Reducing stomatal density by expression of a synthetic Epidermal Patterning Factor increases leaf intrinsic water use efficiency and reduces plant water use in a C4 crop.

Enhancing crop water use efficiency (WUE) is a key target trait for climatic resilience and expanding cultivation on marginal lands. Engineering lower stomatal density to reduce stomatal conductance (gs) has improved WUE in multiple C3 crop species. However, reducing gs in C3 species often reduces photosynthetic carbon gain. A different response is expected in C4 plants because they possess specialized anatomy and biochemistry which concentrates CO2 at the site of fixation. This modifies the photosynthesis (AN) relationship with intracellular CO2 concentration (ci) so that photosynthesis is CO2-saturated and reductions in gs are unlikely to limit AN. To test this hypothesis, genetic strategies were investigated to reduce stomatal density in the C4 crop sorghum. Constitutive expression of a synthetic epidermal patterning factor (EPF) transgenic allele in sorghum, led to reduced stomatal densities, reduced gs, reduced plant water use and avoidance of stress during a period of water deprivation. In addition, moderate reduction in stomatal density did not increase stomatal limitation to AN. However, these positive outcomes were associated with negative pleiotropic effects on reproductive development and photosynthetic capacity. Avoiding pleiotropy by targeting expression of the transgene to specific tissues could provide a pathway to improved agronomic outcomes.

Expression of AtWRI1 and AtDGAT1 during soybean embryo development influences oil and carbohydrate metabolism.

Soybean oil is one of the most consumed vegetable oils worldwide. Genetic improvement of its concentration in seeds has been historically pursued due to its direct association with its market value. Engineering attempts aiming to increase soybean seed oil presented different degrees of success that varied with the genetic design and the specific variety considered. Understanding the embryo's responses to the genetic modifications introduced, is a critical step to successful approaches. In this work, the metabolic and transcriptional responses to AtWRI1 and AtDGAT1 expression in soybean seeds were evaluated. AtWRI1 is a master regulator of fatty acid (FA) biosynthesis, and AtDGAT1 encodes an enzyme catalysing the final and rate-limiting step of triacylglycerides biosynthesis. The events expressing these genes in the embryo did not show an increase in total FA content, but they responded with changes in the oil and carbohydrate composition. Transcriptomic studies revealed a down-regulation of genes putatively encoding for oil body packaging proteins, and a strong induction of genes annotated as lipases and FA biosynthesis inhibitors. Novel putative AtWRI1 targets, presenting an AW-box in the upstream region of the genes, were identified by comparison with an event that harbours only AtWRI1. Lastly, targeted metabolomics analysis showed that carbon from sugar phosphates could be used for FA competing pathways, such as starch and cell wall polysaccharides, contributing to the restriction in oil accumulation. These results allowed the identification of key cellular processes that need to be considered to break the embryo's natural restriction to uncontrolled seed lipid increase.

Transgenic Wheat Harboring an RNAi Element Confers Dual Resistance Against Synergistically Interacting Wheat Streak Mosaic Virus and Triticum Mosaic Virus.

Wheat streak mosaic virus (WSMV) and triticum mosaic virus (TriMV) are economically important viruses of wheat (Triticum aestivum L.), causing significant yield losses in the Great Plains region of the United States. These two viruses are transmitted by wheat curl mites, which often leads to mixed infections with synergistic interaction in grower fields that exacerbates yield losses. Development of dual-resistant wheat lines would provide effective control of these two viruses. In this study, a genetic resistance strategy employing an RNA interference (RNAi) approach was implemented by assembling a hairpin element composed of a 202-bp (404-bp in total) stem sequence of the NIb (replicase) gene from each of WSMV and TriMV in tandem and of an intron sequence in the loop. The derived RNAi element was cloned into a binary vector and was used to transform spring wheat genotype CB037. Phenotyping of T1 lineages across eight independent transgenic events for resistance revealed that i) two of the transgenic events provided resistance to WSMV and TriMV, ii) four events provided resistance to either WSMV or TriMV, and iii) no resistance was found in two other events. T2 populations derived from the two events classified as dual-resistant were subsequently monitored for stability of the resistance phenotype through the T4 generation. The resistance phenotype in these events was temperature-dependent, with a complete dual resistance at temperatures ≥25°C and an increasingly susceptible response at temperatures below 25°C. Northern blot hybridization of total RNA from transgenic wheat revealed that virus-specific small RNAs (vsRNAs) accumulated progressively with an increase in temperature, with no detectable levels of vsRNA accumulation at 20°C. Thus, the resistance phenotype of wheat harboring an RNAi element was correlated with accumulation of vsRNAs, and the generation of vsRNAs can be used as a molecular marker for the prediction of resistant phenotypes of transgenic plants at a specific temperature.

