In Vitro Growth Conditions Boost Plant Lipid Remodelling and Influence Their Composition

Acyl-lipids are vital components for all life functions of plants. They are widely studied using often in vitro conditions to determine inter alia the impact of genetic modifications and the description of biochemical and physiological functions of enzymes responsible for acyl-lipid metabolism. What is currently lacking is knowledge of if these results also hold in real environments—in in vivo conditions. Our study focused on the comparative analysis of both in vitro and in vivo growth conditions and their impact on the acyl-lipid metabolism of Camelina sativa leaves. The results indicate that in vitro conditions significantly decreased the lipid contents and influenced their composition. In in vitro conditions, galactolipid and trienoic acid (16:3 and 18:3) contents significantly declined, indicating the impairment of the prokaryotic pathway. Discrepancies also exist in the case of acyl-CoA:lysophospholipid acyltransferases (LPLATs). Their activity increased about 2–7 times in in vitro conditions compared to in vivo. In vitro conditions also substantially changed LPLATs’ preferences towards acyl-CoA. Additionally, the acyl editing process was three times more efficient in in vitro leaves. The provided evidence suggests that the results of acyl-lipid research from in vitro conditions may not completely reflect and be directly applicable in real growth environments.

EPIP-Evoked Modifications of Redox, Lipid, and Pectin Homeostasis in the Abscission Zone of Lupine Flowers

Yellow lupine is a great model for abscission-related research given that excessive flower abortion reduces its yield. It has been previously shown that the EPIP peptide, a fragment of LlIDL (INFLORESCENCE DEFICIENT IN ABSCISSION) amino-acid sequence, is a sufficient molecule to induce flower abortion, however, the question remains: What are the exact changes evoked by this peptide locally in abscission zone (AZ) cells? Therefore, we used EPIP peptide to monitor specific modifications accompanied by early steps of flower abscission directly in the AZ. EPIP stimulates the downstream elements of the pathway—HAESA and MITOGEN-ACTIVATED PROTEIN KINASE6 and induces cellular symptoms indicating AZ activation. The EPIP treatment disrupts redox homeostasis, involving the accumulation of H2O2 and upregulation of the enzymatic antioxidant system including superoxide dismutase, catalase, and ascorbate peroxidase. A weakening of the cell wall structure in response to EPIP is reflected by pectin demethylation, while a changing pattern of fatty acids and acyl lipids composition suggests a modification of lipid metabolism. Notably, the formation of a signaling molecule—phosphatidic acid is induced locally in EPIP-treated AZ. Collectively, all these changes indicate the switching of several metabolic and signaling pathways directly in the AZ in response to EPIP, which inevitably leads to flower abscission.

A conserved evolutionary mechanism permits Δ9 desaturation of very-long-chain fatty acyl lipids

Δ9 fatty acyl desaturases introduce a cis–double bond between C9 and C10 of saturated fatty acyl chains. From the crystal structure of the mouse stearoyl-CoA desaturase (mSCD1) it was proposed that Tyr-104, a surface residue located at the distal end of the fatty acyl binding pocket plays a key role in specifying 18C selectivity. We created mSCD1-Y104G to test the hypothesis that eliminating this bulky side chain would create an opening and permit the substrate's methyl end to protrude through the enzyme into the lipid bilayer, facilitating the desaturation of very-long-chain (VLC) substrates. Consistent with this hypothesis, Y104G acquired the ability to desaturate 24C and 26C acyl-CoAs while maintaining its Δ9-regioselectivity. We also investigated two distantly related very-long-chain fatty acyl (VLCFA) desaturases from Arabidopsis, ADS1.2 and ADS1.4, which have Ala and Gly, respectively, in place of the gatekeeping Tyr found in mSCD1. Substitution of Tyr for Ala and Gly in ADS1.2 and ADS1.4, respectively, blocked their ability to desaturate VLCFAs. Further, we identified a pair of fungal desaturase homologs which contained either an Ile or a Gly at this location and showed that only the Gly-containing desaturase was capable of very-long-chain desaturation. The conserved desaturase architecture wherein a surface residue with a single bulky side chain forms the end of the substrate binding cavity predisposes them to single amino acid substitutions that enable a switch between long- and very-long-chain selectivity. The data presented here show that such changes have independently occurred multiple times during evolution.

