Dissecting molecular and physiological response mechanisms to high solar
radiation in cyanic and acyanic leaves: a case study on red and green basil
Massimiliano Tattini1* (massimiliano.tattini@ipsp.cnr.it) Federico Sebastiani1 (federico.sebastiani@ipsp.cnr.it) Cecilia Brunetti2,3 (cbrunetti@ivalsa.cnr.it) Alessio Fini2 (alessio.fini@unifi.it) Sara Torre1 (sara.torre@ipsp.cnr.it) Antonella Gori2 (antonella.gori@unifi.it) Mauro Centritto3 (mauro.centritto@cnr.it) Francesco Ferrini2 (francesco.ferrini@unifi.it) Marco Landi4 (marco.landi@forunipi.it) Lucia Guidi4 (lucia.guidi@unipi.it)
1 National Research Council of Italy, Dept. of Biology, Agriculture and Food Sciences, Institute for Sustainable Plant Protection, I- 50019 Sesto Fiorentino, Florence, Italy,
2 Department of Agri-Food Production and Environmental Sciences, University of Florence, Viale delle Idee 30, I-50019, Sesto Fiorentino, Florence, Italy
3 National Research Council of Italy, Dept. of Biology, Agriculture and Food Sciences, Trees and Timber Institute, I- 50019 Sesto Fiorentino, Florence, Italy
4 Department of Agriculture, Food and Environment, University of Pisa, I-56124 Pisa, Italy *Corresponding author: Massimiliano Tattini; Phone +39 055 4574038
Highlight
The attenuation of blue-green light by epidermal anthocyanins evokes shade-avoidance responses, affects leaf development and gas exchange performance, the biosynthesis and catabolism of MEP-derived products, and the biosynthesis of flavonols
Running title: Response mechanisms to high sunlight in red and green leaves Submission date: February 22 2017
Total words: 6267 /5852 3 Tables; 7 Figures
4 Tables and 1 Figure in Supplementary Material 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Abstract
Photosynthetic performance, the expression of genes involved in light signaling, and in the biosynthesis of isoprenoids and phenylpropanoids were analyzed in green (TIG) and red (RR) basil to dissect physiological and molecular response mechanisms to high sunlight. We show that attenuation of blue-green light by epidermal anthocyanins evokes shade-avoidance responses with consequential effects on leaf morpho-anatomical traits and gas exchange performance: RR had lower mesophyll conductance, partially compensated by less effective control of stomata movements in comparison to TIG. Photosynthesis declined more in TIG than in RR facing high sunlight, because of larger stomatal limitations and transient impairment of PSII photochemistry. The MEP pathway mostly promoted the synthesis and de-epoxidation of violaxanthin-cycle pigments in TIG and of neoxanthin and lutein in RR. This allowed green leaves to process effectively the excess of radiant energy, and red leaves to optimize light harvesting and photoprotection. Enhanced GE de-glucosylation and reduced ABA-oxidation, but not superior de-novo ABA synthesis, sustained the greater stomata closure observed in TIG than in RR. Our study reveals competition between anthocyanin and flavonol biosynthesis. This raises the possibility that red individuals, while better protected against supernumerary photons over the visible-waveband, might suffer more than green morphs from excessive UV-radiation.
Key words: ABA-oxidation; ABA-GE de-glucosylation; anthocyanin vs. flavonol biosynthesis; light-responsive transcription factors; MEP and phenylpropanoid pathways; mesophyll and stomatal conductance; RNA-Seq and qT-RT PCR; shade avoidance responses.
Introduction
After more than three decades of extensive research, whether red individuals perform better than green individuals when exposed to excessive solar radiation, remains a highly debated matter (Hughes, 2011; Landi et al., 2015). There are several reasons responsible for these ‘apparently’ unsuccessfully experiments. Firstly, red colouring of leaves is a transient or a permanent condition. Leaves of some species are red during the juvenile phase (Liakopoulos et al., 2006; Zeliou et al., 2009) or become red during autumn-winter (i.e., ‘winter reddening’, Kytridis et al., 2008; Hughes, 2011). In other species, genotypes display leaves that are red throughout the entire life cycle (Kyparissis et al., 2007; Tattini et
al., 2014; Logan et al., 2015). Secondly, anthocyanins may be located in different leaf tissues.
Anthocyanins occur in both the adaxial and the abaxial epidermis (Hughes and Smith, 2007; Tattini et 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56
al., 2014), in just one of the two epidermises (Hughes et al., 2005; Hughes and Smith, 2007), or may be
distributed in the mesophyll (Gould and Quinn, 1999; Kytridis and Manetas, 2006). Thirdly, “not all anthocyanins are born equal” (Kovinich et al., 2014) and, consequently, have different abilities to absorb the visible wavelengths of the solar spectrum (Merzlyak et al., 2008; Jordheim et al., 2016).
Strikingly different experimental set-ups, which include both plant material and growth conditions, add further complexity in providing conclusive evidence about the photoprotective role of anthocyanins. Usually, the photosynthetic machinery is better protected in cyanic than in acyanic leaves under transient exposure to severe light excess (Feild, et al., 2001; Hughes and Smith, 2007). Photosystem II (PSII) photochemistry also recovers faster in red than in green leaves during relief from photoinhibitory light (Landi et al., 2014; Logan et al., 2015). More contradictory observations stem from studies conducted on long-term basis in the field (Steyn et al., 2002; Gould, 2004). Photosynthesis, at saturating light, did not vary in a range of co-occurring winter-red and winter-green angiosperms (Hughes and Smith, 2007; Hughes, 2011), as also observed in high light-grown red and green basil (Tattini et al., 2014). In contrast, red individuals of Cistus creticus and Pistacia lentiscus displayed inherent photosynthetic inferiority, and incurred greater risk of photoinhibition than did green individuals (Kytridis et al., 2008; Zeliou et al., 2009; Nikoforou et al., 2011).
