Background: Alamethicin is a membrane-active peptide isolated from the beneficial root-colonising fungus Trichoderma viride. This peptide can insert into membranes to form voltage-dependent pores. We have previously shown that alamethicin efficiently permeabilises the plasma membrane, mitochondria and plastids of cultured plant cells. In the present investigation, tobacco cells (Nicotiana tabacum L. cv Bright Yellow-2) were pre-treated with elicitors of defence responses to study whether this would affect permeabilisation.Results: Oxygen consumption experiments showed that added cellulase, already upon a limited cell wall digestion, induced a cellular resistance to alamethicin permeabilisation. This effect could not be elicited by xylanase or bacterial elicitors such as flg22 or elf18. The induction of alamethicin resistance was independent of novel protein synthesis. Also, the permeabilisation was unaffected by the membrane-depolarising agent FCCP. As judged by lipid analyses, isolated plasma membranes from cellulase-pretreated tobacco cells contained less negatively charged phospholipids ( PS and PI), yet higher ratios of membrane lipid fatty acid to sterol and to protein, as compared to control membranes.Conclusion: We suggest that altered membrane lipid composition as induced by cellulase activity may render the cells resistant to alamethicin. This induced resistance could reflect a natural process where the plant cells alter their sensitivity to membrane pore-forming agents secreted by Trichoderma spp. to attack other microorganisms, and thus adding to the beneficial effect that Trichoderma has for plant root growth. Furthermore, our data extends previous reports on artificial membranes on the importance of lipid packing and charge for alamethicin permeabilisation to in vivo conditions.

Diversity and Evolution of Plastids and Their Genomes.- The Chloroplast Envelope Proteome and Lipidome.- The Chloroplast Protein Import Apparatus, Its Components, and Their Roles.- Chloroplast Membrane Lipid Biosynthesis and Transport.- The Role of Metabolite Transporters in Integrating Chloroplasts with the Metabolic Network of Plant Cells.- Retrograde Signalling.- Plastid Division Regulation and Interactions with the Environment.- Chloroplast Photorelocation Movement.- A Sentinel Role for Plastids.

As in other eukaryotes, plant plasma membranes contain sphingolipids, phospholipids, and free sterols. In addition, plant plasma membranes also contain sterol derivatives and usually <5 mol% of a galactolipid, digalactosyldiacylglycerol (DGDG). We earlier reported that compared to fully fertilized oats (Avena sativa), oats cultivated without phosphate replaced up to 70 mol% of the root plasma membrane phospholipids with DGDG. Here, we investigated the implications of a high DGDG content on membrane properties. The phospholipid-to-DGDG replacement almost exclusively occurred in the cytosolic leaflet, where DGDG constituted up to one-third of the lipids. In the apoplastic (exoplasmic) leaflet, as well as in rafts, phospholipids were not replaced by DGDG, but by acylated sterol glycosides. Liposome studies revealed that the chain ordering in free sterol/phospholipid mixtures clearly decreased when > 5mol% DGDG was included. As both the apoplastic plasma membrane leaflet (probably the major water permeability barrier) and rafts both contain only trace amounts of DGDG, we conclude that this lipid class is not compatible with membrane functions requiring a high degree of lipid order. By not replacing phospholipids site specifically with DGDG, negative functional effects of this lipid in the plasma membrane are avoided.-Tjellstrom, H., Hellgren, L. I., Wieslander, A., Sandelius, A. S. Lipid asymmetry in plant plasma membranes: phosphate deficiency-induced phospholipid replacement is restricted to the cytosolic leaflet. FASEB J. 24, 1128-1138 (2010). www.fasebj.org

It is well established that phosphate deficiency induces the replacement of membrane phospholipid with non-phosphorous lipids in extra-plastidial membranes (e.g. plasma membrane, tonoplast, mitochondria). The predominant replacement lipid is digalactosyl diacylglycerol (DGDG). This paper reports that the phospholipid-to-DGDG replacement is reversible, and that when oat seedlings are re-supplied with radio-labelled phosphate, it is initially recovered primarily in phosphatidylcholine (PC). Within 2 d, the shoot contains more than half of the lipid-associated radiolabel, reflecting phosphate translocation. Oat was also cultivated in different concentrations of phosphate and the DGDG/PC ratio in roots and phospholipase activities in isolated plasma membranes was assayed after different times of cultivation. The DGDG/PC ratio in root tissue correlated more closely with plasma membrane-localized phospholipase D, yielding phosphatidic acid (PA), than with plasma membrane-localized PA phosphatase, the activity that results in a decreased proportion of phospolipids. The lipid degradation data did not reflect a significant involvement of phospholipase C, although a putative phospholipase C analogue, non-specific phospholipase C4 (NPC4), was present in oat roots. The correlation between increased phospholipase D activity and DGDG/PC ratio is consistent with a model where phospholipid-to-DGDG replacement involves formation of PA that readily is removed from the plasma membrane for further degradation elsewhere.

