Differential gene expression in response to water deficit in leaf and root tissues of soybean genotypes with contrasting tolerance profiles.

Water deficit is one of the major limitations to soybean production worldwide, yet the genetic basis of drought-responsive mechanisms in crops remains poorly understood. In order to study the gene expression patterns in leaves and roots of soybean, two contrasting genotypes, Embrapa 48 (drought-tolerant) and BR 16 (drought-sensitive), were evaluated under moderate and severe water deficit. Transcription factors from the AP2/EREBP and WRKY families were investigated. Embrapa 48 showed 770 more up-regulated genes than BR 16, in eight categories. In general, leaves presented more differentially expressed genes (DEGs) than roots. Embrapa 48 responded to water deficit faster than BR 16, presenting a greater number of DEGs since the first signs of drought. Embrapa 48 exhibited initial modulation of genes associated with stress, while maintaining the level of the ones related to basic functions. The genes expressed exclusively in the drought-tolerant cultivar, belonging to the category of dehydration responsive genes, and the ones with a contrasting expression pattern between the genotypes are examples of important candidates to confer tolerance to plants. Finally, this study identified genes of the AP2/EREBP and WRKY families related to drought tolerance.

Genome-wide analysis of the transcriptional response to drought stress in root and leaf of common bean.

Genes related to the response to drought stress in leaf and root tissue of drought-susceptible (DS) and tolerant (DT) genotypes were characterized by RNA-Seq. In total, 54,750 transcripts, representative of 28,590 genes, were identified; of these, 1,648 were of high-fidelity (merge of 12 libraries) and described for the first time in the Andean germplasm. From the 1,239 differentially expressed genes (DEGs), 458 were identified in DT, with a predominance of genes in categories of oxidative stress, response to stimulus and kinase activity. Most genes related to oxidation-reduction terms in roots were early triggered in DT (T75) compared to DS (T150) suggestive of a mechanism of tolerance by reducing the damage from ROS. Among the KEGG enriched by DEGs up-regulated in DT leaves, two related to the formation of Sulfur-containing compounds, which are known for their involvement in tolerance to abiotic stresses, were common to all treatments. Through qPCR, 88.64% of the DEGs were validated. A total of 151,283 variants were identified and functional effects estimated for 85,780. The raw data files were submitted to the NCBI database. A transcriptome map revealed new genes and isoforms under drought. These results supports a better understanding of the drought tolerance mechanisms in beans.

Daytime soybean transcriptome fluctuations during water deficit stress.

BACKGROUND: Since drought can seriously affect plant growth and development and little is known about how the oscillations of gene expression during the drought stress-acclimation response in soybean is affected, we applied Illumina technology to sequence 36 cDNA libraries synthesized from control and drought-stressed soybean plants to verify the dynamic changes in gene expression during a 24-h time course. Cycling variables were measured from the expression data to determine the putative circadian rhythm regulation of gene expression. RESULTS: We identified 4866 genes differentially expressed in soybean plants in response to water deficit. Of these genes, 3715 were differentially expressed during the light period, from which approximately 9.55% were observed in both light and darkness. We found 887 genes that were either up- or down-regulated in different periods of the day. Of 54,175 predicted soybean genes, 35.52% exhibited expression oscillations in a 24 h period. This number increased to 39.23% when plants were submitted to water deficit. Major differences in gene expression were observed in the control plants from late day (ZT16) until predawn (ZT20) periods, indicating that gene expression oscillates during the course of 24 h in normal development. Under water deficit, dissimilarity increased in all time-periods, indicating that the applied stress influenced gene expression. Such differences in plants under stress were primarily observed in ZT0 (early morning) to ZT8 (late day) and also from ZT4 to ZT12. Stress-related pathways were triggered in response to water deficit primarily during midday, when more genes were up-regulated compared to early morning. Additionally, genes known to be involved in secondary metabolism and hormone signaling were also expressed in the dark period. CONCLUSIONS: Gene expression networks can be dynamically shaped to acclimate plant metabolism under environmental stressful conditions. We have identified putative cycling genes that are expressed in soybean leaves under normal developmental conditions and genes whose expression oscillates under conditions of water deficit. These results suggest that time of day, as well as light and temperature oscillations that occur considerably affect the regulation of water deficit stress response in soybean plants.

