Detection of cytokinins and auxin in plant tissues using histochemistry and immunocytochemistry

Abstract

We report a new method for histochemical localization of cytokinins (CKs) in plant tissues based on bromophenol blue/silver nitrate staining. The method was validated by immunohistochemistry using anti-trans-zeatin riboside antibody. Indole-3-acetic acid (auxin, IAA) was localized by anti-IAA antibody in plant tissues as a proof for IAA histolocalization. We used root sections, because they are major sites of CKs synthesis, and insect galls of Piptadenia gonoacantha that accumulate IAA. Immunostaining confirmed the presence of zeatin and sites of accumulation of IAA indicated by histochemistry. The colors developed by histochemical reactions in free-hand sections of plant tissues were similar to those obtained by thin layer chromatography (TLC), which reinforced the reactive sites of zeatin. The histochemical method for detecting CKs is useful for galls and roots, whereas IAA detection is more efficient for gall tissues. Therefore, galls constitute a useful model for validating histochemical techniques due to their rapid cell cycles and relatively high accumulation of plant hormones.

Enzyme‐mediated metabolism in nutritive tissues of galls induced by Ditylenchus gallaeformans (Nematoda: Anguinidae)

“…Fresh non‐galled leaves and galls (n = 3; three repetitions) were transversely sectioned with razor blades and submitted to Erlich’s reagent (1% 4‐(dimethylamine)benzaldehyde in 1 m HCl; w/v) for 5 min at room temperature and mounted on slides using the same reagent for detection of auxins (Leopold & Plummer ; Bedetti et al , ). The method of Erlich specifically detects indole acetic acid (IAA) as pink/purple colour, and the same reagent may stain some IAA–phenol complexes, resulting in different colours: olive for IAA–catechol, brown for IAA–chlorogenic acid and olive/yellow for IAA–caffeic acid complexes (Leopold & Plummer ; Bedetti et al ).…”

“…The method of Erlich specifically detects indole acetic acid (IAA) as pink/purple colour, and the same reagent may stain some IAA–phenol complexes, resulting in different colours: olive for IAA–catechol, brown for IAA–chlorogenic acid and olive/yellow for IAA–caffeic acid complexes (Leopold & Plummer ; Bedetti et al ). Other anatomical sections (n = 3) were submitted to 0.5% silver nitrate in absolute ethanol for 5 min, followed by incubation in 0.2% bromophenol blue plus 0.15% silver nitrate in an absolute ethanol:butyl acetate mixture (1:1) for 5 min at room temperature, washed in absolute ethanol (three times) and mounted on slides using absolute ethanol for detection of cytokinins (Touchstone ; Bedetti et al ). The slides were immediately analysed and photographed under a light microscope (Leica DFT 7000T).…”

“…Abnormal growth induced by galling organisms on plants is often mediated by increased levels of auxins (IAA, indole acetic acid) and cytokinins, which ultimately lead to formation of galls via cell hypertrophy and hyperplasia (Byers et al ; Mapes & Davies ,; Yamaguchi et al ; Tooker & Helms ; Favery et al ). In fact, histochemical and immunocytochemical analyses of plant hormones have demonstrated the presence of IAA and cytokinins in insect‐induced galls, mainly in sites where cell division, growth and differentiation are evident (Bedetti et al , , ; Suzuki et al ).…”

“…Similarly, invertase activity was detected in nutritive tissues of Cecidomyiidae galls, which are the innermost cell layers that limit the larval chamber (Oliveira et al , ; Bragança et al ). This pattern suggests that cytokinins and invertases co‐occur and are related to the acceleration of cell cycles in galls (Bedetti et al ), especially in sites of constant cell replication (such as the nutritive tissues), even in mature galls (Ferreira & Isaias ; Isaias et al ).…”

“…Root and shoot nematode galls, as insect and mite galls, usually have outer tissue compartments ( sensu Bragança et al ) with hypertrophied cells that accumulate starch and polyphenols (Ferreira et al , , ; Isaias et al ). The accumulation of polyphenols in the outer gall compartments inhibits the activity of IAA oxidase, leading to the accumulation of IAA and, consequently, to cell hypertrophy (Bedetti et al , , ; Suzuki et al ). Outer tissue compartments constitute a common storage tissue (CST) with starch accumulation in galls induced by Ditylenchus gallaeformans (Nematoda: Anguinidae) on leaves and reproductive meristems of Miconia spp.…”