ACCEPTED MANUSCRIPT

Comparison of the Effects of Nano-Iron Fertilizer with Iron-Chelate on Growth

Parameters and Some Biochemical Properties of Catharanthus Roseus

Mehri Askary, 1* Mohammad Reza Amirjani1 and Tahereh Saberi2

1
Department of Biology, Faculty of Sciences, Arak University, Arak 38156-8-8349, Iran

2
M.Sc Student of Plant Physiology, Biology Department, Arak University.

Address correspondence to Mehri Askary: m-askary@araku.ac.ir

ABSTRACT

Nano fertilizer supply of nutrients to the plant is compounds to replace conventional fertilizers.

Iron (Fe) is one of the essential elements for plant growth and plays an important role in the

photosynthetic reactions. Plants treated with different concentrations (0, 5, 10, 20, 30 and 40µM)

of iron oxide nanoparticles (Fe 2O3) for 70 days. Fe2O3 nanoparticles significantly increased

growth parameters, photosynthetic pigments and total protein contents. The maximum amounts

of growth parameters, photosynthetic pigments and protein contents were obtained in 30µM of

Fe2O3 and minimum values of these parameters were found in 0µM. Highest value of total

alkaloid content was obtained in 0µM of Fe 2O3 and lowest value of it was observed in control

1
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

plants. Iron oxide nanoparticles increased potassium, phosphorus and iron absorption but didn’t

show a significantly effect on sodium content.

Keywords

Alkaloid, Fe2O3 nanoparticles, pigments, Protein, Periwinkel or Vinca

2
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

INTRODUCTION

Iron is one of the essential micronutrients for plant growth (Armin et al., 2014). Among all the

micro-nutrients, plants need to iron than other. Iron is a cofactor for approximately 140 enzymes

that catalyze exclusive biochemical reactions (Mohamadipoor et al., 2013). Iron is an essential

element for the suitable functioning of many metabolic and enzymatic processes related to electron

transport, nitrogen fixation, synthesis of deoxyribonucleic acid (DNA) and hormone synthesis,

therefore plays an important role in plant metabolism (Fernandez et al., 2008). Plants have two

main problems with iron as a free ion: its insolubility and its toxicity. To ensure iron absorption

from soil and to avoid from iron excess in the cells, iron uptake and homeostasis are highly

controlled (Hell and Stephan, 2003).

Iron deficiency is an abiotic stress that affects many plants in areas of the world. This

physiological disarrange is often found in plants that grown in calcareous and alkaline soils.

Although iron is abundant in the earth's crust but iron availability to plants is limited due to the

very low solubility of iron (Fe+3) oxides under aerobic conditions (Fernandez et al., 2008). Fe+2 is

partly soluble but is easily oxidized by atmospheric oxygen. The solubility of Fe+3 decreases

dramatically with increasing pH values due to hydrolysation (as iron(III) hydroxide),

polymerization and sedimentation with inorganic anions. While free Fe +3is soluble up to

106M at pH 3.3, this concentration is only 10-17M Fe(III) at pH 7 (Neilands et al., 1987).

However, plants require between 10-4 and 10-8 M Fe(III) (Hell and Stephan, 2003), thus plants

grown in calcareous, high pH soils showed Fe deficiency chlorosis. Iron deficiency extremely

alters the physiology of plants. Reduction of plant fresh and dry weight has been found

3
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

associated with leaf interveinal chlorosis and necrotic that starting from the shoot apex. Iron

is a necessary element for the formation of chlorophyll, therefore deficiency or deactivation

of iron in plants reduced chlorophyll, leading to reduced photosynthesis (Fernandez et al., 2008).

