Biology subjects

Seung, D.

Publications and source records attributed to Seung, D..

18 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

A novel TaNF-YC10-TaNF-YB1-TabHLH95 module coordinates starch biosynthesis in wheat endosperm

Wheat grain weight and flour quality largely depend on starch biosynthesis, yet the mechanisms by which transcription factors coordinate this process remain poorly understood. In this study, using an integrative strategy that combines genome-wide association analysis with yeast two-hybrid library screening, we identify TaNF-YC10, a Nuclear Factor Y transcription factor, as a positive regulator of starch accumulation in the wheat endosperm. Loss of TaNF-YC10 reduces starch content and alters starch granule size distribution, whereas overexpression enhances starch accumulation and increases grain weight. TaNF-YC10 binds and activates core starch biosynthetic-related genes, including AGPL1, GBSS1, YUC11, and NF-YB7, and forms higher-order transcriptional complexes with TaNF-YB1 and TabHLH95 to coordinate multiple regulatory pathways. TaNF-YC10-A1-Hap2 is associated with higher starch content and thousand grain weight and has been selected during wheat breeding in China. Collectively, our findings establish TaNF-YC10 as a pivotal transcriptional hub in starch regulation and highlight its potential as a target for genetic improvement of grain yield in wheat.

plant biology↗

bioRxiv2025

Diploid potato lines for the study and improvement of starch metabolism and structure

Diploid potato breeding enables faster genetic improvement via selection against deleterious alleles in inbred lines, unlike breeding by intercrossing tetraploid varieties. Starch is the major source of calories in potato tubers, but the starch properties of diploid lines have rarely been reported. In this study, we provide a comprehensive characterisation of tuber and starch properties in two diploid lines that are early isolates from the Solynta breeding program, B26 and B100, and their F1 hybrids. B100 produced fewer, but larger tubers compared to B26, and both diploid lines produced tubers that are smaller than the tetraploid variety, Clearwater Russet. The low tuber yield of B100 correlates with its high self-compatibility and fruit production. Pruning of fruits in B100 significantly increased total tuber yield per plant by stimulating more tuber initiations, but had no effect on average tuber weight, starch content or starch structure. Among the diploid, hybrid and tetraploid lines examined, there were no differences in the total starch content of tubers. Although amylopectin structure and amylose content were similar between the two diploid lines and the tetraploid comparison, B26 had elevated levels of resistant starch and a striking elongated granule morphology. Our results showcase the variation in source-sink relations and starch structure in diploid potato breeding material, demonstrating their potential for research into the genetics underpinning metabolic and quality traits.

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

Starch synthesis in Brachypodium distachyon endosperm occurs in dynamic, connected amyloplast compartments

The morphology of starch granules is a major determinant of the functional and nutritional properties of starch and is highly variable among cereal species. Much of this morphological variation stems from differences in the spatial and temporal patterns of starch granule initiation in amyloplasts during grain development. Simple granules are thought to arise from a single initiation per amyloplast (e.g., in Brachypodium distachyon), whereas compound granules develop from multiple initiations per amyloplast (e.g., in rice). Using live-cell imaging in transgenic Brachypodium lines expressing a fluorescent amyloplast reporter construct, we discovered that simple-type starch granules in Brachypodium can arise from multiple initiations per amyloplast. The amyloplasts showed dynamic changes in their structure, the formation of stromules, and the movement of both the amyloplast within the cell and the starch granules within the amyloplasts. Our results suggest complex and pleiomorphic amyloplast organisation and mobility that can influence starch granule formation and morphology. This goes beyond the existing one granule, one amyloplast model for simple-type granules, and advances our understanding of both amyloplast biogenesis and granule formation.

plant biology↗

bioRxiv2025

LIKE EARLY STARVATION is involved in the regulation of starch initiation in potato (Solanum tuberosum cv. Desiree) tubers

Starch initiation has been intensively investigated. In tubers of Solanum tuberosum, it is controlled by a heteromultimeric isoamylase complex composed of ISA1 and ISA2. Moreover, two non-catalytic starch binding proteins (i.e., ESV1 and LESV) regulate leaf starch metabolism in Arabidopsis. Both proteins interact with starch glucans through a C-terminal antiparallel {beta}-sheet domain and differ by the presence of a 115 amino acid N-terminal overhang in LESV. Here we report the CRIPSR/Cas9 inactivation of LESV or ESV1 in potato and the characterization of the respective tuber starches. Starch from esv1 mutants was unaffected whereas granule diameter was severely reduced in tubers of lesv plants. This was negatively correlated to the number of granules, coherent with an altered starch initiation phenotype. Strikingly, scanning electron microscopy of purified starches revealed that lesv plants phenocopy isa1 and isa2 antisense lines described 20 years ago. A physical interaction between LESV and ISA1 was previously demonstrated in rice. Here, we confirmed this interaction in potato using yeast two-hybrid assays, providing molecular evidence for their functional association in potato. This interaction supports the hypothesis that LESV acts in concert with the ISA1/ISA2 complex, likely regulating the early steps of glucan organization required for proper starch granule formation.

