Prof. Lipid Res. eel. 31, No. 1, pp. 53-63, 1992
0163-7827/92/$15.00 © 1992 Pergamon Press pk
Printed in Great Britain. All fights reserved
INOSITOL-CONTAINING LIPIDS IN HIGHER PLANTS ALISTAIR M. HETHERINGTON* and BJffRN K. DROBAK~f *Division of Biological Sciences, IEBS, Lancaster University, Lancaster LAI 4YQ, U.K. tDepartment of Cell Biology, John Innes Centrefor Plant Science Research, Colney Lane, Norwich NR4 7UH, U.K.
CONTENTS 53 53 54 54 54 55 55 57 59
ABBREVIXTIONS I. INTRODUCTION II. PHOSPHATIDYLINOSITOL A. Structure and cellular distribution B. Biosynthesisand metabolism IlL POLYPHOSPHOINO$1TIDI~ A. Structure and cellular distribution B. Biosynthesisand metabolism IV. INOSITOL LYSOPHOSPHOLIPIDS
V. GLYCOPHOSPHosPmNC, OIJPn>S
60
61 62 62
VI. CONCLUSIONSAND FUTURE PROSPECTS ACKNOWLrd~EMENTS
R~ntL~Cm
ABBREVIATIONS CDP-DAG Cystosine diphosphate diacylglycerol GPS Giycophosphosphingolipid IAA Indole-3-acetic acid PC Phosphatidylcholine Ptdlns Phosphatidylinositol PtdIns(4)P Phosphatidyl(4)phosphate Ptdlns(4,5)P2 Phosphatidyl(4,5)bisphosphate I. INTRODUCTION Interest in the biochemistry and physiology of inositol and inositol-containing compounds in plants is not a new phenomenon. It is thus more than 100 years ago that Pf¢ffer first isolated phytic acid (inositol (1,2,3,4,5,6) hexakisphosphate) from seeds. In spite of considerable effort by plant scientists over the years, comparatively little is still known about this group of compounds, but that situation is beginning to change. Until 1983/84 the emphasis in this area of research was mainly on inositol phosphates involved in phytic acid metabolism, indole-3-acetic acid (IAA) esters of myo-inositol and inositol containing lipids such as phosphatidylinositol and the glycophosphosphingolipids. Around 1983 a major breakthrough was made by researchers in the mammalian field who discovered that a group of inositol-containing phospholipids (the phosphoinositides) play a central role in the cellular perception and transduction of a wide variety of extracellular signals. It is now generally recognised that these inositol lipids have similar functions in organisms as disparate as slime moulds, yeast, blowflies and humans. I In addition to the role played in transmembrane signalling inositol-containing lipids appear to have several additional important functions, these include: regulation of enzymic activity, anchoring of proteins to membranes and modulation of cytoskeletal dynamics. 2~2'49 The discovery of the mammalian phosphoinositidc systems has caused a major upsurge in interest in inositol lipids and inositol phosphates in all eukaryotes and has led plant scientists to ask the questions: "Is a similar system involved in transmembrane signalling in plant cells?" and "Do inositol-containing lipids have other physiological functions in plants?" Although definitive answers to these questions have yet to be found, it is becoming 53
.54
A.M. HETHERINGTONand B. K, DR~BAK
increasingly likely that when they are, they will be affirmative. One of the main problems in the characterisation of the plant phosphoinositide system and other aspects of inositol metabolism is the presence of a vast array of inositol-containing compounds in plant cells. It should be noted that there are 63 isomers of phosphorylated myo-inositol, and other isomers of inositol are known to occur in plant tissues. In addition to these several IAA- and methyl esters of myo-inositol, myo-inositol glycosides and at least 10-15 inositol-containing lipids (several with unknown chemical structures) have been shown to be present in various plant cells. In the present context we will focus on the inositol-containing lipids. It is not our intention to present an exhaustive review but rather to give a brief overview of the current state of knowledge and provide an entrance into the literature in this rapidly developing area of plant science. II. P H O S P H A T I D Y L I N O S I T O L
A. Structure and Cellular Distribution
Phosphatidylinositol [1-(3-sn-phosphatidyl)-L-myoinositol] (PtdIns) (Fig. 1) is one of the major phospholipids of plant membranes. It has been reported that PtdIns may comprise up to 19% of the total phospholipid of the plasma membrane, while in contrast chloroplasts contain comparatively small amounts (1-2% of the total phospholipid). In common with most plant phospholipids a saturated fatty acid such as palmitic is often found at the sn- 1 position on PtdIns while the acyl group at the sn-2 position is unsaturated (e.g. linoleic), s' Available evidence would suggest that PtdIns has an asymmetric distribution within the membrane. 9 The reviews by Harwood 2s and Mudd 6~ provide detailed information on the distribution and fatty acid composition of this lipid within different plants, plant parts and among various types of membrane. B. Biosynthesis and Metabolism
The biosynthesis of PtdIns has recently been reviewed by Moore ~ and for full details it is recommended that the reader consult this comprehensive account. However, in outline there are two routes to the synthesis of PtdIns; the first is catalysed by CDP-DAG: inositol 3-phosphatidyltransferase (EC 2.7.8.11) and uses cytosine diphosphate diacylglycerol (CDP-DAG) as substrate and Mn 2+ as a cofactor. 6,2°'s2'53'74'7sThe second mechanism is an inositol exchange catalysed by PtdIns myo-inositol phosphatidyltransferase. ~'74 Both reactions are likely to occur in the endoplasmic reticulum 55'7s'74 and possibly the Golgi complex. 6s o
0
?2.0
o
II - C - F~
II C H 2 - O - C - R'
!
0
II
R'-C-O-CH
O
I
w
CH~--O- P--O-
I
R'-~-O-C
A , ~,
.
O
[
II
t
I~' C H 2 - O - C - R~ [
R- c - o -CH
B ,
[
O
I[
CH2-O-- P-- O-
CH2--O - P - - O -
o
oII
"c
I
OH
OH
oH
(a) Ptdlns
(b) Ptdlns(4)P
(c) Ptdlns(4,5)P 2
Fso. I. The structures of (a) phosphatidylinositol (Ptdlns), (b) phosphatidylinositol(4)phosphate, (PtdIng4)P) and (¢) phosphatidylinositol(4,5)bisphosphatc, (Ptdlns(4,5)P2). In these figures R' and R" represents two fatty acyl residues esterified respectively at the sn-I and sn-2 positions on the inositol phospholipid. For further details see the text. Reaction A is catalys©d by Ptdlns kinasc, reaction B by Ptdlns(4)P kinase, reaction C by Ptdlns(4,5)P2 phosphatase and reaction D by Ptdlns(4)P phosphatase.
lnositol-containing lipids
55
PtdIns is a substrate for a number of lipid degrading enzymes. The best studied is the nonspecific acyl hydrolase from potato tubers. 3a For recent reviews of this group of enzymes the reader should consult Huang 3s'36 (but see also Section IV), The activity of phospholipases A1 (EC 3.1.1.32), A2 (EC 3.1.1.4) and B (EC 3.1.1.5) are not well characterised in plants and currently little is known about their role in PtdIns catabolism (see Section IV). Unusually, among the membrane phospholipids PtdIns does not appear to be a substrate for phospholipase D (EC 3.1.4.4). 23 The activity of phospholipase C towards Ptdlns and the polyphosphoinositides will be considered in Section III.B.
