- Email: [email protected]
Fusicoccin — a key to multiple 14-3-3 locks?
science reviews Interactions (Hennecke, H. and Verma, D.P.S., eds), pp. 336-342, Kluwer 27 Sagan, M. et al. (1995) Selection of nodulation and mycorrhizal mutants in the model plant Medicago truncatula (Gaertn.) after x-ray mutagenesis, Plant Sci. 111, 63-71 28 Shirtliffe, S.J. and Vessey, J.K. (1996) A nodulation (Nod+/Fix-) mutant ofPhaseolus vulgaris L. has nodule-like structures lacking peripheral vascular bundles (Pvb-.) and is resistant to mycorrhizal infection (Myc-), Plant Sci. 118, 209-220 29 Bradbury, S.M., Peterson, R.L. and Bowley, S.R. (1991) Interaction between three alfalfa nodulation genotypes and two Glomus species, New Phytol. 119, 115-120 30 Gollotte, A. et al. (1993) Cellular localization and cytochemical probing of resistance reactions to arbuscular mycorrhizal fungi in a 'locus a' mycmutant ofPisam sativum L., Planta 191, 112-122 31 Koide, R.T. and Schreiner, K.P. (1992) Regulation of the vesiculararbuscular mycorrhiza] symbiosis, Annu. Rev. Plant Physiol. Plant Mol. Biol. 43, 557-581 32 Giovannetti, M. et al. (1996) Analysis of factors involved in fungal recognition responses to host-derived signals by arbuscular mycorrhizal fungi, New Phytol. 133, 65-71 33 B~card, G. and Fortin, J.A. (1988) Early events ofvesicular-arbuscular mycorrhiza formation on Ri T-DNA transformed roots, New Phytol. 108, 211-218 34 Harrison, M.J. and Dixon, R.A. (1993) Isoflavonoid accumulation and expression of defense gene transcripts during the establishment of vesicular-arbuscular mycorrhizal associations in roots of Medicago truncatula, Mol. Plant-Microbe Interact. 6, 643-654 35 Xie, Z-P. et al. (1995) Rhizobial nodulation factors stimulate mycorrhizal colonization of nedulating and nounodulatingsoybeans, Plant Physiol. 108, 1519-1525 36 B6card, G. et al. (1995) Flavonoids are not necessary plant signals in arbuscular mycorrhizal symbiosis, Mol. Plant-Microbe Interact. 8, 252-258 37 Douds, D.D., Jr, Nagahashi, G. and Abney, G.D. (1996) The differential effects of cell wall-associated phenolics, cell walls, and cytosolic phenolics of host and non-host roots on the growth of two species of AM fungi, New Phytol. 133,289-294 38 Fry, S. et al. (1993) Oligosaccharides as signals and substrates in the plant cell wall, Plant Physiol. 103, 1-5
39 Beyrle, H. (1995) The role of phytohormones in the fimction and biology
of mycorrhizas, in Mycorhiza: Structure, Function, Molecular Biology and Biotechnology (Varma, A. and Hock, B., eds), pp. 365-391, Springer 40 Smith, S.E. and Smith, F.A. (1990) Structure and function of the interfaces in biotrophic symbiosis as they relate to nutrient transport, New Phytol. 114, 1-38 41 Pearson, J.N. and Jakobsen, I. (1993) Symbiotic exchange of carbon and phosphorus between cucumber and three arbuscular mycorrhizal fungi, New Phytol. 124, 481-488 42 Jakobsen, I. (1995) Transport of phosphorus and carbon in VA mycorrhizas, in Mycorhiza: Structure, Function, Molecular Biology and Biotechnology (Varma, A. and Hock, B., eds), pp. 297-325, Springer 43 Ravnskov, S. and Jakobsen, I. (1995) Functional compatibility in arbuscular mycorrhizas measured as hyphal P transport to the plant, New Phytol. 129, 611-618 44 Harrison, M.J. and van Buuren, M.L. (1995) A phosphate transporter from the mycorrhizal fungus Glomas versiforme, Nature 378, 626-629 45 Muchhal, U.S., Pardo, J.M. and Raghathama, K.G. (1996) Phosphate transporters from the higher plant Arabidopsis thaliana, Proc. Natl. Acad. Sci. U. S. A. 93, 10519-10523 46 Schwab, S.M., Menge, J.A. and Tinker, P.B. (1991) Regulation of nutrient transfer between host and fungus in vesicular-arbuscular mycorrhizas, New Phytol. 117, 387-398 47 Shachar-Hill, Y. et al. (1995) Partitioning of intermediary carbon metabolism in vesicular-arbuscular mycorrhizal leek, Plant Physiol. 108, 2979-2995 48 Harrison, M.J. (1996) A sugar transporter from Medicago truncatula: altered expression pattern in roots during vesicular-arbuscular (VA) mycorrhizal associations, Plant J. 9, 491-503 49 Smith, S.E. (1993) Transport at the mycorrhizal interface, Mycorrhiza News 5, 1-3 50 Taiz, L. and Zeiger, E. (1991) Plant Physiology, Benjamin/Cumming 51 Ingold, C.T. and Hudson, H.J. (1993) The Biology of Fungi, Chapman & Hall
Ma~ia ~ Hardson is at the Samuel Roberts Noble Foundation,
Fusicoccin - a key to multiple 14-3-3 !0 Cks ?
DoBoer
Plants under attack from pathogens have a range of defence responses at their disposal to fight off the infecting organism. The fungus Fusicoccum amygdali, however, appears to have found a major weakness in the plant defence barrier. A key role in the fungal attack is played by a molecule produced by and named after the fungus: fusicoccin. Recently, it has become clear that fusicoccin targets 14-3-3 proteins, which are at the cross-point of a huge array of signalling and regulatory pathways. Progress has n o w been made in understanding h o w 14-3-3 proteins and fusicoccin are involved in the regulation of key enzymes, such as the plasma membrane H*-ATPase and soluble nitrate reductase. Research on the in vivo effects of fusicoccin may lead to the identification of n e w 14-3-3 target proteins. 'nfection by the fungus Fusicoccum amygdali eventually kills the host - peach (Prunuspersica) or almond (Prunus .amygdalus) trees - but its mode of action is subtle. The fungus excretes fusicoccin, a diterpene glucoside (Ref. 1) (Fig. 1), into the apoplast of an infected leaf, which spreads via the transpiration stream throughout the rest of the 60
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plant. When fusicoccin reaches a guard cell pair (i.e. a stoma) it triggers a signalling pathway leading to a widening of the stomatal pore (Fig. 2). Unlike other signals that stimulate stomatal opening (e.g. light, CO 2 and auxin), the effect of fusicoccin is irreversible. The uncontrolled loss of water eventually results in wilting of the leaf. Although the fungus
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is host specific, fusicoccin is not, and it affects a system common to most plant cells. Thus it also stimulates cell elongation, breaks seed dormancy and stimulates rhizogenesis4'~. At the cellular level, the most obvious responses are a rapid (delay of 10-30 s) hyperpolarization of the membrane potential, and acidification of the cytoplasm6, followed by acidification of the medium and an increase in K÷-uptake. In order to understand how fusicoccin triggers these responses, research has focused on the fusicoccin receptor, and the plasma membrane-located H+-ATPase.
/•
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16 ~-- O .... CH3
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Fig. 1. Structure of fusicoccin A, a metabolite of the fungus Fusicoccum amygdali, and cotylenin A, a metabolite of the fungus Cladosporium. Besides these major metabolites, the fungi produce a range of structurally related compounds (see Ref. 2). Structural requirements for biological activity of the fusicoccin molecule are: an unsubstituted hydroxyl group on C-8; the conformation at C-3 and C-9; and the conformation of the eight-membered ring. The importance of the glycosyl moiety is secondary to that of the carbotricyclic system. The aglycones of fusicoccin and cotylenin (cotylenol) still compete with fusicoccin A for binding and have biological activity3.
The fusicoccin receptor: a 14-3-3 protein The purification and identification of the fusicoccin receptor has a long history (Ref. 7), beginning almost 20 years ago with the first published report of a membrane-bound receptor with high affinity for [3H]fusicoccin. In 1989, the protein was purified to homogeneity as a 30 kDa doublet, and finally in 1994 peptide digests of the purified receptor were sequenced and identified as a member (or members) of the 14-3-3 protein familys-l°. Although the evidence that 14-3-3 dimers form the functional fusicoccin receptor is very strong, an as yet unsolved problem is that labelling by azido-fusicoccin identifies a 90 kDa doublet in maize membranes 7. Either the 90 kDa proteins are in the vicinity of the fusicoccin-binding pocket of the 14-3-3 protein, and are therefore an artefact of labelling by azido-fusicoccin, or they represent a second class of fusicoccin receptors. The identity of the receptor came as a surprise, because prevailing dogma had been that the fusicoccin-binding site was extracellular and that the receptor was an integral membrane protein. The primary structure of 14-3-3 proteins shows that they are hydrophilic and lack membrane-spanning domains (confirmed by the crystal structure11). Hence the binding site for fusicoccin must be intracellular, a conclusion corroborated by an earlier report that the hydrolytic site of the H+-ATPase and the fusicoccin-binding site of the fusicoccin receptor are at the same face of the plasma membrane 12. Moreover, fusicoccin is a rather hydrophobic molecule, and the biological activity of fusicoccin conjugates decreases with increasing polarity (see Ref. 13). The location of the binding site and hypotheses about fusicoccin signalling must be reconsidered in the light of new evidence. The plasma membrane H*-ATPase Plant cells have an H+-pumping enzyme in the plasma membrane, which converts ATP into an electrical and pH gradient across the membrane. Channels and carriers use these gradients to catalyse essential functions, such as
Fusicoccin
Fig. 2. Induction of stomatal opening in the lower epidermis of a leaf from Tradescantia by 10 5 M fusicoccin in the dark. A lipophilic fluorescent dye, DiOC6, was used to visualize the distribution of endoplasmic reticulum and mitochondria; the cell wall around the pore is also stained by the dye. Images were taken by confocal scanning microscopy. Relief was introduced by computerized embossing.
