STEM CELLS AND REGENERATION RESEARCH ARTICLE

Cytokinin signalling regulates organ identity via the AHK4 receptorin ArabidopsisMarketa Pernisova1,2,*,§, Martina Grochova1, Tomas Konecny1, Lenka Plackova3, Danka Harustiakova4,Tatsuo Kakimoto5, Marcus G. Heisler2,‡, Ondrej Novak3 and Jan Hejatko1,§

ABSTRACTMutual interactions of the phytohormones, cytokinins and auxindetermine root or shoot identity during postembryonic de novoorganogenesis in plants. However, our understanding of the role ofhormonal metabolism and perception during early stages of cell fatereprogramming is still elusive. Here we show that auxin activates rootformation, whereas cytokinins mediate early loss of the root identity,primordia disorganisation and initiation of shoot development.Exogenous and endogenous cytokinins influence the initiation ofnewly formed organs, as well as the pace of organ development.The process of de novo shoot apical meristem establishment isaccompanied by accumulation of endogenous cytokinins, differentialregulation of genes for individual cytokinin receptors, strong activationof AHK4-mediated signalling and induction of the shoot-specifichomeodomain regulator WUSCHEL. The last is associated withupregulation of isopentenyladenine-type cytokinins, revealing highershoot-forming potential when compared with trans-zeatin. Moreover,AHK4-controlled cytokinin signalling negatively regulates the rootstem cell organiserWUSCHEL RELATED HOMEOBOX 5 in the rootquiescent centre. We propose an important role for endogenouscytokinin biosynthesis and AHK4-mediated cytokinin signalling in thecontrol of de novo-induced organ identity.

KEY WORDS: Arabidopsis, Cytokinin metabolism,Cytokinin signalling, De novo organogenesis, Root, Shoot

INTRODUCTIONThe ability to undergo postembryonic de novo organogenesis is oneof the most important adaptation strategies of plants. The capacity toform new tissues and organs during postembryonic development ispresent in cells with high regeneration potential. These cells areunder the control of regulators that mediate specific spatiotemporalchanges in their highly responsive developmental programmes.Phytohormones, auxin and cytokinins have long been known to bekey factors controlling de novo organ formation from plant explants

(Skoog andMiller, 1957). In the presence of auxin alone or in mediawith a high auxin-to-cytokinin concentration ratio, growth of theroots is initiated from various plant tissues (Atta et al., 2009;Pernisová et al., 2009; Sugimoto et al., 2010). However, if theauxin-to-cytokinin ratio is reversed (i.e. if the cytokininconcentration is higher than that of auxin), shoots are formed(Skoog and Miller, 1957).

Recently, substantial progress has been achieved inunderstanding the molecular mechanisms underlying the ability ofplants to induce and respecify the identity of newly formed organs.In Arabidopsis, the two-step protocol for de novo organogenesis iswell established (Che et al., 2006; Sugimoto andMeyerowitz, 2013;Valvekens et al., 1988). In the first step, the competence of anexplant to regenerate organs is acquired by cultivation on auxin-richcallus-inducing medium (CIM). Importantly, the organ primordiainduced during the cultivation on CIM from the explants of theshoot origin share plenty of anatomical and molecular determinantswith roots and have been shown to be induced via the root-specificdevelopmental pathway (Atta et al., 2009; Sugimoto et al., 2010).Subsequent cultivation of the organogenesis-competent calli onauxin-supplemented root-inducing medium (RIM) promotesgrowth of roots. Compared with this, the high cytokinin contentin the shoot-inducing medium (SIM) promotes shoot regeneration(Atta et al., 2009; Kareem et al., 2015; Sugimoto et al., 2010;Valvekens et al., 1988). Alternatively, de novo shoot apicalmeristem formation can occur directly via redifferentiation ofauxin-induced lateral root primordia into shoot meristems bycytokinin application without intermediate callus formation(Chatfield et al., 2013; Kareem et al., 2016; Rosspopoff et al.,2017).

In plant postembryonic development, growth is largely controlledby the activity of apical (shoot and root) and lateral (procambium,cambium and axillary) meristems. Meristems maintain a pool oftotipotent (stem) cells that divide and differentiate, allowing for theformation of new plant organs and tissues (reviewed by Greb andLohmann, 2016). In the shoot apical meristem, the localisation ofundifferentiated cells in the stem cell niche is controlled by theexpression of homeodomain transcription factor WUSCHEL (WUS)in the organising centre (Mayer et al., 1998; Schoof et al., 2000).WUS function is essential for shoot apical meristem development(Gallois et al., 2004; Gordon et al., 2007) and wus mutants fail toregenerate shoots in vitro (Zhang et al., 2017). Similarly, the WUShomologue WUSCHEL RELATED HOMEOBOX 5 (WOX5)regulates root stem cell maintenance in the stem cell niche adjacentto the quiescent centre, an analogue of the shoot organising centre inthe root apicalmeristem and the location ofWOX5 expression (Blilouet al., 2005; Pi et al., 2015; Sarkar et al., 2007). Expression anddifferential regulation of the WUS gene family starts early duringembryogenesis (Haecker et al., 2004), and WUS and WOX5 can beconsidered as shoot- and root-specific reporters, respectively.Received 30 January 2018; Accepted 22 June 2018

1CEITEC – Central European Institute of Technology and Functional Genomics andProteomics, NCBR, Faculty of Science, Masaryk University, Brno 62500, CzechRepublic. 2European Molecular Biology Laboratory, Heidelberg 69117, Germany.3Laboratory of Growth Regulators, CRH, Institute of Experimental Botany AS CRand Faculty of Science of Palacký University, Olomouc 78371, Czech Republic.4Institute of Biostatistics and Analyses, Faculty of Medicine and ResearchCentre forToxic Compounds in the Environment, Faculty of Science, Masaryk University, Brno62500, Czech Republic. 5Department of Biological Sciences, Graduate School ofScience, Osaka University, Osaka 560-0043 , Japan.*Present address: Laboratoire Reproduction et Developpement des Plantes,ENS de Lyon, CNRS, Lyon 69364, France. ‡Present address: Faculty of Science,The University of Sydney, Sydney, NSW 2006, Australia.

