UVR8 mediated plant protective responses under low UV-B radiation leading to photosynthetic acclimation

Journal of Photochemistry and Photobiology B: Biology xxx (2014) xxx–xxx

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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol

UVR8 mediated plant protective responses under low UV-B radiation leading to photosynthetic acclimation Suruchi Singh, S.B. Agrawal ⇑, Madhoolika Agrawal Laboratory of Air Pollution and Global Climate Change, Department of Botany, Banaras Hindu University, Varanasi 221005, India

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Article history: Received 30 November 2013 Received in revised form 26 March 2014 Accepted 31 March 2014 Available online xxxx Keywords: UV-B UVR8 SIG5 ELIP Photomorphogenesis Photosynthesis

a b s t r a c t The UV-B photoreceptor UVR8 regulates the expression of several genes leading to acclimation responses in plants. Direct role of UVR8 in maintaining the photosynthesis is not defined but it is known to increase the expression of some chloroplastic proteins like SIG5 and ELIP. It provides indirect protection to photosynthesis by regulating the synthesis of secondary metabolites and photomorphogenesis. Signaling cascades controlled by UVR8 mediate many protective responses thus promotes plant acclimation against stress and secures its survival. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Plants being immovable are inevitably exposed to sunlight for carrying out major physiological and developmental processes. Toxic fraction of sunlight i.e. UV-B (280–320 nm) reaches to the surface of the Earth. This got exaggerated by the progressive thinning of the stratosphere ozone (O3) layer as a result of human activities [1]. UV-B is well known to damage DNA, proteins, photosynthetic machinery and processes and arrests cell cycle [2]. To cope with this problem, plants have adopted several strategies in due course of time like changes in the levels of cellular UV-B absorbing metabolites, UV-B reflective properties and increases in leaf thickness. There are several observations in the scientific literature regulating physiological processes operating in plants being impaired by exposure to high levels of ambient UV-B [3]. The expression of genes involved in prevention and repair of UVB damage is initiated by exposure of plants to relatively low doses of UV-B [4]. A key protein that regulates these genes expression responses is UV RESISTANCE LOCUS8 (UVR8). Several class of plant photoreceptor have evolved that monitor light ranging from ultraviolet-B (UV-B) to the near infrared and allow maximum adaptation to light [5]. Plant perception of UV-B radiation as an environmental stimulus is known to affect growth and development [6]. By adopting forward genetic approaches several mutant screens relying on sensitivity to UV-B radiation, ⇑ Corresponding author. Tel.: +91 542 2368156; fax: +91 542 2368174. E-mail address: [email protected] (S.B. Agrawal).

genes involved in UV-B signaling cascade have been characterized [7]. The identified principal genes of mediators of UV-B photomorphogenic responses were COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1) and HY5 (ELONGATED HYPOCOTYL 5) and UVR, which is recently identified as a UV-B receptor [8,9]. UVR8 contains sequence similarity to the human guanine nucleotide exchange factor ‘‘Regulator of Chromatin Condensation’’ (RCC1) [10], however, available evidence suggests that RCC1 and UVR8 differ in their activity and function [11]. Dimers of UVR8 are monomerised upon UV-B perception and subsequently interact directly with the multifunctional E3 ubiquitin ligase COP1 and thus transduce the required signal [12,9]. The WD40 repeats also known as betatransducin repeats of UVR8 are thought to be responsible for UVB perception [9]. Supporting this notion, the protease resistant core domain of UVR8 retained the same ability as the full-length protein to undergo an UV-B induced, dimer-to-monomer switch. To elucidate the mechanism of UV-B perception by UVR8, its core domain was crystallized by Wu et al. [13]. Intriguingly, UV-B irradiation resulted in cracking of these crystals, indicating that the UVR8 core domain retained the ability to sense UV-B in the crystals. The core domain of variants W285F and W285A crystals failed to crack even after prolonged UV-B irradiation consistent with the loss of UV-B responsiveness [9]. UVR8 is a seven-bladed ß-propeller protein that makes use of tryptophan residues intrinsic to the protein as chromophore for UV-B absorption with a primary role as established for trp285 [14,13]. UVR8 absorbs strongly at 280 nm, as expected from its complement of aromatic residues (14 Trp, 10 Tyr and 8 Phe per 440 residue

http://dx.doi.org/10.1016/j.jphotobiol.2014.03.026 1011-1344/Ó 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: S. Singh et al., UVR8 mediated plant protective responses under low UV-B radiation leading to photosynthetic acclimation, J. Photochem. Photobiol. B: Biol. (2014), http://dx.doi.org/10.1016/j.jphotobiol.2014.03.026

