1568
RESEARCH ARTICLE
DEVELOPMENT AND STEM CELLS
Development 139, 1568-1576 (2012) doi:10.1242/dev.079426
© 2012. Published by The Company of Biologists Ltd
STUNTED mediates the control of cell proliferation by GA in
Arabidopsis
Li Yen Candy Lee1,2,*, Xingliang Hou1,*, Lei Fang1, Shuguo Fan3, Prakash P. Kumar1,2 and Hao Yu1,2,‡
SUMMARY
Gibberellins (GA) are an important family of plant growth regulators, which are essential for many aspects of plant growth and
development. In the GA signaling pathway, the action of GA is opposed by a group of DELLA family repressors, such as RGA.
Although the mechanisms of action of the DELLA proteins have been studied in great detail, the effectors that act downstream
of DELLA proteins and bring about GA-responsive growth and development remain largely unknown. In this study, we have
characterized STUNTED (STU), a receptor-like cytoplasmic kinase (RLCK) VI family gene, which is ubiquitously detectable in all the
tissues examined. RGA activity and GA signaling specifically mediate the levels of STU transcripts in shoot apices that contain
actively dividing cells. stu-1 loss-of-function mutants exhibit retarded growth in many aspects of plant development. During the
vegetative phase, stu-1 seedlings develop smaller leaves and shorter roots than wild-type seedlings, while during the reproductive
phase, stu-1 exhibits delayed floral transition and lower fertility. The reduced stature of stu-1 partly results from a reduction in
cell proliferation. Furthermore, we present evidence that STU serves as an important regulator mediating the control of cell
proliferation by GA possibly through two cyclin-dependent kinase inhibitors, SIM and SMR1. Taken together, our results suggest
that STU acts downstream of RGA and promotes cell proliferation in the GA pathway.
INTRODUCTION
Plants show massive variations in stature and genes involved in the
control of plant architecture have been recently categorized into
three classes, those affecting hormone metabolism and signaling,
transcription and other regulatory factors, and cell cycle regulators
(Busov et al., 2008). The ability to manipulate plant stature is a
valuable asset in agriculture. For example, dwarf cultivars of wheat
and rice that are less prone to lodging in adverse weather
conditions and have higher yield were crucial to the success of the
Green Revolution. These cultivars have been found to be impaired
in gibberellin (GA) biosynthesis or signaling, indicating the
importance of GA in determining plant stature (Peng et al., 1999;
Monna et al., 2002; Sasaki et al., 2002; Spielmeyer et al., 2002).
GA consists of a large family of plant growth regulators that
control many developmental processes. The Arabidopsis GA
biosynthesis mutant ga1-3 is severely dwarfed (Koornneef and
Vanderveen, 1980). In addition, ga1-3 is non-germinating,
produces dark-green leaves, and shows reduced apical dominance
and defects in flowering and floral organ development (Koornneef
and Vanderveen, 1980; Koornneef et al., 1983; Silverstone et al.,
1997; Goto and Pharis, 1999).
DELLA proteins are the major repressors of GA-responsive
growth and belong to the plant-specific GRAS protein family of
regulatory proteins (Pysh et al., 1999). In Arabidopsis, GA binds to
its receptor GID1, which enhances GID1 interaction with DELLA
1
Department of Biological Sciences, Faculty of Sciences, National University of
Singapore, 14 Science Drive 4, Singapore 117543. 2Temasek Life Sciences
Laboratory, 1 Research Link, National University of Singapore, Singapore 117604.
3
Department of Chemistry and Life Science, Chuxiong Normal University, Chuxiong
675000, China.
*These authors contributed equally to this work
Author for correspondence ([email protected])
‡
Accepted 21 February 2012
proteins (Griffiths et al., 2006). This interaction inactivates DELLA
repressor function in different manners (Schwechheimer and Willige,
2009). In a well-studied proteolytic degradation manner, DELLA
proteins are targeted to an SCF-E3 ubiquitin ligase by the F-box
protein SLY1, leading to their subsequent ubiquitylation and
degradation by the 26S proteasome (McGinnis et al., 2003; Dill et
al., 2004; Fu et al., 2004). This lifts the inhibitory effect of DELLAs
and results in GA-responsive growth and development.
