Chez les eucaryotes, la voie de protéolyse Ub/ protéasome 26S est responsable de la dégradation sélective de la plupart des protéines intracellulaires. Cette dégradation par le protéasome 26S est initiée par une polyubiquitination de la protéine réalisée grâce à l’action d’une cascade enzymatique impliquant 3 types d'enzymes nommées « ubiquitin-activating enzyme » (E1), « ubiquitin-conjugating enzyme » (E2) et « ubiquitin-protein ligase » (E3). Il existe différentes classes d’ubiquitines ligases (E3), parmi lesquelles la plus connue est le complexe SCF (Skp1-Cullin-F-box). La protéine SKP1 fixe à la fois la Culline et la F-box qui va reconnaitre spécifiquement la protéine cible. Contrairement aux protistes, les champignons et certains vertébrés qui possèdent un unique gène SKP1 fonctionnel, de nombreux animaux et espèces de plantes présentent plusieurs SKP1 homologues. Vingt et un et trente deux gènes SKP1 ont été décrits respectivement chez Arabidopsis thaliana et Oryza sativa. En dépit de l’importance du complexe SCF, chez le riz, peu de travaux décrivent les interactions entre les dizaines de protéines « SKP1-like » et les centaines de protéines F-box. Dans un premier temps, nous avons collecté et analysé les séquences de 288 gènes « SKP1-like » appartenant à 17 espèces, dont la mousse Physcomitrella patens, cinq monocotylédones et 11 eudicotylédones. Les analyses structurales et phylogénétiques de ces gènes indiquent qu’ils peuvent être divisés en différentes sous-familles. Nos analyses ont montré qu’OSK1 et OSK20 chez le riz constituent une classe de gènes SKP1 à intron unique conservé. Dans un deuxième temps, nous avons étudié le profil d’expression des gènes « SKP1-like » chez le riz. Notre investigation sur le nombre d’EST a montré que les gènes OSK1 et OSK20 sont les plus largement représentés dans les bases de données EST publiques. La méta-analyse de l’expression des gènes « SKP1-like » chez le riz, indique que les gènes OSK présentent des profils d'expression hétérogènes selon les tissus et les conditions physiologiques. Les résultats des intearctions protéine-protéine en double hybride ont révélé que les protéines OSK présentent différentes capacités d’interactions avec les protéines F-box. Cependant, OSK1 et OSK20 semblent interagir avec la plupart des protéines F-box testées. Les études de localisation subcellulaire ont indiqué que OSK1 et OSK20 sont des protéines nucléaires et cytosoliques. En se basant sur les divers résultats obtenus dans ce travail, nous pouvons suggérer que chez le riz, les gènes OSK1 et OSK20 sont fonctionnellement équivalents aux gènes ASK1 et ASK2 chez Arabidopsis thaliana. Nous pouvons également proposer les équivalents de ces gènes chez les autres espèces végétales dont le génome a été séquencé. / In eukaryotes, the ubiquitin Ub/26S proteasome pathway is responsible for the selective degradation of most intracellular proteins. This cellular process is initiated by protein polyubiquitination mediated by a three-step cascade involving: an ubiquitin-activating enzyme (E1), an ubiquitin-conjugating enzyme (E2) and an ubiquitin-protein ligase (E3). The E3 ubiquitin ligases contain several classes, among which the best-known are Skp1-Cullin-F-box (SCF) complexes. The SKP1 protein binds both Cullin and F-box which recognizes specifically the target proteins. Whereas protists, fungi and some vertebrates have a single functional SKP1 gene, many animal and plant species possess multiple SKP1 homologues. Twenty one and thirty-two SKP1-related genes have been described respectively in the Arabidopsis and Oryza sativa genome. Despite the importance of the SCF complex, there have been a few reports of systematic surveys of interactions between the dozens of SKP1-like proteins and the hundreds of F-box proteins in rice. In a first step, we retrieved and analyzed 288 SKP1-like genes belonging to 17 species including the moss Physcomitrella patens, five monocots and 11 eudicots. Structural and phylogenetic analysis of rice OSK genes and other plant SKP1-like genes have indicated that the different members of the plant SKP1 can be split into different subfamily. Our analyses indicated that OSK1 and OSK20 belong to a class of SKP1 genes that contain one intron at a conserved position. In a second step, we studied expression profiles of the rice Skp1-like genes. Our EST survey indicated that OSK1 and OSK20 are the most widely represented genes in public EST databases. Meta-analysis of the expression of rice SKP1-like genes indicated that OSK genes exhibit an expression profile that was heterogeneous in terms of tissues, conditions and overall intensity. Yeast two-hybrid results revealed that OSK proteins display a differing ability to interact with F-box proteins. However, OSK1 and OSK20 seemed to interact with most F-box proteins tested. Subcellular localization studies indicated that OSK1 and OSK20 are nuclear and cytosolic proteins. Based on the results obtained in this study, we can suggest that rice OSK1 and OSK20 are likely to have similar functions as do the Arabidopsis ASK1 and ASK2 genes. Similarly, we suggest a list of functional equivalent in the other sequenced plant genomes.

