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Industrial Crops and Products 54 (2014) 266–271

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Industrial Crops and Products

jo u r n al homep age: www.elsev ier .com/ locate / indcrop

hotosynthetic response of in vitro guayule plants in low and highights and the role of non-photochemical quenching in plantcclimation

atpal Turana, Shashi Kumara,∗, Katrina Cornishb,∗∗

International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, IndiaDepartments of Horticulture and Crop Science, and Food, Agricultural and Biological Engineering, The Ohio State University, 1680 Madison Avenue,ooster, OH 44691-4096, USA

r t i c l e i n f o

rticle history:eceived 7 October 2013eceived in revised form 13 January 2014ccepted 13 January 2014vailable online 26 February 2014

eywords:igh light

a b s t r a c t

Guayule (Parthenium argentatum L.) is a hypoallergenic latex-producing rather recalcitrant crop. Duringin vitro regeneration, the growth and the photosynthetic response of guayule is strongly affected by lightintensities. We have used chlorophyll a (Chl-a) fluorescence to study the photosynthetic responses ofin vitro grown guayule plants under low light (100 �mol m−2 s−1) and high light (1250 �mol m−2 s−1).In high light (HL), the shoot length was reduced and fresh and dry weights were enhanced, contraryto low light (LL) plant response. Total chlorophyll (Chl) and carotenoid contents based on fresh weightor leaf area were reduced by about 50% in HL compared to LL. Although maximum efficiency (Fv/Fm) of

ow lighteactive oxygen speciesatural rubberhihuahuan desert

photosystem II (PSII) in the dark, electron transport rate (ETR-I), and quantum yield of photosystem I(PSI) were unaffected, the electron transport rate (ETR-II), quantum yield of PSII and non-photochemicalquenching (NPQ) were ∼78–88% higher in HL than LL. There were no significant differences observed inmalondialdehyde (MDA) content during regeneration of plants in either HL or LL. The higher NPQ in HLgrown plants than LL grown plants suggests that NPQ plays an important role in photoprotection duringacclimation of guayule plants when exposed to HL.

. Introduction

Guayule (Parthenium argentatum) is a shrub native to the Chi-uahuan desert of Texas and Mexico. It is an alternate specialtyubber crop and recalcitrant species, which provides hypoaller-enic natural rubber suitable for medical applications. It is currentlynder research and development for rubber production (Dong et al.,006; van Beilen and Poirier, 2007) and commercially cultivated inrizona, USA.

Light is one of the main factors that substantially affect plantrowth and photosynthetic rate (Kangasjarvi et al., 2012). Inature, light varies in both intensity and spectral composition,

Abbreviations: Chl, chlorophyll; ETR, electron transport rate; F0, minimum flu-rescence yield in dark adapted leaf; F ′

0, the minimal fluorescence yield in lightdapted leaf; F, mmaximum fluorescence yield in dark adapted leaf; F ′

m, maximumuorescence yield in light adapted leaf; Fv/Fm, maximum quantum yield of PSII;PQ, non-photochemical quenching; MDA, malondialdehyde; PSI, Photosystem I;SII, photosystem II; qp, photochemical quenching.∗ Corresponding author.

∗∗ Corresponding author. Tel.: +1 330 263 3982.E-mail addresses: skrhode@icgeb.res.in (S. Kumar),

ornish.19@osu.edu (K. Cornish).

926-6690/$ – see front matter © 2014 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.indcrop.2014.01.022

© 2014 Elsevier B.V. All rights reserved.

which compels plants to survive by keeping the balance betweenlight absorption required for photosynthesis and photoprotec-tion (Wagner et al., 2006). Light energy is mainly utilized byplant pigments (chlorophyll and carotenoids), and protein com-plexes photosystem II (PSII) and photosystem I (PSI). When theamount of light energy absorbed by chlorophyll (Chl) exceedsthe capacity of the photochemical process, singlet Chl (1Chl*)molecules remain in an excited state long enough to cause theirtransition to reactive triplet Chl (3Chl*) molecules (Barber andAndersson, 1992). These may then react with oxygen in the chloro-plast, creating singlet oxygen radicals, which further react withmembrane lipids and proteins resulting in photoinhibition and/orirreversible photo-oxidative damage (reviewed in Logan et al.,2006; Krieger-Liszkay et al., 2008; Triantaphylidès and Havaux,2009).

