cGMP in ozone and NO dependent responses

Abstract

We have recently reported that ozone (O₃) can inhibit mitochondrial respiration and induce activation of the alternative oxidase (AOX) pathway and in particular AOX1a in tobacco. While O₃ causes mitochondrial H₂O₂, early leaf nitric oxide (NO) as well as transient ethylene (ET) accumulation, the levels of jasmonic acid and 12-oxo-phytodienoic acid remained unchanged. It was shown that both, NO and ET dependent pathways can induce AOX1a transcription by O₃. AOX plays a role in reducing reactive oxygen species (ROS) which in turn are linked to biotic and abiotic plant stresses, much like the second messengers guanosine 3′, 5′-cyclic monophosphate (cGMP). The goal is to unravel specific cGMP signatures and induction pathways downstream from O₃ and NO, including transcription of AOX1a. Here we propose that some late (>3 h) responses to NO, e.g., the accumulation of phenylalanine lyase (PAL) transcripts, are critically cGMP dependent, while the early (<2 h) responses, including AOX1a induction are not.

A Role for Aox in Plant Stress Responses

It is conceivable that a function of the AOX pathway is to reduce the formation of ROS whereby increased activity of AOX could relieve the mitochondrial cytochrome pathway and prevent harmful hyper-reduction, reducing the formation of damaging radicals.^1,2 ROS generation is also involved in biotic and abiotic stresses responses in plants. While AOX abundance and AOX activity are low in unstressed plants, alternative respiration is enhanced after various developmental or environmental stimuli, e.g., under conditions such as wounding and plant disease¹ thus implicating AOX in stress alleviation. The nuclear gene that encodes AOX in tobacco (Nicotiana tabacum; AOX1) is rapidly induced in cultured cells when the cytochrome pathway is specifically inhibited by antimycin A.³

Addition of salicylic acid (SA) to tobacco cell suspensions or intact leaves also induces gene the expression of AOX associated genes⁴ further linking AOX to plant stress responses. More recently NO has been identified as a key molecule that interacts with ROS in a number of ways, either as a crucial partner in determining cell fate or in signaling in response to developmental and stress-related conditions. NO appears to be involved in controlling or modulating various aspects of plant pathogen resistance, growth, development, and senescence, as well as stomatal movement.⁵

In NO treated Arabidopsis cell cultures expression is strongly induced, resulting in increased respiration through the alternative pathway.⁶ Furthermore, AOX1a expression is affected in the Arabidopsis ctr-1 mutant thus pointing to ethylene (ET) dependence.⁷

Different signaling molecules have been found to be involved in AOX induction, but their interactions during environmental stresses remain unresolved. Analyses of Arabidopsis mutants have produced a substantial body of information implicating SA, NO, ROS, jasmonic acid (JA), and ET as major endogenous signals in defense responses. However, much less information is available for other plant species. Our previous studies demonstrated that O₃ treatment induces SA and H₂O₂ accumulation in the O₃-sensitive tobacco BelW3 cultivar,⁸ and the activation of a cell death program⁹ as well as the induction of the AOX pathway by O₃ in tobacco.^10,11

Early (1–1.5 h) production of NO in fumigated leaves was detected with a NO-specific fluorescence concomitant with H₂O₂ accumulation in mitochondria and ET evolution was recorded during O₃ exposure. In summary, while it is established that NO is the main upstream signaling molecule involved in AOX1a expression, which is coordinately activated by ET, the exact nature of the downstream signaling pathway remains to be elucidated.

Linking cGMP to Stress Responses

It is becoming increasingly clear that the cyclic nucleotide cGMP has an important role in many diverse biological processes^12–14 including responses to both abiotic and biotic stresses,^15,16 and NO dependent signaling^5,17,18 as well as the regulation of transcription.¹⁹

It is interesting to note, that the time for cGMP increases does vary considerably from 15 min in response to NaCl and osmotica¹⁵ to 2 h after application of an NO donor,¹⁶ and 5 h in response to a gravitropic stimulus.²⁰ This can be taken as an indication for the complex and differentiated role of cGMP as signaling molecule. Our current experiments suggest that in Tobacco significant rises in cGMP levels occur only >3 h after NO fumigation. This would suggest that cGMP has no role in NO dependent AOX1a induction. Inspecting microarray data in the public domain we noted that in Arabidopsis thaliana AOX1a (At3g22370) is not significantly induced by cGMP neither after 2 nor 5 h¹⁹ and this observation concurs with our own findings²¹ whereas within the same timeframe PAL is (Fig. 1).

Figure 1.

Time-course of NO, H₂O₂, ET, SA and cGMP accumulation after fumigation with O₃ in BelW3 tobacco. Gray arrows indicate established relationships between the signalling molecules, white arrows are proposed hypothetical links. The small arrows indicate the time of first significant accumulation of the respective molecules after fumigation with O₃. The response to O₃ includes a cascade of signal molecules leading to the expression of early and late defense genes. In this model early and NO responsive gene expression (AOX1a) does not depend on cGMP, while the late transcriptional response (e.g., of PAL) is at least in parts critically dependent on cGMP.

Outlook

We are currently performing an extended series of experiments that will resolve the temporal, spatial and stimulus specific induction patterns of O₃ and NO dependent cGMP transients and link them to the cGMP dependent transcriptome and proteome. Based on both the available microarray data and our preliminary results, we hypothesise, that early (<3 hours) O3 and NO responsive gene expression including AOX1a does not depend on cGMP, while the late transcriptional response (>3 hours) is at least in parts critically dependent on cGMP. An example for a late response would be e.g., PAL (Fig. 1). In addition, we foresee that cGMP also has an important late role in responding to homeostatic disturbances caused by biotic and abiotic stresses. Such a role might require prolonged elevation of cytosolic cGMP levels—rather than short transients—which in turn can activate cGMP dependent ion channels. A function of cGMP in long-term ionic regulation after stress is also entirely compatible with the finding of cGMP dependent transcriptional activation of ion channel encoding genes.¹⁹

Addendum to: Ederli L, Morettini R, Borgogni A, Westernack C, Miersch O, Reale L, Ferranti F, Tosti N, Pasqualini S. Interaction between nitric oxide and ethylene in the induction of alternative oxidase in ozone-treated Tobacco plants. Plant Physiol. 2006;142:595–608.