Plant Physiology Preview. Published on December 7, 2015, as DOI:10.1104/pp.15.00650
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Running head: Cytokinin degradation in nodule development
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Author for correspondence:
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Jens Stougaard
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Department of Molecular Biology and Genetics
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Aarhus University
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Gustav Wieds Vej 10, Aarhus C, 8000, Denmark
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+45 87 15 55 04
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[email protected]
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Copyright 2015 by the American Society of Plant Biologists
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Title:
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CYTOKININ OXIDASE/DEHYDROGENASE3 maintains cytokinin homeostasis during root and nodule
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development in Lotus japonicus
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Dugald E Reida, Anne B Heckmanna, Ondřej Novákb, Simon Kellya and Jens Stougaarda
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a
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Genetics, Aarhus University, Gustav Wieds Vej 10, Aarhus C, 8000, Denmark
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b
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University and Institute of Experimental Botany, Academy of Sciences of the Czech Republic, CZ-78371,
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Olomouc, Czech Republic
Centre for Carbohydrate Recognition and Signalling (CARB), Department of Molecular Biology and Laboratory of Growth Regulators and Department of Chemical Biology and Genetics, Palacký
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50 word summary:
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Cytokinin signalling is regulated during nodulation to balance organogenesis with root growth and
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rhizobial infection. In Lotus japonicus the Ckx3 gene is induced during nodule initiation and
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characterization of ckx3 mutants show that cytokinin degradation by CKX3 regulates this process through
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negative regulation of cytokinin levels as measured by LC-MS/MS.
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One sentence summary:
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A Cytokinin oxidase/dehydrogenase in Lotus japonicus regulates cytokinin levels to prevent inhibition of
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root growth and rhizobial infection during symbiosis.
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Author contributions:
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D.R. Designed and performed experiments and wrote the manuscript; A.H. Designed and performed
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experiments; O.N. Performed cytokinin measurement experiments; S.K. Performed experiments; J.S.
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Conceived experiments and complemented the writing
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Financial sources:
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This work was supported by the Danish National Research Foundation grant no. DNRF79, the ERC
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Advanced Grant 268523 and the Ministry of Education, Youth and Sports of the Czech Republic, the
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‘‘Návrat’’ program LK21306
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ABH Present address:
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Arla Foods Ingredients, Sønderhøj 10, 8260 Viby J, Denmark ([email protected])
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Author for correspondence:
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Jens Stougaard ([email protected])
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Abstract
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Cytokinins are required for symbiotic nodule development in legumes and cytokinin signalling responses
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occur locally in nodule primordia and in developing nodules. Here we show that the Lotus japonicus Ckx3
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cytokinin oxidase/dehydrogenase gene is induced by Nod factor during the early phase of nodule
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initiation. At the cellular level, pCkx3::YFP reporter-gene studies revealed that the Ckx3 promoter is
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active during the first cortical cell divisions of the nodule primordium and in growing nodules. Cytokinin
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measurements in ckx3 mutants confirmed that CKX3 activity negatively regulates root cytokinin levels.
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Particularly tZ and DHZ type cytokinins in both inoculated and uninoculated roots were elevated in ckx3
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mutants suggesting that these are targets for degradation by the CKX3 cytokinin oxidase/dehydrogenase.
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The effect of CKX3 on the positive and negative roles of cytokinin in nodule development, infection and
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regulation was further clarified using ckx3 insertion mutants. Phenotypic analysis indicated that ckx3
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mutants have reduced nodulation, infection thread formation and root growth. We also identify a role for
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cytokinin in regulating nodulation and nitrogen fixation in response to nitrate as ckx3 phenotypes are
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exaggerated at increased nitrate levels. Together, these findings show that cytokinin accumulation is
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tightly regulated during nodulation in order to balance the requirement for cell divisions with negative
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regulatory effects of cytokinin on infection events and root development.
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Introduction
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To alleviate nitrogen-limiting conditions, legumes enter symbiotic relationships with rhizobia allowing
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the host plant to acquire fixed nitrogen. Establishment of this symbiosis requires coordinated reinitiation
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of cell divisions and organogenesis to form the nodule. The plant hormone cytokinin plays a central role
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during nodule organogenesis and several components involved in cytokinin signalling have been
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identified during nodulation, primarily in the model legumes (Frugier et al., 2008; Desbrosses and
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Stougaard, 2011). Ectopic application of cytokinin or the snf2 gain-of-function mutation in the Lotus
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japonicus HISTIDINE KINASE1 (LHK1) cytokinin receptor is sufficient to induce cell division and
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nodule primordia in the absence of bacteria (Bauer et al., 1996; Fang and Hirsch, 1998; Tirichine et al.,
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2007; Heckmann et al., 2011). Cytokinin perception is also a requirement for nodule organogenesis as L.
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japonicus lhk1 and M. truncatula cre1 receptor mutants cause impaired symbiotic events and nodulation
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is abolished in the L. japonicus lhk1/lhk1a/lhk3 triple mutant (Gonzalez-Rizzo et al., 2006; Murray et al.,
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2007; Plet et al., 2011; Held et al., 2014). Cytokinin signalling also plays a negative regulatory role in
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rhizobia infection as the lhk1-1 mutant exhibits hyper-infection despite the reduced organogenesis
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(Murray et al., 2007). Downstream cytokinin responses are orchestrated by response regulators, which are
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induced during nodulation (Lohar et al., 2004; Gonzalez-Rizzo et al., 2006; Lohar et al., 2006; Tirichine
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et al., 2007; Op den Camp et al., 2011). Cytokinin signalling output as determined with the synthetic two-
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component signalling sensor (TCS; Müller and Sheen, 2008) has been shown in cortical and pericycle
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cells in response to lipo-chitooligosaccharide Nod factors in M. truncatula and the dividing cells of the
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developing nodule in L. japonicus (Held et al., 2014; van Zeijl et al., 2015). The onset of cortical cell
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divisions requires reprogramming of already differentiated cortical cells and is associated with local auxin
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signalling (Mathesius et al., 2000; Suzaki et al., 2012) and initiation of endoreduplication (Suzaki et al.,
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2014; Yoon et al., 2014). This endocycling may be directly induced by cytokinin as cytokinin controls
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entry into endoreduplication (Takahashi et al., 2013).
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Development of a mature nitrogen-fixing nodule is accomplished by coordination of nodule
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organogenesis and the pathway controlling rhizobial infection (Madsen et al., 2010). Upstream of
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cytokinin perception, nodulation signalling involves decoding of calcium influx and spiking events by the
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CALCIUM AND CALMODULIN DEPENDENT KINASE (CCaMK) (Lévy et al., 2004; Sieberer et al.,
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2009). Autoactive variants of CCaMK and its phosphorylation target CYCLOPS are sufficient to trigger
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downstream nodulation signalling and spontaneous nodules (Tirichine et al., 2006a; Singh et al., 2014).
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Downstream of LHK1, nodule organogenesis requires NODULE INCEPTION (NIN) (Schauser et al.,
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1999; Marsh et al., 2007) and the GRAS transcription factors NSP1 and NSP2 (Kaló et al., 2005; Smit et
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al., 2005; Heckmann et al., 2006; Murakami et al., 2007; Hirsch et al., 2009). These transcription factors
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are also required in the rhizobia infection pathway. Activation of NIN is a central function of cytokinin
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activity (Tirichine et al., 2007; Heckmann et al., 2011) and NIN overexpression is also sufficient for
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spontaneous initiation of cell divisions, which is dependent on the NUCLEAR FACTOR Y
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transcriptional activators NF-YA1 and NF-YB1 in L. japonicus (Soyano et al., 2013). Activation of
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nodulation signalling by cytokinin and NIN also induces systemic inhibition of nodulation as it directly
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activates nodule suppressive CLE peptides (Mortier et al., 2012; Soyano et al., 2014) which systemically
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regulate nodulation (Okamoto et al., 2009; Mortier et al., 2010; Reid et al., 2011a; Saur et al., 2011). CLE
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peptide
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HYPERNODULATION
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TRANSFERASE (IPT3) dependent cytokinin biosynthesis in the shoot which negatively regulates
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nodulation (Krusell et al., 2002; Nishimura et al., 2002; Okamoto et al., 2013; Sasaki et al., 2014). LHK1
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was not required for this negative regulatory role of cytokinin but it was dependent on the function of the
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Kelch repeat-containing F-box protein, TOO MUCH LOVE in the root (Takahara et al., 2013; Sasaki et
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al., 2014).
dependent
nodule
regulation
AND
in L.
ABERRANT
japonicus ROOT1
requires
(HAR1)
the
and
LRR
induces
receptor
kinase
ISOPENTENYL
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The early nodulation signalling pathway directly induces cytokinin biosynthesis as Nod factor
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induced cytokinin accumulation, observed at 3 h in wild-type roots was not detected in the Mtdmi3
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(ccamk) background (van Zeijl et al., 2015). Several cytokinin biosynthesis genes have been identified as
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contributing to cytokinin pools during nodule development including LjIPT3 and two M. truncatula
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LONELY GUYs which directly activate cytokinin nucleotides (Chen et al., 2014; Mortier et al., 2014).
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However, the processes controlling cytokinin levels and their cell autonomous or non-cell autonomous
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effects is poorly understood. Studies in non-legumes, primarily Arabidopsis, have shown that regulation
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of active cytokinin pools occurs through reversible glycosylation, conversion to cytokinin nucleotides by
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adenine phosphoribosyl transferase genes and through irreversible breakdown by cytokinin
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oxidase/dehydrogenases (CKX) (Sakakibara, 2006). Ckx gene expression is enhanced by cytokinin
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signalling and shows expression patterns similar to Ipt genes indicating a requirement for finely
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regulating cytokinin accumulation (Schmülling et al., 2003; Werner et al., 2003; Werner et al., 2006).
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Overexpression of CKX encoding genes can create dominant reduction in cytokinin levels and has been
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used to examine the role of cytokinin in root development (Werner et al., 2003; Lohar et al., 2004;
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Werner et al., 2010). However, loss-of-function ckx mutations in Arabidopsis have not been shown to
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affect root development.
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In order for proper nodule development to progress without secondary effects on root growth, it is
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assumed cytokinin must be released in a tightly controlled spatial and temporal manner. The availability
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of the LORE1 insertion population makes L. japonicus an ideal system for reverse genetics (Fukai et al.,
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2012; Urbański et al., 2012) and here we make use of this resource to address the role of cytokinin
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breakdown during nodule development. We identify two insertion alleles in LjCkx3 and show that this
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gene is critical for maintaining cytokinin homeostasis required for efficient symbiotic infection,
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organogenesis and nitrate dependent regulation of nodulation.
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Results
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Lotus japonicus encodes nine cytokinin oxidase/dehydrogenase genes
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In higher plants, cytokinin oxidase/dehydrogenase is encoded by multigene families. To establish the
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complexity of the family in legumes we first searched the available genome and EST sequences of L.
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japonicus and the M. truncatula genome (v4.0; Young et al., 2011). Nine non-redundant CKX sequences
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were found in both. In order to construct a phylogeny, all amino acid sequences of the two legume species
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and Arabidopsis were aligned and we then named the L. japonicus genes according to the nearest of the
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seven Arabidopsis homologues (Fig. 1).
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Given the roles of cytokinin in both rhizobia infection events and initiation of organogenesis we
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sought to identify LjCkx genes regulated during the early phases of nodule establishment. Searching
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publicly available gene expression data (Høgslund et al., 2009; accessed via ljgea.noble.org, Verdier et
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al., 2013) revealed that two of the LjCkx genes showed expression patterns strongly correlated with
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symbiotic development. LjCkx3 (probe ID TM0914.20_at) was induced in the nodulation susceptible
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zone one day after inoculation and in developing nodules, while LjCkx4 (TM0914.24_at) was expressed
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later in mature nodules. The affymetrix data also indicated that the induction of Ckx3 expression was
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dependent on NFR1 and NFR5 but independent of NIN. Given the early Nod factor dependent expression
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pattern we decided to focus further attention on LjCkx3.
