The transcription factor NO TRANSMITTING TRACT / WIP2 regulates cytokinin homeostasis in Arabidopsis

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Becerra-García , View ORCID Profile José E. Cruz-Valderrama , View ORCID Profile Humberto Herrera-Ubaldo , View ORCID Profile Ricardo A. Chávez Montes , View ORCID Profile Maurizio Di Marzo , View ORCID Profile Lucia Colombo , Martin Sagasser , View ORCID Profile Rüdiger Simon , View ORCID Profile Ondrej Novak , View ORCID Profile Stefan de Folter , View ORCID Profile Nayelli Marsch-Martínez doi: https://doi.org/10.1101/2025.04.03.645522 David Díaz-Ramírez 1 Department of Biotechnology and Biochemistry, Center for Research and Advanced Studies of the National Polytechnic Institute (Cinvestav) , km 9.6 Libramiento Norte, Carretera Irapuato-León, Irapuato, Guanajuato, Mexico Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for David Díaz-Ramírez Edgar Demesa-Arevalo 2 Heinrich-Heine Universität , Düsseldorf, Germany 3 Cluster of Excellence on Plant Sciences, Heinrich Heine University , Düsseldorf, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Edgar Demesa-Arevalo Yolanda Durán-Medina 1 Department of Biotechnology and Biochemistry, Center for Research and Advanced Studies of the National Polytechnic Institute (Cinvestav) , km 9.6 Libramiento Norte, Carretera Irapuato-León, Irapuato, Guanajuato, Mexico Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rosa E. Becerra-García 1 Department of Biotechnology and Biochemistry, Center for Research and Advanced Studies of the National Polytechnic Institute (Cinvestav) , km 9.6 Libramiento Norte, Carretera Irapuato-León, Irapuato, Guanajuato, Mexico 4 Advanced Genomics Unit (UGA-Langebio), Center for Research and Advanced Studies of the National Polytechnic Institute (Cinvestav) , km 9.6 Libramiento Norte, Carretera Irapuato-León, Irapuato, Guanajuato, Mexico Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rosa E. Becerra-García José E. Cruz-Valderrama 4 Advanced Genomics Unit (UGA-Langebio), Center for Research and Advanced Studies of the National Polytechnic Institute (Cinvestav) , km 9.6 Libramiento Norte, Carretera Irapuato-León, Irapuato, Guanajuato, Mexico Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for José E. Cruz-Valderrama Humberto Herrera-Ubaldo 4 Advanced Genomics Unit (UGA-Langebio), Center for Research and Advanced Studies of the National Polytechnic Institute (Cinvestav) , km 9.6 Libramiento Norte, Carretera Irapuato-León, Irapuato, Guanajuato, Mexico Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Humberto Herrera-Ubaldo Ricardo A. Chávez Montes 4 Advanced Genomics Unit (UGA-Langebio), Center for Research and Advanced Studies of the National Polytechnic Institute (Cinvestav) , km 9.6 Libramiento Norte, Carretera Irapuato-León, Irapuato, Guanajuato, Mexico Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ricardo A. Chávez Montes Maurizio Di Marzo 5 Department of Biosciences, University of Milan , Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Maurizio Di Marzo Lucia Colombo 5 Department of Biosciences, University of Milan , Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Lucia Colombo Martin Sagasser 6 Center for Biotechnology, University of Bielefeld , Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rüdiger Simon 2 Heinrich-Heine Universität , Düsseldorf, Germany 3 Cluster of Excellence on Plant Sciences, Heinrich Heine University , Düsseldorf, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rüdiger Simon Ondrej Novak 7 Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany, Czech Academy of Sciences, and Faculty of Science, Palacký University , Czechia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ondrej Novak Stefan de Folter 4 Advanced Genomics Unit (UGA-Langebio), Center for Research and Advanced Studies of the National Polytechnic Institute (Cinvestav) , km 9.6 Libramiento Norte, Carretera Irapuato-León, Irapuato, Guanajuato, Mexico Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Stefan de Folter Nayelli Marsch-Martínez 1 Department of Biotechnology and Biochemistry, Center for Research and Advanced Studies of the National Polytechnic Institute (Cinvestav) , km 9.6 Libramiento Norte, Carretera Irapuato-León, Irapuato, Guanajuato, Mexico Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nayelli Marsch-Martínez For correspondence: nayelli.marsch{at}cinvestav.mx Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract NO TRANSMITTING TRACT / WIP2 (NTT/WIP2) is a zinc finger transcription factor that alters developmental programs in Arabidopsis when overexpressed. Loss of NTT function, alone or in combination with closely related genes, leads to developmental defects in gynoecia and roots, respectively. Its overexpression phenotype resembles the effects of cytokinin treatments, suggesting a potential connection to this hormonal pathway. To further investigate this connection, we conducted cytokinin content and response evaluations, and transcriptome analyses in an inducible NTT line. Alterations in cytokinin content and response, as evaluated with the TCSn::GFP line, were observed. In both cases, as in the global expression analyses, the observed changes were dependent on the tissue analyzed. Moreover, Y1H and transactivation assays suggested direct NTT binding to the IPT5 , AHP6 and CKX7 , regulatory regions. We propose that NTT, among other mechanisms, regulates development through the modulation of cytokinin homeostasis at different levels, including its local concentration. Since NTT is a target of AUXIN RESPONSE FACTOR 5 / MONOPTEROS, the results presented here indicate that NTT connects an auxin input to a change in cytokinin levels. Introduction The transcription factor WIP2 / NO TRANSMITTING TRACT (NTT) belongs to the WIP subfamily of zinc finger proteins that can localize in the nucleus and share a domain comprising four zinc fingers in tandem, which initiates with the amino acids Trp, Ile, Pro (WIP, hence the name, Sagasser et al ., 2001 ). This subfamily includes six members in Arabidopsis thaliana (Appelhagen et al., 2010). WIP2/NTT is expressed in different tissues during plant development, including the early embryo, shoot apical meristem (SAM), root apical meristem (RAM), vasculature and gynoecium, among others ( Crawford et al ., 2007 , 2015 ; Chung et al ., 2013 ; Herrera-Ubaldo et al., 2019 ; Marsch-Martínez et al ., 2014 ). Loss and gain of function alleles of WIP2/NTT in Arabidopsis have revealed several functions. During the development of the gynoecium, the female reproductive organ in flowers, WIP2/NTT participates in the establishment of the transmitting tract, an internal tissue that facilitates pollen tube growth through the ovary to reach and fertilize ovules. Loss of WIP2/NTT function affects the development of this tissue causing partial sterility. It was therefore named NO TRANSMITTING TRACT ( Crawford et al., 2007) and will be addressed as NTT through the rest of the text for simplicity. Alterations in NTT expression also affect replum and valve margin development ( Chung et al ., 2013 ; Marsch-Martínez et al ., 2014 ). NTT, in combination with two other WIP members, WIP4 and WIP5, are key regulators of embryo development and RAM establishment. In the absence of these three proteins, seedlings lack roots (Crawford et al., 2015 ). Although mutant phenotypes indicate that NTT regulates developmental pathways, we are just starting to elucidate its mechanisms of action. Most NTT targets have been studied in the context of gynoecium and fruit development (Marsch-Martinez et al ., 2014; Herrera-Ubaldo et al ., 2019 ). NTT is able to physically interact with many key gynoecium and fruit development regulators, such as REPLUMLESS (RPL), FRUITFUL (FUL), SHATTERPROOF1 and 2 (SHP1 and 2), SEEDSTICK (STK) and others (Marsch-Martinez et al ., 2014; Herrera-Ubaldo et al., 2019). Together, STK and NTT regulate genes involved in cell wall polysaccharide metabolism and lipid synthesis or transport, and some of them lead to altered gynoecium phenotypes when mutated ( Herrera-Ubaldo et al ., 2019 ). Ren et al . (2018) explored the function of a WIP gene in Gerbera hybrida , finding that it acts as a transcriptional repressor and represses cell expansion in petals ( Ren et al ., 2018 ). In Arabidopsis, ChIP-seq and RNA-seq approaches using inducible lines of NTT and related gene WIP1/TRANSPARENT TESTA 1 (TT1) , gave more insights into binding sites and targets of these two WIP transcription factors (Gomez-Roldan et al., 2020). The phenotype of ntt wip4 wip5 ( nww ) triple mutant seedlings is similar to the monopteros ( mp ) mutant phenotype, and the expression of NTT is lost in mp. MP is AUXIN RESPONSE FACTOR 5 (ARF5), a transcription factor that mediates the transcriptional response to auxin ( Crawford et al ., 2015 ). ChIP assays revealed that MP binds to auxin response elements (AuxREs) in the NTT genomic region (and similar regions in WIP4 and WIP5), suggesting that these WIPs act downstream the auxin response. They are not required for the initial activation of the DR5 auxin response marker during embryo development. However, DR5 expression is reduced at later stages, and a pulse of auxin can rescue the rootless phenotype in nww mutants, although these roots develop in a highly abnormal way ( Crawford et al ., 2015 ). On the other hand, NTT physically interacts with cytokinin type-A Arabidopsis Response Regulators (ARR4 and ARR16; Dortay et al ., 2008 ). Moreover, NTT also interacts with SHOOTMERISTEMLESS (STM) and binds to the promoter of BREVIPEDICELLUS ( BP ) (Marsch-Martinez et al ., 2014), and both transcription factors have been reported to activate the cytokinin pathway ( Yanai et al ., 2005 ). In addition, cytokinin treatments rescue replum development in otherwise replum-less ntt replumless (rpl) double mutants ( Zuñiga-Mayo et al ., 2018 ), suggesting that cytokinins may be related to or play a role in NTT function. Cytokinins are phytohormones involved in the regulation of many developmental processes, such as cell proliferation, root differentiation, leaf expansion, and senescence, among others ( Hwang et al ., 2012 ; Marquez-Lopez et al., 2019; Wybouw and De Rybel, 2019 ). Cytokinins are synthesized mainly by ISOPENTENYL TRANSFERASES (IPT) and LONELY GUY (LOG) enzymes (Kuroha et al., 2009; Miyawaki et al ., 2006 ; Schaller et al ., 2015 ; Duran-Medina et al ., 2017; Wybouw and De Rybel, 2019 ). Cytokinins are then sensed by histidine kinases (AHKs), which autophosphorylate and then transfer the phosphate to histidine phosphotransferases (AHPs) (Hwang et al., 2012; Kieber and Schaller, 2018 ). The AHPs then transfer the phosphate to the type-B ARRs, the response regulators that promote the transcription of the genes in response to cytokinin, including the negative regulators type-A ARRs ( Kieber and Schaller, 2018 ). AHP6 is an exception because it does not transfer the phosphate and acts as a negative regulator of the pathway ( Mähönen et al ., 2006 ). Cytokinin inactivation is carried out through degradation by cytokinin oxidases (CKXs) which catalyze an irreversible oxidative breakdown ( Werner et al ., 2001 , 2003 ), and by N- or O-glucosylation by cytokinin glucosyl transferases (Chen et al., 2021). Degradation and N-glucosylation are irreversible, while O-glucosylation is a reversible inactivation process (Chen et al., 2021). Given the key role that cytokinins play in development, the central question of this work was to find out whether NTT regulates cytokinin genes. We found that NTT regulates genes that participate at different steps of the cytokinin pathway, influencing cytokinin content and response. Materials and methods Plant material, growth conditions and genotyping The Arabidopsis thaliana lines used in this study are all in the Columbia (Col-0) background. Plants were grown under greenhouse or growth chamber conditions (22 °C, 16 hours of light) in soil or in vitro, in ½ MS medium supplemented with 0.5 % sugar and 1 % agar. The following lines were used in this work: the dexamethasone inducible line 35S::NTT-GR , the recombineering line gNTT-YPET, nww triple mutant ( Crawford et al ., 2015 ), 35S::NTT ( Marsch-Martínez et al ., 2014 ) , pIPT5::GUS (Dello Ioio et al., 2007) , pAHP6::GFP (Mähönen et al., 2006), ckx7 (GABI-Kat; Kleinboelting et al., 2012), TCSn::GFP (Zürcher et al., 2013) and we generated 35S::NTT-GR ckx7, 35S::NTT-GR x pIPT5::GUS, 35S::NTT-GR x pAHP6::GFP and 35S::NTT-GR x TCSn::GFP through genetic crosses. Primer sequences for genotyping are in Table S1. The Nicotiana benthamiana plants used for the NanoLuc transactivation assays were grown in a 16 h light/8 h dark photoperiod at 25 °C for six weeks. Induction of NTT activity and cytokinin treatments Most NTT inductions were performed on in vitro -grown 35S::NTT-GR seedlings, at four days after germination (dag) by directly applying the solutions to the plants on the plates and removing the liquid after two minutes. 