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Background Small 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. Results The 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. Conclusions Validation 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.

The expanding cultivation of grains to meet agro-industry demands, the implementation of more efficient, sustainable, and integrative management practices capable of mitigating the selection of resistant pests and weeds becomes necessary. Due to the intensive application of chemical herbicides, in addition to environmental impacts, led to the development of resistance mechanisms in weed populations, such as sourgrass ( Digitaria insularis ). These mechanisms complicate management efforts, escalate production costs, and diminish productivity. In this context, RNA interference (RNAi) technology has emerged as a promising molecular tool for the targeted control of weeds, owing to its specificity in the post-transcriptional silencing of vital genes. This study investigated the application of RNAi technology for the suppression of 5-enolpyruvylshikimate-3-phosphate synthase ( EPSPS ) gene expression in D. insularis , a weed species of significant agronomic importance in Latin America. A double-stranded RNA (dsRNA) sequence, specifically designed for the EPSPS gene, was synthesized via bacterial fermentation with a strain of E. coli HT115, extracted using TRIzol™ and purified with phenol: chloroform: isoamyl alcohol, and applied topically in D. insularis leaves. The subsequent phenotypic and molecular effects were evaluated. The spray application of the dsRNA resulted in a 44% reduction in the plant's shoot dry mass and a 75% reduction in the number of tillers, thereby indicating consistent physiological impacts due to gene silencing. Quantitative reverse transcription PCR (qRT-PCR) analysis confirmed a significant suppression of EPSPS transcript levels following treatment, suggesting partial gene silencing. These findings collectively demonstrate the efficacy of RNAi in modulating gene expression in D. insularis , thereby underscoring its potential as a sustainable biotechnological strategy for the development of novel weed control methodologies.

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 (1Ab58), (1Bb2193), and (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.

Soybean (Glycine max) is one of the major crops worldwide and flooding stress affects the production and expansion of cultivated areas. Oxygen is essential for mitochondrial aerobic respiration to supply the energy demand of plant cells. Because oxygen diffusion in water is 10,000 times lower than in air, partial (hypoxic) or total (anoxic) oxygen deficiency is important component of flooding. Even when oxygen is externally available, oxygen deficiency frequently occurs in bulky, dense or metabolically active tissues such as phloem, meristems, seeds, and fruits. In this study, we analyzed conserved and divergent root transcriptional responses between flood-tolerant Embrapa 45 and flood-sensitive BR 4 soybean cultivars under hypoxic stress conditions with RNA-seq. To understand how soybean genes evolve and respond to hypoxia, stable and differentially expressed genes were characterized structurally and compositionally comparing its mechanistic relationship. Between cultivars, Embrapa 45 showed less up- and more down-regulated genes, and stronger induction of phosphoglucomutase (Glyma05g34790), unknown protein related to N-terminal protein myristoylation (Glyma06g03430), protein suppressor of phyA-105 (Glyma06g37080), and fibrillin (Glyma10g32620). RNA-seq and qRT-PCR analysis of non-symbiotic hemoglobin (Glyma11g12980) indicated divergence in gene structure between cultivars. Transcriptional changes for genes in amino acids and derivative metabolic process suggest involvement of amino acids metabolism in tRNA modifications, translation accuracy/efficiency, and endoplasmic reticulum stress in both cultivars under hypoxia. Gene groups differed in promoter TATA box, ABREs (ABA-responsive elements), and CRT/DREs (C-repeat/dehydration-responsive elements) frequency. Gene groups also differed in structure, composition, and codon usage, indicating biological significances. Additional data suggests that cis-acting ABRE elements can mediate gene expression independent of ABA in soybean roots under hypoxia.

Soybean is one of the most common grain crops worldwide, representing an important protein and oil source. Although genetic variability in the chemical composition of grains is seen in soybean, the mean levels of proteins have remained stagnant or, in some cases, have decreased over time, arousing concern in the agricultural industry. Furthermore, environmental conditions influence the chemical composition of grains. Thus, the present study evaluated the effect of water deficit (WD) induced at the vegetative period (vegetative stress (VS)) and reproductive period (reproductive stress (RS)) on the protein and oil contents of grains in different soybean genotypes. Yield and its components were evaluated to evaluate the interrelation of these traits. The experiment was completed over three crop seasons under field conditions in Londrina, Paraná (PR), Brazil. WD was induced using rainout shelters and then stress treatments with irrigated and non-irrigated conditions were compared. WD negatively affected yield and its components. All evaluated genotypes showed similar responses for oil and protein contents under different water conditions. Higher protein content and lower oil content were observed in grains under RS. Such a relationship was not equally established under VS. Additionally, negative relationships between protein and oil content and between protein content and yield were confirmed.

The yield and protein content of soybean seeds are challenging traits for breeding since they have a negative correlation. To solve this issue, it is essential to understand the protein transport in soybean seeds. Here we performed an analysis of 3 datasets containing transcriptional data from soybean seeds and pods, without any treatment, aiming to select genes related to the transport of organic nitrogen compounds. We identified 69 transporters, and among them, seven were chosen to be validated by qRT-PCR. Seeds and pods of soybean cultivars BRS 232 and BRS 284, respectively, presenting higher and lower protein content, were assayed under normal growth condition. Results showed in BRS 232 soybean cultivar, a positive correlation between seed protein content and gene expression for five out of the seven genes analyzed. These nitrogen compounds transporters can play an essential role in the storage of proteins, thus increases the protein content of soybean seeds and contributes to the decrease the negative correlation between yield and protein content of soybean seeds.

