Ozone and nitric oxide induce cGMP-dependent and -independent transcription of defence genes in tobacco

Authors


Author for correspondence:
Stefania Pasqualini
Tel:+39 (0)755856413
Fax:+39 (0)755856598
Email: spas@unipg.it

Summary

  • • Here, we analyse the temporal signatures of ozone (O3)-induced hydrogen peroxide (H2O2) and nitric oxide (NO) and the role of the second messenger guanosine 3′,5′-cyclic monophosphate (cGMP) in transcriptional changes of genes diagnostic for biotic and abiotic stress responses.
  • • Within 90 min O3 induced H2O2 and NO peaks and we demonstrate that NO donors cause rapid H2O2 accumulation in tobacco (Nicotiana tabacum) leaf. Ozone also causes highly significant, late (> 2 h) and sustained cGMP increases, suggesting that the second messenger may not be required in all early (< 2 h) responses to O3, but is essential and sufficient for the induction of some O3-dependent pathways.
  • • This hypothesis was tested resolving the time course of O3-induced transcript accumulation of alternative oxidase (AOX1a), glutathione peroxidase (GPX), aminocyclopropancarboxylic acid synthase (ACS2) that is critical for the synthesis of ethylene, phenylalanine ammonia lyase (PALa) and the pathogenesis-related protein PR1a.
  • • The data show that early O3 and NO caused transcriptional activation of the scavenger encoding proteins AOX1a, GPX and the induction of ethylene production through ACS2 are cGMP independent. By contrast, the early response of PALa and the late response of PR1a show critical dependence on cGMP.

Introduction

An ever-increasing number of reports implicate the second messenger guanosine 3′,5′-cyclic monophosphate (cGMP) in many different physiological responses in higher plants (Newton & Smith, 2004; Meier & Gehring, 2006). They include the gating of K+ channels (Hoshi, 1995; Leng et al., 1999; Ma & Berkowitz, 2007), responses to NaCl, drought (Donaldson et al., 2004) and biotic stresses (Durner et al., 1998), stomatal guard cell regulation (Pharmawati et al., 1998a; Cousson, 2001), gravitropic stimuli (Hu et al., 2005) and the induction of gravity-directed cell polarity (Salmi et al., 2007). In addition, cGMP has been implicated as second messenger in auxin (Pagnussat et al., 2002), gibberellic acid (Penson et al., 1996) and kinetin-dependent signalling (Pharmawati et al., 1998b).

It is noteworthy that the time for cytosolic cGMP increases varies considerably from < 5 min in response to plant natriuretic peptides (Pharmawati et al., 1998b) to 15 min in response to NaCl and osmotica (Donaldson et al., 2004), 2 h after application of a nitric oxide (NO) donor (Durner et al., 1998) and 5 h in response to a gravitropic stimulus (Hu et al., 2005). This can be taken as an indication of the complex and diverse roles of cGMP as signalling molecule and might imply that cGMP has a role as a rapid transducer as well as in sustaining long-term adaptive responses.

Furthermore, a cGMP-dependent transcriptome has been obtained (Maathuis, 2006) and the presence of genes encoding monovalent cation transporters such as nonselective ion channels and cation/H+ antiporters can provide a functional link to biotic and abiotic stress responses. Considering that so many highly diverse processes in higher plants are critically dependent on cGMP, it is somewhat surprising that only two Arabidopsis molecules have been reported that have guanylyl cyclase (GC) activity (Ludidi & Gehring, 2003; Kwezi et al., 2007), particularly so, since the unicellular green alga Chlamydomonas reinhardtii contains > 100 nucleotide cyclases with 22 different domain combinations involving 13 different domain partners (Meier et al., 2007) including the H-NOX domain responsible for NO sensing in animal soluble GCs (Boon et al., 2005).

While it remains unclear how NO is synthesized in planta, many biological processes including xylogenesis, programmed cell death, pathogen defence, flowering, stomatal closure, and gravitropism are NO dependent (for review see Shapiro, 2005; Neill et al., 2007). In addition, we have demonstrated that NO plays a pivotal role in the response of tobacco plants to O3 (Ederli et al., 2006, 2008) and in particular, that NO is indispensable for the activation of the alternative oxidase gene (AOX1a) encoding a protein involved in the scavenging of mitochondrial reactive oxygen species (ROS). Nitric oxide may signal via multi-domain sensor molecules where an H-NOX domain (Boon et al., 2005) combines with a GC domain, as in many annotated soluble GCs, thus linking NO to increasing GC activity. An alternative NO signalling pathway may involve reversible post-translational protein S-nitrosylation (Grennan, 2007). In summary, while the link between NO and cGMP dependent signalling is well established (Neill et al., 2003; Prado et al., 2004; Delledonne, 2005; Salmi et al., 2007) the biochemical nature of the link remains to be discovered.