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Phylogenetic analysis showed that LjCKX3 was most closely related to two M. truncatula genes
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(Medtr4g126150 and Medtr2g039410) recently reported to show induced expression in response to
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Sinorhizobium meliloti Nod factor (van Zeijl et al., 2015). Further analysis indicated LjCkx3 comprises
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five exons annotated by RNAseq analysis and encodes a predicted signal peptide (Fig. 1; SignalP 4.1 D-
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score 0.668, Petersen et al., 2011), while the mature protein has predicted CK and FAD binding domains,
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which together form the active site characteristic of CKX proteins (Malito et al., 2004). LjCKX7 was
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most closely related to MtCKX1 (Medtr1g015410), which was previously reported to be expressed during
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nodule development in M. truncatula (Ariel et al., 2012).
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Ckx3 is expressed during nodule initiation and development
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To confirm the publicly available Affymetrix data, we inoculated with M. loti or applied purified Nod
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factor to roots of 10 d-old plants and conducted quantitative RT-PCR. This showed Ckx3 expression was
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induced by M. loti R7A and purified Nod factor within 8 h and increased further at 24 h (Fig. 2). In
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Arabidopsis, Ckx gene expression is induced by cytokinin application (Werner et al., 2006). Therefore, to
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determine whether Ckx3 also responds to cytokinin independent of rhizobia, we conducted a time course
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following treatment with the synthetic cytokinin 6-Benzylaminopurine (BAP). Within this 12 hours time
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series, Ckx3 expression was induced by cytokinin within 6 hours (Fig. S1).
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To further clarify the spatio-temporal expression patterns, we cloned promoter sequences
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corresponding to approximately 1 kb and 2kb upstream of Ckx3 and fused these to a nuclear-localised
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triple-YFP (tYFPnls) reporter, which is readily visualised relative to autofluorescence in L. japonicus.
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Confocal microscopy revealed that the response patterns were indistinguishable between the 1 kb and 2
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kb promoter fragments (Fig 3, Fig. S7). Expression of pCkx3::tYFPnls was observed in the cells
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corresponding to high cytokinin activity in the meristematic zone of the root tip (Zürcher et al., 2013)
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irrespective of inoculation status (Fig. 3A). YFP was also observed in the central root cylinder in both
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inoculated and uninoculated roots (Fig. 3B, 3D). Sectioning further confirmed that the central root
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cylinder expression observed in whole mounts corresponds to expression in pericycle cells adjacent to
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xylem cells and protoxylem (Fig. 3C). Cross sections of roots inoculated with M. loti expressing DsRed
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revealed expression occurs in the dividing cells of the root cortex (nodule primordium) and pericycle
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during nodule primordium development and is sustained in the cortical cells of more mature growing
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nodules. In mature nodules with differentiated bacteroids, expression was localised to the cells
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surrounding the infected tissue (Fig. 3). YFP expression was not identified in the epidermal cells of
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transgenic roots in response to inoculation. The observed expression patterns driven by the Ckx3 promoter
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are consistent with known cytokinin response domains at the root tip and nodule primordia reported
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elsewhere (Müller and Sheen, 2008; Held et al., 2014) and taken together with the real-time results
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reported here indicate the promoter fragment likely captures the essential cytokinin response elements
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responsible for Ckx3 expression patterns. We further confirmed the cytokinin responsiveness of the
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promoter by treating pCKX3::tYFPnls roots with BAP. Within 3 hours, this treatment induced expression
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of the tYFPnls reporter in the cortex of the root, while vascular expression was indistinguishable from
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untreated roots (Fig. S6).
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Identification of Ckx3 mutant alleles
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To identify mutants in Ckx3, we searched the publicly available LORE1 insertional mutant resources
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(Fukai et al., 2012; Urbański et al., 2012; Hirakawa et al., 2014; accessed via carb.au.dk/lotus-base/). We
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identified lines with insertions in exon 1 (5497) and exon 3 (17827) of Ckx3 and named these ckx3-1 and
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ckx3-2 respectively (Fig. 1). Searching the LORE1 version 2.5 database showed these lines contained 0
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and 2 known additional LORE1 insertions respectively, none of which were exonic.
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Quantification of cytokinins in Lotus japonicus roots
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To identify the effect of inoculation on cytokinin levels, we quantified isoprenoid type cytokinins in 10-d
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old wild type L. japonicus (Gifu) roots 24 and 72 h after inoculation with M. loti R7A or a Nod factor
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defective R7AnodC mutant compared to mock treated whole roots. All isoprenoid-type cytokinins were
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detected as bases, ribosides, and nucleotide metabolites (Supp. table 1). N-glucosides and O-glucosides of
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tZ and DHZ were under detection limits. This analysis showed DHZ and iP cytokinin bases and tZ, cZ,
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DHZ and iP ribosides were all increased 24 h post-inoculation with R7A relative to R7AnodC inoculated
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roots (Fig. 4; Table S1). tZ ribosides showing the most significant change (1.88-fold increased) while iP
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type cytokinins were the most abundant cytokinin species detected. After 72 h, the tZ, DHZ and iP
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cytokinin bases and ribosides remained significantly increased relative to the nodC inoculated roots. At
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72 h, levels of tZ were approximately 2-fold higher in R7A inoculated roots compared to R7AnodC while
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iP was approximately 1.5-fold increased.
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CKX proteins are known to cleave tZ and iP bases and ribosides with varying affinities (Galuszka
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et al., 2007). To confirm whether CKX3 has a biologically relevant role in degrading cytokinin in L.
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japonicus roots, we compared cytokinin concentrations in the ckx3-2 mutant relative to Gifu across the
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same conditions. We found both tZ and DHZ base and riboside levels to be significantly increased in the
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mutants across treatment groups at both 24 and 72 hpi (Fig. 4, S5). In contrast, cZ and iP type cytokinins
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were not significantly altered, or slightly decreased in the mutants relative to Gifu (Fig. 4, S5). In M. loti
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R7A inoculated ckx3-2 roots, tZR were the only species increased at both 24 and 72 h relative to Gifu,
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while DHZ and tZ were increased significantly in the mutants at 24 and 72 h respectively.
238 239
Elevated cytokinin in ckx3 decreases nodulation efficiency
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To determine the effect of the elevated cytokinin levels in ckx3 mutants on nodule development, we grew
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the plants on vertical filter paper covered agar slopes with the roots shielded from light, which allows
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continued observation of nodule developmental phenotypes and kinetics. Both ckx3 alleles developed
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nodules normal in appearance however the number of nodules formed was significantly reduced at all
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timepoints (Fig. 5). To confirm these data in a controlled glasshouse environment, we grew plants in open
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vermiculite pots in nitrate free conditions and found both alleles showed significantly reduced nodulation
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5 w after inoculation (Figure S2).
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Given that reduced cytokinin signalling in L. japonicus causes hyperinfection (Murray et al.,
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2007), we counted infection threads formed on the mutants 10 d after inoculation with a M. loti strain
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expressing DsRED. We found the number of infection threads formed was significantly reduced in both
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alleles (Fig. 5B). Cytokinin can induce ethylene production and is thought to act largely through ethylene
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in repressing root growth. The ethylene synthesis inhibitor aminoethoxyvinylglycine (AVG) can rescue
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root growth inhibition in the presence of elevated endogenous or ectopic cytokinin in legumes (Wopereis
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et al., 2000; Ferguson et al., 2005). We therefore repeated the IT counts with 10-8 M AVG supplemented
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in the media. AVG treatment was sufficient to rescue the reduced infection thread phenotypes in both
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ckx3 alleles, increasing IT numbers close to wild-type levels (Fig. 5C). To further identify the infection
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phenotypes of the ckx3 mutants, we counted infection threads on plants grown in the presence of 2 mM
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KNO3 or 10-8 M BAP. These results showed that while both nitrate and BAP treatment can significantly
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reduce infection events, the reduced infection levels of ckx3 is not further impaired upon treatment (Fig.
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5D).
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Ectopic application of cytokinin or the snf2 gain-of-function mutation has previously been shown
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to be sufficient to initiate nodule organogenesis in L. japonicus (Tirichine et al., 2007; Heckmann et al.,
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2011). To determine if the elevated cytokinin levels in ckx3 mutants was sufficient to trigger nodule
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organogenesis, we grew mutants on nitrate free agar slants in the absence of rhizobia but did not observe
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spontaneous organogenesis in these conditions. To determine if the elevated cytokinin in ckx3 plants
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might be inhibitory to spontaneous nodule development we grew the mutants in the presence of 10-8 M
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BAP. Spontaneous nodule organogenesis was observed in both Gifu and ckx3 mutants in these conditions
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(Fig. S3).
269 270
Cytokinin plays a role in nitrate regulation of nodulation and nitrogen fixation
271 272
Cytokinin biosynthesis in Arabidopsis, in particular through IPT3, is known to be an important means of
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regulating plant development in response to environmental signals, including nitrate (Takei et al., 2004;
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Sakakibara et al., 2006; Ruffel et al., 2011). Since nodulation is negatively regulated by nitrogen,
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especially nitrate, we investigated whether cytokinin might link nitrogen regulation and nodulation. ckx3
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mutant plants were grown under elevated nitrate conditions and the nodulation phenotype observed.
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Interestingly, increased nitrate concentration accentuated the nodulation phenotypes. To quantify this
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effect, we grew Gifu and ckx3-2 under different nitrate regimes and counted the number of red nitrogen-
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fixing nodules, white non-fixing nodules and assayed nitrogen fixation activity from whole roots (Fig 6B)
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and individual nodules (Fig. 6C) using the acetylene reduction assay (ARA). This showed that while
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ckx3-2 had reduced nodulation but formed normal red fixing nodules in nitrate free conditions, it was
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more sensitive to increased nitrate than Gifu (Fig. 6). Growth on 2 mM KNO3 significantly reduced total
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nodule numbers, red nodules and the ARA activity of Gifu but was reduced significantly more in ckx3-2
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(Fig. 6A). Gifu continued to form a small number of pink-red nitrogen-fixing nodules (confirmed by
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ARA activity) at 5 mM KNO3, however this concentration was completely inhibitory to the development
286
of red nodules and nitrogen fixation in ckx3-2 (Fig. 6). To confirm this effect of cytokinin on nitrogen
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fixation, we also grew the plants on media supplemented with 10-8 M BAP and found nodulation and
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nitrogen fixation to be significantly reduced in both Gifu and ckx3-2 (Fig. 6). Acetylene reduction on a
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per nodule basis indicated that this response is likely at earlier infection and nodule organogenesis stages
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as individual nodules formed on BAP treated roots continued to show wild-type acetylene reduction
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activity (Fig 6C). Nodule sections (Fig. 6 J to O) showed that those nodules that did form on nitrate or
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BAP treated roots were colonised by rhizobia expressing the DsRed marker. This included the small
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nodules formed on ckx3 mutants at 5 mM KNO3 despite the white appearance and near-complete
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reduction in acetylene reduction activity.