The ability of anthocyanins to absorb over the blue-green region of the solar spectrum (Feild et
al., 2001) may profoundly alter cryptochrome-dependent photomorphogenesis in red morphs and evoke
shade avoidance responses (Keller et al., 2011; Keuskamp et al., 2011; Zhang and Folta, 2012; Pedmale et al., 2016). Red individuals are less plastic indeed than green individuals to changes in sunlight availability, sometimes referred as to the ‘shade-syndrome’ displayed red leaves (Manetas et
al., 2003). Consistently, red morphs display leaves that are thinner and with less compact mesophyll
than the leaves of green morphs when fully exposed to solar radiation (Kyparissis et al., 2007; Lan et
al., 2011; Tattini et al., 2014). The relative abilities of green and red individuals to modify leaf
morpho-anatomical traits (e.g., leaf thickness and mesophyll compactness) depending on light availability (not only to transmit blue-green light at different rates in the leaf), may largely affect their gas exchange performance (Terashima et al., 2011). The issue has not received attention, but compelling evidence emphasizes the role of mesophyll conductance to CO2 (gm), which strongly
depends on the mesophyll anatomy (Flexas et al., 2016), in limiting photosynthesis in shade- or sun-adapted plants, particularly when challenged by an excess of solar irradiance (Fini et al., 2016; Peguero-Pina et al., 2017). 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87
The attenuation of solar radiation by epidermal anthocyanins may also affect the biosynthesis of photosynthetic and non-photosynthetic pigments. While the concentration of chlorophyll is usually higher, the biosynthesis and de-epoxidation of violaxanthin-cycle (VAZ) pigments is frequently lower in red than in green leaves (Steyn et al., 2002; Hughes, 2011; Landi et al., 2015). This apparently reduces the capacity of red leaves to process excess energy (e.g., through nonphotochemical quenching, NPQ, Demmig-Adams et al., 2012) and limit photo-oxidative stress in the chloroplast (Esteban et al., 2015) in comparison to green leaves. On the contrary, there is contrasting evidence about the phenylpropanoid metabolism in cyanic and acyanic leaves (Stich et al., 1992; Hughes et al., 2010; Luo
et al., 2016). In a few instances, red morphs display more active phenylpropanoid biosynthesis than
green morphs (Gould et al., 2002; Neill et al., 2002; Hada et al., 2003). Nonetheless, red basil invested considerably smaller amounts of fresh assimilated carbon to phenylpropanoid, particularly towards colourless flavonoid biosynthesis during shade-to-sun acclimation as compared to green basil (Tattini
et al., 2014).
In our study, analyses were conducted of photosynthetic performance, targeted transcriptomics and metabolomics to dissect molecular and physiological response mechanisms to high solar irradiance in green (‘Tigullio’, TG) and red (‘Red Rubin’, RR) basil growing during Mediterranean summer. We performed measurements of gas exchange, including response curves of CO2 assimilation to changes in photosynthetic photon flux density (AN/PPFD), intercellular (AN/Ci) and chloroplast CO2 concentration (AN/Cc), and of chlorophyll a fluorescence kinetics. Expression analysis of genes regulating photo-morphogenesis, as well as the biosynthesis of isoprenoids and phenylpropanoids was conducted using RNA-seq and qRT-PCR. Individual carotenoids and phenylpropanoids, free-ABA and its glucoside ester (ABA-GE) were quantified using HPLC-DAD-MS analysis. To the best of our knowledge, this is the first report dissecting the response mechanisms, from the molecular to the whole-leaf level, of cyanic and acyanic individuals to high solar irradiance.
Materials and methods
Plant material and growth conditions
Three-week-old potted green (‘Tigullio’, TIG) and red basil (‘Red Rubin’, RR), were grown under natural sunlight irradiance in Florence, Italy (43°46’N, 11°15’E) under minimum/maximum temperatures of 18.5 ± 1.6/32.5 ± 2.4 °C (mean ± SD, n = 28) over a four-week-period in July. Photosynthetic photon flux density (estimated over the 400–700 nm solar waveband) at midday was 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117
1850 ± 155 µmol quanta m-2 s-1 (mean ± SD, n = 28). Four-week-old leaves, newly developed in full sunlight were used for measurements.
Gas exchange and chlorophyll a fluorescence kinetics
Diurnal changes in CO2 assimilation (AN) and stomatal conductance (gs) were recorded in situ from
08:30 to 17:30 h with a LI-6400 portable photosynthesis system (LI-Cor, Lincoln, NE, USA) operating at 400 µmol mol−1 ambient CO
2 concentration. Daily carbon gain was calculated as described in Tattini
et al. (2004). Response curves of CO2 assimilation to changes in photosynthetic photon flux density
(AN/PPFD) and substomatal CO2 concentration (AN/Ci) were obtained as reported recently in Fini et al.
(2016). AN/Ci curves were performed in leaves exposed at saturating PPFD (1200 or 900 µmol quanta
m-2 s-1 for RR or TIG, respectively). In the cuvette, leaves were kept at a relative humidity of 55-60% and at a temperature of 30 °C. The apparent maximum rate of carboxylation by Rubisco (Vc,max (Ci))
was calculated from AN/Ci curves as reported in Fini et al. (2016).
