Starch phosphorylase acts as a pivotal scaffold connecting starch metabolism enzymes with photosystem I subunits

Abstract In a previous study, we proposed that starch phosphorylase (Pho1) interacts with PsaC, a core subunit of photosystem I (PSI), although it was not clear whether the interaction was a direct 1:1 relationship or by indirect association with another factor. Here, we validated the Pho1-PsaC interaction in vivo using bimolecular fluorescence complementation (BiFC) analysis. Pulldown assays with wildtype, BMF136, and the Pho1-null plants revealed that GST-Pho1 consistently associates with PsaC and the starch disproportionating enzyme (Dpe1) across all genotypes. These results indicate that Pho1 is essential for forming a multi-protein complexes containing PsaC and Dpe1. Yeast 2-hybrid (Y2H) assays further validated the direct Pho1-PsaC interaction and also demonstrated that Pho1 interacts with PsaD, another subunit of PSI, but not with PsaE, indicating that PsaD functions as an additional component of the Pho1-associated heterocomplex. PsaC bounded poorly to the Pho1 catalytically null variants, although PsaC fused to a HaloTag restored interaction with the catalytic null mutants, implying that the HaloTag facilitated or stabilized complex formation. Our results indicate that Pho1 is an essential factor linking starch biosynthesis to photosynthesis through its ability to form multi-protein complexes with starch metabolism enzymes and PSI subunits.