Cytokinin-Modulated Gene Expression in Excised Pumpkin Cotyledons

Abstract

Comparison of two-dimensional polyacrylamide gel electrophoretic maps of proteins isolated from benzyladenine-treated and untreated pumpkin (Cucurbita pepo L. cv Halloween) cotyledons showed that the expression of certain proteins is enhanced, induced, or suppressed by the cytokinin treatment. The amount of poly(A)' mRNA isolated from cotyledons incubated with 10' molar benzyladenine for five days was about four-fold over the water-incubated control. The activity of hydroxypyruvate reductase prepared from purified cotyledonous microbodies and analyzed by native gel electrophoresis is proportionally enhanced by sequentially higher concentrations (10' to 101 molar) of benzyladenine. (9,11,12), water uptake (14), increase in fresh weight, and the development of cell organelles (7,12). This wide range of responses is accompanied by an increment ofDNA synthesis (8), induction ofproteins, or enzymes synthesis (9,12). One of the enzyme activities significantly stimulated by the hormone is HPR2 (12). This enzyme catalyzes the conversion ofhydroxypyruvate to glycerate in microbodies, and is involved in the glycolate pathway (16).Despite an abundance of knowledge about the physiological responses of cotyledons to cytokinin treatment, there is no concrete information on the questions of (a) whether the effects of cytokinin on gene expression are exerted by induction, stimulation, and/or suppression of specific protein syntheses, and (b) if the influence of cytokinin on protein or enzyme synthesis is primarily at the pretranslational or posttranslational level.In the present study, we have utilized two-dimensional PAGE to determine if cytokinin modifies the spectrum of polypeptides synthesized during cytokinin-induced cell expansion ofpumpkin cotyledons. We Cotyledon Preparation. Pumpkin seeds of approximately the same size were imbibed for 16 h with running tap water, and surface sterilized for 10 min with 0.5% NaOCl. The seeds were thoroughly rinsed with sterile distilled H20 and were sown on distilled H20-moistened Whatman No. 1 filter papers contained in Petri dishes under aseptic conditions. These Petri dishes were placed in darkness at room temperature (25°C) for 7 d. Cotyledons were then excised and cultured under aseptic conditions for various times in the dark on filter papers in Petri dishes. The dishes contained 10 ml distilled H20, 0.2 mg/ml penicillin G, with or without BA.Preparation of Microbody HPR. The preparation of pumpkin cotyledon microbodies was adapted from a method for spinach microbody preparation described by Huang et al. (10) and modified by Chang and Huang (3) with the exception that the homogenate was filtered through three layers of cheesecloth instead of Nitex cloth. The microbodies were purified by a 30 to 60% (w/v) linear sucrose gradient. Sucrose gradient-purified microbodies were dialyzed against 10 mm Tris-HCI (pH 7.5) overnight to release proteins from microbodies. The dialysate was centrifuged at 100,000g for 1 h, and the supernatant protein solution containing HPR was reduce...

Inhibition of Growth of Cultured Tobacco Cells at Low Concentrations of Lovastatin Is Reversed by Cytokinin

“…Accordingly, researchers have used primary explants (10), whole plants (12), or cytokinin-starved cell suspensions (7,11) in their work on the physiology and molecular biology of cytokinin action. Cytokinin-depleted suspension cultures, however, can be difficult to obtain because many plant cell lines undergo cytokinin habituation (i.e.…”

Cytokinins and auxins control the expression of a gene in Nicotiana plumbaginifolia cells by feedback regulation.

“…The addition of cytokinins to cultured plant material results in the appearance of a number of new mRNAs and proteins (Fosket and Tepfer, 1978;Chen and Leisner, 1985;Chatfield and Armstrong, 1986;Bauw et al, 1987;Chen et al, 1987;Crowell et al, 1990). Certain plant cultures (tobacco and Lemna) respond to cytokinin by greening in the light and accumulating the light-harvesting chlorophyll a/b binding proteins and their mRNAs (Axelos et al, 1984;Teyssendier de Ia Serve et al, 1985;Tobin, 1986, 1988).…”

Induction of Specific mRNAs in Cultured Soybean Cells during Cytokinin or Auxin Starvation

“…For example, cytokinin treatment of Lemna gibba has been shown to cause increases in the levels of the mRNAs that encode the light-harvesting Chl a/b binding protein and the small subunit of ribulose 1,5-bisphosphate carboxylase (9). Cytokinin-induced mRNAs have also been detected in pumpkin cotyledons (4) and soybean suspension cultures (6). Similarly, auxin-induced mRNAs have been reported in tobacco (20,22), pea (21), and soybean (10,13,24).…”