nutrient uptake, osmoregulation and ion homeostasis. Reflecting its physiological importance, the pump is regulated by a variety of factors, including hormones, light, pathogens and cytosolic pH. Among these factors, fusicoccin is the most direct and potent activator of the H+-ATPase. For example, activation can be measured in real time in a single cell using the patch-clamp method (Fig. 3), a technique surprisingly little used thus far to unravel the mechanism of pump activation. Biochemical approaches have clearly indicated that the C-terminus is the target for fusicoccin action (see Ref. 7). Based on the similarity of in vitro characteristics
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14
12
10
8 C (D
FC 6
4
2
0
I
0
10
I
I
20 30 Time (min)
I
I
40
50
Fig. 3. Fusiocccin (10 ~ M) induced proton pump current measured in a barley leaf protoplast, using the patch-clamp technique in the whole cell configuration. The voltage was clamped to 0 mV with symmetrical potassium chloride solutions at either side of the membrane; the pump was fueled with 5 mM MgATP. The inset diagram indicates how the patch-clamping system was set up. Courtesy ofS.A. Vogelzang.
of a trypsin-treated (resulting in the loss of the C-terminal 7-10 kDa tail) and fusicoccin-treated pump, it was concluded that addition of fusicoccin in vivo results in 'displacement' of the C-terminus. Now the key question is how fusicoccin induces such a conformational change in the pump - this change is retained even after isolation of the pump from its native environment. Progress in understanding the way mammalian 14-3-3 proteins control the activity of target enzymes reveals a common principle: the 14-3-3 protein binds tightly to the target enzyme, and a phosphorylated motif (Arg-x-x-phosphoSer-xPro, where phosphoSer represents a phosphorylated serine, and x represents any amino acid) is essential for binding 1~. Does this same principle hold for 14-3-3 protein-mediated pump activation by fusicoccin? The answer is that it does, for the following reasons: • Recently, good evidence was presented for the role of dephosphorylation in pump activation during the incompatible interaction between tomato cells and the fungal pathogen Cladosporium fulvum ~4. The phosphatase responsible for dephosphorylation is inhibited by okadaic acid (a potent inhibitor of phosphatase 1 and phosphatase 2A) and stimulated by activated G proteins ~4. Rephosphorylation (and inactivation) involves two kinases: one has phosphokinase C-like properties (i.e. it is inhibited by calphostin C and chelerythrin, and activated by phorbol ester); and a second is related to the CaMKII kinase family~4. It should be noted that fungi of the genus Cladosporium produce cotylenin (Fig. 1), a toxin with fusicoccimlike properties. • Solubilized ATPase and [3H]fusicoccin binding activity cosediment in a sucrose gradient in the presence of ATP and fusicoccin ~5. The finding that a strict correlation exists between the amount of h~sicoccin bound to the plasma membrane and the activation state of the pump is an additional 62
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argument in favour of stoichiometric coupling between ATPase and the fusicoccin receptor 1~. Moreover, the ATPase can be purified to near homogeneity (as a 104 kDa polypeptide) in the absence of ATP, but in the presence of ATP a 30 kDa protein copurifies with the ATPase 17. Only under the latter conditions was pump stability preserved. All these results can be interpreted as a phosphorylationdependent coupling between the ATPase and the 30 kDa fusicoccin receptor. This hypothesis was recently tested TM. Addition of a synthetic phosphopeptide, corresponding to the phosphorylated 14-3-3 protein binding motif from Raf-kinase, to plasma membranes from radish seedlings, activated the pump as if it had been treated with fusicoccin. Addition of the same peptide, but lacking the phosphate group, had no effect. Treatment with phosphatase 2A also resulted in pump activation is, thus confirming the results by Xing et al. 14. Based on this evidence, it seems likely that, by analogy with the mechanism known from mammalian 14-3-3 proteins, phosphorylation of the pump is the switch that controls the association with a regulatory protein, in this case the fusicoccin 14-3-3 receptor. However, the ATPase and 14-3-3 protein may be part of a large supra-molecular complex, because the dimeric saddle-shaped structure of 14-3-3 proteins 11'~9 might allow them to assemble into larger structures. The copurification of the ATPase with 70 and 30 kDa polypeptides~7, and coimmunoprecipitation of the phosphorylated ATPase with 70, 45, 30 and 20 kDa polypeptides 2°, suggests the presence of a supra-molecular complex. A model for fusicoccin-induced activation of the H*.ATPase
A model for fusicoccin-induced pump activation has been presented is wherein activation is achieved through a dissociation of the pump-14-3-3 protein complex. However, evidence indicates that fusicoccin stabilizes the coupling between the ATPase and fusicoccin receptor (Refs 8, 10, 15 and 16; also F. Johansson, unpublished). A model incorporating fusicoccininduced association between the pump and fusicoccin receptor is shown in Fig. 4. In this model, the 14-3-3 protein forms a %ridge' between two domains of the pump (e.g. located in the C-terminus and central loop), by analogy with the model for the Raf kinase-14-3-3 protein interaction 1'. In this 'dual interaction' mode, pump activity is low but stable; the pure enzyme is unstable 17. Activation by fusicoccin, phosphatase 2A, or the phosphoSer259-Raf-1 peptide is is explained by a dissociation of the 14-3-3 protein from one domain, which thereby unfolds and activates the ATPase. Subsequent pump dimerization or oligomerization 21'22could explain the reported increase in affinity between the fusicoccin receptor and the membrane after addition of fusicoccin in vivo (Refs 8 and 10; also F. Johansson, unpublished). The latter mechanism is suggested by analogy with the activation of Raf kinase by Ras-GTP, caused by 14-3-3 protein-mediated dimerization and oligomerization of Raf (Ref. 23). The bridge model may hold true for other 14-3-3 target proteins, because it has been suggested that 14-3-3 binding to nitrate reductase occurs both at phosphoSer543 and the amino terminal tail is. The effect of the phosphoSer259-Raf-1 peptide on pump activityis suggests that ATPase sequences contain the consensus 14-3-3 protein-binding motif Arg-x-x-phosphoSer-x-Pro. Aitken has suggested that the motif around Ser699 (Arg-ValLys-Pro-Ser699-Pro-Thr-Pro in AHA3) is involved in 14-3-3 protein coupling 11. However, according to the topographic model for the pump, this domain is in an extracellular loop22.
reviews This implies that either the currently accepted topographic model for the pump needs revision or that other domains containing a phosphorylation motif are involved in the 14-3-3 protein coupling. Putative phosphorylation motifs are present in the C-terminus close to the autoinhibitory domain (RELSg°3EI in AHA3) and in the large cytoplasmic loop (RGAS61VDI)and these domains may play a role in pump regulation. The presence of a 14-3-3 binding site in the C-terminus of the pump has recently been suggested after it was shown that removal of the C-terminus released 14-3-3 proteins in soluble f o r m 24.
H+
H+
PP2Aphi""'osphatas~.~ ~ Kina~j/~ Lowactivity
Highactivity
Fig. 4. Model for the mechanism of H+-ATPase activation involving 14-3-3 protein and fusicoccin (FC). In the steady-state, 14-3-3 protein binds to and stabilizes the phosphorylated H÷-ATPase [filled circles indicate putative phosphorylated 14-3-3 protein binding domains 1 (RGAS~lTDI)and 2 (RELSg°3EI);open circles indicate the dephosphorylated domains]. Under these conditions, the activity of the pump is downregulated. Pump activation can be achieved through: in vivo activation of a phosphatase (by infection with the fungal pathogen Cladosporium)14; addition of phosphatase-2A (PP2A) (Ref. 18);binding of fusicoccin to 14-3-3 proteinTM (which may result in pump dimerization); or addition ofphosphopeptide 259-Raf-1 (Ref. 18).
