RESUMO
Development of leaf margins is an important process in leaf morphogenesis. CIN-clade TCP (TEOSINTE BRANCHED1/CYCLOIDEA/PCF) transcription factors are known to have redundant roles in specifying leaf margins, but the specific mechanisms through which individual TCP genes function remain elusive. In this study, we report that the CIN-TCP gene TCP5 is involved in repressing the initiation and outgrowth of leaf serrations by activating two key regulators of margin development, the Class II KNOX factor KNAT3 and BEL-like SAW1. Specifically, TCP5 directly promotes the transcription of KNAT3 and indirectly activates the expression of SAW1. We also show that TCP5 regulates KNAT3 and SAW1 in a temporal- and spatial- specific manner that is largely in accordance with the progress of formation of serrations. This regulation might serve as a key mechanism in patterning margin morphogenesis and in sculpting the final form of the leaf.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Folhas de Planta/crescimento & desenvolvimento , Fatores de Transcrição , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Plant leaves are the main photosynthetic organ of plants and they occur in an array of different shapes. Leaf shape is determined by morphogenesis whereby patterning of the leaf margin can result in interspaced leaf serrations, lobes, or leaflets, depending on the species, developmental stage, and in some instances the environment. In Arabidopsis, mutations in the homeodomain transcription factors SAW1 and SAW2 result in more prominent leaf margin serrations. Here we show that serrations appear precociously in the saw1 saw2 mutant. The pattern of auxin maxima, and of PIN1 and CUC2 expression, which form a feedback loop that drives serration outgrowth, is altered in saw1 saw2 and correlates with precocious serration initiation. SAW1 is not expressed in the outer epidermal cell layer where PIN1 convergence points generate auxin maxima. Instead, SAW1 is expressed on the adaxial side of the leaf and expression in this domain is sufficient for function. We suggest that SAW1 and SAW2 repress serration initiation and outgrowth by promoting the transition to a determinate fate in the leaf margin.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Homeodomínio/metabolismo , Folhas de Planta/anatomia & histologia , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/genética , Ácidos Indolacéticos , Mutação , Fatores de Transcrição/genéticaRESUMO
SAW1 is required for efficient removal by the Rad1-Rad10 nuclease of 3' non-homologous DNA ends (flaps) formed as intermediates during two modes of double-strand break (DSB) repair in S. cerevisiae, single-strand annealing (SSA) and synthesis-dependent strand annealing. Saw1 was shown in vitro to bind flaps with high affinity, but displayed diminished affinity when flaps were short (<30 deoxynucleotides [nt]), consistent with it not being required for short flap cleavage. Accordingly, this study, using in vivo fluorescence microscopy showed that SAW1 was not required for recruitment of Rad10-YFP to DNA DSBs during their repair by SSA when the flaps were ~10 nt. In contrast, recruitment of Rad10-YFP to DSBs when flaps were ~500 nt did require SAW1 in G1 phase of cell cycle. Interestingly, we observed a substantial increase in colocalization of Saw1-CFP and Rad10-YFP at DSBs when short flaps were formed during repair, especially in G1, indicating significant recruitment of Saw1 despite there being no requirement for Saw1 to recruit Rad10. Saw1-CFP was seldom observed at DSBs without Rad10-YFP. Together, these results support a model in which Saw1 and Rad1-Rad10 are recruited as a complex to short and long flaps in all phases of cell cycle, but that Saw1 is only required for recruitment of Rad1-Rad10 to DSBs when long flaps are formed in G1.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Quebras de DNA de Cadeia DuplaRESUMO
SAW1, coding for Saw1, is required for single-strand annealing (SSA) DNA double-strand break (DSB) repair in Saccharomycescerevisiae. Saw1 physically associates with Rad1 and Rad52 and recruits the Rad1-Rad10 endonuclease. Herein we show by fluorescence microscopy that SAW1 is similarly required for recruitment of Rad10 to sites of Synthesis-Dependent Strand Annealing (SDSA) and associates with sites of SDSA repair in a manner temporally overlapped with Rad10. The magnitude of induction of colocalized Saw1-CFP/Rad10-YFP/DSB-RFP foci in SDSA is more dramatic in S and G2 phase cells than in M phase, consistent with the known mechanism of SDSA. We observed a substantial fraction of foci in which Rad10 was localized to the repair site without Saw1, but few DSB sites that contained Saw1 without Rad10. Together these data are consistent with a model in which Saw1 recruits Rad1-Rad10 to SDSA sites, possibly even binding as a protein-protein complex, but departs the repair site in advance of Rad1-Rad10.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA Bacteriano/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Deleção de Genes , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/análise , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismoRESUMO
SAW1 is required by the Rad1-Rad10 nuclease for efficient removal of 3' non-homologous DNA ends (flaps) formed as intermediates during two modes of double-strand break repair in S. cerevisiae, single-strand annealing (SSA) and synthesis-dependent strand annealing (SDSA). Saw1 was shown in vitro to exhibit increasing affinity for flap DNAs as flap lengths varied from 0 to 40 deoxynucleotides (nt) with almost no binding observed when flaps were shorter than 10 nt. Accordingly, our prior in vivo fluorescence microscopy investigation showed that SAW1 was not required for recruitment of Rad10-YFP to DNA double-strand breaks (DSBs) when flaps were â¼10 nt, but it was required when flaps were â¼500 nt in G1 phase of the cell cycle. We were curious whether we would also observe an increased requirement of SAW1 for Rad10 recruitment in vivo as flaps varied from â¼20 to 50 nt, as was shown in vitro. In this investigation, we utilized SSA substrates that generate 20, 30, and 50 nt flaps in vivo in fluorescence microscopy assays and determined that SAW1 becomes increasingly necessary for SSA starting at about â¼20 nt and is completely required at â¼50 nt. Quantitative PCR experiments corroborate these results by demonstrating that repair product formation decreases in the absence of SAW1 as flap length increases. Experiments with strains containing fluorescently labeled Saw1 (Saw1-CFP) show that Saw1 localizes with Rad10 at SSA foci and that about half of the foci containing Rad10 at DSBs do not contain Saw1. Colocalization patterns of Saw1-CFP are consistent regardless of the flap length of the substrate and are roughly similar in all phases of the cell cycle. Together, these data show that Saw1 becomes increasingly important for Rad1-Rad10 recruitment and SSA repair in the â¼20-50 nt flap range, and Saw1 is present at repair sites even when not required and may depart the repair site ahead of Rad1-Rad10.