RESEARCH PAPER
Phenotypic differences determine drought stress responses in
ecotypes of Arundo donax adapted to different environments
Mastaneh Ahrar1,2, Dilyana Doneva3, Massimiliano Tattini4, Cecilia Brunetti5,6, Antonella Gori7,Mirco Rodeghiero8, Georg Wohlfahrt2, Franco Biasioli9, Claudio Varotto1, Francesco Loreto10 and
Violeta Velikova1,3,*
1 Department of Biodiversity and Molecular Ecology, Research and Innovation Centre, Fondazione Edmund Mach, S. Michele all’Adige,
Trento, Italy
2 Institute of Ecology, University of Innsbruck, Innsbruck, Austria
3 Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, Sofia, Bulgaria
4 The National Research Council of Italy (CNR), Institute for Sustainable Plant Protection, Sesto Fiorentino, Florence, Italy 5 The National Research Council of Italy (CNR), Trees and Timber Institute, Sesto Fiorentino, Florence, Italy
6 Department of Plant, Soil and Environmental Sciences, University of Florence, Florence, Italy
7 Department of Agri-Food Production and Environmental Sciences, University of Florence, Florence, Italy
8 Department of Sustainable Agro-Ecosystems and Bioresources, Research and Innovation Centre, Fondazione Edmund Mach, S.
Michele all’Adige, Trento, Italy
9 Department of Food Quality and Nutrition, Volatile Compound Facility, Research and Innovation Centre, Fondazione Edmund Mach, S.
Michele all’Adige, Trento, Italy
10 The National Research Council of Italy (CNR), Department of Biology, Agriculture and Food Science, Rome, Italy
*Correspondence: violet@bio21.bas.bg
Received 16 January 2017; Editorial decision 22 March 2017; Accepted 5 April 2017 Editor: Roland Pieruschka, Forschungszentrum Jülich
Abstract
Arundo donax has been identified as an important biomass and biofuel crop. Yet, there has been little research on photosynthetic and metabolic traits, which sustain the high productivity of A. donax under drought conditions. This study determined phenotypic differences between two A. donax ecotypes coming from stands with contrasting adap-tation to dry climate. We hypothesized that the Bulgarian (BG) ecotype, adapted to drier conditions, exhibits greater drought tolerance than the Italian (IT) ecotype, adapted to a more mesic environment. Under well-watered conditions the BG ecotype was characterized by higher photosynthesis, mesophyll conductance, intrinsic water use efficiency, PSII efficiency, isoprene emission rate and carotenoids, whereas the IT ecotype showed higher levels of hydroxycin-namates. Photosynthesis of water-stressed plants was mainly limited by diffusional resistance to CO2 in BG, and by
biochemistry in IT. Recovery of photosynthesis was more rapid and complete in BG than in IT, which may indicate bet-ter stability of the photosynthetic apparatus associated to enhanced induction of volatile and non-volatile isoprenoids and phenylpropanoid biosynthesis. This study shows that a large phenotypic plasticity among A. donax ecotypes exists, and may be exploited to compensate for the low genetic variability of this species when selecting plant pro-ductivity in constrained environments.
Key words: Arundo donax, Calvin cycle, drought stress, ecotype, isoprene, phenylpropanoids, photosynthesis.
© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com
Abbreviations: AN, net photosynthetic rate; Cc, chloroplast CO2 concentration; Ci, intercellular CO2 concentration; DES, xanthophyll de-epoxidation state; FTSW,
fraction of transpirable soil water; gm, mesophyll conductance to CO2; gs, stomatal conductance to CO2; iWUE, intrinsic water use efficiency; Jmax, maximum rate of
photosynthetic electron transport; NPQ, non-photochemical quenching; qP, photochemical quenching of Chl fluorescence; ROS, reactive oxygen species; Rubisco,
ribulose1,5 bisphosphate carboxylase/oxygenase; RuBP, ribulose 1,5-bisphosphate; TPU, triose phosphate utilization; Vcmax, maximum carboxylation rate; ΦPSII,
quantum yield of PSII photochemistry.
Introduction
Drought stress is one of the major environmental factors affecting plant growth and productivity worldwide. It is pre-dicted that periods of severe drought will significantly increase in the near future, especially as a consequence of intense heat waves (Trnka et al., 2011; Beringer et al., 2011). Decline in photosynthesis is an early response of plants challenged by drought stress (Lawlor and Cornic, 2002; Munns, 2002; Signarbieux and Feller, 2011), as a consequence of diffusive and metabolic limitations (Cornic, 2000; Centritto et al., 2003; Flexas et al., 2004; Chaves et al., 2009, 2011). Reductions in stomatal (gs) and mesophyll (gm) conductance to CO2 mostly constrain photosynthesis at early stages of drought, because of the reduced CO2 availability at Rubisco sites (Loreto et al., 1994; Flexas et al., 2004). Metabolic impairments become progressively more relevant in limiting photosynthesis as drought stress progresses (Long and Bernacchi, 2003; Chaves
et al., 2011). Under severe drought the electron transport rate may largely exceed the demand of the Calvin cycle, and over-supply of excited electrons generates reactive oxygen species (ROS) at large rates, thus exposing plants to photo-oxidative stress (Silva et al., 2010; Hernández et al., 2012).
Plants have evolved a highly integrated network of ‘second-ary’ defense mechanisms aimed at avoiding the generation of ROS, or detoxifying ROS once they are formed. Secondary metabolites may play a primary role in the response mecha-nisms to drought-induced photo-oxidative stress, particularly in fast-growing species evolved in humid areas of the world, in which the suite of morpho-anatomical traits is unable to efficiently limit the flux and the management of excess pho-tons in the leaf (Tattini et al., 2015 and other references therein). Hygrophilic plants usually emit isoprene at high rates under well-watered conditions (Loreto et al., 2014), and isoprene emission is even transiently stimulated at early stages of drought stress imposition (Brüggemann and Schnitzler, 2002; Beckett et al., 2012), or after recovering from drought (Sharkey and Loreto, 1993). Often, reduced water availability does not have notable effects on isoprene emission even under severe drought conditions. Isoprene emission remains stable or shows only small decreases, while photosynthesis is largely depressed (Sharkey and Loreto, 1993; Funk et al., 2004; Pegoraro et al., 2004; Brilli et al., 2007). There is evidence that isoprene biosynthesis is indeed stimulated by reductions in substomatal CO2 concentration and that plants invest increasing amounts of carbon in isoprene biosynthesis when photosynthesis declines to a greater degree than electron transport rate (Brilli et al., 2007; Morfopoulos et al., 2014). Isoprene has been extensively shown to contribute to stress resistance, by regulating ROS formation and improving the stability of thylakoid membranes (Loreto and Velikova, 2001; Vickers et al., 2009a; Velikova et al., 2011, 2012, 2015), and this also includes drought stress (Ryan et al., 2014; Tattini
et al., 2014).
The function of ROS quenchers is generally ascribed to non-volatile isoprenoids, such as carotenoids. β-Carotene and, to a much lesser extent, zeaxanthin have been recently shown to be oxidized by singlet oxygen, and β-carotene oxidation
products may have a role in chloroplast-to-nucleus (retro-grade) signaling, thus further improving resistance to photo-oxidative stress (Ramel et al., 2012). Recent evidence suggests a potential relationship between isoprene and phenylpropa-noid biosynthesis in a range of species (Beckett et al., 2012; Tattini et al., 2014, 2015; Velikova et al., 2016). Suppression of isoprene biosynthesis in poplar significantly repressed the biosynthesis of phenylpropanoids, such as condensed tannins and flavonoids, and exposed plants to more severe oxidative stress than wild-type poplars during transient drought stress (Behnke et al., 2010). Consistently, the biosynthesis of phe-nylpropanoids increased much more in isoprene-emitting than in non-emitting lines of tobacco in response to drought stress, which may in part explain the superior drought stress resistance displayed by isoprene emitters (Ryan et al., 2014; Tattini et al., 2014). Volatile and non-volatile isoprenoids and phenylpropanoids may play complementary ‘antioxidant’ roles depending on the extent to which drought stress con-strains the usage of radiant energy for CO2 assimilation, on both a daily and a seasonal basis (Brunetti et al., 2015; Tattini
et al., 2015). Exploring the combined contribution of isopre-noids and phenylpropaisopre-noids in plant responses to drought stress is a timely research issue in view of projected increases in air temperature coupled with severe, transient or prolonged periods of soil water scarcity in the near future (IPCC 2013).