Common iron fertilizers used to reduce deficiency syndromes contain iron(II) sulfate heptahydrate

(FeSO4.7H2O) or iron chelates (Hell and Stephan, 2003). Iron chelate (for example Fe-

ethylenediaminetetraacetic acid (EDTA)) is absorbed by plants however it depends to soil’s

conditions especially soil pH (Mohamadipoor et al., 2013). 30% of the arable land worldwide

consists of calcareous and alkaline soils. This limitation cannot easily be overcome using iron-

containing fertilizers, because iron availability is aproblem of solubility and not of abundance

(Guerinot, 2001; Hell and Stephan, 2003). Nowdays, Nano-Fe fertilizer can be used as a rich

source of iron for plants, since it gradual release of Fe in a wide pH range (pH 3 to 11). One

advantage of this nanofertilizer is that ethylene compound is not used in its structure (Armin et

al., 2014). Nano compounds rapidly and completely absorbed by plants and supply it's nutrients

deficiency and needs (Harsini et al., 2014). The use of nano fertilizer leads to an increased

efficiency of the elements, reduce soil toxicity, reduction of negative effects caused by the

excessive consumption of fertilizers and reduce the frequency of application of fertilizers

(Harsini et al., 2014).

Vinca or Catharanthu sroseus L. is one of the important medicinal plants belong to the family

Apocynaceae that produce terpenoids indole alkaloids including vincristine and vinblastine that

used in cancer chemotherapy. Roots of vinca are the main source of an anti-hypertension

alkaloid ajmalicine (Jaleel and Salem, 2010). Vinca is very sensitive to pH and shows rapid

micro-nutrient shortages when grown in soil with a pH above 6.3. Iron absorb is particularly

4
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

difficult for vinca when pH is higher than 5.8 (Thomas et al., 2012).

This research was carried out to determine suitable type of iron fertilizer and to evaluate the

effects of different concentrations of nano-Fe fertilizer on Catharanthus roseus.

MATERIALS AND METHODS

Plant Culture and Maintenance

After germination of sterilized seeds of Catharanthus roseus L., 3-days seedlings were cultured in

plastic vases with mixture of soil and perlite (1:1). The soil was a clay loam (pH 7.6) with 0.03%

nitrogen, 4 ppm absorbable phosphorus, 100 ppm absorbable potassium, 7.6 ppm available Fe, 1

ppm zinc, 0.2 ppm copper and 0.6 ppm manganese. Plants were maintained under 25/20°C

day/night temperatures with the 12 hours photoperiod. Irrigation was done weekly with 100 ml

complete Hoagland solution (containing iron-chelate (Fe-EDTA) for control plants) or 100 ml

Hoagland solution without iron-chelate and containing different concentrations of iron(III) oxide

(Fe2O3) nanoparticles (0, 5, 10, 20, 30 and 40µM) (Adamski et al., 2012; Dokhe et al., 2013).

Plants under 0 µM of Fe2O3 is not received iron in 70-day period. Iron oxide nanoparticles

(Fe2O3) were prepared from Pishgaman Company located in Mashhad, Iran. Different

concentrations of Fe2O3 nanoparticles were prepared by Prasad et al. (2012) method.

Measurements

Every 9 days (in days 18, 27, 36, 45, 54, 63 and 70), shoot height of plants were measured. After

the final harvest of 70 days plants, root length, stem and root fresh weights were measured. Dry

weight of stem and root were obtained by drying samples in an oven for 24 h at 75 °C until

5
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

constant weight. Determination of chlorophyll a, chlorophyll b and total chlorophyll (by

spectrophotometry at 663 and 645 nm, following the method of Arnon, 1949), carotenoids

content (Lichtenthaler and Welburn, 1983 spectrophotometry method) were done. Total protein

content was measured using the method of Bradford (1976) by spectrophotometry at 595 nm.

Bovine serum albumin was used as the standard. Potassium and sodium contents were

measured by flame photometry (atom emission spectroscopy (AES)). For this, samples (0.2 g)

dry ashed at 600°C for 2 h and dissolved in 1 ml hydrochloric acid. Volume of solutions is

brought to 25 ml by double distilled water and the supernatant was used for analysis (Wang and

Zhao, 1995). The contents of phosphorus (by spectrophotometry at 450 nm using the Creus et

al., 2004 method) and iron (by Celik et al., 2004 method) were determined. Total alkaloids

were measured at 254 nm by spectrophotometry (UV- Visible) method following the protocol of

Ataei-Azimi et al. (2008).

