Valesca Pandolfi

New Insights in the Sugarcane Transcriptome Responding to Drought Stress as Revealed by Supersage

In the scope of the present work, four SuperSAGE libraries have been generated, using bulked root tissues from four drought-tolerant accessions as compared with four bulked sensitive genotypes, aiming to generate a panel of differentially expressed stress-responsive genes. Both groups were submitted to 24 hours of water deficit stress. The SuperSAGE libraries produced 8,787,315 tags (26 bp) that, after exclusion of singlets, allowed the identification of 205,975 unitags. Most relevant BlastN matches comprised 567,420 tags, regarding 75,404 unitags with 164,860 different ESTs. To optimize the annotation efficiency, the Gene Ontology (GO) categorization was carried out for 186,191 ESTs (BlastN against Uniprot-SwissProt), permitting the categorization of 118,208 ESTs (63.5%). In an attempt to elect a group of the best tags to be validated by RTqPCR, the GO categorization of the tag-related ESTs allowed the in silico identification of 213 upregulated unitags responding basically to abiotic stresses, from which 145 presented no hits after BlastN analysis, probably concerning new genes still uncovered in previous studies. The present report analyzes the sugarcane transcriptome under drought stress, using a combination of high-throughput transcriptome profiling by SuperSAGE with the Solexa sequencing technology, allowing the identification of potential target genes during the stress response.

Expression dynamics and genome distribution of osmoprotectants in soybean: identifying important components to face abiotic stress

BackgroundDespite the importance of osmoprotectants, no previous in silico evaluation of high throughput data is available for higher plants. The present approach aimed at the identification and annotation of osmoprotectant-related sequences applied to short transcripts from a soybean HT-SuperSAGE (High Throughput Super Serial Analysis of Gene Expression; 26-bp tags) database, and also its comparison with other transcriptomic and genomic data available from different sources.MethodsA curated set of osmoprotectants related sequences was generated using text mining and selected seed sequences for identification of the respective transcripts and proteins in higher plants. To test the efficiency of the seed sequences, these were aligned against four HT-SuperSAGE contrasting libraries generated by our group using soybean tolerant and sensible plants against water deficit, considering only differentially expressed transcripts (p ≤ 0.05). Identified transcripts from soybean and their respective tags were aligned and anchored against the soybean virtual genome.ResultsThe workflow applied resulted in a set including 1,996 seed sequences that allowed the identification of 36 differentially expressed genes related to the biosynthesis of osmoprotectants [Proline (P5CS: 4, P5CR: 2), Trehalose (TPS1: 9, TPPB: 1), Glycine betaine (BADH: 4) and Myo- inositol (MIPS: 7, INPS1: 8)], also mapped in silico in the soybean genome (25 loci). Another approach considered matches using Arabidopsis full length sequences as seed sequences, and allowed the identification of 124 osmoprotectant-related sequences, matching ~10.500 tags anchored in the soybean virtual chromosomes. Osmoprotectant-related genes appeared clustered in all soybean chromosomes, with higher density in some subterminal regions and synteny among some chromosome pairs.ConclusionsSoybean presents all searched osmoprotectant categories with some important members differentially expressed among the comparisons considered (drought tolerant or sensible vs. control; tolerant vs. sensible), allowing the identification of interesting candidates for biotechnological inferences. The identified tags aligned to corresponding genes that matched 19 soybean chromosomes. Osmoprotectant-related genes are not regularly distributed in the soybean genome, but clustered in some regions near the chromosome terminals, with some redundant clusters in different chromosomes indicating their involvement in previous duplication and rearrangements events. The seed sequences, transcripts and map represent the first transversal evaluation for osmoprotectant-related genes and may be easily applied to other plants of interest.

Overall picture of expressed Heat Shock Factors in Glycine max, Lotus japonicus and Medicago truncatula

Heat shock (HS) leads to the activation of molecular mechanisms, known as HS-response, that prevent damage and enhance survival under stress. Plants have a flexible and specialized network of Heat Shock Factors (HSFs), which are transcription factors that induce the expression of heat shock proteins. The present work aimed to identify and characterize the Glycine max HSF repertory in the Soybean Genome Project (GENOSOJA platform), comparing them with other legumes (Medicago truncatula and Lotus japonicus) in view of current knowledge of Arabidopsis thaliana. The HSF characterization in leguminous plants led to the identification of 25, 19 and 21 candidate ESTs in soybean, Lotus and Medicago, respectively. A search in the SuperSAGE libraries revealed 68 tags distributed in seven HSF gene types. From the total number of obtained tags, more than 70% were related to root tissues (water deficit stress libraries vs. controls), indicating their role in abiotic stress responses, since the root is the first tissue to sense and respond to abiotic stress. Moreover, as heat stress is related to the pressure of dryness, a higher HSF expression was expected at the water deficit libraries. On the other hand, expressive HSF candidates were obtained from the library inoculated with Asian Soybean Rust, inferring crosstalk among genes associated with abiotic and biotic stresses. Evolutionary relationships among sequences were consistent with different HSF classes and subclasses. Expression profiling indicated that regulation of specific genes is associated with the stage of plant development and also with stimuli from other abiotic stresses pointing to the maintenance of HSF expression at a basal level in soybean, favoring its activation under heat-stress conditions.

