The Plant Journal (2014) 78, 359–371

doi: 10.1111/tpj.12477

Overexpression of the cytosolic cytokinin oxidase/ dehydrogenase (CKX7) from Arabidopsis causes specific changes in root growth and xylem differentiation € lling1 and Tomas Werner1,* Ireen Ko€ llmer1, Ondrej Novak2, Miroslav Strnad2, Thomas Schmu 1 Institute of Biology/Applied Genetics, Dahlem Centre of Plant Sciences (DCPS), Freie Universita€ t Berlin, Albrecht-Thaer-Weg 6, D-14195 Berlin, Germany, and 2 Laboratory of Growth Regulators, Palacky University and Institute of Experimental Botany ASCR, Slechtitelu 11, CZ-78371 Olomouc, Czech Republic Received 13 March 2013; revised 30 January 2014; accepted 6 February 2014; published online 17 February 2014. *For correspondence (e-mail [email protected]).

SUMMARY Degradation of the plant hormone cytokinin is catalyzed by cytokinin oxidase/dehydrogenase (CKX) enzymes. The Arabidopsis thaliana genome encodes seven CKX proteins which differ in subcellular localization and substrate specificity. Here we analyze the CKX7 gene, which to the best of our knowledge has not yet been studied. pCKX7:GUS expression was detected in the vasculature, the transmitting tissue and the mature embryo sac. A CKX7–GFP fusion protein localized to the cytosol, which is unique among all CKX family members. 35S:CKX7-expressing plants developed short, early terminating primary roots with smaller apical meristems, contrasting with plants overexpressing other CKX genes. The vascular bundles of 35S:CKX7 primary roots contained only protoxylem elements, thus resembling the wol mutant of the CRE1/AHK4 receptor gene. We show that CRE1/AHK4 activity is required to establish the CKX7 overexpression phenotype. Several cytokinin metabolites, in particular cis-zeatin (cZ) and N-glucoside cytokinins, were depleted stronger in 35S:CKX7 plants compared with plants overexpressing other CKX genes. Interestingly, enhanced protoxylem formation together with reduced primary root growth was also found in the cZ-deficient tRNA isopentenyltransferase mutant ipt2,9. However, different cytokinins were similarly efficient in suppressing 35S:CKX7 and ipt2,9 vascular phenotypes. Therefore, we hypothesize that the pool of cytosolic cytokinins is particularly relevant in the root procambium where it mediates the differentiation of vascular tissues through CRE1/AHK4. Taken together, the distinct consequences of CKX7 overexpression indicate that the cellular compartmentalization of cytokinin degradation and substrate preference of CKX isoforms are relevant parameters that define the activities of the hormone. Keywords: cytokinin, cytokinin oxidase/dehydrogenase, cytosol, root meristem, xylem differentiation, plant development, Arabidopsis thaliana.

INTRODUCTION The plant hormone cytokinin has important regulatory functions during various developmental and physiological € lling, 2009; Hwang plant processes (Werner and Schmu et al., 2012). In roots, cytokinin controls several aspects relevant for growth, architecture and functions. In the early phase of embryogenesis in Arabidopsis, a transcriptional interaction between cytokinin and auxin signaling components defines the activity of stem cells in the embryonic © 2014 The Authors. The Plant Journal © 2014 John Wiley & Sons Ltd

€ ller and Sheen, 2008). During post-embryonic root (Mu development, cytokinin negatively controls root elongation and branching (Werner et al., 2001, 2003; Mason et al., 2005; Riefler et al., 2006; Laplaze et al., 2007). Root elongation is largely determined by the activity of the primary root apical meristem, and it has been shown that cytokinin plays a key role in the control of cell differentiation in the transition zone between the proximal meristem 359

€ llmer et al. 360 Ireen Ko and the zone of cellular elongation (Werner et al., 2003; Dello Ioio et al., 2007). This control involves an antagonistic interaction with auxin (Dello Ioio et al., 2008; R uzicka et al., 2009) and a regulatory loop between the transcription factor PHABULOSA and cytokinin biosynthesis (Dello Ioio et al., 2012). The plant vascular system is composed of xylem and phloem, and these conductive tissues are derived from pluripotent stem cells of the procambium and cambium. Cytokinin has been recognized as key regulator of cambial activity, procambium maintenance and development of the vasculature (Miyashima et al., 2013). A strong reduction of cytokinin signaling leads to a severely reduced number of procambial cells in the embryo and root of Arabidopsis; this is associated with an aberrant specification of all the vascular cell files in the root as protoxylem and causes pre€ nen et al., 2000; mature termination of root growth (Ma€ho Hutchison et al., 2006; Yokoyama et al., 2007; Argyros et al., 2008). The maintenance of procambial cell divisions in the Arabidopsis root is regulated by the redundant activity of the three Arabidopsis histidine kinase receptors AHK2, AHK3 and CRE1/AHK4 (Higuchi et al., 2004; Nishimura et al., 2004). The wooden leg (wol) mutation in CRE1/ AHK4 mimics the cytokinin-unbound state of the receptor and confers constitutive phosphatase activity, thus acting in a dominant negative fashion by depleting phosphate € nen from the downstream signaling components (Ma€ho et al., 2006a). The pseudophosphotransfer protein AHP6 is € nen et al., a negative regulator of cytokinin signaling (Ma€ho 2006b) and the eukaryotic translation initiation factor eIF5A-2/FBR12 further modulates cytokinin activities during protoxylem specification (Ren et al., 2013). Recent findings have indicated an important role for long-distance basipetal transport of cytokinin through the phloem in controlling vascular patterning in roots via inhibitory interaction with auxin (Bishopp et al., 2011a,b). Furthermore, CLE peptides were reported to inhibit the formation of protoxylem in Arabidopsis roots through a CLAVATA2-dependent pathway by enhancing cytokinin signaling through the repression of A-type Arabidopsis response regulator (ARR) genes (Kondo et al., 2011). The main naturally occurring cytokinins are N6-prenylated adenine derivatives, such as zeatin (Z), N6-(D2-isopentenyl)adenine (iP) and dihydrozeatin (DZ), whereby the nature of the N6-attached chain can markedly influence the biological activity of the cytokinin. The isoprenoid cytokinin zeatin occurs as a trans- (tZ) and cis-isomer (cZ) with distinct relative abundance in different plant species (Gajdosova et al., 2011). Based on results from classical bioassays (Schmitz et al., 1972; Gajdosova et al., 2011) and cytokinin receptor-binding and activity studies (Spıchal et al., 2004; Romanov et al., 2005), cZ is generally considered as the inactive or weakly active isoform and no specific function has been so far attributed to this cytoki-

