© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 2441-2445 doi:10.1242/dev.107946
RESEARCH REPORT
Endoreduplication-mediated initiation of symbiotic organ
development in Lotus japonicus
Takuya Suzaki1,2,*, Momoyo Ito1, Emiko Yoro1,2, Shusei Sato3, Hideki Hirakawa4, Naoya Takeda1,2 and
Masayoshi Kawaguchi1,2
Many leguminous plants have a unique ability to reset and alter the
fate of differentiated root cortical cells to form new organs of nitrogenfixing root nodules during legume-Rhizobium symbiosis. Recent
genetic studies on the role of cytokinin signaling reveal that activation
of cytokinin signaling is crucial to the nodule organogenesis process.
However, the genetic mechanism underlying the initiation of nodule
organogenesis is poorly understood due to the low number of genes
that have been identified. Here, we have identified a novel
nodulation-deficient mutant named vagrant infection thread 1
(vag1) after suppressor mutant screening of spontaneous nodule
formation 2, a cytokinin receptor gain-of-function mutant in Lotus
japonicus. The VAG1 gene encodes a protein that is putatively
orthologous to Arabidopsis ROOT HAIRLESS 1/HYPOCOTYL 7, a
component of the plant DNA topoisomerase VI that is involved in the
control of endoreduplication. Nodule phenotype of the vag1 mutant
shows that VAG1 is required for the ploidy-dependent cell growth
of rhizobial-infected cells. Furthermore, VAG1 mediates the onset of
endoreduplication in cortical cells during early nodule development,
which may be essential for the initiation of cortical cell proliferation that
leads to nodule primordium formation. In addition, cortical infection is
severely impaired in the vag1 mutants, whereas the epidermal infection
threads formation is normal. This suggests that the VAG1-mediated
endoreduplication of cortical cells may be required for the guidance of
symbiotic bacteria to host meristematic cells.
KEY WORDS: Endoreduplication, Lotus japonicus, Root nodule
development, Topoisomerase VI
INTRODUCTION
In response to appropriate inductive conditions, plants have the
capacity to form new organs from differentiated cells. Root nodulation
is a form of such de novo organogenesis that occurs predominantly in
leguminous plants (Crespi and Frugier, 2008). During early nodule
development, a rhizobia-derived nodulation factor induces the
dedifferentiation of root cortical cells. The activated cortical cells
then proliferate to form the primordium of the symbiotic nitrogenfixing root nodule (Yang et al., 1994; Geurts and Bisseling, 2002;
Oldroyd et al., 2011). Recent identification and functional analyses of
the putative cytokinin receptors Lotus japonicus LOTUS HISTIDINE
KINASE 1 (LHK1) and Medicago truncatula CYTOKININ
RESPONSE 1 have led to a greater understanding of how the
1
2
National Institute for Basic Biology, Okazaki 444-8585, Japan. School of Life
Science, The Graduate University for Advanced Studies, Okazaki 444-8585,
3
Japan. Graduate School of Life Sciences, Tohoku University, Sendai 980-8577,
4
Japan. Kazusa DNA Research Institute, Kisarazu 292-0812, Japan.
*Author for correspondence ([email protected])
Received 14 January 2014; Accepted 22 April 2014
activation of cytokinin signaling is crucial to the initiation of nodule
organogenesis (Gonzalez-Rizzo et al., 2006; Murray et al., 2007;
Tirichine et al., 2007). In particular, it has been shown that in the
L. japonicus spontaneous nodule formation 2 (snf2) mutant, which
possesses a gain-of-function form of LHK1, confers the constitutive
activation of cytokinin signaling, resulting in the formation of
spontaneous nodule-like structures in the absence of rhizobia
(Tirichine et al., 2007).
Normal nodulation is achieved by interactive processes involving
infection by rhizobia and nodule organogenesis (Madsen et al.,
2010); the complexity of these interactions has made it difficult to
study the regulation of these mechanisms separately. The use of
spontaneous nodules, however, appears to be an efficient approach
for focusing on nodule organogenesis because the effect of the
rhizobial infection process can be excluded from spontaneous
nodule development (Tirichine et al., 2006). Spontaneous nodule
formation is suppressed by the mutation of any of the three putative
transcription factors NODULE INCEPTION, NODULATION
SIGNALING PATHWAY 1 (NSP1) and NSP2; these factors are
involved in nodule organogenesis (Tirichine et al., 2007). But our
understanding of the nodule initiation process is still limited because
of the limited number of genes that have been identified.
