RESEARCH ARTICLE 1275
Development 137, 1275-1284 (2010) doi:10.1242/dev.039594
© 2010. Published by The Company of Biologists Ltd
Homologues of the Arabidopsis thaliana SHI/STY/LRP1 genes
control auxin biosynthesis and affect growth and
development in the moss Physcomitrella patens
D. Magnus Eklund1,*, Mattias Thelander1,*, Katarina Landberg1, Veronika Ståldal1, Anders Nilsson2,
Monika Johansson1,2,†, Isabel Valsecchi1, Eric R. A. Pederson1, Mariusz Kowalczyk3, Karin Ljung3, Hans Ronne2
and Eva Sundberg1,‡
SUMMARY
The plant hormone auxin plays fundamental roles in vascular plants. Although exogenous auxin also stimulates developmental
transitions and growth in non-vascular plants, the effects of manipulating endogenous auxin levels have thus far not been
reported. Here, we have altered the levels and sites of auxin production and accumulation in the moss Physcomitrella patens by
changing the expression level of homologues of the Arabidopsis SHI/STY family proteins, which are positive regulators of auxin
biosynthesis genes. Constitutive expression of PpSHI1 resulted in elevated auxin levels, increased and ectopic expression of the
auxin response reporter GmGH3pro:GUS, and in an increased caulonema/chloronema ratio, an effect also induced by exogenous
auxin application. In addition, we observed premature ageing and necrosis in cells ectopically expressing PpSHI1. Knockout of either
of the two PpSHI genes resulted in reduced auxin levels and auxin biosynthesis rates in leafy shoots, reduced internode elongation,
delayed ageing, a decreased caulonema/chloronema ratio and an increased number of axillary hairs, which constitute potential
auxin biosynthesis sites. Some of the identified auxin functions appear to be analogous in vascular and non-vascular plants.
Furthermore, the spatiotemporal expression of the PpSHI genes and GmGH3pro:GUS strongly overlap, suggesting that local auxin
biosynthesis is important for the regulation of auxin peak formation in non-vascular plants.
INTRODUCTION
The most important naturally occurring auxin, indole-3-acetic acid
(IAA), is essential throughout the life cycle of vascular plants and is
required for embryo axis formation, root apical meristem patterning,
organ positioning and initiation, tissue differentiation and
phototropic and gravitropic responses (Dolan, 1998; Friml et al.,
2003; Kaufman et al., 1995; Marchant et al., 1999; Mattsson et al.,
1999; Sabatini et al., 1999; Went, 1974). Auxin is believed to
mediate instructions through concentration gradients and local auxin
maxima created by the combined forces of auxin synthesis and polar
auxin transport (PAT) (Teale et al., 2006). Although Grieneisen and
co-workers showed that transport of auxin is crucial for auxin
maxima formation in root tips (Grieneisen et al., 2007), recent
studies on auxin biosynthesis regulation have convincingly
demonstrated the importance of local biosynthesis in establishing
and maintaining auxin maxima throughout plant development
(Cheng et al., 2006; Cheng et al., 2007; Ikeda et al., 2009; Stepanova
et al., 2008; Tao et al., 2008). There are at least four pathways for
1
Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish
University of Agricultural Sciences, Box 7080, S-750 07 Uppsala, Sweden.
Department of Medical Biochemistry and Microbiology, Uppsala University, Box
582, S-751 23 Uppsala, Sweden. 3Umeå Plant Science Centre, Department of Forest
Genetics and Plant Physiology, Swedish University of Agricultural Sciences, S-901 83
Umeå, Sweden.
2
*These authors contributed equally to this work
†
Present address: Department of Food Science, Swedish University of Agricultural
Sciences, Box 7051, 75007 Uppsala, Sweden
‡
Author for correspondence ([email protected])
Accepted 12 February 2010
biosynthesis of IAA in seed plants, one of which is tryptophan
independent, the other three tryptophan dependent (for reviews see
Chandler, 2009; Lau et al., 2008). The different pathways appear to
function non-redundantly because they produce IAA in different
spatiotemporal patterns, for different purposes and in response to
different stimuli (Tao et al., 2008). This suggests that the
spatiotemporal regulation of auxin biosynthesis plays an important
role in auxin gradient or peak formation. We have previously
demonstrated that SHI/STY family proteins in Arabidopsis induce
the activity of auxin biosynthesis genes (e.g. YUC4), thus playing
important roles in the positioning of local auxin maxima (Sohlberg
et al., 2006; Ståldal et al., 2008; Eklund et al., 2010).
Auxin also functions as a hormone in bryophytes, and IAA
application triggers physiological responses such as the chloronemato-caulonema transition (Johri and Desai, 1973), stem elongation
(Fujita et al., 2008) and rhizoid emergence from the leafy shoot
(Ashton et al., 1979). With the completion of the genome sequence
of the moss Physcomitrella patens, it became clear that genes
involved in auxin transport, perception and signalling in seed plants
are present also in mosses (Rensing et al., 2008). This remarkable
conservation between plants separated by 450 million years of
evolution suggests that this machinery was already in place when
early plants colonised land (Cooke et al., 2002).
The P. patens genome contains four putative PIN-type auxin
efflux facilitator genes (Floyd and Bowman, 2007), which suggests
the presence of active PAT in mosses. Whereas some studies have
reported that PAT inhibitors have effects on gametophyte
development (Geier et al., 1990; Rose and Bopp, 1983; Rose et al.,
1983; Schwuchow et al., 2001), a recent study of five different
mosses suggested that long-range PAT occurs in the subordinate
DEVELOPMENT
KEY WORDS: Physcomitrella patens, SHI, STY, LRP1, Auxin, Moss
1276 RESEARCH ARTICLE
MATERIALS AND METHODS
Growth conditions
Conditions for growth and subculturing and BCD media were as described
(Thelander et al., 2007).
Gene identification and cloning
The P. patens ssp. patens draft genome (v1.1) was screened for SHI/STY,
TAA1, YUCCA and CYP79B2/3 homologues using TBLASTN with query
sequences from Arabidopsis. SHI/STY loci were also searched for in the S.
moellendorffii (v1.0), C. reinhardtii (v3.0), O. lucimarinus (v2.0), O. tauri
(v2.0) and V. carteri f. Nagariensis (v1.0) genomes deposited at the Joint
Genome Institute (JGI). The PpSHI1 cDNA (Kuusk et al., 2006) was used
to design primers for amplification of the PpSHI1 gene, which was cloned
into pCR2.1-TOPO (Invitrogen, Lidingö, Sweden) and sequenced.
