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. 2020 Aug;32(8):2543-2565.
doi: 10.1105/tpc.19.00946. Epub 2020 May 29.

STARCH SYNTHASE5, a Noncanonical Starch Synthase-Like Protein, Promotes Starch Granule Initiation in Arabidopsis

Melanie R Abt  1 , Barbara Pfister  1 , Mayank Sharma  1 , Simona Eicke  1 , Léo Bürgy  1 , Isabel Neale  1 , David Seung  1 , Samuel C Zeeman  2
Affiliations

STARCH SYNTHASE5, a Noncanonical Starch Synthase-Like Protein, Promotes Starch Granule Initiation in Arabidopsis

Melanie R Abt et al. Plant Cell. 2020 Aug.

Abstract

What determines the number of starch granules in plastids is an enigmatic aspect of starch metabolism. Several structurally and functionally diverse proteins have been implicated in the granule initiation process in Arabidopsis (Arabidopsis thaliana), with each protein exerting a varying degree of influence. Here, we show that a conserved starch synthase-like protein, STARCH SYNTHASE5 (SS5), regulates the number of starch granules that form in Arabidopsis chloroplasts. Among the starch synthases, SS5 is most closely related to SS4, a major determinant of granule initiation and morphology. However, unlike SS4 and the other starch synthases, SS5 is a noncanonical isoform that lacks catalytic glycosyltransferase activity. Nevertheless, loss of SS5 reduces starch granule numbers that form per chloroplast in Arabidopsis, and ss5 mutant starch granules are larger than wild-type granules. Like SS4, SS5 has a conserved putative surface binding site for glucans and also interacts with MYOSIN-RESEMBLING CHLOROPLAST PROTEIN, a proposed structural protein influential in starch granule initiation. Phenotypic analysis of a suite of double mutants lacking both SS5 and other proteins implicated in starch granule initiation allows us to propose how SS5 may act in this process.

