SEARCH

SEARCH BY CITATION

Keywords:

  • ultraviolet-B radiation;
  • Arabidopsis thaliana;
  • mutants;
  • signal transduction

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

In plants, low fluences of ultraviolet-B radiation (UV-B; 280–320 nm) trigger the activation of protection mechanisms and various photomorphogenic responses. We established a novel screen to isolate Arabidopsis T-DNA mutants (uli, UV-B light insensitive) with reduced sensitivity towards UV-B light-mediated inhibition of hypocotyl growth. One of the mutants, uli3, shows also a reduced sensitivity of UV-B-induced gene expression events and was, therefore, chosen for further investigation. The ULI3 gene encodes an 80-kDa protein with potential domains for heme- and diacylglycerol-binding. In transiently transfected protoplasts, the ULI3:GFP fusion protein is localised in the cytoplasm but also adjacent to membranes. In etiolated Arabidopsis seedlings irradiated by UV-B, ULI3 mRNA expression is strongly up-regulated. This is caused by elevated transcription as demonstrated using stable transformants where a GUS-reporter was driven by the ULI3-promoter. ULI3 is preferentially expressed in the outer cell layers in leaves, stems and flowers, but not in roots. There is evidence that ULI3 represents a specific component involved in UV-B-mediated signal transduction in higher plants.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Over the past decades, rising UV-B fluence rates at the earth's surface caused by stratospheric ozone depletion have substantially increased interest in understanding UV-B light signal perception and transduction. High UV-B fluence rates directly damage DNA, membranes and proteins in all organisms. However, low UV-B fluence rates can stimulate distinct mechanisms counteracting such damaging effects as well as UV-B responses that are not related to UV-protection (Herrlich et al., 1997). Some of these processes, which are specifically activated by irradiation in the UV-B range (280–320 nm) are mediated by signal transduction cascades acting independently of DNA damage (Herrlich et al., 1997; Jansen et al., 1998). This has been shown for UV-B-induced cytosolic signalling in mammalian and yeast cells (Devary et al., 1993; Engelberg et al., 1994). Increasing evidence including data from Arabidopsis mutants defective in DNA repair (Kim et al., 1998) indicates that in plants UV-B signalling can also act independently from DNA damage (Batschauer et al., 1996; Frohnmeyer et al., 1999; Green and Fluhr, 1995). The best characterised UV-B-mediated responses in Arabidopsis thaliana are the inhibition of hypocotyl growth in seedlings, the biosynthesis of UV-absorptive secondary metabolites such as flavonoids or sinnapate esters and the concomitant stimulation of related gene expression events (Boccalandro et al., 2001; Christie and Jenkins, 1996; Kim et al., 1998; Landry et al., 1995; Li et al., 1993).

The isolation of Arabidopsis mutants with altered sensitivity to a given wavelength in the visible spectrum of sunlight has been a powerful approach in the understanding of photomorphogenesis. These mutants were either defective in the corresponding photoreceptor or in related signal transduction elements (Cashmore et al., 1999; Dieterle et al., 2001; Smith, 2000). Some of the known photoreceptors have some influence on UV-A- (320–360 nm) and, to some extent even on UV-B-mediated responses. Especially the photoreceptors detecting B/UV-A wavelenghts but also the red-absorbing phytochromes seem to be involved in modulating UV-B responses as has been shown, for example, for the UV-B-induced expression of the chalcone synthase in various plant species (Frohnmeyer et al., 1992; Fuglevand et al., 1996; Ohl et al., 1989) and for the opening of cotelydons in Arabidopsis seedlings (Boccalandro et al., 2001). With respect to UV-B wavelength genetic screens for loci affecting the UV-B responsiveness in A. thaliana focused on mutants with defects in their DNA damage repair system or in the synthesis of phenolic sunscreens (Landry et al., 1995; Landry et al., 1997; Li et al., 1993). As white light supplemented with UV-B was used for the selection of these mutants, the simultaneous activation of other photoreceptor systems is likely. This might have excluded the isolation of mutants with defects in UV-B perception or related signal transduction. However, these screens were intended to isolate UV-B hypersensitive mutants with reduced tolerance to UV-stress, which are valuable tools to investigate UV-B-mediated damage responses. In a different approach, Bieza and Lois (2001) described the isolation of a dominant UV-B tolerant mutant exhibiting increased levels of phenolic sunscreens. No other UV-related phenotype has been observed in this mutant.

However, no mutant impaired in the perception or the transduction of UV-B has been described so far. Therefore, we ventured to isolate UV-B hyposensitive mutants by irradiating etiolated seedlings from Arabidopsis T-DNA collections with repetitive low fluence UV-B pulses. In the present work we describe one of the mutants recovered from this screen, uli3 (UV-light-insensitive3) that was impaired in several UV-B responses and, therefore, analysed in more detail.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Identification of UV-B hyposensitive uli mutants

To set up the screen the UV-B dose had to be optimised such that it inhibited hypocotyl elongation without triggering damage responses. For this purpose Arabidopsis seedlings that had been grown for 24 h in darkness, were irradiated with UV-B pulses with constant fluence rates for 2–10 min and then returned to darkness. This UV-B treatment was repeated 24 h later and the final length of the hypocotyls was measured 24 h after this second treatment (Figures 1a,b). Under these conditions hypocotyl growth in the wild type was inhibited in correlation with the duration of the UV-B pulse (Figure 1b). Pulses of 10 min were most effective in terms of growth inhibition but caused also obvious damage such as curling of hypocotyls and reduced root growth (not shown). In contrast, 5 min UV-B pulses were sufficient to cause about 40–50% inhibition of hypocotyl growth without visible signs of DNA damage. To assess the influence of DNA photolyases or the B/UV-A photoreceptors, we tested the effect of UV-A irradiation applied directly after the UV-B pulses (Figure 1a). However, one hour of UV-A irradiation applied by itself or in addition to the UV-B pulses did not affect hypocotyl length of wild-type seedlings under our experimental conditions (Figure 1b). Nevertheless, for the screening (Figure 1a) the UV-A treatment was included to ensure the activation of photolyases that remove residual DNA damage.

image

Figure 1. UV-irradiation program and hypocotyl growth responses in seedlings of wild-type Arabidopsis and corresponding photoreceptor-deficient mutants.