A biologia da reprodução sexual de plantas é um campo de pesquisa de grande importância, já que a maioria dos alimentos consumidos pelo homem é composta de partes reprodutivas das plantas (frutos e sementes), oriundas do desenvolvimento de partes do pistilo fertilizado. Em Nicotiana tabacum, identificou-se um gene específico de estigma/estilete, SCI1 (Stigma/style Cell-cycle Inhibitor 1), que atua na inibição da proliferação celular (DePaoli et al., 2011). Através de ensaios de pull-down, verificou-se a interação da proteína SCI1 com uma proteína quinase dependente de ciclina (CDK) (Strini, dados não publicados). Este trabalho visou à caracterização dessa nova CDK, ortóloga da CDKG;2 de Arabidopsis. A sequência correspondente de N. tabacum (NtCDKG;2) foi amplificada por PCR, a partir de cDNAs de estigmas/estiletes, clonada e sequenciada, o que permitiu a confirmação de sua identidade. A expressão de NtCDKG;2 foi analisada nos diferentes órgãos vegetativos e reprodutivos, por qRT-PCR, o que evidenciou um perfil de expressão ubíqua. Ao estudar o perfil de expressão desse gene nos estigmas/estiletes dos doze estádios de desenvolvimento floral de N. tabacum, observa-se que NtCDKG;2 é mais expresso nos estádios tardios do desenvolvimento em direção à antese, indicando uma função importante de sua proteína ao final do desenvolvimento do pistilo. Análises de expressão de NtCDKG;2 em estigmas/estiletes, de plantas de N. tabacum com produção aumentada do hormônio auxina no pistilo, sugerem que NtCDKG;2 é regulado transcricionalmente por esse hormônio. A expressão transiente da proteína de fusão NtCDKG;2-GFP, em folhas de N. tabacum, evidenciou a localização nuclear da proteína em estudo. Também foram geradas plantas transgênicas estáveis com superexpressão e com silenciamento por RNAi de NtCDKG;2. Apesar dos altos níveis de transcritos de NtCDKG;2 nas plantas de superexpressão e dos baixos níveis nas plantas silenciadas, não foram observadas alterações fenotípicas macroscópicas nessas plantas. Adicionalmente, obteve-se a expressão da proteína NtCDKG;2, fusionada a uma tag de histidina em sua porção N-terminal, em células de Escherichia coliBL21(DE3)CodonPlusRP. Através dos estudos realizados neste trabalho e análises conjuntas da literatura, é possível propor que NtCDKG;2 codifique uma proteína que está envolvida no controle do ciclo celular nos estigmas/estiletes de N. tabacum. / The biology of plant sexual reproduction is a research field of great importance, since most of the food consumed by humans is composed of plant reproductive parts (fruits and seeds), originated by the development of fertilized pistil parts. In Nicotiana tabacum, it was identified a stigma/style-specific gene, SCI1 (Stigma/style Cell-cycle Inhibitor 1), which acts in the inhibition of cell proliferation (DePaoli et al., 2011). Through pull down assays, the interaction of the SCI1 protein with a cyclin-dependent protein kinase (CDK) was verified (Strini, unpublished). This work aimed the characterization of this new CDK, orthologous to the Arabidopsis CDKG;2. The N. tabacum corresponding sequence (NtCDKG;2) was PCR amplified, from stigmas/styles cDNAs, cloned and sequenced, which allowed the confirmation of its identity. The NtCDKG;2 expression was analyzed in the different vegetative and reproductive organs, by qRT-PCR, evidentiating an ubiquitous expression pattern. Studying the expression pattern of this gene in stigmas/styles of the twelve stages of N. tabacum flower development, it was observed that NtCDKG;2 is more expressed at the later developmental stages towards anthesis, indicating an important function of its protein in the end of pistil development. NtCDKG;2 expression analyses in stigmas/styles of N. tabacum plants with an enhanced auxin production in the pistil suggest that NtCDKG;2 is transcriptionally regulated by this hormone. The transient expression of the fusion protein NtCDKG;2-GFP, in N. tabacum leaves, evidentiated the nuclear localization of the studied protein. Stable transgenic plants overexpressing and silencing NtCDKG;2 by RNAi were also generated. Despite the high transcript levels in the plants overexpressing NtCDKG;2 and the low transcript levels in the silencing plants, macroscopic phenotypic alterations were not observed on these plants. Additionally, the expression of the NtCDKG;2 protein, with a histidine tag fused in its N-terminal, was obtained in Escherichia coli BL21(DE3)CodonPlusRP cells. Through studies performed on this work and literature analyses, it is possible to propose that NtCDKG;2 encodes a protein that is involved in the control of cell cycle at the N. tabacum stigmas/styles.