§Authors for correspondence (pernisov@sci.muni.cz; hejatko@sci.muni.cz)

M.P., 0000-0002-5803-2879; J.H., 0000-0002-2622-6046

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Cytokinins control plant development by activating a multistepphosphorelay signalling pathway. Cytokinins recognise theCHASE domain of the sensor histidine kinases ARABIDOPSISHISTIDINE KINASE (AHK)2, AHK3 or AHK4/WOL/CRE1(Higuchi et al., 2004; Inoue et al., 2001; Mahonen et al., 2000;Nishimura et al., 2004; Ueguchi et al., 2001). The signal issubsequently transferred via small cytoplasmic ARABIDOPSISHISTIDINE-CONTAINING PHOSPHOTRANSFER proteins(AHPs) (Hutchison et al., 2006) to ARABIDOPSIS RESPONSEregulators (ARRs) (Suzuki et al., 1998; Tanaka et al., 2004). Type BARRs (ARRs-B) are GARP transcription factors controlling theexpression of cytokinin-regulated genes, including type-A ARRs(ARRs-A) (Mason et al., 2004; Sakai et al., 2001). The targetbinding sequence of ARRs-B served to develop the cytokinin-responsive two-component signalling (TCS) reporter (Müller andSheen, 2008; Zurcher et al., 2013). ARRs-A are cytokinin primaryresponse genes that are quickly upregulated by cytokinins even inthe absence of translation (Brandstatter and Kieber, 1998;D’Agostino et al., 2000). In parallel, ARRs-A inhibit thecytokinin signalling pathway, generating a negative-feedback loop(Hwang and Sheen, 2001; To et al., 2004).The levels of the two major types of endogenous cytokinins, N6-

(Δ2-isopentenyl)-adenine (iP) and trans-zeatin (tZ) are undercontrol of complex metabolic network. In Arabidopsis, the initialstep of cytokinin biosynthesis is catalysed by isopentenyltransferase(IPT), and involves the transfer of a prenyl moiety fromdimethylallyl diphosphate to adenosine triphosphate ordiphosphate to form iP ribotides (Kakimoto, 2001; Takei et al.,2001). iP ribotides could be hydroxylated by the cytochrome P450mono-oxygenases CYP735A1/CYP735A2, resulting in theformation of tZ-type cytokinins (Takei et al., 2004). Directconversion of cytokinin ribotides to active free bases is catalysedby a nucleoside 5′-monophosphate phosphoribohydrolase namedLONELYGUY (LOG) (Kurakawa et al., 2007; Kuroha et al., 2009).Besides the biosynthesis, the level of active cytokinins is regulatedby glycosylation and other conjugations of cytokinin bases. Thesemetabolites serve as storage, transport and inactivated cytokininforms. tZ-type cytokinins with a hydroxylated side chain can bereversibly glycosylated by the UGT85A1 enzyme (Hou et al.,2004). O-glycosylation is one of the first reactions to exogenouscytokinin application and produces non-active cytokinins that canbe re-activated by β-glucosidases (Brzobohaty et al., 1993).Cytokinin O-glucosides could serve as a storage pool and readilyavailable source of active free bases (Kiran et al., 2012). On theother hand, cytokinin glycosylation at the N7 and N9 positions ofthe adenine moiety catalysed by UGT76C1 and UGT76C2 enzymes(Hou et al., 2004; Wang et al., 2011) is irreversible, and therefore N-glucosides were proposed to represent a permanent deactivation inthe cytokinin lifetime (Parker and Letham, 1973). Irreversiblecytokinin degradation is catalysed by the cytokinin oxidase/dehydrogenase (CKX) (Schmülling et al., 2003) that cleavesunsaturated N6-side chains from tZ- and iP-type cytokinins (Jonesand Schreiber, 1997). Biosynthesis, modifications and degradationmaintain cytokinin homeostasis, balancing optimal phytohormonelevels during plant growth and development.Here, we present a model describing the role of endogenous

cytokinin biosynthesis and cytokinin signalling in the regulation ofde novo organ formation. Using the one-step hypocotyl explantassay, we demonstrate that cytokinins inhibit auxin-induced organestablishment and mediate the early disorganisation of organprimordia that is associated with switch in the primordia identity.We show that the de novo organogenesis is associated with

differential regulation of cytokinin metabolism and perception, andthat the effects of endogenous cytokinins are mediated via a specificcytokinin receptor to control de novo organ formation.

RESULTSCytokinin induces early root-to-shoot organ respecificationUsing a one-step protocol, it has been previously demonstrated thatauxin induces de novo root formation along hypocotyl explants,whereas cytokinins alone are not able to induce any organogenicresponse. However, cytokinins are able to modulate the auxin-induced organogenesis in a concentration-dependent manner(Pernisová et al., 2009), leading to shoot formation after long-term cultivation. Here, we focused on early events (the first 5 days ofcultivation), when the decision to form a root or a shoot takes place.Hypocotyls of 5-day-old etiolated Arabidopsis seedlings wereexcised and placed on media supplemented with a range ofphytohormone concentrations; cytokinins were represented bykinetin and auxins by 1-naphthaleneacetic acid (NAA). Two typesof cytokinin-containing media were used: K300, containing 100 ng/ml of NAA and 300 ng/ml of kinetin (the lowest tested cytokininconcentration that leads to the loss of root morphology of the auxin-induced organs; Pernisová et al., 2009); and K1000, with the sameNAA concentration (100 ng/ml) and 1000 ng/ml of kinetin. Usingour protocol, both K300 and K1000 were able to induce shootformation, the latter with higher efficiency (Fig. S1). The non-natural cytokinin kinetin (Kamínek, 2015) was selected to allow thepossible changes in endogenous cytokinin levels to be distinguishedduring the organogenic response. Moreover, CKX activity has beenfound to be upregulated in response to exogenous cytokininapplication (Gelová et al., 2018; Pernisová et al., 2009; Werneret al., 2006) and kinetin has been described as a poor substrate forCKX enzymes (Frébortová et al., 2004; Galuszka et al., 2007;Popelková et al., 2006). Thus, exogenously applied kinetin is notconsidered to be cleaved by endogenous CKX activity, or by CKX3in Pro35S:AtCKX3 line, allowing us to determine functionalimportance of differences in endogenous cytokinin levels duringthe organogenic response.

