Download SABRE is required for stabilization of root hair

Physiologia Plantarum 153: 440–453. 2015
ISSN 0031-9317
SABRE is required for stabilization of root hair patterning
in Arabidopsis thaliana
Stefano Pietraa,† , Patricia Langa,†,‡ and Markus Grebea,b,*
a
b
Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, Umeå SE-90187, Sweden
Institute of Biochemistry and Biology, Plant Physiology, University of Potsdam, Potsdam-Golm DE-14476, Germany
Correspondence
*Corresponding author,
e-mail: [email protected]
Received 18 March 2014;
revised 18 June 2014
doi:10.1111/ppl.12257
Patterned differentiation of distinct cell types is essential for the development
of multicellular organisms. The root epidermis of Arabidopsis thaliana is
composed of alternating files of root hair and non-hair cells and represents a
model system for studying the control of cell-fate acquisition. Epidermal cell
fate is regulated by a network of genes that translate positional information
from the underlying cortical cell layer into a specific pattern of differentiated
cells. While much is known about the genes of this network, new players
continue to be discovered. Here we show that the SABRE (SAB) gene, known
to mediate microtubule organization, anisotropic cell growth and planar
polarity, has an effect on root epidermal hair cell patterning. Loss of SAB
function results in ectopic root hair formation and destabilizes the expression
of cell fate and differentiation markers in the root epidermis, including
expression of the WEREWOLF (WER) and GLABRA2 (GL2) genes. Double
mutant analysis reveal that wer and caprice (cpc) mutants, defective in core
components of the epidermal patterning pathway, genetically interact with
sab. This suggests that SAB may act on epidermal patterning upstream of WER
and CPC. Hence, we provide evidence for a role of SAB in root epidermal
patterning by affecting cell-fate stabilization. Our work opens the door for
future studies addressing SAB-dependent functions of the cytoskeleton during
root epidermal patterning.
Introduction
Multicellular organisms comprise a variety of cells with
diverse shapes and functions. In order for coordinated
development of multicellular organisms to occur, it is
therefore essential to differentiate sets of distinct cell
types based on spatial and developmental cues. The
establishment of different fates in a seemingly homogeneous population of cells based on their relative
positions is referred to as pattern formation (Jürgens
et al. 1991). Patterned cell-fate specification has been
extensively studied in plants by analyzing the controlled
differentiation of root and leaf epidermal cells into
root hairs and trichomes, respectively (Marks 1997,
†
These authors contributed equally to this work.
Present address: Department of Molecular Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 37-39,
Tübingen DE-72076, Germany.
‡
Abbreviations – COBL9, COBRA-LIKE 9; Col, Columbia; CPC, CAPRICE; ERH2, ECTOPIC ROOT HAIR 2; ERH3, ECTOPIC ROOT
HAIR 3; GFP, green fluorescent protein; GL2, GLABRA2; GUS, 𝛽-glucuronidase; JKD, JACKDAW; kuq, kreuz und quer; MS,
Murashige & Skoog; NTF, nuclear targeting fusion; RHD6, ROOT HAIR DEFECTIVE 6; SAB, SABRE; SCM, SCRAMBLED; SCZ,
SCHIZORIZA; SEM, scanning electron microscopy; WER, WEREWOLF.
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© 2014 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.
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Hülskamp 2004, Dolan 2006, Schiefelbein et al. 2009).
These model systems provide a clear morphological
read-out to dissect the regulatory mechanisms behind
their spatial distribution (Dolan et al. 1994, Larkin et al.
1996).
The root epidermis of Arabidopsis thaliana is composed of parallel files of cells that multiply by transversal
cell divisions (Dolan et al. 1993). Each file acquires a
specific fate based on positional information from the
layer of cortical cells underneath: cells overlying the
cleft between two cortical cells are considered to be in
hair (H) position and generally produce a root hair at
maturation, thus being called trichoblasts; cells on top
of one single cortical cell are referred to as non-hair
(N) cells and usually do not produce any hairs, being
designated as atrichoblasts (Dolan and Costa 2001).
Clonal propagation of cell files throughout the root
ensures that the striped pattern of alternating cell fates
is maintained, although sporadic longitudinal divisions
can give rise to file duplications, increasing the number
of epidermal files (Berger et al. 1998).
In the leaf epidermis, trichomes are distributed evenly
at a specific distance from each other (Larkin et al.
1997). In this ‘de novo patterning’, regular spacing is
not achieved through signaling from underlying cells, but
relies on cell to cell interactions that promote trichome
differentiation in one cell while simultaneously repressing it in its neighbors (Schnittger et al. 1999).
Despite substantial differences, the pathways that
govern patterning of root hairs and trichomes are based
on similar gene networks and regulatory mechanisms,
which have been extensively studied in Arabidopsis by
molecular genetics and modeling (Schiefelbein et al.
2009, Balkunde et al. 2010, Benítez et al. 2011). Fate
selection revolves around an activator protein complex
that includes the MYB transcription factor WEREWOLF
(WER) in roots and its homolog GLABRA1 in leaves
(Ishida et al. 2008). The complex induces expression
of the homeobox transcription factor GLABRA2 (GL2),
which mediates formation of hairless cells in the root
whereas it has the opposite effect of initiating trichome
outgrowth in leaves (Rerie et al. 1994, Di Cristina et al.
1996, Masucci et al. 1996). Functionality of the activator
complex is negatively regulated by the small MYB-like
DNA-binding protein CAPRICE (CPC) and its homologues through a mechanism ensuring lateral inhibition;
these inhibitors are synthesized specifically in non-hair
cells and trichomes but then relocalize to neighboring
cells where they determine root-hair or trichome-less
fate (Wada et al. 2002, Wester et al. 2009).
While in trichomes other factors like the control of
endoreplication participate in the regulation of epidermal patterning (Bramsiepe et al. 2010), an important
Physiol. Plant. 153, 2015
contribution to breaking cell homogeneity in the root
epidermis comes from positional cues originating from
the cortex. Cortical signals, whose molecular nature is
still unknown, are likely perceived by the receptor-like
kinase SCRAMBLED (SCM), which reduces levels of the
WER transcription factor specifically in H cells (Kwak
and Schiefelbein 2007). Upstream of the patterning
gene network, the JACKDAW (JKD) zinc finger protein
operates from the cortex and through SCM to induce
asymmetric distribution of fate determinants in epidermal cells, thereby directing cell-fate specification in a
non-cell-autonomous way (Hassan et al. 2010). Another
factor acting non-cell-autonomously on specification
of epidermal fate is the heat shock transcription factor
SCHIZORIZA (SCZ), which is expressed in ground tissue
stem cells, endodermis and cortex and controls cell-fate
segregation (Mylona et al. 2002, ten Hove et al. 2010).
