Transgenic rice seed synthesizing diverse flavonoids at high levels: a new platform for flavonoid production with associated health benefits

Flavonoids possess diverse health‐promoting benefits but are nearly absent from rice, because most of the genes encoding enzymes for flavonoid biosynthesis are not expressed in rice seeds. In the present study, a transgenic rice plant producing several classes of flavonoids in seeds was developed by introducing multiple genes encoding enzymes involved in flavonoid synthesis, from phenylalanine to the target flavonoids, into rice. Rice accumulating naringenin was developed by introducing phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS) genes. Rice producing other classes of flavonoids, kaempferol, genistein, and apigenin, was developed by introducing, together with PAL and CHS, genes encoding flavonol synthase/flavanone‐3‐hydroxylase, isoflavone synthase, and flavone synthases, respectively. The endosperm‐specific GluB‐1 promoter or embryo‐ and aleurone‐specific 18‐kDa oleosin promoters were used to express these biosynthetic genes in seed. The target flavonoids of naringenin, kaempferol, genistein, and apigenin were highly accumulated in each transgenic rice, respectively. Furthermore, tricin was accumulated by introducing hydroxylase and methyltransferase, demonstrating that modification to flavonoid backbones can be also well manipulated in rice seeds. The flavonoids accumulated as both aglycones and several types of glycosides, and flavonoids in the endosperm were deposited into PB‐II‐type protein bodies. Therefore, these rice seeds provide an ideal platform for the production of particular flavonoids due to efficient glycosylation, the presence of appropriate organelles for flavonoid accumulation, and the small effect of endogenous enzymes on the production of flavonoids by exogenous enzymes.     

Metabolic engineering of flavonoids in tomato (Solanum lycopersicum): the potential for metabolomics

Flavonoids comprise a large and diverse group of polyphenolic plant secondary metabolites. In plants, flavonoids play important roles in many biological processes such as pigmentation of flowers, fruits and vegetables, plant-pathogen interactions, fertility and protection against UV light. Being natural plant compounds, flavonoids are an integral part of the human diet and there is increasing evidence that dietary polyphenols are likely candidates for the observed beneficial effects of a diet rich in fruits and vegetables on the prevention of several chronic diseases. Within the plant kingdom, and even within a single plant species, there is a large variation in the levels and composition of flavonoids. This variation is often due to specific mutations in flavonoid-related genes leading to quantitative and qualitative differences in metabolic profiles. The use of such specific flavonoid mutants with easily scorable, visible phenotypes has led to the isolation and characterisation of many structural and regulatory genes involved in the flavonoid biosynthetic pathway from different plant species. These genes have been used to engineer the flavonoid biosynthetic pathway in both model and crop plant species, not only from a fundamental perspective, but also in order to alter important agronomic traits, such as flower and fruit colour, resistance, nutritional value. This review describes the advances made in engineering the flavonoid pathway in tomato (Solanum lycopersicum). Three different approaches will be described; (I) Increasing endogenous tomato flavonoids using structural or regulatory genes; (II) Blocking specific steps in the flavonoid pathway by RNA interference strategies; and (III) Production of novel tomato flavonoids by introducing novel branches of the flavonoid pathway. Metabolite profiling is an essential tool to analyse the effects of pathway engineering approaches, not only to analyse the effect on the flavonoid composition itself, but also on other related or unrelated metabolic pathways. Metabolomics will therefore play an increasingly important role in revealing a more complete picture of metabolic perturbation and will provide additional novel insights into the effect of the introduced genes and the role of flavonoids in plant physiology and development.     

Tradeoff between biomass and flavonoid accumulation in white clover reflects contrasting plant strategies

An outdoor study was conducted to examine relationships between plant productivity and stress-protective phenolic plant metabolites. Twenty-two populations of the pasture legume white clover were grown for 4½ months during spring and summer in Palmerston North, New Zealand. The major phenolic compounds identified and quantified by HPLC analysis were glycosides of the flavonoids quercetin and kaempferol. Multivariate analysis revealed a trade-off between flavonoid accumulation and plant productivity attributes. White clover populations with high biomass production, large leaves and thick tap roots showed low levels of quercetin glycoside accumulation and low quercetin:kaempferol ratios, while the opposite was true for less productive populations. The latter included stress-resistant ecotypes from Turkey and China, and the analysis also identified highly significant positive relationships of quercetin glycoside accumulation with plant morphology (root:shoot ratio). Importantly, a high degree of genetic variation was detected for most of the measured traits. These findings suggest merit for considering flavonoids such as quercetin as potential selection criteria in the genetic improvement of white clover and other crops.     