It has been reported that a reconstituted H+-ATPase-fusicoccin receptor system is still sensitive to fusicoccin7. This is easily reconciled with the model if it is assumed that the pump and receptor can reassociate during reconstitution. However, the Nuclear 14-3-3 proteins and fusicoccin observation that a purified H÷-ATPase free of 14-3-3 protein The earliest assigned function for plant 14-3-3 proteins retains the characteristics of a fusicoccin-activated pump 2~ was the association with transcription factor complexes has not been explained. binding to G box elements in the promoters of genes activated by abscisic acid (ABA) and stress 1~. The 14-3-3 proNitrate reductase and fusicoccin teins do not bind directly to the DNA, but to G box binding NADH:nitrate reductase (NR) is a soluble cytosolic enzyme factors (GBF), which interact directly with the DNA. Phoscatalyzing the reduction of nitrate to nitrite, which is a rate- phorylation of GBF increases its binding to DNA (Ref. 28) limiting step in nitrate assimilation. The NR enzyme is sub- and the presence in GBF of putative phosphoserine 14-3-3 ject to multivalent control at the level of gene expression, and protein-binding motifs 11implies that phosphorylation-depenpost-translational modification in response to nitrate, light dent association of 14-3-3 proteins and GBF may be part of a and osmotic stress. Post-translational modifications of NR kinase-regulated activation of the DNA-binding complex 19. involve reversible phosphorylation, and recefitly Ser543 has In this respect, it is important to note that fusicoccin can been identified as the major regulatory phosphorylation antagonize the action of ABA in many respects, as observed site 26. However, phosphorylation of NR, although necessary, in stomatal movement and seed germination. If fusicoccin is not sufficient for inactivation. The phosphorylated form of can indeed affect both membrane bound and cytosolic 14-3-3 NR binds to an inhibitor protein (NIP), and this event ren- protein isoforms, then there is no reason to assume that it ders the enzyme inactive. The NIP protein has created much cannot bind to the nuclear-located 14-3-3 proteins, thus excitement, because two groups have demonstrated that it interfering with ABA-induced gene activation. belongs to the 14-3-3 familyls'26. The phosphorylated motif in NR involved in coupling to 14-3-3 (RTAS~43TP) fits well with The search for other fusicoccin/14-3-3 targets the consensus 14-3-3 binding motif found in all 14-3-3 target The number of 14-3-3 target proteins found in mamproteins 1~. Indeed, the phosphoSer259-Raf peptide competed malian cells is rapidly increasing, and presently includes 15 effectively with NIP14-3-3, thereby activating NR (Ref. 18). individual proteins (not counting different isoforms11). An Moreover, incubation of NIP14-3-3 with fusicoccin prevented even greater number of 14-3-3 protein isoforms has been the inhibitory action of NIP14-3-3 on the activity of NR (Ref. identified in plants, and it therefore seems safe to assume 18). If this effect of fusicoccin can be demonstrated in vivo as that we have only just begun to identify the full range of 14well, then there are a number of intriguing implications. The 3-3 protein targets in plants. Although a physical association first of these is that fusicoccin must bind to soluble 14-3-3 between two proteins can be demonstrated using a range of isoforms. Thus far this has not been demonstrated, but biochemical and molecular techniques, the identification of experiments have yet to test whether the phosphorylated an 'unknown' target remains difficult. Therefore, we have to status of either the 14-3-3 target proteins (conditional to cou- make an educated guess as to where to look for target propling) or the 14-3-3 proteins themselves is essential for fusic- teins. Several presently unexplained fusicoccin effects may occin binding. A second implication is that the activity status help us to do so. of NR (reflecting the need for nitrate uptake into the cell) may be transduced through a common set of 14-3-3 isoforms Cytoplasmic acidification to the plasma membrane H+-ATPase, allowing the pump to Measurements with pH-sensitive microelectrodes indicate provide the proton motive force necessary for uptake of that, within 1 min of adding fusicoccin, the cytoplasm starts nitrate through an H÷/NQ- cotransport system. This sugges- to acidify29. This timescale fits well with the fusicoccintion is supported by the finding that the addition of nitrate induced increase in the incorporation of 14CO2 into malate 3°. increases the activity of the H+-ATPase in the plasma mem- However, intuitively it would be anticipated that pump actibrane of maize roots 2~. vation would result in an alkalinization of the cytoplasm.
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inward and outward rectifying K÷ channels, indepenMalic enzyme dent of changes in memMalate Phosphoenolpyruvate / ~Oxaloacetate brane potential 82. Indeed, these channels have a com/ mon voltage-independent mode of control involving an CO2 + OHCO2 + OHokadaic acid-sensitive phosphatase 33. The okadaic acidsensitive phosphatase (type Fig. 5. The biochemical pH-stat31. An increase in the activity of phosphoenolpyruvate carboxyl1 or 2A) is a recurrent ase or malate dehydrogenase, as well as a reduced activity of malic enzyme, can result in the theme in 14-3-3 protein accumulation of malate and a lowering of cytoplasmic pH. action 14'1sand, in view of the effects of fusicoccin, it seems worthwhile assessing the role of 14-3-3 proteins in ion channel regulation in plants. Phosphoenolpyruvate carboxylase
Malate dehydrogenase
~
Pyruvate
Tissue bending A curious effect of fusicoccin is the induction of a bending response in bean hypocotyls and epicotyls when fusicoccin is administered in the transpiration stream ~4. Tissue bending involves differential cell growth, a process controlled by auxin and ethylene. Recently, an ethylene response gene, hlsl ('hookless'), essential for differential cell elongation in the hypocotyl of Arabidopsis, was isolated. The HLS1 protein shows significant sequence similarity to a class of N-acetyltransferases85. Interestingly, an N-acetyltransferase in mammalian cells, responsible for the acetylation of serotonin (a hormone with structural Fig. 6. The role of 14-3-3 proteins and fusicoccin (FC) in plant signal transsimilarity to auxin) binds to 14-3-3 proteins 11. duction. Proteins known to interact with 14-3-3 protein and/or to be affected If the activity of HLS1 is controlled by a 14-3by fusicoccin are the H+-ATPase, nitrate reductase (NR) and G box binding 3 protein, like its mammalian counterpart, factor (GBF). In vivo effects of fusicoccin indicate that 14-3-3 proteins might then this could explain the fusicoccin-induced be involved with: enzymes regulating the biochemical pH-stat~'3°; K÷bending of the bean hypocoty134. Polar auxin channels32'~; ethylene signalling intermediates34-~6(ESI); and phytochrome transport is involved in hypocotyl hook curvasignalling intermediates87 (PSI). Abbreviations: PP, protein phosphatase; PK, protein kinase. ture 3~ and acetylation of auxin by HLS1 may affect redistribution of auxin and thus tissue bending. However, fusicoccin could also affect tissue bending by interference with CTR1 Could it be that the change in the pH of the cytoplasm is not ('constitutive triple response'), another protein that is essenrelated to pump activation (as suggested by Felle 6) and tial for ethylene signalling. The CTR1 protein is a member of instead is the result of a direct effect of fusicoccin on the bio- the Raf family of protein kinases ~6. The ctrl mutant is a conchemical pH-stat? The biochemical pH-stat is an important stitutive triple response mutant, indicating that the CTR1 mechanism for controlling cytoplasmic pH, key enzymes protein is a negative regulator of the ethylene response pathbeing phosphoenolpyruvate carboxylase, malate dehydroge- way. In mammalian cells, Raf kinase is coupled to and connase and malic enzyme31 (Fig. 5). A fusicoccin-induced trolled by 14-3-3 protein isoforms'l'~9; association of the plant change in the pH optimum of one of these enzymes could Raf homotogue CTR1 with 14-3-3 protein could explain the easily account for the accumulation of malate and a change fusicoccin-induced tissue bending. The CTR1 protein conin cytoplasmic pH. If so, the mechanism involved might be tains a motif for serine/threonine kinases - Arg-Asp-Leuanalogous to the fusicoccin-induced and 14-3-3 protein- Lys-Ser-Pro (Ref. 36) - which may act as a binding motif for mediated change in pH sensitivity of the H+-ATPase and a 14-3-3 protein. nitrate reductase.
Phytochrome-mediatedseed germination Increased K +uptake Initially, increased K+ uptake was explained as an effect secondary to pump activation: the membrane potential becomes more negative, and voltage-gated inward K÷ channels respond with a higher activity. However, a detailed study using the voltage clamp technique on guard cells showed a fusicoccin-induced alteration of the activity of 64
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Germination of certain seeds is a light-dependent process mediated by phytochrome. Fusicoccin can substitute for the red-light activated form of phytochrome (P~r) (Ref. 37). Importautly, far-red light antagonizes fusicoccin action just as it antagonizes red-light irradiation (B. De Boer and M.W. De Graaf, unpublished). These experiments suggest that the phytochrome and fusicoccin-signalling pathway share one or
mlmillmnmlB reviews more intermediates. One hypothesis is that phytochrome and 14-3-3 protein interact in a fashion dependent on light and fusicoccin. The presence of putative 14-3-3 binding motifs from Arabidopsis in PhyA and PhyE (Arg-Leu-Gln-Ser-LeuPro), and PhyC (Arg-Ser-Ser-Ser-Lys-Pro and Arg-Trp-LysSer-Vat-Pro), warrants testing of this hypothesis. ~
The elusiveendogenous ligand Why do plants have a high-affinity receptor for a molecule that they are unlikely to encounter? The most attractive hypothesis is that fusicoccin mimics an endogenous molecule. Hormones such as auxin, gibberellic acid, abscisic acid, cytokinin and ethylene do not compete with fusicoccin for binding, but plant extracts do contain molecules that compete effectively with [3H]fusicoccin-binding to plasma membranes or fusicoccin antibodies (see Ref. 5). One of the competing molecules extracted from plants has been identified as fusicoccin A (Ref. 5) (see Fig. 1). However, plant extracts do contain molecules competing for [3H]fusicoccin binding that differ from fusicoccin in their chromatographic behaviour and in vivo effect. These molecules might be derivatives with a fusicoccin core structure 5 (just as a plant contains numerous derivatives of gibberellic acid), but it may be that the molecules are structurally very different from fusicoccin. For example, cis-unsaturated fatty acids, such as arachidonic acid and linolenic acid (products of phospholipase A2 activity) are very effective inhibitors of the binding of [3H]fusicoccin to the fusicoccin receptor in the plasma membrane. The ICs0 values of these inhibitors (i.e. inhibitor concentrations at which enzyme activity or binding activity is 50% inhibited) are 30 ~M3s'39. Conclusion Fusicoccin started as a 'tool for plant physiologists'4, but has now become a key used by biochemists, plant physiologists and molecular biologists alike to open doors to as yet unknown regulatory pathways. The identification of the fusicoccin receptor as a 14-3-3 protein corroborates the conclusion that fusicoccin interferes with a system that is at the crossroads of various signalling and regulatory pathways (see Fig. 6). Much of the understanding of fusicoccin and 14-3-3 protein action is still in its infancy, but the massive growth in information about 14-3-3 proteins 19 suggests that gaps in our knowledge will be filled quite rapidly. The mechanism of fusicoccin action is also likely to have implications for research into nitrate assimilation, signal transduction of abscisic acid, auxin and ethylene, and phytochrome-mediated responses.
