5 Enzymes in the Arbuscular Mycorrhizal Symbiosis Jose ´ Manuel Garcı ´ a-Garrido, Juan Antonio Ocampo, and Inmaculada Garcı ´ a-Romera Estacio ´ n Experimental del Zaidı ´ n, CSIC, Granada, Spain I. INTRODUCTION Terrestrial fungi can adopt different life strategies to exploit nutrient sources. They grow as saprotrophs on simple or complex organic substrates, or they can establish a nutritional relationship with higher plants, either as biotrophs or as necrotrophs. Mycorrhizal associa- tions are the most important mutualistic biotrophic interactions (1). Over 80% of vascular flowering plants are capable of entering into symbiotic associations with arbuscular mycor- rhizal (AM) fungi (2). The fungi that form these associations are members of the zygomycetes, and the current classification places them all into one order, Glomales (3). They are strictly depen- dent on their host plant to complete their life cycle, whereas other mycorrhizal fungi, such as ericoid fungi, can be grown in pure culture (4–6). The AM association is a relatively nonspecific, highly compatible, long-lasting mutuality from which both partners derive benefit. The plant supplies the fungus with carbon, on which it is entirely dependent. The fungal contribution is more complex. Although it is clear that the fungi assist the plant with the acquisition of phosphate and other mineral nutrients from the soil, AM fungi also may influence the plant’s resistance to invading pathogens (7). In addition to its ecological significance, the association also may have applications in agriculture. This is particularly important for developing more sustainable systems (8) because mycorrhizae create an intimate link between the soil and the plant and may be manipulated to improve plant nutrition efficiency and soil conservation. The interaction begins when fungal hyphae arising from spores in the soil and on adjacent colonized roots or hyphae contact the root surface. Here they differentiate to form appressoria and penetrate the root. The formation of appressoria is one of the first morphological signs that recognition between the plant and the fungus has occurred. Once inside the root, the fungus may grow both inter- and intracellularly throughout the cortex, but AM fungi do not invade the vascular or the meristematic regions. The types of internal structures that develop depend on the plant/fungal combination and may include intracellu- lar differentiated hyphae called arbuscules and/or intracellular coils (9). Wall-like mate- rial containing proteins and polysaccharides is deposited by the continuous host plas- Copyright © 2002 Marcel Dekker, Inc. malemma against the wall of the fungus, forming an interfacial matrix or apoplast (10). Although the fungal hypha penetrates the cortical cell wall to form arbuscules within the cell, it does not penetrate the plant plasma membrane, which extends to surround the arbuscule (11). Arbuscules die after a few days encased in host cell wall material. The senescence of arbuscules does not affect the development of the residual mycelia, which continue to grow and form arbuscules in other parenchymal cells. The complex interaction at the cellular and molecular level that has resulted in a functional AM symbio- sis must be based on highly evolved physiological and genetic coordination between fun- gus and host. The variety of factors that act immediately before and after contact of an AM fungus with a root surface and might influence the success of root colonization is quite broad. However, fungal development within the host may be modulated by the ability of fungus and host to produce enzymes. The purpose of this chapter is to discuss the role of enzymes in the penetration and development of the fungus inside the plant root. II. ENZYMATIC MECHANISMS OF PENETRATION AND FORMATION OF THE SYMBIOSIS The mechanisms by which endomycorrhizal fungi enter and spread through host tissues are unknown. Different steps in the infection process (e.g., formation of entry points, inter- and intracellular colonization) necessitate the growth of hyphae along the middle lamella or through cell walls of the host root. Only localized changes in wall texture have been observed as endomycorrhizal fungi penetrate epidermal cells or develop through the mid- dle lamella of parenchymal tissue, suggesting that wall-degrading enzyme activities within host tissues are very limited (12). Biotrophic fungi usually are thought to penetrate host tissues mechanically. It has been calculated that high pressure can be generated by appressoria of Magnaporte grisea (a nonmycorrhizal fungus) at the penetration point (13). This mechanical pressure allows the fungus to perforate the host wall through formation of a penetration peg. Some wall components, such as melanin, are considered to play an important role in the increase of hydrostatic pressure since they act to trap solutes within