Abstract 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.

Soybean [Glycine max (L.) Merril], one of the most important crop species in the world, is very susceptible to abiotic and biotic stress. Soybean plants have developed a variety of molecular mechanisms that help them survive stressful conditions. Hybrid proline-rich proteins (HyPRPs) constitute a family of cell-wall proteins with a variable N-terminal domain and conserved C-terminal domain that is phylogenetically related to non-specific lipid transfer proteins. Members of the HyPRP family are involved in basic cellular processes and their expression and activity are modulated by environmental factors. In this study, microarray analysis and real time RT-qPCR were used to identify putative HyPRP genes in the soybean genome and to assess their expression in different plant tissues. Some of the genes were also analyzed by time-course real time RT-qPCR in response to infection by Phakopsora pachyrhizi, the causal agent of Asian soybean rust disease. Our findings indicate that the time of induction of a defense pathway is crucial in triggering the soybean resistance response to P. pachyrhizi. This is the first study to identify the soybean HyPRP group B family and to analyze disease-responsive GmHyPRP during infection by P. pachyrhizi.

Environmental stresses caused by either abiotic or biotic factors greatly affect agriculture. As for soybean [Glycine max (L.) Merril], one of the most important crop species in the world, the situation is not different. In order to deal with these stresses, plants have evolved a variety of sophisticated molecular mechanisms, to which the transcriptional regulation of target-genes by transcription factors is crucial. Even though the involvement of several transcription factor families has been widely reported in stress response, there still is a lot to be uncovered, especially in soybean. Therefore, the objective of this study was to investigate the role of bHLH and trihelix-GT transcription factors in soybean responses to environmental stresses. Gene annotation, data mining for stress response, and phylogenetic analysis of members from both families are presented herein. At least 45 bHLH (from subgroup 25) and 63 trihelix-GT putative genes reside in the soybean genome. Among them, at least 14 bHLH and 11 trihelix-GT seem to be involved in responses to abiotic/biotic stresses. Phylogenetic analysis successfully clustered these with members from other plant species. Nevertheless, bHLH and trihelix-GT genes encompass almost three times more members in soybean than in Arabidopsis or rice, with many of these grouping into new clades with no apparent near orthologs in the other analyzed species. Our results represent an important step towards unraveling the functional roles of plant bHLH and trihelix-GT transcription factors in response to environmental cues.

Transgenic plants represent an invaluable tool for molecular, genetic, biochemical and physiological studies by gene overexpression or silencing, transposon-based mutagenesis, protein sub-cellular localization and/or promoter characterization as well as a breakthrough for breeding programs, allowing the production of novel and genetically diverse genotypes. However, the stable transformation of soybean cannot yet be considered to be routine because it depends on the ability to combine efficient transformation and regeneration techniques. Two methods have been used with relative success to produce completely and stably transformed plants: particle bombardment and the Agrobacterium tumefaciens system. In addition, transformation by Agrobacterium rhizogenes has been used as a powerful tool for functional studies. Most available information on gene function is based on heterologous expression systems. However, as the activity of many promoters or proteins frequently depends on specific interactions that only occur in homologous backgrounds, a final confirmation based on a homologous expression system is desirable. With respect to soybean biotech improvement, transgenic lines with agronomical, nutritional and pharmaceutical traits have been obtained, including herbicide-tolerant soybeans, which represented the principal biotech crop in 2011, occupying 47% of the global biotech area.

O objetivo deste trabalho foi identificar cultivares brasileiras de soja (Glycine max) com capacidade de resposta aos estímulos da cultura in vitro para a indução de embriões somáticos primários, proliferação de embriões secundários e regeneração de plantas. Foram observadas diferenças para cada estádio entre as oito cultivares testadas. Foram selecionadas duas cultivares, 'IAS-5' e BRSMG 68 Vencedora, para avaliação do potencial dos embriões quanto à diferenciação e conversão em plantas. Essas cultivares tiveram altas taxas de indução, proliferação embriogênica repetitiva e baixa germinação precoce dos embriões, no experimento inicial. Foi investigado o efeito da adição de ácido abscísico (ABA) e carvão sobre a regeneração. Os resultados mostraram que a adição de ABA ao meio de proliferação e de ABA e carvão ativado ao meio de maturação aumentaram as taxas de diferenciação dos embriões, o que resultou em maior número de plantas regeneradas. A cultivar BRSMG 68 Vencedora foi identificada como genótipo com alto potencial para indução e proliferação de embriões, bem como para regeneração de plantas. O potencial dessa cultivar quanto à embriogênese somática foi similar ao observado para a cultivar IAS-5, atualmente utilizada para transformação de soja no Brasil. A BRSMG 68 Vencedora pode ser um genótipo alternativo para a engenharia genética de soja via protocolos de embriogênese somática.