During plant development, final organ size is dependent on
growth rate and duration. At the cellular level, growth rate is the
result of the interplay between cell proliferation and expansion.
Cell expansion directly increases organ size, whereas cell division
supplies the cells for expansion. By varying the length and pace of
mitotic activity, the total number of cells and therefore organ size
is altered. In leaf morphogenesis, growth is divided into two
phases. In the initial proliferative phase, cell number increases
rapidly, whereas cell size remains relatively constant. In the second
phase, cell division rate decreases and eventually ceases
accompanied by a dramatic increase in cell size (Donnelly et al.,
1999; Beemster et al., 2006). GA regulates cell elongation through
stimulating the destruction of DELLA repressors in various tissues,
including hypocotyls, stamens, stems and roots (Yang et al., 1996;
Cowling and Harberd, 1999; Cheng et al., 2004; Ubeda-Tomas et
al., 2008). Recently, a novel function of GA in regulating cell
production has also been discovered (Achard et al., 2009; UbedaTomas et al., 2009). GA regulates cell proliferation by removing
DELLA proteins in a subset of root meristem cells. Furthermore,
GA controls cell cycle activity in the root meristem by modulating
mRNA levels of several cell cycle inhibitors via a DELLAdependent mechanism. Therefore, GA regulates both cell expansion
and division by nullifying the negative effects of DELLA
repressors.
The Arabidopsis genome contains more than 600 genes
encoding receptor-like kinases (RLKs) (Shiu and Bleecker, 2001),
which are generally composed of an extracellular domain, a single
DEVELOPMENT
KEY WORDS: Gibberellins, DELLA proteins, Cell proliferation, Kinase
STU mediates cell proliferation
MATERIALS AND METHODS
Plant material and growth conditions
Arabidopsis thaliana plants were grown under long days at 22°C. stu-1
(SALK_039301) and sim-1 (CS23884) are in Columbia (Col) background,
whereas all the other genetic materials used in this study are in Landsberg
erecta (Ler) background. stu-1 (Ler) was created through backcrossing stu1 (Col) to Ler three times. Generation of ga1-3 gai-t6 rgl1-1 rgl2-1 rga-t2
and ga1-3 rgl2-1 rga-t2 35S:RGA-GR was as previously described (Yu et
al., 2004a). GA3 was used for all GA treatment in this study.
Expression analysis
Total RNAs were extracted using the FavorPre Plant Total RNA Mini Kit
(Favorgen) and reverse transcribed using the SuperScript RT-PCR System
(Invitrogen). Quantitative real-time PCR was performed on three
independently collected samples using the CFX384 real-time system (BioRad). The relative gene expression was calculated as previously described
(Liu et al., 2007). Semi-quantitative PCR was performed as previously
described (Yu et al., 2004a). For both quantitative real-time PCR and semiquantitative PCR, the -tubulin gene (TUB2) was amplified as an internal
control. Primers used for gene expression analysis are listed in
supplementary material Table S1. GUS staining and quantitative analysis
of GUS activity were carried out as previously described (Jefferson et al.,
1987). The concentration of dexamethasone, GA or PAC used for GUS
expression analyses was 10 M.
Plasmid construction
To construct STU:GUS, a 1.91 kb STU 5⬘ upstream sequence was amplified
and cloned into HY107 (Liu et al., 2007). A 4.5 kb STU genomic fragment
including the 1.91 kb 5⬘ upstream sequence was amplified and cloned into
pGreen-6HA (Li et al., 2008) to obtain an in-frame fusion of STU:STU6HA. To construct 35S:GFP-STU, the coding regions of GFP and STU
were cloned into pGreen-35S (Yu et al., 2004a). The amiR-stu construct
was generated according to the instructions on the web-based tool at
http://wmd.weigelworld.org (Schwab et al., 2006). 35S:STU was produced
by cloning the coding region of STU into pGreen-35S. 35S:RGA-6HA was
generated by cloning the RGA-coding region into pGreen-35S-6HA (Liu et
al., 2008). All primer sequences used for vector construction are listed in
supplementary material Table S1.
