Plant plasma membrane

The plant PM is a boundary between the cytoplasm and apoplast, delineating distinct biochemical conditions on each side. It is a complex structure whose intrinsic components, lipids, and proteins, are heterogeneously distributed, creating dynamic membrane domains (Gronnier et al., 2018; Jaillais and Ott, 2020) that regulate the flow of compounds and signals into and out of the protoplast. The lipid-to-protein mass ratio of a plant PM is 1.3 (Cacas et al., 2016) but on a molar basis, there is only one

protein per 50–100 molecules of lipids. A given plant PM can contain several thousand different proteins (e.g., more than 3000 protein identifications were obtained from the PM purified from rice leaves, Cao et al., 2016). The analysis of lipidomes is challenging (Orešič, 2011). In general, thousands of different lipids are predicted to occur in plant PMs (Yetukuri et al., 2008). A high density of membrane proteins influences the order of nearby lipids (Tieleman et al., 1998; Corradi et al., 2019).

PM proteins are involved in multiple functions. For instance, transporters, receptors, enzymes involved in CW synthesis, and proteins involved in the vesicular transport all work side by side to mediate inorganic mineral nutrient uptake, maintain pH homeostasis, regulate osmotic gradients, mediate signals, and synthesise CWs (Wang et al. 2018). Membrane transporters and channels are the gatekeepers of the PM as well as other membranes. Together with vesicular transport and diffusion, they are responsible for creating gradients of solutes, for mediating the transmission of information by passing messenger molecules, and for taking in and exporting nutrients and metabolites (Schulz et al., 2011).

Channels like aquaporins (PIP family members for H2O, urea, CO2), anion channels (multiple for NO^3-, Cl^-, SO42-, PO43- malate, citrate), and cation channels (multiple for K⁺, Ca²⁺) are all fundamental components of the PM (Schulz, 2011; Hedrich, 2012;

Maurel et al., 2015; Pantoja, 2021). Pumps, secondary active transporters, and ion channels are all well represented at the PM. P-ATPases are single-unit membrane proteins that hydrolyze ATP to form phosphorylated intermediates during transport, and these can be inhibited by vanadate (Gallagher and Leonard, 1982). Multiple cations (H⁺, Ca²⁺, Zn²⁺, Co²⁺, Cd²⁺, and Cu⁺) serve as substrates (Palmgren et al., 2011;

Pedersen et al., 2012). Bioenergetics at the PM relies largely on H⁺ export by the P- ATPases (Wegner and Shabala, 2020) although in some cells, vacuolar H⁺- pyrophosphatases (V-PPases) are located at the PM in addition to the tonoplast (Cosse and Seidel, 2021). H⁺ export at the PM is accompanied by ion transport through multiple, differentially gated, differentially selective ion channels for cations and anions (Hedrich, 2012; Pantoja, 2021). In addition, H⁺ generation in the apoplast and H⁺ scavenging in the cytosol both have an effect on the gradient and, as a whole, this complex system creates and regulates the pH gradient and membrane potential at the PM (Wegner and Shabala, 2020). Secondary active transporters at the PM rely largely on H⁺ gradients. In addition to secondary active transporters, ABC transporters are

present at the PM as well as at all the endomembranes, and have a plethora of substrates and biological roles, as discussed above. Receptor-like kinases (RLKs) of the PM, including wall-associated kinases (WAKs) and receptor-like proteins (RLPs), can sense signals on the apoplastic side of the PM and mediate information between the cell and its environment (Kanneganti and Gupta, 2008; He et al., 2018). In addition, arabinogalactan proteins are sometimes anchored to the PM at the apoplastic side (Ellis et al., 2010). The PM and the CW are connected to each other through PM proteins. For example, formin1 has been shown to connect the CW through the PM to the cytoskeleton in Arabidopsis (Martinière et al., 2011) and CSCs that synthesise cellulose connect the PM to the CW (Baluška et al., 2003; Liu et al., 2015). A unique feature of the plant PM is that the turgor pressure presses it against the CW meaning that lateral diffusion of many PM proteins is minimal (Martinière et al., 2012).

Membrane lipids influence the diffusion of substances, the fluidity and physical properties of the membrane, the integrity of the cell, the movement and location of membrane proteins, the signalling, etc. (Cassim et al., 2019). The three main lipid groups, glycerolipids (phospholipids and glycolipids), sterols, and sphingolipids, are all represented at the PM. The PM lipid content varies between species and organs but compared to other membranes of the cell, the PM has a higher content of sterols and sphingolipids (Furt et al., 2011). Phosphatidylcholine and phosphatidylethanolamine are the main structural phospholipids of plant PMs, with a lower content of other structural phospholipids phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and phosphatidic acid (Furt et al., 2011). The only glycolipid at the PM, digalactosyl diacylglycerol, has a role in replacing some of the phospholipids during phosphorus deprivation (Russo et al., 2007). In addition to structural glycerolipids, phosphoinositides are important in plant signalling (Heilmann 2016).

Plant sterols (phytosterols) can be found as free forms and with glycosylations and acylations. Phytosterols are said to increase PM permeability (Furt et al., 2011). Plants have more than 500 different sphingolipids (Pata et al., 2010) and their roles range from membrane organisation and dynamics to signalling in abiotic and biotic stress (Michaelson et al., 2016; Huby et al., 2020). The lipid composition of a single PM is not uniform. There is asymmetry in the PM meaning that the inner leaflet and outer leaflets can have different lipid composition (Gronnier et al., 2018). In addition, the PM has variable regions, called membrane microdomains or membrane nanodomains,

that vary both in lipid and protein composition from surrounding membrane areas (Gronnier et al., 2018).

To study both lipids and proteins in the PM, enriched membrane preparations can be useful. A method called the two-phase partitioning system (Widell et al. 1982; Larsson et al., 1994) has been used for the preparation of enriched PM fractions from plant tissues. In this method, plant material is ground and combined with a buffer solution where broken membranes spontaneously form vesicles. These vesicles are separated from cellular debris by filtration and centrifugation and are called microsomal vesicles (MF). PM vesicles are then separated by mixing MF with water-soluble compounds, dextran, and polyethylene glycol (PEG) in correct salt and buffer concentrations.

Right-side-out PM vesicles partition to the upper phase (UP) with PEG whereas the other cellular membranes together with inside-out PM vesicles remain in the lower phase (LP) with Dextran due to the surface properties of the vesicles (Widell et al., 1982). The purification method needs optimisation for each tissue to achieve optimal separation and yield of PM membranes. An optimised two-phase system for Norway spruce tissues of developing xylem, developing phloem, and lignin-forming cultured cells has yielded a membrane fraction that contains enriched PM and tonoplast, and a small amount of other cellular membranes (Kärkönen et al., 2014; Väisänen et al., 2018).