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Highlight Tobacco MPK4L is activated by wounding and ozone exposure in a pathway independent to that of MPK4 and plays a role in the control of stomatal aperture., The mitogen-activated protein kinases (MAPKs/MPKs) are important factors in the regulation of signal transduction in response to biotic and abiotic stresses. Previously, we characterized a MAPK from tobacco, Nicotiana tabacum MPK4 (NtMPK4). Here, we found a highly homologous gene, NtMPK4-like (NtMPK4L), in tobacco as well as other species in Solanaceae and Gramineae. Deduced amino acid sequences of their translation products carried MEY motifs instead of conserved TXY motifs of the MAPK family. We isolated the full length NtMPK4L gene and examined the physiological functions of NtMPK4L. We revealed that NtMPK4L was activated by wounding, like NtMPK4. However, a constitutively active salicylic acid-induced protein kinase kinase (SIPKKEE), which phosphorylates NtMPK4, did not phosphorylate NtMPK4L. Moreover, a tyrosine residue in the MEY motif was not involved in NtMPK4L activation. We also found that NtMPK4L-silenced plants showed rapid transpiration caused by remarkably open stomata. In addition, NtMPK4L-silenced plants completely lost the ability to close stomata upon ozone treatment and were highly sensitive to ozone, suggesting that this atypical MAPK plays a role in ozone tolerance through stomatal regulation.

Los begomovirus (familia Geminiviridae) son virus de DNA cadena sencilla circular que causan enfermedades que constituyen un factor limitante para la producción de cultivos de interés agronómico tales como tomate. Las arvenses o malezas juegan un papel fundamental en la enfermedad geminiviral al servir como hospederos naturales para esta especie de virus. Previamente, nuestro grupo de trabajo aisló cuatro nuevos begomovirus de arvenses colectadas en cultivos de tomate: Verbena sp., Hybanthus attenuatus, Croton hirtus y Rhynchosia minima. El objetivo de este trabajo fue determinar la capacidad emergente de éstos geminivirus para generar infección en solanáceas (tomate y tabaco). Se realizó una extracción de DNA total de las arvenses, se detectó por PCR la presencia de begomovirus y se empleó la técnica de RCA para aumentar el título viral. El DNA begomoviral amplificado fue inoculado en plantas de tomate y tabaco mediante biobalística de baja presión. Las plantas de tomate fueron crecidas en materas en invernadero: 2 ensayos independientes con 10 repeticiones -plantas para cada virus. Las plantas de tabaco fueron crecidas in vitro en medio MS: 1 ensayo con 6 repeticiones- plantas para cada virus. Como control negativo (testigo) se evaluaron plantas de tomate y tabaco sin inocular. Se evaluaron síntomas de manera visual. La replicacion y movimiento del virus se realizó mediante PCR en hojas jóvenes sistémicas. Los resultados indican la presencia de begomovirus en todas las plantas de tomate inoculadas con los aislados virales de las arvenses Hybanthus attenuatus y Verbena sp., obteniendo un 100% de infección viral. Para los ensayos de inoculación con los aislados virales de las arvenses Croton hirtus y Rhynchosia minima se presentó un porcentaje de infección del 90%. Para los ensayos en las plántulas de tabaco se detectó la presencia de begomovirus en todas las plántulas inoculadas obteniendo un 100% de infección. No se presentaron síntomas propios de la enfermedad viral en las plántulas de tomate y tabaco durante los tiempos evaluados. Estos resultados evidencian la capacidad de adaptación a nuevos hospederos que tiene esta familia de virus, lo que incrementa la posibilidad del surgimiento de nuevas variantes virales con mayor patogenicidad. La no presencia de síntomas evidentes es importante ya que los resultados de infección arrojan valores altos, que indican que los virus se están replicando y moviendo, convirtiendo a estos cultivos como fuente de inoculo; razón por la cual se hace necesario establecer estrategias de manejo que no permitan la propagación de estos begomovirus a las plantas cultivadas. //Abstract: Begomoviruses (family Geminiviridae) are circular single-stranded DNA viruses that cause diseases that are a limiting factor for the production of crops of interest agronomic such as tomato. The weeds or weeds play a fundamental role in the geminiviral disease by serving as natural hosts for this species of virus. Previously, our work group isolated four new begomoviruses collected from tomato crops: Verbena sp., Hybanthus attenuatus, Croton hirtus and Rhynchosia minima. The objective of this work was to determine the emergent capacity of these geminiviruses to generate infection in solanaceae (tomato and tobacco). Total DNA extraction from the weeds was performed, the presence of begomovirus was detected by PCR and the RCA technique was used to increase the viral titre. Amplified begomoviral DNA was inoculated in tomato and tobacco plants by low pressure biobalistics. The tomato plants were grown in greenhouses: 2 independent trials with 10 replicates - plants for each virus. Tobacco plants were grown in vitro in MS: 1 assay with 6 replicates - plants for each virus. Noninoculated tomato and tobacco plants were evaluated as negative control (control). Visual symptoms were assessed. Virus replication and movement were performed by PCR on systemic young leaves. The results indicate the presence of begomovirus in all tomato plants inoculated with the viral isolates Hybanthus attenuatus and Verbena sp., obtaining a 100% viral infection. For the inoculation tests with the viral isolates of Croton hirtus and Rhynchosia minima a percentage of infection of 90% was presented. For the tests in the tobacco seedlings the presence of begomovirus was detected in all inoculated seedlings obtaining a 100% of infection. There were no typical symptoms of viral disease in tomato and tobacco seedlings during the evaluated times. These results show the capacity of adaptation to new hosts that have this family of viruses, which increases the possibility of the emergence of new viral variants with greater pathogenicity. The absence of obvious symptoms is important since the results of infection show high values, indicating that the viruses are being replicated and moving, turning these cultures as an inoculum source; which is why it is necessary to establish management strategies that do not allow the propagation of these begomoviruses to the cultivated plants. Maestría

