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. 2021 Nov 26;12(1):6944.
doi: 10.1038/s41467-021-27151-5.

Coalescence and directed anisotropic growth of starch granule initials in subdomains of Arabidopsis thaliana chloroplasts

Léo Bürgy  1 , Simona Eicke  1 , Christophe Kopp  2 , Camilla Jenny  1 , Kuan Jen Lu  1 , Stephane Escrig  2 , Anders Meibom  2   3 , Samuel C Zeeman  4
Affiliations

Coalescence and directed anisotropic growth of starch granule initials in subdomains of Arabidopsis thaliana chloroplasts

Léo Bürgy et al. Nat Commun. .

Abstract

Living cells orchestrate enzyme activities to produce myriads of biopolymers but cell-biological understanding of such processes is scarce. Starch, a plant biopolymer forming discrete, semi-crystalline granules within plastids, plays a central role in glucose storage, which is fundamental to life. Combining complementary imaging techniques and Arabidopsis genetics we reveal that, in chloroplasts, multiple starch granules initiate in stromal pockets between thylakoid membranes. These initials coalesce, then grow anisotropically to form lenticular granules. The major starch polymer, amylopectin, is synthesized at the granule surface, while the minor amylose component is deposited internally. The non-enzymatic domain of STARCH SYNTHASE 4, which controls the protein's localization, is required for anisotropic growth. These results present us with a conceptual framework for understanding the biosynthesis of this key nutrient.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. De-starched chloroplasts exhibit parallel initiation of starch granules shortly after dawn.
Chloroplast sections from an SBF-SEM stack (with 50-nm Z-resolution, inverted SEM images) sampled from the mesophyll of fully expanded leaves of 35-day old Arabidopsis plants grown in a 12 h:12 h diel regime. Starch granules appear as dark-gray disks between thylakoid membranes. a–d Representative chloroplast sections from plants harvested (a) at the end of a regular 12-h night, with a remaining starch granule (white arrowhead), (b) after 15 min light, (c) after 30 min light, and (d) after 8 h light. e Series of images (with 100-nm steps in the Z axis) from the sample stack in b. f–i Representative chloroplast sections from plants exposed to a prolonged night and harvested (f) at the end of the 4-h night extension; arrowheads indicate stromal spaces with a floccular appearance (g) after 15 min light, (h) after 30 min light, and (i) after 8 h light; arrowhead indicates parallel surfaces of abutting granules. j Serial sections (with 100-nm steps in the Z axis) from the sample stack in g. Note the numerous starch initials. Scale bars: 2 µm. Further data are given in Supplementary Fig. 1 and Supplementary Movies 1–4.
Fig. 2
Fig. 2. Imaging of entire chloroplasts to determine the exact number of starch granules.
Tomographic reconstruction from SBF-SEM image stacks of entire chloroplasts from plants sampled after a prolonged night and after 15 min, 30 min, and 8 h light. a The number of granules per chloroplast. b The number of starch-containing pockets per chloroplast. c Number of granules in each cluster category. Data in a–c are expressed as frequency distributions (bins of 4, including 0 in a and b, excluding 0 in c), where the height of the bar represents the frequencies within each bin. For each time point, two plants from two independent experiments were examined. In total, 22, 21, and 27 chloroplasts were examined for the 15-min, 30-min, and 8-h time points, respectively. d 3D-renderings of representative chloroplasts (non-inverted SEM images) with the pockets (yellow) and their starch granules (violet). Scale bars: 2 µm. e The volume (top), surface area (middle), and the surface area-to-volume ratio (SA:V; bottom) for the total starch in each chloroplast. In total, 8, 8, and 12 chloroplasts were examined for the 15-min, 30-min, and 8-h time points, respectively, to determine these values.
Fig. 3
Fig. 3. Coalescence of starch granule initials tracked with stable isotope labeling, EM, and NanoSIMS imaging.
Plants subjected to a 4-h night extension were labeled with a pulse of 13CO2 for 15 min in the light, then harvested immediately (a), or after a chase of 15 min (b), 45 min (c), or 4 h (d) in normal air in the light. In each case, samples were fixed and embedded for EM and NanoSIMS imaging. The electron micrograph (left, inverted SEM image) and the enrichment map (right), and their overlay (center) are shown. The 13C-enrichment is reported as per mille. Note the multiple labeled initials in each stromal space, with an increasing degree of coalescence during the chase in normal air. Scale bar: 2 µm.
Fig. 4
Fig. 4. Anisotropic starch granule expansion revealed by stable isotope labeling, EM, and NanoSIMS imaging.
a Plants subjected to a 4-h night extension photosynthesized for 45 min in normal air, were labeled with a pulse of 13CO2 for 15 min, and then harvested immediately for EM and NanoSIMS imaging, as in Fig. 3. b Plants subjected to a normal night were labeled with a pulse of 13CO2 for 1 h at midday then harvested immediately. Scale bars: 2 µm. c Quantification of 13C-enrichment on the surface of six granules depicted in b. The probing regions (depicted in red in the inset, right) were defined manually with masks over the δ13C map. Individual circular profiles were extracted anticlockwise from the starting point (yellow). Light blue segments highlight the parts of the profiles corresponding to the granule margins, where most enrichment is observed. Horizontal lines show the mean enrichment for each profile.
Fig. 5
Fig. 5. Starch granule initiation and growth are controlled by STARCH SYNTHASE 4.
Plants subjected to a normal night were labeled with a pulse of 13CO2 for 1 h at midday then harvested immediately for EM and NanoSIMS imaging, as in Fig. 3. a Chloroplasts from the ss4 mutant. b Chloroplasts from the ss4 mutant expressing a self-glycosylating glycogen synthase (GS) from A. tumefaciens. c Chloroplasts expressing the GS fused with the N-terminal of SS4. Scale bars: 2 µm. For additional examples, see supplementary Fig. 2g–i. For analysis of the patterns of 13C-enrichment, see Supplementary Fig. 6.
Fig. 6
Fig. 6. GRANULE-BOUND STARCH SYNTHASE synthesizes amylose in the granule cores.
Plants subjected to a normal night were labeled with a pulse of 13CO2 for 1 h at midday then harvested immediately for EM (not shown) and NanoSIMS imaging. Scale bars: 2 µm. a 13C-enrichment map (left) of starch granules within wild-type chloroplasts. Manually masking the granule surfaces revealed enrichment specifically in the cores (right; note different 13C-enrichment scales). b 13C-enrichment map of starch granules (left) and their cores (right) within chloroplasts of the gbss mutant. c Quantification of the 13C-enrichment of granule cores and surfaces overlaid with boxplots, where the minima, maxima of the whiskers represent 2.5% and 97.5%, the bounds of box, 25% and 75%, and the center denotes 50% of the posterior. N = 259 and 269 granules for the wild type and gbss mutant, respectively, in each case from three biological replicates.
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