Transcriptomic and proteomic profiling of maize embryos exposed to camptothecin

<p>Abstract</p> <p>Background</p> <p>Camptothecin is a plant alkaloid that specifically binds topoisomerase I, inhibiting its activity and inducing double stranded breaks in DNA, activating the cell responses to DNA damage and, in response to severe treatments, triggering cell death.</p> <p>Results</p> <p>Comparative transcriptomic and proteomic analyses of maize embryos that had been exposed to camptothecin were conducted. Under the conditions used in this study, camptothecin did not induce extensive degradation in the genomic DNA but induced the transcription of genes involved in DNA repair and repressed genes involved in cell division. Camptothecin also affected the accumulation of several proteins involved in the stress response and induced the activity of certain calcium-dependent nucleases. We also detected changes in the expression and accumulation of different genes and proteins involved in post-translational regulatory processes.</p> <p>Conclusions</p> <p>This study identified several genes and proteins that participate in DNA damage responses in plants. Some of them may be involved in general responses to stress, but others are candidate genes for specific involvement in DNA repair. Our results open a number of new avenues for researching and improving plant resistance to DNA injury.</p

Role of Plant-Specific N-Terminal Domain of Maize CK2β1 Subunit in CK2β Functions and Holoenzyme Regulation

Protein kinase CK2 is a highly pleiotropic Ser/Thr kinase ubiquituous in eukaryotic organisms. CK2 is organized as a heterotetrameric enzyme composed of two types of subunits: the catalytic (CK2α) and the regulatory (CK2β). The CK2β subunits enhance the stability, activity and specificity of the holoenzyme, but they can also perform functions independently of the CK2 tetramer. CK2β regulatory subunits in plants differ from their animal or yeast counterparts, since they present an additional specific N-terminal extension of about 90 aminoacids that shares no homology with any previously characterized functional domain. Sequence analysis of the N-terminal domain of land plant CK2β subunit sequences reveals its arrangement through short, conserved motifs, some of them including CK2 autophosphorylation sites. By using maize CK2β1 and a deleted version (ΔNCK2β1) lacking the N-terminal domain, we have demonstrated that CK2β1 is autophosphorylated within the N-terminal domain. Moreover, the holoenzyme composed with CK2α1/ΔNCK2β1 is able to phosphorylate different substrates more efficiently than CK2α1/CK2β1 or CK2α alone. Transient overexpression of CK2β1 and ΔNCK2β1 fused to GFP in different plant systems show that the presence of N-terminal domain enhances aggregation in nuclear speckles and stabilizes the protein against proteasome degradation. Finally, bimolecular fluorescence complementation (BiFC) assays show the nuclear and cytoplasmic location of the plant CK2 holoenzyme, in contrast to the individual CK2α/β subunits mainly observed in the nucleus. All together, our results support the hypothesis that the plant-specific N-terminal domain of CK2β subunits is involved in the down-regulation of the CK2 holoenzyme activity and in the stabilization of CK2β1 protein. In summary, the whole amount of data shown in this work suggests that this domain was acquired by plants for regulatory purposes

Anàlisis proteómicos y caracterización molecular de proteínas implicadas en la respuesta a estrés hídrico en plantas

