RESUMEN
The promoter of the RECEPTOR-LIKE CYTOPLASMIC KINASE VI_A2 (RLCK VI_A2) gene contains nine binding sites for the REPLUMLESS (RPL) transcription factor. In agreement, the expression of the kinase gene was strongly downregulated in the rpl-4 mutant. Comparing phenotypes of loss-of-function mutants, it was revealed that both genes are involved in stem growth, phyllotaxis, organization of the vascular tissues, and the replum, highlighting potential functional interactions. The expression of the RLCKVI_A2 gene from the constitutive 35S promoter could not complement the rpl-4 phenotypes but exhibited a dominant positive effect on stem growth and affected vascular differentiation and organization. The results also indicated that the number of vascular bundles is regulated independently from stem thickness. Although our study cannot demonstrate a direct link between the RPL and RLVKVI_A2 genes, it highlights the significance of the proper developmental regulation of the RLCKVI_A2 promoter for balanced stem development.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Frutas , Regulación de la Expresión Génica de las Plantas , Regiones Promotoras Genéticas , Factores de Transcripción , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Frutas/genética , Frutas/crecimiento & desarrollo , Frutas/metabolismo , Brotes de la Planta/crecimiento & desarrollo , Brotes de la Planta/genética , Brotes de la Planta/metabolismoRESUMEN
Mitochondria are important sources of energy in plants and are implicated in coordination of a number of metabolic and physiological processes including stabilization of redox balance, synthesis and turnover of a number of metabolites, and control of programmed cell death. Mitochondrial electron transport chain (mETC) is the backbone of the energy producing process which can influence other processes as well. Accumulating evidence suggests that mETC can affect responses to environmental stimuli and modulate tolerance to extreme conditions such as drought or salinity. Screening for stress responses of 13 Arabidopsis mitochondria-related T-DNA insertion mutants, we identified ndufs8.2-1 which has an increased ability to withstand osmotic and oxidative stresses compared to wild type plants. Insertion in ndufs8.2-1 disrupted the gene that encodes the NADH dehydrogenase [ubiquinone] fragment S subunit 8 (NDUFS8) a component of Complex I of mETC. ndufs8.2-1 tolerated reduced water availability, retained photosynthetic activity and recovered from severe water stress with higher efficiency compared to wild type plants. Several mitochondrial functions were altered in the mutant including oxygen consumption, ROS production, ATP and ADP content as well as activities of genes encoding alternative oxidase 1A (AOX1A) and various alternative NAD(P)H dehydrogenases (ND). Our results suggest that in the absence of NDUFS8.2 stress-induced ROS generation is restrained leading to reduced oxidative damage and improved tolerance to water deficiency. mETC components can be implicated in redox and energy homeostasis and modulate responses to stresses associated with reduced water availability.
Asunto(s)
Arabidopsis , Mitocondrias , Especies Reactivas de Oxígeno/metabolismo , Mitocondrias/metabolismo , Oxidación-Reducción , Arabidopsis/metabolismo , Fotosíntesis , Regulación de la Expresión Génica de las PlantasRESUMEN
Nitric oxide (NO) is a versatile signal molecule that mediates environmental and hormonal signals orchestrating plant development. NO may act via reversible S-nitrosation of proteins during which an NO moiety is added to a cysteine thiol to form an S-nitrosothiol. In plants, several proteins implicated in hormonal signaling have been reported to undergo S-nitrosation. Here, we report that the Arabidopsis ROP2 GTPase is a further potential target of NO-mediated regulation. The ROP2 GTPase was found to be required for the root shortening effect of NO. NO inhibits primary root growth by altering the abundance and distribution of the PIN1 auxin efflux carrier protein and lowering the accumulation of auxin in the root meristem. In rop2-1 insertion mutants, however, wild-type-like root size of the NO-treated roots were maintained in agreement with wild-type-like PIN1 abundance in the meristem. The ROP2 GTPase was shown to be S-nitrosated in vitro, suggesting that NO might directly regulate the GTPase. The potential mechanisms of NO-mediated ROP2 GTPase regulation and ROP2-mediated NO signaling in the primary root meristem are discussed.