RESULTS AND DISCUSSION
vag1 suppresses snf2-dependent spontaneous nodule
formation
To investigate the genetic mechanism involved in the onset of nodule
organogenesis, we performed a screen for genetic suppressors of the
snf2 spontaneous nodulation phenotype. A recessive semi-dwarf
mutant named vagrant infection thread 1 (vag1) was identified, and
the number of spontaneous nodules reduced in the vag1 snf2 double
mutant compared with the snf2 single mutant (Fig. 1A). The number
of spontaneous nodules of snf2 increased in the presence of the
hypernodulating hypernodulation aberrant root formation 1 (har1)
mutation as previously shown (Tirichine et al., 2007), but the
enhanced spontaneous nodulation was strikingly suppressed by
the vag1 mutation (Fig. 1A). In the vag1 single mutant, the
initiation of nodule organogenesis was severely attenuated after
inoculation of its rhizobial symbiont Mesorhizobium loti (Fig. 1B,C),
without substantially altering overall root architecture (supplementary
material Fig. S1A,B). The vag1 mutant appeared to respond normally
to cytokinin, as shoot and root growth of the mutant, as well as of the
wild type (WT), were inhibited in the presence of high cytokinin
concentrations (supplementary material Fig. S2), suggesting that the
vag1 mutant acts as a suppressor of snf2 without affecting cytokinin
signaling in shoot and root growth.
During nodule development, rhizobia invade host cortical cells
via a specialized structure called the infection thread (IT), which
originates in a root hair cell (Murray, 2011). In WT, ITs penetrated
the root hair and ramified within inner cortical cells (Fig. 1D;
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DEVELOPMENT
ABSTRACT
RESEARCH REPORT
Development (2014) 141, 2441-2445 doi:10.1242/dev.107946
supplementary material Fig. S3). By contrast, ITs that formed in
the vag1 mutant appeared to penetrate root hair cells normally but
failed to ramify and reach cortical cells (Fig. 1E,F; supplementary
material Fig. S3), suggesting that the mutant infection threads are
blocked at the epidermal-cortical interface. Normal nodule
development is achieved by interactive processes involving
nodule organogenesis and IT formation (Madsen et al., 2010).
To examine the two processes separately, we focused on the
cyclops mutant, in which, despite a premature arrest of IT
elongation in root hairs, the initiation of nodule organogenesis is
nonetheless induced (Yano et al., 2008). In the vag1 cyclops-6
double mutant, although ITs showed the cyclops-type premature
arrest phenotype, the initiation of nodule organogenesis was
significantly suppressed (supplementary material Fig. S4),
suggesting that the impairment of nodule initiation in the vag1
mutant is due to a defect in the nodule organogenesis process
rather than its aberrant IT progression.
VAG1 encodes a putative component of plant DNA
topoisomerase VI
Map-based cloning identified a gene that is responsible for the vag1
mutation (supplementary material Fig. S5). The vag1 mutation carries
a G-to-A nucleotide substitution in the splice donor site of intron 9 in
the gene, causing intron mis-splicing (Fig. 2A-C). The mutant
nodulation phenotype of vag1 was rescued when a genomic fragment
containing the wild-type gene was introduced into the mutant by hairy
root transformation (Fig. 2D-F). Moreover, we isolated another
mutant carrying a mutation in the VAG1 gene (hereafter we denote the
original and the new vag1 mutant vag1-1 and vag1-2, respectively). In
vag1-2, there is one base deletion in exon 1 producing the emergence
of a considerably premature stop codon (Fig. 2A,B). Nodule initiation
was completely compromised in the vag1-2 mutant (Fig. 1B). The
expression of VAG1 seemed to be unaffected by M. loti infection
(supplementary material Fig. S6A). To determine the spatial
expression pattern of VAG1, we first identified a functional VAG1
promoter that could rescue the vag1 mutation when VAG1-coding
sequence was expressed under the control of the promoter (Fig. 2F).
