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Do UV-A radiation and blue light during growth prime leaves to cope with acute high-light in photoreceptor mutants of Arabidopsis thaliana ?

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We studied how plants acclimated to growing conditions that included combinations of blue light and ultraviolet‐A (UV‐A) radiation, and whether their growing environment affected their photosynthetic capacity during and after a brief period of acute high light (as might happen during an under‐canopy sunfleck). Arabidopsis thaliana Landsberg erecta wild‐type were compared with mutants lacking functional blue‐light‐and‐UV photoreceptors: phototropin 1PHOT1, cryptochromes (CRY1 and CRY2) and UV RESISTANT LOCUS 8 (uvr8). This was achieved using LED lamps in a controlled environment to create treatments with or without blue light, in a split‐plot design with or without UV‐A radiation. We compared the accumulation of phenolic compounds under growth conditions and after exposure to 30 minutes of high light at the end of the experiment (46 days), and likewise measured the operational efficiency of photosystem II (φPSII a proxy for photosynthetic performance) and dark‐adapted maximum quantum yield (Fv/Fm to assess PSII damage). Our results indicate that cryptochromes are the main photoreceptors regulating phenolic‐compound accumulation in response to blue light and UV‐A radiation, and a lack of functional cryptochromes impairs photosynthetic performance under high light. Our findings also reveal a role for UVR8 in accumulating flavonoids in response to a low UV‐A dose. Interestingly, phototropin 1 partially‐mediated constitutive accumulation of phenolic compounds in the absence of blue light. Low irradiance blue light and UV‐A did not improve φPSII and Fv/Fm upon our acute high light treatment, however CRYs played an important role in ameliorating high‐light stress.
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Do UV-A radiation and blue light during growth prime leaves to cope with acute high-light in
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photoreceptor mutants of Arabidopsis thaliana?
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Craig C. Brelsford1, Luis O. Morales1, Jakub Nezval2, Titta K. Kotilainen1, Saara M. Hartikainen1,
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Pedro J. Aphalo1 and T. Matthew Robson1
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Affiliations
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1 Organismal and Evolutionary Biology Research Programme, Viikki Plant Science Center (ViPS),
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Faculty of Biological and Environmental Sciences, 00014, University of Helsinki, Finland.
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2 Faculty of Science, University of Ostrava, 30. dubna 22, 701 03 Ostrava, Czech Republic.
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Correspondence email: matthew.robson@helsinki.fi
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Abstract
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We studied how plants acclimated to growing conditions that included combinations of blue light and
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ultraviolet-A (UV-A) radiation, and whether their growing environment affected their photosynthetic
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capacity during and after a brief period of acute high light (as might happen during an under-canopy
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sunfleck). Arabidopsis thaliana wild-type Landsberg erecta (WT) were compared with mutants lacking
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functional blue-light-and-UV photoreceptors: phototropin 1 (phot1); cryptochromes (cry1 and cry2) and
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UV RESISTANT LOCUS 8 (uvr8). This was achieved using LED lamps in a controlled environment to create
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treatments with or without blue light, in a split-plot design with or without UV-A radiation. We
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compared the accumulation of phenolic compounds under growth conditions and after exposure to 30
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minutes of high light at the end of the experiment (46 days), and likewise measured the operational
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efficiency of photosystem II (ϕPSII a proxy for photosynthetic performance) and dark-adapted maximum
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quantum yield (Fv/Fm to assess PSII damage). Our results indicate that cryptochromes are the main
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photoreceptors regulating phenolic-compound accumulation in response to blue light and UV-A
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radiation, and a lack of functional cryptochromes impairs photosynthetic performance under high light.
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Our findings also reveal a role for UVR8 in accumulating flavonoids in response to a low UV-A dose.
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Interestingly, phototropin 1 partially-mediated constitutive accumulation of phenolic compounds in the
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absence of blue light. Low irradiance blue light and UV-A did not facilitate higher ϕPSII and Fv/Fm to our
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acute high light treatment, however CRYs played an important role in ameliorating high-light stress.
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Abbreviations
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CHS, chalcone synthase ; cry1 cry2, cryptochrome 1,2; ϕPSII, Operating efficiency of photosystem II;
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Fv/Fm , Maximum quantum efficiency of PSII; BL, Blue light 420-490nm; B:G ratio of blue:green
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light defined as 420-490:500-570nm, HPLC-DAD, High performance liquid chromatography coupled
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with a diode array detector; PAR, Photosynthetically active radiation (400nm-700nm); phot1, phototropin
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1 mutant; PSII, Photosystem II; R:FR, Red : Far-red light ratio defined as 655-665:725-735m; SAS,
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Shade avoidance syndrome; UV, Ultraviolet radiation 100-400nm; UV-A, Ultraviolet A radiation 315-
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400nm; UV-B, Ultraviolet B radiation 280-315nm; uvr8-2, UV Resistance locus 8-2 mutant; WT,
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Landsberg erecta wild type; VPD, Vapour pressure deficit.
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Introduction
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Plants experience a dynamic and heterogeneous light environment, where spectral composition and
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irradiance can change depending upon the time of day, and of year, as well as the surrounding vegetation
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(Constabel and Lieffers 1996, Montgomery and Chazdon 2001). To maintain a positive carbon balance,
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plants beneath canopies must respond to changes in solar radiation throughout the growing season
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(Augspurger 2003, Lopez et al. 2008, Dion et al. 2016). As well as reduced irradiance during spring due
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to canopy closure, differential attenuation of the blue (Casal 2013a; who define blue as 400 − 500 nm) and
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UV-A regions affect spectral composition in the understorey (Grant et al. 2005). Spring bud burst of
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deciduous species in temperate and boreal forests results in a reduction in total under-canopy irradiance
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(Richardson and O’Keefe 2009) and a change in its spectral composition; reductions in the R:FR and
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blue:green ratios, and an increase in the UV:PAR ratio reaching the understorey (Flint and Caldwell 1998,
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Leuchner et al. 2011, Urban et al. 2012, Dengel et al. 2015). In response to increases in irradiance, plants
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produce phenolic compounds such as anthocyanins and phenolic acids (Agati et al. 2012). For instance,
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flavonoid glycosides can serve as sun-screens accumulating in the vacuoles, and bound to cell walls, of
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leaf epidermal cells protecting the mesophyll from photo-bleaching and photosynthetic damage (Jansen et
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al. 1998, Barnes et al. 2008). Flavonoids absorb UV radiation, whilst also functioning as antioxidants
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preventing damage from reactive oxygen species (Agati and Tattini, 2010). While the low UV irradiance
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under most canopies is unlikely to be harmful to plants, it might be exploited as a cue to optimise growth
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and defence to suit the environment (Mazza and Ballaré 2015).
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Plants monitor their light environment through perception of specific regions of the spectrum, and
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coordinate a response that adjusts their growth strategy to suit the conditions (Casal 2013a, Casal 2013b).
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Different photoreceptors react to specific regions of the spectrum but often interact permitting the plant to
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be sensitive to complex changes in spectral irradiance (Heijde and Ulm, 2012). Plant photoreceptors cry1
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and cry2, and phot1 and phot2, primarily absorb photons in the blue and UV-A regions of the spectrum
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(Briggs and Huala 1999, Banerjee and Batschauer 2005). Of these, phot1 and phot2, are involved in
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stomatal opening, tropism and hypocotyl elongation responses (Casal 2000). Specifically, phot1 promotes
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cryptochrome-mediated accumulation of anthocyanins in response to blue light (Kang et al. 2008), and
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phot2 regulates the chloroplast-avoidance response to high light in angiosperms (Briggs and Christie
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2002, Litthauer et al. 2015). To date, neither phot1 nor phot2 has been reported to regulate the pathways
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responsible for the accumulation of flavonoids. Photoreceptors, cry1 and cry2, alongside phyA and phyB
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detect the light cues which entrain the circadian clock (Somers et al. 1998). Hypocotyl elongation,
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seedling development, as well as the accumulation of flavonoids and anthocyanins are also regulated by
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cry1 and cry2 (Kubasek et al. 1992; Casal 2000, Shalitin et al. 2002). There is also evidence that cry1
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interacts with phyB, which detects changes in R:FR ratio in the presence of neighbouring plants, eliciting
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the SAS response (Ballaré et al. 1987, , Ballaré et al. 1990 Keller et al. 2011). Perception of UV-B
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through UVR8 attenuates the SAS by supressing stem elongation, in an antagonistic response to that
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triggered by phyB under FR (Mazza and Ballaré 2015). UVR8 also promotes the pathways for
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accumulation of flavonoids in response to UV-B radiation under controlled conditions (Kliebenstein et al.
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2002) and at the solar UV-B irradiance occurring outdoors during July in Finland (Morales et al. 2013).
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UV-B radiation has consistently been found to up-regulate the accumulation of flavonoids in plants,
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both in controlled experiments using UV-B lamps (Ballaré et al. 1995, Demkura & Ballaré 2012), and in
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experiments conducted outdoors under solar UV-B radiation (Flint et al. 1985, Krizek et al. 1998, Morales
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et al. 2013). Although flavonoid accumulation in A. thaliana in response to UV-B has been well studied,
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less is known about the influence of the blue and UV-A regions of the spectrum on this response (Christie
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and Jenkins 1996, Fuglevand et al. 1996, Morales et al. 2013). UV-A radiation constitutes a greater
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proportion of the received solar irradiance than UV-B radiation, but is not attenuated by the ozone layer so
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has not been subject to such intensive research. There is evidence that some flavonoid groups are
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regulated mainly by UV-A and others predominately by UV-B (Kotilainen et al. 2008, Siipola et al. 2015,
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Verdaguer et al. 2017). For instance, in an outdoor experiment filtering out solar UV-A and UV-B,
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Kotilainen et al. (2008) found specific UV-A and UV-B effects on the accumulation of flavonols, flavones
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and cinnamic acids in Betula pendula and Alnus incana. Similarly, in an outdoor experiment filtering out
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different wavelengths of solar radiation, blue light had stronger effects than UV-A radiation on the
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accumulation of epidermal flavonol content in pea (Pisum sativum) (Siipola et al. 2015). Nevertheless,
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both UV-A radiation and blue light can elicit flavonoid-accumulation to varying degrees. Morales et al.
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(2013) suggested that the UV-A/blue light signalling pathway, most likely mediated by crys, maybe
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interacting with UVR8 to modulate UV-A responses such as flavonoid accumulation. However, there has
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been little research comparing the responses of plants to UV-A radiation with and without blue light, to
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examine whether these interactions between photoreceptor responses involve redundancy or fine-tuning
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between blue and UV-A radiation to regulate flavonoid accumulation.
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Although PAR defines the spectral region of radiation useful for photosynthesis (McCree 1981),
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beyond this region UV-A radiation has also been reported to increase the rate of photosynthesis in over
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100 plant species across a variety of life forms (Turnbull et al. 2013), including A. thaliana (Bilger et al.
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2007). Chlorophylls (Chl) a and b can absorb radiation within the UV-A region (Lang and Lichtenthaler
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1991, McCree 1981), supporting the suggestion that this region also has the potential to drive
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photosynthesis. However, epidermal screening by UV-absorbing compounds such as flavonoids often
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drastically reduces the amount of UV-A radiation reaching Chl a and Chl b in the mesophyll (Bilger et al.
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1997). This means that UV-A radiation may only be exploited as an alternative energy source for
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photosynthesis in low light environments when plants have low concentrations of epidermal UV-
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absorbing compounds (Štroch et al. 2015).
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UV-A radiation can have deleterious effects on photosynthesis (Jansen et al. 1998, Booij-James et
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al. 2000). Both UV-A and UV-B radiation can cause damage to the PSII protein complex and reduce
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quantum efficiency (Jansen et al. 1998); in doing so they increase photoinhibition of PSII more, per
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photon, than excess PAR (Turcsányi and Vass 2000). However this effect is somewhat counterbalanced by
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the fact that, UV-A radiation, as well as blue light, also enhance DNA-repair (Jansen et al. 1998, Booij-
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James et al. 2000). Entrainment of the circadian rhythm of the photosynthetic capacity of PSII under low
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or dynamic light conditions requires phot1 and phot2, acting through mechanisms that are yet to be fully
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elucidated (Litthauer et al. 2015). The role of phot2 in the chloroplast avoidance response, permits
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avoidance of high-light stress, allowing damage to PSII to be redistributed within the leaves (Davis and
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Hangarter 2012). In response to blue light, CRY1 and CRY2 increase the expression of psbA and psbD
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(Thum et al. 2001, Tsunoyama et al. 2004, Onda et al. 2008). These genes promote the production of the
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D1 and D2 proteins of PSII (Marder et al. 1987) which are needed to repair damage to PSII (Christopher
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and Mullet 1994). Similarly, UVR8 can induce production of the D2 protein, though not D1 protein in
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PSII, in response to UV-B radiation (Davey et al. 2012). However, little is known about the roles of
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UVR8, CRYs and PHOTs in regulating the photosynthetic capacity of PSII in response to UV-A radiation.
