Exploring the Proteomic Profile of Soybean Bran: Unlocking the Potential for Improving Protein Quality and Quantity.

Soybean is a rich source of vegetal protein for both animal and human consumption. Despite the high levels of protein in soybean seeds, industrial processing to obtain soybean bran significantly decreases the final protein content of the byproducts. To overcome this problem, cultivars with higher protein contents must be developed. However, selecting the target proteins is difficult because of the lack of information on the proteome profile of soybean bran. Therefore, this study obtained the comparative proteomic profiles of both natural coatless seeds and defatted bran from an elite tropical-soybean cultivar. Thus, their extracts were characterized using LC-MS/MS and a total of 550 proteins were identified. Among these, 526 proteins were detected in coatless seeds and 319 proteins in defatted bran. Moreover, a total of 139 proteins were identified as presenting different levels of content in coatless seeds and defatted bran. Among them, only 46 were retained after the seed processing. These proteins were clustered in several important metabolic pathways, such as amino-acid biosynthesis, sugar biosynthesis, and antioxidant activity, meaning that they could act as targets for bioactive products or genome editing to improve protein quality and quantity in soybean grains. These findings can enhance our understanding regarding protein robustness for both soybean crops and the commercial bran improvement because target proteins must remain intact after processing and must be bioactive when overexpressed. Overall, the soybean bran proteomic profile was explored for the first time, providing a valuable catalogue of target proteins that can tolerate the industrial process.

Flowering process in soybean under water deficit conditions: A review on genetic aspects.

Soybean is a key crop in many countries, being used from human food to the animal industry due to its nutritional properties. Financially, the grain chain moves large sums of money into the economy of producing countries. However, like other agricultural commodities around the world, it can have its final yield seriously compromised by abiotic environmental stressors, like drought. As flowers imply in pods and in grains inside it to minimize damages caused by water restriction, researchers have focused on understanding flowering-process related genes and their interactions. Here a review dedicated to the soybean flowering process and gene network involved in it is presented, describing gene interactions and how genes act in this complex mechanism, also ruled by environmental triggers such as day-light and circadian cycle. The objective was to gather information and insights on the soybean flowering process, aiming to provide knowledge useful to assist in the development of drought-tolerant soybean lines, minimizing losses due to delays or anticipation of flowering and, consequently, restraining financial and productivity losses.

Overexpression of AtNCED3 gene improved drought tolerance in soybean in greenhouse and field conditions.

Water deficit is an important climatic problem that can impair agriculture yield and economy. Genetically modified soybean plants containing the AtNCED3 gene were obtained aiming drought-tolerance improvement. The NCED3 gene encodes a 9-cis-epoxycarotenoid dioxygenase (NCED, EC 1.13.11.51), an important enzyme in abscisic acid biosynthesis. ABA activates the expression of drought-responsive genes, in water-deficit conditions, targeting defense mechanisms and enabling plants to survive under low water availability. Results from greenhouse experiments showed that the transgene AtNCED3 and the endogenous genes GmAREB1, GmPP2C, GmSnRK2 and GmAAO3 presented higher expression under water deficit (WD) in the event 2Ha11 than in WT-plants. No significant correlation was observed between the plant materials and WD conditions for growth parameters; however, gas exchange measurements decreased in the GM event, which also showed 80% higher intrinsic water use when compared to WT plants. In crop season 2015/16, event 2Ha11 showed higher total number of pods, higher number of pods with seeds and yield than WT plants. ABA concentration was also higher in GM plants under WD. These results obtained in field screenings suggest that AtNCED3 soybean plants might outperform under drought, reducing economic and yield losses, thus being a good candidate line to be incorporated in the soybean-breeding program to develop drought-tolerant cultivars.

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.

Daily heliotropic movements assist gas exchange and productive responses in DREB1A soybean plants under drought stress in the greenhouse.

