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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.

Steviol glucosides, such as stevioside and rebaudioside A, are natural products roughly 200-fold sweeter than sugar and are used as natural, noncaloric sweeteners. Biosynthesis of rebaudioside A, and other related stevia glucosides, involves formation of the steviol diterpenoid followed by a series of glycosylations catalyzed by uridine diphosphate (UDP)-dependent glucosyltransferases. UGT76G1 from Stevia rebaudiana catalyzes the formation of the branched-chain glucoside that defines the stevia molecule and is critical for its high-intensity sweetness. Here, we report the 3D structure of the UDP-glucosyltransferase UGT76G1, including a complex of the protein with UDP and rebaudioside A bound in the active site. The X-ray crystal structure and biochemical analysis of site-directed mutants identifies a catalytic histidine and how the acceptor site of UGT76G1 achieves regioselectivity for branched-glucoside synthesis. The active site accommodates a two-glucosyl side chain and provides a site for addition of a third sugar molecule to the C3′ position of the first C13 sugar group of stevioside. This structure provides insight on the glycosylation of other naturally occurring sweeteners, such as the mogrosides from monk fruit, and a possible template for engineering of steviol biosynthesis.

Background Tea is one of the most popular non-alcoholic beverages worldwide. However, the tea plant, Camellia sinensis , is difficult to culture in vitro , to transform, and has a large genome, rendering little genomic information available. Recent advances in large-scale RNA sequencing (RNA-seq) provide a fast, cost-effective, and reliable approach to generate large expression datasets for functional genomic analysis, which is especially suitable for non-model species with un-sequenced genomes. Results Using high-throughput Illumina RNA-seq, the transcriptome from poly (A) + RNA of C. sinensis was analyzed at an unprecedented depth (2.59 gigabase pairs). Approximate 34.5 million reads were obtained, trimmed, and assembled into 127,094 unigenes, with an average length of 355 bp and an N50 of 506 bp, which consisted of 788 contig clusters and 126,306 singletons. This number of unigenes was 10-fold higher than existing C. sinensis sequences deposited in GenBank (as of August 2010). Sequence similarity analyses against six public databases (Uniprot, NR and COGs at NCBI, Pfam, InterPro and KEGG) found 55,088 unigenes that could be annotated with gene descriptions, conserved protein domains, or gene ontology terms. Some of the unigenes were assigned to putative metabolic pathways. Targeted searches using these annotations identified the majority of genes associated with several primary metabolic pathways and natural product pathways that are important to tea quality, such as flavonoid, theanine and caffeine biosynthesis pathways. Novel candidate genes of these secondary pathways were discovered. Comparisons with four previously prepared cDNA libraries revealed that this transcriptome dataset has both a high degree of consistency with previous EST data and an approximate 20 times increase in coverage. Thirteen unigenes related to theanine and flavonoid synthesis were validated. Their expression patterns in different organs of the tea plant were analyzed by RT-PCR and quantitative real time PCR (qRT-PCR). Conclusions An extensive transcriptome dataset has been obtained from the deep sequencing of tea plant. The coverage of the transcriptome is comprehensive enough to discover all known genes of several major metabolic pathways. This transcriptome dataset can serve as an important public information platform for gene expression, genomics, and functional genomic studies in C. sinensis .

Strawberry (Fragariaxananassa Duch), a fruit of economic and nutritional importance, is also a model species for fleshy fruits and genomics in Rosaceae. Strawberry fruit quality at different harvest stages is a function of the fruit's metabolite content, which results from physiological changes during fruit growth and ripening. In order to investigate strawberry fruit development, untargeted (GC-MS) and targeted (HPLC) metabolic profiling analyses were conducted. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were employed to explore the non-polar and polar metabolite profiles from fruit samples at seven developmental stages. Different cluster patterns and a broad range of metabolites that exerted influence on cluster formation of metabolite profiles were observed. Significant changes in metabolite levels were found in both fruits turning red and fruits over-ripening in comparison with red-ripening fruits. The levels of free amino acids decreased gradually before the red-ripening stage, but increased significantly in the over-ripening stage. Metabolite correlation and network analysis revealed the interdependencies of individual metabolites and metabolic pathways. Activities of several metabolic pathways, including ester biosynthesis, the tricarboxylic acid cycle, the shikimate pathway, and amino acid metabolism, shifted during fruit growth and ripening. These results not only confirmed published metabolic data but also revealed new insights into strawberry fruit composition and metabolite changes, thus demonstrating the value of metabolomics as a functional genomics tool in characterizing the mechanism of fruit quality formation, a key developmental stage in most economically important fruit crops.

