1992 Raskin Review

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Annu. Rev. Plant Physiol. Plant Mol. Bioi. 1992.43:439-463 Copyright © 1992 by Annual Reviews Inc. All rights reserved ROLE OF SALICYLIC ACID IN PLANTS I. Raskin AgBiotech Center, Rutgers University, Cook College, PO Box 231, New Brunswick, New Jersey 08903-0231 K EY WORDS: aspirin, systemic acquired resistance, hypersensitive reaction, pathogen resistance, thermogenesis, heat production CONTENTS INTRO D U CT ION ... .... .... ...... ... .. ... .. ....... ....... . . . ............. .. .............. . .. ... . . .... 439 History of Salicylates . . .......................................................................... 439 General Properties of Salicylic Acid.... ... ............. . . . .. .... . ............ . .. . . ........... 441 Salicylic Acid Levels in Plants .. .. ........... ... . . . ....... .............. ...... ..... . . . ......... 442 EFFECTS OF EXO GENOUS SALICYLIC ACID ............ . . .. .............. . .. . ........... 442 Salicylic Acid and Flowering . ...... ...... .. . .. . . .. ............. .. ................ . . . . . ......... 442 Allelopathic Properties of Salicylic Acid : Effect on Membranes and Ion Uptake. . . . 444 Other Effects of Exogenously Applied Salicylic Acid .... .... .......... . . . . ... ..... . .... ... 445 SALICYLIC A CID A ND H EAT PRODU CTIO N IN PLANTS .... . . ..... . . ... . . ... . .... ... 445 Thermogenic Plants and Search for Calorigen. ... . .. .. .. . . . . ... ....... . .. 445 Salicylic Acid : A Natural Inducer of Thermogenesis in Arum Lilies .. .. ..... .......... 446 SALICYLIC ACID A ND D IS EAS E RES ISTA NCE............. ... ...... . ......... . .......... 447 Disease Resistance in Plants: Effects of Salicylic Acid ... . . . .............. . .. . ... . ....... 447 Salicylic Acid : A Likely Signal for Disease Resistance in Plants. .... .. . . . . . ... . . . ... .. 450 SALICYLIC ACID BIO S Y NTHES IS IN PLANTS . .. . .... .. . ... ............ .. . . . . . . .. . ..... .. 451 Biosynthetic Pathway. . . .............. .... . .. . . .... . .. .. . ... ................. .. . .. . . .. . .......... . 451 Biosynthetic Enzymes.... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......... 453 SALICYLIC ACID M ETABOLISM . . . . .. . ....... . .............................................. 454 M I CRO BIAL PRODU CT IO N OF SALICYLIC ACID ........................................ 454 CONCLUDING REMARKS . . ....... . . . . . . . . . . .. . . . .... . ....... . . . ...... ............ . ... . . .. . . . . INTRODUCTION Histo of SaUcy/ates 455 Centuries before medical scientists identified the numerous therapeutic effects of salicylates, the inhabitants of the Old and New Worlds independently 439 0066-4294/92/0601-0439$02.00 Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 1992.43:439-463. Downloaded from www.annualreviews.org by Universidade de Sao Paulo (USP) on 06/21/11. For personal use only.

Transcript of 1992 Raskin Review

Annu. Rev. Plant Physiol. Plant Mol. Bioi. 1992.43:439-463 Copyright © 1992 by Annual Reviews Inc. All rights reserved

ROLE OF SALICYLIC ACID IN

PLANTS

I. Raskin

AgBiotech Center, Rutgers University, Cook College, PO Box 231, New Brunswick, New Jersey 08903-0231

K EY WORDS: aspirin, systemic acquired resistance, hypersensitive reaction, pathogen resistance, thermogenesis, heat production

CONTENTS

INTRODUCTION . . . . . . . .... . . . . . . ... . . . . . .. . . . . . . . ........ .. . . . . . . . . ..... . . . . . . . . . . . . . . .. . . . . . . . . . . . . 439 History of Salicylates .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 439 General Properties of Salicylic Acid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441 Salicylic Acid Levels in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 442

EFFECTS OF EXOGENOUS SALICYLIC A CID . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 442 Salicylic Acid and Flowering . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 442 Allelopathic Properties of Salicylic Acid : Effect on Membranes and Ion Uptake. .. . 444 Other Effects of Exogenously Applied Salicylic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445

SALICYLIC ACID A ND H EAT PRODU CTION IN PLANTS . . . . . . . . . . . . . . . . . . . .. . . . . . . . . 445 Thermogenic Plants and Search for Calorigen. . . . . . . .. . . . . . . . . . ...... . . . . 445 Salicylic Acid : A Natural Inducer of Thermogenesis in Arum Lilies . . . . . . . . . .... . . . . . . 446

SALICYLIC ACID A ND D IS EAS E RES ISTANCE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447 Disease Resistance in Plants: Effects of Salicylic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447 Salicylic Acid : A Likely Signal for Disease Resistance in Plants. .... . . . . . . . . . . . .. . . . .. 450

SALICYLIC ACID BIOSYNTHES IS IN PLANTS . .. . . ... .. . . . . . . . . . . . . ...... . . . . . . . . . . . . . . .. 451 Biosynthetic Pathway. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451 Biosynthetic Enzymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453

SALICYLIC ACID M ETABOLISM . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454

MI CRO BIAL PRODU CT IO N OF SALICYLIC ACID . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454

CO NCLUDING REMARKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

INTRODUCTION

History of SaUcy/ates

455

Centuries before medical scientists identified the numerous therapeutic effects of salicylates, the inhabitants of the Old and New Worlds independently

439 0066-4294/92/0601-0439$02.00

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discovered that the leaves and bark of the willow tree cured aches and fevers. In the 4th century Be Hippocrates gave willow leaves to women to chew as a pain reliever during childbirth. Long before Columbus, North American Indians used mashed extracts of willow tree bark as compresses to relieve pain.