The LATERAL ROOT DENSITY gene regulates root growth during water stress in wheat.

Drought stress is the major limiting factor in agriculture. Wheat, which is the most widely grown crop in the world, is predominantly cultivated in drought-prone rainfed environments. Since roots play a critical role in water uptake, root response to water limitations is an important component for enhancing wheat adaptation. In an effort to discover novel genetic sources for improving wheat adaptation, we characterized a wheat translocation line with a chromosomal segment from Agropyron elongatum, a wild relative of wheat, which unlike common wheat maintains root growth under limited-water conditions. By exploring the root transcriptome data, we found that reduced transcript level of LATERAL ROOT DENSITY (LRD) gene under limited water in the Agropyron translocation line confers it the ability to maintain root growth. The Agropyron allele of LRD is down-regulated in response to water limitation in contrast with the wheat LRD allele, which is up-regulated by water deficit stress. Suppression of LRD expression in wheat RNAi plants confers the ability to maintain root growth under water limitation. We show that exogenous gibberellic acid (GA) promotes lateral root growth and present evidence for the role of GA in mediating the differential regulation of LRD between the common wheat and the Agropyron alleles under water stress. Suppression of LRD also had a positive pleiotropic effect on grain size and number under optimal growth conditions. Collectively, our findings suggest that LRD can be potentially useful for improving wheat response to water stress and altering yield components.

Molecular and phenotypic characterization of transgenic wheat and sorghum events expressing the barley alanine aminotransferase.

MAIN CONCLUSION: The expression of a barley alanine aminotransferase gene impacts agronomic outcomes in a C3 crop, wheat. The use of nitrogen-based fertilizers has become one of the major agronomic inputs in crop production systems. Strategies to enhance nitrogen assimilation and flux in planta are being pursued through the introduction of novel genetic alleles. Here an Agrobacterium-mediated approach was employed to introduce the alanine aminotransferase from barley (Hordeum vulgare), HvAlaAT, into wheat (Triticum aestivum) and sorghum (Sorghum bicolor), regulated by either constitutive or root preferred promoter elements. Plants harboring the transgenic HvAlaAT alleles displayed increased alanine aminotransferase (alt) activity. The enhanced alt activity impacted height, tillering and significantly boosted vegetative biomass relative to controls in wheat evaluated under hydroponic conditions, where the phenotypic outcome across these parameters varied relative to time of year study was conducted. Constitutive expression of HvAlaAT translated to elevation in wheat grain yield under field conditions. In sorghum, expression of HvAlaAT enhanced enzymatic activity, but no changes in phenotypic outcomes were observed. Taken together these results suggest that positive agronomic outcomes can be achieved through enhanced alt activity in a C3 crop, wheat. However, the variability observed across experiments under greenhouse conditions implies the phenotypic outcomes imparted by the HvAlaAT allele in wheat may be impacted by environment.

Genome-wide expression analysis of soybean NF-Y genes reveals potential function in development and drought response.

Nuclear factor-Y (NF-Y), a heterotrimeric transcription factor, is composed of NF-YA, NF-YB and NF-YC proteins. In plants, there are usually more than 10 genes for each family and their members have been identified to be key regulators in many developmental and physiological processes controlling gametogenesis, embryogenesis, nodule development, seed development, abscisic acid (ABA) signaling, flowering time, primary root elongation, blue light responses, endoplasmic reticulum (ER) stress response and drought tolerance. Taking the advantages of the recent soybean genome draft and information on functional characterizations of nuclear factor Y (NF-Y) transcription factor family in plants, we identified 21 GmNF-YA, 32 GmNF-YB, and 15 GmNF-YC genes in the soybean (Glycine max) genome. Phylogenetic analyses show that soybean's proteins share strong homology to Arabidopsis and many of them are closely related to functionally characterized NF-Y in plants. Expression analysis in various tissues of flower, leaf, root, seeds of different developmental stages, root hairs under rhizobium inoculation, and drought-treated roots and leaves revealed that certain groups of soybean NF-Y are likely involved in specific developmental and stress responses. This study provides extensive evaluation of the soybean NF-Y family and is particularly useful for further functional characterization of GmNF-Y proteins in seed development, nodulation and drought adaptation of soybean.

