Iron deficiency is an important environmental factor restricting plant productivity. Selecting tolerant genotypes is one of the possible ways to solve this problem. Many studies reported the effects of Fe deficiency on photosynthesis and anti-oxidative defense system. Yet, there is little information available on the use of these attributes as selective criteria. In the present study, we aim to determine some physiological and biochemical traits conferring Fe deficiency tolerance at leaf level in two lines of Medicago ciliaris. Our results showed that Fe deprivation had a lowering effect on photosynthesis (chlorophyll, photosynthetic electron transport activity and chlorophyll fluorescence) in both lines studied. However, the sensitive line TN8.7 was more affected. Hydrogen peroxide concentration was negatively correlated with the activities of antioxidant enzymes and with the concentration of some non-enzymatic antioxidant. The tolerant line TN11.11 was characterized by a more efficient antioxidant defense system in comparison with the sensitive line TN8.7. The main conclusion of this study is that photosynthesis and antioxidant defense system could be used as physiological and biochemical indicators of Fe deficiency tolerance in Medicago ciliaris plants.
Under natural conditions, plants are exposed to many constraints that significantly reduce their growth and pro- ductivity. Iron (Fe) deficiency is considered as a major factor threatening plant development in arid and semi- arid regions, especially in calcareous soils leading to the appearance of Fe chlorosis symptoms in plants [
Iron deficiency affects plant morphological and physiological responses because of the fundamental role of Fe in several biological processes. In fact, Fe is a constituent of electron transport chains of both mitochondria and chloroplasts. As a result of its absence, a decrease in the activity of electron transport [
In previous works [
Two lines of Medicago ciliaris L. created by three generations of spontaneous selfing in the greenhouse were used: TN11.11 and TN8.7. Seeds were obtained from the Laboratory of Legumes, Biotechnology Center of Borj-Cedria (CBBC), Tunisia.
Seeds were germinated and grown for 3 days in Agriperlite moistened with 0.1 mM CaSO4. Three-day-old seedlings were transferred to a half strength aerated hydroponic nutrient solution for 7 days and then similar sized seedlings were selected and cultured as groups of 10 plants in 10 L of full strength aerated hydroponic nutrient solution. The composition of the nutrient solution was: 1.25 mM Ca (NO3)2, 1.25 mM KNO3, 0.5 mM MgSO4, 0.25 mM KH2PO4 and 10 µM H3BO3, 1 µM MnSO4, 0.5 µM ZnSO4, 0.05 µM (NH4)6Mo7O24 and 0.4 µM CuSO4.
Two treatments were applied: −Fe (0 mM) and +Fe (0.03 mM). Both media have a pH around 6.2. Fe was supplied as Fe (III)-EDTA. The solution was renewed every 5 days. Plants were maintained in a growth chamber with a 16/8 h day/night, 24˚C/18˚C regime and a relative humidity of 70%.
Bivalent Fe content was determined according to the method of [
Eight plants per treatment were used to determine total chlorophyll content of young leaves according to the method of [
Thylakoid membranes were isolated from leaves as described by [
Iron-sufficient and Fe-deficient M. ciliaris leaves were adapted to dark conditions for 1h at room temperature before experiments. Chla fluorescence was measured in leaf discs using a PAM 2000 fluoremeter (Heinz Walz, Germany). The measurements were performed at room temperature (25˚C). F0 was measured by switching on the modulated light at 0.6 kHz; PPFD was less than 0.1 µ E m−2∙s−1 at the leaf surface. Fm was measured at 20 kHz with a 1s pulse of 6000 µ E m−2∙s−1 of white light. The initial fluorescence (F0) and the maximal fluorescence were determined after dark acclimation. The variable fluorescence (Fv) was taken from the formula: Fv = Fm − F0 and the parameter used were Fv/Fm and 1/F0 − 1/Fm [
The H2O2 level was calorimetrically measured as described by [
Volatile aldehydes were extracted in 5 ml of 2% (v/v) ethanol at 25˚C for 2 hours, under constant stirring using 0.3 g leaf fresh tissues. A 1 mL aliquot was transferred to a test tube containing 1 mL of 0.1% (w/v) 3-methyl-2- benzothiazolinone hydrazone (MBTH) before the addition of 2.5 mL of FeCl36H2O solution (0.23% (w/v)). After 5 min of incubation, 6 mL of acetone were added and the absorbance was read at 635 nm [
Lipid peroxidation was determined as described by [
All operations were carried out at 4˚C. Leaves were homogenised with quartz sand, liquid nitrogen and 10% (w/w) polyvinylpyrolidone (PVP) in a grinding medium (1:2.5, w:v) prepared as described below, and centrifuged at 12000 g for 30 min at 4˚C.
