RESEARCH ARTICLE 1531
Development 138, 1531-1539 (2011) doi:10.1242/dev.058495
© 2011. Published by The Company of Biologists Ltd
Strigolactones regulate protonema branching and act as a
quorum sensing-like signal in the moss Physcomitrella patens
Hélène Proust1, Beate Hoffmann1, Xiaonan Xie2, Kaori Yoneyama2, Didier G. Schaefer3, Koichi Yoneyama2,
Fabien Nogué1 and Catherine Rameau1,*
SUMMARY
Strigolactones are a novel class of plant hormones controlling shoot branching in seed plants. They also signal host root proximity
during symbiotic and parasitic interactions. To gain a better understanding of the origin of strigolactone functions, we
characterised a moss mutant strongly affected in strigolactone biosynthesis following deletion of the CAROTENOID CLEAVAGE
DIOXYGENASE 8 (CCD8) gene. Here, we show that wild-type Physcomitrella patens produces and releases strigolactones into the
medium where they control branching of protonemal filaments and colony extension. We further show that Ppccd8 mutant
colonies fail to sense the proximity of neighbouring colonies, which in wild-type plants causes the arrest of colony extension. The
mutant phenotype is rescued when grown in the proximity of wild-type colonies, by exogenous supply of synthetic strigolactones
or by ectopic expression of seed plant CCD8. Thus, our data demonstrate for the first time that Bryophytes (P. patens) produce
strigolactones that act as signalling factors controlling developmental and potentially ecophysiological processes. We propose
that in P. patens, strigolactones are reminiscent of quorum-sensing molecules used by bacteria to communicate with one another.
INTRODUCTION
When land was colonised, plant morphology significantly
diversified (Kenrick and Crane, 1997b; Langdale, 2008). The
underlying developmental mechanisms of this diversification
during evolution are still poorly understood. Shoot branching is one
of the key innovations that led to more complex architecture.
Recently, strigolactones were shown to represent a novel class of
plant hormones controlling this process (Gomez-Roldan et al.,
2008; Umehara et al., 2008). The same molecules act in the
rhizosphere as signalling molecules in establishing the symbiotic
relationship between arbuscular mycorrhizal fungi and over 80%
of extant land plants (Akiyama et al., 2005; Besserer et al., 2006)
and in stimulating seed germination of root parasitic weeds
(Bouwmeester et al., 2007). Arbuscular mycorrhizal symbiosis
evolved for 460 million years before the appearance of roots and
is coincident with the appearance of the first bryophyte-like land
plants (Remy et al., 1994). In seed plants, strigolactone-deficient
mutants in pea (Fig. 1A), Arabidopsis thaliana and rice show an
increased branching phenotype (Gomez-Roldan et al., 2008;
Umehara et al., 2008). To gain a better understanding of the origin
of branching in seed plants and to explore strigolactone functions
in early land plants, we investigated whether strigolactones are
produced in the moss Physcomitrella patens and what roles they
play in a non vascular plant.
Strigolactones are apocarotenoids (Matusova et al., 2005) and
the first step in their biosynthesis is catalysed by CAROTENOID
CLEAVAGE DIOXYGENASES (CCD). The CCDs are an ancient
family of enzymes with members identified in bacteria, plants and
animals (Auldridge et al., 2006). In Arabidopsis and pea, the CCD7
and CCD8 enzymes are encoded by the branching genes
MAX3/RMS5 (Booker et al., 2004; Johnson et al., 2006) and
MAX4/RMS1 (Sorefan et al., 2003), respectively, and are both
involved in strigolactone biosynthesis (Gomez-Roldan et al., 2008;
Umehara et al., 2008).
The model moss Physcomitrella patens provides unique genetic
and developmental facilities to undertake evolutionary studies in
plant biology (Cove et al., 2006; Lang et al., 2008; Schaefer, 2002).
Its life cycle is dominated by the haploid gametophyte that displays
a succession of simple and well described developmental
transitions that leads from the germinating spore to the colony of
differentiated leafy shoots carrying mature diploid sporophytes
(Cove et al., 2006).
We generated a mutant in the moss homolog of the strigolactone
biosynthesis gene CCD8. Here, we demonstrate that in P. patens,
PpCCD8 is involved in strigolactone biosynthesis and regulates the
branching of filament and colony extension. Furthermore, our data
demonstrate that in wild-type (WT) P. patens, secreted
strigolactones are directly involved in the regulation of colony
extension in response to internal cues or population density. This
novel function of strigolactones in plants is reminiscent of quorumsensing signalling processes that are well described in bacteria
(Fuqua and Greenberg, 2002; Ng and Bassler, 2009), and we
propose that moss uses strigolactones as quorum-sensing molecules
to regulate colonial growth in distinct environmental conditions.
MATERIALS AND METHODS
1
Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech, Centre de VersaillesGrignon, 78026 Versailles Cedex, France. 2Weed Science Center, Utsunomiya
University, Utsunomiya 321-8505, Japan. 3Institute of Biology, Laboratory of Cell and
Molecular Biology, University of Neuchatel, CH-2009 Neuchatel, Switzerland.