nin in Arabidopsis. However, recent studies have demonstrated that cZ is an active cytokinin in other plant species, such as rice or maize (Yonekura-Sakakibara et al., 2004; Kudo et al., 2012). Whereas iP- and tZ-type cytokinins are synthesized by ADP/ATP isopentenyltransferases (IPTs) transferring a prenyl moiety from dimethylallyl diphosphate to adenosine phosphate, cZ-type cytokinins are degradation products of cis-hydroxy tRNAs, which are formed by two tRNA IPT enzymes, IPT2 and IPT9 (Kasahara et al., 2004; Miyawaki et al., 2006). Cytokinin degradation is catalyzed by cytokinin oxidase/dehydrogenase (CKX) enzymes, and seven genes code for these proteins € lling et al., 2003). Individual CKX in Arabidopsis (Schmu proteins differ in their subcellular localizations and the isoforms from Arabidopsis analyzed thus far have been shown to be associated with the secretory pathway (Werner et al., 2003). Some CKX proteins are best at degrading free bases and ribosides of iP and tZ, but others prefer glucosides or nucleotides as a substrate (Galuszka et al., 2007; Kowalska et al., 2010). Several isoforms, including CKX1, CKX5 and CKX7, display good affinities towards cZ (Gajdosova et al., 2011). In this work, we characterize the expression of the CKX7 gene of Arabidopsis and showed a cytosolic localization of the encoded protein. We demonstrate that the overexpression of this cytosolic CKX isoform causes an early termination of root growth associated with defects in vascular development and we hypothesize that the cytosolic cytokinin pool may be particularly relevant in specific tissue types such as the root procambium. RESULTS CKX7 is expressed in the vascular tissue of seedlings and in the female gametophyte The previously characterized CKX genes (CKX1 to CKX6) of Arabidopsis were shown to be differentially expressed at different times in the life of the plant in specific and largely non-overlapping domains (Werner et al., 2003; Bartrina et al., 2011). To study the expression of CKX7, a 2-kb promoter region of CKX7 was used to drive the expression of the GUS reporter gene. In Arabidopsis plants stably transformed with the CKX7:GUS construct, GUS activity was detected at two developmental time points. First, the CKX7 promoter became active about 1– 2 days after germination (DAG) in the vasculature of roots, hypocotyls and cotyledons (Figure 1a,b). The activity was also detected in the vasculature of young true leaves and it became weaker as the individual leaves expanded. The expression was mainly associated with vascular cells (Figure 1c). No GUS activity was detectable during later vegetative stages. In flowers, CKX7: GUS expression was detected in the transmitting tissue of the gynoecium prior to pollination (Figure 1d). Fur-

© 2014 The Authors. The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 359–371

Cytosolic cytokinin degradation and root development 361 thermore, GUS activity was observed in the mature embryo sac and its maximum activity was associated with the egg cell and the synergids (Figure 1e). Weaker activity was occasionally also detected in the central cell. CKX7:GUS activity ceased after pollination and was not detectable in the developing embryo. In some lines, the activity occasionally extended beyond the pollination stage and weak GUS staining was detected in the developing endosperm. The CKX7 protein is localized in the cytosol Sequence analysis of the predicted CKX7 protein revealed no obvious N-terminal targeting sequence or nuclear localization signal, suggesting that it could be localized in € lling et al., 2003). To experimentally the cytosol (Schmu determine the subcellular localization of the CKX7 protein, we fused green fluorescent protein (GFP) to the C-terminus of CKX7 and expressed the fusion protein under the control of the cauliflower mosaic virus 35S promoter in stably transformed Arabidopsis plants. Using confocal microscopy, CKX7–GFP fluorescence was detected in the

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cytosol surrounding the central vacuole and the signal was clearly separated from the cell membrane (Figure 2). The same fluorescence pattern was observed in control plants expressing the untargeted GFP. All individual transgenic lines expressing the CKX7–GFP fusion phenocopied the corresponding 35S:CKX7-overexpressing lines (see below), demonstrating that the fusion protein was functional. CKX7-overexpressing plants have an increased CKX enzymatic activity, causing cytokinin deficiency To study the biological activity of CKX7, we generated and analyzed in detail transgenic Arabidopsis plants expressing CKX7 or a CKX7–GFP fusion gene under the control of the constitutive 35S promoter. Both constructs are called 35S: CKX7 in the following unless specified otherwise. Selected 35S:CKX7 transgenic plants showed enhanced expression

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(b) Figure 2. Cytosolic localization of the CKX7-GFP fusion protein. The GFP signal is detected in the cytosol of 35S:GFP transgenic control plants (a) and 35S:CKX7–GFP transgenic plants (b). The GFP fluorescence in root epidermal cells of 5-day-old seedlings was visualized by confocal laser scanning microscopy. Scale bar = 10 lm.

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Figure 1. The GUS activity in CKX7:GUS transgenic Arabidopsis plants. (a–e) Expression domains of the CKX7 gene were examined by histochemical localization of b-glucuronidase activity in transgenic plants expressing the GUS reporter gene under the control of the CKX7 promoter. In seedlings, CKX7:GUS was active in the vasculature of the primary root (a) and shoot organs (b). (c) Vascular cells in the distal tip of the third rosette leaf 9 days after germination. During later developmental stages, CKX7:GUS was expressed in the transmitting tissue (d) and in the mature embryo sac with the highest activity associated with the egg cell and synergids (e). Scale bar = 100 lm (a, c, d, e) and 1 mm (b). in, integument; ec, egg cell; sc, synergids; mi, micropyle; cc, central cell.

Figure 3. Gene expression analysis and CKX enzymatic activity in 35S: CKX7 and 35S:CKX7–GFP transgenic Arabidopsis seedlings. (a) CKX7 transcript levels are enhanced in 35S:CKX7 and 35S:CKX7–GFP Arabidopsis seedlings compared with the wild type (Col-0). Total RNA from 7-day-old seedlings grown in vitro was used for RNA gel blot analysis. The CKX7 transcript is not detectable in wild-type plants. The ethidium bromidestained 25S rRNA is shown as a loading control. (b) Increase in total CKX enzyme activity in CKX7 transgenic lines. The CKX activity was measured with 0.5 mM N6-(D2-isopentenyl)adenine (iP) as a substrate. The activity in the wild type (Col-0) was close to the detection limit of the method. The same plant material as in (a) was used. Mean values
SD (n ≥ 3) are shown. Student’s t-test was used to compare values with the wild type. *P < 0.05; **P < 0.01.

