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Pc104745-109975 360.370

D.J. (2007a). Glutathione conjugates in the vacuole Halkier, B.A., and Glawischnig, E. (2006).
involvement in camalexin biosynthesis through are degraded by g-glutamyl transpeptidase GGT3 CYP71B15 (PAD3) catalyzes the final step in conjugation of indole-3-carboxylic acid and in Arabidopsis. Plant J. 49: 878–888.
camalexin biosynthesis. Plant Physiol. 141: cysteine and upregulation of camalexin bio- Parisy, V., Poinssot, B., Owsianowski, L., Buchala, synthesis genes. J. Integr. Plant Biol. 54: A., Glazebrook, J., and Mauch, F. (2007).
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synthetase highlights the importance of glutathione (2012). The rise of chemodiversity in plants.
in disease resistance of Arabidopsis. Plant J. 49: Su, T.B., Xu, J.A., Li, Y.A., Lei, L., Zhao, L., Yang, H.L., Feng, J.D., Liu, G.Q., and Ren, Winter, D., Vinegar, B., Nahal, H., Ammar, Saga, H., Ogawa, T., Kai, K., Suzuki, H., D.T. (2011). Glutathione-indole-3-acetonitrile is R., Wilson, G.V., and Provart, N.J. (2007).
required for camalexin biosynthesis in Arabi- An “Electronic Fluorescent Pictograph” Ohta, D. (2012). Identification and character- dopsis thaliana. Plant Cell 23: 364–380.
browser for exploring and analyzing large- ization of ANAC042, a transcription factor Wada, K., Hiratake, J., Irie, M., Okada, T., family gene involved in the regulation of camalexin biosynthesis in Arabidopsis. Mol.
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Escherichia coli g-glutamyltranspeptidase in (1999). Arabidopsis PAD3, a gene required for Saito, K., Hirai, M.Y., and Yonekura-Sakakibara, complex with azaserine and acivicin: novel camalexin biosynthesis, encodes a putative K. (2008). Decoding genes with coexpression mechanistic implication for inhibition by gluta- cytochrome P450 monooxygenase. Plant Cell networks and metabolomics - ‘Majority report by mine antagonists. J. Mol. Biol. 380: 361–372.
precogs’. Trends Plant Sci. 13: 36–43.
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biosynthesis: Metabolism and trafficking. Annu.
Arabidopsis acetyl-amido synthetase GH3.5 Reply: Complexity in Camalexin Biosynthesis accumulation of multiple glutathione S- camalexin pathway in Arabidopsis.
subunits of GGTs from bacteria and animal.
cides with camalexin production;GSTF6 overexpression increased and GSTF6 et al., 2011) may reflect inhibition of GGPs only 4.8 to 9.8% sequence identities with Arabidopsis thaliana GSTF6 expressed in acivicin has been widely used as a specific yeast cells catalyzed GSH(IAN) formation; inhibitor of GGT activity for in vitro and in conserved residues involving in the inter- action of GGTs with acivicin are present in Arabidopsis GGTs, but not GGPs. There- biosynthetic pathway; and, finally, g-glu- binding of acivicin with Arabidopsis GGTs acid residues in substrate binding pocket.
inhibits GGT activity. However, experimen- data and the expression patterns of these GGTs from bacteria and animal cells (Wada hypothesis of Møldrup et al. (2013) that genes, we suggest that (1) GSH is the Cys et al., 2008). For example, Escherichia coli GGT binds acivicin through the formation of inhibits GGP’s activities. For example, we thesis, (2) the conjugation of GSH with IAN a covalent bond between the Og atom of its binding with the GGPs, the binding affinity, dihydroisoxazole ring of acivicin and also and/or the crystal structure of GGP-acivicin camalexin biosynthesis. The Letter to the alternative explanation for reduced cama- Flores et al., 2011) argue that g-glutamyl lexin production following acivicin treat- 2008). To show if Arabidopsis GGTs and ment, suggesting that “termination of all GGT activity could arrest glutathione and www.plantcell.org/cgi/doi/10.1105/tpc.113.109975 glutathione conjugates in the vacuole and Figure 1. Multiple Sequence Alignment of Arabidopsis GGT1, GGT2, GGP1, GGP3, and GGTs from Other Organisms.
