2. LRP positioning is affected by CK

Densities of LRPs, emerged LRs and total LRs are frequently quantified traits that help characterise the root system architecture. Inhibition of LR formation by exogenous CK treatments was shown to be dose-dependent (Chang et al., Reference Chang, Ramireddy and Schmulling2013). Ubiquitous ectopic expression of genes involved in CK biosynthesis and metabolism, such as IPT, LOG and CKX, have independently confirmed this aspect of CK action (Kuderova et al., Reference Kuderova, Urbankova, Valkova, Malbeck, Brzobohaty, Nemethova and Hejatko2008; Kuroha et al., Reference Kuroha, Tokunaga, Kojima, Ueda, Ishida, Nagawa, Fukuda, Sugimoto and Sakakibara2009; Werner et al., Reference Werner, Motyka, Laucou, Smets, Van Onckelen and Schmülling2003).

More targeted manipulation of endogenous CK levels with the use of GAL4-GFP enhancer trap lines, which can drive the expression of target genes in defined plant tissues, provided further insights into where this spatial control occurred. By employing J0121 GAL4-GFP enhancer trap line, Laplaze et al. (Reference Laplaze, Benkova, Casimiro, Maes, Vanneste, Swarup, Weijers, Calvo, Parizot, Herrera-Rodriguez, Offringa, Graham, Doumas, Friml, Bogusz, Beeckman and Bennett2007) observed decreased LRP density in plants expressing an Agrobacterium IPT in XPP cells. These results were later partially confirmed, with the phenotypic inconsistencies attributed to the different growth conditions used in the two studies. Specifically, Bielach et al. (Reference Bielach, Podlesakova, Marhavy, Duclercq, Cuesta, Muller, Grunewald, Tarkowski and Benkova2012) first detected GFP signal in XPP cells that had already left the basal meristem of the same J0121 line, which likely explained the lack of an LRP density phenotype due to delayed expression of the Agrobacterium IPT gene. In addition, they reported the clear expression of GFP in LRPs of the J0121 line (Bielach et al., Reference Bielach, Podlesakova, Marhavy, Duclercq, Cuesta, Muller, Grunewald, Tarkowski and Benkova2012). This shows that care should be taken when using such driver lines (or conventional promoter fragments), as expression patterns may be considerably affected by growth conditions. Based on IPT expression in other GAL4-GFP enhancer trap lines, Bielach et al. (Reference Bielach, Podlesakova, Marhavy, Duclercq, Cuesta, Muller, Grunewald, Tarkowski and Benkova2012) found that regardless of the targeted tissue, strong IPT induction in the basal meristem most efficiently decreased LRP density. Such responsiveness to CK signalling early in the root was not surprising, as root tips had been proposed to integrate information about soil quality and accordingly adjust the frequency of LR initiation events in order to optimise the uptake of water and nutrients (Xuan et al., Reference Xuan, Band, Kumpf, Van Damme, Parizot, De Rop, Opdenacker, Moller, Skorzinski, Njo, De Rybel, Audenaert, Nowack, Vanneste and Beeckman2016). It is yet to be demonstrated whether the resulting iP ribotides from different tissues in the basal meristem act in a non-cell autonomous way or become immediately converted to the active CK bases that then travel and trigger cellular responses in XPP cells.

Auxin efflux carrier PIN-FORMED 7 (PIN7) is a potential downstream regulator of CK effects since its expression was reported to be upregulated in response to exogenous CK treatment and pin7 mutants displayed an increased density of LRP initiation compared to wild type plants (Benkova et al., Reference Benkova, Michniewicz, Sauer, Teichmann, Seifertova, Jurgens and Friml2003; Bishopp et al., Reference Bishopp, Help, El-Showk, Weijers, Scheres, Friml, Benkova, Mahonen and Helariutta2011; Marhavy et al., Reference Marhavy, Vanstraelen, De Rybel, Zhaojun, Bennett, Beeckman and Benkova2013; Ruzicka et al., Reference Ruzicka, Simaskova, Duclercq, Petrasek, Zazimalova, Simon, Friml, Van Montagu and Benkova2009; Simaskova et al., Reference Simaskova, O'Brien, Khan, Van Noorden, Otvos, Vieten, De Clercq, Van Haperen, Cuesta, Hoyerova, Vanneste, Marhavy, Wabnik, Van Breusegem, Nowack, Murphy, Friml, Weijers, Beeckman and Benkova2015). PIN7 expression was detected in the central root cap, stele and LRP initials, and the frequency of FC specification was perturbed in pin7 plants (Marhavy et al., Reference Marhavy, Vanstraelen, De Rybel, Zhaojun, Bennett, Beeckman and Benkova2013; Simaskova et al., Reference Simaskova, O'Brien, Khan, Van Noorden, Otvos, Vieten, De Clercq, Van Haperen, Cuesta, Hoyerova, Vanneste, Marhavy, Wabnik, Van Breusegem, Nowack, Murphy, Friml, Weijers, Beeckman and Benkova2015). CYTOKININ RESPONSE FACTORS (CRFs), which mediate transcriptional control downstream of CK, regulate PINs via a specific PIN CYTOKININ RESPONSE ELEMENT (PCRE) domain. Removal of the PCRE from the PIN7 promoter reduced the sensitivity of LR initiation and development to exogenous CK (Simaskova et al., Reference Simaskova, O'Brien, Khan, Van Noorden, Otvos, Vieten, De Clercq, Van Haperen, Cuesta, Hoyerova, Vanneste, Marhavy, Wabnik, Van Breusegem, Nowack, Murphy, Friml, Weijers, Beeckman and Benkova2015). In contrast to these observations, another study found that CK treatment significantly reduces the expression of PIN7 in the vascular tissue through induction of SHY2 via the AHK3/ARR1 two-component signalling pathway (Dello Ioio et al., Reference Dello Ioio, Nakamura, Moubayidin, Perilli, Taniguchi, Morita, Aoyama, Costantino and Sabatini2008). Additional studies will be required to better understand these opposite results.