Plants have evolved different mechanisms to protect the chloro-plast from photo-oxidative damage (reviewed in Krieger-Liszkayet al., 2008; Takahashi and Badger, 2011). These include enzymaticand non-enzymatic antioxidants (glutathione, ascorbate, ascorbate

peroxidase, superoxide dismutase, etc.), as well as scavengers ofexcess light energy (carotenoids). Any process that inhibits theuse of absorbed light energy relative to its rate of influx willcause ‘excitation pressure’ on the photosystem, that is, more light

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S. Turan et al. / Industrial Crop

nergy is absorbed than can be processed by photochemistry sohe reaction centers become reduced or ‘closed’ (Huner et al.,998).

Chl-a fluorescence kinetics is an informative tool for studyinghe effects of different environmental stresses on photosynthesisKalaji et al., 2012). It is one of the main methods used to investigatehe function of PSII and its reaction to changes in the environmentnd growth conditions (Desotgiu et al., 2012). Among the variousarameters, non-photochemical quenching (NPQ) has commonlyeen used to measure the dissipation of excess excitation energy aseat. Heat dissipation occurrs in the antennae (Belgio et al., 2012) or

n the reaction center (Wilhelm and Selmar, 2011). The basic prin-iple of NPQ operation is the same in all chloroplast-containingpecies but differences occur in its mechanistic regulation (Kanat al., 2012). Light-induced formation of NPQ is activated by a pHecrease in the thylakoid lumen, which further activates the violax-nthin de-epoxidase to convert violaxanthin to zeaxanthin (Jahnsnd Holzwarth, 2012). The protonation of the PsbS protein is alson essential element of NPQ mechanism in higher plants (Li et al.,004). Generally, in excess light, the rate of NPQ increases, butigh light (HL) stress may also reduce NPQ, which is indicative of

rreversible damage to the PSII reaction center and/or antennae.The in vitro regeneration of plants is a crucial technique in crop

evelopment, but is affected by light intensities. Here we have usedow and high light (100 and 1250 �mol m−2 s−1) to discern thecclimatory response in guayule during its in vitro regeneration.ince Chl-a fluorescence is a good marker for estimating the pho-osynthetic rate under varying light intensity, the outcome of thistudy should be helpful in understanding high light acclimatoryechanisms and photosynthetic regulation in recalcitrant in vitro

rown plants.

. Materials and methods

.1. Plant material and growth conditions

P. argentatum (guayule) shoot-regenerated cultures were useds an experimental material. Guayule seeds (P. argentatum cv.anAridus-2, (Panaridus, Casa Grande, Arizona, USA)) were ster-lized by treating with 70% ethanol for 30 s, then with 0.5%aOCl + Tween 20 for 3 min, and then washed 5–6 times with steril-

zed autoclaved water. Seeds were germinated in glass jars on 0.5×S (MS basal salts) medium with 15 g/L sucrose, 100 mg/L inosi-

ol, 0.5 mg/L Thiamine-HCl, 8 g/L agar and pH 7.0 under low light100 �mol m−2 s−1) and at 27 ± 1 ◦C temp. Cultures were main-ained through shoot apex culture and rooted on medium (0.5× MSasal salts) containing 15 g/L sucrose, 100 mg/L inositol, 0.5 mg/Lhiamine-HCl, 0.1 mg/L Indole-3-butyric acid, 8 g/L agar and pH.0. Shoots were grown for fifteen days to develop roots and sub-equently plants were either maintained at 100 �mol m−2 s−1 (LL)r transferred to 1250 �mol m−2 s−1 (HL) for acclimation. The LLnd HL levels chosen based on previous published reports forhe different plant species. The solid state (SSL) lamps (DesignIn-ova, New Delhi, India) were used as the light source. Accordingo vendor’s photobiosim, natural sun light was mimicked forhe plant cultures using specific dominant wavelengths modu-ated in the analog domain after eliminating the IR, UV and EMIight spectra. Specific wavelength generating semiconductor mul-ichips were used to mix up the 30% light from the completepectrum with the 70% of dominant wavelengths in the PARegion (i.e. 400–470 nm and 625–700 nm). The Solid state light

