Proteomics Reveals an Increase in the Abundance of Glycolytic and Ethanolic Fermentation Enzymes in Developing Sugarcane Culms During Sucrose Accumulation.

Sugarcane is an economically important crop contributing to the sugar and ethanol production of the world with 80 and 40%, respectively. Despite its importance as the main crop for sugar production, the mechanisms involved in the regulation of sucrose accumulation in sugarcane culms are still poorly understood. The aim of this work was to compare the quantitative changes of proteins in juvenile and maturing internodes at three stages of plant development. Label-free shotgun proteomics was used for protein profiling and quantification in internodes 5 (I5) and 9 (I9) of 4-, 7-, and 10-month-old-plants (4M, 7M, and 10M, respectively). The I9/I5 ratio was used to assess the differences in the abundance of common proteins at each stage of internode development. I9 of 4M plants showed statistically significant increases in the abundance of several enzymes of the glycolytic pathway and proteoforms of alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC). The changes in content of the enzymes were followed by major increases of proteins related to O2 transport like hemoglobin 2, ROS scavenging enzymes, and enzymes involved in the ascorbate/glutatione system. Besides, intermediates from tricarboxylic acid cycle (TCA) were reduced in I9-4M, indicating that the increase in abundance of several enzymes involved in glycolysis, pentose phosphate cycle, and TCA, might be responsible for higher metabolic flux, reducing its metabolites content. The results observed in I9-4M indicate that hypoxia might be the main cause of the increased flux of glycolysis and ethanolic fermentation to supply ATP and reducing power for plant growth, mitigating the reduction in mitochondrial respiration due to the low oxygen availability inside the culm. As the plant matured and sucrose accumulated to high levels in the culms, the proteins involved in glycolysis, ethanolic fermentation, and primary carbon metabolism were significantly reduced.

Revealing the high variability on nonconserved core and mobile elements of Austropuccinia psidii and other rust mitochondrial genomes.

Mitochondrial genomes are highly conserved in many fungal groups, and they can help characterize the phylogenetic relationships and evolutionary biology of plant pathogenic fungi. Rust fungi are among the most devastating diseases for economically important crops around the world. Here, we report the complete sequence and annotation of the mitochondrial genome of Austropuccinia psidii (syn. Puccinia psidii), the causal agent of myrtle rust. We performed a phylogenomic analysis including the complete mitochondrial sequences from other rust fungi. The genome composed of 93.299 bp has 73 predicted genes, 33 of which encoded nonconserved proteins (ncORFs), representing almost 45% of all predicted genes. A. psidii mtDNA is one of the largest rust mtDNA sequenced to date, most likely due to the abundance of ncORFs. Among them, 33% were within intronic regions of diverse intron groups. Mobile genetic elements invading intron sequences may have played significant roles in size but not shaping of the rust mitochondrial genome structure. The mtDNAs from rust fungi are highly syntenic. Phylogenetic inferences with 14 concatenated mitochondrial proteins encoded by the core genes placed A. psidii according to phylogenetic analysis based on 18S rDNA. Interestingly, cox1, the gene with the greatest number of introns, provided phylogenies not congruent with the core set. For the first time, we identified the proteins encoded by three A. psidii ncORFs using proteomics analyses. Also, the orf208 encoded a transmembrane protein repressed during in vitro morphogenesis. To the best of our knowledge, we presented the first report of a complete mtDNA sequence of a member of the family Sphaerophragmiacea.

Network Analyses and Data Integration of Proteomics and Metabolomics From Leaves of Two Contrasting Varieties of Sugarcane in Response to Drought.

Uncovering the molecular mechanisms involved in the responses of crops to drought is crucial to understand and enhance drought tolerance mechanisms. Sugarcane (Saccharum spp.) is an important commercial crop cultivated mainly in tropical and subtropical areas for sucrose and ethanol production. Usually, drought tolerance has been investigated by single omics analysis (e.g. global transcripts identification). Here we combine label-free quantitative proteomics and metabolomics data (GC-TOF-MS), using a network-based approach, to understand how two contrasting commercial varieties of sugarcane, CTC15 (tolerant) and SP90-3414 (susceptible), adjust their leaf metabolism in response to drought. To this aim, we propose the utilization of regularized canonical correlation analysis (rCCA), which is a modification of classical CCA, and explores the linear relationships between two datasets of quantitative variables from the same experimental units, with a threshold set to 0.99. Light curves revealed that after 4 days of drought, the susceptible variety had its photosynthetic capacity already significantly reduced, while the tolerant variety did not show major reduction. Upon 12 days of drought, photosynthesis in the susceptible plants was completely reduced, while the tolerant variety was at a third of its rate under control conditions. Network analysis of proteins and metabolites revealed that different biological process had a stronger impact in each variety (e.g. translation in CTC15, generation of precursor metabolites, response to stress and energy in SP90-3414). Our results provide a reference data set and demonstrate that rCCA can be a powerful tool to infer experimentally metabolite-protein or protein-metabolite associations to understand plant biology.

