RESUMEN
A plant-specific gene was cloned from melon fruit. This gene was named downward leaf curling (CmDLC) based on the phenotype of transgenic Arabidopsis plants overexpressing the gene. This expression level of this gene was especially upregulated during melon fruit enlargement. Overexpression of CmDLC in Arabidopsis resulted in dwarfism and narrow, epinastically curled leaves. These phenotypes were found to be caused by a reduction in cell number and cell size on the adaxial and abaxial sides of the epidermis, with a greater reduction on the abaxial side of the leaves. These phenotypic characteristics, combined with the more wavy morphology of epidermal cells in overexpression lines, indicate that CmDLC overexpression affects cell elongation and cell morphology. To investigate intracellular protein localization, a CmDLC-GFP fusion protein was made and expressed in onion epidermal cells. This protein was observed to be preferentially localized close to the cell membrane. Thus, we report here a new plant-specific gene that is localized to the cell membrane and that controls leaf cell number, size and morphology.
Asunto(s)
Arabidopsis/genética , Cucumis melo/genética , Proteínas de la Membrana/genética , Hojas de la Planta/genética , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Arabidopsis/citología , Arabidopsis/crecimiento & desarrollo , Clonación Molecular , Cucumis melo/citología , Cucumis melo/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Datos de Secuencia Molecular , Cebollas/citología , Cebollas/genética , Cebollas/metabolismo , Fenotipo , Hojas de la Planta/citología , Hojas de la Planta/crecimiento & desarrollo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Calpain, a calcium-dependent cysteine protease, plays an essential role in basic cellular processes in animal cells, including cell proliferation, apoptosis, and differentiation. NbDEK encodes the calpain homolog of N. benthamiana. In this study, virus-induced gene silencing (VIGS) of NbDEK resulted in arrested organ development and hyperplasia in all the major plant organs examined. The epidermal layers of the leaves and stems were covered with hyperproliferating cell masses, and stomata and trichome development was severely inhibited. During flower development, a single dome-like structure was grown from the flower meristem to generate a large cylinder-shaped flower lacking any floral organs. At the cellular level, cell division was sustained in tissues that were otherwise already differentiated, and cell differentiation was severely hampered. NbDEK is ubiquitously expressed in all the plant tissues examined. In the abnormal organs of the NbDEK VIGS lines, protein levels of D-type cyclins (CycD)2, CycD3, and proliferating cell nuclear antigen (PCNA) were greatly elevated, and transcription of E2F (E2 promoter binding factor), E2F-regulated genes, retinoblastoma (Rb), and KNOTTED1 (KN1)-type homeobox genes was also stimulated. These results suggest that phytocalpain is a key regulator of cell proliferation and differentiation during plant organogenesis, and that it acts partly by controlling the CycD/Rb pathway.