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Journal of Stress Physiology & Biochemistry, Vol. 10 No. 1 2014, pp. 268-279 ISSN 1997-0838 Original Text Copyright © 2014 by Msilini, Guesmi, Chebbi, Amdouni, Lachaal and Ouerghi
ORIGINAL ARTICLE
Strictly NO3- Nutrition Alleviates Iron Deficiency Chlorosis in Arabidopsis thaliana Plants.
Najoua Msilini, Ines Guesmi, Mohamed Chebbi, Thouraya Amdouni, Mokhtar Lachaal, and Zeineb Ouerghi
Unite de Physiologie et de Biochimie de la Tolerance au Sel chez les Plantes, Faculte des Sciences de Tunis, Campus Universitaire, 2092 Tunis, Tunisia.
Tel: 216 71 872 600, Fax: 216 71 885 48 *E-Mail: msilininajoua@yahoo.fr
Received November 12, 2013
The effects of NO3" nutrition on iron deficiency responses were investigated in Arabidopsis thaliana. Plants were grown with or without 5 ^M Fe, and with NO3" alone or a mixture of NO3" and NH4+. The results indicated that, NO3" nutrition induced higher dry matter production, regardless the Fe concentration. Fe deficiency reduced growth activity, photosynthetic pigment concentration and Fe content of plants, whatever the N forms. This decrease was more pronounced in plants grown with mixed N source; those plants presented the highest EL and MDA and anthocyanin contents compared to plants grown under Fe sufficient conditions. In iron free-solutions, with NO3" as the sole nitrogen source, enhanced FC-R activity in the roots was observed. However, in the presence of NH4+, plants displayed some decrease in in FC-R and PEPC activities. The presence of NH4+ modified typical Fe stress responses in Arabidopsis thaliana plants.
Key words: Arabidopsis thaliana, iron deficiency, nitric nutrition
ORIGINAL ARTICLE
Strictly NO3- Nutrition Alleviates Iron Deficiency Chlorosis in Arabidopsis thaliana Plants.
Najoua Msilini, Ines Guesmi, Mohamed Chebbi, Thouraya Amdouni, Mokhtar Lachaal, and Zeineb Ouerghi
Unite de Physiologie et de Biochimie de la Tolerance au Sel chez les Plantes, Faculte des Sciences de Tunis, Campus Universitaire, 2092 Tunis, Tunisia.
Tel: 216 71 872 600, Fax: 216 71 885 48 *E-Mail: msilininajoua@yahoo.fr
Received November 12, 2013
The effects of NO3" nutrition on iron deficiency responses were investigated in Arabidopsis thaliana. Plants were grown with or without 5 ^M Fe, and with NO3" alone or a mixture of NO3" and NH4+. The results indicated that, NO3" nutrition induced higher dry matter production, regardless the Fe concentration. Fe deficiency reduced growth activity, photosynthetic pigment concentration and Fe content of plants, whatever the N forms. This decrease was more pronounced in plants grown with mixed N source; those plants presented the highest EL and MDA and anthocyanin contents compared to plants grown under Fe sufficient conditions. In iron free-solutions, with NO3" as the sole nitrogen source, enhanced FC-R activity in the roots was observed. However, in the presence of NH4+, plants displayed some decrease in in FC-R and PEPC activities. The presence of NH4+ modified typical Fe stress responses in Arabidopsis thaliana plants.
Key words: Arabidopsis thaliana, iron deficiency, nitric nutrition
Among abiotic stresses, iron deficiency is one of severe problems in worldwide agricultural production. Iron deficiency changed plant morphological traits (Briat 2007), decreased plant dry matter, leaf area and crop yield (Rabhi et al. 2007; Zocchi et al. 2007; Zaharieva et al. 2004). Around third of arable land on earth are affected by iron deficiency and the problems becoming an increasing threat to agriculture globally (Mtimet
2001). To overcome this constraint, plants have evolved specific adaptive mechanisms for iron
acquisition, classified into two distinct "strategies". The response to Fe deficiency of dicotyledonous and non-graminaceous monocotyledonous plants, involves a series of morpho-physiological and biochemical changes (Zaharieva et al. 2004) depicted as "strategy I". The main morphological changes are the increase of lateral root formation and the emergence of root hairs and transfer cells, increasing in this way the root surface and consequently Fe uptake (Schmidt et al. 2000). The strategy I response includes also a proton excretion
by roots, which lowers the rhizosphere pH, and an increase in the capacity to reduce Fe3+ (less soluble) to Fe2+ (more soluble) by a Fe(III)-chelate reductase (FCR) (Kim and Guerinot 2007). The reduction step, prior to Fe2+ uptake, has been shown to be critical for Fe uptake from Fe-deficient soil (Kim and Guerinot 2007).
