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At least 55 records · Page 3

Data for "Viral Delivery of Recombinases Activates Heritable Genetic Switches in Plants"

Viral vectors provide an increasingly versatile platform for transformation-free reagent delivery to plants. RNA viral vectors can be used to induce gene silencing, overexpress proteins, or introduce gene editing reagents; however, they are often constrained by carrying capacity or restricted tropism in germline cells. Site-specific recombinases that catalyze precise genetic rearrangements are powerful tools for genome engineering that vary in size and, potentially, efficacy in plants. In this work, we show that viral vectors based on tobacco rattle virus (TRV) deliver and stably express four recombinases ranging in size from ∼0.6 to ∼1.5 kb and achieve simultaneous marker removal and reporter activation through targeted excision in transgenic Nicotiana benthamiana lines. TRV vectors with Cre, FLP, CinH, and Integrase13 efficiently mediated recombination in infected somatic tissue and led to heritable modifications at high frequency. An excision-activated Ruby reporter enabled simple and high-resolution tracing of infected cell lineages without the need for molecular genotyping. Together, our experiments broaden the scope of viral recombinase delivery and offer insights into infection dynamics that may be useful in developing future viral vectors.

gene editing↗

Viral delivery of recombinases activates heritable genetic switches in plants

Viral vectors provide an increasingly versatile platform for transformation-free reagent delivery to plants. RNA viral vectors can be used to induce gene silencing, overexpress proteins, or introduce gene editing reagents; however, they are often constrained by carrying capacity or restricted tropism in germline cells. Site-specific recombinases that catalyze precise genetic rearrangements are powerful tools for genome engineering that vary in size and, potentially, efficacy in plants. In this work, we show that viral vectors based on tobacco rattle virus (TRV) deliver and stably express four recombinases ranging in size from ~0.6kb to ~1.5kb and achieve simultaneous marker removal and reporter activation through targeted excision in transgenic Nicotiana benthamiana lines. TRV vectors with Cre, FLP, CinH, and Integrase13 efficiently mediated recombination in infected somatic tissue and led to heritable modifications at high frequency. Here, an excision-activated Ruby reporter enabled simple and high-resolution tracing of infected cell lineages without the need for molecular genotyping. Together, our experiments broaden the scope of viral recombinase delivery and offer insights into infection dynamics that may be useful in developing future viral vectors.

59 BASIC BIOLOGICAL SCIENCES↗

A split ribozyme system for in vivo plant RNA imaging and genetic engineering

RNA plays a central role in plants, governing various cellular and physiological processes. Monitoring its dynamic abundance provides a discerning understanding of molecular mechanisms underlying plant responses to internal (developmental) and external (environmental) stimuli, paving the way for advances in plant biotechnology to engineer crops with improved resilience, quality and productivity. In general, traditional methods for analysis of RNA abundance in plants require destructive, labour-intensive and time-consuming assays. To overcome these limitations, we developed a transformative innovation for in vivo RNA imaging in plants. Specifically, we established a synthetic split ribozyme system that converts various RNA signals to orthogonal protein outputs, enabling in vivo visualisation of various RNA signals in plants. We demonstrated the utility of this system in transient expression experiments (i.e., leaf infiltration in Nicotiana benthamiana ) to detect RNAs derived from transgenes and tobacco rattle virus, respectively. Also, we successfully engineered a split ribozyme-based biosensor in Arabidopsis thaliana for in vivo visualisation of endogenous gene expression at the cellular level, demonstrating the feasibility of multi-scale (e.g., cellular and tissue level) RNA imaging in plants. Furthermore, we developed a platform for easy incorporation of different protein outputs, allowing for flexible choice of reporters to optimise the detection of target RNAs.

59 BASIC BIOLOGICAL SCIENCES↗

Systems of artificial lighting at the Phytotron of Plant Breeding and Genetic Institute (Odessa)

At the Odessa Phytotron we have installed over 50 climatic chambers and cabinets made by various companies of the United States, Canada, Germany and U.S.S.R. They employ different light sources including Sylvania fluorescent lamps of various types, fluorescent lamps produced in the former Soviet Union with a special luminophore, ordinary tungsten lamps, xenon, mercury, mercury-iodide, sodium, etc. Our objective in lighting is that the intensity distribution over the wave lengths should be maximal in the photosynthetically active part of the spectrum and minimal in the IR part to avoid plant sterilization. Phytotrons are extremely energy consuming entities, and the large part of their energy consumption falls into the lighting category in our electric bills. Therefore, we are in a constant search of the processes to reduce energy. However, the main way to increase effectiveness would be the development of new types of light sources, which would come close to the threshold of 150 to 200 lumens per watt.

