1292 RESEARCH ARTICLE
Development 140, 1292-1300 (2013) doi:10.1242/dev.090761
© 2013. Published by The Company of Biologists Ltd
Identification of SHRUBBY, a SHORT-ROOT and SCARECROW
interacting protein that controls root growth and radial
patterning
Koji Koizumi* and Kimberly L. Gallagher‡
SUMMARY
The timing and extent of cell division is particularly important for the growth and development of multicellular organisms. Roots of
the model organism Arabidopsis thaliana have been widely studied as a paradigm for organ development in plants. In the Arabidopsis
root, the plant-specific GRAS family transcription factors SHORT-ROOT (SHR) and SCARECROW (SCR) are key regulators of root growth
and of the asymmetric cell divisions that separate the ground tissue into two separate layers: the endodermis and cortex. To elucidate
the role of SHR in root development, we identified 17 SHR-interacting proteins. Among those isolated was At5g24740, which we
named SHRUBBY (SHBY). SHBY is a vacuolar sorting protein with similarity to the gene responsible for Cohen syndrome in humans.
Hypomorphic alleles of shby caused poor root growth, decreased meristematic activity and defects in radial patterning that are
characterized by an increase in the number of cell divisions in the ground tissue that lead to extra cells in the cortex and endodermis,
as well as additional cell layers. Analysis of genetic and molecular markers indicates that SHBY acts in a pathway that partially overlaps
with SHR, SCR, PLETHORA1 and PLETHORA2 (PLT1 and PLT2). The shby-1 root phenotype was partially phenocopied by treatment of
wild-type roots with the proteosome inhibitor MG132 or the gibberellic acid (GA) synthesis inhibitor paclobutrazol (PAC). Our results
indicate that SHBY controls root growth downstream of GA in part through the regulation of SHR and SCR.
INTRODUCTION
The root of Arabidopsis thaliana has a simple, consistent and welldefined pattern of cell division and growth that makes it a highly
tractable system for studying cellular development (Dolan et al.,
1993). The primary root is composed of concentric rings of tissues
arranged in a radial axis that surrounds a central cylinder of vascular
and procambial cells. From the outside inwards, these rings of
tissues are: the epidermis, the cortex, the endodermis (collectively
the cortex and endodermis make up the ground tissue) and the
pericycle. Overlaid upon the radial organization of the root are three
longitudinal zones that describe cellular behavior; classically, these
are the meristem, the elongation zone and the differentiation zone.
In the Arabidopsis root, the meristem is generally defined as a
region of the root apex that extends basally from the quiescent
center (QC) to the first set of elongated cells in the cortex. This
region includes the initial cells (stem cells) that surround the QC
and the transit amplifying cells. Above the meristem, cells begin a
phase of rapid cell elongation that precedes cellular differentiation.
The boundaries of these domains are plastic and are largely
regulated by antagonistic interactions between different phytohormones, which regulate the expression of key transcription
factors. Here, we describe SHRUBBY (SHBY), a SHORT-ROOT
(SHR)- and SCARECROW (SCR)-interacting protein. Mutations
in SHBY affect both the longitudinal and radial organization of the
root, through regulation of SHR, SCR, PLETHORA1 (PLT1) and
PLETHORA 2 (PLT2) levels, and gibberellin signaling.
121 Carolyn Lynch Laboratories, Department of Biology, University of Pennsylvania,
Philadelphia, PA 19104, USA.
*Present address: Biology Resources Section, Okinawa Institute of Science and
Technology (OIST) Graduate University, Okinawa 904-0495, Japan
‡
Author for correspondence ([email protected])
Accepted 9 January 2013
In the root meristem, cell divisions occur in two groups of cells: in
the initials that surround the quiescent center (QC) cells; and in a
population of transit amplifying cells that are derivatives of the initials
(Dolan et al., 1993). The maintenance of cell division in these
populations is dependent upon the presence of a functional QC. Loss
of the QC results in premature termination of root growth (van den
Berg et al., 1997; Sabatini et al., 2003; Xu et al., 2006). The
expression of two sets of partially overlapping transcription factors,
SHR and SCR, and the PLT proteins is required for maintaining the
QC (Di Laurenzio et al., 1996; Helariutta et al., 2000; Sabatini et al.,
2003; Aida et al., 2004). Both SHR and SCR are interacting members
of the GRAS family of transcription factors (Cui et al., 2007; Welch
et al., 2007). The SHR protein is expressed in the stele, and moves
from the stele into the surrounding cells. In the endodermis and the
QC, SHR turns on the expression of SCR, which in turn reinforces its
own expression specifically in the QC (Helariutta et al., 2000). Loss
of either SHR or SCR expression results in the formation of a short
root that fails to maintain the QC and meristem.
Also expressed in the QC and root meristem are the PLT proteins.
The PLTs are AP2 class transcription factors, the expression of
which in the root meristem converges upon the QC and the
surrounding initial cells (Aida et al., 2004; Galinha et al., 2007).
The PLT1 protein is at its highest level in the QC and in the
surrounding initials, with a weaker protein gradient extending
basally into the stele. PLT2 also shows strong expression in the QC
and the initials, but its expression basally is maintained at higher
levels in the cortex and epidermis than in the stele. Loss of both
PLT1 and PLT2 expression results in the loss of QC markers,
differentiation of the initial cells and formation of root hairs at the
root tip, indicating that the PLTs function in both the maintenance
and the activity of the QC and initials (Aida et al., 2004). Both PLT1
and PLT2 function in a dose-dependent manner, with high levels of
PLT required to maintain stem cell fates (Galinha et al., 2007).
DEVELOPMENT
KEY WORDS: SHORT-ROOT, SCARECROW, PLETHORA, Arabidopsis root development, Mg132, SHRUBBY
RESEARCH ARTICLE 1293
SHBY regulates root growth
MATERIALS AND METHODS
Yeast 2-hybrid assays
Yeast 2-hybrid assays were contracted to Hybrigenics (Paris, France) using
a prey library made from cDNAs derived from 1-week-old Arabidopsis
seedlings. The SHR bait construct and the second prey library are as
described elsewhere (Koizumi et al., 2011).