The auxin-induced primordia were recognised as early as the firstday of cultivation of hypocotyl explants on auxin-supplementedmedium (K0: kinetin 0 ng/ml+NAA 100 ng/ml; Fig. 1). Similar tothe results achieved when using a two-step protocol (Sugimoto et al.,2010), the root identity of the auxin-induced primordia in our one-step setup was confirmed by the early activation of the root-specificreporters PLETHORA (PLT) 2, PLT3 or SCARECROW (SCR) at day1 and WOX5 at day 2 (Fig. 1A, Figs S2, S3). In contrast, on thecytokinin-supplemented shoot-inducing media (K300, K1000), thesignal of ProWOX5:GFP as well as the other root-specific markerswas downregulated and became undetectable in media with highcytokinin concentration (K1000) latest from the day 3 onwards(Fig. 1A, Figs S2 and S3). Simultaneously, the root primordiachanged their morphology, losing their characteristic root primordiapattern. The shoot-specific ProWUS:tdTomato signal appearedsolely in disorganised primordia at the day 3 of cultivation(Fig. 1A), suggesting primordia identity respecification. NeitherWUS signal outside the organ primordia and/or in the well-organisedprimordia, nor its co-occurrencewithWOX5 signal was detectable inhypocotyl explants carrying both WOX5 and WUS reporters on allmedia tested. Well-differentiated shoots with newly formed leaveswere easily recognisable after 21 days of cultivation on media withboth cytokinin concentrations (K300 and K1000, Fig. S1).

To investigate auxin and cytokinin signalling outputs duringearly events leading to organ identity respecification, we prepared a

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line carrying the auxin signalling reporter pDR5rev::3XVENUS-N7(Wabnik et al., 2013) together with the cytokinin-responsive two-component signalling sensor TCSn::GFP (Zurcher et al., 2013)( pDR5rev::3XVENUS-N7/TCSn::GFP). On the medium withauxin alone (K0), auxin signalling was upregulated throughout thenewly formed primordia on day 1 (Fig. 1B). In later development,DR5 signal shifted to the columella cells of the newly formed root tip,mimicking the situation in the intact root. However, a broad DR5expression domain remained detectable at the base of the newlyformed roots. The cytokinin signalling-responsive TCS signal wasfirst detectable in the vascular tissue at the base of the primordia atday 2 and became delimited to the vasculature and columella/lateralroot cap of the newly formed roots. Cytokinin concentration-dependent downregulation of auxin signalling in the organprimordia was apparent even at the first day of cultivation at K300/K1000. Starting with day 3, the loss of clearly specified DR5maxima

was associated with gradual primordia disorganisation on bothcytokinin-rich media types. The cytokinin signalling reporter TCSn::GFP was strongly induced in disorganised primordia from the day 3onwards (Fig. 1B). Moreover, high level of TCSn::GFP expressioncolocalised with ProWUS:tdTomato in disorganised primordia, asassayed in the TCSn::GFP/ProWUS:tdTomato line (Fig. 1C).

Altogether, we show that, similar to previous reports employingthe two-step protocol (Atta et al., 2009; Kareem et al., 2015;Sugimoto et al., 2010; Valvekens et al., 1988), the one-stepprocedure allows the regeneration of shoots from hypocotylexplants from the auxin-induced organ precursors of root identity.Exogenous cytokinins quickly downregulate auxin signalling in aconcentration-dependent manner and induce early loss of theprimordia root identity. The process of cytokinin-induced organrespecification is connected with loss of the primordia organisationand strong activation of cytokinin signalling.

Fig. 1. Cytokinin induces early root-to-shoot organ respecification.(A) Expression of ProWOX5:GFP andProWUS:tdTomato, reflecting root orshoot identity of newly formed organs,respectively, during the first 5 days ofexplant cultivation. There is earlyinhibition of root-specific WOX5 andactivation of shoot-specific WUS in thecytokinin-rich (K300 and K1000)media. Asterisks indicate disorganisedprimordia. (B) Auxin signallingoutput visualised using thepDR5rev::3XVENUS-N7 reporter.There is an early concentration-dependent inhibition of auxin signallingby cytokinins on day 1 and strongactivation of cytokinin signalling in thedisorganised primordia (asterisks).(C) High cytokinin signalling activityvisualised using TCSn::GFP increasesfrom day 3 in the disorganisedprimordia where it colocalises withWUS expression (arrowheads).Asterisks indicate disorganisedprimordia. K0, kinetin 0 ng/ml+NAA100 ng/ml; K300, kinetin 300 ng/ml+NAA 100 ng/ml; K1000, kinetin1000 ng/ml+NAA 100 ng/ml. Scalebars: 50 µm.

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Cytokinins inhibit de novo primordia initiation anddifferentiation, and promote in vitro primordiadisorganisationThe impact of both endogenous and exogenous cytokinins wasexamined using a detailed morphological analysis of de novo-induced organs. In the wild type, all primordia were primed duringthe first 24 h of explant cultivation and no increase in the totalprimordia number was detected during further cultivation up today 5 (Fig. 2A). Adding exogenous cytokinin into medium (K300)strongly decreased primordia number at day 1, suggesting negativecytokinin effect on primordia initiation (Fig. 2A, Table S1A). Toevaluate the further progress of primordia development, individualdevelopmental stages were scored by employing the classificationpreviously introduced for the lateral root formation (Malamy andBenfey, 1997) (Fig. S4). On day 1, the distribution of individualdevelopmental stages per hypocotyl explant was comparablewith orwithout cytokinin (Fig. 2B, Table S1B). Hereafter, on mediawithout cytokinin (K0), approximately half of the primordiareached developmental stages V+ at day 3 and maturated to rootsat day 5. In comparison, on the shoot-forming media (K300), mostof the primordia stopped growth after reaching stage IV. Only someof them grew further, giving rise to disorganised primordia and veryfew roots (Fig. 2B).The role of endogenous cytokinins in the de novo organogenic

