Defense-related proteins in higher plants.

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DEFENSE-RELATED PROTEINS IN

Annu. Rev. Biochem. 1990.59:873-907. Downloaded from www.annualreviews.org Access provided by East Carolina University on 01/12/18. For personal use only.

HIGH:ER PLANTS Dianna J. Bowles Department of Biochemistry, University of Leeds, United Kingdom KEY WORDS:

plant defense gene regulation, plant-pathogen interactions, local and systemic cell signaling, protein biosynthesis and processing, endomembrane traffic and targetting.

CONTENTS 1. 1ntrodllction .................................................................................. . . 2. Activation of Defense Responses ......................................................... . . 3. Classes of Defense-Related Proteins ... . . ......... . . . .. . . . . . . . . . . . . . .. . . ... .... . . . . . . .. . . .

" ..

4.

""

Core enzymes of phenylpropanoid metabolism ........ . . . . . . . . . . . . . . . . . . . . . . . . . Proteins that Change the Properties of the Extracellular Matrix .................. Extensins: hydroxyproline-rich glycoproteins ..............................

.

,

Glycine-rich proteins ....................................................................... .

Peroxidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cinnamyl alcohol dehydrogenase . . . . . .. .. . . . . . . .. . ... . .. ...

....... .... . .... ....... ...

... .

5.

6.

Callose synthetase............................. .............................................. . Proteins Associated with Deterrence and Antimicrobial Activif)l . .................. . . Endohydrolases ........ .. ....... .... h o n ns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . li . Late enzymes of phytoalexin biosynthesis............................................... . Additional Defense-Related Proteins .........................................." . . . . . . . . . . Pathogenesis-related proteins .......... ........ . . . . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . . . . Proteins encoded by wun 1 and wun 2................................................ . . Proteins encoded by win 1 and win 2 ...................................................

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7. 8.

Elicitor-induced products . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Coordination of Defense Responses """""""" . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . Concluding Remarks... . .. .. . . . ..

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1. Introduction Plants are sedentary organisms. Their need to obtain the full spectrum of

nutrients from the environment has led to a maximization of surface area to 873

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absorb the raw materials . These extensive aerial and subterranean boundaries with the environment are vulnerable to pathogens and can be readily affected by adverse conditions. Survival in a changing environment necessitates rapid responses to external stimuli , whether the signals arise from other organisms and predators, or from conditions such as water-logging, high temperatures, and drought . Plants use these environmental signals as developmental signals. One consequence is that plant development is plastic: developmental regula tion is not restricted to the embryonic and juvenile stages of the life cycle, but is a continuing process intimately associated with growth. Recent evidence has established that many of the plant genes expressed naturally at specific times of vegetative and reproductive growth can also be activated at other times elsewhere in the plant in response to adverse environ mental stimuli. Sometimes, it is the same gene that responds to these de velopmental and environmental signals; at other times, closely related genes within a multigene family are expressed . Two stages in the plant' s life cycle associated with reproductive capacity are highly protected. Thus, organs of storage and regenerative capacity , such as tubers of the potato, contain a diverse array of deterrents and toxic proteins . Similarly, the seeds of flowering plants provide a means of nutritional support and protection for the embryo, but represent a highly vulnerable stage be tween generations. Desiccation is one mechanism to resist adverse conditions , but seeds also contain many defense-related proteins t o counter attack b y pathogens and deter pests and herbivores. Many o f the proteins that accumu late naturally in storage organs and seeds are those that are induced to accumulate elsewhere during a defense response . Given the requirement to protect large surface areas , a strategy has evolved in which the ability to distinguish foreign from self resides in cells throughout the organism. One consequence of these recognition events can be the elicita tion of a defense response. Plant cells are surrounded by walls: a dynamic system of polymers that both reflects and determines the differentiation of the protoplast they encompass. This extracellular matrix is continuous with a system of intercellular air spaces and with the xylem. In total, this space is termed the apoplast and is known to perform a central role in the defense strategy of the organism, since it both provides a continuous network within the plant body and is sited at the interface with the environment. Pathogens that intend to colonize an intact plant must enter via the apoplast. Many pathogens use the apoplast as an internal long-distance transport route and others never leave it. The apoplast is now recognized to be both the site at which signals originate to elicit defense responses and the site at which many defense-related products accumulate. The apoplastic surface of the dermal tissue system in direct contact with the environment is waterproofed to act as a diffusion barrier. Physical injury leads


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to a wound response that involves reestablishment of the diffusion barrier, repair of the damage , and stimulation of new growth and differentiation . This has to be a rapid and coordinated process in the local region of injury, since wound sites are known to be important entry points for many pathogens, particularly viruses. However, through research on defense responses, it is now recognized that local events in the immediate zone of injury or pathogen invasion aliso trigger systemic events. These systemic events can be extremely rapid and generally involve a massive amplification in the response, since local changes occurring within one cell or small group of cells produce systemic changes throughout an entire organ system(s) . Some classes of defense-related products accumulate only at the local sites, whereas others accumulate locally and systemically. There is good evidence that both local and systemic responses involve the coordinate regulation of gene expression: i.e. different defense genes are activated simultaneously within the same cell . Sometimes these encode a set of enzymes that function within a specific metabolic pathway , but genes encoding structurally and functionally diverse products can also be activated. Comparison of the kinetics of local expression of defense genes in susceptible and resistant plant cultivars suggests that a major determinant of success in a resistance strategy lies in its speed. But there is also evidence that different classes of genes, even in a resistance strategy, are expressed sequentially and the chronology of expression may be common to many pathogens invading a diverse array of plant species. An intensive research effort has focussed on the study of local responses. By contrast, systemic responses are only just beginning to be investigated at the molecular level, but they are already known to underlie the phenomenon of acquired resistance: the means by which an interaction with one pathogen/symbiont can influence the outcome of a subsequent challenge by the same or a different organism. Many of the specialized areas within molecular plant pathology have been extensively reviewed over recent years. The reader will be directed to these reviews at relevant points in the text. The purpose of this review is to integrate areas of research that have not been discussed in the same context previously, to provide an update of information, and to introduce an exciting and fast moving field to plant biochemists not directly involved in the area and those of other disciplines who have limited experience of working with plants. 2. Activation of Defense Responses

During a naturally occurring defense response, the primary stimulus will be a second organism or some form of stress . There is insufficient space in this review to consider the whole spectrum of responses to environmental stress . Instead, the reader is referred to reviews ( l , 2) for the heat-shock response, (3) for the anaerobic response, and (4 and refs therein) for an introduction to a


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class of proteins that may function in water stress . Since the plant's response to physical injury per se shares many features with the responses to a second organism, wound-activated genes and their products will be considered here. In plant-pathogen interactions, it is often the case that different host cultivars exhibit specific responses to different physiological races of the pathogen . From genetic analyses of these cultivar race-specific interactions, it has become apparent that one gene in the host for resistance is complemented by a single gene in the pathogen for avirulence (reviewed, 5). This 'gene-for gene' hypothesis holds true irrespective of the number of loci in the host that confer resistance . In many cases , an incompatible interaction is characterized by the hypersensitive re�ponse (HR) involving rapid death of the cell(s) in the immediate vicinity of the pathogen and the triggering of a wide range of inducible defense genes. This response is now recognized to be widespread, occurring as an expression of incompatibility between host plants and pathogens whether the second organism is fungal, bacterial, viral, or a nematode. One of the most studied phenomena during the expression of localized resistance, is the accumulation of low-molecular-weight anti microbial compounds known as phytoalexins. The induction of enzymes involved in the synthesis of these antibiotics, and the genes encoding these

enzymes, has provided a useful system for defining the sequence of events that lead from the initial recognition event to transcription of the inducible genes (reviewed, 6). From these studies it has become apparent that the system can be activated at any number of levels from application of a pathogen through to in vitro induction of a defined step in the transduction pathway linking the primary stimuli to changes in gene expression. Considerable information has arisen from the use of elicitors. Some of these elicitors that activate plant defense genes are race specific, i . e . they mimic the gene-for-gene response in being able to induce a response only in host cultivars on which that race of pathogen is avirulent. These are however the exception, and most are race nonspecific. Elicitor activity was once defined mainly in the context of a bioassay leading to phytoalexin accumula tion (reviewed, 7). Now the term is used much more broadly to include the activation of defense genes such as those encoding hydrolytic enzymes, structural proteins of the wall, and enzyme inhibitors, etc. Work from Alber sheim's lab and others (reviewed, 8 , 9) led to the awareness that fragments of structural polysaccharides could act as elicitors. Thus, phytoalexin accumula tion could be induced in soybean cotyledons by the application of a highly defined p-heptaglucoside derived from hydrolysis of wall polymers of the fungus Phytophthora infestans f.s.p. glycinea (10). But, similar induction could also arise by application of a highly defined oligogalacturonide derived from hydrolysis of a plant cell wall pectin (1\). These and similar findings (12, 13), as well as knowledge of the sequential activation of both plant and pathogen hydrolases during an interaction in vivo