*Author for correspondence ([email protected])
Accepted 28 January 2011
P. patens growth conditions and treatments
The Gransden wild-type strain was used and conditions for cultures were
as described (Ashton and Cove, 1977; Ashton et al., 1979; Trouiller et al.,
2006). For phenotypic analysis, spores were grown on minimal PP-NO3
medium (Ashton et al., 1979). Sporogenesis was performed in Magenta
vessels in which 15-day-old wild-type and mutant colonies were grown for
2 months, then irrigated with sterile water and transferred to growth
DEVELOPMENT
KEY WORDS: Physcomitrella, Strigolactones, CCD8
1532 RESEARCH ARTICLE
Phylogenetic analysis
Evolutionary relationships between CCD proteins were analysed using a
selection of proteins from different databases aligned with the program
CLUSTALX (Thompson et al., 1997). The evolutionary history was
inferred using the Neighbour-Joining method (Saitou and Nei, 1987) and
the optimal tree was generated. Phylogenetic analyses were conducted in
MEGA4 (Tamura et al., 2007).
Generation of Ppccd8, Ppccd8::OsACTpro:RMS1 and
PpCCD8pro:GUS strains
Moss protoplasts were transformed and transgenic strains were selected as
described previously (Trouiller et al., 2006). Transformants were selected
on G418 (50 g/ml) (Duchefa GO 175, Haarlem, The Netherlands). Stable
disruptants of the PpCCD8 gene were confirmed by PCR using primers
specific for the PpCCD8 sequence outside of the transformation construct.
A clean deletion in the PpCCD8 gene was obtained by transient expression
of the Cre recombinase as described previously (Trouiller et al., 2006). The
recombinant locus was analysed by PCR with the same primers (see Fig.
S1 in the supplementary material). Several independent deletion strains
with similar phenotype were obtained and one of them, Ppccd8, was used
for subsequent analyses.
OsACTpro:RMS1 and PpCCD8pro:GUS were linearised with SacII
before transformation, and transformants were selected on hygromycin (25
g/ml). To confirm the presence of constructs in stable transformants, PCR
amplification with specific primers designed on sequences inside and outside
the construct was performed (see Fig. S2B-F in the supplementary material).
Quantification of strigolactones released by wild type and
Ppccd8⌬ mutant
A 2855 bp PCR amplified PpCCD8 genomic fragment (scaffold 14:
2616535-2619389) was cloned into vector pCRII Blunt TOPO
(Invitrogen). To disrupt the PpCCD8 gene, a 664 bp 3⬘ targeting fragment
(scaffold 14: 2616135-2616798) was first recovered by enzyme digestion
with XmnI and BamHI, and inserted into BamHI- and SmaI-digested
pBNRF, which carries a loxP-neoR-loxP selectable marker (Schaefer et al.,
2010). The 925 bp 5⬘ targeting fragment (scaffold 14: 2618287-2619211),
obtained by digestion with AcyI and BseJI, was then inserted between the
ClaI/HpaI sites of vector pBNRF carrying the 3⬘ targeting fragment to
generate pPpccd8-KO (see Fig. S1 in the supplementary material).
Approximately 1 g of 7-day-old protonemata from the wild type or
Ppccd8⌬ mutant was used to inoculate 150 ml of BCD medium in a 300
ml flask on a rotary shaker. Cultures were grown under standard light
conditions for 30 days and the growth medium was replaced with fresh
media weekly. Thirty-day-old tissue was transferred to fresh media and
grown for an additional 3 days. Plant material was collected by filtration
and weighed, while the liquid culture medium was collected and extracted
three times with an equal volume of ethyl acetate. The combined ethyl
acetate solutions were washed with 0.2 M K2HPO4 for removal of acidic
compounds, dried over anhydrous MgSO4, filtered and concentrated in
vacuo. These crude extracts were stored in sealed glass vials at 4°C until
use. Strigolactones released to liquid culture media were determined on
LC-MS/MS as described previously (Yoneyama et al., 2008). For
quantitative analysis using LC-MS/MS, orobanchol-d1, orobanchyl acetated1 and [13C2]strigol were used as internal standards. Levels of other
strigolactones were determined as reported previously (Yoneyama et al.,
2008) and their recovery rates were estimated based on that of orobanchyl
acetate-d1. The experiments were conducted with three replicate samples.
OsACTpro-108-HygR
GUS staining of Physcomitrella patens
Primers
The primers used in this study are described in Table S1 in the
supplementary material.
Molecular cloning
Ppccd8-KO construct
The CaMVter terminator fragment was excised from pDH51 (Pietrzak et
al., 1986) using XbaI/HindIII (HindIII site was blunted) and ligated to
pCOR 104 (McElroy et al., 1991) digested by XbaI/SacII (SacII was
blunted) to generate pCOR104-CaMVter. A 1800 pb fragment carrying the
rice ACTIN-1 gene promoter (GenBank: S44221) and the CaMVter was
obtained by digestion of pCOR104-CaMVter vector with XhoI and PvuII
enzymes. This fragment was cloned into SwaI and SalI sites of BS-108
vector which contains a genomic P. patens fragment corresponding to locus
108 (Schaefer and Zryd, 1997) giving the OsACTpro-108 vector. The
35S::hygroR cassette (Hajdukiewicz et al., 1994) was excised of pBKShygro vector with HindIII and EcoRI enzymes, and integrated in the
HindIII site of p-Act-108 vector to generate OsACTpro-108-HygR (see Fig.
S2A in the supplementary material).
OsACTpro:RMS1 construct
The pea RMS1 cDNA (GenBank: AY557341) obtained by PCR was
subcloned in pTOPO vector and inserted in NcoI/SphI sites of OsACTpro108-H (see Fig. S2A,B in the supplementary material).
