RESUMEN
Proteins of the 14-3-3 family have well-defined functions as regulators of plant primary metabolism and ion homeostasis. However, neither their function nor action mechanism in plant hormonal signaling have been fully addressed. Here we show that abscisic acid (ABA) affects both expression and protein levels of five 14-3-3 isoforms in embryonic barley roots. As ABA prolongs the presence of 14-3-3 proteins in the elongating radicle, we tested whether 14-3-3s are instrumental in ABA action using RNA interference. Transient co-expression of 14-3-3 RNAi constructs along with an ABA-responsive promoter showed that each 14-3-3 is functional in generating an ABA response. In a yeast two-hybrid screen, we identified three new 14-3-3 interactors that belong to the ABF protein family. Moreover, using a yeast two-hybrid assay, we show that the transcription factor HvABI5, which binds to cis-acting elements of the ABA-inducible HVA1 promoter, interacts with three of the five 14-3-3s. Our analyses identify two 14-3-3 binding motifs in HvABI5 that are essential for 14-3-3 binding and proper in vivo trans-activation activity of HvABI5. In line with these results, 14-3-3 silencing effectively blocks trans-activation. Our results indicate that 14-3-3 genes/proteins are not only under the control of ABA, but that they control ABA action as well.
Asunto(s)
Proteínas 14-3-3/metabolismo , Ácido Abscísico/farmacología , Germinación/fisiología , Hordeum/metabolismo , Semillas/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/fisiología , Secuencia de Aminoácidos , Sitios de Unión , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Germinación/efectos de los fármacos , Germinación/genética , Glucuronidasa/genética , Glucuronidasa/metabolismo , Hordeum/genética , Datos de Secuencia Molecular , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiología , Regiones Promotoras Genéticas/genética , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferencia de ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Semillas/genética , Semillas/crecimiento & desarrollo , Alineación de Secuencia , Transducción de Señal , Técnicas del Sistema de Dos HíbridosRESUMEN
The highly conserved family of 14-3-3 proteins function in the regulation of a wide variety of cellular processes. The presence of multiple 14-3-3 isoforms and the diversity of cellular processes regulated by 14-3-3 suggest functional isoform specificity of 14-3-3 isoforms in the regulation of target proteins. Indeed, several studies observed differences in affinity and functionality of 14-3-3 isoforms. However, the structural variation by which isoform specificity is accomplished remains unclear. Because other reports suggest that specificity is found in differential expression and availability of 14-3-3 isoforms, we used the nitrate reductase (NR) model system to analyse the availability and functionality of the three barley 14-3-3 isoforms. We found that 14-3-3C is unavailable in dark harvested barley leaf extract and 14-3-3A is functionally not capable to efficiently inhibit NR activity, leaving 14-3-3B as the only characterized isoform able to regulate NR in barley. Further, using site directed mutagenesis, we identified a single amino acid variation (Gly versus Ser) in loop 8 of the 14-3-3 proteins that plays an important role in the observed isoform specificity. Mutating the Gly residue of 14-3-3A to the alternative residue, as found in 14-3-3B and 14-3-3C, turned it into a potent inhibitor of NR activity. Using surface plasmon resonance, we show that the ability of 14-3-3A and the mutated version to inhibit NR activity correlates well with their binding affinity for the 14-3-3 binding motif in the NR protein, indicating involvement of this residue in ligand discrimination. These results suggest that both the availability of 14-3-3 isoforms as well as binding affinity determine isoform-specific regulation of NR activity.
Asunto(s)
Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Hordeum/enzimología , Nitrato-Reductasa/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas 14-3-3/fisiología , Secuencia de Aminoácidos , Sitios de Unión , Hordeum/genética , Hordeum/metabolismo , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas de Plantas/fisiología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiología , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
Germination of seeds proceeds in general in two phases, an initial imbibition phase and a subsequent growth phase. In grasses like barley, the latter phase is evident as the emergence of the embryonic root (radicle). The hormone abscisic acid (ABA) inhibits germination because it prevents the embryo from entering and completing the growth phase. Genetic and physiological studies have identified many steps in the ABA signal transduction cascade, but how it prevents radicle elongation is still not clear. For elongation growth to proceed, uptake of osmotically active substances (mainly K(+)) is essential. Therefore, we have addressed the question of how the activity of K(+) permeable ion channels in the plasma membrane of radicle cells is regulated under conditions of slow (+ABA) and rapid germination (+fusicoccin). We found that ABA arrests radicle growth, inhibits net K(+) uptake and reduces the activity of K(+) (in) channels as measured with the patch-clamp technique. In contrast, fusicoccin (FC), a well-known stimulator of germination, stimulates radicle growth, net K(+) uptake and reduces the activity of K(+) (out) channels. Both types of channels are under the control of 14-3-3 proteins, known as integral components of signal transduction pathways and instrumental in FC action. Intriguingly, 14-3-3 affected both channels in an opposite fashion: whereas K(+) (in) channel activity was fully dependent upon 14-3-3 proteins, K(+) (out) channel activity was reduced by 14-3-3 proteins by 60%. Together with previous data showing that 14-3-3 proteins control the activity of the plasma membrane H(+)-ATPase, this makes 14-3-3 a prime candidate for molecular master regulator of the cellular osmo-pump. Regulation of the osmo-pump activity by ABA and FC is an important mechanism in controlling the growth of the embryonic root during seed germination.