Increasing amyloplast size in wheat endosperm through mutation of PARC6 affects starch granule morphology

Summary The determination of starch granule morphology in plants is poorly understood. The amyloplasts of wheat endosperm contain large discoid A‐type granules and small spherical B‐type granules. To study the influence of amyloplast structure on these distinct morphological types, we isolated a mutant in durum wheat (Triticum turgidum) defective in the plastid division protein PARC6, which had giant plastids in both leaves and endosperm. Endosperm amyloplasts of the mutant contained more A‐ and B‐type granules than those of the wild‐type. The mutant had increased A‐ and B‐type granule size in mature grains, and its A‐type granules had a highly aberrant, lobed surface. This morphological defect was already evident at early stages of grain development and occurred without alterations in polymer structure and composition. Plant growth and grain size, number and starch content were not affected in the mutants despite the large plastid size. Interestingly, mutation of the PARC6 paralog, ARC6, did not increase plastid or starch granule size. We suggest TtPARC6 can complement disrupted TtARC6 function by interacting with PDV2, the outer plastid envelope protein that typically interacts with ARC6 to promote plastid division. We therefore reveal an important role of amyloplast structure in starch granule morphogenesis in wheat.

: KASP-markers for Ttparc6 and Ttarc6 genotyping.Table S2: Codon optimized DNA sequences of TaPARC1-A1, TaARC6-A1, TaPDV1-1-A1, TaPDV1-2-A1 and TaPDV2-A1.Method S1: Phylogenetic analysis and gene models Method S2: Cloning and construct assembly Method S3: Transient transformation of Nicotiana benthamiana Method S4: Gas exchange Method S5: Starch purification, scanning electron microscopy and polarised light microscopy.Method S6: Total starch quantification, starch composition and amylopectin structure Method S7: Analysis of chloroplast morphology Method S8: Microscopic analysis of amyloplast morphology in developing grain The tree with the highest log likelihood (-36614.75) is shown.The percentage of trees out of 1000 bootstraps in which the associated taxa clustered together is shown next to the branches.The tree is drawn to scale, with branch lengths and scale bar representing the number of substitutions per site.Full experimental procedures can be found in Methods S1.   (a-e) Light response curves were measured at ambient CO2 levels (412 μmol m -2 s -1 ).Lines represent the average and ribbons the standard error.The dotted line at A = 30 μmol m -2 s -1 is provided to aid comparison between genotypes.(f) Assimilation rate at ambient light (280 µmol m -2 s -1 ) and high light (2000 µmol m -2 s -1 ).(g) Estimation of Vcmax was extracted from the A/Ci curves using plant ecophys.package(R).(h) Estimation of Jmax was extracted from the A/Ci curves using plant ecophys.package(R).(a-b) Size distribution plots from Coulter counter analysis.The volume of granules at each diameter relative to the total granule volume was quantified using a Coulter counter.Values represent mean (solid line) ± SEM (shading) of three replicates using grains harvested from separate plants.(c-h) Granule size parameters obtained from fitting a log-normal distribution to the B-type granule peak and a normal distribution to the A-type granule peak in the granule size distribution data presented in (a -b).Three biological replicates were analysed: (c, d) A-type granule diameter (in μm).Significant differences under a one-way ANOVA and all pairwise multiple comparison procedures (Tukey's test) are indicated with different letters (p ≤ 0.05).(e, f) B-type granule diameter (in μm).Significant differences under a Kruskal-Wallis one-way ANOVA on ranks and all pairwise multiple comparison procedures (Tukey's test) are indicated with different letters (p ≤ 0.022) for Ttparc6-1 lines.Significant differences under one-way ANOVA and all pairwise multiple comparison procedures (Tukey's test) are indicated with different letters (p ≤ 0.05) for Ttparc6 BC lines.(g, h) B-type granule content by percentage volume.Significant differences under a one-way ANOVA and all pairwise multiple comparison procedures (Tukey's test) are represented with different letters (p ≤ 0.05).(o-q) Granule size parameters obtained from fitting a log-normal distribution to the B-type granule peak and a normal distribution to the A-type granule peak in the granule size distribution data presented in (h).Three biological replicates were analysed: (o) A-type granule diameter (in μm).Significant differences under a one-way ANOVA and all pairwise multiple comparison procedures (Tukey's test) are indicated with different letters (p ≤ 0.02).(p) B-type granule diameter (in μm).Significant differences under a one-way ANOVA and all pairwise multiple comparison procedures (Tukey's test) are indicated with different letters (p ≤ 0.001).(q) B-type granule content by percentage volume.Significant differences under a one-way ANOVA and all pairwise multiple comparison procedures (Tukey's test) are represented with different letters (p ≤ 0.001).For all boxplots, the bottom and top of the box represent the lower and upper quartiles respectively, and the band inside the box represents the median.The ends of the whiskers represent values within 1.5x of the interquartile range, whereas values outside are outliers.(c-d) Rapid Visco Analyser (RVA) analysis of viscosity during gelatinisation.Analyses were conducted using: (c) Purified starch (1.5 g in 25 mL water).Values represent mean (solid line) ± SEM (shading) of three biological replicates, each using starch from grains harvested from a separate plant.The different genotypes produced viscographs that were generally similar, and there was no obvious difference in peak viscosity or the holding strength during cooling.Slight differences between genotypes were observed during cooling (retrogradation) and final viscosity, but these were not consistent for either Ttparc6 double mutants or wild-type controls.(d) Whole flour (5 g in 25 mL water), where flour was produced by pooling a minimum of 3 biological replicates per genotype.The viscographs were more variable among genotypes, and there was no consistent effect that could be attributed to the mutant genotype.Methods S1: Phylogenetic analysis and gene models PARC6, ARC6, PDV1 and PDV2 protein sequences were retrieved from Ensembl Plants and Phytozome (Goodstein et al., 2012;Yates et al., 2022).Proteins were aligned using ClustalW in MEGA7.Phylogenetic analysis was conducted in MEGA7 (Kumar et al., 2016) using the Maximum Likelihood method based on the JTT matrix-based model (Jones et al 1992).Initial tree(s) for the heuristic search were obtained automatically by applying Neighbour-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value.
Gene models were taken from Ensembl Plants and domains were annotated using Interpro (Yates et al., 2022;Paysan-Lafosse et al., 2023).