Reporter gene analysis using the promoter ( pVAG1::GFP-NLS)
showed that VAG1 was expressed throughout the root, including
proliferating cortical cells, to form nodules (supplementary material
Fig. S6B-E).
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VAG1 encodes a protein that is putatively orthologous to
Arabidopsis ROOT HAIRLESS 1 (RHL1)/HYPOCOTYL 7
(HYP7) (Schneider et al., 1998; Sugimoto-Shirasu et al., 2005), a
component of the plant DNA topoisomerase VI. In Arabidopsis,
loss-of-function mutants of factors constituting topoisomerase VI
have general defects in ploidy-dependent cell growth, and the
number of highly endoreduplicated cells is reduced in the mutants
(Hartung et al., 2002; Sugimoto-Shirasu et al., 2002, 2005; Yin
et al., 2002), indicating that topoisomerase VI has a function as a
positive regulator of endoreduplication. Although the rhl1 mutation
prevents the formation of root hairs, they were formed normally in
the vag1 mutants (supplementary material Fig. S1C,D). RHL1
could rescue the vag1 mutation when it was expressed under the
control of the VAG1 promoter (Fig. 2F), suggesting that the VAG1
protein has a function similar to RHL1.
As represented by trichomes of the leaf epidermis in Arabidopsis
and by endosperm of the embryo in maize, it is believed that
endoreduplication contributes to increasing plant cell sizes, in
particular to producing terminally differentiated cells (Lee et al.,
2009). In legume-Rhizobium symbiosis, rhizobial-infected cells
of nodules are terminally differentiated as a consequence of
endoreduplication (Kondorosi and Kondorosi, 2004). To assess the
involvement of VAG1 in controlling endoreduplication, we next
investigated the nodule phenotype of vag1. The vag1-1 mutant could
form nodules, although their numbers were small (Fig. 1B). The size of
the nodules formed in the mutant was largely indistinguishable from
those of the WT (Fig. 3A,B). An observation of vag1-1 mutant nodule
sections showed that the sizes of cells located in the inner region of
nodules were smaller and the numbers of potential rhizobia-colonized
infected cells were reduced compared with the WT (Fig. 3C-F).
In addition, the inner region of vag1-1 nodules comprised large
numbers of small rhizobia-infected (as yet uncolonized) cells that
resembled those located at surrounding regions of rhizobia-colonized
infected cells in WT (supplementary material Fig. S7). In wild-type
nodules, flow cytometry revealed that few diploid cells were
detected, and cells underwent up to four rounds of endoreduplication
to reach 32C (Fig. 3G,I). By contrast, vag1-1 nodules comprised
increased proportions of diploid cells, whereas the proportions of
endoreduplicated cells (>4C) were significantly reduced (Fig. 3H,I).
The ploidy level of uninoculated vag1 roots was indistinguishable from
that of WT (supplementary material Fig. S1E-G).
DEVELOPMENT
Fig. 1. The effect of the vag1 mutation on
nodulation. (A) The number of spontaneous
nodules present 35 days after germination in the
absence of rhizobia (n=12-22) (*P<0.001). (B) The
number of nodules present 21 days after inoculation
(dai) of rhizobia (n=14). (C) Shoot and root
phenotype of WT (left) and vag1-1 (right) at 21 dai.
Arrowheads indicate nodules. (D-F) IT phenotype of
WT (D) and vag1-1 (E,F) at 7 dai with M. loti
MAFF303099, which constitutively expresses a
DsRED reporter gene. Red areas (DsRED
fluorescence) indicate the presence of rhizobia. ITs
were observed from side (D,F) and front (E) views of
infected roots. Error bars indicate s.e.m. Scale bars:
1 cm in C; 100 μm in D-F.
Fig. 2. Structure of the VAG1 gene. (A) Exon-intron structure of the VAG1
gene. Boxes indicate exons. Arrowheads indicate locations of the vag1
mutations. Arrows indicate locations of primer sets used for RT-PCR analysis
in C. (B) Sites of mutations in the vag1 mutants. (C) RT-PCR analysis of the
VAG1 gene. UBIQUITIN (UBQ) was used as the RNA loading control. cDNAs
were prepared from total RNAs from whorl roots. (D,E) Complementation of
the vag1-1 nodulation phenotype. Representative transgenic hairy roots of
L. japonicus carrying a control empty vector (D) and a 10.9 kb genomic
fragment encompassing the entire VAG1 locus (E). Transgenic roots were
identified by the expression of GFP-LjLTI6b. (F) Average nodule numbers in
the vag1-1 mutants with transgenic roots containing respective constructs.
