Glucosinolates and the clubroot disease: defense compounds or auxin precursors?
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- Ludwig-Müller, J. Phytochem Rev (2009) 8: 135. doi:10.1007/s11101-008-9096-2
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The clubroot disease is caused by the obligate biotrophic protist Plasmodiophora brassicae and is one of the most damaging for the family of Brassicaceae. Since many economically important crops belong to this plant family, the understanding of mechanisms how the disease is developing, are of high importance. Glucosinolates, a group of secondary plant products in the family of Brassicaceae, have long been associated with clubroot disease symptoms. Measurements showed that several glucosinolates are induced in root galls. While aliphatic glucosinolates are regarded as defense compounds, analysis of Brassica cultivars as well as Arabidopsis thaliana mutants provided correlative evidence between disease severity and indole glucosinolate content. The latter have been discussed as precursors for auxin biosynthesis. Since high auxin levels are associated with large root galls, indole glucosinolates could contribute directly or indirectly to the extent of disease development. Transcriptome and proteome experiments have revealed evidence for the involvement of genes from the glucosinolate and auxin pathway in gall formation. These data have been complemented by expression and mutant analysis. It can be concluded that regulation of glucosinolate and IAA biosynthesis might differ in Brassica and Arabidopsis.
KeywordsAuxinBrassicaceaeClubroot diseaseGlucosinolatesPlasmodiophora brassicae
Myrosinase binding protein
Myrosinase associated protein
Quantitative trait loci
Development of the clubroot disease
Life cycle of Plasmodiophora brassicae
The importance of plant hormones during the clubroot disease
During the secondary infection cycle cell divisions occur and later large hypertrophied cells harbouring secondary plasmodia and resting spores can be observed (for more detailed review see Ludwig-Müller and Schuller 2008). Plant hormones, i.e. auxins and cytokinins have been associated with these phenomena (e.g. Devos et al. 2005; Siemens et al. 2006). The availability of hormone-responsive reporter genes in Arabidopsis enabled the localization of enhanced auxin and cytokinin responsiveness to the part of the root where gall formation occurs (Siemens et al. 2006). An increase in free and conjugated IAA levels was reported for Arabidopsis and Brassica, although at different time points during infection, making comparisons difficult (Ludwig-Müller et al. 1993, 1996, 1999a; Grsic et al. 1999; Grsic-Rausch et al. 2000, Devos et al. 2005). However, it is not entirely clear whether the pathogen or the host plant produces the hormones. Although evidence for auxin production by the pathogen is not available, it has been shown that plasmodia of P. brassicae are able to make small amounts of cytokinins (Müller and Hilgenberg 1986). In addition to the cytokinin production by the pathogen, the capacity of the host plant to degrade cytokinins is downregulated in root galls of Arabidopsis (Siemens et al. 2006). Transcriptome data have revealed that also other plant hormones might play a role as signals because genes involved in biosynthesis, metabolism, signalling or response were differentially regulated in gall tissue compared to controls (Siemens et al. 2006). Particularly interesting is the finding that jasmonic acid (JA) is upregulated in Brassica rapa roots during club formation because JA is able to induce indole glucosinolates and also nitrilase (Grsic et al. 1999; Ludwig-Müller et al. 1997), thereby possibly linking auxin and jasmonate metabolism in clubs.
Role of aliphatic and aromatic GSL during clubroot
In an investigation on the host range of P. brassicae in various non-Brassica species further evidence for the possible dual roles of aromatic GSL during club formation was presented (Ludwig-Müller et al. 1999b). Severity of symptom development was correlated with certain glucosinolates in one species, while the increase in other GSL might be regarded as defense response. In the glucosinolate-containing non-Brassicas, Tropaeolum majus and Carica papaya, the concentrations of benzylglucosinolate were increased markedly in roots inoculated with P. brassicae, compared with the controls. Slight gall formation was observed in T. majus (Ludwig-Müller et al. 1999b) and it was hypothesized that benzyl-GSL could act as precursor for phenylacetic acid (PAA; see Fig. 4), which has auxin activity in T. majus (Ludwig-Müller and Cohen 2002). Also, Lepidium sativum, which has a high benzyl-GSL content (Ludwig-Müller et al. 1999b) was able to form clubs after P. brassicae infection (Butcher et al. 1976) and in root galls of L. sativum phenylacetonitrile was detected, which would be the direct precursor of PAA. However, in Reseda alba roots, the total glucosinolate content decreased after inoculation with P. brassicae compared with the controls whereas high root concentrations of 2-OH-2-phenylethylglucosinolate compared with low root indole glucosinolates in this species may limit P. brassicae infection and development.