Estresses abióticos como seca, alta salinidade e baixas temperaturas, afetam o crescimento e a produtividade em culturas de interesse comercial como o feijoeiro comum. Proteínas DREB (Dehydration Responsive Element Binding) são fatores de transcrição que regulam genes específicos envolvidos na tolerância ao estresse abiótico. Para determinar como as plantas toleram condições ambientais adversas, variedades tolerantes, biologia molecular e bioinformática podem ser aplicadas para identificar e caracterizar genes que controlam mecanismos de adaptação a estresses. Baseado nas informações disponíveis nos bancos de dados públicos, a sequência da Orf completa do gene Phvul.009G029600.1| PACid:27146455 contendo 1062 pb foi encontrada e usada para o desenho dos primers e para o sequenciamento. A nova sequência é muito similar ao AtRAP2.4 e foi nomeada como PvDREB6A, segundo a análise filogenética. Ferramentas de predição mostraram que a sequência apresenta 354 aminoácidos e possui uma cópia do domínio AP2, que se dobra em uma estrutura com três ?-folhas e uma ?-hélice apresentando resíduos importantes e motivos específicos de reconhecimento e de ligação ao DNA. Além disso, um peptídeo trânsito foi detectado na porção N-terminal com um sítio de clivagem no resíduo 52. A interação deste fator de transcrição com seu domínio de ligação ao DNA foi validada por Electro Mobility Shift Assay (EMSA). A localização subcelular da proteína foi realizada e expressão da Green Fluorescent Proteín (GFP) foi detectada no núcleo. A transformação genética para a superexpressão do gene PvDREB6A em plantas de Arabidopsis thaliana Columbia-0 e mutantes nocaute para o gene AtRAP2.4 (Salk_020767C) foi realizada. Quatro eventos com cópia única e melhor expressão do gene PvDREB6A denominados Col-0/pFEC2.1 #1, Salk_020767C/pFEC2.1 #13.1, Salk_020767C/ pFEC2.1 #19.7 e Salk_020767C/ pFEC2.1 #23.7, foram selecionados. O evento Salk_020767C/pFEC2.1 #23.7 mostrou melhor expressão do gene PvDREB6A e foi visualizado sob luz UV. A análise funcional revelou que as plantas transgênicas submetidas ao déficit hídrico, à alta salinidade e ao frio, apresentaram maior taxa de sobrevivência. Plantas transgênicas superexpressando o gene PvDREB6A apresentaram menor taxa de desidratação e de vazamento de eletrólitos quando submetidas a estresses abióticos. Uma análise da expressão de genes relacionados à tolerância foi conduzida. A quantificação revelou que a expressão de 18 genes: AtDC1.2, AtUSP, AtKIN1, AtERF69, AtGolS3, AtMT2A, AtCAP160, AtNTR1.7, AtGPR7, AtPDC2, AtLTI78, AtCOR15a, AtCOR15b, AtCOR47, AtCOR413, AtLEA6, AtLEA9 e AtLEA14, relacionados a tolerância a seca, sal e frio foram up-regulated devido à superexpressão do gene PvDREB6A de feijoeiro nas plantas transgênicas / Abiotic stresses like drought, high salinity and low temperatures affect growth and productivity in crops of economic interest such as common bean. DREB (Dehydration Responsive Element Binding) proteins are transcription factors that activate specific genes involved in tolerance to abiotic stress. To generate new information on the research for drought and other abiotic stresses, tolerant varieties, molecular biology and bioinformatics can be applied to identify and characterize genes that control plant defense and adaptation mechanisms to water deprivation, to excessive salt and to high/low temperature. Based on public databases, a common bean DREB sequence was found and an in silico study was carried out. A complete Orf sequence Phvul.009G029600.1 |PACid:27146455 containing 1062 bp was found and used for primer design and sequencing. The new sequence was very similar to AtRAP2.4 and named as PvDREB6A, according to phylogenetic analysis. Prediction tools showed that the deduced 354 aa sequence has one copy of the AP2 domain, folding in a three ?