**4. CKX activity and endogenous phytohormone content in transgenic**  *AtCKX* **centaury plants**

All centaury transgenic *AtCKX1* and *AtCKX2* shoots and roots showed increased CKX activity. Two to ten times higher CKX activity was detected in roots compared to shoots, which corresponds with previously demonstrated data in other plants such as tobacco [113], maize and barley [41, 64, 114]. In analogy to *AtCKX*overexpressing *Arabidopsis* plants [55] the higher CKX activity was detected in shoots and roots of *AtCKX2* than *AtCKX1* centaury lines.

As the CKX represents a valuable regulatory enzyme in CK metabolism, CKX activity directly affects the CK amount in plant tissues [115, 116], the contents and profile of endogenous CKs in centaury non-transformed and *AtCKX* transgenic plants have been determined. In general, centaury shoots grown *in vitro* produced higher content of CKs than roots [104]. Considering production and dominant location of CKs in the roots this distribution of endogenous CKs may be a consequence of *in vitro* conditions itself. The roots of centaury plants overexpressing *AtCKX* transgenes showed higher content of total CKs in comparison to non-transformed ones [84]. Previous investigations on *AtCKX* overexpressing *Arabidopsis* and tobacco plants revealed that increased CKX activity affected on the reduction of different endogenous CK metabolites [44, 55, 79]. Theoretically, increased CKX activity should cause a decline in the total CKs amount. Regardless of this, overexpression of *AtCKX* genes did not contribute to lowered total CKs content in transgenic centaury plants but rather to an alteration in the spectrum of particular CK types. In analogy, transgenic potato plants overexpressing *AtCKX* genes were not found to show decreased total endogenous CK contents while bioactive CK forms were predominantly reduced [82, 83]. Accordingly, although enhanced CKX activity results in more substantial CK degradation in plant tissues it might simultaneously serve as an indirect signal for the plant cell to intensify the biosynthesis of CKs. It may lead to higher accumulation of total endogenous CKs and seems to be a mechanism of re-establishment and maintenance of CK homeostasis in plants [117].

Beside the total CKs contents, an altered amounts of individual endogenous CKs groups were specific for*AtCKX* transgenic centaury plants [104]. The reported considerable differences in endogenous CK spectra might reflect the distinct position and/or the number of transgene copies. The most specific characteristic of transgenic centaury plants was a remarkable reduction of bioactive CK forms, including free bases and their corresponding ribosides. Similarly, the *AtCKX* transgenic tobacco and potato plants also showed decreased contents of bioactive CKs [79, 82, 83, 118, 119]. In transgenic centaury plants, the reduction of bioactive CKs was organspecific. Thus, considerably declined levels of bioactive CK forms were found in the *AtCKX* shoots although their CKX activity was not enhanced. On the other hand, a significant reduction of bioactive CKs levels was shown in *AtCKX* roots together with an increased CKX activity. A possible explanation is that CKs are probably more degraded in roots, the main location of biosynthesis, and accordingly, a smaller content of bioactive CKs is further transported to centaury shoots. Another one attribute of the *AtCKX* transgenic centaury plants was represented by increased amounts of CK storage forms (*O*-glucosides) and/or of irreversibly inactive (or weakly active)CK *N*-glucosides [104]. This is in accordance with recent literature data demonstrating the same CK pattern in *AtCKX1* transgenic potato plants [83]. Simultaneously, the concentrations of CK precursors in transgenic centaury plants were higher than those of bioactive CKs, which also corresponds with the potato plants [82].

To summarize, the *AtCKX* transgenic centaury plants are characterized by altered CK profiles with reduced levels of bioactive CK forms and, at the same time, increased amounts of storage forms (CK *O*-glucosides), inactive (or weakly active)

#### *Transgenic* AtCKX *Centaury Plants Grown* In Vitro *DOI: http://dx.doi.org/10.5772/intechopen.94836*

forms (CK *N*-glucosides) andCK nucleotides. Previous investigations showed that overexpression of *AtCKX* transgene increased the production of storage CK derivatives in transgenic potato plants [81, 82]. It can be assumed that introduction of *AtCKX* transgenes into centaury genome altered CK metabolism in a way leading to the increased production of endogenous CK conjugates. Simultaneously, the level of bioactive CK forms seems to be controlled, besides the CKX activity, by additional regulatory mechanisms involved in CK metabolism and transport throughout the plant tissues. Considering that bioactive CKs are the preferred substrates of CKX, it is also possible that plant tissues activate new CK biosynthetic pathways and further initiate conversion to storage compounds, deactivation forms and/or CK nucleotides.

Numerous physiological and developmental processes in plants are regulated by the cooperation of CKs and auxins [120–123]. Overexpression of *AtCKX* transgenes in centaury plants altered not only the CK homeostasis, but at the same time it affected metabolism of auxins. In transgenic centaury plants, the auxin indole-3-acetic acid (IAA) concentration was significantly decreased compared to the control. In analogy, previous literature data uncovered that an enhancement of the CKX activity led to the decline of endogenous CK levels and simultaneously to the reduction of endogenous IAA content in *A. thaliana* [79, 122]. In contrast, increased IAA concentration was reported in the *AtCKX1*-overexpressing potato plants [83]. These findings support the fact that the balance between CKs and auxins represents the most crucial factor in the shoot and root development in plants [55, 79].

In transgenic *AtCKX* centaury roots, the IAA/bioactive CKs ratio was lowered in comparison to control. It might be a consequence of a reduced regeneration capacity of transgenic centaury roots [84]. Considering that endogenous IAA content was lowered in the roots, it is presumable that *AtCKX*-overexpression weakened the regeneration of shoots in solid roots culture. In transgenic centaury shoots, the IAA/bioactive CKs ratios varied considerably. Contrary to the centaury plants, endogenous IAA content was significantly increased in the shoots and roots of transgenic *AtCKX1* potato [83].

An altered CK metabolism resulting from the overexpression of *AtCKX* transgenes definitely affected IAA content and IAA/bioactive CKs ratio in transformed centaury plants. Different biosynthetic capacity for CK and IAA production could be a reason for the organ-specific differences among *AtCKX* transgenic plants.
