Discovery of genes that regulate the concentrations of anthocyanin and proanthocyanidin antioxidants in purple and red rice varieties

Authors: Pinson, Shannon; Chen, Ming Hsuan; Grunden, Eric; Everette, Jace; Jackson, Aaron; Edwards, Jeremy

Submitted to: Rice Technical Working Group Meeting Proceedings
Publication Type: Proceedings
Publication Acceptance Date: 2/23/2023
Publication Date: 1/30/2024
Citation: Pinson, S.R., Chen, M., Grunden, E., Everette, J.D., Jackson, A.K., Edwards, J. 2024. Discovery of genes that regulate the concentrations of anthocyanin and proanthocyanidin antioxidants in purple and red rice varieties [abstract]. Proceedings of 39th Rice Technical Working Group, Hot Springs, Arkansas, February 20-23, 2023. p. 63-64.

Interpretive Summary:

Technical Abstract: Anthocyanins and proanthocyanidins are the antioxidant pigmented flavonoids that give blueberries, cranberries and red wine their color and health-beneficial attributes. Rice grains with purple colored bran also contain anthocyanins, and red-bran rice contains proanthocyanidins. Previous work determined that the genes Pb and Rc are transcription factors that turn on and off the biosynthesis of anthocyanins (purple) and proanthocyanidins (red), respectively, and molecular gene tags for Pb and Rc were developed. The focus of this study was to determine how the concentrations of these two pigmented flavonoids are regulated in rice grains after activation by Pb and Rc. We mapped quantitative trait loci (QTLs) affecting the variable grain concentrations of anthocyanin and proanthocyanidin in F5 and F6 recombinant inbred lines (RILs) derived from crossing white pericarp ‘IR36ae’ with ‘242’, which produces both anthocyanins and proanthocyanidins in its pericarp. QTLs for total anthocyanin concentration (TAC) were mapped using two years of grains from 60 RILs molecularly determined to be fixed PbPbrcrc or PbPbRcRc, while proanthocyanidin content (PA) QTLs were mapped using 75 RILs that were PbPbRcRc or pbpbRcRc. The RILs were genotyped for approximately 1400 SNPs using the IRRI 1K-Rice Custom Amplicon (1k-RiCA) V4 Panel and the LSU500 Panel. Both TAC and PA independently mapped to a 1.5 Mb QTL region on chromosome 3 between RM3400 (at 15.8 Mb) and RM15123 (17.3 Mb), named qPR3. Across two years, qPR3 explained 36.3% of variance in TAC and 35.8% in PA variance not attributable to Pb or Rc. The qPR3 region encompasses Kala3, (LOC_Os03g29614) a MYB transcription factor previously known to regulate purple grain characteristics, and now found associated with both TAC and PA. QTL analysis of TAC data also indicated a QTL with lesser effect on chromosome 7 that encompassed the anthocyanidins 3-O-glucosyltransferase (AGT) (LOC_Os07g32620) gene. Analysis of PA identified a 2nd QTL between 18.0 and 18.5 Mb on chromosome 5, qPR5. No specific candidate gene was identified for qPR5 because none of the genes in this region had an annotated gene function predicted to impact concentrations of pigmented flavonoids. Because anthocyanins and proanthocyanidins share a large portion of their biosynthetic pathways, we included in our study investigation of the impact of Rc, which activates synthesis of proanthocyanidins, on TAC, and conversely studied the impact of Pb, which activates synthesis of anthocyanins, on PA. To study the effect of Rc on TAC, we derived four sets of PbPbRcRc vs PbPbrcrc NILs by self-pollinating RILs found to be PbPbRcrc in the F3 to F5 generation. The RcRc progeny NILs were found to have 2.1 - 4.5x more TAC over their rcrc counterparts. Similarly, study of seven sets of PbPbRcRc vs. pbpbRcRc NIL sets showed that PbPb NILs had 1.3 to 2.0x more PA than their pbpb counterparts. These data revealed a mutual enhancement, not a trade-off between these compounds that share precursors. This suggests that Pb and Rc upregulate genes in the shared pathway as they activate TAC and PA synthesis, respectively. In summary, we identified three antioxidant-enhancing QTLs (qPR3, qPR5, qPR7) and provide genetic markers with which to incorporate these regions into nutritionally-enhanced rice varieties. Our findings also indicate that combining the Pb and Rc genes will increase concentrations of health-beneficial antioxidants beyond the combined additive effects of the individual genes due to mutual enhancement of both pigmented antioxidant compounds.