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Journal of Plant Physiology 185 (2015) 75–83

Contents lists available at ScienceDirect

Journal of Plant Physiology

journa l homepage: www.e lsev ier .com/ locate / jp lph

hysiology

rbuscular mycorrhizal symbiosis ameliorates the optimum quantumield of photosystem II and reduces non-photochemical quenching inice plants subjected to salt stress

osa Porcel a, Susana Redondo-Gómez b, Enrique Mateos-Naranjo b, Ricardo Aroca a,osalva Garcia c, Juan Manuel Ruiz-Lozano a,∗

Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín (CSIC), Profesor Albareda n◦ 1, 18008 Granada, SpainDepartamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, Apartado 1095, 41080 Sevilla, SpainFacultad de Estudios Superiores Zaragoza, Universidad Nacional Autónoma, Mexico

r t i c l e i n f o

rticle history:eceived 12 June 2015eceived in revised form 15 July 2015ccepted 17 July 2015vailable online 31 July 2015

eywords:rbuscular mycorrhizal symbiosison-photochemical quenchingptimum quantum yieldryza sativahotosystem IIalt stress

a b s t r a c t

Rice is the most important food crop in the world and is a primary source of food for more than halfof the world population. However, salinity is considered the most common abiotic stress reducing itsproductivity. Soil salinity inhibits photosynthetic processes, which can induce an over-reduction of thereaction centres in photosystem II (PSII), damaging the photosynthetic machinery. The arbuscular myc-orrhizal (AM) symbiosis may improve host plant tolerance to salinity, but it is not clear how the AMsymbiosis affects the plant photosynthetic capacity, particularly the efficiency of PSII. This study aimedat determining the influence of the AM symbiosis on the performance of PSII in rice plants subjectedto salinity. Photosynthetic activity, plant gas-exchange parameters, accumulation of photosynthetic pig-ments and rubisco activity and gene expression were also measured in order to analyse comprehensivelythe response of the photosynthetic processes to AM symbiosis and salinity. Results showed that the AMsymbiosis enhanced the actual quantum yield of PSII photochemistry and reduced the quantum yield ofnon-photochemical quenching in rice plants subjected to salinity. AM rice plants maintained higher netphotosynthetic rate, stomatal conductance and transpiration rate than nonAM plants. Thus, we propose

that AM rice plants had a higher photochemical efficiency for CO2 fixation and solar energy utilizationand this increases plant salt tolerance by preventing the injury to the photosystems reaction centres andby allowing a better utilization of light energy in photochemical processes. All these processes trans-lated into higher photosynthetic and rubisco activities in AM rice plants and improved plant biomassproduction under salinity.

© 2015 Elsevier GmbH. All rights reserved.

. Introduction

Rice (Oryza sativa L.) is the most important food crop in theorld and is a primary source of food for more than half of theorld population (Kumar et al., 2013). According to FAO (2005),orld agriculture should produce 70% more food for an additional

.3 billion people by 2050. However, rice is a salt sensitive cropnd salinity is considered the most common abiotic stress reduc-

ng its productivity (Kumar et al., 2013). Indeed, salinity is a majornd increasing environmental problem, affecting over 6% of theotal land area of the world. Thus, investigating different strategies

∗ Corresponding author. Fax: +34 958 129600.E-mail address: juanmanuel.ruiz@eez.csic.es (J.M. Ruiz-Lozano).

ttp://dx.doi.org/10.1016/j.jplph.2015.07.006176-1617/© 2015 Elsevier GmbH. All rights reserved.

to improve rice productivity under salinity is an important chal-lenge to cope with reduced food production due to excessive soilsalinization. Several studies have shown that the arbuscular mycor-rhizal (AM) symbiosis can alleviate salt stress in different host plantspecies (For reviews see Evelin et al., 2009; Ruiz-Lozano et al., 2012;Augé et al., 2014).

Soil salinity leads to a decrease in crop production due, amongother processes, to inhibition of photosynthetic processes (Pitmanand Läuchli, 2002). Indeed, salinity inhibits specific enzymesinvolved for the synthesis of photosynthetic pigments, causing areduction in plant chlorophyll content (Giri and Mukerji, 2004;Murkute et al., 2006; Sheng et al., 2008). Moreover, the lower-

ing of the photosynthetic rate caused by salt stress can inducean over-reduction of the reaction centres in photosystem II (PSII)and this may damage the photosynthetic machinery if the plant

