CYTOKININ OXIDASE/DEHYDROGENASE3 Maintains
Cytokinin Homeostasis during Root and Nodule
Development in Lotus japonicus1[OPEN]
Dugald E. Reid, Anne B. Heckmann 2, Ondrej Novák, Simon Kelly, and Jens Stougaard*
Centre for Carbohydrate Recognition and Signalling (CARB), Department of Molecular Biology and Genetics,
Aarhus University, Gustav Wieds Vej 10, Aarhus C, 8000, Denmark (D.E.R., A.B.H., S.K., J.S.); and Laboratory of
Growth Regulators and Department of Chemical Biology and Genetics, Palacký University and Institute of
Experimental Botany, Academy of Sciences of the Czech Republic, CZ-78371, Olomouc, Czech Republic (O.N.)
ORCID IDs: 0000-0001-9291-9775 (D.E.R.); 0000-0003-3452-0154 (O.N.); 0000-0002-9312-2685 (J.S.).
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).
1
This work was supported by the Danish National Research Foundation grant no. DNRF79, the ERC Advanced Grant 268523 and the
Ministry of Education, Youth and Sports of the Czech Republic, the
‘‘Návrat’’ program LK21306.
2
Present address: Arla Foods Ingredients, Sønderhøj 10, 8260 Viby
J, Denmark
* Address correspondence to [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article, in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Jens Stougaard ([email protected]).
D.R. designed and performed experiments and wrote the manuscript; A.H. designed and performed experiments; O.N. performed
cytokinin measurement experiments; S.K. performed experiments;
and J.S. conceived experiments and complemented the writing.
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1060
Ectopic application of cytokinin or the snf2 gain-offunction 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; GonzalezRizzo 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 lipochitooligosaccharide 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
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Cytokinin Degradation in Nodule Development
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; Urba
nski 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).
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
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Figure 1. 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.
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
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Cytokinin Degradation in Nodule Development
(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).
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).
Identification of Ckx3 Mutant Alleles
To identify mutants in Ckx3, we searched the publicly
available LORE1 insertional mutant resources (Fukai
et al., 2012; Urba
nski 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
Figure 2. 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 6 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 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
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Reid et al.
Figure 3. 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 mM). C,
Cortex; E, endodermis; P, pericycle; X, xylem; Ep,
epidermis; IT, Infection Thread; V, Vascular cylinder.
Bar, 100 mM.
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
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Cytokinin Degradation in Nodule Development
Figure 4. Cytokinin free base and riboside levels in L. japonicus roots following inoculation with M. loti. A, trans-Zeatin. B, transZeatin 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 6 95% CI for n = 4 biological replicates. P-values as determined by Wilcoxon rank-sum testing are indicated by *,0.05.
2-fold higher in R7A inoculated roots compared to
R7AnodC, while iP was approximately 1.5-fold
increased.
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).
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 1028 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 wildtype 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 1028 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).
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Reid et al.
Cytokinin Plays a Role in Nitrate Regulation of
Nodulation and Nitrogen Fixation
Figure 5. 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
(1028 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 (1028 M). Values represent mean 6 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.
Ectopic application of cytokinin or the snf2 gain-offunction 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 1028 M BAP. Spontaneous nodule organogenesis was observed in both Gifu and ckx3 mutants in
these conditions (Supplemental Fig. S3).
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 1028 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.
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
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Cytokinin Degradation in Nodule Development
Figure 6. Sensitivity to nitrate regulation of nodulation in ckx3 mutants. A, Number of red Fix+ nodules (red bars) and white Fixnodules (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, 1028 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 1028 M BAP. M. ckx3-1 nitrate free. N, ckx3-1 5 mM
KNO3. O, ckx3-1 1028 M BAP. Values represent mean 6 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 ranksum testing and are indicated by *,0.05.
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).
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
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Reid et al.
Figure 7. 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 6
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.
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
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Cytokinin Degradation in Nodule Development
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; R
uzicka 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.,
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Reid 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.
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, 1028 BAP or AVG as described for each experiment. Nod factor
treatment was carried out on plates with 1028 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.
CONCLUSIONS
We found that inoculation with M. loti causes an increase in root cytokinin levels and a resulting upregulation 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
(Urba
nski 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
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 (1028 M), Nod factor (1028 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 103/0.3 and 203/0.5. Laser
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Cytokinin Degradation in Nodule Development
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 mM sections using a Leica VT
1000 S vibratome before imaging with the confocal microscope.
Supplemental Figure S7. Activity of LjCkx3 2kb promoter during root and
nodule development.
Supplemental Figure S8. Absence of lateral root phenotypes of Ljckx3
mutants.
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
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.
Received May 1, 2015; accepted December 4, 2015; published December 7, 2015.
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 ml 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 ml 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
Supplemental Table S1. Complete cytokinin quantification profile for Gifu
and ckx3-2 following inoculation.
Supplemental Table S2. Oligonucleotide primer sequences used in this
study
Supplemental Table S3. Ckx gene ID and accession numbers for L. japonicus, M. truncatula and Arabidopsis.
Supplemental Figure S1. Effect of ectopic cytokinin application on Ckx3
mRNA levels.
Supplemental Figure S2. Nodulation phenotypes of ckx3 mutants grown
in open pot conditions.
Supplemental Figure S3. BAP induced spontaneous nodules.
Supplemental Figure S4.Depiction of quantification used for root development.
Supplemental Figure S5. Cytokinin base and riboside concentrations in
Ljckx3-2 relative to Gifu in the same treatment conditions.
Supplemental Figure S6. Effect of ectopic cytokinin application on pCkx3::
tYFPnls as determined with confocal microscopy.
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