In Arabidopsis thaliana, mutants which had been isolated for their altered leaf GSL pattern (Haughn et al. 1991), were used to investigate the relation of aliphatic GSL and P. brassicae infection. Two alkenyl-GSL were only detectable in infected roots of mutants and wild type and total aliphatic GSL were only slightly increased in clubroots. Consequently, a mutant in a gene encoding an enzyme involved in the biosynthesis of aliphatic GSL (gsm1-1; Kroymann et al. 2001) did not show any tolerance to clubroot (Ludwig-Müller et al. 1999a). Such results could indicate that in host plants, even though the GSL metabolism was increased as a sign of defense response, the pathogen might be well adapted.
Since no direct experimental information is available on the control of aliphatic GSL synthesis during clubroot, transcriptome data from clubroot development in Arabidopsis were re-analyzed for the expression of genes involved in aliphatic GSL synthesis (Fig. 3a). In this experiment two distinct developmental stages of clubroot disease were analyzed (Siemens et al. 2006). At an early time point (10 dpi) no disease symptoms were visible and the pathogen was mostly at the stage of young vegetative plasmodia. A second time point was characterized by clearly visible clubs and in root sections plasmodia, sporangia and resting spores of the pathogen could be found (23 dpi). The genes selected included the methylthioalkymalate synthases MAM1 and MAM3 (=MAM-L) which are involved in chain elongation of methionine (Textor et al. 2007), as well as the cytochrome P450 enzymes CYP79F1/F2 involved in aldoxime formation (Chen et al. 2003) and CYP83A1 wich converts the oxime to further metabolites in the pathway (Naur et al. 2003). The CYP79F transcripts were not significantly deregulated in clubroots, which was also the case for MAM1. However, MAM3 and CYP83A1 were regulated in a similar manner. Both were upregulated at an early time point where symptoms were not yet visible, but the tissue already colonized by the pathogen (for more details see Siemens et al. 2006). At a time point where gall formation was clearly visible the two transcripts were then downregulated. It could be hypothesized that upregulation at the first time point is a transient defense reaction of the plant which is later downregulated by the pathogen by yet unkown signals to circumvent the production of toxic compounds.
The role of indole glucosinolates during clubroot
Variations in indole glucosinolates and disease development
A hypothesis which has been brought forward several decades ago indicated a role for indole GSL as precursors for IAA production during clubroot (Butcher et al. 1974). It was emphasized that if plants have large amounts of indole GSL, then after tissue disruption also large amounts of IAA would be produced, thus resulting in susceptible plants. This hypothesis has provoked the reverse assumption, namely that plants with low indole GSL levels and therefore a lower capacity for IAA synthesis by turnover of indole GSL, should be more tolerant to clubroot formation. However, conflicting results have been published on this topic. For example Butcher et al. (1974) and Ockendon and Buczacki (1979) found clear correlations between resistance and low indole glucosinolate content in Brassicaceae. Chong et al. (1981, 1984) confirmed these results with cabbage breeding lines, whereas Mullin et al. (1980) were not able to correlate the indole glucosinolate content and the resistance to clubroot. Comparing two resistant and two susceptible Brassica rapa cultivars prominent differences in the indole glucosinolate pattern was found (Ludwig-Müller et al. 1997). Mainly the two susceptible cultivars reacted with an increase in the indole glucosinolates after infection with Plasmodiophora brassicae 14 and 20 dpi in roots, whereas there was no difference between infected and control roots of the two resistant cultivars. However, all cultivars tested showed increased glucosinolate levels after treatment with salicylic acid (SA) and jasmonic acid (JA). JA induced mainly the indole glucosinolates in the leaves, whereas SA induced the indole glucosinolates also in the roots of both cultivars. Also, the transcript for myrosinase and the activity of nitrilase with IAN as substrate were induced by JA in Brassica linking jasmonate and auxin synthesis (Grsic et al. 1999). It should be noted that both JA (Grsic et al. 1999) and SA (Ludwig-Müller et al. 1995) were increased in root galls compared to control roots during stages were galls were clearly visible making these two compounds good candidates for signals involved in the induction of GSL synthesis during the clubroot disease.
