- Email: [email protected]
Molecular analysis of phospholipase D
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reviews 27 Jones, R.L. and Armstrong, J.E. (1971) Evidence for osmotic regulation of hydrolytic enzyme production in germinating barley seeds, Plant Physiol. 48, 137-142 28 Yu, S-M. et aL (1991) Metabolic derepression of a-amylase gene expression in suspension-cultured cells of rice, J. Biol. Chem. 266, 21131-21137 29 Karrer, E.E. and Rodriguez, R.L. (1992) Metabolic regulation of rice a-amylase and sucrose synthase genes inplanta, Plant J. 2, 517-523 30 Huang, N. et al. (1993) Metabolic regulation of a-amylase gene expression in transgenic cell cultures of rice (Oryza sativa L.), Plant Mol. Biol. 23, 737-747 31 Chan, M-T., Chao, Y-C. and Yu, S-M. (1994) Novel gene expression system for plant cells based on induction of a-amylase promoter by carbohydrate starvation, J. Biol. Chem. 269, 17635-17641 32 Mitsunaga, S., Rodriguez, R.L. and Yamaguchi, J. (1994) Sequencespecific interactions of a nuclear protein factor with the promoter region of a rice gene for a-amylase, RAmy3D, Nucleic Acids Res. 22, 1948-1953 33 Mitsui, T., Yotsushima, K. and Nabekura, Y. (1995) A cell biochemical study on sugar-controlled a-amylase secretion in rice, in Current Topics in Plant Physiology (VoL14) (Pontis, H.G., Salerno, G.L. and Echeverria, E.J., eds), pp. 254-265, American Society of Plant Physiologists 34 Bush, D.S. (1995) Calcium regulation in plant cells and its role in signaling, Annu. Rev. Plant Physiol. Plant Mol. Biol. 46, 95-122 35 Gilroy, S. and Jones, R.L. (1992) Gibberellic acid and abscisic acid coordinately regulate cytoplasmic calcium and secretory activity in barley ateurone protoplasts, Proc. Natl. Acad. Sci. U. S. A. 89, 3591-3595
36 Sheu, J-J. et al. (1994) Control of transcription and mRNA turnover as mechanisms of metabolic repression of a-amylase gene expression, Plant J. 5, 655-664 37 Zorec, R. and Tester, M. (1992) Cytoplasmic calcium stimulates exocytosis in a plant secretory cell, Biophys. J. 63, 864-867 38 Akazawa, T. and Miyata, S. (1982) Biosynthesis and secretion of a-amylase and other hydrolases in germinating cereal seeds, Essays Biochem. 18, 40-78 39 Akazawa, T. and Hara-Nishimura, I. (1985) Topographic aspects of biosynthesis, extracellular secretion, and intracellular storage of proteins in plant cells, Annu. Rev. Plant Physiol. 36, 441-472 40 Ranjhan, S., Karrer, E.E. and Rodrignez, R.L. (1992) Localizing a-amylase gene expression in germinated rice grains, Plant Cell Physiol. 33, 73-79 41 Itoh, IQ et al. (1995) Developmental and hormonal regulation of rice a-amylase (RAmylA)-gusA fusion genes in transgenic rice seeds, Plant Physiol. 107, 25-31
Toshiaki Mitsui* is at the Graduate School of Science and Technology, Niigata University, Niigata 950-21, Japan; Kimiko:lt0h is at the Faculty of Agricul!ure, Niigata University, Niigata 950-21 .,,Japan. *Author for correspondence (tel +81 25 262 6641: fax +81 25 263 1659; e-mail [email protected]~).
Molecular analysis of phospholipase D × Phospholipase D has been proposed to play important roles in cellular processes ranging from the generation of second messengers to membrane degradation. Recent studies in plants have revealed the presence of different genes with multiple isoforms. Conserved motifs include those likely to be involved in catalysis and a Ca2÷/phospholipid-binding domain that is potentially responsible for the activation of the enzyme. Two cloned phospholipase D proteins, designated a and 9, display distinct properties in their dependence on Ca~÷and polyphosphoinositides for activity. Thus, isoforms of phospholipase D in plants may be subject to unique control mechanisms and have distinct cellular functions.
p
hospholipases are grouped into four major classes phospholipase At, phospholipase A2, phospholipase C and phospholipase D - according to the site of cleavage of phospholipids. Of these, phospholipase D appears to be the most prevalent in plant tissues. It catalyzes the hydrolysis of the terminal phosphodiester bond of glycerophospholipids to generate phosphatidic acid and a hydrophilic free head group, such as choline (Fig. 1). Phospholipase D has been implicated in a broad range of cellular processes. Early studies suggested that increased activity was the first step in a lipolytic cascade in membrane deterioration during senescence, ageing and stress injuries 1. Recent reports indicate that phospholipase D-catalyzed hydrolysis plays a pivotal role in transmembrane signaling and cellular regulation 2'3.The enzyme in mammalian cells has been proposed to mediate many processes, including cell proliferation, receptor-mediated exocytosis, a respiratory burst, actin polymerization and membrane trafficking (Fig. 1). The finding that phosphatidic acid serves as an intracellutar and © 1997 Elsevier Science Ltd
extracellular messenger has brought a flurry of research on the role and regulation of the phospholipase D in signal transduction pathways of animal and yeast systems 2-5. Intriguing observations also suggest that the enzyme may contribute to signaling cascades in plants ~-s. This review summarizes the current understanding of phospholipase D at the molecular level.
Multiple genes and structural heterogeneity of phospholipase D The cDNA for an intracellular phospholipase D in eukaryotes was first cloned from castor bean 9. Southern blot analysis revealed that other homologous genes were also present in the plant genome 1°. The sequence of the castor bean cDNA was used to identify several Arabidopsis expressed-sequence-tagged cDNAs entered on sequence databases as putative, incomplete phospholipase D clones. Two full-length cDNAs have been isolated using a nested polymerase chain reaction, rapid amplification of 5'-end PII $1360-1385(97)01059-5
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reviews Arabidopsis, placing them in the phospholipase Da class (Fig. 2). The phospholipase D~ amino acid sequences of castor bean and Arabidopsis are about Fleceptor 80% identical, and those of monocotyf-.~ ledons are about 90% identical. The G protein l-yrosine kinase Calcium PIPe amino acid sequence of phospholipase D~ from Arabidopsis is about 45-50% Phospholipase C Proteinkinase C k ~ u ~ p h o l i p ~ . j identical to the phospholipase D~s from Arabidopsis, castor bean, maize, and rice. In addition, the ~ and ~ forms Phosphotipid of the protein differ substantially in their isoelectric point (pI) and size 9'11'12'17. The phospholipase D~s from different plant species range in size from 808 to 812 amino acids, whereas phospholipase D~ consists of . . . . . . . . . fPhosphati6ic~___~ Lgsophosphatidic + Fatty D,~.y~y,y~.~u, . . . .... ~ acid ac,d Phosphatidylalcohol 968 amino acids. The a forms in plants all have acidic pIs (around pH 5-6), but the ~ form has a basic pI of 7.9 (Ref. 12). A comparison of the deduced ~ar resp~) amino acid sequences within the Nterminal amino acid sequences of puriFig. 