100 µM dexamethasone (DEX), 100 µM DEX + 30 µM cycloheximide (CHX), or mock solutions were used (Fig. S2). The DEX solution was prepared from a sterile 20 mM stock, first dissolved in ethanol, and then diluted with sterile water. To evaluate the induction response in the crosses, seeds were germinated directly in solid medium supplemented with 10 µM DEX. For induction under cytokinin treatments, seedlings were transplanted to medium containing 10 µM DEX and 10 µM 6-benzylaminopurine (BAP, dissolved in NaOH) or mock medium. For cytokinin treatments, four dag Col-0 seedlings (shown in Fig. 1 ) were transplanted onto medium containing 10 µM BAP. Download figure Open in new tab Figure 1. NTT OE phenotypes, cytokinin quantification and protein localization. (A-E) Representative images of 11 dag seedlings: (A) Mock treated 35S::NTT-GR line. (B) DEX induced 35S::NTT-GR. (C) NTT overexpression line ( 35S::NTT line). (D) Mock treated Col-0 seedling. (E) Col-0 seedlings after BAP treatment. Red arrows indicate leaf margins, and the irregularities observed under treatments or NTT overexpression. (F-J) Quantification of different types of cytokinin content in 6 dag Col-0 and 35S::NTT-GR seedlings (root and shoot tissues were collected and analyzed separately, 2 dai, 4 dag seedlings): (F) Total cytokinins (ck), (G-J) Total (ribosides, glucosides, and free bases) (G) isopentenyl adenosine (iP) type ck; (H) trans-zeatin (tZ) types; (I) cis-zeatin (cZ) types; and (J) Dihydro-zeatin (DHZ) types. (K) Differences in root growth between mock and Dex treated Col-0 and 35S::NTT-GR seedlings. Only the DEX treated 35S::NTT-GR line is different from the rest. (F-K) Student’s t-test, * p ≤ 0.01, ** p ≤ 0. 001. (L-P) gNTT-YPET accumulation pattern: (L) in quiescent center, initials and columella in RAM; (M) In initiation of lateral roots and companion cells (white arrows) in root vasculature; (N) surrounding SAM (boundary cells, white arrows) and primordia (1-2 dag seedlings); (O) in companion cells in aerial vasculature (white arrow) and leaf epidermis; and (P) in the carpel marginal meristem (CMM), in a stage 6 gynoecium. L-O: 5 dag seedlings. Counterstaining L-O: SR2200, P: PI. Scale bars 1mm (A-E) and 20 µm (L-P). Cytokinin quantification Quantification of CK metabolites was performed as described in ( Svačinová et al ., 2012 ). Four dag seedlings (Col-0 or 35S::NTT-GR ) were treated with DEX or mock as described previously. After a week of treatment, around 30 mg of shoots and roots of the treated and mock seedlings were harvested separately (four independent replicates), flash-frozen in liquid nitrogen, and stored at -80°C. Extraction, purification and quantification were done as described in (Duran-Medina et al ., 2025; Svačinová et al., 2012). RNA extraction Aerial tissue (shoots) and roots of the treated seedlings were harvested separately for RNA extraction 30 min after the treatment with DEX and CHX, or eight hours after the treatment with DEX, and their respective controls were also harvested at the same times (three replicates, around 10 seedlings for aerial tissue, and around 20 for root tissue). RNA was extracted and purified using Quick RNA miniprep (Zymo Research, Orange, CA, USA). RNA quality assessment, and library construction were performed by Macrogen (Seoul, South Korea). RNA-seq, alignment, quantification, and differential expression analysis RNA-seq was carried out at Macrogen using the TruSeq Stranded mRNA LT Sample Prep Kit and Illumina 100 base paired-end sequencing. The data was deposited at the European Nucleotide Archive under the study accession number PRJEB40086. After quality control using FastQC ( https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ), pseudo-alignment and quantification in paired-end strand-specific mode versus the Arabidopsis Araport11 ( Cheng et al ., 2017 ) cDNA sequences (version 2016) was done using Kallisto (version 0.46.1; Bray et al ., 2016 ). Kallisto data gene-level summarization and pre-processing was done with txt import ( Soneson et al ., 2016 ), and differential expression analysis was done with edgeR ( Robinson et al ., 2009 ; McCarthy et al ., 2012 ) under R ( R Core Team, 2022 ; https://www.R-project.org/ ). Gene Ontology categories enrichment analysis Gene Ontology biological process categories enrichment analysis was done using the R package SetRank (“SetRank: Advanced Gene Set Enrichment Analysis. R package version 1.1.0. https://CRAN.R-/package=SetRank ”; Simillion et al ., 2017 ) using the Gene Ontology gene association file for Arabidopsis (dated 01-02-2019, available at the Gene Ontology Consortium website (Ashburner et al., 2000; The Gene Ontology Consortium, 2019 ) in SetRank format as the annotation file, the complete p-value-ranked edgeR tables as the query, and all expressed genes in either root or aerial tissue as the universe. We considered a gene as expressed if it had a value of at least 1 TPM across all samples of the corresponding tissue. RT-qPCR Confirmation of gene expression was made by RT-qPCR, using the One-Step RT-qPCR Kit (Nzytech) in a Step One TM thermocycler (Applied Biosystems), including 3 biological and technical replicates (tissue was collected in the same conditions as for the RNAseq experiment). Expression levels were normalized to ACTIN2. Relative expression was calculated with the ΔΔCt method ( Livak and Schmittgen, 2001 ). The primers used are listed in Table S1. GUS Staining Samples were collected and stained in GUS solution (0.5% triton, 2.5 mM potassium ferricyanide, 2.5 mM potassium ferrocyanide, 1 M EDTA and 2 mM X-Gluc in 50 mM NaPO4), at 37 °C overnight. Afterwards, plants were cleared in 70 % ethanol to easily distinguish coloration. Whole-mount immunostaining Immunostaining was performed as follows: 1 dag seedlings were fixed as described by Tran et al . (2021) but using MTSB instead of PBS ( Herrera-Ubaldo et al ., 2023 ). Cuticle solubilization was carried out following Pasternak et al . (2015) . Samples then were digested for 27 minutes at 22°C, as in Tran et al . (2021) , followed by permeabilization according to Pasternak et al . (2015) . After this, samples were blocked for 30 minutes in 4% BSA before incubation with the primary antibodies (ab) overnight. The protocol was continued as described by Tran et al . (2021) for days 2 and 3 (washing and incubation with secondary ab). Anti-trans-zeatin riboside (Cat. 004 0312, Olchemim) or anti-N6-isopentenyladenosine (Cat. 004 0172, Olchemim), both raised in rabbit, were used as primary ab. As a technical control, we used Pol II S2p primary ab, raised in mouse (Cat. C15200005, Diagenode; Fig. S7E). Secondary ab used were Alexa Fluor plus 488 (anti-mouse, Cat. A32723, Invitrogen) and Alexa Fluor 568 (anti-rabbit, Cat. A-11036, Invitrogen). Imaging Confocal imaging was obtained using a Zeiss LSM800 or LSM880 confocal microscope. For gNTT-YPET , seedlings were cleared using the ClearSee protocol (Kurihara et al ., 2015) and stained with SCRI Renaissance 2200 (SR2200). Renaissance imaging was performed using a 405 nm laser for excitation (ex), with emission(em) detected between 410–511 nm. For YPET fluorescence ex: 514 nm, em: 519–541 nm. For 35S::NTT-GR x TCSn::GFP and 35S::NTT-GR x pAHP6::GFP crosses, seedlings were directly mounted in propidium iodide (PI, 1 µg/ml). PI ex: 561 nm, em: 617–700 nm. GFP (ex: 488 nm, em 525– 570 nm). For immunostaining, Alexa Fluor 568 ex: 561 nm, em: 586–626 nm. Alexa Fluor 488 ex: 488 nm em: 506–537 nm. All confocal imaging was performed using multi-tracking to detect each fluorophore independently. For phenotyping and GUS expression pattern analyses, images were acquired with a Zeiss Stemi 2000-C stereo microscope, coupled with axiocam ERc 5s camera (Carl Zeiss). Y1H assays Binding of NTT to the promoter regions of putative target genes was tested in a Yeast one-hybrid system (Y1H), based on the Matchmaker Gold Yeast One-Hybrid protocol ( https://www.takarabio.com ). Promoter regions of IPT5, KAN1, CKX7, AHP6 and PUP10 ( Fig. 7A ) were amplified using primers indicated in Table S1, cloned in an Entry vector (pENTR-D topo, Invitrogen), and recombined with CZN1810, a Gateway-compatible version of pAbAi (Clontech; Danisman et al , 2012 )., the next steps were done as described in Duran-Medina et al ., 2025. The background growth for each bait clone is shown in Fig. S5. NTT as prey clone (AD-NTT, pDEST22, Invitrogen; Lozano-Sotomayor et al ., 2016 ) in the haploid PJ69-A yeast was mated with the bait clones and grown in YPAD medium. Diploid yeasts (promoter + AD-NTT) were selected on SD GLUC medium lacking tryptophan and uracil. To test protein-DNA interactions, diploid yeasts representing the possible combinations were plated on SD GLUC medium lacking tryptophan and uracil, with different concentrations of AbA, 250 ng mL -1 for KAN1, CKX7, IPT5-A, PUP10 and IPT5 ; 300 ng mL -1 for AHP6-A ; 700 ng mL -1 for AHP6 . Yeast growth was scored on the sixth day of incubation at 30°C. Assays of NanoLuc activity in plant extracts The same promoters of IPT5, CKX7, and AHP6 used for Y1H were cloned into pGWB601-NL3F10H ( Urquiza-García and Millar, 2019 ) using LR clonase mix (Invitrogen), to generate the reporter constructs pAHP6::NL, pIPT5::NL, and pCKX7::NL , which then were transformed into Agrobacterium tumefaciens GV3101. The assays were conducted following the methodology described by Becerra-García et al . (2023) . Statistical Analyses For all comparisons made, like root length, cytokinin contents, RT-qPCR, and NanoLuc, statistical analysis were performed using the Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001). All plots were made using R Studio ( R Studio Team, 2020 ; http://www.rstudio.com/ ) . Results NTT alters plant shape and is expressed in roots and shoots NTT constitutive overexpression alters the architecture of Arabidopsis organs and plants. One of these alterations is the development of irregular leaf margins, such as the formation of pronounced serrations or projections at the leaf borders ( Fig. 1C ). Similarly, induction of NTT activity by dexamethasone (DEX) in a 35S::NTT -GR line, leads to increased serrations or projections in leaf margins that are visible one week after induction ( Fig. 1A, B ). Besides the leaf phenotypes, a more immediate evident effect is the growth inhibition of the main root ( Fig. 1K ) and increased lateral root development (Fig. S1). The induced phenotype lasts for days but is not permanent. Many plants recovered a wild-type phenotype two weeks after the induction. Then, only about 40% of the induced plants still showed severe phenotype alterations. We considered that the induction phenotypes presented some similarities to cytokinin treated Arabidopsis seedlings ( Fig. 1D, E ). This contributed to the question of whether the cytokinin pathway was among the processes or pathways that were affected and possibly regulated by NTT. NTT alters CK content in seedlings To obtain information about cytokinin levels in the NTT overexpressors, we compared the cytokinin content between DEX-treated wild-type and DEX-induced NTT-GR plants. We used plants that had been induced for 1 week and analyzed shoots and roots separately. We found clear changes in cytokinin content after NTT induction, and interestingly, the changes in CK content in roots and shoots were different ( Fig. 1 F-J, Table S2). Total cytokinin content increased evidently in induced roots, and the total cytokinin species that increased were mostly ribosides, nucleotides, and glucosides. In shoots, fewer statistical differences in total cytokinin content were detected. When different cytokinin types were analyzed (table S2), again, most changes were observed in the root tissue. There, total isopentenyl (iP), trans-zeatin (tZ) and dihydrozeatin (DHZ) cytokinins