Durante esta tesis doctoral se han desarrollado técnicas bioquímicas analíticas y/o separativas de detección y cuantificación de proteínas en diferentes proteomas de plantas. Los avances tanto en gradientes inmobilizados de proteínas, como en espectrometría de masas nos han llevado a la puesta a punto de técnicas de electroforesis bidimensional (2DE), o lo que denominamos Proteómica de Expresión, así como de nuevas técnicas de cromatografía líquida bidimensional. Nuestro trabajo se ha centrado en la caracterización de los diferentes proteomas de variedades cultivares tolerantes y sensibles a sequía de embriones de trigo y arroz que nos han servido como modelos en nuestros estudios. La identificación de proteínas acumuladas diferencialmente en las variedades analizadas nos ha permitido establecer mecanismos de regulación propios en cada variedad frente a estímulos percibidos por déficit hídrico. Además, en las variedades de trigo hemos identificado marcadores moleculares de sequía. Entre las proteínas acumuladas diferencialmente en situaciones de estrés abiótico tanto en trigo como en arroz hemos identificado proteínas LEAs (Late Embriogenesis Abundant). Debido a la importancia de la función de estas proteínas en respuesta a estrés abiótico, se ha llevado a cabo un estudio más profundo de la regulación de estas proteínas. Así, realizamos un análisis de fosfoproteoma de proteínas LEAs en Arabidopsis thaliana, donde encontramos un número importantes de estas proteínas fosforiladas, poniendo de manifiesto la importancia de esta modificación post-traduccional en la regulación de este grupo de proteínas con funciones tan diversas. Además, se ha comprobado in silico e in vitro que estas proteínas se fosforilan por la proteína quinasa CK2. En la última parte de este trabajo nos hemos centrado en la caracterización evolutiva, molecular, estructural y funcional de la subunidad reguladora CK2ß1 de la proteína quinasa CK2 de maíz. CK2 se ha descrito a lo largo de los últimos años como una quinasa ubicua que interviene en diferentes procesos fisiológicos en la planta. Previamente, en nuestro laboratorio se demostró la implicación de CK2 en la respuesta a estrés osmótico, mediante la fosforilación de la proteína RAB17. En este trabajo nos centramos en el estudio de las características específicas de CK2 en plantas y más concretamente en el estudio de sus subunidades reguladoras. Hemos realizado análisis enfocados al conocimiento de la estructura de la subunidad reguladora CK21 de maíz. Una de las características específicas de CK2 en plantas incluye la presencia de un dominio de unos 80-90 aminoácidos en el extremo N-terminal en las subunidades reguladoras CK2s. Debido a que este dominio es exclusivo de plantas y no presenta homología con ninguna otra proteína conocida, se han realizado diferentes aproximaciones con el objetivo de investigar su funcionalidad en plantas. Llegamos a la conclusión de que probablemente este dominio fue adquirido por las plantas por motivos de regulación ya que afecta a la estabilidad de la proteína y a la actividad enzimática del holoenzima.Peer reviewe

Proteomic analysis of wheat embryos with 2-DE and liquid-phase chromatography (ProteomeLab PF-2D) — A wider perspective of the proteome

Cereal embryos are a model system to study desiccation tolerance due to their ability to survive extreme water loss during late embryogenesis. To identify proteins accumulating in mature embryos which can be used as potential markers for dehydration tolerance, we compared the embryo proteome from two durum wheat genotypes (Triticum durum Desf.), Mahmoudi (salt and drought sensitive) and Om Rabia3 (salt and drought tolerant). Total protein extracts from wheat embryos were analyzed by using conventional 2-DE and ProteomeLab PF-2D. Analysis using different pH ranges showed that a larger number of fractions were solved by LC, than by conventional 2-DE at extreme technical pHs (pH 4.0–5.0 and pH 6.5–8.0). In contrast, at intermediate pHs (pH 5.0–6.5), resolution was better in 2-DE gels. The two techniques were used in parallel to analyze total protein extracts from embryos of the two wheat varieties. Several proteins belonging to the seed storage family, LEA-type/heat shock proteins, enzyme metabolism and radical scavengers were identified by analysis of trypsin digested peptides via mass spectrometry. These proteins accumulate in different amounts in embryos of tolerant and sensitive wheat varieties. The differences in expression pattern were further validated by enzyme activity, western blotting analysis and correlated with their corresponding mRNA expression by RT-PCR analyses for the corresponding protein. We suggest that the differential expression pattern could be used as a basis for a biochemical screen of tolerance/sensitivity to drought and salt stress in wheat embryos and germplasm.This work was supported by grants BIO2006-04101 from MCYT (Spain) to MP and PCI Tunisia-Spain 75-04/R2T from AECI to MP and KM.Peer reviewe

