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1.
J Plant Res ; 126(1): 131-9, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22847900

RESUMEN

Plants have developed certain adaptive responses to environmental stresses that cause adverse effects on growth. To identify genes involved in the adaptive mechanisms, we constructed a large population of transgenic Arabidopsis expressing rice full-length cDNAs, and performed gain-of-function screening under high-salinity stress. In this study, we identified a rice R2R3-type MYB transcription factor gene, JAmyb, as a gene whose overexpression causes tolerance to high salinity. JAmyb overexpression in transgenic Arabidopsis improved tolerance to high-salinity stress during seed germination, seedling growth, and root elongation. In rice seedlings, JAmyb expression was induced by high-salinity and high-osmotic stresses and reactive oxygen species (ROS), suggesting that JAmyb is responsible for abiotic stress response. Microarray analysis showed that the overexpression of JAmyb stimulates the expression of several defense-associated genes, some of which have been predicted to be involved in osmotic adjustment, ROS removal, and ion homeostasis. Several transcription factors involved in the jasmonate (JA)-mediated stress response are also regulated by JAmyb. JAmyb has been reported to be associated with disease response. Our observations suggest that JAmyb plays a role in JA-mediated abiotic stress response in addition to biotic stress response in rice.


Asunto(s)
Adaptación Fisiológica/genética , Arabidopsis/genética , Oryza/genética , Tolerancia a la Sal/genética , Estrés Fisiológico/genética , Factores de Transcripción/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Germinación/genética , Homeostasis/genética , Presión Osmótica/fisiología , Raíces de Plantas/crecimiento & desarrollo , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Especies Reactivas de Oxígeno , Salinidad , Plantones/crecimiento & desarrollo , Semillas/crecimiento & desarrollo , Cloruro de Sodio/metabolismo
2.
PLoS One ; 7(10): e46805, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23071642

RESUMEN

Brassinosteroids (BRs) affect a wide range of developmental processes in plants and compromised production or signalling of BRs causes severe growth defects. To identify new regulators of plant organ growth, we searched the Arabidopsis FOX (Full-length cDNA Over-eXpressor gene) collection for mutants with altered organ size and isolated two overexpression lines that display typical BR deficient dwarf phenotypes. The phenotype of these lines, caused by an overexpression of a putative acyltransferase gene PIZZA (PIZ), was partly rescued by supplying exogenous brassinolide (BL) and castasterone (CS), indicating that endogenous BR levels are rate-limiting for the growth of PIZ overexpression lines. Our transcript analysis further showed that PIZ overexpression leads to an elevated expression of genes involved in BR biosynthesis and a reduced expression of BR inactivating hydroxylases, a transcriptional response typical to low BR levels. Taking the advantage of relatively high endogenous BR accumulation in a mild bri1-301 background, we found that overexpression of PIZ results in moderately reduced levels of BL and CS and a strong reduction of typhasterol (TY) and 6-deoxocastasterone (6-deoxoCS), suggesting a role of PIZ in BR metabolism. We tested a set of potential substrates in vitro for heterologously expressed PIZ and confirmed its acyltransferase activity with BL, CS and TY. The PIZ gene is expressed in various tissues but as reported for other genes involved in BR metabolism, the loss-of-function mutants did not display obvious growth phenotypes under standard growth conditions. Together, our data suggest that PIZ can modify BRs by acylation and that these properties might help modulating endogenous BR levels in Arabidopsis.


Asunto(s)
Aciltransferasas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Brasinoesteroides/metabolismo , Acilación , Aciltransferasas/clasificación , Aciltransferasas/genética , Secuencia de Aminoácidos , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/clasificación , Proteínas de Arabidopsis/genética , Vías Biosintéticas , Brasinoesteroides/química , Brasinoesteroides/farmacología , Colestanoles/farmacología , Flores/efectos de los fármacos , Flores/genética , Flores/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Estructura Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Filogenia , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Plantones/efectos de los fármacos , Plantones/genética , Plantones/metabolismo , Homología de Secuencia de Aminoácido , Esteroides Heterocíclicos/farmacología
3.
Proc Natl Acad Sci U S A ; 109(24): 9653-8, 2012 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-22645333