The predicted climate changes may have particular signifi-cance for the cultivation of strong isoprene emitting crops, such as fast growing plants commonly used for biomass (or biofuel) production (Ashworth et al., 2012, 2013; Loreto and Fineschi, 2015; Porter et al., 2012). Here we studied Arundo
donax, a C3 perennial grass that originated in Asia and spread in the Mediterranean region, USА, China, Australia and Southern Africa (Lewandowski et al., 2003), which is among the most promising biofuel and bioethanol crops. Specifically, we compared the physiological and metabolic adjustements in response to drought stress of two A. donax populations, one from Sesto Fiorentino, Florence, Tuscany (Italy) and the other from Srebarna, Silistra (Bulgaria). The Bulgarian (BG) ecotype experiences yearly warmer air temperature and lower precipation than the Italian (IT) ecotype. Since A. donax is an asexually reproducing species due to seed sterility, it is sug-gested that the two selected plant populations are genetically identical (Ahmad et al., 2008; Mariani et al., 2010), but have evolved phenotypic differences under different environmental conditions, and hereinafter in the text they will be referred to as ‘ecotypes’. We hypothesized that the BG ecotype exhibits greater drought tolerance than the IT ecotype, and that this is associated with phenotypic adjustments. The main goal of the present research was to understand how physiological traits in two A. donax ecotypes contribute to their response to drought stress and ability to recover. Our specific aims were to (1) quantify differences in photosynthesis and pho-tosynthetic limitations, and (2) investigate possible relation-ships between isoprene emission and biosynthesis of other secondary metabolites (carotenoids and phenylpropanoids) in drought-stressed ecotypes.
Materials and methods
Plant material and growth conditionsArundo donax rhizomes were collected from natural habitats in
Sesto Fiorentino, Florence, Italy (IT) (mean summer temperature of ~23 °C and ~800 mm annual precipitation, 43°50′N 11°12′E) and Srebarna, Silistra, Bulgaria (BG) (mean summer temperature of ~25 °C and ~500 mm annual precipitation) (44°06′N 27°04′E). Potted plants were grown in a climatic chamber at 25/20 °C day/ night temperature, 12 h photoperiod, 300 µmol m−2 s−1
photosyn-thetic photon flux density (PPFD), 70–80% relative air humid-ity and 400 µmol mol−1 ambient CO
2 concentration over a 4-week
period. Drought stress was imposed by withholding water, and the fraction of transpirable soil water (FTSW, %) was measured as described in Brilli et al. (2007). Gas exchange and isoprene emis-sion were measured at different stages during the experiment: (1) at control conditions before the onset of drought stress (FTSW=98%), (2) during the drought exposure at mild stress (FTSW=45%), (3) during the drought exposure at severe stress (FTSW=28%), and (4) after re-watering (recovery to 96% of FTSW). The time lapse to reach the severe stress (28% FTSW) was 4–6 weeks in different plants. Plant recovery was assessed after 2 weeks at FTSW=96%, i.e. after the values of stomatal conductance remained stable for 5 days. Measurements were carried out on the fourth fully expanded leaf from the top. The relative leaf water content (RWC, %) was meas-ured as (FW–DW)/(TW–DW)×100, where FW is the fresh weight, DW is the dry weight after oven drying the leaf at 80 °C until con-stant weight is reached, and TW is the turgid weight of the leaf (leaf kept in distilled water for 24 h in the dark).
Growth and biomass measurements
The specific leaf area (SLA, the ratio of leaf area to leaf dry weight, expressed as mm2 mg−1 DW) was measured under control conditions
(98% FTSW) and after re-watering following the drought stress (96% FTSW). Leaf discs were cut from the third and fourth leaf of five different plants. Leaf segment dry weight was obtained after oven drying at 80 °C for 48 h. At the end of the experiment, leaf number, stem height, and stem and leaf weights were measured in control and re-watered plants. The above-ground biomass was calculated by summing the stem and leaf dry weights for each plant.
Gas exchange and chlorophyll fluorescence measurements
Exchanges of CO2 and water vapor were measured with a portable
leaf gas exchange system (LI-6400, Li-Cor, Lincoln, NE, USA), and gas-exchange parameters were calculated with the instrument soft-ware. The middle part of the leaf was placed inside the 6 cm2 gas
exchange cuvette, and exposed to a constant flow (300 µmol s−1) of
synthetic air (21% O2, 79% N2, and 400 μmol mol−1 CO2). During
all measurements, temperature and light in the leaf chamber were maintained at 30 °C and 1000 µmol m−2 s−1 PPFD, respectively.
The relative humidity was set at 45–50%. The intrinsic water use efficiency (iWUE) was calculated as the ratio between net photo-synthetic rate (AN) and stomatal conductance (gs). To parametrize
photosynthetic limitations, the response of photosynthesis to changing intercellular CO2 concentrations (AN–Ci curve) was
ana-lysed (Farquhar et al., 1980). Leaves in the cuvette were exposed to a range of CO2 mole fractions from 50 to 1800 µmol mol−1, and all gas
exchange parameters were recorded after reaching the steady-state for each CO2 concentration, typically 5–10 min after the change in
ambient CO2 concentration (Ca) (Centritto et al. 2003). The method
of Sharkey et al. (2007) was used to calculate the maximum car-boxylation rate allowed by ribulose1,5 bisphosphate carboxylase/ oxygenase (Rubisco) (Vcmax, µmol m−2 s−1), the maximum rate of
photosynthetic electron transport based on NADPH requirement for ribulose 1,5-bisphosphate (RuBP) regeneration (Jmax, µmol m−2
s−1), and the triose phosphate utilization (TPU, µmol m−2 s−1).
Chlorophyll fluorescence was measured by an FMS1 modulated chlorophyll fluorometer (Hansatech Instruments, King’s Lynn, UK). Plants were dark adapted for 20 min prior to the determination of
Fo and Fm (minimum and maximum fluorescence, respectively).
The maximum quantum yield of PSII photochemistry (Fv/Fm) was
determined in dark-adapted leaves after application of a saturat-ing pulse of 0.8 s with 3000 µmol photons m−2 s−1. Then the leaves
were exposed to actinic light of 400 µmol m−2 s−1 in order to obtain
chlorophyll fluorescence in light-adapted leaves. The quantum yield of PSII photochemistry (ΦPSII) was calculated using the formula
ΦPSII=(Fm′–Fs)/Fm′ (Genty et al., 1989), where Fm′ is the maximum
fluorescence in the light-adapted leaves and Fs is the steady-state
fluorescence in the light. The non-photochemical dissipation of absorbed light energy (NPQ) was determined using the equation NPQ=(Fm–Fm′)/Fm′ (Bilger and Björkman, 1991).
Isoprene analysis
Isoprene emission from A. donax leaves was analysed by gas chro-matography–mass spectrometry (GC-MS) as detailed in Beckett
et al. (2012). Briefly, part of the air flow out of the leaf cuvette was
directed into a silicosteel cartridge packed with 200 mg of Tenax (Markes International Ltd, Llantrisant, UK). A volume of 2 l of air at a flow rate of 200 ml min−1 was sampled before removing
the trap. The cartridges were analysed with a Perkin Elmer Clarus 580 gas chromatograph coupled with a Perkin Elmer Clarus 560S mass selective detector (Perkin Elmer Inc., Waltham, MA, USA), as reported in Velikova et al. (2016).
Additionally to GC-MS, isoprene emission was measured online by a high-sensitivity proton transfer reaction mass spectrometer (PTR-MS; Ionicon Analytik GmbH, Innsbruck, Austria). After reaching steady-state photosynthesis, 0.1 l min−1 of the cuvette
outflow was directed to the PTR-MS inlet by a T-connection. The operating parameters of the PTR-MS were set at 80 °C drift tube temperature, 550 V drift voltage and 2 mbar drift tube pressure, cor-responding to an electric field strength (E)/gas number density (N) ratio of about 140 Td (where 1 Td=10−17 V cm2). The ion signal
at m/z 69 corresponded to isoprene. Calibration of the instrument was performed by using a standard gas mixture (GCU; Ionicon Analytik GmbH, Innsbruck, Austria). The emission rate was calcu-lated as described in Ahrar et al. (2015). Carbon lost as isoprene (%) was calculated as the ratio of isoprene emission to photosynthesis. The internal concentration of isoprene was calculated as in Singsaas
et al. (1997) to take into account drought-induced changes in
stoma-tal conductance.
