Biology subjects

Fahy, B.

Publications and source records attributed to Fahy, B..

8 recordsLinked to original sources

bioRxiv2026

Plastidial Phosphorylase (Pho1a) is the dominant glucosyltransferase regulating starch granule initiation in potato tubers

Starch granule initiation involves the extension of maltooligosaccharide primers by glucosyltransferases. STARCH SYNTHASE 4 (SS4) plays a central role in almost all examined plant species, while the plastidial PHOSPHORYLASE 1 (Pho1) also plays an important role in some species, including rice and wheat. In Arabidopsis, an additional enzymatically inactive homolog of SS4, STARCH SYNTHASE 5 (SS5) contributes to starch granule initiation. To elucidate the mechanism of starch granule initiation in potato tubers, we used CRISPR/Cas9 to generate ss4, ss5, and pho1a knockout mutants in the commercial tetraploid 'Clearwater Russet', to systematically investigate their contribution to granule initiation. In ss4 and ss5 tubers, starch granule size and morphology were unaltered relative to the wild type, suggesting that SS4 and SS5 are dispensable for normal granule initiation in potato tubers. In contrast, pho1a tubers had compound starch granules that arose from multiple initiations, greatly reduced granule size, and highly variable granule morphologies. Affinity pull-down to find Pho1a interaction partners identified LIKE EARLY STARVATION (LESV), although yeast 2-hybrid assays did not show direct protein-protein binding. When expressed alone in Nicotiana benthamiana leaves, Pho1a located to the chloroplast stroma, but when expressed alongside LESV, both proteins co-located on starch granules. This co-localisation, alongside the similar accumulation of small starch granules when LESV is knocked out in tubers, suggest a possible functional interaction in planta. These findings position Pho1a as the central glucosyltransferase in starch granule initiation in Clearwater Russet tubers, where it acts together with LESV.

plant biology↗

bioRxiv2026

Two redundant paralogs of MAR1-BINDING FILAMENT PROTEIN affect B-type granule initiation in wheat

Starch synthesis in wheat endosperm involves the initiation of large A-type starch granules during early grain development, followed by small B-type granules in later grain development. It is established that MAR-BINDING FILAMENT-LIKE PROTEIN 1 (MFP1) plays an important role in granule initiation in Arabidopsis chloroplasts, but how it influences A- and B-type initiations in wheat amyloplasts is not known. We discovered that due to a gene duplication in cereals, wheat contains two MFP1 paralogs, MFP1.1 and MFP1.2, which are both expressed in the developing endosperm. We generated a series of durum wheat mutants defective in all homoeologs of either MFP1.1 or MFP1.2, or both. While starch granule size distributions and granule morphology of mfp1.1 and mfp1.2 mutants were identical to those of the wild-type, the mfp1.1 mfp1.2 mutants had fewer, but larger B-type granules - suggesting that the two paralogs play redundant roles in B-type granule initiation. Consistent with this, both paralogs interacted with B-GRANULE CONTENT 1 (BGC1), a key protein required for proper B-type granule initiation in wheat, and both paralogs could partially complement defects in starch initiation in the Arabidopsis mfp1 mutant. Our work demonstrates that MFP1 is required for establishing correct starch granule number in non-photosynthetic amyloplasts, but its role in wheat is limited to B-type granule initiation. One-sentence summaryWheat has two MFP1 paralogs that interact with the granule initiation protein, BGC1 and influence B-type granule initiation in non-photosynthetic amyloplasts of endosperm.

plant biology↗

bioRxiv2026

Wheat mutants lacking Starch Synthase 1 have altered starch composition and cell wall content

Starch Synthase 1 (SS1) participates in the synthesis of amylopectin. To determine its role in hexaploid bread wheat, we selected and combined TILLING mutations for each homoeologue to create two independent SS1-deficient lines. The lines, which have different combinations of ss1 mutations, both lacked SS1 protein. Both lines exhibited mild but significant changes to starch phenotype, including fewer short amylopectin chains, a slight increase in B-type starch granules and a modest increase in amylose content. Lack of SS1 also led to changes in the thermal properties of starch measured by differential scanning calorimetry including reduced enthalpy, and increased gelatinization temperature. Despite the changes to starch properties, the starch contents of the mutant lines compared to wild types were within the normal range, as were grain weight and protein content. However, the concentration of total- and water-extractable arabinoxylan, and MLG, were increased in white flour compared to wild-type controls. HighlightLack of SS1 led to changes in starch molecular structure and thermal behavior. Starch content and grain weight were normal but cell wall polysaccharides in white flour were increased.