Acknowledgements, Work in the author's group was supported by grants from the Technical (STW 790.43.850) and Life Sciences (SLW 805.22.765) Foundation and from the European Community (INTAS grant no. 94-4358). I am grateful to Nico Blijleven for assisting with the preparation of Fig. 2 and all members of my lab, past and present, for their support. Furthermore I would like to thank colleagues in the field of fusicoccin and 14:3-3 research for providing preprints of papers in press. References 1 Ballio, A. et al. (1964) Fusicoccin: a new wilting toxin produced by Fusicoccum amygdali Del., Nature 203,279 2 Muromtsev, G.S. et al. (1994) Occurrence of fusicoccanes in plants and fungi, J. Plant Growth Regul. 13, 39-49
3 Ballio, A., Federico, R. and Scalorbi, D. (1981) Fusicoccin structure-activity relationships: in vitro binding to microsomal preparations of maize coleoptiles, Physiol. Plant. 52, 471-475 4 Marrb, E. (1979) Fusicoccin: a tool in plant physiology, Annu. Rev. Plant Physiol. 30, 273-288 5 Muromtsev, G.S. (1996) Is fusicoccin a new phytohormone? Russ. J. Plant Physiol. 43, 421-433 6 Felle, H.H. (1996) Control of cytoplasmic pH under anoxic conditions and its implication for plasma membrane proton transport in Medicago sativa root hairs, J. Exp. Bot. 47, 967-973 7 Aducci, P. et al. (1995) Fusicoccin receptors: perception and transduction of the fusicoccin signal, J. Exp. Bot. 46, 1463-1478 8 Korthout, H.A.A.J. and De Boer, A.H. (1994) A fusicoccin-binding protein belongs to the family of 14-3-3 brain protein homologs, Plant Cell 6, 1681-1692 9 Marra, M. et al. (1994) The 30 kD protein present in purified receptor preparation is a 14-3-3-like protein, Plant Physiol. 106, 1497-1501 10 Oecking, C., Eckerson, C. and Weiler, E.W. (1994) The fusicoccin receptor of plants is a member of the 14-3-3 superfamily of eukaryotic regulatory proteins, FEBS Lett. 352, 163-166 11 Aitken, A. (1996) 14-3-3 and its possible role in coordinating multiple signalling pathways, Trends Cell Biol. 6, 341-347 12 De Boer, A.H. et al. (1987) Sohibilization of the fusicoccin receptor and a protein kinase from highly purified plasma membrane from oat roots (Wirtz, K.W.A., ed.), pp. 181-190, Plenum Publishing 13 De Boer, A.H. and Korthout, H.A.A.J. (1996) 14-3-3 homologues play a central role in the fusicoccin signal transduction pathway, J. Plant Growth Regul. 18, 99-105 14 Xing, T., Higgins, V.J. and Bhimwald, E. (1996) Regulation of plant defense response to fungal pathogens: two types of protein kinases in the reversible phosphorylation of the host plasma membrane H÷-ATPase, Plant Cell 8, 555-564 15 Cocucci, M.C. and MarrY, E. (1991) Co-sedimentation of one form of plasma membrane H+-ATPase and of the fusicoccin receptor from radish microsomes, Plant Sci. 73, 45-54 16 De Michelis, M.I. et al. (1996) Fusicoccin binding to its plasma membrane receptor and the activation of the plasma membrane H÷-ATPase, Plant Physiol. 110, 957-964 17 Johansson, F., Sommarin, M. and Larsson, C. (1994) Rapid purification of the plasma membrane H+-ATPase in its non-activated form using FPLC, Physiol. Plant. 92, 389-396 18 Moorhead, G. et al. (1996) Phosphorylated nitrate reductase from spinach leaves is inhibited by 14-3-3 proteins and activated by fusicoccin, Curr. Biol. 6, 1104-1113 19 Ferl, R.J. (1996) 14-3-3 proteins and signal transduction, Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 49-73 20 Suzuki, Y.S., Wang, Y. and Takemoto, J.Y. (1992) Syringomycinstimulated phosphorylation of the plasma membrane H+-ATPase from red beet storage tissue, Plant Physiol. 99, 1314-1320 21 Briskin, D.P. (1990) The plasma membrane H+-ATPase of higher plant cells: biochemistry and transport function, Biochem. Biophys. Acta 1019, 95-109 22 Kasame, K. and Sakakibara, Y. (1995) The plasma membrane H÷-ATPase from higher plants: functional reconstitution into liposomes and its regulation by phospholipids, Plant Sci. 111, 117-131 23 Farrar, M.A., Alberola-lla, J. and Perlmutter, R.M. (1996) Activation of the Raf-1 kinase cascade by coumermycin-induced dimerization, Nature 383, 178-181 24 Piotrowski, M. et al. (1996) Functional architecture of the fusicoccin receptor, Plant Physiol. Biochem. Special Issue, 14 25 Marra, M. et al. (1995) The H÷-ATPase purified from maize root plasma membranes retains fusicoccin in vivo activation, FEBS Lett. 382, 293-296 26 Bachmann, M. et al. (1996) The inhibitor protein of phosphorylated nitrate reductase from spinach (Spinacia oleracea) leaves is a 14-3-3 protein, FEBS Lett. 387, 127-131 27 Santi, S. et al. (1995) Plasma membrane H÷-ATPase in maize roots induced for NO3- uptake, Plant Physiol. 109, 1277-1283 28 Klimczak, L.I., Schindler, U. and Cashmore, A.R. (1992) DNA binding activity of the Arabidopsis G-box binding factor GBF1 is stimulated by phosphorylation by casein kinase II from broccoli, Plant Cell 4, 87-98 February 1997, Vol. 2, No, 2
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reviews 29 Felle, H.H. et al. (1986) Indole-3-acetic acid and fusicoccin cause
cytosolic acidification of corn coleoptiles, Proc. Natl. Acad. Sci. U. S. A. 83, 8992-8995 30 Johnson, K.D. and Rayle, D.L. (1976) Enhancement of CO2 uptake in Avena coleoptiles by fusicoccin, Plant Physiol. 57, 806-811 31 Smith, F.A. and Raven, J.A. (1979) Intracellular pH and its regulation, Annu. Rev. Plant Physiol. 30, 289-311 32 Blatt, M.R. and Clint, G.M. (1989) Mechanisms of fusicoccin action: kinetic modification and inactivation of K÷ channels in guard cells, Planta 178, 509-523 33 Thiel, G. and Blatt, M.R. (1994) Phosphatase antagonist okadaic acid inhibits steady-state currents in guard cells of Vicia faba, Plant J. 5, 727-733 34 Lavee, S. and Cleland, R.E. (1993) Responses of intact and excised young bean plants to fusicoccin, J. Plant Growth Regul. 12, 255-262 35 Lehman, A., Black, R. and Ecker, J.R. (1996) HOOKLESS 1, an ethylene response gene, is required for differential cell elongation in the Arabidopsis hypocotyl, Cell 85, 183-194
36 Kieber, J.J. et al. (1993) CTR1, a negative regulator of the ethylene response pathway inArabidopsis, encodes a member of the Raf family of protein kinases, Cell 72, 427-441 37 Lado, P., Rasi-Caldoguo, F. and Colombo, R. (1974) Promoting effect of fusicoccin on seed germination, Physiol. Plant. 31, 149-152 38 Aducci, P. et al. (1993) Phospholipase A2 affects the activity of fusicoccin receptors, FEBS Lett. 320, 173-176 39 Van der Hoeven, P.C.J. et al. (1996) A calcium and free fatty acidmodulated protein kinase as putative effector of the fusicoccin 14-3-3 receptor, Plant Physiol. 111, 857-865
Lipid-transfer proteins: a puzzling family of plant proteins ,eoo°C Lipid-transfer proteins are small, basic proteins, and have been purified from various plant sources. They are able to transfer lipids between membranes in vitro and, on the basis of this, were initially thought to participate in the intracellular flux of lipids during membrane synthesis. However, the finding that these proteins are located i n the cell wall and can be secreted has led to the suggestion that they are not required for intracellu!ar lipid transport. Instead, they may be involved in cutin biosynthesis, surface wax formation, pathogen-defence reactions, or the adaptation of plants to environmental changes. ipids have to move from their sites of biosynthesis, mainly in the endoplasmic reticulum, to other cellular organelles, such as mitochondria and chloroplasts 1. The search for lipid carrier proteins led to the discovery of lipid-transfer proteins (LTPs) in a variety ofmonocotyledons and dicotyledons. These proteins are characterized by their ability to transfer phospholipids between membranes and to bind fatty acids in vitro. Recent nuclear magnetic resonance (NMR) and X-ray diffraction studies have confirmed that, structurally, LTPs are well suited to this task. However, none of these observations prove involvement in the intracellular flux of lipids. Moreover, in the light of recent observations on the expression of several LTP genes, the proposed cytoplasmic role for LTPs now appears unlikely and other novel roles, taking into account the extracellular location of the proteins, have been proposed.
