Ultraviolet-B radiation stimulates downward leaf curling in Arabidopsis thaliana

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

PLAPHY4115_proof ■ 17 December 2014 ■ 1/9

Plant Physiology and Biochemistry xxx (2014) 1e9

Contents lists available at ScienceDirect

Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy

Ultraviolet-B radiation stimulates downward leaf curling in Arabidopsis thaliana Q4

Ana Carolina Fierro a, Olivier Leroux b, Barbara De Coninck c, d, Bruno P.A. Cammue c, d, Kathleen Marchal a, c, e, Els Prinsen f, Dominique Van Der Straeten g, Filip Vandenbussche g, * a

Department of Information Technology, IMinds, Faculty of Sciences, Ghent University, B-9000 Ghent, Belgium Department of Biology, Ghent University, KL Ledeganckstraat 35, B-9000 Ghent, Belgium Centre of Microbial and Plant Genetics, KU Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium d Department of Plant Systems Biology, VIB, 9052 Ghent, Belgium e Department of Plant Biotechnology and Bioinformatics, Faculty of Sciences, Ghent University, B-9000 Ghent, Belgium f Department of Biology, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerpen, Belgium g Laboratory of Functional Plant Biology, Department of Physiology, Ghent University, KL Ledeganckstraat 35, B-9000 Ghent, Belgium b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 September 2014 Accepted 10 December 2014 Available online xxx

Plants are very well adapted to growth in ultraviolet-B (UV-B) containing light. In Arabidopsis thaliana, many of these adaptations are mediated by the UV-B receptor UV RESISTANCE LOCUS 8 (UVR8). Using small amounts of supplementary UV-B light, we observed changes in the shape of rosette leaf blades. Wild type plants show more pronounced epinasty of the blade edges, while this is not the case in uvr8 mutant plants. The UVR8 effect thus mimics the effect of phytochrome (phy) B in red light. In addition, a meta-analysis of transcriptome data indicates that the UVR8 and phyB signaling pathways have over 70% of gene regulation in common. Moreover, in low levels of supplementary UV-B light, mutant analysis revealed that phyB signaling is necessary for epinasty of the blade edges. Analysis of auxin levels and the auxin signal reporter DR5::GUS suggest that the epinasty relies on altered auxin distribution, keeping auxin at the leaf blade edges in the presence of UV-B. Together, our results suggest a co-action of phyB and UVR8 signaling, with auxin as a downstream factor. © 2014 Published by Elsevier Masson SAS.

Keywords: UV-B Arabidopsis Phytochrome Phototropin Auxin phyB UVR8

1. Introduction Being sessile organisms, plants are able to gather and process information of their light environment. During evolution, they have developed the capacity of detecting light from a variety of spectral wavebands, by using distinct photoreceptors. These include the mainly red and far red absorbing phytochromes, the blue and UV-A light absorbing phototropins, cryptochromes and ZEITLUPE (ZTL), FLAVIN BINDING KELCH REPEAT F-BOX 1 (FKF1), LOV KELCH PROTEIN 2 (LKP2) type receptors (Kami et al., 2010). Fairly recently, a

* Corresponding author. E-mail addresses: carolina.fi[email protected] (A.C. Fierro), Olivier.Leroux@ ugent.be (O. Leroux), [email protected] (B. De Coninck), [email protected] (B.P.A. Cammue), kathleen.marchal@intec. ugent.be (K. Marchal), [email protected] (E. Prinsen), Dominique. [email protected] (D. Van Der Straeten), [email protected] (F. Vandenbussche).

UV-B specific photoreceptor, UV-RESISTANCE LOCUS 8 (UVR8), has been identified. In Arabidopsis, the main processes associated with the UVR8 signaling pathway comprise photoprotection by pigment emainly flavonoid e accumulation, inhibition of elongation, reduction of rosette expansion, regulation of the circadian clock, but also resistance to Botrytis cinerea (reviewed in (Jenkins, 2014); (Hectors et al., 2007; Wargent et al., 2009)). Since good light conditions are indispensable for efficient photosynthesis, most plants are able to adapt their morphology for optimal light harvesting. For instance, in order to attain favorable light conditions, they direct their photosynthetic organs towards the light source (Hohm et al., 2013). Bending can occur in stems, but also in petioles and leaf blades (de Carbonnel et al., 2010). Leaf flattening and curling of the edges of the leaf blade in white light occur as an interaction of phytochrome and phototropin action (Kozuka et al., 2013; Johansson and Hughes, 2014). In monochromatic red light that mainly stimulates phytochrome activity, edges curl downward (epinasty), away from the light (Inoue et al.,

http://dx.doi.org/10.1016/j.plaphy.2014.12.012 0981-9428/© 2014 Published by Elsevier Masson SAS.

Please cite this article in press as: Fierro, A.C., et al., Ultraviolet-B radiation stimulates downward leaf curling in Arabidopsis thaliana, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.12.012

55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

PLAPHY4115_proof ■ 17 December 2014 ■ 2/9

2

A.C. Fierro et al. / Plant Physiology and Biochemistry xxx (2014) 1e9

2008). In addition, phytochrome B (phyB) mutants have flatter leaves than wild type in white light, indicating that phyB signaling stimulates downward curling of the leaf blade edges. Moreover, PHYTOCHROME INTERACTING FACTORS (PIFs) are acting downstream of phyB and stimulate downward leaf curling (Johansson and Hughes, 2014). By contrast, phototropin action makes the leaves flatter, hence partly overruling the phytochrome stimulated downward curling (Kozuka et al., 2013; Johansson and Hughes, 2014; Inoue et al., 2008). Leaf curling is a case of differential growth. Differential growth responses in plant organs are very often controlled by the plant hormone auxin. Mutations in proteins regulating auxin transport, biosynthesis or transcription factors that stimulate the auxin response, yield defects and abnormalities in differential growth. For instance, AUXIN RESPONSE FACTOR 7 (ARF7), and ARF19 are homologous proteins that are specifically involved in differential growth (Okushima et al., 2005). Interestingly, in leaf blades, arf7/ massugu 1/non phototropic hypocotyl 4 mutations cause allele dependent leaf curling phenotypes (Watahiki and Yamamoto, 1997; Stowe-Evans et al., 1998). In general, auxin mutations often confer a leaf curling phenotype, including mutations in AUX/IAA genes, biosynthesis genes and transporters (Li et al., 2007; Bainbridge et al., 2008). These observations suggest that auxin has a pivotal role in controlling leaf curling. Moreover, auxin appears to work downstream of the photoreceptors regulating leaf curling. Recently, phyB was speculated to promote downward leaf curling by suppressing the expression of auxin related genes (Kozuka et al., 2013). Also the photomorphogenic effects of UV-B have been linked frequently with auxin (Jansen, 2002; Hectors et al., 2012). In Arabidopsis seedlings, UV-B through UVR8 counteracts the shade avoidance phenotype in part by lowering auxin biosynthesis gene expression (Hayes et al., 2014). It was also demonstrated that the UVR8 pathway can affect differential growth in seedlings and that the auxin signal is higher at the more elongating side (Vandenbussche et al., 2014). In addition, transcript profiling suggests that also the UVR8 photoreceptor in part functions through the downregulation of auxin inducible genes (Vandenbussche et al., 2014; Favory et al., 2009). Leaf curling by increased levels of UV-B occurs in some plants, and is likely due to uneven inhibition of growth although the mechanisms involved remain elusive (Jansen et al., 1998; Wilson and Greenberg, 1993). Such a UV-B effect has not been described for Arabidopsis, and to date it remains to be seen whether UV-B affects leaf blade curling in Arabidopsis rosettes. Despite clear roles for phototropins and phytochrome B in regulating leaf blade curling respectively in blue and red light, the mechanisms by which UV-B affects this process are still unknown. Here we investigate the role of UV-B in leaf curling and present evidence that low levels of UV-B cause epinasty of the blade edges through the function of UVR8.

