The source and accessibility of cytokinins to regulate plant development is largely determined by their metabolic machinery. The initial step of cytokinin biosynthesis is catalyzed by the enzyme isopentenyltransferase (IPT) and involves the transfer of a prenyl moiety from dimethylallyl diphosphate to ATP or ADP to form N⁶-isopentenyladenine (iP) ribotides (Kakimoto, 2001; Takei et al., 2001). The major initial products can be hydroxylated to trans-zeatin (tZ)-type cytokinins by the action of cytochrome P450 mono-oxygenases CYP735A1 and CYP735A2 (Takei et al., 2004). The tZ-types are most probably converted by zeatin reductase to DHZ-type (Martin et al., 1989; Gaudinova et al., 2005) but the corresponding gene has not yet been identified. Cytokinins of the cis-zeatin (cZ)-type arise from tRNA degradation (Kakimoto, 2001; Miyawaki et al., 2006). Direct conversion of cytokinin ribotides to active free bases is catalysed by a nucleoside 5’-monophosphate phosphoribohydrolase named LONELY GUY (LOG) which plays a pivotal role in regulating cytokinin activity during plant growth (Kurakawa et al., 2007; Kuroha et al., 2009). Free cytokinin bases and cytokinin ribosides can be converted back to ribotides by the action of adenine phosphoribosyl transferases (APT) (Lee and Moffatt, 1993; Allen et al., 2002) and adenosine kinases (ADK) (Moffatt et al., 2000), respectively. Cytokinin deactivation readily occurs via O- and N-glycosylation catalysed by uridine diphosphate glycosyltransferases (UGT) (Hou et al., 2004). Cytokinin O-glucosides can be converted to the active free bases by β-glucosidase (Brzobohaty et al., 1993). Irreversible cytokinin degradation is catalysed by the cytokinin oxidase (CKX) (Bilyeu et al., 2001; Galuszka et al., 2001), which cleaves unsaturated N⁶-side chains from tZ and iP-type cytokinins (Jones and Schreiber, 1997). The combined regulation of biosynthesis, modifications and degradation maintains cytokinin homeostasis, keeping phytohormone levels optimal at each stage as well as in each tissue throughout plant growth and development.

Cytokinins trigger multistep phosphorelay signaling by binding to their cognate receptors. In Arabidopsis, three receptors ARABIDOPSIS HISTIDINE KINASE AHK2, AHK3 or AHK4/WOL/CRE1 (Inoue et al., 2001; Higuchi et al., 2004; Nishimura et al., 2004) sense cytokinins via their CHASE domains (Anantharaman and Aravind, 2001). This signal is sequentially transferred to ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEINS (AHPs; AHP1-5) (Hutchison et al., 2006) and then to ARABIDOPSIS RESPONSE REGULATORS (ARRs) (Suzuki et al., 1998; Tanaka et al., 2004). The one exception is AHP6, which lacks a histidine and attenuates cytokinin signaling (Mahonen et al., 2006). Type-A ARRs (ARR3-9, 15-17) represent cytokinin primary response genes and are promptly upregulated by cytokinins, while simultaneously inhibiting the cytokinin signaling pathway, thus creating a negative feedback loop (Hwang and Sheen, 2001; To et al., 2004). Type-B ARRs (ARR1, 2, 10-14, 18-21) contain a DNA binding domain and control the expression of cytokinin-regulated genes, including type-A ARRs (Sakai et al., 2000; Mason et al., 2005). A third class, type-C ARRs, consists of only two members (ARR22, 24) which are structurally related to type-A ARRs, but their transcription is not induced by cytokinins (Kiba et al., 2004). Moreover, AHPs also interact with a set of cytokinin-regulated transcription factors, the CYTOKININ RESPONSE FACTORS (CRFs) (Rashotte et al., 2006; Cutcliffe et al., 2011), thus providing additional fine-tuning of cytokinin signaling output.

Plants show a remarkable plasticity in their growth over their entire lifetime, which sets them apart from mammals. In plants, two apical meristematic systems localized at opposite ends of the plant body axis – the shoot apical meristem (SAM) and the root apical meristem (RAM) – are established during embryogenesis. These meristems are responsible for giving rise to the entire plant body – both the root and the areal parts. The proper functioning of meristems is dependent on the presence of a small number of undifferentiated pluripotent stem cells, which are located in specific environments called stem cell niches (reviewed in Greb and Lohmann, 2016).