Mesophyll conductance to CO2 (gm) was estimated using the variable J method (Harley et al., 1992)
according to the equation:
gm =
AN
C i−Γ∗[J f +8( A+R day)]
J f −4 ( A+R day )
(1)
where Γ* is the apparent CO2 compensation point to photorespiration, Jf is the rate of linear electron transport estimated from chlorophyll fluorescence, and (AN + Rday) is the gross photosynthetic rate (including non-photorespiratory respiration). Γ* was calculated using the Rubisco specific factor (τ) estimated for annual herbs (91.3 at 25 °C, see Galmés et al., 2005) as:
Γ* = 0.5 O/τ (2)
where O is the oxygen concentration.
Jf was calculated as:
Jf = ΦPSII ×·α × 0.5 × PPFD (3)
where α, the leaf absorptance, measured with a spectroradiometer equipped with an integrating sphere (LI-Cor 1800-12S), was 0.9 in ‘Red Rubin’ and 0.85 in ‘Tigullio’; the factor 0.5 was chosen assuming an equal distribution of absorbed photons between PSI and PSII. After every measurement, it was 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149
verified that Jf corresponded to electron transport rate under non-photorespiratory conditions (i.e. lowering O2 concentration at 1%) estimated by gas exchange (Loreto et al., 1992; Laisk and Loreto, 1996). The CO2 concentration in the chloroplasts (Cc) was then calculated as:
Cc = Ci AN/gm (4)
Mesophyll conductance (gm) measured in each specific step of the curve, allowed calculate Cc from the
corresponding Ci value, and draw response curves of CO2 assimilation to chloroplastic CO2 concentration (AN/Cc curves), from which actual maximum rate of carboxylation by Rubisco (Vc,max,
(Cc)) was calculated according to Sharkey et al. (2007).
A modulated Chl a fluorescence analysis was conducted on dark-adapted (over a 40-min period) leaves sampled at different hours of the day using a PAM-2000 fluorometer (Walz, Effeltrich, Germany) connected to a Walz 2030-B leaf-clip holder through a Walz 2010-F fiber optic cable. The maximum efficiency of photosystem II (PSII) photochemistry was calculated as Fv/Fm = (Fm − F0)/Fm,
where Fv is the variable fluorescence and Fm is the maximum fluorescence of dark-adapted leaves. The
minimal fluorescence, F0, was measured using a modulated light pulse < 1 μmol m-2 s-1, to avoid
appreciable variable fluorescence. Fm and F′m were determined at 20 kHz using a 0.8-s saturating light
pulse of white light at 8000 μmol m-2 s-1. F′
m, the maximum fluorescence in light conditions, was
estimated at 900 or 1200 μmol quanta m-2 s-1 in TIG and RR, respectively (based on PPFDs at which saturation of photosynthesis occurred in TIG and RR). PSII quantum yield in the light (ΦPSII) and nonphotochemical quenching [NPQ = (Fm/F′m) − 1] were estimated using the saturation pulse method of Schreiber et al. (1986), and calculated according to Genty et al. (1989) and Bilger and Björkman (1990), respectively.
RNA-Seq analysis
Gene expression levels were calculated using the RPKM (Reads per Kilobase per Million Mapped reads) method (Mortazavi et al., 2008), by mapping raw reads of green and red basil to the basil reference transcriptome. The reference transcriptome of sweet basil was assembled using the Illumina platform as detailed in Torre et al. (2016). Sequence reads have been deposited at the Sequence Read Archive (SRA) of the National Centre of Biotechnology Information (NCBI, Bethesda, MD, USA) under the accession number SRA313233.
Gene expression analysis by quantitative Real-Time PCR (qRT-PCR)
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Total RNA (2 μg), treated with RNAse-free DNaseI (Invitrogen Ambion, Carlsbad, CA, USA), was used as the template for cDNA synthesis, performed using SuperScript® VILO cDNA Synthesis Kit (Invitrogen), as previously reported (Torre et al., 2016). qRT-PCR analysis was carried out in a StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA, USA) using SYBR green technology (Power SYBR green PCR Master Mix; Applied Biosystems) according to the manufacturer’s instruction, under the following cycling conditions: 10 min at 95°C, 40 cycles of 15 s at 95°C, 1 min at 60°C. Amplification specificity was checked by the analysis of dissociation curve. Transcripts abundance, expressed as normalized relative quantities (NRQs), was calculated by geometric normalization against the mean of three basil housekeeping genes, namely actin (ACT), tubulin (TUB) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Vandesompele et al., 2002). The primer sequences used for qRT-PCR are listed in Supplementary Table S1.
Analysis of photosynthetic pigments, abscisic acid and phenylpropanoids
Individual carotenoids were identified and quantified as reported in Tattini et al. (2014). Freeze dried leaf material (200 mg) was extracted with 2 × 5 mL acetone (added with 0.5 g L-1 CaCO
3) and 15 µL of supernatant injected into a Perkin Elmer Flexar liquid chromatograph equipped with a quaternary 200Q/410 pump and an LC 200 diode array detector (DAD) (all from Perkin Elmer, Bradford, CT, USA). Photosynthetic pigments were separated in a 250 × 4.6 mm Agilent Zorbax SB-C18 (5 µm) column operating at 30°C, eluted for 18 min with a linear gradient solvent system, at a flow rate of 1 mL min-1, from 100% CH
3CN/MeOH (95/5 with 0.05% of triethylamine) to 100% MeOH/ethyl acetate (6.8/3.2). Violaxanthin cycle pigments (violaxanthin, antheraxanthin, zeaxanthin, collectively named VAZ), neoxanthin, lutein, and β-carotene, were identified using visible spectral characteristics and retention times. Individual carotenoids and chlorophylls were calibrated using authentic standards from Extrasynthese (Lyon-Nord, Genay, France) and Sigma Aldrich (Sigma-Aldrich Italia, Milan, Italy), respectively.
