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. 2015 Dec 7;170(2):1060–1074. doi: 10.1104/pp.15.00650

CYTOKININ OXIDASE/DEHYDROGENASE3 Maintains Cytokinin Homeostasis during Root and Nodule Development in Lotus japonicus1[OPEN]

Dugald E Reid 1,2, Anne B Heckmann 1,2,2, Ondřej Novák 1,2, Simon Kelly 1,2, Jens Stougaard 1,2,*
PMCID: PMC4734552  PMID: 26644503

A Cytokinin oxidase/dehydrogenase in Lotus japonicus regulates cytokinin levels to prevent inhibition of root growth and rhizobial infection during symbiosis.

Abstract

Cytokinins are required for symbiotic nodule development in legumes, and cytokinin signaling responses occur locally in nodule primordia and in developing nodules. Here, we show that the Lotus japonicus Ckx3 cytokinin oxidase/dehydrogenase gene is induced by Nod factor during the early phase of nodule initiation. At the cellular level, pCkx3::YFP reporter-gene studies revealed that the Ckx3 promoter is active during the first cortical cell divisions of the nodule primordium and in growing nodules. Cytokinin measurements in ckx3 mutants confirmed that CKX3 activity negatively regulates root cytokinin levels. Particularly, tZ and DHZ type cytokinins in both inoculated and uninoculated roots were elevated in ckx3 mutants, suggesting that these are targets for degradation by the CKX3 cytokinin oxidase/dehydrogenase. The effect of CKX3 on the positive and negative roles of cytokinin in nodule development, infection and regulation was further clarified using ckx3 insertion mutants. Phenotypic analysis indicated that ckx3 mutants have reduced nodulation, infection thread formation and root growth. We also identify a role for cytokinin in regulating nodulation and nitrogen fixation in response to nitrate as ckx3 phenotypes are exaggerated at increased nitrate levels. Together, these findings show that cytokinin accumulation is tightly regulated during nodulation in order to balance the requirement for cell divisions with negative regulatory effects of cytokinin on infection events and root development.

To alleviate nitrogen-limiting conditions, legumes enter symbiotic relationships with rhizobia, allowing the host plant to acquire fixed nitrogen. Establishment of this symbiosis requires coordinated reinitiation of cell divisions and organogenesis to form the nodule. The plant hormone cytokinin plays a central role during nodule organogenesis, and several components involved in cytokinin signaling have been identified during nodulation, primarily in the model legumes (Frugier et al., 2008; Desbrosses and Stougaard, 2011). Ectopic application of cytokinin or the snf2 gain-of-function mutation in the Lotus japonicus HISTIDINE KINASE1 (LHK1) cytokinin receptor is sufficient to induce cell division and nodule primordia in the absence of bacteria (Bauer et al., 1996; Fang and Hirsch, 1998; Tirichine et al., 2007; Heckmann et al., 2011). Cytokinin perception is also a requirement for nodule organogenesis, as L. japonicus lhk1 and M. truncatula cre1 receptor mutants cause impaired symbiotic events and nodulation is abolished in the L. japonicus lhk1/lhk1a/lhk3 triple mutant (Gonzalez-Rizzo et al., 2006; Murray et al., 2007; Plet et al., 2011; Held et al., 2014). Cytokinin signaling also plays a negative regulatory role in rhizobia infection, as the lhk1-1 mutant exhibits hyper-infection despite the reduced organogenesis (Murray et al., 2007). Downstream cytokinin responses are orchestrated by response regulators, which are induced during nodulation (Lohar et al., 2004; Gonzalez-Rizzo et al., 2006; Lohar et al., 2006; Tirichine et al., 2007; Op den Camp et al., 2011). Cytokinin signaling output as determined with the synthetic two-component signaling sensor (TCS; Müller and Sheen, 2008) has been shown in cortical and pericycle cells in response to lipo-chitooligosaccharide Nod factors in M. truncatula and the dividing cells of the developing nodule in L. japonicus (Held et al., 2014; van Zeijl et al., 2015). The onset of cortical cell divisions requires reprogramming of already differentiated cortical cells and is associated with local auxin signaling (Mathesius et al., 2000; Suzaki et al., 2012) and initiation of endoreduplication (Suzaki et al., 2014; Yoon et al., 2014). This endocycling may be directly induced by cytokinin as cytokinin controls entry into endoreduplication (Takahashi et al., 2013).

Development of a mature nitrogen-fixing nodule is accomplished by coordination of nodule organogenesis and the pathway controlling rhizobial infection (Madsen et al., 2010). Upstream of cytokinin perception, nodulation signaling involves decoding of calcium influx and spiking events by the CALCIUM/CALMODULIN DEPENDENT KINASE (CCaMK) (Lévy et al., 2004; Sieberer et al., 2009). Autoactive variants of CCaMK and its phosphorylation target CYCLOPS are sufficient to trigger downstream nodulation signaling and spontaneous nodules (Tirichine et al., 2006a; Singh et al., 2014). Downstream of LHK1, nodule organogenesis requires NODULE INCEPTION (NIN) (Schauser et al., 1999; Marsh et al., 2007) and the GRAS transcription factors NSP1 and NSP2 (Kaló et al., 2005; Smit et al., 2005; Heckmann et al., 2006; Murakami et al., 2007; Hirsch et al., 2009). These transcription factors are also required in the rhizobia infection pathway. Activation of NIN is a central function of cytokinin activity (Tirichine et al., 2007; Heckmann et al., 2011), and NIN overexpression is also sufficient for spontaneous initiation of cell divisions, which is dependent on the NUCLEAR FACTOR Y transcriptional activators NF-YA1 and NF-YB1 in L. japonicus (Soyano et al., 2013). Activation of nodulation signaling by cytokinin and NIN also induces systemic inhibition of nodulation as it directly activates nodule suppressive CLE peptides (Mortier et al., 2012; Soyano et al., 2014), which systemically regulate nodulation (Okamoto et al., 2009; Mortier et al., 2010; Reid et al., 2011a; Saur et al., 2011). CLE peptide dependent nodule regulation in L. japonicus requires the LRR receptor kinase HYPERNODULATION AND ABERRANT ROOT1 (HAR1) and induces ISOPENTENYL TRANSFERASE (IPT3) dependent cytokinin biosynthesis in the shoot, which negatively regulates nodulation (Krusell et al., 2002; Nishimura et al., 2002; Okamoto et al., 2013; Sasaki et al., 2014). LHK1 was not required for this negative regulatory role of cytokinin, but it was dependent on the function of the Kelch repeat-containing F-box protein, TOO MUCH LOVE in the root (Takahara et al., 2013; Sasaki et al., 2014).

