Identification of plant cell wall mutants by means of a forward chemical genetic approach using hydrolases.
A previously undescribed forward chemical genetic screen using hydrolases affecting the extracellular matrix is introduced. The developed screen takes advantage of the power of chemical genetics and combines it with the known substrate specificity of glycosylhydrolases, resulting in the selection of conditional mutants that exhibit structural defects in their extracellular matrix. Identification of the responsible genetic locus in those mutants significantly extends our knowledge of genes involved in the biosynthesis, metabolism, signaling, and functionality of components of the extracellular matrix. The method is exemplified by a screen of mutagenized Arabidopsis plants subjected to growth in liquid culture in the presence of a xyloglucanase, an enzyme acting on the major cross-linking glycan found in the extracellular matrix of this plant. Using this hydrolase-based screen, dozens of plant cell wall mutants (xeg mutants) were identified, leading to the identification of 23 genetic loci that affect plant cell walls. One of the identified loci is XEG113, encoding a family 77 glycosyltransferase (GT77). Detailed analysis of the wall of this mutant indicated that its extensins, structural glyocoproteins present in walls, are underarabinosylated. Xeg-113 plants exhibit more elongated hypocotyls than WT, providing genetic evidence that plant O-glycosylation--more specifically, extensin arabinosylation--is important for cell elongation.
Gille
Sascha
S
Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA.
Hänsel
Ulrike
U
Ziemann
Mark
M
Pauly
Markus
M
D017360:Arabidopsis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics; D005020:Ethyl Methanesulfonate / Q000633:toxicity; D018506:Gene Expression Regulation, Plant; D005816:Genetic Complementation Test; D005821:Genetic Techniques; D005936:Glucans / Q000737:chemistry / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism*; D018527:Hydroponics / Q000379:methods; D006909:Hydroxyproline / Q000378:metabolism; D007150:Immunohistochemistry; D009005:Monosaccharides / Q000378:metabolism; D016296:Mutagenesis / Q000187:drug effects; D016254:Mutagenesis, Insertional; D009154:Mutation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D036226:Seedlings / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014990:Xylans / Q000737:chemistry / Q000378:metabolism
D004269:DNA, Bacterial; D005936:Glucans; D009005:Monosaccharides; C036483:T-DNA; D014990:Xylans; C029353:xyloglucan; D005020:Ethyl Methanesulfonate; D006867:Hydrolases; D006026:Glycoside Hydrolases; C072818:xyloglucan endo(1-4)-beta-D-glucanase; D006909:Hydroxyproline