Symbiotic root nodules in leguminous plants result from interaction between the plant and nitrogen-fixing rhizobia bacteria. There are two major types of legume nodules, determinate and indeterminate. Determinate nodules do not have a persistent meristem, while indeterminate nodules have a persistent meristem. Auxin is thought to play a role in the development of both these types of nodules. However, inhibition of rootward auxin transport at the site of nodule initiation is crucial for the development of indeterminate nodules but not determinate nodules. Using the synthetic auxin-responsive DR5 promoter in soybean (Glycine max), we show that there is relatively low auxin activity during determinate nodule initiation and that it is restricted to the nodule periphery subsequently during development. To examine if and what role auxin plays in determinate nodule development, we generated soybean composite plants with altered sensitivity to auxin. We overexpressed microRNA393 to silence the auxin receptor gene family, and these roots were hyposensitive to auxin. These roots nodulated normally, suggesting that only minimal/reduced auxin signaling is required for determinate nodule development. We overexpressed microRNA160 to silence a set of repressor auxin response factor transcription factors, and these roots were hypersensitive to auxin. These roots were not impaired in epidermal responses to rhizobia but had significantly reduced nodule primordium formation, suggesting that auxin hypersensitivity inhibits nodule development. These roots were also hyposensitive to cytokinin and had attenuated expression of key nodulation-associated transcription factors known to be regulated by cytokinin. We propose a regulatory feedback loop involving auxin and cytokinin during nodulation.

Background microRNAs (miRNAs) are key regulators of gene expression and play important roles in many aspects of plant biology. The role(s) of miRNAs in nitrogen-fixing root nodules of leguminous plants such as soybean is not well understood. We examined a library of small RNAs from Bradyrhizobium japonicum -inoculated soybean roots and identified novel miRNAs. In order to enhance our understanding of miRNA evolution, diversification and function, we classified all known soybean miRNAs based on their phylogenetic conservation (conserved, legume- and soybean-specific miRNAs) and examined their genome organization, family characteristics and target diversity. We predicted targets of these miRNAs and experimentally validated several of them. We also examined organ-specific expression of selected miRNAs and their targets. Results We identified 120 previously unknown miRNA genes from soybean including 5 novel miRNA families. In the soybean genome, genes encoding miRNAs are primarily intergenic and a small percentage were intragenic or less than 1000 bp from a protein-coding gene, suggesting potential co-regulation between the miRNA and its parent gene. Difference in number and orientation of tandemly duplicated miRNA genes between orthologous genomic loci indicated continuous evolution and diversification. Conserved miRNA families are often larger in size and produce less diverse mature miRNAs than legume- and soybean-specific families. In addition, the majority of conserved and legume-specific miRNA families produce 21 nt long mature miRNAs with distinct nucleotide distribution and regulate a more conserved set of target mRNAs compared to soybean-specific families. A set of nodule-specific target mRNAs and their cognate regulatory miRNAs had inverse expression between root and nodule tissues suggesting that spatial restriction of target gene transcripts by miRNAs might govern nodule-specific gene expression in soybean. Conclusions Genome organization of soybean miRNAs suggests that they are actively evolving. Distinct family characteristics of soybean miRNAs suggest continuous diversification of function. Inverse organ-specific expression between selected miRNAs and their targets in the roots and nodules, suggested a potential role for these miRNAs in regulating nodule development.

Background Flavonoid 3′,5′-hydroxylase (F3′5′H), an important branch point enzyme in tea plant flavan-3-ol synthesis, belongs to the CYP75A subfamily and catalyzes the conversion of flavones, flavanones, dihydroflavonols and flavonols into 3′,4′,5′-hydroxylated derivatives. However, whether B-ring hydroxylation occurs at the level of flavanones and/or dihydroflavonols, in vivo remains unknown. Results The Camellia sinensis F3′5′H ( CsF3′5′H ) gene was isolated from tea cDNA library. Expression pattern analysis revealed that CsF3′5′H expression was tissue specific, very high in the buds and extremely low in the roots. CsF3′5′H expression was enhanced by light and sucrose. Over-expression of CsF3′5′H produced new-delphinidin derivatives, and increased the cyanidin derivative content of corollas of transgenic tobacco plants, resulting in the deeper transgenic plant flower color. Heterologous expressions of CsF3′5′H in yeast were carried out to demonstrate the function of CsF3′5′H enzyme in vitro . Heterologous expression of the modified CsF3′5′H ( CsF3′5′H gene fused with Vitis vinifera signal peptide , FSI) revealed that 4′-hydroxylated flavanone (naringenin, N) is the optimum substrate for CsF3′5′H, and was efficiently converted into both 3′4′- and 3′4′5′-forms. The ratio of 3′4′5′- to 3′4′-hydroxylated products in FSI transgenic cells was significantly higher than VvF3′5′H cells. Conclusions CsF3′5′H is a key controller of tri-hydroxyl flavan-3-ol synthesis in tea plants, which can effectively convert 4′-hydroxylated flavanone into 3′4′5′- and/or 3′4′-hydroxylated products. These findings provide animportant basis for further studies of flavonoid biosynthesis in tea plants. Such studies would help accelerate flavonoid metabolic engineering in order to increase B-ring tri-hydroxyl product yields.

