A Thiol Protease and an Anionic Peroxidase Are Induced by Lowering Cytokinins during Callus Growth in Petunia

Abstract

We previously identified a group of proteins that increase early i n Petunia hybrida calli subcultured on a low-cytokinin medium, unlike the calli subcultured on a high-cytokinin medium. The calli on the low-cytokinin medium do not regenerate (J.-P. Renaudin, C. Tournaire, B. Teyssendier de Ia Serve [1991] Physiol Plant 82: 48-56). Two of these proteins, P21 and P17, have been identified by peptide sequencing and cloned. P21 is highly homologous to a group of thiol proteases, including barley aleurain, rice oryzain y, Arabidopsis SACZ, and mammalian cathepsin H. P17 is highly homologous to a group of anionic peroxidases from potato and tomato. A study of their expression in t w o P. hybrida lines, PC6 and St40, which differ in their ability to regenerate, showed that the genes for P21 and P17 are differentially expressed depending on the type and the age of the organ, with the highest expression in senescing leaves and in aged calli. The data are in favor of these genes being associated with an early step of senescence, which may be due, i n part, to a reduction i n total cytokinin. The two fetunia lines are, thus, functionally different concerning the action of cytokinin in two developmental phenomena: in vitro organogenesis and senescence.In vitro organogenesis is a complex, developmental pathway of plant tissues under the control of a large range of effectors, including, notably, growth substances, nutrients, and light (Thorpe, 1980). The duration of the biological response, spanning days and even weeks, and the paucity of genetic studies have notably contributed to our limited understanding of the control of in vitro organogenesis. Cytokinins are considered key regulators of the formation of vegetative buds in vitro, both when they are added in vitro (Skoog and Miller, 1957) and when they are synthesized endogenously (Estruch et al., 1991; Li et al., 1992). Our knowledge of cytokinin function still lags behind that of the other plant growth substances, although some progress has been made in this area recently (Binns, 1994).We have developed a biological system to search for molecular markers of cytokinin action during in vitro organogenesis (Renaudin et al., 1990). Microcalli grown from Petunia kybrida mesophyll protoplasts were induced to regenerate * Corresponding author; e-mail renaudin@msdos.ensam.inra.fr; fax 33-67-52-57-37. buds, roots, or further calli according to the absolute concentrations of auxin and cytokinin added to the culture medium, and the effect of cytokinin on vegetative bud formation was studied (Renaudin et al., 1990;Traas et al., 1990). The continuous presence of cytokinin was required for 2 weeks, until bud primordia were formed. Vegetative buds became visible at the end of the 3rd week of culture. The growth of calli during the first 2 weeks occurred to the same extent when the cytokinin concentration was lowered 10-fold, but primordium induction and vegetative bud regeneration were triggered only by the highest cytokinin concentration.2D SDS-PAGE patterns of total protein of Petu...

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Cytosolic acidification but not auxin at physiological concentration is an activator of MAP kinases in tobacco cells

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A study of cotyledon senescence in cucumber (Cucumis sativus L.) based on expressed sequence tags and gene expression

“…For example, levels of a thiol protease (P21) homologous SAG2 increase in Petunia calli subcultures produced on Iow-cytokinin media. Likewise, differential expression of the anionic peroxidase (P17) gene is highly correlated with the degree of leaf senescence as well as the age of the calli (Tournaire et al, 1996). Therefore, antioxidant enzyme genes, such as those for peroxidase, are associated with an early stage of senescence that may be due, in part, to a reduction in total cytokinin levels in the target organ (Sen 11-6 and Fig.…”