The functions of the vast majority of genes encoding R2R3 MYB domain proteins remain unknown. The closely related MYB33 and MYB65 genes of Arabidopsis thaliana have high sequence similarity to the barley (Hordeum vulgare) GAMYB gene. T-DNA insertional mutants were isolated for both genes, and a myb33 myb65 double mutant was defective in anther development. In myb33 myb65 anthers, the tapetum undergoes hypertrophy at the pollen mother cell stage, resulting in premeiotic abortion of pollen development. However, myb33 myb65 sterility was conditional, where fertility increased both under higher light or lower temperature conditions. Thus, MYB33/MYB65 facilitate, but are not essential for, anther development. Neither single mutant displayed a phenotype, implying that MYB33 and MYB65 are functionally redundant. Consistent with functional redundancy, promoter-b-glucuronidase (GUS) fusions of MYB33 and MYB65 gave identical expression patterns in flowers (sepals, style, receptacle, anther filaments, and connective but not in anthers themselves), shoot apices, and root tips. By contrast, expression of a MYB33:GUS translational fusion in flowers was solely in young anthers (consistent with the male sterile phenotype), and no staining was seen in shoot meristems or root tips. A microRNA target sequence is present in the MYB genes, and mutating this sequence in the MYB33:GUS fusion results in an expanded expression pattern, in tissues similar to that observed in the promoter-GUS lines, implying that the microRNA target sequence is restricting MYB33 expression. Arabidopsis transformed with MYB33 containing the mutated microRNA target had dramatic pleiotrophic developmental defects, suggesting that restricting MYB33 expression, especially in the shoot apices, is essential for proper plant development. INTRODUCTIONWith >120 genes in the Arabidopsis thaliana genome, the R2R3-MYB gene family has been identified as one of the most abundant classes of transcription factors in plants (Stracke et al., 2001). They are involved in a diverse range of processes, including controlling cell shape, disease resistance, regulating secondary metabolism, and hormone signal transduction (Jin and Martin, 1999). One MYB gene from barley (Hordeum vulgare), HvGAMYB, is involved in gibberellin (GA) signaling in the aleurone (Gubler et al., 1995). Here, expression of HvGAMYB is upregulated by GA, where it then binds to the TAACAAA motif of a barley high-pI a-amylase promoter, a motif that plays an important role in the GA-regulated expression of the a-amylase gene (Skriver et al., 1991;Gubler and Jacobsen, 1992;Lanahan et al., 1992). In transient assays, constitutive expression of HvGAMYB is sufficient to activate the a-amylase promoter (Gubler et al., 1995) and the promoters of other aleurone GAregulated genes that are required for the mobilization of endosperm reserves (Cercó s et al., 1999;Gubler et al., 1999). Thus, in regards to expression of these genes in aleurone layers, transient expression of HvGAMYB has the same effect as GA application. Fr...

SummaryThe Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene encodes a putative seed-specific condensing enzyme. It is the first of four enzyme activities that comprise the microsomal fatty acid elongase (FAE) involved in the biosynthesis of very-long-chain fatty acids (VLCFAs). FAEI has been expressed in yeast and in tissues of Arabidopsis and tobacco, where significant quantities of VLCFAs are not found. The introduction of FAE1 alone in these systems is sufficient for the production of VLCFAs, for wherever FAE1 was expressed, VLCFAs accumulated. These results indicate that FAE1 is the rate-limiting enzyme for VLCFA biosynthesis in Arabidopsis seed, because introduction of extra copies of FAE1 resulted in higher levels of the VLCFAs. Furthermore, the condensing enzyme is the activity of the elongase that determines the acyl chain length of the VLCFAs produced. In contrast, it appears that the other three enzyme activities of the elongase are found ubiquitously throughout the plant, are not rate-limiting and play no role in the control of VLCFA synthesis. The ability of yeast containing FAE1 to synthesize VLCFAs suggests that the expression and the acyl chain length specificity of the condensing enzyme, along with the apparent broad specificities of the other three FAE activities, may be a universal eukaryotic mechanism for regulating the amounts and acyl chain length of VLCFAs synthesized.

SummaryWe have investigated the relationship between seed dormancy and abscisic acid (ABA) metabolism in the monocot barley and the dicot Arabidopsis. Whether dormant (D) or non-dormant (ND), dry seed of Arabidopsis and embryos of dry barley grains all had similarly high levels of ABA. ABA levels decreased rapidly upon imbibition, although they fell further in ND than in D. Gene expression profiles were determined in Arabidopsis for key ABA biosynthetic [the 9-cis epoxycarotenoid dioxygenase gene family] and ABA catabolic [the ABA 8¢-hydroxylase gene family (CYP707A)] genes. Of these, only the AtCYP707A2 gene was differentially expressed between D and ND seeds, being expressed to a much higher level in ND seeds. Similarly, a barley CYP707 homologue, (HvABA8¢OH-1) was expressed to a much higher level in embryos from ND grains than from D grains. Consistent with this, in situ hybridization studies showed HvABA8¢OH-1 mRNA expression was stronger in embryos from ND grains. Surprisingly, the signal was confined in the coleorhiza, suggesting that this tissue plays a key role in dormancy release. Constitutive expression of a CYP707A gene in transgenic Arabidopsis resulted in decreased ABA content in mature dry seeds and a much shorter after-ripening period to overcome dormancy. Conversely, mutating the CYP707A2 gene resulted in seeds that required longer afterripening to break dormancy. Our results point to a pivotal role for the ABA 8¢-hydroxylase gene in controlling dormancy and that the action of this enzyme may be confined to a particular organ as in the coleorhiza of cereals.

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