Analysis of carotenoids and phenylpropanoids
Carotenoids were identified and quantified as reported in Tattini
et al. (2014). Lyophilized leaf material was extracted with 2 × 5 ml
acetone (added with 0.5 g l−1 CaCO
3) and injected (15 µl) into a
Perkin Elmer Flexar liquid chromatograph equipped with a quater-nary 200Q/410 pump and an LC 200 diode array detector (DAD) (all from Perkin Elmer, Branford, CT, USA). Photosynthetic pig-ments 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%
CH3CN/methanol (95/5 with 0.05% of triethylamine) to 100%
methanol/ethyl acetate (6.8/3.2). Xanthophyll cycle pigments (vio-laxanthin, anteraxanthin, and zeaxanthin, collectively named VAZ), eoxanthin, 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, respectively, as previously reported (Tattini et al., 2014).
Individual phenylpropanoids were extracted and purified follow-ing the protocol of Tattini et al. (2014). Briefly, 100 mg lyophilized leaf material was extracted with 3 × 2 ml of 75% C2H5OH/H2O
(pH was adjusted to 2.5 with formic acid). The supernatant was
partitioned with 2 × 6 ml n-hexane, reduced to dryness and rinsed with 0.5 ml of CH3OH/H2O, and a 10 µl aliquot injected into the
Perkin Elmer liquid chromatography unit reported above. The sepa-ration of phenylpropanoids was carried out by using a 250 × 4.6 mm Polaris C-18 Ether column (5 µm) kept at 30 °C. The eluent was H2O
(with addition of 0.1% triethylamine and pH adjusted to 2.5 with H3PO4) and CH3CN. Phenylpropanoids were separated using a
lin-ear gradient solvent system as reported in Tattini et al. (2004) and identified using retention times and UV-spectral characteristics of authentic standards (Extrasynthese), as well as by mass spectromet-ric data (details given in Tattini et al., 2014).
Statistical analyses
Data were analysed using repeated measures with ANOVA (Girden, 1992). Ecotype (ECO) was the between-subjects factor, and water treatment (WT) was the within-subjects factor. Gas exchange, PSII performance, and isoprene emission were estimated on plants at 98, 45, and 28% of FTSW, before and during drought stress, and at 96% FSTW during re-watering. Biochemical analyses were performed on plants at 98% (before stress), 28% (during severe stress) and 96% FTSW (during re-watering). Differences between ecotypes at each water treatment were analysed using Student’s t-test. Asterisks indi-cate significant differences: *P<0.05, **P<0.001, ***P<0.0001.
Results
Effect of drought on gas exchange and PSII performance
The BG and the IT ecotypes of A. donax examined in our study differed significantly for the majority of the photosyn-thesis-related traits (Table 1; Figs 1 and 2). This resulted from both constitutively different gas exchange performance (i.e. in plants under well-watered conditions, FTSW of 98%) and significant ecotype×water-treatment interaction effects.
Photosynthesis was higher in BG than that in IT under well-watered conditions, and drought negatively affected AN to a greater extent in IT (–84%) than in BG (–69%) (Fig. 1A). Photosynthesis recovered similarly and incompletely in BG (67%) and IT (62%) during re-watering. Stomatal conduct-ance was not significantly different in well-watered ecotypes (Fig. 1B). However, drought stress significantly inhibited gs of both ecotypes, and gs of IT was significantly more affected than in BG, so that the intrinsic water use efficiency (iWUE) became much higher in BG than in IT under severe stress (Fig. 1D). Recovery of gs was incomplete and very similar in the two ecotypes (Fig. 1B). Notably, BG and IT also dis-played large differences in the gm to CO2, which was higher in BG than in IT, irrespective of the water treatment (Fig. 1C). Differences in gm did not correspond to variations in the chlo-roplast CO2 concentration (Cc) under well-watered conditions (both before stress and after re-watering), whereas Cc was higher in BG than in IT at mild and severe drought (Fig. 1F). In contrast, Ci differed much less than Cc between examined ecotypes, and only significantly dropped in IT exposed to severe drought (Fig. 1E).
In order to better understand the factors limiting pho-tosynthesis in A. donax ecotypes exposed to drought, the parameters derived from the responses of AN to changes in Ci (AN–Ci curves) were analysed. Apparent maximum carboxy-lation rate (Vcmax, Fig. 1G) and maximum electron transport
rate (Jmax, Fig. 1H) did not vary between ecotypes under well-watered conditions, but declined more and recovered less in IT than in BG during drought stress progression and re-watering, respectively. A similar (though less evident) trend was also observed for limitation of photosynthesis by triose-phosphate use (TPU, Fig. 1I).
The photochemical quenching of Chl fluorescence (qP, Fig. 2A) and the actual efficiency of PSII photochemistry (ΦPSII, Fig. 2B) were constitutively greater in BG than in IT, Table 1. Summary of two-way analysis of variance (ANOVA) for
the effects of ecotype (ECO) and water treatment (WT), with their interaction factor (ECO×WT) on physiological- and biochemical-related traits in Arundo donax plants
Abbreviations as shown in the text. Data were analysed using repeated measures with ANOVA. Ecotype (ECO) was the between-subjects factor, and water treatment (WT) was the within-between-subjects factor. Gas exchange and isoprene emission were estimated on plants at 98, 45, and 28% of fraction of transpirable soil water (FTSW), before and during drought stress, and at 96% FSTW during re-watering (total degree of freedom, d.f.=39). Chlorophyll fluorescence-derived parameters and biochemical analyses were performed on plants at 98% (before stress), 28% (during severe stress), and 96% (during re-watering) FTSW (d.f.=17). *P<0.05; **P<0.001; ***P<0.0001. n.s., not significant. ETR, electron transport rate.
Trait FECO FWT FECO×WT
AN 139.1*** 219.3*** 0.9 n.s. gs 16.7*** 320.9*** 9.6** gm 125.5*** 89.1*** 12.1** Ci 0.7 n.s. 11.2** 12.9** Cc 14.9*** 52.3*** 5.4* iWUE 22.3*** 124.4*** 17.1*** Vcmax 66.1*** 243.5*** 18.2*** Jmax 61.8*** 178.6*** 5.6* TPU 28.9*** 78.1*** 2.8 n.s. Fv/Fm 0.8 n.s. 12.4** 0.3 n.s. ΦPSII 49.7*** 73.4*** 2.3 n.s. qP 27.3*** 49.7*** 1.6 n.s. NPQ 41.6*** 38.9*** 5.9* ETR 46.4*** 74.8*** 1.7 n.s. Isoprene 113.0*** 119.8*** 11.8** Chltot 0.5 n.s. 66.4*** 8.9* Cartot 8.5* 70.0*** 18.6**
Cartot/Chltot 3.4 n.s. 20.7*** 0.7 n.s.
Neoxanthin 10.1** 64.4*** 15.7** Violaxanthin (V) 4.0 n.s. 95.9*** 1.9 n.s. Antheraxanthin (A) 9.1* 79.9*** 12.8** Zeaxanthin (Z) 81.1*** 219.7*** 59.4*** Lutein 1.6 n.s. 13.2** 5.9* β-Carotene 32.1*** 59.1*** 36.1*** VAZ/Chltot 3.5 n.s. 29.4*** 5.7* DES=(0.5A+Z)/(VAZ) 4.2 n.s. 198.5*** 21.6*** Ferulic acid 26.8*** 0.2 n.s. 14.1** Caffeic acid 19.8*** 22.4*** 24.4*** Apigenin glycosides 41.5*** 11.3** 10.4** Luteolin glycosides 4.0 n.s. 85.9*** 5.2* Total phenylpropanoids 3.0 n.s. 65.6*** 10.7**
and this difference was observed also when both qP and ΦPSII declined because of severe drought, and when the parameters slightly recovered after re-watering (Fig. 2A, B). The abil-ity to dissipate thermal energy, estimated by the non-photo-chemical quenching of Chl fluorescence (NPQ, Fig. 2C), was significantly higher in IT than in BG under both well-watered and drought-stressed conditions, and did not differ signifi-cantly between ecotypes after re-watering.
Effect of drought on isoprene, photosynthetic pigments, and phenylpropanoids
The BG ecotype differed strongly from the IT ecotype in the rates of isoprene emission (Table 1). Isoprene emission, which was higher in well-watered BG than IT plants, either significantly increased (in BG) or remained constant (in IT)
when drought was mild (Fig. 3A). However, isoprene emis-sion was severely inhibited in both ecotypes when drought was severe, and recovered after re-watering only in BG. The internal concentration of isoprene increased during drought stress progression in both ecotypes, and declined again after re-watering, when it became significantly higher in BG than in IT (Fig. 3B).
