Dehydration response in Stylosanthes scabra: Transcriptional, biochemical, and physiological modulations.

Stylosanthes scabra, popularly known as stylo, is native to the Brazilian Caatinga semiarid region and stands out as a drought-tolerant shrub forage crop. This work provides information about the plant response during the first 48 h of water deficit, followed by a rehydration treatment. Besides root transcriptomics data, 13 physiological or biochemical parameters were scrutinized. Additionally, RNA-Seq annotated transcripts not associated with the "Viridiplantae" clade were taxonomically categorized. It was found that S. scabra quickly perceives and recovers from the oscillations of the imposed water regime. Physiologically, mechanisms that minimize evapotranspiration or protect the photosynthetic apparatus stood out. Biochemically, it was found that the root tissue invests in synthesizing compounds that can act as osmolytes (proline and sugars), emphasizing the importance of osmoregulation to water deficit acclimation. Consistently, transcriptome and qPCR analyses showed that a set of enriched biological processes with upregulated (UR) transcripts were involved in protective functions against reactive oxygen species or encoding enzymes of important metabolic pathways, which might contribute to S. scabra response to water deficit. Additionally, several UR kinases and transcription factors were identified. Finally, in an innovative approach, some naturally occurring microbial groups (such as Schizosaccharomyces, Bradyrhizobium, etc.) were identified in the S. scabra roots. This study reveals insights into the physiological, biochemical, and molecular mechanisms underlying the S. scabra response to water deficit and provides candidate genes that may be useful in developing drought-tolerant crop varieties through biotechnological applications.

Transcriptome of Cenostigma pyramidale roots, a woody legume, under different salt stress times.

Salinity stress has a significant impact on the gain of plant biomass. Our study provides the first root transcriptome of Cenostigma pyramidale, a tolerant woody legume from a tropical dry forest, under three different salt stress times (30 min, 2 h, and 11 days). The transcriptome was assembled using the RNA sequencing (RNA-Seq) de novo pipeline from GenPipes. We observed 932, 804, and 3157 upregulated differentially expressed genes (DEGs) and 164, 273, and 1332 downregulated DEGs for salt over 30 min, 2 h, and 11 days, respectively. For DEGs annotated with the Viridiplantae clade in the early stress periods, the response to salt stress was mainly achieved by stabilizing homeostasis of such ions like Na+ and K+ , signaling by Ca2+ , transcription factor modulation, water transport, and oxidative stress. For salt stress at 11 days, we observed a higher modulation of transcription factors including the WRKY, MYB, bHLH, NAC, HSF, and AP2-EREBP families, as well as DEGs involved in hormonal responses, water transport, sugar metabolism, proline, and reactive oxygen scavenging mechanisms. Five selected DEGs (K+ transporter, aquaporin, glutathione S-transferase, cyclic nucleotide-gated channel, and superoxide dismutase) were validated by qPCR. Our results indicated that C. pyramidale had an early perception of salt stress modulating ionic channels and transporters, and as the stress progressed, the focus turned to the antioxidant system, aquaporins, and complex hormone responses. The results of this first root transcriptome provide clues on how this native species modulate gene expression to achieve salt stress tolerance.

Importance of inositols and their derivatives in cowpea under root dehydration: An omics perspective.