III. P O L Y P H O S P H O I N O S I T I D E S
A. Structure and Cellular Distribution
The presence of phosphorylated species of PtdIns in higher plants was first reported around 1985.7°That no such reports can be found in earlier literature may be due to several factors. The most likely is that the traditional Folch-type lipid extractions which have been employed by most plant researchers are unsuited for the extraction of highly polar phospholipids. If near neutral/high salt "upper phase" solutions are used to wash chloroform/methanol rich extracts the polyphosphoinositides have a strong tendency to migrate into the polar phase which is commonly discarded. Only if acidified extraction solvents are used will the deprotonation of the phosphate ester groups of the polyphosphoinositides be suppressed sufficiently for these compounds to remain in the non-polar extraction solvent during washes. The first experiments which suggested the presence of polyphosphoinositides in plant tissues were carried out using radiolabelled cells and tissues as lipid source, acidified extraction systems and separation of lipids by T L C . 5'13'29'7° It was found that two radiolabelled lipids had chromatographic properties closely resembling those of mammalian phosphatidylinositol(4)phosphate (PtdIns(4)P) and phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2). Direct chemical determination of the abundance of polyphosphoinositides in plant cells and tissues has so far not been carried out so the best estimates come from isotope labelling experiments using tissues in which near isotopic equilibrium has been reached. Such experiments have given ratios of Ptdlns: PtdIns(4)P: PtdIns(4,5)P2 ranging from 300:17:1 to 10:1 : 1.67'7° In most of these experiments lipids have been separated by TLC and the identification of polyphosphoinositides has been indirect and based on co-chromatography of labelled compounds with authentic mammalian polyphosphoinositide standards. Whereas such an approach is often adequate for studies of mammalian phosphoinositide metabolism the same is not true for plants. Drobak et al. ~3using a triple-isotope approach found that when labelled phospholipids from suspension cultured tomato cells were separated by TLC and the radioactivity derived from 3H-glycerol, 3H-inositol and 32p-orthophosphate respectively determined in compounds co-chromatographing with authentic standards, discrepancies existed. Whereas the ratio of incorporated phosphate:inositol: glycerol in the compound cochromatographing with the PtdIns(4)P standard was very close to the ratio expected on the basis of the chemical structure and biosynthetic pathways of PtdIns(4)P the same was not true for labelled material co-chromatographing with PtdIns(4,5)P2. Only a minute amount of radioactivity derived from 3H-glycerol could be detected and ratios of phosphate:inositol were close to 1:1. These findings led the authors to conclude that: (1) the majority of the compounds co-migrating with authentic PtdIns(4,5)P2 was not Ptdlns(4,5)P2 but more likely a glycophosphosphingolipid (see Section V) and (2) if such compounds are generally present in plant tissues the amount of PtdIns(4,5)P2 reported by various groups using similar methods of analyses are likely to have been considerably overestimated. Experiments where glycerophosphoinositides or inositol phosphate derivatives of phosphoinositides from equilibrium labelled plant tissues have been separated by HPLC (a procedure which largely overcomes problems of co-chromatography of polyphosphoinositide derivatives and other inositol derivatives) indicate that
56
A.M. HETr~mNGTONand B. K. DROBAK
Ptdlns(4,5)P2 is likely not to constitute more than 0.05% of total phospholipids or 0.5% of phosphoinositides. ~3'39 Determination of the structure of the plant polyphosphoinositides was carried out by Irvine and co-workers39 using the strategy illustrated in Fig. 2. Deacylation ( O N acylation) of polyphosphoinositides followed by removal of the glycerol backbone (mild periodate/dimethylhydrazine) results in the formation of inositol polyphosphates (this procedure is equivalent to the action of phospholipase C). If Ptdlns(4)P and Ptdlns(4,5)P2 are the parent molecules, D-myo-inositol(1,4,5)trisphosphate and D-myo-inositol(1,4) bisphosphate will be the resulting hydrolysis products. Treatment of inositol phosphates with periodate results in the cleavage of C--C bonds in the inositol ring between adjacent C-atoms if they are both carrying hydroxyls. Thus, all inositol bisphosphates will be attacked by periodate as will all inositol trisphosphates which do not have all three phosphomonoesters in meta positions. When the aldehyde derivatives of inositolbis/trisphosphates formed by periodate treatment are reduced (borohydride) and dephosphorylated the resulting products are alditols (except if inositol bisphosphates with phosphomonoester groups in para positions or inositol trisphosphates with phosphomonoester groups in meta positions are used as starting material in which case the reaction products are either destroyed in the process or will be myo-inositol). As illustrated in Fig. 2 the alditol derived from D-myo-inositol(1,4,5)trisphosphate by this procedure is D-iditol. Irvine et al. 39 found that the alditol obtained from the conversion of PtdlnsP2 isolated from pea leaf discs indeed was iditol and that the iditol was resistant to L-iditol dehydrogenase. This only leaves two possible structures open for the InsP3 derived from the PtdlnsP 2 namely Ins(1,4,5)P 3 or Ins(1,4,6)P 2. Upon incubation of the InsP3 with Ins(1,4,5)P3-5- phosphatase from human red blood cell membranes the plant derived InsP3 was hydrolysed with kinetics similar to authentic Ins(1,4,5)P~ confirming that the parent polyphosphoinositide is Ptdlns(4,5)P2. The InsP2 fraction derived from pea PtdlnsP co-chromatographed on HPLC with authentic Ins(1,4)P2 and when subjected to periodate oxidation, reduction and dephosphorylation yielded no noncyclic alditols. This suggests that the phosphomonoesters are para, and given the linkage of the D-1 phosphate in Ptdlns to the glycerol moiety, they must be in the 1 and 4 position and the plant Ptdlns be Ptdlns(4)P. The structures of Ptdlns(4)P and Ptdlns(4,5)P2 are shown in Fig. 1. Ptdlns(4,5)P2
~
O-N Transacylatlon
GPtdlns(4,5)P2 k Periodate/Olmethylhydrazlne o
HOPO- O 0 ~r- OPO-
_
-%o-~
Ins(1,4,SlP3
~eriodate o HO56. . 1 . ~ , ~
o IA
-O~O~ H
CH20H OH~ OH~
OH OH
/
0
"4 ° "
BorohydrldelDephosphor ylatlon
Flo. 2. Strategy used by Irvine and ¢0 -wor kers 39 to det©rmine the structure of plant polyphosphoinositides. For full details see the text.
Inositol-containing lipids
57
A number of novel phosphoinositides have recently been discovered in certain mammalian cells. 76 The structures of these lipids have been shown to be phosphatidylinositol(3)phosphate, phosphatidylinositol(3,4)bisphosphate and phosphatidylinositol(3,4,5)trisphosphate. In intact neutrophils activated by the formyl lx'ptide f-Met-Leu-Phe a phosphatidylinositol(4,5)bisphosphate-3-kinase seems to be responsible for agonist stimulated formation of polyphosphoinositides phosphorylated in the 3 position of the inositol moiety. 76 There is currently no evidence for the presence of such polyphosphoinositides in plant tissues (B. K. Dmbak, unpublished data) but a more detailed search in a wider range of plant tissues may change this situation. Although extensive surveys of the fatty acid composition of the polyphosphoinositides have not been carried out, the data at hand suggest that the predominant fatty acids in Ptdlns(4)P and Ptdlns(4,5)P2 are palmitate and linoleate) 3'7°'81 This is consistent with a synthetic pathway involving direct phosphorylation of Ptdlns. The precise subcellular localisation of plant polyphosphoinositides is currently not fully clarified but results from labelling experiments employing both intact cells and membrane fractions enriched in plasmalemma vesicles suggests that polyphosphoinositides, as in mammalian cells, are associated with the plasma membrane, 69'7° although, recent reports suggest that smaller amounts may be associated with organdies such as nuclei. 32 This assumption is supported by studies on the subcellular distribution of the phosphoinositide kinases. In isolated membrane fractions from hypocotyls of dark grown soybean, both Ptdlns and PtdlnsP hydroxykinases are associated predominantly with the plasma membrane whereas only negligible activity of these enzymes is found in the tonoplast, nuclei, mitochondria and plastid fractions. 7° It is clear that a more thorough investigation of the subcellular distribution of polyphosphoinositides is needed, in particular in the light of recent findings that PtdlnsP in mammalian cells can be synthesised via an intracellular route. 44 Problems facing plant scientists in pursuing this line of work are not only associated with the chemical analysis of small amounts of polyphosphoinositides but also with the production of membrane fractions of sufficient purity. B. Biosynthesis and Metabolism
In mammalian cells Ptdlns(4,5)P2 is formed by a two step phosphorylation of Ptdlns. Ptdlns is first phosphorylated in the 4 position of the inositol ring resulting in the formation of PtdIns(4)P. This lipid is then further phosphorylated in the 5 position yielding Ptdlns(4,5)Pv Two phosphohydrolases work concomitantly with the 4- and 5-hydroxykinase so Ptdlns(4)P and Ptdlns(4,5)P2 are constantly being formed and degraded (Fig. 1). This is termed the "futile" phosphoinositide cycle, but it is now obvious that it is anything but futile. That polyphosphoinositides are synthesised in a similar manner in plants is likely, but is as yet not proven. The most convincing evidence favouring such a scenario comes from in vitro studies using isolated membrane fractions as enzyme source and endogenous and exogenous phosphoinositides as substrates. Sommarin and Sandelius 75 thus demonstrated the presence of both Ptdlns and PtdIns(4)P hydroxykinases in purified plasma membranes from wheat. They found that both kinases were able to phosphorylate both endogenous and exogenous substrates and that the activity was modulated by detergents (Triton X 100, 0.1-0.3%). It is likely that the cause of the increase in kinase activity observed in the presence of both exogenous substrates and detergents is dual in nature, as detergents may affect both the form in which the substrate is presented to the enzyme and enzyme latency. Both the PtdIns and PtdIns(4)P kinase have absolute requirements for ATP and divalent cations--with a preference for Mg 2+. Maximum activity of the polyphosphoinositide kinases in wheat plasma membranes occurred at approximately 1 mM ATP and the Km was determined to be 0.2 m i . 75 Kamada and Muto 4t have demonstrated that submicromolar concentrations of calcium inhibit both PtdIns and PtdIns(4)P kinases and have suggested that the effect of calcium may be part of a regulatory system involved in transmembrane signalling.