Methods S2: Cloning and construct assembly
To generate the transgenic wheat amyloplast reporter lines, we modified a construct design from Matsushima and Hirano (2019), to use a codon-optimised mCherry coding sequence (rather than GFP in the original citation) downstream of the OsWaxy transit peptide sequence (sequence in Table S2).This fusion sequence, flanked by attB1 and attB2 recombination sites, was synthesised as a gBlocks fragment (IDT) and recombined into the Gateway entry vector pDONR221 using Gateway BP clonase II (Invitrogen, Thermo Fisher Scientific).The cTPmCherry coding sequence was then recombined using Gateway LR clonase II (Invitrogen, Thermo Fisher Scientific) into a modified pGGG vector (Hayta et al., 2021), pGGG_AH_Ubi_GW_NosT, encoding for a Hygromycin resistance gene driven by an actin promoter (AH), a gateway cassette for gateway recombination (GW) downstream of the ZmUbiquitin promoter (Ubi) and upstream of a Nos terminator (NosT).

Methods S3: Transient transformation of Nicotiana benthamiana
Nicotiana benthamiana plants were transiently transformed using Agrobacterium tumefaciens (GV3101) carrying the respective constructs.The bacteria were grown at 28°C for 48 h.Cultures were resuspended in MMA buffer (10 mM MES pH 5.6, 10 mM MgCl2, 0.1 mM acetosyringone) at an optical density of 1.0 at 600 nm for confocal microscopy and of 0.3 (0.2 for p19) at 600 nm for protein extraction, and infiltrated into the abaxial side of the leaf using a syringe.Leaves were harvested for confocal microscopy and protein extraction 48-72 h after infiltration.

Methods S4: Gas exchange
Gas exchange measurements were made using an LI-6800P portable photosynthesis system (Li-COR) 40-46 days after germination on the fully expanded flag leaves in the glasshouse (CO2 concentration ca.412 ppm, light intensity ca.280 µmol m -2 s -1 , temperature ca 21°C) as described in Watson-Lazowski et al. (2022).The responses of the CO2 assimilation rate to step increases in light intensity (AQ) were measured under constant CO2 conditions (412 ppm).AQ measurements were taken after acclimation of 60-120s at increasing light intensities (0,20,50,75,100,150,200,500,750,1000,1200,1500,1800,2000 µmol m -2 s -1 ).The response of the CO2 assimilation rate to step increases of intra-cellular CO2 (A/Ci) was measured at saturating light (2000 µmol m -2 s -1 ).The A/Ci curves measured at decreasing and increasing CO2 steps of 400, 300, 200, 100, 50, 0, 400, 400, 600, 800, 1000, 1200 ppm.Maximum rates of carboxylation (Vcmax) and electron transport (Jmax) were calculated from A/Ci curves using the 'Plantecophys' package in R by fitting the raw data to a Farquhar, von Caemmerer, and Berry photosynthesis model (Farquhar et al., 1980;Duursma, 2015).AQ and A/Ci curves were measured consecutively.Before carrying out A/Ci curves, leaves were allowed to stabilise for 20 min at maximal light intensity (2000 µmol m -2 s -1 ).All measurements were taken 8-14 h after the end of the night.For mature grains, three grains per sample were soaked overnight in double distilled water (ddH2O) at 4°C, then homogenized in a mortar and pestle with additional ddH2O.Developing grains were snap frozen in liquid nitrogen at harvest and stored at -80°C.Seeds were thawed immediately before endosperm dissection, and endosperms were homogenized in ddH2O using a ball mill at 30 Hz for 1.5 minutes.For large amounts of starch, mature grains were first milled into flour (Cyclone Mill Twister, Retsch).Homogenates were filtered through a 100 µm nylon mesh, centrifuged and the pellet was resuspended in 90% (v/v) Percoll, 50 mM Tris-HCl, pH 8.
Granule size distribution was analysed and plotted in relative volume/diameter using the Multisizer 4e Coulter counter (Beckman Coulter).fitted with a 70 µm aperture, operating on either total count mode (measuring a minimum of 500,000 particles) or volumetric mode (measuring a minimum of 1 mL starch suspension).Measurements were conducted with logarithmic bin spacing and were corrected for bin width for presentation on a linear x-axis.Aand B-type granule diameters as well as B-granule contents were extracted by fitting distribution models to the data.Python script available at: https://github.com/DavidSeungLab/Coulter-Counter-Data-Analysis.
The morphology of starch granules was examined by scanning electron microscopy, using a Nova NanoSEM 450 (FEI) scanning electron microscope and the Leica DM6000 microscope for polarised light microscopy.Images were processed using ImageJ software (http://rsbweb.nih.gov/ij/) and Adobe Photoshop 2020.