The extraction buffer for superoxide dismutase (SOD) and guaiacol-POD (POD) contained 220 mM Tris-HCl (pH 7.4), 250 mM sucrose, 50 mM KCl, 1 mM MgCl2, 1% β mercaptoethanol and 0.01% (w/v) phenylmethylsulfonylfluoride (PMSF) [
Superoxide dismutase activity was measured according to the method of [
Peroxidase activity was tested using guaiacol as reducing phenolic substrate. The reaction medium contained 20 mM Na-acetate (pH 5.0), 30 mM H2O2, 2 mM guaiacol and an appropriate amount of enzyme extract. The rate of guaiacol oxidation was recorded at 470 nm [
Ascorbate peroxidase activity was determined following the decrease in absorbance at 290 nm due to the oxidation of ascorbic acid in the first 30 s from the beginning of the reaction, using the extinction coefficient of 2.8 mM−1∙cm−1 for ascorbate. The reaction medium contained 0.5 mM reduced ascorbate (ASA), 0.1 mM H2O2, 1 mM EDTA and 0.1 M HEPES-KOH buffer (pH 7.8) [
Non-enzymatic antioxidant activity, ascorbate and glutathione content.
Total non-enzymatic antioxidant activity was measured using the Ferric Reducing Ability of Plasma (FRAP) assay. Leaf tissue (0.5 g) was homogenized in 5 ml methanol. The homogenate was first filtered and then centrifuged at 12,800 g for 2 min. The FRAP assay was performed with FRAP reagent, i.e. 1 mM 2,4,6-tripyridyl-2- triazine (TPTZ) and 20 mM ferric chloride in 0.25 M sodium acetate (pH 3.6). An aliquot of 100 ml of leaf extract (0.5 g/10 mL in methanol) was added to 2 mL of FRAP reagent and thoroughly mixed. Next, the mixture was left at 20˚C for 5 min and the absorbance was measured at 593 nm. Calibration was obtained by means of a standard curve (25 - 1600 mM ferrous iron) using freshly prepared ammonium ferrous sulfate [
Extraction of ascorbate and glutathione
Leaf samples were ground in a cold mortar with liquid nitrogen and homogenized with 25 mM sulphuric acid (800 µl/400 mg fresh weight). The homogenate was centrifuged at 16,700 g for 30 min at 4˚C, and the supernatant was collected for analysis of ascorbate and glutathione.
Quantification of reduced (ASC) and oxidized (DHA) ascorbate
The assay is based on the reduction of Fe3+ to Fe2+ by ascorbic acid [
Quantification of reduced (GSH) and oxidized (GSSG) glutathione
The contents of GSH and GSSG were determined as described by [
A two-way analysis of variance (ANOVA), with lines and treatments as factors, was performed for the whole data using the STATI-CF statistical program. Means were compared using the Newman-Keuls test at the p<0.05 level when significant differences were found. Data shown are means of five replicates for each treatment.
Medicago ciliaris plants grown under Fe deficiency conditions for 10 days developed symptoms of chlorosis. These visual manifestations were observed in both lines (TN8.7 and TN11.11) and were concomitant with a significant reduction in chlorophyll concentration (
Iron deficiency affected significantly the whole chain photoelectron transport rate of both genotypes TN8.7 and TN11.11 (
The measurement of chlorophyll fluorescence as the ratio Fv/Fm revealed that at the beginning of the experiment, this parameter was similar in the control leaves of both genotypes (
For all samples, lipid peroxidation estimated by the MDA-TBA complex concentration was evaluated (
. Bivalent iron content (µmol∙g−1 DW) and chlorophyll concentration (mg∙g−1 FW) of two lines of Medicago ciliaris plants grown on a control nutrient solution (+Fe) or in the absence of iron (−Fe) during the treatment period (10 days). Values followed by different letters are significantly different at P < 0.05 according to Newman-Keuls
Chlorophyll content | Bivalent iron | |
---|---|---|
TN11.11 | ||
Treatments | ||
+Fe | 4.1 ± 0.6 b | 3.02 ± 0.3 a |
−Fe | 2.9 ± 0.7 c | 2.32 ± 0.5 b |
TN8.7 | ||
Treatments | ||
+Fe | 4.9 ± 0.7 a | 3.14 ± 0.2 a |
−Fe | 2.4 ± 0.4 cd | 1.98 ± 0.4 c |
. Evolution of fluorescence parameters measured in leaves of two lines of Medicago ciliaris plants grown on a control nutrient solution (+Fe) or in the absence of iron (−Fe) during the treatment period (10 days). Values followed by different letters are significantly different at P < 0.05 according to Newman-Keuls
F0 | Fv | Fv/Fm | 1/F0 − 1/Fm | |
---|---|---|---|---|
TN11.11 | ||||
+Fe | 2.6 ± 0.01 b | 10.4 ± 0.10 a | 0.81 ± 0.04 a | 0.30 ± 0.03 a |