RESUMEN

Movement of the plant hormone abscisic acid (ABA) within plants has been documented; however, the molecular mechanisms that regulate ABA transport are not fully understood. By using a modified yeast two-hybrid system, we screened Arabidopsis cDNAs capable of inducing interactions between the ABA receptor PYR/PYL/RCAR and PP2C protein phosphatase under low ABA concentrations. By using this approach, we identified four members of the NRT1/PTR family as candidates for ABA importers. Transport assays in yeast and insect cells demonstrated that at least one of the candidates ABA-IMPORTING TRANSPORTER (AIT) 1, which had been characterized as the low-affinity nitrate transporter NRT1.2, mediates cellular ABA uptake. Compared with WT, the ait1/nrt1.2 mutants were less sensitive to exogenously applied ABA during seed germination and/or postgermination growth, whereas overexpression of AIT1/NRT1.2 resulted in ABA hypersensitivity in the same conditions. Interestingly, the inflorescence stems of ait1/nrt1.2 had a lower surface temperature than those of the WT because of excess water loss from open stomata. We detected promoter activities of AIT1/NRT1.2 around vascular tissues in inflorescence stems, leaves, and roots. These data suggest that the function of AIT1/NRT1.2 as an ABA importer at the site of ABA biosynthesis is important for the regulation of stomatal aperture in inflorescence stems.


Asunto(s)
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Técnicas del Sistema de Dos Híbridos
4.
Methods Mol Biol ; 847: 75-90, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22351001

RESUMEN

As a result of the progress in sequencing technology, many plant genomes have now been determined. Functional genomics is required to clarify gene function in many of these species. To identify useful genes easily and quickly, we have developed a FOX (full-length cDNA overexpressor) hunting system in which full-length cDNAs are overexpressed in Arabidopsis plants. This system was applied to high-throughput analysis of rice genes through heterologous expression in Arabidopsis (rice FOX Arabidopsis lines). We demonstrated that it is possible to carry out high-throughput analysis of gene function by utilizing rice FOX Arabidopsis lines. In this protocol, we describe how to isolate candidate rice FOX Arabidopsis lines and to determine the rice fl-cDNA that is responsible for the observed phenotype.


Asunto(s)
Arabidopsis/genética , Arabidopsis/metabolismo , ADN Complementario , Genes de Plantas , Oryza/genética , Agrobacterium/genética , Escherichia coli/genética , Ferredoxina-NADP Reductasa/biosíntesis , Ferredoxina-NADP Reductasa/genética , Regulación de la Expresión Génica de las Plantas , Biblioteca de Genes , Técnicas de Transferencia de Gen , Genoma de Planta , Ensayos Analíticos de Alto Rendimiento , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Regiones Promotoras Genéticas , Transformación Genética , Ubiquitina/genética
5.
Plant Physiol ; 157(1): 96-108, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21734114

RESUMEN

Ferredoxin-NADP(+)-oxidoreductase (FNR) mediates electron transfer between ferredoxin (Fd) and NADP(+); therefore, it is a key enzyme that provides the reducing power used in the Calvin cycle. Other than FNR, nitrite reductase, sulfite reductase, glutamate synthase, and Fd-thioredoxin reductase also accept electrons from Fd, an electron carrier protein in the stroma. Therefore, the regulation of electron partitioning in the chloroplast is important for photosynthesis and other metabolic pathways. The regulatory mechanism of electron partitioning, however, remains to be elucidated. We found, by taking advantage of a gain-of-function approach, that expression of two rice (Oryza sativa) full-length cDNAs of leaf-type FNRs (OsLFNR1 and OsLFNR2) led to altered chlorophyll fluorescence and growth in Arabidopsis (Arabidopsis thaliana) and rice. We revealed that overexpression of the OsLFNR1 and OsLFNR2 full-length cDNAs resulted in distinct phenotypes despite the high sequence similarity between them. Expression of OsLFNR1 affected the nitrogen assimilation pathway without inhibition of photosynthesis under normal conditions. On the other hand, OsLFNR2 expression led to the impairment of photosynthetic linear electron transport as well as Fd-dependent cyclic electron flow around photosystem I. The endogenous protein level of OsLFNR was found to be suppressed in both OsLFNR1- and OsLFNR2-overexpressing rice plants, leading to changes in the stoichiometry of the two LFNR isoforms within the thylakoid and soluble fractions. Thus, we propose that the stoichiometry of two LFNR isoforms plays an important role in electron partitioning between carbon fixation and nitrogen assimilation.


