Evaluation of the primary metabolism of Eucalyptus grandis cambial region and bark

O gênero Eucalyptus é uma das principais espécies arbóreas comercialmente plantadas em todo o mundo. Apresenta inúmeras características favoráveis para a produção e comercialização de sua madeira, tais como o rápido crescimento, rotação de ciclo curto e produção de biomassa renovável, com potencial para geração de biocombustível. Apesar de sua grande importância econômica, pouco é conhecido sobre os processos moleculares que envolvem a formação da madeira e casca, principalmente no que diz respeito ao metabolismo primário. Ademais, não se têm muitas informações sobre mudanças moleculares que ocorrem durante a formação desses tecidos em resposta a variações sazonais. Sabe-se que a região cambial das árvores apresenta maior atividade metabólica durante o verão, quando comparada ao inverno. Deste modo, visando compreender mudanças dinâmicas ao nível transcricional, protéico e metabólico, principalmente em relação ao metabolismo primário, o presente trabalho teve por objetivo comparar os tecidos da região cambial e casca, coletados em diferentes períodos climáticos (verão e inverno). A expressão do padrão transcricional foi analisada por PCR em tempo real, seguido de normalização e análise estatística, usando os programas NormFinder e Rest, respectivamente. O perfil protéico foi obtido por eletroforese bidimensional (2D-PAGE), seguido de análise por espectrometria de massas associada a cromatografia líquida (LC-MS/MS) e posterior identificação das proteínas pelo programa Mascot Daemon. Já o perfil metabólico foi obtido por espectrometria de massas associada à cromatografia gasosa (GC/MS), com posterior análise dos picos identificados pelo programa MatLab. Em seguida, as análises estatísticas foram geradas pelo programa SIMCA P+ e \"R\". Para os transcritos, foi possível observar tanto para a região cambial quanto para a casca padrão diferencial na expressão dos genes analisados, principalmente aqueles atuantes na glicólise, durante os dois períodos sazonais. Quanto ao perfil protéico, foram identificadas um total de 77 e 75 proteínas estatisticamente significativas na região cambial e casca, respectivamente. Proteínas pertencentes ao metabolismo primário foram identificadas em ambos os tecidos. Metabólitos relacionados ao metabolismo de açúcares também foram encontrados.Eucalyptus genus is the most widely planted hardwood crop in the world because of its superior growth, broad adaptability and multipurpose wood properties. In today\'s \"new carbon economy\", eucalypts are receiving attention as fast-growing, short-rotation, renewable biomass crop for energy production. In spite of its economical importance, little information is available about the molecular changes that occur in primary metabolism in the wood and bark forming tissues. Furthermore, there is less information about molecular changes that occur during wood and bark formation in response to seasonal variation. It\'s known that Eucalyptus cambial region presents higher metabolic activity in summer than in winter. Thus, in order to observe the dynamic changes in transcript, protein and metabolite levels, mainly related to primary metabolism, we compared cambial tissue and bark collected in two different seasons (summer high temp + high rainfall) and winter (lower temp and little rainfall). Transcript expression patterns were analyzed by Real-Time PCR, normalization chosen by NormFinder and statistical analysis carried out using REST. The protein profile was obtained by bidimensional electrophoresis (2D-PAGE) followed by liquid chromatography associated with mass spectrometry (LC-MS/MS) and analyzed by Mascot Daemon. Metabolite profile was obtained by GC-TOF/MS, peaks were analyzed in MatLab and statistical analyses were done using SIMCA and \"R\". The results obtained with transcripts indicate differential gene expression in the cambial region and bark during summer and winter. A total of 77 and 75 proteins in cambium and bark, respectively, presented statistically significant alterations and were identified and classified into functional categories. We identified many proteins from primary metabolism. Metabolites from carbohydrate metabolism were also identified

Genome-Wide Analysis of Lipoxygenase (LOX) Genes in Angiosperms

Lipoxygenases (LOXs) are enzymes that catalyze the addition of an oxygen molecule to unsaturated fatty acids, thus forming hydroperoxides. In plants, these enzymes are encoded by a multigene family found in several organs with varying activity patterns, by which they are classified as LOX9 or LOX13. They are involved in several physiological functions, such as growth, fruit development, and plant defense. Despite several studies on genes of the LOX family in plants, most studies are restricted to a single species or a few closely related species. This study aimed to analyze the diversity, evolution, and expression of LOX genes in angiosperm species. We identified 247 LOX genes among 23 species of angiosperms and basal plants. Phylogenetic analyses identified clades supporting LOX13 and two main clades for LOX9: LOX9_A and LOX9_B. Eudicot species such as Tarenaya hassleriana, Capsella rubella, and Arabidopsis thaliana did not present LOX9_B genes; however, LOX9_B was present in all monocots used in this study. We identified that there were potential new subcellular localization patterns and conserved residues of oxidation for LOX9 and LOX13 yet unexplored. In summary, our study provides a basis for the further functional and evolutionary study of lipoxygenases in angiosperms

Physiological Responses to Drought, Salinity, and Heat Stress in Plants: A Review