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(reviewed, 1 4) , led to the notion of exogenous and endogenous elicitors: the former arising from hydrolysis of pathogen components by plant enzymes, the latter from hydrolysis of plant components by pathogen enzymes. In parallel to these studies, cell wall fragments were also discovered to play a role in the wound response of the Solanaceae, when it was shown that one of the active components of a total leaf tissue hydrolysate (PIIF) was a fragment of pectin (9, 15). Since the wound response does not involve a second organism, the results implied that release of bioactive fragments can be caused by 'self' hydrolases (such as those released into the wall from the vacuole on cellular injury and consequent loss of compartmentalization/turgor) . Current! y, many different elicitors and combinations are in use to study defense responses (reviewed , 6). These range from culture filtrates to defined enzymes, from PlIF and cell wall hydrolysates to highly characterized pep tides and oligosaccharides. Unfortunately, due to the specific interests of different n:search groups, the full spectrum of 'defense responses' evoked by any single defined elicitor has not yet been studied. In addition to elicitors derived from glycoconjugates and proteins, many additional compounds are known to activate defense genes. These include salicylic and related hydroxy-benzoic acids (reviewed, 1 6 , 17) H202 (released on elicitor treatment, 18), glutathione ( 1 9 , 20) , arachidonic acid (21) , and vanadate (22, 23). Their relevance in a defense response in vivo is as yet unknown, but it is envisaged that they may represent specific links in , mimic, or interfere with the signal transduction pathway , and as such can bypass the initial recognition events in the same way as calcium!A23 1 87 and phorbol esters have: been shown to be effective in soybean and carrot, respectively (24, 25). It is also increasingly apparent that the classical growth regulators may be involved at some causal level in defense-related signaling, in particu lar, ethylene and abscisic acid (reviewed 26, 27) . In the following discussion on defense-related proteins, reference is made to the use of one or many of the above means of studying a particular 'defense response. ' 3. Classes of Defense-Related Proteins

Defense-related proteins will be divided for ease of discussion into three classes bas.ed on their role in defense responses. The first class involves products that directly change the properties of the extracellular matrix and thereby affect the defense status of the plant by strengthening , repairing , or altering the wall environment. Proteins within this class include the structural proteins: hydroxyproline-rich glycoproteins (HRGPs) and glycine-rich pro teins (GRP:;;) , as well as the wide range of enzymes involved in the construc tion and/or modification of other wall polymers including suberin, lignin, wall-bound phenolics, and callose.


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The second class involves defense-related proteins that act directly as deterrents, exhibit antimicrobial activities , or catalyze the synthesis of anti microbial products. These include proteins that function as enzyme inhibitors , such as amylase(s) and proteinase inhibitors; toxic proteins, such as lectins and thionins; hydrolases such as chitinases, /31 ,3 glucanases, and proteinases; and enzymes involved in the synthesis of oxidized phenolics, tannins, 0quinones, and the low-molecular-weight antimicrobial compounds: phyto alexins. The third class involves those proteins whose appearance can be correlated with a defense response, but which are of unknown function. Examples include members of the family of proteins defined initially as 'pathogenesis related' (PR), proteins such as the products of the genes wun 1 and win I that accumulate in a wound response, and examples of novel products that occur in response to elicitor treatment. A function has recently been assigned to certain PR-proteins that have been identified as /3l , 3-glucanases and chiti nases. In these examples, the products will be discussed within the appropri ate classes. In a defense situation in vivo , a coordinated response is generally induced such that an array of products accumulate . The integration of communication networks leading to defined temporal and spatial patterns of defense gene expression is considered in a later section . Key enzymes that catalyze core reactions in phenylpropanoid metabolism will be described before discussion of the different classes of proteins. This is necessary since the reactions are common to a range of pathways leading to functionally diverse defense-related products. The area is well reviewed (28, 29), and only a summary will be given here to provide a framework for later discussions. As described in (28), the various branch pathways leading to products such as lignin , suberin , wall-bound phenolics, flavonoids, etc, derive their building units from three core reactions. These reactions are fundamental to processes in normal growth and development as well as in defense responses. Accord ingly , the regulation of, particularly the first enzyme, the entry point into all of these different pathways, is complex. This enzyme, phenylalanine ammo nia lyase (PAL), catalyzes the deamination of L-phenylalanine to yield trans cinnamic acid and NHt. CORE ENZYMES OF PHENYLPROPANOID METABOLISM

PAL Details of the enzyme have come primarily from studies on parsley, bean, and potato (6, 28) , but PAL, purified from bean, is the best character ized; the enzyme is known to be a tetramer, and contains two active sites per molecule, with a subunit Mr of 77000 (30, 3 1 ). PAL enzymes from all sources are purified as multiple isomeric forms, produced both from multiple RNA



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species and posttranslational modifications. In bean, the isoforms exhibit different Kms for phenylalanine; the forms with low Km are preferentially induced by fungal elicitor (30). Recently, the structure of the genes encoding PAL in parsley (32) and bean (33) has been analyzed in detail . In parsley, PAL is encoded by a small family of at least four genes. Three genes (designated PAL l , PAL2, PAL3) were studied and each was found to respond to UV light, wounding, and elicitor. In cultured cells, the elicitor effect was both greater and more rapid than the effect of UV light. Wounding of root tissue also caused increases in transcript levels corresponding to each of the three genes. Interestingly, DNA footprint ing has shown that distinct but overlapping elements within the 5' promoter region of PAL l could be correlated with induction by two different stimuli: elicitor and UV light. Immunogold labeling showed that there was a local accumulation of PAL around infection sites following fungal spore inocula tion of parsley seedlings (34) . There are three divergent classes of PAL genes within the bean genome that are differentially regulated during development and in response to environ mental stimuli (33). For example, while all classes (designated gPALl , gPAL2 and gPAL3) are induced by wounding of the hypocotyl, only the former two are activated by fungal elicitor. The tissue- and cell-specific activity of the gPAL2 promoter has recently been analyzed in transgenic tobacco and potato expressing PAL-GUS fusion proteins (35). This analysis showed that the same promoter region was able to confer developmental specificity (regions of xylem differentiation and development; epidermal cells, trichomes, and root hairs; petals) and wound inducibility (40 h postwounding, the layer adjacent to the wound surface; 100 h, developing periderm) . Thus, in each location identified, activity of the promoter could be correlated with known biochemical events involving phenylpropanoid metabolites. The cinnamic acid formed by the action of PAL is the precursor of all phenylpropanoids. It is converted by the second of the core enzymes, coumar ic acid 4-hydroxylase (C4H) to p-coumaric acid. At this point, a range of alternative reactions can occur. f3-Coumaric acid can be converted sequential ly through caffeic acid to ferulic acid (catalyzed by SAM: caffeic acid O-methyl transferase) and to synapic acid. Each of these can enter a diverse array of pathways. But, any one of coumaric , ferulic , and synapic acids can be convert,ed by the third core enzyme of general phenylpropanoid metabo lism, to their corresponding coenzyme A esters . This core enzyme, 4coumarate CoA ligase (4CL), has also been extensively studied. Most is known about the parsley enzyme that exists in two isomeric forms encoded by two single-copy genes that are 97-99% homologous (36).

4CL


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Each isomer consists of a single polypeptide chain; the isomers differ in only three aa residues and exhibit virtually identical catalytic activities (37). In contrast, with 4CL in soybean, different isoforms are differentially involved in flavonoid or lignin biosynthesis (38). Induction of 4CL gene expression by UV light and elicitor was found to be coordinately regulated with that of PAL (32). Following the action of 4CL, pathway divergence can again occur, since the esters are entry points into pathways that include lignin and flavonoid biosynthesis. Incorporation of metabolites into lignin involves the sequential actions of cinnamoyl CoA reductase and cinnamyl alcohol dehydrogenase (CAD) to give cinnamyl alcohols , the direct precursors of lignin and sub strates for peroxidases . These later reactions in lignin biosynthesis are dis cussed in the context of products that change the extracellular matrix. The first committed step into flavonoid biosynthesis involves the action of the enzyme chalcone synthase (CHS) on 4-coumaryl CoA. Although this is not a core enzyme of phenylpropanoid metabolism per se, it does represent the entry point into all flavonoid metabolism and is therefore again a common step in the biosynthesis of many diverse products important both in develop ment and in defense responses (reviewed, 39, 40). In parsley, the CHS gene occurs in only one copy, although the two alleles differ in size by the insertion of a 927-bp transposonlike fragment in the promoter region of one allele relative to the other (4 1). In bean and other legumes , CHS serves a multiple function, since it is involved both in the different branch pathways leading to flavone and isoflavone derivatives and in the generation of 5' -deoxy and 5' -oxy flavonoids. The complexity of function is reflected in the relatively high number of CHS genes in bean (42) and soybean (43) compared to parsley and other nonlegumes . The induction of flavonoid biosynthesis in parsley is light dependent (44, 45), and CHS activation has been recently shown to involve a stable blue-light-derived signal (46). The entire sequence ,of events from CHS gene activation to flavonoid glycoside accumulation is localized specifically in the epidermis (45). This surface-specific expression is compatible with the putative role of flavonoids in protection against UV. Parsley CHS gene activation is efficient ly light induced, and 5' flanking sequences involved in light activation have been identified (47), but the gene is insensitive to other environmental stimuli, induding fungal elicitor and pathogen infection. By contrast, several CHS genes in bean can be differentially activated by elicitor and by wound ing, and the activity of CHS in these instances can be correlated with the accumulation of isoflavonoid phytoalexins (42). Recently, the activity of a bean CHS promoter has been studied in elec troporated soybean protoplasts and shown to be responsive both to a fungal elicitor and to glutathione (20). The regulation of CHS genes in soybean is CHS



