Flooding is a frequent environmental stress that reduces soybean (
) growth and grain yield in many producing areas in the world, such as, e.g., in the United States, Southeast Asia and Southern ...Brazil. In these regions, soybean is frequently cultivated in lowland areas by rotating with rice (
), which provides numerous technical, economic and environmental benefits. Given these realities, this work aimed to characterize physiological responses, identify genes differentially expressed under flooding stress in Brazilian soybean genotypes with contrasting flooding tolerance, and select SNPs with potential use for marker-assisted selection. Soybean cultivars TECIRGA 6070 (flooding tolerant) and FUNDACEP 62 (flooding sensitive) were grown up to the V6 growth stage and then flooding stress was imposed. Total RNA was extracted from leaves 24 h after the stress was imposed and sequenced. In total, 421 induced and 291 repressed genes were identified in both genotypes. TECIRGA 6070 presented 284 and 460 genes up- and down-regulated, respectively, under flooding conditions. Of those, 100 and 148 genes were exclusively up- and down-regulated, respectively, in the tolerant genotype. Based on the RNA sequencing data, SNPs in differentially expressed genes in response to flooding stress were identified. Finally, 38 SNPs, located in genes with functional annotation for response to abiotic stresses, were found in TECIRGA 6070 and absent in FUNDACEP 62. To validate them, 22 SNPs were selected for designing KASP assays that were used to genotype a panel of 11 contrasting genotypes with known phenotypes. In addition, the phenotypic and grain yield impacts were analyzed in four field experiments using a panel of 166 Brazilian soybean genotypes. Five SNPs possibly related to flooding tolerance in Brazilian soybean genotypes were identified. The information generated from this research will be useful to develop soybean genotypes adapted to poorly drained soils or areas subject to flooding.
Osmotin is an important multifunctional protein related to plant stress responses and is classified into the thaumatin-like protein (TLP) family. Using genome-wide and phylogenetic approaches, we ...investigated osmotin origin and diversification across plant TLP evolution. Genomic and protein in silico analysis tools were also accessed and considered for the study conclusions. Phylogenetic analysis including a total of 722 sequences from 32 Viridiplantae species allowed the identification of an osmotin group that includes all previously characterized osmotins. Based on the phylogenetic tree results, it is evident that the osmotin group emerged from spermatophytes. Phylogenetic separation and gene expansion could be accounted for by an exclusive motif composition and organization that emerged and was maintained following tandem and block duplications as well as natural selection. The TLP family conserved residues and structures that were also identified in the sequences of the osmotin group, thus suggesting their maintenance for defense responses. The gene expression of Arabidopsis and rice putative osmotins reinforces its roles during stress response.
Potato (Solanum tuberosum) yield has increased dramatically over the last 50 years and this has been achieved by a combination of improved agronomy and biotechnology efforts. Gene studies are taking ...place to improve new qualities and develop new cultivars. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a bench-marking analytical tool for gene expression analysis, but its accuracy is highly dependent on a reliable normalization strategy of an invariant reference genes. For this reason, the goal of this work was to select and validate reference genes for transcriptional analysis of edible tubers of potato. To do so, RT-qPCR primers were designed for ten genes with relatively stable expression in potato tubers as observed in RNA-Seq experiments. Primers were designed across exon boundaries to avoid genomic DNA contamination. Differences were observed in the ranking of candidate genes identified by geNorm, NormFinder and BestKeeper algorithms. The ranks determined by geNorm and NormFinder were very similar and for all samples the most stable candidates were C2, exocyst complex component sec3 (SEC3) and ATCUL3/ATCUL3A/CUL3/CUL3A (CUL3A). According to BestKeeper, the importin alpha and ubiquitin-associated/ts-n genes were the most stable. Three genes were selected as reference genes for potato edible tubers in RT-qPCR studies. The first one, called C2, was selected in common by NormFinder and geNorm, the second one is SEC3, selected by NormFinder, and the third one is CUL3A, selected by geNorm. Appropriate reference genes identified in this work will help to improve the accuracy of gene expression quantification analyses by taking into account differences that may be observed in RNA quality or reverse transcription efficiency across the samples.
