LPEATs Tailor Plant Phospholipid Composition through Adjusting Substrate Preferences to Temperature

Acyl-CoA:lysophosphatidylethanolamine acyltransferases (LPEATs) are known as enzymes utilizing acyl-CoAs and lysophospholipids to produce phosphatidylethanolamine. Recently, it has been discovered that they are also involved in the growth regulation of Arabidopsis thaliana. In our study we investigated expression of each Camelina sativa LPEAT isoform and their behavior in response to temperature changes. In order to conduct a more extensive biochemical evaluation we focused both on LPEAT enzymes present in microsomal fractions from C. sativa plant tissues, and on cloned CsLPEAT isoforms expressed in yeast system. Phylogenetic analyses revealed that CsLPEAT1c and CsLPEAT2c originated from Camelina hispida, whereas other isoforms originated from Camelina neglecta. The expression ratio of all CsLPEAT1 isoforms to all CsLPEAT2 isoforms was higher in seeds than in other tissues. The isoforms also displayed divergent substrate specificities in utilization of LPE; CsLPEAT1 preferred 18:1-LPE, whereas CsLPEAT2 preferred 18:2-LPE. Unlike CsLPEAT1, CsLPEAT2 isoforms were specific towards very-long-chain fatty acids. Above all, we discovered that temperature strongly regulates LPEATs activity and substrate specificity towards different acyl donors, making LPEATs sort of a sensor of external thermal changes. We observed the presented findings not only for LPEAT activity in plant-derived microsomal fractions, but also for yeast-expressed individual CsLPEAT isoforms

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

Phospholipid:diacylglycerol acyltransferase1-overexpression stimulates lipid turnover, oil production and fitness in cold-grown plants

Abstract Background Extensive population growth and climate change accelerate the search for alternative ways of plant-based biomass, biofuel and feed production. Here, we focus on hitherto unknow, new promising cold-stimulated function of phospholipid:diacylglycerol acyltransferase1 (PDAT1) – an enzyme catalyzing the last step of triacylglycerol (TAG) biosynthesis. Result Overexpression of AtPDAT1 boosted seed yield by 160% in Arabidopsis plants exposed to long-term cold compared to standard conditions. Such seeds increased both their weight and acyl-lipids content. This work also elucidates PDAT1’s role in leaves, which was previously unclear. Aerial parts of AtPDAT1-overexpressing plants were characterized by accelerated growth at early and vegetative stages of development and by biomass weighing three times more than control. Overexpression of PDAT1 increased the expression of SUGAR-DEPENDENT1 (SDP1) TAG lipase and enhanced lipid remodeling, driving lipid turnover and influencing biomass increment. This effect was especially pronounced in cold conditions, where the elevated synergistic expression of PDAT1 and SDP1 resulted in double biomass increase compared to standard conditions. Elevated phospholipid remodeling also enhanced autophagy flux in AtPDAT1-overexpresing lines subjected to cold, despite the overall diminished autophagy intensity in cold conditions. Conclusions Our data suggest that PDAT1 promotes greater vitality in cold-exposed plants, stimulates their longevity and boosts oilseed oil production at low temperature

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)

Subcellular Localization of Acyl-CoA: Lysophosphatidylethanolamine Acyltransferases (LPEATs) and the Effects of Knocking-Out and Overexpression of Their Genes on Autophagy Markers Level and Life Span of A. thaliana

Arabidopsis thaliana possesses two acyl-CoA:lysophosphatidylethanolamine acyltransferases, LPEAT1 and LPEAT2, which are encoded by At1g80950 and At2g45670 genes, respectively. Both single lpeat2 mutant and double lpeat1 lpeat2 mutant plants exhibit a variety of conspicuous phenotypes, including dwarfed growth. Confocal microscopic analysis of tobacco suspension-cultured cells transiently transformed with green fluorescent protein-tagged versions of LPEAT1 or LPEAT2 revealed that LPEAT1 is localized to the endoplasmic reticulum (ER), whereas LPEAT2 is localized to both Golgi and late endosomes. Considering that the primary product of the reaction catalyzed by LPEATs is phosphatidylethanolamine, which is known to be covalently conjugated with autophagy-related protein ATG8 during a key step of the formation of autophagosomes, we investigated the requirements for LPEATs to engage in autophagic activity in Arabidopsis. Knocking out of either or both LPEAT genes led to enhanced accumulation of the autophagic adaptor protein NBR1 and decreased levels of both ATG8a mRNA and total ATG8 protein. Moreover, we detected significantly fewer membrane objects in the vacuoles of lpeat1 lpeat2 double mutant mesophyll cells than in vacuoles of control plants. However, contrary to what has been reported on autophagy deficient plants, the lpeat mutants displayed a prolonged life span compared to wild type, including delayed senescence