In vitro shoot organogenesis and hormone response are affected by the altered levels of Brassica napus meristem genes

Arabidopsis shoot meristem activity is regulated by a molecular network involving the participation of several components, including SHOOTMERISTEMLESS (STM), CLAVATA1 (CLV1), and ZWILLE (ZLL). In an effort to identify the role of these genes during in vitro shoot formation Brassica and Arabidopsis plants were transformed with the Brassica napus (Bn) STM, CLV1, ZLL1 and ZLL2 identified in previous work [1]. In both systems shoot organogenesis was promoted by the over-expression of BnSTM, BnZLL1, and BnZLL2, and repressed by the over-expression of BnCLV1. This distinct regulation, analogous to that occurring during in vivo meristem formation where STM and ZLL encourage stem cell formation while CLV1 accelerates transition to differentiation, suggests similar regulatory mechanisms governing shoot formation in vivo and in vitro. While the BnZLL1 and BnZLL2 induction of shoot organogenesis correlated only to changes in auxin signaling, BnSTM and BnCLV1 evoked major transcriptional alterations in cytokinin response. Besides increasing the transcript levels of two cytokinin receptors, ARABIDOPSIS HISTIDINE KINASE4 (AHK4) and CYTOKININ INDEPENDENT KINASE (CKI1), ectopic expression of BnSTM induced Type-B ARABIDOPSIS RESPONSE REGULATORS (ARRs) and repressed Type-A ARRs. Opposite transcriptional patterns occurred in explants over-expressing BnCLV1, characterized by a decreased ability to produce shoots. The role played by Type-A and Type-B ARRs during shoot organogenesis was further examined using a genetic approach which revealed the requirement of ARR12 for the BnSTM positive regulation of shoot organogenesis. Collectively these results expand our knowledge on the function of meristem genes, and provide new tools for enhancing in vitro propagation systems.

► Altered expression of Brassica meristem genes affect the in vitro shoot organogenic process in a similar fashion to that described during in vivo meristem development.
► SHOOTMERISTEMLESS and CLAVATA1 seem to competitively regulate shoot production in culture, with the former gene promoting the process and the latter inhibiting shoot production.
► The effects described in (2) are associated with a precise regulation of Type-A and Type-B RESPONSE REGULATORS which mediate cytokinin response in the tissue.
► ARR12 is required for the BnSTM positive regulation of shoot organogenesis.