The Plant Journal (2014) 79, 92–105
doi: 10.1111/tpj.12541
Comparative analysis of Arabidopsis UGT74 glucosyltransferases reveals a special role of UGT74C1 in glucosinolate biosynthesis C. Douglas Grubb1,*, Brandon J. Zipp2,†, Jakub Kopycki1,‡, Melvin Schubert1, Marcel Quint1, Eng-Kiat Lim3,§, Dianna J. Bowles3, M. Soledade C. Pedras4 and Steffen Abel1,2,5 1 Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle, Germany, 2 Department of Plant Sciences, University of California, Davis, CA 95616, USA, 3 Department of Biology, University of York, York Y010 5DD, UK, 4 Department of Chemistry, University of Saskatchewan, Saskatoon SK S7N 5C9, Canada, and 5 Institute of Biochemistry and Biotechnology, Martin Luther University, Halle-Wittenberg, 06120 Halle, Germany Received 20 February 2014; revised 3 April 2014; accepted 23 April 2014; published online 29 April 2014. *For correspondence (e-mail [email protected]). † Present address: Stevia First Corporation, Yuba City, CA 95993, USA. ‡ Present address: Department of Plant Physiology, Justus Liebig University, 35390 Giessen, Germany. § Present address: Center for Immunology and Infection, Department of Biology and Hull York Medical School (Area 12), University of York, York Y010 5DD, UK.
SUMMARY The study of glucosinolates and their regulation has provided a powerful framework for the exploration of fundamental questions about the function, evolution, and ecological significance of plant natural products, but uncertainties about their metabolism remain. Previous work has identified one thiohydroximate S-glucosyltransferase, UGT74B1, with an important role in the core pathway, but also made clear that this enzyme functions redundantly and cannot be the sole UDP-glucose dependent glucosyltransferase (UGT) in glucosinolate synthesis. Here, we present the results of a nearly comprehensive in vitro activity screen of recombinant Arabidopsis Family 1 UGTs, which implicate other members of the UGT74 clade as candidate glucosinolate biosynthetic enzymes. Systematic genetic analysis of this clade indicates that UGT74C1 plays a special role in the synthesis of aliphatic glucosinolates, a conclusion strongly supported by phylogenetic and gene expression analyses. Finally, the ability of UGT74C1 to complement phenotypes and chemotypes of the ugt74b1-2 knockout mutant and to express thiohydroximate UGT activity in planta provides conclusive evidence for UGT74C1 being an accessory enzyme in glucosinolate biosynthesis with a potential function during plant adaptation to environmental challenge. Keywords: Arabidopsis thaliana, secondary metabolism, thiohydroximate, Family 1 glycosyltransferase, UGT74 clade, UGT74B1.
INTRODUCTION Secondary metabolism is a dominant factor that mediates evolutionary and ecological relationships between heterotrophs and autotrophs, and serves as the ultimate source of numerous drugs used in medicine, facts that explain why study in this area has been and continues to be of central importance in biology. Glucosinolates are a class of sulfur-rich metabolites almost exclusive to the Brassicales (Agerbirk and Olsen, 2012). Because of their importance for human health (Dinkova-Kostova and Kostov, 2012), plant defense against herbivores and innate immunity (Lip€ fer and Boland, 2012), glucosinolates ka et al., 2010; Mitho have been the focus of major research efforts facilitated by 92
the powerful genetic resources and molecular tools available for the reference plant, Arabidopsis thaliana (Sønderby et al., 2010). Glucosinolate biosynthesis comprises up to three stages. First, the parental amino acid may undergo one or more rounds of chain elongation via a series of reactions analogous to those converting valine to leucine. The distribution of chain-length in the final products is controlled by genotype as well genotype 9 environment interactions (Burow et al., 2010). Next, the (homo)amino acids enter the core pathway common to all glucosinolates, which can be interpreted as an initial activation, wherein the (homo)amino © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd
UGT74C1 acts in glucosinolate synthesis 93 acid is converted to a highly reactive nitrile oxide. The subsequent series of detoxification steps generates the stable glucosinolates, which are N-sulfated S-glucosyl thiohydroximates (Figure 1). Finally, the structural diversity of these compounds can be further increased by side-chain modifications, including oxygenation, hydroxylation, dehydrogenation, benzoylation and methoxylation, among others (Grubb and Abel, 2006; Halkier and Gershenzon, 2006; Sønderby et al., 2010). In Arabidopsis, the major glucosinolates are aliphatic, derived from methionine (Met,) or indolic derived from tryptophan (Trp); again, the distribution of final products is under genetic and environmental control (Burow et al., 2010).
