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Keywords:

  • Arabidopsis thaliana;
  • cell development;
  • endopolyploidy;
  • morphogenesis;
  • UV-B;
  • UVR8

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • • 
    Responses specific to ultraviolet B (UV-B) wavelengths are still poorly understood, both in terms of initial signalling and effects on morphogenesis. Arabidopsis thaliana UV RESISTANCE LOCUS8 (UVR8) is the only known UV-B specific signalling component, but the role of UVR8 in leaf morphogenesis is unknown.
  • • 
    The regulatory effects of UVR8 on leaf morphogenesis at a range of supplementary UV-B doses were characterized, revealing both UVR8-dependent and independent responses to UV irradiation.
  • • 
    Inhibition of epidermal cell division in response to UV-B is largely independent of UVR8. However, overall leaf growth under UV-B irradiation in wild-type plants is enhanced compared with a uvr8 mutant because of a UVR8-dependent compensatory increase of cell area in wild-type plants. UVR8 was also required for the regulation of endopolyploidy in response to UV-B, and the uvr8 mutant also has a lower density of stomata than the wild type in the presence of UV-B, indicating that UVR8 has a regulatory role in other developmental events.
  • • 
    Our findings show that, in addition to regulating UV-protective gene expression responses, UVR8 is involved in controlling aspects of leaf growth and morphogenesis. This work extends our understanding of how UV-B response is orchestrated at the whole-plant level.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

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.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant material and growth conditions

Seeds of A. thaliana (L.) Heynh. ecotype Landsberg erecta (Ler) and uvr8–2 (also Ler; described in detail in Brown et al., 2005) were propagated on a growth medium of 4 : 1 peat-based compost (SHL multi-purpose; William Sinclair Horticulture, Knottingley, UK) and horticultural silver sand (Silvaperl; William Sinclair Horticulture) in 51 mm circular acrylic pots (Richard Sankey & Son Ltd, Nottingham, UK). The standard light : dark regime for maximum vegetative stage leaf size was 10 h : 14 h, photosynthetically active radiation (PAR) flux was 300 µmol m−2 s−1, with a constant temperature of 20°C ± 2, RH: 55% ± 2. Plants were grown to a minimum age of 15 d after germination before commencement of supplementary UV-B treatments.

Experimental growth conditions and UV-B exposure conditions

The UV-B treatments for all lines were applied in a group of three controlled environment growth cabinets (Microclima 1750; Snijders Scientific B.V., Tilburg, Netherlands), which contained a series of PAR sources: (20 × Sylvania Luxline Plus, FH024W/T5 840, 550 mm; 10 × Sylvania Luxline Plus, FH054/T5 840, 1150 mm; 6 × Sylvania BriteGro, F58W/T8 2023, 1514 mm; all CEC Technology, Glasgow, UK), delivering a PAR flux of 300 ± 20 µmol m−2 s−1. The light : dark regime (both PAR and UV) was 10 h : 14 h with day : night temperatures of 21 : 18° ± 2°C, RH: 60%. Ultraviolet B was provided by three UV-B tubes (Q-Panel 313; Q-Panel Laboratory Products, Bolton, UK) wrapped in 0.13 mm cellulose diacetate (CA) film (Clarifoil; Courtaulds Ltd, Derby, UK) in order to exclude all wavelengths below 290 nm, with new quantities of pre-aged CA film installed into the growth chambers and rescanned on a routine basis throughout exposure periods. The separation between the UV-B+ and control dose region of each cabinet was maximized by wrapping one end of each supplementary UV-B tube with a commercial-grade UV-opaque polyethylene film (BPI Agri Ltd., Stockton on Tees, UK) and in addition, a sheet of clear polyester (Lee Filters, Andover, UK) was fitted between chamber treatment regions in order to prevent wavelengths below 320 nm reaching the control area of the chamber bench. Where each cabinet was typically used to deliver one of three differing UV doses during an experiment, doses were moved between cabinets on a per experiment basis to avoid any positional/microclimatic bias. All UV treatments were quantified using a double monochromator scanning spectroradiometer (model SR991-v7; Macam Photometrics, Livingston, UK). The UV treatments were determined using the generalized plant action spectrum (Caldwell, 1971), with UV-B doses ranging from below to above local (54°N) maximum ambient UV fluxes, which equated to a UV-B dose range of 0–25 kJ m−2 d−1, equivalent to a fluence rate of 0–5.2 µmol m−2 s−1 UV-B.