-sheets and one ?-helix structure, and presenting important residues and motifs for DNA contacting and binding specificity. In addition, a chloroplast transit peptide was detected at the N-terminal region with cleavage site in the 52 residue. Binding activity of this transcription factor was validated by Electro Mobility Shift Assay (EMSA). Subcellular localization was verified by transient expression of PvDREB6A::GFP in Nicotiana benthamiana and the expression of GFP was detected at the nucleus. Genetic transformation for overexpression of PvDREB6A gene in Arabidopsis thaliana wild type and knockout mutant for AtRAP2.4 gene was conducted. Four single copy events with better expression of the PvDREB6A named Col-0/pFEC2.1 #1, Salk_020767C/pFEC2.1 #13.1, Salk_020767C/pFEC2.1 #19.7 and Salk_020767C/pFEC2.1 #23.7 were selected. The event Salk_020767C/pFEC2.1 #23.7 showed the best expression of PvDREB6A and was visualized under UV light. Functional analysis, revealed that transgenic plants under water deficit, high salt, and cold showed higher survival rate. Transgenic plants overexpressing the PvDREB6A exhibited lower water loss rate and electrolyte leakage rate under abiotic stress. A gene expression analysis with tolerant-related genes was conduted. The quantification revealed that 18 genes, AtDC1.2, AtUSP, AtKIN1, AtERF69, AtGolS3, AtMT2A, AtCAP160, AtNTR1.7, AtGPR7, AtPDC2, AtLTI78, AtCOR15a, AtCOR15b, AtCOR47, AtCOR413, AtLEA6, AtLEA9 e AtLEA14, related to drought, salt and cold tolerance, were up-regulated due to the overexpression of PvDREB6A from common bean in the transgenic plants

Les aminoacyl-ARNt synthétases catalysent la formation des aminoacyl-ARNt, utilisés lors de la synthèse protéique et peuvent également former des complexes multi-synthétasiques (MSC). Chez S. cerevisiae, le complexe AME associe les glutamyl- et méthionyl-ARNt synthétases à la protéine d’ancrage Arc1 et joue un rôle primordial dans la coordination de l’expression des génomes nucléaire et mitochondrial. Tous les composants de ce MSC sont multi-localisés et assurent des fonctions essentielles dans d’autres compartiments. Pour étudier ces localisations multiples, nous avons élaboré un outil, basé sur la Split-GFP, qui nous permet de visualiser spécifiquement la fraction organellaire d’une protéine multi-localisée. Pour cela, la GFP a été séparée en deux fragments : i) β1-10, restreint à un compartiment subcellulaire et ii) β11, fusionné aux protéines d’intérêts. Cet outil nous a permis d’étudier diverses relocalisations, ainsi que de délimiter des signaux d’import. / Aminoacyl-tRNA synthetases catalyze aminoacyl-tRNA formation, required for protein synthesis but can also associate into multi-synthetase complexes (MSC). In S. cerevisiae, the AME complex contains glutamyl- and methionyl-tRNA synthetases bound to the anchor protein Arc1 and is responsible for the coordination of nuclear and mitochondrial genome expression. The three MSC partners are multi-localized and present simultaneously in several compartments. The detection of the organellar pools of these multilocalized proteins in vivo is difficult, since they are mainly cytosolic. Therefore, we engineered a split-GFP based localization tool that allows us to specifically visualize organellar fractions of multi-localized proteins. To do so, GFP was split into two parts: β1-10, restricted to a subcellular compartment and β11, fused to the protein of interest. This tool allowed us to study relocalization of cytosolic proteins and characterize targeting signals.