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↗

bioRxiv2024

A curve fitting method for analysing starch granule size distributions in cereals

Background and ObjectivesThe size distribution of starch granules is an important factor determining functional and nutritional properties of starch. However, a simple, standardised method for their analysis is lacking. Here, we developed an approach for estimating granule size parameters using a Python script that fits curves to volumetric granule size distributions generated using a Coulter counter. FindingsThe bimodal size distribution of starch from most wheat and barley cultivars could be best described with a mixed distribution curve. A log-normal distribution was fitted to the small B-type granules, and a normal distribution was fitted to the large A-type granules, allowing estimation of their relative abundance and size parameters, despite their overlapping size distributions. However, the optimal fitting is altered in wheat mutants with large perturbations in B-type granule content. In maize and rice, which have unimodal granule size distributions, size parameters were calculated by fitting a single normal distribution. ConclusionsCurve fitting is an effective approach for estimating starch granule size parameters in diverse cereals, particularly the Triticeae with A- and B-type granules. Significance and noveltyWe provide new tools and guidelines for the quantitative analysis of granule size in cereals.

plant biology↗

bioRxiv2023

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

The pyrenoid is a chloroplastic microcompartment in which most algae and some terrestrial plants condense the primary carboxylase, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) as part of a CO2-concentrating mechanism that improves the efficiency of CO2 capture. Engineering a pyrenoid-based CO2-concentrating mechanism (pCCM) into C3 crop plants is a promising strategy to enhance yield capacities and resilience to the changing climate. Many pyrenoids are characterized by a sheath of starch plates that is proposed to act as a barrier to limit CO2 diffusion. Recently, we have reconstituted a phase-separated proto-pyrenoid Rubisco matrix in the model C3 plant Arabidopsis thaliana using proteins from the alga with the most well studied pyrenoid, Chlamydomonas reinhardtii (1). Here we describe the impact of introducing the Chlamydomonas proteins StArch Granules Abnormal 1 (SAGA1) and SAGA2, which are associated with the regulation of pyrenoid starch biogenesis and morphology. We show that SAGA1 localizes to the proto-pyrenoid in engineered Arabidopsis plants, which results in the formation of atypical spherical starch granules enclosed within the proto-pyrenoid condensate and adjacent plate-like granules that partially cover the condensate, but without modifying the total amount of chloroplastic starch accrued. Additional expression of SAGA2 further increases the proportion of starch synthesised as adjacent plate-like granules that fully encircle the proto-pyrenoid. Our findings pave the way to assembling a diffusion barrier as part of a functional pCCM in vascular plants, whilst also advancing our understanding of the roles of SAGA1 and SAGA2 in starch sheath formation and opening novel avenues for engineering starch morphology.

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↗

bioRxiv2023

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

Starch granule morphology is a major factor determining the functional and nutritional properties of starch. Here, we reveal amyloplast structure plays an important role in starch granule morphogenesis in wheat endosperm. Wheat amyloplasts contain large discoid A-type granules and small spherical B-type granules. We isolated a mutant in durum wheat defective in the plastid division protein PARC6, which had increased plastid size in both leaves and endosperm. Endosperm amyloplasts of the mutant contained more A- and B-type granules than those of the wild type. In mature grains, the mutant had larger A- and B-type granules than the wild type, and its A-type granules had a highly aberrant, lobed surface. This defect in granule morphology was already evident at early stages of grain development, when granule size was identical between the mutant and the wild type, and occurred without obvious alterations in starch polymer structure and composition. Plant growth and photosynthetic efficiency, as well as the size, number and starch content of grains, were not affected in the Ttparc6 mutants despite the large changes in plastid size. Interestingly, mutation of the PARC6 paralog, ARC6, in durum wheat did not increase plastid or starch granule size. We suggest this is because 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 propose that amyloplast compartment size and available stromal volume play important roles in determining starch granule size, shape and number per amyloplast.

