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. 1998 Jul;10(7):1135–1149. doi: 10.1105/tpc.10.7.1135

Functional complementation of anthocyanin sequestration in the vacuole by widely divergent glutathione S-transferases.

M R Alfenito 1, E Souer 1, C D Goodman 1, R Buell 1, J Mol 1, R Koes 1, V Walbot 1
PMCID: PMC144053  PMID: 9668133

Abstract

Glutathione S-transferases (GSTs) traditionally have been studied in plants and other organisms for their ability to detoxify chemically diverse herbicides and other toxic organic compounds. Anthocyanins are among the few endogenous substrates of plant GSTs that have been identified. The Bronze2 (Bz2) gene encodes a type III GST and performs the last genetically defined step of the maize anthocyanin pigment pathway. This step is the conjugation of glutathione to cyanidin 3-glucoside (C3G). Glutathionated C3G is transported to the vacuole via a tonoplast Mg-ATP-requiring glutathione pump (GS-X pump). Genetically, the comparable step in the petunia anthocyanin pathway is controlled by the Anthocyanin9 (An9) gene. An9 was cloned by transposon tagging and found to encode a type I plant GST. Bz2 and An9 have evolved independently from distinct types of GSTs, but each is regulated by the conserved transcriptional activators of the anthocyanin pathway. Here, a phylogenetic analysis is presented, with special consideration given to the origin of these genes and their relaxed substrate requirements. In particle bombardment tests, An9 and Bz2 functionally complement both mutants. Among several other GSTs tested, only soybean GmGST26A (previously called GmHsp26A and GH2/4) and maize GSTIII were found to confer vacuolar sequestration of anthocyanin. Previously, these genes had not been associated with the anthocyanin pathway. Requirements for An9 and Bz2 gene function were investigated by sequencing functional and nonfunctional germinal revertants of an9-T3529, bz2::Ds, and bz2::Mu1.

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Selected References

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  1. Bartling D., Radzio R., Steiner U., Weiler E. W. A glutathione S-transferase with glutathione-peroxidase activity from Arabidopsis thaliana. Molecular cloning and functional characterization. Eur J Biochem. 1993 Sep 1;216(2):579–586. doi: 10.1111/j.1432-1033.1993.tb18177.x. [DOI] [PubMed] [Google Scholar]
  2. Christensen A. H., Quail P. H. Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res. 1996 May;5(3):213–218. doi: 10.1007/BF01969712. [DOI] [PubMed] [Google Scholar]
  3. Czarnecka E., Nagao R. T., Key J. L., Gurley W. B. Characterization of Gmhsp26-A, a stress gene encoding a divergent heat shock protein of soybean: heavy-metal-induced inhibition of intron processing. Mol Cell Biol. 1988 Mar;8(3):1113–1122. doi: 10.1128/mcb.8.3.1113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dooner H. K., Robbins T. P., Jorgensen R. A. Genetic and developmental control of anthocyanin biosynthesis. Annu Rev Genet. 1991;25:173–199. doi: 10.1146/annurev.ge.25.120191.001133. [DOI] [PubMed] [Google Scholar]
  5. Droog F. N., Hooykaas P. J., Libbenga K. R., van der Zaal E. J. Proteins encoded by an auxin-regulated gene family of tobacco share limited but significant homology with glutathione S-transferases and one member indeed shows in vitro GST activity. Plant Mol Biol. 1993 Mar;21(6):965–972. doi: 10.1007/BF00023595. [DOI] [PubMed] [Google Scholar]
  6. Droog FNJ., Hooykaas PJJ., Van Der Zaal B. J. 2,4-Dichlorophenoxyacetic Acid and Related Chlorinated Compounds Inhibit Two Auxin-Regulated Type-III Tobacco Glutathione S-Transferases. Plant Physiol. 1995 Apr;107(4):1139–1146. doi: 10.1104/pp.107.4.1139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dudler R., Hertig C., Rebmann G., Bull J., Mauch F. A pathogen-induced wheat gene encodes a protein homologous to glutathione-S-transferases. Mol Plant Microbe Interact. 1991 Jan-Feb;4(1):14–18. doi: 10.1094/mpmi-4-014. [DOI] [PubMed] [Google Scholar]