RESUMEN
The plant-specific receptor-like cytoplasmic kinases (RLCKs) form a large, poorly characterized family. Members of the RLCK VI_A class of dicots have a unique characteristic: their activity is regulated by Rho-of-plants (ROP) GTPases. The biological function of one of these kinases was investigated using a T-DNA insertion mutant and RNA interference. Loss of RLCK VI_A2 function resulted in restricted cell expansion and seedling growth. Although these phenotypes could be rescued by exogenous gibberellin, the mutant did not exhibit lower levels of active gibberellins nor decreased gibberellin sensitivity. Transcriptome analysis confirmed that gibberellin is not the direct target of the kinase; its absence rather affected the metabolism and signalling of other hormones such as auxin. It is hypothesized that gibberellins and the RLCK VI_A2 kinase act in parallel to regulate cell expansion and plant growth. Gene expression studies also indicated that the kinase might have an overlapping role with the transcription factor circuit (PIF4-BZR1-ARF6) controlling skotomorphogenesis-related hypocotyl/cotyledon elongation. Furthermore, the transcriptomic changes revealed that the loss of RLCK VI_A2 function alters cellular processes that are associated with cell membranes, take place at the cell periphery or in the apoplast, and are related to cellular transport and/or cell wall reorganisation.
Asunto(s)
Arabidopsis/genética , Cotiledón/genética , Regulación de la Expresión Génica de las Plantas , Hipocótilo/genética , Proteínas Serina-Treonina Quinasas/genética , Plantones/genética , Arabidopsis/efectos de los fármacos , Arabidopsis/enzimología , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Cotiledón/efectos de los fármacos , Cotiledón/enzimología , Cotiledón/crecimiento & desarrollo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Giberelinas/metabolismo , Giberelinas/farmacología , Hipocótilo/efectos de los fármacos , Hipocótilo/enzimología , Hipocótilo/crecimiento & desarrollo , Ácidos Indolacéticos/metabolismo , Ácidos Indolacéticos/farmacología , Mutagénesis Insercional , Reguladores del Crecimiento de las Plantas/farmacología , Plantas Modificadas Genéticamente , Proteínas Serina-Treonina Quinasas/metabolismo , Plantones/efectos de los fármacos , Plantones/enzimología , Plantones/crecimiento & desarrollo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
The fine tuning of hormone (e.g., auxin and gibberellin) levels and hormone signaling is required for maintaining normal embryogenesis. Embryo polarity, for example, is ensured by the directional movement of auxin that is controlled by various types of auxin transporters. Here, we present pieces of evidence for the auxin-gibberellic acid (GA) hormonal crosstalk during embryo development and the regulatory role of the Arabidopsis thaliana Calcium-Dependent Protein Kinase-Related Kinase 5 (AtCRK5) in this regard. It is pointed out that the embryogenesis of the Atcrk5-1 mutant is delayed in comparison to the wild type. This delay is accompanied with a decrease in the levels of GA and auxin, as well as the abundance of the polar auxin transport (PAT) proteins PIN1, PIN4, and PIN7 in the mutant embryos. We have previously showed that AtCRK5 can regulate the PIN2 and PIN3 proteins either directly by phosphorylation or indirectly affecting the GA level during the root gravitropic and hypocotyl hook bending responses. In this manuscript, we provide evidence that the AtCRK5 protein kinase can in vitro phosphorylate the hydrophilic loops of additional PIN proteins that are important for embryogenesis. We propose that AtCRK5 can govern embryo development in Arabidopsis through the fine tuning of auxin-GA level and the accumulation of certain polar auxin transport proteins.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Germinación , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Superficie Celular/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica de las Plantas , Giberelinas/análisis , Giberelinas/metabolismo , Ácidos Indolacéticos/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Semillas/anatomía & histología , Semillas/crecimiento & desarrollo , Semillas/metabolismoRESUMEN