Malnutrition affects the pressor response to microinjection of L-glutamate into the RVLM of awake rats.

Post-weaning protein malnutrition is often related to the development of cardiovascular and metabolic diseases in humans, as well to changed content of neurotransmitters in the central nervous system under experimental conditions. The rostral ventrolateral medulla (RVLM) is a bulbar region that contains sympathetic premotor neurons; the excitatory amino acid L-glutamate seems to be the main neurotransmitter at this level. The aim of the present study was to evaluate the possible change in the L-glutamate sensitivity of the RVLM neurons of malnourished animals. Male Fischer rats were divided into two groups: control (n = 15) and malnourished (n = 19). Four days before the experiments, guide cannulas were implanted bilaterally in direction of the RVLM for microinjection of L-glutamate. Twenty-four hours before the experiments, the femoral artery was cannulated for cardiovascular recordings. The results showed that the baseline heart rate increased in malnourished compared to control animals (412.18 ± 16.03 bpm vs. 370.74 ± 9.59 bpm, respectively). Malnourished animals presented a dissimilar concentration-dependent pressor response curve to L-glutamate and an attenuated baroreflex gain. Our results suggest that post-weaning protein restriction affects glutamatergic neurotransmission of the baroreflex at the RVLM level.

Transcriptome-Wide Identification of Reference Genes for Expression Analysis of Soybean Responses to Drought Stress along the Day.

The soybean transcriptome displays strong variation along the day in optimal growth conditions and also in response to adverse circumstances, like drought stress. However, no study conducted to date has presented suitable reference genes, with stable expression along the day, for relative gene expression quantification in combined studies on drought stress and diurnal oscillations. Recently, water deficit responses have been associated with circadian clock oscillations at the transcription level, revealing the existence of hitherto unknown processes and increasing the demand for studies on plant responses to drought stress and its oscillation during the day. We performed data mining from a transcriptome-wide background using microarrays and RNA-seq databases to select an unpublished set of candidate reference genes, specifically chosen for the normalization of gene expression in studies on soybean under both drought stress and diurnal oscillations. Experimental validation and stability analysis in soybean plants submitted to drought stress and sampled during a 24 h timecourse showed that four of these newer reference genes (FYVE, NUDIX, Golgin-84 and CYST) indeed exhibited greater expression stability than the conventionally used housekeeping genes (ELF1-ß and ß-actin) under these conditions. We also demonstrated the effect of using reference candidate genes with different stability values to normalize the relative expression data from a drought-inducible soybean gene (DREB5) evaluated in different periods of the day.

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.

Expression patterns of GmAP2/EREB-like transcription factors involved in soybean responses to water deficit.

Soybean farming has faced several losses in productivity due to drought events in the last few decades. However, plants have molecular mechanisms to prevent and protect against water deficit injuries, and transcription factors play an important role in triggering different defense mechanisms. Understanding the expression patterns of transcription factors in response to water deficit and to environmental diurnal changes is very important for unveiling water deficit stress tolerance mechanisms. Here, we analyzed the expression patterns of ten APETALA2/Ethylene Responsive Element Binding-like (AP2/EREB-like) transcription factors in two soybean genotypes (BR16: drought-sensitive; and Embrapa 48: drought-tolerant). According to phylogenetic and domain analyses, these genes can be included in the DREB and ERF subfamilies. We also analyzed a GmDRIP-like gene that encodes a DREB negative regulator. We detected the up-regulation of 9 GmAP2/EREB-like genes and identified transcriptional differences that were dependent on the levels of the stress applied and the tissue type analyzed (the expression of the GmDREB1F-like gene, for example, was four times higher in roots than in leaves). The GmDRIP-like gene was not induced by water deficit in BR16 during the longest periods of stress, but was significantly induced in Embrapa 48; this suggests a possible genetic/molecular difference between the responses of these cultivars to water deficit stress. Additionally, RNAseq gene expression analysis over a 24-h time course indicates that the expression patterns of several GmDREB-like genes are subject to oscillation over the course of the day, indicating a possible circadian regulation.

