J. Plant Biochemistry & Biotechnology Vol. 1 , 27-31, January 1992 Purification and Immunological Detection of Phytochrome from Wheat Coleoptiles MK Malik, VK Sharma, JP Khurana and SC Maheshwari* Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi-11 0021, India Phytochrome was partially purified from dark grown wheat coleoptiles by ammonium sulfate precipitation, hydroxyapatite column chromatography and polyethylene glycol precipitation, with a yield of about 27%. Phytochrome prepared by this method contains a dominant species having a molecular weight of 124 kO on 50S-PAGE. This 124 kO polypeptide is recognized by the antiserum raised against partially purified wheat phytochrome in rabbit. Oat anti-phytochrome polyclonal antibodies can also cross-react with phytochrome purified from wheat as assayed by ELISA and immunoblotting. Key words: ELISA, immunoblotting, phytochrome, wheat. Light is the energy source on which the plants and ulti- mately all living beings depend. However, in addition to its utilization in the process of photosynthesis, light plays an important regulatory role also in plant growth and de- velopment. Indeed, there now seem to be multiple pho- toreceptors that allow light to cue plant responses. One of these photoreceptors is phytochrome, a chromopro- tein, that absorbs in the red and far-red regions of the visible spectrum and governs a whole array of respon- ses starting from seed germination through senescence (1). However, till date, molecular mechanism of phy- tochrome action is unknown. In order to understand the mechanism by which phytochrome regulates these di- verse responses, considerable attention has been fo- cussed on the physicochemical properties of the phy- tochrome molecule itself. The purification of phytochrome in an undegraded and undenatured form has been a daunting task. The history of phytochrome purification and characterization demon- strates the hindrances faced in attainment of this goal (2). Although the probable proteinaceous nature of the photoreceptor was demonstrated along with its spec- trophotometric measurement in vivo (3), only 24 years later it became possible to purify the phytochrome molecule from oat with properties identical to those of native phytochrome (4, 5). Thereafter, the native phytochrome molecule has been purified from pea (6), rye (7), and zucchini (8), and a detailed characterization of this molecule from oat and to a lesser extent from pea has begun. However, extensive characterization of phytochrome from one or two plant species may yield misleading informa- tion, since at least some of the properties observed may be unique only to the molecule from that particular species 'Corresponding author (9). A search for molecular properties conserved among phytochromes from evolutionarily divergent plant species may be potentially useful in understanding the structure- function relationship. Employing immunological meth ods, several common features among phytochromes from different plant species have already been identified, including the presence of conserved epitopes (9-11) and domains whose protease sensitivity is modulated by the spectral form of the molecule (12). In the present investigation, an attempt has been made to purify and characterize phytochrome from etio- lated wheat coleoptiles. Materials and Methods Plant material and growth conditions - For phytochrome isolation, seeds of wheat (Triticum aestivum var. CPAN 1676) were soaked overnight in running tap water, in dark, and then sown on moist absorbent cotton in plastic trays. The seedlings were grown for 4 days in complete darkness, at 27 ± 1°C. Phytochrome purification - For purification of phy- tochrome, the method developed by Vierstra and Quail (4) was followed with some modifications. Etiolated wheat coleoptiles (500 g fresh wt) were homogenized in 500 ml of buffer containing 100 mM Tris, 140 mM ammonium sulfate, 10 mM EDTA, 50% ethylene glycol, 20 mM sodium metabisulfite (added fresh), 4 mMphenylmethane- sulfonyl fluoride (PMSF) (added just prior to use from a stock of 200 mM) and 20,000 kallikrein-inactivating unitsl I aprotinin (added just before use); final pH 8.3. The homogenized extract was filtered through two layers of muslin cloth, Polyethylenimine solution (10%v/v), pH7.8, was added (10 mill extract) and stirred for 15 min. The extract was centrifuged at 13,000 xg for 20 min. Additionall PMSF (at a final concentration of 2 mM) was added to