The story of salicylates has been summarized by Weissmann (162). French and German scientists pursuing folklore concerning the healing powers of willow bark competed to isolate the active principle of willow bark. In 1828 Johann Buchner, working in Munich, successfully isolated a tiny amount of salicin-a salicyl alcohol glucoside, which was the major salicylate in willow bark. The name salicylic acid (SA), from the Latin Salix, a willow tree, was given to this active ingredient by Raffaele Piria in 1838. During the 19th century SA and other salicylates, mainly methyl esters and glucosides easily convertible to SA, were isolated from a variety of plants, including spirea and wintergreen. The first commercial production of synthetic SA began in Germany in 1874. Aspirin, a trade name for acetylsalicylic acid, was in­troduced by the Bayer Company in 1898 and rapidly became one of the world's best-selling drugs. Aspirin was as effective as SA and caused much less irritation of the human digestive system.

Americans consume over 16,000 tons of aspirin tablets annually at a cost of about $2 billion a year. Writes Weissmann (162), "At the lowest doses-less than one tablet a day-aspirin can be used to treat and prevent heart attacks and to prevent cerebral thrombosis. Two to six tablets a day (one to three grams) are useful for reducing pain and fever. And much higher doses (four to eight grams a day) reduce the redness and swelling of joints in diseases such as rheumatic fever, gout and rheumatoid arthritis . . . . Salicylates can dissolve corns on the toes, provoke loss of uric acid from kidneys and kill bacteria in vitro. " The medicinal mechanism of action of salicylates is a subject of continual debate. The initial Nobel-prize-winning hypothesis suggested that salicylates act by blocking the synthesis of inflamation-causing prostaglandins from arachidonic acid (152). A more recent explanation of many clinical effects of salicylates is based on their ability to disrupt cellular interactions, preventing the activation of neutrophils and other cells involved in the first stages of inflammation and blood clotting (I).

Aspirin undergoes spontaneous hydrolysis to SA even in aqueous solutions (108). In blood plasma the hydrolysis of aspirin is catalyzed by nonspecific arylesterases (58, 109). Since arylesterases are found in most living tissues, it is reasonable to assume that in plants, as in animals, exogenously applied aspirin is rapidly converted to salicylic acid. This assumption is supported by the observation that in all studied cases the effect of aspirin in plants was similar to that of SA (see below). Many plant scientists have used aspirin and SA interchangeably in their experiments, despite the fact that aspirin has not been identified as a natural plant product. The important difference between

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SALICYLIC ACID IN PLANTS 441

aspirin and SA lies in the ability of aspirin to donate an acetyl group in trans acetylation reactions, thereby blocking prostaglandin biosynthesis in animals. However, since no prostaglandins have been identified in plants, the relevance of this reaction to plants is not known.

General Properties of Salicylic Acid

Salicylic acid belongs to an extraordinary diverse group of plant phenolics usually defined as substances that possess an aromatic ring bearing a hydroxyl group or its functional derivative. Plant phenolics have often been referred to as secondary metabolites. The term "secondary" implied that such compounds were only of minor importance to the plant and could sometimes be equated with waste products. This notion has been gradually replaced, however, by the view that many phenolic compounds play an essential role in the regula­tion of plant growth, development, and interaction with other organisms (56).

For example, phenolics are essential for the biosynthesis of lignin, an important structural component of plant cell walls. Furthermore, phenolics, most notably phytoalexins (54), have been associated with the chemical defenses of plants against microbes, insects, and herbivores (104). Several phenolics function as allelopathic compounds influencing germination and growth of neighboring plants (31). Phenolic molecules produced by plant roots are essential for germination, haustorium formation, and host attach­ment in parasitic Striga species (94). Experimental evidence increasingly suggests that phenolics function as signals in plant-microbe interactions. Agrobacterium tumefaciens virulence gene expression was shown to be acti­vated specifically by the plant-produced phenolic compounds acetosyringone and a-hydroxyacetosyringone ( 143). Species-specific flavonoids exuded from legume roots and seeds are essential for the induction of the nod genes of Bradyrhizobium and Rhizobium species (92).

Free SA is a crystalline powder that melts at 157-l59°C. It is moderately soluble in water and very soluble in polar organic solvents. The pH of a saturated aqueous solution of SA is 2.4. SA fluoresces at 412 nm when excited at 301 nm, and this property can be used to detect this compound in a number of plant systems (122, 123). A procedure for the extraction and HPLC analysis of SA content in plant tissues adapted from (124) can be found in Yalpani et al ( 170). Antibodies against SA have been prepared but have not been tested in plants (10).

According to a recently developed mathematical model (63, 75) the physi­cal properties of S A [pKa = 2.98 (107), log Kow (octanollwater partitioning coefficient) = 2.26 (55)] are nearly ideal for long-distance transport in the phloem. Therefore, unless free SA is actively transported, metabolized, or conjugated, it should translocate rapidly from the point of initial application or synthesis to distant tissues.

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Salicylic Acid Levels in Plants

The presence of SA in plants, long suggested (48 , 120), has been confirmed by several investigators using modern analytical technics (7, 24, 101). A comprehensive survey of SA in the leaves and reproductive structures of 34 agronomically important species confirmed the ubiquitous distribution of this compound in plants (123). Rice, crabgrass, green foxtail, barley, and soybean had SA levels in excess of 1 /J-g g-l fresh weight. The highest levels of SA were recorded in the inflorescences of thermogenic plants and in plants infected with necrotizing pathogens (see below).

EFFECTS OF EXOGENOUS SALICYLIC ACID

Salicylic Acid and Flowering Most people learn of the effects of salicylic acid on flowering from the finding

that a tablet of aspirin dissolved in water will make cut flowers last longer. The origin of this observation could not be traced to the scientific literature. However, some indications of the mechanisms by which SA may increase flower longevity can be found in the discovery that SA inhibits ethylene biosynthesis in pear cell suspension culture by blocking the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene (87). This inhibition was greatest between pH 3.5 and 4.5 and had an apparent Iso value (concentration that inhibited ethylene production by 50%) of 40 /J-M after 3 hr incubation (130). Among 22 related phenolic compounds tested, only aspirin showed levels of inhibition similar to that of SA (88). In contrast to the results obtained in the pear cell suspension, nonphytotoxic levels of SA did not affect ethylene formation in soybean cuttings (lISa). It is also unlikely that the endogenous levels of SA in the floral tissues are high enough to affect ethylene formation in the flower tissue. In addition, ethylene is not always involved in flower senescence. While aspirin will delay the senescence of roses, this effect is likely due to the acidification of the medium used to feed cut flowers and can be duplicated with other organic acids (D. Kuiper, unpublished information).