the appressoria, causing water to be absorbed because of the increasing osmotic gradient (13). Most phytopathogenic fungi and bacteria are known to produce enzymes that de- grade pectin, cellulolytic, and hemicellulolytic substances (14). These hydrolytic enzymes play a fundamental role in pathogenesis (15,16). Polygalacturonase plays multiple roles during infection; this enzyme allows the fungus to colonize the host tissues and to obtain nutrients from the degradation of complex pectic substrates. Concomitantly, polygalactur- onases can produce oligogalacturonides, which elicit plant defense response (17). Many of the enzymes that degrade pectic, cellulolytic, and hemicellulolytic sub- stances are produced by the plants themselves, including the fruits, epicotyls, cotyledons, and other growing tissues (18–20). Research is scarce on these enzymes in plant roots, and on their mode of action in the process of penetration and development of symbiotic microorganisms (21). Infection of roots by other mutualistic microorganisms, such as Rhi- zobium and Azospirillum species, appears to be mediated by cell wall–hydrolyzing en- zymes (22–24). The observation that arbuscular mycorrhizal (AM) fungi penetrate the plant cell wall at the site of contact during the establishment of intracellular symbiosis (25) indicates that Copyright © 2002 Marcel Dekker, Inc. hydrolytic enzymes may be involved in the AM colonization process. However, since AM fungi have not yet been cultured axenically in the absence of plant roots, it is difficult to confirm the production of hydrolytic enzymes by AM fungi or their possible participation in the colonization of roots. This is because of the very low levels of enzyme produced, as occurs with the other mutualistic microorganisms (24). Investigations have demon- strated the production of pectinase, cellulase, and xyloglucanase (5,6,26–32) from the external hyphae and the mycorrhizal roots. It seems that mycorrhizal fungi colonize the root tissues of their host plant by a combination of mechanical and enzymatic mechanisms (33,34). A very weak and localized production of hydrolytic enzymes by AM fungi might ensure that viability of the host is maintained and defense responses are not triggered, allowing compatibility between plant and fungi (17). The primary (growing) cell walls of plants are rigid yet dynamic structures com- posed of roughly equal quantities (around 30% for each) of cellulose, hemicellulosic, and pectic polysaccharides, plus about 10% glycoproteins (hydroxyproline-rich glycoproteins and enzymes) and a small proportion of phenolic compounds (35,36). The cell wall com- prises a crystalline microfibrillar array of cellulose embedded in an amorphous mass of pectic and hemicellulose materials. The AM fungi hydrolyze these cellular complexes in a very organized manner to make their entry into the root cortical cells (37). The mode of action of some of the important enzymes and the role of these enzymes in the penetration of the fungus inside the plant root are discussed later. A. Cellulases Cellulose is the best known of all plant cell wall polysaccharides. It is particularly abundant in secondary cell walls and accounts for 20%–30% of the dry mass of most primary cell walls (38). Chemically, cellulose is a linear β-4-linked d-glucan that provides the mechani- cal strength of plant cell walls. Cellulose self-associates by intermolecular hydrogen bond- ing to form microfibrils of at least 36 glucan chains and becomes strongly associated with hemicellulose in the cell wall. Indeed, it has been suggested that the diameter of the cellu- lose microfibril may be determined, at least in part, by the binding of hemicellulose during cellulose synthesis, which prevents combining of small microfibrils into larger bun- dles (39). Cellulases comprise a number of extracellular β-1,4-glucanases. Endohydrolases randomly disrupt internal linkages throughout β-1,4-glucan chains, producing glucose, cellobiose, and high-molecular-weight fractions. Exohydrolases or β-1,4-cellobiohydro- lases act only on the exposed ends of β-1,4-glucan chains releasing the disaccharide cello- biose (17). β-Glucosidase and cellobiohydrolase also are part of the cellulase complex of some microorganisms. Because of its crystalline nature, native cellulose is degraded slowly. Plant pathologists generally have thought that cellulases are not particularly impor- tant in pathogenesis since extensive cellulose degradation typically occurs only late in infection, if at all. However, when the major endoglucanase genes of the phytopathogenic bacteria Pseudomonas solanacearum and Xanthomonas campestris pv. compestris were disrupted, virulence decreased (14). Extracts of arbuscular mycorrhizal fungus (AMF) spores and external mycelium of G. mosseae have been shown to have endo- and exoglucanase activities (27). The enzyme activities in spores and external