Plant materials and growth conditions
Arabidopsis thaliana Columbia lines (Col-0) and the heterozygous siblings
of the shby-1 allele were used as wild type. Homozygous T-DNA insertions
(http://abrc.osu.edu/) were identified using PCR (supplementary material
Table S3). Other lines used in this study are described in Table S3. Plants
were grown vertically on 0.5× MS medium containing 0.05% (w/v) Mes
(pH 5.7), 1% (w/v) sucrose and 0.5% (w/v) Phytagel (Sigma-Aldrich) in
growth chambers at 23°C with 16-hour light and 8-hour dark photoperiods.
Plasmid construction and transformation
Gateway pDONR P4P1R plasmid containing the En7 promoter (Heidstra et.
al 2004) and pDONR221 with ER-localized GFP (erGFP) were recombined
into dpGreen BarT (Lee et al., 2006) using Gateway protocols
(Invitrogen/Life Technologies). The resulting binary vector was introduced
into Agrobacterium strain GV3101-pSoup-pMP. Standard protocols were
used for transformation.
Microscopy and imaging
Root cross-sections and confocal images were prepared as described
previously (Koizumi et al., 2000; Koizumi et al., 2011). The contrast and
brightness of some images were adjusted using Adobe Photoshop 7. For
fluorescent quantification, identical settings were used to collect images on
the same day for both the wild-type and mutant plants homozygous for the
GFP or YFP marker. Intensity levels were measured using ImageJ software
(http://rsbweb.nih.gov/ij/) on unmodified root images. SHR movement was
examined as described previously (Koizumi et al., 2011). Root meristem
cell number and meristem lengths were measured on DIC images of cleared
roots using ImageJ. Cell production rate was calculated using cell flux
assays as described in Beemster and Baskin (Beemster and Baskin, 1998).
Whole-mount in situ hybridization was carried out as described previously
(Hejátko et al., 2006). The region between the F3 and R3 primers
(supplementary material Fig. S2A; Table S3) was used for making the sense
and antisense probes.
Drug treatment
For PAC treatment, seeds were germinated for 30 hours on MS plates and
individually transferred to MS plates supplemented with 2 µM PAC. Roots
were then examined at day 7. For short-term (6 hours) PAC treatment, 5day-old seedlings were transferred to MS plates supplemented with 10 µM
PAC. For GA3 treatment, seeds were sown on MS plates, including 10 μM
GA3 and examined at day 5. For MG132 treatment, seeds were either sown
on MS plates, including 10 μM MG132 or transferred at day 4 for short-term
(24 hours) MG132 treatment.
Quantitative RT-PCR
Total RNA was isolated from 5-day-old roots and cDNA was synthesized
as described previously (Koizumi et al., 2011). Semi-quantitative (q) RTPCR was performed using standard PCR techniques. RNA levels were
normalized to EIF4A. Real-time qRT-PCR was performed using Power
SYBR Green PCR master mix (Applied Biosystems) and Step One Plus
Real-time PCR System. RNA levels were normalized using UBQ10
(Sozzani, et al., 2010) as a standard. Primers used are listed in
supplementary material Table S3.
Bimolecular fluorescence complementation (BiFC)
Full-length cDNAs were used for all BiFC experiments with the exception
of SHBY for which part of the cDNA that included the DUF1162 region
(which was the region found to interact with SHR by Y2H) was amplified
(supplementary material Table S3) from a root cDNA library by PCR and
recombined into pDONR221 (Invitrogen/Life Technologies). Assays were
performed in onion epidermal cells using pBatTL, BiFC vectors and
procedures described elsewhere (Uhrig et al., 2007). A minimum of five
fluorescent cells after a single bombardment was regarded as a positive
interaction.
RESULTS
Using a modified version of the SHR protein that maintains the
ability to move and rescue shr-2 mutants (Koizumi et al., 2011),
yeast two-hybrid (Y2H) screens were conducted. The screening of
DEVELOPMENT
Outside of the QC, SHR and SCR are required for radial
patterning of the root (Di Laurenzio et al., 1996; Helariutta et al.,
2000). In the meristem, formative divisions in the initials give rise
to, or maintain, distinct cell layers. For example, the cortical
endodermal initials (CEI) that flank the QC divide asymmetrically
to produce daughter (CED) cells that divide periclinally to produce
the endodermis and cortex. Periclinal division of the CED is
controlled by SHR and SCR. Both of these genes induce the
expression of a D-type cyclin, CYCD6;1, in the CED cell, which
leads to the asymmetric periclinal cell division that produces the
endodermis and cortex (Sozzani et al., 2010). Following this
asymmetric division, both SHR and SCR are rapidly degraded in
the cortex cell, thus preventing further periclinal cell divisions
(Heidstra et al., 2004). Loss-of-function mutations in SHR or SCR
result in the production of a single ground tissue layer between the
pericycle and the epidermis.
Later in root development, both SHR and SCR control the
periclinal cell divisions in the endodermis that produce middle cortex
(MC) (Baum et al., 2002; Paquette and Benfey, 2005). Between days
7 and 14 the endodermis begins to divide periclinally and
asymmetrically to produce another layer of ground tissue, the MC.
In contrast to their similar roles in the formation of the endodermis
and cortex, SHR and SCR play different roles in the production of
MC. SCR inhibits the divisions in the endodermis that produce the
MC, whereas SHR is required for them. The result is that MC forms
precociously in plants that lack SCR function (Paquette and Benfey,
2005); it never forms in shr-2 mutants. However, MC forms
precociously in shr-2 heterozygotes or in shr hypomorphs (Koizumi
et al., 2012). It is likely that SHR promotes formation of MC via
activation of CYCD6;1, as Sozzanni et al. (Sozzanni et al., 2010)
showed expression of CYCD6;1 in endodermal cells that form MC,
and a reduction in MC-generating cell divisions in cycd6;1 mutants.
GA signaling also regulates formation of MC. Inhibition of GA
signaling in either wild-type or scr roots hastens the formation of MC
(Heo et al., 2011; Paquette and Benfey, 2005).
