CAN COPPER AND ZINC IN DIFFERENT CHEMICAL FORMS AMELIORATE THE EFFECTS OF IRON-DEFICIENCY IN PHASEOLUS PLANTS?
Abstract
In this research, the differentiation between the effects of ionic (CuSO4), (Zn SO4) and chelated various forms of copper and zinc Cu (II) HEDTA and Zn (II) HEDTA, while HEDTA is N- (hydroxyethyl) ethylene diamine triacetic acid, exercised at the micromolar concentration in the nutrient solution of phaseolus vulgaris plants grown hydroponically under the event of iron insufficiency (- Fe) were inspected. Plant variants (– Fe +2 µM Cu2+) and (– Fe+ 20 µM Zn2+) with considerably vigorous chlorosis were scrutinized for the implementing to pull after the recuperation, of leaf greening formed after dealing with Cu(II)HEDTA in the two variances, mostly for the recently created leaf, as it has manifest with chlorophyll assessment. Changes in plasma layer reductase tendency (PMRA) in- roots after remediation with ionic or chelated copper were perceived in (+Fe) and (– Fe) plants. Adversity in an arrangement of elements in a particular form motivates a noteworthy accretion of ferric-chelate reductase power (with the substrate of Fe (III) HEDTA).Corneous utilization of cupric particles in arrangements of iron-inadequate plants brought about an emotional restraint of Fe (III) HEDTA. The cupric-chelate Cu (II) HEDTA, linked to similar substantiation in arrangements with (– Fe) plants, preserve the high solicitation of plasma layer ferric-chelate reductase activity. The remediation with Cu (II) HEDTA elevate the expansion and root PMRA and additionally other iron-deficiency response of phaseolus plants. Regard to cell reinforcement compounds, measurements of 20 ?M of Zn altogether developed action of the protein superoxide dismutase and GPX.
Key words: Phaseolus Vulgaris- Fe deficiency- Fe chelates reductase activity- Cu+2, Zn+2 – Cu-HEDTA-Zn-HEDTA.
INTRODUCTION
Copper and iron are important plant micronutrients with similar redox-properties and control in their mobilization, uptake and translocation. The regulation of Fe and Cu homeostasis in plant cells under sub-optimal growth conditions is extremely important for plant productivity. Iron deficiency chlorosis is a limiting factor for plant growth and yield and is spread in different crops, mainly in alkaline and carbonate soils, due to the insolubility of iron oxides and hydroxides (Schmidt 1999). Additionally, Fe-insufficiency stress can be combined with increased level of copper moving in plant tissues by Fungicide spraying against diseases (Babalakova et al. 2005).
Ferric-chelate reductase is the ultimate thoughtful redox enzyme; it is an integration membrane protein pertinence to a family of flavoproteins that convey electrons from cytosolic NADH to extracellular electron acceptors through FAD and heme set (Robinson et al. 1999; Curie and Briat 2003). Beside an altitude creation of ferric-chelate reductase activity (FeChRA) in roots of iron-insufficient plants, iron deficiency dicotyledonous plants such as phaseolus promote diverse adaptive morphological and biochemical mechanization to ameliorate iron procuration in soil solutions (Fe-deficiency stress responses; Raboti et al. 1995; Espen et al. 2000).plants, and (iii) to study the activity of antioxidant enzymes activities in control and iron-deficient plants.
3. RESULTS AND DISCUSSION
3.1.Effect of chemical forms on symptoms of iron deficiency in leaves
The developing plants were shown in a 12-day feeding solution without iron and copper ions (2 ?mol) yellowing in the first and second leaves compared to control.
Fig.1: Effect of chemical forms on iron deficiency symptoms in leaves
Data given in (Figure 1) show that phaseolus plants are grown for 12 days without iron in the nutrient solution progressing reasonable chlorosis on the first and second leaves, with chlorophyll dropping in comparison with (+Fe) plants. The presence of 2.00 µM cupric ions in the solution of Fe-insufficient plants imparted to the manifestation of very potent chlorosis (Fig.1). The same or even higher drops in chlorophyll concentration of Fe-deficient plants were found with 20 µM Zn-ions supply. Chelated forms of Cu and Zn, exercised continuously on Fe-deficient plants conserved, the pigment content to a higher level in comparison with the control (–Fe) plants. Regardless of very strong chlorosis in variant (–Fe+Cu) before treatment with Cu (II) HEDTA treatment of phaseolus brought to some retardation of leaf green. Using the same experimental for the other variant (–Fe+Zn) with pale-yellow first leaf, some distinction after implementation of cupric- and zinc-chelates were received (Fig.2). The favorable effect on leaf greening was a singularity with the implementation of Cu(II)HEDTA feeding created smaller, but still positive effect, and Zn(II)HEDTA supply did not ameliorate leaf chlorosis (Figure 1). The duration of our experiment might be not sufficient to remedy leaf chlorosis of Phaseolus, treated with zinc-chelate. Other authors received that plant roots treated with zinc did not show promote response like copper 23 (Bartha et al., 2005). Zn (II) HEDTA supply was without greening effect on Phaseolus leaves. Chelated copper and greening of iron-deficient phaseolus remobilization for improved chlorophyll biosynthesis in treated Fe-deficient plants was shown in (Fig. 3-1 and 5). Recently it has been reported that copper ions in nutrient solution encourage Fe Ch-reductase of pea plants, whereas, zinc ions do not offer the prompt response 23-24-25(Bartha et al., 2005; Bethke et al., 2006 and Sarath et al., 2006).
