# A novel mechanism of auxin habituation: upregulation of auxin receptor TRANSPORT INHIBITOR RESPONSE 1 allows cell proliferation independent of external auxin

## TL;DR

This study reveals that tobacco cell lines can proliferate without external auxin by increasing their auxin receptor levels, a novel adaptation mechanism.

## Contribution

The discovery of TIR1 gene amplification as a novel mechanism for auxin habituation in tobacco cell lines.

## Key findings

- BY-2H cell line shows TIR1 gene amplification leading to auxin receptor upregulation.
- VBI-2 cell line shows epigenetic habituation via MADS-domain transcription factors.
- Inducible TIR1 overexpression allows auxin-dependent cell lines to proliferate without exogenous auxin.

## Abstract

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.

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.