Acclimation to UV-B radiation and visible light in Lactuca sativa involves up-regulation of photosynthetic performance and orchestration of metabolome-wide responses

This article is protected by copyright. All rights reserved.

[UV-SI] 1

2

Title: Acclimation to UV-B radiation and visible light in Lactuca sativa 3

involves upregulation of photosynthetic performance and orchestration 4

of metabolome-wide responses1 5

6

Authors: Wargent J.J.1*, Nelson B.C.W.1, McGhie T.K.2, Barnes P.W.3 7

8

1Institute of Agriculture & Environment, Massey University, Palmerston North, New 9

Zealand 10

2The New Zealand Institute for Plant & Food Research, Palmerston North, New Zealand 11

3Department of Biological Sciences, Loyola University, New Orleans, USA 12

13

*Corresponding author: [email protected] 14

15

16

17

18

19

20

21

22

23

24

This article has been accepted for publication and undergone full peer review but has not been through the

copyediting, typesetting, pagination and proofreading process, which may lead to differences between this

version and the Version of Record. Please cite this article as doi: 10.1111/pce.12392

Acc

epte

d A

rticl

e


25

26

ABSTRACT 27

UV-B radiation is often viewed as a source of stress for higher plants. In particular, 28

photosynthetic function has been described as a common target for UV-B impairment, yet 29

as our understanding of UV-B photomorphogenesis increases, there are opportunities to 30

expand the emerging paradigm of regulatory UV response. Lactuca sativa is an important 31

dietary crop species, and is often subjected to rapid sunlight exposure at field transfer. 32

Acclimation to UV-B and visible light conditions in L. sativa was dissected using gas 33

exchange and chlorophyll fluorescence measurements, in addition to non-destructive 34

assessments of UV epidermal shielding (SUV). Following UV-B treatment, seedlings were 35

subjected to wide-range metabolomic analysis using LC-QTOF-HRMS. During the 36

acclimation period, net photosynthetic rate increased in UV-treated plants, epidermal UV 37

shielding increased in both sub-sets of plants transferred to the acclimatory conditions 38

(UV+/UV- plants) and Fv/Fm declined slightly in UV+/UV- plants. Metabolomic analysis 39

revealed that a key group of secondary compounds was upregulated by higher light 40

conditions, yet several of these compounds were elevated further by UV-B. In conclusion, 41

acclimation to UV-B involves co-protection from the effects of visible light, and responses 42

to UV-B radiation at a photosynthetic level may not be consistently viewed as damaging to 43

plant development. 44

45

Keyword index: UV-B radiation, Lactuca sativa, acclimation, photosynthesis, 46

photoprotection 47

48

49

50

Acc

epte

d A

rticl

e


51

52

INTRODUCTION 53

Ultraviolet (UV) radiation is an energetic driver of numerous components of plant function. 54

Despite routine reporting of injurious effects of UV-B radiation (290-320 nm) upon cellular 55

and molecular function (Frohnmeyer, 1999, Jordan, 1996), plants in nature rarely display 56

signs of significant damage routinely inflicted by UV-B. This is thought to be due to aspects 57

of the diverse and formidable suite of responses to UV plants have evolved (Jenkins, 58

2007), including wide-ranging up-regulation of phenolic metabolism (Rozema et al., 2002, 59

Stracke et al., 2010), thought to act as an effective sun-screen, and inhibition of leaf 60

elongation, plus other alterations in plant morphology (Hectors et al., 2010, Wargent et al., 61

2009b). Taken together with the recent confirmation of the UVR8-COP1-HY5 UV-B 62

photoreception pathway [UVR8: UV RESISTANCE LOCUS 8; COP1: CONSTITUTIVELY 63

PHOTOMORPHOGENIC 1; HY5: ELONGATED HYPOCOTYL 5] (Brown et al., 2005, 64

Christie et al., 2012, Favory et al., 2009, Rizzini et al., 2011), UV-B photobiology is 65

currently presented with an opportunity to extend our understanding of plant responses to 66

UV radiation into a new phase, where routine regulation of plant development by UV-B 67

photomorphogenesis can be considered in equal fashion to that of the day-to-day 68

adjustments mediated by red and blue light cues (Briggs & Christie, 2002, Franklin & 69

Whitelam, 2004). Equally, understanding the breadth of regulatory UV response may 70

underpin advances towards a sustained food security (Wargent & Jordan, 2013). 71

Contrary to past dogma that photosystem competence and ensuing photosynthetic 72

performance can be highly vulnerable to UV-B (Allen et al., 1997, Mackerness et al., 1998, 73

Takahashi et al., 2010), there is now growing evidence that under realistic exposure 74

fluxes, UV radiation can protect against limitations to photosynthetic performance. For 75

example, several studies have implicated the role of UV in the induction of enhanced 76 Acc

epte

d A

rticl

e


photoprotection against high-light or long-wave UV radiation (Bolink et al., 2001, Hakala-77

Yatkin et al., 2010, Poulson et al., 2002), and it has been suggested that UV-B exposure 78

may enhance recovery from a photoprotected state (Xu & Gao, 2010). Recently, we 79

showed that in the crop species Lactuca sativa, exposing seedlings to a moderately high 80

dose of UV-B increased net photosynthetic rate. In addition, maximum quantum efficiency 81

of PSII was elevated in UV-B treated plants following exposure to high-light and 82

temperature conditions, as compared to seedlings maintained under visible light (Wargent 83

et al., 2011). From a mechanistic perspective, it is known that the UV-B photoreceptor 84

UVR8 is required for the regulation of photosynthetic efficiency in Arabidopsis in response 85

to UV-B (Davey et al., 2012), yet the basic regulatory network which may underpin UV-86

mediated photosynthetic competency remains poorly described. One central component of 87

UV response which has been linked to photoprotection is that of stimulated secondary 88

metabolic activity. UV induces a broad range of plant metabolites (Bassman, 2004), yet 89

compounds have often been investigated individually in studies, with knowledge gaps still 90

evident (Jansen et al., 2008). For example, despite technological advances, there are still 91

very few larger-scale metabolomic studies of UV response to date. At the same time, 92

various roles of UV-induced metabolites have been suggested, largely focused on the 93

shikimate-derived phenylpropanoids, including UV-absorption (Bornman & Vogelmann, 94

1991, Day et al., 1993), herbivore feeding deterrence (Izaguirre et al., 2007), reactive 95

oxygen scavenging (Hideg et al., 2012), and induced resistance to fungal pathogen attack 96

(Demkura & Ballare, 2012). In terms of applying our understanding of UV-mediated effects 97

on primary and secondary metabolism to extend our knowledge of plant development, one 98

potential criticism of many UV-B photobiological studies is that exposure of plants to UV-B 99

often follows a ‘sudden shock’ approach, whereby no ‘acclimation’ phase of steadily 100

increased UV-B exposure is included. However, understanding inducible UV acclimation 101

provides valuable information within the context of agriculture. Many crops are initially 102 Acc

epte

d A

rticl

e


propagated in the complete absence of UV-B wavelengths, and are then transferred into 103

the field after a period of initial seedling growth, therefore leading to the constant sudden 104

exposure of many crop plants to UV. This aspect of UV photomorphogenesis is now 105

receiving some attention; for example, the UV-B photoreceptor UVR8 has a clear role in 106

orchestrating phenolic metabolism when plants are exposed to ambient sunlight (Morales 107

et al., 2013), and controlled exposure of Arabidopsis seedlings to narrowband UV-B 108

revealed rapid changes in expression profile of several hundred UVR8-dependent gene 109

transcripts, many of which are related to phenylpropanoid metabolism (Favory et al., 110

