Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology Official Journal of the Societa Botanica Italiana
ISSN: 1126-3504 (Print) 1724-5575 (Online) Journal homepage: http://www.tandfonline.com/loi/tplb20
Effect of salt stress on photosynthesis and chlorophyll fluorescence in Medicago truncatula Rim Najar, Samir Aydi, Sameh Sassi-Aydi, Abdennabi Zarai & Chedly Abdelly To cite this article: Rim Najar, Samir Aydi, Sameh Sassi-Aydi, Abdennabi Zarai & Chedly Abdelly (2018): Effect of salt stress on photosynthesis and chlorophyll fluorescence in Medicago truncatula, Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology, DOI: 10.1080/11263504.2018.1461701 To link to this article: https://doi.org/10.1080/11263504.2018.1461701
Published online: 24 Apr 2018.
Submit your article to this journal
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tplb20
Plant Biosystems – An International Journal Dealing with all Aspects of Plant Biology, 2018 https://doi.org/10.1080/11263504.2018.1461701
Effect of salt stress on photosynthesis and chlorophyll fluorescence in Medicago truncatula Rim Najara, Samir Aydia,b , Sameh Sassi-Aydic, Abdennabi Zaraia and Chedly Abdellya a
Laboratory of Extremophile Plants, Centre of Biotechnology of Borj Cedria (CBBC), Hammam-Lif, Tunisia; bFaculty of Sciences, Department of Life Sciences, University of Gabes, Gabes, Tunisia; cResearch Unit Biodiversity and Valorization of Bioresources in Arid Zones, Faculty of Sciences of Gabes, University of Gabes, Gabes, Tunisia
ABSTRACT
In the present study, photosynthetic parameters including gas exchanges, pigment contents, and chlorophyll fluorescence, were compared in two contrasting local Medicago truncatula lines TN6.18 and TN8.20, in response to salt added to the nutrient solution. Plants were cultivated under symbiotic nitrogen fixation (SNF) after inoculation with a reference strain Sinorhizobium meliloti 2011, a very tolerant strain to salinity (700 mM NaCl), and grown in a controlled glasshouse. On one month old plants (with active SNF), salt treatment (75 mM NaCl) was gradually applied. Photosynthesis, assimilating pigments and chlorophyll fluorescence were monitored throughout the experiment during both short and long terms, compared to control (non-saline) conditions. A genotypic variation in salt tolerance was found; TN6.18 was the more sensitive to salinity. The relative tolerance of TN8.20 was concomitant with the highest photochemical quenching coefficient (qP) affecting the maximum quantum yield of PSII (Y); the real quantum yield (ɸexc) was the most affected in the sensitive line. Moreover, stomatal and PSII reaction centers activities differed clearly between the studied lines. We found that the effect of salinity on photosynthesis of M. truncatula was related to PSII activity reduction rather than to stomatal conductance limitation. Photosynthesis was reduced by the inhibition of CO2 assimilation caused by PSII damage. This was clearly estimated by the Y, ɸexc and especially by the quantum yield of electron transport of PSII (ΦPSII). Thus, on the basis of our results on the two local M. truncatula lines, we recommend the use of chlorophyll fluorescence as non-destructive screening method to discriminate susceptible and resistant legumes to salt stress.
Introduction Salinity adversely affects plant growth and development, with nearly 20% of the world cultivated area and about half of the world irrigated lands being affected by salt stress (Sairam and Tyagi 2004). This problem is more relevant in semiarid regions with low rainfall and high evaporative demand, which strongly contribute to increase soil salinization (Viégas et al. 2001). In these regions, the problem of soil secondary salinization is exacerbated by the use of low-quality water associated with inadequate techniques of soil management (Ferreira-Silva et al. 2009). Photosynthesis, as one of the most important physiological processes, provides 90% of the plant dry matter (Steduto et al. 2000). The photosynthetic capacity of plants grown under saline conditions is depressed depending on type of salinity, duration of treatment, species and plant age (Sultana et al. 1999; Steduto et al. 2000; Hester et al. 2001; Koyro 2006). Many studies have concluded that the reduction in photosynthesis in response to salinity is, to some extent, the result of reduced stomatal conductance and, consequently, restriction of the availability of CO2 for carboxylation (Everard et al.1994; Chartzoulakis et al. 1995; Koyro 2006).