Glucosylation of the thiohydroxamic acid intermediate is the penultimate reaction of the core pathway. We previously showed that Arabidopsis UGT74B1, a UDP-glucosedependent glucosyltransferase (UGT) of plant Family 1 glycosyltransferases (GTs), is the major enzyme catalyzing this reaction in vivo (Grubb et al., 2004). Nevertheless, mutant plants lacking functional UGT74B1 still accumulate substantial levels of glucosinolates, indicating that at least one additional enzyme is capable of thiohydroximate glucosylation. Several reports of co-regulation of the closely related UGT74C1 with glucosinolate pathway-related genes, particularly with those involved in aliphatic glucosinolate synthesis, have predicted UGT74C1 as a candidate (Gachon et al., 2005; Hirai et al., 2007; Mikkelsen et al., 2010). However, there is as yet no direct evidence for its in vivo function. Arabidopsis thaliana contains 107 members of Family 1 UGTs, which share the highly conserved plant-specific UGT signature motif and glucosylate a large array of small molecules (Bowles et al., 2006; Osmani et al., 2009). We therefore set out to systematically search for and identify the additional UGT(s) that function(s) in glucosinolate synthesis. Our results from complementary biochemical and genetic approaches clearly demonstrate that UGT74C1 acts as an accessory UGT in the glucosinolate core pathway. RESULTS Screening recombinant Arabidopsis family 1 UGT enzymes We expressed almost all A. thaliana Family 1 UGTs as recombinant C-terminal GST-fusions in E. coli, which we affinity-purified (Lim et al., 1998, 2002) and tested for activity against phenylacetothiohydroximate (PATH) as described previously for UGT74B1 (Grubb et al., 2004). As expected, amongst the 93 recombinant enzymes assayed (Table S1), the UGT74B1–GST (glutathione S-transferase) protein displayed the highest activity toward PATH under the three pH conditions tested (pH 6, pH 7 and pH 8). Interestingly, all analysed members of clade UGT74 showed activity toward PATH (Figure 2), ranging from approximately 50% (UGT74F1) to 5% (UGT74E2) relative to UGT74B1, with the exception of UGT74E1, which was not expressed. Minor activities (2–8%) were recorded for recombinant UGT enzymes 88A1, 75B1, 71B6, 73B1, and 73B3. The remaining 81 UGT–GST proteins expressed only trace ( 30) and showed the highest affinity (Km < 20 lM) for both substrates, while the other four enzymes displayed low activities (kcat < 0.2) and affinities (Km > 100 lM). Nevertheless, a relatively inefficient enzyme could provide substantial activity if expressed at sufficiently high level. We therefore compared UGT74 gene expression in wild-type and ugt74b1 knockout plants to test if any clade member is overexpressed as a consequence of the metabolic block in ugt74b1. We did not observe significantly higher UGT74 mRNA levels, indicating that compensatory changes in UGT74 expression are not a likely explanation for the residual glucosinolate content of ugt74b1 plants (Figure S2a). Thus, while we could confirm that UGT74 members are particularly apt in thiohydroximate glucosylation relative to the other 86 UGTs tested, no particular candidate for a new glucosinolate biosynthetic enzyme emerged from this in vitro approach.
Figure 2. Relative activities of recombinant UGT74 enzymes. Ninety-three purified Arabidopsis Family 1 UGT–GST fusion proteins were assayed with UDP-glucose and phenylacetothiohydroximate (PATH) as the thiohydroximate aglycone (see Experimental procedures and Table S1). Activities of UGT74 clade members are given relative to UGT74B1–GST, the most active recombinant UGT enzyme toward PATH.
(Figure S1): 4-methylthiobutylthiohydroximate (MTBTH) and 1-methylindolyl-3-acetothiohydroximate (MIATH). The first compound is the natural precursor of the aliphatic 4methylsulfinylbutyl glucosinolate (S4), the most abundant glucosinolate in leaves of Col-0, while the latter is a quasinatural product and an analogue of the thiohydroximate precursor of all indolic glucosinolates, which can be incorporated into 1-methylindolyl-3-methyl glucosinolate (Pedras et al., 2009). Unfortunately, we could not further evaluate our lead candidate, UGT74C1, because bacterial preparations of the enzyme never consistently showed activity above background, despite extensive exploration of variations in culture conditions, purification protocols, metal co-factors, sugar-nucleotide donor substrates, and reaction buffer composition. The UGT74C1–GST construct of the initial screen expressed very low activity in subsequent experiments, and successful transient expression of (His)6–UGT74C1 in tobacco leaves (Nicotiana benthamiana) resulted in inactive preparations, while the corresponding UGT74B1 control was highly active in this system, results we cannot explain at present. The remaining five purified (His)6–UGT74 enzymes showed significant activity above background and establishment of pseudo-single substrate conditions allowed the
Mutational analysis of clade UGT74 implicates UGT74C1 in glucosinolate synthesis We next examined phenotypes caused by loss-of-function alleles of all UGT74 genes. Figure 3(a) illustrates the positions of insertional and point mutations. Analysis of mRNA levels in the wild-type and mutant lines by quantitative RTPCR confirmed that all ugt74 lines are likely functional knockouts (Figure S2b). Initial characterization of the single mutants revealed, with the exception of ugt74b1 lines, no obvious morphological differences from the wild-type. Slightly elevated accumulation of aliphatic glucosinolates was measured for ugt74e2-1, inconsistent with the idea that this gene plays a direct role in thiohydroximate metabolism (Figure 4a). Thus, we concluded that if any UGT74 member plays such a role, it may be masked by the activity of UGT74B1. We therefore generated double homozygous mutants of each line with either ugt74b1-1 (Ws-0) in the case of ugt74f1 or with ugt74b1-2 (Col-0) for the remaining ugt74 mutants. We previously characterized both ugt74b1 alleles, which cause dwarfism, partial sterility, and a low-glucosinolate chemotype (Grubb et al., 2004). Both wild-type accessions
Table 1 Kinetic parameters of recombinant UGT74 proteins toward MIATH and MTBTH MIATH
MTBTH
Protein
kcat (sec1)
UGT74B1 UGT74C1 UGT74D1 UGT74E2 UGT74F1 UGT74F2
36.5 n.d. 0.11 0.009 0.182 0.021
26.2
0.06 0.010 0.028 0.012
Km app (lM) 17.2 n.d. 158 232 235 130
7.1
90 136 39 161
kcat (sec1) 2.28 n.d. 0.0303 0.0164 0.0267 n.d.
