three chalcones that differ in their hydroxylation pattern. N-bromosuccinimide, but also naringenin and phloretin, inhibited the purified enzyme considerably. This is the first report on a bacterial chalcone isomerase. The physiological function of the purified enzyme is unclear, but an involvement in the conversion of the flavanone naringenin to the chalcone is proposed.     

Changes in Fructose 2,6-Bisphosphate Levels in Green Pepper (Capsicum annuum L.) Fruit in Response to Temperature

The regulatory metabolite, fructose 2,6-bisphosphate (Fru 2,6-P₂) was found in green pepper (Capsicum annuum L.). The Fru 2,6-P₂ level was found to: (a) rise rapidly in response to heat; (b) drop rapidly, followed by recovery, in response to cold storage of fruit and, (c) oscillate during cold storage of fruit. The possible existence of a relationship between chilling injury and Fru 2,6-P₂ is considered.