Depletion of the stratospheric ozone layer results in increased levels of the sun’s ultraviolet-B (UV-B) radiation (280–315 nm) at the Earth’s surface. This influx of short-wave photons with high energy implies serious effects for all living organisms. For instance, UV-B can damage DNA, proteins and membrane lipids, and induce defence reactions. Plants, being sessile and photosynthetic organisms, are constantly exposed to this harmful radiation. Numerous UV-B effects have been reported, affecting plants on both the molecular and the ecosystem level (Caldwell et al. 1998). The damage by UV-B irradiation can be the result of both direct action on the cellular components and indirect action through the induction of formation of chemical adducts such as reactive oxygen species (ROS; Brosché & Strid 2003; Barta et al. 2004). To resist the damaging UV-B radiation, plants have developed mechanisms of protection and repair that have been extensively studied over the years, such as accumulation of UV-B-absorbing compounds (Harborne & Williams 2000) and repair of UV-B-induced DNA damage (Sancar 2003). At the molecular level, natural and relatively low doses of UV-B radiation result in significant changes in gene expression (Broschéet al. 2002; Casati & Walbot 2003; Ulm et al. 2004). The diversity of the metabolic and signalling pathways affected by UV-B is evidence for their overlapping and close interactions. ROS and mitogen-activated protein kinases (MAPKs) could act as convergence points in these networks.
Different kinds of ROS were detected in plant tissues as a result of UV-B stress (Hideg & Vass 1996; Barta et al. 2004). In the context of the present work, we examined the role of superoxide anion O2• − as a signalling molecule during UV-B stress. Superoxide anions are short-lived radicals that readily undergo either spontaneous or enzymatic dismutation yielding hydrogen peroxide (H2O2). Therefore, at least partially, the detectable H2O2 has O2• − as the primary source, and the effects observed as the result of H2O2 production can be due to O2• − dependent signalling.
The enzyme NADPH oxidase is one of the potential sources of H2O2 in plants. The involvement of H2O2 and NADPH oxidase in UV-B signalling was suggested by both pharmacological studies (A.-H.-Mackerness et al. 2001) as well as direct measurements of NADPH-oxidase activity (Rao, Paliyath & Ormrod 1996) and NADPH-oxidase mRNA transcript levels (Casati & Walbot 2003). Genetically controlled production of O2• − through NADPH oxidase induction has been implicated as a signal in a wide range of biotic and abiotic stress responses, as well as in the regulation of cell expansion and plant development (Mittler 2002; Foreman et al. 2003). An NADPH oxidase complex homologous to that of activated mammalian phagocytes was suggested to be a likely source of O2• − and H2O2, and the apoplastic oxidative burst in plants (Torres et al. 1998). Ten NADPH oxidase genes were identified in Arabidopsis, of which two (AtRBOHD and AtRBOHF) are expressed in leaves. Should ROS act as a second messenger in signal transduction pathways, it would be able to transfer the perceived information into a change in gene expression. This could partially be achieved by interaction with MAPK cascades.
MAPK signalling pathways were reported to be actively involved in transducing oxidative signalling (Kovtun et al. 2000). Several reports have demonstrated the activation of MAPKs by H2O2 (Desikan et al. 1999; Grant, Yun and Loake 2000) in Arabidopsis. H2O2 activates AtMPK3 and AtMPK6 via Arabidopsis NPK1-related protein kinase 1 (ANP1; Kovtun et al. 2000) and strongly induces expression of nucleotide diphosphate kinase NDPK2 gene in Arabidopsis (Moon et al. 2003). Involvement of MAPKs in UV-B signalling and the convergence of different signalling pathways at the level of MAPKs were demonstrated by Holley et al. (2003) in suspension cell culture of the wild tomato species Lycopersicon peruvianum. Two highly homologous MAPKs, LeMPK1 and LeMPK2, were found to be activated in response to different stresses, including UV-B radiation, while an additional MAPK, LeMPK3, was activated by UV-B radiation only. Irradiation of evacuolated parsley protoplasts with UV stimulated a change in the phosphorylation pattern within seconds (Harter et al. 1994). A pharmacological approach was used by Christie & Jenkins (1996) to demonstrate the involvement of protein phosphorylation in regulation of CHS and PAL gene expression after UV-B and UV-A/blue irradiation. Addition of serine/threonine protein kinase inhibitors to Arabidopsis cell suspension cultures resulted in decreased CHS gene expression. At the same time, it was shown that protein phosphatase inhibitors lead to the opposite result.
Phosphatases are responsible for dephosphorylation of components of MAPK cascades and regulation of the magnitude and the duration of their activation and thereby the signal. Another way of modulation of MAPK activity is by direct interaction of phosphatases with H2O2 (Wu, Kwon & Rhee 1998). Oxidation of receptor-directed protein tyrosine phosphatases by ROS is one of the mechanisms of UV-B signal transduction in animal cells (Gross et al. 1999).
The first direct evidence of involvement of MKPs in genotoxic stress relief was presented by Ulm et al. (2001). The conditional mutant mkp1 was hypersensitive to UV-C and methyl methansulphonate, and insensitive to other abiotic stresses, such as osmotic stress or ROS (paraquat and H2O2). The disrupted gene appeared to be an Arabidopsis homologue of mitogen-activated protein kinase phosphatase (AtMKP1). It is interesting to note that MKP1 interacts with MPK3, 4 and 6 as was shown by using a yeast two-hybrid assay (Ulm et al. 2002).
Sequencing of the Arabidopsis genome revealed the existence of 20 genes encoding MAPKs. The number of known MKPs is five at the most (Ulm et al. 2002). The disproportion in the numbers of MAPKs and MKPs suggests the involvement of one MKP in regulation of several MAPKs. In this way, MKPs presumably can serve as integration points for several different signals.
The evidence found in the literature prompted us to study genetically the involvement of MKP1 and NADPH oxidase in UV-B signal transduction. In the present paper, we examine whether the Arabidopsis thaliana homologues of the human respiratory burst oxidase, and a homologue of MAPK phosphatase MKP1, are involved in UV-B signalling. We used the atrbohF, atrbohD and atrbohDF mutants to address the role of NADPH oxidase and the mkp1 mutant and its complemented lines to address the role of the MKP1. We characterize the effects of UV-B on regulation of expression of UV-B molecular markers in these mutants and in the corresponding wild-type plants. In addition, we investigate the morphologic phenotypes and the oxidative status of the plants as the consequence of exposure to UV-B radiation.