Post-synthetic modification of plant cell walls by expression of microbial hydrolases in the apoplast

Abstract

The systematic creation of defined cell wall modifications in the model plant Arabidopsis thaliana by expression of microbial hydrolases with known specific activities is a promising approach to examine the impacts of cell wall composition and structure on both plant fitness and cell wall recalcitrance. Moreover, this approach allows the direct evaluation in living plants of hydrolase specificity, which can differ from in vitro specificity. To express genes encoding microbial hydrolases in A. thaliana, and target the hydrolases to the apoplast compartment, we constructed an expression cassette composed of the Cauliflower Mosaic Virus 35S RNA promoter, the A. thaliana β-expansin signal peptide, and the fluorescent marker protein YFP. Using this construct we successfully introduced into Colombia-0 plants three Aspergillus nidulans hydrolases, β-xylosidase/α-arabinosidase, feruloyl esterase, acetylxylan esterase, and a Xanthomonas oryzae putative a-L: -arabinofuranosidase. Fusion with YFP permitted quick and easy screening of transformants, detection of apoplastic localization, and protein size confirmation. Compared to wild-type Col-0, all transgenic lines showed a significant increase in the corresponding hydrolytic activity in the apoplast and changes in cell wall composition. Examination of hydrolytic activity in the transgenic plants also showed, for the first time, that the X. oryzae gene indeed encoded an enzyme with α-L: -arabinofuranosidase activity. None of the transgenic plants showed a visible phenotype; however, the induced compositional changes increased the degradability of biomass from plants expressing feruloyl esterase and β-xylosidase/α-arabinosidase. Our results demonstrate the viability of creating a set of transgenic A. thaliana plants with modified cell walls to use as a toolset for investigation of how cell wall composition contributes to recalcitrance and affects plant fitness.

Arabidopsis and Brachypodium distachyon Transgenic Plants Expressing Aspergillus nidulans Acetylesterases Have Decreased Degree of Polysaccharide Acetylation and Increased Resistance to Pathogens

“…Earlier, we reported the preparation of Arabidopsis plants expressing AnAXE and demonstrated that a signal peptide fused to the N terminus of the hydrolase ensures its delivery to the apoplast, and a fluorescent protein fused to the C terminus of the hydrolase does not affect its activity (Pogorelko et al, 2011). In this study, we constructed new versatile transformation vectors and expressed AnAXE and AnRAE esterases in the apoplast of Arabidopsis and Brachypodium plants characterized by type I and type II cell walls, respectively.…”

“…Apoplastic fluids from aerial plant parts were extracted as described previously (Pogorelko et al, 2011). Immunoblot analyses were performed according to Magi and Liberatori (2005).…”

“…Recently, plant cell wall polysaccharides have attracted significant attention because they are a major source of reduced carbon for bioethanol production, which has the potential to supply part of our fossil fuel demand (Jordan et al, 2012). Direct modification of cell wall polysaccharides can increase the efficiency of biomass digestibility and improve saccharification during ethanol production (Lionetti et al, 2010;Pogorelko et al, 2011;Fursova et al, 2012;Jung et al, 2012;Song et al, 2012). However, modification of cell wall polysaccharides may also affect important cell wall functions, and the plant cell must maintain the functional integrity of the wall during morphogenesis and exposure to environmental cues (Hamman, 2012).…”

Cell Wall Proteomics as a Means to Identify Target Genes to Improve Second‐Generation Biofuel Production

“…"y altering transcription factors in A. thaliana, it was possible both to increase cellulose and decrease lignin content [7 ] and improve secondary cell wall synthesis in ibre cells [80]. In addition, the golden pot may be near transgenic A. thaliana expressing microbial hydrolases showed no visible changes in phenotype and increased wall degradability [81]. "n alternative to decrease the transgenic debate and perhaps optimize eiciency could be altering the expression of the own plant enzymes generating a genetically modiied plant, but not a transgenic one.…”

Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases

“…A CE7 AcXE gene has recently been modified by site-directed mutagenesis to create a perhydrolytic enzyme with improved activity for the production of perhydrolytic acids applicable in laundry care and disinfectant formulations [163]. AcXEs have also been employed in deacetylation of other acetylated polysaccharides [40] as well as in the post-synthetic (in planta) deacetylation of xylan to enhance lignocellulose saccharification [7,8,164,165]. In synthetic carbohydrate chemistry, AcXEs can potentially be harnessed in catalyzing regioselective transacetylation reactions [23,24,166].…”