  8. Gaillard C., Dufaud A., Tommasini R., Kreuz K., Amrhein N., Martinoia E. A herbicide antidote (safener) induces the activity of both the herbicide detoxifying enzyme and of a vacuolar transporter for the detoxified herbicide. FEBS Lett. 1994 Sep 26;352(2):219–221. doi: 10.1016/0014-5793(94)00961-9. [DOI] [PubMed] [Google Scholar]
  9. Gerats A. G., Huits H., Vrijlandt E., Maraña C., Souer E., Beld M. Molecular characterization of a nonautonomous transposable element (dTph1) of petunia. Plant Cell. 1990 Nov;2(11):1121–1128. doi: 10.1105/tpc.2.11.1121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Grove G., Zarlengo R. P., Timmerman K. P., Li N. Q., Tam M. F., Tu C. P. Characterization and heterospecific expression of cDNA clones of genes in the maize GSH S-transferase multigene family. Nucleic Acids Res. 1988 Jan 25;16(2):425–438. doi: 10.1093/nar/16.2.425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hagen G., Uhrhammer N., Guilfoyle T. J. Regulation of expression of an auxin-induced soybean sequence by cadmium. J Biol Chem. 1988 May 5;263(13):6442–6446. [PubMed] [Google Scholar]
  12. Holt D. C., Lay V. J., Clarke E. D., Dinsmore A., Jepson I., Bright S. W., Greenland A. J. Characterization of the safener-induced glutathione S-transferase isoform II from maize. Planta. 1995;196(2):295–302. doi: 10.1007/BF00201388. [DOI] [PubMed] [Google Scholar]
  13. Holton T. A., Cornish E. C. Genetics and Biochemistry of Anthocyanin Biosynthesis. Plant Cell. 1995 Jul;7(7):1071–1083. doi: 10.1105/tpc.7.7.1071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Itzhaki H., Woodson W. R. Characterization of an ethylene-responsive glutathione S-transferase gene cluster in carnation. Plant Mol Biol. 1993 Apr;22(1):43–58. doi: 10.1007/BF00038994. [DOI] [PubMed] [Google Scholar]
  15. Jepson I., Lay V. J., Holt D. C., Bright S. W., Greenland A. J. Cloning and characterization of maize herbicide safener-induced cDNAs encoding subunits of glutathione S-transferase isoforms I, II and IV. Plant Mol Biol. 1994 Dec;26(6):1855–1866. doi: 10.1007/BF00019498. [DOI] [PubMed] [Google Scholar]
  16. Kiyosue T., Yamaguchi-Shinozaki K., Shinozaki K. Characterization of two cDNAs (ERD11 and ERD13) for dehydration-inducible genes that encode putative glutathione S-transferases in Arabidopsis thaliana L. FEBS Lett. 1993 Dec 6;335(2):189–192. doi: 10.1016/0014-5793(93)80727-c. [DOI] [PubMed] [Google Scholar]
  17. Kroon J., Souer E., de Graaff A., Xue Y., Mol J., Koes R. Cloning and structural analysis of the anthocyanin pigmentation locus Rt of Petunia hybrida: characterization of insertion sequences in two mutant alleles. Plant J. 1994 Jan;5(1):69–80. doi: 10.1046/j.1365-313x.1994.5010069.x. [DOI] [PubMed] [Google Scholar]
  18. Kutchan T. M., Hochberger A. Nucleotide Sequence of a cDNA Encoding a Constitutively Expressed Glutathione S-Transferase from Cell Suspension Cultures of Silene cucubalus. Plant Physiol. 1992 Jun;99(2):789–790. doi: 10.1104/pp.99.2.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lee H. C., Toung Y. P., Tu Y. S., Tu C. P. A molecular genetic approach for the identification of essential residues in human glutathione S-transferase function in Escherichia coli. J Biol Chem. 1995 Jan 6;270(1):99–109. doi: 10.1074/jbc.270.1.99. [DOI] [PubMed] [Google Scholar]
  20. Levy A. A., Walbot V. Regulation of the timing of transposable element excision during maize development. Science. 1990 Jun 22;248(4962):1534–1537. doi: 10.1126/science.2163107. [DOI] [PubMed] [Google Scholar]
  21. Li Z. S., Alfenito M., Rea P. A., Walbot V., Dixon R. A. Vacuolar uptake of the phytoalexin medicarpin by the glutathione conjugate pump. Phytochemistry. 1997 Jun;45(4):689–693. doi: 10.1016/s0031-9422(97)00031-9. [DOI] [PubMed] [Google Scholar]