3.2. Effect of chemical forms of copper and zinc on root dry weights
Relying on the chemical form of copper and zinc the diminution of root dry weight under conditions of iron inanition was observed. Data given in (Figure 2) indicate that the decrease was observed in root dry weight in Fe deficiency stressed plants. Rebate of the growth dry weight with morphological changes, discriminatory of iron insufficiency, was also spotted (Figs 1&2).
The concentration of 2?M cupric chelate shows remarkable growth elaboration of Fe-deficient phaseolus plants, as compared to the noteworthy decrease of root growth weight at the same concentration of ionic copper (Figure2). The same trend was noticed due to the presence of Zn. Zn HEDTA amended the root growth weight and deny the nutritional disorders and consequently, caused increases in the uptake of nutrients by the roots 26(El-Fouly et al. 2002). These findings are in agreement with those obtained by 27-28-29-30 (Din and Flowers, 2002; Al-Ansari, 2003; Salama et al., 2014 &2015). It was pointed that zinc enforcement for output enhances biomass product and plant zinc concentration 31 (Subbaiah et al., 2016). Our results coincide with those acquired, that offers a significant augmentation with respect to the control in plant biomass and Zn content in the leaves of Phaseolus at low doses of Zn (Figures 1and 2). One of the most widely utilized points of stress is plant biomass 32 (Rios et al., 2009).
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Figure 2. Effect of chemical forms of copper and zinc on dry weights
3.3. Effect of long term treatment of ionic Cu and Zn 2 micromol and Cu and Zn HEDTA 20micromol on micronutrients concentration
Data offered in (Fig. 3) show that under iron feeding conditions presence of Cu+2and Zn+2 noteworthy raise in micronutrients concentration. However, under Fe deficiency conditions plants showed a reduction in Fe, Zn, and Cu concentration as parallel with plants feeding with chelated copper and zinc under Fe deficiency. In spite of, intensive research proposition this may be due to the part of chelation in the mechanization of mineral uptake and translocation and metabolism in plants (El Fouly et al., 2002, 2010).Also, may be due to the reaction of copper ion to compose stabilized convention and to participate in redox reactions at the plasma membrane 33-34 (Guinn and Joham 1963; Taylor and Foy 1985). Our results exhibit significant differences with Zn HEDTA, respect to the control.
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Figure 3: Effect of long term of chemical forms on micronutrients concentration
3.4. Effect of long term of chemical forms of copper and zinc on pigments content.
The concentrations of the photosynthetic pigment of phaseolus plants grown in presence or absence of Fe are shown in (Table 1). In general, the plants in a non-iron feed solution performed in a significant decrease in the chlorophyll (A) -chlorophyll (B) and chlorophyll (A + B) leaf content with the appearance of yellowing in the leaves with the apparition of yellowing in new leaves (Fig. 1). The addition of 2.0 micromole copper metals or the addition of 20 micromol zinc metal in the absence of iron procured to a significant decrease in the content of chlorophyll with the occurrence of pale yellowing in modern leaves (Figure 1). The addition of Cu-HEDTA 2.0 µM or 20 µM of Zn-HEDTA resulted in a significant increase in leaf content of chlorophyll A, B and T-Chl (A + B) compared to non-iron treatment. Zn HEDTA might improve the tolerance of Phaseolus plants by restoring the main photosynthetic pigments. The similar results were reported by Salama et al., (2015), that chlorophylls in photosynthetic membranes could protect the photosynthetic system from excessive ROS by quenching of singlet oxygen and other radicals under stress conditions. Cu (II)HEDTA elucidates that it could collaborate in the process of greening by remobilization of apoplastic and cellular iron pools (Fig.1).In spite of very vigorous, chlorosis in variant (–Fe+Cu) before treatment with Cu (II)HEDTA, leaf greening started soon after enforcement Cu and Zn HEDTA. Cu(II)HEDTA supply created smaller effects, but Zn (II)HEDTA supply did not ameliorate leaf chlorosis (Fig.2). The duration of our experiment might be not sufficient to remedy leaf chlorosis of Phaseolus, treated with zinc-chelate (Bartha et al., 2005).