Statistical Analysis

The experiment was conducted at the research laboratory of Arak university of Iran, based on

randomized complete design with three replications in 2013. Data was subjected to analysis of

variance using SPSS software (Chicago, IL) and means were compared using Duncan test at

P=0.05.

6
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

RESULTS

Growth Parameters

The results showed that Fe2O3 nanoparticles had significant effects (P< 0.01) on shoot height of

plants in all development periods (9, 18, 27, 36, 45, 54, 63 and 70 days). Shoot height increased

with increasing concentration of Fe2O3 (Table 1). Highest value of shoot height of 70 days plants

was observed in 30µM Fe2O3 (9.51 cm) and lowest value of it was in 0µM (5.17 cm).

Root length, stem and root fresh and dry weights of 70-days plants increased significantly

(P<0.01) with increasing Fe2O3 nanoparticles concentrations (of 5 to 30 µM) in comparison with

controls (Fe-chelate, complete Hoagland). In 30 µM, root length increased by 3 fold compared to

controls. Stem fresh and dry weights increased by 2.16 and 2.08 fold compared to controls

respectively in 30 µM Fe2O3 nanoparticles. This increase was observed 3.1 and 2.8 fold for root

fresh and dry weights respectively in 30 µM. Between 30 and 40 µM of Fe2O3 didn’t show

significant difference. Root length, stem and root fresh and dry weights decreased in 0 µM of

Fe2O3 compared to control plants. This reduction was evaluated 33% for root length, 66.66% for

stem fresh weight, 67% for root fresh weight, 58.33% for stem dry and 60% for root dry weight

(Table 2).

7
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

Photosynthetic Pigments and Protein Contents

Chlorophyll a, chlorophyll b, total chlorophyll, carotenoids and protein contents increased

significantly under Fe2O3 nanoparticles treatment (5 to 30 µM). These parameters decreased in 0

µM of Fe2O3 in comparison with controls (Fe-chelate). Highest values of chlorophyll a (3.37

mg/g fresh matter), b (1.63 mg/g fresh matter), total chlorophyll (4.91 mg/g fresh matter),

carotenoids (17.69 mg/g fresh matter) and protein (9.87 mg/g fresh matter) were observed in 30

µM of Fe2O3. Lowest values of these parameters were observed in 0 µM Fe 2O3. There wasn’t

significant difference between 30 and 40 µM of Fe 2O3. Protein contents increased by 11.91%,

25.78%, 32.50% and 52.78 in 5, 10, 20 and 30 µMof Fe2O3as compared to controls (Figure 1).

Total Alkaloid Content

Results showed that Fe2O3 application had significant effect (P<0.01) on total alkaloid content.

Alkaloid contents increased significantly in 0 µMof Fe2O3 as compared to control plants (under

complete Hoagland contains Fe-chelate). Highest and lowest values of alkaloid were observed

in plants under 0 µM of Fe2O3and control plants, respectively. Alkaloid content increased

significantly with increasing concentration of Fe2O3 nanoparticles. This increase was evaluated

192%, 22.47%, 21.34%, 64%, 116% and 115% in 0, 5, 10, 20, 30 and 40 µM of Fe2O3 in

compared with controls, respectively (Figure 2).

8
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

Element contents: Potassium, phosphorus and iron contents of vinca were significantly affected

by Fe2O3 nanoparticles, whereas Fe 2O3 had no significant effect on sodium content. Maximum

amounts of these elements were observed in 30 µM of Fe 2O3 with 168%, 212% and 69.71%

increase in potassium, phosphorus and iron contents, respectively. Potassium, phosphorus and

iron contents decreased in 0 µM of Fe2O3. Minimum amounts of these elements were obtained in

plants under 0 µM (Figure 3).

DISCUSSION

Nano-particles have high reactivity because of more specific surface area, more density of

reactive areas, or increased reactivity of these areas on the particle surfaces. These features

streamline absorption of fertilizers and pesticides that produced in nano scale (Sheykhbaglou et

al., 2010). Nano-particles can transport within plant body with subsequent interactions with

biomolecules, such as nucleic acids, proteins including enzymes, and cell structures, such as cell

walls and biomembranes (Krystofova et al., 2013). Iron nanofertilizer can be considered as an

enriched and trusted source of bivalent iron for plant because of its high stability and gradual

release of Fe in a wide pH range (pH 3 to 11) (Moghadam et al., 2012).

