Journal of Plant Physiology 185 (2015) 75–83
Contents lists available at ScienceDirect
Journal of Plant Physiology
journal homepage: www.elsevier.com/locate/jplph
Physiology
Arbuscular mycorrhizal symbiosis ameliorates the optimum quantum
yield of photosystem II and reduces non-photochemical quenching in
rice plants subjected to salt stress
Rosa Porcel a , Susana Redondo-Gómez b , Enrique Mateos-Naranjo b , Ricardo Aroca a ,
Rosalva 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, Spain
Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, Apartado 1095, 41080 Sevilla, Spain
c
Facultad de Estudios Superiores Zaragoza, Universidad Nacional Autónoma, Mexico
a
b
a r t i c l e
i n f o
Article history:
Received 12 June 2015
Received in revised form 15 July 2015
Accepted 17 July 2015
Available online 31 July 2015
Keywords:
Arbuscular mycorrhizal symbiosis
Non-photochemical quenching
Optimum quantum yield
Oryza sativa
Photosystem II
Salt 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 half
of the world population. However, salinity is considered the most common abiotic stress reducing its
productivity. Soil salinity inhibits photosynthetic processes, which can induce an over-reduction of the
reaction centres in photosystem II (PSII), damaging the photosynthetic machinery. The arbuscular mycorrhizal (AM) symbiosis may improve host plant tolerance to salinity, but it is not clear how the AM
symbiosis affects the plant photosynthetic capacity, particularly the efficiency of PSII. This study aimed
at determining the influence of the AM symbiosis on the performance of PSII in rice plants subjected
to salinity. Photosynthetic activity, plant gas-exchange parameters, accumulation of photosynthetic pigments and rubisco activity and gene expression were also measured in order to analyse comprehensively
the response of the photosynthetic processes to AM symbiosis and salinity. Results showed that the AM
symbiosis enhanced the actual quantum yield of PSII photochemistry and reduced the quantum yield of
non-photochemical quenching in rice plants subjected to salinity. AM rice plants maintained higher net
photosynthetic 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 utilization
and this increases plant salt tolerance by preventing the injury to the photosystems reaction centres and
by allowing a better utilization of light energy in photochemical processes. All these processes translated into higher photosynthetic and rubisco activities in AM rice plants and improved plant biomass
production under salinity.
© 2015 Elsevier GmbH. All rights reserved.
1. Introduction
Rice (Oryza sativa L.) is the most important food crop in the
world and is a primary source of food for more than half of the
world population (Kumar et al., 2013). According to FAO (2005),
world agriculture should produce 70% more food for an additional
2.3 billion people by 2050. However, rice is a salt sensitive crop
and salinity is considered the most common abiotic stress reducing its productivity (Kumar et al., 2013). Indeed, salinity is a major
and increasing environmental problem, affecting over 6% of the
total land area of the world. Thus, investigating different strategies
∗ Corresponding author. Fax: +34 958 129600.
E-mail address: [email protected] (J.M. Ruiz-Lozano).
http://dx.doi.org/10.1016/j.jplph.2015.07.006
0176-1617/© 2015 Elsevier GmbH. All rights reserved.
to improve rice productivity under salinity is an important challenge to cope with reduced food production due to excessive soil
salinization. Several studies have shown that the arbuscular mycorrhizal (AM) symbiosis can alleviate salt stress in different host plant
species (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, among
other processes, to inhibition of photosynthetic processes (Pitman
and Läuchli, 2002). Indeed, salinity inhibits specific enzymes
involved for the synthesis of photosynthetic pigments, causing a
reduction in plant chlorophyll content (Giri and Mukerji, 2004;
Murkute et al., 2006; Sheng et al., 2008). Moreover, the lowering of the photosynthetic rate caused by salt stress can induce
an over-reduction of the reaction centres in photosystem II (PSII)
and this may damage the photosynthetic machinery if the plant
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R. Porcel et al. / Journal of Plant Physiology 185 (2015) 75–83
is unable to dissipate the excess energy (Baker, 2008). Indeed, the
light energy absorbed by chlorophyll molecules can be used either
to drive photosynthesis, it can be re-emitted as light-chlorophyll
fluorescence or the excess energy can be dissipated as heat. These
three processes occur in a competitive way, so that any increase
in the efficiency of one will decrease the yield of the other two
(Maxwell and Johnson, 2000; Harbinson, 2013). Thus, the ability of
the plant to dissipate or not the excess energy can be quantified by
measuring the chlorophyll a fluorescence.