Since the publication of the complete sequence of the human genome in 2003 there has been great interest in exploring the functions of the proteins encoded by the genes. To reveal the function of each and every protein, investigation of protein localization at the subcellular level has become a central focus in this research area, since the localization and function of a protein is closely related. The objective of the studies presented in this doctoral thesis was to systematically explore the human proteome at the subcellular level using bioimaging and to develop techniques for validation of the results obtained. A common imaging technique for protein detection is immunofluorescence (IF), where antibodies are used to target proteins in fixated cells. A fixation protocol suitable for large-scale IF studies was developed and optimized to work for a broad set of proteins. As the technique relies on antibodies, validation of their specificity to the target protein is crucial. A platform based on siRNA gene silencing in combination with IF was set-up to evaluate antibody specificity by quantitative image analysis before and after suppression of its target protein. As a proof of concept, the platform was then used for validation of 75 antibodies, proving it to be applicable for validation of antibodies in a systematic manner. Because of the fixation, there is a common concern about how well IF data reflects the in vivo subcellular distribution of proteins. To address this, 500 proteins were tagged with green fluorescent protein (GFP) and used to compare protein localization results between IF to those achieved using GFP tagged proteins in live cells. It was concluded that protein localization data from fixated cells satisfactory represented the situation in vivo and together exhibit a powerful approach for confirming localizations of yet uncharacterized proteins. Finally, a global analysis based on IF data of approximately 20 % of the human proteome was performed, providing a first overview of the subcellular landscape in three different cell lines. It was found that the intracellular distribution of proteins is complex, with many proteins occurring in several organelles. The results also confirmed the close relationship between protein function and localization, which in a way further strengthens the accuracy of the IF approach for detection of proteins at the subcellular level. / <p>QC 20121017</p> / The Human Protein Atlas

Aberrant subcellular localization of mutant transmembrane receptors is increasingly acknowledged as a possible mechanism for an altered signaling quality leading to transformation. There is evidence that mutated receptor tyrosine kinases of subclass III, for example the platelet-derived growth factor receptor (PDGFR) and KIT-protein, are aberrantly localized in human cancers. In order to further analyze this phenomenon, we investigated the localization of FLT3, a subclass III receptor tyrosine kinase frequently mutated in leukemia. By immunofluorescence staining and confocal laser scanning microscopy we found that in retrovirally transduced COS7 cells, wild type FLT3 receptor protein is localized primarily at the cell surface. In contrast, a mutant FLT3 receptor protein with an internal tandem duplication (ITD) accumulates in a perinuclear region and is not detectable at the plasma membrane. Surprisingly, and in contrast to previously published data, intracellular FLT3-ITD accumulation could neither be detected in the endoplasmic reticulum (ER) nor in the Golgi apparatus. Furthermore, transient overexpression per se leads to accumulation of wild type FLT3 receptor protein in the ER in addition to surface localization, probably due to inefficient intracellular transport by the overloaded sorting machinery of the secretory pathway. Based on our data and the immature glycosylation pattern of FLT3-ITD, we speculate that the mutant protein resides most probably in an unidentified compartment of the secretory pathway between the ER and the Golgi apparatus. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Estresses abióticos como seca, alta salinidade e baixas temperaturas, afetam o crescimento e a produtividade em culturas de interesse comercial como o feijoeiro comum. Proteínas DREB (Dehydration Responsive Element Binding) são fatores de transcrição que regulam genes específicos envolvidos na tolerância ao estresse abiótico. Para determinar como as plantas toleram condições ambientais adversas, variedades tolerantes, biologia molecular e bioinformática podem ser aplicadas para identificar e caracterizar genes que controlam mecanismos de adaptação a estresses. Baseado nas informações disponíveis nos bancos de dados públicos, a sequência da Orf completa do gene Phvul.009G029600.1| PACid:27146455 contendo 1062 pb foi encontrada e usada para o desenho dos primers e para o sequenciamento. A nova sequência é muito similar ao AtRAP2.4 e foi nomeada como PvDREB6A, segundo a análise filogenética. Ferramentas de predição mostraram que a sequência apresenta 354 aminoácidos e possui uma cópia do domínio AP2, que se dobra em uma estrutura com três ?