295 296
Ckx3 regulates cytokinin levels affecting root meristem elongation and differentiation
297 298
To investigate the role of Ckx3 in root development, we measured total root length in Gifu and ckx3
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mutants. We found that the ckx3 mutants showed significantly reduced root length relative to Gifu at 20 d
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after germination (Fig. 7A). To determine the basis of this reduced root growth, we investigated the zones
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of cell proliferation and elongation as well as the differentiation zone at the root tip. The region from the
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root tip to the first emerging root hairs includes the zones of proliferation and elongation, while the zone
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of differentiation is defined by the emergence and growth of root hairs (Williamson et al., 2001; Jones et
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al., 2009; Petricka et al., 2012). We therefore measured the distance between the root tip and first root
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hair as a measure of proliferative and elongation zone length and found the ckx3 mutants exhibit
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significantly reduced root tip length (Fig 7B). To evaluate the effect of ckx3 mutation on root hair
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emergence in the differentiation zone, we measured the angle created by the emergence of root hairs
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immediately behind the meristem (Fig. S4). This analysis showed that the angle was significantly greater
309
in ckx3 indicating more rapid differentiation and/or a reduced differentiation zone length (Fig. 7C).
310 311
Discussion
312 313
Cytokinin signalling must be finely regulated during nodulation in order to balance the positive role
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during nodule organogenesis with the negative effect in symbiotic infection and crosstalk with other
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hormones. Nod factor induced cytokinin accumulation plays a crucial role in the induction of early
316
nodulation responses (van Zeijl et al., 2015) and is perceived partially redundantly by the LHK receptors
317
in L. japonicus (Held et al., 2014). Ckx encoding genes expressed during nodule development have also
318
been implicated in regulation of cytokinin levels and signalling during nodulation (Held et al., 2008; Ariel
319
et al., 2012; van Zeijl et al., 2015) however the lack of well-defined mutants made the determination of
320
their precise role during nodulation difficult. Here we identify LORE1 insertion mutants in LjCkx3, which
321
exhibit reduced nodulation, rhizobia infection and root growth and establish a role for Ckx genes during
322
symbiosis. We show that regulation of cytokinin accumulation through breakdown of cytokinin by
323
LjCKX3 plays a role in maintaining efficient nodule development. Our gene expression data and
324
cytokinin measurements showed that cytokinin signalling rapidly induces expression of Ckx3 in order to
325
restrict cytokinin accumulation. This may serve to avoid over-stimulation of cell division, maintain
326
cytokinin signalling autonomy for neighbouring cells and/or stimulation of negative feedback
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mechanisms such as the effects of ethylene on infection.
328
Our data also highlight the extensive crosstalk between cytokinin and ethylene. Cytokinin
329
signalling negatively regulates infection since mutations in Lhk1 results in hyperinfection (Murray et al.,
330
2007) while ethylene also inhibits infection (Penmetsa and Cook, 1997; Penmetsa et al., 2008). Consistent
331
with these results, we found the reduced infection phenotypes of the ckx3 mutants could be rescued by
332
AVG treatment, indicating cytokinin degradation is critical in preventing over stimulation of ethylene
333
dependent inhibition of symbiotic infection. This interpretation is supported by results showing that
334
cytokinin induces and stabilises ACC synthase, the rate limiting step in ethylene biosynthesis (Chae et al.,
335
2003; El-Showk et al., 2013). In return, ethylene can regulate cytokinin signalling through the type-A
336
ARRs (Shi et al., 2012).
337 338
CKX3 primarily regulates tZ levels
339 340
Our quantification of isoprenoid cytokinins showed that Ckx3 regulates root cytokinin levels in vivo. The
341
elevated levels of tZ and DHZ type cytokinins in ckx3-2 suggest these are either the species most
342
susceptible to CKX3 degradation or that accumulate in response to CKX activity on other cytokinins.
343
Biochemical studies have shown Arabidopsis CKX genes expressed in a heterologous Nicotiana tabacum
344
system have highest activity against trans-Zeatin (tZ) and isopentenyl adenine (iP) type cytokinins while
345
cis-Zeatin (cZ) and dihydrozeatin (DHZ) are resistant to cleavage (Galuszka et al., 2007). While CKX
346
was shown to have limited activity against DHZ in one study, tZ may be converted to DHZ by zeatin
347
reductase, especially in the absence of CKX activity (Gaudinová et al., 2005). DHZ does not have strong
348
activity in Arabidopsis and has been suggested to act as a storage or transport form of cytokinin (Mok and
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349
Mok, 2001). tZ may therefore be the primary target of CKX3 and increased DHZ is a direct result of high
350
tZ levels. In Arabidopsis the three AHK receptors maintain specificity in cytokinin response through both
351
expression domains and differing affinities to the cytokinin ligands, with tZ having high affinity binding
352
against AHK2, AHK3 and AHK4 while iP shows strong affinities against AHK2 and AHK4 (Romanov et
353
al., 2006; Stolz et al., 2011). The LHK1, LHK1a and LHK3 receptors in L. japonicus have been shown to
354
functionally restore cytokinin sensitivity in Yeast or E. coli heterologous system assays, however no data
355
is available on their cytokinin binding specificity (Murray et al., 2007; Held et al., 2014). IPT3 dependent
356
synthesis of iP type cytokinin in the shoot has been shown to negatively regulate nodule numbers in L.
357
japonicus (Sasaki et al., 2014). Our study provides further evidence that increased cytokinin levels can
358
negatively regulate infection and organogenesis events and that cytokinin levels are therefore finely
359
regulated to maintain efficient nodulation.
360
We found the accumulation of cytokinin bases was dependent on Nod factor signalling as the
361
R7AnodC mutant failed to initiate the responses observed for R7A wild type strain. This is consistent
362
with the results showing Nod factor induction of cytokinin in M. truncatula (van Zeijl et al., 2015), albeit
363
at later timepoints in our experiments. The Nod factor treatment reported in M. truncatula was found to
364
induce accumulation of iP, iPR and tZ type cytokinins (van Zeijl et al., 2015). We also found iP and iPR
365
to be induced by M. loti inoculation on L. japonicus at both 24 h and 72 h, whereas tZ was only increased
366
at 72 h. We also found significant increases in tZR, DHZ and DHZR at both 24 h and 72 h after M. loti
367
inoculation. These additional cytokinins identified in our studies may result from differences in Nod
368
factor and rhizobia responses or result from the later time-points we examined relative to the early Nod
369
factor responses reported. Furthermore, it is possible that cytokinin inter-conversion occurs following
370
initial synthesis or that between species differences exist in cytokinin responses to rhizobia. We found
371
that although the cytokinin pool is under negative feedback by CKX3 which is induced within 8 h of
372
inoculation, elevated cytokinin is maintained during the first three days following inoculation, though
373
likely in a tightly spatially restricted manner. TCS expression in L. japonicus showed cytokinin signalling
374
domains were restricted to the dividing cells of the nodule primordia while Nod factor responses in M.
375
truncatula triggered a more widespread cortical and pericycle response (Held et al., 2014; van Zeijl et al.,
376
2015). Further analysis at the early points during symbiotic interaction is required to confirm whether
377
rhizobia induce cytokinin signalling in a wide region as is observed for Nod factor treatment or through
378
more restricted biosynthesis and associated degradation in order to restrict cytokinin signalling and
379
induction of nodulation foci to a small number of defined cells.
380 381
Local restriction of cytokinin accumulation is required for efficient infection
382
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383
Our data showing a Nod factor dependent (Fig. 2A) and nodule primordia localised (Fig. 3E,F)
384
expression pattern for Ckx3 indicate that cytokinin signal induction is transient and must be tightly
385
regulated to avoid negative effects. This is consistent with data indicating that increased cytokinin
386
signalling in Ljsnf2 or through ectopic MtLog overexpression reduces nodulation (Tirichine et al., 2007;
387
Mortier et al., 2014). Most Arabidopsis CKX have predicted signal peptides and are proposed to localise
388
to the apoplast or vacuoles (Werner et al., 2003; Kowalska et al., 2010). We also found LjCKX3 has a
389
predicted secretion signal and is therefore likely degrading cytokinin in the extracellular space.
390
Cytokinin has been proposed as a non-cell autonomous signal during nodulation and restriction of
391
extracellular cytokinin would therefore be important for regulation of this signalling. We did not observe
392
epidermal expression of Ckx3 and it remains unresolved whether alternative Ckx genes may be expressed
393
here or whether cytokinin biosynthesis in the epidermis is sufficient to produce a non-cell autonomous
394
cytokinin signal to initiate cell divisions in the cortex. The close correlation of Ckx3 expression in nodule
395
primordia cells and correlation with areas of high cytokinin response in the root tip are strongly correlated
396
to the signalling domains of TCS in legumes and Arabidopsis respectively (TCS; Müller and Sheen,
397
2008; Zürcher et al., 2013; Held et al., 2014). Furthermore, it is likely that the role of CKX3 in regulating
398
cytokinin levels in response to Nod factor is conserved in legumes as two closely related homologues in
399
M. truncatula were both shown to respond to Nod factor treatment (van Zeijl et al., 2015). The induction
400
of spontaneous nodules through elevated cytokinin signalling continues to maintain defined foci rather
401
than widespread induction of cell divisions (Tirichine et al., 2006b; Tirichine et al., 2007; Heckmann et
402
al., 2011). Our observations also suggest unknown mechanisms outside of cytokinin signalling might be
403
required to define cells competent for division and to maintain these divisions to a limited nodule foci as
404
we did not observe persistent cell divisions or abnormally shaped nodules in ckx3 mutants. Maintaining
405
organised cell divisions during nodule development therefore results from the coordination of nodulation
406
specific transcriptional networks with hormones required for cell specification and division.
407
Cytokinins are known to alter root meristem size (Dello Ioio et al., 2007). This inhibition is
408
dependent on ethylene in Arabidopsis and L. japonicus (Wopereis et al., 2000; Růžička et al., 2009).
409
Ethylene is produced during nodulation (Ligero et al., 1986) and our work finds the breakdown of
410
cytokinin is required to prevent the resulting inhibition of root growth and infection. Further analysis of
411
the genetics of ethylene induction in the common symbiosis pathway will help to clarify the observed
412
cross talk and regulatory functions. Pericycle and root tip expression of Ckx3 was independent of
413
inoculation, indicating that the evolutionary ancestral role of CKX3 is likely in regulation of root
414
development. However, no root phenotypes for ckx3 mutants have been reported in Arabidopsis, while
415
ckx3 ckx5 double mutants have altered shoot inflorescence meristem size (Bartrina et al., 2011). Pericycle
416
cells in Medicago truncatula maintain the ability to divide in order to initiate lateral root and nodule
14
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417
primordia (Xiao et al., 2014). In Arabidopsis, Ckx genes are expressed during lateral root primordia
418
initiation (Werner et al., 2003) and expression of Ckx in the xylem pole pericycle cells can increase lateral
419
root density (Laplaze et al., 2007). While we observed expression of Ckx3 in pericycle cells, it was not
420
universal in pericycle cells. The expression of Ckx3 in a subset of pericycle cells may play a role in
421
priming cells and determining their susceptibility to undergo division. It would be interesting to determine
422
if the cells with Ckx3 expression are correlated with the radial or longitudinal positioning of nodule
423
primordia sites. Improved spatio-temporal expression analysis using promoter-YFP stable lines may
424
provide a means to determine whether cytokinin degradation plays a role in radial nodule positioning and
425
the crosstalk with ethylene in this process during nodule development.
426 427
Cytokinin regulates nodulation in response to environmental signals
428 429
Legumes regulate nodulation locally and systemically in response to environmental cues, including
430
nitrate, in order to balance fixed nitrogen from symbiosis with other nitrogen sources (Reid et al., 2011b).