The early nodulation signaling pathway directly induces cytokinin biosynthesis as Nod factor induced cytokinin accumulation, observed at 3 h in wild-type roots, and was not detected in the Mtdmi3 (ccamk) background (van Zeijl et al., 2015). Several cytokinin biosynthesis genes have been identified as contributing to cytokinin pools during nodule development including LjIPT3 and two M. truncatula LONELY GUYs, which directly activate cytokinin nucleotides (Chen et al., 2014; Mortier et al., 2014). However, the processes controlling cytokinin levels and their cell autonomous or non-cell autonomous effects is poorly understood. Studies in non-legumes, primarily Arabidopsis (Arabidopsis thaliana), have shown that regulation of active cytokinin pools occurs through reversible glycosylation, conversion to cytokinin nucleotides by adenine phosphoribosyl transferase genes, and through irreversible breakdown by cytokinin oxidase/dehydrogenases (CKX) (Sakakibara, 2006). Ckx gene expression is enhanced by cytokinin signaling and shows expression patterns similar to Ipt genes, indicating a requirement for finely regulating cytokinin accumulation (Schmülling et al., 2003; Werner et al., 2003; Werner et al., 2006). Overexpression of CKX encoding genes can create dominant reduction in cytokinin levels and has been used to examine the role of cytokinin in root development (Werner et al., 2003; Lohar et al., 2004; Werner et al., 2010). However, loss-of-function ckx mutations in Arabidopsis have not been shown to affect root development.

In order for proper nodule development to progress without secondary effects on root growth, it is assumed cytokinin must be released in a tightly controlled spatial and temporal manner. The availability of the LORE1 insertion population makes L. japonicus an ideal system for reverse genetics (Fukai et al., 2012; Urbański et al., 2012), and here, we make use of this resource to address the role of cytokinin breakdown during nodule development. We identify two insertion alleles in LjCkx3 and show that this gene is critical for maintaining cytokinin homeostasis required for efficient symbiotic infection, organogenesis, and nitrate-dependent regulation of nodulation.

RESULTS

Lotus japonicus Encodes Nine Cytokinin Oxidase/Dehydrogenase Genes

In higher plants, cytokinin oxidase/dehydrogenase is encoded by multigene families. To establish the complexity of the family in legumes, we first searched the available genome and EST sequences of L. japonicus and the M. truncatula genome (v4.0; Young et al., 2011). Nine nonredundant CKX sequences were found in both. In order to construct a phylogeny, all amino acid sequences of the two legume species and Arabidopsis were aligned, and we then named the L. japonicus genes according to the nearest of the seven Arabidopsis homologs (Fig. 1).

Figure 1.

Figure 1.

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Lotus japonicus CKX family. A, CKX phylogeny assembled by alignment of the L. japonicus, M. truncatula, and Arabidopsis amino acid sequences. LjCKX3 (red) is most closely related to two M. truncatula genes, which are induced by Nod factor application (van Zeijl et al., 2015). Bootstrap values are shown for each node based on 1,000 replications. B, LjCkx3 comprises five exons and encodes a predicted signal peptide (SP), cytokinin (CK bind), and FAD binding domains. Lines containing LORE1 insertions were characterized in the first and third exons of LjCkx3.

Given the roles of cytokinin in both rhizobia infection events and initiation of organogenesis, we sought to identify LjCkx genes regulated during the early phases of nodule establishment. Searching publicly available gene expression data (Høgslund et al., 2009; accessed via ljgea.noble.org (Verdier et al., 2013) revealed that two of the LjCkx genes showed expression patterns strongly correlated with symbiotic development. LjCkx3 (probe ID TM0914.20_at) was induced in the nodulation susceptible zone one day after inoculation and in developing nodules, while LjCkx4 (TM0914.24_at) was expressed later in mature nodules. The affymetrix data also indicated that the induction of Ckx3 expression was dependent on NFR1 and NFR5 but independent of NIN. Given the early Nod-factor-dependent expression pattern, we decided to focus further attention on LjCkx3.

Phylogenetic analysis showed that LjCKX3 was most closely related to two M. truncatula genes (Medtr4g126150 and Medtr2g039410) recently reported to show induced expression in response to Sinorhizobium meliloti Nod factor (van Zeijl et al., 2015). Further analysis indicated LjCkx3 comprises five exons annotated by RNAseq analysis and encodes a predicted signal peptide (Fig. 1; SignalP 4.1 d-score 0.668, Petersen et al., 2011), while the mature protein has predicted CK and FAD binding domains, which together form the active site characteristic of CKX proteins (Malito et al., 2004). LjCKX7 was most closely related to MtCKX1 (Medtr1g015410), which was previously reported to be expressed during nodule development in M. truncatula (Ariel et al., 2012).

Ckx3 Is Expressed during Nodule Initiation and Development

To confirm the publicly available Affymetrix data, we inoculated with M. loti or applied purified Nod factor to roots of 10-d-old plants and conducted quantitative reverse transcription (RT)-PCR. This showed Ckx3 expression was induced by M. loti R7A and purified Nod factor within 8 h and increased further at 24 h (Fig. 2). In Arabidopsis, Ckx gene expression is induced by cytokinin application (Werner et al., 2006). Therefore, to determine whether Ckx3 also responds to cytokinin independent of rhizobia, we conducted a time course following treatment with the synthetic cytokinin 6-Benzylaminopurine (BAP). Within this 12 h time series, Ckx3 expression was induced by cytokinin within 6 h (Supplemental Fig. S1).

