Overexpression of AtNCED3 gene improved drought tolerance in soybean in greenhouse and field conditions.

Water deficit is an important climatic problem that can impair agriculture yield and economy. Genetically modified soybean plants containing the AtNCED3 gene were obtained aiming drought-tolerance improvement. The NCED3 gene encodes a 9-cis-epoxycarotenoid dioxygenase (NCED, EC 1.13.11.51), an important enzyme in abscisic acid biosynthesis. ABA activates the expression of drought-responsive genes, in water-deficit conditions, targeting defense mechanisms and enabling plants to survive under low water availability. Results from greenhouse experiments showed that the transgene AtNCED3 and the endogenous genes GmAREB1, GmPP2C, GmSnRK2 and GmAAO3 presented higher expression under water deficit (WD) in the event 2Ha11 than in WT-plants. No significant correlation was observed between the plant materials and WD conditions for growth parameters; however, gas exchange measurements decreased in the GM event, which also showed 80% higher intrinsic water use when compared to WT plants. In crop season 2015/16, event 2Ha11 showed higher total number of pods, higher number of pods with seeds and yield than WT plants. ABA concentration was also higher in GM plants under WD. These results obtained in field screenings suggest that AtNCED3 soybean plants might outperform under drought, reducing economic and yield losses, thus being a good candidate line to be incorporated in the soybean-breeding program to develop drought-tolerant cultivars.

Expression of the CCCH-tandem zinc finger protein gene OsTZF5 under a stress-inducible promoter mitigates the effect of drought stress on rice grain yield under field conditions.

Increasing drought resistance without sacrificing grain yield remains an ongoing challenge in crop improvement. In this study, we report that Oryza sativa CCCH-tandem zinc finger protein 5 (OsTZF5) can confer drought resistance and increase grain yield in transgenic rice plants. Expression of OsTZF5 was induced by abscisic acid, dehydration and cold stress. Upon stress, OsTZF5-GFP localized to the cytoplasm and cytoplasmic foci. Transgenic rice plants overexpressing OsTZF5 under the constitutive maize ubiquitin promoter exhibited improved survival under drought but also growth retardation. By introducing OsTZF5 behind the stress-responsive OsNAC6 promoter in two commercial upland cultivars, Curinga and NERICA4, we obtained transgenic plants that showed no growth retardation. Moreover, these plants exhibited significantly increased grain yield compared to non-transgenic cultivars in different confined field drought environments. Physiological analysis indicated that OsTZF5 promoted both drought tolerance and drought avoidance. Collectively, our results provide strong evidence that OsTZF5 is a useful biotechnological tool to minimize yield losses in rice grown under drought conditions.

Metabolic alterations in conventional and genetically modified soybean plants with GmDREB2A;2 FL and GmDREB2A;2 CA transcription factors during water deficit.

Water deficit is one of the main abiotic stress that affects plant growth and productivity. The GmDREB2A;2 (Glyma14g06080) gene is an important transcription factor involved in regulating the plants' responses under water deficit. In previous studies, soybean plants overexpressing full-length (GmDREB2A;2 FL) and constitutively active (GmDREB2A;2 CA) forms of the GmDREB2A;2 gene, presented higher tolerance to water deficit when compared with the conventional cultivar BRS 283. Therefore, identifying the changes in metabolite profile in these tolerant genotypes can contribute to the understanding of the metabolic pathways involved in the tolerance mechanism. In this work, the metabolic changes in roots and leaves of genetically modified (GM) soybean plants subjected to water deficit were elucidated by 1H-NMR spectroscopy. Three events were analyzed, one containing the gene in FL form (GmDREB2A;2 FL) and two presenting its CA form (GmDREB2A;2 CA-1 and GmDREB2A;2 CA-2) and compared with the conventional cultivar BRS 283. The results indicated different responses between leaves and roots for all genotypes. Most of these metabolic variations were related to carbohydrate and amino acid pathways. BRS 283 stood out with higher accumulation of amino acids in leaves under water deficit. The results also showed that the events GmDREB2A;2 FL and GmDREB2A;2 CA-1 presented higher concentrations of ß-glucose and fructose in leaves, whereas BRS 283 accumulated more sucrose and pinitol. In roots, the GM events accumulated higher ß-glucose, fructose, asparagine and phenylalanine, when compared with the conventional cultivar. These insights can add information on how the transcription factor (TF) DREB2A acts in soybean plants triggering and controlling a network of complex responses to drought.

Endophytic bacterial microbiome associated with leaves of genetically modified (AtAREB1) and conventional (BR 16) soybean plants.

Plant leaves (phyllosphere) have a great potential for colonization and microbial growth, consisting of a dynamic environment in which several factors can interfere with the microbial population structure. The use of genetically modified (GM) plants has introduced several traits in agriculture, such as the improvement of plant drought tolerance, as observed in the AtAREB1 transcription factor overexpression in soybean (Glycine max L. Merrill). The present study aimed at investigating the taxonomic and functional profile of the leaf microbial community of bacteria found in GM (drought-tolerant event 1Ea2939) and conventional (BR 16) soybean plants. Bacterial DNA was extracted from leaf samples collected from each genotype and used for microbial diversity and richness analysis through the MiSeq Illumina platform. Functional prediction was performed using the PICRUSt tool and the STAMP v 2.1.3 software. The obtainment of the GM event 1Ea2939 showed minimum effects on the microbial community and in the potential for chemical-genetic communication, i.e. in the potential for symbiotic and/or mutualistic interaction between plants and their natural microbiota.