were increased when the induced NTT-GR was compared to DEX-treated wild-type roots. From them, the content of almost all iP, tZ and DHZ derivatives (ribosides, nucleotides, and glucosides) increased, as also occurred with the biologically active tZ and DHZ. The exceptions were free iP, iP9G, and DHZ9G, which did not present a statistically significant change. From all the cytokinins analyzed, cis-zeatin (cZ) and all its quantified derivatives remained unchanged in the root. In the shoot, cZ riboside some DHZ glucosides content increased, while iP glucosides (iP7G and iP9G) diminished. In summary, NTT induction caused different effects in cytokinin content in the distinct tissues, mainly an increase on cytokinin levels in root tissues. NTT induction causes general and specific changes in gene expression To search for genes related to the cytokinin pathway, and to obtain a global view of the genes that could be regulated directly or indirectly by NTT, we performed comparative RNA-seq analyses. In young plants, gNTT-YPET reporter lines ( Crawford et al ., 2015 ) revealed NTT expression at the root tip ( Fig. 1L , incipient and very young lateral roots ( Fig. 1M ), and in the regions around the shoot apical meristem ( Fig. 1N ) and in vasculature of the hypocotyl, cotyledons ( Fig. 1O ), roots ( Fig 1M , arrows) and leaves (Chung et al., 2013; Marsch-Martinez et al ., 2014; Crawford et al ., 2015 ; Herrera-Ubaldo et al ., 2019 ). Expression at the carpel marginal meristem was also observed ( Fig. 1P ). However, despite expression in these tissues, due to redundancy ( Crawford et al ., 2015 ; Du et al., 2022 ), the loss of NTT/WIP2 function visibly affects only specific tissues of the gynoecium (transmitting tract and replum, Crawford et al., 2007 , Marsch-Martínez et al ., 2014 ), and leaf size at specific developmental stages ( Diaz-Ramirez et al., 2022 ). Moreover, the triple nww mutant is severely affected, since very young plants lack roots and can only be recovered by auxin treatments ( Crawford et al ., 2015 ). To avoid redundancy issues, an NTT-GR inducible line was used for the RNA-seq analyses. Moreover, since NTT is present in different tissues, we analyzed aerial and root tissues separately to obtain information about the regulation it exerts in these different contexts. We were also interested in investigating the regulation over time, discriminating between “early response genes”, putatively direct targets, and “late” or secondary targets. The experiment was carried out using 35S::NTT-GR seedlings four days after germination (dag), treated with a DEX or with a mock solution. Roots and shoots were collected 30 min or 8 h after DEX treatment, according to the table in Fig. S2. To reduce the chance of downstream regulation of genes by transcription factors that were induced or repressed by NTT, the translation inhibitor cycloheximide was included in the treatment of the samples that were collected at 30 min. Differential gene expression analyses were done between induced and the non-induced samples for each tissue and each time point ( Fig 2A ). 30 min after NTT induction we found 1546 differentially expressed genes in aerial tissue and 1302 in roots, and at 8 h we found 1057 and 1945, respectively ( Fig 2A ). Some DEGs were common in different tissue types and times, and 49 genes were found to be differentially expressed in all conditions. Download figure Open in new tab Figure 2. Analyses of differentially expressed genes (DEGs) obtained through RNA-seq analysis of roots and roots 30 minutes and 8 hours after NTT induction. (A) UpSet plot of DEGs found in the different conditions. Set size indicates the number of DEGs in shoots (aerial) and roots at 30 min or 8 hours after induction. Intersection size indicates the number of DEGs in the corresponding intersection. Only DEGs exclusive of a comparison or DEGs shared between same tissues or same induction times comparisons are shown. (B) Bubble plot of hormone-related GO Biological Process categories enrichment analysis. Bubble size and color represent the false discovery rate, with larger and darker points indicating lower FDR values. (C) Heatmap of the log2FC values for cytokinin-related DEGs. The full DEGs dataset is presented in Supplementary Dataset S1. We noticed that in the case of roots there was a greater number of downregulated than upregulated genes. Conversely, in the aerial tissue there were more upregulated genes, about 70% of the genes, while almost 60% of the genes were downregulated in roots 30 min after induction (Dataset S1). Considering only DEGs with a log2 fold change of 2 and greater, these percentages increase, making more than 90% upregulated in aerial tissue (8 h), contrasting with more than 80% downregulated in roots (at 30 min). Some of the genes that were found to be differentially expressed are genes related to development, such as CUP SHAPED COTYLEDON 1 and 2 ( CUC1 and 2, Aida et al., 1997), the knotted1-like genes KNAT1 , 4 , 6 and 7 ( Hake et al ., 2004 ; Bharathan et al ., 1999 ) , ASYMMETRIC LEAVES 2 (AS2, Semiarti et al ., 2001 ), KANADI (KAN, Bowman et al., 2001 ), WUSCHEL-related homeobox 4 (WOX4, Ji et al ., 2010 ), SHORT ROOT (SHR, Helariutta et al ., 2000 ) , and NGATHA3 (NGA3, Trigueros et al., 2009 ) , among others, and are very interesting as candidate NTT targets (Fig. S3B). Most were up or downregulated in specific tissues or at specific times, except for SHR, downregulated in 3 comparisons, and KAN, downregulated in all 4 comparisons. Interestingly, NTT itself was also found to be downregulated after induction in three comparisons (Fig S3B). Next, a gene ontology (GO) enrichment analysis was done using both the complete lists and separating the up and downregulated genes. When exploring enrichment of DEGs belonging to certain GO categories in the differentially expressed gene datasets, we noticed that some were only enriched in the root or aerial tissue datasets, or at those obtained at earlier or later times, and others were found to be enriched in all (Dataset S2). Some of these categories included genes related to different hormonal pathways, from ethylene to auxin, gibberellin, jasmonate, abscisic acid, salicylic acid and cytokinin ( Fig. 2B , S3A). NTT affects the expression of cytokinin genes We then explored the possible regulatory role of NTT in the cytokinin pathway. Moreover, considering that NTT is a target of the ARF5/MP, and that the auxin and cytokinin pathways are frequently related, we also searched for auxin-related genes (Fig. S3C), besides cytokinin-related genes ( Fig. 2C ) in the DEG lists of roots and shoots at the two times. The DEG lists contained several genes corresponding to these hormonal pathways, and were related to biosynthetic, transport, signaling and response activities. For most of them, NTT affected their expression in a tissue and time-after-induction manner. The auxin-related genes included those responsible for the transcriptional response to auxin, Auxin Response Factors, and their interactors Aux/IAA proteins ( Roosjen et al ., 2018 ; Powers and Strader, 2019 ). Interestingly, ARF5/MP was found to be upregulated in the root 8 h after NTT induction. Moreover, besides Aux/IAA genes, differences in expression were detected in many genes belonging to the two other classes of early/primary auxin response genes, Gretchen Hagen 3 (GH3) and Small Auxin Upregulated RNAs ( SAURs) ( Hagen and Guilfoyle, 2002 ). Finally, genes coding for the auxin receptors AUXIN-SIGNALING F-BOX (AFB) 2 and 3 ( Prigge et al ., 2020 ), for the PIN1, 3, 5 and PILs transporters, and for the biosynthetic enzyme YUCCA9, also changed their expression in some of the tissues after NTT induction. Most changes in auxin gene expression were from low to moderate, with IAA5, GH3.1 and GH3.3 being the most upregulated of the auxin-related genes, while the most downregulated genes were SAURs . On the other hand, some cytokinin-related genes presented a greater change in expression upon NTT induction ( Fig. 2C ). For this pathway, again genes involved in different processes were differentially expressed, some were up and some downregulated in a tissue and time-specific fashion, indicating that the effect of NTT in this pathway is complex. These genes included the WOL/AHK4 cytokinin receptor ( Mähönen et al ., 2000 ; Yamada et al., 2001), the downstream phosphotransfer or pseudo-phosphotransfer proteins AHP4 and AHP6 ( Mähönen et al ., 2006 ), and the transcriptional regulators type-B ARRs ARR1, ARR10 and ARR11 ( Hill et al ., 2013 ). The expression of the negative regulator KISS ME DEADLY 1 (KMD1 ), that targets type-B ARRs for degradation ( Kim et al ., 2013 ), and some type-A ARR negative regulators ( To et al ., 2004 ), including ARR5, 6 and 15, whose repression is important for SAM development (Liebfried et al ., 2005), was also altered. Cytokinin transporter genes such as ABCG14 ( Zhang et al ., 2014 ) and PUPs (reviewed in Durán-Medina et al ., 2017 ) were also affected. Cytokinin metabolism genes also presented changes in expression. Related to biosynthesis, we found ISOPENTENYL TRANSFERASE 5 ( IPT5 ; Miyawaki et al., 2004) and LONELY GUY 4 and 7 ( LOG4 and 7, Tokunaga et al ., 2012 ). Related to cytokinin inactivation, genes coding for the N-glucosyltransferase UGT76C2 ( Wang et al ., 2011 ), and the genes that encode for enzymes responsible for cytokinin degradation CKX4 and 7 ( Werner et al ., 2001 , 2003 ; Köllmer et al ., 2014 ) presented expression changes upon NTT induction. In general, cytokinin-related genes presented greater changes in expression than auxin-related genes. We decided to analyze IPT5 , AHP6 and CKX7 as genes that act at different steps of the cytokinin pathway and could act downstream NTT, because of their reported expression patterns ( Miyawaki et al ., 2004 ; Atta et al ., 2009 ; Köllmer et al ., 2014 ) already suggested some coincidences with NTT expression. IPT5 has been reported to be expressed in incipient lateral roots ( Miyawaki et al ., 2004 ), like NTT . Moreover, the transcriptional fusion reporter line pIPT5::GUS showed expression in the vasculature of different tissues and the SAM, where NTT expression was also detected. The pAHP6::GFP reporter line shows expression in the root vasculature (Mähönen et al., 2006; Moreira et al., 2013). Finally, CKX7 is expressed in the transmitting tract and the vasculature of different organs ( Köllmer et al ., 2014 ), including the replum ( Di Marzo et al., 2020 ), where NTT is also expressed ( Chung et al ., 2013 ; Marsch-Martínez et al ., 2014 , and Fig. 1 M, O). NTT induction causes an increase in IPT5 expression Given the increase in cytokinin content detected upon NTT induction, we first focused on studying the cytokinin biosynthesis gene IPT5 . We analyzed the changes in its expression after NTT induction by RT-qPCR and found an increase in IPT5 expression as occurred in the transcriptome analysis. The ratio of the increase was also comparable to the ratio found in the transcriptome analyses ( Fig. 3A ). Download figure Open in new tab Figure 3. Increase of IPT5 expression after NTT induction in RT-qPCR and genetic assays. (A) IPT5 relative expression at 30 min and 8 h after NTT induction in DEX treated (+DEX, dark green) and mock treated (-DEX, light green) root tissue. The y-axis indicates the fold change. Three biological replicates were used for each measurement. Values are given as mean ± SE. Asterisks above the bars indicate significant differences between the respective values (* p<0.05, ** p < 0.01, Student’s t-test). (B-H) 35S::NTT-GR x pIPT5::GUS seedlings mock treated or induced with DEX and stained 48 h after induction. (B-D) seedlings that were geminated directly on either mock (B, C) or DEX (D), supplemented medium and stained 7 dag. Red arrow indicating absence of staining in main root (B) and staining in the tip of a lateral root (C); (D) seedling germinated directly in media with DEX, red arrows indicate staining in elongation zone and at the hypocotyl-root junction, barely detected in non-induced plants. (E, G) mock treated seedlings, showing staining in SAM and slightly in cotyledon vasculature; (F, H) seedlings induced with DEX. The red arrow indicates GUS staining at the hypocotyl-root junction, similar to D. Also, there is an increase of staining in vasculature. G and H are a close ups from E, F respectively. Scale