Chloroplasts and their surrounding cell are highly interdependent. One example is lipid metabolism, where the cell depends on its chloroplasts to provide fatty acids for lipid synthesis in the endoplasmic reticulum (ER) and in turn, chloroplasts rely on import of lipid precursors from the ER. Despite its fundamental importance, the route for lipid trafficking into and out of chloroplasts remains unknown. Biochemical studies of plant membrane lipid metabolism have suggested the possibility of lipid transport at membrane contact sites (MCSs) between the ER and chloroplasts. With the aid of optical manipulation, we recently could present physical evidence for this association. Leaf protoplasts isolated from Arabidopsis thaliana expressing green fluorescent protein (GFP) in the ER lumen were observed by confocal microscopy. A laser scalpel was used to rupture the protoplasts. ER fragments associated with the released chloroplasts could be stretched out by optical tweezers but remained attached to the chloroplast surface, even when a stretching force of 400 pN was applied. We thus provided the first physical evidence for MCSs between two membranes and we propose for the ER-chloroplast pair, that such tight associations are involved in bidirectional lipid trafficking between the two compartments.

Eukaryote cells depend on membrane lipid trafficking from biogenic membranes, like the endoplasmic reticulum (ER), to other membranes in the cell. Two major routes for membrane lipid transport are recognized: vesicular trafficking and lipid transfer at zones of close contact between membranes. Specific ER regions involved in such membrane contact sites (MCSs) have been isolated, and lipid transfer at MCSs as well as protein-protein interactions between the partaking membranes have been demonstrated (reviewed by Holthuis, J. C. M., and Levine, T. P. (2005) Nat. Rev. 6, 209-220). Here we present the first demonstration of the physical association between membranes involved in MCSs: by using optical imaging and manipulation, strong attracting forces between ER and chloroplasts are revealed. We used Arabidopsis thaliana expressing green fluorescent protein in the ER lumen and observed leaf protoplasts by confocal microscopy. The ER network was evident, with ER branch end points apparently localized at chloroplast surfaces. After rupture of a protoplast using a laser scalpel, the cell content was released. ER fragments remained attached to the released chloroplasts and could be stretched out by optical tweezers. The applied force, 400 pN, could not drag a chloroplast free from its attached ER, which could reflect protein-protein interactions at the ER-chloroplast MCSs. As chloroplasts rely on import of ER-synthesized lipids, we propose that lipid transfer occurs at these MCSs. We suggest that lipid transfer at the MCSs also occurs in the opposite direction, for example to channel plastid-synthesized acyl groups to supply substrates for ER-localized synthesis of membrane and storage lipids.

Background: The phospholipids of the plant plasma membrane are synthesized in the endoplasmic reticulum (ER). The majority of these lipids reach the plasma membrane independently of the secretory vesicular pathway. Phospholipid delivery to the mitochondria and chloroplasts of plant cells also bypasses the secretory pathway and here it has been proposed that lysophospholipids are transported at contact sites between specific regions of the ER and the respective organelle, followed by lysophospholipid acylation in the target organelle. To test the hypothesis that a corresponding mechanism operates to transport phospholipids to the plasma membrane outside the secretory pathway, we investigated whether lysolipid acylation occurs also in the plant plasma membrane and whether this membrane, like the chloroplasts and mitochondria, is in close contact with the ER.Results: The plant plasma membrane readily incorporated the acyl chain of acyl-CoA into phospholipids. Oleic acid was preferred over palmitic acid as substrate and acyl incorporation occurred predominantly into phosphatidylcholine (PC). Phospholipase A(2) stimulated the reaction, as did exogenous lysoPC when administered in above critical micellar concentrations. AgNO3 was inhibitory. The lysophospholipid acylation reaction was higher in a membrane fraction that could be washed off the isolated plasma membranes after repeated freezing and thawing cycles in a medium with lowered pH. This fraction exhibited several ER-like characteristics. When plasma membranes isolated from transgenic Arabidopsis expressing green fluorescent protein in the ER lumen were observed by confocal microscopy, membranes of ER origin were associated with the isolated plasma membranes.Conclusion: We conclude that a lysoPC acylation activity is associated with plant plasma membranes and cannot exclude a PC transacylase activity. It is highly plausible that the enzyme(s) resides in a fraction of the ER, closely associated with the plasma membrane, or in both. We suggest that this fraction might be the equivalent of the mitochondria associated membrane of ER origin that delivers phospholipids to the mitochondria, and to the recently isolated ER-derived membrane fraction that is in close contact with chloroplasts. The in situ function of the lysoPC acylation/PC transacylase activity is unknown, but involvement in lipid delivery from the ER to the plasma membrane is suggested.

