Arbuscular mycorrhizal symbiosis ameliorates the optimum quantum yield of photosystem II and reduces non-photochemical quenching in rice plants subjected to salt stress

Rosa Porcel1, Susana Redondo-Gómez2, Enrique Mateos-Naranjo2, Ricardo Aroca1, Rosalva Garcia3 and Juan Manuel Ruiz-Lozano1*

1Departamento de Microbiología del Suelo y Sistemas Simbióticos. Estación Experimental del Zaidín (CSIC). Profesor Albareda nº 1, 18008 Granada, Spain.

2Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, Apartado 1095, 41080 Sevilla, Spain

3Facultad de Estudios Superiores Zaragoza. Universidad Nacional Autónoma. México

* Corresponding author: Dr. Juan Manuel Ruiz-Lozano.

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ABSTRACT

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.

Keywords: Arbuscular mycorrhizal symbiosis, non-photochemical quenching, optimum quantum yield, Oriza sativa, photosystem II, salt stress

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 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 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 semi-quantification 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 (ΦPSII), as well as, quantum yield of non-photochemical quenching (ΦNPQ) (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.

Materials and methods

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, 36º59’34’’N; 3º34’47’’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 (100ºC 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 (Oryza 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, 36º45´24´´N 02º13´17´´W), which is an area with serious problems of salinity and affected by desertification. The AMF species was Claroideoglomus 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 mm) of the AM inocula in order to provide a general microbial population free of AM propagules.

Growth Conditions

The experiment was carried out under glasshouse conditions with temperatures ranging from 19 to 25ºC, 16/8 light/dark period, and a relative humidity of 50-60%. At the leaf level, a photosynthetic photon flux density of 800 μmol 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, 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.

Parameters measured

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

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).

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 mmol mol-1, temperature of 25/30 oC, 50 ± 5% relative humidity and a PFD of 1000 mmol 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 (H2O transpired)]. Measurements were made at midday.

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 mmol 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 mmol m-2 s-1 for 1.6 ms) 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 mmol 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).

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 mmol m-2 s-1 for 0.7s was then used to produce maximum fluorescence yield (Fm’) by temporarily inhibiting PSII photochemistry. Using fluorescence parameters determined in both light- and dark-adapted states, the following were calculated: actual quantum yield of PSII photochemistry [ΦPSII = (Fm’ – Fs)/Fm’] (Genty et al., 1989) and quantum yield of non-photochemical quenching, which is the regulatory light-induced non-photochemical quenching [ΦNPQ = (Fs/Fm’) – (Fs/Fm)] (Lazár, 2015). ΦPSII relates to achieved efficiency in a plant under a given treatment and indicates the proportion of absorbed energy being used in photochemistry, while ΦNPQ provides an indication of the amount of energy that is dissipated in the form of heat (Maxwell and Johnson, 2000).