1. The activation of phospholipase D, and pathways taken by phospholipase fled phospholipase D~s reveals the presD-catalyzed reaction products in signal transduetion. The question mark indicates ence of a leader peptide of 30-46 amino a possible pathway that has not yet been shown to occur. Abbreviations: PIP~, phosacids 9''~. This peptide does not display phatidylinositol 4,5-bisphosphate. the typical characteristics of a signal peptide for targeting. Localization studies indicate that phospholipase D~ in cDNA and screening of cDNA libraries 11'12.The names phos- castor bean is associated with the plasma membrane and pholipase Da and phospholipase D~ have been proposed for intracellular membranes including the tonoplast, but it is not the two Arabidopsis proteins based on their sequences and found in association with chloroplasts or mitochondria is. catalytic properties 12''3. The two proteins are the products of The cloning of a castor bean phospholipase D~ facilitated separate genes, and there is evidence for additional phos- the identification ofphospholipase D genes and cDNAs from pholipase D gene(s) in Arabidopsis ~'~2, indicating a multi- yeast 19-21, Caenorhabditis elegans 22 and human 23. The gene family. eukaryotic phospholipase D gene family shares several Studies have also shown the occurrence of multiple common homology domains 24 (Fig. 3). Alignment of these forms of phospholipase D at the protein level. Three vari- sequences reveals two groups, separating the plant proteins ants of the protein from castor bean were resolved by from those from other sources (Fig. 2). Within the plant nondenaturing polyacrylamide gel electrophoresis, isoelec- group, phospholipase D~ forms a subgroup distinct from tric focusing and size exclusion chromatography ~4. Such that of phospholipase Das from Arabidopsis, castor bean, structural heterogeneity has been found in other dicoty- maize and rice. Phylogenetic analysis in the unrooted phyloledon and monocotyledon plants s'~. The appearance of some genies groups the Arabidopsis phospholipase D~ with the variants is associated with specific growth stages and con- yeast and human phospholipase D proteins '2. The phyloditions, suggesting that they may have different cellular genic groupings derived from sequence comparisons are functions. In castor bean and rice, one variant is constitu- supported by comparing the calculated pIs and catalytic tive, whereas the appearance of other variants is associated properties of these proteins. These analyses show that the with specific conditions such as rapid growth, wounding and and ~ forms are evolutionarily divergent, and that the senescences''4'~. Analyses of these variants from castor bean form is more closely related to the proteins cloned from indicated that the catalytic units are the product of the yeast and human than the ~ form is. same gene, that encoding phospholipase D~. The postPhospholipase D cDNAs cloned from different sources all transcriptional and/or post-translational events that lead to contain the HxKxxxD motif, which is found twice within each the formation of these variants have not yet been studied. sequence24(Fig. 3). The duplicated HxKxxxD motifs were also Taken together, these results indicate that isoforms of phos- observed in two other phospholipid-metabolizing enzymes, pholipase D in plants can be derived from different genes, by bacterial phosphatidylserine synthase and cardiolipin synpost-transcriptional or translational modification, and/or a thase 24. The absolute conservation of these amino acids sugcombination of these factors. gests that the His, Lys and Asp residues at these positions are in the active site. The same residues in a homologous protein Comparison and putative catalytic motifs of family also suggest that the proteins might share similar catalytic mechanisms. Of those proteins, the reaction mechanism phospholipase D sequences Complete phospholipase D sequences in plants are avail- of the bacterial phosphatidylserine synthase has been studied able from castor bean 9, Arabidopsis ~'1~, rice and maize 1~. in detail. The analysis has shown that the hydrolysis of the The proteins from castor bean, rice and maize share a high P-O bond occurs via a two-step, 'ping-pong' reaction mechadegree of sequence similarity with phospholipase D~ of nism involving a phosphatidylated enzyme intermediate 24. Stimulus
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reviews Biochemical properties of different phospholipase D proteins
~
Maize
Phospholipase Da is the best-characterized plant phosRice pholipase D, and has been purified from species including cabbage, peanut, soybean, castor bean and rice ~'~-~. Phospholipase D~ is a novel form of the enzyme that has not been isolated in its native state from plants. The presence of ~ar~t/::idboff~i~a-form the ~-form activity was initially documented in transgenic Arabidopsis in which the ~ form was suppressed by antiArabidopsis,~-form sense expressionS% Phospholipase D~ catalyzes the hydrolysis of phosphatidylcholine, phosphatidylethanolamine and Caenorhabditiselegans phosphatidylglycerol, but not phosphatidylinositol and phosphatidylserine TM. Phospholipase D~ also catalyzes the Human cleavage of phosphatidylcholineml", but its activity towards other phospholipids has not been assessed. Yeast Phospholipase De and phospholipase D~ can also catalyze the transfer of the phosphatidyl group to a primary Fig. 2. Comparison of phospholipase D amino acid alcohol to form a phosphatidylalcohol9,m~, a reaction sequences. The diagram shows a dendrogram of clustering referred to as transphosphatidylation (Fig. 1). The producrelationships among sequences from Arabidopsis, castor tion of phosphatidylalcohol has been widely used as an indibean, maize, rice, human, Caenorhabditis elegans and yeast. The amino acid sequences were compared using the cator of phospholipase D activity, because this process is PILEUP program from the University of Wisconsin unique to the enzyme and phosphatidylalcohol undergoes Genetics Computer Group (UWGCG) software package. no further metabolic steps. However, the absence of transphosphatidylation activity has also been reported for both a plant phosphatidylinositol-specific phospholipase D (Ref. 28) and a yeast phospholipase D t h a t prefers The activation ofphospholipase D by polyphosphoinositides is phosphatidylethanolamine and phosphatidylserine to of physiological relevance because, in addition to being an important precursor for second messengers, PIP 2 itself moduphosphatidylcholine~t Although both the ~ and ~ forms of phospholipase D use lates the function of various proteins. In animal tissues, the phosphatidylcholine as a substrate, the conditions for their activation of PIP2-regulated phospholipase D requires active hydrolysis are strikingly different. The distinct property of PIP 2 synthesis 3°. Phosphatidic acid, the lipid product of phosthe ~ form is its requirement for millimolar Ca ~÷ concen- pholipase D hydrolysis, increases PIP 2 synthesis via its abiltrations for optimal activity in vitro ~, and the highest activ- ity to stimulate PIP 5-kinase. A positive feedback loop ity from castor bean occurs at around 50 mM Ca ~÷ (Fig. 4). between phospholipase D and PIP kinase has been proposed; In contrast, the ~ form is fully active at micromolar concen- such a loop would promote a rapid synthesis of phosphatidic trations of Ca ~÷. Phospholipase D~ activity increases at acid and PIP 2. The membrane-fusion property of phospha~idic nanomolar to micromolar Ca 2÷ concentrations, and tapers off at the millimolar level (Fig. 4). The distinct proCalcium/phospholipidCatalytic motifs binding, C2 domain files of Ca2+-dependence between the First HKD Second HKD and ~ forms suggest that changes in ..c-c intracellular Ca 2÷could act as a regulator ~ifferentially to activate the isoSyn-bl forms. However, the exact role of Ca 2÷ Arabidopsis,~-form I T S ~ P Y V S V S V (16) N P V W M Q H F on the function of the enzyme remains D ~T K ~ I L DI to be defined. Arabidopsis,a-form G E T R L Y A T I D L (15) N P K W Y E S F Another major difference between G V S ~ L Y A T I D L (16) N P R W Y E S F Castor bean the a and ~ forms of phospholipase D is n ~
/ \/ \
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1000 E T E '5 E
(z-form / / ~ 800
I~-form
600
C3 O.. O cQ_
400
200
7 0
, -8
-7
-6
-5
-4
-3
-2
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Fig. 4. Effect of Ca ~÷ concentration on phospholipase De and D~ activities. Phospholipases D~ and D~ were expressed in Escherichia coli from the castor bean phospholipase De cDNA and Arabidopsis phospholipases D~ cDNA, respectively, and assayed for phosphatidylcholine hydrolysis in lipid vesicles composed of phosphatidylethanelamine, phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine12'13.
acid would contribute to the fusion of donor and accepter membranes in vesicular trafficking ~°.