Overexpression of AtDREB1D transcription factor improves drought tolerance in soybean.

Drought is one of the major abiotic stresses that affect productivity in soybean (Glycine max L.) Several genes induced by drought stress include functional genes and regulatory transcription factors. The Arabidopsis thaliana DREB1D transcription factor driven by the constitutive and ABA-inducible promoters was introduced into soybean through Agrobacterium tumefaciens-mediated gene transfer. Several transgenic lines were generated and molecular analysis was performed to confirm transgene integration. Transgenic plants with an ABA-inducible promoter showed a 1.5- to two-fold increase of transgene expression under severe stress conditions. Under well-watered conditions, transgenic plants with constitutive and ABA-inducible promoters showed reduced total leaf area and shoot biomass compared to non-transgenic plants. No significant differences in root length or root biomass were observed between transgenic and non-transgenic plants under non-stress conditions. When subjected to gradual water deficit, transgenic plants maintained higher relative water content because the transgenic lines used water more slowly as a result of reduced total leaf area. This caused them to wilt slower than non-transgenic plants. Transgenic plants showed differential drought tolerance responses with a significantly higher survival rate compared to non-transgenic plants when subjected to comparable severe water-deficit conditions. Moreover, the transgenic plants also showed improved drought tolerance by maintaining 17-24 % greater leaf cell membrane stability compared to non-transgenic plants. The results demonstrate the feasibility of engineering soybean for enhanced drought tolerance by expressing stress-responsive genes.

Introduction of Genome Editing Reagents and Genotyping of Derived Edited Alleles in Soybean (Glycine max (L.) Merr.).

Cas9-based genome editing is a powerful genetic tool for loci specifically targeted for genome modification. This chapter describes up-to-date protocols using Cas9-based genome editing technology, including vector construction with GoldenBraid assembly, Agrobacterium-mediated soybean transformation, and identification of editing in the genome.

Molecular characterization and functional analysis of Glycine max sterol methyl transferase 2 genes involved in plant membrane sterol biosynthesis.

Sterol C24 methyltransferase (SMT2) genes governing the pattern of phytosterols synthesized in higher plants have been studied in Glycine seedlings and wild-type and engineered Arabidopsis thaliana plants. The SMT2 genes of soybean (SMT2-1 and SMT2-2) previously cloned and characterized (Neelakandan et al. 2009) were shown to complement the SMT deficient cvp1 mutant Arabidopsis plants, consistent with their role in regulation of 24-alkyl sterol-controlled plant physiology. Further analysis of these genes showed that environmental cues, including dehydration, cold, and abscisic acid induced differential changes in transcript levels of the SMT2 during soybean seedling growth. Sterol analyses of transgenic Arabidopsis seeds originating in variant constructs of AtHMGR1, GmSMT1, and GmSMT2 engineered in seeds showed relevant modifications in the ratio of 24-methyl to 24-ethyl sterol in the direction of sitosterol formation. To provide insight into the structural features of the sterol gene that affects transcript regulation, the upstream promoter sequences of soybean SMT2 genes were cloned and characterized. Sequence analysis revealed several important cis-elements and transcription factor binding sites. The analysis of promoter-GUS fusions in transgenic Arabidopsis plants revealed shared and distinct expression features in different developmental stages and tissues. The data are interpreted to imply that SMT2 is an important contributor to normal plant growth and development.

Stearidonic-Enriched Soybean Oil Modulates Obesity, Glucose Metabolism, and Fatty Acid Profiles Independently of Akkermansia muciniphila.