To elucidate the function of SHR in root growth and patterning,
we isolated 17 proteins that interact with SHR. Among the isolated
proteins were the bona fide SHR interacting proteins SCR,
MAGPIE (MPG) and JACKDAW (JKD) (Cui et al., 2007; Welch et
al., 2007), and multiple related zinc-finger domain transcription
factors. We concentrated our analysis on an uncharacterized protein,
SHBY with similarity to the gene responsible for Cohen syndrome
in humans (Velayos-Baeza et al., 2004). Mutations in SHBY reduced
meristem activity and increased cell divisions in the ground tissue.
We show that SHBY interacts with SHR and SCR, and regulates
the levels of SHR, SCR, PLT1 and PLT2 proteins. In turn, SHR and
SCR also regulate the levels of SHBY, with shr-2 and scr-4 mutants
showing considerably reduced SHBY expression. The overall
phenotype of the shby mutants suggests roles in protein turnover
and in GA signaling. Indeed the shby-1 root phenotype is partially
phenocopied by growth of wild-type roots on low doses of MG132
or of the GA inhibitor paclobutrazol (PAC). In addition shby-1
mutants are insensitive to GA and PAC, suggesting that SHBY is
required for GA signaling. SHBY therefore provides a link between
the SHR/SCR pathway, the PLTs and GA.
two different Arabidopsis cDNA libraries identified 17 SHR
interacting proteins (supplementary material Table S1). These
proteins fell broadly into two major classes: regulation of
transcription and membrane trafficking. Included in the group of
transcriptional regulators were the verified SHR-interacting proteins
SCR, JKD and MGP. From this full list of SHR-interacting proteins,
we selected candidates for further analysis that had not been
extensively characterized and had T-DNA insertion lines available.
In addition, we performed bimolecular fluorescence
complementation (BiFC) assays on a subset of these new proteins
to verify protein interaction; five of the six proteins tested (not
including SCR, JKD and MGP) showed an interaction in planta
(supplementary material Table S1). Mutations in At1g03860,
At2g02080, At3g46620, At4g29950 and At5g12430 caused no
obvious root growth or cellular patterning defects, indicating that
they are not essential for SHR function. As previously published,
sec5a and sec5b single mutants appeared normal; attempts to make
double mutants between sec5a and sec5b were unsuccessful (Hála
et al., 2008). Therefore, from this screen we narrowed our focus to
At5g24740, which had an obvious mutant phenotype (Fig. 1A;
supplementary material Fig. S1), and showed interaction with both
the SHR and SCR proteins (Fig. 1B).
Phenotype of the shby mutants
At5g24740 is a single gene in Arabidopsis (TAIR BLAST AA) and
is classified as a vacuolar sorting protein with chorein-N, ATG_C,
multiple MRS6 homology and a DUF1162 domain (supplementary
material Fig. S2A) (Bowers and Stevens, 2005; Kanehisa and Goto,
2000; Kanehisa et al., 2006; Kanehisa et al., 2010). These domains
are found in vacuolar sorting proteins from yeast to human
(Marchler-Bauer et al., 2011). Mutations in these proteins are
associated with defects in intracellular trafficking, protein turnover
and Cohen syndrome in humans (Velayos-Baeza et al., 2004).
Arabidopsis seedlings homozygous for mutations in At5g24740
all had increased branching and a shrubby appearance
(supplementary material Fig. S1A,B). We therefore named
At5g24740 as SHRUBBY. Plants heterozygous for shby
Fig. 1. shby mutants show defective root growth. (A) Five-day-old
seedlings of wild-type and shby homozygotes. (B) Result of BiFC analysis
using the DUF1162 domain of the SHBY cDNA (supplementary material
Fig. S2A) and full-length SHR or SCR. Fluorescence indicates proteinprotein interaction. (C-H) DIC images of (C) wild-type, (D) shby-1, (E) shr-2,
(F) shby-1 shr-2, (G) scr-4 and (H) shby-1 scr-4 root meristems at day 5. Black
arrowheads indicate the beginning of the elongation zone. The white
arrowheads indicate the QC. Scale bars: 1 cm in A; 100 μm in C-H.
Quantification of C to H is shown in supplementary material Fig. S3C,D.
Development 140 (6)
(SHBY/shby-1, SHBY/shby-2, SHBY/shby-3 or SHBY/shby-4) were
indistinguishable from wild type. The progeny of self-fertilized
SHBY/shby-1 plants segregated 3:1 for the shby phenotype,
indicating that the shby mutations are monogenic and recessive. All
of the shby mutants developed a significantly shorter root than wild
type (Fig. 1A; supplementary material Fig. S3A), with the shby-1
homozygotes being the shortest (Fig. 1A; supplementary material
Fig. S3A). shby-1 seedlings were therefore used for a more
comprehensive analysis of the shby phenotype. Overall, shby-1
plants were smaller than controls, had dark green curled leaves, late
bolting (supplementary material Fig. S1C) and abnormal shortpetaled flowers with the gynoecium extending from the unopened
flower bud (supplementary material Fig. S1D). shby-1 plants were
both male and female sterile. The stamens produced no visible
pollen on their surface (supplementary material Fig. S1E), and even
when the stigma was manually pollinated with wild-type pollen, no
seeds were produced.
There are two predicted splicing variants of SHBY. The first
contains the first 15 exons of the full-length cRNA with part of
intron 15 (in supplementary material Fig. S2A from the ATG to the
first stop codon). The second transcript contains all 46 exons that
encode the 3462 amino acid protein. Real-time and semiquantitative RT-PCR on shby-1 roots revealed no decrease in the
signal upstream of the T-DNA insertion, but a significant decrease
downstream of the T-DNA, suggesting that the T-DNA insertion in
shby-1 resulted in a truncated mRNA (supplementary material Fig.
S2B,D). Based upon the position of the insertions in each of the
four shby alleles, it is unlikely that the shorter transcript is affected
by any of these insertions and therefore that any of the four shby
alleles are true nulls. However, using primers homologous to the 5⬘
end of the transcript and intron 15, which is absent in the longer
transcript, no product was detected in either wild-type or shby-1
roots. Likewise, there were no significant differences in the
expression of products amplified by primers complementary to the
N terminus (1832-1946; supplementary material Fig. S2A,C) or to
the C terminus (16668-16776; supplementary material Fig. S2A,C)
in the roots of wild-type plants, suggesting that there is little or no
expression of the short transcript in the root.
