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

ABSTRACT: PHS1 is a plastidial α-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. Further, using a double mutant defective in both PHS1 and BGC1, we show that PHS1 is exclusively involved in B-type granule initiation. Grain size and starch content were not affected by the mutations. In leaves, the total starch content and number of starch granules per chloroplast 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 by distinct biochemical mechanisms, where PHS1 plays an exclusive role in B-type granule initiation.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Triticum Turgidum

TISSUE(S): Endosperm

SUBMITTER: Gerhard Saalbach

LAB HEAD: David Seung

PROVIDER: PXD042396 | Pride | 2024-01-22

REPOSITORIES: Pride

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Project description:Hordeum vulgare (barley) hordoindolines (HINs), HINa, HINb1 and HINb2, are orthologous proteins of wheat puroindolines (PINs) that are small, basic, cysteine-rich seed-specific proteins and responsible for grain hardness. Grain hardness, is, next to its protein content, a major quality trait. In barley, HINb is most highly expressed in the mid-stage developed endosperm and is associated with both major endosperm texture and grain hardness. However, data required tounderstand the spatio-temporal dynamics of HIN transcripts and HIN protein regulation during grain filling processes are missing. Using reverse transcription quantitative PCR (RT-qPCR) and proteomics we analyzed HIN transcript and HIN protein abundance from whole seeds (WSs) at four ((6 days after pollination (dap), 10 dap, 12 dap and ≥ 20 dap)) as well as from aleurone, subaleurone and starchy endosperm at two (12 dap and ≥ 20 dap) developmental stages. At the WS level, results from RT-qPCR, proteomics and western blot showed a continuous increase of HIN transcript and HIN protein abundance across these four developmental stages. Miroscopic studies revealed HIN localization mainly at the vacuolar membrane in the aleurone, at protein bodies (PBs) in subaleurone and at the periphery of starch granules in the starchy endosperm. Laser microdissetion (LMD) proteomic analyses identified HINb2 as the most prominent HIN protein in starchy endosperm at ≥ 20 dap. Additionally, our quantification data revealed a poor correlation between transcript and protein levels of HINs in subaleurone during development. Here, we correlated data achieved by RT-qPCR, proteomics and microscopy that reveal different expression and localization pattern of HINs in each layer during barley endosperm development. This indicats a contribution of each tissue to the regulation of HINs during grain filling. The effect of the high protein abundance of HINs in the starchy endosperm and their localization at the periphery of starch granules at late development stages at the high end-product quality is discussed. Understanding the spatio-temporal regulated HINs is essential to improve barley quality traits for high end-product quality, as hard texture of the barley grain is regulated by the ratio between HINb/HINa.

			Action	DRS
	230309_03.msf	Msf
	230309_03.mzML	Mzml
	230309_03.mzid.gz	Mzid
	230309_03.raw	Raw
	230309_04.raw	Raw

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

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