The concentrations of photosynthetic pigments under-went significant ecotype×water-treatment interaction effects (Fig. 4). Chlorophyll declined more in response to severe drought, but partially recovered in BG, whereas the drop of chlorophyll was more permanent in re-watered IT (Fig. 4A). Carotenoids, which were in greater concentration in well-watered BG than IT, declined to a significantly greater degree in BG than IT because of severe drought (Fig. 4C), mostly because of large decreases in β-carotene (Fig. 4G) and
Fig. 1. Photosynthesis (AN, A), stomatal conductance (gs, B), mesophyll conductance to CO2 (gm, C), intrinsic water use efficiency (iWUE, D), intercellular
CO2 concentration (Ci, E), chloroplast CO2 concentration (Cc, F), apparent maximum carboxylation rate (Vcmax, G), maximum rate of photosynthetic
electron transport (Jmax, H), and limitation of photosynthesis by triose phosphate use (TPU, I) in BG (open bars) and IT (grey bars) ecotypes of A. donax.
Measurements were carried out in well-watered plants, during drought stress and after re-watering, as indicated by the fraction of transpirable soil water. Data are means±SD (n=5). Analysis of variance (ANOVA) with between- and within-subject factors (summary given in Table 1) was carried out to assess statistical significance of mean differences. Asterisks denote significant differences (P<0.05) between BG and IT ecotypes at each fraction of transpirable soil water (FTSW): *P<0.05; **P<0.001; ***P<0.0001.
especially lutein (Fig. 4E) concentrations. β-Carotene did not recover after re-watering in BG, and remained unchanged in IT. The level of lutein was almost restored in re-watered BG,
but it was lower in re-watered IT compared with pre-stress values. The concentration of violaxanthin (Fig. 4D) and neoxanthin (Fig. 4I) also decreased significantly in response to drought, in both ecotypes. However, the concentration of xanthophylls dropped less than chlorophyll during drought stress, which resulted in an increase of xanthophylls on a chlorophyll basis, especially in BG (Fig. 4B). Drought stress promoted xanthophyll de-epoxidation (DES, Fig. 4J), par-ticularly in IT. The greater biosynthesis of antheraxanthin (Fig. 4F), and particularly of zeaxanthin (Fig. 4H) in IT than in BG was not accompanied by similar differences in the concentration of violaxanthin, which was equally reduced in the two ecotypes during sever stress and after recovery. Xanthophyll-cycle pigments were mostly epoxized by the end of re-watering in both BG and IT, reaching pre-stress values (Fig. 4J).
Hydroxycinnamic acid derivatives were in higher con-centration in IT than in BG under well-watered conditions, whereas the opposite was observed for the concentration of
Fig. 2. Photochemical quenching (qP, A), actual efficiency of PSII
photochemistry (ΦPSII, B), and non-photochemical quenching (NPQ, C) in
BG (open bars) and IT (grey bars) ecotypes of A. donax. Measurements were carried out in well-watered plants, during drought stress and after re-watering, as indicated by the fraction of transpirable soil water. Data are means±SD (n=5). Statistical treatment of data is given in Table 1. Asterisks denote significant differences (P<0.05) between BG and IT ecotypes at each fraction of transpirable soil water (FTSW): *P<0.05, **P<0.001, ***P<0.0001.
Fig. 3. The isoprene emission rate (A) and the internal isoprene
concentration (B) in BG (open bars) and IT (grey bars) ecotypes of A. donax. Measurements were carried out in well-watered plants, during drought stress and after re-watering, as indicated by the fraction of transpirable soil water. Data are means±SD (n=5). Statistical treatment of data is given in Table 1. Asterisks denote significant differences (P<0.05) between BG and IT ecotypes at each fraction of transpirable soil water (FTSW): *P<0.05, **P<0.001, ***P<0.0001.
flavonoids (Fig. 5). Overall the concentration of hydroxycin-namates (Fig. 5A, B) varied less than the concentration of flavonoids (Fig. 5C, D) because of drought stress. Among hydroxycinnamates, the concentration of caffeic acid deriva-tives increased particularly in drought-stressed BG, while it did not vary in IT (Fig. 5B). Among the flavonoids, the biosynthesis of luteolin derivatives was greatly enhanced by drought stress in both ecotypes (Fig. 5C), whereas apigenin derivatives were only enhanced by the stress in the BG ecotype (Fig. 5D). Re-watering largely restored the concentration of phenylpropanoids observed before stress.
Plant morphology and biomass accumulation
Changes of morphological parameters (stem height, leaf number, SLA) and biomass accumulation were followed in plants that were not stressed or recovered from stress. No sig-nificant differences in either of these parameters between BG and IT ecotypes were observed following the drought-stress treatment (Supplementary Fig. S1 at JXB online), which indi-cates that the stress was not sufficiently acute to impair plant growth.
Discussion
Ecotypes of A. donax are phenotypically different under well-watered conditions
The two ecotypes of A. donax that were investigated in this study showed different physiological and metabolic features when grown under well-watered conditions. Photosynthesis was significantly higher in the BG than in the IT ecotype. Interestingly, gm was significantly higher in the BG ecotype, whereas no differences between the two ecotypes in terms of
gs, Ci, and Cc were observed. There is evidence that the meso-phyll diffusion conductance strongly depends on leaf ana-tomical and structural features (Tosens et al., 2012; Adachi
et al., 2013; Xiong et al., 2017). The two ecotypes did not show any differences in their foliar anatomical structure (data not shown). These were not expected, as it was demonstrated that drought stress did not cause any change in leaf anatomy of plants belonging to the Arundinoideae family (Velikova
et al., 2016). Observed differences in gm could be due to cell wall thickness or other parameters that were not measured. Indeed, gm mostly correlates with chloroplast exposed surface to leaf area ratio and cell wall thickness (Evans and Loreto, 2000; Tomás et al., 2013) Alternatively, different levels of aquaporins, or different concentrations of carbonic anhy-drase in the stroma, could contribute to set different gm in BG and IT ecotypes, as both are involved in the regulation CO2 transfer toward chloroplasts (Perez-Martin et al., 2014).
iWUE is an important eco-physiological trait under drought conditions, which can provide a reliable assessment of how effectively water is saved by plants coping with drought stress (Medrano et al., 2009). In our study, iWUE of well-watered plants was lower in IT than in BG, suggesting that the ecotype adapted to the drier environment developed a more efficient water saving strategy. However, generally adaptation to dry
environments involves reduced stomatal aperture, reduced mesophyll conductance, and reduced photosynthesis. The conductances of BG leaves were similar and often higher than those of IT leaves. Therefore, the higher iWUE simply indicates an inherently higher capacity of photosynthesis of the BG ecotype under non-stressful conditions. The reported iWUE values are similar to those observed by Nackley et al. (2014) in a greenhouse experiment. However, when compared with the recent report of Webster et al. (2016) on naturally occurring A. donax stands in southern Portugal, the ecotypes tested here had lower iWUE under control conditions, pos-sibly due to their high gs. The BG ecotype was also character-ized by higher efficiency of PSII photochemistry and lower NPQ under well-watered conditions. This is in line with the better photosynthetic performance observed here, and with a lower need to dissipate light energy non-photochemically.
The higher levels of isoprenoids (isoprene, antheraxan-thin and especially β-carotene) might also indicate a higher activity of the methylerythritol 4-phosphate (MEP) path-way in the BG ecotype (Cazzonelli and Pogson, 2010). In unstressed conditions, the same relative carbon investment into isoprene was made by the two ecotypes, corresponding to about 0.4% of the photosynthetic carbon. However, in absolute terms, more carbon was allocated to the MEP path-way of BG plants. We interpret this as a constitutive trait that may provide rapid protection against occasional stresses. We know that the BG ecotype grows in a more challenging envi-ronment compared with the IT ecotype (see ‘Introduction’). Thus, the higher biosynthesis of isoprenoids may be adaptive, protecting the photosynthetic apparatus when unfavorable conditions develop (Velikova et al., 2012). In contrast, the IT ecotype seems to invest more into phenylpropanoids, namely hydroxycinnamic acids. Since hydroxycinnamates are well known powerful antioxidants with diverse physiological roles (for review, Teixeira et al., 2013; Macoy et al., 2015), we again interpret their higher presence in IT as an adaptive trait, per-haps an alternative to isoprenoids. The switch between the two protecting mechanisms might be related to their biosyn-thesis and concentration in the leaf mesophyll. Perhaps the BG ecotype has developed a better strategy to protect leaf chloroplasts and the photosynthetic apparatus, whereas the IT ecotype must develop antioxidants that preserve the entire leaf integrity against different stresses.
