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Rediscoveringleafopticalproperties:NewinsightsintoplantacclimationtosolarUVradiation

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Plant Physiology and Biochemistry 93 (2015) 94e100

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Plant Physiology and Biochemistry

journal homepage: www.elsevier .com/locate/plaphy

Rediscovering leaf optical properties: New insights into plantacclimation to solar UV radiation

Paul W. Barnes a, *, Stephan D. Flint b, Ronald J. Ryel c, Mark A. Tobler a, Anne E. Barkley a,Jason J. Wargent d

a Department of Biological Sciences and Environment Program, Loyola University New Orleans, 6363 St. Charles Avenue, New Orleans, LA 70118, USAb Department of Forest, Rangeland and Fire Sciences, UIPO 441135, University of Idaho, Moscow, ID 83844-1135, USAc Department of Wildland Resources, Utah State University, 5230 Old Main Hill, Logan, UT 84322-5230, USAd Institute of Agriculture & Environment, Massey University, Private Bag 11222, Palmerston North 4442, New Zealand

a r t i c l e i n f o

Article history:Received 1 October 2014Accepted 17 November 2014Available online 18 November 2014

Keywords:Chlorophyll fluorescenceEpidermal UV transmittanceFlavonoidsIntegrating sphereMicroprobesUV-BUV-A

Abbreviations: FBL, chlorophyll fluorescence indufluorescence yield; FUV, chlorophyll fluorescence indratio of variable to maximum fluorescence; PAR, photo(400e700 nm); PFD, photon flux density (400e70transmittance; UV, ultraviolet radiation (280e400 nmtion (315e400 nm); UV-B, ultraviolet-B radiation (28* Corresponding author.

E-mail address: pwbarnes@loyno.edu (P.W. Barne

http://dx.doi.org/10.1016/j.plaphy.2014.11.0150981-9428/© 2014 Elsevier Masson SAS. All rights re

a b s t r a c t

The accumulation of UV-absorbing compounds (flavonoids and other phenylpropanoid derivatives) andresultant decrease in the UV transmittance of the epidermis in leaves (TUV), is a primary protectivemechanism against the potentially deleterious effects of UV radiation and is a critical component of theoverall acclimation response of plants to changing UV environments. Traditional measurements of TUVwere laborious, time-consuming and destructive or invasive, thus limiting their ability to efficiently makemultiple measurements of the optical properties of plants in the field. The development of rapid,nondestructive optical methods of determining TUV has permitted the examination of UV optical prop-erties of leaves with increased replication, on a finer time scale, and enabled repeated sampling of thesame leaf over time. This technology has therefore allowed for studies examining acclimation responsesto UV in plants in ways not previously possible. Here we provide a brief review of these earlier studiesexamining leaf UV optical properties and some of their important contributions, describe the principlesby which the newer non-invasive measurements of epidermal UV transmittance are made, and highlightseveral case studies that reveal how this technique is providing new insights into this UV acclimationresponse in plants, which is far more plastic and dynamic than previously thought.

© 2014 Elsevier Masson SAS. All rights reserved.

1. Introduction

Ultraviolet radiation (UV; 280e400 nm) represents a relativelysmall, but important part of solar spectrum for higher plants.Exposure to UV, especially the shorter wavelengths in the UV-Bregion (280e315 nm), has the potential to result in a number ofdeleterious effects in plants, including disruption of the integrityand function of biological macromolecules (DNA, proteins andlipids), oxidative damage, the partial inhibition of photosynthesis,and ultimately reduction in growth and productivity (Ballar�e et al.,

ced by blue light; Fo, initialuced by UV radiation; Fv/Fm,synthetically active radiation0 nm); TUV, epidermal UV); UV-A, ultraviolet-A radia-

0e315 nm).

s).

served.

