Project description:Plants employ photosynthesis to produce sugars for supporting their growth. During photosynthesis, an enzyme Ribulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco) combines its substrate Ribulose 1,5 bisphosphate (RuBP) with CO2 to produce phosphoglycerate (PGA). Alongside, Rubisco also takes up O2 and produce 2-phosphoglycolate (2-PG), a toxic compound broken down into PGA through photorespiration. Photorespiration is not only a resource-demanding process but also results in CO2 loss which affects photosynthetic efficiency in C3 plants. Here, we propose to circumvent photorespiration by adopting the cyanobacterial glycolate decarboxylation pathway into C3 plants. For that, we have integrated the cyanobacterial glycolate decarboxylation pathway into a kinetic model of C3 photosynthetic pathway to evaluate its impact on photosynthesis and photorespiration. Our results show that the cyanobacterial glycolate decarboxylation bypass model exhibits a 10% increase in net photosynthetic rate (A) in comparison with C3 model. Moreover, an increased supply of intercellular CO2 (Ci) from the bypass resulted in a 54.8% increase in PGA while reducing photorespiratory intermediates including glycolate (-?49%) and serine (-?32%). The bypass model, at default conditions, also elucidated a decline in phosphate-based metabolites including RuBP (-?61.3%). The C3 model at elevated level of inorganic phosphate (Pi), exhibited a significant change in RuBP (+?355%) and PGA (-?98%) which is attributable to the low availability of Ci. Whereas, at elevated Pi, the bypass model exhibited an increase of 73.1% and 33.9% in PGA and RuBP, respectively. Therefore, we deduce a synergistic effect of elevation in CO2 and Pi pool on photosynthesis. We also evaluated the integrative action of CO2, Pi, and Rubisco carboxylation activity (Vcmax) on A and observed that their simultaneous increase raised A by 26%, in the bypass model. Taken together, the study potentiates engineering of cyanobacterial decarboxylation pathway in C3 plants to bypass photorespiration thereby increasing the overall efficiency of photosynthesis.
Project description:Synthetically designed alternative photorespiratory pathways increase the biomass of tobacco and rice plants. Likewise, some in planta-tested synthetic carbon-concentrating cycles (CCCs) hold promise to increase plant biomass while diminishing atmospheric carbon dioxide burden. Taking these individual contributions into account, we hypothesize that the integration of bypasses and CCCs will further increase plant productivity. To test this in silico, we reconstructed a metabolic model by integrating photorespiration and photosynthesis with the synthetically designed alternative pathway 3 (AP3) enzymes and transporters. We calculated fluxes of the native plant system and those of AP3 combined with the inhibition of the glycolate/glycerate transporter by using the YANAsquare package. The activity values corresponding to each enzyme in photosynthesis, photorespiration, and for synthetically designed alternative pathways were estimated. Next, we modeled the effect of the crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA cycle (CETCH), which is a set of natural and synthetically designed enzymes that fix CO₂ manifold more than the native Calvin-Benson-Bassham (CBB) cycle. We compared estimated fluxes across various pathways in the native model and under an introduced CETCH cycle. Moreover, we combined CETCH and AP3-w/plgg1RNAi, and calculated the fluxes. We anticipate higher carbon dioxide-harvesting potential in plants with an AP3 bypass and CETCH-AP3 combination. We discuss the in vivo implementation of these strategies for the improvement of C3 plants and in natural high carbon harvesters.
Project description:Hydrogen peroxide (H2O2) can act as a signaling molecule that influences various aspects of plant growth and development, including stress signaling and cell death. To unravel the molecular mechanisms that regulate the response towards an impact of increased H2O2 levels in plant cells, we focused on the photorespiration-dependent peroxisomal H2O2 production in Arabidopsis thaliana mutants lacking CATALASE2 (CAT2) activity (cat2-2).
Project description:BACKGROUND:Camelina sativa is an oilseed crop with great potential for biofuel production on marginal land. The seed oil from camelina has been converted to jet fuel and improved fuel efficiency in commercial and military test flights. Hydrogenation-derived renewable diesel from camelina is environmentally superior to that from canola due to lower agricultural inputs, and the seed meal is FDA approved for animal consumption. However, relatively low yield makes its farming less profitable. Our study is aimed at increasing camelina seed yield by reducing carbon loss from photorespiration via a photorespiratory bypass. Genes encoding three enzymes of the Escherichia coli glycolate catabolic pathway were introduced: glycolate dehydrogenase (GDH), glyoxylate carboxyligase (GCL) and tartronic semialdehyde reductase (TSR). These enzymes compete for the photorespiratory substrate, glycolate, convert it to glycerate within the chloroplasts, and reduce photorespiration. As a by-product of the reaction, CO2 is released in the chloroplast, which increases photosynthesis. Camelina plants were transformed with either partial bypass (GDH), or full bypass (GDH, GCL and TSR) genes. Transgenic plants were evaluated for physiological and metabolic traits. RESULTS:Expressing the photorespiratory bypass genes in camelina reduced photorespiration and increased photosynthesis in both partial and full bypass expressing lines. Expression of partial bypass increased seed yield by 50-57 %, while expression of full bypass increased seed yield by 57-73 %, with no loss in seed quality. The transgenic plants also showed increased vegetative biomass and faster development; they flowered, set seed and reached seed maturity about 1 week earlier than WT. At the transcriptional level, transgenic plants showed differential expression in categories such as respiration, amino acid biosynthesis and fatty acid metabolism. The increased growth of the bypass transgenics compared to WT was only observed in ambient or low CO2 conditions, but not in elevated CO2 conditions. CONCLUSIONS:The photorespiratory bypass is an effective approach to increase photosynthetic productivity in camelina. By reducing photorespiratory losses and increasing photosynthetic CO2 fixation rates, transgenic plants show dramatic increases in seed yield. Because photorespiration causes losses in productivity of most C3 plants, the bypass approach may have significant impact on increasing agricultural productivity for C3 crops.
