GRDC Functional Genomics Program: "Growth and End-use Quality of Cereals"
1 Plant Cell Biology Research Centre, School of Botany, The University of Melbourne, VIC 3010
2 Department of Plant Science, Adelaide University, Waite Campus, Glen Osmond SA 5064
The plant cell wall is a complex, diverse and dynamic entity, that changes throughout the processes of cell division, growth and differentiation. While cell walls are of central importance in plant growth and development, they also represent the single major determinant of plant structure, resistance to pathogens, quality of plant-based foods and the properties of fibres and fuels.
We are using barley suspension culture cells to study cell wall synthesis during wall regeneration by protoplasts and elongating coleoptiles to study wall expansion. We have focussed our efforts on the CesA/Csl superfamily as likely candidate genes for the major cell wall polysaccharide synthases.
To date we have identified CesAs and Csls belonging to the C and D family and will describe their expression patterns in barley tissues.
There are two types of cell walls found in higher plants. Type I cell walls are found in dicots, gymnosperms and most monocots, and type II walls are found in grasses and their close relatives (Bacic et al., 1988; Carpita and Gibeaut, 1993; Carpita, 1996). Cellulose, a linear (1,4)-β-D-glucan,
is the main load-bearing polysaccharide in both types of walls. In the type II wall, noncellulosic-polysaccharides such as glucuronoarabinoxylans and ‘mixed-linkage’ (1,3;1,4)-β-D-glucans form a matrix that holds the cellulose microfibrils together. These polysaccharides are only found in type II walls, and even though they make up a relatively minor part of the total weight of cereal grains, they have a surprisingly large influence on the quality of grain.
The structural features of the polysaccharides in type II walls have been well characterised; however little is known about their synthesis and deposition. This is primarily because to date no cell wall polysaccharide synthase has been isolated. In plants, the first step in identifying the catalytic sub unit of cellulose synthase was the cloning of candidate cDNAs from a cotton fibre cDNA library (GhCesA1 and GhCesA2). The encoded protein sequences contained three domains with similarity to bacterial cellulose synthase genes (Pear et al., 1996). Cutler and Somerville (1997) and Saxena and Brown (1997) described a family of related higher plant genes that showed similarity to the CesA sequences. These genes, now known as the Csl (cellulose synthase-like) genes, have been classified into 6 subfamilies, named CslA, CslB, CslC, CslD, CslE and CslG. They speculated that the six classes of Csl genes may either contribute to the synthesis of non-cellulosic polysaccharides, or they may also have a role in the synthesis of cellulose (Richmond and Somerville, 2000).
The aim of this study is to clone CesA and Csl genes from the C and D families in barley. Representatives from these families will be analysed to determine expression and possible function.
Initially, gene isolation was achieved by PCR on barley cDNA preparations and screening of a barley tissue culture cDNA library. Primers were designed to conserved regions of alignments of CesA and Csl sequences such as the D,D,D, QxxRW motifs from other plant species, or to particular barley ESTs. Information on the CesA, CslC and CslD gene sequences was obtained from the website maintained by Dr. Todd Richmond (http://cellwall.stanford.edu). Isolated fragments were cloned and sequenced, and confirmed as CesA, CslC or CslD by BLAST searches. Genes are being isolated by screening a barley BAC library and sequencing the genomic clone. RFLP analysis of barley mapping populations from the breeding program at the University of Adelaide will be used to identify the location of these genes in the barley genome.
Northern blotting and PCR have been used to identify tissues expressing isolated genes. Recombinant peptides made from fragments of the CslC and CslD sequences have been expressed in Escherichia coli, and were used to generate polyclonal antibodies in rabbits. These antibodies will enable immunolocalisation of Csl proteins in vivo.
Functional analysis will involve a range of systems. These will include gain-of-function by transformation of CesA and Csl genes into different plant species or yeast cells. Loss-of-function in barley will also be achieved by gene silencing techniques such as dsRNAi.
We have isolated more than 7 CesA genes, 3 CslC genes and 2 CslD genes from barley. Some of these genes are full length, and efforts to complete the missing sequences from other genes are continuing.
Northern analysis has been unsuccessful, but RT-PCR has revealed expression sites of the genes in a variety of barley tissues, including stem, root, spike and suspension culture cells.
Rabbit antibodies have been generated to CslC and CslD recombinant E. coli proteins. Immunomicroscopy will be used to determine the subcellular localisation of proteins in barley protoplasts and elongating coleoptiles.
The work presented here is at an early stage, but the potential is exciting. The isolation of CesA and related genes from barley will allow comparison with the corresponding gene families in other plant species. The comparison with dicots species such as Arabidopsis will be especially interesting, because important differences between the enzymes involved in the synthesis of type I and type II cell walls may become apparent.
This work is supported by a Functional Genomics grants from the Grains Research and Development Corporation.
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2. Carpita NC (1996) Structure and biogenesis of the cell wall of grasses. Annual Review of Plant Physiology and Plant Molecular Biology, 47:445-476.
3. Carpita NC, and Gibeaut DM (1993) Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. Plant Journal, 3:1-30.
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4. Pear JR, Kawagoe Y, Schreckengost WE, Delmer DP, and Stalker DM (1996) Higher plants contain homologs of the bacterial celA genes encoding the catalytic subunit of cellulose synthase. PNAS USA, 93(22):12637-12642.
5. Richmond TA, and Somerville CR (2000) The Cellulose Synthase Superfamily. Plant Physiology, 124(2): 495-498.
6. Saxena IM and Brown, RM (1997) Identification of cellulose synthase(s) in higher plants: sequence analysis of processive beta-glycosyltransferases with the common motif 'D,D,D35Q(R,Q)XRW'. Cellulose, 4:33-49.