Meristem activity in the shby-1 mutant
To examine the root growth phenotype in the shby-1 mutants, we
measured meristem size, meristem cell number, and both cell size
and cell production in shby-1 and wild-type roots (Fig. 1C,D;
supplementary material Fig. S3B). Both the size of the meristem
and the number of cortex cells in the meristem of shby-1 roots was
reduced compared with wild type. The growth curves for the shby1 and wild-type roots and root meristems were also different
(supplementary material Fig. S3A,B). Over a 4-day period (from
days 3-7) the wild-type meristem doubled in size and the number of
cortex cells increased by 70%. This means that in wild type, new
and longer cells were added to the meristem during the first week
of root growth. In the shby-1 roots, the size of the meristem
increased by only 10% over the same 4-day period and the number
of cortex cells was increased by 33%. In contrast to wild-type roots,
the average size of the cortex cells in the shby-1 meristem decreased
over the first week of growth. To investigate whether the short root
phenotype of shby-1 was caused by reduced cell expansion or cell
division, or both, we conducted cell flux assays (Beemster and
Baskin, 1998). Cell production in shby-1 was approximately half of
what we measured in wild type (supplementary material Fig. S3B).
The length of the mature cortex cells in shby-1 was also reduced
(wild type=212.3±5.6 µm, n=10; shby-1=168.0±8.7 µm, n=10; t-
DEVELOPMENT
1294 RESEARCH ARTICLE
Fig. 2. shby-1 affects expression of QC markers. (A-J) Expression of
(A,B) pWOX5:GFP, (C,D) pPLT1:PLT1-YFP, (E,F) pPLT2:PLT2-YFP, (G,H) pSHR:SHRGFP and (I,J) pSCR:SCR-GFP in wild-type and shby-1 root tips at day 5. The
graphs in D,F illustrate the differences in expression between wild-type
(left bar) and shby-1 (right bar) roots. **P<0.01 (t-test). (D) Wild type, n=10;
shby-1, n=13. (F) Wild type, n=5; shby-1, n=7.
test, P=0.005), but not to the same extent as cell production
(supplementary material Fig. S3B). These results suggest that
impaired root growth in shby-1 is the product of both reduced cell
production and reduced cell expansion.
As the QC in shby-1 mutants appeared disorganized and defects
in root growth often correlate with abnormal QC function, we
examined expression of the QC markers, WUSCHEL RELATED
HOMEOBOX 5 (WOX5), PLT1, PLT2, SHR and SCR in wild-type
and shby-1 roots. To examine the QC cells, we counted the number
of pWOX5:GFP-expressing cells in serial confocal sections in the
meristem (Fig. 2A,B). In 5-day-old wild-type roots, we counted 4.6
(±0.2, n=18) GFP-positive cells on average, whereas in shby-1,
there were 5.8 (±0.3, n=17, t-test, P=0.004). These results are
consistent with cell divisions in the QC leading to a weak expansion
of the WOX5-expression domain, which is associated with a
reduction in QC identity (Sarkar et al., 2007). These results suggest
QC defects in the shby-1 mutants.
We next examined the expression of pPLT1:PLT1-YFP and
pPLT2:PLT2-YFP in wild-type and shby-1 mutants. Both PLT1 and
PLT2 act in a concentration-dependent manner in the root meristem
to promote stem cell fate and cell divisions (Galinha et al., 2007).
The levels of pPLT1:PLT1-YFP and pPLT2:PLT2-YFP were
markedly reduced in shby-1 compared with wild type (Fig. 2C-F).
These results suggest that loss of SHBY activity affects either the
expression and/or stability of PLT1 and PLT2. However, the overall
domain of PLT1 and PLT2 expression was not altered in shby-1. We
also examined both SCR and SHR expression specifically in the
QC and found that the levels were reduced (Fig. 2G-J). This was
more pronounced for SCR-GFP, than for SHR-GFP, but in both
RESEARCH ARTICLE 1295
Fig. 3. shby-1 mutants have ectopic cell divisions in the ground
tissue. (A-C) Longitudinal confocal sections of the root meristem in 5day-old seedlings. The area in the dashed box is shown at higher
magnification. Compared with wild type (A), in shby-1 (B,C) there is an
expansion of the ground tissue from two to three cell layers (numbered).
In B, the extra cell layer arises in the cortex; in C it comes from the
endodermis. Arrows indicate ectopic cell divisions. (D-G) Expression of
the cortex-specific marker pCo2:RE3xYFP (D,E) and the endodermalspecific promoter pEn7:erGFP (F,G) in wild type and shby-1 mutants
indicates that the extra cell layers in shby-1 are cortex, independent of the
origin of the new layer. Arrows indicate ectopic cell divisions. C, cortex; E,
endodermis.
cases the reduction was statistically significant compared with the
levels in wild-type roots. Collectively, these results show that SHBY
is required to maintain SHR, SCR, PLT1 and PLT2 at wild-type
levels in the QC, and therefore suggest a role for SHBY in meristem
maintenance.
Ground tissue formation in the shby-1 root
In addition to regulating the meristematic activity of the root, both
SHR and SCR play roles in root patterning (Di Laurenzio et al.,
1996; Helariutta et al., 2000). We therefore examined the patterning
of the ground tissue in the shby-1 root compared with wild type
Wild-type roots contain single layers of endodermis and cortex, each
of which is composed of eight cells (Fig. 3A; Fig. 6A) (Dolan et al.,
1993). The shby-1 mutant root contained additional ground tissue
layers and an increase in the number of cells in each layer
(Fig. 3B,C; Fig. 6B). This increase in the number of cell layers was
not uniform throughout the root, leading to different numbers of
layers in different regions of the root (Fig. 3B,C). In addition, based
upon morphology, the extra cell layers in shby-1 arose from either
the cortex (Fig. 3B) or the endodermis (Fig. 3C). Expression
analysis of early markers of cortex (pCo2:RE3xYFP) and
endodermis (pEn7:erGFP) showed that expression of pEn7:erGFP
was not maintained outside of the endodermis, whereas
pCo2:RE3xYFP was often detected strongly in more than one cell
layer. These results indicate that the extra cell layers in shby-1 adopt
a cortex cell identity (Fig. 3D-G) (Heidstra et al., 2004).