The Ca2*/phospholipid-binding C2 domain A unique Ca2÷/phospholipid-binding fold termed the C2 (or CalB) domain was first identified in Ca2+-dependent protein kinase C isoforms 31'32. This domain is present in several different proteins involved in signal transduction and membrane trafficking, including intracellular phospholipase A2 and PIP2-phospholipase C isoforms. The three-dimensional structures of the C2 domains from the neuronal protein synaptotagmin and protein kinase C have recently been resolved by Xray crystallography and nuclear magnetic resonance 32'33.The domains are comprised of an eight-B-sheet-strand sandwich containing 4-5 acidic residues involved in Ca2÷binding. Plant phospholipase D sequences contain a calcium/phospholipid-binding C2 domain near their N-termini 12'31. The structure of the ~ form is more similar to the well-characterized C2 domain of other proteins than that of the form ~2. The two most highly conserved segments in different C2-containing proteins are PYV and NPVFNExF (Ref. 32). These two regions are largely hydrophobic and have been suggested to maintain the structural integrity of the C2 fold. In phospholipase D~, the first segment is completely conserved and the second segment, NPVWMQHF, is largely the same, with some conservative amino acid substitutions (Fig. 3). Furthermore, the ~ form contains the conserved acidic residues that serve to coordinate Ca2÷-binding. In contrast, two of the acidic residues in the ~ form are substituted with positively charged or neutral amino acids ~2 (Fig. 3). The substitutions within the C2 Ca2÷-binding site of phospholipase D~ sequences indicate a loss of affinity for Ca 2÷. This raises the interesting question as to whether differences in the C2 domain underlie the different Ca2÷ 264
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requirements observed for the ~ and ~ forms. As already described, the difference in the amount of Ca 2÷required for activity is one of the most distinct in vitro properties that distinguishes phospholipase Da and D~ (Fig. 4). The finding of a C2 domain provides important insights into the mechanism of cellular activation of phospholipase D in plants. Many C2 domains mediate Ca2+-dependent phospholipid binding 32'33. Such binding could lead to a Ca 2÷dependent intracellular translocation between the cytosol and membranes. Proteins such as protein kinase C and phospholipase A2 are activated when associated with membranes 2. Indeed, activation of plant phospholipase D through intracellular translocation has been proposed in a study involving wound-induced lipid hydrolysis in castor bean 7. The study showed that wounding a leaf at one site induced a transient increase of membrane-associated phospholipase D in the whole leaf and that this membrane association was promoted by calcium. Further analysis of the C2 domain should clarify the role of Ca ~÷in the regulation and catalytic mechanisms of different isoforms ofphospholipase D.
Structure and expression of the phospholipase DoLgene The phospholipase D~ gene of castor bean consists of four exons separated by three introns, one of which is in the 5'untranslated region 1°. It is unclear whether this intron plays a role in regulating the transcription of the gene. The availability of the 5'-flanking region will allow further analysis of cis and trans elements controlling promoter activity and expression. The expression ofphospholipase Da has been investigated in terms of the accumulation of the transcript and protein. The gene is expressed in various tissues, but the levels of expression differ greatly. During seed development, expression is strong in early to middle stages, decreases at late stages and is undetectable in mature dry seeds 17'34. During germination, gene expression of the ~ form is detectable 1 d after imbibition. In seedlings of castor bean, the transcript accumulates to much higher levels in hypocotyls than endosperm ~. In general, the levels of activity, protein and gene expression of phospholipase D~ are high in young and metabolically more active tissues. In addition, the expression of the gene is modulated by phytohormones 1G.Different temporal induction patterns of phospholipase D gene expression were observed in rice leaves undergoing pathogen resistance and susceptibility interactions s. Cellular regulation and manipulation of phospholipase D The cellular control of phospholipase D is complex and involves several mechanisms. The mammalian enzyme is regulated by a variety of factors, including Ca 2÷flux, protein kinase C, G proteins (particularly small G proteins such as ADP-ribosylation factors and Rho), receptor-linked tyrosine kinases, PIP 2 and oleic acidu5 (Fig. 1). Protein phospholipase D inhibitors have also been identified 35. Current data indicate that plant phospholipase D is likely to be regulated by gene expression TM, translocation between membranes and cytosol7, Ca 2÷, PIP 2 (Refs 12 and 13) and G proteins 6. The requirement for different membrane lipids and Ca 2+ concentrations for the activation of different phospholipase D proteins in vitro suggests that these may also be key factors that differentially regulate the isoforms in rive. Most functional studies of phospholipase D in recent years have centered on its participation in transmembrane signaling and vesicular trafficking. The prevalent hypothesis
reviews in mammalian systems is that the enzyme is an integral part of the signaling network, involving various phospholipases, and that its activation initiates and/or potentiates signaling responses 2'3. Its direct lipid product, phosphatidic acid, is a mitogen and can stimulate several signal-transducing enzymes such as PIP2-phospholipase C and protein kinase C (Refs 2,3 and 5). In addition, phosphatidic acid can be further metabolized to diacylglycerol, lysophosphatidic acid and free fatty acids, all of which are biologically active compounds (Fig. 1). In plants, phosphatidic acid can also be converted to diacylglycerolpyrophosphate via phosphatidic acid kinase 36. Activation of plant phospholipase D in vivo occurs in response to treatment with mastoparan and alcohols, or wounding and pathogen attack 6-s. Such activation leads to the synthesis of lipid messengers as a part of physiological responses. For example, phosphatidic acid in Chlamydomonas has been proposed to be a mediator of defiagellation 6. One powerful way to study the cellular function of phospholipase D in an organism is genetically to manipulate its expression. This is exemplified by recent findings in yeast, which have demonstrated that phospholipase D is required for meiosis. Previous genetic analysis established that the gene SP014 is required for sporulation 37. Yeasts with a SP014 mutation are arrested at the second phase of meiosis and fail to sporulate. The sequence similarity between SP014 and the castor bean phospholipase D facilitated the identification of the yeast SP014 gene as a phospholipase D (Refs 19-21). Phospholipase D is now proposed to be essential in yeast meiotic signaling. In plants, expression of the phospholipase De gene was suppressed in Arabidopsis by introducing an antisense cDNA to phospholipase De (Ref. 13). The antisense plants possessed <5% e-form activity in leaves, but retained normal levels of PIPs-dependent, p-form activity. Transgenic tobacco overexpressing the castor bean phospholipase D has also been produced 38. These transgenic plants should prove to be instrumental in resolving the cellular roles of the different forms of the enzyme.
Future prospects Recent studies have established the presence of multiple phospholipase D proteins and genes in plants. The biochemical properties of the proteins suggest that these isoforms are regulated differently and that they may be involved in different cellular processes. Phospholipase De appears to be more prevalent than phospholipase D~ (Ref. 13), and a comparison of its catalytic properties with those of the mammalian and yeast enzymes suggests that it is unique to plants. On the other hand, the ~ form shares some properties with the recently cloned human and yeast phospholipase D proteins. The phospholipase D cloned from yeast is required for meiosis, whereas the cloned human phospholipase D is thought to be involved in membrane trafficking and secretion 4. Whether phospholipase D is involved in reproduction and vesicular trafficking in plants has yet to be assessed. The presence of distinct phospholipase D genes and isoforms raises many interesting questions: • Are the genes expressed differentially in different tissues, cell types and/or growth stages? • Do the isoforms have different intracellular locations, and how is each regulated? • Do the isoforms have different substrate specificities and preferences?
• Are all or some genetically redundant, or does each isoform have unique cellular functions? Ultimately, the issue is the physiological role of phospholipase D in plants. Increasing evidence indicates that it is involved in producing second messengers in various signaling cascades 2-s. It is also possible that it is involved in lipid turnover and membrane remodeling, because phosphatidic acid is a central intermediate in glycerolipid biosynthesis. Plants lacking individual phospholipase D forms will be powerful tools with which to establish the cellular roles of the different isoforms. It may be necessary to generate plants that lack two or more isoforms to resolve functions if some genetic redundancy exists among the multiple phospholipase D genes. The cloning of distinct forms of phospholipase D from Arabidopsis has made it possible to achieve such genetic manipulation in plants.