58
A.M. HETHERINGTON and B. K. DRBBAK
In vivo experiments suggest that the rate of turnover of PtdIns(4)P is very fast in many plant tissues. Drzbak et al. 13 found that after short incubation times (i.e. 30 min) with 32p-orthophosphate that more than 30% of total label incorporated into phospholipids in suspension cultured tomato cells was in PtdIns(4)P. Although the position of the label in the PtdIns(4)P molecule was not determined it is likely that it was predominantly in the 4-phosphomonoester indicating a highly active PtdIns (4) hydroxykinase. A highly active PtdIns (4) hydroxykinase when seen in conjunction with the comparatively low chemical levels of PtdIns(4)P suggest that this lipid is metabolized further with some speed. The relative importance of hydroxykinases, phosphomonoesterases and phospholipases in this process remains to be clarified. Little is currently known about the rate of turnover of PtdIns(4,5)P2 in vivo. The main problem in the study of PtdIns(4,5)P2 metabolism is the very low chemical levels of this lipid but the high rate of 32p incorporation into the 4-phosphomonoester of PtdIns(4)P and the possibility of distinct cellular phosphoinositide pools further complicates the assessment of changes in specific activity of the PtdIns(4,5)P2 5-monoester from simple pulse/chase experiments. 32p incorporation data from experiments using brinjal leaves however suggests that the rate of turnover of PtdIns(4,5)P2 may be considerably higher than that of the main phospholipid pool in this tissue. 82 A more thorough investigation of this aspect of polyphosphoinositide metabolism is clearly needed before generalisations can be made. The inositol phospholipid specific phospholipase(s) C in plants have been the subject of a considerable amount of research in recent years. Soluble enzymes which preferentially hydrolyse PtdIns in wheat, 4s celery, cauliflower, onion and daffodil, 38'47pollen tubes 3~ and soybean hypocotyls66 have been described. Membrane associated forms of phospholipases C have also been reported to be present in pollen, 3°'31soy and bushbean 66and wheat plasma membrane. ~ Where investigated, it has been found that there is a dependency on mM concentrations of free calcium ions for full activity of the PtdIns specific form of the enzyme. Available evidence would suggest that the polyphosphoinositide specific forms of the enzyme are fully activated by micromolar concentrations of calcium ions. The plasma membrane associated enzyme from wheat ~ was fully activated at 10 # M free calcium (using PtdIns(4)P and PtdIns(4,5)P2 as substrates). Similar results were reported in an oat root plasma membrane preparation by Tate et al. 79 In this case the enzyme hydrolysed PtdIns(4,5)P2 and PtdIns(4)P respectively, at 10 and 4 times the rate of PtdIns hydrolysis. Again the enzyme was fully activated by #M concentrations of free calcium. A similar calcium requirement was reported in studies of the release of inositol phosphates from soybean membranes. 2 These results are important, as #M concentrations are close to the levels of cytosolic free calcium determined in vivo in a number of plant cells. 24'46It has been suggested 79 that the studieP 7,66which report full activation of the particulate form of the enzyme by mM free calcium may have resulted from the contamination of these preparations by the soluble PtdIns specific form of the enzyme which is known to require this concentration of calcium for activation. As yet there has been no direct investigation of whether the polyphosphoinositide specific phospholipase C is regulated by a mechanism involving receptor occupancy and GTP-binding proteins as is known to be the case in mammalian cells. Published studies give conflicting evidence with regard to the involvement of GTP-binding proteins. No indication of their participation in the regulation of the membrane associated phospholipase C activity in wheat, ~ soybean2 or the tissues studied by McMurray and Irvine 47 has been found. However, Dillenschneider et al) ° reported that guanine nucleotides stimulated the release of inositol phosphates from A c e r pseudoplatanus membranes. More direct evidence that GTP binding proteins may be involved in the regulation of these enzymes comes from work on the marine microalgae Dunaliella salina where it has recently been reported that the membrane associated calcium regulated phospholipase C which hydrolyses PtdIns(4,5)P2 was stimulated by the addition of GTP. 16
Inositol-containing lipids
59
The low chemical levels of the polyphosphoinositides in plant membranes and the high rate of metabolism of PtdIns(4)P (and perhaps PtdIns(4,5)P2) suggests that in addition to a structural function these lipids may have other roles in cellular metabolism. The function of polyphosphoinositides in plant cells which has received the most attention recently is their potential role in signal transduction as precursors for the second messengers Ins(1,4,5)P3 and 1,2-DAG. Upon stimulation of mammalian cells by selected agonists known to induce a rise in intracellular Ca 2+, PtdIns(4,5)P2, localised in the inner leaftlet of the plasma membrane, is hydrolysed by the enzyme(s) phospholipase C (phosphoinositidase C). This results in the formation of two molecules: 1,2-diacylglycerol (DAG) and Ins(1,4,5)trisphosphate (Ins(1,4,5)P3). DAG remains in the membrane matrix where it activates a protein kinase (protein kinase C). Ins(1,4,5)P3 on the other hand, being highly polar diffuses from the plasma membrane to intracellular membrane bounded Ca 2+ stores where it induces a release of Ca 2+ into the cytosol by opening specific calcium channels. Activation of mammalian cells can in this way result in bifurcated cellular signal transduction pathways involving both the activation of protein kinase C and a rise in cytosolic Ca 2+. The use of two transduction strands opens the possibility for cross-talk and results in a high degree of flexibility. It is now clear that many equivalents to the mammalian PI system also exist in plant cells. In addition to the phosphoinositide kinases/phosphatases and the phosphoinositide specific phospholipase(s) C several other components of the mammalian phosphoinositide system have been found also to be present in plant cells. These include: the ability of Ins(1,4,5)P3 to release sequestered Ca 2+ from intracellular stores, 12'7~presence of enzymes capable of rapidly metabolising Ins(1,4,5)P3 into both higher and lower inositol phosphates ~5'4°'51 and the existence of enzymes bearing some resemblance to protein kinase C. 1s,63,72In the last few years reports have appeared suggesting an involvement of the plant phosphoinositide system in a wide variety of transmembrane signalling processes such as plant pathogen interactions, 14 light induced leaf movements, 57'5s'59 control of turgor in stomatal guard cells 3'25'~ and the response to phytohormones, mg'26,62,uFor a more detailed discussion of these areas the reader should consult recent reviews. 4,11'~7,27'43'56'~° Another possible function for the polyphosphoinositides is that they may be capable of modulating enzyme activity. Memon e t al. 5° thus found that both PtdIns(4)P and PtdIns(4,5)P2 were capable of increasing the vanadate sensitive ATPase activity associated with the plasma membrane and they later demonstrated a correlation between rapid light induced changes in phosphoinositide kinase activity and H + ATPase activity in plasma membranes from sunflower hypocotyls. 49 A different aspect of phosphoinositide function which, as yet, has not been demonstrated in plant cells but is receiving increased attention in mammalian research is the interaction of phosphoinositides with a number of actin-binding proteins such as gelsolin, gcap39, cofilin, destrin and profilin. 2] It has been shown that the in v i t r o actions of the binding proteins can be inhibited by polyphosphoinositides and that phosphoinositide hydrolysis by phosphoinositidase C is sensitive to the presence of actin binding proteins. Recently, it has been demonstrated that profilin is present in plant cells, s° It will be very interesting to see whether polyphosphoinositides are also involved in such interactions in plants. IV. I N O S I T O L
LYSOPHOSPHOLIPIDS
It has recently been suggested that the lyso-derivatives of inositol phospholipids may play a second messenger role in plant cells, s3 Lyso-phospholipids are formed through the activity of phospholipases A~ (removes the fatty acid at the sn-1 position on the glycerol backbone) and A2 (removes the fatty acid at the sn-2 position). 35'36 With the exception of a phospholipase A2 which hydrolysed phosphatidylcholine (PC) to lyso-PC and a free fatty acid 65 (Wheeler and Boss, unpublished results) s3 which suggest that plants possess a PtdIns specific phospholipase A2 active over a pH range from 5.5-7 in the presence of calcium, these enzymes have not been well characterised. Most work has been carried out on the non-specific acyl hydrolases which catalyse the formation of
60
A . M . HETHERINGTON and B. K. DgOBAK
deacylated lipids. 35'36A soluble non-specific acyl hydrolase which exhibits activity towards Ptdlns has been described in potato tubers) 3 What is not clear yet is whether the activity of this enzyme will generate inositol lysophospholipids. When phosphatidylcholine is the substrate lyso derivatives are not found among the products of the reaction catalysed by the non-specific acyl hydrolase, whereas, when the glycoglycerolipid monogalactosyldiacylglycerol is used a monoacyl derivative is formed. 33"35 That the other lysophospholipids may have important physiological functions is demonstrated by the findings that in oat plasma membrane vesicles lyso-PC is capable of regulating H ÷ ATPase activity 64'65and other lyso-lipids have been reported to regulate protein kinase activity: 5
V. G L Y C O P H O S P H O S P H I N G O L I P I D S
In addition to the inositol containing phospholipids based on glycerol, plants also contain inositol phospholipids based on the long chain amino alcohols phytosphingosine (4-D-hydroxy sphinganine) or dehydrophytosphingosine (4-D-hydroxy-8-sphingenine). Unlike the other plant glycerophospholipids (with the exception of N-acyl phosphatidylcholine), the sphingolipids have their fatty acid linked through an amide bond to the amino group on C2 of the amino alcohol. Interestingly, it has been reported that 2-hydroxy derivatives of long chain fatty acids such as tetracosanoate (24: 0) are associated with glycophosphosphingolipids. 7 The significance of this observation remains to be determined. Glycolipids based on phytosphingosine which contain both inositol and phosphate were first identified by Carter and co-workers in the 1950s. This work has been continued by Lester and co-workers using more sophisticated analytical procedures than were available to the Illinois group. The general structure of the glycophosphosphingolipids is illustrated in Fig. 3. Both phytosphingosine and dehydrophytosphingosine based variants have been reported, however all contain inositol linked via a phosphodiester linkage to the ceramide and via a glycosidic bond to a chain of sugar residues of variable composition. In the older literature this group of compounds were frequently referred to as "phytoglycolipids". However, Laine and Hsieh 4~ suggest that in order to avoid possible confusion with other glycosphingolipids which have sugars attached directly to the ceramide that the term glycophosphosphingolipid (GPS) is used to distinguish these compounds from other plant glycosphingolipids. We will adopt this terminology. The structure of a number of the plant GPSs have been reported 42'77 and two of these are presented in Fig. 4. In addition to higher plants GPSs have also been identified in fungi
.