Highlight By generating a large set of transgenic and transplastomic knock-down lines in tobacco, the phenotypic effects of impaired chloroplast protease function on plant growth and development, leaf morphology, pigmentation, and photosynthesis were characterized., Protein degradation in chloroplasts is carried out by a set of proteases that eliminate misfolded, damaged, or superfluous proteins. The ATP-dependent caseinolytic protease (Clp) is the most complex protease in plastids and has been implicated mainly in stromal protein degradation. In contrast, FtsH, a thylakoid membrane-associated metalloprotease, is believed to participate mainly in the degradation of thylakoidal proteins. To determine the role of specific Clp and FtsH subunits in plant growth and development, RNAi lines targeting at least one subunit of each Clp ring and FtsH were generated in tobacco. In addition, mutation of the translation initiation codon was employed to down-regulate expression of the plastid-encoded ClpP1 subunit. These protease lines cover a broad range of reductions at the transcript and protein levels of the targeted genes. A wide spectrum of phenotypes was obtained, including pigment deficiency, alterations in leaf development, leaf variegations, and impaired photosynthesis. When knock-down lines for the different protease subunits were compared, both common and specific phenotypes were observed, suggesting distinct functions of at least some subunits. Our work provides a well-characterized collection of knock-down lines for plastid proteases in tobacco and reveals the importance of the Clp protease in physiology and plant development.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:20:29Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:34:28Z : No. of bitstreams: 1 2-s2.0-0036703817.pdf: 565138 bytes, checksum: a14678a1391703ac0b80109630298044 (MD5) Made available in DSpace on 2014-05-27T11:20:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2002-08-01 In order to identify genes expressed in the pistil that may have a role in the reproduction process, we have established an expressed sequence tags project to randomly sequence clones from a Nicotiana tabacum stigma/style cDNA library. A cDNA clone (MTL-8) showing high sequence similarity to genes encoding glycine-rich RNA-binding proteins was chosen for further characterization. Based on the extensive identity of MTL-8 to the RGP-1a sequence of N. sylvestris, a primer was defined to extend the 5′ sequence of MTL-8 by RT-PCR from stigma/style RNAs. The amplification product was sequenced and it was confirmed that MTL-8 corresponds to an mRNA encoding a glycine-rich RNA-binding protein. Two transcripts of different sizes and expression patterns were identified when the MTL-8 cDNA insert was used as a probe in RNA blots. The largest is 1,100 nucleotides (nt) long and markedly predominant in ovaries. The smaller transcript, with 600 nt, is ubiquitous to the vegetative and reproductive organs analyzed (roots, stems, leaves, sepals, petals, stamens, stigmas/styles and ovaries). Plants submitted to stress (wounding, virus infection and ethylene treatment) presented an increased level of the 600-nt transcript in leaves, especially after tobacco necrosis virus infection. In contrast, the level of the 1,100-nt transcript seems to be unaffected by the stress conditions tested. Results of Southern blot experiments have suggested that MTL-8 is present in one or two copies in the tobacco genome. Our results suggest that the shorter transcript is related to stress while the larger one is a flower predominant and nonstress-inducible messenger. Departamento de Biologia Faculdade de Filosofia Universidade de São Paulo, Ribeirão Preto, SP Fac. de Ciencias Agrarias e Vet. Univ. Estadual Julio de Mesquita, Jaboticabal, SP Departamento de Genética Fac. de Medicina de Ribeirao Preto Universidade de São Paulo, Ribeirão Preto, SP Dept. de Ciencias Farmaceuticas Faculdade de Ciencias Farmaceuticas Universidade de São Paulo, Ribeirão Preto, SP