Improvements in photosynthetic activity or water use efficiency have been reported in AM plants growing under salt stress
(Sheng et al., 2008; Zuccarini and Okurowska, 2008; Hajiboland
et al., 2010) or under drought stress (Birhane et al., 2012; Liu
et al., 2015). Nevertheless, few studies have investigated so far
the influence of AM fungi on leaf photochemical properties under
salt stress. Sheng et al. (2008) found that the AM symbiosis
improved the photosynthetic capacity of maize plants, mainly
by regulating the energy bifurcation between photochemical and
non-photochemical events and elevating the efficiency of photochemistry and non-photochemistry of PSII. However, Sheng et al.
(2008) attributed the influence of the AM symbiosis on maize photosynthetic capacity to a mycorrhiza-mediated enhancement of
plant water status, rather than to a direct influence on the efficiency of PSII. Hajiboland et al. (2010) found that mycorrhization
improved photosynthetic activity in tomato plants through both,
elevating stomatal conductance and protecting PSII photochemical processes against salinity. However, authors suggested that AM
colonization acted only on maintenance of photochemical capacity in stressed leaves and did not increase its potential for energy
trapping, since the enhancement of the PSII photochemistry by AM
fungi did not occur in plants not subjected to salt stress. Other studies have shown modulation of PSII efficiency by the AM symbiosis in
rose, pistachio and poplar plants subjected to drought (Pinior et al.,
2005; Bagheri et al., 2011; Liu et al., 2015), in citrus plants growing
in low-zinc soil (Chen et al., 2014), as well as, under non-stressful
conditions in maize and black locust seedlings (Rai et al., 2008;
Zhu et al., 2014). Nevertheless, so far, it is not clear how the AM
symbiosis affects the plant photosynthetic capacity, particularly
the efficiency of photosystem II in plants subjected to salinity.
The activity of enzymes involved in carbon assimilation such
as the ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco)
is also determinant for plant photosynthetic efficiency (Masumoto
et al., 2005; Goicoechea et al., 2014). In a study with grapevine
plants, Valentine et al. (2006) found that AM plants subjected
to drought had higher rubisco activity and water use efficiency
than non AM plants. However, these authors did not directly
measure the enzymatic activity; they calculated rubisco activity
from CO2 response curves measured with an infrared gas analyzer
(IRGA). More recently, Goicoechea et al. (2014) carried out a semiquantification of the large (RLS) and small (RSS) rubisco subunits
in alfalfa with no significant differences between AM and nonAM
plants. However, to our knowledge, no data are available about
direct enzymatic rubisco activity measured in AM plants subjected
to salinity. Thus, this aspect deserves to be examined in studies
dealing with salt stress alleviation by the AM symbiosis, in combination with molecular studies aimed at evaluating the expression
pattern of genes encoding for the small (rbcS) and large (rbcL)
rubisco subunits (Tsutsumi et al., 2008).
The present study aimed at determining the influence of the
AM symbiosis on the performance of PSII in rice plants subjected to increasing salinity levels. Thus, chlorophyll a fluorescence
was measured to calculate the maximum quantum efficiency
of PSII photochemistry (Fv /Fm ), actual quantum yield of photosystem II photochemistry (PSII ), as well as, quantum yield of
non-photochemical quenching (NPQ ) (Lazár, 2015). Photosynthetic activity, plant gas-exchange parameters, accumulation of
photosynthetic pigments, activity of rubisco enzyme and expression of rubisco-encoding genes were also quantified in order to
analyse comprehensively the response of the photosynthetic processes in rice to AM symbiosis and salinity. The starting hypotesis
is that the AM symbiosis will alter the photosynthetic capacity of
rice plants by changing plant gas-exchange parameters and performance of photosystem II.
2. Materials and methods
2.1. Experimental design
The experiment consisted of a randomized complete block
design with two inoculation treatments: (1) non-mycorrhizal
control plants, (2) plants inoculated with the AM fungus
Claroideoglomus etunicatum (isolate EEZ 163). There were 30 replicates of each inoculation treatment, totalling 60 pots (two plants
per pot), so that ten pots of each inoculation treatment were grown
under non-saline conditions throughout the entire experiment,
while ten pots per treatment were subjected to 75 mM of NaCl
for four weeks and the remaining ten pots per treatment were
subjected 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 the
original soil was 0.2 dS m−1 .
Three indica rice (O. sativa L.) seedlings (cv puntal), previously
germinated on sand, were sown in pots containing 900 g of the same
soil/sand/vermiculite mixture as described above and thinned to
two seedlings per pot after three days.
2.3. Inoculation treatments
Mycorrhizal inoculum was bulked in an open-pot culture of
Zea mays L. and consisted of soil, spores, mycelia and infected
root fragments. The AM fungus used in this study had been previously isolated from Cabo de Gata Natural Park (Almería, Spain,
36◦ 45 24 N 02◦ 13 17 W), which is an area with serious problems
of salinity and affected by desertification. The AMF species was C.
etunicatum (isolate EEZ 163), previously characterized as an efficient AM fungus under salinity (Estrada et al., 2013a,b). Appropriate
amounts of the inoculum containing about 700 infective propagules (according to the most probable number test), were added to
the corresponding pots at sowing time just below rice seedlings.