A. donax ecotypes display different strategies to cope
with drought stress
Indeed, the studied ecotypes also exhibited differences in their responses to drought. Photosynthesis was slightly, but significantly, less affected by mild drought in the BG than in the IT ecotype. Photosynthesis inhibition at earlier stages of drought was mainly due to reduced stomatal conductance in both ecotypes, in turn leading to diffusive limitations asso-ciated with reduced availability of CO2 at the carboxylation sites (Flexas et al., 2004; Grassi and Magnani, 2005; Erismann
et al., 2008; Chaves et al., 2009). In fact, the Ci reduction in the leaves of both ecotypes under mild drought, albeit not statistically significant, was consistent with stomatal closure
Fig. 4. The concentrations of total chlorophyll (A), the concentration of xanthophyll pigments relative to total chlorophyll (B), total (C) and individual
carotenoids (D–I), and the xanthophyll de-epoxidation state (J) in BG (open bars) and IT (grey bars) ecotypes of A. donax. Measurements were carried out in well-watered plants, during drought stress and after re-watering, as indicated by the fraction of transpirable soil water. Data are means±SD (n=5). Statistical treatment of data is given in Table 1. Asterisks denote significant differences (P<0.05) between BG and IT ecotypes at each fraction of transpirable soil water (FTSW): *P<0.05, **P<0.001, ***P<0.0001.
inducing increased diffusive limitations to CO2 entry, and suggested no onset of biochemical limitations (Cornic, 2000). A further reduction of Ci was only observed in BG leaves under severe drought stress, consistent with a progres-sive control of photosynthesis by diffuprogres-sive limitation. On the other hand, in severely drought-stressed IT leaves, stomatal closure did not cause a further reduction of Ci, which may indicate the onset of metabolic limitations of photosynthesis (Lawlor and Cornic, 2002; Centritto et al., 2003), and a lower adaptation of this ecotype to drought. Metabolic impairment of photosynthesis is often seen in plants that are not adapted to drought (Ashraf et al., 2004; Ashraf and Harris, 2013). It may be caused by a decrease in Rubisco carboxylation (Parry
et al., 2002) or by reduced regeneration of RUBP (Medrano
et al., 2002). In fact, our A. donax ecotypes exhibited simi-lar trends of changes in photosynthetic parameters associ-ated with photosynthetic limitations. Both ecotypes showed reduced Vcmax, Jmax and TPU under drought, and the reduc-tion was particularly strong in severely stressed leaves of the IT ecotype. Vcmax reduction indicates substantial reduction of Rubisco activity (Centritto et al., 2003). Jmax reduction suggests lower RUBP regeneration and energy-limited pho-tosynthesis. TPU reduction indicates accumulation of triose phosphate in the chloroplasts, which in turn decreases the availability of inorganic phosphate to the chloroplasts (Foyer and Noctor, 2000; Drozdova et al., 2004).
In the present study, iWUE increased in both ecotypes with the onset of drought, which indicates a strong conservative
control of stomatal conductance on the water losses by tran-spiration (Ma et al., 2014; Velikova et al., 2016). The values of iWUE observed under drought in A. donax were higher than in other herbaceous species (25–70 µmol mol−1) (Medrano et al., 2009), especially in the case of BG, which highlights the potential of A. donax to cope with soil water deficit and withstand large changes of water availability through stoma-tal closure. A very tight control of the water resource in A.
donax was also previously reported (Sánchez et al., 2015). When the drought became severe, iWUE increased strongly only in the BG ecotype, suggesting a better strategy of water use (Bacelar et al., 2012), and a better adaptation to reduced water supply in this ecotype (Ares et al., 2000; Chaves et al., 2009).
The significant stimulation of isoprene emission under mild drought in the BG ecotype could indicate activation of the MEP pathway for protection of photosynthesis from drought-induced ROS (Fig. 3A). Our results are consistent with previous studies with transgenic plants (Behnke et al., 2007; Ryan et al., 2014; Tattini et al., 2014) and support the general antioxidant role of isoprene under abiotic stress (Loreto and Velikova, 2001; Vickers et al., 2009b; Velikova
et al., 2011; Selmar and Kleinwächter, 2013). Under severe drought conditions, isoprene emission dropped significantly, similar to photosynthesis. Generally, the reduction of iso-prene emission under drought is mainly the result of limita-tion in carbon and energy supply through photosynthesis for isoprene biosynthesis (Brilli et al., 2007; Fortunati et al., 2008;
Fig. 5. The concentrations of hydroxycinnamic acid (A, B) and flavonoid (C, D) derivatives in BG (open bars) and IT (grey bars) ecotypes of A. donax.
Measurements were carried out in well-watered plants, during drought stress and after re-watering, as indicated by the fraction of transpirable soil water. Data are means±SD (n=5). Statistical treatment of data is given in Table 1. Asterisks denote significant differences (P<0.05) between BG and IT ecotypes at each fraction of transpirable soil water (FTSW): *P<0.05, **P<0.001, ***P<0.0001.
Tattini et al., 2014). Alternative carbon sources such as xylem-transported glucose (Kreuzwieser et al., 2002; Schnitzler
et al., 2004), chloroplastic starch (Karl et al., 2002) or refixa-tion of CO2 generated by light respiration (Anderson et al., 1998; Loreto et al., 2004) can contribute to provide carbon for isoprene when photosynthesis is totally inhibited. However, in the case of the Arundo ecotypes examined here, photosyn-thesis was not completely suppressed under drought, and we argue that carbon sources other than photosynthesis might have not been activated for isoprene biosynthesis. In fact, the isoprene emission drop may have been largely caused by stomatal closure, and isoprene production might have con-tinued at high rates, as the calculated internal concentration of isoprene was very high in severely stressed leaves of both ecotypes.
Severe drought also caused substantial changes in carot-enoids (Fig. 4) and penylpropanoids (Fig. 5) of both A.
donax ecotypes. The IT ecotype exhibited a stronger increase
of antheraxanthin and zeaxanthin and a consequently much higher deepoxidation of xanthophylls, suggesting that this ecotype was exposed to higher oxidative pressure (Munné-Bosch and Peñuelas, 2004). The stronger increase of zeaxan-thin concentration in the IT compared with the BG ecotype could contribute to the quenching of the singlet oxygen excited state of chlorophyll and to NPQ formation (Havaux
et al., 2007). Moreover, higher zeaxanthin in the IT ecotype may have increased the rigidity of thylakoid membranes, and reduced peroxidative damage under drought stress (Havaux
et al., 1996, 2007). β-Carotene decreased much more in the BG than in the IT drought-stressed ecotype. This result is at odds with the proposed overall stimulation of the MEP pathway in the drought-resistant BG ecotype. It may indicate a preferen-tial use (and oxidation) of β-carotene as an antioxidant dur-ing drought, especially when the stress is severe (Telfer, 2002). In the light of the hypothesized role of isoprene in providing protection against lipid peroxidation as a result of oxidative damage, and facilitating membrane stabilization during abi-otic stress (Loreto and Velikova, 2001; Vickers et al., 2009a; Velikova et al., 2011), it could be surmised that zeaxanthin has a predominant role in the ecotype with lower ability to pro-duce isoprene (IT), and that the two ecotypes have contrasting strategies of using their isoprenoids. The negative correlation between isoprene—zeaxanthin and isoprene—NPQ was seen in plants with genetically downregulated isoprene emission (Behnke et al., 2009; Velikova et al., 2015). Native non-iso-prene emitting species are also characterized by higher levels of zeaxanthin and NPQ (Velikova et al., 2016). As a general hypothesis, volatile isoprenoids might protect photosynthesis under mild stress while xanthophylls might be more effective when photosynthesis is largely inhibited and the oxidation potential is higher (Beckett et al., 2012). However, the pool of isoprene increased in both ecotypes under severe stress, whereas the pool of xantophylls expressed on a chlorophyll basis (VAZ/Chltot) was overall significantly higher in the BG than in the IT drought-stressed ecotype, suggesting that the improved resistance to drought in BG comes from coopera-tion between volatile and non-volatile isoprenoids.
The concentration of phenylpropanoids was significantly stimulated by severe drought in both ecotypes, but flavonoids
were specifically more stimulated in the BG than in the IT ecotype. The biosynthesis of flavonoids is upregulated under the same conditions that activate antioxidant enzymes, con-firming their ROS-scavenging function when plants are exposed to prolonged stress conditions (Agati et al., 2011). Our results suggest that accumulation of flavonoids may favor drought stress tolerance in A. donax plants (Hernández
et al., 2004) in cooperation with other secondary metabolites, namely isoprenoids (Tattini et al., 2004). The higher accumu-lation of flavonoids and hydroxycinnamic acids, especially in the BG ecotype, could also act as photoprotection, reducing the excess light absorbed by drought-stressed leaves.
