Characterization of Soybean Events with Enhanced Expression of the Microtubule-Associated Protein 65-1 (MAP65-1).

Microtubule-associated protein 65-1 (MAP65-1) protein plays an essential role in plant cellular dynamics through impacting stabilization of the cytoskeleton by serving as a crosslinker of microtubules. The role of MAP65-1 in plants has been associated with phenotypic outcomes in response to various environmental stresses. The Arabidopsis MAP65-1 (AtMAP65-1) is a known virulence target of plant bacterial pathogens and is thus a component of plant immunity. Soybean events were generated that carry transgenic alleles for both AtMAP65-1 and GmMAP65-1, the soybean AtMAP65-1 homolog, under control of cauliflower mosaic virus 35S promoter. Both AtMAP65-1 and GmMAP65-1 transgenic soybeans are more resistant to challenges by the soybean bacterial pathogen Pseudomonas syringae pv. glycinea and the oomycete pathogen Phytophthora sojae, but not the soybean cyst nematode, Heterodera glycines. Soybean plants expressing AtMAP65-1 and GmMAP65-1 also display a tolerance to the herbicide oryzalin, which has a mode of action to destabilize microtubules. In addition, GmMAP65-1-expressing soybean plants show reduced cytosol ion leakage under freezing conditions, hinting that ectopic expression of GmMAP65-1 may enhance cold tolerance in soybean. Taken together, overexpression of AtMAP65-1 and GmMAP65-1 confers tolerance of soybean plants to various biotic and abiotic stresses. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Editing the 19 kDa alpha-zein gene family generates non-opaque2-based quality protein maize.

Maize grain is deficient in lysine. While the opaque2 mutation increases grain lysine, o2 is a transcription factor that regulates a wide network of genes beyond zeins, which leads to pleiotropic and often negative effects. Additionally, the drastic reduction in 19 kDa and 22 kDa alpha-zeins causes a floury kernel, unsuitable for agricultural use. Quality protein maize (QPM) overcame the undesirable kernel texture through the introgression of modifying alleles. However, QPM still lacks a functional o2 transcription factor, which has a penalty on non-lysine amino acids due to the o2 mutation. CRISPR/cas9 gives researchers the ability to directly target genes of interest. In this paper, gene editing was used to specifically target the 19 kDa alpha zein gene family. This allows for proteome rebalancing to occur without an o2 mutation and without a total alpha-zein knockout. The results showed that editing some, but not all, of the 19 kDa zeins resulted in up to 30% more lysine. An edited line displayed an increase of 30% over the wild type. While not quite the 55% lysine increase displayed by QPM, the line had little collateral impact on other amino acid levels compared to QPM. Additionally, the edited line containing a partially reduced 19 kDa showed an advantage in kernel texture that had a complete 19 kDa knockout. These results serve as proof of concept that editing the 19 kDa alpha-zein family alone can enhance lysine while retaining vitreous endosperm and a functional O2 transcription factor.

Greater aperture counteracts effects of reduced stomatal density on WUE: a case study on sugarcane and meta-analysis.

Stomata regulate CO2 and water vapor exchange between leaves and the atmosphere. Stomata are a target for engineering to improve crop intrinsic water use efficiency (iWUE). One example is by expressing genes that lower stomatal density (SD) and reduce stomatal conductance (gsw). However, the quantitative relationship between reduced SD, gsw, and the mechanisms underlying it is poorly understood. We addressed this knowledge gap using low-SD sugarcane (Saccharum spp. hybrid) as a case study alongside a meta-analysis of data from 10 species. Transgenic expression of EPIDERMAL PATTERNING FACTOR 2 from Sorghum bicolor (SbEFP2) in sugarcane reduced SD by 26-38% but did not affect gsw compared to wildtype. Further, no changes occurred in stomatal complex size or proxies for photosynthetic capacity. Measurements of gas exchange at low CO2 concentrations that promote complete stomatal opening to normalize aperture size between genotypes were combined with modeling of maximum gsw from anatomical data. These data suggest that increased stomatal aperture is the only possible explanation for maintaining gsw when SD is reduced. Meta-analysis across C3 dicots, C3 monocots, and C4 monocots revealed engineered reductions in SD are strongly correlated with lower gsw (r2=0.60-0.98), but this response is damped relative to the change in anatomy.