H
,-C - C A OR 1
G,¥cAm-,mos.
o.
o - P- o
i
2
3
c.:c
4
H
- .c - c
6H
-cc.,
8--
-'
ore,
NH OH OH
I
C=O I HO-C
i
I
CH3
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CERAMIDE
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PHOSPHORYLCERAMIDE
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FIG. 3. General structure of a glycophosphosphingolipid. The fatty acyl group is typically mono-hydroxylated and saturated.
61
Inositol-containing lipids
Man(=l-2)~ GIcNAc(od_4)GIcUA(aI_6),/In°s(1-0-ph°sph°~'yl)ceramide (a)
GIcNAcp(od-4)GIcUAp(al-2)lnos(1-0-phosphoryl)ceramide (b)
FIG. 4. Structures of two plant glycophosphosphingolipids from (a) corn, determined by Carter et al. s (with modifications according to Lain¢ and Hsieh42) and (b) tobacco, as reported by Hsieh et a l Y T h e following abbreviations are used: Man = Mannose; Inos = Inositol; GlcNAc ffi NAcetylglucosamine; and GIcUA = (31ucuronic acid.
and algae. There have been no reports of their occurrence in animals. 77 Methods for the analysis of plant GPSs have been reviewed by Laine and Hsieh 42 and for full details this comprehensive account should be consulted. Currently, there is no information about the biosynthesis or subcellular distribution of this group of compounds. From their structure and analogy to animal glycosphingolipids it seems likely that they are membrane lipids. Whether they have a role in cell surface recognition phenomena as has been found for the animal sialic acid containing glycosphingolipid, gangliosides, 37 remains to be determined. It is obvious that much work on biosynthesis, subcellular location and structure of GPSs is required before physiological roles can be assigned to this enigmatic group of compounds.
VI. C O N C L U S I O N S
AND
FUTURE
PROSPECTS
Although much has been learned about inositol-containing lipids in plants over the years it is clear that our present knowledge only represents the tip of an iceberg. With regard to the polyphosphoinositides a thorough investigation of their in ewe metabolism and subcellular distribution would be timely. In spite of the low levels of polyphosphoinositides in plant tissues, techniques are now available which allow accurate determination of rates of turnover of various parts of these molecules. Also we clearly need further insight into mechanism of action of the group of enzymes which are responsible for their metabolism. Once this information is available it should be considerably easier to carry out appropriate experiments to elucidate the physiological role(s) of phosphoinositides in plant cells. One problem which seems to be particularly associated with plant science is the plethora of experimental systems currently under investigation. The dilution of research effort which results does not help rapid progress and makes the authentication of results difficult. Progress will continue to be slow until some general agreement upon appropriate model systems for use in these experiments can be reached. The benefits of such an approach are obvious when one considers the advances which have been made through the study of e.g. E. co/i or HeLa cells. Another group of compounds which obviously deserve attention are the giycophosphosphingolipids. Further structural and distributional studies on the GPSs would be a prerequisite to determining whether these compounds have a role in cell recognition or are analogous in function to the animal phosphatidylinositol giycans. The rapid development of new and more powerful analytical tools, the progress of molecular biology and the discovery of the eukaryotic phosphoinositide system will hopefully mean that research on inositol-containing lipids in plants, far from being the esoteric pursuit of a few devotees, becomes a field attracting the interest of a wide range of lipid biochemists. There is little doubt that this area of lipidology can provide both exciting and rewarding challenges for some considerable time to come.
62
A.M. HETHERINGTONand B.K. DRI3BAK
Acknowledgements--We both gratefully acknowledge the Agriculture and Food Research Council (AFRC) of the UK for providing support to our laboratories during the course of writing this review. AMH is also grateful to The Gatsby Charitable Foundation for providing additional support.
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BimRIDGE,M. J. and IRVl~m,R. F. Nature 341, 197-205 0989). BI~N, M. and HANr~, D. E. Plant Sci. 69, 147-155 (1990). BLATr, M. R., THmL, G. and T R E N ~ , D. M. Nature 346, 766-769 (1990). Boss, W. F. In Second Messengers in Plant Growth and Development, pp. 29-56 (Boss, W. F. and MORRO, D. J., eds) Alan R. Liss, New York, 1989. Boss, W. F. and MASSEL,M. Biochem. Biophys. Res. Commun. 132, 1018-1023 (1985). CARMAN,G. M. and FELDER,S. M. J. Food Bioehem. 3, 89-102 (1979). CARTER,H. E. and KOOB,J. L. J. Lipid Res. 10, 363-369 (1969). CARTER,H. E., STROSACH,D. R. and HAWTHORn'E,J. N. Biochemistry 8, 383-388 (1969). CI4~-q~BOROUGH,T. M. and MooRE, T. S. Plant. Physiol. 65, 1076-1080 (1980). DILLENSCHNEIDER,M., HETHERINGTON,A. M., GRAZIANA,A., ALIBERT, G., BERTA, P., HAIECH, J. and RANJEVA,R. FEBS Lett. 2,08, 413-417 (1986). DI~BAK, B. K. Essays Biochem. 26, 27-37 (1991). DROBAK,B. K. and FEROUSON,I. B. Biochem. Biophys. Res. Comman. 130, 1241-1246 (1985). DROBAK,B. K., I~RGUSON,I. B., DAWSON,A. P. and IRVl~rE,R. F. Plant Physiol. 87, 217-222 (1988). DReBAK,B. K. and ROBERTS,K. In Molecular Plant Pathology--A Practical Approach (BOWl.B, D., ed.) IRL Press (in press). DRg~BAK,B. K., WATKINS,P. A. C., CHATTAWAY,J. A., ROBERTS,K. and DAWSON,A. P. Plant Physiol. 95, 412-419 (1991). EINSPAHR,K. J., P r ~ , T. L. and THOMPSON,G. A. JR Plant Physiol. 90, Ill5-1120 (1989). ErNSl'Atm, K. J. and THOMPSON,G. A. JR Plant Physiol. 93, 361-366 (1990). ELLIOTT,D. C. and SrdNNER, J. D. Phytochemistry/~, 39--44 (1986). EI"rLINGF.R,C. and LF~t~, J. Nature 331, 176-178 (1988). FEILD,M. and MOORE,T. S. Plant Physiol. 86, 83S (1988). FERGUSON,J. E. and HANt~W, M. R. Curr. Opinion Cell Biol. 3, 206-202 (1991). F~RousoN, M. A. J. and WmUAMS,A. F. Annu. Rev. Biochem. 57, 285-320 (1988). GALLmRD,T. In The Biochemistry of Plants, Vol. 4, pp. 85-116 (STufF, P. K., ccl.), Academic Press, New York, 1980. GmROY, S., HuG~,% W. A. and TREWAVAS,A. J. Plant Physiol. 90, 482-491 (1989). GmROY, S., READ,N. D. and TI~WAVAS,A. J. Nature 343, 769-771 (1990). GRABOWSKI,L., HELM,S. and WAONER,K. G. Plant Sci. 75, 33-38 (1991). HANKE, D. S. Ann. Rev. Plant Physiol. Plant Mol. Biol. (in press). HARWOOD,J. L. In The Biochemistry of Plants, Vol. 4, pp. 1-55 (STutaPF, P. K., ed.) Academic Press, New York, 1980. HEtM, S. and WAOt,mR, K. G. Biochem. Biophys. Res. Commun. 134, 1175-1181 (1986). HE~PER, J. P. F. G., DEGROOT,P. F., LINrdNS, H. F. and JACKSON,J. F. Phytochemistry 25, 2193-2199 (1986). I ~ P E R , J. P. F. G., Hm~taSKF.RK,J. W. M. and V~RKAm', J. H. Physiol. Plant. 71, 120-126 (1987). HENDmX,K. W., QASEFA,H. A. and Boss, W. F. Protoplasma 151, 62-72 (1989). HmAYA~aA,O., MATSUDA,H., TAKEDA,H., MAENAKA,K. and TAK^TSUKA,H. Biochim. Biophys. Acta 384, 127-137 (1975). HSmH, T. C.-Y., LAI~, R. A. J. and LEs~R, R. L. Biochemistry 17, 3575-3581 (1978). HUANO,A. H. C. In The Biochemistry of Plants, Vol. 9, pp. 91-119 (STUMPF,P. K., ed.) Academic Press, New York, 1987. HUA~G, A. H. C. In Methods in Plant Biochemistry, Vol. 3, pp. 219-227 (LEA, P. J., ed.) Academic Press, New York, 1990. IOARISHI,Y., NOJIRI,H., HANAI,N. and HAKOMORI,S.-I. Methods EnzymoL 179, 521-541 (1989). IRv~r¢l~,R. F., LETCrlr.R,A. J. and DAWSON,R. M. C. Biochem..L 192, 279-283 (1980). IRV~mZ,R. F., LSTCHr.R,A. J., LA~DER,D. J., Dlt~AK, B. K., DAWSON,A. P. and MUSORAW,A. Plant Physiol. 89, 888--892 (1989). JOSEI'rLS. K., Esce, T. and BONN~R,W. D. Biochem. J. 264, 851-856 (1989). KAMADA,Y. and Muro, S. Biochim. Biophys. Acta 1093, 72-79 (1991). LA~N~,R. A. and HSmH, T. C.-Y. Methods Enzymol. 138, 186-195 (1987). LmtLE, L. Plant MoL Biol. 15, 647-658 (1990). L U N G , O. A. and JEROIL,B. FEBS Lett. 240, 171-176 (1988). MA.R'nr,~-BsstON,G. and SCHr.RER,G. F. E. J. Biol. Chem. 264, 18052-18059 (1989). McAINSH, M. R., BROWNLEE,C. and HETHERINGTON,A. M. Nature 343, 186-188 (1990). McMURRAY, W. C. and IRVlNE,R. F. Biochem. J. 249, 877--881 (1988). M ~ N , P.-M., S O l e m N , M., SANDmJUS,A. S. and J~RO~L,B. FEBS Lett. 2,23, 87-91 (1987). M~ON, A. R. and Boss, W. F. J. Biol. Chem. 265, 14817-14821 (1990). MI~ON, A. R., CtmN, Q. and Boss, W. F. Biochem. Biophys. Res. Comman. 162, 1295-1301 (1989). M~ON, A. R., RnqcoN, M. and Boss, W. F. Plant Physiol. 91, 477-480 (1989). MooRE, T. S. JR Annu. Rev. Plant Physiol. 33, 235-259 (1982). MooRE, T. S. JR Methods. Enzymol. 148, 585-596 (1987). MooRE, T. S. JR In Inositol Metabolism in Plants, pp. 107-I 12 (Mom~, D. J., Boss, W. F. and Loswus, F. A., eds) Wiley-Liss, New York, 1990. MooRE, T. S. JR, LORD, J. M., KAOAWA,T. and B~¢mts, H. Plant Physiol. 52, 50-53 (1973).