PpSHI1 in vitro mutagenesis
The BamHI site in exon 3 of PpSHI1 was mutated to AGATCC using
primers 5⬘Gex3A and 3⬘Gex3A (see Table S1 in the supplementary
material) together with gene-specific primers. The PCR products were
combined in another PCR to fuse the two fragments. To mutate the BamHI
site in intron 2 to GGATCT we used primers 5⬘Gint2A and 3⬘Gint2A. In a
final PCR, the fragments containing the two mutations were fused. The
resulting fragment was cloned into pCRII-TOPO T/A (Invitrogen) to
produce plasmid pPpSHI1GexintA. The PpSHI1 cDNA was mutated using
a similar strategy.
GUS staining and fluorescence microscopy
GUS staining was performed according to Jefferson (Jefferson, 1987) but
with 2 mM ferri/ferrocyanide. Tissue was stained at room temperature for
2-24 hours before destaining in 70% ethanol and analysis using a stereo
dissection microscope (Nikon SMZ1500) with a Nikon DS-Fi1 camera and
NIS-Elemens D2.30 imaging software. GFP fluorescence was analysed with
a Zeiss Axioskop 2 Mot microscope with an AxioCam HR camera and
AxioVision software. GFP was detected using a FITC filter,
autofluorescence using a TRITC filter and Hoechst 33342 using a DAPI
filter.
PpSHI1pro:PpSHI1-GFP and PpSHI2pro:PpSHI2-GUS lines
The vector pPpGUS was constructed by inserting a GUS-TNOS1
BamHI/EcoRI fragment from plasmid pBI101.3 (Clontech, Stockholm,
Sweden) into the BamHI/EcoRI sites of pCR-BluntII-TOPO (Invitrogen),
after which a P35S-NPTII-T35S neomycin resistance EcoRI cassette from
plasmid pMT164 (Thelander et al., 2007) was inserted into the EcoRI site
of the product. Plasmid pPpGFP was created using the same strategy with a
GFP-TNOS1 BamHI/EcoRI fragment from plasmid psmRS-GFP (Davis
and Vierstra, 1996).
A PpSHI1 fragment amplified from pPpSHI1GexintA using primers
165BamHIIIF and 165BamHIR (see Table S1 in the supplementary
material) was inserted into the BamHI site of pPpGFP. Another fragment
directly downstream of the stop codon was amplified with primers 5⬘PpUTR
and 3⬘PpUTR and inserted between the NotI and XhoI sites of the previous
product, creating plasmid pPpSHI1-iGFP. Similarly, a PpSHI2 genomic
fragment terminating immediately prior to the stop codon was amplified
with primers 1652BglIIF and 1652BglIIR and inserted into the BamHI site
of the vector pPpGUS. A NotI/XbaI fragment in the resulting product was
exchanged for a genomic fragment amplified with primers 3UTR2NotIF and
3UTR2XbaIR and covering the 3⬘UTR of PpSHI2 to produce plasmid
pPpSHI2-iGUS.
The targeting constructs pPpSHI1-iGFP and pPpSHI2-iGUS (see Fig.
S3A,B in the supplementary material) were released by XbaI digestion prior
to transformation (Schaefer et al., 1991). Transformants were selected on 50
g/ml G418 (G9516, Sigma, Stockholm, Sweden). PCR genotyping of
stable transformants was performed with primers shown in Fig. S3A,B and
Table S1 in the supplementary material.
PpSHI1 overexpression
PpSHI1 was amplified from cDNA using primers PpSHIox5⬘ and
PpSHIox3⬘ (see Table S1 in the supplementary material) and inserted into
the ApaI site of pCMAK1 creating pPpSHI1ox1. pPpSHI1ox1 was NotIlinearised before transformation. Transformants were selected on 50 g/ml
zeocine (Invitrogen).
PpSHI1 and PpSHI2 gene disruption
Two PpSHI1 targeting constructs were used. For construct 1, a PpSHI1
fragment was amplified using primers PpSHI1-5A and PpSHI1-3A (see
Table S1 in the supplementary material) and cloned into the SpeI site of
pBluescript SK+ (Stratagene), creating plasmid pBluSKSHI1. The
hygromycin resistance cassette from plasmid pMT123 (Thelander et al.,
2004) was released by HindIII/SpeI cleavage, T4 polished and cloned
between the blunted NcoI and SnaBI sites of pBluSKSHI1, creating plasmid
pPpKOSHI1. For construct 2, two PpSHI1 genomic fragments were
amplified with primers SHI55 and SHI53 and with SHI35 and SHI33 and
cloned between the NotI and XbaI and the XhoI and KpnI sites, respectively,
of pMT123, to produce plasmid pPpKOSHI1b. For the PpSHI2 targeting
construct, PpSHI2 5⬘ and 3⬘ fragments were amplified with primers
PpSHI255 and PpSHI253 and with PpSHI235 and PpSHI233, respectively.
The 5⬘ fragment was cut with SalI and inserted into the XhoI site of pMT164
(Thelander et al., 2007), whereas the 3⬘ fragment was cut with XbaI/SpeI and
inserted into the SpeI site of the resulting product, creating plasmid
pPpKOSHI2. Disruption constructs were linearised using SpeI
(pPpKOSHI1), NotI/KpnI (pPpKOSHI1b) or BamHI/XhoI (pPpKOSHI2)
before transformation (Schaefer et al., 1991). Transformants were selected
on hygromycin (pPpKOSHI1 and pPpKOSHI1b) or G418 (pPpKOSHI2).
Stable transformants were analysed by PCR and RT-PCR as described in
Fig. S5 in the supplementary material with primers positioned as shown in
Fig. S4 in the supplementary material. Flow cytometry analysis by Plant
Cytometry Services (Netherlands) confirmed that all knockout lines were
haploid.
