RESUMEN
Germ cells are indispensable carriers of genetic information from one generation to the next. In contrast to the well-understood process in animals, information on the mechanism of germ cell initiation in plants is very limited. SPOROCYTELESS/NOZZLE was previously identified as an essential regulator of diploid germ cell (archesporial cell) differentiation in the stamens and ovules of Arabidopsis (Arabidopsis thaliana). Although SPOROCYTELESS (SPL) transcription is activated by the floral organ identity regulator AGAMOUS and epigenetically regulated by SET DOMAIN GROUP2, little is known about the regulation of the SPL protein. Here, we report that the protein kinases MPK3 and MPK6 can both interact with SPL in vitro and in vivo and can phosphorylate the SPL protein in vitro. In addition, phosphorylation of the SPL protein by MPK3/6 is required for SPL function in the Arabidopsis anther, as measured by its effect on archesporial cell differentiation. We further demonstrate that phosphorylation enhances SPL protein stability. This work not only uncovers the importance of SPL phosphorylation for its regulatory role in Arabidopsis anther development, but also supports the hypothesis that the regulation of precise spatiotemporal patterning of germ cell initiation and that differentiation is achieved progressively through multiple levels of regulation, including transcriptional and posttranslational modification.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Flores/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Diferenciación Celular/genética , Flores/genética , Flores/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Células Germinativas de las Plantas/citología , Células Germinativas de las Plantas/metabolismo , Inmunohistoquímica , Microscopía Fluorescente , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Mutación , Proteínas Nucleares/genética , Fosforilación , Plantas Modificadas Genéticamente , Unión Proteica , Estabilidad Proteica , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The involvement of cytoskeleton-related proteins in regulating mitochondrial respiration has been revealed in mammalian cells. However, it is unclear if there is a relationship between the microtubule-based motor protein kinesin and mitochondrial respiration. In this research, we demonstrate that a plant-specific kinesin, Kinesin-like protein 1 (KP1; At KIN14 h), is involved in respiratory regulation during seed germination at a low temperature. Using in vitro biochemical methods and in vivo transgenic cell observations, we demonstrate that KP1 is able to localize to mitochondria via its tail domain (C terminus) and specifically interacts with a mitochondrial outer membrane protein, voltage-dependent anion channel 3 (VDAC3). Targeting of the KP1-tail to mitochondria is dependent on the presence of VDAC3. When grown at 4° C, KP1 dominant-negative mutants (TAILOEs) and vdac3 mutants exhibited a higher seed germination frequency. All germinating seeds of the kp1 and vdac3 mutants had increased oxygen consumption; the respiration balance between the cytochrome pathway and the alternative oxidase pathway was disrupted, and the ATP level was reduced. We conclude that the plant-specific kinesin, KP1, specifically interacts with VDAC3 on the mitochondrial outer membrane and that both KP1 and VDAC3 regulate aerobic respiration during seed germination at low temperature.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Germinación , Cinesinas/metabolismo , Proteínas Mitocondriales/metabolismo , Canales Aniónicos Dependientes del Voltaje/metabolismo , Adenosina Trifosfato/análisis , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Respiración de la Célula , Citrato (si)-Sintasa/análisis , Frío , Cinesinas/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Oxígeno/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Semillas/crecimiento & desarrollo , Semillas/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , TransgenesRESUMEN
A pea actin isoform PEAc1 with green fluorescent protein (GFP) fusion to its C-terminus and His-tag to its N-terminus, was expressed in prokaryotic cells in soluble form, and highly purified with Ni-Chelating Sepharose Fast Flow column. The purified fusion protein (PEAc1-GFP) efficiently inhibited DNase I activities before polymerization, and activated the myosin Mg-ATPase activities after polymerization. The PEAc1-GFP also polymerized into green fluorescent filamentous structures with a critical concentration of 0.75 uM. These filamentous structures were labeled by TRITC-phalloidin, a specific agent for staining actin microfilaments, and identified as having 9 nm diameters by negative staining. These results indicated that PEAc1 preserved the essential characteristics of actin even with His-tag and GFP fusion, suggesting a promising potential to use GFP fusion protein in obtaining soluble plant actin isoform to analyze its physical and biochemical properties in vitro. The PEAc1-GFP was also expressed in tobacco BY2 cells, which offers a new pathway for further studying its distribution and function in vivo.