In mutants defective in either SCM or JKD function the
differentiation of cells in the epidermis is more irregular
with respect to their position over the cortex, while roots
of scz mutants develop more hairs and lack the alternation of hair and non-hair files (Mylona et al. 2002, ten
Hove et al. 2010).
In addition to the core genes of the epidermal patterning network, several other genes are involved in the specification and initiation of root hairs. Some of them belong
to pathways of hormone biosynthesis and signal transduction (Schiefelbein 2000, Grierson et al. 2001, Guimil
and Dunand 2006). Ethylene and auxin have long been
known to affect the development of root hairs (Masucci
and Schiefelbein 1994, Masucci and Schiefelbein 1996)
and, so far, all studies have placed their action just before
hair outgrowth and downstream of the patterning mechanism (Dolan 2001). Interestingly, auxin and ethylene,
together with other genes required for hair initiation
such as ROOT HAIR DEFECTIVE 6 (RHD6; Masucci
and Schiefelbein 1994), are also main players in the
establishment of planar polarity of root hairs (Masucci
and Schiefelbein 1994, Fischer et al. 2006). This process
controls coordinated hair emergence in a specific position within trichoblasts over the whole root epidermal
layer (Fischer et al. 2006). In this study, we analyze epidermal patterning of mutants defective in SABRE (SAB),
a large gene implicated in planar polarity of root hair
positioning that encodes for a 290 kDa protein with conserved domains of unknown function (Pietra et al. 2013).
SAB has originally been isolated and characterized as a
gene required for correct root morphogenesis and cell
expansion (Benfey et al. 1993, Aeschbacher et al. 1995).
Recently, SAB was shown to genetically interact with the
gene encoding the microtubule-associated protein cytoplasmic linker protein 170 (CLIP170)-associated protein
(CLASP). The two genes interact to stabilize orientation of
441
cell division planes and mitotic microtubule structures,
organization of cortical microtubules, and establishment
of planar polarity on both the root and leaf surface
(Pietra et al. 2013). However, a role of SAB in cell-fate
specification has not been addressed. Here, we uncover
and characterize a defect in epidermal patterning of sab
mutants that leads to the formation of ectopic root hairs
but does not alter the density of leaf trichomes. Unstable
expression of cell fate and differentiation markers in sab
mutants as well as genetic interaction of SAB with WER
and CPC, core components of the patterning pathway,
suggest that SAB function acts on the regulatory network
controlling root epidermal cell fate.
Materials and methods
Plant material and growth conditions
Arabidopsis thaliana ecotype Columbia (Col) was
used as wild-type control. The following mutant and
transgenic lines were obtained from the Nottingham
Arabidopsis Stock Centre (NASC) or the corresponding
authors of the cited publications: sab-5 and sabkuq
(Pietra et al. 2013), pGL2:NTF (Deal and Henikoff
2010), p35S:mCherry-TUA5 (Gutierrez et al. 2009,
Sampathkumar et al. 2011), pGL2:GUS (Szymanski
et al. 1998), pWER:ER-GFP and wer-1 (Lee and Schiefelbein 1999), pCOBL9:GFP (Brady et al. 2007), and cpc-1
(Wada et al. 1997). Seeds were surface sterilized and
stratified at 4∘ C for 3 days prior to plating on Murashige
& Skoog (MS) medium (1× MS salts, 1% sucrose, 1 M
morpholinoethanesulphonic acid, 0.8% plant agar, pH
5.7) (Fischer et al. 2006). Plates were grown vertically
under long-day conditions (16 h of 150 μmol m−2 s−1
light at 22∘ C and 8 h of dark at 18∘ C) with 60% humidity,
and seedlings were analyzed after 5 days.
Genotyping and generation of double mutants
The sab-5 and sabkuq mutants were genotyped as previously described (Pietra et al. 2013), and homozygous
mutant seedlings for analyses were phenotypically
identified from the progeny of heterozygous plants. F2
seedlings from the crosses between sab-5 and either
cpc-1 or wer-1 were selected for displaying the cpc-1
or wer-1 phenotype. They were genotyped for sab-5
heterozygosity and then tested to confirm cpc-1 or
wer-1 homozygosity using the gene-specific primers
CPC_F (TCT TGT CTT GTG AAT TAA GGA GAG G)
and CPC_R (CAG GTT AGA GAC TCT TT TCT CTC
G) or WER_F (CTT CAT TTG CTC AAA ATT AGT TTC
TC) and WER_R (TGA ACC CAA AGT GAA CTC AAG
TAG). Polymerase chain reaction (PCR) products of
442
WER_F and WER_R were digested with MboI allowing
genotyping with a cleaved amplified polymorphism
(CAPS) marker. Progeny of the selected plants showed
166/674 (24.6%) and 161/704 (22.9%) seedlings with
a phenotype clearly different from cpc-1 and wer-1,
respectively, and these were considered double mutants
and used in the following analyses.
Light microscopy
Bright field microscopy images of 5-day-old seedlings
and root close-ups were acquired with a Leica MZ 9.5
stereomicroscope (Leica Microsystems, Wetzlar, Germany) equipped with a Leica DC 300 digital camera.
𝛽-Glucuronidase (GUS)-stained roots and roots for hair
patterning quantification were visualized with the 2.5×
or 40× objectives of an Axioplan 2 microscope (Zeiss,
Göttingen, Germany) equipped with Nomarski optics.
Images were acquired with an Axiocam digital camera
(Zeiss) using the AXIOVS40 V 4.8.2.0 software (Zeiss),
and processed with IMAGEJ (http://imagej.nih.gov/ij/) and
ADOBE PHOTOSHOP CS4 (Adobe Systems, San Jose, CA).
Quantitative analysis of root hair patterning
Seedlings were grown for 5 days and then mounted
on microscope slides in clearing solution (80 g chloral
hydrate, 30 ml glycerol and 10 ml dH2 O). Analysis
was performed on three independent experiments per
genotype, using at least 10 seedlings per experiment.