(a) Sketch of the irradiation program used to optimise UV-irradiation.

(b) Hypocotyl length of 3-day-old Arabidopsis wild-type seedlings (Ws) kept in darkness (D) or exposed for 2, 5 or 10 min to UV-B (4.8 W m−2) per day (UV-B). After irradiation the seedlings were either directly transferred to darkness (UV-B [RIGHTWARDS ARROW] D) or 1 h UV-A (6.0 W m−2) was applied after the UV-B pulse before dark incubation (UV-B [RIGHTWARDS ARROW] UV-A). For the UV-B exposure with subsequent UV-A irradiation a 1 h UV-A irradiation per day (UV-A) serves as control. Error bars give standard errors (n = 90 for each condition).

(c) Hypocotyl length of wild type (Ler), phytochrome-deficient (phyA, phyB, phyAphyB) and blue-light (cry1) photoreceptor mutants under screening conditions (UV-B) corresponding to 5 min UV-B pulses per day as described in B. Control (D, UV-A). Error bars give standard errors (n = 90).

Download figure to PowerPoint

In order to investigate if other photoreceptors of the phytochrome or cryptochrome families are activated by the UV-B pulse regime and affect the hypocotyl response, we irradiated different photoreceptor-deficient mutants according to our screening program (5-min UV-B followed by 1 h UV-A per day) and measured their hypocotyl length after the second dark period (Figures 1a,c). Under our experimental conditions, the inhibition of hypocotyl growth by UV-B is obviously not mediated by phytochromes and B/UV-A photoreceptors, as the photoreceptor-defective mutants phyA, phyB, phyA/phyB and cry1 exhibited similar hypocotyl growth as the wild type (Figure 1c). Subsequent experiments, using nph1, cry1cry2 and phyAphyBcry1 mutants, confirmed that low doses of UV-B are not mediated by these photoreceptors (data not shown). These data, which are confirmed by recent physiological studies (Boccalandro et al., 2001), suggest strongly that the UV-B-mediated inhibition of hypocotyl growth in etiolated seedlings is exclusively dependent on the action of a UV-B photoreceptor.

For the screening, we choose 5-min pulse irradiation given every 24 h. Under these conditions, the daily exposure to UV-B is equivalent to a UV-B dose of 0.26 kJ UV-BBE m−2 day−1 which is lower than the UV-B exposure currently encountered in the Northern mid-latitudes (Ries et al., 2000a). The UV-B pulse irradiation regime (including a subsequent 1 h UV-A exposure) was used to screen a population of 8000 T-DNA mutagenised plants and led to the isolation of four different UV-B hyposensitive lines (Figure 2a, uli). All mutant lines were found to be recessive (data not shown) and all exhibited longer hypocotyls than the corresponding wild type after exposure to UV-B (Figure 2a). We then tested the efficiency of long and short wavelengths within the UV-B range on hypocotyl growth inhibition in the mutants and the wild type (Figure 2b). We found that in contrast to wild-type seedlings uli mutants are insensitive to UV-B longer than 295 nm, since removal of wavelengths shorter than 295 nm from the light source by using cut-off filters resulted in an almost complete loss of growth inhibition. Moreover, the residual inhibition of hypocotyl elongation in uli mutants is completely due to UV-B wavelength below 295 nm and under these conditions still much less pronounced than in the corresponding wild type (Figure 2b). The hyposensitivity of the mutants is specific for UV-B wavelengths and not mediated by other photoreceptors, as no phenotypes with long hypocotyls were detectable in dark-grown seedlings after exposure to continuous UV-A, blue, red or far-red light (Figure 2c).

image

Figure 2. Phenotypes of uli mutants after UV-B pulse irradiation and after continuous irradiation with longer wavelengths.

(a) Phenotype of wild type (Ws) and uli seedlings grown in darkness and irradiated with 5 min UV-B (4.8 W m−2) corresponding to 0.26 kJ UV-BBE m−2 h−1) followed by 1 h UV-A (6.0 W m−2) per day as described in Figure 1(b).

(b) Hypocotyl lengths of wild type (Ws), uli1.1, uli1.2, uli2 and uli3 after UV-B or UV-A treatment or after growth in darkness. The light treatment was performed with 5 min UV-B pulses under the conditions described in Figure 1(a). Different ranges of UV-B wavelengths (>280, >295, >305, >327 nm) were created by using cut-off filters.

(c) Relative hypocotyl length of wild type and uli mutants after 3 day continuous irradiation with UV-A (cUV-A), red (cR), far-red (cFR) or blue (cB) light. The hypocotyl length of each line is given relative to the length in darkness that was set as 1. All data (n = 90) represent mean and error bars give standard errors.

Download figure to PowerPoint

uli3 is affected in several UV-B-mediated responses

A mutation that affects upstream elements of UV-B signalling is expected to impair several UV-B-mediated responses, whereas mutations that are located downstream in the signalling cascade should not alter other UV-B responses. We therefore tested, whether the uli lines are impaired in UV-B responses other than hypocotyl growth. As shown below, we found that this criterion holds only for uli3, whereas in the other uli mutants the phenotype was limited to hypocotyl growth (not shown). We, therefore, focused on the uli3 mutant. Initial characterisation of the other three mutants showed that ULI1.1 and ULI1.2 were allelic with single T-DNA insertions linked to the phenotype of uli1 at the same locus on chromosome 2 (BAC clone F15L11). Data bank searches indicated an ORF with low homology to a putative non-LTR retroelement reverse transcriptase near the T-DNA insertion site. The T-DNA insertion site of ULI2 is still unknown.