Figure 2.

Figure 2.

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Effect of ectopic Nod factor application or M. loti inoculation on Ckx3 mRNA levels. A, Relative expression levels following Nod factor treatment. B, Relative expression levels following inoculation with M. loti R7A. Values are relative to mock treatment and indicate mean ± 95% CI for n = 3. P-values were calculated using Wilcoxon rank-sum testing between mock and treatment groups and are indicated by *<0.05.

To further clarify the spatio-temporal expression patterns, we cloned promoter sequences corresponding to approximately 1 kb and 2 kb upstream of Ckx3 and fused these to a nuclear-localized triple-YFP (tYFPnls) reporter, which is readily visualized relative to autofluorescence in L. japonicus. Confocal microscopy revealed that the response patterns were indistinguishable between the 1 kb and 2 kb promoter fragments (Fig. 3; Supplemental Fig. S7). Expression of pCkx3::tYFPnls was observed in the cells corresponding to high cytokinin activity in the meristematic zone of the root tip (Zürcher et al., 2013) irrespective of inoculation status (Fig. 3A). YFP was also observed in the central root cylinder in both inoculated and uninoculated roots (Fig. 3, B and D). Sectioning further confirmed that the central root cylinder expression observed in whole mounts corresponds to expression in pericycle cells adjacent to xylem cells and protoxylem (Fig. 3C). Cross sections of roots inoculated with M. loti expressing DsRed revealed expression occurs in the dividing cells of the root cortex (nodule primordium) and pericycle during nodule primordium development and is sustained in the cortical cells of more mature growing nodules. In mature nodules with differentiated bacteroids, expression was localized to the cells surrounding the infected tissue (Fig. 3). YFP expression was not identified in the epidermal cells of transgenic roots in response to inoculation. The observed expression patterns driven by the Ckx3 promoter are consistent with known cytokinin response domains at the root tip and nodule primordia reported elsewhere (Müller and Sheen, 2008; Held et al., 2014) and, taken together with the real-time results reported here, indicate the promoter fragment likely captures the essential cytokinin response elements responsible for Ckx3 expression patterns. We further confirmed the cytokinin responsiveness of the promoter by treating the 2 kb pCKX3::tYFPnls roots with BAP. Within 3 h, this treatment induced expression of the tYFPnls reporter in the cortex of the root, while vascular expression was indistinguishable from untreated roots (Supplemental Fig. S6).

Figure 3.

Figure 3.

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Activity of LjCkx3 1 kb promoter during root and nodule development. Spatio-temporal expression driven by the Ckx3 promoter was determined in hairy roots using confocal microscopy with a LjCkx3::tYFPnls reporter (nuclear-localized, green). A to C are uninoculated, while d to H are inoculated with M. loti expressing DsRED (magenta) A, The meristematic zone at the root tip independent of inoculation. B, Expression in uninoculated roots. C, Root cross-section showing expression in pericycle adjacent to xylem poles and in protoxylem. D, Expression in the central vascular cylinder of inoculated roots. E, Nodule primordia at 5 d after inoculation with M. loti. Note the expression associated with dividing cells in the cortex and pericycle. F, Nodule primordia 5 dpi. G, Cortex of growing nodules. H, Periphery of fixing nodules. A, B, and D are whole mounts, while others show sections (80–100 µM). C, Cortex; E, endodermis; P, pericycle; X, xylem; Ep, epidermis; IT, Infection Thread; V, Vascular cylinder. Bar, 100 µM.

Identification of Ckx3 Mutant Alleles

To identify mutants in Ckx3, we searched the publicly available LORE1 insertional mutant resources (Fukai et al., 2012; Urbański et al., 2012; Hirakawa et al., 2014; accessed via carb.au.dk/lotus-base/). We identified lines with insertions in exon 1 (5497) and exon 3 (17827) of Ckx3 and named these ckx3-1 and ckx3-2, respectively (Fig. 1). Searching the LORE1 version 2.5 database showed these lines contained 0 and 2 known additional LORE1 insertions respectively, none of which were exonic.

Quantification of Cytokinins in Lotus japonicus Roots

To identify the effect of inoculation on cytokinin levels, we quantified isoprenoid type cytokinins in 10-d old wild type L. japonicus (Gifu) roots 24 and 72 h after inoculation with M. loti R7A or a Nod factor defective R7AnodC mutant compared to mock-treated whole roots. All isoprenoid-type cytokinins were detected as bases, ribosides, and nucleotide metabolites (Supplemental Table S1). N-glucosides and O-glucosides of tZ and DHZ were under detection limits. This analysis showed DHZ and iP cytokinin bases and tZ, cZ, DHZ, and iP ribosides were all increased 24 h postinoculation with R7A relative to R7AnodC inoculated roots (Fig. 4; Supplemental Table S1). tZ ribosides showing the most significant change (1.88-fold increased), while iP type cytokinins were the most abundant cytokinin species detected. After 72 h, the tZ, DHZ, and iP cytokinin bases and ribosides remained significantly increased relative to the nodC inoculated roots. At 72 h, levels of tZ were approximately 2-fold higher in R7A inoculated roots compared to R7AnodC, while iP was approximately 1.5-fold increased.

Figure 4.

Figure 4.

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Cytokinin free base and riboside levels in L. japonicus roots following inoculation with M. loti. A, trans-Zeatin. B, trans-Zeatin Riboside. C, cis-Zeatin. D, cis-Zeatin Riboside. E, Dihydrozeatin. F, Dihydrozeatin Riboside. G, Isopentenyladenine. H, Isopentenyladenine Riboside. Concentrations were measured in mock (white bars) M. loti R7AnodC (striped bars) and M. loti R7A (checked bars) treated Lotus japonicus Gifu and ckx3-2 whole roots 24 h and 72 h after treatment as indicated. Stars represent comparisons to R7AnodC for wild-type bars and between ckx3-2 and wild type under the same treatment group for ckx3-2 bars. Values represent mean ± 95% CI for n = 4 biological replicates. P-values as determined by Wilcoxon rank-sum testing are indicated by *<0.05.