PpCCD8pro:GUS construct
This construct was made in the OsACTpro-108-H vector. A 1616 bp
amplified fragment of the PpCCD8 promoter (scaffold 14: 26191862620801) was cloned behind the GUS:Noster (Fourgoux-Nicol et al., 1999)
cassette of the pAF1 vector digested with SacII/HindIII. A fragment
containing PpCCD8pro:GUS:Noster was excised using SacII and NcoI,
blunted and used to replace the deleted rice actin promoter CaMVter
cassette of the OsACTpro-108-H vector with KpnI sites (see Fig. S2E in
the supplementary material).
Colonies developed from ground tissues were incubated for 12 hours at
37°C in 0.8 mM 5-bromo-4-chloro-3-indolyl--D-glucuronide, 2 mM
potassium ferrocyanide, 2 mM potassium ferricyanide and 100 mM
phosphate buffer (pH 7).
Gene expression analysis
Total RNA was extracted using the RNeasy plant mini kit (Qiagen. Ref
74904). cDNA was synthesised from 2 g of total RNA with RevertAid H
Minus M-MuLV Reverse Transcriptase (Fermentas) and dT(18) primer.
Quantitative PCR was performed in a Mastercycle ep realplex (Eppendorf)
with Realmaster mix SYBR ROX 2.5⫻ (5 PRIME). PpAPT, which encodes
an ADENOSINE PHOSPHORIBOSYL TRANSFERASE, was used as the
constitutive gene (Trouiller et al., 2006). Relative expression of PpCCD was
established with the formula 2–Ct(PpCCD)–Ct(PpAPT). Three biological
replicates were carried out with two technical replicates for each sample.
RESULTS
Characterisation of P. patens strigolactone
biosynthesis genes
Unique CCD7-like and CCD8-like sequences were found in the
genome of P. patens that were designated PpCCD7 (GenBank
Accession Number HM007803) and PpCCD8 (GenBank
Accession Number HM007802). The predicted moss proteins
share 45% and 57% amino acid identity with CCD7 and
CCD8 from Arabidopsis, respectively. Phylogenetic analyses
demonstrated that they grouped with their respective AtCCD
DEVELOPMENT
chambers at 15°C with 8 hours of light per day and a quantum irradiance
of 15 E m–2 s–1. Maturation of spore capsules was followed visually. The
differentiation of caulonemata and buds was followed using a binocular
(Nikon SMZ1000). For a precise estimation of caulonema differentiation,
colonies of different ages were transferred in darkness on minimal medium
supplemented with 5 g/l glucose for 11 days. Because of negative
gravitropism, Petri dishes were kept upside-down in the dark for an easy
observation and counting of caulonemal filaments. In the cross-feeding
experiment, the central colony was transferred to the medium 10 days after
the four surrounding colonies. In the density experiment, to eliminate a
putative starvation effect, the experiment was carried out on large Petri
dishes (12 cm) and glucose (0.5%) was added in another similar
experiment (not shown). Cell measurements were performed with ImageJ
software (http://rsb.info.nih.gov/ij/).
Development 138 (8)
Strigolactones function in moss
RESEARCH ARTICLE 1533
clades (see Fig. S3 in the supplementary material). Two other
genes involved in strigolactone biosynthesis have been identified
by genetic analyses: MAX1 in Arabidopsis, which encodes a
member of the CYP450 family; CYP711A1 (Booker et al.,
2005); and the rice D27 gene, which encodes an iron-containing
protein (Lin et al., 2009). No MAX1 homologue was identified
in the P. patens genome, whereas several homologues for D27
were found, one (XP001767010) encoding a protein with 47%
of identity with D27 from rice.
To characterise the function of the CCD8 gene in P. patens, we
generated a Ppccd8 mutant in which genomic sequences
encoding part of exon V, exons VI and VII were deleted. RT-PCR
analyses confirmed the absence of a full-length PpCCD8 transcript
in the mutant, showing that the Ppccd8 strain is a null allele (see
Fig. S1 in the supplementary material). The Ppccd8 strain was
fertile and we used its selfed progeny to analyse single sporederived colonies in all the experiments described below. Each
colony should, thus, be considered as an individual sibling and not
as a clonal replicate.
Ppccd8 strains display several developmental
phenotypes
Following spore germination, P. patens colonies develop into
photosynthetic primary chloronemal filaments that further
differentiate into caulonemal filaments implicated in rapid radial
extension of the colony (Fig. 1B). Most caulonemal subapical cells
further form secondary filaments and some buds (Fig. 1B) that will
develop into leafy gametophores. The developmental fate of these
caulonemal side branch initials is rather constant in P. patens, with
most of them developing into secondary chloronemata and only a
few percentage into new caulonemata or buds (Cove et al., 2006).
Under our growth conditions, spore germination occurred
significantly earlier in Ppccd8 spores than in the wild type (see
Fig. S4A in the supplementary material) and was associated with a
higher frequency of multipolar germination patterns (Fig. 1C,D).
Branching of primary chloronemata was also higher in the mutant
during this early developmental stage. The percentage of lateral
cells was higher in the mutant (37.2%) than in the wild type
(10.8%) 93 hours after spore transfer on the medium. This increase
in lateral cell number is not due to early germination of the
Ppccd8 mutant as the percentage was still higher for the mutant
at 81 hours (25.6%) (see Fig. S4B in the supplementary material).