Inositol-containing lipids
63
56. M o ~ , D. J., Boss, W. F. and LOEWUS,F. A. (eds) Inositol Metabolism in Plants, p. 393, Wiley-Liss, New York, 1990. 57. MOgSE, M. J., CRAIN, R. C. and SAltER, R. L. Plant Physiol. 89, 640-644 (1987). 58. MORSE,M. J., C ~ N , R. C. and SATYR, R. L. Proc. Natl. Acad. S¢i. (U.S.A.) g4, 7075-7078 (1987). 59. MORSE,M. J., CaiN, R. C., COT~,G. G. and SATT~R,R. L. Plant Physiol. m , 724--727 (1989). 60. MORSE,M. J., SATTF.~,R. L., CRAIN, R. C. and COTI~,G. G. Physiol. Plant. 76, 118-121 (1989). 61. MUDD,J. B. In The Biochemistry of Plants, Vol. 4, pp. 250-282 (STUMPF,P. K., ed.) Academic Press, New York, 1980. 62. MURTHY,P. P. N., RENDEgS,J. M. and ~ ' ~ , L. M. Plant Physiol. 91, 1266-1269 (1989). 63. Ot.AH, Z. and Kxss, Z. FEBS Lett. 195, 33-37 (1986). 64. PALMGSEN,M. G. and SOMMAIUN,M. Plant Physiol. 90, 1009-1014 (1989). 65. PALMOSEN,M. G., SOMMAPaN,M., ULVSKOVand JOROENSEN,P. L. Physiol. Plant, 74, 11-19 (1988). 66. PFAFFMA~, H., HARTMANN,E., BRIGHTMAN,A. O. and Momu~, D. J. Plant Physiol. 85, 1151-1155 (1987). 67. PaNCON,M. and Boss, W. F. In Inositol Metabolism in Plants, pp. 173-200 (Mop,t~, D. J., Boss, W. F. and Loewus, F., eds) Wiley-Liss, New York, 1990. 68. SANDEUUS,A. S. and MotetS, D. J. Plant Physiol. 84, 1022-1027 (1987). 69. SA~EUUS, A. S. and SOMM~PJN,M. FEBS Lett. 201, 282-286 (1986). 70. SANDELIUS,A. S. and SOMMARXN,M. In lnositol Metabolism in Plants, pp. 131-161 (MoRtaL D. J., Boss, W. F. and LOEWUS,F., eds), Wiley-Liss, New York, 1990. 71. SCHAFER,A. F., BYOI~VE, S., MATZENAUER,S. and MARM~,D. FEBS Lett. 187, 25-28 (1985). 72. Scm.rMAKER,K. S. and SZE, H. J. Biol. Chem. 262, 3944-3946 (1987). 73. SEXTON,J. C. and MoosE, T. S. Plant Physiol. 67, 978-980 (1978). 74. SEXTON,J. C. and MOOSE,T. S. Plant Physiol. 68, 18-22 (1981). 75. S o ~ N , M. and SANDELIUS,A. S. Biochem. Biophys. Acta 958, 268-278 (1987). 76. ST~I'H~NS,L. R., HUOH~, K. T. and IRVlNE,R. F. Nature 351, 33-39 (1991). 77. STULTS,C. L. M., SW'~L~, C. C. and MACHr~q,B. A. Methods Enzymoi. 179, 167-214 (1989). 78. SUMIDA,S. and MUDD, J. B. Plant Physiol. 45, 712-718 (1970). 79. TATE,B. F., SCHALLEg,G. E., SUSSMAN,M. R. and O~JN, R. C. Plant Physiol. 91, 1275-1279 (1989). 80. VALENTA,R. M., DUCHENE,K., PETTENBU't~GF.R,C., SILLABER,P., VALENT,P., BETmLt~M, M., BRF.ITEh'aACH, A., RUMPOLD,D., KRAFT,D. and SCH~NER,O. Science 2 ~ , 557-560 (1991). 81. VAN BREENAN, R. B., WHEELER, J. and Boss, W. F. Lipids 25, 328-334 (1990). 82. WAGH,S., MENON,K. K. G. and NATARAJAN,V. Biochim. Biophys. Acta 962, 178-185 (1988). 83. Wm~LER,J. J. and Boss, W. F. In Inositol Metabolism in Plants, pp. 163-172 (Molutt, D. J., Boss, W. F. and LoEwus, F. A., eds), Wiley-Liss, New York, 1990. 84. ZaELL, B. and WALTER-BAcK,C. J. Plant Physiol. 133, 353-360 (1988).
JPLR 31/1--E
0163-7827/92/$15.00 © 1992 Pergamon Press pk
Printed in Great Britain. All fights reserved
INOSITOL-CONTAINING LIPIDS IN HIGHER PLANTS ALISTAIR M. HETHERINGTON* and BJffRN K. DROBAK~f *Division of Biological Sciences, IEBS, Lancaster University, Lancaster LAI 4YQ, U.K. tDepartment of Cell Biology, John Innes Centrefor Plant Science Research, Colney Lane, Norwich NR4 7UH, U.K.
CONTENTS 53 53 54 54 54 55 55 57 59
ABBREVIXTIONS I. INTRODUCTION II. PHOSPHATIDYLINOSITOL A. Structure and cellular distribution B. Biosynthesisand metabolism IlL POLYPHOSPHOINO$1TIDI~ A. Structure and cellular distribution B. Biosynthesisand metabolism IV. INOSITOL LYSOPHOSPHOLIPIDS
V. GLYCOPHOSPHosPmNC, OIJPn>S
60
61 62 62
VI. CONCLUSIONSAND FUTURE PROSPECTS ACKNOWLrd~EMENTS
R~ntL~Cm
ABBREVIATIONS CDP-DAG Cystosine diphosphate diacylglycerol GPS Giycophosphosphingolipid IAA Indole-3-acetic acid PC Phosphatidylcholine Ptdlns Phosphatidylinositol PtdIns(4)P Phosphatidyl(4)phosphate Ptdlns(4,5)P2 Phosphatidyl(4,5)bisphosphate I. INTRODUCTION Interest in the biochemistry and physiology of inositol and inositol-containing compounds in plants is not a new phenomenon. It is thus more than 100 years ago that Pf¢ffer first isolated phytic acid (inositol (1,2,3,4,5,6) hexakisphosphate) from seeds. In spite of considerable effort by plant scientists over the years, comparatively little is still known about this group of compounds, but that situation is beginning to change. Until 1983/84 the emphasis in this area of research was mainly on inositol phosphates involved in phytic acid metabolism, indole-3-acetic acid (IAA) esters of myo-inositol and inositol containing lipids such as phosphatidylinositol and the glycophosphosphingolipids. Around 1983 a major breakthrough was made by researchers in the mammalian field who discovered that a group of inositol-containing phospholipids (the phosphoinositides) play a central role in the cellular perception and transduction of a wide variety of extracellular signals. It is now generally recognised that these inositol lipids have similar functions in organisms as disparate as slime moulds, yeast, blowflies and humans. I In addition to the role played in transmembrane signalling inositol-containing lipids appear to have several additional important functions, these include: regulation of enzymic activity, anchoring of proteins to membranes and modulation of cytoskeletal dynamics. 2~2'49 The discovery of the mammalian phosphoinositidc systems has caused a major upsurge in interest in inositol lipids and inositol phosphates in all eukaryotes and has led plant scientists to ask the questions: "Is a similar system involved in transmembrane signalling in plant cells?" and "Do inositol-containing lipids have other physiological functions in plants?" Although definitive answers to these questions have yet to be found, it is becoming 53
.54
A.M. HETHERINGTONand B. K, DR~BAK
increasingly likely that when they are, they will be affirmative. One of the main problems in the characterisation of the plant phosphoinositide system and other aspects of inositol metabolism is the presence of a vast array of inositol-containing compounds in plant cells. It should be noted that there are 63 isomers of phosphorylated myo-inositol, and other isomers of inositol are known to occur in plant tissues. In addition to these several IAA- and methyl esters of myo-inositol, myo-inositol glycosides and at least 10-15 inositol-containing lipids (several with unknown chemical structures) have been shown to be present in various plant cells. In the present context we will focus on the inositol-containing lipids. It is not our intention to present an exhaustive review but rather to give a brief overview of the current state of knowledge and provide an entrance into the literature in this rapidly developing area of plant science. II. P H O S P H A T I D Y L I N O S I T O L
A. Structure and Cellular Distribution
Phosphatidylinositol [1-(3-sn-phosphatidyl)-L-myoinositol] (PtdIns) (Fig. 1) is one of the major phospholipids of plant membranes. It has been reported that PtdIns may comprise up to 19% of the total phospholipid of the plasma membrane, while in contrast chloroplasts contain comparatively small amounts (1-2% of the total phospholipid). In common with most plant phospholipids a saturated fatty acid such as palmitic is often found at the sn- 1 position on PtdIns while the acyl group at the sn-2 position is unsaturated (e.g. linoleic), s' Available evidence would suggest that PtdIns has an asymmetric distribution within the membrane. 9 The reviews by Harwood 2s and Mudd 6~ provide detailed information on the distribution and fatty acid composition of this lipid within different plants, plant parts and among various types of membrane. B. Biosynthesis and Metabolism
The biosynthesis of PtdIns has recently been reviewed by Moore ~ and for full details it is recommended that the reader consult this comprehensive account. However, in outline there are two routes to the synthesis of PtdIns; the first is catalysed by CDP-DAG: inositol 3-phosphatidyltransferase (EC 2.7.8.11) and uses cytosine diphosphate diacylglycerol (CDP-DAG) as substrate and Mn 2+ as a cofactor. 6,2°'s2'53'74'7sThe second mechanism is an inositol exchange catalysed by PtdIns myo-inositol phosphatidyltransferase. ~'74 Both reactions are likely to occur in the endoplasmic reticulum 55'7s'74 and possibly the Golgi complex. 6s o
0
?2.0
o
II - C - F~
II C H 2 - O - C - R'
!