Methods S6: Total starch quantification, starch composition and amylopectin structure
Grain starch quantification was performed using the Total Starch Assay kit (K-TSTA; Megazyme): Flour (milled in ball mill: 5-10 mg) was suspended in 20 µL 80% ethanol and incubated with 500 µL thermostable α-amylase in 100 mM sodium acetate buffer, pH 5, on a shaking thermomixer at 99 °C and 1400 rpm for 7 min.Amyloglucosidase was added and incubated on a shaking thermomixer at 50 °C and 1000 rpm for 35 min.Samples were centrifuged at 20,800g for 10 min and glucose content was measured in the supernatant using the hexokinase/glucose-6phosphate dehydrogenase assay (Roche, Basel, Switzerland) to calculate starch content in glucose equivalents.Amylose content was determined using an iodine-binding method on starch granules dispersed in water, adapted from Washington et al., (2000).Briefly: 1 mg of purified starch (as in S4) was resuspended in 200 µL water, mixed with 200 µL 2 M NaOH solution and incubated at room temperature overnight.The starch slurry was neutralised with 400 µL 1 M HCl. 5 µL of the starch suspension were diluted in 220 µL water and 25 µL Lugol solution (Sigma Life Science).
Absorbance was measured at 620 nm and 535 nm and Amylose content was calculated as described in Washington et al., (2000).Amylopectin chain length distribution was quantified using High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) on a Dionex ICS-5000-PAD fitted with a PA-100 column (Thermo).The preparation of debranched samples was carried out as described in Streb et al., (2008).

Methods S7: Analysis of chloroplast morphology
For the analysis of mesophyll chloroplast morphology: separation of mesophyll cells was performed according to Pyke and Leech (1991) with adjustments: Leaf segments of the leaf tip of the 3 rd fully developed leaf were harvested into 10% formaldehyde solution (Sigma) in PBS (v/v) and incubated in the dark for 2 h.Formaldehyde solution was replaced by 0.1 M Na2EDTA, pH 9 and samples were incubated with shaking at 100 rpm and 60 °C for 2 h.Cells were separated by carefully knocking the coverslip during mounting.Mesophyll chloroplasts were imaged using the LSM800 (Zeiss) or the TCS SP8X (Leica) using a 40.0x or 63.0x water immersion objective.Chlorophyll autofluorescence was excited using a white light laser set to 555 nm, 576 nm or 630 nm and emission was detected at 651 nm to 750 nm using a hybrid detector or Airyscan.

Methods S8: Microscopic analysis of amyloplast morphology in developing grain
For the analysis of endosperm amyloplast morphology using confocal microscopy: Developing grain of amyloplast reporter lines (see above) were harvested at 16 DAF, embedded in 4% low melting agarose and sectioned into 150 µm cross sections using the vibratome VT1000s (Leica).
Images were acquired immediately after sectioning on the LSM800 using a 63.0 x oil immersion objective (Zeiss).mCherry signal was excited at 561 nm and emission was detected at 562 nm to 623 nm (605 nm).
For analysis of endosperm amyloplast morphology using transmission electron microscopy (TEM), samples were prepared and imaged as described in Chen et al. (2022a): Developing grain (16 DAF) were harvested into 2.5% glutaraldehyde in 0.05 M sodium cacodylate, pH 7.4.Samples were post-fixed in 1% (w/v) osmium tetroxide (OsO4) in 0.05 M sodium cacodylate for 2 h at room temperature, dehydrated in ethanol and infiltrated with LR White resin (Agar Scientific, Stansted, UK), using an EM TP embedding machine (Leica, Milton Keynes, UK).LR White blocks were polymerised at 60°C for 16h.For transmission electron microscopy (TEM) ultrathin sections (ca.90 nm) were cut with a diamond knife and placed onto formvar and carbon coated copper grids (EM Resolutions, Sheffield, UK).The sections were stained using 2% (w/v) uranyl acetate for 1 h and 1% (w/v) lead citrate for 1 min, washed in water and air dried.Sections were imaged on a Talos 200C TEM (FEI) at 200 kV and a OneView 4K x 4K camera (Gatan, Warrendale, PA, USA).
All images in this manuscript were processed using the ImageJ software (http://rsbweb.nih.gov/ij/) and Adobe Photoshop 2020.Chloroplast images were additionally processed using the Zeiss ZEN software

Figure S1 :
Figure S1: Molecular phylogenetic analysis of ARC6 and PARC6 gene families.Figure S2: TaARC6 gene models and chloroplast morphology of the Ttarc6 mutant.Figure S3: Growth and seed phenotype of Ttparc6 single homeolog mutants.Figure S4: Photosynthesis parameters of Ttparc6 mutant plants.Figure S5: Size distribution of purified starch granules from mature grains of the Ttparc6 single mutants.Figure S6: Plant growth, grain morphology and starch phenotypes of the Ttparc6 mutant expressing the cTPmCherry amyloplast marker.Figure S7: Analysis of properties of TtPARC6-deficient Triticum turgidum starch.Figure S8: Size distribution of starch granules of the Ttarc6 mutant and TtARC6 and TtPARC6 expression patterns during endosperm development.Figure S9: Molecular phylogenetic analysis of PDV1 and PDV2 gene families and ARC6 mutant protein alignment.Table S1: KASP-markers for Ttparc6 and Ttarc6 genotyping.Table S2: Codon optimized DNA sequences of TaPARC1-A1, TaARC6-A1, TaPDV1-1-A1, TaPDV1-2-A1 and TaPDV2-A1.Method S1: Phylogenetic analysis and gene models Method S2: Cloning and construct assembly Method S3: Transient transformation of Nicotiana benthamiana Method S4: Gas exchange Method S5: Starch purification, scanning electron microscopy and polarised light microscopy.Method S6: Total starch quantification, starch composition and amylopectin structure Method S7: Analysis of chloroplast morphology Method S8: Microscopic analysis of amyloplast morphology in developing grain

