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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.

 

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. 

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.

 

ACYL CARRIER PROTEIN4 (ACP4) is the most abundant ACP isoform in Arabidopsis (Arabidopsis thaliana) leaves and acts as a scaffold for de novo fatty acid biosynthesis and as a substrate for acyl-ACP-utilizing enzymes. Recently, ACP4 was found to interact with a protein-designated plastid RHOMBOID LIKE10 (RBL10) that affects chloroplast monogalactosyldiacylglycerol (MGDG) biosynthesis, but the cellular function of this interaction remains to be explored. Here, we generated and characterized acp4 rbl10 double mutants to explore whether ACP4 and RBL10 directly interact in influencing chloroplast lipid metabolism. Alterations in the content and molecular species of chloroplast lipids such as MGDG and phosphatidylglycerol were observed in the acp4 and rbl10 mutants, which are likely associated with the changes in the size and profiles of diacylglycerol (DAG), phosphatidic acid (PA), and acyl-ACP precursor pools. ACP4 contributed to the size and profile of the acyl-ACP pool and interacted with acyl-ACP-utilizing enzymes, as expected for its role in fatty acid biosynthesis and chloroplast lipid assembly. RBL10 appeared to be involved in the conversion of PA to DAG precursors for MGDG biosynthesis as evidenced by the increased 34:x PA and decreased 34:x DAG in the rbl10 mutant and the slow turnover of radiolabeled PA in isolated chloroplasts fed with [14C] acetate. Interestingly, the impaired PA turnover in rbl10 was partially reversed in the acp4 rbl10 double mutant. Collectively, this study shows that ACP4 and RBL10 affect chloroplast lipid biosynthesis by modulating substrate precursor pools and appear to act independently.

 

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

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.

 

The cell membrane plays a pivotal role in protecting bacteria against external threats, such as antibiotics. Cationic phospholipids such as lysyl-phosphatidyglycerol (L-PG) resist the action of cationic antimicrobial peptides through electrostatic repulsion.

 

Pollen germination is an essential process for pollen tube growth, pollination, and therefore seed production in flowering plants, and it requires energy either from remobilization of stored carbon sources, such as lipids and starches, or from secreted exudates from the stigma. Transcriptome analysis fromin vitropollen germination previously showed that 14 GO terms, including metabolism and energy, were overrepresented inArabidopsis. However, little is understood about global changes in carbohydrate and energy-related metabolites during the transition from mature pollen grain to hydrated pollen, a prerequisite to pollen germination, in most plants, includingArabidopsis. In this study, we investigated differential metabolic pathway enrichment among mature, hydrated, and germinated pollen using an untargeted metabolomic approach. Integration of publicly available transcriptome data with metabolomic data generated as a part of this study revealed starch and sucrose metabolism increased significantly during pollen hydration and germination. We analyzed in detail alterations in central metabolism, focusing on soluble carbohydrates, non-esterified fatty acids, glycerophospholipids, and glycerolipids. We found that several metabolites, including palmitic acid, oleic acid, linolenic acid, quercetin, luteolin/kaempferol, and γ-aminobutyric acid (GABA), were elevated in hydrated pollen, suggesting a potential role in activating pollen tube emergence. The metabolite levels of mature, hydrated, and germinated pollen, presented in this work provide insights on the molecular basis of pollen germination.

 

In developing soybean seeds, carbon is partitioned between oil, protein and carbohydrates. Here, we demonstrate that suppression of lipase-mediated turnover of triacylglycerols (TAG) during late seed development increases fatty acid content and decreases the presence of undigestible oligosaccharides. During late stages of embryo development, the fatty acid content of soybean seed decreases while the levels of the oligosaccharides raffinose and stachyose increase. Three soybean genes orthologous to the Arabidopsis lipase gene SUGAR-DEPENDENT1 ( SDP1 ) are upregulated at this time. Suppression of these genes resulted in higher oil levels, with lipid levels in the best lines exceeding 24% of seed weight. In addition, lipase-suppressed lines produced larger seeds compared to wild-type plants, resulting in increases of over 20% in total lipid per seed. Levels of raffinose and stachyose were lower in the transgenic lines, with average reductions of 15% in total raffinose family oligosaccharides observed. Despite the increase in oil, protein content was not negatively impacted and trended higher in the transgenic lines. These results are consistent with a role for SDP1 in turning over TAG to supply carbon for other needs, including the synthesis of oligosaccharides, and offer new strategies to further improve the composition of soybean seeds. 

Abstract Assessing central carbon metabolism in plants can be challenging due to the dynamic range in pool sizes, with low levels of important phosphorylated sugars relative to more abundant sugars and organic acids. Here, we report a sensitive liquid chromatography–mass spectrometry method for analysing central metabolites on a hybrid column, where both anion-exchange and hydrophilic interaction chromatography (HILIC) ligands are embedded in the stationary phase. The liquid chromatography method was developed for enhanced selectivity of 27 central metabolites in a single run with sensitivity at femtomole levels observed for most phosphorylated sugars. The method resolved phosphorylated hexose, pentose, and triose isomers that are otherwise challenging. Compared with a standard HILIC approach, these metabolites had improved peak areas using our approach due to ion enhancement or low ion suppression in the biological sample matrix. The approach was applied to investigate metabolism in high lipid-producing tobacco leaves that exhibited increased levels of acetyl-CoA, a precursor for oil biosynthesis. The application of the method to isotopologue detection and quantification was considered through evaluating 13C-labeled seeds from Camelina sativa. The method provides a means to analyse intermediates more comprehensively in central metabolism of plant tissues.