bars 0.1 mm in (B, C), 1 mm in (E, F), and 0.5 mm in (D, G, H). We crossed an IPT5 reporter line ( pIPT 5:: GUS, Dello Ioio et al., 2007) with the NTT inducible line to evaluate its spatial expression upon NTT induction. In 35S::NTT-GR x pIPT 5:: GUS seedlings, we observed an increase in GUS staining 48 hours after NTT induction ( Fig. 3B-H ). In the shoot, the increase in pIPT 5:: GUS expression was detected particularly in the SAM and in the vasculature of developing leaves ( Fig. 3E-H ). Also, we observed GUS staining at the base of the hypocotyl, which was barely detected in non-induced plants ( Fig. 3D, F ). Moreover, strong GUS staining was detected in the root elongation zone in seedlings that were directly germinated in DEX-supplemented medium, contrasting with no staining in this zone in non-induced seedlings ( Fig. 3B-D ). Therefore, IPT5 expression showed a clear increase after NTT induction. This increase appeared to be specific to certain tissues, and more intense in roots. NTT affects cytokinin response After finding that NTT could alter cytokinin content and IPT5 expression, we then evaluated the effect of NTT on cytokinin signaling. Since AHP6 was one of the signaling genes with changed expression, we explored its expression after NTT induction both by RT-qPCR and using the pAHP6::GFP reporter line (Fig. S4). When root tips of non-induced and induced seedlings were visualized, we observed a decrease in the GFP signal in the induced seedlings (Fig. S4A-D), the same tendency as the transcriptome and RT-qPCR analyses (Fig. S4E). Because the transcriptome analyses revealed that more signaling genes were also affected by NTT induction, we explored the overall effect of NTT induction in the cytokinin pathway in roots, using a cross of the NTT inducible line with the TCSn::GFP cytokinin reporter line (Zurcher et al ., 2013). We analyzed cytokinin transcriptional response 48 h after NTT induction and observed that in the meristem cell elongation transition zone, TCSn::GFP signal was visible in the vascular bundle and epidermis of induced roots but absent in mock-treated roots ( Fig. 4A, B ). The same occurred in the mature region of the root, where the GFP signal was observed in the vascular bundle and epidermis of induced plants, but barely detected in non-induced plants ( Fig. 4C, D ). Conversely, at the root apical meristem, though slight differences were observed, the TCSn::GFP pattern was similar in mock and DEX treated plants, with a clear signal in the lateral root cap, columella, stele initials, and the cells above them ( Fig. 4E, F , Zürcher et al., 2013). However, 7 days after induction, no TCSn::GFP signal was visible in the root apical meristem anymore, and meristem organization was altered ( Fig. 4G ). Nevertheless, in these same roots, but at upper regions, the TCSn::GFP signal was still visible in newly formed lateral root primordia ( Fig. 4H ). Download figure Open in new tab Figure 4. Cytokinin signaling ( TCSn::GFP ) response to NTT induction. (A-H) 4 dag 35S::NTT-GR x TCSn::GF seedlings either mock or DEX treated, observed 2 dai (A-F) or 7 dai (G-H). (A) In mock treated roots, no signal is observed beyond the meristematic zone; (B) In contrast, expression is detected in the vasculature between the proliferation and transition domains in induced plants 48 h after induction (2 dai). (C, D) In differentiated cells in mature roots, no expression can be seen in mock treated plants (C), while expression is detected in DEX treated seedlings 2 dai (D). (E) Mock treated RAM observed two days after treatment. (F) Changes in TCSn expression in DEX treated roots are almost imperceptible in the root tip 2 dai, but an expansion of the signal upwards in the second external cell layer was detected when compared to mock treated roots (white arrow). (G) More evident changes can be noticed 1 week after induction, when the morphology of the RAM seems clearly different, and the signal is lost in DEX treated plants. (H) This same plant still shows expression in young lateral roots. Scale bars in (A, B) represent 50 µm, and in the rest of the panels 25 µm. Therefore, cytokinin signaling is affected after NTT induction in diverse ways depending on the tissue and time after induction, with some regions increasing and others decreasing CK response. CKX7 mediates part of the NTT induction phenotype Intriguingly, even though different cytokinin biosynthesis genes were upregulated and CK content was increased, also the expression of cytokinin degradation or inactivation genes increased after NTT induction. Moreover, CK signaling increased or decreased depending on the tissue, suggesting a complex effect of NTT on the CK pathway. CKX7, that encodes a cytokinin degradation enzyme, was found among the genes that were differentially expressed. It was upregulated in three of the four conditions and showed the greatest upregulation compared to other auxin- or cytokinin-related genes. When its expression was evaluated by RT-qPCR, a clear increase was again detected ( Fig. 5A ). We found puzzling that a cytokinin degradation gene was upregulated by NTT along with IPT5 , and we investigated whether CKX7 could also be involved in the NTT induction phenotype. For this, we analyzed the effect of the loss of CKX7 function in the 35S::NTT-GR phenotype. Download figure Open in new tab Figure 5. NTT induction phenotype in ckx7 seedlings. (A) CKX7 relative expression at 30 min and 8 h after treatment in root tissue treated with DEX (+DEX, dark yellow) or mock-treated (-DEX, light yellow). The y-axis indicates the fold change. Three biological replicates were used for each measurement. Values are shown as mean ± SE. Asterisks above the bars indicate significant differences (*p<0.05, Student’s t - test). (B-I) Mock of DEX treated 35S::NTT-GR compared to 35S::NTT-GR x ckx7 plants. Shoots of 35S::NTT-GR (B, C) and 35S::NTT-GR x ckx7 (F, G) seedlings, were either mock treated (B, F), or induced with DEX (C, G). Roots of 35S::NTT-GR (D, E) and 35S::NTT-GR x ckx7 (H, I) seedlings, either mock treated (D, H), or induced with DEX (E, I). Red arrows highlight irregularities in leaf margins or callus formation in roots. Scale bars represent 1 mm in all images. Uninduced 35S::NTT-GR and 35S::NTT-GR ckx7 plants were very similar and resembled wild-type plants ( Fig. 5B, F ). As expected, DEX-induced 35S::NTT-GR plants developed leaf abnormalities as shown before ( Fig. 1B , 5C). Strikingly, the ckx7 mutation largely suppressed this effect. The leaves of the plants did not form pronounced leaf projections or serrations when induced, although they still presented shorter petioles ( Fig. 5G ). Most leaves in the 35S::NTT-GR ckx7 individuals examined after induction did not present serrations at all or only very slight serrations. Therefore, CKX7 activity appears to be required for the irregular leaf margin phenotype caused by NTT induction. Moreover, the leaves of induced 35S::NTT-GR ckx7 were wider and larger than those of induced 35S::NTT-GR ( Fig. 5C, G ). Interestingly, induced 35S::NTT-GR ckx7 plants presented an unexpected root phenotype, not observed in induced 35S::NTT-GR plants ( Fig. 5D, E, H, I ). DEX-treated 35S::NTT-GR ckx7 roots developed white calli, clearly visible 16 days after induction, at the positions where secondary roots would usually emerge ( Fig. 5 I). Therefore, CKX7 prevents the formation of calli in the roots of induced plants, probably by locally counteracting the increased CK content. We also tested the effect of adding cytokinins (BAP) to both 35S::NTT-GR and 35S::NTT-GR ckx7 plants. Uninduced 35S::NTT-GR that were treated with BAP produced serrated leaves with short petioles and a profusion of trichomes (Fig. S6A). However, uninduced 35S::NTT-GR ckx7 were differently affected by the cytokinin treatment. Most of their leaves also presented serrations and shorter petioles, but they were evidently smaller, and some seedlings presented one larger leaf (Fig. S6E), suggesting that 35S::NTT-GR ckx7 seedlings display an increased response to the supplemented cytokinin, as would be expected. When 35S::NTT-GR seedlings grown on cytokinin-supplemented medium were induced by DEX, they developed much smaller leaves, shorter petioles, and more severe leaf serrations or projections at the leaf margin, than those grown on normal medium (Fig. S6A, B). 35S::NTT-GR ckx7 plants under the same conditions also developed leaves with very short petioles (Fig. S6F), but these leaves were evidently wider and larger than those of cytokinin treated 35S::NTT-GR plants. Instead of serrations, their edges presented undulations, leading to an atypical shape of lettuce-like appearance (Fig. S6F). The undulations and trichomes were more evident (Fig. S6F) than those of induced 35S::NTT-GR ckx7 plants without cytokinins ( Fig. 6G ). Download figure Open in new tab Figure 6. Cytokinin immunolocalization in root tips. Whole-mount root tips of 1 dag Col-0 (A, C), nww (B, D) and 35S::NTT-GR (E-H) seedlings, either mock treated (E, G), or induced with DEX (F, H). LUT images of the localization of trans-Zeatin-riboside (A, B, E, F), or Isopentenyl adenosine (C, D, G and H). Scale bars represent 20 µm. None of the uninduced 35S::NTT-GR ckx7, 35S::NTT-GR, or wild-type plants produced calli when treated with cytokinins (Fig. S6C, G). When induced 35S::NTT- GR seedlings were treated with cytokinin, only one in 40 plants developed calli (Fig. S6D), in contrast to 40 out of 40 induced, cytokinin-treated, 35S::NTT-GR ckx7 plants (Fig. S6H). Taken together, the results indicate that CKX7 acts downstream of NTT and plays a significant role in the phenotypes that NTT induction causes in the plants, possibly by modulating the cytokinin content locally. The CK localization pattern is altered in nww mutants and after NTT induction After observing the changes in overall cytokinin content and the relevance of CKX7 in the NTT induction phenotype, which would affect local cytokinin content, we sought to explore cytokinin localization in the mutant and the inducible overexpression backgrounds. NTT expression is localized to a few cells in the meristem of the young developing root ( Fig 1L ), where it plays an essential but redundant function with WIP4 and WIP5 ( Crawford et al ., 2015 ). Therefore, the triple mutant lacks roots ( Crawford et al ., 2015 ), which can be distinguished in very young seedlings. Therefore, we analyzed the cytokinin localization pattern by immunolocalization at the root tip in young wild-type and inducible overexpressors, or the equivalent region in the nww mutant ( Fig. 6 ). Trans-zeatin and isopentenyl adenine content was changed in roots after NTT induction. Moreover, trans-zeatin content is reduced to only 9% of the normal wild-type content in plants that overexpress CKX7 (Kollmer et al ., 2014). Therefore, we used anti-trans-zeatin riboside and anti-N6-isopentenyladenosine antibodies to assess their localization. Interestingly, the pattern of cytokinin distribution in the root tip was clearly affected both in the mutants and the induced lines. Wild-type ( Fig. 6A, C ) and 35S::NTT-GR non-induced ( Fig. 6E, G ) roots had a similar pattern of cytokinin distribution. In both, trans-zeatin riboside and isopentenyl adenosine were abundant above the quiescent center of the meristem and relatively low below it and in the columella. In the nww mutant, however, they were more homogeneously distributed towards the apex, and reduced towards the top ( Fig. 6B, D ), compared to wild-type roots ( Fig. 6A, C ). In the case of the induced 35S::NTT-GR plants, they presented a homogeneous distribution in the whole root tip, which already displayed an altered size and morphology ( Fig. 6 F, H), compared to non-induced plants ( Fig. 6 E, G). In conclusion, both the mutant and the inducible overexpressor line showed clear alterations in the distribution of the immunodetected cytokinin types. NTT binds to the promoter of cytokinin-related genes The effects of NTT in cytokinin homeostasis and the expression of cytokinin-related genes were very clear. Because the translation inhibitor cycloheximide was used in the transcriptome and RT-qPCR analyses at 30 min, some differentially expressed genes could be direct NTT targets. We tested whether the regulatory regions of the three selected genes ( IPT5 , AHP6 and CKX7 ) were bound by NTT, first using Y1H assays. We