Asunto(s)
Arabidopsis/genética , Ferredoxina-NADP Reductasa/metabolismo , Isoenzimas/metabolismo , Oryza/enzimología , Hojas de la Planta/enzimología , Secuencia de Aminoácidos , Clorofila/metabolismo , ADN Complementario , Transporte de Electrón , Ferredoxina-NADP Reductasa/química , Ferredoxina-NADP Reductasa/genética , Perfilación de la Expresión Génica , Genes de Plantas , Datos de Secuencia Molecular , Oryza/genética , ARN Mensajero/genética , Homología de Secuencia de Aminoácido , Espectrometría de Fluorescencia
6.
Methods Mol Biol ; 729: 183-97, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21365491

RESUMEN

Generation and characterization of mutants are important for the investigation of gene function. Gain-of-function technology is one of the most useful approaches for the systematic production of mutant resources. Full-length cDNAs have been collected from various plant species and have become important resources for functional genomics. We have developed a novel gain-of-function technology for the identification of gene function using a full-length cDNA library, and this system has been named as FOX hunting system (Full-length cDNA Over-eXpressing gene hunting system). In this system, full-length cDNAs are randomly expressed in Arabidopsis. We also generated rice FOX Arabidopsis lines in which full-length cDNAs from rice were expressed in Arabidopsis, and we demonstrated that gene function derived from heterologous organisms can be analyzed systematically using the FOX hunting approach. In this protocol, we describe the process of generating Arabidopsis mutants expressing rice full-length cDNA libraries and the methods of identifying genes from the isolated mutants.


Asunto(s)
Biblioteca de Genes , Genes de Plantas , Ingeniería Genética/métodos , Plantas Modificadas Genéticamente/genética , Arabidopsis/genética , ADN Complementario/genética , ADN de Plantas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Mutación , Oryza/genética , Fenotipo , Rhizobium/genética , Rhizobium/metabolismo , Análisis de Secuencia
7.
Methods Mol Biol ; 678: 77-89, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-20931374

RESUMEN

Full-length cDNAs (fl-cDNAs) are important resources for the characterization of gene function, since they contain all the information required for the production of functional RNAs and proteins. Large sets of fl-cDNA clones have been collected from several plant species and have become available for functional genomic analysis. We have developed a system for the identification of gene function by screening for transgenic plants ectopically expressing fl-cDNAs and named it the FOX (fl-cDNA overexpressor gene) hunting system. This system can be applied to almost all plant species without prior knowledge of their genome sequences because only fl-cDNAs are required. For utilization of the FOX hunting system, Agrobacterium libraries and Arabidopsis seeds carrying rice and Arabidopsis fl-cDNAs are available. Here, we will describe the procedure followed in the FOX hunting system from the generation of expression vectors carrying fl-cDNAs to the confirmation of phenotype in retransformed plants.


Asunto(s)
ADN Complementario/genética , Plantas Modificadas Genéticamente/genética , Arabidopsis/genética , Electroporación , Vectores Genéticos/genética , Modelos Biológicos , Oryza/genética , Reacción en Cadena de la Polimerasa , Rhizobium/genética , Transformación Genética/genética
8.
Plant Biotechnol J ; 9(4): 466-85, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-20955180

RESUMEN

Approximately 20,000 of the rice-FOX Arabidopsis transgenic lines, which overexpress 13,000 rice full-length cDNAs at random in Arabidopsis, were screened for bacterial disease resistance by dip inoculation with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). The identities of the overexpressed genes were determined in 72 lines that showed consistent resistance after three independent screens. Pst DC3000 resistance was verified for 19 genes by characterizing other independent Arabidopsis lines for the same genes in the original rice-FOX hunting population or obtained by reintroducing the genes into ecotype Columbia by floral dip transformation. Thirteen lines of these 72 selections were also resistant to the fungal pathogen Colletotrichum higginsianum. Eight genes that conferred resistance to Pst DC3000 in Arabidopsis have been introduced into rice for overexpression, and transformants were evaluated for resistance to the rice bacterial pathogen, Xanthomonas oryzae pv. oryzae. One of the transgenic rice lines was highly resistant to Xanthomonas oryzae pv. oryzae. Interestingly, this line also showed remarkably high resistance to Magnaporthe grisea, the fungal pathogen causing rice blast, which is the most devastating rice disease in many countries. The causal rice gene, encoding a putative receptor-like cytoplasmic kinase, was therefore designated as BROAD-SPECTRUM RESISTANCE 1. Our results demonstrate the utility of the rice-FOX Arabidopsis lines as a tool for the identification of genes involved in plant defence and suggest the presence of a defence mechanism common between monocots and dicots.