2011; Jordan, 2002). Because of these effects, plants have developedmechanisms to detect UV and then protect and repair sensitivetargets from direct and indirect UV-induced damage (Jenkins,2009; Rizzini et al., 2011; Jansen and Bornman, 2012). One of themost commonly reported protective responses of plants to UV isthe induction of flavonoids and other phenylpropanoids (Searleset al., 2001; Li et al., 2010). These compounds appear to havemultiple functions in photoprotection in plants (Agati et al., 2013).Some flavonoids accumulate within mesophyll tissue and arethought to function as antioxidants, thereby reducing oxidativedamage that can be induced by UV-B and high PAR (Olsson et al.,1998; Tattini et al., 2005). Other compounds, including certainflavonols and hydroxycinnamic acids (HCAs), are known to functionas UV “sunscreens” and accumulate in epidermal tissue where theyeffectively reduce the penetration of UV to the underlying photo-synthetic mesophyll (Mazza et al., 2000; Bidel et al., 2007; Li et al.,1993). Perhaps because of the efficiency of these and other pro-tective measures, negative effects of ambient or realistically-

Fig. 1. Principle of measurement of epidermal UV transmittance using chlorophyllfluorescence. The technique involves the near simultaneous measurement of chloro-phyll fluorescence induced by both UV (FUV) and visible (e.g., bluegreen (BG); FBG)radiation. For typical green leaves, FUV is influenced by the amount of UV-absorbingcompounds in the epidermis whereas FBG is not. Fluorescence induced by BG (FBG)thus serves as a reference to account for variation in chlorophyll content and chloro-plast distribution in the underlying mesophyll. Provided certain precautions andstandards are utilized, the ratio of FUV to FBG provides a robust estimate of TUV orconcentrations of epidermal UV-absorbing compounds.

P.W. Barnes et al. / Plant Physiology and Biochemistry 93 (2015) 94e100 95

enhanced solar UV-B on plant growth, photosynthesis and pro-ductivity are generally small or difficult to detect in field-grownplants (Searles et al., 2001; Newsham and Robinson, 2009).

The ability of plants to accumulate these protective UV-absorbing compounds and change their optical properties (pri-marily epidermal transmittance) in response to days or weeks ofexposure to UV-B radiation has long been known (Caldwell et al.,1983). Indeed, a meta-analysis of 62 studies has shown that theincreased production of UV-B-absorbing compounds is one of themost commonly observed responses of field-grown plants to UV-B-supplementation simulating ozone depletion (Searles et al., 2001).This acclimation response entails a measurable energetic/carboncost (Snell et al., 2009; Guidi et al., 2011), varies with species andgenotype [e.g., (Day et al., 1992; Randriamanana et al., 2015)], isinfluenced by other environmental factors (e.g., UV-A(315e400 nm), visible light (400e700 nm) and temperature(Guidi et al., 2011; Bilger et al., 2007; Flint et al., 2004; Gotz et al.,2010; Siipola et al., 2015)) and is linked with cross-tolerance toother abiotic and biotic stresses (e.g., drought and herbivory (Mazzaet al., 2013; Bandurska et al., 2013)).

Until recently, the determination of epidermal UV transmittance(TUV), the optical property of primary interest, has involvedmeasuring the transmittance of epidermal peels using an inte-grating sphere [e.g., (Robberecht and Caldwell, 1978)] or measuringUV penetration into leaves with microprobes [e.g., (Vogelmann,1989)]. However, these techniques are laborious, time-consumingand destructive or at least invasive. The development of rapid,nondestructive optical methods of determining TUV (UVA-PAM,(Kolb et al., 2005); Dualex/Multiplex, (Goulas et al., 2004)) haspermitted the examination of UV optical properties of leaves withincreased replication, on a finer time scale, and enabled repeatedsampling of the same leaf over time. This technology has thusallowed for studies examining acclimation responses to UV inplants in ways not previously possible. Here we provide a briefoverview of the earlier studies examining leaf UV optical propertiesand point to some of their major contributions, describe the prin-ciples by which the newer non-invasive measurements ofepidermal UV transmittance are made, including some of thenecessary precautions, and highlight several case studies thatreveal how this technique is providing new insights into the plasticand dynamic nature of this UV acclimation response in plants.

2. Early approaches and findings

Many of the initial studies on leaf UV optical properties exam-ined the natural variation in TUV that exists among species or eco-types and the anatomical factors that influence this filteringmechanism. The comprehensive companion studies by Caldwell,Robberecht and Billings (Caldwell et al., 1980) and Robberecht,Caldwell and Billings (Robberecht et al., 1980) showed that the UVtransmittance of epidermal peels from plants originating from lowUV-B environments (i.e., low elevation sites in the Arctic) wasgenerally higher and more variable than those of plants from highUV-B environments (i.e., high elevation equatorial alpine habitats).Results from these, and follow-up comparative UV-sensitivitystudies of Arctic-alpine plants (Caldwell et al., 1982; Barnes et al.,1987), provided strong circumstantial evidence that solar UV wasindeed an important ecological and selective factor along this lat-itudinal gradient. Species surveys were also conducted by Day,Vogelmann and DeLucia (Day et al., 1992) and others [e.g., (Qi et al.,2010)] using microprobes, and these studies documented consid-erable variation in the UV screening capabilities amongmajor plantgrowth forms (grasses, broad-leaved herbaceous species andneedle-leaved conifers). These differences were related to variationin the levels of UV-absorbing compounds as well as leaf anatomical