mycelium indicated which types of enzymes are found in mycorrhizae during root colonization. Endo- and exoglucanase activities increased in plants colonized by AMF when G. mosseae was in its logarithmic stage of growth (40). No Copyright © 2002 Marcel Dekker, Inc. relationship was found between number of vesicles and endo- and exoglucanase activities, although the maximum hydrolytic activities coincided with the beginning of entry point formation and arbuscule development (40). Endoglucanases are present in noncolonized roots during growth and development (41). Several electrophoretic bands of endoglucanase activity observed in colonized plants had the same mobility as in noncolonized plants; however, some of these bands were present at earlier stages of plant growth in mycorrhizal plants than in nonmycorrhizal plants (27). The presence of bands different from those observed in nonmycorrhizal roots or external mycelia suggests that some of this activity may be induced by the fungus in the plant (Fig. 1). These findings indicate that endoglucanases produced by either the plant or the AM fungus may be involved in the process of host wall degradation. Some of the endoglucanase activity can be attributed to the extramatrical phase of the AM fungi since at least one of the endoglucanase activities found in the external mycelium and in the mycorrhizal root extracts showed the same electrophoretic mobility (42,43) (Fig. 1). En- doglucanase (EC 3.2.1.4) was purified from roots of onion (Allium cepa) colonized by G. mosseae. The endoglucanase has a relative molecular weight of about 27 kD and behaves as a monomer in its native form (44). B. Pectinases Pectins and related polysaccharides provide a protective material between plant cells. The term pectin encompasses a complex group of polysaccharides, some of which may be structural domains of larger, more complex molecules. The classic pectin fraction from Figure 1 Nondenaturing polyacrylamide gradient gel electrophoresis of cellulase on 4–12% acryl- amide. Lane 1, extract from non-AM onion roots; lane 2, extracts from AM onion roots; lane 3, extracts from external mycelium of Glomus mosseae. Copyright © 2002 Marcel Dekker, Inc. oat seedlings contains 23% galacturonic acid and earlier it was thought that pectin con- sisted solely of α-d-1,4-linked galacturonic acid residues. Today, all evidence suggests that other sugars are covalently attached to the polygalacturonide backbone and that other sugars may even form an integral part of the main chain (45,46). Pectinolysis is carried out by a complex of enzymes (pectinases), which include endo- and exopectate lyase (PL), endo- and exopolygalacturonase (PG), and pectin meth- ylesterase (PME). Degradation of pectin was reported for a sterile ericoid mycelium isolated from Calluna vulgaris by Perotto and associates (4) and Cairney and Burke (47). A wide range of ericoid fungi from different geographic regions was capable of growing on pectin as a sole carbon source. Ericoid fungi seem to use polygalacturonase during their saprotrophic life. Attempts to demonstrate pectinase in extracts from AM tissues have not been suc- cessful (48). However, catabolic repression experiments by Garcı ´ a-Romera and colleagues (49) showed that pectolytic enzymes may be involved in the process of root colonization by AM fungi. The spores and external mycelium of G. mosseae possess a complex of pectinolytic (pectin esterase [PE], pectin lyase [PL], pectatolyase [PNL], polygalacturo- nase [PG], and polymethylgalacturonase [PMG]) activities (26). The production of hy- drolytic enzymes was studied during the process of penetration and development by G. mosseae in plant roots (29). The PE activity was consistently higher throughout the process of root colonization in plants inoculated with AM fungi than in controls. PE is thought to facilitate the action of the other pectinase enzymes (50). PMG and PNL (pectinolytic) activities were higher during the logarithmic stage of AM development in plants inoculated with the fungus than in nonmycorrhizal plants. The increase in fungal structures that pene- trate the cell wall during the logarithmic stage of root colonization may explain the in- crease in PMG and PNL activities at this time. However, PG and PL (pectolytic) activities in AM plants were similar to those in controls throughout the experiment (29). The lack of differences in these degradative enzymes is not, however, conclusive evidence that they do not participate in the colonization process. It may indicate that PG and PL are involved during other stages of development (i.e., appressoria formation and penetration) in view of the presence of these enzymes in the extracts of spores and external mycelium of AM fungi (26). The simultaneous presence of polygalacturonase produced by the fungus and of pectins secreted by the plant in the interfacial matrix suggests that the