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Cell- and tissue-specific localization and regulation of the epithiospecifier protein in Arabidopsis thaliana

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Abstract

The glucosinolate-myrosinase system found in plants of the order Brassicales is one of the best studied plant defense systems. Hydrolysis of the physiologically inert glucosinolates by hydrolytic enzymes called myrosinases, which only occurs upon tissue disruption, leads to the formation of biologically active compounds. The chemical nature of the hydrolysis products depends on the presence or absence of supplementary proteins, such as epithiospecifier proteins (ESPs). ESPs promote the formation of epithionitriles and simple nitriles at the expense of the corresponding isothiocyanates which are formed through spontaneous rearrangement of the aglucone core structure. While isothiocyanates are toxic to a wide range of organisms, including insects, the ecological significance of nitrile formation and thus the role of ESP in plant-insect interactions is unclear. Here, we identified ESP-expressing cells in various organs and several developmental stages of different Arabidopsis thaliana ecotypes by immunolocalization. In the ecotype Landsberg erecta, ESP was found to be consistently present in the epidermal cells of all aerial parts except the anthers and in S-cells of the stem below the inflorescence. Analyses of ESP expression by quantitative real-time PCR, Western blotting, and ESP activity assays suggest that plants control the outcome of glucosinolate hydrolysis by regulation of ESP at both the transcriptional and the post-transcriptional levels. The localization of ESP in the epidermal cell layers of leaves, stems and reproductive organs supports the hypothesis that this protein has a specific function in defense against herbivores and pathogens.

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Abbreviations

DAPI:

4′,6-diamidino-2-phenylindole

ESP:

epithiospecifier protein

FID:

flame ionization detection

qRT-PCR:

quantitative real-time PCR

RP2LS:

ribosomal protein 2, large subunit

References

  • Andréasson E, Jørgensen LB (2003) Localization of plant myrosinases and glucosinolates. In: Romeo JT (eds) Integrative phytochemistry: from ethnobotany to molecular ecology, recent advances in phytochemistry, vol 37. Elsevier, Amsterdam, pp 79–99

    Google Scholar 

  • Andréasson E, Jørgensen LB et al (2001) Different myrosinase and idioblast distribution in Arabidopsis and Brassica napus. Plant Physiol 127:1750–1763

    Article  PubMed  Google Scholar 

  • Barth C, Jander G (2006) Arabidopsis myrosinases TGG1 and TGG2 have redundant function in glucosinolate breakdown and insect defense. Plant J 46:549–562

    Article  PubMed  CAS  Google Scholar 

  • Bernardi R, Negri A et al (2000) Isolation of the epithiospecifier protein from oil-rape (Brassica napus ssp oleifera) seed and its characterization. FEBS Lett 467:296–298

    Article  PubMed  CAS  Google Scholar 

  • Broun P (2005) Transcriptional control of flavonoid biosynthesis: a complex network of conserved regulators involved in multiple aspects of differentiation in Arabidopsis. Curr Opin Plant Biol 8:272–279

    Article  PubMed  CAS  Google Scholar 

  • Brown PD, Tokuhisa JG et al (2003) Variation of glucosinolate accumulation among different organs and developmental stages of Arabidopsis thaliana. Phytochemistry 62:471–481

    Article  PubMed  CAS  Google Scholar 

  • Burow M, Markert J et al (2006a) Comparative biochemical characterization of nitrile-forming proteins from plants and insects that alter myrosinase-catalyzed hydrolysis of glucosinolates. FEBS J 273:2432–2446

    Article  CAS  Google Scholar 

  • Burow M, Müller R et al (2006b) Altered glucosinolate hydrolysis in genetically engineered Arabidopsis thaliana and its influence on the larval development of Spodoptera littoralis. J Chem Ecol 32(11):2333–2349

    Article  CAS  Google Scholar 

  • Eriksson S, Ek B et al (2001) Identification and characterization of soluble and insoluble myrosinase isoenzymes in different organs of Sinapis alba. Physiol Plant 111:353–364