Gene expression analysis is increasingly important in biological research, with reverse transcription-quantitative PCR (RT-qPCR) becoming the method of choice for high-throughput and accurate ...expression profiling of selected genes. Considering the increased sensitivity, reproducibility and large dynamic range of this method, the requirements for proper internal reference gene(s) for relative expression normalization have become much more stringent. Given the increasing interest in the functional genomics of Eucalyptus, we sought to identify and experimentally verify suitable reference genes for the normalization of gene expression associated with the flower, leaf and xylem of six species of the genus. We selected 50 genes that exhibited the least variation in microarrays of E. grandis leaves and xylem, and E. globulus xylem. We further performed the experimental analysis using RT-qPCR for six Eucalyptus species and three different organs/tissues. Employing algorithms geNorm and NormFinder, we assessed the gene expression stability of eight candidate new reference genes. Classic housekeeping genes were also included in the analysis. The stability profiles of candidate genes were in very good agreement. PCR results proved that the expression of novel Eucons04, Eucons08 and Eucons21 genes was the most stable in all Eucalyptus organs/tissues and species studied. We showed that the combination of these genes as references when measuring the expression of a test gene results in more reliable patterns of expression than traditional housekeeping genes. Hence, novel Eucons04, Eucons08 and Eucons21 genes are the best suitable references for the normalization of expression studies in the Eucalyptus genus.
The drought response is a genome-wide phenomenon that, due to its complexity, is hard to achieve a “universal” response requiring an analysis method that takes this characteristic into account. Here, ...the Transcriptogramer tool was used to (i) produce a
Glycine max
ordering list based on protein–protein associations and (ii) analyze transcriptome data from soybean roots submitted to dehydration for 1, 6, and 12 h in a genome-wide scale. Sixteen, five, and six gene ontology (GO) categories were differentially expressed at 1, 6, and 12 h of drought stress, respectively. The most enriched GO categories are cell division, cell cycle, cell wall organization, stress responses, hormone signaling pathways, phosphorelay signal transduction, and regulation of gene expression. In total, 274 genes were differentially expressed (DE). The list includes 15 calcium-binding proteins, 17 TIFY, 3 ABA receptors, 5 E2 ubiquitin-conjugatin enzymes, 2 E3 ligases, 4 proteasome regulatory subunits, 1 chloroplastic superoxide dismutase, 1 peroxidase, and 10 thioredoxin-encoding genes already known to be related to drought response. The Transcriptogramer has evinced a global reprogrammed transcriptome and revealed that the most observable effects of water deficit occur during the first hour.
Osmotins are multifunctional proteins belonging to the thaumatin-like family related to plant stress responses. To better understand the functions of soybean osmotins in drought stress response, the ...current study presents the characterisation of four previously described proteins and a novel putative soybean osmotin (GmOLPa-like). Gene and protein structure as well as gene expression analyses were conducted on different tissues and developmental stages of two soybean cultivars with varying dehydration sensitivities (BR16 and EMB48 are highly and slightly sensitive, respectively). The analysed osmotin sequences share the conserved amino acid signature and 3D structure of the thaumatin-like family. Some differences were observed in the conserved regions of protein sequences and in the electrostatic surface potential. P21-like present the most similar electrostatic potential to osmotins previously characterised as promoters of drought tolerance in
Nicotiana tabacum
and
Solanum nigrum
. Gene expression analysis indicated that soybean osmotins were differentially expressed in different organs (leaves and roots), developmental stages (R1 and V3), and cultivars in response to dehydration. In addition, under dehydration conditions, the highest level of gene expression was detected for GmOLPa-like and P21-like osmotins in the leaves and roots, respectively, of the less drought sensitive cultivar. Altogether, the results suggest an involvement of these genes in drought stress tolerance.