Evaluation of the functional role of the maize Glossy2 and Glossy2-like genes in cuticular lipid deposition

ABSTRACTPlant epidermal cells express unique molecular machinery that juxtapose the assembly of intracellular lipid components and the unique extracellular cuticular lipids that are unidirectionally secreted to plant surfaces. In maize (Zea mays L.), mutations at the glossy2 (gl2) locus affect the deposition of extracellular cuticular lipids. Sequence-based genome scanning identified a novel gl2 homolog in the maize genome, Gl2-like. Sequence homology identifies that both the Gl2-like and Gl2 genes are members of the BAHD superfamily of acyltransferases, with close sequence homology to the Arabidopsis CER2 gene. Transgenic experiments demonstrate that Gl2-like and Gl2 functionally complement the Arabidopsis cer2 mutation, with differential impacts on the cuticular lipids and the lipidome of the plant, particularly affecting the longer alkyl chain acyl lipids, particularly at the 32-carbon chain length. Site-directed mutagenesis of the putative BAHD catalytic HXXXDX-motif indicates that Gl2-like requires this catalytic capability to fully complement the cer2 function, but Gl2 can accomplish this without the need for this catalytic motif. These findings demonstrate that both Gl2 and Gl2-like overlap in their cuticular lipid function, however the two genes have evolutionary diverged to acquire non-overlapping functions.One-sentence summaryTransgenesis dissection of the functional roles of the maize Glossy2 and Glossy2-Like genes in cuticular lipid deposition.

Comprehensive Circulatory Metabolomics in ME/CFS Reveals Disrupted Metabolism of Acyl Lipids and Steroids

The latest worldwide prevalence rate projects that over 65 million patients suffer from myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), an illness with known effects on the functioning of the immune and nervous systems. We performed an extensive metabolomics analysis on the plasma of 52 female subjects, equally sampled between controls and ME/CFS patients, which delivered data for about 1750 blood compounds spanning 20 super-pathways, subdivided into 113 sub-pathways. Statistical analysis combined with pathway enrichment analysis points to a few disrupted metabolic pathways containing many unexplored compounds. The most intriguing finding concerns acyl cholines, belonging to the fatty acid metabolism sub-pathway of lipids, for which all compounds are consistently reduced in two distinct ME/CFS patient cohorts. We compiled the extremely limited knowledge about these compounds and regard them as promising in the quest to explain many of the ME/CFS symptoms. Another class of lipids with far-reaching activity on virtually all organ systems are steroids; androgenic, progestin, and corticosteroids are broadly reduced in our patient cohort. We also report on lower dipeptides and elevated sphingolipids abundance in patients compared to controls. Disturbances in the metabolism of many of these molecules can be linked to the profound organ system symptoms endured by ME/CFS patients.

Characterization of Intact Eukaryotic Cells with Subcellular Spatial Resolution by Photothermal-Induced Resonance Infrared Spectroscopy and Imaging

Photothermal-induced resonance (PTIR) spectroscopy and imaging with infrared light has seen increasing application in the molecular spectroscopy of biological samples. The appeal of the technique lies in its capability to provide information about IR light absorption at a spatial resolution better than that allowed by light diffraction, typically below 100 nm. In the present work, we tested the capability of the technique to perform measurements with subcellular resolution on intact eukaryotic cells, without drying or fixing. We demonstrate the possibility of obtaining PTIR images and spectra from the nucleus and multiple organelles with high resolution, better than that allowed by diffraction with infrared light. We obtain particularly strong signal from bands typically assigned to acyl lipids and proteins. We also show that while a stronger signal is obtained from some subcellular structures, other large subcellular components provide a weaker or undetectable PTIR response. The mechanism that underlies such variability in response is presently unclear. We propose and discuss different possibilities, addressing thermomechanical, geometrical, and electrical properties of the sample and the presence of cellular water, from which the difference in response may arise.

Corrected and Republished from: Activation Status-Coupled Transient S-Acylation Determines Membrane Partitioning of a Plant Rho-Related GTPase

ABSTRACT ROPs or RACs are plant Rho-related GTPases implicated in the regulation of a multitude of signaling pathways that function at the plasma membrane via posttranslational lipid modifications. The relationships between ROP activation status and membrane localization has not been established. Here, we show that endogenous ROPs, as well as a transgenic His6-green fluorescent protein (GFP)-Arabidopsis thaliana ROP6 (AtROP6) fusion protein, were partitioned between Triton X-100-soluble and -insoluble membranes. In contrast, the His6-GFP-Atrop6CA activated mutant accumulated exclusively in detergent-resistant membranes. GDP induced accumulation of ROPs in Triton-soluble membranes, whereas GTPγS induced accumulation of ROPs in detergent-resistant membranes. Recombinant wild-type and constitutively active AtROP6 proteins were purified from Arabidopsis plants, and in turn, their lipids were cleaved and analyzed by gas chromatography-coupled mass spectrometry. In Triton-soluble membranes, the wild-type AtROP6 was only prenylated, primarily by geranylgeranyl. The activated AtROP6 that accumulated in detergent-resistant membranes was modified by prenyl and acyl lipids, identified as palmitic and stearic acids. Consistently, activated His6-GFP-Atrop6CAmS156, in which C156 was mutated into serine, accumulated in Triton-soluble membranes. These findings show that upon GTP binding and activation, AtROP6, and possibly other ROPs, are transiently S-acylated, inducing their partitioning into detergent-resistant membranes.