Non-mycorrhizal control plants received the same amount of autoclaved mycorrhizal inocula together with a 10 ml aliquot of a filtrate
(<20 ␮m) of the AM inocula in order to provide a general microbial
population free of AM propagules.
2.4. Growth conditions
The experiment was carried out under glasshouse conditions
with temperatures ranging from 19 to 25 ◦ C, 16/8 light/dark period,
and a relative humidity of 50–60%. At the leaf level, a photosynthetic
photon flux density of 800 ␮mol m−2 s−1 was measured with a light
meter (LICOR, Lincoln, NE, USA, model LI-188B). Water was supplied daily to the entire period of plant growth to avoid any drought
effect. Plants were established for five weeks prior to salinization to
allow adequate plant and symbiotic establishment. After that time,
R. Porcel et al. / Journal of Plant Physiology 185 (2015) 75–83
a group of plants were kept under non-saline solutions, by irrigating
with water until the end of the experiment (0 mM NaCl), while two
groups of each inoculation treatments were watered with an aqueous solution containing 75 or 150 mM NaCl, respectively. Plants
were maintained under these conditions for additional four weeks.
During this period, plants received each week 10 ml per pot of
Hoagland nutrient solution containing only ¼ P concentration to
avoid inhibition of AM root colonization. At the end of the experiment, the electrical conductivities in the soil:sand:vermiculite
mixture used as growing substrate were 0.5, 3.4 and 6.3 dS m−1
for the salt levels of 0, 75, and 150 mM NaCl, respectively.
2.5. Parameters measured
2.5.1. Biomass production
At harvest (60 days after planting), the shoot and root system were separated and the shoot dry weight (SDW) and root dry
weight (RDW) was measured after drying in a forced hot-air oven
at 70 ◦ C for two days. The shoot water content was calculated as
(FW-DW)/FW (Marulanda et al., 2007), and expressed as g H2 O per
g of FW.
2.5.2. Symbiotic development
The percentage of mycorrhizal root infection in maize plants
was estimated by visual observation of fungal colonization after
clearing washed roots in 10% KOH and staining with 0.05% trypan
blue in lactic acid (v/v), as described by Phillips and Hayman (1970).
The extent of mycorrhizal colonization was calculated according to
the gridline intersect method (Giovannetti and Mosse, 1980).
2.5.3. Plant gas-exchange and photosynthetic parameters
Measurements were taken on the second youngest leaf from
each plant (n = 7, per treatment) using an infrared gas analyzer in
an open system (LCI-portable, ADC system, England). Net photosynthetic rate (A), intercellular CO2 concentration (Ci) and stomatal
conductance (Gs) were all determined at an ambient CO2 concentration of 390 ␮mol mol−1 , temperature of 25/30 ◦ C, 50 ± 5%
relative humidity and a PFD of 1000 ␮mol m−2 s−1 . A, Gs and transpiration rate (E) were calculated using standard formulae from
Von Caemmerer and Farquhar (1981). Intrinsic water-use efficiency
(iWUE) was calculated as the ratio between A and Gs [mmol (CO2
assimilated) mol−1 (H2 O transpired)]. Measurements were made
at midday.
2.5.4. Chlorophyll fluorescence parameters
Chlorophyll fluorescence was measured as described by
Redondo-Gómez et al. (2010) using a portable modulated fluorimeter (FMS-2, Hansatech Instruments Ltd., UK). Measurements
were made on 10 plants per treatment (n = 10). Light- and
dark-adapted fluorescence parameters were measured at midday (1400 ␮mol m−2 s−1 ) to investigate whether salt concentration
affected the sensitivity of plants to photoinhibition (Maxwell and
Johnson, 2000).
Plants were dark-adapted for 30 min, using leaf clips designed
for this purpose. Minimal fluorescence in the dark-adapted state
(F0 ) was measured using a modulated pulse (<0.05 ␮mol m−2 s−1
for 1.6 ␮s) which was too small to induce significant physiological
changes in the plant. The stored data were averages taken over a
1.6-s period. Maximal fluorescence in this state (Fm ) was measured
after applying a saturating actinic pulse of 18,000 ␮mol m−2 s−1 for
0.7 s. Values of variable fluorescence (Fv = Fm − F0 ) and maximum
quantum efficiency of PSII photochemistry (Fv /Fm ) were calculated
from F0 and Fm . According to Maxwell and Johnson (2000), Fv /Fm
reflects the potential maximum efficiency of PSII (i.e. the quantum
efficiency if all PSII centres were open).