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S. Singh et al. / Journal of Photochemistry and Photobiology B: Biology xxx (2014) xxx–xxx

monomer). Lack of any bound cofactor to photoactive purified UVR8 demonstrates that the reversible property of dimer dissociation is a property intrinsic to the protein. Arabidopsis mutants lacking UVR8 suffer necrosis when exposed to high ambient levels of UV-B because they lack UV-protection [15]. The knockout mutants that were deficient in metabolites, which are known for photoprotective functions such as flavonoids, phenylpropanoid, ascorbate and tocopherol displayed UV-B sensitive phenotypic changes alongside an enhancement of gene expression of UV-B responsive genes. The UV-B light insensitive (uli) mutants exhibited reduced hypocotyls growth inhibition and CHS gene expression compared with wild type seedlings after exposure to UV-B [16]. Higher plants are known to adapt a range of protective strategies in response to UV-B including increases in leaf thickness, UV-B reflective properties, increases in antioxidants and cellular levels of UV-B absorbing metabolites. Specific pathways mediating physiological and molecular responses of Arabidopsis to low level of UV-B involve the bZIP transcription factor HY5, the E3 ubiquitin ligase COP1 and the ß-propeller UV RESISTANCE LOCUS 8 (UVR8) [8,17]. In the UV-B signaling transduction cascade, the bZIP transcription factor HY5, a mediator of several photomorphogenic pathways is required for UV-B mediated gene expression [9]. 2. Repression of photomorphogenesis Heijde and Ulm [18] described that a WD40 repeat proteins, REPRESSOR OF UV-B PHOTOMORPHOGENESIS (RUP)1 AND RUP2 as negative feedback regulators of the UV-B signaling cascade [19]. The RUP1 (385 aa) and RUP2 (368 aa) proteins are highly homologous with 63% identity in an overlap of 349 amino acids. Both proteins consist of seven WD40 repeats. RUP1 and RUP2YFP fusion proteins localize to both nucleus and cytoplasm [19]. RUP proteins influence the balance between the UV-B induced growth inhibition and defense measures as a result of UV-B specific signaling. The direct interaction of RUP1 and RUP2 with UVR8 suggests that their repressive mechanism takes place at the photoreceptor level [19]. The RUP1 and RUP2 mechanism of action may be by preventing monomerization and/or facilitating UVR8 redimerisation upon UVB exposure (Fig. 1). Heijde and Ulm [18] showed strong impairment of rup1rup2 double mutants in UVR8 dimer recovery. This clearly showed that, without RUP1 and RUP2, the ability of

monomerized UVR8 to revert the inactive dimeric state is compromised. The two RUP proteins show significant sequence conservation in their seven WD40-repeat domains with the COP1 and SUPPRESOR OF PHYTOCHROME A (SPA1 and SPA2) proteins, including a conserved 16-amino acid DWD [DDB(Damaged DNA binding protein)1-binding WD40] motif [20]. The ubiquitin-proteasome pathway regulates the concentration and conformation of many cellular proteins in response to changes in physiological conditions. This pathway consists of a cascade of three activities performed by E1 (uniquitin-activating), E2 (ubiquitin-conjugating) and E3 (ubiguitin-ligase) enzymes [21]. CULLIN4 (CUL4), a recently identified regulator of photomorphogenesis, links the three COP complexes (the COP1-SPA complexes, CSN and the CDD complex) in the ubiquitination/proteasome mediated degradation of key photomorphogenesis promoting factors [22]. CUL4 is an important member of Cullin family and serves as a scaffold in CUL4-DDB1-based ubiquitin ligases to regulate many processes, including cell proliferation, DNA repair and genome integrity by ubiquitinating key regulators [23]. UV irradiation induces two major classes of DNA damage products: cyclobutane-pyrimidine dimmers (CPDs) and 6-4 photoproducts (6-4 PPs). Plants recover from these lesions through photoreactivation and light-independent pathways. In Arabidopsis thaliana, UV Resistance 2 (UVR2), encodes a photolyase and is specific for CPD damage repair [24], while UVR3, another type of photolyase protein, functions in 6-4 PP damage repair [25]. Among the DNA damage repair pathways, the nucleotide excision repair (NER) pathway in mammalian cells is involved in both global genome repair (GGR) and transcription coupled repair (TCR). Classical GGR and TCR factors include Damage-Specific DNA Binding Protein 2 (DDB2) and Cockayne Syndrome A (CSA) proteins, which are the substrate receptors for the CUL4-DDB1 E3 ligase [26]. CUL4DDB1DDB2 regulates the ubiquitination of the xeroderma pigmentosa complementation group C protein [27] while CUL4-DDB1CSA regulates the ubiquitination of CSB [28] in the DNA damage repair process. A CUL4-DDB1 complex has recently been identified in plants and functions in many biological processes [29]. It has been shown that Arabidopsis mutant lacking DDB2 enhanced the sensitivity to UV-radiation [30] and that over expression of Arabidopsis DDB1A increases plant tolerance to UV [31]. DDB2 associates with the CUL4-DDB1A complex to form an E3 ligase that modulates GGR-type DNA damage repair upon UV stress [32].