0
II
R'-C-O-CH
O
I
w
CH~--O- P--O-
I
R'-~-O-C
A , ~,
.
O
[
II
t
I~' C H 2 - O - C - R~ [
R- c - o -CH
B ,
[
O
I[
CH2-O-- P-- O-
CH2--O - P - - O -
o
oII
"c
I
OH
OH
oH
(a) Ptdlns
(b) Ptdlns(4)P
(c) Ptdlns(4,5)P 2
Fso. I. The structures of (a) phosphatidylinositol (Ptdlns), (b) phosphatidylinositol(4)phosphate, (PtdIng4)P) and (¢) phosphatidylinositol(4,5)bisphosphatc, (Ptdlns(4,5)P2). In these figures R' and R" represents two fatty acyl residues esterified respectively at the sn-I and sn-2 positions on the inositol phospholipid. For further details see the text. Reaction A is catalys©d by Ptdlns kinasc, reaction B by Ptdlns(4)P kinase, reaction C by Ptdlns(4,5)P2 phosphatase and reaction D by Ptdlns(4)P phosphatase.
lnositol-containing lipids
55
PtdIns is a substrate for a number of lipid degrading enzymes. The best studied is the nonspecific acyl hydrolase from potato tubers. 3a For recent reviews of this group of enzymes the reader should consult Huang 3s'36 (but see also Section IV), The activity of phospholipases A1 (EC 3.1.1.32), A2 (EC 3.1.1.4) and B (EC 3.1.1.5) are not well characterised in plants and currently little is known about their role in PtdIns catabolism (see Section IV). Unusually, among the membrane phospholipids PtdIns does not appear to be a substrate for phospholipase D (EC 3.1.4.4). 23 The activity of phospholipase C towards Ptdlns and the polyphosphoinositides will be considered in Section III.B.
III. P O L Y P H O S P H O I N O S I T I D E S
A. Structure and Cellular Distribution
The presence of phosphorylated species of PtdIns in higher plants was first reported around 1985.7°That no such reports can be found in earlier literature may be due to several factors. The most likely is that the traditional Folch-type lipid extractions which have been employed by most plant researchers are unsuited for the extraction of highly polar phospholipids. If near neutral/high salt "upper phase" solutions are used to wash chloroform/methanol rich extracts the polyphosphoinositides have a strong tendency to migrate into the polar phase which is commonly discarded. Only if acidified extraction solvents are used will the deprotonation of the phosphate ester groups of the polyphosphoinositides be suppressed sufficiently for these compounds to remain in the non-polar extraction solvent during washes. The first experiments which suggested the presence of polyphosphoinositides in plant tissues were carried out using radiolabelled cells and tissues as lipid source, acidified extraction systems and separation of lipids by T L C . 5'13'29'7° It was found that two radiolabelled lipids had chromatographic properties closely resembling those of mammalian phosphatidylinositol(4)phosphate (PtdIns(4)P) and phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2). Direct chemical determination of the abundance of polyphosphoinositides in plant cells and tissues has so far not been carried out so the best estimates come from isotope labelling experiments using tissues in which near isotopic equilibrium has been reached. Such experiments have given ratios of Ptdlns: PtdIns(4)P: PtdIns(4,5)P2 ranging from 300:17:1 to 10:1 : 1.67'7° In most of these experiments lipids have been separated by TLC and the identification of polyphosphoinositides has been indirect and based on co-chromatography of labelled compounds with authentic mammalian polyphosphoinositide standards. Whereas such an approach is often adequate for studies of mammalian phosphoinositide metabolism the same is not true for plants. Drobak et al. ~3using a triple-isotope approach found that when labelled phospholipids from suspension cultured tomato cells were separated by TLC and the radioactivity derived from 3H-glycerol, 3H-inositol and 32p-orthophosphate respectively determined in compounds co-chromatographing with authentic standards, discrepancies existed. Whereas the ratio of incorporated phosphate:inositol: glycerol in the compound cochromatographing with the PtdIns(4)P standard was very close to the ratio expected on the basis of the chemical structure and biosynthetic pathways of PtdIns(4)P the same was not true for labelled material co-chromatographing with PtdIns(4,5)P2. Only a minute amount of radioactivity derived from 3H-glycerol could be detected and ratios of phosphate:inositol were close to 1:1. These findings led the authors to conclude that: (1) the majority of the compounds co-migrating with authentic PtdIns(4,5)P2 was not Ptdlns(4,5)P2 but more likely a glycophosphosphingolipid (see Section V) and (2) if such compounds are generally present in plant tissues the amount of PtdIns(4,5)P2 reported by various groups using similar methods of analyses are likely to have been considerably overestimated. Experiments where glycerophosphoinositides or inositol phosphate derivatives of phosphoinositides from equilibrium labelled plant tissues have been separated by HPLC (a procedure which largely overcomes problems of co-chromatography of polyphosphoinositide derivatives and other inositol derivatives) indicate that
56
A.M. HETr~mNGTONand B. K. DROBAK
Ptdlns(4,5)P2 is likely not to constitute more than 0.05% of total phospholipids or 0.5% of phosphoinositides. ~3'39 Determination of the structure of the plant polyphosphoinositides was carried out by Irvine and co-workers39 using the strategy illustrated in Fig. 2. Deacylation ( O N acylation) of polyphosphoinositides followed by removal of the glycerol backbone (mild periodate/dimethylhydrazine) results in the formation of inositol polyphosphates (this procedure is equivalent to the action of phospholipase C). If Ptdlns(4)P and Ptdlns(4,5)P2 are the parent molecules, D-myo-inositol(1,4,5)trisphosphate and D-myo-inositol(1,4) bisphosphate will be the resulting hydrolysis products. Treatment of inositol phosphates with periodate results in the cleavage of C--C bonds in the inositol ring between adjacent C-atoms if they are both carrying hydroxyls. Thus, all inositol bisphosphates will be attacked by periodate as will all inositol trisphosphates which do not have all three phosphomonoesters in meta positions. When the aldehyde derivatives of inositolbis/trisphosphates formed by periodate treatment are reduced (borohydride) and dephosphorylated the resulting products are alditols (except if inositol bisphosphates with phosphomonoester groups in para positions or inositol trisphosphates with phosphomonoester groups in meta positions are used as starting material in which case the reaction products are either destroyed in the process or will be myo-inositol). As illustrated in Fig. 2 the alditol derived from D-myo-inositol(1,4,5)trisphosphate by this procedure is D-iditol. Irvine et al. 39 found that the alditol obtained from the conversion of PtdlnsP2 isolated from pea leaf discs indeed was iditol and that the iditol was resistant to L-iditol dehydrogenase. This only leaves two possible structures open for the InsP3 derived from the PtdlnsP 2 namely Ins(1,4,5)P 3 or Ins(1,4,6)P 2. Upon incubation of the InsP3 with Ins(1,4,5)P3-5- phosphatase from human red blood cell membranes the plant derived InsP3 was hydrolysed with kinetics similar to authentic Ins(1,4,5)P~ confirming that the parent polyphosphoinositide is Ptdlns(4,5)P2. The InsP2 fraction derived from pea PtdlnsP co-chromatographed on HPLC with authentic Ins(1,4)P2 and when subjected to periodate oxidation, reduction and dephosphorylation yielded no noncyclic alditols. This suggests that the phosphomonoesters are para, and given the linkage of the D-1 phosphate in Ptdlns to the glycerol moiety, they must be in the 1 and 4 position and the plant Ptdlns be Ptdlns(4)P. The structures of Ptdlns(4)P and Ptdlns(4,5)P2 are shown in Fig. 1. Ptdlns(4,5)P2
~
O-N Transacylatlon
GPtdlns(4,5)P2 k Periodate/Olmethylhydrazlne o
HOPO- O 0 ~r- OPO-
_
-%o-~
Ins(1,4,SlP3
~eriodate o HO56. . 1 . ~ , ~
o IA
-O~O~ H
CH20H OH~ OH~
OH OH
/
0
"4 ° "
BorohydrldelDephosphor ylatlon
Flo. 2. Strategy used by Irvine and ¢0 -wor kers 39 to det©rmine the structure of plant polyphosphoinositides. For full details see the text.
