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

Abstract

Starch 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.(2) We 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.(3) SS4 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.(4) We 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.

Loss of PROTEIN TARGETING TO STARCH 2 has variable effects on starch synthesis across organs and species

“…To compare the role of PTST2 in leaves of different grass species, we obtained mutants defective in PTST2 orthologues of barley ( Hordeum vulgare ; Hv ), durum wheat ( Triticum turgidum ; Tt ), and Brachypodium distachyon ( Bd ). The barley ( Hvflo6 ) and wheat mutants ( Ttbgc1 ) are those described in Saito et al (2018) and Hawkins et al (2021) , respectively, whereas the Brachypodium Bdptst2 mutant carrying an insertion in exon 1 was isolated from a T-DNA mutant collection ( Supplementary Fig. S1A–C ).…”

“…Although PTST2 is required for normal granule initiation in the endosperm of wheat and barley ( Chia et al , 2020 ; Hawkins et al , 2021 ), its role in leaves has not yet been explored. In our study, Hvflo6 and Ttbgc1 mutants had distinct starch phenotypes in the leaves.…”

“…The wild-type control used was the unmutagenized Nubet background. The Triticum turgidum (tetraploid durum wheat) mutant was bgc1-3 ( Ttbgc1 ; in cultivar Kronos) ( Hawkins et al , 2021 ). This line has a premature stop codon mutation in the A genome copy and a splice site mutation in the B genome copy.…”

“…Using an EM TP embedding machine (Leica, Milton Keynes, UK), samples were post-fixed in 1% (w/v) OsO 4 in 0.05 M sodium cacodylate for 1 h at room temperature, dehydrated in ethanol, and infiltrated with LR White resin (London Resin Company). LR White blocks were polymerized at 60 °C for 16 h. For light microscopy, semi-thin sections (~0.5 µm) from the blocks were stained with periodic acid–Schiff’s reagent as described in Hawkins et al (2021) and/or 0.5% (w/v) toluidine blue for 30 s, prior to mounting in Histomount for visualization. Sections were imaged with a DM6000 (Leica) or AxioObserver Z1 (Zeiss) microscope.…”

Starch biosynthesis in cereal endosperms: An updated review over the last decade

“…Gradually decreasing the expression of SBEs can lead to the formation of various heterogeneous starch granules, including polygonal, aggregate, elongated, and hollow granules ( Wang et al., 2018 ). Studies in wheat indicated that SSIV and BGC1 are required for proper granule initiation, as TaSSIVb-D mutation led to a decrease in starch granules per chloroplast ( Guo et al., 2017 ), and mutation of either SSIV or BGC1 resulted in multiple initiations of amyloplasts and the formation of compound granules ( Hawkins et al., 2021 ). Mutation of SSIVb (SSIV-2) or SSIIIa did not show even a slight effect on starch granule morphology in the rice endosperm, but their combination resulted in small and spherical starch granules ( Toyosawa et al., 2016 ).…”