For multiple-input multiple-output (MIMO) wireless communication system with minimum mean square error (MMSE) detection, a new scheme of power allocation between pilot and data symbols is investigated under MMSE channel estimation in this paper. First, we propose a novel soft-output MMSE MIMO detector by taking into consideration the channel estimation error. Then, through the application of random matrix theorem, we propose an efficient scheme for power allocation between pilot and data symbols which maximizes the lower bound of postprocessing signal-to-interference-and-noise ratio (SINR) for MIMO systems with equal number of transmitter and receiver antennas. We have proven the existence and uniqueness of the proposed optimal power allocation settings. Furthermore, our analysis shows that the proposed power allocation is also valid and applicable for those MIMO systems with unequal number of transmitter and receiver antennas. Finally, our extensive simulation results have validated this novel power allocation scheme.

A microarray study was conducted on transcriptome changes of Norton berry skin during the period of 37 to 127 days after bloom, which represents berry developmental phases from herbaceous growth to full ripeness. Samples of six berry developmental stages were collected. Analysis of the microarray data revealed that a total of 3,352 probe sets exhibited significant differences at transcript levels, with two-fold changes between at least two developmental stages. Expression profiles of defense-related genes showed a dynamic modulation of nucleotide-binding site-leucine-rich repeat (NBS-LRR) resistance genes and pathogenesis-related (PR) genes during berry development. Transcript levels of PR-1 in Norton berry skin clearly increased during the ripening phase. As in other grapevines, genes of the phenylpropanoid pathway were up-regulated in Norton as the berry developed. The most noticeable was the steady increase of transcript levels of stilbene synthase genes. Transcriptional patterns of six MYB transcription factors and eleven structural genes of the flavonoid pathway and profiles of anthocyanins and proanthocyanidins (PAs) during berry skin development were analyzed comparatively in Norton and Cabernet Sauvignon. Transcriptional patterns of MYB5A and MYB5B were similar during berry development between the two varieties, but those of MYBPA1 and MYBPA2 were strikingly different, demonstrating that the general flavonoid pathways are regulated under different MYB factors. The data showed that there were higher transcript levels of the genes encoding flavonoid-3'-O-hydroxylase (F3'H), flavonoid-3',5'-hydroxylase (F3'5'H), leucoanthocyanidin dioxygenase (LDOX), UDP-glucose:flavonoid 3'-O-glucosyltransferase (UFGT), anthocyanidin reductase (ANR), leucoanthocyanidin reductase (LAR) 1 and LAR2 in berry skin of Norton than in those of Cabernet Sauvignon. It was also found that the total amount of anthocyanins was markedly higher in Norton than in Cabernet Sauvignon berry skin at harvest, and five anthocyanin derivatives and three PA compounds exhibited distinctive accumulation patterns in Norton berry skin. Conclusions This study provides an overview of the transcriptome changes and the flavonoid profiles in the berry skin of Norton, an important North American wine grape, during berry development. The steady increase of transcripts of PR-1 and stilbene synthase genes likely contributes to the developmentally regulated resistance during ripening of Norton berries. More studies are required to address the precise role of each stilbene synthase gene in berry development and disease resistance. Transcriptional regulation of MYBA1, MYBA2, MYB5A and MYBPA1 as well as expression levels of their putative targets F3'H, F3'5'H, LDOX, UFGT, ANR, LAR1, and LAR2 are highly correlated with the characteristic anthocyanin and PA profiles in Norton berry skin. These results reveal a unique pattern of the regulation of transcription and biosynthesis pathways underlying the viticultural and enological characteristics of Norton grape, and yield new insights into the understanding of the flavonoid pathway in non-vinifera grape varieties.

Tea leaves contain abundant flavan-3-ols, which include dihydroxylated and trihydroxylated catechins. Flavonoid 3′-hydroxylase (F3′H: EC 1.14.13.21) is one of the enzymes in the establishment of the hydroxylation pattern. A gene encoding F3′H, designated as CsF3′H, was isolated from Camellia sinensis with a homology-based cloning technique and deposited in the GenBank (GenBank ID: KT180309). Bioinformatic analysis revealed that CsF3′H was highly homologous with the characterized F3′Hs from other plant species. Four conserved cytochrome P450-featured motifs and three F3′H-specific conserved motifs were discovered in the protein sequence of CsF3′H. Enzymatic analysis of the heterologously expressed CsF3′H in yeast demonstrated that tea F3′H catalyzed the 3′-hydroxylation of naringenin, dihydrokaempferol and kaempferol. Apparent Km values for these substrates were 17.08, 143.64 and 68.06 μM, and their apparent Vmax values were 0.98, 0.19 and 0.44 pM·min−1, respectively. Transcription level of CsF3′H in the new shoots, during tea seed germination was measured, along with that of other key genes for flavonoid biosynthesis using real-time PCR technique. The changes in 3′,4′-flavan-3-ols, 3′,4′,5′-flavan-3-ols and flavan-3-ols, were consistent with the expression level of CsF3′H and other related genes in the leaves. In the study of nitrogen supply for the tea plant growth, our results showed the expression level of CsF3′H and all other tested genes increased in response to nitrogen depletion after 12 days of treatment, in agreement with a corresponding increase in 3′,4′-catechins, 3′,4′,5′-catechins and flavan 3-ols content in the leaves. All these results suggest the importance of CsF3′H in the biosynthesis of 3′,4′-catechins, 3′,4′,5′-catechins and flavan 3-ols in tea leaves.