431
This regulation occurs by both HAR1 dependent and independent mechanisms. In Arabidopsis, IPT3
432
expression is known to be induced by nitrate and cytokinin levels are increased as a result (Takei et al.,
433
2004). Cytokinin thus plays a central role in regulating responses to the environment (Sakakibara et al.,
434
2006; Ruffel et al., 2011). We found the increased cytokinin levels in Ljckx3 mutants enhanced the
435
susceptibility to negative effects of nitrate on nodulation and nitrogen fixation. This indicates cytokinin
436
plays a role in regulating nitrogen fixation. The strong inhibition of nitrogen fixation relative to
437
nodulation indicates that nitrogen fixation and nodule organogenesis possess both common and
438
independent regulatory mechanisms in response to available nitrogen. This is supported by the fact
439
hypernodulation mutants show reduced nitrogen fixation on a per nodule basis (Carroll et al., 1985; Jeudy
440
et al., 2010). Whether the cytokinin regulation of nitrogen fixation occurs locally or systemically in
441
response to nitrate and how this integrates with the HAR1 mediated regulation of nodulation remains to
442
be resolved. Nitrate inhibition of nodulation has previously been shown to involve ethylene biosynthesis
443
as it can be alleviated by AVG treatment, however it is not known if this involves local or systemic
444
ethylene responses (Ligero et al., 1991). Ethylene is also thought to regulate cytokinin signalling in both
445
environmental responses and nodulation signalling (Shi et al., 2012; van Zeijl et al., 2015). Together with
446
our findings, this is consistent with cytokinin and its crosstalk with ethylene playing a significant role in
447
the inhibition of nodulation and nitrogen fixation by nitrate.
448 449
Conclusions
450
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451
We found that inoculation with M. loti causes an increase in root cytokinin levels and a resulting
452
upregulation of negative feedback through cleavage of active cytokinins, particularly tZ, by CKX3.
453
CKX3 acts to restrict the pool of active cytokinin and prevents the resulting stimulation of ethylene and
454
negative effects on nodule organogenesis, nitrogen fixation, and infection thread development. Expression
455
of Ckx3 in the root tip ensures homeostasis of cytokinin in the meristematic zone in order to balance cell
456
elongation with differentiation and root hair outgrowth. Overall, these results confirm the importance of
457
cytokinin in maintaining effective nodulation and identify a new negative regulator of this signalling.
458
Future efforts to elucidate the roles of crosstalk of cytokinin with other plant hormones, particularly auxin
459
(Breakspear et al., 2014) and ethylene (Ferguson and Mathesius, 2014) will assist in understanding the
460
cellular mechanism involved in nodule development.
461 462
Materials and Methods
463 464
Plant and bacteria genotypes
465 466
Lotus japonicus ecotype Gifu was used in all experiments (Handberg and Stougaard, 1992). Homozygous
467
LORE1 inserts were genotyped with allele specific primers in combination with the P2 internal LORE1
468
primer as described (Urbański et al., 2012). Primer sequences were obtained from the LORE1 resource
469
page (carb.au.dk/lotus-base) or designed in the same region if amplification was unsuccessful (Table S2).
470
M. loti R7A and the Nod factor defective nodC variant (Rodpothong et al., 2009) were diluted to an
471
inoculum density of OD600 = 0.01. For infection thread counting, the M. loti MAFF303099 strain carrying
472
chromosomal DsRed insertion was used (Maekawa et al., 2009).
473 474
Plant and bacteria growth conditions
475 476
For nodulation assays and IT counts, 3 d-old seedlings were transferred to vertical plates with filter paper
477
on 1.4 % agar noble containing quarter-strength B&D nutrients (Broughton and Dilworth, 1971) in the
478
presence or absence of KNO3, 10-8 BAP or AVG as described for each experiment. Nod factor treatment
479
was carried out on plates with 10-8 M. loti R7A Nod factor pipetted directly onto roots. Infection threads
480
were counted 10 d after inoculation by placing whole roots on microscope slides to allow counting of the
481
root surface contacting the growth plates. Hairy roots were induced by infection of 6 d-old seedlings
482
growing on vertical 0.8 % Phytagel (Sigma) plates with half-strength B5 salts and vitamins as described
483
(Stougaard et al., 1987; Hansen et al., 1989; Stougaard, 1995). Three weeks after infection, primary roots
484
were removed and the chimeric plants transferred to plastic magenta boxes containing 1:4
16
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485
leca:vermiculite mix. For phenotyping in open pots, 3 d-old seedlings were transferred to pots containing
486
vermiculite watered with nitrate free half-strength B&D.
487 488
Bioinformatics
489 490
The amino acid translations of the representative gene models for Arabidopsis Ckx genes were obtained
491
from TAIR for BLAST queries against L. japonicus resources at NCBI and Lotus base (carb.au.dk/lotus-
492
base). M. truncatula sequences were obtained by searching Mt v4 at Phytozome.org. The gene phylogeny
493
was drawn based on alignment of the amino acid sequences of all three species and subsequent bootstrap
494
analysis based on 1000 replications using ClustalX (Larkin et al., 2007). Cytokinin and FAD binding
495
domains within CKX3 were identified by BLAST query against the NCBI Conserved Domain Database
496
(Marchler-Bauer et al., 2015). Signal peptide prediction was carried out using SignalP 4.1 (Petersen et al.,
497
2011). Microarray data was identified and analysed using the L. japonicus gene expression atlas (Verdier
498
et al., 2013) by identifying probes against LjCkx genes with BLAST.
499 500
Cloning
501 502
Primers for cloning a Ckx3 promoter sequence corresponding to approximately 1 kb upstream sequence
503
were designed against the Lj2.5 genome and amplification carried out from MG-20 genomic DNA while
504
the 2kb promoter fragment was synthesised according to MG-20 genomic sequence. The Ckx3 promoter
505
fragment was subsequenctly cloned by TOPO cloning into the Gateway compatible pDONR vector (Life
506
Technologies). tYFPnls was constructed by amplifying a tYFP cDNA with primers including a C-terminal
507
nuclear localisation signal (Takeda et al., 2012) and excision of the largest of three resulting bands before
508
cloning into a pIV10 vector (Stougaard, 1995) modified to accept Gateway promoter clones.
509 510
Quantitative RT-PCR
511 512
For expression analysis in roots, plants were grown and Nod factor or BAP applied as described
513
previously (Heckmann et al., 2011). mRNA was isolated from BAP (10-8 M), Nod factor (10-8 M) or
514
R7A (OD600=0.02) treated roots using Dynabeads mRNA DIRECTTM kit (Invitrogen). RevertAid M-
515
MuLV Reverse Transcriptase (Fermentas) was used for cDNA synthesis. All cDNA samples were tested
516
for genomic DNA contamination using primers specific for the NIN gene promoter (Lohmann et al.,
517
2010). A Lightcycler480 instrument and Lightcycler480 SYBR Green I master (Roche Diagnostics
518
GmbH) was used for the real time quantitative PCR. ATP-synthase (ATP), Ubiquitin-conjugating enzyme
17
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519
(UBC) and Protein phosphatase 2A (PP2A) were used as reference genes (Czechowski et al., 2005). The
520
relative quantification software (Roche) was used to calculate the normalized efficiency-corrected relative
521
transcript levels. The geometric mean of the relative transcript levels for the three biological (each
522
consisting of 10 plants) and three technical repetitions and the corresponding upper and lower 95%
523
confidence were calculated (Vandesompele et al., 2002). Primer sequences are listed in Table S2.
524 525
Microscopy
526 527
Microscopy was performed with a Zeiss LSM 510 Meta Confocal Microscope. Objective lenses were
528
Zeiss Plan-Neofluar 10x/0.3 and 20x/0.5. Laser excitation was at 488 nm for YFP and 543 nm for DsRed
529
and emission filters were 505-550 nm for YFP and 585-615 nm for DsRed. For sections, live roots were
530
embedded in 3% agarose and cut to 80-100 µM sections using a Leica VT 1000 S vibratome before
531
imaging with the confocal microscope.
532 533
Statistical analysis
534 535
Statistical analysis was carried out using GraphPad Prism software. Comparison of multiple groups
536
included ANOVA followed by Tukey post-hoc testing to determine statistical significance. Students T-
537
test or Wilcoxon rank-sum test was used to determine differences when making single comparisons. All
538
data is plotted as mean with 95% CI for the indicated number of biological replicates.
539 540
Acetylene reduction assay
541 542
Acetylene was produced by reaction of calcium carbide with water. The resulting gas was collected and
543
diluted to 2% in stoppered glass vials. For the assay, 250 µl air was removed from the 5 ml glass GC vials
544
containing whole nodulated roots 14 d after inoculation and replaced with equal volume of 2% acetylene.
545
Samples were incubated 30 min before quantification of ethylene conversion using a SensorSense
546
(Nijmegen, NL) ETD-300 ethylene detector operating in sample mode with 2.5 L/h flow rate and 6-
547
minute detection time.
548 549
Endogenous cytokinin measurements
550 551
For cytokinin analysis, plants were grown on filter paper covered agar slants as described above. 10 d-old
552
plants were inoculated before whole roots were harvested at 24 h or 72 h after treatment. Prepared
18
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553
biological quadruplicates were extracted and purified using the method published previously (Novák et
554
al., 2008) with some minor modifications. 10-20 mg FW were extracted in 1 ml of modified Bieleski
555
buffer (60% MeOH, 10% HCOOH and 30% H2O) together with a cocktail of 23 stable isotope-labeled
556
CK internal standards (0.5 pmol of CK bases, ribosides, N-glucosides, 1 pmol of O-glucosides and
557
nucleotides) to check recovery during purification and to validate the determination. The samples were
558
purified using a combination of C18 (100 mg/1ml) and MCX cartridges (30 mg/1ml) and immunoaffinity
559
chromatography (IAC) based on wide-range specific monoclonal antibodies against cytokinins (Faiss et
560
al., 1997). The eluates from the IAC columns were evaporated to dryness and dissolved in 20 µl of the
561
mobile phase used for quantitative analysis. The samples were analyzed by the LC-MS/MS system
562
consisting of an ACQUITY UPLC® System (Waters) and Xevo™ TQ-S (Waters) triple quadrupole mass
563
spectrometer. Quantification was obtained using a multiple reaction-monitoring (MRM) mode of selected
564
precursor ions and the appropriate product ion.
565 566
Accession numbers
567 568
Genbank accession numbers: LjCkx1, KR296932; LjCkx2, KR296933; LjCkx3, KR296934; LjCkx4,
569
KR296935; LjCkx5, KR296936; LjCkx6, KR296937; LjCkx7, KR296938; LjCkx8, KR296939; LjCkx9,
570
KR296940
571 572
Figure Legends
573 574
Figure 1. Lotus japonicus CKX family. A, CKX phylogeny assembled by alignment of the L. japonicus,
575
M. truncatula and A. thaliana amino acid sequences. LjCKX3 (red) is most closely related to two M.
576
truncatula genes, which are induced by Nod factorapplication (van Zeijl et al., 2015). Bootstrap values
577
are shown for each node based on 1000 replications. B, LjCkx3 comprises five exons and encodes a
578
predicted signal peptide (SP), cytokinin (CK bind) and FAD binding domains. Lines containing LORE1
579
insertions were characterised in the first and third exons of LjCkx3.
580 581
Figure 2. Effect of ectopic Nod factor application or M. loti inoculation on Ckx3 mRNA levels. A,
582
Relative expression levels following Nod factor treatment. B, Relative expression levels following
583
inoculation with M. loti R7A. Values are relative to mock treatment and indicate mean ± 95% CI for n=3.
