New Aspect of H₂O₂ Signaling

Abstract

Plant defense is based on a complex response triggered by unfavorable external impacts. The redox state of the cells and its temporal alteration, the oxidative burst, is an important regulatory element of this defense response. Data collected during the last years have caused us to change the previous, strongly simplified theory on signaling which had been based on a speculative, rather sequential mechanism. In the framework of signal transduction, H₂O₂ signaling pathway(s) is/are only a special part of signal transduction but interacting with other pathways it/they influence the whole transducting system in several points. Our results show that in complexity and in basic regulatory mechanisms (transients, oscillation, tuning, signaling pattern) H₂O₂ signaling is comparable with other pathways, of which we have more detailed cognition, and our present knowledge makes developing a new theory on this aspect necessary.

Introduction

Plants have developed advanced response to struggle with adverse biotic and abiotic impacts of their environment. One component of this complex defense response is the oxidative burst, production of “excess” reactive oxygen species (ROSs)^1,2 leading to the expression of defense genes³ and characteristic changes of metabolism.⁴ Because of its relative long lifetime and medium reactivity, H₂O₂ plays a central role in the defense mechanism:⁴ as a second messenger, H₂O₂ acts directly in the signal transduction system while as a potent oxidative factor it has a particular effect on the redox state of the cell and modifies several outcomes of regulation and metabolism indirectly.²

In case of general developmental^5,6 and physiological⁷ processes and defense responses called forth by different pathogens or stresses, there is a number of H₂O₂ sources: e.g., superoxide dismutase, cell wall bounded peroxidases, photosynthesis, disordered function of mitochondria,^2,4,8 probably with different timing and regulation. In addition, H₂O₂ generation byplays occur because of the cross-talk of other signaling pathways.^4,9 Our results also point to the fact that the previously described one or two H₂O₂ peaks^1,10,11 after triggering the plant cells are only part of the whole oxidative response manifested through H₂O₂ production.¹² A potential reason for the contradictory results is the different manner of measurements, namely, authors established the H₂O₂ generation activity from the supernatant (extracellular space)^9,12,13 or the cells^5,12,14 of the used model. Similarly, there are obscurities in the H₂O₂ quantification, inasmuch as, by implication, authors accept the measured values as general rates; however, there are some indications that at least in some cases strong polarization exists in H₂O₂ formation within the cell.^{5,6,12,15–17} The produced H₂O₂ acts in the framework of a multi-component redox system of the plant cell² and the scavenging system effectively modulates the influence of oxidative factors.^2,4 Glutathion,¹⁷ ascorbate^19,20 and other enzymatic and nonenzymatic reductive systems^2,4,9 together operate with H₂O₂ and other ROS species^{2,4,9,17–20} to maintain the redox state and redox sensing regulates metabolism on the basis of the cumulative effect of the individual redox components.^2,4

In their paper, Trewavas and Malhó²¹ pointed out the complexity of Ca²⁺ signaling, its multiple-influential feature and its connections with other transduction pathways. They emphasized that spatial and timing characteristics of “unit” signals act in concert to create the actual Ca²⁺ fingerprint. In 2002, Trewavas²² went one step further when he summarized in his excellent work the characterization of regulation, thereby giving a quintessential description of it.

In this addendum, we discuss our previous results focusing on the regulatory characteristics of H₂O₂ measured and suggest the intrinsic regulatory characters of it.

Timing of H₂O₂ Formation

Similarly to other second messengers (e.g., cAMP,^23,24 Ca^{2+ 21}), H₂O₂ shows significant changes in its concentration after various stress situations. We have detected a number of H₂O₂ transients in fungal elicitor challenged Rubia tinctorum suspension cultures. Most of the authors observed alterations in H₂O₂ levels, one or two maxima, during the first hour of elicitor treatments or pathogen-host interactions.^3,8,10,11 Only some papers implied three or more transients.^12,13 Our results show that there is a rapid but not short H₂O₂ accumulation and an evident oscillation is detected not ending even at 48 hours after treatments. However, time-points, durations and amplitudes of the transients induced by the fungal cell wall hydrolysate, jasmonic acid (JA) and salicylic acid (SA) treatments were different during the prolonged challenge. Figure 1A shows the summarized, log scaled data of treatment effects on H₂O₂ level measured in the cells up to 48 hours after exposition to elicitors. Vitality of cells decreased during the experiments but up to 48 hours it was not significant. Changes of H₂O₂ level in the supernatant were obvious but the fine and quick alterations were not detectable. These facts point to the importance of data collecting methods and some uncertainty regarding the earlier published final conclusions.

Figure 1.

