Search for: All records

Abstract The capacity to leverage high resolution mass spectrometry (HRMS) with transient isotope labeling experiments is an untapped opportunity to derive insights on context-specific metabolism, that is difficult to assess quantitatively. Tools are needed to comprehensively mine isotopologue information in an automated, high-throughput way without errors. We describe a tool, Stable Isotope-assisted Metabolomics for Pathway Elucidation (SIMPEL), to simplify analysis and interpretation of isotope-enriched HRMS datasets. The efficacy ofSIMPELis demonstrated through examples of central carbon and lipid metabolism. In the first description, a dual-isotope labeling experiment is paired withSIMPELand isotopically nonstationary metabolic flux analysis (INST-MFA) to resolve fluxes in central metabolism that would be otherwise challenging to quantify. In the second example,SIMPELwas paired with HRMS-based lipidomics data to describe lipid metabolism based on a single labeling experiment. Available as an R package,SIMPELextends metabolomics analyses to include isotopologue signatures necessary to quantify metabolic flux. 

Abstract FatPlants, an open-access, web-based database, consolidates data, annotations, analysis results, and visualizations of lipid-related genes, proteins, and metabolic pathways in plants. Serving as a minable resource, FatPlants offers a user-friendly interface for facilitating studies into the regulation of plant lipid metabolism and supporting breeding efforts aimed at increasing crop oil content. This web resource, developed using data derived from our own research, curated from public resources, and gleaned from academic literature, comprises information on known fatty-acid-related proteins, genes, and pathways in multiple plants, with an emphasis on Glycine max, Arabidopsis thaliana, and Camelina sativa. Furthermore, the platform includes machine-learning based methods and navigation tools designed to aid in characterizing metabolic pathways and protein interactions. Comprehensive gene and protein information cards, a Basic Local Alignment Search Tool search function, similar structure search capacities from AphaFold, and ChatGPT-based query for protein information are additional features. Database URL: https://www.fatplants.net/ 

Summary Metabolic flux analysis (MFA) is a valuable tool for quantifying cellular phenotypes and to guide plant metabolic engineering. By introducing stable isotopic tracers and employing mathematical models, MFA can quantify the rates of metabolic reactions through biochemical pathways. Recent applications of isotopically nonstationary MFA (INST‐MFA) to plants have elucidated nonintuitive metabolism in leaves under optimal and stress conditions, described coupled fluxes for fast‐growing algae, and produced a synergistic multi‐organ flux map that is a first in MFA for any biological system. These insights could not be elucidated through other approaches and show the potential of INST‐MFA to correct an oversimplified understanding of plant metabolism. 

Abstract Engineering plant vegetative tissue to accumulate triacylglycerols (TAG, e.g. oil) can increase the amount of oil harvested per acre to levels that exceed current oilseed crops. Engineered tobacco (Nicotiana tabacum) lines that accumulate 15% to 30% oil of leaf dry weight resulted in starkly different metabolic phenotypes. In-depth analysis of the leaf lipid accumulation and 14CO2 tracking describe metabolic adaptations to the leaf oil engineering. An oil-for-membrane lipid tradeoff in the 15% oil line (referred to as HO) was surprisingly not further exacerbated when lipid production was enhanced to 30% (LEAFY COTYLEDON 2 (LEC2) line). The HO line exhibited a futile cycle that limited TAG yield through exchange with starch, altered carbon flux into various metabolite pools and end products, and suggested interference of the glyoxylate cycle with photorespiration that limited CO2 assimilation by 50%. In contrast, inclusion of the LEC2 transcription factor in tobacco improved TAG stability, alleviated the TAG-to-starch futile cycle, and recovered CO2 assimilation and plant growth comparable to wild type but with much higher lipid levels in leaves. Thus, the unstable production of storage reserves and futile cycling limit vegetative oil engineering approaches. The capacity to overcome futile cycles and maintain enhanced stable TAG levels in LEC2 demonstrated the importance of considering unanticipated metabolic adaptations while engineering vegetative oil crops. 

Summary Mosses hold a unique position in plant evolution and are crucial for protecting natural, long‐term carbon storage systems such as permafrost and bogs. Due to small stature, mosses grow close to the soil surface and are exposed to high levels of CO₂, produced by soil respiration. However, the impact of elevated CO₂(eCO₂) levels on mosses remains underexplored.We determined the growth responses of the mossPhyscomitrium patensto eCO₂in combination with different nitrogen levels and characterized the underlying physiological and metabolic changes.Three distinct growth characteristics, an early transition to caulonema, the development of longer, highly pigmented rhizoids, and increased biomass, define the phenotypic responses ofP. patensto eCO₂. Elevated CO₂impacts growth by enhancing the level of a sugar signaling metabolite, T6P. The quantity and form of nitrogen source influences these metabolic and phenotypic changes. Under eCO₂,P. patensexhibits a diffused growth pattern in the presence of nitrate, but ammonium supplementation results in dense growth with tall gametophores, demonstrating high phenotypic plasticity under different environments.These results provide a framework for comparing the eCO₂responses ofP. patenswith other plant groups and provide crucial insights into moss growth that may benefit climate change models. 

