LPEATs Tailor Plant Phospholipid Composition through Adjusting Substrate Preferences to Temperature

Abstract

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.

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

“…The addition of 18:1-CoA to the reaction mixture caused higher acyl editing intensity compared with the addition of 18:2-CoA and especially 18:3-CoA. Similar observations were noticed previously in assays with microsomal fractions of C. sativa seeds [21,22] and leaves [23]. Thus, this seems to be a commonplace phenomenon.…”

“…Enzyme assay determining the activity and the substrate specificity of the three acyl-CoA:lysophospholipid acyltransferases—LPAAT, LPCAT and LPEAT—was conducted on microsomal fractions derived from in vivo and in vitro C. sativa leaves. For enzymatic reactions, previously established parameters for these enzymes present in seeds and leaves were used [ 21 , 23 ]. As acyl donors, ten various acyl-CoAs were used: decanoyl-CoA ([ 14 C]10:0-CoA), lauroyl-CoA ([ 14 C]12:0-CoA), myristoyl-CoA ([ 14 C]14:0-CoA), palmitoyl-CoA ([ 14 C]16:0-CoA), stearoyl-CoA ([ 14 C]18:0-CoA), oleoyl-CoA ([ 14 C]18:1-CoA), linoleoyl-CoA ([ 14 C]18:2-CoA), linolenoyl-CoA ([ 14 C]18:3-CoA), eicosenoyl-CoA ([ 14 C]20:1-CoA) and erucoyl-CoA ([ 14 C]22:1-CoA).…”

“…For measurement of the intensity of remodelling of PC, PE and PA and the effect of different acyl-CoAs on this process, to the reaction mixture aliquots of microsomal fractions containing 10 nmol of endogenous PC together with 10 nmol of oleoyl-CoA ([ 14 C]18:1-CoA), linoleoyl-CoA ([ 14 C]18:2-CoA) or linolenoyl-CoA ([ 14 C]18:3-CoA) and 1 mg of BSA were added. The reaction mixtures were filled up to 100 µL with 40 mM potassium buffer (pH 7.2) and incubated at 30 °C with continuous shaking (1250 rpm) for 5 and 60 min (modified method described by Klińska et al [ 23 ]). Reactions were stopped as described above for LPLAT activity assays.…”

“…Enzyme assay determining the activity and the substrate specificity of the three acyl-CoA:lysophospholipid acyltransferases-LPAAT, LPCAT and LPEAT-was conducted on microsomal fractions derived from in vivo and in vitro C. sativa leaves. For enzymatic reactions, previously established parameters for these enzymes present in seeds and leaves were used [21,23] For determination of the activity of LPCAT, LPEAT and LPAAT type of enzymes in the prepared microsomal fractions, to the reaction mixtures we added, respectively: 5 nmol of exogenous sn-1-18:1-lysophosphatidylcholine, 5 nmol sn-1-18:1-lysophosphatidylethanolamine or 5 nmol of sn-1-18:1-lysophoshatidic acid together with 5 nmol of appropriate [ 14 C]acyl-CoA and aliquots of microsomal fractions (equivalent to 0.2 nmol and 0.5 nmol of the endogenous PC). Reaction mixtures were filled up to 100 µL with 40 mM potassium buffer (pH 7.2).…”

“…So far, only the role of LPEATs has been investigated. It was shown by Klińska et al [ 23 ] that the activity and substrate specificity of LPEAT enzymes present in leaves are strongly regulated by temperature, which makes them a sensor of external thermal changes. Information concerning the remodelling of galactolipids is practically unavailable.…”