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1.
Plant Cell Rep ; 37(4): 627-639, 2018 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-29340786

RESUMEN

KEY MESSAGE: Several amino acid motifs required for Rop-dependent activity were found to form a common surface on RLCKVI_A kinases. This indicates a unique mechanism for Rho-type GTPase-mediated kinase activation in plants. Rho-of-plants (Rop) G-proteins are implicated in the regulation of various cellular processes, including cell growth, cell polarity, hormonal and pathogen responses. Our knowledge about the signalling pathways downstream of Rops is continuously increasing. However, there are still substantial gaps in this knowledge. One reason for this is that these pathways are considerably different from those described for yeast and/or animal Rho-type GTPases. Among others, plants lack all Rho/Rac/Cdc42-activated kinase families. Only a small group of plant-specific receptor-like cytoplasmic kinases (RLCK VI_A) has been shown to exhibit Rop-binding-dependent in vitro activity. These kinases do not carry any known GTPase-binding motifs. Based on the sequence comparison of the Rop-activated RLCK VI_A and the closely related but constitutively active RLCK VI_B kinases, several distinguishing amino acid residues/motifs were identified. All but one of these were found to be required for the Rop-mediated regulation of the in vitro activity of two RLCK VI_A kinases. Structural modelling indicated that these motifs might form a common Rop-binding surface. Based on in silico data mining, kinases that have the identified Rop-binding motifs are present in Embryophyta but not in unicellular green algae. It can, therefore, be supposed that Rops recruited these plant-specific kinases for signalling at an early stage of land plant evolution.


Asunto(s)
Proteínas Algáceas/genética , Secuencias de Aminoácidos/genética , Proteínas de Unión al GTP/genética , Proteínas de Plantas/genética , Proteínas Quinasas/genética , Proteínas Algáceas/metabolismo , Secuencia de Aminoácidos , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Simulación por Computador , Proteínas de Unión al GTP/metabolismo , Modelos Moleculares , Fosforilación , Proteínas de Plantas/metabolismo , Unión Proteica , Dominios Proteicos , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos Híbridos
2.
Plant Sci ; 267: 124-134, 2018 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-29362091

RESUMEN

Plant nucleosome assembly protein-related proteins (NRPs) are histone chaperons involved in nucleosome turnover. Despite this basic cellular function, the Arabidopsis nrp1-1 nrp2-1 knock out mutant has been reported to exhibit only mild seedling root phenotypes and to significantly affect the expression of only few hundred genes Zhu et al. (2006). Here we report that NRP loss-of-function as well as the ectopic overexpression of At NRP1 significantly affected the growth, development, and the pathogen response of Arabidopsis plants under short day conditions. The nrp1-1 nrp2-1 mutant grew faster and flowered weeks earlier than the wild type and the overexpressor. The latter developed slower and flowered at a lower number of leaves than the mutant and the wild type. Moreover, the mutant was more sensitive, the overexpressor was more tolerant to pathogen-induced necrosis correlating with their more adult and juvenile character, respectively. Transcriptomic comparison of mature non-bolting plants agreed with the phenotypes. The presented and other published data indicate that although NRPs might not be absolutely required for normal plant growth and development, their level needs to be controlled to allow the epigenetic coordination of metabolic, growth, defence and developmental processes during the acclimation to unfavourable growth conditions such as short days.


Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/fisiología , Flores/genética , Chaperonas Moleculares/genética , Aclimatación , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/inmunología , Proteínas de Arabidopsis/metabolismo , Flores/crecimiento & desarrollo , Chaperonas Moleculares/metabolismo , Fenotipo , Inmunidad de la Planta
3.
PLoS One ; 12(11): e0187094, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29121646

RESUMEN

The small GTPases of the Rho family comprising RhoA, Rac1 and Cdc42 function as molecular switches controlling several essential biochemical pathways in eukaryotic cells. Their activity is cycling between an active GTP-bound and an inactive GDP-bound conformation. The exchange of GDP to GTP is catalyzed by guanine nucleotide exchange factors (GEFs). Here we report a novel regulatory mechanism of Rac1 activity, which is controlled by a phosphomimetic (Ser179Glu) mutant of syndecan-4 (SDC4). SDC4 is a ubiquitously expressed transmembrane, heparan sulfate proteoglycan. In this study we show that the Ser179Glu mutant binds strongly Tiam1, a Rac1-GEF reducing Rac1-GTP by 3-fold in MCF-7 breast adenocarcinoma cells. Mutational analysis unravels the PDZ interaction between SDC4 and Tiam1 is indispensable for the suppression of the Rac1 activity. Neither of the SDC4 interactions is effective alone to block the Rac1 activity, on the contrary, lack of either of interactions can increase the activity of Rac1, therefore the Rac1 activity is the resultant of the inhibitory and stimulatory effects. In addition, SDC4 can bind and tether RhoGDI1 (GDP-dissociation inhibitor 1) to the membrane. Expression of the phosphomimetic SDC4 results in the accumulation of the Rac1-RhoGDI1 complex. Co-immunoprecipitation assays (co-IP-s) reveal that SDC4 can form complexes with RhoGDI1. Together, the regulation of the basal activity of Rac1 is fine tuned and SDC4 is implicated in multiple ways.


