Characterisation of Cellulose Synthase Like F6 (CslF6) Mutants Shows Altered Carbon Metabolism in β-D-(1,3;1,4)-Glucan Deficient Grain in Brachypodium distachyon

Abstract

Brachypodium distachyon is a small, fast growing grass species in the Pooideae subfamily that has become established as a model for other temperate cereals of agricultural significance, such as barley (Hordeum vulgare) and wheat (Triticum aestivum). The unusually high content in whole grains of β-D-(1,3;1,4)-glucan or mixed linkage glucan (MLG), considered a valuable dietary fibre due to its increased solubility in water compared with cellulose, makes B. distachyon an attractive model for these polysaccharides. The carbohydrate composition of grain in B. distachyon is interesting not only in understanding the synthesis of MLG, but more broadly in the mechanism(s) of carbon partitioning in cereal grains. Several mutants in the major MLG synthase, cellulose synthase like (CSL) F6, were identified in a screen of a TILLING population that show a loss of function in vitro. Surprisingly, loss of cslf6 synthase capacity appears to have a severe impact on survival, growth, and development in B. distachyon in contrast to equivalent mutants in barley and rice. One mutant, A656T, which showed milder growth impacts in heterozygotes shows a 21% (w/w) reduction in average grain MLG and more than doubling of starch compared with wildtype. The endosperm architecture of grains with the A656T mutation is altered, with a reduction in wall thickness and increased deposition of starch in larger granules than typical of wildtype B. distachyon. Together these changes demonstrate an alteration in the carbon storage of cslf6 mutant grains in response to reduced MLG synthase capacity and a possible cross-regulation with starch synthesis which should be a focus in future work in composition of these grains. The consequences of these findings for the use of B. distachyon as a model species for understanding MLG synthesis, and more broadly the implications for improving the nutritional value of cereal grains through alteration of soluble dietary fibre content are discussed.

Keywords: 3;1; 4)-glucan; Brachypodium distachyon; cell wall; endosperm; starch; β-D-(1.

FIGURE 1

Region within the catalytic domain of BdCSLF6 which was screened for mutations by NGS in the TILLING population. A schematic of the protein sequence includes; predicted transmembrane helices (TMH) (black); the characteristic catalytic domain DD, DxD, xED, and QxxRW motifs (dark grey); plant conserved region (PCR) and class-specific region (CSR); and key conserved structural motifs (light grey). TMH5 is shown in dark grey and inverted commas as it is predicted by modelling to lie adjacent to, rather than traversing, the membrane based on homology to interfacial helix (IF) 3 in the BcsA crystal structure (Morgan et al., 2013) (model of BdCSLF6 shown in Figure 4). Due to lack of homology to BcsA, and protease protection assays showing topology such that the N- and C-termini are both cytosolic (Kim et al., 2015), TMH8 is also not predicted to traverse the membrane (dark grey, inverted commas). The amino acid sequence of the target region is shown, from residue T526 to F679, residues within the CSR (Sethaphong et al., 2013; Dimitroff et al., 2016) indicated with grey shading and the xED and QxxRW motifs in bold. Boxed residues signify mutations identified in the screen that give rise to changes in the amino acid sequence.

FIGURE 2

A656T 7175 mutants after 2 months of growth compared to wildtype Bd21-3. (A) A wildtype Bd21-3 individual (left) compared to a M6 null (centre) and heterozygous individual (right) which has entered the stem elongation phase. (B) Homozygous A656T mutant at 2 months old compared to a wildtype Bd21-3, both of which have reached their final heights, and (C) detail of compact arrangement of leaves yet to emerge from the sheath. (D) Timing of entry into key developmental stages on a modified BBCH scale (Hong et al., 2011); from germination and leaf formation (0–1); elongation of stem and tillering (2–3); heading and booting (4–5); to grain filling and senescence (6–9).