structures (Day, 1993). Other studies, again with microprobes,showed that the UV penetration into leaves could be influenced notonly by the epidermis but also by surface features, such as waxesand trichomes (Karabourniotis et al., 1999; Karabourniotis andBornman, 1999).

The use of microprobes further allowed for the three-dimensional examination of internal UV-filtering in leaves(Alenius et al., 1995) and these studies revealed that the epidermiswas non-uniform in its UV filtering effect (Day et al., 1993). Thisheterogeneity in UV penetration into leaves resulted from differ-ences in UV transmittance among cellular components (e.g., vacu-oles vs. cell walls), the spatial patterns of epidermal tissue (e.g., thedistribution and arrangement of stomatal guard cells vs. otherepidermal cells), and the optical focusing and reflective propertiesof different cells and tissues (Karabourniotis et al., 2000;Vogelmann et al., 1996).

Both integrating sphere [e.g., (Robberecht and Caldwell, 1983)]and microprobe techniques [e.g., (Sullivan et al., 1996; Olsson et al.,1999; Cen and Bornman, 1993)] were used to demonstrate that TUVdecreased (and UV-absorbing compounds increased) in response toUV-B exposure when plants were grown under elevated UV-B,indicating that many plants possessed the potential to acclimateto a range of UV exposures including enhanced UV-B levels asso-ciated with ozone depletion.

3. Chlorophyll fluorescence as a non-invasive probe of leafoptical properties

3.1. Principles and tests of the technique's validity

In 1997 Bilger, Veit, Schreiber and Schreiber (Bilger et al., 1997)introduced a non-invasive technique to measure TUV based onchlorophyll fluorescence. This approach provided indirect esti-mates of TUV bymeasuring the fluorescence yield of chlorophyll (Fo,l > 650 nm) induced by UV and blue-green (BG) radiation (Fig. 1).The technique is based on the premise that both UV and BG

Fig. 2. Variation in adaxial epidermal UV transmittance in leaves occupying differentlight environments within a single canopy of quaking aspen (Populus tremuloides)growing under field conditions near Logan, Utah, USA. Adapted from (Barnes et al.,2013a).

P.W. Barnes et al. / Plant Physiology and Biochemistry 93 (2015) 94e10096

radiation can induce chlorophyll fluorescence and that reduction inthe penetration of UV to the mesophyll (i.e., from UV-absorbingcompounds in the epidermis) would reduce UV-induced fluores-cence (FUV). Fluorescence induced by BG (FBG), which is notabsorbed by UV-absorbing compounds in the epidermis, serves as areference to account for variation in chlorophyll content andchanges in chloroplast orientation/distribution. Ideally, true valuesof epidermal UV transmittance are obtained by electronicallyadjusting the output from the BG source such that FUV/FBG is unityfor an epidermis-free leaf of the species being measured. As it is notpossible to readily remove the epidermis for most species, FUV/FBGvalues are normally expressed relative to artificial standards thatmimic the optical properties of epidermis-free leaves.

Initially, measurements of FUV/FBG weremade using a laboratorysystem that was equipped with a Xenon lamp (i.e., XE-PAM; (Bilgeret al., 1997; Schreiber et al., 1993)). While this system allowed forthe use of different excitation wavebands in the UV (UV-B or UV-A)and visible by using different optical filters, it was not field portableand measurements could only be made on detached leaf samplesrather than intact leaves. Nonetheless, TUV measured with thissystem compared well with measurements made on epidermalpeels with an integrating sphere (Barnes et al., 2000) or spectro-photometer (Markstadter et al., 2001; Bilger et al., 2001). In addi-tion, variation in TUV within a species as determined by chlorophyllfluorescence was found to be highly correlated (r2 ¼ 0.76e0.90)with variation in whole-leaf UV absorbing compounds (Barneset al., 2000; Ryel et al., 2010) and epidermal UV-absorbing com-pounds (Bilger et al., 2001).