Effect of SHBY on SHR and SCR localization
In the root meristem, the SHR protein is expressed in the stele and
moves into the endodermis. In the stele, the SHR protein localizes
both to the nucleus and cytoplasm, whereas in the endodermis the
protein is exclusively localized in the nucleus (Fig. 4A) (Nakajima
et al., 2001; Gallagher et al., 2004; Cui et al., 2007; Gallagher and
DEVELOPMENT
SHBY regulates root growth
Development 140 (6)
Fig. 4. Expression of SHR and SCR in the shby-1 mutant.
(A-H) Expression of (A,B) pSHR:SHR-GFP, (C,D) pSHR:erGFP, (E,F) pSCR:erYFP
and (G,H) pSCR:SCR-GFP in the meristems of wild-type and shby-1 roots at
day 5. In E-H, the region outlined is shown at high magnification. Arrows
indicate regions of abnormal division. E, endodermis; C, cortex. For
comparison, real-time data of whole root (cullet to root cap)
measurements of SHR and SCR mRNA in shby-1 and wild type are shown
in supplementary material Fig. S5A.
Fig. 5. Effects of the shby mutation, MG132 and PAC on the
maintenance of the root meristem and divisions in the ground
tissue. (A,B) pCYCD6;1:GFP-GUS in day 5 wild-type and shby-1 roots.
(C) A wild-type root grown with 10 μM MG132 for 5 days. Arrows indicate
regions of abnormal division. (D-F) Localization of SCR-GFP in roots grown
for 24 hours in 10 μM MG132. Arrowheads indicate SCR-GFP perdurance in
the middle ground tissue layer. (G,H) Expression of pCYCD6;1:GFP-GUS in 7
day-old roots. (G) Control root and (H) root treated for 6 days with PAC. E,
endodermis; C, cortex. Arrows in H indicate SCR-GFP.
Benfey, 2009). To examine SHR-GFP in the shby-1 mutants, we
examined levels of GFP fluorescence in both the pSHR:SHR-GFP
and pSHR:erGFP fusions in wild type and shby-1 mutants. Overall,
the patterns of pSHR:SHR-GFP and pSHR:erGFP expression did
not differ between wild-type and shby-1 roots (Fig. 4A-D).
However, compared with wild type, the expression levels of
pSHR:erGFP and the pSHR:SHR-GFP in shby-1 were 80% (t-test,
P=0.005; wild type, n=15; shby-1, n=17) and 60% (t-test, P=0.0008;
wild type, n=20; shby-1, n=18) of wild-type levels, respectively, for
the same transgenes. We saw no obvious changes in the subcellular
localization of SHR-GFP in either the stele tissue or the endodermis
in the shby-1 roots compared with wild type. These results suggest
that SHBY affects SHR both at the level of the protein and at the
level of the transcript, but not protein localization.
To test whether shby-1 has any affect on the movement of SHR
from the stele into the endodermis, we measured the level of the
SHR-GFP signal in the endodermis as a percentage of the stele
signal, as this is a good indication of SHR movement (Gallagher
and Benfey, 2009; Koizumi et al., 2011). In wild-type roots, the
SHR-GFP signal in the endodermis is generally 1.2-1.5, depending
on the age of the root and the growth conditions. When we
examined the ratio in shby-1 roots and their wild-type siblings, we
saw no significant differences between these two groups. The
average ratio of endodermal signal to stele signal was 1.2 in wild
type (n=27) and 1.29 in shby-1 (n=31), indicating that SHBY does
not play an essential role in SHR movement.
To examine SCR expression in shby-1 mutants, we crossed the
pSCR:erGFP and pSCR:SCR-GFP markers lines into shby-1
mutants. In wild type, SCR:erGFP is highly expressed in the
endodermis (Fig. 4E). SCR-GFP expressed from the same promoter
is found at high levels in the endodermis and to a lesser extent in the
cortex (Fig. 4G) (Gallagher et al., 2004; Wysocka-Diller et al., 2000).
In shby-1 roots, SCR:erGFP was also expressed in the endodermis,
but at only 55% of what we saw in wild type (Fig. 4F) (t-test,
P=0.009; wild type, n=8; shby-1, n=8). By contrast, the levels of
pSCR:SCR-GFP in the endodermis of shby-1 were not different than
wild type (Fig. 4G,H) (t-test, P=0.596; wild type, n=15; shby-1,
n=19), so although expression levels were decreased based upon the
pSCR:erGFP results, the protein levels were not. In addition, in shby1 roots, SCR-GFP was frequently detected at higher levels in the
cortex relative to the endodermis when compared with wild-type roots
(t-test, P=0.02; shby-1, n=18; wild type, n=10; Fig. 4H shows an
example of this). This effect on SCR-GFP was particularly true in
regions of the root where abnormally oriented or ectopic divisions
had occurred. In pSCR:erGFP lines, erGFP was present in the middle
ground tissue layer immediately following division of the
endodermis, but was not maintained (Fig. 4F). Likewise following
the division that produces the endodermis and cortex layers,
pSCR:erGFP was not maintained in the cortex. As the SCR protein
was found at higher than wild-type levels in the cortex and middle
ground tissue layer, these results suggest that SCR is not efficiently
lost following the asymmetric division that generates the endodermis
and cortex or the middle ground tissue layer in the shby-1 mutants.