fungus might use pectins as a food source (31), as suggested by Dexheimer and coworkers (51). Wall-degrading pectic enzymes uniquely associated with the interface of fine arbus- cule branches may contribute to the interference of wall formation of the host plant. Active H ϩ adenosine triphosphatase (ATPase) on fungal and plant membranes bordering the inter- face suggest that protons accumulate in the interfacial matrix and the resulting change in pH also could contribute to wall loosening (52). C. Hemicellulases Hemicelluloses are an integral part of all plant cell walls and form about 25% of the total dry weight of annuals and up to 40% in woody species. Hemicelluloses consist of chains of sugars in nonfibrillar organization that are linked to cellulose microfibrils by weak hydrogen bonds. In dicot primary walls, the major hemicelluloses are neutral xyloglucans and acidic arabinoxylans; in monocots they are acidic arabinoxylans and neutral β-(1-3,1- Copyright © 2002 Marcel Dekker, Inc. 4)-glucans(53).Xyloglucansareβ-1,4-glucanswithsidechainsthatcanhydrogenbond tocellulosemicrofibrils,cross-linkingthemandrestrainingcellexpansion.Inadditionto astructuralrole,xyloglucanscanbehydrolyzedbyhydrolyticenzymes,andtheoligosac- charidesproducedmayactassignalmolecules(15,54). Theplantcellwallcontainsglucanasesandglycosidasesthathydrolyzexyloglucan intomonosaccharides.Endo-β-1,4-glucanaseactivityisresponsibleforthefirststepof degradationwherebythexyloglucanisendohydrolyzedintolargefragmentsandexo-1,4- glucanaseactivityliberateslow-molecular-weightfractionsfromtheendsoflongpolysac- charidechains(41).Theproductionofhemicellulolyticenzymeshasbeenobservednot onlyinparasitesbutalsoinmutualisticmicroorganismssuchasRhizobiumspecies(24) andarbuscularmycorrhiza(28). Endoxyloglucanaseactivityincreasesduringgrowthanddevelopmentofroots(55). Thisactivitywasconsistentlyhigheratthebeginningofcolonizationandthelogarithmic stageofdevelopmentofmycorrhizalfungus(55).Theincreaseinfungalstructuresthat penetratethecellwallduringthelogarithmicstageofrootcolonizationmayexplainthe increaseinthedifferentactivitiesatthistime(56).Theevolutionofendoxyloglucanase activitiesinplantsparalleledthechangesintheexternalmycelium.Therewere,however, bandsofxyloglucanaseactivityinnonmycorrhizalrootsthatwereabsentinmycorrhizal roots;thatmaysuggestqualitativeinhibitionbythefungusofsomeplantactivity.Inhibi- tionofplantproteinsynthesisbyAMfungihasbeenobservedinseveralplant–AMfungi associations(57,58). III.ENZYMESINTHEPHYSIOLOGYOFTHEASSOCIATION A.PhosphorusUptake Itnowisestablishedthatmycorrhizalcolonizationcanenhancetheuptakefromsoilof solubleinorganicPbyplantroots(59).Althoughparticularlyimportantinlow-Psoils, anincreasedrateofPuptakecanoccuroverarangeofsoilPlevelsevenwhenmycorrhizal growthresponsesnolongeroccur.TheenhancedPuptakebymycorrhizalplantsismost likelytheresultoftheexternalfungalhyphae’sactingasanextensionoftherootsystem, therebyprovidingamoreefficient(moreextensiveandbetterdistributed)absorbingsur- faceforuptakeofnutrientsfromthesoilandfortranslocationtothehostroot(60).External hyphaeofAMfungimustabsorborthophosphate(Pi)byactivetransport(59,61).They haveanactiveH ϩ -ATPaseintheplasmamembranethatwouldbecapableofgenerating therequiredproton-motiveforcetodriveH ϩ -phosphatecotransport,andPcertainlyis accumulatedtohighconcentration(62). Polyphosphate(poly-P)isamajorPreserveinmanyfungianditaccumulatesin vacuolesofAMfungi(63).Transferofmycorrhizalrootsfromlow-tohigh-Pmedia resultsinarapidaccumulationofpoly-P(64).Enzymesofpoly-Psynthesishavebeen foundinmycorrhizaltissue(63,65).Polyphosphatekinase,whichcatalyzesthetransfer oftheterminalphosphatefromATPtopoly-P,wasdetectedinbothexternalhyphaeand mycorrhizalrootsbutnotinuninfectedroots,indicatingthatpoly-Pcanbesynthesized onlybythefungalcomponentofthemycorrhiza. AlthoughitnowseemslikelythatPistranslocatedbyprotoplasmicstreaminginto theintraradicalhyphaeaspoly-P(66),littleisyetknownofthebiochemicalmechanisms involved.Thetransportthroughthehyphaeandunloadingstepswithinthearbusculemay belinkedtopoly-Pmetabolism(Fig.2).Highproportionoflong-chainpoly-Ptototal Copyright © 2002 Marcel Dekker, Inc. Figure 2 Enzymes involved in P transport in AM roots. poly-P was observed in the external hyphae, and short-chain poly-P was higher in the internal hyphae (67). Long-chain poly-P seems to be more efficient in transporting Pi from the extraradical to the intraradical part of the fungi. Activity of enzymes of polyphosphate breakdown (exopolyphosphatase and endopolyphosphatase) is greater in mycorrhizal roots than in uninfected roots (65). Both enzymes have been detected in extracts of internal hyphae, but not those of external hyphae. The long-chain poly-P may be partly hydrolyzed into short-chain poly-P with endopolyphosphatase. Depolymerized short-chain poly-P may be hydrolyzed further with exopolyphosphatase to liberate Pi (67). Alternatively, the