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We had three major aims in this experiment. Firstly, to test the respective roles of blue and UV-A
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radiation in the accumulation of flavonoids in plants growing in a low-to-moderate light environment (c
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168 μmol m-2 s-1 of PAR) and to identify those photoreceptors responsible for this response. Secondly, to
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determine whether growth under these blue light and UV-A radiation treatments provided pre-emptive
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acclimation to high-light (priming), and lastly whether flavonoid content was correlated with the
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protection of PSII. To address these questions, we examined wild type (WT) Ler and mutants deficient in
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UVR8, CRY1CRY2 and PHOT1 activity. We hypothesised that the absorption tail of UVR8 into the UV-
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A region would enable it to mediate flavonoid accumulation and promote quantum efficiency of PSII in
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response to UV-A radiation. We expected cryptochromes to increase leaf flavonoid content and promote
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the quantum efficiency of PSII under blue light and UV-A radiation, and for this also to benefit PSII under
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high-light exposure. Lastly, we considered that phot1 in plants growing under blue light and UV-A
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radiation might promote cry-mediated anthocyanin accumulation and thus increase the overall phenolic
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content.
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Materials and Methods
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Plant Material
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All genotypes used in this experiment had the background accession Arabidopsis thaliana
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Landsberg erecta. All seeds were produced simultaneously from plants grown under the same standard
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conditions in the growth rooms and greenhouses at the University of Helsinki, Viikki campus. The
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following photoreceptor mutants were included in the experiments: phot1 (Inada et al. 2004), cry1 cry2
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(Mazzella et al. 2001), and uvr8-2 (Brown et al. 2005).
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Light treatments
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The experiment was set up in six individual compartments of equal size (97 cm wide × 57 cm deep × 57
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cm high) in a temperature-controlled room. These were arranged into three blocks, with each block
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containing two compartments, each randomly allocated a light treatment (Blue Light / No Blue Light,
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hereafter referred to as BL and no BL treatments). This meant that in total three of the six compartments
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received a broad spectrum of irradiance from an array of LED lamps (Valoya AP67, 400-750nm, PAR 168
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µmol m-2 s-1 plus 32 µmol m-2 s-1 far red) and in the other three compartments blue light was attenuated by
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wrapping film Rosco #313 Canary Yellow (Westlighting, Helsinki, Finland) around the LED lamps (no
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BL): the height of lamps used in either treatment was adjusted to compensate for the lack of blue light and
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equalise the PAR received across the treatments (SI Appendix 1 Fig. S1, Table S1). All compartments
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were protected from extraneous light with white-black plastic film that blocked visible and UV radiation.
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The number of layers of these plastic sheets was also adjusted to help equalise temperature across the
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treatments (SI Appendix 1 Table S2). The UV-A treatment was applied as a split-plot factor by dividing
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each of the six compartments in two halves with a curtain of Rosco #226 film (Westlighting, Helsinki,
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Finland) attenuating all UV-A radiation (λ <400 nm): one half of each compartment received UV-A
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radiation from high power LED arrays with peak emission at 365 nm (mean and SE of 15.0 ± 0.6 µmol m-
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2 s-1: Z1-Z1-10UV00, LED Engin, San Jose, CA, USA). Spectral irradiance measurements were made in
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each compartment with an array spectroradiometer (Maya 2000Pro, Ocean Optics Inc., Dunedin, Florida,
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USA). The BL and UV-A treatments were chosen to approximate the ratio of UV-A: BL and PAR
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irradiance that we measured at noon in under-canopy shade in a Betula stand in southern Finland (Fig. 1,
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example of under-canopy spectral irradiance, SI Appendix 1 Table S3 and Table S4). The broad-spectrum
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lamps were kept on a 10-hour photoperiod from 08:00 to 18:00, whereas the UV-A LEDs were kept on for
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a 4-hour period centred around ‘noon’ from 10:30 to 14:30, when UV-A irradiance is at its highest in
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natural sunlight (Flint and Caldwell, 1998).
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Fig. 1. Spectral photon irradiance on 4th June 2016 at solar noon in the understorey of a Betula stand at
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Lammi Biological Station, Finland (130 masl, 61°03'14.3"N 25°02'14.2"E). Irradiance measured in a
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sunfleck (solid line) is compared with shade caused by canopy foliage (dashed line).
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Growing conditions
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Five seeds per 6-×-6 cm pot were sown directly into the growing substrate; well-soaked 1:1 pre-
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fertilised peat to vermiculite (Agra-vermiculite; Pull Rhenen, TX Rhenen, the Netherlands) a thin layer
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of peat was sieved at 2 mm gauge to provide a smooth surface giving good contact with the germinating
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seeds. After sowing, pots were placed for one day in darkness at 15°C to allow the seeds to become fully
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imbibed, then for three days the pots were under the light treatments at 5°C-day / 2°C-night to facilitate
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cold stratification, before increasing to reasonable spring-time temperature for northern temperate
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latitudes of 15°C day / 10°C night (± SE 0.24°C), with a VPD of 0.17 kPa day/ 0.18 kPa night during
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germination and subsequent growth. Temperature was continuously monitored with iButton sensors
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(Maxim Integrated, San Jose, United States) for the entire duration of the experiment to check that
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temperature was consistent among all compartments (SI Appendix 1 Table S2).
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After germination when the first pair of true leaves became visible, the seedlings were thinned to
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one plant per pot, leaving 288 plants in the experiment: 6 plants × 5 genotypes × 6 compartments × 2 split
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with or without UV-A radiation. The soil moisture was monitored daily, and maintained by adding c 50 ml
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per pot every 5-7 days. Plants were rotated within compartments to attenuate any unknown gradients in
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temperature, relative humidity, and irradiance.
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Acute high-light exposure
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Plants from each growth condition were randomly separated into two groups, a control group
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remaining under the growth conditions, and a group which would be exposed to an acute high-light-stress
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treatment. Each group contained 6 plants per treatment combination (genotype × blue × UV-A × high-
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light) divided in equal numbers per block.
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High-light treatments were made in a temperature-controlled greenhouse compartment at 25°C. The
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lamps (Osram Powerstar HQI-E 400W/D stadium lamps, Osram GmbH, Munich, Germany) warmed up
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for at least 1 hr prior to implementing the treatment to ensure their emission was stable, and 75-mm deep
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baths of flowing water, placed between the lamps and plants, served as a heat sink reducing the warming
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effect of the lamps. Plants were kept underneath the lamps for exactly 30 min, at which point the quantum
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yield of PSII was measured before they were returned to their growing conditions. Photon irradiance
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incident on the leaves of plants receiving the high-light treatment was 1800-2100 μmol-1 m-2 s-1 PAR (SI
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Appendix 1 Figure S3, Table S5).
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Non-invasive measurements of UV-screening and leaf chlorophyll content
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A Dualex Scientific+ device (Force-A TM, Paris, France) was used to make non-destructive
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measurements of leaf pigments based on their optical properties. The leaf chlorophyll content is
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calculated by the Dualex device based on the difference in transmission through the leaf in the near infra-
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red at 710 nm and 850 nm (NIR), and in the red regions of the spectrum (Cerovic et al. 2012). The
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relationship between leaf chlorophyll content (μg cm-2) and the chlorophyll-specific absorption index
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measured with the Dualex Scientific+ was linear across the range of values we obtained (Robson et al.
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unpublished data, Parry et al. 2014). The relative leaf adaxial-epidermal UV-A-absorbance at 375 nm is
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calculated by the Dualex device based on chlorophyll fluorescence at 375 nm relative to chlorophyll
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fluorescence in the red region of the spectrum while accounting for leaf chlorophyll content (Cerovic et
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al. 2012). There is a linear relationship between this index of UV-absorbance at 375 nm and epidermal
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flavonol content for the range of values that we report (Robson et al. unpublished data, also reviewed by
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Julkunen-Tiitto et al. 2015).
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Dualex measurements were made from all plants from both control and high-light groups, 45 days
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after germination under the growing light conditions of the experiment between 15:30 and 18:00. A
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second set of Dualex measurements were then taken three hours after the end of the high-light exposure
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(day 46) back in the experimental compartments of the growth room. Both sets of measurements were
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made on the same two fully-expanded mature non-senescent unshaded leaves per plant that were
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horizontal to the light source at the point of measurement. Plants were all rosettes with no visible signs of
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development beyond their vegetative state at the time of measurement.
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Measurements of the quantum yield of PSII
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Quantum yield at midday (day 45), of same two leaves per plant selected for Dualex measurements,
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was measured with a PAM fluorometer (mini-PAM, Heinz-Walz GmbH, Effeltrich, Germany). We used
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ϕPSII (calculated as Fq`/Fm`; Murchie and Lawson, 2013) as an indicator of the operating efficiency of
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PSII during the day for plants under the light treatments (day 45). It was possible to compare the Fq`/Fm`
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of leaves measured in ambient conditions under the light treatments since the photon irradiance was
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matched to be as similar as possible in each treatment combination. A second set of Fq`/Fm` measurements
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were made the following day (day 46) between 10:00 and 12:00 for the control group under growing light
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treatments (as before), and between 12:00 and 13:50 for the test group under high-light exposure. To
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assess damage to PSII, Fv/Fm of all plants was measured from the same leaves as ϕPSII measurements
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after they had been dark-adapted for at least 30 min on day 45 and on day 46 at 19:00 hrs.
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Quantitative Analysis of Soluble Phenolic Compounds
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HPLC Analysis
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Directly following the Dualex measurements after the high-light exposure (day 46), the leaves used
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for these optical assessments were harvested from both control and high-light plants. Only those leaves, or
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parts of leaves, that were unshaded by other leaves in the rosette were selected for biochemical extraction
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of soluble (i.e. non-cell-wall bound) phenolic compounds. Likewise, the petiole and proximal lamina
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section were excluded ensuring that the sampled leaves had actually received their respective radiation
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treatments, following Julkunen-Tiitto et al. (2015). After each sample was harvested, they were weighed
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ensuring that there was at least 100 mg fresh weight of each sample, and immediately frozen in liquid
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nitrogen inside Al-foil packets and stored at -80°C. Samples were lyophilised in the dark and transported
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on dry ice for HPLC and UV/Vis analyses. Leaves were ground in 3-ml 40% methanol using a pestle and
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mortar, before being placed in an ultrasonic bath (Ultrasonic compact cleaner UC 006 DM1, Tesla, CZ),
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and sonicated for 5 min. The samples were then centrifuged for 3 min at 6000 RPM, equivalent of 3461
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RCF (EBA 20 Hettich Zentrifugen, Germany). 1 ml of supernatant was filtered through a 0.2 μm filter
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(Premium Syringe Filters, Agilent, USA) and used for HPLC-DAD analysis with an Agilent 1200 HPLC
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system (Agilent Technologies, USA). Each extract for HPLC-DAD (1 ml) was collected in a vial, and 5
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μl of sample was injected into the HPLC for each analysis. Separation was performed using a Hypersil
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Gold (C18, 50 x 2.1 mm column, with 1.9 μm particles, Thermofisher scientific, San Jose, CA, USA)
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chromatographic column which was tempered to 30 °C during the separation process. Two mobile phases
264
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were used, mobile phase A consisted of 5% acetonitrile, and mobile phase B consisted of 80%
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acetonitrile, and both phases were acidified using methanoic acid with a ratio of 1:999 v/v. Flow of the
266
mobile phases was 0.3 ml min-1. Compounds were detected at 270, 314 and 360 nm. For the purpose of
267
quantification of phenolic compounds, and for particular flavonoids, peaks on an optical chromatogram
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detected at 360 nm were manually integrated. Peak areas were adjusted against the fresh weight of leaves
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and used to determined relative content of compounds in samples. The spectra of phenolic compounds
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were measured across a spectral interval of 190-750 nm. The identification of compounds was done with
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an UltiMate 3000 HPLC system (Thermofisher Scientific, USA San Jose, CA) followed by Q-TOF mass
272
spectrometer (micrOTOF-QII, Bruker Daltonics, Germany) by comparing the retention time order, UV-
273
VIS absorption spectra, and the mass per charge ratio of the mother and fragment ions of each phenolic
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compound against values from the literature (SI Appendix 2). The compound identity was subsequently
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assigned to individual peaks detected during quantitative HPLC-DAD analysis according to their
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retention time and UV-VIS absorption spectra similarity (SI Appendix 2).
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Data Analysis
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The effects of genotype, blue light, UV-A radiation, and high-light exposure and their interaction
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were tested using analysis of variance (ANOVA). All statistical tests were done in R version 3.2.2 (2016,
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The R Foundation for Statistical Computing, Vienna, Austria). Data were analysed using a linear mixed-
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effects model (LME), which was fitted using the NLME package (Pinheiro and Bates 2000). The function
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‘weights=varPower’ was used to reduce heterogeneity of variance in the model.
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A split-plot experimental design was used with BL/no BL the main effect factor, UV-A/no UV-A
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the split-plot factor, and genotype nested within the split-plot factor. At the end of the experiment, high
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light was also included in the model. There were three replicates of each treatment combination for each
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genotype, corresponding to the number of split-compartments receiving the same treatment.
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The six compartments were divided into three blocks based on the arrangement of pairs of BL and
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no BL treatments. Block was used as a random factor in the model. Multiple comparisons were only
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tested when treatment effects were p < 0.05 using a fit-contrast comparison to test for differences between
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each genotype and the WT. Holm’s correction was used to adjust p values for multiple testing. The R
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package ggplot2 (Wickham and Chang 2013) was used to construct all figures. Pearson’s correlation was
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used to test the relationship of epidermal flavonoids with ϕPSII and Fv/Fm, as well as leaf chlorophyll with
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ϕPSII and Fv/Fm.
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Results
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The accumulation of flavonoids is regulated by CRYs under BL but both CRYs and UVR8 under
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UV-A radiation
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Non-invasive measurements of UV-screening
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Genotypes differed in their adaxial leaf flavonol content, as assessed by Dualex, (p<0.001, SI
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Appendix 1 Table S6A). Although the effect of BL was marginally non-significant overall (p=0.062),
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there was a significant interactive effect of BL and genotype on UV-screening (Genotype × BL: p<0.001).
