Anthocyanins cause purple, brown or red colors in various tissues of rice plants, but the specific determinant factors and regulatory systems for anthocyanin biosynthesis in almost all tissues remain largely unknown. In the present study, we mapped and isolated two complementary genes, OsC1 encoding a R2R3-MYB transcriptional factor and OsDFR encoding a dihydroflavonol 4-reductase, which are responsible for the purple coloration of apiculi and stigmas in indica cultivar Xieqingzao by the map-based cloning strategy. We also identified two tissue-specific pigmentation genes, OsPa for apiculi and OsPs for stigmas, by phylogenetic analysis of all anthocyanin biosynthesis-associated bHLH transcriptional factors in maize and rice, CRISPR/Cas9 knockout and transcriptional expression analysis. The OsC1, OsPa and OsPs proteins are all localized in the nucleus while the OsDFR protein is localized in the nucleus and cytoplasm, and the OsC1 and OsDFR genes are preferentially strongly expressed in both purple-colored tissues while the OsPa and OsPs genes are preferentially strongly expressed in apiculi and stigmas, respectively. OsC1 specifically interacts with OsPa or OsPs to activate OsDFR and other anthocyanin biosynthesis genes, resulting in purple-colored apiculi or stigmas. OsC1 itself does not produce color but can produce brown apiculi when functioning together with OsPa. Loss of function of OsDFR alone leads to brown apiculi and straw-white stigmas. Genotyping and phenotyping of a panel of 176 rice accessions revealed diverse genotypic combinations of OsC1, OsDFR, OsPa and OsPs that enable accurate prediction of their apiculus and stigma pigmentation phenotypes, thus validating the general applicability of the OsC1-OsDFR-OsPa and OsC1-OsDFR-OsPs models to natural populations. Our findings disclosed the biological functions of OsC1, OsPa and OsPs, and shed light on the specific regulatory systems of anthocyanin biosynthesis in apiculi and stigmas, a further step in understanding the regulatory network of anthocyanin biosynthesis in rice.

花色苷在水稻植物的各种组织中引起紫色，棕色或红色，但是在几乎所有组织中花色苷生物合成的具体决定因素和调节系统仍然未知。在本研究中，我们映射并分离两个互补的基因，OSC1编码R2R3-MYB转录因子和OsDFR编码二氢黄酮醇4-还原酶，这是负责apiculi和柱头在紫色着色籼稻品种协青早由基于地图克隆策略。我们还确定了两个组织特异性的色素沉着基因，OSPA为apiculi和互联网服务商对于柱头，通过系统发育分析玉米和水稻中所有与花色苷生物合成相关的bHLH转录因子，进行CRISPR / Cas9敲除和转录表达分析。OsC1，OsPa和OsPs蛋白都位于细胞核中，而OsDFR蛋白则位于细胞核和细胞质中，OsC1和OsDFR基因在紫色组织中优先表达，而OsPa和OsPs基因则优先表达分别以尖顶和柱头表达。OsC1与OsPa或OsPs特异性相互作用，以激活OsDFR和其他花色苷生物合成基因，从而导致紫色的尖头或柱头。OsC1与OsPa一起使用时，本身不会产生颜色，但会产生褐色的毛细血管。仅OsDFR的功能丧失会导致褐色的尖头和稻草的白色柱头。对176个水稻种质的基因分型和表型分析显示，OsC1，OsDFR，OsPa和OsPs的基因型不同，能够准确预测其蜂culus和柱头色素表型，从而验证了OsC1 - OsDFR - OsPa和OsC1 - Os的普遍适用性-操作系统自然人口的模型。我们的发现揭示了OsC1，OsPa和OsPs的生物学功能，并揭示了尖峰和柱头中花色苷生物合成的特定调控系统，这是理解水稻花色苷生物合成调控网络的又一步。