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      Enhanced Photosynthetic Efficiency for Increased Carbon Assimilation and Woody Biomass Production in Engineered Hybrid Poplar

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          Abstract

          Increasing CO2 levels in the atmosphere and the resulting negative impacts of climate change have compelled global efforts to achieve carbon neutrality or negativity. Most such efforts focus on carbon sequestration through chemical or physical approaches. Harnessing the power of synthetic biology to enhance the natural ability of carbon sequestration in plants, especially non-annuals, provides a biological approach to further reduce CO2 levels in the air. Here, we selected a photorespiration bypass pathway and tested its effectiveness on photosynthetic enhancement in a hybrid poplar, INRA717-IB4. The design includes an RNAi strategy to reduce the transportation of the photorespiration byproduct, glycolate, out of chloroplast and a shunt pathway to metabolize the retained glycolate back to CO2 for fixation through the Calvin-Benson cycle. Molecular and physiological data collected from two separate growth experiments indicate that transgenic plants expressing genes in the photorespiration bypass pathway have increased photosynthetic efficiency, leading to faster plant growth and elevated biomass production. One lead transgenic event accumulated 35%–53% more above-ground dry biomass over four months of growth in a controlled environment. Our results provide a proof of concept for engineering trees to help combat climate change.

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          Construct design for efficient, effective and high-throughput gene silencing in plants.

          Post-transcriptional silencing of plant genes using anti-sense or co-suppression constructs usually results in only a modest proportion of silenced individuals. Recent work has demonstrated the potential for constructs encoding self-complementary 'hairpin' RNA (hpRNA) to efficiently silence genes. In this study we examine design rules for efficient gene silencing, in terms of both the proportion of independent transgenic plants showing silencing, and the degree of silencing. Using hpRNA constructs containing sense/anti-sense arms ranging from 98 to 853 nt gave efficient silencing in a wide range of plant species, and inclusion of an intron in these constructs had a consistently enhancing effect. Intron-containing constructs (ihpRNA) generally gave 90-100% of independent transgenic plants showing silencing. The degree of silencing with these constructs was much greater than that obtained using either co-suppression or anti-sense constructs. We have made a generic vector, pHANNIBAL, that allows a simple, single PCR product from a gene of interest to be easily converted into a highly effective ihpRNA silencing construct. We have also created a high-throughput vector, pHELLSGATE, that should facilitate the cloning of gene libraries or large numbers of defined genes, such as those in EST collections, using an in vitro recombinase system. This system may facilitate the large-scale determination and discovery of plant gene functions in the same way as RNAi is being used to examine gene function in Caenorhabditis elegans.
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            Macromolecular modeling and design in Rosetta: recent methods and frameworks

            The Rosetta software for macromolecular modeling, docking and design is extensively used in laboratories worldwide. During two decades of development by a community of laboratories at more than 60 institutions, Rosetta has been continuously refactored and extended. Its advantages are its performance and interoperability between broad modeling capabilities. Here we review tools developed in the last 5 years, including over 80 methods. We discuss improvements to the score function, user interfaces and usability. Rosetta is available at http://www.rosettacommons.org.
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              Synthetic glycolate metabolism pathways stimulate crop growth and productivity in the field

              Photorespiration is required in C3 plants to metabolize toxic glycolate formed when ribulose-1,5-bisphosphate carboxylase-oxygenase oxygenates rather than carboxylates ribulose-1,5-bisphosphate. Depending on growing temperatures, photorespiration can reduce yields by 20 to 50% in C3 crops. Inspired by earlier work, we installed into tobacco chloroplasts synthetic glycolate metabolic pathways that are thought to be more efficient than the native pathway. Flux through the synthetic pathways was maximized by inhibiting glycolate export from the chloroplast. The synthetic pathways tested improved photosynthetic quantum yield by 20%. Numerous homozygous transgenic lines increased biomass productivity between 19 and 37% in replicated field trials. These results show that engineering alternative glycolate metabolic pathways into crop chloroplasts while inhibiting glycolate export into the native pathway can drive increases in C3 crop yield under agricultural field conditions.
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                Author and article information

                Contributors
                (View ORCID Profile)
                (View ORCID Profile)
                Journal
                Forests
                Forests
                MDPI AG
                1999-4907
                April 2023
                April 18 2023
                : 14
                : 4
                : 827
                Article
                10.3390/f14040827
                84de372c-ede0-48d1-8e85-07e168078401
                © 2023

                https://creativecommons.org/licenses/by/4.0/

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