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Similarly, while UV-A had no significant effect overall (p=0.217), there was a significant Genotype × UV-
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A interaction (p= 0.006).
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These interactions indicate that the response to each of BL and UV-A radiation differed among
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genotypes. BL significantly increased UV-screening in uvr8-2, phot1 and WT (respectively p=0.022,
309
p=0.034, p=0.020; Fig. 2), but had no effect compared with no BL in cry1 cry2 (p=0.430). In the presence
310
of BL, cry1 cry2 had the lowest epidermal flavonol contents of all the genotypes, and was 24.5% lower
311
than WT (p<0.001, Table 1, Fig. 2).
312
Considering those treatments without BL, epidermal flavonol content in the upper epidermis of
313
phot1 plants was lower than that of WT, in both treatments with and without UV-A radiation (p=0.001,
314
p=0.021, respectively). In contrast, uvr8-2 grown in the absence of both BL and UV-A, had higher leaf
315
epidermal flavonol content than WT (p=0.007), and cry1 cry2 was not significantly higher than WT
316
(p=0.422, Fig. 2). In the treatment receiving UV-A radiation but not BL, there was no significant
317
difference in the epidermal flavonol content in either uvr8-2 or cry1 cry2 compared to the WT (p=0.290,
318
p=0.934).
319
Growth under the UV-A radiation treatments caused a decrease in epidermal flavonol content
320
compared with no UV-A (irrespective of BL) in cry1 cry2, and uvr8-2 (p<0.002, p=0.022), and had no
321
effect on phot1 or WT (p=0.931, p= 0.256). After a 4 h period back in their growing conditions, the 30
322
min of high-light exposure had caused no significant effect on the leaf adaxial epidermal anthocyanin or
323
flavonol content (p=0.895, p=0.305 Fig. 2 & SI Appendix 1 Fig. S3), but leaf chlorophyll content was
324
6.8% higher in plants exposed to high-light (p=0.026, SI Appendix 1 Fig. S4).
325
326
12
327
Fig. 2. Epidermal flavonol content estimated through epidermal UV-absorbance at 375 nm with Dualex.
328
Means ( 1 SE) are displayed for four A. thaliana genotypes grown under Blue Light (BL) / No Blue
329
Light (No BL) treatments in a split-plot with UV-A (Δ) or without UV-A () radiation.
330
331
332
Epidermal anthocyanin content followed the opposite trends to epidermal flavonol content in
333
general, hence in plants with low epidermal flavonol content the epidermal anthocyanin content tended to
334
be high, but a genotype-specific difference in this relationship was not detected (Table 1, SI Appendix 1
335
Figure S5). This pattern also meant that epidermal anthocyanin content was higher in the no BL than to
336
the BL treatments, however the relative differences between genotypes and treatments were much smaller
337
than for epidermal flavonol content (Table 1, Fig. 2). Leaf chlorophyll content tended to co-vary in the
338
same direction as epidermal flavonoids among genotypes and light treatments (Table 1). All genotypes
339
apart from cry1cry2 had higher chlorophyll content in the BL treatment than the no BL treatment (Table
340
1), whereas the effect of UV-A radiation on chlorophyll content was non-significant (SI Appendix 1 Table
341
S6B).
342
343
Table 1. Chlorophyll content, upper epidermal anthocyanin content, and absorption at 375nm detected
344
spectrophotometrically, ϕPSII and Fv/Fm under controlled conditions, ϕPSII and Fv/Fm under high-light
345
13
conditions for different A. thaliana genotypes under split-plot conditions of Blue/No Blue, with and
346
without UV-A. Chlorophyll and anthocyanin content measured with Dualex. Mean ± 1 standard error.
347
348
349
Qualitative analysis of the effects of blue light and UV-A radiation on leaf phenolic compounds
350
The same nine phenolic compounds were present in all our treatments at concentrations above the
351
detection limits, allowing their quantification by HPLC-DAD (listed in Table 2). Of these nine
352
compounds, four were confirmed to be kaempferol derivatives, three were sinapic-acid derivatives, and
353
two could not be positively identified.
354
Genotype had a significant effect overall on the sum of all measured phenolic compounds per unit
355
fresh weight (henceforth, phenolic content; p=0.002, Fig. 3). BL and UV-A did not have a significant
356
effect overall on total phenolic content (p=0.158, p=0.124), however, genotype and BL had a significant
357
interactive effect (p=0.004) following a similar pattern in the direction and size of effects to the leaf
358
adaxial-epidermal flavonol content measured with the Dualex. BL significantly increased total phenolic
359
content in phot1, compared with the no BL treatment (p=0.012, Fig. 3, Table 2), but had no significant
360
effect on any other genotypes (Table 2). Pairwise comparisons of genotypes revealed that in the BL only
361
cry1 cry2 had significantly lower total phenolic content than WT (p<0.001), but no other significant
362
differences between genotypes and WT (Fig. 3, SI Appendix 1 Table S7). High-light treatment had no
363
significant effect on the total phenolic content of leaves sampled 4 h after the exposure (SI Appendix 1
364
Table S8A-K).
365
Genotype
LED
UV-A
UV/Vis Absorption
Spectrophotometry
Chlorophyll Content
Anthocyanin Content
phot1
BL
No UV-A
17.22
1.02
19.94
0.01
0.180
0.003
cry1 cry2
BL
No UV-A
18.21
1.25
24.25
0.01
0.185
0.004
uvr8-2
BL
No UV-A
21.97
1.48
22.85
0.01
0.187
0.003
WT
BL
No UV-A
20.48
1.60
25.19
0.01
0.175
0.003
phot1
BL
UV-A
16.83
1.89
18.15
0.02
0.180
0.004
cry1 cry2
BL
UV-A
15.35
2.27
24.23
0.01
0.198
0.004
uvr8-2
BL
UV-A
15.32
2.03
24.71
0.01
0.174
0.003
WT
BL
UV-A
19.99
2.61
23.94
0.01
0.173
0.003
phot1
No BL
No UV-A
10.84
1.45
19.45
0.01
0.194
0.004
cry1 cry2
No BL
No UV-A
14.84
1.17
16.62
0.01
0.196
0.005
uvr8-2
No BL
No UV-A
19.13
1.89
20.41
0.01
0.198
0.005
WT
No BL
No UV-A
14.62
0.85
20.44
0.01
0.191
0.003
phot1
No BL
UV-A
9.22
1.03
18.18
0.01
0.198
0.005
cry1 cry2
No BL
UV-A
12.48
1.30
17.85
0.01
0.205
0.007
uvr8-2
No BL
UV-A
14.23
0.80
19.72
0.01
0.192
0.004
WT
No BL
UV-A
13.40
0.89
20.74
0.01
0.189
0.003
14
366
367
Fig. 3. Integrated area of the sum of total phenolic compounds per fresh weight in mg. Means ( 1 SE) are
368
displayed for four A. thaliana genotypes grown under Blue Light (BL) / No Blue Light (No BL)
369
treatments in a split-plot with UV-A (Δ) or without UV-A () radiation. Measurements were taken on day
370
46 after germination.
371
372
The response to growth under BL of the content of total kaempferol derivatives per unit fresh
373
weight was dependent on genotype, given that genotype had a significant effect overall (p=0.001), and
374
there was a significant interaction between genotype and BL (p=0.027: Table 2). However BL and UV-A
375
radiation had no significant overall effect on kaempferol content (p=0.161, p=0.094). Pairwise
376
comparisons also showed that when cry1 cry2 was grown under BL, it had 11.4% lower total kaempferol
377
content than WT (p=0.040: Table 2), but phot1 and uvr8-2 did not differ significantly from WT (p=0.097,
378
p=0.453). The accumulation of kaempferol derivatives appeared to be partly regulated by phot1 in the
379
absence of BL, as only phot1 had a significantly lower kaempferol content than WT in the no BL; a
380
difference of 12%.
381
As with kaempferol derivatives, the concentration of the total sinapic-acid derivatives per unit fresh
382
weight differed among genotypes (p<0.001). BL had no significant effect overall on sinapic-acid content
383
15
(p=0.154), but this was because the genotypes responded in contrasting ways to BL (significant
384
interactive effect; p<0.001). UV-A radiation had no significant overall effect on sinapic-acid content
385
(p=0.210). Under the BL treatment, phot1 mutants had a significantly higher total sinapic-acid content
386
than without BL (p=0.023), but the other genotypes did not differ (Table 2). Pairwise comparisons of
387
sinapic-acid contents between the WT and the photoreceptor mutants, showed that under BL leaves of
388
cry1 cry2 and phot1 had a significantly lower content of sinapic-acid derivatives than those of WT
389
(p<0.001, p=0.012). In the no BL treatment, phot1 had significantly lower sinapic-acid content than WT
390
(p=0.006), with all other genotypes showing no significant differences from WT (Table 2).
391
Among the individual phenolic compounds identified, genotype had a significant effect on the
392
concentrations of sinapoyl glucose (p<0.001), sinapoyl tartronate (p<0.001), kaempferol 3-O-rha-glu 7-O-
393
rha (p<0.001), kaempferol 3-O-glu-glu 7-O-rha (p=0.002) and kaempferol 3-O-glu 7-O-rha (p=0.014).
394
The response of these compounds to UV-A, and the interaction between BL and genotype, were consistent
395
in their direction and extent with the responses of the total content of phenolic compounds described
396
above (Table 2, SI Appendix 1 , Table S7). Although we report differences in the content of these various
397
phenolic compounds among our treatments, as described above, we did not detect a change in the
398
composition of phenolic compounds between treatments.
399
400
401
402
403
404
405
406
407
408
409
410
411
16
Table 2. The concentration of total phenolic compounds, total kaempferol derivatives, total sinapic-acid derivatives, sinapoyl glucose, sinapoyl
412
tartronate, kaempferol 3-O-rha-glu-7-O-rha, kaempferol 3-O-glu-glu-7-O-rha, kaempferol 3-O-glu 7-O-rha, sinapoyl malate and kaempferol 3-O-
413
rhamnoside 7-O-rhamnoside. Means (± 1 SE) are displayed for four A. thaliana genotypes grown under Blue Light / No Blue Light treatments in a
414
split-plot with or without UV-A radiation. Measurements were taken on day 46 after germination. The content of phenolic compounds is given as
415
the integrated peak area in arbitrary units per FW mg-1. Means are from three replicate compartments per treatment combination.
416
LED
BL
BL
No BL
No BL
UVA
NoUV-A
UV-A
NoUV-A
UV-A
Genotype
phot1
cry1
cry2
uvr8-2
WT
phot1
cry1
cry2
uvr8-2
WT
phot1
cry1
cry2
uvr8-2
WT
phot1
cry1
cry2
uvr8-2
WT
Total phenolic
compounds
52.72
± 5.04
54.91
± 1.98
64.73
± 4.24
61.61
± 3.55
40.74
± 5.58
50.96
± 3.50
58.23
± 6.85
59.49
± 8.00
47.52
± 3.37
39.17
± 5.50
49.41
± 5.03
48.27
±13.20
40.15
± 3.79
31.66
± 2.97
40.13
± 2.35
38.01
± 3.25
Total
kaempferols
25.96
± 2.40
24.02
± 0.90
28.91
± 1.86
26.81
± 1.33
20.10
± 2.35
22.48
± 3.28
26.25
± 3.09
25.69
± 3.69
21.18
± 1.43
15.85
± 3.07
21.31
± 2.07
21.84
± 2.15
18.29
± 1.73
13.46
± 4.31
17.31
± 1.09
16.74
± 1.65
Total sinapic
acids
17.35
± 0.94
18.62
± 0.66
21.35
± 1.43
20.88
± 1.34
14.85
± 1.95
17.17
± 1.20
19.07
± 2.24
20.36
± 2.63
15.86
± 1.21
12.58
± 1.56
16.72
± 1.76
15.84
± 1.40
13.32
± 1.35
9.52
± 1.02
13.67
± 0.75
12.77
± 0.99
Sinapoyl
glucose
5.88
± 0.45
6.18
± 0.32
7.28
± 0.51
7.21
± 0.55
4.87
± 0.76
5.69
± 0.54
6.19
± 0.72
7.23
± 0.84
5.19
± 0.45
3.52
± 0.35
6.19
± 0.69
5.14
± 0.42
4.04
± 0.51
3.45
± 0.31
4.89
± 0.27
4.36
± 0.34
Sinapoyl
tartronate
5.91
± 0.46
6.29
± 0.29
7.19
± 0.50
7.24
± 0.54
4.79
± 0.71
5.74
± 0.51
6.22
± 0.73
7.08
± 0.80
5.14
± 0.44
3.74
± 0.37
6.09
± 0.67
5.17
± 0.43
4.18
± 0.49
3.70
± 0.31
4.77
± 0.27
4.32
± 0.33
Kaempferol 3-
O-rha-glu-7-O-
rha
5.64
± 0.25
6.23
± 0.30
6.90
± 0.39
7.09
± 0.49
4.88
± 0.69
6.59
± 1.01
6.33
± 0.72
6.91
± 0.84
5.20
± 0.41
3.91
± 0.38
5.90
± 0.64
5.16
± 1.85
4.39
± 0.46
3.30
± 0.22
4.73
± 0.28
4.24
± 0.35
Kaempferol 3-
O-glu-glu-7-O-
rha
6.44
± 0.58
6.12
± 0.22
7.16
± 0.47
6.77
± 0.39
5.04
± 0.63
5.67
± 0.38
6.48
± 0.76
6.50
± 0.89
5.32
± 0.38
4.52
± 0.63
5.35
± 0.56
5.33
± 0.49
4.52
± 0.41
3.34
± 0.33
4.37
± 0.28
4.20
± 0.38
Kaempferol 3-
O-glu 7-O-rha
6.50
± 0.60
5.98
± 0.23
9.79
± 0.48
6.67
± 0.35
5.04
± 0.59
6.31
± 0.81
6.70
± 0.79
8.08
± 0.88
5.33
± 0.37
4.66
± 0.73
5.27
± 0.56
6.66
± 0.53
4.50
± 0.45
4.37
± 0.31
4.31
± 0.29
4.18
± 0.40
Sinapoyl
malate
6.95
± 0.70
6.15
± 0.33
6.87
± 0.43
6.43
± 0.32
5.19
± 0.52
6.28
± 0.69
6.65
± 0.86
6.05
± 1.00
5.53
± 0.45
5.33
± 1.07
4.44
± 0.41
6.19
± 0.80
5.10
± 0.66
3.73
± 0.32
3.94
± 0.24
4.09
± 0.48
Kaempferol 3-
O-rha 7-O-rha
6.34
± 0.76
5.67
± 0.36
7.21
± 0.43
6.28
± 0.23
5.14
± 0.48
6.11
± 0.79
6.13
± 0.86
5.94
± 1.23
5.34
± 0.39
3.83
± 0.83
5.11
± 0.44
6.29
± 0.69
4.74
± 0.56
3.73
± 1.95
3.91
± 0.28
4.13
± 0.65
17
417
Blue light increases the quantum yield of photosystem II via CRYs under growth conditions
418
To investigate the role of each class of photoreceptors in maintaining PSII function, we first
419
measured ϕPSII, and the maximum quantum yield (Fv/Fm) of dark-adapted plants, under their growing
420
conditions to serve as a baseline to compare against ϕPSII under high light and Fv/Fm after high-light
421
exposure.