Acknowledgements Work in the author's lab is supported by grants from the National Science Foundation, US Dept of Agriculture, Pioneer Hi-bred International and the Kansas Board of Agriculture. The author thanks Dr J.E. Leach and K. Pappan for critically reading the manuscript. This is contribution 97-313-J of the Kansas Agricultural Experiment Station. References 1 Paliyath, G. and Droillard, M.J. (1992) The mechanisms of membrane deterioration and disassembly during senescence, Plant Biochem. Physiol. 30, 789-812 2 Exton, J.H. (1994) Phosphatidylcholine breakdown and signal transduction, Biochim. Biophys. Acta 1212, 26-42 3 Natarajan, V., ed. (1996) Phospholipase D and signal transduction in mammalian cells, Chem. Phys. Lipids 80, 1-147 4 Morris, A.J., Engebrecht, A. and Frohman, A.A. (1996) Structure and regulation of phospholipase D, Trends Pharmacol. Sci. 17, 182-185 5 English, D. (1996) Phosphatidic acid: a lipid messenger involved in intracellular and extracellular signalling, Cell. Signal. 8, 341-347 6 Munnik, T. et al. (1995) G protein activation stimulates phospholipase D signaling in plants, Plant Cell 7, 2197-2210 7 Ryu, S.B. and Wang, X. (1996) Activation of phospholipase D and the possible mechanism of activation in wound-induced lipid hydrolysis in castor bean leaves, Biochim. Biophys. Acta 1303, 243-250 8 Young, S.A., Wang, X. and Leach, J.E. (1996) Changes in the plasma membrane distribution of rice phospholipase D during resistant interactions with Xanthomonas oryzae pv oryzae, Plant Cell 8, 1079-1090 9 Wang, X., Xu, L. and Zheng, L. (1994) Cloning and expression of phosphatidylcholine-hydrolyzing phospholipase D from Ricinus communis L., J. Biol. Chem. 269, 20312-20317 10 Xu, L. et al. (1996) Structure and analysis of phospholipase D gene from Ricinus communis L., Plant Mol. Biol. 32, 767-771 11 Dyer, J.H., Zheng, L. and Wang, X. (1995) Cloning and nucleotide sequence of a cDNA encoding phospholipase D from Arabidopsis (Accession No. U36381) (PGR 95-096), Plant Physiol. 109, 1497 12 Pappan, K. et al. (1997) Molecular cloning and functional analysis of polyphosphoinositide-dependent phospholipase D, PLD~, from Arabidopsis. J. Biol. Chem. 272, 7055-7061 13 Pappan, K., Zheng, S. and Wang, Y~(1997) Identification and characterization of a novel phospholipase D that requires polyphosphoinositide and submicromolar calcium for activity in Arabidopsis, J. Biol. Chem. 272, 7048-7054 14 Dyer, J.H., Ryu, S.B. and Wang, X. (1994) Multiple forms of phospholipase D following germination and during leaf development of castor bean, Plant Physiol. 105, 715-724 July 1997, Vol, 2, No, 7
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29 Mayr, J.A., Kohlwein,S.D. and Paltauf, F. (1996) Identification of a novel, Ca2÷-dependentphospholipaseD with preferencefor phosphatidylserine and phosphatidyethanolaminein Saccharemyces cerevisiae, FEBS Lett. 393, 236-240 30 Liscovitch,M. et al. (1995) Phosphatidylinositol4,5-bisphosphate synthesis is required for activation of phospholipaseD in U937 cells, J. Biol. Chem. 270, 5130-5135 31 Ponting, C P. and Parker, P.J. (1996) Extending the C2 domain ihmily: C2s in PKCs ~, e, 0, 0, phospholipases,GAPs and perforin, Protein Sci. 5, 162-166 32 Sutton, R.B. et al. (1995) Stz-actm'eof the first C2 domain of synaptotagmin: a novel Ca2Vphospholipid-bindingfold, Cell 80, 929-938 33 Shao, X. et al. (1996) Bipartite Ca2+-bindingmotif in C2 domains of synaptotagmin and protein kinase C, Science 273, 248-251 34 Ryu, S.B. and Wang, X. (1996) Changes in phospholipaseD expressionin soybeans during seed developmentand germination, J. Am. Oil Chem. Soc. 73, 1171-1176 35 Kim,J.H. et al. (1996) Inhibition of phospholipaseD by a protein factor from bovinebrain cytosol,J. Biol. Chem. 271, 25213-25219 36 Mmmik, T. et al. (1996) Identificationof diacylglycerolpyrophosphate as a novel metabolicproduct of phosphatidic acid during G-protein activation in plants, J. Biol. Chem. 271, 15708-15715 37 Honigberg,S.M., Conicella,C. and Esposito,R.E. (1992) Commitment to meiosis in Saccharamyces cerevisiae: involvementof the SP014 gene, Genetics 130, 703-716 38 Wang, X. et al. (1997) Characterization of phospholipaseD-overexpressed and suppressed transgenic tobaccoand Arabidopsis, in Physiology, Biochemistry, and Molecular Biology of Plant Lipids (Williams,J.P., Khan, M.U. and Lem, N.W., eds), pp. 345-347, Kluwer
Xuemin Wang is at the Dept of Biochemistry, Kansas State University, Manhattan, KS 66506, USA (tel +1 913 532 6422; fax +1 9t3 532 7278; e-mail [email protected]).
Salicylic acid and disease resistance in plants o ,
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Daniel F. K essig Plants have evolved complex mechanisms to defend themselves against pathogens, and thus a great deal of attention has been directed towards elucidating the molecular nature of resistance. Salicylic acid has been s h o w n to be a signaling molecule involved in both local defense reactions at infection sites and the i n d u c t i o n of systemic resistance. Although it is still unclear w h e t h e r this compound can serve as a long-distance messenger signaling the presence of a pathogen, its synthesis and accumulation are important requirements for defense responses. Recent advances have further established the key role of the signal transduction pathways d e p e n d e n t on salicylic acid.