The first caulonemal cells differentiated ~7 days after spore
germination, followed by the formation of the first buds at day 15;
timing of chloronemata to caulonemata transition was similar in
both wild type and Ppccd8 mutant with a higher number of
caulonemata in the mutant very likely to be due to higher branching
of chloronemal cells (see Fig. S4C in the supplementary material).
Fifteen days after spore germination, branching (i.e. production of
secondary filaments or new buds) of caulonemata protruding from
DEVELOPMENT
Fig. 1. CCD8 controls shoot branching in pea and the synthesis of a diffusible signal regulating caulonema branching and colony
extension in P. patens. (A)Wild-type (left) and rms1 (Psccd8) (right) pea plants. (B)Caulonemata (ca), secondary chloronemata (ch) and bud (bud)
from a 18-day-old wild-type colony. (C,D)Development of wild-type (C) and Ppccd8⌬ (D) colonies, 56 to 106 hours after spore transfer to the medium.
(E-H)Wild-type (E,F) and Ppccd8⌬ (G,H) colony, periphery of 25-day-old colonies (E,G), 40-day-old colonies (F,H). (I-L)Cross-feeding experiment with a
30-day-old central colony surrounded by four 40-day-old colonies. Scale bars: 200m in B; 100m in C,D; 500m in E,G; 1 cm in F,H-L.
the colonies was similar in both genotypes (see Fig. S4F in the
supplementary material). Until day 21, diameter of mutant colonies
(measured from one caulonema extremity to the other on the
opposite side of the colony) are comparable with the diameter of
wild-type colonies (see Fig. S4G in the supplementary material).
Moreover, size of chloronemal cells for Ppccd8 is not
significantly different from wild type and length of caulonemal
cells is slightly higher in Ppccd8 compared with wild type (see
Fig. S4D,E in the supplementary material). Taken together, these
results suggested that rate of apical cell division was not increased
in the Ppccd8 mutant and that the higher branching observed in
the mutant was not linked with a general higher cell division rate.
A sudden and significant difference appeared 20 days after spore
germination: the diameter of wild-type colonies reached a plateau,
while mutant colonies continued to extend (Fig. 1F,H; see Fig. S4G
in the supplementary material). At this stage, all caulonemal-type
filaments, a mixture of caulonemata and rhizoids, protruding from
the wild-type colonies were unbranched (Fig. 1E) and had stopped
to elongate. By contrast, elongation and branching of Ppccd8
caulonemata continued up to 45 days after spore germination
giving rise to much larger colonies (Fig. 1G; see Fig. S4F,G in the
supplementary material).
In both genotypes, the first buds appeared ~8 days after
caulonema differentiation. Size of gametophores was affected in
Ppccd8 compared with wild type. Mutant gametophores were in
average half the size of wild type, had reduced number of leaves
and shorter internodes after 45 days (see Fig. S4H-J in the
supplementary material). Yet leaves were normally shaped and
displayed normal phyllotaxy, and the general architecture of the
leafy shoot was unaffected. Most importantly, we did not observe
any increase in gametophore branching, and all Ppccd8
gametophores were formed by a single stem as in wild type (data
not shown). Gametophores bearing a capsule were less frequent in
the mutant (0.55 sporophyte/gametophore in wild type versus 0.04
in Ppccd8). Nevertheless, capsules from mutant contained viable
spores but in reduced numbers compared with wild type (see Fig.
S4K,L in the supplementary material).
These results indicated that while cells and organs forming
gametophyte and sporophyte were not altered, except in size,
control of protonema branching and colony extension was strongly
affected in Ppccd8.
A diffusible signal secreted by wild-type colony
complements Ppccd8 phenotype
In P. patens, hormones such as auxins and cytokinins are secreted
in the medium to control the protonemal stage of gametophyte
development (Cove et al., 2006). To assess whether the PpCCD8
gene is involved in the biosynthesis of a signal secreted into the
medium, we performed a series of cross-feeding assays.
After 2 weeks of co-culture, the Ppccd8 colony surrounded by
wild-type colonies displayed a wild-type phenotype with a strong
decrease in colony diameter (Fig. 1I-L; see Fig. S5 in the
supplementary material). These results suggested that wild-type
moss produces a signal that diffuses into the medium and
complements the phenotype of Ppccd8. This signal inhibits
colony extension and filament branching. The mutant does not
appear to be producing this inhibitor that accounts for its
developmental phenotype.
When a wild-type colony was surrounded by Ppccd8 colonies,
a slight but significant increase in the diameter of the central wildtype colony was observed (Student’s t-test, P<0.01) (see Fig. S5 in
the supplementary material). The central wild-type colony also had
Development 138 (8)
an effect on the surrounding Ppccd8 colonies. The mutant colonies
grew asymmetrically owing to reduced extension on the side closest
to the wild-type colony (Fig. 1K). The difference in diameter of the
wild-type colony when surrounded by either WT or mutant colonies
also indicated that the colony stopped extending once the signal
reached a certain threshold. This is attained faster when surrounding
colonies also participate in the production of the signal.
Feedback regulation of PpCCD7 and PpCCD8
In several seed plants, CCD7 and CCD8 transcript levels are
upregulated in strigolactone-deficient mutants suggesting that
negative feedback controls strigolactone biosynthesis (Beveridge
and Kyozuka, 2010). In rice, application of the synthetic
strigolactone GR24 to the strigolactone deficient ccd8 (d10) mutant
restored CCD8 transcript levels to wild type (Umehara et al.,
2008).
