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Plant responses to ultraviolet B (UV-B) radiation (280–315 nm) are numerous, resulting in rapid and typically permanent alteration to numerous aspects of plant form, physiology and biochemistry. Such responses include inhibition of growth (Liu et al., 1995; Searles et al., 2001), changes in biomass allocation (McCloud & Berenbaum, 1999), induction of UV-absorbing secondary metabolism (e.g. foliar pigmentation; Krizek et al., 1998), knock-on effects influencing various ecological processes (e.g. insect herbivory and litter decomposition) and effects on plant pathogens (Newsham et al., 1997; Paul et al., 2005; Foggo et al., 2007). While such phenomena have been previously studied with reference to concerns over stratospheric ozone depletion (Ballare et al., 2001; Flint et al., 2003), a recent refocus towards the effects of environmentally relevant UV-B doses has provided an opportunity to investigate the mechanistic basis for such well-defined plant responses.
Plant responses to light are usually the culmination of both initial perception of specific spectral qualities and complex signalling cascades, with observed effects varying according to fluence rate, irradiance or dose, and wavelength of exposure. Responses to red/far-red light, mediated by phytochromes and blue/UV-A light, mediated by cryptochromes and phototropins (Smith, 1995; Briggs & Olney, 2001; Chen et al., 2004) are relatively well defined. By contrast, initial response events specific to UV-B wavelengths, possibly mediated by an as yet unidentified UV-B photoreceptor are still not completely understood, although genetic mutants are now providing reliable tools for dissecting this process. As many macromolecules, proteins and other cellular components are targets for UV-B, there are numerous possibilities regarding the mechanisms by which plants mediate responses specific to UV-B. DNA is known to readily absorb UV-B wavelengths, and UV-B has been shown to play a causal role in the formation of photoproducts such as cyclobutane pyrimidine dimers (CPDs) (Giordano et al., 2004), which has led to some speculation that DNA could act as a primary receptor for UV-B (Kucera et al., 2003). However, UV-B wavelengths commonly associated with DNA damage are much shorter than those involved in photomorphogenic responses at lower UV-B fluxes (Ensminger, 1993; Frohnmeyer, 1999), shifting the emphasis from high flux, short wavelength, stress-inducing UV-B responses to those mechanisms thought to constitute regulatory roles in photomorphogenesis. Several plant signalling components are thought to play a role in UV-B responses, such as NADPH oxidase-derived reactive oxygen species (ROS) (Kalbina & Strid, 2006), jasmonic acid (Mackerness et al., 1999), nitric oxide (Izaguirre et al., 2007) and mitogen-activated protein kinases (MAPKs) (Holley et al., 2003), although it is likely that such components form part of a general multiple-stress response network, such as those regulating wound and defence-signalling (Stratmann, 2003), and are therefore unlikely to be specific to UV-B.
Ultraviolet B-specific responses have proved more difficult to characterize, and studies have usually focused on components upstream of commonly observed whole-plant responses, such as the increase of phenolic pigmentation and inhibition of hypocotyl elongation (Kim et al., 1998; Jenkins et al., 2001; Suesslin & Frohnmeyer, 2003; Ulm & Nagy, 2005; Jenkins & Brown, 2007). The role of phenolic pigments, such as flavonoids acting as UV-absorbing sun-screens to protect against DNA damage initiated by UV-B, is now well established. For example, Zea mays lines deficient in flavonoid accumulation exhibit increased CPD formation in response to UV-B (Stapleton & Walbot, 1994) and, conversely, induction of UV-B-absorbing compounds shield DNA from damage (Mazza et al., 2000; Rozema et al., 2002). Many upstream components could act as regulators of UV-B response, although it has been demonstrated that several putative mechanisms are responsive to other stimuli in addition to UV-B (Bieza & Lois, 2001; Wade et al., 2001). Transcription of key genes in the flavonoid biosynthesis pathway, such as CHALCONE SYNTHASE (CHS), is retained in both cryptochrome and phytochrome light perception mutants when exposed to UV-B (Wade et al., 2001; Brosche & Strid, 2003; Ulm et al., 2004), indicating that an independent UV-B signalling pathway culminating in flavonoid biosynthesis does exist. In terms of growth inhibition, several UV-B light sensitive (uli) mutants have been isolated that are altered in the UV-B-mediated inhibition of hypocotyl elongation (Suesslin & Frohnmeyer, 2003), although the function of the ULI3 gene remains unknown.
