Glutathione S-transferase isoenzymes in plant defense reactions against stress and disease

1. Glutathione S-transferase isoenzymes in plant defense reactions against stress and disease Gábor Gullner Plant Protection Institute Hungarian Academy of Sciences Budapest, Hungary

2. Glutathione S-transferase (GST) activity: first detected in maize Frear and Swanson, 1970 + glutathione + HCl atrazine conjugate High GST activity: maize, sorghum

3. Glutathione (GSH) Glu Cys Gly SH -L-glutamyl-L-cysteinyl-glycine - most important non-protein thiol compound ~1 mM in cytosol - very reactive sulfhydryl group - detoxification reactions - antioxidant

4. Herbicide detoxification Several herbicides are rapidly metabolized via glutathione conjugation in plants. - different GST isoenzymes exist in plants Often GSTs are the basis of herbicide selectivity: - crop plants (maize): high GST activity - competing weeds: low GST activity

5. Herbicide detoxification by GSTs Triazines: atrazine, simazine Chloroacetanilides: acetochlor, alachlor, metolachlor Thiocarbamates: EPTC Nitrodiphenylethers: acifluorfen, fluorodifen Sulfonylureas: chlorimuron ethyl Others: chlorfenprop-methyl, metribuzin

6. Transgenic tobacco Transformation of tobacco with a maize gene encoding the GST27 isoenzyme conferred tolerance to chloroacetanilide herbicides (Jepson et al., 1997)

7. Herbicide safeners (antidotes) Wide range of synthetic compounds They increase the herbicide tolerance in crop plants (usually in cereals) Mode of action: Selective induction of GST isoenzymes participating in herbicide detoxification

8. Other toxic GST substrates 1,2-dichloro-4-nitrobenzene 1-chloro-2,4-dinitrobenzene 4-nitropyridine-N-oxide p-nitrophenethyl-bromide p-nitrobenzyl chloride bromosulfophthalein

9. Classical definition of GSTs: GSTs catalyze the conjugation of glutathione to a wide range of toxic and hydrophobic compounds large isoenzyme family predominantly cytosolic in maize: 1-2% of all soluble proteins endogenous, physiological substrates??

10. Transport to the vacuoles ATP-dependent membrane pumps recognize the glutathione S-conjugates and transfer them into the vacuoles Glutathione conjugation ”tags” the xenobiotic substrates for recognition by membrane pumps

11. Measurement of total glutathione S-transferase (GST) activity:spectrophotometrically at 340 nm conjugate CDNB Best model substrate: 1-chloro-2,4-dinitrobenzene (CDNB)

12. Separation of GST isoenzymes by HPLC in wheat extracts Crude extract (NH4)2SO4 precipitation affinity chromatography on GSH-agarose column RP-HPLC UV detection (214 nm)

13. Old history (1990s): GST isoenzymes in maize Two subunits Homo- and heterodimer molecules constitutive or inducible

14. GSTs today Maize: 42 isoenzymes EST (expressed sequence tag) database BLAST searching (Du Pont Company, USA) Arabidopsis thaliana: 47 isoenzymes McGonigle et al., Plant Physiol. 124, 1105-1120 (2000)

15. Classification of GST isoenzymes Based on: intron-exon gene structure amino acid sequence similarity immunological cross reactivity Phi class: 2 introns, major maize GSTs Zeta class: 9 introns Tau class: 1 intron, auxin inducible Theta class: 6 introns Edwards et al., 2000

16. GSTs in endogenous metabolism GSTs are not only stress- and detoxification enzymes Other activities: GST has high affinity for auxins and cytokinins: ligandin function, hormone homeostasis (storage)? auxin carrier, transport between cell compartments anthocyanin sequestration in the vacuoles

17. Structure of GST proteins X-ray crystallography: Arabidopsis Phi class GST Two subunits, both with two distinct domains Active site: G-site: conserved GSH binding site (in N terminal domain - in red) H-site: binding pocket for hydrophobic cosubstrates - serine residue activates GSH for conjugation

18. Inducibility of GSTs H2O2 and other ROS xenobiotics safeners heavy metals cold, heat drought plant hormones pathogen infections salicylic acid (SA)

19. Peroxidase activity of GST R-OOH + 2 GSH  R-OH + GS-SG + H2O Detoxification of organic hydroperoxides Tolerance against oxidative stress Hydrogen peroxide is not a substrate Glutathione peroxidase - different enzyme

20. GST activity in cold-hardened cereals Cold-hardening: gradually decreasing temperatures for 7 weeks between 14 and 3 C  increasing frost resistance Control: 17 C Strong induction But: no correlation between GST induction and frost tolerance Janda et al., Plant Science 164, 301-6 (2003)