Asunto(s)
Arabidopsis/genética , Arabidopsis/microbiología , Oryza/genética , Oryza/microbiología , Enfermedades de las Plantas/genética , Pseudomonas syringae/patogenicidad , Arabidopsis/enzimología , Clonación Molecular , Colletotrichum/patogenicidad , Regulación de la Expresión Génica de las Plantas , Variación Genética , Inmunidad Innata , Magnaporthe/patogenicidad , Oryza/enzimología , Enfermedades de las Plantas/microbiología , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/microbiología , Transgenes , Xanthomonas/patogenicidad
9.
J Exp Bot ; 62(2): 557-69, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20876334

RESUMEN

Environmental stresses are major factors in limiting plant growth and crop production. To find genes improving salt tolerance, the screening of a large population of transgenic Arabidopsis thaliana that expressed rice full-length cDNAs under salinity stress is reported here. In this study one of the isolated salt-tolerant lines, R07303 was analysed in detail. An uncharacterized rice gene CHLOROPLAST PROTEIN-ENHANCING STRESS TOLERANCE (OsCEST) was integrated in R07303. Newly constructed transgenic Arabidopsis that overexpressed OsCEST or its Arabidopsis homologue AtCEST showed improved tolerance to salinity stress. OsCEST and AtCEST were mainly transcribed in photosynthetic tissues. Green fluorescent protein-fused OsCEST and AtCEST proteins were localized to the chloroplast in the Arabidopsis leaf protoplasts. CEST-overexpressing Arabidopsis showed enhanced tolerance not only to salt stress but also to drought stress, high-temperature stress, and paraquat, which causes photooxidative stress. Under saline conditions, overexpression of CESTs modulated the stress-induced impairment of photosynthetic activity and the peroxidation of lipids. Reduced expression of AtCEST because of double-stranded RNA interference resulted in the impairment of photosynthetic activity, the reduction of green pigment, defects in chloroplast development, and growth retardation under light. This paper discusses the relationship between the chloroplast protein CEST and photooxidative damage.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Cloroplastos/metabolismo , Oryza/genética , Estrés Oxidativo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/fisiología , Secuencia de Aminoácidos , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Cloroplastos/química , Cloroplastos/genética , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Calor , Luz , Datos de Secuencia Molecular , Oryza/metabolismo , Estrés Oxidativo/efectos de la radiación , Fotosíntesis , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/química , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/efectos de la radiación , Tolerancia a la Sal , Alineación de Secuencia , Estrés Fisiológico
10.
Plant Cell Physiol ; 52(2): 265-73, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21186176

RESUMEN

Identification of gene function is important not only for basic research but also for applied science, especially with regard to improvements in crop production. For rapid and efficient elucidation of useful traits, we developed a system named FOX hunting (Full-length cDNA Over-eXpressor gene hunting) using full-length cDNAs (fl-cDNAs). A heterologous expression approach provides a solution for the high-throughput characterization of gene functions in agricultural plant species. Since fl-cDNAs contain all the information of functional mRNAs and proteins, we introduced rice fl-cDNAs into Arabidopsis plants for systematic gain-of-function mutation. We generated >30,000 independent Arabidopsis transgenic lines expressing rice fl-cDNAs (rice FOX Arabidopsis mutant lines). These rice FOX Arabidopsis lines were screened systematically for various criteria such as morphology, photosynthesis, UV resistance, element composition, plant hormone profile, metabolite profile/fingerprinting, bacterial resistance, and heat and salt tolerance. The information obtained from these screenings was compiled into a database named 'RiceFOX'. This database contains around 18,000 records of rice FOX Arabidopsis lines and allows users to search against all the observed results, ranging from morphological to invisible traits. The number of searchable items is approximately 100; moreover, the rice FOX Arabidopsis lines can be searched by rice and Arabidopsis gene/protein identifiers, sequence similarity to the introduced rice fl-cDNA and traits. The RiceFOX database is available at http://ricefox.psc.riken.jp/.