Before commercial release, new potato (Solanum tuberosum) varieties must be evaluated for content of toxic compounds such as glycoalkaloids (GAs), which are potent poisons. GA biosynthesis proceeds ...via the cholesterol pathway to α-chaconine and α-solanine. The goal of this study was to evaluate the relationship between total glycoalkaloid (TGA) content and the expression of GAME, SGT1, and SGT3 genes in potato tubers. TGA content was measured by HPLC–MS, and reverse transcription quantitative polymerase chain reactions were performed to determine the relative expression of GAME, SGT1, and SGT3 genes. We searched for cis-elements of the transcription start site using the PlantPAN database. There was a relationship between TGA content and the relative expression of GAME, SGT1, and SGT3 genes in potato tubers. Putative promoter regions showed the presence of several cis-elements related to biotic and abiotic stresses and light. These findings provide an important step toward understanding TGA regulation and variation in potato tubers.
In plants, ureases have been related to urea degradation, to defense against pathogenic fungi and phytophagous insects, and to the soybean−Bradyrhizobium japonicum symbiosis. Two urease isoforms have ...been described for soybean: the embryo-specific, encoded by Eu1 gene, and the ubiquitous urease, encoded by Eu4. A third urease-encoding locus exists in the completed soybean genome. The gene was designated Eu5 and the putative product of its ORF as SBU-III. Phylogenetic analysis shows that 41 plant, moss and algal ureases have diverged from a common ancestor protein, but ureases from monocots, eudicots and ancient species have evolved independently. Genomes of ancient organisms present a single urease-encoding gene and urease-encoding gene duplication has occurred independently along the evolution of some eudicot species. SBU-III has a shorter amino acid sequence, since many gaps are found when compared to other sequences. A mutation in a highly conserved amino acid residue suggests absence of ureolytic activity, but the overall protein architecture remains very similar to the other ureases. The expression profile of urease-encoding genes in different organs and developmental stages was determined by RT-qPCR. Eu5 transcripts were detected in seeds one day after dormancy break, roots of young plants and embryos of developing seeds. Eu1 and Eu4 transcripts were found in all analyzed organs, but Eu4 expression was more prominent in seeds one day after dormancy break whereas Eu1 predominated in developing seeds. The evidence suggests that SBU-III may not be involved in nitrogen availability to plants, but it could be involved in other biological role(s).
•The new urease-encoding gene was designated Eu5 and its product SBU-III.•SBU-III has a shorter amino acid sequence since many gaps are found.•A mutation in a highly conserved amino acid residue suggests absence of ureolytic activity.•Eu5 transcripts were detected in seeds, roots of young plants and embryos of developing seeds.•SBU-III is probably not involved in nitrogen availability in soybean.
Drought is today, and perhaps even more in the future, the main challenge for grain crops, resulting in a drastic yield reduction. Thus, it is of great interest to obtain soybean genotypes tolerant ...to water deficit. The drought tolerance trait is difficult to obtain through classical breeding due to its polygenic basis. In this context, genetic engineering is presented as a way to achieve this attribute. The ability to modulate the expression of many genes placed the transcription factors as promising biotechnological targets to develop stress tolerant cultivars. The WRKY proteins form a large family of transcription factors that are involved in important physiological and biochemical processes in plants, including the response to water deficit. In this study, the expression pattern determined by qPCR showed that, GmWRKY6, GmWRKY46, GmWRKY56, GmWRKY106 and GmWRKY149 genes are differentially expressed between a drought tolerant and a susceptible soybean genotype in water stress conditions. The in silico promoter and coexpression analysis indicate that these genes act in a stress physiological background.
•The soybean WRKY6, 46, 56, 106 and 149 genes presented a differential expression under water deficits conditions.•The putative promoters of the genes analyzed are enriched of cis-elements which regulate the ABA-induced gene expression.•The genes coexpressed with the WRKY genes analyzed are involved in stress response mechanisms.•The GmWRKY56 and GmWRKY106 coexpression networks showed broad overlap.•The GmWRKY106 emerges as candidate for genetic engineering strategies aiming to develop cultivars tolerant to dehydration.
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