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 ( ) was identified in 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 ( ) remains unknown. Here, we investigate the family members in soybean and functionally characterize a homologous gene. The constitutive overexpression of delayed flowering in Arabidopsis, showing the functional conservation among plants as part of the flowering control machinery. We also show that alters the expression of Arabidopsis key flowering time genes ( and ), and this down-regulation is the likely cause of flowering delay phenotypes. Furthermore, we identified the network genes to infer the participation of 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.

Soybean is the primary source of vegetable protein and is used for various purposes, mainly to feed animals. This crop can have diverse seed coat colors, varying from yellow, black, brown, and green to bicolor. Black seed coat cultivars have already been assigned as favorable for both seed and grain production. Thus, this work aimed to identify genes associated with soybean seed quality by comparing the transcriptomes of soybean seeds with contrasting seed coat colors. The results from RNA-seq analyses were validated with real-time PCR using the cultivar BRS 715A (black seed coat) and the cultivars BRS 413 RR and DM 6563 IPRO (yellow seed coat). We found 318 genes differentially expressed in all cultivars (freshly harvested seeds and seeds stored in cold chamber). From the in silico analysis of the transcriptomes, the following genes were selected and validated with RT-qPCR: ACS1, ACSF3, CYP90A1, CYP710A1, HCT, CBL, and SAHH. These genes are genes induced in the black seed coat cultivar and are part of pathways responsible for ethylene, lipid, brassinosteroid, lignin, and sulfur amino acid biosynthesis. The BRSMG 715A gene has almost 4times more lignin than the yellow seed coat cultivars. These attributes are related to the BRSMG 715A cultivar’s higher seed quality, which translates to more longevity and resistance to moisture and mechanical damage. Future silencing studies may evaluate the knockout of these genes to better understand the biology of soybean seeds with black seed coat.

The present study aimed to identify the relationship between lignin content in soybean pods, pod dehiscence rate, gene expression from phenylpropanoid pathway, cell wall lignification, and deterioration of seed due to weather. To evaluate the weathering deterioration, an experiment simulating 150 mm of rainfall was performed in greenhouse conditions. Gene expression was assessed by RT-qPCR. Results showed that soybean cultivars BRS Jiripoca and BRS 388 RR considered tolerant to weathering deterioration, presented higher levels of lignin content in pods. The data showed that dehiscence rate was neither a trait involved in weathering deterioration tolerance nor in lignin content in soybean pods. Lignin content and weathering deterioration were highly negatively correlated, probably due to changes in cell wall permeability to water accordingly to lignin level. Higher expression levels of genes involved in lignin biosynthesis and cell wall lignification were identified in both tolerant cultivars, however, with opposite expression profiles and in different developmental phases, suggesting a possible temporal strategy to cope with environmental adverse conditions.

Background Despite the importance of osmoprotectants, no previous in silico evaluation of high throughput data is available for higher plants. The present approach aimed at the identification and annotation of osmoprotectant-related sequences applied to short transcripts from a soybean HT-SuperSAGE (High Throughput Super Serial Analysis of Gene Expression; 26-bp tags) database, and also its comparison with other transcriptomic and genomic data available from different sources. Methods A curated set of osmoprotectants related sequences was generated using text mining and selected seed sequences for identification of the respective transcripts and proteins in higher plants. To test the efficiency of the seed sequences, these were aligned against four HT-SuperSAGE contrasting libraries generated by our group using soybean tolerant and sensible plants against water deficit, considering only differentially expressed transcripts (p ≤ 0.05). Identified transcripts from soybean and their respective tags were aligned and anchored against the soybean virtual genome. Results The workflow applied resulted in a set including 1,996 seed sequences that allowed the identification of 36 differentially expressed genes related to the biosynthesis of osmoprotectants [Proline ( P5CS : 4, P5CR : 2), Trehalose ( TPS1 : 9, TPPB : 1), Glycine betaine ( BADH : 4) and Myo- inositol ( MIPS : 7, INPS1 : 8)], also mapped in silico in the soybean genome (25 loci). Another approach considered matches using Arabidopsis full length sequences as seed sequences, and allowed the identification of 124 osmoprotectant-related sequences, matching ~10.500 tags anchored in the soybean virtual chromosomes. Osmoprotectant-related genes appeared clustered in all soybean chromosomes, with higher density in some subterminal regions and synteny among some chromosome pairs. Conclusions Soybean presents all searched osmoprotectant categories with some important members differentially expressed among the comparisons considered (drought tolerant or sensible vs . control; tolerant vs . sensible), allowing the identification of interesting candidates for biotechnological inferences. The identified tags aligned to corresponding genes that matched 19 soybean chromosomes. Osmoprotectant-related genes are not regularly distributed in the soybean genome, but clustered in some regions near the chromosome terminals, with some redundant clusters in different chromosomes indicating their involvement in previous duplication and rearrangements events. The seed sequences, transcripts and map represent the first transversal evaluation for osmoprotectant-related genes and may be easily applied to other plants of interest.