77
The same leaf of each plant was used to measure light-adapted
parameters. Steady-state fluorescence yield (Fs ) was recorded
under ambient light conditions. A saturating actinic pulse of
18,000 ␮mol m−2 s−1 for 0.7 s was then used to produce maximum
fluorescence yield (Fm ) by temporarily inhibiting PSII photochemistry. Using fluorescence parameters determined in both lightand dark-adapted states, the following were calculated: actual
quantum yield of PSII photochemistry [PSII = (Fm – Fs )/Fm ] (Genty
et al., 1989) and quantum yield of non-photochemical quenching, which is the regulatory light-induced non-photochemical
quenching [NPQ = (Fs /Fm ) – (Fs /Fm )] (Lazár, 2015). PSII relates to
achieved efficiency in a plant under a given treatment and indicates
the proportion of absorbed energy being used in photochemistry,
while NPQ provides an indication of the amount of energy that is
dissipated in the form of heat (Maxwell and Johnson, 2000).
2.5.5. Pigments concentrations in leaves
Photosynthetic pigments of five leaves per treatment were
extracted using 0.1 g of fresh material in 5 ml 80% aqueous acetone. After filtering, 1 ml of the suspension was diluted with a
further 2 ml acetone, and chlorophyll a (Chl a), chlorophyll b (Chl b)
and carotenoid (Cx + c) content were determined with a Hitachi U2001 spectrophotometer (Hitachi Ltd., Japan), at 663.2, 646.8 and
470.0 nm. The concentrations of pigments were calculated according to the formula provided by Lichtenthaler (1987).
2.5.6. Determination of rubisco activity
Ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39)
activity was measured following the method described by Lilley
and Walker (1974) with slight modifications described by Aroca
et al. (2003). Leaf samples of 0.1 g FW were homogeneized in a
cold mortar using an extraction buffer containing 50 mM potassium phosphate buffer (pH 7.8), 1 mM EDTA, 8 mM MgCl2 , 5 mM
DTT (daily prepared) and 1% PVPP. The extract was clarified by
centrifugation at 26,850 × g for 10 min at 4 ◦ C. Soluble protein
was determined by the dye binding microassay (Bio-Rad, Madrid,
Spain) using BSA as the standard (Bradford 1976). Rubisco total
activity was calculated after incubating the leaf extract in 10 mM
NaCO3 H over 10 min in order to activate all rubisco protein. An
aliquot of 100 ␮l of the supernatant was added to the reaction
mixture (2 ml) containing 50 mM HEPES buffer, 20 mM MgCl2 ,
0.6 mM ribulose-biphosphate, 0.2 mM NADH+ , 5 mM ATP, 5 mM
phosphocreatine, 4.8 units of creatine phosphokinase, 4.8 units
of glyceraldehyde-3-phosphate dehydrogenase and 4.8 units of
phosphoglyceric phosphokinase. Enzyme activity was determined
by measuring the oxidation of NADH+ , monitoring the decrease
of absorbance at 340 nm and 25 ◦ C for 4 min, using a U-1900
spectrophotometer (Hitachi Instruments, San José, CA, USA). Two
blanks, one without the enzyme extract and the other without
NADH+ were used as controls.
2.5.7. RNA extraction, synthesis of cDNA and gene expression
analyses
RNA was extracted from rice root and leaf samples by a phenol/chloroform extraction method followed by precipitation with
LiCl and stored at −80 ◦ C. The RNA was subjected to DNase
treatment and reverse-transcription using the QuantiTect Reverse
Transcription Kit (Qiagen), following the instructions provided by
manufacturer. To rule out the possibility of a genomic DNA contamination, all the cDNA sets were checked by running control PCR
reactions with aliquots of the same RNA that have been subjected
to the DNase treatment but not to the reverse transcription step.
Gene expression analyses were carried out by quantitative
reverse transcription (qRT)-PCR using an iCycler iQ apparatus (BioRad, Hercules, CA, U.S.A.). Individual real-time RT-PCR reactions
were assembled with oligonucleotide primers (0.15 ␮M each),
78
R. Porcel et al. / Journal of Plant Physiology 185 (2015) 75–83
Table 1
Primers used in q-RT-PCR.
