Branched oligosaccharides cause atypical starch granule initiation in Arabidopsis chloroplasts

Plant chloroplasts store starch during the day, which acts as a source of carbohydrates and energy at night. Starch granule initiation relies on the elongation of malto-oligosaccharide primers. In Arabidopsis thaliana, PROTEIN TARGETING TO STARCH 2 (PTST2) and STARCH SYNTHASE 4 (SS4) are essential for the selective binding and elongation of malto-oligosaccharide primers, respectively, and very few granules are initiated in their absence. However, the precise origin and metabolism of the primers remain unknown. Potential origins of malto-oligosaccharide primers include de novo biosynthesis or their release from existing starch granules. For example, the endoamylase α-AMYLASE 3 (AMY3) can cleave a range of malto-oligosaccharides from the granule surface during starch degradation at night, some of which are branched. In the Arabidopsis double mutant deficient in the two debranching enzymes ISOAMYLASE 3 (ISA3) and LIMIT DEXTRINASE (LDA), branched malto-oligosaccharides accumulate in the chloroplast stroma. Here, we reveal that the isa3 lda double mutant shows a substantial increase in granule number per chloroplast, caused by these branched malto-oligosaccharides. The amy3 isa3 lda triple mutant, which lacks branched malto-oligosaccharides, has far fewer granules than isa3 lda, and its granule numbers are barely higher than in the wild type. Plants lacking both ISA3 and LDA and either PTST2 or SS4 show granule over-initiation, indicating that this process occurs independently of the recently described granule initiation pathway. Our findings provide insight into how and where starch granules are initiated. This knowledge can be used to alter granule number and morphological characteristics, traits known to affect starch properties.