Harlequin (hlq) : an arabidopsis mutant that ectopically expresses Dc3-GUS and shows defects in cell wall morphogenesis

Plant growth and development is regulated by complex interactions among different hormonal, developmental, and environmental signaling pathways. Isolation of mutants in these processes is a powerful approach to dissect unknown mechanisms in regulatory networks. The plant hormones abscisic acid (ABA) and auxin are involved in vegetative, developmental and environmental growth responses, including cell division and elongation. The uidA (β-glucuronidase; GUS) reporter gene driven by the carrot (Daucus carota) Late Embryogenesis-Abundant Dc3 promoter in the roots of transgenic Arabidopsis thaliana seedlings is ABA- , drought- and auxin-inducible in a tissue specific manner. An ethyl methane sulfonate induced M2 mutant population of abi2/abi2 homozygous plants of a line that carries two independent Dc3-GUS reporter genes was screened for mutants with altered expression of the Dc3-GUS transgene upon induction with ABA. Of these, one mutant class showed ectopic expression of Dc3-GUS in a chequered pattern and was named harlequin (hlq). Genetic analysis revealed that the hlq gene was inherited as a single-gene, nuclear, recessive mutation. The physiological ABA responses of the hlq mutant tested in both abi2 and Ler backgrounds were normal and the mutant phenotype does not require the presence of abi2-1 mutant allele for its expression. Further morphological characterization revealed that the hlq mutant is an extreme dwarf and sterile. Response of hlq to various plant growth regulators were not different from the wild type and did not rescue the dwarf phenotype, suggesting that hlq is not perturbed in hormone homeostasis. The hlq mutant has sparse root-hairs and abnormal leaf trichomes. Scanning Electron Micrographs shows missing / collapsed epidermal cells both in the roots and the hypocotyl. The radial swelling of the root zone of elongation was similar to that of cellulose deficient mutants or suggesting cell wall deficiency. There was ectopic deposition of callose on the roots and the callose content was up to five times that of the wild type and the cellulose content was twice as much. Ruthenium red staining showed ectopic deposition of pectin in the interstitial spaces apart from the cell wall in hlq mutants suggesting defective Golgi vesicle transport or secretion. The hlq callus was also more friable than the wild type, suggesting defective pectin composition that is needed for cell-to-cell adhesion. The cell wall associated enzymes like, Pectin Methyl Esterase activity was normal in hlq but the cell wall peroxidase showed abnormalities. The wild type cell wall peroxidase was absent in the hlq mutants but there was appearance of three more extraneous peroxidases, whose functions are unknown. EST Microarray profiling of hlq total RNA showed the upregulation of stress-responsive genes and down regulation of some genes involved in cell-wall biogenesis. It is proposed that, the loss of extensibility of the cell wall due to callose deposition to supplement defective pectin matrix, would elicit stress responses by expressing GUS in those tissues and also result in a dwarf phenotype. The hlq locus has been mapped to a 220kb interval on chromosome IIl. Further cloning and characterization of the hlq gene may enhance our knowledge on wall biogenesis and provide insights into the relationship of cell wall signaling and hormone-regulated gene expression in plant growth and development