In Arabidopsis thaliana ecotype Columbia indole GSL were also increased during clubroot development (Ludwig-Müller et al. 1999a). Two mutant lines with altered GSL content (Haughn et al. 1991), named lines tu3 and tu8 proved to be particularly useful to study the involvement of glucosinolates and auxin in clubroot disease (see also below). Both showed reduced symptom development compared to the wild type, but only in tu8 indolic GSL decreased while in tu3 they were unchanged compared to wild type (Ludwig-Müller et al. 1999a). The role of indole glucosinolates, and more specifically their breakdown products, as potential precursors of IAA, therefore does not appear to be a simple relationship. Other major factors that need to be considered include the flux through the glucosinolate biosynthetic and degradative pathways (specifically the rate of indole GSL and 1-MeOH-indole-GL biosynthesis versus the rate of myrosinase-catalyzed degradation), and the rate of nitrilase-catalyzed conversion of IAN to IAA (see below).
Regulation of auxin and indole GSL biosynthesis and metabolism during clubroot
Work on the enzymes and genes involved in the biosynthesis has more concentrated on auxin synthesis/indole GSL metabolism than in the first steps of indole GSL biosynthesis. For a recent review on auxin biosynthesis and metabolism see e.g. Woodward and Bartel (2005). The general pathways for GSL biosynthesis have been described elsewhere in detail (Grubb and Abel 2006; Halkier and Gershenzon 2006). Microarray analysis (Siemens et al. 2006) has revealed that several genes coding for proteins involved in the biosynthesis of indole GSL and IAA are differentially regulated during the disease in Arabidopsis thaliana (Fig. 3b). It is noteworthy that only two early indole GSL biosynthetic genes (CYP79B2/B3; Hull et al. 2000) converting tryptophan to indole-3-acetaldoxime (IAOx) were upregulated at an early time point, whereas later genes in the pathway such as SUR2 (Barlier et al. 2000), SUR1 (Mikkelsen et al. 2004), and UGT74B1 (Grubb et al. 2004) were not regulated or actually downregulated (Fig. 3b). Only those involved directly in IAA biosynthesis were upregulated later, indicating that the increase in IAA via the indole GSL/nitrilase pathway is important only during late stages of gall development. These stages are accompanied by hypertrophied cells harbouring plasmodia and resting spores of the pathogen (Figs. 1, 2).
The first enzyme necessary for the formation of indole-3-acetonitrile (IAN) from indole GSL is myrosinase. While myrosinase genes were not upregulated in Arabidopsis according to microarray analysis, proteome analysis found a myrosinase upregulated during early inoculation events (Devos et al. 2006). Contrary, in Brassica rapa, the expression level of myrosinase was increased in infected roots compared to controls (Grsic et al. 1999). It can be speculated that myrosinase expression and IAN formation are dependent on different endogenous and exogenous signals and that an apparent up- or down-regulation might not reflect the situation in planta, where maybe only few infected cells contribute to IAN synthesis. Up to now there is no evidence for the involvement of other myrosinase associated proteins or for epithiospecifier protein in clubroot development.