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6 R. Porcel et al. / Journal of Pl

s unable to dissipate the excess energy (Baker, 2008). Indeed, theight energy absorbed by chlorophyll molecules can be used eithero drive photosynthesis, it can be re-emitted as light-chlorophylluorescence or the excess energy can be dissipated as heat. Thesehree processes occur in a competitive way, so that any increasen the efficiency of one will decrease the yield of the other twoMaxwell and Johnson, 2000; Harbinson, 2013). Thus, the ability ofhe plant to dissipate or not the excess energy can be quantified by

easuring the chlorophyll a fluorescence.Improvements in photosynthetic activity or water use effi-

iency have been reported in AM plants growing under salt stressSheng et al., 2008; Zuccarini and Okurowska, 2008; Hajibolandt al., 2010) or under drought stress (Birhane et al., 2012; Liut al., 2015). Nevertheless, few studies have investigated so farhe influence of AM fungi on leaf photochemical properties underalt stress. Sheng et al. (2008) found that the AM symbiosismproved the photosynthetic capacity of maize plants, mainlyy regulating the energy bifurcation between photochemical andon-photochemical events and elevating the efficiency of photo-hemistry and non-photochemistry of PSII. However, Sheng et al.2008) attributed the influence of the AM symbiosis on maize pho-osynthetic capacity to a mycorrhiza-mediated enhancement oflant water status, rather than to a direct influence on the effi-iency of PSII. Hajiboland et al. (2010) found that mycorrhizationmproved photosynthetic activity in tomato plants through both,levating stomatal conductance and protecting PSII photochemi-al processes against salinity. However, authors suggested that AMolonization acted only on maintenance of photochemical capac-ty in stressed leaves and did not increase its potential for energyrapping, since the enhancement of the PSII photochemistry by AMungi did not occur in plants not subjected to salt stress. Other stud-es have shown modulation of PSII efficiency by the AM symbiosis inose, pistachio and poplar plants subjected to drought (Pinior et al.,005; Bagheri et al., 2011; Liu et al., 2015), in citrus plants growing

n low-zinc soil (Chen et al., 2014), as well as, under non-stressfulonditions in maize and black locust seedlings (Rai et al., 2008;hu et al., 2014). Nevertheless, so far, it is not clear how the AMymbiosis affects the plant photosynthetic capacity, particularlyhe efficiency of photosystem II in plants subjected to salinity.

The activity of enzymes involved in carbon assimilation suchs the ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco)s also determinant for plant photosynthetic efficiency (Masumotot al., 2005; Goicoechea et al., 2014). In a study with grapevinelants, Valentine et al. (2006) found that AM plants subjectedo drought had higher rubisco activity and water use efficiencyhan non AM plants. However, these authors did not directly

easure the enzymatic activity; they calculated rubisco activityrom CO2 response curves measured with an infrared gas analyzerIRGA). More recently, Goicoechea et al. (2014) carried out a semi-uantification of the large (RLS) and small (RSS) rubisco subunits

n alfalfa with no significant differences between AM and nonAMlants. However, to our knowledge, no data are available aboutirect enzymatic rubisco activity measured in AM plants subjectedo salinity. Thus, this aspect deserves to be examined in studiesealing with salt stress alleviation by the AM symbiosis, in combi-ation with molecular studies aimed at evaluating the expressionattern of genes encoding for the small (rbcS) and large (rbcL)ubisco subunits (Tsutsumi et al., 2008).

The present study aimed at determining the influence of theM symbiosis on the performance of PSII in rice plants sub-

ected to increasing salinity levels. Thus, chlorophyll a fluorescenceas measured to calculate the maximum quantum efficiency

f PSII photochemistry (Fv/Fm), actual quantum yield of photo-ystem II photochemistry (�PSII), as well as, quantum yield ofon-photochemical quenching (�NPQ) (Lazár, 2015). Photosyn-hetic activity, plant gas-exchange parameters, accumulation of

ysiology 185 (2015) 75–83

photosynthetic pigments, activity of rubisco enzyme and expres-sion of rubisco-encoding genes were also quantified in order toanalyse comprehensively the response of the photosynthetic pro-cesses in rice to AM symbiosis and salinity. The starting hypotesisis that the AM symbiosis will alter the photosynthetic capacity ofrice plants by changing plant gas-exchange parameters and perfor-mance of photosystem II.

2. Materials and methods

2.1. Experimental design

The experiment consisted of a randomized complete blockdesign with two inoculation treatments: (1) non-mycorrhizalcontrol plants, (2) plants inoculated with the AM fungusClaroideoglomus etunicatum (isolate EEZ 163). There were 30 repli-cates of each inoculation treatment, totalling 60 pots (two plantsper pot), so that ten pots of each inoculation treatment were grownunder non-saline conditions throughout the entire experiment,while ten pots per treatment were subjected to 75 mM of NaClfor four weeks and the remaining ten pots per treatment weresubjected to 150 mM of NaCl for four weeks.

2.2. Soil and biological materials

Loamy soil was collected from Granada province (Spain,36◦59′34′′N; 3◦34′47′′W), sieved (5 mm), diluted with quartz-sand(<2 mm) and with vermiculite (1:1:1, soil:sand:vermiculite, v/v/v)and sterilized by steaming (100 ◦C for 1 h on 3 consecutive days).The original soil had a pH of 8.2 [measured in water 1:5 (w/v)];1.5 % organic matter, nutrient concentrations (g kg−1): N, 1.9; P, 1(NaHCO3-extractable P); K, 6.9. The electrical conductivity of theoriginal soil was 0.2 dS m−1.