Gene name
OsUBQ5 Forw
OsUBQ5 Rev
OsrbcL Forw
OsrbcL Rev
OsrbcS Forw
OsrbcS Rev
Accession
Gene description
AK061988
Housekeeping ubiquitin 5
L24073
Chloroplast rubisco large subunit
AY445627
Chloroplast rubisco small subunit
Annealing temperature (◦ C)
Primer sequence
5 -ACCACTTCGACCGCCACTACT-3
5 -ACGCCTAAGCCTGCTGGTT-3
5 -AGTTGAACAAATACGGTCGTC-3
5 -CGGCTAGACACTCATAACATGC-3
5 -GCAGTTGATTAGCTTCATCGC-3
5 -TATTATAGGCAGCAATCCACC-3
56
56
56
10 ␮l of 2× KAPA SYBR® FAST qPCR Kit Master Mix (Kapa Biosystems, Boston, Massachusetts, USA) plus 1 ␮l of a 1:10 dilution
of each corresponding cDNA in a final volume of 20 ␮l. The PCR
program consisted in a 4 min incubation at 95 ◦ C to activate the
hot-start recombinant Taq DNA polymerase, followed by 32 cycles
of 30 s at 95 ◦ C, 30 s at 56 ◦ C and 40 s at 72 ◦ C, where the fluorescence
signal was measured. The specificity of the PCR amplification procedure was checked with a heat dissociation protocol (from 60 ◦ C to
100 ◦ C) after the final cycle of the PCR. Standardization was carried
out based on the expression of the rice ubiquitin gene (accession
AK061988) in each sample. Primers used for qPCR experiments are
presented in Table 1. The relative abundances of transcripts were
calculated by using the 2− Ct method (Livak and Schmittgen,
2001). Experiments were repeated three times, with the threshold cycle (Ct) determined in triplicate, using cDNAs that originated
from RNAs extracted from three different biological samples.
2.6. Statistical analysis
Statistical analysis was performed using SPSS 19.0 statistical
program (SPSS Inc., Chicago, IL, USA). Data were subjected to analysis of variance (ANOVA) with inoculation treatment, salt levels and
their interactions as sources of variation. Post hoc comparison with
the Duncan’s multiple range test (Duncan, 1955) was used to find
out differences between groups with P < 0.05 as the significance
cut-off.
3. Results
3.1. Plant biomass production, shoot water content and symbiotic
development
AM plants produced higher shoot fresh and dry biomass than
nonAM plants at whatever salt level assayed (Fig. 1A and B, Table 2).
The increase in shoot dry weight ranged from 97% under non-saline
conditions to 156% under 150 mM NaCl. In nonAM plants, salinity
decreased the shoot biomass production similarly at both salt levels. In AM plants shoot biomass decreased under 75 mM NaCl, but
it was not significantly reduced at 150 mM NaCl.
The shoot water content was similar in AM and nonAM plants
under non-saline and under 75 mM NaCl (Fig. 1C, Table 2), but at
150 mM NaCl it decreased significantly in nonAM plants, reaching
lower values than in AM plants.
The mycorrhizal root length achieved in rice roots was 49%
under non-saline conditions, 59% under 75 mM NaCl and 63% under
150 mM NaCl. No AM colonization was found in uninoculated rice
plants.
3.2. Plant gas exchange parameters and photosynthetic pigments
concentrations
The net photosynthetic rate (A), stomatal conductance (Gs) and
transpiration rate (E) were higher in AM plants than in nonAM ones
at 0 and 150 mM NaCl, while differences were not significant at
75 mM NaCl (Fig. 2A, B, and D, Table 2). The application of 150 mM
NaCl decreased A and E both in AM and in nonAM plants as com-
Fig. 1. (A, B) Shoot fresh and dry weights and (C) shoot water content in rice plants
cultivated under non-saline conditions (0 mM) or subjected to 75 mM or 150 mM
NaCl. Plants remained as uninoculated controls (black columns) or were inoculated with the arbuscular mycorrhizal fungus Claroideoglomus etunicatum (white
columns). Bars represent mean ± standard error. Different letters indicate significant
differences (p < 0.05) (n = 15).
pared to non-saline conditions. The application of 75 mM NaCl also
reduced E in AM plants. Finally, Gs was significantly reduced in AM
plants at both saline levels. In spite of this reduction, at 150 mM
NaCl, AM plants maintained higher E and Gs values than nonAM
plants.
The intrinsic water use efficiency (iWUE) was similar in AM and
nonAM plants at whatever salt level assayed (Fig. 2C, Table 2). It
only increased as consequence of salt application at 150 mM NaCl.
R. Porcel et al. / Journal of Plant Physiology 185 (2015) 75–83
79
Table 2
Significance of sources of variation for each parameter after two-way ANOVA
analyses.
Shoot FW
Shoot DW
Shoot WC
A
Gs
iWUE
E
Chl a
Chl b
Cx + c
Fv /Fm
ϕPSII
ϕNPQ
Rubisco activity
Protein content
OsrbcS
OsrbcL
AM
S
AM × S
***
***
***
***
*
*
***
**
ns
ns
ns
ns
ns
ns
***
**
ns
*
***
**
*
*
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
*
***
**
***
**
ns
ns
***
**
ns
ns
ns
ns
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 and
carotenoids did not significantly change with salt levels or with
the AM treatment (Fig. 3A, B, and C, Table 2). Significant differences
between AM and nonAM plants were only found for chlorophyll
a concentration at 150 mM NaCl. In the rest of treatments pigments concentrations were similar in AM and nonAM plants, but
carotenoids concentration diminished by 75 mM NaCl treatment in
AM plants.