© 2014 The Authors. The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 359–371

€ llmer et al. 362 Ireen Ko of the transgene compared with wild-type plants in which CKX7 transcripts were not detectable by RNA gel blot analysis (Figure 3a). The transcript levels were significantly higher in plants expressing the CKX7–GFP fusion gene compared with CKX7, suggesting stabilization of the chimeric transcript. The CKX activity was increased in all 35S:CKX7 transgenic lines in comparison with the wild type (Figure 3b) and correlated with the level of transgene expression. In the weaker expressing lines 35S:CKX7-92 and 35S:CKX7-93, CKX activity was increased about four times, whereas in the strongly expressing line 35S:CKX7GFP-26 the increase was about 300-fold compared with the wild type. These results demonstrate that CKX7 in Arabidopsis possesses canonical cytokinin-degrading enzyme activity. Next we analyzed the effects of the enhanced CKX7 activity on the content of endogenous cytokinin in seedlings and compared this with the cytokinin content in wildtype seedlings as well as with transgenic seedlings expressing other CKX isoforms with different subcellular localizations and biochemical characteristics (35S:CKX1 and 35S:CKX2; Werner et al., 2003). This comparison may indicate the substrate preferences of the analyzed CKX isoforms and/or the accessibility of different cytokinin substrates for the different CKX enzymes. Collectively, 35S: Table 1 Cytokinin content in Arabidopsis plants overproducing CKX1, CKX2 or CKX7 compared with the wild type Cytokinin/line

35S:CKX1

35S:CKX2

35S:CKX7a

tZ cZ iP tZR cZR iPR tZ9G cZ9G iP9G tZOG cZOG tZRP cZRP iPRP

6 31 45 2 40 31 4 19 7 5 41 17 14 33

1 15 3 2 123 63 3 57 6 4 55 9 144 81

9 3 16 17 53 105 6 n.d. n.d. 10 18 32 141 125

The concentration of cytokinin metabolites was measured in 6day-old seedlings of lines 35S:CKX1-11, 35S:CKX2-9 and 35S: CKX7-GFP-26, and expressed as a percentage of wild-type values. Absolute values and statistical analysis are shown in Table S1. tZ, trans-zeatin; cZ, cis-zeatin; iP, N6-(D2-isopentenyl)adenine; tZR, tZ riboside; cZR, cZ riboside; iPR, iP riboside; tZ9G, tZ 9-N-glucoside; cZ9G, cZ 9-N-glucoside; iP9G, iP 9-N-glucoside; tZOG, tZ Oglucoside; cZOG, cZ O-glucoside; tZRP, tZR 50 -phosphate; cZRP, cZR 50 -phosphate; iPRP, iPR 50 -phosphate; n.d., concentration below the detection limit. a Comparison of cytokinin contents between line 35S:CKX7-93 and the wild type is shown in Table S1.

CKX7 expression resulted in a 30% reduction in the total cytokinin content compared with the wild type. The levels of most of the main cytokinin metabolites were reduced below 20% compared with the respective metabolites in the wild type (Table 1 and Table S1 in Supporting Information). A similar reduction in the total cytokinin content was detected in seedlings expressing 35S:CKX2 (35% reduction) and a stronger reduction (77%) was detected in 35S: CKX1 plants. Interestingly, significant differences in the relative content of several individual cytokinin metabolites were detected among the lines expressing individual CKX isoforms. For example, the content of tZR, iPR and cytokinin nucleotides (tZRP and iPRP) in 35S:CKX7 was less reduced than in 35S:CKX1 and 35S:CKX2 seedlings. In contrast, cZ-type cytokinins (cZ and cZ9G) and iP9G were significantly more strongly reduced in 35S:CKX7 plants, suggesting a high affinity of CKX7 to these substrates or a better access to them due to the cytosolic localization. Distinct phenotypic changes of CKX7-overexpressing Arabidopsis plants The reduction of cytokinin content in CKX7-overexpressing seedlings resulted in specific changes in seedling morphology and development. 35S:CKX7 seedlings germinated normally and looked similar to the wild type early after germination. However, the primary root did not develop any lateral roots and stopped growing approximately three DAG at a length of about 5 mm (Figure 4). This determinate root phenotype was observed in all of about 150 independent T1 plants harboring either 35S:CKX7 or 35S: CKX7–GFP. The phenotype was independent of the degree of increase in CKX activity, indicating that a small increase in CKX7 expression is sufficient to cause these changes. In accordance with this, it was found that the cytokinin content was reduced to a similar degree in strongly (35S: CKX7–GFP-26) and weakly (35S:CKX7-93) expressing lines (Table S1). This root phenotype was strongly contrasting with the enhanced root elongation of plants overproducing other CKX isoforms (Werner et al., 2003). The first true leaves in 35S:CKX7 seedlings stayed small, grew epinastic and displayed visible stress symptoms such as yellowing and accumulation of anthocyanin (Figure 4a and Figure S1), suggesting poor nutrient uptake from the medium. The growth arrest of the 35S:CKX7 shoot lasted for about 1 week until the formation of adventitious roots at the hypocotyl–root junction was initiated, leading to the formation of a root system and the recovery of shoot growth (Figure 4c). During later development, only 35S: CKX7–GFP transgenic lines with a strongly increased CKX activity showed differences in shoot morphology (Figure 4d). The size of the leaves and the plant height were slightly reduced and a few more lateral branches and fewer flowers were produced compared with the wild type (Figure 4d).

© 2014 The Authors. The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 359–371

Cytosolic cytokinin degradation and root development 363

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Figure 4. Phenotype of CKX7-overexpressing Arabidopsis plants. (a) Comparison of growth of wild-type (left) and 35S:CKX7 (line 35S:CKX793; right) seedlings 11 days after germination (DAG). (b) Primary root length of wild-type (Col-0) and 35S:CKX7 lines 11 DAG. (c) Initiation of adventitious root formation and progression of shoot development in 35S:CKX7 plants from 14 DAG onwards. (d) Shoots of CKX7-overexpressing plants compared with wild type at 30 DAG. Scale bar = 1 cm. Asterisks in (a) and (c) mark the end of the mutant primary root.