Plant Antimicrobial Peptides: An Overview of SuperSAGE Transcriptional Profile and a Functional Review

Defensin, thionin and lipid transfer protein (LTP) gene families, which antimicrobial activity has an attractive use in protein engineering and transgenic production of agronomical important plants, have been here functionally reviewed. Also, a transcriptional overview of a set of plant SuperSAGE libraries and analysis looking for 26 bp tags possibly annotated for those families is presented. Tags differentially expressed (p = 0.05) or constitutively transcribed were identified from leaves or roots SuperSAGE libraries from important Brazilian plant species [cowpea (Vigna unguiculata (L.) Walp.), soybean (Glycine max (L.) Merr.) and modern sugarcane hybrids (Saccharum spp.)] submitted to abiotic [salt (100 mM NaCl) or drought] or biotic stresses [fungus inoculation (Phakopsora pachyrhizi; Asiatic Soyben Rust phytopathogen)]. The diverse transcriptional patterns observed, probably related to the variable range of targets and functions involved, could be the first step to unravel the antimicrobial peptide world and the plant stress response relationship. Moreover, SuperSAGE opens the opportunity to find some SNPs or even rare transcript that could be important on plant stress resistance mechanisms. Putative defensin or LTP identified by SuperSAGE following a specific plant treatment or physiological condition could be useful for future use in genetic improvement of plants.

Early transcriptional response of soybean contrasting accessions to root dehydration.

Drought is a significant constraint to yield increase in soybean. The early perception of water deprivation is critical for recruitment of genes that promote plant tolerance. DeepSuperSAGE libraries, including one control and a bulk of six stress times imposed (from 25 to 150 min of root dehydration) for drought-tolerant and sensitive soybean accessions, allowed to identify new molecular targets for drought tolerance. The survey uncovered 120,770 unique transcripts expressed by the contrasting accessions. Of these, 57,610 aligned with known cDNA sequences, allowing the annotation of 32,373 unitags. A total of 1,127 unitags were up-regulated only in the tolerant accession, whereas 1,557 were up-regulated in both as compared to their controls. An expression profile concerning the most representative Gene Ontology (GO) categories for the tolerant accession revealed the expression “protein binding” as the most represented for “Molecular Function”, whereas CDPK and CBL were the most up-regulated protein families in this category. Furthermore, particular genes expressed different isoforms according to the accession, showing the potential to operate in the distinction of physiological behaviors. Besides, heat maps comprising GO categories related to abiotic stress response and the unitags regulation observed in the expression contrasts covering tolerant and sensitive accessions, revealed the unitags potential for plant breeding. Candidate genes related to “hormone response” (LOX, ERF1b, XET), “water response” (PUB, BMY), “salt stress response” (WRKY, MYB) and “oxidative stress response” (PER) figured among the most promising molecular targets. Additionally, nine transcripts (HMGR, XET, WRKY20, RAP2-4, EREBP, NAC3, PER, GPX5 and BMY) validated by RT-qPCR (four different time points) confirmed their differential expression and pointed that already after 25 minutes a transcriptional reorganization started in response to the new condition, with important differences between both accessions.

Identification of Plant Protein Kinases in Response to Abiotic and Biotic Stresses Using SuperSAGE

Plants are sessile organisms subjected to many environmental adversities. For their survival they must sense and respond to biotic and abiotic stresses efficiently. During this process, protein kinases are essential in the perception of environmental stimuli, triggering signaling cascades. Kinases are among the largest and most important gene families for biotechnological purposes, bringing many challenges to the bioinformaticians due to the combination of conserved domains besides diversified regions. Cowpea [Vigna unguiculata (L.) Walp.] is an important legume that is adapted to different agroclimatic conditions, including drought, humidity and a range of temperatures. For this crop, the association of the SuperSAGE method with high-throughput sequencing technology would generate reliable transcriptome profiles with millions of tags counted and statistically analyzed. An approach evaluating biotic and abiotic stresses was carried out generating over 13 million cowpea SuperSAGE tags available from leaves/roots of plants under abiotic (mechanical injury and salinity) or biotic (CABMV, Cowpea aphid born mosaic virus) stresses. The annotation and identification of tags linked by BlastN to previously well described ESTs, allowed the posterior identification of kinases. The annotation efficiency depended on the database used, with the KEGG figuring as a good source for annotated ESTs especially when complemented by an independent Gene Ontology categorization, as well as the Gene Index using selected species. The use of different approaches allowed the identification of 1,350 kinase candidates considering biotic libraries and 2,268 regarding abiotic libraries, based on a combination of both, adequate descriptions and GO terms. Additional searches in kinase specific databases allowed the identification of a relatively low number of additional kinases, uncovering the lack of kinase databases for non-model organisms, especially plants. Concerning the kinase families, a total of 713 potential kinases were classified into 13 families of the CMGC and STE groups. Concerning the differentially expressed kinases, 169 of the 713 potential kinases were identified (p < 0.05), 100 up- and 69 down-regulated when comparing distinct libraries, allowing the generation of a comprehensive panel of the differentially expressed kinases under biotic and abiotic stresses in a non-model plant as cowpea.

