Genetic and biochemical analyses of Arabidopsis thaliana glycosyl hydrolase family 43 enzymes A and B

Faisal Anwar Chaudhry, Purdue University

Abstract

Plants have adapted a timed mechanism to release seeds from a dormant state to avoid unfavorable environmental conditions in order to establish a healthy plant and be able to reproduce. The release of seed dormancy to initiate germination involves the transcriptional regulation of β-1,3-glucanases. A possible substrate of β-1,3-glucanase activity is callose, which can be found in the endosperm of several plant seeds. However, the biochemical activities and substrate specificities of seed-expressed β-1,3-glucanases have not been thoroughly investigated. In this study, two enzyme isoforms from Arabidopsis thaliana grouped in Glycosyl Hydrolase Family 43 (Carbohydrate Active EnZyme database, www.cazy.org) have been characterized. Phylogenetic analysis reveals a closer similarity of known plant GH43 sequences to putative bacterial β-1,3-glucanases rather than to arabinanases, xylosidases, or galactosidases. AtGH43A and AtGH43B transcripts are relatively evenly expressed in both vegetative and reproductive plant tissues. The single- and double-mutants do not show significant visual phenotypes, even under various stress stimuli, yet a delay in seed germination is observed in the atgh43b mutant and, to a lesser extent, in the double mutant. Linkage analysis reveals a two-fold increase in the 3-gal linkage in aerial and root cell walls. Recombinant proteins expressed in Nicotiana tabacum BY-2 demonstrate activity by releasing glucose monosaccharides from purified Arabidopsis aerial cell walls in a time-dependent manner. AtGH43B enzyme has measurable activity against laminarin (β-1,3-glucan), and β-gentiobiose (β-1,6-glucan), but not to cellobiose (β-1,4-glucan) or sophorose (β-1,2-glucan). It is postulated that AtGH43A and AtGH43B may target callose (a 1,3-linked glucose polymer) or a 1,6-glucan linkage in AGPs or glycosylated protein which may affect seed germination. It is also possible that AtGH43 enzymes may work in concert with other hydrolytic enzymes specific against other substrates, such as galactosidases, which may explain the increase in 3-gal linkages in aerial and root cell walls isolated from GH43 mutants.

Degree

Ph.D.

Advisors

McCann, Purdue University.

Subject Area

Biochemistry

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