The concentrations of free-ABA and ABA-glucoside (ABA-GE) were determined in 200 mg of freeze-dried leaf tissue, ground in liquid nitrogen, to which 50 ng of deuterated abscisic acid (d6-ABA) and 50 ng of deuterated ABA-GE (d5-ABA-GE, from National Research Council of Canada, Saskatoon, SK, Canada) were added. The tissue was extracted with 3 mL CH3OH/H2O (50/50 adjusted to pH 2.5 with HCOOH) for 30 min at 4°C, and the supernatant partitioned with 3 × 3 mL n-hexane. The CH3OH/H2O fraction was loaded onto Sep-Pak C18 cartridges (Waters, Massachusetts, USA), 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210
which were washed with 2 mL pH 2.5 H2O, and then eluted with 1.2 mL ethyl acetate. The eluate, dried under nitrogen, was rinsed with 500 µL pH 2.5 CH3OH/H2O. Aliquots of 3 µL were injected in a LC-ESI-MS/MS equipment, consisting of an Agilent LC1200 chromatograph coupled with an Agilent 6410 triple quadrupole MS detector equipped with an ESI source (all from Agilent Technologies, Santa Clara, CA, USA), operating in negative ion mode. Free-ABA (ABA) and ABA-glucoside (ABA-GE) were separated in an Agilent Poroshell C18 column (3.0 × 100 mm, 2.7 µm), eluted with a linear gradient solvent, at a flow rate of 0.3 mL min-1, from 95% H
2O (with 0.1 % of HCOOH, solvent A) to 100% CH3CN/MeOH (50/50, with 0.1 % of HCOOH, solvent B) over a 30-min run. Quantification of free-ABA and ABA-GE was conducted in multiple reaction mode (MRM) as reported in López-Carbonell et al. (2009).
Identification and quantification of phenylpropanoids, namely caffeic acid derivatives, both colorless flavonoids and anthocyanins was carried out following the protocol reported in Tattini et al. (2014) with few modifications. Freeze dried leaf tissue (300 mg) was extracted with 3 × 6 mL 70% EtOH adjusted to pH 2 with formic acid (HCOOH). The supernatant was partitioned with 3 × 5 mL n-hexane, reduced to dryness under vacuum and rinsed with CH3OH/H2O (50/50, pH 2). Aliquots of 5µL were injected into the Flexar liquid chromatograph unit reported above. Metabolites were separated in a 4.6 × 150 mm Poroshell 120 SB-C18 column (2.7 µm) (Agilent Technologies, Milan, Italy), operating at 30°C, and eluted at a flow rate of 0.6 mL min-1 with a linear gradient solvent system consisting of H
2O (with 3% HCOOH, solvent A), CH3OH (with 3% HCOOH, solvent B) and CH3CN (with 3% HCOOH, solvent C). The chromatographic run lasted 45 min: 0-5 min 74% A, 8% B, 8% C; 5-35 min 56% A, 22% B, 22% C; 35-45 min 20% A, 40% B, 40% C. Individual phenylpropanoids were identified based on their retention times, UV-spectral characteristics and mass-spectrometric data. Quantification of anthocyanins was performed at 530 nm using calibration curves of cyanidin 3-O-glucoside and pelargonidin 3-O-glucoside (Extrasynthese). Quantification of caffeic acid derivatives (caftaric, chicoric and rosmarinic acids) was performed at 330 nm using the calibration curve of rosmarinic acid (Extrasynthese). Quantification of a luteolin derivative was performed at 345 nm using the calibration curve of luteolin 7-O-glucoside (Extrasynthese). HPMS analysis was carried out in a LC-MS-8030 triple quadrupole mass spectrometer operating in the electrospray ionization (ESI) mode and a Nexera HPLC system (all from Shimadzu, Kyoto, Japan). The mass spectrometer operated in negative ion scan mode for caffeic acid derivatives and colorless flavonoid detection, and in positive ion scan mode for anthocyanin detection. Product ion spectra were obtained using Argon as collision gas at a 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241
pressure of 230 kPa. Identification and quantification of quercetin 3-O-rutinoside was performed by HPLC-MS-MS in MRM, since the flavonol concentration was at detection limits in HPLC-DAD. Flavonoids located on adaxial and abaxial surfaces and in the epidermises of leaves (referred as to Epidermal flavonoids throughout the paper) were optically estimated in vivo using the Multiplex 2 (FORCE-A, Orsay, France) portable fluorimetric sensor, as detailed in Agati et al. (2011). The Chl fluorescence signals under red light excitation (λexc = 625 nm, FRFR) and UV-excitation (λexc = 375 nm, FRFUV) were used to calculate the content of epidermal flavonoids, as:
Epidermal flavonoids = FRFR (FRFUV)-1 (5)
Experimental design and statistics
The experiment was performed using a completely randomized design with 32 plants per cultivar. Diurnal variations in gas exchange, chlorophyll fluorescence kinetics, the concentrations of isoprenoids and phenylpropanoids was performed on four replicate plants, sampled at 08:30, 12:00, 14:30 and 17:30 h. Each replicate for metabolite analysis consisted of three leaves. Five replicate plants per cultivar, sampled between 10:00-14:30 h on two consecutive days, were used for drawing AN/Ci, AN/Cc,
and AN/PPFD curves. Data of diurnal measurements were analyzed with repeated measures ANOVA
with cultivar considered to be the between-subjects effect and daily hour as the within-subjects effect (SPSS v.20, IBM, NY, USA). Parameters derived from AN/Ci and AN/Cc curves were subjected to a
one-way ANOVA. Since the concentrations of individual phenylpropanoids did not significantly vary during the day, data were pooled together and subjected to a one-way ANOVA. Epidermal flavonoids were measured of six replicate plants (two leaves per plant) at different hours of the day and data pooled together prior to statistical analysis. Significant differences among means were estimated at the 5% (P < 0.05) level, using the Tukey’s test. RNA-seq was performed on five plants (two leaves per plant) per cultivar sampled at each individual daily hour and pooled together prior to analysis. Statistical treatment of data was performed with the Z-test (Kal et al., 1999) with LOG2 fold change > ±1 and adjusted P-value < 0.05, to identify genes with significantly different expression between green and red basil morphs (CLC Genomic Workbench, Qiagen, Hilden, Germany). Quantitative Real-Time-PCR analysis was performed in triplicate, and data are reported as mean ± SD.
