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ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. GLUCOSINOLATES
  5. GLUCOSINOLATES AND INSECT INTERACTIONS
  6. GLUCOSINOLATES AND PATHOGEN INTERACTIONS
  7. CAMALEXIN AND PATHOGEN INTERACTIONS
  8. PHENYLPROPANOIDS AND UV-B RESISTANCE
  9. FLAVONOID AGLUCONES AND AUXIN TRANSPORT
  10. TERPENOIDS AND PLANT/INSECT INTERACTIONS
  11. FUTURE AVENUES
  12. REFERENCES

Arabidopsis thaliana is a successful model plant for studying wide-ranging topics including plant development, genetics and pathogen resistance. In addition, significant research has been conducted in the area of secondary metabolite biochemical genetics. The secondary metabolites in Arabidopsis include glucosinolates, terpenoids, phenylpropanoids, the alkaloid-like camalexin, and other uncharacterized compounds. The genetic tools developed in studying secondary metabolite biochemistry are now being used to study how secondary metabolites control various biological processes. This includes compounds involved in plant/insect and plant/pathogen interactions, compounds preventing UV-B damage, and compounds involved in hormone homeostasis. This review will describe what light Arabidopsis is shedding on the biological and ecological importance of specific secondary metabolites.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. GLUCOSINOLATES
  5. GLUCOSINOLATES AND INSECT INTERACTIONS
  6. GLUCOSINOLATES AND PATHOGEN INTERACTIONS
  7. CAMALEXIN AND PATHOGEN INTERACTIONS
  8. PHENYLPROPANOIDS AND UV-B RESISTANCE
  9. FLAVONOID AGLUCONES AND AUXIN TRANSPORT
  10. TERPENOIDS AND PLANT/INSECT INTERACTIONS
  11. FUTURE AVENUES
  12. REFERENCES

In 1891, following Stahls work on plant biochemistry, Kössel suggested a distinction between basic and secondary metabolism (Stahl 1888). Basic metabolism refers to the anabolic and catabolic processes required for respiration, nutrient assimilation, and growth/development, namely those processes required for cell maintenance and proliferation. In contrast, secondary metabolism refers to compounds present in specialized cells that are not necessary for the cells survival but are thought to be required for the plants survival in the environment. These compounds are believed to aid plant fitness by preventing insect herbivory and pathogen attack as well as aiding reproduction through providing pollinator attraction as either floral scent or colouration. This requirement for secondary metabolites to have highly diverse biological activities has led plants to accumulate a vast catalogue of compounds. The catalogue in vascular plants is at least several hundred thousand secondary metabolites (Wink 1988).

Most of the secondary metabolite structural diversity is generated by differentially modifying common backbone structures, with the derived compounds having potentially divergent biological activities. Differential modification of common backbone structures can alter the biological activity of a number of plant hormones and secondary metabolites including auxins, glucosinolates, gibberellins and phenylpropanoid derivatives (Kobayashi et al. 1993; Du, Vanloon & Renwick 1995; Salah et al. 1995; Morton et al. 2000; Facchini 2001). This modification of common backbone structures or precursors produces approximately 12 000 known alkaloid structures (Facchini 2001). One explanation for this modular diversity is that selection favours plants with newly derived defences when insects or other pests have evolved the ability to overcome existing defences (Ehrlich & Raven 1964). New defensive compounds can be synthesized by structurally modifying a toxic compound to evade the pest's counter-defence while maintaining the compounds toxic activity. The reiteration of this process over millennia may explain the vast range of plant secondary metabolic chemistry. In addition to aiding plant survival, this chemical diversity has led to the development of numerous medical treatments and assisted in significant nutritional advancements (Caporale 1995; Morton et al. 2000). The biological impact of small molecular changes is significant enough that the pharmaceutical industry is creating combinatorial chemistry technologies to generate the same structural diversity in vitro (Garret & Workman 1999; Leonard, Deisseroth & Austin 2001). Despite the biological and medical importance of structural variation, little is known about how plants generate this variation, why it has arisen and whether it impacts plant biology.

Arabidopsis thaliana is a well-established model plant with a complete genomic sequence and a diverse secondary metabolite arsenal (Fig. 1). This arsenal is comprised of anthocyanins, flavonoids, sinapoyl esters, glucosinolates, terpenoids, camalexin, and other tryptophan derivatives (Chapple et al. 1994; Van Poecke, Posthumus & Dicke 2001; Chen et al. 2003). The numbers of identified and structurally verified compounds for each class are listed in Fig. 1. Arabidopsis secondary metabolite research has primarily focused on utilizing genetics to identify the biosynthetic genes required for the compound's production (Zhou, Tootle & Glazebrook 1999; Wittstock & Halkier 2000; Hansen et al. 2001; Kliebenstein et al. 2001a; Lambrix et al. 2001). This is well illustrated in the long-established phenylpropanoid genetics field (Chapple et al. 1994; Shirley et al. 1995; Lehfeldt et al. 2000). In recent years, the tools that Arabidopsis provides for studying secondary metabolites have been applied towards testing specific theories about the biological activities of secondary metabolites. This ranges from studies investigating how a secondary metabolite alters plant/insect or pathogen interactions to studies on the ecological and evolutionary impact of secondary metabolites. This review will focus on what these studies in Arabidopsis are revealing about the biological role of secondary metabolites .

