The majority of organs in plants are not established until after germination, when pluripotent stem cells in the growing apices give rise to daughter cells that proliferate and subsequently differentiate into new tissues and organ primordia. This remarkable capacity is not only restricted to the meristem, since maturing cells in many organs can also rapidly alter their identity depending on the cues they receive. One general feature of plant cell differentiation is a change in cell wall composition at the cell surface. Historically, this has been viewed as a downstream response to primary cues controlling differentiation, but a closer inspection of the wall suggests that it may play a much more active role. Specific polymers within the wall can act as substrates for modifications that impact receptor binding, signal mobility, and cell flexibility. Therefore, far from being a static barrier, the cell wall and its constituent polysaccharides can dictate signal transmission and perception, and directly contribute to a cell’s capacity to differentiate. In this review, we re-visit the role of plant cell wall-related genes and polysaccharides during various stages of development, with a particular focus on how changes in cell wall machinery accompany the exit of cells from the stem cell niche.

The aleurone is a critical component of the cereal seed and is located at the periphery of the starchy endosperm. During germination, the aleurone is responsible for releasing hydrolytic enzymes that degrade cell wall polysaccharides and starch granules, which is a key requirement for barley malt production. Inter- and intra-species differences in aleurone layer number have been identified in the cereals but the significance of this variation during seed development and germination remains unclear. In this study, natural variation in mature aleurone features was examined in a panel of 33 Hordeum vulgare (barley) genotypes. Differences were identified in the number of aleurone cell layers, the transverse thickness of the aleurone and the proportion of aleurone relative to starchy endosperm. In addition, variation was identified in the activity of hydrolytic enzymes that are associated with germination. Notably, activity of the free fraction of β-amylase (BMY), but not the bound fraction, was increased at grain maturity in barley varieties possessing more aleurone. Laser capture microdissection (LCM) and transcriptional profiling confirmed that HvBMY1 is the most abundant BMY gene in developing grain and accumulates in the aleurone during early stages of grain fill. The results reveal a link between molecular pathways influencing early aleurone development and increased levels of free β-amylase enzyme, potentially highlighting the aleurone as a repository of free β-amylase at grain maturity.

Summary: In cereal species, grain size is influenced by growth of the ovule integuments (seed coat), the spikelet hull (lemma and palea) and the filial endosperm. Whether a highly conserved ovule tissue, the nucellus, has any impact on grain size has remained unclear. Immunolabelling revealed that the barley nucellus comprises two distinct cell types that differ in terms of cell wall homogalacturonan (HG) accumulation. Transcriptional profiling of the nucellus identified two pectin methylesterase (PME) genes, OVULE PECTIN MODIFIER 1 (OPM1) and OPM2, which are expressed in the unfertilized ovule but absent from the seed. Ovules from an opm1 opm2 mutant and plants expressing an ovule‐specific pectin methylesterase inhibitor (PMEI), exhibit reduced HG accumulation. This results in changes to ovule cell size and shape and ovules that are longer than wild‐type (WT) controls. At grain maturity, this is manifested as significantly longer grain. These findings indicate that cell wall composition during ovule development acts to limit ovule and seed growth. The investigation of ovule PME and PMEI activity reveals an unexpected role of maternal tissues in controlling grain growth before fertilization, one that has been lacking from models exploring improvements in grain size.

In cereal grain, sucrose is converted into storage carbohydrates: mainly starch, fructan, and mixed-linkage (1,3;1,4)-β-glucan (MLG). Previously, endosperm-specific overexpression of the HvCslF6 gene in hull-less barley was shown to result in high MLG and low starch content in mature grains. Morphological changes included inwardly elongated aleurone cells, irregular cell shapes of peripheral endosperm, and smaller starch granules of starchy endosperm. Here we explored the physiological basis for these defects by investigating how changes in carbohydrate composition of developing grain impact mature grain morphology. Augmented MLG coincided with increased levels of soluble carbohydrates in the cavity and endosperm at the storage phase. Transcript levels of genes relating to cell wall, starch, sucrose, and fructan metabolism were perturbed in all tissues. The cell walls of endosperm transfer cells (ETCs) in transgenic grain were thinner and showed reduced mannan labelling relative to the wild type. At the early storage phase, ruptures of the non-uniformly developed ETCs and disorganization of adjacent endosperm cells were observed. Soluble sugars accumulated in the developing grain cavity, suggesting a disturbance of carbohydrate flow from the cavity towards the endosperm, resulting in a shrunken mature grain phenotype. Our findings demonstrate the importance of regulating carbohydrate partitioning in maintenance of grain cellularization and filling processes.

The female germline of flowering plants develops within a niche of sporophytic (somatic) ovule cells, also referred to as the nucellus. How niche cells maintain their own somatic developmental programme, yet support the development of adjoining germline cells, remains largely unknown. Here we report that MADS31, a conserved MADS-box transcription factor from the B-sister subclass, is a potent regulator of niche cell identity. In barley, MADS31 is preferentially expressed in nucellar cells directly adjoining the germline, and loss-of-function mads31 mutants exhibit deformed and disorganized nucellar cells, leading to impaired germline development and partial female sterility. Remarkably similar phenotypes are observed in mads31 mutants in wheat, suggesting functional conservation within the Triticeae tribe. Molecular assays indicate that MADS31 encodes a potent transcriptional repressor, targeting genes in the ovule that are normally active in the seed. One prominent target of MADS31 is NRPD4b, a seed-expressed component of RNA polymerase IV/V that is involved in epigenetic regulation. NRPD4b is directly repressed by MADS31 in vivo and is derepressed in mads31 ovules, while overexpression of NRPD4b recapitulates the mads31 ovule phenotype. Thus, repression of NRPD4b by MADS31 is required to maintain ovule niche functionality. Our findings reveal a new mechanism by which somatic ovule tissues maintain their identity and support germline development before transitioning to the post-fertilization programme.

We profiled the grain oligosaccharide content of 154 two-row spring barley genotypes and quantified 27 compounds, mainly fructans, that exhibited differential abundance. Clustering revealed two major profile groups where the ‘high’ set contained greater amounts of sugar monomers, sucrose and overall fructans, but lower fructosylraffinose. GWAS identified a significant association for the variability of two fructan types; neoseries-DP7 and inulin-DP9 which showed increased strength when a compound-ratio GWAS was applied. Gene models within this region included five fructan biosynthesis genes, of which three ( fructan:fructan 1-fructosyltransferase , s ucrose:sucrose 1-fructosyltransferase , and s ucrose:fructan 6-fructosyltransferase) have already been described. The remaining two, 6(G)-fructosyltransferase and vacuolar invertase1 have not previously been linked to fructan biosynthesis in barley and showed expression patterns distinct from those of the other three genes, including exclusive expression of 6(G)-fructosyltransferase in outer grain tissues at the storage phase. From exome capture data several SNPs related to inulin- and neoseries-type fructan variability were identified in fructan:fructan 1-fructosyltransferase and 6(G)-fructosyltransferase genes Co-expression analyses uncovered potential regulators of fructan biosynthesis including transcription factors. Our results provide evidence for the distinct biosynthesis of neoseries-type fructans during barley grain maturation plus new gene candidates likely involved in the differential biosynthesis of the various fructan types. Highlight: Grain fructan profiles in barley are more complex than previously expected and variations in a diversity panel relate to a genomic region where fructan biosynthesis genes cluster.