2009). Regardless, our understanding of the complex process of UV-B acclimation from a 111

temporal and whole-plant perspective is still limited. 112

Here, we extend our understanding of the rapid nature of UV-B plant response in a 113

dissection of physiological and metabolomic responses in Lactuca sativa, a species of 114

significant dietary nutritional importance. We show for the first time, the progression of 115

rapid UV-B and visible light acclimation at a leaf gas exchange and epidermal sun-116

screening level, as underpinned by metabolomic change. 117

118

MATERIALS & METHODS 119

Plant growth & UV treatments 120

Seeds of Lactuca sativa ‘Expedition’ (Rijk Zwaan) were germinated in vermiculite, and 121

then transferred after 9 d into a standard potting soil. Seedlings were grown to an age of 122

21 d under standard glasshouse conditions, where leaf size then allowed for gas exchange 123

measurements. Our standard glasshouse conditions consisted of a 160 m2 glasshouse 124

situated at the Massey University Plant Growth Unit, Palmerston North, New Zealand (-125

40.42, 175.58) where air temperature was maintained at 20°C±4, and relative humidity of 126

70%±10. No UV-B or substantial short-wave UV-A radiation penetrated the glass cladding 127

of the house (Fig. 1), and at the time of year the experiments were carried out (January-128 Acc

epte

d A

rticl

e


March), mean photoperiod for the glasshouse conditions was 14:10 hr day/night. 129

Photosynthetically active radiation (PAR: 400-700 nm) was quantified in both glasshouse 130

and controlled environment chamber environments using a datalogger equipped with four 131

LI-190SZ quantum PAR sensors (Li-Cor Inc, Lincoln, NE, USA). In total, three days 132

sampling of the PAR environment was undertaken in both experimental environments, with 133

additional checks as required. In glasshouse conditions, mean maximum PAR per day was 134

1999 µmol m-2 s-1 ± 56 (S.D.), and mean integrated PAR (total PAR day-1, to allow 135

comparison with growth chamber values) was 53 mol m-2 day-1 ± 11.2 (S.D.). For UV-B 136

exposure, plants were placed into a modified controlled environment chamber (Contherm 137

630, Contherm Scientific Ltd, Petone, New Zealand). Environmental conditions in the 138

chamber were maintained at: 14:10 hr day/night length; 21°C/18°C day/night temperature; 139

75% relative humidity. PAR light sources in the growth chamber consisted of a bank of 400 140

W metal halide lamps (Phillips HPI T Plus, Eindhoven, Netherlands). The PAR light system 141

was at a set-point of 750 µmol m-2 s-1, providing a mean integrated PAR at plant canopy 142

height of 54 mol m-2 day-1 ± 1.8 (S.D.), which was markedly close to the integrated PAR 143

level in the glasshouse conditions. The growth chamber was equipped with UV-B 144

fluorescent tubes (Q-Panel 313, Q-Lab Corp, Cleveland, USA), wrapped in filtered with 145

0.13-mm-thick cellulose diacetate (Clarifoil,Courtaulds Ltd, Derby, UK) to remove 146

wavelengths <290 nm (Fig. 1). The chamber was split into a UV+ zone and UV- zone 147

separated by a central curtain of UV-B opaque film (Fig. 1) (Lumivar, BPI Visqueen, 148

Ardeer, UK), with air allowed to circulate freely between the two zones. For the no UV 149

zone, those lengths of UV-B tubes were wrapped in the same UV-B opaque film, with UV+ 150

and UV- zones alternated regularly between experiments. UV-B treatments were 151

quantified at plant canopy height with an Optronics 756 spectroradiometer equipped with 152

integrating sphere, providing a biologically effective UV dose of 10 kJ m-2 day-1 as 153

weighted with the Caldwell (1971) generalized plant action spectrum, normalized to unity 154 Acc

epte

d A

rticl

e


at 300 nm, which would be typically observed under New Zealand summer conditions. 155

Spectroradiometric scans of the controlled environment chamber confirmed that the 156

biologically effective UV dose was <0.01 kJ m-2 day-1 in the UV- zone. In order to compare 157

changes in the plants between growth chamber conditions and the propagation 158

environment, a proportion of experimental plants were maintained throughout the 159

experiment under the standard glasshouse conditions. Under GH conditions, the 160

biologically effective UV dose was <0.01 kJ m-2 day-1. For metabolomic studies, the foliage 161

of entire seedlings was snap frozen in liquid N2, and stored at -80°C for subsequent 162

analysis, with sampling undertaken (1) immediately prior to the point of transfer into the 163

controlled environment chamber, 21 d after seed sowing [‘GH D0’], (2) 6 d after transfer of 164

seedlings into the chamber according to treatment [‘UV- D6’, ‘UV+ D6’], in addition to 165

those seedlings which had been maintained under standard glasshouse conditions [‘GH 166

D6’]. 167

168

Gas exchange & chlorophyll fluorescence 169

All measurements were non-destructively taken from the second true leaf of each 170

seedling, and were taken on several days between Day 0 (i.e. prior to transfer of seedlings 171

from the glasshouse to the growth chamber) to Day 6 following transfer of seedlings into 172

the growth chamber. Gas exchange measurements were carried out with an LI-6400 infra-173

red gas analyser (Li-Cor Inc, Lincoln, NE, USA); where cuvette conditions were maintained 174

at a saturating 1000 µmol m-2 s-1 PAR, 75% relative humidity, 20°C cuvette block 175

temperature, and 400 ppm of CO2. During measurements, PAR was provided by a LI-COR 176

RGB LED light source housed within the cuvette, and leaves were maintained in the 177

cuvette for at least five minutes to reach steady-state prior to measurement. Parameters 178

measured included light-saturated net exchange rate of CO2 (light-saturated net 179

photosynthetic rate or Pmax) and H2O (transpiration rate or E), stomatal conductance (gs), 180 Acc

epte

d A

rticl

e


and intercellular CO2 mole fraction (internal CO2 or Ci). Chlorophyll fluorescence analysis 181

of maximum quantum efficiency of PSII photochemistry (Fv/Fm) was spot-measured using 182

a Plant Efficiency Analyser (Hansatech, UK), with leaves allowed to dark-adapt for 20 183

minutes before measurement. Non‐invasive measurements of epidermal UV shielding 184

(SUV) were made with a portable pulse amplitude modulation (Xe-PAM) chlorophyll 185

fluorometer (UVA‐PAM; Gademann Instruments, Würzburg, Germany). This instrument 186

provides indirect estimates of epidermal UV-A transmittance/shielding by measuring the 187

fluorescence yield of chlorophyll (Fo; λ > 650 nm) induced by UV-A (UV; 375 nm) and blue 188

(BL; 470 nm) radiation, as outlined by Kolb et al. (2005) and following the precautions and 189

procedures of Barnes et al. (2008) and Barnes et al. (2013). 190

191

Our dataset is composed of two identical repeat experiments, with data analysed using 192

repeated measures Analysis of Variance (ANOVA) (SPSS statistics v20, IBM, US), where 193

effects of experimental treatment, experimental day, and experimental iteration were 194

analysed. All gas exchange, chlorophyll fluorescence, and epidermal UV shielding 195

measurements were taken concurrently and close to midday on each sampling day, with a 196

replicate from each treatment sampled in a rotated blocking pattern throughout. Ten plants 197

were measured on each sampling occasion from each of the three treatments. Individual 198

plants were the unit of replication within each experimental iteration within ANOVA (n per 199

iteration =10, total replicates within each treatment per day from both iterations: n=20). 200

Plants were routinely re-arranged under within each treatment on a regular basis. 201

202

Metabolomic analysis by LC-QTOF-HRMS 203

Metabolomic profiling was carried out using liquid chromatography high resolution mass 204

spectrometry (LC-HRMS). LC-HRMS grade acetonitrile and methanol was from Fischer 205

