A Case Study from the Overexpression of OsTZF5, Encoding a CCCH Tandem Zinc Finger Protein, in Rice Plants Across Nineteen Yield Trials.

BACKGROUND: Development of transgenic rice overexpressing transcription factors involved in drought response has been previously reported to confer drought tolerance and therefore represents a means of crop improvement. We transformed lowland rice IR64 with OsTZF5, encoding a CCCH-tandem zinc finger protein, under the control of the rice LIP9 stress-inducible promoter and compared the drought response of transgenic lines and nulls to IR64 in successive screenhouse paddy and field trials up to the T6 generation. RESULTS: Compared to the well-watered conditions, the level of drought stress across experiments varied from a minimum of - 25 to - 75 kPa at a soil depth of 30 cm which reduced biomass by 30-55% and grain yield by 1-92%, presenting a range of drought severities. OsTZF5 transgenic lines showed high yield advantage under drought over IR64 in early generations, which was related to shorter time to flowering, lower shoot biomass and higher harvest index. However, the increases in values for yield and related traits in the transgenics became smaller over successive generations despite continued detection of drought-induced transgene expression as conferred by the LIP9 promoter. The decreased advantage of the transgenics over generations tended to coincide with increased levels of homozygosity. Background cleaning of the transgenic lines as well as introgression of the transgene into an IR64 line containing major-effect drought yield QTLs, which were evaluated starting at the BC3F1 and BC2F3 generation, respectively, did not result in consistently increased yield under drought as compared to the respective checks. CONCLUSIONS: Although we cannot conclusively explain the genetic factors behind the loss of yield advantage of the transgenics under drought across generations, our results help in distinguishing among potential drought tolerance mechanisms related to effectiveness of the transgenics, since early flowering and harvest index most closely reflected the levels of yield advantage in the transgenics across generations while reduced biomass did not.

A Case of End-stage Renal Disease in Which Diazoxide was Effective in Treating Hypoglycemia Caused by Late Dumping Syndrome after Gastrectomy.

We herein report a case in which diazoxide was effective in treating reactive hypoglycemia caused by late dumping syndrome in a patient with ESRD. A 50-year-old man with end-stage renal disease (ESRD) and a history of gastrectomy underwent hemodialysis. Although he was administered voglibose to treat recurrent reactive hypoglycemia caused by late dumping syndrome, he had difficulty continuing treatment because of gastrointestinal side effects. When he began diazoxide treatment, the reactive hypoglycemia improved. The dose was gradually increased with no apparent side effects, and the hypoglycemic attacks disappeared one year after the start of treatment.

OsTZF1, a CCCH-tandem zinc finger protein gene, driven under own promoter produces no pleiotropic effects and confers salt and drought tolerance in rice.

Different abiotic stresses induce OsTZF1, a tandem CCCH-type zinc finger domain gene, in rice. Here, we report that transgenic rice plants overexpressing OsTZF1 under own promoter (POsTZF1:OsTZF1-OX [for overexpression]) transferred to soil showed normal growth similar to vector control plants. The POsTZF1:OsTZF1-OX produced normal leaves without any lesion mimic phenotype and exhibited normal seed setting. The POsTZF1:OsTZF1-OX plants showed significantly increased tolerance to salt and drought stresses and enhanced post stress recovery. Microarray analysis revealed a total of 846 genes up-regulated and 360 genes down-regulated in POsTZF1:OsTZF1-OX salt-treated plants. Microarray analysis of POsTZF1:OsTZF1-OX plants showed the regulation of many abiotic stress tolerance genes. These results suggest that OsTZF1-OX under own promoter show abiotic stress tolerance and produces no pleiotropic effect on phenotype of transgenic rice plant.

Constitutive expression of Arabidopsis bZIP transcription factor AREB1 activates cross-signaling responses in soybean under drought and flooding stresses.