In uli3 mutants, the expression of UV-B-induced genes was determined in more detail in seedlings that were either kept in darkness or irradiated for 3 days with UV-A or with UV-B supplemented with UV-A. The result of a typical gene expression experiment is shown in Figure 3(a). In the wild type, UV-B strongly induced the accumulation of chalcone synthase (CHS) mRNA whereas this stimulation was strongly reduced in uli3. The residual induction of CHS expression in the mutant might be due to the action of other photoreceptors that are activated by UV-B due to the long irradiation period (72 h). It has been shown that continuous irradiation of etiolated seedlings with UV-B can induce phytochromes to a photoequilibrium of 50–70% (Jabben et al., 1982). The reduced CHS expression in uli3 is mirrored by a significantly reduced anthocyanin content, although this effect was less pronounced as compared to CHS expression (Figure 3b). UV-A irradiation alone caused a weak CHS stimulation in wild-type seedlings which was reduced in uli3 (Figure 3a). This reduced CHS expression was not reflected by anthocyanin levels (Figure 3b). However, UV-A irradiation resulted in a less pronounced difference between wild type and mutant. This finding suggests that the impaired response of uli3 is specific for UV-B. Under the same irradiation regime, we also examined the expression of PR-1 and NDPK1a, which are involved in pathogen defence and in histidine biosynthesis (Green and Fluhr, 1995; Zimmermann et al., 1999). Consistent with the results of the CHS stimulation, both genes were strongly induced in the wild type upon UV-B irradiation while their expression was considerably reduced in uli3 seedlings (Figure 3a). Remarkably, the induction of PR1 was confined to UV-B while UV-A was not effective. The corresponding mRNA of this gene with a very tight UV-B requirement was hardly detectable in UV-B treated uli3. NDPK1a mRNA was chosen as an intermediate gene with a weak constitutive expression in dark-grown seedlings, which is stimulated by UV-B and UV-A after long-term treatment. Similar to CHS, the mRNA accumulation was reduced in the uli3 mutant and the reduction was more pronounced when UV-B was present as compared to UV-A alone (Figure 3a).

image

Figure 3. UV-B-specific gene expression and anthocyanin content of Ws and uli3.

(a) Representative Northern blot analysis of chalcone synthase (CHS), pathogen-related protein1 (PR-1) and nucleoside diphosphate kinase1a (NDPK1a) mRNA expression. After induction of germination the seedlings were grown for 3 days (D) in darkness, in continuous UV-A supplemented with UV-B (305 nm cut-off; 1.9 kJ UV-BBE m−2 h−1) or in continuous UV-A light (6.0 W m−2). The light sources are described in more detail in the experimental procedures. Equal amounts of total RNA (15 µg) were separated on an agarose gel, then transferred to a membrane and probed with radioactively labelled cDNA.

(b) Anthocyanin levels from 100 seedlings irradiated as described in (a) were determined and given as A535. Data show the mean and standard error of four independent experiments.

Download figure to PowerPoint

Identification of the uli3 locus

The genomic fragment flanking the T-DNA in uli3 was isolated by plasmid rescue as described in Experimental Procedures. The sequence is identical to the P1 clone MMN10 and is located 22 kb from the ARA3 gene on chromosome 5 (Figure 4a). Database analyses indicate that ULI3 shares an 80% homology to a sequence on chromosome 4, while no other homologies were found in other organisms. The mutant uli3 allele contains a single T-DNA insertion 120 bp upstream of an open-reading frame (Figure 4a). The ORF of ULI3 lacks any introns and comprises 2133 bp (GenBank accession no. AF321919) as confirmed by Northern blot analyses (Figure 5a) and by the description of an expressed sequence tag (AV537547), encoding only 514 bp of the ULI3 3′ region.

image

Figure 4. Identity of the ULI3 gene and complementation of the uli3 mutation.

(a) Localisation of the T-DNA in the ULI3-promoter. The diagram depicts (from top to bottom) the position of the ULI3 locus on Chromosome 5; the sequenced P1 clone MMN10 containing this locus (ARA12 + 3: RAS-related proteins); the T-DNA insertion site in the promoter region of ULI3, the uli3 gene and the structure of the protein.

(b) Representative phenotypes of wild type (Ws), of uli3, uli3 lines transformed with the genomic ULI3 gene (ULI3:ULI3; five lines in total) and uli3 lines transformed with a construct for the overexpression of ULI3 cDNA (35S:ULI3; 25 lines in total) after UV-B irradiation under screening conditions. All seedlings were grown for 24 h in darkness and then irradiated with 5 min UV-B pulses followed by 1 h UV-A as described in Figure 1(b).

(c) Hypocotyl length under the conditions described in (B). Data (n = 90) represent mean and error bars give the standard error. According to the student t-test the values of uli3 and of the complementing lines are significantly different at a confidence interval 99%.

Download figure to PowerPoint

image

Figure 5. UV-induced ULI3 expression in seedlings and subcellular localisation of the ULI3:GFP fusion protein in protoplasts.

(a) RNA gel-blot analysis of ULI3 mRNA accumulation in wild type (Ws) and uli3 seedlings. Seedlings were grown in darkness for 3 days (D) or transferred to continuous UV-B supplemented UV-A (305 nm cut-off; 1.9 kJ UV-BBE m−2 h−1) or UV-A light (6.0 W m−2) and equal amounts of total RNA were analysed as described in Figure 3.

(b) RNA gel-blot analysis of ULI3 mRNA accumulation in wild-type seedlings after UV-B pulse irradiation. The seedlings were etiolated for 3 days (D) or etiolated seedlings were irradiated with two UV-B pulses (5 min day−1) and then transferred to darkness (UV-B [RIGHTWARDS ARROW] D) corresponding to the conditions of the screening. RNA was extracted 2, 4, 8 or 24 h after the second UV-B pulse and total RNA (15 µg) was transferred and probed as described (Figure 3).