Here the aim was to characterize the role of cGMP in O3- and NO-dependent transcriptional responses of genes with a role in plant defence and a particular emphasis is on resolving the temporal signature of the second messenger.

Materials and Methods

Plant material

The tobacco (Nicotiana tabacum L. cv. BelW3) seeds were kindly provided by J. Nicholson (NCSU Crop Science Department, Raleigh, NC, USA). Growth chamber conditions were: 14 h photoperiod, photosynthetic photon fluence rate (PPFR) of 120 µmol m2 s1, day/night air temperature of 25°C/20°C, and relative humidity 60–75%. The fourth leaf from the apex of four treated and four untreated (controls) 12-wk-old plants were used in all experiments and the experiments were replicated four times.

O3 treatment

Plants were exposed to 150 nl l−1 O3 or to filtered air in Plexiglas chambers (0.32 m3) under light with a PPFR of 400 µmol m−2 s−1 for up to 5 h (from 08 : 00 h to 13 : 00 h). The O3 produced by UV irradiation (OEG50L lamp; Helios Italquartz s.r.l., Milan, Italy) was continuously monitored with a UV photometric O3 analyser (Thermo Electron Corporation, Franklin, MA, USA). At different times during the 5 h of O3 treatment and at 8 h and 24 h after starting ozone fumigation, the fourth leaves were sampled, frozen under liquid nitrogen and stored at −80°C until H2O2 determination and transcript analysis. For NO quantification fresh material was frozen under liquid N2 and immediately analysed. To reduce NO and cGMP accumulation, before the onset of O3 fumigation, the leaves were painted with 200 µm of the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) or 200 µm of the inhibitor of soluble GCs 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ).

Treatment of tobacco discs with chemicals

To evaluate the NO induced H2O2 accumulation, leaf discs (10 mm diameter) were floated in 5 mm sodium nitroprusside (SNP) or 1 U ml1 of nitric oxide synthase (NOS) (Durner et al., 1998). NOS (Calbiochem-Inalco, Milan, Italy) was obtained by expressing recombinant constitutive rat neuronal NOS in Spodoptera frugiperda cells using a baculovirus expression system. For enzymatic production of NO by NOS the following mixture was prepared: NOS (5 units) in 5 ml of 40 mm N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) (pH 7.4), containing all cofactors and substrates of NOS (5 µm oxyhaemoglobin, 50 µm l-arginine, 100 µm CaCl2, 20 µg ml1 calmodulin, 120 µm NADPH, 12 µm tetrahydro-l-biopterin and 100 µm dithiothreitol (DTT)). Solutions of 10 mm K-phosphate buffer (pH 7.4) or substrate/cofactor mix lacking NOS were used as controls for SNP and NOS, respectively. The H2O2 was also quantified in leaf discs floated on NO-donors and 1 mm of the NO quencher cPTIO. After 1 h and 2 h incubation at 37°C under light (PPFR 400 µmol m2 s1), leaf discs were sampled and H2O2 measured. To evaluate the H2O2-induced NO accumulation 100, 250 and 500 mm H2O2 or H2O as control were injected with a syringe into intercellular space of tobacco leaf (Jabs et al., 1996) and NO was quantified by haemoglobin assay in the tissue after 15, 30, 45 and 60 min. To evaluate transcript modulation AOX1a, PR1a, PALa, GPX and ACS2, leaf discs were floated for 1 h in the following solutions: 10 µm of the cGMP analogue 8-Br-cGMP, 5 mm SNP, 1 mm cPTIO, 200 µm ODQ and H2O as control. After the incubation the leaf discs were transferred in H2O and sampled for RNA extraction after 2, 3, 5 and 24 h. To test cGMP dependence of ethylene (ET) evolution after NO donor treatment, leaf discs were painted with 200 µm, 500 µm and 1000 µm ODQ alone or together with 5 mm SNP. As control, leaf discs were treated with H2O. After 2 h ET accumulation was measured.

Quantification of NO by haemoglobin assay

The NO accumulation was assayed and calculated by following the conversion of oxyhaemoglobin (HbO2) to methaemoglobin (MetHb) spectrophotometrically at 401 nm and 421 nm, using an extinction coefficient of 77 mm−1 cm1 (A401 HbO2, A421 MetHb) (Murphy & Noack, 1994). Oxyhaemoglobin was prepared as detailed previously (Clarke & Higgins, 2000). Leaf tissue (150 mg FW) was snap frozen and homogenized in a mortar with 100 mm K-phosphate buffer (pH 7.0) and 0.6% (w : v) insoluble polyvinylpolypyrrolidone. The extract was clarified by adding powdered activated carbon and centrifuged at 11 000 g for 10 min at 4°C. The supernatant was filtered through a PTFE Millipore membrane (0.45 µm) and immediately assayed for NO. Five minutes before oxyhaemoglobin addition, samples were pretreated with catalase (100 U) and superoxide dismutase (100 U) to remove ROS. To evaluate the percentage of recovery of NO during extraction, 1 mm SNP, which releases 5 µm NO at room temperature and under light (PPFR 400 µmol m2 s1), was added to leaf tissue and the measured recovery after Millipore filtration ranged from 68 to 75%.

