SAGA1 and SAGA2 promote starch formation around proto-pyrenoids in Arabidopsis chloroplasts

Significance Engineering a pyrenoid-based CO2 concentrating mechanism (pCCM) into land plants could improve crop yield potential and resilience to fluctuating climates. Modeling predicts that an efficient pCCM requires a barrier around the pyrenoid to limit CO2 diffusion away from Rubisco. In many species, including the model algae Chlamydomonas, this barrier consists of a sheath of starch plates. Here, we have engineered starch plate-like granules around the proto-pyrenoid in Arabidopsis. This work represents a critical step forward toward achieving a functional pCCM in plants that could ameliorate food security concerns.

protein-protein interactions between SAGA2 and the small subunit 1 of Rubisco in Chlamydomonas (CrRbcS1; Cre02.g120100), the large subunits of Rubisco (CrRbcL) and EPYC1.Control column shows growth on CSM-L-W, whilst interaction columns show growth on CSM-L-W-H dropout media (weak), and with 10 mM 3-aminotriazole (3-AT) inhibitor (strong).C. Y2H assays showing the interaction strength between SAGA2 and EPYC1 with mutations in various residues of the α-helix.Interaction strength was scored by growth on increasing concentrations of 3-AT as in Atkinson et al. (26).The EPYC1 α-helix KO is a full length EPYC1 in which each of five α-helix sequences was replaced by alanine residues.In the subsequent samples just one repeat of EPYC1 is tested for interaction (Rep1, corresponding to amino acid residues 28-76 of the full-length protein), with one or more residues substituted.D. SAGA2 was split into 5 regions, with region 3 further subdivided into three parts (A-C), to test for interaction with EPYC1.
E. Model of one of the repeat α-helix sequences of EPYC1 (residues 63-72) with hydrophobic residues shown in orange and charged residues shown in red.A comparison of the residues that interact with CrRbcS (19) and SAGA2 (from data in C) is shown.F. Predicted structure of SAGA2 (Alphafold2), with sections of region 3 highlighted in magenta (A), green (B) and orange (C), respectively.G. Predicted SAGA2 structure coloured by confidence score according to the Predicted Local Difference Distance Test (pLDDT), where red = high and blue = low.Please note that SAGA2 is predicted to contain several intrinsically disordered regions (IDRs), and the structural predictions of AlphaFold can be unreliable for IDRs.

Figure S2 .
Figure S2.Lighter-staining regions in EP1S1-1 are not stained by osmication.Following fixation EP1S1-1 leaf samples were stained with 1% (w/v) osmium tetroxide in 0.05 M sodium cacodylate for 45 minutes.Samples were washed three times with phosphate buffered saline, before being dehydrated and embedded as normal.Membranes such as the visible grana stacks were stained black during this process, whilst the patterned structures in the condensate matrix remained lighter-coloured.Scale bar = 0.5 µm.

Figure S3 .
Figure S3.Total starch area and representative images in EP1S1-1 over time.A. Average of total areas for stroma, adjacent and enclosed starch granules in EP1S1-1 plants over time from Fig. 2G.The averages at each time point represents the mean ± SEM of 22-39 images.Letters indicate significant difference (p < 0.05) as determined by one-way ANOVA followed by a Kruskal-Wallis post-hoc test.B. Starch degradation rates plotted for each starch type showing total starch area (left) and proportional starch area (right).Trendlines are fitted to the data and the values for the slopes given under each graph.C. Representative TEM images of starch abundance over time.

Figure S5 .
Figure S5.Additional representative TEM images for each of the three EP1S1S2 lines.See Fig. 3 for transgene expression levels.Scale bar = 0.5 µm.

Figure S6 .
Figure S6.Additional starch granule parameters from area analysis of TEM images.A. The number of distinct condensates observed per chloroplast in each line.B. Proportion of total starch granules per chloroplast that are associated with the condensate (adjacent or enclosed granules) compared to those that were located in the stroma (stromal granules).C. Total number of starch granules per chloroplast classified as stromal, adjacent or enclosed.D. Total number of starch granules per chloroplast.Error bars show the mean ± SEM of 24-48 chloroplasts per line.

Figure S9 .
Figure S9.Fresh weight and rosette areas of S1 and EP1S1 lines.A. Fresh weight of S1 and EP1S1 lines and their azygous (Az) segregants 32 days after germination.B. Rosette expansion for S1 and EP1S1 lines measured over 30 days post germination.Error bars show the mean ± SEM of 12-21 individual rosettes.