Differentiated non-consecutive epidermal cells were
randomly selected over the whole root, avoiding the
region of transition between root and hypocotyl. Cells
were scored for whether they carried a root hair (or
root-hair bulge) or not, and their position with respect
to the underlying cortical cell layer was determined by
focusing through the entire epidermal layer: cells on top
of a single cortical cell file were considered in N position, while cells on the cleft between two cortical cell
files were considered in H position (Berger et al. 1998).
Significance of the differences between genotypes was
then analyzed by Fisher’s exact test.
Scanning electron microscopy of leaves
The third true leaf of 21-day-old Col and sab-5 plants
was processed for scanning electron microscopy (SEM)
as previously described (Pietra et al. 2013) and examined with a Stereoscan 360iXP microscope (Cambridge
Instruments, Cambridge, UK). For each sample, three
micrographs were recorded over the adaxial leaf surface:
one overview image for counting the total number of
trichomes and measuring the leaf area, and two images
Physiol. Plant. 153, 2015
at higher magnification for estimating the number of
cells per leaf. The latter were close-ups of 300 × 400 μm
regions located at one quarter and three quarters of the
distance between the leaf tip and the petiole, halfway
between central vein and leaf margin. Micrographs from
at least 10 samples per genotype were analyzed; number
of trichomes and cells were counted manually and leaf
outlines were drawn using the polygon selection tool of
IMAGEJ to calculate leaf area. Data were processed with
Microsoft Excel.
Analysis of pGL2:GUS activity
GUS activity was histochemically detected as previously
described (Men et al. 2008). In brief, 5-day-old whole
seedlings were stained for 8 h at 37∘ C, fixed for 1 h
in 4% paraformaldehyde, cleared with 8:3:1 chloral
hydrate:glycerol:distilled water and mounted on microscope slides for observation. To determine the frequency
of gain or loss of pGL2:GUS activity, roots of sab-5
mutants were analyzed from the first rapidly elongating
cells to the transition zone between root and hypocotyl.
In the wild-type counting was done on a 4.6 mm long
section of the root starting from the meristem, a region
1.25 times the average length of a sab-5 mutant root.
As epidermal cells are on average about 25% shorter
in sab-5 mutants compared with wild-type (Pietra et al.
2013), this allowed analyzing an equivalent number of
cells, compensating for the differences in root length
and cell elongation between wild-type and sab-5. Ten
seedlings per genotype for each of two experiments
were analyzed. A loss or gain of pGL2:GUS activity was
scored as a single event regardless of how many consecutive cells in one file it involved. Re-establishment
of the original status of pGL2:GUS activity after a fate
switch was not considered as an additional switching
event, while independent alternations in the same file
were counted individually.
Confocal laser scanning microscopy
Live imaging of lines expressing fluorescent fusions was
performed with a Zeiss LSM780 spectral confocal laser
scanning microscopy (CLSM) system mounted on a Zeiss
Axio Observer Z1 inverted microscope. Five-day-old
seedlings were mounted on cover slips in MS medium
and observed with a water-corrected C-Apochromat
40× 1.2 numerical aperture (NA) objective (M27, Zeiss).
Laser excitation lines were 488 nm for green fluorescent
protein (GFP) [and nuclear targeting fusion (NTF)] and
561 nm for mCherry, emission filters were 493–556 nm
for GFP (and NTF) and 586–647 nm for mCherry. To
Physiol. Plant. 153, 2015
determine the frequency of fate switches in lines expressing the pGL2:NTF marker, 10 roots of each genotype
were analyzed from the meristem to the transition zone
between root and hypocotyl, and scoring was done
following the same criteria as for pGL2:GUS. For top
views and cross-sections of lines expressing fate or
differentiation markers and mCherry-TUA5 (TUBULIN
ALPHA-5), Z stacks of planes at 0.45 μm distance were
acquired over the periclinal face of roots. Top views were
maximum intensity projections of the stacks, while for
cross-sections slices of 3–50 μm were cut transversally
out of the stacks and rotated three-dimensionally. Images
were processed and analyzed using IMAGEJ and ADOBE
PHOTOSHOP CS4, and assembled in Adobe Illustrator CS3
(Adobe Systems).
Statistical analysis
Significance of the differences between the number
of root hair- and non-hair cells found in H position
and N position in different genotypes were assessed by
Fisher’s exact test with populations of >150 cells for
each position from at least 30 roots and three independent experiments, and significance level P < 0.05 (http://
quantitativeskills.com/sisa/statistics/fisher.htm). Analysis
of the number of cell fate switches detected with the
pGL2:GUS and pGL2:NTF reporters in wild-type and
sab-5 roots was performed by two-tailed, unpaired
t-test with equal variance and significance level P < 0.05
(computed with Microsoft Excel), with n = 3 independent
experiments of 10 seedlings per genotype.
Results
sab mutants have defects in root hair patterning
The Arabidopsis root epidermis, with its regular pattern
of hair-forming trichoblast files alternating with one
or two files of hairless atrichoblasts, provides an ideal
model to study epidermal cell-fate determination (Dolan
et al. 1994; Fig. 1A). Here, we investigated whether the
SAB gene is involved in patterning of epidermal cells.
Benfey et al. (1993) reported that sab mutations do not
cause abnormalities in radial symmetry and number of
epidermal cells in the roots. Intriguingly, observation of
sab mutant roots revealed an increased amount of hairs
ectopically growing in non-hair files right next to other
hair-bearing cells (Fig. 1B, C, arrowheads). In order
to quantify the sab epidermal patterning defects, we
calculated the proportion of cells that displayed a hair in
either H position (over the anticlinal walls of two cortical
cells) or N position (over the outer periclinal wall of a
single cortical cell) for Col wild-type and two sab mutant
443
A
D
Col
E
Col
B
Col
F
sab-5
C
sab-5
G
sabkuq
sab-5
Fig. 1. sab mutants are defective in root hair patterning, but not in
leaf trichome spacing. (A–C) Bright field microscopy images of roots of
5-day-old (A) Col, (B) sab-5 and (C) sabkuq seedlings. Regular alternation
of hair- and non-hair cell files in (A) Col is compromised in (B–C) sab
mutants, where root hairs can emerge from adjacent files (arrowheads).
(D–G) Scanning electron microscopy images of the third true leaf of
21-day-old (D–E) Col and (F–G) sab-5 plants. (D, F) Overall views of the
adaxial leaf surface and (E, G) close-ups of epidermal cells located at one
quarter of the distance between the leaf tip and the petiole, halfway
between the central vein and leaf margin. Note, different total leaf area
and cell density between Col and sab-5 (see Table 2 for quantification).