Quantification of H2O2

Frozen leaves (200 mg FW) were ground to a powder under liquid N2 and homogenized with 1 ml of 0.2 m HClO4. The extract was kept on ice for 5 min and centrifuged at 10 000 g for 10 min at 4°C. The acidic supernatant was neutralized to pH 7.0–8.0 with 0.2 m NH4OH pH 9.5 and centrifuged at 3000 g for 2 min to sediment the insoluble material. The recovered supernatant was passed through column of AG 1-X8 resin (Bio-Rad, Hercules, CA, USA) and was eluted with distilled water. The H2O2 concentrations were determined spectrophotometrically by xylenol orange assay, as reported previously (Pasqualini et al., 2003). To evaluate the percentage of recovery of H2O2 during extraction and purification of samples, 2 µmol H2O2 were added to leaf tissue. Recovery after column elution ranged from 90 to 98%.

Cell viability

Cell death was evaluated by histochemical analysis using trypan blue. For trypan blue staining, samples were covered with an alcoholic lactophenol trypan blue mixture (30 ml of ethanol, 10 g of phenol, 10 ml of water, 10 ml of glycerol, 10 ml of lactic acid and 10 mg of trypan blue), placed in a boiling water bath for 2 min, left at room temperature for 1 h, then transferred into a chloral hydrate solution (2.5 g ml1) and boiled for 20 min to destain. After multiple changes of chloral hydrate solution to reduce the background, samples were equilibrated with 50% glycerol, mounted and observed with a stereomicroscope.

Determination of ET

The determination of ET from leaf segments was performed as detailed previously (Ederli et al., 2006).

Quantification of cGMP

Frozen tissue (200 mg) was ground to a fine powder, added to 2 ml of ice-cold 6% trichloroacetic acid and the homogenate centrifuged at 4000 g for 15 min. The aqueous extract was extracted four times in five volumes of diethyl ether, dried under vacuum at 25°C overnight and stored at −70°C. Cyclic GMP was determined according to the manufacturer's instructions for the cGMP Assay System (Amersham Bioscience, Piscataway, NJ, USA).

Cyanide analysis

Since is well-documented that both NO and cyanide (CN) are released during the decomposition of SNP (Bethke et al., 2006) and cyanide has been reported to transcriptionally activate AOX gene in tobacco (Sabar et al., 2000), we measured CN developed from 5 mm SNP solution with a cyanide detection kit (Model CYN-3; Hach Company, Loveland, CO, USA). Cyanide was undetectable after 1 h, which is the incubation time used to induce transcription in leaf discs (see the Supporting Information, Fig. S1). Significant CN concentrations were detected only after 5 h incubation and peaked at 24 h (Fig. S1).

Isolation and analysis of RNA

Total RNA was extracted from frozen, homogenized leaf tissue (100–150 mg FW) using NucleoSpin RNA Plant (Macherey-Nagel, Düren, Germany). One microgram of total RNA was reverse transcribed for 1 h at 42°C with 200 units of SuperscriptII RT (Invitrogen, Carlsbad, CA, USA) in 10 mm DTT, 0.4 mm of each dNTP and 0.5 µg oligo dT12–18 primer (Invitrogen, Carlsbad, CA). The cDNA was used for polymerase chain reaction (PCR) with 1 unit Taq polymerase in 0.2 mm of each dNTP and 10 µm of the respective primers (see Table S1). The amplification steps were: initial heating at 94°C for 75 s, denaturation at 94°C for 5 s, annealing for 20 s, 72°C for 45 s and a final extension at 72°C for 7 min. The authenticity of the PCR products was checked by two directional sequencing using an ABI Prism 310 Genetic Analyzer (Perkin Elmer Life and Analytical Sciences, Boston, MA, USA). For quantification, filters were scanned and band intensities were determined with image analysis software.

Results

To test if O3 triggers an oxidative burst, we quantified intracellular H2O2 accumulation after fumigation of whole plants. A highly significant transient increase of H2O2 to > 2.0 µmol g1 FW was registered after 1.5 h of O3 fumigation. Subsequently, the levels declined but remain significantly higher than the control for up to 5 h (Fig. 1a). After 5 h of treatment the intracellular H2O2 had returned to control levels of 0.53 µmol g1 FW.

