Norman Neumaier

Drought Tolerance Characteristics of Brazilian Soybean Cultivars

Abstract Drought is one of the major constraints for soybean production in Brazil. Seed yield of ten Brazilian soybean cultivarssheltered from rain (drought stress) for one month after the first flowering was examined over two growing seasons in the field in Londrina, Brazil. The drought tolerance on the basis of seed yield varied with the cultivar, and the yield ranking among cultivars was nearly the same across two years. In cultivars with higher drought tolerance, crop growth rate (CGR)during the drought stress period was higher than in other cultivars. They also maintained a larger leaf area during the stress period. Although reproductive development was retarded by the drought stress, it tended to be retarded less in drought-tolerant cultivars. The information obtained in this research may be useful for breeding drought-tolerant cultivars or selecting diverse germplasms of soybean cultivars.

Modeling nitrogen accumulation and use by soybean

Abstract Simulation of soybean growth and yield requires an accurate representation of nitrogen accumulation and distribution in the developing crop. Approaches to simulate nitrogen accumulation by soybean have been complicated by the need to account for both soil nitrate uptake and symbiotic nitrogen fixation. Past approaches to simulating soybean have generally been empirical requiring ‘calibration’ for each new environment. Recently, Jamieson and Semonov [Field Crops Res. 68 (2000) 21] proposed a relatively simple approach for simulating nitrogen accumulation by wheat based on the nitrogen demand imposed separately by the development of leaf area and the growth of stems. This paper assesses this approach for soybean. This approach was further simplified by assuming that nitrogen availability to the plant was limited only by soil moisture conditions and a maximum uptake rate and that it was not necessary to distinguish between nitrate uptake and symbiotic nitrogen fixation. The simplified model generally resulted in simulations that corresponded to experimental observations on nitrogen and mass accumulation through the growing season, and on yield over a number of years. Sensitivity analysis of the model indicated that storage of nitrogen in the stem might be a key trait in increasing soybean yield potential.

Soybean physiology and gene expression during drought.

Soybean genotypes MG/BR46 (Conquista) and BR16, drought-tolerant and -sensitive, respectively, were compared in terms of morphophysiological and gene-expression responses to water stress during two stages of development. Gene-expression analysis showed differential responses in Gmdreb1a and Gmpip1b mRNA expression within 30 days of water-deficit initiation in MG/BR46 (Conquista) plants. Within 45 days of initiating stress, Gmp5cs and Gmpip1b had relatively higher expression. Initially, BR16 showed increased expression only for Gmdreb1a, and later (45 days) for Gmp5cs, Gmdefensin and Gmpip1b. Only BR16 presented down-regulated expression of genes, such as Gmp5cs and Gmpip1b, 30 days after the onset of moisture stress, and Gmgols after 45 days of stress. The faster perception of water stress in MG/BR46 (Conquista) and the better maintenance of up-regulated gene expression than in the sensitive BR16 genotype imply mechanisms by which the former is better adapted to tolerate moisture deficiency.

Expression patterns of GmAP2/EREB-like transcription factors involved in soybean responses to water deficit.

Soybean farming has faced several losses in productivity due to drought events in the last few decades. However, plants have molecular mechanisms to prevent and protect against water deficit injuries, and transcription factors play an important role in triggering different defense mechanisms. Understanding the expression patterns of transcription factors in response to water deficit and to environmental diurnal changes is very important for unveiling water deficit stress tolerance mechanisms. Here, we analyzed the expression patterns of ten APETALA2/Ethylene Responsive Element Binding-like (AP2/EREB-like) transcription factors in two soybean genotypes (BR16: drought-sensitive; and Embrapa 48: drought-tolerant). According to phylogenetic and domain analyses, these genes can be included in the DREB and ERF subfamilies. We also analyzed a GmDRIP-like gene that encodes a DREB negative regulator. We detected the up-regulation of 9 GmAP2/EREB-like genes and identified transcriptional differences that were dependent on the levels of the stress applied and the tissue type analyzed (the expression of the GmDREB1F-like gene, for example, was four times higher in roots than in leaves). The GmDRIP-like gene was not induced by water deficit in BR16 during the longest periods of stress, but was significantly induced in Embrapa 48; this suggests a possible genetic/molecular difference between the responses of these cultivars to water deficit stress. Additionally, RNAseq gene expression analysis over a 24-h time course indicates that the expression patterns of several GmDREB-like genes are subject to oscillation over the course of the day, indicating a possible circadian regulation.

Subtractive libraries for prospecting differentially expressed genes in the soybean under water deficit

Soybean has a wide range of applications in the industry and, due to its crop potential, its improvement is widely desirable. During drought conditions, soybean crops suffer significant losses in productivity. Therefore, understanding the responses of the soybean under this stress is an effective way of targeting crop improvement techniques. In this study, we employed the Suppressive Subtractive Hybridization (SSH) technique to investigate differentially expressed genes under water deficit conditions. Embrapa 48 and BR 16 soybean lines, known as drought-tolerant and -sensitive, respectively, were grown hydroponically and subjected to different short-term periods of stress by withholding the nutrient solution. Using this approach, we have identified genes expressed during the early response to water deficit in roots and leaves. These genes were compared among the lines to assess probable differences in the plant transcriptomes. In general, similar biochemical processes were predominant in both cultivars; however, there were more considerable differences between roots and leaves of Embrapa 48. Moreover, we present here a fast, clean and straightforward method to obtain drought-stressed root tissues and a large enriched collection of transcripts expressed by soybean plants under water deficit that can be useful for further studies towards the understanding of plant responses to stress.