Seedling establishment following germination requires the fine tuning of plant hormone levels including that of auxin. Directional movement of auxin has a central role in the associated processes, among others, in hypocotyl hook development. Regulated auxin transport is ensured by several transporters (PINs, AUX1, ABCB) and their tight cooperation. Here we describe the regulatory role of the Arabidopsis thaliana CRK5 protein kinase during hypocotyl hook formation/opening influencing auxin transport and the auxin-ethylene-GA hormonal crosstalk. It was found that the Atcrk5-1 mutant exhibits an impaired hypocotyl hook establishment phenotype resulting only in limited bending in the dark. The Atcrk5-1 mutant proved to be deficient in the maintenance of local auxin accumulation at the concave side of the hypocotyl hook as demonstrated by decreased fluorescence of the auxin sensor DR5::GFP. Abundance of the polar auxin transport (PAT) proteins PIN3, PIN7, and AUX1 were also decreased in the Atcrk5-1 hypocotyl hook. The AtCRK5 protein kinase was reported to regulate PIN2 protein activity by phosphorylation during the root gravitropic response. Here it is shown that AtCRK5 can also phosphorylate in vitro the hydrophilic loops of PIN3. We propose that AtCRK5 may regulate hypocotyl hook formation in Arabidopsis thaliana through the phosphorylation of polar auxin transport (PAT) proteins, the fine tuning of auxin transport, and consequently the coordination of auxin-ethylene-GA levels.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Hipocótilo/fisiología , Morfogénesis , Desarrollo de la Planta , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Superficie Celular/metabolismo , Arabidopsis/efectos de los fármacos , Biomarcadores , Regulación de la Expresión Génica de las Plantas , Genes Reporteros , Germinación , Morfogénesis/efectos de los fármacos , Morfogénesis/genética , Fenotipo , Fosforilación , Desarrollo de la Planta/efectos de los fármacos , Desarrollo de la Planta/genética , Transducción de Señal , Xantonas/farmacologíaRESUMEN
The Calcium-Dependent Protein Kinase (CDPK)-Related Kinase family (CRKs) consists of eight members in Arabidopsis. Recently, AtCRK5 was shown to play a direct role in the regulation of root gravitropic response involving polar auxin transport (PAT). However, limited information is available about the function of the other AtCRK genes. Here, we report a comparative analysis of the Arabidopsis CRK genes, including transcription regulation, intracellular localization, and biological function. AtCRK transcripts were detectable in all organs tested and a considerable variation in transcript levels was detected among them. Most AtCRK proteins localized at the plasma membrane as revealed by microscopic analysis of 35S::cCRK-GFP (Green Fluorescence Protein) expressing plants or protoplasts. Interestingly, 35S::cCRK1-GFP and 35S::cCRK7-GFP had a dual localization pattern which was associated with plasma membrane and endomembrane structures, as well. Analysis of T-DNA insertion mutants revealed that AtCRK genes are important for root growth and control of gravitropic responses in roots and hypocotyls. While Atcrk mutants were indistinguishable from wild type plants in short days, Atcrk1-1 mutant had serious growth defects under continuous illumination. Semi-dwarf phenotype of Atcrk1-1 was accompanied with chlorophyll depletion, disturbed photosynthesis, accumulation of singlet oxygen, and enhanced cell death in photosynthetic tissues. AtCRK1 is therefore important to maintain cellular homeostasis during continuous illumination.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Unión al Calcio/genética , Fotosíntesis , Proteínas Quinasas/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Unión al Calcio/metabolismo , Membrana Celular/metabolismo , Clorofila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Hipocótilo/genética , Hipocótilo/crecimiento & desarrollo , Hipocótilo/metabolismo , Mutación , Fenotipo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Proteínas Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Luz SolarRESUMEN