0.79 0.0037 0.0019 0.0046
Km app (lM) 1.48 n.d. 462 159 110 >300
0.55 67 36 39
The mean of three independent determinations is given standard deviation (SD); n.d. – activity not detectable. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 79, 92–105
UGT74C1 acts in glucosinolate synthesis 95
(a)
(a)
(b) (b)
(c)
Figure 3. Loss-of-function alleles of UGT74 genes and their phenotypes in ugt74b1-null backgrounds. (a) Map of T-DNA insertion and mutation sites in UGT74 genes. Exons are depicted as white block arrows and non-translated regions as black lines. The grey box designates the position of the critical UGT signature motif. TDNA insertion sites are marked with a solid black triangle or, for the gene trap line GT1305 (ugt74c1-1), a white triangle. A previously characterized loss-of-function allele (ugt74f2-i1a) generated by ethyl methanesulfonate (EMS) mutagenesis (*) was used for UGT74F2 (Quiel and Bender, 2003). (b) Wild-type plants (Ws-0, Col-0) and indicated knockout lines after 4 weeks of growth on soil. (c) Phenotypes of 3-week-old ugt74b1 knockout lines, alone or in combination with knockouts of other UGT74 members. The two mutants in the leftmost column are in accession Ws-0, all others are in Col-0.
and their corresponding single ugt74b1 mutants are shown in Figure 3(b). All double mutants closely resemble ugt74b1, with one dramatic exception: the ugt74b1-2
Figure 4. Glucosinolate profiles of ugt74 loss-of function lines. (a) Aliphatic and indolic glucosinolate content of the seven single ugt74 mutants relative to the respective wild-type (1 = 100%). Significant differences relative to wild-type (P < 0.05, n = 5) are marked (*). (b) Aliphatic and indolic glucosinolates of ugt74b1 double mutants relative to the respective ugt74b1 background (1 = 100%): ugt74b1-1 for the ugt74b1-1 ugt74f1 double mutant, ugt74b1-2 for all other combinations. Significant differences (P < 0.05, n = 5) are marked (*).
ugt74c1-2 double mutant shows a strongly additive phenotype, producing extremely small plants with only a few small leaves (Figure 3b,c). While these plants persist for a few weeks on soil and eventually produce floral structures, they are completely sterile. The glucosinolate chemotype of ugt74b1-2 ugt74c1-2 plants also stands in stark contrast to those of the other double mutants (Figure 4b). While indolic glucosinolates are not affected relative to ugt74b1-2, the content of aliphatic glucosinolates is reduced an additional 70%, again implicating UGT74C1 specifically in the synthesis of Met-derived glucosinolates. Among the other double mutants, ugt74b1-2 ugt74d1 shows a approximately 2-fold increase in indolic glucosinolates. Although interesting, this result is inconsistent with direct a role of UGT74D1 in thiohydroximate metabolism and was not pursued further. Loss of UGT74C1 in ugt74b1 plants causes seedling lethality We maintained the ugt74b1-2 (+/) ugt74c1-2 (/) line, which is fertile and indistinguishable from the wild-type,
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 79, 92–105
96 C. D. Grubb et al. Figure 5. Phenotypic details of single and double ugt74c1-2 and ugt74b1-2 mutants in comparison with wild-type (Col-0) and sur1 plants grown in axenic conditions. (a, b) Growth on horizontal and vertical agar plates (2 weeks). (c) Germination for 2.5 days in the dark. (d) Plant development for 2 months. The inset shows a futile attempt at flowering by a ugt74b1-2 ugt74c1-2 plant.
(a)
(b) studied plant growth under axenic conditions. We included the sur1 line for comparison because loss of the SUR1 C-S lyase catalyzing the reaction prior to the glucosylation step causes failure of glucosinolate synthesis, overproduction of auxin, and seedling lethality (Boerjan et al., 1995; Mikkelsen et al., 2004). When grown on agar, ugt74b1-2 ugt74c1-2 plants are significantly smaller than ugt74b1-2 and similar in size to sur1 seedlings (Figure 5). The double mutant is extremely stunted and produces very small, curly and chlorotic leaves. Unlike sur1 seedlings, it occasionally develops a short inflorescence with petite sterile flowers. The double mutant maintains a plant-like habit with recognizable organs after an extended period of growth, while sur1 seedlings completely degenerate into callus tissue (Figure 5d). Loss of UGT74C1 in ugt74b1 plants does not mimic the high-auxin phenotype of sur1
(c)
(d)
as a source of double homozygous mutant seedlings. We reasoned that their poor growth on soil might be caused by interactions with hostile organisms and therefore
We reported previously that loss of UGT74B1 causes morphological alterations that are consistent with auxin overproduction, which we confirmed by indole-3-acetic acid (IAA) measurements (Grubb et al., 2004). Interestingly, although ugt74b1-2 ugt74c1-2 seedlings are significantly smaller than ugt74b1-2 and more similar to sur1 plants during early stages of development, they do not share the extreme high-auxin phenotype of sur1. The root system of the double mutant, albeit significantly underdeveloped relative to wild-type and ugt74c1-2 seedlings, is comparable with the root system of ugt74b1-2 plants, but clearly differs from that of the sur1 mutant (Figure 5b). In contrast with sur1 plants, ugt74b1-2 ugt74c1-2 seedlings produce excessive roots and callus-like protrusions at the hypocotyl-root junction only after extended growth on agar (>1 month). When grown under conditions to promote adventitious rooting (Grubb et al., 2004), the double mutant is again more similar to ugt74b1-2 than to sur1 seedlings (Figure S3). When germinated in the dark, ugt74b1-2 seedlings display a moderately de-etiolated phenotype, which is not exaggerated in the ugt74b1-2 ugt74c1-2 double mutant (Figure 5c). Although these data suggest that loss of UGT74C1 activity does not further elevate auxin production in the ugt74b1-2 background, we measured significantly elevated auxin levels relative to wild-type and ugt74b1-2. However, precursors of jasmonic acid and ethylene are also highly increased, suggesting general perturbation of hormone homeostasis in the extremely dwarfed double mutant (Table S2).