Stem cells of SAM are located in the central zone (CZ) at the shoot apex (Fletcher et al., 1999). Stem cells undergo very slow cell division keeping the stem cell pool constant (Laux, 2003). Daughter cells on the periphery are pushed out of the CZ to neighboring zones, where they undergo faster cell divisions and subsequently differentiate giving rise to specific plant tissues and organs (Schoof et al., 2000; Reddy et al., 2004). In Arabidopsis, CZ comprises three layers called L1, L2, and L3, where cells from each layer have a different identity. Cells in the L1 and L2 layers divide anticlinally giving rise to leaf and flower primordia. L3 cells divide anticlinally and periclinally, leading to internal tissue production. A distinct group of cells that controls CZ activity, called the organizing center (OC), is located below the CZ, and furnishes signals to block differentiation, thereby keeping stem cells undifferentiated (reviewed in Greb and Lohmann, 2016).

For proper plant body development, SAM must perpetuate itself. This demands general regulation factors. Homeodomain transcription factor WUSCHEL (WUS) plays a central role in SAM maintenance as a necessary and sufficient regulator of stem cells (reviewed in Lopes et al., 2021). The wus mutation causes defects in the shoot meristem at all developmental stages in Arabidopsis (Laux et al., 1996; Mayer et al., 1998). In cooperation with the CLAVATA3 (CLV3) peptide, WUS preserves the stem cell niche ( Figure 1A ). Originating in the OC, WUS migrates through plasmodesmata to the CZ, where it directly activates CLV3 expression (Yadav et al., 2011; Daum et al., 2014). The CLV3 peptide is perceived by leucine rich repeat (LRR)-based receptors, either by homomers of LRR-receptor-like kinases, such as CLAVATA1 (CLV1) (Ogawa et al., 2008), or by complexes of LRRs with membrane-bound kinases or pseudokinases, such as CLAVATA2 (CLV2) and CORYNE (CRN) (Bleckmann et al., 2009; Guo et al., 2010). CLV3 binding to dedicated receptors results in the activation of a signaling cascade that regulates WUS activity. Thus, the WUS-CLV interaction establishes a self-organizing feedback loop maintaining equilibrium between cell division and cell differentiation in the SAM (Fletcher et al., 1999; Schoof et al., 2000). Additionally, two CLV3-related peptides CLE16 and CLE17 contribute to SAM stem cell maintenance and organ production independently from CLV3. Their signal is precepted by BARELY ANY MERISTEM (BAM) and act upstream of WUS (Dao et al., 2022).

Recently, the subcellular – particularly the nucleolar versus cytoplasmic – distribution of WUS was revealed to be an important factor in stem cell niche maintenance. In L1 and L2 layers, CLV3 inhibits WUS export from the nucleus thus blocking WUS diffusion into surrounding cells. Simultaneously, CLV3 represses WUS expression resulting in lower WUS concentrations in the nucleus than is needed for the activation of CLV3 expression. Transport of WUS from the nucleus to the cytoplasm can be enabled by direct interaction of WUS with EXPORTIN proteins via the EAR-like domain (Plong et al., 2021). The EAR-like domain could serve as a nuclear export signal and may also be required for destabilizing WUS in the cytoplasm. Mutations in the WUS EAR-like domain leads to an enlarged SAM, which was attributed to the higher pool of cytoplasmically-stable WUS available for diffusion into adjacent cells (Rodriguez et al., 2016).

Another level of WUS action control arises from the ability of WUS to form heterodimers with other proteins ( Figure 1B ). In the L1 and L2 layers, WUS interacts with SHOOTMERISTEMLESS (STM), a transcription factor (Long et al., 1996) required for maintaining the proliferative state of the meristematic cells (Vollbrecht et al., 2000). The WUS-STM interaction strengthens its binding to the CLV3 promoter and enhances CLV3 expression (Su et al., 2020). On the other hand, WUS can create heterodimers with proteins from HAIRY MERISTEM (HAM) family leading to the repression of CLV3 expression in the L3 layer (Zhou et al., 2015; Zhou et al., 2018b).