Abiotic stress, such as drought and flooding, are responsible for considerable losses in grain production worldwide. Soybean, the main cultivated oilseed in the world, is sensitive to both stresses. Plant molecular mechanisms answer via crosstalk of several signaling pathways, in which particular genes can respond to different stresses. Previous studies confirmed that overexpression of transcription factor AtAREB1 confers drought tolerance in soybean. However, plants containing this gene have not yet been tested under flooding. Thus, the objective of this study was to characterize genetically modified (GM) soybean plants overexpressing AtAREB1 under drought and flooding conditions in comparison to its genetic background. Physiological and biochemical measurements were performed. In addition, the expression level of genes commonly activated under both stresses was evaluated. The results supported the role of the AtAREB1 gene in conferring tolerance to water deficit in soybeans. Furthermore, under flooding, the GM line was efficient in maintaining a higher photosynthetic rate, intrinsic efficiency in water use, and instantaneous carboxylation efficiency, resulting in higher grain yield under stress. The GM line also presented higher protein content, lower concentration of hydrogen peroxide, and lower expression levels of genes related to fermentative metabolism and alanine biosynthesis. These results indicate that in addition to drought stress, plants overexpressing AtAREB1 exhibited better performance under flooding when compared to the non-GM line, suggesting a cross-signaling response to both abiotic factors.

Genetic and Molecular Mechanisms Conferring Heat Stress Tolerance in Tomato Plants.

Climate change is a major threat to global food security. Changes in climate can directly impact food systems by reducing the production and genetic diversity of crops and their wild relatives, thereby restricting future options for breeding improved varieties and reducing the ability to adapt crops to future challenges. The global surface temperature is predicted to rise by an average of 0.3°C during the next decade, and the Paris Agreement (Paris Climate Accords) aims to limit global warming to below an average of 2°C, preferably to 1.5°C compared to pre-industrial levels. Even if the goal of the Paris Agreement can be met, the predicted rise in temperatures will increase the likelihood of extreme weather events, including heatwaves, making heat stress (HS) a major global abiotic stress factor for many crops. HS can have adverse effects on plant morphology, physiology, and biochemistry during all stages of vegetative and reproductive development. In fruiting vegetables, even moderate HS reduces fruit set and yields, and high temperatures may result in poor fruit quality. In this review, we emphasize the effects of abiotic stress, especially at high temperatures, on crop plants, such as tomatoes, touching upon key processes determining plant growth and yield. Specifically, we investigated the molecular mechanisms involved in HS tolerance and the challenges of developing heat-tolerant tomato varieties. Finally, we discuss a strategy for effectively improving the heat tolerance of vegetable crops.

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.

Overexpression of an Arabidopsis thaliana galactinol synthase gene improves drought tolerance in transgenic rice and increased grain yield in the field.

Drought stress has often caused significant decreases in crop production which could be associated with global warming. Enhancing drought tolerance without a grain yield penalty has been a great challenge in crop improvement. Here, we report the Arabidopsis thaliana galactinol synthase 2 gene (AtGolS2) was able to confer drought tolerance and increase grain yield in two different rice (Oryza sativa) genotypes under dry field conditions. The developed transgenic lines expressing AtGolS2 under the control of the constitutive maize ubiquitin promoter (Ubi:AtGolS2) also had higher levels of galactinol than the non-transgenic control. The increased grain yield of the transgenic rice under drought conditions was related to a higher number of panicles, grain fertility and biomass. Extensive confined field trials using Ubi:AtGolS2 transgenic lines in Curinga, tropical japonica and NERICA4, interspecific hybrid across two different seasons and environments revealed the verified lines have the proven field drought tolerance of the Ubi:AtGolS2 transgenic rice. The amended drought tolerance was associated with higher relative water content of leaves, higher photosynthesis activity, lesser reduction in plant growth and faster recovering ability. Collectively, our results provide strong evidence that AtGolS2 is a useful biotechnological tool to reduce grain yield losses in rice beyond genetic differences under field drought stress.

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.

Insulin Autoimmune Syndrome Accompanied by Multiple Myeloma.

In 1981, a 48-year old man was diagnosed with insulin autoimmune syndrome. In 2005, he experienced a substantial increase in his monoclonal insulin antibody levels; in 2006 and 2007, serum monoclonal gammopathy and an 11% marrow plasmacyte ratio were confirmed. In 2012, asymptomatic multiple myeloma was diagnosed based on an increased γ-globulin fraction and serum M-protein (IgG) levels. The insulin antibody binding rate was 75.4% in 2005 and 78.8% in 2012. In 2012, he was hospitalized for ileus and died. Autopsy identified multiple myeloma and no endocrinological tumors in the pancreas.

SNAC-As, stress-responsive NAC transcription factors, mediate ABA-inducible leaf senescence.