Scale bars = (A–C, E, G) 100 μm, (D, F) 1 mm.
alleles, sab-5 and sabkreuz und quer (sabkuq ; Table 1). In Col
roots about 73% of the cells in H position carried a hair,
and this percentage was similar in mutant roots (65.2%
for sab-5 and 66.5% for sabkuq ). However, sab-5 (20.5%)
and sabkuq (19.2%) mutants had more than four times as
many hair cells in N position than Col (4.6%), indicating
that in sab-5 and sabkuq a considerable number of cells
in the N position did not follow their developmental program, resulting in a higher amount of ectopic hairs than
in the wild-type. This strongly suggested that sab mutants
have a patterning defect in the root epidermis, and that
SAB might be required for either the specification or
execution of epidermal cell fate.
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Leaf trichome density is not affected
in sab-5 mutants
Several determinants of root epidermal cell fate such as
GLABRA 2, GLABRA 3, TRANSPARENT TESTA GLABRA 1,
TRIPTYCHON and CAPRICE are also involved in controlling patterning of trichomes on the leaf epidermis
(Koornneef 1981, Hülskamp et al. 1994, Wada et al.
1997). The presence of a defect in root hair patterning
of sab mutants raised the question as to whether the
SAB gene may as well be involved in specification and
spacing of leaf trichomes.
To this end, we acquired SEM images of the third
true leaf of 21-day-old Col wild-type and sab-5 mutant
plants (Fig. 1D, F) and calculated the average number
of trichomes per leaf, revealing that sab-5 leaves had
almost half as many trichomes as wild-type (Table 2).
In order to compensate for the smaller size of mutant
leaves (Aeschbacher et al. 1995, Pietra et al. 2013) and
a potential difference in cell density between sab-5 and
wild-type, we measured total leaf areas and counted the
number of epidermal cells in 0.12 mm2 regions on the
adaxial surface of wild-type and sab-5 leaves (Fig. 1E,
G; Table 2). We confirmed that wild-type leaves are over
four times larger than those of sab-5, and uncovered a
much higher density of cells in the sab-5 mutant leaf
epidermis. This allowed us to estimate the total number
of epidermal cells per leaf in wild-type and sab-5 and
determine the average number of cells per trichome
in each of the two genotypes (Table 2). Strikingly, this
ratio was very similar between the two, suggesting that
in sab mutants the relative amount of leaf epidermal
cells that develop into trichomes does not differ from
the wild-type. Thus, SAB does not likely have a role in
regulating the density of trichome placement in the leaf
epidermis suggesting that its effect on hair patterning may
be root-specific.
Cell file divisions and subsequent cell-fate
duplications do not account for increased ectopic
hair formation in sab-5
Root epidermal cells typically divide transversely to
the direction of root growth, generating clonal files of
cells that all originate from the same initial. However,
as already observed by Berger et al. (1998), in rare
cases one longitudinal division can determine a file
duplication, initiating two daughter clones of cells. As
the SAB gene has been shown to have an important role
in cell division plane orientation (Pietra et al. 2013),
we tested if a higher number of aberrant longitudinal
divisions and a preferential duplication of the H cell
fate could explain the increased number of hair cells
observed in sab mutants. We counted the number
Physiol. Plant. 153, 2015
Table 1. sab-mutant roots display hair-patterning defects. Percentage of root hair and non-hair epidermal cells in H position (on top of the border
between two cortical cell files) and N position (on top of one single cortical cell file) in 5-day-old seedlings of wild-type Col and two sab mutant alleles.
Average value and standard deviation between three independent experiments are displayed. In H position: P = 0.0693 Col vs sab-5, P = 0.1025 Col
vs sabkuq . In N position: P = 2.3 × 10−6 Col vs sab-5, P = 2.3 × 10−5 Col vs sabkuq by Fisher’s exact test, n indicated in parenthesis.
Cells in the H position
Col
sab-5
sabkuq
Cells in the N position
Hair cells
(%)
Non-hair cells
(%)
Hair cells
(%)
Non-hair cells
(%)
73.2 ± 1.8 (249)
65.2 ± 5.1 (118)
66.5 ± 2.6 (141)
26.8 ± 1.8 (91)
34.8 ± 5.1 (63)
33.5 ± 2.6 (71)
4.6 ± 2.8 (9)
20.5 ± 7.1 (40)
19.2 ± 8.2 (30)
95.4 ± 2.8 (185)
79.5 ± 7.1 (155)
80.8 ± 8.2 (126)
Table 2. Trichome patterning is unaffected in sab-5 mutants. Quantification of the number of trichomes and epidermal cells on the adaxial face of the third true leaf of 21-day-old wild-type Col and sab-5
plants through analysis of SEM images, and subsequent calculation of
the cells/trichome ratio. Number of trichomes per leaf represents average ± standard deviation from 21 and 27 leaves for Col and sab-5,
respectively. Total leaf area and number of epidermal cells represent average ± standard deviation from 10 leaves.
Col
Trichomes per leaf
Total leaf area
Epidermal cells per
0.12 mm2 leaf area
Estimated epidermal cells
per leaf
Average epidermal cells
per trichome
90 ± 20.68
50.66 ± 12.72 mm2
22 ± 15.01
sab-5
49 ± 12.52
11.41 ± 1.70 mm2
53 ± 14.60
9057
5049
101
104
of file duplications in wild-type and sab-5, and used
the fluorescent pGL2:NTF marker (Deal and Henikoff
2010) and the pGL2:GUS reporter construct (Szymanski et al. 1998), which are specifically expressed in
atrichoblast cells (Masucci et al. 1996), to assess the
identity of epidermal cells in files that had undergone
duplication.
In 30 wild-type seedlings that expressed pGL2:GUS
we observed 17 file duplication events, while in the
same number of seedlings with the sab-5 phenotype we
only found 15. These results indicated that mutations
in SAB do not induce a higher number of longitudinal
divisions in the root meristem. Moreover, when considering the identity of files undergoing division, 16 of 17
duplications in the wild-type took place in H files and in
most cases (n = 14/16) gave rise to one H and one N file
(Fig. 2A, B), with the exception of two events in which
both daughter files acquired H fate (Fig. 2C). On the
other hand, only 9 of 15 duplications in sab-5-looking
seedlings occurred in H files, and also in this case they
mainly produced one H and one N file (n = 8/9; Fig. 2D,
E), except for one event that resulted in two N files.