CKX proteins are known to cleave tZ and iP bases and ribosides with varying affinities (Galuszka et al., 2007). To confirm whether CKX3 has a biologically relevant role in degrading cytokinin in L. japonicus roots, we compared cytokinin concentrations in the ckx3-2 mutant relative to Gifu across the same conditions. We found both tZ and DHZ base and riboside levels to be significantly increased in the mutants across treatment groups at both 24 and 72 hpi (Fig. 4; Supplemental Fig. S5). In contrast, cZ and iP type cytokinins were not significantly altered, or slightly decreased, in the mutants relative to Gifu (Fig. 4; Supplemental Fig. S5). In M. loti R7A inoculated ckx3-2 roots, tZR were the only species increased at both 24 and 72 h relative to Gifu, while DHZ and tZ were increased significantly in the mutants at 24 and 72 h, respectively.

Elevated Cytokinin in ckx3 Decreases Nodulation Efficiency

To determine the effect of the elevated cytokinin levels in ckx3 mutants on nodule development, we grew the plants on vertical filter-paper-covered agar slopes with the roots shielded from light, which allows continued observation of nodule developmental phenotypes and kinetics. Both ckx3 alleles developed nodules normal in appearance; however, the number of nodules formed was significantly reduced at all time points (Fig. 5). To confirm these data in a controlled glasshouse environment, we grew plants in open vermiculite pots in nitrate-free conditions and found both alleles showed significantly reduced nodulation 5 w after inoculation (Supplemental Fig. S2).

Figure 5.

Figure 5.

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Symbiotic phenotypes of Ljckx3 mutants. A, Number of nodules per plant 7 to 14 d after inoculation for Gifu, ckx3-1, and ckx3-2 grown on agar slants supplemented with 1 mm KNO3 and inoculated with M. loti R7A. B, Number of infection threads formed per root 10 d after inoculation with M. loti expressing DsRED. C, Number of infection threads formed on plants grown on agar slants supplemented with AVG (10−8 M) 10 d after inoculation with M. loti expressing DsRED. D, Number of infection threads formed on plants grown in the presence of nitrate and BAP (10−8 M). Values represent mean ± 95% CI for n = 30–49 for nodule counts and n = 9 to 10 for IT counts. Statistical comparisons are shown between wild type and ckx3 mutants in A and B and between treatments and control for each genotype in C and D. P-values were calculated using ANOVA and Tukey posthoc testing and are indicated by *<0.05, **<0.01, ***<0.001.

Given that reduced cytokinin signaling in L. japonicus causes hyperinfection (Murray et al., 2007), we counted infection threads formed on the mutants 10 d after inoculation with a M. loti strain expressing DsRED. We found the number of infection threads formed was significantly reduced in both alleles (Fig. 5B). Cytokinin can induce ethylene production and is thought to act largely through ethylene in repressing root growth. The ethylene synthesis inhibitor aminoethoxyvinyl-glycine (AVG) can rescue root growth inhibition in the presence of elevated endogenous or ectopic cytokinin in legumes (Wopereis et al., 2000; Ferguson et al., 2005). We therefore repeated the IT counts with 10−8 M AVG supplemented in the media. AVG treatment was sufficient to rescue the reduced infection thread phenotypes in both ckx3 alleles, increasing IT numbers close to wild-type levels (Fig. 5C). To further identify the infection phenotypes of the ckx3 mutants, we counted infection threads on plants grown in the presence of 2 mm KNO3 or 10−8 M BAP. These results showed that while both nitrate and BAP treatment can significantly reduce infection events, the reduced infection levels of ckx3 is not further impaired upon treatment (Fig. 5D).

Ectopic application of cytokinin or the snf2 gain-of-function mutation has previously been shown to be sufficient to initiate nodule organogenesis in L. japonicus (Tirichine et al., 2007; Heckmann et al., 2011). To determine if the elevated cytokinin levels in ckx3 mutants was sufficient to trigger nodule organogenesis, we grew mutants on nitrate-free agar slants in the absence of rhizobia but did not observe spontaneous organogenesis in these conditions. To determine if the elevated cytokinin in ckx3 plants might be inhibitory to spontaneous nodule development, we grew the mutants in the presence of 10−8 M BAP. Spontaneous nodule organogenesis was observed in both Gifu and ckx3 mutants in these conditions (Supplemental Fig. S3).

Cytokinin Plays a Role in Nitrate Regulation of Nodulation and Nitrogen Fixation

Cytokinin biosynthesis in Arabidopsis, in particular through IPT3, is known to be an important means of regulating plant development in response to environmental signals, including nitrate (Takei et al., 2004; Sakakibara et al., 2006; Ruffel et al., 2011). Since nodulation is negatively regulated by nitrogen, especially nitrate, we investigated whether cytokinin might link nitrogen regulation and nodulation. ckx3 mutant plants were grown under elevated nitrate conditions and the nodulation phenotype observed. Interestingly, increased nitrate concentration accentuated the nodulation phenotypes. To quantify this effect, we grew Gifu and ckx3-2 under different nitrate regimes and counted the number of red nitrogen-fixing nodules, white nonfixing nodules (Fig. 6A) and assayed nitrogen fixation activity from whole roots (Fig. 6B) and individual nodules (Fig. 6C) using the acetylene reduction assay (ARA). This showed that while ckx3-2 had reduced nodulation but formed normal red fixing nodules in nitrate-free conditions, it was more sensitive to increased nitrate than Gifu (Fig. 6, A, D–G). Growth on 2 mm KNO3 significantly reduced total nodule numbers, red nodules, and the ARA activity of Gifu but was reduced significantly more in ckx3-2 (Fig. 6A). Gifu continued to form a small number of pink-red nitrogen-fixing nodules (confirmed by ARA activity) at 5 mm KNO3, however this concentration was completely inhibitory to the development of red nodules and nitrogen fixation in ckx3-2 (Fig. 6A, F, G). To confirm this effect of cytokinin on nitrogen fixation, we also grew the plants on media supplemented with 10−8 M BAP and found nodulation and nitrogen fixation to be significantly reduced in both Gifu and ckx3-2 (Fig. 6A, H, I). Acetylene reduction on a per nodule basis indicated that this response is likely at earlier infection and nodule organogenesis stages as individual nodules formed on BAP-treated roots continued to show normal acetylene reduction activity (Fig. 6C). Nodule sections (Fig. 6, J–O) showed that those nodules that did form on nitrate or BAP-treated roots were colonized by rhizobia expressing the DsRed marker. This included the small nodules formed on ckx3 mutants at 5 mm KNO3 despite the white appearance and near-complete reduction in acetylene reduction activity.