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 79, 92–105
UGT74C1 acts in glucosinolate synthesis 97 Loss of UGT74C1 in ugt74b1 plants affects primarily aliphatic glucosinolates We determined glucosinolate composition in shoots of 3week-old wild-type, ugt74c1-2, ugt74b1-2, ugt74b1-2 ugt74c1-2, and sur1 plants (Figure 6). Similar to the ugt74b1-1 allele (Grubb et al., 2004), the ugt74b1-2 mutation decreases total glucosinolates by about 30% due to lower content of both aliphatic and indolic derivatives.
(a)
(b)
(c)
Figure 6. Details of the glucosinolate chemotypes of single and double ugt74c1 and ugt74b1 mutants in comparison with wild-type and sur1 plants (3-week-old). (a) Content of total, aliphatic and indolic glucosinolates in the shoot. (b) Content of the major Met-derived glucosinolates: 3-methylsulfinylpropyl glucosinolate (S3), 4-methylsulfinylbutyl glucosinolate (S4), and 8-methylsulfinyloctyl glucosinolate (S8). (c) Content of major Trp-derived glucosinolates: indolyl-3-methyl glucosinolate (IM), 4-methoxyindolyl-3-methyl glucosinolate (4IM), and 1-methoxyindolyl-3-methyl glucosinolate (1IM). Mean values [standard error (SE)] are given (n = 10). An asterisk indicates a statistically significant difference (P < 0.05) with respect to the wild-type (for ugt74c1-2) or to ugt74b1-2 (for the ugt74b1-2 ugt74c1-2 double mutant). Similar results were obtained for two additional experiments with 2- or 3-week-old plants.
Interestingly, while the glucosinolate chemotype of the ugt74c1-2 single mutant is barely distinguishable from the wild-type, levels of all major Met-derived glucosinolates are dramatically reduced in the ugt74b1-2 ugt74c1-2 double mutant. The reduction of total aliphatic glucosinolates by about 80% in ugt74b1-2 ugt74c1-2 plants clearly exceeds the effect of the ugt74b1-2 mutation (30%). As in the single ugt74c1-2 mutant, loss of UGT74C1 in the ugt74b1-2 background differentially affects aliphatic glucosinolates depending on the length of the side chain (Figure 6b). While the content of 3-methylsulfinylpropyl glucosinolate (S3) is reduced by about half, 8-methylsulfinyloctyl glucosinolate (S8) is nearly abolished, with the S4 compound exhibiting an intermediate reduction. Thus, UGT74C1 seems to prefer aliphatic substrates with longer side-chains in vivo. In contrast, content of Trp-derived glucosinolates is quite similar between the ugt74b1-2 and ugt74b1-2 ugt74c1-2 lines (Figure 6c). While the level of indolyl-3methyl glucosinolate (IM) is lower by about 50% in both lines relative to wild-type, content of 4-methoxyindolyl-3methyl glucosinolate (4IM) is largely unaffected and maintained at wild-type level. The content of 1-methoxyindolyl3-methyl glucosinolate (1IM) is reduced significantly in the double mutant relative to ugt74b1-2 plants. However, the absolute magnitude of this change is miniscule compared with the change in aliphatic glucosinolates. Because the genetic evidence implicates UGT74C1 in glucosinolate synthesis, we searched in more detail for a chemotype in the ugt74c1-2 single mutant. To this end, we examined the glucosinolate profiles of cauline leaves, flowers, fully expanded green siliques and seeds; none of these profiles revealed any significant differences compared with the wild-type (Figure S4). Therefore, the primary role of UGT74C1 in glucosinolate synthesis seems to be unrelated to the developmental stage of adult plants. In summary, the genetic analysis points to a role of UGT74C1 in glucosinolate biosynthesis that is limited to its aliphatic branch. This conclusion is consistent with the phenotypic data that do not indicate enhancement of the high-auxin phenotype of ugt74b1-2 plants by loss of UGT74C1, as would be expected if UGT74C1 also glucosylates indolic thiohydroximates. Consistent with previous reports (Mikkelsen et al., 2004), glucosinolate production in sur1 plants is reduced severely (
doi: 10.1111/tpj.12541
Comparative analysis of Arabidopsis UGT74 glucosyltransferases reveals a special role of UGT74C1 in glucosinolate biosynthesis C. Douglas Grubb1,*, Brandon J. Zipp2,†, Jakub Kopycki1,‡, Melvin Schubert1, Marcel Quint1, Eng-Kiat Lim3,§, Dianna J. Bowles3, M. Soledade C. Pedras4 and Steffen Abel1,2,5 1 Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle, Germany, 2 Department of Plant Sciences, University of California, Davis, CA 95616, USA, 3 Department of Biology, University of York, York Y010 5DD, UK, 4 Department of Chemistry, University of Saskatchewan, Saskatoon SK S7N 5C9, Canada, and 5 Institute of Biochemistry and Biotechnology, Martin Luther University, Halle-Wittenberg, 06120 Halle, Germany Received 20 February 2014; revised 3 April 2014; accepted 23 April 2014; published online 29 April 2014. *For correspondence (e-mail [email protected]). † Present address: Stevia First Corporation, Yuba City, CA 95993, USA. ‡ Present address: Department of Plant Physiology, Justus Liebig University, 35390 Giessen, Germany. § Present address: Center for Immunology and Infection, Department of Biology and Hull York Medical School (Area 12), University of York, York Y010 5DD, UK.