Asunto(s)
Arabidopsis/genética , ADN Complementario/genética , Bases de Datos Genéticas , Oryza/genética , Arabidopsis/metabolismo , Análisis por Conglomerados , ADN de Plantas/genética , Genoma de Planta , Internet , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Análisis de Secuencia de ADN , Interfaz Usuario-Computador
11.
Plant Cell Rep ; 30(1): 113-24, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21076835

RESUMEN

In our previous study, a transgenic tomato line that expressed the MIR gene under control of the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator (tNOS) produced the taste-modifying protein miraculin (MIR). However, the concentration of MIR in the tomatoes was lower than that in the MIR gene's native miracle fruit. To increase MIR production, the native MIR terminator (tMIR) was used and a synthetic gene encoding MIR protein (sMIR) was designed to optimize its codon usage for tomato. Four different combinations of these genes and terminators (MIR-tNOS, MIR-tMIR, sMIR-tNOS and sMIR-tMIR) were constructed and used for transformation. The average MIR concentrations in MIR-tNOS, MIR-tMIR, sMIR-tNOS and sMIR-tMIR fruits were 131, 197, 128 and 287 µg/g fresh weight, respectively. The MIR concentrations using tMIR were higher than those using tNOS. The highest MIR accumulation was detected in sMIR-tMIR fruits. On the other hand, the MIR concentration was largely unaffected by sMIR-tNOS. The expression levels of both MIR and sMIR mRNAs terminated by tMIR tended to be higher than those terminated by tNOS. Read-through mRNA transcripts terminated by tNOS were much longer than those terminated by tMIR. These results suggest that tMIR enhances mRNA expression and permits the multiplier effect of optimized codon usage.


Asunto(s)
Codón/genética , Genes de Plantas/genética , Glicoproteínas/metabolismo , Proteínas Recombinantes/metabolismo , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Regiones Terminadoras Genéticas/genética , Secuencia de Bases , Southern Blotting , Frutas/genética , Regulación de la Expresión Génica de las Plantas , Glucuronidasa/metabolismo , Glicoproteínas/genética , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente , Poliadenilación/genética , Transcripción Genética , Transgenes/genética
12.
J Plant Physiol ; 168(2): 181-7, 2011 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-21106274

RESUMEN

Using the full-length cDNA overexpressor (FOX) gene-hunting system, we have generated 130 Arabidopsis FOX-superroot lines in bird's-foot trefoil (Lotus corniculatus) for the systematic functional analysis of genes expressed in roots and for the selection of induced mutants with interesting root growth characteristics. We used the Arabidopsis-FOX Agrobacterium library (constructed by ligating pBIG2113SF) for the Agrobacterium-mediated transformation of superroots (SR) and the subsequent selection of gain-of-function mutants with ectopically expressed Arabidopsis genes. The original superroot culture of L. corniculatus is a unique host system displaying fast root growth in vitro, allowing continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely hormone-free culture conditions. Several of the Arabidopsis FOX-superroot lines show interesting deviations from normal growth and morphology of roots from SR-plants, such as differences in pigmentation, growth rate, length or diameter. Some of these mutations are of potential agricultural interest. Genomic PCR analysis revealed that 100 (76.9%) out of the 130 transgenic lines showed the amplification of single fragments. Sequence analysis of the PCR fragments from these 100 lines identified full-length cDNA in 74 of them. Forty-three out of 74 full-length cDNA carried known genes. The Arabidopsis FOX-superroot lines of L. corniculatus, produced in this study, expand the FOX hunting system and provide a new tool for the genetic analysis and control of root growth in a leguminous forage plant.


Asunto(s)
Arabidopsis/genética , Lotus/crecimiento & desarrollo , Lotus/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/genética , ADN Complementario/genética , Reacción en Cadena de la Polimerasa , Rhizobium/genética
13.
BMC Plant Biol ; 10: 57, 2010 Apr 02.
Artículo en Inglés | MEDLINE | ID: mdl-20359373