Asunto(s)
Mutación/genética , Sindecano-4/genética , Sindecano-4/metabolismo , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T/química , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Secuencia de Aminoácidos , Humanos , Células MCF-7 , Modelos Biológicos , Dominios PDZ , Unión Proteica , Proteína Quinasa C-alfa/metabolismo , Sindecano-4/química , Quinasas p21 Activadas/metabolismo , Inhibidor alfa de Disociación del Nucleótido Guanina rho/metabolismo
4.
Plant Physiol ; 165(1): 319-34, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24676858

RESUMEN

Heat shock factors (HSFs) are principal regulators of plant responses to several abiotic stresses. Here, we show that estradiol-dependent induction of HSFA4A confers enhanced tolerance to salt and oxidative agents, whereas inactivation of HSFA4A results in hypersensitivity to salt stress in Arabidopsis (Arabidopsis thaliana). Estradiol induction of HSFA4A in transgenic plants decreases, while the knockout hsfa4a mutation elevates hydrogen peroxide accumulation and lipid peroxidation. Overexpression of HSFA4A alters the transcription of a large set of genes regulated by oxidative stress. In yeast (Saccharomyces cerevisiae) two-hybrid and bimolecular fluorescence complementation assays, HSFA4A shows homomeric interaction, which is reduced by alanine replacement of three conserved cysteine residues. HSFA4A interacts with mitogen-activated protein kinases MPK3 and MPK6 in yeast and plant cells. MPK3 and MPK6 phosphorylate HSFA4A in vitro on three distinct sites, serine-309 being the major phosphorylation site. Activation of the MPK3 and MPK6 mitogen-activated protein kinase pathway led to the transcriptional activation of the HEAT SHOCK PROTEIN17.6A gene. In agreement that mutation of serine-309 to alanine strongly diminished phosphorylation of HSFA4A, it also strongly reduced the transcriptional activation of HEAT SHOCK PROTEIN17.6A. These data suggest that HSFA4A is a substrate of the MPK3/MPK6 signaling and that it regulates stress responses in Arabidopsis.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Estrés Oxidativo , Tolerancia a la Sal , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Células Cultivadas , ADN Bacteriano/genética , Estradiol/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas , Datos de Secuencia Molecular , Mutagénesis Insercional/genética , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Fosforilación/efectos de los fármacos , Plantas Modificadas Genéticamente , Unión Proteica/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Salinidad , Tolerancia a la Sal/efectos de los fármacos , Tolerancia a la Sal/genética , Cloruro de Sodio/farmacología , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Transcripción Genética/efectos de los fármacos , Transformación Genética/efectos de los fármacos
5.
Plant Cell Rep ; 32(3): 339-48, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23160639

RESUMEN

KEY MESSAGE : Wheat genes differentially expressed in the egg cell before and after fertilization were identified. The data support zygotic gene activation before the first cell division in wheat. To have an insight into fertilization-induced gene expression, cDNA libraries have been prepared from isolated wheat egg cells and one-celled zygotes. Two-hundred and twenty-six egg cell and 253 zygote-expressed EST sequences were determined. Most of the represented transcripts were detected in the wheat egg cell or zygote transcriptome at the first time. Expression analysis of fourteen of the identified genes and three controls was carried out by real-time quantitative PCR. The preferential expression of all investigated genes in the female gametophyte-derived samples (egg cells, zygotes, two-celled proembryos, and basal ovule parts with synergids) in comparison to the anthers, and the leaves were verified. Three genes with putative signaling/regulatory functions were expressed at a low level in the egg cell but exhibited increased (2-to-33-fold) relative expression in the zygote and the proembryo. Genes with high EST abundance in cDNA libraries exhibited strong expression in the egg cell and the zygote, while the ones coding for unknown or hypothetical proteins exhibited differential expression patterns with preferential transcript accumulation in egg cells and/or zygotes. The obtained data support the activation of the zygotic genome before the first cell division in wheat.