Scientific; dimethyl sulfoxide (DMSO) was from Sigma-Aldrich. Ultrapure water was 206 Acc

epte

d A

rticl

e


obtained from a Milli-Q Synthesis system (Millipore). Foliar material was homogenised in 207

liquid N2 and weighed out to an equal mass of 150 mg for each sample. Each of the 208

powdered leaf samples were extracted overnight at 1°C with 1.5 mL ethanol/MQ/formic 209

acid (80/20/1 v/v/v). Samples were diluted with methanol before analysis by LC-HRMS. 210

The LC-HRMS system was composed of a Dionex Ultimate® 3000 Rapid Separation LC 211

and a micrOTOF QII mass spectrometer (Bruker Daltonics, Bremen, Germany) fitted with 212

an electrospray ion source. The LC contained a SRD-3400 solvent rack/degasser, HPR-213

3400RS binary pump, WPS-3000RS thermostated autosampler, and a TCC-3000RS 214

thermostated column compartment. Metabolite separation was achieved using two UHPLC 215

columns connected in series. The columns were both ZorbaxTM SB-C18 2.1 x 100 mm, 216

1.8 µm (Agilent, Melbourne, Australia) and were maintained at 60 °C. The flow was 350 217

µL/min. Solvents were A = 100% acetronitrile, and B = 0.4% formic acid. The gradient 218

was: 10%A, 90% B, 0-0.5 min; linear gradient to 35% A, 65%B, 0.5-18 min; linear gradient 219

to 100% A, 18-25 min; composition held at 100% A, 25-28 min; linear gradient to 10% A, 220

90% B, 28-28.2 min; to return to the initial conditions before another sample injection at 31 221

min. The injection volume for samples and standards was 1 μL. The micrOTOF QII source 222

parameters were: temperature 225 ºC; drying N2 flow 6 L/min; nebulizer N2 1.5 bar, 223

endplate offset -500V, mass range 100-2000 Da, acquired were acquired at 2 scans/s. 224

Negative ion electrospray was used with a capillary voltage of +3500 V. Post-acquisition 225

internal mass calibration used sodium formate clusters with the sodium formate delivered 226

by a syringe pump at the start of each chromatographic analysis. 227

228

Metabolomic data analysis 229

The metabolites present in each analysis were extracted from the raw MS data files using 230

the Find Molecular Features algorithm contained in the DataAnalysis (Bruker Daltonics, 231

Bremen, Germany) software package. The molecular features detected for all analyses 232 Acc

epte

d A

rticl

e


were then combined into a single bucket table using ProfileAnalysis (Bruker Daltonics, 233

Bremen, Germany) (Supplementary File Appendix 1). This data table contains molecular 234

features, tagged with an accurate mass/retention time data, as rows with the analyses 235

arranged as columns. Following analysis, discrete molecular features were submitted to 236

Principle Component Analysis (PCA) in ProfileAnalysis for identification of treatment-237

dependent differences. To identify metabolites that differ between treatments, the t-test 238

function in ProfileAnalysis was used, and the results visualised using volcano plots. 239

Metabolites that had a fold change of greater than 1.5 with a P<0.05 were considered for 240

further investigation. To obtain more precise quantitative data for metabolites of interest, 241

the accurate masses and retention times were used with QuantAnalysis (Bruker Daltonics, 242

Bremen, Germany) software to obtain peak areas from exact ion chromatograms (EICs) 243

for each metabolite. Replicate data from each treatment were then analysed by one-way 244

ANOVA, along with Tukey post-hoc testing for homogeneity. For each analysis point 245

according to treatment and day, 6-7 replicate seedlings were collected and analysed as 246

above within a single experimental iteration. 247

248

RESULTS 249

Photosynthetic competency increases during UV-B acclimation 250

In order to dissect the process of UV-B acclimation, we transferred young L. sativa plants 251

from a standard glasshouse propagation environment into growth chamber conditions 252

which provided fixed PAR levels, and the presence or absence of supplementary UV-B 253

radiation. Prior to transfer of seedlings from our glasshouse environment [‘GH’] into the 254

growth chamber, we observed values for maximum quantum efficiency of PSII 255

photochemistry (Fv/Fm) well within the normal range for healthy plants (~0.83; Figure 2A). 256

Also, in order to indirectly yet non-destructively monitor leaf UV-shielding, we employed a 257

Xe UVA-PAM fluorometer, which indicated that % UV shielding values were extremely low 258 Acc

epte

d A

rticl

e


(<5%; Fig. 2C) prior to transfer from our propagation conditions. Within 24 hrs following 259

transfer of seedlings into the PAR light and UV-B+/- conditions of the growth chamber, we 260

observed a decrease in Fv/Fm in UV+ and UV- seedlings compared to GH plants (P<0.001; 261

Fig. 2A), while GH plants retained Fv/Fm values >0.8 for the duration of the experiment. By 262

Day 2 of the experiment (i.e. 48 hrs after transfer), Fv/Fm of our UV+ plants, which were 263

exposed to supplementary UV-B radiation (10 kJ m-2 day-1), was a little further reduced 264

compared to UV- plants (P=0.08; Fig. 2A), but Fv/Fm values recovered quickly in UV+ 265

plants, and by Day 6 of the experiment, both UV+/- plants were exhibiting values of 0.78, 266

which was still reduced compared to GH plants (GH=0.83; P<0.001; Fig. 2A). Over the 267

course of the transfer phase of the experiment, net photosynthesis in UV+ plants was 268

increased compared to UV- plants (Fig. 2B; P<0.001), and photosynthetic rate was also 269

increased in UV- plants compared to GH plants (Fig. 2B; P<0.001). There was some 270

variation between experimental iteration (Repeated measures for experiment factor: 271

P<0.001), yet photosynthetic rate increased in UV+ plants during the duration of the 272

transfer phase, less so in UV- plants; UV+ photosynthetic rates were still significantly 273

higher than those of UV- and GH plants by the end of the experiment on Day 6 (Fig. 2B; 274

P<0.001). In terms of other gas exchange variables, transpiration rate was significantly 275

higher in UV+ plants during the six day transfer phase compared to both UV- and GH 276

plants (Fig. 2D; Repeated measures for treatment factor: P<0.001, Repeated measures for 277

experiment factor: P<0.001), and transpiration rate was also higher in UV+ plants 278

compared to UV- plants on Day 6 of the experiments (Fig. 2D; P<0.001). There were no 279

significant differences in stomatal conductance (Fig. 2E) or internal CO2 values during the 280

six days of the transfer phase, or on any one single day of the experiments (Fig. 2F). In 281

addition to responses related to photosynthetic competency, there were marked increases 282

in UV shielding observed in both UV+ and UV- plants following transfer at Day 0 (Fig. 2C; 283

P<0.001). After just 48 hrs of UV-B exposure, UV+ plants were experiencing higher UV 284 Acc

epte

d A

rticl

e


shielding than UV- plants (Fig. 2C; P<0.001), and by the end of the transfer phase, % 285

shielding values were 75%, 57% and 3% for UV+, UV-, and GH plants respectively (Fig 286

2C; P<0.001). 287

288

Leaf metabolomic responses involve acclimation to visible light & UV-B radiation 289

In order to investigate the acclimation of plants to UV-B at a metabolomic level, foliar 290

material was snap frozen at D0 and D6 of the transfer phase of the study, according to 291

treatment, and was subjected to metabolomic investigation via LC-QTOF-HRMS. Our 292

metabolomic approach quantified >1300 discrete molecular features across our sample 293

pool in L. sativa seedlings, irrespective of treatment (Supplementary File Appendix 1). 294

Those discrete molecular features were then submitted to PCA analysis for identification of 295

treatment-dependent differences prior to further investigation. To initially screen for 296

metabolites that may differ between treatments, the t-test function in ProfileAnalysis was 297

used, and metabolites that had a fold change of greater than 1.5 with a P<0.05 were 298

considered for further investigation. Molecular features are listed according to treatment 299

comparison and p-value results in Supplementary File Appendix 1. Analysis by standard t-300

test revealed numbers of molecular features were subject to significant fold-change 301

between all treatment comparisons. When comparing our GH control seedlings from Day 0 302