DEVELOPMENT
sporophyte generation, but not in the gametophyte leafy shoot
(Fujita et al., 2008). Consequently, it appears likely that tightly
controlled local auxin biosynthesis is important in mosses, and that
increased knowledge about these processes could shed light on the
ancestral mechanisms for creation of auxin maxima in plants. We
searched the P. patens genome sequence for auxin biosynthesis
genes and found homologues of six Arabidopsis thaliana (At)
YUCCA (AtYUC) (Gallavotti et al., 2008; Rensing et al., 2008) and
four AtTAA1 genes, which encode enzymes proposed to act in two
separate tryptophan-dependent pathways (Stepanova et al., 2008;
Tao et al., 2008; Zhao et al., 2001). By contrast, although numerous
cytochrome P450-related genes exist in moss (Rensing et al., 2008),
we were unable to find a gene with a deduced protein sequence
clustering within the CYP79 subgroup, which harbours enzymes
that catalyze the conversion of tryptophan to indole-3-acetaldomixe
in Arabidopsis (Werck-Reichhart et al., 2002). This suggests that at
least two tryptophan-dependent pathways for auxin biosynthesis are
likely to exist in P. patens.
Here, we report the functional characterisation of PpSHI1 and
PpSHI2, two highly similar P. patens members of the SHI/STY gene
family. Our data show that the PpSHI genes are co-expressed with
an auxin response reporter construct, and that a knockout of either
PpSHI gene results in reduced auxin biosynthesis rates and
accumulation in the apical end of leafy shoots, suggesting that the
PpSHI genes might induce auxin biosynthesis at their expression
sites. The phenotypic effects of PpSHI knockouts are consistent with
the expected effects of reduced endogenous auxin production. We
further show that constitutive expression of PpSHI1 results in
elevated IAA levels and some phenotypic abnormalities normally
associated with high auxin levels. These experiments also revealed
that PpSHI1 can induce necrosis when ectopically expressed.
Finally, our results suggest a possible role for axillary hairs in auxin
production or secretion.
Development 137 (8)
Auxin concentration and biosynthesis rate measurements
Moss colonies grown for 23 days on cellophane-overlaid BCD media
(PpSHI1ox) or apices from 50 gametophores grown for 7 weeks on BCD
media (Ppshi1-1, Ppshi2-1) were harvested from three independent
biological replicates. For IAA synthesis measurements, gametophores were
incubated with liquid BCD medium containing 30% 2H2O for 24 hours
before apices were harvested. 13C6-IAA (500 pg, internal standard) was
added to each sample before extraction. Extraction and purification of
samples were performed as described (Andersen et al., 2008). IAA
quantification and biosynthesis measurements were performed by combined
gas chromatography-mass spectrometry (GC-MS) (Edlund et al., 1995;
Ljung et al., 2005).
Phenotypic analysis
For phenotypic analysis, small protonemata pieces (~1 mm in diameter)
were inoculated onto BCD plates and grown under standard conditions.
Colonies or individual gametophores harvested with intact rhizoid bundles
were photographed using the stereo dissection microscope described above.
To visualise axillary hairs and rhizoid initials, detached gametophores were
incubated in 0.05% Evan’s Blue (46160, Fluka, Stockholm, Sweden)
dissolved in PBS (pH 8.0) for 1-2 hours and then washed in PBS.
Transgene copy number determinations
Real-time PCR was performed using the IQ5 Detection System (BioRad,
Sundbyberg, Sweden). We used 10 minutes at 95°C followed by 35 cycles
of 10 seconds at 95°C and 20 seconds at 60°C (amplification), or 71 cycles
of 1 minute at 95°C, 1 minute at 60°C and 10 seconds at 60°C (dissociation).
1⫻DyNAmo Flash SYBR Green qPCR Kit solution (Finnzymes,
Stockholm, Sweden), each primer at 0.3 M, 1 mM EDTA, 0.1 mg/ml BSA
and 8 ng of genomic DNA were mixed in a final volume of 20 l. PpSHI1
amplification with primers FcgSHI1 and RcgSHI1 (132 bp) was normalised
against PpSKI1 (Thelander et al., 2007) amplification with primers FgSKI1
and RgSKI1 (150 bp) (see Table S1 in the supplementary material).
Specificity was verified by melting curve and gel analysis. Standard curves
based on a dilution series of genomic wild-type DNA confirmed that both
targets were amplified with efficiencies close to 100%. For each line, three
biological repeats with three technical repeats were assayed using both
primer pairs. CtPpSKI1–PpSHI1 was first calculated for technical replicates
from both the PpSHI1ox and wild-type (WT) lines, after which
CtPpSHI1ox–WT values were calculated. The wild type contains one PpSHI1
copy and copy number in the PpSHI1ox lines therefore equals
2Ct(PpSHI1ox–WT).
Southern blot analysis
DNA extractions, blotting and hybridisation were performed as described
(Murén et al., 2009) using 0.4 g protonemata and subsequently 10 g of
DNA per restriction digest. Probe templates were PCR amplified with
primers PpSHI1-5⬘probe and PpSHI1-3⬘probe and with PpSHI2-5⬘probe
and PpSHI1-3⬘probe, respectively (see Table S1 in the supplementary
material), and cloned into pCR-BluntII-TOPO, from which they were
excised with EcoRI and isolated by gel purification prior to DIG labelling
using the DIG High Prime DNA Labelling and Detection Kit (Roche).
RESULTS
The SHI/STY gene family may be unique to
streptophytes
All members of the SHI/STY gene family possess two conserved
regions that encode a zinc-finger domain (Fridborg et al., 1999) and
the unique IGGH domain (Fridborg et al., 2001). The family has a
comparatively high copy number in seed plants, as exemplified by
the ten genes in Arabidopsis (Kuusk et al., 2006), whereas the
lycophyte Selaginella moellendorffii, which belongs to a primitive
group of vascular plants, has three genes, and the moss P. patens
two, suggesting that the SHI/STY gene copy number in
embryophytes correlates with their morphological complexity. A
survey of the draft genome sequences of the algal species
Chlamydomonas reinhardtii, Ostreococcus lucimarinus,
RESEARCH ARTICLE 1277
Ostreococcus tauri and Volvox carteri failed to identify any SHI/STY
genes, indicating that the family is not present in chlorophytes and
is thus restricted to streptophytes, or even embryophytes, as it is still
unclear whether they exist in charophyte algae. Hence, the presence
of SHI/STY genes might correlate with the appearance of the
TIR/AFB receptor and the ARF response genes that have so far only
been found in embryophytes (Lau et al., 2009).
A phylogenetic analysis shows that the two moss SHI genes are
highly similar and that they, like many of the Arabidopsis SHI/STY
genes, are likely to result from a recent gene duplication (see Fig.
S1A in the supplementary material) (Kuusk et al., 2006).
