Effects of loss of CO₂-Responsive CCT Protein interaction with 14-3-3 proteins and its ectopic expression on starch synthesis in rice.

-Responsive CCT Protein (CRCT) is a transcription factor that positively regulates the expression of starch synthesis-related genes in the vegetative organs of rice. CRCT interacts with 14-3-3 proteins. Yeast two-hybrid analysis showed that the CRCT with the S217A mutation was unable to interact with CRCT. This mutated CRCT was introduced into the CRCT knockout rice. The transcript of CRCT-S217A was detected significantly by qRT-PCR, but not by immunoblotting. Nevertheless, starch accumulation and the expression of starch synthesis-related genes in the leaf sheath of the transformants were higher than those of the CRCT knockout line. These results suggest the possibility that 14-3-3 proteins may stabilize CRCT through their interaction, while free unstable CRCT can promote starch synthesis. CRCT was shown to be highly expressed in the vascular bundle and is hypothesized to move to the starch-accumulating storage parenchyma cells in leaf sheaths. To investigate this hypothesis, we generated transgenic lines expressing CRCT ectopically in the CRCT knockout line background. Transgenic lines expressing CRCT using Sucrose transpor1 promoter, known to express specifically in vascular tissues, and the Chlorophyll a/b binding protein promoter, known to express highly in photosynthetic tissues, showed recovered starch accumulation in the leaf sheath. These results suggest that CRCT can move from an unspecified cell to storage parenchyma cells via the vascular bundle to regulate starch synthesis. This study can provide new insight into the regulation mechanism of starch biosynthesis in the vegetative organs.