[‘D0’] of the experiment to Day 6 [‘D6’], 52 molecular features exceeded our cut-off of a 303

fold change of 1.5, and 34 features were subject to significant change according to 304

treatment via t-test at P<0.05 (Fig. 3A). Of those 34 features, 30 were over-represented in 305

GH D6 seedlings compared to GH D0 samples, and 4 features decreased in abundance 306

during the time the seedlings remained in glasshouse conditions (Fig. 3A). Following 307

transfer of seedlings into our controlled conditions, plants remained under our three 308

regimes for six days. At the end of the transfer period, there were 75 discrete features at a 309

fold change >1.5 in seedlings maintained under GH D6/UV- D6 conditions, and 54 310 Acc

epte

d A

rticl

e


features were significantly changed at P<0.05 (Fig. 3B). Of these, 34 features were 311

increased in UV- seedlings, and 20 features were over-represented in GH D6 plants (Fig. 312

3B). When comparing UV treated plants to those maintained in glasshouse conditions 313

during the six day experiment, there were 110 molecular features at a fold change of >1.5 314

[GH D6 vs UV+ D6], and 80 features significantly changed at P<0.05. Within that group, 28 315

features were relatively increased in UV+ D6 plants, and 52 features were increased in GH 316

D6 plants (Fig. 3C). Yet only 4 of those 52 features upregulated in GH D6 plants exceeded 317

a fold change of >2, as compared to 20 out of the 28 features in UV+ plants where fold 318

changes exceeded >2. When metabolites were compared purely on the basis of UV 319

treatment, there were 16 discrete molecular features at a fold change of >1.5 [UV- D6 vs 320

UV+ D6], and 9 features significantly changed at P<0.05. Of those, eight features were 321

over-represented in UV+ plants, as compared to just one feature in UV- plants (Fig. 3D). 322

Following this preliminary analysis, molecular features which were significantly 323

over-represented according to treatment comparison were filtered again to exclude those 324

features indistinguishable from signal noise. Accurate masses and retention times were 325

then used to obtain peak areas from exact ion chromatograms (EICs) for each metabolite. 326

We then undertook an identification procedure, based on assigning accurate masses to 327

elemental composition and thus elucidating molecular formulae. An emphasis was placed 328

on profiling compounds where an LC-QTOF-HRMS signal was detected for the majority or 329

preferably all replicate samples. Following this filtering and formulae assignment 330

procedure, ten metabolites were remaining in our pool for detailed examination (Table 1). 331

Nomenclature approaches were then carried out for those ten compounds, using the 332

database resource ChemSpider (www.ChemSpider.com), resulting in common names 333

being assigned to six of our ten metabolites (Table 1). There were clear fold increases in 334

the abundance of the majority of the metabolites when comparing Day 0 and GH D6 plants 335

with those transferred into UV- and UV+ conditions (Table 1), with the exception of 336 Acc

epte

d A

rticl

e


C22H18O11 and C41H72O9, where there was far less difference between GH D6 seedlings 337

and UV- /UV+ plants. In order to quantitatively examine the differences between the 338

effects of treatments by the end of the experiment [Day 6], replicate data from each 339

treatment were compared using one-way ANOVA (p-values are presented in Fig. 4). In 340

terms of responses to the UV-B treatment, there were significant increases in luteolin 7-341

glucuronide (Fig. 4A), quercetin 3-(6''-malonyl-glucoside) (Fig. 4B), and quercetin 3-342

glucuronide (Fig. 4J), in addition to the unnamed compounds C21H20O11 (Fig. 4F) and 343

C30H30O21 (Fig. 4I). Intriguingly, the unnamed C21H20O11 exhibited the largest fold change 344

in UV+ plants compared to the UV- treatment, and GH treatments. In comparison, two of 345

our final pool of metabolites were significantly elevated on the basis of the controlled 346

conditions (i.e. UV+/UV-) vs the glasshouse regime. These included diethyl 1,10-347

decanedioate (Fig. 4C), and chlorogenic acid (Fig. 4D). Only one of the compounds in the 348

pool was significantly increased in UV- plants compared to UV+ plants and GH plants; p-349

coumaryl quinic acid (Fig. 4E). The two unnamed compounds C22H18O11 (Fig. 4G) and 350

C41H72O9 (Fig. 4H) made large contributions to the overall metabolite pool in L. sativa 351

seedlings, but were not significantly different in terms of treatment effects. Both of these 352

compounds were elevated in all Day 6 samples compared to our Day 0 starting point. The 353

relative difference in the total sum of all peak areas for our metabolite pool at Day 6 354

between UV- and UV+ plants was 21% (Table 1), which compares closely to the 18% 355

difference in UV shielding values observed between UV- and UV+ plants by Day 6 of the 356

experiment (Fig. 2C). 357

358

DISCUSSION 359

Previously, we showed that realistic levels of UV-B radiation can potentially up-regulate 360

photosynthetic performance in an important vegetable species, and that such responses 361

may be linked to photoprotective processes when plants are challenged with high PAR 362 Acc

epte

d A

rticl

e


fluxes (Wargent et al., 2011). We believe the current study is one of the first detailed 363

metabolomic investigations of UV-B plant response, and certainly the first time such 364

responses have been investigated concordantly alongside measurements of leaf gas 365

exchange, photosynthetic efficiency, and leaf shielding protection. Responses to UV 366

radiation have received good attention to date with regard to UV-B signal transduction 367

(Heijde & Ulm, 2012, Jenkins, 2009), and observations of key physiological outcomes, 368

including reduction in leaf expansion (Hectors et al., 2010, Wargent et al., 2009b), and 369

induction of secondary metabolism, yet we have very limited information regarding the 370

temporal, physiological and biochemical nature of UV-B acclimation. Kusano and 371

colleagues (2011) recently suggested that shorter-term responses to UV-B at a 372

metabolomic and transcriptome level were central to a strategy employed in Arabidopsis to 373

channel carbon flux into the shikimate pathway for sunscreening bio-accumulation. 374

Specifically, changes in intermediates of the tricarboxylic (TCA) cycle were suggested as a 375

possible link to changes in energy provision for enhanced phenylpropanoid activity. While 376

this study drew interesting linkages between primary and secondary metabolism function 377

within UV-B response cascades in Arabidopsis, the authors employed highly unnatural UV 378

treatments, indicating that extending our knowledge of early events in UV-B acclimation is 379

worthy of increased attention. 380

381

UV-B regulates photosynthetic performance in L. sativa 382

Our results further underpin the concept that responses to UV radiation at a photosynthetic 383

level may not be consistently viewed as negative or damaging to plant development. While 384

there is still little information regarding the mechanistic changes driving UV-B mediated 385

increases in photosynthesis, recent work on the woody shrub Pimelea ligustrina 386

demonstrated that UV-A radiation increased in situ photosynthetic rates in P. ligustrina by 387

