Biophysical analysis of the plant-specific GIPC sphingolipids reveals multiple modes of membrane regulation

Abstract

The plant plasma membrane (PM) is an essential barrier between the cell and the external environment, controlling signal perception and transmission. It consists of an asymmetrical lipid bilayer made up of three different lipid classes: sphingolipids, sterols, and phospholipids. The glycosyl inositol phosphoryl ceramides (GIPCs), representing up to 40% of total sphingolipids, are assumed to be almost exclusively in the outer leaflet of the PM. However, their biological role and properties are poorly defined. In this study, we investigated the role of GIPCs in membrane organization. Because GIPCs are not commercially available, we developed a protocol to extract and isolate GIPC-enriched fractions from eudicots (cauliflower and tobacco) and monocots (leek and rice). Lipidomic analysis confirmed the presence of trihydroxylated long chain bases and 2-hydroxylated very long-chain fatty acids up to 26 carbon atoms. The glycan head groups of the GIPCs from monocots and dicots were analyzed by gas chromatograph–mass spectrometry, revealing different sugar moieties. Multiple biophysics tools, namely Langmuir monolayer, ζ-Potential, light scattering, neutron reflectivity, solid state 2H-NMR, and molecular modeling, were used to investigate the physical properties of the GIPCs, as well as their interaction with free and conjugated phytosterols. We showed that GIPCs increase the thickness and electronegativity of model membranes, interact differentially with the different phytosterols species, and regulate the gel-to-fluid phase transition during temperature variations. These results unveil the multiple roles played by GIPCs in the plant PM.

Sphingolipids mediate polar sorting of PIN2 through phosphoinositide consumption at the trans-Golgi network

“…Quantification of sphingolipids was performed as described in 75 , 76 . Briefly, lipids were extracted in 3 ml of propan-2-ol:hexane:water 2.75:1:1.25, incubated at 60 °C for 20 min, centrifuged and the supernatant was collected.…”

Biosynthesis and Functions of Very-Long-Chain Fatty Acids in the Responses of Plants to Abiotic and Biotic Stresses

“…Plant sphingolipids constitute a highly complex lipid class since they are composed of up to 9 different LCBs, 32 different fatty acids and various polar heads, which still need to be completely described in terms of exact sugar composition, kind, and stereochemistry of bounds. If several LC-MS methods based on multiple reaction monitoring mass spectrometry scan have been described to analyze the full sphingolipidome of plants [ 9 , 103 , 104 , 105 , 106 , 107 ], only relative quantification can be performed, and suitable standards for plant GIPCs are still lacking so adequate methods still need to be developed.…”

“…This particularity, easily detectable by GC-MS after silylation, facilitated sphingolipids purification and analysis in model plants [ 100 ]. The presence of VLCFA, 2-hydroxylated or not seems universal in all plant GIPCs analyzed [ 11 , 107 ]. In arabidopsis, two fatty acid 2-hydroxylases (FAH) have been described: AtFAH1, which preferentially adds a hydroxyl group to a VLCFA, and AtFAH2, which prefers 16:0 [ 114 ].…”

“…Although phytosterols were proposed a while ago to be essential for regulating the fluidity of plant plasma membrane in response to changing temperatures [ 129 ], solid state 2H-NMR recently showed that GIPC have also a high propensity to regulate the fluidity of the PM when temperature varies [ 107 ]. In fact, subtle changes in sphingolipids can induce radical effects on the development of arabidopsis when exposed to abiotic stress such as cold.…”

Diversity in sphingolipid metabolism across land plants

“…These studies of cell cultures have consistently revealed an increase in the extent of glycosylation when compared with developed organs containing multiple cell and tissue types; enrichment of detergent-insoluble membranes from BY-2 cells revealed that GIPCs could constitute up to 60% of the lipids in this fraction. The roles of GIPCs in the functionality of these membrane domains is poorly understood, but an exciting and growing field ( Gronnier et al , 2016 ; Mamode Cassim et al ., 2019 , 2021 ).…”