Figure 1.

Hydrogen peroxide (H2O2) and nitric oxide (NO) accumulation in ozonated leaves of tobacco (Nicotiana tabacum). H2O2 (a) and NO (b) accumulation in leaf extracts from control (closed circles) and fumigated (open circles) plants. Plants were fumigated with 150 nl l1 O3 for 5 h or were maintained in filtered air for the same period. Insets: the average ± SE of two experiments in which the sampling was performed at 15 min to best resolve the time-course of H2O2 and NO accumulation. Significance of variance for one-way anova is indicated: *, P ≤ 0.05; **, P ≤ 0.01.

Simultaneous spectrophotometric monitoring of NO accumulation revealed that it significantly increased after 1 h, with a highly significant peak at 1.5 h where the level increased > 7 times from 0.13 to 0.91 nmol g1 FW min1. At ≥ 3 h NO concentrations reverted to pretreatment levels (Fig. 1b). There was a temporal overlap between the O3-dependent H2O2 and the NO response signature with the latter showing an earlier onset and reversion to the base line.

To determine whether O3 induces H2O2 and NO transients independently rather than being causally linked, we used a foliar disc assay. First, to test if NO can induce H2O2 increases, foliar discs were exposed to 5 mm SNP or recombinant rat neuronal NOS synthase. The result showed that both can induce highly significant H2O2 accumulation in leaf discs (Fig. 2a) and while 5 mm SNP triggers a larger accumulation of H2O2, the response to 1 U ml1 NOS was more rapid. Confirmation that H2O2 accumulation was caused by NO came from the demonstration that the NO scavenger cPTIO suppressed both SNP- and NOS-induced H2O2 accumulation (Fig. 2a). Second, we tested if H2O2 could induce NO accumulation. Since H2O2 is rapidly degraded by endogenous scavengers, we quantified tissue H2O2 levels after 15 min treatment with different concentrations of H2O2 (Fig. 2b) and noted that a concentration as low as 100 mm H2O2 induced highly significant increases. However, H2O2 concentrations as high as 500 mm failed to significantly alter tissue NO levels (Fig. 2c) indicating that, in this experimental system, H2O2 does not induce NO accumulation in tobacco leaf. In order to exclude the possibility that the elevated H2O2 and SNP concentrations used to induce NO accumulation caused cell death and suppression of enzymatic NO production, we assayed cell viability with trypan blue and found no evidence that suggested that viability was affected (Fig. S2). Conversely, 5 h of O3 fumigation induced cell death which was not uniformly distributed throughout the leaf, but appeared as spots (Fig. S2).

Figure 2.

Hydrogen peroxide (H2O2) and nitric oxide (NO) accumulation in leaf tobacco (Nicotiana tabacum) discs. (a) H2O2 concentration measured in tobacco leaf discs floated for 1 h and 2 h with the NO donors 5 mm sodium nitroprusside (SNP) and 1 U ml1 nitric oxide synthase (NOS, lighter tinted bars), with 10 mm K-phosphate buffer (pH 7.4) or substrate/cofactor mix lacking NOS, respectively, as control for SNP and NOS (closed bars) or with 5 mm SNP + 1 mm 2–4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) or 1 U ml1 NOS + 1 mm cPTIO (darker tinted bars). (b) H2O2 concentration measured in leaf discs after 15 min from the infiltration with 100, 250 and 500 mm H2O2 or H2O as control. (c) The NO content measured in leaf discs by haemoglobin assay at different time-points after injection with different H2O2 concentration. The data are the mean ± SE from four independent experiments. Significance of variance for one-way anova: **, P ≤ 0.01.

To elucidate whether the second messenger cGMP has a role in the O3-induced responses we measured cellular cGMP levels during fumigation. A highly significant rise of cGMP level was detected after 3 h and significantly elevated levels persisted over the next 2 h (Fig. 3a). Moreover, the guanylate cyclase inhibitor ODQ at 200 µm suppressed the O3-induced cGMP accumulation (Fig. 3a). It is noteworthy that the cGMP elevation follows both the O3-induced H2O2 and NO peaks. Given that NO is an essential signal molecule for the activation of the mitochondrial AOX1a (Ederli et al., 2006, 2008), we were interested to resolve whether cGMP has a role in the transcriptional activation of this gene even though significant NO dependent cGMP increases above basal levels were registered only after the onset of O3-dependent AOX1a transcript accumulation (≥ 1.5 h) (Ederli et al., 2006).

Figure 3.