3. CK regulates LR initiation and spacing

Although mutants affected in CK biosynthesis may not display a strongly increased LRP density compared to wild type, they often exhibit more closely spaced LRPs and recruit more LRFCs upon auxin treatment, suggesting that CK also affects LR formation in the differentiation zone (Bielach et al., Reference Bielach, Podlesakova, Marhavy, Duclercq, Cuesta, Muller, Grunewald, Tarkowski and Benkova2012; Chang et al., Reference Chang, Ramireddy and Schmulling2015). Using the fluorescent CK-signalling reporter TCS::GFP (Muller & Sheen, Reference Muller and Sheen2008) to monitor CK responses in cells and tissues involved in LR organogenesis, no fluorescence signal was observed in the root zone corresponding to the developmental window, encompassing LRFCs and Stages I–III. The expression of TCS::GFP was enhanced in XPP cells in between already initiated LRPs (Figure 1). This response was correlated with LR formation, since TCS expression could only be observed in the few pericycle cells adjacent to an already initiated LRP (Bielach et al., Reference Bielach, Podlesakova, Marhavy, Duclercq, Cuesta, Muller, Grunewald, Tarkowski and Benkova2012; Marhavy et al., Reference Marhavy, Duclercq, Weller, Feraru, Bielach, Offringa, Friml, Schwechheimer, Murphy and Benkova2014). It has been hypothesised that the CK molecules perceived by these cells originated both from themselves and the neighbouring LRP, and that this CK signalling prevented them from entering the LR developmental programme, that is, CK could be a positional cue regulating LR spacing (Bielach et al., Reference Bielach, Podlesakova, Marhavy, Duclercq, Cuesta, Muller, Grunewald, Tarkowski and Benkova2012; Chang et al., Reference Chang, Ramireddy and Schmulling2015). We would also like to point out that an improved version of the CK reporter, TCS new (TCSn), was developed, which was reported to be more sensitive to phosphorelay signalling in Arabidopsis. Plant expressing TCSn::GFP showed stronger GFP fluorescence in the root meristem and vasculature compared to TCS::GFP, whereas their expression patterns were qualitatively very similar during the LR development (Zürcher et al., Reference Zürcher, Tavor-Deslex, Lituiev, Enkerli, Tarr and Muller2013). However, a study comparing the two CK reporters in the LR developmental window has not been reported.

Figure 1. Schematic illustration of the expression of cytokinin (CK)-signalling reporters in primary root and an overview of CK metabolism, transport and signalling in a cell of Stage I lateral root primordium (LRP). Fluorescent CK-signalling reporters TCS::GFP and TCSn::ntd Tomato were used to monitor CK responses in primary root (Bielach et al., Reference Bielach, Podlesakova, Marhavy, Duclercq, Cuesta, Muller, Grunewald, Tarkowski and Benkova2012; Marhavy et al., Reference Marhavy, Duclercq, Weller, Feraru, Bielach, Offringa, Friml, Schwechheimer, Murphy and Benkova2014; Montesinos et al., Reference Montesinos, Abuzeineh, Kopf, Juanes-Garcia, Otvos, Petrasek, Sixt and Benkova2020). Here, we focus on a stage I LRP to illustrate a scenario where a cell that expresses CK receptors and biosynthesis genes might suppress CK signalling and serve as a potential source of the hormone to neighbouring pericycle cells. Plasmodesmata represent one of the potential ways through which CK might reach the neighbouring cells, although more research is needed to confirm such mode of CK transport. ARABIDOPSIS HISTIDINE KINASES (AHKs) are depicted at both the endoplasmatic reticulum (ER) and plasma membranes, although the latter localization has yet to be shown in the LRP cells. In the case of CK binding, AHKs can be autophosphorylated at a conserved His residue (H) and this phosphate is then carried over to a conserved Asp (D). Exact downstream pathways that could mediate transcriptional dependent and -independent effects of CK signalling in stage I LRPs are unknown. Arrows with a solid line indicate chemical reaction, arrows with a dashed line indicate movement. Additional abbreviations: LR, lateral root; AHP, ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN; ARR-A/B, type-A/B ARABIDOPSIS RESPONSE REGULATOR; PUP, PURINE PERMEASE; CKX, CYTOKININ OXIDASE; IPT, ADENOSINE PHOSPHATE-ISOPENTENYLTRANSFERASE; LOG, LONELY GUY; ADP, adenosine diphosphate; ATP, adenosine triphosphate. The drawing of the primary root is a modified version from Peret (Reference Peret2017), while the illustration of CK signalling circuitry is modified from on a published schematic (Hwang et al., Reference Hwang, Sheen and Muller2012).