Figure 6.

Figure 6.

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Sensitivity to nitrate regulation of nodulation in ckx3 mutants. A, Number of red Fix+ nodules (red bars) and white Fix- nodules (white bars) formed on Gifu (plain bars) and ckx3-2 (patterned bars) roots 11 d after inoculation with M. loti R7A. Plants were grown on agar slants supplemented with the indicated KNO3 or BAP concentrations. B and C, Box plots of acetylene reduction assay measurements conducted on whole roots (B) or individual nodules (C) 14 dpi in same conditions as above. D to I, Indicative photographs of Gifu and ckx3-2 plants under different conditions. D and E, Nitrate-free conditions. F and G, 5 mm KNO3. H and I, 10−8 M BAP. J to O, nodule sections of Gifu and ckx3-1 grown under different conditions and inoculated with M. Loti MAFF303099 DsRED. J, Gifu nitrate free. K, Gifu 5 mm KNO3. L, Gifu 10−8 M BAP. M. ckx3-1 nitrate free. N, ckx3-1 5 mm KNO3. O, ckx3-1 10−8 M BAP. Values represent mean ± 95% CI for n = 16–29 for nodule counts. ARA was conducted on n = 6–9 roots or nodules. P-values in A indicate comparison of red nodule numbers and were calculated using ANOVA and Tukey posthoc testing. P-values in B and C indicate comparison of Gifu and ckx3-1 for each treatment and were calculated using Wilcoxon rank-sum testing and are indicated by *<0.05.

Ckx3 Regulates Cytokinin Levels Affecting Root Meristem Elongation and Differentiation

To investigate the role of Ckx3 in root development, we measured total root length in Gifu and ckx3 mutants. We found that the ckx3 mutants showed significantly reduced root length relative to Gifu at 20 d after germination (Fig. 7A). To determine the basis of this reduced root growth, we investigated the zones of cell proliferation and elongation, as well as the differentiation zone at the root tip. The region from the root tip to the first emerging root hairs includes the zones of proliferation and elongation, while the zone of differentiation is defined by the emergence and growth of root hairs (Williamson et al., 2001; Jones et al., 2009; Petricka et al., 2012). We therefore measured the distance between the root tip and first root hair as a measure of proliferative and elongation zone length and found the ckx3 mutants exhibit significantly reduced root tip length (Fig. 7B). To evaluate the effect of ckx3 mutation on root hair emergence in the differentiation zone, we measured the angle created by the emergence of root hairs immediately behind the meristem (Supplemental Fig. S4). This analysis showed that the angle was significantly greater in ckx3, indicating more rapid differentiation and/or a reduced differentiation zone length (Fig. 7C).

Figure 7.

Figure 7.

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Root development phenotypes of ckx3 mutants. A, Root length 20 d after germination. B, Length from the root tip to the first emerging root hair. C, Root hair development determined by the angle enclosing emerging root hairs (see Supplemental Fig. S4). Values represent mean ± 95% CI for n = 27–34 for root lengths and n = 15 for root tip and root hair measures. P-values were calculated using ANOVA and Tukey posthoc testing and are indicated by *<0.05, **<0.01, ***<0.001.

DISCUSSION

Cytokinin signaling must be finely regulated during nodulation in order to balance the positive role during nodule organogenesis with the negative effect in symbiotic infection and crosstalk with other hormones. Nod-factor-induced cytokinin accumulation plays a crucial role in the induction of early nodulation responses (van Zeijl et al., 2015) and is perceived partially redundantly by the LHK receptors in L. japonicus (Held et al., 2014). Ckx encoding genes expressed during nodule development have also been implicated in regulation of cytokinin levels and signaling during nodulation (Held et al., 2008; Ariel et al., 2012; van Zeijl et al., 2015); however, the lack of well-defined mutants made the determination of their precise role during nodulation difficult. Here, we identify LORE1 insertion mutants in LjCkx3, which exhibit reduced nodulation, rhizobia infection, and root growth and establish a role for Ckx genes during symbiosis. We show that regulation of cytokinin accumulation through breakdown of cytokinin by LjCKX3 plays a role in maintaining efficient nodule development. Our gene expression data and cytokinin measurements showed that cytokinin signaling rapidly induces expression of Ckx3 in order to restrict cytokinin accumulation. This may serve to avoid over-stimulation of cell division, maintain cytokinin signaling autonomy for neighboring cells and/or stimulation of negative feedback mechanisms such as the effects of ethylene on infection.

Our data also highlight the extensive crosstalk between cytokinin and ethylene. Cytokinin signaling negatively regulates infection since mutations in Lhk1 results in hyperinfection (Murray et al., 2007), while ethylene also inhibits infection (Penmetsa and Cook, 1997; Penmetsa et al., 2008). Consistent with these results, we found the reduced infection phenotypes of the ckx3 mutants could be rescued by AVG treatment, indicating cytokinin degradation is critical in preventing over-stimulation of ethylene-dependent inhibition of symbiotic infection. This interpretation is supported by results showing that cytokinin induces and stabilizes ACC synthase, the rate limiting step in ethylene biosynthesis (Chae et al., 2003; El-Showk et al., 2013). In return, ethylene can regulate cytokinin signaling through the type-A ARRs (Shi et al., 2012).