This work presents a robust analysis of the inositols (INSs) and raffinose family oligosaccharides (RFOs) pathways, using genomic and transcriptomic tools in cowpea under root dehydration. Nineteen (~70%) of the 26 scrutinized enzymes presented transcriptional up-regulation in at least one treatment time. The transcriptional orchestration allowed categorization of the analyzed enzymes as time-independent (those showing the same regulation throughout the assay) and time-dependent (those showing different transcriptional regulation over time). It is suggested that up-regulated time-independent enzymes (INSs: myo-inositol oxygenase, inositol-tetrakisphosphate 1-kinase 3, phosphatidylinositol 4-phosphate 5-kinase 4-like, 1-phosphatidylinositol-3-phosphate 5-kinase, phosphoinositide phospholipase C, and non-specific phospholipase C; RFOs: α-galactosidase, invertase, and raffinose synthase) actively participate in the reorganization of cowpea molecular physiology under the applied stress. In turn, time-dependent enzymes, especially those up-regulated in some of the treatment times (INSs: inositol-pentakisphosphate 2-kinase, phosphatidylinositol 4-kinase, phosphatidylinositol synthase, multiple inositol polyphosphate phosphatase 1, methylmalonate-semialdehyde dehydrogenase, triosephosphate isomerase, myo-inositol-3-phosphate synthase, phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and protein-tyrosine-phosphatase, and phosphatidylinositol 3-kinase; RFOs: galactinol synthase) seem to participate in fine-tuning of the molecular physiology, helping the cowpea plants to acclimatize under dehydration stress. Not all loci encoding the studied enzymes were expressed during the assay; most of the expressed ones exhibited a variable transcriptional profile in the different treatment times. Genes of the INSs and RFOs pathways showed high orthology with analyzed Phaseoleae members, suggesting a relevant role within this legume group. Regarding the promoter regions of INSs and RFOs genes, some bona fide cis-regulatory elements were identified in association with seven transcription factor families (AP2-EFR, Dof-type, MADS-box, bZIP, CPP, ZF-HD, and GATA-type). Members of INSs and RFOs pathways potentially participate in other processes regulated by these proteins in cowpea.

Reference genes for quantitative real-time PCR normalization of Cenostigma pyramidale roots under salt stress and mycorrhizal association.

Cenostigma pyramidale is a native legume of the Brazilian semiarid region which performs symbiotic association with arbuscular mycorrhizal fungi (AMF), being an excellent model for studying genes associated with tolerance against abiotic and biotic stresses. In RT-qPCR approach, the use of reference genes is mandatory to avoid incorrect interpretation of the relative expression. This study evaluated the stability of ten candidate reference genes (CRGs) from C. pyramidale root tissues under salt stress (three collection times) and associated with AMF (three different times of salinity). The de novo transcriptome was obtained via RNA-Seq sequencing. Three algorithms were used to calculate the stability of CRGs under different conditions: (i) global (Salt, Salt+AMF, AMF and Control, and collection times), (ii) only non-inoculated plants, and (iii) AMF (only inoculated plants). HAG2, SAC1, aRP3 were the most stable CRGs for global and AMF assays, whereas HAG2, SAC1, RHS1 were the best for salt stress assay. This CRGs were used to validate the relative expression of two up-regulated transcripts in Salt2h (RAP2-3 and PIN8). Our study provides the first set of reference genes for C. pyramidale under salinity and AMF, supporting future researches on gene expression with this species.

The endophytome (plant-associated microbiome): methodological approaches, biological aspects, and biotech applications.

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.

Salt tolerance of Calotropis procera begins with immediate regulation of aquaporin activity in the root system.

The ability to respond quickly to salt stress can determine the tolerance level of a species. Here, we test how rapidly the roots of Calotropis procera react to high salinity conditions. In the first 24 h after saline exposure, the plants reduced stomatal conductance, increased CO2 assimilation, and water use efficiency. Thus, the root tissue showed an immediate increase in soluble sugars, free amino acid, and soluble protein contents. Twelve aquaporins showed differential gene expression in the roots of C. procera under salinity. Transcriptional upregulation was observed only after 2 h, with greater induction of CpTIP1.4 (fourfold). Transcriptional downregulation, in turn, occurred mainly after 8 h, with the largest associated with CpPIP1.2 (fourfold). C. procera plants responded quickly to high saline levels. Our results showed a strong stomatal control associated with high free amino acid and soluble sugar contents, regulated aquaporin expression in roots, and supported the high performance of the root system of C. procera under salinity. Moreover, this species was able to maintain a lower Na+/K+ ratio in the leaves compared to that of the roots of stressed plants. The first response of the root system, after immediate contact with saline solution, present an interesting scenario to discuss. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-00957-9.