Table 1: Effect of long term of ionic Cu and Zn 2 micromol and Cu and Zn HEDTA 20 µMol on chlorophyll (a),(b) and T(a+b) and antioxidant enzymes activity
Variants
Chla
mg/gfw Chlb
mg/gfw T-chl
mg/gfw GPx
EU/gfw SOD
mg/gfw
FNS(+Fe) Control
1.436 a
0.369 a 1.809a 176a 31a
Cu+2
1.120 b 0.258 a 1.38b 272b 28ab
Cu-ch
1.980 c 0.412 c 2.392c 79c 30b
Zn+2
1.950 c 0.412 c 2.36c 133d 34b
Zn-ch
0.996 c 0.389 b 1.39c 156e 43c
LSD 0.05 0.20 0.049 0.14 4.44 3.81
FNS(-Fe) Control
0.335 a 0.260 c 0.595a 230a 39a
Cu+2
0.219 b 0.210 a 0.429b 245c 34b
Cu-ch
0.711 c 0.430 d 1.141c 116c 42ab
Zn+2
0.210 d 0.248 b 0.458d 220b 39a
Zn-ch
0.780 e 0.426 c 1.206e 97d 44ab
LSD 0.05 0.13 0.041 0.11 3.57 4.40
3.5. Effect of long term of chemical forms of copper and zinc on antioxidant enzymes activities.
The amelioration of stress tolerance under Fe deficiency is often related to an increase in the activity of antioxidant enzymes which can prominence in an increase in antioxidant enzyme activities, which in turn conserve plants from oxidative stress caused by Fe deficiency. As long as, increased SOD activity in phaseolus elucidate that this species of plant has the capacity to naturalize to high levels of ROS by promoting an antioxidant defense system. SOD is an important member of the cell conservative antioxidant system. This enzyme catalyzes the dismutation of the superoxide anion into H2O2 plus molecular oxygen 35 (?lesak, ?lesak, Libik, & Miszalski, 2008). Under Fe deficiency SOD activity significantly increased in leaves accordingly lower Zn HEDTA (Table 1). These increased resulted highly significant with respect to the control. Data presented in Table (1) shows that under normal conditions of (+Fe) treatment presence of ionic Cu+2 significantly increased GPX enzyme activity. However, SOD did not affect. While the presence of Cu-HEDTA or Zn-HEDTA significantly decreased the activities of both enzymes. AGX activity showed remarkable increase under Fe deficiency (Table. 2). However, the presence of Cu+2 showed a marked increase in GPX enzyme activity as compared with either control or (-Fe) treatment.
3.6. Effect of long term of chemical forms on Fe-Chelate reductase activity of phaseolus seedlings (Nebraska).
As shown in (Table 2) the depression of iron at age 12days 2.76 ?mol / g fresh weight by means of the increased value of ferric –chelate-reductase-activity (Fe III reductase) as the recipient, of electrons on the plasmid wall of the roots. Remediation of plants under iron deficiency with 2 ?mol leads to a diminution, in the efficiency of ferric-chelate reductase activity (Fe (III) HEDTA from 2.76 to 1.45 ?mol / g fresh weight parallel to the control treatment of ionic zinc 20 ?mol which gave the same effectiveness. The treatment of zinc-lead to a relief in ferric -chelate reductase activity (Fe (III) HEDTA) 1.55 ?m / g FW under iron deficiency conditions compared to control. Fe insufficiency gives elevation to similar changes in the ferric reductase activities and clarifies the existence of redox proteins with similar characteristic at PM.Altitude activation of ferric reductase in the roots of Fe-deficient plants might be linked to the same extent with increased copper uptake under iron starvation (Herbic et al. 1996).
The reconsider repressive effect of ionic copper on the plant root reducing capability after the invention of Fe deficiency assured previously acquire results (Alcantara et al.1994; Romera et al. 1997; Schmidt et al. 1997). The implementation of RA in Fe-deficient plants was related to pH changes of the nutrient solutions during iron starvation and copper remediation. Enforcement of ionic copper initiates to inhibit the t release of protons by roots of Fe-deficient plants from the first day of solution change (Babalakova et al. 2005) and this inhibition correlated with the high inhibition of FeChRA by ionic copper. At the same time, chelated copper enforcement encourages the H+ throwing by the roots of Fe-deficient phaseolus plants.Elevation of acidification of the intermedium and alimentary the high level of FeChRA in significant plants because of the enzyme is pH sensitive 38(Wei et al. 1997; Schmidt 1999).
Table 2: Effect of long term of chemical forms of copper and zinc on pigments content of phaseolus seedlings (Nebraska).
Variants Fe(III) HEDTA-RA 0.265 a Control FNS(+Fe)
0.251a 2µM Cu+2 0.270 b 2uMCµHEDTA 0.435a 20µM Zn+2 0.448b 20µMZn HEDTA 0.038 LSD at 0.05
2.760c Control FNS(-Fe)
1.450d 2µM Cu+2 1.920a 2uMCµHEDTA 1.550e 20µM Zn+2 4.160b 20µMZn HEDTA 0.378 LSD at 0.05
The chemical various forms of exercise copper and zinc used to parallel their effects in control (+Fe) and Fe-deficient (–Fe) plants have distinct electrical charges. Copper in copper (II) sulfate form a Cupric-hexahydrate cation, Cu (II) (OH2)62+, in aqueous solution and keep the ionic characteristic, of copper. When HEDTA is added to aqueous solution of copper (II) sulfate, HEDTA forms a strong chelate with Cu (II), hydroxyethyl ethylenediamine-triacetato cuprate Cu (II) HEDTA2- with anionic character41 (Coombes et al. 1978)