sought predicted NTT binding sites in the promoters and coding regions ( Persikov and Singh, 2014 ; Herrera-Ubaldo et al., 2019) of these genes and cloned genomic fragments containing them ( Fig. 7A ). We also tested other promoter fragments. For IPT5 , we tested a 1945 bp promoter, a 70 bp promoter fragment ( IPT 5-A), and a 435 bp fragment inside the coding region that contains two predicted binding sites ( IPT 5-B). The AHP6 promoter presented a single site, 720 bp upstream of the ATG. We tested a 1900 bp promoter fragment, and a 450 fragment around the predicted site ( AHP6 -A). For the CKX7 regulatory region, we used a fragment comprising 969 bp upstream the ATG, containing two predicted sites ( Fig. 7A ). Download figure Open in new tab Figure 7. Y1H and NanoLuc assays to test NTT binding and regulation of candidate target genes. (A) Schematic representation of the tested genomic regions, with large promoter fragments shown in green and short or internal fragments in orange. PBS: Putative Binding Site. (B) Y1H analyses of NTT protein interaction with the target promoters and fragments depicted in (A). (C) NanoLuc assays. The constructs used as effector and reporters are indicated in the diagrams. The graph presents relative NanoLuc activity, detected in each combination. Values are given as mean ± SE and asterisks indicate a significant difference (* p ≤ 0.01, ** p ≤ 0.001, Student’s t-test, RLU: Relative Luminescence Units). We found that NTT binds the three complete promoters of IPT5, CKX7 and AHP6 ( Fig. 7B ). NTT also bound to the short IPT 5-A fragment (Fig. S5). The other fragment, IPT5-B , could not be tested due to autoactivation (Fig. S5). Moreover, NTT bound to the AHP6-A fragment ( AHP6 -A, Fig. 7B ). Therefore, it appears that NTT could be directly regulating the expression of CKX7, IPT5 and AHP6. To further confirm other differentially expressed genes, we also evaluated NTT binding to the promoters of KAN , which was downregulated in all samples, and PUP10 , a cytokinin transporter downregulated in roots at both early and late times. NTT also bound to these promoters in the Y1H test (Fig. S5). Next, we validated and explored the regulation of NTT on IPT5, CKX7 and AHP6 promoters in planta using a transient luciferase assay. The same complete IPT5, CKX7 and AHP6 promoters used for the Y1H assay were fused to NanoLuc ( Urquiza-García and Millar, 2019 ; Becerra-García et al ., 2023 ). These promoters were transiently expressed in tobacco leaves, in combination with a 35S::NTT construct ( Fig. 7C ). For pIPT5 and pCKX7 , a clear activation of expression was revealed by a marked increase in bioluminescence when 35S::NTT was co-expressed with the promoter constructs ( Fig. 7C ). This indicates that NTT positively regulates these promoters in this system. These genes were found to increase their expression in the transcriptome data and by RT-qPCR 30 min after NTT induction, suggesting that they may be target genes positively regulated by NTT. In the case of the AHP6 promoter, although there was an increase in NanoLuc activity when coexpressed with NTT, it was not statistically significant, This gene was downregulated in the root, in the transcriptome analyses and RT-qPCRs performed in induced seedlings and may be downregulated or upregulated in other tissues and stages. Together, the results of this work indicate that NTT can modulate the cytokinin pathway at different steps in a complex fashion. Discussion In this work, we investigated whether the NTT/WIP2 transcription factor regulates genes involved in the cytokinin pathway. Some phenotypes of NTT overexpression lines are similar to those of plants that have been grown in cytokinin-supplemented medium ( Chung et al ., 2013 ; Marsch-Martínez et al ., 2014 ; Skalák et al ., 2019 ; Dello Ioio et al ., 2007 ; Liu et al ., 2022 ; Werner et al., 2003). Also, cytokinin treatments can recover replum development in the Arabidopsis rpl ntt double mutant, which lacks this tissue ( Zuñiga-Mayo et al ., 2018 ). Moreover, STK, an interacting partner of NTT ( Herrera-Ubaldo et al ., 2019 ) regulates cytokinin levels during fruit development (Di Marzo et al., 2020), and NTT/WIP2 has been found to interact with cytokinin signaling system components ( Dortay et al ., 2008 ). In addition, NTT/WIP2 is a target of ARF5/MP, thought to act downstream auxin signaling ( Crawford et al ., 2015 ), and the auxin and cytokinin pathways are frequently closely related ( Schaller et al ., 2015 ). When revising published expression patters of the cytokinin response TCS marker (Muller and Sheen, 2008), there seemed to be coincidences with reported NTT expression during very early stages of root development ( Crawford et al ., 2015 ), the development of the gynoecium, particularly in the inner tissues ( Crawford et al ., 2007 ; Marsch-Martinez et al ., 2012), and the vasculature (as revealed by the TCSn::GFP line; Zürcher et al ., 2013 ; Skalák et al ., 2019 ). Accordingly, changes in cytokinin content were found in seedlings where the activity of NTT was induced. Curiously, the changes in content were different depending on the tissue (roots or shoots), suggesting that NTT could have a distinct, tissue-specific effect on cytokinin content. NTT affects cytokinin genes and has a tissue-specific effect on gene expression Global expression analyses provided information about candidate cytokinin genes that could be modulated by NTT. An inducible NTT line was used because of the high degree of redundancy of NTT with other WIP family members ( Du et al ., 2022 ) and the severe phenotype of combined mutations ( Crawford et al ., 2015 ). Interestingly, the differentially expressed cytokinin genes participate in a positive (such as the biosynthetic enzymes IPT5 or LOG4 or the type-B ARRs) or a negative way (such as cytokinin degradation enzymes CKX7, or the negative signaling components AHP6, or type-A ARRs) in cytokinin metabolism, signaling or response. Moreover, the changes in expression of cytokinin-related genes were higher than for genes belonging to the auxin pathway. This suggests that NTT could modulate the cytokinin pathway at diverse steps and possibly have a stronger effect in it than in the auxin pathway at a global level. Regarding the overall data obtained from the RNA-seq experiments, many interesting DEGs were found and worth of further analyses in future studies, like CUC1 and 2, KNAT1, 4, 6 and 7, AS2, KAN, WOX4, SHR and NGA3 . The data obtained from roots and shoots was analyzed separately, and some DEGs were found in both tissues at both times, suggesting that they may be directly or indirectly regulated by NTT independently of the tissue. One example of these common DEGs was KAN (Fig. S3B, Bowman et al., 2001). Its promoter is bound by NTT in Y1H experiments (Fig. S5) and was also found to be downregulated and a direct target of NTT (Gomez-Roldán et al ., 2020) and the closely related WIP5 (O’Malley et al., 2016). However, we also found DEGs, the majority, for which the change in expression was dependent on the tissue and time after induction. Therefore, NTT might have tissue-specific activity and affect genes differently depending on the context. In the GO category enrichment analyses, again, both common and tissue specific categories were present, and some were similar to those found in other reports. For example, in Gerbera hybrida 45-day-old flower bud clusters that overexpress GhWIP2, an NTT homologue, GhWIP2 was reported to act as a repressor, suppressing cell expansion during organ growth by modulating crosstalk between gibberellins, abscisic acid, and auxin (Ren et al., 2018). Categories related to these hormones were also enriched in our transcriptome comparisons. WIP1 and 2 proteins have been reported to integrate hormonal signals to regulate organ growth and adaptation to stress ( Gomez-Roldan et al ., 2020 ). Most cell wall, development, growth, and morphogenesis categories were enriched among downregulated genes in whole seedling samples of inducible WIP1 and WIP2/NTT lines analyzed by Gomez-Roldán et al ., 2020. In contrast, when the tissues were analyzed separately (this work), cell wall, development and morphogenesis categories were prominently present in the root 8 h downregulated genes but were less prominent in aerial tissue DEGs. Stress categories, enriched among the upregulated genes in whole seedlings (Gomez-Roldán et al ., 2020), were also frequent in both tissues (this work). However, while genes belonging to general hormone categories were present in all conditions, those belonging to specific hormonal categories, such as jasmonic acid, ethylene, gibberellins, cytokinins and abscisic acid, are enriched differently in the distinct tissues and times (Dataset S2). Therefore, NTT may regulate genes and processes both in a general and a tissue-specific (young shoot or root, as observed for changes in cytokinin content), or maybe even stage-specific manner. This may also occur in other organs and tissues, such as the gynoecium, the vasculature, or specific cells at certain stages, like during early embryo development. NTT modulates cytokinin homeostasis Since our aim was to study the effect of NTT on the cytokinin pathway, we focused on cytokinin genes, particularly those with reported expression in tissues where NTT is also expressed, such as transmitting tract, vasculature and young roots, and where cytokinin homeostasis plays an important role ( Miyawaki et al ., 2004 ; Atta et al ., 2009 ; Köllmer et al ., 2014 ; Miyawaki et al ., 2004 ; Mähönen et al., 2006; Moreira et al., 2013; Köllmer et al ., 2014 ; Di-Marzo et al ., 2020; Muller and Sheen, 2008; Chung et al ., 2013 ; Marsch-Martínez et al ., 2012 and 2014; Zurcher et al., 2013). Cytokinin levels are the result of the balance between biosynthesis and degradation or inactivation pathways ( Kieber and Schaller, 2018 ), and transport impacting their distribution among different tissues ( Hirose et al ., 2008 ). For biosynthesis, the rate-limiting step is catalyzed by ADP/ATP isopentenyl transferases (IPTs, Miyawaki et al ., 2004 ). IPT increased expression results in higher levels of iP ( Kakimoto, 2001 ; Sun et al ., 2003 ; Zubko et al ., 2002 ), while in ipt mutants, iP and tZ-type cytokinin species are reduced ( Miyawaki et al ., 2006 ). IPT5 was increased upon NTT induction, and accordingly, a higher content of total, and specially iP, tZ and DHZ (a reduced form of tZ) types, was detected in the roots of induced plants. Interestingly, even though the inducible construct contains a 35S promoter, pIPT5::GUS expression increased only in specific regions of the plant, suggesting that IPT5 , and possibly many other target genes, are subject to tissue specific regulation. The expression pattern of the cytokinin response marker TCS was also affected by NTT induction. Cytokinins are perceived by membrane receptors which start a signaling phosphorelay cascade that activates type-B ARR transcription factors ( Kieber and Schaller, 2018 ). The TCS signal both increased and appeared in ectopic regions or decreased and disappeared from its normal regions of expression, depending on the region of the tissue, root in our case, and time after induction, again suggesting cytokinin homeostasis is affected after NTT induction in different ways depending on the tissue and time after induction. The TCS marker reports the transcriptional response to cytokinin, reflecting both content and signaling activity (Muller and Sheen, 2008; Zürcher et al ., 2013 ). Therefore, the observed changes could be due to expression differences in genes acting at different steps of the pathway, as found in the transcriptome comparisons. One of these genes is AHP6 , which plays an important inhibitory role in the signaling cascade (Mähönen et al., 2006), and its expression was confirmed to be altered upon NTT induction. Cytokinins could be synthesized locally, transported, act as an autocrine or paracrine signals, and be catabolized at nearby or distant sites ( Hirose et al ., 2008 ). Moreover, cytokinins can have opposite roles depending on the developmental stage of an organ or tissue ( Skalák et al ., 2019 ; Di Marzo et al ., 2020 ). Therefore, regions where cytokinin was crucial, may require cytokinin degradation to further proceed in their development. For degradation, the cytokinin catabolic enzymes CKX are key in controlling