Three indica rice (O. sativa L.) seedlings (cv puntal), previouslygerminated on sand, were sown in pots containing 900 g of the samesoil/sand/vermiculite mixture as described above and thinned totwo seedlings per pot after three days.

2.3. Inoculation treatments

Mycorrhizal inoculum was bulked in an open-pot culture ofZea mays L. and consisted of soil, spores, mycelia and infectedroot fragments. The AM fungus used in this study had been pre-viously isolated from Cabo de Gata Natural Park (Almería, Spain,36◦45′24′′N 02◦13′17′′W), which is an area with serious problemsof salinity and affected by desertification. The AMF species was C.etunicatum (isolate EEZ 163), previously characterized as an effi-cient AM fungus under salinity (Estrada et al., 2013a,b). Appropriateamounts of the inoculum containing about 700 infective propag-ules (according to the most probable number test), were added tothe corresponding pots at sowing time just below rice seedlings.Non-mycorrhizal control plants received the same amount of auto-claved mycorrhizal inocula together with a 10 ml aliquot of a filtrate(<20 �m) of the AM inocula in order to provide a general microbialpopulation free of AM propagules.

2.4. Growth conditions

The experiment was carried out under glasshouse conditionswith temperatures ranging from 19 to 25 ◦C, 16/8 light/dark period,and a relative humidity of 50–60%. At the leaf level, a photosyntheticphoton flux density of 800 �mol m−2 s−1 was measured with a light

meter (LICOR, Lincoln, NE, USA, model LI-188B). Water was sup-plied daily to the entire period of plant growth to avoid any droughteffect. Plants were established for five weeks prior to salinization toallow adequate plant and symbiotic establishment. After that time,

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group of plants were kept under non-saline solutions, by irrigatingith water until the end of the experiment (0 mM NaCl), while two

roups of each inoculation treatments were watered with an aque-us solution containing 75 or 150 mM NaCl, respectively. Plantsere maintained under these conditions for additional four weeks.uring this period, plants received each week 10 ml per pot ofoagland nutrient solution containing only ¼ P concentration tovoid inhibition of AM root colonization. At the end of the exper-ment, the electrical conductivities in the soil:sand:vermiculite

ixture used as growing substrate were 0.5, 3.4 and 6.3 dS m−1

or the salt levels of 0, 75, and 150 mM NaCl, respectively.

.5. Parameters measured

.5.1. Biomass productionAt harvest (60 days after planting), the shoot and root sys-

em were separated and the shoot dry weight (SDW) and root dryeight (RDW) was measured after drying in a forced hot-air oven

t 70 ◦C for two days. The shoot water content was calculated asFW-DW)/FW (Marulanda et al., 2007), and expressed as g H2O per

of FW.

.5.2. Symbiotic developmentThe percentage of mycorrhizal root infection in maize plants

as estimated by visual observation of fungal colonization afterlearing washed roots in 10% KOH and staining with 0.05% trypanlue in lactic acid (v/v), as described by Phillips and Hayman (1970).he extent of mycorrhizal colonization was calculated according tohe gridline intersect method (Giovannetti and Mosse, 1980).

.5.3. Plant gas-exchange and photosynthetic parametersMeasurements were taken on the second youngest leaf from

ach plant (n = 7, per treatment) using an infrared gas analyzer inn open system (LCI-portable, ADC system, England). Net photosyn-hetic rate (A), intercellular CO2 concentration (Ci) and stomatalonductance (Gs) were all determined at an ambient CO2 con-entration of 390 �mol mol−1, temperature of 25/30 ◦C, 50 ± 5%elative humidity and a PFD of 1000 �mol m−2 s−1. A, Gs and tran-piration rate (E) were calculated using standard formulae fromon Caemmerer and Farquhar (1981). Intrinsic water-use efficiency

iWUE) was calculated as the ratio between A and Gs [mmol (CO2ssimilated) mol−1 (H2O transpired)]. Measurements were madet midday.

.5.4. Chlorophyll fluorescence parametersChlorophyll fluorescence was measured as described by

edondo-Gómez et al. (2010) using a portable modulated fluo-imeter (FMS-2, Hansatech Instruments Ltd., UK). Measurementsere made on 10 plants per treatment (n = 10). Light- and

ark-adapted fluorescence parameters were measured at mid-ay (1400 �mol m−2 s−1) to investigate whether salt concentrationffected the sensitivity of plants to photoinhibition (Maxwell andohnson, 2000).