Figure S2 :
Figure S1: Molecular phylogenetic analysis of ARC6 and PARC6 gene families.Figure S2: TaARC6 gene models and chloroplast morphology of the Ttarc6 mutant.Figure S3: Growth and seed phenotype of Ttparc6 single homeolog mutants.Figure S4: Photosynthesis parameters of Ttparc6 mutant plants.Figure S5: Size distribution of purified starch granules from mature grains of the Ttparc6 single mutants.Figure S6: Plant growth, grain morphology and starch phenotypes of the Ttparc6 mutant expressing the cTPmCherry amyloplast marker.Figure S7: Analysis of properties of TtPARC6-deficient Triticum turgidum starch.Figure S8: Size distribution of starch granules of the Ttarc6 mutant and TtARC6 and TtPARC6 expression patterns during endosperm development.Figure S9: Molecular phylogenetic analysis of PDV1 and PDV2 gene families and ARC6 mutant protein alignment.Table S1: KASP-markers for Ttparc6 and Ttarc6 genotyping.Table S2: Codon optimized DNA sequences of TaPARC1-A1, TaARC6-A1, TaPDV1-1-A1, TaPDV1-2-A1 and TaPDV2-A1.Method S1: Phylogenetic analysis and gene models Method S2: Cloning and construct assembly Method S3: Transient transformation of Nicotiana benthamiana Method S4: Gas exchange Method S5: Starch purification, scanning electron microscopy and polarised light microscopy.Method S6: Total starch quantification, starch composition and amylopectin structure Method S7: Analysis of chloroplast morphology Method S8: Microscopic analysis of amyloplast morphology in developing grain

Figure S3 :
Figure S1: Molecular phylogenetic analysis of ARC6 and PARC6 gene families.Figure S2: TaARC6 gene models and chloroplast morphology of the Ttarc6 mutant.Figure S3: Growth and seed phenotype of Ttparc6 single homeolog mutants.Figure S4: Photosynthesis parameters of Ttparc6 mutant plants.Figure S5: Size distribution of purified starch granules from mature grains of the Ttparc6 single mutants.Figure S6: Plant growth, grain morphology and starch phenotypes of the Ttparc6 mutant expressing the cTPmCherry amyloplast marker.Figure S7: Analysis of properties of TtPARC6-deficient Triticum turgidum starch.Figure S8: Size distribution of starch granules of the Ttarc6 mutant and TtARC6 and TtPARC6 expression patterns during endosperm development.Figure S9: Molecular phylogenetic analysis of PDV1 and PDV2 gene families and ARC6 mutant protein alignment.Table S1: KASP-markers for Ttparc6 and Ttarc6 genotyping.Table S2: Codon optimized DNA sequences of TaPARC1-A1, TaARC6-A1, TaPDV1-1-A1, TaPDV1-2-A1 and TaPDV2-A1.Method S1: Phylogenetic analysis and gene models Method S2: Cloning and construct assembly Method S3: Transient transformation of Nicotiana benthamiana Method S4: Gas exchange Method S5: Starch purification, scanning electron microscopy and polarised light microscopy.Method S6: Total starch quantification, starch composition and amylopectin structure Method S7: Analysis of chloroplast morphology Method S8: Microscopic analysis of amyloplast morphology in developing grain

Figure S4 :
Figure S1: Molecular phylogenetic analysis of ARC6 and PARC6 gene families.Figure S2: TaARC6 gene models and chloroplast morphology of the Ttarc6 mutant.Figure S3: Growth and seed phenotype of Ttparc6 single homeolog mutants.Figure S4: Photosynthesis parameters of Ttparc6 mutant plants.Figure S5: Size distribution of purified starch granules from mature grains of the Ttparc6 single mutants.Figure S6: Plant growth, grain morphology and starch phenotypes of the Ttparc6 mutant expressing the cTPmCherry amyloplast marker.Figure S7: Analysis of properties of TtPARC6-deficient Triticum turgidum starch.Figure S8: Size distribution of starch granules of the Ttarc6 mutant and TtARC6 and TtPARC6 expression patterns during endosperm development.Figure S9: Molecular phylogenetic analysis of PDV1 and PDV2 gene families and ARC6 mutant protein alignment.Table S1: KASP-markers for Ttparc6 and Ttarc6 genotyping.Table S2: Codon optimized DNA sequences of TaPARC1-A1, TaARC6-A1, TaPDV1-1-A1, TaPDV1-2-A1 and TaPDV2-A1.Method S1: Phylogenetic analysis and gene models Method S2: Cloning and construct assembly Method S3: Transient transformation of Nicotiana benthamiana Method S4: Gas exchange Method S5: Starch purification, scanning electron microscopy and polarised light microscopy.Method S6: Total starch quantification, starch composition and amylopectin structure Method S7: Analysis of chloroplast morphology Method S8: Microscopic analysis of amyloplast morphology in developing grain