cytokinin levels in plant tissues ( Werner et al ., 2001 ). CKX7 presented a high change in expression in the global expression analyses, RT-qPCRs and transactivation assays. From the members of the Arabidopsis CKX family, it is the only localized at the cytosol ( Werner et al ., 2001 , 2003 ; Köllmer et al., 2014) and has been proposed to have unique access to a specific subcellular pool of cytokinin, relevant in tissues such as the root procambium (Köllmer et al., 2014). Its overexpression causes root inhibition and a higher reduction on c Z-type cytokinins ( c Z and c Z9G) and iP9G, in comparison to CKX1 and CKX2 (Köllmer et al., 2014). Interestingly, after NTT induction, while the content of most cytokinin types increased in induced roots, cZ species were not, and iP, iP7G and iP9G content was even reduced in shoots. In Arabidopsis, CKX7 is expressed in the transmitting tract and root and shoot vasculature ( Köllmer et al ., 2014 ), two regions where NTT is also expressed. Moreover, CKX7 has been recently reported to be a target of STK, an NTT protein interactor ( Di Marzo et al ., 2020 ; Herrera-Ubaldo et al ., 2019 ), suggesting that CKX7 could be a common downstream target gene of both transcription factors. The expression found in the cycloheximide-treated samples of the transcriptome, RT-qPCR, transactivation, and Y1H assays also support this. Interestingly, the lack of CKX7 function in the NTT inducible line led to a clear reduction in the induction phenotype, indicating that at least partially, NTT acts through CKX7 . The reduction of serrations was unexpected, and we speculate that the local degradation of cytokinins by CKX7 contributes to patterns of cytokinin accumulation that are involved in the formation of serrations or morphological changes in leaf edges. Moreover, the appearance of calli in NTT induced ckx7 roots could be related to the reduced capacity of the plants to counteract the cytokinin excess promoted by NTT, in the absence of CKX7 function. NTT modulates the localization pattern of cytokinins The auxin-cytokinin interplay has been compared to the yin-yang concept of complementarity ( Schaller et al ., 2015 ). They are connected both positively and negatively and together guide the development of the early embryo (Muller and Sheen, 2008), the vasculature in roots (Mähönen et al., 2006; De Rybel et al., 2014; Ohashi-Ito et al., 2014), or the gynoecium (Marsch-Martinez et al., 2014; Zuñiga-Mayo et al., 2014), among others. We found that NTT, an ARF5/MP target ( Crawford et al ., 2015 ), regulates cytokinin related genes, suggesting that it can link these two pathways ( Fig. 8 ). Callus development is another process in which both auxin and cytokinin play a crucial role ( Skoog and Miller, 1957 ). The formation of calli in the induced 35S::NTT-GR ckx7 line could point to the connection between both pathways. Moreover, interestingly, in ovules, a different ARF5/MP isoform is expressed in cells with low auxin and high cytokinin concentration ( Cucinotta et al ., 2021 ), suggesting that MP can be related to both. Download figure Open in new tab Figure 8. A model of NTT regulation. Proposed model of NTT acting as a link between the auxin and the cytokinin pathways. Additional stimuli beyond auxin and ARF5/MP may regulate NTT expression. NTT modulates the expression of genes involved in cytokinin biosynthesis, signaling, and degradation, among others. Another MP target, TARGET OF MONOPTEROS 5 ( TMO5 ), with TMO5-LIKE1 ( T5L ) and their interacting partner LONESOME HIGHWAY (LHW), activate the expression of the cytokinin genes LOG4 and AHP6 (Ohashi-Ito et al., 2007, 2014; De Rybel et al ., 2014 ). These genes, together with CKX3 , lead to the creation of a local, nonresponding cytokinin source within the root vascular tissue that is essential for its proper development ( Ohashi-Ito et al ., 2014 ; De Rybel et al., 2015; Yang et al ., 2021 ). We speculate that a similar situation, albeit through the regulation of different genes, could occur for NTT. CKX7 upregulation, together with IPT5 and AHP6 modulation, if simultaneous, may generate cytokinin differential regions, either in content or in response within the NTT-responsive regions. CKXs have been proposed to participate in the maintenance of cytokinin local developmental fields ( Werner et al ., 2001 ). Moreover, Köllmer et al., 2014 noticed that both pCKX7 and the cytokinin reporter TCSn::GFP (Zürcher et al., 2013) are expressed in an unequal distribution in the embryo sac and already proposed that CKX7 could establish a cytokinin gradient in the cytoplasm (Köllmer et al., 2014). It would be interesting to explore these possibilities. Another hypothetical scenario to explain the activation of both IPT5 and CKX7 by NTT could be a short temporal delay between their activation. Interestingly, some of the tissues where TCSn::GFP and NTT coincide present early cytokinin signaling that is lost at later stages, like at the medial domain of the gynoecium (Marsch-Martinez et al ., 2014; Reyes-Olalde et al., 2017), or the first division of the hypophysis in the embryo, after which one cell retains, and one loses TCS signal (Muller and Sheen, 2008). NTT is expressed in these two tissues ( Crawford et al ., 2007 , 2015 ) and could first activate IPT5 to increase cytokinin content, and then CKX7 to decrease it when required. Nevertheless, even if the mechanism by which the regulation of the cytokinin pathway by NTT works is not yet clear, it is evident that NTT impacts cytokinin distribution. The immunolocalization assays performed for two cytokinin species in the nww mutant and the inducible line, showed clear changes in their distribution pattern. The distribution of specific CK species is the result of production in certain tissues, transport, and modification in the same or in other tissues, as CK species are interconverted into one another ( Mok and Mok, 2001 ; Hirose et al ., 2008 ), and the distribution of active forms will impact the cell transcriptional response ( Kieber and Schaller, 2018 ). Though IPT5 and CKX7 may play a prominent role, it is possible that PUP10 (whose promoter is bound by NTT), LOG4 and 7, AHP6, ARRs, KMD1 , and WOL/AHK4, genes that were differentially expressed upon NTT induction, among others, could also be involved in the effect of NTT in the cytokinin distribution and transcriptional response. We speculate that the action of NTT in the tissues where it naturally acts, might be also dependent on the tissue and its developmental stage, such as the cells that will give rise to the root meristem, the transmitting tract, and possibly and vasculature, for which a role has not been described, but expression has been observed ( Chung et al ., 2013 ; Diaz-Ramirez et al ., 2022 ). In this regard, CKX7, AHP6, or AHK4/WOL, KAN , have been reported to also be expressed in vascular tissues or the pericycle (Mähönen et al., 2000, 2006; Emery et al., 2003; Eshed et al., 2004; Hawker and Bowman, 2004 ; Köllmer et al., 2014). IPT5 was also expressed in the vasculature. Moreover, the fact that the nww mutant is defective in hypocotyl vasculature formation (Crawford et al., 2015) may support this hypothesis. In conclusion, based on cytokinin quantification, global expression, binding, activation and genetic analyses, together with cytokinin immunolocalization in the mutant and overexpression background, we propose that NTT regulates cytokinin genes and can act as a link between the auxin and cytokinin pathways ( Fig. 8 ). It will be very interesting to further investigate the tissues and developmental stages where this modulation occurs, and whether it results in the generation of cytokinin fields, delayed gene activation or other mechanisms that guide development. Finally, we provide new shoot and root transcriptomics data at different time points, which indicate that NTT modulates a variety of processes, some in a general, and some in a tissue and time-specific fashion. Declarations Competing interests The authors declare that they have no competing interests. Funding The study was financed by the National Council of Science and Technology (CONACyT now SECIHTI), grant CB-2015-255069 and CF-2023-G-219. DDR was supported by Ph.D. fellowship 254467 and postdoc fellowship 386470, REBG by fellowship 747047, and JECV and YDM were supported by postdoc fellowships, all from CONACyT. DDR was also supported by a DAAD fellowship during a short stay in the University of Bielefeld at the group of MS and in the HHU at the group of RS. SDF acknowledges the CONACyT grants: FC-2015-2/1061, INFR-2015-253504 and CB-2017-2018-A1-S-10126, and the Marcos Moshinsky Foundation. Author’s contributions DDR and NMM conceptualized the research. DDR, NMM, REBG, HHU, YDM, EDA and JECV designed the experiments. ECV, REBG, DDR, HHU, and YDM performed the experiments. RCM performed Bioinformatic analyses. EDA contributed with confocal imaging. ON, MS, MDM, RS, LC and SdF contributed experimental resources and analyzed data. DDR and NMM contributed to manuscript writing and reviewing. NMM obtained financial resources and supervised the project. All authors read and approved the manuscript. SUPPLEMENTARY DATA Supplementary Figures Figure S1. Root phenotype of induced 35S::NTT-GR seedlings. Figure S2. Global expression experiment workflow. Figure S3. Bubble plot of GO enriched categories and heatmap of developmental and auxin-related differentially expressed genes. Figure S4. Change in expression of AHP6 after NTT induction. Figure S5. Y1H Autoactivation assays, and pKAN and pPUP10 experiments. Figure S6. NTT induction phenotype in ckx7 seedlings, treated with cytokinin. Figure S7. Root phenotypes and immunolocalization technical control. Supplementary Tables Table S1 Primers used in this work. Table S2 Cytokinin profile in shoot and root of Col-0 and 35S::NTT-GR line, under Mock or DEX treatment (pmol/g FW; Mean ±SE). Supplementary Datasets Dataset S1. Differentially Expressed Genes found in the Global Expression Analyses in different tissues. Dataset S2. Gene Ontology enrichment analyses. Acknowledgements We thank Brian Crawford for plant material and Andrew Millar for NanoLuc plasmids. We thank Andrea Gomez Felipe and Vincent Cerbantez Bueno for sharing some promoter fragments and Juan Carlos Ochoa for his help with RT-qPCR experiments. References Aida , M. , Ishida , T. , Fukaki , H. , Fujisawa , H. , and Tasaka , M . 1997 . Genes involved in organ separation in Arabidopsis: An analysis of the cup-shaped cotyledon mutant . The Plant Cell 9 : 841 – 857 . OpenUrl Abstract / FREE Full Text Appelhagen , I. , Huep , G. , Lu , G.H. , Strompen , G. , Weisshaar , B. , and Sagasser , M . 2010 . Weird fingers: Functional analysis of WIP domain proteins . FEBS Letters . 584 : 3116 – 3122 . OpenUrl CrossRef PubMed Ashburner , M. , Ball , C. , Blake , J. A. , Botstein , D. , Butler , H. , Cherry , J. M. , Davis , A. P. , Dolinski , K. , Dwight , S. S. , Eppig , J. T. , Harris , M. A. , Hill , D. P. , Issel-Tarver , L. , Kasarskis , A. , Lewis , S. , Matese , J. C. , Richardson , J. E. , Ringwald , M. , Rubin , G. M. , and Sherlock , G . 2000 . Gene Ontology: tool for the unification of biology . Nature Genetics 25 , 25 – 29 . OpenUrl CrossRef PubMed Web of Science ↵ Atta , R. , Laurens , L. , Boucheron-Dubuisson , E. , Guivarc’h , A. , Carnero , E. , Giraudat-Pautot , V. , Rech , P. , and Chriqui , D . 2009 . Pluripotency of Arabidopsis xylem pericycle underlies shoot regeneration from root and hypocotyl explants grown in vitro . The Plant Journal . 57 : 626 – 644 . OpenUrl CrossRef PubMed Web of Science ↵ Becerra-García , R. E. , Cruz-Valderrama , J. E. , Cerbantez-Bueno , V. E. , Marsch-Martínez , N. , and de Folter , S . 2023 . A NanoLuc-Based Transactivation Assay in Plants . Methods in Molecular Biology 2686 , 553 – 565 . OpenUrl CrossRef PubMed ↵ Bharathan , G. , Janssen , B. J. , Kellogg , E. A. , and Sinha , N . 1999 . Phylogenetic relationships and evolution of the KNOTTED class of plant homeodomain proteins . Molecular Biology and Evolution , 16 ( 4 ), 553 – 563 . OpenUrl CrossRef PubMed Web of Science ↵ Bowman , J.L. , Eshed , Y. , and Baum , S.F . 2001 . Establishment of polarity in angiosperm lateral organs . Trends in Genetics . 18 : 134 – 141 . OpenUrl ↵ Bray , N. L. , Pimentel , H. , Melsted , P. , and Pachter , L . 