Impact of Altered Cell Wall Composition on Saccharification Efficiency in Stem Tissue of Arabidopsis RABA GTPase-Deficient Knockout Mutants

Impact of Altered Cell Wall Composition on Saccharification Efficiency in Stem Tissue of Arabidopsis RABA GTPase-Deficient Knockout Mutants Daniel Lunn 1 Roger Ibbett 1 Gregory A. Tucker 1 Grantley W. Lycett 1 0 ) School of Biosciences, University of Nottingham , Sutton Bonington Campus, Loughborough LE12 5RD , UK 1 Present Address: D. Lunn Institute of Biological Chemistry, Washington State University , Pullman, WA 99163 , USA Use of biomass for second-generation biofuel production is severely hindered by the inherent recalcitrance of the plant cell wall to digestion. Trafficking is crucial for compartmentalisation within the cell. This process is partly regulated by small Rab GTPase proteins. In particular, control of trafficking to the cell wall is regulated through the RABA clade. Manipulation of this regulatory system offers tantalising opportunities for manipulation of cell wall composition and hence recalcitrance. Trafficking-defective rabA mutants have already been shown to impact cell wall composition. To study the impacts of these mutants on cell wall digestion, we developed a saccharification process for Arabidopsis based on the hot water method. We then showed that following pre-treatment, stems from the T-DNA knockouts of the three RABA4 genes expressed in Arabidopsis stem show an increased sugar release on saccharification. These rabA4 mutants have been shown to impact the Bhemicellulose-rich^ fraction during cell wall fractionation. Furthermore, we go on to show that these mutant lines also show increased sugar release when subjected to saccharification without pre-treatment. Finally, we used X-ray diffraction to show that rabA4 mutants had no impact on cellulose crystallinity, thus supporting the hypothesis that the increases in saccharification were not due to alterations of the cellulose microfibrils but were a direct effect of reduced hemicellulose levels. We also present data to show that the growth characteristics of these plants were unaffected. The data obtained from these lines are most easily explained by the reported alteration in hemicellulose increasing pre-treatment efficiency. Rab GTPase; Vesicle trafficking; Cell wall; Biofuels; Saccharification; Hemicellulose - An increasing demand globally for finite fossil fuels and concern over greenhouse gas emissions from their combustion has justified an increase in bioenergy research. Traditionally, bioenergy was considered expensive and uneconomic; however, an ever-rising oil price has redressed the balance in favour of bioenergy [1]. Mounting concerns about the sustainability of bioenergy crops and food security have focused attention on using non-food biomass. These secondgeneration feed stocks employ lignocellulosic biomass to create biofuel [2]. However, the cell wall has evolved to defend the organism from animal, viral, fungal and bacterial attack [3] and, as a result, requires pre-treatment before the hydrolysis step in the production process [4]. Due to this requirement, two solutions have been explored through the literature. The first of these focuses on efficiency of pre-treatment, and the second focuses on exploration of genetic modification to increase susceptibility to pre-treatment and/or enzymatic hydrolysis. Currently, in the literature, various types of pretreatment for Arabidopsis have been described, which tend to use treatment with acids, alkalis and hot water [58], and a number of protocols for high-throughput analysis have also been described [9, 10]. However, a consensus throughout the community has yet to be established. The second line of research has been the exploration of gene knockouts which could increase the susceptibility to enzyme hydrolysis. Most of the work in this field has focused on genes affecting lignin and xylan composition [68, 1113]. However, direct interference with cell wall biosynthesis machinery could have an adverse effect on the agronomic properties of the crop. Due to this, knockout or knockdown approaches to inhibiting genes encoding components of the trafficking machinery may be a viable alternative, as they often exhibit a high degree of redundancy within their gene families. In eukaryotes, Rab GTPases regulate vesicle trafficking, and in Arabidopsis, 57 homologues have been identified and grouped into clades (RABA, RABB, RABC, etc.) based upon sequence homology [14]. The RABA clade has 26 members and has been further divided into subclades (RABA1, RABA2, RABA3, etc.) with individual proteins identified by lowercase letters (RABA1a, RABA1b, etc.). Members of the RABA clade regulate trafficking to the cell wall through the trans-Golgi network (TGN), by acting as molecular switches for vesicle docking [15]. In Arabidopsis, RABA2 and RABA3 proteins have been associated with trafficking to the cell plate [16], and in tomato, inhibition of a RABA1 orthologue has been shown to impact pectin levels in fruit [17]. More recently, three independent lines with T-DNA knockouts in the three different RABA1 genes (RABA1a, RABA1c and RABA1d) that are expressed in stem tissue were tested and each showed similar levels of reduction in the level of pectin. Similarly, consistent results were obtained for reductions in the cellulose and hemicellulose fractions in different rabA2 and rabA4 mutant lines, respectively [18]. What is particularly interesting from the work carried out by Lunn and colleagues [18] is that the gene knockouts described in that study impacted on cell wall composition without directly affecting cell wall biosynthetic machinery. Here, we show that rabA4 knockout mutants exhibited an increased sugar release upon saccharification, both with and without pre-treatment. Materials and Methods Arabidopsis thaliana Col-1 and mutant lines are listed in Table 1. Each of the genes expressed in stem tissue of Arabidopsis was identified, and one T-DNA knockout mutant line was used for each gene. The characterisation of these lines List of rabA mutant lines used AGI ID no. for NASC ID no. for gene Arabidopsis line a Gene nomenclature according to Rutherford and Moore [14] and the confirmation of the lack of an RNA transcript has been described previously [18]. Arabidopsis Growth and Phenotyping Plants were grown under glass in the summer. Glasshouse conditions were as follows: 22 C with 16-h light and 8-h dark period, light intensity of 150 mol/m2/s. For compositional and digestibility assays, 50 plants of each line were placed in a randomised block structure. Stem material from the 50 plants was pooled at the senescent stage for analysis. Plants were grown in three successive months and treated as triplicates. Phenotype analysis was conducted on three replicates grown on separate occasions, using a randomised block structure, with ten plants each comprising one replicate. Phenotypic analysis was carried out based on the methodology described by Boyes et al. [19]. Dry, senescent Arabidopsis stem samples were milled to a particle size of 700 (...truncated)