Inositol-containing lipids
57
A number of novel phosphoinositides have recently been discovered in certain mammalian cells. 76 The structures of these lipids have been shown to be phosphatidylinositol(3)phosphate, phosphatidylinositol(3,4)bisphosphate and phosphatidylinositol(3,4,5)trisphosphate. In intact neutrophils activated by the formyl lx'ptide f-Met-Leu-Phe a phosphatidylinositol(4,5)bisphosphate-3-kinase seems to be responsible for agonist stimulated formation of polyphosphoinositides phosphorylated in the 3 position of the inositol moiety. 76 There is currently no evidence for the presence of such polyphosphoinositides in plant tissues (B. K. Dmbak, unpublished data) but a more detailed search in a wider range of plant tissues may change this situation. Although extensive surveys of the fatty acid composition of the polyphosphoinositides have not been carried out, the data at hand suggest that the predominant fatty acids in Ptdlns(4)P and Ptdlns(4,5)P2 are palmitate and linoleate) 3'7°'81 This is consistent with a synthetic pathway involving direct phosphorylation of Ptdlns. The precise subcellular localisation of plant polyphosphoinositides is currently not fully clarified but results from labelling experiments employing both intact cells and membrane fractions enriched in plasmalemma vesicles suggests that polyphosphoinositides, as in mammalian cells, are associated with the plasma membrane, 69'7° although, recent reports suggest that smaller amounts may be associated with organdies such as nuclei. 32 This assumption is supported by studies on the subcellular distribution of the phosphoinositide kinases. In isolated membrane fractions from hypocotyls of dark grown soybean, both Ptdlns and PtdlnsP hydroxykinases are associated predominantly with the plasma membrane whereas only negligible activity of these enzymes is found in the tonoplast, nuclei, mitochondria and plastid fractions. 7° It is clear that a more thorough investigation of the subcellular distribution of polyphosphoinositides is needed, in particular in the light of recent findings that PtdlnsP in mammalian cells can be synthesised via an intracellular route. 44 Problems facing plant scientists in pursuing this line of work are not only associated with the chemical analysis of small amounts of polyphosphoinositides but also with the production of membrane fractions of sufficient purity. B. Biosynthesis and Metabolism
In mammalian cells Ptdlns(4,5)P2 is formed by a two step phosphorylation of Ptdlns. Ptdlns is first phosphorylated in the 4 position of the inositol ring resulting in the formation of PtdIns(4)P. This lipid is then further phosphorylated in the 5 position yielding Ptdlns(4,5)Pv Two phosphohydrolases work concomitantly with the 4- and 5-hydroxykinase so Ptdlns(4)P and Ptdlns(4,5)P2 are constantly being formed and degraded (Fig. 1). This is termed the "futile" phosphoinositide cycle, but it is now obvious that it is anything but futile. That polyphosphoinositides are synthesised in a similar manner in plants is likely, but is as yet not proven. The most convincing evidence favouring such a scenario comes from in vitro studies using isolated membrane fractions as enzyme source and endogenous and exogenous phosphoinositides as substrates. Sommarin and Sandelius 75 thus demonstrated the presence of both Ptdlns and PtdIns(4)P hydroxykinases in purified plasma membranes from wheat. They found that both kinases were able to phosphorylate both endogenous and exogenous substrates and that the activity was modulated by detergents (Triton X 100, 0.1-0.3%). It is likely that the cause of the increase in kinase activity observed in the presence of both exogenous substrates and detergents is dual in nature, as detergents may affect both the form in which the substrate is presented to the enzyme and enzyme latency. Both the PtdIns and PtdIns(4)P kinase have absolute requirements for ATP and divalent cations--with a preference for Mg 2+. Maximum activity of the polyphosphoinositide kinases in wheat plasma membranes occurred at approximately 1 mM ATP and the Km was determined to be 0.2 m i . 75 Kamada and Muto 4t have demonstrated that submicromolar concentrations of calcium inhibit both PtdIns and PtdIns(4)P kinases and have suggested that the effect of calcium may be part of a regulatory system involved in transmembrane signalling.
58
A.M. HETHERINGTON and B. K. DRBBAK
In vivo experiments suggest that the rate of turnover of PtdIns(4)P is very fast in many plant tissues. Drzbak et al. 13 found that after short incubation times (i.e. 30 min) with 32p-orthophosphate that more than 30% of total label incorporated into phospholipids in suspension cultured tomato cells was in PtdIns(4)P. Although the position of the label in the PtdIns(4)P molecule was not determined it is likely that it was predominantly in the 4-phosphomonoester indicating a highly active PtdIns (4) hydroxykinase. A highly active PtdIns (4) hydroxykinase when seen in conjunction with the comparatively low chemical levels of PtdIns(4)P suggest that this lipid is metabolized further with some speed. The relative importance of hydroxykinases, phosphomonoesterases and phospholipases in this process remains to be clarified. Little is currently known about the rate of turnover of PtdIns(4,5)P2 in vivo. The main problem in the study of PtdIns(4,5)P2 metabolism is the very low chemical levels of this lipid but the high rate of 32p incorporation into the 4-phosphomonoester of PtdIns(4)P and the possibility of distinct cellular phosphoinositide pools further complicates the assessment of changes in specific activity of the PtdIns(4,5)P2 5-monoester from simple pulse/chase experiments. 32p incorporation data from experiments using brinjal leaves however suggests that the rate of turnover of PtdIns(4,5)P2 may be considerably higher than that of the main phospholipid pool in this tissue. 82 A more thorough investigation of this aspect of polyphosphoinositide metabolism is clearly needed before generalisations can be made. The inositol phospholipid specific phospholipase(s) C in plants have been the subject of a considerable amount of research in recent years. Soluble enzymes which preferentially hydrolyse PtdIns in wheat, 4s celery, cauliflower, onion and daffodil, 38'47pollen tubes 3~ and soybean hypocotyls66 have been described. Membrane associated forms of phospholipases C have also been reported to be present in pollen, 3°'31soy and bushbean 66and wheat plasma membrane. ~ Where investigated, it has been found that there is a dependency on mM concentrations of free calcium ions for full activity of the PtdIns specific form of the enzyme. Available evidence would suggest that the polyphosphoinositide specific forms of the enzyme are fully activated by micromolar concentrations of calcium ions. The plasma membrane associated enzyme from wheat ~ was fully activated at 10 # M free calcium (using PtdIns(4)P and PtdIns(4,5)P2 as substrates). Similar results were reported in an oat root plasma membrane preparation by Tate et al. 79 In this case the enzyme hydrolysed PtdIns(4,5)P2 and PtdIns(4)P respectively, at 10 and 4 times the rate of PtdIns hydrolysis. Again the enzyme was fully activated by #M concentrations of free calcium. A similar calcium requirement was reported in studies of the release of inositol phosphates from soybean membranes. 2 These results are important, as #M concentrations are close to the levels of cytosolic free calcium determined in vivo in a number of plant cells. 24'46It has been suggested 79 that the studieP 7,66which report full activation of the particulate form of the enzyme by mM free calcium may have resulted from the contamination of these preparations by the soluble PtdIns specific form of the enzyme which is known to require this concentration of calcium for activation. As yet there has been no direct investigation of whether the polyphosphoinositide specific phospholipase C is regulated by a mechanism involving receptor occupancy and GTP-binding proteins as is known to be the case in mammalian cells. Published studies give conflicting evidence with regard to the involvement of GTP-binding proteins. No indication of their participation in the regulation of the membrane associated phospholipase C activity in wheat, ~ soybean2 or the tissues studied by McMurray and Irvine 47 has been found. However, Dillenschneider et al) ° reported that guanine nucleotides stimulated the release of inositol phosphates from A c e r pseudoplatanus membranes. More direct evidence that GTP binding proteins may be involved in the regulation of these enzymes comes from work on the marine microalgae Dunaliella salina where it has recently been reported that the membrane associated calcium regulated phospholipase C which hydrolyses PtdIns(4,5)P2 was stimulated by the addition of GTP. 16
Inositol-containing lipids
59