(c,d) Subcellular localisation of an ULI3:GFP in transiently transformed parsley protoplasts. Representative images of a ULI3:GFP expressing protoplasts (c) and the corresponding bright field image (d) are shown.

Download figure to PowerPoint

The predicted 80-kDa ULI3 protein (Figure 4a) exhibits 27% amino acid homology to a human 80-kDa diacylglycerol kinase. In fact, the N-terminus contains one C1-domain known to bind diacylglycerol (DAG; Caloca et al. 1999; Oancea and Meyer, 1998). However, no obvious kinase domain could be detected by homology search of the ULI3 sequence. In addition to the DAG-binding domain, the N-terminus harbours two binding sites for heme chromophores known from cytochrome P450 proteins (Chapple, 1998; Figure 4a). However, the homology of ULI3 to cytochrome P450 proteins is confined to these binding sites. The C-terminus of the predicted ULI3 protein contains an aspartic acid-rich domain that has been described to participate in protein–protein interaction in various plant proteins involved in transcriptional regulation (Ludwig et al., 1989; Wu et al., 2000).

The uli3 mutation could be complemented by transformation with a 35S-promoter:ULI3-ORF:NOS-terminator construct as well as by a genomic fragment including the putative ULI3-promoter and terminator regions. uli3 lines transformed with these constructs exhibited an enhanced UV-B light sensitivity and are indistinguishable from wild-type seedlings under screening conditions (Figures 4b,c). Thus both, the genomic fragment and also an overexpressed ULI3-ORF restored the wild-type phenotype.

The expression of ULI3 is stimulated by UV-B

While the ULI3 ORF remains intact in the mutant, the close insertion of the T-DNA adjacent to the 5′-end of the translational ATG destroys the integrity of the promoter architecture. As a consequence, ULI3 mRNA expression is not detectable in dark-grown and light-treated uli3 mutants (Figure 5a). In contrast, ULI3 is already expressed at a low level in dark-grown wild-type seedlings and is strongly stimulated after continuous irradiation with UV-B. Irradiation by UV-A alone led to a less pronounced induction of ULI3 mRNA (Figure 5a). In addition, the transcription of ULI3 was also stimulated in wild-type seedlings grown under screening conditions (Figure 5b). Kinetic studies under these conditions showed, that the amount of ULI3 mRNA increases within 2 h after the second UV-B pulse and remained elevated during the subsequent 24 h.

ULI3 is localised in the cytoplasm and adjacent to the plasma membrane

To investigate the subcellular localisation of the protein, the coding region of ULI3 was translationally fused to green fluorescent protein (GFP) and transiently transfected into parsley protoplasts. ULI3:GFP appeared mainly in the cytoplasm and also seemed to be attached to plasma membranes while the fusion protein was extruded from the nucleus (Figures 5c,d). Since ULI3 encodes a conserved 50 amino acid stretch with all features of a C1 domain and DAG binds to the C1 domain of human protein kinases binding to cellular membranes (Caloca et al., 1999) this intracellular distribution of ULI3:GFP might be related to the C1 domain of ULI3 (Caloca et al., 1999; Oancea and Meyer, 1998).

UV-B pulses induce the ULI3-promoter

To analyse the light regulation and tissue specificity of ULI3 expression Arabidopsis wild-type plants were transformed with a full-length ULI3-promoter fused to a β-glucuronidase (GUS) reporter gene. Histochemical analyses (Figure 6a) and quantitative GUS assays (Figure 6b) confirmed the mRNA accumulation studies (Figure 5), as the ULI3-promoter:GUS fusion was strongly induced by UV-B in both cases. As shown in Figure 6(a), ULI3:GUS accumulated in hypocotyls and cotelydons of seedlings that were irradiated under the conditions of our UV-B screen. Consistent with the Northern analysis (Figure 5), a weak enzyme activity could be detected already in dark-grown seedlings (Figure 6a), and UV-B pulses resulted in a fourfold increase of GUS activity 72 h after the onset of irradiation (Figure 6b). In contrast, other light qualities (UV-A, B, R, FR) given as light pulses under the same time schedule failed to stimulate ULI3-driven GUS activity (Figure 6b). These data show that ULI3 expression in etiolated Arabidopsis seedlings is exclusively induced by UV-B.

image

Figure 6. Enzyme activity of β-glucuronidase under control of the ULI3-promotor in transgenic Arabidopsis seedlings.

(a) Histochemical staining of 3-day-old seedlings grown in darkness for 3 days or irradiated with two UV-B pulses (5 min day−1) and then transferred to darkness (UV-B) as described for the screening conditions.

(b) GUS enzyme activity in 3-day-old seedlings. The seedlings were etiolated for grown in darkness for 3 days (D) or were irradiated with two light pulses (5 min day−1) with the corresponding wavelength as indicated and then transferred to darkness. All seedlings were harvested 24 h after the second light pulse. Data represent mean of three independent experiments with three different transgenic lines and error bars denote SEM.

Download figure to PowerPoint

The ULI3-promoter is active in all organs except roots

The expression of ULI3 was UV-B specific only in young seedlings, as plants that has been cultivated for 6 weeks in the greenhouse showed significant ULI3-promoter:GUS activity in leaves, flowers and stems but not in roots (Figure 7). Within the leaves, the GUS reporter was preferentially found in veins and trichomes (Figure 7a). In flowers, the expression was strong in veins, anthers and pollen, whereas the stylum was only stained in the apical part. No enzyme activity could be detected in the stigma and in the lower part of the pistil (Figure 7d). In stems, ULI3:GUS showed a homogenous distribution (Figure 7c). However, the reporter gene was not expressed in roots (Figure 7b).

image

Figure 7. Histochemical staining of adult transgenic ULI3-promoter:GUS Arabidopsis plants. (a) Leaves (b) roots (c) stems (d) flowers. Plants were grown for 6 weeks under white light conditions (16 h light/8 h dark cycles) and the different organs were stained as described in Experimental procedures. The inserts at the right bottom show the GUS staining of untransformed wild-type plants.

