Use of 59Fe Isotope in Iron Chlorosis for Fodder Sorghum Bicolor R K Dadhich 1, R P Sharma 2, S M Kumawat 3, G Singh 4, M P Sahu 5

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1 Noto-are : Agriculture Use of 59Fe Isotope in Iron Chlorosis for Fodder Sorghum Bicolor R K Dadhich 1, R P Sharma 2, S M Kumawat 3, G Singh 4, M P Sahu 5 1 National Seeds Corporation Limited(Govt of India Undertaking) Ramnagar Varanasi Uttar Pradesh INDIA2 National Bureau of Soil Survey and Land Use Planning, R C Udaipur Bohara Ganesh ji Road, University Campus Udaipur Rajasthan INDIA3 Swami Keshwanand Rajasthan Agricultural University Bikaner Rajasthan INDIA4 Swami Keshwanand Rajasthan Agricultural University Bikaner Rajasthan INDIA5 Swami Keshwanand Rajasthan Agricultural University Bikaner Rajasthan INDIA Abstract The effect of foliar spray of different chemicals on the sorghum cultivar KH-333 in four type nutrient medium viz; normal ph, high ph, high bicarbonate (10 mm) and, high ph (8.0) &high bicarbonate (10 mm) on biochemical parameters and the recovery of iron chlorosis in fodder sorghum was examined. The effect of iron chlorosis and sulphydryl compounds on the antioxidant enzymes and active iron content to see whether sorghum plants are able to cope up with iron stress by foliar treatment with iron compounds and thio biomolecules. The activity of some enzymes involved in antioxidant defense and thiols associated mechanism has been studied. The activity of antioxidant defense system related enzymes (APOX, GPOX, CAT and SOD) were found higher by foliar spray of 0.2% FeSO4 and chelating agents. Activity of active iron content was recorded higher with treatment of iron and thiol compounds. In another experiment, Fe absorption and transport was measured over 72 hour, using 59Fe labelled 0.01mM FeSO4, 0.05 mm FeSO4, 0.01 mm Fe-EDDHA and 0.05 mm Fe-EDDHA(pH 6.3). The plant parts (Root and shoot) were radio assayed in a liquid scintillation counter. The absorption and transport of Fe from FeSO4 are found to be higher than from Fe- EDDHA in plant (root and shoot). It is also shown that very less quantity is transported to the shoot. 1. Introduction Iron deficiency symptom chlorosis is one of the oldest plant nutritional disorders and yet continues to be the most difficult to control. However, Fe present in the available form in soil system has been the crucial factor in most cases. Kliman was the first to detect Fe2 in the root epidermis and postulate that Fe3 was reduced to Fe2 before absorption. Subsequent discovery and identification of soybean and other crop cultivars differing in their capacity to tolerate Fe deficiency stimulated further research towards understanding the mechanisms of tolerance in them. Brown and coworkers studied the mechanisms of conversion of Fe3 to Fe2 by Fe-efficient plants and proposed the following sequence of events in Fe-stress response: (i) onset of chlorosis in the younger leaves, (ii) H ion excretion and decrease of ph of the root medium,(iii) release of reductant chemicals by the roots, (iv) solublization of Fe3 and absorption by roots, (v) greening of the chlorotic leaves, (vi) increase of ph, and (vii) the disappearance of reductants. Olsen and others examined in detail the mechanism of Fe3 reduction through the release of chemical substances named as reductants in tomato and provided the first evidence that the chemicals like chlrosgenic acid and caffeic acid are excreted by the roots with the onset of chlorosis. Using 14C-labelled caffeic acid and 59Fe, they also showed that caffeic acid was reabsorbed by plants and the absorption of Fe2 was enhanced several folds. The finding was attributed to the chelation of Fe2 and movement of the chelated species into the roots. Recent studies have sown that graminaceous and non-graminaceous spp. have different mechanisms for Fe acquisition. These mechanisms have been classified as strategy-i and strategy-ii. Briefly, Strategy I plants (dicots and non-graminaceous monocots) obtain Fe from the rhizosphere by first reducing FeIII to FeII through the action of membrane-bound FeIII-chelate reductase. Iron reduction is then followed by uptake of FeII into root cells by metal ion trans- Copyright c 2012 Noto-are. All rights reserved. ISSN