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l a n t s r e a c t to p a t h o g e n a t t a c k by activating an elabor a t e defense m e c h a n i s m t h a t acts both locally a n d systemically. In m a n y cases, local resistance is manifested as a h y p e r s e n s i t i v e response, which is c h a r a c t e r i z e d by the d e v e l o p m e n t of lesions t h a t r e s t r i c t p a t h o g e n growth and/or s p r e a d 1 (Fig. 1). Associated w i t h the h y p e r s e n s i t i v e r e s p o n s e is t h e induction of a diverse group of defenser e l a t e d genes. The p r o d u c t s of m a n y of t h e s e genes p l a y i m p o r t a n t roles in c o n t a i n i n g p a t h o g e n growth, e i t h e r 266
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indirectly, by h e l p i n g to reinforce t h e defense capabilities of h o s t cell walls, or directly, by p r o v i d i n g a n t i m i c r o b i a l e n z y m e s and secondary m e t a b o l i t e s (Fig. 2). These products include cell w a l l polymers, such as lignin a n d suberin, as well as p h e n y l p r o p a n o i d s a n d phytoalexins. Several faroilies of p a t h o g e n e s i s - r e l a t e d (PR) proteins are also induced d u r i n g the h y p e r s e n s i t i v e response. Some of t h e s e proteins a r e hydrolytic enzymes [e.g. ~ - l , 3 - g l u c a n a s e s (PR-2) a n d c h i t i n a s e s (PR-3)], b u t t h e functions of o t h e r PR proteins
© 1997 ElsevierScience Ltd
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reviews 27 Jones, R.L. and Armstrong, J.E. (1971) Evidence for osmotic regulation of hydrolytic enzyme production in germinating barley seeds, Plant Physiol. 48, 137-142 28 Yu, S-M. et aL (1991) Metabolic derepression of a-amylase gene expression in suspension-cultured cells of rice, J. Biol. Chem. 266, 21131-21137 29 Karrer, E.E. and Rodriguez, R.L. (1992) Metabolic regulation of rice a-amylase and sucrose synthase genes inplanta, Plant J. 2, 517-523 30 Huang, N. et al. (1993) Metabolic regulation of a-amylase gene expression in transgenic cell cultures of rice (Oryza sativa L.), Plant Mol. Biol. 23, 737-747 31 Chan, M-T., Chao, Y-C. and Yu, S-M. (1994) Novel gene expression system for plant cells based on induction of a-amylase promoter by carbohydrate starvation, J. Biol. Chem. 269, 17635-17641 32 Mitsunaga, S., Rodriguez, R.L. and Yamaguchi, J. (1994) Sequencespecific interactions of a nuclear protein factor with the promoter region of a rice gene for a-amylase, RAmy3D, Nucleic Acids Res. 22, 1948-1953 33 Mitsui, T., Yotsushima, K. and Nabekura, Y. (1995) A cell biochemical study on sugar-controlled a-amylase secretion in rice, in Current Topics in Plant Physiology (VoL14) (Pontis, H.G., Salerno, G.L. and Echeverria, E.J., eds), pp. 254-265, American Society of Plant Physiologists 34 Bush, D.S. (1995) Calcium regulation in plant cells and its role in signaling, Annu. Rev. Plant Physiol. Plant Mol. Biol. 46, 95-122 35 Gilroy, S. and Jones, R.L. (1992) Gibberellic acid and abscisic acid coordinately regulate cytoplasmic calcium and secretory activity in barley ateurone protoplasts, Proc. Natl. Acad. Sci. U. S. A. 89, 3591-3595
36 Sheu, J-J. et al. (1994) Control of transcription and mRNA turnover as mechanisms of metabolic repression of a-amylase gene expression, Plant J. 5, 655-664 37 Zorec, R. and Tester, M. (1992) Cytoplasmic calcium stimulates exocytosis in a plant secretory cell, Biophys. J. 63, 864-867 38 Akazawa, T. and Miyata, S. (1982) Biosynthesis and secretion of a-amylase and other hydrolases in germinating cereal seeds, Essays Biochem. 18, 40-78 39 Akazawa, T. and Hara-Nishimura, I. (1985) Topographic aspects of biosynthesis, extracellular secretion, and intracellular storage of proteins in plant cells, Annu. Rev. Plant Physiol. 36, 441-472 40 Ranjhan, S., Karrer, E.E. and Rodrignez, R.L. (1992) Localizing a-amylase gene expression in germinated rice grains, Plant Cell Physiol. 33, 73-79 41 Itoh, IQ et al. (1995) Developmental and hormonal regulation of rice a-amylase (RAmylA)-gusA fusion genes in transgenic rice seeds, Plant Physiol. 107, 25-31
Toshiaki Mitsui* is at the Graduate School of Science and Technology, Niigata University, Niigata 950-21, Japan; Kimiko:lt0h is at the Faculty of Agricul!ure, Niigata University, Niigata 950-21 .,,Japan. *Author for correspondence (tel +81 25 262 6641: fax +81 25 263 1659; e-mail [email protected]~).
Molecular analysis of phospholipase D × Phospholipase D has been proposed to play important roles in cellular processes ranging from the generation of second messengers to membrane degradation. Recent studies in plants have revealed the presence of different genes with multiple isoforms. Conserved motifs include those likely to be involved in catalysis and a Ca2÷/phospholipid-binding domain that is potentially responsible for the activation of the enzyme. Two cloned phospholipase D proteins, designated a and 9, display distinct properties in their dependence on Ca~÷and polyphosphoinositides for activity. Thus, isoforms of phospholipase D in plants may be subject to unique control mechanisms and have distinct cellular functions.
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hospholipases are grouped into four major classes phospholipase At, phospholipase A2, phospholipase C and phospholipase D - according to the site of cleavage of phospholipids. Of these, phospholipase D appears to be the most prevalent in plant tissues. It catalyzes the hydrolysis of the terminal phosphodiester bond of glycerophospholipids to generate phosphatidic acid and a hydrophilic free head group, such as choline (Fig. 1). Phospholipase D has been implicated in a broad range of cellular processes. Early studies suggested that increased activity was the first step in a lipolytic cascade in membrane deterioration during senescence, ageing and stress injuries 1. Recent reports indicate that phospholipase D-catalyzed hydrolysis plays a pivotal role in transmembrane signaling and cellular regulation 2'3.The enzyme in mammalian cells has been proposed to mediate many processes, including cell proliferation, receptor-mediated exocytosis, a respiratory burst, actin polymerization and membrane trafficking (Fig. 1). The finding that phosphatidic acid serves as an intracellutar and © 1997 Elsevier Science Ltd
extracellular messenger has brought a flurry of research on the role and regulation of the phospholipase D in signal transduction pathways of animal and yeast systems 2-5. Intriguing observations also suggest that the enzyme may contribute to signaling cascades in plants ~-s. This review summarizes the current understanding of phospholipase D at the molecular level.
Multiple genes and structural heterogeneity of phospholipase D The cDNA for an intracellular phospholipase D in eukaryotes was first cloned from castor bean 9. Southern blot analysis revealed that other homologous genes were also present in the plant genome 1°. The sequence of the castor bean cDNA was used to identify several Arabidopsis expressed-sequence-tagged cDNAs entered on sequence databases as putative, incomplete phospholipase D clones. Two full-length cDNAs have been isolated using a nested polymerase chain reaction, rapid amplification of 5'-end PII $1360-1385(97)01059-5
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reviews Arabidopsis, placing them in the phospholipase Da class (Fig. 2). The phospholipase D~ amino acid sequences of castor bean and Arabidopsis are about Fleceptor 80% identical, and those of monocotyf-.~ ledons are about 90% identical. The G protein l-yrosine kinase Calcium PIPe amino acid sequence of phospholipase D~ from Arabidopsis is about 45-50% Phospholipase C Proteinkinase C k ~ u ~ p h o l i p ~ . j identical to the phospholipase D~s from Arabidopsis, castor bean, maize, and rice. In addition, the ~ and ~ forms Phosphotipid of the protein differ substantially in their isoelectric point (pI) and size 9'11'12'17. The phospholipase D~s from different plant species range in size from 808 to 812 amino acids, whereas phospholipase D~ consists of . . . . . . . . . fPhosphati6ic~___~ Lgsophosphatidic + Fatty D,~.y~y,y~.~u, . . . .... ~ acid ac,d Phosphatidylalcohol 968 amino acids. The a forms in plants all have acidic pIs (around pH 5-6), but the ~ form has a basic pI of 7.9 (Ref. 12). A comparison of the deduced ~ar resp~) amino acid sequences within the Nterminal amino acid sequences of puriFig. 