584
P-values were calculated using Wilcoxon rank-sum testing between mock and treatment groups and are
585
indicated by *
1
Running head: Cytokinin degradation in nodule development
2 3
Author for correspondence:
4
Jens Stougaard
5
Department of Molecular Biology and Genetics
6
Aarhus University
7
Gustav Wieds Vej 10, Aarhus C, 8000, Denmark
8
+45 87 15 55 04
9
[email protected]
10
1
Downloaded from www.plantphysiol.org on February 4, 2016 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Copyright 2015 by the American Society of Plant Biologists
11
Title:
12
CYTOKININ OXIDASE/DEHYDROGENASE3 maintains cytokinin homeostasis during root and nodule
13
development in Lotus japonicus
14 15
Dugald E Reida, Anne B Heckmanna, Ondřej Novákb, Simon Kellya and Jens Stougaarda
16 17
a
18
Genetics, Aarhus University, Gustav Wieds Vej 10, Aarhus C, 8000, Denmark
19
b
20
University and Institute of Experimental Botany, Academy of Sciences of the Czech Republic, CZ-78371,
21
Olomouc, Czech Republic
Centre for Carbohydrate Recognition and Signalling (CARB), Department of Molecular Biology and Laboratory of Growth Regulators and Department of Chemical Biology and Genetics, Palacký
22 23
50 word summary:
24
Cytokinin signalling is regulated during nodulation to balance organogenesis with root growth and
25
rhizobial infection. In Lotus japonicus the Ckx3 gene is induced during nodule initiation and
26
characterization of ckx3 mutants show that cytokinin degradation by CKX3 regulates this process through
27
negative regulation of cytokinin levels as measured by LC-MS/MS.
28 29 30
One sentence summary:
31
A Cytokinin oxidase/dehydrogenase in Lotus japonicus regulates cytokinin levels to prevent inhibition of
32
root growth and rhizobial infection during symbiosis.
33 34
Author contributions:
35
D.R. Designed and performed experiments and wrote the manuscript; A.H. Designed and performed
36
experiments; O.N. Performed cytokinin measurement experiments; S.K. Performed experiments; J.S.
37
Conceived experiments and complemented the writing
38 39
2
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40
Financial sources:
41
This work was supported by the Danish National Research Foundation grant no. DNRF79, the ERC
42
Advanced Grant 268523 and the Ministry of Education, Youth and Sports of the Czech Republic, the
43
‘‘Návrat’’ program LK21306
44 45
ABH Present address:
46
Arla Foods Ingredients, Sønderhøj 10, 8260 Viby J, Denmark ([email protected])
47 48
Author for correspondence:
49
Jens Stougaard ([email protected])
50 51 52 53 54 55 56
3
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57
Abstract
58 59
Cytokinins are required for symbiotic nodule development in legumes and cytokinin signalling responses
60
occur locally in nodule primordia and in developing nodules. Here we show that the Lotus japonicus Ckx3
61
cytokinin oxidase/dehydrogenase gene is induced by Nod factor during the early phase of nodule
62
initiation. At the cellular level, pCkx3::YFP reporter-gene studies revealed that the Ckx3 promoter is
63
active during the first cortical cell divisions of the nodule primordium and in growing nodules. Cytokinin
64
measurements in ckx3 mutants confirmed that CKX3 activity negatively regulates root cytokinin levels.
65
Particularly tZ and DHZ type cytokinins in both inoculated and uninoculated roots were elevated in ckx3
66
mutants suggesting that these are targets for degradation by the CKX3 cytokinin oxidase/dehydrogenase.
67
The effect of CKX3 on the positive and negative roles of cytokinin in nodule development, infection and
68
regulation was further clarified using ckx3 insertion mutants. Phenotypic analysis indicated that ckx3
69
mutants have reduced nodulation, infection thread formation and root growth. We also identify a role for
70
cytokinin in regulating nodulation and nitrogen fixation in response to nitrate as ckx3 phenotypes are
71
exaggerated at increased nitrate levels. Together, these findings show that cytokinin accumulation is
72
tightly regulated during nodulation in order to balance the requirement for cell divisions with negative
73
regulatory effects of cytokinin on infection events and root development.
74 75 76
4
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77
Introduction
78 79
To alleviate nitrogen-limiting conditions, legumes enter symbiotic relationships with rhizobia allowing
80
the host plant to acquire fixed nitrogen. Establishment of this symbiosis requires coordinated reinitiation
81
of cell divisions and organogenesis to form the nodule. The plant hormone cytokinin plays a central role
82
during nodule organogenesis and several components involved in cytokinin signalling have been
83
identified during nodulation, primarily in the model legumes (Frugier et al., 2008; Desbrosses and
84
Stougaard, 2011). Ectopic application of cytokinin or the snf2 gain-of-function mutation in the Lotus
85
japonicus HISTIDINE KINASE1 (LHK1) cytokinin receptor is sufficient to induce cell division and
86
nodule primordia in the absence of bacteria (Bauer et al., 1996; Fang and Hirsch, 1998; Tirichine et al.,
87
2007; Heckmann et al., 2011). Cytokinin perception is also a requirement for nodule organogenesis as L.
88
japonicus lhk1 and M. truncatula cre1 receptor mutants cause impaired symbiotic events and nodulation
89
is abolished in the L. japonicus lhk1/lhk1a/lhk3 triple mutant (Gonzalez-Rizzo et al., 2006; Murray et al.,
90
2007; Plet et al., 2011; Held et al., 2014). Cytokinin signalling also plays a negative regulatory role in
91
rhizobia infection as the lhk1-1 mutant exhibits hyper-infection despite the reduced organogenesis
92
(Murray et al., 2007). Downstream cytokinin responses are orchestrated by response regulators, which are
93
induced during nodulation (Lohar et al., 2004; Gonzalez-Rizzo et al., 2006; Lohar et al., 2006; Tirichine
94
et al., 2007; Op den Camp et al., 2011). Cytokinin signalling output as determined with the synthetic two-
95
component signalling sensor (TCS; Müller and Sheen, 2008) has been shown in cortical and pericycle
96
cells in response to lipo-chitooligosaccharide Nod factors in M. truncatula and the dividing cells of the
97
developing nodule in L. japonicus (Held et al., 2014; van Zeijl et al., 2015). The onset of cortical cell
98
divisions requires reprogramming of already differentiated cortical cells and is associated with local auxin
99
signalling (Mathesius et al., 2000; Suzaki et al., 2012) and initiation of endoreduplication (Suzaki et al.,
100
2014; Yoon et al., 2014). This endocycling may be directly induced by cytokinin as cytokinin controls
101
entry into endoreduplication (Takahashi et al., 2013).
102
Development of a mature nitrogen-fixing nodule is accomplished by coordination of nodule
103
organogenesis and the pathway controlling rhizobial infection (Madsen et al., 2010). Upstream of
104
cytokinin perception, nodulation signalling involves decoding of calcium influx and spiking events by the
105
CALCIUM AND CALMODULIN DEPENDENT KINASE (CCaMK) (Lévy et al., 2004; Sieberer et al.,
106
2009). Autoactive variants of CCaMK and its phosphorylation target CYCLOPS are sufficient to trigger
107
downstream nodulation signalling and spontaneous nodules (Tirichine et al., 2006a; Singh et al., 2014).
108
Downstream of LHK1, nodule organogenesis requires NODULE INCEPTION (NIN) (Schauser et al.,
109
1999; Marsh et al., 2007) and the GRAS transcription factors NSP1 and NSP2 (Kaló et al., 2005; Smit et
110
al., 2005; Heckmann et al., 2006; Murakami et al., 2007; Hirsch et al., 2009). These transcription factors
5
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111
are also required in the rhizobia infection pathway. Activation of NIN is a central function of cytokinin
112
activity (Tirichine et al., 2007; Heckmann et al., 2011) and NIN overexpression is also sufficient for
113
spontaneous initiation of cell divisions, which is dependent on the NUCLEAR FACTOR Y
114
transcriptional activators NF-YA1 and NF-YB1 in L. japonicus (Soyano et al., 2013). Activation of
115
nodulation signalling by cytokinin and NIN also induces systemic inhibition of nodulation as it directly
116
activates nodule suppressive CLE peptides (Mortier et al., 2012; Soyano et al., 2014) which systemically
117
regulate nodulation (Okamoto et al., 2009; Mortier et al., 2010; Reid et al., 2011a; Saur et al., 2011). CLE
118
peptide
119
HYPERNODULATION
120
TRANSFERASE (IPT3) dependent cytokinin biosynthesis in the shoot which negatively regulates
121
nodulation (Krusell et al., 2002; Nishimura et al., 2002; Okamoto et al., 2013; Sasaki et al., 2014). LHK1
122
was not required for this negative regulatory role of cytokinin but it was dependent on the function of the
123
Kelch repeat-containing F-box protein, TOO MUCH LOVE in the root (Takahara et al., 2013; Sasaki et
124
al., 2014).
dependent
nodule
regulation
AND
in L.
ABERRANT
japonicus ROOT1
requires
(HAR1)
the
and
LRR
induces
receptor
kinase
ISOPENTENYL
125
The early nodulation signalling pathway directly induces cytokinin biosynthesis as Nod factor
126
induced cytokinin accumulation, observed at 3 h in wild-type roots was not detected in the Mtdmi3
127
(ccamk) background (van Zeijl et al., 2015). Several cytokinin biosynthesis genes have been identified as
128
contributing to cytokinin pools during nodule development including LjIPT3 and two M. truncatula
129
LONELY GUYs which directly activate cytokinin nucleotides (Chen et al., 2014; Mortier et al., 2014).
130
However, the processes controlling cytokinin levels and their cell autonomous or non-cell autonomous
131
effects is poorly understood. Studies in non-legumes, primarily Arabidopsis, have shown that regulation
132
of active cytokinin pools occurs through reversible glycosylation, conversion to cytokinin nucleotides by
133
adenine phosphoribosyl transferase genes and through irreversible breakdown by cytokinin
134
oxidase/dehydrogenases (CKX) (Sakakibara, 2006). Ckx gene expression is enhanced by cytokinin
135
signalling and shows expression patterns similar to Ipt genes indicating a requirement for finely
136
regulating cytokinin accumulation (Schmülling et al., 2003; Werner et al., 2003; Werner et al., 2006).
137
Overexpression of CKX encoding genes can create dominant reduction in cytokinin levels and has been
138
used to examine the role of cytokinin in root development (Werner et al., 2003; Lohar et al., 2004;
139
Werner et al., 2010). However, loss-of-function ckx mutations in Arabidopsis have not been shown to
140
affect root development.
141
In order for proper nodule development to progress without secondary effects on root growth, it is
142
assumed cytokinin must be released in a tightly controlled spatial and temporal manner. The availability
143
of the LORE1 insertion population makes L. japonicus an ideal system for reverse genetics (Fukai et al.,
144
2012; Urbański et al., 2012) and here we make use of this resource to address the role of cytokinin
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145
breakdown during nodule development. We identify two insertion alleles in LjCkx3 and show that this
146
gene is critical for maintaining cytokinin homeostasis required for efficient symbiotic infection,
147
organogenesis and nitrate dependent regulation of nodulation.
148 149
Results
150 151
Lotus japonicus encodes nine cytokinin oxidase/dehydrogenase genes
152 153
In higher plants, cytokinin oxidase/dehydrogenase is encoded by multigene families. To establish the
154
complexity of the family in legumes we first searched the available genome and EST sequences of L.
155
japonicus and the M. truncatula genome (v4.0; Young et al., 2011). Nine non-redundant CKX sequences
156
were found in both. In order to construct a phylogeny, all amino acid sequences of the two legume species
157
and Arabidopsis were aligned and we then named the L. japonicus genes according to the nearest of the
158
seven Arabidopsis homologues (Fig. 1).
159
Given the roles of cytokinin in both rhizobia infection events and initiation of organogenesis we
160
sought to identify LjCkx genes regulated during the early phases of nodule establishment. Searching
161
publicly available gene expression data (Høgslund et al., 2009; accessed via ljgea.noble.org, Verdier et
162
al., 2013) revealed that two of the LjCkx genes showed expression patterns strongly correlated with
163
symbiotic development. LjCkx3 (probe ID TM0914.20_at) was induced in the nodulation susceptible
164
zone one day after inoculation and in developing nodules, while LjCkx4 (TM0914.24_at) was expressed
165
later in mature nodules. The affymetrix data also indicated that the induction of Ckx3 expression was
166
dependent on NFR1 and NFR5 but independent of NIN. Given the early Nod factor dependent expression
167
pattern we decided to focus further attention on LjCkx3.