Plants were dark-adapted for 30 min, using leaf clips designedor this purpose. Minimal fluorescence in the dark-adapted stateF0) was measured using a modulated pulse (<0.05 �mol m−2 s−1

or 1.6 �s) which was too small to induce significant physiologicalhanges in the plant. The stored data were averages taken over a.6-s period. Maximal fluorescence in this state (Fm) was measuredfter applying a saturating actinic pulse of 18,000 �mol m−2 s−1 for.7 s. Values of variable fluorescence (Fv = Fm − F0) and maximum

uantum efficiency of PSII photochemistry (Fv/Fm) were calculated

rom F0 and Fm. According to Maxwell and Johnson (2000), Fv/Fm

eflects the potential maximum efficiency of PSII (i.e. the quantumfficiency if all PSII centres were open).

ysiology 185 (2015) 75–83 77

The same leaf of each plant was used to measure light-adaptedparameters. Steady-state fluorescence yield (Fs) was recordedunder ambient light conditions. A saturating actinic pulse of18,000 �mol m−2 s−1 for 0.7 s was then used to produce maximumfluorescence yield (Fm

′) by temporarily inhibiting PSII photochem-istry. Using fluorescence parameters determined in both light-and dark-adapted states, the following were calculated: actualquantum yield of PSII photochemistry [�PSII = (Fm

′ – Fs)/Fm′] (Genty

et al., 1989) and quantum yield of non-photochemical quench-ing, which is the regulatory light-induced non-photochemicalquenching [�NPQ = (Fs/Fm

′) – (Fs/Fm)] (Lazár, 2015). �PSII relates toachieved efficiency in a plant under a given treatment and indicatesthe proportion of absorbed energy being used in photochemistry,while �NPQ provides an indication of the amount of energy that isdissipated in the form of heat (Maxwell and Johnson, 2000).

2.5.5. Pigments concentrations in leavesPhotosynthetic pigments of five leaves per treatment were

extracted using 0.1 g of fresh material in 5 ml 80% aqueous ace-tone. After filtering, 1 ml of the suspension was diluted with afurther 2 ml acetone, and chlorophyll a (Chl a), chlorophyll b (Chl b)and carotenoid (Cx + c) content were determined with a Hitachi U-2001 spectrophotometer (Hitachi Ltd., Japan), at 663.2, 646.8 and470.0 nm. The concentrations of pigments were calculated accord-ing to the formula provided by Lichtenthaler (1987).

2.5.6. Determination of rubisco activityRibulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39)

activity was measured following the method described by Lilleyand Walker (1974) with slight modifications described by Arocaet al. (2003). Leaf samples of 0.1 g FW were homogeneized in acold mortar using an extraction buffer containing 50 mM potas-sium phosphate buffer (pH 7.8), 1 mM EDTA, 8 mM MgCl2, 5 mMDTT (daily prepared) and 1% PVPP. The extract was clarified bycentrifugation at 26,850 × g for 10 min at 4 ◦C. Soluble proteinwas determined by the dye binding microassay (Bio-Rad, Madrid,Spain) using BSA as the standard (Bradford 1976). Rubisco totalactivity was calculated after incubating the leaf extract in 10 mMNaCO3H over 10 min in order to activate all rubisco protein. Analiquot of 100 �l of the supernatant was added to the reactionmixture (2 ml) containing 50 mM HEPES buffer, 20 mM MgCl2,0.6 mM ribulose-biphosphate, 0.2 mM NADH+, 5 mM ATP, 5 mMphosphocreatine, 4.8 units of creatine phosphokinase, 4.8 unitsof glyceraldehyde-3-phosphate dehydrogenase and 4.8 units ofphosphoglyceric phosphokinase. Enzyme activity was determinedby measuring the oxidation of NADH+, monitoring the decreaseof absorbance at 340 nm and 25 ◦C for 4 min, using a U-1900spectrophotometer (Hitachi Instruments, San José, CA, USA). Twoblanks, one without the enzyme extract and the other withoutNADH+ were used as controls.

2.5.7. RNA extraction, synthesis of cDNA and gene expressionanalyses

RNA was extracted from rice root and leaf samples by a phe-nol/chloroform extraction method followed by precipitation withLiCl and stored at −80 ◦C. The RNA was subjected to DNasetreatment and reverse-transcription using the QuantiTect ReverseTranscription Kit (Qiagen), following the instructions provided bymanufacturer. To rule out the possibility of a genomic DNA con-tamination, all the cDNA sets were checked by running control PCRreactions with aliquots of the same RNA that have been subjectedto the DNase treatment but not to the reverse transcription step.

Gene expression analyses were carried out by quantitativereverse transcription (qRT)-PCR using an iCycler iQ apparatus (Bio-Rad, Hercules, CA, U.S.A.). Individual real-time RT-PCR reactionswere assembled with oligonucleotide primers (0.15 �M each),

78 R. Porcel et al. / Journal of Plant Physiology 185 (2015) 75–83

Table 1Primers used in q-RT-PCR.