Initial field tests of this technique showed that it could indeeddetect treatment differences in TUV in cultivated plants (soybean;Glycine max) grown under near-ambient and sub-ambient solarUV-B in a temperate location (Barnes et al., 2000). Tests of thistechnique in native plants growing in a high-latitude cold envi-ronment (Tierra del Fuego) however showed that artifacts couldarise when exposure to UV induced the production of anthocyaninsthat absorb blue-green radiation, which then alters the referenceFBG signal and thus the FUV:FBG ratio (Barnes et al., 2000). Theseproblems can be detected by extracting and quantifying both an-thocyanins and UV-absorbing compounds following fluorescencemeasurements and/or by analyzing the individual fluorescencesignals (i.e., FUV and FBG) as well as the FUV:FBG ratio. Instrumentsthat utilize longer wavelength reference excitation sources (i.e., theDualex) are less susceptible to this potential interferencewith blue-green absorbing pigments (see below). Indeed, the Dualex instru-ment can be used to quantify anthocyanins as well as UV-screeningcompounds. While there is the potential that surface features (e.g.,waxes, trichomes) could influence these measurements, we knowof no studies that have explored this possibility. Hence, our dis-cussion here is restricted to typical “green” leaves.

The development of fully field-portable fluorescence units (i.e.,the UVA-PAM (Kolb et al., 2005) and the Dualex (Goulas et al.,2004)) enabled, for the first time, non-destructive measurementsof UV sunscreen protection in leaves of plants growing in the field.In general, the principles of measurement between these twosystems are the same, but there are differences in the approachesused to excite and detect chlorophyll fluorescence (Pfündel et al.,2007) and the wavelengths used in the reference beam (theUVA-PAM utilizes blue light (lmax ¼ 470 nm) whereas the Dualexuses red light (lmax ¼ 655 nm)). For technical reasons, both ofthese field-portable systems use UV-A (lmax ¼ 375 nm) ratherthan UV-B as the UV excitation waveband, and there is no optionto select different excitation wavelengths within the UV-A. Hence,any epidermal compound that absorbs radiation at this wave-length will influence these measurements. Within a species,epidermal UV transmittance measured in the UV-A is usually

highly correlated with that in the UV-B (Barnes et al., 2000) someasurements with these systems do provide information on UV-B as well as UV-A screening. Parallel measurements of UV-B andUV-A excitation (with systems such as the XE-PAM) can howeverprovide information on the specific type of compounds involved inthe UV-filtering process (i.e., flavonols vs. HCAs) because of dif-ferences in the absorbance properties of these compounds (Bilgeret al., 2001). In general, comparisons between the UVA-PAM andDualex have shown good agreement in data obtained from greenleaves, but the UVA-PAM consistently reported higher epidermalUV screening than the Dualex in red leaves that possessed highanthocyanin contents, for the reasons described above (Pfündelet al., 2007).

3.2. Spatial patterns of UV protection

Findings from these new technologies have confirmed resultsfrom previous studies using traditional techniques but have alsoprovided new insights into the patterns and temporal dynamics ofUV acclimation in plants. For example, at broad spatial scales, re-sults from studies using the UVA-PAM have shown that TUV canvary significantly within a species along natural UV-B gradients(i.e., elevation (Ruhland et al., 2013) and latitude (Nybakken et al.,2004)), but considerable variation often exists between species inthis response. For example, along a UV-elevation gradient spanningca. 4000 m in Hawaii, TUV showed a strong linear decrease withincreasing elevation in the exotic herbaceous species mullein(Verbascum thapsus) but not in the native shrub ohelo (Vacciniumreticulatum), which showed low transmittances at all elevations (R.Ryel, S. Flint, and P. Barnes, unpublished data). At much smallerspatial scales, it has been shown that UV screening varies withinindividual leaves of the grass Hordeum vulgare, depending ondevelopmental stage of leaf sections (Wagner et al., 2003), and thatconsiderable variation in TUV (4e98%) can exist within a singlecanopy of a quaking aspen (Populus tremuloides) tree (Fig. 2;(Barnes et al., 2013a)). For aspen, the strong non-linear relationshipbetween TUV and light conditions within the canopy (Fig. 2) sug-gests however that most of the leaves in the canopy, with theexception of those in deep shade, would be reasonably well pro-tected fromUV insult (i.e., TUV < 15%). Howapplicable this finding isfor other trees or other growth forms with different canopy archi-tectures (e.g., grasses and broad-leaved herbaceous plants) isunknown.