Growth Parameters

In present study, growth parameters such as shoot height, root length and weight parameters

decreased in 0 µM nano-iron but increased in different concentrations of nano- iron. Peyvandi et al.

9
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

(2011) studied effects of different concentrations of iron-chelate (1.5, 4.5 and 7.5 Kg.ha-1) and

nano-iron fertilizer (1, 3 and 5 Kg.ha-1) on Ocimum basilicum and resulted that maximum

amounts of growth parameters such as shoot and root fresh and dry weights were observed in

plants under nano-iron fertilizer treatment. Study of Fe2O3 effects on growth parameters of

peanut showed that all of growth parameters increased significantly under Fe2O3 treatment (Xiu-

mei et al., 2005). Positive effects of nano-iron fertilizer on growth parameters were observed in

Spinacia oleracea (Moghadam et al., 2012). With production of nano fertilizers, these compounds

rapidly absorb by plants and provide essential and required nutrients for plants. Therefore

increase in plant growth occurs with use of nano materials (Mohamadipoor et al., 2013).

Photosynthetic Pigments and Protein Contents

Iron plays important role in plant metabolism especially in chlorophyll synthesis which is

essential for plant photosynthesis. Iron is a key structural component in many enzymes such as

catalases and peroxidases as well as flavoproteins (Boorboori et al., 2012). Iron is an integral part

of chlorophyll and is important for its synthesis. Fe chlorosis decreases the level of chlorophyll

and other carotenoids in all plant species and leads to decreased photosynthesis. Among the

different processes of photosynthesis, photosystem II is more susceptible to Fe chlorosis than

photosystem I. Fe chlorosis decreases all components of the electron transport chain. With

respect to the direct involvement of Fe in protein synthesis, Fe deficiency decreases total

proteins including ribulose-1,5-bisphosphate (rubisco), which plays a crucial role in the carbon

cycle of C3 plants (Prasad, 2003).

10
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

In this study, photosynthetic pigments and protein content of vinca plants increased with

increasing Fe2O3 concentration but these parameters decreased in Fe deficiency (0 µM of Fe2O3).

Treatment of nano iron fertilizer in different concentrations causes significant increase in content

of chlorophyll a and b compared to iron chelate (Peyvandi et al., 2011). Nenova (2006) reported

that chlorophyll value is very sensitive to Fe application and expressed that a decrease in

photosynthetic pigments under Fe deficiency followed the same trend as growth degradation.

Such results were observed in Beta vulgaris (Rombola et al., 2005).

In Phaseolus vulgaris under nano-iron spraying, protein content increased significantly (Jahanara

et al., 2013). Plant with having availability to micronutrients has further efficient use of nitrogen

in the soil and then protein synthesis increases. On the other hand, micronutrients such as Fe and

zinc cooperativein the structure of proteins and also in nitrogen metabolism, therefore may cause

to increase the protein amount (Monsef-Afshar et al., 2012).

Total Alkaloid Content

In this study, alkaloid content of vinca plants increased under Fe 2O3 treatment. This may be

due to increasing of nitrogen uptake by nano-iron particles that is essential for the

biosynthesis of alkaloids. Foliar application of iron on Catharanthus roseus leaves caused an

increase in alkaloid content (Lakshimi et al., 1993). In adequate levels of Fe induce a range of

morphological and metabolic changes required to resistance to the resultant stress and to

maintain Fe homeostasis. However, maximum amount of alkaloid was observed in Fe deficiency

(0 µM Fe2O3) in this study. Iron deficiency affected on secondary metabolism, such as alkaloid

11
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

metabolism (Khandakar et al., 2013). Khandakar et al. (2013) reported that Fe deficiency increased

alkaloid content of Hyoscyamus albus.