Leaf senescence is the terminal phenotype of plant leaf development, and ethylene is a major plant hormone inducing leaf senescence. Recent studies have shown that abscisic acid (ABA) also induces leaf senescence. However, the detailed mechanisms of ABA-induced leaf senescence remain unclear. We focused on the A subfamily of stress-responsive NAC (SNAC-A) transcription factors, the expression of which is induced by abiotic stresses, particularly ABA. Gene expression analysis revealed that seven SNAC-A genes including ANAC055, ANAC019, ANAC072/RD26, ANAC002/ATAF1, ANAC081/ATAF2, ANAC102 and ANAC032 were induced by long-term treatment with ABA and/or during age-dependent senescence. The SNAC-A septuple mutant clearly showed retardation of ABA-inducible leaf senescence. Microarray analysis indicated that SNAC-As induce ABA- and senescence-inducible genes. In addition, comparison of the expression profiles of the downstream genes of SNAC-As and ABA-responsive element (ABRE)-binding protein (AREB)/ABRE-binding factor (ABF) (AREB/ABFs) indicates that SNAC-As induce a different set of ABA-inducible genes from those mediated by AREB/ABFs. These results suggest that SNAC-As play crucial roles in ABA-induced leaf senescence signaling. We also discuss the function of SNAC-As in the transcriptional change of leaf senescence as well as in ABA response under abiotic stress conditions.

The transcriptional regulatory network in the drought response and its crosstalk in abiotic stress responses including drought, cold, and heat.

Drought negatively impacts plant growth and the productivity of crops around the world. Understanding the molecular mechanisms in the drought response is important for improvement of drought tolerance using molecular techniques. In plants, abscisic acid (ABA) is accumulated under osmotic stress conditions caused by drought, and has a key role in stress responses and tolerance. Comprehensive molecular analyses have shown that ABA regulates the expression of many genes under osmotic stress conditions, and the ABA-responsive element (ABRE) is the major cis-element for ABA-responsive gene expression. Transcription factors (TFs) are master regulators of gene expression. ABRE-binding protein and ABRE-binding factor TFs control gene expression in an ABA-dependent manner. SNF1-related protein kinases 2, group A 2C-type protein phosphatases, and ABA receptors were shown to control the ABA signaling pathway. ABA-independent signaling pathways such as dehydration-responsive element-binding protein TFs and NAC TFs are also involved in stress responses including drought, heat, and cold. Recent studies have suggested that there are interactions between the major ABA signaling pathway and other signaling factors in stress responses. The important roles of these TFs in crosstalk among abiotic stress responses will be discussed. Control of ABA or stress signaling factor expression can improve tolerance to environmental stresses. Recent studies using crops have shown that stress-specific overexpression of TFs improves drought tolerance and grain yield compared with controls in the field.

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.

Phenotyping soybean plants transformed with rd29A:AtDREB1A for drought tolerance in the greenhouse and field.

The development of drought tolerant plants is a high priority because the area suffering from drought is expected to increase in the future due to global warming. One strategy for the development of drought tolerance is to genetically engineer plants with transcription factors (TFs) that regulate the expression of several genes related to abiotic stress defense responses. This work assessed the performance of soybean plants overexpressing the TF DREB1A under drought conditions in the field and in the greenhouse. Drought was simulated in the greenhouse by progressively drying the soil of pot cultures of the P58 and P1142 lines. In the field, the performance of the P58 line and of 09D-0077, a cross between the cultivars BR16 and P58, was evaluated under four different water regimes: irrigation, natural drought (no irrigation) and water stress created using rain-out shelters in the vegetative or reproductive stages. Although the dehydration-responsive element-binding protein (DREB) plants did not outperform the cultivar BR16 in terms of yield, some yield components were increased when drought was introduced during the vegetative stage, such as the number of seeds, the number of pods with seeds and the total number of pods. The greenhouse data suggest that the higher survival rates of DREB plants are because of lower water use due to lower transpiration rates under well watered conditions. Further studies are needed to better characterize the soil and atmospheric conditions under which these plants may outperform the non-transformed parental plants.

Comparative functional analysis of six drought-responsive promoters in transgenic rice.

