Immunocytochemical localization of cytokinins in Craigella tomato and a sideshootless mutant

Abstract

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.

The POLARIS Gene of Arabidopsis Encodes a Predicted Peptide Required for Correct Root Growth and Leaf Vascular Patterning

“…Because cytokinins are synthesized in root tips and are at relatively high concentrations in roots (Sossountov et al, 1988;Chiappetta et al, 2001), we can speculate that a mechanism exists to reduce the sensitivity of cells at the root tip to cytokinins to suppress their growth-inhibitory effects. This system may involve the gene STUNTED PLANT1 (Baskin et al, 1995), and PLS also may encode a component of this mechanism.…”

“…For example, cytokinins can act as important regulators of cell division and appear to be synthesized in root tips, although exogenous cytokinins can inhibit root growth (Sossountov et al, 1988;Riou-Khamlichi et al, 1999;Chiappetta et al, 2001). Cytokinins can act antagonistically with auxin in some developmental contexts, such as in lateral root development, but they also can act synergistically, as in ethylene biosynthesis (Vogel et al, 1998).…”

EXORDIUM– a gene expressed in proliferating cells and with a role in meristem function, identified by promoter trapping in Arabidopsis

“…They are synthesised in root tips (Chiappetta et al, 2001;Sossountov et al, 1988), and might be expected to limit the level of EXO expression in the meristem, since Northern analysis showed downregulation of EXO transcription by cytokinins. Therefore, EXO expression may be antagonistically upregulated by auxin and downregulated by cytokinin in the meristem, to modulate its level of expression.…”

Hormone Levels and Apical Dominance in the Aquatic Fern Marsilea drummondii A. Br

“…The relative distribution of Z + ZR per bud also showed a steep downward gradient as for IAA. We have recently shown (18) by immunocytochemical methods that tomato inhibited buds are well correlated with very low contents of endogenous CKs in their quiescent meristematic cells, and we concluded that CKs are necessary for axillary bud growth. Thus, as for IAA, the distribution of Z-type CKs within the buds fits well with growth activity: actively growing buds contained the highest levels of these hormones.…”

“…Therefore, it seems important to analyze the subcellular localization of the hormone. This important question can be answered by the use of immunocytochemical methods to localize plant hormones as initiated in our laboratory (17)(18)(19) to delineate the possible role of a regulator not based on its tissue or organ concentration.…”