RESUMEN

BACKGROUND: Reminiscent of their free-living cyanobacterial ancestor, chloroplasts proliferate by division coupled with the partition of nucleoids (DNA-protein complexes). Division of the chloroplast envelope membrane is performed by constriction of the ring structures at the division site. During division, nucleoids also change their shape and are distributed essentially equally to the daughter chloroplasts. Although several components of the envelope division machinery have been identified and characterized, little is known about the molecular components/mechanisms underlying the change of the nucleoid structure. RESULTS: In order to identify new factors that are involved in the chloroplast division, we isolated Arabidopsis thaliana chloroplast division mutants from a pool of random cDNA-overexpressed lines. We found that the overexpression of a previously uncharacterized gene (AtYLMG1-1) of cyanobacterial origin results in the formation of an irregular network of chloroplast nucleoids, along with a defect in chloroplast division. In contrast, knockdown of AtYLMG1-1 resulted in a concentration of the nucleoids into a few large structures, but did not affect chloroplast division. Immunofluorescence microscopy showed that AtYLMG1-1 localizes in small puncta on thylakoid membranes, to which a subset of nucleoids colocalize. In addition, in the cyanobacterium Synechococcus elongates, overexpression and deletion of ylmG also displayed defects in nucleoid structure and cell division. CONCLUSIONS: These results suggest that the proper distribution of nucleoids requires the YlmG protein, and the mechanism is conserved between cyanobacteria and chloroplasts. Given that ylmG exists in a cell division gene cluster downstream of ftsZ in gram-positive bacteria and that ylmG overexpression impaired the chloroplast division, the nucleoid partitioning by YlmG might be related to chloroplast and cyanobacterial division processes.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Bacterianas/metabolismo , Cloroplastos/metabolismo , ADN/metabolismo , Synechococcus/metabolismo , Secuencia de Aminoácidos , Arabidopsis/citología , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , División Celular , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Datos de Secuencia Molecular , Familia de Multigenes/genética , Mutación/genética , Fenotipo , Filogenia , Transporte de Proteínas
14.
Mol Plant ; 3(1): 125-42, 2010 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20085895

RESUMEN

Plant metabolomics developed as a powerful tool to examine gene functions and to gain deeper insight into the physiology of the plant cell. In this study, we screened Arabidopsis lines overexpressing rice full-length (FL) cDNAs (rice FOX Arabidopsis lines) using a gas chromatography-time-of-flight mass spectrometry (GC-TOF/MS)-based technique to identify rice genes that caused metabolic changes. This screening system allows fast and reliable identification of candidate lines showing altered metabolite profiles. We performed metabolomic and transcriptomic analysis of a rice FOX Arabidopsis line that harbored the FL cDNA of the rice ortholog of the Lateral Organ Boundaries (LOB) Domain (LBD)/Asymmetric Leaves2-like (ASL) gene of Arabidopsis, At-LBD37/ASL39. The investigated rice FOX Arabidopsis line showed prominent changes in the levels of metabolites related to nitrogen metabolism. The transcriptomic data as well as the results from the metabolite analysis of the Arabidopsis At-LBD37/ASL39-overexpressor plants were consistent with these findings. Furthermore, the metabolomic and transcriptomic analysis of the Os-LBD37/ASL39-overexpressing rice plants indicated that Os-LBD37/ASL39 is associated with processes related to nitrogen metabolism in rice. Thus, the combination of a metabolomics-based screening method and a gain-of-function approach is useful for rapid characterization of novel genes in both Arabidopsis and rice.


Asunto(s)
Arabidopsis/metabolismo , Nitrógeno/metabolismo , Oryza/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Arabidopsis/genética , Cromatografía de Gases , Espectrometría de Masas , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Filogenia , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/clasificación , Plantas Modificadas Genéticamente/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
15.
Plant Cell ; 21(8): 2307-22, 2009 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-19717615

RESUMEN

Leaf trichomes in Arabidopsis thaliana develop through several distinct cellular processes, such as patterning, differentiation, and growth. Although recent studies have identified several key transcription factors as regulating early patterning and differentiation steps, it is still largely unknown how these regulatory proteins mediate subsequent trichome development, which is accompanied by rapid cell growth and branching. Here, we report a novel trichome mutation in Arabidopsis, which in contrast with previously identified mutants, increases trichome cell size without altering its overall patterning or branching. We show that the corresponding gene encodes a GT-2-LIKE1 (GTL1) protein, a member of the trihelix transcription factor family. GTL1 is present within the nucleus during the postbranching stages of trichome development, and its loss of function leads to an increase in the nuclear DNA content only in trichomes that have completed branching. Our data further demonstrate that the gtl1 mutation modifies the expression of several cell cycle genes and partially rescues the ploidy defects in the cyclin-dependent kinase inhibitor mutant siamese. Taken together, this study provides the genetic evidence for the requirement of transcriptional regulation in the repression of ploidy-dependent plant cell growth as well as for an involvement of GTL trihelix proteins in this regulation.


Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/citología , Arabidopsis/metabolismo , Hojas de la Planta/citología , Hojas de la Planta/metabolismo , Ploidias , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Caulimovirus/genética , Aumento de la Célula , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Microscopía Fluorescente , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente/citología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Regiones Promotoras Genéticas/genética , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
16.
Plant J ; 60(5): 852-64, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19702666

RESUMEN

Plants are able to sense and respond to changes in the balance between carbon (C) and nitrogen (N) metabolite availability, known as the C/N response. During the transition to photoautotrophic growth following germination, growth of seedlings is arrested if a high external C/N ratio is detected. To clarify the mechanisms for C/N sensing and signaling during this transition period, we screened a large collection of FOX transgenic plants, overexpressing full-length cDNAs, for individuals able to continue post-germinative growth under severe C/N stress. One line, cni1-D (carbon/nitrogen insensitive 1-dominant), was shown to have a suppressed sensitivity to C/N conditions at both the physiological and molecular level. The CNI1 cDNA encoded a predicted RING-type ubiquitin ligase previously annotated as ATL31. Overexpression of ATL31 was confirmed to be responsible for the cni1-D phenotype, and a knock-out of this gene resulted in hypersensitivity to C/N conditions during post-germinative growth. The ATL31 protein was confirmed to contain ubiquitin ligase activity using an in vitro assay system. Moreover, removal of this ubiquitin ligase activity from the overexpressed protein resulted in the loss of the mutant phenotype. Taken together, these data demonstrated that CNI1/ATL31 activity is required for the plant C/N response during seedling growth transition.


Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/enzimología , Carbono/metabolismo , Nitrógeno/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/análisis , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Germinación , Proteínas Fluorescentes Verdes/análisis , Mutación , Cebollas/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Proteínas Recombinantes de Fusión/análisis , Plantones/enzimología , Plantones/genética , Plantones/crecimiento & desarrollo , Ubiquitina-Proteína Ligasas/análisis , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación
17.
Plant Mol Biol ; 71(4-5): 391-402, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19653105

RESUMEN

Plant growth and crop production are limited by environmental stress. We used a large population of transgenic Arabidopsis expressing rice full-length cDNAs to isolate the rice genes that improve the tolerance of plants to environmental stress. By sowing T2 seeds of the transgenic lines under conditions of salinity stress, the salt-tolerant line R07047 was isolated. It expressed a rice gene, OsSMCP1, which encodes a small protein with a single C2 domain, a Ca(2+)-dependent membrane-targeting domain. Retransformation of wild-type Arabidopsis revealed that OsSMCP1 is responsible for conferring the salt tolerance. It is particularly interesting that R07047 and newly constructed OsSMCP1-overexpressing Arabidopsis showed enhanced tolerance not only to high salinity but also to osmotic, dehydrative, and oxidative stresses. Furthermore, R07047 showed improved resistance to Pseudomonas syringae. The OsSMCP1 expression in rice is constitutive. Particle-bombardment-mediated transient expression analysis revealed that OsSMCP1 is targeted to plastids in rice epidermal cells. It induced overexpression of several nuclear encoded genes, including the stress-associated genes, in transgenic Arabidopsis. No marked morphological change or growth retardation was observed in R07047 or retransformants. For molecular breeding to improve the tolerance of crops against environmental stress, OsSMCP1 is a promising candidate.


Asunto(s)
Arabidopsis/fisiología , Sequías , Regulación de la Expresión Génica de las Plantas , Oryza/genética , Presión Osmótica , Proteínas de Plantas/fisiología , Plantas Modificadas Genéticamente/fisiología , Secuencia de Aminoácidos , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/microbiología , Secuencia de Bases , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/genética , Peróxido de Hidrógeno/farmacología , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo/efectos de los fármacos , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/microbiología , Brotes de la Planta/efectos de los fármacos , Brotes de la Planta/genética , Brotes de la Planta/crecimiento & desarrollo , Brotes de la Planta/microbiología , Plantas Modificadas Genéticamente/efectos de los fármacos , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/microbiología , Pseudomonas syringae/patogenicidad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sales (Química)/farmacología , Alineación de Secuencia
18.
Plant Cell ; 21(6): 1769-80, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19567705

