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The research received financial support of FACEPE (Fundação de Amparo à Pesquisa do Estado de Pernambuco, Brazil) CNPq (Conselho Nacional de Desenvolvimento Científico Tecnológico, no. 313581/2020-7, 442019/2019-0, and 433931/2018-3) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, no. 88881.507105/2020-01) throught research funding and fellowships grants.

Cowpea (Vigna unguiculata (L.) Walp.) is an important legume cultivated mainly in regions with limited water availability across the African and American continents. Its productivity is significantly affected by environmental stresses. Thaumatin-like proteins (TLPs), which belong to the PR-5 (pathogenesis-related 5) protein family, are known to be responsive to both biotic and abiotic stresses. However, their role remains controversial, with some TLPs associated with plant defense (particularly against fungal infections) and others associated with abiotic stresses response. In this study, we evaluated the structural diversity and gene expression of TLPs in cowpea (VuTLPs) under different stress conditions, including biotic [mechanical injury followed by inoculation with Cowpea Aphid-borne Mosaic Virus (CABMV) or Cowpea Severe Mosaic Virus (CPSMV)] and abiotic (root dehydration). Genomic anchoring of VuTLPs revealed 34 loci encoding these proteins. Neighbor- joining analysis clustered the VuTLPs into three distinct groups. We identified 15 segmental duplication and 6 tandem duplication gene pairs, with the majority of VuTLP genes found to be under purifying selection. Promoter analysis associated VuTLPs with bHLH, Dof-type, and MYB- related transcription factors, supporting their diverse roles. Diversity in VuTLP function was also observed in their expression profiles under the studied stress conditions. Gene expression data showed that most VuTLPs are recruited within the first minutes after biotic stress imposition. For the root dehydration assay, the most transcripts were up-regulated 150 min post-stress. Moreover, the gene expression data suggested that VuTLPs exhibit functional specialization depending on the stress condition, highlighting their diverse roles and biotechnological potential.

Stylosanthes scabra, a legume native to the Brazilian semiarid region, exhibits remarkable drought tolerance and represents a valuable model for studying molecular adaptation in legumes. Transcription factors of the AP2/ERF superfamily play central roles in plant development and stress response. This study aimed to identify and characterize AP2/ERF genes in Stylosanthes scabra and to analyze their transcriptional response to root dehydration. Candidate genes were identified through a Hidden Markov Model (HMM) search using the AP2 domain profile (PF00847), followed by validation of conserved domains, physicochemical characterization, prediction of subcellular localization, phylogenetic and structural analyses, and functional annotation. A total of 295 AP2/ERF proteins were identified and designated as SscAP2/ERF, most of which were predicted to be localized in the nucleus. These proteins exhibited a wide range of molecular weights and isoelectric points, reflecting structural diversity, and were classified into four subfamilies: AP2, ERF, DREB, and RAV. Functional annotation revealed predominant roles in DNA binding and transcriptional regulation, while promoter analysis identified numerous stress-related cis-elements. A total of 32 transcripts were differentially expressed under 24 h of water deficit, and four selected genes had their expression patterns validated by qPCR. These findings provide new insights into the AP2/ERF gene subfamily in Stylosanthes scabra and lay the groundwork for future biotechnological approaches to enhance stress tolerance in legumes.

Even before the perception or interaction with pathogens, plants rely on constitutively guardian molecules, often specific to tissue or stage, with further expression after contact with the pathogen. These guardians include small molecules as antimicrobial peptides (AMPs), generally cysteine-rich, functioning to prevent pathogen establishment. Some of these AMPs are shared among eukaryotes (eg, defensins and cyclotides), others are plant specific (eg, snakins), while some are specific to certain plant families (such as heveins). When compared with other organisms, plants tend to present a higher amount of AMP isoforms due to gene duplications or polyploidy, an occurrence possibly also associated with the sessile habit of plants, which prevents them from evading biotic and environmental stresses. Therefore, plants arise as a rich resource for new AMPs. As these molecules are difficult to retrieve from databases using simple sequence alignments, a description of their characteristics and in silico (bioinformatics) approaches used to retrieve them is provided, considering resources and databases available. The possibilities and applications based on tools versus database approaches are considerable and have been so far underestimated.

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.

One of the most challenging aspects of RT-qPCR data analysis is the identification of reliable reference genes. Ideally, they should be neither induced nor repressed under different experimental conditions. To date, few reference genes have been adequately studied for sugarcane (Saccharum spp.) using statistical approaches. In this work, six candidate genes (αTUB, GAPDH, H1, SAMDC, UBQ, and 25S rRNA) were tested for gene expression normalization of sugarcane root tissues from drought-tolerant and -sensitive accessions after continuous dehydration (24 h). By undergoing different approaches (GeNorm, NormFinder, and BestKeeper), it was shown that most of them could be used in combinations for normalization purposes, with the exception of SAMDC. Nevertheless three of them (H1, αTUB, and GAPDH) were considered the most reliable reference genes. Their suitability as reference genes validated the expression profiles of two targets (AS and PFPα1), related to SuperSAGE unitags, in agreement with results revealed by previous in silico analysis. The other two sugarcane unitags (ACC oxidase and PIP1-1), after salt stress (100 mM NaCl), presented their expressions validated in the same way. In conclusion, these reference genes will be useful for dissecting gene expression in sugarcane roots under abiotic stress, especially in transcriptomic studies using SuperSAGE or RNAseq approaches.