Epidermal cell size/number and stomatal assays

At the end of the UV-B treatment period (13 d), the youngest leaf at full expansion from each plant was removed in order to measure adaxial epidermal cell size using the dental rubber impression technique (Weyers & Johansen, 1985; Poole et al., 1996). The procedure involved first covering the leaf surface with dental impression material at the central region of the lamina avoiding the mid-vein (Xantopren VL; Dental Linkline, Leigh, UK) thus creating an imprint of the epidermal surface area. Once the material had set (1–2 min) the leaf was peeled away. Acrylic-based varnish was used to produce a translucent positive replica from the negative rubber impression. Images of the epidermal impressions were taken at ×100 magnification with a light microscope fitted with a CCD digital camera (Spot Insight; Diagnostic Instruments Inc., Sterling Heights, MI, USA), with cell size, cell number per unit area, stomatal density and stomatal index analysed using Image Pro Plus v4.5 (Media Cybernetics Inc., Bethesda, MD, USA). In order to calculate the number of epidermal cells per leaf, final leaf area of the imprint leaves was also determined at the point of harvest.

Quantification of whole-plant leaf area

At the end of the UV-B treatment period plants were defoliated and whole-plant leaf area determined using a LI-COR LI-3000A area meter (Li-Cor Inc., Lincoln, NE, USA).

Endopolyploidy analysis

Comparisons of nuclear DNA content in uvr8-2 and Ler plants were analysed using flow cytometry (FCM). The fifth rosette leaf at 15 dpi (days post leaf initiation) was chopped finely with a razor blade in 500 µl of extraction buffer (Partec, Münster, Germany) and filtered through a 30 µm mesh (Partec). The sample was frozen in liquid N2 and stored at –70°C. Before analysis, frozen samples were left to thaw at room temperature and then 1 ml Cystain UV staining solution (Partec) was added. Endopolyploidy analysis was performed with a PAS II Ploidy analyser (Partec) using an arc-lamp. The instrument was calibrated using 3 µm calibration beads (Ex: 488 nm; Partec) for the alignment of the argon ion laser and trout erythrocytes for the arc-lamp (excitation for UV < 420 nm). Coefficients of variation (CV values) of < 4.0 for the calibration beads (main peaks for both forward scatter and side scatter) and < 2.0 for the trout erythrocytes were considered satisfactory for analysis. A total of 20 000 events were counted in each run at an average speed of 50 events per second. The CV value for most of the peaks obtained was < 4. The left cut-off point was set at 150 and the right cut-off at 999.9. All the data was acquired on a logarithmic amplification (Log3) scale unless otherwise stated. In the analyses presented here, three biological replicates were assessed and the number of counts of each peak was obtained by manual gating.

Data analysis

Leaf area data is shown from four comparable experiments across the dose–response range (5–10 biological replicates per dose per genotype according to particular experiment), with nonlinear regression analyses performed with Prism graphpad version 4 (GraphPad Software, San Diego, CA, USA). Leaf area, cell size and cell number data were all compared using one-way and two-way analysis of variance (results from all anova tests are listed in the Supporting Information, Table S1), where cell size/number data is representative of two experimental repeats, with means of five biological replicates presented per genotype per treatment. Endopolyploidy data are from three comparable experiments, where experimental means were used as replicates (two or three biological replicates per experiment), with χ2 comparisons used for endopolyploidy profiles between genotype/treatments, and one-way analysis of variance for pairwise comparisons of endopolyploidy responses between genotypes at individual endopolyploidy regions (all using SPSS v15.0; SPSS Inc, Chicago, USA).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Regulation of leaf growth morphogenesis by UVR8 in response to UV-B