The activity of meristematic tissues is regulated by many factors, with a crucial role played by phytohormones. Cytokinins are important regulators of SAM function and maintenance, and their abundance depends on the biosynthetic machinery. One of the rate-limiting enzymes of cytokinin biosynthesis IPT7 is triggered by STM localized in SAM (Yanai et al., 2005) ( Figure 1A ). The L1 layer is thought to be the source of active cytokinins as the expression of another cytokinin biosynthetic gene LOG4 was detected there (Chickarmane et al., 2012). It is hypothesized that active cytokinins move from the L1 layer into inner cell layers, where they affect SAM function. Moreover, LOG3 was showed to localize to developing primordia. Further, basic helix-loop-helix (bHLH) transcription factor heterodimers of TARGET OF MONOPTEROS5 (TMO5) and LONESOME HIGHWAY (LHW) subclades were suggested to act as a general regulator of cell proliferation in all meristems of Arabidopsis. Heterodimer complexes could regulate meristems due to heterodimer variations between subclade members, leading to diversification of target gene expression (Mor et al., 2022). The TMO5-LHW heterodimer directly regulates several enzymes of cytokinin metabolism (e.g., LOG3, LOG4, CKX3) (De Rybel et al., 2014; Yang et al., 2021) thus impacting endogenous cytokinin levels. In SAM, the phenotype and gene expression pattern of TMO5 and LHW subclade members suggest additional heterodimer combinations can affect LOG expression. LOG3 and LOG4 reporter signals are completely missing in the lhw single mutant suggesting that the LHW transcription factor is essential for LOG3 and LOG4 expression (Mor et al., 2022). Additionally, the cytokinin degrading CKX3 is expressed in a similar pattern as WUS, while CKX5 is also present in several other areas. The ckx3 ckx5 double mutant forms larger meristems with an expanded WUS domain (Bartrina et al., 2011), probably because of higher endogenous cytokinin levels.

To be functional, cytokinins need to be “visible”. The cytokinin receptors AHK2, AHK3 and AHK4 are expressed in the inner tissues of SAM including OC, but not in the L1 and L2 layers. Therefore, it seems that it is mainly the L3 layer that is the active site for cytokinin perception (Gordon et al., 2009; Chickarmane et al., 2012). The presence of cytokinins triggers cytokinin signaling leading to the activation of type-B ARRs ( Figure 1A ). The transcription factors ARR1, ARR10 and ARR12 bind directly to the WUS promoter and induce WUS expression (Meng et al., 2017; Zubo et al., 2017). The arr1 arr10 arr12 triple mutant forms a much smaller SAM compared to WT recalling that of the ahk2 ahk3 ahk4 triple mutant (Ishida et al., 2008), which points to a fundamental role for ARR1, ARR10 and ARR12 in SAM maintenance. Moreover, ARR1 stabilizes WUS (Snipes et al., 2018), thus setting up positive feedback between cytokinins and WUS.

Apart from activating WUS, type-B ARRs also activate type-A ARRs (Sakai et al., 2000), thus providing negative feedback regulation of cytokinin signaling (Hwang and Sheen, 2001; To et al., 2004). ARR1, ARR2 and ARR10 are responsible for activating ARR6, with ARR2 being the most effective (Hwang and Sheen, 2001). On the other hand, several type-A ARRs, particularly ARR5, ARR6, ARR7 and ARR15, are repressed by WUS, thereby potentiating cytokinin signaling. A constitutively active form of ARR7 causes significant defects in SAM function with arrested meristem in severe cases (Leibfried et al., 2005). Consistent with this, miRNA mediated attenuation of ARR7 and ARR15 expression results in SAM expansion and a severe reduction of CLV3 mRNA, pointing to ARR7 and ARR15 control of SAM maintenance by regulating CLV3 expression (Zhao et al., 2010).

It seems that local auxin accumulation is another important factor for proper functioning of ARR7 and ARR15, as revealed by their elevated transcripts in the auxin biosynthesis yucca (yuc) mutants, the auxin transport pinformed 1 (pin1) mutant, or following treatment by the auxin transport inhibitor NPA. The negative regulation of ARR7 and ARR15 by auxin is partially mediated by signaling through AUXIN RESPONSE FACTOR5/MONOPTEROS (ARF5/MP) (Zhao et al., 2010). Other targets of ARF5 involved in SAM activity and maintenance have been identified in Arabidopsis, e.g., AHP6 (Besnard et al., 2014), TMOs (Schlereth et al., 2010), DORNRÖSCHEN/ENHANCER OF SHOOT REGENERATION1 (DRN/ESR1) (Cole et al., 2009), and interestingly ARF5 itself (Lau et al., 2011). The role of auxin signaling in SAM regulation and maintenance has been reviewed recently (Pernisová and Vernoux, 2021) and we will not cover this topic in more detail here.

