Membrane Remodelling In Phosphorus-Deficient Plants

Introduction Lipid bilayers divide the eukaryotic cell into different compartments. Furthermore, lipids are major constituents of the plasma membrane, the outer boundary of the cell. Each membrane is characterised by a unique set of lipids including phospholipids, glycolipids, sterols and sphingolipids. Plant cells contain large amounts of the two galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), and of the sulfolipid sulfoquinovosyldiacylglycerol (SQDG). Together with the phospholipid phosphatidylglycerol (PG), which is found in chloroplasts and in extraplastidial compartments, these four lipids are the main constituents of the thylakoid membranes (Siegenthaler, 1998). The characteristic lipid composition of thylakoids is conserved in all organisms performing oxygenic photosynthesis (land plants, green algae, cyanobacteria), where these lipids play important roles in photosynthetic electron transfer (Wada & Murata, 1998). A further phospholipid, phosphatidylcholine (PC), is present in the outer chloroplast envelope, but absent from thylakoids (Moreau et al., 1998). The characteristics of membrane lipids are further determined by their fatty acids bound to the sn-1 and sn-2 positions of the glycerol moiety. The combinational possibilities of different fatty acids result in a high diversity of molecular species for each lipid class. The plastids are the site of de novo fatty acid biosynthesis, with 16:0 and 18:1 as the main products (Ohlrogge et al., 1979). Fatty acids are used for the synthesis of plastid-derived lipids (prokaryotic pathway), or are exported to the endoplasmic reticulum (ER) where they are incorporated into ER-derived extraplastidial lipids (eukaryotic pathway). Lipid precursors assembled at the ER can also be returned to the plastids for the formation of ER-derived plastidial lipids. Plant fatty acids are characterised by a large number of double bonds. Palmitoyl (16:0) groups are desaturated to 16:1 and 16:3 in the chloroplast, and 18:1 is desaturated to 18:2 and 18:3 in the chloroplast and at the ER. These desaturation reactions occur on acyl groups bound to glycerolipids (Ohlrogge & Browse, 1995). The fatty acid profile of each lipid represents a signature allowing the assignment to different compartments or pathways. The origin of glycerolipids can be determined by their fatty acids bound to the sn-2 position of the glycerol backbone. Glycerolipids from the prokaryotic pathway contain exclusively C16 at sn-2 and are only found in the plastids, while glycerolipids from the eukaryotic pathway are characterised by C18 fatty acids at sn-2 and are present in extraplastidial and plastidial membranes (Ohlrogge & Browse, 1995). Plants such as Arabidopsis thaliana employ both pathways for glycerolipid synthesis, and are designated 16:3 plants because of the presence of 16:3 in the galactolipids. Plants synthesizing exclusively eukaryotic glycerolipids lacking 16:3 are termed 18:3 plants.

Membrane lipid remodelling during phosphate deprivation Lipids play crucial roles in the plant’s ability to survive and acclimatise to environmental changes. Inorganic phosphate (Pi) is an essential macronutrient for plant growth and development (Raghothama, 1999). Many soils are characterised by low Pi availability (Chapter 1) (Fang et al., 2009; Vance et al., 2003), and plants have developed different strategies to respond to Pi deprivation, including morphological, physiological and metabolic acclimatisation mechanisms to enhance Pi acquisition and mobilisation (Fang et al., 2009; Vance et al., 2003). Arabidopsis responds to Pi deprivation with a twofold increase in root proliferation accompanied by a reduced rosette leaf growth (Li et al., 2006). Plants can mobilise Pi from endogenous P-containing resources such as RNA, organic phosphate-esters, and phospholipids; indeed, about one-third of the organic-P in leaves of Pi fertilised plants is bound to phospholipids (Poirier et al., 1991). Their substitution with lipids that do not contain phosphorus saves Pi, and enables plants to survive on low-P soils. DGDG plays a prominent role in lipid remodelling under Pi deprivation, as it serves as a surrogate for phospholipids in plastidial and extraplastidial compartments. The lipid response during Pi deprivation differs between leaves and roots. Due to the abundance of photosynthetic membranes the leaves contain large proportions of galactolipids; thus, during Pi deprivation DGDG levels increase about twofold in Arabidopsis leaves, accompanied by an equivalent decrease in phospholipids (Hartel & Benning, 2000; Hartel et al., 2001; Kelly et al., 2003; Li et al., 2006). The galactolipid concentration in roots is low, because root plastids lack internal membranes. In contrast to leaves, the increase in DGDG level in roots during Pi deprivation is much greater, about eight- to tenfold. Most of the extra DGDG formed in the roots is extraplastidial, and the DGDG increase is not sufficient to substitute for the decrease in phospholipids, leading to a reduction in total lipid concentration by 42% (Li et al., 2006). The experimental strategy for Pi deficiency includes the transfer of plants to low-Pi soil or an agarose medium. The pho1 mutant of Arabidopsis represents an alternative, genetic model to investigate Pi deprivation (Poirier et al., 1991). A single recessive mutation in the PHO1 locus leads to a block of Pi transport from the roots to the shoots. The aerial parts of pho1 plants show symptoms of Pi deprivation, including reduced growth and alterations of lipid composition, while photosynthesis is not affected (Hartel et al., 1998). The plasma membrane is a prominent extraplastidial target for the replacement of phospholipids with glycolipids during Pi deprivation, as shown for oat (Avena sativa) (Andersson et al., 2003). In non-stressed oat, DGDG is present in the plasma membrane as a minor constituent (<5%) (Tjellstrom et al., 2010), but after four weeks of Pi deprivation the DGDG concentration in the plasma membrane of shoots and roots is increased to 46% and 70%, respectively. The fatty acid composition of the extraplastidial DGDG resembles that of total root DGDG, with 16:0 and 18:2 dominating over 18:3. However, 18:3, which is highly abundant in chloroplasts, is the major fatty acid in the DGDG of shoots (Andersson et al., 2003; Russo et al., 2007). In oat (18:3 plant), all 16:0 in galactolipids is linked to the sn-1 position of glycerol. DGDG is also found in the tonoplast and the mitochondria of Pi-deprived plants (Andersson et al., 2005; Jouhet et al., 2004). In addition, DGDG occurs in the peribacteriod membrane surrounding rhizobial bacteria in nitrogen-fixing root nodules of legumes (Gaude et al., 2004). To date, no reports have been made on the occurrence of DGDG in the ER or the Golgi.