Plant size, shape and color are important parameters of plants, which have traditionally been measured by destructive and time-consuming methods. Non-destructive image analysis is an increasingly popular technology to characterize plant development in time. High throughput automatic phenotyping platforms can simultaneously analyze multiple morphological and physiological parameters of hundreds or thousands of plants. Such platforms are, however, expensive and are not affordable for many laboratories. Moreover, determination of basic parameters is sufficient for most studies. Here we describe a non-invasive method, which simultaneously measures basic morphological and physiological parameters of in vitro cultured plants. Changes of plant size, shape and color is monitored by repeated photography with a commercial digital camera using neutral white background. Images are analyzed with the MatLab-based computer application PlantSize, which simultaneously calculates several parameters including rosette size, convex area, convex ratio, chlorophyll and anthocyanin contents of all plants identified on the image. Numerical data are exported in MS Excel-compatible format. Subsequent data processing provides information on growth rates, chlorophyll and anthocyanin contents. Proof-of-concept validation of the imaging technology was demonstrated by revealing small but significant differences between wild type and transgenic Arabidopsis plants overexpressing the HSFA4A transcription factor or the hsfa4a knockout mutant, subjected to different stress conditions. While HSFA4A overexpression was associated with better growth, higher chlorophyll and lower anthocyanin content in saline conditions, the knockout hsfa4a mutant showed hypersensitivity to various stresses. Morphological differences were revealed by comparing rosette size, shape and color of wild type plants with phytochrome B (phyB-9) mutant. While the technology was developed with Arabidopsis plants, it is suitable to characterize plants of other species including crops, in a simple, affordable and fast way. PlantSize is publicly available (http://www.brc.hu/pub/psize/index.html).
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 HíbridosRESUMEN
Extremophile plants are valuable sources of genes conferring tolerance traits, which can be explored to improve stress tolerance of crops. Lepidium crassifolium is a halophytic relative of the model plant Arabidopsis thaliana, and displays tolerance to salt, osmotic and oxidative stresses. We have employed the modified Conditional cDNA Overexpression System to transfer a cDNA library from L. crassifolium to the glycophyte A. thaliana. By screening for salt, osmotic and oxidative stress tolerance through in vitro growth assays and non-destructive chlorophyll fluorescence imaging, 20 Arabidopsis lines were identified with superior performance under restrictive conditions. Several cDNA inserts were cloned and confirmed to be responsible for the enhanced tolerance by analysing independent transgenic lines. Examples include full-length cDNAs encoding proteins with high homologies to GDSL-lipase/esterase or acyl CoA-binding protein or proteins without known function, which could confer tolerance to one or several stress conditions. Our results confirm that random gene transfer from stress tolerant to sensitive plant species is a valuable tool to discover novel genes with potential for biotechnological applications.
Asunto(s)
Deshidratación , Técnicas Genéticas , Lepidium/genética , Estrés Oxidativo , Plantas Tolerantes a la Sal/genética , Arabidopsis , Biblioteca de Genes , Genes de Plantas , ParaquatRESUMEN
To investigate the mechanism of phytochrome action in vivo, NtPHYB, AtPHYB and phyD:green fluorescent protein (GFP) were overexpressed in Nicotiana plumbaginifolia and Arabidopsis thaliana. The expression of 35S:NtPHYB:GFP and 35S:AtPHYB:GFP complemented the tobacco hgl2 and Arabidopsis phyB-9 mutations, whereas the 35S:AtPHYD:GFP only rescued the hgl2 mutant. All three fusion proteins are transported into the nucleus in all genetic backgrounds. These data indicate that AtPHYD:GFP is biologically active and functions as the main red light receptor in transgenic tobacco, and establish an experimental system for the functional analysis of this elusive photoreceptor in vivo.