Several metabolic changes have also been described in Fe-deficienct roots, including an increase in the activity of phosphoenolpyruvate carboxylase (PEPC) accompanied with an accumulation of organic acids in roots, mainly malate and citrate (Abadia et al. 2000). PEPC catalyses the carboxylation of
phosphoenolpyruvate (PEP) to oxaloacetate. The latter is reduced to malate via the cytosolic enzyme, malate dehydrogenase, then malate is transported to mitochondria through the malate-oxaloacetate shuttle and converted into citrate by citrate synthase (Andaluz et al. 2002). The role of organic acid accumulation in Fe deficiency response is mainly related to Fe transport to shoots as Fe citrate in xylem sap (Stephan 2002).
Nitrogen plays a pivotal role in the inorganic nutrition of plants and hence in determining growth. The form of N supply, to a great extent, controls the uptake ratio of cations and anions and thus, influences dry matter production and root rhizosphere and apoplastic pH (Mengel 1995; Marschner 1995). It has been reported that the uptake of NH4+ by plant roots leads to a further acidification of plant root zone (Marschner 1995; Von Wiren et al. 2001) and enhances Fe solubility. In contrast, in soils with high pH, where nitrate is the predominant N source, the uptake of NO3-anions results in an alkalinization of the rhizosphere (Marschner 1995; Touraine et al. 2001) limiting in this way Fe availability to roots (Lindsay 1984).
Nitrate regime not only increases soil pH but also induces high leaf apoplastic pH (Kosegarten and Englisch 1994; Kosegarten et al. 2001) which reduces leaf FCR activity, resulting in Fe inactivation in rosette leaves (Mengel et al. 1994).
The aim of this work was to study the effect of nitric regime under Fe-deficient conditions on growth characteristics and physiological responses in Arabidopsis thaliana (isolate Col).
MATERIALS AND METHODS
Plant material and growth conditions
Seeds of A. thaliana, Col, were sown in pots containing a mixture of sand and peat (1V: 2V). After germination, seedlings were grown in a growth chamber under controlled conditions (Gibeaut et al. 1997): 12 h daily light, 150 ^mol m-2 s-1 photon flux density; 22/18°C day/night temperature regime, and 60/80% day/night relative humidity regime. They were irrigated with distilled water for 8 days then with a complete nutrient solution containing 5 ^M Fe-EDTA (Gay and Hauk 1994). After 3 weeks, they were transferred into 300 ml plastic pots and acclimated over one week. Then, four treatments were started. Plants were separated in 4 lots, each one placed on one of the following nutrient solutions: T1: 100% NO3- N + 5 HM Fe; T2: 100% NO3-N + 0 nM Fe; T3: 50% NO3-N:50% NH4+ N + 5 ^M Fe; T4: 50% NO3- N:50% NH4+ N + 0 ^M Fe. The medium was weekly-renewed and the plants were harvested after 10 days of treatment.
Pigment concentrations
For each treatment, six plants were used, and individually treated. Fresh leaves of each plant were separately incubated in the dark for 72 h at 4°C in acetone 80% (v/v). Absorbance of each of the acetone extracts was measured with a DU 640
Beckman spectrophotometer. Concentrations of chlorophyll and total carotenoids were calculated using the equations proposed by Lichtenthaler (1988). Anthocyanin levels were estimated in methanolic extracts form A530 and A653 as indicated by Murray and Hackett (1991).
Ferrous iron content
Bivalent iron (Fe2+) was extracted following the method of Liorente et al. (1976). Plant materials were washed with distilled water, dried at 70 °C for 72 h, weighed then ground with a mortar. The obtained powder samples were digested using 1N HCl solution. Fe2+ content was determined by atomic absorption spectrophotometry.
Electrolyte leakage
Electrolyte leakage was determined as described by Dionisio-Sese and Tobita (1998). Leaf samples (approximately 200 mg FW) were submerged into 10 ml distilled water and kept at 32°C over 2 h. Then, the initial electrical conductivity of the medium (EC1) was measured. After the conductivity measurement, the leaf tissues were killed by autoclaving at 121°C for 20 min to release all electrolytes, cooled to 25°C, and then the final electrical conductivity (EC2) was measured. The electrolyte leakage (EL) was calculated as EL = 100 EC1/EC2.
