HIG1/MYB51 is a regulator of glucosinolate biosynthetic pathways
Substantial progress has been achieved in the understanding of glucosinolate biosynthesis using forward and reverse genetics in the model plant A. thaliana. However, little is known about underlying regulatory mechanisms and genes that coordinate and control glucosinolate compound accumulation during development and in response to environmental challenges. Recently, the first regulatory factors involved in the control of glucosinolate biosynthesis, including IQD1 were described (Levy et al., 2005; see Introduction). In addition, the R2R-MYB transcription factor ATR1/MYB34, which is known to activate tryptophan biosynthetic genes (Bender and Fink, 1998), has been reported to act as a regulator of indolic glucosinolate homeostasis (Celenza et al., 2005).
Here, we present HIG1/MYB51 as a new regulator of glucosinolate biosynthesis identified as an activation-tagged mutant (HPC1-1D, HIG1-1D), that was initially isolated in a screen for mutants with altered accumulation of secondary metabolites (Schneider et al., 2005). The chemotype caused by the dominant HIG1-1D allele, namely the accumulation of I3M, was shown to be caused by 4x35S enhancer-mediated overexpression of the R2R3-MYB transcription factor HIG1/MYB51. 4x35S enhancer elements have been demonstrated to increase quantitatively the original expression pattern of a gene, and not to lead to constitutive overexpression (Neff et al., 1999; van der Graaff et al., 2003). Remarkably, ectopic overexpression of HIG1/MYB51 in the wild-type background could phenocopy the chemotype of the original activation-tagged line HIG1-1D (Figure 2). On the other hand, a T-DNA insertion allele of HIG1/MYB51 (hig1-1) caused a significant reduction in I3M levels, and this chemotype could be counteracted by overexpression of HIG1/MYB51 (Figure S3). Thus, HIG1/MYB51 represents an important, but certainly not the only, regulatory component controlling indolic glucosinolate biosynthesis in A. thaliana. Notably, neither the hig1-1 mutant nor the HIG1-1D or HIG1/MYB51 overexpressing plants showed significant changes in IAA levels or aberrant growth phenotypes indicative for distorted auxin levels (Figure 5).
We also demonstrate that genes involved in the biosynthesis of tryptophan and indolic glucosinolate biosynthesis, i.e. DHS1, TSB1, CYP79B2, CYP79B3, CYP83B1 and AtST5a, are activated by HIG1/MYB51 in trans (Figure 4). Thus, HIG1/MYB51 appears to serve as a general activator of both tryptophan synthesis genes and tryptophan secondary metabolism genes. A co-regulation of indolic glucosinolate pathway enzymes and tryptophan biosynthetic enzymes has been observed by analyzing microarray data derived from different stress experiments (Gachon et al., 2005). Hence, the activation of the tryptophan biosynthetic pathway seems to be required for providing sufficient levels of the precursor tryptophan for the increased formation of indolic glucosinolates in response to stress.
The level of the main short-chain aliphatic glucosinolate 4MSOB is lower in HIG1-1D and in HIG1/MYB51 overexpression lines compared with the wild-type (Figures 2 and 6). It thus appears that HIG1/MYB51 possesses opposite effects on the biosynthetic pathways of indolic and aliphatic glucosinolates. One may speculate that the decreased accumulation of methionine-derived glucosinolates may result from a metabolic crosstalk between both branches of glucosinolate biosynthesis, by which a distinct ratio of the different glucosinolates is maintained. The ‘limiting electron hypothesis’, which has been recently proposed, could serve as an explanation for these observations (Grubb and Abel, 2006): the competition of cytochrome P450 monooxygenases involved in aliphatic (CYP79F1, CYP79F2 and CYP83A1) and indolic glucosinolate (CYP79B2, CYP79B3 and CYP83B1) biosynthetic pathways for electrons to reduce dioxygen to water could be the reason for a reciprocal negative feedback regulation between both branches of glucosinolate biosynthesis.
