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.

Transcript levels of the Arabidopsis R2R3-AtMYB102 transcription factor gene, previously named AtM4, are rapidly induced by osmotic stress or abscisic acid (ABA) treatment. Reporter gene expression studies revealed that in addition, wounding is required for full induction of the gene. Histochemical analysis showed a local ␤-glucuronidase induction around the wounding site, especially in veins. In ABA-treated plants, wounding-induced ␤-glucuronidase activity could be mimicked by the wound signaling compound methyl jasmonate. In silico studies of the AtMYB102 promoter sequence and its close homolog AtMYB74 demonstrated several conserved putative stress regulatory elements such as an ABA-responsive element, its coupling element 1 (CE1), and a W box. Interestingly, further studies showed that the 5Ј-untranslated region is essential for the osmotic stress and wounding induced expression of the AtMYB102 gene. This 5Ј-untranslated region contains putative conserved regulatory elements such as a second W box and an overlapping MYB-binding element. These studies suggest that AtMYB102 expression depends on and integrates signals derived from both wounding and osmotic stress.Abscisic acid (ABA) is the major plant hormone in water stress signaling. ABA regulates plant water balance and osmotic stress tolerance. The role of ABA in the osmotic stress responses has been studied extensively, and the availability of ABA-deficient and -insensitive mutants especially has been most helpful in these studies (Koornneef et al., 1998). Several of these mutants show severe wilting even upon a mild water stress treatments, supporting the role of ABA under stress conditions. ABA levels increase in wilting leaves due to the de novo synthesis of the carotenoid cleavage enzyme 9-cis-epoxycarotenoid dioxygenase (NCED), VP14 in maize (Zea mays). This enzyme catalyzes what appears to be the ratelimiting step in ABA biosynthesis (Tan et al., 1997). A clear correlation in Phaseolus vulgaris was found between NCED (PvNCED1) mRNA expression, NCED protein levels, and ABA levels in dehydrated leaves and roots, indicating the important role of NCED in stress ABA biosynthesis. Moreover, overexpression of the Arabidopsis AtNCED3 results in the accumulation of ABA (Iuchi et al., 2001).Two separate groups of stress-responsive genes are induced during osmotic stress: the "early response genes" and the "delayed-response genes" (Kiyosue et al., 1994;Yamaguchi-Shinozaki and Shinozaki, 1994). The early response genes can be induced within minutes. Expression is often transient and is independent of protein synthesis. These genes typically encode transcription factors that activate downstream delayed-response genes. The latter constitute the vast majority of the stress-responsive genes, which are activated by stress more slowly but often show sustained expression. In osmotically stressed Arabidopsis plants, ABA accumulation starts after approximately 2 h after the treatment (Kiyosue et al., 1994). Several genes respond already within this time period to dehydra...

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.

In Arabidopsis thaliana the R2R3-MYB transcription factor family consists of over 100 members and is implicated in many biological processes, such as plant development, metabolism, senescence, and defense. The R2R3-MYB transcription factor gene AtMYB102 has been shown to respond to salt stress, ABA, JA, and wounding, suggesting that AtMYB102 plays a role in the response of plants to dehydration after wounding. Here, we studied the role of AtMYB102 in the response of A. thaliana to feeding by larvae of the white cabbage butterfly Pieris rapae. A. thaliana reporter lines expressing GUS under control of the AtMYB102 promoter revealed that AtMYB102 is expressed locally at the feeding sites of herbivore-damaged leaves, but not systemically in uninfested plant parts. Knockout AtMYB102 transposon-insertion mutant plants (myb102) allowed a faster development of P. rapae caterpillars than wild-type Col-0 plants. Moreover, the number of caterpillars that had developed into pupae within 14 days was significantly higher on myb102, indicating that in wild-type plants AtMYB102 contributes to basal resistance against P. rapae feeding. Microarray analysis of wild-type Col-0 and AtMYB102 overexpressing 35S::MYB102 plants revealed a large number of differentially expressed genes. Besides several defense-related genes, a relatively large number of genes is associated with cell wall modifications.

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...

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The oecurrenee and .lecumulation of /J-1, 3-glucanase and chitinase in seedling roots of spruce {Picea ahies) following challenge by the root-rot pathogen Heterobasidion annosum were studied. Chitinase activity inereased 2-3 fold following inoculation, whereas no signifieant inerease in the activity levels of glucanasc was recorded during infection. With TEM immunogold labelling, the enzymes were localized in protein aggregates in host tissues and in the cell walls of intercellular hyphae. Gold particles were sparse and irregularly distributed within host-cell walls. Only minor labelling was observed on hyphaf walls coated with electron-dense materials. The labelling intensity increased with infeetion time and was always higher than in non-infected seedling roots. Wlien this experiment was repeated using root samples inoeulated with the saprophyte Phlebiopsis gigantea, a similar labelling pattern was obsei-ved. The cross reactivity of antisera raised .against sugar-beet ehitinase and glucanase with spruceroot enzyme extracts was demonstrated using dot-blot assays and ELISA.U. S.