Rice production is greatly affected by environmental stresses such as drought and high salinity. Transgenic rice plants tolerant to such stresses are expected to be produced. Stress-responsive promoters with low expression under normal growth conditions are needed to minimize the adverse effects of stress-tolerance genes on rice growth. We performed expression analyses of drought-responsive genes in rice plants using a microarray, and selected LIP9, OsNAC6, OsLEA14a, OsRAB16D, OsLEA3-1, and Oshox24 for promoter analysis. Transient assays using the promoters indicated that AREB/ABF (abscisic acid (ABA)-responsive element-binding protein/ABA-binding factor) transcription factors enhanced expressions of these genes. We generated transgenic rice plants containing each promoter and the ß-glucuronidase (GUS) reporter gene. GUS assays revealed that the LIP9 and OsNAC6 promoters were induced by drought, high salinity, and ABA treatment, and both promoters showed strong activity under normal growth conditions in the root. The other promoters were strongly induced by stresses and ABA, but showed low activity under normal growth conditions. In seeds, GUS staining showed that Oshox24 expression was low and expressions of the other genes were high. Transgenic rice plants overexpressing OsNAC6 under the control of the Oshox24 promoter showed increased tolerance to drought and high salinity, and no growth defects. These data suggest that the Oshox24 promoter is useful to overexpress stress-tolerance genes without adversely affecting growth.

ABA signaling in stress-response and seed development.

KEY MESSAGE : We review the recent progress on ABA signaling, especially ABA signaling for ABA-dependent gene expression, including the AREB/ABF regulon, SnRK2 protein kinase, 2C-type protein phosphatases and ABA receptors. Drought negatively impacts plant growth and the productivity of crops. Drought causes osmotic stress to organisms, and the osmotic stress causes dehydration in plant cells. Abscisic acid (ABA) is produced under osmotic stress conditions, and it plays an important role in the stress response and tolerance of plants. ABA regulates many genes under osmotic stress conditions. It also regulates gene expression during seed development and germination. The ABA-responsive element (ABRE) is the major cis-element for ABA-responsive gene expression. ABRE-binding protein (AREB)/ABRE-binding factor (ABF) transcription factors (TFs) regulate ABRE-dependent gene expression. Other TFs are also involved in ABA-responsive gene expression. SNF1-related protein kinases 2 are the key regulators of ABA signaling including the AREB/ABF regulon. Recently, ABA receptors and group A 2C-type protein phosphatases were shown to govern the ABA signaling pathway. Moreover, recent studies have suggested that there are interactions between the major ABA signaling pathway and other signaling factors in stress-response and seed development. The control of the expression of ABA signaling factors may improve tolerance to environmental stresses.

OsTZF1, a CCCH-tandem zinc finger protein, confers delayed senescence and stress tolerance in rice by regulating stress-related genes.

OsTZF1 is a member of the CCCH-type zinc finger gene family in rice (Oryza sativa). Expression of OsTZF1 was induced by drought, high-salt stress, and hydrogen peroxide. OsTZF1 gene expression was also induced by abscisic acid, methyl jasmonate, and salicylic acid. Histochemical activity of ß-glucuronidase in transgenic rice plants containing the promoter of OsTZF1 fused with ß-glucuronidase was observed in callus, coleoptile, young leaf, and panicle tissues. Upon stress, OsTZF1-green fluorescent protein localization was observed in the cytoplasm and cytoplasmic foci. Transgenic rice plants overexpressing OsTZF1 driven by a maize (Zea mays) ubiquitin promoter (Ubi:OsTZF1-OX [for overexpression]) exhibited delayed seed germination, growth retardation at the seedling stage, and delayed leaf senescence. RNA interference (RNAi) knocked-down plants (OsTZF1-RNAi) showed early seed germination, enhanced seedling growth, and early leaf senescence compared with controls. Ubi:OsTZF1-OX plants showed improved tolerance to high-salt and drought stresses and vice versa for OsTZF1-RNAi plants. Microarray analysis revealed that genes related to stress, reactive oxygen species homeostasis, and metal homeostasis were regulated in the Ubi:OsTZF1-OX plants. RNA-binding assays indicated that OsTZF1 binds to U-rich regions in the 3' untranslated region of messenger RNAs, suggesting that OsTZF1 might be associated with RNA metabolism of stress-responsive genes. OsTZF1 may serve as a useful biotechnological tool for the improvement of stress tolerance in various plants through the control of RNA metabolism of stress-responsive genes.