Particle bombardment
35S:GFP-STU plasmid was mixed with DNAdel gold carrier particles
(Seashell Technology) and precipitated following the manufacturer’s
instructions. Bombardment was carried out using the Biolistic PDS1000/He particle gun (Bio-Rad) as previously described (Yu et al., 2000).
Prior to bombardment, onion cells were treated with 400 mM sorbitol on
MS medium for 4 hours. After bombardment, they were placed on MS
medium and incubated overnight in the dark at room temperature before
viewing.
Analysis of Arabidopsis growth stage
Arabidopsis wild-type, stu-1 and 35S:STU seedlings grown on soil were
observed throughout the life of the plants and the growth stages reached
were noted every day. Number of days refers to days after sowing (das).
Growth stages are defined as previously described (Boyes et al., 2001).
Analysis of leaf growth
Measurements of leaf area, cell area and total cell numbers were carried
out on the first true leaf pair of 12 seedlings grown on soil at 10 and 22
das, as previously described (Achard et al., 2009).
ChIP assay
Eight-day-old seedlings of ga1-3 gai-t6 rgl1-1 rgl2-1 rga-t2 35S:RGA-6HA
were fixed and used for ChIP assays as previously described (Liu et al.,
2008). Total chromatin before pull down was used as an input control
against DNA recovered after binding to anti-HA antibody (Sigma). Ten
pairs of primers (supplementary material Table S1) distributed along the
STU genomic region were designed for measuring DNA enrichment.
Flow cytometric analysis
The first true leaves of 3-week-old wild type, stu-1, sim-1 and sim-1 stu-1
plants were collected for flow cytometry analysis as previously described
(Arumuganathan and Earle, 1991). The ploidy levels were analyzed with
the CyAn ADP analyzer (Beckman-Coulter, Roissy, France) using Summit
v4.3 software.
RESULTS
STU is downregulated by RGA in the GA signaling
pathway
We performed a microarray analysis to identify immediate
targets of RGA in Arabidopsis flower development (Hou et al.,
2008) and selected STU (At2g16750) from a list of genes whose
expression were altered by the induced RGA activity. To
understand further the regulation of STU expression in the GA
signaling pathway, we examined its expression in seedlings of
various mutant backgrounds (Fig. 1A). There was no change in
STU transcript levels between wild-type and rga-t2 seedlings,
possibly because RGA activity was very low or abolished in
either wild-type or rga-t2 seedlings, respectively (Silverstone et
al., 2001; Ariizumi et al., 2008). In the GA-deficient mutant ga13, in which RGA protein was relatively stabilized, STU
transcription was closely controlled by RGA activity. In the
absence of RGA in ga1-3 rga-t2, STU was significantly
upregulated (Fig. 1A). This indicates that RGA activity
suppresses STU expression in the whole seedlings, which is
similar to the scenario in flower development (Hou et al., 2008).
Next, we examined the response of STU expression to GA
treatment in wild-type and ga1-3 seedlings, and found that STU
transcript levels were significantly upregulated by GA in ga1-3
(Fig. 1B). This suggests that GA treatment, which stimulates the
degradation of DELLA proteins, promotes STU expression.
To validate the regulation of STU by RGA, we studied STU
expression in an established steroid-inducible functional version of
RGA (RGA-GR) in ga1-3 rgl2-1 rga-t2 (Yu et al., 2004b). The
time course expression of STU from dexamethasone- and mocktreated ga1-3 rgl2-1 rga-t2 35S:RGA-GR seedlings was examined
DEVELOPMENT
transmembrane span and a cytoplasmic region containing a
conserved kinase domain. Receptor-like cytoplasmic kinases
(RLCKs), which lack a transmembrane or extracellular domain, are
a subfamily of RLKs (Shiu and Bleecker, 2003). They make up
~25% of RLKs and are further divided into eight classes numbered
RLCK I to VIII. The subfamily of class VI RLCKs consists of 14
members (Jurca et al., 2008). Although the members in this
subfamily from different plant species have been found to interact
with a group of plant-specific signaling regulators, the Rho-type
GTPases (Molendijk et al., 2008; Dorjgotov et al., 2009), their
biological functions are largely unknown.