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Figure 1. Secondary metabolite classes in Arabidopsis. The major secondary metabolite classes in Arabidopsis are listed along with a rough estimate of the current number of structurally characterized secondary metabolite related compounds in each class. A representative of each major class is shown; phenylpropanoids, dihydrokaempferol; glucosinolates, 4-methoxy-indol3-ylmethyl glucosinolate; Terpenoids; phytoalexins/alkaloids, camalexin.

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GLUCOSINOLATES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. GLUCOSINOLATES
  5. GLUCOSINOLATES AND INSECT INTERACTIONS
  6. GLUCOSINOLATES AND PATHOGEN INTERACTIONS
  7. CAMALEXIN AND PATHOGEN INTERACTIONS
  8. PHENYLPROPANOIDS AND UV-B RESISTANCE
  9. FLAVONOID AGLUCONES AND AUXIN TRANSPORT
  10. TERPENOIDS AND PLANT/INSECT INTERACTIONS
  11. FUTURE AVENUES
  12. REFERENCES

Glucosinolates are the largest known group of Arabidopsis secondary metabolites, with more than 36 different structures described (Hogge et al. 1988; Brown et al. 2003). They are synthesized from a variety of typical protein amino acids (methionine, tryptophan and phenylalanine) and their chain-elongated analogues. The first step in glucosinolate biosynthesis is the oxidation of the amino group to an oxime moiety by amino acid-specific cytochrome P450 monooxygenases that are encoded by the CYP79 gene family (Du et al. 1995; Wittstock & Halkier 2000). After thiohydroximate formation, sequential glucose and sulplate transfer creates the basic glucosinolate skeleton (Halkier & Du 1997).

The initially formed glucosinolate can undergo a variety of subsequent side chain modifications. For example, the C4 dihomomethionine-derived glucosinolate backbone can be modified into seven different glucosinolates in Arabidopsis (Fig. 2). As most of these modifications are sequential, each step is catalysed by a different enzyme that appears to have independently evolved (Kliebenstein et al. 2001a; Kroymann et al. 2001). Variation in the presence or absence of these enzymes lead to the production of dramatically different glucosinolate profiles via altered side-chain modification (Kliebenstein et al. 2001c).This variation also provides genetic tools to test the in planta biological activities of the differentially modified glucosinolates (Kliebenstein, Figuth & Mitchell-Olds 2002a; Kliebenstein, Gershenzon & Mitchell-Olds 2001b).

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Figure 2. Naturally variable enzymatic loci controlling glucosinolate structure. (A) Four of the five major loci impacting short chain methionine-derived glucosinolates. n is equal to either 1 or 2 carbons depending upon the allelic status at the GS-Elong locus not shown. GS-OHP can only act on 3 carbon side glucosinolates while GS-OH can only act on 4 carbon side chain glucosinolates. The compounds names are as follows (a) 3 or 4-methylthio glucosinolate; (b) 3 or 4-methylsulphinyl glucosinolate (3-MSO or 4-MSO); (c) 3-hydroxypropyl glucosinolate (3-OHP); (d) Allyl (3C) or But-3-enyl (4C, But) glucosinolate; (e) R/S 2-hydroxy-but-3-enyl glucosinolate. (B) ESP control of glucosinolate hydrolysis. Spont. = spontaneous reaction. R = side chain structure, reference the side chains in part A. The compound names are as follows (i) isothiocyanates (ITC); (ii) epithionitriles (epi); (iii) nitrile (nit).

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The potential biological activity of a glucosinolate is fully realized upon hydrolysis by the enzyme myrosinase, a thioglucosidase (Fig. 2B). Glucosinolates and myrosinases are spatially separated within the plant (Cole 1976; Bones & Rossiter 1996), but are brought into contact by tissue damage yielding a variety of hydrolysis products including nitriles, isothiocyanates, thiocyanates, oxazolidine-2-thiones and epithionitriles (Fig. 2B) (Bones & Rossiter 1996; Halkier & Du 1997). Work in Arabidopsis and Brassica has shown that the type of hydrolysis products formed depends on the chemical nature of the parent glucosinolate side chain and the presence of various myrosinase-associated proteins (Bernardi et al. 2000; Foo et al. 2000; Lambrix et al. 2001). In Arabidopsis, one of these proteins promotes nitrile formation. Called the epithiospecifier protein (ESP), its presence during the hydrolysis of alkenyl glucosinolates leads to the formation of epithionitriles instead of isothiocyanates by transfer of the sulphur atom from the basic glucosinolate backbone to the terminal alkene residue of the side chain (Fig. 2B) (Lambrix et al. 2001). During non-alkenyl glucosinolate hydrolysis, ESP promotes simple nitrile formation (Lambrix et al. 2001).