SUMMARY The study of glucosinolates and their regulation has provided a powerful framework for the exploration of fundamental questions about the function, evolution, and ecological significance of plant natural products, but uncertainties about their metabolism remain. Previous work has identified one thiohydroximate S-glucosyltransferase, UGT74B1, with an important role in the core pathway, but also made clear that this enzyme functions redundantly and cannot be the sole UDP-glucose dependent glucosyltransferase (UGT) in glucosinolate synthesis. Here, we present the results of a nearly comprehensive in vitro activity screen of recombinant Arabidopsis Family 1 UGTs, which implicate other members of the UGT74 clade as candidate glucosinolate biosynthetic enzymes. Systematic genetic analysis of this clade indicates that UGT74C1 plays a special role in the synthesis of aliphatic glucosinolates, a conclusion strongly supported by phylogenetic and gene expression analyses. Finally, the ability of UGT74C1 to complement phenotypes and chemotypes of the ugt74b1-2 knockout mutant and to express thiohydroximate UGT activity in planta provides conclusive evidence for UGT74C1 being an accessory enzyme in glucosinolate biosynthesis with a potential function during plant adaptation to environmental challenge. Keywords: Arabidopsis thaliana, secondary metabolism, thiohydroximate, Family 1 glycosyltransferase, UGT74 clade, UGT74B1.
INTRODUCTION Secondary metabolism is a dominant factor that mediates evolutionary and ecological relationships between heterotrophs and autotrophs, and serves as the ultimate source of numerous drugs used in medicine, facts that explain why study in this area has been and continues to be of central importance in biology. Glucosinolates are a class of sulfur-rich metabolites almost exclusive to the Brassicales (Agerbirk and Olsen, 2012). Because of their importance for human health (Dinkova-Kostova and Kostov, 2012), plant defense against herbivores and innate immunity (Lip€ fer and Boland, 2012), glucosinolates ka et al., 2010; Mitho have been the focus of major research efforts facilitated by 92
the powerful genetic resources and molecular tools available for the reference plant, Arabidopsis thaliana (Sønderby et al., 2010). Glucosinolate biosynthesis comprises up to three stages. First, the parental amino acid may undergo one or more rounds of chain elongation via a series of reactions analogous to those converting valine to leucine. The distribution of chain-length in the final products is controlled by genotype as well genotype 9 environment interactions (Burow et al., 2010). Next, the (homo)amino acids enter the core pathway common to all glucosinolates, which can be interpreted as an initial activation, wherein the (homo)amino © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd
UGT74C1 acts in glucosinolate synthesis 93 acid is converted to a highly reactive nitrile oxide. The subsequent series of detoxification steps generates the stable glucosinolates, which are N-sulfated S-glucosyl thiohydroximates (Figure 1). Finally, the structural diversity of these compounds can be further increased by side-chain modifications, including oxygenation, hydroxylation, dehydrogenation, benzoylation and methoxylation, among others (Grubb and Abel, 2006; Halkier and Gershenzon, 2006; Sønderby et al., 2010). In Arabidopsis, the major glucosinolates are aliphatic, derived from methionine (Met,) or indolic derived from tryptophan (Trp); again, the distribution of final products is under genetic and environmental control (Burow et al., 2010).