    Article  PubMed  CAS  Google Scholar 

  • Fahey JW, Zalcmann AT et al (2001) The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry 56:5–51

    Article  PubMed  CAS  Google Scholar 

  • Fieldsend J, Milford GFJ (1994) Changes in glucosinolates during crop development in single-low and double-low genotypes of winter oilseed rape (Brassica napus). 1. Production and distribution in vegetative tissues and developing pods during development and potential role in the recycling of sulfur within the crop. Ann Appl Biol 124:531–542

    CAS  Google Scholar 

  • Foo HL, Gronning LM et al (2000) Purification and characterisation of epithiospecifier protein from Brassica napus: enzymic intramolecular sulphur addition within alkenyl thiohydroximates derived from alkenyl glucosinolate hydrolysis. FEBS Lett 468:243–246

    Article  PubMed  CAS  Google Scholar 

  • Griffiths DW, Deighton N et al (2001) Identification of glucosinolates on the leaf surface of plants from the Cruciferae and other closely related species. Phytochemistry 57:693–700

    Article  PubMed  CAS  Google Scholar 

  • Halkier BA, Gershenzon J (2006) Biology and biochemistry of glucosinolates. Ann Rev Plant Biol 57:303–333

    Article  CAS  Google Scholar 

  • Hause B, Stenzel I et al (2000) Tissue-specific oxylipin signature of tomato flowers: allene oxide cyclase is highly expressed in distinct flower organs and vascular bundles. Plant J 24:113–126

    Article  PubMed  CAS  Google Scholar 

  • Höglund AS, Jones AM et al (2002) An antigen expressed during plant vascular development crossreacts with antibodies towards KLH (keyhole limpet hemocyanin). J Histochem Cytochem 50:999–1004

    PubMed  Google Scholar 

  • Husebye H, Chadchawan S et al (2002) Guard cell- and phloem idioblast-specific expression of thioglucoside glucohydrolase 1 (myrosinase) in Arabidopsis. Plant Physiol 128:1180–1188

    Article  PubMed  CAS  Google Scholar 

  • Jander G, Cui JP et al (2001) The TASTY locus on chromosome 1 of Arabidopsis affects feeding of the insect herbivore Trichoplusia ni. Plant Physiol 126:890–898

    Article  PubMed  CAS  Google Scholar 

  • Kelly PJ, Bones A et al (1998) Sub-cellular immunolocalization of the glucosinolate sinigrin in seedlings of Brassica juncea. Planta 206:370–377

    Article  PubMed  CAS  Google Scholar 

  • Kliebenstein DJ, Gershenzon J et al (2001a) Comparative quantitative trait loci mapping of aliphatic, indolic and benzylic glucosinolate production in Arabidopsis thaliana leaves and seeds. Genetics 159:359–370

    CAS  Google Scholar 

  • Kliebenstein DJ, Kroymann J et al (2001b) Genetic control of natural variation in Arabidopsis glucosinolate accumulation. Plant Physiol 126:811–825

    Article  CAS  Google Scholar 

  • Kliebenstein DJ, Kroymann J et al (2005) The glucosinolate-myrosinase system in an ecological and evolutionary context. Curr Opin Plant Biol 8:264–271

    Article  PubMed  CAS  Google Scholar 

  • Koroleva OA, Davies A et al (2000) Identification of a new glucosinolate-rich cell type in Arabidopsis flower stalk. Plant Physiol 124:599–608

    Article  PubMed  CAS  Google Scholar 

  • Kroymann J, Textor S et al (2001) A gene controlling variation in Arabidopsis glucosinolate composition is part of the methionine chain elongation pathway. Plant Physiol 127:1077–1088

    Article  PubMed  CAS  Google Scholar 

  • Lambrix VM, Reichelt M et al (2001) The Arabidopsis epithiospecifier protein promotes the hydrolysis of glucosinolates to nitriles and influences Trichoplusia ni herbivory. Plant Cell 13:2793–2807

