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

Abstract

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.

Cytokinin is perceived by three sensor histidine kinases, including ARABIDOPSIS HISTIDINE KINASE2 (AHK2), AHK3, and AHK4, and the binding of cytokinin to AHKs allows autophosphorylation of their receiver domains (Liu et al., 2019). Subsequently, the phosphoryl group is transferred to HISTIDINE PHOSPHOTRANSFER PROTEINs (AHPs) that translocate into the nucleus to activate type-B ARRs in a phosphorylation-dependent manner (To & Kieber, 2008). This main regulatory axis of cytokinin signaling is involved in diverse aspects of plant growth and development, especially shoot development (Leibfried et al., 2005; Riefler et al., 2006; Gordon et al., 2009). For example, the cytokinin signaling circuit determines SAM growth, and genetic mutations of cytokinin receptors and signaling components lead to reduced SAM size (Higuchi et al., 2004; Kurakawa et al., 2007). Consistently, the type-B ARRs directly bind to and activate the WUS gene, which is a pivotal regulator for SAM maintenance (Meng et al., 2017; Zhang et al., 2017). The cytokinin signaling axis is also critical for de novo shoot regeneration during in vitro plant regeneration. Upon the incubation on SIM, cytokinin signaling is provoked and stimulates shoot stem cell establishment and shoot emergence. In particular, the type-B ARR transcription factors directly activate WUS expression in callus to establish the shoot progenitor (Meng et al., 2017). In support, the arr1 arr12 mutant exhibits defects in shoot regeneration from callus, which can be rescued by ectopic expression of WUS (Meng et al., 2017).

The cytokinin-responsive ESR1 protein is also known as a core regulator of de novo shoot regeneration, because the loss-of-function mutants exhibit completely impaired shoot regeneration (Iwase et al., 2017; Fig. 1d,e), although its position on the cytokinin signaling network has been elusive. Here, we demonstrate that ESR1 forms a positive feedback loop with type-B ARRs. ESR1 directly activates type-B ARRs, which also directly bind to the ESR1 locus to promote expression. The ESR1–type-B ARR module reinforces cytokinin responses, and thus, the progression of de novo shoot regeneration with enhanced WUS expression. In support, both esr1-2 and arr1 arr12 mutants exhibited complete defects in shoot regeneration (Meng et al., 2017; Ogura et al., 2023). Consistent with the previous studies showing that a positive feedback loop strengthens stimuli-responsive signal transduction (Pandey et al., 2018; Ohashi-Ito et al., 2019), the positive feedback loop constituted by ESR1 and type-B ARRs likely maximizes cytokinin responses, as evidenced by their interdependence in de novo shoot regeneration. Furthermore, the mutual activation of ESR1 and type-B ARRs is also crucial for full activation of WUS expression. They form a protein complex and directly bind to the WUS locus, synergistically promoting expression. ESR1 is particularly important for specifying the WUS promoter region, where both ESR1 and type-B ARRs bind. Binding of type-B ARRs to WUS was diminished in the esr1-2 mutant (Fig. S13), whereas type-B arr mutants had a relatively lower impact in ESR1 binding to WUS (Fig. S13). This may account for the complete loss of regeneration capacity in esr1-2 mutant overexpressing type-B ARR gene (Fig. 4a,b). In addition, we suspected that the role of ESR1 in shoot stem cell specification is likely dependent on cell types. The ESR1–type-B ARR module is expressed mainly in the outer cell layer in callus, where shoot meristem niches are formed, at a later stage of SIM incubation. Considering that the constitutive expression of ESR1 throughout callus sometimes represses de novo shoot regeneration (Temman et al., 2023), the cell type-specific function of ESR1 may explain the controversial effect of ESR1 on shoot regeneration (Banno et al., 2001; Iwase et al., 2017; Temman et al., 2023).

The role of ESR1 in cytokinin signaling also appears to be related to wound-induced callus formation. Wound responses depend on cytokinin signaling to trigger cell cycle reentry (Dewitte et al., 2007; Ikeuchi et al., 2017). Consistently, wound-induced callus formation is impaired in cytokinin signaling mutants, such as the arr1 arr12 double mutant (Ikeuchi et al., 2017). Indeed, the wound-responsive WIND1 transcription factor, a key player of wound-induced cellular reprogramming, acts through cytokinin signaling, as WIND1-induced callus formation is compromised in arr1 arr12 mutants (Iwase et al., 2011). Given that WIND1 directly activates the ESR1 gene (Iwase et al., 2017), whose product binds to type-B ARR loci to promote expression, WIND1 regulation of cytokinin signaling may depend on ESR1 during wound-induced cellular reprograming. In agreement with the integrative role of ESR1 in wounding and cytokinin signaling, esr1 mutants have severe defects in wound-induced callus formation (Iwase et al., 2017). Furthermore, considering that ESR1 is also involved in protoplast regeneration (Xu et al., 2021), the ESR1–ARR module is anticipated to be important for a wide range of cytokinin-dependent plant regeneration. Based on the importance of ESR1 in plant regeneration as well as its conservation across a wide range of plant species (Xu et al., 2021; Larriba et al., 2022), our knowledge can be widely applied to improve the efficiency of various in vitro tissue culture applications, because a low efficiency of de novo shoot organogenesis is a key hurdle in protoplast regeneration, in vitro organogenesis, and crop transformation.

None declared.

PJS conceived and designed the study. KL, HY and O-SP conducted the experiments. PJS and KL wrote the manuscript. All authors read and approved the manuscript. KL and HY contributed equally to this work.

The New Phytologist Foundation remains neutral with regard to jurisdictional claims in maps and in any institutional affiliations.