2.2. Plant growth conditions Plants were grown on Jiffy pellets (Jiffy, Norway) with 1/ 500 N,P,K fertilizer Wuxal 8-8-6 (Optimagro, Aventis) at first watering, and in subsequent days supplemented with tap water. The growth room had 10 h/14 h light/dark cycles and temperature was 22
C. Light was delivered mainly from above, as the walls of the growth room were painted in non-reflective black. Cool white light (Philips TL reflex) was provided at a photosynthetic photon flux density of 75 mmol m2 s1. At day 20, when the second leaf pair is developing, UV-B treatment started. UV-B treatment was performed using TL12 lamps (Philips, Eindhoven), electronically dimmed to the final required intensity of 0.200 W/m2 under cellulose acetate filter. During 3e4 weeks, UV-B light was provided for 2 h around midday at 0.2 W/m2, yielding a daily dose of 1.44 kJ. UV-B was measured with a PMA2100 with specific UV-B detector (solar light, Glenside, Pennsylvania). UV-B light was filtered through a cellulose acetate filter (UV-B treatment) to remove residual UV-C or a UV-blocking polycarbonate filter (Rachow Kunststoff-folien Gmbh, Germany) to remove all UV light.

2.3. Biometry of leaf blades and preparation of sections Using an electronic vernier caliper, the distance between the edges was measured halfway the leaf blade in curled condition and compared with the distance between the edges halfway the leaf blade along the adaxial side of the unrolled leaf. Subsequently, the values were transformed as such: the degree of epinasty ¼ 1  (distance between leaf edges/distance between leaf edges of unrolled leaf); A flat leaf has value ¼ 0. When leaf blades were hyponastic, the value was multiplied by 1, thus becoming negative. Epinastic leave blades have positive values. To obtain median transverse sections, leaves were cut in half (perpendicular to the primary vein) and fixed in 3.7% (v/v) formaldehyde, 50% (v/v) ethanol, and 5% (v/v) glacial acetic acid. Paraplast embedding was performed following dehydration in ascending ethanol/tertbutanol series and Paraplast Xtra (Sigma) infiltration. 7 mm-thick sections were cut with a Microm HM360 (Microm International GmbH, Germany) rotary microtome and collected on Polysine slides (Menzel GmbH, Germany). Slides were deparaffinized using xylene and hydrated in graded ethanol series. Fully hydrated slides were stained with 0.05% (w/v) toluidine blue O (Merck, Darmstadt, Germany, C.I. No. 52040) in 0.1 (w/v) Na2B4O7, dehydrated in graded ethanol series and mounted in Euparal (Carl Roth, Germany). Sections were observed with a Nikon Eclipse E600 microscope and images recorded with a Nikon digital camera DXM1200 were stitched using Photoshop CS3.

2.4. Infection with B. cinerea 2. Methods 2.1. Plant materials Col-0, Ler, arf7-1, arf7-1 arf19-1, nph4-1 arf19-1, and pif1-1 pif3-7 pif4-2 pif5-3 and phyB-9 were purchased at NASC (Nottingham, UK). Aux1 lax1 lax2 lax3 and PIF5 OEX were a kind gift from prof. M. Bennett (University of Nottingham)and Prof. Fankhauser (University of Lausanne) respectively. Uvr8-6 was kindly provided by Prof. Roman Ulm (University of Geneva) and 35S::PID-21 by Dr. Remko Offringa (Universiteit Leiden). Uvr8-1 (Kliebenstein et al., 2002) and pin3-3 (Friml et al., 2002) are as described. Abcb1abcb19 mutants were a gift from prof M. Geisler (University of Fribourg).

Cultivation and spore harvesting of B. cinerea strain B05.10 (provided by Rudi Aerts, Katholieke Hogeschool Kempen, Belgium) was performed as described previously (Broekaert et al., 1990). A 5 mL drop of a B. cinerea spore suspension (5  105/mL in ½ Potato Dextrose Broth (Difco)) was inoculated onto three leaves per plant. Plants were kept in transparent sealed boxes to retain almost 100% humidity after inoculation. Disease symptoms were scored by measuring the diameters of the necrotic lesions on day 2 and 3 post inoculation (dpi). Twenty-two plants per line and condition, divided over 4 boxes, were analyzed. Statistical analysis was performed by analysis of variance (ANOVA). The Tukey HSD test was applied for multiple comparisons of group means.