12%, a response which was attributed to the excitation of chlorophyll a by UV-A directly 388 Acc

epte

d A

rticl

e


(Turnbull et al., 2013). While we did observe a concordant elevation of transpiration rate in 389

our study, the sequence of events which leads to elevated Pmax in response to UV-B 390

remains unresolved. UV-B photomorphogenesis has been associated with changes in 391

stomatal density (Kakani et al., 2003, Wargent et al., 2009a), yet in the present study, we 392

have seen little morphological change during the short acclimation period, and so stomatal 393

patterning is unlikely to explain our findings. While cuvette conditions were very similar 394

indeed to the chamber conditions, PAR levels were at a higher, saturating flux for gas 395

exchange measurements (i.e. an increase in ~400 µmol m-2 s-1), which may further 396

underpin a concept of UV-mediated photoprotection. It has been suggested that UVR8 397

may play a regulatory role in photosynthetic performance in response to UV-B, possibly 398

involving additional encoding of chloroplastic proteins (Davey et al., 2012), yet the 399

regulatory components remain somewhat elusive. In other species of agronomic 400

importance, such as Vitis vinifera, hallmark changes in secondary metabolism can be 401

linked to UV-B exposure levels (Gregan et al., 2012, Schultz, 2000), yet at the same time, 402

photosynthetic responses to UV-B are certainly equivocal. For example, Berli et al. (2013) 403

recently showed that UV-B exposure of grapevines for 90 d led to reduced stomatal 404

conductance and photosynthetic rates, in addition to reductions in leaf area, and increases 405

in leaf thickness. Our six day UV-B exposure period did not lead to changes in leaf area, 406

shoot fresh weight or dry weight in either experimental iteration (data not shown), which is 407

not particularly surprising given our focus on the relatively brief acclimation phase. 408

Nonetheless, more attention is clearly needed, particularly with regard to differentiating 409

those complex interactions of leaf size and morphology alongside regulation of gas 410

exchange. 411

412

Metabolomic responses to UV-B can overlap with high light acclimation 413 Acc

epte

d A

rticl

e


Our metabolomic approach determined a large number of molecular features in L. sativa, 414

yet the marked contributions to leaf metabolomic profile appear to be made by a smaller 415

group of compounds. The majority of those named compounds which were elevated by 416

UV-B are shikimate acid pathway derivatives, and so likely fulfil sunscreening roles in 417

leaves. The myricetin derivative, luteolin 7-glucuronide, is a flavonol-type flavonoid found 418

in various grapes, berries and vegetables, and has been previously associated with UV 419

response, notably in Salix sp. (Tegelberg & Julkunen-Tiitto, 2001). The flavonol quercetin 420

3-(6''-malonyl-glucoside) has received limited attention in the UV-B literature to date, but 421

has been previously found in green leaved cultivars of L. sativa (DuPont et al., 2000). 422

Quercetin 3-glucuronide is a flavonol known to increase in mature strawberry fruit in 423

response to UV-B (Josuttis et al., 2010), and is linked to human health benefits, partly due 424

to the likelihood of bioavailability when consumed (Boots et al., 2008, Wittig et al., 2001). 425

Overall, UV shielding values are consistent with the relative differences in the abundance 426

of these key metabolites in UV- and UV+ treated plants, suggesting that the flavonoids do 427

indeed account for the majority of UV screening effects in our study. 428

The implications for greater understanding of UV-B response from an agronomic 429

perspective are now receiving greater attention (Ballare et al., 2012, Wargent & Jordan, 430

2013), and our study included a comparison of UV-/UV+ seedlings with a glasshouse 431

regime. Interestingly, while the overarching photosynthetic responses were observed 432

solely in UV-treated plants, there were clear and substantial changes in leaf metabolite 433

composition when comparing plants transferred from glasshouse conditions to the UV-434

/UV+ regime. While conditions in our GH treatment were not completely identical to UV-435

/UV+ conditions, integrated daily PAR values between GH and UV-/UV+ were very similar. 436

In terms of spectral quality, under typical clear sky conditions, red to far red ratios (R:FR; 437

660 nm/730 nm) were relatively comparable between the glasshouse and chamber 438

conditions (R:FRglasshouse = 1.3; R:FRchamber = 1.5), and both sets of conditions included 439 Acc

epte

d A

rticl

e


moderately high levels of long-wave UV-A radiation. In addition to blue and red 440

wavelengths, the growth chamber also provided notable irradiance peaking at 535 nm, 441

which was not present in glasshouse conditions at such maxima. Anthocyanins readily 442

absorb green light (Lee & Gould, 2002, Neill & Gould, 1999), and it is therefore possible 443

that such spectral elements in the UV-/UV+ conditions may also account for some of our 444

changes in metabolite response. Moreover, in the absence of protective anthocyanins, 445

green light can potentially lead to photosystem stress (Zhang et al., 2010), which may 446

explain the reduction in Fv/Fm upon transfer to growth chamber conditions. Yet at the same 447

time, UV-/UV+ plants were clearly not impaired following transfer in terms of Pmax. Eight of 448

the ten metabolites examined in detail were elevated to some extent in response to 449

glasshouse transfer, and of those, there were two named compounds which did not 450

respond further to UV-B treatment. The phenylpropanoid-derived chlorogenic acid is an 451

ester of caffeic acid, and has been associated with UV-screening in tomato (Cle et al., 452

2008), and has also been linked to herbivore feeding responses (Izaguirre et al., 2007). 453

Diethyl 1,10-decanedioate (or the synonym of sebacic acid) has received no attention as a 454

plant metabolite previously, and the high m-Sigma value attributed to this elemental 455

composition (Table 1) indicates likely high error in this identification. Only one of the 456

compounds in the pool was significantly increased in UV- plants compared to UV+ and 457

glasshouse plants. The hydroxycinnamic p-coumaryl quinic acid may be associated with 458

elements of lignin metabolism and resistance to pathogen attack (Franke et al., 2002). 459

Two of our unnamed compounds (C22H18O11 and C41H72O9) made large contributions to 460

the overall metabolite pool in Day 6 plants, but were not significantly different in terms of 461

treatment effects, suggesting that those compounds may be a component of development 462

and growth in L. sativa. 463

464

Conclusion 465 Acc

epte

d A

rticl

e


Overall, our results indicate that those secondary compounds responsible for the 466

acclimation response to UV-B partly share a role in acclimating to changes in the PAR 467

environment. This conclusion adds support to the concept that large discrete pools of 468

metabolites may not be mobilized in response to environmental cues such as UV radiation, 469

but rather that compounds may be multi-purpose in nature. In a recent field survey of UV 470

shielding in response to differing UV and PAR conditions, overall PAR levels were seen as 471

the main determinant of UV shielding levels as compared to effects attributable to UV 472

radiation alone (Barnes et al., 2013). At the same time, the authors observed that both UV-473

A and UV-B were required for maximal sun acclimation in previously unexposed leaves. 474

Certainly, dynamic control of secondary metabolism in response to environmental factors 475

and plant productivity levels would provide an efficient strategy for plants, and while 476

plasticity of metabolite biosynthesis and availability is evident in those systems studies to 477

date (Vogt, 2010), more integrated understanding of plant UV-B response is needed. From 478

an agricultural perspective, many vegetable crops are initially cultivated within indoor 479

environments, or are transplanted outside following young growth indoors. Understanding 480

UV-B acclimation can arguably provide greater prediction of plant responses to sudden 481

sunlight exposure, or the ability to induce desired responses for food production. In 482

conclusion, our findings support the new paradigm of agronomically exploitative benefits 483

related to UV-B plant response, and offers a new sequence of physiological and metabolic 484

change driven by UV-B in young unexposed plants. This suggests that an as-yet 485

unidentified mechanism rapidly promotes photosynthetic performance following UV-B 486

exposure, and it could be speculated that increased availability of resources may then be 487

used for metabolite synthesis and subsequent UV-B acclimation. Future investigation of 488

this chain of events could offer further mechanistic understanding of UV-B 489

photomorphogenesis, and provide markers for enhanced food crop stress resilience and 490

productivity. 491 Acc

epte

d A

rticl

e


492

493

ACKNOWLEDGEMENTS 494

We are grateful to the Institute of Agriculture and Environment, Massey University, for 495

sponsoring this work with a summer scholarship to B.C.W.N., and to the Massey 496

University Research Fund for an award to J.J.W. Support to P.W.B. was provided by the 497

J.H. Mullahy Endowment in Environmental Biology at Loyola University New Orleans. We 498

also thank two anonymous referees for their constructive comments on our manuscript. 499

500

REFERENCES 501

Allen D.J., McKee I.F., Farage P.K. & Baker N.R. (1997) Analysis of limitations to CO2 502

assimilation on exposure of leaves of two Brassica napus cultivars to UV-B. Plant 503