GLUCOSINOLATES AND INSECT INTERACTIONS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. GLUCOSINOLATES
  5. GLUCOSINOLATES AND INSECT INTERACTIONS
  6. GLUCOSINOLATES AND PATHOGEN INTERACTIONS
  7. CAMALEXIN AND PATHOGEN INTERACTIONS
  8. PHENYLPROPANOIDS AND UV-B RESISTANCE
  9. FLAVONOID AGLUCONES AND AUXIN TRANSPORT
  10. TERPENOIDS AND PLANT/INSECT INTERACTIONS
  11. FUTURE AVENUES
  12. REFERENCES

Arabidopsis glucosinolate type, accumulation and hydrolysis are highly variable in wild-collected accessions. Glucosinolate type and hydrolysis are controlled by genetic variation for the presence/absence of a number of enzymes that function in a sequential pathway (Kliebenstein et al. 2001c; Lambrix et al. 2001). This generates a system whereby a deleterious mutation in one of the enzymes or a cross-pollination event could lead to a rapid and dramatic glucosinolate profile shift from one generation to the next (Fig. 2). Thus, when a herbivore or pathogen obtains resistance to the current glucosinolate profile, a single mutational or out-crossing event could rapidly generate a new profile without the need for de novo evolution of a new enzymatic capacity (Kliebenstein et al. 2001c). This modular control of structural diversity may maximize the glucosinolate fitness benefit while minimizing the potential for an insect or pathogen to obtain complete resistance to the glucosinolate/myrosinase system.

One caveat to the above model is that the different glucosinolates must have disparate biological activities. For if they all had the same activity, a single defence mechanism in the insect would bypass the entire modular evolution scheme. Evidence for diverse glucosinolate biological activities was obtained by analysing glucosinolate variation and its impact on Trichoplusia ni resistance, a generalist insect herbivore (Kliebenstein, Pedersen & Mitchell-Olds 2002b). This study utilized two different recombinant inbred populations varying for both biosynthetic and hydrolytic structure (Fig. 3A). All of the genes controlling the variation in biosynthesis and hydrolysis co-localized with the insect resistance QTL and could possibly explain all but one of the QTL controlling insect herbivory (Lambrix et al. 2001; Kliebenstein et al. 2002b). The impact of the biosynthetic and hydolysis QTLs on Trichoplusia ni herbivory were verified using an independent F2 cross between the Da(1)-12 and Ei-2 ecotypes (Lambrix et al. 2001). One of these QTL was shown to alter resistance to Spodoptera exigua herbivory, another generalist insect, and mapped to a 50 kb interval overlying the MAM gene which controls the length of the glucosinolate side-chain (Kroymann et al. 2003).

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Figure 3. Ranking in planta glucosinolate impact on Trichoplusia ni herbivory via genetics. (A) Genetic crosses tested for Trichoplusia ni herbivory and their corresponding glucosinolate genotypes. The loci refer to the same loci as in Fig. 2. + = functional, – = non-functional. (B) List of major glucosinolates obtained as single constituents in specific Recombinant Inbred Lines via transgressive segregation and ranking of their relative impact on Trichoplusi ni herbivory.

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Assuming that the candidate gene identities are correct, it is possible to use this genetic cross to rank the impact of the various Arabidopsis glucosinolate breakdown products on Trichoplusia ni resistance (Fig. 3B (Lambrix et al. 2001; Kliebenstein et al. 2002b). This analysis showed that nitriles are less deterrent than the corresponding isothiocyanate to Trichoplusia ni but that the epithionitriles were equal to the isothiocyanates (Fig. 3B). Further, the alkenyl side chains were more deterrent than the non-alkenyl. Interestingly, the impact of side-chain length was dependent upon the chemical nature of the side chain. For non-alkenyl glucosinolates, 4 carbon side chains were more deterrent than 3 carbon, whereas the reverse was true in alkenyl glucosinolates with the 3 carbon allyl glucosinolate being a more efficient deterrent than the 4 carbon but-3-enyl (Fig. 3B). The development of transgenic lines containing all combinations of these genes would allow for this ranking to be confirmed as well as rapidly expanded to a number of other insects and pathogens. This is currently a very difficult and expensive task as only three of the 36 compounds are commercially available.