Nodulation phenotypes were evaluated at 14 dai (n=7-10). Error bars
indicate s.e.m.
VAG1-mediated endoreduplication is crucial to the initiation
of nodule organogenesis
An observation of the attenuation of nodule initiation in the vag1
mutant and VAG1 implication in endoreduplication of infected
cells within nodules led us to postulate that VAG1-mediated
endoreduplication is required for the onset of nodule organogenesis.
We previously produced transgenic L. japonicus plants, in which
nuclear localized GFP is expressed under the control of a synthetic
auxin-responsive element, DR5 (Suzaki et al., 2012). Use of these
plants enabled us to monitor the nuclei of cortical cells relevant to the
nodulation cell lineage. As a result, we observed that nuclei of
the outermost cortical cells immediately before initiation of division
were larger than those of the surrounding cortical cells in WT (Fig. 4A).
The enlarged nuclei were continuously observed after the initiation of
cortical cell proliferation (Fig. 4B,C). In addition, ITs appeared to
elongate towards the cortical cells with the enlarged nuclei (Fig. 4A-C).
By contrast, in the vag1-1 mutants there were no enlarged nuclei in
cortical cells beneath root hairs with ITs and no cortical cell division
Development (2014) 141, 2441-2445 doi:10.1242/dev.107946
Fig. 3. The effect of the vag1 mutation on nodule structure. (A,B) Nodule
phenotypes of WT (A) and the vag1-1 mutant (B) at 21 dai. (C-F) Sections
through nodules of WT (C,E) and vag1-1 (D,F) at 21 dai with rhizobia that
constitutively express the lacZ reporter gene. (E,F) Rhizobia-colonized infected
cells located at the inner region of nodules. X-Gal-stained blue areas indicate
the presence of rhizobia. (G-I) Flow cytometry of wild-type (G) and vag1-1 (H)
nodules at 21 dai. Average distributions of nuclei (I). For each measurement, at
least 10,000 cells derived from five nodules were analyzed. Error bars indicate
s.e.m. Scale bars: 1 mm in A,B; 100 μm in C,D; 20 μm in E,F.
was induced (Fig. 4D,E), despite the induction of auxin response
(supplementary material Fig. S8). As DNA size is reflected in nuclear
size (Bourdon et al., 2011; Iwata et al., 2011), we next estimated
the DNA size of the enlarged nuclei by quantifying the size of
DAPI-labeled nuclei. In WT, the size of the enlarged nuclei was more
than 8-fold larger compared with those of root cap cells (2C control)
(supplementary material Fig. S9), suggesting that their DNA size
reaches up to at least 16C. In the vag1 mutants, the size of nuclei in
cortical cells beneath root hairs with IT was largely comparable to that
of cortical cells of uninoculated roots (supplementary material Fig. S9).
In plants, CCS52A isoforms are necessary and sufficient for the
progress of endocycling (Cebolla et al., 1999; Larson-Rabin et al.,
2009; Mathieu-Rivet et al., 2010; Baloban et al., 2013), which skips the
mitotic phase and re-enters the S phase without cytokinesis, resulting in
the duplication of the nuclear DNA content. In M. truncatula,
MtCCS52A is shown to be expressed before endoreduplication and to
regulate endoreduplication of infected cells within nodules (Vinardell
et al., 2003). We isolated two L. japonicus CCS52A1 genes
(LjCCS52A1-LIKE1 and LjCCS52A1-LIKE2) with high similarity to
Arabidopsis CCS52A1. Promoter-GUS reporter analysis and in situ
hybridization revealed that the two genes were expressed within
nodules (supplementary material Fig. S10). The genes were also
expressed in dividing cortical cells during early nodule development
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Development (2014) 141, 2441-2445 doi:10.1242/dev.107946
Fig. 4. The effect of the vag1 mutation on early nodule
development. (A-E) Initiation of nodule organogenesis in DR5::
GFP-NLS/WT (A-C) and DR5::GFP-NLS/vag1-1 (D,E) transgenic
plants. Roots were observed at 3 (A-E, except C) and 5 (C) dai
with rhizobia that constitutively express DsRED. Auxin response
patterns during nodulation were visualized by GFP fluorescence
(green). Nuclei were stained with DAPI (blue). Arrowheads
indicate enlarged nuclei. For fluorescence analysis, at least 20
plants were analyzed at each developmental stage in three
independent experiments. (F) X-Gluc staining of a root region
containing dividing cortical cells in wild-type transgenic hairy roots
containing the pLjCCS52A1-LIKE2::GUS construct at 5 dai.