Fig. 1. A schematic presentation of ultraviolet-B induced, UVR8-mediated signaling cascades.

Please cite this article in press as: S. Singh et al., UVR8 mediated plant protective responses under low UV-B radiation leading to photosynthetic acclimation, J. Photochem. Photobiol. B: Biol. (2014), http://dx.doi.org/10.1016/j.jphotobiol.2014.03.026

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3. Mechanism of action of UVR8

4. UV-B and gene expressions

The low levels of UV-B mediate the translocation of UVR8 protein from the cytosol into the nucleus [33]. UVR8 interacts with the WD40-repeat domain of COP1, which is closely linked to downstream UV-B specific responses [12]. One of the constitutive photomorphogenesis/de-etiolated/fusca (COP/DET/FUS) genes, COP1 encodes ring-finger type ubiquitin E3 ligase, which is responsible for the ubiquitylation of photomorphogenesis promoting factors such as HY5 and LAF1 in darkness [34]. In Arabidopsis, the E3 ubiquitin ligase activity of the COP1 complex was supported by direct interaction with the COP9 signalosome and the CDD (COP10, DDB1, DET1) complexes [35]. In vivo, COP1 acts as a large protein complex of 700 kDa [36] A further dozen of the COP/DET/FUS genes define the COP9 signalosome (CSN), which is a nuclear enriched protein complex showing homology with the 26S proteasome. Peng et al. [37] showed that the CSN interacts with the 26S proteasome. COP10 was originally identified as negative regulator of photomorphogenesis essential for COP1-mediated degradation of HY5 [38]. DET1 is also a negative regulator of photomorphogenesis [39]. COP10 belongs to a family of ubiquitin E2 variant (UEV) proteins that contain the ubiquitin-conjugating motif (ubc) but lacks a critical cysteine residue required for conjugation [40]. UEVs have been shown to function in numerous cellular processes, including post replicative DNA repair and control of the cell cycle [41]. In plants, the E3 ubiquitin ligase COP1 is a multifunctional protein best known for its role as a repressor of photomorphogenesis [42], evidenced by cop1 mutant seedlings. COP1 functionality is highly modular, as seen in Arabidopsis [43]. COP1 contains nuclear import and export signals, and, in plants, its subcellular localization is regulated by light [43]. At the molecular level, COP1 targets different photomorphogenesis-promoting transcription factors for degradation in the dark and HY5 is amongst them [36]. Upon activation of photoreceptors by visible light, COP1 is activated and physically separated from HY5 by nuclear exclusion, allowing HY5 stabilization and activation of light responsive genes [44]. In addition to HY5, COP1 was shown to target two other photomorphogenesis-promoting transcription factors, LONG AFTER FAR-RED LIGHT1 and LONG HYPOCOTYL IN FAR-RED1 (HFR1) and the photoreceptor, phytochrome A for ubiquitination and proteolysis [45]. However, difference in COP1 function lie under UV-B from that in visible light including promotive versus repressive function [8]. The reorganization from the CUL4-DDB1-COP1-SPA E3 apparatus to UVR8-COP1-SPA complex(es) upon UV-B irradiation achieves a functional switch of COP1 from repressing (under visible light) to promoting photomorphogenesis (under UV-B) [46]. Under natural sunlight the UVA/blue light signaling pathway mediated by cryptochrome interacts with UVR8 to modulate UV-A responses in the presence of UV-B [47]. There is a speculation that COP1 could be playing a crucial role in this interaction as COP1 is involved in light responses mediated by cryptochromes, phytochromes and UVR8 and its WD40 domain is a common point of interaction [18]. Favory et al. [12] showed that the interaction of UVR8 and COP1 under UV. Further, mutational analysis indicates that nuclear exclusion of COP1 is a rate-limiting step for the establishment of photomorphogenic development [48]. cop1 mutants display light-grown phenotypes even in complete darkness, including short hypocotyls, open cotyledons, elevated pigment levels. UVR8 was shown to be associated with chromatin of UV-B responsive genes including the HY5 promoter region, probably by interaction with histones (H2B) and not with DNA directly [15,49] and the association of UVR8 with chromatin does not require the UV-B exposure. However, the possibility of increase in the association of UVR8 under UV-B cannot be ruled out. Chromatin immunoprecipitation experiments suggested that the histone modification is important in the regulation of transcription by UV-B [49].