The roots were immersed 5 min in a solution of CaCl2 (0.5 mM), then incubated in 25 ml of distilled water for one hour in the dark and the EC1 values were measured. Subsequently, they were transferred to 25 ml of boiling distilled water for 5 minutes and the EC2 values were measured. The EL was calculated from EL= EC1/ (EC1+EC2).
Lipid peroxidation
The level of lipid peroxidation was determined by a procedure based on the method of Heath and
Packer (1968). 0.5 g fresh leaves were ground in 5 mL of ice-cold phosphate buffer solution (0.05 mM, pH 7.8) containing 1% PVP. The homogenate was centrifuged at 10,000 g for 30 min. 2 mL of supernatant was mixed with 2 mL of thiobarbituric acid (TBA) (0.5% TBA, 20% TCA). The mixture was heated at 100°C for 30 min, chilled on ice, and then centrifuged at 1000 g for 10 min. Absorbance of the supernatant was measured at 532 nm and adjusted for non-specific absorbance at 510 nm and 560 nm. Root Fe(III)-reductase activity
Fe(III)-chelate reductase activity was estimated as in vivo reduction of Fe(III)-EDTA by intact plant roots. The formation of the red Fe(II)-bathophenanthrolinedisulphonate (BPDS) complex was followed by measuring its absorbance at 535 nm (Chaney et al. 1972). The reaction was performed for 30 min with BPDS (0.3 mM) and Fe(III)-Na-EDTA (0.1 mM) in full-strength nutrient solution, buffered with 10 mM MES-KOH (pH 5.5). Enzyme extraction and assay
Fresh leaf or root samples were ground in a mortar with 100 mM Tris-bicine (pH 8.0) containing 1 mM ethylenediaminetetraacetic acid (EDTA), 5% glycerol (v/v), 5 mM MgCl2, 1% mercaptoethanol (v/v), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 5% polyvinylpyrrolidone (PVP) (w/v of sample FW). After centrifugation at 12000 X g for 20 min at 4°C, the supernatant was collected and enzyme activities were immediately measured.
The activities of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) and Rubisco (Rubisco E.C.4.1.1.39) were assayed according to Ouerghi et al. (2000). PEPC reaction mixture contained 100 mM Tris-bicine (pH 8.0), 5 mM MgCl2, 1 mM DTT, 5 mM NaHCO3, 0.2 mM NADH, 4 mM phosphoenolpyruvate, 5 enzyme units of
malate dehydrogenase (MDH). The crude extract (100 ^l) was added to the reaction medium then the activity was monitored at 340 nm for 15 min. Statistics
Statistical analysis was performed with Statistica™ software, using two-way ANOVA and Newman-Keuls test for post-hoc mean comparison. The ANOVA was performed over the whole set of data
RESULTS
Plant aspect and growth
Chlorosis symptoms appeared first on the fifth day after the beginning of the treatments in the young leaves of plants grown without Fe (0 ^M Fe) with whatever the N form. After 10 days of treatment, chlorosis intensity of plants grown without Fe and N source as 50% NO3-:50% NH4+ N was higher than those of plant grown without Fe and N as 100% NO3-.
At the end of treatment, plants grown with 5 ^M Fe and 100% NO3- showed significantly higher total dry weight compared to plants grown with 5 ^M Fe and 50% NO3-:50% NH4+ (Fig. 1). Iron deficiency reduced dry weight of plants whatever the N source. This reduction was more pronounced for plants grown with 50% NO3-:50% NH4+ (Fig. 1).
Fe content
Root and shoot Fe concentration of Arabidopsis plants grown without Fe (0 ^M Fe) was found to be significantly lower than in plants with 5 ^M Fe, regardless of the N form (Fig. 2) Whereas, regardless of the Fe level, Fe concentration in plants grown with 100% NO3- was significantly higher than that of plants gown with both forms of N.
Pigments contents
A significant reduction in chlorophyll content was found in Fe-deficient plants as compared to Fe-
sufficient plants. This effect was more pronounced in NH4+ fed plants (Fig. 3). Fe deficiency did not affect carotenoids content whatever the N form (Fig. 3). Anthocyanins content was significantly induced by iron deficiency when plants are grown with % NO3-:50% NH4+. However, an opposite effect was observed in NO3- treated (Fig. 4).