Project description:Hydrogen peroxide (H2O2) can act as a signaling molecule that influences various aspects of plant growth and development, including stress signaling and cell death. To unravel the molecular mechanisms that regulate the response towards an impact of increased H2O2 levels in plant cells, we focused on the photorespiration-dependent peroxisomal H2O2 production in Arabidopsis thaliana mutants lacking CATALASE2 (CAT2) activity (cat2-2). Unstressed and stressed samples of cat2-2 in triplicate
Project description:It is well established that the plastidic isoform of glutamine synthetase (GS2) is the enzyme in charge of photorespiratory ammonium reassimilation in plants. The metabolic events associated to photorespiratory NH4(+) accumulation were analyzed in a Lotus japonicus photorespiratory mutant lacking GS2. The mutant plants accumulated high levels of NH4(+) when photorespiration was active, followed by a sudden drop in the levels of this compound. In this paper it was examined the possible existence of enzymatic pathways alternative to GS2 that could account for this decline in the photorespiratory ammonium. Induction of genes encoding for cytosolic glutamine synthetase (GS1), glutamate dehydrogenase (GDH) and asparagine synthetase (ASN) was observed in the mutant in correspondence with the diminishment of NH4(+). Measurements of gene expression, polypeptide levels, enzyme activity and metabolite levels were carried out in leaf samples from WT and mutant plants after different periods of time under active photorespiratory conditions. In the case of asparagine synthetase it was not possible to determine enzyme activity and polypeptide content; however, an increased asparagine content in parallel with the induction of ASN gene expression was detected in the mutant plants. This increase in asparagine levels took place concomitantly with an increase in glutamine due to the induction of cytosolic GS1 in the mutant, thus revealing a major role of cytosolic GS1 in the reassimilation and detoxification of photorespiratory NH4(+) when the plastidic GS2 isoform is lacking. Moreover, a diminishment in glutamate levels was observed, that may be explained by the induction of NAD(H)-dependent GDH activity.
Project description:The broad application of single-cell RNA profiling in plants has been hindered by the prerequisite of protoplasting that requires digesting the cell walls from different types of plant tissues. Here, we present a protoplasting-free approach, flsnRNA-seq, for large-scale full-length RNA profiling at a single-nucleus level in plants using isolated nuclei. Combined with 10x Genomics and Nanopore long-read sequencing, we validate the robustness of this approach in Arabidopsis root cells and the developing endosperm. Sequencing results demonstrate that it allows for uncovering alternative splicing and polyadenylation-related RNA isoform information at the single-cell level, which facilitates characterizing cell identities.
Project description:The effect of drought on plant isoprene emission varies tremendously across species and environments. It was recently shown that an increased ratio of photosynthetic electron transport rate (ETR) to net carbon assimilation rate (NAR) consistently supported increased emission under drought. In this commentary, we highlight some of the physiological aspects of drought tolerance that are central to the observed variability. We briefly discuss some of the issues that must be addressed in order to refine our understanding of plant isoprene emission response to drought and increasing global temperature.
Project description:Using a chromatin regulator-focused shRNA library, we found that suppression of sex determining region Y-box 10 (SOX10) in melanoma causes resistance to BRAF and MEK inhibitors. To investigate how SOX10 loss leads to drug resistance, we performed transcriptome sequencing (RNAseq) of both parental A375 (Ctrl. PLKO) and A375-SOX10KD (shSOX10-1, shSOX10-2) cells. To ask directly whether SOX10 is involved indrug resistance in BRAF(V600E) melanoma patients, we isolated RNA from paired biopsies from melanoma patients (pre- and post- treatment) , that had gained BRAF or MEK inhibitor resistance . We performed RNAseq analysis to determine changes in transcriptome upon drug resistance. Investigate genes regulated by SOX10 and differntial gene expression between pre- and post-treatment biopsies. We use short hairpin RNA to suppression SOX10 in A375 cells and cells were harvested with trizol reagent for RNA isolation. For paired biopsies (patient samples) we collected the first biopsy before the initiation of treatment and the second biopsy after drug resistance developed. RNA was isolated from FFPE samples and subjected for RNA sequencing.