Inhibition in leaf growth is a commonly observed response to UV-B in numerous plant species (Liu et al., 1995; Searles et al., 2001), but there is no current evidence of the role that UVR8 may play in any inhibition response. The uvr8-2 mutant (Brown et al., 2005) and wild type were grown under a range of supplementary UV-B doses of 0–25 kJ m−2 d−1 for a typical exposure period of 15 d, and assessments of mean whole-rosette leaf area from several experiments showed no consistent differences between wild-type and uvr8-2 plants in the absence of supplementary UV-B (anovagenotype: P > 0.05; data not shown). When exposed to a range of supplementary UV-B doses in the physiological range that plants might be expected to experience in nature, mean whole-rosette leaf area in both uvr8-2 and wild-type plants was significantly smaller across the range of UV-B doses (anovauv-b: P < 0.001; Fig. 1), with uvr8-2 plants and wild-type plants, respectively, 87% and 63% smaller than controls at maximum supplementary UV-B dose (25 kJ m−2 d−1). The uvr8-2 plants were 25–50% smaller than wild-type plants above 10 kJ m−2 d−1 (anovagenotype: P < 0.001; Fig. 1 ). In addition, the uvr8 mutant exhibited gross foliar damage at doses above 10 kJ m−2 d−1 (Fig. 1a). Because of the nonlinear nature of both uvr8-2 and wild-type rosette leaf area dose responses, an inverse sigmoid curve was used to further describe the relationship in both lines according to UV-B dose, with uvr8-2 plants showing greater reductions in leaf area than wild-type at UV-B doses over 6.7 kJ m−2 d−1 (Fig. 1b), with a plateau in both dose responses at 10 kJ m−2 d−1 and above (Fig. 1b). We therefore conclude that the UVR8-dependent signalling pathway is a vital component in both regulating and protecting leaf expansion from the effects of UV-B.

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Figure 1. Effects of ultraviolet B (UV-B) on leaf growth. (a) UV-B sensitivity assay. Arabidopsis thaliana wild-type Ler and uvr8 mutant plants were grown under a regime of 5–15 kJ m−2 d−1 of supplementary UV-B and 300 ± 20 µmol m−2 s−1 of white light for 15 d. Untreated control plants (UV-B–) were grown under identical conditions without UV-B. (b) UV-B effects on whole plant leaf area of wild-type and uvr8 mutant plants represented as per cent of zero UV-B control plants. Ler (closed circles) and uvr8-2 (open circles) plants were grown under a regime of 5–25 kJ m−2 d−1 of supplementary UV-B and 300 µmol m−2 s−1 of white light for a typical exposure period of 15 d. Untreated control plants were grown under identical conditions without UV-B. Nonlinear regressions are expressed as a LogEC50 relationship (Y = BOTTOM + (TOP-BOTTOM)/(1 + 10(X – logEC50)). Data presented are an average of 5–10 biological replicates according to experiment ± SE. Leaf area (% of control) was significantly less in the uvr8 mutant (two-way anova P < 0.001) than wild-type plants, and both genotypes were significantly affected by UV-B dose (P < 0.001). All supplementary UV-B doses are weighted according to Caldwell's generalized plant action spectrum (Caldwell, 1971).

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Regulation of compensatory epidermal cell growth by UVR8 in response to UV-B

Previous studies of the underlying mechanisms of UV-mediated leaf growth inhibition have focused on the cellular development of the epidermis (Gonzalez et al., 1998; Nogues et al., 1998), primarily because of the well-established role of the epidermis in controlling leaf growth and shape (Dale, 1988; Savaldi-Goldstein et al., 2007). In order to assess number of epidermal cells per leaf in addition to epidermal cell size, wild-type and mutant plants were grown under 10 kJ m−2 d−1 supplementary UV-B for 13 d (Fig. 2a). There were no differences in the average number of epidermal cells per leaf (anovauv-b–: P > 0.05; Fig. 2b) or in average epidermal cell area (anovauv-b–: P > 0.05; Fig. 2 c) between uvr8-2 and wild-type plants in the absence of UV-B. Ultraviolet B significantly reduced the number of epidermal cells per leaf by about 1.6-fold and about 2.2-fold in both uvr8-2 and wild-type respectively (anovauv-b: P < 0.001; Fig. 2b), which suggests that typical UV-B mediated effects on epidermal cell division are largely independent of UVR8. In contrast to epidermal cell number, epidermal cell area was significantly increased by supplementary UV-B in wild-type plants (anovaler: P < 0.05; Fig. 2c). While epidermal cells of wild-type plants were significantly larger than those of uvr8-2 plants following UV-B treatment (anovauv-b: P < 0.05; Fig. 2c), epidermal cell area of uvr8-2 plants was not significantly different between treatments (anovauvr8: P > 0.05; Fig. 2c). This indicates that UVR8 is required for the UV-B-stimulated increase in cell area observed in the wild-type. In conclusion, the observed reductions in leaf area in both wild type and uvr8 in Arabidopsis are caused principally by the marked decrease in epidermal cell number per leaf, thus indicating that such a reduction is substantially independent of UVR8. However, the uvr8 mutant has smaller leaves than the wild type in response to UV-B because the general (UVR8-independent) decrease in cell number is compounded by the (UVR8-dependent) lack of stimulation of increased epidermal cell area by UV-B absent in uvr8.

