A plastidic starch biosynthetic pathway defines columella-specific carbon metabolism in rice root tips

Starch biosynthesis in rice ( Oryza sativa ) is precisely regulated in an organ-specific manner, but the molecular mechanism operating this pathway in root-tip columella cells has remained unclear. Here, we elucidated the starch biosynthetic pathway in rice root tips using CRISPR/Cas9-mediated mutagenesis, Lugol’s iodine staining, expression analysis, anatomical validation, and quantitative analysis of starch and soluble sugar contents. Systematic genetic screening revealed that disrupting genes encoding individual large (AGPL) or small (AGPS) subunits of ADP-glucose pyrophosphorylase (AGPase) did not affect columella starch accumulation, indicating functional redundancy. Expression profiling and double-knockout analysis demonstrated that starch biosynthesis in columella amyloplasts specifically requires the plastidic AGPase subunits OsAGPL1, OsAGPL4, OsAGPS1, and OsAGPS2a, whereas cytosolic AGPase subunits are dispensable for this pathway. Subunit-specific disruption further showed that OsAGPS2a, but not OsAGPS2b, is essential for starch formation in columella cells, establishing the exclusive requirement for plastidic AGPase function in this tissue. We also show that glucose-6-phosphate (Glc-6-P) import into columella amyloplasts is mediated by Glc-6-P/Phosphate Translocator 1 (OsGPT1), whereas OsGPT2 paralogs do not appear to contribute to this process. Loss of OsGPT1 function markedly diminished, but did not completely abolish, starch accumulation. These changes were accompanied by lower soluble sugar levels, suggesting that disrupting plastidic starch biosynthesis impairs sink strength in root-tip tissues. Analysis of resin-embedded sections confirmed that starch deposition is restricted to columella amyloplasts and is dependent on plastidic AGPase and OsGPT1 function. Together, our results define a complete plastidic starch biosynthetic pathway in rice root-tip columella cells and establish this tissue as a distinct, locally specialized metabolic sink within the organ-specific carbon partitioning network of rice.