2 porters. Reductase and transporter activities are inducible in roots under Fe deficiency. Furthermore, these plants release more protons when Fedeficient, thereby lowering the rhizosphere ph and increasing Fe-solubility. Strategy II plants, which includes all of the grassess, release FeIII-binding compounds called phytosiderophores into the surrounding soil that bind iron and are then taken up into the roots (Marschner,1995). The yellow plant some times regained by addition of iron. The reasons however, might be that nitrate reductase, nitrite reductase and glutathione synthase three essential enzymes in the normal assimilation of nitrogen all contain iron. Iron deficiency stress could produce Radical Oxygen generating System, which could inactivate Fe S cluster are the central to plant metabolism and photosynthesis. Stress situations cause increased production of toxic oxygen derivatives. To counteract the toxicity of active oxygen species (AOS), a complex antioxidative defense system composed of both nonenzymatic and enzymatic constituents is present in the plant cell. In response to increase production of oxygen radicals capacity of the antioxidative defense system is increased by the most situation the response is moderate (Foyer et al, 1994, Rios Gonzalez, 2000). Plants have developed a complex antioxidative defense system that includes reduced glutathione(gsh), ascorbic acid carotenoids and enzymes that protect the plants against oxidative damage (Dalton, 1995). The project basically deals with effect of high ph, bicarbonate ions and iron deficiency on biochemical parameters in sorghum and use 59Fe in iron chlorosis in fodder sorghum. Therefore, it was felt necessary to examine the effect of foliar spray of different chemicals in normal ph, high ph, Dadhich, Sharma, Kumawat, Singh, Sahu high bicarbonate and high ph &high bicarbonate medium on biochemical parameters and the recovery of iron chlorosis in fodder sorghum. Keeping in view of the above considerations the present project report entitled Iron chlorosis in fodder sorghum and Use of 59Fe in iron chlorosis in fodder sorghum give details the effort taken in laboratory towards the fulfillment of the above objectives. 2. Materials and Methods Seeds of sorghum hybrid KH-333 were germinated in aerated distilled water and grown in trays over 0.1mM CaSO4 solution in the dark for 3 days. The young seedlings were then transplanted and further grown in half strength nutrient medium of Fe for 10 days. The same seedlings were transplanted in full strength nutrient medium of Fe for 13 days. The plants were grown under 12 hour photo period (17000 lux) and 25oC temperature with aeration following to the methodology described by Kannan (1980). Then after the same seedlings were transferred in following four different nutrient medium: Normal ph(6.3), High ph (8.0), High bicarbonate(10 mm), Normal ph(6.3) &High bicarbonate(10 mm). Plants were grown for one week in these nutrient medium. Visual scoring and ph readings were noted every day after transplanting the plants in various nutrient mediums. Photographs were also taken before and after the foliar spray. The foliar spray of following chemicals were done after one week of transferring of plants in four different nutrient medium viz; Control, Foliar spray of 0.2 % FeSO4, Foliar spray of 0.1 % Fe-EDDHA, Foliar spray of 0.2 % FeSO4 0.05% TU, Foliar spray of 0.2 % FeSO4 0.05% CA. Extracts for determina- 2 tion of CAT, APOX and GPOX activities were prepared from 1 g of leaves homogenized under ice-cold conditions in 5 ml of extraction buffer, containing 50 mm phosphate buffer, ph=7.4, 1 mm EDTA, 1 g PVP and 0.5% (v/v) TX-100 at 40C. The homogenates were centrifuged at rpm for 20 minute and supernatant was used for the assays (Costa et. al, 2002). CAT activity(um/mg protein/min) was determined in the homogenates by measuring the decrease in absorbance at 240 nm. APOX activity was measured