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

Abstract

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.

Figure 1.

PTST2 and PTST3 Are Chloroplast-Localized CBM48-Containing Proteins with Coiled Coils. (A) Schematic illustration of domains in the PTST proteins. The chloroplast transit peptides (TP) and the CBM48 domains are shown in green and violet, respectively. Coiled coil (CC)-containing regions are shown in blue. The locations of coiled coils predicted using COILS analysis with a 14-amino acid prediction window are shown with an indication of probability score (from 0 to 1, where 1 is the highest probability). (B) Phylogenetic tree of PTST proteins. The tree was constructed using the neighbor-joining method, and clades corresponding to PTST1-, PTST2-, and PTST3-like sequences are highlighted in pink, yellow, and cyan, respectively. Branches in red, green, and blue represent sequences from Bryophytes, Pteridophytes, and monocots, respectively. Bootstrap values greater than 50 are shown above or underneath the branches. The amino acid alignment used to generate this tree is provided in Supplemental Figure 1.

Figure 2.

Localization of Arabidopsis PTST2-YFP and PTST3-YFP. The fusion proteins were transiently expressed in either wild-type N. sylvestris or its starchless pgm mutant. YFP and chlorophyll fluorescence were imaged using confocal microscopy. All panels are shown at the same scale. Bar = 5 µm.

Figure 3.

Starch Granules in Chloroplasts of Arabidopsis Mutants Carrying T-DNA Insertions in PTST2 or PTST3. (A) Schematic illustration of the exon-intron structure of PTST2 and PTST3 genes. Exons are represented with blue boxes, while the pale-blue boxes represent the 5′ and 3′ untranslated regions. The position of the translation start codon (ATG) and the stop codon (TGA) are shown with black arrows. Red arrowheads represent the position of T-DNA insertions. (B) Immunoblot detection of PTST2 and PTST3 proteins in total protein extracts from Arabidopsis leaves. Panels from top to bottom show immunoblots with antisera raised against Arabidopsis PTST2, PTST3, SS4, and ISA1, respectively. An equal amount of total protein (30 µg) was loaded in each lane. The leftmost lanes on the PTST2 and PTST3 immunoblot panels were loaded with the respective recombinant (recomb.) protein (0.01 µg), expressed in E. coli, and purified. The wild-type controls for the ptst2-6 and ptst3-1 mutants are Nossen (No) and Ler, respectively. All other mutants are in the Col background. The migration of molecular mass markers (in kD) is indicated on the left of each panel. (C) Observation of starch granules in chloroplasts of mutants using light microscopy. Leaf segments were excised from young leaves (as shown in Figure 4B) at the end of the day and embedded in resin for sectioning. Thin sections were stained with toluidine blue. Similar starch granule phenotypes were observed in at least three independent experiments performed on different batches of plants, and representative images are shown. Bars = 10 µm.

Figure 4.

Phenotype of the ptst2 ptst3 Double Mutant. The ptst2-3 and ptst3-5 alleles were used for all experiments in this figure. (A) Photographs showing the appearance of ptst2, ptst3 and double mutant rosettes, compared with ss4 rosettes. (B) Iodine staining of starch in rosettes harvested at the end of day. The lighter-staining areas at the center of ptst2, ptst2 ptst3, and ss4 rosettes are indicated with red arrows. The blue arrows indicate examples of young leaves taken for light microscopy analysis. (C) Starch turnover over a 12-h-day/12-h-night cycle. Entire rosettes were harvested at the indicated time points and starch content was determined. Each value represents the mean ±se of measurements made on n = 4 to 6 rosettes. All data were collected within a single experiment. For clarity, data from the single mutants are presented on the left panel, and data from the double mutant and ss4 are presented on the right panel. The same data from the wild type are shown in both panels.

Figure 5.