In addition to glucosinolate structure, glucosinolate amount also negatively impacted generalist herbivory for both Trichoplusia ni and Spodoptera exigua (Kliebenstein et al. 2002b; Kroymann et al. 2003); lines with higher glucosinolate levels were more resistant to herbivory than were lines with lower glucosinolates. In contrast, glucosinolate variation was ineffective in preventing herbivory by the specialist Plutella xylostella (Kliebenstein et al. 2002b; Kroymann et al. 2003). In fact, there was a significant positive correlation between glucosinolate levels and Plutella xylostella herbivory suggesting that glucosinolates were acting as feeding stimulants (personal communication; Mitchell-Olds, T., Max Planck Institute for Chemical Ecology, Jena, Germany; Kliebenstein et al. 2002b).

The previous work suggests that glucosinolates are required for Arabidopsis insect resistance. However, these papers focus on the interaction of Arabidopsis with lepidopteran larvae that probably do not present a significant herbivory load on wild Arabidopsis. Support for glucosinolates protecting Arabidopsis in an ecological context comes from a study looking at the fitness cost of glucosinolate production (Mauricio 1998). The glucosinolate level was negatively correlated with herbivory damage suggesting that the laboratory studies of glucosinolate action are representative of their role in the wild. This study also showed that, in the absence of herbivores, plants with high glucosinolate levels had lower fitness than those with low glucosinolate accumulation. Thus, there is a cost of glucosinolate production, and this cost is countered by the benefit of decreased herbivore damage. Only total glucosinolate levels were measured and not the glucosinolate type or hydrolysis products (Mauricio 1998). As total glucosinolate was not the most significant factor in the laboratory studies, it would be highly interesting to repeat this field experiment with lines specifically varying for the different glucosinolate and hydrolysis products. This could lead to a better understanding of the biological basis for glucosinolate variation maintenance in Arabidopsis.

GLUCOSINOLATES AND PATHOGEN INTERACTIONS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. GLUCOSINOLATES
  5. GLUCOSINOLATES AND INSECT INTERACTIONS
  6. GLUCOSINOLATES AND PATHOGEN INTERACTIONS
  7. CAMALEXIN AND PATHOGEN INTERACTIONS
  8. PHENYLPROPANOIDS AND UV-B RESISTANCE
  9. FLAVONOID AGLUCONES AND AUXIN TRANSPORT
  10. TERPENOIDS AND PLANT/INSECT INTERACTIONS
  11. FUTURE AVENUES
  12. REFERENCES

In addition to controlling plant/insect interactions, glucosinolates have long been hypothesized to provide resistance against fungal and bacterial pathogens. In vitro assays have long showed that purified glucosinolate breakdown products are toxic to a wide range of bacteria, fungi and nematodes. This has been extended to showing that the major glucosinolate breakdown product, 4-methylsulphinylisothiocyanate (4-MSOITC), in the Col-0 accession displays in vitro toxicity to assorted fungi and bacteria (Tierens et al. 2001). All biotrophic pathogens, both bacterial and fungal, were sensitive to 4-MSOITC in vitro. The only resistant organisms were the necrotrophic fungi, Botrytis cinerea and Alternaria brassicola. This contrast between biotrophs and necrotrophs could be predicted because tissue disruption is necessary to mix the glucosinolate and myrosinase. Necrotrophs by definition cause and require tissue disruption and thereby should be more exposed to glucosinolate breakdown products than are biotrophs that do not cause tissue disruption. Thus, necrotrophs would be under selective pressure to evolve defences against the glucosinolate toxins.

To test the in vitro results, the authors utilized a 4-MSOITC-deficient mutant and compared growth between the mutant and wild-type Arabidopsis. This showed that the biotrophs, in spite of their in vitro 4-MSOITC sensitivity, were unaffected in vivo by 4-MSOITC (Tierens et al. 2001). Likewise, the 4-MSOITC-insensitive necrotrophs showed no difference in growth between the wild-type and mutant plants as expected. In contrast, the one in vitro 4-MSOITC-sensitive necrotrophic fungus, Fusarium oxysporum, grew better on the 4-MSOITC-deficient mutant, indicating that Arabidopsis glucosinolates provide some resistance to some pathogens. Further, this implies that biotrophic pathogens may not be exposed to glucosinolates whereas necrotrophic pathogenicity may be partially controlled by the glucosinolate content of the plant and the glucosinolate sensitivity of the pathogen (Tierens et al. 2001). Further avenues to investigate this relationship between glucosinolates and necrotroph pathogenicity would be to utilize genetic lines with altered glucosinolate biosynthesis or breakdown to test if 4-MSOITC is the most effective antibiotic glucosinolate or if others are more powerful.