Chernozubov, Adolf↗

Loss of function alleles of PtEPSP-TF and its regulatory targets in rice

The present disclosure provides genetically modified plants, plant cells and plant tissues that show reduced lignin content as compared to a control plant which was not genetically modified. In addition, the disclosure provides methods of regulating lignin content in a plant. The disclosure also provides methods of producing bioproducts using the genetically modified plants of the instant disclosure.

Xie, Meng↗

Metabolic flux, metabolite, and transcript analysis uncover reprogramming of metabolism toward higher seed oil

Overexpression of WRINKLED1 (WRI1), a master regulator of glycolysis and fatty acid biosynthesis, together with DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1), which catalyzes the final step of triacylglycerol assembly, is a promising strategy for enhancing seed oil content. However, how these regulators coordinate system-wide metabolic reprogramming at the levels of gene expression, metabolite pools, and fluxes remains poorly understood. To address this, we performed 13 C-metabolic flux analysis, metabolomics, and transcriptomics on in vitro cultured pennycress (Thlaspi arvense L.) embryos overexpressing the native WRI1 and DGAT1 homologs. Here, in cultured embryos, WRI1/DGAT1 overexpression increased triacylglycerol accumulation by 28% while reducing protein content by 34%, relative to the wild type. Embryos showed ∼20-fold and 50-fold upregulation of WRI1 and DGAT1 along with induction of WRI1 target genes in glycolysis and fatty acid biosynthesis. Genes associated with photosynthesis and Calvin cycle functions were also upregulated, whereas genes encoding ribosomal proteins and seed storage proteins were strongly repressed, consistent with the observed lipid–protein tradeoff. Flux analysis revealed that enhanced triacylglycerol biosynthesis is supported by increased flux through the Rubisco shunt and cytosolic pyruvate kinase, while the oxidative pentose phosphate pathway and malic enzyme contributed little to NADPH or pyruvate supply. Metabolomic profiling revealed extensive perturbations in glycolytic intermediates, tricarboxylic acid cycle metabolites, and amino acids. In plant grown seeds, WRI1/DGAT1 lines also showed a modest but significant increase in total lipid content. Collectively, these findings reveal how WRI1 and DGAT1 reprogram central metabolism to enhance oil accumulation, with relevance to mature seeds.

59 BASIC BIOLOGICAL SCIENCES↗

Physiological and genetic characterization of plant growth and gravitropism in LED light sources

Among the many problems of growing plants in completely controlled environments, such as those anticipated for the space station and the CELSS program, is the need to provide light that is both adequate for photosynthesis and of proper quality for normal growth and development. NASA scientists and engineers have recently become interested in the possibility of utilizing densely packed, solid state, light emitting diodes (LED's) as a source for this light. Unlike more conventional incandescent or electrical discharge lamps, these sources are highly monochromatic and lack energy in spectral regions thought to be important for normal plant development. In addition, a recent observation by NASA scientist has suggested that infra-red LED's, that are routinely used as photographic safelights for plants grown in darkness, may interact with the ability of plants to detect gravity. In order to establish how plants respond to light from these LED light sources we carried out a series of experiments with known pigment mutants of the model mustard plant, Arabidopsis thaliana, growing in either a gravity field or on a clinostat to simulate a micro-gravity environment. Results indicate that only red light from the 665 nm LED's disrupts the ability of normal wildtype seedlings to detect a gravity stimulus. There was no consistent effect found for the far-red (735 nm) LED's or either of the infrared (880 nm or 935 nm) LED sources but both showed some effect in one or more of the genotypes tested. Of these five members of the phytochrome multigene family in Arabidopsis, only the phytochrome B pigment mutant (hy3) lacked the ability to detect gravity under all conditions. There was no effect of either micro-gravity (clinostat) or the infra-red LED's on the light induced inhibition of hypocotyl elongation. Measurements of the pigment phytochrome in oats also showed no photoconversion by 15 min irradiations with the infra-red LED's. We conclude that phytochrome B is required for the perception of gravity and that only red light is able to disrupt this perception. The infra-red LED's also do not appear to interact with gravity perception in Arabidopsis, but caution should be exercised if infra-red LED's are to be used as photographic safelights for these types of experiments.