Number of Starch Granules per Chloroplast in the ptst2 and ptst3 Single and Double Mutants. The ptst2-3 and ptst3-5 alleles were used for all experiments in this figure. (A) Starch granules in chloroplasts observed with light microscopy in leaf sections stained with toluidine blue. Leaves were harvested at the end of the day. Similar phenotypes were observed in three independent experiments performed on different batches of plants. Representative images are shown. Bars = 10 µm. (B) Quantification of granule sections within chloroplasts. Light micrographs were acquired as in (A), and the number of starch granules observed within each chloroplast section was counted. Histograms show the frequency of chloroplasts containing a given number of granule sections relative to the total number of chloroplasts analyzed (n = 540 for each genotype). Equal numbers of chloroplasts were analyzed in leaves from three different plants for each genotype. Arrows indicate the bin containing the median value.

Figure 6.

Starch Granule Morphology in the ptst2 and ptst3 Single and Double Mutants. Granules were purified from entire 4-week-old rosettes harvested at the end of the day (∼60 rosettes per genotype) and observed using scanning electron microscopy. The same sample prepared from each genotype was imaged at three different magnifications, 2000× (upper panels, bars = 5 µm), 5000× (middle panels, bars = 2 µm), and 15,000× (lower panels, bars = 1 µm).

Figure 7.

ADP-Glucose Accumulation in ptst2 and ptst2 ptst3 Mutants. Entire rosettes of 4-week-old plants were harvested at the end of the day and metabolites were extracted with the chloroform/methanol procedure. ADP-glucose was quantified using UHPLC-MS/MS. Values represent mean ±se of n = 4 rosettes.

Figure 8.

Immunoprecipitation Experiments for Interaction Partners of PTST2 and PTST3. The antibodies (Ab) used for detection and the migration of molecular mass markers (in kD) are indicated on the left of all immunoblot panels. (A) Immunoprecipitation of PTST2-YFP and PTST3-YFP expressed in Arabidopsis. Proteins extracted from leaf tissue (Input) were incubated with beads conjugated to YFP antibodies. The bound proteins were eluted (anti-YFP IP), and proteins in the input and IP fractions were detected by immunoblotting. (B) Immunoprecipitation of SS4-RFP expressed in Arabidopsis. The experiment was conducted as for (A), except that anti-RFP beads were used. SS4, PTST2, and PTST3 proteins in the input and IP fractions were detected by immunoblotting. (C) Immunoprecipitation of YFP-tagged PTST proteins coexpressed in N. benthamiana leaves with HA-tagged SS4 protein. A truncated version of SS4 lacking the N-terminal coiled coils (SS4 ∆CC) was used. Immunoprecipitation was conducted as for (A). Epitope-tagged proteins in the Input and IP fractions were detected using immunoblotting. (D) Immunoprecipitation of PTST proteins tagged with TAP-tags, coexpressed in N. benthamiana leaves with CFP-tagged GBSS protein. The experiment was conducted as described for (C), except using anti-myc beads, which bind the c-myc epitopes in the TAP tag. (E) Immunoprecipitation of YFP-tagged PTST3 coexpressed in N. benthamiana leaves with HA-tagged PTST2 protein. The experiment was conducted as described for (A). Epitope-tagged proteins in the input and IP fractions were detected using anti-YFP (upper panels) and anti-HA (lower panels).

Figure 9.

Effect of PTST2 Overexpression on Starch Granule Numbers. (A) Starch granules in chloroplasts of transgenic Arabidopsis lines overexpressing PTST2-YFP under the control of the 35S promoter. Leaves harvested at the end of the day were sectioned and stained with toluidine blue prior to observation by light microscopy. Similar starch granule phenotypes were observed in at least three independent experiments performed on different batches of plants (either on plants from the T2 or T3 generation). Representative images from T3 homozygous plants are shown. Bars = 10 µm. (B) Immunoblots showing the level of transgene expression. The antibodies (Ab) used for detection and the migration positions of molecular mass markers (in kD) are indicated on the left of each panel. An equal amount of total protein was loaded in each lane: 10 µg for anti-YFP and anti-PTST2 blots; 20 µg for anti-PTST3 and anti-SS4 blots. (C) Starch granules in chloroplasts of transgenic Arabidopsis lines expressing PTST2-YFP under the control of the 35S promoter, as in (A), but in the ss4 mutant background. Similar starch granule phenotypes were observed in a second, independent experiment performed on a different batch of plants. Representative images are shown. The line in the wild-type background is from the T3 generation and homozygous for the insertion. The lines in the ss4 background are from the T2 generation (either homo- or heterozygous for the insertion). Bars = 10 µm. (D) Immunoblot with anti-YFP showing the levels of transgene expression in ss4/35S_pro:PTST2-YFP lines. Protein extracts were produced from the same plants analyzed in (C). Equal fresh weight (equivalent to 1.8 mg) was loaded per lane.