422
The Fv/Fm of dark-adapted plants under their growing conditions differed significantly with
423
genotype (p=0.012) and BL (p=0.049), but not UV-A (p=0.314). Genotypes differed in their response of
424
Fv/Fm to BL, producing a significant interaction between genotype and BL (p=0.036). BL significantly
425
increased Fv/Fm compared with no BL in WT and phot1 (p=0.037, p=0.030), but had no significant effect
426
on cry1 cry2 nor uvr8-2 (p=0.301, p=0.109). This result meant that cry1 cry2 had a significantly lower
427
Fv/Fm than WT under the BL treatment (p<0.001), while the other genotypes were not significantly
428
different from the WT (Fig. 4)
429
430
Fig. 4. Maximum quantum yield of PSII (Fv/Fm) measured in dark-adapted leaves. Means ( 1 SE) are
431
displayed for four A. thaliana genotypes grown under Blue Light (BL) / No Blue Light (No BL)
432
treatments in a split-plot with UV-A (Δ) or without UV-A () radiation. Measurements were taken under
433
growth conditions.
434
18
435
Fig. 5. Operating efficiency of PSII measured as ϕPSII. Means ( 1 SE) are displayed for four A. thaliana
436
genotypes grown under Blue Light (BL) / No Blue Light (No BL) treatments in a split-plot with UV-A (Δ)
437
or without UV-A () radiation. Measurements were taken under growth conditions.
438
439
440
Genotype and BL also significantly affected ϕPSII under growth conditions (p<0.001, p=0.010,
441
Fig. 5); UV-A had no significant effect (p=0.169). BL significantly increased ϕPSII in phot1, uvr8-2 and
442
WT (p=0.028, p=0.016, p=0.031), but had no significant effect on cry1 cry2 (p=0.215). In the BL
443
treatment, cry1 cry2 had the lowest ϕPSII of all genotypes (p<0.001), but no other genotypes were
444
significantly different from WT (Fig. 5). When grown in the absence of BL, phot1 had significantly lower
445
ϕPSII in comparison to WT (p=0.001), however there were no other significant differences from the WT
446
among genotypes in the no BL treatment (Table S9).
447
448
19
449
Fig. 6. Operating efficiency of PSII measured as ϕPSII. Means ( 1 SE) are displayed for four A. thaliana
450
genotypes grown under Blue Light (BL) / No Blue Light (No BL) treatments in a split-plot with UV-A (Δ)
451
or without UV-A () radiation. Measurements were taken after high-light conditions.
452
20
453
Fig. 7. Maximum quantum yield of PSII (Fv/Fm) measured in dark-adapted leaves. Means ( 1 SE) are
454
displayed for four A. thaliana genotypes grown under Blue Light (BL) / No Blue Light (No BL)
455
treatments in a split-plot with UV-A (Δ) or without UV-A () radiation. Measurements were taken after
456
high-light conditions.
457
458
459
Under 30-min saturating high-light treatment, ϕPSII was reduced, but also became more variable
460
within genotypes and treatment combinations, compared with plants not receiving high light. This may
461
partially explain why, although ϕPSII under high light differed with genotype (p<0.001), previous growth
462
under BL or UV-A had no significant overall effect, nor interactive effects with genotype (Fig. 6; Table 1).
463
Among genotypes, cry1 cry2 had the lowest ϕPSII under high light and was the only mutant to
464
significantly differ from the WT (p=0.007). The Fv/Fm measured in dark-adapted leaves 4 hrs after the end
465
of the acute high-light exposure was not significantly affected by genotype, BL nor UV-A (Fig. 7,
466
p=0.080, p=0.234, p=0.338, respectively).
467
468
469
470
21
Discussion
471
Cryptochromes and UVR8 both regulate the accumulation of photoprotective pigments in response
472
to low doses of UV-A radiation during growth.
473
Our experiment examined how growth under broad spectrum irradiance treatments with and
474
without BL and UV-A radiation affected photoprotection and flavonoid accumulation. Under these
475
conditions, based on a ratio of UV-A:BL similar to that in understorey shade, BL had a relatively large
476
effect compared with UV-A on leaf epidermal UV-screening by flavonoids which we could mainly
477
attribute to regulation by cryptochromes. Within each genotype, the effects of BL and UV-A radiation on
478
phenolic content quantified from leaf extracts were consistent with those for UV-epidermal screening by
479
flavonols assessed optically, however among genotypes this relationship was not as consistent (Table 2,
480
Figs. 2 & 3). Considered together, the increase in optically-assessed flavonol content in plants grown
481
under BL, as well as several phenolic compounds quantified from leaf extracts, can be attributed to the
482
role of cryptochromes in promoting the accumulation of phenolic compounds in response to blue light.
483
Recent studies reporting large increases in the accumulation of phenolic compounds and flavonoids
484
caused by blue light have been conducted outdoors in experiments compared against attenuated solar blue
485
light (Siipola et al. 2015), or in controlled conditions using high irradiances of blue light, together with
486
PAR equivalent to full sunlight (Hoffman et al. 2015, Taulavuori et al. 2016). However, even with the
487
moderate PAR in our experiment, our BL treatment had a strong effect on phenolic accumulation which
488
outweighed that of our UV-A radiation treatment. From our results, we can assert that functional CRY1
489
and CRY2, promote photoprotective, as well as photomorphogenic responses to blue light
490
(Vandenbussche et al. 2005, Keller et al. 2011). Similarly, the small increase we report in chlorophyll
491
content in response to BL can also be attributed to the role of functional CRYs (Table 1). CRY 1 has been
492
reported to maintain a high concentration of chlorophyll in A. thaliana seedlings exposed to 24 hrs high
493
light (Kleine et al. 2007), and to increase chlorophyll concentration in the roots of A. thaliana in response
494
to blue light (Usami et al. 2004). Surprisingly, our results are the first that we are aware of to find that
495
CRYs mediate an increase in chlorophyll content in the leaves of A. thaliana grown under blue light.
496
Although it has already been shown that phot1 promotes cry-mediated accumulation of
497
anthocyanins (Kang et al. 2008), hitherto there was no evidence that phot1 elicits the accumulation of
498
flavonoids in the absence of blue light. Our result, that epidermal flavonol content was significantly lower
499
in phot1 when BL was attenuated, suggests that phot1 may contribute to the baseline accumulation of
500
flavonoids in the absence of (or at very low irradiances of) blue light. This does not necessarily imply that
501
phot1 itself promotes pathways that can mediate flavonoid accumulation, maybe phot1 simply affects the
502
metabolism of the plant in such a way that directs investment into the production of flavonoids. In this
503
respect, it would be interesting to know how phot interacts with other photoreceptors to regulate
504
22
constitutive flavonoid production.
505
Based on previous solar UV-attenuation studies outdoors, we hypothesized that UV-A radiation
506
would increase flavonol content in our WT plants (Ibdah et al. 2002 [attenuated 50% solar <360 nm],
507
Kotilainen et al. 2008, Kotilainen et al. 2009, Morales et al. 2013 [all three attenuated 50% solar
508
<400nm]). However, our UV-A treatment did not increase epidermal flavonol content assessed using
509
Dualex. There are several possible explanations for this discrepancy between our results and UV-A
510
supplementation studies. We used a UV-A LED source with a narrow peak at 365 nm, whereas the
511
majority of UV-A-supplementation studies which have used UV-A fluorescent lamps with a broader
512
spectrum across the UV-A region (Joshi et al. 2007 [330-390 nm], Victório et al. 2011 [320-400 nm],
513
Štroch et al 2015 [350-400 nm]). The absorption spectra of all the photoreceptors considered here, and
514
additionally phytochrome, absorb in the UV-A region (Shinomura et al. 1996, Briggs and Huala 1999,
515
Heijde and Ulm, 2012). This potential for interactions among photoreceptors may imply that responses
516
elicited are highly wavelength dependent within the UV-A region. Alternatively, it is possible that the
517
plants acclimated to the low but ecologically-realistic UV-A irradiance used in this experiment (15 μmol
518
m-2 s-1: Fig. 1, SI Appendix 1 Tables S1, S2, S3) throughout their growth, or that it was not high enough to
519
elicit a flavonoid response in WT. Transmittance to the chloroplasts of non-damaging amounts of UV-A
520
radiation may even be useful since it can drive photosynthesis (Bilger et al. 2007, Turnbull et al. 2013,
521
Štroch et al 2015), being absorbed by chlorophyll and inducing chlorophyll fluorescence (McCree 1981,
522
Lang and Lichtenthaler 1991). The potential benefits of UV-A radiation for photosynthesis could be more
523
pronounced in shaded understorey conditions, where light is often limiting and the UV-A irradiance is
524
considered unlikely to be high enough to induce stress or photodamage (Štroch et al 2015 Casal 2013b)
525
but is proportionally enriched compared to PAR (Flint and Caldwell 1998, Leuchner et al. 2011, Urban et
526
al. 2012, Dengel et al. 2015).
527
Although PHOT1 absorbs UV-A radiation, and has been shown to have an action spectrum for
528
tropism in the UV-A region, our UV-A treatment did not affect either the epidermal flavonol content or
529
leaf phenolic content of phot1 mutants. It has previously been suggested that the flavonoid response to BL
530
and UV-A radiation in A. thaliana is most likely to be largely driven by CRY1 and CRY2, which control
531
CHS expression and the subsequent flavonoid biosynthesis (Jenkins et al. 2001). Our results provide
532
evidence that cryptochromes and UVR8 (but not phototropin1) are both involved in eliciting flavonoids in
533
response to UV-A radiation.
534
535
536
537
538
23
Cryptochromes promote accumulation of soluble leaf phenolic compounds in response to both blue
539
light and UV-A radiation, whereas UVR8 promotes accumulation of sinapoyl glucose in response to
540
UV-A radiation
541
The composition of phenolic compounds, as opposed the total phenolic content, is often reported to
542
change in response to blue light and UV-A radiation (Morales et al. 2010, Morales et al. 2013, Siipola et
543
al. 2015). A coherent change in composition would involve the conversion of kaempferols to quercetin-
544
derivatives which have higher ROS-scavenging activity, along with sinapic-acid derivatives such as
545
sinapate esters, which absorb more UV radiation in epidermal cells (Sheahan, 1996, Götz et al. 2010,
546
Agati et al. 2012, Csepregi et al. 2016). In contrast to this expectation, our treatments did not produce
547
detectable changes in the composition of kaempferol and sinapic-acid derivatives, only affecting their
548
content. The directional effects of BL and UV-A on leaf phenolic content were consistent with the changes
549
in epidermal flavonoid content (Figs. 2 & 3), although to varying degrees of significance (SI Appendix 1
550
Table S6A, S7 and Tables S8A-K). Such a result is consistent with studies that report a change in the total
551
content of phenolic compounds in response to UV-A, as opposed to a change in their composition (Maffei
552
et al. 1999, Lee et al. 2014). Cryptochromes have been reported to mediate a general increase in flavonoid
553
accumulation in response to blue light (Ouzounis et al. 2015, Taulavuori et al. 2016), and blue light can
554
have species-specific effects on the total concentration of phenolic-acid derivatives (Ouzounis et al. 2015,
555
Taulavuori et al. 2016). There has been to-date little evidence of a specific contribution of cryptochromes
556
to the accumulation of particular groups of phenolic compounds in response to BL as well as to UV-A
557
radiation. Here, we found that cryptochromes contribute to the accumulation of total phenolic-acid
558
derivatives, several individual sinapic-acid derivatives and kaempferol derivatives in response to growth
559
under BL, as well as the accumulation of sinapoyl glucose and kaempferol 3-O-glu-glu-7-O-rha in
560
response to growth under UV-A radiation.