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also complex, but unlike bean, it seems that only one gene (CHSl) is activated in seedlings in response to UV-treatment or elicitor (43). The remaining five genes encoding CHS products in soybean do not appear to be expressed in seedlings, even though their activity in soybean suspension cultures in response to elicitor treatment has been shown (48, 49) . In terestingly, in soybean seedlings, the pattern of CHS gene expression with respect to timing and transcript level was dependent on the organ system challenged by the stimuli: roots, stems, and cotyledons respond differently. Also, the plant was able to distinguish between challenge by a pathogen versus a symbiont, since the former induced CHS , whereas the latter did not (43). The different classes of defense-related proteins summarized earlier will now be discussed. With the exception of enzymes involved in phytoalexin synthesis , these proteins are located in one of two sites. Either they are found outside of the cell: within the apoplast or as a plasma membrane component, or they are located inside the cell as a component of the vacuole or specialized form of the vacuole, the protein body. Since neither site is cytoplasmic , the protein must enter the endomembrane system for transport to the surface or to the intracellular compartment. The processes involved in cotranslational segregation of proteins destined to become membrane components , lumenal products of intracellular compartments, and secretory components have been reviewed elsewhere (50-52). Although the signals involved in the initial segregation into the endomembrane system at the rough endoplasmic reticu lum are well characterized, as yet little is known of the targeting events in plants that lead to retention at intracellular loci, secretion out of the cell, or highly spe.;ific and localized deposition within the wall matrix. 4. Proteins that Change the Properties of the Extracellular Matrix

As described in the introduction , the apoplast plays a crucial role in defense. The properties of the extracellular matrix can be affected in any number of ways involving changed composition and changes in the number of non covalent and/or covalent interpolymer associations. Structural proteins of the matrix are discussed first, followed by enzymes involved in wall metab olism . EXTENSINS: HYDROXYPROLINE-RICH GLYCOPROTEINS These structural proteins have been purified from a number of plant species and tissues, and their general properties have been the subject of a recent review (53). The proteins are thought to play a central role in the primary wall organization, but are known also to accumulate in response to pathogen invasion and wounding. All extensins characterized to date are highly glycosylated, basic proteins.


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For example, the salt-extractable cell wall glycoprotein purified from carrot (54) consists of 35% protein and 65% carbohydrate. The bulk of the sugar (97%) is L-arabinose, the remainder being D-galactose. Most of the Hyp is found within repeating pentapeptide sequences of Ser (HYP)4' and is glycosy lated with short oligomers (3-4 residues) of arabinose. The galactose is linked to serine residues . Secondary structure determination revealed extensin to be completely in the polyproline II conformation (55), which was mainly lost on deglycosylation, implying that the glycosyl residues reinforce the structure. Studies on tomato extensin monomers indicate the proteins are highly periodic , composed of distinct glycosylated and nonglycosylated domains (56). Extensin polypeptides are synthesized within the cell and secreted as soluble monomers into the wall matrix where they become progressively insolubilized in muro into a highly cross-linked network (57-59). Hydroxyla tion of the peptidyl proline is a posttranslational event and the prolyl hydroxy lases have been localized in the endoplasmic reticulum (60), whereas arabi nosyl and galactosyl transferases act within the Golgi complex (6 1). The predicted translation product of a carrot extension gene contains an N terminal extension, typical of a signal peptide (62). Insolubilization within the wall is thought to occur via a progressive increase in inter- and intra molecular cross-linking involving isodityrosine bridges catalyzed by per oxidase(s). It is generally agreed that extensin polypeptides (HRGPs) are encoded by a multigene family, although it has been suggested that alternative splicing of rnRNAs encoded by a single gene also occurs (53). Considerable evidence indicates that the expression of HRGP gene(s) is regulated both de velopmentally and by environmental stimuli that include wounding and pathogen invasion. Also, enzymes involved in the processing of the extensin protein are induced by elicitor treatment (6 1 , 63). In studies on carrot, extensin transcript levels increased 50- 1 00-fold on wounding cold-stored roots, but only 2-3-fold if actively growing roots were injured (64, 65). Similarly, elicitor treatment of carrot suspension--cultured cells also led to an increase in the level of extensin transcripts (66). The effect of wound induction in tomato depended on the tissue, since HRGP transcripts increased in response to stem wounding, but not leaf wounding (67). Complex temporal changes in HRGP transcript abundance were observed on wounding bean hypocotyls. The pattern was interpreted as the differential temporal regulation of individual HRGP genes: one (Hyp 3 . 6) was rapidly activated within 1 .5 h, with transcript levels remaining high through 1 2- 1 8 h and declining by 24 h; two others (Hyp 2 . 1 3 and Hyp 4. 1 ) were activated more slowly with maximal transcript levels remaining through 24 h (68). Differential expression of these three genes was also observed following pathogen invasion, when Hyp 3 .6 was only weakly induced by the fungus,



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Hyp 2 . 1 3 was induced more strongly in the compatible interaction, and Hyp 4 . 1 was induced in both compatible and incompatible interactions. The rate of induction for all genes was more rapid in the incompatible interactions. Interestingly, in this response , systemic as well as local induction occurred (68, 69) but was restricted to Hyp 3 . 6 and 4 . 1 . It was subsequently shown that changes in level of HRGP transcripts were due to transcriptional activation (70). Related studies have shown that ethylene can cause the accumulation of HRGP protein in several plant species (7 1 ) and the activation of HRGP gene(s) in carrot (e .g. 64). However, the effects of ethylene are different from those of wounding, since the former stimulus leads to the accumulation of two transcripts in carrot cells (1. 8 and 4. 0) and the latter leads to three ( 1 . 8, 4.0, and 1 . 5 kb) . It seems that one gene produces two transcripts ( 1 .8 and 1 . 5 kb) through the use of alternate transcription start sites within its promoter (62, 64). This implies that ethylene and wounding are distinct signals in the carrot system, leading to differential activation of the same gene. As yet, there is no evidence of the spatial regulation of HRGP gene expression within the locality of a wound or infection site, nor in systemically responding tissues. This family of wall proteins is characterized by a high (60%) glycine content. The predicted structure of the polypeptide from a cloned petunia gene indicates the entire sequence can be represented as (Gly-X)n, when X is frequently glycine (72) . To date , GRPs have been isolated from a number of plant species (73 , 74) , and genes encoding GRPs have been cloned (72, 75-77) and expressed as gene fusion constructs in transgenic plants (78) . The predicted translation products of two bean GRP genes (designated GRP 1 . 8; GRP 1 .0), as well as that of the petunia gene, all have N-terminal extensions resembling typical signal peptides. It has been suggested that the unexpectedly high degree of homology in the COOH domain of the leader sequences on the bean precursors might be important in cell wall targeting (77, 79). However, as yet, nothing is known of the processing events during synthesis of these proteins in vivo. Regulation of GRP gene expression by developmental and environmental signals has. been shown in two species: petunia and bean. In petunia, tran script levels were higher in young leaf and stem tissue compared with old, but rapidly increased (within five min) in all tissues in response to wounding (72). In bean , a similar differential expression was found in young versus mature stems, but high levels were found in roots irrespective of age . A low level of transient expression (4--1 2 h) was detected in mature stems following wound induction (77). The activity of the bean GRP promoter was analyzed in more detail using GRP-GUS gene fusions in transgenic tobacco (78) . Only a small

