Function Search in a Large Transcription Factor Gene Family in Arabidopsis: Assessing the Potential of Reverse Genetics to Identify Insertional Mutations in R2R3 MYB Genes

Abstract

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 Ͼ 90 family members, and the map positions and expression profiles of Ͼ 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-DNAinsertion 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. INTRODUCTIONThe assignment of biological function to the large number of genes that have now been sequenced, with new sequence data being compiled rapidly, is currently one of the most challenging goals in biology. Genetic analysis, particularly the effects of loss-of-function mutations, is of central importance to achieving this goal. In plants, unlike yeast, targeted gene disruption is laborious and inefficient (Kempin et al., 1997). Gene silencing by antisense or sense suppression is also a common approach to studying plant gene function (Kooter and Mol, 1993;Baulcombe, 1996), but the specificity and extent of gene disruption through such methods have not been extensively tested, 1 These authors contributed equally to this work. 2 Current address: CPRO-DLO, Department of Molecular Biology, Droevendaalsesteeg 1, 6700 AA Wageningen, The Netherlands. 3 Current address: Laboratoire de Radiobiologie Vegetale DEVM, CEA, Cadarache, 13108 St. Paul-lez Durance Cedex, France. 4 To whom correspondence should be addressed. E-mail bevan@ bbsrc.ac.uk; fax 44-1603-505725. 1828The Plant Cell so the analysis and interpretation of suppression sometimes have proved difficult (van der Krol et al., 1990;Höfgen et al., 1994).T-DNA and transposable elements can alter gene function upon insertion into coding or regulatory sequences (Feldmann, 1991;Martienssen, 1998). Many lines of Arabidopsis harboring either T-DNA or transposon insertions have been generated (Koncz et al., 1992;Azpiroz-Leehan and Feldmann, 1997;Bouchez and Höfte, 1998;Martienssen, 1998;Wisman et al., 1998aWisman et al., , 1998b. Individual lines carrying insertions within a gene of interest can be identified by polymerase chain reaction (PCR) by using a gene-sp...

Identification of conserved gene structures and carboxy-terminal motifs in the Myb gene family of Arabidopsis and Oryza sativa L. ssp. indica

“…In all, 47 insertion mutants were detected in 36 distinct Myb genes by screening a total of 73 genes. However, none of the insertions gave rise to morphological phenotypes visible in soil-grown plants [11]. The redundancy of Myb genes may diminish the efficiency of the molecular approach by complementation of function.…”

Fine mapping and candidate gene prediction of the photoperiod and thermo-sensitive genic male sterile gene pms1(t) in rice

“…LOC_Os07g12130, as a pms1(t) candidate gene, encodes a Myb-like protein with a DNA binding domain. Myb proteins are the largest superfamily of transcription factors that play a regulating role in the process of development and defense reaction, such as regulation of plant cell shape, growth regulators, and responses to stress (Meissner et al, 1999). A new Myb-like gene (AtMYB103) showed a high level of mRNA expression in the early development of anther tapetum and middle-level performance in Arabidopsis thaliana (Li et al, 1999).…”

Developmental gene regulation during tomato fruit ripening and in-vitro sepal morphogenesis

“…In Arabidopsis more than 92 MYB genes have been described [38]. In plants, MYB genes regulate secondary metabolism, cell morphology, and signal transduction in plant growth and pathogen defense [39].…”

“…Further research is needed to determine the function of this transcription factor. However, in gene-disruption experiments, Meissner et al [38] found no obvious phenotypes in 32 homozygous insertion lines of 26 genes. Even more intensive greenhouse and plate-based screening failed to find a phenotype for most of these plants, possibly indicating redundancy [38].…”