IAN can further be converted to IAA by the enzyme nitrilase. In Arabidopsis there is a family of four nitrilases of which three are capable to convert IAN to IAA (Bartel and Fink 1994; Hillebrand et al. 1998). It was shown that nitrilase 1 and 2 are increased in developing root galls during later stages by Northern analysis, promoter-GUS studies (Grsic-Rausch et al. 2000) and microarray analysis (Siemens et al. 2006; see also Fig. 3b). Immunolocalization confirmed nitrilase protein in hypertrophied cells harbouring sporulating plasmodia (Grsic-Rausch et al. 2000). These data indicate that in Arabidopsis nitrilase is associated with late stages of clubroot development. Early in vivo labeling studies showed that IAN is specifically converted to IAA from indole GSL in infected Brassica roots, whereas wounding induced the formation of different metabolites (Rausch et al. 1983). Also, increased nitrilase activity has been associated with increased IAA content and root gall development in different Brassicaceae (Rausch et al. 1981b; Grsic et al. 1999; Ugajin et al. 2003; Ishikawa et al. 2007a). However, Northern blot analysis did not show any differences in the mRNA levels of nitrilase in B. rapa (Chinese cabbage) during gall formation (Bischoff et al. 1995) but the probes used in this study were not gene specific and might have therefore missed subtle differences. This hypothesis is likely, based on results from Ishikawa et al. (2007a) who showed that in B. rapa (turnip) nitrilase was differentially expressed in clubroots. Both, nitrilase 1 from turnip and Chinese cabbage convert IAN to IAA, but high Km values indicate that IAN might not be the natural substrate. Instead, phenylacetonitrile and phenylpropionitrile are highly converted to the corresponding acid, indicating maybe a different or additional role for nitrilase in clubroot formation (Grsic et al. 1998; Ishikawa et al. 2007a). Whether this might be the conversion of nitriles to nontoxic compounds or whether phenylacetic acid is produced which might also act as auxin (see above) is not clear. However, alternatives for the biosynthesis of IAA in Brassica root galls have been reported. For Brassica rapa two different possibilities for the conversion of IAOx to IAA have been demonstrated on an enzymatic level. Ludwig-Müller and Hilgenberg (1990) described an enzymatic activity capable to catalyze the reaction from IAOx to IAN and Helmlinger et al. (1987) described an activity converting IAOx to indole-3-acetaldehyde (IAAld). Both routes bypass indole GSL. However, the contribution of these pathways to IAA synthesis in clubroots has not been investigated. IAAld can be further converted to IAA in Arabidopsis by aldehyde oxidases (Seo et al. 1998). Aldehyde oxidase was also induced in Brassica clubroots (Ando et al. 2006). In comparison, Arabidopsis did not show such an induction according to microarray data (Fig. 3b). In B. rapa (turnip) in addition to nitrilase the conversion of indole-3-acetamide to IAA, a pathway initially described for plant pathogenic microorganisms such as Agrobacterium tumefaciens (Inzé et al. 1984) was increased in clubroots on an enzymatic level (Ishikawa et al. 2007b). Amidase is also present in Arabidopsis (Pollmann et al. 2003), but does not contribute to IAA levels in root galls according to transcriptome data (Fig. 3b). Direct evidence for the contribution of other genes involved in auxin biosynthesis such as the YUCCA family (Zhao et al. 2001) has yet to be presented.
It should be noted that other compounds related to the IAOx/indole GSL pathway such as the phytoalexin camalexin were also induced during clubroot (Siemens et al. 2008), thus maybe diverting the metabolic flow away from indole GSL. The accumulation of IAN after clubroot infection could, for example, result in the competition of the two different pathways for one substrate (Nafisi et al. 2007). One protein in this pathway (CYP71A12/A13) could play a dual role in the formation of IAN which is converted either further to IAA or camalexin (Nafisi et al. 2007). The gene for this protein was also highly induced in root galls (Fig. 3b). On the other hand the cytochrome P450 CYP71B15 (PAD3), which is also involved in camalexin biosynthesis (Zhou et al. 1999), is probably not directly involved in clubroot formation, because the mutant pad3 did not show tolerant phenotypes (Siemens et al. 2002), even though the corresponding gene was found to be upregulated in transcriptome analysis (Fig. 3b; Siemens et al. 2006, 2008). Alternatively, IAN could be channeled within each pathway or IAN could be only converted to IAA when excess IAN accumulates (Nafisi et al. 2007). The latter idea may explain why the expression of NIT1 and NIT2 is induced by exogenous IAN (Grsic et al. 1998). In Arabidopsis it has not been shown whether the two pools of IAN are interchangeable. In Brassica juncea it was shown that IAN was most likely produced from trpytophan via IAOx after infection with Phoma lingam, but no evidence for the further metabolism of IAN was provided (Pedras et al. 2002).
