The Arabidopsis GAMYB-Like Genes, MYB33 and MYB65, Are MicroRNA-Regulated Genes That Redundantly Facilitate Anther Development

Abstract

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...

The Gibberellin perception system evolved to regulate a pre-existing GAMYB-mediated system during land plant evolution

“…2c-e), where the sporopollenin material is produced for the outer wall development [9][10][11][21][22][23] . Considering that GAMYBs, CYP703As and GA biosynthesis genes in flowering plants are also co-expressed in the tapetal cells and microspores 1,8,12,[24][25][26] , these observations indicate that SmGAMYB activity in these tissues of Selaginella is required to mediate GA signalling during the outer wall formation of microspores.…”

Floral patterning defects induced by Arabidopsis APETALA2 and microRNA172 expression in Nicotiana benthamiana

“…Recent studies have uncovered the role of microRNAs (miRNAs), ~21-nucleotide noncoding RNAs, as regulators of floral patterning gene expression in Arabidopsis flower development (Aukerman and Sakai 2003;Schmid et al 2003;Achard et al 2004;Chen 2004;Mallory et al 2004a;Baker et al 2005;Millar and Gubler 2005). Plant miRNAs regulate cognate protein coding RNAs through either transcript cleavage or translational inhibition or both (reviewed in Bartel and Bartel 2003;Carrington and Ambros 2003;Bartel 2004;Dugas and Bartel 2004;He and Hannon 2004;Mallory and Vaucheret 2004;Kidner and Martienssen 2005;Chen 2005;Valencia-Sanchez et al 2006).…”

Duplication and expression analysis of multicopy miRNA gene family members in Arabidopsis and rice

“…Recently, more and more plant miRNAs have been reported for their specialized developmental functions, such as miR159, miR-JAW, miR160, miR162, miR165/166, miR168, and miR172. These miRNAs are involved in flower anther development [21], rosette leaf curvature [22], organ polarity [23,24], floral organ identity and flowering time [25,26], auxin response [27], and feedback regulation of the miRNA pathway [19,28].…”