In untreated plants, LR initiation is restricted to a relatively narrow developmental window, implying that there is a regulatory mechanism to control such patterning. These unknown regulatory mechanisms operate within the zone distal to the youngest LRP and determine the competence of XPP cells to launch the LR developmental programme (Dubrovsky et al., Reference Dubrovsky, Gambetta, Hernandez-Barrera, Shishkova and Gonzalez2006). When wild type plants were treated with 1-naphthaleneacetic acid (NAA; 1μM) for 25 hours, LRP initiation was induced between already recruited LRFCs and stage I LRPs, that is, additional initiation events were limited mainly to the early differentiation zone. Interestingly, in the ipt3,ipt5,ipt7 triple mutant, the same auxin treatment induced LRP initiation along the whole root. Similar results were observed in the arr1,arr11 double mutant, which lacks two B-type ARRs that mediate the signal transfer downstream of the CK receptors and were expressed in the pericycle and elsewhere in roots. In this experiment, Bielach et al. (Reference Bielach, Podlesakova, Marhavy, Duclercq, Cuesta, Muller, Grunewald, Tarkowski and Benkova2012) also found that in the arr1,arr11 double mutant, 11.6% of LRPs were misplaced (two LRPs separated by one pericycle cell) compared to 1.8% in wild type. Thus, they proposed that CK activity in pericycle cells might be important to prevent LR initiation outside the early differentiation zone and in close proximity to existing LRPs. In agreement with this hypothesis, Chang et al. (Reference Chang, Ramireddy and Schmulling2013) reported that LRP and LR density showed an increase in response to auxin treatment in the ahk2,ahk3 double mutant compared to wild type plants.

The same authors published a model predicting that the suppression of LR initiation in XPP cells neighbouring existing LRFCs was a combined effect of local CK synthesis and by the neighbouring cells (Chang et al., Reference Chang, Ramireddy and Schmulling2015). Of the genes involved in CK biosynthesis, IPT5 and LOG4 were shown to be expressed in most cells of LRPs, from Stage I until emergence (Figure 1) (Chang et al., Reference Chang, Ramireddy and Schmulling2015). Therefore, LRPs themselves could be a source of iP nucleobases. Besides LRPs and XPP cells, LOG4 was also expressed in the xylem and xylem pole endodermal cells in the root tip (De Rybel et al., Reference De Rybel, Adibi, Breda, Wendrich, Smit, Novak, Yamaguchi, Yoshida, Van Isterdael, Palovaara, Nijsse, Boekschoten, Hooiveld, Beeckman, Wagner, Ljung, Fleck and Weijers2014). log4 mutants displayed more abnormally positioned LRPs, separated by zero, one or two XPP cells, compared to wild-type roots. LRP spacing was not significantly altered in ipt5 mutants, probably due to a higher degree of functional redundancy in this gene family. In addition to LRPs, IPT5 was also shown to be expressed in the pericycle (Chang et al., Reference Chang, Ramireddy and Schmulling2015). Other root-expressed members of this gene family are IPT3, expressed in the phloem and pericycle, and IPT7, which was expressed both in the endodermis in the elongation zone and in the phloem (Miyawaki et al., Reference Miyawaki, Matsumoto-Kitano and Kakimoto2004; Takei et al., Reference Takei, Ueda, Aoki, Kuromori, Hirayama, Shinozaki, Yamaya and Sakakibara2004b). ipt3,ipt5 double mutants had almost as many abnormally positioned LRPs as observed in log4 mutants, and this phenotype was further enhanced in roots of ipt3,ipt5,ipt7 mutants. Interestingly, LR spacing phenotype was also observed in a cyp735a2 mutant. CYP735A2 is expressed in the phloem and pericycle, but not in LRPs, and converts the products of IPT enzymes into the tZ ribotides (Chang et al., Reference Chang, Ramireddy and Schmulling2015; Takei et al., Reference Takei, Yamaya and Sakakibara2004a).

Considering that these CK biosynthesis genes have an expression pattern that is not restricted to LRPs or the immediately surrounding XPP cells, it can only be speculated about the mechanisms that prevent LR initiation in cells surrounding newly recruited LRFCs. Moreover, the observed abnormal LR positioning phenotypes in log4, ipt3,ipt5,ipt7 and cyp735a2 mutants were very subtle and had a low penetrance, that is, the majority of LRPs did not have abnormal positions compared to wild-type roots. Thus, although important knowledge has been gathered, additional experiments are required to decipher the molecular mechanism underlying CK-mediated LR spacing. Using the DR5pro::LUCIFERASE system, it would be interesting to see whether the altered spacing is already visible when pre-branching sites are specified, or if this is caused by a signalling event that occurs later in the root. Other pathways controlling LRP spacing were recently reviewed by Cavallari et al. (Reference Cavallari, Artner and Benkova2021).

