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.

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.

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.

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.

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.

The OCL3 promoter from Sorghum bicolor directs gene expression to abscission and nutrient-transfer zones at the bases of floral organs.

BACKGROUND AND AIMS: During seed fill in cereals, nutrients are symplasmically unloaded to vascular parenchyma in ovules, but thereafter nutrient transport is less certain. In Zea mays, two mechanisms of nutrient passage through the chalaza and nucellus have been hypothesized, apoplasmic and symplasmic. In a recent study, nutrients first passed non-selectively to the chalazal apoplasm and were then selectively absorbed by the nucellus before being released to the endosperm apoplasm. This study reports that the promoter of OUTER CELL LAYER3 (PSbOCL3) from Sorghum bicolor (sorghum) directs gene expression to chalazal cells where the apoplasmic barrier is thought to form. The aims were to elucidate PSbOCL3 expression patterns in sorghum and relate them to processes of nutrient pathway development in kernels and to recognized functions of the homeodomain-leucine zipper (HD-Zip) IV transcription factor family to which the promoter belongs. METHODS: PSbOCL3 was cloned and transformed into sorghum as a promoter-GUS (ß-glucuronidase) construct. Plant tissues from control and transformed plants were then stained for GUS, and kernels were cleared and characterized using differential interference contrast microscopy. KEY RESULTS: A symplasmic disconnect between the chalaza and nucellus during seed fill is inferred by the combination of two phenomena: differentiation of a distinct nucellar epidermis adjacent to the chalaza, and lysis of GUS-stained chalazal cells immediately proximal to the nucellar epidermis. Compression of the GUS-stained chalazal cells during kernel maturation produced the kernel abscission zone (closing layer). CONCLUSIONS: The results suggest that the HD-Zip IV transcription factor SbOCL3 regulates kernel nutrition and abscission. The latter is consistent with evidence that members of this transcription factor group regulate silique abscission and dehiscence in Arabidopsis thaliana. Collectively, the findings suggest that processes of floral organ abscission are conserved among angiosperms and may in some respects differ from processes of leaf abscission.

Activity of the Arabidopsis RD29A and RD29B promoter elements in soybean under water stress.

The constitutive and drought-induced activities of the Arabidopsis thaliana RD29A and RD29B promoters were monitored in soybean (Glycine max (L.) Merr.] via fusions with the visual marker gene ß-glucuronidase (GUS). Physiological responses of soybean plants were monitored over 9 days of water deprivation under greenhouse conditions. Data were used to select appropriate time points to monitor the activities of the respective promoter elements. Qualitative and quantitative assays for GUS expression were conducted in root and leaf tissues, from plants under well-watered and dry-down conditions. Both RD29A and RD29B promoters were significantly activated in soybean plants subjected to dry-down conditions. However, a low level of constitutive promoter activity was also observed in both root and leaves of plants under well-watered conditions. GUS expression was notably higher in roots than in leaves. These observations suggest that the respective drought-responsive regulatory elements present in the RD29X promoters may be useful in controlling targeted transgenes to mitigate abiotic stress in soybean, provided the transgene under control of these promoters does not invoke agronomic penalties with leaky expression when no abiotic stress is imposed.

The chloroplast triggers developmental reprogramming when mutS HOMOLOG1 is suppressed in plants.

Multicellular eukaryotes demonstrate nongenetic, heritable phenotypic versatility in their adaptation to environmental changes. This inclusive inheritance is composed of interacting epigenetic, maternal, and environmental factors. Yet-unidentified maternal effects can have a pronounced influence on plant phenotypic adaptation to changing environmental conditions. To explore the control of phenotypy in higher plants, we examined the effect of a single plant nuclear gene on the expression and transmission of phenotypic variability in Arabidopsis (Arabidopsis thaliana). MutS HOMOLOG1 (MSH1) is a plant-specific nuclear gene product that functions in both mitochondria and plastids to maintain genome stability. RNA interference suppression of the gene elicits strikingly similar programmed changes in plant growth pattern in six different plant species, changes subsequently heritable independent of the RNA interference transgene. The altered phenotypes reflect multiple pathways that are known to participate in adaptation, including altered phytohormone effects for dwarfed growth and reduced internode elongation, enhanced branching, reduced stomatal density, altered leaf morphology, delayed flowering, and extended juvenility, with conversion to perennial growth pattern in short days. Some of these effects are partially reversed with the application of gibberellic acid. Genetic hemicomplementation experiments show that this phenotypic plasticity derives from changes in chloroplast state. Our results suggest that suppression of MSH1, which occurs under several forms of abiotic stress, triggers a plastidial response process that involves nongenetic inheritance.

A high-oleic-acid and low-palmitic-acid soybean: agronomic performance and evaluation as a feedstock for biodiesel.

Phenotypic characterization of soybean event 335-13, which possesses oil with an increased oleic acid content (> 85%) and reduced palmitic acid content (< 5%), was conducted across multiple environments during 2004 and 2005. Under these conditions, the stability of the novel fatty acid profile of the oil was not influenced by environment. Importantly, the novel soybean event 335-13 was not compromised in yield in both irrigated and non-irrigated production schemes. Moreover, seed characteristics, including total oil and protein, as well as amino acid profile, were not altered as a result of the large shift in the fatty acid profile. The novel oil trait was inherited in a simple Mendelian fashion. The event 335-13 was also evaluated as a feedstock for biodiesel. Extruded oil from event 335-13 produced a biodiesel with improved cold flow and enhanced oxidative stability, two critical fuel parameters that can limit the utility of this renewable transportation fuel.

Enhancing soybean photosynthetic CO2 assimilation using a cyanobacterial membrane protein, ictB.

Soybean C3 photosynthesis can suffer a severe loss in efficiency due to photorespiration and the lack of a carbon concentrating mechanism (CCM) such as those present in other plant species or cyanobacteria. Transgenic soybean (Glycine max cv. Thorne) plants constitutively expressing cyanobacterial ictB (inorganic carbon transporter B) gene were generated using Agrobacterium-mediated transformation. Although more recent data suggest that ictB does not actively transport HCO3-/CO2, there is nevertheless mounting evidence that transformation with this gene can increase higher plant photosynthesis. The hypothesis that expression of the ictB gene would improve photosynthesis, biomass production and seed yield in soybean was tested, in two independent replicated greenhouse and field trials. Results showed significant increases in photosynthetic CO2 uptake (Anet) and dry mass in transgenic relative to wild type (WT) control plants in both the greenhouse and field trials. Transgenic plants also showed increased photosynthetic rates and biomass production during a drought mimic study. The findings presented herein demonstrate that ictB, as a single-gene, contributes to enhancement in various yield parameters in a major commodity crop and point to the significant role that biotechnological approaches to increasing photosynthetic efficiency can play in helping to meet increased global demands for food.

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.

Sorghum (Sorghum bicolor).

Agrobacterium-mediated transformation of sorghum (Sorghum bicolor L. Moench) targeting immature embryo explants is a route to introduce transgenic alleles into the crop. The protocol requires maintenance of quality stock plants under greenhouse conditions for a constant supply of immature embryo explants. This is typically carried out by a regular sowing of seeds, minimal use of pesticides, and monitoring of plants to document pollen dispersal and bagging of heads. The time frame from explant inoculation to establishment of a primary transgenic event in the greenhouse typically ranges from 4 to 6 months. Seed set in the primary transformants is comparable to greenhouse-grown stock plants, with the majority of the transgenic alleles being inherited as a single functional locus.