-folhas e uma ?-hélice apresentando resíduos importantes e motivos específicos de reconhecimento e de ligação ao DNA. Além disso, um peptídeo trânsito foi detectado na porção N-terminal com um sítio de clivagem no resíduo 52. A interação deste fator de transcrição com seu domínio de ligação ao DNA foi validada por Electro Mobility Shift Assay (EMSA). A localização subcelular da proteína foi realizada e expressão da Green Fluorescent Proteín (GFP) foi detectada no núcleo. A transformação genética para a superexpressão do gene PvDREB6A em plantas de Arabidopsis thaliana Columbia-0 e mutantes nocaute para o gene AtRAP2.4 (Salk_020767C) foi realizada. Quatro eventos com cópia única e melhor expressão do gene PvDREB6A denominados Col-0/pFEC2.1 #1, Salk_020767C/pFEC2.1 #13.1, Salk_020767C/ pFEC2.1 #19.7 e Salk_020767C/ pFEC2.1 #23.7, foram selecionados. O evento Salk_020767C/pFEC2.1 #23.7 mostrou melhor expressão do gene PvDREB6A e foi visualizado sob luz UV. A análise funcional revelou que as plantas transgênicas submetidas ao déficit hídrico, à alta salinidade e ao frio, apresentaram maior taxa de sobrevivência. Plantas transgênicas superexpressando o gene PvDREB6A apresentaram menor taxa de desidratação e de vazamento de eletrólitos quando submetidas a estresses abióticos. Uma análise da expressão de genes relacionados à tolerância foi conduzida. A quantificação revelou que a expressão de 18 genes: AtDC1.2, AtUSP, AtKIN1, AtERF69, AtGolS3, AtMT2A, AtCAP160, AtNTR1.7, AtGPR7, AtPDC2, AtLTI78, AtCOR15a, AtCOR15b, AtCOR47, AtCOR413, AtLEA6, AtLEA9 e AtLEA14, relacionados a tolerância a seca, sal e frio foram up-regulated devido à superexpressão do gene PvDREB6A de feijoeiro nas plantas transgênicas / Abiotic stresses like drought, high salinity and low temperatures affect growth and productivity in crops of economic interest such as common bean. DREB (Dehydration Responsive Element Binding) proteins are transcription factors that activate specific genes involved in tolerance to abiotic stress. To generate new information on the research for drought and other abiotic stresses, tolerant varieties, molecular biology and bioinformatics can be applied to identify and characterize genes that control plant defense and adaptation mechanisms to water deprivation, to excessive salt and to high/low temperature. Based on public databases, a common bean DREB sequence was found and an in silico study was carried out. A complete Orf sequence Phvul.009G029600.1 |PACid:27146455 containing 1062 bp was found and used for primer design and sequencing. The new sequence was very similar to AtRAP2.4 and named as PvDREB6A, according to phylogenetic analysis. Prediction tools showed that the deduced 354 aa sequence has one copy of the AP2 domain, folding in a three ?-sheets and one ?-helix structure, and presenting important residues and motifs for DNA contacting and binding specificity. In addition, a chloroplast transit peptide was detected at the N-terminal region with cleavage site in the 52 residue. Binding activity of this transcription factor was validated by Electro Mobility Shift Assay (EMSA). Subcellular localization was verified by transient expression of PvDREB6A::GFP in Nicotiana benthamiana and the expression of GFP was detected at the nucleus. Genetic transformation for overexpression of PvDREB6A gene in Arabidopsis thaliana wild type and knockout mutant for AtRAP2.4 gene was conducted. Four single copy events with better expression of the PvDREB6A named Col-0/pFEC2.1 #1, Salk_020767C/pFEC2.1 #13.1, Salk_020767C/pFEC2.1 #19.7 and Salk_020767C/pFEC2.1 #23.7 were selected. The event Salk_020767C/pFEC2.1 #23.7 showed the best expression of PvDREB6A and was visualized under UV light. Functional analysis, revealed that transgenic plants under water deficit, high salt, and cold showed higher survival rate. Transgenic plants overexpressing the PvDREB6A exhibited lower water loss rate and electrolyte leakage rate under abiotic stress. A gene expression analysis with tolerant-related genes was conduted. The quantification revealed that 18 genes, AtDC1.2, AtUSP, AtKIN1, AtERF69, AtGolS3, AtMT2A, AtCAP160, AtNTR1.7, AtGPR7, AtPDC2, AtLTI78, AtCOR15a, AtCOR15b, AtCOR47, AtCOR413, AtLEA6, AtLEA9 e AtLEA14, related to drought, salt and cold tolerance, were up-regulated due to the overexpression of PvDREB6A from common bean in the transgenic plants