P.W. Barnes et al. / Plant Physiology and Biochemistry 93 (2015) 94e100 97

3.3. Temporal dynamics of UV protection

Perhaps the most significant insights from this technique,however, relate to the temporal aspect of this UV acclimationresponse and the degree to which plants can modulate their UVprotection in response to rapid changes in their UV environment. Inparticular, this new technique has allowed investigators to addressa number of questions that were not possible using traditionaltechniques. These include, 1) How long does this protectiveresponse take to develop in young leaves and what are the impli-cations of time-lags in UV protection for plants exposed to UVduring this developmental phase? 2) Once leaves have maturedand acclimated to specific UV/light conditions can they adjust theirUV shielding (either up or down) in response to sudden changes inUV? 3) Do leaves have the ability to change their optical propertiesin response to rapid changes in solar UV that occurs over a day or asa result of changing cloud cover? And 4) What are the implicationsof rapid changes in UV-absorbing compounds for the timing ofplant responses to other abiotic or biotic stresses that utilize someof these same secondary compounds for acclimation and protection(e.g., are plants more susceptible to herbivore or pathogen attack atdifferent times of day as a result of diurnal changes in flavonoidsand other phenolic compounds)? Addressing these and similarquestions is not only of fundamental interest but has a number ofimportant ecological and agricultural implications for under-standing how plants cope with a changing UV climate resultingfrom stratospheric ozone depletion and climate change (Ballar�eet al., 2011; Bornman et al., 2015).

Fig. 3. Temporal response of greenhouse-grown lettuce (Lactuca sativa) transferred toa high PAR growth chamber environment with or without UV-B. Panel A is the timecourse of adaxial epidermal UV transmittances as determined by a UVA-PAM. Panel Bshows degree of photoinhibition (Fv/Fm; as determined by a portable chlorophyllfluorometer) in the same leaves as measured in Panel A. Data are means ± SE. Adaptedfrom (Wargent et al., 2015).

From an agronomic perspective, many crops are initially prop-agated in greenhouses or shade structures where UV is either ab-sent or very low and following a certain period of growth they arethen transplanted to the field where UV irradiances are muchhigher. This practice can result in a significant transplant “shock”and the potential for UV-induced inhibition of growth and photo-synthesis until plants can sufficiently acclimate to the new solarradiation regime (Bogenrieder and Klein, 1977). For example,studies with leaf lettuce (Lactuca sativa) have recently shown thatleaves developed under typical greenhouse conditions virtuallylack any epidermal UV shielding (TUV > 98%) and that at least 6e8days is required to fully acclimate to UV-B conditions in a growthchamber simulating field conditions (Fig. 3A). Immediatelyfollowing transfer, there was substantial photoinhibition (asmeasured by changes in Fv/Fm; Fig. 3B). However, exposure to UV-Bduring this acclimation phase was found to enhance leaf photo-synthetic carbon uptake and increase a number of key secondarycompounds likely involved in UV photoprotection (i.e., flavonoidsand other derivatives of the shikimate pathway), relative to plantsreceiving no UV-B (Wargent et al., 2015). Thus, exposure to UV-Benhanced the acclimation process in this species and appeared to“harden” plants for growth under field conditions. In this context,UV exposure can thereby provide a significant beneficial effectduring seedling development that could ultimately have importantconsequences for crop vigor and agricultural production (Wargentand Jordan, 2013).