Download figure to PowerPoint

Cross-sections of leaves and stems showed that ULI3:GUS expression was found in several cell layers (Figure 8). However, the ULI3-promoter is preferentially active in the outer cell layers of these organs. In leaves, strong enzyme activity was found in the vascular tissue (Figure 8a). In stems, the expression of the reporter gene seems to be confined to the cortical tissue (Figure 8b).

image

Figure 8. Histochemical staining of transgenic ULI3-promoter:GUS Arabidopsis tissues. Cross-sections from a leaf (a) and a stem (b) are shown. Embedding and staining of the tissues was performed as described in the Experimental procedures and visualised in a light microscope (Zeiss, Oberkochem, Germany; magnification 20×).

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We have isolated several A. thaliana mutants which show reduced inhibition of their hypocotyl growth after irradiation with UV-B pulses. These uli mutants were specifically insensitive to wavelengths between 300 and 320 nm and the lack of responsiveness within this range of the light spectrum is in agreement with described action spectra of UV-B-specific responses in wild-type plants (Jansen et al., 1998). Other known photoreceptors are not affected in uli mutants as they exhibit no long- or short-hypocotyl phenotype after irradiation with FR, R, B and UV-A. These physiological studies show that uli mutants are specifically impaired in UV-B-mediated responses. Our data are in agreement with recent physiological analyses in Arabidopsis, which proofed that the hypocotyl elongation in UV-B irradiated seedlings is mediated by a UV-B sensor and not by known photoreceptor systems (Boccalandro et al., 2001; Kim et al., 1998).

Light-grown uli mutants exhibited no phenotype with respect to their size, leaf-shape and flowering time as it has been described for phytochrome- and blue/UVA-light photoreceptor-deficient mutants (Cashmore et al., 1999; Smith, 2000). This indicates a highly specific function of ULI proteins in UV-B-mediated signalling during early developmental stages.

It is unlikely that the hypocotyl response induced by our screening conditions is caused by UV-B-mediated DNA damage. When we checked the hypocotyl response of the photolyase-deficient uvr2-1 mutant (Landry et al., 1997), which is hypersensitive to UV-B-mediated DNA-damage (Kim et al., 1998), we could not find any difference to the wild type under our screening program (data not shown). In addition, we observed that high UV-B doses produced similar amounts of DNA dimers in uli3 mutants and in wild-type plants (data not shown). Thus, uli3 mutants are not affected in their capacity to repair DNA damage.

In this study, we concentrated on the most promising mutant, uli3, which is impaired in several UV-B-mediated responses including hypocotyl growth and UV-B-induced expression of CHS, PR1 and NDPK1a. This multiple insensitivity of UV-B responses suggests a role of ULI3 either in UV-B signal perception or in early signal transduction. This hypothesis is supported by pharmacological approaches in Arabidopsis that demonstrate that UV-B control of CHS and PR-1 is conveyed by different pathways (Christie and Jenkins, 1996; Green and Fluhr, 1995).

ULI3 mRNA accumulated within 2 h after a UV-B pulse in etiolated seedlings and the expression was restricted to hypocotyls and cotyledons. This temporal and spatial pattern indicates that the long hypocotyl phenotype of uli3 is directly related to ULI3 expression in these organs. Continuous irradiation with white light also stimulated ULI3 expression, especially during later developmental stages with a preferential expression in the outer cell layers of leaves and stems and in some flower organs. Thus, the expression seems to be limited to organs exposed to light (and UV-B) as no ULI3:GUS activity was detectable in roots.

The protein structure of ULI3 does not obviously reveal a potential function as UV-B receptor, since UV-B absorbing chromophores or clusters of UV-B absorbing amino acids are absent in the sequence. The potential heme chromophores of ULI3 would not be suited to absorb specifically UV-B (Chapple, 1998), but might act as electron acceptors during signal transduction. Such a process of electron transfer has been proposed as an early event during UV-B-induced signalling to CHS in Arabidopsis (Long and Jenkins, 1998). ULI3 might represent an electron donor after excitation of a putative UV-B photoreceptor or it might sense UV-B-triggered changes of the cytosolic redox potential. It is well known that heme chromophores are involved in light-dependent electron transition, as has been shown, for instance, for cytochrome P450 reductases and P450 proteins (Chapple, 1998).

Cytochromes P450 are also known to be involved in the biosynthesis of hormones, lipids and secondary metabolites of the plant (Bolwell et al., 1994; Mathews, 1985). As the interaction of light and hormones in photomorphogenic events has been described recently (Garcia-Martinez and Gil, 2001; Yin et al., 2002; Zhao and Chory, 2001), a distinct function of ULI3 in this context cannot be excluded.

ULI3 is localised in the cytoplasm, but propably also partly attached to the plasma membrane, which represents a typical pattern for proteins containing a C1 domain. These proteins bind to membranes via interaction of membrane-located DAG. UV-B treatment of mammalian cell cultures causes the partial translocation of C1 domain-containing protein kinases from the cytosol to membranes, and this membrane attachment seems to be necessary for downstream signalling (Chen et al., 1999). Although only a small portion of the overexpressed ULI3:GFP was bound to the plasma membrane, we did not observe a light-induced translocation of the ULI3:GFP fusion protein, at least in protoplasts. However, the use of this system might be not suitable to detect a partial translocation of the 20–30% of the protein that has been reported for UV-treated animal cells (Chen et al., 1999).