Phenotypic traits underlie different recovery from drought stress in the A. donax ecotypes
Generally, the BG ecotype showed better recovery after re-watering than the IT ecotype. Photosynthesis did not recover completely in either of the ecotypes, and we attribute this effect to long lasting diffusive limitations, as both conduct-ances (gs and gm) and the concentration of CO2 at the car-boxylation site (Cc) remained largely reduced in the two ecotypes. This indicates that stomatal and mesophyll limita-tions might have a strong impact in limiting photosynthesis of A. donax under natural conditions and recurrent drought stress. However, photosynthetic parameters (Vcmax, Jmax and TPU) reached control values only in BG re-watered plants. It is thus suggested that complete recovery from drought only occurs when photosynthetic limitations are diffusional (i.e. in the BG ecotype, where we might have seen complete recovery of photosynthesis once diffusive limitations were overcome), not when a more permanent damage to the photosynthetic machinery occurs (as in the IT ecotype) (Flexas and Medrano, 2002; Flexas et al., 2004). We surmise that metabolic limita-tions were prevented in BG by the large investment in anti-oxidants, and also suggest that volatile and non-volatile isoprenoids might have played an important role in this respect. Isoprene emission and internal pools almost reached the pre-stressed values in re-watered BG, but remained largely inhibited in IT. Higher Jmax in BG after recovery could indi-cate a higher rate of linear electron transport for isoprenoid biosynthesis (Dani et al., 2014). It has been shown that the presence of isoprene enhances the fluidity of thylakoid mem-branes and flow of electrons along photosystems (PS) I and II, especially under stress (Velikova et al., 2011), but also under more physiological conditions (Pollastri et al., 2014). Therefore, we suggest that the observed differences between both ecotypes could be due to a more efficient electron trans-port rate in the BG ecotype with higher isoprene emission under mild drought and recovery (Lawlor and Cornic, 2002; Wentworth et al., 2006; Ashraf and Harris, 2013).
Conclusions
In conclusion, our data show that photosynthetic inhibition of drought-stressed A. donax is largely due to a strong reduc-tion of stomatal and mesophyll conductance, which limits CO2 entry, down-regulates Rubisco and the electron transport
rate, but allows iWUE to increase and resistance to prolonged stress to occur. Analysis of ecotypes adapted to different cli-mate suggests that the metabolic impairment of photosyn-thesis only occurs in the ecotype adapted to mesic conditions (IT). In the BG ecotype, which was adapted to harsher condi-tions, enhanced biosynthesis of isoprenoids might have con-tributed to protecting photosynthetic membranes, avoiding the metabolic damage under severe drought and allowing for a more complete and rapid recovery of parameters deter-mining potential photosynthesis after re-watering (Tattini
et al., 2014, 2015; Brunetti et al., 2015). Considering (i) the increasing global limitation in water resources, (ii) the gen-eral interest in cultivating A. donax and other biofuel crops on marginal land (Pilu et al., 2013), and (iii) the low genetic variability due to Arundo sterility, which makes conventional breeding of new genotypes or varieties impossible (Ahmad
et al., 2008; Mariani et al., 2010; Pilu et al., 2012, 2013), exploring ecotypes with superior performances in drought-stress conditions, possibly associated with higher investments in isoprenoids, might improve the cultivation of A. donax in regions with limited water resources.
Supplementary data
Supplementary data are available at JXB online.
Fig. S1. Stem height, leaf number, specific leaf area and total biomass accumulation in BG and IT ecotypes of
A. donax.
Acknowledgements
This work was supported by the Autonomous Province of Trento (Italy) through the MAN-VIP project (Team 2011 Call approved with provincial government resolution no. 2902 on 14 December 2010; MA and VV) and core funding of the Ecogenomics group (CV). Further support came from the EU FP7 project (ECLAIRE contract 282910), and partly from the Ministry of Education and Science (Bulgaria), contract D01-168/14.07.2014.
References
Adachi S, Nakae T, Uchida M, et al. 2013. The mesophyll anatomy
enhancing CO2 diffusion is a key trait for improving rice photosynthesis.
Journal of Experimental Botany 64, 1061–1072.
Agati G, Biricolti S, Guidi L, Ferrini F, Fini A, Tattini M. 2011. The
biosynthesis of flavonoids is enhanced similarly by UV radiation and root zone salinity in L. vulgare leaves. Journal of Plant Physiology 168,
204–212.
Ahmad R, Liow P, Spencer DF, Jasieniuk M. 2008. Molecular evidence
for a single genetic clone of invasive Arundo donax in the United States. Aquatic Botany 88, 113–120.
Ahrar M, Doneva D, Koleva D, et al. 2015. Isoprene emission in
the monocot Arundineae tribe in relation to functional and structural organization of the photosynthetic apparatus. Environmental and Experimental Botany 119, 87–95.
Anderson MD, Che P, Song J, Nikolau BJ, Wurtele ES. 1998.
3-Methylcrotonyl-coenzyme A carboxylase is a component of the mitochondrial leucine catabolic pathway in plants. Plant Physiology 118,
1127–1138.
Ares A, Fownes JH, Sun W. 2000. Genetic differentiation of intrinsic
water-use efficiency in the Hawaiian native Acacia koa. International Journal of Plant Science 161, 909–915.
Ashraf M, Ashraf MY, Khaliq A, Rha ES. 2004. Growth and leaf gas
exchange characteristics in Dalbergia sissoo Roxb. and D. latifolia Roxb. under water deficit. Photosynthetica 42, 157–160.
Ashraf M, Harris PJC. 2013. Photosynthesis under stressful
environments: An overview. Photosynthetica 51, 163–190.
Ashworth K, Folberth G, Hewitt CN, Wild O. 2012. Impacts of
near-future cultivation of biofuel feedstocks on atmospheric composition and local air quality. Atmospheric Chemistry and Physics 12, 919–939. Ashworth K, Wild O, Hewitt CN. 2013. Impacts of biofuel cultivation on
mortality and crop yields. Nature Climate Change 3, 492–496.
Bacelar EL, Moutinho-Pereira JM, Gonçalves BM, Brito CV, Gomes-Laranjo J, Ferreira HM, Correia CM. 2012. Water use strategies of
plants under drought conditions. In: Aroca R, ed. Plant responses to drought stress. From morphological to molecular features. New York: Springer Verlag, 145–170.
Beckett M, Loreto F, Velikova V, Brunetti C, Di Ferdinando M, Tattini M, Calfapietra C, Farrant JM. 2012. Photosynthetic limitations
and volatile and non-volatile isoprenoids in the poikilochlorophyllous resurrection plant Xerophyta humilis during dehydration and rehydration. Plant, Cell & Environment 35, 2061–2074.
Behnke K, Ehlting B, Teuber M, Bauerfeind M, Louis S, Hänsch R, Polle A, Bohlmann J, Schnitzler JP. 2007. Transgenic, non-isoprene
emitting poplars don’t like it hot. The Plant Journal 51, 485–499. Behnke K, Kleist E, Uerlings R, Wildt J, Rennenberg H, Schnitzler JP. 2009. RNAi-mediated suppression of isoprene biosynthesis in hybrid
poplar impacts ozone tolerance. Tree Physiology 29, 725–736. Behnke K, Loivamäki M, Zimmer I, Rennenberg H, Schnitzler JP, Louis S. 2010. Isoprene emission protects photosynthesis in sunfleck
exposed Grey poplar. Photosynthesis Research 104, 5–17.
Beringer T, Lucht W, Schaphoff S. 2011. Bioenergy production potential
of global biomass plantations under environmental and agricultural constraints. Global Change Biology Bioenergy 3, 299–312.
Bilger W, Björkman O. 1991. Temperature dependence of violaxanthin
de-epoxidation and non-photochemical fluorescence quenching in intact leaves of Gossypium hirsutum L. and Malva parviflora L. Planta 184,
226–234.
Brilli F, Barta C, Fortunati A, Lerdau M, Loreto F, Centritto M. 2007.
Response of isoprene emission and carbon metabolism to drought in white poplar (Populus alba) saplings. The New Phytologist 175, 244–254. Brüggemann N, Schnitzler JP. 2002. Comparison of isoprene emission,
intercellular isoprene concentration and photosynthetic performance in water-limited oak (Quercus pubescens Willd. and Quercus robur L.) saplings. Plant Biology 4, 456–463.
Brunetti C, Guidi L, Sebastiani F, Tattini M. 2015. Isoprenoids and
phenylpropanoids are key components of the antioxidant defense system of plants facing severe excess light stress. Environmental and Experimental Botany 119, 54–62.