reaction catalyzed by polyphosphate kinase is readily reversible, so there is also the possi- bility that poly-P could be hydrolyzed, liberating ATP (63). The Pi (or ATP) so released in the arbuscule then would be transferred into the host (66). Copyright © 2002 Marcel Dekker, Inc. The presence of intense ATPase activity indicates there is a carrier that mediates active transport mechanisms for Pi uptake at the host plasmalemma. ATPase activity has been observed in both plant and fungal plasma membranes and in the interfacial matrix associated with young arbuscules that decreased with senescence of arbuscules (68). A lack of H ϩ -ATPase activity in the host periarbuscular membrane surrounding nonfunc- tional arbuscules has been reported (69). Other enzymes also have been implicated in P metabolism. Mycorrhizal-specific alkaline phosphatase is located in the vacuole of extraradical and intraradical hyphae (70– 72). Maximum activity occurs while infections are young (100% arbuscular), coinciding with the start of the mycorrhizal growth response, but disappears with degeneration and collapse of the arbuscule. This enzyme appears to be of fungal origin. However, the role of alkaline phosphatase in Pi metabolism is still unknown (59,71). The amount of P in soil available to plants is small, about 1% to 5% of the total P content. This finding has led to the suggestion that AM fungi are capable of utilizing insoluble P sources. Organic phosphates in soil may be utilized by plants through the action of phosphatases. Phosphatase activity in soil may originate from the plant roots or from microorganisms (73,74). High levels of acid and alkaline phosphatases have been found in the roots (70) and rhizosphere (75,76) of plants colonized by AM fungi. This increase in phosphatase activity would result in Pi’s being liberated from organic phos- phates immediately adjacent to the cell surface to be captured by the uptake mechanisms of mycorrhizal fungi. Some results have shown exudation of phosphatase by the external hypha and efficient hydrolysis of phytate-P by the phosphatase of mycorrhizal hyphae (76). Acid phosphatase activity release was visually shown as a red-colored ‘‘hyphal print’’ on filter paper treated with napthyl phosphate and Fast Red TR (diazotized 2-amino-5 chlorotoluene 1,5-naphthalene disulphonate) (77). However, other results indicated that the role of fungal phosphatases in P uptake from organic P is not clear: (1) extracellular phosphatase activity of mycorrhizal roots was stimulated in the presence of easily hy- drolyzed substrates (76) but repressed by nonhydrolyzable forms of organic P (Po) (78), (2) no effect of mycorrhiza on specific activity of phosphatase was detected for clover grown in soil amended with 32 P-labeled organic matter (79), (3) the production of phospha- tase varied with the choice of host plant and fungal endophyte (75,78), and no relationship between the level of AM colonization and phosphatase activity in different wheat cultivars has been found (80,81), (4) the addition of P fertilizer and CaCO 3 to soils decreased AM colonization but increased phosphatase activity in the plant rhizosphere (82); and (5) soil microorganims can mineralize organic P and AM hyphae may use Pi derived from their activity (83). Thus the results obtained are conflicting despite much effort (84). One of the most important factors involved in controlling AM colonization of roots is soil and plant P. High P concentrations inhibit mycorrhizal colonization (60,85). The activity of mycorrhiza-specific alkaline phosphatases of Glomus species declines at high P levels (86,87). These observations suggest these enzymes would be involved in the regulation of mycorrhizal colonization of roots by P content of plants (85). However, high soil P concentration decreased AM colonization of roots by Gigaspora species but did not affect alkaline phosphatase activity (72). Thus the mechanism whereby the internal P content of the host regulates mycorrhizal infection is not clear. B. Nitrogen Metabolism Mycorrhizal plants sometimes improved nodulation and N fixation (88), an effect that may be due to enhanced P uptake (89). However, AM contribute to the N nutrition of the Copyright © 2002 Marcel Dekker, Inc. host by assimilation of soil nitrogen (N). The plant growth response to AM colonization may be greater in the presence of NH 4 than NO 3 (59). Ammonium N and nitrate N have different pathways for metabolism, cation-carbox- ylate storage, and pH regulation, and hence they have rather different biochemical and physiological implications for the host (89). Nitrate is reduced, first to nitrite by nitrate reductase, then to ammonium by nitrite reductase. Ammonium N, once inside the cell, becomes directly incorporated into the various pathways for amino acid synthesis. Assimi- lation may be by glutamate dehydrogenase (GDH), or via glutamine synthetase (GS) and glutamate synthase (GOGAT) with the formation of glutamate. GS activity is increased in mycorrhizal root systems, partly as a result of a contribution from the fungi themselves; activity has been detected in fungal tissue separated from mycorrhizal roots (90). Improved P nutrition in the plants resulted in only a small increase in activity, confirming that the fungi have an important contribution and that GS is not related to P nutrition. In contrast, GDH activity showed no direct relationship with colonization (90). This limited evidence suggests that the fungi may have the capacity to assimilate NH ϩ 4 and, in consequence, N is likely to be transferred from fungi to plants in organic form. Nitrate reductase activity has been detected in isolated spores of AM fungi (91,92). There are suggestions that either the AM fungi increase the nitrate reductase activity in the host plant (regardless of the P content) or the AM fungi have enzymatic activity per se (93). The fungal nitrate reductase messenger ribonucleic acid (mRNA) was detected in arbuscules but not in vesicles by in situ hybridization (94). The observation that AM fungi possess the gene coding for assimilatory nitrate reductase does not rule out the possibility that plant root cells mainly reduce nitrate in the AM symbiosis (95). The plant colonized by different AM fungi showed different nitrate reductase activity (93). Nitrate reductase and glutamine synthetase decreased with the age of mycorrhizal plants (96). Nitrite formation catalyzed by nitrate reductase was mainly reduced nicotinamide-adenine dinucleotide phosphate–(NADPH)-dependent in roots of AM colonized plants but not in those nonmycorrhizal plants, a finding consistent with the fact that the nitrate reductases of fungi preferentially utilized NADPH as the reductant (94). These investigations suggest that the fungus in AM-colonized root performs nitrate uptake and nitrate reduction to some degree. Because of its toxicity, the nitrite formed probably is not exported from the fungal to the plant cells. Other enzymes of nitrate assimilation have been described to occur in AM fungi (90). Thus nitrite reductase, glutamate synthetase, and glutamate syn- thase may transform nitrite, and N compounds (e.g., ammonium, glutamine, glutamate) probably are transferred from arbuscules to host cells. C. Carbohydrate Assimilation It is commonly accepted that the AM fungi are obligate symbionts and that carbohydrates are transferred from autotroph to heterotroph. It is likely that the fungus obtains the bulk of its carbon from host sugars; short-term 14 CO 2 labeling experiments have shown transport of photosynthate from the host to the fungus (97). Most (70% to 90%) of the 14 C label present in both the roots and mycelium was in the form of soluble carbohydrates. The carbohydrates, predominantly sucrose, are delivered to the apoplast by the host cell. Then sucrose is hydrolyzed in the apoplastic interface by an acid invertase of plant origin, and the resulting hexoses are absorbed by the fungus (59), and used for trehalose synthesis. Trehalose has been shown to accumulate in both spores and external hyphae of AM fungi (66,98,99). Trehalose was detected in roots of colonized plants but not of control plants (56). Polyphosphates may serve in phosphorylation for the active transport of carbon skele- Copyright © 2002 Marcel Dekker, Inc. tons into the arbuscule from the host either through the ATP produced by degradative polyphosphate kinase action or through a direct phosphorylation of sugars by enzymes of the polyphosphate glucokinase type (64). On the other hand, trehalase has been found in plants (100), and this enzyme increased upon mycorrhizal colonization (101). The biologi- cal function of plant trehalases is unknown, but they might be involved in the degradation of trehalose released from senescent AM fungus. The possible metabolic pathways of carbon utilization in AM are largely uninvesti- gated. Dehydrogenases indicative of glycolysis are found in hyphae, vesicles, arbuscules, and spore germ tubes (102). From this, MacDonald and Lewis (102) have inferred that AM fungi employ the Embden–Meyerhof–Parnas glycolytic scheme, the hexose mono- phosphate shunt (or pentose phosphate cycle), and the tricarboxylic acid cycle. A cyanide-insensitive respiratory pathway has been noted in AM roots (103). Such a pathway of electron transport to oxygen has been established in the sheath tissue of ectomycorrhizal roots (104). This pathway is not coupled to oxidative phosphorylation and may operate when oxidative phosphorylation is reduced by adenosine diphosphate (ADP) limitations. It is likely that the operation of such a pathway would increase the overall utilization of carbohydrates in mycorrhizal tissues (66). IV. ENZYMES IMPLICATED IN THE HOST DEFENSE RESPONSE TO ARBUSCULAR MYCORRHIZAL FUNGAL COLONIZATION Arbuscular mycorrhizal