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An essential role for arabinan breakdown during Arabidopsis seedling establishment

Reference	BB/D521481/1
Principal Investigator / Supervisor	Professor Simon McQueen-Mason
Co-Investigators / Co-Supervisors
Institution	University of York
Department	Biology
Funding type	Research
Value (£)	280,172
Status	Completed
Type	Research Grant
Start date	01/11/2005
End date	30/04/2009
Duration	42 months

Abstract

Oilseed plants such as Arabidopsis store the majority of seed reserves as lipids and proteins and have little or (as is the case in Arabidopsis) no starch. Because they lack starch reserves, it is generally believed that during early seedling establishment the metabolic requirement for sugars is met by the conversion of acetyl units, derived from lipids, to sugars via the glyoxylate cycle and gluconeogenesis. Although starch is the major storage carbohydrate in many seeds, in some plant species cell wall polysaccharides play an important role as storage reserves. We have found that the cell walls of cotyledons in Arabidopsis seeds are highly enriched with arabinose, probably in the form of arabinian, and levels of this sugar decrease markedly during germination and early growth. We have identified two putative endo-arabinanase genes in the Arabidopsis genome, obtained T-DNA insertion mutants for both and produced plants with reduced levels of endo-arabinanase using a targeted post-transcriptional gene silencing approach. Mutant plants show reduced levels of endo-arabinanase activity, supporting the putative enzymatic function of the gene products. These plants share a common phenotype in that they are severely compromised in early seedling establishment, and this defect can be overcome by supplying supplementary sucrose to the germinating seed. Such a phenotype is characteristic of plants carrying mutations affecting storage reserve mobilisation. Preliminary work shows that the levels of storage lipid and its mobilisation are very similar in the growth-arrested mutants to those of wild type plants indicating that they are not compromised in general storage carbon mobilisation. This suggests that there may be a specific requirement for carbohydrates during germination in Arabidopsis that cannot be fully satisfied through gluconeogenesis. An alternative possibility is that arabinan-derived oligosaccharides play a signalling function during germination. The proposed work involves a series of experiments to test the hypothesis that the two putative arabinanase genes are involved in arabinan mobilisation during seedling establishment and to clarify the role of arabinans in the process. Experiments will be undertaken to characterise and compare the structure and composition of cotyledon cell wall polysaccharides and their mobilisation in wild type and mutant plants. In addition, we will compare the profiles of sugar metabolites such as oligosaccharides, sugars and sugar phosphates, in wild type and mutant plants to establish the impacts of the mutations on metabolism. The two genes do not appear to have redundant functions as mutations in both independently cause the sucrose dependant phenotype and this suggests either that they have distinct biochemical specificities, or perhaps are expressed differentially. In order to rest this possibility, recombinant versions of the enzymes will be produced and their enzymatic activity assessed on a range of substrates. We will also test whether each gene can complement the other by expressing each under the control of the others promoter in plants carrying a mutation in the alternate gene and examine the phenotype of double mutants carrying lesions in both genes. Precise expression patterns for the two genes will be determined using promoter-reporter constructs. We will carry out microarray analyses to determine the impact of the mutations on global transcript profiles during seedling establishment. These studies, in combination with the metabolic profiling, will help identify genes likely to be involved in arabinan metabolism or in related signalling pathways, we will isolate mutations in some of these and examine their germination phenotypes.

Summary

Plant cells are surrounded by a complex, polysaccharide-based cell wall that serves many functions in the life of a plant. These functions include the provision of mechanical support, determining the rates and directions of cell growth, providing the sites of cell-to-cell adhesion by which plant tissues are held together, as well as providing the context for much of cell-to-cell communications in plants. In some species, the cell wall also serves as a site for the accumulation of storage reserve polysaccharides that are mobilised during germination. Cell walls are typically composed of cellulose microfibrils embedded in a matrix composed of hemicelluloses and pectins. Arabinans are components of the pectin network but little is known regarding their functional roles. To gain an understanding of the roles of these polymers in cell walls, we have been examining two genes that we believe encode enzymes called arabinanases that degrade arabinans. Arabinans are highly abundant in the cell walls of storage tissues in Arabidopsis where they comprise up to 10 per cent of the dry weight. When we interfere with the normal operation of two putative arabinanase genes in these plants, we find that although seeds can germinate the resulting seedlings cannot grow unless we feed them extra sucrose or glucose. This suggests that, in normal plants, cell wall arabinans may be metabolised to form sugars that can provide energy to the developing seedling until it becomes photosynthetically competent. Another possibility is that arabinan fragments produced by arabinanase action have a signalling role important for normal germination. In the proposed programme of work, we will examine the structure of arabinans in seed storage tissues and look at what happens to these polysaccharides during germination. Both of the putative arabinanase genes appear to be active during germination but mutation in either one independently impairs seedling establishment and this suggests that the two enzymesencoded by these genes may be carrying out a different enzymic reaction to one another. Arabinans may be branched polymers with arabinose sugars bonded at different positions and the two enzymes may thus be active on different bonds in the polymer. We will examine this possibility by looking at the biochemical specificity of the two proteins after producing them in an artificial gene expression system. We will compare the profiles of major sugar metabolites in mutant and wild type plants during germination to help determine the impact of the mutations on metabolism. We will compare the expression levels of almost all of the genes in mutant and wild type plants during germination as this will indicate whether the major effects of the mutations are on metabolic or signalling events, and help identify other genes involved in arabinan processing during germination and isolate mutations in these genes to determine whether they also require sugar supplementation in order to grow normally. Arabinans occur in large quantities in the cell walls of storage tissues of many plant species and it might be that there is a widespread and previously unsuspected role for these polymers in plant germination. The proposed research may therefore prove important to our fundamental understanding of seed physiology and early seedling establishment.

Committee	Closed Committee - Plant & Microbial Sciences (PMS)
Research Topics	Plant Science
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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