In this study, we report the characterization of a RLCK VI
family protein, STUNTED (STU; meaning stunted plant
growth), which was earlier designated as RLCK VI_B4 (Jurca et
al., 2008). STU expression in seedlings is downregulated by
RGA in response to GA. The loss-of-function mutant, stu-1,
displays multiple defects, including a smaller plant stature that
results from a reduction in cell division. In the absence of STU,
the effect of GA signaling on cell division is reduced, whereas
overexpression of STU elevates cell cycle activity. GA promotes
STU expression via degradation of RGA. STU in turn represses
the transcription of two cell cycle inhibitors, SIAMESE (SIM)
and SIAMESE-RELATED 1 (SMR1), to enhance cell division,
thus promoting GA-mediated growth and development. Our
results establish STU as an important regulator that mediates GA
regulation of plant stature.
RESEARCH ARTICLE 1569
1570 RESEARCH ARTICLE
Development 139 (9)
To test whether RGA affects STU expression through directly
associating with the STU promoter, we created 35S:RGA-6HA lines
for chromatin immunoprecipitation (ChIP) analysis. 35S:RGA-6HA
was introduced into ga1-3 gai-t6 rgl1-1 rgl2-1 rga-t2, and a functional
line that mimicked ga1-3 phenotypes was chosen for further ChIP
assays. ChIP analyses did not detect the binding of RGA-6HA to the
STU locus (supplementary material Fig. S2), suggesting that RGA
regulation of STU involves other regulatory factors.
Fig. 1. STU is downregulated by RGA in a GA-dependent manner.
(A)STU expression in 8-day-old wild-type, rga-t2, ga1-3 and ga1-3 rgat2 seedlings. (B)STU expression in 8-day-old wild-type and ga1-3
seedlings mock treated or treated with 10M GA for 4 hours. (C)Time
course expression of STU in 8-day-old ga1-3 rgl2-1 rga-t2 35S:RGA-GR
seedlings. Values were calculated by normalizing the relative mRNA
levels of STU in 10M dexamethasone-treated (DEX) versus mocktreated (MOCK) samples at 0, 2, 4 and 8 hours after treatment. (D)STU
expression in 8-day-old ga1-3 rgl2-1 rga-t2 35S:RGA-GR seedlings
mock treated (MOCK), or treated with 10M dexamethasone (DEX),
10M cycloheximide (CYC) or 10M cycloheximide plus
dexamethasone (CYC+DEX) after 8 hours of treatment. Transcript levels
(A-D) were determined by quantitative real-time PCR analyses. Relative
gene expression levels were normalized against the expression of TUB2.
Asterisks indicate significant changes in gene expression in each pair of
samples (A,B,D) or when compared with the gene expression at 0 hour
(C) (P<0.05, by Student’s t-test). Error bars indicate s.d.
by quantitative real-time PCR at 0, 2, 4 and 8 hours after treatment.
STU expression was downregulated from 4 hours after RGA
induction in dexamethasone-treated seedlings (Fig. 1C). We further
examined STU expression under a combined treatment of
dexamethasone and cycloheximide, an inhibitor of protein
synthesis, at the 8-hour time point. STU exhibited a similar
decreased expression pattern under the combined treatment (Fig.
1D), suggesting that RGA modulates STU expression
independently of protein synthesis and that STU might be an
immediate target of RGA. Thus, repression of STU by RGA occurs
not only in flower development (Hou et al., 2008), but also in
young seedlings.
As STU was differentially expressed in ga1-3 rga-t2 and rga-t2
(Fig. 1A), other DELLA proteins that are stabilized in ga1-3 might
also play a role in affecting STU expression. Thus, we measured
STU expression in various DELLA mutants with ga1-3 background
to examine to what extent different DELLA proteins affect STU
expression (supplementary material Fig. S1). Among the DELLA
proteins investigated, RGA had the strongest effect on STU
expression. GAI had a redundant effect as RGA, whereas RGL1
and RGL2 slightly upregulated STU. These results imply that
although various DELLA proteins affect STU expression, RGA
might be a major regulator of STU.