2016 . Near-optimal probabilistic RNA-seq quantification . Nature Biotechnology , 34 ( 5 ), 525 – 527 . OpenUrl CrossRef PubMed Chen , L. , Zhao , J. , Song , J. , and Jameson , P. E . 2021 . Cytokinin glucosyl transferases, key regulators of cytokinin homeostasis, have potential value for wheat improvement . Plant Biotechnology Journal , 19 ( 5 ), 878 – 896 . OpenUrl CrossRef PubMed ↵ Cheng , C. , Krishnakumar , V. , Chan , A. P. , and Town , C. D . 2017 . Araport11: a complete reannotation of the Arabidopsis thaliana reference genome . The Plant journal . ( 4 ) 789 – 804 . OpenUrl ↵ Chung , K.S. , Lee , J.H. , Lee , J.S. , and Ahn , J.H . 2013 . Fruit indehiscence caused by enhanced expression of NO TRANSMITTING TRACT in Arabidopsis thaliana . Molecular Cell 35 : 519 – 525 . OpenUrl CrossRef ↵ Crawford , B. , Ditta , G. , and Yanofsky , M.F . 2007 . The NTT Gene Is Required for Transmitting-Tract Development in Carpels of Arabidopsis thaliana . Current Biology . 17 : 1101 – 1108 . OpenUrl CrossRef PubMed Web of Science ↵ Crawford , B. , Sewell , J. , Golembeski , G. , Roshan , C. , Long , J. , and Yanofsky , M. F . 2015 . Genetic control of distal stem cell fate within root and embryonic meristems . Science , 347 , 655 – 659 . OpenUrl Abstract / FREE Full Text ↵ Cucinotta , M. , Cavalleri , A. , Guazzotti , A. , Astori , C. , Manrique , S. , Bombarely , A. , Oliveto , S. , Biffo , S. , Weijers , D. , Kater , M. M. , and Colombo , L . 2021 . Alternative Splicing Generates a MONOPTEROS Isoform Required for Ovule Development . Current Biology . 31 : 892 – 899 .e3. OpenUrl CrossRef PubMed ↵ Danisman , S. , van der Wal , F. , Dhondt , S. , Waites , R. , de Folter , S. , Bimbo , A. , van Dijk , A. , Muino , J. M. , Cutri , L. , Dornelas , M. , Angenent , G. , and Immink , R . 2012 . Arabidopsis class I and class II TCP transcription factors regulate jasmonic acid metabolism and leaf development antagonistically . Plant Physiology . 159 : 1511 – 1523 . OpenUrl Abstract / FREE Full Text ↵ De Rybel , B. , Adibi , M. , Breda , A. , Wendrich , J. R. , Smit , M. E. , Novák , O. , Yamaguchi , N. , Yoshida , S. , van Isterdael , G. , Palovaara , J. , Nijsse , B. , Boekschoten , M. v. , Hooiveld , G. , Beeckman , T. , Wagner , D. , Ljung , K. , Fleck , C. , and Weijers , D . 2014 . Integration of growth and patterning during vascular tissue formation in Arabidopsis . Science 345 : 1255215 . OpenUrl Abstract / FREE Full Text De Rybel , B. , Mähönen , A. P. , Helariutta , Y. , and Weijers , D. 2015 . Plant vascular development: from early specification to differentiation . Nature Reviews Molecular Cell Biology , 17 ( 1 ), 30 – 40 . OpenUrl CrossRef PubMed ↵ Dello Ioio , R. , Linhares , F.S. , Scacchi , E. , Casamitjana-Martinez , E. , Heidstra , R. , Costantino , P. , and Sabatini , S . 2007 . Cytokinins Determine Arabidopsis Root-Meristem Size by Controlling Cell Differentiation . Current Biology . 17 : 678 – 682 . OpenUrl CrossRef PubMed Web of Science ↵ Di Marzo , M. , Herrera-Ubaldo , H. , Caporali , E. , Mendes , M.A. , De Folter , S. and Colombo , L . 2020 . SEEDSTICK Controls Arabidopsis Fruit Size by Regulating Cytokinin Levels and FRUITFULL . Cell Reports . 30 : 2846 – 2857 . OpenUrl CrossRef PubMed ↵ Diaz-Ramirez , D. , Diaz-Garcia , U. S. , Magdaleno-Garcia , G. , Huep , G. , Appelhagen , I. , Sagasser , M. , and Marsch-Martinez , N . 2022 . Expression and Functional Analyses of the WIP Gene Family in Arabidopsis . Plants , 11 ( 15 ). OpenUrl ↵ Dortay , H. , Gruhn , N. , Pfeifer , A. , Schwerdtner , M. , Schmülling , T. , and Heyl , A . 2008 . Toward an interaction map of the two-component signaling pathway of arabidopsis thaliana . Journal of Proteome Research . 7 : 3649 – 3660 . OpenUrl CrossRef PubMed Web of Science ↵ Du , Y. , Roldan , M. V. G. , Haraghi , A. , Haili , N. , Izhaq , F. , Verdenaud , M. , Boualem , A. , and Bendahmane , A . 2022 . Spatially expressed WIP genes control Arabidopsis embryonic root development . Nature Plants , 8 ( 6 ), 635 – 645 . OpenUrl CrossRef PubMed ↵ Durán-Medina , Y. , Díaz-Ramírez , D. , and Marsch-Martínez , N . 2017 . Cytokinins on the move . Frontiers in Plant Science . 8 : 146 OpenUrl PubMed Durán-Medina , Y. , Díaz-Ramírez , D. , Herrera-Ubaldo , H. , di Marzo , M. , Gómez Felipe , A. , Cruz-Valderrama , J. E. , Vázquez , C. A. , Guerrero-Largo , H. , Colombo , L. , Novak , O. , de Folter , S. , and Marsch-Martínez , N . 2025 . ESR2 orchestrates cytokinin dynamics leading to developmental reprogramming and green callus formation . Plant Physiology , in press. Emery , J. F. , Floyd , S. K. , Alvarez , J. , Eshed , Y. , Hawker , N. P. , Izhaki , A. , Baum , S. F. , and Bowman , J. L . 2003 . Radial patterning of Arabidopsis shoots by class III HD-ZIP and KANADI genes . Current biology . 13 ( 20 ), 1768 – 1774 . OpenUrl CrossRef PubMed Web of Science Eshed , Y. , Izhaki , A. , Baum , S. F. , Floyd , S. K. , and Bowman , J. L . 2004 . Asymmetric leaf development and blade expansion in Arabidopsis are mediated by KANADI and YABBY activities . Development . 131 ( 12 ), 2997 – 3006 . OpenUrl Abstract / FREE Full Text ↵ Gomez-Roldan , M.V. , Izhaq , F. , Verdenaud , M. , Eleblu , J. , Haraghi , A. , Sommard , V. , Chambrier , P. , Latrasse , D. , Jégu , T. , Benhamed , M. , Szécsi , J. , Bendahmane , M. , Boualem , A. , and Bendahmane , A . 2020 . Integrative genome-wide analysis reveals the role of WIP proteins in inhibition of growth and development . Communications Biology 3 : 1 – 12 . OpenUrl CrossRef PubMed ↵ Hagen , G. , and Guilfoyle , T . 2002 . Auxin-responsive gene expression: genes, promoters, and regulatory factors . Plant Molecular Biology , 49 ( 3-4 ), 373 – 385 . OpenUrl CrossRef PubMed Web of Science ↵ Hake , S. , Smith , H. M. S. , Holtan , H. , Magnani , E. , Mele , G. , and Ramirez , J . 2004 . The role of knox genes in plant development . Annual Review of Cell and Developmental Biology , 20 , 125 – 151 . OpenUrl CrossRef PubMed Web of Science ↵ Hawker , N. and Bowman , J . ( 2004 ). Roles for class III HD-zip and Kanadi genes in arabidopsis root development . Plant Physiology . 135 : 2261 – 2270 . OpenUrl Abstract / FREE Full Text ↵ Helariutta , Y. , Fukaki , H. , Wysocka-Diller , J. , Nakajima , K. , Jung , J. , Sena , G. , Hauser , M. T. , and Benfey , P. N . 2000 . The SHORT-ROOT gene controls radial patterning of the Arabidopsis root through radial signaling . Cell , 101 ( 5 ), 555 – 567 . OpenUrl CrossRef PubMed Web of Science ↵ Herrera-Ubaldo , H. , Lozano-Sotomayor , P. , Ezquer , I. , di Marzo , M. , Montes , R. A. C. , Goḿez-Felipe , A. , Pablo-Villa , J. , Diaz-Ramirez , D. , Ballester , P. , Ferrańdiz , C. , Sagasser , M. , Colombo , L. , Marsch-Martıńez , N. , and de Folter , S . 2019 . New roles of NO TRANSMITTING TRACT and SEEDSTICK during medial domain development in Arabidopsis fruits . Development . 146 : dev172395. ↵ Herrera-Ubaldo , H. , Nájera-González , H. R. , Luna-García , V. , Marsch-Martínez , N. , and de Folter , S . 2023 . Hormone immunolabeling in resin-embedded Arabidopsis tissues . STAR Protocols , 4 ( 3 ), 102514 . OpenUrl CrossRef PubMed ↵ Hill , K. , Mathews , D. E. , Kim , H. J. , Street , I. H. , Wildes , S. L. , Chiang , Y. H. , Mason , M. G. , Alonso , J. M. , Ecker , J. R. , Kieber , J. J. , and Schaller , G. E . 2013 . Functional characterization of type-B response regulators in the Arabidopsis cytokinin response . Plant Physiology 162 : 212 – 224 . OpenUrl Abstract / FREE Full Text ↵ Hirose , N. , Takei , K. , Kuroha , T. , Kamada-Nobusada , T. , Hayashi , H. , and Sakakibara , H . 2008 . Regulation of cytokinin biosynthesis, compartmentalization and translocation . Journal of Experimental Botany , 59 ( 1 ), 75 – 83 . OpenUrl CrossRef PubMed Web of Science ↵ Hwang , I. , Sheen , J. , and Müller , B . 2012 . Cytokinin signaling networks . Annual Review of Plant Biology . 63 : 353 – 380 . OpenUrl CrossRef PubMed Web of Science ↵ Ji , J. , Strable , J. , Shimizu , R. , Koenig , D. , Sinha , N. , and Scanlon , M. J . 2010 . WOX4 promotes procambial development . Plant Physiology , 152 ( 3 ), 1346 – 1356 . OpenUrl Abstract / FREE Full Text ↵ Kakimoto , T . 2001 . Identification of plant cytokinin biosynthetic enzymes as dimethylallyl diphosphate: ATP/ADP isopentenyltransferases . Plant and Cell Physiology , 42 ( 7 ), 677 – 685 . OpenUrl CrossRef PubMed Web of Science ↵ Kieber , J.J. and Schaller , G.E . 2018 . Cytokinin signaling in plant development . Development 145 : 1 – 7 . OpenUrl CrossRef ↵ Kim , H.J. , Chiang , Y.H. , Kieber , J.J. , and Schaller , G.E . 2013 . SCFKMD controls cytokinin signaling by regulating the degradation of type-B response regulators . Proceedings of the National Academy of Sciences 110 : 10028 – 10033 . OpenUrl Abstract / FREE Full Text Kleinboelting , N. , Huep , G. , Kloetgen , A. , Viehoever , P. , and Weisshaar , B . 2012 . GABI-Kat SimpleSearch: New features of the Arabidopsis thaliana T-DNA mutant database . Nucleic Acids Research . 40 . D1211 – D1215 OpenUrl CrossRef PubMed Web of Science ↵ Köllmer , I. , Novák , O. , Strnad , M. , Schmülling , T. , and Werner , T . 2014 . Overexpression of the cytosolic cytokinin oxidase/dehydrogenase (CKX7) from Arabidopsis causes specific changes in root growth and xylem differentiation . The Plant Journal . 78 : 359 – 371 . OpenUrl CrossRef PubMed Web of Science Kuroha , T. , Tokunaga , H. , Kojima , M. , Ueda , N. , Ishida , T. , Nagawa , S. , Fukuda , H. , Sugimoto , K. , and Sakakibara , H . 2009 . Functional analyses of LONELY GUY cytokinin-activating enzymes reveal the importance of the direct activation pathway in Arabidopsis . The Plant Cell 21 : 3152 – 3169 . OpenUrl Abstract / FREE Full Text Leibfried , A. , To , J.P.C. , Busch , W. , Stehling , S. , Kehle , A. , Demar , M. , Kieber , J.J. , and Lohmann , J.U . 2005 . WUSCHEL controls meristem function by direct regulation of cytokinin-inducible response regulators . Nature 438 : 22 – 25 . OpenUrl ↵ Livak , K. J. , and Schmittgen , T. D . 2001 . Analysis of relative gene expression data using real-time quantitative PCR and the 2 -ΔΔCT Method . Methods , 25 ( 4 ), 402 – 408 OpenUrl CrossRef PubMed Web of Science ↵ Liu , S. , Strauss , S. , Adibi , M. , Mosca , G. , Yoshida , S. , Dello Ioio , R. , Runions , A. , Andersen , T. G. , Grossmann , G. , Huijser , P. , Smith , R. S. , and Tsiantis , M . 2022 . Cytokinin promotes growth cessation in the Arabidopsis root . Current Biology : 32 ( 9 ), 1974 – 1985 .e3. OpenUrl CrossRef PubMed ↵ Lozano-Sotomayor , P. , Chávez Montes , R. A. , Silvestre-Vañó , M. , Herrera-Ubaldo , H. , Greco , R. , Pablo-Villa , J. , Galliani , B. M. , Diaz-Ramirez , D. , Weemen , M. , Boutilier , K. , Pereira , A. , Colombo , L. , Madueño , F. , Marsch-Martínez , N. , and de Folter , S . 2016 . Altered expression of the bZIP transcription factor DRINK ME affects growth and reproductive development in Arabidopsis thaliana . The Plant Journal . 88 : 437 – 451 . OpenUrl CrossRef PubMed ↵ Mähönen , A.P. , Bonke , M. , Kauppinen , L. , Riikonen , M. , Benfey , P.N. , and Helariutta , Y . 2000 . A novel two-component hybrid molecule regulates vascular morphogenesis of the Arabidopsis root . Genes & Development 14 : 2938 – 2943 . OpenUrl Abstract / FREE Full Text ↵ Mähönen , A.P. , Bishopp , A. , Higuchi , M. , Nieminen , K.M. , Kinoshita , K. , Törmäkangas , K. , Ikeda , Y. , Oka , A. , Kakimoto , T. , and Helariutta , Y . 2006 . Cytokinin signaling and its inhibitor AHP6 regulate cell fate during vascular development . Science . 311 : 94 – 98 . OpenUrl Abstract / FREE Full Text Márquez-López , R.E. , Quintana-Escobar , A.O. , and Loyola-Vargas , V.M . 2019 . Cytokinins, the Cinderella of plant growth regulators . Phytochemistry Review 18 : 1387 – 1408 . OpenUrl CrossRef ↵ Marsch-Martínez , N. , Ramos-Cruz , D. , Irepan Reyes-Olalde , J. , Lozano-Sotomayor , P. , Zúñiga-Mayo , V. M. , and de Folter , S . 2012 . The role of cytokinin during Arabidopsis gynoecia and fruit morphogenesis and patterning . The Plant Journal , 72 ( 2 ), 222 – 234 . OpenUrl CrossRef PubMed Web of Science ↵ Marsch-Martínez , N. , Zúñiga-Mayo , V. M. , Herrera-Ubaldo , H. , Ouwerkerk , P. B. F. , Pablo-Villa , J. , Lozano-Sotomayor , P. , Greco , R. , Ballester , P. , Balanzá , V. , Kuijt , S. J. H. , Meijer , A. H. , Pereira , A. , Ferrándiz , C. , and de Folter , S . 