Cazzonelli CI, Pogson BJ. 2010. Source to sink: regulation of carotenoid
biosynthesis in plants. Trends in Plant Science 15, 266–274.
Centritto M, Loreto F, Chartzoulakis K. 2003. The use of low [CO2]
to estimate diffusional and non-diffusional limitations of photosynthetic capacity of salt-stressed olive saplings. Plant, Cell & Environment 26,
585–594.
Chaves MM, Costa JM, Saibo NJM. 2011. Recent advances in
photosynthesis under drought and salinity. Advances in Botanical Research 57, 49–104.
Chaves MM, Flexas J, Pinheiro C. 2009. Photosynthesis under drought
and salt stress: regulation mechanisms from whole plant to cell. Annals of Botany 103, 551–560.
Cornic G. 2000. Drought stress inhibits photosynthesis by decreasing
stomatal aperture – not by affecting. Trends in Plant Science 5, 187–188. Dani KG, Jamie IM, Prentice IC, Atwell BJ. 2014. Increased ratio of
electron transport to net assimilation rate supports elevated isoprenoid emission rate in eucalypts under drought. Plant Physiology 166,
1059–1072.
Drozdova IS, Pustovoitova TN, Dzhibladze TG, Barabanshchikova NS, Zhdanova NE, Maevskaya SN, Bukhov NG. 2004. Endogenous
control of photosynthetic activity during progressive drought: influence of final products of photosynthesis. Russian Journal of Plant Physiology 51,
668–675.
Erismann ND, Eduardo C, Tucci MLS. 2008. Photosynthetic limitation
by CO2 diffusion in drought stressed orange leaves on three rootstocks.
Photosynthesis Research 96, 163–172.
Evans JR, Loreto F. 2000. Acquisition and diffusion of CO2 in higher
plant leaves. In: Leegood FC, Sharkey TD, von Caemmerer S, eds. Photosynthesis: physiology and metabolism. Dordrecht: Springer Netherlands, 321–351.
Farquhar GD, von Caemmerer S, Berry JA. 1980. A biochemical model
of photosynthetic CO2 assimilation in leaves of C3 species. Planta 149,
78–90.
Flexas J, Bota J, Loreto F, Cornic G, Sharkey TD. 2004. Diffusive and
metabolic limitations to photosynthesis under drought and salinity in C3
plants. Plant Biology 6, 269–279.
Flexas J, Medrano H. 2002. Drought-inhibition of photosynthesis in C3
plants: stomatal and non-stomatal limitations revisited. Annals of Botany
89, 183–189.
Fortunati A, Barta C, Brilli F, Centritto M, Zimmer I, Schnitzler JP, Loreto F. 2008. Isoprene emission is not temperature-dependent during
and after severe drought-stress: a physiological and biochemical analysis. The Plant Journal 55, 687–697.
Foyer CH, Noctor G. 2000. Oxygen processing in photosynthesis:
regulation and signalling. The New Phytologist 146, 359–388.
Funk JL, Mak JE, Lerdau MT. 2004. Stress-induced changes in carbon
sources for isoprene production in Populus deltoides. Plant, Cell & Environment 27, 747–755.
Genty B, Briantai J-M, Baker NR. 1989. The relationship between
quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochimica et Biophysica Acta 990, 87–92. Girden ER. 1992. ANOVA: Repeated measures. Sage university papers
series on quantitative applications in the social sciences, 84. Newbury Park, CA, USA: Sage.
Grassi G, Magnani F. 2005. Stomatal, mesophyll conductance and
biochemical limitations to photosynthesis as affected by drought and leaf ontogeny in ash and oak trees. Plant, Cell & Environment 28, 834–849. Havaux M, Dall’osto L, Bassi R. 2007. Zeaxanthin has enhanced
antioxidant capacity with respect to all other xanthophylls in Arabidopsis leaves and functions independent of binding to PSII antennae. Plant Physiology 145, 1506–1520.
Havaux M, Tardt F, Ravenel I, Chamu D, Parot P. 1996. Thylakoid
membrane stability to heat stress studied by flash spectroscopic measurements of the electronic shift in intact potato leaves: influence of the xanthophyll content. Plant, Cell & Environment 19, 1359–1368. Hernández I, Alegre L, Munné-Bosch S. 2004. Drought-induced
changes in flavonoids and other low molecular weight antioxidants in Cistus clusii grown under Mediterranean field conditions. Tree Physiology
24, 1303–1311.
Hernández I, Cela J, Alegre L, Munné-Bosch S. 2012. Antioxidant
defences against drought stress. In: Aroca R, ed. Plant responses to drought stress. From morphological to molecular features. New York: Springer Verlag, 231–258.
IPCC. 2013. Intergovernmental Panel on Climate Change, www.ipcc.ch/.
Karl T, Fall R, Rosenstiel TN, Prazeller P, Larsen B, Seufert G, Lindinger W. 2002. On-line analysis of the 13CO
2 labeling of leaf isoprene suggests
multiple subcellular origins of isoprene precursors. Planta 215, 894–905. Kreuzwieser J, Graus M, Wisthaler A, Hansel A, Rennenberg H, Schnitzler JP. 2002. Xylem-transported glucose as an additional carbon
source for leaf isoprene formation in Quercus robur. The New Phytologist
156, 171–178.
Lawlor DW, Cornic G. 2002. Photosynthetic carbon assimilation and
associated metabolism in relation to water deficits in higher plants. Plant, Cell & Environment 25, 275–294.
Lewandowski I, Scurlock JMO, Lindvall E, Christou M. 2003. The
development and current status of perennial rhizomatous grasses as energy crops in the US and Europe. Biomass Bioenergy 25, 335–361. Long SP, Bernacchi CJ. 2003. Gas exchange measurements, what can
they tell us about the underlying limitations to photosynthesis? Procedures and sources of error. Journal of Experimental Botany 54, 2393–2401. Loreto F, Bagnoli F, Calfapietra C, et al. 2014. Isoprenoid emission
in hygrophyte and xerophyte European woody flora: ecological and evolutionary implications. Global Ecology and Biogeography 23, 334–345.
Loreto F, Di Marco G, Tricoli D, Sharkey TD. 1994. Measurements of
mesophyll conductance, photosynthetic electron transport and alternative electron sinks of field grown wheat leaves. Photosynthesis Research 41,
397–403.
Loreto F, Fineschi S. 2015. Reconciling functions and evolution of
isoprene emission in higher plants. The New Phytologist 206, 578–582. Loreto F, Pinelli P, Brancaleoni E, Ciccioli P. 2004. 13C labeling reveals
chloroplastic and extrachloroplastic pools of dimethylallyl pyrophosphate and their contribution to isoprene formation. Plant Physiology 135,
1903–1907.
Loreto F, Velikova V. 2001. Isoprene produced by leaves protects the
photosynthetic apparatus against ozone damage, quenches ozone products, and reduces lipid peroxidation of cellular membranes. Plant Physiology 127, 1781–1787.
Ma F, Xu TT, Ji MF, Zhao CM. 2014. Differential drought tolerance in tree
populations from contrasting elevations. AoB Plants 6, plu069.
Macoy DM, Kim W-Y, Lee SY, Kim MG. 2015. Biosynthesis, physiology,
and functions of hydroxycinamic acids in plants. Plant Biotechnology Reports 9, 269–278.
Mariani C, Cabrini R, Danin A, Piffanelli P, Fricano A, Gomarasca S, Dicandilo M, Grassi F, Soave C. 2010. Origin, diffusion and reproduction
of the giant reed (Arundo donax L.) a promising weedy energy crop. Annals of Applied Biology 157, 191–202.
Medrano H, Escalona JM, Bota J, Gulías J, Flexas J. 2002.
Regulation of photosynthesis of C3 plants in response to progressive
drought: stomatal conductance as a reference parameter. Annals of Botany 89 Spec No, 895–905.
Medrano H, Flexas J, Galmés J. 2009. Variability in water use efficiency
at the leaf level among Mediterranean plants with different growth forms. Plant Soil 317, 17–29.
Morfopoulos C, Sperlich D, Peñuelas J, Filella I, Llusià J, Medlyn BE, Niinemets Ü, Possell M, Sun Z, Prentice IC. 2014. A model of
plant isoprene emission based on available reducing power captures responses to atmospheric CO2. The New Phytologist 203, 125–139.
Munné-Bosch S, Peñuelas J. 2004. Drought-induced oxidative stress
in strawberry tree (Arbutus unedo L.) growing in Mediterranean field conditions. Plant Science 166, 1105–1110.
Munns R. 2002. Comparative physiology of salt and water stress. Plant,
Cell & Environment 25, 239–250.