The low chemical levels of the polyphosphoinositides in plant membranes and the high rate of metabolism of PtdIns(4)P (and perhaps PtdIns(4,5)P2) suggests that in addition to a structural function these lipids may have other roles in cellular metabolism. The function of polyphosphoinositides in plant cells which has received the most attention recently is their potential role in signal transduction as precursors for the second messengers Ins(1,4,5)P3 and 1,2-DAG. Upon stimulation of mammalian cells by selected agonists known to induce a rise in intracellular Ca 2+, PtdIns(4,5)P2, localised in the inner leaftlet of the plasma membrane, is hydrolysed by the enzyme(s) phospholipase C (phosphoinositidase C). This results in the formation of two molecules: 1,2-diacylglycerol (DAG) and Ins(1,4,5)trisphosphate (Ins(1,4,5)P3). DAG remains in the membrane matrix where it activates a protein kinase (protein kinase C). Ins(1,4,5)P3 on the other hand, being highly polar diffuses from the plasma membrane to intracellular membrane bounded Ca 2+ stores where it induces a release of Ca 2+ into the cytosol by opening specific calcium channels. Activation of mammalian cells can in this way result in bifurcated cellular signal transduction pathways involving both the activation of protein kinase C and a rise in cytosolic Ca 2+. The use of two transduction strands opens the possibility for cross-talk and results in a high degree of flexibility. It is now clear that many equivalents to the mammalian PI system also exist in plant cells. In addition to the phosphoinositide kinases/phosphatases and the phosphoinositide specific phospholipase(s) C several other components of the mammalian phosphoinositide system have been found also to be present in plant cells. These include: the ability of Ins(1,4,5)P3 to release sequestered Ca 2+ from intracellular stores, 12'7~presence of enzymes capable of rapidly metabolising Ins(1,4,5)P3 into both higher and lower inositol phosphates ~5'4°'51 and the existence of enzymes bearing some resemblance to protein kinase C. 1s,63,72In the last few years reports have appeared suggesting an involvement of the plant phosphoinositide system in a wide variety of transmembrane signalling processes such as plant pathogen interactions, 14 light induced leaf movements, 57'5s'59 control of turgor in stomatal guard cells 3'25'~ and the response to phytohormones, mg'26,62,uFor a more detailed discussion of these areas the reader should consult recent reviews. 4,11'~7,27'43'56'~° Another possible function for the polyphosphoinositides is that they may be capable of modulating enzyme activity. Memon e t al. 5° thus found that both PtdIns(4)P and PtdIns(4,5)P2 were capable of increasing the vanadate sensitive ATPase activity associated with the plasma membrane and they later demonstrated a correlation between rapid light induced changes in phosphoinositide kinase activity and H + ATPase activity in plasma membranes from sunflower hypocotyls. 49 A different aspect of phosphoinositide function which, as yet, has not been demonstrated in plant cells but is receiving increased attention in mammalian research is the interaction of phosphoinositides with a number of actin-binding proteins such as gelsolin, gcap39, cofilin, destrin and profilin. 2] It has been shown that the in v i t r o actions of the binding proteins can be inhibited by polyphosphoinositides and that phosphoinositide hydrolysis by phosphoinositidase C is sensitive to the presence of actin binding proteins. Recently, it has been demonstrated that profilin is present in plant cells, s° It will be very interesting to see whether polyphosphoinositides are also involved in such interactions in plants. IV. I N O S I T O L
LYSOPHOSPHOLIPIDS
It has recently been suggested that the lyso-derivatives of inositol phospholipids may play a second messenger role in plant cells, s3 Lyso-phospholipids are formed through the activity of phospholipases A~ (removes the fatty acid at the sn-1 position on the glycerol backbone) and A2 (removes the fatty acid at the sn-2 position). 35'36 With the exception of a phospholipase A2 which hydrolysed phosphatidylcholine (PC) to lyso-PC and a free fatty acid 65 (Wheeler and Boss, unpublished results) s3 which suggest that plants possess a PtdIns specific phospholipase A2 active over a pH range from 5.5-7 in the presence of calcium, these enzymes have not been well characterised. Most work has been carried out on the non-specific acyl hydrolases which catalyse the formation of
60
A . M . HETHERINGTON and B. K. DgOBAK
deacylated lipids. 35'36A soluble non-specific acyl hydrolase which exhibits activity towards Ptdlns has been described in potato tubers) 3 What is not clear yet is whether the activity of this enzyme will generate inositol lysophospholipids. When phosphatidylcholine is the substrate lyso derivatives are not found among the products of the reaction catalysed by the non-specific acyl hydrolase, whereas, when the glycoglycerolipid monogalactosyldiacylglycerol is used a monoacyl derivative is formed. 33"35 That the other lysophospholipids may have important physiological functions is demonstrated by the findings that in oat plasma membrane vesicles lyso-PC is capable of regulating H ÷ ATPase activity 64'65and other lyso-lipids have been reported to regulate protein kinase activity: 5
V. G L Y C O P H O S P H O S P H I N G O L I P I D S
In addition to the inositol containing phospholipids based on glycerol, plants also contain inositol phospholipids based on the long chain amino alcohols phytosphingosine (4-D-hydroxy sphinganine) or dehydrophytosphingosine (4-D-hydroxy-8-sphingenine). Unlike the other plant glycerophospholipids (with the exception of N-acyl phosphatidylcholine), the sphingolipids have their fatty acid linked through an amide bond to the amino group on C2 of the amino alcohol. Interestingly, it has been reported that 2-hydroxy derivatives of long chain fatty acids such as tetracosanoate (24: 0) are associated with glycophosphosphingolipids. 7 The significance of this observation remains to be determined. Glycolipids based on phytosphingosine which contain both inositol and phosphate were first identified by Carter and co-workers in the 1950s. This work has been continued by Lester and co-workers using more sophisticated analytical procedures than were available to the Illinois group. The general structure of the glycophosphosphingolipids is illustrated in Fig. 3. Both phytosphingosine and dehydrophytosphingosine based variants have been reported, however all contain inositol linked via a phosphodiester linkage to the ceramide and via a glycosidic bond to a chain of sugar residues of variable composition. In the older literature this group of compounds were frequently referred to as "phytoglycolipids". However, Laine and Hsieh 4~ suggest that in order to avoid possible confusion with other glycosphingolipids which have sugars attached directly to the ceramide that the term glycophosphosphingolipid (GPS) is used to distinguish these compounds from other plant glycosphingolipids. We will adopt this terminology. The structure of a number of the plant GPSs have been reported 42'77 and two of these are presented in Fig. 4. In addition to higher plants GPSs have also been identified in fungi
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H
,-C - C A OR 1
G,¥cAm-,mos.
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o - P- o
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2
3
c.:c
4
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6H
-cc.,
8--
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ore,
NH OH OH
I
C=O I HO-C
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CH3
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CERAMIDE
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PHOSPHORYLCERAMIDE
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FIG. 3. General structure of a glycophosphosphingolipid. The fatty acyl group is typically mono-hydroxylated and saturated.
61
Inositol-containing lipids
Man(=l-2)~ GIcNAc(od_4)GIcUA(aI_6),/In°s(1-0-ph°sph°~'yl)ceramide (a)
GIcNAcp(od-4)GIcUAp(al-2)lnos(1-0-phosphoryl)ceramide (b)
FIG. 4. Structures of two plant glycophosphosphingolipids from (a) corn, determined by Carter et al. s (with modifications according to Lain¢ and Hsieh42) and (b) tobacco, as reported by Hsieh et a l Y T h e following abbreviations are used: Man = Mannose; Inos = Inositol; GlcNAc ffi NAcetylglucosamine; and GIcUA = (31ucuronic acid.
and algae. There have been no reports of their occurrence in animals. 77 Methods for the analysis of plant GPSs have been reviewed by Laine and Hsieh 42 and for full details this comprehensive account should be consulted. Currently, there is no information about the biosynthesis or subcellular distribution of this group of compounds. From their structure and analogy to animal glycosphingolipids it seems likely that they are membrane lipids. Whether they have a role in cell surface recognition phenomena as has been found for the animal sialic acid containing glycosphingolipid, gangliosides, 37 remains to be determined. It is obvious that much work on biosynthesis, subcellular location and structure of GPSs is required before physiological roles can be assigned to this enigmatic group of compounds.
VI. C O N C L U S I O N S
AND
FUTURE
PROSPECTS
Although much has been learned about inositol-containing lipids in plants over the years it is clear that our present knowledge only represents the tip of an iceberg. With regard to the polyphosphoinositides a thorough investigation of their in ewe metabolism and subcellular distribution would be timely. In spite of the low levels of polyphosphoinositides in plant tissues, techniques are now available which allow accurate determination of rates of turnover of various parts of these molecules. Also we clearly need further insight into mechanism of action of the group of enzymes which are responsible for their metabolism. Once this information is available it should be considerably easier to carry out appropriate experiments to elucidate the physiological role(s) of phosphoinositides in plant cells. One problem which seems to be particularly associated with plant science is the plethora of experimental systems currently under investigation. The dilution of research effort which results does not help rapid progress and makes the authentication of results difficult. Progress will continue to be slow until some general agreement upon appropriate model systems for use in these experiments can be reached. The benefits of such an approach are obvious when one considers the advances which have been made through the study of e.g. E. co/i or HeLa cells. Another group of compounds which obviously deserve attention are the giycophosphosphingolipids. Further structural and distributional studies on the GPSs would be a prerequisite to determining whether these compounds have a role in cell recognition or are analogous in function to the animal phosphatidylinositol giycans. The rapid development of new and more powerful analytical tools, the progress of molecular biology and the discovery of the eukaryotic phosphoinositide system will hopefully mean that research on inositol-containing lipids in plants, far from being the esoteric pursuit of a few devotees, becomes a field attracting the interest of a wide range of lipid biochemists. There is little doubt that this area of lipidology can provide both exciting and rewarding challenges for some considerable time to come.
62
A.M. HETHERINGTONand B.K. DRI3BAK
Acknowledgements--We both gratefully acknowledge the Agriculture and Food Research Council (AFRC) of the UK for providing support to our laboratories during the course of writing this review. AMH is also grateful to The Gatsby Charitable Foundation for providing additional support.
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