Molecular dissection of the pathogen-inducible 3-deoxyanthocyanidin biosynthesis pathway in sorghum.

3-Deoxyanthocyanidins are the unique phytoalexins synthesized by sorghum in response to fungal inoculation. They are structurally related to anthocyanins but the final steps of their pathogen-inducible biosynthesis are not fully understood. We have identified new flavonoid structural genes from the recently completed sorghum BTx623 genome sequence. The biochemical functions of the different expressed sorghum genes were established in planta by complementation in the appropriate Arabidopsis transparent testa mutants. There is a family of nine chalcone synthase genes which are all inducible by fungal inoculation in sorghum seedlings. Specific dihydroflavonol 4-reductase (DFR) genes responsive to conditions which stimulated anthocyanin accumulation (SbDFR1) or 3-deoxyanthocyanidin production (SbDFR3) were identified. Recombinant SbDFR1 and SbDFR3 were found to function as typical DFRs by accepting dihydroflavonol substrates. On the other hand, both DFRs showed substantially lower but detectable NADPH-dependent activities toward flavanones. Reduction of flavanones to flavan-4-ols is a reaction step required for 3-deoxyanthocyanidin production. Flavanone 3-hydroxylase (F3H) converts flavanones to dihydroflavonols for anthocyanin biosynthesis. In sorghum seedlings, expression of two F3H genes was either absent or strongly suppressed during the accumulation of 3-deoxyanthocyanidins. Under such conditions, most flavanones are expected to be reduced by the pathogen-induced SbDFR3 for the formation of flavan-4-ols. Our work also revealed that 3-deoxyanthocyanidin accumulation and SbDFR3 expression were induced by methyl jasmonate treatment in sorghum roots but the stimulation effects were antagonized by salicylic acid.

Functional characterization of key structural genes in rice flavonoid biosynthesis.

Rice is a model system for monocot but the molecular features of rice flavonoid biosynthesis have not been extensively characterized. Rice structural gene homologs encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS) were identified by homology searches. Unique differential expression of OsF3H, OsDFR, and OsANS1 controlled by the Pl(w) locus, which contains the R/B-type regulatory genes OSB1 and OSB2, was demonstrated during light-induced anthocyanin accumulation in T65-Plw seedlings. Previously, F3H genes were often considered as early genes co-regulated with CHS and CHI genes in other plants. In selected non-pigmented rice lines, OSB2 is not expressed following illumination while their expressed OSB1sequences all contain the same nucleotide change leading to the T(64) M substitution within the conserved N-terminal interacting domain. Furthermore, the biochemical roles of the expressed rice structural genes (OsCHS1, OsCHI, OsF3H, and OsF3'H) were established in planta for the first time by complementation in the appropriate Arabidopsis transparent testa mutants. Using yeast two-hybrid analysis, OsCHS1 was demonstrated to interact physically with OsF3H, OsF3'H, OsDFR, and OsANS1, suggesting the existence of a macromolecular complex for anthocyanin biosynthesis in rice. Finally, flavones were identified as the major flavonoid class in the non-pigmented T65 seedlings in which the single-copy OsF3H gene was not expressed. Competition between flavone and anthocyanin pathways was evidenced by the significant reduction of tricin accumulation in the T65-Plw seedlings.

Accumulation of isoflavone genistin in transgenic tomato plants overexpressing a soybean isoflavone synthase gene.

Isoflavones are legume-specific flavonoids best known for their potential cancer preventive and phytoestrogenic properties. In this study, we attempted to engineer the isoflavone pathway in the popular fruit crop tomato (Solanum lycopersicum L). Tomato plants were transformed with a soybean (Glycine max L) isoflavone synthase (GmIFS2) cDNA under the control of the cauliflower mosaic virus 35S promoter. LC-MS/MS analysis demonstrated the presence of genistin (genistein 7-O-glucoside) as the major isoflavone metabolite in the transgenic plants. Substantial amounts of genistin (up to 90 nmol/g FW) were found in leaves, while the levels were marginally detectable (less than 0.5 nmol/g FW) in fruit peels. In either case, no drastic variations in endogenous phenolic contents were observed. Fruit peels were found to accumulate high levels of naringenin chalcone, implicating the limitation of naringenin substrates for isoflavone synthesis. Our results suggested that tomato plants could be engineered to produce isoflavones without comprising the levels of endogenous flavonols, which are also health-beneficial, but it may be necessary to enhance the expression levels of chalcone isomerase simultaneously to achieve significant yields in edible tissues such as fruit peels.

