Planta (1984)160:382 384

P l a n t a 9 Springer-Verlag 1984

Short communication

Discrimination between the red- and far-red-absorbing forms of phytochrome from Arena sativa L. by monoclonal antibodies B. Thomas 1 *, S.E. Penn 1, G.W. Butcher 2 and G. Galfre 2 1 Plant Physiology Department, Glasshouse Crops Research Institute, Littlehampton, West Sussex, UK, and 2 Monoclonal Antibody Centre, Agricultural Research Council, Institute of Animal Physiology, Babraham, Cambridge, U K

Abstract. A set of rat monoclonal antibodies (ARC MAC 48 to 52 and 54 to 56), raised to phytochrome from dark-grown seedlings of Arena sativa L. was tested for the ability to discriminate between the red-absorbing (Pr) and far-red-absorbing (Pfr) forms of phytochrome by indirect enzyme-linked immunosorbent assay. MAC 50 bound more strongly to Pfr and MAC 49 and 52 showed preferential binding to Pr from extracts of dark-grown Arena seedlings; MAC 50 also bound more strongly to Pfr from brushite-purified phytochrome. The remainder of the monoclonal antibodies and a rabbit polyclonal antiphytochrome preparation did not discriminate between Pr and Pfr. The results provide evidence for conformational changes in defined regions of the phytochrome apoprotein upon photoconversion. Key words: Arena (phytochrome) - Immunological discrimination Monoclonal antibody Phytochrome (red-, far-red absorbing forms).

(Pfr). Antisera raised to phytochrome typically show similar reactivity with Pr and Pfr (see Pratt 1979 for a review). This could be interpreted as evidence against changes in the phytochrome apoprotein during photoconversion. However, since the specificity of an antiserum represents only the average behaviour of the many distinct antibody species contained in it, changes in only part of the protein might not be detected or be compensated for by changes at other sites. For this reason, attempts to obtain discriminating reagents have been made by preparing monoclonal antibodies (McAb) against phytochrome and examining their specificity for Pr and Pfr. McAb from mice have been raised to phytochrome from Arena, Pisum and Secale (Cordonnier et al. 1983; Nagatani et al. 1983) but these reacted equally well with Pr and Pfr. Here we describe the discrimination between Pr and Pfr in a set of rat McAb raised to Arena phytochrome.

Material and methods

Introduction

Phytochrome is the major photoreceptor concerned with the photoregulation of higher-plant development. It is a chromoprotein which exists in two photo-interconvertible spectral forms, a redabsorbing form (Pr) and a far-red absorbing form * To whom correspondence should be addressed Abbreviations: ELISA=enzyme-linked immunosorbent assay; FR - far-red light; McAb - monoclonal antibody(ies) ; PBS phosphate-buffered saline; Pfr = far-red-absorbing form of phytochrome; Pr-red-absorbing form of phytochrome; R = r e d light; PMSF = phenylmethylsulphonylfluoride

Production of monoclonal antibodies. The McAb were raised to phytochrome (118,000 dalton, A667/A280 = 0.7-0.8) purified from dark-grown Arena sativa L. cv. Saladin by the method described by Smith and Daniels (1981). AO rats were immunised with two injections, separated by 106 d, of 80 gg phytochrome in complete Freunds adjuvant. On day 173 one rat was boosted intravenously with 100 ~g of phytochrome in Dulbecco's phosphate-buffered saline (PBS). Three days later rat spleen cells were fused with Y3Agl.2.3 (Galfre et al. 1979) or YB2/3.0Ag20 (Kilmartin et al. 1982) rat myeloma cells. The fusion and subsequent hybridoma selection were as described by Galfre and Milstein (1981). Cultures secreting anti-phytochrome were identified using an indirect enzyme-linked immunosorbent assay (ELISA) screening procedure. Eight hybridomas (ARC M A C 48 to 52 and 54 to 56) were cloned in agar and culture supernatants used as the source of McAb in these studies.

B. Thomas et al. : Discrimination between Pr and Pfr by monoclonal antibodies Preparation of Pr and Pfr. A soluble preparation of phytochrome was obtained by homogenising 6-d-old dark-grown seedlings of Arena in 50 mM 2-amino-2-(hydroxymethyl)-l,3propanediol (Tris)-HC1 (pH 7.8), 4 mM 2-mercaptoethanol, 4 mM phenylmethylsulphonylfluoride (PMSF) (2:3, w/v) and taking the supernatant after centrifugation at 17,500g for 20 min. Partially purified preparations of phytochrome were obtained by ammonium-sulphate precipitation and brushite chromatography as described by Smith and Daniels (1981). Phytochrome preparations were diluted in PBS-Tween [0.02 M phosphate buffer, 0.15 M NaC1, 0.5% v/v polyoxyethylene sorbitan monolaurate (Tween-20), pH 7.4], 4 mM PMSF and divided into a suitable number of atiquots. Each aliquot was irradiated for 2 min with red (R) or far-red (FR) light or as indicated. Comparison of antibody binding to Pr and Pfr. Assays were carried out in darkness on 96-well microelisa plates (Dynatech, Billinghurst, Sussex UK) and manipulations carried out in dim green safelight. Plates were coated overnight at 4 ~ C with 200 gl per welt of rabbit anti-phytochrome immunoglobulin G (IgG) at 4 ~g ml -~ in 0.05 M sodium-carbonate buffer pH 9.6 (Thomas et al. 1984). The following incubation steps were all performed at 20~ and the wells washed with PBS-Tween after each stage. To each well was added 150 gl of phytochrome preparation as Pr or Pfr. After 2 h incubation this was followed by 150 gl McAb for 1 h and then 150 gl of rabbit anti-rat alkaline-phosphatase conjugate (Miles Laboratories, Slough, UK), diluted 1 : 500 (v/v) in PBS-Tween for 1 h. Bound alkaline-phosphatase activity ( ~ McAb binding) was determined by adding 100 gl of 10% (v/v) diethanolamine-HC1 (pH 9.8) containing I mg ml- x p-nitrophenylphosphate. The reaction was stopped after a suitable length of time by adding 50 gl of 3 M NaOH and the absorbance read at 405 nm. Results of single experiments are presented and have been repeated on a minimum of three occasions.

383

,0y

0.5 o

i

1.0

I

i

i

I

i

i

i

i

i

i

i

i

i

i

2/

MAC 51

E o~ 0.5

Discrimination between the red- and far-red-absorbing forms of phytochrome from Avena sativa L. by monoclonal antibodies.

A set of rat monoclonal antibodies (ARC MAC 48 to 52 and 54 to 56), raised to phytochrome from dark-grown seedlings of Avena sativa L. was tested for ...
296KB Sizes 0 Downloads 0 Views