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Post-embedding immunocytochemical techniques using peroxidase-antiperoxidase or immunoglobulin G-gold as markers were used for the localization of cytokinins (CKs) in two isogenic lines, Craigella (C) and Craigella lateral suppressor (Cls), of tomato Lycopersicon esculentum Mill. Terminal buds, nodes, hypocotyl segments and root tips were submitted to a periodate-borohydride procedure, to obtain the coupling of isopentenyladeosine and zeatin riboside to cellular proteins, followed by a fixative step with a paraformaldehyde and glutaraldehyde mixture. Enzyme-linked immunosorbent assay tests performed on ovalbumin-coated microtitration plates have shown that this method was effective for CK riboside and base coupling to proteins. Paraffin-wax- or Spurr's-resin-embedded sections were cleared of wax or resin before incubation with anti-zeatin riboside or anti-isopentenyladenosine antibodies. The procedure was thoroughly investigated and many controls were done in order to eliminate artefacts. The immunostaining patterns observed along the plants showed a basipetally decreasing gradient of CKs along the stem and in the roots. Immunolabelling was higher in the actively growing regions of the stem bud and root apices. Terminal buds of Cls appeared to be less immunoreactive than C, whereas no differences were detected in root-tip immunolabelling. The staining patterns are consistent with the idea that root and bud apices have a different CK metabolism. The absence of axillary bud formation in Cls is correlated with low CK levels in the organogensis sites.

A solid phase enzyme immunoassay was developed for isopentenyladenine (iP) and isopentenyladenosine (iPA) quantitation in HPLC purified plant extracts. It was performed on antigen-coated microtitration plates on which bound antibodies were indirectly labeled by the means of a biotinylated goat anti-rabbit antibody and an avidin-alkaline phosphatase conjugate. Less than 3 femtomoles of iP or iPA were easily detected and the measuring range extended from 3 femtomole to 1 picomole. The reproducibility has been tested and intra-and interassay variations did not exceed 5.0%. The specificity of iPA antibodies was good, as determined by cross-reactivity measurements with other adenylic compounds.The specificity of the measurements for iP and iPA was demonstrated by analysis of the immunoreactivity of fractions obtained by HPLC separation of a methanolic tobacco leaf extract.Immunoenzymatic assays have been shown to be very powerful tools for plant growth substances analysis (12). Compared with other techniques, they offer the best compromise with regard to the possibility to perform many measurements within a short period (up to several hundreds per day) combined with a very high sensitivity and a very good precision due to the specificity of antibodies. We recently described a solid phase immunoenzymatic method, based upon an indirect antibody labeling system which involves the avidin-biotin interaction complex, for the determination of several plant hormone levels within the same HPLC purified plant extract (1 1). In this paper, we report on the extension of this immunological procedure to the analysis of iP' and iPA in plant samples. The efficiency of the assay is discussed in relation with its sensitivity, its reproducibility, and its specificity. An Thirteen days after sowing they exhibited two fully expanded leaves. Leaves were then excised, dropped into liquid N2, and kept at -40TC until extraction for hormone analysis.Extraction and Purification of Samples. Aliquots ofthe sample (100 mg fresh weight) were homogenized in 5 ml 80:20 (vol:vol) methanol:distilled water containing 40 mg/L butylhydroxytoluol (BHT) as an antioxidant.[3H]iPA (about 1 kBq) was added in the extract which was stirred overnight at 4TC in darkness. The extract was passed through a Millipore prefilter connected with a Sep-Pak Cl 8 cartridge (Waters, USA). The prefilter and the Sep-Pak cartridge were rinsed with 10 ml of 80% methanol. The bulk eluates were reduced to about 50 1l by rotary evaporation, taken up with 1 ml acidified water (0.6 M acetic acid), and injected into a reverse phase HPLC column (ODS C18, Chrompack, The Netherlands). The elution was conducted with a Beckman 114 M HPLC system following an acidified water:methanol gradient (11). Tributylamine (10 mM) added in solvents resulted in an improved iP and iPA separation. Fractions (400 tl, 1 min elution time) were collected in 1.5 ml Treff microcentrifuge test tubes (Treff AG, Switzerland) and evaporated to dryness in a Speed-Vac concentrator (Savant, USA). All fract...

Further study on the localization of abscisic acid (ABA) has been undertaken at the ultrastructural level in Chenopodium polyspermum L. Axillary-bud-bearing nodes on the main axis were fixed with soluble 1-(3-dimethylaminopropyl)-3 ethyl carbodiimide, then postfixed with paraformaldehyde and embedded in Lowicryl K4M at-20° C. Ultrathin sections mounted on grids were successively incubated with rabbit anti-ABA antibodies and with gold-labelled goat anti-rabbit anti-bodies (40 nm particle size). Control sections treated with preimmune rabbit serum and ABA-preabsorbed antibodies were devoid of label. The background staining was very low with this technique. Quantitative analysis of the immunolabelling showed that two main sites of ABA accumulation could be defined: first, plastids in cortical cells and vascular parenchyma cells associated with sieve elements and xylem vessels; second, the cell cytoplasm and nucleus in the axillary bud tip and in procambial strands. In vascular bundles, the cambial cells showed no immunoreactivity. These observations support the hypothesis for the cytoplasmic synthesis of ABA which is subsequently trapped in plastids as cells mature.