immediately in fresh extracts by using a following reaction mixture of 1ml. The H2O2 dependent oxidation of Ascorbate (E=2.8 mm-1 cm- 1) was monitored by the decrease in absorbance at 290 nm. GPOX activity was determined in the homogenates by measuring the increasing in absorbance at 470 nm due to the tetra guaicol (E=26.6 mm-1 cm-1). Total SOD activity was assayed by the inhibition of the photochemical reduction of NBT. The reaction mixture consisted of 50 ul of enzyme extract and 3ml of O2 generating solution (2 um riboflavin, 13 mm methionine, 75 um NBT). Extracts were brought to a final volume with 50 mm potassium phosphate buffer, ph=7.8 and 0.1 mm EDTA. Beakers were shaken and placed 30 cm from light source consisting of six 15 W fluorescent lamps. The reaction was allowed to run for 30 minutes and stopped by switching the light off. Reading absorbance at 560 nm followed the reduction in NBT. Blanks and controls were run the same way but without illumination and enzyme respectively. One unit of SOD was defined as the amount of enzyme, which produced a 50% inhibition of NBT reduction under the assay conditions. Ferrous estimated according to Katyal and Sharma (1980) 1.5% 1,10-

3 orthophenothroline with maintained ph 7.0 with the help of 1N HNO3. Estimation of Proteins was done by Bradford method. In another experiment 2 weeks old plants of sorghum were transferred to distilled water for 24 hour and Fe absorption and transport were measured over 72 hour, using 59Fe labelled 0.01mM FeSO4, 0.05 mm FeSO4, 0.01 mm Fe-EDDHA and 0.05 mm Fe-EDDHA (ph 6.3). For shortterm experiments with excised roots or small plants about 10 uci per litre is an appropriate level. However, in ion uptake experiments, the activity is usually used to get reasonable count rates from materials exposed. Then in this experiment 10 uci per litre level of radioactivity is decided. Five plants were used for each 59Fe labelled 0.01 FeSO4, 0.05 FeSO4, 0.01 Fe-EDDHA and 0.05 Fe-EDDHA medium. One ml medium was taken in vial from each labelled 59Fe medium. Then all four vials were kept in infrared light for getting 59Fe alone. At the end of the uptake period, the roots were placed in 0.05 mm Fe-EDDHA for 30 minutes to remove the free space and the exchangeable Fe. The fresh weight of plant parts (root &shoot) of all the plants from each labelled medium was taken. Plant parts (root &shoot) from each labbled medium was transferred in vials and 10 ml cocktail containin BBOT, Methanol &Toluene was added by dispenser in all the vials. The plant parts (Root and shoot) were radio assayed in a liquid scintillation spectrometer. The CPM readings of 59Fe alone labelled 0.01 FeSO4, 0.05 FeSO4, 0.01 Fe-EDDHA and 0.05 Fe- EDDHA medium were taken. To find out the absolute amount of Fe, reading A, B, C and D of 59Fe alone labelled 0.01 FeSO4, 0.05 FeSO4, 0.01 Fe-EDDHA and 0.05 Fe-EDDHA medium Use of 59Fe Isotope in Iron Chlorosis for Fodder Sorghum Bicolor were calculated by dividing the CPM values of these medium in 10, 50, 10 &50, respectively. The values were expressed as absolute amounts of Fe in nanomole per gm fresh weight from following formula: Absolute Amount of 59Fe (nanomole per gm) = *Standard factor (x)/ Fresh weight of plant part in gm *Where standard factor (x) is obtained by multiplying the calculated readings A, B, C &D in CPM readings of each plant part(root or shoot) of 59Fe labelled 0.01 FeSO4, 0.05 FeSO4, 0.01 Fe-EDDHA and 0.05 Fe-EDDHA medium respectively. 3. Results and Discussions: 3.1 Biochemical Parameters 3.2 Catalase : Data related to table 3 revealed that the activity of catalase was recorded higher with the foliar spray of 0.2% FeSO % Thiourea. However the activity of catalase was higher in the foliar spray of 0.1% Fe-EDDHA in comparison to rest three foliar spray except the foliar spray of 0.2% FeSO % Thiourea under the normal ph 6.3 and without iron application. Under ph 8.0 alone and as well as 10 mm bicarbonate + ph 8.0 treatments, the activity of catalase recorded higher by the foliar application of 0.1% Fe-EDDHA followed by 0.2% FeSO4 and 0.2% FeSO % citric