561
In addition to cryptochromes, functional UVR8 promoted the accumulation of sinapoyl glucose as
562
well as epidermal flavonols in response to UV-A radiation. This result supports the suggestion by Morales
563
et al. (2013) that cryptochromes and UVR8 could modulate the accumulation of phenolic compounds in
564
the UV-A region. One potential reason for plants to have multiple photoreceptors whose absorption
565
spectra lie within the UV-A region is that coordination between crys and UVR8 could provide the optimal
566
composition of phenolic compounds for growth and defense in a dynamic light environment where the
567
ratio of UV radiation to blue light often differs from that of full sunshine (Fig. 1, SI Appendix 1 Table S3).
568
However, we are not yet in a position to identify the signalling mechanisms by which cryptochromes and
569
UVR8 could co-regulate the accumulation of phenolic compounds in general, nor the interconversion of
570
specific compounds from the phenolpropanoid pathway (Wade et al. 2001, Morales et al. 2013).
571
572
24
Moderate blue light but not UV-A radiation during growth enhances the operating efficiency and
573
maximal quantum yield of PSII via cryptochromes
574
The UV-A treatment used in our experiment did not have any significant effect on the operating
575
efficiency of PSII (ϕPSII) nor on the maximum quantum efficiency of PSII (Fv/Fm). Similarly, a study
576
using higher UV-A irradiance on barley (Hordeum vulgare) found no significant effect on Fv/Fm (Štroch et
577
al. 2015). However, under unshaded conditions solar UV-A radiation has been reported to decrease Fv/Fm
578
in the A. thaliana mutant uvr8-1 (Coffey et al. 2017). Increases in Fv/Fm for plants that have been
579
cultivated under blue light have been reported in many plant species (Goins et al. 1997, Matsuda et al.
580
2008, Terfa et al. 2013, Hoffmann et al. 2015), yet to our knowledge this is the first study of the effects of
581
blue light on ϕPSII and Fv/Fm to compare differences amongst A. thaliana photoreceptor mutants cry1
582
cry2, phot1 and uvr8-2. Here, we report that CRYs mediated an increase of both ϕPSII and Fv/Fm during
583
growth under BL, and ϕPSII when subjected to acute high light, suggesting that CRYs play an important
584
role in the induction of photoprotection. It is conceivable that such a mechanism would be useful for a
585
plant during the transition from shade to a sunfleck, which is accompanied by a particularly large shift in
586
blue light. It has been suggested that CRYs could affect leaf morphology in response to blue light in such
587
a way as to moderate the absorption of light passing through the leaf or alter the ratio of absorption
588
between PSI and PSII reaction centres (Murchie and Lawson 2013, Miao et al. 2016). CRYs mediate
589
blue-light activation of D1 and D2 proteins which participate in the maintenance and repair of PSII
590
(Thum et al. 2001, Tsunoyama et al. 2004, Onda et al. 2008), and so may also be integral to the normal
591
functioning of PSII. Such mechanisms could explain the manner by which CRYs increased ϕPSII and
592
Fv/Fm under BL during growth in our experiment.
593
Klem et al. (2015) found the quantum efficiency of PSII to be positively correlated with leaf
594
flavonoid content in H. vulgare under high-light stress. However, in our study there was no overall
595
relationship between ϕPSII or Fv/Fm and with either epidermal UV-screening by flavonoids (SI Appendix
596
1 Fig. S7- Fig. S10) or leaf chlorophyll content (Fig. S11- S14). These results seem to rule out flavonoids
597
as being directly responsible for an improvement in photoprotection in our experiment. Boccalandro et al.
598
(2012) reported a reduced photosynthetic capacity of cry1 cry2 mutants when exposed to high light
599
suggesting that nonstomatal limitations could be responsible: one such limitation is a reduced electron
600
transport rate per unit area (Boonman et al. 2009). Additionally, Kleine et al. (2007) have demonstrated
601
that cry1 is necessary for the activation of 77 genes out of 996 that respond to high light. These included
602
genes responsible for phylloquinone electron acceptors in the PSI reaction centre, which are critical for
603
photosynthetic function, as well as the gene for Vitamin B6 which provides antioxidant activity against
604
ROS stress. All of the above examples and our own results support the contribution of CRYs in initiating
605
those high-light responses in plants, through which a higher ϕPSII and Fv/Fm can be maintained through
606
25
mechanisms besides photoprotection endowed by flavonoids.
607
608
Ecological implications of blue light and UV-A photoreceptor responses
609
Whilst A. thaliana is not an understorey species, as a model plant it has provided insight as to how
610
UVR8 and phytochromes coordinate shade responses (Fraser et al. 2016), as well as optimising flavonoid
611
content beneath a patchy canopy (Mazza and Ballaré 2015). Under controlled conditions, we report
612
important roles for cryptochromes and UVR8 in coordinating the accumulation of phenolic compounds in
613
response to UV-A radiation and blue light at an irradiance that can be found in understorey shade. It
614
remains to be seen whether naturally-occurring forest species would respond similarly to equivalent
615
changes in these wavelength regions.
616
In our experiment, there was a larger effect of blue light increasing flavonoid accumulation than
617
that of UV-A radiation. Although the UV-B irradiance in understorey shade is very low, both UV-A and
618
UV-B radiation are enriched relative to PAR. There is evidence that the photoprotection endowed by
619
flavonoids, especially against high irradiances of UV-B as well as UV-A radiation (Li et al. 1993, Jansen
620
et al. 1998), could constitute a precautionary response to sunflecks by increasing antioxidant capacity
621
(Gould et al.2000). Flavonoid responses to UV radiation are known to be dose dependent (reviewed by
622
Robson et al. 2015). For instance, Morales et al. (2010) reported a dose-dependent response of certain
623
flavonoids to UV-A radiation, which was quadratic under UV-B radiation. Considering the enrichment in
624
UV-B radiation relative to PAR in understorey shade (Flint and Caldwell 1998), the effects of understorey
625
UV-B radiation on flavonoid content and its synergistic effects with blue light would be worthy of
626
investigation since they could be more pronounced than those of UV-A radiation.
627
The epidermal flavonol contents we report were within the range of published values for A.
628
thaliana exposed to solar radiation for 36 hrs (Morales et al. 2013), but lower than the range of values
629
reported in H. vulgare exposed to solar radiation at a higher irradiance of supplemental UV-A than used in
630
our experiment (Klem et al. 2015). The relatively low values of epidermal UVA-absorption in our study
631
maybe one reason we did not find the same correlative relationship between flavonoid content and ϕPSII
632
or Fv/Fm as reported previously by Klem et al. (2015). Although equally, this could reflect species-specific
633
difference in this response for H. vulgare and A. thaliana, as further exemplified by the cultivar-specific
634
differences in Lactuca sativa (Ouzounis et al. 2015). These inconsistencies among species exemplify the
635
need to study the ecological relevance of this response for understorey species, and the physiological
636
mechanisms which allow plants to acclimate and capitalise on transient sunflecks between periods of
637
shade in the understorey.
638
639
640
26
Conclusions
641
Cryptochromes and UVR8 both have regulatory effects on the flavonoid response to UV-A
642
radiation in plants grown under controlled conditions, in light treatments based on a ratio of UV-A: BL
643
and PAR irradiance measured in understorey shade. Flavonol content in the adaxial epidermis did not
644
significantly correlate with ϕPSII or Fv/Fm values, nor did the UV-A radiation and BL treatments
645
noticeably prime leaves to cope with acute high-light. However, it is clear from our results that
646
cryptochromes are required for plants to attain high ϕPSII and Fv/Fm in the presence of blue light during
647
growth, and to maintain a high ϕPSII under high-light exposure. The decreased tolerance to high light in
648
the cry1 cry2 mutant (but not in the uvr8-2 mutant), with no concurrent decrease in epidermal flavonol
649
content, underlines the importance of cryptochromes in acclimation to high light through mechanisms
650
besides photoprotection by flavonoid accumulation.
651
652
Author Contributions
653
Craig Brelsford designed and carried out the experiment, analysed the data and wrote the
654
manuscript. Luis Morales contributed to the writing of and ideas behind the manuscript. Jakub Nezval
655
carried out HPLC analysis, UV-Vis spectrophotometry and contributed to the writing of the manuscript.
656
Titta Kotilainen advised on the experimental design, spectral irradiance measurements, manuscript
657
writing and analysis. Saara Hartikainen helped design, set-up and sampling for the experiment. Pedro
658
Aphalo helped to interpret the results and write the manuscript. T. Matthew Robson proposed the original
659
experiment, supervised the work and contributed throughout the entire process.
660
661
Acknowledgements
662
The Finnish Academy of Sciences funded this project through decisions # 266523 and # 304519 to
663
TMR. We thank Valoya Oy for providing the LED Lamps, David Israel, Neha Rai, Sari Siipola and Fang
664
Wang for advice on plant material and sampling, Gareth Jenkins and Tatsuya Kasai for initially donating
665
uvr8-2 and phot1 seeds, and staff at Lammi Biological Station for their help with field measurements of
666
irradiance. The participation of JN was supported by the Czech Ministry of Education, Youth and Sports
667
Project LO1208 “TEWEP” National Feasibility Programme I and EU structural funding Operational
668
Programme Research and Development for Innovation project # CZ.1.05/2.1.00/19.0388.
669
670
References
671
672
Augspurger CK (2003) Differences in leaf phenology between juvenile and adult trees in a temperate
673
deciduous forest. Tree Physiology 23
674
27
675
Agati G, Tattini M (2010) Multiple functional roles of flavonoids in photoprotection. New Phytologist
676
186: 786-793
677
678
Agati G, Azzarello E, Pollastri S, Tattini M (2012) Flavonoids as antioxidants in plants: location and
679
functional significance. Plant Science 196: 67-76
680
681
Ballaré CL, Sánchez RA, Scopel AL, Casal JJ, Ghersa CM (1987) Early detection of neighbour plants by
682
phytochrome perception of spectral changes in reflected sunlight. Plant, Cell & Environment 10: 551-557
683
684
Ballaré CL, Scopel AL, Sánchez RA (1990) Far-red radiation reflected from adjacent leaves: an early
685
signal of competition in plant canopies. Science 247: 329-332
686
687
Ballaré CL, Barnes PW, Flint SD (1995) Inhibition of hypocotyl elongation by ultravioletB radiation in
688
deetiolating tomato seedlings. Physiologia Plantarum 93: 584-592
689
690
Banerjee R, Batschauer A (2005) Plant blue-light receptors. Planta 220: 498-502
691
692
Barnes PW, Flint SD, Slusser JR, Gao W, Ryel RJ (2008) Diurnal changes in epidermal UV transmittance
693
of plants in naturally high UV environments. Physiologia plantarum 133: 363-372
694
695
Bilger W, Veit, M, Schreiber L, Schreiber U (1997). Measurement of leaf epidermal transmittance of UV
696
radiation by chlorophyll fluorescence. Physiologia plantarum 101: 754-763
697
698
Bilger W, Rolland M, Nybakken, L (2007) UV screening in higher plants induced by low temperature in
699
the absence of UV-B radiation. Photochemical & Photobiological Sciences 6: 190-195
700
701
Boccalandro HE, Giordano CV, Ploschuk EL, Piccoli PN, Bottini R, Casal JJ (2012). Phototropins but not
702
cryptochromes mediate the blue light-specific promotion of stomatal conductance, while both enhance
703
photosynthesis and transpiration under full sunlight. Plant physiology 158: 1475-1484
704
705
Booij-James IS, Dube SK, Jansen MA, Edelman M, Mattoo AK (2000) Ultraviolet-B radiation impacts
706
light-mediated turnover of the photosystem II reaction center heterodimer in Arabidopsis mutants altered
707
in phenolic metabolism. Plant Physiology: 1275-1284
708
28
709
Boonman A, Prinsen E, Voesenek LACJ, Pons TL (2009) Redundant roles of photoreceptors and
710
cytokinins in regulating photosynthetic acclimation to canopy density. Journal of Experimental Botany
711
60: 1179-1190
712
713
Briggs WR, Huala E (1999) Blue-light photoreceptors in higher plants. Annual review of cell and
714
developmental biology 15: 33-62
715
716
Briggs WR, Christie, JM (2002) Phototropins 1 and 2: versatile plant blue-light receptors. Trends in Plant
717
Science, 7: 204-210
718
719
Brown BA, Cloix, C, Jiang GH, Kaiserli E, Herzyk P, Kliebenstein DJ, Jenkins GI (2005) A UV-B-
720
specific signaling component orchestrates plant UV protection. Proceedings of the National Academy of
721
Sciences of the United States of America, 102:18225-18230
722
723
Casal JJ (2013a) Canopy light signals and crop yield in sickness and in health. ISRN Agronomy 2013
724
725
Casal JJ (2013b) Photoreceptor signaling networks in plant responses to shade. Annual review of plant
726
biology 64: 403-427
727
728
Casal JJ (2000) Phytochromes, cryptochromes, phototropin: photoreceptor interactions in plants.