Besides restricting the ability of XPP cells to launch the LR developmental programme, CK signalling in XPP cells might also be required for proper secondary growth establishment. In Arabidopsis roots, secondary development is organised by two meristems: vascular cambium and phellogen/cork cambium (Ragni & Greb, Reference Ragni and Greb2018). The first originates from procambium and the XPP, and the latter from the entire pericycle, although the first formative divisions occur in XPP cells and then extend to the rest of the pericycle (Smetana et al., Reference Smetana, Makila, Lyu, Amiryousefi, Sanchez Rodriguez, Wu, Sole-Gil, Leal Gavarron, Siligato, Miyashima, Roszak, Blomster, Reed, Broholm and Mahonen2019; Wunderling et al., Reference Wunderling, Ripper, Barra-Jimenez, Mahn, Sajak, Targem and Ragni2018). In the ipt3,ipt5,ipt7 triple mutant, which showed LR formation along the whole root upon auxin treatment, secondary root growth was abolished and could be recovered by addition of tZ, in a dose-dependent manner (Bielach et al., Reference Bielach, Podlesakova, Marhavy, Duclercq, Cuesta, Muller, Grunewald, Tarkowski and Benkova2012; Matsumoto-Kitano et al., Reference Matsumoto-Kitano, Kusumoto, Tarkowski, Kinoshita-Tsujimura, Vaclavikova, Miyawaki and Kakimoto2008). Smetana et al. (Reference Smetana, Makila, Lyu, Amiryousefi, Sanchez Rodriguez, Wu, Sole-Gil, Leal Gavarron, Siligato, Miyashima, Roszak, Blomster, Reed, Broholm and Mahonen2019) proposed that a mobile xylem derived signal induced stem-cell identity in the neighbouring cells that give rise to the vascular cambium and that CKs could be these mobile stem-cell-promoting factors. Establishment of the vascular cambium is a prerequisite for XPP cells to be able to start dividing and contributing to radial thickening of the root. It was suggested that a non-cell-autonomous signal triggered by the cambium could render XPP competent to become a secondary meristem (Xiao et al., Reference Xiao, Molina, Wunderling, Ripper, Vermeer and Ragni2020). It could be speculated whether by limiting the developmental window in which XPP cells could be recruited to form LRPs, CKs also enable XPP cells to transition into a new role as secondary meristems.

4. Multiple roles of CK in the LRP outgrowth

The pace of LR development can vary even in two successively initiated primordia. LR initiation, meristem establishment and LR emergence represent growth-control points that can be regulated independently, which provides an additional level of plasticity during the root system development (Dubrovsky et al., Reference Dubrovsky, Gambetta, Hernandez-Barrera, Shishkova and Gonzalez2006). Exogenous CK treatment inhibited LR initiation and arrested growth of LRs at Stages I–IV, while an increase in endogenous CKs resulted in plants with no LRs developed past Stage IV or V (Bielach et al., Reference Bielach, Podlesakova, Marhavy, Duclercq, Cuesta, Muller, Grunewald, Tarkowski and Benkova2012; Chang et al., Reference Chang, Ramireddy and Schmulling2013; Kuderova et al., Reference Kuderova, Urbankova, Valkova, Malbeck, Brzobohaty, Nemethova and Hejatko2008; Laplaze et al., Reference Laplaze, Benkova, Casimiro, Maes, Vanneste, Swarup, Weijers, Calvo, Parizot, Herrera-Rodriguez, Offringa, Graham, Doumas, Friml, Bogusz, Beeckman and Bennett2007; Li et al., Reference Li, Mo, Shou and Wu2006).