response was examined in the Pro35S:AtCKX3 stable transgenicline with depleted endogenous cytokinins via overexpressionof CKX gene (Pernisová et al., 2009). From day 1 onwards, we

detected a higher number of primordia along the hypocotyl explantsin Pro35S:AtCKX3 line when compared with wild type, suggestinga negative role for endogenous cytokinins in primordia initiation(Fig. 2A, Table S1C). Furthermore, the higher frequency of olderprimordia stages on media without cytokinins (K0) indicatedaccelerated primordia growth in the Pro35S:AtCKX3 line whencompared with wild type at all the tested time points (statisticallysignificant differences were scored for category III+ at day 1 and V+at day 3 and 5 (Fig. 2B, Table S1D). When compared with K0 andK300, similar to the situation observed in wild type, Pro35S:AtCKX3 lines also revealed cytokinin-mediated downregulation inthe frequency of primordia at stages V and older (Fig. 2B,Table S1E). Moreover, the percentage of disorganised primordiawas decreased (but statistically insignificant) in the Pro35S:AtCKX3 line (Fig. 2B, Table S1F).

Taken together, these data indicate that both exogenous andendogenous cytokinins affect de novo organogenesis bysuppressing primordia initiation and primordia differentiation,particularly at transition beyond stage IV. Importantly,endogenous cytokinins also seem to contribute to cytokinin-induced primordia disorganisation.

De novo shoot apical meristem establishment isaccompanied by upregulation of iP-type cytokininsTo achieve amore detailed insight into the regulatory rolemediated byendogenous cytokinins, the expression and morphology analyseswere complemented by measurement of endogenous cytokinincontent in hypocotyl explants. The total cytokinin content steadilyincreased during the first 5 days of cultivation. However, a largeincrease was apparent particularly at the third day of cultivation(Fig. 3A, Table S2), correlating with upregulated TCSn::GFPexpression and activation of the ProWUS:tdTomato reporter(Fig. 1). Cytokinin ribosides followed by cytokinin N-glucosidesand ribotides dominantly contributed to the total endogenouscytokinin levels (Fig. S5, Table S2). The partial decrease of

Fig. 2. Cytokinins negatively affect primordia initiation and differentiationand promote primordia disorganisation. (A) Total primordia number isincreased in Pro35S:AtCKX3 explants with decreased endogenous cytokininswhen compared with wild type. Data are mean±s.e. Mann–Whitney U-test:aP<0.05 for wild type compared with Pro35S:AtCKX3 line in the correspondingmedia; bP<0.05 for K300 compared with K0 in a corresponding line.(B) Primordia differentiation is faster in the Pro35S:AtCKX3 line (indicated byhigher proportion of older developmental stages: III+ at day 1 and V+ at days 3and 5) (aP<0.05). Exogenous kinetin (K300) slows down the primordiadevelopmental rate (bP<0.05). For detailed statistical analysis, see Table S1.R, roots; dis, disorganised primordia; K0, kinetin 0 ng/ml+NAA 100 ng/ml;K300, kinetin 300 ng/ml+NAA 100 ng/ml.

Fig. 3. iP-type cytokinins associate with de novo shoot establishment.(A) Levels of endogenous tZ-type cytokinins increase in response to allexogenous cytokinin concentrations whereas iP-type cytokinins areupregulated specifically on shooting media with high cytokinin concentration(K1000). Data aremean±s.d., n=5.Mann–WhitneyU-test: aP<0.05 when day 1is compared with days 2-5 in the corresponding media; bP<0.05 when K300and K1000 are compared with K0 on the same day. CK, cytokinin; iP,isopentenyladenine; tZ, trans-zeatin; K0, kinetin 0 ng/ml+NAA 100 ng/ml;K300, kinetin 300 ng/ml+NAA 100 ng/ml; K1000, kinetin 1000 ng/ml+NAA100 ng/ml. For all data, see Table S2. (B) iP induces shoot regeneration moreefficiently than tZ. Numbers represent the cytokinin concentration in ng/ml.Auxin NAA (100 ng/ml) is present in all samples. Scale bar: 1 cm.

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endogenous cytokinin levels observed in K300 when compared withhypocotyls grown in K0might be a result of upregulated endogenousCKX activity, as reported previously (Pernisová et al., 2009; Werneret al., 2006). Interestingly, although the dynamics of tZ-typecytokinins was comparable at all the three media tested (K0, K300and K1000), the levels of iP-type cytokinins strongly increased,particularly from the day 3 onwards, on shoot-forming mediacontaining a high cytokinin concentration (K1000; Fig. 3A). Thecorrelation of upregulation of iP-type cytokinins with occurrence ofdisorganised primordia and WUS activation might suggest possiblerole for iP in the shoot formation. Accordingly, exogenously appliediP was more efficient at de novo shoot regeneration than was tZ andinduced shoot formation at a lower concentration (Fig. 3B).Overall, our results point to a differential regulation of tZ- and iP-

type cytokinin homeostasis during de novo shoot formation.Although upregulation of tZ-type cytokinins seems to beconnected with organ development in general, the ability ofhypocotyl explants to form de novo shoot apical meristems isassociated with upregulation of iP-type cytokinins.

Cytokinin receptors differentially control de novo primordiadevelopment and organ identityThe specificity of individual cytokinin receptors (AHK2, AHK3,AHK4) in terms of their affinity for individual cytokinins as well asfor tissue-specific upregulation of cytokinin signalling has beendemonstrated previously (Romanov et al., 2006; Stolz et al., 2011).To uncover the role of AHK-mediated cytokinin perception andindividual cytokinin receptors in the early events of de novoorganogenesis, we inspected primordia development in double ahkmutants, in which only one cytokinin receptor was functional. Asmentioned above, in wild type all the primordia were established onday 1 and no further increasewas observed throughout the rest of thecultivation period (Figs 2A and 4A). In contrast, there was anapparent increase in the total number of primordia in ahk3 ahk4 atday 3 and day 5 when compared with day 1; a similar trend,although statistically insignificant, was also observed for ahk2 ahk4(Fig. 4A, Table S3A). All three double mutant lines possessed aninsensitivity to exogenous cytokinin-mediated inhibition ofprimordia initiation (Fig. 4A, Table S3B). The progress throughthe primordia developmental path was faster in ahk2 ahk4 and ahk3ahk4, revealing a higher proportion of primordia at stages III-V onday 1 when compared with wild type (Fig. 4B, blue rectangle;Table S3C), suggesting that AHK4 is a negative regulator of earlyprimordia development. Additionally, disorganised primordiaformed in the presence of exogenous cytokinin were detectedonly in wild type and ahk2 ahk3 lines, where AHK4 is functional(Fig. 4B, green rectangle; Table S3D,E). Accordingly, althoughshoots were formed in wild type and ahk2 ahk3 mutants after21 days of explant cultivation, no shoots were recognised in singleor multiple mutants carrying the ahk4 allele (Fig. 4C, Figs S6, S7).To visualise the spatiotemporal distribution of cytokinin