168
Phylogenetic analysis showed that LjCKX3 was most closely related to two M. truncatula genes
169
(Medtr4g126150 and Medtr2g039410) recently reported to show induced expression in response to
170
Sinorhizobium meliloti Nod factor (van Zeijl et al., 2015). Further analysis indicated LjCkx3 comprises
171
five exons annotated by RNAseq analysis and encodes a predicted signal peptide (Fig. 1; SignalP 4.1 D-
172
score 0.668, Petersen et al., 2011), while the mature protein has predicted CK and FAD binding domains,
173
which together form the active site characteristic of CKX proteins (Malito et al., 2004). LjCKX7 was
174
most closely related to MtCKX1 (Medtr1g015410), which was previously reported to be expressed during
175
nodule development in M. truncatula (Ariel et al., 2012).
176 177
Ckx3 is expressed during nodule initiation and development
178
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179
To confirm the publicly available Affymetrix data, we inoculated with M. loti or applied purified Nod
180
factor to roots of 10 d-old plants and conducted quantitative RT-PCR. This showed Ckx3 expression was
181
induced by M. loti R7A and purified Nod factor within 8 h and increased further at 24 h (Fig. 2). In
182
Arabidopsis, Ckx gene expression is induced by cytokinin application (Werner et al., 2006). Therefore, to
183
determine whether Ckx3 also responds to cytokinin independent of rhizobia, we conducted a time course
184
following treatment with the synthetic cytokinin 6-Benzylaminopurine (BAP). Within this 12 hours time
185
series, Ckx3 expression was induced by cytokinin within 6 hours (Fig. S1).
186
To further clarify the spatio-temporal expression patterns, we cloned promoter sequences
187
corresponding to approximately 1 kb and 2kb upstream of Ckx3 and fused these to a nuclear-localised
188
triple-YFP (tYFPnls) reporter, which is readily visualised relative to autofluorescence in L. japonicus.
189
Confocal microscopy revealed that the response patterns were indistinguishable between the 1 kb and 2
190
kb promoter fragments (Fig 3, Fig. S7). Expression of pCkx3::tYFPnls was observed in the cells
191
corresponding to high cytokinin activity in the meristematic zone of the root tip (Zürcher et al., 2013)
192
irrespective of inoculation status (Fig. 3A). YFP was also observed in the central root cylinder in both
193
inoculated and uninoculated roots (Fig. 3B, 3D). Sectioning further confirmed that the central root
194
cylinder expression observed in whole mounts corresponds to expression in pericycle cells adjacent to
195
xylem cells and protoxylem (Fig. 3C). Cross sections of roots inoculated with M. loti expressing DsRed
196
revealed expression occurs in the dividing cells of the root cortex (nodule primordium) and pericycle
197
during nodule primordium development and is sustained in the cortical cells of more mature growing
198
nodules. In mature nodules with differentiated bacteroids, expression was localised to the cells
199
surrounding the infected tissue (Fig. 3). YFP expression was not identified in the epidermal cells of
200
transgenic roots in response to inoculation. The observed expression patterns driven by the Ckx3 promoter
201
are consistent with known cytokinin response domains at the root tip and nodule primordia reported
202
elsewhere (Müller and Sheen, 2008; Held et al., 2014) and taken together with the real-time results
203
reported here indicate the promoter fragment likely captures the essential cytokinin response elements
204
responsible for Ckx3 expression patterns. We further confirmed the cytokinin responsiveness of the
205
promoter by treating pCKX3::tYFPnls roots with BAP. Within 3 hours, this treatment induced expression
206
of the tYFPnls reporter in the cortex of the root, while vascular expression was indistinguishable from
207
untreated roots (Fig. S6).
208 209
Identification of Ckx3 mutant alleles
210 211
To identify mutants in Ckx3, we searched the publicly available LORE1 insertional mutant resources
212
(Fukai et al., 2012; Urbański et al., 2012; Hirakawa et al., 2014; accessed via carb.au.dk/lotus-base/). We
8
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213
identified lines with insertions in exon 1 (5497) and exon 3 (17827) of Ckx3 and named these ckx3-1 and
214
ckx3-2 respectively (Fig. 1). Searching the LORE1 version 2.5 database showed these lines contained 0
215
and 2 known additional LORE1 insertions respectively, none of which were exonic.
216 217
Quantification of cytokinins in Lotus japonicus roots
218 219
To identify the effect of inoculation on cytokinin levels, we quantified isoprenoid type cytokinins in 10-d
220
old wild type L. japonicus (Gifu) roots 24 and 72 h after inoculation with M. loti R7A or a Nod factor
221
defective R7AnodC mutant compared to mock treated whole roots. All isoprenoid-type cytokinins were
222
detected as bases, ribosides, and nucleotide metabolites (Supp. table 1). N-glucosides and O-glucosides of
223
tZ and DHZ were under detection limits. This analysis showed DHZ and iP cytokinin bases and tZ, cZ,
224
DHZ and iP ribosides were all increased 24 h post-inoculation with R7A relative to R7AnodC inoculated
225
roots (Fig. 4; Table S1). tZ ribosides showing the most significant change (1.88-fold increased) while iP
226
type cytokinins were the most abundant cytokinin species detected. After 72 h, the tZ, DHZ and iP
227
cytokinin bases and ribosides remained significantly increased relative to the nodC inoculated roots. At
228
72 h, levels of tZ were approximately 2-fold higher in R7A inoculated roots compared to R7AnodC while
229
iP was approximately 1.5-fold increased.
230
CKX proteins are known to cleave tZ and iP bases and ribosides with varying affinities (Galuszka
231
et al., 2007). To confirm whether CKX3 has a biologically relevant role in degrading cytokinin in L.
232
japonicus roots, we compared cytokinin concentrations in the ckx3-2 mutant relative to Gifu across the
233
same conditions. We found both tZ and DHZ base and riboside levels to be significantly increased in the
234
mutants across treatment groups at both 24 and 72 hpi (Fig. 4, S5). In contrast, cZ and iP type cytokinins
235
were not significantly altered, or slightly decreased in the mutants relative to Gifu (Fig. 4, S5). In M. loti
236
R7A inoculated ckx3-2 roots, tZR were the only species increased at both 24 and 72 h relative to Gifu,
237
while DHZ and tZ were increased significantly in the mutants at 24 and 72 h respectively.
238 239
Elevated cytokinin in ckx3 decreases nodulation efficiency
240 241
To determine the effect of the elevated cytokinin levels in ckx3 mutants on nodule development, we grew
242
the plants on vertical filter paper covered agar slopes with the roots shielded from light, which allows
243
continued observation of nodule developmental phenotypes and kinetics. Both ckx3 alleles developed
244
nodules normal in appearance however the number of nodules formed was significantly reduced at all
245
timepoints (Fig. 5). To confirm these data in a controlled glasshouse environment, we grew plants in open
246
vermiculite pots in nitrate free conditions and found both alleles showed significantly reduced nodulation
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247
5 w after inoculation (Figure S2).
248
Given that reduced cytokinin signalling in L. japonicus causes hyperinfection (Murray et al.,
249
2007), we counted infection threads formed on the mutants 10 d after inoculation with a M. loti strain
250
expressing DsRED. We found the number of infection threads formed was significantly reduced in both
251
alleles (Fig. 5B). Cytokinin can induce ethylene production and is thought to act largely through ethylene
252
in repressing root growth. The ethylene synthesis inhibitor aminoethoxyvinylglycine (AVG) can rescue
253
root growth inhibition in the presence of elevated endogenous or ectopic cytokinin in legumes (Wopereis
254
et al., 2000; Ferguson et al., 2005). We therefore repeated the IT counts with 10-8 M AVG supplemented
255
in the media. AVG treatment was sufficient to rescue the reduced infection thread phenotypes in both
256
ckx3 alleles, increasing IT numbers close to wild-type levels (Fig. 5C). To further identify the infection
257
phenotypes of the ckx3 mutants, we counted infection threads on plants grown in the presence of 2 mM
258
KNO3 or 10-8 M BAP. These results showed that while both nitrate and BAP treatment can significantly
259
reduce infection events, the reduced infection levels of ckx3 is not further impaired upon treatment (Fig.
260
5D).
261
Ectopic application of cytokinin or the snf2 gain-of-function mutation has previously been shown
262
to be sufficient to initiate nodule organogenesis in L. japonicus (Tirichine et al., 2007; Heckmann et al.,
263
2011). To determine if the elevated cytokinin levels in ckx3 mutants was sufficient to trigger nodule
264
organogenesis, we grew mutants on nitrate free agar slants in the absence of rhizobia but did not observe
265
spontaneous organogenesis in these conditions. To determine if the elevated cytokinin in ckx3 plants
266
might be inhibitory to spontaneous nodule development we grew the mutants in the presence of 10-8 M
267
BAP. Spontaneous nodule organogenesis was observed in both Gifu and ckx3 mutants in these conditions
268
(Fig. S3).
269 270
Cytokinin plays a role in nitrate regulation of nodulation and nitrogen fixation
271 272
Cytokinin biosynthesis in Arabidopsis, in particular through IPT3, is known to be an important means of
273
regulating plant development in response to environmental signals, including nitrate (Takei et al., 2004;
274
Sakakibara et al., 2006; Ruffel et al., 2011). Since nodulation is negatively regulated by nitrogen,
275
especially nitrate, we investigated whether cytokinin might link nitrogen regulation and nodulation. ckx3
276
mutant plants were grown under elevated nitrate conditions and the nodulation phenotype observed.
277
Interestingly, increased nitrate concentration accentuated the nodulation phenotypes. To quantify this
278
effect, we grew Gifu and ckx3-2 under different nitrate regimes and counted the number of red nitrogen-
279
fixing nodules, white non-fixing nodules and assayed nitrogen fixation activity from whole roots (Fig 6B)
280
and individual nodules (Fig. 6C) using the acetylene reduction assay (ARA). This showed that while
10
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281
ckx3-2 had reduced nodulation but formed normal red fixing nodules in nitrate free conditions, it was
282
more sensitive to increased nitrate than Gifu (Fig. 6). Growth on 2 mM KNO3 significantly reduced total
283
nodule numbers, red nodules and the ARA activity of Gifu but was reduced significantly more in ckx3-2
284
(Fig. 6A). Gifu continued to form a small number of pink-red nitrogen-fixing nodules (confirmed by
285
ARA activity) at 5 mM KNO3, however this concentration was completely inhibitory to the development
286
of red nodules and nitrogen fixation in ckx3-2 (Fig. 6). To confirm this effect of cytokinin on nitrogen
287
fixation, we also grew the plants on media supplemented with 10-8 M BAP and found nodulation and
288
nitrogen fixation to be significantly reduced in both Gifu and ckx3-2 (Fig. 6). Acetylene reduction on a
289
per nodule basis indicated that this response is likely at earlier infection and nodule organogenesis stages
290
as individual nodules formed on BAP treated roots continued to show wild-type acetylene reduction
291
activity (Fig 6C). Nodule sections (Fig. 6 J to O) showed that those nodules that did form on nitrate or
292
BAP treated roots were colonised by rhizobia expressing the DsRed marker. This included the small
293
nodules formed on ckx3 mutants at 5 mM KNO3 despite the white appearance and near-complete
294
reduction in acetylene reduction activity.