Evidence has now emerged of a pathway mediating transcriptional responses specific to UV-B in Arabidopsis (Arabidopsis thaliana). The transcription factor ELONGATED HYPOCOTYL5 (HY5), a mediator of several photomorphogenic pathways (Osterlund et al., 2000; Chen et al., 2004), is required for UV-B-mediated gene expression (Ulm et al., 2004; Brown et al., 2005; Oravecz et al., 2006; Brown & Jenkins, 2008), and UV-B induction of HY5 is retained in both cry and phy mutants (Ulm et al., 2004). The CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) protein regulates a range of low-fluence UV-B-mediated gene expression responses in addition to flavonoid accumulation and inhibition of hypocotyl elongation (Oravecz et al., 2006). While COP1 and HY5 have established roles in the regulation of signalling events related to both UV-B and nonUV-B stimuli, the recent characterization of the UV RESISTANCE LOCUS8 (UVR8) signalling component has shown UVR8 to act in the UV-B-specific regulation of gene expression (Brown et al., 2005). Microarray analysis showed that in addition to the majority of flavonoid biosynthesis genes, UVR8 regulates transcription of genes related to terpenoid biosynthesis, photolyase activity and photooxidative repair. The uvr8 mutant was also shown to retain CHS induction by both UV-A and far-red illumination, in addition to nonlight stimuli, thus demonstrating the UV-B specificity of UVR8 in the regulation of CHS induction. It was further demonstrated that UVR8 regulates HY5 gene expression specifically in UV-B (Brown et al., 2005), and there is now evidence that there are at least two genetically distinct UV-B signalling pathways that stimulate gene expression in mature Arabidopsis leaf tissue, of which only one pathway requires UVR8, responding to lower UV-B fluences (Brown & Jenkins, 2008). While both the UVR8-dependent and UVR8-independent pathways function in mutants lacking phytochromes, cryptochromes and phototropins, HY5 is only involved in the regulation of the UVR8-dependent pathway. This is in addition to the HY5 HOMOLOG (HYH) transcription factor, which has also been implicated in the UVR8 signalling pathway (Brown & Jenkins, 2008). While little is still known about initial responses involving UVR8, there is now strong evidence that native UVR8 binds to chromatin in vivo, and that UV-B is not required for this interaction (Cloix & Jenkins, 2008). However, UV-B is required to stimulate UVR8 function in the nucleus, in addition to the nuclear accumulation of UVR8, thus leading to the UV-B induction of the HY5 gene (Kaiserli & Jenkins, 2007). Microarray analysis of Arabidopsis uvr8 has shown that UVR8 is involved in the regulation of a wide range of genes (Brown et al., 2005). This suggests that UVR8 regulates a range of UV-B responses, although the regulatory effects of UVR8 are still poorly understood at the whole plant scale.
Ultraviolet B tolerance is likely to be multifactorial with several putative mechanisms existing to buffer plants from the effects of UV still largely unexplored. One such mechanism is endoreduplication, a particular mode of cell cycle where additional rounds of nuclear DNA replication in the absence of mitosis results in endopolyploidy where somatic nuclei contain multiple copies of DNA. The biological significance of endoreduplication is still under active debate (Sugimoto-Shirasu & Roberts, 2003; Cookson et al., 2006) but there is a strong correlation with increased cell size (Melaragno et al., 1993), particularly during hypocotyl elongation (Gendreau et al., 1997) and trichome growth (Folkers et al., 1997). Endoreduplication is also associated with increased tolerance to a range of abiotic factors (Barow & Meister, 2003). Endopolyploidy has been hypothesized as an adaptive response to UV radiation (Vlieghe et al., 2007), possibly via the resultant increased gene copies which could prevent DNA damage. The Arabidopsis mutant uvi4, which displays increased levels of ploidy has shown increased resistance to UV-B when grown at high UV-B fluxes (Hase et al., 2006), although the mechanisms involved remain poorly defined. Characterizing the role of UVR8 at the cellular level would provide greater understanding of how UVR8 may orchestrate whole-plant responses to UV-B, such as inhibition of leaf growth.
Here we show that UVR8 is required for a UV-B-stimulated compensatory increase in epidermal cell size, while reductions in epidermal cell number in response to UV-B are substantially independent of UVR8, thus demonstrating that UVR8 regulates leaf growth through the control of epidermal cell development. We also report that UVR8 is required for normal progression of endocycle in response to UV-B and has a regulatory role in stomatal differentiation. Such an approach provides not only an assessment of the importance of UVR8 in UV-B responses, but provides an opportunity to develop our understanding of how UV-B response is orchestrated at the whole-plant level.