Expression pattern of STU
To gain insights into the biological function of STU, we
investigated its temporal and spatial expression during Arabidopsis
development. Semi-quantitative RT-PCR was performed with
RNAs extracted from different parts of 6-week-old wild-type
seedlings (Fig. 2A). STU was expressed in all the tissues examined
with the highest expression in roots and lowest expression in open
flowers and siliques.
To further examine the detailed expression patterns of STU in
Arabidopsis development, we transcriptionally fused a 1.91 kb STU
5⬘ upstream sequence to the GUS reporter gene, because a STU
genomic fragment including this upstream sequence (STU:STU6HA) was sufficient to rescue stu-1 phenotypes described below
(supplementary material Fig. S4). Among 18 independent lines of
the transformants harboring STU:GUS, 13 lines displayed similar
GUS staining patterns and one representative line was thus selected
for further investigation.
At 3 days after germination, GUS activity was detected in shoot
apices and roots (Fig. 2B). Intense staining patterns were observed
in the same regions of 1-week-old and 2-week-old seedlings with
gradually increased staining in the leaf vasculature (Fig. 2C,D). In
3-week-old seedlings, GUS staining remained in main
inflorescence apices and was also strongly detected in secondary
inflorescence meristems that were subtended by cauline leaves
(Fig. 2E). GUS staining was strong in the center of the
inflorescence apex and the anthers of floral buds at flower stages 9
to 12 (Fig. 2F). A close examination of longitudinal sections of a
stage 10 flower revealed the specific staining in the tapetum (Fig.
2G). At stage 14, GUS staining was mainly detected in ovules and
pollen (Fig. 2H). Thus, STU is mainly expressed in various tissues
or organs containing actively dividing cells.
To determine the subcellular localization of STU protein, the
green fluorescent protein (GFP) was fused to the N terminus of
STU under the control of the cauliflower mosaic virus (CaMV)
35S promoter. The 35S:GFP-STU construct was introduced into
onion epidermal cells by particle bombardment and the localization
of the fusion protein was analyzed. Like 35S:GFP (Fig. 2K,L),
GFP-STU signals were observed throughout the cell (Fig. 2I,J),
which is similar to the pattern of several characterized RLCKs
(Kong et al., 2007; Molendijk et al., 2008), implying that STU may
DEVELOPMENT
STU encodes a putative RLCK VI family protein
The STU gene consists of 11 exons and 10 introns and encodes a
putative Ser/Thr kinase, belonging to the subfamily of class VI
RLCKs in Arabidopsis (Jurca et al., 2008). In Arabidopsis, the
STU protein shares 69% amino acid identity with its closest
homolog, RLCK VI_B3 (At4g35030). Multiple sequence
alignment revealed a highly conserved kinase domain and an active
site among all the RLCKs compared from different plant species
(supplementary material Fig. S3). In addition, STU has a UspA
domain near its N terminal, which was named after the universal
stress protein of E. coli, UspA, the expression of which is enhanced
upon stress treatment (Nystrom and Neidhardt, 1994).
STU mediates cell proliferation
RESEARCH ARTICLE 1571
Fig. 2. Expression patterns of STU. (A)STU expression in various
tissues of 6-week-old wild-type seedlings. TUB2 was amplified as an
internal control. RL, rosette leaf; CL, cauline leaf; ST, stem; B, floral bud;
O, open flower; S, silique; R, root. (B-E)GUS staining of STU:GUS
seedlings grown on MS medium at 3 days (B), 1 week (C), 2 weeks (D)
and 3 weeks (E) after germination. Arrows in E indicate the positions of
secondary inflorescence meristems. (F)GUS staining of an STU:GUS
inflorescence apex containing floral buds up to flower stage 11 or 12.