Characterisation of the two Physcomitrella
SHI/STY genes
The two Physcomitrella SHI/STY genes share an identical
organisation, with three exons, whereas the Arabidopsis
homologues have two exons (Kuusk et al., 2006). PpSHI1 and
PpSHI2 encode proteins of 289 and 291 amino acids, respectively,
sharing 93% identity (see Fig. S1B in the supplementary
material). The two proteins are 100% identical over the conserved
zinc-finger and IGGH domains and share 79% sequence identity
with the AtLRP1 (At5g12330) protein (see Fig. S1B in the
supplementary material). The regions between the N-termini and
the zinc-finger domains of the two Physcomitrella proteins are
strikingly rich in glutamine (Q) residues and several Arabidopsis
SHI/STY proteins also possess one or more Q-rich regions. Q-rich
regions have been shown to act as transcriptional activation
domains and experimental studies support such a function for
Arabidopsis and Physcomitrella members of the SHI/STY family
(Kuusk et al., 2006; Eklund et al., 2010).
PpSHI1 and PpSHI2 are mainly localised to the
nucleus
PpSHI1 and PpSHI2 share a short region that is enriched for positively
charged amino acids (see Fig. S1B in the supplementary material)
with the majority of the Arabidopsis, rice and poplar SHI/STY gene
products. This sequence resembles a monopartite nuclear localisation
signal (Lange et al., 2007) and strong evidence suggests that AtSTY1
exerts its function in the nucleus (Sohlberg et al., 2006; Eklund et al.,
2010). We therefore transiently expressed a PpSHI1-GFP fusion
protein in Physcomitrella protoplasts and found, as expected, a mainly
nuclear GFP signal (see Fig. S2A-D in the supplementary material).
This result was confirmed using gene targeting to integrate GFP and
GUS reporter genes, in frame, immediately upstream of the stop
codons of the PpSHI genes, resulting in stable PpSHI1pro:PpSHI1GFP and PpSHI2pro:PpSHI2-GUS transformants in which the fusion
proteins are expressed from the native promoters, thus precluding
overexpression artefacts (Fig. 1G-I; see Fig. S2E in the supplementary
material). A nuclear GUS signal was only detected after a short
staining time and when ethanol destaining was omitted. We conclude
that PpSHI1 and PpSHI2 are most likely active in the nucleus.
Cell- and tissue-specific expression of PpSHI1 and
PpSHI2
To determine the spatial and temporal expression patterns of the two
moss SHI/STY genes, we utilised the targeting constructs described
above (see Fig. S3A,B in the supplementary material) to generate
seven independent PpSHI2pro:PpSHI2-GUS lines and a single
PpSHI1pro:PpSHI1-GFP line, all of which were verified by PCR
analysis (see Fig. S3C,D in the supplementary material). All
PpSHI2pro:PpSHI2-GUS lines and the PpSHI1pro:PpSHI1-GFP
line showed the same overall expression pattern.
DEVELOPMENT
SHI genes and auxin biosynthesis in moss
1278 RESEARCH ARTICLE
Development 137 (8)
Fig. 1. PpSHI expression in gametophytes.
(A-F)PpSHI2pro:PpSHI2-GUS activity after
prolonged staining (5-12 hours). (A)GUS-stained
caulonemal filament (arrow), with unstained
chloronemal side branches (arrowhead).
(B)Bud/juvenile gametophore with GUS-stained
young rhizoids (arrowhead) and putative axillary
hairs in the shoot apex (arrow). (C-F)Adult
gametophores. (C)GUS-stained rhizoids
(arrowhead) and axillary hairs (arrow).
(D)Magnification of C, showing the expression
maxima at the rhizoid ends (arrowhead). (E)Apical
gametophore end with intensively stained axillary
hairs (arrows). Inset shows magnified axillary hair.
(F)Picture taken from above, demonstrating the
adaxial position of axillary hairs (arrow) close to the
stem junction. (G-I)PpSHI1pro:PpSHI1-GFP
expression using overlay images with a merge of the
GFP fluorescence (green) and chloroplast
autofluorescence (red). (G)Caulonemal filament
(arrow) exhibiting nuclear PpSHI1-GFP fluorescence,
whereas the chloronemal side branches (arrowhead)
show no fluorescence. (H)The base of an adult
gametophore showing mainly nuclear PpSHI1-GFP
signals in protruding rhizoids. (I)Basal adaxial part of
a detached leaf carrying two axillary hairs (arrows)
with predominantly nuclear PpSHI1-GFP localisation.
PpSHI1/2 expression coincides with sites of auxin
response
The PpSHI1/2 expression pattern in the adult gametophore, with
maxima in rhizoids and axillary hairs, is almost identical to that of a
GmGH3pro:GUS reporter [Gm, Glycine max (soybean)] used to
detect sites of auxin response (Bierfreund et al., 2003; Fujita et al.,
2008). If not coincidental, this correlation could indicate that the
PpSHI genes are induced by auxin, or that their expression is a
prerequisite for the generation of auxin peaks or auxin
responsiveness. In order to discriminate between these alternatives,
we compared the responses of GmGH3pro:GUS and
PpSHI2pro:PpSHI2-GUS lines to exogenous auxin. As previously
reported (Bierfreund et al., 2003; Fujita et al., 2008), the GmGH3
promoter was induced by auxin treatment in a concentrationdependent manner, which resulted in an almost uniform staining of
the entire gametophore, suggesting that all cell types can respond to
auxin (Decker et al., 2006) (Fig. 2). By contrast, GUS expression
from the PpSHI2 promoter remained unaffected by the auxin
treatment (Fig. 2). The fact that wild-type cells and tissues not
expressing the PpSHI genes still exhibited auxin-induced
GmGH3pro:GUS expression indicates that their products are not
required for auxin sensing or signalling, but could be important for
the generation of local auxin peaks.
Phenotypic effects of constitutive PpSHI1
expression
In order to alter the strength and tissue specificity of PpSHI1 activity,
we expressed it from a modified 35S (E7113) promoter (Mitsuhara
et al., 1996). Gene targeting was used to integrate the p35S-PpSHI1
expression cassette into the neutral BS213 locus, which has been
shown not to affect moss growth and development per se when
disrupted (Schaefer and Zryd, 1997). The background line used for
these experiments carried the GmGH3pro:GUS reporter (Bierfreund
et al., 2003), enabling monitoring of the spatial distribution of auxin
activity.
DEVELOPMENT
PpSHI2 was expressed in caulonemal cells, whereas its
expression was extremely low or absent in chloronemal cells (Fig.
