A positive feedback loop of cytokinin signaling ensures efficient de novo shoot regeneration in Arabidopsis

Abstract

Introduction

Plants possess a remarkable ability to regenerate tissues, which enables the healing of wounds and the induction of de novo organogenesis. In vitro plant tissue culture techniques are based on the regenerative capacity of plants and facilitate the reprogramming of differentiated somatic cells into a new organ or even an entire plant (Sugimoto et al., 2010). Differentiated plant tissues are used as explants to generate a pluripotent cell mass, called callus, on auxin-rich callus-inducing medium (CIM) (Ikeuchi et al., 2013; Zhai & Xu, 2021; Yin et al., 2024). Subsequently, the callus undergoes de novo shoot regeneration on cytokinin-rich shoot-inducing medium (SIM) (Che et al., 2007). A particular emphasis has been placed on de novo shoot organogenesis because the low shoot regeneration rate frequently limits in vitro plant regeneration in many species (Ijaz et al., 2012; Zimik & Arumugam, 2017).

Consistent with the fact that de novo shoot regeneration during in vitro tissue culture involves the conversion from callus cells to shoot meristem (Meng et al., 2017; Ogura et al., 2023), key regulators of shoot apical meristem (SAM) establishment are implicated in de novo shoot regeneration (Ikeuchi et al., 2016; Eshed Williams, 2021; Mathew & Prasad, 2021). The PLETHORA 3 (PLT3), PLT5, and PLT7 genes, which are expressed in the whole process of plant regeneration, play a particular role in shoot progenitor formation. Upon transferring to SIM, they are specifically expressed in shoot progenitor cells and promote promeristem formation by activating CUP-SHAPED COTYLEDON 1 (CUC1) and CUC2 (Kareem et al., 2015). The CUC1 and CUC2 proteins are involved in promoting SHOOT MERISTEMLESS (STM) expression and polarizing PIN-FORMED 1 (PIN1) localization to initiate shoot meristem development (Hibara et al., 2003; Bilsborough et al., 2011; Kamiuchi et al., 2014; Kareem et al., 2015). CUC2 also activates the expression of XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE 9 (XTH9) encoding a cell wall-loosening enzyme in nonprogenitor cells and contributes to establishing cell polarity for meristem formation (Varapparambath et al., 2022). Additionally, the main cytokinin regulatory axis is linked to the establishment of shoot stem cells in callus. Type-B ARABIDOPSIS RESPONSE REGULATORs (ARRs), positive regulators of cytokinin signaling, directly promote the expression of WUSCHEL (WUS), which unequivocally regulates the formation of the shoot stem cell niche (Meng et al., 2017; Zhang et al., 2017). Accordingly, mutations in type-B ARRs lead to impaired de novo shoot regeneration (Meng et al., 2017). By contrast, type-A ARRs, which play a negative role in cytokinin signaling, repress de novo shoot regeneration (Buechel et al., 2010).

The APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF)-type transcription factor gene ENHANCER OF SHOOT REGENERATION 1 (ESR1)/DORNROSCHEN (DRN) is involved in diverse aspects of plant regeneration, including wound-induced callus formation and de novo shoot regeneration (Iwase et al., 2017). In particular, during in vitro tissue culture, ESR1 is induced in response to cytokinin and promotes de novo organogenesis from callus (Banno et al., 2001; Iwase et al., 2017). Ectopic expression of ESR1 substantially enhances de novo shoot regeneration, whereas esr1 mutants display reduced de novo shoot formation from calli (Banno et al., 2001; Iwase et al., 2017). Despite the importance of ESR1 in de novo shoot regeneration, its modes of action in the plant cell remain unclear. In this study, we report that ESR1 stimulates cytokinin signaling and ensures efficient de novo shoot regeneration. ESR1 directly activates type-B ARR genes, which ultimately activate WUS. Notably, type-B ARRs also bind to the ESR1 promoter and activate its expression, establishing a positive feedback loop of cytokinin signaling. Collectively, the ESR1–type-B ARR module acts as a crucial player in the process of de novo shoot regeneration by strongly activating cytokinin responses to maximize the plant regeneration efficiency.