CKX3 Primarily Regulates tZ Levels

Our quantification of isoprenoid cytokinins showed that Ckx3 regulates root cytokinin levels in vivo. The elevated levels of tZ and DHZ type cytokinins in ckx3-2 suggest these are either the species most susceptible to CKX3 degradation or that accumulate in response to CKX activity on other cytokinins. Biochemical studies have shown Arabidopsis CKX genes expressed in a heterologous Nicotiana tabacum system have highest activity against trans-Zeatin (tZ) and isopentenyl adenine (iP) type cytokinins while cis-Zeatin (cZ) and dihydrozeatin (DHZ) are resistant to cleavage (Galuszka et al., 2007). While CKX was shown to have limited activity against DHZ in one study, tZ may be converted to DHZ by zeatin reductase, especially in the absence of CKX activity (Gaudinová et al., 2005). DHZ does not have strong activity in Arabidopsis and has been suggested to act as a storage or transport form of cytokinin (Mok and Mok, 2001). tZ may therefore be the primary target of CKX3 and increased DHZ is a direct result of high tZ levels. In Arabidopsis, the three AHK receptors maintain specificity in cytokinin response through both expression domains and differing affinities to the cytokinin ligands, with tZ having high affinity binding against AHK2, AHK3, and AHK4, while iP shows strong affinities against AHK2 and AHK4 (Romanov et al., 2006; Stolz et al., 2011). The LHK1, LHK1a, and LHK3 receptors in L. japonicus have been shown to functionally restore cytokinin sensitivity in Yeast or E. coli heterologous system assays; however, no data are available on their cytokinin binding specificity (Murray et al., 2007; Held et al., 2014). IPT3 dependent synthesis of iP type cytokinin in the shoot has been shown to negatively regulate nodule numbers in L. japonicus (Sasaki et al., 2014). Our study provides further evidence that increased cytokinin levels can negatively regulate infection and organogenesis events and that cytokinin levels are therefore finely regulated to maintain efficient nodulation.

We found the accumulation of cytokinin bases was dependent on Nod factor signaling as the R7AnodC mutant failed to initiate the responses observed for R7A wild-type strain. This is consistent with the results showing Nod factor induction of cytokinin in M. truncatula (van Zeijl et al., 2015), albeit at later timepoints in our experiments. The Nod factor treatment reported in M. truncatula was found to induce accumulation of iP, iPR, and tZ type cytokinins (van Zeijl et al., 2015). We also found iP and iPR to be induced by M. loti inoculation on L. japonicus at both 24 h and 72 h, whereas tZ was only increased at 72 h. We also found significant increases in tZR, DHZ, and DHZR at both 24 h and 72 h after M. loti inoculation. These additional cytokinins identified in our studies may result from differences in Nod factor and rhizobia responses or result from the later time-points we examined relative to the early Nod factor responses reported. Furthermore, it is possible that cytokinin interconversion occurs following initial synthesis or that between-species differences exist in cytokinin responses to rhizobia. We found that although the cytokinin pool is under negative feedback by CKX3, which is induced within 8 h of inoculation, elevated cytokinin is maintained during the first three days following inoculation, though likely in a tightly spatially restricted manner. TCS expression in L. japonicus showed cytokinin signaling domains were restricted to the dividing cells of the nodule primordia, while Nod factor responses in M. truncatula triggered a more widespread cortical and pericycle response (Held et al., 2014; van Zeijl et al., 2015). Further analysis at the early points during symbiotic interaction is required to confirm whether rhizobia induce cytokinin signaling in a wide region as is observed for Nod factor treatment or through more restricted biosynthesis and associated degradation in order to restrict cytokinin signaling and induction of nodulation foci to a small number of defined cells.

Local Restriction of Cytokinin Accumulation Is Required for Efficient Infection

Our data showing a Nod factor dependent (Fig. 2A) and nodule primordia localized (Fig. 3, E and F) expression pattern for Ckx3 indicate that cytokinin signal induction is transient and must be tightly regulated to avoid negative effects. This is consistent with data indicating that increased cytokinin signaling in Ljsnf2 or through ectopic MtLog overexpression reduces nodulation (Tirichine et al., 2007; Mortier et al., 2014). Most Arabidopsis CKX have predicted signal peptides and are proposed to localize to the apoplast or vacuoles (Werner et al., 2003; Kowalska et al., 2010). We also found LjCKX3 has a predicted secretion signal and is therefore likely degrading cytokinin in the extracellular space.

Cytokinin has been proposed as a noncell autonomous signal during nodulation and restriction of extracellular cytokinin would therefore be important for regulation of this signaling. We did not observe epidermal expression of Ckx3, and it remains unresolved whether alternative Ckx genes may be expressed here or whether cytokinin biosynthesis in the epidermis is sufficient to produce a non-cell autonomous cytokinin signal to initiate cell divisions in the cortex. The close correlation of Ckx3 expression in nodule primordia cells and correlation with areas of high cytokinin response in the root tip are strongly correlated to the signaling domains of TCS in legumes and Arabidopsis respectively (TCS; Müller and Sheen, 2008; Zürcher et al., 2013; Held et al., 2014). Furthermore, it is likely that the role of CKX3 in regulating cytokinin levels in response to Nod factor is conserved in legumes as two closely related homologs in M. truncatula were both shown to respond to Nod factor treatment (van Zeijl et al., 2015). The induction of spontaneous nodules through elevated cytokinin signaling continues to maintain defined foci rather than widespread induction of cell divisions (Tirichine et al., 2006b, 2007; Heckmann et al., 2011). Our observations also suggest unknown mechanisms outside of cytokinin signaling might be required to define cells competent for division and to maintain these divisions to a limited nodule foci as we did not observe persistent cell divisions or abnormally shaped nodules in ckx3 mutants. Maintaining organized cell divisions during nodule development therefore results from the coordination of nodulation specific transcriptional networks with hormones required for cell specification and division.