Cell and Environment, 20, 633-640. 504

Ballare C.L., Mazza C.A., Austin A.T. & Pierik R. (2012) Canopy light and plant health. 505

Plant Physiology, 160, 145-155. 506

Barnes P.W., Flint S.D., Slusser J.R., Gao W. & Ryel R.J. (2008) Diurnal changes in 507

epidermal UV transmittance of plants in naturally high UV environments. 508

Physiologia Plantarum, 133, 363-372. 509

Barnes P.W., Kersting A.R., Flint S.D., Beyschlag W. & Ryel R.J. (2013) Adjustments in 510

epidermal UV-transmittance of leaves in sun-shade transitions. Physiologia 511

Plantarum, 149, 200-213. 512

Bassman J.H. (2004) Ecosystem consequences of enhanced solar ultraviolet radiation: 513

Secondary plant metabolites as mediators of multiple trophic interactions in 514

terrestrial plant communities. Photochemistry and Photobiology, 79, 382-398. 515 Acc

epte

d A

rticl

e


Berli F.J., Alonso R., Bressan-Smith R. & Bottini R. (2013) UV-B impairs growth and gas 516

exchange in grapevines grown in high altitude. Physiologia Plantarum, 149, 127-517

140. 518

Bolink E.M., van Schalkwijk I., Posthumus F. & van Hasselt P.R. (2001) Growth under UV-519

B radiation increases tolerance to high-light stress in pea and bean plants. Plant 520

Ecology, 154, 149-156. 521

Boots A.W., Haenen G. & Bast A. (2008) Health effects of quercetin: From antioxidant to 522

nutraceutical. European Journal of Pharmacology, 585, 325-337. 523

Bornman J.F. & Vogelmann T.C. (1991) Effect of UV-B radiation on leaf optical-properties 524

measured with fiber optics. Journal of Experimental Botany, 42, 547-554. 525

Briggs W.R. & Christie J.M. (2002) Phototropins 1 and 2: versatile plant blue-light 526

receptors. Trends in Plant Science, 7, 204-210. 527

Brown B.A., Cloix C., Jiang G.H., Kaiserli E., Herzyk P., Kliebenstein D.J. & Jenkins G.I. 528

(2005) A UV-B-specific signaling component orchestrates plant UV protection. 529

Proceedings of the National Academy of Sciences of the United States of America, 530

102, 18225-18230. 531

Caldwell M.M. (1971) Solar UV radiation and the growth and development of higher plants. 532

Academic Press, New York. 533

Christie J.M., Arvai A.S., Baxter K.J., Heilmann M., Pratt A.J., O'Hara A., Kelly S.M., 534

Hothorn M., Smith B.O., Hitomi K., Jenkins G.I. & Getzoff E.D. (2012) Plant UVR8 535

photoreceptor senses UV-B by tryptophan-mediated disruption of cross-dimer salt 536

bridges. Science, 335, 1492-1496. 537

Cle C., Hill L.M., Niggeweg R., Martin C.R., Guisez Y., Prinsen E. & Jansen M.A.K. (2008) 538

Modulation of chlorogenic acid biosynthesis in Solanum lycopersicum; 539

consequences for phenolic accumulation and UV-tolerance. Phytochemistry, 69, 540

2149-2156. 541 Acc

epte

d A

rticl

e


Davey M.P., Susanti N.I., Wargent J.J., Findlay J.E., Quick W.P., Paul N.D. & Jenkins G.I. 542

(2012) The UV-B photoreceptor UVR8 promotes photosynthetic efficiency in 543

Arabidopsis thaliana exposed to elevated levels of UV-B. Photosynthesis Research, 544

114, 121-131. 545

Day T.A., Martin G. & Vogelmann T.C. (1993) Penetration of UV-B radiation in foliage - 546

evidence that the epidermis behaves as a nonuniform Filter. Plant Cell and 547

Environment, 16, 735-741. 548

Demkura P.V. & Ballare C.L. (2012) UVR8 mediates UV-B-induced Arabidopsis defense 549

responses against Botrytis cinerea by controlling sinapate accumulation. Molecular 550

Plant, 5, 642-652. 551

DuPont M.S., Mondin Z., Williamson G. & Price K.R. (2000) Effect of variety, processing, 552

and storage on the flavonoid glycoside content and composition of lettuce and 553

endive. Journal of Agricultural and Food Chemistry, 48, 3957-3964. 554

Favory J.J., Stec A., Gruber H., Rizzini L., Oravecz A., Funk M., Albert A., Cloix C., 555

Jenkins G.I., Oakeley E.J., Seidlitz H.K., Nagy F. & Ulm R. (2009) Interaction of 556

COP1 and UVR8 regulates UV-B-induced photomorphogenesis and stress 557

acclimation in Arabidopsis. Embo Journal, 28, 591-601. 558

Franke R., Hemm M.R., Denault J.W., Ruegger M.O., Humphreys J.M. & Chapple C. 559

(2002) Changes in secondary metabolism and deposition of an unusual lignin in the 560

ref8 mutant of Arabidopsis. Plant Journal, 30, 47-59. 561

Franklin K.A. & Whitelam G.C. (2004) Light signals, phytochromes and cross-talk with 562

other environmental cues. Journal of Experimental Botany, 55, 271-276. 563

Frohnmeyer H. (1999) In or out - photoreceptors in motion. Trends in Plant Science, 4, 564

294-295. 565

Gregan S.M., Wargent J.J., Liu L., Shinkle J., Hofmann R., Winefield C., Trought M. & 566

Jordan B. (2012) Effects of solar ultraviolet radiation and canopy manipulation on 567 Acc

epte

d A

rticl

e


the biochemical composition of Sauvignon Blanc grapes. Australian Journal of 568

Grape and Wine Research, 18, 227-238. 569

Hakala-Yatkin M., Mantysaari M., Mattila H. & Tyystjarvi E. (2010) Contributions of visible 570

and ultraviolet parts of sunlight to photoinhibition. Plant and Cell Physiology, 51, 571

1745-1753. 572

Hectors K., Jacques E., Prinsen E., Guisez Y., Verbelen J.P., Jansen M.A.K. & Vissenberg 573

K. (2010) UV radiation reduces epidermal cell expansion in leaves of Arabidopsis 574

thaliana. Journal of Experimental Botany, 61, 4339-4349. 575

Heijde M. & Ulm R. (2012) UV-B photoreceptor-mediated signalling in plants. Trends in 576

Plant Science, 17, 230-237. 577

Hideg É., Jansen M.A.K. & Strid Å. (2012) UV-B exposure, ROS, and stress: inseparable 578

companions or loosely linked associates? Trends in Plant Science. 579

Izaguirre M.M., Mazza C.A., Svatos A., Baldwin I.T. & Ballare C.L. (2007) Solar ultraviolet-580

B radiation and insect herbivory trigger partially overlapping phenolic responses in 581

Nicotiana attenuata and Nicotiana longiflora. Annals of Botany, 99, 103-109. 582

Jansen M.A.K., Hectors K., O'Brien N.M., Guisez Y. & Potters G. (2008) Plant stress and 583

human health: Do human consumers benefit from UV-B acclimated crops? Plant 584

Science, 175, 449-458. 585

Jenkins G.I. (2009) Signal transduction in responses to UV-B radiation. Annual Review of 586

Plant Biology, 60, 407-431. 587

Jenkins G.I., Brown, B.A. (2007) UV-B perception and signal transduction. Blackwell 588

Publishing, Oxford. 589

Jordan B.R. (1996) The effects of ultraviolet-B radiation on plants: A molecular 590

perspective. In: Advances in Botanical Research, Vol 22: Incorporating Advances in 591