CAMALEXIN AND PATHOGEN INTERACTIONS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. GLUCOSINOLATES
  5. GLUCOSINOLATES AND INSECT INTERACTIONS
  6. GLUCOSINOLATES AND PATHOGEN INTERACTIONS
  7. CAMALEXIN AND PATHOGEN INTERACTIONS
  8. PHENYLPROPANOIDS AND UV-B RESISTANCE
  9. FLAVONOID AGLUCONES AND AUXIN TRANSPORT
  10. TERPENOIDS AND PLANT/INSECT INTERACTIONS
  11. FUTURE AVENUES
  12. REFERENCES

Another Arabidopsis secondary metabolite involved in plant/pathogen interactions is the indole phytoalexin, camalexin (Fig. 4; Tsuji et al. 1992). This phytoalexin is induced under pathogen attack and by abiotic elicitors that generate reactive oxygen species (Tsuji et al. 1992; Reuber et al. 1998; Roetschi et al. 2001). Camalexin is derived from the tryptophan biosynthetic pathway via an unknown mechanism and only one putative biosynthetic enzyme is known, PAD3 which is a cytochrome P450 (Glazebrook, Rogers & Ausubel 1997; Zhou et al. 1999). Mutations in the PAD3 locus lead to a complete absence of camalexin production and allows for testing the role camalexin plays in providing resistance to various pathogens (Glazebrook et al. 1997; Zhou et al. 1999). A number of other mutations affect camalexin accumulation following pathogen attack in Arabidopsis; however, all of these are pleiotropic signalling mutants and cannot be considered a direct test of the biological role of camalexins (Glazebrook et al. 1997; Jirage et al. 1999). Thus, comparing the growth of a pathogen on pad3 mutants versus wild type is the most direct method available to test the in planta role of camalexins in plant/pathogen interactions.

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Figure 4. Fungal camalexin detoxification. Enzymatic conversion of toxic camalexin to non-toxic metabolites via R. solani and S. sclerotiorum.

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The pad3 camalexin biosynthetic mutant was first isolated in a screen for mutants unable to induce camalexin in response to Pseudomonas syringae pv. maculicola (Glazebrook & Ausubel 1994). However, this mutant did not allow more pathogen growth suggesting that PAD3 and the resulting camalexin do not provide resistance to Pseudomonas syringae nor Xanthomonas campestris (Glazebrook & Ausubel 1994). In contrast to the in planta analysis, Pseudomonas is sensitive to camalexin in vitro. This suggests, as for the glucosinolates, that camalexin requires cell disruption for full activity. In agreement with this, the biotrophic fungi, Hyaloperonospora parasitica and Erysiphe orontii do not show enhanced symptomology on pad3 mutants whereas the necrotrophic fungi, Botrytis cinerea and Alternaria brassicicola do show increased growth on the camalexin-deficient mutant (Glazebrook et al. 1997; Reuber et al. 1998; Thomma et al. 1999; Ferrari et al. 2003; Mert-Turk et al. 2003a). The putative requirement for tissue disruption in camalexin activity requires an analysis of the cellular localization of camalexin.

The role of camalexin in necrotrophic fungal defence should be interpreted with the caveat that fungi can be polymorphic in their ability to detoxify plant toxins. Thus, one fungal isolate may be sensitive to the toxin and the next isolate of that same fungus may be resistant. For example, Botrytis cinerea has variable resistance to the tomato toxin, tomatin (Quidde, Osbourn & Tudzynski 1998). In addition to fungal polymorphism, there are a number of different fungal detoxification mechanisms for camalexin. A Botrytis cinerea relative, Sclerotinia sclerotiorum, can glycosylate camalexin on the indole ring's C-6 or N-1 atom, generating compounds with dramatically lower toxicity (Pedras & Ahiahonu 2002). Rhizoctonia solani can 5-hydroxylate camalexin allowing the compound to be rapidly metabolized (Fig. 4). Further, all of the resulting metabolites have significantly less toxicity than camalexin (Pedras & Khan 1997). Thus, fungi could have polymorphic camalexin defence capacity.

This polymorphic camalexin defence may have already been identified in Arabidopsis/Botrytis interactions. Two Arabidopsis studies differ in their conclusion about Botrytis cinerea camalexin sensitivity in pad3 mutants and to in vitro camalexin exposure (Thomma et al. 1999; Zhou et al. 1999; Ferrari et al. 2003). The major difference between these studies is they used different Botrytis isolates. Possibly one isolate could detoxify camalexin whereas the other could not. However, this remains to be shown in a side-by-side isolate comparison. Thus, to fully understand the role of any toxin in plant/pathogen interactions, the interaction must be tested with numerous pathogen isolates. Because most of the above studies utilized a single isolate, sweeping conclusions cannot be drawn about the role of camalexin in any specific plant/pathogen interaction. Instead, the best summation is that camalexin provides resistance to some isolates of some pathogens. However, this suggests that there are Arabidopsis/pathogen interaction systems whereby natural variation in camalexin production and its detoxification can be studied as a surrogate for the evolutionary arms/race theory on an intraspecific level.