Expression of SHR target CYCD6;1 in the shby-1
mutant
Sozzani et al. (Sozzani et al., 2010) reported that the Arabidopsis Dtype cyclin CYCD6;1 was a direct target of SHR and SCR, and that
this cyclin promoted the formative periclinal cell division that
generates the endodermis and cortex, and the divisions that pattern
the MC. In 5-day-old roots, pCYCD6;1:GFP-GUS was specifically
expressed in the CEI and the CEI-daughter cells (CEDs). By day
10, the expression of CYCD6:1 was expanded into the endodermis
(Sozzani et al., 2010). To understand the relationship between SHBY
and CYCD6;1, we examined the expression pattern of
pCYCD6;1:GFP-GUS in shby-1. Under our growth conditions,
pCYCD6;1:GFP-GUS was expressed in the CEI and CED cells of
5-day-old seedlings (Fig. 5A), and weakly and sporadically
expressed in some cells of the endodermis in 7-day-old seedlings
(Fig. 5G). In the root meristem of 5-day-old shby-1 mutants, GFP
was weakly detected in the CEI and CED cells, moderately
expressed in the endodermis, and strongly expressed in regions of
DEVELOPMENT
1296 RESEARCH ARTICLE
the endodermis that showed amplification of the ground tissue
(Fig. 5B). These result indicate that SHBY plays a role in inhibiting
CYCD6;1 expression in the endodermis.
Recently, Cruz-Ramirez et al. (Cruz-Ramirez et al., 2012)
reported that treatment of roots with the proteosome inhibitor
MG132 resulted in increased SCR, ectopic expression of CYCD6;1
and an increase in the frequency of periclinal cell divisions in the
ground tissue. As this phenotype is very similar to shby-1, and
SHBY is a putative vacuolar-sorting protein, we tested whether we
could phenocopy the loss of SHBY by growing seedlings on media
containing low levels (10 µM) of MG132 for 5 days, or short-term
for 24 hours. Similar to what was reported by Cruz-Ramirez et al.
(Cruz-Ramirez et al., 2012), both treatments showed a significant
increase in the number of ground tissue layers (Fig. 5C; Fig. 6C;
supplementary material Table S2), which correlated with activation
of CYCD6;1 expression in the endodermis. No further increase in
ground tissue layers was seen in the shby-1 mutants treated with
MG132 (supplementary material Table S2). In contrast to CruzRamirez et al. (Cruz-Ramirez et al., 2012), we saw no significant
effects of either the 5-day or 24-hour treatments with MG132 on
the level of SCR-GFP in the endodermis (P=0.93 and P=0.30,
respectively, t-test; control, n=10; 10 µM MG132, n=10). However,
in endodermal cells that had undergone MG132-induced periclinal
cell division, SCR-GFP was often retained in the middle ground
tissue layer (examples shown in Fig. 5D-F), indicating that SCR is
not efficiently degraded. MG132 also decreased root growth and
caused a general disorganization of the cellular patterning of the QC
(boxed region in Fig. 5C). This correlated with an increase in the
domain of pWOX5:GFP expression (supplementary material Fig.
S4A,B) and a reduction in the levels of both PLT1 and PLT2 in the
root meristem (supplementary material Fig. S4C-F) (reduced by
36% and 32%, respectively, at 24 hours; 12 roots were analyzed for
each treatment; and by 39% and 45%, respectively, at day 5; PLT1
control, n=10; 10 µM MG132, n=10; PLT2 control, n=10; 10 µM
MG132, n=10; P<0.01). These results show that many of the shby1 phenotypes can be reproduced by reducing protein turnover.
Genetic interactions between SHR, SCR, SCL3 and
SHBY
As shby-1 mutants have an increase both in the number of ground
tissue layers and changes in SHR and SCR levels, we examined
RESEARCH ARTICLE 1297
genetic interactions between shby-1 (Fig. 6B), shr-2 (Fig. 6D) and
scr-4 (Fig. 6H). For most of the shby-1 double mutant combinations,
the roots were marginally but significantly shorter than the single
mutants alone (supplementary material Fig. S3E), indicating
additive effects on root growth. For the shby-1 shr-2, this correlated
with a reduced meristem cell number compared with the
corresponding single mutants (Fig. 1D-H; supplementary material
Fig. S3C,D).
shr-2 and scr-4 roots make only one ground tissue layer of
approximately eight cells. shr-2 fails to make MC, whereas scr-4
does so precociously. The shby-1 shr-2 double mutants showed
three different phenotypes in roughly equal proportions. A third of
roots had a shr-2 phenotype (Fig. 6E), whereas the other two-thirds
showed either an increase in anticlinal (Fig. 6F) or an increase in
both anticlinal and periclinal cell divisions (Fig. 6G) compared with
the shr-2 single mutant (Fig. 6D), but still significantly fewer cell
divisions (particularly periclinal cell divisions) than in shby-1
(supplementary material Fig. S3F). In the shby-1 scr-4 double
mutants, the shby-1 mutation increased the number of cell layers in
the scr-4 mutants (Fig. 6H,I). shby-1 scr-4 mutants had two layers
of ground tissue instead of one, indicating that SCR is not required
for the ectopic periclinal cell division in shby-1 mutant; however,
the number of ground tissue cells in the shby-1 scr-4 double mutant
was significantly reduced compared with the shby-1 single mutant
(supplementary material Fig. S3F). These results suggest that both
SHR and SCR promote ectopic or precocious cell divisions in shby1, but that neither alone is sufficient for these cell divisions.
As shby-1, shr-2 and scr-4 do not show clear epistatic
relationships, we examined SHBY expression in shr-2 and scr-4
mutants. Whole-mount in situ hybridization with the SHBY sense
and antisense probes in 5-day-old wild-type seedlings showed
strong expression of SHBY in all the cells of the root meristem
extending into the elongation and differentiation zones
(supplementary material Fig. S5C). In shr-2 and scr-4 roots, the
expression pattern of SHBY was fairly similar to wild type; however,
the levels of expression were reduced with scr-4 roots showing
levels of SHBY expression that were intermediate between wild
type and shr-2 (supplementary material Fig. S5C,D). If SHBY levels
are decreased in shr-2 and scr-4 backgrounds, then based upon our
results, PLT1 and PLT2 should also be decreased in shr-2 and scr4 backgrounds. Indeed, both mutants showed reduced PLT1-GFP
Fig. 6. Interactions between shby, shr, scr, scl3, MG132
and PAC treatments. Transverse cross-sections above the
meristem through the roots of single and double mutants.