Plant Pathology (ed J.A. Callow), pp. 97-162. 592 Acc

epte

d A

rticl

e


Josuttis M., Dietrich H., Treutter D., Will F., Linnemannstons L. & Kruger E. (2010) Solar 593

UVB response of bioactives in strawberry (Fragaria x ananassa Duch. L.): A 594

comparison of protected and open-field cultivation. Journal of Agricultural and Food 595

Chemistry, 58, 12692-12702. 596

Kakani V.G., Reddy K.R., Zhao D. & Mohammed A.R. (2003) Effects of ultraviolet-B 597

radiation on cotton (Gossypium hirsutum L.) morphology and anatomy. Annals of 598

Botany, 91, 817-826. 599

Kolb C.A., Schreiber U., Gademann R. & Pfundel E.E. (2005) UV-A screening in plants 600

determined using a new portable fluorimeter. Photosynthetica, 43, 371-377. 601

Kusano M., Tohge T., Fukushima A., Kobayashi M., Hayashi N., Otsuki H., Kondou Y., 602

Goto H., Kawashima M., Matsuda F., Niida R., Matsui M., Saito K. & Fernie A.R. 603

(2011) Metabolomics reveals comprehensive reprogramming involving two 604

independent metabolic responses of Arabidopsis to UV-B light. Plant Journal, 67, 605

354-369. 606

Lee D.W. & Gould K.S. (2002) Why leaves turn red: pigments called anthocyanins 607

probably protect leaves from light damage by direct shielding and by scavenging 608

free radicals, American Scientist, 90, 524–531. 609

Mackerness S.A.H., Surplus S.L., Jordan B.R. & Thomas B. (1998) Effects of 610

supplementary ultraviolet-B radiation on photosynthetic transcripts af different 611

stages of leaf development and light levels in pea (Pisum sativum L.: Role of active 612

oxygen species and antioxidant enzymes. Photochemistry and Photobiology, 68, 613

88-96. 614

Morales L.O., Brosché M., Vainonen J., Jenkins G.I., Wargent J.J., Sipari N., Strid Å., 615

Lindfors A.V., Tegelberg R. & Aphalo P.J. (2013) Multiple roles for UV 616

RESISTANCE LOCUS8 in regulating gene expression and metabolite accumulation 617

in Arabidopsis under solar ultraviolet radiation. Plant Physiology, 161, 744-759. 618 Acc

epte

d A

rticl

e


Neill S.O. & Gould K.S. (1999) Optical properties of leaves in relation to anthocyanin 619

concentration and distribution. Canadian Journal of Botany, 77, 1777–1782. 620

Poulson M.E., Donahue R.A., Konvalinka J. & Boeger M.R.T. (2002) Enhanced tolerance 621

of photosynthesis to high-light and drought stress in Pseudotsuga menziesii 622

seedlings grown in ultraviolet-B radiation. Tree Physiology, 22, 829-838. 623

Rizzini L., Favory J.-J., Cloix C., Faggionato D., O'Hara A., Kaiserli E., Baumeister R., 624

Schaefer E., Nagy F., Jenkins G.I. & Ulm R. (2011) Perception of UV-B by the 625

Arabidopsis UVR8 protein. Science, 332, 103-106. 626

Rozema J., Bjorn L.O., Bornman J.F., Gaberscik A., Hader D.P., Trost T., Germ M., Klisch 627

M., Groniger A., Sinha R.P., Lebert M., He Y.Y., Buffoni-Hall R., de Bakker N.V.J., 628

van de Staaij J. & Meijkamp B.B. (2002) The role of UV-B radiation in aquatic and 629

terrestrial ecosystems - an experimental and functional analysis of the evolution of 630

UV-absorbing compounds. Journal of Photochemistry and Photobiology B-Biology, 631

66, 2-12. 632

Schultz H.R. (2000) Climate change and viticulture: a European perspective on 633

climatology, carbon dioxide and UV-B effects. Australian Journal of Grape and Wine 634

Research, 6, 2-12. 635

Stracke R., Favory J.J., Gruber H., Bartelniewoehner L., Bartels S., Binkert M., Funk M., 636

Weisshaar B. & Ulm R. (2010) The Arabidopsis bZIP transcription factor HY5 637

regulates expression of the PFG1/MYB12 gene in response to light and ultraviolet-B 638

radiation. Plant Cell and Environment, 33, 88-103. 639

Takahashi S., Milward S.E., Yamori W., Evans J.R., Hillier W. & Badger M.R. (2010) The 640

solar action spectrum of Photosystem II Damage. Plant Physiology, 153, 988-993. 641

Tegelberg R. & Julkunen-Tiitto R. (2001) Quantitative changes in secondary metabolites of 642

dark-leaved willow (Salix myrsinifolia) exposed to enhanced ultraviolet-B radiation. 643

Physiologia Plantarum, 113, 541-547. 644 Acc

epte

d A

rticl

e


Turnbull T.L., Barlow A.M. & Adams M.A. (2013) Photosynthetic benefits of ultraviolet-A to 645

Pimelea ligustrina, a woody shrub of sub-alpine Australia. Oecologia, 173, 375-385. 646

Vogt T. (2010) Phenylpropanoid Biosynthesis. Molecular Plant, 3, 2-20. 647

Wargent J.J., Elfadly E.M., Moore J.P. & Paul N.D. (2011) Increased exposure to UV-B 648

radiation during early development leads to enhanced photoprotection and 649

improved long-term performance in Lactuca sativa. Plant Cell and Environment, 34, 650

1401-1413. 651

Wargent J.J., Gegas V.C., Jenkins G.I., Doonan J.H. & Paul N.D. (2009a) UVR8 in 652

Arabidopsis thaliana regulates multiple aspects of cellular differentiation during leaf 653

development in response to ultraviolet B radiation. New Phytologist, 183, 315-326. 654

Wargent J.J. & Jordan B.R. (2013) From ozone depletion to agriculture: understanding the 655

role of UV radiation in sustainable crop production. New Phytologist, 197, 1058-656

1076. 657

Wargent J.J., Moore J.P., Roland Ennos A. & Paul N.D. (2009b) Ultraviolet radiation as a 658

limiting factor in leaf expansion and development. Photochemistry and 659

Photobiology, 85, 279-286. 660

Wittig J., Herderich M., Graefe E.U. & Veit M. (2001) Identification of quercetin 661

glucuronides in human plasma by high-performance liquid chromatography-tandem 662

mass spectrometry. Journal of Chromatography B, 753, 237-243. 663

Xu J.T. & Gao K.S. (2010) UV-A enhanced growth and UV-B induced positive effects in 664

the recovery of photochemical yield in Gracilaria lemaneiformis (Rhodophyta). 665

Journal of Photochemistry and Photobiology B-Biology, 100, 117-122. 666

Zhang, K.M., Yu, H.J., Shi, K., Zhou, Y.H., Yu, J.Q. & Xia, X.J. (2010) Photoprotective 667

roles of anthocyanins in Begonia semperflorens. Plant Science. 179, 202-208. 668

669

670 Acc

epte

d A

rticl

e


671

672

SUPPORTING INFORMATION 673

Appendix 1. Bucket tables for LC-QTOF-HRMS analysis of foliar metabolites. Tables are 674

presented according to each two treatment comparison, fold change according to 675

comparison, t-test values, and signal counts for each replicate seedling sample per 676

treatment. 677

678

679

680

Acc

epte

d A

rticl

e


Table 1. Molecular features in L. sativa discovered by LC-QTOF-HRMS. Features were extracted from the raw MS data files and original 681

bucket table, which was subjected to PCA analysis for identification of treatment-dependent differences. Peak areas from exact ion 682

chromatograms (EICs) were extracted for each metabolite that had a fold change of greater than 1.5 with a P<0.05, prior to assignment 683

of elemental composition. Molecular features are presented with accurate mass/retention times, plus peak areas and treatment-684

dependent fold changes according to treatment and experimental day. n=6-7 replicate seedlings for each individual treatment. 685