fungal penetration and establishment in the host roots involve a complex sequence of events and intracellular modifications that influence root colonization (25). Genotype and environmental factors influence the infection intensity or even the host compatibility and/or resistance (33,105). The key to understanding the phenomenon of compatibility is to study recognition mechanisms and molecules involved in early stages of the AM interaction. In this sense, the formation of appressoria is one of the first morphological signs that recognition be- tween the plant and the fungus has occurred. Some authors suggest that plant defense reactions may occur only after appresorium formation when the fungus has changed its state from saprophytic to infective (106). Although AM fungi are considered as biotrophic microorganisms and biotrophs gen- erally exhibit a high degree of host specificity, most AM fungi that have been studied show little or no specificity and are not thought to induce typical defense responses in host plants. Nevertheless, some plant resistance markers have been investigated in compatible symbiotic AM fungus–root interactions, and the early activation of certain plant defense genes has been shown (105). Since the plant host can elicit a weak defense response to the invading fungus, this may be a natural mechanism to control the number and/or location of infections. Furthermore, some phenomena of suppression of defense responses have been demonstrated in mycorrhizal roots (107,108). Whether this suppression is systemic or restricted to the infected area or whether products of symbiosis-related plant genes sup- press the defense genes directly or through activation of fungal-derived suppressors re- mains to be elucidated. So far, it is not known how the induction/suppression of mecha- nisms associated with plant resistance could participate in the phenomenon of compatibility between plant roots and AM fungi. The investigation of early events and molecules involved in fungal–plant interactions is crucial for a better understanding of symbiosis. Copyright © 2002 Marcel Dekker, Inc. [...]... their role in defense response The first class involves hydrolases such as chitinases and -1 ,3-glucanases that act directly as potent inhibitors of fungal growth The second class involves enzymes related to oxidative stress such as catalases and peroxidases, and the third class consists of key enzymes that catalyze core reactions in phenylpropanoid metabolism A Chitinases and -1 ,3-Glucanases The initiation... peroxidase activity increased linearly with increasing P supply, suggesting a role of peroxidase in limiting AM infection in well-P-nourished plants (140) The analysis of catalase and ascorbate peroxidase activities during the early stage of tobacco–Glomus mosseae interaction revealed transient enhancements of both enzymatic activities in the inoculated plants (141) These increases coincided with the stage of.. .In the following sections, some results obtained from studying the enzymatic activities produced by the host are reviewed and their contribution in the induction/suppression of mechanisms associated with plant resistance and in the control of intraradical fungal growth and maintenance of the symbiotic status is discussed For ease of discussion the defense-related activities are divided into three... the extent, timing, and enzymatic activities and compounds released appear to depend on the plant Copyright © 2002 Marcel Dekker, Inc and fungal genotypes involved In situ localization of transcripts encoding PAL and CHS in mycorrhizal roots showed that the transcripts were discretely localized in cells containing arbuscules ( 155 ) The expression of other gene encoding enzymes in the flavonoid/ isoflavonoids... acid and alkaline phosphatases in onion roots infected by Glomus mosseae (Nicol and Gerd.) New Phytol 82: 127–132, 1979 71 V Gianinazzi-Pearson, E Dumas-Gaudot, S Gianinazzi Proteins and proteins activities in endomycorrhizal symbioses In: A Varma, B Hock, eds Mycorrhiza 2nd ed Berlin: Springer-Verlag, 1999, pp 255 –272 72 CL Boddington, JC Dodd Evidence that differences in phosphate metabolism in mycorrhizas... vascular cylinder (121) Nevertheless, the accumulation of chitinase and -1 ,3-glucanase transcripts has been observed around a number of cortical cells containing arbuscules (120,121), suggesting that the encoded enzyme might be involved in the control of intraradical fungal growth The accumulation of -1 ,3-glucanase mRNA in cells containing arbuscules was modulated by P concentration The higher level of mRNA... suppression was found in roots of soybean plants infected with the more infective isolate of Glomus intraradices (116) In some particular plant–fungal combinations, the initial increase of chitinase activity was not followed by suppression, and higher levels of activity persisted (117) In some cases, no changes in chitinase and -1 ,3-glucanase activities were detected between inoculated and noninoculated plants... 