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Figures

Figure 1.
Figure 1.
SS5 Proteins Differ Substantially from the Canonical Starch Synthases but Localize to the Chloroplast. (A) Protein domain organization of the Arabidopsis soluble starch synthases SS1 to SS5. Note that the transit peptide (cTP) of SS3 is not predicted for the splice form resulting from the longest transcript. The rice SS5 ortholog (Os-SS5) is included because the At-SS5 structure is not representative of the majority of SS5 proteins. The dashed line in At-SS5 indicates the C-terminal deletion relative to the rice ortholog. Locations of amino acid residues and motifs mentioned in the text and Supplemental Figures 3A and 3D are highlighted in red (conserved in all canonical starch synthases) and blue (SS4-specific). Residue identities in each starch synthase isoform are specified. aa, amino acids. (B) Fluorescence images of representative leaf epidermal cells of a transgenic Arabidopsis plant expressing mCitrine-tagged At-SS5 under the control of the endogenous promoter. The images are orthogonal projections of several single images acquired in the Z plane. At-SS5-mCitrine adopts a punctate localization pattern (highlighted with white arrowheads). Bars = 10 μm.
Figure 2.
Figure 2.
SS5 Shares a Putative Surface Carbohydrate Binding Site with SS4. (A) Surface amino acid conservation in SS4 and SS5. The resolved crystal structure of the catalytic domain of At-SS4 (dark blue; Nielsen et al., 2018) was used to model corresponding three-dimensional structures of At-SS5 (cyan) and Os-SS5 (green). Amino acid conservation deduced from multiple sequence alignments of orthologs for each respective isoform was superimposed on the resulting models. The modeled structures are shown with ADP and acarbose (a glucosidase inhibitor used as an acceptor mimic; Nielsen et al., 2018) as ligands and maltose as a generic glucan at the surface binding site (carbon, oxygen, nitrogen, and phosphate atoms in yellow, red, blue, and orange, respectively) shown in the same position for Os-SS5 and At-SS5 as for At-SS4. (B) Carbohydrate binding of recombinant polyhistidine-tagged At-SS5. S, soluble; FW, final wash, I, insoluble; Rep 1 to Rep 3, replicates. The immunoblot shows that some At-SS5 protein (black arrowheads) is detectable in the insoluble fraction when using maize starch as a binding reagent (variable levels, between 3 and 9% of the respective soluble portion). Note that some protein was detected in the insoluble fraction also when using cross-linked dextran beads (Sephadex G-10) as a nonstarch polymer. Further note the presence of a prominent double band, which is likely due to partial loss of the polyhistidine tag, in the soluble fraction.
Figure 3.
Figure 3.
Arabidopsis SS5 Does Not Produce Glucans in Saccharomyces cerevisiae. (A) At-SS1, At-SS2, At-SS3, At-SS4, and At-SS5 without their respective predicted chloroplast transit peptides were expressed in yeast cells purged of their endogenous glycogen-metabolic genes except for the glycogenin genes GLG1 and GLG2 as indicated by Pfister et al. (2016). Arabidopsis branching enzyme(s) and the bacterial ADP-Glc pyrophosphorylase (glgC; for the provision of ADP-Glc) were coexpressed as indicated. Six hours after the induction of heterologous protein expression, cells were stained with iodine to visualize glucan accumulation. Shown are representative light micrographs from two independent experiments that gave identical results. Bar = 10 μm. (B) Representative immunoblot showing that At-SS5 was readily detected in soluble extracts of yeast at the expected size (white arrowhead) with the antibody raised against At-SS5.
Figure 4.
Figure 4.
Arabidopsis SS5 Insertional Mutants Lack SS5. (A) Schematic representation of the At-SS5 genomic sequence (AT5G65685.1). Exons are represented as black boxes, introns as thin lines, and UTRs as gray boxes. Triangles show sites of T-DNA insertions of lines used in this study. Photographs of 25-d-old Arabidopsis rosettes of the respective lines are shown below the gene model. Bar = 2 cm. (B) Confirmation of gene disruption in the T-DNA insertional mutants shown in (A). Upper panels, endpoint RT-PCR on cDNA preparations from wild-type (Col-0) plants and ss5 T-DNA lines. Colors correspond to the amplification products indicated below the gene model in (A). Note the absence of bands spanning the respective insertion sites. Primers used for RT-PCR are described in Supplemental Data Set 2. Lower panels, immunoblot of total leaf protein extracts probed with the antibody raised against At-SS5, showing a weak band corresponding to At-SS5 that is absent in the three T-DNA insertional mutant lines. An At-SS5-YFP anti-YFP immunoprecipitation sample (bait indicated by a black arrowhead) containing endogenous At-SS5 (indicated by a white arrowhead) as a prey was included as an indicator of native At-SS5 migration (ind); this lane was run on the same gel. Actin levels analyzed simultaneously on the blot (in red) served as a loading control.
Figure 5.
Figure 5.
Arabidopsis SS5 Is Expressed to Low Levels in Planta. (A) Expression of At-SS5-mCitrine from a genomic fragment. An immunoblot of total leaf protein extracts is shown. Two independent transgenic lines are shown per background. mCitrine-tagged and endogenous At-SS5 are indicated by black and white arrowheads, respectively. (B) Whole-rosette fluorescent imaging of 2-week-old transgenic plants expressing At-SS5-mCitrine driven by the At-SS5 promoter from a genomic fragment. The same rosette was imaged in both overview (upper two rows) and close-up magnifications (lower two rows). Bars = 500 μm.
Figure 6.
Figure 6.
Arabidopsis SS5 Insertional Mutants Produce Fewer but Larger Starch Granules Per Chloroplast. (A) Starch content in whole Arabidopsis rosettes. Plants were harvested after 12 h of light (end of the day; ED) and after 12 h of dark (end of the night; EN). Error bars represent the se. ***, P < 0.001, based on t tests (n = 4 to 6 biological replicates [rosettes]; see also Supplemental Data Set 3). Individual measurements are shown by red points. FW, fresh weight. (B) Starch granule quantifications in sections of embedded ss5 leaf tissue, represented as histograms of granules per chloroplast section in red with the wild-type (Col-0) distribution underlaid in transparent gray. Individual chloroplast counts (n) are scattered into bins below the histograms with hues differentiating the biological replicates (individually sampled rosettes; N). Representative light micrographs of the respective lines are shown above each histogram. Bar = 10 μm. (C) Scanning electron micrographs of purified wild-type (Col-0) and ss5 starch granules at a magnification of 15,000×. Bar = 4 μm.
Figure 7.
Figure 7.
Complementation of the ss5 Phenotype by SS5 Expression from a Genomic Fragment. Overlay histograms represent the distribution of granule numbers per chloroplast section in each three replicate plants (Col-0 and ss5-1) or three independent T1 transformants per background. Different hues are shown to account for variations between different transgene insertions in this experiment; black outlines represent the mean across all lines or replicates. Individual counts (n) are scattered into bins below the histograms with hues corresponding to the respective transformant/replicate histogram. Representative micrographs of each transformant or control are shown above each histogram. Bar = 10 μm.
Figure 8.
Figure 8.
Arabidopsis SS5 and MRC Interact. (A) At-SS5-YFP was expressed in different genetic backgrounds and immunoprecipitated via the YFP tag (black arrowheads). Shown are immunoblots using antibodies recognizing GFP/YFP, SS5, or MRC. Endogenous At-SS5 (white arrowhead) is enriched in the immunoprecipitate (IP) when the bait is expressed in a wild-type (Col-0) background, whereas At-MRC is enriched when it is expressed in either the wild-type or ss5 mutant background. Two independent transgenic lines are shown per genetic background. Actin, analyzed simultaneously (in red), served as a loading control on anti-GFP and anti-SS5 immunoblots. (B) Immunoprecipitation using At-MRC-mCitrine (yellow arrowhead) as bait. Endogenous At-SS5 (white arrowhead) is enriched in the immunoprecipitate. Note that endogenous MRC runs slightly higher in this blot than in (A), likely due to the polyacrylamide gradient gel used for SDS-PAGE in this case. (C) At-SS5-YFP and At-MRC-CFP were transiently expressed in tobacco leaves. When expressed together, the proteins colocalize to the same puncta (examples indicated by white arrowheads) in the chloroplast. Bars = 10 μm. (D) Deletion of the At-SS5 coiled coil in the At-SS5Δcc-YFP bait abolishes both dimerization and interaction with At-MRC. The Os-SS5-YFP immunoprecipitate contains At-MRC but not endogenous At-SS5. Protein bands are indicated with arrowheads as follows: At-SS5-YFP, black; endogenous At-SS5, white; At-SS5Δcc-YFP, pink; Os-SS5-YFP, blue.
Figure 9.
Figure 9.
SS5 Exerts Its Function through MRC and Acts in the Absence of SS4. (A) Quantification of starch granule content, represented as histograms of counts per chloroplast section, in crosses of ss5-1 and mutants of other proteins involved in starch granule initiation. Shown is the distribution upon loss of SS5 (red) in the wild-type (Col-0) or mutant background (transparent blue). Individual chloroplast counts (n) upon loss of SS5 are scattered into bins below the histogram with different hues representing biological replicates (N). Representative micrographs of the genetic background as well as the respective double mutants are shown above each histogram. Note that plants used for this quantification come from the same experimental batch (and therefore share the same Col-0 and ss5-1 quantifications) as the ones used for Figure 6B. Bar = 10 μm. (B) Whole-rosette measurements of starch content in the ss4 ss5 double mutant (n = 4 to 6 rosettes). Plants were harvested after 12 h of light (end of the day; ED) and after 12 h of dark (end of the night; EN). Individual data points are shown in red. FW, fresh weight. (C) Iodine-stained rosettes harvested at end of the day. Wild types and ss5-1 stain similarly. As in ss4, immature leaves of ss4 ss5 double mutants show very weak staining. Shown are two biological replicates for each genotype. (D) ADP-Glc content measurements of the ss4 ss5 double mutant (n = 3 to 4 rosettes). Entire rosettes were harvested at end of the day, and metabolites were extracted and quantified as described previously by Seung et al. (2016). Individual data points are shown in red. Error bars in (B) and (D) represent the se. Asterisks show statistical significance: *, P < 0.05; **, P < 0.01; and ***, P < 0.001, based on t tests.
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References

