Exogenně aplikovaný rostlinný hormon auxin je nezbytný pro založení buněčných linií v tkáňových kulturách a pro udržení jejich proliferace. Některé buněčné linie však mohou získat schopnost růst i v médiu bez přídavku auxinu – stávají se tzv. habituované. Tato studie se zabývala mechanismy, které tento proces navozují. U dvou nezávisle habituovaných tabákových buněčných linií, BY‐2H a VBI‐2, odvozených od auxin‐závislých linií Bright Yellow (BY‐2) a Virginia Bright Italia (VBI‐0), jsme provedli komplexní profilování metabolismu auxinu, farmakologické testy inhibitorové odezvy a transkriptomické porovnání. Naše výsledky ukazují, že obě habituované linie vyvinuly odlišný mechanismus auxinové autonomie. Zatímco v případě VBI‐2 linie, kde zvýšená exprese genů transkripčních faktorů rodiny MADS naznačuje epigeneticky determinovanou dráhu, v linii BY‐2H bylo identifikováno masivní pomnožení genomové oblasti obsahující gen kodující receptor auxinu TRANSPORT INHIBITOR RESPONSE1 (TIR1), které vedlo k jeho výrazné nadregulaci. Napodobením tohoto jevu expresí indukovatelným TIR1 v auxin‐závislé linii BY‐2 bylo dosaženo schopnosti jejího dělení bez přítomnosti vnějšího auxinu. Kompenzace nedostatku auxinu zvýšením hladiny jeho receptoru je velmi zajímavým jevem. Amplifikace genomové oblasti TIR1 je jedinečným příkladem mikroevoluce v laboratorních podmínkách pod silným selekčním tlakem, a může mít významný potenciál pro biotechnologické aplikace.

Exogenously applied auxins are essential for establishing cell lines in tissue cultures and maintaining their proliferation. Cell lines may develop the ability to proliferate even in media lacking auxin, they may become auxin-habituated. This study investigated the mechanisms underlying this process. Here, we conducted comprehensive auxin metabolic profilings, pharmacological treatments and transcriptomic comparisons in two independently habituated tobacco cell lines, BY-2H and VBI-2, derived from cell lines of cultivars Bright Yellow (BY-2) and Virginia Bright Italia (VBI-0). Our results show that both habituated lines developed different mechanisms of auxin autonomy. In VBI-2, increased expression of MADS-domain transcription factor genes suggests epigenetically determined habituation. By contrast, in BY-2H, genome resequencing identified a massive amplification of the genomic region containing the TRANSPORT INHIBITOR RESPONSE 1 (TIR1) gene, causing its strong upregulation. Mimicking this by inducible overexpression of TIR1 in the auxin-dependent BY-2 line allowed its proliferation in the absence of exogenous auxin. Compensating for auxin deficiency by increasing level of its receptor is a very intriguing phenomenon. The amplification of the TIR1 genomic region is a unique example of in-flask microevolution under strong selection pressure with potential interest for biotechnological applications.

Department of Experimental Plant Biology Faculty of Science Charles University Viničná 5 128 00 Prague 2 Czech Republic

Institute of Experimental Botany of the Czech Academy of Sciences Rozvojová 263 165 00 Prague 6 Czech Republic

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