Targeted mutation of barley (1,3;1,4)-β-glucan synthases reveals complex relationships between the storage and cell wall polysaccharide content

TY - JOUR

T1 - Targeted mutation of barley (1,3;1,4)-β-glucan synthases reveals complex relationships between the storage and cell wall polysaccharide content

AU - Garcia-Gimenez, Guillermo

AU - Barakate, Abdellah

AU - Smith, Pauline

AU - Stephens, Jennifer

AU - Khor, Shi F.

AU - Doblin, Monika S.

AU - Hao, Pengfei

AU - Bacic, Antony

AU - Fincher, Geoffrey B.

AU - Burton, Rachel A.

AU - Waugh, Robbie

AU - Tucker, Matthew R.

AU - Houston, Kelly

N1 - Funding Information: The authors thank Diane Davidson from the Functional Genomics (FUNGEN) facility, The James Hutton Institute, UK for barley transformation. The authors gratefully acknowledge Dr Alison Roberts and Dr Kathryn Wright, The James Hutton Institute (UK), for technical assistance during immunolabelling experiments and confocal imaging. The authors also thank Edwin R. Lampugnani for discussions. RW and Abdellah Barakate acknowledge support from ERC project 669182 ?SHUFFLE? to RW. KH, PS, JS and RW acknowledge support from BBSRC project BB/J014869/1 the Rural & Environment Science & Analytical Services Division of the Scottish Government. RAB, GBF, MRT, MSD and Antony Bacic acknowledge the support of the Australian Research Council Centre of Excellence in Plant Cell Walls Grant CE1101007, and MSD, PH and Antony Bacic acknowledge the support of a startup grant from La Trobe University. Publisher Copyright: © 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd

PY - 2020/11

Y1 - 2020/11

N2 - Barley (Hordeum vulgare L) grain is comparatively rich in (1,3;1,4)-β-glucan, a source of fermentable dietary fibre that protects against various human health conditions. However, low grain (1,3;1,4)-β-glucan content is preferred for brewing and distilling. We took a reverse genetics approach, using CRISPR/Cas9 to generate mutations in members of the Cellulose synthase-like (Csl) gene superfamily that encode known (HvCslF6 and HvCslH1) and putative (HvCslF3 and HvCslF9) (1,3;1,4)-β-glucan synthases. Resultant mutations ranged from single amino acid (aa) substitutions to frameshift mutations causing premature stop codons, and led to specific differences in grain morphology, composition and (1,3;1,4)-β-glucan content. (1,3;1,4)-β-Glucan was absent in the grain of cslf6 knockout lines, whereas cslf9 knockout lines had similar (1,3;1,4)-β-glucan content to wild-type (WT). However, cslf9 mutants showed changes in the abundance of other cell-wall-related monosaccharides compared with WT. Thousand grain weight (TGW), grain length, width and surface area were altered in cslf6 knockouts, and to a lesser extent TGW in cslf9 knockouts. cslf3 and cslh1 mutants had no effect on grain (1,3;1,4)-β-glucan content. Our data indicate that multiple members of the CslF/H family fulfil important functions during grain development but, with the exception of HvCslF6, do not impact the abundance of (1,3;1,4)-β-glucan in mature grain.

AB - Barley (Hordeum vulgare L) grain is comparatively rich in (1,3;1,4)-β-glucan, a source of fermentable dietary fibre that protects against various human health conditions. However, low grain (1,3;1,4)-β-glucan content is preferred for brewing and distilling. We took a reverse genetics approach, using CRISPR/Cas9 to generate mutations in members of the Cellulose synthase-like (Csl) gene superfamily that encode known (HvCslF6 and HvCslH1) and putative (HvCslF3 and HvCslF9) (1,3;1,4)-β-glucan synthases. Resultant mutations ranged from single amino acid (aa) substitutions to frameshift mutations causing premature stop codons, and led to specific differences in grain morphology, composition and (1,3;1,4)-β-glucan content. (1,3;1,4)-β-Glucan was absent in the grain of cslf6 knockout lines, whereas cslf9 knockout lines had similar (1,3;1,4)-β-glucan content to wild-type (WT). However, cslf9 mutants showed changes in the abundance of other cell-wall-related monosaccharides compared with WT. Thousand grain weight (TGW), grain length, width and surface area were altered in cslf6 knockouts, and to a lesser extent TGW in cslf9 knockouts. cslf3 and cslh1 mutants had no effect on grain (1,3;1,4)-β-glucan content. Our data indicate that multiple members of the CslF/H family fulfil important functions during grain development but, with the exception of HvCslF6, do not impact the abundance of (1,3;1,4)-β-glucan in mature grain.

KW - (1,3;1,4)-β-glucan

KW - CRISPR/Cas9

KW - barley

KW - cell walls

KW - gene editing

UR - https://www.scopus.com/pages/publications/85091685056

U2 - 10.1111/tpj.14977

DO - 10.1111/tpj.14977

M3 - Article

C2 - 32890421

SN - 0960-7412

VL - 104

SP - 1009

EP - 1022

JO - Plant Journal

JF - Plant Journal

IS - 4

ER -