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set of cells responded to the wound stimulus in the stem: a ring inside the vascular cylinder restricted to pith-tissue , within 1-2 mm of the wound surface. Interestingly in the transgenic tobacco, expression of the GRP gene peaked 30-45 min after wounding. Since this was much faster than wound regulation of the endogenous gene in bean plants, it is clear that the host plant species determines the kinetics of the wound response. As yet, it is unknown whether the native GRP gene(s) of tobacco (76, 80) that can be activated by virus infection or spraying with aspirin , has an identical highly localized expression. PEROXIDASES These enzymes use HzOz in a range of oxidations. Per oxidases have been studied extensively in higher plants for a number of years, and their activity and isoenzyme pattern can be correlated with any number of growth , developmental , and defense processes. In the present context, per oxidases will be discussed in relation to four defense-related events that occur in the extracellular matrix: (a) clearance of HzOz , (b) suberin synthesis, (c) lignin synthesis, and (d) construction of intermolecular linkages. It is prob able that peroxidases are also involved in other processes , such as the inactivation of host and pathogen enzymes by oxidized phenolics (81). (a) There is good evidence that generation of superoxide radicals is an important feature of the local events that occur in plant-pathogen interactions (82 , 83), and scavenging of the superoxide anion by superoxide dismutase (SOD) with the concurrent production of HZ02 has been reported (84) . Hydroxyl radicals have been implicated in the abiotic elicitation of phytoalex ins in legumes (85). Recently, a very rapid burst of H202 has also been shown to occur on treatment of soybean cultures with fungal elicitor or a defined oligogalacturonide ( 1 9). H202 will be used up in the action of any peroxidase and therefore in a sense cleared from the system , but the rapidity of the c learance demonstrated in ( 1 9) suggests the involvement of a preexisting wall enzyme, rather than activation of peroxidase genes as required in defense related lignin or suberin synthesis (27 , 86, 87). Clearance of H202 may be a defensive step, but equally could be a link in cell signaling either directly as suggested in ( 1 9) or indircctly through lipid peroxidation, a known conse quence of peroxidase action (88 , 89) . (b) The action of a highly anionic peroxidase is thought to be involved in the polmerization of phenolic monomers to generate the aromatic matrix of suberin (90, 9 1 ) . Suberization is a developmentally regulated process, but can also be induced site-specifically within a defense context when a diffusion barrier must be constructed, such as in the locality of a wound or as an added defense within the apoplast (92-94). Wound-induced changes in peroxidase mRNA levels and the genes encoding anionic peroxidase from potato and tomato have recently been studied (27, 87 , 95). The structure of the potato



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enzyme, predicted from cloned eDNA, is that of a protein of 35 1 amino acids (aas) with 9 consensus sites for N-glycosylation (87). The deduced structures of two tomato anionic peroxidases (encoded by TAP 1 and TAP2) showed 96% and 89% homology respectively to the potato enzyme (27). The pre dicted structures of the peroxidase translation products have an N-terminal extension, but it is much longer than usual and consists of a region of 25 aa, typical of signal peptides, followed by a further region of 50 aa, which contains two consensus sites for N-glycosylation (27). To date, an N-terminal extension of equivalent length and similar properties has been found on one other tomato enzyme: polygalacturonase (96), but it is not a general feature of secretory proteins in tomato, nor one of proteins targeted to the wall environ ment (reviewed , 53). As yet, it is unknown whether the anionic peroxidase precursors are processed at the C-terminal, in a manner analogous to horse radish peroxidase C (97). (c) As discussed earlier, the final stages of lignin biosynthesis involve the dehydrogenative polymerization of cinnamyl alcohols to yield phenoxy radi cals that couple nonenzymically to give oligomers of increasing size. The formation of phenoxy radicals is catalzyed by peroxidase (98 , 99). Lignins are highly abundant polymers in the secondary wall of vascular plants, pro gressively replacing water in the wall matrix and increasing the stability, inertness, and strength of the supportive structure. However, lignification is also induced in plant-pathogen responses, and has been correlated both with local resistan ce (reviewed, 81 , 100) and induced systemic resistance (re viewed , 101) . The accumulation of a group of acidic peroxidases, inherited as a single locus ( 1 02), has been correlated with induced resistance in cucumber and melon (103) . Recently, the gene encoding a peroxidase putatively in volved in lignin formation has been cloned from tobacco (86). The predicted size of the mature enzyme is 302 aa, with four consensus sites for N glycosylation; the predicted sequence of the precursor has a typical N terminal extension of 22 aa. (d) The properties of the wall matrix can be greatly affected by covalent cross-linking of polymers. Peroxidases are known to play a crucial role in the formation of these intermolecular cross-links. The process is fundamental to wall organiz,ation during growth and development (104), but an important role can also be envisaged during wound repair and wall strengthening in the immediate locality of an invading pathogen . There are two principle polymer bound phenolic groups that act as substrates for peroxidases. The first is the side-chain of tyrosine, an amino acid in high abundance in HGRPs (reviewed, 53). The second are products derived from p-coumaric acid such as p coumaryl groups, feru1ate, and p-hydroxybenzoate. These phenolics are attached to wall polysaccharides and in particular, pectins have been shown to be feruloylated (lOS). The mechanism that transfers feruloyl groups to pectins

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is extremely site specific

( 1 06),

and is known to occur within minutes of

incorporation of sugar precursors into the polymer, i.e. in the Golgi complex. The consequence of peroxidase action on these wall phenolics is twofold: the glycoproteins become cross-linked via isotyrosines, leading to an extremely stable, insoluble network

( 1 07-1 09),

and the gelling of polysaccharides can

be substantially increased by diferuloyl bridges

( 1 1 0 , 1 1 1 ).

In prinCiple,

regulation of this cross-linking during a defense response could arise from changes in levels of the relevant peroxidases, the temporalispatial availability


of

HzOz in the wall microenvironment, and/or from the degree of feruloyla

tion of the polysaccharides that are secreted. To date, most of the information available on changes in peroxidase gene expression is indirect and involves changes in isoenzyme pattern (reviewed,

95, 1 12).

These data clearly show that the abundance of specific isoenzymes

changes, both at the local site of infection/wounding and systemically. The systemic response is not limited to fungal or bacterial infection, but can be induced in leaves of potato plants by root infection with cyst nematodes, when one of the abundant PR proteins of potato was shown to be a peroxidase

( 1 1 3) .

Using cDNA probes in Northern analyses, mRNA for the peroxidase

involved in suberization was undetectable in unwounded potato tissue, but reached maximal levels in the tubers at four days

(87).

product of TAPI gene increased on wound induction

In tomato, only the

(27).

Expression of

TAPI and the corresponding potato gene was also induced in callus two days after culture on media-containing exogenous ABA

(10-4M) (27),

an agent

previously shown to have an indirect role in promoting suberization A s described i n Section

CINNAMYL ALCOHOL DEHYDROGENASE

( 1 1 4) .

2,

prod

ucts of the reaction catalyzed by this enzyme are the cinnamyl alcohols, the direct precursors of lignin biosynthesis. In bean, the enzyme is encoded by a single gene that has recently been cloned

( 1 1 5) .

The CAD gene is transiently

activated (within 1.5 h) in response to elicitor treatment rapid increases in CAD enzyme activity

( 1 1 6).

( 1 1 5),

leading to

The rapidity of the CAD

response has led to the suggestion that the enzyme may be responsible for the generation of signaling molecules, such as those related to dehydroconiferyl glucosides that exhibit cell-division promoting activity CALLOSE SYNTHETASE

( 1 1 7 , 1 18).

This enzyme catalyzes the formation of the

f3 1 ,3

glucan called callose. The protein is a functional component of the plasma membrane (PM), and in vitro enzyme activity is strictly dependent on Caz+, in the presence of polyamino compounds and/or Mg2+

( 1 19) .

Invariably

associated with the activation of callose synthetase is an efflux of K+, an external alkalinization, and a net Ca2+ influx into the cells

( 1 20).