    Article  PubMed  CAS  Google Scholar 

  • Lenman M, Falk A et al (1993) Differential expression of myrosinase gene families. Plant Physiol 103:703–711

    Article  PubMed  CAS  Google Scholar 

  • Marazzi C, Patrian B et al (2004) Secondary metabolites of the leaf surface affected by sulphur fertilisation and perceived by the cabbage root fly. Chemoecology 14:87–94

    Article  CAS  Google Scholar 

  • Matile P (1980) The mustard oil bomb—Compartmentation of the myrosinase system. Biochem Physiol Pflanzen 175:722–731

    CAS  Google Scholar 

  • Matusheski NV, Swarup R et al (2006) Epithiospecifier protein from broccoli (Brassica oleracea L. ssp italica) inhibits formation of the anticancer agent sulforaphane. J Agric Food Chem 54:2069–2076

    Article  PubMed  CAS  Google Scholar 

  • Müller C, Riederer M (2005) Plant surface properties in chemical ecology. J Chem Ecol 31:2621–2651

    Article  PubMed  CAS  Google Scholar 

  • Petersen BL, Chen SX et al (2002) Composition and content of glucosinolates in developing Arabidopsis thaliana. Planta 214:562–571

    Article  PubMed  CAS  Google Scholar 

  • Porter AJR, Morton AM et al (1991) Variation in the glucosinolate content of oilseed rape (Brassica napus L.) leaves. 1. Effect of leaf age and position. Ann Appl Biol 118:461–467

    Article  CAS  Google Scholar 

  • Rask L, Andréasson E et al (2000) Myrosinase: gene family evolution and herbivore defense in Brassicaceae. Plant Mol Biol 42:93–113

    Article  PubMed  CAS  Google Scholar 

  • Reichelt M, Brown PD et al (2002) Benzoic acid glucosinolate esters and other glucosinolates from Arabidopsis thaliana. Phytochemistry 59:663–671

    Article  PubMed  CAS  Google Scholar 

  • Scanlon JT, Willis DE (1985) Calculation of flame ionization detector relative response factors using the effective carbon number concept. J Chromatogr Sci 23:333–340

    CAS  Google Scholar 

  • Spencer FG, Daxenbichler ME (1980) Gas chromatography-mass spectrometry of nitriles, isothiocyanates and oxazolidinethiones derived from cruciferous glucosinolates. J Sci Food Agric 31:359–367

    Article  CAS  Google Scholar 

  • Thangstad OP, Gilde B et al (2004) Cell specific, cross-species expression of myrosinases in Brassica napus, Arabidopsis thaliana and Nicotiana tabacum. Plant Mol Biol 54:597–611

    Article  PubMed  CAS  Google Scholar 

  • Thies W (1979) Detection and utilization of a glucosinolate sulfohydrolase in the edible snail, Helix pomatia. Naturwissenschaften 66:364–365

    Article  CAS  Google Scholar 

  • Tholl D (2006) Terpene synthases and the regulation, diversity and biological roles of terpene metabolism. Curr Opin Plant Biol 9:297–304

    Article  PubMed  CAS  Google Scholar 

  • Tookey HL (1973) Crambe thioglucoside glucohydrolase (EC 3.2.3.1)—Separation of a protein required for epithiobutane formation. Can J Biochem 51:1654–1660

    Article  PubMed  CAS  Google Scholar 

  • Ueda H, Nishiyama C et al (2006) AtVAM3 is required for normal specification of idioblasts, myrosin cells. Plant Cell Physiol 47:164–175

    Article  PubMed  CAS  Google Scholar 

  • Wei X, Roomans GM et al (1981) Localization of glucosinolates in roots of Sinapis alba using X-ray-microanalysis. Scan Electron Micros 481–488