SSL) is a super set of light that makes use of semiconductorEDs, Polymer LEDs (PLED), and Organic LEDs (OLED), as illu-ination sources instead of plasma (used in fluorescent lamps),

as or electric filaments. All experimental measurements were

Products 54 (2014) 266–271 267

conducted after 15 days of the plants’ transfer to light (LL andHL).

2.2. Chl, carotenoid and protein estimation

Chl and carotenoid contents were estimated from fullyexpanded leaves from the middle portion of the plants as describedby Porra et al. (1989) and Welburn and Lichtenthaler (1984). Proteinquantification was performed according to Bradford (1976).

2.3. Chlorophyll fluorescence and P700 measurement

Chl fluorescence was studied in detached leaves. The Chlfluorescence of PSII and the redox state of P700 were mea-sured at room temperature using the Dual-PAM-100 fluorometer(Walz, Effeltrich, Germany) and Dual-PAM software. Actinic lightfrom a 620 nm light-emitting diode (LED) and blue actinic lightat 100 �mol m−2 s−1 from 460 nm LED arrays was delivered toa guayule leaf for 5 min, along with saturating light pulses(10,000 �mol m−2 s−1) of 300 ms. The guayule leaves immedi-ately after detachment were kept in the dark for 10 min beforethe measurement of minimum (F0) and maximum (Fm) fluores-cence (using saturated flash). Maximum quantum yield of PSII(Fv/Fm) was measured as (Schreiber, 2004). The F ′

m (maximum flu-orescence in light adapted leaf) was measured when the leaveswere illuminated. The quantum yield Y(II) of PSII was measuredas (F ′

m − F)/F ′m (Kramer et al., 2004). F is the steady state fluo-

rescence in a light adapted leaf. The electron transport rate ofPSII (ETRII) was estimated by Dual-PAM software (Walz, Effel-trich, Germany). NPQ was calculated as (Fm − F ′

m)/F ′m (Schreiber

et al., 1986). Photochemical quenching (qp) was calculated as(F ′

m − F)/(F ′m − F ′

0) where F ′0 is the minimal fluorescence in light

adapted leaf.The P700 redox state was also measured by Dual-PAM-100 with

a dual wavelength (830/875 nm) unit (Klughammer and Schreiber,1994; Schreiber and Klughammer, 2008). The P700 signal (P) mayvary from minimum (P700 fully reduced) to maximum (P700 fullyoxidized). The maximum PSI signal (Pm), which is analogous toFm, was determined with application of a saturation pulse afterpre-illumination with far-red light. The minimum P700 signal (P0),analogous to F0, was calculated when complete reduction of PSI wasinduced after the saturation pulse and in the absence of far-red illu-mination. P ′

m was determined similarly to Pm, but with backgroundactinic light instead of far-red illumination. The quantum yield Y(I)of PSI was calculated as (P ′

m − P)/P ′m (Pfündel et al., 2008). Electron

transport rates of PSI (ETRI) were analyzed using the Dual-PAMsoftware.

2.4. Malondialdehyde assay

MDA, a measure of lipid peroxidation and an indicator of oxida-tive stress (Turan and Tripathy, 2013), was analyzed as describedby Dhindsa and Matowe (1981). Leaf tissues were ground in 10 mLof 10% (w/v) TCA and homogenate was centrifuged at 10,000 rpmfor 10 min. Afterward, 2 mL of 10% TCA containing 0.67% (w/v)thiobarbituric acid was added to 2 mL supernatant. The mixturewas incubated at 100 ◦C for 15 min and centrifuged at 10,000 × gat 4 ◦C for 10 min. The OD of the supernatant was measured at

532 nm (corrected for nonspecific turbidity by subtracting theA600). MDA values were calculated using a molar extinction coeffi-cient of 1.56910 5 M−1 cm−1 and expressed relative to the leaf FW(nmol g−1 FW).