Phytoalexin's role in plant defence has been debated for nearly seven decades. This debate oscillates back and forth between whether phytoalexins are the cause or consequence of induced plant pathogen defence (Muller, Meyer & Klikowski 1939; Muller 1961; Peuppke & VanEtten 1976). At first glance, the Arabidopsis/camalexin studies seem to further deepen the mystery behind the debate between cause and consequence as the camalexin phytoalexin only helps Arabidopsis defend itself against a few pathogens and sometimes not even all of the individuals of the same pathogen. However, phytoalexins could be the cause of induced plant pathogen defence against sensitive pathogens whereas against resistance pathogens they are a consequence of induced defences. This suggests that phytoalexins are regulated by non-specific stress-stimulated networks that overlap with a number of pathogen induced networks. Non-specific overlapping regulation appears to be the case as camalexin is induced by most Arabidopsis pathogens, irrespective of the pathogens camalexin sensitivity, as well as UV-B and other abiotic stresses whose regulatory networks overlap with pathogen responses (Tsuji et al. 1992; Glazebrook & Ausubel 1994; Brosche & Strid 2003; Mert-Turk et al. 2003b). In general, the only requirement for camalexin induction is reactive oxygen species production or cell death. The extent to which the non-specific overlapping regulatory theory is undetermined.

PHENYLPROPANOIDS AND UV-B RESISTANCE

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. GLUCOSINOLATES
  5. GLUCOSINOLATES AND INSECT INTERACTIONS
  6. GLUCOSINOLATES AND PATHOGEN INTERACTIONS
  7. CAMALEXIN AND PATHOGEN INTERACTIONS
  8. PHENYLPROPANOIDS AND UV-B RESISTANCE
  9. FLAVONOID AGLUCONES AND AUXIN TRANSPORT
  10. TERPENOIDS AND PLANT/INSECT INTERACTIONS
  11. FUTURE AVENUES
  12. REFERENCES

Arabidopsis makes numerous phenylpropanoid-derived secondary metabolites including sinapoyl esters, flavonol glycosides, anthocyanins and tannins (Chapple et al. 1994). The genetics of this biosynthetic pathway has been thoroughly studied, generating many characterized recessive mutations in genes encoding the pathway enzymes (Koornneef 1981; Chapple et al. 1992; Shirley et al. 1995; Meyer et al. 1996). These mutants make it possible to test specific hypotheses about the impact of various phenylpropanoids upon biological processes. The main biological question that has been asked with these mutants is the role of phenylpropanoids in providing UV-B resistance.

Phenylpropanoids are the major plant secondary metabolite class that absorbs UV-B irradiation. This has led to speculation that one of their major roles was to act as sunscreens to absorb UV-B light. Several studies have looked at UV-B sensitivity in various phenylpropanoid mutants to test this hypothesis (Li et al. 1992; Landry, Chapple & Last 1995; Rao, Paliyath & Ormrod 1996; Sheahan 1996). In these studies, phenylpropanoid-deficient mutants were exposed to UV-B radiation and the relative resistance measured. All phenylpropanoid mutants were more UV-B sensitive in comparison with wild type but there were dramatic UV-B sensitivity differences between these mutants. These studies engendered disparate conclusions ranging from the hydroxycinnamic acid sinapoyl esters being the predominant UV-B protectant (Landry et al. 1995; Sheahan 1996), to flavonol induction being the predominant UV-B protectant (Rao et al. 1996), to both the flavonol and sinapoyl esters providing resistance (Li et al. 1992). One major contributor to these diverse conclusions is that each study investigated a small subset of the available mutants and did not analyse all the available mutants. If these studies are combined, the more likely conclusion is that the importance of hydroxycinnamic acids and flavonols is directly relational to their relative concentrations. In wild-type Arabidopsis the hydroxycinnamic acids are the most abundant phenylpropanoids and thus, provide the most UV-B resistance (Chapple et al. 1992; Landry et al. 1995).