Although LRPs could produce active iP nucleobases and express the CK receptors AHK2, AHK3 and CRE1/AHK4 already at Stage  I, the CK response in this developing organ was reported to be activated only from Stage III and onwards, based on the absence of TSC::GFP signal (Figure 1) (Marhavy et al., Reference Marhavy, Duclercq, Weller, Feraru, Bielach, Offringa, Friml, Schwechheimer, Murphy and Benkova2014). The endoplasmic reticulum (ER) membrane is the principal CK perception site, but a recent study showed that in meristematic cells, pools of ER-located AHK4 could enter the secretory pathway and reach the plasma membrane (Kubiasova et al., Reference Kubiasova, Montesinos, Samajova, Nisler, Mik, Semeradova, Plihalova, Novak, Marhavy, Cavallari, Zalabak, Berka, Dolezal, Galuszka, Samaj, Strnad, Benkova, Plihal and Spichal2020). In parallel, experiments on protoplasts isolated from Arabidopsis roots confirmed CK perception by plasma membrane localised AHK3 and AHK4. Moreover, the CK free bases were detected in apoplastic fractions from roots (Antoniadi et al., Reference Antoniadi, Novak, Gelova, Johnson, Plihal, Simersky, Mik, Vain, Mateo-Bonmati, Karady, Pernisova, Plackova, Opassathian, Hejatko, Robert, Friml, Dolezal, Ljung and Turnbull2020). It was hypothesised that the plasma membrane localised receptors might activate distinct branches of downstream signalling compared to the CK receptors localised in the ER, which would enable control of specific processes in meristematic cells (Kubiasova et al., Reference Kubiasova, Montesinos, Samajova, Nisler, Mik, Semeradova, Plihalova, Novak, Marhavy, Cavallari, Zalabak, Berka, Dolezal, Galuszka, Samaj, Strnad, Benkova, Plihal and Spichal2020). Thus, it is probable that the suppression of CK signalling in young LRPs is a result of both intracellular AHP6 and extracellular CKX6 (Figure 1) (Andersen et al., Reference Andersen, Naseer, Ursache, Wybouw, Smet, De Rybel, Vermeer and Geldner2018; Chang et al., Reference Chang, Ramireddy and Schmulling2013; Moreira et al., Reference Moreira, Bishopp, Carvalho and Campilho2013; Werner et al., Reference Werner, Motyka, Laucou, Smets, Van Onckelen and Schmülling2003). Even though LRP density and distribution of different stages were not significantly altered in ahp6 mutants compared to wild type, there were defects in the orientation of cell divisions in early stages of LR development. It was proposed that AHP6, through repression of CK signalling, enabled a correct localization of auxin efflux carrier PIN1 and thus the formation of the auxin gradient that is required for LRP patterning (Moreira et al., Reference Moreira, Bishopp, Carvalho and Campilho2013). While the suppression of CK signalling was needed in the early stages of LR development, from Stage III onward, proper establishment of PIN1 polarity required an intact CK perception. It was shown that independently of transcription, CK enhanced endocytic recycling of PIN1 at specific polar domains, thereby directing the auxin flux towards the tip of LRPs and promoting their development. This CK-mediated control of auxin distribution was evident in plants treated with exogenous CKs, which caused rapid and excessive depletion of plasma membrane-localised PIN1, leading to an arrest of LR development (Marhavy et al., Reference Marhavy, Bielach, Abas, Abuzeineh, Duclercq, Tanaka, Parezova, Petrasek, Friml, Kleine-Vehn and Benkova2011; Marhavy et al., Reference Marhavy, Duclercq, Weller, Feraru, Bielach, Offringa, Friml, Schwechheimer, Murphy and Benkova2014). Based on the reported specific expression of CKX6 in Stage I LRPs, it would be interesting to investigate the contribution of this gene to the LR development and spacing (Chang et al., Reference Chang, Ramireddy and Schmulling2015).

TRANSPORTER OF IBA1 (TOB1) was recently identified as a tonoplast-localized transporter of the auxin precursor indole-3-butyric acid (IBA) and was shown to limit LR formation. CK treatment resulted in the expansion of TOB1 expression from LRP-flanking regions to the whole primordium, suggesting an additional pathway of CK-mediated regulation of LRP organogenesis (Michniewicz et al., Reference Michniewicz, Ho, Enders, Floro, Damodaran, Gunther, Powers, Frick, Topp, Frommer and Strader2019). The cell cycle could also be a potential target of CK action in LRP outgrowth, since a high concentration of exogenous CK caused an arrest of LRFCs at the G2/M phase (Li et al., Reference Li, Mo, Shou and Wu2006). Transcription of genes specific for the G2/M phase was reported to be mediated by two R1R2R3-MYB transcriptional activators, MYB3R1 and MYB3R4. The corresponding myb3r1,myb3r4 double mutant has fewer cells in the shoot and root apical meristems (SAM and RAM). However, their expression is not limited to just these two tissues, for example, both genes are also expressed in the pericycle (Haga et al., Reference Haga, Kato, Murase, Araki, Kubo, Demura, Suzuki, Muller, Voss, Jurgens and Ito2007). It has been recently shown that CK directly promotes nuclear trafficking of MYB3R4 to activate mitotic cell cycle-related gene expression in the SAM. Interestingly, while CK treatment increased the number of cells in the epidermal (L1) layer of wild-type SAMs, the same CK treatment led to a premature SAM termination or reduction of cells in the L1 layer in the myb3r1,myb3r4 double mutant compared to the control (Yang et al., Reference Yang, Cortijo, Korsbo, Roszak, Schiessl, Gurzadyan, Wightman, Jonsson and Meyerowitz2021). It would be interesting to see if these genes are expressed in LRPs and whether MYB3R4 could also modulate CK signalling responses during LR development.

