SummaryTranscription factors containing a conserved DNA-binding domain similar to that of the proto-oncogene c-myb have been identified in nearly all eukaryotes. MYB-related proteins from plants generally contain two related helix-turnhelix motifs, the R2 and R3 repeats. It was estimated that Arabidopsis thaliana contains more than 100 R2R3-MYB genes. The few cases where functional data are available suggest an important role of these genes in the regulation of secondary metabolism, the control of cell shape, disease resistance, and hormone responses. To determine the full regulatory potential of this large family of regulatory genes, a systematic search for the function of all genes of this family was initiated. Sequence data for more than 90 different A. thaliana R2R3-MYB genes have been obtained. Sequence comparison revealed conserved amino acid motifs shared by subgroups of R2R3-MYB genes in addition to the characteristic DNA-binding domain. No significant clustering of the genes was detected, although they are not uniformly distributed throughout the A. thaliana genome.

More than 92 genes encoding MYB transcription factors of the R2R3 class have been described in Arabidopsis. The functions of a few members of this large gene family have been described, indicating important roles for R2R3 MYB transcription factors in the regulation of secondary metabolism, cell shape, and disease resistance, and in responses to growth regulators and stresses. For the majority of the genes in this family, however, little functional information is available. As the first step to characterizing these genes functionally, the sequences of >90 family members, and the map positions and expression profiles of >60 members, have been determined previously. An important second step in the functional analysis of the MYB family, through a process of reverse genetics that entails the isolation of insertion mutants, is described here. For this purpose, a variety of gene disruption resources has been used, including T-DNA-insertion populations and three distinct populations that harbor transposon insertions. We report the isolation of 47 insertions into 36 distinct MYB genes by screening a total of 73 genes. These defined insertion lines will provide the foundation for subsequent detailed functional analyses for the assignment of specific functions to individual members of the R2R3 MYB gene family.

We have undertaken a systematic reverse genetic approach to understand R2R3-MYB gene function in Arabidopsis. Here, we report the functional characterization of MYB61 based on the phenotype of three independent insertion alleles. Wide-ranging phenotype screens indicated that MYB61 mutants were deficient in seed mucilage extrusion upon imbibition. This phenotype was expressed in the sporophytic tissues of the seed. Deposition and extrusion of the principal component of the mucilage, a relatively unbranched rhamnogalacturonan, were reduced in the MYB61 mutant seed coats. Additional defects in the maturation of the testa epidermal cells suggested a potential deficiency in extracellular secretion in myb61 lines. Consistent with a proposed role in testa development, reverse transcription-polymerase chain reaction analysis showed the highest MYB61 expression in siliques, which was localized to the seed coat by a beta-glucuronidase (GUS) reporter gene fusion. Lower levels of GUS expression were detected in developing vascular tissue. Parallel analysis of the ttg1-1 mutant phenotype indicated that this mutant showed more severe developmental defects than myb61 and suggested that MYB61 may function in a genetic pathway distinct from that of TTG1. The transient nature of seed epidermal characteristics in the ttg1-1 mutant suggested that TTG1 was required for maintenance rather than initiation of testa epidermal differentiation. Germination and seedling establishment were compromised in the myb61 and ttg1-1 mutants under conditions of reduced water potential, suggesting a function for Arabidopsis seed mucilage during germination in dry conditions.

In order to study the mode of action of herbicides we conducted a pilot study analysing phenotype and gene expression of flufenacet- and benfuresate-treated Arabidopsis thaliana (L) Heynhoe plants. Treatments with either herbicide caused phenocopies of the known Arabidopsis mutant fiddlehead, displaying fused organs and the typical fiddlehead-like inflorescence. Herbicide treatments of other plant species, including monocots, also gave rise to analogous organ fusions, indicating the presence of the target in a broad range of plants. Furthermore, many other herbicides with a proposed similar mode of action, eg chloroacetanilides, produced comparable fusion phenotypes in plants. The fiddlehead gene encodes a putative very-long-chain fatty acid elongase (VLCFAE), which corroborates earlier biochemical results pointing to the inhibition of VLCFA synthesis as mode of action of flufenacet. Gene expression profiles of herbicide-treated plants using the first 8247 gene Arabidopsis gene array of Affymetrix provided additional clues in support of inhibition of VLCFA synthesis. We discuss fiddlehead-like elongases as plant specific targets for flufenacet and many other herbicides.

We have undertaken a systematic reverse genetic approach to understand R2R3-MYB gene function in Arabidopsis. Here, we report the functional characterization of MYB61 based on the phenotype of three independent insertion alleles. Wide-ranging phenotype screens indicated that MYB61 mutants were deficient in seed mucilage extrusion upon imbibition. This phenotype was expressed in the sporophytic tissues of the seed. Deposition and extrusion of the principal component of the mucilage, a relatively unbranched rhamnogalacturonan, were reduced in the MYB61 mutant seed coats. Additional defects in the maturation of the testa epidermal cells suggested a potential deficiency in extracellular secretion in myb61 lines. Consistent with a proposed role in testa development, reverse transcription-polymerase chain reaction analysis showed the highest MYB61 expression in siliques, which was localized to the seed coat by a ␤ -glucuronidase ( GUS ) reporter gene fusion. Lower levels of GUS expression were detected in developing vascular tissue. Parallel analysis of the ttg1-1 mutant phenotype indicated that this mutant showed more severe developmental defects than myb61 and suggested that MYB61 may function in a genetic pathway distinct from that of TTG1. The transient nature of seed epidermal characteristics in the ttg1-1 mutant suggested that TTG1 was required for maintenance rather than initiation of testa epidermal differentiation. Germination and seedling establishment were compromised in the myb61 and ttg1-1 mutants under conditions of reduced water potential, suggesting a function for Arabidopsis seed mucilage during germination in dry conditions. fax 44-1603-450025. Article, publication date, and citation information can be found at www.plantcell.org/cgi/