Antimicrobial and structural insights of a new snakin-like peptide isolated from Peltophorum dubium (Fabaceae).

Snakins are antimicrobial peptides (AMPs) found, so far, exclusively in plants, and known to be important in the defense against a wide range of pathogens. Like other plant AMPs, they contain several positively charged amino acids, and an even number of cysteine residues forming disulfide bridges which are considered important for their usual function. Despite its importance, studies on snakin tertiary structure and mode of action are still scarce. In this study, a new snakin-like gene was isolated from the native plant Peltophorum dubium, and its expression was verified in seedlings and adult leaves. The deduced peptide (PdSN1) shows 84% sequence identity with potato snakin-1 mature peptide, with the 12 cysteines characteristic from this peptide family at the GASA domain. The mature PdSN1 coding sequence was successfully expressed in Escherichia coli. The purified recombinant peptide inhibits the growth of important plant and human pathogens, like the economically relevant potato pathogen Streptomyces scabies and the opportunistic fungi Candida albicans and Aspergillus niger. Finally, homology and ab initio modeling techniques coupled to extensive molecular dynamics simulations were used to gain insight on the 3D structure of PdSN1, which exhibited a helix-turn-helix motif conserved in both native and recombinant peptides. We found this motif to be strongly coded in the sequence of PdSN1, as it is stable under different patterns of disulfide bonds connectivity, and even when the 12 cysteines are considered in their reduced form, explaining the previous experimental evidences.

From Genes to Stress Response: Genomic and Transcriptomic Data Suggest the Significance of the Inositol and Raffinose Family Oligosaccharide Pathways in Stylosanthes scabra, Adaptation to the Caatinga Environment.

S. scabra is an important forage and extremophilic plant native to the Brazilian Caatinga semiarid region. It has only recently been subjected to omics-based investigations, and the generated datasets offer insights into biotechnologically significant candidates yet to be thoroughly examined. INSs (inositol and its derivatives) and RFO (raffinose oligosaccharide family) pathways emerge as pivotal candidates, given their critical roles in plant physiology. The mentioned compounds have also been linked to negative impacts on the absorption of nutrients in mammals, affecting overall nutritional intake and metabolism. Therefore, studying these metabolic pathways is important not just for plants but also for animals who depend on them as part of their diet. INS and RFO pathways in S. scabra stood out for their abundance of identified loci and enzymes. The enzymes exhibited genomic redundancy, being encoded by multiple loci and various gene families. The phylogenomic analysis unveiled an expansion of the PIP5K and GolS gene families relative to the immediate S. scabra ancestor. These enzymes are crucial for synthesizing key secondary messengers and the RFO precursor, respectively. Transcriptional control of the studied pathways was associated with DOF-type, C2H2, and BCP1 transcription factors. Identification of biological processes related to INS and RFO metabolic routes in S. scabra highlighted their significance in responding to stressful conditions prevalent in the Caatinga environment. Finally, RNA-Seq and qPCR data revealed the relevant influence of genes of the INS and RFO pathways in the S. scabra response to water deprivation. Our study deciphers the genetics and transcriptomics of the INS and RFO in S. scabra, shedding light on their importance for a Caatinga-native plant and paving the way for future biotechnological applications in this species and beyond.

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

BACKGROUND: Despite 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. METHODS: A 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. RESULTS: The 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. CONCLUSIONS: Soybean 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.

Natural antisense transcripts in plants: a review and identification in soybean infected with Phakopsora pachyrhizi SuperSAGE library.