RT (min)

Mass (m/z)

Identification Formula Measured

mass (m/z)

mDa Diff

mSigma

Peak area Fold change

GH [D0] GH [D6] UV- [D6] UV+ [D6] GHD0/ GHD6

GHD6/ UV-

GHD6/ UV+

UV-/ UV+

5.15 353.087 chlorogenic

acid C16H18O9 353.0878 0.0 2.0 81305.74442 211495.91 392109.4 439674.5893 2.60 1.85 2.08 1.12

5.61 725.120 unknown C30H30O21 725.1200 -0.6 25.6 706.6118644 2964.8249 7390.1126 10149.60596 4.20 2.49 3.42 1.37

7.01 337.092 p-coumaryl quinic acid

C16H18O8 337.0919 1.0 15.2 3824.121059 16736.759 39629.909 33515.94699 4.38 2.37 2.00 0.85

9.76 477.067 quercetin 3-glucuronide

C21H18O13 477.0672 -0.2 3.4 15144.24711 53863.892 156157.94 187238.375 3.73 2.86 3.87 1.35

10.30 461.072 luteolin 7-

glucuronide C21H18O12 461.0717 0.8 5.0 2574.371102 13618.438 46484.282 78683.14174 5.29 3.41 5.78 1.69

10.40 447.093 unknown C21H20O11 447.0919 1.4 24.7 236.4221909 935.28877 2830.2976 7269.868653 3.96 3.03 7.77 2.57

10.76 549.088 quercetin 3-(6''-malonyl-glucoside)

C24H22O15 549.0878 -0.8 9.4 20159.19022 75153.897 214793.99 290517.567 3.73 2.86 3.87 1.35

12.55 457.076 unknown C22H18O11 457.0774 0.6 15.2 2924.724359 8175.4303 8682.7858 11068.50432 2.80 1.06 1.35 1.27

26.29 257.176 diethyl 1,10-decanedioate

C14H26O4 257.1775 -1.7 36.9 1847.48209 3718.8857 6830.46 5799.527832 2.01 1.84 1.56 0.85

26.56 707.512 unknown C41H72O9 707.5111 0.8 38.4 8505.012277 12730.881 15688.104 14375.51325 1.50 1.23 1.13 0.92

686

687 Acc

epte

d A

rticl

e


Figure legends 688

689

Figure 1. Spectral irradiances of experimental conditions. Irradiance measurements were 690

made with a double-scanning spectroradiometer, and describe visible light spectra (main 691

graph) and the UV region (inset graph). Spectra are labeled according to experimental 692

treatment; UVB+ (‘UV+’; dashed line); UVB- (‘UV-‘; unbroken line); glasshouse (‘GH’; thin 693

line); PAR (visible light spectrum for UV+ and UV- conditions; unbroken line). PAR 694

conditions are described by the unbroken line in the main graph at wavelengths >400 nm 695

only. Glasshouse conditions were measured under clear sky conditions. 696

697

Figure 2. Gas exchange parameters, photosynthetic competency, and UV shielding in L. 698

sativa seedlings. Measurements were taken at timed intervals from the day plants were 699

transferred into UV+/UV- conditions (‘Day 0’) for six days. Plants maintained in glasshouse 700

conditions were also sampled during the transfer phase. UV+, open circles; UV-, open 701

squares; GH (glasshouse), closed circles. (a) Quantum efficiency of Photosystem II, 702

(Fv/Fm); (b) net photosynthesis; (c) UV shielding (SUV); (d) transpiration rate; (e) stomatal 703

conductance; (f) internal CO2 concentration. Values are means of two repeated 704

experiments (n=2) ± 1 S.E., where each measurement point consisted of ten plants per 705

treatment per replicate experiment. Days 3 and 5 were sampled once only in different 706

replicate experiments therefore standard errors are not presented. 707

708

Figure 3. Fold change of metabolomic features according to LC-QTOF-HRMS analysis. 709

Following bucket table extraction, molecular features were submitted to Principle 710

Component Analysis (PCA) in ProfileAnalysis for identification of treatment-dependent 711

differences. Volcano plots show relative fold changes (log2) versus standard t-test p-712

values (log10) between paired treatments; (a) GH (glasshouse) Day0/GH D6; (b) GH 713 Acc

epte

d A

rticl

e


D6/UV- D6; (c) GH D6/UV+ D6; (d) UV- D6/UV+ D6. Metabolites with a fold change 714

greater than 1.5 where P<0.05 are represented by open circles, and labels denote the 715

subject treatment for each comparison. Values are means of 6-7 replicate seedlings for 716

each treatment. 717

718

Figure 4. Metabolites in L. sativa in response to UV-B radiation. Following assignment of 719

elemental compositions, metabolites analysed by LC-QTOF-HRMS were compared using 720

one-way ANOVA on the basis of relative change in metabolite abundance according to 721

treatment. Relative change values are from comparisons of treatment metabolite values to 722

GH D0 seedlings at the start of the experiment. (a) luteolin 7-glucuronide; (b) quercetin 3-723

(6''-malonyl-glucoside); (c) diethyl 1,10-decanedioate; (d) chlorogenic acid; (e) p-coumaryl 724

quinic acid; (f) C21H20O11; (g) C22H18O11; (h) C41H72O9; (i) C30H30O21; (j) quercetin 3-725

glucuronide. Values are means of 6-7 replicate seedlings per treatment, and different 726

letters indicate significance according to Tukey at P<0.05. 727

Acc

epte

d A

rticl

e

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

200 300 400 500 600 700

Irrad

ianc

e (W

m-2

s-1

)

Wavelength (nm)

UVB+UVB-PARGH

0.00

0.10

0.20

0.30

270 300 330 360 390

Acc

epte

d A

rticl

e

0

10

20

30

40

50

60

70

80

90

0 1 2 3 4 5 6 7

UV

shie

ldin

g (%

)

0

1

2

3

4

5

6

7

8

9

10

Net

pht

otsy

nthe

sis

(µm

ol m⁻²

s⁻¹

)

0.65

0.7

0.75

0.8

0.85

0.9

F v/F

m

UV+ UV- GH

(a)

(c)

(b)

150

200

250

300

350

400

0 1 2 3 4 5 6 7

Inte

rnal

CO

2 (pp

m)

0

0.05

0.1

0.15

0.2

0.25

Stom

atal

con

duct

ance

(mol

m-2

s-1

)

0

0.2

0.4

0.6

0.8

1

1.2

Tran

spira

tion

(mol

m-2

s-1

)

(d)

(f)

(e)

Time of treatment (days)

Acc

epte

d A

rticl

e

(c)

(b)

(a)

(d)

Acc

epte

d A

rticl

e

02

04

06

08

0GH UV- UV+

LUTEOLIN 7-GLUCURONIDE

b

c

a

01

02

03

04

0

GH UV- UV+

C21H20O11

b

c

a

01

02

03

0

GH UV- UV+

QUERCETIN 3-6'

b

c

a

01

23

45

GH UV- UV+

C22H18O11

abc

bc

ab

02

46

GH UV- UV+

DIETHYL 1,10-DECANEDIOATE

bc

bc

a

01

23

4

GH UV- UV+

C41H72O9

bc

abcac

05

10

15

GH UV- UV+

CHLOROGENIC ACID

b

b

a

01

02

03

0

GH UV- UV+

C30H30O21

b

c

a

05

10

15

GH UV- UV+

P-COUMARYL QUINIC ACID

b

c

a

05

10

15

20

25

GH UV- UV+

QUERCETIN 3-GLUCURONIDE

b

c

a

Re

lative c

hange

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

Acc

epte

d A

rticl

e

Documents

Acclimation to UV-B radiation and visible light in Lactuca sativa involves up-regulation of photosynthetic performance and orchestration of metabolome-wide responses