Alexandre Lima Nepomuceno

Identification of novel soybean microRNAs involved in abiotic and biotic stresses

BackgroundSmall RNAs (19-24 nt) are key regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in eukaryotes. Current studies have demonstrated that microRNAs (miRNAs) act in several plant pathways associated with tissue proliferation, differentiation, and development and in response to abiotic and biotic stresses. In order to identify new miRNAs in soybean and to verify those that are possibly water deficit and rust-stress regulated, eight libraries of small RNAs were constructed and submitted to Solexa sequencing.ResultsThe libraries were developed from drought-sensitive and tolerant seedlings and rust-susceptible and resistant soybeans with or without stressors. Sequencing the library and subsequent analyses detected 256 miRNAs. From this total, we identified 24 families of novel miRNAs that had not been reported before, six families of conserved miRNAs that exist in other plants species, and 22 families previously reported in soybean. We also observed the presence of several isomiRNAs during our analyses. To validate novel miRNAs, we performed RT-qPCR across the eight different libraries. Among the 11 miRNAs analyzed, all showed different expression profiles during biotic and abiotic stresses to soybean. The majority of miRNAs were up-regulated during water deficit stress in the sensitive plants. However, for the tolerant genotype, most of the miRNAs were down regulated. The pattern of miRNAs expression was also different for the distinct genotypes submitted to the pathogen stress. Most miRNAs were down regulated during the fungus infection in the susceptible genotype; however, in the resistant genotype, most miRNAs did not vary during rust attack. A prediction of the putative targets was carried out for conserved and novel miRNAs families.ConclusionsValidation of our results with quantitative RT-qPCR revealed that Solexa sequencing is a powerful tool for miRNA discovery. The identification of differentially expressed plant miRNAs provides molecular evidence for the possible involvement of miRNAs in the process of water deficit- and rust-stress responses.

Molecular mapping of two loci that confer resistance to Asian rust in soybean.

Asian soybean rust (ASR) is caused by the fungal pathogen Phakopsora pachyrhizi Sydow & Sydow. It was first identified in Brazil in 2001 and quickly infected soybean areas in several countries in South America. Primary efforts to combat this disease must involve the development of resistant cultivars. Four distinct genes that confer resistance against ASR have been reported: Rpp1, Rpp2, Rpp3, and Rpp4. However, no cultivar carrying any of those resistance loci has been released. The main objective of this study was to genetically map Rpp2 and Rpp4 resistance genes. Two F2:3 populations, derived from the crosses between the resistant lines PI 230970 (Rpp2), PI 459025 (Rpp4) and the susceptible cultivar BRS 184, were used in this study. The mapping populations and parental lines were inoculated with a field isolate of P. pachyrhizi and evaluated for lesion type as resistant (RB lesions) or susceptible (TAN lesions). The mapping populations were screened with SSR markers, using the bulk segregant analysis (BSA) to expedite the identification of linked markers. Both resistance genes showed an expected segregation ratio for a dominant trait. This study allowed mapping Rpp2 and Rpp4 loci on the linkage groups J and G, respectively. The associated markers will be of great value on marker assisted selection for this trait.

Physiological responses of cotton leaves and roots to water deficit induced by polyethylene glycol

Abstract The characterization of plant water relations is a prerequisite for subsequent selection and genetic manipulation for drought tolerance. To evaluate roots in addition to leaves, a method based on PEG was developed to obtain clean, stressed roots quickly while avoiding toxic effects associated with PEG treatment. A mild water deficit of −0.3 MPa was induced with polyethylene glycol (PEG 6000) around the roots of four cotton ( Gossypium hirsutum ) genotypes with different water deficit tolerances. Treatment with PEG during diurnal dark period avoided toxic effects observed with treatment during the light period. The genotypes examined included: (1) Siokra L-23, a drought-tolerant cultivar; (2) T-1521, a wild-type with significant osmotic adjustment capability; (3) CS-50, a moderately susceptible cultivar; and (4) Stoneville 506, a susceptible cultivar. The water potentials ( ψ w ) and osmotic potentials ( ψ s ) of roots and leaves of stressed and non-stressed plants were determined psychrometrically. In response to the water deficits Siokra L-23 and T-1521 showed 25 and 20% reductions in leaf ψ s , respectively, compared with unstressed controls. At the same time, ψ w of these two genotypes did not change significantly. Conversely, the decrease in the ψ s of CS-50 and Stoneville 506 was not significant, while their ψ w decreased significantly. By maintaining a higher ψ w during stress, the tolerant genotypes were able to maintain photosynthesis, stomatal conductance and relative water content near unstressed control levels. The use of PEG 6000 to maintain a constant ψ w in the root environment provides an efficient method for controlling ψ w while allowing for rapid sampling of clean root tissue for analysis. The results demonstrated that the empirically determined differences in drought tolerance of these cultivars can be related to measurable physiological parameters. These results suggest that physiological monitoring can be an effective tool in germplasm selection and improvement.