Taken together, the overlap between cells with threshold cytokinin levels and cells competent for cytokinin perception presumably occurs at a fixed distance from the cytokinin source (L1 layer), leading to precise WUS activation, and defining the OC domain.

Shoots can be regenerated from explants in in vitro conditions using two approaches. During two-step regeneration, explants are first placed on a callus-inducing medium (CIM), which contains a high concentration of auxin. In the second step, the callus formed on the CIM is transferred to a shoot-inducing medium (SIM), with a high concentration of cytokinins, where shoots develop (Valvekens et al., 1988). The callus maintains some level of cell organization resembling lateral root primordium pointing to the fact that a callus is not a population of undifferentiated cells, but rather a partially differentiated tissue with features resembling the lateral root developmental program (Atta et al., 2009; Sugimoto et al., 2010). Originally, it was thought that during de novo organogenesis, any somatic cell can dedifferentiate and reenter the cell cycle. However, further research uncovered that organogenesis originates from populations of partially differentiated stem cells. One such population is formed of pericycle cells located adjacent to the xylem poles in roots (Dubrovsky et al., 2000; Beeckman et al., 2001). In aerial parts of the plant body, de novo organogenesis originates from pericycle-like cells, which are present around the vasculature of multiple organs throughout the plant body (Sugimoto et al., 2010). Initiation of founder cells is the first step in lateral root development. Acquisition of founder cell status depends on auxin accumulation in pericycle cells. The specification of founder cells is followed by nuclear polarization of two adjacent pericycle cells that divide anticlinally and asymmetrically, forming two large cells and two smaller daughter cells representing stage I of the lateral root primordium (Malamy and Benfey, 1997; Casimiro et al., 2001; Dubrovsky et al., 2001). Recent observations during the early stages of regeneration have shown that the whole process likely does not originate from a single cell, but rather from a group of cells (Subban et al., 2020).

A local auxin maximum is established and auxin signaling is activated in the early stages of founder cell specification ( Figure 2A ). Auxin accumulation in founder cells leads to the degradation of the AUXIN/INDOLE-3-ACETIC ACID INDUCIBLE28 (AUX/IAA28) repressor, which controls the founder cell-specifying GATA23 transcription factor (De Rybel et al., 2010). Auxin also controls the division of founder cells by degrading SOLITARY ROOT/INDOLE 3 ACETIC ACID INDUCIBLE14 (SLR/IAA14), which acts as a repressor of ARF7 and ARF19. The repression of ARF7 and ARF19 is also dependent on PICKLE/SUPRESSOR OF SLR2 (PKL/SSL2). Following SLR/IAA14 degradation, ARF7 and ARF19 positively regulate the expression of LATERAL ORGAN BOUNDARIES-DOMAIN29/ASYMMETRIC LEAVES2-LIKE16 (LBD29/ASL16), LBD16/ASL18 and LBD18/ASL20 (Fukaki et al., 2002; Okushima et al., 2005; Fukaki et al., 2006), that are necessary for the initiation of lateral roots (Okushima et al., 2007; Lee et al., 2009). LBD induction promotes cell cycle progression through the G1 – S checkpoint (Feng et al., 2012). The process of callus formation seems also to be positively regulated by MORE AUXILARY GROWTH2 (MAX2) (Li et al., 2019). In the max2 mutant, LBD33 is less upregulated on CIM medium (Temmerman et al., 2022). LBD33 forms a heterodimer with a LBD18, and this is responsible for the induction of pericycle cell cycle activation via E2Fa transcription factor (Okushima et al., 2007; Berckmans et al., 2011).

Ca²⁺-mediated signaling plays an important role in auxin-controlled initiation of callus formation. CALMODULIN IQ-MOTIF CONTAINING PROTEIN (CaM-IQM) physically interacts with IAA19 in a Ca²⁺-dependent manner, which then destabilizes the repressive interaction of IAA19 with ARF7, promoting callus formation via LBD activity (Zhang et al., 2022). The impact of Cam-IQM on other IAAs or ARFs is unclear so far.