Figure S5 :
Figure S1: Molecular phylogenetic analysis of ARC6 and PARC6 gene families.Figure S2: TaARC6 gene models and chloroplast morphology of the Ttarc6 mutant.Figure S3: Growth and seed phenotype of Ttparc6 single homeolog mutants.Figure S4: Photosynthesis parameters of Ttparc6 mutant plants.Figure S5: Size distribution of purified starch granules from mature grains of the Ttparc6 single mutants.Figure S6: Plant growth, grain morphology and starch phenotypes of the Ttparc6 mutant expressing the cTPmCherry amyloplast marker.Figure S7: Analysis of properties of TtPARC6-deficient Triticum turgidum starch.Figure S8: Size distribution of starch granules of the Ttarc6 mutant and TtARC6 and TtPARC6 expression patterns during endosperm development.Figure S9: Molecular phylogenetic analysis of PDV1 and PDV2 gene families and ARC6 mutant protein alignment.Table S1: KASP-markers for Ttparc6 and Ttarc6 genotyping.Table S2: Codon optimized DNA sequences of TaPARC1-A1, TaARC6-A1, TaPDV1-1-A1, TaPDV1-2-A1 and TaPDV2-A1.Method S1: Phylogenetic analysis and gene models Method S2: Cloning and construct assembly Method S3: Transient transformation of Nicotiana benthamiana Method S4: Gas exchange Method S5: Starch purification, scanning electron microscopy and polarised light microscopy.Method S6: Total starch quantification, starch composition and amylopectin structure Method S7: Analysis of chloroplast morphology Method S8: Microscopic analysis of amyloplast morphology in developing grain

Figure S6 :
Figure S1: Molecular phylogenetic analysis of ARC6 and PARC6 gene families.Figure S2: TaARC6 gene models and chloroplast morphology of the Ttarc6 mutant.Figure S3: Growth and seed phenotype of Ttparc6 single homeolog mutants.Figure S4: Photosynthesis parameters of Ttparc6 mutant plants.Figure S5: Size distribution of purified starch granules from mature grains of the Ttparc6 single mutants.Figure S6: Plant growth, grain morphology and starch phenotypes of the Ttparc6 mutant expressing the cTPmCherry amyloplast marker.Figure S7: Analysis of properties of TtPARC6-deficient Triticum turgidum starch.Figure S8: Size distribution of starch granules of the Ttarc6 mutant and TtARC6 and TtPARC6 expression patterns during endosperm development.Figure S9: Molecular phylogenetic analysis of PDV1 and PDV2 gene families and ARC6 mutant protein alignment.Table S1: KASP-markers for Ttparc6 and Ttarc6 genotyping.Table S2: Codon optimized DNA sequences of TaPARC1-A1, TaARC6-A1, TaPDV1-1-A1, TaPDV1-2-A1 and TaPDV2-A1.Method S1: Phylogenetic analysis and gene models Method S2: Cloning and construct assembly Method S3: Transient transformation of Nicotiana benthamiana Method S4: Gas exchange Method S5: Starch purification, scanning electron microscopy and polarised light microscopy.Method S6: Total starch quantification, starch composition and amylopectin structure Method S7: Analysis of chloroplast morphology Method S8: Microscopic analysis of amyloplast morphology in developing grain

Figure S7 :
Figure S1: Molecular phylogenetic analysis of ARC6 and PARC6 gene families.Figure S2: TaARC6 gene models and chloroplast morphology of the Ttarc6 mutant.Figure S3: Growth and seed phenotype of Ttparc6 single homeolog mutants.Figure S4: Photosynthesis parameters of Ttparc6 mutant plants.Figure S5: Size distribution of purified starch granules from mature grains of the Ttparc6 single mutants.Figure S6: Plant growth, grain morphology and starch phenotypes of the Ttparc6 mutant expressing the cTPmCherry amyloplast marker.Figure S7: Analysis of properties of TtPARC6-deficient Triticum turgidum starch.Figure S8: Size distribution of starch granules of the Ttarc6 mutant and TtARC6 and TtPARC6 expression patterns during endosperm development.Figure S9: Molecular phylogenetic analysis of PDV1 and PDV2 gene families and ARC6 mutant protein alignment.Table S1: KASP-markers for Ttparc6 and Ttarc6 genotyping.Table S2: Codon optimized DNA sequences of TaPARC1-A1, TaARC6-A1, TaPDV1-1-A1, TaPDV1-2-A1 and TaPDV2-A1.Method S1: Phylogenetic analysis and gene models Method S2: Cloning and construct assembly Method S3: Transient transformation of Nicotiana benthamiana Method S4: Gas exchange Method S5: Starch purification, scanning electron microscopy and polarised light microscopy.Method S6: Total starch quantification, starch composition and amylopectin structure Method S7: Analysis of chloroplast morphology Method S8: Microscopic analysis of amyloplast morphology in developing grain

Figure S8 :
Figure S1: Molecular phylogenetic analysis of ARC6 and PARC6 gene families.Figure S2: TaARC6 gene models and chloroplast morphology of the Ttarc6 mutant.Figure S3: Growth and seed phenotype of Ttparc6 single homeolog mutants.Figure S4: Photosynthesis parameters of Ttparc6 mutant plants.Figure S5: Size distribution of purified starch granules from mature grains of the Ttparc6 single mutants.Figure S6: Plant growth, grain morphology and starch phenotypes of the Ttparc6 mutant expressing the cTPmCherry amyloplast marker.Figure S7: Analysis of properties of TtPARC6-deficient Triticum turgidum starch.Figure S8: Size distribution of starch granules of the Ttarc6 mutant and TtARC6 and TtPARC6 expression patterns during endosperm development.Figure S9: Molecular phylogenetic analysis of PDV1 and PDV2 gene families and ARC6 mutant protein alignment.Table S1: KASP-markers for Ttparc6 and Ttarc6 genotyping.Table S2: Codon optimized DNA sequences of TaPARC1-A1, TaARC6-A1, TaPDV1-1-A1, TaPDV1-2-A1 and TaPDV2-A1.Method S1: Phylogenetic analysis and gene models Method S2: Cloning and construct assembly Method S3: Transient transformation of Nicotiana benthamiana Method S4: Gas exchange Method S5: Starch purification, scanning electron microscopy and polarised light microscopy.Method S6: Total starch quantification, starch composition and amylopectin structure Method S7: Analysis of chloroplast morphology Method S8: Microscopic analysis of amyloplast morphology in developing grain