One further debate about the role of phenylpropanoids in providing UV-B defence arises from comparing growth chamber UV-B exposures to natural exposures. The UV-B levels and irradiation spectra between environmental and growth chamber exposure are dramatically different, diminishing the capacity to make direct comparisons. To test for environmental impacts, the ability of wild-type Arabidopsis and tt5 mutant lines, deficient in flavonol glycoside production but not sinapoyl esters, to grow in the wild was compared (Fiscus et al. 1999). Plants were grown in the wild in the presence or absence of Mylar filters that remove UV-B and their fitness compared between the two conditions. Wild-type plants showed very little difference in fitness in the presence or absence of UV-B whereas the tt5 was significantly less fit in the presence of UV-B (Fiscus et al. 1999). This phenotype was even more dramatic when a triple tt5, uvr1, uvr2 mutant was made by removing the flavonol sunscreen compounds and DNA repair mechanisms. Normal solar UV-B irradiation levels killed this genotype within 48 h (Britt & Fiscus 2003). The tt5 mutant was always less fit and smaller than the wild-type suggesting that chalcone isomerase-derived products are important for other physiological processes (Fiscus et al. 1999). Thus, Arabidopsis flavonol glycosides appear important for UV-B defence in the wild as well as potentially impacting other unknown environmental responses. However, tt5′s pleiotropic effects in the absence of UV-B do limit the capacity to state explicitly that this effect is via the phenylpropanoids UV-B absorption.

FLAVONOID AGLUCONES AND AUXIN TRANSPORT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. GLUCOSINOLATES
  5. GLUCOSINOLATES AND INSECT INTERACTIONS
  6. GLUCOSINOLATES AND PATHOGEN INTERACTIONS
  7. CAMALEXIN AND PATHOGEN INTERACTIONS
  8. PHENYLPROPANOIDS AND UV-B RESISTANCE
  9. FLAVONOID AGLUCONES AND AUXIN TRANSPORT
  10. TERPENOIDS AND PLANT/INSECT INTERACTIONS
  11. FUTURE AVENUES
  12. REFERENCES

The flavonol aglucones, kaempferol and quercitin, can regulate polar auxin transport in vitro (Jacobs & Rubery 1988; Faulkner & Rubery 1992). However, the in planta relevance of this phenomenon was not explicitly validated until comparing auxin accumulation between Arabidopsis flavonol mutants. Most Arabidopsis flavonols accumulate as either kaempferol or quercitin glycosides (Li et al. 1992; Graham 1998). However, developing seedlings accumulate significant kaempferol and quercitin aglucone levels (Peer et al. 2001). This accumulation occurs in the same tissues as does auxin and auxin transport protein accumulation (Peer et al. 2001). Thus, aglucones could impact polar auxin transport. To confirm this hypothesis, the auxin allocation in developing wild-type and tt4, a chalcone synthase-deficient mutant, was compared (Shirley et al. 1995; Murphy, Peer & Taiz 2000). The mutant tt4 plants lacking flavonols had altered auxin accumulation patterns which were restored to wild-type via supplementation with the chalcone synthase product, naringenin. Thus, flavonols impact auxin transport. However, the flavonol-deficient seedlings do not have a visible growth alteration. Thus, the developmental impact of flavonol-regulated auxin transport has yet to be ascertained.

TERPENOIDS AND PLANT/INSECT INTERACTIONS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. GLUCOSINOLATES
  5. GLUCOSINOLATES AND INSECT INTERACTIONS
  6. GLUCOSINOLATES AND PATHOGEN INTERACTIONS
  7. CAMALEXIN AND PATHOGEN INTERACTIONS
  8. PHENYLPROPANOIDS AND UV-B RESISTANCE
  9. FLAVONOID AGLUCONES AND AUXIN TRANSPORT
  10. TERPENOIDS AND PLANT/INSECT INTERACTIONS
  11. FUTURE AVENUES
  12. REFERENCES

The completed Arabidopsis genomic sequence revealed the diversity and number of terpene synthase genes; the final tally counted more than 30 Terpene synthase homologues (Aubourg, Lecharny & Bohlmann 2002). These genes are responsible for the conversion of various allylic prenyl diphosphates such as geranyl diphosphate (C10), farnesyl diphosphate (C15) and geranylgeranyl diphosphate (C20) to monoterpenes (C10), sesquiterpenes (C15) and diterpenes (C20). However, the only known terpene-related compounds in Arabidopsis at the time were primary metabolites or hormones such as gibberrelic acids, brassinosteroids and carotenoids. Thus, there were a number of ‘extra’ genes in the genome that could be active or evolutionary remnants.

The first data suggesting that these ‘extra’ genes were actually functional and that there must be unidentified terpene compounds in Arabidopsis came from the cloning and functional characterization of a β-myrcene/(E)-β-ocimene monoterpene synthase from an Arabidopsis cDNA library (Bohlmann et al. 2000). This has since been expanded to include a wound inducible (E)-β-ocimene synthase and several terpenoid synthases involved in flower volatile formation (Bohlmann et al. 2000; Chen et al. 2003). Thus, Arabidopsis has numerous functional terpene synthases with diverse biochemical activities but a limited set of actual terpenes had yet to be identified. An analysis of volatile compounds from Arabidopsis flowers identified nearly 20 mono/sesqui terpenes (Chen et al. 2003). This study also showed that the terpene synthases had diverse organ expression patterns, but that six terpene synthases appeared floral specific and their biochemical activities agreed with the terpene mix present in floral volatiles.