As previously noted, the ability of leaves to adjust their UVshielding in response to UV exposure during leaf development iswell established and studies, such as the one above and others [e.g.,(Morales et al., 2011; Louis et al., 2009)], are providing insights intothe precise time courses of these responses as they relate to UVprotection under field conditions. What is less clear, however, ishow pliable these UV screening properties are in leaves once theyhave matured and developed under one set of conditions and arethen confronted with abrupt changes in these conditions. Abruptchanges in solar UV would routinely occur in understory plantsexposed to periodic sunflecks (Krause et al., 2003a, 2003b) and sunand shade leaves within plant canopies as a result of canopy per-turbations due to herbivory or other factors (Barnes et al., 2013a).Some studies have shown that mature leaves or leaf segments aregenerally unresponsive in their UV-sunscreen capacities whenconfronted with changes in the environment (Bilger et al., 2007;Stroch et al., 2008). Field studies with mature sun and shadeleaves of P. tremuloides and Vicia faba have shown, however, thatthere is considerable flexibility in UV acclimation in mature leaves,at least for those developed under low light conditions (i.e., shadeleaves). Specifically, shade leaveswere shown to decrease TUVwhentransferred to sun environments, but sun leaves did not decreasetheir UV sunscreen protection when exposed to shade conditions(Barnes et al., 2013a). These findings are consistent with earlierfindings which showed that tropical understory plants can respondto abrupt increases in solar UV radiation by increasing UV-absorbing compounds (Krause et al., 2004) and indicate that sub-stantial flexibility in UV protection exists even after leaves havefully developed. This adjustment in UV transmittance of shadeleaves requires both UV-B and UV-A, whereas PAR and UV wereinvolved in the establishment of UV sunscreen protection duringleaf development (Barnes et al., 2013a).

Recent findings using the UVA-PAM have also shown that TUVcan vary on a diurnal basis, with UV protection increasing fromdawn to midday and then decreasing towards sunset (Barnes et al.,2008). The earliest report of daily fluctuations in TUV was byLautenschlager-Fleury (Lautenschlager-Fleury, 1955), whoobserved that the UV-B transmittance in epidermal peels of Viciafabawas low duringmidday on a sunny day but remained relatively

P.W. Barnes et al. / Plant Physiology and Biochemistry 93 (2015) 94e10098

high on a cloudy day. Subsequently, Veit, Bilger, Muhlbauer,Brummet andWinter (Veit et al., 1996) reported substantial middayincreases in flavonoid levels in an alpine fern (Cryptogramma crispa)and a tropical tree (Anacardium excelsum). Since the initial report byBarnes, Flint, Slusser, Gao and Ryel (Barnes et al., 2008) of diurnalchanges in TUV in three plant species growing in the Hawaiianalpine, additional studies have shown that this is a wide-spreadphenomenon among plants species (wild and cultivated) and isassociated with diurnal changes in the amount of UV-absorbingcompounds per leaf area (Fig. 4; (Barnes et al., 2013b)). While notall species exhibit this phenomenon (e.g., Zea mays), some speciessuch as the cultivated herbaceous plant okra (Albelmoschus escu-lentus), exhibit substantial diurnal changes in TUV (Fig. 4). Thisvariation in TUV throughout the day is correlated with solar UV-Band PAR irradiances (r2 ¼ 0.76 and 0.86, respectively for okra; un-published data) and can be disrupted by shading from clouds orartificial shade structures (Barnes et al., 2008). Hence, thesechanges in TUV during the day are not the result of internal, circa-dium rhythms. Moreover, direct measurements on epidermal peelsof V. faba with an integrating sphere have confirmed that rapidchanges (within 1.5 h) in TUV are indeed possible (P. Barnes, un-published data).

The possibility that higher plants can rapidly adjust their UVsunscreen protection in response to daily changes in UV irradiancesmay not be surprising as diurnal fluctuations in UV protection havebeen documented in systems as diverse as human epidermal stemcells (Janich et al., 2013), marine algae (Taira et al., 2004) and

Fig. 4. Diurnal changes in adaxial epidermal UV transmittance and whole-leaf UV-absorbing compounds in okra (Abelmoschus esculentus) grown in pots outside in NewOrleans, Louisiana, USA. Concentrations of UV-absorbing compounds are expressed interms of Absorbance cm�2 leaf area using extraction methods of Barnes, Kersting, Flint,Beyschlag and Ryel (Barnes et al., 2013a). Data were collected over consecutive days inJune and are means ± SE (n ¼ 15).