Cytokinins are known to alter root meristem size (Dello Ioio et al., 2007). This inhibition is dependent on ethylene in Arabidopsis and L. japonicus (Wopereis et al., 2000; Růžička et al., 2009). Ethylene is produced during nodulation (Ligero et al., 1986) and our work finds the breakdown of cytokinin is required to prevent the resulting inhibition of root growth and infection. Further analysis of the genetics of ethylene induction in the common symbiosis pathway will help to clarify the observed cross talk and regulatory functions. Pericycle and root tip expression of Ckx3 was independent of inoculation, indicating that the evolutionary ancestral role of CKX3 is likely in regulation of root development. However, no root phenotypes for ckx3 mutants have been reported in Arabidopsis, while ckx3 ckx5 double mutants have altered shoot inflorescence meristem size (Bartrina et al., 2011). Pericycle cells in Medicago truncatula maintain the ability to divide in order to initiate lateral root and nodule primordia (Xiao et al., 2014). In Arabidopsis, Ckx genes are expressed during lateral root primordia initiation (Werner et al., 2003) and expression of Ckx in the xylem pole pericycle cells can increase lateral root density (Laplaze et al., 2007). While we observed expression of Ckx3 in pericycle cells, it was not universal in pericycle cells. The expression of Ckx3 in a subset of pericycle cells may play a role in priming cells and determining their susceptibility to undergo division. It would be interesting to determine if the cells with Ckx3 expression are correlated with the radial or longitudinal positioning of nodule primordia sites. Improved spatio-temporal expression analysis using promoter-YFP stable lines may provide a means to determine whether cytokinin degradation plays a role in radial nodule positioning and the crosstalk with ethylene in this process during nodule development.

Cytokinin Regulates Nodulation in Response to Environmental Signals

Legumes regulate nodulation locally and systemically in response to environmental cues, including nitrate, in order to balance fixed nitrogen from symbiosis with other nitrogen sources (Reid et al., 2011b). This regulation occurs by both HAR1-dependent and -independent mechanisms. In Arabidopsis, IPT3 expression is known to be induced by nitrate and cytokinin levels are increased as a result (Takei et al., 2004). Cytokinin thus plays a central role in regulating responses to the environment (Sakakibara et al., 2006; Ruffel et al., 2011). We found the increased cytokinin levels in Ljckx3 mutants enhanced the susceptibility to negative effects of nitrate on nodulation and nitrogen fixation. This indicates cytokinin plays a role in regulating nitrogen fixation. The strong inhibition of nitrogen fixation relative to nodulation indicates that nitrogen fixation and nodule organogenesis possess both common and independent regulatory mechanisms in response to available nitrogen. This is supported by the fact hypernodulation mutants show reduced nitrogen fixation on a per-nodule basis (Carroll et al., 1985; Jeudy et al., 2010). Whether the cytokinin regulation of nitrogen fixation occurs locally or systemically in response to nitrate and how this integrates with the HAR1 mediated regulation of nodulation remains to be resolved. Nitrate inhibition of nodulation has previously been shown to involve ethylene biosynthesis as it can be alleviated by AVG treatment; however, it is not known if this involves local or systemic ethylene responses (Ligero et al., 1991). Ethylene is also thought to regulate cytokinin signaling in both environmental responses and nodulation signaling (Shi et al., 2012; van Zeijl et al., 2015). Together with our findings, this is consistent with cytokinin and its crosstalk with ethylene playing a significant role in the inhibition of nodulation and nitrogen fixation by nitrate.

CONCLUSIONS

We found that inoculation with M. loti causes an increase in root cytokinin levels and a resulting up-regulation of negative feedback through cleavage of active cytokinins, particularly tZ, by CKX3. CKX3 acts to restrict the pool of active cytokinin and prevents the resulting stimulation of ethylene and negative effects on nodule organogenesis, nitrogen fixation, and infection thread development. Expression of Ckx3 in the root tip ensures homeostasis of cytokinin in the meristematic zone in order to balance cell elongation with differentiation and root hair outgrowth. Overall, these results confirm the importance of cytokinin in maintaining effective nodulation and identify a new negative regulator of this signaling. Future efforts to elucidate the roles of crosstalk of cytokinin with other plant hormones, particularly auxin (Breakspear et al., 2014) and ethylene (Ferguson and Mathesius, 2014), will assist in understanding the cellular mechanism involved in nodule development.

MATERIALS AND METHODS

Plant and Bacteria Genotypes

Lotus japonicus ecotype Gifu was used in all experiments (Handberg and Stougaard, 1992). Homozygous LORE1 inserts were genotyped with allele specific primers in combination with the P2 internal LORE1 primer as described (Urbański et al., 2012). Primer sequences were obtained from the LORE1 resource page (carb.au.dk/lotus-base) or designed in the same region if amplification was unsuccessful (Supplemental Table S2). M. loti R7A and the Nod factor defective nodC variant (Rodpothong et al., 2009) were diluted to an inoculum density of OD600 = 0.01. For infection thread counting, the M. loti MAFF303099 strain carrying chromosomal DsRed insertion was used (Maekawa et al., 2009).

Plant and Bacteria Growth Conditions

For nodulation assays and IT counts, 3-d-old seedlings were transferred to vertical plates with filter paper on 1.4% agar noble containing quarter-strength B&D nutrients (B&D; Broughton and Dilworth, 1971) in the presence or absence of KNO3, 10−8 BAP or AVG as described for each experiment. Nod factor treatment was carried out on plates with 10−8 M. loti R7A Nod factor pipetted directly onto roots. Infection threads were counted 10 d after inoculation by placing whole roots on microscope slides to allow counting of the root surface contacting the growth plates. Hairy roots were induced by infection of 6-d-old seedlings growing on vertical 0.8% Phytagel (Sigma) plates with half-strength B5 salts and vitamins as described (Stougaard et al., 1987; Hansen et al., 1989; Stougaard, 1995). Three weeks after infection, primary roots were removed and the chimeric plants transferred to plastic magenta boxes containing 1:4 leca:vermiculite mix. For phenotyping in open pots, 3-d-old seedlings were transferred to pots containing vermiculite watered with nitrate free half-strength B&D.