The analysis of mutants and transgenic plants which have altered levels of genes involved in the indole GSL/IAA pathway will help to shed light on the role of these gene products for gall formation. In Arabidopsis many such mutants are available and have been studied. Mutants with altered indole GSL levels (Haughn et al. 1991; see above) showed reduced root galls with fewer pathogen structures (Ludwig-Müller et al. 1999a). This was accompanied by lower IAN and IAA levels, but amazingly, the indole GSL level was not so much reduced. This was a first indication that indole GSL are not the sole contributors to the pathway and thus IAA formation. The mutant tu8 was shown to be not directly connected to indole GSL metabolism because the mutated gene was identified as heterochromatin like protein 1 (Kim et al. 2004; Bennett et al. 2005). Therefore, the phenotypes observed might be rather indirect effects of a chromatin modifying protein and the clubroot/indole GSL/auxin phenotypes observed in tu8 have to remain correlative.
Focus was also placed on changes in nitrilase expression. For example the mutant nit1, defective in the nitrilase 1 gene, showed smaller galls with lesser pathogen structures and this was accompanied by a reduction in free IAA levels in clubroots (Grsic-Rausch et al. 2000). Furthermore, a transgenic plant reduced in nitrilase 2 showed slower development of the root galls (Neuhaus et al. 2000). However, overexpression of nitrilase did not result in altered symptoms after infection with P. brassicae (Neuhaus et al. 2000), implicating that wild type levels of nitrilase are sufficient for gall formation, but if there is a rate limiting step upstream in the pathway has not yet been determined.
Since nitrilase is situated late in the pathway after indole GSL (see Figs. 2, 4), mutants available in the first steps of indole GSL biosynthesis are useful tools to investigate the direct role of these compounds in gall formation. Single mutants of two cytochrome P450 enzymes involved in the synthesis of IAOx (cyp79b2 or cyp79b3) did not result in any phenotype (Siemens et al. 2008). Since the two proteins could substitute for each other, double mutants were tested. The cyp79b2/b3 double mutant is almost completely devoid of indole GSL (Zhao et al. 2002). Surprisingly, the cyp79b2/b3 double mutant also showed normal clubroot symptoms and the levels of free IAA in galls were comparable to wild type (Siemens et al. 2008). These results show that indole GSL are not important in Arabidopsis for gall development. It was therefore concluded that a block in early steps in indole GSL biosynthesis could be overcome in terms of IAA synthesis by other pathways such as the YUCCA pathway (Zhao et al. 2001) or by direct synthesis via IAOx and IAN (Nafisi et al. 2007; see also Fig. 2). However, alterations in late steps can not be compensated, thus resulting in reduced gall size (Fig. 4). The situation could be different in Brassica as shown for other genes as well.
Other auxin related mutants which were investigated for clubroot formation included transport, signaling and homeostasis mutants such as aux1, axr1, axr2, ilr1 (Siemens et al. 2002), tir1, tir3, axr6 (Alix et al. 2007), but they did not show any tolerance to clubroot. However, axr3, also an auxin signaling mutant encoding the Aux/IAA protein IAA17 was partially resistant to clubroot (Alix et al. 2007).