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 malic enzyme reveals subcellular carbon partitioning for storage reserve production in soybeans.

Central metabolism produces amino and fatty acids for protein and lipids that establish seed value. Biosynthesis of storage reserves occurs in multiple organelles that exchange central intermediates including two essential metabolites, malate, and pyruvate that are linked by malic enzyme. Malic enzyme can be active in multiple subcellular compartments, partitioning carbon and reducing equivalents for anabolic and catabolic requirements. Prior studies based on isotopic labeling and steady-state metabolic flux analyses indicated malic enzyme provides carbon for fatty acid biosynthesis in plants, though genetic evidence confirming this role is lacking. We hypothesized that increasing malic enzyme flux would alter carbon partitioning and result in increased lipid levels in soybeans. Homozygous transgenic soybean plants expressing Arabidopsis malic enzyme alleles, targeting the translational products to plastid or outside the plastid during seed development, were verified by transcript and enzyme activity analyses, organelle proteomics, and transient expression assays. Protein, oil, central metabolites, cofactors, and acyl-acyl carrier protein (ACPs) levels were quantified overdevelopment. Amino and fatty acid levels were altered resulting in an increase in lipids by 0.5-2% of seed biomass (i.e. 2-9% change in oil). Subcellular targeting of a single gene product in central metabolism impacts carbon and reducing equivalent partitioning for seed storage reserves in soybeans.

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.

InMut-finder: a software tool for insertion identification in mutagenesis using Nanopore long reads.

BACKGROUND: Biological mutagens (such as transposon) with sequences inserted, play a crucial role to link observed phenotype and genotype in reverse genetic studies. For this reason, accurate and efficient software tools for identifying insertion sites based on the analysis of sequencing reads are desired. RESULTS: We developed a bioinformatics tool, a Finder, to identify genome-wide Insertions in Mutagenesis (named as "InMut-Finder"), based on target sequences and flanking sequences from long reads, such as Oxford Nanopore Sequencing. InMut-Finder succeeded in identify > 100 insertion sites in Medicago truncatula and soybean mutants based on sequencing reads of whole-genome DNA or enriched insertion-site DNA fragments. Insertion sites discovered by InMut-Finder were validated by PCR experiments. CONCLUSION: InMut-Finder is a comprehensive and powerful tool for automated insertion detection from Nanopore long reads. The simplicity, efficiency, and flexibility of InMut-Finder make it a valuable tool for functional genomics and forward and reverse genetics. InMut-Finder was implemented with Perl, R, and Shell scripts, which are independent of the OS. The source code and instructions can be accessed at https://github.com/jsg200830/InMut-Finder .

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.

Metabolic engineering of soybean seeds for enhanced vitamin E tocochromanol content and effects on oil antioxidant properties in polyunsaturated fatty acid-rich germplasm.

Soybean seeds produce oil enriched in oxidatively unstable polyunsaturated fatty acids (PUFAs) and are also a potential biotechnological platform for synthesis of oils with nutritional omega-3 PUFAs. In this study, we engineered soybeans for seed-specific expression of a barley homogentisate geranylgeranyl transferase (HGGT) transgene alone and with a soybean γ-tocopherol methyltransferase (γ-TMT) transgene. Seeds for HGGT-expressing lines had 8- to 10-fold increases in total vitamin E tocochromanols, principally as tocotrienols, with little effect on seed oil or protein concentrations. Tocochromanols were primarily in δ- and γ-forms, which were shifted largely to α- and ß-tocochromanols with γ-TMT co-expression. We tested whether oxidative stability of conventional or PUFA-enhanced soybean oil could be improved by metabolic engineering for increased vitamin E antioxidants. Selected lines were crossed with a stearidonic acid (SDA, 18:4Δ6,9,12,15)-producing line, resulting in progeny with oil enriched in SDA and α- or γ-linoleic acid (ALA, 18:3Δ9,12,15 or GLA, 18:3Δ6,9,12), from transgene segregation. Oil extracted from HGGT-expressing lines had ≥6-fold increase in free radical scavenging activity compared to controls. However, the oxidative stability index of oil from vitamin E-enhanced lines was ~15% lower than that of oil from non-engineered seeds and nearly the same or modestly increased in oil from the GLA, ALA and SDA backgrounds relative to controls. These findings show that soybean is an effective platform for producing high levels of free-radical scavenging vitamin E antioxidants, but this trait may have negative effects on oxidative stability of conventional oil or only modest improvement of the oxidative stability of PUFA-enhanced oil.