Transcriptome analysis of wild-type and uvr8 plants showed that UVR8 regulates over 100 UV-B induced genes [12], including those that involved in sunscreen pigment biosynthesis, other metabolic pathways, DNA repair, protection against oxidative stress and chloroplast function. UVR8 regulates leaf growth through the control of epidermal cell development [50]. UVR8 is required for normal progression of endocycle in response to UV-B and has a regulatory role in stomatal differentiation [50]. UVR8 proteins containing mutations impair UV-B induced photomorphogenesis [12]. Kusano et al. [51] suggested that in early stages of exposure to UVB, plant cells efficiently divert the carbon (mainly maltose) from primary metabolism towards the aromatic amino acid precursors of the phenylpropanoid pathway (Fig. 2). UV-B protectant metabolites can be categorized to different families according to their chemical structural functions: (1) light perception in proportion to aromatic ring, (2) antioxidant activity corresponding to reduction moieties such as phenolic moiety and unsaturated carbon– carbon bonds, and (3) increasing osmotic specific location for sample in vacuole [7]. In flavonoid-less mutants, large metabolic changes and enhancement of UV-B associated transcriptional programs inducing senescence were observed [51]. Molecular characterization of hp-1 and hp-2 tomato mutants, which exhibited exaggerated light responsiveness, revealed that HP1 and HP2 genes encode tomato homologues of the light signal transduction proteins DDB1a and DET1, respectively involved in the Arabidopsis CDD complex (COP10-DDB1a-DET1) [52]. In Arabidopsis CULLIN4 (CUL4) has been demonstrated to directly interact with either the CDD complex or the COP1-SPA complexes in the absence of the CDD complex and to form a heterogenous group of E3 ligases that regulates multiple aspects of the light regulation [21]. Recent studies demonstrated that in tomato, HP1/LeDDB1 and HP2/LeDET1 are essential component of CUL4-based E3 ligase complex, in which LeDDB1 is associated with tomato CUL4 and DET1 [53]. Davey et al. [54] observed more substantial photoinhibition in uvr8 indicating that PSII is more sensitive to UV-B induced damage. They anticipated several reasons for this, first, the mutant would acquire less capacity to prevent or repair DNA damage than wild type. Specifically, uvr8 would have accumulated less UV-B absorbing flavonoids which make PSII more susceptible to UV-B damage [55]. Alternatively, the uvr8 mutant may be less able than wild type to replace damaged PSII reaction centres. The protection offered by plants to photosynthetic machinery against low doses of UV-B through UVR8 regulated gene expression can be direct via induction of chloroplastic proteins and indirect via regulating the phenylpropanoid pathway, photomorphogenesis and other secondary metabolite and DNA repair (Fig 3). The review is therefore written with an objective to trace the UVR8 regulated signaling cascades leading to photosynthetic acclimation/protection against UV-B. Few signature pathway/ component regulated by UVR8 are discussed below.

4.1. Phenylpropanoid pathway HYH and HY5 control expression of a range of key elements for UV-B acclimation, including genes encoding for enzymes of the phenylpropanoid pathway [15]. In the dark, the HY5 protein is turned over in the nucleus by the E3 ubiquitin ligase COP1, a crucial repressor of light signaling [37]. In the light, activation of photoreceptors lead to the inactivation and nucleus exclusion of COP1, allowing HY5 stabilization and activation of light responsive genes [38]. Studies performed in transient expression systems established that UV-B and UV-A/blue induced transcription of CHS and other genes of the phenylpropanoid biosynthesis pathway

Please cite this article in press as: S. Singh et al., UVR8 mediated plant protective responses under low UV-B radiation leading to photosynthetic acclimation, J. Photochem. Photobiol. B: Biol. (2014), http://dx.doi.org/10.1016/j.jphotobiol.2014.03.026

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Fig. 2. Diagrammatic presentation of co-ordinate diversions of carbon from primary metabolic pathway to secondary metabolic pathway under UV-B.