Cyclic GMP generation and AOX1a response. (a) Cyclic GMP levels measured at different time points in leaves of tobacco (Nicotiana tabacum) plants fumigated with 150 nl l1 O3 (open circles), maintained in filtered air for the same period (closed circles) or pretreated with 200 µm 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) before the ozone fumigation (triangles). The data are the means ± SE from four independent experiments. Significance of variance for one-way anova: *, P ≤ 0.05; **, P ≤ 0.01. (b) Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for AOX1a in response to treatment with 5 mm sodium nitroprusside (SNP), 5 mm SNP plus 1 mm 2–4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), 10 µm 8-Br-cGMP, 5 mm SNP plus 200 µm ODQ or H2O as control. Leaf discs were floated for 1 h with chemicals and then transferred in H2O. At the indicated times total RNA was isolated from leaf tissue. Total RNA was reverse transcribed and amplified by RT-PCR. Semi-quantification of mRNA levels loaded in each lane was performed by co-amplification and normalization with an internal standard (actin). Beside each blot, a graph with relative intensities of the signals ± SE is shown.

The cell-permeant analogue 8-Br-cGMP was used to test whether cGMP can activate transcription of AOX1a (Fig. 3b). Despite the fact that treatment with 1 µm 8-Br-cGMP delivers 8233.29 fmol 8-Br-cGMP g1 FW to leaf tissue, no transcriptional induction of AOX1a was detected, while the NO donor SNP induced transcript accumulation after 3 h. Furthermore, the effect of the NO donor was completely suppressed in the presence of the NO scavenger cPTIO, while the presence of ODQ, a selective inhibitor of NO-sensitive soluble GCs, did not affect NO-dependent transcription. It was thus concluded that the NO effect on AOX1a does not depend on a cGMP transient.

Given that cGMP has been established as a key signal component in the NO-dependent PR1a induction (Delledonne et al., 1998; Durner et al., 1998), we tested different 8-Br-cGMP concentrations for inducibility in our system and found that 10 µm 8-Br-cGMP causes the highest induction (Fig. 4). Subsequently, other defence genes were tested for cGMP-dependent transcriptional activation. They included the ROS detoxification enzyme glutathione peroxidase encoding gene (GPX), the aminocyclopropancarboxylic acid synthase gene involved in the synthesis of ethylene (ACS2), a gene encoding a protein with a role in stress-induced secondary metabolite synthesis, phenylalanine ammonia lyase (PALa) and the pathogenesis-related protein 1a (PR1a) (Fig. 5). Induction was monitored over 24 h at 2, 3, 5 and 24 h and GPX and ACS2 (Fig. 5a,b), much like AOX1a, were NO inducible but did not require cGMP increases. PALa and PR1a are also NO inducible but induction is critically cGMP dependent (Fig. 5c,d). The second messenger cGMP is sufficient to induce PALa and PR1a while induction is almost completely suppressed in the presence of the soluble GC inhibitor ODQ (Fig. 5c,d), suggesting a critical role of cGMP in the signalling pathway. The temporal induction profiles of PALa and PR1a are distinctly different, the former shows a transient peak at 3 h, while the latter is elevated above control levels at 24 h only, both in response to NO and 8-Br-cGMP. Since the NO-dependent cGMP levels occur at 3 h, direct induction can only be postulated for PALa.

Figure 4.

Cyclic GMP dependent PR1a transcription. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for PR1a in response to treatment with different concentrations (from 0.05 to 10 µm) of 8-Br-cGMP or with H2O as control. Tobacco (Nicotiana tabacum) leaf discs were floated with 8-Br-cGMP for 24 h and after this time total RNA was isolated from tissue leaf. Total RNA was reverse transcribed and amplified by RT-PCR. Semi-quantification of mRNA levels loaded in each lane was performed by co-amplification and normalization with an internal standard (actin). A graph with relative intensities of the signals ± SE is shown below the blot.

Figure 5.

Nitric oxide (NO)-dependent transcriptional responses. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for GPX (a), ACS2 (b), PALa (c) and PR1a (d) in response to treatment with 5 mm sodium nitroprusside (SNP), 5 mm SNP plus 1 mm 2–4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), 10 µm 8-Br-cGMP, 5 mm SNP plus 200 µm 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) or H2O as control. Tobacco (Nicotiana tabacum) leaf discs were floated for 1 h with chemicals and then transferred in H2O. At the indicated times, total RNA was isolated from tissue leaf. Total RNA was reverse transcribed and amplified by RT-PCR. Semi-quantification of mRNA levels loaded in each lane was performed by co-amplification and normalization with an internal standard (actin). A graph with relative intensities of the signals ± SE is shown beside each blot.