Please cite this article in press as: Fierro, A.C., et al., Ultraviolet-B radiation stimulates downward leaf curling in Arabidopsis thaliana, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.12.012

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

PLAPHY4115_proof ■ 17 December 2014 ■ 3/9

A.C. Fierro et al. / Plant Physiology and Biochemistry xxx (2014) 1e9

2.5. Auxin determination Leaf blades were harvested 1 h after the last UV-B treatment. Following dissection, all material was snap frozen in liquid nitrogen. Plant material was ground in a Magna Lyser (5  15 s, speed 6500 rpm.; Roche Molecular Biochemicals, Mannheim, Germany). Indole-3-acetic acid (IAA) and IAA conjugates were extracted overnight in 80% methanol. An internal standard of 100 pmol [13C6phenyl]-IAA (Cambridge Isotope Laboratory Inc., Andover,MA) was added for recovery and quantification purpose. Samples were purified by solid phase extraction combining anion exchange (DEAESephadex A25, 500 mg, GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and reversed phase cartridges (C18-RP, 500 mg, BondElut Varian) according to (Hectors et al., 2012). An ACQUITY UPLC System combined with an ACQUITY TQD mass spectrometer (Waters, Milford, MA) was used for quantification. Samples were injected on a VanGuard pre-column (BEH C18, 1.7 mm, 2.1  5 mm2; Waters) coupled to a reversed phase column (BEH C18, 1.7 mm, 2.1  50 mm2;Waters). ESI(þ)-MRM mode was used for quantification based on specific diagnostic transitions for IAA and 13C6-IAA (190 > 130 and 196 > 136, respectively). For specific settings and chromatographic conditions see (Hectors et al., 2012). Concentrations were calculated using the principle of isotope dilution. 2.6. Glucuronidase assay 1 h after UV-B treatment, leaf blades of leaf 8 were harvested and put in 90% ice cold acetone for 30 min. Samples were washed with 0.1 M phosphate buffer, and subsequently incubated in GUSbuffer 0.1 M phosphate, 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6, 2 mM X-Gluc (Immunosource, Halle, Belgium) for 24 h at 37
C. Afterwards, the samples were kept for 2 weeks in 70% ethanol. Leaf blades were photographed using a Canon 500D camera mounted on a Stemi SV11 stereomicroscope (Zeiss, Germany). 2.7. Meta-analysis of gene expression data Microarray data preprocessing was performed as follows. Raw microarray data was downloaded from ArrayExpress at EMBL-EBI for the following datasets: E-MEXP-1957, E-GEOD-627, E-GEOD17845, E-GEOD-17159, and E-GEOD-8951. Raw data from selected experiments was normalized using RMA from the Bioconductor affy package. Raw data was not available for E-MEXP-1957 and thus the GC-RMA processed data was used instead. Differentially expressed genes for each dataset and their respective contrasts of interest were obtained with the LIMMA R package (Smyth, 2004) at a False Discovery Rate (FDR) of 5%. Multiple testing correction of pvalues was done using the Benjamini & Hochberg method (Benjamini and Hochberg, 1995). Replicated samples were averaged and log-ratios were calculated for all the contrasts of interest. Gene names and gene descriptions were retrieved from www. arabidopsis.org. Statistical significance for the overlap of differentially expressed genes between 2 contrasts was calculated with a Fisher's exact test. Gene selection was performed as follows: (i) UVB-uvr8-responsive genes were obtained from the dataset E-MEXP-1957. Differentially expressed genes between Col0 samples treated for 1 h with 305 nm cut off filters and Col-0 treated only with 345 nm cut off filters (no UV-B). A minimum log fold-change of 1 was used to restrict the list to most changing genes. No genes were differentially expressed between uvr8 mutant samples at 1 h 305 nm cut off treatment versus no UV-B treatment. For clarity, statistically nondifferentially expressed genes in uvr8 samples with

3

moderate mean log-fold-change (above 0.5) were excluded from the final gene list. (ii) UVB-specific genes were defined as UVR8-responsive genes that are not differentially expressed in any other of the selected contrasts (FDR at 5%). (iii) UVB-specific down-regulated and phyB controlled auxin upregulated genes must fulfill two criteria: First the genes must be down-regulated (i.e. differentially expressed at an FDR of 5% with a negative log fold-change) in at least one of the following contrasts: WT 1 h red, YHB-darkness or pifqdarkness versus wild-type in darkness, or upregulated in leaf blade EOD Fr versus darkness. Secondly, the genes must be up-regulated (i.e. differentially expressed at an FDR of 5% with a positive log fold-change) in: WT auxin/WT none. 3. Results 3.1. UVR8 action confers changes in leaf architecture without increasing resistance to B. cinerea UV-B inhibits leaf expansion (Hectors et al., 2007) and UVR8 was shown to be responsible for a large part of UV-B mediated reduction in size of Arabidopsis leaves (Wargent et al., 2009). We used growth conditions in which light was mainly coming from above, with omission or addition of a low dose of UV-B (1.44 kJ daily). A reduction in leaf blade size due to UV-B was visible, confirming literature data (Fig. 1A). However, we observed an additional trait, in that leaf blades from UV-B treated plants had more epinastic edges than untreated plants (Fig. 1B and C). This was the case for wild type Col-0 and not visible in uvr8-6 mutant lines. In Ler background, typically leaves curled up, and were flattened by the effect of supplemental UV-B, a phenotype absent in uvr8-1 mutants (Fig. 1B and C). It has been suggested that plants often “choose” between regulation of growth and defense (Leone et al., 2014; Belkhadir et al., 2014). To investigate whether our low levels of UV-B could stimulate such defense mechanisms, UV-B pretreated rosettes were inoculated with B. cinerea. The effect on the plants was scored by evaluating the lesion diameter caused by the fungal infection. There was no effect of the UV-treatment on the development of the disease (Fig. 1D). This indicates that the effects of UVR8 signaling on growth can be separated from those in plant defense in low levels of UV-B. Interestingly, at higher doses, under sunlight, UVR8 protects plants from B. cinerea, without affecting plant growth, again pointing to a mechanistic separation of both processes (Demkura and Ballare, 2012). 3.2. Overlaps in UVR8 regulated and red light regulated gene expression Red light through phytochrome signaling (Kozuka et al., 2013; Johansson and Hughes, 2014) and UV-B through UVR8 signaling (Fig. 1) have similar effects on leaf blade curling. Therefore, it is possible that these two pathways control a shared output mechanism that leads to leaf edge epinasty. To unravel the network of possible overlapping components involved, we performed a metaanalysis of micro-array data available at ArrayExpress (European Bioinformatics Institute), using experiments that were designed to study the effects of UV-B and red light. In addition, since nastic responses, such as leaf edge curling, have been frequently documented to be auxin controlled, and since the diminished phyB signaling was shown to be linked with a signature of enhanced auxin response combined with less downward leaf blade curling (Kozuka et al., 2013; Johansson and Hughes, 2014), a dataset on auxin controlled gene expression was also included. Where possible, we selected datasets with short exposure to light or plant

Please cite this article in press as: Fierro, A.C., et al., Ultraviolet-B radiation stimulates downward leaf curling in Arabidopsis thaliana, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.12.012