Root meristem activity is reduced and vascular development altered in 35S:CKX7 plants The primary root was studied in more detail in order to understand its determinate phenotype. Microscopic analysis showed that the diameter of the 35S:CKX7 primary root as well as the size of the root meristem was strongly reduced compared with the wild type (Figure 5). The reduced size of the root meristem was clearly demonstrated by the reduced number of cortex cells counted in a single cell file between the quiescent center and the cell elongation zone (Figure 5b). The size of the root meristem in the wild type increased continuously from germination until 6 DAG when about 33 cells were established. In 35S:CKX7 roots, the number of meristematic cells was comparable to the wild type directly after germination (about 18 cells; Figure 5b) but there was no increase during the later stages. The relative cell number was clearly reduced already at three DAG in the 35S:CKX7 meristem (Figure 5b). The meristem size in

Figure 5. Root meristem size and root diameter are reduced in 35S:CKX7 transgenic plants. (a) Bright-field microscopy images of wild-type (Col-0) and 35S:CKX7 (line 35S:CKX7-93) root meristems. The distance between the transition zone, as indicated by the first elongating cortical cell (black arrowhead), and the quiescent center (white arrowhead) corresponds to the meristem size. (b) Number of cortex cells in root meristems of 35S:CKX7 (line 35S:CKX793) seedlings compared with wild type between 1 and 9 days after germination (DAG). Cells in one cortex cell file between the white and black arrowheads as shown in (a) were counted. The values shown are means
SD (n ≥ 20). The values for three, six and nine DAG differed significantly (Student’s t-test, P < 0.001). Scale bars = 100 lm.

35S:CKX7 plants was reduced at six DAG compared with size at three DAG (Figure 5b), indicating that the differentiating meristematic cells were not replenished due to the low cell division activity in the meristem. These changes correlated temporally with the arrested root elongation. To substantiate the consequences of CKX7 overexpression for cell division, we monitored the activity of a mitotic marker, CYCB1:GUS, in wild-type and 35S:CKX7 root meristems. Owing to a mitotic degradation signal in the protein, the reporter gene visualizes only actively dividing  n-Carmona cells at the G2–M phase of the cell cycle (Colo et al., 1999). The GUS staining showed that cell division activity was already strongly reduced in the root meristem of 35S:CKX7 plants shortly after germination and it declined further at later time points (Figure 6). The GUS activity was almost undetectable in 35S:CKX7 meristems at 5–6 DAG, corresponding to the reduced meristem size and cessation of root growth. Further inspection of the primary root structure showed no difference with respect to overall organization of the

© 2014 The Authors. The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 359–371

€ llmer et al. 364 Ireen Ko meristem or identity of the ground tissues and quiescent center. However, we observed that markedly fewer vascular cell files were present in the central cylinder of the 35S: CKX7 primary root. Fuchsin staining of lignified tissues of the xylem showed clear differences in the differentiation of the xylem (Figure 7). The xylem axis of the wild-type primary root consists of two cell types: protoxylem with spiral or annular secondary cell wall thickenings and more centrally positioned metaxylem with pitted walls (Figure 7a; € nen et al., 2000). In contrast, the reduced vascular Ma€ho cylinder in the 35S:CKX7 primary root contained only protoxylem (Figure 7b). Such an abnormal differentiation of all vascular cell files into protoxylem was previously described for the wol mutants of the CRE1/AHK4 cytokinin € nen et al., 2000; receptor gene (Scheres et al., 1995; Ma€ho  n et al., 2004; Kuroha et al., 2006) as Garcıa-Ponce de Leo well as for mutants with strong cytokinin signaling defects (Higuchi et al., 2004; Nishimura et al., 2004; Hutchison et al., 2006; Yokoyama et al., 2007; Argyros et al., 2008). This suggests that ectopic expression of CKX7 strongly diminished cytokinin activity in the differentiating procambial cells.

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The procambial cell divisions required to generate the vascular cell files in the root are regulated by the redundant activity of the three cytokinin receptors AHK2, AHK3 and CRE1/AHK4. Therefore only simultaneous mutation of all three genes leads to the aberrant vascular development and determinate growth (Higuchi et al., 2004; Nishimura et al., 2004). However, CRE1/AHK4 displays a binary mode of activity: cytokinin binding promotes its kinase activity, whereas in the absence of cytokinin CRE1/AHK4 is a phosphatase that dephosphorylates AHP proteins and thus € nen et al., 2006a). abbreviates cytokinin signaling (Ma€ho Hence, the wol mutation in CRE1/AHK4 that abolishes cytokinin binding (Yamada et al., 2001) causes the same devel-

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Figure 6. Cell division activity visualized by the expression of the CYCB1: GUS marker gene in the root meristem. Activity of the CYCB1:GUS marker gene in the apical root meristems of wild-type (a) and 35S:CKX7 (line 35S:CKX7-93) transgenic seedlings (b) at 1 day after germination (DAG) (left) and five DAG (right). Scale bar = 100 lm.

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The 35S:CKX7 root phenotype is mediated specifically by the CRE1/AHK4 receptor

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Figure 7. Vascular development in primary roots of 35S:CKX7 plants. (a) Wild-type roots show the formation of proto- and metaxylem. (b) Roots of CKX7-overexpressing plants (line 35S:CKX7-93) show exclusive protoxylem differentiation. The confocal images were taken from seedlings stained with fuchsin. mx, metaxylem; px, protoxylem. Scale bar = 20 lm.

Figure 8. Loss of CRE1/AHK4 receptor function rescues the 35S:CKX7 root phenotype. (a) Wild-type, 35S:CKX7 (line 35S:CKX7-GFP-26), 35S:CKX7 cre1-2 and cre12 seedlings grown in vitro for 9 days. (b) Elongation of the primary root in plants depicted in (a). (c) Number of lateral roots in plants depicted in (a). (d) The CKX enzyme activity in extracts of wild-type, 35S:CKX7, 35S:CKX7 cre1-2 and cre1-2 seedlings grown in vitro for 9 days.