Drought stress is one of the most severe environmental constraints on plant production. Under environmental pressures, complex daily heliotropic adjustments of leaflet angles in soybean can help to reduce transpiration losses by diminishing light interception (paraheliotropism), increase diurnal carbon gain in sparse canopies and reduce carbon gain in dense canopies by solar tracking (diaheliotropism). The plant materials studied were cultivar BR 16 and its genetically engineered isoline P58, ectopically overexpressing AtDREB1A, which is involved in abiotic stress responses. We aimed to follow the movements of central and lateral leaflets in vegetative stages V7-V10 and reproductive stages R4-R5, integrating the reversible morphogenetic changes into an estimate of daily plant photosynthesis using three-dimensional modeling, and to analyze the production parameters of BR 16 and P58. The patterns of daily movements of central leaflets of BR 16 in V7-V10 and R4-R5 were similar, expressing fewer diaheliotropic movements under drought stress than under non-limiting water conditions. Daily heliotropic patterns of lateral leaflets in V7-V10 and R4-R5 showed more diaheliotropic movements in drought-stressed P58 plants than in those grown under non-limiting water conditions. Leaf area in R4-R5 was generally higher in P58 than in BR 16. Drought significantly affected gas exchange and vegetative and reproductive architectural features. DREB1A could be involved in various responses to drought stress. Compared with the parental BR 16, P58 copes with drought through better compensation between diaheliotropic and paraheliotropic movements, finer tuning of water-use efficiency, a lower transpiration rate, higher leaf area and higher pod abortion to accomplish the maximum possible grain production under continued drought conditions.

Endophytic bacterial microbiome associated with leaves of genetically modified (AtAREB1) and conventional (BR 16) soybean plants.

Plant leaves (phyllosphere) have a great potential for colonization and microbial growth, consisting of a dynamic environment in which several factors can interfere with the microbial population structure. The use of genetically modified (GM) plants has introduced several traits in agriculture, such as the improvement of plant drought tolerance, as observed in the AtAREB1 transcription factor overexpression in soybean (Glycine max L. Merrill). The present study aimed at investigating the taxonomic and functional profile of the leaf microbial community of bacteria found in GM (drought-tolerant event 1Ea2939) and conventional (BR 16) soybean plants. Bacterial DNA was extracted from leaf samples collected from each genotype and used for microbial diversity and richness analysis through the MiSeq Illumina platform. Functional prediction was performed using the PICRUSt tool and the STAMP v 2.1.3 software. The obtainment of the GM event 1Ea2939 showed minimum effects on the microbial community and in the potential for chemical-genetic communication, i.e. in the potential for symbiotic and/or mutualistic interaction between plants and their natural microbiota.

Functional Characterization of a Putative Glycine max ELF4 in Transgenic Arabidopsis and Its Role during Flowering Control.

Flowering is an important trait in major crops like soybean due to its direct relation to grain production. The circadian clock mediates the perception of seasonal changes in day length and temperature to modulate flowering time. The circadian clock gene EARLY FLOWERING 4 (ELF4) was identified in Arabidopsis thaliana and is believed to play a key role in the integration of photoperiod, circadian regulation, and flowering. The molecular circuitry that comprises the circadian clock and flowering control in soybeans is just beginning to be understood. To date, insufficient information regarding the soybean negative flowering regulators exist, and the biological function of the soybean ELF4 (GmELF4) remains unknown. Here, we investigate the ELF4 family members in soybean and functionally characterize a GmELF4 homologous gene. The constitutive overexpression of GmELF4 delayed flowering in Arabidopsis, showing the ELF4 functional conservation among plants as part of the flowering control machinery. We also show that GmELF4 alters the expression of Arabidopsis key flowering time genes (AtCO and AtFT), and this down-regulation is the likely cause of flowering delay phenotypes. Furthermore, we identified the GmELF4 network genes to infer the participation of GmELF4 in soybeans. The data generated in this study provide original insights for comprehending the role of the soybean circadian clock ELF4 gene as a negative flowering controller.

Characterization of Soybean Genetically Modified for Drought Tolerance in Field Conditions.