268 S. Turan et al. / Industrial Crops and

Table 1Guayule plants were grown in vitro as described in Section 2 initially under LL(100 �mol m−2 s−1) and then 50% of plants were transferred to HL for studying var-ious parameters simultaneously in LL as well as HL (1250 �mol m−2 s−1). Values inparentheses represent standard deviation. For shoot length, fresh weight, dry weightmeasurements 10 replicates (n = 10) were taken and for MDA contents, Fv/Fm andChl measurements 3 replicates (n = 3) were taken.

Parameters LL HL

Shoot length (cm) 14.22 ( ± 1.71) 10.56 ( ± 0.70)*

Fresh weight/shoot (g) 0.42 ( ± 0.07) 0.92 ( ± 0.14)*

Dry weight/shoot (g) 0.029 ( ± 0.004) 0.064 ( ± 0.015)*

Fresh weight/leaf (mg) 41.69 ( ± 3.069) 138.14 ( ± 10.126)*

Dry weight/leaf (mg) 3.164 ( ± 0.219) 9.673 ( ± 0.926)*

MDA content (nmol/g FW) 4.34 ( ± 0.51) 3.92 ( ± 0.25)Fv/Fm 0.763 ( ± 0.025) 0.765 ( ± 0.018)

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Chl a/b ratio 3.32 ( ± 0.062) 3.38 ( ± 0.094)

* Significant differences from LL with P < 0.05.

.5. Statistical analysis

All experiments were carried out with at least 3 or more repli-ates and repeated at least twice. Significant differences wereested using Student’s t-test with P < 0.05.

. Results

.1. Plant growth

P. argentatum shoots grown in vitro for 15 days grew longern the presence of LL than HL (Fig. 1A). The average shoot length

as 14.22 cm in LL and 10.56 cm in HL (Table 1). However, overalleaf area and leaf weight increased in response to HL conditionsompared to LL illumination. Fresh weight per leaf was 41.69 mg±3.069) in LL and 138.14 mg (±10.126) in HL (Table 1). Dry weighter leaf was 3.164 mg (±0.219) in LL and 9.673 mg (±0.926) in HLonditions (Table 1). Leaves were dark green in LL as comparedo HL (Fig. 1B). Average fresh weight per plant’s shoot (excludingoot) observed was 0.425 g in LL and 0.918 g in HL (Table 1). Dryatter per plant’s shoot (excluding root) was higher 0.064 g in HL

nd 0.029 g in LL illuminated plants (Table 1).

.2. Chl and carotenoids contents

Total Chl contents were reduced ∼50% in HL (0.98 mg/g FW)hen compared to LL (1.82 mg/g FW). Similarly, Chl-a and Chl-

concentrations also were higher in LL than HL (Fig. 1C). Thearotenoid concentrations in LL and HL were 0.199 mg/g FW and.104 mg/g FW respectively (∼50% low in HL) (Fig. 1C). The Chl a/batio was unaffected by LL (3.32 ± 0.062) and HL (3.37 ± 0.094) illu-ination.). Chl and carotenoids contents per cm2 leaf area were

lmost double in LL grown plants than those in HL conditionsFig. 1D). Pigments level per leaf was higher in HL grown plant thann LL grown plants. Chl contents per leaf were 96.53 �g in LL and45.38 �g in HL conditions (Fig. 1E). Carotenoids content per whole

eaf was 9.25 �g in LL and 15.93 �g in HL grown plants (Fig. 1E).