1. The activation of phospholipase D, and pathways taken by phospholipase fled phospholipase D~s reveals the presD-catalyzed reaction products in signal transduetion. The question mark indicates ence of a leader peptide of 30-46 amino a possible pathway that has not yet been shown to occur. Abbreviations: PIP~, phosacids 9''~. This peptide does not display phatidylinositol 4,5-bisphosphate. the typical characteristics of a signal peptide for targeting. Localization studies indicate that phospholipase D~ in cDNA and screening of cDNA libraries 11'12.The names phos- castor bean is associated with the plasma membrane and pholipase Da and phospholipase D~ have been proposed for intracellular membranes including the tonoplast, but it is not the two Arabidopsis proteins based on their sequences and found in association with chloroplasts or mitochondria is. catalytic properties 12''3. The two proteins are the products of The cloning of a castor bean phospholipase D~ facilitated separate genes, and there is evidence for additional phos- the identification ofphospholipase D genes and cDNAs from pholipase D gene(s) in Arabidopsis ~'~2, indicating a multi- yeast 19-21, Caenorhabditis elegans 22 and human 23. The gene family. eukaryotic phospholipase D gene family shares several Studies have also shown the occurrence of multiple common homology domains 24 (Fig. 3). Alignment of these forms of phospholipase D at the protein level. Three vari- sequences reveals two groups, separating the plant proteins ants of the protein from castor bean were resolved by from those from other sources (Fig. 2). Within the plant nondenaturing polyacrylamide gel electrophoresis, isoelec- group, phospholipase D~ forms a subgroup distinct from tric focusing and size exclusion chromatography ~4. Such that of phospholipase Das from Arabidopsis, castor bean, structural heterogeneity has been found in other dicoty- maize and rice. Phylogenetic analysis in the unrooted phyloledon and monocotyledon plants s'~. The appearance of some genies groups the Arabidopsis phospholipase D~ with the variants is associated with specific growth stages and con- yeast and human phospholipase D proteins '2. The phyloditions, suggesting that they may have different cellular genic groupings derived from sequence comparisons are functions. In castor bean and rice, one variant is constitu- supported by comparing the calculated pIs and catalytic tive, whereas the appearance of other variants is associated properties of these proteins. These analyses show that the with specific conditions such as rapid growth, wounding and and ~ forms are evolutionarily divergent, and that the senescences''4'~. Analyses of these variants from castor bean form is more closely related to the proteins cloned from indicated that the catalytic units are the product of the yeast and human than the ~ form is. same gene, that encoding phospholipase D~. The postPhospholipase D cDNAs cloned from different sources all transcriptional and/or post-translational events that lead to contain the HxKxxxD motif, which is found twice within each the formation of these variants have not yet been studied. sequence24(Fig. 3). The duplicated HxKxxxD motifs were also Taken together, these results indicate that isoforms of phos- observed in two other phospholipid-metabolizing enzymes, pholipase D in plants can be derived from different genes, by bacterial phosphatidylserine synthase and cardiolipin synpost-transcriptional or translational modification, and/or a thase 24. The absolute conservation of these amino acids sugcombination of these factors. gests that the His, Lys and Asp residues at these positions are in the active site. The same residues in a homologous protein Comparison and putative catalytic motifs of family also suggest that the proteins might share similar catalytic mechanisms. Of those proteins, the reaction mechanism phospholipase D sequences Complete phospholipase D sequences in plants are avail- of the bacterial phosphatidylserine synthase has been studied able from castor bean 9, Arabidopsis ~'1~, rice and maize 1~. in detail. The analysis has shown that the hydrolysis of the The proteins from castor bean, rice and maize share a high P-O bond occurs via a two-step, 'ping-pong' reaction mechadegree of sequence similarity with phospholipase D~ of nism involving a phosphatidylated enzyme intermediate 24. Stimulus
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reviews Biochemical properties of different phospholipase D proteins
~
Maize
Phospholipase Da is the best-characterized plant phosRice pholipase D, and has been purified from species including cabbage, peanut, soybean, castor bean and rice ~'~-~. Phospholipase D~ is a novel form of the enzyme that has not been isolated in its native state from plants. The presence of ~ar~t/::idboff~i~a-form the ~-form activity was initially documented in transgenic Arabidopsis in which the ~ form was suppressed by antiArabidopsis,~-form sense expressionS% Phospholipase D~ catalyzes the hydrolysis of phosphatidylcholine, phosphatidylethanolamine and Caenorhabditiselegans phosphatidylglycerol, but not phosphatidylinositol and phosphatidylserine TM. Phospholipase D~ also catalyzes the Human cleavage of phosphatidylcholineml", but its activity towards other phospholipids has not been assessed. Yeast Phospholipase De and phospholipase D~ can also catalyze the transfer of the phosphatidyl group to a primary Fig. 2. Comparison of phospholipase D amino acid alcohol to form a phosphatidylalcohol9,m~, a reaction sequences. The diagram shows a dendrogram of clustering referred to as transphosphatidylation (Fig. 1). The producrelationships among sequences from Arabidopsis, castor tion of phosphatidylalcohol has been widely used as an indibean, maize, rice, human, Caenorhabditis elegans and yeast. The amino acid sequences were compared using the cator of phospholipase D activity, because this process is PILEUP program from the University of Wisconsin unique to the enzyme and phosphatidylalcohol undergoes Genetics Computer Group (UWGCG) software package. no further metabolic steps. However, the absence of transphosphatidylation activity has also been reported for both a plant phosphatidylinositol-specific phospholipase D (Ref. 28) and a yeast phospholipase D t h a t prefers The activation ofphospholipase D by polyphosphoinositides is phosphatidylethanolamine and phosphatidylserine to of physiological relevance because, in addition to being an important precursor for second messengers, PIP 2 itself moduphosphatidylcholine~t Although both the ~ and ~ forms of phospholipase D use lates the function of various proteins. In animal tissues, the phosphatidylcholine as a substrate, the conditions for their activation of PIP2-regulated phospholipase D requires active hydrolysis are strikingly different. The distinct property of PIP 2 synthesis 3°. Phosphatidic acid, the lipid product of phosthe ~ form is its requirement for millimolar Ca ~÷ concen- pholipase D hydrolysis, increases PIP 2 synthesis via its abiltrations for optimal activity in vitro ~, and the highest activ- ity to stimulate PIP 5-kinase. A positive feedback loop ity from castor bean occurs at around 50 mM Ca ~÷ (Fig. 4). between phospholipase D and PIP kinase has been proposed; In contrast, the ~ form is fully active at micromolar concen- such a loop would promote a rapid synthesis of phosphatidic trations of Ca ~÷. Phospholipase D~ activity increases at acid and PIP 2. The membrane-fusion property of phospha~idic nanomolar to micromolar Ca 2÷ concentrations, and tapers off at the millimolar level (Fig. 4). The distinct proCalcium/phospholipidCatalytic motifs binding, C2 domain files of Ca2+-dependence between the First HKD Second HKD and ~ forms suggest that changes in ..c-c intracellular Ca 2÷could act as a regulator ~ifferentially to activate the isoSyn-bl forms. However, the exact role of Ca 2÷ Arabidopsis,~-form I T S ~ P Y V S V S V (16) N P V W M Q H F on the function of the enzyme remains D ~T K ~ I L DI to be defined. Arabidopsis,a-form G E T R L Y A T I D L (15) N P K W Y E S F Another major difference between G V S ~ L Y A T I D L (16) N P R W Y E S F Castor bean the a and ~ forms of phospholipase D is n ~
/ \/ \
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1000 E T E '5 E
(z-form / / ~ 800
I~-form
600
C3 O.. O cQ_
400
200
7 0
, -8
-7
-6
-5
-4
-3
-2
Log [Ca2+] (M)
Fig. 4. Effect of Ca ~÷ concentration on phospholipase De and D~ activities. Phospholipases D~ and D~ were expressed in Escherichia coli from the castor bean phospholipase De cDNA and Arabidopsis phospholipases D~ cDNA, respectively, and assayed for phosphatidylcholine hydrolysis in lipid vesicles composed of phosphatidylethanelamine, phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine12'13.
acid would contribute to the fusion of donor and accepter membranes in vesicular trafficking ~°.