Gene name Accession Gene description Primer sequence Annealing temperature (◦C)

OsUBQ5 Forw AK061988 Housekeeping ubiquitin 5 5′-ACCACTTCGACCGCCACTACT-3′ 56OsUBQ5 Rev 5′-ACGCCTAAGCCTGCTGGTT-3′

OsrbcL Forw L24073 Chloroplast rubisco large subunit 5′-AGTTGAACAAATACGGTCGTC-3′ 565′-CGGCTAGACACTCATAACATGC-3′

5′-GCAGTTGATTAGCTTCATCGC-3′ 565′-TATTATAGGCAGCAATCCACC-3′

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Fig. 1. (A, B) Shoot fresh and dry weights and (C) shoot water content in rice plantscultivated under non-saline conditions (0 mM) or subjected to 75 mM or 150 mMNaCl. Plants remained as uninoculated controls (black columns) or were inocu-lated with the arbuscular mycorrhizal fungus Claroideoglomus etunicatum (white

OsrbcL Rev

OsrbcS Forw AY445627 Chloroplast rubisco small subunitOsrbcS Rev

0 �l of 2× KAPA SYBR® FAST qPCR Kit Master Mix (Kapa Biosys-ems, Boston, Massachusetts, USA) plus 1 �l of a 1:10 dilutionf each corresponding cDNA in a final volume of 20 �l. The PCRrogram consisted in a 4 min incubation at 95 ◦C to activate theot-start recombinant Taq DNA polymerase, followed by 32 cyclesf 30 s at 95 ◦C, 30 s at 56 ◦C and 40 s at 72 ◦C, where the fluorescenceignal was measured. The specificity of the PCR amplification pro-edure was checked with a heat dissociation protocol (from 60 ◦C to00 ◦C) after the final cycle of the PCR. Standardization was carriedut based on the expression of the rice ubiquitin gene (accessionK061988) in each sample. Primers used for qPCR experiments areresented in Table 1. The relative abundances of transcripts werealculated by using the 2−��Ct method (Livak and Schmittgen,001). Experiments were repeated three times, with the thresh-ld cycle (Ct) determined in triplicate, using cDNAs that originatedrom RNAs extracted from three different biological samples.

.6. Statistical analysis

Statistical analysis was performed using SPSS 19.0 statisticalrogram (SPSS Inc., Chicago, IL, USA). Data were subjected to analy-is of variance (ANOVA) with inoculation treatment, salt levels andheir interactions as sources of variation. Post hoc comparison withhe Duncan’s multiple range test (Duncan, 1955) was used to findut differences between groups with P < 0.05 as the significanceut-off.

. Results

.1. Plant biomass production, shoot water content and symbioticevelopment

AM plants produced higher shoot fresh and dry biomass thanonAM plants at whatever salt level assayed (Fig. 1A and B, Table 2).he increase in shoot dry weight ranged from 97% under non-salineonditions to 156% under 150 mM NaCl. In nonAM plants, salinityecreased the shoot biomass production similarly at both salt lev-ls. In AM plants shoot biomass decreased under 75 mM NaCl, butt was not significantly reduced at 150 mM NaCl.

The shoot water content was similar in AM and nonAM plantsnder non-saline and under 75 mM NaCl (Fig. 1C, Table 2), but at50 mM NaCl it decreased significantly in nonAM plants, reaching

ower values than in AM plants.The mycorrhizal root length achieved in rice roots was 49%

nder non-saline conditions, 59% under 75 mM NaCl and 63% under50 mM NaCl. No AM colonization was found in uninoculated ricelants.

.2. Plant gas exchange parameters and photosynthetic pigmentsoncentrations

The net photosynthetic rate (A), stomatal conductance (Gs) and

ranspiration rate (E) were higher in AM plants than in nonAM onest 0 and 150 mM NaCl, while differences were not significant at5 mM NaCl (Fig. 2A, B, and D, Table 2). The application of 150 mMaCl decreased A and E both in AM and in nonAM plants as com-

columns). Bars represent mean ± standard error. Different letters indicate significantdifferences (p < 0.05) (n = 15).

pared to non-saline conditions. The application of 75 mM NaCl alsoreduced E in AM plants. Finally, Gs was significantly reduced in AMplants at both saline levels. In spite of this reduction, at 150 mMNaCl, AM plants maintained higher E and Gs values than nonAMplants.

The intrinsic water use efficiency (iWUE) was similar in AM andnonAM plants at whatever salt level assayed (Fig. 2C, Table 2). Itonly increased as consequence of salt application at 150 mM NaCl.

R. Porcel et al. / Journal of Plant Physiology 185 (2015) 75–83 79

Fig. 2. (A) Net photosynthetic rate (A), (B) stomatal conductance (Gs), (C) intrinsicwater use efficiency (iWUE) and (D) transpiration rate (E) in rice plants cultivatedunder non-saline conditions (0 mM) or subjected to 75 mM or 150 mM NaCl. Plantsremained as uninoculated controls (black columns) or were inoculated with thearbuscular mycorrhizal fungus Claroideoglomus etunicatum (white columns). Barsrepresent mean ± standard error. Different letters indicate significant differences(p < 0.05) (n = 10).

Table 2Significance of sources of variation for each parameter after two-way ANOVAanalyses.

AM S AM × S

Shoot FW *** *** nsShoot DW *** *** nsShoot WC * * nsA *** ** nsGs *** ** nsiWUE ns * nsE *** ** *

Chl a * ns nsChl b ns ns nsCx + c * ns nsFv/Fm

*** ns nsϕPSII

** ns nsϕNPQ

*** ** nsRubisco activity ns *** **

Protein content ns ns nsOsrbcS ** ns nsOsrbcL ** ** ns

The sources of variation were AM inoculation (AM), salt level (S) and their interaction(AM × S). ns not significant.