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Figure 2. Effects of ultraviolet B (UV-B) on epidermal cell development. Arabidopsis thaliana wild-type Ler and uvr8 mutant plants were grown under a regime of 10 kJ m−2 d−1 of supplementary UV-B (UV-B+) and 300 ± 20 µmol m−2 s−1 of white light for 13 d. Untreated control plants (UV-B–) were grown under identical conditions without UV-B. Supplementary UV-B doses are weighted according to Caldwell's generalized plant action spectrum (Caldwell, 1971). (a) Micrographs of epidermal cell impressions in Ler and uvr8 mutant plants according to UV-B treatment. (b) UV-B effects on epidermal cell number per leaf in youngest fully expanded rosette leaves. Wild-type Ler (closed bars) and uvr8 mutant (open bars) plants both had significantly fewer cells per leaf in the presence of UV-B (two-way anova P < 0.001). (c) UV-B effects on epidermal cell area in youngest fully expanded rosette leaves. Wild-type Ler plants (closed bars) showed a significantly larger average cell area when exposed to UV-B (one-way anova P < 0.05) while average cell area in the uvr8 mutant (open bars) did not significantly change in response to UV-B (P > 0.05). Asterisks indicate significant differences for each genotype in the presence of UV-B when compared with UV-B– treatments: *, P < 0.05; **, P < 0.01. Data presented are an average of five biological replicates ± SE.

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Requirement of UVR8 for normal progression of the endocycle in response to UV-B

Endoreduplication is strongly correlated with increased cell size (Barow & Meister, 2003), and the requirement for UVR8 function for normal cell growth under UV-B could suggest a physiological role for endoreduplication in UV-B response, as suggested by several authors (Vlieghe et al., 2007). We therefore investigated the role that UV-B and the UVR8 locus could play in regulating endocycle function. Wild-type and uvr8-2 plants were grown under 10 kJ m−2 d−1 supplementary UV-B, and the endoreduplication profile determined by means of FCM made in the fifth rosette leaf at 15 d post leaf initiation. In the absence of supplementary UV-B, there were no differences in the endoreduplication profile between uvr8-2 plants and wild-type plants (inline image: P > 0.05; Fig. 3). When exposed to supplementary UV-B, the uvr8 mutant had a significantly different endoreduplication profile than uvr8-2 plants subjected to zero UV-B conditions (inline image: P < 0.001; Fig. 3), with the endoreduplication profile for the uvr8 mutant also significantly different from the wild-type in the presence of supplementary UV-B (inline image: P < 0.001; Fig. 3). In terms of individual levels of endopolyploidy, uvr8-2 plants were defective in progressing towards higher levels of endoreduplication in response to supplementary UV-B, as indicated by a significant accumulation of 8C cells (anova8c uv-b+/–: P < 0.001; Fig. 3), in addition to significant reductions in 32C cells (anova32c uv-b+/–: P < 0.05; Fig. 3). Cells with a 64C content were predictably low (< 1% of total nuclei) in the contribution to overall endopolyploidy profiles of both the uvr8 mutant and wild-type. An effect of UV-B on endoreduplication was observed in the wild-type (inline image: P < 0.001; Fig. 3) across the entire endoreduplication profile as a whole, but there were no significant pairwise differences observed between UV-B+ and UV-B– wild-type plants at individual endoreduplication fractions (anovauv-b+/–: P > 0.05; Table 1). We therefore conclude that UV-B acts as a regulatory influence on the endocycle and that UVR8 is required for normal progression of the endocycle in Arabidopsis as part of the UV-B response.