(G)GUS staining in a longitudinal section of a stage 10 STU:GUS
flower. (H)GUS staining of a flower stage 14 STU:GUS flower. Scale
bars: 5 mm in B-E; 200m in F-H. (I,J)Subcellular localization of
35S:GFP-STU in onion epidermal cells. (K,L)Subcellular localization of
the control 35S:GFP in onion epidermal cells. (I,K)GFP signal.
(J,L)Merging of GFP signal and bright-field image.
exert its function in multiple cell organelles. As 35S:GFP-STU was
able to rescue stu-1 (data not shown), GFP-STU protein is probably
functional.
RGA mediates regulation of STU by GA in shoot
apices
To understand how GA signaling regulates STU expression in
tissues containing actively dividing cells in Arabidopsis, we
monitored GUS staining patterns in shoot apices of 8-day-old
STU:GUS seedlings treated with GA and the GA-biosynthesis
inhibitor paclobutrazol (PAC). GA treatment promoted STU:GUS
activity in shoot apices when compared with mock treatment,
whereas PAC inhibited STU:GUS activity (Fig. 3A,B). These
results are consistent with those from gene expression analysis in
seedlings (Fig. 1B) and shoot apices (Fig. 3B), suggesting that
upregulation of STU by GA occurs in shoot apices of seedlings.
To further confirm that the regulation of STU expression by GA
is mediated through RGA, we examined STU:GUS activity and
STU transcripts in 8-day-old ga1-3 rgl2-1 rga-t2 35S:RGA-GR
stu-1 loss-of-function mutants show multiple
developmental defects
To investigate the biological role of STU in Arabidopsis, we
identified a STU mutant (SALK_039301) containing a T-DNA
insertion in the 6th exon from the Arabidopsis Biological Resource
Centre (ABRC) (Fig. 4A). The presence of the T-DNA insertion was
confirmed by PCR genotyping (data not shown), and semiquantitative RT-PCR revealed that STU transcripts in developing
seedlings were abolished by the presence of the T-DNA
(supplementary material Fig. S5A). As this is the first instance of
identification of a STU null allele, we have named this mutant stu-1.
Compared with wild-type plants, seedling development of stu-1
was retarded, resulting in a smaller plant stature (Fig. 4B). At 4
weeks after germination, corresponding rosette and cauline leaves in
stu-1 were more curled and smaller in area than those in wild-type
plants (Fig. 4C), but the chlorophyll content of the leaves was
unchanged (supplementary material Fig. S6A). stu-1 roots were also
shorter than those of wild-type plants when they were grown on MS
medium (Fig. 4D). At 5 weeks after germination, the first flowers of
wild-type plants were usually open, whereas stu-1 had not yet bolted
(Fig. 4E). In addition to the above-mentioned retarded growth
defects, stu-1 also displayed lower germination rate and reduced
fertility, as observed from its short siliques and deformed pollen
grains (supplementary material Fig. S6B-H). Taken together, stu-1
demonstrates the similar developmental defects to those of GAdeficient mutants (Koornneef and Vanderveen, 1980).
To verify the phenotypes observed in stu-1, we created amiR-stu
knockdown transgenic lines that expressed an artificial microRNA
specifically targeting STU (Fig. 4A). A total of 28 independent
transgenic lines were subsequently obtained, among which 12 lines
were chosen for further analysis of their STU mRNA expression
(supplementary material Fig. S5B). STU mRNA levels were
downregulated in 11 of these lines, among which eight lines (lines
1, 2, 5, 6, 7, 8, 11 and 12) with the lower STU mRNA levels
showed an obvious decrease in plant stature when compared with
wild-type plants. Line 2 (hereafter called amiR-stu), which had the
lowest STU mRNA expression, exhibited a similar stunted
phenotype as stu-1 (Fig. 4B). These results confirm that reduced
STU expression causes the retarded plant growth observed. This is
further corroborated by a complementation test in which a 4.5 kb
STU genomic fragment was able to rescue the stu-1 phenotypes
(supplementary material Figs S4, S7).