Results
242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270Gas exchange and PSII photochemistry
Photosynthesis did not differ between green (TIG) and red (RR) basil on diurnal basis, since total assimilated CO2, calculated by integrating measurements of AN from 08:30 to 17:30 h (Fig. 1A), was
308 ± 24 (mean ± SD, n = 4) in RR and 288 ± 28 mmol m-2 d-1 in TIG, respectively. TIG had either superior capacity in early morning and late afternoon or lower ability during the central daily hours to assimilate carbon as compared to RR (Fig. 1B). This conforms to the observation that saturation of photosynthesis occurred at 900 and 1200 µmol quanta m-2 s-1 PPFD in TIG and RR, respectively, and photoinhibition of photosynthesis was apparent in TIG at PPFD > 1300 µmol quanta m-2 s-1 (Fig. 1A). Stomatal and mesophyll CO2 conductances also differed between TIG and RR. While stomatal conductance (gs) was higher (+65% or +50% when calculated from AN/Ci curves or from daily in situ
measurements of gas exchange, respectively; Table1 and Fig. 1C), mesophyll conductance to CO2 (gm)
was lower (46%) in RR than in TIG (Table 1). Consistently, apparent (Vc,max(Ci)) was lower, whereas actual (Vc,max (Cc)) carboxylation efficiency was higher in RR than in TIG (Table 1). Stomatal conductance varied to greater extent in TIG than in RR on daily basis (Fig. 1C). Intrinsic water use efficiency (iWUE, AN/gs) was much higher in TIG (on average iWUE = 0.071) than in RR
leaves (on average iWUE = 0.042). The morning- to-midday increase in iWUE was markedly higher in TIG (+72%) than in RR (+18%).
Sunlight irradiance affected PSII photochemistry to a greater degree in TIG than in RR leaves, as revealed by diurnal variations in Chla fluorescence-derived parameters (Fig. 2). Maximal efficiency of PSII photochemistry, Fv/Fm, declined more in TIG than in RR, from early morning to midday (Fig. 2A). Fv/Fm quickly recovered in TIG in the early afternoon, but remained significantly lower than in RR. The actual efficiency of PSII photochemistry, ΦPSII, was on average 20% lower and declined more in TIG than in RR from early morning to late afternoon (Fig. 2B). Mechanisms involved in the thermal dissipation of excess radiant energy, as estimated by NPQ (Fig. 2C), did not operate much in RR, even in leaves suffering from a severe excess of sunlight, in contrast to that observed in TIG.
Genes regulating photomorphogenesis
A set of genes involved in light signaling were differentially expressed in red and green basil (Fig. 3, Supplementary Table S2). Three members of the HOMODOMAIN-leucine zipper (HD-ZIP) family of transcription factors, ATHB1, ATHB2 and ATHB12, were overexpressed in RR, as also observed for the expressions of some relevant members of the basic helix-loop-helix (BHLH) family of TFs 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300
(bHLH112, bHLH130-Flowering BHLH4 (FBH4); bHLH135-ACTIVATION-TAGGED BRI1
(BRASSINOSTEROID-INSENSITIVE 1)-SUPPRESSOR 1 (ATBS1); bHLH144-SUPPRESSOR OF ACAULIS5 LIKE3 (SACL3). On the contrary, the expressions of ATHB10 (GLABRA 2), BHLH6
(MYC2) and bHLH150 (ATBS1 INTERACTING FACTOR, AIF1) were significantly higher in TIG.
Genes regulating isoprenoid and phenylpropanoid biosynthesis
The expressions of genes involved in early (D-xylulose-5-phosphate synthase, DXS; 1-deoxy-D-xylulose-5-phosphate reductoisomerase, DXR; geranylgeranyl pyrophosphate synthase; GGPS) and late (phytoene synthase, PSY; violaxanthin de-epoxidase, VDE) steps of the MEP pathway were significantly higher in TIG as compared to RR (Fig. 4, Supplementary Table S3). The expression of zeaxanthin epoxidase (ZEP) was instead lower in TIG than in RR. We did not observe significant differences in the expression of genes involved in de-novo ABA biosynthesis, such as neoxanthin synthase (NSY) and 9-cis-epoxycarotenoid dioxygenase (NCED). On the contrary, the expression of a gene coding for a member of the subfamily1 of β-glucosidases (BGL1, which catalyzes the de-glucosylation of ABA-GE, Lee et al., 2006) was higher in TIG than in RR, the reverse being observed for the expression of two genes regulating the oxidative degradation of ABA (ABA 8’-hydroxylase,
CYP707A1).
Green and red basil also largely differed in the expression of genes involved in the biosynthesis of phenylpropanoids (Figure 5, Supplementary Table S4). In detail, the expression levels of early genes of the general phenylpropanoid metabolism (phenylalanine ammonia-lyase, PAL; 4-coumarate-CoA ligase, 4CL), and most genes involved in flavonoid biosynthesis were higher in RR than in TIG. This included early (chalcone synthase CHS; chalcone-flavonone isomerase CHI; flavanone 3-dioxygenase
FHT; flavonoid 3’-monooxygenase, F3’H) and late genes of the flavonoid pathway (dihydroflavonol
4-reducase DFR; leucoanthocianidin dioxygenase, LDOX; anthocyanidin 3-O-glucosyltransferas, 3GT; anthocyanidin 3-O-glucoside 5-O-glucosyltransferase, 5GT; anthocyanidin 3-O-glucoside 6”-acyltransferase 3AT; anthocyanin 5-O-glucoside-6”’-O-malonyltransferase, 5MAT1). On the contrary, the expression of genes involved in flavonol biosynthesis, i.e. FLS, was higher in TIG than in RR.