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Figure 3. Effects of ultraviolet B (UV-B) on endopolyploidy responses in uvr8-2 plants. Nuclear DNA content was compared at 15 d post leaf initiation (DPI) in 5th true leaves by flow-cytometry (FCM) with number of nuclei/ploidy fraction presented as per cent difference in Arabidopsis thaliana uvr8-2 plants from wild-type Ler plants in terms of total proportional number of nuclei counted according to UV-B treatment, where UV-B– plants (closed bars) were grown in 300 ± 20 mmol m−2 s−1 of white light for 23 d in the absence of supplementary UV-B. The UV-B+ plants (open bars) were grown under identical conditions in addition to 10 kJ m−2 d−1 of supplementary UV-B. Supplementary UV-B doses are weighted according to Caldwell's generalized plant action spectrum (Caldwell, 1971). Asterisks indicate significant differences between UV-B treatments for the uvr8 mutant: *, P < 0.05; ***, P < 0.001. Data presented are an average of three repeated experiments ± SE, with two or three biological replicates per genotype/treatment per experiment.

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Table 1.  Effects of ultraviolet B (UV-B) on endopolyploidy responses in Arabidopsis thaliana wild-type Ler plants
Number of nuclei% Of total endoreduplication
UV-B−± SEUV-B+± SEP
  1. Nuclear DNA content was compared in 15 d post leaf initiation (dpi) in fifth true leaves by flow cytometry (FCM) with number of nuclei/ploidy fraction presented as % of total number of nuclei counted according to UV-B treatment. P values indicate significance for one-way anova comparisons of individual endopolyploidy fractions according to UV-B treatment. Data presented are an average of three repeated experiments ± SE, with two or three biological replicates per genotype/treatment per experiment.

2C29.381.0722.632.32> 0.05
4C26.220.2524.280.87> 0.05
8C23.122.6920.883.54> 0.05
16C15.492.9524.412.12> 0.05
32C5.243.357.224.08> 0.05
64C0.610.410.720.51> 0.05

Regulatory role of UVR8 in stomatal differentiation in response to UV-B

The data shown suggest that UVR8 is required to facilitate aspects of cell differentiation during UV-B exposure. To investigate whether this was a general phenomenon or was specific to the endocycle and pavement cell growth, we examined other cell types, including stomata. Stomata are the developmental endpoint of a mitotically driven pathway, and in Arabidopsis stomata comprise a stoma surrounded by three unequally sized cells (Serna & Fenoll, 2000). Although the role of UV-B in controlling stomatal patterning has been postulated (Holroyd et al., 2002), there is currently little evidence to support such links. We selected stomatal index, or number of stomata per epidermal cell, as a differentiation hallmark during leaf development. Stomatal index was measured by growing wild-type and mutant plants under 10 kJ m−2 d−1 supplementary UV-B for 13 d. Stomatal index provided a reliable indicator of any UV-B- and/or UVR8-led differences in stomatal patterning as stomatal index is independent of epidermal cell area differences mediated by UV-B. There were no differences in stomatal index between uvr8-2 and wild-type control plants (anovauv-b–: P > 0.05; Fig. 4). Ultraviolet B significantly increased stomatal index in wild-type plants (anovaler: P < 0.01; Fig. 4), demonstrating a UV-B-mediated increase in the number of stomata per epidermal cell. In the presence of supplementary UV-B, the stomatal index of uvr8-2 plants was lower than that of wild-type plants (anovauv-b: P < 0.001; Fig. 4), indicating a role for UVR8 in stomatal development. In contrast to the wild-type, the uvr8 mutant has fewer stomata per epidermal cell after UV-B exposure compared with control conditions (anovauvr8-2: P < 0.01; Fig. 4). In conclusion, UVR8 is necessary for the UV-B-mediated increase in stomatal index observed in wild-type plants in response to supplementary UV-B.