perception and signalling, we inspected the expression pattern ofindividual cytokinin receptors and the primary cytokinin responsegene ARR5. Expression of ProAHK2:AHK2-uidA (AHK2-GUS)and ProAHK3:AHK3-uidA (AHK3-GUS) reporters displayed asimilar pattern, being recognisable from day 1 of cultivation(Fig. 5). The signal of both receptors appeared at the base of the rootprimordia and emerged roots, and decreased in disorganisedprimordia. In comparison with AHK2, the signal of AHK3 wasstronger in the provascular tissue of newly developing roots.Primordia on medium with high cytokinin levels (K300, K1000)displayed weaker AHK2 and AHK3 expression when compared

with cytokinin-free medium (K0). However, AHK4 and ARR5reporters (ProWOL::4xYFP and ProARR5:GUS, respectively) wereundetectable or very low in root primordia formed in the absence of

Fig. 4. Cytokinin receptors differentially regulate de novo primordiadevelopment. (A) Total primordia number is increased in ahk2 ahk4 and ahk3ahk4 double mutants when compared with wild type and ahk2 ahk3 on days 3and 5. Data are mean±s.e. Kruskal–Wallis test and Mann–WhitneyU-test withBonferroni correction: aP<0.05 for wild type compared with ahk lines in thecorresponding media; bP<0.05 for K300 compared with K0 in the same line.(B) Frequency of individual primordia developmental stages in wild type anddouble ahkmutants. Primordia develop faster in ahk4 carrying mutants, whichis reflected by the higher proportion of primordia at stage III+ (outlined with ablue rectangle) and the primordia disorganisation in lines with functional AHK4(outlined with a green rectangle). R, roots; dis, disorganised primordia; K0,kinetin 0 ng/ml+NAA 100 ng/ml; K300, kinetin 300 ng/ml+NAA 100 ng/ml. Fordetailed statistical analysis, see Table S3. (C) Shoot regeneration is impaired inmutant lines carrying the ahk4 allele. Scale bar: 1 cm.

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exogenous cytokinins (K0), with ARR5 and weak AHK4 signalbeing detectable in provascular tissue of emerging roots at day4 and 5. However, the signal of both AHK4 and ARR5 wasupregulated by exogenous cytokinins (K300, K1000) fromday 2. Stronger expression of both reporters concentrated indisorganised primordia – a pattern also corresponding to thespatial specificity of TCSn::GFP and overlapping with shoot apicalmeristem-specific ProWUS:tdTomato (Fig. 1). With the exceptionof the columella/lateral root cap on cytokinin-free media (K0), the

expression pattern of AHK4 seems to correlate well with the two-component signalling sensor TCSn::GFP in developing roots, aswell as in disorganised primordia (Figs 1B and 5). Nonetheless, itshould be highlighted here that, in contrast to the AHK2 and AHK3reporters, AHK4 is not translational but transcriptional fusion. Thus,post-transcriptional regulations that potentially influence the finalAHK4 localisation domain cannot be excluded.

These results suggest an important role for the cytokinin receptorAHK4 in the process of in vitro primordia initiation and

Fig. 5. Spatiotemporal expression profile of cytokininreceptors and cytokinin signalling activity during denovo organ formation. Expression of cytokinin receptorsdiffers during de novo organ development. AHK2(ProAHK2:AHK2-uidA) and AHK3 (ProAHK3:AHK3-uidA)display similar expression patterns with stronger AHK3signal. Weak AHK4 (ProWOL::4xYFP) is recognisable inthe stele of newly formed roots in cytokinin-free media (K0),whereas it is upregulated in disorganised primordia startingfrom the third day in cytokinin-rich medium (K300, K1000).Expression of the cytokinin primary response gene ARR5resembles that of two-component signalling (TCS) activity(Fig. 1) and AHK4 expression. K0, kinetin 0 ng/ml+NAA100 ng/ml; K300, kinetin 300 ng/ml+NAA 100 ng/ml;K1000, kinetin 1000 ng/ml+NAA 100 ng/ml.Scale bars: 50 µm.

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differentiation, and indicate a central role for AHK4 in cytokinin-induced primordia respecification and de novo shoot formation.Moreover, our findings uncovered differential regulation ofindividual cytokinin receptors by exogenous kinetin application inthe course of the early organogenic response.

AHK4-mediated cytokinin signalling downregulatesWOX5 inthe rootTo investigate a possible role for cytokinin perception andsignalling in the root apical meristem of intact seedlings, wecrossed single and double ahkmutants with TCSn::GFP, ProARR5:GUS and ProWOX5:GFP reporters. In all combinations carryingthe ahk4mutant allele, the TCSn::GFP and ProARR5:GUS signal ina stele was either very low or undetectable (Fig. 6A). An increase inProWOX5:GFP expression was detected in ahk3 and particularly inahk4 single, and ahk2 ahk4 and ahk3 ahk4 double mutantbackgrounds. In contrast, ahk2 ahk3 mutants displayedattenuation of the ProWOX5:GFP signal (Fig. 6). In all of theassayed mutant backgrounds, theWOX5 reporter activity appears tobe in a negative correlation with the intensity of cytokininsignalling, as assayed by TCSn::GFP and ProARR5:GUSreporters (Fig. 6A). Therefore, AHK4-regulated cytokininsignalling seems to negatively regulate WOX5 expression in thequiescent centre, thus potentially controlling identity and/or activityof the root stem cell niche.