Combination of 2DE and LC for plant proteomics analysis

The use of analytical biochemical techniques with different separation properties allows us to better understand the proteome. To demonstrate this we have used two different methodologies to analyze embryos from a Tunisian cultivar of durum wheat (Triticum durum Desf.), variety Oum Rabiaa. We compared conventional 2D electrophoresis with liquid-phase chromatography. Our results show that a similar number of proteins were detected with both techniques. However, analysis of protein resolution at different pH ranges showed significant differences. By using a large pH gradient we observed that liquid chromatography presents higher resolution at extreme pH, either acidic or basic. Conversely, 2DE is more resolutive at intermediate pH (pH 5–6.5). Taking these results in consideration, we propose that 2DE and liquid chromatography are complementary methods to analyze complex protein extracts and can be used in parallel to acquire a wider perspective and a better understanding of the embryo proteome.This work was supported by Bio 2009-13044-Co2-01 from MICIN.Peer reviewe

A proteomic approach reveals new actors of nodule response to drought in split-root grown pea plants

Drought is considered the more harmful abiotic stress resulting in crops yield loss. Legumes in symbiosis with rhizobia are able to fix atmospheric nitrogen. Biological nitrogen fixation (SNF) is a very sensitive process to drought and limits legumes agricultural productivity. Several factors are known to regulate SNF including oxygen availability to bacteroids, carbon and nitrogen metabolisms; but the signaling pathways leading to SNF inhibition are largely unknown. In this work, we have performed a proteomic approach of pea plants grown in split-root system where one half of the root was well-irrigated and the other was subjected to drought. Water stress locally provoked nodule water potential decrease that led to SNF local inhibition. The proteomic approach revealed 11 and 7 nodule proteins regulated by drought encoded by Pisum sativum and Rhizobium leguminosarum genomes respectively. Among these 18 proteins, 3 proteins related to flavonoid metabolism, 2 to sulfur metabolism and 3 RNA-binding proteins were identified. These proteins could be molecular targets for future studies focused on the improvement of legumes tolerance to drought. Moreover, this work also provides new hints for the deciphering of SNF regulation machinery in nodules.This work has been partially funded by the Spanish National Research and Development Programme (AGL2011-30386-CO2-1 and AGL2011-23738).Peer reviewe

Novel clues on abiotic stress tolerance emerge from embryo proteome analyses of rice varieties with contrasting stress adaptation

Cereal embryos sustain severe water deficit at the final stage of seed maturation. The molecular mechanisms underlying the acquisition of desiccation tolerance in seed embryos are similar to those displayed during water deficit in vegetative tissues. The genetic variation among six rice genotypes adapted to diverse environmental conditions was analysed at the proteome level to get further clues on the mechanisms leading to water-stress tolerance. MS analysis allowed the identification of 28 proteins involved in stress tolerance (late embryogenesis abundant proteins), nutrient reservoir activity, among other proteins implicated in diverse cellular processes potentially related to the stress response (e.g., mitochondrial import translocase). Hierarchical clustering and multidimensional scaling analyses revealed a close relationship between the stress-sensitive genotypes, whereas the stress-tolerant varieties were more distantly related. Besides qualitative and significant quantitative changes in embryo proteins across the distinct varieties, we also found differences at post-translational level. The results indicated that late embryogenesis abundant Rab21 was more strongly phosphorylated in the embryos of the sensitive varieties than in the embryos of the tolerant ones. We propose that the differences found in the phosphorylation status of Rab21 are related to stress tolerance.This work was supported by Fundação para a Ciência e a Tecnologia (FCT) from the Portuguese Ministério da Ciência, Tecnologia e Ensino Superior; by Instituto Tecnologia Química e Biológica (ITQB Research Line ICV382)–Portugal, and the Spanish Ministerio de Educación y Ciencia (Project BIO2003-01133). A.P.F. was supported through the Ph.D. fellowship SFRH/BD/1185/2000 from FCT.Peer reviewe

Functional characterization of DHN-5, a dehydrin showing a differential phosphorylation pattern in two Tunisian durum wheat (Triticum durum Desf.) varieties with marked differences in salt and drought tolerance