In summary, the physiological features of the uli3 mutant resemble those of other Arabidopsis mutants, that had been isolated due to impaired responses to red, far-red or blue light and later found to be affected either in the function of a specific photoreceptor or in signalling triggered by this photoreceptor (Ahmad and Cashmore, 1993; Cashmore et al., 1999; Nagatani et al., 1993; Reed et al., 1993; Somers et al., 1991). Our data place ULI3 close to a UV-B photoreceptor, where it might be activated by electron transfer and in turn activates downstream elements of UV-B signalling by membrane attachment, electron transfer or both.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Plant material, light conditions and screening procedure of uli mutants

For the screening of UV-B insensitive mutants we used a collection of 8000 T-DNA insertion lines of A. thaliana ecotype Wassilewskija (Feldmann and Marks, 1987). Additional A. thaliana mutants defective in phytochromes (phyA-201, phyA-201phyB-5,Nagatani et al., 1993; phyB-5, Reed et al., 1993), in cryptochromes (cry1 = hy4-23 N, Ahmad and Cashmore, 1993; cry1cry2,Cashmore et al., 1999) and in both photoreceptor types (phyAphyBcry1,Hennig et al., 1999) were used to investigate UV-B-mediated hypocotyl growth in etiolated seedlings and were compared to their corresponding ecotype (Landsberg erecta, Ler). The screening program and subsequent hypocotyl growth assays were performed as follows: seeds were sown on four layers of water-soaked filter paper, stratified for 24 h at 8°C in darkness and then irradiated for 24 h with continuous white light (10 W m−2) at room temperature to induce germination. Seedlings were then returned to darkness for 24 h at room temperature and then irradiated daily on two subsequent days with a 5-min UV-B pulse (5.5 W m−2 corresponding to 0.26 kJ UV-BBE m−2 h−1) followed by 1 h UV-A (6.0 W m−2). The hypocotyl length was determined 24 h after the second UV-treatment. For comparison, UV-B irradiation was converted to biologically effective UV-B [kJ biologically effective UV-B (UV-BBE) m−2 s−1] using the general plant action spectrum normalised at 300 nm (Ries et al., 2000a). Blue, red and far-red light sources were as described (Schäfer, 1977).

UV-B radiation was produced by TL40W/12 lamps, UV-A by 36 W/73 lamps (Osram, Germany), a detailed description of the spectra and the cut-off filters is given in Ries et al. (2000b). To remove damaging UV-C and UV-B irradiation cut-off filter glasses (Schott, Germany) with transmission at 280, 295, 305 and 327 nm were used. Continuous UV-B light was supplemented with UV-A by combining two TL40W/12 and one 36 W/73 lamp filtered through a 305-nm cut-off filter (6 W m−2; 305–380 nm). The same light source combined with a 345-nm cut-off filter was used to generate continuous UV-A.

Molecular analysis of uli3

The close linkage of a single-locus T-DNA insertion to the mutant locus was established by analysing the co-segregation of kanamycin-resistance and UV-B hyposensitivity. The uli3 mutant was backcrossed to the wild type (Ws) and the descendants of the F2 progeny were examined. Co-segregation was observed among 19 of these lines. A 5.1-kb fragment of genomic DNA flanking the right border of the T-DNA was cloned by using plasmid rescue techniques (Feldmann, 1992). Genomic DNA was isolated using standard procedures. Fragments flanking the left border were identified by PCR amplification with primers for the left border and ULI3-specific sequences. For the rescue with the ULI3 genomic construct, a 3.8-kb fragment was amplified from genomic DNA by PCR using a 5′ primer containing an EcoRI (5′-GCGAATTCGCAGAGAAGAAGCCCGCTGAGAAG-3′) restriction site and a 3′ primer (5′-CGGGATCCATGCCGGTTTGATCAATCTGTGC-3′). This fragment was cloned into the pGPTV-BAR plant transformation vector (Ueberlacker and Werr, 1996) using the EcoRI and SmaI restriction enzyme sites. To create the 35S-promoter:ULI3-NOS-terminator construct, the ULI3 coding region was amplified from genomic DNA by PCR using primers containing 5′- and 3′-NotI sites. The fragment was subcloned into the pRT-Ο/Not/Asc vector and finally, cloned into the pGPTV-BAR-Asc transformation vector using the AscI restriction sites of the vector (Ueberlacker and Werr, 1996). Plant transformation was performed as described (Bechthold et al., 1993).

Localisation of ULI3

The full-length ULI3 cDNA was cloned between the BamHI/SmaI sites into pMAV4 (Zimmermann et al., 1999) after amplification with gene-specific primers containing the respective restriction sites. Parsley protoplasts were transiently transformed by electroporation (Frohnmeyer et al., 1999) and the localisation of GFP fused to ULI3 was observed by a fluorescence microscope (Zeiss, Oberkochem, Germany).

Northern analysis

Seedlings were either irradiated with the indicated wavelength or kept in darkness for 3 days, then directly frozen in liquid nitrogen and stored at −80°C. RNA was isolated from frozen seedlings and tissue sections of plants using the RNAeasy kit (Qiagen, Hilden, Germany). Northern blots were prepared using 15 µg of total RNA and membranes probed with radioactively labelled ULI3, CHS, NDPK1a, PR1 and 18S DNA fragments according to standard procedures. The experiment was carried out three times.

Anthocyanin concentration

Anthocyanin levels were analysed from 3-day-old dark-grown or irradiated Arabidopsis seedlings following the protocol described in Batschauer et al. (1996). Samples of four independent experiments were determined in duplicate.