LBD16 is controlled by WUCHEL-RELATED HOMEOBOX 11 (WOX11), which is activated shortly after the acquisition of root primordium identity by founder cells (Liu et al., 2014). WOX11 also activates another member of the WOX family, WOX5 (Hu and Xu, 2016), broadly expressed in the subepidermal layer of each callus type (Sugimoto et al., 2010). WOX5 was originally identified as a WUS homolog in SAM and is analogically responsible for the maintenance of the root quiescent center in the stem cell niche (Sarkar et al., 2007; Pi et al., 2015). Moreover, WOX5 directly affects auxin levels in cooperation with the AP2-family transcription factors PLETHORA1 (PLT1) and PLT2 by activating the auxin biosynthetic gene TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) (Zhai and Xu, 2021).

During CIM cultivation, the expression of PLT3, PLT5, and PLT7 is rapidly induced. They control PLT1 and PLT2, leading to the establishment of a pluripotent callus (Kareem et al., 2015). Analysis of the gene regulatory network uncovered PLT3 as a critical node in shoot regeneration (Ikeuchi et al., 2018). Meristem development is also dependent on the PLT1 gradient (Galinha et al., 2007), which indicates the existence of a fine-tuned transcriptional regulatory mechanism. In addition, the activity of PLT3, PLT5, and PLT7 regulates the shoot-promoting factor CUPSHAPED COTYLEDON2 (CUC2) (Kareem et al., 2015), which was shown to promote adventitious shoot formation (Daimon et al., 2003). CUC genes play an important role in the regulation of auxin abundance by controlling the auxin biosynthetic genes YUC1 and YUC4 (Yamada et al., 2022). CUC2 promotes meristem formation by activating the cell wall loosening enzyme (XTH9), whose activity is triggered in cells surrounding the meristem progenitor. This results in a mechanical conflict, triggering cell polarity in the progenitors and the subsequent promotion of meristem formation (Varapparambath et al., 2022).

Callus formation is dependent on cytokinin perception by the receptors AHK2, AHK3 and AHK4. The corresponding mutants exhibit reduced callus size and greening (Li et al., 2019). Moreover, the presence of auxin in CIM leads to strong local up-regulation of AHK4, which predestines future cytokinin-induced WUS expression (Gordon et al., 2009). Auxin signaling is therefore partially responsible for the sensitivity of the whole system to cytokinins. Further cytokinin signal transduction also plays an important role in the acquisition of pluripotency in the callus. The type-B ARR2 and ARR12 interact with the root-specific WOX5 that promotes cytokinin sensitivity of the system (Zhai and Xu, 2021). It seems that type-B ARRs also affect the recalcitrancy of plant tissues. ARR18 expression was much lower in recalcitrant Arabidopsis lines than in highly regenerative ones (Lall et al., 2004). Furthermore, WOX5 also represses type-A ARRs, negative regulators of cytokinin signaling. Overexpression of WOX5 inhibits the expression of ARR5, ARR6 and ARR7. Complementarily, the transcript level of ARR5 was higher in the wox5 mutant on both CIM and SIM (Zhai and Xu, 2021; Lee et al., 2022). The loss of ARR7 function resulted in strong induction of callus formation, which points to an inhibitory role for ARR7 in the acquisition of pluripotency (Buechel et al., 2010). Preincubation on CIM is necessary for the expression and proper function of ARR15, a direct target of ARR2 (Che et al., 2008). Thus, a proper balance between various up- and down-regulators of cytokinin signaling is crucial for the acquisition of pluripotency.

Information about changes in endogenous cytokinin levels in Arabidopsis during processes leading to de novo shoot regeneration in vitro is rather scarce. Arabidopsis hypocotyls cultivated on medium supplemented with kinetin at concentrations of 0, 300 and 1000 ng/ml had comparable dynamics of endogenous tZ-type cytokinins levels, whereas the levels of iP-type cytokinins strongly increased on media containing a high kinetin concentration (1000 ng/ml) (Pernisova et al., 2018). This seems to suggest a possible role for iP-type cytokinins in the development of de novo shoot apical meristems in vitro.