L
Assay, purification and basic properties The determination of LTP activity is a complex process involving two types of membranes (donor and acceptor). The donor membranes are usually liposomes prepared by ultrasonication using a radioactive phospholipid. Acceptor membranes are from mitochondria or any other subcellular fraction that can be separated from liposomes by centrifugation. When the donor and acceptor membranes are incubated in the presence of LTP and then separated, radioactive phospholipid is detected in the acceptor membrane, indicating transfer from the donor membranes. in the absence of LTP, no transfer is observed - the spontaneous movement of phospholipids is very slow. 66
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Alternatively, liposomes used as donor membranes can be prepared from fluorescent lipids, and fluorescence quenching used to measure LTP activity in all extracts. These assays can be performed either with crude extracts or with purified LTPs. LTPs have been purified from various sources of plant tissue (e.g. leaves and seedlings). They are 9-10 kDa in size, and have isoelectric points ranging between 8.8 and 10, depending on the plant source2'~. They are also relatively abundant - on the basis of enzyme-linked immunosorbent assays (ELISAs) and immunoblotting, it has been calculated that LTPs constitute 4% of the soluble proteins extracted from maize seedlings 4. The LTPs have a broad specificity for phospholipids and are able to transfer phosphatidylcholine (PC), phosphatidylinositol (PI) and phosphatidylethanolamine (PE) between various membranes. Galactolipids [e.g. monogalactosyl-diacylglycerol, digalactosyl-diacylglycerol and a sulfolipid (sulphoquinovosyl-diacylglycerol)], but not triacylglycerols, are also transferred 5.
Structure The primary amino acid structures have been determined for the LTPs from maize, castor bean, wheat and rice, and the primary structures of several other LTPs have been deduced from their cDNA sequences (Fig. 1). The proteins consist of 91-95 amino acid residues, differing in sequence but containing eight strictly conserved cysteines forming four disulfide bridges 3'~. Within a given species, great divergence can be observed between the
© 1997 Elsevier Science Ltd
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39 Beyrle, H. (1995) The role of phytohormones in the fimction and biology
of mycorrhizas, in Mycorhiza: Structure, Function, Molecular Biology and Biotechnology (Varma, A. and Hock, B., eds), pp. 365-391, Springer 40 Smith, S.E. and Smith, F.A. (1990) Structure and function of the interfaces in biotrophic symbiosis as they relate to nutrient transport, New Phytol. 114, 1-38 41 Pearson, J.N. and Jakobsen, I. (1993) Symbiotic exchange of carbon and phosphorus between cucumber and three arbuscular mycorrhizal fungi, New Phytol. 124, 481-488 42 Jakobsen, I. (1995) Transport of phosphorus and carbon in VA mycorrhizas, in Mycorhiza: Structure, Function, Molecular Biology and Biotechnology (Varma, A. and Hock, B., eds), pp. 297-325, Springer 43 Ravnskov, S. and Jakobsen, I. (1995) Functional compatibility in arbuscular mycorrhizas measured as hyphal P transport to the plant, New Phytol. 129, 611-618 44 Harrison, M.J. and van Buuren, M.L. (1995) A phosphate transporter from the mycorrhizal fungus Glomas versiforme, Nature 378, 626-629 45 Muchhal, U.S., Pardo, J.M. and Raghathama, K.G. (1996) Phosphate transporters from the higher plant Arabidopsis thaliana, Proc. Natl. Acad. Sci. U. S. A. 93, 10519-10523 46 Schwab, S.M., Menge, J.A. and Tinker, P.B. (1991) Regulation of nutrient transfer between host and fungus in vesicular-arbuscular mycorrhizas, New Phytol. 117, 387-398 47 Shachar-Hill, Y. et al. (1995) Partitioning of intermediary carbon metabolism in vesicular-arbuscular mycorrhizal leek, Plant Physiol. 108, 2979-2995 48 Harrison, M.J. (1996) A sugar transporter from Medicago truncatula: altered expression pattern in roots during vesicular-arbuscular (VA) mycorrhizal associations, Plant J. 9, 491-503 49 Smith, S.E. (1993) Transport at the mycorrhizal interface, Mycorrhiza News 5, 1-3 50 Taiz, L. and Zeiger, E. (1991) Plant Physiology, Benjamin/Cumming 51 Ingold, C.T. and Hudson, H.J. (1993) The Biology of Fungi, Chapman & Hall
Ma~ia ~ Hardson is at the Samuel Roberts Noble Foundation,
Fusicoccin - a key to multiple 14-3-3 !0 Cks ?
DoBoer
Plants under attack from pathogens have a range of defence responses at their disposal to fight off the infecting organism. The fungus Fusicoccum amygdali, however, appears to have found a major weakness in the plant defence barrier. A key role in the fungal attack is played by a molecule produced by and named after the fungus: fusicoccin. Recently, it has become clear that fusicoccin targets 14-3-3 proteins, which are at the cross-point of a huge array of signalling and regulatory pathways. Progress has n o w been made in understanding h o w 14-3-3 proteins and fusicoccin are involved in the regulation of key enzymes, such as the plasma membrane H*-ATPase and soluble nitrate reductase. Research on the in vivo effects of fusicoccin may lead to the identification of n e w 14-3-3 target proteins. 'nfection by the fungus Fusicoccum amygdali eventually kills the host - peach (Prunuspersica) or almond (Prunus .amygdalus) trees - but its mode of action is subtle. The fungus excretes fusicoccin, a diterpene glucoside (Ref. 1) (Fig. 1), into the apoplast of an infected leaf, which spreads via the transpiration stream throughout the rest of the 60
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plant. When fusicoccin reaches a guard cell pair (i.e. a stoma) it triggers a signalling pathway leading to a widening of the stomatal pore (Fig. 2). Unlike other signals that stimulate stomatal opening (e.g. light, CO 2 and auxin), the effect of fusicoccin is irreversible. The uncontrolled loss of water eventually results in wilting of the leaf. Although the fungus
© 1997 Elsevier Science Ltd
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is host specific, fusicoccin is not, and it affects a system common to most plant cells. Thus it also stimulates cell elongation, breaks seed dormancy and stimulates rhizogenesis4'~. At the cellular level, the most obvious responses are a rapid (delay of 10-30 s) hyperpolarization of the membrane potential, and acidification of the cytoplasm6, followed by acidification of the medium and an increase in K÷-uptake. In order to understand how fusicoccin triggers these responses, research has focused on the fusicoccin receptor, and the plasma membrane-located H+-ATPase.
/•
O
CH3
.,\\H 0 ~CH3 HOI ~ ~
HO'"~_O / " ~ /
OjCH3
/ i
HO O H3C 17 ~ E ~ 141.~5 CH3
/7--c.3 -~___~,- A ~
4L_~
o
&H3
108H3 OH
16 ~-- O .... CH3
HO16~--O --CH 3 FusicoccinA
CotyleninA
Fig. 1. Structure of fusicoccin A, a metabolite of the fungus Fusicoccum amygdali, and cotylenin A, a metabolite of the fungus Cladosporium. Besides these major metabolites, the fungi produce a range of structurally related compounds (see Ref. 2). Structural requirements for biological activity of the fusicoccin molecule are: an unsubstituted hydroxyl group on C-8; the conformation at C-3 and C-9; and the conformation of the eight-membered ring. The importance of the glycosyl moiety is secondary to that of the carbotricyclic system. The aglycones of fusicoccin and cotylenin (cotylenol) still compete with fusicoccin A for binding and have biological activity3.
The fusicoccin receptor: a 14-3-3 protein The purification and identification of the fusicoccin receptor has a long history (Ref. 7), beginning almost 20 years ago with the first published report of a membrane-bound receptor with high affinity for [3H]fusicoccin. In 1989, the protein was purified to homogeneity as a 30 kDa doublet, and finally in 1994 peptide digests of the purified receptor were sequenced and identified as a member (or members) of the 14-3-3 protein familys-l°. Although the evidence that 14-3-3 dimers form the functional fusicoccin receptor is very strong, an as yet unsolved problem is that labelling by azido-fusicoccin identifies a 90 kDa doublet in maize membranes 7. Either the 90 kDa proteins are in the vicinity of the fusicoccin-binding pocket of the 14-3-3 protein, and are therefore an artefact of labelling by azido-fusicoccin, or they represent a second class of fusicoccin receptors. The identity of the receptor came as a surprise, because prevailing dogma had been that the fusicoccin-binding site was extracellular and that the receptor was an integral membrane protein. The primary structure of 14-3-3 proteins shows that they are hydrophilic and lack membrane-spanning domains (confirmed by the crystal structure11). Hence the binding site for fusicoccin must be intracellular, a conclusion corroborated by an earlier report that the hydrolytic site of the H+-ATPase and the fusicoccin-binding site of the fusicoccin receptor are at the same face of the plasma membrane 12. Moreover, fusicoccin is a rather hydrophobic molecule, and the biological activity of fusicoccin conjugates decreases with increasing polarity (see Ref. 13). The location of the binding site and hypotheses about fusicoccin signalling must be reconsidered in the light of new evidence. The plasma membrane H*-ATPase Plant cells have an H+-pumping enzyme in the plasma membrane, which converts ATP into an electrical and pH gradient across the membrane. Channels and carriers use these gradients to catalyse essential functions, such as
Fusicoccin
Fig. 2. Induction of stomatal opening in the lower epidermis of a leaf from Tradescantia by 10 5 M fusicoccin in the dark. A lipophilic fluorescent dye, DiOC6, was used to visualize the distribution of endoplasmic reticulum and mitochondria; the cell wall around the pore is also stained by the dye. Images were taken by confocal scanning microscopy. Relief was introduced by computerized embossing.
nutrient uptake, osmoregulation and ion homeostasis. Reflecting its physiological importance, the pump is regulated by a variety of factors, including hormones, light, pathogens and cytosolic pH. Among these factors, fusicoccin is the most direct and potent activator of the H+-ATPase. For example, activation can be measured in real time in a single cell using the patch-clamp method (Fig. 3), a technique surprisingly little used thus far to unravel the mechanism of pump activation. Biochemical approaches have clearly indicated that the C-terminus is the target for fusicoccin action (see Ref. 7). Based on the similarity of in vitro characteristics
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14
12
10
8 C (D
FC 6
4
2
0
I
0
10
I
I
20 30 Time (min)
I
I
40
50
Fig. 3. Fusiocccin (10 ~ M) induced proton pump current measured in a barley leaf protoplast, using the patch-clamp technique in the whole cell configuration. The voltage was clamped to 0 mV with symmetrical potassium chloride solutions at either side of the membrane; the pump was fueled with 5 mM MgATP. The inset diagram indicates how the patch-clamping system was set up. Courtesy ofS.A. Vogelzang.