Expression of PpCCD8 in the wild type was first investigated by
quantitative RT-PCR. The expression level of PpCCD8 was low
but detectable 10 days after spore germination and increased
approximately eightfold between days 15 and 21 to reach a plateau
(see Fig. S6A in the supplementary material). These results show
that PpCCD8 is expressed at low levels in the protonemata and is
upregulated along with the differentiation of new gametophores
where it is more abundantly expressed (see below).
Quantitative RT-PCR further revealed that PpCCD7 displayed a
similar expression profile in both wild-type and Ppccd8 colonies.
Yet PpCCD7 transcript levels were five to 20 times higher in
Ppccd8 than in wild type (see Fig. S6A,B in the supplementary
material). This observation suggests that, in Physcomitrella, the
two CCD genes are co-regulated with feedback control of
PpCCD7, as previously observed in seed plants (Beveridge and
Kyozuka, 2010).
To gain more insight in the regulation of PpCCD8 expression,
the -glucuronidase (GUS) reporter gene driven by PpCCD8
promoter sequence was introduced into locus 108 in wild-type and
Ppccd8 strains. Locus 108 is often used as target sequence as it
does not contain any coding sequence (Schaefer and Zryd, 1997).
GUS expression driven by the PpCCD8 promoter was not
detectable in wild type by histochemical staining (data not shown),
indicating that the expression level of PpCCD8 was very low. By
contrast, GUS staining was visible following overnight incubation
in the Ppccd8 background, and was observed in every
differentiated bud in the protonemata and at the foot of each
gametophore (Fig. 2A,B). These data indicate that PpCCD8
expression is also regulated by feedback control, as observed above
for the PpCCD7 transcript. They further indicate that the biological
activity of PpCCD8 is developmentally regulated and spatially
concentrated at the base of gametophores.
Strigolactone identification and production in
wild type and Ppccd8
The presence of strigolactones in Physcomitrella was tested further
with the analysis of liquid medium in which differentiated wildtype and Ppccd8 tissue were grown for 3 days. As shown for
several seed plants, P. patens exuded a mixture of strigolactones
(see Fig. S7A,B in the supplementary material). The major
strigolactones produced by the wild type were 7-oxoorobanchyl
acetate, orobanchyl acetate, 7-hydroxyorobanchyl acetate, fabacyl
acetate, strigol and orobanchol (Fig. 3A). The mutant produced
only strigol and 7-oxoorobanchyl acetate (Fig. 3A) along with
small amounts of 5-deoxystrigol, which could not be quantified in
either strains. Wild type produced and released approximately 350
DEVELOPMENT
1534 RESEARCH ARTICLE
Strigolactones function in moss
RESEARCH ARTICLE 1535
Fig. 2. PpCCD8 is expressed at the base of each gametophore.
(A)PpCCD8pro:GUS in a 21-day-old Ppccd8⌬ colony. (B)Young
gametophore (enlargement of A as indicated). Scale bars: 500m in A;
100m in B. Arrowheads indicate bud or gametophore.
pg/g fresh weight (FW) of strigolactones in 3 days, while the
mutant produced 40 pg/g FW, less than one-eighth that of wild
type. These data provide the first characterisation of strigolactones
in a moss. They further demonstrate that strigolactone biosynthesis
is severely affected in Ppccd8, indicating the involvement of
PpCCD8 in strigolactone biosynthesis in P. patens and the potential
involvement of the strigolactone pool in the developmental
phenotype described above.
Fig. 3. The signal that regulates colony extension in P. patens is a
strigolactone. (A)Quantification of strigolactones released by wild
type (open bars) and Ppccd8⌬ (black bars) in the growth media for 3
days. Data are presented as mean±s.e.m. (n3).
(B-G)Complementation of Ppccd8⌬ by 1M GR24 application. Twentyfive-day-old colonies (B-D). Periphery of the same colonies (E-G).
ch, chloronema side branches. Scale bars: 1 cm in B-D; 1 mm in E-G.
(H)Expression of PpCCD7 in wild-type (white) and Ppccd8⌬ colonies
(21-day-old) 24 and 48 hours after transfer to new media containing
0M (black), 1M GR24 (blue) or to media on which wild-type (light
grey) or Ppccd8⌬ colonies (dark grey) were grown for 21 days. Error
bars indicate s.e.m., n3. Letters indicate means that differ significantly
(P<0.01, ANOVA test).
1 M GR24. These analyses showed that GR24 downregulated
PpCCD7 expression in Ppccd8 (Fig. 3H). To determine whether
the wild-type diffusible signal identified in cross-feeding
experiments also regulates PpCCD7 expression, we analysed
Ppccd8 samples that were transferred on media where wild-type
or Ppccd8 colonies had grown previously. The same down
regulation of PpCCD7 expression was observed in Ppccd8 tissue
treated with wild-type preconditioned medium (Fig. 3H).