Transgenic ULI3:GUS plants and GUS staining

The ULI3-promoter sequence was isolated by amplification of the sequence located 1,6 kb upstream of the start codon using sequence-specific primers and cloned into the XbaI site of the vector pPCV812 (Koncz et al., 1994). Transformation of Arabidopsis wild-type plants (Ws) was performed as described (Koncz et al., 1994). Histochemical staining and measurements of the β-glucuronidase enzyme activity were performed using standard methods (Jefferson et al., 1986, 1987). For the histochemical staining seedlings were grown under screening conditions or the plants were grown in 8 h dark/16 h white light cycles for 6 weeks. Embedding of the organ sections in paraffin was performed as described by Jackson (1991). GUS enzyme activity was measured with 30 seedlings per experiment.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank I. Abel and S. Fleig for their excellent technical assistance; G. Neuhaus for the gift of Arabidopsis PR-1 and CHS cDNAs; P. Nick and I. Werner for critical reading of the manuscript and the Arabidopsis Stock Center in Nottingham for providing seeds. This work was partially funded by grant FR936/1 from Deutsche Forschungsgemeinschaft.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  • Ahmad, M. and Cashmore, A. (1993) HY4 gene of Arabidopsis thaliana encodes a protein with characteristics of a blue-light photoreceptor. Nature, 366, 162166.
  • Batschauer, A., Rocholl, M., Kaiser, T., Nagatani, A., Furuya, M. and Schäfer, E. (1996) Blue and UV-A light-regulated CHS expression in Arabidopsis independent of phytochrome A and phytochrome B. Plant J. 9, 6369.
  • Bechthold, N., Ellis, J. and Pelletier, G. (1993) In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. CR Acad. Sci. Paris Life Sci. 316, 11941199.
  • Bieza, K. and Lois, R. (2001) An Arabidopsis mutant tolerant to lethal ultraviolet B level shows constitutively elevated accumulation of flavonoids and other phenolics. Plant Phys. 126, 11051115.
  • Boccalandro, H., Mazza, C., Mazzella, M., Casal, J. and Ballaré, C. (2001) Ultraviolet B radiation enhances a phytochrome-B-mediated photomorphogenic response in Arabidopsis. Plant Phys. 126, 780788.
  • Bolwell, G.P., Bozak, K. and Zimmerlin, A. (1994) Plant cytochrome P450. Phytochem. 37, 14911506.
  • Caloca, M.J., Garcia-Bermejo, M.L., Blumberg, P.M. et al. (1999) β2-Chimaerin is a novel target for diacylglycerol: binding properties and changes in subcellular localization mediated by ligand binding to its C1 domain. Proc. Natl Acad. Sci. USA, 96, 1185411859.
  • Cashmore, A., Jarillo, J., Wu, Y.-J. and Liu, D. (1999) Cryptochromes: blue light receptors for plants and animals. Science, 284, 760765.
  • Chapple, C. (1998) Molecular-genetic analysis of plant cytochrome P450-dependent monooxygenases. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 311343.
  • Chen, N., Ma, W., Huang, C. and Dong, Z. (1999) Translocation of protein kinase Cepsilon and protein kinase C-delta to membrane is required for ultraviolet B-induced activation of mitogen-activated protein kinases and apoptosis. J. Biol. Chem. 274, 1538915394.
  • Christie, J. and Jenkins, G. (1996) Distinct UV-B and UV-A/blue light signal transduction pathways induce chalcone synthase gene expression in Arabidopsis cells. Plant Cell, 8, 15551567.
  • Devary, Y., Rosette, C., DiDonato, J. and Karin, M. (1993) NF-kappa B activation by ultraviolet light not dependent on a nuclear signal. Science, 261, 14421445.
  • Dieterle, M., Zhou, Y.-Z., Schäfer, E., Funk, M. and Kretsch, T. (2001) EID1, an F-box protein involved in phytochrome A-specific light signalling. Genes Dev. 15, 939944.
  • Engelberg, D., Klein, C., Martinetto, H., Struhl, K. and Karin, M. (1994) The UV response involving the Ras signalling pathway and AP-1 transcription factors is conserved between yeast and mammals. Cell, 77, 381390.
  • Feldmann, K. (1992) T-DNA insertion mutagenesis in Arabidopsis: seed infection/transformation. In: Methods in Arabidopsis Research (C.Koncz, N.-H.Chua and J.Schell, eds). London: World Scientific, pp. 274289.
  • Feldmann, K.A. and Marks, M.D. (1987) Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana: a non-tissue culture approach. Mol. General Genet. 208, 19.
  • Frohnmeyer, H., Ehmann, B., Kretsch, T., Rocholl, M., Harter, K., Nagatani, A., Furuya, M., Batschauer, A., Hahlbrock, K. and Schäfer, E. (1992) Differential usage of photoreceptors for chalcone synthase gene expression during development. Plant J. 2, 899906.
  • Frohnmeyer, H., Loyall, L., Blatt, M.R. and Grabov, A. (1999) Millisecond UV-B irradiation evokes prolonged elevation of cytosolic-free Ca2+ and stimulates gene expression in transgenic parsley cell cultures. Plant J. 20, 109118.
  • Fuglevand, G., Jackson, J.A. and Jenkins, G.I. (1996) UV-B, UV-A, blue light signal transduction pathways interact synergistically to regulate chalcone synthase gene expression in Arabidopsis. Plant Cell, 8, 23472357.
  • Garcia-Martinez, J.L. and Gil, J. (2001) Light regulation of gibberellin biosynthesis and mode of action. J. Plant Growth Regul. 20, 354368.
  • Green, R. and Fluhr, R. (1995) UV-B-induced PR-1 accumulation is mediated by active oxygen species. Plant Cell, 7, 203212.