Activation

can be caused by treatment of cells with chitosan, a known component of fungal walls and the exoskeleton of insects

(121). The effectiveness of the



887

elicitor increases with its degree of polymerization (d.p.) up to a d.p. of 4000 ( 1 22). This suggests that rather than acting through a specific receptor , the effect of chitosan on callose synthetase arises from damage to the membrane and/or a change in properties caused by interaction of the charged oligosac charide with the negatively charged head groups of membrane phospholipids. The role of the enzyme in surface-mediated defense responses has recently been well reviewed (123, 124). Callose deposition is known to be a very rapid and very localized event in response to pathogen invasion or mechanical injury. For example , studies on soybean indicated that rapid callose deposition was restricted to the zone adjacent to fungal hyphae , and the speed of the response was correlated with incompatability ( 1 25). It is envisaged that transient fluxes or increases in Ca2+ concentrations with localized microenvironments could provide the means to activate the enzyme and produce such localized deposition of callose. As an insurance against wounding, it is to be expected that callose synthetase is present in the PM at all times , being latent until activated by 2 Ca + and membrane damage . It has been suggested that callose synthetase and cellulose synthetase are the same enzyme (126). Thus, there would be one PM-located UDP glucose: glucosyl transferase that , dependent on the microenvironment at the cell surface, transfers glucose to the 4-0H (cellu lose) or 3-0H (callose) of the terminal glucose in a nascent glucan chain. If this model proves correct, switching from one polymer to another would be a highly elegant means of adaptation to circumstance. Turnover of callose has not been widely addressed as yet, but it is thought that {3 1 ,3-glucanases are involved, both those that are constitutively expressed in the wall and presum ably, those that are synthesized de novo and targeted to the waJl in a defense response . 5. Proteins Associated with Deterrence and Antimicrobial Activity

As described in the introduction, many plant proteins that accumulate natural ly in storage organs and seeds are those that are induced to accumulate elsewhere during defense responses. To date , this phenomenon has been well characterized for certain protein families, such as hydrolases and proteinase inhibitors . Much less is known, however, of other toxins and enzyme in hibitors, and virtually nothing is known of the role of lectins, even though as a class they are more abundant than proteinase inhibitors and probably represent the most biochemically characterized of all seed proteins (reviewed , 127, 128). This section mainly reviews recent findings on the known families of defense proteins and the enzymes involved in the synthesis of antimicrobial products . But, since a defensive role for lectins is often suggested, current information on this possibility is first briefly discussed.


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The definition of a lectin as a protein exhibiting multiple noncatalytic binding sites for carbohydrates is 'operational' and provides as much insight into the function of the protein as the definition of an enzyme based on catalytic activity alone. The abundance of lectins in seeds ranges from >30% total protein (e.g. concanavalin A, 129) to less than 0 . 1% [e.g. wheat germ agglutinin (WGA), 130], a range that renders it unlikely the proteins can all be classified per se as storage reserves. Although simple sugars will act as competitive inhibitors , plant lectins can show extraordinary specificity toward the shape of oligosaccharides (128, 131) , and this ability to transmit in formational content makes them obvious candidates for mediators of molecu lar recognition. Recognition events involving plant-encoded receptors seem unlikely , since at seed maturity when levels of the lectins are greatest, only minimal amounts of endogenous glycoconjugates exist with the correct specificity to permit lectin binding (e.g. 132-135). Developmental regulation of lectin gene expression can be complex. For example, recently, the temporal and spatial regulation of the gene encod ing soybean lectin has been studied in depth (136-138). The same gene is expressed to high levels at a specific developmental stage of seed matur ation, and to low but detectable levels in mature roots . Similarly, recent studies on WGA have located lectin transcripts in situ during embryo for mation and in the root system, specifically in the root cap cells of seedlings (139) . The expression of closely related genes to those encoding the seed lectins has been shown for a number of plant species (reviewed, 127), and for some, expression in vegetative tissues is correlated with the seasonal formation of storage reserves (140-142). As yet, however, very few studies have ad dressed the possibility that lectin gene expression can be triggered in vegeta tive tissues by defense-related stimuli. There is one report that potato lectin accumulates on wounding (143). Also, recent studies on wound-induced genes in potato tubers have identified win 1 and win 2 (144) . These will be discussed in the ncxt section, but of relevance to this context is the finding that the proteins they encode contain a highly-"conserved domain of 43 aas, which has previously been identified in proteins that bind N-acetyl glucos amine, such as the lectin WGA (145) and the chitinases (146) . Certain lectins, e.g. Ricin, phytohemagglutin (PHA), and ConA, are known to be toxic (reviewed , 147a), and recently, an amylase inhibitor and the toxic protein arcelin from bcan have been shown to be encoded by genes that are closely homologous to those encoding PHA ( l 47b, c) . But, the presumed antifungal properties of WGA (148) have recently been shown to be due to low-level contamination of the lectin with endochitinase (149) . Studies involving the constitutive expression of lectins in transgenic plants should clarify the role of these proteins in conferring resistance.



889

ENDOHYDROLASES Studies on defense responses have mainly focussed on the endohydrolases that exhibit /31 ,3-glucanase and chitinase activities. Sub strates for these enzymes are common components of the surface structures of pathogens and pests, /3-glucans being major cell wall constituents of common fungal pathogens (reviewed, 150), and chitin an abundant product of micro bial walls and the exoskeleton of insects. There is good evidence that the action of the endohydrolases leads to detrimental effects, such as the inhibi tion of hyphal growth ( 1 49, 1 5 1 , 1 52), as well as the probable release of signaling molecules (/3-g1ucans and chitin/chitosan oligomers) that activate defense genes (153). The proteins have been found in monocots (154-158) and dicots (many species; reviewed, 159) and are known to accumulate under developmental regulation, as well as in response to defense-related stimuli. For a full discussion , the reader is referred to reviews (26, 159, 1 60). Recently, groups 2 and 3 of the tobacco PR-proteins have been identified as hydrolases (16 1 , 1 62). Each tobacco hydrolase exists as acidic and basic species: there are three acidic and one basic /31 , 3-glucanases and two acidic and two basic chitinases. Sequence homology between isoforms of each hydrolase is moderately high. Information on the synthesis of the tobacco hydrolases comes primarily from work on the /3-glucanases. It is known that these are synthesized as precursors with typical N-terminal extensions and the precursor of the basic glucanase has an additional C-terminal extension, which is N-glycosylated during processing, but is proteolytically cleaved to yield the mature protein (163). Targeting of hydrolases follows a clear pattern in tobacco: the acidic forms of both enzymes are secreted into the apoplast, whereas the basic forms accumulate intracellularly at a site presumed to be the vacuole (164) . Equivalent hydrolases have also been well characterized in two other Solanaceae species, potato and tomato: in both, proteins previously described as PR-proteins have been recently identified as m,3 glucanases and chitinases ( 1 65-167). In thesc spccies as in tobacco, thc enzymes exist in acidic and basic isoforms, and there is some evidence that the former are targeted to the apoplast and the latter to the vacuole. Since the m ,3-glucanases and chitinases from all species so far in vestigated exist as isomers, it is probable the two enzymes are each encoded by muitigellle families. This is already known for gene products of barley (156, 1 5 7), bean ( 1 68), and the PR-proteins assigned hydrolase activity in tobacco (reviewed, 164, 169) . Regulation of genes encoding these products is complex, leading to coordinated but differential expression of isoenzymes in response to developmental signals and defense-related stimuli. For example, Mauch et a1 ( 1 70) followed changes in four hydrolases of pea, in response to seed maturation, fungal infection, wounding, and stress (ethylene or chito san). Each stimulus produced a different pattern of response.

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Similar studies showing wound and pathogen induction of chitinases have been carried out on melon ( 17 1 , 1 72) and bean ( 1 73). In bean, activation of chitinase genes was found to be very rapid, l O-fold stimulation being observed within 5 min of elicitor addition. Similarly, chitinase transcripts accumulated in suspension-cultured cells in response to elicitor much more rapidly than transcripts encoding phytoaJexin biosynthetic enzymes (max levels 2 h and 3-4 h, respectively) . Ethylene ( 1 68 1 7 1 , 1 74-- 1 77) , salicylic acid ( 1 64) , auxin , and cytokinin ( 1 78) have all been implicated as signals affecting endohydrolase gene expression, but as yet it is unclear which signal is the most directly relevant to the transduction pathway used during a defense response in vivo. Interestingly, a local stimulus will lead to the accumulation of hydrolases in systemically responding tissue (1 1 3 , 1 79). This has been shown following leaf inoculation of cucumber plants with viral , bacterial , and fungal pathogens ( 179, 1 79a), but also as a systemic response in aerial tissues of potato following root inoculation with cyst nematodes ( 1 1 3) . It has been suggested that hydrolases provide a dual mechanism of defense: the wall-located species being the first line of attack and the vacuolar species the second, when the plant cell membrane is breached ( 1 80) . However, the systemic data imply a third role, not related to the immediate local effects of pathogen invasion but rather to longer-term and generalized protection, such as that underlying acquired resistance.