CONTACT Samir Aydi
[email protected],
[email protected]
© 2018 Società Botanica Italiana
Published online 24 Apr 2018
ARTICLE HISTORY
Received 18 October 2017 Accepted 26 March 2018 KEYWORDS
Model legume; salinity; photosynthesis; assimilating pigment; chlorophyll fluorescence
Stomatal conductance estimates the rate of gas exchange (i.e. carbon dioxide uptake) and transpiration (i.e. water loss) through the leaf stomata as determined by the degree of stomatal aperture (and therefore the physical resistance to the movement of gases between the air and the leaves tissues). Hence, it is a function of the density, size, and degree of stomata opening; with more open stomata allowing greater conductance, and consequently indicating that photosynthesis and transpiration rates are potentially higher. Generally, photosynthesis and transpiration rates reflect and quantify the effects of the environmental stress. As a consequence, the photochemical centers use the electrons from the water photolysis and the two photosystems (PSII and PSI) are connected by a chain of electrons carriers that allows the ATP and NADPH synthesis. A photon of light (quantum) absorbed by any chlorophyll, migrates very quickly through the pigments to the reaction center where the photochemical conversion is being performed. The two pigments activate – two reaction centers – of the two photosystems that trap the absorbed photons, of lower energy. This reflection of light constitutes the fluorescence. The fluorescence contributes thus to dissipate the energy of light
2
R. NAJAR ET AL.
absorbed by the chlorophylls that would be converted into chemical energy. When the emission of electrons is disrupted, the transport chain deteriorated for example by NaCl, the dissipation of energy (heat and fluorescence) increases. The intensity of fluorescence is inversely related to the photochemical yield. Although the fluorescence better reflects the photosynthetic disturbances, this relationship can be considered as a specific intrinsic indicator of the clear reaction of photosynthesis at the level of the PSII. Therefore, the main aim of this study was to comparatively analyze the in vivo changes in gas exchanges for photosynthesis estimation and PSII photochemistry as induced by salt stress in two local Medicago truncatula lines. Moreover, we also tested whether the chlorophyll fluorescence data, as non-destructive method, could be used to discriminate salt tolerant lines or varieties of annual plants.
Material and methods Plant material and culture conditions This study was conducted on a couple of local M. truncatula lines (TN8.20 and TN6.18) originating from the edge of a saline depression, the Sebkha of Soliman and from Tala (a non-saline area), respectively. These lines were purified and characterized in the “Laboratory of Legumes” (CBBC, Hammam-Lif, Tunisia). Seeds were: scarified, surface sterilized, and soaked in sterilized distilled water for 3 h with permanent homogenization. Rehydrated seeds were pre-germinated in 0.9% agar plates, after a vernalization at 4 °C for 24 h (cold treatment to alleviate embryonic dormancy). After 2 days in dark condition at 25 °C, seedlings where transferred on sterilized sand, that was kept at 100 °C for 2 days in an oven to eliminate all micro-organisms, prior to the transfer. The roots of selected uniform seedlings were transferred to a humid soil and were initially added with 1 ml of Sinorhizobium meliloti 2011 inoculum, (a very tolerant strain to salinity 700 mM NaCl) (Jebara et al. 2001), containing approximately 108 cells ml−1. Only during the two initial weeks, plants were irrigated with a diluted (1/4) nutrient solution that contained 0.7 mM K2SO4; 1 mM MgSO4,7H2O; 1.65 mM CaCl2; 15 μmol KH2PO4; 22.5 μM Fe for macronutrients, and 6.6 μM Mn; 4 μM Bo; 1.5 μM Cu; 1.5 μM Zn; 0.1 μM Mo for micronutrients. During the first two weeks, i.e. before nodule functioning, the nutrient solution was supplemented with 2 mM urea (Ribet and Drevon 1996; Krouma et al. 2008). To avoid any osmotic shock, salt was progressively added as 25; 50 and 75 mM NaCl after 2; 4 and 6 days after the transfer, respectively. Plants were irrigated three times per week with nutrient solution and one time with excessive quantity of distilled water to prevent nutrient and salt accumulation in the soil. Plants were grown in a temperature-controlled glasshouse with night/ day temperatures of ca. 20/28 °C and a 16 h photoperiod with additional lights of 500 μmol PAR m−2 s−1.
Photosynthesis parameters Extraction, separation, and quantification of pigments To extract the pigments, 1.5 g of fresh leaf material was collected from each experimental series of plants and ground with pestle and mortar in 10 mL of 80% acetone (CH3COCH3). The homogenate was centrifuged at 1000 g for 15 min and the supernatant
was collected. The residue was re-extracted with 80% acetone. The resulting supernatant solution was saved and used for pigment determination. To quantify the pigments, the absorbance of the solution was measured at 460, 645, and 663 nm using the UV-spectrophotometer. 80% acetone was used as the blank. The concentrations of pigments (in mg/L) were calculated according the following formulas: ( ) ( ) Chlorophyll a = 12.7 A663 − 2.69 A645
( ) ( ) Chlorophyll b = 22.9 A645 − 4.68 A663 ( ) ( ) Total chlorophyll = 20.2 A645 + 8.02 A663 Catorenoï ds = (5(A460)(3.19(Chl a))(130.3(Chl b)))∕200 where Abs645 = absorbance of solution at 645 nm; Abs663 = absorbance of solution at 663 nm, and Abs460 = absorbance of solution at 460 nm.