729
Photochemistry and Photobiology 71: 1-11
730
731
Cerovic ZG, Masdoumier G, Ghozlen NB, Latouche G (2012) A new optical leaf-clip meter for
732
simultaneous nondestructive assessment of leaf chlorophyll and epidermal flavonoids. Physiologia
733
plantarum 146: 251-260
734
735
Christie JM, Jenkins GI (1996) Distinct UV-B and UV-A/blue light signal transduction pathways induce
736
chalcone synthase gene expression in Arabidopsis cells. The Plant Cell 8: 1555-1567
737
738
Christopher DA, Mullet JE (1994) Separate photosensory pathways co-regulate blue light/ultraviolet-A-
739
activated psbD-psbC transcription and light-induced D2 and CP43 degradation in barley (Hordeum
740
vulgare) chloroplasts. Plant Physiology, 104: 1119-1129
741
742
29
Coffey A, Prinsen E, Jansen MAK, Conway J (2017) The UVB photoreceptor UVR8 mediates
743
accumulation of UVabsorbing pigments, but not changes in plant morphology, under outdoor
744
conditions. Plant, Cell & Environment
745
746
Constabel AJ, Lieffers VJ (1996) Seasonal patterns of light transmission through boreal mixedwood
747
canopies. Canadian Journal of Forest Research 26: 1008-1014
748
749
Csepregi K, Neugart S, Schreiner M, Hideg É (2016) Comparative evaluation of total antioxidant
750
capacities of plant polyphenols. Molecules 21: 208
751
752
Davey MP, Susanti NI, Wargent, JJ, Findlay JE, Quick WP, Paul ND, Jenkins GI (2012) The UV-B
753
photoreceptor UVR8 promotes photosynthetic efficiency in Arabidopsis thaliana exposed to elevated
754
levels of UV-B.Photosynthesis research 114: 121-131
755
756
Davis PA, Hangarter RP (2012) Chloroplast movement provides photoprotection to plants by
757
redistributing PSII damage within leaves. Photosynthesis research 112: 153-161
758
759
Dengel S, Grace J, MacArthur A (2015) Transmissivity of solar radiation within a Picea sitchensis stand
760
under various sky conditions. Biogeosciences
761
762
Demkura PV, Ballaré CL (2012) UVR8 mediates UV-B-induced Arabidopsis defense responses against
763
Botrytis cinerea by controlling sinapate accumulation. Molecular plant 5: 642-652
764
765
Dion PP, Brisson J, Fontaine B, Lapointe L (2016) Light acclimation strategies change from summer
766
green to spring ephemeral as wild-leek plants age. American journal of botany 103: 963-970
767
768
Flint SD, Jordan PW, Caldwell MM (1985) Plant protective response to enhanced UVB radiation under
769
field conditions: leaf optical properties and photosynthesis. Photochemistry and Photobiology: 41: 95-99.
770
771
Flint SD, Caldwell MM (1998) Solar UVB and visible radiation in tropical forest gaps: measurements
772
partitioning direct and diffuse radiation. Global change biology 4: 863-870
773
774
Fraser DP, Hayes S, Franklin KA (2016) Photoreceptor crosstalk in shade avoidance. Current opinion in
775
plant biology 33:1-7
776
30
777
Fuglevand G, Jackson JA, Jenkins GI (1996) UV-B, UV-A, and blue light signal transduction pathways
778
interact synergistically to regulate chalcone synthase gene expression in Arabidopsis. The Plant Cell 8:
779
2347-2357
780
781
Goins GD, Yorio NC, Sanwo MM, Brown CS (1997) Photomorphogenesis, photosynthesis, and seed
782
yield of wheat plants grown under red light-emitting diodes (LEDs) with and without supplemental blue
783
lighting. J Exp Bot 48: 14071413
784
785
Gould KS, Markham KR, Smith RH, Goris JJ (2000) Functional role of anthocyanins in the leaves of
786
Quintinia serrata A. Cunn. Journal of Experimental Botany 5: 1107-1115
787
788
Götz M, Albert A, Stich S, Heller W, Scherb H, Krins A, Langebartels C, Seidlitz HK and Ernst D (2010)
789
PAR modulation of the UV-dependent levels of flavonoid metabolites in Arabidopsis thaliana (L.) Heynh.
790
leaf rosettes: cumulative effects after a whole vegetative growth period. Protoplasma 243: 95-103
791
792
Grant RH, Apostol K, Gao W (2005) Biologically effective UV-B exposures of an oak-hickory forest
793
understory during leaf-out. Agricultural and forest meteorology 132: 28-43
794
795
Heijde M, Ulm R (2012) UV-B photoreceptor-mediated signalling in plants. Trends in plant science 17:
796
230-237
797
798
Hoffmann AM, Noga G, Hunsche M (2015) High blue light improves acclimation and photosynthetic
799
recovery of pepper plants exposed to UV stress. Environmental and Experimental Botany 109: 254-263
800
801
Ibdah M, Krins A, Seidlitz HK, Heller W, Strack D, Vogt T (2002) Spectral dependence of flavonol and
802
betacyanin accumulation in Mesembryanthemum crystallinum under enhanced ultraviolet radiation. Plant,
803
Cell & Environment 25: 1145-1154
804
805
Inada S, Ohgishi M, Mayama T, Okada K, Sakai T (2004) RPT2 is a signal transducer involved in
806
phototropic response and stomatal opening by association with phototropin 1 in Arabidopsis thaliana. The
807
Plant Cell 16: 887-896
808
809
Jansen MAK, Gaba V, Greenberg BM (1998) Higher plants and UV-B radiation: balancing damage, repair
810
31
and acclimation. Trends in plant science 3: 131-135
811
812
Jenkins GI, Long JC, Wade HK, Shenton MR, Bibikova TN (2001) UV and blue light signalling:
813
pathways regulating chalcone synthase gene expression in Arabidopsis. New Phytologist 151: 121-131
814
815
Joshi PN, Ramaswamy NK, Iyer RK, Nair JS, Pradhan MK, Gartia S, Biswal B, Biswal, UC (2007)
816
Partial protection of photosynthetic apparatus from UV-B-induced damage by UV-A radiation.
817
Environmental and experimental botany 59: 166-172
818
819
Julkunen-Tiitto R, Nenadis N, Neugart S, Robson TM, Agati G, Vepsäläinen J, Zipoli G, Nybakken L,
820
Winkler B, Jansen MAK (2015) Assessing the response of plant flavonoids to UV radiation: an overview
821
of appropriate techniques. Phytochemistry reviews 14: 273-297
822
823
Kang B, Grancher N, Koyffmann V, Lardemer D, Burney S, & Ahmad, M (2008) Multiple interactions
824
between cryptochrome and phototropin blue-light signalling pathways in Arabidopsis thaliana. Planta
825
227: 1091-1099
826
827
Keller MM., Jaillais Y, Pedmale UV, Moreno JE, Chory J, Ballaré CL (2011). Cryptochrome 1 and
828
phytochrome B control shadeavoidance responses in Arabidopsis via partially independent hormonal
829
cascades. The Plant Journal 67:195-207
830
831
Kleine T, Kindgren P, Benedict C, Hendrickson L, Strand Å (2007) Genome-wide gene expression
832
analysis reveals a critical role for CRYPTOCHROME1 in the response of Arabidopsis to high irradiance.
833
Plant Physiology 144: 1391-1406
834
835
Klem K, Holub P, Štroch M, Nezval J, Špunda V, Tříska J, Jansen MA, Robson TM, Urban O (2015)
836
Ultraviolet and photosynthetically active radiation can both induce photoprotective capacity allowing
837
barley to overcome high radiation stress. Plant Physiology and Biochemistry 93: 74-83.
838
839
Kliebenstein DJ, Lim JE, Landry LG, Last RL (2002) Arabidopsis UVR8 regulates ultraviolet-B signal
840
transduction and tolerance and contains sequence similarity to human regulator of chromatin
841
condensation 1. Plant Physiology 130: 234-243
842
843
32
Kotilainen T, Tegelberg R, Julkunen-Tiitto R, Lindfors A, Aphalo PJ (2008) Metabolite specific effects of
844
solar UVA and UVB on alder and birch leaf phenolics. Global change biology 14: 1294-1304
845
846
Kotilainen T, Venäläinen T, Tegelberg R, Lindfors A, JulkunenTiitto R, Sutinen S, O’Hara RB, Aphalo
847
PJ (2009) Assessment of UV biological spectral weighting functions for phenolic metabolites and growth
848
responses in silver birch seedlings. Photochemistry and photobiology 85: 1346-1355
849
850
Kubasek WL, Shirley BW, McKillop A, Goodman HM, Briggs W, Ausubel FM (1992) Regulation of
851
flavonoid biosynthetic genes in germinating Arabidopsis seedlings. The Plant Cell 4: 1229-1236
852
Lang M and Lichtenthaler HK (1991) Changes in the blue-green and red fluorescence-emission spectra of
853
beech leaves during the autumnal chlorophyll breakdown. Journal of plant physiology 138: 550-553
854
855
Lee MJ, Son JE, Oh MM (2014) Growth and phenolic compounds of Lactuca sativa L. grown in a
856
closedtype plant production system with UVA,B, orC lamp. Journal of the Science of Food and
857
Agriculture 94: 197-204
858
859
Leuchner M, Hertel C, Menzel A (2011) Spatial variability of photosynthetically active radiation in
860
European beech and Norway spruce. Agricultural and forest meteorology 151: 1226-1232
861
862
Li J, Ou-Lee TM, Raba R, Amundson RG, Last RL (1993) Arabidopsis flavonoid mutants are
863
hypersensitive to UV-B irradiation. The Plant Cell Online 5: 171-179
864
865
Litthauer S, Battle MW, Lawson T, Jones MA (2015) Phototropins maintain robust circadian oscillation of
866
PSII operating efficiency under blue light. The Plant Journal 83: 1034-1045
867
868
Lopez OR, Farris-Lopez K, Montgomery RA, Givnish TJ (2008) Leaf phenology in relation to canopy
869
closure in southern Appalachian trees. American Journal of Botany 95: 1395-1407
870
871
Maffei M, Canova D, Bertea CM, Scannerini S (1999) UV-A effects on photomorphogenesis and
872
essential-oil composition in Mentha piperita. Journal of Photochemistry and Photobiology B: Biology 52:
873
105-110
874
875
Marder JB, Chapman DJ, Telfer A, Nixon PJ, Barber J (1987) Identification of psbA and psbD gene
876
products, D1 and D2, as reaction centre proteins of photosystem 2. Plant molecular biology, 9: 325-333
877
33
878
Martínez-García JF, Gallemí M, Molina-Contreras MJ, Llorente B, Bevilaqua MRR, and Quail PH (2014)
879
The shade avoidance syndrome in Arabidopsis: the antagonistic role of phytochrome A and B
880
differentiates vegetation proximity and canopy shade. PloS one 9: e109275
881
882
Matsuda R, Ohashi-Kaneko K, Fujiwara K, Kurata K (2008) Effects of blue light deficiency on
883
acclimation of light energy partitioning in PSII and CO2 assimilation capacity to high irradiance in
884
spinach leaves. Plant and Cell Physiology 49: 664-670
885
886
Mazza CA, Ballaré CL (2015) Photoreceptors UVR8 and phytochrome B cooperate to optimize plant
887
growth and defense in patchy canopies. New Phytologist 207: 4-9
888
889
Mazzella MA, Cerdán PD, Staneloni RJ, Casal JJ (2001) Hierarchical coupling of phytochromes and
890
cryptochromes reconciles stability and light modulation of Arabidopsis development. Development 128:
891
2291-2299
892
893
McCree KJ (1981) Photosynthetically active radiation. In Encyclopedia of Plant Physiology,
894
Physiological Plant Ecology I. Responses to the Physical Environments (Lange OL,Nobel PS,Osmond
895
CB, Ziegler H eds),Vol. 12: 4155. Springer-Verlag, Berlin . ISBN 3-540-10763-0
896
897
Miao YX, Wang XZ, Gao LH, Chen QY, Mei QU (2016) Blue light is more essential than red light for
898
maintaining the activities of photosystem II and I and photosynthetic electron transport capacity in
899
cucumber leaves. Journal of Integrative Agriculture, 15: 87-100
900
901
Montgomery RA, Chazdon, RL (2001) Forest structure, canopy architecture, and light transmittance in
902
tropical wet forests.Ecology 82: 2707-2718
903
904
Morales LO, Tegelberg R, Brosché M, Keinänen M, Lindfors A, Aphalo PJ (2010) Effects of solar UV-A
905
and UV-B radiation on gene expression and phenolic accumulation in Betula pendula leaves. Tree
906
Physiology 30: 923-934
907
908
Morales LO, Brosché M, Vainonen J, Jenkins GI, Wargent JJ, Sipari Nina, Strid Å, Lindfors AV,
909
Tegelberg R,Aphalo PJ (2013) Multiple roles for UV RESISTANCE LOCUS8 in regulating gene
910
expression and metabolite accumulation in Arabidopsis under solar ultraviolet radiation. Plant Physiology
911
34
161: 744-759
912
913
Murchie EH, Lawson T (2013) Chlorophyll fluorescence analysis: a guide to good practice and
914
understanding some new applications. Journal of experimental botany, 64: 3983-3998
915
916
Neff MM, Chory J (1998) Genetic interactions between phytochrome A, phytochrome B and
917
cryptochrome 1 during Arabidopsis development. Plant Physiol 118: 27-35¨
918
919
Onda Y, Yagi Y, Saito Y, Takenaka N, Toyoshima Y (2008) Light induction of Arabidopsis SIG1 and SIG5
920
transcripts in mature leaves: differential roles of cryptochrome 1 and cryptochrome 2 and dual function of
921
SIG5 in the recognition of plastid promoters. The Plant Journal 55: 968-978
922
923
Ouzounis T, Parjikolaei BR, Fretté X, Rosenqvist E, Ottosen C (2015) Predawn and high intensity
924
application of supplemental blue light decreases the quantum yield of PSII and enhances the amount of
925
phenolic acids, flavonoids, and pigments in Lactuca sativa. Frontiers in plant science 6
926
927
Pinheiro JC, Bates DM (2000) Mixed-Effects Models in S and S-Plus. Springer New York. 528
928
929
R Development Core Team (2006) R: A Language and Environment for Statistical Computing. R
930
Foundation for Statistical Computing, Vienna, Austria
931
932
Richardson AD, O’Keefe J (2009) Phenological differences between understory and overstory. In
933
Phenology of ecosystem processes. Springer New York. 87-117
934
935
Robson T, Klem, K, Urban O, Jansen MA (2015) Reinterpreting plant morphological responses to
936
UVB radiation. Plant, cell & environment 38: 856-866
937
938
Shalitin D, Hongyun Y, Todd CM, Maskit M (2002) Regulation of Arabidopsis cryptochrome 2 by blue-
939
light dependent phosphorylation. Nature 417: 763
940
941
Sheahan JJ (1996) Sinapate esters provide greater UV-B attenuation than flavonoids in Arabidopsis
942
thaliana (Brassicaceae). American Journal of Botany 679-686
943
944
35
Shinomura T, Nagatani A, Hanzawa H, Kubota M, Watanabe M, Furuya M (1996) Action spectra for
945
phytochrome A-and B-specific photoinduction of seed germination in Arabidopsis thaliana. Proceedings
946
of the National Academy of Sciences 93: 8129-8133
947
948
Siipola SM, Kotilainen T, Sipari N, Morales LO, Lindfors AV, Robson TM, Aphalo PJ (2015) Epidermal
949
UVA absorbance and wholeleaf flavonoid composition in pea respond more to solar blue light than to
950
solar UV radiation. Plant, cell & environment 38: 941-952
951
952
Somers DE, Devlin PF, Kay SA (1998) Phytochromes and cryptochromes in the entrainment of the
953
Arabidopsis circadian clock. Science 282: 1488-1490
954
955
Štroch M, Materová Z, Vrábl D, Karlický V, Šigut L, Nezval J, Špunda V (2015) Protective effect of UV-
956
A radiation during acclimation of the photosynthetic apparatus to UV-B treatment. Plant Physiology and
957
Biochemistry 96: 96
958
959
Taulavuori K, Hyöky V, Oksanen J, Taulavuori E, Julkunen-Tiitto R (2016) Species-specific differences in
960
synthesis of flavonoids and phenolic acids under increasing periods of enhanced blue light.