The flower-inducing effects of SA were first indicated in an organogenic tobacco tissue culture supplemented with kinetin and indole acetic acid (85). All monohydroxybenzoic acids were found to promote flower bud formation from tobacco callus; SA was active even at 4 /J-M concentration. These observations have never attracted much attention because a number of differ­ent molecules induce flower bud formation in tobacco cell cultures (30). The first suggestion that SA may be involved in the regulation of flowering in plants came from experiments in which aphids were allowed to feed on vegetative and reproductive shoots of the short-day plant Xanthiurn strurnar­urn. It was hypothesized that a phloem-transmissible factor responsible for the induction of flowering could be found in the honeydew excreted by aphids.

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SALICYLIC ACID IN PLANTS 443

Different fractions of honeydew were tested in a bioassay system using Lemna gibba strain G3, a long-day plant, kept under nonphotoinductive light cycle. Flower-inducing as well as flower-inhibiting components were identified in the collected honeydew (23). The regulatory substances were thought to be of plant origin since the honeydew produced by aphids feeding on a synthetic diet lacked any flower-inducing activity. The flower-inducing substance from X. strumarum was identified as SA, which at 5. 6 pM caused a maximal induction of L. gibba flowering (24). SA accelerated flower induction in Lemna, while having little effect on the rate of subsequent flower develop­ment (24, 25). The stimulatory effects of SA on flowering were demonstrated in other species of Lemna, both short- and long-day (24, 25, 70, 160). In addition, SA, aspirin, and related phenolics triggered flowering under non­inductive photoperiods in Spirodela polyrrhiza (71), Spirodela punctata (135), and Wolffia microscopica (73, 147), which belong to other genera of Lemnaceae. Judging from the dependence of SA activity upon pH, it was suggested that the undissociated, neutral form of SA has the greatest florigen­ic activity (159).

The possibility that SA functions as the endogenous regulator of flowering in Xanthium and Lemnaceae was diminished by the fact that SA did not induce flowering in X. strumarum and that the levels of SA were not different in honeydew collected from vegetative and flowering plants. Also no changes in the endogenous levels of SA in vegetative or flowering Lemna have been reported. In addition, SA effect was not specific: A large variety of benzoic acids (159), nonphenolic compounds including chelating agents (112), fer­ricyanide (148), nicotinic acid (39), and cytokinins (37, 72) induced flower­ing in Lemna maintained under a non-inductive photoperiod. Suzuki et al (146) have shown that 2-hydroxy-l-phenyl- l ,4-pcntancdionc and phcnylglox­al purified from Pharbitis purpurea seeds could also induce flowering in Lemna paucicostata. The most extensive analysis of the structure-activity relationship of benzoic acid derivatives on flowering of Lemna paucicostata 151 was performed by Watanabe et al (159). With some exceptions, the increase in the electron-withdrawing ability and a decrease in the size of the benzyl ring substituents correlated with the ability of different benzoic acids to induce flowering. SA exhibited higher activity than predicted from the model.

The reports of the florigenic effects of exogenous SA in Lemnaceae were soon followed by demonstrations of its ability to induce or promote flowering in plants belonging to different families. Aspirin combined with sucrose enhanced flower opening in Oncidium, an ornamental orchid species (60). SA (at 1 and 100 mg I-I, applied to the apexes of the plants) as well as GA3 and f3-naphtol were shown to induce floral bud formation in Impatiens baisamina, a qualitatively short-day plant, under strictly non-inductive photoperiods (110, 142). The effect of SA on the timing of nower bud initiation and on the

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total number of floral buds was synergistic to the GA effect and was accom­panied by increases in total RNA content (79), phosphatases (78), and some non-identified proteins (80) in the vegetative organs of treated plants. Similar synergism between SA and GA3 was observed in the acceleration of flowering in Arabidopsis thaliana (46). Flowering in Pisita stratiotes L. , water lettuce, a species belonging to the Araceae family, was also hastened by the addition of SA to the culture medium (117). However, just as in other species, the effect of SA in P. stratiotes was not specific: GA3 and a chelating agent ethylenediamine-di-o-hydroxyphenylacetic acid (EDDHA) were as effective as SA in inducing flowering under non-inductive conditions. At least in one reported case SA at 0.1 mM inhibited flowering of plantlets of Pharbitis nil

(50). The mechanism by which SA induces flowering in plants is not known.

One hypothesis suggests that SA induces flowering by acting as a chelating agent (113), because the free o-hydroxyl group confers metal chelating activity on benzoic acids. This view is supported by the fact that chelating agents can induce flowering in Lemnaceae (112, 136) and that this induction resembled the flower-inducing effects of SA (118). Nevertheless, the florigenic activity of benzoic acid (38, 160) and other nonchelating phenolics (159) suggests that other flower-inducing mechanisms may be involved. The iron chelating properties of SA could, however, explain the inhibitory effect of SA on the ethylene-forming enzyme (see above), because iron is an essential cofactor for the conversion of l -aminocyclopropane-I-carboxylic acid to ethylene (14a).

Allelopathic Properties of Salicylic Acid: Effect on Membranes and Ion Uptake

Allelopathic chemicals are excreted by some plants to prevent germination or inhibit growth of the neighboring plants (31). It was suggested that SA produced in the rhizosphere of some plants functions as an allelopathic chemical that inhibits growth of the surrounding vegetation. SA reduced shoot dry-weight accumulation of several crop and weed species (137), perhaps by interfering with membrane ion transport in roots (43, 57). In an animal system (molluscan neurons) SA increased the membrane potential (hyperpolariza­tion) by increasing potassium conductance and decreasing chloride con­ductance (90). A subsequent look at plant systems showed that SA at 0.05 mM inhibited phosphate uptake by 54% (43) and substantially reduced potas­sium absorption in barley roots (44). SA also inhibited potassium absorption in oat roots in a pH- and concentration-dependent manner (57). At lower pH SA was a more effective inhibitor of potassium uptake, suggesting that the protonated form of SA is more active than its charged form. In contrast to the case in the animal system the membrane potentials of aged, excised barley

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SALICYLIC ACID IN PLANTS 445

root cells were rapidly depolarized by addition of SA (45). SA caused the collapse of the transmembrane electrochemical potential of mitochondria and the ATP-dependent proton gradient of the tonoplast-enriched vesicles (96).

