ORCID IDs: 0000-0003-3905-3647 (D.S.); 0000-0002-8569-9150 (J.B.); 0000-0001-8682-5668 (J.M.); 0000-0002-4440-1776 (T.B.S.); 0000-0002-9501-7854 (M.Z.); 0000-0002-2791-0915 (S.C.Z.)The molecular mechanism that initiates the synthesis of starch granules is poorly understood. Here, we discovered two plastidial proteins involved in granule initiation in Arabidopsis thaliana leaves. Both contain coiled coils and a family-48 carbohydrate binding module (CBM48) and are homologs of the PROTEIN TARGETING TO STARCH (PTST) protein; thus, we named them PTST2 and PTST3. Chloroplasts in mesophyll cells typically contain five to seven granules, but remarkably, most chloroplasts in ptst2 mutants contained zero or one large granule. Chloroplasts in ptst3 had a slight reduction in granule number compared with the wild type, while those of the ptst2 ptst3 double mutant contained even fewer granules than ptst2. The ptst2 granules were larger but similar in morphology to wild-type granules, but those of the double mutant had an aberrant morphology. Immunoprecipitation showed that PTST2 interacts with STARCH SYNTHASE4 (SS4), which influences granule initiation and morphology. Overexpression of PTST2 resulted in chloroplasts containing many small granules, an effect that was dependent on the presence of SS4. Furthermore, isothermal titration calorimetry revealed that the CBM48 domain of PTST2, which is essential for its function, interacts with long maltooligosaccharides. We propose that PTST2 and PTST3 are critical during granule initiation, as they bind and deliver suitable maltooligosaccharide primers to SS4.

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β-Amylases are key enzymes of transitory starch degradation in chloroplasts, a process that buffers the availability of photosynthetically fixed carbon over the diel cycle to maintain energy levels and plant growth at night. However, during vascular plant evolution, the β-amylase gene family diversified, giving rise to isoforms with different compartmentation and biological activities. Here, we characterized BETA-AMYLASE 9 (BAM9) of Arabidopsis (Arabidopsis thaliana). Among the β-amylases, BAM9 is most closely related to BAM4 but is more widely conserved in plants. BAM9 and BAM4 share features including their plastidial localization and lack of measurable α-1,4-glucan hydrolyzing capacity. BAM4 is a regulator of starch degradation, and bam4 mutants display a starch-excess phenotype. Although bam9 single mutants resemble the wild type, genetic experiments reveal that the loss of BAM9 markedly enhances the starch-excess phenotypes of mutants already impaired in starch degradation. Thus, BAM9 also regulates starch breakdown, but in a different way. Interestingly, BAM9 gene expression is responsive to several environmental changes, while that of BAM4 is not. Further, overexpression of BAM9 in the wild type reduced leaf starch content, but overexpression in bam4 failed to complement fully that mutant’s starch-excess phenotype, suggesting that BAM9 and BAM4 are not redundant. We propose that BAM9 activates starch degradation, helping to manage carbohydrate availability in response to fluctuations in environmental conditions. As such, BAM9 represents an interesting gene target to explore in crop species.