We recently reported that cultivation of oat (Avena sativa L.) without phosphate resulted in plasma membrane phosphoglycerolipids being replaced to a large extent by digalactosyldiacylglycerol (DGDG) (Andersson, M. X., Stridh, M. H., Larsson, K. E., Liljenberg, C., and Sandelius, A. S. ( 2003) FEBS Lett. 537, 128-132). We report here that DGDG is not the only non-phosphorous-containing lipid that replaces phospholipids but that also the content of glucosylceramides and sterolglycosides increased in plasma membranes as a response to phosphate starvation. In addition, phosphate deficiency induced similar changes in lipid composition in the tonoplast. The phospholipid-to-glycolipid replacement apparently did not occur to any greater extent in endoplasmic reticulum, Golgi apparatus, or mitochondrial inner membranes. In contrast to the marked effects on lipid composition, the polypeptide patterns were largely similar between root plasma membranes from well-fertilized and phosphate-limited oat, although the latter condition induced at least four polypeptides, including a chaperone of the HSP80 or HSP90 family, a phosphate transporter, and a bacterial-type phosphoesterase. The latter polypeptide reacted with an antibody raised against a phosphate deficiency-induced phospholipase C from Arabidopsis thaliana (Nakamura, Y., Awai, K., Masuda, T., Yoshioka, Y., Takamiya, K., and Ohta, H. ( 2005) J. Biol. Chem. 280, 7469-7476). In plasma membranes from oat, however, a phospholipase D-type activity and a phosphatidic acid phosphatase were the dominant lipase activities induced by phosphate deficiency. Our results reflect a highly developed plasticity in the lipid composition of the plasma membrane and the tonoplast. In addition, phosphate deficiency-induced alterations in plasma membrane lipid composition may involve different sets of lipid-metabolizing enzymes in different plant tissues or species, at different stages of plant development and/or at different stages of stress adjustments.

Background: The thylakoid membrane of higher plant chloroplasts is made of membrane lipids synthesized in the chloroplast envelope. As the inner envelope membrane and the thylakoid are separated by the aqueous stroma, a system for transporting newly synthesized lipids from the inner envelope membrane to the thylakoid is required. Ultrastructural as well as biochemical studies have indicated that lipid transport inside the chloroplast could be mediated by a system similar in characteristics to vesicular trafficking in the cytosol. If indeed the chloroplast system is related to cytosolic vesicular trafficking systems, a certain degree of sequence conservation between components of the chloroplast and the cytosolic systems could be expected. We used the Arabidopsis thaliana genome and web-based subcellular localization prediction tools to search for chloroplast-localized homologues of cytosolic vesicular trafficking components.Results: Out of the 28952 hypothetical proteins in the A. thaliana genome sequence, 1947 were predicted to be chloroplast-localized by two different subcellular localization predictors. In this chloroplast protein dataset, strong homologues for the main coat proteins of COPII coated cytosolic vesicles were found. Homologues of the small GTPases ARF1 and Sar1 were also found in the chloroplast protein dataset.Conclusion: Our database search approach gives further support to that a system similar to cytosolic vesicular trafficking is operational inside the chloroplast. However, solid biochemical data is needed to support the chloroplast localization of the identified proteins as well as their involvment in intra-chloroplast lipid trafficking.

Here we report that cytosolic phospholipases are involved in the utilization of phosphatidylcholine (PC) as substrate for chloroplast-localized synthesis of monogalactosyldiacylglycerol (MGDG). Isolated chloroplasts were pre-incubated with lysoPC and [14C]18:0-CoA to form [14C]PC. When soluble plant proteins (cytosol) and UDP-galactose were added, [14C] MGDG was formed. An inhibitor of phospholipase D markedly lowered the formation of [14C]MGDG, whereas thermolysin pretreatment of the chloroplasts was without effect. The cytosolic activity resided in the >100-kDa fraction. In a second approach, [14C]PC-containing lipid mixtures were incubated with cytosol. Degradation of [14C]PC to [14C]diacylglycerol was highest when the lipid composition of the mixture mimicked that of the outer chloroplast envelope. We also investigated whether PC of extraplastidic origin could function as substrate for MGDG synthesis. Isolated chloroplasts were incubated with enriched endoplasmic reticulum containing radiolabelled acyl lipids. In the presence of cytosol and UDP-galactose, there was a time-dependent transfer of [14C]PC from this fraction to chloroplasts, where [14C]MGDG was formed. We conclude that chloroplasts recruit cytosolic phospholipase D and phosphatidic acid phosphatase to convert PC to diacylglycerol. Apparently, these lipases do not interact with chloroplast surface proteins, but rather with outer membrane lipids, either for association to the envelope or for substrate presentation.