Summary After 10 days of cultivation at 37 °C, the herbicide resistance encoded by the chimaeric pat 4l gene (coding region from Streptomyces viridochromogenes fused to the 823 bp CaMV35S promoter) was strongly reduced in all of the 27 independent transgenic Nicotiana tabacum SRI lines analyzed. This reversible reduction occurred in sterile and unsterile culture in the first and second generation and even when the overnight temperature was reduced to 24 °C. Neither the enzyme activity, the protein nor the pat 4l specific RNA could be detected in the heat treated plants, regardless of the number of copies and the hemior homozygous state. In contrast to this, the expression of the synthetic pat S coding region fused to the 534bp CaMV35S promoter and coding for essentially the same protein, was stable in heat treated plants. The exchange of the GC rich coding region of the pat 4l gene by the AT rich synthetic DNA fragment carrying the pat S coding region led to the stabilization of the specific RNA steady state level. However, the presence of the transgene-encoded protein at 37 °C could only be achieved by using specific 5′ and 3′ untranslated regions of the synthetic patS gene.

La biotecnología moderna junto con los conocimientos genéticos, se han aplicado en diversas áreas del conocimiento, como en el mejoramiento de las especies en la agricultura, mediante la extracción, purificación, estudio, caracterización y obtención de proteínas contenidas en plantas. La producción de estas proteínas recombinantes se realiza en sistemas biológicos como bacterias y suspensiones de cultivos celulares de plantas. La proteína de interés producida codificada por el gen At1g28290 ( AtAGP31) esta conferida a las células de hipocótilos etiolados de Arabidopsis thaliana del compartimiento llamado pared celular. A fin de realizar un estudio preliminar del proceso de producción de la estructura de la AtAGP31 completa, así como también de versiones incompletas, en este trabajo se obtuvieron diferentes construcciones que permiten la producción de dos proteínas en suspensiones de cultivos celulares de Nicotiana Tabacum Bright Yellow 2 (T-BY2): At1g28290c y At1g28290ps+PAC; y una proteína en E. coli; At1g28290PAC. La proteína recombinante contiene además 6x etiquetas de His en C-terminal que permiten la purificación por cromatografía de afinidad, y el epítopo V5 mediante el cual se realiza el reconocimiento por anticuerpos específicos. Se han establecido las condiciones iniciales de producción de la proteína en la bacteria E. coli las cuales son: 0.002% de arabinosa inducida a 37°C por 4h. Se obtuvo la transformación estable de TBY-2 para el clon 4I.1 perteneciente a la construcción At1g28290c/6His/V5 la cual fue verificada por microscopía de fluorescencia invertida. En el caso de la construcción At1g28290ps+PAC/6His/V5, el clon 1G.1 fue seleccionado de las células A. tumefaciens para usarse en la transformación de T-BY2, la cual esta aun en desarrollo.

