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. 2011 Aug;62(13):4605-16.
doi: 10.1093/jxb/err171. Epub 2011 May 27.

S-Nitrosoglutathione reductase (GSNOR) mediates the biosynthesis of jasmonic acid and ethylene induced by feeding of the insect herbivore Manduca sexta and is important for jasmonate-elicited responses in Nicotiana attenuata

Hendrik Wünsche  1 , Ian T Baldwin, Jianqiang Wu
Affiliations

S-Nitrosoglutathione reductase (GSNOR) mediates the biosynthesis of jasmonic acid and ethylene induced by feeding of the insect herbivore Manduca sexta and is important for jasmonate-elicited responses in Nicotiana attenuata

Hendrik Wünsche et al. J Exp Bot. 2011 Aug.

Abstract

S-nitrosoglutathione reductase (GSNOR) reduces the nitric oxide (NO) adduct S-nitrosoglutathione (GSNO), an essential reservoir for NO bioactivity. In plants, GSNOR has been found to be important in resistance to bacterial and fungal pathogens, but whether it is also involved in plant-herbivore interactions was not known. Using a virus-induced gene silencing (VIGS) system, the activity of GSNOR in a wild tobacco species, Nicotiana attenuata, was knocked down and the function of GSNOR in defence against the insect herbivore Manduca sexta was examined. Silencing GSNOR decreased the herbivory-induced accumulation of jasmonic acid (JA) and ethylene, two important phytohormones regulating plant defence levels, without compromising the activity of two mitogen-activated protein kinases (MAPKs), salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK). Decreased activity of trypsin proteinase inhibitors (TPIs) were detected in GSNOR-silenced plants after simulated M. sexta feeding and bioassays indicated that GSNOR-silenced plants have elevated susceptibility to M. sexta attack. Furthermore, GSNOR is required for methyl jasmonate (MeJA)-induced accumulation of defence-related secondary metabolites (TPI, caffeoylputrescine, and diterpene glycosides) but is not needed for the transcriptional regulation of JAZ3 (jasmonate ZIM-domain 3) and TD (threonine deaminase), indicating that GSNOR mediates certain but not all jasmonate-inducible responses. This work highlights the important role of GSNOR in plant resistance to herbivory and jasmonate signalling and suggests the potential involvement of NO in plant-herbivore interactions. Our data also suggest that GSNOR could be a target of genetic modification for improving crop resistance to herbivores.

