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.

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.

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.

Design of an optimal promoter involved in the heat-induced transcriptional pathway in Arabidopsis, soybean, rice and maize.

Interactions between heat shock (HS) factors (HSFs) and heat shock response elements (HSEs) are important during the heat shock response (HSR) of flora and fauna. Moreover, plant HSFs that are involved in heat stress are also involved in abiotic stresses such as dehydration and cold as well as development, cell differentiation and proliferation. Because the specific combination of HSFs and HSEs involved in plants under heat stress remains unclear, the mechanism of their interaction has not yet been utilized in molecular breeding of plants for climate change. For the study reported herein, we compared the sequences of HS-inducible genes and their promoters in Arabidopsis, soybean, rice and maize and then designed an optimal HS-inducible promoter. Our analyses suggest that, for the four species, the abscisic acid-independent, HSE/HSF-dependent transcriptional pathway plays a major role in HS-inducible gene expression. We found that an 18-bp sequence that includes the HSE has an important role in the HSR, and that those sequences could be classified as representative of monocotyledons or dicotyledons. With the HS-inducible promoter designed based on our bioinformatic predictions, we were able to develop an optimal HS-specific inducible promoter for seedlings or single cells in roots. These findings demonstrate the utility of our HS-specific inducible promoter, which we expect will contribute to molecular breeding efforts and cell-targeted gene expression in specific plant tissues.

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.

Three SnRK2 protein kinases are the main positive regulators of abscisic acid signaling in response to water stress in Arabidopsis.

Responses to water stress are thought to be mediated by transcriptional regulation of gene expression via reversible protein phosphorylation events. Previously, we reported that bZIP (basic-domain leucine zipper)-type AREB/ABF (ABA-responsive element-binding protein/factor) transcription factors are involved in ABA signaling under water stress conditions in Arabidopsis. The AREB1 protein is phosphorylated in vitro by ABA-activated SNF1-related protein kinase 2s (SnRK2s) such as SRK2D/SnRK2.2, SRK2E/SnRK2.6 and SRK2I/SnRK2.3 (SRK2D/E/I). Consistent with this, we now show that SRK2D/E/I and AREB1 co-localize and interact in nuclei in planta. Our results show that unlike srk2d, srk2e and srk2i single and double mutants, srk2d srk2e srk2i (srk2d/e/i) triple mutants exhibit greatly reduced tolerance to drought stress and highly enhanced insensitivity to ABA. Under water stress conditions, ABA- and water stress-dependent gene expression, including that of transcription factors, is globally and drastically impaired, and jasmonic acid (JA)-responsive and flowering genes are up-regulated in srk2d/e/i triple mutants, but not in other single and double mutants. The down-regulated genes in srk2d/e/i and areb/abf triple mutants largely overlap in ABA-dependent expression, supporting the view that SRK2D/E/I regulate AREB/ABFs in ABA signaling in response to water stress. Almost all dehydration-responsive LEA (late embryogenesis abundant) protein genes and group-A PP2C (protein phosphatase 2C) genes are strongly down-regulated in the srk2d/e/i triple mutants. Further, our data show that these group-A PP2Cs, such as HAI1 and ABI1, interact with SRK2D. Together, our results indicate that SRK2D/E/I function as main positive regulators, and suggest that ABA signaling is controlled by the dual modulation of SRK2D/E/I and group-A PP2Cs.

Three Arabidopsis SnRK2 protein kinases, SRK2D/SnRK2.2, SRK2E/SnRK2.6/OST1 and SRK2I/SnRK2.3, involved in ABA signaling are essential for the control of seed development and dormancy.

ABA is an important phytohormone regulating various plant processes, including stress tolerance, seed development and germination. SRK2D/SnRK2.2, SRK2E/SnRK2.6/OST1 and SRK2I/SnRK2.3 are redundant ABA-activated SNF1-related protein kinases 2 (SnRK2s) in Arabidopsis thaliana. We examined the role of these protein kinases in seed development and germination. These SnRK2 proteins were mainly expressed in the nucleus during seed development and germination. The triple mutant (srk2d srk2e srk2i) was sensitive to desiccation and showed severe growth defects during seed development. It exhibited a loss of dormancy and elevated seed ABA content relative to wild-type plants. The severity of these phenotypes was far stronger than that of any single or double SRK2D, SRK2E and SRK2I mutants, including the srk2d srk2i mutant. The triple mutant had greatly reduced phosphorylation activity in in-gel kinase experiments using basic leucine zipper (bZIP) transcription factors including ABI5. Microarray experiments revealed that 48 and 30% of the down-regulated genes in abi5 and abi3 seeds were suppressed in the triple mutant seeds, respectively. Moreover, disruption of the three protein kinases induced global changes in the up-regulation of ABA-repressive gene expression, as well as the down-regulation of ABA-inducible gene expression. These alterations in gene expression result in a loss of dormancy and severe growth defects during seed development. Collectively, these results indicate that SRK2D, SRK2E and SRK2I protein kinases involved in ABA signaling are essential for the control of seed development and dormancy through the extensive control of gene expression.