Transcription factors expressed in soybean roots under drought stress

To gain insight into stress-responsive gene regulation in soybean plants, we identified consensus sequences that could categorize the transcription factors MYBJ7, BZIP50, C2H2, and NAC2 as members of the gene families myb, bzip, c2h2, and nac, respectively. We also investigated the evolutionary relationship of these transcription factors and analyzed their expression levels under drought stress. The NCBI software was used to find the predicted amino acid sequences of the transcription factors, and the Clustal X software was used to align soybean and other plant species sequences. Phylogenetic trees were built using the Mega 4.1 software by neighbor joining and the degree of confidence test by Bootstrap. Expression level studies were carried out using hydroponic culture; the experiments were designed in completely randomized blocks with three repetitions. The blocks consisted of two genotypes, MG/BR46 Conquista (drought-tolerant) and BR16 (drought-sensitive) and the treatments consisted of increasingly long dehydration periods (0, 25, 50, 75, and 100 min). The transcription factors presented domains and/or conserved regions that characterized them as belonging to the bzip, c2h2, myb, and nac families. Based on the phylogenetic trees, it was found that the myb, bzip and nac genes are closely related to myb78, bzip48 and nac2 of soybean and that c2h2 is closely related to c2h2 of Brassica napus. Expression of all genes was in general increased under drought stress in both genotypes. Major differences between genotypes were due to the lowering of the expression of the mybj7 and c2h2 genes in the drought-tolerant variety at some times. Over-expression or silencing of some of these genes has the potential to increase stress tolerance.

Overexpression of the activated form of the AtAREB1 gene (AtAREB1 Delta QT) improves soybean responses to water deficit

Abscisic acid-responsive element binding protein (AREB1) is a basic domain/leucine zipper transcription factor that binds to the abscisic acid (ABA)-responsive element motif in the promoter region of ABA-inducible genes. Because AREB1 is not sufficient to direct the expression of downstream genes under non-stress conditions, an activated form of AREB1 (AREB1ΔQT) was created. Several reports claim that plants overexpressing AREB1 or AREB1ΔQT show improved drought tolerance. In our studies, soybean plants overexpressing AREB1ΔQT were characterized molecularly, and the phenotype and drought response of three lines were accessed under greenhouse conditions. Under conditions of water deficit, the transformed plants presented a higher survival rate (100%) than those of their isoline, cultivar BR 16 (40%). Moreover, the transformed plants displayed better water use efficiency and had a higher number of leaves than their isoline. Because the transgenic plants had higher stomatal conductance than its isoline under well-watered conditions, it was suggested that the enhanced drought response of AREB1ΔQT soybean plants might not be associated with altered transpiration rates mediated by ABA-dependent stomatal closure. However, it is possible that the smaller leaf area of the transgenic plants reduced their transpiration and water use, causing delayed stress onset. The difference in the degree of wilting and percentage of survival between the 35S-AREB1ΔQT and wildtype plants may also be related to the regulation of genes that protect against dehydration because metabolic impairment of photosynthesis, deduced by an increasing internal CO2 concentration, was not observed in the transgenic plants.

Cloning and quantitative expression analysis of drought-induced genes in soybean.

We determined the expression levels of DREB transcription factor (Gmdreb1) and of the genes Gmgols, Gmpip1b, Gmereb, and Gmdefensin in drought-tolerant (MG/BR46-Conquista) and drought-sensitive (BR16) genotypes of soybean, during drought. The trial was carried out in a controlled-environment chamber, set up to provide drought conditions. Sequences of Arabidopsis thaliana DREB-family proteins were used to build a phylogenetic tree through the alignment of the conserved regions near the AP2 domain. We found that Gmdreb1 is similar to Atrap2.1, which is located near the AtDREB1 and AtDREB2 families. The amplified fragment was cloned and sequenced; alignment with the sequence available at Genbank showed total similarity. Expression analysis showed that under drought: a) Gmdreb1 expression increased in leaves and roots of both genotypes and expression level changes occurred that were correlated with the length of the water-deficit period; b) there were increased expression levels of Gmdefensin in roots of MG/BR46; c) expression of Gmgols increased in leaves and roots of the two genotypes; d) Gmpip1b expression generally increased, except in roots of BR16, and e) the same was found for Gmereb, except in roots of MG/BR46.

Morpho-anatomical and micromorphometrical evaluations in soybean genotypes during water stress.

In a greenhouse experiment, morpho-anatomical and micromorphometrical analyses of two soybean cultivars, MG/BR46 (Conquista) and BR16-tolerant and sensitive to drought, respectively-were used to study their water-deficit-tolerance strategies. Drought treatments were applied at reproductive stages from R2 to R7, where evaluations were conducted at 30 days and 45 days after stress started, respectively. The total length of Conquista plants (shoot + root) was greater than of BR16 plants. Pod dry weight was adversely affected due to the lack of moisture, decreasing productivity even of Conquista plants. Both the cultivars had normal development of root hairs; however, it was observed a decrease in the cortex:central cylinder ratio in BR16 stressed for 30 days, and they also showed similar leaflet thickness and stomata distribution. Differences in drought tolerance observed between the two cultivars seemed to be related to factors other than morphological traits since this species has a short lifecycle.

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.