liverworts (Fab�on et al., 2012). How plants achieve these rapidchanges and what significance this is for plant function beyondprotection from UV radiation (Fig. 5) is not yet known. Increasedallocation of carbon to UV-absorbing compounds may have nega-tive consequences for photosynthesis (Sumbele et al., 2012) suchthat reducing UV protection during times of the day when solar UVlevels are low could enhance daily carbon gain. In comparison toplants that maintain high UV sunscreen protection throughout theday, plants with horizontal leaves that exhibit diurnal changes inepidermal UV transmittancewill experience increased UV exposureto the underlying mesophyll both early and late in the day but notat midday (Fig. 5). It is possible that increased UV penetration atthese times may enhance the beneficial effect of UV exposure onphotoinhibition under bright light, which often occurs in themiddle of the day (Wargent et al., 2011). There is evidence that UV-A can drive photosynthesis (Turnbull et al., 2013) and increasedpenetration of UV-A may therefore increase photosynthesis attimes of the day when leaves are light-limited. There is also the

Fig. 5. Calculated plant effective UV-B irradiances received directly beneath theepidermis (i.e., at the top of the underlying mesophyll) in okra for a representativenear-clear summer sky in New Orleans, Louisiana, USA. Panel A shows the diurnalcourse of ambient solar UV-B irradiance and calculated UV-B irradiance just beneaththe adaxial (upper) epidermis for three scenarios: actual diurnal change in adaxialepidermal UV transmittance (triangles), assuming constant dawn epidermal UVtransmittance (squares) and assuming constant midday midday epidermal UV trans-mittance (circle). Panel B shows the integrated daily plant effective UV-B doses for thethree scenarios in Panel A relative to incident solar UV-B. Calculations assumed ahorizontal leaf and no qualitative change in the UV-absorbance spectrum (Fig. 4B).

P.W. Barnes et al. / Plant Physiology and Biochemistry 93 (2015) 94e100 99

possibility that maintaining high levels of flavonoids could interferewith plant growth during nighttime periods. Several of the com-pounds induced by UV (e.g., quercetin, kaempferol) have beenshown to inhibit polar auxin transport (Falcone-Ferreyra et al.,2012). Regardless of the mechanisms and significance of this phe-nomenon, it is now clear that the UV sunscreen acclimationresponse in plants is capable of changing on a much more rapidtime scale than previously observed.

4. Conclusions

The epidermis of leaves is a selective filter of solar radia-tioneabsorbing much of the potentially detrimental solar UV whiletransmitting visible wavelengths (PAR) needed for photosynthesisin the underlying mesophyll. The epidermis is also a variable UVfilter and the accumulation of UV-absorbing compounds (flavo-noids and related phenolics) and resultant decrease in epidermalUV transmittance represents a primarymechanism bywhich plantsacclimate to changing UV environments. Understanding the natureand limitations of this acclimation response is fundamental toevaluating the ecological and agronomic significance of naturalvariation in solar UV that occurs over multiple spatial (e.g., latitudevs. canopy) and temporal scales (e.g., seasonal vs. diurnal changes)and also the significance of alterations in the solar UV climateresulting from ozone depletion and climate change.

The availability of non-invasive optical technologies based onchlorophyll fluorescence is providing new opportunities to exploremany facets of this acclimation response, including its fine scalespatial and temporal dimensions. Recent results confirm certainprevious findings based on traditional techniques but also reveal aUV protective mechanism in plants that is muchmore dynamic andplastic than previously thought. While the mechanistic un-derpinnings of these responses are clear in certain cases (e.g., theadjustments of mature shade leaves to abrupt increases in UVexposure) the basis of other responses (e.g., diurnal changes in UVshielding) are less understood. Additional studies are needed toevaluate the adaptive significance of rapid modulation in UVshielding in plants and the potential implications of a dynamic UVprotection system for other abiotic (drought, nutrient limitationand temperature extremes; [e.g., (Bilger et al., 2007; Petridis et al.,2012; Meyer et al., 2006)]) and biotic factors (herbivore and path-ogen protection; (Ballar�e, 2014; Latouche et al., 2013)) that arecross-linked to this UV acclimation response.

Acknowledgments

The research described here was supported, in part, by fundsfrom the Louisiana Board of Regents (SURE Grant No. LEQSF-EPS(2013)-SURE-85 to Loyola University), a Massey UniversityResearch Fund grant, the US Department of Agriculture UV-Monitoring Program (USDA-CSREES No. 2004-34263-14270 toUtah State University via subcontract with Colorado State Univer-sity), the Utah Agricultural Experiment Station, the Loyola Univer-sity J.H. Mullahy Endowment in Environmental Biology, and aLoyola University Faculty Research Grant. We thank M. Grabner, D.Hackenburg, I. Bottge, and R. Burnett for their assistance.

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