Altogether, these data show that GR24 almost completely
complements the developmental phenotype of Ppccd8 mutant, the
DEVELOPMENT
Ppccd8 phenotype is complemented by
expression of pea CCD8 and by exogenous
strigolactone application
To examine whether expression of CCD8 genes from seed plants
can complement the Ppccd8 phenotype, the pea CCD8 gene
(RMS1) driven by the rice Actin-1 gene promoter was introduced
at locus 108 in the Ppccd8 mutant. Several independent
Ppccd8::OsACTpro:RMS1 strains were obtained that exhibited
the same wild-type developmental pattern: colony extension
stopped after 3 weeks and colonies developed leafy shoots of
normal size. Furthermore, these strains perfectly restored normal
development of Ppccd8 colonies in cross-feeding experiments
(see Fig. S8A,B in the supplementary material). Consistently, semiquantitative RT-PCR indicated that the expression of PpCCD7 was
reduced to wild-type levels in Ppccd8::OsACTpro:RMS1 strains
(see Fig. S8C in the supplementary material). These results provide
genetic evidence that pea RMS1 is able to restore both normal
development and feedback control of PpCCD7 expression in
Ppccd8 mutant. These data suggest that pea and moss CCD8 have
the same biochemical activity that has been conserved during land
plant evolution.
To determine whether the synthetic strigolactone GR24 could
complement the Ppccd8 developmental phenotype, 15-day-old
Ppccd8 colonies were transferred on media containing 1 M of
GR24. After 2 weeks, these colonies showed an intermediate
diameter and were surrounded by unbranched caulonema-type
filaments (Fig. 3B-G). In a dose-response experiment, the
diameters of both wild-type and Ppccd8 colonies were
significantly smaller after 23 days on medium supplemented with
100 nM or 1 M GR24 (test of ANOVA, P<0.01, n12; see Fig.
S9 in the supplementary material). At the same stage, we observed
in both strains that gametophores were larger than in the nonsupplemented controls (Fig. 3B-D). To assess whether GR24 was
able to restore feedback control on PpCCD7 expression, we
analysed its expression in 21-day-old wild-type and Ppccd8
colonies during the 2 days following transfer on media containing
1536 RESEARCH ARTICLE
Development 138 (8)
most striking feature being the complete arrest of secondary
chloronemata formation at the edge of the colonies. They further
demonstrate that both GR24 and the wild-type diffusible signal
downregulate PpCCD7 expression.
DISCUSSION
Conservation of strigolactone biosynthesis among
land plants
The first steps in strigolactone biosynthesis involve the cleavage
of carotenoids by CCD7 and CCD8 (Beveridge and Kyozuka,
2010; Leyser, 2008). The moss genome contains one orthologue
for each corresponding gene and our phylogenetic analysis
suggests that the CCD7 and CCD8 clades evolved before the
separation of the bryophytes and the vascular plants from a
common ancestor 420 million years ago (Kenrick and Crane,
1997b). Mutations in either of these two genes are associated
with strigolactone deficiency in pea and rice (Gomez-Roldan et
al., 2008; Umehara et al., 2008). Our analysis of strigolactone
production in wild type and Ppccd8 demonstrates that PpCCD8
is similarly involved in strigolactone biosynthesis in moss. In
seed plants, the cytochrome P450 MAX1 is necessary for the
synthesis of the shoot branching inhibitor, and grafting
experiments indicated that it acts downstream of the two CCDs
(Booker et al., 2005). Although there is no MAX1 homologue in
the moss genome, P. patens produces strigolactones that were
previously identified in the exudates of seed plants. This
suggests that MAX1 functions in later steps of the active
branching inhibiting hormone biosynthesis, i.e. conversion of
strigolactones to the shoot branching inhibitor. Hence, the
bioactive signal in P. patens and seed plants may be different.
Alternatively, moss may have other P450(s) able to replace the
function of MAX1 for strigolactone biosynthesis.
The Ppccd8 mutant still produced strigolactones, whereas no
strigolactones were detected by LC-MS in the ccd8 mutants of pea
and rice (Gomez-Roldan et al., 2008; Umehara et al., 2008),
suggesting that another strigolactone biosynthesis pathway may be
present in moss. Hence, seven CCD genes other than PpCCD8 are
present in the genome of Physcomitrella (see Fig. S3 in the
supplementary material) that may be able to cleave carotenoids to
produce small amounts of strigolactones. Alternatively, as root
Fig. 4. Strigolactones control colony extension in response to
colony density. (A-D)Thirty-nine-day-old wild-type (top panels) and
Ppccd8⌬ (below panels) colonies grown at different densities. Scale
bars: 1 cm. (E)Diameter of wild-type (blue lines) and Ppccd8⌬ (green
lines) colonies grown in light at different densities, two colonies (white),
four colonies (light grey), nine colonies (dark grey) and 15 colonies
(black) (n12). Data are mean±s.e.m.
exudates from the ccd8 mutants of pea and rice did induce
germination of root parasitic weed seeds, it could suggest that these
mutants may also still produce small amounts of strigolactones
(Gomez-Roldan et al., 2008; Umehara et al., 2008).
Strong upregulation of CCD7 and CCD8 transcription is
generally observed in strigolactone-deficient mutants (Dun et al.,
2009). Our work revealed that transcription levels of both PpCCD7
and PpCCD8 genes were also strongly upregulated in Ppccd8,
indicating that the transcriptional feedback regulation that controls
this biosynthetic pathway is probably conserved among land plants.
We demonstrated that expression of pea CCD8 fully complements
the developmental phenotype of Ppccd8, which provides
evidence for functional complementation of CCD8 proteins in
these distant species. These results showed that the first steps of
strigolactone biosynthesis are conserved among Embryophytes.
However, strigolactone biosynthetic pathway is not fully
characterised, and even in seed plants the biochemical function of
MAX1, for example, is still unknown. A better understanding of
the final steps of the strigolactone biosynthesis pathway should
highlight difference and similarity between land plants.