Accumulation of trans-piceid in sorghum seedlings infected with Colletotrichum sublineolum.

Sorghum SbSTS1, a pathogen inducible gene, was previously demonstrated to encode an enzyme with stilbene synthase activity. In this study, we attempt to identify the stilbene derivatives that accumulate in infected sorghum seedlings after inoculation with the anthracnose pathogen Colletotrichum sublineolum. Scanning for precursor ions that produced the common stilbene aglycones as diagnostic ions was performed in a triple quadrupole mass spectrometer. It was found that infected sorghum seedlings accumulated trans-piceid as the major stilbene metabolite together with an unknown resveratrol derivative. Time-course accumulation of trans-piceid was examined in two sorghum cultivars, DK18 and DK77, which are resistant and susceptible to C. sublineolum, respectively. In both cultivars, trans-piceid was not detected until 48h after inoculation, consistent with the late induction of SbSTS1 reported previously in infected sorghum plants. The levels of trans-piceid detected in DK77 seedlings were approximately three times the levels detected in DK18 seedlings at 120h after inoculation. In vitro assays demonstrated that trans-piceid did not exhibit significant toxicity on conidial germination and mycelial growth of C. sublineolum. Hence trans-piceid alone may not represent an important defense component against the anthracnose pathogen in sorghum seedlings.

Quantitative analysis of anticancer 3-deoxyanthocyanidins in infected sorghum seedlings.

3-Deoxyanthocyanidins are structurally related to the anthocyanin pigments, which are popular as health-promoting phytochemicals. Here, it is demonstrated that the 3-deoxyanthocyanidins are more cytotoxic on human cancer cells than the 3-hydroxylated anthocyanidin analogues. At 200 microM concentration, luteolinidin reduced the viability of HL-60 and HepG2 cells by 90 and 50%, respectively. Sorghum is a major source of 3-deoxyanthocyanidins, which are present as seed pigments and as phytoalexins responding to pathogen attack. On the basis of the collision-induced dissociation spectra of luteolinidin and apigeninidin, an LC-MS/MS method, operating in multiple-reaction monitoring mode, was developed for the specific detection and accurate quantification of these compounds in complex mixtures, which may be difficult to analyze using absorbance measurements. The results demonstrated that inoculated sorghum seedlings could be utilized for convenient and large-scale production of 3-deoxyanthocyanidins. A quantity of almost 270 microg/g (fresh weight) of luteolinidin was produced 72 h after fungal inoculation of 1-week-old seedlings.

Differential expression of two flavonoid 3'-hydroxylase cDNAs involved in biosynthesis of anthocyanin pigments and 3-deoxyanthocyanidin phytoalexins in sorghum.

Three unique sorghum flavonoid 3'-hydroxylase (F3'H) cDNAs (SbF3'H1, SbF3'H2 and SbF3'H3) were discovered through bioinformatics analysis. Their encoded proteins showed >60% identity to the Arabidopsis TT7 (F3'H) protein. Overexpression of SbF3'H1 or SbF3'H2 restored the ability of tt7 mutants to produce 3'-hydroxylated flavonoids, establishing their roles as functional F3'H enzymes. In sorghum mesocotyls, SbF3'H1 expression was involved in light-specific anthocyanin accumulation while SbF3'H2 expression was involved in pathogen-specific 3-deoxyanthocyanidin synthesis. No SbF3'H3 expression was detected in all tissues examined. The sorghum mesocotyls represent a good system for investigation of differential regulation of F3'H genes/alleles responding to different external stimuli.