(G-I) In situ localization of LjCCS52A1-LIKE1 transcripts in
wild-type root regions containing dividing cortical cells at
3 dai (G,I) and 5 dai (H). Antisense (G,H) or sense (I) probes were
used for the detection of LjCCS52A1-LIKE1 transcripts.
Scale bars: 100 μm.
Conclusion and perspectives
In this study, we have shown that topoisomerase VI complex
containing VAG1 has an essential role in regulating the onset of
nodule development. Based on the results, we propose a hypothesis in
which VAG1-mediated endoreduplication of cortical cells may be
required for the initiation of nodule organogenesis (supplementary
material Fig. S11). The primary defect of vag1 could be
endoreduplication preceding cortical cell division, which could lead
to an attenuation of the phase of the cortical cell proliferation. Thus, we
cannot examine the direct effect of the vag1 mutation on the phase of
the cell cycle related to the cortical cell proliferation, or entirely rule
out the possibility that a more general defect in the cell cycle could be
responsible for the vag1 nodulation phenotype. During the nodule
initiation process, the cortical cell sizes with enlarged nuclei are almost
identical to adjacent cortical cells. This observation implies that the
endoreduplication that occurs in this developmental context is
unrelated to cell growth process, although its biological significance
remains elusive. Given that change of ploidy level can be associated
with regulation of gene expression involving epigenetic gene
silencing, as previously shown (Comai et al., 2000; Wang et al.,
2004; Yu et al., 2010), it is possible that the endoreduplication of
cortical cells may be involved in silencing of genes that determine the
state of differentiated cortical cells. This cellular-level epigenetic
control may trigger the activation of sets of genes relevant to the
nodule development. In order to identify a key molecular mechanism
involved in the cell fate conversion, the elucidation of the detailed role
of endoreduplication of cortical cells is required.
In addition, we have shown that ITs penetrate host cells in the
direction of the endoreduplicated cortical cells. Loss of such
endoreduplicated cells in the vag1 mutants causes a misguided
elongation of ITs. As the phenotype of misguided infection is
reminiscent of hyperinfected 1, a loss-of-function mutant of LHK1
(Murray et al., 2007), the onset of endoreduplication may occur
downstream of the cytokinin response. Overall, during root nodule
symbiosis, the endoreduplication of cortical cells may have another
important role of acting as a determinant to guide rhizobia to host
cells (supplementary material Fig. S11).
2444
In Arabidopsis, topoisomerase VI is generally involved in diverse
cell growth processes that are accompanied by increased DNA
levels. It is known that the pattern of ploidy-dependent cell growth is
different among plant species; this may account for the observations
that non-symbiotic shoot and root development is largely normal in
the vag1 mutants. Currently, the detailed molecular mechanism that
links topoisomerase VI to endoreduplication remains unknown. We
show that the expression patterns of VAG1 and LjCCS52A1-LIKE
genes overlap during nodule development. Further investigation of
the potential interactions among the genes will be needed to
elucidate how topoisomerase VI achieves progress of the endocycle.
MATERIALS AND METHODS
Plant materials and growth conditions
The Miyakojima MG-20 ecotype of L. japonicus was used as WT in this study.