Metabolic profiles of planktonic and biofilm cells of Candida orthopsilosis.

AIM: This study aims to understand which Candida orthopsilosis protein aids fungus adaptation upon its switching from planktonic growth to biofilm. MATERIALS & METHODS: Ion mobility separation within mass spectrometry analysis combination were used. RESULTS: Proteins mapped for different biosynthetic pathways showed that selective ribosome autophagy might occur in biofilms. Glucose, used as a carbon source in the glycolytic flux, changed to glycogen and trehalose. CONCLUSION: Candida orthopsilosis expresses proteins that combine a variety of mechanisms to provide yeasts with the means to adjust the catalytic properties of enzymes. Adjustment of the enzymes helps modulate the biosynthesis/degradation rates of the available nutrients, in order to control and coordinate the metabolic pathways that enable cells to express an adequate response to nutrient availability.

Label-Free Quantitative Proteomic Analysis of Puccinia psidii Uredospores Reveals Differences of Fungal Populations Infecting Eucalyptus and Guava.

Puccinia psidii 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 P. psidii 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 P. psidii s.l. uredospores from two contrasting populations: guava fruits (PpGuava) and eucalyptus leaves (PpEucalyptus). NanoUPLC-MSE was used to generate peptide spectra that were matched to the UniProt Puccinia genera sequences (UniProt database) resulting in the first proteomic analysis of the phytopathogenic fungus P. psidii. 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.

Cell wall proteomics of sugarcane cell suspension cultures.

The use of cell walls to produce cellulosic ethanol from sugarcane bagasse is a new challenge. A better knowledge of proteins involved in cell wall remodelling is essential to improve the saccharification processes. Cell suspension cultures were used for this first cell wall proteomics study of sugarcane. Proteins extracted from cell walls were identified using an adapted protocol. They were extracted using 0.2 M CaCl2 and 2 M LiCl after purification of cell walls. The proteins were then identified by the innovative nanoACQUITY UPLC MS/MS technology and bioinformatics using the translated SUCEST EST cluster database of sugarcane. The experiments were reproduced three times. Since Sorghum bicolor is the closest plant with a fully sequenced genome, homologous proteins were searched for to complete the annotation of proteins, that is, prediction of subcellular localization and functional domains. Altogether, 69 different proteins predicted to be secreted were identified among 377 proteins. The reproducibility of the experiments is discussed. These proteins were distributed into eight functional classes. Oxidoreductases such as peroxidases were well represented, whereas glycoside hydrolases were scarce. This work provides information about the proteins that could be manipulated through genetic transformation, to increase second-generation ethanol production.

SAGE transcript profiling of the juvenile cambial region of Eucalyptus grandis.

Despite the importance of Eucalyptus spp. in the pulp and paper industry, functional genomic approaches have only recently been applied to understand wood formation in this genus. We attempted to establish a global view of gene expression in the juvenile cambial region of Eucalyptus grandis Hill ex Maiden. The expression profile was obtained from serial analysis of gene expression (SAGE) library data produced from 3- and 6-year-old trees. Fourteen-base expressed sequence tags (ESTs) were searched against public Eucalyptus ESTs and annotated with GenBank. Altogether 43,304 tags were generated producing 3066 unigenes with three or more copies each, 445 with a putative identity, 215 with unknown function and 2406 without an EST match. The expression profile of the juvenile cambial region revealed the presence of highly frequent transcripts related to general metabolism and energy metabolism, cellular processes, transport, structural components and information pathways. We made a quantitative analysis of a large number of genes involved in the biosynthesis of cellulose, pectin, hemicellulose and lignin. Our findings provide insight into the expression of functionally related genes involved in juvenile wood formation in young fast-growing E. grandis trees.

Proteomic analysis of the cambial region in juvenile Eucalyptus grandis at three ages.

Recent advances in genomics and proteomics have provided an excellent opportunity to understand complex biological processes such as wood formation at the gene and protein levels. The aim of this work was to describe the proteins participating in the processes involved in juvenile wood formation by isolating proteins from the cambial region of Eucalyptus grandis, at three ages of growth (6-month-old seedlings, 3- and 6-year-old trees), and also to identify proteins differentially expressed. Using a 2-D-LC-MS/MS strategy we identified a total of 240 proteins, with 54 corresponding spots being present in at least two ages. Overall, nine proteins classified into the functional categories of metabolism, cellular processes, and macromolecular metabolism showed significant changes in expression. Proteins were classified into seven main functional categories, with metabolism representing 35.2% of the total proteins identified. The comparison of the reference maps showed not only differences in the expression pattern of individual proteins at each age, but also among isoforms. The results described in this paper provide a dynamic view of the proteins involved in the formation of juvenile wood in E. grandis.