The tu8 Mutation of Arabidopsis thaliana Encoding a Heterochromatin Protein 1 Homolog Causes Defects in the Induction of Secondary Metabolite Biosynthesis

Abstract

The tu8 mutant of Arabidopsis thaliana (L.) Heynh. was previously described as deficient in pathogen-induced auxin and glucosinolate (GSL) accumulation, as well as in heat-induced accumulation of cytosolic Hsp90, the latter feature was accom„panied by reduced thermotolerance at higher temperatures. The mutated gene was shown to be a novel allele ofTERMINAL FLOWER2, encoding the only Arabidopsis homolog for heterochromatin protein 1 ([Kim et al., 2004]). In this report, we investigated the influence of heat stress on auxin and GSL content, as well as the accumulation of several secondary metabolites derived from the phenylpropanoid pathway, including anthocyanins and sinapine derivatives, in the mutant tu8. tu8 had less sinapine and sinapoyl esters compared to the wild type. In addition, the induction of sinapine by heat shock in Columbia was not found in tu8. Anthocyanins were also induced by heat stress in wild type plants, whereas tu8 showed only slight induction of these compounds and only at higher temperatures. GSLs were induced at higher temperatures in the wild type, but induction was absent in tu8. Transcript levels known to be involved in IAA/glucosinolate synthesis and metabolism (nitrilase and myrosinase) were examined and both showed developmental regulation, while only nitrilase mRNA levels differed between wild type and mutant seedlings. Treatment of Columbia and tu8 with jasmonic acid (JA), a known inducer of glucosinolates, showed differences between wild type and tu8 with respect to induction of individual GSLs and anthocyanins. However, the transcript level of the TU8/TFL2 gene after heat shock and jasmonate treatment did not change. Loss of function or altered function in the heterochromatin protein most likely lead to the pleiotropic phenotype observed for the tu8 mutant.

Key words

Arabidopsis - auxin - glucosinolate - anthocyanin - heat shock - heterochromatin protein 1 homolog - jasmonic acid - phenylpropanoid pathway - sinapine derivatives.