In wild-type roots, we observed longitudinal divisions
Physiol. Plant. 153, 2015
in N files only once (n = 1/17); in contrast, longitudinal divisions in N files represented 40% of the events
in sab-5 (n = 6/15) and in all cases led to a duplication
of the N fate (Fig. 2F). These findings clearly indicated
that sab-5 mutants did not have a bias toward divisions
that would increase the number of H cells but on the
contrary showed more duplications of N fate than
wild-type seedlings. Taken together, file duplications are
unlikely to account for the higher occurrence of ectopic
hairs in sab mutants, and the effect of SAB on epidermal pattering may not directly depend on SAB function
during the orientation of cell division.
sab-5 affects stable expression of fate
and differentiation markers in elongated root
epidermal cells
Root epidermal cell files acquire their hair or non-hair
fate early in the elongation zone (Dolan et al. 1994). This
fate is then generally maintained in the whole clonal
progeny of the early cells, and translates in the homogeneous expression of fate markers throughout an entire
cell file (Masucci et al. 1996, Lee and Schiefelbein
1999). We could confirm this homogeneous expression within files when observing the N-fate markers
pGL2:NTF and pGL2:GUS in the epidermis of wild-type
roots: here files of cells that consistently expressed the
marker alternated with files of cells that did not show
any marker expression (Fig. 3A, B; Fig. S1A, Supporting
information). However, in rare instances we noticed one
or more cells of different fate compared with the other
cells of the file they belonged to, causing two consecutive cells of the same file to display opposite marker
expression (Fig. S1B). When studying expression of the
markers in sab-5-looking roots we repeatedly observed
similar fate alternations, leading to cells expressing
N-fate markers in trichoblast files and cells not expressing them in atrichoblast files (Figs 3C–E and S1C, D).
To assess whether fate alterations were more frequent
in sab-5 than in wild-type, we quantified their number
analyzing 10 seedlings expressing the pGL2:NTF marker
445
A
B
C
wt
D
wt
E
wt
F
sab-5
sab-5
sab-5
Fig. 2. Duplication of root epidermal cell files can lead to differential cell
fates in daughter files. (A, D) Live imaging and (B, C, E, F) GUS staining of
(A–C) wild-type (wt) and (D, F) sab-5 seedlings expressing N-fate markers. (A, D) Confocal laser scanning microscopy images of meristematic
root epidermal cells where N fate is indicated by expression of the nuclear
pGL2:NTF marker gene (green) and cell outlines are labeled with the
tubulin marker mCherry-TUBULIN ALPHA-5 (mCherry-TUA5; magenta).
Note the duplication of H files resulting in one H file and one N file,
which in D is composed of only three cells. (B, C, E, F) Representative differential interference contrast (DIC) images from a total of 30 wild-type
and 30 sab-5-looking seedlings expressing the pGL2:GUS marker, where
blue color indicates N fate. (B, E) H file dividing into one H file and one
N file; representative images of 14 and 8 similar divisions observed in
wild-type and sab-5, respectively. (C) Duplication of an H file into two
H daughter files, representative of two images from wild-type seedlings.
Note that the two daughter files return one file after only four cells. (F)
Division of an N file resulting in the duplication of the N fate, representative of events observed once in wild-type and six times in sab-5. One
additional event (not shown) involved a sab-5 H file dividing into two N
files. Arrowheads indicate file divisions and return of duplicated files to
one file. Scale bars = 10 μm.
and 20 seedlings carrying the pGL2:GUS reporter for
each genotype. Strikingly, in all experiments the number
of alternations in seedlings with the sab-5 phenotype
was almost double than in wild-type, adding up to
significantly different totals of 124 and 73 events,
446
respectively (P = 0.00135 by t-test; n = 3 experiments
of 10 seedlings each). Hence, SAB was required for
stabilizing the expression of the pGL2 promoter in the
root epidermis, although it was still unclear whether this
role extended to epidermal fate specification in general.
In order to test if the transcriptional fusion of another
positive regulator of non-hair cell fate was affected in a
similar way, we analyzed expression of the pWER:GFP
marker (Lee and Schiefelbein 1999) in wild-type and
sab-5 roots. Strikingly, also in this case the number of
fate alternations was substantially higher in the sab-5
mutant compared with wild-type (Fig. 3F–J), supporting the hypothesis that overall stabilization of N fate
was impaired in sab-5. Finally, to test whether cells with
H fate were also influenced by the lack of SAB activity, we examined expression of the pCOBRA-LIKE 9:GFP
marker, which is specifically active in hair cells [pCOBL9
(COBRA-LIKE 9):GFP; Brady et al. 2007]. Here, we similarly observed a much more irregular expression pattern
in epidermal cell files of sab-5-mutant roots, when compared with the uniform and stable activity of the promoter in wild-type roots (Fig. 3K–O). Taken together,
these results indicated that lack of SAB function destabilizes the file-specific expression of cell fate and differentiation markers in N and H cell files. Our findings suggest
that SAB function may either contribute to determine or
to reinforce cell-fate specification in the root epidermis.
Genetic interactions between SAB and WER or
CPC, two core regulators of root epidermal cell fate
To further test SAB function in root epidermal cell-fate
determination, we investigated whether sab mutants
genetically interact with mutants of genes known to regulate epidermal cell patterning. The WER MYB protein is
one of the core components of the complex that induces
N fate in root epidermal cells, and loss-of-function
wer-1 mutants display strongly increased formation of
hairs in N position, resulting in characteristic ‘hairy’
seedlings (Lee and Schiefelbein 1999; Fig. 4A–B, G–H).
