Liliane Marcia Mertz-Henning

Characterization of Molecular and Physiological Responses Under Water Deficit of Genetically Modified Soybean Plants Overexpressing the AtAREB1 Transcription Factor

Drought is one of the major factors limiting crop productivity worldwide. Currently, the techniques of genetic engineering are powerful tools for the development of drought-tolerant plants, once they allow for the modification of expression patterns of genes responsive to drought. Within this context, transcription factors recognize specific DNA sequences in the regulatory region of target genes, and thereby regulate their expression. AREB is a transcription factor in the basic leucine zipper family, which binds to the ABRE element in the promoter region of genes induced by abscisic acid and drought. In this study, soybean plants transformed with the 35S:AtAREB1 construct were submitted to drought under greenhouse conditions. AtAREB1 expression was observed in the transgenic lines 1Ea2939 and 1Eb2889, but not in the event 1Ea15 and, under control of the CaMV 35S promoter, did not cause dwarfism and resulted in a higher survival rate of transformed plants after drought and rehydration. Moreover, 1Ea2939 and 1Eb2889 plants presented a greater total number of pods and seeds and increased dry matter content of seeds. The best performance of the transgenic lines 1Ea2939 and 1Eb2889 relative to BR 16 plants (wild type) and to event 1Ea15 might be related to mechanisms of drought prevention through reduced stomatal conductance and leaf transpiration under control conditions. Changes in the expression profile of phosphatases and kinases may also be involved. Such results suggest that the constitutive overexpression of the transcription factor AtAREB1 leads to an improved capacity of the soybean crop to cope with drought with no yield losses.

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.

Molecular, physiological, and agronomical characterization, in greenhouse and in field conditions, of soybean plants genetically modified with AtGolS2 gene for drought tolerance

Water deficit may occur at any stage of crop development, affecting productivity and causing economic losses. In response to drought, raffinose family oligosaccharides (RFOs) are accumulated in plant tissues stabilizing and protecting cell membranes and keeping the vital functions. The enzyme galactinol synthase (GolS, EC 2.4.1.123) catalyzes the first step in the biosynthesis of RFOs. In our study, soybean events overexpressing 35S:AtGolS2 were molecularly, physiological, and agronomical characterized, under drought simulated in greenhouse and in field conditions during the crop season 2014/2015. The conventional soybean cultivar BRS 184 was transformed and five positive events were obtained. Four events transmitted the transgene to further generations and in the events 2Ia1 and 2Ia4, two to four copies of AtGols2 gene were observed. Results in greenhouse showed that the overexpression of AtGolS2 in genetically modified (GM) plants led to increased galactinol transcripts, probably resulting in changes in carbohydrate metabolism. Accumulation of these transcripts that may have acted as osmoprotectors, lead to higher drought tolerance and survival rate of 2Ia4 plants. In addition, in field conditions, higher yield was observed for 2Ia4 plants under irrigated (IRR) and non-irrigated (NIRR) treatments. This result can be due to the increased synthesis of RFOs even under well-watered conditions. This field screening showed promising results for drought tolerance, suggesting that 2Ia4 plants may be useful in a breeding program for the development of drought-tolerant plants. However, additional studies are needed in further crop seasons and other sites to better characterize how these plants may outperform the WT plants under water deficit.

Insights into soybean transcriptome reconfiguration under hypoxic stress: Functional, regulatory, structural, and compositional characterization.

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.

Application of Biotechnology to Generate Drought-Tolerant Soybean Plants in Brazil: Development of Genetic Engineering Technology of Crops with Stress Tolerance Against Degradation of Global Environment

Brazil is the second largest soybean-producing country, but yields have recently been unstable because of droughts. The objective of this project was to develop drought-tolerant soybean lines based on information from earlier molecular studies involving model plants. We also searched the soybean genome for genes conferring drought tolerance and elucidated the mechanisms regulating the identified genes. Based on our findings, we generated new soybean lines, which were then evaluated under greenhouse and field conditions to identify the most drought-tolerant lines. We analyzed the functions of drought tolerance genes in Arabidopsis thaliana and identified soybean genes exhibiting similar properties. We also comprehensively investigated soybean gene expression levels in stressed plants. Additionally, we determined the best combinations of drought tolerance genes and promoters and introduced these combinations into soybean cells using biolistic and Agrobacterium tumefaciens-based methods. We evaluated the stress tolerance of the resulting transgenic plants in a greenhouse and in the field and observed that some transgenic soybean lines exhibited increased drought tolerance. We developed a new technique for generating genetically modified soybean lines that are more tolerant to environmental stresses such as drought. These lines may be useful for mitigating the effects of climate changes. The developed technique and generated transgenic soybean lines may help stabilize or increase soybean production in Brazil.

Integrating High‐Resolution and Solid‐State Magic Angle Spinning NMR Spectroscopy and a Transcriptomic Analysis of Soybean Tissues in Response to Water Deficiency

INTRODUCTION Solid-state NMR (SSNMR) spectroscopy methods provide chemical environment and ultrastructural details that are not easily accessible by other non-destructive, high-resolution spectral techniques. High-resolution magic angle spinning (HR-MAS) has been widely used to obtain the metabolic profile of a heterogeneous sample, combining the resolution enhancement provided by MAS in SSNMR with the shimming and locking procedures in liquid-state NMR. OBJECTIVE In this work, we explored the feasibility of using the HR-MAS and SSNMR techniques to identify metabolic changes in soybean leaves subjected to water-deficient conditions. METHODOLOGY Control and water-deficient soybean leaves were analysed using one-dimensional (1D) HR-MAS and SSNMR. Total RNA was extracted from the leaves for the transcriptomic analysis. RESULTS The 1 H HR-MAS and CP-MAS 13 C{1 H} spectra of soybean leaves grown with and without water deficiency stress revealed striking differences in metabolites. A total of 30 metabolites were identified, and the impact of water deficiency on the metabolite profile of soybean leaves was to induce amino acid synthesis. High expression levels of genes required for amino acid biosynthesis were highly correlated with the compounds identified by 1 H HR-MAS. CONCLUSIONS The integration of the 1 H HR-MAS and SSNMR spectra with the transcriptomic data provided a complete picture of the major changes in the metabolic profile of soybeans in response to water deficiency. Copyright