A biologia da reprodução sexual de plantas é um campo de pesquisa de grande importância, já que a maioria dos alimentos consumidos pelo homem é composta de partes reprodutivas das plantas (frutos e sementes), oriundas do desenvolvimento de partes do pistilo fertilizado. Em Nicotiana tabacum, identificou-se um gene específico de estigma/estilete, SCI1 (Stigma/style Cell-cycle Inhibitor 1), que atua na inibição da proliferação celular (DePaoli et al., 2011). Através de ensaios de pull-down, verificou-se a interação da proteína SCI1 com uma proteína quinase dependente de ciclina (CDK) (Strini, dados não publicados). Este trabalho visou à caracterização dessa nova CDK, ortóloga da CDKG;2 de Arabidopsis. A sequência correspondente de N. tabacum (NtCDKG;2) foi amplificada por PCR, a partir de cDNAs de estigmas/estiletes, clonada e sequenciada, o que permitiu a confirmação de sua identidade. A expressão de NtCDKG;2 foi analisada nos diferentes órgãos vegetativos e reprodutivos, por qRT-PCR, o que evidenciou um perfil de expressão ubíqua. Ao estudar o perfil de expressão desse gene nos estigmas/estiletes dos doze estádios de desenvolvimento floral de N. tabacum, observa-se que NtCDKG;2 é mais expresso nos estádios tardios do desenvolvimento em direção à antese, indicando uma função importante de sua proteína ao final do desenvolvimento do pistilo. Análises de expressão de NtCDKG;2 em estigmas/estiletes, de plantas de N. tabacum com produção aumentada do hormônio auxina no pistilo, sugerem que NtCDKG;2 é regulado transcricionalmente por esse hormônio. A expressão transiente da proteína de fusão NtCDKG;2-GFP, em folhas de N. tabacum, evidenciou a localização nuclear da proteína em estudo. Também foram geradas plantas transgênicas estáveis com superexpressão e com silenciamento por RNAi de NtCDKG;2. Apesar dos altos níveis de transcritos de NtCDKG;2 nas plantas de superexpressão e dos baixos níveis nas plantas silenciadas, não foram observadas alterações fenotípicas macroscópicas nessas plantas. Adicionalmente, obteve-se a expressão da proteína NtCDKG;2, fusionada a uma tag de histidina em sua porção N-terminal, em células de Escherichia coliBL21(DE3)CodonPlusRP. Através dos estudos realizados neste trabalho e análises conjuntas da literatura, é possível propor que NtCDKG;2 codifique uma proteína que está envolvida no controle do ciclo celular nos estigmas/estiletes de N. tabacum. / The biology of plant sexual reproduction is a research field of great importance, since most of the food consumed by humans is composed of plant reproductive parts (fruits and seeds), originated by the development of fertilized pistil parts. In Nicotiana tabacum, it was identified a stigma/style-specific gene, SCI1 (Stigma/style Cell-cycle Inhibitor 1), which acts in the inhibition of cell proliferation (DePaoli et al., 2011). Through pull down assays, the interaction of the SCI1 protein with a cyclin-dependent protein kinase (CDK) was verified (Strini, unpublished). This work aimed the characterization of this new CDK, orthologous to the Arabidopsis CDKG;2. The N. tabacum corresponding sequence (NtCDKG;2) was PCR amplified, from stigmas/styles cDNAs, cloned and sequenced, which allowed the confirmation of its identity. The NtCDKG;2 expression was analyzed in the different vegetative and reproductive organs, by qRT-PCR, evidentiating an ubiquitous expression pattern. Studying the expression pattern of this gene in stigmas/styles of the twelve stages of N. tabacum flower development, it was observed that NtCDKG;2 is more expressed at the later developmental stages towards anthesis, indicating an important function of its protein in the end of pistil development. NtCDKG;2 expression analyses in stigmas/styles of N. tabacum plants with an enhanced auxin production in the pistil suggest that NtCDKG;2 is transcriptionally regulated by this hormone. The transient expression of the fusion protein NtCDKG;2-GFP, in N. tabacum leaves, evidentiated the nuclear localization of the studied protein. Stable transgenic plants overexpressing and silencing NtCDKG;2 by RNAi were also generated. Despite the high transcript levels in the plants overexpressing NtCDKG;2 and the low transcript levels in the silencing plants, macroscopic phenotypic alterations were not observed on these plants. Additionally, the expression of the NtCDKG;2 protein, with a histidine tag fused in its N-terminal, was obtained in Escherichia coli BL21(DE3)CodonPlusRP cells. Through studies performed on this work and literature analyses, it is possible to propose that NtCDKG;2 encodes a protein that is involved in the control of cell cycle at the N. tabacum stigmas/styles.