We also created transgenic lines overexpressing STU under the
control of 35S promoter. A total of 29 independent transgenic lines
were obtained, among which nine lines were chosen for further
analysis of STU expression (supplementary material Fig. S5C).
STU mRNA levels increased in eight of these lines, among which
six showed a slight increase in plant stature as compared with wildtype plants. Line 1 (hereafter called 35S:STU) with the highest STU
mRNA expression was selected for further analysis (Fig. 4B).
To analyze further the plant growth affected by STU, we
performed a detailed phenotypic analysis of wild-type, 35S:STU
and stu-1 plants at two stages each during vegetative and
reproductive development following the system previously
DEVELOPMENT
seedlings mock treated or treated with dexamethasone. Induction
of RGA activity at 4 hours after dexamethasone treatment reduced
STU:GUS activity and STU expression in shoot apices when
compared with those after mock treatment (Fig. 3C,D). This
demonstrates the repression of STU expression by RGA in shoot
apices, and substantiates that RGA mediates the regulation of STU
expression by GA.
Fig. 3. GA regulates STU expression in shoot apices through RGA.
(A)GUS expression in shoot apices of 8-day-old STU:GUS seedlings
mock treated (MOCK), or treated with 10M GA or 10M PAC for 24
hours. Right panels show enlarged views of the shoot apices shown on
the left. Scale bars: 1 mm. (B)Quantitative analysis of STU expression
upon GA and PAC treatment. Upper panel shows STU expression in
shoot apices of 8-day-old wild-type seedlings treated as shown in A.
Lower panel shows GUS activity in shoot apices of 8-day-old STU:GUS
seedlings treated as shown in A. (C)GUS expression in shoot apices of
8-day-old ga1-3 rgl2-1 rga-t2 35S:RGA-GR STU:GUS seedlings mocktreated (MOCK) or treated with dexamethasone (DEX) for 4 hours.
Right panels show enlarged views of the shoot apices shown on the
left. Scale bars: 1 mm. (D)Quantitative analysis of STU expression in
response to RGA activity. Upper panel shows STU expression in shoot
apices of 8-day-old ga1-3 rgl2-1 rga-t2 35S:RGA-GR seedlings treated
as shown in C. Lower panel shows GUS activity in shoot apices of 8day-old ga1-3 rgl2-1 rga-t2 35S:RGA-GR STU:GUS seedlings treated as
shown in C. In B,D, STU expression levels were determined by
quantitative real-time PCR analyses and normalized against the
expression of TUB2. GUS activity values were scored from at least 15
plants of each treatment. Asterisks indicate significant changes in gene
expression or GUS activity when compared with mock treatment
(P<0.05, by Student’s t-test). Error bars indicate s.d.
developed (Boyes et al., 2001). As shown in Fig. 4F, the vegetative
stages 1.02 and 1.10 indicate the number of days after germination
at which the 2nd and 10th rosette leaves are greater than 1 mm in
length, respectively. The reproductive stage 5.10 defines the time
at which the first floral bud becomes visible, whereas stage 6.00 is
the point at which the first flower is open. Our results showed that
all wild-type, stu-1 and 35S:STU plants took ~10 days to reach
stage 1.02, despite their differences in stature (Fig. 4F). Wild-type,
stu-1 and 35S:STU plants showed slight differences in the average
number of days taken to reach stage 1.10. The differences in their
Development 139 (9)
Fig. 4. Loss of STU function causes retarded Arabidopsis growth.
(A)Schematic diagram indicating the T-DNA insertion site in stu-1
(SALK_039301) and the target region of amiR-stu. Exons are
represented by boxes and introns by lines. Grey boxes represent the
untranslated regions. (B)Comparison of wild-type, stu-1, amiR-stu (line
2) and 35S:STU (line 1) seedlings at 18 days after germination.