Other Effects of Exogenously Applied Salicylic Acid

SA at concentrations of I and 10 mM significantly reduced transpiration in kidney bean (Phaseolus vulgaris) leaves (83) and in epidermal strips of Commelina communis (84). Because of the high concentration of SA used, it is unlikely that this effect has any significance for the physiological regulation of stomatal behavior. In later experiments SA and other phenolic acids reversed ABA-induced stomatal closure (121) and leaf abscission in kidney beans (6). SA and related phenolic compounds also antagonized the growth­inhibitory effects of ABA in radish seedlings (126) and in Amaranthus caudatus seedlings (127). Other effects of SA on plant development include increasing the pod number and yield in mung beans (140) and increasing the height and grain number of cheena millet (Panicum miliaceum) (27). In combination with indoleacetic acid (IAA), SA at 0. 1 mM stimulated adventitious root initiation in mung beans (76). At 0. 01-0.1 mM con­centrations SA increased the in vivo activity of nitrate reductase in maize seedlings (67). It was suggested that SA increased nitrate reductase activity indirectly, by protecting the enzyme from inactivation.

SALICYLIC ACID AND HEAT PRODUCTION IN

PLANTS

Thermogenic Plants and Search for Calorigen

Thermogenicity (heat production) in plants, first described by Lamarck in 1778 (81) for the genus Arum, is now known to occur in the male reproductive structures of cycads and in the flowers or inflorescences of some angiosperm species belonging to the families Annonaceae, Araceae, Aristolochiaceae, Cyclanthaceae, Nymphaeaceae, and Pal mae (100). The heating is believed to be associated with a large increase in the cyanide-resistant nonphosphorylat­ing electron transport pathway unique to plant mitochondria (68, 100, 153). The increase in this alternative respiratory pathway is so dramatic that oxygen consumption in the inflorescences of Arum lilies at the peak of heat produc­tion is as high as that of a hummingbird in flight (82). In addition to the activation of the alternative oxidase, thermogenicity involves activation of the glycolytic and Krebs cycle enzymes that provide substrates for this remark­able metabolic explosion.

In one of the Arum lilies, Sauromatum guttatum Schott (voodoo lily), the inflorescence develops from a large corm and can reach 80 em in height. Early on the day of anthesis, a large bract (spathe) that surrounds the central column of the inflorescence (spadix) unfolds to expose the upper part of the

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spadix known as the appendix. Soon thereafter, the appendix starts to gener­ate heat, which facilitates the volatilization of foul-smelling amines and indoles (141) attractive to insect pollinators. By early afternoon the tempera­ture of the appendix can increase by 14°C above ambient, but it returns to ambient in the evening. The second thermogenic episode in the lower spadix starts late at night and ends the following morning after maximum tempera­ture increases of more than lOoC. In 1937 Van Herk (154, 155) suggested that the burst of metabolic activity in the appendix of the voodoo lily is triggered by "calorigen," a water-soluble substance produced in the male (staminate) flower primordia located just below the appendix. Van Herk believed that calorigen began to enter the appendix on the day preceding the day of anthesis. Van Herk's ideas have encountered some skepticism, partially because attempts to isolate and characterize calorigen were not successful until recently (15, 20).

Salicylic Acid: A Natural Inducer of Thermogenesis in Arum Lilies

In 1987 an attempt to identify the elusive calorigen ended in success. Mass spectroscopic analysis of highly purified calorigen extracted from the male flowers of voodoo lily indicated the presence of SA (122). Application of salicylic acid at 0. 13 ILg g-I fresh weight to sections of the immature appendix led to temperature increases of as much as l 2°C. These increases duplicated the temperature increases produced by the crude calorigen extract both in magnitude and timing, indicating that SA is calorigen. The sensitivity of appendix tissue to salicylic acid increased daily with the approach of anthesis and was controlled by the photoperiod. On the day preceding the day of blooming the levels of SA in the appendix of the voodoo lily increased almost 100-fold, reaching the level of 1 ILg g-I fresh weight (124). The level of SA in the appendix began to rise in the afternoon and reached its maximum late in the evening, while the maximum accumulation of SA in the lower spadix occurred late at night. The concentration of SA in both thermogenic tissues promptly returned to basal, pre-blooming levels at the end of the thermogenic periods. The observed kinetics of SA accumulation in the appen­dix was consistent with the original suggestion by Van Herk (see above) that calorigen is made in the male flowers and moves to the appendix during the day preceding the day of blooming. Of 33 analogs of SA tested, only 2,6-dihydroxybenzoic acid and aspirin were thermogenic. The activity of 2,6-dihydroxybenzoic acid exceeded that of SA. SA, 2,6-dihydroxybenzoic acid, and aspirin also induce the production of large quantities of amines and indoles on the first day of blooming (122). The thermogenic effect of SA could not be separated from its odor-producing effect, suggesting that the transduction pathways for these processes are closely linked.

The levels of SA determined during heat production in thermogenic in-

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SALICYLIC ACID IN PLANTS 447

florescences of five other aroid species, and in male cones of four thermogen­ic cycads exceeded 1 J1-g g-I fresh weight (123). However, SA was not detected in the thermogenic flowers of the water lily, Victoria regia Lindle (Nymphaeaceae), and Bactris major Facq (Palmae).

The nuclear gene from Sauromatum gutta tum encoding the alternative oxidase protein with the calculated molecular mass of 38.9 kDa was recently isolated and characterized (128). Both calorigen extract (34) and SA (35) cause the induction of the alternative oxidase gene, providing additional confirmation of the chemical identity of calorigen. In at least one case, not related to thermogenic plants, SA was shown to induce the activity of alternative oxidase in Chlamydomonas (47). While the mechanism involved in SA induction of alternative respiration is being unraveled, the mechanism by which SA induces glycolysis, Krebs cycle, and odor production during the thermogenic syndrome still remains a mystery.

