Homologs of PROTEIN TARGETING TO STARCH Control Starch Granule Initiation in Arabidopsis Leaves

Abstract

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.

Distinct Functions of STARCH SYNTHASE 4 Domains in Starch Granule Formation

“…Like ss4 mutants, the ptst2 mutants have reduced numbers of starch granules, and this phenotype was exacerbated by loss of another homolog (PTST3; Seung et al, 2017). PTST2 was proposed to bind long malto-oligosaccharides with its carbohydrate-binding module and provide them as substrates to SS4 (Seung et al, 2017). This idea is broadly consistent with other observations that mutations affecting malto-oligosaccharide metabolism affect starch granule initiation (Malinova et al, 2017;Satoh et al, 2008;Seung et al, 2016).…”

“…2A). This is presumably because SS4C does not have selfpriming activity (Szydlowski et al, 2009) and rather relies on interaction with the recently described PTST2 and/or PTST3 proteins to deliver its substrates (Seung et al, 2017). Second, our YFP fluorescence data revealed that SS4C was uniformly distributed in chloroplasts, unlike the granule-surface localization of GS (Fig.…”

“…For example, an external helix on the catalytic domain of GBSS enables it to engage in a coiled-coil-mediated interaction with PROTEIN TARGETING TO STARCH (PTST), which helps localize GBSS to the starch granule (Seung et al, 2015). Similarly, SS4 was recently shown to bind to PTST2, a protein related to PTST that also contains a carbohydrate-binding module and predicted coiled-coil motifs (Seung et al, 2017). This interaction appeared to involve the catalytic domain of SS4, although the exact interaction site between SS4 and PTST2 has not yet been identified.…”

“…This interaction appeared to involve the catalytic domain of SS4, although the exact interaction site between SS4 and PTST2 has not yet been identified. Like ss4 mutants, the ptst2 mutants have reduced numbers of starch granules, and this phenotype was exacerbated by loss of another homolog (PTST3; Seung et al, 2017). PTST2 was proposed to bind long malto-oligosaccharides with its carbohydrate-binding module and provide them as substrates to SS4 (Seung et al, 2017).…”

“…SS4 is presumed to determine the number of starch granules in a chloroplast by synthesizing specific amylopectin precursors (Roldán et al, 2007;Szydlowski et al, 2009), a role facilitated by its interaction with PTST2 (Seung et al, 2017). This emerging model contrasts with the priming of glycogen synthesis in other organisms.…”