Element Contents

In present study, elements of vinca such as potassium, phosphorus and iron increased with

Fe2O3 nanoparticles application. Effects of iron oxide nanoparticles on peanut plants were

evaluated. The results showed that iron oxide nanoparticles caused an increase in element

contents such as potassium and phosphorus (Xiu-mei et al., 2005). Treatment of nano-iron on

Vigna unguiculata was significant and cause of increase Fe contents of leaves. Whereas did not

have significant effect on leaf phosphorus or seed iron (Monsef-Afshar et al., 2012). It was

reported that Fe treatment, increase the potassium content in leaves and seeds of lentil and

cowpea under Fe deficiency, significantly. Fe deficiency cause reduced energy availability and

decreased the active absorption of anions in root cells that reduces absorption of cautions such as

potassium (Mahmoudi et al., 2005). Foliar application of different concentrations (0, 0.5, 1 and

1.5 per 1000) of nano-iron on Vigna unguiculata caused an increase in leaf iron contents.

Concentration of 1 per 1000 Fe leads to highly increase in leaf iron content compared to the

other treatments and between this treatment and 1.5 per 1000 Fe there were no significant

difference (Monsef-Afshar et al., 2012). Taghavi et al. (2005) reported that the highest potassium

value was observed in strawberry leaf under 1 and 2 per 1000 concentrations of Fe. The P

concentrations were higher in leaves that were exposed to the 4.5 and 9.0 mmol L-1Fe treatments.

In this study, contents of potassium, phosphorus and iron decreased under Fe deficiency (0 µM of

12
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

Fe2O3). Iron deficiency influences inseveral metabolic processes, leads to nutrient imbalances in

plants and is responsible for significant decreases in growth and yield (Pestana et al., 2010).

Lopez-Millan et al., (2001) reported that iron deficiency induced changes in the mineral nutrient

composition of sugar beet leaves. Iron-deficient leaves had 50 and 37% decreases in total P and K

concentrations, respectively. In Beta vulgaris plants under iron deficiency, leaf P and K

concentrations were lower than those of the controls both at 7 and 17 days plants (Rombola et al.,

2005).

CONCLUSION

Our findings from this study suggest that suitable type of iron fertilizer for Catharanthus roseus

is nano-Fe fertilizer. Concentration of 30 µM nano-Fe fertilizer has better effect on some

parameters such as growth parameters, photosynthetic pigments, protein, alkaloid and element

contents of Catharanthus roseus. There was no statistical difference between 30 µM and 40 µM

of nano-Fe concentration for most parameters.

13
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

REFERENCES

Adamski, J. M., R. Danieloski, S. Deuner, E. J. B. Braga, L. A. S. de Castro, and J. A. Peters.

2012. Responses to excess iron in sweet potato: impacts on growth, enzyme activities,

mineral concentrations, and anatomy. Acta Physiologiae Plantarum 34(2): 1827-1836.

Armin, M., S. Akbari and S. Mashhadi. 2014. Effect of time and concentration of nano-Fe foliar

application on yield and yield components of wheat. International Journal of

Biosciences 4(9): 69-75.

Arnon, D. I. 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris.

Plant Physiology 24: 1-15.

Ataei-Azimi, A., B. D. Hashemloian, H. Ebrahimzadeh, and A. Majd. 2008. High in vitro

production of ant-canceric indole alkaloids from periwinkle (Catharanthus roseus) tissue

culture. African Journal of Biotechnology 7(16): 2834-2839.

Boorboori, M. R., D. Eradatmand-Asli, and M. M. Tehrani. 2012. Effect of micronutrient

application by different methods on yield, morphological traits and grain protein percentage of

barley (Hordeum vulgare L.) in greenhouse conditions. Revista Científica UDO Agrícola

12 (1): 127-134.

Bradford, M.M. 1976. A Rapid and Sensitive Method for the Quantitation of Microgram

Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical

Biochemistry 74: 248-254.

Celik, M., C. Tureli, M. Celik, Y. Yanar, U. Erdem and A. Kucukgulmez. 2004. Fatty acid

composition of the blue crab (Callinecte ssapidus Rathbun, 1896) in the North Eastern

Mediterranean. Food Chemistry 88: 271-273.

14
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

Creus, C. M., R. J. Sueldo and C. A. Barassi. 2004. Water relations in Azospirillum-inoculated

wheat seedlings under osmotic stress.Canadian Journal of Botany 76: 238-244.