Early transcriptional response of soybean contrasting accessions to root dehydration.

Drought is a significant constraint to yield increase in soybean. The early perception of water deprivation is critical for recruitment of genes that promote plant tolerance. DeepSuperSAGE libraries, including one control and a bulk of six stress times imposed (from 25 to 150 min of root dehydration) for drought-tolerant and sensitive soybean accessions, allowed to identify new molecular targets for drought tolerance. The survey uncovered 120,770 unique transcripts expressed by the contrasting accessions. Of these, 57,610 aligned with known cDNA sequences, allowing the annotation of 32,373 unitags. A total of 1,127 unitags were up-regulated only in the tolerant accession, whereas 1,557 were up-regulated in both as compared to their controls. An expression profile concerning the most representative Gene Ontology (GO) categories for the tolerant accession revealed the expression "protein binding" as the most represented for "Molecular Function", whereas CDPK and CBL were the most up-regulated protein families in this category. Furthermore, particular genes expressed different isoforms according to the accession, showing the potential to operate in the distinction of physiological behaviors. Besides, heat maps comprising GO categories related to abiotic stress response and the unitags regulation observed in the expression contrasts covering tolerant and sensitive accessions, revealed the unitags potential for plant breeding. Candidate genes related to "hormone response" (LOX, ERF1b, XET), "water response" (PUB, BMY), "salt stress response" (WRKY, MYB) and "oxidative stress response" (PER) figured among the most promising molecular targets. Additionally, nine transcripts (HMGR, XET, WRKY20, RAP2-4, EREBP, NAC3, PER, GPX5 and BMY) validated by RT-qPCR (four different time points) confirmed their differential expression and pointed that already after 25 minutes a transcriptional reorganization started in response to the new condition, with important differences between both accessions.

Subtractive libraries for prospecting differentially expressed genes in the soybean under water deficit.

Soybean has a wide range of applications in the industry and, due to its crop potential, its improvement is widely desirable. During drought conditions, soybean crops suffer significant losses in productivity. Therefore, understanding the responses of the soybean under this stress is an effective way of targeting crop improvement techniques. In this study, we employed the Suppressive Subtractive Hybridization (SSH) technique to investigate differentially expressed genes under water deficit conditions. Embrapa 48 and BR 16 soybean lines, known as drought-tolerant and -sensitive, respectively, were grown hydroponically and subjected to different short-term periods of stress by withholding the nutrient solution. Using this approach, we have identified genes expressed during the early response to water deficit in roots and leaves. These genes were compared among the lines to assess probable differences in the plant transcriptomes. In general, similar biochemical processes were predominant in both cultivars; however, there were more considerable differences between roots and leaves of Embrapa 48. Moreover, we present here a fast, clean and straightforward method to obtain drought-stressed root tissues and a large enriched collection of transcripts expressed by soybean plants under water deficit that can be useful for further studies towards the understanding of plant responses to stress.

Malnutrition affects the pressor response to microinjection of L-glutamate into the RVLM of awake rats

Post-weaning protein malnutrition is often related to the development of cardiovascular and metabolic diseases in humans, as well to changed content of neurotransmitters in the central nervous system under experimental conditions. The rostral ventrolateral medulla (RVLM) is a bulbar region that contains sympathetic premotor neurons; the excitatory amino acid L-glutamate seems to be the main neurotransmitter at this level. The aim of the present study was to evaluate the possible change in the L-glutamate sensitivity of the RVLM neurons of malnourished animals. Male Fischer rats were divided into two groups: control (n = 15) and malnourished (n = 19). Four days before the experiments, guide cannulas were implanted bilaterally in direction of the RVLM for microinjection of L-glutamate. Twenty-four hours before the experiments, the femoral artery was cannulated for cardiovascular recordings. The results showed that the baseline heart rate increased in malnourished compared to control animals (412.18 ± 16.03 bpm vs. 370.74 ± 9.59 bpm, respectively). Malnourished animals presented a dissimilar concentration-dependent pressor response curve to L-glutamate and an attenuated baroreflex gain. Our results suggest that post-weaning protein restriction affects glutamatergic neurotransmission of the baroreflex at the RVLM level.