Le virus de la mosaïque jaune du navet (TYMV) possède un génome monopartite constitué d’ARN de polarité positive codant pour trois protéines, dont seule la polyprotéine 206K est indispensable à la réplication virale.Elle subit une maturation protéolytique, générant les protéines 140K et 66K, localisées au niveau de l’enveloppe des chloroplastes, siège de la réplication virale.Adressée aux chloroplastes, la protéine 140K y recrute la 66K et se comporte comme une protéine intégrale membranaire.Le domaine d’adressage aux chloroplastes (DAC) de la protéine 140K a été défini grâce à la transfection et à des protoplastes d’Arabidopsis thaliana par différentes constructions codantpour des versions délétées de la protéine fusionnées à l’EGFP, et à leur observation en microscopie confocale. Le DAC comprend deux hélices alpha amphipathiques dont la présence a été attestée par dichroïsme circulaire. Leur nécessité pour la localisation aux chloroplastes, l’association aux membranes et la réplication virale, a été étudiée. Différents patterns de distribution subcellulaire de la protéine 140K ont été observés. Ils sont corrélés au taux d’expression de la protéine. Sa dimérisation a également été démontrée.L’implication d’autres résidus du DAC dans la localisation subcellulaire, la dimérisation et la réplication virale, a également été recherchée. / Turnip yellow mosaic virus (TYMV) is a positive single-stranded RNA virus. Among the three ORFs encoded by the TYMV genome, 206K is the only protein required for viral replication. It is cleaved into 140K and 66K, which are both present at the chloroplast envelope membrane, where viral replication takes place.The 140K protein is targeted to chloroplasts, where it recruits 66K, and behaves as an integral membrane protein. The chloroplast targeting domain (DAC) of the 140K protein was defined using Arabidopsis thaliana protoplasts transfected by various constructs encoding deleted versions of 140Kfused to EGFP and subsequent confocal microscopy. The DAC comprises two amphipathic alpha helices, as confirmed by circular dichroism. Their involvement in chloroplast localisation and membrane association has been assessed, as well as their contribution to viral replication.We observed different subcellular distribution patterns of 140K protein, which correlate with the expression level of the protein. Its capability to dimerize has also been demonstrated.The involvement of other DAC residues in subcellular localisation, dimerization and viral replication has been studied.

Aberrant subcellular localization of mutant transmembrane receptors is increasingly acknowledged as a possible mechanism for an altered signaling quality leading to transformation. There is evidence that mutated receptor tyrosine kinases of subclass III, for example the platelet-derived growth factor receptor (PDGFR) and KIT-protein, are aberrantly localized in human cancers. In order to further analyze this phenomenon, we investigated the localization of FLT3, a subclass III receptor tyrosine kinase frequently mutated in leukemia. By immunofluorescence staining and confocal laser scanning microscopy we found that in retrovirally transduced COS7 cells, wild type FLT3 receptor protein is localized primarily at the cell surface. In contrast, a mutant FLT3 receptor protein with an internal tandem duplication (ITD) accumulates in a perinuclear region and is not detectable at the plasma membrane. Surprisingly, and in contrast to previously published data, intracellular FLT3-ITD accumulation could neither be detected in the endoplasmic reticulum (ER) nor in the Golgi apparatus. Furthermore, transient overexpression per se leads to accumulation of wild type FLT3 receptor protein in the ER in addition to surface localization, probably due to inefficient intracellular transport by the overloaded sorting machinery of the secretory pathway. Based on our data and the immature glycosylation pattern of FLT3-ITD, we speculate that the mutant protein resides most probably in an unidentified compartment of the secretory pathway between the ER and the Golgi apparatus. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.