The Ca2*/phospholipid-binding C2 domain A unique Ca2÷/phospholipid-binding fold termed the C2 (or CalB) domain was first identified in Ca2+-dependent protein kinase C isoforms 31'32. This domain is present in several different proteins involved in signal transduction and membrane trafficking, including intracellular phospholipase A2 and PIP2-phospholipase C isoforms. The three-dimensional structures of the C2 domains from the neuronal protein synaptotagmin and protein kinase C have recently been resolved by Xray crystallography and nuclear magnetic resonance 32'33.The domains are comprised of an eight-B-sheet-strand sandwich containing 4-5 acidic residues involved in Ca2÷binding. Plant phospholipase D sequences contain a calcium/phospholipid-binding C2 domain near their N-termini 12'31. The structure of the ~ form is more similar to the well-characterized C2 domain of other proteins than that of the form ~2. The two most highly conserved segments in different C2-containing proteins are PYV and NPVFNExF (Ref. 32). These two regions are largely hydrophobic and have been suggested to maintain the structural integrity of the C2 fold. In phospholipase D~, the first segment is completely conserved and the second segment, NPVWMQHF, is largely the same, with some conservative amino acid substitutions (Fig. 3). Furthermore, the ~ form contains the conserved acidic residues that serve to coordinate Ca2÷-binding. In contrast, two of the acidic residues in the ~ form are substituted with positively charged or neutral amino acids ~2 (Fig. 3). The substitutions within the C2 Ca2÷-binding site of phospholipase D~ sequences indicate a loss of affinity for Ca 2÷. This raises the interesting question as to whether differences in the C2 domain underlie the different Ca2÷ 264
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requirements observed for the ~ and ~ forms. As already described, the difference in the amount of Ca 2÷required for activity is one of the most distinct in vitro properties that distinguishes phospholipase Da and D~ (Fig. 4). The finding of a C2 domain provides important insights into the mechanism of cellular activation of phospholipase D in plants. Many C2 domains mediate Ca2+-dependent phospholipid binding 32'33. Such binding could lead to a Ca 2÷dependent intracellular translocation between the cytosol and membranes. Proteins such as protein kinase C and phospholipase A2 are activated when associated with membranes 2. Indeed, activation of plant phospholipase D through intracellular translocation has been proposed in a study involving wound-induced lipid hydrolysis in castor bean 7. The study showed that wounding a leaf at one site induced a transient increase of membrane-associated phospholipase D in the whole leaf and that this membrane association was promoted by calcium. Further analysis of the C2 domain should clarify the role of Ca ~÷in the regulation and catalytic mechanisms of different isoforms ofphospholipase D.
Structure and expression of the phospholipase DoLgene The phospholipase D~ gene of castor bean consists of four exons separated by three introns, one of which is in the 5'untranslated region 1°. It is unclear whether this intron plays a role in regulating the transcription of the gene. The availability of the 5'-flanking region will allow further analysis of cis and trans elements controlling promoter activity and expression. The expression ofphospholipase Da has been investigated in terms of the accumulation of the transcript and protein. The gene is expressed in various tissues, but the levels of expression differ greatly. During seed development, expression is strong in early to middle stages, decreases at late stages and is undetectable in mature dry seeds 17'34. During germination, gene expression of the ~ form is detectable 1 d after imbibition. In seedlings of castor bean, the transcript accumulates to much higher levels in hypocotyls than endosperm ~. In general, the levels of activity, protein and gene expression of phospholipase D~ are high in young and metabolically more active tissues. In addition, the expression of the gene is modulated by phytohormones 1G.Different temporal induction patterns of phospholipase D gene expression were observed in rice leaves undergoing pathogen resistance and susceptibility interactions s. Cellular regulation and manipulation of phospholipase D The cellular control of phospholipase D is complex and involves several mechanisms. The mammalian enzyme is regulated by a variety of factors, including Ca 2÷flux, protein kinase C, G proteins (particularly small G proteins such as ADP-ribosylation factors and Rho), receptor-linked tyrosine kinases, PIP 2 and oleic acidu5 (Fig. 1). Protein phospholipase D inhibitors have also been identified 35. Current data indicate that plant phospholipase D is likely to be regulated by gene expression TM, translocation between membranes and cytosol7, Ca 2÷, PIP 2 (Refs 12 and 13) and G proteins 6. The requirement for different membrane lipids and Ca 2+ concentrations for the activation of different phospholipase D proteins in vitro suggests that these may also be key factors that differentially regulate the isoforms in rive. Most functional studies of phospholipase D in recent years have centered on its participation in transmembrane signaling and vesicular trafficking. The prevalent hypothesis
reviews in mammalian systems is that the enzyme is an integral part of the signaling network, involving various phospholipases, and that its activation initiates and/or potentiates signaling responses 2'3. Its direct lipid product, phosphatidic acid, is a mitogen and can stimulate several signal-transducing enzymes such as PIP2-phospholipase C and protein kinase C (Refs 2,3 and 5). In addition, phosphatidic acid can be further metabolized to diacylglycerol, lysophosphatidic acid and free fatty acids, all of which are biologically active compounds (Fig. 1). In plants, phosphatidic acid can also be converted to diacylglycerolpyrophosphate via phosphatidic acid kinase 36. Activation of plant phospholipase D in vivo occurs in response to treatment with mastoparan and alcohols, or wounding and pathogen attack 6-s. Such activation leads to the synthesis of lipid messengers as a part of physiological responses. For example, phosphatidic acid in Chlamydomonas has been proposed to be a mediator of defiagellation 6. One powerful way to study the cellular function of phospholipase D in an organism is genetically to manipulate its expression. This is exemplified by recent findings in yeast, which have demonstrated that phospholipase D is required for meiosis. Previous genetic analysis established that the gene SP014 is required for sporulation 37. Yeasts with a SP014 mutation are arrested at the second phase of meiosis and fail to sporulate. The sequence similarity between SP014 and the castor bean phospholipase D facilitated the identification of the yeast SP014 gene as a phospholipase D (Refs 19-21). Phospholipase D is now proposed to be essential in yeast meiotic signaling. In plants, expression of the phospholipase De gene was suppressed in Arabidopsis by introducing an antisense cDNA to phospholipase De (Ref. 13). The antisense plants possessed <5% e-form activity in leaves, but retained normal levels of PIPs-dependent, p-form activity. Transgenic tobacco overexpressing the castor bean phospholipase D has also been produced 38. These transgenic plants should prove to be instrumental in resolving the cellular roles of the different forms of the enzyme.
Future prospects Recent studies have established the presence of multiple phospholipase D proteins and genes in plants. The biochemical properties of the proteins suggest that these isoforms are regulated differently and that they may be involved in different cellular processes. Phospholipase De appears to be more prevalent than phospholipase D~ (Ref. 13), and a comparison of its catalytic properties with those of the mammalian and yeast enzymes suggests that it is unique to plants. On the other hand, the ~ form shares some properties with the recently cloned human and yeast phospholipase D proteins. The phospholipase D cloned from yeast is required for meiosis, whereas the cloned human phospholipase D is thought to be involved in membrane trafficking and secretion 4. Whether phospholipase D is involved in reproduction and vesicular trafficking in plants has yet to be assessed. The presence of distinct phospholipase D genes and isoforms raises many interesting questions: • Are the genes expressed differentially in different tissues, cell types and/or growth stages? • Do the isoforms have different intracellular locations, and how is each regulated? • Do the isoforms have different substrate specificities and preferences?
• Are all or some genetically redundant, or does each isoform have unique cellular functions? Ultimately, the issue is the physiological role of phospholipase D in plants. Increasing evidence indicates that it is involved in producing second messengers in various signaling cascades 2-s. It is also possible that it is involved in lipid turnover and membrane remodeling, because phosphatidic acid is a central intermediate in glycerolipid biosynthesis. Plants lacking individual phospholipase D forms will be powerful tools with which to establish the cellular roles of the different isoforms. It may be necessary to generate plants that lack two or more isoforms to resolve functions if some genetic redundancy exists among the multiple phospholipase D genes. The cloning of distinct forms of phospholipase D from Arabidopsis has made it possible to achieve such genetic manipulation in plants.