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

PLAPHY4115_proof ■ 17 December 2014 ■ 4/9

4

A.C. Fierro et al. / Plant Physiology and Biochemistry xxx (2014) 1e9

Fig. 1. Phenotypes of 38 day old UV-B treated rosettes. (A) Surface area of leaf 8 of wild type and uvr8 mutant. Error bars indicate SD, n  7. * indicates statistically significant difference from non-treated control (p < 0.05). (B) Photographs of median transverse sections through leaf 8. (C) Degree of leaf 8 epinasty in wild type (Col, Ler) and uvr8 mutant plants. Error bars indicate SD, n  7. * indicates statistically significant difference from non-treated control (p < 0.05). (D) Analysis of B. cinerea infection of UV-B pretreated plants, 2 and 3 days after inoculation (dpi). Error bars indicate SE, n ¼ 4.

hormone. Two sets of long term constant dark conditions were also included, where either the effect of a light-independent active form of phyB (YHB) or the absence of four of the PIFs (pif1,3,4,5) was studied. In particular, we focused on possible gene expression overlaps between regulation by UVR8-signaling (Favory et al., 2009), red light and phyB effects (Leivar et al., 2012; Hu et al., 2009), association with leaf blades with low phyB activity (Kozuka et al., 2013), and auxin regulation by ARF7 and ARF19 (Okushima et al., 2005). The heat maps resulting from the hierarchical clustering reveal a very strong overlap between UVR8 and phyB controlled genes in Arabidopsis seedlings, with over 70% (p-value ¼ 2.04e138) of UVR8 regulated genes being under phyB control as well (Fig. 2; supplementary table 1). Only a small number of genes (cluster1 and 2) of various functions appeared UVR8 specific (Table 1). The phyB signaling effect was very clear in the YHB samples that shared most of gene regulation with the 1 h red light treatment of the wild type, whereas the effect of the quadruple pif1,3,4,5 mutation was much less visible (Fig. 2). This could be associated with the strength of the phenotypes of both type of plant lines, with YHB having a stronger cop1-like phenotype, or the role of these particular PIFs mainly in prolonged light exposures (Hu et al., 2009; Leivar et al., 2009, 2008a). Moreover, redundancy with other PIFs may also come

into play (Leivar et al., 2008b). Candidate genes of which the product can stimulate downward leaf curling, should be found among those that are reduced at the transcript level by UV-B through UVR8 and elevated by inactivation of phyB in the leaf, or elevated by UV-B through UVR8 and reduced by inactivation of phyB in the leaf. One cluster (cluster 3) in particular consisted of genes that were downregulated by UVR8 signaling, yet induced upon end of day far-red treatment, which inhibits phyB activity. This cluster showed many genes in common with ARF7 and ARF19 dependent auxin induction (Table 2). These data indicate a participation of the plant hormone auxin in generating both UVR8 and phyB responses.

3.3. Phytochrome signaling components and adequate auxin responses are necessary for the UV-B effect on leaf blade curling The transcriptome meta-analysis suggests that somehow phyB and auxin signaling are involved in the UV-B leaf curling response. First, to investigate the role of phytochrome signaling more directly, a number of mutants were grown in the absence and presence of UV-B. phyB mutants and PIF5 OVEREXPRESSOR (PIF5 OEX) plants had constitutive upward curling (Fig. 3). pif1,3,4,5 mutants had downward curling which was only slightly enhanced by UV-B. These data

Please cite this article in press as: Fierro, A.C., et al., Ultraviolet-B radiation stimulates downward leaf curling in Arabidopsis thaliana, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.12.012

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

PLAPHY4115_proof ■ 17 December 2014 ■ 5/9

A.C. Fierro et al. / Plant Physiology and Biochemistry xxx (2014) 1e9

5

Fig. 2. Heat map resulting from the micro-array meta-analysis of UV-B, red light and auxin related experiments. Genes were preselected for UVR8 regulation. Inductions are shown in red, repressions in blue; white indicates no significant change. Values in the color key represent log fold changes. On the left: grey bars: UV-B down, up by inactivation of phyB, orange bars: UVR8 specific genes. WT: wild type Col-0, YHB: light independent active phyB; pifq: quadruple pif1,3,4,5 mutant, FR: 2 h End-of-day Far Red light treatment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

indicate that phyB signaling through modulation of PIF proteins is necessary for leaf curling in UV-B. Second, to further explore the auxin metabolism in leaf blades under these conditions, the blade of leaf 8 was harvested 30 min after the treatment on day 38, and auxin extracted and determined using UPLC/MSeMS. No differences were found in total levels of free auxin, nor in the levels of conjugated auxins in wild type plants treated with UV-B versus untreated controls (Fig. 4A and B). Also, no effect of uvr8 mutations was visible. If auxin in any way would be involved in the generation of the phenotype, this should be visible using a marker for auxin signaling. The activity of glucuronidase expressed under control of the synthetic auxin inducible DR5 promoter was used to evaluate the effect of UV-B on the auxin signal. DR5 promoter activity was found mainly in the hydathodes in leaf blades of control plants (Fig. 4C). In UV-B exposed plants, a very clear blue stained band along the edge of the leaf blade was visible (Fig. 4C). This indicates that a larger area around the leaf edges contains a high auxin signal upon UV-B treatment. Together with the auxin measurements, this may point to a difference in auxin distribution, or a local difference in auxin sensitivity by signal modulation. We next investigated a number of auxin signaling and transport mutants for their leaf curling phenotype (Fig. 4D). Arf7 mutants had epinastic leaf edges, but UV-B was able to enhance this phenotype. The arf7arf19 double mutants had equally constitutively epinastic leaf edges irrespective of the presence of UV-B. In addition, aux1 lax2 lax2 lax3 mutants, defective in all auxin influx carriers also showed severe epinastic edges, with only a relatively small enhancement by UV-B. Plants with combined mutations in the auxin efflux proteins abcb1 and abcb19 caused epinastic leaves and did not respond to UV-B. Pin3-3 mutants and 35S::PID plants, known to have phenotypes associated with diminished auxin efflux (Friml et al., 2002; Benjamins et al., 2001), had overall slightly less downward curling of leaf edges, but were still responsive to UV-B albeit to a lesser extent than the wild type. Together these

observations are indicative for the necessity of correct auxin transport for leaf curling in general, including the regulation by UVB. 4. Discussion 4.1. UV-B regulates leaf curling similarly as red light We found that low levels of UV-B promote downward leaf curling in Arabidopsis leaf blades in an UVR8 dependent manner. Downward curling or epinasty of leaf edges also occurs in monochromatic red light and is believed to be a phyB effect (Kozuka et al., 2013; Johansson and Hughes, 2014). Hence UV-B and red light affect leaf curling similarly. It remains to be seen how UVR8 and phyB are mechanistically connected, but downstream both pathways have many genes regulated in common (Fig. 2). Moreover, our mutant analysis has shown that phyB and the well-known phytochrome downstream components PIFs are necessary for the UV-B response (Fig. 3A). These data indicate that UV-B signaling, in this situation, may be just enhancing phyB effects. A similar sort of interplay can be found during shade avoidance, where UVR8 signaling counteracts the diminished activity of phyB (Hayes et al., 2014). Co-action of UV-B and phyB signaling has also been observed during de-etiolation of seedlings. Cotyledon opening in red light is enhanced by UV-B pretreatment, and relies on a process that is phyB dependent (Boccalandro et al., 2001). For the latter, to date, involvement of UVR8 has not yet been demonstrated. The above evidence for connections between the phyB and UVR8 pathway may lead the way to more profound studies on mechanistic photosignaling interactions. Such interactions are indeed not uncommon in plants and can even occur at the receptor level. Phototropins were shown to interact physically with phytochromes (Jaedicke et al., 2012), cryptochrome 1 interacts with phyA (Ahmad et al., 1998) and cryptochrome 2 interacts with phyB (Mas et al., 2000). However, the interactions between phyB and UVR8