−Fe | 2.7 ± 0.03 b | 4.5 ± 0.20 b | 0.62 ± 0.01 b | 0.23 ± 0.02 b |
TN8.7 | ||||
+Fe | 2.4 ± 0.04 b | 9.7 ± 0.15 a | 0.74 ± 0.02 a | 0.34 ± 0.02 a |
−Fe | 3.8 ± 0.02 a | 2.7 ± 0.08 b | 0.49 ± 0.01 c | 0.08 ± 0.03 c |
Photo-electron transport rate of two lines of Medicago ciliaris plants grown on a control nutrient solution (+Fe) or in the absence of iron (−Fe) during the treatment period (10 days). Values followed by different letters are significantly different at P < 0.05 according to Newman-Keuls
Leaf malondialdehyde content (MDA) of two lines of Medicago ciliaris plants grown on a control nutrient solution (+Fe) or in the absence of iron (−Fe) during the treatment period (10 days). Values followed by different letters are significantly different at P < 0.05 according to Newman-Keuls
The concentration of volatile aldehydes as an indicator of oxidative stress degree (mg∙g−1 FW) was determined (
The leaf tissue content of H2O2, another signal of oxidative stress, was measured. Hydrogen peroxide content significantly increased in both genotypes grown under Fe deprivation (
Total SOD activity was significantly stimulated under Fe deficiency in TN8.7 (+32% respect to the control), while in TN11.11 it was increased only by 13% (
Leaf volatile aldehyde content of two lines of Medicago ciliaris plants grown on a control nutrient solution (+Fe) or in the absence of iron (−Fe) during the treatment period (10 days). Values followed by different letters are significantly different at P < 0.05 according to Newman-Keuls
H2O2 content in leaves of two lines of Medicago ciliaris plants grown on a control nutrient solution (+Fe) or in the absence of iron (−Fe) during the treatment period (10 days). Values followed by different letters are significantly different at P < 0.05 according to Newman-Keuls
. Enzymatic activity of total superoxide dismutase (SOD) and its different isoforms in two lines of Medicago ciliaris plants grown on a control nutrient solution (+Fe) or in the absence of iron (−Fe) during the treatment period (10 days). One enzymatic unit is defined as the amount of enzyme inhibiting 50% of NBT-photoreduction. Values followed by different letters are significantly different at P < 0.05 according to Newman-Keuls
Total SOD | CuZn-SOD | Mn-SOD | Fe-SOD | |
---|---|---|---|---|
TN11.11 | ||||
+Fe | 101.33 ± 3 bc | 33.33 ± 2 c | 40 ± 1 c | 28 ± 1b |
−Fe | 115.36 ± 4 b | 57.66 ± 8 b | 57.33 ± 2 b | nd c |
TN8.7 | ||||
+Fe | 97 ± 11 d | 30.33 ± 1 c | 32.66 ± 4 d | 34 ± 2 a |
−Fe | 128.33 ± 4 a | 60.66 ± 2 a | 67.66 ± 4 a | nd c |
KCN-insensitive) was detected only in plants of both genotypes grown in the presence of Fe in the nutrient solution (
Data related to POD activity demonstrated a significant decrease under Fe deficiency conditions respect to the control in both lines (−23% and −14% for TN8.7 and TN11.11 respectively). Ascorbic peroxidase exhibited the strongest decrease under Fe deficiency respect to the control in both genotypes, being −45% and −32% in TN8.7 and TN11.11 respectively (
Leaf antioxidant activity (FRAP) of the two lines was presented in
. Enzymatic activity of guaiacol peroxidase (POD) and ascorbate peroxidase (APx) in two lines of Medicago ciliaris plants grown on a control nutrient solution (+Fe) or in the absence of iron (−Fe) during the treatment period (10 days). Enzyme activity of G-POD and APx is expressed as µmol guaiacol or ascorbate consumed min−1∙mg∙prot−1, respectively. Values followed by different letters are significantly different at P < 0.05 according to Newman-Keuls
POD | APX | |
---|---|---|
TN11.11 | ||
+Fe | 13.96 ± 2.21 a | 11.23 ± 1.11 a |
−Fe | 11.91 ± 1.15 b | 7.57 ± 0.9 b |
TN8.7 | ||
+Fe | 13.93 ± 0.13 a | 10.76 ± 0.54 a |
−Fe | 10.66 ± 0.22 c | 5.91 ± 0.7 c |
. FRAP assay, changes in ascorbate (reduced (ASC) and oxidized (DHA) ascorbate)and glutathione(reduced (GSH) and oxidized (GSSG) glutathione) concentration in leaves of two lines of Medicago ciliaris plants grown on a control nutrient solution (+Fe) or in the absence of iron (−Fe) during the treatment period (10 days). Values followed by different letters are significantly different at P < 0.05 according to Newman-Keuls
TN11.11 | TN8.7 | |||
---|---|---|---|---|
+Fe | −Fe | +Fe | −Fe | |
FRAP µmol∙g−1 FW | 784 ± 4.43 a | 801 ± 3.65 a | 791 ± 5.92 a | 657 ± 4.98 b |
Ascorbate content | ||||