Developmental and colonial defects of
strigolactone deficient P. patens
Detailed analysis of the strigolactone-deficient mutant showed that
PpCCD8 controls moss development from the germination stage
with an earlier spore germination, higher number of primary
chloronemata developing from the spore and higher branching of
chloronemata in Ppccd8. This indicates that the strigolactone
pathway is already involved in the spore development to control
specific cellular process. The apparent different phenotypes may
DEVELOPMENT
Colony extension responds to colony density in
wild-type moss but not in Ppccd8
To analyse further the threshold effect of signal accumulation in the
medium, we tested if by varying the density of colonies, the
diameter of wild-type colonies changed and examined the effect on
mutant colonies. We followed the evolution of wild-type and
Ppccd8 colonies inoculated at four different densities (Fig. 4AD). At high densities, the Ppccd8 colonies started to merge at 28
days and at 39 days, the surface of the Petri dish was completely
covered by mutant colonies (Fig. 4C,D). Consequently, after 28
days we were unable to measure colony diameter for Ppccd8. By
contrast, at 39 days, regardless of the initial colony density, wildtype colonies showed a well-delimited round surface (Fig. 4A-D).
ANOVA confirmed a significant effect of density on diameter at 28
days in wild type (P<0.001) but not in Ppccd8. The diameter of
wild-type colonies was significantly higher at density 2 and 4
colonies per plate, and reached a plateau at 39 days for the two
highest densities (Fig. 4E). These data show that in P. patens,
strigolactones accumulate in the medium as the number of
gametophores increases to a threshold concentration to control
colony extension in response to colony density.
RESEARCH ARTICLE 1537
result from the same cellular mechanism of higher rates of lateral
cell differentiation and/or division and would need further
investigation.
The subsequent development of colonies was similar in both
wild type and Ppccd8 until 3 weeks after germination, indicating
that strigolactones are not required to establish the normal
developmental pattern of colonies. This is consistent with the
absence of detectable morphological alteration in the mutant.
Under our growth conditions, and for a density of 15 to 20
colonies per Petri dish, wild-type colonies stop to extent and to
differentiate a branched protonema after 3 weeks. By contrast,
Ppccd8 colonies maintain their extension with a continuous
production of secondary chloronemata and buds on caulonemata.
The decrease in gametophore size, in number of gametophore
bearing a capsule and in spore number per capsule observed in
strigolactone-deficient Ppccd8 should be seen as a trophic
response to higher intra-colony competition for resources
between gametophores. Similarly, in seed plants, the high
branching of strigolactone-deficient ccd8 mutants results in an
increase in the number of flowers per plant with a concomitant
decrease in flower size and seed number per plant (Snowden et
al., 2005).
propose that when a ‘quorum’ of strigolactone producing
gametophores is reached, the whole colony and the neighbouring
colonies respond by halting their extension.
The idea of chemical interactions between mosses regulating
spreading of colonies is a long-standing observation for people
working with mosses. A diffusible signal called ‘Factor H’, first
identified in Funaria hygrometrica, was postulated to account for
the maintenance of a clear zone between moss colonies or clones
that did not involve competition for nutrients (Bopp, 1959; Klein,
1967). Factor H, which may be a complex of substances, was
known to inhibit caulonema growth and lateral spread and to
promote bud formation (Bopp and Knoop, 1974; Klein, 1967).
Watson (Watson, 1981) proposed that such diffusible substances
have developmental as well as ecological implications and may be
possible determinant of moss community structure in nature.
Strigolactone could be good candidates for the caulonema growth
and lateral spread inhibition effect of the factor H. However,
strigolactones have no inductive effect on bud formation and rather
favour the simultaneous growth of gametophores already
differentiated in the population. Thus, more extensive work is
needed to asses whether strigolactones could be one component of
the factor H.
Strigolactone activity in moss are reminiscent of
quorum-sensing signal in bacteria
In wild type, the arrest of colony extension is sudden and involves
a coordinated response from the whole colony. This response is
developmentally associated with a complete arrest of caulonema
branching and elongation. By contrast, despite producing a small
amount of strigolactones, Ppccd8 colonies continue to extend. All
these results suggest that wild-type colonies released strigolactones
or derived molecules into the medium to control colony extension.
These data also indicate that a minimal threshold must be reached
for the colony to respond to strigolactones and imply that
strigolactones are synthesised and degraded in media as this
threshold is never reached by Ppccd8 colonies. Strigolactones are
characterised by a tricyclic lactone (ABC part) connected, via an
enol ether bridge, to a butyrolactone group (the D-ring) (Yoneyama
et al., 2009). They are known to be short-lived in the rhizosphere
owing to this labile ether bond that spontaneously hydrolyses in
water (Akiyama and Hayashi, 2006; Akiyama et al., 2010). This
instability creates a steep concentration gradient that has been
suggested to be a reliable indicator of the proximity of a host root
(Parniske, 2008).