Bioinformatics

The amino acid translations of the representative gene models for Arabidopsis Ckx genes were obtained from TAIR for BLAST queries against L. japonicus resources at NCBI and Lotus base (carb.au.dk/lotus-base). M. truncatula sequences were obtained by searching Mt v4 at Phytozome.org. The gene phylogeny was drawn based on alignment of the amino acid sequences of all three species and subsequent bootstrap analysis based on 1000 replications using ClustalX (Larkin et al., 2007). Cytokinin and FAD binding domains within CKX3 were identified by BLAST query against the NCBI Conserved Domain Database (Marchler-Bauer et al., 2015). Signal peptide prediction was carried out using SignalP 4.1 (Petersen et al., 2011). Microarray data were identified and analyzed using the L. japonicus gene expression atlas (Verdier et al., 2013) by identifying probes against LjCkx genes with BLAST.

Cloning

Primers for cloning a Ckx3 promoter sequence corresponding to approximately 1 kb upstream sequence were designed against the Lj2.5 genome and amplification carried out from MG-20 genomic DNA, while the 2kb promoter fragment was synthesized according to MG-20 genomic sequence. The Ckx3 promoter fragment was subsequenctly cloned by TOPO cloning into the Gateway compatible pDONR vector (Life Technologies). tYFPnls was constructed by amplifying a tYFP cDNA with primers including a C-terminal nuclear localization signal (Takeda et al., 2012) and excision of the largest of three resulting bands before cloning into a pIV10 vector (Stougaard, 1995) modified to accept Gateway promoter clones.

Quantitative RT-PCR

For expression analysis in roots, plants were grown and Nod factor or BAP applied as described previously (Heckmann et al., 2011). mRNA was isolated from BAP (10−8 M), Nod factor (10−8 M) or R7A (OD600=0.02) treated roots using Dynabeads mRNA DIRECT kit (Invitrogen). RevertAid M-MuLV Reverse Transcriptase (Fermentas) was used for cDNA synthesis. All cDNA samples were tested for genomic DNA contamination using primers specific for the NIN gene promoter (Lohmann et al., 2010). A Lightcycler480 instrument and Lightcycler480 SYBR Green I master (Roche Diagnostics GmbH) was used for the real-time quantitative PCR. ATP-synthase (ATP), Ubiquitin-conjugating enzyme (UBC), and Protein phosphatase 2A (PP2A) were used as reference genes (Czechowski et al., 2005). The relative quantification software (Roche) was used to calculate the normalized efficiency-corrected relative transcript levels. The geometric mean of the relative transcript levels for the three biological (each consisting of 10 plants) and three technical repetitions and the corresponding upper and lower 95% confidence were calculated (Vandesompele et al., 2002). Primer sequences are listed in Supplemental Table S2.

Microscopy

Microscopy was performed with a Zeiss LSM 510 Meta Confocal Microscope. Objective lenses were Zeiss Plan-Neofluar 10×/0.3 and 20×/0.5. Laser excitation was at 488 nm for YFP and 543 nm for DsRed and emission filters were 505–550 nm for YFP and 585–615 nm for DsRed. For sections, live roots were embedded in 3% agarose and cut to 80–100 µM sections using a Leica VT 1000 S vibratome before imaging with the confocal microscope.

Statistical Analysis

Statistical analysis was carried out using GraphPad Prism software. Comparison of multiple groups included ANOVA followed by Tukey posthoc testing to determine statistical significance. Students t-test or Wilcoxon rank-sum test was used to determine differences when making single comparisons. All data are plotted as mean with 95% CI for the indicated number of biological replicates.

Acetylene Reduction Assay

Acetylene was produced by reaction of calcium carbide with water. The resulting gas was collected and diluted to 2% in stoppered glass vials. For the assay, 250 µl air was removed from the 5 ml glass GC vials containing whole nodulated roots 14 d after inoculation and replaced with equal volume of 2% acetylene. Samples were incubated 30 min before quantification of ethylene conversion using a SensorSense (Nijmegen, NL) ETD-300 ethylene detector operating in sample mode with 2.5 L/h flow rate and 6-min detection time.

Endogenous Cytokinin Measurements

For cytokinin analysis, plants were grown on filter paper covered agar slants as described above. Ten-day-old plants were inoculated before whole roots were harvested at 24 h or 72 h after treatment. Prepared biological quadruplicates were extracted and purified using the method published previously (Novák et al., 2008) with some minor modifications. Ten to twenty mg FW were extracted in 1 ml of modified Bieleski buffer (60% MeOH, 10% HCOOH, and 30% H2O) together with a cocktail of 23 stable isotope-labeled CK internal standards (0.5 pmol of CK bases, ribosides, N-glucosides, 1 pmol of O-glucosides and nucleotides) to check recovery during purification and to validate the determination. The samples were purified using a combination of C18 (100 mg/1mL) and MCX cartridges (30 mg/1mL) and immunoaffinity chromatography (IAC) based on wide-range specific monoclonal antibodies against cytokinins (Faiss et al., 1997). The eluates from the IAC columns were evaporated to dryness and dissolved in 20 µl of the mobile phase used for quantitative analysis. The samples were analyzed by the LC-MS/MS system consisting of an ACQUITY UPLC System (Waters) and Xevo TQ-S (Waters) triple quadrupole mass spectrometer. Quantification was obtained using a multiple reaction-monitoring mode of selected precursor ions and the appropriate product ion.

GenBank accession numbers: LjCkx1, KR296932; LjCkx2, KR296933; LjCkx3, KR296934; LjCkx4, KR296935; LjCkx5, KR296936; LjCkx6, KR296937; LjCkx7, KR296938; LjCkx8, KR296939; LjCkx9, KR296940

Supplemental Data

Supplementary Material

Supplemental Data
supp_170_2_1060__index.html (1.2KB, html)

Acknowledgments

We thank Finn Pedersen for glasshouse assistance, Niels Sandal for assistance with plant materials, Katharina Markmann and Dennis Holt for cDNA, Anna Jurkiewicz for microscopy assistance, Clive Ronson and John Sullivan for purified Nod factor, and Eva Hirnerová and Michaela Glosová for technical assistance in cytokinin analysis.

Glossary

TCS

two-component signaling sensor

BAP

6-Benzylaminopurine

AVG

aminoethoxyvinyl-Gly

ARA

acetylene reduction assay

Footnotes

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