Please cite this article in press as: Fierro, A.C., et al., Ultraviolet-B radiation stimulates downward leaf curling in Arabidopsis thaliana, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.12.012

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

PLAPHY4115_proof ■ 17 December 2014 ■ 6/9

6

A.C. Fierro et al. / Plant Physiology and Biochemistry xxx (2014) 1e9

Table 1 Cluster 1 and 2 type genes: regulated by UVR8 but not by phyB signaling. Tair. ID

Short_description

AT5G56620 NAC domain containing protein 99 AT1G77360 Tetratricopeptide repeat (TPR)-like superfamily protein AT5G44160 C2H2-like zinc finger protein AT5G19200 NAD(P)-binding Rossmann-fold superfamily protein AT3G58120 Basic-leucine zipper (bZIP) transcription factor family protein AT1G74190 Receptor like protein 15 AT3G15760 AT2G45800 GATA type zinc finger transcription factor family protein AT2G39950 AT2G17270 Phosphate transporter 3; 3

Symbol NAC099, anac099 APPR6 AtIDD8, IDD8, NUC TSC10B ATBZIP61, BZIP61 RLP15, AtRLP15 PLIM2a

MPT1, PHT3; 3

AT4G27652 AT1G05100 Mitogen-activated protein kinase kinase kinase 18 MAPKKK18 AT3G30845 AT2G36970 UDP-Glycosyltransferase superfamily protein AT5G66985 AT2G34610 AT1G65870 Disease resistance-responsive (dirigent-like protein) family protein AT1G34290 Receptor like protein 5 RLP5, AtRLP5 AT2G34360 MATE efflux family protein AT3G17150 Plant invertase/pectin methylesterase inhibitor superfamily protein AT1G66540 Cytochrome P450 superfamily protein AT4G15100 Serine carboxypeptidase-like 30 scpl30 AT1G23010 Cupredoxin superfamily protein LPR1 AT2G30730 Protein kinase superfamily protein AT1G08270

signaling are possibly at the level of the PIFs. It was shown recently that COP1, a component of UVR8 signaling, and phyB interact physically with the transcription factor PIF3-like 1 (PIL1), with COP1 stimulating degradation and phyB accumulation of PIL1 (Luo et al., 2014). PIL1 then negatively affects transcriptional activity of other PIFs, thus causing photomorphogenesis. As it is believed that UVR8 inhibits COP1 E3-ligase function resulting in stabilization of its target photomorphogenesis promoting transcription factors (Huang et al., 2013), this may explain the enhancement of phyB effects.

Table 2 Cluster 3 type genes: downregulated by 1 h UV-B, upregulated by 2 h auxin in an ARF7 and ARF19 dependent manner. Tair. ID

Short_description

symbol

AT1G29440 SAUR-like auxin-responsive protein family AT1G29450 SAUR-like auxin-responsive protein family AT1G29460 SAUR-like auxin-responsive protein family AT1G29500 SAUR-like auxin-responsive protein family AT1G29510 SAUR-like auxin-responsive protein family AT2G26710 Cytochrome P450 superfamily protein AT2G40610 Expansin A8

SAUR63

AT3G03830 SAUR-like auxin-responsive protein family AT3G03840 SAUR-like auxin-responsive protein family AT3G14370 Protein kinase superfamily protein AT3G42800 AT3G50340 AT3G60390 Homeobox-leucine zipper protein 3 AT3G63440 Cytokinin oxidase/dehydrogenase 6 AT4G09890 Protein of unknown function (DUF3511) AT4G16515 AT4G31910 HXXXD-type acyl-transferase family protein AT4G32280 Indole-3-acetic acid inducible 29 AT5G18060 SAUR-like auxin-responsive protein family AT5G47370 Homeobox-leucine zipper protein 4 (HB-4)/HD-ZIP protein AT5G62280 Protein of unknown function (DUF1442)

SAUR68 CYP734A1, BAS1, CYP72B1 ATEXPA8, ATHEXP ALPHA 1.11, ATEXP8, EXPA8, EXP8 SAUR28 SAUR27 WAG2

HAT3 CKX6, ATCKX6, ATCKX7

RGF6, CLEL 6, GLV1 BAT1, PIZ IAA29 SAUR23 HAT2

4.2. Possible ecological relevance of UV-B mediated downward leaf curling In white light, the phyB effect on leaf blades is usually masked by the action of phototropins, which orchestrate the flattening of the leaf in order to optimize photosynthesis. Inhibition of epinasty by phototropins is especially useful when low levels of photosynthetically active radiation (PAR) is present and the leaf should expose as much as possible a large surface directly to incoming light. By contrast, detection of levels of UV-B above a certain threshold may function as a signal for presence of or for anticipating satisfactory or superfluous PAR. Exposure to excess light is undesirable for plants, as it causes production of potentially harmful reactive oxygen species (Horton, 2012). Leaf curling thus prevents overexposure of much of the leaf tissue to UV-rich sunlight. Interestingly, since especially the edges curl downward, the hydathodes are also more protected from direct light. It has been shown in the past that UV-B radiation can lead to photodestruction of auxin (Ros and Tevini, 1995). The hydathodes are known to have

Fig. 3. Degree of epinasty of leaf 7 edges in wild type (Col) and selected phytochrome signaling mutants: phyB-9, pif1,3,4,5, PIF5 OEX. Error bars indicate SD, n  7. Statistically significant difference from untreated control: *: p < 0.05, **:p < 0.01.

elevated expression of auxin biosynthesis genes and auxin signaling (Aloni et al., 2003; Wang et al., 2011; Esteve-Bruna et al., 2013). Auxin by itself is a very important growth regulator for plant architecture. Hence, such a mechanism may protect these potentially important sources of auxin both from damage of oxidative stress and direct photodestruction caused by excess light and significant UV-B levels.

