RESUMEN
Here, we report a subtilase gene (SBT1.1) specifically expressed in the endosperm of Medicago truncatula and Pisum sativum seeds during development, which is located at a chromosomal position coinciding with a seed weight quantitative trait locus (QTL). Association studies between SBT1.1 polymorphisms and seed weights in ecotype collections provided further evidence for linkage disequilibrium between the SBT1.1 locus and a seed weight locus. To investigate the possible contribution of SBT1.1 to the control of seed weight, a search for TILLING (Targeting Induced Local Lesions in Genomes) mutants was performed. An inspection of seed phenotype revealed a decreased weight and area of the sbt1.1 mutant seeds, thus inferring a role of SBT1.1 in the control of seed size in the forage and grain legume species. Microscopic analyses of the embryo, representing the major part of the seed, revealed a reduced number of cells in the MtP330S mutant, but no significant variation in cell size. SBT1.1 is therefore most likely to be involved in the control of cotyledon cell number, rather than cell expansion, during seed development. This raises the hypothesis of a role of SBT1.1 in the regulation of seed size by providing molecules that can act as signals to control cell division within the embryo.
Asunto(s)
Endospermo/enzimología , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/metabolismo , Semillas/crecimiento & desarrollo , Subtilisinas/metabolismo , Recuento de Células , División Celular , Endospermo/crecimiento & desarrollo , Regulación Enzimológica de la Expresión Génica , Genes de Plantas , Desequilibrio de Ligamiento , Medicago truncatula/enzimología , Medicago truncatula/genética , Medicago truncatula/crecimiento & desarrollo , Mutación , Pisum sativum/enzimología , Pisum sativum/genética , Pisum sativum/crecimiento & desarrollo , Fenotipo , Filogenia , Proteínas de Plantas/genética , Sitios de Carácter Cuantitativo , Semillas/enzimología , Subtilisinas/genéticaRESUMEN
Transposable elements have been widely used as mutagens in many organisms. Among them, the maize transposable element En/Spm has been shown to transpose efficiently in several plant species including the model plant Arabidopsis, where it has been used for large-scale mutagenesis. To determine whether we could use this transposon as a mutagen in the model legume plant Medicago truncatula, we tested the activity of the autonomous element, as well as two defective elements, in this plant, and in Arabidopsis as a positive control. In agreement with previous reports, we observed efficient excision of the autonomous En/Spm element in A. thaliana. This element was also active in M. truncatula, but the transposition activity was low and was apparently restricted to the tissue culture step necessary for the production of transgenic plants. The activity of one of the defective transposable elements, dSpm, was very low in A. thaliana and even lower in M. truncatula. The use of different sources of transposases suggested that this defect in transposition was associated with the dSpm element itself. Transposition of the other defective element, I6078, was also detected in M. truncatula, but, as observed with the autonomous element, transposition events were very rare and occurred during tissue culture. These results suggest that the En/Spm element is rapidly inactivated in the regenerated plants and their progeny, and therefore is not suitable for routine insertion mutagenesis in M. truncatula.
Asunto(s)
Arabidopsis/genética , Elementos Transponibles de ADN , Medicago/genética , Zea mays/genética , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Cartilla de ADN/química , ADN Bacteriano/genética , ADN de Plantas/genética , Glucuronidasa/genética , Glucuronidasa/metabolismo , Kanamicina/farmacología , Medicago/crecimiento & desarrollo , Datos de Secuencia Molecular , Mutagénesis Insercional , Fenotipo , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Recombinación Genética , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Transformación Genética , Transposasas/metabolismoRESUMEN
Proteins of the closely related homeodomain-leucine zipper (HD-Zip) families I and II in plants are putative transcription factors that interact with similar pseudopalindromic DNA recognition sites. We have previously described the Oshox1 gene from rice, which encodes an HD-Zip II protein. To identify further rice HD-Zip proteins, one-hybrid screens were performed in yeast strains containing a HIS3 reporter gene with upstream HD-Zip recognition sites. This resulted in the isolation of six new cDNAs encoding HD-Zip proteins belonging to family I (Oshox4, -5, -6) or family II (Oshox2, -3, -7). In transient assays, using rice suspension-cultured cells transformed by particle bombardment, we showed previously that Oshox1 can transcriptionally repress the activity of reporter gene constructs with upstream HD-Zip binding sites. Here, we confirm the repression properties of Oshox1 by showing that the repression function can be conferred on a heterologous DNA-binding domain. This portable functional domain (residues 1-155) is located proximal to the HD-Zip domain. In yeast, the same region of the Oshox1 protein was found to confer transcriptional activation instead of repression, pointing to the possibility that cell type-specific factors may determine the functional properties of the Oshox1 protein in rice. Like Oshox1, another HD-Zip family II protein (Oshox3) was also found to function as a transcriptional repressor in rice cells. In contrast, two HD-Zip I family proteins (Oshox4 and -5) appeared to act as activators in both rice and yeast cells. Results of two-hybrid assays and electrophoretic mobility shift assays strongly suggest that all HD-Zip proteins of families I and II can form homodimers and also heterodimers with all HD-Zip proteins of the same family. Heterodimerization across the HD-Zip families I and II apparently does not to occur.