plant biology↗

bioRxiv2025

Targeting granule initiation and amyloplast structure to create giant starch granules in wheat

Starch granule size influences the functional, digestive, and processing qualities of starch, but its genetic control in plants is poorly understood. Here, we demonstrate that both space and substrate constraints limit starch granule size in wheat and provide an approach to achieve substantial increases in granule size. Wheat starch typically contains large A-type and small B-type granules. To increase A-type granule size, we explored the effect of mutations in the plastid division component PARALOG OF ARC 6 (PARC6), which increases amyloplast size and therefore the space available for granule growth, and in B-GRANULE CONTENT 1 (BGC1), which reduces the number of granule initiations and thus competition between growing granules for space and substrates. While parc6 and bgc1 single mutants had only modest increases in A-type granule size, the parc6 bgc1 double mutant produced striking giant granules that were more than double the size of typical A-type granules. The increase in granule size in parc6 bgc1 was reproducible in both the glasshouse and field, and had no detectable effect on plant growth, grain size and starch composition or content. We demonstrate that the size increase affects a range of functional properties, including viscosity and pasting temperature. Overall, by targeting both constraints, we created a new class of giant cereal starch that has not been previously observed in nature, with altered physicochemical properties that can be used in food and industrial applications. Significance statementWe provide a major advance in understanding the factors determining starch granule size in plants - a trait that strongly influences starch functionality. Starch granules from cereals are typically smaller than those of most root/tuber crops, rarely exceeding 30 {micro}m. We created a novel cereal starch in wheat that exceeds this size range. By simultaneously increasing amyloplast size and reducing granule initiations, we increased space for granule growth and reduced competition between growing granules for substrates. This resulted in a new class of cereal starch where nearly 50% of the starch volume were in granules greater than 30 {micro}m, and some exceeded 50 {micro}m. These giant granules had altered physicochemical properties that could find novel application in food and industry.

plant biology↗

bioRxiv2025

Tuning starch granule size distributions in durum wheat using genetic variation at a single locus

The size distribution of starch granules in wheat grains influences bread- and pasta-making quality, as well as nutritional properties. Here, we demonstrate that in durum wheat, wide variation in starch granule size distributions can be induced through missense mutations at a single genetic locus encoding the MYOSIN RESEMBLING CHLOROPLAST PROTEIN on chromosome 6A (TtMRC-A1). We isolated 29 independent TILLING mutants in durum cultivar Kronos, each harbouring a different missense mutation that causes an amino acid substitution in the MRC protein. Compared to the B-type granule content of wild-type Kronos (24%), six of the missense lines had significant increases in B-type granule content (33-42%), although not to the extent observed in the mrc-1 mutant (58%) which carries a premature stop codon mutation. Notably, one missense line had significantly decreased B-type granule content (15%), demonstrating that mutations in TtMRC-A1 can achieve both increases and decreases in B-type granule content. In these lines, A-type granule size decreased as B-type granule content increased, and Rapid Visco Analysis on selected lines demonstrated that both B-type granule content and A-type granule size strongly correlated with pasting parameters (e.g., peak viscosity and pasting temperature). However, strong correlations between pasting properties and A-type granule size were still observed after removing most of the B-type granules via sieving, indicating that A-type granule size is the primary contributor to the observed variation in pasting properties. Overall, we demonstrate that mutations at TtMRC-A1 can greatly extend the range of granule size distributions in durum wheat, creating useful alterations in starch properties. Key MessageDifferent missense mutations in TtMRC-A1 can be used to fine tune granule size distributions in durum wheat grains, creating useful alterations in starch properties.

plant biology↗

bioRxiv2023

Initiation of B-type starch granules in wheat endosperm requires the plastidial α-glucan phosphorylase PHS1