Strikingly, the hig1-1 null allele caused not only reduced levels of indolic glucosinolates, but also of short-chain aliphatic glucosinolates, resulting in a reduction of the total glucosinolate content by approximately 65% (Figure 2). On the other hand, HIG1/MYB51 overexpression in both the wild-type and the hig1-1 mutant background led to the expected increase in the level of indolic glucosinolates, with aliphatic glucosinolates remaining at low levels (Figure 2; Figure S3). The reason for this is not known yet but might point to a regulatory interplay between HIG1/MYB51 and other factors involved in the control of both indolic and aliphatic glucosinolate biosynthetic genes. It is feasible, for example, that the activity of putative regulators of aliphatic glucosinolate biosynthesis and corresponding biosynthetic genes is altered in the hig1-1 mutant.
Roles of HIG1/MYB51, ATR1/MYB34 and MYB122 in the regulation of glucosinolate biosynthesis are divergent
A. thaliana mutants defective in the early steps of the glucosinolate biosynthetic pathway, or overexpressing these enzymes, all show an altered morphology reminiscent of IAA overexpression or IAA repression phenotypes, respectively (Zhao et al., 2002; Barlier et al., 2000; Bak et al., 2001; Reintanz et al., 2001). These observations can be attributed to the fact that IAOx as a product of CYP79B2 and CYP79B3 actions is an important intermediate of both the indolic glucosinolate and IAA pathways (Figure 3). Overexpression of the transcription factor AtDof1.1 causes changes in glucosinolate profiles, but also affects auxin accumulation (Skirycz et al., 2006). Overexpression of ATR1/MYB34 was shown earlier to result in a hyperactivation of indolic glucosinolate pathway genes, accompanied by only a modest elevation of IAA levels with no impact on plant growth (Celenza et al., 2005).
The question of how glucosinolate biosynthesis is regulated (for example, in response to biotic stresses) without a strong impact on auxin biosynthesis is still open. It does not appear implicitly favourable to plants to alter IAA biosynthesis when an increased biosynthesis of indolic glucosinolates is required. Our data demonstrate that even though both IAA and indolic glucosinolate biosynthetic pathways share common enzymes, both pathways can be specifically regulated to allow appropriate plant responses to environmental challenges. HIG1/MYB51 can activate genes both upstream and downstream of IAOx, leading to increased indolic glucosinolate levels without a significant effect on IAA contents (Figures 2, 5b and 6). Even though ATR1/MYB34 can partially rescue the low-indolic glucosinolate hig1-1 mutant chemotype, overexpression of ATR1/MYB34 in the hig1-1 or wild-type background in our hands led to up to sevenfold higher IAA levels, reflected by pronounced high-IAA growth phenotypes (Figure 5a). In addition, all ATR1/MYB34 overexpressing plants were defective in the development of generative organs, the root system, as well as the vegetative biomass. In contrast, HIG1/MYB51 overexpressors and hig1-1 plants display unaltered morphology compared with the wild-type, and also the IAA levels of these plants are, if at all, only slightly altered (Figure 5).
Remarkably, ASA1 is not trans-activated by HIG1/MYB51 (Figure 4), and the upregulation of ASA1 therefore does not seem to be required for an increased biosynthesis of indolic glucosinolates. However, ASA1 appears to play a pivotal role in the regulation of auxin biosynthesis. For example, the ethylene-triggered activation of auxin biosynthesis was recently shown to be strictly dependent on ASA1. Likewise, the high-IAA phenotype of sur1 and sur2 mutants with defects in C-S lyase and CYP83B1 functions, respectively, is suppressed in mutants defective in ASA1/WEI2 function (Stepanova et al., 2005). Also, these results support the hypothesis that ATR1/MYB34 overexpression, which does cause increased steady-state levels of ASA1 transcripts (Celenza et al., 2005; Figure 6c), might primarily be linked to the regulation of IAA homeostasis.
In addition, the observation that ATR1/MYB34 does not show any expression in vegetative parts of the plant, e.g. rosette leaves, argues against a major role of ATR1/MYB34 in the control of indolic glucosinolate biosynthesis in leaves of adult plants (Figure S5; AtGenExpress data analyzed using Genevestigator, http://www.genevestigator.ethz.ch/).