RESUMEN

In most algae, the chloroplast division rate is held constant to maintain the proper number of chloroplasts per cell. By contrast, land plants evolved cell and chloroplast differentiation systems in which the size and number of chloroplasts change along with their respective cellular function by regulation of the division rate. Here, we show that PLASTID DIVISION (PDV) proteins, land plant-specific components of the division apparatus, determine the rate of chloroplast division. Overexpression of PDV proteins in the angiosperm Arabidopsis thaliana and the moss Physcomitrella patens increased the number but decreased the size of chloroplasts; reduction of PDV levels resulted in the opposite effect. The level of PDV proteins, but not other division components, decreased during leaf development, during which the chloroplast division rate also decreased. Exogenous cytokinins or overexpression of the cytokinin-responsive transcription factor CYTOKININ RESPONSE FACTOR2 increased the chloroplast division rate, where PDV proteins, but not other components of the division apparatus, were upregulated. These results suggest that the integration of PDV proteins into the division machinery enabled land plant cells to change chloroplast size and number in accord with the fate of cell differentiation.


Asunto(s)
Arabidopsis/metabolismo , Bryopsida/metabolismo , Cloroplastos/metabolismo , Proteínas de Plantas/fisiología , Arabidopsis/genética , Arabidopsis/ultraestructura , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiología , Bryopsida/genética , Bryopsida/ultraestructura , Cloroplastos/genética , Cloroplastos/ultraestructura , Modelos Biológicos , Datos de Secuencia Molecular , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/ultraestructura , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
19.
J Plant Res ; 122(6): 633-43, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19618250

RESUMEN

Plants have evolved intricate mechanisms to respond and adapt to a wide variety of biotic and abiotic stresses in their environment. The Arabidopsis DEAR1 (DREB and EAR motif protein 1; At3g50260) gene encodes a protein containing significant homology to the DREB1/CBF (dehydration-responsive element binding protein 1/C-repeat binding factor) domain and the EAR (ethylene response factor-associated amphiphilic repression) motif. We show here that DEAR1 mRNA accumulates in response to both pathogen infection and cold treatment. Transgenic Arabidopsis overexpressing DEAR1 (DEAR1ox) showed a dwarf phenotype and lesion-like cell death, together with constitutive expression of PR genes and accumulation of salicylic acid. DEAR1ox also showed more limited P. syringae pathogen growth compared to wild-type, consistent with an activated defense phenotype. In addition, transient expression experiments revealed that the DEAR1 protein represses DRE/CRT (dehydration-responsive element/C-repeat)-dependent transcription, which is regulated by low temperature. Furthermore, the induction of DREB1/CBF family genes by cold treatment was suppressed in DEAR1ox, leading to a reduction in freezing tolerance. These results suggest that DEAR1 has an upstream regulatory role in mediating crosstalk between signaling pathways for biotic and abiotic stress responses.


Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/fisiología , Congelación , Proteínas Represoras/fisiología , Estrés Fisiológico , Factores de Transcripción/genética , Transcripción Genética/fisiología , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Clonación Molecular , Perfilación de la Expresión Génica , Datos de Secuencia Molecular , Proteínas Represoras/química , Homología de Secuencia de Aminoácido
20.
Planta ; 229(5): 1065-75, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19225807

RESUMEN

Environmental stresses limit plant growth and crop production worldwide. We attempted to isolate rice genes involved in conferring tolerance to environmental stresses by using a transgenic Arabidopsis population expressing full-length cDNAs of rice. Among these lines, a thermotolerant line, R08946, was detected. The rice cDNA inserted in R08946 encoded a NAC transcription factor, ONAC063. This protein was localized in the nucleus and showed transactivation activity at the C-terminus. ONAC063 expression was not induced by high-temperature but highly induced by high-salinity in rice roots. High-osmotic pressure and reactive oxygen species levels also induced ONAC063 expression. The seeds of ONAC063-expressing transgenic Arabidopsis showed enhanced tolerance to high-salinity and osmotic pressure. Microarray and real-time reverse transcription-polymerase chain reaction analyses showed upregulated expression of some salinity-inducible genes, including the amylase gene AMY1, in ONAC063-expressing transgenic Arabidopsis. Thus, ONAC063 may play an important role in eliciting responses to high-salinity stress.


Asunto(s)
Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Ambiente , Oryza/genética , Tolerancia a la Sal/genética , Cloruro de Sodio/farmacología , Estrés Fisiológico/genética , Secuencia de Aminoácidos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas , Proteínas Fluorescentes Verdes/metabolismo , Datos de Secuencia Molecular , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/genética , Plantas Modificadas Genéticamente , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Salinidad , Estrés Fisiológico/efectos de los fármacos , Temperatura , Activación Transcripcional/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos
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