2014 . The NTT transcription factor promotes replum development in Arabidopsis fruits . The Plant Journal , 80 , 69 – 81 . OpenUrl CrossRef PubMed ↵ McCarthy , D. J. , Chen , Y. , and Smyth , G. K . 2012 . Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation . Nucleic Acids Research , 40 ( 10 ), 4288 – 4297 . OpenUrl CrossRef PubMed Web of Science ↵ Miyawaki , K. , Matsumoto-Kitano , M. , and Kakimoto , T . 2004 . Expression of cytokinin biosynthetic isopentenyltransferase genes in Arabidopsis: Tissue specificity and regulation by auxin, cytokinin, and nitrate . The Plant Journal . 37 : 128 – 138 . OpenUrl CrossRef PubMed Web of Science ↵ Miyawaki , K. , Tarkowski , P. , Matsumoto-Kitano , M. , Kato , T. , Sato , S. , Tarkowska , D. , Tabata , S. , Sandberg , G. , and Kakimoto , T . 2006 . Roles of Arabidopsis ATP/ADP isopentenyltransferases and tRNA isopentenyltransferases in cytokinin biosynthesis . Proceedings of the National Academy of Sciences , 103 ( 44 ), 16598 – 16603 . OpenUrl Abstract / FREE Full Text ↵ Mok , D. W. S. , and Mok , M. C . 2001 . Cytokinin metabolism and action . Annual Review of Plant Physiology and Plant Molecular Biology , 52 , 89 – 118 . OpenUrl CrossRef PubMed Web of Science Moreira , S. , Bishopp , A. , Carvalho , H. , and Campilho , A . 2013 . AHP6 inhibits cytokinin signaling to regulate the orientation of pericycle cell division during lateral root initiation . PLoS One 8 ( 2 ): e56370 . OpenUrl CrossRef PubMed Müller , B. , and Sheen , J . 2008 . Cytokinin and auxin interaction in root stem-cell specification during early embryogenesis . Nature , 453 ( 7198 ), 1094 – 1097 . OpenUrl CrossRef PubMed Web of Science O’Malley , R.C. , Huang , S.C. , Song , L. , Lewsey , M.G. , Bartlett , A. , Nery , J.R. , Galli , M. , Gallavotti , A. , and Ecker , J.R . 2016 . Cistrome and epicistrome features shape the regulatory DNA landscape . Cell 166 : 1598 . OpenUrl CrossRef PubMed Ohashi-Ito , K. and Bergmann , D.C . 2007 . Regulation of the Arabidopsis root vascular initial population by Lonesome highway . Development 134 : 2959 – 2968 . OpenUrl Abstract / FREE Full Text ↵ Ohashi-Ito , K. , Saegusa , M. , Iwamoto , K. , Oda , Y. , Katayama , H. , Kojima , M. , Sakakibara , H. , and Fukuda , H . 2014 . A bHLH complex activates vascular cell division via cytokinin action in root apical meristem . Current Biology 24 : 2053 – 2058 . OpenUrl CrossRef PubMed ↵ Pasternak , T. , Tietz , O. , Rapp , K. , Begheldo , M. , Nitschke , R. , Ruperti , B. , and Palme , K . 2015 . Protocol: an improved and universal procedure for whole mount immunolocalization in plants . Plant Methods , 11 ( 1 ). ↵ Persikov , A. V and Singh , M . 2014 . De novo prediction of DNA-binding specificities for Cys2His 2 zinc finger proteins . Nucleic Acids Research . 42 : 97 – 108 . OpenUrl CrossRef PubMed Web of Science ↵ Prigge , M. J. , Platre , M. , Kadakia , N. , Zhang , Y. , Greenham , K. , Szutu , W. , Pandey , B. K. , Bhosale , R. A. , Bennett , M. J. , Busch , W. , and Estelle , M . 2020 . Genetic analysis of the arabidopsis TIR1/AFB auxin receptors reveals both overlapping and specialized functions . Elife 9 . e54740 . OpenUrl CrossRef PubMed ↵ Powers , S. K. , and Strader , L. C . 2019 . Regulation of auxin transcriptional responses . Developmental Dynamics 249 : 4 , 483 – 495 OpenUrl PubMed ↵ R Core Team . 2022 . R: A language and environment for statistical computing . R foundation for statistical computing, Vienna, Austria . URL https://www.R-project.org/ . ↵ R Studio Team . 2020 . RStudio: integrated development for R. RStudio, PBC, Boston, MA URL http://www.rstudio.com/ ↵ Ren , G. , Li , L. , Huang , Y. , Wang , Y. , Zhang , W. , Zheng , R. , Zhong , C. , and Wang , X . 2018 . GhWIP2, a WIP zinc finger protein, suppresses cell expansion in Gerbera hybrida by mediating crosstalk between gibberellin, abscisic acid, and auxin . The New phytologist , 219 ( 2 ), 728 – 742 . OpenUrl CrossRef PubMed Reyes-Olalde , J. I. , Zúñiga-Mayo , V.M. , Serwatowska , J. , Chavez Montes , R. A. , Lozano-Sotomayor , P. , Herrera-Ubaldo , H. , Gonzalez-Aguilera , K. L. , Ballester , P. , Ripoll , J. J. , Ezquer , I. , Paolo , D. , Heyl , A. , Colombo , L. , Yanofsky , M. F. , Ferrandiz , C. , Marsch-Martínez , N. , and de Folter , S . 2017 . The bHLH transcription factor SPATULA enables cytokinin signaling, and both activate auxin biosynthesis and transport genes at the medial domain of the gynoecium . PLoS genetics , 13 ( 4 ), e1006726 . OpenUrl CrossRef ↵ Robinson , M. D. , McCarthy , D. J. , and Smyth , G. K . 2009 . edgeR: A Bioconductor package for differential expression analysis of digital gene expression data . Bioinformatics , 26 ( 1 ), 139 – 140 . OpenUrl CrossRef PubMed Web of Science ↵ Roosjen , M. , Paque , S. , and Weijers , D . 2018 . Auxin Response Factors: Output control in auxin biology . Journal of Experimental Botany . 69 : 179 – 188 . OpenUrl CrossRef PubMed ↵ Sagasser , M. , Lu , G.H. , Hahlbrock , K. , and Weisshaar , B . 2001 . A. thaliana Transparent testa 1 is involved in seed coat development and defines the WIP subfamily of plant zinc finger proteins . Genes & Development 16 : 138 – 149 . OpenUrl ↵ Schaller , G.E. , Bishopp , A. , and Kieber , J.J . 2015 . The yin-yang of hormones: Cytokinin and auxin interactions in plant development . The Plant Cell 27 : 44 – 63 . OpenUrl Abstract / FREE Full Text ↵ Semiarti , E. , Ueno , Y. , Tsukaya , H. , Iwakawa , H. , Machida , C. , and Machida , Y . 2001 . The ASYMMETRIC LEAVES2 gene of Arabidopsis thaliana regulates formation of a symmetric lamina, establishment of venation and repression of meristem-related homeobox genes in leaves . Development , 128 ( 10 ), 1771 – 1783 . OpenUrl Abstract ↵ Simillion , C. , Liechti , R. , Lischer , H. E. L. , Ioannidis , V. , and Bruggmann , R . 2017 . Avoiding the pitfalls of gene set enrichment analysis with SetRank . BMC Bioinformatics , 18 ( 1 ), 1 – 14 . OpenUrl CrossRef PubMed ↵ Skalák , J. , Vercruyssen , L. , Claeys , H. , Hradilová , J. , Černý , M. , Novák , O. , Plačková , L. , Saiz-Fernández , I. , Skaláková , P. , Coppens , F. , Dhondt , S. , Koukalová , Š. , Zouhar , J. , Inzé , D. , and Brzobohatý , B . 2019 . Multifaceted activity of cytokinin in leaf development shapes its size and structure in Arabidopsis . The Plant Journal . 97 : 805 – 824 . OpenUrl CrossRef PubMed ↵ Skoog F. and Miller C . 1957 . Chemical regulation of growth and organ formation in plant tissues cultured in vitro . Symposia of the Society for Experimental Biology 11 : 118 – 30 OpenUrl PubMed ↵ Soneson , C. , Love , M. I. , and Robinson , M. D . 2016 . Differential analyses for RNA-seq: Transcript-level estimates improve gene-level inferences . F1000 Research , 4 , 1 – 22 . OpenUrl CrossRef ↵ Sun , J. , Niu , Q. W. , Tarkowski , P. , Zheng , B. , Tarkowska , D. , Sandberg , G. , Chua , N. H. , and Zuo , J . 2003 . The arabidopsis AtIPT8/PGA22 gene encodes an isopentenyl transferase that is involved in de novo cytokinin biosynthesis . Plant Physiology , 131 ( 1 ), 167 – 176 . OpenUrl Abstract / FREE Full Text ↵ Svačinová , J. , Novák , O. , Plačková , L. , Lenobel , R. , Holík , J. , Strnad , M. , and Doležal , K . 2012 . A new approach for cytokinin isolation from Arabidopsis tissues using miniaturized purification: pipette tip solid-phase extraction . Plant Methods , 8 ( 1 ), 1 – 14 . OpenUrl CrossRef PubMed ↵ The Gene Ontology Consortium . 2019 . The Gene Ontology Resource: 20 years and still GOing strong . Nucleic Acids Research . 47 : D330 – D338 . OpenUrl CrossRef PubMed ↵ To , J.P.C. , Haberer , G. , Ferreira , F.J. , Derue , J. , Schaller , G.E. , Alonso , J.M. , Ecker , J.R. , and Kieber , J.J . 2004 . Type-A Arabidopsis Response Regulators are partially redundant negative regulators of cytokinin signaling . The Plant Cell 16 : 658 – 671 . OpenUrl Abstract / FREE Full Text ↵ Tokunaga , H. , Kojima , M. , Kuroha , T. , Ishida , T. , Sugimoto , K. , Kiba , T. , and Sakakibara , H . 2012 . Arabidopsis lonely guy (LOG) multiple mutants reveal a central role of the LOG-dependent pathway in cytokinin activation . The Plant Journal . 69 : 355 – 365 . OpenUrl CrossRef PubMed Web of Science ↵ Tran , T. M. , Demesa-Arevalo , E. , Kitagawa , M. , Garcia-Aguilar , M. , Grimanelli , D. , and Jackson , D . 2021 . An optimized whole-mount immunofluorescence method for shoot apices . Current Protocols , 1 ( 4 ). Trigueros , M. , Navarrete-Gómez , M. , Sato , S. , Christensen , S. K. , Pelaz , S. , Weigel , D. , Yanofsky , M. F. , and Ferrándiza , C . 2009 . The NGATHA genes direct style development in the Arabidopsis gynoecium . The Plant Cell , 21 ( 5 ), 1394 – 1409 . OpenUrl Abstract / FREE Full Text ↵ Urquiza-García , U. and Millar , A.J . 2019 . Expanding the bioluminescent reporter toolkit for plant science with NanoLuc . Plant Methods 15 : 15 , 68 . OpenUrl CrossRef PubMed ↵ Wang , J. , Ma , X. , Kojima , M. , Sakakibara , H. , and Hou , B . 2011 . N -Glucosyltransferase UGT76C2 is involved in cytokinin homeostasis and cytokinin response in Arabidopsis thaliana . Plant and Cell Physiology 52 : 2200 – 2213 . OpenUrl CrossRef PubMed Web of Science ↵ Werner , T. , Motyka , V. , Strnad , M. , and Schmülling , T . 2001 . Regulation of plant growth by cytokinin . Proceedings of the National Academy of Sciences 98 : 10487 – 10492 . OpenUrl Abstract / FREE Full Text ↵ Werner , T. , Motyka , V. , Laucou , V. , Smets , R. , Van Onckelen , H. , and Schmülling , T . 2003 . Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity . The Plant Cell 15 : 2532 – 2550 . OpenUrl Abstract / FREE Full Text ↵ Wybouw , B. and De Rybel , B . 2019 . Cytokinin – A developing story . Trends in Plant Science . 24 : 177 – 185 . OpenUrl CrossRef PubMed Yamada , H. , Suzuki , T. , Terada , K. , Takei , K. , Ishikawa , K. , Yamashino , T. , and Mizuno , T . 2001 . The Arabidopsis AHK4 Histidine Kinase is a cytokinin-binding receptor that transduces cytokinin signals across the membrane . Plant and Cell Physiology 42 : 1017 – 1023 . OpenUrl CrossRef PubMed Web of Science ↵ Yanai , O. , Shani , E. , Dolezal , K. , Tarkowski , P. , Sablowski , R. , Sandberg , G. , Samach , A. , and Ori , N . 2005 . Arabidopsis KNOXI Proteins Activate Cytokinin Biosynthesis . Current Biology , 15 ( 17 ), 1566 – 1571 . OpenUrl CrossRef PubMed Web of Science ↵ Yang , B. J. , Minne , M. , Brunoni , F. , Plačková , L. , Petřík , I. , Sun , Y. , Nolf , J. , Smet , W. , Verstaen , K. , Wendrich , J. R. , Eekhout , T. , Hoyerová , K. , van Isterdael , G. , Haustraete , J. , Bishopp , A. , Farcot , E. , Novák , O. , Saeys , Y. , and de Rybel , B . 2021 . Non-cell autonomous and spatiotemporal signalling from a tissue organizer orchestrates root vascular development . Nature Plants 7:11, 7 ( 11 ), 1485 – 1494 . OpenUrl CrossRef PubMed ↵ Zhang , K. , Novak , O. , Wei , Z. , Gou , M. , Zhang , X. , Yu , Y. , Yang , H. , Cai , Y. , Strnad , M. , and Liu , C.J . 2014 . Arabidopsis ABCG14 protein controls the acropetal translocation of root-synthesized cytokinins . Nature Communications . 5 : 3274 . OpenUrl CrossRef PubMed ↵ Zubko , E. , Adams , C. J. , Macháèková , I. , Malbeck , J. , Scollan , C. , and Meyer , P . 2002 . Activation tagging identifies a gene from Petunia hybrida responsible for the production of active cytokinins in plants . The Plant Journal , 29 ( 6 ), 797 – 808 . OpenUrl CrossRef PubMed Web of Science Zúñiga-Mayo , V.M. , Reyes-Olalde , J.I. , Marsch-Martinez , N. , and de Folter , S. 2014 . Cytokinin treatments affect the apical-basal patterning of the Arabidopsis gynoecium and resemble the effects of polar auxin transport inhibition . Frontiers in Plant Science . 5 : 1 – 8 . OpenUrl ↵ Zuñiga-Mayo , V. M. , Baños-Bayardo , C. R. , Díaz-Ramírez , D. , Marsch-Martínez , N. , and de Folter , S . 2018 . Conserved and novel responses to cytokinin treatments during flower and fruit development in Brassica napus and Arabidopsis thaliana . Scientific Reports 8:1, 8 ( 1 ), 1 – 10 . OpenUrl PubMed ↵ Zürcher , E. , Tavor-Deslex , D. , Lituiev , D. , Enkerli , K. , Tarr , P.T. , and Müller , B . 2013 . A robust and sensitive synthetic sensor to monitor the transcriptional output of the cytokinin signaling network in planta . Plant Physiology . 161 : 1066 – 1075 . OpenUrl Abstract / FREE Full Text View the discussion thread. Back to top Previous Next Posted April 06, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. 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