The discovery of the role of SA in the flowering of thermogenic plants was the first demonstration of an important regulatory role played by endogenous SA. The study ended a 50-year-long search for calorigen and laid the founda­tion for ongoing investigations of other processes in plants that may be regulated by SA. This discovery also moved SA research from the stage of phenomenological observations to serious attempts to understand the mech­anisms of its action.

It is important to remember that both heat and scent production are integral parts of flowering in thermogenic plants. Considering the numerous reports on the induction of flowering by SA (see above) it is tempting to speculate that endogenous SA may play a role in the regulation of certain events in flowering of plants that are not overtly thermogenic.

SALICYLIC ACID AND DISEASE RESISTANCE

Disease Resistance in Plants: Effects of Salicylic Acid

Some disease-resistant plants restrict the spread of fungal, bacterial, or viral pathogens to a small area around the point of initial penetration where a necrotic lesion appears. This protective cell suicide is referred to as the hypersensitive reaction (HR). The HR may lead to acquired resistance, defined as a resistance to subsequent pathogen attack that develops after the initial inoculation with lesion-forming viruses, bacteria, and fungi. Acquired resistance was first discovered in Dianthus barbatus infected by carnation mosaic virus (42). Subsequently acquired resistance was subdivided into local acquired resistance (LAR), detected in the vicinity of the HR lesions (131), and systemic acquired resistance, detected in the uninoculated, pathogen-free parts of the plants ( l32). Systemic acquired resistance develops in a variety of plant-pathogen interactions, is detected several days after the initial infection,

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448 RASKIN

could last for several weeks, and is effective against a broad range of pathogens that may be unrelated to the inducing organism (97, 166).

Commonly associated with HR and systemic acquired resistance is the systemic synthesis of several families of serologically distinct, low-molecu­lar-weight, pathogenesis-related (PR) proteins (14, 18). The function of the PR-I family of proteins, PR-l a, Ib, Ie, remains to be elucidated. The PR-2 family of proteins has {3-1,3-g1ucanase activity (69), while PR-3 proteins are chitinases (86). The localization, timing of appearance, and functions of at least some PR-proteins suggest their possible involvement in acquired resis­tance. However, definitive proof that the induction of PR-proteins causes acquired resistance is still lacking.

Resistance to pathogens and the production of at least some PR-proteins in plants can be induced by SA or acetylsalicylic acid, even in the absence of pathogenic organisms. The discovery of a protective function of salicylates was made in 1979, in Xanthi-nc tobacco (Nicotiana tabacum). Xanthi-nc tobacco contains the "N" gene, which originates from Nicotiana glutinosa and confers HR response to tobacco mosaic virus (TMV) (61). Injection of tobacco leaves with SA (0. 01 % solution) and aspirin (0.02% solution), as well as spraying and watering of tobacco plants with aspirin prior to the TMV inoculation, caused a dramatic reduction in lesion number ( 165). In addition to decreasing the lesion number, SA reduced the lesion size in Xanthi-nc tobacco (169). Currently, most researchers consider the reduction in the size of HR lesion a more direct and reproducible measure of increased resistance than the reduction in lesion number. Salicylate treatments also resulted in the induction of PR-I proteins in treated leaves. Subsequently, aspirin was shown to induce resistance and PR-I proteins in five different cultivars of Nicotiana tabacum (5). The level of PR-protein induction and TMV protection increased with increasing aspirin concentrations (168). PR-protein synthesis in tobacco began after a lag of about 18 hr in leaf discs treated with SA. The induction of PR-proteins in tobacco leaf discs was transcriptionally regulated (99, I l l). mRNAs for basic and acidic isozymes of {3-1 ,3-glucanase, a member of the PR-2 group, were strongly induced after TMV inoculation or SA treatment of tobacco (91). Similarly in cucumber, extracellular endochitinase, a member of the PR-3 group of proteins, is induced systemically following viral, bacterial, or fungal infection (102, 106). This protein can be effectively induced by SA at the level of RNA accumulation (103). Aspirin-induced synthesis of PR-1 proteins in Xanthi-nc tobacco is controlled at the transcrip­tional level (17).

Exogenously applied SA induces PR-proteins mainly at the site of applica­tion, in contrast to pathogens that induce PR-proteins systemically. In tobac­co, SA strongly induced mRNAs encoding acidic, and basic PR-l proteins, acidic glucanases and basic chitinases. SA induction of basic glucanase,

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SALICYLIC ACID IN PLANTS 449

acidic chitinase, and PR-protein S, homologous to the sweet-tasting thauma­tin, was significantly below that caused by TMV (13). However, a recent comprehensive study utilizing modern molecular approaches showed that nine classes of PR-protein mRNAs that are induced during the development of systemic acquired resistance to TMV in tobacco can be induced by SA to a similar degree (I58a). In TMV-susceptible Nicotiana tobacum cv. Samsun containing "n" allele, TMV does not trigger the induction of PR-proteins and HR. Instead, the virus spreads systemically, causing the characteristic mosaic in younger leaves. However, aspirin induced PR-proteins in Samsun tobacco and simultaneously reduced the spread and total accumulation of TMV (167). Ethylene, l -aminocydopropane-l -carboxylic acid (a direct precursor of ethylene), and various chemical treatments that result in the production of stress ethylene are also capable of inducing at least some PR-proteins (157, 158). In addition to SA and aspirin, only 2,6-dihydroxybenzoic acid was capable of directly inducing PR-proteins and resistance to viruses, without increasing ethylene biosynthesis (157). This observation is particularly in­teresting since 2,6-dihydroxybenzoic acid is the only other chemical, in addition to SA and aspirin, that induced thermogenesis in voodoo lily appen­dices ( 124).

SA or aspirin applied directly to inoculated tissues induces PR-proteins and some form of pathogen resistance in most systems studied, with the notable exception of soybean (129). Even when watered onto the soil, SA reduced the size of tobacco necrosis virus (TNV) lesions in tobacco (116). Salicylate reduced the symptoms of tobacco necrosis virus (TNV) infection in asparagus bean by 90% (115) and induced PR-proteins and resistance to alfalfa mosaic virus in bean and cowpea (62). SA also inhibited the replication of alfalfa mosaic virus in cowpea protoplasts by up to 90%, depending on the mode of application (62). At present, because of the remaining confusion in the definition of PR-proteins and the need for more research it is difficult to provide a qualitative and quantitative comparison between the PR-proteins induced by SA and necrotizing pathogens. We still do not know to what extent SA-induced resistance is based on the induction of PR-proteins. It is certainly possible that SA activates other resistance mechanisms.

