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Identification of plant cell wall mutants by means of a forward chemical genetic approach using hydrolases.

ABSTRACT: 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.

SUBMITTER: Gille S

PROVIDER: S-EPMC2731844 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:In pursuing our long-term goals of identifying causal genes for mutant phenotypes in maize, we have developed a new, phenotype-to-genotype approach for transposon-based resources, and used this to identify candidate genes that co-segregate with visible kernel mutants. The strategy incorporates a redesigned Mu-seq protocol (sequence-based, transposon mapping) for high-throughput identification of individual plants carrying Mu insertions. Forward-genetic Mu-seq also involves a genetic pipeline for generating families that segregate for mutants of interest, and grid designs for concurrent analysis of genotypes in multiple families. Critically, this approach not only eliminates gene-specific PCR genotyping, but also profiles all Mu-insertions in hundreds of individuals simultaneously. Here, we employ this scalable approach to study 12 families that showed Mendelian segregation of visible seed mutants. These families were analyzed in parallel, and 7 showed clear co-segregation between the selected phenotype and a Mu insertion in a specific gene. Results were confirmed by PCR. Mutant genes that associated with kernel phenotypes include those encoding: a new allele of Whirly1 (a transcription factor with high affinity for organellar and single-stranded DNA), a predicted splicing factor with a KH domain, a small protein with unknown function, a putative mitochondrial transcription-termination factor, and three proteins with pentatricopeptide repeat domains (predicted mitochondrial). Identification of such associations allows mutants to be prioritized for subsequent research based on their functional annotations. Forward-genetic Mu-seq also allows a systematic dissection of mutant classes with similar phenotypes. In the present work, a high proportion of kernel phenotypes were associated with mutations affecting organellar gene transcription and processing, highlighting the importance and non-redundance of genes controlling these aspects of seed development.

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Identification of plant cell wall mutants by means of a forward chemical genetic approach using hydrolases.

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