,

THIONINS These are a family of small basic proteins that have been identi fied in a number of monocots and dicots (reviewed, 1 8 1 , 1 82) but are best characterized in barley. Originally extracted from the endosperm of seeds and known to exhibit toxic properties to a range of organisms ( 1 82- 1 85) , they were assumed to play a protective role. More recently, other members of the multi gene family have been shown to be leaf specific ( 1 86 , 1 87) and are induced to accumulate in response to pathogen challenge and stress ( 1 88 , 1 89). Thionins would appear to have two subcellular locations: the endosperm specific group are targeted to the protein bodies ( 1 90) and the leaf-specific group targeted to the wall ( 1 88) . Despite this apparent difference, the pre cursor form of all thionins is almost identical and provides no insight into the different targeting mechanisms. Thus, the mature proteins all have an Mr 5000, whereas the precursors are much larger (�Mr 1 5 ,000) and possess both an N-terminal extension of 28 aas typical of leader sequences , and a massive C-terminal extension that is surprisingly homologous in all thionin precursors analyzed to date ( 1 87 , 1 9 1 , 1 92). The location of the leaf-specific thionins was determined by immunolabeling ( 1 88). Interestingly, the middle lamella was not stained; the antigens were restricted to a discrete layer adjacent to the



89 1

PM. Thionins are not extractable into intercellular wash fluids under buffer conditions that were able to extract PR-proteins from a dicot ( 1 89) . In barley, in excess of 50 copies of thionin genes exist per haploid genome, all of them confined to chromosome 6 ( 1 88). In normal development, leaf-specific thionin gene expression is down-regulated by light ( 1 86-1 88) , but challenge of nonetiolated green seedlings with a pathogen (188), or treatment of plants with chemical agents known to induce stress ( 1 89), leads to the accumulation of thionin mRNA transcripts. The response was slow (peaking 44 h postchal lenge), and both the timing and level of induction were identical for compat ible and incompatible interactions. The mode of action of thionins in a defense response may be related to their toxicity and/or their ability to exhibit thioredoxin activity ( 193). Protein inhibitors of enzymes with potential defensive roles against pests and pathogens are found throughout the plant kingdom, and the reader is referred to reviews ( l 47a, 194, 195) for a full discussion. This text will focus on two families of inhibitors: those that share homology to thaumatin, and the proteinase inhibitors. PROTEINS Thaumatin is the trivial name given to an intensely s weet protein found in the fruits of a tropical shrub ( 1 96) . The protein shares extensive homology to a bifunctional inhibitor from maize ( 1 97) that shows potent in vitro activity against bovine trypsin and a-amylase. Interestingly, the acidic isoforms of a group of tobacco PR proteins also show the same level of homology to thaumatin (198-200) , suggesting that they may also express inhibitory properties. The basic isoform of this group of tobacco PR-proteins (20 1 ) is thought to be equivalent to a salt-induced protein in tomato (202) , and both are correlated with osmotic adaptation. It seems that the same pattern of targeting is found with these proteins in tobacco as with the hydrolases: the acidic forms are secreted to the apoplast, and the basic to the vacuole (reviewed, 1 64, 20 1 ) . Accumulation of the tobacco protein can be induced by viral infection but not by aspirin (80).

THAUMATINLIKE

This discussion focusses mainly on the serine PROTEINASE INHIBITORS proteinase inhibitor 1 and 2 families, since they have been studied in detail as inducible defense-related proteins. General reviews include (203-205) . In hibitors of each family exist in isomeric forms, encoded by multiple genes that are under developmental regulation and that respond to defense-related stimuli. Thus, the proteins accumulate to abundant levels in storage organs and seeds, but PI l and 2 genes can be activated by wounding and pathogens. Induction in defense responses is particularly interesting, since it can occur both at the local site of injury and systemically. A detrimental effect of these families of PIs on insect larvae has been shown (206-210), but a wider


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BOWLES

protective role is envisaged , given the abundance of serine proteinases in digestive tracts and/or secretions of herbivores, pathogens, and pests. To date , proteins of the inhibitor 1 family have been identified in several plant species, including the Solanaceae and monocots (reviewed, 203 , 2 1 1 , 2 1 2), but the PI that is wound induced in leaf tissue of tomato has been extensively studied . The mature protein accumulates in the vacuole (2 1 3) and exhibits exceptionally slow rates of tumover (214) . The predicted structure of the precursor indicated an N-terminal extension, in which the first 23 aas was typical of a signal peptide and the next 19 aas contained a high proportion of charged residues (2 1 5) . Since the N-terminal sequence of the mature vacuolar protein ( f>ubunit Mr 7800) was known, co- and post-translational proteolytic processing to remove the entire extension was envisaged. The predicted structure of a homologous inhibitor 1 from potatoes was deduced from a genomic sequence, and the precursor of that protein was found again to have an extended N-terminal region (216) . However, it is unlikely that the 19 aa (tomato) and 1 3 aa (potato) sequences per se represent a vacuolar targeting signal , since an equivalent domain is absent from the precursor of inhibitor 2 of tomato, even though its final destination is also vacuolar (2 1 3 , 2 1 7) . Extensive studies have also been carried out o n the inhibitor 2 protein isolated from potato, including the identification and sequencing of its gene (2 1 8 , 2 1 9) . Small multi gene families encode inhibitor 1 and 2 proteins i n potato and tomato (reviewed , 220), but comparatively little is known of the expression of the different members of each family since most attention has centered on the genes that are wound inducible. Recently it has been shown that genes encoding isomers of inhibitors 1 and 2 are developmentally regulated during the ripening of wild tomato fruit (22 1 ) , and an inhibitor 1 gene is activated by ethylene during fruit ripening of a modem tomato variety (222). But, as yet no systematic analysis with gene-specific probes has been carried out. The regulation of PI gene expression during the response of potato and tomato plants to wounding, elicitors, or pathogens, has been followed at several levels including changes in PI activity (223-226), protein (reviewed, 220) , translation products (227), mRNA (2 1 8 , 228-230) , and promoter activity in transgenic plants (231-234). To date, PI gene activation is the most useful model to study long-range signaling in plants , since the systemic response is well established, having been known for almost 20 years (reviewed, 205). More recent findings, only, are summarized here . In tomato, it is known that inhibitor 1 and 2 transcripts are undetectable in unwounded leaves, but rapidly accumulate at the local site of leaf injury with maximum levels at 8 h (228) . Multiple wounding, whether continuous (every 3 h) or intermittent (lag of 15 h) further increases mRNA levels (228) . The timing of the response at the local site differs from that in the systemically ,



893

responding tissue, when translation products for the PIs showed only a transient abundance (from 4 to 1 0 h) in unwounded leaves (227). The ability of the plants to respond to injury or oligosaccharides could be abolished by pretreatment with aspirin or fusicoccin (223, 224). In these systems, the induction of PI activity could be correlated with changes in membrane potential ( 225) and more long-range electrical events (226). Recent studies on potato have focussed more on inhibitor 2 gene expres sion, when local and systemic increases in abundance of mRNA transcripts are known to occur on leaf wounding and tuber wounding (218 , 229) . Interestingly, mRNA levels do not increase in the root system nor the lower stem region of the plants during the systemic response (229). This has been confirmed in later studies and extended to show that root wounding, whether mechanical or during invasion with cyst nematodes, is capable of inducing systemic increases elsewhere in the plant (230). A comparable pattern of loeal and systemic induction was shown when the potato inhibitor 2 gene was either expressed in entirety in tobacco plants (23 1 ) or as a CAT fusion construct (232, 233) . In both studies, the 3 ' flanking regions as well as the 5 ' upstream region of the gene were implicated in regulation . Since the pattern of expres sion was identical in the heterologous transgenic system, the signaling events linking injury to transcriptional activities were conserved between potato and tobacco. More recently , the 5 ' region alone was found sufficient to confer wound inducibility: promoter activity was studied in transgenic potato plants expressing an inhibitor-GUS fusion protein (234). The same promoter region of a specific PI gene was found to be able to confer developmental specificity (tuber-specific expression) and wound inducibility. In wounded leaf tissue, promoter activity led to GUS expression throughout the mesophyll and epidermis, but interestingly in systemically responding tissue, expression was highest only in the immediate region of the vascular tissue and decreased as the distance from the veins increased. The local and systemic regulation of PI gene expression is not limited to inhibitors of the I and 2 families . In studies on alfalfa, a PI of the Bowman Birk family showed a typical pattern of accumulation in response to injury (235), and PI activity increased rapidly in melon in response to fungal infection (236). Since constitutive expression of a Bowman-Birk class of PI from cow pea in transgenic tobacco led to enhanced resistance to insect larvae (237), it is probable that research into this family of defense-related genes and their products will increase for many years to come, particularly since the cow pea inhibitor conferred resistance to a tobacco pest. LATE ENZYMES OF PHYTOALEXIN BIOSYNTHESIS As discussed in the section on Activation uf Defense Responses (Section 2), plants synthesize low-molecular-weight antimicrobial compounds during localized resistance