Fig. 3. Proposed model for UVR8 mediated possible protective mechanisms against UV-B.

is mediated by multiple light regulatory units (LRUs) [56]. The HY5 protein is able to bind CHS promoter sequences in vitro [57] and in vivo [58]. In the dark, COP1 degrades HY5 protein (and HY5homolog HYH), but in response to light perception this degradation is prevented [59]. In response to UV-B however, HY5 gene expression is re-activated and the HY5 protein re-accumulates, requiring functional UVR8 and COP1 [12]. Several studies confirm that UVR8 mediated pathways, UV-B induction of CHS and other

genes is UV-B specific and it does not overlap with non-specific signaling pathways. Regulation of synthesis of UV-B protective compounds is transcriptional as has been observed in many plant species [60]. The transcriptional control of the enzymes of the phenylpropanoid metabolism is partially characterized. To protect the sensitive photosynthetic system and susceptible macromolecules, plants screen out UV-B with flavonoids deposited in the vacuoles of epidermal cells. Thus, the UV-B absorbing

Please cite this article in press as: S. Singh et al., UVR8 mediated plant protective responses under low UV-B radiation leading to photosynthetic acclimation, J. Photochem. Photobiol. B: Biol. (2014), http://dx.doi.org/10.1016/j.jphotobiol.2014.03.026

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capacity of flavonoids is utilized [61]. The flavonoid biosynthesis pathway is a branch of the general phenylpropanoid pathway, with the first step catalyzed by chalcone synthase (CHS). Further reactions catalyzed by chalcone isomerase (CHI), flavones-3-hydroxylase (F30 H) and flavonol synthase (FLS) which lead to the formation of quercetin and kaempferol that are further glycosylated by several glycotransferases [62]. These flavonol glycosides have strong antioxidant capacity and strong UV-B absorbance properties. Flavonoid biosynthesis pathway is regulated at the transcriptional level, both of the regulators and the biosynthetic genes [63]. Transcriptional regulation for the promoter of the chalcone synthase gene (CHS) has been undertaken most intensively in detail. Genomic DNA footprinting and promoter dissection using transient gene expression in parsley protoplasts have defined cisacting elements involved in light-dependent expression from the CHS promoter [64]. A 52-bp CHS promoter region, designated as light responsive units (LRUs), consisting of two cis-acting elements, an AGCT-containing element (ACE) and a MYB recognition element (MRE), confer responsiveness to UV-B containing white light.

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UVR8 mediates the accumulation of several transcripts encoding chloroplastic proteins in response to UV-B including SIG5, which encodes the plastid RNA polymerase sigma factor that regulates psbD expression and hence the synthesis of the D2 polypeptide [65] and EARLY LIGHT-INDUCABLE PROTEIN 1 (ELIP1) (Fig. 4). ELIP1 is one of the major light responsive genes which leads to tolerance to photoinhibition and photooxidative stress [66]. ELIPs are thylakoid proteins encoded by nuclear genes and expressed in plants exposed to stress [67]. Cross-linking experiments have demonstrated that ELIP interacts with components of photosystem II reaction centre, especially with the D1 protein [68]. Blocking the photosynthetic electron flow and the degradation of D1 protein by addition of DCMU under high light exposure does not interfere with ELIP synthesis [69]. ELIPs are synthesized in cytoplasm, imported into the chloroplast and inserted in thylakoids via a pathway involving cpSRP43 [70]. ELIP is stable in the thylakoids of the leaves exposed to UV-B. ELIPs have three transmembrane domains (I, II and III), with the I and III a-helices showing a high homology with the corresponding helices of Cab (Chlorophyll a/b binding) proteins [71]. Comparison of the amino acid sequences between ELIPs and Cabs shows that ELIPs contain four putative chlorophyll binding residues in the helices I and III [72]. Unlike the Cab proteins which are constitutively expressed in thylakoids, ELIPs show transient accumulation. Corresponding transcripts and ELIPs protein are induced in the first hours of greening of etiolated seedlings [73], when the photosynthetic system was more susceptible to photooxidative stress [74]. Although, Adamska et al. [69] reported that UV-B does not induce ELIP transcription but prevents its degradation and indicates that UV-B acts at more than one level in the regulation of ELIP turnover. In mature plants they are absent until the plants are exposed to stress like UV-B [69]. This inducible property with the ability to bind the pigments, suggests that ELIPs may have photoprotective function.