3.3. Chlorophyll fluorescence parameters
The maximum efficiency of PSII (Fv /Fm ) was little affected by
mycorrhization and salinity. This parameter was higher in nonAM
plants under non-saline conditions and at 75 mM NaCl (Fig. 4A,
Table 2). However, at 150 mM NaCl the values were similar in AM
and nonAM plants, because it increased in the later.
The actual quantum yield of PSII photochemistry (PSII ) was
similar in AM and nonAM plants under non-saline conditions, but
resulted higher in AM plants than in nonAM ones at both saline
levels (Fig. 4B, Table 2). The increase in PSII due to mycorrhization
was 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 similar levels under salinity than under non-saline conditions (Fig. 4C,
Table 2). In contrast, NPQ increased in nonAM plants as consequence of salinity. Moreover, at 75 and 150 mM NaCl, AM plants
exhibited significantly lower NPQ than nonAM plants. The reduction ranged from 30% under 75 mM NaCl to 40% under 150 mM
NaCl.
Fig. 2. (A) Net photosynthetic rate (A), (B) stomatal conductance (Gs), (C) intrinsic
water use efficiency (iWUE) and (D) transpiration rate (E) in rice plants cultivated
under non-saline conditions (0 mM) or subjected to 75 mM or 150 mM NaCl. Plants
remained as uninoculated controls (black columns) or were inoculated with the
arbuscular mycorrhizal fungus Claroideoglomus etunicatum (white columns). Bars
represent mean ± standard error. Different letters indicate significant differences
(p < 0.05) (n = 10).
3.4. Rubisco enzyme activity
The activity of rubisco was unaffected by salinity in nonAM
plants, which maintained similar activity at all salinity levels
(Fig. 5A, Table 2). In contrast, in AM plants the rubisco activity
increased with increasing salinity and was significantly higher than
in nonAM plants at 75 and at 150 mM NaCl. Thus, at 75 mM NaCl
the activity enhancement by mycorrhizal presence was by 76% and
at 150 mM NaCl the increase was by 196%. Moreover, in AM plants
at 150 mM NaCl the rubisco activity was 96% higher than at 75 mM
NaCl.
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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 uninoculated
controls (black columns) or were inoculated with the arbuscular mycorrhizal fungus
Claroideoglomus etunicatum (white columns). Bars represent mean ± standard error.
Different letters indicate significant differences (p < 0.05) (n = 10).
The total soluble protein content in leaves was not significantly
affected by either the AM inoculation or the salt treatment (Fig. 5B,
Table 2).
3.5. Expression of genes encoding small (rbcS) and large (rbcL)
rubisco subunits
Fig. 4. (A) Maximum efficiency of photosystem II (Fv /Fm ), (B) actual quantum yield of
PSII photochemistry (PSII ) and (C) quantum yield of non-photochemical quenching
(NPQ ) in rice plants cultivated under non-saline conditions (0 mM) or subjected to
75 mM or 150 mM NaCl. Plants remained as uninoculated controls (black columns)
or were inoculated with the arbuscular mycorrhizal fungus Claroideoglomus etunicatum (white columns). Bars represent mean ± standard error. Different letters
indicate significant differences (p < 0.05) (n = 10).
medium increased. At 150 mM NaCl, the decline in gene expression
in nonAM plants was significant (by 51%) as compared to non-saline
conditions. Nevertheless, at 75 and 150 mM NaCl, AM and nonAM
plants exhibited similar rbcL gene expression levels.
4. Discussion
Both, in AM and in nonAM plants, no significant differences were
found in the expression of rbcS gene as a consequence of salinity
(Fig. 6A, Table 2). However, AM colonization affected the expression
of this gene under 75 and 150 mM NaCl. Thus, AM plants reduced
the expression of this gene by 44% and 38%, respectively.
In the case of rbcL gene, its expression was maintained constant
in AM plants at whatever salt level, being significantly lower (by
47%) than in nonAM plants under non-saline conditions (Fig. 6B,
Table 2). NonAM plants exhibited the maximum expression under
non saline conditions and it declined progressively as salinity in the
It is well known that salt stress can decrease photosynthetic
ability in plants, which leads to a decrease in crop production
(Sheng et al., 2008). Plant biomass production is an integrative measurement of plant performance under many types of abiotic stress
conditions 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 well
colonized by C. etunicatum and the level or root colonization rose
with increased salinity. This effect has been related with enhanced
R. Porcel et al. / Journal of Plant Physiology 185 (2015) 75–83
Fig. 5. (A) Total rubisco enzymatic activity, (B) total soluble leaf proteins in rice
plants cultivated under non-saline conditions (0 mM) or subjected to 75 mM or
150 mM NaCl. Plants remained as uninoculated controls (black columns) or were
inoculated with the arbuscular mycorrhizal fungus Claroideoglomus etunicatum
(white columns). Bars represent mean ± standard error. Different letters indicate
significant differences (p < 0.05) (n = 5).