Asunto(s)
Regulación de la Expresión Génica de las Plantas/genética , Óvulo Vegetal/genética , Triticum/genética , Etiquetas de Secuencia Expresada , Fertilización , Expresión Génica , Perfilación de la Expresión Génica , Biblioteca de Genes , Genoma de Planta/genética , Especificidad de Órganos , Reacción en Cadena en Tiempo Real de la Polimerasa , Semillas/genética , Cigoto
6.
Plant Physiol ; 159(1): 311-20, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22415513

RESUMEN

Certain plant receptor-like cytoplasmic kinases were reported to interact with small monomeric G-proteins of the RHO of plant (ROP; also called RAC) family in planta and to be activated by this interaction in vitro. We identified a barley (Hordeum vulgare) partial cDNA of a ROP binding protein kinase (HvRBK1) in yeast (Saccharomyces cerevisiae) two-hybrid screenings with barley HvROP bait proteins. Protein interaction of the constitutively activated (CA) barley HvROPs CA HvRACB and CA HvRAC1 with full-length HvRBK1 was verified in yeast and in planta. Green fluorescent protein-tagged HvRBK1 appears in the cytoplasm and nucleoplasm, but CA HvRACB or CA HvRAC1 can recruit green fluorescent protein-HvRBK1 to the cell periphery. Barley HvRBK1 is an active kinase in vitro, and activity is enhanced by CA HvRACB or GTP-loaded HvRAC1. Hence, HvRBK1 might act downstream of active HvROPs. Transient-induced gene silencing of barley HvRBK1 supported penetration by the parasitic fungus Blumeria graminis f. sp. hordei, suggesting a function of the protein in basal disease resistance. Transient knockdown of HvRBK1 also influenced the stability of cortical microtubules in barley epidermal cells. Hence, HvRBK1 might function in basal resistance to powdery mildew by influencing microtubule organization.


Asunto(s)
Ascomicetos/patogenicidad , Resistencia a la Enfermedad , Hordeum/microbiología , Microtúbulos/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , ADN Complementario/genética , ADN Complementario/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Genes de Plantas , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hordeum/enzimología , Hordeum/genética , Microtúbulos/genética , Datos de Secuencia Molecular , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Epidermis de la Planta/genética , Epidermis de la Planta/metabolismo , Inmunidad de la Planta , Proteínas de Plantas/genética , Mapeo de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
7.
Plant Physiol Biochem ; 52: 162-8, 2012 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-22285370

RESUMEN

SET/I(2)(PP2A), a member of the family of nucleosome assembly proteins (NAPs), has been previously described as a multifunctional protein inhibiting protein phosphatase 2A (PP2A)-mediated histone H3((pSer10)) dephosphorylation during the heat shock response in animal cells. In the present work we demonstrate that its plant orthologs, designated as NAP-related proteins (NRPs), have a similar in vitro biochemical activity and interact with PP2A and histone H3((pSer10))in vivo. Although heat shock gene promoters were found to be associated with histone H3((pSer10))-marked chromatin following a high temperature treatment, heat shock gene expression was not affected in NRP-deficient mutant Arabidopsis thaliana (L.) plantlets. These observations indicate that NRPs are potential regulators of histone dephosphorylation in plants, but they are dispensable for gene expression reorganization in response to heat shock.


Asunto(s)
Arabidopsis/enzimología , Proteínas de Choque Térmico/genética , Medicago sativa/enzimología , Nucleosomas/metabolismo , Proteína Fosfatasa 2/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Anticuerpos , Arabidopsis/genética , Ensamble y Desensamble de Cromatina , Inhibidores Enzimáticos , Expresión Génica , Histonas/genética , Histonas/metabolismo , Calor , Medicago sativa/genética , Datos de Secuencia Molecular , Mutación , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiones Promotoras Genéticas/genética , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Conejos , Alineación de Secuencia
8.
Plant J ; 66(4): 669-79, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21309864

RESUMEN

Plant ROP (Rho of plants) proteins form a unique subgroup within the family of Rho-type small G-proteins of eukaryotes. In this paper we demonstrate that the phosphomimetic mutation of a serine residue conserved in all Rho proteins affects the signaling properties of plant ROPs. We found that the S74E mutation in Medicago ROP6 and Arabidopsis ROP4 prevented the binding of these proteins to their plant-specific upstream activator the plant-specific ROP nucleotide exchanger (PRONE)-domain-containing RopGEF (guanine nucleotide exchange factor) protein and abolished the PRONE-mediated nucleotide exchange reaction in vitro. Structural modeling supported the hypothesis that potential phosphorylation of the S74 residue interferes with the binding of the PRONE-domain to the adjacent plant-specific R76 residue which plays an important role in functional ROP-PRONE interaction. Moreover, we show that while the binding of constitutively active MsROP6 to the effector protein RIC (ROP-interactive CRIB-motif-containing protein) was not affected by the S74E mutation, the capability of this mutated protein to bind and activate the RRK1 kinase in vitro was reduced. These observations are in agreement with the morphology of tobacco pollen tubes expressing mutant forms of yellow fluorescent protein (YFP):MsROP6. The S74E mutation in MsROP6 had no influence on pollen tube morphology and attenuated the phenotype of a constitutively active form of MsROP6. The presented Medicago and Arabidopsis data support the notion that the phosphorylation of the serine residue in ROPs corresponding to S74 in Medicago ROP6 could be a general principle for regulating ROP activation and signaling in plants.