Glucosylation of the thiohydroxamic acid intermediate is the penultimate reaction of the core pathway. We previously showed that Arabidopsis UGT74B1, a UDP-glucosedependent glucosyltransferase (UGT) of plant Family 1 glycosyltransferases (GTs), is the major enzyme catalyzing this reaction in vivo (Grubb et al., 2004). Nevertheless, mutant plants lacking functional UGT74B1 still accumulate substantial levels of glucosinolates, indicating that at least one additional enzyme is capable of thiohydroximate glucosylation. Several reports of co-regulation of the closely related UGT74C1 with glucosinolate pathway-related genes, particularly with those involved in aliphatic glucosinolate synthesis, have predicted UGT74C1 as a candidate (Gachon et al., 2005; Hirai et al., 2007; Mikkelsen et al., 2010). However, there is as yet no direct evidence for its in vivo function. Arabidopsis thaliana contains 107 members of Family 1 UGTs, which share the highly conserved plant-specific UGT signature motif and glucosylate a large array of small molecules (Bowles et al., 2006; Osmani et al., 2009). We therefore set out to systematically search for and identify the additional UGT(s) that function(s) in glucosinolate synthesis. Our results from complementary biochemical and genetic approaches clearly demonstrate that UGT74C1 acts as an accessory UGT in the glucosinolate core pathway. RESULTS Screening recombinant Arabidopsis family 1 UGT enzymes We expressed almost all A. thaliana Family 1 UGTs as recombinant C-terminal GST-fusions in E. coli, which we affinity-purified (Lim et al., 1998, 2002) and tested for activity against phenylacetothiohydroximate (PATH) as described previously for UGT74B1 (Grubb et al., 2004). As expected, amongst the 93 recombinant enzymes assayed (Table S1), the UGT74B1–GST (glutathione S-transferase) protein displayed the highest activity toward PATH under the three pH conditions tested (pH 6, pH 7 and pH 8). Interestingly, all analysed members of clade UGT74 showed activity toward PATH (Figure 2), ranging from approximately 50% (UGT74F1) to 5% (UGT74E2) relative to UGT74B1, with the exception of UGT74E1, which was not expressed. Minor activities (2–8%) were recorded for recombinant UGT enzymes 88A1, 75B1, 71B6, 73B1, and 73B3. The remaining 81 UGT–GST proteins expressed only trace ( 30) and showed the highest affinity (Km < 20 lM) for both substrates, while the other four enzymes displayed low activities (kcat < 0.2) and affinities (Km > 100 lM). Nevertheless, a relatively inefficient enzyme could provide substantial activity if expressed at sufficiently high level. We therefore compared UGT74 gene expression in wild-type and ugt74b1 knockout plants to test if any clade member is overexpressed as a consequence of the metabolic block in ugt74b1. We did not observe significantly higher UGT74 mRNA levels, indicating that compensatory changes in UGT74 expression are not a likely explanation for the residual glucosinolate content of ugt74b1 plants (Figure S2a). Thus, while we could confirm that UGT74 members are particularly apt in thiohydroximate glucosylation relative to the other 86 UGTs tested, no particular candidate for a new glucosinolate biosynthetic enzyme emerged from this in vitro approach.
Figure 2. Relative activities of recombinant UGT74 enzymes. Ninety-three purified Arabidopsis Family 1 UGT–GST fusion proteins were assayed with UDP-glucose and phenylacetothiohydroximate (PATH) as the thiohydroximate aglycone (see Experimental procedures and Table S1). Activities of UGT74 clade members are given relative to UGT74B1–GST, the most active recombinant UGT enzyme toward PATH.
(Figure S1): 4-methylthiobutylthiohydroximate (MTBTH) and 1-methylindolyl-3-acetothiohydroximate (MIATH). The first compound is the natural precursor of the aliphatic 4methylsulfinylbutyl glucosinolate (S4), the most abundant glucosinolate in leaves of Col-0, while the latter is a quasinatural product and an analogue of the thiohydroximate precursor of all indolic glucosinolates, which can be incorporated into 1-methylindolyl-3-methyl glucosinolate (Pedras et al., 2009). Unfortunately, we could not further evaluate our lead candidate, UGT74C1, because bacterial preparations of the enzyme never consistently showed activity above background, despite extensive exploration of variations in culture conditions, purification protocols, metal co-factors, sugar-nucleotide donor substrates, and reaction buffer composition. The UGT74C1–GST construct of the initial screen expressed very low activity in subsequent experiments, and successful transient expression of (His)6–UGT74C1 in tobacco leaves (Nicotiana benthamiana) resulted in inactive preparations, while the corresponding UGT74B1 control was highly active in this system, results we cannot explain at present. The remaining five purified (His)6–UGT74 enzymes showed significant activity above background and establishment of pseudo-single substrate conditions allowed the
Mutational analysis of clade UGT74 implicates UGT74C1 in glucosinolate synthesis We next examined phenotypes caused by loss-of-function alleles of all UGT74 genes. Figure 3(a) illustrates the positions of insertional and point mutations. Analysis of mRNA levels in the wild-type and mutant lines by quantitative RTPCR confirmed that all ugt74 lines are likely functional knockouts (Figure S2b). Initial characterization of the single mutants revealed, with the exception of ugt74b1 lines, no obvious morphological differences from the wild-type. Slightly elevated accumulation of aliphatic glucosinolates was measured for ugt74e2-1, inconsistent with the idea that this gene plays a direct role in thiohydroximate metabolism (Figure 4a). Thus, we concluded that if any UGT74 member plays such a role, it may be masked by the activity of UGT74B1. We therefore generated double homozygous mutants of each line with either ugt74b1-1 (Ws-0) in the case of ugt74f1 or with ugt74b1-2 (Col-0) for the remaining ugt74 mutants. We previously characterized both ugt74b1 alleles, which cause dwarfism, partial sterility, and a low-glucosinolate chemotype (Grubb et al., 2004). Both wild-type accessions
Table 1 Kinetic parameters of recombinant UGT74 proteins toward MIATH and MTBTH MIATH
MTBTH
Protein
kcat (sec1)
UGT74B1 UGT74C1 UGT74D1 UGT74E2 UGT74F1 UGT74F2
36.5 n.d. 0.11 0.009 0.182 0.021
26.2
0.06 0.010 0.028 0.012
Km app (lM) 17.2 n.d. 158 232 235 130
7.1
90 136 39 161
kcat (sec1) 2.28 n.d. 0.0303 0.0164 0.0267 n.d.