Water-deficit stress caused by drought and soil salinization adversely affects plant growth and crop productivity. Dehydrins are involved in the adaptation to water and osmotic stress. We have identified a wheat dehydrin named DHN-5 that is closely related to the maize RAB17. The full-length cDNA of Dhn-5 gene encodes a putative protein of 227 amino acids and contains 2 conserved lysine-rich-K-segment (EKKGIMDKIKEKLPG) repeats preceded by a stretch of eight serine residues, characteristic of group 2 LEA family. The Northern blot analyses showed a strong accumulation of Dhn-5 transcript in mature wheat embryos and to a lesser extent in ABA and salt-treated seedlings. Interestingly, DHN-5 protein accumulated differentially in two Tunisian durum wheat (Triticum durum Desf.) varieties with marked differences in salt and drought tolerance. By using specific dehydrin antibodies and 2D immunoblot analysis on proteins extracted from mature embryos in these two varieties, a differential phosphorylation pattern of DHN-5 was observed. In the resistant variety (R), beside a basic protein spot, a series of acidic spots were detected whereas in the sensitive variety (S) the acidic spots were weakly detectable. These acidic forms correspond to highly phosphorylated forms of DHN-5, which can be removed by alkaline phosphatase treatment. Accumulation of phosphorylated DHN-5 mainly in the R variety suggests a role of P-DHN-5 in preservation of cell integrity during late embryogenesis and desiccation. Subcellular localization of the DHN-5:GFP fusion protein indicated that DHN-5 would be primarily nuclear, suggesting a nuclear role in wheat osmotic stress response. The observed differential phosphorylation pattern of DHN-5 in the resistant and sensitive wheat varieties could be used as a basis for a molecular screen of tolerance/sensitivity to drought and salt stresses in wheat germplasm.This work was supported jointly by grants from the Ministry of Scientific Research, Technology and Development of Competencies, Tunisia and the Agence Espagnole de cooperation Internationale (AECI) Officina Técnica de Cooperación, Spain.Peer reviewe

Transcriptomic and proteomic profiling of maize embryos exposed to camptothecin

Camptothecin is a plant alkaloid that specifically binds topoisomerase I, inhibiting its activity and inducing double stranded breaks in DNA, activating the cell responses to DNA damage and, in response to severe treatments, triggering cell death. Comparative transcriptomic and proteomic analyses of maize embryos that had been exposed to camptothecin were conducted. Under the conditions used in this study, camptothecin did not induce extensive degradation in the genomic DNA but induced the transcription of genes involved in DNA repair and repressed genes involved in cell division. Camptothecin also affected the accumulation of several proteins involved in the stress response and induced the activity of certain calcium-dependent nucleases. We also detected changes in the expression and accumulation of different genes and proteins involved in post-translational regulatory processes. This study identified several genes and proteins that participate in DNA damage responses in plants. Some of them may be involved in general responses to stress, but others are candidate genes for specific involvement in DNA repair. Our results open a number of new avenues for researching and improving plant resistance to DNA injury

Specific characteristics of CK2β regulatory subunits in plants

In all eukaryotes, the typical CK2 holoenzyme is an heterotetramer composed of two catalytic (CK2α and CK2α′) and two regulatory (CK2β) subunits. One of the distinctive traits of plant CK2 is that they present a greater number of genes encoding for CK2α/β subunits than animals or yeasts, for instance, in Arabidopsis and maize both CK2α/β subunits belong to multigenic families composed by up to four genes. Here, we conducted a genome-wide survey examining 34 different plant genomes in order to investigate if the multigenic property of CK2β genes is a common feature through the entire plant kingdom. Also, at the level of structure, the plant CK2β regulatory subunits present distinctive features as (i) they lack about 20 aminoacids in the C-terminal domain, (ii) they present a specific N-terminal extension of about 90 aminoacids that shares no homology with any previously characterized functional domain, and (iii) the acidic loop region is poorly conserved at the aminoacid level. Since there is no data about CK2β or holoenzyme structure in plants, in this study, we use human CK2β as a template to predict a structure for Zea mays CK2β1 by homology modeling and we discuss about possible structural changes in the acidic loop region that could affect the enzyme regulation.M.R. was financed by I3P-CSIC2006 and CONSOLIDER (CSD2007-00057) from MICINN (Spain), I.C. V–B. by predoctoral fellowship FPI2007 from MICINN (Spain) and L. C–P. by Juan de la Cierva Programme, MICINN (Spain). This work was supported by grant BIO2009-13044-CO2-01 from MICINN (Spain).Peer reviewe