© 2014 The Authors. The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 359–371

Cytosolic cytokinin degradation and root development 365 opmental defects as displayed by triple receptor mutants. To test whether the phosphatase activity of CRE1/AHK4 might be causally involved in the establishment of the 35S: CKX7 phenotype, we introgressed a loss-of-function allele of CRE1/AHK4, cre1-2 (Inoue et al., 2001), in the 35S:CKX7 background and analyzed root development. Figure 8(a) shows that cre1-2 almost completely suppressed the 35S: CKX7 short root phenotype. Likewise, loss of CRE1/AHK4 function rescued the development of the vascular system, lateral root formation and normal seedling growth. Enzyme measurements showed that 35S:CKX7 activity was not significantly changed in the cre1-2 background (Figure 8b). In contrast, introgression of ahk2 or ahk3 mutant alleles in the 35S:CKX7 plants did not rescue the primary root phenotype (Figure S2), indicating that the CRE1/AHK4 activity mediated most if not all of the 35S:CKX7 phenotypic changes. This further suggests that the vascular phenotype was specifically caused by the phosphatase activity of CRE1/AHK4. To test how cytokinin activity was altered by cytokinin deficiency in 35S:CKX7 transgenic plants, we compared the expression of the cytokinin marker gene ARR5:GUS (D’Agostino et al., 2000) in wild type and in 35S:CKX7 transgenic plants (Figure 9). In the wild-type root, ARR5: GUS expression was highest in the columella root cap and procambial region of the meristem, and it became progressively stronger during the time course of development (Figure 9a). In comparison, ARR5:GUS activity was weaker directly after germination and at all tested time points in 35S:CKX7 roots (Figure 9b), indicating reduced cytokinin activity. Furthermore, ARR5:GUS staining was only detectable in columella cells but it was completely missing in the procambium and vascular cylinder of 35S: CKX7 roots even after prolonged staining. Provided that the reduction of the total cytokinin content in 35S:CKX7 plants was weaker than in other CKX overexpressers, the absence of ARR5:GUS expression in the procambium indicated a particularly strong reduction in the cytokinin content or a depletion of specific cytokinin metabolites in this tissue.

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cZ-deficientipt 2,9 plants develop shorter primary roots with aberrant vasculature The remarkably strong reduction in the concentration of several cZ-type cytokinins in the 35S:CKX7 background (Table 1) led us to test the eventual involvement of these cytokinin metabolites during root development and their possible relevance for establishing the wol-like phenotype. Whereas iP- and tZ-type cytokinins are synthesized by ADP/ATP IPTs, cZ-cytokinins are degradation products of cis-hydroxy tRNAs, which are formed by two tRNA IPT enzymes, IPT2 and IPT9 (Miyawaki et al., 2006). The consequences of loss of cZ production in the ipt2,9 double mutant for plant development have not been addressed in detail. In agreement with a previous report, our quantification of endogenous cytokinins showed that all cZ-type cytokinins, except for cZOG, were completely absent in ipt2,9 plants (Miyawaki et al., 2006; Table S1). Figure 10 shows that the elongation of the primary root was strongly reduced by about 70% in these mutants compared with the wild-type control (Figure 10a,b). Microscopic analysis revealed that the short-root phenotype of ipt2,9 plants correlated with a smaller size of the root meristem which contained fewer meristematic cells than the wild type (a)

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Figure 9. Reduced ARR5:GUS expression in the 35S:CKX7 transgenic background. ARR5:GUS expression in wild-type plants (a) and 35S:CKX7 transgenic plants (b) at 1 day after germination (DAG) (left) and six DAG (right). Scale bar = 100 lm. Line 35S:CKX7-93 was used.

Figure 10. Root growth and development in ipt2,9 double mutant plants. (a) Wild-type and ipt2,9 seedlings grown in vitro for 9 days. (b–d) Morphometric analysis of root growth and development. (b) Primary root elongation between 2 and 9 days after germination (DAG), (c) number of emerged lateral roots (LR) at nine DAG, (d) lateral root density. The values shown are means
SD (n ≥ 50). Both mutant lines differed significantly from the wild type for all parameters measured (Student’s t-test, P < 0.001). (e) Bright-field microscopy images of wild-type and ipt2,9 root meristems. The distance between the transition zone, as indicated by the first elongating cortical cells (black arrowheads) and the quiescent center (white arrowhead) corresponds to the meristem size. Scale bar = 1 cm (a) and 100 lm (e).

© 2014 The Authors. The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 359–371

€ llmer et al. 366 Ireen Ko (Figure 10e). The transition zone was positioned more distally, indicating an earlier onset of cell differentiation (Figure 10e). In comparison with the wild type, ipt2,9 roots were markedly thinner. The formation of lateral roots was decreased by about 50% in ipt2,9 plants, but the lateral root density was not reduced (Figure 10c,d). In contrast, both root elongation and lateral root formation in ipt3,5,7 – a multiple ADP/ATP ipt mutant with reduced iP- and tZ-type cytokinin content – were slightly increased, in agreement with a previous report (Figure 10b,c; Miyawaki et al., 2006). Next we analyzed whether IPT2 and IPT9 are also required for the correct differentiation of root vascular tissue. Histological studies revealed that the pattern of differentiation was altered in ipt2,9 roots, with extra protoxylem cell files being frequently established. Two continuous protoxylem cell files were typically observed in wild-type roots and only sporadically short stretches of extra protoxylem were observed (7%, n = 52). This pattern of differentiation was altered in ipt2,9 roots which established one to four ectopic protoxylem cell files in 68% of cases (n = 55; Figure 11). These cell files were often extended over a longer distance. Similarly, ectopic development of one or two protoxylem files was observed in the double cytokinin receptor mutant cre1 ahk3, with 100% frequency € nen et al., 2006b). Notwithstanding the (Figure 11; Ma€ho

apparent similarity, there were subtle differences between the ipt2,9 and cre1 ahk3 mutants. First, although the frequency of ectopic protoxylem formation per plant was higher in cre1 ahk3 than in ipt2,9 (Figure 11b), the number of ectopic protoxylem files was often lower (up to two extra files in cre1 ahk3 versus up to four extra files in ipt2,9). Secondly, whereas the ectopic protoxylem file(s) often continued over a long distance or even the entire root length in cre1 ahk3, the ectopic protoxylem formation was more erratic in ipt2,9. The highest number of extra protoxylem files was often observed close to the root tip in the differentiation zone. These files continued over a certain distance but were eventually terminated and the protoxylem/metaxylem pattern was partially normalized (Figure S3). Importantly, there was no difference in protoxylem development in ipt3,5,7 roots, supporting the hypothesis that protoxylem differentiation in the root procambium could be predominantly controlled by tRNA IPTs. cis-Zeatin is not a specific regulator of root growth and vascular differentiation To test if the reduction of specific cytokinin metabolites is causally linked to developmental changes in 35S:CKX7 and