Deitzer, Gerald F.↗

Molecular genetic analysis of plant gravitropism

The analysis of mutants is a powerful approach for elucidating the components of complex biological processes. A growing number of mutants have been isolated which affect plant gravitropism and the classes of mutants found thus far provide important information about the gravity response mechanism. The wide variety of mutants isolated, especially in Arabidopsis, indicates that gravitropism is a complex, multi-step process. The existence of mutants altered in either root gravitropism alone, shoot gravitropism alone, or both indicates that the root and shoot gravitropic mechanisms have both separate and common steps. Reduced starch mutants have confirmed the role of amyloplasts in sensing the gravity signal. The hormone auxin is thought to act as the transducing signal between the sites of gravity perception (the starch parenchyma cells surrounding the vascular tissue in shoots and the columella cells of root caps) and asymmetric growth (the epidermal cells of the elongation zone(s) of each organ). To date, all mutants that are resistant to high concentrations of auxin have also been found to exhibit a reduced gravitropic response, thus supporting the role of auxin. Not all gravitropic mutants are auxin-resistant, however, indicating that there are additional steps which do not involve auxin. Studies with mutants of tomato which exhibit either reduced or reversed gravitropic responses further support the role of auxin redistribution in gravitropism and suggest that both red light and cytokinin interact with gravitropism through controlling lateral auxin transport. Plant responses to gravity thus likely involve changes in both auxin transport and sensitivity.

Review↗

Which Plant Traits Increase Soil Carbon Sequestration? Empirical Evidence From a Long‐Term Poplar Genetic Diversity Trial

Plants play a key role in mediating soil response to global change, and breeding or engineering crops to increase soil organic carbon (SOC) storage is a potential route to land-based carbon dioxide removal in agricultural systems. However, due to limited observational datasets plus shifting paradigms of SOC stabilization, it is unclear which plant traits are most important for enhancing different types of soil organic matter. Existing long-term common gardens of genetically diverse plant populations may provide an opportunity to evaluate biological controls on SOC, separate from environmental or management variability. Here we report on soil and root chemical data collected for 24 genotypes within a 13-year-old common garden in northwestern Oregon planted with a large natural variant population of Populus trichocarpa. Fractionating surface soil (0–15 cm) revealed substantial variation in stocks of mineral-associated organic matter (MAOM; 18–67 t C/ha) and particulate organic matter (POM; 2–22 t C/ha). Tree genotype explained 24% and 26% of the MAOM and POM stock variability, respectively, after controlling for background variability. We found minimal association between SOC concentration and either aboveground tree productivity or root biomass recalcitrance (C/N ratios and lignin content). In contrast, root elemental content appeared influential for MAOM-C concentration, which showed a strong positive association with root aluminum (Al) and a strong negative association with root boron (B) and magnesium (Mg). Furthermore, root concentrations of these elements were highly heritable (57%–78%) and not simply a reflection of background variation in soil elemental concentrations. We estimate that surface SOC stocks under these 24 genotypes have diverged at rates of up to 1.2–4.3 t C/ha/year. These results suggest that long-term genetic diversity trials have value for elucidating biological controls on soil organic matter dynamics, and that traits associated with root elemental content may be a useful target for enhancing biosequestration.

biomass recalcitrance↗

Arabidopsis alcohol dehydrogenase expression in both shoots and roots is conditioned by root growth environment

It is widely accepted that the Arabidopsis Adh (alcohol dehydrogenase) gene is constitutively expressed at low levels in the roots of young plants grown on agar media, and that the expression level is greatly induced by anoxic or hypoxic stresses. We questioned whether the agar medium itself created an anaerobic environment for the roots upon their growing into the gel. beta-Glucuronidase (GUS) expression driven by the Adh promoter was examined by growing transgenic Arabidopsis plants in different growing systems. Whereas roots grown on horizontal-positioned plates showed high Adh/GUS expression levels, roots from vertical-positioned plates had no Adh/GUS expression. Additional results indicate that growth on vertical plates closely mimics the Adh/GUS expression observed for soil-grown seedlings, and that growth on horizontal plates results in induction of high Adh/GUS expression that is consistent with hypoxic or anoxic conditions within the agar of the root zone. Adh/GUS expression in the shoot apex is also highly induced by root penetration of the agar medium. This induction of Adh/GUS in shoot apex and roots is due, at least in part, to mechanisms involving Ca2+ signal transduction.

