Targeted Analysis of the Plant Lipidome by UPLC-NanoESI-MS/MS

Herrfurth, Cornelia; Liu, Yi-Tse; Feußner, Ivo

doi:10.1007/978-1-0716-1362-7_9

Cited by 17 publications

(17 citation statements)

References 27 publications

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“…Lipid species were analysed as described in Herrfurth et al. (2021). Leaf disks were freeze‐dried and then pulverized with small metal beads by shaking on a vortex.…”

Section: Methodsmentioning

confidence: 99%

Metabolic priming in G6PDH isoenzyme‐replaced tobacco lines improves stress tolerance and seed yields via altering assimilate partitioning

Scharte,

Hassa,

Herrfurth

et al. 2023

The Plant Journal

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SUMMARYWe investigated the basis for better performance of transgenic Nicotiana tabacum plants with G6PDH‐isoenzyme replacement in the cytosol (Xanthi::cP2::cytRNAi, Scharte et al., 2009). After six generations of selfing, infiltration of Phytophthora nicotianae zoospores into source leaves confirmed that defence responses (ROS, callose) are accelerated, showing as fast cell death of the infected tissue. Yet, stress‐related hormone profiles resembled susceptible Xanthi and not resistant cultivar SNN, hinting at mainly metabolic adjustments in the transgenic lines. Leaves of non‐stressed plants contained twofold elevated fructose‐2,6‐bisphosphate (F2,6P2) levels, leading to partial sugar retention (soluble sugars, starch) and elevated hexose‐to‐sucrose ratios, but also more lipids. Above‐ground biomass lay in between susceptible Xanthi and resistant SNN, with photo‐assimilates preferentially allocated to inflorescences. Seeds were heavier with higher lipid‐to‐carbohydrate ratios, resulting in increased harvest yields ‐ also under water limitation. Abiotic stress tolerance (salt, drought) was improved during germination, and in floated leaf disks of non‐stressed plants. In leaves of salt‐watered plants, proline accumulated to higher levels during illumination, concomitant with efficient NADP(H) use and recycling. Non‐stressed plants showed enhanced PSII‐induction kinetics (upon dark–light transition) with little differences at the stationary phase. Leaf exudates contained 10% less sucrose, similar amino acids, but more fatty acids – especially in the light. Export of specific fatty acids via the phloem may contribute to both, earlier flowering and higher seed yields of the Xanthi‐cP2 lines. Apparently, metabolic priming by F2,6P2—combined with sustained NADP(H) turnover—bypasses the genetically fixed growth–defence trade‐off, rendering tobacco plants more stress‐resilient and productive.

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“…Lipid species were analysed as described in Herrfurth et al. (2021). Leaf disks were freeze‐dried and then pulverized with small metal beads by shaking on a vortex.…”

Section: Methodsmentioning

confidence: 99%

Metabolic priming in G6PDH isoenzyme‐replaced tobacco lines improves stress tolerance and seed yields via altering assimilate partitioning

Scharte,

Hassa,

Herrfurth

et al. 2023

The Plant Journal

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“…Analysis of lipids by UPLC-nano ESI-MS/MS molecular species analysis was performed as previously described, with some modifications ( Tarazona et al., 2015 ; Herrfurth et al., 2021 ). The analysis was started by UPLC using an ACQUITY UPLC ® I-class system (Waters Corporation, Milford, MA, USA) equipped with an ACQUITY UPLC ® HSS T3 column (100 mm × 1 mm, 1 μm; Waters Corporation, Milford, MA, USA).…”

Section: Methodsmentioning

confidence: 99%

Plastidic membrane lipids are oxidized by a lipoxygenase in Lobosphaera incisa

Djian¹,

Feussner²,

Herrfurth³

et al. 2022

Front. Plant Sci.

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Green microalgae can accumulate neutral lipids, as part of a general lipid remodeling mechanism under stress such as nitrogen starvation. Lobosphaera incisa is of special interest because of its unique TAG acyl chain composition, especially 20:4 (n-6) can reach up to 21% of dry weight after nitrogen starvation. In order to identify factors that may influence the accumulation of polyunsaturated fatty acids (PUFAs), we identified recently a linoleate 13-lipoxygenase (LiLOX). It shares highest identity with plastidic enzymes from vascular plants and is induced upon nitrogen starvation. Here, we confirmed the localization of LiLOX in the stroma of plastids via transient expression in epithelial onion cells. In order to further characterize this enzyme, we focused on the identification of the endogenous substrate of LiLOX. In this regard, an ex vivo enzymatic assay, coupled with non-targeted analysis via mass spectrometry allowed the identification of MGDG, DGDG and PC as three substrate candidates, later confirmed via in vitro assays. Further investigation revealed that LiLOX has preferences towards the lipid class MGDG, which seems in agreement with its localization in the galactolipid rich plastid. Altogether, this study shows the first characterization of plastidic LOX from green algae, showing preference for MGDGs. However, lipidomics analysis did neither reveal an endogenous LiLOX product nor the final end product of MGDG oxidation. Nevertheless, the latter is a key to understanding the role of this enzyme and since its expression is highest during the degradation of the plastidic membrane, it is tempting to assume its involvement in this process.

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“…This provided cleaner background and removed signals that could otherwise be misinterpreted as false positives in our sphingolipid analysis. This followed a protocol (Herrfurth et al, 2021) adapted from previous work (Markham and Jaworski, 2007).…”

Section: Methylamine Treatment Of Lipid Extractsmentioning

confidence: 99%

Viable mutants of essential genes inPhyscomitrium patensas tools for studying primary metabolic processes

Haslam,

Herrfurth,

Feussner

2023

Preprint

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Sphingolipids are essential components of plant cells, which have been notoriously difficult to study in part due to pleiotropic or lethal knock-out mutant phenotypes. By relying on alternative end-joining of double stranded breaks, we successfully used CRISPR/Cas9 mutagenesis to generate a population of diverse, viable mutant alleles of genes required for sphingolipid assembly in totipotent protoplasts of the moss Physcomitrium patens. We targeted the INOSITOL PHOSPHORYLCERAMIDE SYNTHASE (IPCS) gene family, which catalyzes the committed step in the synthesis of glycosyl inositol phosphorylceramides (GIPCs), the most abundant class of sphingolipids found in plants. We isolated knock-out single mutants and knock-down higher-order mutants showing a spectrum of deficiencies in GIPC content. Remarkably, we also identified two mutant alleles accumulating inositol phosphorylceramides, the direct products of IPCS activity, and provide our best explanation for this unexpected phenotype. Our approach is broadly applicable for studying essential genes and gene families, and for obtaining unusual lesions within a gene of interest.

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Targeted Analysis of the Plant Lipidome by UPLC-NanoESI-MS/MS

Cited by 17 publications

References 27 publications

Metabolic priming in G6PDH isoenzyme‐replaced tobacco lines improves stress tolerance and seed yields via altering assimilate partitioning

Metabolic priming in G6PDH isoenzyme‐replaced tobacco lines improves stress tolerance and seed yields via altering assimilate partitioning

Plastidic membrane lipids are oxidized by a lipoxygenase in Lobosphaera incisa

Viable mutants of essential genes inPhyscomitrium patensas tools for studying primary metabolic processes

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