To regenerate a shoot, cells first acquire competence for organogenesis on CIM with a high concentration of auxin. The calli are then exposed to a high concentration of cytokinins on SIM, which promotes the acquisition of shoot meristem properties and subsequent shoot organogenesis (Christianson and Warnick, 1983). A rapid downregulation of root-specific markers occurs shortly after transferring the callus to SIM. Root specific WOX5 expression in the middle cell layer of callus is gradually reduced within 48 hours after transfer to SIM (Zhai and Xu, 2021). Accordingly, the ProWOX5:GFP signal is downregulated in a cytokinin concentration-dependent manner (Pernisova et al., 2018). Expression of LBD16, activated by WOX11, is also reduced after transfer to SIM (Liu et al., 2018b). Shoot-specific genes are activated at the same time as root-specific genes become attenuated. Of the transcription factors involved in shoot regeneration, WUS and STM play a key role in the initiation of shoot organogenesis (Hibara et al., 2003; Gordon et al., 2009; Chatfield et al., 2013). The shoot-specific ProWUS:tdTomato signal was detectable as soon as three days on cytokinin-rich media (Pernisova et al., 2018). Accordingly, wus mutants fail to regenerate shoots in vitro (Zhang et al., 2017b). Another member of the WOX family, WOX14 can be an important factor in shoot regeneration. A WOX14 overexpressor was able to regenerate shoots on cytokinin free medium. The ProWOX14:GUS reporter line exhibited WOX14 activity on SIM, but not on CIM (Wang et al., 2023a). However, the molecular mechanisms behind these results have yet to be uncovered.

The activity of general regulators requires the interplay and achievement of a fine balance of auxin and cytokinin function at several levels. For successful shoot regeneration, auxin must first trigger a program leading to the establishment of pluripotent status, while the presence of cytokinins modulates the organogenic response leading to shoot formation. Cytokinins quickly downregulate auxin signaling in a concentration-dependent manner, leading to a loss of root identity (Pernisova et al., 2018). The perturbation of auxin signaling was revealed to be crucial for the future induction of shoot regeneration on SIM (Ohbayashi et al., 2022). Auxin signaling can be affected by reducing the source, e.g., by decreased biosynthesis or disrupted transport. Cytokinin signaling suppresses the expression of auxin biosynthetic genes in the central region of the forming meristem, which can lead to decreased auxin levels ( Figure 3 ). The transcription factors ARR1, ARR10, and ARR12 interact with YUC promoters leading to the inhibition of YUC1 and YUC4 expression (Meng et al., 2017). Application of auxin biosynthesis inhibitors or polar auxin transport inhibitors on CIM enhanced subsequent shoot formation on SIM (Ohbayashi et al., 2022). Also, cytokinins affect auxin distribution by regulating the expression of auxin transporters from the PIN FORMED (PIN) family (Pernisová et al., 2009). CRFs acting downstream of cytokinin perception, participate in shoot regeneration as well. Shoot formation is induced in crf2 mutants and diminished in crf5 mutants during in vitro regeneration (Rashotte et al., 2006). The expression of CRF2 is dependent on a crucial auxin signaling component ARF5 (Ckurshumova et al., 2014), thus connecting cytokinin and auxin signaling pathways. The activity of ARF5 is negatively regulated by IAA12, and in the presence of auxin, ARF5 repression is compromised by the presence of ARF4, which competes with ARF5 in binding to IAA12. ARF4 compromises the level of free IAA12, thus maintaining the activity of ARF5 (Zhang et al., 2021). Another member of auxin signaling, ARF3 acts as a negative regulator of de novo organ regeneration. It binds directly to the promoter of IPT5, which disrupts the cytokinin biosynthesis pathway (Cheng et al., 2012). ARF3 binding to the IPT5 promoter requires high auxin concentration and is thus attenuated on SIM, leading to the activation of cytokinin biosynthesis. Taken together, fine-tuning between the effects of auxins and cytokinins is fundamental for proper plant organ regeneration.