Figure S9 :
Figure S1: Molecular phylogenetic analysis of ARC6 and PARC6 gene families.Figure S2: TaARC6 gene models and chloroplast morphology of the Ttarc6 mutant.Figure S3: Growth and seed phenotype of Ttparc6 single homeolog mutants.Figure S4: Photosynthesis parameters of Ttparc6 mutant plants.Figure S5: Size distribution of purified starch granules from mature grains of the Ttparc6 single mutants.Figure S6: Plant growth, grain morphology and starch phenotypes of the Ttparc6 mutant expressing the cTPmCherry amyloplast marker.Figure S7: Analysis of properties of TtPARC6-deficient Triticum turgidum starch.Figure S8: Size distribution of starch granules of the Ttarc6 mutant and TtARC6 and TtPARC6 expression patterns during endosperm development.Figure S9: Molecular phylogenetic analysis of PDV1 and PDV2 gene families and ARC6 mutant protein alignment.Table S1: KASP-markers for Ttparc6 and Ttarc6 genotyping.Table S2: Codon optimized DNA sequences of TaPARC1-A1, TaARC6-A1, TaPDV1-1-A1, TaPDV1-2-A1 and TaPDV2-A1.Method S1: Phylogenetic analysis and gene models Method S2: Cloning and construct assembly Method S3: Transient transformation of Nicotiana benthamiana Method S4: Gas exchange Method S5: Starch purification, scanning electron microscopy and polarised light microscopy.Method S6: Total starch quantification, starch composition and amylopectin structure Method S7: Analysis of chloroplast morphology Method S8: Microscopic analysis of amyloplast morphology in developing grain

Figure S1 :
Figure S1: Molecular phylogenetic analysis of ARC6 and PARC6 gene families.The tree with the highest log likelihood (-36614.75) is shown.The percentage of trees out of 1000 bootstraps in which the associated taxa clustered together is shown next to the branches.The tree is drawn to scale, with branch lengths and scale bar representing the number of substitutions per site.Full experimental procedures can be found in Methods S1.

Figure S2 :
Figure S2: Wheat TaARC6 gene models and chloroplast morphology of the Ttarc6 durum wheat mutant.(a) Schematic illustration of the gene models for the canonical transcripts of TaARC6-A1, -B1 and -D1 in bread wheat.Exons are represented as purple boxes and UTRs are represented as white boxes.Mutation sites in K3404 and K2205 are indicated by black lines and the resulting amino acid to stop codon (*) substitutions are annotated.Regions encoding domains are indicated by black horizontal lines (TM: Transmembrane, IMS: Inter Membrane Space).(b-c) Images of mesophyll-cell chloroplasts in the third leaf of Ttarc6 mutants seedlings.Images were acquired using confocal microscopy and are Z-projections of image stacks.Chlorophyll auto-fluorescence of the chloroplasts is shown in cyan.Bars = 10 μm.

Figure S3 :
Figure S3: Growth and seed phenotype of Ttparc6 single homeolog mutants of durum wheat.(a, b) Photographs of 8-week-old Ttparc6 double and single mutants (Ttparc6-1 aabb, Ttparc6-1 aaBB and Ttparc6-1 AAbb) and the corresponding negative segregant (Ttparc6-1 AABB); and Ttparc6 backcrossed double and single mutant (Ttparc6 BC aabb, Ttparc6 BC aaBB and Ttparc6 BC AAbb) and the corresponding negative segregant (Ttparc6 BC AABB) and WT wheat (cv Kronos) plants.Bars = 10 cm.(c-j) Images of mesophyll-cell chloroplasts in the third leaf of Ttarc6 mutant seedlings.Images were acquired using confocal microscopy.Chlorophyll auto-fluorescence in the chloroplasts is shown in cyan.Bars = 10 μm.(k) Photograph of 10 representative mature grains per genotype.Bar = 1 cm.(l) The number of tillers per plant (Tiller no.) of mature plants (n = 6 -19 per genotype).Significant differences between the lines as determined by Kurskal-Wallis One Way ANOVA on the Ranks all pairwise multiple comparison (Dunn's Method) (p ≤ 0.009) are represented by different letters.(m) Total grain weight harvested per plant (in g).Dots represent the total grain weight of individual plants (n = 6-19).Significant differences under a one-way ANOVA and all pairwise multiple comparison procedures (Tukey's test) are indicated with different letters (P ≤ 0.001).(n) Thousand grain weight (TGW) (in g).Dots represent calculated TGW of individual plants (n = 7-19) per genotype.Significant differences under a one-way ANOVA and all pairwise multiple comparison procedures (Tukey's test) are represented by different letters (p ≤ 0.001).(o) Grain size measured as seed area (in mm²).Dots represent measurements for seeds of 3-19 plants per genotype.Significant differences under one-way ANOVA and all pairwise multiple comparison procedures (Tukey's test) are represented by different letters (p ≤ 0.05).(p)Total starch content as % (w/w).3 technical replicates of 2 biological replicates per genotype.Significant differences under a one-way ANOVA and all pairwise multiple comparison procedures (Tukey's test) are represented by different letters (p ≤ 0.05).(q) Amylose content [% of total starch].Dots represent 3 technical replicates of 3 biological replicates.Significant differences between the lines as determined by a one-way ANOVA on the ranks and all pairwise multiple comparison (Tukey's test) are represented by different letters (p ≤ 0.001).For all boxplots, the bottom and top of the box represent the lower and upper quartiles respectively, and the band inside the box represents the median.The ends of the whiskers represent values within 1.5x of the interquartile range, whereas values outside are outliers.