(a)

(b)

(c)

(d)

(a)

(b)

Figure 11. Ectopic protoxylem formation in the primary root of the ipt2,9 mutant. (a) Protoxylem formation in the wild type, ipt2,9, cre1 ahk3, and ipt3,5,7. The Arabidopsis wild-type root has a central xylem axis mostly with a single protoxylem cell file at each marginal position (px1 and px2 indicated by arrowheads). ipt2,9 roots often develop two or three protoxylem files (arrowheads) at each position. Two images of the same area at different focal planes are shown for ipt2,9 to visualize both protoxylem positions. Bright-field/differential interference contrast images were taken in a root zone before metaxylem differentiation. Scale bar = 20 lm. (b) The frequency of ectopic protoxylem files shown in (a); n ≥ 50.

Figure 12. Analysis of cytokinin sensitivity in 35S:CKX7 and ipt2,9 roots. (a) Elongation of the primary root of 35S:CKX7 seedlings grown on medium supplemented with various concentrations of cis-zeatin (cZ), trans-zeatin (tZ) and benzyladenine (BA; n > 40). (b) Number of protoxylem (px) and metaxylem (mx) cell files in 35S:CKX7 roots grown on medium supplemented with cZ and tZ. Roots on control medium contained only protoxylem; ≥7 files per root (n > 30). (c) Elongation of the primary root of ipt2,9 seedlings grown on medium supplemented with various concentrations of cZ, tZ and BA (n > 40). (d) Quantitative analysis of the extra protoxylem phenotype in ipt2,9 roots grown on medium supplemented with cZ and tZ (n > 30).

© 2014 The Authors. The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 359–371

Cytosolic cytokinin degradation and root development 367 ipt2,9 plants, different cytokinins were tested for their capability to complement these changes. The elongation of primary roots in 35S:CKX7 plants could be weakly, and to a similar extend, complemented by the highest concentrations of cZ, tZ and benzyladenine (BA; Figure 12a). However, the primary root length was still very short in comparison with the wild type. In contrast, Figure 12(b) shows that the root vascular phenotype in 35S:CKX7 was fully rescued by exogenous cytokinin, as the number of protoxylem cell files was suppressed and the formation of metaxylem restored in a dose-dependent manner (Figure 12b). Both zeatin isomers could suppress the wollike vascular pattern; however, tZ was more effective already at lower concentrations compared with cZ. In ipt2,9 plants, all tested cytokinins had only a small effect on primary root elongation at lower concentrations; the highest tested concentration had an inhibitory effect (Figure 12c). However, ectopic protoxylem formation in ipt2,9 plants was suppressed by low cytokinin concentrations (Figure 12d). Interestingly, in the cZ-deficient ipt2,9 mutant, tZ was also effective in suppressing ectopic protoxylem formation, at even lower concentrations than cZ (Figure 12d). These results suggest that, at least in the case of regulation of vascular development in the Arabidopsis root, cZ and tZ have similar activities. DISCUSSION CKX7 is distinct from all other CKX genes of Arabidopsis This work has revealed several distinct properties of the Arabidopsis CKX7 gene and the corresponding protein. One peculiarity is the cytosolic localization of CKX7. Indeed, the cytosolic localization is unique among the seven members of the CKX protein family in Arabidopsis. The CKX1, CKX2 and CKX3 proteins were shown to be localized to different compartments of the secretory pathway (Werner et al., 2003). The subcellular localizations of CKX4, CKX5 and CKX6 have not yet been analysed experimentally, but bioinformatic analyses strongly predict their hydrophobic N-termini to contain targeting signals for the secretory pathway as well. Interestingly, sequence analyses predict that other plant genomes also encode only a single cytosolic CKX isoform but numer ous CKX proteins for the secretory pathway (Smehilov a et al., 2009; Gu et al., 2010). Phylogenetic analysis has revealed that CKX7 is the Arabidopsis CKX family member that is most closely related to CKX-like proteins from € lling et al., phytopathogenic bacteria, green algae (Schmu 2003) and lower land plants (Frebort et al., 2011). This suggests that cytosolic CKX7 is an evolutionarily ancient isoform which may have retained specific functions. The location of CKX7 in the cytosol implies that it has access to a specific subcellular pool of cytokinin that differs from all other CKX proteins.

The expression of the CKX7 gene is also distinguished from other CKX genes of Arabidopsis (Werner et al., 2003) as it is expressed in specific tissues, namely the vasculature of young seedlings and the developing embryo sac, with maximum expression in the egg cell and the synergids. Whether CKX7 participates in the recently described cytokinin-mediated control of female gametophyte development (Kinoshita-Tsujimura and Kakimoto, 2011; Bencivenga et al., 2012; Cheng et al., 2013) needs to be analyzed. However, its apparently unequal distribution in the embryo sac makes it a prime candidate for establishing a cytokinin gradient within the cytoplasmic continuum of € rcher et al., 2013). the embryo sac syncytium (Zu Overexpression of CKX7 indicates a functional relevance of the cytosolic cytokinin pool The most prominent effects of 35S:CKX7 expression were early termination of primary root elongation, complete suppression of lateral root initiation and aberrations of root vascular development similar to those observed in the wol mutant and triple cytokinin receptor mutant (Ma€ho€ nen et al., 2000; Higuchi et al., 2004; Nishimura et al., 2004). This is a very surprising result because 35S-driven overexpression of several other CKX genes from Arabidopsis and other species caused enhanced elongation of the primary root and increased root branching (Werner et al., 2001, 2003; Yang et al., 2003; Galuszka et al., 2004). These opposite phenotypic consequences cannot be explained by the degree of cytokinin depletion, since the cytokinin metabolite profile of 35S:CKX7 plants showed a similar or even weaker overall cytokinin reduction compared with 35S: CKX2 and 35S:CKX1 plants, respectively. A detailed comparison of cytokinin metabolite profiles revealed that cZ, cZ9G and iP9G were the only metabolites which were significantly more strongly reduced in 35S:CKX7 seedlings than in both 35S:CKX1 and 35S:CKX2 lines. This result is generally in good agreement with the substrate preferences of the individual Arabidopsis CKX proteins (Galuszka et al., 2007; Kowalska et al., 2010), among which CKX7 possesses a substantially higher activity towards cZ than to tZ (Gajdosova et al., 2011). We tested the possibility that the specific depletion of cZ cytokinins could be causal for the 35S: CKX7 root phenotype. However, exogenously supplied tZ was even slightly more effective than cZ in suppressing primary root elongation and protoxylem formation. This led us to conclude that the wol-like phenotype in 35S:CKX7 roots was primarily caused by the reduction in the cytokinin concentration in the cytosol. The fact that the shoot growth of 35S:CKX7 plants was less affected than in the 35S:CKX1 line, for example, suggests that the concentration of the cytosolic cytokinin fraction is a particularly relevant parameter in certain tissues such as the root procambium. It should be noted that misexpression of the CKX2 gene can phenocopy the root defects of wol and 35S:CKX7; however,