Photosynthetic pigments, abscisic acid and phenylpropanoids
Green basil leaves had significantly lower (37%) Chltot concentration, higher both Chla to Chlb (+80%) ratio and total carotenoid concentration (Cartot +17%, Table 2) in comparison to red basil leaves. In 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329
detail, the concentrations of β-carotene (Fig. 6B) and VAZ (Fig. 6, C-E) were higher, whereas the concentrations of lutein (Fig. 3A) and neoxanthin (Fig. 6F) were lower in TIG than in RR. The pool of VAZ was steeply higher (+118%) in TIG than in RR when expressed on chlorophyll basis (Fig. 6G). While the concentration of carotenoids and the VAZ pool did not vary much in RR, the concentration of β-carotene and VAZ, particularly of the de-epoxided forms antheraxanthin and zeaxanthin varied considerably during the day in TIG.
The concentration of abscisic acid also differed between red and green basil. Leaves of TIG had greater concentration of both free-ABA (+103%, Fig. 7A) and ABA-GE (+24% Fig. 7B) than RR leaves. The daily trend of foliar free-ABA concentration was consistent with that of stomatal conductance, with the lowest or the highest concentrations observed in early morning or late afternoon, respectively. Morning to midday increases in free-ABA concentration (+136% in TIG, +61% in RR) and in the ratio of free-ABA to ABA-GE (+84% in TIG, +20% in RR) were markedly higher in TIG than in RR.
Irrespective of genotype, we did not observe diurnal changes in the whole-leaf concentration of phenylpropanoids as well as in the content of epidermal flavonoids. The data of the time course experiments have been pooled together prior to statistical analysis (Table 3). The concentration of colourless phenylpropanoids was 20% higher in TIG than in RR leaves, as the result of major increases in the concentrations of colourless flavonoids (+118%). Similarly, the level of epidermal flavonoids, as monitored by in vivo measurements of absorbance at 375 nm by our chlorophyll fluorescence-based methodology, was higher (+49%) in TIG than in RR. Anthocyanins detected in red basil were mostly cyanidin derivatives, both coumaroyl and malonyl derivatives of cyanidin 3-O-glucoside. Pelargonidin 3-O-derivatives accounted for 9% of the total anthocyanin pool.
Discussion
The data from our study offers a comprehensive picture of the effects of a constitutive epidermal cyanic filter on a suite of molecular and physiological traits that are responsible for the differential acclimation mechanisms to high solar irradiance in red and green individuals.
Blue light attenuation by epidermal anthocyanins evoke shade avoidance response and sustain higher photosynthetic performance in cyanic than in acyanic basil leaves exposed to excess light
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Attenuation of blue light may simulate the shade avoidance response usually displayed by plants growing in low R/FR light (Keuskamp et al., 2011; Pedmale et al., 2016; Supplementary Fig. S1). Markers of shade avoidance responses, such as the three members of the HD-ZIP transcription factor family, ATHB1, ATHB2 and ATHB12, and the two members of the BHLH family of TFs, bHLH 135 and bHLH 144 (Ciolfi et al., 2013), were highly expressed indeed in RR. ATHB1 and ATHB2, whose expressions decrease in high green light (Zhang and Folta, 2012) or increase in low FR/R light (Steindler et al., 1999; Clitwood et al., 2015) may alter leaf cell fate, causing defects in palisade parenchyma development (Ayoama et al., 1995). Overexpression of ATHB-12 also regulates leaf growth by promoting cell expansion, and reducing the number of palisade cells (Hur et al., 2015).
BHLH 135 (SACL3) and BHLH 144 (ATBS1) genes also negatively affect cell proliferation, and Arabidopsis lines overexpressing ATBS1 display light hyposensitive phenotypes (Castelain et al.,
2012). Consistent with the light hyposensitivity usually displayed by red individuals, the expression of
BHLH 150, which antagonizes the regulatory effects of ATBS1 (Wang et al., 2009) was poorly
expressed indeed in RR. The expression of the BHLH6 gene (MYC2), a key component of cryptochrome-mediated responses (Gupta et al., 2014), was also low in shade-type RR leaves (Chico et
al., 2014). MYC2 has been shown to tightly regulate the whole-plant morphology, as plants
overexpressing MYC2 display shorter internode length and more lateral shoot branches (Yadav et al., 2005; Gupta et al., 2014), as usually observed in sun-adapted green plants, including sweet basil (Tattini et al., 2014). Therefore, data of our study confirm that blue light-induced shade avoidance response is inherently morpho-anatomical in its nature (Keller et al., 2011, Supplementary Fig. S1), and add conclusive evidence to previous observation that RR is almost unresponsive to changes in solar irradiance at the level of the leaf and the whole-plant (Tattini et al., 2014).