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Figure 4. Ultraviolet B (UV-B) effects on stomatal differentiation. Arabidopsis thaliana wild-type Ler and uvr8 mutant plants were grown under a regime of 10 kJ m−2 d−1 of supplementary UV-B (UV-B+) and 300 ± 20 µmol m−2 s−1 of white light for 13 d. Untreated control plants (UV-B–) were grown under identical conditions without UV-B with stomatal index assessed in the youngest fully expanded rosette leaf. Supplementary UV-B doses are weighted according to Caldwell's generalized plant action spectrum (Caldwell, 1971). Wild-type Ler (closed bars) and uvr8 mutant (open bars) plants had significantly different average numbers of stomata per epidermal cell according to both UV-B dose and genotype (anova UV-B/genotype interaction P < 0.001). Asterisks indicate significant differences for each genotype in the presence of UV-B when compared with UVB– treatments: *, P < 0.05. Data presented are an average of five biological replicates ± SE.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

In this report, we show that UVR8 is required to maintain leaf expansion under UV-B exposure and this can be explained completely by its effects on cellular differentiation. UVR8 is the only known UV-B-specific signalling component, and the functions of UVR8 in the cell in addition to the pattern of gene expression orchestrated by UVR8 are now becoming better understood. This study has characterized key regulatory roles of the UVR8 signalling component in UV-B whole-plant response (Fig. 5), particularly in terms of leaf photomorphogenesis, and we show that UVR8 controls aspects of leaf expansion as a result of effects on UV-B-mediated compensatory epidermal cell growth. We show that UVR8 is necessary for normal progression of endocycle function as part of UV-B adaptive processes and provide evidence for a UVR8-mediated increase in stomatal index observed in response to supplementary UV-B. Our findings suggest that UV-B, via UVR8, acts to regulate several key developmental plant responses, and adds further evidence to the increasing view that UV-B is not necessarily a damage-inducing source of stress, but instead acts as an important environmental cue in higher plants.

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Figure 5. Model showing the UVR8-regulated and nonUVR8 regulated components of the ultraviolet B (UV-B) response in Arabidopsis leaves. Epidermal cell division is inhibited by UV-B radiation independently of UVR8. UVR8 regulates normal progression of the endocycle in addition to compensatory increases in epidermal cell size and promotion of stomatal development in response to UV-B.

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Regulation of key aspects of leaf morphogenesis by the UVR8 signalling pathway in response to UV-B

The role of UV-B in influencing leaf morphogenesis is still poorly understood, both in terms of those molecular events that might initiate changes in leaf development (Jansen, 2002), and subsequent effects on whole-leaf expansion (Paul & Gwynn-Jones, 2003). Our results show that the leaves of uvr8-2 plants were much smaller than those of the wild type, and this difference became evident at doses of between 6.7 kJ m−2 d−1 and 10 kJ m−2 d−1. It is unlikely that the relative growth reduction in the uvr8 mutant was a result of gross UV-B damage, which is only evident at doses upwards of 15 kJ m−2 d−1 (Fig. 1a). This suggests that the reduction in leaf size in the uvr8 mutant was caused by an altered UV-B leaf-morphogenic response over the lower dose range.

Exposure to UV-B leads to a dose-dependent reduction of leaf size in a diverse range of plant species (Liu et al., 1995; Nogues et al., 1998; Searles et al., 2001; Barkan et al., 2006), most likely by inhibition of epidermal cell expansion and cell division (Gonzalez et al., 1998; Nogues et al., 1998). Here we show that UV-B significantly reduced the number of epidermal cells per leaf in both uvr8 2 and wild type, which indicates that most of the UV-mediated leaf area reduction in wild-type Arabidopsis is explained by changes in cell division, and that this aspect of UV-B response is independent of UVR8. In contrast to those observed findings concerning epidermal cell division, epidermal cell area was significantly increased by supplementary UV-B in wild-type plants, but not in uvr8-2 plants, indicating that UVR8 is required for the UV-B stimulated increase in cell area observed in the wild-type. Although inhibition of growth is a commonly observed whole-leaf response to UV-B (Liu et al., 1995; Searles et al., 2001), there is increasing evidence for various compensatory growth mechanisms in response to reductions in cell number that occur in response to changes in cell volume and/or cell number during differing phases of leaf growth (Hemerly et al., 1995; Tsukaya, 2003). Endoreduplication is considered to be one mechanism involved in compensatory growth (Cookson et al., 2006), and endoreduplication responses such as those observed in our study may play a vital role in leaf photomorphogenesis, buffering plants from the more extreme effects of UV-B wavelengths. The molecular mechanisms underlying compensatory organ growth are still obscure but our data reveal that UVR8 is essential to maintain organ size in the presence of UV-B.