References

• 1 Aagaard L., Laible G., Selenko P., Schmid M., Dorn R., Schotta G., Kuhfittig S., Wolf A., Lebersorger A., Singh P. B., Reuter G., Jenuwein T.. Functional mammalian homologues of the Drosophila PEV-modifier Su(var) 3-9 encode centromere-associated proteins which complex with the heterochromatin component M31.  EMBO Journal. (1999);  18 1923-1938
• 47 Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman G. J.. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.  Nucleic Acids Research. (1997);  25 3389-3402
• 2 Bennett R., Ludwig-Müller J., Kiddle G., Hilgenberg W., Wallsgrove R.. Developmental regulation of aldoxime formation in seedlings and mature plants of Chinese cabbage (Brassica campestris) and oilseed rape (Brassica napus): Glucosinolate and IAA biosynthetic enzymes.  Planta. (1995);  196 239-244
• 3 Bischoff M., Löw R., Grsic S., Rausch T., Hilgenberg W., Ludwig-Müller J.. Infection with the obligate biotroph Plasmodiophora brassicae, the causal agent of the clubroot disease, does not affect expression of NIT1/2-related nitrilases in roots of Chinese cabbage.  Journal of Plant Physiology. (1995);  147 341-345
• 4 Bloor S. J., Abrahams S.. The structure of the major anthocyanin in Arabidopsis thaliana. .  Phytochemistry. (2002);  59 343-346
• 5 Bodnaryk R. P.. Potent effect of jasmonates on indole glucosinolates in oilseed rape and mustard.  Phytochemistry. (1994);  35 301-306
• 6 Brown P. D., Tokuhisa J. G., Reichelt M., Gershenzon J.. Variation of glucosinolate accumulation among different organs and developmental stages of Arabidopsis thaliana. .  Phytochemistry. (2003);  62 471-481
• 7 Butcher D. N., El-Tigani S., Ingram D. S.. The role of indole glucosinolates in the clubroot disease of the Cruciferae.  Physiological Plant Pathology. (1974);  4 127-141
• 8 Campanella J. J., Kumburis N. C., Singh V. S., Ludwig-Müller J.. Fine genetic mapping and isolation of the Arabidopsis tu8 mutant. Plant Biology 2001, Providence, RI, USA, http://216.133.76.127/pb2001/public/P33/0217.html. (2001)
• 9 Campanella J. J., Ludwig-Müller J., Bakllamaja V., Sharma V., Cartier A.. ILR1 and sILR1 IAA amidohydrolase homologs differ in expression pattern and substrate specificity.  Plant Growth Regulation. (2003);  41 215-223
• 10 Daxenbichler M. E., Spencer G. F., Carlson D. G., Rose G. B., Brinker A. M., Powell R. G.. Glucosinolate composition of seeds from 297 species of wild plants.  Phytochemistry. (1991);  30 2623-2638
• 11 Doughty K. J., Porter A. J. R., Morton A. M., Kiddle G., Bock C. H., Wallsgrove R. M.. Variation in the glucosinolate content of oilseed rape leaves. 11. Response to infection by Alternaria brassicae (Berk) Sacc.  Annals of Applied Biology. (1991);  118 469-477
• 12 Doughty K. J., Kiddle G. A., Pye B. J., Wallsgrove R. M., Pickett J. A.. Selective induction of glucosinolates in oilseed rape leaves by methyl jasmonate.  Phytochemistry. (1995);  38 347-350
• 13 Eissenberg J. C., Elgin S. C.. The HP1 protein family: getting a grip on chromatin.  Current Opinion in Genetics and Development. (2000);  10 204-210
• 14 Ellis R. J., van der Vries S. M.. Molecular chaperones.  Annual Review of Biochemistry. (1991);  60 321-347
• 15 Fahey J., Zalcmann A. T., Talalay P.. The chemical diversity and distribution of glucosinolates and isothiocyanates among plants.  Phytochemistry. (2001);  56 5-51
• 16 Forreiter C., Nover L.. The heat stress response and the concept of molecular chaperones.  Journal of Bioscience. (1998);  23 287-302
• 17 Furuta K., Chan E. K., Kiyosawa K., Reimer G., Luderschmidt C., Tan E. M.. Heterochromatin protein HP1Hsbeta (p25beta) and its localization with centromeres in mitosis.  Chromosoma. (1997);  106 11-19
• 18 Gaudin V., Libault M., Pouteau S., Juul T., Zhao G., Lefebvre D., Grandjean O.. Mutations in LIKE HETEROCHROMATIN PROTEIN 1 affect flowering time and plant architechture in Arabidopsis. .  Development. (2001);  128 4847-4858
• 19 Goujon T., Sibout R., Pollet B., Maba B., Nussaume L., Bechtold N., Lu F., Ralph J., Mila I., Barrière Y., Lapierre C., Jouanin L.. A new Arabidopsis thaliana mutant deficient in the expression of O-methyltransferase impacts lignins and sinapoyl esters.  Plant Molecular Biology. (2003);  51 973-989
• 20 Grsic S., Sauerteig S., Neuhaus K., Albrecht M., Rossiter J., Ludwig-Müller J.. Physiological analysis of transgenic Arabidopsis thaliana plants expressing one nitrilase isoform in sense or antisense direction.  Journal of Plant Physiology. (1998);  153 446-456
• 21 Grsic S., Kirchheim B., Pieper K., Fritsch M., Hilgenberg W., Ludwig-Müller J.. Auxin biosynthesis in clubroot diseased Chinese cabbage plants and induction by jasmonic acid.  Physiologia Plantarum. (1999);  105 521-531
• 22 Haughn G. W., Davin L., Giblin M., Underhill E. W.. Biochemical genetics of plant secondary metabolites in Arabidopsis thaliana. The glucosinolates.  Plant Physiolology. (1991);  97 217-226