Possible transcriptional control of glucosinolate synthesis during clubroot
Transcriptional control is an additional level to be considered for the regulation of metabolites from the indole glucosinolate pathway during clubroot formation. Since there are no experimental data available on transcriptional control of GSL synthesis during clubroot development, microarray data from Arabidopsis (Siemens et al. 2006) have been re-evaluated for expression levels of some transcription factors involved in aliphatic and indole GSL biosynthesis. A transcription factor (OBP2; At1g0640) involved in controlling a downstream step of IAOx to indole-GSL conversion via induction of CYP83B1 (SUR2) was identified (Skirycz et al. 2006), but this zinc finger protein was slightly repressed according to transcriptome analysis at the second time point (−1.1/−2.0). Another transcription factor of the MYB family (ATR1; At5g60890) activates expression of a tryptophan synthesis gene as well as the tryptophan-metabolizing genes CYP79B2, CYP79B3, and CYP83B1 (Celenza et al. 2005). This transcription factor was slightly increased according to transcriptome analysis at the first time point, but repressed at the second time point (1.9/−2.1). A close homolog to ATR1 was identified as another transcription factor involved in the control of indole glucosinolate biosynthesis (Gigolashvili et al. 2007a). The MYB51/HIG1 (HIG = high indolic glucosinolates) gene (At1g18570) was downregulated at the first time point in the microarray and not regulated at the second time point (−2.9/−1.2). If the increase in indole GSL levels plays a role during clubroot development, the downregulation of transcription factors might indicate that transcriptional control of indole GSL synthesis is possibly not an important mechanism. Also the synthesis of aliphatic GSL is controlled by MYB transcription factors (Gigolashvili et al. 2007b, 2008, this issue). MYB28/HAG1 (HAG = high aliphatic glucosinolates) transcripts (At5g61420) (Gigolashvili et al. 2007b) were upregulated at the early time point and slightly downregulated at the second time point (2.0/−1.6) according to transcriptome data. MYB29/HAG3 (At5g07690) and MYB76/HAG2 (At5g07700) transcription factors were also involved in controlling aliphatic GSL (Gigolashvili et al. 2008). Interestingly, it could be shown that the two latter transcription factors not only positively controlled aliphatic GSL content but negatively regulated indole GSL levels (Gigolashvili et al. 2008). Effects of aliphatic GSL on indole GSL have already been reported in the bushy mutant (CYP79F1) with lower aliphatic GSL levels, but the indole GSL were increased (Reintanz et al. 2001). However, trancript levels did not change dramatically according to microarray data for MYB29/HAG3 (1.2/1.7) and for MYB76/HAG2 (1.1/1.5). Either the pathway is not strictly under transcriptional control, or the transcription factors are regulated only in a few infected cells which would not show up in the analysis of the whole root tissue. This problem might be overcome in the future by using single cell analysis methods.
The question initially asked is still open to some extent. GSL and their metabolites may play a role as defense compounds also in the clubroot disease via degradation by myrosinase and associated proteins, but they are not essential factors responsible for resistance. Since transcripts for proteins involved in the biosynthesis of aliphatic GSL from methionine are partially upregulated, the role of aliphatic GSL as defense compounds is likely (see Fig. 4). Using Arabidopsis it could be feasible to introduce corresponding genes and investigate transgenic plants for altered responses to clubroot. On the other hand, the contribution of indole GSL to IAA biosynthesis and thus their contribution as possible link to gall size is only indirect, at least in Arabidopsis. IAN as precursor might be more directly linked to IAA, especially during late stages of gall growth, whereas other pathways must be operating in early stages to contribute to an increase in IAA. However, there are increasingly more mutants available in pathways for aliphatic and indolic glucosinolates which could be tested for tolerance or resistance against the clubroot pathogen. Also, in such mutant lines alternative auxin biosynthesis pathways could be investigated which would substitue for a missing pathway. Finally, the differences between the model plant Arabidopsis and Brassica species in terms of gene regulation or enzymatic activity have to be taken into consideration. Still, Arabidopsis is an important model for studying clubroot disease, because candidate genes possibly involved in gall formation can be much easier assessed than in Brassica. On the other hand, differences seen so far can be also studied involving for example Brassica microarrays which are now becoming available.
Work in the authors laboratory was funded by the Saxonian Ministery of Environment and Geology and the Deutsche Forschungsgemeinschaft.