Dokhe, S. A., P. Mahajan, R. Kamble and A. Khanna. 2013. Effect of nanoparticles suspension on

the growth of mung (Vigna radiata) seedlings by foliar spray method. Nanotechnology

Development 3(1): 1-5.

Fernandez, V., T. Eichert, V. Del Rio, G. Lَpez-Casado, J. A. Heredia-Guerrero, A. Abadía, A.

Heredia and J. Abadía. 2008. Leaf structural changes associated with iron deficiency

chlorosis in field-grown pear and peach: physiological implications. Plant and Soil 311:

161-172.

Guerinot, M.L. 2001. Improving rice yields-ironing out the details. Nature Biotechnology

19(5):417-418.

Harsini, M.G., H. Habibi and G. H. Talaei. 2014. Study the effects of iron nano chelated

fertilizers foliar application on yield and yield components of new line of wheat cold

region of Kermanshah provence. Agricultural Advances 3(4) 95-102.

Hell, R. and U. W. Stephan. 2003. Iron uptake, trafficking and homeostasis in plants. Planta 216:

541-551.

Jahanara, F., M. Sadeghi and M. Ashouri. 2013. Effect of Nano-Iron (Fe) Fertilization and

Rhizobium leguminosarum on the Qualitative and Quantitative Traits of Phaseolus vulgaris

Genotypes. International Journal of Agriculture and Crop Sciences 5 (6): 572-578.

Jaleel, C. A. and M. A. Salem. 2010. Fate of biochemical components of Catharanthus roseus

after treatment with different plant growth regulators. Australian Journal of

15
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

Agricultural Engineering (AJAE) 1(2):45-53.

Khandakar, J., I. Haraguchi, K. Yamaguchi and Y. Kitamura. 2013. A small-scale proteomic

approach reveals a survival strategy, including a reduction in alkaloid biosynthesis, in

Hyoscyamus albus roots subjected to iron deficiency. Frontiers in Plant Science, Plant

Nutrition 4(331): 1-13.

Krystofova, O., J. Sochor, O. Zitka, P. Babula, V. Kudrle, V. Adam and R. Kizek. 2013.

Effect of Magnetic Nanoparticles on Tobacco BY-2 Cell Suspension Culture. International

Journal of Environmental Research and Public Health 10: 47-71.

Lakshimi, S. R. A., J. Ranatunge, S. Wijerante, P. Fernando and K. Wickramasinghe. 1993.

Optimising Alkalioid Yield in Catharanthus roseus. National Science Council of Sri

Lanka 21(1): 63-71.

Lichtenthaler, H. K. and A. R. Wellburn. 1983. Determinations of total carotenoids and

chlorophylls a and b of leaf extracts in different solvents. Biochemical Society

Transactions 11:591-592.

Lopez-Millan, A. F., F. Morales, A. Abadı´a and J. Abadı´a. 2001. Changes induced by Fe

deficiency and Fe resupply in the organic acid metabolism of sugar beet (Beta vulgaris)

leaves. Physiologia Plantarum 112: 31-38.

Mahmoudi, H., R. Ksouria, M. Gharsallia, M. Lachaa. 2005. Differences in responses to iron

deficiency between two legumes: lentil (Lens culinaris L.) and chickpea (Cicer

arietinum L.) Plant Physiology 162: 1237-1245.

Moghadam, A., H. Vattani, N. Baghaei and N. Keshavarz. 2012. Effect of Different Levels of

Fertilizer Nano_Iron Chelates on Growth and Yield Characteristics of Two Varieties of

16
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

Spinach (Spinacia oleracea L.): Varamin 88 and Viroflay. Research Journal of Applied

Sciences, Engineering and Technology 4(12): 4813-4818.

Mohamadipoor, R., S. Sedaghathoor and A. Mahboub-Khomami. 2013. Effect of application of

iron fertilizers in two methods 'foliar and soil application' on growth characteristics of

Spathyphyllum illusion. European Journal of Experimental Biology 3(1): 232-240.

Monsef-Afshar, R., H. Hadi and A. Pirzad. 2012. Effect of Nano-iron foliar application on

qualitative and quantitative characteristics of cowpea, under end season drought stress.