acid, respectively. Under bicarbonate (10 mm) nutrient medium, catalase activity was increased due to the foliar spray of 0.2% FeSO % citric acid followed by 0.1% Fe- EDDHA over to 0.2% FeSO % Thiourea as well as control Ascorbate peroxidase (APOX) activity: Data shown in table 4 indicated that the activity of APOX was recorded higher by the foliar spray of 0.2% FeSO4 0.05% citric acid followed by 0.2% FeSO4 0.05%Thiourea compared to 0.1%Fe- EDDHA as well as 0.2% FeSO4 alone but activity of APOX was recorded lower in control at normal ph 6.3 and without iron application. Activity of APOX with foliar spray of 0.2% FeSO4 0.05% citric acid registered higher followed by 0.1% Fe- EDDHA over to 0.2% FeSO4 0.05%Thiourea as well as 0.2% FeSO4 alone but had higher activity compared to control under ph 8.0 alone and 10 mm bicarbonate ph 8.0 treatments. Whereas in 10 mm bicarbonate treatment, foliar spray of 0.2% FeSO4 0.05%Thiourea registered better after that 0.2% FeSO4 0.05% citric acid over to control as well as 0.1% Fe-EDDHA. 3.4 Guaiacol peroxidase (GPOX) activity: It is explicit from the data presented in table 4 showed that the activity of guaiacol peroxidase was recorded higher in case of foliar spray of 0.2% FeSO %Thiourea followed by control over the 0.1% Fe-EDDHA as well as 0.2% FeSO4 and lowest activity was registered with foliar application of 0.2% FeSO % citric acid at normal ph (6.3) of hydroponics solution and with out iron application. At ph 8.0 of the nutrient solution, the activity of guaiacol peroxidase was registered superior with the foliar spray of 0.2% FeSO % citric acid after that 0.2% FeSO4 alone foliar spray over to 0.2% FeSO %Thiourea as well as 0.1% Fe-EDDHA. In hydroponics set, which contained 10mM bicarbonate, 0.1% Fe-EDDHA proved best followed by 0.2% FeSO %Thiourea over to 0.2%

4 FeSO % citric acid as well as control. Under ph 8.0 alone and 10 mm bicarbonate + ph 8.0 medium, the foliar spray of 0.2% FeSO % citric acid recorded highest activity of guaiacol peroxidase followed by 0.1% Fe-EDDHA over to control as well as rest foliar sprays. 3.5 Superoxide dismutase activity (SOD) : Data shows in table 3 revealed that the SOD activity recorded highest by the foliar spray of 0.1% Fe-EDDHA followed by 0.2% FeSO4 0.05% Thiourea and 0.2% FeSO4 0.05% citric acid over the 0.2% FeSO4 as well as control at ph 6.3 without iron application. At ph 8.0, activity of SOD was registered highest with foliar application of 0.2% FeSO4 alone followed by 0.1% Fe-EDDHA compared to 0.2% FeSO4 0.05%Thiourea as well as 0.2% FeSO4 0.05% citric acid. SOD activity under set of 10 mm bicarbonate recorded highest in control than 0.2% FeSO4 0.05%Thiourea over to 0.1% Fe-EDDHA and 0.2% FeSO4 0.05% citric acid. Under the set of ph 8.0 along with 10 mm bicarbonate 0.1% Fe-EDDHA recorded highest activity of SOD followed by control over to 0.2% FeSO4 0.05% citric acid as well as 0.2% FeSO4 0.05%Thiourea. 3.6 Ferrous Content : Data representing to ferrous content is presented in table 5 which indicated that highest ferrous content was recorded in green leaves which were sprayed by 0.2% FeSO4 alone, followed by 0.2% FeSO4 0.05%Thiourea over to 0.2% FeSO4 0.05% citric acid as well as 0.1% Fe-EDDHA and control registered least content of ferrous. At ph 8.0, 0.2% FeSO4 0.05%Thiourea were recorded highest content of ferrous followed by 0.2% FeSO4 0.05% citric acid compared to 0.1% Fe-EDDHA Dadhich, Sharma, Kumawat, Singh, Sahu as well as 0.2% FeSO4 alone but yet both were superior to control. In the set of 10 mm bicabonate, again 0.2% FeSO4 0.05%Thiourea proved better than others. But other treatments also showed more or less similar ferrous content except control. The combination set of ph 8.0 along with 10 mm bicarbonate showed highest content of ferrous by the application of 0.2% FeSO4 0.05%Thiourea than 0.2% FeSO4 0.05% citric acid and 0.2% FeSO4 alone over to control. 