of a trypsin-treated (resulting in the loss of the C-terminal 7-10 kDa tail) and fusicoccin-treated pump, it was concluded that addition of fusicoccin in vivo results in 'displacement' of the C-terminus. Now the key question is how fusicoccin induces such a conformational change in the pump - this change is retained even after isolation of the pump from its native environment. Progress in understanding the way mammalian 14-3-3 proteins control the activity of target enzymes reveals a common principle: the 14-3-3 protein binds tightly to the target enzyme, and a phosphorylated motif (Arg-x-x-phosphoSer-xPro, where phosphoSer represents a phosphorylated serine, and x represents any amino acid) is essential for binding 1~. Does this same principle hold for 14-3-3 protein-mediated pump activation by fusicoccin? The answer is that it does, for the following reasons: • Recently, good evidence was presented for the role of dephosphorylation in pump activation during the incompatible interaction between tomato cells and the fungal pathogen Cladosporium fulvum ~4. The phosphatase responsible for dephosphorylation is inhibited by okadaic acid (a potent inhibitor of phosphatase 1 and phosphatase 2A) and stimulated by activated G proteins ~4. Rephosphorylation (and inactivation) involves two kinases: one has phosphokinase C-like properties (i.e. it is inhibited by calphostin C and chelerythrin, and activated by phorbol ester); and a second is related to the CaMKII kinase family~4. It should be noted that fungi of the genus Cladosporium produce cotylenin (Fig. 1), a toxin with fusicoccimlike properties. • Solubilized ATPase and [3H]fusicoccin binding activity cosediment in a sucrose gradient in the presence of ATP and fusicoccin ~5. The finding that a strict correlation exists between the amount of h~sicoccin bound to the plasma membrane and the activation state of the pump is an additional 62
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argument in favour of stoichiometric coupling between ATPase and the fusicoccin receptor 1~. Moreover, the ATPase can be purified to near homogeneity (as a 104 kDa polypeptide) in the absence of ATP, but in the presence of ATP a 30 kDa protein copurifies with the ATPase 17. Only under the latter conditions was pump stability preserved. All these results can be interpreted as a phosphorylationdependent coupling between the ATPase and the 30 kDa fusicoccin receptor. This hypothesis was recently tested TM. Addition of a synthetic phosphopeptide, corresponding to the phosphorylated 14-3-3 protein binding motif from Raf-kinase, to plasma membranes from radish seedlings, activated the pump as if it had been treated with fusicoccin. Addition of the same peptide, but lacking the phosphate group, had no effect. Treatment with phosphatase 2A also resulted in pump activation is, thus confirming the results by Xing et al. 14. Based on this evidence, it seems likely that, by analogy with the mechanism known from mammalian 14-3-3 proteins, phosphorylation of the pump is the switch that controls the association with a regulatory protein, in this case the fusicoccin 14-3-3 receptor. However, the ATPase and 14-3-3 protein may be part of a large supra-molecular complex, because the dimeric saddle-shaped structure of 14-3-3 proteins 11'~9 might allow them to assemble into larger structures. The copurification of the ATPase with 70 and 30 kDa polypeptides~7, and coimmunoprecipitation of the phosphorylated ATPase with 70, 45, 30 and 20 kDa polypeptides 2°, suggests the presence of a supra-molecular complex. A model for fusicoccin-induced activation of the H*.ATPase
A model for fusicoccin-induced pump activation has been presented is wherein activation is achieved through a dissociation of the pump-14-3-3 protein complex. However, evidence indicates that fusicoccin stabilizes the coupling between the ATPase and fusicoccin receptor (Refs 8, 10, 15 and 16; also F. Johansson, unpublished). A model incorporating fusicoccininduced association between the pump and fusicoccin receptor is shown in Fig. 4. In this model, the 14-3-3 protein forms a %ridge' between two domains of the pump (e.g. located in the C-terminus and central loop), by analogy with the model for the Raf kinase-14-3-3 protein interaction 1'. In this 'dual interaction' mode, pump activity is low but stable; the pure enzyme is unstable 17. Activation by fusicoccin, phosphatase 2A, or the phosphoSer259-Raf-1 peptide is is explained by a dissociation of the 14-3-3 protein from one domain, which thereby unfolds and activates the ATPase. Subsequent pump dimerization or oligomerization 21'22could explain the reported increase in affinity between the fusicoccin receptor and the membrane after addition of fusicoccin in vivo (Refs 8 and 10; also F. Johansson, unpublished). The latter mechanism is suggested by analogy with the activation of Raf kinase by Ras-GTP, caused by 14-3-3 protein-mediated dimerization and oligomerization of Raf (Ref. 23). The bridge model may hold true for other 14-3-3 target proteins, because it has been suggested that 14-3-3 binding to nitrate reductase occurs both at phosphoSer543 and the amino terminal tail is. The effect of the phosphoSer259-Raf-1 peptide on pump activityis suggests that ATPase sequences contain the consensus 14-3-3 protein-binding motif Arg-x-x-phosphoSer-x-Pro. Aitken has suggested that the motif around Ser699 (Arg-ValLys-Pro-Ser699-Pro-Thr-Pro in AHA3) is involved in 14-3-3 protein coupling 11. However, according to the topographic model for the pump, this domain is in an extracellular loop22.
reviews This implies that either the currently accepted topographic model for the pump needs revision or that other domains containing a phosphorylation motif are involved in the 14-3-3 protein coupling. Putative phosphorylation motifs are present in the C-terminus close to the autoinhibitory domain (RELSg°3EI in AHA3) and in the large cytoplasmic loop (RGAS61VDI)and these domains may play a role in pump regulation. The presence of a 14-3-3 binding site in the C-terminus of the pump has recently been suggested after it was shown that removal of the C-terminus released 14-3-3 proteins in soluble f o r m 24.
H+
H+
PP2Aphi""'osphatas~.~ ~ Kina~j/~ Lowactivity
Highactivity
Fig. 4. Model for the mechanism of H+-ATPase activation involving 14-3-3 protein and fusicoccin (FC). In the steady-state, 14-3-3 protein binds to and stabilizes the phosphorylated H÷-ATPase [filled circles indicate putative phosphorylated 14-3-3 protein binding domains 1 (RGAS~lTDI)and 2 (RELSg°3EI);open circles indicate the dephosphorylated domains]. Under these conditions, the activity of the pump is downregulated. Pump activation can be achieved through: in vivo activation of a phosphatase (by infection with the fungal pathogen Cladosporium)14; addition of phosphatase-2A (PP2A) (Ref. 18);binding of fusicoccin to 14-3-3 proteinTM (which may result in pump dimerization); or addition ofphosphopeptide 259-Raf-1 (Ref. 18).
It has been reported that a reconstituted H+-ATPase-fusicoccin receptor system is still sensitive to fusicoccin7. This is easily reconciled with the model if it is assumed that the pump and receptor can reassociate during reconstitution. However, the Nuclear 14-3-3 proteins and fusicoccin observation that a purified H÷-ATPase free of 14-3-3 protein The earliest assigned function for plant 14-3-3 proteins retains the characteristics of a fusicoccin-activated pump 2~ was the association with transcription factor complexes has not been explained. binding to G box elements in the promoters of genes activated by abscisic acid (ABA) and stress 1~. The 14-3-3 proNitrate reductase and fusicoccin teins do not bind directly to the DNA, but to G box binding NADH:nitrate reductase (NR) is a soluble cytosolic enzyme factors (GBF), which interact directly with the DNA. Phoscatalyzing the reduction of nitrate to nitrite, which is a rate- phorylation of GBF increases its binding to DNA (Ref. 28) limiting step in nitrate assimilation. The NR enzyme is sub- and the presence in GBF of putative phosphoserine 14-3-3 ject to multivalent control at the level of gene expression, and protein-binding motifs 11implies that phosphorylation-depenpost-translational modification in response to nitrate, light dent association of 14-3-3 proteins and GBF may be part of a and osmotic stress. Post-translational modifications of NR kinase-regulated activation of the DNA-binding complex 19. involve reversible phosphorylation, and recefitly Ser543 has In this respect, it is important to note that fusicoccin can been identified as the major regulatory phosphorylation antagonize the action of ABA in many respects, as observed site 26. However, phosphorylation of NR, although necessary, in stomatal movement and seed germination. If fusicoccin is not sufficient for inactivation. The phosphorylated form of can indeed affect both membrane bound and cytosolic 14-3-3 NR binds to an inhibitor protein (NIP), and this event ren- protein isoforms, then there is no reason to assume that it ders the enzyme inactive. The NIP protein has created much cannot bind to the nuclear-located 14-3-3 proteins, thus excitement, because two groups have demonstrated that it interfering with ABA-induced gene activation. belongs to the 14-3-3 familyls'26. The phosphorylated motif in NR involved in coupling to 14-3-3 (RTAS~43TP) fits well with The search for other fusicoccin/14-3-3 targets the consensus 14-3-3 binding motif found in all 14-3-3 target The number of 14-3-3 target proteins found in mamproteins 1~. Indeed, the phosphoSer259-Raf peptide competed malian cells is rapidly increasing, and presently includes 15 effectively with NIP14-3-3, thereby activating NR (Ref. 18). individual proteins (not counting different isoforms11). An Moreover, incubation of NIP14-3-3 with fusicoccin prevented even greater number of 14-3-3 protein isoforms has been the inhibitory action of NIP14-3-3 on the activity of NR (Ref. identified in plants, and it therefore seems safe to assume 18). If this effect of fusicoccin can be demonstrated in vivo as that we have only just begun to identify the full range of 14well, then there are a number of intriguing implications. The 3-3 protein targets in plants. Although a physical association first of these is that fusicoccin must bind to soluble 14-3-3 between two proteins can be demonstrated using a range of isoforms. Thus far this has not been demonstrated, but biochemical and molecular techniques, the identification of experiments have yet to test whether the phosphorylated an 'unknown' target remains difficult. Therefore, we have to status of either the 14-3-3 target proteins (conditional to cou- make an educated guess as to where to look for target propling) or the 14-3-3 proteins themselves is essential for fusic- teins. Several presently unexplained fusicoccin effects may occin binding. A second implication is that the activity status help us to do so. of NR (reflecting the need for nitrate uptake into the cell) may be transduced through a common set of 14-3-3 isoforms Cytoplasmic acidification to the plasma membrane H+-ATPase, allowing the pump to Measurements with pH-sensitive microelectrodes indicate provide the proton motive force necessary for uptake of that, within 1 min of adding fusicoccin, the cytoplasm starts nitrate through an H÷/NQ- cotransport system. This sugges- to acidify29. This timescale fits well with the fusicoccintion is supported by the finding that the addition of nitrate induced increase in the incorporation of 14CO2 into malate 3°. increases the activity of the H+-ATPase in the plasma mem- However, intuitively it would be anticipated that pump actibrane of maize roots 2~. vation would result in an alkalinization of the cytoplasm.
