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Báo cáo khoa học: "Study of endogenous plant growth Douglas fir I. Cytokinin analysis"

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Study of endogenous plant growth substances in Douglas fir I. Cytokinin analysis M. Bonnet-Masimbert 2 N. Imbault P. Doumas C. Joseph 1 Laboratoire des Composes Ph6noliques, Université d’Orl6ans, BP 6769, 45067 Orleans Cedex 02, and 2 INRA, Station dAm6lioration des Arbres Forestiers, Ardon, 45i60 Clivet, France Introduction Cytokinin isolation extracted with 80% methanol in Cytokinins were buffer (pH 7.2). After concentration, phosphate To ascertain the part played by a natural the extracts were passed through a diethylami- substance in a biological phenomenon, it is noethyl-cellulose column and purified either on necessary to follow the endogenous evolu- an immunoaffinity (IA) column (as described tion of this compound during the induction below) octadecylsilica one. Cytokinins or on an of the process. This is a real problem with then separated by high-performance liquid were plant growth substances (PGS). Indeed, chromatography (HPLC) using a reverse phase column (MacDonald et al., 1981) and measured their very low concentrations in tissues either by UV absorption or by ELISA or RIA (as make PGS difficult to quantify. Because of reported below). their sensitivity and specificity, immuno- logical methods have been adapted to the analysis of PGS and enable, in some cases, measurements at the level of a Immunological methods single organ, as reported for principally For IAC and EL procedures, monoclonal SA J herbaceous species (Weiler, 1984). In this antibodies were raised against cytokinins conju- paper, some of their applications to the gated to bovine serum albumin (MacDonald woody plant, Douglas fir (Pseudotsuga and Morris, 198!i). IA columns of 1 ml each menziesii Mirb.), are presented: purifica- contained equal amounts of anti-ribosylzeatin n tion by immunoaffinity chromatography (anti-RZ) and anti-isopenteny!adenosine (anti- IPA) antibodies coupled to a cellulose matrix. (IAC) and measurement by an enzyme- With this mixture of antibodies, IAC was linked immunosorbent assay (ELISA) or a performed according to MacDonald and Morris radioimmunoassay (RIA). (1985). Thus, the usual cytokinin bases and ribosides were recognized. ELISA was perform- ed as described in Bataille et al. (1987); detec- tion limit and range were 15 pg and 20-5000 Materials and Methods pg, respectively. RIA was done according to MacDonald et al. (1981) using polyclonal anti- cytokinin antibodies; detection limit and range Material here were, 50 pg and 100-5000 pg, respec- The sexual buds of study was performed on tively. Douglas fir.
Results plant material. However, the detection limit by UV absorpi:ion (254 nm) after IAC was only 1-5 ng. For small samples, the more 1 shows the HPLC chromatogram of Fig. sensitive ELISA or RIA (15 or 50 pg) could extract from a female bud of Douglas one be used. Thus, despite the inherent diffi- fir subjected to IAC (B) or not (A). IAC culties of the woody material, PGS analy- cleared the extract of UV absorbing com- sis is possible and practical at the organ pounds. Further cytokinin quantification level, where physiological performed by RIA on HPLC fractions processes One application of this possibility demonstrated no significant losses of occur. was illustrated by the study of Imbault et these PGS through iAC. Therefore, IAC, al. (1988), which showed the intervention which retained only immunologically reac- of IP and IPA in Douglas fir flowering. tive compounds, acted as a selective filter enabling quantification by integration of the peaks. In Fig. 2, radioimmunohistograms of References HPLC male (A) and female (B) bud ex- tracts are represented. A RZ-like sub- Bataille A., Dournas P., Zaerr J.B. & Morris R.O. stance exists in both male and female (1987) Comparison of ELISA and RIA for cyto- buds and quantities were very similar. kinin analysis. Plant Physiol. 83 (suppl.), 96 a peak, called C, which did Furthermore, Imbault N., Tardieu L, Joseph C., Zaerr J.B. & not co-chromatograph with any cytokinin Bonnet-Masimbert M. (1988) Possible role of standard, was only present in female bud isopentenyladenine and isopentenyladenosine in flowering of Pseudotsuga menziesii: endo- extracts. Thus, this measurement method genous variations and exogenous applications. made it possible to determine molecules Plant. Physiol. Biochem. 26, 289-295 other than the standard ones. These MacDonald E.M.S. & Morris R.O. (1985) Isola- results were confirmed by ELISA. tion of cytokinins by immunoaffinity chroma- tography and analysis by high-performance li- quid chromatography-radioimmunoassay. Me- thods Enzymol. 110, 347-358 Discussion and Conclusion MacDonald E.M.S., Akiyoshi D.E. & Morris R.O. (1981) Combined high-performance liquid chro- matography-radioimmunoassay for cytokinins. To the evolution of cytokinins in study J. Chromatogr. 214, 101-109 fir tissues, Douglas immunological Weiler E.W. (1984) Immunoassay of plant methods can be used. Because of their growth regulators. Annu. Rev. Plant Physiol. 35, sensitivity, they need only low quantities of 85-95

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