Lipid Rafts in Higher Plant Cells

Abstract

A large body of evidence from the past decade supports the existence of functional microdomains in membranes of animal and yeast cells, which play important roles in protein sorting, signal transduction, or infection by pathogens. They are based on the dynamic clustering of sphingolipids and cholesterol or ergosterol and are characterized by their insolubility, at low temperature, in nonionic detergents. Here we show that similar microdomains also exist in plant plasma membrane isolated from both tobacco leaves and BY2 cells. Tobacco lipid rafts were found to be greatly enriched in a sphingolipid, identified as glycosylceramide, as well as in a mixture of stigmasterol, sitosterol, 24-methylcholesterol, and cholesterol. Phospho-and glycoglycerolipids of the plasma membrane were largely excluded from lipid rafts. Membrane proteins were separated by oneand two-dimensional gel electrophoresis and identified by tandem mass spectrometry or use of specific antibody. The data clearly indicate that tobacco microdomains are able to recruit a specific set of the plasma membrane proteins and exclude others. We demonstrate the recruitment of the NADPH oxidase after elicitation by cryptogein and the presence of the small G protein NtRac5, a negative regulator of NADPH oxidase, in lipid rafts.A new aspect of the lipid bilayer organization has arisen from biophysical and biochemical studies performed with animal cells for several years. Indeed, lipids are not uniformly miscible, but lateral separation of specific lipid species leads to the formation of specialized phase domains also called "lipid rafts" (1). The main role in the process of domain organization is played by sterols and sphingolipids, these latter interacting together through weak interaction between aliphatic chains stabilized by the presence of saturated alkyl chains, voids between sphingolipids being filled by sterols (for a review, see Ref.2). The cholesterol-sphingolipid-enriched domain formation is also enhanced by the fact that sphingolipids have higher melting temperatures than phospholipids. Regions between rafts are occupied by phospholipids, with unsaturated fatty acids forming a liquid-crystalline phase, whereas lipid rafts that contain more saturated aliphatic chains form a liquidordered phase. In model and biological membranes, the formation of the liquid-ordered phase correlates with resistance to solubilization by nonionic detergent such as Triton X-100 at 4°C and buoyancy at specific density in a sucrose gradient (3). Thus, isolation of detergent-insoluble membranes (DIM) 1 or detergent-insoluble glycolipid-enriched membrane domains is one of the most widely used methods for studying lipid rafts.In animal cells, these membrane domains act, for example, as sorting devices for the accumulation of acylated, glycosylphosphatidylinositol-anchored, palmitoylated signaling molecules that selectively locate in these domains. Tyrosine kinases of the Src family protein, heterotrimeric and small G-proteins, as well as phosphoinositides have been proved to be...

Recruitment and interaction dynamics of plant penetration resistance components in a plasma membrane microdomain

“…Consistent with this idea of multiple PM microdomains recruiting different sets of proteins, is the observation that lipid rafts isolated from healthy tobacco were capable of recruiting aquaporins, whereas both tested barley aquaporin isoforms, known to be expressed in the leaf epidermis, were excluded from the Bgh-triggered microdomain (ref. 22, this study, and Fig. 5).…”

“…Moreover, lack of FRAP in the Bgh-triggered microdomain is also reminiscent of lipid rafts in animal cells (20,21). Although very little is known about lipid rafts in plants, biochemical evidence for the existence of lipid raft-mediated compartmentalization of PM proteins has been recently obtained by analysis of PMs derived from healthy tobacco (22). These lipid rafts were greatly enriched in glycosylceramide and a mixture of plant sterols (stigmasterol, sitosterol, 24-methylcholesterol, and cholesterol).…”

“…This finding is surprising because apoplastic oxidative microbursts (revealed by hydrogen peroxide accumulation) are known to take place underneath both primary and AGTs, demonstrating that each germ tube is capable of eliciting a subcellular defense response at the cell periphery of attacked host cells (17,23). Although the enzyme(s) generating these oxidative microbursts have not been conclusively identified in barley (24,25), it is interesting that treatment of tobacco BY2 cells with cryptogein, a 10-kDa protein secreted by the phytoparasitic oomycete Phytophthora cryptogea, resulted in the accumulation of the NADPH oxidase NtrbohD and its presumed negative regulator, NtRac5, components of the oxi- dative burst machinery, in PM lipid rafts (22). One possibility is that unknown Bgh effector(s), secreted by both primary and AGTs, trigger the localization of the oxidative burst machinery underneath both fungal structures, whereas focal accumulation of MLO and ROR2 beneath Bgh AGTs might be due to effectors released specifically by appressoria.…”

Cinnamoyl-CoA reductase, a key enzyme in lignin biosynthesis, is an effector of small GTPase Rac in defense signaling in rice

“…Our recent experiments show that the Rac GTPase regulates the Rboh enzymatic activity by direct interaction (unpublished results). In tobacco, Rac GTPase and Rboh have been shown to be colocalized in lipid rafts at PM (43). The Rboh-mediated ROS are most likely to be used to polymerize monolignols in the cell wall.…”

Loss of the Transit Peptide and an Increase in Gene Expression of an Ancestral Chloroplastic Carbonic Anhydrase Were Instrumental in the Evolution of the Cytosolic C4 Carbonic Anhydrase in Flaveria

“…Interestingly, cytosolic b-CAs have been found associated with the plasma membranes of several plant species (Utsunomiya and Muto, 1993;Santoni et al, 1998;Kawamura and Uemura, 2003;Alexandersson et al, 2004;Mongrand et al, 2004;Benschop et al, 2007;Mitra et al, 2007), and AtbCA4 specifically localizes to Arabidopsis cell membranes (Fabre et al, 2007). Mongrand et al (2004) showed that tobacco plasma membrane lipid rafts were enriched in a cytosolic b-CA and an aquaporin, NtAQP1, which has CO 2 transport activity (Uehlein et al, 2003) and decreases the resistance of the envelope to CO 2 (Uehlein et al, 2008). b-CA has also been implicated as a factor aiding the conductance of CO 2 through the leaf (Cowan, 1986).…”