*

p < 0.05.** p < 0.01.

*** p < 0.001.

The concentrations of chlorophyll a, chlorophyll b andcarotenoids did not significantly change with salt levels or withthe AM treatment (Fig. 3A, B, and C, Table 2). Significant differencesbetween AM and nonAM plants were only found for chlorophylla concentration at 150 mM NaCl. In the rest of treatments pig-ments concentrations were similar in AM and nonAM plants, butcarotenoids concentration diminished by 75 mM NaCl treatment inAM plants.

3.3. Chlorophyll fluorescence parameters

The maximum efficiency of PSII (Fv/Fm) was little affected bymycorrhization and salinity. This parameter was higher in nonAMplants under non-saline conditions and at 75 mM NaCl (Fig. 4A,Table 2). However, at 150 mM NaCl the values were similar in AMand nonAM plants, because it increased in the later.

The actual quantum yield of PSII photochemistry (�PSII) wassimilar in AM and nonAM plants under non-saline conditions, butresulted higher in AM plants than in nonAM ones at both salinelevels (Fig. 4B, Table 2). The increase in �PSII due to mycorrhizationwas by 25% at 75 mM NaCl and by 34% at 150 mM NaCl.

The quantum yield of non-photochemical quenching (�NPQ)was unaffected by salinity in AM plants, which maintained simi-lar levels under salinity than under non-saline conditions (Fig. 4C,Table 2). In contrast, �NPQ increased in nonAM plants as conse-quence of salinity. Moreover, at 75 and 150 mM NaCl, AM plantsexhibited significantly lower �NPQ than nonAM plants. The reduc-tion ranged from 30% under 75 mM NaCl to 40% under 150 mMNaCl.

3.4. Rubisco enzyme activity

The activity of rubisco was unaffected by salinity in nonAMplants, which maintained similar activity at all salinity levels(Fig. 5A, Table 2). In contrast, in AM plants the rubisco activityincreased with increasing salinity and was significantly higher thanin nonAM plants at 75 and at 150 mM NaCl. Thus, at 75 mM NaCl

the activity enhancement by mycorrhizal presence was by 76% andat 150 mM NaCl the increase was by 196%. Moreover, in AM plantsat 150 mM NaCl the rubisco activity was 96% higher than at 75 mMNaCl.

80 R. Porcel et al. / Journal of Plant Physiology 185 (2015) 75–83

Fig. 3. (A) Chlorophyll a content (Chl a), (B) Chlorophyll b content (Chl b) and (C)carotenoids content (Cx + c) in rice plants cultivated under non-saline conditions(0 mM) or subjected to 75 mM or 150 mM NaCl. Plants remained as uninoculatedcCD

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Fig. 4. (A) Maximum efficiency of photosystem II (Fv/Fm), (B) actual quantum yield ofPSII photochemistry (�PSII) and (C) quantum yield of non-photochemical quenching(�NPQ) in rice plants cultivated under non-saline conditions (0 mM) or subjected to75 mM or 150 mM NaCl. Plants remained as uninoculated controls (black columns)

ontrols (black columns) or were inoculated with the arbuscular mycorrhizal fungus

laroideoglomus etunicatum (white columns). Bars represent mean ± standard error.ifferent letters indicate significant differences (p < 0.05) (n = 10).

The total soluble protein content in leaves was not significantlyffected by either the AM inoculation or the salt treatment (Fig. 5B,able 2).

.5. Expression of genes encoding small (rbcS) and large (rbcL)ubisco subunits

Both, in AM and in nonAM plants, no significant differences wereound in the expression of rbcS gene as a consequence of salinityFig. 6A, Table 2). However, AM colonization affected the expressionf this gene under 75 and 150 mM NaCl. Thus, AM plants reducedhe expression of this gene by 44% and 38%, respectively.

In the case of rbcL gene, its expression was maintained constant

n AM plants at whatever salt level, being significantly lower (by7%) than in nonAM plants under non-saline conditions (Fig. 6B,able 2). NonAM plants exhibited the maximum expression underon saline conditions and it declined progressively as salinity in the

or were inoculated with the arbuscular mycorrhizal fungus Claroideoglomus etu-nicatum (white columns). Bars represent mean ± standard error. Different lettersindicate significant differences (p < 0.05) (n = 10).

medium increased. At 150 mM NaCl, the decline in gene expressionin nonAM plants was significant (by 51%) as compared to non-salineconditions. Nevertheless, at 75 and 150 mM NaCl, AM and nonAMplants exhibited similar rbcL gene expression levels.