Figure S4 :
Figure S4: Photosynthesis parameters of Ttparc6 durum wheat mutants.Light response and A/Ci curves were measured on the flag leaf of 40-46 day old plants in three separate plants (n = 3).Experimental procedures are in Methods S4.(a-e)Light response curves were measured at ambient CO2 levels (412 μmol m -2 s -1 ).Lines represent the average and ribbons the standard error.The dotted line at A = 30 μmol m -2 s -1 is provided to aid comparison between genotypes.(f) Assimilation rate at ambient light (280 µmol m -2 s -1 ) and high light (2000 µmol m -2 s -1 ).(g) Estimation of Vcmax was extracted from the A/Ci curves using plant ecophys.package(R).(h) Estimation of Jmax was extracted from the A/Ci curves using plant ecophys.package(R).

Figure S5 :
Figure S5: Size distribution of purified starch granules from mature grains of the durum wheat Ttparc6 single mutants.(a-b) Size distribution plots from Coulter counter analysis.The volume of granules at each diameter relative to the total granule volume was quantified using a Coulter counter.Values represent mean (solid line) ± SEM (shading) of three replicates using grains harvested from separate plants.(c-h) Granule size parameters obtained from fitting a log-normal distribution to the B-type granule peak and a normal distribution to the A-type granule peak in the granule size distribution data presented in (a -b).Three biological replicates were analysed: (c, d) A-type granule diameter (in μm).Significant differences under a one-way ANOVA and all pairwise multiple comparison procedures (Tukey's test) are indicated with different letters (p ≤ 0.05).(e, f) B-type granule diameter (in μm).Significant differences under a Kruskal-Wallis one-way ANOVA on ranks and all pairwise multiple comparison procedures (Tukey's test) are indicated with different letters (p ≤ 0.022) for Ttparc6-1 lines.Significant differences under one-way ANOVA and all pairwise multiple comparison procedures (Tukey's test) are indicated with different letters (p ≤ 0.05) for Ttparc6 BC lines.(g, h) B-type granule content by percentage volume.Significant differences under a one-way ANOVA and all pairwise multiple comparison procedures (Tukey's test) are represented with different letters (p ≤ 0.05).(i-m) Scanning Electron Microscopy of purified starch granules.Bars = 10 μm.(n-r) Polarised light microscopy of purified starch granules.Bars = 10 μm.
Figure S5: Size distribution of purified starch granules from mature grains of the durum wheat Ttparc6 single mutants.(a-b) Size distribution plots from Coulter counter analysis.The volume of granules at each diameter relative to the total granule volume was quantified using a Coulter counter.Values represent mean (solid line) ± SEM (shading) of three replicates using grains harvested from separate plants.(c-h) Granule size parameters obtained from fitting a log-normal distribution to the B-type granule peak and a normal distribution to the A-type granule peak in the granule size distribution data presented in (a -b).Three biological replicates were analysed: (c, d) A-type granule diameter (in μm).Significant differences under a one-way ANOVA and all pairwise multiple comparison procedures (Tukey's test) are indicated with different letters (p ≤ 0.05).(e, f) B-type granule diameter (in μm).Significant differences under a Kruskal-Wallis one-way ANOVA on ranks and all pairwise multiple comparison procedures (Tukey's test) are indicated with different letters (p ≤ 0.022) for Ttparc6-1 lines.Significant differences under one-way ANOVA and all pairwise multiple comparison procedures (Tukey's test) are indicated with different letters (p ≤ 0.05) for Ttparc6 BC lines.(g, h) B-type granule content by percentage volume.Significant differences under a one-way ANOVA and all pairwise multiple comparison procedures (Tukey's test) are represented with different letters (p ≤ 0.05).(i-m) Scanning Electron Microscopy of purified starch granules.Bars = 10 μm.(n-r) Polarised light microscopy of purified starch granules.Bars = 10 μm.