The conversion of phospholipids into DGDG encompasses the initial hydrolysis of the P-containing polar head groups from phospholipids, particularly PC, by phospholipases (Cruz-Ramırez et al., 2006; Li et al., 2006). Phosphate is subsequently mobilised from the polar head groups by phosphatases. A number of genes were identified showing transcriptional induction upon Pi deficiency (Hammond et al., 2004; Mission et al., 2005; Morcuende et al., 2007; Wu et al., 2003). Many Pi-starvation-induced genes encode phospholipid-hydrolysing enzymes, including phospholipases D (PLD), C (PLC), A1 (PLA1), A2 (PLA2), and B (PLB). PLD and PLC activities are involved in the formation of PA and DAG, respectively (Wang, 2000). The phospholipases PLA and PLB hydrolyse the sn-1 ester bond of phospholipids to yield free fatty acids and lysophospholipids, or sequentially remove the two fatty acids from phospholipids, respectively. These enzymes are involved in the decrease in phospholipid concentration under Pi starvation.

Phosphate deprivation leads to the reduction of all phospholipids, independent of their bilayer-forming or non-bilayer-forming characteristics. DGDG as a bilayer-forming lipid is able to replace the bilayer-forming lipids PC, phosphatidylinositol (PI), and PG. The capacity to replace the nonbilayer-forming lipid phosphatidylethanolamine (PE) with DGDG might be limited, as a certain ratio of bilayer- to non-bilayer-forming lipids needs to be maintained for correct membrane functions. The non-bilayer lipid MGDG which, in principle, could substitute for PE, is not involved in lipid remodelling during Pi deprivation. MGDG the precursor for DGDG synthesis, but its levels do not increase and it remains in the chloroplast during Pi deprivation. The process of phospholipid replacement with DGDG under Pi deprivation is reversible when Pi supplementation is increased (Tjellstrom et al., 2008).

Sulfolipid (SQDG) and PG are minor components of the photosynthetic membranes (Table 9.1), but due to their anionic charge they are required to maintain the surface charge of thylakoid membranes. Although SQDG and PG can mutually substitute for each other in the thylakoids (Frentzen, 2004; Yu & Benning, 2003), the diacylglycerol (DAG) backbone of PG does not serve as precursor for SQDG synthesis. The structure of PG is characterised by its unique 16:1Δ3trans fatty acid at sn-2 (Dorne & Heinz, 1989), while the main SQDG molecular species is 18:3/16:0-SQDG (18:3 at sn-1, 16:0 at sn-2), and to a lesser amount 18:3/18:3-SQDG (Hartel et al., 1998; Okazaki et al., 2009; Welti et al., 2003). Recently, a further anionic glycolipid, glucuronosyldiaclyglycerol (GlcADG), was discovered in Arabidopsis which is synthesised under Pi deprivation in the chloroplasts (Okazakiet al., 2013). At present it is unknown whether GlcADG is exported to extraplastidial membranes for the replacement of phospholipids.

The regulation of lipid remodelling during Pi deprivation is mostly exerted by changes in gene expression. During Pi limitation, the expression of many genes involved in phospholipid degradation and galactolipid and sulfolipid synthesis is induced (Mission et al., 2005; Morcuende et al., 2007). It is generally accepted that an increased mRNA level is translated into an elevated protein amount, resulting in increased enzymatic activity. For many enzymes of lipid synthesis, antibodies are not available and enzyme assays are challenging. Therefore, the presumed increases in protein amount or enzyme activity after Pi deprivation have not been experimentally confirmed. It is clear that further regulatory mechanisms need to be taken into account, including posttranslational modifications (PTMs) such as phosphorylation, regulation by calcium or magnesium, or interactions with phytohormones. It should also be noted that the subcellular localisation of lipids and lipid-modifying enzymes, as well as lipid trafficking, contribute to lipid metabolism during Pi deprivation.