International Research Journal of Applied and Basic Sciences 3 (8): 1709-1717.

Neilands, J. B., K. Konopka, B. Schwyn, M. Coy, T. Francis, B. H. Paw, A. Bagg. 1987.

Comparative biochemistry of microbial iron assimilation. In Iron transport in microbes,

plants and animals, eds. G. Winkelmann, D. van der Helm and J. B. Neilands, 3-34.

Weinheim: VCH.

Nenova, V. 2006. Effect of iron supply on growth and photosystem II efficiency of Pea plants.

Plant Physiology, Special Issue 81-90.

Pestana, M., A. de-Varennes, M. G. Miguel, P. J. Correia. 2010. Consequences of iron

deficiency on fruit quality in citrus and strawberry. Environmentally Friendly and Safe

Technologies for Quality of Fruits and Vegetables. Faro: Universidade do Algarve.

Peyvandi, M., H. Parande and M. Mirza. 2011. Comparison of nano Fe chelate with Fe

chelate effect on growth parameters and antioxidant enzymes activity of Ocimum

basilicum. New Cellular and Molecular Biotechnology Journal 1 (4): 89-98.

Prasad, P. V. V. 2003. Plant Nutrition: Iron Chlorosis. In: Encyclopedia of Applied Plant

Sciences, eds. B. Thomas, D. J. Murphy and B.G. Murray, 649-656. London, UK: Elsevier.

17
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

Prasad, T. N. V. K. V., P. Sudhakar, Y. Sreenivasulu, P. Latha, V. Munaswamy, k. Raja Reddy,

T. S. Sreeprasad, P. R. Sajanla and T. Pradeep. 2012. Effect of nano scale zink oxid

particle on the germination growth and yield of peanut. Journal of plant Nutrition

35(6):905-927.

Rombola, A. D., Y. Gogorcena, A. Larbi, F. Morales, E. Baldi, B. Marangoni, M. Tagliavini and

J. Abad´ıa. 2005. Iron deficiency-induced changes in carbon fixation and leaf

elemental composition of sugar beet (Beta vulgaris) plants. Plant and Soil 271: 39-45.

Sheykhbaglou, R., M. Sedghi, M. Tajbakhsh-Shishevan, R. Seyed-Sharifi. 2010. Effects of

Nano-Iron Oxide Particles on Agronomic Traits of Soybean. Notulae Scientia

Biologicae 2(2): 112-113.

Taghavi, T., M. Babalar, A. Ebadi, H. Ebrahimzadeh. 2005. Effect of different levels of iron and

on elements amount and strawberry yield. Iranian Agricultural Sciences 1065-1073: 36 (5).

Thomas, P., J. Woodward, F. Stegelin and B. Pennisi. 2012. A Guide for Commercial

Production of Vinca. U.S.: The University of Georgia Cooperative Extension.

Wang, B. S. and K. F. Zhao. 1995. Comparison of extractive methods of Na +, K+ in wheat

leave. Plant Physiology Communications 31(1): 50-52.

Xiu-mei, L., Z. Fu-dao, F. Zhao-bin, Z. Shu-qing, H. Xu-sheng, W. Ru-fang, and W. Yu-jun.

2005. Effects of nano-ferric oxide on the growth and nutrients absorption of peanut.

Plant Nutrition and Fertilizer Science 11(4): 551-555.

18
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

Table 1.Effects of Fe2O3 and Fe-chelate (complete Hogland as a control (H)) on shoot height

in developmental periodsof 9, 18, 27, 36, 45, 54, 63 and 70 days. Values within a row with a

letter in common are not significantly different according to Duncan’s test’.

