Biosynthesis and Transfer of α-Elostearic Acid In Vivo in <i>Momordica charantia</i> L. Developing Seeds and In Vitro in Microsomal Fractions of These Seeds

The research concerned the efficiency of biosynthesis and transfer to triacylglycerols (TAG) of α-eleostearic acid (αESA). The experiments were carried out on developing seeds of Momordica charantia L. and on microsomal fractions obtained from these seeds. The seeds from in vivo conditions were collected 20, 23, 26 and 33 days after pollination (DAP) and used for lipid extraction and further analyses. Microsomal fractions were prepared from seeds at 26 DAP. The most intensive lipid accumulation occurred between 20 and 26 DAP, but continued up to 33 DAP. The most abundant lipid fraction was TAG; up to 98% of total acyl lipids at 33 DAP. The synthesised in vivo αESA was very efficiently transferred to TAG and constituted about 60% of its total fatty acids in 33 DAP. The content of αESA in polar lipids (containing, among others, phosphatidylcholine—the place of αESA biosynthesis) was very low. The biosynthesis of αESA in vitro (assays with microsomal fractions and [14C]-labelled substrates) in the presence of NADPH was fairly intensive (about 60% of the corresponding intensity in vivo) when linolenic acid was used as a substrate. Contrary to the in vivo condition, most of the synthesised in vitro αESA remained in phosphatidylcholine

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

Diatoms and Plants Acyl-CoA:lysophosphatidylcholine Acyltransferases (LPCATs) Exhibit Diverse Substrate Specificity and Biochemical Properties

The search of the Phaeodactylum tricornutum genome database revealed the existence of six genes potentially encoding lysophospholipid acyltransferases. One of these genes, Phatr3_J20460, after introduction to yeast ale1 mutant disrupted in the LPCAT gene, produced a very active acyl-CoA:lysophosphatidylcholine (LPCAT) enzyme. Using in vitro assays applying different radioactive and non-radioactive substrates and microsomal fractions from such yeast, we have characterized the biochemical properties and substrate specificities of this PtLPCAT1. We have found that the substrate specificity of this enzyme indicates that it can completely supply phosphatidylcholine (PC) with all fatty acids connected with a biosynthetic pathway of very long-chain polyunsaturated fatty acids (VLC-PUFAs) used further for the desaturation process. Additionally, we have shown that biochemical properties of the PtLPCAT1 in comparison to plant LPCATs are in some cases similar (such as the dependency of its activity on pH value), differ moderately (such as in response to temperature changes), or express completely different properties (such as in reaction to calcium and magnesium ions or toward some acyl-CoA with 20C polyunsaturated fatty acids). Moreover, the obtained results suggest that cloned “Phatr3_J20460” gene can be useful in oilseeds plant engineering toward efficient production of VLC-PUFA as LPCAT it encodes can (contrary to plant LPCATs) introduce 20:4-CoA (n-3) to PC for further desaturation to 20:5 (EPA, eicosapentaenoic acid)

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

Molecular Characterization of Two Lysophospholipid:acyl-CoA Acyltransferases Belonging to the MBOAT Family in Nicotiana benthamiana.