DISCUSSIONRespecification of organ primordia identity is associatedwith cytokinin-induced loss of primordia organisationSeveral recent studies show that formation of pluripotent callusduring the two-step protocol, i.e. during cultivation on auxin-richCIM, requires activation of key root trait determinants, including

members of PLT gene family and/or regulators necessary for thelateral root formation, e.g. ALF4 (Kareem et al., 2015; Sugimotoet al., 2010). During later cultivation on the SIM, cytokinins inhibitthe activity of those root determinants and activate the shoot-determining factors, includingWUS (Atta et al., 2009; Gordon et al.,2007, 2009; Sugimoto et al., 2010). Our detailed morphologicalanalysis suggests that cytokinins inhibit both de novo organprimordia initiation as well as their later differentiation, as most ofthe primordia did not succeed in proceeding beyond stage IV oncytokinin-rich medium. In plants, lateral root primordia werereported to be more sensitive to exogenous cytokinin at earlydevelopmental stages (Bielach et al., 2012; Laplaze et al., 2007). Inaddition, endogenous cytokinin overproduction in plants leads toprimordia growth inhibition preferentially in stages II-IV (Bielachet al., 2012; Kuderová et al., 2008). Similar observations wereobtained during direct conversion of lateral root primordia intoshoots (Rosspopoff et al., 2017). In that work, the young rootprimordia (up to stage V) were shown to be incompetent to thecytokinin-induced identity switch and stopped their growth soonafter transplanting to the cytokinin-rich media. Interestingly, thetransition of stage IV to stage V coincides with the initiation ofWOX5 expression in the root primordia (Goh et al., 2016;Rosspopoff et al., 2017) grown in the cytokinin-free media that isquickly inactivated by exogenous cytokinins in the shoot-inducingmedia.

In the shooting media from day 3 onwards, we observed thegradual primordia disorganisation that associated with strongupregulation of cytokinin signalling that spatially overlaps theWUS expression domain. It is not clear whether the cytokinin-induced structural disorganisation has any functional meaning inthe process of identity switching and/or differentiation status ofthe tissue or whether it is merely a collateral consequence of

Fig. 6. Cytokinin signalling in stele affects WOX5 expression in the quiescent centre via the AHK4 receptor. (A) The cytokinin response visualisedusingTCSn::GFPandProARR5:GUS diminishes in stele of ahk4 single and doublemutants (arrows). The attenuation of cytokinin signalling in the stele correlateswith an increase in ProWOX5:GFP expression in the root quiescent centre. Scale bars: 100 µm (TCSn::GFP, ProARR5:GUS); 10 µm (ProWOX5:GFP).(B) WOX5 signal intensity increases in all ahk4 single and double mutants. Data are mean±s.d., n=20. Statistically significant differences (t-test) are indicated:*P=0.05 and ***P=0.001. (C) WOX5 is expressed specifically in the root quiescent centre. Scale bar: 10 μm.

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cytokinin-induced loss of the auxin patterning, possibly viamisregulation of auxin transport (Pernisová et al., 2009).However, the identity switch, exemplified by WUS activation, wasobserved solely in the disorganising primordia. Thus, in our one-step system, the cytokinin-induced primordia disorganisation seemsto be tightly associated with the competence to change their identity.Nonetheless, although WUS is essential for de novo shoot

regeneration (Zhang et al., 2017), activation ofWUS alone does notseem to be sufficient for the switch in the organ identity. In thepresence of high kinetin concentrations, WUS could also beactivated in the non-growing trunk regions of root explants, whichare unable to regenerate into shoots (Sugimoto et al., 2010).Similarly, WUS was activated at stage V in the non-competentlateral root primordia that were not developing into the shoots whentransplanted to the shoot-inducing media (Rosspopoff et al., 2017).This implies the existence of a developmental- and tissue-specificcontext necessary for both cytokinin-mediated WUS activation andWUS-dependent induction of a change in organ identity. Recently,the type-B ARRs ARR1, ARR2, ARR10 and ARR12 have beenimplicated in directly interacting with WUS promoter (Dai et al.,2017;Meng et al., 2017;Wang et al., 2017; Zhang et al., 2017; Zuboet al., 2017). However, similar to our study, showing a much largerTCSn:GFP domain when compared with highly focused WUSactivity (Fig. 1C), the expression domains of ARR1, ARR10 andARR12 were also larger than that of WUS (Meng et al., 2017).Furthermore, WUS activity, although weaker, was detectable andnormally positioned in the arr1 arr10 arr12 background, implyingthe existence of other factors controlling WUS expression (Menget al., 2017). The regulatory elements that allow cytokinin-inducedupregulation ofWUS specifically in the disorganising primordia andthe tissue-specific determinants that act downstream of WUS in theroot-to-shoot identity switch therefore remain to be identified.

Root-to-shoot induction associates with specificity in theendogenous cytokinin production and signallingThe measurement of endogenous cytokinin levels revealedconnection between shoot apical meristem establishment and iP-type cytokinin production. A possible explanation could beexpression specificity of cytochrome P450 monooxygenaseCYP735A2, which mediates the formation of tZ cytokinin typesvia iP hydroxylation. Expression of CYP735A2 was found to occurdominantly in the roots. Furthermore, activity of CYP735A2 in theroots was strongly inducible by cytokinins, including its substrateiP, whereas a similar response was not observable in the shoot(Takei et al., 2004). Thus, the specific upregulation of iP typecytokinins on the shoot-forming media might reflect low activity ofCYP735A2, thus leaving most of the produced cytokinins in theirnon-hydroxylated form.Our results showed that exogenously applied iP induced in vitro

shoot regeneration at a much lower concentration in comparison withthat of tZ (Fig. 3B). Our data also suggest that AHK4plays a dominantrole in mediating the cytokinin-induced organ disorganisation andrespecification. Furthermore, AHK4 seems to be negative regulator ofWOX5 in the root, whereas bothAHK2 andAHK3might haveweakereffect on WOX5 expression (Fig. 6). Accordingly, although AHK4receptor recognises both tZ and iP with comparable affinity(Romanov et al., 2006; Spíchal et al., 2004), AHK3 recognises iPwith ∼100 times lower efficiency than tZ (Romanov et al., 2006). Inline with our observations suggesting kinetin as being the leastefficient ‘shooting’ cytokinin when compared with iP and tZ, kinetinwas found to have significantly lower activity than iP or tZ in anAHK4-mediated β-galactosidase assay (Spíchal et al., 2004).