The vag1-1 mutant was isolated from the M2 generation of snf2 plants that had
been mutagenized with ethylmethane sulfonate (EMS) (Miyazawa et al., 2010;
Suzaki et al., 2013). The vag1-2 mutants were isolated by a screening of
nodulation-deficient mutants using EMS-treated MG-20 plants, which were
provided by Legume Base. DR5::GFP-NLS/vag1-1 plants were produced by
crossing DR5::GFP-NLS transgenic plants (Suzaki et al., 2012) with vag1-1
plants. For the analyses of rhizobial-induced nodulation or spontaneous
nodulation, plants were grown with or without M. loti MAFF 303099,
respectively, as previously described (Suzaki et al., 2013).
Constructs and transformation of L. japonicus
The primers used for PCR are listed in supplementary material Table S1. For the
complementation analysis, a 10.9 kb genomic DNA fragment including the
VAG1 candidate gene was amplified by PCR from wild-type genomic DNA.
This fragment included 3.0 kb of sequence directly upstream of the initiation
codon, and was cloned into pCAMBIA1300-GFP-LjLTI6b (Suzaki et al.,
2012). For the VAG1 expression analysis, a gateway-cassette (GW) fragment
was cloned into pCAMBIA1300-GFP-LjLTI6b to create the new binary vector
pCAMBIA1300-GW-GFP-LjLTI6b. Next, 3.0 kb of sequence directly
upstream of the initiation codon of VAG1 was amplified by PCR and cloned
into pCAMBIA1300-GW-GFP-LjLTI6b to create the vector pCAMBIA1300pVAG1-GW-GFP-LjLTI6b. VAG1- or RHL1-coding sequence (cds) was
amplified by PCR from a template cDNA prepared from wild-type L. japonicus
or Arabidopsis Col-0 plants, respectively, and cloned into a pENTR/D-TOPO
vector (Invitrogen). The VAG1 and RHL1 cds in pENTR/D-TOPO and
GFP-NLS in pDONR221 (Suzaki et al., 2012) were inserted downstream
of the VAG1 promoter by LR recombination reactions. To create the
DEVELOPMENT
(Fig. 4F-I), indicating that the progress of endocycling could occur
during the nodule-initiation process.
pLjCCS52A1-LIKE2::GUS construct, 2.8 kb of sequence directly upstream of
the initiation codon of LjCCS52A1-LIKE2 was amplified by PCR and cloned
into pCAMBIA1300-GW-GFP-LjLTI6b to create the vector pCAMBIA1300pLjCCS52A1-LIKE2-GW-GFP-LjLTI6b. The β-glucuronidase (GUS) gene
in pENTR-gus (Invitrogen) was inserted downstream of the LjCCS52A1LIKE2 promoter by the LR recombination reaction. The recombinant plasmids
were introduced into Agrobacterium rhizogenes strain AR1193 and were
transformed into roots of L. japonicus plants by a hairy-root transformation
method previously described (Okamoto et al., 2009).
Flow cytometry
Nodules were removed from wild-type or vag1-1 roots, cut into pieces with
razor blades in extraction buffer (Partec) and incubated for 2 min at room
temperature. The suspension was filtered through a 30 µm Celltrix filter (Partec)
and stained with staining buffer (Partec). Flow cytometry was performed using
a Ploidy Analyzer PA (Partec). Data are shown as mean±s.e.m. of three
biological replicates.
Accession numbers
Sequence data from this report can be found in the GenBank/EMBL
data libraries under the following accession numbers: VAG1, AB871650;
RHL1, At1g48380; LjCCS52A1-LIKE1, AB871651; LjCCS52A1LIKE2, AB871652; CCS52A1, At4g22910.
Acknowledgements
We thank Sachiko Tanaka, Michiko Ichikawa, Yuko Ogawa and Kiyoshi Tatematsu
for technical support; and Makoto Hayashi for providing M. loti MAFF303099
expressing DsRED. This work was supported by NIBB Core Research Facilities and
NIBB Model Plant Research Facility.
Competing interests
The authors declare no competing financial interests.
Author contributions
T.S. and M.I. designed research and analyzed data; T.S., M.I., E.Y., S.S., H.H. and
N.T. performed research; T.S., M.I. and M.K. wrote the paper.
Funding
This work was supported by the Ministry of Education, Culture, Sports, Science and
Technology Grants-in-Aid for Scientific Research on Innovative Area [25114519 to
T.S. and 22128006 to M.K.].
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.107946/-/DC1
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