3.7 Visual Scoring and Changing in ph due to foliar spray of chemicals in various nutrient medium: In Normal ph (6.3) and %ufffd%ufffdfe nutrient medium: It is explicit from table 2, photograph-1 that recovery in iron chlorosis due to the foliar spray of 0.2 % FeSO4 &0.1%Fe- EDDHA was higher then other spray treatments. A reduction in ph of nutrient is also found (Table 2) In High ph (8.0) and Fe nutrient medium: Data depicted in photograph-2 shows that greening was occurred due to the foliar application of chemical treatments. Almost similar and better results were obtained with foliar spray of 0.1%Fe- EDDHA, 0.2% FeSO4 0.05% TU and 0.2% FeSO4 0.05% citric acid. It was also noted from table 2 that the reduction in ph after given the nutrient medium application was rapid and after foliar spray was slight. In High Bicarbonate (10 mm) and Fe nutrient medium: Data depicted in table 2 shows that the foliar spray of different chemicals was able to recover the chlorosis. Almost similar effect was seen from the foliar spray of chemicals. In case of change in ph in nutrient medium, slight increase and slight decrease in ph after application of nutrient medium and after foliar spray of 4 chemicals, respectively (Table 3). In High Bicarbonate (10 mm) and High ph (8.0) and Fe nutrient medium: All plants were recovered after one week of foliar spray of different chemicals. Almost similar recovery was observed from different foliar spray of chemicals. A regular decrease in ph was observed due to the application of different foliar spray except the foliar spray of 0.2% FeSO4 0.05% TU (Table 3). 3.8 Absorption and transport of 59Fe in sorghum : The table 1 shown the amount of absorption and transport of 59Fe during a 72 hour period in 59Fe labelled 0.01mM FeSO4, 0.05 mm FeSO4, 0.01 mm Fe-EDDHA and 0.05 mm Fe-EDDHA mediums. The absorption and transport of Fe from FeSO4 are found to be higher than from Fe- EDDHA in both root and shoot of the plant. The amount of 59Fe was found higher in their respective higher labelled medium either FeSO4 or Fe- EDDHA. It is also shown that very less quantity is transported to the shoot. This emphasizes the point that Fe-tolerance and susceptibility are not dependent on the rate of uptake; on the other hand, it may be related to the Fe utilization, following absorption and transport to the leaves. 3.9 Conclusion Activity of active iron content was recorded higher with treatment of iron and thiol compounds.the activity of antioxidant defense system related enzymes (APOX, GPOX, CAT and SOD) were found higher by foliar spray of 0.2% FeSO4 and chelating agents. The absorption and transport of Fe from FeSO4 are found to be higher than from Fe-EDDHA in plant (root and shoot).

5 Fig. 1: medium Response at Normal ph (6.3) and Fe nutrient Use of 59Fe Isotope in Iron Chlorosis for Fodder Sorghum Bicolor Fig. 2: Response at High ph (8.0) and +Fe nutrient medium References [1], Brown, J.C., Iron and Plants. In: Iron, University Park Press, Baltimore,, Vol., No ,,. [2], Costa, H., Gallego, S.M., Tomaro, M.L., Effect of UV-B radiation on antioxidant defense system in sunower cotyledons. Plant Sci.,, Vol. 162, No., ,. [3], Dalton DA (1995) Antioxidant defenses of plants and fungi. In S Ahmad, ed, Oxidative Stress and Antioxidant Defenses in Biology. Chapman &Hall, New York,, Vol., No., ,. [4], Foyer CH, Lopez-Delgado H, Dat JF, Scott IM Hydrogen peroxideand glutathioneassociated mechanisms of acclimatory stress tolerance and signalling. Physiol. Plant.,, Vol. 100, No., 24154,. [5], Kannan, S Differences in iron stress response and iron uptake in some sorghum varieties. Journal of Plant Nutrition.,, Vol. 2, No., ,. [6], Katyal, J.C. and Sharma, B.D. (1980) A new technology of plant analysis to resolve iron chlorosis, Plant and Soil.,, Vol. 55, No., ,. [7], Marschner, H (1995). Mineral Nutrition of Higher Plants, Ed 2. Academic Press San Diego.,, Vol., No.,,. [8], Olsen, R.A., Bennet, J.H., Blume, Q and Brown, J.C. (1981). Chemical respects of the Fe stress response mechanism in tomatoes. J. Plant Nutrition.,, Vol. 3, No., ,. [9], Rios-Gonzalez K, Erdei L, Lips SH (2002). The activity of antioxidant enzymes in maize and sunflower seedlings as affected by salinity and different nitrogen sources. Plant Science.,, Vol. 162, No., ,. 5