Figure S6 :
Figure S6: Plant growth, grain morphology and starch phenotypes of the Ttparc6 durum wheat mutant expressing the cTPmCherry amyloplast marker.(a) Photograph of 10 representative mature grains per genotype.Bar = 1 cm.(b) Photograph of 8-week old transgenic cTPmCherry overexpressing mutant plants and corresponding negative segregants (Taparc6-2 + cTPmCherry aabb and Taparc6-2 + cTPmCherry AABB) and WT (cv Kronos) plants.Bar = 10 cm.(c) The number of tillers per plant (Tiller no.) of mature plants (n = 10 -16 per genotype).Significant differences between the lines as determined by Kurskal-Wallis One Way ANOVA on the Ranks all pairwise multiple comparison (Dunn's Method) (p ≤ 0.007) are represented by different letters.(d) Thousand grain weight (TGW) (in g).Dots represent calculated TGW of individual plants (n = 10 -16 per genotype).There were no significant differences under a one-way ANOVA.(e) Grain size measured as seed area (in mm²).Dots represent measurements for seeds of 10-16 individual plants per genotype.Significant differences under a one-way ANOVA all pairwise multiple comparison procedures (Tukey's Test) are represented by different letters (p ≤ 0.05).(f) Total starch content as % (w/w).3 technical replicates of 2 biological replicates per genotype.Significant differences under a one-way ANOVA and all pairwise multiple comparison procedures (Tukey's Test) are represented by different letters (p ≤ 0.002).(g) Amylose content [% of total starch].Dots represent 3 technical replicates of 3 biological replicates.There were no significant differences between the lines determined by Kurskal-Wallis One Way ANOVA on the Ranks (p=0.317).(h) Size distribution plots from Coulter counter analysis.The volume of granules at each diameter relative to the total granule volume was quantified using a Coulter counter.Values represent mean (solid line) ± SEM (shading) of three biological replicates.(i-k) Scanning Electron Microscopy of purified starch granules from mature grain.Bars = 10 μm.(l-n) Polarised light microscopy of purified starch granules from mature grain.Bars = 10 μm.(o-q) Granule size parameters obtained from fitting a log-normal distribution to the B-type granule peak and a normal distribution to the A-type granule peak in the granule size distribution data presented in (h).Three biological replicates were analysed: (o) A-type granule diameter (in μm).Significant differences under a one-way ANOVA and all pairwise multiple comparison procedures (Tukey's test) are indicated with different letters (p ≤ 0.02).(p) B-type granule diameter (in μm).Significant differences under a one-way ANOVA and all pairwise multiple comparison procedures (Tukey's test) are indicated with different letters (p ≤ 0.001).(q) B-type granule content by percentage volume.Significant differences under a one-way ANOVA and all pairwise multiple comparison procedures (Tukey's test) are represented with different letters (p ≤ 0.001).For all boxplots, the bottom and top of the box represent the lower and upper quartiles respectively, and the band inside the box represents the median.The ends of the whiskers represent values within 1.5x of the interquartile range, whereas values outside are outliers.

Figure S7 :
Figure S7: Analysis of properties of TtPARC6-deficient durum wheat starch.(a) Amylose content [% of total starch] per genotype.Dots represent 3 technical replicates of 3 biological replicates.The bottom and top of the box represent the lower and upper quartiles respectively, and the band inside the box represents the median.The ends of the whiskers represent values within 1.5x of the interquartile range, whereas values outside are outliers.Significant differences between the lines as determined by Kurskal-Wallis One Way ANOVA on the Ranks all pairwise multiple comparison (Tukey's test) (p ≤ 0.001) are indicated with different letters.(b) Amylopectin chain length distributions.Starch was purified from grains and debranched with isoamylase prior to analysis using HPAEC-PAD.The y-axis represents the relative percentage of chains at each DP.Each line represents the average of three replicates per genotype (each using starch from grains harvested from a separate plant), and the shading represents the SEM.(c-d)Rapid Visco Analyser (RVA) analysis of viscosity during gelatinisation.Analyses were conducted using: (c) Purified starch (1.5 g in 25 mL water).Values represent mean (solid line) ± SEM (shading) of three biological replicates, each using starch from grains harvested from a separate plant.The different genotypes produced viscographs that were generally similar, and there was no obvious difference in peak viscosity or the holding strength during cooling.Slight differences between genotypes were observed during cooling (retrogradation) and final viscosity, but these were not consistent for either Ttparc6 double mutants or wild-type controls.(d) Whole flour (5 g in 25 mL water), where flour was produced by pooling a minimum of 3 biological replicates per genotype.The viscographs were more variable among genotypes, and there was no consistent effect that could be attributed to the mutant genotype.

Figure S8 :
Figure S8: Size distribution of starch granules of the durum wheat Ttarc6 mutant and TtARC6 and TtPARC6 expression patterns during endosperm development.(a) Size distribution of purified starch granules from mature grain.The volume of granules at each diameter relative to the total granule volume was quantified using a Coulter counter.Values represent mean (solid line) ± SEM (shading) of three biological replicates.(b-j) Average TPM values (Transcript per million) of the PARC6, ARC6, PDV1-1, PDV1-2 and PDV2 homeologs in the durum wheat endosperm.Values are the mean ± SEM of the n = 3 replicates.Normalised values were retrieved from Chen et al. (2022b).

Figure S9 .
Figure S9.Molecular phylogenetic analysis of PDV1 and PDV2 gene families and ARC6 mutant protein alignment.(a) Schematic representation of multiple protein sequence alignment of wheat and Arabidopsis ARC6 protein sequences.Black lines indicate aligned sequence, gaps represent gaps in alignment.Degree of conservation is represented below, where red indicates high conservation and blue indicates low conservation.Brown arrows represent the region encoding the annotated transmembrane domain.Sequences and mutant sequences were retrieved from Ensembl plants andGlynn et al. (2008).(b) Molecular phylogenetic tree of the PDV gene family was constructed from an amino acid alignment using the Maximum Likelihood method based on the JTT matrix-based model.The tree with the highest log likelihood(-20825.25) is shown.The percentage of trees out of 1000 bootstraps in which the associated taxa clustered together is shown next to the branches.The tree is drawn to scale, with branch lengths and scale bar representing the number of substitutions per site.Full experimental procedures can be found in Methods S1.