  22. Li Z. S., Zhen R. G., Rea P. A. 1-Chloro-2,4-Dinitrobenzene-Elicited Increase in Vacuolar Glutathione-S-Conjugate Transport Activity. Plant Physiol. 1995 Sep;109(1):177–185. doi: 10.1104/pp.109.1.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lu Y. P., Li Z. S., Rea P. A. AtMRP1 gene of Arabidopsis encodes a glutathione S-conjugate pump: isolation and functional definition of a plant ATP-binding cassette transporter gene. Proc Natl Acad Sci U S A. 1997 Jul 22;94(15):8243–8248. doi: 10.1073/pnas.94.15.8243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ludwig S. R., Bowen B., Beach L., Wessler S. R. A regulatory gene as a novel visible marker for maize transformation. Science. 1990 Jan 26;247(4941):449–450. doi: 10.1126/science.247.4941.449. [DOI] [PubMed] [Google Scholar]
  25. Luehrsen K. R., de Wet J. R., Walbot V. Transient expression analysis in plants using firefly luciferase reporter gene. Methods Enzymol. 1992;216:397–414. doi: 10.1016/0076-6879(92)16037-k. [DOI] [PubMed] [Google Scholar]
  26. Marrs K. A., Alfenito M. R., Lloyd A. M., Walbot V. A glutathione S-transferase involved in vacuolar transfer encoded by the maize gene Bronze-2. Nature. 1995 Jun 1;375(6530):397–400. doi: 10.1038/375397a0. [DOI] [PubMed] [Google Scholar]
  27. Marrs K. A., Walbot V. Expression and RNA splicing of the maize glutathione S-transferase Bronze2 gene is regulated by cadmium and other stresses. Plant Physiol. 1997 Jan;113(1):93–102. doi: 10.1104/pp.113.1.93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Marrs Kathleen A. THE FUNCTIONS AND REGULATION OF GLUTATHIONE S-TRANSFERASES IN PLANTS. Annu Rev Plant Physiol Plant Mol Biol. 1996 Jun;47(NaN):127–158. doi: 10.1146/annurev.arplant.47.1.127. [DOI] [PubMed] [Google Scholar]
  29. McCLINTOCK B. Chromosome organization and genic expression. Cold Spring Harb Symp Quant Biol. 1951;16:13–47. doi: 10.1101/sqb.1951.016.01.004. [DOI] [PubMed] [Google Scholar]
  30. McCormick S. Pigment synthesis in maize aleurone from precursors fed to anthocyanin mutants. Biochem Genet. 1978 Aug;16(7-8):777–785. doi: 10.1007/BF00484735. [DOI] [PubMed] [Google Scholar]
  31. Meyer R. C., Jr, Goldsbrough P. B., Woodson W. R. An ethylene-responsive flower senescence-related gene from carnation encodes a protein homologous to glutathione S-transferases. Plant Mol Biol. 1991 Aug;17(2):277–281. doi: 10.1007/BF00039505. [DOI] [PubMed] [Google Scholar]
  32. Müller M., Meijer C., Zaman G. J., Borst P., Scheper R. J., Mulder N. H., de Vries E. G., Jansen P. L. Overexpression of the gene encoding the multidrug resistance-associated protein results in increased ATP-dependent glutathione S-conjugate transport. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):13033–13037. doi: 10.1073/pnas.91.26.13033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nash J., Walbot V. Bronze-2 Gene Expression and Intron Splicing Patterns in Cells and Tissues of Zea mays L. Plant Physiol. 1992 Sep;100(1):464–471. doi: 10.1104/pp.100.1.464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Paulusma C. C., Bosma P. J., Zaman G. J., Bakker C. T., Otter M., Scheffer G. L., Scheper R. J., Borst P., Oude Elferink R. P. Congenital jaundice in rats with a mutation in a multidrug resistance-associated protein gene. Science. 1996 Feb 23;271(5252):1126–1128. doi: 10.1126/science.271.5252.1126. [DOI] [PubMed] [Google Scholar]
  35. Pemble S. E., Taylor J. B. An evolutionary perspective on glutathione transferases inferred from class-theta glutathione transferase cDNA sequences. Biochem J. 1992 Nov 1;287(Pt 3):957–963. doi: 10.1042/bj2870957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Quattrocchio F., Wing J. F., Leppen HTC., Mol JNM., Koes R. E. Regulatory Genes Controlling Anthocyanin Pigmentation Are Functionally Conserved among Plant Species and Have Distinct Sets of Target Genes. Plant Cell. 1993 Nov;5(11):1497–1512. doi: 10.1105/tpc.5.11.1497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Quattrocchio F., Wing J. F., van der Woude K., Mol J. N., Koes R. Analysis of bHLH and MYB domain proteins: species-specific regulatory differences are caused by divergent evolution of target anthocyanin genes. Plant J. 1998 Feb;13(4):475–488. doi: 10.1046/j.1365-313x.1998.00046.x. [DOI] [PubMed] [Google Scholar]
