Molecular marker expression in NADPH oxidase mutants
ROS, in particular superoxide and H2O2, are considered to be important signalling molecules in both animal and plant cells. The involvement of ROS in UV-B signalling has been studied using pharmacological approaches (A.-H.-Mackerness et al. 2001). It was argued that O2• − and H2O2 act as second messengers in up-regulation of PR and defensin (PDF1.2) genes and down-regulation of photosynthetic genes (LHCB). The sources for the ROS involved in regulation of different molecular markers were not the same, according to the same study. PDF1.2 expression was probably regulated through the activity of peroxidases and involve O2• − directly, whereas regulation of PR-1 and LHCB transcription was suggested to be mediated by ROS of clearly distinct origin, namely H2O2 derived from O2• −.
Plasma membrane NADPH oxidase is an important source of O2• − and H2O2. Knockout experiments demonstrated that the AtRBOHF and AtRBOHD genes (encoding two out of 10 NADPH oxidase genes) were required for H2O2 generation during bacterial and pathogen challenge in Arabidopsis (Torres, Dangl & Jones 2002). Using a reverse genetics approach and a number of molecular markers (CHS, PYROA and MEB5.2 in addition to those mentioned earlier), we here investigated the involvement of NADPH oxidase in ROS-mediated UV-B signalling. Compared with the high UV-B levels used by A.-H.-Mackerness et al. (2001), the levels of UV irradiation used in our experiments were comparable with ambient levels. At such low fluence rates of UV irradiation, we did not observe any significant expression of PDF1.2 and PR-1 genes, either in the atrboh mutants or wild type.
As can be seen from Fig. 2d and e, there are no significant differences in regulation of the LHCB1*3 and PR-5 genes by UV-B between Col-0, and atrbohD and atrbohF mutants. This means that low-level UV-B regulation of these genes is not accomplished through NADPH oxidase. Either this regulation is totally ROS-independent, or another distinct pool of ROS not produced by NADPH oxidase participates in the regulation. Compared with Surplus et al. (1998) and A.-H.-Mackerness et al. (2001), the conclusions at first seem contrasting. However, our view that LHCB1*3 gene expression is not influenced by specific H2O2 production through NADPH oxidase is based on short-term low-level experiments, whereas the data of A.-H.-Mackerness et al. (2001) were produced in experiments carried out for extended periods of time (4–16-fold longer than ours) at a 2.6-fold higher UV-B level. The long exposure time at the higher level most likely resulted in ROS non-specifically produced through interaction of the UV-B with cellular oxygen-containing constituents, which in turn, as a secondary effect, depressed mRNA levels for LHCB. For a discussion on specific and non-specific regulation of genes during UV-B exposure, compare with Brosché & Strid (2003).
The levels of CHS mRNA transcripts in Col-0 and both atrboh single mutants were the same after 3 h of UV-B exposure, whereas the double mutant atrbohDF showed a CHS expression half of that in Col-0 and the single mutants (Fig. 2a). Our results suggest a quantitative role of ROS produced by NADPH oxidase in the UV-B regulation of CHS, fine-tuning the expression level of this gene. However, qualitatively, CHS expression can be induced in the absence of functional NADPH oxidase. Jenkins et al. (2001) have previously argued that ROS are not involved in regulation of CHS gene expression in Arabidopsis. The basis for this is primarily that addition of H2O2 itself to Arabidopsis cell cultures or the inhibition of catalase during normal metabolism in these cultures with the inhibitor 3-amino-1,2,4-triazole did not induce CHS expression. These data do not necessarily contradict our results because we have shown that the existence of a functional NADPH oxidase is not a prerequisite for CHS expression but instead enhances the accumulation of CHS mRNA, that is, H2O2 may not be the trigger of UV-B-induced CHS transcription but functions to amplify the expression. In addition, the ROS scavengers pyrrolidine carbamate and N-acetylcysteine, added to the Arabidopsis cell cultures, did not alter UV-dependent CHS expression (Jenkins et al. 2001), which would be anticipated keeping our data in mind. There are a number of plausible explanations for this discrepancy. One evident reason could be that the uptake of these pharmacological substances was small in the cell cultures, or that they did not scavenge the particular H2O2 pool that is responsible for UV-signalling. However, the most evident explanation would instead be that the difference in tissue differentiation state, cell cultures (Jenkins et al. 2001) and leaves of plants (our study), respectively, influenced the regulation of the CHS gene. Indeed, CHS regulation is known to be strongly influenced by, for instance, the developmental stage of an Arabidopsis plant. Phytochrome induces CHS induction in very young seedlings only, whereas UV-B and blue light regulation occurs also in mature plants (Jenkins et al. 2001). Therefore, we believe that using whole Arabidopsis mutant plants in our studies mimics a situation that is more closely resembling the physiological state than using cell cultures.
For the MEB5.2 gene, the expression pattern was very similar to the one for CHS (Fig. 2c), and the same points of discussion also hold for this gene.
The expression of the PYROA gene decreased in all three mutants (single mutants atrbohD, atrbohF and double mutant atrbohDF) compared with Col-0 (Fig. 2b). The expression pattern of this gene, as the result of UV-B exposure, clearly indicates the involvement of NADPH oxidase-dependent regulation of the levels of PYROA expression, although, again, substantial PYROA expression is found also in the absence of functional NADPH oxidase. However, for regulation of this molecular marker, the redundancy of the two mutant AtRBOH genes was not as clear as for the CHS and MEB5.2 markers.
