Pattern of Deposition of Cell Wall Polysaccharides and Transcript Abundance of Related Cell Wall Synthesis Genes during Differentiation in Barley Endosperm

Abstract

Immunolabeling, combined with chemical analyses and transcript profiling, have provided a comprehensive temporal and spatial picture of the deposition and modification of cell wall polysaccharides during barley (Hordeum vulgare) grain development, from endosperm cellularization at 3 d after pollination (DAP) through differentiation to the mature grain at 38 DAP. (1→3)-β-d-Glucan appears transiently during cellularization but reappears in patches in the subaleurone cell walls around 20 DAP. (1→3, 1→4)-β-Glucan, the most abundant polysaccharide of the mature barley grain, accumulates throughout development. Arabino-(1-4)-β-d-xylan is deposited significantly earlier than we previously reported. This was attributable to the initial deposition of the polysaccharide in a highly substituted form that was not recognized by antibodies commonly used to detect arabino-(1-4)-β-d-xylans in sections of plant material. The epitopes needed for antibody recognition were exposed by pretreatment of sections with α-l-arabinofuranosidase; this procedure showed that arabino-(1-4)-β-d-xylans were deposited as early as 5 DAP and highlighted their changing structures during endosperm development. By 28 DAP labeling of hetero-(1→4)-β-d-mannan is observed in the walls of the starchy endosperm but not in the aleurone walls. Although absent in mature endosperm cell walls we now show that xyloglucan is present transiently from 3 until about 6 DAP and disappears by 8 DAP. Quantitative reverse transcription-polymerase chain reaction of transcripts for GLUCAN SYNTHASE-LIKE, Cellulose Synthase, and CELLULOSE SYNTHASE-LIKE genes were consistent with the patterns of polysaccharide deposition. Transcript profiling of some members from the Carbohydrate-Active Enzymes database glycosyl transferase families GT61, GT47, and GT43, previously implicated in arabino-(1-4)-β-d-xylan biosynthesis, confirms their presence during grain development.

The Dynamics of Transcript Abundance during Cellularization of Developing Barley Endosperm

“…S5, A, B, C, D, E, and F9), LAM (Supplemental Table S9), and RNAseq (see Fig. 4; Burton et al, 2008;Sreenivasulu et al, 2008;Mayer et al, 2012;Wilson et al, 2012;Thiel et al, 2012). The comparisons showed that it was indeed possible to produce consistent transcript profiles with different methods and to accurately reproduce the conditions of grain development.…”

“…These criteria could be met through comparisons of the progress observed here for barley grain development with previously published studies in which identical conditions were used (Wilson et al, 2006(Wilson et al, , 2012. Moreover, it was possible to compare transcript profiles generated in this work by 44 K custom microarray analyses with data generated by quantitative RT-PCR, 22 K microarrays (Supplemental Fig.…”

“…As the grain fills and expands, normal cell division continues and further differentiation occurs. In developing barley grain, aleurone cells are easily recognizable at 10 DAP and a histologically distinct subaleurone layer of starchy endosperm cells becomes evident by 14 DAP (Becraft and Asuncion-Crabb, 2000;Wilson et al, 2012). The aleurone cells remain triploid and living in the mature grain, but the starchy endosperm cells become polyploid through endoreduplication and finally undergo programmed cell death and are nonliving in the mature grain (Young and Gallie, 2000).…”

“…In addition, we wished to define regulatory genes that might influence these unique cellular and biochemical processes through the identification of transcription factor (TF) genes that are cotranscribed with genes known to mediate cell-wall polysaccharide synthesis. The availability of information on the temporal and spatial appearance of wall polysaccharides during barley endosperm development (Wilson et al, 2006(Wilson et al, , 2012 has allowed the transcript analyses to be linked to morphological data and hence to other key events in grain development.…”

Toward Industrially Feasible Methods for Following the Process of Manufacturing Cellulose Nanofibers

“…Cellulose, the most common biopolymer on Earth (Klemm et al 2005;Wilson et al 2012;Brinchi et al 2013), is composed of β-(14)-D-glucopyranose units (Meshitsuka and Isogai 1995) linearly arranged into elementary fibrils with lateral dimensions of 3 to 5 nm (Meier 1962;Ohad and Danon 1964;Heyn 1969;Blackwell and Kolpak 1975) and lengths of a few microns (Klemm et al 2005(Klemm et al , 2011Siró and Plackett 2010). These fibrils have segments with lower crystallinity and higher reactivity (Blackwell and Kolpak 1975;Henriksson et al 2007b;Pääkkö et al 2007).…”