21. ”Futile” gene induction? Ozone fumigation of tobacco caused a strong induction of basic -1,3-glucanase and chitinase activities and mRNA levels Pathogenesis-related proteins Function?? Sandermann et al., 1996

22. Tobacco overexpressing GST:increased stress tolerance Transformation: Chimeric Nt 107 gene encoding GST under control of an enhanced CaMV promoter Increased stress tolerance against - salt stress - chilling stress Reduced lipid peroxidation Enhanced growth of seedlings during stress Roxas et al., 2000

23. Injection of salicylic acid (0,8 mM) into leaves of Xanthi-nc tobacco % of control GST activity Days after treatment

24. Induction of GSTs by salicylic acid (SA) SA-responsive element in the promoter of several GSTs: activation sequence-1 (as-1) approx. 20 base pairs, contains two TGACG motifs that bind basic/Leu zipper transcription factors

25. Salicylic acid auxin methyl jasmonate reactive oxygen species (ROS) as-1 binds nuclear transcription factors GST induction

26. Induction of GST genesafter oxidative stress oxidative stress response elementcoding region DNA-protein binding nuclear factor

27. Activation of nuclear transcription factors: phosphorylation OH O P ROS inactive protein active protein binding to as-1

28. Activation of nuclear transcription factors: disulfide bridge formation SH SH S S ROS inactive protein active protein binding to as-1

29. Similar factors in animal cells nuclear factor-B AP-1 transcription factor

30. Detection of DNA-protein binding: gel mobility shift assay 1. isolation of nuclear proteins 2. incubation with 32P-labeled DNA probe containing promoter element 3. polyacrylamide gel electrophoresis of DNA probes with and without nuclear proteins 4. autoradiography

31. as-1 as-1 as-1 as-1 CaMV 35S GUS reporter gene Activation of as-1 element in a transgenic tobacco construct 1 mM SA, light  accumulation of ROS  as-1 promoter element can bind nuclear protein Antioxidants inhibit the SA induced gene expression (Hidalgo et al., 2001; Garretón et a., 2002)

32. GUS reporter gene -glucuronidase: hydrolyzes a wide range of -glucuronides Widely used in histochemical studies Low endogenous activity in plants Substrate: X-Gluc 5-bromo-4-chloro-3-indolyl--glucuronide Cleavage, oxidation of indole derivative  dimerization  formation of insoluble indigo dye

33. Glutathione S-transferase (GST) in wheat after fungal infection Dudler et al., 1991 Winter wheat was infected by barley powdery mildew (Blumeria graminis f.sp. hordei) Resistance against wheat powdery mildew Accumulation of mRNA encoding GST (29 kD protein) This GST was specifically induced by infection Its role ??

34. GST inductions in plant-pathogen interactions Hahn és Strittmatter, 1994 Potato leaf - fungus (Phytophthora infestans) Greenberg et al., 1994; Ádám et al., 1997 Bean, Arabidopsis - Pseudomonas syringae pv. phaseolicola Venisse és mtsai., 2001 Tobacco, pear - Erwinia amylovora

35. Xanthi-nc tobacco infected with tobacco mosaic virus (TMV) incompatible interaction

36. Induction of GST activity in tobaccoleaves infected with tobacco mosaic virus Spectrophotometric analysis (340 nm) - total GST activity Fodor et al., Plant Physiol. 114, 1443-1451 (1997)

37. Induction of GST by the monoterpene oil S-carvone in Xanthi-nc tobacco leaf discs GST activity % caraway, dill Days after treatment

38. Visible symptoms of TMV infection in Xanthi-nc tobacco leaf discs after (S)-carvone pretreatment 30 25 Number of lesions 20 15 ** 10 5 0 TMV (S)-carvone + TMV

39. Virus concentrationswith and without (S)-carvonepretreatment by ELISA test 140 120 arbitrary units 100 80 60 40 20 0 TMV (S)-carvone + TMV

40. Number of necrotic lesions Control (wild) tobacco 107 Transgenic tobaco 39 Effect of GST on the number of necrotic lesions Wagner and Mauch, 2000 Xanthi-nc tobacco plants were transformed by a wheat GST gene, which can be induced by fungal infection - CaMV 35S promoter

41. Glutatione S-transferase isoenzymes are able to suppress the development of necrotic lesions by an unknown mechanism.

42. TMV infection:

43. The role of glutathione S-transferase isoenzymes in infected plants is the detoxification of toxic lipid hydroperoxides, which are produced during lipid peroxidation.

44. Future plans: • Study of the induction of specific GST isoenzyme(s) in TMV infected tobacco leaves on mRNA level by RT-PCR RT-PCR: excellent tool to study gene inductions - isolation of total mRNA from virus infected leaves - synthesis of cDNAs by reverse transcriptase - PCR with GST specific or degenerate primers - sequencing - cloning into bacteria, functional studies

45. Plant Protection Institute, Budapest