The use of microRNAs as reference genes for quantitative polymerase chain reaction in soybean

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a robust and widely applied technique used to investigate gene expression. However, for correct analysis and interpretation of results, the choice of a suitable gene to use as an internal control is a crucial factor. These genes, such as housekeeping genes, should have a constant expression level in different tissues and across different conditions. The advances in genome sequencing have provided high-throughput gene expression analysis and have contributed to the identification of new genes, including microRNAs (miRNAs). The miRNAs are fundamental regulatory genes of eukaryotic genomes, acting on several biological functions. In this study, miRNA expression stability was investigated in different soybean tissues and genotypes as well as after abiotic or biotic stress treatments. The present study represents the first investigation into the suitability of miRNAs as housekeeping genes in plants. The transcript stability of 10 miRNAs was compared to those of six previously reported housekeeping genes for the soybean. In this study, we provide evidence that the expression stabilities of miR156b and miR1520d were the highest across the soybean experiments. Furthermore, these miRNAs genes were more stable than the most commonly protein-coding genes used in soybean gene expression studies involving RT-qPCR.

Drought Tolerance Characteristics of Brazilian Soybean Cultivars

Abstract Drought is one of the major constraints for soybean production in Brazil. Seed yield of ten Brazilian soybean cultivarssheltered from rain (drought stress) for one month after the first flowering was examined over two growing seasons in the field in Londrina, Brazil. The drought tolerance on the basis of seed yield varied with the cultivar, and the yield ranking among cultivars was nearly the same across two years. In cultivars with higher drought tolerance, crop growth rate (CGR)during the drought stress period was higher than in other cultivars. They also maintained a larger leaf area during the stress period. Although reproductive development was retarded by the drought stress, it tended to be retarded less in drought-tolerant cultivars. The information obtained in this research may be useful for breeding drought-tolerant cultivars or selecting diverse germplasms of soybean cultivars.

Genome-wide analysis of the Hsp20 gene family in soybean: comprehensive sequence, genomic organization and expression profile analysis under abiotic and biotic stresses

BackgroundThe Hsp 20 genes are associated with stress caused by HS and other abiotic factors, but have recently been found to be associated with the response to biotic stresses. These genes represent the most abundant class among the HSPs in plants, but little is known about this gene family in soybean. Because of their apparent multifunctionality, these proteins are promising targets for developing crop varieties that are better adapted to biotic and abiotic stresses. Thus, in the present study an in silico identification of GmHsp 20 gene family members was performed, and the genes were characterized and subjected to in vivo expression analysis under biotic and abiotic stresses.ResultsA search of the available soybean genome databases revealed 51 gene models as potential GmHsp 20 candidates. The 51 GmHsp 20 genes were distributed across a total of 15 subfamilies where a specific predicted secondary structure was identified. Based on in vivo analysis, only 47 soybean Hsp 20 genes were responsive to heat shock stress. Among the GmHsp 20 genes that were potentials HSR, five were also cold-induced, and another five, in addition to one GmAcd gene, were responsive to Meloidogyne javanica infection. Furthermore, one predicted GmHsp 20 was shown to be responsive only to nematode infection; no expression change was detected under other stress conditions. Some of the biotic stress-responsive GmHsp 20 genes exhibited a divergent expression pattern between resistant and susceptible soybean genotypes under M. javanica infection. The putative regulatory elements presenting some conservation level in the GmHsp 20 promoters included HSE, W-box, CAAT box, and TA-rich elements. Some of these putative elements showed a unique occurrence pattern among genes responsive to nematode infection.ConclusionsThe evolution of Hsp 20 family in soybean genome has most likely involved a total of 23 gene duplications. The obtained expression profiles revealed that the majority of the 51 GmHsp 20 candidates are induced under HT, but other members of this family could also be involved in normal cellular functions, unrelated to HT. Some of the GmHsp 20 genes might be specialized to respond to nematode stress, and the predicted promoter structure of these genes seems to have a particular conserved pattern related to their biological function.

Modeling nitrogen accumulation and use by soybean

Abstract Simulation of soybean growth and yield requires an accurate representation of nitrogen accumulation and distribution in the developing crop. Approaches to simulate nitrogen accumulation by soybean have been complicated by the need to account for both soil nitrate uptake and symbiotic nitrogen fixation. Past approaches to simulating soybean have generally been empirical requiring ‘calibration’ for each new environment. Recently, Jamieson and Semonov [Field Crops Res. 68 (2000) 21] proposed a relatively simple approach for simulating nitrogen accumulation by wheat based on the nitrogen demand imposed separately by the development of leaf area and the growth of stems. This paper assesses this approach for soybean. This approach was further simplified by assuming that nitrogen availability to the plant was limited only by soil moisture conditions and a maximum uptake rate and that it was not necessary to distinguish between nitrate uptake and symbiotic nitrogen fixation. The simplified model generally resulted in simulations that corresponded to experimental observations on nitrogen and mass accumulation through the growing season, and on yield over a number of years. Sensitivity analysis of the model indicated that storage of nitrogen in the stem might be a key trait in increasing soybean yield potential.