1A). Buds and juvenile and adult gametophores showed staining
restricted to the emerging rhizoids and to distinct spots in the apex,
which could represent axillary hair primordia (Fig. 1B,C). In
rhizoids, PpSHI2 expression was detected already in the primordia
(not shown), and subsequently peaked in mitotically active apical
cells of developing rhizoids (Fig. 1C,D). Expression was stronger in
short compared with longer rhizoids, and staining appeared similar
in basal and mid-stem rhizoids [rhizoid definition according to
Sakakibara et al. (Sakakibara et al., 2003)]. We did not see any
evidence of a corresponding pattern in the morphologically similar
caulonemata, in which PpSHI2 appeared to be relatively evenly
expressed regardless of the position, age and mitotic activity of the
cells.
Furthermore, one to four PpSHI2-expressing axillary hairs were
found adaxially and basally on each side of the midrib of young
leaves (Fig. 1E,F). At the gametophore apex, stained cells, which
most likely represent young axillary hairs, were evident already in
the mass of mitotically active cells, surrounded by leaf primordia
(Fig. 1E). The abundance of PpSHI2-expressing axillary hairs
appeared to decrease gradually as the more basal parts of a
gametophore were examined. Juvenile adventitious gametophores
that occasionally grow out from the epidermal cell layer of an adult
gametophore also expressed PpSHI2 (not shown). Very little or no
GUS staining was detected in the stems and leaves of the
gametophores.
The PpSHI1-GFP expression pattern of the single PpSHI1GFP line was very similar to that of PpSHI2-GUS: signals were
detected in caulonema, rhizoids and axillary hairs, whereas
chloronemal cells showed no or very low-level signals (Fig. 1GI). In all cases, the PpSHI1-GFP product was mainly localised to
the cell nuclei. Together, our results suggest that PpSHI1 and
PpSHI2 have identical expression patterns in the vegetative
gametophyte.
SHI genes and auxin biosynthesis in moss
RESEARCH ARTICLE 1279
Ten stable transformants were generated, and although the
PpSHI1 expression level varied, it was enhanced in all five lines
examined (see Fig. S4 in the supplementary material). Gene
targeting in P. patens frequently results in the integration of multiple
transgenes into the targeted locus (Kamisugi et al., 2006) and using
a genomic real-time PCR approach we detected different transgene
copy numbers in the five lines. Surprisingly, the correlation between
transgene copy number and expression level was negative,
indicating co-suppression activity (see Fig. S4 in the supplementary
material). The expression levels in high-copy-number lines never
dropped below that of the control line, though, suggesting that cosuppression might be transgene specific and that the native PpSHI1
copy escapes downregulation. We also found a negative correlation
between the expression of PpSHI2 and PpSHI1 (see Fig. S4 in the
supplementary material), suggesting that PpSHI2 could be feedback
inhibited by elevated PpSHI1 expression. Given the overlap in
expression patterns, it appears likely that in wild-type moss,
feedback inhibition may be mediated by both PpSHI1/2 proteins and
that the PpSHI protein level, directly or indirectly (but not via
auxin), affects the activity of both genes. Alternatively, the
endogenous genes might be co-suppressed by the transgenes.
The transgenic lines exhibited a distinct set of phenotypes that
correlated roughly with the degree of PpSHI1 expression. Hence,
the phenotypic differences to the background line were subtle in
PpSHI1ox-3, moderate in PpSHI1ox-5, and striking in PpSHI1ox-1,
PpSHI1ox-2 and PpSHI1ox-4. At the protonemal stage, caulonemata
growth was stimulated while chloronemata growth was suppressed,
resulting in low-density star-like colonies (Fig. 3). The stimulation
of caulonema and suppression of chloronema formation are well
known effects of external auxin treatment (Ashton et al., 1979),
suggesting that the PpSHI1ox lines might exhibit increased auxin
levels or auxin hypersensitivity. The PpSHIox caulonemata was
hyperpigmented (Fig. 3A,B,G,H), which is another known effect of
auxin treatment (Ashton et al., 1979). Interestingly, the formation of
chloronemata from subapical caulonemal side branch initials at the
periphery of the colony appeared to be normal. Instead, the reduction
in chloronemata resulted from growth suppression and even
regression in the central older part of the colony. Thus, aged
chloronemal cells in the PpSHI1ox lines appeared to undergo
Fig. 3. Phenotypes caused by PpSHI1 overexpression.
(A-J)Representative images of PpSHI1ox-2 (B,D,F,H,J) and the control
line (A,C,E,G,I). (A,B)10-day-old colonies. (C,D)20-day-old colonies.
(E,F)Adult gametophores. (G,H)Caulonema with chloronemal side
branches derived from the central, aged part of colony. (I,J)Margin of
adult basal leaves.
necrosis: they lost chloroplasts, accumulated a water-soluble
pigment and became increasingly vacuolated, resulting in what
appeared to be empty shells (Fig. 3G,H).
Bud formation and subsequent transition into juvenile
gametophores were induced earlier in PpSHIox lines than in the
control (Fig. 3A,B). Whereas apical young leaves appeared normal,
DEVELOPMENT
Fig. 2. Auxin responsiveness of the GmGH3, but not the PpSHI2,
promoter. Mature gametophores carrying the PpSHI2pro:PpSHI2-GUS
(left) or GmGH3pro:GUS (right) reporters were harvested and
submerged in water (top) or 10M 1-NAA (auxin analogue, bottom)
for 24 hours prior to staining for GUS activity (blue) in order to test the
auxin responsiveness of the reporter genes.
1280 RESEARCH ARTICLE
Development 137 (8)
Fig. 4. PpSHI1 overexpression correlates with auxin levels and the spatial distribution of auxin activity. (A)Bar chart showing IAA
concentration [in ng per gram fresh weight (FW)] in four different PpSHI1ox lines and the control line. Each data point is the average of six
independent samples. Error bars represent s.d.; *, P<0.05 (by Student’s t-test, as compared with the control line). (B,C)GmGH3pro:GUS activity in
the control line (B) and in a PpSHI1-overexpressing line (C). Note the more abundant stem and rhizoid staining and the scattered leaf staining in the
latter.
mature leaves in the basal part of the transgenic gametophores wilted
(Fig. 3E,F), resulting in smaller and less developed shoots (Fig. 3AF). As described for aged chloronemata above, the majority of
mature leaf cells showed a reduced number of chloroplasts, pigment
accumulation and appeared to be dead or dying (Fig. 3I,J). The latter
impression was reinforced by our finding that leaves from the older,
basal part of the PpSHI1-overexpressing gametophores failed to
regenerate when excised and placed on ammonium tartrate-enriched
BCD media.