© 2014 The Authors. The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 359–371

€ llmer et al. 368 Ireen Ko the CRE1 promoter, conferring presumably a stronger expression level in procambium than the 35S promoter, is € nen et al., 2006b). This required to elicit this effect (Ma€ho shows that, at least in these cells, the cytosolic cytokinin pool is more relevant than the cytokinin pool controlled by CKX2. The strong effect of 35S:CKX7 expression on activity of the root procambium was supported by a complete absence of ARR5:GUS signal in this tissue. In contrast, although 35S:CKX1 expression caused a strong reduction of all measured cytokinins, the ARR5:GUS activity in the procambium was reduced only weakly (Werner et al., 2003). The cZ-type cytokinins contribute to the regulation of xylem specification A detailed phenotypic analysis of the ipt2,9 double mutant, which completely lacks cZ-type cytokinins but contains wild-type levels of iP- and tZ-type cytokinins (Miyawaki et al., 2006), was another approach to test the role of cZtype cytokinins in the regulation of procambial cell proliferation and vascular differentiation in the Arabidopsis root. We showed that this mutant frequently initiates ectopic protoxylem differentiation similarly to cytokinin signaling € nen et al., 2006b). However, this differentiamutants (Ma€ho tion was not exclusive as in 35S:CKX7 plants and wol mutants, indicating that cZ-type cytokinins play an important, but not exclusive, role in suppressing protoxylem specification, suggesting that other cytokinins contribute to this control circuit as well. This was supported by the observation that both zeatin isomers could suppress the ectopic protoxylem in ipt2,9 roots, similar to the case in 35S:CKX7. Therefore, although the xylem patterning defect in ipt2,9 roots is apparently caused by the absence of cZ-type cytokinins, cZ does not have an exclusive capacity to regulate this process. Based on the fact that only the ipt2,9 and not adenylate-IPT mutants displayed an aberrant xylem phenotype, we conclude that cZ deficiency may also significantly contribute to the wol-like phenotype in 35S:CKX7 plants. The cZ-type cytokinins regulating cell proliferation and vascular specification in the root procambium may be produced locally in this tissue or come from elsewhere in the plant. There is support for both possibilities. Although the overall expression of IPT2 and IPT9 genes is more ubiquitous in comparison to ADP/ATP IPT genes, they are both highly expressed in the root apical meristem (Miyawaki et al., 2004), indicating that the source for cZ cytokinins may overlap with their site of action. However, cZ- and iP-type cytokinins are also the major cytokinin metabolites found in the phloem (Hirose et al., 2008) and it has been shown that a reduction in phloem cytokinin levels can perturb the root vascular pattern (Bishopp et al., 2011b). This would support a function of phloem-derived cZ-type cytokinins in regulating the differentiation of the root vasculature. It is notable that cZ is more abundant than tZ in seeds and germinating seedlings of Arabidopsis

(Gajdosova et al., 2011), which might suggest a relevant function of this cytokinin for the embryonic and early postembryonic procambial cell proliferation controlled by CRE1/AHK4 (Ma€ho€ nen et al., 2000). Besides being deficient in cZ-type cytokinins, the ipt2,9 mutant also lacks isopentenyl- and cis-hydroxy isopentenyl-tRNA (Miyawaki et al., 2006). The roles of many tRNA modifications in both prokaryotes and eukaryotes are not fully understood, but there is evidence that these modifications affect the tRNA function by, for example, influencing translational efficiency and fidelity (El Yacoubi et al., 2012). It is thus important to note that some of the phenotypic changes observed in the ipt2,9 mutant may be primarily caused by the altered activity of certain tRNAs. Indeed, we were unable to rescue the reduced meristem size, primary root elongation and lateral root formation by exogenous cZ and other cytokinins. CKX7 activity depends on the receptor CRE1/AHK4 We showed that the introgression of a loss-of-function allele of CRE1/AHK4 largely rescued the developmental aberrations in the 35S:CKX7 root. This shows that CKX7 activity depends on CRE1/AHK4 which acts negatively on cytokinin signaling in 35S:CKX7 plants. This negative activity is comparable with that of the wol mutation, which eliminates cytokinin binding (Yamada et al., 2001) and thereby causes domination of the negatively acting phosphatase € nen et al., 2006a). Mutation of activity of CRE1/AHK4 (Ma€ho AHK2 or AHK3 did not alleviate the 35S:CKX7 phenotype, which is in agreement with the predominant activity of CRE1/AHK4 in the root procambium and the phosphatase activity specific to this receptor (Ma€ho€ nen et al., 2006a). Why the cytosolic cytokinin pool is particularly relevant in conjunction with CRE1/AHK4 receptor activity remains unclear. However, this work showed that differentially active cytokinin pools exist in plant cells and that compartmentalization of cytokinin metabolism is relevant for cytokinin action and plant development. The fact that the degradation of cytokinin at different sites of the plant cell results in partially different quantitative and/or qualitative changes in plant growth and development supports the notion that different signaling components may be involved. In this regard, it will be important to understand the role of intracellular cytokinin transport for the cellular compartmentalization of this hormone. EXPERIMENTAL PROCEDURES Plasmid construction and plant transformation The CKX7 gene (At5g21482) was amplified from genomic DNA of Arabidopsis thaliana Columbia (Col-0) with the DNA polymerase Bio-X-Act-long (Bioline, http://www.bioline.com/) using the oligonucleotides L1/KpnI-fw (50 -cggggtaccACACACACACCAAAATGATAGCTT-30 ) and L2/SalI-rev (50 -cgggtcgacAATATGAGGGGTCAAA