Overexpression of class 1 plant hemoglobin genes enhances symbiotic nitrogen fixation activity between Mesorhizobium loti and Lotus japonicus.

Plant hemoglobins (Hbs) have been divided into three groups: class 1, class 2, and truncated Hbs. The various physiological functions of class 1 Hb include its role as a modulator of nitric oxide (NO) levels in plants. To gain more insight into the functions of class 1 Hbs, we investigated the physical properties of LjHb1 and AfHb1, class 1 Hbs of a model legume Lotus japonicus and an actinorhizal plant Alnus firma, respectively. Spectrophotometric analysis showed that the recombinant form of the LjHb1 and AfHb1 proteins reacted with NO. The localization of LjHb1 expression was correlated with the site of NO production. Overexpression of LjHb1 and AfHb1 by transformed hairy roots caused changes in symbiosis with rhizobia. The number of nodules formed on hairy roots overexpressing LjHb1 or AfHb1 increased compared with that on untransformed hairy roots. Furthermore, nitrogenase activity as acetylene-reduction activity (ARA) of LjHb1- or AfHb1-overexpressing nodules was higher than that of the vector control nodules. Microscopic observation with a NO-specific fluorescent dye suggested that the NO level in LjHb1- and AfHb1-overexpressing nodules was lower than that of control nodules. Exogenous application of a NO scavenger enhanced ARA in L. japonicus nodules, whereas a NO donor inhibited ARA. These results suggest that the basal level of NO in nodules inhibits nitrogen fixation, and overexpression of class 1 Hbs enhances symbiotic nitrogen fixation activity by removing NO as an inhibitor of nitrogenase.

Genetics of symbiosis in Lotus japonicus: recombinant inbred lines, comparative genetic maps, and map position of 35 symbiotic loci.

Development of molecular tools for the analysis of the plant genetic contribution to rhizobial and mycorrhizal symbiosis has provided major advances in our understanding of plant-microbe interactions, and several key symbiotic genes have been identified and characterized. In order to increase the efficiency of genetic analysis in the model legume Lotus japonicus, we present here a selection of improved genetic tools. The two genetic linkage maps previously developed from an interspecific cross between L. japonicus Gifu and L. filicaulis, and an intraspecific cross between the two ecotypes L. japonicus Gifu and L. japonicus MG-20, were aligned through a set of anchor markers. Regions of linkage groups, where genetic resolution is obtained preferentially using one or the other parental combination, are highlighted. Additional genetic resolution and stabilized mapping populations were obtained in recombinant inbred lines derived by a single seed descent from the two populations. For faster mapping of new loci, a selection of reliable markers spread over the chromosome arms provides a common framework for more efficient identification of new alleles and new symbiotic loci among uncharacterized mutant lines. Combining resources from the Lotus community, map positions of a large collection of symbiotic loci are provided together with alleles and closely linked molecular markers. Altogether, this establishes a common genetic resource for Lotus spp. A web-based version will enable this resource to be curated and updated regularly.

A nucleoporin is required for induction of Ca2+ spiking in legume nodule development and essential for rhizobial and fungal symbiosis.

Nuclear-cytoplasmic partitioning and traffic between cytoplasmic and nuclear compartments are fundamental processes in eukaryotic cells. Nuclear pore complexes mediate transport of proteins, RNAs and ribonucleoprotein particles in and out of the nucleus. Here we present positional cloning of a plant nucleoporin gene, Nup133, essential for a symbiotic signal transduction pathway shared by Rhizobium bacteria and mycorrhizal fungi. Mutation of Nup133 results in a temperature sensitive nodulation deficient phenotype and absence of mycorrhizal colonization. Root nodules developing with reduced frequency at permissive temperatures are ineffective and electron microscopy show that Rhizobium bacteria are not released from infection threads. Measurement of ion fluxes using a calcium-sensitive dye show that Nup133 is required for the Ca2+ spiking normally detectable within minutes after application of purified rhizobial Nod-factor signal molecules to root hairs. Localization of NUP133 in the nuclear envelope of root cells and root hair cells shown with enhanced yellow fluorescent protein fusion proteins suggests a novel role for NUP133 nucleoporins in a rapid nuclear-cytoplasmic communication after host-plant recognition of symbiotic microbes. Our results identify a component of an intriguing signal process requiring interaction at the cell plasma membrane and at intracellular nuclear and plastid organelle-membranes to induce a second messenger.