NASA Discipline Plant Biology↗

Site-directed mutagenesis of serine 158 demonstrates its role in spinach leaf sucrose-phosphate synthase modulation

Site-directed mutagenesis of spinach sucrose-phosphate synthase (SPS) was performed to investigate the role of Ser158 in the modulation of spinach leaf SPS. Tobacco plants expressing the spinach wild-type (WT), S158A, S158T and S157F/S158E SPS transgenes were produced. Expression of transgenes appeared not to reduce expression of the tobacco host SPS. SPS activity in the WT and the S158T SPS transgenics showed light/dark modulation, whereas the S158A and S157F/S158E mutants were not similarly light/dark modulated: the S158A mutant enzyme was not inactivated in the dark, and the S157F/S158E was not activated in the light. The inability to modulate the activity of the S158A mutant enzyme by protein phosphorylation was demonstrated in vitro. The WT spinach enzyme immunopurified from dark transgenic tobacco leaves had a low initial activation state, and could be activated by PP2A and subsequently inactivated by SPS-kinase plus ATP. Rapid purification of the S158A mutant enzyme from dark leaves of transgenic plants using spinach-specific monoclonal antibodies yielded enzyme that had a high initial activation state, and pre-incubation with leaf PP2A or ATP plus SPS-kinase (the PKIII enzyme) caused little modulation of activity. The results demonstrate the regulatory significance of Ser158 as the major site responsible for dark inactivation of spinach SPS in vivo, and indicate that the significance of phosphorylation is the introduction of a negative charge at the Ser158 position.

Non-NASA Center↗

Multi-trait multi-environment genomic prediction strategies for Miscanthus sacchariflorus

Genomic selection holds the potential to serve as a strategic tool to enhance the genetic gain of complex traits in Miscanthus breeding programs. The development of improved cultivars requires their assessment for various traits across diverse environments to ensure suitable overall performance. Hence, the multi-trait multi-environment (MTME) genomic prediction (GP) models offer an opportunity to improve selection accuracy. This study aims to evaluate the potential of five GP models: (1) three MTME models including genotype-by-trait-by-environment interaction (G×E×T) and (2) two single-trait multi-environment (STME) models (with and without G×E interaction). A Miscanthus sacchariflorus population comprising 336 genotypes evaluated in three environments and scored for four traits (biomass yield YDY, total culm number TCM, average internode length AIL, and culm node number CNN) was analyzed. The predictive ability of the models was evaluated considering three cross-validation schemes resembling realistic scenarios (CV1: predicting new genotypes, CVP: predicting missing traits in a given environment, and CV2: predicting partially observed genotypes). On average, in all cross-validation schemes compared to the STME the predictive ability of the MTME models was 10% to 70% higher for TCM and AIL. On the other hand, for YDY and CNN, both STME models performed similarly or slightly better (between 5 to 64%) than the MTME models in most environments. While the MTME models were not successful for all traits when compared to their STME counterparts, MTME models improved the prediction of the performance of genotypes that were untested across environments or lacked trait information in a specific environment. Overall, our study suggests that MTME GP models can be implemented in Miscanthus breeding programs to improve the predictive ability of the complex traits, shorten breeding cycles, and accelerate selection decisions.

genomic prediction (GP)↗

Optimizing resource allocation in Miscanthus breeding via sparse testing designs for genomic prediction