Natural antisense ranscripts (NAT) are RNA molecules complementary to other endogenous RNAs. They are capable of regulating the expression of target genes at different levels (transcription, mRNA stability, translation, etc.). Such a property makes them ideal for interventions in organisms' metabolism. The present study reviewed plant NAT aspects, including features, availability and genesis, conservation and distribution, coding capacity, NAT pair expression, and functions. Besides, an in silico identification of NATs pairs was presented, using deepSuperSAGE libraries of soybean infected or not with Phakopsora pachyrhizi. Results showed that around 1/3 of the 77,903 predicted trans-NATs (by PlantsNATsDB database) detected had unitags mapped in both sequences of each pair. The same 1/3 of the 436 foreseen cis-NATs showed unitags anchored in both sequences of the related pairs. For those unitags mapped in NAT pairs, a modulation expression was assigned as upregulated, downregulated, or constitutive, based on the statistical analysis (P < 0.05). As a result, the infected treatment promoted the expression of 2,313 trans-NATs pairs comprising unitags exclusively from that library (1,326 pairs had unitags only found in the mock library). To understand the regulation of these NAT pairs could be a key aspect in the ASR plant response.

Bridging the Gap: Combining Genomics and Transcriptomics Approaches to Understand Stylosanthes scabra, an Orphan Legume from the Brazilian Caatinga.

Stylosanthes scabra is a scientifically orphaned legume found in the Brazilian Caatinga biome (a semi-arid environment). This work utilized omics approaches to investigate some ecophysiological aspects of stress tolerance/resistance in S. scabra, study its genomic landscape, and predict potential metabolic pathways. Considering its high-confidence conceptual proteome, 1694 (~2.6%) proteins were associated with resistance proteins, some of which were found in soybean QTL regions that confer resistance to Asian soybean rust. S. scabra was also found to be a potential source of terpenes, as biosynthetic gene clusters associated with terpene biosynthesis were identified in its genome. The analysis revealed that mobile elements comprised approximately 59% of the sequenced genome. In the remaining 41% of the sections, some of the 22,681 protein-coding gene families were categorized into two informational groups: those that were specific to S. scabra and those that expanded significantly compared to their immediate ancestor. Biological process enrichment analyses indicated that these gene families play fundamental roles in the adaptation of S. scabra to extreme environments. Additionally, phylogenomic analysis indicated a close evolutionary relationship between the genera Stylosanthes and Arachis. Finally, this study found a high number (57) of aquaporin-encoding loci in the S. scabra genome. RNA-Seq and qPCR data suggested that the PIP subfamily may play a key role in the species' adaptation to water deficit conditions. Overall, these results provide valuable insights into S. scabra biology and a wealth of gene/transcript information for future legume omics studies.

Unlocking Cowpea's Defense Responses: Conserved Transcriptional Signatures in the Battle against CABMV and CPSMV Viruses.

Cowpea aphid-borne mosaic virus (CABMV) and Cowpea severe mosaic virus (CPSMV) threaten cowpea commercial production. This study aimed to analyze Conserved Transcriptional Signatures (CTS) in cowpea's genotypes that are resistant to these viruses. CTS covered up- (UR) or down-regulated (DR) cowpea transcripts in response to CABMV and CPSMV mechanical inoculations. The conservation of cowpea's UR defense response was primarily observed with the one hpi treatments, with decreased CTS representatives as time elapsed. This suggests that cowpea utilizes generic mechanisms during its early interaction with the studied viruses, and subsequently employs more specialized strategies for each viral agent. The potential action of the CTS-UR emphasizes the importance of redox balance, ethylene and jasmonic acid pathways. Additionally, the CTS-UR provides evidence for the involvement of R genes, PR proteins, and PRRs receptors-extensively investigated in combating bacterial and fungal pathogens-in the defense against viral inoculation. AP2-ERF, WRKY, and MYB transcription factors, as well as PIP aquaporins and MAPK cascades, also emerged as significant molecular players. The presented work represents the first study investigating conserved mechanisms in the cowpea defense response to viral inoculations, highlighting relevant processes for initial defense responses.

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.