19 95 B Dassi, E Dumas-Gaudot, A Asselin, C Richard, S Gianinazzi Chitinase and -1 ,3-glucanase isoforms expressed in pea roots inoculated with arbuscular mycorrhizal or pathogenic fungi Eur J Plant Pathol 102:1 05 108, 1996 MJ Pozo, E Dumas-Gaudot, S Slezack, C Cordier, A Asselin, S Gianinazzi, V Gianinazzi´ Pearson, C Azcon-Aguilar, JM Barea Induction of new chitinase isoforms in tomato roots during interactions... found, depending on the specific interaction of Medicago and fungal species (107,111, 154 , 155 ) Some of these compounds stimulated hyphal growth ( 154 ) Formononetin was found to accumulate in Medicago sativa roots in the presence of the fungus Glomus intraradix, before fungal penetration and colonization (111) The analysis of enzymatic activities and accumulation of mRNA transcripts encoding enzymes of flavonoid/... initiation of chitinase and other hydrolase activities is predominantly one of the coordinated and widespread mechanisms of plant defense against pathogen attack There is good evidence that the action of the endohydrolases leads to detrimental effects, such as the inhibition of hyphal growth by invading fungi, as well as the probable release of signaling molecules (β-glucans and chitin/chitosan oligomers) . Dekker, Inc. Figure 2 Enzymes involved in P transport in AM roots. poly-P was observed in the external hyphae, and short-chain poly-P was higher in the internal hyphae (67). Long-chain poly-P seems. Inc. 4)-glucans (53 ).Xyloglucansare -1 ,4-glucanswithsidechainsthatcanhydrogenbond tocellulosemicrofibrils,cross-linkingthemandrestrainingcellexpansion.Inadditionto astructuralrole,xyloglucanscanbehydrolyzedbyhydrolyticenzymes,andtheoligosac- charidesproducedmayactassignalmolecules( 15, 54). Theplantcellwallcontainsglucanasesandglycosidasesthathydrolyzexyloglucan intomonosaccharides.Endo- -1 ,4-glucanaseactivityisresponsibleforthefirststepof degradationwherebythexyloglucanisendohydrolyzedintolargefragmentsandexo-1, 4- glucanaseactivityliberateslow-molecular-weightfractionsfromtheendsoflongpolysac- charidechains(41).Theproductionofhemicellulolyticenzymeshasbeenobservednot onlyinparasitesbutalsoinmutualisticmicroorganismssuchasRhizobiumspecies(24) andarbuscularmycorrhiza(28). Endoxyloglucanaseactivityincreasesduringgrowthanddevelopmentofroots (55 ). Thisactivitywasconsistentlyhigheratthebeginningofcolonizationandthelogarithmic stageofdevelopmentofmycorrhizalfungus (55 ).Theincreaseinfungalstructuresthat penetratethecellwallduringthelogarithmicstageofrootcolonizationmayexplainthe increaseinthedifferentactivitiesatthistime (56 ).Theevolutionofendoxyloglucanase activitiesinplantsparalleledthechangesintheexternalmycelium.Therewere,however, bandsofxyloglucanaseactivityinnonmycorrhizalrootsthatwereabsentinmycorrhizal roots;thatmaysuggestqualitativeinhibitionbythefungusofsomeplantactivity.Inhibi- tionofplantproteinsynthesisbyAMfungihasbeenobservedinseveralplant–AMfungi associations (57 ,58 ). III.ENZYMESINTHEPHYSIOLOGYOFTHEASSOCIATION A.PhosphorusUptake Itnowisestablishedthatmycorrhizalcolonizationcanenhancetheuptakefromsoilof solubleinorganicPbyplantroots (59 ).Althoughparticularlyimportantinlow-Psoils, anincreasedrateofPuptakecanoccuroverarangeofsoilPlevelsevenwhenmycorrhizal growthresponsesnolongeroccur.TheenhancedPuptakebymycorrhizalplantsismost likelytheresultoftheexternalfungalhyphae’sactingasanextensionoftherootsystem, therebyprovidingamoreefficient(moreextensiveandbetterdistributed)absorbingsur- faceforuptakeofnutrientsfromthesoilandfortranslocationtothehostroot(60).External hyphaeofAMfungimustabsorborthophosphate(Pi)byactivetransport (59 ,61).They haveanactiveH ϩ -ATPaseintheplasmamembranethatwouldbecapableofgenerating therequiredproton-motiveforcetodriveH ϩ -phosphatecotransport,andPcertainlyis accumulatedtohighconcentration(62). Polyphosphate(poly-P)isamajorPreserveinmanyfungianditaccumulatesin vacuolesofAMfungi(63).Transferofmycorrhizalrootsfromlow-tohigh-Pmedia resultsinarapidaccumulationofpoly-P(64).Enzymesofpoly-Psynthesishavebeen foundinmycorrhizaltissue(63, 65) .Polyphosphatekinase,whichcatalyzesthetransfer oftheterminalphosphatefromATPtopoly-P,wasdetectedinbothexternalhyphaeand mycorrhizalrootsbutnotinuninfectedroots,indicatingthatpoly-Pcanbesynthesized onlybythefungalcomponentofthemycorrhiza. AlthoughitnowseemslikelythatPistranslocatedbyprotoplasmicstreaminginto theintraradicalhyphaeaspoly-P(66),littleisyetknownofthebiochemicalmechanisms involved.Thetransportthroughthehyphaeandunloadingstepswithinthearbusculemay belinkedtopoly-Pmetabolism(Fig.2).Highproportionoflong-chainpoly-Ptototal Copyright. Wall-like mate- rial containing proteins and polysaccharides is deposited by the continuous host plas- Copyright © 2002 Marcel Dekker, Inc. malemma against the wall of the fungus, forming an interfacial