961
Environmental and Experimental Botany 121 (2016): 145-150
962
963
Terfa MT, Solhaug KA, Gislerød HR, Olsen JE, Torre S (2013) A high proportion of blue light increases
964
the photosynthesis capacity and leaf formation rate of Rosa× hybrida but does not affect time to flower
965
opening. Physiologia plantarum 148: 146-159
966
967
Turcsányi E, Vass I (2000) Inhibition of photosynthetic electron transport by UVA radiation targets the
968
photosystem II complex. Photochemistry and Photobiology 72: 513-520
969
970
Thum KE, Kim M, Christopher DA, Mullet JE (2001) Cryptochrome 1, cryptochrome 2, and
971
phytochrome A co-activate the chloroplast psbD blue lightresponsive promoter. The Plant Cell 13: 2747-
972
2760
973
974
Turnbull TL, Barlow AM, Adams MA (2013) Photosynthetic benefits of ultraviolet-A to Pimelea
975
ligustrina, a woody shrub of sub-alpine Australia. Oecologia 173: 375-385
976
977
Tsunoyama Y, Yoko I, Kazuya M, Maki K, Yoichi N, Go T, Yoshinori T, Takashi (2004) Blue light-
978
36
induced transcription of plastid-encoded psbD gene is mediated by a nuclear-encoded transcription
979
initiation factor, AtSig5. Proceedings of the National Academy of Sciences of the United States of
980
America, 101: 3304-3309
981
982
Urban O, Klem K, Ač A, Havránková K, Holišová P, Navratil M, Zitová M, Kozlová K, Pokorný R,
983
Šprtová M, Tomášková I (2012) Impact of clear and cloudy sky conditions on the vertical distribution of
984
photosynthetic CO2 uptake within a spruce canopy. Functional Ecology 26: 46-55
985
986
Usami T, Mochizuki N, Kondo M, Nishimura M, Nagatani A (2004) Cryptochromes and phytochromes
987
synergistically regulate Arabidopsis root greening under blue light. Plant and Cell Physiology 45: 1798-
988
1808.
989
990
Vandenbussche F, Pierik R, Millenaar FF, Voesenek LA, Van Der Straeten D (2005) Reaching out of the
991
shade. Current opinion in plant biology 8: 462-468
992
993
Verdaguer, D., Jansen, M. A., Llorens, L., Morales, L. O., & Neugart, S. (2017). UV-A radiation effects on
994
higher plants: Exploring the known unknown. Plant Science 255: 72-81.
995
996
Victório CP, LealCosta MV, Schwartz Tavares E, Machado Kuster R, Salgueiro Lage, CL (2011) Effects
997
of supplemental UV-A on the development, anatomy and metabolite production of Phyllanthus tenellus
998
cultured in vitro. Photochemistry and Photobiology 87: 685-689
999
1000
Wade HK, Bibikova TN, Valentine WJ, Jenkins GI (2001) Interactions within a network of phytochrome,
1001
cryptochrome and UV-B phototransduction pathways regulate chalcone synthase gene expression in
1002
Arabidopsis leaf tissue. The Plant Journal 25: 675-685
1003
1004
Wang H, Gu M, Cui J, Shi K, Zhou Y, Yu J (2009) Effects of light quality on CO2 assimilation,
1005
chlorophyll-fluorescence quenching, expression of Calvin cycle genes and carbohydrate accumulation in
1006
Cucumis sativus. Journal of Photochemistry and Photobiology B: Biology 96: 30-37
1007
1008
Wickham H, Chang W (2013) An implementation of the Grammar of Graphics. Version: 0.9.3.1
1009
1010
1011
1012
... UV-B and UV-A radiation stimulate PheC synthesis via the UV Resistance Locus 8 [24]. High intensities of PAR also induce PheC accumulation [25], especially blue light, which is perceived via cryptochromes (CRYs) [26,27]. Although phytochromes (PHY) are active primarily in the red region of light spectra [28], it was recently found that phytochromes in their active state (Pfr form) absorb photons also in the blue region [29,30]; thus, a role of PHY in blue-light-dependent regulation of PheCs synthesis cannot be excluded. ...
... In this context, the activation of phenylpropanoid and flavonoid pathways followed by the accumulation PheCs in leaf tissues could be classified as one of the many blue-light-induced acclimation responses of plants to high/excessive PAR. Since PheCs are relatively stable in leaf tissues [77], blue light could be used as an instrument for priming the plants against photo-oxidative stress [26] and should be further studied as a factor which increases plant tolerance against other environmental stresses interconnected with high ROS production (i.e., cross-tolerance) [89]. The importance of PheCs increases in conditions impairing the function of AOX enzymes (strong UV, high or low temperature, heavy metals) which are more sensitive to degradation/inactivation due to their protein nature. ...
Article
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Photosynthetically active radiation (PAR) is an important environmental cue inducing the production of many secondary metabolites involved in plant oxidative stress avoidance and tolerance. To examine the complex role of PAR irradiance and specific spectral components on the accumulation of phenolic compounds (PheCs), we acclimated spring barley (Hordeum vulgare) to different spectral qualities (white, blue, green, red) at three irradiances (100, 200, 400 µmol m−2 s−1). We confirmed that blue light irradiance is essential for the accumulation of PheCs in secondary barley leaves (in UV-lacking conditions), which underpins the importance of photoreceptor signals (especially cryptochrome). Increasing blue light irradiance most effectively induced the accumulation of B-dihydroxylated flavonoids, probably due to the significantly enhanced expression of the F3’H gene. These changes in PheC metabolism led to a steeper increase in antioxidant activity than epidermal UV-A shielding in leaf extracts containing PheCs. In addition, we examined the possible role of miRNAs in the complex regulation of gene expression related to PheC biosynthesis.
... Plants produce photoprotective pigments such as carotenoids and flavonoids in response to high-light stress and UV radiation (Agati et al., 2020;Agati & Tattini, 2010). There is increasing evidence that flavonoid accumulation is mediated through cryptochromes (CRYs) in response to blue light and down to 350-nm wavelength of UV-A radiation, while UV Resistance Locus 8 (UVR8) largely dictates plant responses to UV-B radiation and up to 350-nm wavelength of UV-A radiation (Brelsford, Morales, et al., 2019a;Rai et al., 2019;Rai et al., 2020). The flavonoids in plant leaves partly function as antioxidants but can be broadly separated into flavonols/flavones which also screen UV-radiation, and anthocyanins which absorb blue-green light, and to a lesser-extent UV radiation (Agati & Tattini, 2010). ...
... grown in full sunlight. Likewise, in a growth room, simulated understorey blue light increased leaf adaxial epidermal flavonol content via CRY photoreceptors under controlled PAR conditions(Brelsford, Morales, et al., 2019a). Accordingly, much of the reduction in flavonols during canopy closure in summer (Figure 4A), could be attributable to the reduction in blue light reaching the understorey. ...
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Forest understorey plants receive most sunlight in springtime before canopy closure, and in autumn following leaf‐fall. We hypothesized that plant species must adjust their phenological and photoprotective strategies in response to large changes in the spectral composition of the sunlight they receive. Here, we identified how plant species growing in northern deciduous and evergreen forest understoreys differ in their response to blue light and ultraviolet (UV) radiation according to their functional strategy. We installed filters in a forest understorey in southern Finland, to create the following treatments attenuating: UV radiation < 350 nm, all UV radiation (< 400 nm), all blue light and UV radiation (< 500 nm), and a transparent control. In eight species, representing different functional strategies, we assessed leaf optical properties, phenology, and epidermal flavonoid contents over two years. Blue light accelerated leaf senescence in all species measured in the understorey, apart from Quercus robur seedlings, whereas UV radiation only accelerated leaf senescence in Acer platanoides seedlings. More light‐demanding species accumulated flavonols in response to seasonal changes in light quality compared to shade‐tolerant and wintergreen species and were particularly responsive to blue light. Reduction of blue and UV radiation under shade reveals an important role for microclimatic effects on autumn phenology and leaf photoprotection. An extension of canopy cover under climate change, and its associated suppression of understorey blue light and UV radiation, may delay leaf senescence for understorey species with an autumn niche. This article is protected by copyright. All rights reserved.
... UV-A and UV-B wavelengths are known to regulate the synthesis of some classes of plant metabolites via wavelength-specific photoreceptors (such as UVR8, phototropin, and cryptochrome) [69,70]. For example, biosynthesis of flavonoids and other phenolics is co-regulated through the photoreceptors UVR8 [12,16,71] and cryptochrome [72]. This study shows that the accumulation of carotenoids under UV radiation is not dependent on one particular photoreceptor. ...
Article
Full-text available
UV-B and UV-A radiation are natural components of solar radiation that can cause plant stress, as well as induce a range of acclimatory responses mediated by photoreceptors. UV-mediated accumulation of flavonoids and glucosinolates is well documented, but much less is known about UV effects on carotenoid content. Carotenoids are involved in a range of plant physiological processes, including photoprotection of the photosynthetic machinery. UV-induced changes in carotenoid profile were quantified in plants (Arabidopsis thaliana) exposed for up to ten days to supplemental UV radiation under growth chamber conditions. UV induces specific changes in carotenoid profile, including increases in antheraxanthin, neoxanthin, violaxanthin and lutein contents in leaves. The extent of induction was dependent on exposure duration. No individual UV-B (UVR8) or UV-A (Cryptochrome or Phototropin) photoreceptor was found to mediate this induction. Remarkably, UV-induced accumulation of violaxanthin could not be linked to protection of the photosynthetic machinery from UV damage, questioning the functional relevance of this UV response. Here, it is argued that plants exploit UV radiation as a proxy for other stressors. Thus, it is speculated that the function of UV-induced alterations in carotenoid profile is not UV protection, but rather protection against other environmental stressors such as high intensity visible light that will normally accompany UV radiation.
... UVA-radiation can effectively induce cryptochrome (CRY) and phototropin (PHOT) signal transduction [35]. In Arabidopsis thaliana, UVR8 is generally activated by UVB (280-315 nm) radiation, and it also responds to UVA (365 nm, 15 μmol/m 2 /s) radiation and promotes the production of antioxidants [36]. Additionally, UVR8 presented UVA-dose dependency, with the most apparent upregulation obtained with UV6 treatment, as well as a relatively tender but also significant up-regulation caused by UV12 ( Figure 10). ...