295 296
Ckx3 regulates cytokinin levels affecting root meristem elongation and differentiation
297 298
To investigate the role of Ckx3 in root development, we measured total root length in Gifu and ckx3
299
mutants. We found that the ckx3 mutants showed significantly reduced root length relative to Gifu at 20 d
300
after germination (Fig. 7A). To determine the basis of this reduced root growth, we investigated the zones
301
of cell proliferation and elongation as well as the differentiation zone at the root tip. The region from the
302
root tip to the first emerging root hairs includes the zones of proliferation and elongation, while the zone
303
of differentiation is defined by the emergence and growth of root hairs (Williamson et al., 2001; Jones et
304
al., 2009; Petricka et al., 2012). We therefore measured the distance between the root tip and first root
305
hair as a measure of proliferative and elongation zone length and found the ckx3 mutants exhibit
306
significantly reduced root tip length (Fig 7B). To evaluate the effect of ckx3 mutation on root hair
307
emergence in the differentiation zone, we measured the angle created by the emergence of root hairs
308
immediately behind the meristem (Fig. S4). This analysis showed that the angle was significantly greater
309
in ckx3 indicating more rapid differentiation and/or a reduced differentiation zone length (Fig. 7C).
310 311
Discussion
312 313
Cytokinin signalling must be finely regulated during nodulation in order to balance the positive role
314
during nodule organogenesis with the negative effect in symbiotic infection and crosstalk with other
11
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315
hormones. Nod factor induced cytokinin accumulation plays a crucial role in the induction of early
316
nodulation responses (van Zeijl et al., 2015) and is perceived partially redundantly by the LHK receptors
317
in L. japonicus (Held et al., 2014). Ckx encoding genes expressed during nodule development have also
318
been implicated in regulation of cytokinin levels and signalling during nodulation (Held et al., 2008; Ariel
319
et al., 2012; van Zeijl et al., 2015) however the lack of well-defined mutants made the determination of
320
their precise role during nodulation difficult. Here we identify LORE1 insertion mutants in LjCkx3, which
321
exhibit reduced nodulation, rhizobia infection and root growth and establish a role for Ckx genes during
322
symbiosis. We show that regulation of cytokinin accumulation through breakdown of cytokinin by
323
LjCKX3 plays a role in maintaining efficient nodule development. Our gene expression data and
324
cytokinin measurements showed that cytokinin signalling rapidly induces expression of Ckx3 in order to
325
restrict cytokinin accumulation. This may serve to avoid over-stimulation of cell division, maintain
326
cytokinin signalling autonomy for neighbouring cells and/or stimulation of negative feedback
327
mechanisms such as the effects of ethylene on infection.
328
Our data also highlight the extensive crosstalk between cytokinin and ethylene. Cytokinin
329
signalling negatively regulates infection since mutations in Lhk1 results in hyperinfection (Murray et al.,
330
2007) while ethylene also inhibits infection (Penmetsa and Cook, 1997; Penmetsa et al., 2008). Consistent
331
with these results, we found the reduced infection phenotypes of the ckx3 mutants could be rescued by
332
AVG treatment, indicating cytokinin degradation is critical in preventing over stimulation of ethylene
333
dependent inhibition of symbiotic infection. This interpretation is supported by results showing that
334
cytokinin induces and stabilises ACC synthase, the rate limiting step in ethylene biosynthesis (Chae et al.,
335
2003; El-Showk et al., 2013). In return, ethylene can regulate cytokinin signalling through the type-A
336
ARRs (Shi et al., 2012).
337 338
CKX3 primarily regulates tZ levels
339 340
Our quantification of isoprenoid cytokinins showed that Ckx3 regulates root cytokinin levels in vivo. The
341
elevated levels of tZ and DHZ type cytokinins in ckx3-2 suggest these are either the species most
342
susceptible to CKX3 degradation or that accumulate in response to CKX activity on other cytokinins.
343
Biochemical studies have shown Arabidopsis CKX genes expressed in a heterologous Nicotiana tabacum
344
system have highest activity against trans-Zeatin (tZ) and isopentenyl adenine (iP) type cytokinins while
345
cis-Zeatin (cZ) and dihydrozeatin (DHZ) are resistant to cleavage (Galuszka et al., 2007). While CKX
346
was shown to have limited activity against DHZ in one study, tZ may be converted to DHZ by zeatin
347
reductase, especially in the absence of CKX activity (Gaudinová et al., 2005). DHZ does not have strong
348
activity in Arabidopsis and has been suggested to act as a storage or transport form of cytokinin (Mok and
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349
Mok, 2001). tZ may therefore be the primary target of CKX3 and increased DHZ is a direct result of high
350
tZ levels. In Arabidopsis the three AHK receptors maintain specificity in cytokinin response through both
351
expression domains and differing affinities to the cytokinin ligands, with tZ having high affinity binding
352
against AHK2, AHK3 and AHK4 while iP shows strong affinities against AHK2 and AHK4 (Romanov et
353
al., 2006; Stolz et al., 2011). The LHK1, LHK1a and LHK3 receptors in L. japonicus have been shown to
354
functionally restore cytokinin sensitivity in Yeast or E. coli heterologous system assays, however no data
355
is available on their cytokinin binding specificity (Murray et al., 2007; Held et al., 2014). IPT3 dependent
356
synthesis of iP type cytokinin in the shoot has been shown to negatively regulate nodule numbers in L.
357
japonicus (Sasaki et al., 2014). Our study provides further evidence that increased cytokinin levels can
358
negatively regulate infection and organogenesis events and that cytokinin levels are therefore finely
359
regulated to maintain efficient nodulation.
360
We found the accumulation of cytokinin bases was dependent on Nod factor signalling as the
361
R7AnodC mutant failed to initiate the responses observed for R7A wild type strain. This is consistent
362
with the results showing Nod factor induction of cytokinin in M. truncatula (van Zeijl et al., 2015), albeit
363
at later timepoints in our experiments. The Nod factor treatment reported in M. truncatula was found to
364
induce accumulation of iP, iPR and tZ type cytokinins (van Zeijl et al., 2015). We also found iP and iPR
365
to be induced by M. loti inoculation on L. japonicus at both 24 h and 72 h, whereas tZ was only increased
366
at 72 h. We also found significant increases in tZR, DHZ and DHZR at both 24 h and 72 h after M. loti
367
inoculation. These additional cytokinins identified in our studies may result from differences in Nod
368
factor and rhizobia responses or result from the later time-points we examined relative to the early Nod
369
factor responses reported. Furthermore, it is possible that cytokinin inter-conversion occurs following
370
initial synthesis or that between species differences exist in cytokinin responses to rhizobia. We found
371
that although the cytokinin pool is under negative feedback by CKX3 which is induced within 8 h of
372
inoculation, elevated cytokinin is maintained during the first three days following inoculation, though
373
likely in a tightly spatially restricted manner. TCS expression in L. japonicus showed cytokinin signalling
374
domains were restricted to the dividing cells of the nodule primordia while Nod factor responses in M.
375
truncatula triggered a more widespread cortical and pericycle response (Held et al., 2014; van Zeijl et al.,
376
2015). Further analysis at the early points during symbiotic interaction is required to confirm whether
377
rhizobia induce cytokinin signalling in a wide region as is observed for Nod factor treatment or through
378
more restricted biosynthesis and associated degradation in order to restrict cytokinin signalling and
379
induction of nodulation foci to a small number of defined cells.
380 381
Local restriction of cytokinin accumulation is required for efficient infection
382
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383
Our data showing a Nod factor dependent (Fig. 2A) and nodule primordia localised (Fig. 3E,F)
384
expression pattern for Ckx3 indicate that cytokinin signal induction is transient and must be tightly
385
regulated to avoid negative effects. This is consistent with data indicating that increased cytokinin
386
signalling in Ljsnf2 or through ectopic MtLog overexpression reduces nodulation (Tirichine et al., 2007;
387
Mortier et al., 2014). Most Arabidopsis CKX have predicted signal peptides and are proposed to localise
388
to the apoplast or vacuoles (Werner et al., 2003; Kowalska et al., 2010). We also found LjCKX3 has a
389
predicted secretion signal and is therefore likely degrading cytokinin in the extracellular space.
390
Cytokinin has been proposed as a non-cell autonomous signal during nodulation and restriction of
391
extracellular cytokinin would therefore be important for regulation of this signalling. We did not observe
392
epidermal expression of Ckx3 and it remains unresolved whether alternative Ckx genes may be expressed
393
here or whether cytokinin biosynthesis in the epidermis is sufficient to produce a non-cell autonomous
394
cytokinin signal to initiate cell divisions in the cortex. The close correlation of Ckx3 expression in nodule
395
primordia cells and correlation with areas of high cytokinin response in the root tip are strongly correlated
396
to the signalling domains of TCS in legumes and Arabidopsis respectively (TCS; Müller and Sheen,
397
2008; Zürcher et al., 2013; Held et al., 2014). Furthermore, it is likely that the role of CKX3 in regulating
398
cytokinin levels in response to Nod factor is conserved in legumes as two closely related homologues in
399
M. truncatula were both shown to respond to Nod factor treatment (van Zeijl et al., 2015). The induction
400
of spontaneous nodules through elevated cytokinin signalling continues to maintain defined foci rather
401
than widespread induction of cell divisions (Tirichine et al., 2006b; Tirichine et al., 2007; Heckmann et
402
al., 2011). Our observations also suggest unknown mechanisms outside of cytokinin signalling might be
403
required to define cells competent for division and to maintain these divisions to a limited nodule foci as
404
we did not observe persistent cell divisions or abnormally shaped nodules in ckx3 mutants. Maintaining
405
organised cell divisions during nodule development therefore results from the coordination of nodulation
406
specific transcriptional networks with hormones required for cell specification and division.
407
Cytokinins are known to alter root meristem size (Dello Ioio et al., 2007). This inhibition is
408
dependent on ethylene in Arabidopsis and L. japonicus (Wopereis et al., 2000; Růžička et al., 2009).
409
Ethylene is produced during nodulation (Ligero et al., 1986) and our work finds the breakdown of
410
cytokinin is required to prevent the resulting inhibition of root growth and infection. Further analysis of
411
the genetics of ethylene induction in the common symbiosis pathway will help to clarify the observed
412
cross talk and regulatory functions. Pericycle and root tip expression of Ckx3 was independent of
413
inoculation, indicating that the evolutionary ancestral role of CKX3 is likely in regulation of root
414
development. However, no root phenotypes for ckx3 mutants have been reported in Arabidopsis, while
415
ckx3 ckx5 double mutants have altered shoot inflorescence meristem size (Bartrina et al., 2011). Pericycle
416
cells in Medicago truncatula maintain the ability to divide in order to initiate lateral root and nodule
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417
primordia (Xiao et al., 2014). In Arabidopsis, Ckx genes are expressed during lateral root primordia
418
initiation (Werner et al., 2003) and expression of Ckx in the xylem pole pericycle cells can increase lateral
419
root density (Laplaze et al., 2007). While we observed expression of Ckx3 in pericycle cells, it was not
420
universal in pericycle cells. The expression of Ckx3 in a subset of pericycle cells may play a role in
421
priming cells and determining their susceptibility to undergo division. It would be interesting to determine
422
if the cells with Ckx3 expression are correlated with the radial or longitudinal positioning of nodule
423
primordia sites. Improved spatio-temporal expression analysis using promoter-YFP stable lines may
424
provide a means to determine whether cytokinin degradation plays a role in radial nodule positioning and
425
the crosstalk with ethylene in this process during nodule development.
426 427
Cytokinin regulates nodulation in response to environmental signals
428 429
Legumes regulate nodulation locally and systemically in response to environmental cues, including
430
nitrate, in order to balance fixed nitrogen from symbiosis with other nitrogen sources (Reid et al., 2011b).
