Intrachloroplast Localization of65Zn and63Ni in a Zn-tolerant Plant,Ocimum basilicumBenth

Abstract

Intrachloroplast localization studies of 63Zn and 63Ni were made in a Zn-tolerant plant Ocimum basilicum var. purpurascens Benth. in order to investigate the mechanism and specificity of metal tolerance. The isotopes were supplied in solution to the roots 14 d before fractionation. It was observed that 65Zn activity was comparatively greater in the chloroplast envelope membranes and stroma than the 63Ni; and 63Ni was largely found in the lamellar and stroma fractions. Further analysis of lamellae revealed that photosystem II (PS II) particles were richer in radioactivity than photosystem I (PS I) particles. The photochemical events of photosynthesis were less affected in Zn-treated plants than in the Ni-treated plants. The changed levels of the electron transport chain intermediates including cytochromes, plastocyanin and ferredoxin provide supporting evidence for the localization studies. The activity of carbonic anhydrase, a zinc metalloprotein, was increased in Zn-treated plants with increase in nutrient concentration, indicating the binding of zinc to a protein moiety in the chloroplasts. INTRODUCTION The phenomenon of heavy metal tolerance in plants growing either on the mines or on the mine-devastated environments has been of interest for several decades (Prat, 1934; Bradshaw, 1952). It is claimed to be a genetically controlled character that is specific to the contaminant metal (Antonovics, Bradshaw, and Turner, 1971; Gartside and McNeilly, 1974a; Karataglis, 1980). However heavy metal tolerances and their specificity, is also reported in plants growing on normal soils (Gartside and McNeilly, 1974b; McNaughton, Folsom, Lee, Park, Price, Roeder, Schmitz, and Stockwell, 1974; Veeranjaneyulu and Das, 1981a). O. basilicum var. purpurascens growing on normal soils has been reported to be tolerant to high concentrations of zinc or copper in the soil and to be susceptible to cobalt or nickel (Veeranjaneyulu and Das, 1981b). The mechanisms giving metal tolerance in plants are poorly understood. The present study attempts to find an explanation of the mechanism and specificity of zinc tolerance by studying the intrachloroplast localization of 63Zn and 63Ni in 0. basilicum. MATERIALS AND METHODS Six-week-old plants of O. basilicum var. purpurascens were grown in Hewitt's full nutrient culture solution (Hewitt, 1966) in a growth chamber at 27 °C, under a light intensity of 150 W m~2 with a 12 h photoperiod for 7 d. The nutrient solutions were changed every 2 d. Later the roots were washed in distilled water, rinsed in 0-02 M CaCl2 solution and again in distilled water. Then plants were 1 Present address and to whom correspondence should be sent: Department of Biosciences, Sri Krishnadevaraya University, Anantapur-515 003, India. This content downloaded from 157.55.39.3 on Tue, 15 Nov 2016 04:02:51 UTC All use subject to http://about.jstor.org/terms 1162 Veeranjaneyulu and Das—Chloroplast Localization of Metals transferred to salt solutions containing Zn (200 pC' 1_I, 4-0 parts 10-6 Zn) or 63Ni (30 //Ci l-1, 1-0 part 10-6 Ni) prepared in 0-05% (w/v) Ca(N03)2 solution. Chloroplast isolation, fractionation and radioactivity measurements The leaves were harvested after 14 d and washed. Intact chloroplasts were isolated in 0-05 M HEPES buffer, pH 7-3, 0-33 M sorbitol, 1-0 mM MgCl2 and 0-01 M NaCl and were fractionated into envelopes, lamellae and stroma according to Mackender and Leech (1974). Subchloroplast fragments rich in PS I and PS II particles were obtained according to Boardman (1971) by differential centrifugation in a Spinco Model L ultracentrifuge. Radioactivity was measured in a gas flow proportional counter after digesting the sample with H2S04 and HN03. Photochemical reactions and electron transport chain intermediates The photochemical reactions were studied in isolated chloroplasts at 25 ± 1 °C by exposing the reaction medium to light, 200 W m-2 according to Raghavendra and Das (1976). 2,6-Dichlorophenol indophenol (DCIP) reduction, a measure of PS II, was measured by following the decrease in absorbance at 620 nm in a reaction mixture (3-0 ml) containing 0-05 M HEPES buffer, pH 7-5, 20//M DCIP, 1 -0 mM MgCl2, 20 mM NaCl and the chloroplasts (10 pg chl ml-1). NADP reduction with water as the electron donor was studied with the reaction mixture (3-0 ml) having 10 mM HEPES buffer, pH 7-8, 25 mM NaCl, 1-0 mM MgCl2, 0-5 mM NADP, 5-0 pM spinach ferredoxin and chloroplasts (10 pg chl ml-1). Soon after switching the lights off, 0-3 ml of 1-0 N NaOH was added to the reaction vessels to avoid the re-oxidation of NADPH formed. NADP reduction in the presence of DCIP (60 /iM) and ascorbate (2-5 mM) was used to measure PS I activity in the above reaction mixture with the addition also of 1-25 //M DCMU. Cytochromes were estimated according to Bendall, Davenport, and Hill (1971). Plastocyanin was estimated by the method of Katoh (1971) and ferredoxin by the method of Buchanan and Arnon (1971). Chlorophyll content was estimated according to Arnon (1949) in a Beckman DU2 spectrophotometer and carbonic anhydrase activity according to Everson (1970).