ASC + DHA | 48.19 ± 4.34 b | 89.41 ± 4.67 a | 45.17 ± 4.62 b | 38.74 ± 3.81 c |
ASC | 34.65 ± 3.87 b | 75.76 ± 3.95 a | 30.54 ± 3.71 b | 25.71 ± 3.94 c |
DHA | 13.54 ± 2.53 a | 13.65 ± 1.98 a | 14.63 ± 2.73 a | 13.03 ± 1.63 a |
ASC/DHA | 2.56 ± 3.62 b | 5.55 ± 0.76 a | 2.09 ± 0.86 b | 1.97 ± 0.61 b |
Glutathione content | ||||
GSH + GSSG | 22.06 ± 3.62 a | 21.89 ± 2.84 a | 24.13 ± 3.63 a | 19.36 ± 2.83 b |
GSH | 13.53 ± 1.96 b | 13.87 ± 1.73 b | 15.52 ± 1.94 a | 10.65 ± 1.83 c |
GSSG | 8.53 ± 0.75 a | 8.02 ± 1.04 a | 8.61 ± 0.76 a | 8.71 ± 0.98 a |
GSH/GSSG | 1.59 ± 0.09 a | 1.73 ± 0.12 a | 1.80 ± 0.08 a | 1.22 ± 0.05 a |
In TN8.7 line, both the total ascorbate concentration and the reduced form significantly decreased while the oxidized form did not change under Fe starvation. Nevertheless, no modification of the ASC /DHA ratio was noted under Fe deficiency stress. Similarly, the total glutathione pool and its reduced form decreased in Fe- deficient leaves (respectively −19% and −25% respect to the control), whereas the oxidized form (GSSG) was similar to the control. Also in this case, the GSH/GSSH ratio remained constant (
In the present study, we investigate possible mechanisms responsible for Fe deficiency tolerance at leaf level in two Medicago ciliaris lines differing in their tolerance to Fe deficiency. The genotype TN11.11 is native of a calcareous soil, characterized by its high pH causing Fe deficiency, and is known for its ability to deal with such severe conditions. The genotype TN8.7, colonizing a soil poor in lime content where the pH is lower, is considered to be more sensitive and less efficient in responding to Fe deficiency [
Based on the leaf responses to Fe deficiency, significant differences were observed between the two lines studied. In fact, the majority of these responses, including leaf chlorophyll and Fe concentrations, fluorescence parameters and antioxidative defence were genotype-dependant. Our results showed that the photosynthetic parameters, leaf Fe content and antioxidant system of the sensitive line TN8.7 were more affected than those of the tolerant TN11.11 line by Fe starvation. These recent findings reinforced our previous work focused in root responses of the same Medicago ciliaris lines [
Chlorosis is a phenomenon which appears in young leaves and is often known as the first visual sign of Fe deficiency in plants. It is associated not only with a loss of chlorophyll content, since several steps of its biosynthesis are Fe-depend, but also with changes in the expression and assembly of other components of the photosynthetic apparatus [
The impact of Fe deficiency stress on photosynthetic apparatus mainly by impairing the electron transport chain functionality both at the mitochondrion and at the chloroplast level [
Aldehydes are intermediates in several fundamental metabolism pathways for carbohydrates, vitamins, steroids, amino acids, and lipids [
According to [
Ascorbate (Asc) and glutathione (GSH) are important metabolites in plants. Their most prominent and best established functions are their crucial antioxidant roles in the Asc-GSH cycle [
According to the literature, many studies are focused on Fe deficiency tolerance in plant. Nevertheless, there are no well-defined plant indicators for tolerance to this abiotic stress at leaf level. This is the first paper studying the biochemical (MDA, enzymatic and non-enzymatic antioxidant system) and physiological (photosynthetic electron transport of the whole chain and the chlorophyll a fluorescence parameters) traits in Fe-stressed Medicago ciliaris leaves. Our results showed significant changes in these parameters in both lines considered: TN11.11, which is tolerant to Fe deficiency and TN8.7 which is susceptible. Only the tolerant line TN11.11 showed an efficient enzymatic and non-enzymatic antioxidant defence system in order to limit ROS production under Fe deficiency stress and consequently to prevent over reduction of the photosynthetic electron transport chain. The main output of this study is that the parameters here considered can be used as precious criteria for predicting plant performance under Fe deficiency stress and for screening tolerant genotypes at leaf level.
This work was supported by grants from the Tunisian Ministry of Higher Education, Scientific Research (LR10- CBBC02).