• 23 Hemm M. R., Ruegger M. O., Chapple C.. The Arabidopsis ref2 mutant is defective in the gene encoding CYP83A1 and shows both phenylpropanoid and glucosinolate phenotypes.  Plant Cell. (2003);  15 179-194
• 24 Ilić N., Normanly J., Cohen J. D.. Quantification of free plus conjugated indoleacetic acid in Arabidopsis requires correction for nonenzymatic conversion of indolic nitriles.  Plant Physiology. (1996);  111 781-788
• 25 Jones D. O., Cowell I. G., Singh P. B.. Mammalian chromodomain proteins: their role in genome organization and expression.  Bioessays. (2000);  22 124-137
• 26 Kim J. H., Durrett T. P., Las R. L., Jander G.. Characterization of the Arabidopsis TU8 glucosinolate mutation, an allele of TERMINAL FLOWER2. .  Plant Molecular Biolology. (2004);  54 671-682
• 27 Kotake T., Takada S., Nakahigashi K., Ohto M., Goto K.. Arabidopsis TERMINAL FLOWER2 gene encodes a heterochromatin protein 1 homolog and repesses both FLOWERING LOCUS T to regulate flowering time and several floral homeotic genes.  Plant and Cell Physiology. (2003);  44 555-564
• 28 Kreig D.. Ethyl methanesulfonate-induced reversion of bacteriophage T4r II mutants.  Genetics. (1963);  48 561-580
• 29 Lindquist S.. The heat shock response.  Annual Review of Biochemistry. (1986);  55 1151-1191
• 30 Löw R., Rausch T.. Sensitive nonradioactive Northern Blots using alkaline transfer of total RNA and PCR-amplified biotinylated probes.  Biotechniques. (1994);  17 1026-1030
• 31 Ludwig-Müller J., Hilgenberg W.. Tryptophan oxidizing enzyme and basic peroxidase isoenzymes in Arabidopsis thaliana (L.) Heynh: Are they identical?.  Plant and Cell Physiology. (1992);  33 1115-1125
• 32 Ludwig-Müller J., Schubert B., Pieper K., Ihmig S., Hilgenberg W.. Glucosinolate content in susceptible and tolerant Chinese cabbage varieties during the development of the clubroot disease.  Phytochemistry. (1997);  44 407-414
• 33 Ludwig-Müller J., Pieper K., Ruppel M., Cohen J. D., Epstein E., Kiddle G., Bennett R.. Indole glucosinolate and auxin biosynthesis in Arabidopsis thaliana L. glucosinolate mutants and the development of the clubroot disease.  Planta. (1999);  208 409-419
• 34 Ludwig-Müller J., Krishna P., Forreiter C.. A glucosinolate mutant of Arabidopsis thaliana is thermosensitive and defective in cytosolic Hsp90 expression after heat stress.  Plant Physiology. (2000);  123 949-958
• 35 Mellon F. A., Bennett R. N., Holst B., Williamson G.. Intact glucosinolate analysis in plant extracts by programmed cone voltage electrospray LC/MS: performance and comparison with LC/MS/MS methods.  Analytical Biochemistry. (2002);  306 83-91
• 36 Mikkelsen M. D., Petersen B. L., Glawischnig E., Jensen A. B., Andreasson E., Halkier B. A.. Modulation of CYP79 genes and glucosinolate profiles in Arabidopsis by defense signaling pathways.  Plant Physiology. (2003);  131 298-308
• 37 Murashige T., Skoog F.. A revised medium for rapid growth and bioassays with tobacco tissue cultures.  Physiologia Plantarum. (1962);  15 473-497
• 38 Paro R., Hogness D. S.. The Polycomb protein shares a homologous domain with a heterochromatin-associated protein of Drosophila. .  Proceedings of the National Academy of Sciences of the USA. (1991);  88 263-267
• 39 Reintanz B., Lehnen M., Reichelt M., Gershenzon J., Kowalczyk M., Sandberg G., Godde M., Uhl R., Palme K.. bus, a bushy Arabidopsis CYP79F1 knockout mutant with abolished synthesis of short-chain aliphatic glucosinolates.  Plant Cell. (2001);  13 351-367
• 40 Rogers S. O., Bendich A.J.. Extraction of DNA from milligram amounts of fresh herbarium and mummified tissues.  Plant Molecular Biology. (1985);  5 69-76
• 41 Rosa E. A. S., Heaney R. K., Fenwick G. R., Portas C. A. M.. Glucosinolates in crop plants.  Horticultural Review. (1997);  19 99-215
• 42 Sambrook J., Fritsch E. F., Maniatis T.. Molecular Cloning, A Laboratory Manual. New York; Cold Spring Harbour Laboratory Press (1989)
• 43 Verhoeven D. T. H., Verhagen H., Goldbohm R. A., van der Brandt P. A., van Poppel G. A.. A review of mechanisms underlying anticarcinogenicity by Brassica vegetables.  Chemical-Biological Interaction. (1997);  103 79-129
• 44 Wallsgrove R. M., Doughty K., Bennett R. N.. Glucosinolates. Singh, B. J., ed. Plant Amino Acids: Biochemistry and Biotechnology. New York; Marcel Dekker Inc. (1999): 523-562
• 45 Wenke T., Ludwig-Müller J.. Genetic and physiological characterization of the glucosinolate deficient A. thaliana tu8 mutant. Deutsche Botanikertagung, Freiburg, Germany. (2002)
• 46 Zhao Y., Hull A. K., Gupta N. R., Goss K. A., Alonso J., Ecker J. E., Normanly J., Chory J., Celenza J. C.. Trp-dependent auxin biosynthesis in Arabidopsis: involvement of cytochrome P450s CYP79B2 and CYP79B3.  Genes and Development. (2002);  16 3100-3112

Editor: E. Pichersky