(A-C) Both the (B) shby-1 mutation and (C) treatment with
MG132 cause an increase in the number of ground tissue
layers compared with wild type (A). (D-G) One-third of the
shby-1 shr-2 mutants (E) were identical to the shr-2 mutants
(D). Of the remaining two-thirds (F,G), some showed an
increase in anticlinal cell divisions (F) and the smallest class
showed both an increase in periclinal and anticlinal cell
divisions (G) . (H,I) All of the shby-1 scr-4 double mutants (I)
(n=17) showed an increase in the number of cell layers
compared with the scr-4 mutant (H). (J,K) The shby-1 scl3-1
double mutant (K) has an increased number of ground tissue
cells compared with the scl3-1 mutant (J).
(L) Treatment of scr-4 roots with PAC phenocopied the
cellular patterning of the shby-1 scr-4 double mutant roots.
Quantification is shown in supplementary material Fig. S3F
and/or Table S2.
DEVELOPMENT
SHBY regulates root growth
(49% and 53% of wild type in shr-2 and scr-4, respectively) and
PLT2-GFP (47% and 37% of wild type in shr-2 and scr-4,
respectively) expression (supplementary material Fig. S4G-J).
These results suggest that SHBY provides a link between the
SHR/SCR and PLT pathways.
Role of gibberellin in SHBY function
Although the shby-1 phenotype was partially phenocopied by
treating roots with MG132, the overall phenotype of the shby-1
mutants [dwarfed, bushy plants with dark green leaves and delayed
flowering (supplementary material Fig. S1)] indicates defects in GA
signaling. Likewise, the phenotype of the shby-1 scr-4 roots (Fig. 6I)
is reminiscent of the scarecrow-like 3 (scl3) scr-5 double mutant
phenotype reported by Heo et al. (Heo et al., 2011). SCL3 promotes
GA signaling in the regulation of ground tissue patterning. To
determine whether SHBY functions in the GA pathway, we
examined the shby-1 phenotype under three different conditions that
affect GA signaling. First, we made double mutants between scl31 and shby-1, and then tested the sensitivity of the shby-1 mutants
to PAC (an inhibitor of GA biosynthesis) treatment and addition of
GA3 to the growth medium. In otherwise wild-type roots, mutations
in SCL3 increase the frequency of periclinal cell divisions in the
ground tissue (Heo et al., 2011); however, not quite to the same
degree as shby-1 (Fig. 6J; supplementary material Fig. S3F). In
shby-1 scl3-1 double mutants, the number of ground tissue cells was
moderately increased compared with scl3 single mutants, but
decreased compared with shby-1 (Fig. 6K; supplementary material
Fig. S3F), suggesting that SHBY pathways partially overlap with
SCL3. To test the effects of inhibition of GA, we treated wild-type
and shby-1 mutants with PAC, and examined ground tissue
patterning. PAC had no effect on shby-1 (supplementary material
Table S2), indicating that shby-1 is insensitive to PAC. Likewise,
treatment with GA3 also failed to elicit a response in shby-1 mutants,
indicating that SHBY is required for GA3-induced signaling.
The above results suggest that shby-1 mutants have reduced GA
signaling. To test whether we could phenocopy the effects of shby-1
by inhibiting GA, we treated wild-type seedlings with PAC. The
expression of pPLT1:PLT1-YFP and pPLT2:PLT2-YFP were not
repressed by PAC as they are in the shby-1 mutants; however,
treatment with GA3 caused moderate upregulation of pPLT2:PLT2YFP (supplementary material Fig. S4K,L), suggesting that at least
PLT2 is responsive to GA. As previously published, the shr-2 mutants
were insensitive to PAC (Paquette and Benfey, 2005). Interestingly,
treatment of scr-4 seedlings with 2.0 μM PAC phenocopied the shby1 scr-4 double mutant (Fig. 6L; supplementary material Table S2):
there were now two ground tissue layers in the PAC-treated scr-4
roots. In addition, treatment of the CYCD6;1:GFP-GUS roots with
PAC caused an increase in expression of the transgene (Fig. 5H). PAC
treatment had no effect on cell divisions in the cycd6;1 lines
(supplementary material Table S2), indicating that PAC exerts its
effect on ground tissue patterning via CYCD6;1.
As the shby-1 mutants resemble GA-deficient mutants and are
insensitive to GA3 application (supplementary material Table S2),
we attempted to rescue the shby-1 phenotype downstream of GA
by crossing into the shby-1 background the recessive GA
INSENSITIVE (gai-2) allele, which allows GA signaling in the
absence of GA. We examined 72 F2 seedlings and recovered the
expected numbers of gai-2 and shby-1 single mutants, but never
recovered the gai-2 shby-1 double mutant. In an F2 population of
471, there was a total of 28 ungerminated seeds, which could
represent the gai-2 shby-1 double mutants; however, attempts to
genotype these seeds were unsuccessful. Collectively, these results
Development 140 (6)
show that PAC can partially phenocopy the shby-1 patterning
phenotype and suggest that SHBY promotes GA signaling
downstream of GA synthesis, as shby-1 mutants are insensitive to
application of GA3.
DISCUSSION
In the present study, we identified 17 SHR-interacting proteins,
including the verified SHR-interacting proteins SCR, JKD and
MGP (Cui et al., 2007; Welch et al., 2007). In the group of other
proteins, we were able to verify the interaction of SHR with five of
the six tested. However, none of the single insertion lines for these
genes (with the exception of SHBY) had a single mutant phenotype.
Among the SHR-interacting proteins was SHBY, a vacuolar sorting
protein with similarity to VPS13 (COH1) (Marchler-Bauer et al.,
2011). Velayos-Baeza et al. (Velayos-Baeza et al., 2004) reported
homology in the N terminus between SHBY and yeast VPS13p.
The human homologs of VPS13p are mutated in choreaacanthocytosis and Cohen syndrome (Kolehmainen et al., 2003;
Rampoldi et al., 2001). Functional analysis of VPS13b in yeast
showed that it is involved in the trafficking of membrane proteins
between the trans-Golgi network and the prevacuolar
compartment/late endosome (Brickner et al., 2001; Redding et al.,
1996), suggesting roles in protein recycling.