Our analyses further demonstrate that strigolactones act as
diffusible signals that regulate colonial extension in response to
population densities (Fig. 4A-D). In wild type, the final diameter
of 45-day-old colonies is negatively correlated with the number of
colonies per plate and this correlation completely disappears for
Ppccd8 colonies (Fig. 4E). Based on these features, as well as on
the fact that strigolactones are low molecular weight molecules that
diffuse in the medium, we propose that strigolactones behave as
quorum sensing-like molecules. Quorum sensing is a common, if
not universal, cell-to-cell communication process in the microbial
world. It is mediated by low molecular weight molecules, the most
common signals in bacteria being acyl-homoserine-lactones (Ng
and Bassler, 2009), that accumulate in the environment depending
on the density of the organisms synthesising these molecules
(Fuqua and Greenberg, 2002). When a quorum-sensing signal
molecule reaches a threshold concentration, i.e. a threshold cell
density, the entire population responds, usually through the
coordinated expression of specific target genes. By analogy, we
Possible role of strigolactone in early land plant
evolution
The colonisation of land by plants is associated with a large
number of developmental, morphological, anatomical and chemical
innovations (Kenrick and Crane, 1997b; Langdale, 2008).
Arbuscular mycorrhizal symbiosis is an association between one of
the oldest group of fungi, the Glomales, and plants. The fungal
partner provides nutrients, essentially phosphate, and water to the
plant that brings carbohydrates to the fungi (Parniske, 2008). This
symbiosis has been found in almost all major lineages of extant
land plants and is considered as one of the major innovations that
contributed to the colonisation of land by plants (Kenrick and
Crane, 1997b; Renzaglia et al., 2000). Mycorrhization is tightly
associated with strigolactone biosynthesis (Akiyama et al., 2005;
Akiyama et al., 2010; Gomez-Roldan et al., 2008). Despite the fact
that P. patens, like most mosses, is not mycorrhizal, other
bryophytes, liverworts that are considered the earliest-diverging
lineage of land plants, and hornworts are mycorrhizal (Read et al.,
2000; Wang and Qiu, 2006; Wang et al., 2010). The evolutionary
homology of symbiotic interactions between gametophytes of
liverworts and hornworts and mycorrhizal fungi, and those between
sporophytes of vascular plants and the fungi has been demonstrated
recently (Wang et al., 2010). These data and our demonstration that
strigolactones are signalling molecules in the bryophyte P. patens
support the hypothesis that strigolactones are very ancient
molecules that have played a crucial role in the ecological
adaptation of plants to terrestrial environment. Further studies with
Marchantia, a mycorrhizal liverwort recently used in biological
studies (Bowman et al., 2007; Chiyoda et al., 2008; Ishizaki et al.,
2008,) would confirm the role of strigolactones in the establishment
of arbuscular mycorrizal symbiosis in bryophytes.
Land plants (embryophytes) evolved from streptophyte green
algae, a small group of freshwater algae ranging from unicellular
flagellates (Mesostigma) to complex filamentous thalli with
branching (Charales) (Becker and Marin, 2009). No CCD7 or
CCD8 homologues have been found in streptophyte green algae
EST databases, which could mean that strigolactones arose very
early in the land plant lineage. However, no complete genome is
currently available for streptophyte green algae (Becker and Marin,
DEVELOPMENT
Strigolactones function in moss
2009; Timme and Delwiche, 2010) and whether they are able to
synthesize strigolactones would give some clue on the earliest
role(s) of strigolactones during plant evolution.
In this work, we have shown that strigolactones repress
protonema branching in P. patens that is the differentiation of a
lateral cell from subapical cells. Branching in seed plants involved
several steps from the initiation of axillary meristems at the axils
of leaves, their subsequent development in axillary buds that will
remain in a dormant state or will outgrow to produce a branch.
Strigolactones control specifically the maintenance in a dormant
state of this particular organ, the axillary bud. It is quite intriguing
that strigolactones control branching of the gametophyte in moss
and branching of the sporophyte in seed plants. P. patens genome
analysis and its comparison with the genome of Arabidopsis have
suggested that genes controlling gametophyte development have
been recruited for sporophyte development (Nishiyama et al., 2003;
Rensing et al., 2008). Whether common molecular actors are
involved to control gametophyte (protonema) or sporophyte
branching in response to strigolactones will be revealed by future
studies. However the term ‘branching’ can be misleading and, for
example, it is very likely that sporophyte branching due to
dichotomy observed in many early taxa, involved different
mechanisms from what is currently known in seed plants (Kenrick
and Crane, 1997a; Langdale, 2008). Thus, more data will be
needed in different taxa in order to decipher branching mechanisms
in plants (Boyce, 2010). Nevertheless, this work is a first step in
the understanding of the evolution of branching patterns and
contributes to the wonderful story of how a small family of
molecules has been co-opted during evolution to control chemical
interactions between organisms and whole-plant architecture.
Acknowledgements
We thank F. D. Boyer for provided GR24, K. Mori for strigol, T. Asami for [2,313
C2]strigol, and K. Akiyama for [6’-2H]orobanchol and [6’-2H]orobanchyl
acetate. H.P. was supported by a PhD fellowship from the Ministère de
l’Education Nationale, de la Recherche et de la Technologie (MENRT). Work
from the C.R. group was supported by ANR (Agence Nationale de la
Recherche, project ANR.05.BLAN.0262), and work from the K.Y. group was
supported by KAKANHI (18208010, 2109111) and Program for Promotion of
Basic and Applied Researches for Innovations in Bio-oriented Industry. We
thank Y. Dessaux for helpful discussions; H. Höfte, Y. Dessaux, G. Pelletier and
S. Bonhomme for their comments on the manuscript; and M. Sawabe for her
assistance.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.058495/-/DC1
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DEVELOPMENT
Strigolactones function in moss