.3. Photosynthetic parameters related to PSII and PSI

When Chl-a fluorescence was used as noninvasive tool to studyhe photosynthetic activity of PSII and PSI, the electron transportate (ETR II) of PSII was higher in HL (50.6 �mol m−2 s−1) than in LL28.3 �mol m−2 s−1) at photosynthetically active radiations (PAR)bove 200 �mol m−2 s−1 (Fig. 2A). The quantum yield of PSII was

ower in LL (0.0755) when compared to HL (0.142) (Fig. 2B). The QAeduction state of PSII, estimated using the fluorescence parameter

− qp, was 13% higher in LL (0.897) than HL (0.792) (Fig. 2C). NPQas 78% higher in HL (0.87) compared to LL (0.48) at highest PAR

Products 54 (2014) 266–271

(Fig. 2D). Thus, HL considerably altered the light-induced thermalenergy dissipation capacity, determined as NPQ (Fig. 2D), withoutaffecting the Fv/Fm ratio (Table 1). The electron transport rate (ETR I)of PSI in HL and LL was 64.8 �mol m−2 s−1 and 61.23 �mol m−2 s−1

respectively at highest PAR used (Fig. 2E). The quantitative yieldof PSI at the highest PAR was 0.182 (±0.042) and 0.158 (±0.0192),respectively, in HL and LL (Fig. 2F). Thus, PSI parameters were notmuch affected by light treatment.

3.4. Lipid peroxidation

The lipid peroxidation assay indicated 4.38 (±0.51) and 3.92(±0.25) nmol MDA/g FW in LL and HL, respectively (Table 1).

4. Discussion

Plants have different mechanisms to cope with excess lightstress during photosynthesis (Wagner et al., 2006). Under low lightintensity, most of the absorbed light usually can be used for photo-synthesis, but under relatively high light intensity, only part of theabsorbed light can be used. Here we have used Chl fluorescence toelucidate how NPQ mediated photoprotection in our in vitro grownguayule plants by studying comparative photosynthetic parame-ters in LL and HL.

The pigments, Chl and carotenoids, content on basis of per mgFW and on the basis of per whole leaf and on basis of per cm2 leafarea were lower in HL (∼50%) as compared to those in LL grownplants (Fig. 1C, D) which may be due to less synthesis or breakdownof pigments in HL conditions. Biswal et al. (2012) have suggestedthat less accumulation of Chl in tobacco plants grown under highlight may be due to downregulation of Chl biosynthetic enzymes.Chl degrades faster in high light than in low light, effectively reduc-ing the half-life of Chl (Vavilin and Vermaas, 2007). Reduction inleaf Chl content (Fig. 1C, D) is usually observed with HL acclima-tion in a wide range of species (Matsubara et al., 2009; Biswalet al., 2012). When Arabidopsis plants are grown under constant HL,they accumulated less Chl but more PSII along with smaller light-harvesting antennae compared to the plants grown in LL (Ballottariet al., 2007). However, in our study, no significant change in Chl a/bratio was observed, which indicates that antenna size may have notbeen affected in guayule plants. Higher Chl and carotenoid con-tents on a per leaf basis in HL as compared to LL in vitro grownguayule plants (Fig. 1E) may be due to its adaptive potential to HLbecause as pigment content per cm2 leaf area is low in HL grownguayule plants. HL grown guayule plants have leaves with largerarea than grown in LL (Fig. 1B) to utilize more light for photo-synthesis. Plants appear to be growing healthier in HL than in LL(Fig. 1A).

Guayule is a shrub native to Chihuahuan desert, which mighthave naturally selected for maintaining high photosyntheticpotential under adverse conditions like light and/or temperaturefluctuations as present in desert environment. As in vitro plantstry to mimic field grown plants, the high photosynthetic poten-tial might have been reflected in the in vitro grown plants as thehigher electron transport rate in PSII under HL conditions (Fig. 2A).Similar results were observed, i.e. increases in electron transportrate in response to HL, in tobacco plants (Biswal et al., 2012). Like-wise, higher QA oxidation in HL than in LL grown plants, which wasindicated by lower 1 − qP in HL (Fig. 2C), indicated a better lightutilization by PSII in HL, in agreement with other reports (Alteret al., 2012). The greater increase in dry matter per plant and/or

per leaf in HL than in LL (Table 1) directly indicates improved plantgrowth performance and adaptability of in vitro grown guayule toHL. Further, a major acclimatory response in guayule was charac-terized by up-regulation of the NPQ capacity and measured as heat