ing a hydrophobic core in the 3D HTH structure [75]. MYB proteins can be divided into different classes depending on the number of adjacent repeats. The three repeats of the prototypic MYB protein c-Myb are referred to as R1, R2 and R3. Numerous R2R3-MYB proteins have been characterized by genetic approaches and found to be involved in the control of plant specific processes, including (i) primary and secondary metabolism, (ii) cell fate and identity (iii) developmental process, and (iv) responses to biotic and abiotic stresses. One subgroup in particular was recently identified as flavonol-specific activators of flavonoid biosynthesis during plant development, namely the SG7/PRODUCTION OF FLAVONOL GLYCOSIDES (PFG) family constituted of PFG1/MYB12, PFG2/MYB11 and PFG3,/MYB111 [76]. Indeed, PFG1/MYB12 was shown to regulate the CHS expression by binding with the MRE element [77]. The Arabidopsis seedlings of triple mutant myb11 myb12 myb111 do not form flavonoids under standard growth conditions while the anthocyanin accumulation was not affected. HY5 regulation of PFG1 (PRODUCTION OF FLAVONOL GLYCOSIDES/AtMYB12) gene expression is recently discovered [78]. Stracke et al. [78] provided evidence for HY5 as the major regulator of PFG1/MYB12 gene activation in response to UV-B radiation, directly targeting a specific PFG1/MYB12 promoter region containing putative HY5 basal binding motifs. The PFG family of MYB transcription factor is the only regulator of the flavonol pathway [79]. Both HY5 and MYB12 bind to promoters of common target genes such as CHS; these two transcriptional regulators might cooperate at the promoter site. HY5 binding to the MYB12 promoter is involved in the early UV-B response. Subsequently, HY5 regulates downstream genes like CHS, in combination with MYB12 [79]. Stracke et al. [76] proposed that the cooperation of HY5 and MYB12 leads to the full response and optimal regulation of flavonoid synthesis. Li et al. [80] reported that flavonoid-less mutants, tt4 (CHS, chalcone synthase) and tt5 (CHI, chalcone isomerase) mutants revealed hypersensitive phenotypic responses to UV-B irradiation. Kusano et al. [81] also observed clear up regulation under UV-B of the structural genes of flavonoid biosynthesis: TT4, TT5, TT6 and UGT78D2. Analysis of A. thaliana lines mutant for the AtMYB4 gene has demonstrated that AtMYB4 acts as a negative regulator of hydroxycinnamic acid metabolism, principally through regulating the expression of the gene encoding cinnamate-4-hydroxylyase. Expression of AtMYB4 is reduced by different environment conditions including UV-B. Like this, it brings about a derepression of C4H gene expression, resulting in higher synthesis of protecting sinapate esters. A mutant of AtMYB4 that produces significantly higher levels of sinapate esters in its leaves also shows improved tolerance to UV-B treatment. It is interesting that the transcriptional response to UV-B that operates through AtMYB4 is focused on the regulation of expression of the C4H gene. There has been considerable debate as to which phenylpropanoids are involved in the response to UV-B and which provide the most effective UV-B screening and C4H is active in the synthesis of both sinapate esters and flavonoids. Hydroxylation catalyzed by C4H shifts the UV-B absorbance spectrum of the hydroxycinnamic acid intermediates toward longer wavelengths to provide compounds that are effective in absorbing biologically relevant wavelengths of UV [82].

4.3. MYB and flavonoids

4.4. Vitamin B6

MYB proteins are characterized by a highly conserved DNAbinding domain: the MYB domain. This domain generally consists of up to four imperfect amino acid sequence repeats (R) of about 52 amino acids, each forming three a-helices. The second and third helices of each repeat build a helix-turn-helix (HTH) structure with three tryptophan regularly spaced or hydrophobic residues, form-

Vitamin B6, similar to vitamins C and E, show antioxidant activity and may be an important component of cellular antioxidant defenses. Denslow et al. [83] have shown that B6 vitamins are efficient quenchers of both singlet oxygen and superoxide and thus have antioxidant capacity [84]. PDX1 and PDX2 are the two genes involved for vitamin B6 production [85]. These studies showed that

4.2. Chloroplastic proteins

Please cite this article in press as: S. Singh et al., UVR8 mediated plant protective responses under low UV-B radiation leading to photosynthetic acclimation, J. Photochem. Photobiol. B: Biol. (2014), http://dx.doi.org/10.1016/j.jphotobiol.2014.03.026