SCOPE: Previous studies have suggested that diets rich in omega-3 and low in omega-6 long-chain polyunsaturated fatty acids (PUFAs) can limit the development of metabolic syndrome (MetS). Transgenic soybeans yielding oils enriched for omega-3 PUFAs represent a new and readily-available option for incorporating omega-3 PUFAs into diets to provide health benefits. METHODS AND RESULTS: Transgenic soybean oils, enriched for either stearidonic acid (SDA) or eicosapentaenoic acid (EPA), are incorporated into diets to test their effects on limiting the development of MetS in a mouse model of diet-induced obesity. Supplementation with SDA- but not EPA-enriched oils improved features of MetS compared to feeding a control wild-type oil. Because previous studies have linked the gut microorganism Akkermansia muciniphila to the metabolic effects of feeding omega-3 PUFAs, the causal contribution of A. muciniphila to mediating the metabolic benefits provided by SDA-enriched diets is investigated. Although A. muciniphila is not required for SDA-induced metabolic improvements, this microorganism does modulate levels of saturated and mono-unsaturated fatty acids in host adipose tissues. CONCLUSION: Together, these findings support the utilization of SDA-enriched diets to modulate weight gain, glucose metabolism, and fatty acid profiles of liver and adipose tissue.

Molecular characterization of stress-inducible GmNAC genes in soybean.

Drought is detrimental to plant growth and development, and often results in significant losses to the yields of economically important crops such as soybeans (Glycine max L.). NAC transcription factors (TFs), which consist of a large family of plant-specific TFs, have been reported to enhance drought tolerance in a number of plants. In this study, 31 unigenes that contain the complete open reading frames encoding GmNAC proteins were identified and cloned from soybean. Analysis of C-terminal regulatory domain using yeast one-hybrid system indicated that among 31 GmNAC proteins, 28 have transcriptional activation activity. Expression analysis of these GmNAC genes showed that they are differentially expressed in different organs, suggesting that they have diverse functions during plant growth and development. To search for the drought-inducible GmNAC genes, we prescreened and re-confirmed by quantitative real-time PCR analysis that nine GmNAC genes are induced by dehydration stress with differential induction levels in both shoot and root. The expression profiles of these nine GmNAC genes were also examined under other stresses such as high salinity, cold and with abscisic acid hormone treatments. Phylogenetic analysis of the GmNAC proteins with previously reported drought-inducible NAC proteins of Arabidopsis and rice revealed that the nine drought-inducible GmNAC proteins belong to the "stress-inducible" NAC group. The results of this systematic analysis of the GmNAC family will provide novel tools and resources for the development of improved drought tolerant transgenic soybean cultivars.

Expression of the Maize Dof1 Transcription Factor in Wheat and Sorghum.

Nitrogen is essential for plant growth and development. Improving the ability of plants to acquire and assimilate nitrogen more efficiently is a key agronomic parameter that will augment sustainability in agriculture. A transcription factor approach was pursued to address improvement of nitrogen use efficiency in two major commodity crops. To this end, the Zea mays Dof1 (ZmDof1) transcription factor was expressed in both wheat (Triticum aestivum) and sorghum (Sorghum bicolor) either constitutively, UBI4 promoter from sugarcane, or in a tissue specific fashion via the maize rbcS1 promoter. The primary transcription activation target of ZmDof1, phosphoenolpyruvate carboxylase (PEPC), is observed in transgenic wheat events. Expression ZmDof1 under control of the rbcs1 promoter translates to increase in biomass and yield components in wheat. However, constitutive expression of ZmDof1 led to the down-regulation of genes involved in photosynthesis and the functional apparatus of chloroplasts, and an outcome that negatively impacts photosynthesis, height, and biomass in wheat. Similar patterns were also observed in sorghum transgenic events harboring the constitutive expression cassette of ZmDof1. These results indicate that transcription factor strategies to boost agronomic phenotypic outcomes in crops need to consider expression patterns of the genetic elements to be introduced.

Functional analysis of water stress-responsive soybean GmNAC003 and GmNAC004 transcription factors in lateral root development in arabidopsis.

