RESUMEN
In most flowering plant species, pollination and fertilization occur during the hot summer, so plants must have evolved a mechanism that ensures normal growth of their pollen tubes at high temperatures. Despite its importance to plant reproduction, little is known about the molecular basis of thermotolerance in pollen tubes. Here we report the identification and characterization of a novel Arabidopsis gene, Thermosensitive Male Sterile 1 (TMS1), which plays an important role in thermotolerance of pollen tubes. TMS1 encodes a Hsp40-homologous protein with a DnaJ domain and an a_ERdj5_C domain found in protein disulfide isomerases (PDI). Purified TMS1 expressed in Escherichia coli (BL21 DE3) had the reductive activity of PDI. TMS1 was expressed in pollen grains, pollen tubes and other vegetative tissues, including leaves, stems and roots. Heat shock treatment at 37 degrees C increased its expression levels in growing pollen tubes as well as in vegetative tissues. A knockout mutation in TMS1 grown at 30 degrees C had greatly retarded pollen tube growth in the transmitting tract, resulting in a significant reduction in male fertility. Our study suggests that TMS1 is required for thermotolerance of pollen tubes in Arabidopsis, possibly by functioning as a co-molecular chaperone.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Choque Térmico/metabolismo , Infertilidad Vegetal/genética , Tubo Polínico/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Clonación Molecular , Escherichia coli/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Prueba de Complementación Genética , Proteínas de Choque Térmico/genética , Respuesta al Choque Térmico/genética , Calor , Mutación , Fenotipo , Tubo Polínico/crecimiento & desarrollo , ARN de Planta/metabolismoRESUMEN
Previously, we reported that the TAPETUM DETERMINANT1 (TPD1) gene is required for specialization of tapetal cells in the Arabidopsis (Arabidopsis thaliana) anther. The tpd1 mutant is phenotypically identical to the excess microsporocytes1 (ems1)/extra sporogenous cells (exs) mutant. The TPD1 and EMS1/EXS genes may function in the same developmental pathway in the Arabidopsis anther. Here, we further report that overexpression of TPD1 alters the cell fates in the Arabidopsis carpel and tapetum. When TPD1 was expressed ectopically in the wild-type Arabidopsis carpel, the number of cells in the carpel increased significantly, showing that the ectopic expression of TPD1 protein could activate the cell division in the carpel. Furthermore, the genetic analysis showed that the activation of cell division in the transgenic carpel by TPD1 was dependent on EMS1/EXS, as it did not happen in the ems1/exs mutant. This result further suggests that TPD1 regulates cell fates in coordination with EMS1/EXS. Moreover, overexpression of TPD1 in tapetal cells also delayed the degeneration of tapetum. The TPD1 may function not only in the specialization of tapetal cells but also in the maintenance of tapetal cell fate.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Arabidopsis/metabolismo , Flores/citología , Flores/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Diferenciación Celular , División Celular , Flores/genética , Expresión Génica , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Mutación , Plantas Modificadas Genéticamente , Regiones Promotoras GenéticasRESUMEN
In flowering plants, penetration of the pollen tube through stigma, style, and transmitting tract is essential for delivery of sperm nuclei to the egg cells embedded deeply within female tissues. Despite its importance in plant reproduction, little is known about the underlying molecular mechanisms that regulate the navigation of the pollen tube through the stigma, style, and transmitting tract. Here, we report the identification and characterization of an Arabidopsis thaliana gene, VANGUARD1 (VGD1) that encodes a pectin methylesterase (PME)-homologous protein of 595 amino acids and is required for enhancing the growth of pollen tubes in the style and transmitting tract tissues. VGD1 was expressed specifically in pollen grain and the pollen tube. The VGD1 protein was distributed throughout the pollen grain and pollen tube, including the plasma membrane and cell wall. Functional interruption of VGD1 reduced PME activity in the pollen to 82% of the wild type and greatly retarded the growth of the pollen tube in the style and transmitting tract, resulting in a significant reduction of male fertility. In addition, the vgd1 pollen tubes were unstable and burst more frequently when germinated and grown on in vitro culture medium, compared with wild-type pollen tubes. Our study suggests that the VGD1 product is required for growth of the pollen tube, possibly via modifying the cell wall and enhancing the interaction of the pollen tube with the female style and transmitting tract tissues.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Hidrolasas de Éster Carboxílico/genética , Flores/crecimiento & desarrollo , Polen/crecimiento & desarrollo , Secuencia de Aminoácidos , Mapeo Cromosómico , Clonación Molecular , Flores/genética , Prueba de Complementación Genética , Germinación , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Mutación , Fenotipo , Polen/genética , Polen/ultraestructura , Proteínas Recombinantes de Fusión/genética , Homología de Secuencia de AminoácidoRESUMEN
The plant life cycle involves an alternation of generations between sporophyte and gametophyte. Currently, the genes and pathways involved in gametophytic development and function in flowering plants remain largely unknown. A large-scale mutant screen of Ds transposon insertion lines was employed to identify 130 mutants of Arabidopsis thaliana with defects in female gametophyte development and function. A wide variety of mutant phenotypes were observed, ranging from defects in different stages of early embryo sac development to mutants with apparently normal embryo sacs, but exhibiting defects in processes such as pollen tube guidance, fertilization or early embryo development. Unexpectedly, nearly half of the mutants isolated in this study were found to be primarily defective in post-fertilization processes dependent on the maternal allele, suggesting that genes expressed from the female gametophyte or the maternal genome play a major role in the early development of plant embryos. Sequence identification of the genes disrupted in the mutants revealed genes involved in protein degradation, cell death, signal transduction and transcriptional regulation required for embryo sac development, fertilization and early embryogenesis. These results provide a first comprehensive overview of the genes and gene products involved in female gametophyte development and function within a flowering plant.
Asunto(s)
Arabidopsis/citología , Arabidopsis/genética , Gametogénesis/genética , Gametogénesis/fisiología , Genes de Plantas/genética , Arabidopsis/embriología , Fertilización/genética , Mutación/genética , FenotipoRESUMEN
In flowering plants, pollen formation depends on the differentiation and interaction of two cell types in the anther: the reproductive cells, called microsporocytes, and somatic cells that form the tapetum. The microsporocytes generate microspores, whereas the tapetal cells support the development of microspores into mature pollen grains. Despite their importance to plant reproduction, little is known about the underlying genetic mechanisms that regulate the differentiation and interaction of these highly specialized cells in the anther. Here, we report the identification and characterization of a novel tapetum determinant1 (TPD1) gene that is required for the specialization of tapetal cells in the Arabidopsis anther. Analysis of the male-sterile mutant, tpd1, showed that functional interruption of TPD1 caused the precursors of tapetal cells to differentiate and develop into microsporocytes instead of tapetum. As a results, extra microsporocytes were formed and tapetum was absent in developing tpd1 anthers. Molecular cloning of TPD1 revealed that it encodes a small protein of 176 amino acids. In addition, tpd1 was phenotypically similar to excess microsporocytes1/extra sporogenous cells (ems1/exs) single and tpd1 ems1/exs double mutants. These data suggest that the TPD1 product plays an important role in the differentiation of tapetal cells, possibly in coordination with the EMS1/EXS gene product, a Leu-rich repeat receptor protein kinase.