As activity of the pWER promoter was affected by
mutation of the SAB gene (Fig. 3F–J), we generated
sab-5;wer-1 double mutants and analyzed their patterning phenotype to test for potential functional genetic
interaction. sab-5 seedlings showed reduced growth and
shorter, swollen roots compared with Col wild-type
(Pietra et al. 2013; Fig. 4A, D), and the overall phenotype
of sab-5;wer-1 double mutants was very similar to that of
sab-5 mutants (Fig. 4D, E). However, closer observation
of double-mutant roots revealed a clearly higher amount
of ectopic hairs (Fig. 4J, K). Accordingly, quantitative
analysis confirmed a significant difference in the number of ectopic root hairs in N position between sab-5 and
Physiol. Plant. 153, 2015
pCOBL9:GFP;mCherry-TUA5
pWER:GFP;mCherry-TUA5
pGL2:NTF;mCherry-TUA5
A
C
D
before
*
sab-5
E
after
wt
B
*
wt
sab-5
sab-5
F
H
I
before
*
*
sab-5
J
after
*
wt
*
G
wt
sab-5
sab-5
K
M
N
before
*
*
*
sab-5
O
*
wt
*
after
*
L
wt
sab-5
sab-5
Fig. 3. SAB stabilizes expression of cell fate and differentiation markers along root epidermal cell files. (A–E) Live imaging of (A, B) wild-type and
(C–E) sab-5 mutant seedlings expressing the N-fate nuclear marker pGL2:NTF (green). (F–J) Visualization of the endoplasmic reticulum (ER)-localized
N-fate marker pWER:GFP (green) in epidermal cells of (F, G) wild-type and (H–J) sab-5 seedlings. (K–O) Imaging of the hair-cell differentiation marker
pCOBL9:GFP (green) in roots of (K, L) wild-type and (M–O) sab-5 seedlings. All seedlings also express the tubulin marker mCherry-TUA5 (magenta),
indicating cell outlines. (A, C, F, H, K, M) Top views showing the stable expression of fate and differentiation markers along meristematic cell files of
wild-type roots and the disturbed patterning of the markers in cell files of sab-5 mutants. (B, D–E, G, I–J, L, N–O) Cross-sections displaying regular
patterning of wild-type epidermal cells with respect to the underlying cortical layer, as opposed to differences in fate between cells of the same file
displayed in consecutive sections of sab-5 seedlings. Upper images (before) show marker expression before an observed difference in fate, and lower
images (after) display a section of the same seedling taken closer to the root tip. Differential fate of the cells marked by an asterisk, which belong to
the same cell file. (I, J) Dotted lines with arrowheads indicate the slightly twisted orientation of cells in sab-5 mutant roots. Scale bars = 20 μm.
both wer-1 and sab-5;wer-1 mutants (Table 3), while no
significant difference was observed between wer-1 and
the double mutant (Table 3). The number of hairs in H
position did not differ between sab-5 and wer-1, and was
thus not indicative for evaluating interaction between the
two mutants. Together, these findings suggested that the
Physiol. Plant. 153, 2015
wer-1 defect in non-hair epidermal cell-fate specification
is epistatic to the phenotype of sab-5 mutants.
In order to also address potential genetic interaction
between SAB and a gene promoting H fate of root epidermal cells, we crossed sab-5 with cpc-1, a knock-out
mutant defective in the positive regulator of hair fate
447
A
Col
B
C
wer-1
cpc-1
D
G
J
sab-5
Col
sab-5
E
H
K
sab-5;
wer-1
wer-1
sab-5;
wer-1
F
I
L
sab-5;
cpc-1
cpc-1
sab-5;
cpc-1
Fig. 4. Double mutant analysis reveals genetic interaction of WER and CPC with SAB in root hair patterning. Seedling and root hair patterning
phenotypes of sab-5;wer-1 and sab-5;cpc-1 double mutants. (A–F) Five-day-old seedlings of (A) Col wild-type, of (B) wer-1, (C) cpc-1 and (D) sab-5
single mutants, and of (E) sab-5;wer-1 and (F) sab-5;cpc-1 double mutants; small seedlings with curved cotyledons and short, thick root in sab-5;wer-1
and sab-5;cpc-1, similar to sab-5. (G–L) Bright field microscopy images of the root epidermis of 5-day-old (G) Col, (H) wer-1, (I) cpc-1, (J) sab-5,
(K) sab-5;wer-1 and (L) sab-5;cpc-1 seedlings. wer-1 and sab-5 mutants display an increased number of ectopic root hairs, although to different
extents, while cpc-1 mutants almost completely lack hairs throughout the whole root (see Table 3 for quantification). Note how, with respect to root
hair patterning, sab-5;wer-1 and sab-5;cpc-1 double mutants closely resemble wer-1 and cpc-1 single mutants, respectively, rather than sab-5. Scale
bars = (A–C) 1 mm, (D–F) 500 μm, (G–L) 100 μm.
Table 3. Genetic relationship of wer-1 and cpc-1 with sab-5 in root-hair patterning. Percentage of root hair and non-hair epidermal cells in H and N
positions in 5-day-old sab-5, wer-1, cpc-1 single-mutant and sab-5;wer-1, sab-5;cpc-1 double-mutant seedlings. Average value and standard deviation
among three independent experiments are displayed. In H position: P = 0.0522 sab-5 vs wer-1, P = 5.9 × 10−24 sab-5 vs cpc-1, P = 0.6380 sab-5 vs
sab-5;wer-1, P = 2.0 × 10−30 sab-5 vs sab-5;cpc-1, P = 0.0114 wer-1 vs sab-5;wer-1, P = 0.3734 cpc-1 vs sab-5;cpc-1. In N position: P = 4.9 × 10−10
sab-5 vs wer-1, P = 0 sab-5 vs cpc-1, P = 2.5 × 10−7 sab-5 vs sab-5;wer-1, P = 0 sab-5 vs sab-5;cpc-1, P = 0.2960 wer-1 vs sab-5;wer-1, P = 1 cpc-1 vs
sab-5;cpc-1 by Fisher’s exact test, n indicated in parenthesis.
Cells in the H position
sab-5
wer-1
cpc-1
sab-5;wer-1
sab-5;cpc-1
Hair cells (%)
Non-hair cells (%)
Hair cells (%)
Non-hair cells (%)
75.7 ± 3.2 (143)
84.2 ± 1.7 (155)
23.1 ± 4.1 (39)
73.2 ± 8.3 (139)
19.2 ± 2.7 (39)
24.3 ± 3.2 (46)
15.8 ± 1.7 (29)
76.9 ± 4.1 (130)
26.8 ± 8.3 (51)
80.8 ± 2.7 (164)
24.0 ± 4.7 (46)
55.6 ± 1.9 (100)
1.7 ± 1.8 (3)
49.7 ± 2.7 (93)
1.4 ± 1.0 (3)
76.0 ± 4.7 (146)
44.4 ± 1.9 (80)
98.3 ± 1.8 (175)
50.3 ± 2.7 (94)
98.6 ± 1.0 (214)
CPC (Wada et al. 1997). The cpc-1 mutation causes the
formation of only a few and irregularly distributed hairs
(Wada et al. 1997; Fig. 4C, I), which was strikingly and
significantly different compared with the sab-5 phenotype when analyzing the proportion of both H cells and
N cells bearing root hairs (Table 3). Intriguingly, as in
the case of sab-5;wer-1 double mutants, sab-5;cpc-1
seedlings had an overall phenotype resembling that of
448
Cells in the N position
sab-5 single mutants, i.e. impaired growth and short
bloated roots (Fig. 4F). However, all sab-5;cpc-1 mutants
displayed extremely few root hairs (Fig. 4L), in a manner highly similar to the cpc-1 mutant. Quantitative
analysis demonstrated that while the percentage of hair
cells in H and N position was significantly different
between sab-5;cpc-1 and sab-5, no difference could
be detected between sab-5;cpc-1 and cpc-1 (Table 3).