4. Discussion

It is well known that salt stress can decrease photosyntheticability in plants, which leads to a decrease in crop production(Sheng et al., 2008). Plant biomass production is an integrative mea-surement of plant performance under many types of abiotic stressconditions and the symbiotic efficiency of AMF has been measured

in terms of plant growth improvement (see reviews by Evelin et al.,2009; Ruiz-Lozano et al., 2012). In this study, rice plants were wellcolonized by C. etunicatum and the level or root colonization rosewith increased salinity. This effect has been related with enhanced

R. Porcel et al. / Journal of Plant Ph

Fig. 5. (A) Total rubisco enzymatic activity, (B) total soluble leaf proteins in riceplants cultivated under non-saline conditions (0 mM) or subjected to 75 mM or150 mM NaCl. Plants remained as uninoculated controls (black columns) or wereinoculated with the arbuscular mycorrhizal fungus Claroideoglomus etunicatum(white columns). Bars represent mean ± standard error. Different letters indicatesignificant differences (p < 0.05) (n = 5).

Fig. 6. (A) Expression of Rbcs gene and (B) Rbcl gene in shoots of rice plants cultivatedunder non-saline conditions (0 mM) or subjected to 75 mM or 150 mM NaCl. Plantsremained as uninoculated controls (black columns) or were inoculated with thearbuscular mycorrhizal fungus Claroideoglomus etunicatum (white columns). Barsrepresent mean ± standard error. Different letters indicate significant differences(p < 0.05) (n = 4).

ysiology 185 (2015) 75–83 81

strigolactone production by plants subjected to salt stress (Arocaet al., 2013). It is also shown that AM rice plants produced highershoot fresh and dry biomass than nonAM plants, specially under150 mM NaCl. Moreover, salinity decreased the shoot biomass pro-duction in nonAM plants, while in AM plants shoot biomass onlydecreased transiently at 75 mM NaCl but it was not significantlyreduced at 150 mM NaCl. These results agree with several reportson salt stress alleviation by AM symbiosis (for reviews see Evelinet al., 2009; Dodd and Pérez-Alfocea, 2012; Porcel et al., 2012; Ruiz-Lozano et al., 2012).

The reduction in plant biomass production has been linked,among others, to direct effects of salinity on the plant photo-synthetic capacity. Primarily, salinity affects photosynthetic CO2assimilation because the osmotic component of salt stress reducesstomatal conductance. This, in turn, results in low CO2 supply torubisco. In a second phase, salinity might cause biochemical andphotochemical effects on photosynthesis (Duarte et al., 2013). Thus,the impairment in CO2 assimilation induces accumulation of excessenergy that if is not quenched may lead to excess electron accu-mulation from the photochemical phase in thylakoid membranes,particularly in the presence of high light intensity. This effect maylead to over-reduction of the reaction centres of PSII, causing dam-age to the photosynthetic apparatus (Redondo-Gómez et al., 2010).Plants have several strategies to protect the photosystems againstphotoinhibition and photodamage, including down-regulation inlight harvesting, excess energy dissipation by non-photochemicalquenching or cyclic electron flow (Lima-Neto et al., 2014).

In this study, salinity reduced net photosynthetic rate, stomatalconductance and transpiration in AM and in nonAM rice plants, sug-gesting that the reduction of photosynthetic rate was likely causedby stomatal limitations (Chen et al., 2014). However, AM rice plantsmaintained higher net photosynthetic rate, stomatal conductanceand transpiration rate than nonAM plants both under non-salineconditions and under 150 mM NaCl. Increased stomatal conduc-tance and transpiration in AM plants did not negatively influencedplant performance. The increase of gas exchange by the AM symbio-sis has been related to alterations of host plant hormonal levels andwith the enhanced uptake and translocation of water (Ebel et al.,1997; Goicoechea et al., 1997; Sheng et al., 2008; Ruiz-Lozano andAroca, 2010) and would normally translate into increased photo-synthesis (Birhane et al., 2012). On the other hand, the stimulationof carbohydrate transport and metabolism between source and sinktissues has been proposed as a mechanism to alleviate metabolicinhibitions of photosynthesis, avoiding thus photoinhibition dueto salinity (Dodd and Pérez-Alfocea, 2012). Plant roots become astrong sink for carbohydrates when colonized by AM fungi, as thesefungi can consume up to 20% of the host photosynthate (Feng et al.,2002; Heinemeyer et al., 2006). Thus, AM fungi modulate thesesource–sink relations by enhancing exchange of carbohydrates andmineral nutrients and can stimulate the rate of photosynthesis suf-ficiently to compensate for fungal carbon requirements (Kaschuket al., 2009; Dodd and Pérez-Alfocea, 2012). Indeed, in this studyat 150 mM NaCl the total soluble sugar content in roots was sig-nificantly higher in AM plants than in nonAM ones. No significantdifferences were found in the other saline levels (data not shown).