February 1997,Vol2,.No.2 63
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inward and outward rectifying K÷ channels, indepenMalic enzyme dent of changes in memMalate Phosphoenolpyruvate / ~Oxaloacetate brane potential 82. Indeed, these channels have a com/ mon voltage-independent mode of control involving an CO2 + OHCO2 + OHokadaic acid-sensitive phosphatase 33. The okadaic acidsensitive phosphatase (type Fig. 5. The biochemical pH-stat31. An increase in the activity of phosphoenolpyruvate carboxyl1 or 2A) is a recurrent ase or malate dehydrogenase, as well as a reduced activity of malic enzyme, can result in the theme in 14-3-3 protein accumulation of malate and a lowering of cytoplasmic pH. action 14'1sand, in view of the effects of fusicoccin, it seems worthwhile assessing the role of 14-3-3 proteins in ion channel regulation in plants. Phosphoenolpyruvate carboxylase
Malate dehydrogenase
~
Pyruvate
Tissue bending A curious effect of fusicoccin is the induction of a bending response in bean hypocotyls and epicotyls when fusicoccin is administered in the transpiration stream ~4. Tissue bending involves differential cell growth, a process controlled by auxin and ethylene. Recently, an ethylene response gene, hlsl ('hookless'), essential for differential cell elongation in the hypocotyl of Arabidopsis, was isolated. The HLS1 protein shows significant sequence similarity to a class of N-acetyltransferases85. Interestingly, an N-acetyltransferase in mammalian cells, responsible for the acetylation of serotonin (a hormone with structural Fig. 6. The role of 14-3-3 proteins and fusicoccin (FC) in plant signal transsimilarity to auxin) binds to 14-3-3 proteins 11. duction. Proteins known to interact with 14-3-3 protein and/or to be affected If the activity of HLS1 is controlled by a 14-3by fusicoccin are the H+-ATPase, nitrate reductase (NR) and G box binding 3 protein, like its mammalian counterpart, factor (GBF). In vivo effects of fusicoccin indicate that 14-3-3 proteins might then this could explain the fusicoccin-induced be involved with: enzymes regulating the biochemical pH-stat~'3°; K÷bending of the bean hypocoty134. Polar auxin channels32'~; ethylene signalling intermediates34-~6(ESI); and phytochrome transport is involved in hypocotyl hook curvasignalling intermediates87 (PSI). Abbreviations: PP, protein phosphatase; PK, protein kinase. ture 3~ and acetylation of auxin by HLS1 may affect redistribution of auxin and thus tissue bending. However, fusicoccin could also affect tissue bending by interference with CTR1 Could it be that the change in the pH of the cytoplasm is not ('constitutive triple response'), another protein that is essenrelated to pump activation (as suggested by Felle 6) and tial for ethylene signalling. The CTR1 protein is a member of instead is the result of a direct effect of fusicoccin on the bio- the Raf family of protein kinases ~6. The ctrl mutant is a conchemical pH-stat? The biochemical pH-stat is an important stitutive triple response mutant, indicating that the CTR1 mechanism for controlling cytoplasmic pH, key enzymes protein is a negative regulator of the ethylene response pathbeing phosphoenolpyruvate carboxylase, malate dehydroge- way. In mammalian cells, Raf kinase is coupled to and connase and malic enzyme31 (Fig. 5). A fusicoccin-induced trolled by 14-3-3 protein isoforms'l'~9; association of the plant change in the pH optimum of one of these enzymes could Raf homotogue CTR1 with 14-3-3 protein could explain the easily account for the accumulation of malate and a change fusicoccin-induced tissue bending. The CTR1 protein conin cytoplasmic pH. If so, the mechanism involved might be tains a motif for serine/threonine kinases - Arg-Asp-Leuanalogous to the fusicoccin-induced and 14-3-3 protein- Lys-Ser-Pro (Ref. 36) - which may act as a binding motif for mediated change in pH sensitivity of the H+-ATPase and a 14-3-3 protein. nitrate reductase.
Phytochrome-mediatedseed germination Increased K +uptake Initially, increased K+ uptake was explained as an effect secondary to pump activation: the membrane potential becomes more negative, and voltage-gated inward K÷ channels respond with a higher activity. However, a detailed study using the voltage clamp technique on guard cells showed a fusicoccin-induced alteration of the activity of 64
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Germination of certain seeds is a light-dependent process mediated by phytochrome. Fusicoccin can substitute for the red-light activated form of phytochrome (P~r) (Ref. 37). Importautly, far-red light antagonizes fusicoccin action just as it antagonizes red-light irradiation (B. De Boer and M.W. De Graaf, unpublished). These experiments suggest that the phytochrome and fusicoccin-signalling pathway share one or
mlmillmnmlB reviews more intermediates. One hypothesis is that phytochrome and 14-3-3 protein interact in a fashion dependent on light and fusicoccin. The presence of putative 14-3-3 binding motifs from Arabidopsis in PhyA and PhyE (Arg-Leu-Gln-Ser-LeuPro), and PhyC (Arg-Ser-Ser-Ser-Lys-Pro and Arg-Trp-LysSer-Vat-Pro), warrants testing of this hypothesis. ~
The elusiveendogenous ligand Why do plants have a high-affinity receptor for a molecule that they are unlikely to encounter? The most attractive hypothesis is that fusicoccin mimics an endogenous molecule. Hormones such as auxin, gibberellic acid, abscisic acid, cytokinin and ethylene do not compete with fusicoccin for binding, but plant extracts do contain molecules that compete effectively with [3H]fusicoccin-binding to plasma membranes or fusicoccin antibodies (see Ref. 5). One of the competing molecules extracted from plants has been identified as fusicoccin A (Ref. 5) (see Fig. 1). However, plant extracts do contain molecules competing for [3H]fusicoccin binding that differ from fusicoccin in their chromatographic behaviour and in vivo effect. These molecules might be derivatives with a fusicoccin core structure 5 (just as a plant contains numerous derivatives of gibberellic acid), but it may be that the molecules are structurally very different from fusicoccin. For example, cis-unsaturated fatty acids, such as arachidonic acid and linolenic acid (products of phospholipase A2 activity) are very effective inhibitors of the binding of [3H]fusicoccin to the fusicoccin receptor in the plasma membrane. The ICs0 values of these inhibitors (i.e. inhibitor concentrations at which enzyme activity or binding activity is 50% inhibited) are 30 ~M3s'39. Conclusion Fusicoccin started as a 'tool for plant physiologists'4, but has now become a key used by biochemists, plant physiologists and molecular biologists alike to open doors to as yet unknown regulatory pathways. The identification of the fusicoccin receptor as a 14-3-3 protein corroborates the conclusion that fusicoccin interferes with a system that is at the crossroads of various signalling and regulatory pathways (see Fig. 6). Much of the understanding of fusicoccin and 14-3-3 protein action is still in its infancy, but the massive growth in information about 14-3-3 proteins 19 suggests that gaps in our knowledge will be filled quite rapidly. The mechanism of fusicoccin action is also likely to have implications for research into nitrate assimilation, signal transduction of abscisic acid, auxin and ethylene, and phytochrome-mediated responses.