The WRKY transcription factor family in cowpea: Genomic characterization and transcriptomic profiling under root dehydration.

Cowpea [Vigna unguiculata (L.) Walp.] is one of the most tolerant legume crops to drought and salt stresses. WRKY transcription factor (TF) family members stand out among plant transcriptional regulators related to abiotic stress tolerance. However, little information is currently available on the expression of the cowpea WRKY gene family (VuWRKY) in response to water deficit. Thus, we analyzed genomic and transcriptomic data from cowpea to identify VuWRKY members and characterize their structure and transcriptional response under root dehydration stress. Ninety-two complete VuWRKY genes were found in the cowpea genome based on their domain characteristics. They were clustered into three groups: I (15 members), II (58), and III (16), while three genes were unclassified. Domain analysis of the encoded proteins identified four major variants of the conserved heptapeptide motif WRKYGQK. In silico analysis of VuWRKY gene promoters identified eight candidate binding motifs of cis-regulatory elements, regulated mainly by six TF families associated with abiotic stress responses. Ninety-seven VuWRKY modulated splicing variants associated with 55 VuWRKY genes were identified via RNA-Seq analysis available at the Cowpea Genomics Consortium (CpGC) database. qPCR analyses showed that 22 genes are induced under root dehydration, with VuWRKY18, 21, and 75 exhibiting the most significant induction levels. Given their central role in activating signal transduction cascades in abiotic stress response, the data provide a foundation for the targeted modification of specific VuWRKY family members to improve drought tolerance in this important climate-resilient legume in the developing world and beyond.

Plant Thaumatin-like Proteins: Function, Evolution and Biotechnological Applications.

Thaumatin-like proteins (TLPs) are a highly complex protein family associated with host defense and developmental processes in plants, animals, and fungi. They are highly diverse in angiosperms, for which they are classified as the PR-5 (Pathogenesis-Related-5) protein family. In plants, TLPs have a variety of properties associated with their structural diversity. They are mostly associated with responses to biotic stresses, in addition to some predicted activities under drought and osmotic stresses. The present review covers aspects related to the structure, evolution, gene expression, and biotechnological potential of TLPs. The efficiency of the discovery of new TLPs is below its potential, considering the availability of omics data. Furthermore, we present an exemplary bioinformatics annotation procedure that was applied to cowpea (Vigna unguiculata) transcriptome, including libraries of two tissues (root and leaf), and two stress types (biotic/abiotic) generated using different sequencing approaches. Even without using genomic sequences, the pipeline uncovered 56 TLP candidates in both tissues and stresses. Interestingly, abiotic stress (root dehydration) was associated with a high number of modulated TLP isoforms. The nomenclature used so far for TLPs was also evaluated, considering TLP structure and possible functions identified to date. It is clear that plant TLPs are promising candidates for breeding purposes and for plant transformation aiming a better performance under biotic and abiotic stresses. The development of new therapeutic drugs against human fungal pathogens also deserves attention. Despite that, applications derived from TLP molecules are still below their potential, as it is evident in our review.

Reference genes selection for Calotropis procera under different salt stress conditions.

Calotropis procera is a perennial Asian shrub with significant adaptation to adverse climate conditions and poor soils. Given its increased salt and drought stress tolerance, C. procera stands out as a powerful candidate to provide alternative genetic resources for biotechnological approaches. The qPCR (real-time quantitative polymerase chain reaction), widely recognized among the most accurate methods for quantifying gene expression, demands suitable reference genes (RGs) to avoid over- or underestimations of the relative expression and incorrect interpretation. This study aimed at evaluating the stability of ten RGs for normalization of gene expression of root and leaf of C. procera under different salt stress conditions and different collection times. The selected RGs were used on expression analysis of three target genes. Three independent experiments were carried out in greenhouse with young plants: i) Leaf100 = leaf samples collected 30 min, 2 h, 8 h and 45 days after NaCl-stress (100 mM NaCl); ii) Root50 and iii) Root200 = root samples collected 30 min, 2 h, 8 h and 1day after NaCl-stress (50 and 200 mM NaCl, respectively). Stability rank among the three algorithms used showed high agreement for the four most stable RGs. The four most stable RGs showed high congruence among all combination of collection time, for each software studied, with minor disagreements. CYP23 was the best RG (rank of top four) for all experimental conditions (Leaf100, Root50, and Root200). Using appropriated RGs, we validated the relative expression level of three differentially expressed target genes (NAC78, CNBL4, and ND1) in Leaf100 and Root200 samples. This study provides the first selection of stable reference genes for C. procera under salinity. Our results emphasize the need for caution when evaluating the stability RGs under different amplitude of variable factors.