The factors determining the differential role of AHK2 and AHK3on the one hand and AHK4 on the other in the regulation ofWOX5in the root quiescent centre remain to be identified. Specificity of theAHK intracellular domains to AHPs, the downstream members ofthe multistep phosphorelay pathway, as shown in the case of anothersensor histidine kinase, CKI1 (Pekárová et al., 2011), together withdifferential expression and cytokinin responsiveness during de novoshoot formation (Fig. 5) might be one of the possible mechanisms.Moreover, in contrast to AHK2 and AHK3, AHK4 has been shownto have phosphatase activity in the absence of cytokinins (Mähönenet al., 2006), which might also contribute to the specific type ofregulation mediated by AHK4 in the control of de novo shootformation.

Amodel for the cytokinin-induced change in organ identity inthe single-step protocolBased on the aforementioned findings, we propose that, in the one-step approach, auxin induces formation of root primordia, possiblyactivating root-specific developmental pathways, as previouslydemonstrated in the two-step protocols. The process of primordiainitiation is under the negative control of cytokinins (both endo- andexogenous). The process of organ primordia formation is associatedwith endogenous cytokinin production that seems to have anautoregulatory role in inhibiting further primordia formation, aspreviously demonstrated in the root (Bielach et al., 2012); thisnegative effect seems to be dominantly mediated by AHK4 (Fig. 7).Moreover, the expression of individual cytokinin receptors seems tobe under differential cytokinin control, possibly also contributing tothe final signalling output. The root primordia reaching the transitionto stage V become susceptible to exogenous cytokinins that, viaAHK4, preferentially mediate the loss of the root identity, asevidenced by the downregulation of root reporters. Primordiarespecification, which is possibly accompanied by downregulationofCYP735A2 activity, is associated with upregulation of endogenousiP-type cytokinins that further contribute to the upregulation ofAHK4-mediated cytokinin signalling and to the loss of root primordiaarchitecture. Activated cytokinin signalling, directly or indirectly andin a cooperation with the yet unknown factors acting in a tissue- anddevelopmental-specific context, mediate induction of WUSspecifically in the disorganised primordia, leading to the acquisition

Fig. 7. A model for cytokinin-mediated regulation of de novo root andshoot regeneration.Auxin-induced de novo organogenesis is associatedwithendogenous cytokinin production. Both endogenous and exogenouscytokinins inhibit auxin-induced primordia initiation via the AHK4 receptor. Incytokinin-rich media (K300 and K1000), AHK4-mediated signalling attenuatesWOX5 expression, induces primordia disorganisation and initiates WUSexpression specifically in the disorganised organ primordia. CK, cytokinin; K0,kinetin 0 ng/ml+NAA 100 ng/ml; K1000, kinetin 1000 ng/ml+NAA 100 ng/ml.

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of shoot identity and the activation of the downstream developmentalcascade, which allows de novo shoot formation.

MATERIALS AND METHODSPlant materialUnless otherwise mentioned, all plant material used was from Arabidopsisthaliana, ecotype Col (NASCN60000). The transgenic or mutant lines havebeen described previously: Pro35S:AtCKX3 (Pernisová et al., 2009),pDR5rev::3XVENUS-N7 (Wabnik et al., 2013), TCSn::GFP (Zurcher et al.,2013), ProARR5:GUS (Che et al., 2002), ProWOX5:GFP (Blilou et al.,2005), ProWOL::4xYFP (Marques-Bueno et al., 2016), ProAHK3:AHK3-uidA (Dello Ioio et al., 2007), ProSCR:GFP-SCR (Gallagher et al., 2004),ProPLT2:PLT2-YFP and ProPLT3:PLT3-YFP (Galinha et al., 2007), ahk4-1 (Ueguchi et al., 2001), cre1-2 (Inoue et al., 2001), and ahk2-5, ahk3-7,ahk2-5 ahk3-7, ahk2-5 cre1-2, ahk3-7 cre1-2 and ahk2-5 ahk3-7 cre1-2(Riefler et al., 2006).

pDR5rev::3XVENUS-N7 and TCSn::GFP, ProWOX5:GFP andProWUS:tdTomato, TCSn::GFP and ProWUS:tdTomato were crossed anddouble homozygous lines were analysed. Mutant lines ahk2-5, ahk3-7,cre1-2, ahk2-5 ahk3-7, ahk2-5 cre1-2 and ahk3-7 cre1-2 were crossedwith reporters TCSn::GFP, ProARR5:GUS and ProWOX5:GFP, andhomozygous lines were used in experiments.

Growth conditionsThe growth media used comprised 0.5 Murashige and Skoog medium(Duchefa) with 1% sucrose (Lach-Ner) and 0.8% Plant agar (Duchefa), pH5.7 adjusted by KOH. Plants were cultivated in growth chambers (CLF PlantClimatics) under long-day conditions (16 h light/8 h dark) at 21°C in Petridishes or in soil, with a light intensity of 150 μM m−2 s−1 and 40% relativehumidity.

GenotypingPrimers for genotyping of ahk2-5 and ahk3-7 have been publishedpreviously (Riefler et al., 2006). New cre1-2 specific primers weredesigned as follows: CRE1-2 for (5′-CTCTTTTGTTCTTGAATTCGC-3′); CRE1-2 rev (5′-ATCCTGCAACATTCTAGCTC-3′); and cre1-2 in (5′-ATAACGCTGCGGACATCTAC-3′).

Three primers for each gene were optimised in one PCR reaction. For allthree genes, the following conditions were used: 94°C for 1 min 30 s; 40×(94°C for 15 s, 56°C for 30 s and 72°C for 40 s); and 72°C for 7 min.