Lipid peroxidation and electrolyte leakage
The extent of the Fe-induced oxidative damage was assessed by measuring the MDA formation and electrolyte leakage (Fig. 5). Amount of electrolyte leakage increased under iron deficiency conditions only for plants grown with both forms of N. However, in plant grown in nitric medium, electrolyte leakage was not affected by iron deficiency (Fig. 5A). Similarly, Fe deficiency increased MDA contents in plants grown with 50% NO3-:50% NH4+, whereas MDA content was not significantly affected by iron deficiency if NO3- was used as the sole nitrogen source (Fig. 5B).
Nutrient solution acidification
The changes in pH depended primarily on nitrogen source. In the fact, NO3- uptake increases the pH of the outer solution, whereas NH4+ uptake doesn't affects pH of the medium. When plants are grown without Fe, the pH of the nutrient solution remained stable (Fig. 6A).
Root ferric chelate reducing activity
The highest level of FCR activity was recorded in plant grown in presence of Fe and with 50% NO3-:50% NH4+. Whereas, elimination of Fe from the growth medium significantly decreased FCR activity, in these conditions. In plants grown with 100% NO3-, iron deficiency had a significant positive effect on FCR activity (Fig. 6B).
PEPC activity in roots extracts
The activity of PEPC, enzyme involved in organic
acid metabolism was measured in the root tips of PEPC activity in NH4+ fed plants, whereas no
the plants from all treatments after 10 days of Fe significant effect in NO3- treated plants was
starvation. Fe deficiency significantly decreased observed (Fig. 7).
Figure 1 : Shoot (A) and Root (B) dry weights of Arabidopsis thaliana plants grown during 10 days with 0 ^M Fe(III)-EDTA or Б ^M Fe(III)-EDTA, and with NO3- as the only source of nitrogen or with a mixed NH4+/NO3- supply. Bars are means of 8 replicates ± SE. Bars labelled by the same letter are not statistically different according to the ANOVA test at P < 0.0Б.
Figure 2. Shoot (A) and Root (B) Fe content of Arabidopsis thaliana plants grown during 10 days with 0 ^M Fe(III)-EDTA or 5 ^M Fe(III)-EDTA, and with NO3- as the only source of nitrogen or with a mixed NH4+/NO3- supply. Bars are means of 6 replicates ± SE. Bars labelled by the same letter are not statistically different according to the ANOVA test at P < 0.05.
Figure S. Chlorophyll and carotenoid contents in shoot of Arabidopsis thaliana plants grown during 10 days with 0 ^M Fe(III)-EDTA or Б ^M Fe(III)-EDTA, and with NO3- as the only source of nitrogen or with a mixed NH4+/NO3- supply. Bars are means of 6 replicates ± SE. Bars labelled by the same letter are not statistically different according to the ANOVA test at P < 0.0Б.
Figure 4. Anthocyanins content in shoot of Arabidopsis thaliana plants grown during 10 days with 0 ^M Fe(III)-EDTA or 5 ^M Fe(III)-EDTA, and with NO3- as the only source of nitrogen or with a mixed NH4+/ NO3- supply. Bars are means of 6 replicates ± SE. Bars labelled by the same letter are not statistically different according to the ANOVA test at P < 0.05.
Figure 5. Electrolyte leakage (A) and MDA content (B) in shoot of Arabidopsis thaliana plants grown during 10 days with 0 ^M Fe(III)-EDTA or 5 ^M Fe(III)-EDTA, and with NO3- as the only source of nitrogen or with a mixed NH4+/ NO3- supply. Bars are means of 6 replicates ± SE. Bars labelled by the same letter are not statistically different according to the ANOVA test at P < 0.05.
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Figure 6. pH values of the culture media (A) and Fe-reductase activity in root (B) of Arabidopsis thaliana plants grown during 10 days with 0 ^M Fe(III)-EDTA or 5 ^M Fe(III)-EDTA, and with NO3- as the only source of nitrogen or with a mixed NH4+/ NO3- supply. Bars are means of 6 replicates ± SE. Bars labelled by the same letter are not statistically different according to the ANOVA test at P < 0.05.
Figure 7. PEPC activity in root of Arabidopsis thaliana plants grown during 10 days with 0 ^M Fe(III)-EDTA or 5 ^M Fe(III)-EDTA, and with NO3- as the only source of nitrogen or with a mixed NH4+/NO3-supply. Bars are means of 6 replicates ± SE. Bars labelled by the same letter are not statistically different according to the ANOVA test at P < 0.05.
