Plastidial Phosphorylase (Pho1a) is the dominant glucosyltransferase regulating starch granule initiation in potato tubers

Starch granule initiation involves the extension of maltooligosaccharide primers by glucosyltransferases. STARCH SYNTHASE 4 (SS4) plays a central role in almost all examined plant species, while the plastidial PHOSPHORYLASE 1 (Pho1) also plays an important role in some species, including rice and wheat. In Arabidopsis, an additional enzymatically inactive homolog of SS4, STARCH SYNTHASE 5 (SS5) contributes to starch granule initiation. To elucidate the mechanism of starch granule initiation in potato tubers, we used CRISPR/Cas9 to generate ss4, ss5, and pho1a knockout mutants in the commercial tetraploid 'Clearwater Russet', to systematically investigate their contribution to granule initiation. In ss4 and ss5 tubers, starch granule size and morphology were unaltered relative to the wild type, suggesting that SS4 and SS5 are dispensable for normal granule initiation in potato tubers. In contrast, pho1a tubers had compound starch granules that arose from multiple initiations, greatly reduced granule size, and highly variable granule morphologies. Affinity pull-down to find Pho1a interaction partners identified LIKE EARLY STARVATION (LESV), although yeast 2-hybrid assays did not show direct protein-protein binding. When expressed alone in Nicotiana benthamiana leaves, Pho1a located to the chloroplast stroma, but when expressed alongside LESV, both proteins co-located on starch granules. This co-localisation, alongside the similar accumulation of small starch granules when LESV is knocked out in tubers, suggest a possible functional interaction in planta. These findings position Pho1a as the central glucosyltransferase in starch granule initiation in Clearwater Russet tubers, where it acts together with LESV.