Transcription of the AOX1a, GPX, ACS2, PALa and PR1a genes are induced by O3 (Fig. 6). Since AOX1a is NO induced (Fig. 3b) and NO accumulates in fumigated leaves (Fig. 1b), we tested whether O3-induced AOX1a upregulation can be prevented in ozonated plants pretreated with the NO quencher cPTIO. The result (Fig. 6b) indicated that AOX1a mRNA accumulation was completely abolished in NO-suppressed (+cPTIO) plants, thus suggesting that NO is indispensable for AOX1a induction. Similar to AOX1a, induction of the other NO-responsive genes (e.g. GPX, ACS2, PALa and PR1a) was also suppressed when the plants were pre-treated with cPTIO (data not shown).

Figure 6.

Ozone (O3) dependent transcriptional responses. (a) Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for AOX1a, GPX, ACS2, PALa, and PR1a genes from O3-treated tobacco (Nicotiana tabacum) plants (150 nl l1 O3 for 5 h). Plants were sampled before O3 fumigation (0 h) and after 1, 2, 3, 4, 5, 8 and 24 h from the start of fumigation. Total RNA was reverse transcribed and amplified by RT-PCR. Semi-quantification of mRNA levels loaded in each lane was performed by co-amplification and normalization with an internal standard (actin). A graph with relative intensities of the signals ± SE is shown beside each blot. (b) Changes in levels of the AOX1a transcript from O3-fumigated plants after treatment with the NO scavenger 2–4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO, closed bars). Leaves were brushed with 200 µm cPTIO 1.5 h before fumigation. This treatment was repeated three times during the fumigation. Control plants were brushed with distilled water (open bars). Sampling for mRNA analysis was done before and after 1, 2 and 5 h after the start of fumigation. Total RNA was reverse transcribed and amplified by RT-PCR. A graph with relative intensities of the signals ± SE is shown under the blots.

In summary, the genes that encode proteins with a role in scavenging or ET-dependent signalling (AOX1a, GPX and ACS2) show cGMP independent induction, while those encoding proteins with a role in defence-related secondary metabolism (PALa and PR1a) appear to be cGMP dependent. We also noted that PALa increases are observed 2 h after the onset of O3 fumigation at a time when H2O2 levels have peaked (Fig. 1a) but before cGMP levels are detectable above control (Fig. 3a) and that the induction shows biphasic behaviour within the first 24 h. The first transient between 2 h and 6 h was previously reported in BelW3 (Ogawa et al., 2005).

In both whole plants and leaf discs NO induces ET synthesis via the activation of ACS2 (Ederli et al., 2006). This NO-mediated effect on ET synthesis appears not to be directly dependent on cGMP (Fig. 5b). To further clarify the relationship between cGMP and ET, we floated foliar discs in 8-Br-cGMP and measured ET accumulation (Fig. 7a). The results demonstrates that no ET evolution occurs in the presence of 10 µm 8-Br-cGMP alone over the 5 h experimental time, indicating that cGMP is not sufficient for ET synthesis. However, when we treated tobacco leaf discs with SNP in the presence of ODQ we found that the NO-induced ET evolution was drastically inhibited in an ODQ concentration-dependent manner (Fig. 7b). The same result was obtained when the plants were pretreated with ODQ before the ozone fumigation. In this case too, no ET evolution was detected in fumigated plants, despite a peak of ET after 5 h of O3 fumigation in plant without ODQ (data not shown).

Figure 7.

Cyclic GMP dependence of ethylene (ET) evolution. (a) Ethylene emission from pieces of tobacco (Nicotiana tabacum) leaf after infiltration for the time indicated with 10 µm 8-Br-cGMP (open circles) or with H2O (closed circles) as control. The data are the mean ± SE from four independent experiments. (b) Ethylene emission by leaf pieces after 2 h from application of 5 mm SNP, 200, 500 and 1000 µm 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) or ODQ (200, 500 and 1000 µm) + 5 mm sodium nitroprusside (SNP). The data are the means ± SE from two independent experiments.

Discussion

The second messenger cGMP is increasingly recognized as an important signalling molecule that is involved in a diverse number of physiological responses in plants. Similar to the second messenger Ca2+, we are starting to see different signatures associated with response types, ranging from < 1 min (Donaldson et al., 2004), to rapid transients with a peak at < 5 min after the stimulus (Pharmawati et al., 1998b), to the delayed more sustained response with significant increases occurring after 2 h and remaining elevated after 3 h (Durner et al., 1998) post NOS stimulus. The two different response types are indicative for an involvement of cGMP in both rapid signal transduction and modulation of ion channels (Hoshi, 1995; Leng et al., 1999) and late long-term adaptive responses sustaining changes in the transcriptome (Maathuis, 2006) and the proteome.