  38. REDDY G. M., COE E. H., Jr Inter-tissue complemention: a simple technique for direct analysis of gene-action sequence. Science. 1962 Oct 12;138(3537):149–150. doi: 10.1126/science.138.3537.149. [DOI] [PubMed] [Google Scholar]
  39. Reinemer P., Prade L., Hof P., Neuefeind T., Huber R., Zettl R., Palme K., Schell J., Koelln I., Bartunik H. D. Three-dimensional structure of glutathione S-transferase from Arabidopsis thaliana at 2.2 A resolution: structural characterization of herbicide-conjugating plant glutathione S-transferases and a novel active site architecture. J Mol Biol. 1996 Jan 19;255(2):289–309. doi: 10.1006/jmbi.1996.0024. [DOI] [PubMed] [Google Scholar]
  40. Renckens S., De Greve H., Beltrán-Herrera J., Toong L. T., Deboeck F., De Rycke R., Van Montagu M., Hernalsteens J. P. Insertion mutagenesis and study of transposable elements using a new unstable virescent seedling allele for isolation of haploid petunia lines. Plant J. 1996 Sep;10(3):533–544. doi: 10.1046/j.1365-313x.1996.10030533.x. [DOI] [PubMed] [Google Scholar]
  41. Souer E., van Houwelingen A., Kloos D., Mol J., Koes R. The no apical meristem gene of Petunia is required for pattern formation in embryos and flowers and is expressed at meristem and primordia boundaries. Cell. 1996 Apr 19;85(2):159–170. doi: 10.1016/s0092-8674(00)81093-4. [DOI] [PubMed] [Google Scholar]
  42. Takahashi Y., Hasezawa S., Kusaba M., Nagata T. Expression of the auxin-regulated parA gene in transgenic tobacco and nuclear localization of its gene products. Planta. 1995;196(1):111–117. doi: 10.1007/BF00193224. [DOI] [PubMed] [Google Scholar]
  43. Takahashi Y., Kuroda H., Tanaka T., Machida Y., Takebe I., Nagata T. Isolation of an auxin-regulated gene cDNA expressed during the transition from G0 to S phase in tobacco mesophyll protoplasts. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9279–9283. doi: 10.1073/pnas.86.23.9279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Takahashi Y., Nagata T. parB: an auxin-regulated gene encoding glutathione S-transferase. Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):56–59. doi: 10.1073/pnas.89.1.56. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Taylor J. L., Fritzemeier K. H., Häuser I., Kombrink E., Rohwer F., Schröder M., Strittmatter G., Hahlbrock K. Structural analysis and activation by fungal infection of a gene encoding a pathogenesis-related protein in potato. Mol Plant Microbe Interact. 1990 Mar-Apr;3(2):72–77. [PubMed] [Google Scholar]
  46. Wan Y., Lemaux P. G. Generation of Large Numbers of Independently Transformed Fertile Barley Plants. Plant Physiol. 1994 Jan;104(1):37–48. doi: 10.1104/pp.104.1.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zettl R., Schell J., Palme K. Photoaffinity labeling of Arabidopsis thaliana plasma membrane vesicles by 5-azido-[7-3H]indole-3-acetic acid: identification of a glutathione S-transferase. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):689–693. doi: 10.1073/pnas.91.2.689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zhou J., Goldsbrough P. B. An Arabidopsis gene with homology to glutathione S-transferases is regulated by ethylene. Plant Mol Biol. 1993 Jun;22(3):517–523. doi: 10.1007/BF00015980. [DOI] [PubMed] [Google Scholar]
  49. van Houwelingen A., Souer E., Spelt K., Kloos D., Mol J., Koes R. Analysis of flower pigmentation mutants generated by random transposon mutagenesis in Petunia hybrida. Plant J. 1998 Jan;13(1):39–50. doi: 10.1046/j.1365-313x.1998.00005.x. [DOI] [PubMed] [Google Scholar]
  50. van Tunen A. J., Koes R. E., Spelt C. E., van der Krol A. R., Stuitje A. R., Mol J. N. Cloning of the two chalcone flavanone isomerase genes from Petunia hybrida: coordinate, light-regulated and differential expression of flavonoid genes. EMBO J. 1988 May;7(5):1257–1263. doi: 10.1002/j.1460-2075.1988.tb02939.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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