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.

Induced over-expression of AtDREB2A CA improves drought tolerance in sugarcane.

Drought is one of the most challenging agricultural issues limiting sustainable sugarcane production and, in some cases, yield losses caused by drought are nearly 50%. DREB proteins play vital regulatory roles in abiotic stress responses in plants. The transcription factor DREB2A interacts with a cis-acting DRE sequence to activate the expression of downstream genes that are involved in drought-, salt- and heat-stress response in Arabidopsis thaliana. In the present study, we evaluated the effects of stress-inducible over-expression of AtDREB2A CA on gene expression, leaf water potential (ΨL), relative water content (RWC), sucrose content and gas exchanges of sugarcane plants submitted to a four-days water deficit treatment in a rhizotron-grown root system. The plants were also phenotyped by scanning the roots and measuring morphological parameters of the shoot. The stress-inducible expression of AtDREB2A CA in transgenic sugarcane led to the up-regulation of genes involved in plant response to drought stress. The transgenic plants maintained higher RWC and ΨL over 4 days after withholding water and had higher photosynthetic rates until the 3rd day of water-deficit. Induced expression of AtDREB2A CA in sugarcane increased sucrose levels and improved bud sprouting of the transgenic plants. Our results indicate that induced expression of AtDREB2A CA in sugarcane enhanced its drought tolerance without biomass penalty.

Introduction of the rd29A:AtDREB2A CA gene into soybean (Glycine max L. Merril) and its molecular characterization in leaves and roots during dehydration.

The loss of soybean yield to Brazilian producers because of a water deficit in the 2011-2012 season was 12.9%. To reduce such losses, molecular biology techniques, including plant transformation, can be used to insert genes of interest into conventional soybean cultivars to produce lines that are more tolerant to drought. The abscisic acid (ABA)-independent Dehydration Responsive Element Binding (DREB) gene family has been used to obtain plants with increased tolerance to abiotic stresses. In the present study, the rd29A:AtDREB2A CA gene from Arabidopsis thaliana was inserted into soybean using biolistics. Seventy-eight genetically modified (GM) soybean lines containing 2-17 copies of the AtDREB2A CA gene were produced. Two GM soybean lines (P1397 and P2193) were analyzed to assess the differential expression of the AtDREB2A CA transgene in leaves and roots submitted to various dehydration treatments. Both GM lines exhibited high expression of the transgene, with the roots of P2193 showing the highest expression levels during water deficit. Physiological parameters examined during water deficit confirmed the induction of stress. This analysis of AtDREB2A CA expression in GM soybean indicated that line P2193 had the greatest stability and highest expression in roots during water deficit-induced stress.

Phenotyping transgenic wheat for drought resistance.

Realistic experimental protocols to screen for drought adaptation in controlled conditions are crucial if high throughput phenotyping is to be used for the identification of high performance lines, and is especially important in the evaluation of transgenes where stringent biosecurity measures restrict the frequency of open field trials. Transgenic DREB1A-wheat events were selected under greenhouse conditions by evaluating survival and recovery under severe drought (SURV) as well as for water use efficiency (WUE). Greenhouse experiments confirmed the advantages of transgenic events in recovery after severe water stress. Under field conditions, the group of transgenic lines did not generally outperform the controls in terms of grain yield under water deficit. However, the events selected for WUE were identified as lines that combine an acceptable yield-even higher yield (WUE-11) under well irrigated conditions-and stable performance across the different environments generated by the experimental treatments.

Stress-induced expression in wheat of the Arabidopsis thaliana DREB1A gene delays water stress symptoms under greenhouse conditions.

One of the major environmental factors limiting plant productivity is lack of water. This is especially true for the major cereals maize, rice, and wheat, which demonstrate a range of susceptibility to moisture deficit. Although conventional breeding and marker-assisted selection are being used to develop varieties more tolerant to water stress, these methods are time and resource consuming and germplasm dependent. Genetic engineering is attractive because of its potential to improve abiotic stress tolerance more rapidly. Transcription factors have been shown to produce multiple phenotypic alterations, many of which are involved in stress responses. DREB1A, a transcription factor that recognizes dehydration response elements, has been shown in Arabidopsis thaliana to play a crucial role in promoting the expression of drought-tolerance genes. In our efforts to enhance drought tolerance in wheat, the A. thaliana DREB1A gene was placed under control of a stress-inducible promoter from the rd29A gene and transferred via biolistic transformation into bread wheat. Plants expressing the DREB1A gene demonstrated substantial resistance to water stress in comparison with checks under experimental greenhouse conditions, manifested by a 10-day delay in wilting when water was withheld.