Article
Full-text available
Ultraviolet-A (UVA) (315–400 nm) is an essential environmental signal that regulates plant development and affects phytochemicals biosynthesis, including glucosinolate biosynthesis. The effects of different UVA (380 ± 10 nm, 40 μmol/m2/s) exposure durations, including 0 h/d (UV0), 6 h/d (UV6) and 12 h/d (UV12), on the growth and phytochemicals of Chinese kale (Brassica alboglabra) under white 250 μmol/m2/s LEDs were investigated. UVA exposure of different durations influenced the growth and phytochemicals biosynthesis of Chinese kale. Prolonging UVA irradiation throughout the growth cycle positively affected the growth and the development of Chinese kale, with evident increases in the dry weights of shoots and roots, plant height, stem diameter, specific leaf weight and flower budding rate. The application of UVA increased the soluble sugar content, whereas higher flavonoid content and antioxidant capacity (FRAP) and lower nitrate content were only observed in Chinese kale exposed to UV6 treatment. Besides, the qPCR assay showed that supplemental UVA-radiation exposure up-regulated the gene expressions of UVR8, transcription factors genes and genes related to the glucosinolate biosynthesis pathway, thereby promoting the accumulation of glucosinolates. Therefore, supplemental UVA-radiation exposure for 12 h/d was more conducive to plant growth, while supplemental UVA-radiation exposure for 6 h/d was better for phytochemical biosynthesis in Chinese kale in an artificial-light plant factory.
... electron transport capacity, antenna molecules per reaction centre). Although regulation through feedback plays a key role, once again, pigment composition, photosynthetic antenna size, and abundance of photosynthetic enzymes are also regulated through the interaction of multiple photoreceptors (Anderson et al., 1995;Brelsford et al., 2018;Rai et al., 2020). ...
Preprint
In this review we discuss the different factors affecting the use efficiency of light considering quantum properties of light, pigment light absorption, whole-leaf light absorption, the action spectrum of photosynthesis and allocation of biomass to leaf area, and how of these processes are regulated by the color of light.
... A recent study identifies a set of high light-responsive genes that protect plants against high light by dynamically regulating hormones, anthocyanin, photosynthesis, photoreceptors, and phytochrome-interacting factors (Huang et al., 2019). Another study reveals that cryptochromes CRY1 responds to high light through photoprotection mechanisms and flavonoid accumulation (Brelsford et al., 2019). Therefore, it is of great significance to study mechanisms of maintaining the normal physiological function of cells by dissipating excess excitation energy when plants are exposed to low temperature stress. ...
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The light-harvesting chlorophyll a/b-binding proteins (Lhcb) constitute the antenna system of the photosynthetic apparatus that has an essential function in photosynthesis and modulating stress responsiveness. In this study, we identified 35 Lhcb genes (BnLhcbs) in the rapeseed (Brassica napus L.) genome, which were clustered into 8 groups. The BnLhcb genes were distributed on 15 chromosomes of rapeseed; the subsequent analysis of gene structures showed that these members were highly conserved. Based on the importance of Lhcbs for plant defense against abiotic stresses, responses of BnLhcbs to cold stress were analyzed using cold-tolerant and -sensitive rapeseed cultivars. The BnLhcb genes exhibited distinct expression patterns, among which, BnLhcb3.4 was dramatically induced in the tolerant cultivar and down-regulated in the sensitive cultivar. Furthermore, the cDNA sequences of the BnLhcb3.4 gene was successfully cloned, the subcellular investigation confirmed that BnLhcb3.4 localized in chloroplast. The transgenic analysis indicated that overexpression of the BnLhcb3.4 gene significantly enhanced the freezing tolerance of transgenic Arabidopsis, together with increased abscisic acid (ABA) sensitivity. In addition, a set of ABA-responsive genes were altered in the transgenic plants. Taken together, these data demonstrate that BnLhcb3.4 may contribute to cold tolerance by evolving ABA signaling pathway.
Chapter
Among the different types of abiotic stress, one of the most important is irradiation stress. Ultraviolet (UV) irradiation represents between 7 and 9% of total solar radiation, and and is composed between 100 and 400 nm of the electromagnetic spectrum. It is mainly divided into UV-A (315–400 nm), UV-B (280–320 nm), and UV-C (200–280 nm). This type of irradiation can affect DNA, proteins, and plant cells. This could affect the development, morphology, and photosynthesis. To counteract these harmful effects, plants use their defense mechanisms, mainly the production of secondary metabolites, among which phenolic compounds stand out (phenolic acids, flavonoids, stilbenes, tannins, lignins, and coumarins). These are of great interest for its antioxidant, anticancer, antimicrobial properties, among others. However, the responses in the increase of the different phenolic compounds will depend on factors such as intensity, type of UV light and exposure time, which is reflected in the different investigations carried out on this topic. In this sense, it is interesting to review the variety of phenolic compounds that have been reported through induction by UV irradiation.
Chapter
Plants are continually exposed to various environmental extremities during their growing period. As such plants have to constantly struggle with different abiotic and biotic factors. Biotic factors can be controlled to a certain extent through the application of pesticides or by adopting various crop protection techniques. But the adverse impacts of abiotic stress elements such as drought, high temperature, salinity, heavy rainfall, snowfall, UV radiations, hazardous chemicals, air pollutants, etc. are very difficult to manage. Plants usually adopt various mechanisms involving alteration in anatomical, physiological, biochemical functions, or regulation of different stress-responsive genes, signalling pathways etc. Abiotic stresses cause modifications in plant metabolism that leads to enhanced production of different secondary metabolites like polyamines, phenol, proline etc. which, in turn, act directly or indirectly to build up abiotic stress tolerance by activating different stress response systems. Starch, the major reserve material of plants plays a key role in stress mitigation. Plants remobilize their reserve starch during stress conditions to provide energy. This chapter aims to discuss briefly how plants perceive different kinds of stresses, transduce early signals within their system, elicit different types of responses, or how these stress responses are determined genetically. Attempts have also been made to illustrate what options would be helpful to attain agricultural sustainability through the mitigation of stresses.
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UV-A light has different effects on the growth and bioactive compounds of vegetables, medicinal plants, and other crops. The purpose of this study was to determine the effects of short-term irradiation with UV-A light-emitting diodes (LEDs) on the growth and bioactive substances of kale (Brassica oleracea var. acephala). Two-week-old kale seedlings were cultivated for 3 weeks in a plant factory illuminated with LEDs (red:white:blue = 8:1:1) of 150 μmol m−2 s−1 photosynthetic photon flux density. Then, the plants were continuously exposed to five peak wavelengths (365, 375, 385, 395, and 405 nm) of UV-A LEDs with an energy of 30 W m−2 in addition to the background lighting for 7 days. Treatments with 395 and 405 nm wavelengths increased most of the assessed growth characteristics and photosynthetic rates compared to the control after 7 days of treatment. The maximum quantum efficiency of photosystem II (Fv/Fm) value started to decrease after 1 day of treatment, and after 5 days, we detected an increase in the concentration of reactive oxygen species with a decrease in the wavelength of the UV-A light treatment. There were increases in the total phenolic and flavonoid contents and antioxidant levels of kale plants subjected to all of the UV-A LEDs compared with control plants after 7 days. Our observations indicated that phenylalanine ammonia lyase (PAL) and chalcone synthase gene expression and PAL enzyme activity were upregulated by the UV-A LED treatments, although no significant differences among treatments were detected. Collectively, our results indicate that kale biomass and bioactive compounds can be enhanced through supplementary UV-A radiation, with 405-nm LEDs having the best effects, thereby suggesting that it would be beneficial to conduct additional research on the spectral threshold between UV-A and deep-blue light.
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Ultraviolet (UV) radiation is an important environmental factor for plant communities; however, plant responses to solar UV are not fully understood. Here, we report differential effects of solar UV-A and UV-B radiation on the expression of flavonoid pathway genes and phenolic accumulation in leaves of Betula pendula Roth (silver birch) seedlings grown outdoors. Plants were exposed for 30 days to six UV treatments created using three types of plastic film. Epidermal flavonoids measured in vivo decreased when UV-B was excluded. In addition, the concentrations of six flavonoids determined by high-performance liquid chromatography-mass spectrometry declined linearly with UV-B exclusion, and transcripts of PAL and HYH measured by quantitative real-time polymerase chain reaction were expressed at lower levels. UV-A linearly regulated the accumulation of quercetin-3-galactoside and quercetin-3-arabinopyranoside and had a quadratic effect on HYH expression. Furthermore, there were strong positive correlations between PAL expression and accumulation of four flavonols under the UV treatments. Our findings in silver birch contribute to a more detailed understanding of plant responses to solar UV radiation at both molecular and metabolite levels.
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Mutants affected in flavonoid (tt4) or sinapate ester (fah1) biosynthesis were used to assess the relative importance of these phenolic UV photoprotectants in Arabidopsis. Flavonoid and sinapate ester absorption was more specific for UV-B than major nonphenolic chromophores in crude extracts. A new method of evaluating phenolic UV-B attenuation was developed using fluorescence analysis. When excited by UV-B, sinapate ester containing leaves and cotyledons had enhanced sinapate ester fluorescence and reduced chlorophyll fluorescence relative to those without sinapate esters. Although fluorescence analysis gave no evidence of UV-B attenuation by flavonoids, enhanced chlorophyll and protein loss were observed upon UV-B exposure in flavonoid-deficient leaves, suggesting they have another mechanism of UV-B protection. The hydroxycinnamates have been largely ignored as UV-B attenuating pigments, and the results indicate that greater attention should be paid to their role in attenuating UV-B.
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UVB radiation is biologically active; in plants, it can induce a range of molecular, biochemical, morphological and developmental responses. Although much progress has been made in elucidating UVB perception and signalling pathways under controlled laboratory conditions, understanding of the adaptive, ecological role of UVB responses is still very limited. In this study, we looked at the functional role of UVR8 under outdoor light conditions, by studying growth, photosynthetic competence and accumulation of UV absorbing pigments in a mutant lacking functional UVR8 protein. It was found that the influence of UVB on morphology is restricted to the summer, and is independent of UVR8. In contrast, UVB had an effect on the content of UV-absorbing pigments and the maximal efficiency of photosystem II of photosynthesis in the uvr8-1 mutant throughout the year. It is concluded that the UVR8 photoreceptor plays a role throughout the year, in the temperate climate zone, even when UVB levels are relatively low.
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Chlorophyll fluorescence analysis has become one of the most powerful and widely used techniques available to plant physiologists and ecophysiologists. This review aims to provide an introduction for the novice into the methodology and applications of chlorophyll fluorescence. After a brief introduction into the theoretical background of the technique, the methodology and some of the technical pitfalls that can be encountered are explained. A selection of examples is then used to illustrate the types of information that fluorescence can provide.
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Identifying factors that influence variation in light availability within forested ecosystems represents an important component in our understanding of the complex determinants of tree seedling regeneration. We assessed the influence of forest structure and canopy tree architecture on spatial heterogeneity of understory light availability in three old-growth and three second-growth forests in lowland Costa Rica. Forest structure and understory light availability were measured within forest types using contiguous 10 × 10 m quadrats along three 130–160 m transects in each stand. Two 20 × 60 m plots in each forest type were sampled more intensively, including vertical profiles of light availability from 1 to 9 m height. Mean diffuse light transmittance increased from 2% at 1 m height to over 10% at 9 m height and did not differ significantly between forest types at any height. However, the relationships among height classes differed between forest types. Second-growth plots showed a negative spatial autocorr...
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Ultraviolet-A radiation (UV-A: 315–400 nm) is a component of solar radiation that exerts a wide range of physiological responses in plants. Currently, field attenuation experiments are the most reliable source of information on the effects of UV-A. Common plant responses to UV-A include both inhibitory and stimulatory effects on biomass accumulation and morphology. UV-A effects on biomass accumulation can differ from those on root: shoot ratio, and distinct responses are described for different leaf tissues. In this paper we analysed inhibitory and enhancing effects of UV-A on photosynthesis, as well as activation of photoprotective responses, including UV-absorbing pigments. UV-A-induced leaf flavonoids are highly compound-specific and species-dependent. Many of the effects on growth and development exerted by UV-A are distinct to those triggered by UV-B and vary considerably in terms of the direction the response takes. Such differences may reflect diverse UV-perception mechanisms with multiple photoreceptors operating in the UV-A range and/or variations in the experimental approaches used. This review highlights a role that various photoreceptors (UVR8, phototropins, phytochromes and cryptochromes) may play in plant responses to UV-A when dose, wavelength and other conditions are taken into account.
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Premise of the study: Spring-ephemeral forest-herbs emerge early to take advantage of the high-light conditions preceding canopy closure; they complete their life cycle in a few weeks, then senesce as the tree canopy closes. Summer greens acclimate their leaves to shade and thus manage to maintain a net carbon gain throughout summer. Differences in phenology among life stages within a species have been reported in tree saplings, whose leaf activity may extend beyond the period of shade conditions caused by mature trees. Similar phenological acclimation has seldom been studied in forest herbs. Methods: We compared wild-leek bulb growth and leaf phenology among plants from seedling to maturity and from under 4 to 60% natural light availability. We also compared leaf chlorophyll content and chl a/b ratio among seedlings and adult plants in a natural population as an indicator of photosynthetic capacity and acclimation to light environment. Key results: Overall, younger plants senesced later than mature ones. Increasing light availability delayed senescence in mature plants, while hastening seedling senescence. In natural populations, only seedlings acclimated to the natural reduction in light availability through time. Conclusions: Wild-leek seedlings exhibit a summer-green phenology, whereas mature plants behave as true spring ephemerals. Growth appears to be more source-limited in seedlings than in mature plants. This modulation of phenological strategy, if confirmed in other species, would require a review of the current classification of species as either spring ephemerals, summer greens, wintergreens, or evergreens.