Hypocotyl explants assayPlants were cultivated for 1 day in the light and 5 days in the dark in Petridishes with Murashige and Skoog medium including Gamborg B5 vitamins(Duchefa), with 1% sucrose (Lach-Ner) and 0.3% Phytagel (Sigma), pH 5.7adjusted by KOH. Etiolated hypocotyls were isolated by removingcotyledons and roots, and were placed on Petri dishes. Hypocotyl explantcultivation medium contained Murashige and Skoog medium includingGamborg B5 vitamins (Duchefa), with 1% sucrose, 0.3% Phytagel (Sigma)and 1 mg/l biotin (Duchefa), pH 5.7 adjusted byKOH, and enriched with thephytohormones 1-naphthalene acetic acid (NAA; Sigma), kinetin (Sigma),trans-zeatin (tZ; OlChemIm) or isopentenyladenine (iP; OlChemIm) atthe appropriate concentrations. Hypocotyl explants were cultivatedunder continuous light conditions at 21°C, with a light intensity of150 μM m−2 s−1 and 40% relative humidity.

MicroscopyDIC microscopy was performed on an Olympus BX61 microscope equippedwith ×10, ×20 and ×40 air objectives and a DP70 CCD camera. Confocalmicroscopy was carried out on two microscopes: an inverted ZeissObserver.Z1 equipped with a LSM780 confocal unit and ×20 air objectiveand ×40 water immersion objective; and an upright LEICA DM 2500 withTCS SPE confocal unit and ×40 air objective. Excitation and detection offluorophores were configured as follows: GFP was excited at 488 nm anddetected at 490-530 nm; YFP and VENUS were excited at 514 nm anddetected at 520-560 nm; tdTomatowas excited at 561 nm and detected at 570-630 nm. For microscopic analyses, 10-12 hypocotyl explants were inspected.

Analysis of primordium developmental stagesFor morphological analyses of primordia developmental stages, 10-12hypocotyl explants were inspected. Developmental stages were determinedand evaluated based on lateral root developmental stages (Malamy andBenfey, 1997).

Image analysisSignal intensity measurements were carried out using the softwareaccompanying the confocal microscopes: ZEN (Carl ZeissMicroImaging), LAS AF lite (Leica Microsystems CMS) and analySIS^D(Olympus Soft Imaging Solutions). ProWOX5:GFP signal intensities weremeasured as an average grey scale values, ranging from 0 to 4096.

Histochemical stainingAHK2-GUS, AHK3-GUS and pARR5:GUS hypocotyl explants orseedlings were stained in 0.1 M sodium phosphate buffer (pH 7.0)containing 0.1% X-GlcA sodium salt (Duchefa), 1 mM K3[Fe(CN)6],1 mMK4[Fe(CN)6] and 0.05% Triton X-100 for 1 h (ProARR5:GUS) or 2 h(AHK2-GUS, AHK3-GUS) at 37°C and were incubated overnight in 80%(vol/vol) ethanol at room temperature. Tissue clearing was conducted aspreviously described (Malamy and Benfey, 1997).

Measurements of endogenous cytokininsQuantification of cytokinin metabolites was performed according to themethod described by Svacinová et al. (2012), including modificationsdescribed by Antoniadi et al. (2015). For details, see the supplementaryMaterials and Methods.

Accession numbersThe AGI codes (www.arabidopsis.org) of loci used for genotypingand reporter line preparation are as follows: AHK2 (AT5G35750),AHK3 (AT1G27320), AHK4 (AT2G01830), ARR5 (AT3G48100), WOX5(AT3G11260), WUS (AT2G17950), CKX3 (AT5G56970), SCR(AT3G54220), PLT1 (AT3G20840) and PLT3 (AT5G10510).

Statistical analysisStatistical analysis was performed with Statistica, version 13 (Dell) byemploying non-parametric Mann–Whitney U-test, Kruskal–Wallis andFisher’s exact tests. For details, see the supplementaryMaterials andMethods.

ProWUS:tdTomato line preparationThe ProWUS::tdTomato-N7 transgene was constructed by gateway-mediated cloning. For details, see the supplementaryMaterials andMethods.

AcknowledgementsThe work was greatly supported, both materially and intellectually, by Paul T. Tarrand Elliot M. Meyerowitz, Division of Biology and Biological Engineering, CaliforniaInstitute of Technology, Pasadena, CA, USA. We thank Bruno Muller for the TCSn:GFP, Jirı Friml for the pDR5rev::3XVENUS-N7, Philip N. Benfey for the ProSCR:GFP-SCR, and Ben Scheres for the ProWOX5:GFP, ProPLT2:PLT2-YFP andProPLT3:PLT3-YFP lines. The Plant Sciences Core Facility of CEITEC MasarykUniversity is acknowledged for plant and explant cultivation.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: M.P., J.H.; Methodology: M.P., D.H., T. Kakimoto, M.G.H., O.N.;Formal analysis: M.P., M.G., T. Konecny, L.P., D.H., T. Kakimoto, M.G.H., O.N.;Investigation: M.P., J.H.; Resources: M.P., T. Kakimoto, J.H.; Data curation: M.P.,M.G., T. Konecny, L.P., D.H., O.N.; Writing - original draft: M.P., J.H.; Writing - review& editing: M.P., J.H.; Visualization: M.P.; Supervision: M.P., O.N., J.H.; Projectadministration: M.P.; Funding acquisition: M.P., O.N., J.H., T. Kakimoto.

FundingThis work was supported by the Grantova Agentura Ceske Republiky [GP14-30004P to M.P. and T. Konecny, and 13-25280S to J.H.], the Ministerstvo Skolstvı,Mladeze a Telovýchovy [CEITEC 2020 (LQ1601) to M.P., M.M. and J.H.; LH14104to M.P. and J.H.; National Program for Sustainability I, LO1204 to L.P. and O.N.;

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LM2015062 Czech-BioImaging], the EU Seventh Framework Programme[286154 – SYLICA to M.P.] and a Japan Society for the Promotion of Sciencegrant [25113006 to T. Kakimoto].

Supplementary informationSupplementary information available online athttp://dev.biologists.org/lookup/doi/10.1242/dev.163907.supplemental

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