H₂O₂ accumulation in elicited Rubia tinctorum suspension culture cells. (A) Timing of H₂O₂ formation in untreated and elicited cultures. Mock (Mock), Botrytis cinerea fungal cell wall hydrolysate (Bot), jasmonic acid (JA), salicylic acid (SA). The 0.1 value of the log scale indicate 0 minuntes, other time-points are 2, 4, 6, 8, 10, 20, 30, 40, 50, 60, 120, 240, 480, 720, 1140, 2160, 2880 minutes. (B) Ce perhydroxids detected after 30 min fungal elicitor treatment. (C) Ce precipitates in the cell wall after 8 hours fungal elicitor treatment. v, vacuole; t, tonoplast; cw, cell wall; m, mitochondrion; Bar: 200 nm.

Tuning System

The observed oscillation of H₂O₂ level shows interesting minimal values between the maxima, which are significantly lower than the start level. These transient minima are general features of transduction systems and constitute the lifeblood of signal transduction regulation. ^21,24,25 The accelerated production and “normal” metabolism of the signaling compound do not create such a profile except when there is only a temporary balance between the enhanced production and increased metabolic activity acting in concern but being out of mesh. Oscillation of H₂O₂ level in a wide range between maximal and minimal values is a result of the momentary rate of H₂O₂ generating processes and scavenging systems of the cell. In this way, apart from oxidative burst events there are also antioxidant dominancy periods in the redox state. The origin of these maxima and minima is rather poorly understood because of the lack of exact timing observations on the different H₂O₂ formation and scavenging elements. However, according to Trewavas' signaling system description, the observed features suggest a strictly fine-tuned mechanism in the case of H₂O₂ signaling also.

Location of H₂O₂ Generation

Fluorescence microscopic and cytochemical (Ce and DAB) investigations revealed topography of stress or pathogen attack caused H₂O₂ production in some model systems.^3,13,15–17 H₂O₂ accumulation also occurred in spots during certain developmental processes (fiber secondary wall differentiation, lignification of xylem^5,6). In cells after osmotic stress, the main H₂O₂ generation place was the tonoplast but to some extent H₂O₂ production was cytoplasmic endosomes.²⁶ In our elicitor treated R. tinctorum cells, H₂O₂ formation appeared locally in the plasma membrane and cytoplasm at short-term (30 minute) sampling while later (one to eight hours) Ce perhydroxids were deposited mainly in the cell wall (Fig. 1B and C, respectively). The appearance of precipitate also differed. Cytoplasmic and plasma membrane related precipitates were rough and more or less vesicular or seemed to be of vesicular origin but cytochemical reaction resulted deposits were finely dispersed in the cell wall. The observed local feature of diaminobenzidine or Ce precipitates (or fluorescence signal) is reasonable and regular after pathogen attacks^15–17,27 but in case of elicitor induced defense responses of suspension culture cells the triggering signals were direction independent. Interestingly, H₂O₂ generation seemed to be also a local event in these cells, affecting only a restricted cell area: apart from the visible electron density gradient, it was also proved by the sharp decrease of Ce content of the nearby cell wall regions detected by electron energy loss spectroscopy (EELS).

Conclusions

It is obvious from the above-mentioned facts that H₂O₂ utilizing metabolism and signaling pathways are multifunctional and complex systems. H₂O₂ formation and metabolism as well as their regulation comprise universal regulatory elements (feedback and/or feed-forward loops, amplification, transients, fine-tuning, oscillation, etc) and in several points there are cross-talk possibilities between the H₂O₂ pathway(s) and other signaling routes. Its/their coexistence and cooperation with various pathways does/do not exclude triggering or strong moderation of other transduction pathways (e.g., lipid, Ca²⁺, MAPK kinase signaling refs. ^{1, 2, 24} and ^28–30). The coordinating effect can be manifested through influence on the redox state and may regulate processes in a cell, organ, organism or even between organisms (pathogenic or symbiotic partner and host interaction).¹³

The timing, tuning and localization features of H₂O₂ signaling elements point to the multi-source H₂O₂ total amount and the multi-element scavenging system interacting in a harmonized manner. An important principle is that signaling events are not only sequential even in the framework of one signaling pathway.^2,4,22 The fine-tuning mechanism contains at least a few elements from the generation and scavenging side also. These elements and their regulation events are highly coordinated. H₂O₂ generation and scavenging system interaction and their regulation in cross-talk with other signal transduction pathways create a signal specific pattern resulting in a characteristic outcome after triggering. H₂O₂ signaling apart from other signaling pathways is also related to H₂O₂ utilizing metabolic processes (e.g., lignification, cell wall protein cross-link formation),²⁸ which influence the signal amount. The relatively low average H₂O₂ level in the living systems queried its real antimicrobial effect¹⁵ but taking into consideration the strong spatial limit of H₂O₂ production, the local concentration of H₂O₂ might be high enough to injure microbes.

Addendum to: Bóka K, Orbán N, Kristóf Z. Dynamics and Localization of H2O2 Production in Elicited Plant Cells. Protoplasma. 2007;230:89–97. doi: 10.1007/s00709-006-0225-8.