In rare instances, SA suppresses certain resistance mechanisms. Tomato wounding or treatment with pectic fragments causes the systemic accumula­tion of proteinase inhibitor, which may provide some protection against insect predation. This response can be inhibited by pretreatment of plants with aspirin or salicylic acid (29). Resistance of carnation cuttings to Phytophthora parasitica can be induced by dipping the cuttings in fungal extract containing elicitors. When SA (0. 05 to 0. 2 mM) was added to the Phytophthora parasiti­ca extract the synthesis of phytoalexins in the cuttings was totally inhibited and the cuttings remained susceptible to the subsequent infections (119).

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450 RASKIN

Salicylic Acid: A Likely Signal for Disease Resistance in Plants Since systemic acquired resistance can be induced by localized infections, the existence of a systemic signal that activates PR-proteins and/or other resist­ance mechanisms has been hypothesized for at least 25 years (77, 95, l33). Evidence from stem girdling and grafting experiments suggests that the putative signal moves through the phloem tissue of the vascular system of the plant (41, 52).

The observations that exogenous SA applications induced resistance and PR-proteins in plants, that SA is an important endogenous messenger in thermogenic plants, and the development of analytical methods to quantify its endogenous levels in plant tissues (124) prepared the way to test the possibil­ity that SA is an endogenous messenger that activates important elements of host resistance to pathogens. The single-gene inheritance of TMV resistance in tobacco provided a suitable experimental system in which to investigate this possibility.

SA levels in TMV -resistant (Xanthi-nc), but not susceptible (Xanthi) tobac­co increase almost 50-fold, to 1 IJ-g g-l fresh weight, in TMV -inoculated leaves and lO-fold in uninfected leaves of the same plant (98). Induction of PR-l genes paralleled the rise in SA levels. While TMV induced PR-proteins only in Xanthi-nc tobacco, SA was effective in both Xanthi "n" and Xanthi-nc "N" plants. By feeding SA to excised leaves of Xanthi-nc (NN) tobacco it was shown that the observed increase in endogenous SA levels is sufficient for the systemic induction of PR-l proteins (170) and increased resistance to TMV (I. Raskin, unpublished information). TMV infection becomes systemic and Xanthi-nc plants fail to accumulate PR-l proteins at 32°C (156, 167). This loss of HR at high temperature was associated with an inability to accumulate SA. However, spraying leaves with SA induced PR- l proteins at both 24°C and 32°C.

SA was also exported from the primary site of infection to the uninfected tissues (170). When leaves of Xanthi-nc tobacco were excised 24 hr after TMV inoculation and exudates from the cut petioles collected, the increase in endogenous SA in TMV -inoculated leaves paralleled SA levels in exudates. Exudation and leaf accumulation of SA were proportional to TMV concentra­tion and were higher in light than in darkness. Different components of TMV were compared for their ability to induce SA accumulation and exudation: Three different aggregate states of coat protein failed to induce SA, but unencapsidated viral RNA elicited SA accumulation in leaves and phloem (170). Mechanical leaf injury did not stimulate SA production and exudation. The highest concentration of free SA was observed in and around hypersensi­tive lesions (I. Raskin, unpublished information). Only free SA was detected in the phloem and virus-free leaves of TMV-inoculated Xanthi-nc tobacco. These results support the hypothesis that SA acts as an endogenous signal in

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SALICYLIC ACID IN PLANTS 451

systemic induction of PR-l proteins and some components of systemic ac­quired resistance in NN tobacco (I. Raskin, unpublished information).

Another set of experiments has demonstrated that a fluorescent metabolite identified as SA increased dramatically in the phloem of cucumber plants inoculated with tobacco necrosis virus or the fungal pathogen Colletotrichum lagenarium (l05). Levels of SA increased transiently after inoculation, with a peak reached before systemic acquired resistance was detected. However, analysis of phloem exudate from cucumber leaves demonstrated that the earliest detectable increases in SA occurred 8 hr after inoculation with Pseudomonas syringae pv. syringae (125). Despite this the systemic accumu­lation of SA was observed even when the inoculated leaf remained attached to the plant for only 4 hr. While supporting the role of SA as a component of the transduction pathway leading to resistance, these results suggest that another chemical signal may be required for the systemic accumulation of SA. However, the relatively insensitive analytical method employed in this study leaves open the possibility that the amount of SA exuded from the inoculated leaf in the first 4 hr was sufficiently large for this compound to function as a primary transmissible signal in systemic acquired resistance.

Very recently, several observations on the molecular action of SA have been made. The AS-l element, located between the -85 to -65 region of the cauliflower mosaic virus 35S promoter was shown to be highly responsive to SA. A 10-20-fold induction of GUS mRNA was obtained by treating transgenic tobacco containing an AS-l GUS fusion with SA. The induction was rapid and insensitive to cycloheximide (N.-H. Chua, unpublished in­formation).

An SA binding protein with an apparent mass of 650 kDa and apparent Kd for SA of 14 JLM has been identified in tobacco (21). 2,6-Dixydroxybenzoic acid and aspirin competed with SA for binding. The SA-binding protein was suggested to be involved in detecting and transducing the SA signal activating disease-resistance responses. However, in the absence of a functional assay or a genetic system for the study of the receptor function of the identified binding proteins it is not yet possible to assign biological significance to the observed binding.

SALICYLIC ACID BIOSYNTHESIS IN PLANTS

Biosynthetic Pathway

Any future effort to manipulate the levels of SA depends on an understanding of SA biosynthesis. The most important mechanism for formation of benzoic acids in plants is the side-chain degradation of cinnamic acids (51, 59), which are important intermediates in the shikimic acid pathway. Therefore, a priori, SA (ortho-hydroxybenzoic acid) could be viewed as a derivative of cinnamic

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( ) H,N/coal

Phenylalanine n ( ) reOOH

trans-Cinnamic acid

� � Lignin, flavonoids, xanthones, phenolics, benzoic acids, other natural products

Shikimic acid < � rCOOH

? , "

·�I OH ""

...

