Analysis and application of regulatory genes controlling anthocyanin accumulation in rice

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The Purple leaf (Pl) locus of rice (Oryza sativa L.) affects regulation of anthocyanin biosynthesis in various plant tissues. The tissue-specific patterns of anthocyanin pigmentation, together with the syntenic relationship, indicate that the rice Pl locus may play a role in the anthocyanin pathway similar to the maize R/B loci. We isolated two cDNAs OSB1 and OSB2 showing significant identity to the basic helix-loop-helix (bHLH) proteins found in the maize R gene family. Characterization of the corresponding genomic region revealed that the sequence identical to a 5'-portion of OSB2 existed 〜10-kb downstream of the OSB1 coding region. OSB2 lacks a conserved C-terminal domain. RFLP analyses using an F_2 population indicate that both genes co-segregate with the purple leaf phenotype. To examine if these cDNAs actually promote anthocyanin accumulation in vivo, we developed a transient complementation assay by which we showed that the anthocyanin pathway is inducible by OSB1 or OSB2. These … More results suggest that the Pl^w allele may be complex and composed of at least two genes encoding bHLH proteins.We introduced OSB1 and OSB2 in rice plants under the control of a constitutive CaMV 35S promoter. The resulting trangenic plants showed anthocyanin accumulation in vegetative tissues. Further examination revealed that anthocyanin is also present in seed tissues, but it is limited to seed coat and pericarp and not accumulated in embryo and endosperm. It is postulated that other regulatory components may be required for inducing anthocyanin pathway in embryo and endosperm.In addition to Purple leaf, there are other loci genetically known to control anthocyanin pigmentation in rice. An example is Activator (A) that has been suggested to correspond to DFR gene, based on the synteny between rice and maize genomes. To examine this, we employed our transient assay and showed that anthocyanin pigments were inducible when DFR was introduced together with OSB1 and OSB2. The result indicated that A encodes DFR. Less