The aim of this work was to identify Brazilian soybean (Glycine max) genotypes with potential to respond to in vitro culture stimuli for primary somatic embryo induction, secondary embryo proliferation and plant regeneration. Differences among eight tested cultivars were observed at each stage. Two cultivars, IAS-5 and BRSMG 68 Vencedora, were selected for the evaluation of the capacity for embryo differentiation and plant regeneration. These cultivars had high embryo induction frequencies, repetitive embryogenic proliferation, and low precocious embryo germination in the initial experiment. The effect of abscisic acid (ABA) and charcoal addition on plant regeneration was investigated. The addition of ABA to proliferation medium and of ABA and activated charcoal to maturation medium increased embryo differentiation rates, which resulted in a higher number of regenerated plants. The BRSMG 68 Vencedora cultivar was found to have a high potential for embryo induction, embryo proliferation and plant regeneration. The potential of this cultivar for somatic embryogenesis was similar to that observed for cultivar IAS-5, which is currently used for soybean transformation in Brazil. BRSMG 68 Vencedora may be a good alternative genotype for soybean genetic engineering via somatic embryogenesis protocols.

Foi avaliada a influência de diferentes tratamentos com antibióticos durante a transformação genética de soja. Inicialmente, desenvolveu-se um estudo para identificar como diferentes tratamentos com antibióticos afetam o tecido embriogênico da soja. O efeito da carbenicilina a 500 mg L-1 foi genótipo-dependente. Esse antibiótico não afetou a sobrevivência dos embriões da cv. IAS5, enquanto a proliferação dos embriões da cv. Bragg foi três vezes maior quando comparada com o controle. Por outro lado, cefotaxima, a 350 e 500 mg L-1, causou a morte dos tecidos embriogênicos de ambas as cultivares. Por fim, a associação de cefotaxima (250 mg L-1) + vancomicina (250 mg L-1) não afetou negativamente os embriões somáticos das cultivares testadas durante os 63 dias de tratamento. Posteriormente, foi realizado um segundo experimento para determinar a eficiência de diferentes tratamentos com antibióticos na supressão da linhagem LBA4404 de Agrobacterium tumefaciens durante a transformação genética. Nas condições da cultura, a carbenicilina, a 500 e 1000 mg L-1, não foi eficiente para a supressão de Agrobacterium. Por outro lado, os tratamentos com cefotaxima, 350 e 500 mg L-1, ou cefotaxima + vancomicina suprimiram eficientemente Agrobacterium após 49 dias. Os dados dos dois experimentos sugerem a cefotaxima + vancomicina por 49-63 dias como o tratamento mais apropriado. Esse é o primeiro registro do efeito de antibióticos sobre tecidos de soja. Foi identificada uma combinação de antibióticos que suprimiram A. tumefaciens com efeitos fitotóxicos mínimos, podendo ser recomendada para a otimização do método de transformação genética de soja mediada por Agrobacterium.

The influence of different antibiotic treatments in soybean genetic transformation was evaluated. First, an assay was performed to verify how different antibiotic treatments affect soybean embryogenic tissues. The effect of carbenicillin at 500 mg L-1 was genotype-dependent. This antibiotic did not affect embryo survival of cv. IAS5, but a three-fold increase of embryo proliferation was observed for cv. Bragg, when compared to the control. On the other hand, cefotaxime at 350 and 500 mg L-1 caused death of embryogenic tissues of both cultivars. Finally, the association of cefotaxime (250 mg L-1) + vancomycin (250 mg L-1) did not affect negatively the somatic embryos of tested cultivars until 63 days of treatment. Thereafter, a second experiment was carried out to determine the efficacy of different antibiotic treatments in suppressing LBA4404 Agrobacterium tumefaciens strain in genetic transformation. On tissue culture conditions, carbenicillin at 500 and 1000 mg L-1 was not active against Agrobacterium. On the other hand, treatments with cefotaxime at 350 and 500 mg L-1, and cefotaxime + vancomycin efficiently suppressed Agrobacterium during 49 days. Data of both experiments suggested cefotaxime + vancomycin for 49-63 days as the most appropriate treatment. This is the first work reporting the effect of antibiotics on soybean tissues. By identifying an antibiotic combination that suppressed A. tumefaciens with minimal phytotoxic effects, we are able to recommend it for improvement of soybean Agrobacterium-mediated transformation procedure.