FIGURE 3

Heterologous expression of BdCSLF6 variants present in TILLING lines in N. benthamiana, and analysis of the MLG content in resulting AIR preparations. (A) Representative immunoblot probed with the CSLF6 antibody of microsomal membranes prepared from transformed N. benthamiana leaves with either the native BdCSLF6 or mutant sequence, harvested 2 DPI compared with a no infiltration control (NIC) (40 μg total protein). Dark grey arrow indicates expected size of the full length BdCSLF6 protein at 105 kDa, and light grey arrow shows expected size of the W614* mutant which results in a truncated protein (approximately 67.5 kDa). (B) MLG content as a percentage of the dry weight of AIR preparations from N. benthamiana leaves (left y-axis), normalised to the abundance of wildtype BdCSLF6 protein (the same sample was run on each immunoblot), for variants which produced amounts detectable by HPAEC-PAD (n = 3, ± SE). Ratios between the amount of DP3 and DP4 are shown above each total percentage (± SE) (right y-axis). (C) Example of the HPAEC-PAD elution profile of lichenase digested AIR for variants found to produce MLG compared to a barley flour control (Megazyme); and (D) variants where no lichenase released products were detected. Characteristic oligosaccharide series with DP3, 4, and 5 indicated above peaks (C,D).

FIGURE 4

Homology model of the mutant BdCSLF6 A656T protein showing the variant residue relative to the active site. Model shown in two rotations (A), the relative position of the plasma membrane indicated with grey lines. Last two Glc residues of the nascent polymer, the acceptor, and recently transferred donor Glc are shown for illustrative purposes, which are joined by a β-(1,4)-linkage and shown relative to the UDP donor as captured in the structure described by Morgan et al. (2013). (A) The UDP donor is still bound in the active site, coordinated by a Mg²⁺ ion shown in brown. The region which corresponds to the NGS screen for mutations in the TILLING population is indicated in blue excluding the CSR. (B) Enlarged view of the region of the donor binding pocket in relation to the UDP and nascent chain where the A656T mutation models. The native Ala at position 656 is shown in green, compared to the Thr substitution in red (B). The mutation is present in a flexible region between α helices 9 and 10 and is within 5 Å of several residues in the loop behind, G459, V460, and D461, shown in purple (B).

FIGURE 5

Comparison of MLG content and deposition in mature grains between wild type Bd21-3, Null 7175, and A656T 7175 lines. Fluorescent antibody labelling of MLG on resin sections with identical light intensity and exposure times (A–I). Transverse sections of whole, mature grain at 18–20 DAP from the Bd21-3 (A), Null 7175 (D), and A656T 7175 (G) lines, scale bars shown at 200 μm, nucellar epidermis (ne), and endosperm (e) are indicated. Labelling is evident in the walls of endosperm cells (B,E,H) with adjacent interference contrast brightfield images shown for cellular detail (C,F,I), scale bars show 20 μm. (J) Average cell areas (μm²) of endosperm cells for Bd21-3 (n = 837), Null 7175 (n = 1,261) and A656T 7175 (n = 1,103) (± SE). (K) Average cell wall thickness of endosperm cells from Bd21-3 (n = 21), Null 7175 (n = 40) and A656T 7175 (n = 53).

FIGURE 6

Starch granules in the endosperm of wildtype Bd21-3 grain compared with Null 7175, and A656T 7175, in different sections from the same grains shown for MLG (Figure 5). (A,E,I) TBO staining showing general architecture of grains in transverse sections, collected at maturity (18–20 DAP). Lugol staining for starch in whole grains of Bd21-3 (B), Null 7175 (F), and A656T 7175 (J). Scale for images of whole grains shown as 200 μm. Detail showing starch grains stained with Lugol within cells, indicated with black arrows, at the periphery of the grain including aleurone (al) (C,G,K), and in the central endosperm (e) for Bd21-3 (D), Null 7175 (H), and A656T 7175 (L) lines. A 20 μm scale is shown for the higher magnifications (C,D,G,H,K,L).

FIGURE 7

TEM and immunogold labelling of first internode of stems from all lines upon entering elongation phase at 3 weeks post germination. Cell walls of central pith cells labelled with antibodies for MLG (A,D,G,J), cellulose (CBM3a) (B,E,H,K), and branched xylans (LM11) (C,F,I,L). The wildtype Bd21-3, Null 7175, heterozygous A656T-7175, and homozygous mutant are compared as shown on the left. Scale bars shown as 200 nm.