,;'

o-Cownaric acid "-

.. ?

< �OOOH

\" I \0- " OH � J eOOH

' Salicylic acid

Benzoic acid

Figure 1 Proposed pathway for salicylic acid biosynthesis in plants.

acid. The conversion of cinnamic acid to SA is likely to proceed via one of two pathways outlined in Figure 1. These pathways differ in the order of l3-oxidation and ortho-hydroxylation reactions and could operate in­dependently in plants.

The suggestion that both pathways may be operational in plants carne from a published observation that infection of young tomato plants with Agrobac­terium tumefaciens increased the ortho-hydroxylation of cinnamic acid to o-coumaric acid, followed by its l3-oxidation to SA. In non-infected plants, the cinnamic acid � benzoic acid � SA pathway was most active (19). The existence of both pathways in plants was also suggested by EI-Basyouni et al (32), who found that hydroxybenzoic acids are synthesized in a variety of plants from the corresponding hydroxycinnamic acids. However, precursor feeding experiments have shown that in Catalpa ovata l3-oxidation is the last step in the formation of benzoic acids from the corresponding cinnamic acids (171). Also, feeding the young leaves of Gaultheria procumbens with 14C_ benzoic, or 14C-cinnamic acid resulted in formation of labeled SA (33), suggesting that hydroxylation patterns of hydroxybenzoic acids in plants could be established before and after l3-oxidation.

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Biosynthetic Enzymes

SALICYLIC ACID IN PLANTS 453

Most studies of cinnamate hydroxylation have used trans-cinnamate-4-hydroxylase, which converts trans-cinnamic acid to para-coumaric acid. This reaction is the first step in the biosynthesis of lignin, flavonoids, and ubi­quinone (16). This enzyme was first discovered in pea seedlings (134) and later localized in the microsomal fraction of Quercus pedunculata (2, 3). Trans-cinnamate-4-hydroxylase was immunocharacterized in Pisum sativum micro somes and mitochondria using polyclonal antibodies raised against Pseudomonas putida camphor hydroxylase (144). Recently this enzyme was purified from micro somes of manganese-induced Jerusalem artichoke tuber tissue (39a). A high-performance liquid chromatography protocol (HPLC) for assaying trans-cinnamate-4-hydroxylase in plants was developed (12). The enzymatic activity that converts trans-cinnamic acid to 2-hydroxy-cinnamic (o-coumaric) acid has been isolated from the chloroplasts of Melilotus alba (40). However, this result could not be reproduced by the same laboratory at a later date (2Sa). Another instance of ortho-hydroxylation of trans-cinnamic acid was described for Petunia hybrida chloroplasts (121a). Both subclasses of trans-cinnamate-hydroxylase are almost certainly NADPH-dependent cytochrome P-4S0 monoxygenases and represent the most prominent P-4S0 activity detectable in plants (144). Other known plant P-450 monoxygenases catalyze gibberellin biosynthesis, lO-hydroxygeraniol formation, and herbi­cide metabolism (164). At this time there is no information on the monoxyge­nase that may be involved in SA synthesis from cinnamic acid.

Enzymatic activity that catalyzes f3-oxidation of cinnamic acid to benzoic acid in Quercus pedunculata was reported by Alibert & Ranjeva in 1971 (2) and then by Alibert et al in 1972 (3). Enzymes that convert benzoic acid or o-coumaric acid to SA have not been isolated. However, the seemingly physiologically relevant in vitro system that converts para-coumaric acid to para-hydroxybenzoic acid was found to be associated with mitochondrial membranes of roots of Cucumis sativum (53).

It is not known whether conjugated pools of these precursors play a significant role in the accumulation of SA during HR. The potential presence of such conjugates is suggested by the isolation of a uridine diphosphoglu­cose:trans-cinnamate glucosyltransferase that could also catalyze the glucosylation of o-coumaric acid from sweet potato roots (138). Conversion of 14C-benzoic acid to benzoyl f3-D-glucose by Helianthus hypocotyls has been reported by KHimbt (74).

More work is needed to confirm the mechanism and kinetics of SA biosynthesis from cinnamic acid and to purify the enzymes involved in SA biosynthesis in vivo. The discovery of the important regulatory role played by this compound in plants should give this research a high priority. Such studies will eventually lead to the cloning of SA biosynthetic enzymes from plants.

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SALICYLIC ACID METABOLISM

Esters or glucosides of a variety of substituted benzoic acids are known to occur in higher plants (49, 66). When fed to plants, hydroxybenzoic acids form O-glucosides unless they contain an ortho-hydroxyl group that forms a glucose ester (26). So far the only exception to this rule was found in cell suspension cultures of soybean and mung bean, which exclusively formed glucose-esters of SA (9). Helianthus hypocotyls incubated with carboxyl­labeled benzoic acid formed small amounts of SA and relatively larger amounts of l3-glucosido-salicylic acid (74). Recently, glucosyltransferase activity that catalyzes the formation of o-O-I3-D-glucosylbenzoic acid (13-glucosido-salicylic acid) from SA and UDP-glucose was partially purified from suspension cultures of Mallotus japonicus (149) and from roots of oat seedlings (8). Both enzymes were highly inducible by SA.

Metabolic inactivation of SA may result from additional hydroxylation of the aromatic ring. Thus, 2,3-dihydroxybenzoic (o-pyrocatechuic) acid and 2,5-dihydroxybenzoic (gentisic) acid have been isolated from Astilbe sinensis

and tomato plants fed 14C-cinnamic acid or 14C-benzoic acid ( 1 1 , 19). Tobacco leaves metabolize SA rapidly. Levels of SA in excised Xanthi-nc

leaves fed with SA through the cut petioles increased rapidly and reached a maximum after 3 hr (I. Raskin, unpublished information). Thereafter, SA levels decreased by 80% and remained constant for the next 48 hr. Data obtained from TMV-inoculated tobacco are consistent with the observations made in other plants (see above) that l3-glucosyl-salicylic acid is a major derivative of SA. Treatment of leaf extracts from the areas containing TMV­induced HR lesions with l3-glucosidase increased levels of free SA up to lO-fold, suggesting the presence of glucosyl-SA (I. Raskin, unpublished information). Levels of SA in extracts from mock-inoculated leaves treated with l3-glucosidase-treated or buffer control were not significantly different and only slightly above the detection level. I3-Glucosidase treatment did not increase SA levels in phloem exudates or in the upper uninoculated leaves during systemic acquired resistance, suggesting that SA is transported as a free acid.