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Fig. 4. Model showing induction of ELIP and SIG5 under low UV-B radiation.

pyridoxine (PDX1) and PDX2 proteins form a complex having glutamine amidotransferase activity, with PDX2 protein serving as a glutaminase. This reaction utilizes glutamine plus either dihydroacetone phosphoate or glyceraldehyde-3-phosphate and produces pyridoxal-50 -phosphate. PDX2 extracts an ammonia group from glutamine that is incorporated into the product [86] and PDX1 accepts this ammonium group and synthesizes the final product [87]. By using PDX1 specific antibodies, Ristilä et al. [88] showed an increased level of the PDX1 protein in plants exposed to UV-B. Ristilä et al. [88] showed that the transcription of the PDX1.3 gene is accomplished by low level of UV-B radiation through a putative receptor. This receptor would regulate PDX1.3 expression via the UVR8 and COP1 components since corresponding mutants lack UV-B induction of PDX1.3 [12]. PDX1 or pyridoxine in turn is needed for the wild type regulation of PR-5. The UV-B dependent down-regulation of the photosynthetic LHCB13 gene is commonly low in pdx1.3. 4.5. Brassinosteroids Brassinosteroids (BR) are growth-promoting steroid hormones that regulate many physiological and developmental processes in plants. BR mutant plants display a wide range of phenotypes, which includes dwarfism, curly and dark leaves, delayed flowering and senescence, etc. Shimada et al. [89] studied the biosynthetic pathways leading to brassinolide (active form of BR) using cultured cells of Catharathus roseus and proposed the two alternative biosynthetic pathways (Fig. 5). Shimada et al. [89] isolated AtBR6ox gene and suggested that it belongs to the P450 superfamily. They also demonstrated that AtBR6ox are involved in C-6 oxidation and hence the corresponding proteins were designated BR-6-oxidases. Several mutants, such as constitutive photomorphogenesis and dwarfism (cpd) have been shown to be defective in the BR-specific pathway [90]. Such BR-mutant dwarfs exhibit short robust stems, reduced fertility and prolonged life cycle. The CPD gene was shown to encode a cytochrome P450 steroid hydroxylating enzyme (CYP90A1). cpd mutant, known to abolish the production of a CYP450, shows elevated levels of CHS mRNA in white light. Contrary to this, the transcripts levels for pathogenesis related protein PR-1, PR-2 and PR-5 in cpd mutants were lowered compared with those in wild type plants [91]. The mRNA levels for CHS, MEB5.2, PYROA, PR-5 and chalcone isomerase in cpd mutants were severely reduced under UV-B [92]. PR-5, PYROA and MEB5.2 were shown to be regulated by low levels of UV-B [92]. PYROA encodes a protein involved in biosynthesis of pyridoxine or vitamin B6 [93]. Pyridoxine is also a cofactor for enzymes partici-

pating in amino acid metabolism. PYROA is putatively required for resistance against singlet oxygen radicals [93]. MEB5.2 is a gene with an unknown function but showed strongest induction pattern in DNA microarray screening of induced genes in UV-B exposed Arabidopsis [94].

5. Co-expression networks and metabolomics: high throughput decoding of gene function Genes involved in metabolic processes are co-regulated thus coexpressed under the control of a shared regulatory system. To study trascriptionally regulated gene networks, a co-expression analysis was conducted using the vast publically available microarray data [95]. To construct UV-B responsive gene network related to secondary metabolic pathway, co-expression network analysis was performed using 17 genes of UV-B signaling cascade (Table 1). This co-expression analysis revealed 9 subclusters (Fig. 6) which were connected to each other through acquainted signaling cascades. Cluster 1 is related with brassinosteroid-6-oxidase 2, short chain dehydrogenase/rehydrogenase and brassinosteroids. Short chain dehydrogenase/reductases are enzymes of almost 250 residue subunits catalyzing NAD(P)(H)-dependent oxidation– reduction reactions. SDRs substrate range from alcohol, sugars, steroids and aromatic compounds to xenobiotics. The N-terminal region binds the coenzyme NAD(H) or NADP(H), while the C-terminal region constitute the substrate binding part. Cluster 2 is with the synthesis of vitamin B6. Cluster 3 consisted of CRY3, TT6, TT4, TT7, UGT78D2, CM1, PAL1, TT5, TT3, FS1, PKT2, 4CL3 and ROL1. Chalcone isomerase deficient tt5 lines is already quite UV-B hypersensitive under growth chamber conditions due to accumulation of flavonoids and sinapate esters [96]. Kliebenstein et al. [10] reported that uvr-8 mutation blocks the UV-B mediated induction of CHS mRNA and protein, as well as reduces flavonoid and anthocyanin pigment. In contrast to the positive action of UVR8, AtMYB4, a transcription factor involved in regulating the phenylpropanoid metabolism in response to UV-B, is a negative regulator. This subcluster deals with the biosynthesis of flavonol. This cluster connects directly with cluster 4. Cluster 4 deals with protection against photooxidative damage (ELIP proteins) and for genome integrity and UV-B (DDB2) and transport related genes like POP1, NAP9. Sugar isomerase (SIS) of cluster 4 are phosphosugar isomerases that can catalyze the isomerization of not only phosphosugar but also of monosaccharides. Strong interaction between cluster 3 and 4 reflects strong correlation between different protective responses against UV-B (see Fig. 6).