Expression of the Arabidopsis WRINKLED 1 transcription factor leads to higher accumulation of palmitate in soybean seed.

Soybean (Glycine max [L.] Merr.) is a commodity crop highly valued for its protein and oil content. The high percentage of polyunsaturated fatty acids in soybean oil results in low oxidative stability, which is a key parameter for usage in baking, high temperature frying applications, and affects shelf life of packaged products containing soybean oil. Introduction of a seed-specific expression cassette carrying the Arabidopsis transcription factor WRINKLED1 (AtWRI1) into soybean, led to seed oil with levels of palmitate up to approximately 20%. Stacking of the AtWRI1 transgenic allele with a transgenic locus harbouring the mangosteen steroyl-ACP thioesterase (GmFatA) resulted in oil with total saturates up to 30%. The creation of a triple stack in soybean, wherein the AtWRI1 and GmFatA alleles were combined with a FAD2-1 silencing allele led to the synthesis of an oil with 28% saturates and approximately 60% oleate. Constructs were then assembled that carry a dual FAD2-1 silencing element/GmFatA expression cassette, alone or combined with an AtWRI1 cassette. These plasmids are designated pPTN1289 and pPTN1301, respectively. Transgenic events carrying the T-DNA of pPTN1289 displayed an oil with stearate levels between 18% and 25%, and oleate in the upper 60%, with reduced palmitate (<5%). While soybean events harboring transgenic alleles of pPTN1301 had similar levels of stearic and oleate levels as that of the pPTRN1289 events, but with levels of palmitate closer to wild type. The modified fatty acid composition results in an oil with higher oxidative stability, and functionality attributes for end use in baking applications.

Mapping of transgenic alleles in soybean using a nanopore-based sequencing strategy.

Transgenic technology was developed to introduce transgenes into various organisms to validate gene function and add genetic variations >40 years ago. However, the identification of the transgene insertion position is still challenging in organisms with complex genomes. Here, we report a nanopore-based method to map the insertion position of a Ds transposable element originating in maize in the soybean genome. In this method, an oligo probe is used to capture the DNA fragments containing the Ds element from pooled DNA samples of transgenic soybean plants. The Ds element-enriched DNAs are then sequenced using the MinION-based platform of Nanopore. This method allowed us to rapidly map the Ds insertion positions in 51 transgenic soybean lines through a single sequencing run. This strategy is high throughput, convenient, reliable, and cost-efficient. The transgenic allele mapping protocol can be easily translated to other eukaryotes with complex genomes.

A novel R3 MYB transcriptional repressor associated with the loss of floral pigmentation in Iochroma.

Losses of floral pigmentation represent one of the most common evolutionary transitions in flower color, yet the genetic basis for these changes has been elucidated in only a handful of cases. Here we used crossing studies, bulk-segregant RNA sequencing, phylogenetic analyses and functional tests to identify the gene(s) responsible for the transition to white flowers in Iochroma loxense. Crosses between I. loxense and its blue-flowered sister species, I. cyaneum, suggested that a single locus controls the flower color difference and that the white allele causes a nearly complete loss of pigmentation. Examining sequence variation across phenotypic pools from the crosses, we found that alleles at a novel R3 MYB transcription factor were tightly associated with flower color variation. This gene, which we term MYBL1, falls into a class of MYB transcriptional repressors and, accordingly, higher expression of this gene is associated with downregulation of multiple anthocyanin pigment pathway genes. We confirmed the repressive function of MYBL1 through stable transformation of Nicotiana. The mechanism underlying the evolution of white flowers in I. loxense differs from that uncovered in previous studies, pointing to multiple mechanisms for achieving fixed transitions in flower color intensity.

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.

Towards the development of a sustainable soya bean-based feedstock for aquaculture.