International audience; Leaf hydraulic conductance (Kleaf) plays a significant part in plant-water regulation. In walnut leaves, Kleaf is stimulated by irradiance and closely relates to the accumulation of JrPIP2s aquaporin transcripts, but it is independent of stomatal aperture. To provide an insight into the early molecular events occurred during light-induced Kleaf, a large-scale transcriptomic analysis consisting of the cDNA-amplified fragment length polymorphism (AFLP) was carried out on walnut leaves maintained under irradiance or in darkness. Of the total 12 000 transcript-derived fragments (TDFs) obtained using cDNA-AFLP with 128 primer pairs, 187 TDFs were selected after sequencing, and only 93 (49 %) that had been ascribed known functions through BLAST searching of the GenBank databases. Most of these TDFs correspond to genes whose protein products are involved in cellular regulation (57.9 %) and global metabolism (39.8 %). To validate cDNA-AFLP expression patterns, 30 TDFs were further analyzed using real-time quantitative polymerase chain reaction. Moreover, exposure of leaves to irradiance was accompanied by the modification of the Ca2+-signaling pathway, ubiquitin-proteasome pathway, vesicle trafficking process and expression of multiple transcription factors.

We used next generation sequencing to characterize and compare the genomes of the recently derived allotetraploid, Nicotiana tabacum (

Rate and extent of spontaneous and induced chromosome loss have been determined at the callus level of somatic hybrids of mutants of Solanum tuberosum and Nicotiana plumbaginifolia. AEC (amino ethyl cystein) resistance in potato and Nitrate-Reductase deficiency in N. plumbaginifolia have been used as genetic markers and chromosome morphology as a cytological marker. In this combination, development of hybrid callus was late and slow. Only a limited number of non-regenerable hybrid calli have become available. Chromosome loss could clearly be established in these hybrids from loss of markers and from chromosome cytology. Loss of markers occurred independently.The best conditions to induce loss of chromosomal material in donor cells by irradiation were found by cytological investigations. A very drastic reduction in chromosome transfer by fusion could be effected by irradiation of plant tissue and subsequent preparation of protoplasts after a few days. Following fusion, hybrid callus was recovered with the potato genome drastically reduced. The amount of loss was deduced from the presence of a few fragments in metaphase cells or from interphase nuclei after in situ hybridization with a repetitive potato DNA probe.

International audience; This paper establishes relationships between two aspects of ribosomal DNA (rDNA) biology: epigenetic silencing of rDNA loci; and homogenization leading to concerted evolution. Here, we examined rDNA inheritance and expression patterns in three natural Nicotiana allopolyploids (closest living descendants of diploid parents are given), N. rustica (N. paniculata x N. undulata), N. tabacum (N. sylvestris x N. tomentosiformis) and N. arentsii (N. undulata x N. wigandioides), and synthetic F-1 hybrids and allopolyploids. The extent of interlocus rDNA homogenization decreased in the direction N. arentsii > N. tabacum > N. rustica. The persistence of parental rDNA units in one of the subgenomes was associated with their transcription inactivity and likely heterochromatization. Of synthetic hybrids and polyploids only N. paniculata x N. undulata showed strong uniparental transcriptional silencing of rDNA triggered already in F-1. Epigenetic patterns of expression established early in allopolyploid nucleus formation may render units susceptible or resistant to homogenization over longer time-frames. We propose that nucleolus-associated transcription leaves rDNA units vulnerable to homogenization, while epigenetically inactivated units, well-separated from the nucleolus, remain unconverted.