Gas exchanges Leaf gas exchanges were carried out on fully developed leaves under the following conditions: an atmospheric pressure of 101 kPa; an atmospheric CO2 concentration of 530–570 ppm; a relative humidity of 40–45%; a light intensity of 1000 μmol PAR m−2 s−1, and a leaves temperature of 22 ± 0.5 °C. Net photosynthesis rate (PN), expressed in μmol (CO2) m−2 s−1; stomatal conductance (gs), expressed in moles (H2O) m−2 s−1; transpiration rate (E), expressed in mmol (H2O) m−2s−1 and intercellular CO2 concentration (Ci) expressed in μmol (CO2) mol–1 or ppm, were measured by a portable CI-340 Handheld Photosynthesis System (CI340 Bio-Science, Inc., USA). This measurement system provides automatic control that is independent of environmental conditions of the interior of the leaf chamber. The water use efficiency (WUE [μmol (CO2) mmol (H2O)–1]) for photosynthesis activity was estimated by the ratio of PN and E values.
Chlorophyll fluorescence Quantifications of the chlorophyll fluorescence were performed using an OS30p+ Hand Held Chlorophyll Fluorometer (OptiSciences.Inc), on plants previously adapted to the darkness during 30–40 min. As a result of the application of a pulse of saturating light, fluorescence increases the fundamental state Fo (all reaction centers are open) toward a maximum level Fm (all reaction centers are closed). This situation allows us to determine the maximum photochemical quantum yield of PSII (designated Y) as follows:
Y = Fv∕Fm = (Fm − Fo)∕Fm. Just after the transfer of plants in continuous light, we can measure the efficiency of quantum open centers as follows: ( ) Φexc = Fv� ∕Fm� = Fm� − Fo� ∕Fm� . The coefficient of the photochemical quenching (photochemical quenching) allows us to estimate the proportion of reaction centers open in the PSII, as follows: ( ) ( ) qP = Fm� − Fs ∕ Fm� − F�0 .
PLANT BIOSYSTEMS – AN INTERNATIONAL JOURNAL DEALING WITH ALL ASPECTS OF PLANT BIOLOGY
PN; µmol.m-2. s -1
35
40
(B)
(A)
35
30
30
25
25
20
20 15
15 10
Control
5
Treated
0
0
5
10
15
Control Treated 20
25
0
5
10
15
20
10
PN; µmol.m-2. s -1
40
3
5 25
0 DAT
Figure 1. Effect of salinity on photosynthetic assimilation rate (PN: μmol CO2 m−2 s−1) in two local M. truncatula lines TN6.18 (A) and TN8.20 (B) cultivated under symbiotic nitrogen fixation condition and irrigated during 28 days with nutrient solution with (treated) and without (control) 75 mM NaCl. DAT: days after treatment. Plants were 49 days old. Results are the mean of nine replicates ± S.D.
(A)
0.8
0.9
(B)
0.8
0.7
0.7
0.6
0.6
Control
0.5
0.5
Treated
0.4
0.3 0.2
0
5
10
15
20
0.4
Control Treated 25
0
5
10
15
20
0.3 25
E; mmol.m-2. s-1
E; mmol.m-2. s-1
0.9
0.2 DAT
Figure 2. Variation of leaves transpiration rate (E: mmol (H2O) m−2 s−1) in two local lines of M truncatula TN6.18 (A) and TN8.20 (B), during 28 days of salt treatment. DAT: days after treatment. Results are the mean of nine replicates ± S.D
The dissipated energy in heat form NPQ (non-photochemical quenching) is given by the ratio: (Fm-Fm′)/Fm′. The quantum yield of electron transport of PSII; designated ɸPSII, estimate the efficiency of all the reaction centers of PSII in the light. It determines the quantum yield of the photochemistry: ɸPSII = (Fm′–Fs)/Fm′ or even ɸPSII = Φexc . qP.
Statistical analysis Data were subjected to analysis of variance and comparison of means by ANOVA test. The analysis of variance and the lowest standard deviation (LSD) of the means (n = 8) were used to determine the significance (p