Photosynthesis, antioxidant activities and transcriptional responses in two sugarcane (Saccharum officinarum L.) cultivars under salt stress

This study analyzes changes in gene expression and the biochemical and physiological properties of the antioxidant system in the leaves of two sugarcane cultivars under salt stress. In both salt-stressed cultivars, no alteration in the foliar nitrogen content was observed; however, there was a reduction in the phosphorus and potassium levels and an increase in the sodium and chloride concentrations. There was also a reduction in gas exchange on the third day under salt stress. Although the content of soluble sugars remained stable in both species, there was a decrease in free amino acids. However, only the RB872552 cultivar displayed a lower leaf protein content compared to the control. The salt stress resulted in higher superoxide dismutase and l-ascorbate peroxidase activities, but only for the RB92579 cultivar. On the other hand, both cultivars were able to maintain lower malondialdehyde contents than the control plants. The gene expression analysis revealed down-regulated expression levels, including the levels of those enzymes linked to higher activities under salt stress. Our results showed that gene induction and leaf antioxidative cycle enzyme activity do not occur at the same time. The variations in gene expression and physiological responses are also discussed.

In silico identification of known osmotic stress responsive genes from Arabidopsis in soybean and Medicago

Plants experience various environmental stresses, but tolerance to these adverse conditions is a very complex phenomenon. The present research aimed to evaluate a set of genes involved in osmotic response, comparing soybean and medicago with the well-described Arabidopsis thaliana model plant. Based on 103 Arabidopsis proteins from 27 categories of osmotic stress response, comparative analyses against Genosoja and Medicago truncatula databases allowed the identification of 1,088 soybean and 1,210 Medicago sequences. The analysis showed a high number of sequences and high diversity, comprising genes from all categories in both organisms. Genes with unknown function were among the most representative, followed by transcription factors, ion transport proteins, water channel, plant defense, protein degradation, cellular structure, organization & biogenesis and senescence. An analysis of sequences with unknown function allowed the annotation of 174 soybean and 217 Medicago sequences, most of them concerning transcription factors. However, for about 30% of the sequences no function could be attributed using in silico procedures. The establishment of a gene set involved in osmotic stress responses in soybean and barrel medic will help to better understand the survival mechanisms for this type of stress condition in legumes.

Characterization of a new defensin from cowpea (Vigna unguiculata (L.) Walp.).

Using Phaseoleae defensins available in databases, a putative defensin gene was isolated in cowpea (Vigna unguiculata (L.) Walp.) and cloned from genomic cowpea DNA. The putative mature defensin sequence displays the characteristic defensins residues arrangement, secondary and tertiary structures were predicted and splicing analysis was performed. Using RT-PCR, defensin expression and differences in response to biotic stimuli between infected and non infected plants were tested.

An overall evaluation of the resistance (R) and pathogenesis-related (PR) superfamilies in soybean, as compared with Medicago and Arabidopsis

Plants have the ability to recognize and respond to a multitude of pathogens, resulting in a massive reprogramming of the plant to activate defense responses including Resistance (R) and Pathogenesis-Related (PR) genes. Abiotic stresses can also activate PR genes and enhance pathogen resistance, representing valuable genes for breeding purposes. The present work offers an overview of soybean R and PR genes present in the GENOSOJA (Brazilian Soybean Genome Consortium) platform, regarding their structure, abundance, evolution and role in the plant-pathogen metabolic pathway, as compared with Medicago and Arabidopsis. Searches revealed 3,065 R candidates (756 in Soybean, 1,142 in Medicago and 1,167 in Arabidopsis), and PR candidates matching to 1,261 sequences (310, 585 and 366 for the three species, respectively). The identified transcripts were also evaluated regarding their expression pattern in 65 libraries, showing prevalence in seeds and developing tissues. Upon consulting the SuperSAGE libraries, 1,072 R and 481 PR tags were identified in association with the different libraries. Multiple alignments were generated for Xa21 and PR-2 genes, allowing inferences about their evolution. The results revealed interesting insights regarding the variability and complexity of defense genes in soybean, as compared with Medicago and Arabidopsis.