Were investigated the time of solarization, in the disinfestations of beds almaciguents, toward produce plantules of tobacco in sector of Juan guerra - Region San Martin, during September to December of the 2001 . The design utilize was the DBCA with 12 treatments, 3 witness absolutes and another with bromide of methyl. Were solarize the substratum (60% soil clay, sandy and franc and 40% humus the worm ), during 1 O, 20, 30, and 40 days, being used plastics transparent of 80 um of thickness; being compared with witness without solarize and another with bromide of methyl to dose of O, 15 lb/m2 . During the time that lasted the solarization they registered the temperatures of the soil and of the atmosphere. Were realization evaluations to the substratum with emphasis in nematodes and mushrooms, and to level of almacigue presence of overgrowths, weight humid and weight dry of the seedling. The results were expressed in percentages in the different evaluations. To from of 20, 30 and 40 days of solarization were achieved reductions considerate of nematodes, (Pratylenchus sp., Paratylenchus sp., Tylenchus sp.) of 99% to 100% in the different treatments, and mushrooms pathogens, (Phytophthora, sp Fusarium sp.) of 60% to 98%, and overgrowths monocotyledonous and dicotyledonous, (Amaranthus sp., Euphorbia sp., Cyperus sp, Rotboella sp) of 55% to 97%, the weight humid and dry off they obtained increments in weight to more day of solarization respectively. The treatment with bromide of methyl their control was of 90% to 100%. The antecedents generals indicate that the method solarization allows lower the occurrence of microorganism's pathogens and overgrowths. The results obtained demonstrate that with the solarization were can gain a control adequate the diverse plagues in the disinfestations of beds of tobacco. In connection with the analysis economic, it indicates that the treatment with 40 days was who obtained an major B/C of 1,21 in connection with the other treatments evaluated. Se investigó el tiempo de solarización en la desinfestación de camas almacigueras para producir plántulas de tabaco en el sector de Juan guerra- Región san Martín. Durante Setiembre a diciembre del 2001. El substrato (60% suelo arci.lloso, arenoso y franco y 40% humus de lombriz), durante 1 O, 20, 30, y 40 días, utilizándose plásticos transparentes de 80 um de espesor; comparándose con testigos sin solarizar y otro con bromuro de metilo a dosis de 0, 15 lb/m2 . Durante el tiempo que duró la solarización se registraron las temperaturas del suelo y del ambiente. Se realizaron evaluaciones al substrato con énfasis en nematodos y hongos, a nivel de almácigo presencia de malezas, peso húmedo y peso seco de los lechuguinos. Los resultados se expresaron en porcentajes de control en las diferentes evaluaciones. A partir de 20, 30, y 40 días de solarización se lograron reducciones considerables de nematodos (Pratylenchus sp., Paratylenchus sp., Tylenchus sp.), de 99 a 100% en los diferentes tratamientos, y hongos patógenos (Phytophthora, Fusarium sp.) de 60 a 98%, y malezas monocotiledóneas y dicotiledóneas (Amaranthus sp., Euphorbia sp., Cyperus sp, Rotboella sp) de 55 a 97%, los pesos húmedo y seco obtuvieron incrementos en pesos a mayor día de solarización respectivamente. El tratamiento con bromuro de metilo su control fue de 90 a 100%. Los antecedentes generales indican que el método de solarización permite bajar la ocurrencia de microorganismos patógenos y malezas. Los resultados obtenidos demuestran que con la solarización se puede lograr un control adecuado de diversas plagas en la desinfestación de camas de tabaco. En relación con el análisis económico, nos indica que él tratamiento con 40 días fue quien obtuvo un mayor B/C de 1,21 en relación con los demás tratamientos evaluados.

“Nicotina" es el género botánico con que los científicos han designado una de las plantas más explotadas en el mundo industrial, que ha sido en todos los tiempos fuente de riqueza para los países que la producen y aun para aquellos que la importan, pero que, mediante su industrialización, obtienen de ella pingües beneficios. Entre nosotros ha dado origen a la verdadera industria autóctona y, tras ingentes luchas, ha llegado a ser explotada de una manera científica y con una organización que es envidiada por muchos países de ultramar. En Colombia, sin una verdadera clasificación científica de los tabacos, diferenciamos sin embargo, las cualidades de cada uno de ellos por medio de los procedimientos un poco empíricos, de distinguirlos de acuerdo con las regiones productoras. Como base de nuestra industria tabacalera, el tabaco Santander se ha impuesto definitivamente, debido a sus excelentes cualidades; le siguen en importancia los del Tolima, Valle, Cauca, Nariño, Huila y Antioquia, figurando en un renglón totalmente separado el tabaco de Bolívar, que muy poca o ninguna influencia tiene en el desarrollo de nuestra industria y que sólo constituye un renglón importante bajo el aspecto de exportación, ya que su precio sumamente bajo es el único que permite competir en los mercados extranjeros con calidades superiores, pero que económicamente no pueden ofrecerse en idénticas condiciones.