  • Wittstock U, Kliebenstein DJ et al (2003) Glucosinolate hydrolysis and its impact on generalist and specialist insect herbivores. In: Romeo JT (eds) Integrative phytochemistry: from ethnobotany to molecular ecology, recent advances in phytochemistry, vol 37. Elsevier, Amsterdam, pp 101–126

    Google Scholar 

  • Xue JP, Pihlgren U et al (1993) Temporal, cell-specific, and tissue-preferential expression of myrosinase genes during embryo and seedling development in Sinapis alba. Planta 191:95–101

    Article  CAS  Google Scholar 

  • Zabala MD, Grant M et al (2005) Characterisation of recombinant epithiospecifier protein and its over-expression in Arabidopsis thaliana. Phytochemistry 66:859–867

    Article  CAS  Google Scholar 

  • Zhang Z-Y, Ober JA et al (2006) The gene controlling the quantitative trait locus epithiospecifier modifier 1 alters glucosinolate hydrolysis and insect resistance in Arabidopsis. Plant Cell 18:1524–1536

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We thank Andrea Bergner for technical assistance, Michael Reichelt for providing intact glucosinolates, and the Max Planck Society for financial support.

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Author notes

  1. Meike Burow & Ute Wittstock

    Present address: Institut für Pharmazeutische Biologie, Technische Universität Braunschweig, Mendelssohnstr. 1, D-38106 , Braunschweig, Germany

Authors and Affiliations

  1. Department of Biochemistry, Max Planck Institute for Chemical Ecology, Beutenberg Campus, Hans-Knöll-Str. 8, D-07745 , Jena, Germany

    Meike Burow, Margaret Rice, Jonathan Gershenzon & Ute Wittstock

  2. California Polytechnic State University, San Luis Obispo, CA, 93407, USA

    Margaret Rice

  3. Leibniz Institute for Plant Biochemistry, Weinberg 3, D-06120 , Halle, Germany

    Bettina Hause

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  1. Meike Burow
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  3. Bettina Hause
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  4. Jonathan Gershenzon
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  5. Ute Wittstock
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Corresponding author

Correspondence to Ute Wittstock.

Additional information

Meike Burow and Margaret Rice contributed equally to this work.

Electronic supplementary material

Supplemental Figure 1

Expression analysis of ESP at the protein level in different organs of 7-week-old A. thalianaLer. For Western blot analysis using a polyclonal peptide-antibody against the ESP from A. thaliana Ler, equal amounts of protein were separated on SDS-polyacrylamide gels. (Lane 1, protein marker; lane 2, purified recombinant ESP (with Strep-tag; 2 μg); Lane 3, older rosette leaves; Lane 4, youngest fully expanded rosette leaves; Lane 5, stem; Lane 6, cauline leaves; Lane 7, flowers) (PDF 27 kb)

Supplemental Figure 2

Immunolocalization of ESP in developing seeds of A. thalianaLer. (A) Autofluorescence in a cross section incubated with preimmune serum. (B) Specific labelling (green fluorescence) by the anti-ESP antibody in the epidermis of the seed coat. Bars represent 25 µm in both micrographs (PDF 2, 630 kb)

Supplemental Figure 3

Subcellular localization of ESP in A. thalianaLer. Leaf (A, B) and flower stalk (C, D) cross sections of A. thaliana Ler. were immunolabelled with a polyclonal antibody against the ESP from A. thaliana Ler. (1:2,000) followed by staining of DNA with DAPI. The occurrence of ESP (A, C) is visualized immunocytochemically by AlexaFluor488 (green). B and D show the identical sections as in A and C, respectively, illuminated for the DAPI-stained nuclei and plastids (blue). Note the occurrence of ESP within the cytoplasm and the nuclei of epidermal cells. Bars represent 10 µm in all micrographs (PDF 1,995 kb)

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Burow, M., Rice, M., Hause, B. et al. Cell- and tissue-specific localization and regulation of the epithiospecifier protein in Arabidopsis thaliana . Plant Mol Biol 64, 173–185 (2007). https://doi.org/10.1007/s11103-007-9143-1

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