Given that in plants cGMP has been reported to increase in response to NO (Durner et al., 1998), and that O3 induces increases in NO levels, we tested whether O3 induction was also causing downstream cGMP elevations. Since O3-dependent NO elevations occur after 1 h, and considering previously reported NO dependent cGMP transients (Durner et al., 1998), one might expect that subsequent cGMP increases would occur 2 h later and exactly mirror the timing of previous studies. Consistent with these expectations, we did not observe a short early cGMP transient; however a highly significant cGMP increase was detected after 3 h of O3 treatment which corresponds to the delayed, late and sustained second type of response (Fig. 3a).

This finding raises a number of major questions such as: What is the cascade of events leading to O3-induced elevated cGMP levels? How is cGMP generated? and What are the consequent cGMP dependent responses? The answer to the first question is complicated by the fact that O3 triggers transient increases in both cellular levels of H2O2 and NO, both peaking at 90 min with the onset of significant changes in NO levels occurring 15 min before the changes in cellular H2O2 (Fig. 1a,b). The fact that the H2O2 transients can also be induced by the NO donors (Fig. 2a) would suggest that O3 can activate NO-dependent and -independent pathways, both contributing to the generation of H2O2 possibly leading to potentiation of the response. We have also ascertained that NO increases are independent of H2O2 levels and likely occur upstream of H2O2 since H2O2 increases do not affect NO levels (Fig. 2c). It is noteworthy that this sequence – NO increases preceding H2O2 increases – has also been reported in response to an avirulent Pseudomonas syringae strain in Arabidopsis suspension culture cells (Clarke et al., 2000). However, our results are not in agreement with Zhang et al. (2007) who show that exogenous application of H2O2 to maize leaves induced NO generation. The discrepancy between the studies may be related to the different methods of NO detection employed. Zhang et al. (2007) reported that NO detection after H2O2 application was performed using the NO-sensitive dye DAF-2DA, and this is likely to lead to NO overestimation when oxidant and NO are cosynthesized (Jourd’heuil, 2002), while in our experiments NO was detected spectrophotometrically with a haemoglobin assay.

To assess whether transcriptional changes resulting from O3 treatment of our target genes –AOX1a, GPX, ACS2, PALa and PR1a– were NO and/or cGMP dependent, we monitored their transcription in the presence of various combinations of the following agonists and antagonists: the NO donor SNP, the NO scavenger cPTIO, the cell permeant cGMP analogue 8-Br-cGMP and the soluble GC inhibitor ODQ (Figs 3b, 5).

In the case of AOX1a, we observed that NO-dependent transcription remained unaffected by the inhibition of soluble GCs and the gene was not induced by the cell-permeant cGMP analogue. It is thus concluded that cGMP has no direct role in the transcriptional regulation of AOX1a (Fig. 3b). The response to NO shows a distinct transient with transcripts accumulating above control levels only 3 h after SNP application.

The responses of GPX and ACS2 are similar in that they are NO dependent but cGMP independent; however, in SNP-treated leaf discs the transcript levels stayed higher than the control for the duration of the monitored period of 24 h (Fig. 5a,b). Taken together, two genes that encode proteins with a role in alleviating oxidative stress –AOX1a and GPX– are both induced rapidly and cGMP independently. AOX1a directly prevents the mitochondrial oxidative stress while GPX may be critical for the oxidative damage protection of the chloroplast in particular. The third gene, which is not directly dependent on cGMP, is ACS2. Aminocyclopropancarboxylic acid synthase has a role in ET biosynthesis, and it is known to increase in response to O3 in tobacco (Ogawa et al., 2005; Ederli et al., 2006), Arabidopsis (Rao et al., 2002) and citrus plants (Iglesias et al., 2006). However, when we treated tobacco leaf discs with the NO donor SNP and the soluble GC inhibitor ODQ it was found that NO-induced ET evolution was suppressed (Fig. 7b), suggesting that transcription of ACS2 is not dependent on cGMP but downstream processes (e.g. post-transcriptional processes) involved in ET synthesis may critically depend on cGMP.

By contrast, NO-induced PALa and PR1a transcript accumulation were both cGMP dependent, with the former showing a transient increase after 3 h while the latter showed a delayed response at 24 h (Fig. 5c,d). We report an early O3 activation of PALa, starting after 2 h of fumigation with peaks between 3 and 5 h, and a late activation after 24 h. This early O3-dependent onset is consistent with salicylic acid (SA) accumulation after 5–7 h fumigation (Pasqualini et al., 2002) since in tobacco PAL is a precursor of SA, whereas in Arabidopsis thaliana isochorismate synthase (ICS) is responsible for SA synthesis (Wildermuth et al., 2001). The secondary late activation of PALa at 24 h after the onset of O3 fumigation or at 96 h after challenge with the virulent strain of P. syringae was also observed in Arabidopsis (data not shown). However, the biological role remains unresolved. It is conceivable that while the early induction is responsible for SA synthesis, late induction could be the consequence of necrotic processes (Kangasjarvi et al., 1994). Incidentally, the late and second PALa induction in response to a challenge with avirulent pathogens has been reported previously in Arabidopsis (Zeier et al., 2004), implying that induction of PAL by O3 may be mechanistically similar to the pathogen defence responses. We also noted that PALa induction initiates before the massive cGMP accumulation and it is conceivable that it may be caused by H2O2 (Desikan et al., 1998).