0.79 0.0037 0.0019 0.0046
Km app (lM) 1.48 n.d. 462 159 110 >300
0.55 67 36 39
The mean of three independent determinations is given standard deviation (SD); n.d. – activity not detectable. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 79, 92–105
UGT74C1 acts in glucosinolate synthesis 95
(a)
(a)
(b) (b)
(c)
Figure 3. Loss-of-function alleles of UGT74 genes and their phenotypes in ugt74b1-null backgrounds. (a) Map of T-DNA insertion and mutation sites in UGT74 genes. Exons are depicted as white block arrows and non-translated regions as black lines. The grey box designates the position of the critical UGT signature motif. TDNA insertion sites are marked with a solid black triangle or, for the gene trap line GT1305 (ugt74c1-1), a white triangle. A previously characterized loss-of-function allele (ugt74f2-i1a) generated by ethyl methanesulfonate (EMS) mutagenesis (*) was used for UGT74F2 (Quiel and Bender, 2003). (b) Wild-type plants (Ws-0, Col-0) and indicated knockout lines after 4 weeks of growth on soil. (c) Phenotypes of 3-week-old ugt74b1 knockout lines, alone or in combination with knockouts of other UGT74 members. The two mutants in the leftmost column are in accession Ws-0, all others are in Col-0.
and their corresponding single ugt74b1 mutants are shown in Figure 3(b). All double mutants closely resemble ugt74b1, with one dramatic exception: the ugt74b1-2
Figure 4. Glucosinolate profiles of ugt74 loss-of function lines. (a) Aliphatic and indolic glucosinolate content of the seven single ugt74 mutants relative to the respective wild-type (1 = 100%). Significant differences relative to wild-type (P < 0.05, n = 5) are marked (*). (b) Aliphatic and indolic glucosinolates of ugt74b1 double mutants relative to the respective ugt74b1 background (1 = 100%): ugt74b1-1 for the ugt74b1-1 ugt74f1 double mutant, ugt74b1-2 for all other combinations. Significant differences (P < 0.05, n = 5) are marked (*).
ugt74c1-2 double mutant shows a strongly additive phenotype, producing extremely small plants with only a few small leaves (Figure 3b,c). While these plants persist for a few weeks on soil and eventually produce floral structures, they are completely sterile. The glucosinolate chemotype of ugt74b1-2 ugt74c1-2 plants also stands in stark contrast to those of the other double mutants (Figure 4b). While indolic glucosinolates are not affected relative to ugt74b1-2, the content of aliphatic glucosinolates is reduced an additional 70%, again implicating UGT74C1 specifically in the synthesis of Met-derived glucosinolates. Among the other double mutants, ugt74b1-2 ugt74d1 shows a approximately 2-fold increase in indolic glucosinolates. Although interesting, this result is inconsistent with direct a role of UGT74D1 in thiohydroximate metabolism and was not pursued further. Loss of UGT74C1 in ugt74b1 plants causes seedling lethality We maintained the ugt74b1-2 (+/) ugt74c1-2 (/) line, which is fertile and indistinguishable from the wild-type,
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 79, 92–105
96 C. D. Grubb et al. Figure 5. Phenotypic details of single and double ugt74c1-2 and ugt74b1-2 mutants in comparison with wild-type (Col-0) and sur1 plants grown in axenic conditions. (a, b) Growth on horizontal and vertical agar plates (2 weeks). (c) Germination for 2.5 days in the dark. (d) Plant development for 2 months. The inset shows a futile attempt at flowering by a ugt74b1-2 ugt74c1-2 plant.
(a)
(b) studied plant growth under axenic conditions. We included the sur1 line for comparison because loss of the SUR1 C-S lyase catalyzing the reaction prior to the glucosylation step causes failure of glucosinolate synthesis, overproduction of auxin, and seedling lethality (Boerjan et al., 1995; Mikkelsen et al., 2004). When grown on agar, ugt74b1-2 ugt74c1-2 plants are significantly smaller than ugt74b1-2 and similar in size to sur1 seedlings (Figure 5). The double mutant is extremely stunted and produces very small, curly and chlorotic leaves. Unlike sur1 seedlings, it occasionally develops a short inflorescence with petite sterile flowers. The double mutant maintains a plant-like habit with recognizable organs after an extended period of growth, while sur1 seedlings completely degenerate into callus tissue (Figure 5d). Loss of UGT74C1 in ugt74b1 plants does not mimic the high-auxin phenotype of sur1
(c)
(d)
as a source of double homozygous mutant seedlings. We reasoned that their poor growth on soil might be caused by interactions with hostile organisms and therefore
We reported previously that loss of UGT74B1 causes morphological alterations that are consistent with auxin overproduction, which we confirmed by indole-3-acetic acid (IAA) measurements (Grubb et al., 2004). Interestingly, although ugt74b1-2 ugt74c1-2 seedlings are significantly smaller than ugt74b1-2 and more similar to sur1 plants during early stages of development, they do not share the extreme high-auxin phenotype of sur1. The root system of the double mutant, albeit significantly underdeveloped relative to wild-type and ugt74c1-2 seedlings, is comparable with the root system of ugt74b1-2 plants, but clearly differs from that of the sur1 mutant (Figure 5b). In contrast with sur1 plants, ugt74b1-2 ugt74c1-2 seedlings produce excessive roots and callus-like protrusions at the hypocotyl-root junction only after extended growth on agar (>1 month). When grown under conditions to promote adventitious rooting (Grubb et al., 2004), the double mutant is again more similar to ugt74b1-2 than to sur1 seedlings (Figure S3). When germinated in the dark, ugt74b1-2 seedlings display a moderately de-etiolated phenotype, which is not exaggerated in the ugt74b1-2 ugt74c1-2 double mutant (Figure 5c). Although these data suggest that loss of UGT74C1 activity does not further elevate auxin production in the ugt74b1-2 background, we measured significantly elevated auxin levels relative to wild-type and ugt74b1-2. However, precursors of jasmonic acid and ethylene are also highly increased, suggesting general perturbation of hormone homeostasis in the extremely dwarfed double mutant (Table S2).