Acknowledgements Work in the author's lab is supported by grants from the National Science Foundation, US Dept of Agriculture, Pioneer Hi-bred International and the Kansas Board of Agriculture. The author thanks Dr J.E. Leach and K. Pappan for critically reading the manuscript. This is contribution 97-313-J of the Kansas Agricultural Experiment Station. References 1 Paliyath, G. and Droillard, M.J. (1992) The mechanisms of membrane deterioration and disassembly during senescence, Plant Biochem. Physiol. 30, 789-812 2 Exton, J.H. (1994) Phosphatidylcholine breakdown and signal transduction, Biochim. Biophys. Acta 1212, 26-42 3 Natarajan, V., ed. (1996) Phospholipase D and signal transduction in mammalian cells, Chem. Phys. Lipids 80, 1-147 4 Morris, A.J., Engebrecht, A. and Frohman, A.A. (1996) Structure and regulation of phospholipase D, Trends Pharmacol. Sci. 17, 182-185 5 English, D. (1996) Phosphatidic acid: a lipid messenger involved in intracellular and extracellular signalling, Cell. Signal. 8, 341-347 6 Munnik, T. et al. (1995) G protein activation stimulates phospholipase D signaling in plants, Plant Cell 7, 2197-2210 7 Ryu, S.B. and Wang, X. (1996) Activation of phospholipase D and the possible mechanism of activation in wound-induced lipid hydrolysis in castor bean leaves, Biochim. Biophys. Acta 1303, 243-250 8 Young, S.A., Wang, X. and Leach, J.E. (1996) Changes in the plasma membrane distribution of rice phospholipase D during resistant interactions with Xanthomonas oryzae pv oryzae, Plant Cell 8, 1079-1090 9 Wang, X., Xu, L. and Zheng, L. (1994) Cloning and expression of phosphatidylcholine-hydrolyzing phospholipase D from Ricinus communis L., J. Biol. Chem. 269, 20312-20317 10 Xu, L. et al. (1996) Structure and analysis of phospholipase D gene from Ricinus communis L., Plant Mol. Biol. 32, 767-771 11 Dyer, J.H., Zheng, L. and Wang, X. (1995) Cloning and nucleotide sequence of a cDNA encoding phospholipase D from Arabidopsis (Accession No. U36381) (PGR 95-096), Plant Physiol. 109, 1497 12 Pappan, K. et al. (1997) Molecular cloning and functional analysis of polyphosphoinositide-dependent phospholipase D, PLD~, from Arabidopsis. J. Biol. Chem. 272, 7055-7061 13 Pappan, K., Zheng, S. and Wang, Y~(1997) Identification and characterization of a novel phospholipase D that requires polyphosphoinositide and submicromolar calcium for activity in Arabidopsis, J. Biol. Chem. 272, 7048-7054 14 Dyer, J.H., Ryu, S.B. and Wang, X. (1994) Multiple forms of phospholipase D following germination and during leaf development of castor bean, Plant Physiol. 105, 715-724 July 1997, Vol, 2, No, 7
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29 Mayr, J.A., Kohlwein,S.D. and Paltauf, F. (1996) Identification of a novel, Ca2÷-dependentphospholipaseD with preferencefor phosphatidylserine and phosphatidyethanolaminein Saccharemyces cerevisiae, FEBS Lett. 393, 236-240 30 Liscovitch,M. et al. (1995) Phosphatidylinositol4,5-bisphosphate synthesis is required for activation of phospholipaseD in U937 cells, J. Biol. Chem. 270, 5130-5135 31 Ponting, C P. and Parker, P.J. (1996) Extending the C2 domain ihmily: C2s in PKCs ~, e, 0, 0, phospholipases,GAPs and perforin, Protein Sci. 5, 162-166 32 Sutton, R.B. et al. (1995) Stz-actm'eof the first C2 domain of synaptotagmin: a novel Ca2Vphospholipid-bindingfold, Cell 80, 929-938 33 Shao, X. et al. (1996) Bipartite Ca2+-bindingmotif in C2 domains of synaptotagmin and protein kinase C, Science 273, 248-251 34 Ryu, S.B. and Wang, X. (1996) Changes in phospholipaseD expressionin soybeans during seed developmentand germination, J. Am. Oil Chem. Soc. 73, 1171-1176 35 Kim,J.H. et al. (1996) Inhibition of phospholipaseD by a protein factor from bovinebrain cytosol,J. Biol. Chem. 271, 25213-25219 36 Mmmik, T. et al. (1996) Identificationof diacylglycerolpyrophosphate as a novel metabolicproduct of phosphatidic acid during G-protein activation in plants, J. Biol. Chem. 271, 15708-15715 37 Honigberg,S.M., Conicella,C. and Esposito,R.E. (1992) Commitment to meiosis in Saccharamyces cerevisiae: involvementof the SP014 gene, Genetics 130, 703-716 38 Wang, X. et al. (1997) Characterization of phospholipaseD-overexpressed and suppressed transgenic tobaccoand Arabidopsis, in Physiology, Biochemistry, and Molecular Biology of Plant Lipids (Williams,J.P., Khan, M.U. and Lem, N.W., eds), pp. 345-347, Kluwer
Xuemin Wang is at the Dept of Biochemistry, Kansas State University, Manhattan, KS 66506, USA (tel +1 913 532 6422; fax +1 9t3 532 7278; e-mail [email protected]).
Salicylic acid and disease resistance in plants o ,
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Daniel F. K essig Plants have evolved complex mechanisms to defend themselves against pathogens, and thus a great deal of attention has been directed towards elucidating the molecular nature of resistance. Salicylic acid has been s h o w n to be a signaling molecule involved in both local defense reactions at infection sites and the i n d u c t i o n of systemic resistance. Although it is still unclear w h e t h e r this compound can serve as a long-distance messenger signaling the presence of a pathogen, its synthesis and accumulation are important requirements for defense responses. Recent advances have further established the key role of the signal transduction pathways d e p e n d e n t on salicylic acid.
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l a n t s r e a c t to p a t h o g e n a t t a c k by activating an elabor a t e defense m e c h a n i s m t h a t acts both locally a n d systemically. In m a n y cases, local resistance is manifested as a h y p e r s e n s i t i v e response, which is c h a r a c t e r i z e d by the d e v e l o p m e n t of lesions t h a t r e s t r i c t p a t h o g e n growth and/or s p r e a d 1 (Fig. 1). Associated w i t h the h y p e r s e n s i t i v e r e s p o n s e is t h e induction of a diverse group of defenser e l a t e d genes. The p r o d u c t s of m a n y of t h e s e genes p l a y i m p o r t a n t roles in c o n t a i n i n g p a t h o g e n growth, e i t h e r 266
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indirectly, by h e l p i n g to reinforce t h e defense capabilities of h o s t cell walls, or directly, by p r o v i d i n g a n t i m i c r o b i a l e n z y m e s and secondary m e t a b o l i t e s (Fig. 2). These products include cell w a l l polymers, such as lignin a n d suberin, as well as p h e n y l p r o p a n o i d s a n d phytoalexins. Several faroilies of p a t h o g e n e s i s - r e l a t e d (PR) proteins are also induced d u r i n g the h y p e r s e n s i t i v e response. Some of t h e s e proteins a r e hydrolytic enzymes [e.g. ~ - l , 3 - g l u c a n a s e s (PR-2) a n d c h i t i n a s e s (PR-3)], b u t t h e functions of o t h e r PR proteins
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