We have also analysed Arabidopsis microarray data of the above genes including isochorismate synthase (ICS1) in response to 1 h and 3 h H2O2 exposure and 6 h O3 exposure (Fig. 8a). In the case of H2O2, the most pronounced response is seen by ACS2 linking H2O2 to the synthesis and responses to ET. The anti-oxidation protecting AOX1A and GPX6 are also induced whereas PAL1 and PR1a are upregulated after 3 h only. Ozone at 6 h causes transcript accumulation of all genes and notably ICS1 responsible for SA synthesis in Arabidopsis. The fact that SA may slightly reduce ICS1 expression (Fig. 8b, inset) suggests a negative feedback regulation of SA production, while the stimulation of GPX6, ACS2, AOX1A, PAL1 and PR1a by SA is indicative for a role in a more sustained response for the protection of organelles as well as ET synthesis and consequent downstream responses. Inspecting the Arabidopsis cGMP-dependent transcriptome (supplementary data (Maathuis, 2006)) we noted that cGMP leads to an increase in ICS transcript levels both at 2 h and 5 h thus making the response similar to that in the tobacco model where we propose that cGMP can cause SA increases through the transcriptional activation of PAL.

Figure 8.

Defence gene induction in Arabidopsis thaliana and tobacco (Nicotiana tabacum). (a) The results are the fold changes (log2) in expression of GPX6 (At4g11600), ACS2 (At4g11280), AOX1A (At3g22370), PAL1 (At2g37040), ICS1 (AT1g74710) and PR1a (At2g14580) in A. thaliana in response to 6 h of O3 (500 nl l1; light tinted bars) (NASCArray, ID O3–26, n = 3), 1 h of 20 mm H2O2 treatment (dark tinted bars; n = 3) or 3 h of 10 mm H2O2 treatment (closed bars; n = 3) (E-GEOD −3709). Error bars represent standard errors of the mean. (b) Model of O3-induced responses in BelW3 tobacco plants. The solid box indicates genes directly involved in early oxidation protection. The genes that can be induced by cGMP and the compounds for which the synthesis is cGMP-dependent are in an empty box. Dotted arrows are experimentally unconfirmed links. Inset: fold change (log2) in expression of the Arabidopsis GPX6 (1), ACS2 (2), AOX1A (3), PAL1 (4), ICS1 (5) and PR1a (6) in response to 3 h treatment with 10 µm salicylic acid (SA) (TAIR ATGenExpress, salicylic acid-ME00364; n = 2).

While cGMP is accepted as an important second messenger in higher plants, the search for molecules that can catalyse the synthesis of cGMP from GTP in higher plants is complicated by the fact that blast searches with GC domains from either higher or lower eukaryotes do not yield any positive hits, suggesting that higher plants have evolved unique GCs where only the catalytic centre (Liu et al., 1997) contains any degree of conservation (Ludidi & Gehring, 2003).

We have learned from animal GCs that essentially two distinctly different GC domain architectures exist – that of soluble and that of particulate GCs. The former typically have a highly conserved NO-binding site consisting of an H-NOX domain and play a key role in NO sensing and signal transduction; the latter serve as transmembrane receptors where the GC domain located next to a kinase domain is in the cytosol (Meier et al., 2007). Since we report that NO-dependent induction of PALa and PR1a are inhibited by ODQ (Fig. 5c,d), a selective inhibitor of NO-sensitive soluble GCs, we might expect that the GCs involved are activated by NO and possibly contain an H-NOX domain. Such dual domain enzymes common in animals are also annotated in Chlamydomonas reinhardti but have not been reported in higher plants (Meier et al., 2007). There are, however, proteins in higher plants that contain the catalytic core of the H-NOX domain (Hx{12}Px{14,16}YxSxR) and it remains to be seen if any of them are directly or indirectly associated with NO binding and the generation of cGMP.

Accession number

Sequence data from this article can be found in the NCBI/GenBank data libraries under the following accession numbers: S71335 (AOX1a), AJ005002 (ACS2), AB008199 (PALa), AB041518 (GPX), X06361 (PR1a) and AF15640 (actin).

Acknowledgements

This research was supported by a grant to S.P. from MIUR (National Project, PRIN 2005, 2005075991_004). S.M. and C.G. are supported by the South African National Research foundation.

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