In contrast to the exogenous CK treatments, manipulation of endogenous CK levels suggested that, unlike XPP cells, young LRPs are not as sensitive to this hormone (Laplaze et al., Reference Laplaze, Benkova, Casimiro, Maes, Vanneste, Swarup, Weijers, Calvo, Parizot, Herrera-Rodriguez, Offringa, Graham, Doumas, Friml, Bogusz, Beeckman and Bennett2007). Increased CK levels in the early stages of LRP development caused LR growth defects in some GAL4-GFP enhancer trap lines, while they were absent in others (Bielach et al., Reference Bielach, Podlesakova, Marhavy, Duclercq, Cuesta, Muller, Grunewald, Tarkowski and Benkova2012; Laplaze et al., Reference Laplaze, Benkova, Casimiro, Maes, Vanneste, Swarup, Weijers, Calvo, Parizot, Herrera-Rodriguez, Offringa, Graham, Doumas, Friml, Bogusz, Beeckman and Bennett2007). Since they used an Agrobacterium IPT that can catalyse the first step in CK synthesis, the subsequent activity of LOG enzymes would still be required to synthesise active nucleobases. Therefore, LOG4 levels in LRP might have acted as a bottle-neck of CK synthesis in these experiments, possibly masking developmental defects. Additionally, cell cycle arrest and PIN1 depletion upon exogenous CK treatment were absent in cre1/ahk4 mutants (Li et al., Reference Li, Mo, Shou and Wu2006; Marhavy et al., Reference Marhavy, Bielach, Abas, Abuzeineh, Duclercq, Tanaka, Parezova, Petrasek, Friml, Kleine-Vehn and Benkova2011). Considering that a portion of CRE1/AHK4 receptors has been shown to reside on the plasma membrane of meristematic cells and might activate distinct branches of signalling downstream CK perception compared to those localised to the ER, it is possible that young LRPs were insensitive to endogenously increased CK levels because the produced CKs remained inside the cells. Our knowledge about the molecular mechanisms involved in CK transport is still incomplete, although potential efflux and influx transporters have been identified (Duran-Medina et al., Reference Duran-Medina, Diaz-Ramirez and Marsch-Martinez2017; Liu et al., Reference Liu, Zhao and Zhang2019; Nedved et al., Reference Nedved, Hosek, Klima and Hoyerova2021).

The only characterised CK efflux carrier facilitating the transport of CK across the plasma membrane is ATP-BINDING CASSETTE G14 (ABCG14), which is expressed primarily in the pericycle and stele of roots, but it was not revealed whether ABGC14 is also expressed in LRPs. Depending on the study, abcg14 mutant seedlings had either shorter or longer primary roots compared to wild type, without differences in LRP densities being reported (Ko et al., Reference Ko, Kang, Kiba, Park, Kojima, Do, Kim, Kwon, Endler, Song, Martinoia, Sakakibara and Lee2014; Zhang et al., Reference Zhang, Novak, Wei, Gou, Zhang, Yu, Yang, Cai, Strnad and Liu2014). Other genes implicated in CK transport are PURINE PERMEASES (PUPs), AZA-GUANINE RESISTANT (AZG) family and EQUILIBRATIVE NUCLEOSIDE TRANSPORTERS (ENTs) (Burkle et al., Reference Burkle, Cedzich, Dopke, Stransky, Okumoto, Gillissen, Kuhn and Frommer2003; Gillissen et al., Reference Gillissen, Burkle, Andre, Kuhn, Rentsch, Brandl and Frommer2000; Hirose et al., Reference Hirose, Takei, Kuroha, Kamada-Nobusada, Hayashi and Sakakibara2008; Tessi et al., Reference Tessi, Brumm, Winklbauer, Schumacher, Pettinari, Lescano, Gonzalez, Wanke, Maurino, Harter and Desimone2021a; Reference Tessi, Shahriari, Maurino, Meissner, Novak, Pasternak, Schumacher, Flubacher, Nautscher, Williams, Kazimierczak, Strnad, Thumfart, Palme, Desimone and Teale2021b; Zürcher et al., Reference Zürcher, Liu, di Donato, Geisler and Muller2016). Besides free CKs, members of the first two families could transport other purines, while ENTs were able to transport nucleosides as well as CK ribosides. Of the three characterised PUPs, which all demonstrated direct CK uptake activity in different assays, only PUP14 was reported to be expressed in LRPs, localizing to the plasma membrane (Figure 1) (Burkle et al., Reference Burkle, Cedzich, Dopke, Stransky, Okumoto, Gillissen, Kuhn and Frommer2003; Zürcher et al., Reference Zürcher, Liu, di Donato, Geisler and Muller2016). Inducible knockdown of PUP14 expression resulted in shorter roots and suppressed LR formation (Zürcher et al., Reference Zürcher, Liu, di Donato, Geisler and Muller2016). It would be important to use the CRISPR-Cas9 technology to generate pup14 knockout mutants and check whether these plants show similar phenotypes. Likewise, it would be interesting to elucidate the expression patterns of other members of the PUP family in the context of LR formation and to reveal their subcellular localization, as it was recently reported that two rice PUPs localise to the ER (Xiao et al., Reference Xiao, Liu, Zhang, Gao, Liu, Xu, Che, Wang, Tong and Chu2019; Xiao et al., Reference Xiao, Molina, Wunderling, Ripper, Vermeer and Ragni2020). The AZG family has only two members in Arabidopsis, AZG1 expressed in vasculature of already emerged LRs, and AZG2 expressed in a few cortical and epidermal cells overlying LRPs (Tessi et al., Reference Tessi, Brumm, Winklbauer, Schumacher, Pettinari, Lescano, Gonzalez, Wanke, Maurino, Harter and Desimone2021a; Reference Tessi, Shahriari, Maurino, Meissner, Novak, Pasternak, Schumacher, Flubacher, Nautscher, Williams, Kazimierczak, Strnad, Thumfart, Palme, Desimone and Teale2021b). AZG2 was proposed to function as a CK diffusion facilitator functioning in both transport directions across ER and plasma membranes. However, it was not shown to which compartment AGZ2 predominantly localises in the cells overlying LRPs, as only transcriptional reporters were used to study the expression during LR emergence. azg2 roots had a slightly increased number of Stages VI to VIII LRP compared to control plants, but this difference was not significant (Tessi et al., Reference Tessi, Brumm, Winklbauer, Schumacher, Pettinari, Lescano, Gonzalez, Wanke, Maurino, Harter and Desimone2021a). With regard to the ENT family, ENT3 and ENT6 could participate in CK riboside transport, with both genes being expressed in the root vasculature (Hirose et al., Reference Hirose, Takei, Kuroha, Kamada-Nobusada, Hayashi and Sakakibara2008; Korobova et al., Reference Korobova, Kuluev, Mohlmann, Veselov and Kudoyarova2021). Finally, naturally occurring CKs possess relatively hydrophobic side-chains and their diffusion from the cytoplasm to the apoplast cannot be ruled out (Nedved et al., Reference Nedved, Hosek, Klima and Hoyerova2021).