PHS1 is a plastidial -glucan phosphorylase that can elongate and degrade maltooligosaccharides (MOS), but its exact physiological role in plants is poorly understood. Here, we discover a specialised role of PHS1 in establishing the unique bimodal characteristic of starch granules in the wheat endosperm. Wheat endosperm contains large A-type granules that initiate at early grain development, and small B-type granules that initiate in later grain development. We demonstrate that PHS1 interacts with BGC1 - a carbohydrate-binding protein essential for normal B-type granule initiation. Mutants of tetraploid durum wheat deficient in all homeologs of PHS1 had normal A-type granules, but fewer and larger B-type granules. Grain size and starch content were not affected by the mutations. Further, by assessing granule numbers during grain development in the phs1 mutant, and using a double mutant defective in both PHS1 and BGC1, we demonstrate that PHS1 is exclusively involved in B-type granule initiation. The total starch content and number of starch granules per chloroplast in leaves were not affected by loss of PHS1, suggesting that its role in granule initiation in wheat is limited to the endosperm. We therefore propose that the initiation of A- and B-type granules occur via distinct biochemical mechanisms, where PHS1 plays an exclusive role in B-type granule initiation.

plant biology↗

bioRxiv2022

Contrasting roles of the wheat MRC in promoting starch granule initiation in leaves and repressing the onset of B-type granule initiation in the endosperm

The spatial and temporal patterns by which starch granules initiate vary greatly between species and organs, but molecular factors that contribute to these diverse patterns are poorly understood. We reveal distinct organ-specific roles of the MYOSIN-RESEMBLING CHLOROPLAST PROTEIN (MRC) in regulating granule initiation in the endosperm and leaves of wheat. We isolated three independent TILLING mutants of tetraploid wheat (Triticum turgidum cv. Kronos) with premature stop or missense mutations in the A-genome homeolog, which we showed to be the only active homeolog in tetraploid wheat due to a disruption of the B-genome homeolog. Wheat endosperm contains both large A-type granules initiated during early grain development, and small B-type granules that initiate about 10 - 15 days later. The mrc mutants had significantly smaller A-type granules and a higher relative volume of B-type granules in the endosperm than the wild type. Whereas B-type granules initiated 15 - 20 days post anthesis (dpa) in the wild-type, they appeared as early as 10 dpa in the mrc-1 mutant, suggesting a role for MRC in suppressing B-type granule initiation during early grain development. By contrast, MRC promotes granule initiation in leaves: mutants carrying premature stop mutations in MRC had fewer granules per chloroplast than the wild type. These contrasting roles of MRC among wheat organs provide new insight into functional diversification of granule initiation proteins, and suggest that they may facilitate the diverse patterns of granule initiation observed across species and organs.

plant biology↗

bioRxiv2021

STARCH SYNTHASE 4 is required for normal starch granule initiation in amyloplasts of wheat endosperm

O_LIStarch granule initiation is poorly understood at the molecular level. The glucosyltransferase, STARCH SYNTHASE 4 (SS4), plays a central role in granule initiation in Arabidopsis leaves, but its function in cereal endosperms is unknown. We investigated the role of SS4 in wheat, which has a distinct spatiotemporal pattern of granule initiation during grain development. C_LIO_LIWe generated TILLING mutants in tetraploid wheat (Triticum turgidum) that are defective in both SS4 homoeologs. The morphology of endosperm starch was examined in developing and mature grains. C_LIO_LISS4 deficiency led to severe alterations in endosperm starch granule morphology. During early grain development, while the wild type initiated single A-type granules per amyloplast, most amyloplasts in the mutant formed compound granules due to multiple initiations. This phenotype was similar to mutants deficient in B-GRANULE CONTENT 1 (BGC1). SS4 deficiency also reduced starch content in leaves and pollen grains. C_LIO_LIWe propose that SS4 and BGC1 are required for the proper control of granule initiation during early grain development that leads to a single A-type granule per amyloplast. The absence of either protein results in a variable number of initiations per amyloplast and compound granule formation. C_LI

plant biology↗