PpSHI1 overexpression causes elevated auxin
levels and activity
In order to test whether PpSHI1 overexpression could be linked to
changes in auxin homeostasis, we measured IAA levels in 23-dayold colonies of the control line and of PpSHI1ox-2, PpSHI1ox-3,
PpSHI1ox-4 and PpSHI1ox-5. Three transgenic lines exhibited
dramatically elevated IAA levels, with an average 14.5-fold increase
compared with the control (Fig. 4A). The fourth line, PpSHI1ox-3,
which did not show a significant increase, had only moderately
elevated PpSHI1 expression (see Fig. S4 in the supplementary
material) and a very subtle phenotype.
We also assayed GmGH3pro:GUS activity in the PpSHI1ox lines
to identify changes in auxin activity (Fig. 4B,C). In agreement with
previous reports (Bierfreund et al., 2003; Fujita et al., 2008),
GmGH3pro:GUS was expressed primarily in the base of the stem, in
rhizoids and in axillary hairs in the wild type. PpSHI1 overexpression
resulted in increased stem and rhizoid staining, in terms of signal
strength, penetrance and distribution. Furthermore, a weak but clear
staining of some leaf cells was also detected in PpSHI1ox lines, but
never in the control line. However, we did not observe any increase in
axillary hair staining. The differences observed generally correlated
with PpSHI1 expression levels (see Fig. S4 in the supplementary
material), being more striking in the highly expressing lines.
Taken together, the auxin and GmGH3pro:GUS expression levels
strongly suggest that PpSHI1 overexpression causes an
overproduction of auxin.
Developmental abnormalities and reduced auxin
biosynthesis in Ppshi1 and Ppshi2 knockout
mutants
We next produced two PpSHI1 (Ppshi1-1, Ppshi1-2) and two
PpSHI2 (Ppshi2-1, Ppshi2-2) gene disruption lines by homologous
recombination using the targeting constructs shown in Fig. S5A,B
in the supplementary material. We used PCR to confirm that the
wild-type loci had been properly targeted (see Fig. S5C,D in the
supplementary material) and verified the loss of the corresponding
transcripts by RT-PCR (see Fig. S5E in the supplementary material).
Southern blots confirmed that the correct locus had been targeted in
Phenotype
(A) Colony diameter (mm)
(B) Gametophores/colony
(C) Shoot length (mm)
(D) Leaves
(E) Internode length (mm)
(F) Rhizoids
(G) Most apical rhizoid
(H) Rhizoid length, 3w (mm)
(I) Rhizoid length, 5w (mm)
(J) Rhizoid length, 7w (mm)
(K) Thick brown rhizoids
(L) Axillary hairs
Wild type
Ppshi1-1
Ppshi1-2
Ppshi2-1
Ppshi2-2
10.6±1.0
48.1±9.4
6.7±0.7
28.0±4.6
0.24±0.04
126±27
6.8±1.3
5.3±1.2
5.1±0.5
6.2±0.9
19.8±6.6
2.7±0.9
8.7±0.7*
31.0±5.2*
3.1±1.1*
28.0±3.0
0.11±0.04*
152±38
13.2±2.9*
5.6±1.0
7.3±0.8*
9.8±1.1*
39.4±12.9*
4.6±1.0*
9.2±0.8*
36.0±4.5*
3.2±1.0*
27.8±3.3
0.12±0.03*
107±22
13.7±2.2*
n.d.
n.d.
n.d.
36.2±10.2*
4.7±0.9*
8.6±0.6*
34.7±5.4*
2.5±0.2*
28.2±2.6
0.09±0.01*
108±19
17.7±2.2*
5.75±0.9
7.6±1.1*
9.9±0.9*
31.5±6.9*
4.5±1.3*
8.0±0.4*
27.7±3.2*
3.2±0.5*
25.6±3.1
0.13±0.03*
125±30
12.5±1.7*
n.d.
n.d.
n.d.
47.9±8.9*
5.2±1.0*
Two- (A,B) and nine- (C-G,K,L) week-old colonies. Means and s.d. are based on the analysis of 10-12 colonies or gametophores. (A) Colony diameter calculated as the mean
of three measurements made at 45°, 90° and 135° angles. (B) Average number of gametophores per colony. (C) Average length of the shoot stem (measured after leaves had
been detached). (D) Average number of juvenile and adult leaves per gametophore. (E) Internode length calculated using the values in rows C and D. (F) Average number of
basal and mid-stem rhizoids per gametophore. (G) Average number of leaves above the most apical rhizoid. (H-J) Average length of the rhizoid bundle. Colony age is
indicated as number of weeks (w). (K) Average number of thick brown rhizoids per gametophore. (L) Average number of axillary hairs associated with each of the ten apicalmost leaves.
*P<0.001 (by Student’s t-test, as compared with wild type).
DEVELOPMENT
Table 1. Phenotypic data for knockout lines
each line and showed that Ppshi1-1 lacked additional integration
sites, whereas the other three lines also carried a small number of
line-specific random insertions (see Fig. S6 in the supplementary
material). Thus, the essentially identical phenotypes displayed by
these four lines apparently depend on the one genetic alteration they
have in common: the targeting of one of two redundant PpSHI loci.
This indicates that the two genes provide similar functions and that
one gene alone is insufficient to support wild-type growth and
development. We also found three lines that appeared to be PpSHI1
knockouts based on both PCR and RT-PCR data (not shown), but
which were phenotypically indistinguishable from the wild type, a
fact for which we currently lack an explanation.
We were unable to PCR amplify the targeted locus in any of the
four knockout lines using flanking primers (see Fig. S5F in the
supplementary material). This was probably due to the presence of
tandem repeats of the transforming DNA at the insertion site, a
common outcome after gene targeting in P. patens owing to in vivo
concatenation prior to integration (Kamisugi et al., 2006). Southern
blots confirmed this, but also revealed the inclusion of vector DNA
in some of the repeats. Concatenation can be the result of either
recombination between microhomologies, preferentially those
longer than 12 bp, or of ligation of compatible sticky ends (Murén
et al., 2009). None of our targeting fragments possesses direct
repeats longer than 12 bp, and as we mainly used different restriction
enzymes for their release from the vector, the vector was most likely
used as a linker to produce the tandem repeats.