Please cite this article in press as: Fierro, A.C., et al., Ultraviolet-B radiation stimulates downward leaf curling in Arabidopsis thaliana, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.12.012

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

PLAPHY4115_proof ■ 17 December 2014 ■ 7/9

A.C. Fierro et al. / Plant Physiology and Biochemistry xxx (2014) 1e9

7

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 Fig. 4. The plant hormone auxin in UV-B-treated rosette leaf blades. (A) Free auxin content of leaf 8 blades in wild type (Col, Ler) and uvr8 mutant, in control and UV-B treatment 95 conditions. Error bars are SD, n ¼ 8. Error bars are SD, n ¼ 8. (B) As (A) but for conjugated auxins. (C) Photograph of the abaxial side of leaf blade 8 of a DR5::GUS rosette grown in the 96 absence (control) or the presence of UV-B (þUV-B) and subsequently subjected to a glucuronidase assay. (D) Degree of leaf 8 epinasty in wild type (Col) and auxin mutants. Error 97 bars indicate SD, n  7. Statistically significant difference from untreated control: **: p < 0.01; *: p < 0.05. 98 99 very specific range of differential auxin concentrations across the 4.3. How can auxin mediate the UV-B generated phenotype? 100 leaf, occurring in natural conditions of ambient light. 101 Alternatively, as suggested by our data, auxin transport may be The flatter leaves in phyB mutants have been speculated to be a 102 affected in an as yet mechanistically unknown manner. The pattern result of stronger auxin signaling (Kozuka et al., 2013). Conversely, 103 of DR5:GUS activity (Fig. 4C (Hectors et al., 2012)) suggests that downward inclination of leaf edges could be the result of dimin104 transport from the edges/hydathodes to the center of the leaf is ished auxin signaling. Our data seem to confirm this reasoning. 105 inhibited in UV-B. UV-B inducible flavonols accumulate at the First, just like phyB, UVR8 is responsible for downward leaf curling 106 adaxial side of the leaf and have been suggested as inhibitors of and reduces auxin inducible gene expression (Figs. 1 and 2). Sec107 auxin transport (Yin et al., 2013; Hectors et al., 2014). Hence, it is ond, arf7arf19 loss of function mutants that confer diminished 108 possible that the auxin in UV-B treated plants remains at the leaf auxin signaling have downward leaf curling (Figs. 2 and 3). In 109 margins, causing expansion of the adaxial side and causing the leaf addition, downward leaf curling has been related with auxin 110 to curve downward. However, this hypothesis is not easy to inactivation and auxin insensitivity previously (Cheng et al., 2004; 111 reconcile with the phenotype of the arf7 mutants. ARF7 has an Jin et al., 2013). However, the picture appears more complex than 112 overall transcriptional expression in leaf primordia (Hardtke et al., this. In the past, both up- and down-curling of leaves has been 113 2004). One would expect that the leaf in an arf7 mutant curls up, linked with increased auxin signaling in the leaf edges (Song et al., 114 since the gene is expressed at the adaxial side where the auxin 2012). Moreover, the gain of function Dof5.1-D mutants display 115 transport is inhibited by flavonols, unless we assume that in a flat reduced auxin responses at the molecular level, while having up116 leaf situation ARF7 is somehow more active at the abaxial side. In curled leaves (Kim et al., 2010). Also, mutants overproducing 117 that case, again the most obvious cause would be a differential auxins were found to have downward leaf curling and many gain of 118 function mutations in aux/iaa genes, considered to be auxin regulation by specific AUX/IAAs. 119 insensitive, have upcurling of leaves (Li et al., 2007). These seem120 ingly contradictory observations could be the result of suboptimal 121 Acknowledgements and supra-optimal concentrations for auxin to stimulate elonga122 tion, in combination with the differentiality and levels of gene 123 The authors would like to thank Profs. Bennett, Offringa, Fankexpression of the ARF and AUX/IAA genes involved. AUX/IAA co124 hauser and Ulm for sharing the aux1lax2lax3lax4, 35S::PID, PIF5 OEX receptors for auxin differ in their ability to bind auxin, and may 125 and uvr8-6 respectively. This work was supported by grants from hence be responsive to different auxin concentrations (Villalobos the Research Foundation Flanders (G.0656.13N) and Ghent Uni- Q1 126 et al., 2012). Beyond this, their spatial expression pattern, along 127 versity to DVDS. This work was performed in the frame of the COSTwith that of their target ARFs will affect whether one side of a tissue 128 Action program [FA0906] and supported by the Research Foundawill elongate more than another. Hence, the ARFs responsible for 129 tion Flanders (FWO) [grant W0.038.04N] to EP and DVDS. BDC acregulating leaf curling in wild type plants may be regulated by a 130 knowledges the receipt of a postdoctoral fellowship from the Please cite this article in press as: Fierro, A.C., et al., Ultraviolet-B radiation stimulates downward leaf curling in Arabidopsis thaliana, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.12.012

1 2 3 4 5 6 7 8 Q2 9 10 11 12 13 14 15 16 17 18 19 Q3 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