Fig. 6. (A) Expression of Rbcs gene and (B) Rbcl gene in shoots of rice plants cultivated
under non-saline conditions (0 mM) or subjected to 75 mM or 150 mM NaCl. Plants
remained as uninoculated controls (black columns) or were inoculated with the
arbuscular mycorrhizal fungus Claroideoglomus etunicatum (white columns). Bars
represent mean ± standard error. Different letters indicate significant differences
(p < 0.05) (n = 4).
81
strigolactone production by plants subjected to salt stress (Aroca
et al., 2013). It is also shown that AM rice plants produced higher
shoot fresh and dry biomass than nonAM plants, specially under
150 mM NaCl. Moreover, salinity decreased the shoot biomass production in nonAM plants, while in AM plants shoot biomass only
decreased transiently at 75 mM NaCl but it was not significantly
reduced at 150 mM NaCl. These results agree with several reports
on salt stress alleviation by AM symbiosis (for reviews see Evelin
et al., 2009; Dodd and Pérez-Alfocea, 2012; Porcel et al., 2012; RuizLozano et al., 2012).
The reduction in plant biomass production has been linked,
among others, to direct effects of salinity on the plant photosynthetic capacity. Primarily, salinity affects photosynthetic CO2
assimilation because the osmotic component of salt stress reduces
stomatal conductance. This, in turn, results in low CO2 supply to
rubisco. In a second phase, salinity might cause biochemical and
photochemical effects on photosynthesis (Duarte et al., 2013). Thus,
the impairment in CO2 assimilation induces accumulation of excess
energy that if is not quenched may lead to excess electron accumulation from the photochemical phase in thylakoid membranes,
particularly in the presence of high light intensity. This effect may
lead to over-reduction of the reaction centres of PSII, causing damage to the photosynthetic apparatus (Redondo-Gómez et al., 2010).
Plants have several strategies to protect the photosystems against
photoinhibition and photodamage, including down-regulation in
light harvesting, excess energy dissipation by non-photochemical
quenching or cyclic electron flow (Lima-Neto et al., 2014).
In this study, salinity reduced net photosynthetic rate, stomatal
conductance and transpiration in AM and in nonAM rice plants, suggesting that the reduction of photosynthetic rate was likely caused
by stomatal limitations (Chen et al., 2014). However, AM rice plants
maintained higher net photosynthetic rate, stomatal conductance
and transpiration rate than nonAM plants both under non-saline
conditions and under 150 mM NaCl. Increased stomatal conductance and transpiration in AM plants did not negatively influenced
plant performance. The increase of gas exchange by the AM symbiosis has been related to alterations of host plant hormonal levels and
with the enhanced uptake and translocation of water (Ebel et al.,
1997; Goicoechea et al., 1997; Sheng et al., 2008; Ruiz-Lozano and
Aroca, 2010) and would normally translate into increased photosynthesis (Birhane et al., 2012). On the other hand, the stimulation
of carbohydrate transport and metabolism between source and sink
tissues has been proposed as a mechanism to alleviate metabolic
inhibitions of photosynthesis, avoiding thus photoinhibition due
to salinity (Dodd and Pérez-Alfocea, 2012). Plant roots become a
strong sink for carbohydrates when colonized by AM fungi, as these
fungi can consume up to 20% of the host photosynthate (Feng et al.,
2002; Heinemeyer et al., 2006). Thus, AM fungi modulate these
source–sink relations by enhancing exchange of carbohydrates and
mineral nutrients and can stimulate the rate of photosynthesis sufficiently to compensate for fungal carbon requirements (Kaschuk
et al., 2009; Dodd and Pérez-Alfocea, 2012). Indeed, in this study
at 150 mM NaCl the total soluble sugar content in roots was significantly higher in AM plants than in nonAM ones. No significant
differences were found in the other saline levels (data not shown).
Chlorophyll content is a key factor for plant photosynthesis and
closely reflects the photosynthetic ability of plants such as rice
(Takai et al., 2010). Enhanced chlorophyll concentration has been
described in AM plants (Giri and Mukerji, 2004; Sannazzaro et al.,
2006; Colla et al., 2008). In the present study, a higher chlorophyll
a concentration was found in AM rice plants subjected to 150 mM
NaCl. For the other photosynthetic pigments measured the increase
was not significant. Enhanced chlorophyll content in AM plants has
been related to increased P and Mg uptake (Zhu et al., 2014). AM rice
plants also displayed higher rubisco activity under all salt levels,
demonstrating a lower metabolic limitation of photosynthesis than
82
R. Porcel et al. / Journal of Plant Physiology 185 (2015) 75–83
nonAM plants (Lima-Neto et al., 2014). Rubisco activity is a parameter that is well correlated with CO2 assimilation (Sanz-Sáez et al.,
2013). Thus, the enhanced net photosynthetic activity of AM plants
could be also due to non-stomatal factors such as higher chlorophyll a content and rubisco activity (Chen et al., 2014). However,
the rubisco enzymatic activity did not correlate with expression
of genes encoding for small (rbcS) and large (rcbL) rubisco subunits. The rbcS gene reduced its expression in AM plants under 75
and 150 mM NaCl and the rbcL gene expression was similar in AM
and nonAM plants at 75 and 150 mM NaCl, while its expression
was lower in AM plants under non-saline conditions. In any case,
Sanz-Sáez et al. (2013) also found that rubisco activity was not coordinated with gene expression, possibly due to a lag between gene
transcription and protein translation.