The colony diameter and emerging gametophore number were
slightly reduced in young protonemal colonies of the knockout lines
(Table 1; Fig. 5A). Both these effects appeared to result from a less
efficient protrusion of caulonema filaments, which promote the
radial spread of colonies (Cove et al., 2006) and serve as precursors
for gametophore emergence (Ashton et al., 1979; Lehnert and Bopp,
1983). The reduced size and compact appearance of Ppshi1 and
Ppshi2 colonies were retained in older colonies.
The gametophore stem length was reduced at least 2-fold in all
four mutants, whereas the leaf number remained unaffected,
revealing that internode lengths were significantly reduced (Table
1). This phenotype, and the apparent inhibition of caulonema
proliferation, are consistent with decreased auxin levels, as treatment
with exogenous auxin produces the opposite phenotypes. Indeed, we
found that the IAA level and biosynthesis rate were both reduced in
the actively growing apical parts of Ppshi1 and Ppshi2
gametophores (Fig. 5C,D), suggesting that the PpSHI genes
normally promote auxin biosynthesis. Somewhat surprisingly,
though, the total number of rhizoids in the mutants was unaffected,
suggesting that the auxin level might not be reduced below the
threshold level for rhizoid initiation in the basal stem. Instead, a
successive increase in the difference in rhizoid length between
mutants and wild type suggested that mutant rhizoids have an
increased growth rate (Table 1) and most likely also a delayed
elongation arrest (Fig. 5A). The mutants also displayed an unusually
high proportion of thick and dark brown rhizoids (Table 1).
The occurrence of axillary hairs and emerging mid-stem rhizoids
in leaf axils of Evans Blue-stained gametophores was examined
(Table 1). Whereas in wild type the most apical mid-stem rhizoids are
found associated with a leaf axil that has an average of 6.8 discernible
leaves above it, the corresponding number was between 12.5 and
17.7 for the mutants, revealing a more basal mid-stem rhizoid
localisation. We also found an unexpected increase in the number of
axillary hairs in the mutant lines. The average number of hairs per
axil for the ten apical-most leaves varied between 4.5 and 5.2 in the
mutants, whereas there were only 2.7 hairs per axil in the wild type.
RESEARCH ARTICLE 1281
Fig. 5. Ppshi1-1 and Ppshi2-1 single-knockout phenotypes and
IAA levels. (A)Colony morphology. The left and middle columns show
2- and 6-week-old colonies, respectively, whereas the right-hand
column shows vertical slices through 7-week-old colonies of mutants
and wild type. (B)Adult gametophores with intact rhizoid bundles. The
same magnifications were used when comparing wild-type and
knockout lines. (C,D)IAA concentration (C) and IAA biosynthesis rate
(D) in apices from Ppshi1-1, Ppshi2-1 and the wild type as determined
by GC-MS analysis. The IAA biosynthesis rate was calculated as the
ratio between labelled IAA [m/z (203+204)], measured as the
incorporation of one to two deuterium atoms into the IAA molecule,
and unlabelled IAA [m/z (202)], and corrections were made for the
contribution of natural isotopic abundances to m/z 203 and m/z 204 as
described (Ljung et al., 2005). Each data point is the average of three
independent samples; error bars represent s.d.; *, P<0.05 (by Student’s
t-test, as compared with the wild type).
DEVELOPMENT
SHI genes and auxin biosynthesis in moss
Finally, we noticed a putative delay of ageing in the Ppshi1 and
Ppshi2 knockout lines. Thus, gametophores in the central part of
old wild-type colonies had a wilty phenotype with brownish stems
and leaves. By contrast, Ppshi1 or Ppshi2 mutant colonies of the
same age had mainly green and healthy gametophores, regardless
of whether a central or peripheral part of the colony was
examined.
DISCUSSION
Here, we have for the first time actively modified the endogenous
auxin levels in P. patens, providing new tools for studies of the role
of auxin in bryophytes. The changes in auxin levels were achieved
by altering the expression of the two PpSHI genes, and our data
strongly suggest that these genes cooperatively induce IAA
biosynthesis, at least in the gametophyte.
The PpSHI genes act as positive regulators of
auxin biosynthesis
The IAA level was increased more than 10-fold in lines substantially
overexpressing PpSHI1. This is consistent with previous results
from AtSTY1 overexpression (Sohlberg et al., 2006), suggesting a
conserved role of SHI/STY genes in auxin homeostasis. In
accordance, GmGH3pro:GUS expression was significantly
increased in PpSHI1ox rhizoids and gametophore stems, and
expanded to cells outside the normal GmGH3 expression domain,
causing a uniform staining of both rhizoids and gametophore stems,
as well as the staining of some leaf cells. Reduced IAA levels and
biosynthesis rates in gametophore shoots of PpSHI1 and PpSHI2
knockout lines provide additional support for the hypothesis that
SHI/STY proteins act as positive spatiotemporal regulators of auxin
biosynthesis not only in Arabidopsis, but also in moss. It would be
most interesting to investigate whether the PpSHI genes and AtSTY1
act through homologous auxin biosynthesis target genes, but our
experience from Arabidopsis (Sohlberg et al., 2006) suggests that
we need inducible overexpressor lines for such studies in order to
avoid looking at secondary effects, and we do not have access to
such moss lines yet.
Overlapping PpSHI1/2 and GmGH3 expression
suggests that local auxin biosynthesis contributes
to the auxin response maxima
The PpSHI1pro:PpSHI1-GFP and PpSHI2pro:PpSHI2-GUS
reporters were expressed in caulonema, at rhizoid initiation sites, in
rhizoids with a specific preference for apical cells, and in axillary
hairs, suggesting that these are the sites of auxin biosynthesis. They
also represent sites of high auxin activity in moss (Bierfreund et al.,
2003; Fujita et al., 2008), strongly implying that auxin response
positions are at least partially determined by spatiotemporal control
of auxin biosynthesis, in which the PpSHI genes most likely play an
important role. The one exception to the overlap between PpSHI and
auxin response activity was in developing rhizoids, where
GmGH3pro:GUS had a basal and PpSHI an apical expression
maximum. Interestingly, this apparent discrepancy could support the
existence of basipetal auxin transport in rhizoids, as suggested by
Rose and Bopp (Rose and Bopp, 1983).