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Figures

Fig. 1.
Fig. 1.
NaGSNOR transcript accumulation and enzyme activity after wounding and simulated herbivory. Transition leaves of N. attenuata rosette plants were wounded with a pattern wheel, and were subsequently treated with 20 μl of water (W+W) or 20 μl of M. sexta oral secretions (W+OS). Samples were harvested after the indicated times. (A) Transcript levels (mean ±SE) of NaGSNOR were measured with qPCR. (B) Activity (mean ±SE) of NaGSNOR. Stars indicate significantly different levels between treated and non-treated samples (Student's t test; *P ≤0.05; ***P ≤0.001; n=5).
Fig. 2.
Fig. 2.
NaGSNOR-VIGS plants have highly diminished transcript levels of NaGSNOR and strongly reduced GSNOR activity. N. attenuata plants were infiltrated with Agrobacterium carrying pTV00 or a pTV-NaGSNOR to generate EV and NaGSNOR-VIGS plants, respectively. (A) Transcript levels (mean ±SE) of NaGSNOR and (B) GSNOR activity (mean ±SE) were determined in EV and NaGSNOR-VIGS plants. Stars indicate significantly different levels between EV and NaGSNOR-VIGS plants (Student's t test; ***P ≤0.001; n=5).
Fig. 3.
Fig. 3.
Wounding- and simulated herbivory-induced levels of phytohormones in EV and NaGSNOR-VIGS plants. EV and NaGSNOR-VIGS plants were wounded with a pattern wheel and were subsequently treated with 20 μl of water (W+W) or 20 μl of M. sexta oral secretions (OS) (W+OS). (A) JA, (B) JA-Ile, and (C) SA contents (mean ±SE) were measured on a HPLC-MS/MS. (D) Ethylene (mean ±SE) emitted from non-treated (Cont) and W+OS-treated EV and NaGSNOR-VIGS plants. Stars indicate significantly different levels between EV and NaGSNOR-VIGS plants (Student's t test; *P ≤0.05; ***P ≤0.001; n=5).
Fig. 4.
Fig. 4.
Silencing NaGSNOR does not impair wounding- and simulated herbivory-induced MAPK activity in N. attenuata. EV and NaGSNOR-VIGS plants were wounded with a pattern wheel and were subsequently applied with 20 μl of water (W+W) or 20 μl of M. sexta oral secretions (OS) (W+OS). Samples were harvested after the indicated times. An in-gel kinase activity assay (upper panel) was performed to detect the activity of SIPK and WIPK. Replicated samples were run on a SDS-PAGE gel, and this gel was thereafter stained with Coomassie Brilliant Blue (CBB) for visualization of equal loading (lower panel).
Fig. 5.
Fig. 5.
Accumulation of herbivore defense-related secondary metabolites in EV and NaGSNOR-VIGS plants. Leaves of EV and NaGSNOR-VIGS plants were wounded with a pattern wheel, and were thereafter applied with 20 μl of water (W+W) or 20 μl of M. sexta oral secretions (W+OS). The activity of NaTPI (A), contents of caffeoylputrescine (CP) (B), diterpene glycosides (DTGs) (C), and nicotine (D) (mean ±SE) were determined in EV and NaGSNOR-VIGS plants 3 d after treatments; non-treated plants served as controls (Cont). Star indicates significantly different levels between EV and NaGSNOR-VIGS plants (Student's t test; *P ≤0.05; n=5).
Fig. 6.
Fig. 6.
Silencing NaGSNOR in N. attenuata compromises plant resistance to insect herbivore, M. sexta. Neonate M. sexta larvae were placed on rosette-staged EV and NaGSNOR-VIGS plants and larval masses (mean ±SE) were measured after 4, 9, and 14 d. Stars indicate significantly different larval masses between those fed on EV and on NaGSNOR-VIGS plants (Student's t test; *, P ≤0.05; ***P ≤0.001; n=30).
Fig. 7.
Fig. 7.
Herbivore defence-related secondary metabolites in EV and NaGSNOR-VIGS plants after methyl jasmonate treatment. EV and NaGSNOR-VIGS plants were applied with lanolin pastes (20 μl) containing 5 mg ml−1 methyl jasmonate (MJ) or pastes of pure lanolin (Lan) (20 μl) for comparisons. The activity of NaTPI (A), contents of caffeoylputrescine (CP) (B), diterpene glycosides (DTGs) (C), and nicotine (D) (mean ±SE) were determined in EV and NaGSNOR-VIGS plants 3 d after treatments. Stars indicate significantly different levels between EV and NaGSNOR-VIGS plants (Student's t test; *P ≤0.05; n=5).
Fig. 8.
Fig. 8.
Transcript levels of NaTPI, NaJAZ3, and NaTD in methyl jasmonate-treated EV and NaGSNOR-VIGS plants. EV and NaGSNOR-VIGS plants were applied with lanolin pastes (20 μl) containing 5 mg ml−1 methyl jasmonate (MJ), or pastes of pure lanolin (Lan) (20 μl) for comparison. The transcript levels of NaTPI (A), NaJAZ3 (B), and NaTD (C) (mean ±SE) were determined in EV and NaGSNOR-VIGS plants 8 h after treatments. Stars indicate significantly different levels between EV and NaGSNOR-VIGS plants (Student's t test; *P ≤0.05; n=5).
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