Acknowledgements, Work in the author's group was supported by grants from the Technical (STW 790.43.850) and Life Sciences (SLW 805.22.765) Foundation and from the European Community (INTAS grant no. 94-4358). I am grateful to Nico Blijleven for assisting with the preparation of Fig. 2 and all members of my lab, past and present, for their support. Furthermore I would like to thank colleagues in the field of fusicoccin and 14:3-3 research for providing preprints of papers in press. References 1 Ballio, A. et al. (1964) Fusicoccin: a new wilting toxin produced by Fusicoccum amygdali Del., Nature 203,279 2 Muromtsev, G.S. et al. (1994) Occurrence of fusicoccanes in plants and fungi, J. Plant Growth Regul. 13, 39-49
3 Ballio, A., Federico, R. and Scalorbi, D. (1981) Fusicoccin structure-activity relationships: in vitro binding to microsomal preparations of maize coleoptiles, Physiol. Plant. 52, 471-475 4 Marrb, E. (1979) Fusicoccin: a tool in plant physiology, Annu. Rev. Plant Physiol. 30, 273-288 5 Muromtsev, G.S. (1996) Is fusicoccin a new phytohormone? Russ. J. Plant Physiol. 43, 421-433 6 Felle, H.H. (1996) Control of cytoplasmic pH under anoxic conditions and its implication for plasma membrane proton transport in Medicago sativa root hairs, J. Exp. Bot. 47, 967-973 7 Aducci, P. et al. (1995) Fusicoccin receptors: perception and transduction of the fusicoccin signal, J. Exp. Bot. 46, 1463-1478 8 Korthout, H.A.A.J. and De Boer, A.H. (1994) A fusicoccin-binding protein belongs to the family of 14-3-3 brain protein homologs, Plant Cell 6, 1681-1692 9 Marra, M. et al. (1994) The 30 kD protein present in purified receptor preparation is a 14-3-3-like protein, Plant Physiol. 106, 1497-1501 10 Oecking, C., Eckerson, C. and Weiler, E.W. (1994) The fusicoccin receptor of plants is a member of the 14-3-3 superfamily of eukaryotic regulatory proteins, FEBS Lett. 352, 163-166 11 Aitken, A. (1996) 14-3-3 and its possible role in coordinating multiple signalling pathways, Trends Cell Biol. 6, 341-347 12 De Boer, A.H. et al. (1987) Sohibilization of the fusicoccin receptor and a protein kinase from highly purified plasma membrane from oat roots (Wirtz, K.W.A., ed.), pp. 181-190, Plenum Publishing 13 De Boer, A.H. and Korthout, H.A.A.J. (1996) 14-3-3 homologues play a central role in the fusicoccin signal transduction pathway, J. Plant Growth Regul. 18, 99-105 14 Xing, T., Higgins, V.J. and Bhimwald, E. (1996) Regulation of plant defense response to fungal pathogens: two types of protein kinases in the reversible phosphorylation of the host plasma membrane H÷-ATPase, Plant Cell 8, 555-564 15 Cocucci, M.C. and MarrY, E. (1991) Co-sedimentation of one form of plasma membrane H+-ATPase and of the fusicoccin receptor from radish microsomes, Plant Sci. 73, 45-54 16 De Michelis, M.I. et al. (1996) Fusicoccin binding to its plasma membrane receptor and the activation of the plasma membrane H÷-ATPase, Plant Physiol. 110, 957-964 17 Johansson, F., Sommarin, M. and Larsson, C. (1994) Rapid purification of the plasma membrane H+-ATPase in its non-activated form using FPLC, Physiol. Plant. 92, 389-396 18 Moorhead, G. et al. (1996) Phosphorylated nitrate reductase from spinach leaves is inhibited by 14-3-3 proteins and activated by fusicoccin, Curr. Biol. 6, 1104-1113 19 Ferl, R.J. (1996) 14-3-3 proteins and signal transduction, Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 49-73 20 Suzuki, Y.S., Wang, Y. and Takemoto, J.Y. (1992) Syringomycinstimulated phosphorylation of the plasma membrane H+-ATPase from red beet storage tissue, Plant Physiol. 99, 1314-1320 21 Briskin, D.P. (1990) The plasma membrane H+-ATPase of higher plant cells: biochemistry and transport function, Biochem. Biophys. Acta 1019, 95-109 22 Kasame, K. and Sakakibara, Y. (1995) The plasma membrane H÷-ATPase from higher plants: functional reconstitution into liposomes and its regulation by phospholipids, Plant Sci. 111, 117-131 23 Farrar, M.A., Alberola-lla, J. and Perlmutter, R.M. (1996) Activation of the Raf-1 kinase cascade by coumermycin-induced dimerization, Nature 383, 178-181 24 Piotrowski, M. et al. (1996) Functional architecture of the fusicoccin receptor, Plant Physiol. Biochem. Special Issue, 14 25 Marra, M. et al. (1995) The H÷-ATPase purified from maize root plasma membranes retains fusicoccin in vivo activation, FEBS Lett. 382, 293-296 26 Bachmann, M. et al. (1996) The inhibitor protein of phosphorylated nitrate reductase from spinach (Spinacia oleracea) leaves is a 14-3-3 protein, FEBS Lett. 387, 127-131 27 Santi, S. et al. (1995) Plasma membrane H÷-ATPase in maize roots induced for NO3- uptake, Plant Physiol. 109, 1277-1283 28 Klimczak, L.I., Schindler, U. and Cashmore, A.R. (1992) DNA binding activity of the Arabidopsis G-box binding factor GBF1 is stimulated by phosphorylation by casein kinase II from broccoli, Plant Cell 4, 87-98 February 1997, Vol. 2, No, 2
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reviews 29 Felle, H.H. et al. (1986) Indole-3-acetic acid and fusicoccin cause
cytosolic acidification of corn coleoptiles, Proc. Natl. Acad. Sci. U. S. A. 83, 8992-8995 30 Johnson, K.D. and Rayle, D.L. (1976) Enhancement of CO2 uptake in Avena coleoptiles by fusicoccin, Plant Physiol. 57, 806-811 31 Smith, F.A. and Raven, J.A. (1979) Intracellular pH and its regulation, Annu. Rev. Plant Physiol. 30, 289-311 32 Blatt, M.R. and Clint, G.M. (1989) Mechanisms of fusicoccin action: kinetic modification and inactivation of K÷ channels in guard cells, Planta 178, 509-523 33 Thiel, G. and Blatt, M.R. (1994) Phosphatase antagonist okadaic acid inhibits steady-state currents in guard cells of Vicia faba, Plant J. 5, 727-733 34 Lavee, S. and Cleland, R.E. (1993) Responses of intact and excised young bean plants to fusicoccin, J. Plant Growth Regul. 12, 255-262 35 Lehman, A., Black, R. and Ecker, J.R. (1996) HOOKLESS 1, an ethylene response gene, is required for differential cell elongation in the Arabidopsis hypocotyl, Cell 85, 183-194
36 Kieber, J.J. et al. (1993) CTR1, a negative regulator of the ethylene response pathway inArabidopsis, encodes a member of the Raf family of protein kinases, Cell 72, 427-441 37 Lado, P., Rasi-Caldoguo, F. and Colombo, R. (1974) Promoting effect of fusicoccin on seed germination, Physiol. Plant. 31, 149-152 38 Aducci, P. et al. (1993) Phospholipase A2 affects the activity of fusicoccin receptors, FEBS Lett. 320, 173-176 39 Van der Hoeven, P.C.J. et al. (1996) A calcium and free fatty acidmodulated protein kinase as putative effector of the fusicoccin 14-3-3 receptor, Plant Physiol. 111, 857-865
Lipid-transfer proteins: a puzzling family of plant proteins ,eoo°C Lipid-transfer proteins are small, basic proteins, and have been purified from various plant sources. They are able to transfer lipids between membranes in vitro and, on the basis of this, were initially thought to participate in the intracellular flux of lipids during membrane synthesis. However, the finding that these proteins are located i n the cell wall and can be secreted has led to the suggestion that they are not required for intracellu!ar lipid transport. Instead, they may be involved in cutin biosynthesis, surface wax formation, pathogen-defence reactions, or the adaptation of plants to environmental changes. ipids have to move from their sites of biosynthesis, mainly in the endoplasmic reticulum, to other cellular organelles, such as mitochondria and chloroplasts 1. The search for lipid carrier proteins led to the discovery of lipid-transfer proteins (LTPs) in a variety ofmonocotyledons and dicotyledons. These proteins are characterized by their ability to transfer phospholipids between membranes and to bind fatty acids in vitro. Recent nuclear magnetic resonance (NMR) and X-ray diffraction studies have confirmed that, structurally, LTPs are well suited to this task. However, none of these observations prove involvement in the intracellular flux of lipids. Moreover, in the light of recent observations on the expression of several LTP genes, the proposed cytoplasmic role for LTPs now appears unlikely and other novel roles, taking into account the extracellular location of the proteins, have been proposed.
L
Assay, purification and basic properties The determination of LTP activity is a complex process involving two types of membranes (donor and acceptor). The donor membranes are usually liposomes prepared by ultrasonication using a radioactive phospholipid. Acceptor membranes are from mitochondria or any other subcellular fraction that can be separated from liposomes by centrifugation. When the donor and acceptor membranes are incubated in the presence of LTP and then separated, radioactive phospholipid is detected in the acceptor membrane, indicating transfer from the donor membranes. in the absence of LTP, no transfer is observed - the spontaneous movement of phospholipids is very slow. 66
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Alternatively, liposomes used as donor membranes can be prepared from fluorescent lipids, and fluorescence quenching used to measure LTP activity in all extracts. These assays can be performed either with crude extracts or with purified LTPs. LTPs have been purified from various sources of plant tissue (e.g. leaves and seedlings). They are 9-10 kDa in size, and have isoelectric points ranging between 8.8 and 10, depending on the plant source2'~. They are also relatively abundant - on the basis of enzyme-linked immunosorbent assays (ELISAs) and immunoblotting, it has been calculated that LTPs constitute 4% of the soluble proteins extracted from maize seedlings 4. The LTPs have a broad specificity for phospholipids and are able to transfer phosphatidylcholine (PC), phosphatidylinositol (PI) and phosphatidylethanolamine (PE) between various membranes. Galactolipids [e.g. monogalactosyl-diacylglycerol, digalactosyl-diacylglycerol and a sulfolipid (sulphoquinovosyl-diacylglycerol)], but not triacylglycerols, are also transferred 5.
Structure The primary amino acid structures have been determined for the LTPs from maize, castor bean, wheat and rice, and the primary structures of several other LTPs have been deduced from their cDNA sequences (Fig. 1). The proteins consist of 91-95 amino acid residues, differing in sequence but containing eight strictly conserved cysteines forming four disulfide bridges 3'~. Within a given species, great divergence can be observed between the
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