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Background Due to cowpea ability to fix nitrogen in poor soils and relative tolerance to drought and salt stresses, efforts have been directed to identifying genes and pathways that confer stress tolerance in this species. Real-time quantitative PCR (qPCR) has been widely used as the most reliable method to measure gene expression, due to its high accuracy and specificity. In the present study, nine candidate reference genes were rigorously tested for their application in normalization of qPCR data onto roots of four distinct cowpea accessions under two abiotic stresses: root dehydration and salt (NaCl, 100 mM). In addition, the regulation of four target transcripts, under the same referred conditions was also scrutinized. Results geNorm, NormFinder, BestKeeper, and ΔCt method results indicated a set of three statistically validated RGs for each stress condition: (I) root dehydration (actin, ubiquitin-conjugating enzyme E2 variant 1D, and a Phaseolus vulgaris unknown gene— UNK ), and (II) salt (ubiquitin-conjugating enzyme E2 variant 1D, F-box protein, and UNK ). The expression profile of the target transcripts suggests that flavonoids are important players in the cowpea response to the abiotic stresses analyzed, since chalcone isomerase and chalcone synthase were up-regulated in the tolerant and sensitive accessions. A lipid transfer protein also participates in the cowpea tolerance mechanisms to root dehydration and salt stress. The referred transcript was up-regulated in the two tolerant accessions and presented no differential expression in the sensitive counterparts. Chitinase B, in turn, generally related to plant defense, was an important target transcript under salt stress, being up-regulated at the tolerant, and down-regulated in the sensitive accession. Conclusions Reference genes suitable for qPCR analyses in cowpea under root dehydration and salt stress were identified. This action will lead to a more accurate and reliable analysis of gene expression on this species. Additionally, the results obtained in this study may guide future research on gene expression in cowpea under other abiotic stress types that impose osmotic imbalance. The target genes analyzed, in turn, deserve functional evaluation due to their transcriptional regulation under stresses and biotechnological potential.

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.

Similar to other organisms, plants establish interactions with a variety of microorganisms in their natural environment. The plant microbiome occupies the host plant’s tissues, either internally or on its surfaces, showing interactions that can assist in its growth, development, and adaptation to face environmental stresses. The advance of metagenomics and metatranscriptomics approaches has strongly driven the study and recognition of plant microbiome impacts. Research in this regard provides comprehensive information about the taxonomic and functional aspects of microbial plant communities, contributing to a better understanding of their dynamics. Evidence of the plant microbiome’s functional potential has boosted its exploitation to develop more ecological and sustainable agricultural practices that impact human health. Although microbial inoculants’ development and use are promising to revolutionize crop production, interdisciplinary studies are needed to identify new candidates and promote effective practical applications. On the other hand, there are challenges in understanding and analyzing complex data generated within a plant microbiome project’s scope. This review presents aspects about the complex structuring and assembly of the microbiome in the host plant’s tissues, metagenomics, and metatranscriptomics approaches for its understanding, covering descriptions of recent studies concerning metagenomics to characterize the microbiome of non-model plants under different aspects. Studies involving bio-inoculants, isolated from plant microbial communities, capable of assisting in crops’ productivity, are also reviewed.

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.

The present work represents a pioneering effort, being the first to analyze genomic and transcriptomic data from Vigna unguiculata (cowpea) kinases. We evaluated the cowpea kinome considering its genome-wide distribution and structural characteristics (at the gene and protein levels), sequence evolution, conservation among Viridiplantae species, and gene expression in three cowpea genotypes under different stress situations, including biotic (injury followed by virus inoculation—CABMV or CPSMV) and abiotic (root dehydration). The structural features of cowpea kinases (VuPKs) indicated that 1,293 bona fide VuPKs covered 20 groups and 118 different families. The RLK-Pelle was the largest group, with 908 members. Insights on the mechanisms of VuPK genomic expansion and conservation among Viridiplantae species indicated dispersed and tandem duplications as major forces for VuPKs’ distribution pattern and high orthology indexes and synteny with other legume species, respectively. K a / K s ratios showed that almost all (91%) of the tandem duplication events were under purifying selection. Candidate cis -regulatory elements were associated with different transcription factors (TFs) in the promoter regions of the RLK-Pelle group. C2H2 TFs were closely associated with the promoter regions of almost all scrutinized families for the mentioned group. At the transcriptional level, it was suggested that VuPK up-regulation was stress, genotype, or tissue dependent (or a combination of them). The most prominent families in responding (up-regulation) to all the analyzed stresses were RLK-Pelle_DLSV and CAMK_CAMKL-CHK1. Concerning root dehydration, it was suggested that the up-regulated VuPKs are associated with ABA hormone signaling, auxin hormone transport, and potassium ion metabolism. Additionally, up-regulated VuPKs under root dehydration potentially assist in a critical physiological strategy of the studied cowpea genotype in this assay, with activation of defense mechanisms against biotic stress while responding to root dehydration. This study provides the foundation for further studies on the evolution and molecular function of VuPKs.

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.

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.

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.

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.

Physic nut (Jatropha curcas L.), a small oleaginous tree spontaneously occurring in arid and semi-arid tropical regions, is a sustainable and renewable energy source for biodiesel. However, the J. curcas yield in such areas should consider soil salinity and its consequences. Transcription factor (TF) proteins recognize cis-regulatory elements in promoters of genes to be expressed. In the present work, differentially expressed genes (DEGs) encoding putative TFs from physic nut plants responding to NaCl (150 mM), after 3 h of exposition, covered 23 TF families. The expressed profiles of members from AP2/ERF and NAC families basically showed induction after the salt stimulus, while members of bHLH, FHY3-FAR1, and ARF families presented repression. Concerning the induced TF DEGs, the gene ontology (GO) enrichment analysis highlighted terms related to abiotic stress responses, while those terms representing the repressed TF DEGs stood out the basal metabolism. In turn, the TF enrichment analysis predicted those TFs targeting promoters of induced TF DEGs. Some of the enriched TFs may be good candidates as transgenes in transgenic events. Also, RT-qPCR analyses validated the up-regulation of six TF DEGs (RAV1, ERF9, ZAT12, PTI5, MYB340, and BZIP4) of eight candidates selected from the expressed TFome. The generated data could help breeders to better understand the molecular basis of physic nut plants responding to salinity, to select potential candidates for transgenic studies, as well as to develop functional molecular markers to assist selection steps in breeding programs.