Natural variation of ZmCGT1 is responsible for isoorientin accumulation in maize silk

Maize (Zea mays L.) silk contains high levels of flavonoids and is widely used to promote human health. Isoorientin, a natural C-glycoside flavone abundant in maize silk, has attracted considerable attention due to its potential value. Although different classes of flavonoid have been well characterized in plants, the genes involved in the biosynthesis of isoorientin in maize are largely unknown. Here, we used targeted metabolic profiling of isoorientin on the silks in an association panel consisting of 294 maize inbred lines. We identified the gene ZmCGT1 by genome-wide association analysis. The ZmCGT1 protein was characterized as a 2-hydroxyflavanone C-glycosyltransferase that can C-glycosylate 2-hydroxyflavanone to form flavone-C-glycoside after dehydration. Moreover, ZmCGT1 overexpression increased isoorientin levels and RNA interference-mediated ZmCGT1 knockdown decreased accumulation of isoorientin in maize silk. Further, two nucleotide polymorphisms, A502C and A1022G, which led to amino acid changes I168L and E341G, respectively, were identified to be functional polymorphisms responsible for the natural variation in isoorientin levels. In summary, we identified the gene ZmCGT1, which plays an important role in isoorientin biosynthesis, providing insights into the genetic basis of the natural variation in isoorientin levels in maize silk. The identified favorable CG allele of ZmCGT1 may be further used for genetic improvement of nutritional quality in maize.     

The flavonoid biosynthetic pathway in plants: Function and evolution

Flavonoids are a class of low molecular weight phenolic compounds that is widely distributed in the plant kingdom. They exhibit a diverse spectrum of biological functions and play an important role in the interaction between plants and their environment. Flavonoids not only protect the plant from the harmful effects of UV irradiation but also play a crucial role in the sexual reproduction process. A special class of flavonoid polymers, the tannins, plays a structural role in the plant. Yet other classes of flavonoids, flavonols and anthocyanins, have been implicated in the attraction of pollinators. Certain flavonoids participate in the interaction between plants and other organisms such as symbiotic bacteria and parasites. This raises the intriguing question as to how these different compounds arose and evolved. Based on taxonomy and molecular analysis of gene expression patterns it is possible to deduce a putative sequence of acquisition of the different branches of the biosynthetic pathway and their regulators.     

A chemical complementation approach reveals genes and interactions of flavonoids with other pathways

In addition to the classical functions of flavonoids in the response to biotic/abiotic stress conditions, these phenolic compounds have been implicated in the modulation of various developmental processes. These findings suggest that flavonoids are more integral components of the plant signaling machinery than traditionally recognized. To understand how flux through the flavonoid pathway affects plant cellular processes, we used wild‐type and chalcone isomerase mutant (transparent testa 5, tt5) seedlings grown under anthocyanin inductive conditions, in the presence or absence of the flavonoid intermediate naringenin, the product of the chalcone isomerase enzyme. Because flavonoid biosynthetic genes are expressed under anthocyanin inductive conditions regardless of whether anthocyanins are formed or not, this system provides an excellent opportunity to specifically investigate the molecular changes associated with increased flux through the flavonoid pathway. By assessing genome‐wide mRNA accumulation changes in naringenin‐treated and untreated tt5 and wild‐type seedlings, we identified a flavonoid‐responsive gene set associated with cellular trafficking, stress responses and cellular signaling. Jasmonate biosynthetic genes were highly represented among the signaling pathways induced by increased flux through the flavonoid pathway. In contrast to studies showing a role for flavonoids in the control of auxin transport, no effect on auxin‐responsive genes was observed. Taken together, our data suggest that Arabidopsis can sense flavonoids as a signal for multiple fundamental cellular processes.     

Flavonoid races in lasthenia (Compositae)

Nine of the 13 taxa ofLasthenia in which two or more populations were examined for flavonoid constituents exhibited interpopulation variation in these constituents. In certain species entire classes of compounds were present in some races and absent from others. Some of these biochemical differences are due to the failure of certain steps to occur in the biosynthesis of various flavonoids from precursor compounds. Another pattern of variation involves intraspecific differences in the nature of flavonoid glycosides that are produced. In view of the close biosynthetic relationships among all the flavonoids produced byLasthenia, the genetic differences among the flavonoid races of a species may be small. Whether or not these biochemical differences have any adaptive significance is problematical at present.     