Phenotypic effects of reduced PpSHI expression
are consistent with a reduction in endogenous
auxin levels
Since moss lines with verified reductions in the endogenous auxin
levels have not been described previously, it is not known what
phenotypic effects such alterations would cause. However, one
Development 137 (8)
might expect effects, i.e. reduced caulonema formation and
gametophore internode elongation, opposite to those resulting
from exogenous IAA application (Ashton et al., 1979; Fujita et al.,
2008; Johri and Desai, 1973). Indeed, the Ppshi1 and Ppshi2
single-knockout mutants possessed these expected traits. In
addition, they produced fewer gametophores per colony,
supporting previous assumptions that gametophore number is
dependent on auxin levels (Ashton et al., 1979). Interestingly, the
mutant colonies and gametophores stayed green and healthy much
longer than in the wild type and the rhizoid growth period appeared
significantly extended, suggesting a delay in growth arrest and
perhaps also ageing. Rhizoids are tip-growing filaments that have
rooting functions similar to the root hairs of vascular plants
(Menand et al., 2007). Rhizoid initiation is stimulated by
exogenous auxin (Sakakibara et al., 2003), but to our surprise we
found that the number of rhizoids in the Ppshi1 and Ppshi2
mutants was unaffected, suggesting that the auxin levels in the
rhizoid-forming stem epidermal cells remained above the
threshold level required for rhizoid formation. However, the more
basal position of mid-stem rhizoids in the mutant lines might be a
consequence of reduced auxin levels at least in the apical axillary
hair-dense part of the leafy shoot, as exogenous auxin induces a
more apical positioning of mid-stem rhizoids in wild-type
gametophores (Sakakibara et al., 2003). Furthermore, the number
of axillary hairs was significantly increased in the knockout
mutants. This suggests that the reduced PpSHI expression might
induce a compensatory programme that strives to provide the shoot
with more auxin-producing units. These data are intriguing, and
further studies of the regulatory pathways for axillary hair
formation would be very interesting.
No Ppshi1 Ppshi2 double-knockout mutants were recovered,
despite several independent attempts to make such lines, suggesting
that the PpSHI genes together perform an essential function during
the early stages of protoplast regeneration and/or colony formation.
This, together with the fact that the single-knockout lines have
identical phenotypes, strongly suggests that the two PpSHI genes act
in concert in a dose-dependent manner. The activity of the SHI/STY
proteins in Arabidopsis is also very dose dependent, and a dominantnegative suppressor construct results in seedling lethality (Kuusk et
al., 2006; Eklund et al., 2010), suggesting that the Arabidopsis
SHI/STY proteins together perform essential functions during early
development.
Ectopic PpSHI activity promotes premature cell
death
Apart from affecting the caulonemata-to-chloronemata ratio
positively, PpSHI1 overexpression also appears to induce
premature cell death in chloronemata and leafy shoots, most likely
through elevated auxin levels. Necrotic cells, exhibiting pigment
accumulation, a reduced number of chloroplasts and a loss of
regeneration capacity, were identified in older chloronemata and
leaves. Since we were unable to detect PpSHI1pro:PpSHI1-GFP,
PpSHI2pro:PpSHI2-GUS or GmGH3pro:GUS expression in leaf
cells of wild-type control lines, we conclude that the necrotic
effects are likely to be due to ectopic misexpression of the PpSHI1
protein, suggesting that leaf cells normally represent low auxin
zones, and that the ectopic PpSHI1 expression might induce
premature cell death in leaf cells. This might well apply also to
chloronemata, which are morphologically very similar to leaf cells.
Accordingly, the observed excess of caulonemata in the PpSHI1overexpression lines could, at least in part, be due to selective loss
of chloronemata.
DEVELOPMENT
1282 RESEARCH ARTICLE
Auxin appears to regulate similar developmental
decisions in vascular plants and bryophytes
Analogous to its function in vascular plants, auxin plays a role in aerial
tissue elongation in moss, as exemplified by its role in the induction
of gametophore stem elongation (our data) (Fujita et al., 2008). In
vascular plants, auxin accumulation also occurs at organ initiation
sites, both during aerial organ development and the initiation of lateral
root primordia (Benkova et al., 2003). Our data and studies using
auxin reporter constructs (Bierfreund et al., 2003; Fujita et al., 2008)
suggest that auxin in moss accumulates in rhizoid initial cells, but not
in leaf initials. Instead, the PpSHI genes and auxin response reporter
genes are expressed in axillary hairs, which are positioned close to the
lateral meristem-like sites from which mid-stem rhizoids and
adventitious shoots emerge. Their structural complexity varies
between moss species, but in P. patens we have found them to
normally consist of one large protruding cell anchored to the
epidermal cell layer via one or two small connector cells (Fig. 1F,
inset). Axillary hairs from some species secrete mucilage (Ligrone,
1986), but little else is known about their function. The mucilage has
been proposed to protect young leaves from desiccation (Schofield
and Hebant, 1984), but the hairs might also have additional functions.
It is tempting to speculate that axillary hairs contribute to the
regulation of lateral organ formation. A possible mechanism is the
secretion of auxin into the mucilage, from where it could be
transported to, and thus affect, nearby cells. We found numerous
axillary hairs associated with very young leaf primordia close to both
apical and lateral meristem-like regions, and Harrison et al. (Harrison
et al., 2009) have shown that an axillary hair is initiated on the young
bud, prior to the formation of the first leaf primordia, suggesting the
possibility that axillary hairs contribute to the regulation of organ
initiation, positioning, growth and/or differentiation. The organ in
flowering plants, which occupies a position seemingly analogous to
that of axillary hairs in mosses, is the stipule. Although future studies
will be needed to show whether stipules and axillary hairs are
functionally analogous, or even homologous, we note that stipules in
Arabidopsis represent potential sites of auxin production and
accumulation, based on the expression of auxin response markers
(Aloni et al., 2003) as well as of SHI/STY gene family members
(Fridborg et al., 2001) and their proposed targets (Cheng et al., 2007).
In conclusion, our results show that manipulations of endogenous
auxin levels are useful tools for elucidating the functional roles of
auxin in moss. We were able to test hypotheses from previous work
that used exogenous application of auxin, and were able to identify
new phenotypes that are most likely caused by ectopic auxin
production as well as by the reduction in auxin production in specific
tissues.
Acknowledgements
We thank Mitsuyasu Hasebe for the pCMAK1 plasmid, Eva Decker for the
GmGH3pro:GUS strains and Sofia Eitrem and Roger Granbom for technical
assistance. This work was supported by grants from the Carl Tryggers
Foundation to M.T., from the Swedish Research Councils Formas and VR to
E.S. and from Formas to H.R.
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.039594/-/DC1
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