Because of its sensitivity to abiotic stresses, PSII activity has been
widely used to study response and adaptation to stress by plants
(Strasser et al., 2000). When the metabolism of a plant is disturbed
by biotic or abiotic stresses, redundant energy hast to be dissipated
in order to avoid damage of plant tissues. Dissipation results via
non-photochemical processes like heat or chlorophyll fluorescence
(Pinior et al., 2005). The parameters of Chl fluorescence reflect accurately photosynthetic ability and energy conversion efficiency (Zhu
et al., 2014). The maximum quantum yield of primary photochemistry (Fv /Fm ) reflects the potential quantum efficiency of PSII and is
used as an index of plant photosynthetic performance, with optimal values for most plant species of around 0.83 (Björkman and
Demmig, 1987). In this study, values of Fv /Fm ranged from 0.68 and
0.78 in the different treatments, which suggests that the performance of the photosynthetic apparatus was not at the optimum
level (Bagheri et al., 2011). However, data also showed that under
salinity the actual quantum yield of PSII photochemistry (PSII ) was
enhanced by AM symbiosis. The increase in PSII due to mycorrhization was by 25% and 34% at 75 and 150 mM NaCl, respectively.
At the same time, NPQ was significantly lower in AM plants under
both saline levels. The reduction ranged from 30% under 75 mM
NaCl to 40% under 150 mM NaCl. Moreover, NPQ increased in
nonAM plants as consequence of salinity, but resulted unaffected
by salinity in AM plants. Normally, NPQ increases as a mechanism
to protect the leaf from light-induced damage, but this means that
photochemical processes are reduced proportionally (Maxwell and
Johnson, 2000; Baker, 2008; Lazár, 2015). Sheng et al. (2008) found
that AM symbiosis triggered the regulation of energy bifurcation
between photochemical and non-photochemical events. Thus, our
data indicate that AM rice plants had a higher photochemical efficiency for CO2 fixation and solar energy utilization and that AM
inoculation would reduce the light-induced damage due to salinity
(Zhu et al., 2014). As a result, AM symbiosis increases salt tolerance
in rice plants by preventing the injury to the photosystems reaction centres and by allowing a better utilization of light energy in
photochemical processes (Pinior et al., 2005; Bagheri et al., 2011),
reducing at the same time light energy dissipation as heat. In agreement with our results, Sheng et al. (2008) found that salt stress
could destroy PSII reaction centres or disrupt electron transport in
photosynthetic apparatus of AM and nonAM maize plants subjected
to several salinity levels, but the toxic influence of salinity on maize
PSII reaction centres were mitigated by the AM symbiosis. These
effects of the AM symbiosis enhancing PSII and reducing NPQ
may be related to the sink stimulation of AM symbiosis. Kaschuk
et al. (2009) showed that the carbon sink stregth due to the fungal
presence in the plant root stimulates the host plant, increasing the
photosynthetic rate. In this context, Chen et al. (2014) found that
sink strength of AM symbiosis could alleviate the negative effects of
low-Zn on PSII. Moreover, it has been found in citrus plants that AM
symbiosis reduced leaf temperature under drought stress conditions (Wu and Xia, 2006). Authors related this finding with a lower
resistance in AM plants to vapour transfer from inside the leaves to
the atmosphere.
In conclusion, the present results show that the AM symbiosis enhances the actual quantum yield of PSII photochemistry and
reduces NPQ in rice plants subjected to salinity. Thus, we propose that AM rice plants had a higher photochemical efficiency for
CO2 fixation and solar energy utilization and this increases plant
salt tolerance by preventing the injury to the photosystems reaction centres and by allowing a better utilization of light energy in
photochemical processes, reducing at the same time light energy
dissipation as heat. All these processes translated into a higher photosynthetic and rubisco activities in AM rice plants and improved
plant biomass production under salinity.
Acknowledgments
This work was financed by a research project supported by Junta
de Andalucía (Spain). Project P11-CVI-7107. We would like to thank
Michael O’shea for proofreading the document.
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