Soya bean (Glycine max (L.) Merr.) is sought after for both its oil and protein components. Genetic approaches to add value to either component are ongoing efforts in soya bean breeding and molecular biology programmes. The former is the primary vegetable oil consumed in the world. Hence, its primary usage is in direct human consumption. As a means to increase its utility in feed applications, thereby expanding the market of soya bean coproducts, we investigated the simultaneous displacement of marine ingredients in aquafeeds with soya bean-based protein and a high Omega-3 fatty acid soya bean oil, enriched with alpha-linolenic and stearidonic acids, in both steelhead trout (Oncorhynchus mykiss) and Kampachi (Seriola rivoliana). Communicated herein are aquafeed formulations with major reduction in marine ingredients that translates to more total Omega-3 fatty acids in harvested flesh. Building off of these findings, subsequent efforts were directed towards a genetic strategy that would translate to a prototype design of an optimal identity-preserved soya bean-based feedstock for aquaculture, whereby a multigene stack approach for the targeted synthesis of two value-added output traits, eicosapentaenoic acid and the ketocarotenoid, astaxanthin, were introduced into the crop. To this end, the systematic introduction of seven transgenic cassettes into soya bean, and the molecular and phenotypic evaluation of the derived novel events are described.

Expression of cyanobacterial FBP/SBPase in soybean prevents yield depression under future climate conditions.

Predictions suggest that current crop production needs to double by 2050 to meet global food and energy demands. Based on theory and experimental studies, overexpression of the photosynthetic enzyme sedoheptulose-1,7-bisphosphatase (SBPase) is expected to enhance C3 crop photosynthesis and yields. Here we test how expression of the cyanobacterial, bifunctional fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) affects carbon assimilation and seed yield (SY) in a major crop (soybean, Glycine max). For three growing seasons, wild-type (WT) and FBP/SBPase-expressing (FS) plants were grown in the field under ambient (400 µmol mol−1) and elevated (600 µmol mol−1) CO2 concentrations [CO2] and under ambient and elevated temperatures (+2.7 °C during daytime, +3.4 °C at night) at the SoyFACE research site. Across treatments, FS plants had significantly higher carbon assimilation (4­14%), Vc,max (5­8%), and Jmax (4­8%). Under ambient [CO2], elevated temperature led to significant reductions of SY of both genotypes by 19­31%. However, under elevated [CO2] and elevated temperature, FS plants maintained SY levels, while the WT showed significant reductions between 11% and 22% compared with plants under elevated [CO2] alone. These results show that the manipulation of the photosynthetic carbon reduction cycle can mitigate the effects of future high CO2 and high temperature environments on soybean yield.

An evaluation of new and established methods to determine T-DNA copy number and homozygosity in transgenic plants.

Stable transformation of plants is a powerful tool for hypothesis testing. A rapid and reliable evaluation method of the transgenic allele for copy number and homozygosity is vital in analysing these transformations. Here the suitability of Southern blot analysis, thermal asymmetric interlaced (TAIL-)PCR, quantitative (q)PCR and digital droplet (dd)PCR to estimate T-DNA copy number, locus complexity and homozygosity were compared in transgenic tobacco. Southern blot analysis and ddPCR on three generations of transgenic offspring with contrasting zygosity and copy number were entirely consistent, whereas TAIL-PCR often underestimated copy number. qPCR deviated considerably from the Southern blot results and had lower precision and higher variability than ddPCR. Comparison of segregation analyses and ddPCR of T1 progeny from 26 T0 plants showed that at least 19% of the lines carried multiple T-DNA insertions per locus, which can lead to unstable transgene expression. Segregation analyses failed to detect these multiple copies, presumably because of their close linkage. This shows the importance of routine T-DNA copy number estimation. Based on our results, ddPCR is the most suitable method, because it is as reliable as Southern blot analysis yet much faster. A protocol for this application of ddPCR to large plant genomes is provided.

Metabolic engineering of oilseed crops to produce high levels of novel acetyl glyceride oils with reduced viscosity, freezing point and calorific value.