Considering our incomplete knowledge on CK transport and different branches of CK signalling in young LRPs, discrepancies between experiments employing exogenous CK treatments and those manipulating endogenous CK levels cannot be easily resolved. Another proposed explanation concerns the fact that while the affinity of AHKs to their ligands is around 1–40 nM, relatively high CK concentrations (more than 50 nM 6-benzylaminopurine) were required to cause a strong cell cycle inhibition and altered cellular patterning in LRPs (Chang et al., Reference Chang, Ramireddy and Schmulling2013; Lomin et al., Reference Lomin, Krivosheev, Steklov, Arkhipov, Osolodkin, Schmulling and Romanov2015). Moreover, the concentrations of iP and tZ in root extracts were in the range from 0.4 to 2.6 pmol/g fresh weight (Svacinova et al., Reference Svacinova, Novak, Plackova, Lenobel, Holik, Strnad and Dolezal2012). Thus, Chang et al. (Reference Chang, Ramireddy and Schmulling2013) suggested that the experiments employing high CK treatments might not be informative about the physiologically relevant CK effects. However, local CK concentration in LRPs could be higher than those measurements in root extracts, as CKs are unevenly distributed in organs at either the tissue or cellular level. More targeted measurements of CK concentration in LRPs, like what was achieved for different cell types in the root apex, could advance this discussion, assuming that isolation of single cells from differentiated tissues is feasible (Antoniadi et al., Reference Antoniadi, Plackova, Simonovik, Dolezal, Turnbull, Ljung and Novak2015).

5. CK signalling in the overlying endodermis

LR emergence starts with the first formative cell divisions leading to a Stage I LRP, as the newly formed organ needs to overcome the mechanical constraints imposed by the overlying cell layers. In order to make way for the emerging LRP, neighbouring endodermal cells lose volume and/or alter their shape and relinquish their tight junction-like diffusion barrier called the Casparian strip. These accommodating responses are crucial for LR development and are regulated by AUX/IAA SHORT HYPOCOTYL 2 (SHY2)-mediated auxin signalling in differentiated endodermal cells (Vermeer et al., Reference Vermeer, von Wangenheim, Barberon, Lee, Stelzer, Maizel and Geldner2014). It has been proposed that auxin coordinates reorganization of the cytoskeleton in the pericycle and endodermis, however, important knowledge on the remodelling of endodermis is still missing (Vilches Barro et al., Reference Vilches Barro, Stockle, Thellmann, Ruiz-Duarte, Bald, Louveaux, von Born, Denninger, Goh, Fukaki, Vermeer and Maizel2019). Interestingly, the CK signalling reporter, TCS::GFP, was shown to be also induced in the endodermal cells overlying the early-stage LRPs (Figure  1) (Bielach et al., Reference Bielach, Podlesakova, Marhavy, Duclercq, Cuesta, Muller, Grunewald, Tarkowski and Benkova2012). This suggests that the same cells that display SHY2-mediated auxin signalling, also experience CK signalling. In the transition zone of the primary root, it was reported that CK could antagonise auxin by inducing the expression of SHY2 (Dello Ioio et al., Reference Dello Ioio, Nakamura, Moubayidin, Perilli, Taniguchi, Morita, Aoyama, Costantino and Sabatini2008). However, it is unknown if similar auxin-cytokinin interactions exist in the endodermis during LR emergence. Recently, it was shown that CK affects the dynamics of the microtubule cytoskeleton and counteracts auxin-driven microtubule rearrangements in epidermal cells of the primary root (Montesinos et al., Reference Montesinos, Abuzeineh, Kopf, Juanes-Garcia, Otvos, Petrasek, Sixt and Benkova2020). If cortical microtubules contribute to the remodelling of endodermis, it would be interesting to see whether and how auxin and CK affect their organization and dynamics. For example, does CK signalling in the endodermis affect spatial accommodating responses by stabilising the microtubule cytoskeleton, or does it have a completely different role? The endodermal cells overlying LRPs also undergo dynamic suberisation. First, suberin is deposited, then it is removed and replaced by another polymer, cutin (Berhin et al., Reference Berhin, de Bellis, Franke, Buono, Nowack and Nawrath2019; Ursache et al., Reference Ursache, De Jesus Vieira Teixeira, Denervaud Tendon, Gully, De Bellis, Schmid-Siegert, Grube Andersen, Shekhar, Calderon, Pradervand, Nawrath, Geldner and Vermeer2021). However, unlike in the rest of the endodermis, suberisation around LR emergence sites was reported to be independent of CK signalling (Andersen et al., Reference Andersen, Naseer, Ursache, Wybouw, Smet, De Rybel, Vermeer and Geldner2018).

