The Anaphase-Promoting Complex Is a Dual Integrator That Regulates Both MicroRNA-Mediated Transcriptional Regulation ofCyclin B1and Degradation of Cyclin B1 duringArabidopsisMale Gametophyte Development  

Abstract

The anaphase-promoting complex/cyclosome (APC/C), an essential ubiquitin protein ligase, regulates mitotic progression and exit by enhancing degradation of cell cycle regulatory proteins, such as CYCB1;1, whose transcripts are upregulated by DUO POLLEN1 (DUO1). DUO1 is required for cell division in male gametophytes and is a target of microRNA 159 (miR159) in Arabidopsis thaliana. Whether APC/C is required for DUO1-dependent CYCB1;1 regulation is unknown. Mutants in both APC8 and APC13 had pleiotrophic phenotypes resembling those of mutants affecting microRNA biogenesis. We show that these apc/c mutants had reduced miR159 levels and increased DUO1 and CYCB1;1 transcript levels and that APC/C is required to recruit RNA polymerase II to MIR159 promoters. Thus, in addition to its role in degrading CYCB1;1, APC/C stimulates production of miR159, which downregulates DUO1 expression, leading to reduced CYCB1;1 transcription. Both MIR159 and APC8-yellow fluorescent protein accumulated in unicellular microspores and bicellular pollen but decreased in tricellular pollen, suggesting that spatial and temporal regulation of miR159 by APC/C ensures mitotic progression. Consistent with this, the percentage of mature pollen with no or single sperm-like cells increased in apc/c mutants and plants overexpressing APC8 partially mimicked the duo1 phenotype. Thus, APC/C is an integrator that regulates both microRNA-mediated transcriptional regulation of CYCB1;1 and degradation of CYCB1;1.

SAMBA, a plant-specific anaphase-promoting complex/cyclosome regulator is involved in early development and A-type cyclin stabilization

“…Here, we show that samba is defective in mitosis I of pollen development, resulting in pollen without sperm nuclei. Mutants of APC8 and APC13 (29,46) show phenotypes similar to those described here for the samba plants. Both mutants are affected in pollen development, leading to an increased proportion of uninucleated mature pollen, indicating that the APC/C is required during the first mitosis of the male gametophytic development.…”

“…Mutations in most APC/C subunits affect female and/or male gametogenesis (24)(25)(26)(27)(28)(29). Here, we show that samba is defective in mitosis I of pollen development, resulting in pollen without sperm nuclei.…”

“…In all cases, except for APC8, the analysis of the mutants revealed female gametophytic defects, probably as a consequence of the inability to degrade mitotic cyclins (24)(25)(26)(27)(28). APC8 was shown to be involved in male gametogenesis (29). Moreover, reduced expression levels of APC6 (10) or APC10 (10, 28) exhibited several developmental abnormalities, including defects in vascular development, whereas an APC4 loss-of-function mutant was defective in embryogenesis (26).…”

An EAR-Dependent Regulatory Module Promotes Male Germ Cell Division and Sperm Fertility in Arabidopsis

“…Furthermore, the DUO1 promoter is sufficient to direct male germline-specific expression, suggesting that miR159-mediated regulation of DUO1 may be restricted to the male germline (Brownfield et al, 2009a). Finally, increased expression of DUO1 has been associated with reduced levels of APC/C and miR159 (Zheng et al, 2011). We thus speculate that DAZ1/DAZ2 might stimulate DUO1 turnover via miR159-mediated regulation, consistent with the low levels of APC/C in mutant duo1 and daz1 daz2 germ cells (Figures 5F to 5J).…”

Arabidopsis Tetraspanins Are Confined to Discrete Expression Domains and Cell Types in Reproductive Tissues and Form Homo- and Heterodimers When Expressed in Yeast

“…For transcriptional fusions, the native tetraspanin promoters were amplified from genomic DNA by PCR (Supplemental Table S3) to generate Gateway entry clones that were then recombined into the Gateway-compatible version (Zheng et al, 2011) of the pGreenII-based vector NLS3xeGFPnopaline synthase terminator (Takada and Jürgens, 2007). These constructs were introduced into Agrobacterium tumefaciens strain GV3101 harboring the pGreenII helper plasmid pSOUP and transformed in Arabidopsis qrt1-2 Columbia plants using the floral dipping method (Clough, 2005).…”