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events to pathogen invasion. The area has been thoroughly reviewed (6, 238), and recent results involving enzymes used in the initial steps of isoflavanoid phytoalexin biosynthesis have been summarized in Section 2. Less is known of the enzymes involved in later stages that are specific for isoflavonoid or furanocoumarin phytoalexins (reviewed, 239) . Similarly, little is known of the second class of phytoalexins, the tcrpenoids. All terpenoids are formed from acetyl CoA, but the pathways leading from polyprenyl pyrophosphates to the different phytoalexins are complicated since at least three intracellular compartments have been shown to be capable of terpenoid biosynthesis (reviewed, 240). A terpenoid system that is readily amenable to molecular studies is the formation of casbene, the trivial name given to a diterpene phytoalexin in Ricinus communis . Casbene synthase, catalyzing the formation of the cyclic diterpene from geranyl-geranyl pyrophosphate , has been purified and is located in proplastids (24 1 ) . Enzyme activity is absent from healthy tissue, but rapidly increases in response to a pathogen polygalacturonase (242) or a defined oligosaccharide ( 1 2). Interestingly , the size of the oligosac charide was critical for the elicitation of the defense response. 6. Additional Defense-Related Proteins PATHOGENESIS-RELATED PROTEINS Gene products now called PR proteins were first identified in tobacco following infection of the plants with tobacco mosaic virus (TMV). The proteins were grouped into a family based on acid solubility, resistance to proteinases, and coordinate accumulation in ' response to the viral-induced necrotic lesions. Initially, the molecular species were analyzed by native gel electrophoresis , and nomenclature was based on the resolution of that separation technique. It was then found that proteins corresponding in character to those defined as pathogenesis-related could be recovered in wash fluids from the intercellular spaces of the leaves. This led to the notion that analysis of wash fluids could provide a direct indicator of the accumulation of PR-proteins. But more recent work suggests that different members of the family are targeted to two distinct locations: the wall and the vacuole. To add to the difficulties of nomenclature, I -D and 2-D SDS-PAGE has superseded earlier techniques, and resolution of many more molecular species has been achieved. PR-proteins based on some or all of the above characteristics and homology at the aa or nucleic acid level, have been found in many species of plant and can be induced to accumulate in response to a diverse range of pathogens and biotic and abiotic elicitors. Despite years of work, the precise role of the PR-family in defense re sponses remains elusive, although increasingly, properties are being assigned to individual members, such as the GRPs and the endohydrolases and the enzyme inhibitors described earlier. A role for PR-proteins in viral resistance



895

was initially suggested, but the constitutive expression of PR 1 , GRP, or the "thaumatinlike" protein individually in transgenic tobacco had no effect in conferring viral resistance, although the effect of combined expression was not tested (243) . At present, it is considered more likely that the proteins are involved in general defense, and certainly their wide-ranging modes of action, from inhibitors and enzymes to structural wall proteins, make them ideally suited in defense strategies against 'all-comers . ' For a detailed discussion of PR-proteins, the reader is referred to the following reviews ( 1 6, 1 7, 26, 1 64, 244, 245). PR-proteins with known properties have been discussed earlier in this review. This section summarizes current understanding of the remaining PR-proteins from tobacco and other species. Currently, there are five recognized groups of PR-proteins in tobacco (updated, 164). The family known as the PR- l proteins have been extensively studied, but no function has as yet been assigned . Although PR- l proteins exist in acidic and basic forms, most work has focussed on the former species, where >90% structural homology has been found between l a, 1 b, and I e , deduced from sequencing cDNA and genomic clones (246-250). Only 67% homology was found between those and the basic forms (25 1-253). The proteins are synthesized as precursors (24�254) with an N-terminal exten sion of 30 aa, preceding the sequence of the mature protein of Mr 1 5 ,000. Interestingly, precursors of the basic PR- l species have an additional C terminal extension of 36 aa (25 1 ) . The acidic forms are targeted to the apoplast, and an extracellular location has been confirmed at light and elec tron microscopy level (255-257). Within the wall matrix, the proteins are located in the middle lamella but unexpectedly, were also found in xylem elements, suggesting diffusion had occurred within the apoplast. Studies on the regulation of PR· l gene expression have shown the local and systemic accumulation of PR- l transcripts in response to viral infection (254-256) , but the response is slow: maximal levels, three days (local site) and eight days (systemic site) (246). Direct induction of PR transcripts by spraying with salicylic acid (5 mM) is more rapid with maximal levels at two days (258). There is some evidence to suggest developmental regulation of PR- l genes occurs, since PR- 1 proteins were found at high levels in yellow senescent leaves of uninfected tobacco plants (259) . An homologous protein to the basic PR- l of tobacco has been identified in tomato (260). The protein, known as P- 14, has been located in the in tercellular spaces (26 1 ) . Similarly, virus-inducible proteins, serologically related to tobacco PR- I proteins, have been found in numerous plant species including the monocots, maize, and barley (262, 263), and additional PR proteins have also been identified recently in maize (264). Work on PR proteins in tomato extends beyond characterization of P- 1 4 and the endohy drolases described earlier, since a wide range of novel proteins (apoplastic


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and/or acid-extractable) can be induced following virus infection and spraying with ethephon (265a-c). A recent detailed analysis of the tomato PR-proteins has revealed their local and systemic accumulation in response to leaf inocula tion with fungal spores and has identified several as enzymes: in particular, P-45 is a peroxidase (265b, c) . There is some evidence that salicylic acid treatment induces PR-proteins in tomato under conditions in which the same compound inhibits the PI response of the plants to wounding or to oligosac charides, suggesting the event affected by salicylic acid may be common to the induction of both defense responses ( 1 89 , 223). To date, several studies have investigated PR-proteins in potato ( 1 1 3 , 1 65 , 267-269) . Some o f these proteins have been identified as endohydrolases ( 1 65) and peroxidases ( 1 1 3) , but many more, as yet uncharacterized, can be induced by a range of pathogens as well as by direct spraying with aspirin (268). PROTEINS ENCODED B Y wun 1 AND wun 2 Clones encoding wun 1 and wun 2 were initially identified in a cDNA library prepared from wounded potato tubers (270) . mRN A levels rapidly increased at the local site of injury, accumulating within 30 min and reaching maximal levels between 4 and 24 h . The expression o f the wun 1 gene in potato and the activity o f the wun 1 promoter have since been studied in more detail (27 1 ) . Potato leaves were detached and sprayed with H20 or with different strains of a fungal pathogen. Although wun 1 transcript levels increased in all treatments within 2 h following detachment, abundance was maintained only in leaves undergoing a compatible interaction characterized by cell collapse and death . Interestingly, in transient expression assays, the activity of the promoter was as high in rice protoplasts as in potato protoplasts, even though no wun I cross-hybridization was detected to rice DNA or RNA. This implies that the trans-acting factors necessary for wun 1 expression are conserved in both monocots and dicots and may be used to regulate a variety of genes . Also it should be noted that in protoplasts, the promoter appeared to be switched on to maximal levels without the need for induction , suggesting that the conditions for protoplast preparation/maintenance were sufficient alone to trigger the system.

win 1 AND win 2 A cDNA clone complementary to a wound-induced transcript from potato tubers (272) was used to isolate a single genomic clone containing win 1 and win 2 genes ( 144). The genes encode two homologous cysteine-rich proteins which, as described earlier, contain N-terminal domains of 43 aa also found in N-acetyl-glucosamine binding proteins . The predicted structures of both precursors show an N terminal extension of 25 aa, typical of a signal peptide, that precedes the conserved sugar-binding domain. The genes are members of a small multi gene family that exists also in tobacco and tomato ( 1 44) . Interestingly , the

PROTEINS ENCODED BY


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two genes show differential patterns of expression when analyzed 20 h following local wounding: win I transcripts accumulated only in stem and leaf tissue but not in roots and tubers, whereas win 2 transcripts accumulated in all organs , albeit to low levels only in the roots . Elicitor-activated genes (designated ELl genes) have recent ly been identified in parsley using an induction system involving addition of heat-released elicitor to suspension-cultured cells (273). Nuclear run-off RNA was used as a hybridization probe for differential screening and isolation of clones from an elicitor-induced cDNA library (prepared 3 h posttreatment) . Four groups of ELl-genes were identified, based on different temporal activa tion in response to elicitor treatment. Maximal transcription occurred from 0 . 5 to 1 . 0 h (group A), I to 5 h (group B), 2 . 5 to 4 . 5 h (group C), and 5 to 8 h (group D) . The most abundant (group B) corresponds to the ' PR- I ' gene previously shown , using in situ localization, to be activated in parsley leaves in the vicinity of fungal infection sites (274). As yet, proteins encoded by these genes have not been characterized.


ELI PRODUCTS

ELICITOR-1NDUCED PRODUCTS Studies similar to the above, but using different plant species and pathogens/elicitors , have also been carried out and have been reviewed in (6, 7, 9) . The results described in (275 , 276) empha size the importance of using a highly defined elicitor. In bean suspension cultures, different elicitor-active subfractions prepared from culture filtrates (by ion-exchange and ConA affinity chromatography) induced different pat terns of response analyzed by translatable mRNA species (275). Similarly, carrot elicitors either prepared in vitro or released by hydrolases in vivo, produced quite different responses in suspension cultures (276). Application of PIIF to excised tomato plants also produced a very different spectrum of response to that evoked by endogenous systemic signals released on wound ing, even though PI genes were activated in both instances (227).