We have studied the interaction of GTP and its slowly-hydrolysable analogue, GTPγS, with isolated chloroplast envelope, stroma and thylakoids from spinach (Spinacia oleracea). We used a filter assay to monitor [35S]GTPγS-dependent labelling of the chloroplast fractions. All fractions were labelled by [35S]GTPγS and the labelling was competed by unlabelled GTPγS, GTP and GDP over ATP and GMP. The label associated with the chloroplast fractions was, at least partly, covalent thiophosphate labelling of proteins as well as traces of lipid labelling. The pattern of [35S]GTPγS-dependent protein thiophosphorylation differed from [35S]ATPγS dependent thiophosphorylation as well as from [γ-32P]GTP and [γ-32P]ATP dependent protein phosphorylation. Incubation of chloroplast fractions with [γ-32P]GTP also resulted in heavy labelling of several lipids and lipid phosphorylation was markedly more prominent than lipid thiophosphorylation. The results are discussed in relation to the possible roles of guanine nucleotides in the regulation of thylakoid biogenesis.

The plasma membranes of oat normally resemble those of other eukaryotes in containing mainly phospholipids and sterols. We here report the novel finding that the galactolipid digalactosyldiacylglycerol (DGDG) can constitute a substantial proportion of oat plasma membrane lipids, in both shoots and roots. When oat was cultivated under severe phosphate limitation, up to 70% of the plasma membrane phosphoglycerolipids were replaced by DGDG. Our finding not only reflects a far more developed potential for plasticity in plasma membrane lipid composition than often assumed, but also merits interest in the context of the limited phosphate availability in many soils.

Proteins binding guanosine triphosphate (GTP) have emerged as important regulators in several cellular processes in plants. To investigate any role of such proteins in chloroplast functions, we subjected envelope, stroma and thylakoid fractions isolated from spinach chloroplasts to two different GTP-binding assays. With both methods, we detected GTP-specific binding only in the envelope fraction. Two chloroplast envelope proteins with the apparent molecular weights of 30.5 and 33.5 kDa, respectively, bound [alpha-32P]GTP after SDS-PAGE followed by electroblotting onto a PVDF-membrane and renaturation. Both proteins were intrinsic proteins located in the outer chloroplast envelope. Also, when the fractions were incubated with [alpha-32P]GTP, followed by periodate oxidation and borohydride reduction to cross-link GTP to proteins, two proteins in the envelope fraction, of apparent molecular weights of 28 and 39 kDa, appeared to specifically bind GTP. When agents that stimulate heterotrimeric G-proteins, cholera toxin or the mastoparan analogue mas7, were added to isolated chloroplast envelope, the binding of radiolabelled GTP to the 39 kDa protein, a protein of the inner chloroplast envelope, was stimulated, whereas GTP-binding of the 28 kDa protein, a protein of the outer envelope, was unchanged. Mas7 also stimulated synthesis of monogalactosyl diacylglycerol in isolated chloroplast envelope. The occurrence and regulation of GTP-binding proteins in the chloroplast envelope suggests that GTP-binding proteins could be involved in communication with the extraplastidic compartment during chloroplast biogenesis and development.

Plasma membranes were isolated from leaves of 16‐day‐old garden pea, Pisum sativum L., that had been grown in the absence or presence of 65 nl l−1 ozone for 4 days prior to membrane isolation. Plasma membranes from ozone‐fumigated plants contained significantly more acyl lipids per protein than those from leaves of plants grown in filtered air on a molar/weight ratio. The ratio between the major acyl lipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), also increased due to the ozone fumigation, while the fatty acid unsaturation level was unaltered in total plasma membrane acyl lipids, as well as in PC and PE. The amount of free sterols per protein was unaltered, but the percentage of campesterol increased, concomitant with a decrease in stigmasterol. The dynamical properties of the isolated plasma membranes were assessed using Laurdan fluorescence spectroscopy, which monitors water penetration and mobility at the hydrophilic‐hydrophobic interface of the membrane. At 0°C, the molecular mobility was slightly lower in plasma membranes from ozone‐fumigated plants than in plasma membranes from control plants, possibly reflecting the increased PE/PC, campesterol/stigmasterol and lipid/protein ratios, and suggesting that ozone‐fumigated pea plants may be more susceptible to freezing injuries.