6. CK regulates angular growth of emerged LRs

The angle at which LRs emerge from the primary root is another root architectural trait that affects plant performance and was recently found to be regulated by CK. Upon emergence at a 90° angle, LRs start perceiving gravity and other environmental cues, like hypoxia, to establish the gravitropic set point angle (GSA). CK signalling at the upper flank of emerged LRs integrates these environmental signals and interferes with cellular elongation and proliferation through transcriptional regulators, such as CRF2 and CRF3. Hence, CK presents an anti-gravitropic component in emerged LRs that controls the radial expansion of the root system (Waidmann et al., Reference Waidmann, Ruiz Rosquete, Scholler, Sarkel, Lindner, LaRue, Petrik, Dunser, Martopawiro, Sasidharan, Novak, Wabnik, Dinneny and Kleine-Vehn2019). Interestingly, cold stress, which is known to decrease LR numbers and GSA, was also shown to induce the expression of CRF2 and CRF3 in primary roots, LRPs and emerged LRs. The cold signal for expressing CRF2 was mediated by a subset of the TCS system (AHK2, AHK3, AHPs, ARR1, ARR10 and ARR12), while CRF3 was upregulated via TCS-independent pathways. Both transcription factors were necessary for LR initiation in plants exposed to low temperatures, whereas their involvement in establishing GSA under cold stress were not reported (Jeon et al., Reference Jeon, Cho, Lee, Van Binh and Kim2016).

7. Concluding remarks

Auxin is a master regulator of LR organogenesis that can integrate a variety of environmental signals, thus coordinating the endogenous plant developmental programme with exogenous inputs from the environment (Cavallari et al., Reference Cavallari, Artner and Benkova2021). Nonetheless, it is clear that auxin is not the only crucial regulator, with CK affecting every step of LR development. To date, major progress has been made in our understanding of the CK-mediated control of developmental processes. Still, some questions remain open. Further insights could arise from the advances in imaging that allow quantification of shapes, volume and changes in the (an)isotropy of growing cells. To better understand CK transport during plant development, a similar biosensor to the recently described AuxSens that enables direct quantification of auxin in living cells would be a major breakthrough (Herud-Sikimic et al., Reference Herud-Sikimic, Stiel, Kolb, Shanmugaratnam, Berendzen, Feldhaus, Hocker and Jurgens2021). Since there is a strong interaction between CK and auxin signalling and responses, it is important to monitor their levels simultaneously when possible. It is likely that cellular decisions require a certain CK to auxin ratio. In addition, the use of cell type-specific promoters to specifically image or manipulate particular tissues will be instrumental to better understand how hormone signalling affects organ development. Likewise, these promoters are essential for the CRISPR system that generates tissue-specific knockouts, CRISPR-TSKO. This approach has already been proven efficient in creating somatic mutations in LRFCs resulting in LR-specific knock-outs of genes that also have roles in other developmental processes. Moreover, by linking it to promoters that are active a defined stages of LR development, or inducible expression systems, TSKO could be used to target genes at specific stages of LR development (Decaestecker et al., Reference Decaestecker, Buono, Pfeiffer, Vangheluwe, Jourquin, Karimi, Van Isterdael, Beeckman, Nowack and Jacobs2019). Uncovering new regulators of LR development could also be prompted by the high-throughput single-cell RNA sequencing methods of the root, although it could be a challenge to isolate single cells from differentiated tissues (Denyer et al., Reference Denyer, Ma, Klesen, Scacchi, Nieselt and Timmermans2019; Wang et al., Reference Wang, Ye, Lyu, Ursache, Loytynoja and Mahonen2020; Wendrich et al., Reference Wendrich, Yang, Vandamme, Verstaen, Smet, Van de Velde, Minne, Wybouw, Mor, Arents, Nolf, Van Duyse, Van Isterdael, Maere, Saeys and De Rybel2020).