One well-known activity of floral terpenoid volatiles is that of insect attractance to encourage cross-pollination (Doudareva & Pichersky 2000). Thus, it is possible that these floral volatile terpenes may function as insect attractants in Arabidopsis flowers. This is a slight conundrum as the prevailing understanding is that Arabidopsis is a selfing species. However, several lines of data indicate that out-crossing occurs in the wild and this may be via insect pollination. Estimates of cross-pollination frequency are as high as 2% in outdoor-grown Arabidopsis inbred lines (Snape & Lawrence 1971). This same study observed several insect species visiting Arabidopsis flowers. Together this suggests that insects may function as low frequency out-crossing vectors in wild Arabidopsis thaliana populations. Recent genomic analyses of Arabidopsis variation have also identified a moderate level of heterozygosity in some wild Arabidopsis populations.

Another theory for the role of Arabidopsis terpenes focuses on the wound inducibility of (E)-β-ocimene monoterpene synthase and (E)-β-ocimene (Faldt et al. 2003). (E)-β-ocimene is a common constituent of wound- and insect-inducible volatile blends in diverse plant species (Turlings, Tumlinson & Lewis 1990; Kessler & Baldwin 2001). (E)-β-ocimene may function in a tritrophic relationship whereby it attracts natural enemies of the insect herbivore (Kessler & Baldwin 2001). Alternatively (E)-β-ocimene has also been shown to induce gene expression in lima beans and may act as a signalling molecule (Arimura et al. 2000). Wound- and herbivore-induced volatiles in Arabidopsis can attract parasitoids but the specific volatile component responsible for this attraction has not been identified (Van Poecke et al. 2001). However, either the cross-pollination model or plant–insect interaction models have yet to be functionally tested or verified. This should be possible using knockouts of the various terpene synthases and investigating the impacts on cross-pollination or tritrophic interactions.

FUTURE AVENUES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. GLUCOSINOLATES
  5. GLUCOSINOLATES AND INSECT INTERACTIONS
  6. GLUCOSINOLATES AND PATHOGEN INTERACTIONS
  7. CAMALEXIN AND PATHOGEN INTERACTIONS
  8. PHENYLPROPANOIDS AND UV-B RESISTANCE
  9. FLAVONOID AGLUCONES AND AUXIN TRANSPORT
  10. TERPENOIDS AND PLANT/INSECT INTERACTIONS
  11. FUTURE AVENUES
  12. REFERENCES

Studies in Arabidopsis have provided significant progress towards understanding the biological role of different secondary metabolites. However, there are a large number of unanswered questions remaining. A complete understanding of Arabidopsis secondary metabolites will require identifying the complete Arabidopsis secondary metabolome. With each ensuing study, the number of secondary metabolites identified in Arabidopsis keeps growing (Hagemeier et al. 2001; Reichelt et al. 2002; Chen et al. 2003; Walker et al. 2003). A number of these compounds are unexpected and their function unknown. This and the large number of genes that by predicted amino acid sequence are involved in secondary metabolism suggests that there is a much larger than expected secondary metabolome in Arabidopsis. Identifying these compounds and their function is an important task for the future. Some other important questions for the future are listed below.

  • 1
    Where do the different secondary metabolites accumulate in the plant?
  • 2
    What is more important – structure, amount, regulation or localization – in determining biological activity?
  • 3
    How pervasive is cross-talk between the secondary metabolite biosynthetic pathways?
  • 4
    What is the ecological importance of each secondary metabolite?
  • 5
    Does secondary metabolite natural variation impact plant fitness or interactions with the environment?
  • 6
    How does plant variation in secondary metabolite biosynthesis impact detoxification variation in the attacking pest?

Answering these questions will require combining diverse research techniques including mass-spectrometry/nuclear magnetic resonance, cell biology, ecology, evolution, genetics, biochemistry and other yet to be developed technologies.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. GLUCOSINOLATES
  5. GLUCOSINOLATES AND INSECT INTERACTIONS
  6. GLUCOSINOLATES AND PATHOGEN INTERACTIONS
  7. CAMALEXIN AND PATHOGEN INTERACTIONS
  8. PHENYLPROPANOIDS AND UV-B RESISTANCE
  9. FLAVONOID AGLUCONES AND AUXIN TRANSPORT
  10. TERPENOIDS AND PLANT/INSECT INTERACTIONS
  11. FUTURE AVENUES
  12. REFERENCES
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