Physiol. Plant. 153, 2015
Hence, we concluded that the cpc-1 mutation is able
to completely suppress the sab-5 root hair patterning
phenotype, indicating an epistatic relationship similar to
the one discovered for wer-1. Thus, our findings suggest
that SAB genetically interacts with WER and CPC to
determine root epidermal hair cell fate and its stable,
ordered patterning in the root epidermis.
Discussion
Epidermal cell differentiation in Arabidopsis is controlled
by a well-studied network of genes that directs emergence of trichomes and root hairs in a regular pattern.
Some of the components that induce root hair initiation are also involved in determining planar polarity of
hair positioning, however, a few of those have a role in
epidermal cell-fate determination. In this study, we discover that the SAB gene, recently shown to be a player in
planar polarity establishment, does also affect root hair
patterning. SAB interacts genetically with core genes of
the patterning pathway and lack of SAB function affects
the expression of cell fate markers, suggesting that SAB
function is required for stable cell-fate patterning in the
root epidermis.
SAB affects patterning in the root epidermis
Our analyses of root hair-forming epidermal cells clearly
revealed that sab mutants have an increased amount of
ectopic hairs in the N position. This is similar to what has
been shown for a number of mutants in genes involved in
N fate specification, including GL2 and WER (Di Cristina
et al. 1996, Masucci et al. 1996, Lee and Schiefelbein
1999), and indicates that SAB has an effect on patterning
of root epidermal cells.
The excessive number of ectopic hairs in sab mutants
cannot be justified by an increased amount of H fate
duplications in epidermal cell files of sab roots, as the
frequency of file duplications in sab mutants was even
lower than in wild-type. When considering the fate of
files affected by duplications, in control seedlings 94%
of the files were in H position; this is in line with the
findings of Berger et al. (1998), who reported that these
events are up to 20 times more frequent in H files than in
N files. Surprisingly, however, in sab mutants only 60%
of the duplications occurred in H files, suggesting that
SAB might also function in selection of the fate of cells
undergoing aberrant longitudinal divisions. A similar
defect has been observed in mutants defective in the core
pathway of epidermal cell-fate specification (Berger et al.
1998), and is consistent with an involvement of SAB in
epidermal patterning.
Physiol. Plant. 153, 2015
Intriguingly, ectopic hairs are frequently found in
mutants of the ECTOPIC ROOT HAIR 3 (ERH3) gene,
which encodes a p60 katanin involved in microtubule
severing and re-organization (Webb et al. 2002). Strikingly, SAB also directs microtubule organization, and
similarly to ERH3 it is required for proper arrangement
of mitotic microtubule structures, division plane orientation and branching of leaf trichomes. In addition, katanin
mutants have defects in anisotropic growth of cells in
the shoot apical meristem, resulting in abnormal tissue
morphogenesis (Uyttewaal et al. 2012). This suggests
analogies in the way ERH3/KATANIN and SAB affect
epidermal patterning, consistent with the view that SAB
function in cell-fate determination may be based on its
effects on microtubules.
Furthermore, the impaired regulation of anisotropic
cell growth is equally obvious in erh3 roots, which are
radially swollen compared with wild-type (Schneider
et al. 1997). This is true as well for the ectopic root hair
2/pom-pom 1 mutant, which also shows an increase of
ectopic root hairs (erh2/pom1; Schneider et al. 1997),
indicating a possible link between root hair initiation
and radial cell expansion. This hypothesis is interesting considering that the increased radial dimension of
erh2/pom1 is mainly due to abnormal expansion of the
cortical cell layer (Hauser et al. 1995), which is also
a morphological feature of sab mutants (Benfey et al.
1993). Therefore, SAB function during anisotropic cell
expansion, particularly of cortical cells, may have an
influence on epidermal patterning, although this would
require future testing.
Furthermore, enlargement of cortical cells is caused by
iron deficiency in plants (Schmidt 1999) and Fe-deficient
conditions are known to increase root hair formation in
N position, an adaptation that is also typical of reduced
phosphorus bioavailability (Schmidt and Schikora 2001).
Interestingly, sab mutants exhibit enhanced sensitivity to
P starvation (Yu et al. 2012). Thus, a higher amount of
ectopic hairs could theoretically be explained by a hyperactive physiological response inducing hair outgrowth
even under normal P availability. Such a scenario, however, could neither explain the unstable expression of
fate markers in sab mutants nor the epistasis of core patterning pathway components over SAB, because P and
other environmental factors have been found to act on
epidermal cell differentiation downstream of the patterning pathway (Müller and Schmidt 2004).
Similarly, it is unlikely that induction of ectopic root
hairs in sab is the consequence of an effect on ethylene
signaling. SAB has been proposed to act antagonistically
to ethylene (Aeschbacher et al. 1995, Yu et al. 2012), and
ethylene is required for promoting root hair outgrowth
(Tanimoto et al. 1995), so it may be argued that loss of
449
SAB function could induce ethylene signaling and emergence of hairs in N position. However, again, ethylene
affects root hair differentiation downstream of GL2 and of
the patterning gene network (Masucci and Schiefelbein
1996). Therefore, the effect of sab on fate markers and its
genetic interaction with wer and cpc are not consistent
with such a scenario. Taken together, our data show that
SAB is a novel gene that plays a role in establishment of
root epidermal cell fate likely acting upstream of WER
and CPC function, and future work can reveal the exact
mechanisms involved.