    1. Alonso J.M., et al. (2003). Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301: 653–657. - PubMed
    1. Arrivault S., Guenther M., Ivakov A., Feil R., Vosloh D., van Dongen J.T., Sulpice R., Stitt M.(2009). Use of reverse-phase liquid chromatography, linked to tandem mass spectrometry, to profile the Calvin cycle and other metabolic intermediates in Arabidopsis rosettes at different carbon dioxide concentrations. Plant J. 59: 826–839. - PubMed
    1. Baerenfaller K., Hirsch-Hoffmann M., Svozil J., Hull R., Russenberger D., Bischof S., Lu Q., Gruissem W., Baginsky S.(2011). pep2pro: A new tool for comprehensive proteome data analysis to reveal information about organ-specific proteomes in Arabidopsis thaliana. Integr. Biol. 3: 225–237. - PubMed
    1. Chia T., Chirico M., King R., Ramirez-Gonzalez R., Saccomanno B., Seung D., Simmonds J., Trick M., Uauy C., Verhoeven T., Trafford K.(2020). A carbohydrate-binding protein, B-GRANULE CONTENT 1, influences starch granule size distribution in a dose-dependent manner in polyploid wheat. J. Exp. Bot. 71: 105–115. - PubMed
    1. Crooks G.E., Hon G., Chandonia J.-M., Brenner S.E.(2004). WebLogo: A sequence logo generator. Genome Res. 14: 1188–1190. - PMC - PubMed

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