In the remodeling pathway for the synthesis of phosphatidylcholine (PC), acyl-CoA-dependent lysophosphatidylcholine (lysoPC) acyltransferase (LPCAT) catalyzes the reacylation of lysoPC. A number of genes encoding LPCATs have been cloned and characterized from several plants in recent years. Using Arabidopsis and other plant LPCAT sequences to screen the genome database of Nicotiana benthamiana, we identified two cDNAs encoding the putative tobacco LPCATs (NbLPCAT1 and NbLPCAT2). Both of them were predicted to encode a protein of 463 amino acids with high similarity to LPCATs from other plants. Protein sequence features such as the presence of at least eight putative transmembrane regions, four highly conserved signature motifs and several invariant residues indicate that NbLPCATs belong to the membrane bound O-acyltransferase family. Lysophospholipid acyltransferase activity of NbLPCATs was confirmed by testing lyso-platelet-activating factor (lysoPAF) sensitivity through heterologous expression of each full-length cDNA in a yeast mutant Y02431 (lca1△) disrupted in endogenous LPCAT enzyme activity. Analysis of fatty acid profiles of phospholipids from the NbLPCAT-expressing yeast mutant Y02431 cultures supplemented with polyunsaturated fatty acids suggested more incorporation of linoleic acid (18:2n6, LA) and α-linolenic acid (18:3n3, ALA) into PC compared to yeast mutant harbouring empty vector. In vitro enzymatic assay demonstrated that NbLPCAT1had high lysoPC acyltransferase activity with a clear preference for α-linolenoyl-CoA (18:3), while NbLPCAT2 showed a high lysophosphatidic acid (lysoPA) acyltransferase activity towards α-linolenoyl-CoA and a weak lysoPC acyltransferase activity. Tissue-specific expression analysis showed a ubiquitous expression of NbLPCAT1 and NbLPCAT2 in roots, stems, leaves, flowers and seeds, and a strong expression in developing flowers. This is the first report on the cloning and characterization of lysophospholipid acyltransferases from N. benthamiana

Predicted transmembrane domains for <i>N</i>. <i>benthamiana</i> LPCAT sequences.

<p>The TMHMM web tools of the Center for Biological Sequence Analysis, Technical University of Denmark TMHMM Server plot the probability of the ALDH sequence forming a membrane-spanning helix (0–1.0 on the y-axis). The transmembrane regions are shown in red, whereas regions of those sequences predicted to be located inside or outside the membrane are shown in blue and pink, respectively.</p

Distribution of exogenously supplemented ALA (A) or LA (B) in phospholipids from cultures of yeast mutant Y02431 (<i>lca1</i>△) expressing NbLPCAT or harbouring the pESC-Ura empty vector.

<p>Cultures were supplemented with 500 μM PUFA in the presence of 1% (w/v) Tween 40. The value was expressed as the amount of PUFA (mg, isolated from PC, PE, or PI) per g dry cell weight (mg/g). The data represent the mean±S.E. of three measurements. ALA, α-linolenic acid (18:3n3); LA, linoleic acid (18:2n6); PUFA, polyunsaturated fatty acid.</p

LysoPAF sensitivity test for yeast mutant Y02431 (<i>lca1</i>△) expressing NbLPCAT or harbouring the pESC-Ura empty vector.

<p>Yeast cells grown overnight and induced for the expression of NbLPCAT for 24 h were suspended in sterile distilled water and adjusted to an OD₆₀₀ of 2, 1, 0.5, and 0.1. The resulting 2 μL yeast solution was spotted on a SC-Ura agar plate containing 5, 10, 25, and 30 μg/mL lysoPAF. The growth of yeast cells was evaluated after 72 h at 28°C.</p

Expression patterns of <i>NbLPCAT</i> genes in roots (A,B), stems (C,D), flowers and seeds (E,F) during different developing periods of tissue-cultivated <i>N</i>. <i>benthamiana</i>.

<p>The expression levels of <i>NbLPCAT</i>s were analyzed by the real-time quantitative RT-PCR method. X-axis indicates different developing periods and y-axis indicates relative expression levels. Gene encoding elongation factor 1α signal was used as the reference gene. Error bars represent standard deviations of mean value from three technical replicates. W, week.</p

Sequence alignment of NbLPCATs with the related LPCATs from higher plants.

<p>The amino acid sequences of NbLPCATs were aligned, using the software Clustal X v1.83 with those of characterized LPCATs from <i>B</i>. <i>napus</i> and <i>A</i>. <i>thaliana</i>. The Jalview v2.8.2 program was used to highlight the homology between LPCAT protein sequences. Conserved motifs and the putative ER signal are boxed. Invariant residues are marked with black triangle stars.</p