Diurnal oscillations of soybean circadian clock and drought responsive genes.

Rhythms produced by the endogenous circadian clock play a critical role in allowing plants to respond and adapt to the environment. While there is a well-established regulatory link between the circadian clock and responses to abiotic stress in model plants, little is known of the circadian system in crop species like soybean. This study examines how drought impacts diurnal oscillation of both drought responsive and circadian clock genes in soybean. Drought stress induced marked changes in gene expression of several circadian clock-like components, such as LCL1-, GmELF4- and PRR-like genes, which had reduced expression in stressed plants. The same conditions produced a phase advance of expression for the GmTOC1-like, GmLUX-like and GmPRR7-like genes. Similarly, the rhythmic expression pattern of the soybean drought-responsive genes DREB-, bZIP-, GOLS-, RAB18- and Remorin-like changed significantly after plant exposure to drought. In silico analysis of promoter regions of these genes revealed the presence of cis-elements associated both with stress and circadian clock regulation. Furthermore, some soybean genes with upstream ABRE elements were responsive to abscisic acid treatment. Our results indicate that some connection between the drought response and the circadian clock may exist in soybean since (i) drought stress affects gene expression of circadian clock components and (ii) several stress responsive genes display diurnal oscillation in soybeans.

Induced over-expression of AtDREB2A CA improves drought tolerance in sugarcane

Drought is one of the most challenging agricultural issues limiting sustainable sugarcane production and, in some cases, yield losses caused by drought are nearly 50%. DREB proteins play vital regulatory roles in abiotic stress responses in plants. The transcription factor DREB2A interacts with a cis-acting DRE sequence to activate the expression of downstream genes that are involved in drought-, salt- and heat-stress response in Arabidopsis thaliana. In the present study, we evaluated the effects of stress-inducible over-expression of AtDREB2A CA on gene expression, leaf water potential (ΨL), relative water content (RWC), sucrose content and gas exchanges of sugarcane plants submitted to a four-days water deficit treatment in a rhizotron-grown root system. The plants were also phenotyped by scanning the roots and measuring morphological parameters of the shoot. The stress-inducible expression of AtDREB2A CA in transgenic sugarcane led to the up-regulation of genes involved in plant response to drought stress. The transgenic plants maintained higher RWC and ΨL over 4 days after withholding water and had higher photosynthetic rates until the 3rd day of water-deficit. Induced expression of AtDREB2A CA in sugarcane increased sucrose levels and improved bud sprouting of the transgenic plants. Our results indicate that induced expression of AtDREB2A CA in sugarcane enhanced its drought tolerance without biomass penalty.

Implications of ethylene biosynthesis and signaling in soybean drought stress tolerance

BackgroundEthylene is a phytohormone known for inducing a triple response in seedlings, leaf abscission and other responses to various stresses. Several studies in model plants have evaluated the importance of this hormone in crosstalk signaling with different metabolic pathways, in addition to responses to biotic stresses. However, the mechanism of action in plants of agricultural interest, such as soybean, and its participation in abiotic stresses remain unclear.ResultsThe studies presented in this work allowed for the identification of 176 soybean genes described elsewhere for ethylene biosynthesis (108 genes) and signal transduction (68 genes). A model to predict these routes in soybean was proposed, and it had great representability compared to those described for Arabidopsis thaliana and Oryza sativa. Furthermore, analysis of putative gene promoters from soybean gene orthologs permitted the identification of 29 families of cis-acting elements. These elements are essential for ethylene-mediated regulation and its possible crosstalk with other signaling pathways mediated by other plant hormones.From genes that are differentially expressed in the transcriptome database, we analyzed the relative expression of some selected genes in resistant and tolerant soybean plants subjected to water deficit. The differential expression of a set of five soybean ethylene-related genes (MAT, ACS, ACO, ETR and CTR) was validated with RT-qPCR experiments, which confirmed variations in the expression of these soybean target genes, as identified in the transcriptome database. In particular, two families of ethylene biosynthesis genes (ACS and ACO) were upregulated under these experimental conditions, whereas CTR (involved in ethylene signal transduction) was downregulated. In the same samples, high levels of ethylene production were detected and were directly correlated with the free fraction levels of ethylene’s precursor. Thus, the combination of these data indicated the involvement of ethylene biosynthesis and signaling in soybean responses to water stress.ConclusionsThe in silico analysis, combined with the quantification of ethylene production (and its precursor) and RT-qPCR experiments, allowed for a better understanding of the importance of ethylene at a molecular level in this crop as well as its role in the response to abiotic stresses. In summary, all of the data presented here suggested that soybean responses to water stress could be regulated by a crosstalk network among different signaling pathways, which might involve various phytohormones, such as auxins, ABA and jasmonic acid. The integration of in silico and physiological data could also contribute to the application of biotechnological strategies to the development of improved cultivars with regard to different stresses, such as the isolation of stress-specific plant promoters.