In contrast to ATR1/MYB34, MYB122 could not rescue the low-indolic glucosinolate chemotype of the hig1-1 mutant (Figure 6b). Likewise, MYB122 overexpression did not cause a high-IAA phenotype in hig1-1 mutants, which raises the question about the role of MYB122 in the tryptophan pathway. Overexpression of MYB122 in the wild-type background, however, led to the enhanced transcription of several tryptophan pathway genes, including ASA1, TSB1, CYP79B2, CYP79B3 and CYP83B1, along with elevated auxin levels (Figures 5b and 6). A number of scenarios may explain this result. Increased IAA production could cause a positive feedback on the expression of these genes. Alternatively, MYB122 could directly enhance the expression of these genes, although only in concert with HIG1/MYB51. In addition, or alternatively, HIG1/MYB51 and/or MYB122 could act as activators or repressors, depending on the context, in a similar way as it has been shown for MYB4 (Jin et al., 2000).
It can be concluded that ATR1/MYB34, HIG1/MYB51 and MYB122 apparently have different roles in the regulation of indolic glucosinolate and IAA biosynthetic pathways. All three factors have the potential to upregulate glucosinolate biosynthetic pathway genes, e.g. TSB1, CYP79B2 and CYP79B3, and can positively regulate indolic glucosinolate accumulation. However, along with increased levels of indolic glucosinolates, overexpression of ATR1/MYB34 and MYB122 led to high-IAA phenotypes. Only the overexpression of HIG1/MYB51 led additionally to the activation of genes further downstream of CYP83B1, i.e. of UGT74B1 and AtST5a. These plants did not exhibit an aberrant growth phenotype. Obviously, the next step is a detailed analysis of the interplay of subgroup 12 R2R3-MYB factors, and potentially other factors, in controlling distinct but partially overlapping sets of target genes from IAA and glucosinolate biosynthetic pathways.
HIG1/MYB51 is expressed at sites of indolic glucosinolate accumulation and plays a role in biotic stress responses
The expression of glucosinolate biosynthetic genes in A. thaliana, e.g. that of CYP79B2, UGT74B1, CYP79F1 and CYP79F2, and also of recently discovered regulators of glucosinolate biosynthesis, IQD1 and AtDof1.1, often appeared to be restricted to vascular tissues (Mikkelsen et al., 2000; Grubb et al., 2004; Reintanz et al., 2001; Levy et al., 2005; Skirycz et al., 2006). HIG1/MYB51 expression overlaps with the expression of these genes but, in addition, is also present in the mesophyll of mature rosette leaves, the pavement cells of young rosette leaves and trichomes (Figure 7). Thus, HIG1/MYB51 expression correlates at least to a great extent with the sites of indolic glucosinolates biosynthesis and accumulation, an observation that is in accordance with the role of HIG1/MYB51 as a positive regulator of this pathway.
Environmental stimuli such as herbivore attack or wounding are known to have a great impact on the regulation of glucosinolate biosynthesis. Several glucosinolate biosynthetic pathway genes were shown to be induced upon mechanical stimuli or hormone treatment (Brader et al., 2001; Kliebenstein et al., 2002b; Mikkelsen et al., 2003; Mewis et al., 2005). Here we show that expression of HIG1/MYB51, but not of ATR1/MYB34, in leaves is rapidly induced by wounding or touch (Figure 8). It is tempting to speculate that HIG1/MYB51 is a key player in the signal transduction chain leading from the touch perception output to an increased biosynthesis of indolic glucosinolates, thereby rendering the plant more resistant to herbivores. It could indeed be shown that generalist herbivores avoided the HIG1-1D line with higher contents of indolic glucosinolates in dual-choice assays (Figure 9). Furthermore, QTL (quantitative trait locus) mapping analyses in A. thaliana (Kliebenstein et al., 2002a) provided data concerning a QTL controlling herbivore resistance in a Ler × Col population, which could be mapped on chromosome I between 14 and 28 cM. This QTL nicely fits to the position of the locus At1g18570 encoding HIG1/MYB51. This finding adds further evidence to the assignment of HIG1/MYB51 as an important regulatory component in controlling glucosinolate biosynthesis upon biotic challenges.
Until now it was assumed that the common pathway for the biosynthesis of both IAA and indolic glucosinolates in plants from the Brassicaceae family is simultaneously regulated by ATR1/MYB34. As shown in this study, HIG1/MYB51 obviously represents a key component in controlling the mechanical-induced regulation of the indolic glucosinolate biosynthetic pathway in A. thaliana without affecting IAA biosynthesis and plant morphology.