The coding sequences of all genes were aligned by ClustalW (MegAlign program DNAStar). Sequences corresponding to the S-subunits of GGTs frombacteria and animals are presented. Identical residues in all GGTs are boxed. The residues that interact with acivicin in E. coli GGT are indicated by anasterisk above the alignment.
extracellular space” (Møldrup et al., 2013).
in the camalexin secretion. Most recently, porters in Arabidopsis, are reported to be Møldrup et al. (2013) also questioned the uole and cytosol for GSTF6 (Carter et al., our experiments (Su et al., 2011). They note lexin biosynthetic pathway to be cytosolic based on the cytosolic localization of some after inhibition of GGT activity or elimination known enzymes in the pathway. Their point 2011; Møldrup et al., 2013) will be difficult of GGT gene expression is possibly due to of view is that enzymes in a biosynthetic pathway usually are colocalized temporally secretion. In fact, the liquid media system and spatially (Møldrup et al., 2013). Be- camalexin biosynthetic pathway. It is a gen- erally accepted viewpoint that enzymes in a biosynthetic pathway might be located in et al., 2008; Su et al., 2011) and by other groups (Bednarek et al., 2005; Ren et al., ing to this viewpoint, the different localiza- as described by Xu et al. (2008). The camalexin inliquid media and GSH(IAN) in seedlings weremeasured as described (Su et al., 2011).
Sequence data from this article can be found in theArabidopsis Genome Initiative or GenBank/EMBLdatabases under the following accession numbers:MKK9, At1g73500; GGT1, At4g39640; GGT2,At4g39650; Escherichia coli GGT, NP_417904;Helicobacter pylori GGT, NP_207909; humanGGT, NM_005265; pig GGT, NM_214030; ratGGT, GGP3, At4g30550. T-DNA insertion lines usedhere are as follows: ggt1-1 (SAIL_1178_C01) andggt2-1 (SAIL_6_G02).
Figure 2. Camalexin and GSH(IAN) production in MKK9DD, Columbia Wild-Type, ggt1-1, and ggt2-1Seedlings.
Twelve-day-old seedlings were either treated with dexamethasone (DEX) or inoculated with Botrytiscinerea spores. Twelve hours after dexamethasone treatment or 16 h after inoculation with B. cinerea,the seedlings were used for GSH(IAN) extraction and the media were used for camalexin measurement.
This work was supported by grants from the Right axis, peak areas for GSH(IAN) (mass-to-charge ratio [M1H]1 ¼ 462.17); left axis, camalexin National Natural Science Foundation of China content. Data represent the means 6 SD of three biological replicates. Asterisks indicate statistically (No. 31030010 and 31125006 to D.R., No.
significant differences between wild-type (WT) and GGT mutant seedlings. **P , 0.01 (paired sample t 31000127 to Y.L., and No. 30771124 to H.Y.).
likelihood that a given enzyme is involved in a particular pathway in planta,” as noted by T.S., Y.L., and H.Y. performed the experiments.
T.S., Y.L., H.Y., and D.R. wrote the article.
colocalization cannot be taken as evidence that such enzymes do not work in the same camalexin than the wild type after patho- Another observation that led Møldrup et al.
(2013) to argue that GGPs instead of GGTs are involving in camalexin biosynthesis is that (IAN) in GGT mutants in our previous work ported that GH3.5, a multifunctional acetyl- (Su et al., 2011). However, we have since amido synthetase, is involved in camalexin measured the contents of GSH(IAN) in MKK9 biosynthesis through catalyzing the conju- hyperactive mutants (as MKK9DD), Columbia gation of indole-3-carboxylic acid with Cys.
camalexin biosynthesis. We found that GSH biosynthetic pathway. Current data there- (IAN) accumulated in the wild type and in Badri, D.V., Chaparro, J.M., Manter, D.K., MKK9DD, ggt1, and ggt2 mutants undergoing Martinoia, E., and Vivanco, J.M. (2012).
camalexin biosynthesis. Figure 2 shows that GSH(IAN) accumulation is positively corre- chosen by Arabidopsis plants likely de- porters in root exudation of phytoalexins, lated with camalexin production. The result signals, and in disease resistance. Front. Plant substrate [e.g., GSH(IAN)] in order to produce Oldham, N.J., and Hahlbrock, K. (2005).
As shown in Figure 2 of Møldrup et al.
Structural complexity, differential response toinfection, and tissue specificity of indolic and (2013), both the ggt1 and ggt2 mutants Arabidopsis roots. Plant Physiol. 138: 1058– wild type after AgNO3 treatment. However, Arabidopsis thaliana wild type (ecotype Columbia- Carter, C., Pan, S., Zouhar, J., Avila, E.L., biological repeats in their experiments, the 0), ggt1-1 and ggt2-1 mutants, and MKK9DD authors suggest that there are no signifi- transgenic seedlings were all grown and treated vegetative vacuole proteome of Arabidopsis thaliana reveals predicted and unexpected B.A. (2013). Assigning gene function in bio- Su, T., Xu, J., Li, Y., Lei, L., Zhao, L., Yang, H., proteins. Plant Cell 16: 3285–3303.
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Reply: Complexity in Camalexin Biosynthesis
Tongbing Su, Yuan Li, Hailian Yang and Dongtao Ren ; originally published online February 28, 2013; This information is current as of December 13, 2013 References
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