Index Different concentrations of Fe2O3 (μM)

Age H 0 5 10 20 30 40

(day)

Shoot 9 3.87d 1.73e 4.57c 4.50c 5.43b 6.33a 6.17a

height 18 4.38d 2.32e 5.07c 5.00c 5.93b 6.85a 6.66a

(cm) 27 4.70d 2.64e 5.41c 5.45c 6.37b 7.23a 7.08a

36 4.9d 2.96e 5.66c 5.60c 6.53b 7.47a 7.26a

45 5.32d 3.37e 6.06c 6.00c 6.98b 7.83a 7.60a

54 5.90d 3.96e 6.68c 6.93c 7.55b 8.44a 8.26a

63 6.50d 4.57e 7.27c 7.58c 8.14b 9.03a 8.91a

70 7.00d 5.17e 8.07c 8.64c 8.68b 9.51a 9.43a

19
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

Table 2. Effects of Fe2O3 nanoparticles and Fe-chelate (complete Hoagland as a control) on root

length, stem and root fresh and dry weights 70-days plants. Values within a row with a letter

in common are not significantly different according to Duncan’s test’.

Index Different concentrations of Fe2O3 (μM)

H 0 5 10 20 30 50

Root length (cm) 9.50f 6.33g 12.50e 14.80d 19.33c 28.50a 25.66b

Stem fresh weight (g) 0.12c 0.04d 0.13c 0.18b 0.19b 0.26a 0.27a

Stem dry weight (g) 0.012c 0.005d 0.011c 0.018b 0.017b 0.025a 0.023a

Root fresh weight (g) 0.09c 0.03d 0.11c 0.17b 0.16b 0.28a 0.27a

Root dry weight (g) 0.01c 0.004d 0.011c 0.016b 0.017b\ 0.028a 0.027a

20
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

4 2
3.5 a a a b a a
3 b 1.5
2.5 c

Chlb mg/gFW
Chla mg/gFW

d b
2 1 c
1.5 f d
1 0.5 e
e f
0.5
0 0
H 0 5 10 20 30 40 H 0 5 10 20 30 40
Fe2O3(µM) Fe2O3(µM)

6 20 a
c a a d a
5 b
15 b
Carotenoids mg/gFW
b
4 c
c
ChlT mg/gFW

d
3 d 10 e
2 e
1 f 5
0 0
H 0 5 10 20 30 40 H 0 5 10 20 30 40
Fe2O3(µM) Fe2O3(µM)

12
e a a
10 b
c c
8
protein mg/gFW

d
6 e
4
2
0
H 0 5 10 20 30 40
Fe2O3(µM)

Figure 1. Effects of different concentrations of Fe2O3 nanoparticles (0, 5, 10, 20, 30 and 40 µM)

and complete Hoagland contains Fe-chelate as a control (H) on chlorophyll a (a), chlorophyll b

(b), total chlorophyll (c), carotenoids (d) and protein (e) contents of leaves of 70-days plants.

Values within a graph with a letter in common are not significantly different as determined by

21
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

Duncan’s test.

3
a
2.5
Total alkaloids mg/g FW

b b
2
c
1.5
d d
1 e

0.5

0
H 0 5 10 20 30 40
Fe2O3(µM)

Figure 2. Effects of different concentrations of Fe2O3 nanoparticles (0, 5, 10, 20, 30 and 40 µM)

and complete Hoagland contains Fe-chelate as a control (H) on total alkaloid contents of

leaves of 70-days plants. Values with a letter in common are not significantly different as

determined by Duncan’s test.

22
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

0.16
0.16 a a
b
0.14 a
0.14 a
Potassium mg/g DW

0.12

Phosphorus mg/g DW
0.12
0.1 b 0.1
cd bc
0.08 cd 0.08 b b
b
0.06 d 0.06
c
0.04 0.04
e
0.02 d
0.02
0 0
H 0 5 10 20 30 40 H 0 5 10 20 30 40
Fe2O3(µM) Fe2O3(µM)

500 a a
450
c
a a
400
350 a
300 a
Fe μg/g DW

250
200 b
150
100
50
0
H 0 5 10 20 30 40
Fe2O3(µM)

Figure 3. Effects of different concentrations of Fe2O3 nanoparticles (0, 5, 10, 20, 30 and 40 µM)

and complete Hoagland contains Fe-chelate as a control (H) on potassium (a), phosphorus (b)

and iron (c) contents of leaves of 70-days plants. Values within a graph with a letter in common

are not significantly different as determined by Duncan’s test.

23
ACCEPTED MANUSCRIPT