Molecular marker expression in the mkp1 mutant
It has been shown that MAPK cascades can be activated by both UV-B irradiation and H2O2 (Harter et al. 1994; Christie & Jenkins 1996; Kovtun et al. 2000). Because H2O2 generation occurs in response to a number of different stresses and because certain defence responses to such stresses are shared, MAPK cascades could serve as a branching point in a complex signalling network. Given the importance of MAPK signalling cascades, it is clear that their activity must be tightly regulated. Evidence for the involvement of MKP1 in the integration and fine-tuning of plant responses to osmotic and genotoxic stresses was obtained by Ulm et al. (2002). In the present study, we tested whether MKP1 is also involved in regulation of UV-B-dependent gene expression of molecular markers.
The responses of different molecular markers to supplementary UV-B in the mkp1 mutant, in the Ws-4 wild type, and in lines 6 and 10 (mkp1 complemented by ectopic expression of MKP1) are presented in Fig. 3. Under our experimental conditions, we did not observe any differences between these lines with respect to the expression patterns for LHCB1*3 and CHS. These results show that regulation of CHS and LHCB1*3 is independent of MKP1 at low UV-B levels.
A slight decrease in PYROA and MEB5.2 mRNA transcript levels in the complemented lines compared with wild type and knockout mutant was observed. However, the most significant differences were found in expression of PDF1.2, PR-1 and PR-5. We observed constitutive expression of these markers in mkp1, lines 6 and 10, but no expression in the wild-type strain. The expression was not affected by UV-B at the low fluence rates used in this study.
The constitutive expression of PR-1, PR-5 and PDF1.2 genes in mkp1 suggests the involvement of MKP1 in repression of the expression of these genes and the release of transcription at higher phosphorylation levels of MAPKs. Surprisingly, we observed the same expression pattern of these genes in both the knockout mutant and the complemented lines overexpressing AtMKP1, that is, both knockout and overexpression of MKP1 resulted in constitutive activation of PR and PDF genes. The observed action of the MKP1 is to some extent similar to a ‘double jeopardy’ scenario described by Samuel & Ellis (2002). They demonstrated that both overexpression and suppression of a redox-activated MAPK (SIPK) in tobacco plants gave rise to ozone sensitivity. Furthermore, transgenes overexpressing a non-phosphorylatable version of SIPK were not sensitive. Therefore, our data suggest that a balanced phosphorylation level of MAPKs is necessary for tight regulation of PDF1.2, PR-1 and PR-5 transcription, whereas both highly phosphorylated MAPKs (mkp1 mutant) and non-phosphorylated MAPKs (MKP1 overexpressing lines 6 and 10) result in derepression of expression of these genes (Fig. 5).
Figure 5. A scheme showing the suggested relationship between genotype, phosphorylation level and the phenotype with respect to the transcriptional control on PR and PDF genes. MAP, mitogen-activated protein.
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Samuel & Ellis (2002) also performed an analysis of the MAPK activation profiles that revealed an interplay between SIPK and wound-induced protein kinase (WIPK). Prolonged activation of SIPK was seen in the SIPK-overexpressing genotype, without WIPK activation, whereas strong activation of WIPK was observed in the SIPK-suppressed genotype. Therefore, an extension of our study would be a detailed study of the protein levels and the phosphrylation levels of each different Arabidopsis MAPK ortholog in the wild type, the mkp1 mutant and the two MKP1 overexpressing mutant lines 6 and 10. Such a large effort would decipher the gene expression pattern observed by us and reveal important mechanisms behind gene expression regulation by phosphorylated proteins.
Taken together, our results show the involvement of MKP1 in more general stress responses similar to those induced by pathogen attack as judged by the results on PR-1, PR-5 and PDF1.2 expression, and the absence of regulation (or very insignificant regulation) of more UV-B-specific molecular markers (CHS, PYROA and MEB5.2; Brosché & Strid 2003).
H2O2 accumulation and UV-B-dependent phenotypes
Staining for H2O2 in atrboh mutants and Col-0 wild type showed an increase in H2O2 accumulation in the wild type as a result of UV-B (Fig. 4a and Table 2). None of the three atrboh mutants showed any evident differences in H2O2 levels in leaves between UV-A and UV-A+B-exposed plants. The absolute H2O2 levels were comparable in all strains tested in the absence of UV-B. These results confirm the suggestion that NADPH oxidase contributes to H2O2 accumulation under UV-B exposure.
The phenotype studies of atrboh mutants and the Col-0 background ecotype clearly revealed the involvement of the AtRBOHD and AtRBOHF genes in UV-B responses. All tested atrboh mutants showed statistically significant decreases in fresh and dry weights, leaf area and rosette diameter as the result of UV-B exposure, whereas the same morphological traits remained unaffected in Col-0 (Table 3). These results strongly suggest NADPH oxidase as an important enzyme for protection against UV-B stress in plants.
Knockout of the MKP1 gene also resulted in growth retardation. Statistically significant decreases in leaf area and rosette size (Table 4) were observed in the mkp1 mutant but neither in the complemented line, nor in the wild type. Furthermore, we observed such an additional phenotypical response in young seedlings of the mkp1 mutant in response to UV-B that was not observed in mature plants. One-week-old seedlings exposed to very low fluence rate (UVBE,300 = 0.07 W m−2) revealed visible signs of bleaching (Fig. 4a). This implies that the involvement of MKP1 in stress relief is dependent on the developmental stage of the plants.