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.

Background The rationale for gathering information from plants procuring nitrogen through symbiotic interactions controlled by a common genetic program for a sustainable biofuel production is the high energy demanding application of synthetic nitrogen fertilizers. We curated sequence information publicly available for the biofuel plant sugarcane, performed an analysis of the common SYM pathway known to control symbiosis in other plants, and provide results, sequences and literature links as an online database. Methods Sugarcane sequences and informations were downloaded from the nucEST database, cleaned and trimmed with seqclean, assembled with TGICL plus translating mapping method, and annotated. The annotation is based on BLAST searches against a local formatted plant Uniprot90 generated with CD-HIT for functional assignment, rpsBLAST to CDD database for conserved domain analysis, and BLAST search to sorghum's for Gene Ontology (GO) assignment. Gene expression was normalized according the Unigene standard, presented as ESTs/100 kb. Protein sequences known in the SYM pathway were used as queries to search the SymGRASS sequence database. Additionally, antimicrobial peptides described in the PhytAMP database served as queries to retrieve and generate expression profiles of these defense genes in the libraries compared to the libraries obtained under symbiotic interactions. Results We describe the SymGRASS, a database of sugarcane orthologous genes involved in arbuscular mycorrhiza (AM) and root nodule (RN) symbiosis. The database aggregates knowledge about sequences, tissues, organ, developmental stages and experimental conditions, and provides annotation and level of gene expression for sugarcane transcripts and SYM orthologous genes in sugarcane through a web interface. Several candidate genes were found for all nodes in the pathway, and interestingly a set of symbiosis specific genes was found. Conclusions The knowledge integrated in SymGRASS may guide studies on molecular, cellular and physiological mechanisms by which sugarcane controls the establishment and efficiency of endophytic associations. We believe that the candidate sequences for the SYM pathway together with the pool of exclusively expressed tentative consensus (TC) sequences are crucial for the design of molecular studies to unravel the mechanisms controlling the establishment of symbioses in sugarcane, ultimately serving as a basis for the improvement of grass crops.

The present work is a pioneer study specifically addressing the aquaporin transcripts in sugarcane transcriptomes. Representatives of the four aquaporin subfamilies (PIP, TIP, SIP, and NIP), already described for higher plants, were identified. Forty-two distinct aquaporin isoforms were expressed in four HT-SuperSAGE libraries from sugarcane roots of drought-tolerant and -sensitive genotypes, respectively. At least 10 different potential aquaporin isoform targets and their respective unitags were considered to be promising for future studies and especially for the development of molecular markers for plant breeding. From those 10 isoforms, four (SoPIP2-4, SoPIP2-6, OsPIP2-4, and SsPIP1-1) showed distinct responses towards drought, with divergent expressions between the bulks from tolerant and sensitive genotypes, when they were compared under normal and stress conditions. Two targets (SsPIP1-1 and SoPIP1-3/PIP1-4) were selected for validation via RT-qPCR and their expression patterns as detected by HT-SuperSAGE were confirmed. The employed validation strategy revealed that different genotypes share the same tolerant or sensitive phenotype, respectively, but may use different routes for stress acclimation, indicating the aquaporin transcription in sugarcane to be potentially genotype-specific.

Histone methyltransferases (SDGs) and demethylases (JMJs) are well-established regulators of transcriptional responses in plants under adverse conditions. This study characterized SDG and JMJ enzymes in the cowpea (Vigna unguiculata) genome and analyzed their expression patterns under various stress conditions, including root dehydration and mechanical injury followed by CABMV or CPSMV inoculation. A total of 47 VuSDG genes were identified in the cowpea genome and classified into seven distinct classes: I, II, III, IV, V, VI, and VII. Additionally, 26 VuJMJ-coding genes were identified and categorized into their respective groups: Jmj-only, JMJD6, KDM3, KDM5, and KDM4. Analysis of gene expansion mechanisms for the studied loci revealed a predominance of dispersed duplication and WGD/segmental duplication events, with Ka/Ks ratios indicating that all WGD/segmental duplications are under purifying selection. Furthermore, a high degree of conservation was observed for these loci across species, with legumes displaying the highest conservation rates. Cis-Regulatory Element analysis of VuSDG and VuJMJ gene promoters revealed associations with Dof-type and bZIP transcription factors, both of which are known to play roles in plant stress responses and developmental processes. Differential expression patterns were observed for VuSDG and VuJMJ genes under the studied stress conditions, with the highest number of upregulated transcripts detected during the root dehydration assay. Our expression data suggest that as the referred stress persists, the tolerant cowpea accession decreases methylation activity on target histones and shifts towards enhanced demethylation. This dynamic balance between histone methylation and demethylation may regulate the expression of genes linked to dehydration tolerance. During the mechanical injury and viral inoculation assays, VuSDG and VuJMJ transcripts were upregulated exclusively within 60 min after the initial mechanical injury combined with CABMV or CPSMV inoculation, indicating an early role for these enzymes in the plant’s defense response to pathogen exposure. The current study presents a detailed analysis of histone modifiers in cowpea and indicates their role as important epigenetic regulators modulating stress tolerance.