Ivana Č. Dragićević

Cytokinin Profiles of AtCKX2-Overexpressing Potato Plants and the Impact of Altered Cytokinin Homeostasis on Tuberization In Vitro

Genes encoding cytokinin oxidase/dehydrogenase (CKX) enzymes have been used lately to study cytokinin homeostasis in a variety of plant species. In this study AtCKX2-overexpressing potato plants were engineered and grown in vitro as a model system to investigate the effects of altered cytokinin levels on tuber formation and tuber size. Protein extracts from shoots and roots of transformed potato plants exhibited higher CKX activity compared to control plants. Total endogenous cytokinin levels were generally not decreased in AtCKX2 overexpressors. However, levels of bioactive cytokinins were markedly lowered, which was accompanied by increased levels of O- and N-glucosides in some transgenic lines. The AtCKX2-overexpressing plants displayed reduced shoot growth but other symptoms of the “cytokinin deficiency syndrome” were not recorded. The transgenic plants were able to produce tubers in noninducing conditions. In inducing conditions they developed larger tubers than control. Tubers were also formed on a greater portion of the analyzed AtCKX2 plants, but with a lower number of tubers per plant compared to control. Taken together, our data suggest that cytokinins cannot be regarded simply as positive or negative regulators of tuberization, at least in vitro. Interactions with other plant hormones that play an important role in control of tuberization, such as gibberellins, should be further studied in detail.

Endogenous Phytohormones in Spontaneously Regenerated Centaurium erythraea Rafn. Plants Grown In Vitro

Phytohormones are important regulators of numerous developmental and physiological processes in plants. Spontaneous morphogenesis of the common centaury (Centaurium erythraea Rafn.) is possible on nutrition medium without addition of any plant growth regulator depending solely on endogenous phytohormone levels. Thus, this plant species represents a very good model system for the investigation of numerous physiological processes under phytohormonal control in vitro. We analysed the total amount of endogenous cytokinins (CKs) including the contents of their individual groups in shoots and roots of C. erythraea plants grown in vitro. The total amount of endogenous CKs was 1.4 times higher in shoots than in roots. Inactive or weakly active N-glucosides found to predominate in both organs of centaury plants, whereas free bases and O-glucosides represented only a small portion of the total CK pool. Consequently, centaury roots showed higher IAA content as well as IAA/free CK base ratios compared to shoots. Centaury tissues also showed increased levels of “stress hormones”. In contrast to SA, considerably higher levels of ABA were found in centaury shoots than in roots. Our results could serve as a basis for understanding and elucidating spontaneous de novo shoot organogenesis and further plant regeneration of C. erythraea in vitro.

Changes in cytokinin content and altered cytokinin homeostasis in AtCKX1 and AtCKX2-overexpressing centaury (Centaurium erythraea Rafn.) plants grown in vitro

The plant hormones cytokinins (CKs) regulate a number of physiological processes. Their homeostasis is controlled by the rate of de novo synthesis and the rate of catabolism. The aim of this work was to analyze the content of total as well as individual groups of endogenous CKs in AtCKX1 and AtCKX2-overexpressing centaury (Centaurium erythraea Rafn.) plants grown in vitro. Transgenic CKX plants represent a suitable model system for studying physiological and morphological processes controlled by CKs. In this work we clearly demonstrate a significant effect of AtCKX transgenes on CK metabolism in transgenic centaury plants. However, shoots and roots of only one AtCKX1 line and three AtCKX2 lines with a significant reduction of bioactive CKs were obtained. We also show that changes in the CKs metabolism considerably affected endogenous indole-3-acetic acid (IAA) levels in plant tissues. All analyzed transgenic AtCKX centaury lines exhibited decreased amount of endogenous IAA in shoots as well as in roots. Consequently, the IAA/bioactive CK forms ratios showed a significant variation in the shoots and roots of all analyzed AtCKX centaury transformants.