Phenotyping high-biomass perennial crops is laborious and the rate of genetic gain in conventional perennial crop breeding programs is typically low. So, it is especially important to identify methods that produce efficiency gains in the breeding process. Miscanthus is a C4 perennial grass with favorable characteristics for producing biomass as a feedstock for biofuels and diverse bio-based products. Increasing biomass yield will increase profitability and environmental benefits, so it is a key target for Miscanthus breeding. In addition, the identification of well-adapted genotypes across a wide range of environmental conditions requires the establishment of multi-environment trials (METs). Sparse testing is a genomic prediction-based strategy that reduces the phenotyping costs in METs by selecting a subset of genotypes to evaluate in a subset of environments and then predicts the performance of the unobserved genotype-environment combinations. A Miscanthus sacchariflorus (MSA) population comprising 336 genotypes observed across three environments was analyzed implementing sparse testing designs. Three prediction models considering main effects (environments, genotypes, genomic) and interaction effects (genotype-by-environment; G×E interaction) were implemented for forecasting dry biomass yield (YDY), total culm (TCM), average internode length (AIL), and culm node number (CNN). Multiple calibration sets based on different compositions and sizes were considered to evaluate performance in terms of the predictive ability (PA) and the mean square error (MSE) for a fixed testing set size. The training set size ranged from 52 to 112 to predict a fixed set of 224 unobserved genotypes across all three environments. The results showed that the model accounting for G×E interaction consistently presented the highest PA and the lowest MSE: for CNN (PA: ~0.77, MSE: ~0.5) and YDY (PA: ~0.70, MSE: ~1.3) while for TCM and AIL these ranged from ~0.28 to 0.41 and ~1.3 to 4.3, respectively. Overall, varying training sets and allocation strategies did not affect PA and MSE, with 52 non-overlapping and 0 overlapping genotypes per environment as the optimal cost-effective allocation framework. This suggests that implementing sparse testing designs could significantly reduce phenotyping costs by fivefold, without compromising PA in breeding programs for perennial crops such as Miscanthus.

Miscanthus sacchariflorus (MSA)↗

Enhanced microbial production of protocatechuate from engineered sorghum using an integrated feedstock-to-product conversion technology

Building a stronger bioeconomy requires production capabilities that are largely generated through microbial genetic engineering. Plant feedstocks can additionally be genetically engineered to generate desirable feedstock traits and provide precursors for direct microbial conversion into desired products. The oleaginous yeast Rhodosporidium toruloides is a promising organism for this type of conversion as it can grow on a wide range of deconstructed biomass and consume a variety of carbon sources. Here, we leveraged R. toruloides native p-coumaric acid consumption pathway to accumulate protocatechuate (PCA) from 4-hydroxybenzoate (4HBA) released from a sorghum feedstock line genetically engineered to overproduce 4HBA. We did so by generating and evaluating an R. toruloides strain that accumulates PCA, RSΔ12623. We then show that at two scales a cholinium lysinate pretreatment with enzymatic saccharification successfully extracts 95% of the 4HBA from the engineered sorghum biomass while producing deconstructed lignin that can be more efficiently depolymerized in a subsequent thermochemical reaction. We also demonstrate that strain RSΔ12623 can convert more than 95% of 4HBA to PCA while consuming >95% of the glucose and >80% of the xylose present in sorghum hydrolysates. Finally, to evaluate the scalability of such fermentations, we conducted the conversion of 4HBA to PCA in a 2 L bioreactor under controlled conditions. Importantly, this work demonstrates the potential of purposefully producing aromatic precursors in planta that can be liberated during biomass deconstruction for direct microbial conversion to desirable bioproducts.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Transgenics in crops

With rapid world population growth and declining availability of fresh water and arable land, a new technology is urgently needed to enhance agricultural productivity. Recent discoveries in the field of crop transgenics clearly demonstrate the great potential of this technology for increasing food production and improving food quality while preserving the environment for future generations. In this review, we briefly discuss some of the recent achievements in crop improvement that have been made using gene transfer technology.

Review↗

Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants

An efficient protocol for the production of transgenic Brassica napus cv. Westar plants was developed by optimizing two important parameters: preconditioning time and co-cultivation time. Agrobacterium tumefaciens-mediated transformation was performed using hypocotyls as explant tissue. Two variants of a green fluorescent protein (GFP)-encoding gene--mGFP5-ER and eGFP--both under the constitutive expression of the cauliflower mosaic virus 35S promoter, were used for the experiments. Optimizing the preconditioning time to 72 h and co-cultivation time with Agrobacterium to 48 h provided the increase in the transformation efficiency from a baseline of 4% to 25%. With mGFP5-ER, the transformation rate was 17% and with eGFP it was 25%. Transgenic shoots were selected on 200 mg/l kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 10 g/l sucrose and 0.5 mg/l indole butyric acid in the presence of kanamycin.

NASA Discipline Life Sciences Technologies↗