Once regarded as mere membrane building blocks, lipids are now recognized as diverse and intricate players that mold the functions, identities, and responses of cellular membranes. Although the interactions of lipids with integral and peripheral membrane proteins are crucial for their localization, activity, and function, how proteins bind lipids is still far from being thoroughly explored. Describing and characterizing these dynamic protein-lipid interactions is thus essential to understanding the membrane-associated processes. Here we review the current range of experimental techniques employed to study plant protein-lipid interactions, integrating various methods. We summarize the principles, advantages, and limitations of classical in vitro biochemical approaches, including protein-lipid overlays and various liposome binding assays, and complement them with in vivo microscopic techniques centered around the use of genetically encoded lipid sensors and pharmacological or genetic membrane lipid manipulation tools. We also highlight several emerging techniques still awaiting their advancement into plant membrane research and emphasize the need to use complementary experimental strategies as key for elucidating the mechanistic roles of protein-lipid interactions in plant cell biology.

• Keywords
• Genetically encoded biosensors, lipid manipulation, membrane lipid imaging, microscopy, peripheral membrane proteins, protein–lipid interactions,
• MeSH
• Cell Membrane * metabolism   MeSH
• Membrane Lipids metabolism   MeSH
• Membrane Proteins metabolism   MeSH
• Plant Proteins * metabolism   MeSH
• Plants metabolism   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Membrane Lipids MeSH
• Membrane Proteins MeSH
• Plant Proteins * MeSH

Pollen germination and subsequent pollen tube elongation are essential for successful land plant reproduction. These processes are achieved through well-documented activation of membrane trafficking and cell metabolism. Despite this, our knowledge of the dynamics of cellular phospholipids remains scarce. Here we present the turnover of the glycerolipid composition during the establishment of cell polarity and elongation processes in tobacco pollen and show the lipid composition of pollen plasma membrane-enriched fraction for the first time. To achieve this, we have combined several techniques, such as lipidomics, plasma membrane isolation, and live-cell microscopy, and performed a study with different time points during the pollen germination and pollen tube growth. Our results showed that tobacco pollen tubes undergo substantial changes in their whole-cell lipid composition during the pollen germination and growth, finding differences in most of the glycerolipids analyzed. Notably, while lysophospholipid levels decrease during germination and growth, phosphatidic acid increases significantly at cell polarity establishment and continues with similar abundance in cell elongation. We corroborated these findings by measuring several phospholipase activities in situ. We also observed that lysophospholipids and phosphatidic acid are more abundant in the plasma membrane-enriched fraction than that in the whole cell. Our results support the important role for the phosphatidic acid in the establishment and maintenance of cellular polarity in tobacco pollen tubes and indicate that plasma membrane lysophospholipids may be involved in pollen germination.

• Keywords
• lipidomics, phosphatidic acid, phospholipid, plasma membrane, pollen, pollen tube, tip growth, tobacco,
• Publication type
• Journal Article MeSH