The low morpho-anatomical plasticity usually displayed by red leaves in response to light irradiance (Tattini et al., 2014; Landi et al., 2015) is consistent with the significantly lower mesophyll conductance to CO2 observed in RR than in TIG, since gm is usually low in shaded leaves (Flexas et al.,
2008; Peguero-Pina et al., 2016). Red basil partly compensated for the inherently lower gm through a
lower control of gs, since gtot did not differ much between TIG and RR (gtot was 46.9 in RR and 55.5 in
TIG, data not shown). The ability of anthocyanins to absorb more efficiently the green than the blue solar wavelengths, may help to explain the higher gs observed in RR, as green light opposes blue
light-induced stomata opening (Talbott et al., 2002; Kim et al., 2004). A high gm to gs ratio has been
previously observed in thick leaves with densely packed mesophyll (Niinemets et al., 2009; Peguero-358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388
Pina et al., 2017), to compensate for high stomatal resistance to CO2 diffusion. This may represent an effective physiological adjustment to increase iWUE (Flexas et al., 2016; Peguero-Pina et al., 2017), particularly during the hottest hours of the day. This is consistent with the green-leafed TIG in our study, which displayed markedly greater gm to gs ratio and iWUE as compared to red-leafed RR.
The absorption of solar blue wavelengths by epidermal anthocyanins was responsible for the greater capacity of RR to limit morning-to-midday inhibition in PSII photochemistry as compared to RR, as blue light may severely damage the oxygen-evolving complex in PSII (Takahashi and Murata, 2008). This, when coupled with the lower midday depression in gs, is consistent with the higher
photosynthesis of RR than of TIG under photoinhibitory light conditions. On the contrary, absorption of blue-green light by epidermal anthocyanins makes RR leaves less able than TIG leaves to use radiant energy for CO2 assimilation at relatively low solar irradiance (Gould et al., 2002; Logan et al., 2015). Therefore, our study adds strong support to previous suggestions that epidermal anthocyanins are effective photoprotective pigments (Feild et al., 2001; Hughes, 2011; Landi et al., 2015).
Light attenuation by epidermal anthocyanins affects the biosynthesis and catabolism of MEP-derived products
The higher expression of early (DXS and DXR) and late (GGPS and PSY) genes of the MEP pathway in TIG as compared to RR conforms to the notion that light is one of the primary environmental drivers for isoprenoid biosynthesis (Cordoba et al., 2009; Pokhilko et al., 2015). DXS, which is a rate limiting step for the MEP pathway flow (Wright et al., 2014), is among the most responsive MEP genes to light irradiance (Cordoba et al., 2009). An increased expression of PSY may also trigger an increased supply of MEP-derived precursors via post-transcriptional up-regulation of DXS (Rodriguez-Villalon et al., 2009), further promoting the biosynthesis of late MEP-derived products (Cozzonelli and Pogson, 2010). This is consistent with the significantly higher concentration of carotenoids, particularly of VAZ pigments detected in TIG than in RR leaves. As the ratio of β-carotene to lutein was markedly higher in TIG (1.34) than in RR (0.79), it is suggested that the light-induced activation of the MEP pathway in green basil specifically promoted VAZ biosynthesis and de-epoxidation (Lichtenthaler, 2007), thus sustaining NPQ (Demmig-Adams et al., 2012; Esteban et al., 2015). We hypothesize that zeaxanthin contributed only in part to NPQ in TIG leaves under photoinhibitory light conditions, probably performing additional antioxidant functions in the chloroplast (Havaux et al., 2007; Peguero-Pina et al., 2013; Tattini et al., 2015), since the VAZ pool exceeded the potential binding sites in antenna proteins 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418
(Esteban et al., 2015). In contrast, the MEP pathway mostly sustained the biosynthesis of chlorophyll and PSII light-harvesting complexes (LHCII) in shade-type RR leaves and, consequently, promoted mostly the biosynthesis of neoxanthin and lutein to optimize light harvesting and photoprotection (Mozzo et al., 2008; Horton, 2012).
In our study, the superior stomatal regulation in TIG correlates well with the levels of foliar free-ABA, as expected for plants that grew under optimal irrigation. The higher levels of foliar ABA observed in TIG than in RR, unlikely originated from a superior de-novo ABA synthesis through the plastidial MEP pathway: The expression of 9-cis epoxycarotenoid oxygenase (NCED) and neoxanthin synthase (NSY) did not vary between the two basil genotypes (Nambara and Marion-Poll, 2005; Galvez-Valdivieso et al., 2009; Finkelstein, 2013). It is conceivable that in leaves challenged by a severe light excess, violaxanthin primarily supports zeaxanthin biosynthesis, rather than constituting a substrate for neoxanthin and/or ABA biosynthesis (Lichtenthaler, 2007; Ruiz-Sola and Rodriguez-Concepcion, 2012). Data of our study indicate that de-glucosylation of ABA-GE (Lee et al., 2006) likely sustained the high accumulation of free-ABA in green basil leaves. The expression of β-glucosidase1 (BGL1) and the ratio of free-ABA to ABA-GE were markedly higher indeed in TIG as compared to RR. Since the expression of genes involved in the first committed (oxidation) step of the ABA catabolic pathway (ABAH, CYP707A1) was largely lower in TIG in comparison to RR, it is suggested that reduced ABA-oxidation also contributed to enhance the levels of free-ABA in green basil leaves. Our suggestion is corroborated by the notion that high light affects ABA degradation more than de-novo ABA synthesis (Kraepiel et al., 1994; Talmann, 2004), and that high blue light represses the expression of CYP707A1 (Barrero et al., 2014).
Light attenuation by epidermal anthocyanins represses the biosynthesis of colourless flavonoids
We have found that early genes of the general phenylpropanoid metabolism (PAL and 4CL), as well as early (CHS, CHI, FHT, F3’H) and late (DFR, LDOX) genes of the flavonoid branch pathway are largely over-expressed in RR. However, the concentrations of colourless flavonoids, particularly of luteolin 7-O and quercetin 3-O-derivatives were higher in TIG than in RR, adding further support to recent findings that blue light is particularly effective in promoting the biosynthesis of flavonols (Siipola et al., 2015; Barnes et al., 2016).
