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Urease catalyzes the hydrolysis of urea to ammonia and carbon dioxide. The ammonia (nitrogen (N) product of urease activity) is incorporated into organic compounds. Thus, urease is involved in N remobilization, as well as in primary N assimilation. Two urease isoforms have been described for soybean: the embryo-specific, encoded by the Eu1 gene, and the ubiquitous urease, encoded by Eu4. A third urease-encoding gene was recently identified, designated Eu5, which encodes the putative protein product SBU-III. The present study aimed to evaluate the contribution of soybean ureases to seed germination and plant development. Analyses were performed using Eu1/Eu4/Eu5-co-suppressed transgenic plants and mutants of the Eu1 and Eu4 urease structural genes, as well as a urease-null mutant (eu3-a) that activates neither the ubiquitous nor embryo-specific ureases. The co-suppressed plants presented a developmental delay during the first month after germination; shoots and roots were significantly smaller and lighter. Slower development was observed for the double eu1-a/eu4-a mutant and the eu3-a single mutant. The N content in transgenic plants was significantly lower than in non-transgenic plants. Among the mutants, eu3-a presented the lowest and eu1-a the highest N content. Altogether, these results indicate that increased ureolytic activity plays an important role in plant development.

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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.

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Flooding is a frequent environmental stress that reduces soybean (Glycine max) 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 (Oryza sativa), 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.

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