PLAPHY4115_proof ■ 17 December 2014 ■ 8/9

8

A.C. Fierro et al. / Plant Physiology and Biochemistry xxx (2014) 1e9

Research Foundation Flanders (FWO) (FWO/12A7213N). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2014.12.012. References Ahmad, M., Jarillo, J.A., Smirnova, O., Cashmore, A.R., 1998. The CRY1 blue light photoreceptor of Arabidopsis interacts with phytochrome A in vitro. Mol. Cell 1, 939e948. Aloni, R., Schwalm, K., Langhans, M., Ullrich, C.I., 2003. Gradual shifts in sites of freeauxin production during leaf-primordium development and their role in vascular differentiation and leaf morphogenesis in Arabidopsis. Planta 216, 841e853. Bainbridge, K., Guyomarc'h, S., Bayer, E., Swarup, R., Bennett, M., Mandel, T., Kuhlemeier, C., 2008. Auxin influx carriers stabilize phyllotactic patterning. Gene Dev. 22, 810e823. Belkhadir, Y., Yang, L., Hetzel, J., Dangl, J.L., Chory, J., 2014. The growth-defense pivot: crisis management in plants mediated by LRR-RK surface receptors. Trends Biochem. Sci.. Benjamini, Y., Hochberg, Y., 1995. Controlling the false discovery rate e a practical and powerful approach to multiple testing. J. Roy. Stat. Soc. B Met. 57, 289e300. Benjamins, R., Quint, A., Weijers, D., Hooykaas, P., Offringa, R., 2001. The PINOID protein kinase regulates organ development in Arabidopsis by enhancing polar auxin transport. Development 128, 4057e4067. Boccalandro, H.E., Mazza, C.A., Mazzella, M.A., Casal, J.J., Ballare, C.L., 2001. Ultraviolet B radiation enhances a phytochrome-B-mediated photomorphogenic response in Arabidopsis. Plant Physiol. 126, 780e788. Broekaert, W.F., Terras, F.R.G., Cammue, B.P.A., Vanderleyden, J., 1990. An automated quantitative assay for fungal growth-inhibition. FEMS Microbiol. Lett. 69, 55e59. Cheng, Y.F., Dai, X.H., Zhao, Y., 2004. AtCAND1, a HEAT-repeat protein that participates in auxin signaling in Arabidopsis. Plant Physiol. 135, 1020e1026. de Carbonnel, M., Davis, P., Roelfsema, M.R.G., Inoue, S., Schepens, I., Lariguet, P., Geisler, M., Shimazaki, K., Hangarter, R., Fankhauser, C., 2010. The Arabidopsis PHYTOCHROME KINASE SUBSTRATE2 protein is a phototropin signaling element that regulates leaf flattening and leaf positioning. Plant Physiol. 152, 1391e1405. Demkura, P.V., Ballare, C.L., 2012. UVR8 mediates UV-B-induced Arabidopsis defense responses against Botrytis cinerea by controlling sinapate accumulation. Mol. Plant 5, 642e652. Esteve-Bruna, D., Perez-Perez, J.M., Ponce, M.R., Micol, J.L., 2013. incurvata13, a Novel allele of AUXIN RESISTANT6, reveals a specific role for auxin and the SCF complex in Arabidopsis embryogenesis, vascular specification, and leaf flatness. Plant Physiol. 161, 1303e1320. Favory, J.J., Stec, A., Gruber, H., Rizzini, L., Oravecz, A., Funk, M., Albert, A., Cloix, C., Jenkins, G.I., Oakeley, E.J., Seidlitz, H.K., Nagy, F., Ulm, R., 2009. Interaction of COP1 and UVR8 regulates UV-B-induced photomorphogenesis and stress acclimation in Arabidopsis. Embo J. 28, 591e601. Friml, J., Wisniewska, J., Benkova, E., Mendgen, K., Palme, K., 2002. Lateral relocation of auxin efflux regulator PIN3 mediates tropism in Arabidopsis. Nature 415, 806e809. Hardtke, C.S., Ckurshumova, W., Vidaurre, D.P., Singh, S.A., Stamatiou, G., Tiwari, S.B., Hagen, G., Guilfoyle, T.J., Berleth, T., 2004. Overlapping and nonredundant functions of the Arabidopsis auxin response factors MONOPTEROS and NONPHOTOTROPIC HYPOCOTYL 4. Development 131, 1089e1100. Hayes, S., Velanis, C.N., Jenkins, G.I., Franklin, K.A., 2014. UV-B detected by the UVR8 photoreceptor antagonizes auxin signaling and plant shade avoidance. Proc. Natl. Acad. Sci. U. S. A. 111, 11894e11899. Hectors, K., Prinsen, E., De Coen, W., Jansen, M.A.K., Guisez, Y., 2007. Arabidopsis thaliana plants acclimated to low dose rates of ultraviolet B radiation show specific changes in morphology and gene expression in the absence of stress symptoms. New Phytol. 175, 255e270. Hectors, K., van Oevelen, S., Guisez, Y., Prinsen, E., Jansen, M.A.K., 2012. The phytohormone auxin is a component of the regulatory system that controls UVmediated accumulation of flavonoids and UV-induced morphogenesis. Physiol. Plant. 145, 594e603. Hectors, K., Van Oevelen, S., Geuns, J., Guisez, Y., Jansen, M.A., Prinsen, E., 2014. Dynamic changes in plant secondary metabolites during UV acclimation in Arabidopsis thaliana. Physiol. Plant.. Hohm, T., Preuten, T., Fankhauser, C., 2013. Phototropism: translating light into directional growth. Am. J. Bot. 100, 47e59. Horton, P., 2012. Optimization of light harvesting and photoprotection: molecular mechanisms and physiological consequences. Philos. Trans. R. Soc. B 367, 3455e3465. Hu, W., Su, Y.S., Lagarias, J.C., 2009. A light-independent allele of phytochrome B faithfully recapitulates photomorphogenic transcriptional networks. Mol. Plant 2, 166e182. Huang, X., Ouyang, X.H., Yang, P.Y., Lau, O.S., Chen, L.B., Wei, N., Deng, X.W., 2013. Conversion from CUL4-based COP1-SPA E3 apparatus to UVR8-COP1-SPA