Chlorophyll content is a key factor for plant photosynthesis andclosely reflects the photosynthetic ability of plants such as rice(Takai et al., 2010). Enhanced chlorophyll concentration has beendescribed in AM plants (Giri and Mukerji, 2004; Sannazzaro et al.,2006; Colla et al., 2008). In the present study, a higher chlorophylla concentration was found in AM rice plants subjected to 150 mMNaCl. For the other photosynthetic pigments measured the increase

was not significant. Enhanced chlorophyll content in AM plants hasbeen related to increased P and Mg uptake (Zhu et al., 2014). AM riceplants also displayed higher rubisco activity under all salt levels,demonstrating a lower metabolic limitation of photosynthesis than

8 ant Ph

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2 R. Porcel et al. / Journal of Pl

onAM plants (Lima-Neto et al., 2014). Rubisco activity is a param-ter that is well correlated with CO2 assimilation (Sanz-Sáez et al.,013). Thus, the enhanced net photosynthetic activity of AM plantsould be also due to non-stomatal factors such as higher chloro-hyll a content and rubisco activity (Chen et al., 2014). However,he rubisco enzymatic activity did not correlate with expressionf genes encoding for small (rbcS) and large (rcbL) rubisco sub-nits. The rbcS gene reduced its expression in AM plants under 75nd 150 mM NaCl and the rbcL gene expression was similar in AMnd nonAM plants at 75 and 150 mM NaCl, while its expressionas lower in AM plants under non-saline conditions. In any case,

anz-Sáez et al. (2013) also found that rubisco activity was not coor-inated with gene expression, possibly due to a lag between generanscription and protein translation.

Because of its sensitivity to abiotic stresses, PSII activity has beenidely used to study response and adaptation to stress by plants

Strasser et al., 2000). When the metabolism of a plant is disturbedy biotic or abiotic stresses, redundant energy hast to be dissipated

n order to avoid damage of plant tissues. Dissipation results viaon-photochemical processes like heat or chlorophyll fluorescencePinior et al., 2005). The parameters of Chl fluorescence reflect accu-ately photosynthetic ability and energy conversion efficiency (Zhut al., 2014). The maximum quantum yield of primary photochem-stry (Fv/Fm) reflects the potential quantum efficiency of PSII and issed as an index of plant photosynthetic performance, with opti-al values for most plant species of around 0.83 (Björkman andemmig, 1987). In this study, values of Fv/Fm ranged from 0.68 and.78 in the different treatments, which suggests that the perfor-ance of the photosynthetic apparatus was not at the optimum

evel (Bagheri et al., 2011). However, data also showed that underalinity the actual quantum yield of PSII photochemistry (�PSII) wasnhanced by AM symbiosis. The increase in �PSII due to mycor-hization was by 25% and 34% at 75 and 150 mM NaCl, respectively.t the same time, �NPQ was significantly lower in AM plants underoth saline levels. The reduction ranged from 30% under 75 mMaCl to 40% under 150 mM NaCl. Moreover, �NPQ increased inonAM plants as consequence of salinity, but resulted unaffectedy salinity in AM plants. Normally, �NPQ increases as a mechanismo protect the leaf from light-induced damage, but this means thathotochemical processes are reduced proportionally (Maxwell and

ohnson, 2000; Baker, 2008; Lazár, 2015). Sheng et al. (2008) foundhat AM symbiosis triggered the regulation of energy bifurcationetween photochemical and non-photochemical events. Thus, ourata indicate that AM rice plants had a higher photochemical effi-iency for CO2 fixation and solar energy utilization and that AMnoculation would reduce the light-induced damage due to salinityZhu et al., 2014). As a result, AM symbiosis increases salt tolerancen rice plants by preventing the injury to the photosystems reac-ion centres and by allowing a better utilization of light energy inhotochemical processes (Pinior et al., 2005; Bagheri et al., 2011),educing at the same time light energy dissipation as heat. In agree-

ent with our results, Sheng et al. (2008) found that salt stressould destroy PSII reaction centres or disrupt electron transport inhotosynthetic apparatus of AM and nonAM maize plants subjectedo several salinity levels, but the toxic influence of salinity on maizeSII reaction centres were mitigated by the AM symbiosis. Theseffects of the AM symbiosis enhancing �PSII and reducing �NPQay be related to the sink stimulation of AM symbiosis. Kaschuk

t al. (2009) showed that the carbon sink stregth due to the fungalresence in the plant root stimulates the host plant, increasing thehotosynthetic rate. In this context, Chen et al. (2014) found thatink strength of AM symbiosis could alleviate the negative effects of

ow-Zn on PSII. Moreover, it has been found in citrus plants that AMymbiosis reduced leaf temperature under drought stress condi-ions (Wu and Xia, 2006). Authors related this finding with a lower

ysiology 185 (2015) 75–83

resistance in AM plants to vapour transfer from inside the leaves tothe atmosphere.

In conclusion, the present results show that the AM symbio-sis enhances the actual quantum yield of PSII photochemistry andreduces �NPQ in rice plants subjected to salinity. Thus, we pro-pose that AM rice plants had a higher photochemical efficiency forCO2 fixation and solar energy utilization and this increases plantsalt tolerance by preventing the injury to the photosystems reac-tion centres and by allowing a better utilization of light energy inphotochemical processes, reducing at the same time light energydissipation as heat. All these processes translated into a higher pho-tosynthetic and rubisco activities in AM rice plants and improvedplant biomass production under salinity.

Acknowledgments

This work was financed by a research project supported by Juntade Andalucía (Spain). Project P11-CVI-7107. We would like to thankMichael O’shea for proofreading the document.

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