Requirement of UVR8 for normal progression of the endocycle in response to UV-B

Endoreduplication is increasingly being seen as an important trait in plant adaptation to a range of environmental factors (Jovtchev et al., 2007; Vlieghe et al., 2007), and in many species successive rounds of DNA replication are an important aspect of cell growth and differentiation (Sugimoto-Shirasu & Roberts, 2003; Cookson et al., 2006). There is some existing evidence that longer wavelengths of light can influence endocycle progression. The red/far-red light signalling pathway, mediated by phytochrome, affects cell growth and endoreduplication in other plant tissues such as the hypocotyl, and progression of endocycle not usually observed in wild-type Arabidopsis occurs in phytochrome A mutants when grown in continuous red light (Gendreau et al., 1997). Here, we have shown that the uvr8 mutant is defective in progressing into higher levels of endoreduplication in response to supplementary UV-B, as indicated by an accumulation of 8C cells, and reduced numbers of 32C cells (Fig. 3). While the differences observed in cell size in our study are unlikely to be solely attributable to differences in endopolyploidy, there is a strong positive correlation between nuclear volume and cell size in Arabidopsis (Jovtchev et al., 2006), and it is clear from our results that UVR8 plays an important role in maintaining normal endocycle progression in the presence of UV-B, suggesting that correct progression through the endocycle might be a necessary requirement for the UVR8-dependent compensatory cell growth that is observed in response to UV-B treatment. Increased resistance to UV-B was also associated with increased levels of endopolyploidy in the Arabidopsis mutant uvi4 when grown at high doses of supplementary UV-B (Hase et al., 2006). Here we show that UVR8 plays an important role in regulating endopolyploidy as an adaptive mechanism, and although some direct influence of UV-B on overall endoreduplication profiles was observed in the wild-type, such responses were modest in nature when compared with the mutant, but nonetheless clearly indicate that endoreduplication warrants future attention as a new and important area of UV-B response. It is also likely that other aspects of UVR8 signalling could be integral to the ploidy response (e.g. Arabidopsis ecotypes exhibiting naturally high levels of ploidy have been observed to have significantly higher constitutive levels of UV-B-absorbing secondary compounds; J. Wargent & N. Paul, unpublished), and it has been suggested that endoreduplication could increase transcription of genes responsible for production of UV-B screening compounds (Vlieghe et al., 2007).

UV-B-mediated control of stomatal differentiation in Arabidopsis by a UVR8-dependent signalling pathway

Stomatal patterning is known to be sensitive to various environmental conditions in a range of plant species (Martin & Glover, 2007). However, no previous studies have established any mechanism by which UV-B may regulate stomatal development. Whilst the observed increases in stomatal density in the uvr8 mutant is most likely a consequence of the previously discussed reductions in epidermal cell size, the UV-B-mediated reduction in stomatal index in uvr8-2 in addition to the observed increase in stomatal index in wild-type Arabidopsis (Fig. 4) indicates that UVR8 is required to maintain the relative density of stomatal pores in the presence of UV-B radiation. Although there is now evidence that other environmental factors can influence stomatal patterning and development (e.g. changes in atmospheric CO2; Woodward, 1987; Brownlee, 2001), those few studies that have examined UV-B-mediated effects on stomatal number vary, with increases and decreases in both stomatal index and density (Dai et al., 1995; Kakani et al., 2003a) reported for different species. Shading of photosynthetically active radiation has been associated with decreases in stomatal index in wild-type Arabidopsis (Lake et al., 2001), perhaps mediated by systemic signalling (Coupe et al., 2006). In addition, changes in light quality could also drive patterning responses with increases in stomatal index, as observed in Vigna sinensis plants when grown under continuous red light (Schoch et al., 1984). There is some evidence to suggest that signalling cascades involving UV-B and physiological factors such as epicuticular wax content and composition may regulate stomatal number (Gray et al., 2000; Holroyd et al., 2002), and previous work in several species has provided evidence that UV-B influences epicuticular wax composition and can increase leaf wax content (Gonzalez et al., 1996; Gordon et al., 1998), presumably as a screening mechanism to reduce UV-B transmittance through the leaf. Increases in stomatal index in addition to UV-B-mediated increases in epidermal wax content have been observed by Kakani et al. (2003b).