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The results of our study offer additional evidence of a negative relation between flavonol and anthocyanin biosynthesis (Noda et al., 2004; Lotkowska et al., 2015; Luo et al., 2016). The synthesis of flavonols and anthocyanins originates indeed from the very same intermediate substrates, i.e. dihydro-flavonols (Davies et al., 2003; Yuan et al., 2016), and FLS or DFR synthesize flavonols or anthocyanins, respectively. In this study, the negligible transcript abundance of DFR in TIG likely favored quercetin 3-O-rutinoside biosynthesis as compared to RR (Tian et al. 2015). The higher expression of the HD-ZIP transcription factor GLABRA 2, which exclusively regulates the expression of ‘late’ genes of anthocyanin biosynthesis, suggests that the competition between flavonol and anthocyanin biosynthesis occurs at the level of the C2-C3 bond in the dihydro-flavonoid skeleton (Tian
et al., 2015; Wang et al., 2015; Yuan et al., 2016).
Conclusions
The attenuation of blue light by epidermal anthocyanins profoundly alters the expression of a suite of light responsive genes, and evokes shade avoidance response. This experiment suggests that the shade avoidance syndrome in red individuals primarily involve genes regulating leaf cell development (Supplementary Fig. S1), and reveals the consequential effects on gas exchange performance. We have shown here that the shade avoidance syndrome in red basil is responsible for the lower mesophyll conductance to CO2. The effects of blue-green light attenuation by epidermal anthocyanins on the leaf functional anatomy has received scarce attention, but the issue is of the greatest significance to reconcile contrasting results about the photosynthetic performance of red vs. green individuals growing in full sunlight.
Our study gives new insights on the molecular events that regulate the biosynthesis of MEP-derived products in red and green individuals. We confirm that in green leaves the MEP pathway mostly sustains VAZ synthesis and de-epoxidation to process effectively the excess of radiant energy. Consequently, green leaves activate routes alternative to de-novo ABA synthesis (through the plastidial MEP pathway) to enhance the levels of free-ABA and tightly regulate stomatal conductance under photoinhibitory light conditions. Data of our study are consistent with, and may help to explain previous observations that high light promotes de-glucosylation of ABA-GE and depress ABA oxidative degradation, but does not affect de-novo ABA synthesis.
We offer evidence of a strong competition between flavonol and anthocyanin biosynthesis. This raises the possibility that constitutively red individuals, while better protected against supernumerary 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477
photons over the visible portion of the solar spectrum, might suffer more than green morphs from an excess of the shortest solar wavelengths, because of a reduced capacity to absorb UV-B radiation and repair UV-B-induced DNA damage. The issue has ecological significance for the successful acclimation of red morphs to high doses of UV-radiation, and open possibilities for new experimentation in respect of the changing UV climate caused by the depletion of stratospheric ozone and rapid climate change.
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Supplementary Data
Table S1. Primers used in this research
Table S2. Transcript abundance of light-responsive genes Table S3. Transcript abundance of MEP genes
Table S4. Transcript abundance of phenylpropanoid genes Figure S1. Light microscopy analysis of red and green basil leaf
Acknowledgments
We thank Dr Matthew Haworth for the critical reading of the manuscript, and Dr Cristina Giordano for performing light microscopy analysis. Work in the authors’ lab. was supported by Ministero dell’Istruzione, dell’Università e della Ricerca of Italy: PRIN 2010-2011 projects “PRO-ROOT” and “TreeCity”, and by Uniser Consortium, Pistoia.
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Table 1. Carboxylation efficiency (Vc,max) based on intercellular (Ci) or
chloroplast (Cc) CO2 concentration, stomatal (gs) and mesophyll
conductance (gm) to CO2, intercellular (Ci) and chloroplast (Cc) CO2
concentration in red (Red Rubin) and green (Tigullio) basil leaves. Measurements were conducted on four-week-old leaves developed at full sunlight. Data are the mean ± SD (n = 5), and those in a row not accompanied by the same letter differ significantly at P < 0.05, using Tukey’s test.
Parameter Red Rubin Tigullio
Vc,max (Ci) (µmol m-2 s-1) 63.2 ± 5.4 b 77.9 ± 7.0 a Vc,max (Cc) (µmol m-2 s-1) 153.9 ± 11.1 a 122.7 ± 12.5 b gs (mmol m-2 s-1) 178.5 ± 11.6 a 104.5 ± 9.2 b gm (mmol m-2 s-1) 62.8 ± 8.8 b 115.8 ± 10.6 a Ci (µmol mol-1) 263.5 ± 11.0 a 218.7 ± 17.4 b Cc (µmol mol-1) 67.6 ± 5.8 b 111.3 ± 10.3 a 744 745 746 747 748 749 750 751 752
Table 2. The concentrations (µmol g-1 DW) of total chlorophyll (Chltot) and total carotenoids (Cartot), and the ratio of Chla to Chl b (Chla/Chlb) in red (Red Rubin, RR) and green (Tigullio, TIG) leaves at the end of a four-week period of exposure to full solar irradiance. Leaves were sampled at 08:30, 12:00, 14:30, and 17:30 h, and data have been pooled together prior to statistical analysis. Data are the mean ± SD (n = 16) and those in a row not accompanied by the same letter differ significantly at the 5% level, using Tukey’s test.
Red Rubin Tigullio
Chltot 5.84 ± 0.83 a 3.66 ± 0.52 b Chla/Chlb 2.08 ± 0.12 b 2.94 ± 0.14 a Cartot 1.14 ± 0.06 b 1.32 ± 0.08 a 753 754 755 756 757 758 759 760 761 762 763