© 2014 The Authors. The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 359–371

Cytosolic cytokinin degradation and root development 369 GAGACCTA-30 ). The resulting gene fragment was cloned into pCR2.1-TOPO (Invitrogen, http://www.invitrogen.com/), generating pCR2.1-CKX7. The gene sequence was verified by sequencing and subcloned in the KpnI and SalI sites of pBINHygTx (Gatz et al., 1992) downstream of the 35S promoter. To generate the 35S:CKX7–GFP fusion gene, the CKX7 gene was amplified from pCR2.1-CKX7 using the oligonucleotides L1/ KpnI-fw and CKX7-Stop/XhoI-rev (50 -gcctcgagAGAGACCTATTGAAAATC-30 ) which mutated the stop codon into a serin codon (TCG). The KpnI/XhoI fragment was subcloned in the vector pBinSMGFP (Werner et al., 2003) creating the CKX7–GFP fusion gene under control of the 35S promoter. A 2-kb promoter fragment of CKX7 was amplified by PCR using the oligonucleotides pCKX7SmaI50 -fw (50 -cgcccgggTTTTCTACTGGAACAACACAATTT TT-30 ) and pCKX7SmaI30 -rev (50 -cgcccgggTGTGTGATTGTGTGTAAATGCTAAAT-30 ). The resulting fragment was cloned into the SmaI site of pBluescript II SK(+), sequenced and subsequently subcloned into the SmaI site of the binary vector pGPTV-BAR (Becker et al., 1992) upstream of the uidA reporter gene. The binary vectors were transformed via Agrobacterium tumefaciens GV3101 (pMP90) into A. thaliana Col-0 according to the standard protocol (Clough and Bent, 1998). Primary transformants were selected on half-strength MS medium (Murashige and Skoog, 1962) containing 1% sucrose and supplemented either with 15 mg L1 hygromycin B for 35S:CKX7- and 35S:CKX7-GFPexpressing plants or with 12 mg L1 phosphinotricin for CKX7: GUS transformants.

Plant material and growth conditions CYCB1:GUS, ARR5:GUS and 35S:GFP transgenic plants were  n-Carmona et al., 1999; D’Agostino described previously (Colo et al., 2000; Werner et al., 2003) as well as lines 35S:CKX1-11 and 35S:CKX2-9 (Werner et al., 2003). The CRE1/AHK4 T-DNA insertion mutant line cre1-2 was kindly provided by T. Kakimoto (Inoue et al., 2001). Plants were grown in the greenhouse on soil and in vitro at 19–22°C under long-day conditions (16 h light/8 h dark). For in vitro assays, seeds were surface-sterilized and cold treated at 4°C for 3 days in the dark and then exposed to white light (about 75 lmol m2 sec1). Seedlings were grown on plates containing half strength MS medium with 1% sucrose.

for 1–6 h at room temperature (20 °C). The length of the cortical cells was measured from microscopy pictures using SCION IMAGE software (http://www.scioncorp.com).

Confocal microscopy Lignified xylem tissue was stained as described previously € nen et al., 2000). Seedlings were incubated in 0.01% basic (Ma€ho fuchsin for 3 min at room temperature and, if necessary, destained at room temperature in 70% ethanol. Fuchsin and GFP fluorescence was analyzed by confocal microscopy (Leica TCS SP2, Leica, http://www.leica.com/) using the 488 nm line of the argon laser for excitation. Emission was recorded between 543 and 627 nm and 500 and 530 nm, respectively.

Measurement of CKX activity The enzymatic activity of CKX in plant material was measured using a modified end-point method (Frebort et al., 2002). The reactions were set up as described in Galuszka et al. (2007) using 0.2 ml of plant extract and 0.5 mM iP as a substrate. Reactions were incubated at 37°C for 2–20 h. The concentration of the produced Schiff base was determined using the molar absorption coefficient e352 = 15.2 mM1 cm1 (Frebort et al., 2002) and expressed as the amount of cleaved iP per total protein (Bradford, 1976) and reaction time.

Quantification of endogenous cytokinins Each sample contained about 1 g of 6-day-old seedlings grown in vitro. Three independent biological replicates were analysed for each genotype. Extraction, purification and quantification of endogenous cytokinins was performed according to the method described by Novak et al. (2003) including the modification described in Riefler et al. (2006).

ACKNOWLEDGEMENTS We thank the Deutsche Forschungsgemeinschaft (DFG) for financial support in the frame of the priority program ‘Molecular analysis of phytohormone action’ (SPP 1067).

CONFLICT OF INTEREST

RNA extraction and RNA gel blot analysis

The authors have no conflict of interest to declare.

Total RNA was extracted from 7-day-old seedlings grown in vitro and RNA gel blot analysis was performed as described (Brenner et al., 2005). Radioactive-labelled DNA probe corresponding to CKX7 cDNA was prepared using the Prime-It II Random Primer Labeling Kit (Stratagene, http://www.stratagene.com/). A phosphor imager (Storm 860; Molecular Dynamics, http://www.gelifescienc es.com) was used for signal detection.

SUPPORTING INFORMATION

The GUS staining and light microscopy procedure The GUS expression analysis was carried out as described previously (Werner et al., 2003). For microscopic inspection, tissues were cleared as described (Malamy and Benfey, 1997). Staining was observed with a stereomicroscope (SZX12; Olympus, http:// www.olympus.com/) or a microscope (Axioskop 2 plus with AxioCam ICc3 camera; Zeiss, http://www.zeiss.com/). Similar GUS activity patterns were detected in several independent transgenic lines harboring the CKX7:GUS construct. For microscopic inspection of root meristems, seedlings were fixed in a 1:6 mixture of acetic acid and ethanol for 2–4 h at room temperature and cleared with chloralhydrate:glycerol:H2O (8:1:2)

Additional Supporting Information may be found in the online version of this article. Figure S1. Detailed view of CKX7-overexpressing Arabidopsis plants. Figure S2. Loss of AHK2 and AHK3 function does not alter the 35S:CKX7 root phenotype. Figure S3. Ectopic protoxylem formation in the primary root of the ipt2,9 mutant. Table S1. Cytokinin content in plants overexpressing different CKX genes and in the ipt2,9 mutant.

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© 2014 The Authors. The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 359–371