In Arabidopsis, NAC (NAM, ATAF and CUC) transcription factors have been found to promote lateral root number through the auxin signaling pathway. In the present study, the role of water stress-inducible soybean GmNAC003 and GmNAC004 genes in the enhancement of lateral root development under water deficit conditions was investigated. Both genes were highly expressed in roots, leaves and flowers of soybean and were strongly induced by water stress and moderately induced by a treatment with abscisic acid (ABA). They showed a slight response to treatment with 2,4-dichlorophenoxyacetic acid (2,4-D). The transgenic Arabidopsis plants overexpressing GmNAC004 showed an increase in lateral root number and length under non-stress conditions and maintained higher lateral root number and length under mild water stress conditions compared to the wild-type (WT), while the transgenic plants overexpressing GmNAC003 did not show any response. However, LR development of GmNAC004 transgenic Arabidopsis plants was not enhanced in the water-stressed compared to the well-watered treatment. In the treatment with ABA, LR density of the GmNAC004 transgenic Arabidopsis was less suppressed than that of the WT, suggesting that GmNAC004 counteracts ABA-induced inhibition of lateral root development. In the treatment with 2,4-D, lateral root density was enhanced in both GmNAC004 transgenic Arabidopsis and WT plants but the promotion was higher in the transgenic plants. Conversely, in the treatment with naphthylphthalamic acid (NPA), lateral root density was inhibited and there was no difference in the phenotype of the GmNAC004 transgenic Arabidopsis and WT plants, indicating that auxin is required for the action of GmNAC004. Transcript analysis for a number of known auxin and ABA related genes showed that GmNAC004's role may suppress ABA signaling but promote auxin signaling to increase lateral root development in the Arabidopsis heterologous system.

Molecular characterization of Glycine max squalene synthase genes in seed phytosterol biosynthesis.

The reaction catalyzed by squalene synthase (EC.2.5.1.21) that converts two molecules of farnesyl pyrophosphate to squalene represents a crucial branch point of the isoprenoid pathway in diverting carbon flux towards the biosynthesis of sterols. In the present study two soybean squalene synthase genes, GmSQS1 and GmSQS2, were identified in the soybean genome and functionally characterized for their roles in sterol biosynthesis. Both genes encode a deduced protein of 413 amino acids. Complementation assays showed that the two genes were able to convert yeast sterol auxotrophy erg9 mutant to sterol prototrophy. Expression of GmSQS1 and GmSQS2 was ubiquitous in roots, stem, leaves, flower and young seeds of soybean, however GmSQS1 transcript was preferential in roots while GmSQS2 transcript was more in leaves. Their expression was lower in response to dehydration treatments suggesting they might be negative regulators of water stress adaptation. Transgenic Arabidopsis plants overexpressing GmSQS1 driven by either constitutive or seed-specific promoters showed increases in the major end product sterols: campesterol, sitosterol and stigmasterol, which resulted in up to 50% increase in total sterol content in the seeds. The increase in the end product sterols by GmSQS1 overexpression was at the level achievable by previously reported overexpression of individual or combination of other key enzymes in the sterol pathway. Together the data demonstrate that soybean SQS genes play an important role in diverting carbon flux to the biosynthesis of the end product sterols in the seeds.

Evaluation of candidate reference genes for normalization of quantitative RT-PCR in soybean tissues under various abiotic stress conditions.

Quantitative RT-PCR can be a very sensitive and powerful technique for measuring differential gene expression. Changes in gene expression induced by abiotic stresses are complex and multifaceted, which make determining stably expressed genes for data normalization difficult. To identify the most suitable reference genes for abiotic stress studies in soybean, 13 candidate genes collected from literature were evaluated for stability of expression under dehydration, high salinity, cold and ABA (abscisic acid) treatments using delta CT and geNorm approaches. Validation of reference genes indicated that the best reference genes are tissue- and stress-dependent. With respect to dehydration treatment, the Fbox/ABC, Fbox/60s gene pairs were found to have the highest expression stability in the root and shoot tissues of soybean seedlings, respectively. Fbox and 60s genes are the most suitable reference genes across dehydrated root and shoot tissues. Under salt stress the ELF1b/IDE and Fbox/ELF1b are the most stably expressed gene pairs in roots and shoots, respectively, while 60s/Fbox is the best gene pair in both tissues. For studying cold stress in roots or shoots, IDE/60s and Fbox/Act27 are good reference gene pairs, respectively. With regard to gene expression analysis under ABA treatment in either roots, shoots or across these tissues, 60s/ELF1b, ELF1b/Fbox and 60s/ELF1b are the most suitable reference genes, respectively. The expression of ELF1b/60s, 60s/Fbox and 60s/Fbox genes was most stable in roots, shoots and both tissues, respectively, under various stresses studied. Among the genes tested, 60s was found to be the best reference gene in different tissues and under various stress conditions. The highly ranked reference genes identified from this study were proved to be capable of detecting subtle differences in expression rates that otherwise would be missed if a less stable reference gene was used.