Bruno Sotta

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.

Crosstalk between reactive oxygen species and hormonal signalling pathways regulates grain dormancy in barley

Seed dormancy, defined as the inability to germinate under favourable conditions, is controlled by abscisic acid (ABA) and gibberellins (GAs). Phytohormone signalling interacts with reactive oxygen species (ROS) signalling regarding diverse aspects of plant physiology and is assumed to be important in dormancy alleviation. Using dormant barley grains that do not germinate at 30 °C in darkness, we analysed ROS content and ROS-processing systems, ABA content and metabolism, GA-responsive genes and genes involved in GA metabolism in response to hydrogen peroxide (H₂O₂) treatment. During after-ripening, the ROS content in the embryo was not affected, while the antioxidant glutathione (GSH) was gradually converted to glutathione disulphide (GSSG). ABA treatment up-regulated catalase activity through transcriptional activation of HvCAT2. Exogenous H₂O₂ partially alleviated dormancy although it was associated with a small increase in embryonic ABA content related to a slight induction of HvNCED transcripts. H₂O₂ treatment did not affect ABA sensitivity but up-regulated the expression of HvExpA11 (GA-induced gene), inhibited the expression of HvGA2ox3 involved in GA catabolism and enhanced the expression of HvGA20ox1 implicated in GA synthesis. In barley, H₂O₂ could be implicated in dormancy alleviation through activation of GA signalling and synthesis rather than repression of ABA signalling.

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.

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.

Changes in indole-3-acetic acid levels during tomato (Lycopersicon esculentum Mill.) seed development

SummaryThe changes in the level of indole-3-acetic acid (IAA) were investigated in seeds and fruit tissues-placenta and mesocarp-during tomato (Lycopersicon esculentum Mill.) zygotic embryogenesis, which was characterized through eight morphological embryo stages [from globular (stage 1) to mature embryo (stage 8)]. In whole seeds, IAA levels increased mainly at stage 3 (young torpedo) and at stage 5 (late torpedo stage). As the seed matured and dehydrated, IAA levels decreased and showed a new distribution pattern within seed structures, preferentially in endosperm tissue. IAA contents in fruit tissues were lower but followed the same pattern as those of seeds. These data support the hypothesis of IAA biosynthesis in seeds with a transient role of the endosperm at the end of embryo development and suggest a role of IAA in fruit and seed growth. Moreover a comparison of IAA and ABA changes suggests that IAA could be especially necessary for the beginning of embryo growth, whereas ABA could act mainly at the end of the growth phase.

Arabidopsis thaliana lipid phosphate phosphatase 2 is involved in abscisic acid signalling in leaves

Lipid phosphate phosphatases (LPPs, E.C. 3.1.3.4) catalyse the dephosphorylation of diacylglycerol pyrophosphate (DGPP) and phosphatidic acid (PA), which are secondary messengers in abscisic acid (ABA) signalling. In this study, we investigated the effect of ABA on the expression of AtLPP genes as they encode putative ABA-signalling partners. We observed that AtLPP2 expression was down-regulated by ABA and we performed experiments on Atlpp2-2, an AtLPP2 knockout mutant, to determine whether AtLPP2 was involved in ABA signalling. We observed that Atlpp2-2 plantlets contained about twice as much PA as the wild-type Col-0 and exhibited higher PA kinase (PAK) activity than Col-0 plants. In addition, we showed that ABA stimulated diacylglycerol kinase (DGK) activity independently of AtLPP2 activity but that the ABA-stimulation of PAK activity recorded in Col-0 was dependent on AtLPP2. In order to evaluate the involvement of AtLPP2 activity in guard cell function, we measured the ABA sensitivity of Atlpp2-2 stomata. The inhibition of stomatal opening was less sensitive to ABA in Atlpp2-2 than in Col-0. Watered and water-stressed plants of the two genotypes accumulated ABA to the same extent, thus leading us to consider Atlpp2-2 an ABA-signalling mutant. Taken together our observations show that AtLPP2 is a part of ABA signalling and participate to the regulation of stomatal movements.

TL-DNA transformation decreases ABA level

The endogenous levels of ABA were measured in Agrobacterium rhizogenes A4 Tl -DNA transformed oilseed rape (Brassica napus L. var. oleifera cv. Brutor and cv. Drakkar), cabbage (Brassica oleracea). A4 transformed tobacco (Nicotiana tabacum cv. Xanthi) and their normal counterparts, using high performance liquid chromatography and enzyme-liked immunosorbent assay. Measurements were made on different plant tissues (i. e. floral stem, terminal bud, young leaf, mature leaf, root and root tips) and ABA levels were compared in unstressed and osmotically stressed oilseed rape plants (cv. Brutor). In unstressed Plants. in each of the 5 independent transformation events studied, a significant reduction (about 65% of control) in ABA concentration was observed in all transformed plants. When subjected to an osmotic stress, TL transformed Brutor plants showed a higher ABA accumulation than untransformed plants. The change in ABA content as a consequence of TL -DNA transformation is discussed with regard to phenotype, drought resistance and adaptability.

Evidence for ACD5 ceramide kinase activity involvement in Arabidopsis response to cold stress

Although sphingolipids emerged as important signals for plant response to low temperature, investigations have been limited so far to the function of long-chain base intermediates. The formation and function of ceramide phosphates (Cer-Ps) in chilled Arabidopsis were explored. Cer-Ps were analysed by thin layer chromatography (TLC) following in vivo metabolic radiolabelling. Ceramide kinase activity, gene expression and growth phenotype were determined in unstressed and cold-stressed wild type (WT) and Arabidopsis ceramide kinase mutant acd5. A rapid and transient formation of Cer-P occurs in cold-stressed WT Arabidopsis plantlets and cultured cells, which is strongly impaired in acd5 mutant. Although concomitant, Cer-P formation is independent of long-chain base phosphate (LCB-P) formation. No variation of ceramide kinase activity was measured in vitro in WT plantlets upon cold stress but the activity in acd5 mutant was further reduced by cold stress. At the seedling stage, acd5 response to cold was similar to that of WT. Nevertheless, acd5 seed germination was hypersensitive to cold and abscisic acid (ABA), and ABA-dependent gene expression was modified in acd5 seeds when germinated at low temperature. Our data involve for the first time Cer-P and ACD5 in low temperature response and further underline the complexity of sphingolipid signalling operating during cold stress.