Regulatory Roles of PURPLE FRUIT 1 and OsPa in Pigmentation of Rice Grain

Purple rice has long been treasured for health benefits, but the molecular mechanism of its grain pigmentation remains to be fully understood. Previous evidence shows that a bHLH gene, OsB2, is over-expressed in purple-rice's caryopsis to cause its increased pigmentation and a MYB gene, OsMYB3, decreases the pigmentation when knocked out in purple rice. Nonetheless, these transcription factors are inadequate to explain why hull color of purple rice can vary independently from that of caryopsis. We show here that more than two regulatory (MYB/bHLH/WDR (MBW)) complexes are involved in grain pigmentation of purple rice. A previously unsuspected MYB, encoded by PURPLE FRUIT 1 (PF1), and OsPa (bHLH), also participate in the grain pigmentation. Along with OsB2 and OsMYB3, they form redundant yet differential MBW complexes in purple-rice grain to regulate not only structural genes of the anthocyanin pathway but also OsLAR of the proanthocyanidin pathway. In the developing caryopses, co-expression patterns of OsPF1 and OsB2 can better predict accumulation process of anthocyanins than those of OsMYB3 and OsB2. Disrupting OsPF1 or OsPa via CRISPR/Cas9 in purple rice leads to reduced pigmentations of caryopsis and hulls, with OsPa's knockout-effect on hull color particularly pronouncing. When paired with OsPa and OsTTG1 to form the MBW complex, OsPF1 can initiate a stronger activation of OsCHI but weaker one of OsLAR than OsMYB3 under the same condition. Compared to significantly enhanced expression levels of OsB2 and OsPa in grains of purple rice, transcriptions of OsPF1 and OsMYB3 remain non-differentiated between straw-white and purple grains at the same developmental stage. The new advances, along with the previously known, have enabled a mechanistic illustration of pigmentations of caryopsis and hulls in purple rice, which may guide future breeding of purple rice in creating varieties of desired grain anthocyanins to improve human health.