Drought is one of the most stressful environmental factor causing yield and economic losses in many soybean-producing regions. In the last decades, transcription factors (TFs) are being used to develop genetically modified plants more tolerant to abiotic stresses. Dehydration responsive element binding (DREB) and ABA-responsive element-binding (AREB) TFs were introduced in soybean showing improved drought tolerance, under controlled conditions. However, these results may not be representative of the way in which plants behave over the entire season in the real field situation. Thus, the objectives of this study were to analyze agronomical traits and physiological parameters of AtDREB1A (1Ab58), AtDREB2CA (1Bb2193), and AtAREB1 (1Ea2939) GM lines under irrigated (IRR) and non-irrigated (NIRR) conditions in a field experiment, over two crop seasons and quantify transgene and drought-responsive genes expression. Results from season 2013/2014 revealed that line 1Ea2939 showed higher intrinsic water use and leaf area index. Lines 1Ab58 and 1Bb2193 showed a similar behavior to wild-type plants in relation to chlorophyll content. Oil and protein contents were not affected in transgenic lines in NIRR conditions. Lodging, due to plentiful rain, impaired yield from the 1Ea2939 line in IRR conditions. qPCR results confirmed the expression of the inserted TFs and drought-responsive endogenous genes. No differences were identified in the field experiment performed in crop season 2014/2015, probably due to the optimum rainfall volume during the cycle. These field screenings showed promising results for drought tolerance. However, additional studies are needed in further crop seasons and other sites to better characterize how these plants may outperform the WT under field water deficit.

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.

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.

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.

Phenotyping soybean plants transformed with rd29A:AtDREB1A for drought tolerance in the greenhouse and field.

The development of drought tolerant plants is a high priority because the area suffering from drought is expected to increase in the future due to global warming. One strategy for the development of drought tolerance is to genetically engineer plants with transcription factors (TFs) that regulate the expression of several genes related to abiotic stress defense responses. This work assessed the performance of soybean plants overexpressing the TF DREB1A under drought conditions in the field and in the greenhouse. Drought was simulated in the greenhouse by progressively drying the soil of pot cultures of the P58 and P1142 lines. In the field, the performance of the P58 line and of 09D-0077, a cross between the cultivars BR16 and P58, was evaluated under four different water regimes: irrigation, natural drought (no irrigation) and water stress created using rain-out shelters in the vegetative or reproductive stages. Although the dehydration-responsive element-binding protein (DREB) plants did not outperform the cultivar BR16 in terms of yield, some yield components were increased when drought was introduced during the vegetative stage, such as the number of seeds, the number of pods with seeds and the total number of pods. The greenhouse data suggest that the higher survival rates of DREB plants are because of lower water use due to lower transpiration rates under well watered conditions. Further studies are needed to better characterize the soil and atmospheric conditions under which these plants may outperform the non-transformed parental plants.

Introduction of the rd29A:AtDREB2A CA gene into soybean (Glycine max L. Merril) and its molecular characterization in leaves and roots during dehydration.

The loss of soybean yield to Brazilian producers because of a water deficit in the 2011-2012 season was 12.9%. To reduce such losses, molecular biology techniques, including plant transformation, can be used to insert genes of interest into conventional soybean cultivars to produce lines that are more tolerant to drought. The abscisic acid (ABA)-independent Dehydration Responsive Element Binding (DREB) gene family has been used to obtain plants with increased tolerance to abiotic stresses. In the present study, the rd29A:AtDREB2A CA gene from Arabidopsis thaliana was inserted into soybean using biolistics. Seventy-eight genetically modified (GM) soybean lines containing 2-17 copies of the AtDREB2A CA gene were produced. Two GM soybean lines (P1397 and P2193) were analyzed to assess the differential expression of the AtDREB2A CA transgene in leaves and roots submitted to various dehydration treatments. Both GM lines exhibited high expression of the transgene, with the roots of P2193 showing the highest expression levels during water deficit. Physiological parameters examined during water deficit confirmed the induction of stress. This analysis of AtDREB2A CA expression in GM soybean indicated that line P2193 had the greatest stability and highest expression in roots during water deficit-induced stress.