S. Turan et al. / Industrial Crops and Products 54 (2014) 266–271 269

Fig. 1. Plants were grown in vitro under LL (100 �mol m−2 s−1) and HL irradiance (1250 �mol m−2 s−1) described in Section 2. (A) Shoot portion of plants at the time ofm f planc , per wr differ

ekeimM

easurement in HL and LL conditions. (B) Leaf morphology from middle portion oarotenoids contents on the basis of (C) per mg leaf FW (D), per 1 cm2 leaf area (E)eplicates and error bar represents standard deviation. Asterisks denote significant

nergy dissipation in HL in comparison to LL (Fig. 2D). It is wellnown that zeaxanthin accumulates under high light stress (Beisel

t al., 2010). High light stress usually is associated with an increasen lipid peroxidation but, in the present investigation, there is not

uch difference in lipid peroxidation in LL and HL as indicated byDA levels (Table 1). This lack of difference may due to efficient

t which were taken for study in LL and HL conditions. Total Chl, Chl a, Chl b andhole leaf in guayule in LL and HL grown plants. Each data point is an average of 3

ences from LL with P < 0.05.

NPQ observed in our study and/or due to some other antioxidativemechanisms (antioxidative enzymes, ascorbate, glutathione or

tocopherols) which need to be further investigated. Zeaxanthin,which is known to increase in HL stress in plants, can also actsas a scavenger of ROSs and thus avoid lipid peroxidation (Havauxet al., 2007; Zhang et al., 2012).

270 S. Turan et al. / Industrial Crops and Products 54 (2014) 266–271

Fig. 2. Plants were grown under LL (100 �mol m−2 s−1) and HL (1250 �mol m−2 s−1) irradiance as described in Section 2. Chl-a fluorescence was used for measuring (A) ETR IIof PSII, (B) quantum yield of PSII, (C) 1 − q , (D) NPQ of PSII, (E) ETR I of PSI, and (F) quantum yield of PSI. Each data point is an average of 3 replicates and error bar representss

nptiPGaltd

P

tandard deviation. Asterisks denote significant differences from LL with P < 0.05.

The induction of NPQ coupled with enhanced PSII activity oro effect on PSI (Fig. 2E and F) indicates that guayule plants werehotoprotected by the NPQ mechanism, which helps in acclima-ion of plants to high irradiation without increase in oxidativenjury or lipid peroxidation. PSI is relatively more stable thanSII and is less affected by high irradiances (Huang et al., 2010).erotto et al. (2012) have shown that NPQ helps in long term

cclimation to high light in Physcomitrella patens, and mutantsacking in NPQ show enhanced photosensitivity to high light inhis moss. As Chl b is rich in the antenna of PSII, an increase orecline in the Chl a/b ratio will indicate smaller and larger antenna

size, respectively. Also biosynthesis of pigments is found to becoordinated with that of the LHC apoproteins (Johanningmeierand Howell, 1984). The lower Chl content per unit leaf area inHL relative to LL leaves (Fig. 1C) suggests that total numbers ofPSII per leaf area may be less during HL illumination than inLL.

Thus, HL acclimation of guayule plants appears to be mediated

by NPQ quenching without an increase in lipid-peroxidation. Thisshows that guayule can be used as a model to understand the NPQquenching mechanism. There is further need to investigate whatcomponents of NPQ quenching are involved in photoprotection and

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ow PSII and PSI reaction center and/or antenna related protein’sxpression varies under LL and HL. Moreover, use of different spec-ral intensity will be interesting and informative in a future study.

cknowledgement

This research work is supported by OSU and PanAridus fundingo ICGEB (Grant #OSU/Shashi/11/438).

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