A large‐scale gene regulatory network for rice endosperm starch biosynthesis and its application in genetic improvement of rice quality

Rice (Oryza sativa L.) is one of the most important food crops. Starch is the main substance of rice endosperm and largely determines the grain quality and yield. Starch biosynthesis in endosperm is very complex, requiring a series of enzymes which are also regulated by many transcription factors (TFs). But until now, the large-scale regulatory network for rice endosperm starch biosynthesis has not been established. Here, we constructed a rice endosperm starch biosynthesis regulatory network comprised of 277 TFs and 15 starch synthesis enzyme-encoding genes (SSEGs) using DNA affinity chromatography/pull-down combined with liquid chromatography-mass spectrometry (DNA pull-down and LC-MS). In this regulatory network, each SSEG is directly regulated by 7-46 TFs. Based on this network, we found a new pathway 'ABA-OsABI5-OsERF44-SSEGs' that regulates rice endosperm starch biosynthesis. We also knocked out five TFs targeting the key amylose synthesis enzyme gene OsGBSSI in japonica rice 'Nipponbare' background and found that all mutants had moderately decreased amylose content (AC) in endosperm and improved eating and cooking quality (ECQ). Notably, the knockout of OsSPL7 and OsB3 improves the ECQ without compromising the rice appearance quality, which was further validated in the indica rice 'Zhongjiazao17' background. In summary, this gene regulatory network for rice endosperm starch biosynthesis established here will provide important theoretical and practical guidance for the genetic improvement of rice quality.