The effect of SAB on epidermal patterning
is restricted to the root
The gene network that determines cell fate and regular
spacing of Arabidopsis leaf trichomes closely resembles
the one directing patterning of epidermal non-hair fate in
roots. Accordingly, many mutants that display aberrant
root hair patterning display opposite defects in trichome
density; mutants with fewer root hairs have an increased
number of trichomes and vice versa. We therefore speculated that the newly discovered effect of SAB on root
epidermal patterning could be extended to leaf trichome
patterning. This hypothesis was appealing, as SAB has a
role in branching of leaf trichomes and establishment of
planar polarity in leaves as well as in roots (Pietra et al.
2013). However, quantitative analysis revealed a very
similar number of epidermal cells per trichome in sab
mutant leaves compared with the wild-type, indicating
that SAB does not contribute to leaf trichome density or
patterning.
SAB may act upstream of WER and CPC
in patterning of root epidermal cell fate
Valuable information on the relationship between SAB
and the core network of epidermal cell differentiation
came from studying the expression of fate makers in
sab background and the genetic interaction of sab with
mutants in components of the patterning pathway.
Expression of reporters marking either H or N cells
in the root became more randomized upon loss of SAB
function. The pCOBL9:GFP reporter is active in tip
growth of root hairs (Brady et al. 2007), and destabilization of its expression suggested not only that SAB affects
H cells as well as N cells, but also that SAB function has
an effect on selection of cell fate, and not on hair initiation or outgrowth downstream of COBL9. The analysis of
promoter GL2 reporter lines further supported this idea,
considering that GL2 transcription is a target of the central activator complex that regulates epidermal cell fate
(Schiefelbein et al. 2009); as lack of SAB activity induced
450
changes in GL2 expression, SAB is likely to act upstream
of GL2. Finally, we observed that similar expression of
pWER:GFP was less stable in sab mutants, which could
either imply that SAB functions earlier than WER or that it
feeds on WER transcription. Of the three genes analyzed,
WER is the one acting earliest in fate determination, and
destabilization of its expression could explain the GL2
and COBL9 marker phenotypes, further supporting an
effect of SAB upstream of these latter components.
However, when analyzing the effect of sab on WER
expression it is hard to explain the root hair patterning
defect of sab mutants; the increased number of fate
alternations in sab was not consistently affecting one or
the other cell fate, and thus did not account for a higher
amount of ectopic hairs in N position. Hence, SAB seems
to affect maintenance of the same fate within cell files
rather than the specification of one or the other cell fate.
Nevertheless, additional clues on the effect of SAB on
cell-fate determination came from studying its genetic
interaction with the WER and CPC genes. Overall
seedling phenotypes of the sab;wer and sab;cpc double
mutants closely resembled those of sab single mutants,
indicating that with respect to its roles in seedling
morphology SAB is epistatic to WER and CPC (Fig. 4).
Strikingly, however, root hair patterning defects of wer
and cpc mutants were epistatic to those of sab in
non-hair and both hair and non-hair cells, respectively
(Table 3). This suggested that SAB may function upstream
of WER and CPC, the two components of the patterning
gene network. Furthermore, the observation that mutants
lacking both SAB and CPC activity produce very few
hairs favors the possibility that the activator complex
is functional even in the absence of SAB, consistent
with the idea that SAB is not part of the core patterning
pathway.
When considering the effect of the wer and cpc mutations on sab seedlings, there are some intriguing similarities to what has been described for the receptor-like
kinase SCM: scm;wer and scm;cpc double mutants have
phenotypes undistinguishable from wer and cpc, respectively (Kwak and Schiefelbein 2007). Loss-of-function
scm mutants display a higher number of non-hair cells in
the H position, in addition to increased frequency of hair
cells in the N position (Kwak et al. 2005), a feature they
share with erh3 mutants and mutants defective in the
zinc-finger protein gene JKD (Webb et al. 2002, Hassan
et al. 2010). Moreover, expression of promoter GL2 and
of a pWER reporter constructs is perturbed in scm and
jkd, resembling the unstable patterns observed in sab
mutants (Kwak et al. 2005, Hassan et al. 2010; Fig. 3).
The SAB protein is expressed in all root cell types (Pietra
et al. 2013) and could therefore cell-autonomously
affect stable hair patterning in epidermal cells, although
Physiol. Plant. 153, 2015
an additional non-cell-autonomous function from cortex cells cannot be excluded. For example, JKD acts
non-cell-autonomously on epidermal patterning from
the cortical layer (Hassan et al. 2010). However, these
considerations support the idea that, similar to SCM
and JKD, SAB functions in stabilizing fate acquisition
operating upstream of the core patterning pathway.
Taken together, SAB likely acts upstream of the core
epidermal cell-fate pathway to stabilize patterning of the
root epidermis. Defects of sab mutants in anisotropic
cell expansion and microtubule organization suggest
that SAB could influence epidermal patterning by
affecting microtubule-mediated anisotropic cell growth.
Roles of SAB have been described in both root epidermal and cortical cells, and although SAB might act
cell-autonomously on hair and non-hair fate specification, future analyses are needed to assess whether its
functions in the cortex contribute to epidermal patterning. Our findings reveal SAB as a prime example of a
gene required for stabilization of epidermal cell fate
and planar polarity of root hair positioning. They thus
stimulate further studies on the interactions between
these two processes and on the role of the microtubule
cytoskeleton during epidermal patterning.
Author contributions
M. G. conceived the project; M. G., S. P. and P.
L. designed experiments and interpreted experimental
results; P. L. and S. P. prepared experimental material and
performed the experiments; P. L. performed the majority
of experiments under supervision of S. P. and M. G.; S. P.
and M. G. wrote the manuscript; all authors commented
on and edited the manuscript.
Acknowledgements – We thank P. Benfey, D. W. Ehrhardt,
S. Henikoff, S. Persson and J. Schiefelbein for sharing published materials, and P. Hörstedt at Umeå Core Facility
Electron Microscopy for providing excellent SEM services
including SEM micrographs. We acknowledge the Nottingham Arabidopsis Stock Centre for providing seed stocks. We
are grateful to A. Gustavsson, C. Kiefer and M. Nakamura for
critical reading of the manuscript and for useful comments.
The work on SAB was supported by grants 2009-4846 and
2013–4405 from the Swedish Research Council (Vetenskapsrådet) to M. G.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Fig S1. SAB function contributes to stable expression of
the N-fate marker pGL2:GUS in the root epidermis.
Edited by Y. Helariutta
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