The regulation of SAM formation during shoot regeneration requires WUS as a central player. WUS represents one of the first shoot-specific markers active in SAM-forming cells (Pernisova et al., 2018). Loss of WUS function leads to a severe disruption of shoot regeneration (Gordon et al., 2007; Chatfield et al., 2013). In the later stages of shoot regeneration, WUS activity can be observed only in the regions where the AHK4 cytokinin receptor was previously expressed during incubation on CIM (Gordon et al., 2009). Areas with an active AHK4 during CIM incubation also define the future positions of auxin transport protein PIN1 upregulated during SIM incubation (Gordon et al., 2007; Atta et al., 2009; Gordon et al., 2009). Application of the histidine kinase inhibitor TSCA during the first four days of SIM incubation leads to the inhibition of shoot regeneration in Arabidopsis. This inhibition seems to be at least partially connected to the disruption of cytokinin signaling via AHK4, AHK3, AHP3 and AHP5 (Lardon et al., 2022). Overall, AHK4 seems to be pivotal in the cytokinin-mediated regulation of organ identity affecting shoot-specific WUS as well as root-specific WOX5 activity (Pernisova et al., 2018).

Other members of the cytokinin signaling pathway – response regulators – are also involved in shoot regeneration. arr1 mutants generate more shoots than the wild type. It was shown that ARR1 binds to the IAA17 promoter, inducing its expression during SIM cultivation, leading to the indirect WUS inhibition and disruption of shoot regeneration (Liu et al., 2020). Another cytokinin-related transcription factor ARR12 promotes the CLV3 expression. Additionally, ARR1 causes repression of CLV3 in an ARR12 dependent manner. It was revealed that ARR1 and ARR12 compete in binding to the CLV3 promoter, which regulates its activity (Liu et al., 2020). Moreover, ARR12 positively regulates WUS by binding to its promoter and activating expression (Dai et al., 2017). Thus, ARR12 seems to be a critical player in shoot stem cell niche maintenance and shoot regeneration. Additionally, the ectopic expression of ARR2 and ARR11 gives rise to in vitro shoot regeneration in the absence of exogenous cytokinins (Hwang and Sheen, 2001; Imamura et al., 2003) as seen also with cytokinin-independent1 (CKI1) (Kakimoto, 1996).

On the other hand, the activity of type-A ARRs (negative regulators of cytokinin signaling) usually results in a reduction of shoot regeneration (Buechel et al., 2010). ARR1 binds directly to the promoters of type-A ARR5 and ARR7, which causes their repression. On SIM, ATXR2 is responsible for H3K336me3 deposition at ARR5 and ARR7 promoters thus disrupting ARR1 binding and activating type-A ARRs (Lee et al., 2021). ARR8 seems to also have an impact on shoot regeneration capacity. ARR8 overexpression abolishes SAM formation (Osakabe et al., 2002).

WUS and CLV3 activity and subsequent shoot regeneration are dependent also on abiotic factors, such as light or temperature. It was shown that ELONGATED HYPOCOTYL (HY5) a transcription factor of light signaling is responsible for inhibition of CLV3 and WUS by direct interaction with their promoter. Indirect inhibition of WUS and CLV3 is achieved through the interaction between HY5 and ARR12 (Dai et al., 2022). Temperature affects shoot regeneration competency by depleting the histone variant H2A.Z which acts as a repressor of de novo shoot organogenesis and is present at 17°C but removed at 27°C (Lambolez et al., 2022).

ALTERED MERISTEM PROGRAM 1 (AMP1) is a putative Glu carboxypeptidase present in Arabidopsis. Defective AMP1 results in the formation of enlarged shoot meristems, a larger stem cell pool and a higher rate of leaf formation (Huang et al., 2015). AMP1 affects stem cell niche patterning by controlling the downstream HD-ZIPIII/RAP2.6 transcription factor module. RAP2.6L is under direct transcriptional control of HD-ZIP III transcription factors via their binding to the RAP2.6L promoter. The presence of HD-ZIP III is elevated in the amp1 mutant. AMP1, therefore, seems to limit SAM activity via miRNA-dependent control of HD-ZIP III transcription factors and subsequent RAP2.6L activity (Yang et al., 2018). The regulatory pathways of HD-ZIP III activity also comprise auxin activity. A novel chemical inhibitor of polar auxin transport ZIC2 (LITTLE ZIPPER3 inducing compound) promotes shoot regeneration and subsequent RAP2.6L elevation in an HD-ZIP III-dependent manner (Yang et al., 2022). Moreover, previous research suggests that oxygen also works as a signal, as its spatial distribution affects the proteolysis of LITTLE ZIPPER 2 (ZPR2), controlling the activity of HD-ZIP III transcription factors (Weits et al., 2019). These findings together lift the lid on the exquisite complexity of the HD-ZIP III transcription factor regulatory network in the process of shoot regeneration.