Coordination of Defense Responses

The preceding sections have introduced the range of proteins currently un der investigation within the context of plant defense responses. From the data it is clear that a variety of plant species, experimental/assay systems , and primary stimuli are in use. In vivo, the response of plant cells to pathogen invasion is likely to be a highly coordinated network of events involving exchange and recognition of signals that lead to local and long-range activation of genes. It is possible that each signal triggers more than one response in order to coordinate events such as those involved in containment, repair, and defense. However, this is difficult to determine experimentally unless a highly defined elicitor is used to trigger the system.


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It is apparent that even pure oligogalacturonides can have different effects depending on their degree of polymerization: the trisaccharides being capable of inducing PI gene activation ( 16) and PAL (277a) , whereas oligomers greater than a d.p. of 8 are required for induction of glyceollin accumulation in soybean ( 1 1 ) and casbene in castor bean ( 1 2). Recently , it has also been shown that only the larger oligogalacturonides are capable of influencing morphogenetic programs in the tobacco explant system (277b, 278) . Sim ilarly, although small oligomers of chitosan have diverse biological effects, polymers are much more effective in triggering callose synthetase ( 1 22). The potential complexity arising from the addition of an ill-defined mixture of elicitors is illustrated both by the different effects of elicitor subfractions (275) and the synergistic effects arising from the addition of two elicitors simultaneously (279) . Also , it has recently been shown that addition of a purified elicitor to parsley cells actually switches off the transcription of some defense genes encoding enzymes of pathways that compete for substrates (280). The use of this purified glycoprotein should prove to be one means of identifying the responses elicited by a single molecular species . This is particularly so since bioactivity is retained following protein hydrolysis, thereby providing the means to correlate elicitor activity with a defined peptide sequence. Similar studies should be possible using glycoconjugates, although the complexities of polysaccharides, the fragments derived from them, and the analytical systems for structural determination, make the approach inherently more complicated. To date, within an organized plant tissue, the most rapid defense-related response is callose deposition ( 1 23). Membrane damage can trigger the response ( 1 22) , but equally it is known that small changes in membrane potential can affect the capacity of the membrane to synthesize cellulose or callose ( 1 26). Several elicitors are known to induce changes in ion transport (24, 1 20, 2 8 1 -284) and rapid, but different effects of small and large oligoga lacturonides on membrane potential have been demonstrated using in tracellular electrodes (225). Similarly, agents that affect ion transport such as aspirin , vanadate , and fusicoccin, modulate defense-related responses , and this has led to suggestions that depolarization or hyperpolarization may be early causal events in elicitor transduction pathways (22 , 223-226, 265b, 285). The relationship of these ion fluxes at the cell surface to those involving changes in intracellular calcium (24, 280) and protein phosphorylation (286, 287) remains to be understood. In principle, events such as these could arise either from "nonspecific" changes in membrane permeability (e .g. damaging action of hydroxyl radicals, binding of pathogen-encoded toxins, etc) or from the highly-specific interaction of an elicitor signal with its receptor. The bioactivity of structurally specific and hydrophilic signals such as oligo saccharides necessarily implies the existence of recognition events in-


899

volving receptors. Binding sites for gluean elicitors have been identified in soybean cells and microsomal membranes (288a, b), but to date no receptor has been biochemically characterized . As a consequence, we do not yet know whether activation of defense response genes in a particular cell by the recognition of different elicitors will involve the use of one or multiple transduction pathways . The components of several different signaling systems are known to exist in plant cells, for example: phospholipase C (289-29 1); inositol phospholipids and inositol phosphates implicated in calcium mobilization (292-298); lysophospholipids (reviewed, 299); protein kinase C (300-302) ; calmodulin (reviewed, 303); and annex ins (304, 305). Also, we do not yet know the mechanism underlying the induction and coordination of long-range signaling and systemic activation of defense genes. In some systemic responses, such as the induction of endohydrolases, certain PR-proteins , and products of the win 1 and win 2 genes, ethylene is known to act as a signal (26). However, this need not imply that ethylene or its precursors are the long-range signal. In the wound-response of the SoLa naceae, it was originally thought that oligosaccharides represented the long range signal for systemic gene activation , but it is now accepted as probable that they do not move from the local injury site (306). These two long-range responses involving PI and PR gene activation differ from one another in several respects . First, systemic PI gene induction is much faster, occurring within hours of local injury, whereas systemic PR gene is characteristically much slower, occurring only 5-8 days after application of the local stimulus. Second, different signals have been implicated in the two responses: ethylene does not induce PIs (307, 308) and oligosaccharides do not induce PRs. Indeed, aspirin inhibits the PI response but triggers the PR response (223). Yet, in spite of these differences, the cellular route taken by the systemic signal during the long-range activation of win 2 (M. Bevan, personal com munication) and PI 2 (234) is known to be the same: the channel of parenchy ma cells associated with the vascular tissue. This suggests that different systemic si.gnals may use a common long-range transport route in the plant. Altemative:ly, it is possible that there is a common form of systemic signal that triggers the activation of different defense genes because of the involve ment of different local effectors . The vascular parenchyma have been pre viously identified both as the channel taken during auxin transport (309, 3 10), and that taken by electrical signals in the well characterized long-range response of Mimosa (3 1 1) .


-

8 . Concluding Remarks

Advances in our understanding of plant defense responses have increased rapidly ove:r the last five years , and the rate of progress will probably gather momentum even more over the next five. What is particularly encouraging is


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that the experimental systems in use provide a wealth of information beyond the remit of plant molecular pathology. Through the use of in situ hybridiza tion and the expression of defined genes and/or their promoters in transgenic plants, evidence is becoming available on the chronology and very precise spatial regulation of local and systemic events induced by a stimulus. This in tum provides a means of identifying the cell types that respond and the means of determining which events may be under coordinated control. The research is also opening up an awareness of the apoplast and showing it to be both a reservoir of regulatory signals and a place of considerable spatial order in which deposition and modification of polymers can be restricted to specific microenvironments . Signaling is another area that has benefited from research on defense responses and certainly, within the next few years , we may be in a position to identify the sequence of events leading from a defined extracellular signal through to transcription of a target gene. Many proteins are known to be synthesized at the same time in a cell and yet are transported to very different functional locations . These observations illustrate the im portance of targeting, both for our understanding of cellular organization and for the expression of foreign proteins in defined intra- or extra-cellular loci within transgenic plants. Research on defense responses has already identified a wide number of proteins and genes suitable for detailed analysis of targeting signals. In total , the range of expertise in use, the interdisciplinary nature of much of the work, and the impact of the results on our understanding of the molecular organization of plants, make this an exciting and important area. ACKNOWLEDGMENTS

This work was supported by the Agriculture and Food Research Council (AFRC) and the Science and Engineering Research Council (SERC). Paula Duncan and Maggi Rose are thanked for typing the manuscript and Keith Roberts, Sarah GUff, Helen Doherty, and Bjorn Drobak are thanked for helpful discussions. Literature Cited 1 . Schoff!, F . , Baumann, G . , Raschke. E . • Bevan. M . 1 986. Phi/os. Trans. R . Soc. London, Ser. B 3 1 4:453-68 2. Schoff], F . , Baumann, G . , Raschke, E. 1 988. In Plant Gene Research

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Phospholipid transfer proteins from higher plants.

Inositol-containing lipids in higher plants.

Metabolic repression of transcription in higher plants.

Hormonal control of transcription in higher plants.

[Poly- and metaphosphates in higher plants (Lemnaceae)].

Chemically induced aneuploidy in higher plants.

Toxic proteins in plants.

Heat shock proteins from cell cultures of higher plants and their somatic hybrids.

[Potential carcinogenic contents of higher plants].

Nuclear ribonucleoprotein particles of higher plants.

Synthesis of sulphoquinovosyl diacylglycerol by higher plants.

Antimutagenicity of secondary metabolites from higher plants.

Rapidly labeled intermediates in DNA replication in higher plants.

Amino acid metabolism in nongrowing environments in higher plants.

Fat metabolism in higher plants. Differential incorporation of acyl-coenzymes A and acyl-acyl carrier proteins into plant microsomal lipids.

Polypeptide composition of bacterial cyclic diguanylic acid-dependent cellulose synthase and the occurrence of immunologically crossreacting proteins in higher plants.

Physiological Functions of the COPI Complex in Higher Plants.

The trafficking of the cellulose synthase complex in higher plants.

Multiple forms of NADPH-cytochrome P450 reductase in higher plants.

Integration of photosynthetic carbon and nitrogen metabolism in higher plants.

Acetyl groups in cell-wall preparations from higher plants.

Metabolic costs of terpenoid accumulation in higher plants.

Uptake and metabolism of 2,4,6-trinitrotoluene in higher plants.

State 2 changes in higher plants and algae.