  • Hennig, L., Poppe, C., Unger, S. and Schäfer, E. (1999) Control of hypocotyl elongation in Arabidopsis thaliana by photoreceptor interaction. Planta, 208, 257263.
  • Herrlich, P., Blattner, C., Knebel, A., Bender, K. and Rahmsdorf, H. (1997) Nuclear and non-nuclear targets of genotoxic agents in the induction of gene expression. Shared principles in yeast, rodents, man and plants. Biol. Chem. 378, 12171229.
  • Jabben, M., Beggs, C. and Schäfer, E. (1982) Dependence of Pfr/Ptot-ratios on light quality and light quantity. Photochem. Photobiol. 35, 709712.
  • Jackson, D. (1991) In-Situ Hybridisation in Plants. Molecular Plant Pathology: a Practical Approach (Bowles, D.J., Gurr, S.J. and McPherson, M., eds). Oxford: Oxford University Press.
  • Jansen, M., Gaba, V. and Greenberg, B. (1998) Higher plants and UV-B radiation: balancing damage, repair and acclimation. Trends Plant Sci. 3, 131135.
  • Jefferson, R.A., Burgess, S.M. and Hirsh, D. (1986) β-Glucuronidase from Escherichia coli as a gene fusion marker. Proc. Natl Acad. Sci. USA, 83, 84478451.
  • Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 39013907.
  • Kim, B.C., Tenessen, D. and Last, R. (1998) UV-B-induced photomorphogenesis in Arabidopsis thaliana. Plant J. 15, 667674.
  • Koncz, C., Martini, N., Szabados, L., Hrouda, M., Bachmair, A. and Schell, J. (1994) Specialized vectors for gene tagging and expression studies. In: Plant Molecular Biology Manual (Gelvin, B.S. and Schilperoort, R.A., eds). The Netherlands, Dordrecht: Kluwer Academic Press, pp. 122.
  • Landry, L., Chapple, C. and Last, R. (1995) Arabidopsis mutants lacking phenolic sunscreens exhibit enhanced ultraviolet-B injury and oxidative damage. Plant Physiol. 109, 11591166.
  • Landry, L., Stapleton, A., Lim, J., Hoffman, P., Hays, J., Walbot, V. and Last, R. (1997) An Arabidopsis photolyase mutant is hypersensitive to ultraviolet-B radiation. Proc. Natl Acad. Sci. USA, 94, 328332.
  • Li, J., Ou-Lee, T., Raba, R., Amundson, R. and Last, R. (1993) Arabidopsis flavonoid mutants are hypersensitive to UV-B irradiation. Plant Cell, 5, 171179.
  • Long, J.C. and Jenkins, G.I. (1998) Involvement of plasma membrane redox activity and calcium homeostasis in the UV-B and UV-A/blue light induction of gene expression in Arabidopsis. Plant Cell, 10, 20772086.
  • Ludwig, S.R., Habera, L.F., Dellaporta, S.L. and Wessler, S.R. (1989) Lc, a member of the maize R. gene family responsible for tissue-specific anthocyanin production, encodes a protein similar to transcriptional activators and contains the myc-homology region. Proc. Natl Acad. Sci. USA, 86, 70928006.
  • Mathews, F.S. (1985) The structure, function and evolution of cytochromes. Progr. Biophys. Mol. Biol. 45, 156.
  • Nagatani, A., Reed, J. and Chory, J. (1993) Isolation and initial characterisation of Arabidopsis mutants that are deficient in phytochrome A. Plant Physiol. 102, 269277.
  • Oancea, E. and Meyer, T. (1998) Protein kinase C as a molecular machine for decoding calcium and diacylglycerol signals. Cell, 95, 307318.
  • Ohl, S., Hahlbrock, K. and Schäfer, E. (1989) A stable blue-light-derived signal modulates ultraviolet-light-induced activation of the chalcone synthase gene in cultured parsley cells. Planta, 177, 228236.
  • Reed, J., Nagpal, P., Poole, D., Furuya, M. and Chory, J. (1993) Mutations in the gene for the red–far-red light receptor phytochrome B alter cell elongation and physiological responses throughout Arabidopsis development. Plant Cell, 5, 147157.
  • Ries, G., Buchholz, G., Frohnmeyer, H. and Hohn, B. (2000b) UV-damage-mediated induction of homologous recombination in Arabidopsis is dependent on photosynthetically active radiation. Proc. Natl Acad. Sci. USA, 97, 1342513429.
  • Ries, G., Heller, W., Puchta, H., Sandermann, H., Seidlitz, H.K. and Hohn, B. (2000a) Elevated UV-B radiation reduces genome stability in plants. Nature, 406, 98101.
  • Schäfer, E. (1977) Kunstlicht und Pflanzenzucht. In: Optische Strahlungsquellen (AlbrechtH., ed.). Grafenau: Lexika-Verlag, pp. 249266.
  • Smith, H. (2000) Phytochromes and light signal perception by plants – an emerging synthesis. Nature, 407, 585591.
  • Somers, D.E., Sharrock, R.A., Tepperman, J.M. and Quail, P.H. (1991) The hy3 long hypocotyl mutant of Arabidopsis is deficient in phytochrome B. Plant Cell, 3, 12631274.
  • Ueberlacker, B. and Werr, W. (1996) Vectors with rare-cutter restriction enzyme sites for expression of open-reading frames in transgenic plants. Mol. Breed, 2, 293295.
  • Wu, K., Tian, L., Malik, K., Brown, D. and Miki, B. (2000) Functional analysis of HD2 histone deacetylase homologues in Arabidopsis thaliana. Plant J. 22, 1927.
  • Yin, Y., Cheong, H., Friedrichsen, D., Zhao, Y., Hu, J., Mora-Garcia, S. and Chory, J. (2002) A crucial role for the putative Arabidopsis topoisomerase VI in plant growth and development. Proc. Natl Acad. Sci. USA, 99, 1019110196.
  • Zhao, Y. and Chory, J. (2001) A link between the light and gibberellin signaling cascades. Dev. Cell. Sep, 1 (3), 315316.
  • Zimmermann, S., Baumann, A., Jaekel, K., Marbach, I., Engelberg, D. and Frohnmeyer, H. (1999) UV responsive genes of Arabidopsis thaliana revealed by similarity to the GCN4 mediated UV response in yeast. J. Biol. Chem. 274, 1701717024.

GenBank accession number ULI3AF321919.