Mechanisms of habituation for exogenous auxin-independent proliferation of tobacco cells

Pavel Jelíneka, Karel Müllerb, Petre I. Dobrevb, Roberta Vaculíkováb, Zuzana Vondrákováb, Barbora Svobodováb, Julie Talpováb, Anita Bírošíkováb, Vojtěch Čermáka, Adéla Přibylováa, Eliška Kobercováa, Lukáš Fischera, Jan Petrášeka

a Department of Experimental Plant Biology, Faculty of Science, Charles University, Viničná 5, 128 44 Prague 2, Czech Republic b Institute of Experimental Botany, the Academy of Sciences of the Czech Republic, Rozvojová 263,165 02 Prague 6, Czech Republic

Auxins are essential phytohormones that are involved in the regulation of the plant cell cycle. In vitro cultured plant cells require exogenous auxin for proliferation. However, they may acquire the ability to survive and proliferate in auxin-deprived media through the phenomenon of habituation. In this study, we sought to understand the mechanisms behind the auxin habituation of two tobacco cell lines. We compared two pairs of lines derived from two tobacco cultivars; BY-2 x BY-2H and VBI-0 x VBI-2b. In each pair, we had one line dependent on exogenous auxin (BY-2, VBI-0) and one habituated (BY-2H, VBI-2b). To describe the changes between auxin-dependent and habituated lines, we used a set of approaches such as transcriptome analysis (RNAseq), genomic sequence analysis, and phytohormonome analysis. By combining these approaches, we were able to show that the proliferation of both habituated lines is still dependent on endogenously synthesised auxin. However, each culture possesses different mechanisms. The BY-2H line contains an increased number of copies of the auxin receptor TIR1 in its genome resulting in 200-fold higher expression. We were able to localise the copies of TIR1 on different chromosomes. We were also able to detect higher levels of endogenous IAA in BY-2H compared to the BY-2 line. On the other hand, VBI-2b habituation hypothetically depends on a decreased expression of EMF2, a component of the PRC2 complex, which probably leads to an increased expression of a set of MADS-box transcription factors. Our focus now is to provide functional evidence for both hypotheses.

Supported by CSF grant n. 23-07813S and GAUK n. 414022