The relationship between the cell cycle and stomatal development is still little understood, and while there are few studies, it has been established that mature guard cells express genes associated with competence for cell division (Serna & Fenoll, 1997), and B-type cyclin-dependent kinases (CDKs) have been shown to regulate stomatal differentiation (Boudolf et al., 2004). Similarly, the role of hormonal control in cell development, as regulated by UV-B, is still poorly understood, and could involve both UVR8-dependent and UVR8-independent pathways. Several plant growth regulators are known to influence stomatal patterning, including abscisic acid (ABA) (Bradford et al., 1983), cytokinins (Wang et al., 2001) and ethylene (Serna & Fenoll, 1997). Although the role of auxin in promoting cell fate (Pekker et al., 2005) in addition to UV-B-mediated changes in auxin transport and catabolism via changes in flavonoid metabolism (Jansen, 2002) are becoming better understood, a fully elucidated role for UV-B in stomatal development is yet to be established.

UVR8 and whole-plant responses to UV-B radiation

From the experiments presented, we conclude that UVR8 regulates three related aspects of cell development in response to UV-B: (1) progression of typical endoreduplication events; (2) regulation of compensatory increases in epidermal cell size; and (3) promotion of stomatal development. Little is known about the effects of UV-B on endocycle, cell differentiation and cell expansion during leaf growth, particularly at the molecular level, although UV-B has been associated with changes in mitotic index (Hopkins et al., 2002). UVR8 has some sequence similarity to human REGULATOR OF CHROMATIN CONDENSATION 1 (RCC1) (Kliebenstein et al., 2002), a regulatory component of the cell cycle and mitosis (Renault et al., 2001), and although UVR8 is not a functional homologue of RCC1 (Brown et al., 2005) our findings indicate that UV-B may play an important role in regulating the cell cycle of plants.

This study shows that UVR8 is a key signalling component in the whole-plant response to UV-B, regulating important morphogenic processes in the leaf. Furthermore, it is evident that the role of UVR8 extends beyond the positive regulation of gene expression associated with phenylpropanoid metabolism and other UV-protective responses established by previous microarray analysis (Brown et al., 2005). This research raises the possibility that UVR8 regulates further processes in response to UV-B at the whole-plant level. For example, UVR8 may regulate other aspects of morphogenesis or could mediate cross-protection in response to biotic and abiotic factors. Many compounds active in UV-B response have other functions, such as defence against herbivore attack (Bassman, 2004) and pathogens (Treutter, 2005). Hence it is becoming clear that UV-B is not a source of damaging stress in nature, but is an environmental cue that enables plants to regulate protection against several factors, including UV-B itself, high light, drought and herbivory. It is unclear how such response networks are coordinated in natural solar conditions, where photosynthetically active radiation and longer UV-A wavelengths are at far higher fluxes than those achievable in laboratory conditions. Further study of these factors will be of vital importance in the future, not just to characterize the UVR8 pathway and its significance in UV-B morphogenesis, but also to help us further understand the role that UV-B plays in regulating key aspects of terrestrial ecosystem function.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was supported by the UK Horticultural Development Council (grant no. CP 26) and by the European Commission Framework Programme 6, (Integrated Project: AGRON-OMICS – LSHG-CT-2006–037704). Our thanks go to Joanna Heaton (Department of Biological Sciences, Lancaster University, UK) for technical assistance.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Table S1 ANOVA results from all one-way and two-way comparisons of epidermal cell number per leaf, epidermal cell area and stomatal index

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