Nackley LL, Vogt KA, Kim S-H. 2014. Arundo donax water use
and photosynthetic responses to elevated CO2. Agricultural Water
Management 136, 13–22.
Parry MA, Andralojc PJ, Khan S, Lea PJ, Keys AJ. 2002. Rubisco
activity: effects of drought stress. Annals of Botany 89 Spec No, 833–839. Pegoraro E, Rey A, Greenberg J, Harley P, Grace J, Malhi Y, Guenther AB. 2004. Effect of drought on isoprene emission rates from
leaves of Quercus virginiana Mill. Atmospheric Environment 38, 6149–6156. Perez-Martin A, Michelazzo C, Torres-Ruiz JM, Flexas J, Fernández JE, Sebastiani L, Diaz-Espejo A. 2014. Regulation of photosynthesis
and stomatal and mesophyll conductance under water stress and recovery in olive trees: correlation with gene expression of carbonic anhydrase and aquaporins. Journal of Experimental Botany 65, 3143–3156.
Pilu R, Bucci A, Badone FC, Landoni M. 2012. Giant reed (Arundo
donax L.): A weed plant or a promising energy crop? African Journal of Biotechnology 11, 9163–9174.
Pilu R, Manca A, Landoni M. 2013. Arundo donax as an energy crop:
pros and cons of the utilization of this perennial plant. Maydica 58, 54–59. Pollastri S, Tsonev T, Loreto F. 2014. Isoprene improves photochemical
efficiency and enhances heat dissipation in plants at physiological temperatures. Journal of Experimental Botany 65, 1565–1570. Porter WC, Barsanti KC, Baughman EC, Rosenstiel TN. 2012.
Considering the air quality impacts of bioenergy crop production: a case study involving Arundo donax. Environmental Science and Technology 46,
9777–9784.
Ramel F, Birtic S, Ginies C, Soubigou-Taconnat L, Triantaphylidès C, Havaux M. 2012. Carotenoid oxidation products are stress signals that
mediate gene responses to singlet oxygen in plants. Proceedings of the National Academy of Sciences, USA 109, 5535–5540.
Ryan AC, Hewitt CN, Possell M, Vickers CE, Purnell A, Mullineaux PM, Davies WJ, Dodd IC. 2014. Isoprene emission protects
photosynthesis but reduces plant productivity during drought in transgenic tobacco (Nicotiana tabacum) plants. The New Phytologist
201, 205–216.
Sánchez E, Scordia D, Lino G, Arias C, Cosentino SL, Nogués S.
2015. Salinity and water stress effects on biomass production in different Arundo donax L. clones. BioEnergy Research 8, 1461–1479.
Schnitzler JP, Graus M, Kreuzwieser J, Heizmann U, Rennenberg H, Wisthaler A, Hansel A. 2004. Contribution of different carbon sources to
isoprene biosynthesis in poplar leaves. Plant Physiology 135, 152–160. Selmar D, Kleinwächter M. 2013. Stress enhances the synthesis of
secondary plant products: the impact of stress-related over-reduction on the accumulation of natural products. Plant & Cell Physiology 54,
817–826.
Sharkey TD, Bernacchi CJ, Farquhar GD, Singsaas EL. 2007. Fitting
photosynthetic carbon dioxide response curves for C3 leaves. Plant, Cell &
Environment 30, 1035–1040.
Sharkey TD, Loreto F. 1993. Water stress, temperature, and light effects
on the capacity for isoprene emission and photosynthesis of kudzu leaves. Oecologia 95, 328–333.
Signarbieux C, Feller U. 2011. Non-stomatal limitations of
photosynthesis in grassland species under artificial drought in the field. Environment and Experimental Botany 71, 192–197.
Silva EN, Ferreira-Silva SL, Fontenele Ade V, Ribeiro RV, Viégas RA, Silveira JA. 2010. Photosynthetic changes and protective mechanisms
against oxidative damage subjected to isolated and combined drought and heat stresses in Jatropha curcas plants. Journal of Plant Physiology
167, 1157–1164.
Singsaas EL, Lerdau M, Winter K, Sharkey TD. 1997. Isoprene
increases thermotolerance of isoprene-emitting species. Plant Physiology
115, 1413–1420.
Tattini M, Galardi C, Pinelli P, Massai R, Remorini D, Agati G. 2004.
Differential accumulation of flavonoids and hydroxycinnamates in leaves of Ligustrum vulgare under excess light and drought stress. The New Phytologist 163, 547–561.
Tattini M, Loreto F, Fini A, Guidi L, Brunetti C, Velikova V, Gori A, Ferrini F. 2015. Isoprenoids and phenylpropanoids are part of
the antioxidant defense orchestrated daily by drought-stressed Platanus×acerifolia plants during Mediterranean summers. The New Phytologist 207, 613–626.
Tattini M, Velikova V, Vickers C, Brunetti C, Di Ferdinando M, Trivellini A, Fineschi S, Agati G, Ferrini F, Loreto F. 2014.
Isoprene production in transgenic tobacco alters isoprenoid, non-structural carbohydrate and phenylpropanoid metabolism, and protects photosynthesis from drought stress. Plant, Cell & Environment 37,
1950–1964.
Teixeira J, Gaspar A, Garrido EM, Garrido J, Borges F. 2013.
Hydroxycinamic acid antioxidants: an electrochemical overview. BioMed Research International 2013, 251754.
Telfer A. 2002. What is beta-carotene doing in the photosystem II reaction
centre? Philosophical transactions of the Royal Society of London. Series B, Biological Sciences 357, 1431–1439; discussion 1439.
Tomás M, Flexas J, Copolovici L, Galmés J, Hallik L, Medrano H, Ribas-Carbó M, Tosens T, Vislap V, Niinemets Ü. 2013. Importance
of leaf anatomy in determining mesophyll diffusion conductance to CO2
across species: quantitative limitations and scaling up by models. Journal of Experimental Botany 64, 2269–2281.
Tosens T, Niinemets U, Vislap V, Eichelmann H, Castro Díez P.
2012. Developmental changes in mesophyll diffusion conductance and photosynthetic capacity under different light and water availabilities in Populus tremula: how structure constrains function. Plant, Cell & Environment 35, 839–856.
Trnka M, Olesen JE, Kersebaum KC, et al. 2011. Agroclimatic
conditions in Europe under climate change. Global Change Biology 17,
2298–2318.
Velikova V, Sharkey TD, Loreto F. 2012. Stabilization of thylakoid
membranes in isoprene-emitting plants reduces formation of reactive oxygen species. Plant Signaling & Behavior 7, 139–141.
Velikova V, Brunetti C, Tattini M, et al. 2016. Physiological
significance of isoprenoids and phenylpropanoids in drought response of Arundinoideae species with contrasting habitats and metabolism. Plant, Cell & Environment 39, 2185–2197.
Velikova V, Müller C, Ghirardo A, Rock TM, Aichler M, Walch A, Schmitt-Kopplin P, Schnitzler JP. 2015. Knocking down of isoprene
emission modifies the lipid matrix of thylakoid membranes and influences the chloroplast ultrastructure in poplar. Plant Physiology 168, 859–870. Velikova V, Várkonyi Z, Szabó M, et al. 2011. Increased thermostability
of thylakoid membranes in isoprene-emitting leaves probed with three biophysical techniques. Plant Physiology 157, 905–916.
Vickers CE, Gershenzon J, Lerdau MT, Loreto F. 2009a. A unified
mechanism of action for volatile isoprenoids in plant abiotic stress. Nature Chemical Biology 5, 283–291.
Vickers CE, Possell M, Cojocariu CI, Velikova VB, Laothawornkitkul J, Ryan A, Mullineaux PM, Nicholas Hewitt C. 2009b. Isoprene
synthesis protects transgenic tobacco plants from oxidative stress. Plant, Cell & Environment 32, 520–531.
Webster RJ, Driever SM, Kromdijk J, et al. 2016. High C3
photosynthetic capacity and high intrinsic water use efficiency underlies the high productivity of the bioenergy grass Arundo donax. Scientific Reports 6, 20694.
Wentworth M, Murchie EH, Gray JE, Villegas D, Pastenes C, Pinto M, Horton P. 2006. Differential adaptation of two varieties of common
bean to abiotic stress: II. Acclimation of photosynthesis. Journal of Experimental Botany 57, 699–709.
Xiong D, Flexas J, Yu T, Peng S, Huang J. 2017. Leaf anatomy
mediates coordination of leaf hydraulic conductance and mesophyll conductance to CO2 in Oryza. The New Phytologist 213, 572–583.