Metabolic engineering of flavonoids in plants and microorganisms

Over 9,000 flavonoid compounds have been found in various plants, comprising one of the largest families of natural products. Flavonoids are an essential factor in plant interactions with the environment, often serving as the first line of defense against UV irradiation and pathogen attacks. Flavonoids are also major nutritional compounds in foods and beverages, with demonstrated health benefits. Some flavonoids are potent antioxidants, and specific flavonoid compounds are beneficial in many physiological and pharmacological processes. Therefore, engineering of flavonoid biosynthesis in plants or in microorganisms has significant scientific and economical importance. Construction of biosynthetic pathways in heterologous systems offers promising results for large-scale flavonoid production by fermentation or bioconversion. Genomics and metabolomics now offer unprecedented tools for detailed understanding of the engineered transgenic organism and for developing novel technologies to further increase flavonoid production yields. We summarize some of the recent metabolic engineering strategies in plants and microorganisms, with a focus on applications of metabolic flux analysis. We are confident that these engineering approaches will lead to successful industrial flavonoid production in the near future.     

Significance of flavonoids in plant resistance and enhancement of their biosynthesis

The roles of flavonoids in plant defence against pathogens, herbivores, and environmental stress are reviewed and their significant contribution to plant resistance is discussed. The induction of flavonoids is of particular interest for gathering evidence of their roles. Tools are mentioned which may enhance flavonoid biosynthesis and accumulation. These include metabolic engineering and UV light. The induction of defence-related flavonoids is modified by other determining factors and competition between growth and secondary metabolism may exist. In an evolutionary context, stress-related oxidative pressure may have been a major trigger for the distribution and abundance of flavonoids. UV protection is one of their most significant, or even the most significant, functional role for flavonoids. The multi-functionality of these compounds, however, often complicates the interpretation of experimental results but, overall, it supports the importance of flavonoids.     

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.     

Geographic variation of flavonoids in Phlox carolina

Non-anthocyanin flavonoid expression was determined among 64 continuous range and nine disjunct range populations of Phlox carolina. The species contains four flavone nuclei and a total of 25 derivatives, most of which are $\text{C-}\text{glycosides}$. Nearly all populations express unique flavone patterns. Using a qualitative similarity index of minimum biosynthetic step distance (MBSD), the average population contains only ca. 50% of the overall substitutional diversity. The separate ranges are distinctive in the % occurrence of most compounds but only weakly divergent in comparative mean MBSD. Proximity and MBSD are significantly correlated overall but local variability is extensive and no discrete racism is apparent. Within the continuous range 18 compounds are distributed in independent but overlapping gradients and 13 of these extend consistently through the disjunct range. The patch-work continuum of flavone variation is the apparent consequence of dissection and contraction of a historically more widespread distribution. Other general aspects of the variation patterns are discussed.     

Biosynthesis of 5-deoxyflavanones in microorganisms

Flavanones are the common precursors of plant polyphenolic compounds collectively known as flavonoids. Leguminous plants have evolved a distinct class of flavanone molecules, known as 5-deoxyflavanones that play important roles in their symbiotic interactions. A four-step metabolic circuit was constructed in Escherichia coli with plant genes from heterologous origins: 4-coumarate:coenzyme A ligase from Petroselinum crispum, chalcone synthases (CHS) from Medicago sativa and Petunia x hybrida and chalcone reductase and chalcone isomerase from M. sativa. Evaluation of the different recombinant strains in shake flask experiments demonstrated that P. hybrida rather than M. sativa CHS resulted in the highest liquiritigenin production levels in glucose minimal medium, starting from precursor p-coumaric acid. Expression of the same recombinant pathway in Saccharomyces cerevisiae resulted in the accumulation of both 5-hydroxyflavanone and 5-deoxyflavanone, with the yields of the later lower than that achieved in E. coli. Other phenylpropanoid acid precursors, such as cinnamic acid and caffeic acid could also be metabolized through the recombinant pathway, yielding corresponding 5-deoxyflavanone compounds. The construction of such recombinant strains for 5-deoxyflavanone biosynthesis offers an alternative way to biochemically characterize flavonoid biosynthetic enzymes and promising production platforms for the biosynthesis of such high-value natural products.