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Identification and characterization of genes involved in Arabidopsis thaliana cell wall acetylation

  • Author(s): de Souza, Amancio Jose
  • Advisor(s): Pauly, Markus
  • et al.
Abstract

Most non-cellulosic plant cell wall polysaccharides including the hemicellulose xyloglucan and the pectic polysaccharides can be O-acetylated. This feature has direct significance in the use of these polymers in the food and biofuel industry. For example, increased pectin acetylation can reduce its gelling abilities and is hence detrimental in its application as a food thickener or emulsifier. In general, plant biomass with wall polymers with high acetate content can negatively influence biomass hydrolysis by fungal enzymes and interfere with downstream fermentation by yeasts.

Genetic and biochemical approaches were used to identify genes involved in wall polymer acetylation in the model species Arabidopsis. A forward genetics approach was able to identify wall acetylation differences within a population of Arabidopsis ecotypes in both total wall acetate and xyloglucan O-acetylation. One of these naturally occurring mutant alleles of a recently identified putative xyloglucan acetyltransferase (AXY4) was found in the Ty-0 ecotype. Ty-0 completely lacks xyloglucan acetyl-substituents in roots and leaves, suggesting that the lack of XyG acetylation does not compromise the fitness of the ecotype in its ecological niche. In an attempt to further characterize the AXY4 protein, and characterize peptide domains involved in cell wall polymer recognition, a functional domain swap approach was utilized without success. A biochemical approach to identify and purify a putative xyloglucan acetylesterase was attempted also without success. Another way to modulate wall acetylation is by apoplastic plant acetylesterases, which naturally remove acetyl-substituents on polymers. To characterize genes in the Pectin acetyl esterase gene family in Arabidopsis thaliana, reverse genetics was used and lead to the identification of two genes responsible for altering wall acetate levels of pectins. Further pectin fractionation experiments suggest that PAE8 and PAE9 act predominantly but distinctively on pectic rhamnogalacturonan I. Pectin acetylesterase activity was demonstrated in vitro for the proteins encoded by PAE8 and PAE9. Through genetic means it is possible to increase wall acetate content, but this modification lead to a reduction in Arabidopsis inflorescence size giving insights about the function of acetylation of pectins.

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