plant biology↗

bioRxiv2022

Development of a high-throughput starch digestibility assay reveals wide variation among the A. E. Watkins wheat landrace collection

Breeding for less digestible starch in wheat can improve the health impact of bread and other wheat foods. Based on an established in vitro starch digestibility assay by Edwards et al. (2019) we developed a high-throughput assay to measure starch digestibility in hydrothermally processed samples for use in forward genetic approaches. Digestibility of purified starch from maize and wheat was measured using both methods and produced comparable results. Using the high-throughput assay, we estimated starch digestibility of 118 wheat landraces from the core Watkins collection and found wide variation across lines and elite UK varieties, (20% to 40% and 31% to 44% starch digested after 90 minutes respectively). Sieved flour fractions and purified starch for selected lines showed altered starch digestibility profiles compared with wholemeal flour, suggesting that matrix properties of flour rather than intrinsic properties of starch granules conferred the low starch digestibility observed.

biochemistry↗

bioRxiv2022

AtFZL is required for correct starch granule morphology in Arabidopsis chloroplasts

The control of starch granule number and morphology in plastids is poorly understood. Here, we demonstrate that AtFZL, a protein involved in thylakoid membrane organisation, is required for correct starch granule morphology in Arabidopsis. Leaves of mutants lacking AtFZL had the same starch content as wild-type leaves, but their starch granules were smaller and had a distinct, uneven surface morphology. Most chloroplasts in the mutant were larger than those of the wild type. However, the difference in chloroplast size could not explain the difference in granule size and shape in the Atfzl mutants, since other mutants with larger chloroplasts than the wild type (arc mutants) had granules that were similar in size and shape to wild-type granules. As reported previously, the Atfzl mutant had aberrant thylakoid organisation. We found that this phenomenon was particularly pronounced in regions surrounding starch granules. The location of the thylakoid-bound granule initiation protein MFP1 was unaffected in the Atfzl mutant. We propose that AtFZL affects starch granule size and shape by influencing thylakoid organisation at the periphery of starch granules. Our results are consistent with an important role for thylakoid architecture in determining granule morphology.

plant biology↗

bioRxiv2022

Gene expression profile of the developing endosperm in durum wheat provides insight into starch biosynthesis

Durum wheat (Triticum turgidum subsp. durum) is widely grown for pasta production, and more recently, is gaining additional interest due to its resilience to warm, dry climates and its use as an experimental model for wheat research. Like in bread wheat, the starch and protein accumulated in the endosperm during grain development are the primary contributors to the calorific value of durum grains. To enable further research into endosperm development and storage reserve synthesis, we generated a high-quality transcriptomics dataset from developing endosperms of variety Kronos, to complement the extensive mutant resources available for this variety. Endosperms were dissected from grains harvested at eight timepoints during grain development (6 to 30 days post anthesis (dpa)), then RNA sequencing was used to profile the transcriptome at each stage. The largest changes in gene expression profile were observed between the earlier timepoints, prior to 15 dpa. We detected a total of 29,925 genes that were significantly differentially expressed between at least two timepoints, and clustering analysis revealed nine distinct expression patterns. We demonstrate the potential of our dataset to provide new insights into key processes that occur during endosperm development, using starch metabolism as an example. Overall, we provide a valuable resource for studying endosperm development in this increasingly important crop species.

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↗

bioRxiv2019

A carbohydrate-binding protein, FLOURY ENDOSPERM 6 influences the initiation of A- and B-type starch granules in wheat.

Previously, we identified a quantitative trait locus on the group 4 chromosomes of Aegilops and bread wheat that controls B-type starch-granule content. Here, we identify a candidate gene by fine-mapping in Aegilops and confirm its function using wheat TILLING mutants. This gene is orthologous to the FLOURY ENDOSPERM 6 (FLO6) gene of rice and barley and the PTST2 gene of Arabidopsis. In Triticeae endosperm, reduction in the gene dose of functional FLO6 alleles results in reduction, or loss, of B-granules. This is due to repression of granule initiation in late-grain development, but has no deleterious impact on the synthesis of A-granules. The complete absence of functional FLO6, however, results in reduced numbers of normal A-type and B-type granules and the production of highly-abnormal granules that vary in size and shape. This polymorphous starch seen in a wheat flo6 triple mutant is similar to that observed in the barley mutant Franubet. Analysis of Franubet (fractured Nubet) starch suggests that the mutant A-granules are not fractured but compound, due to stimulation of granule initiation in plastids during early-grain development. Thus, in different situations in Triticeae, FLO6 either stimulates or represses granule initiation.

plant biology↗