Seed oils have proved recalcitrant to modification for the production of industrially useful lipids. Here, we demonstrate the successful metabolic engineering and subsequent field production of an oilseed crop with the highest accumulation of unusual oil achieved so far in transgenic plants. Previously, expression of the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene in wild-type Arabidopsis seeds resulted in the accumulation of 45 mol% of unusual 3-acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) in the seed oil (Durrett et al., 2010 PNAS 107:9464). Expression of EaDAcT in dgat1 mutants compromised in their ability to synthesize regular triacylglycerols increased acetyl-TAGs to 65 mol%. Camelina and soybean transformed with the EaDAcT gene accumulate acetyl-triacylglycerols (acetyl-TAGs) at up to 70 mol% of seed oil. A similar strategy of coexpression of EaDAcT together with RNAi suppression of DGAT1 increased acetyl-TAG levels to up to 85 mol% in field-grown transgenic Camelina. Additionally, total moles of triacylglycerol (TAG) per seed increased 20%. Analysis of the acetyl-TAG fraction revealed a twofold reduction in very long chain fatty acids (VLCFA), consistent with their displacement from the sn-3 position by acetate. Seed germination remained high, and seedlings were able to metabolize the stored acetyl-TAGs as rapidly as regular triacylglycerols. Viscosity, freezing point and caloric content of the Camelina acetyl-TAG oils were reduced, enabling use of this oil in several nonfood and food applications.

Expression of Chlorovirus MT325 aquaglyceroporin (aqpv1) in tobacco and its role in mitigating drought stress.

MAIN CONCLUSIONS: A Chlorovirus aquaglyceroporin expressed in tobacco is localized to the plastid and plasma membranes. Transgenic events display improved response to water deficit. Necrosis in adult stage plants is observed. Aquaglyceroporins are a subclass of the water channel aquaporin proteins (AQPs) that transport glycerol along with other small molecules transcellular in addition to water. In the studies communicated herein, we analyzed the expression of the aquaglyceroporin gene designated, aqpv1, from Chlorovirus MT325, in tobacco (Nicotiana tabacum), along with phenotypic changes induced by aqpv1 expression in planta. Interestingly, aqpv1 expression under control of either a constitutive or a root-preferred promoter, triggered local lesion formation in older leaves, which progressed significantly after induction of flowering. Fusion of aqpv1 with GFP suggests that the protein localized to the plasmalemma, and potentially with plastid and endoplasmic reticulum membranes. Physiological characterizations of transgenic plants during juvenile stage growth were monitored for potential mitigation to water dry-down (i.e., drought) and recovery. Phenotypic analyses on drought mimic/recovery of juvenile transgenic plants that expressed a functional aqpv1 transgene had higher photosynthetic rates, stomatal conductance, and water use efficiency, along with maximum carboxylation and electron transport rates when compared to control plants. These physiological attributes permitted the juvenile aqpv1 transgenic plants to perform better under drought-mimicked conditions and hastened recovery following re-watering. This drought mitigation effect is linked to the ability of the transgenic plants to maintain cell turgor.

Stacking of a stearoyl-ACP thioesterase with a dual-silenced palmitoyl-ACP thioesterase and ∆12 fatty acid desaturase in transgenic soybean.

Soybean (Glycine max (L.) Merr) is valued for both its protein and oil, whose seed is composed of 40% and 20% of each component, respectively. Given its high percentage of polyunsaturated fatty acids, linoleic acid and linolenic acid, soybean oil oxidative stability is relatively poor. Historically food processors have employed a partial hydrogenation process to soybean oil as a means to improve both the oxidative stability and functionality in end-use applications. However, the hydrogenation process leads to the formation of trans-fats, which are associated with negative cardiovascular health. As a means to circumvent the need for the hydrogenation process, genetic approaches are being pursued to improve oil quality in oilseeds. In this regard, we report here on the introduction of the mangosteen (Garcinia mangostana) stearoyl-ACP thioesterase into soybean and the subsequent stacking with an event that is dual-silenced in palmitoyl-ACP thioesterase and ∆12 fatty acid desaturase expression in a seed-specific fashion. Phenotypic analyses on transgenic soybean expressing the mangosteen stearoyl-ACP thioesterase revealed increases in seed stearic acid levels up to 17%. The subsequent stacked with a soybean event silenced in both palmitoyl-ACP thioesterase and ∆12 fatty acid desaturase activity, resulted in a seed lipid phenotype of approximately 11%-19% stearate and approximately 70% oleate. The oil profile created by the stack was maintained for four generations under greenhouse conditions and a fifth generation under a field environment. However, in generation six and seven under field conditions, the oleate levels decreased to 30%-40%, while the stearic level remained elevated.