On the world stage, the increase in temperatures due to global warming is already a reality that has become one of the main challenges faced by the scientific community. Since agriculture is highly dependent on climatic conditions, it may suffer a great impact in the short term if no measures are taken to adapt and mitigate the agricultural system. Plant responses to abiotic stresses have been the subject of research by numerous groups worldwide. Initially, these studies were concentrated on model plants, and, later, they expanded their studies in several economically important crops such as rice, corn, soybeans, coffee, and others. However, agronomic evaluations for the launching of cultivars and the classical genetic improvement process focus, above all, on productivity, historically leaving factors such as tolerance to abiotic stresses in the background. Considering the importance of the impact that abiotic stresses can have on agriculture in the short term, new strategies are currently being sought and adopted in breeding programs to understand the physiological, biochemical, and molecular responses to environmental disturbances in plants of agronomic interest, thus ensuring the world food security. Moreover, integration of these approaches is bringing new insights on breeding. We will discuss how water deficit, high temperatures, and salinity exert effects on plants

A metabolomic study of <i>Gomphrena agrestis</i> in Brazilian Cerrado suggests drought-adaptive strategies on metabolism

Drought is the main factor that limits the distribution and productivity of plant species. In the Brazilian Cerrado, the vegetation is adapted to a seasonal climate with long- and short-term periods of drought. To analyze the metabolic strategies under such conditions, a metabolomic approach was used to characterize Gomphrena agrestis Mart. (Amaranthaceae) a native species that grows under natural conditions, in a rock-field area. Roots and leaves material from native specimens were sampled along different seasons of the year and LC–MS and GC–MS analyzed for multiple chemical constituents. The datasets derived from the different measurements were combined and evaluated using multivariate analysis. Principal component analysis was used to obtain an overview of the samples and identify outliers. Later, the data was analyzed with orthogonal projection to latent structures discriminant analysis to obtain valid models that could explain the metabolite variations in the different seasons. Two hundred and eighty metabolites were annotated, generating a unique database to characterize metabolic strategies used to cope with the effects of drought. The accumulation of fructans in the thickened roots is consistent with the storage of carbons during the rainy season to support the energy demand during a long period of drought. The accumulation of Abscisic acid, sugars and sugar alcohols, phenolics, and pigment in the leaves suggests physiological adaptations. To cope with long-term drought, the data suggests that tissue water status and storage of reserves are important to support plant survival and regrowth. However, during short-term drought, osmoregulation and oxidative protection seems to be essential, probably to support the maintenance of active photosynthesis

Label-Free Quantitative Proteomic Analysis of <i>Puccinia psidii</i> Uredospores Reveals Differences of Fungal Populations Infecting Eucalyptus and Guava

<div><p><i>Puccinia psidii</i> sensu lato (s.l.) is the causal agent of eucalyptus and guava rust, but it also attacks a wide range of plant species from the myrtle family, resulting in a significant genetic and physiological variability among populations accessed from different hosts. The uredospores are crucial to <i>P</i>. <i>psidii</i> dissemination in the field. Although they are important for the fungal pathogenesis, their molecular characterization has been poorly studied. In this work, we report the first in-depth proteomic analysis of <i>P</i>. <i>psidii</i> s.l. uredospores from two contrasting populations: guava fruits (PpGuava) and eucalyptus leaves (PpEucalyptus). NanoUPLC-MS^E was used to generate peptide spectra that were matched to the UniProt <i>Puccinia</i> genera sequences (UniProt database) resulting in the first proteomic analysis of the phytopathogenic fungus <i>P</i>. <i>psidii</i>. Three hundred and fourty proteins were detected and quantified using Label free proteomics. A significant number of unique proteins were found for each sample, others were significantly more or less abundant, according to the fungal populations. In PpGuava population, many proteins correlated with fungal virulence, such as malate dehydrogenase, proteossomes subunits, enolases and others were increased. On the other hand, PpEucalyptus proteins involved in biogenesis, protein folding and translocation were increased, supporting the physiological variability of the fungal populations according to their protein reservoirs and specific host interaction strategies.</p></div

Proteins identified from <i>P</i>. <i>psidii</i> uredospore populations.

<p>The proteins exclusively found in PpGuava (right) and PpEucalyptus (left) and common to both populations (center). Of the common proteins, 25 and 120 whose abundance were increased in PpEucalyptus and PpGuava, respectively.</p

Morphological and viability analysis of <i>Puccinia psidii</i> uredospores.

<p><i>P</i>. <i>psidii</i> uredospores from <i>E</i>. <i>grandis</i><b>(A)</b> and <i>P</i>. <i>guajava</i><b>(B)</b> exhibit similar morphology and germination viability, respectively <b>(C and D)</b>.The arrows indicate the fungal germ tube in both uredospore populations, 24 hours after inoculation in water-agar medium. Light microscopy images of PpEucalyptus and PpGuava uredospores are shown at 100 X (A and B) and 200 X (C and D) magnification. Scale bar: 20 μm in A and B, 50 μm in C and D.</p

Gene ontology of biological process terms in the proteomic analysis.

<p>Bar graph represents the ratio of % composition of term in the proteomic data.</p

Eucalyptus infection by guava and eucalyptus rust.

<p>Symptoms induced by inoculation of <i>P</i>. <i>psidii</i> uredospores from PpEucalyptus <b>(A)</b> on <i>E</i>. <i>grandis</i> variety D901. This clone is rust susceptible when grown under field conditions. The white arrows indicate the fungal pustules. The leaves are shown 15 days after inoculation.The PpGuava populations <b>(B)</b> and control <b>(C)</b> did not show typical rust symptoms.</p