The Transcriptional Modulation of Inositols and Raffinose Family Oligosaccharides Pathways in Plants — An (A)Biotic Stress Perspective

Among the multifunctional molecules that participate in processes of plant tolerance/resistance to stresses, inositol (Ins) and its derivatives (phosphorylated, methylated, oxygenated, and Raffinose Family Oligosaccharides) have attracted the attention of researchers. These compounds represent versatile and dynamic signaling molecules and osmolytes in all eukaryotes. Due to the impacts related to Ins and its derivatives in a plant cell, assays have been conducted to understand how these biomolecules affect plant physiology. Thus, overexpression or knockout of Ins-related genes has been shown as interesting strategies for generating more efficient plants capable of growing under stress conditions. In this chapter, studies using molecular tools are presented, and the impacts of their results are discussed based on the plant stress tolerance/resistance. Furthermore, an informative panel is provided with transcriptional modulation of genes related to Ins and its derivatives expressed in plants under stress. There is a gap involving about two dozen enzymes associated with the synthesis of Ins-related compounds that have not been adequately studied, and they represent an area of high biotechnological potential

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

Validation of Novel Reference Genes for Reverse Transcription Quantitative Real-Time PCR in Drought-Stressed Sugarcane

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

Análise IN SILICO DE EST-SSR em Phaseolus vulgaris E Glycine max E Trasferibilidade de Marcadores para Vigna unguiculata

Bancos de dados de seqüências expressas representam uma fonte potencialmente valorosa para o desenvolvimento de marcadores moleculares. Neste trabalho, estudos foram realizados para o desenvolvimento de marcadores para aplicação no melhoramento de feijão-caupi, feijão-comum e soja. Foram analisadas 10.880 ESTs de feijão-comum e 116.965 ESTs de soja. Um total de 331 e 4553 SSRs foram identificados nas seqüências de feijão-comum e soja, respectivamente. Trinucleotídeos (45,62 %) foram as mais abundantes em feijãocomum, enquanto que os dinucletídeos (49,67 %), em soja. A densidade média variou de um SSR a cada 22 Kb (feijão-comum) a um SSR a cada 14,68 Kb (soja). Repetições diméricas AG/GA / TC/CT e triméricas AAG/AGA/GAA / TTC/TCT/CTT foram as mais abundantes em ambas espécies. Um total de 153 e 1928 pares de primers EST-SSRs foram propostos para feijão-comum e para soja, respectivamente. Desses, sinteizaram-se 20 para soja, dos quais apenas nove amplificaram (sendo quatro de tamanho esperado e, desses, dois polimórficos) e dois foram transferíveis. Para feijão-comum sintetizaram-se 22, dos quais 15 amplificaram, sendo que 11 apresentaram tamanho esperado e 10 desses foram polimórficos (PIC médio de 0,50); 11 dos funcionais foram transferidos para feijão-caupi, sendo oito de tamanho esperado e cinco desses, polimórficos (PIC médio de 0,36). Análises detalhadas dos amplicons seqüenciados determinaram que as extensões dos motivos de SSRs foram variáveis e que as regiões flanqueadoras dessas repetições foram geralmente bem conservadas, confirmando o sucesso da transferibilidade entre feijão-comum e feijão-caup

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

Abstract 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

Validation of novel reference genes for reverse transcription quantitative real-time PCR in drought-stressed sugarcane

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

Expression analysis of sugarcane aquaporin genes under water deficit

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

Bridging the Gap: Combining Genomics and Transcriptomics Approaches to Understand <i>Stylosanthes scabra</i>, 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

Lipid transfer proteins (Ltps)- structure, diversity and roles beyond antimicrobial activity

Lipid transfer proteins (LTPs) are among the most promising plant-exclusive antimicrobial peptides (AMPs). They figure among the most challenging AMPs from the point of view of their structural diversity, functions and biotechnological applications. This review presents a current picture of the LTP research, addressing not only their structural, evolutionary and further predicted functional aspects. Traditionally, LTPs have been identified by their direct isolation by biochemical techniques, whereas omics data and bioinformatics deserve special attention for their potential to bring new insights. In this context, new possible functions have been identified revealing that LTPs are actually multipurpose, with many additional predicted roles. Despite some challenges due to the toxicity and allergenicity of LTPs, a systematic review and search in patent databases, indicate promising perspectives for the biotechnological use of LTPs in human health and also plant defense.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