complexes underlies a distinct biochemical function of COP1 under UV-B. Proc. Natl. Acad. Sci. U. S. A. 110, 16669e16674. Inoue, S., Kinoshita, T., Takemiya, A., Doi, M., Shimazaki, K., 2008. Leaf positioning of Arabidopsis in response to blue light. Mol. Plant 1, 15e26. Jaedicke, K., Lichtenthaler, A.L., Meyberg, R., Zeidler, M., Hughes, J., 2012. A phytochrome-phototropin light signaling complex at the plasma membrane. Proc. Natl. Acad. Sci. U. S. A. 109, 12231e12236. Jansen, M.A.K., 2002. Ultraviolet-B radiation effects on plants: induction of morphogenic responses. Physiol. Plant. 116, 423e429. Jansen, M.A.K., Gaba, V., Greenberg, B.M., 1998. Higher plants and UV-B radiation: balancing damage, repair and acclimation. Trends Plant Sci. 3, 131e135. Jenkins, G.I., 2014. The UV-B photoreceptor UVR8: from structure to physiology. Plant Cell 26, 21e37. Jin, S.H., Ma, X.M., Han, P., Wang, B., Sun, Y.G., Zhang, G.Z., Li, Y.J., Hou, B.K., 2013. UGT74D1 is a novel auxin glycosyltransferase from Arabidopsis thaliana. PLoS One 8. Johansson, H., Hughes, J., 2014. Nuclear phytochrome B regulates leaf flattening through phytochrome interacting factors. Mol. Plant. Kami, C., Lorrain, S., Hornitschek, P., Fankhauser, C., 2010. Light-regulated plant growth and development. Curr. Top. Dev. Biol. 91, 29e66. Kim, H.S., Kim, S.J., Abbasi, N., Bressan, R.A., Yun, D.J., Yoo, S.D., Kwon, S.Y., Choi, S.B., 2010. The DOF transcription factor Dof5.1 influences leaf axial patterning by promoting revoluta transcription in Arabidopsis. Plant J. 64, 524e535. Kliebenstein, D.J., Lim, J.E., Landry, L.G., Last, R.L., 2002. Arabidopsis UVR8 regulates ultraviolet-B signal transduction and tolerance and contains sequence similarity to human regulator of chromatin condensation 1. Plant Physiol. 130, 234e243. Kozuka, T., Suetsugu, N., Wada, M., Nagatani, A., 2013. Antagonistic regulation of leaf flattening by phytochrome B and phototropin in Arabidopsis thaliana. Plant Cell Physiol. 54, 69e79. Leivar, P., Monte, E., Oka, Y., Liu, T., Carle, C., Castillon, A., Huq, E., Quail, P.H., 2008. Multiple phytochrome-interacting bHLH transcription factors repress premature seedling photomorphogenesis in darkness. Curr. Biol. 18, 1815e1823. Leivar, P., Monte, E., Al-Sady, B., Carle, C., Storer, A., Alonso, J.M., Ecker, J.R., Quail, P.H., 2008. The Arabidopsis phytochrome-interacting factor PIF7, together with PIF3 and PIF4, regulates responses to prolonged red light by modulating phyB levels. Plant Cell 20, 337e352. Leivar, P., Tepperman, J.M., Monte, E., Calderon, R.H., Liu, T.L., Quail, P.H., 2009. Definition of early transcriptional circuitry involved in light-induced reversal of pif-imposed repression of photomorphogenesis in young Arabidopsis seedlings. Plant Cell 21, 3535e3553. Leivar, P., Tepperman, J.M., Cohn, M.M., Monte, E., Al-Sady, B., Erickson, E., Quail, P.H., 2012. Dynamic antagonism between phytochromes and PIF family basic helix-loop-helix factors induces selective reciprocal responses to light and shade in a rapidly responsive transcriptional network in Arabidopsis. Plant Cell 24, 1398e1419. Leone, M., Keller, M.M., Cerrudo, I., Ballare, C.L., 2014. To grow or defend? Low red: far-red ratios reduce jasmonate sensitivity in Arabidopsis seedlings by promoting DELLA degradation and increasing JAZ10 stability. New Phytol.. Li, L.C., Kang, D.M., Chen, Z.L., Qu, L.J., 2007. Hormonal regulation of leaf morphogenesis in Arabidopsis. J. Integr. Plant Biol. 49, 75e80. Luo, Q., Lian, H.L., He, S.B., Li, L., Jia, K.P., Yang, H.Q., 2014. COP1 and phyB physically interact with PIL1 to regulate its stability and photomorphogenic development in Arabidopsis. Plant Cell. 26, 2441e2456. Mas, P., Devlin, P.F., Panda, S., Kay, S.A., 2000. Functional interaction of phytochrome B and cryptochrome 2. Nature 408, 207e211. Okushima, Y., Overvoorde, P.J., Arima, K., Alonso, J.M., Chan, A., Chang, C., Ecker, J.R., Hughes, B., Lui, A., Nguyen, D., Onodera, C., Quach, H., Smith, A., Yu, G.X., Theologis, A., 2005. Functional genomic analysis of the AUXIN RESPONSE FACTOR gene family members in Arabidopsis thaliana: unique and overlapping functions of ARF7 and ARF19. Plant Cell 17, 444e463. Ros, J., Tevini, M., 1995. Interaction of Uv-radiation and iaa during growth of seedlings and hypocotyl segments of sunflower. J. Plant Physiol. 146, 295e302. Smyth, G.K., 2004. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3. Article3. Song, J.B., Huang, S.Q., Dalmay, T., Yang, Z.M., 2012. Regulation of leaf morphology by microRNA394 and its target LEAF CURLING RESPONSIVENESS. Plant Cell Physiol. 53, 1283e1294. Stowe-Evans, E.L., Harper, R.M., Motchoulski, A.V., Liscum, E., 1998. NPH4, a conditional modulator of auxin-dependent differential growth responses in Arabidopsis. Plant Physiol. 118, 1265e1275. Vandenbussche, F., Tilbrook, K., Fierro, A.C., Marchal, K., Poelman, D., Van der Straeten, D., Ulm, R., 2014. Photoreceptor-mediated bending towards UV-B in Arabidopsis. Mol. Plant 7, 1041e1052. Villalobos, L.I.A.C., Lee, S., De Oliveira, C., Ivetac, A., Brandt, W., Armitage, L., Sheard, L.B., Tan, X., Parry, G., Mao, H.B., Zheng, N., Napier, R., Kepinski, S., Estelle, M., 2012. A combinatorial TIR1/AFB-Aux/IAA co-receptor system for differential sensing of auxin. Nat. Chem. Biol. 8, 477e485. Wang, W., Xu, B., Wang, H., Li, J.Q., Huang, H., Xu, L., 2011. YUCCA genes are expressed in response to leaf adaxial-abaxial juxtaposition and are required for leaf margin development. Plant Physiol. 157, 1805e1819. Wargent, J.J., Gegas, V.C., Jenkins, G.I., Doonan, J.H., Paul, N.D., 2009. UVR8 in Arabidopsis thaliana regulates multiple aspects of cellular differentiation during leaf development in response to ultraviolet B radiation. New Phytol. 183,

Please cite this article in press as: Fierro, A.C., et al., Ultraviolet-B radiation stimulates downward leaf curling in Arabidopsis thaliana, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.12.012

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5

PLAPHY4115_proof ■ 17 December 2014 ■ 9/9

A.C. Fierro et al. / Plant Physiology and Biochemistry xxx (2014) 1e9 315e326. Watahiki, M.K., Yamamoto, K.T., 1997. The massugu1 mutation of Arabidopsis identified with failure of auxin-induced growth curvature of hypocotyl confers auxin insensitivity to hypocotyl and leaf. Plant Physiol. 115, 419e426. Wilson, M.I., Greenberg, B.M., 1993. Specificity and photomorphogenic nature of

9

ultraviolet-B-induced cotyledon curling in Brassica-napus L. Plant Physiol. 102, 671e677. Yin, R., Han, K., Heller, W., Albert, A., Dobrev, P.I., Zazimalova, E., Schaffner, A.R., 2013. Kaempferol 3-O-rhamnoside-7-O-rhamnoside is an endogenous flavonol inhibitor of polar auxin transport in Arabidopsis shoots. New Phytol..

Please cite this article in press as: Fierro, A.C., et al., Ultraviolet-B radiation stimulates downward leaf curling in Arabidopsis thaliana, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.12.012

6 7 8 9 10