DISCUSSION
The results from this study showed that the interaction between different Fe levels and N forms in the nutrient solution had a significant effect on vegetative growth, nutrient concentration and biochemical response in Arabidopsis plants. Concerning the total growth, plants supplied with N as 100% NO3- produce significantly higher dry matter than plants with mixed N nutrition. A similar result has been recorded in tomato plants (Claussen
2002) who have noted that tomato plants cultivated on a nitric medium produced higher dry weight than plants cultivated in a mixed medium. Moreover, Fe deprivation reduced plants growth whatever the N form, this reduction was more pronounced for plants grown in the presence of NH4+ in the medium culture.
On the other hand, Fe deficiency caused iron chlorosis in Arabidopsis plants of both N forms. Chlorosis symptom was accompanied by a significantly decrease of chlorophyll content in the shoots. The impact of iron deficiency, on chlorophyll formation has been reported in several studies. For example, bicarbonate presence in the medium
significantly reduced chlorophyll concentration in sunflower leaves (Gharsalli and Hajji 1992) as well as in two peach rootstocks (Molassiotis et al. 2006). These findings are explained by the primordial role of Fe2+ on the formation of the chlorophyll precursors d-aminolevulinic acid and protochlorophyllide (Marschner 1995).
The shoot and root Fe content was significantly higher in plants grown with sufficient Fe and 100% NO3- compared either to plants with sufficient Fe and both form of N or without Fe and whatever N form. Similar findings were showed by Zornoza and Gonzalez (1998) who found that the presence of 20% N as NH4+ reduced the uptake of Fe, by spinach plants. Contrary to our findings, (Assimakopoulou, 2006) demonstrated that Fe concentration increased with the addition of mixed N nutrition. Regardless of the N form, the Fe deprivation from the nutrient solution significantly decreased shoot and root Fe concentration.
It is well known that, Fe deficiency induces oxidative stress in dicotyledonous species which causes disturbance of expression and activity of diverse antioxidant enzymes (Ranieri et al. 2001). In
this work we assessed oxidative stress by measuring the anthocyanin and MDA content and electrolyte leakage. Under Fe deficiency conditions, MDA content and electrolyte leakage increased markedly only for plant grown with mixed N nutrition, suggesting that cell membrane stability was affected under theses conditions. Lipid peroxidation of membranes was accompanied by an increase of anthocyanin content, in the same conditions. According to (Sgherri et al. 2004) anthocyanin production, known as a hallmark of plant stress, allows the plant to develop resistance to a number of environmental stresses, especially oxidative damages.
Acidification of the rhizosphere is known to be part of the mechanism by which some dicotyledonous plants respond to Fe starvation. This acidification has been attributed to activation of the root plasma membrane H+-ATPase (Dell'Orto et al. 2000b). When grown without Fe, regardless of the nitrogen source, pH of the medium was not affected. Whereas, a slight increase in pH was observed when plants were supplied with NO3-. It has been shown that medium acidification and root FeIII reducing capacities are related to plants adaptation to Fe deficiency (Brancadoro et al. 1995; Schmidt 2006; Zaharieva et al. 2004). Under our growth conditions, we did not observe root acidification under Fe-deficient conditions, however, Fe-chelate reductase activity was significantly increased in Fe-deficient plants fed with NO3-. On the opposite when supplied with NH4+ under Fe-deficient conditions, activity of FCR was significantly reduced. These results are in agreement with those of Rivero et al. (2003) who found higher FCR activity in the roots of both tomato and watermelon plants treated with NO3- in comparison with those treated with NH4+.
Kosegarten et al. (2004) and Nikolic and Romheld (2003) demonstrated close relationships between the form of nitrogen in the nutrient solution, root apoplast pH and FC-R activity in Helianthus annuus L.. Based on these relationships, the stimulation of FC-R activity may be linked to the decrease in pH associated with NH4+ uptake. Nevertheless, our results are not in agreement with the expected effect of various nitrogen forms on Fe3+ reduction. In addition, the application of nitrate in the nutrient solution did not affect PEPC activity. In contrast, a significant decrease of this activity was observed under ammoniacal regime.
Our results showed that nitrogen uptake and metabolism interfere with Fe metabolism in Arabidopsis plants. In a mixed medium (50% NO3-:50% NH4+), growth activity and membrane integrity of Arabidopsis was greatly affected by iron deficiency. In these conditions, some typical biochemical responses to Fe deficiency (medium acidification, root FC-R and PEPC activity) are not induced in Arabidopsis plants. However, plants fed with 100% NO3- showed a better behavior towards iron deificiency. In the fact, growth activity was less affected and an induction of root FC-R activity in the roots when subjected to Fe deficiency in these conditions.
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