Plant Aquaporins: Diversity, Evolution and Biotechnological Applications.

The plasma membrane forms a permeable barrier that separates the cytoplasm from the external environment, defining the physical and chemical limits in each cell in all organisms. The movement of molecules and ions into and out of cells is controlled by the plasma membrane as a critical process for cell stability and survival, maintaining essential differences between the composition of the extracellular fluid and the cytosol. In this process aquaporins (AQPs) figure as important actors, comprising highly conserved membrane proteins that carry water, glycerol and other hydrophilic molecules through biomembranes, including the cell wall and membranes of cytoplasmic organelles. While mammals have 15 types of AQPs described so far (displaying 18 paralogs), a single plant species can present more than 120 isoforms, providing transport of different types of solutes. Such aquaporins may be present in the whole plant or can be associated with different tissues or situations, including biotic and especially abiotic stresses, such as drought, salinity or tolerance to soils rich in heavy metals, for instance. The present review addresses several aspects of plant aquaporins, from their structure, classification, and function, to in silico methodologies for their analysis and identification in transcriptomes and genomes. Aspects of evolution and diversification of AQPs (with a focus on plants) are approached for the first time with the aid of the LCA (Last Common Ancestor) analysis. Finally, the main practical applications involving the use of AQPs are discussed, including patents and future perspectives involving this important protein family.

Cowpea and abiotic stresses: identification of reference genes for transcriptional profiling by qPCR.

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.

Snakin: Structure, Roles and Applications of a Plant Antimicrobial Peptide.

Snakins are plant antimicrobial peptides (AMPs) of the Snakin/GASA family, formed by three distinct regions: an N-terminal signal peptide; a variable site; and the GASA domain in the Cterminal region composed by twelve conserved cysteine residues that contribute to the biochemical stability of the molecule. These peptides are known to play different roles in response to a variety of biotic (i.e., induced by bacteria, fungi and nematode pathogens) and abiotic (salinity, drought and ROS) stressors, as well as in crosstalk promoted by plant hormones, with emphasis on abscisic and salicylic acid (ABA and SA, respectively). Such properties make snakin/GASA members promising biotechnological sources for potential therapeutic and agricultural applications. However, information regarding their tertiary structure, mode of action and function are not yet completely elucidated. The present review presents aspects of snakin structure, expression, functional studies and perspectives about the potential applications for agricultural and medical purposes.

Heterologous Expression Systems for Plant Defensin Expression: Examples of Success and Pitfalls.

Defensins are a superfamily of antimicrobial peptides, present in vertebrates, invertebrates, fungi and plants, suggesting that they appeared prior to the divergence in eukaryotes. The destitution of toxicity to mammalian cells of plant defensins has led to a new research ground, i.e., their potential medical use against human infectious diseases. Isolating defensins from natural sources, like plant tissues, can be time-consuming, labor intensive and usually present low yields. Strategies for large-scale production of purified active defensins have been employed using heterologous expression systems (HES) for defensin production, usually based in E. coli system. Like any other technology, HES present limitations and drawbacks demanding a careful experimental design prior the system selection. This review is proposed to discuss some of the major concerns when choosing to heterologously express plant defensins, with special attention on bacterial expression systems.