In humans, SA is for the most part metabolized by the kidneys where it is partially conjugated to form salicyluric acid and possibly SA glucuronides (89).

MICROBIAL PRODUCTION OF SALICYLIC ACID

Various microorganisms produce SA (59, 161) primarily via chorismic acid­an important intermediate of the shikimic acid pathway. The rates of micro­bial SA biosynthesis and excretion can be substantial. Mycobacterium smeg-

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SALICYLIC ACID IN PLANTS 455

matis produces 3.6 mg SA g-l dry weight of cells in one day (64, 65). Pseudomonas aeruginosa produces SA as an intermediate in the biosynthesis of pyochelin, a phenolate siderophore, which plays an important part in its interactions with the mammalian nutritional system (4). Microorganisms associated with crop plants are also capable of synthesizing and excreting SA. Since plant residues and root exudates are good substrates for microbial SA production (22), large amounts of SA can be found in the soil samples taken from the rhizosphere. For example, rhizospheric soils of Zea mays and Phaseolus aureus (mung bean) contained 3 1 and 141 /Lg SA per 100 g soil, respectively, while SA levels in nonrhizospheric soil were either lower or nondetectable (114). It has been suggested that various benzoic acids in the rhizosphere serve a protective or allelopathic function (see above). The production of SA by phyllospheric or endophytic microorganisms has not been examined.

CONCLUDING REMARKS

Centuries have passed since the healing substance from the willow bark was shown to have value not only for humans but for the plants that synthesize it. Surprisingly, some of the SA effects in plants are also associated with reduction of disease symptoms. We still do not know whether there are any connections between the therapeutic effects of salicylates in plants and those in animals. The available data suggest that SA is a likely inducer of PR­proteins during systemic acquired resistance and local acquired resistance. SA is also the most promising candidate as the endogenous activator of resistance to pathogens in tobacco and cucumber, whether via activation of PR-proteins and/or via some other mechanisms that remain to be identified. However, the role of SA as a primary transmissible signal in systemic acquired resistance has not been established. Although the levels of SA increase systemically following the initial inoculation with necrotizing pathogens, we still do not know whether these increases can be explained by the documented phloem export of SA from the inoculated leaves. The possibility remains that they are produced by another highly mobile signal molecule that precedes SA in the transduction pathway of the resistance response.

The role of SA as a calorigen in Saromatum guttatum has been established with a fair degree of certainty. However, we still do not understand the biochemical connection between the action of SA in plant disease resistance and its thermogenic and odor-producing effects in Arum lilies. It is possible that these processes branch off from a single transduction pathway initiated by SA. The increases in respiration routinely observed during HR response may provide some clues to the common origin of both processes (139, 145). A relation may also exist between the regulatory role of SA in thermogenesis

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456 RASKIN

and its flower-inducing effects in Lemna and other plants. In some thermogenic plants SA regulates several crucial processes in the flowering sequence. It is conceivable that SA may also regulate some yet unidentified events in the flowering of non-overtly thermogenic species. An important challenge for future research is to search for these events. Since SA is a potent inducer of PR-proteins (see above), reports on the appearance of PR-proteins in healthy tobacco flowers (93) and leaves from flowering plants (36) may provide an interesting lead in the investigation of the universal role of SA in flowering.

The growing appreciation of the role of SA in plants may bring some practical applications. Manipulating the level of SA in plants may be a promising area for the application of biotechnology to crop protection. The biosynthesis of SA is likely to be a two-step enzymatic process (see above) , and the levels of SA sufficient for the activation of PR proteins and some disease resistance are not phytotoxic (I. Raskin, unpublished information). Increases in endogenous SA may be achieved via enhancing transcription and translation of the genes for SA biosynthesis or by blocking the expression of genes involved in SA metabolism. These genes may be of plant or bacterial origin. In either case, transgenic plants with elevated SA levels may be the first step in the creation of crops with increased resistance to agronomically important pathogens.

Although the various physiological and biochemical effects of SA applica­tion to plants have been known for a long time the regulatory role of endogenous SA was only established in 1 987, when SA was implicated as an endogenous regulator in plant thermogenesis ( 122). Since that time the stand­ing of SA among other regulatory molecules remains uncertain. The classical definition of a plant hormone suggests that it is an organic substance that acts in small quantities at some distance from the site of its synthesis ( 1 50, 1 63).

This definition borrows heavily from the definition of animal hormones, and its applicability to plants is often questioned ( l5 1 ). A more recent and probably more universal definition simply states that a plant hormone is a "natural compound in plants with an ability to affect physiological processes at concentrations far below those where either nutrients or vitamins would affect these processes" (28). All the information on the role of SA in thermo­genesis and disease resistance suggests that under these criteria SA qualifies as a plant hormone. In addition to being transported from the male flowers during the blooming of Sauromatum guttatum and from the leaves inoculated with necrotizing pathogens, SA acts at very low concentrations. Even at 100

ng g- I fresh weight SA triggers dramatic metabolic explosion in Sauromatum guttatum ( 1 22) and induces PR -proteins in tobacco leaves ( 1 70). However, only time and further research can tell whether SA will receive the title of plant hormone thus far bestowed on only five other molecules.

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ACKNOWLEDGMENTS

SALICYLIC ACID IN PLANTS 457

I thank V. Shulaev for helping to put this manuscript together. I am grateful to P. Day, A. Enyedi, Y. Kapulnik, P. Silverman, and N. Yalpani for their comments on the manuscript. This review is based on literature available prior to August 1991, from which only some of the important works were cited. Financial support from the New Jersey Commission for Science and Technol­ogy, US Department of Agriculture/Competitive Research Grants Office, and Division of Energy Biosciences of US Department of Energy.

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