431
This regulation occurs by both HAR1 dependent and independent mechanisms. In Arabidopsis, IPT3
432
expression is known to be induced by nitrate and cytokinin levels are increased as a result (Takei et al.,
433
2004). Cytokinin thus plays a central role in regulating responses to the environment (Sakakibara et al.,
434
2006; Ruffel et al., 2011). We found the increased cytokinin levels in Ljckx3 mutants enhanced the
435
susceptibility to negative effects of nitrate on nodulation and nitrogen fixation. This indicates cytokinin
436
plays a role in regulating nitrogen fixation. The strong inhibition of nitrogen fixation relative to
437
nodulation indicates that nitrogen fixation and nodule organogenesis possess both common and
438
independent regulatory mechanisms in response to available nitrogen. This is supported by the fact
439
hypernodulation mutants show reduced nitrogen fixation on a per nodule basis (Carroll et al., 1985; Jeudy
440
et al., 2010). Whether the cytokinin regulation of nitrogen fixation occurs locally or systemically in
441
response to nitrate and how this integrates with the HAR1 mediated regulation of nodulation remains to
442
be resolved. Nitrate inhibition of nodulation has previously been shown to involve ethylene biosynthesis
443
as it can be alleviated by AVG treatment, however it is not known if this involves local or systemic
444
ethylene responses (Ligero et al., 1991). Ethylene is also thought to regulate cytokinin signalling in both
445
environmental responses and nodulation signalling (Shi et al., 2012; van Zeijl et al., 2015). Together with
446
our findings, this is consistent with cytokinin and its crosstalk with ethylene playing a significant role in
447
the inhibition of nodulation and nitrogen fixation by nitrate.
448 449
Conclusions
450
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451
We found that inoculation with M. loti causes an increase in root cytokinin levels and a resulting
452
upregulation of negative feedback through cleavage of active cytokinins, particularly tZ, by CKX3.
453
CKX3 acts to restrict the pool of active cytokinin and prevents the resulting stimulation of ethylene and
454
negative effects on nodule organogenesis, nitrogen fixation, and infection thread development. Expression
455
of Ckx3 in the root tip ensures homeostasis of cytokinin in the meristematic zone in order to balance cell
456
elongation with differentiation and root hair outgrowth. Overall, these results confirm the importance of
457
cytokinin in maintaining effective nodulation and identify a new negative regulator of this signalling.
458
Future efforts to elucidate the roles of crosstalk of cytokinin with other plant hormones, particularly auxin
459
(Breakspear et al., 2014) and ethylene (Ferguson and Mathesius, 2014) will assist in understanding the
460
cellular mechanism involved in nodule development.
461 462
Materials and Methods
463 464
Plant and bacteria genotypes
465 466
Lotus japonicus ecotype Gifu was used in all experiments (Handberg and Stougaard, 1992). Homozygous
467
LORE1 inserts were genotyped with allele specific primers in combination with the P2 internal LORE1
468
primer as described (Urbański et al., 2012). Primer sequences were obtained from the LORE1 resource
469
page (carb.au.dk/lotus-base) or designed in the same region if amplification was unsuccessful (Table S2).
470
M. loti R7A and the Nod factor defective nodC variant (Rodpothong et al., 2009) were diluted to an
471
inoculum density of OD600 = 0.01. For infection thread counting, the M. loti MAFF303099 strain carrying
472
chromosomal DsRed insertion was used (Maekawa et al., 2009).
473 474
Plant and bacteria growth conditions
475 476
For nodulation assays and IT counts, 3 d-old seedlings were transferred to vertical plates with filter paper
477
on 1.4 % agar noble containing quarter-strength B&D nutrients (Broughton and Dilworth, 1971) in the
478
presence or absence of KNO3, 10-8 BAP or AVG as described for each experiment. Nod factor treatment
479
was carried out on plates with 10-8 M. loti R7A Nod factor pipetted directly onto roots. Infection threads
480
were counted 10 d after inoculation by placing whole roots on microscope slides to allow counting of the
481
root surface contacting the growth plates. Hairy roots were induced by infection of 6 d-old seedlings
482
growing on vertical 0.8 % Phytagel (Sigma) plates with half-strength B5 salts and vitamins as described
483
(Stougaard et al., 1987; Hansen et al., 1989; Stougaard, 1995). Three weeks after infection, primary roots
484
were removed and the chimeric plants transferred to plastic magenta boxes containing 1:4
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485
leca:vermiculite mix. For phenotyping in open pots, 3 d-old seedlings were transferred to pots containing
486
vermiculite watered with nitrate free half-strength B&D.
487 488
Bioinformatics
489 490
The amino acid translations of the representative gene models for Arabidopsis Ckx genes were obtained
491
from TAIR for BLAST queries against L. japonicus resources at NCBI and Lotus base (carb.au.dk/lotus-
492
base). M. truncatula sequences were obtained by searching Mt v4 at Phytozome.org. The gene phylogeny
493
was drawn based on alignment of the amino acid sequences of all three species and subsequent bootstrap
494
analysis based on 1000 replications using ClustalX (Larkin et al., 2007). Cytokinin and FAD binding
495
domains within CKX3 were identified by BLAST query against the NCBI Conserved Domain Database
496
(Marchler-Bauer et al., 2015). Signal peptide prediction was carried out using SignalP 4.1 (Petersen et al.,
497
2011). Microarray data was identified and analysed using the L. japonicus gene expression atlas (Verdier
498
et al., 2013) by identifying probes against LjCkx genes with BLAST.
499 500
Cloning
501 502
Primers for cloning a Ckx3 promoter sequence corresponding to approximately 1 kb upstream sequence
503
were designed against the Lj2.5 genome and amplification carried out from MG-20 genomic DNA while
504
the 2kb promoter fragment was synthesised according to MG-20 genomic sequence. The Ckx3 promoter
505
fragment was subsequenctly cloned by TOPO cloning into the Gateway compatible pDONR vector (Life
506
Technologies). tYFPnls was constructed by amplifying a tYFP cDNA with primers including a C-terminal
507
nuclear localisation signal (Takeda et al., 2012) and excision of the largest of three resulting bands before
508
cloning into a pIV10 vector (Stougaard, 1995) modified to accept Gateway promoter clones.
509 510
Quantitative RT-PCR
511 512
For expression analysis in roots, plants were grown and Nod factor or BAP applied as described
513
previously (Heckmann et al., 2011). mRNA was isolated from BAP (10-8 M), Nod factor (10-8 M) or
514
R7A (OD600=0.02) treated roots using Dynabeads mRNA DIRECTTM kit (Invitrogen). RevertAid M-
515
MuLV Reverse Transcriptase (Fermentas) was used for cDNA synthesis. All cDNA samples were tested
516
for genomic DNA contamination using primers specific for the NIN gene promoter (Lohmann et al.,
517
2010). A Lightcycler480 instrument and Lightcycler480 SYBR Green I master (Roche Diagnostics
518
GmbH) was used for the real time quantitative PCR. ATP-synthase (ATP), Ubiquitin-conjugating enzyme
17
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519
(UBC) and Protein phosphatase 2A (PP2A) were used as reference genes (Czechowski et al., 2005). The
520
relative quantification software (Roche) was used to calculate the normalized efficiency-corrected relative
521
transcript levels. The geometric mean of the relative transcript levels for the three biological (each
522
consisting of 10 plants) and three technical repetitions and the corresponding upper and lower 95%
523
confidence were calculated (Vandesompele et al., 2002). Primer sequences are listed in Table S2.
524 525
Microscopy
526 527
Microscopy was performed with a Zeiss LSM 510 Meta Confocal Microscope. Objective lenses were
528
Zeiss Plan-Neofluar 10x/0.3 and 20x/0.5. Laser excitation was at 488 nm for YFP and 543 nm for DsRed
529
and emission filters were 505-550 nm for YFP and 585-615 nm for DsRed. For sections, live roots were
530
embedded in 3% agarose and cut to 80-100 µM sections using a Leica VT 1000 S vibratome before
531
imaging with the confocal microscope.
532 533
Statistical analysis
534 535
Statistical analysis was carried out using GraphPad Prism software. Comparison of multiple groups
536
included ANOVA followed by Tukey post-hoc testing to determine statistical significance. Students T-
537
test or Wilcoxon rank-sum test was used to determine differences when making single comparisons. All
538
data is plotted as mean with 95% CI for the indicated number of biological replicates.
539 540
Acetylene reduction assay
541 542
Acetylene was produced by reaction of calcium carbide with water. The resulting gas was collected and
543
diluted to 2% in stoppered glass vials. For the assay, 250 µl air was removed from the 5 ml glass GC vials
544
containing whole nodulated roots 14 d after inoculation and replaced with equal volume of 2% acetylene.
545
Samples were incubated 30 min before quantification of ethylene conversion using a SensorSense
546
(Nijmegen, NL) ETD-300 ethylene detector operating in sample mode with 2.5 L/h flow rate and 6-
547
minute detection time.
548 549
Endogenous cytokinin measurements
550 551
For cytokinin analysis, plants were grown on filter paper covered agar slants as described above. 10 d-old
552
plants were inoculated before whole roots were harvested at 24 h or 72 h after treatment. Prepared
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553
biological quadruplicates were extracted and purified using the method published previously (Novák et
554
al., 2008) with some minor modifications. 10-20 mg FW were extracted in 1 ml of modified Bieleski
555
buffer (60% MeOH, 10% HCOOH and 30% H2O) together with a cocktail of 23 stable isotope-labeled
556
CK internal standards (0.5 pmol of CK bases, ribosides, N-glucosides, 1 pmol of O-glucosides and
557
nucleotides) to check recovery during purification and to validate the determination. The samples were
558
purified using a combination of C18 (100 mg/1ml) and MCX cartridges (30 mg/1ml) and immunoaffinity
559
chromatography (IAC) based on wide-range specific monoclonal antibodies against cytokinins (Faiss et
560
al., 1997). The eluates from the IAC columns were evaporated to dryness and dissolved in 20 µl of the
561
mobile phase used for quantitative analysis. The samples were analyzed by the LC-MS/MS system
562
consisting of an ACQUITY UPLC® System (Waters) and Xevo™ TQ-S (Waters) triple quadrupole mass
563
spectrometer. Quantification was obtained using a multiple reaction-monitoring (MRM) mode of selected
564
precursor ions and the appropriate product ion.
565 566
Accession numbers
567 568
Genbank accession numbers: LjCkx1, KR296932; LjCkx2, KR296933; LjCkx3, KR296934; LjCkx4,
569
KR296935; LjCkx5, KR296936; LjCkx6, KR296937; LjCkx7, KR296938; LjCkx8, KR296939; LjCkx9,
570
KR296940
571 572
Figure Legends
573 574
Figure 1. Lotus japonicus CKX family. A, CKX phylogeny assembled by alignment of the L. japonicus,
575
M. truncatula and A. thaliana amino acid sequences. LjCKX3 (red) is most closely related to two M.
576
truncatula genes, which are induced by Nod factorapplication (van Zeijl et al., 2015). Bootstrap values
577
are shown for each node based on 1000 replications. B, LjCkx3 comprises five exons and encodes a
578
predicted signal peptide (SP), cytokinin (CK bind) and FAD binding domains. Lines containing LORE1
579
insertions were characterised in the first and third exons of LjCkx3.
580 581
Figure 2. Effect of ectopic Nod factor application or M. loti inoculation on Ckx3 mRNA levels. A,
582
Relative expression levels following Nod factor treatment. B, Relative expression levels following
583
inoculation with M. loti R7A. Values are relative to mock treatment and indicate mean ± 95% CI for n=3.
584
P-values were calculated using Wilcoxon rank-sum testing between mock and treatment groups and are
585
indicated by *