Figure 10.

The Role of the Glucan Binding CBM48 Domain in PTST2. (A) Glucan binding assay using recombinant His-PTST2 against intact waxy maize starch granules. Unbound proteins are in the soluble fraction (S), while bound proteins are in the starch pellet (P). The final wash fraction was also loaded (W). The 2×W→A variant of PTST2 carries two Trp-to-Ala substitutions in both conserved glucan binding Trp residues within the CBM48 (Trp-475 and Trp-510). Similar results were obtained in two other independent experiments. (B) Levels of PTST2-YFP and the 2×W→A variant expressed under the control of the 35S promoter in transgenic Arabidopsis lines. An equal amount of protein (30 µg) was loaded per lane, and immunoblots were probed with the PTST2 antibody (upper panel) and the actin antibody as a loading control (lower panel). (C) Starch granules in chloroplasts overexpressing either the wild type or 2×W→A variant of PTST2. Leaves harvested at the end of the day were sectioned and stained with toluidine blue prior to observation under light microscopy. The lines are from the T3 generation and homozygous for the transgene. Bars = 10 µm. (D) Quantification of granule sections within chloroplasts. Light micrographs were acquired as in (C), and the number of starch granules observed within each chloroplast section was counted. Histograms show the frequency of chloroplasts containing a given number of granule sections relative to the total number of chloroplasts analyzed (n = 705 for each line). Equal numbers of chloroplasts were analyzed in leaves from three different plants for each genotype. Arrows indicate the bin containing the median value.

Figure 11.

ITC Analysis of Interactions between PTST2 and Different Soluble Glucans. (A) ITC profile of maltoheptaose (blue) and β-cyclodextrin (black) injected into a solution of the full-length His-PTST2 protein. The upper panels show the raw calorimetric data. The bottom plots are integrated heats as a function of the glucan/PTST2 molar ratio. The best fit for β-cyclodextrin was obtained from a nonlinear least squares method using a one-site binding model for two significantly distant N-values. Heats of dilution without PTST2 protein have been obtained from independent titration experiments (Supplemental Figure 7A). Reactions are exothermic. (B) As for (A), but showing maltoheptaose (blue) and β-cyclodextrin (black) injected into the isolated CBM48 domain. (C) As for (A), but showing maltodecaose injected into the full-length His-PTST2 protein.

Figure 12.

A Model for PTST2 Function in Granule Initiation. In an initial step (A), short MOS (such as maltose and maltotriose) is elongated into longer MOS. This is likely to be a stochastic process, and the amount of longer MOS available will be influenced by the rate of MOS elongation by the SS isoforms and phosphorylase, as well as the rate of MOS degradation by starch-degrading enzymes. Once PTST2 recognizes a long MOS through its CBM48 domain, it can recruit SS4 to the substrate (B), allowing SS4 to further elongate the MOS (C). PTST2 could then dissociate from the MOS, becoming available to interact with other SS4 and MOS molecules (D). Since SS4 can also dimerize, several MOS molecules may be recruited by PTST2 for elongation within close proximity of each other, which could increase the efficiency at which crystalline glucans are formed (E). The other starch biosynthesis enzymes (SS isoforms, branching enzymes, and isoamylase) then further elaborate the structure of these substrates until a starch granule is formed (F).