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 79, 92–105
UGT74C1 acts in glucosinolate synthesis 97 Loss of UGT74C1 in ugt74b1 plants affects primarily aliphatic glucosinolates We determined glucosinolate composition in shoots of 3week-old wild-type, ugt74c1-2, ugt74b1-2, ugt74b1-2 ugt74c1-2, and sur1 plants (Figure 6). Similar to the ugt74b1-1 allele (Grubb et al., 2004), the ugt74b1-2 mutation decreases total glucosinolates by about 30% due to lower content of both aliphatic and indolic derivatives.
(a)
(b)
(c)
Figure 6. Details of the glucosinolate chemotypes of single and double ugt74c1 and ugt74b1 mutants in comparison with wild-type and sur1 plants (3-week-old). (a) Content of total, aliphatic and indolic glucosinolates in the shoot. (b) Content of the major Met-derived glucosinolates: 3-methylsulfinylpropyl glucosinolate (S3), 4-methylsulfinylbutyl glucosinolate (S4), and 8-methylsulfinyloctyl glucosinolate (S8). (c) Content of major Trp-derived glucosinolates: indolyl-3-methyl glucosinolate (IM), 4-methoxyindolyl-3-methyl glucosinolate (4IM), and 1-methoxyindolyl-3-methyl glucosinolate (1IM). Mean values [standard error (SE)] are given (n = 10). An asterisk indicates a statistically significant difference (P < 0.05) with respect to the wild-type (for ugt74c1-2) or to ugt74b1-2 (for the ugt74b1-2 ugt74c1-2 double mutant). Similar results were obtained for two additional experiments with 2- or 3-week-old plants.
Interestingly, while the glucosinolate chemotype of the ugt74c1-2 single mutant is barely distinguishable from the wild-type, levels of all major Met-derived glucosinolates are dramatically reduced in the ugt74b1-2 ugt74c1-2 double mutant. The reduction of total aliphatic glucosinolates by about 80% in ugt74b1-2 ugt74c1-2 plants clearly exceeds the effect of the ugt74b1-2 mutation (30%). As in the single ugt74c1-2 mutant, loss of UGT74C1 in the ugt74b1-2 background differentially affects aliphatic glucosinolates depending on the length of the side chain (Figure 6b). While the content of 3-methylsulfinylpropyl glucosinolate (S3) is reduced by about half, 8-methylsulfinyloctyl glucosinolate (S8) is nearly abolished, with the S4 compound exhibiting an intermediate reduction. Thus, UGT74C1 seems to prefer aliphatic substrates with longer side-chains in vivo. In contrast, content of Trp-derived glucosinolates is quite similar between the ugt74b1-2 and ugt74b1-2 ugt74c1-2 lines (Figure 6c). While the level of indolyl-3methyl glucosinolate (IM) is lower by about 50% in both lines relative to wild-type, content of 4-methoxyindolyl-3methyl glucosinolate (4IM) is largely unaffected and maintained at wild-type level. The content of 1-methoxyindolyl3-methyl glucosinolate (1IM) is reduced significantly in the double mutant relative to ugt74b1-2 plants. However, the absolute magnitude of this change is miniscule compared with the change in aliphatic glucosinolates. Because the genetic evidence implicates UGT74C1 in glucosinolate synthesis, we searched in more detail for a chemotype in the ugt74c1-2 single mutant. To this end, we examined the glucosinolate profiles of cauline leaves, flowers, fully expanded green siliques and seeds; none of these profiles revealed any significant differences compared with the wild-type (Figure S4). Therefore, the primary role of UGT74C1 in glucosinolate synthesis seems to be unrelated to the developmental stage of adult plants. In summary, the genetic analysis points to a role of UGT74C1 in glucosinolate biosynthesis that is limited to its aliphatic branch. This conclusion is consistent with the phenotypic data that do not indicate enhancement of the high-auxin phenotype of ugt74b1-2 plants by loss of UGT74C1, as would be expected if UGT74C1 also glucosylates indolic thiohydroximates. Consistent with previous reports (Mikkelsen et al., 2004), glucosinolate production in sur1 plants is reduced severely (