Asunto(s)
Arabidopsis/genética , Medicago truncatula/genética , Proteínas de Plantas/metabolismo , Serina/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Clonación Molecular , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Medicago truncatula/anatomía & histología , Medicago truncatula/metabolismo , Modelos Moleculares , Mutación , Fosforilación , Proteínas de Plantas/genética , Polen/anatomía & histología , Polen/genética , Unión Proteica , Mapeo de Interacción de Proteínas , Proteínas Recombinantes/metabolismo , Serina/genética , Transducción de Señal , Nicotiana/genética
9.
Biochim Biophys Acta ; 1759(1-2): 108-15, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-16603258

RESUMEN

Three cDNA clones coding for Medicago sativa Rop GTPases have been isolated. The represented genes could be assigned to various linkage groups by genetic mapping. They were expressed in all investigated plant organs, although at different level. Relative gene expression patterns in response to Sinorhizobium infection of roots as well as during somatic embryogenesis indicated their differential participation in these processes. DNA sequences coding for altogether six different Medicago sp. Rop GTPases could be identified in sequence databases. Based on their homology to each other and to their Arabidopsis counterparts, a unified nomenclature is suggested for Medicago Rop GTPases.


Asunto(s)
Medicago sativa/genética , Proteínas de Unión al GTP rho/genética , Mapeo Cromosómico , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Desarrollo Embrionario , Regulación de la Expresión Génica de las Plantas/fisiología , Medicago sativa/enzimología , Proteínas de Plantas/genética , Estructuras de las Plantas/embriología , Estructuras de las Plantas/genética , Estructuras de las Plantas/microbiología , Sinorhizobium , Terminología como Asunto
10.
Plant J ; 43(6): 849-60, 2005 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16146524

RESUMEN

It is now well established that nitric oxide (NO) serves as a signaling molecule in plant cells. In this paper experimental data are presented which indicate that NO can stimulate the activation of cell division and embryogenic cell formation in leaf protoplast-derived cells of alfalfa in the presence of auxin. It was found that various NO-releasing compounds promoted auxin-dependent division (as shown by incorporation of bromodeoxyuridine) of leaf protoplast-derived alfalfa cells. In contrast, application of NO scavenger or NO synthesis inhibitor inhibited the same process. Both the promotion and the inhibition of cell cycle activation correlated with the amount and activity of the cognate alfalfa p34cdc2 protein Medsa;CDKA;1,2. The effect of l-NG-monomethyl-L-arginine (L-NMMA) was transient, and protoplast-derived cells spending more than 3 days in culture become insensitive to the inhibitor as far as cell cycle progression was concerned. L-NMMA had no effect on the cell cycle parameters of cycling suspension-cultured cells, but had a moderate transient inhibitory effect on cells re-entering the cell cycle following phosphate starvation. Cycling cultured cells, however, could respond to NO, as indicated by the sodium nitroprusside (SNP)- and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO)-dependent accumulation of the ferritin protein. Based on these observations, it is hypothesized that L-NMMA-sensitive generation of NO is involved in the activation, but not the progression of the plant cell division cycle. In addition, SNP promoted and L-NMMA delayed the exogenous auxin [2,4-dichlorophenoxyacetic acid (2,4-D)] concentration-dependent formation of embryogenic cell clusters expressing the MsSERK1 gene; this further supports a link between auxin- and NO-dependent signaling pathways in plant cells.


Asunto(s)
Ciclo Celular/fisiología , Ácidos Indolacéticos/fisiología , Medicago sativa/fisiología , Óxido Nítrico/fisiología , Semillas/fisiología , Ácido 2,4-Diclorofenoxiacético/farmacología , Células Cultivadas , Herbicidas/farmacología , Medicago sativa/citología , Medicago sativa/efectos de los fármacos , Medicago sativa/embriología , Semillas/citología , omega-N-Metilarginina/farmacología
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