Lucienne Sossountzov

A biotin-avidin-based enzyme immunoassay to quantify three phytohormones: auxin, abscisic acid and zeatin-riboside

Abstract Avidin-biotin complexes have been used in an ELISA assay of three phytohormones: auxin (IAA), abscisic acid (ABA) and zeatin-riboside (ZR). Anti-hormone antibodies were elicited in rabbits immunized with hormone-bovine serum albumin conjugates. Microtitration plates coated with antigen protein were used in the ELISA method. The competition between plate-bound and free (standards or samples) antigen for a limiting amount of specific antibody results in a variable level of antibody adsorbed to the wells after washes. Such antibodies were labelled in two steps: first, incubation with biotinylated goat anti-rabbit IgG and, second, incubation with avidin-alkaline phosphatase conjugate. Bound ezyme activity was measured spectrophotometrically with p-nitrophenylphosphate and standard curves and ZR. The detection limit was 3–5 pg of plant hormone and the working range was between 10–1000 pg. This compares favourably with the best systems of analysis described in the literature. To avoid unwanted cross-reactions, a rapid and efficient HPLC purification step was included prior to measuring IAA, ABA and ZR in the same plant extract. About 100 mg fresh weight of a tomato sample could be analyzed by this technique.

Immunocytochemical localization of cytokinins in Craigella tomato and a sideshootless mutant.

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 Spurrs-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.

Arrest of somatic embryo development in grapevine: histological characterization and the effect of ABA, BAP and zeatin in stimulating plantlet development

In an effort to understand the causes of arrest of somatic embryo development, generally observed in grapevine (Vitis sp.), histological studies were undertaken, using two cultivars (CH76 and 41B) which differ in their ability to develop into plants. Embryos with a high conversion rate (70%; CH76) formed a well-structured and functional shoot apex between two thread-like cotyledons. In contrast, embryos with a low conversion rate (10%; 41B) formed a normal root apex but lacked a well-structured shoot apex and developed a wide range of aberrant forms in the intercotyledonary area: uncontrolled cellular proliferation, formation of adventitious buds, over-growth of cotyledonary or leaf meristems. ABA increased the conversion rate of 41B embryos from 10% to 20%, but failed to improve embryo morphology. Zeatin and BAP promoted growth of 41B somatic embryos, but generated a high level of abnormalities and failed to improve conversion rate. Applied in combination with ABA, these PGRs increased the frequency of cotyledonary embryos, but decreased the conversion rate.

Immunoelectron-microscopy localization of abscisic acid with colloidal gold on Lowicryl-embedded tissues of Chnopodium polyspermum L.

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.

Comparative quantitation of abscisic acid in plant extracts by gas-liquid chromatography and an enzyme-linked immunosorbent assay using the avidin-biotin system

In this report we describe an enzyme-linked immunosorbent assay (ELISA) for the quantitation of abscisic acid (ABA) in plant extracts. A microtitration plate is coated with an ABA-protein complex. The ABA, standard or sample, is then added to each well with a limiting quantity of rabbit anti-ABA antibodies. During the following incubation period, antibodies bind either to free or to bound ABA on the plates. After washing, bound antibodies are indirectly labelled in two steps by the means of biotinylated goat antirabbit immunoglobulin-G antibodies which act as a link between rabbit anti-ABA antibodies and an avidin-alkaline phosphatase complex. The relative enzyme activity bound is measured spectrophotometrically. The detection limit of this method is 5 pg ABA and the measuring range extends to 10 ng. Gas-liquid-chromatography controls, with an electron capture detector, show a good correlation with ELISA results obtained using extracts of Lycopersicon esculentum, Nicotiana tabacum and Pseudotsuga menziesii samples purified by high-performance liquid chromatography. This provides a good argument for the accuracy of the immunoenzymatic method. The indirect labelling of antibodies, with the avidin-biotin amplifying system, should make this technique suitable for the quantitation of other plant growth substances against which specific antibodies are available.

Abscissic acid localization by light microscopic immunohistochemistry in Chenopodium polyspermum L. Effect of water stress.

An indirect immunohistochemical technique was developed using a rabbit anti-abscissic acid (ABA) serum and the soluble peroxidase-antiperoxidase (PAP) complex for the localization of endogenous ABA in the aerial parts of Chenopodium. Terminal bud, axillary bud bearing nodes, and adult leaves were prefixed by a soluble carbodiimide to obtain the coupling of ABA on cellular proteins and postfixed by a conventional mixture of aldehydes. They were then embedded in paraffin or in plastic. Numerous controls were carried out on sections and on a model system to test the validity of the technique. Based on the staining patterns observed along the plant, an apico-basal gradient of ABA was revealed. In the older buds, ABA was mainly concentrated in the quiescent meristematic cells of the apex. Phloem cells of the main axis and chloroplasts of the leaves were specifically labeled. No reaction product was visualized in the parenchyma cells or in the cambial zone. Water stress, which is known to increase ABA content, induced an increase of immunoreactivity within the same compartments. This physiological test validates the stain.

DNA synthesis in excised tobacco leaves after bromodeoxyuridine incorporation: immunohistochemical detection in semi-thin spurr sections.

We describe a method for localizing replicating cells in detached tobacco leaves allowed to root. The proposed protocol has shown that formalin fixation and Spurr embedding of petiole bases can be used for demonstrating DNA synthesis after bromodeoxyuridine (BrdU) incorporation. The incorporated BrdU was immunologically visualized. After resin removal, different procedures of DNA denaturation and protease digestion were tested. Combined hydrolysis with 4 N HCl for 10 min at room temperature and digestion with 0.4% pepsin for 15 min at 37 degrees C led to the best reproducible results, with either the peroxidase or the gold detection system. The method is rapid and sensitive, with precise resolution. It can be used at the light and electron microscopic levels. Its potential application is to elucidate in the same organ the role of cytokinins, a class of plant growth regulators, in dividing cells and to define the chronology of their biosynthesis in roots in relation to DNA synthesis.