Abstract The basal level of the plant defense hormone jasmonate (JA) in unstressed leaves is low, but wounding causes its near instantaneous increase. How JA biosynthesis is initiated is uncertain, but the lipolysis step that generates fatty acid precursors is generally considered to be the first step. Here, we used a series of physiological, pharmacological, genetic, and kinetic analyses of gene expression and hormone profiling to demonstrate that the early spiking of JA upon wounding does not depend on the expression of JA biosynthetic genes in Arabidopsis (Arabidopsis thaliana). Using a transgenic system, we showed how decoupling the responses to wounding and JA prevents the perpetual synthesis of JA in wounded leaves. We then used DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1) as a model wound-responsive lipase to demonstrate that although its transient expression in leaves can elicit JA biosynthesis to a low level, an additional level of activation is triggered by wounding, which causes massive accumulation of JA. This wound-triggered boosting effect of DAD1-mediated JA synthesis can happen directly in damaged leaves or indirectly in undamaged remote leaves by the systemically transmitted wound signal. Finally, protein stability of DAD1 was influenced by wounding, α-linolenic acid, and mutation in its catalytic site. Together, the data support mechanisms that are independent of gene transcription and translation to initiate the rapid JA burst in wounded leaves and demonstrate how transient expression of the lipase can be used to reveal changes occurring at the level of activity and stability of the key lipolytic step. 

Abstract In plants, light-dependent activation of de novo fatty acid synthesis (FAS) is partially mediated by acetyl-CoA carboxylase (ACCase), the first committed step for this pathway. However, it is not fully understood how plants control light-dependent FAS regulation to meet the cellular demand for acyl chains. We report here the identification of a gene family encoding for three small plastidial proteins of the envelope membrane that interact with the α-carboxyltransferase (α-CT) subunit of ACCase and participate in an original mechanism restraining FAS in the light. Light enhances the interaction between carboxyltransferase interactors (CTIs) and α-CT, which in turn attenuates carbon flux into FAS. Knockouts for CTI exhibit higher rates of FAS and marked increase in absolute triacylglycerol levels in leaves, more than 4-fold higher than in wild-type plants. Furthermore, WRINKLED1, a master transcriptional regulator of FAS, positively regulatesCTI1expression by direct binding to its promoter. This study reveals that in addition to light-dependent activation, “envelope docking” of ACCase permits fine-tuning of fatty acid supply during the plant life cycle. 

Generating new strategies to improve plant performance and yield in crop plants becomes increasingly relevant with ongoing and predicted global climate changes. E3 ligases that function as key regulators within the ubiquitin proteasome pathway often are involved in abiotic stress responses, development, and metabolism in plants. The aim of this research was to transiently downregulate an E3 ligase that uses BTB/POZ-MATH proteins as substrate adaptors in a tissue-specific manner. Interfering with the E3 ligase at the seedling stage and in developing seeds results in increased salt-stress tolerance and elevated fatty acid levels, respectively. This novel approach can help to improve specific traits in crop plants to maintain sustainable agriculture. 

A unique feature of plastid phosphatidylglycerol (PG) is a trans-double bond specifically at the sn-2 position of 16C fatty acid (16:1t- PG), which is catalyzed by FATTY ACID DESATURASE 4 (FAD4). To offer additional insights about the in vivo roles of FAD4 and its product 16:1t-PG, FAD4 overexpression lines (OX-FAD4s) were generated in Arabidopsis thaliana Columbia ecotype. When grown under continuous light condition, the fad4-2 and OX-FAD4s plants exhibited higher growth rates compared to WT control. Total lipids were isolated from Col, fad4-2, and OX-FAD4_2 plants, and polar lipids quantified by lipidomic profiling. We found that disrupting FAD4 expression altered prokaryotic and eukaryotic PG content and composition. Prokaryotic and eukaryotic monogalactosyl diacylglycerol (MGDG) was up-regulated in OX-FAD4 plants but not in fad4-2 mutant. We propose that 16:1t-PG homeostasis in plastid envelope membranes may coordinate plant growth and stress response by restricting photoassimilate export from the chloroplast. 

Flux analysis indicates that camelina pod photosynthesis contributes to seed yield.