We isolated and characterized four different alleles of shby and
found that SHBY is required for normal root growth. The roots of
shby mutants were consistently shorter than wild type. Analysis of
the shby-1 mutant showed that the reduced root growth was the
result of reduced cell elongation and reduced cell production. Part
of the meristem defect in shby-1 can be explained by a decrease in
the levels of SHR and SCR in the QC. However, as the shr-2 and
scr-4 mutants are not clearly epistatic to shby-1 with respect to root
growth and meristem cell number, other pathways must also be
affected in shby-1. Consistent with this, we see a strong decrease in
PLT1 and PLT2 levels in the root meristem of shby-1 mutants. The
SHR/SCR and PLT pathways represent two partially independent
pathways that are required to maintain the function of the stem cell
niche in the Arabidopsis root. The reduction of PLT1 and PLT2, and
of SHR and SCR in the QC of the shby-1 mutants suggests that
SHBY acts in both pathways to maintain expression at wild-type
levels. Consistent with its role in root growth and meristem
maintenance, SHBY shows high expression in the root meristem in
a domain that overlaps with PLT1, PLT2, SHR and SCR.
Previous results by Aida et al. (Aida et al., 2004) suggested that
the expression of PLT1 mRNA was moderately decreased in both
the shr and scr mutants. We were able to confirm these results at the
level of the protein for both PLT1 and PLT2 using the PLT-GFP
marker lines. Interestingly, we find that the levels of SHBY
expression are also reduced in both the shr-2 and scr-4 mutants. As
PLT1 and PLT2 expression were both reduced in shby-1, a decrease
in SHBY in the shr and scr mutants may explain the reduced PLT1
and PLT2 expression. In this scenario, the reduced levels of SHBY
in the shr-2 and scr-4 mutant backgrounds result in the observed
decrease in PLT1 and PLT2. Another possibility is that the reduction
in SHR in shby-1 directly affects PLT levels. However, we think
that this is unlikely, as the level of SHR in the shby-1 mutant was
60% of the wild type and plants heterozygous for shr-2 have no
obvious meristem maintenance phenotypes (Koizumi et al., 2012).
Therefore, SHBY may provide a link between the SHR/SCR and the
PLT pathways in maintenance of root growth.
The other obvious phenotype of the shby-1 roots is the production
of extra ground tissue layers, as well as extra cells in each layer.
The ectopic periclinal cell divisions seen in shby-1 mutants could
DEVELOPMENT
1298 RESEARCH ARTICLE
point to a misregulation of MC formation. In wild-type plants, roots
begin making MC at around days 7-14. In these roots, MC forms
first in endodermal cells that lie in the xylem axis and then proceeds
circumferentially until all of the endodermal cells have divided
(Baum et al., 2002; Paquette and Benfey, 2005). The periclinal cell
divisions in the shby mutants are present by day 3 and they do not
follow a regular pattern; instead, they form randomly, which
indicates that in shby-1 roots the patterning of MC may be affected.
However, the effects of shby-1 on the patterning of the ground tissue
are not limited to the endodermis; they also occur in the cortex and
are either periclinal or anticlinal, which is inconsistent with
formation of MC. In addition the formation of MC is entirely
dependent upon functional SHR. This was not true for the cell
divisions in shby-1. Based upon the double mutant analysis, neither
SHR nor SCR was essential for the divisions in shby-1, although
both the shr-2 and scr-4 mutations reduced the number of ground
tissue layers in shby-1. Collectively, these results suggest that the
role of SHBY in patterning of the ground tissue extends beyond
only the regulation of MC and beyond SHR and SCR.
Although SHR was restricted to the endodermis and we saw no
expression of pSCR:erGFP outside of the endodermis, there was
an increase in SCR-GFP in the cortex cell layer, particularly in the
middle ground tissue layer following periclinal cell division. As
SHBY is a putative vacuolar-sorting protein that interacts with SCR,
this may reflect a reduction in the normal degradation of SCR
following periclinal cell divisions in the ground tissue of shby-1
roots. Consistent with a role for SHBY in protein turnover, there
was an overall decrease in SCR transcription in shby-1 without a
concomitant decrease in SCR protein levels. Likewise, we could
phenocopy the effects of shby-1 on SCR-GFP levels outside of the
endodermis by treating roots with the proteosome inhibitor MG132.
Interestingly, in the MG132-treated roots there were ectopic
divisions in the ground tissue, and expression of the
pCYCD6;1:GFP-GUS marker in the ground tissue beyond the
initial cells and their immediate daughters; this reflects what we
observed in shby-1 roots. Treatment of wild-type roots with MG132
also phenocopied the effects of shby-1 on PLT1 and PLT2 levels,
and the QC defects. These results are consistent with shby-1 playing
a role in protein degradation in the root meristem and in the
regulation of cell division and QC maintenance.
Both the aerial and the root phenotypes of shby seedlings suggested
defects in GA synthesis, perception or signal transduction (Ariizumi
and Steber, 2007; Ariizumi et al., 2008; Heo et al., 2011; Tyler et al.,
2004; Wen and Chang, 2002; Zhang et al., 2007). In the root, GA acts
in a partially overlapping pathway with SCR to inhibit asymmetric
cell divisions in the ground tissue. In the meristem, GA promotes
auxin signaling which maintains the meristem. To test whether
shby-1 seedlings are defective in GA signaling, we treated the plants
with PAC or GA and found that shby-1 is insensitive to both
treatments and that aspects of the shby phenotype could be induced
in wild-type roots by treatment with PAC. These results suggest that
SHBY acts downstream of GA biosynthesis either in GA perception
or in GA signaling. The precise function of SHBY in the GA pathway
remains to be elucidated. However, SHBY provides a connection
between GA, SHR and PLT signaling in the root meristem.
Acknowledgements
We thank R. Schlarp, K. Miyahara and J. Ugochukwu for technical assistance;
R. Sozzani for providing the pCYCD6;1:GUS-GFP seeds; T. P. Sun for providing
the gai-2 seeds; and R. Sozzani, C. Perin and D. Wagner for critical comments
on the manuscript. The Salk Institute Genomic Analysis Laboratory provided
the sequence-indexed Arabidopsis T-DNA insertion mutants. N. Matsumoto
made the pSHR:erGFP line.
RESEARCH ARTICLE 1299
Funding
This research was funded by the National Science Foundation [0920327 to
K.L.G. that partially supports K.K.].
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.090761/-/DC1
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