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S. Singh et al. / Journal of Photochemistry and Photobiology B: Biology xxx (2014) xxx–xxx Table 1 Details about the studied genes in the co-expression network analysis. Genes

Annotation

References

DWF3 (Dwarf 3)

Encodes a member of the CP90A family, a cytochrome P450 monooxygenase Encodes a p450 enzyme that catalyzes the last reaction in the production of Brassinolide Catalyzes metabolism of quinones and carbonyls Phosphosugar isomerization Encodes flavonone-3-hydroxylase Encodes chalcone synthase Encodes a cytochrome P450 superfamily protein required for flavonoid-30 -hydroxylase activity Encodes a anthocyanidin 3-o-glucosyltransferase Catalyzes a key step in shikimate pathway Catalyzes the conversion of phenylalanine to trans-cinnamic acid Chalcone-flavanone isomerase family protein: catalyzes the conversion of chalcones into flavanones Catalyzes formation of flavonols from dihydroflavanols Encodes a UDP-L-Rhamnose synthase involved in the biosynthesis of UDP-L-rhamnose from UDP-D-Glucose

Sävenstrand et al. [92]

Encodes an isoform of 4-coumarate:CoA ligase involved in the last step of phenylpropanoid pathway Dihydroflavonol-4-reductase

Kimura et al. [99]

R6O2 (Brassinosteroid-6-oxidase 2) SDR1 (Short chain dehydrogenase/reductase) SIS (Sugar isomerase) TT6 (Transparent testa 6) TT4 (Transparent testa 4) TT7 (Transparent testa 7) UGT78D2 (UDP-glycosyl transferase 78D2) CM1 (Chorismate mutase 1) PAL (Phenylalanine ammonia lyase) TT5 (Transparent testa 5) FLS (Flavonol synthase 1) ROL1 (Repressor of LRX1)/RHM1 (Rhamnose biosynthesis)/ATRHM1 (Arabidopsis thaliana Rhamnose B1) 4CL3 (4-coumarate:CoA ligase) TT3 (Transparent testa 3)/DFR

Choe et al. [90] Scherbak et al. [97] Jenkins [11] Li et al. [80] Li et al. [80] Ryan et al. [98] Kimura et al. [99] Tohge et al. [7] Gitz et al. [100] Landry et al. [96] Ferreyra et al. [101] Kuhn et al. [102]

Mazza et al. [103]

Mevalonate (MVA)

Episterol

5-Dehydroepisterol

Cathasterone cpd

6-Oxocampestenol

Teasterone

Campesterol

Campesterol

6-Deoxocathastererone cpd

3-Dehydroteasterone

6-Deoxoteasterone

Typhasterol 3-Dehydro-6-deoxoteasterone 6-Deoxotyphasterol Castasterone

6α-hydroxycastasterone

6-Deoxocastasterone

Brassinolide

Perception

Signal Transduction Fig. 5. Schematic presentation of two alternative biosynthetic pathways leading to brassinolide synthesis.

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S. Singh et al. / Journal of Photochemistry and Photobiology B: Biology xxx (2014) xxx–xxx

Fig. 6. Co-expression network analysis of UV-B influenced metabolic genes.

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Please cite this article in press as: S. Singh et al., UVR8 mediated plant protective responses under low UV-B radiation leading to photosynthetic acclimation, J. Photochem. Photobiol. B: Biol. (2014), http://dx.doi.org/10.1016/j.jphotobiol.2014.03.026