(C)Comparison of corresponding leaves of 4-week-old wild-type and
stu-1 seedlings. Except for the cauline leaf pair on the extreme right, all
the leaves displayed are rosette leaves. Scale bar: 1 cm. (D)Comparison
of wild-type and stu-1 seedlings grown on MS medium at 8 days after
germination. (E)Comparison of wild-type and stu-1 at 5 weeks after
germination. (F)Comparison of the growth of wild-type, stu-1 and
35S:STU through developmental stages 1.02, 1.10, 5.10 and 6.00. At
least 15 plants of each genotype were analyzed. Asterisks indicate
significant differences in growth when compared with wild type
(P<0.05, by Student’s t-test). Error bars indicate s.d.
growth were more obvious at stages 5.10 and 6.00. In wild-type
plants, the first flower buds emerged at around 23 days, whereas
the corresponding days in stu-1 and 35S:STU were 30 and 20,
respectively. The number of days at which the first flowers were
open in wild type, stu-1 and 35S:STU are 32, 37 and 30,
respectively. Consistently, the differences in rosette radius and plant
height of various STU transgenic lines and mutants also support
that STU affects plant stature (supplementary material Fig. S7).
STU mediates the control of cell division by GA
To understand the difference in plant stature between wild-type and
stu-1 plants, we investigated cell division and elongation, the two
cellular processes responsible for growth, by scanning electron
microscopy of the first true leaves at 10 and 22 days after sowing
(das) (supplementary material Fig. S8). At 10 das when leaf growth
is mainly linked to cell proliferation (Donnelly et al., 1999;
DEVELOPMENT
1572 RESEARCH ARTICLE
STU mediates cell proliferation
RESEARCH ARTICLE 1573
Beemster et al., 2006), leaf area of stu-1 was only 61% of that of
wild-type plants (Fig. 5A). Individual cell area in stu-1 remained
largely unchanged, whereas cell number in stu-1 was 31% less than
in wild-type plants. At 22 das, when cell expansion is the major
cause of leaf growth (Donnelly et al., 1999; Beemster et al., 2006),
individual cell area of wild-type and stu-1 plants were still similar,
whereas leaf area and cell number in stu-1 were reduced by 26%
and 25% when compared with wild-type plants, respectively (Fig.
5A). Thus, these results demonstrate that a reduction in cell
proliferation is the main reason for the reduced leaf area of stu-1.
We then crossed a Dbox CYCB1;1-GUS expressing line with
stu-1 to study the role of STU in cell division. The CYCLIN B1
(CYCB1;1)-GUS reporter is expressed in cells during the G2-M
phase of cell cycle, thus allowing us to monitor mitotic activity
(Colon-Carmona et al., 1999). The root meristem was examined for
the difference in cell division between wild-type and stu-1 plants.
Based on the intensity and extent of GUS staining and quantitative
analysis of GUS activity, mitotic activity was evidently reduced in
root meristems of stu-1 when compared with wild-type plants
under mock treatment (Fig. 5B,C). In wild-type root meristems,
GA treatment increased the number of dividing cells, whereas PAC
treatment significantly decreased mitotic activity (Fig. 5B,C). On
the contrary, similar treatments in stu-1 had less effect on the
change of cell division (Fig. 5B,C), suggesting that GA mediates
plant cell division via STU.
To quantify the observed differences in cell division, we
measured the transcript levels of CYCB1;1 and KNOLLE, two
genes known to be expressed during mitosis (supplementary
material Fig. S9A). At 8 days after germination, transcript levels of
CYCB1;1 and KNOLLE in stu-1 were reduced by approximately
two-thirds and one-half, respectively, when compared with wildtype plants, substantiating that STU mediates cell proliferation rate.
Consistent with the change of CYCB1;1 and KNOLLE expression
in stu-1, the root meristem size, which was measured as the number
of cells between the quiescent center and the first elongating cell,
was reduced in stu-1 at both 3 and 5 days after germination
(supplementary material Fig. S9B,C).
To confirm that STU mediates the control of plant stature by GA,
we examined whether increased STU activity is able to rescue GAdeficient phenotypes. Hence, we created 35S:STU in the Ler
DEVELOPMENT
Fig. 5. STU affects cell proliferation.
(A)Comparison of leaf blade area, average
area of a cell and total cell number of the
first true leaf pair of wild type and stu-1 at
10 and 22 days after sowing (das). Asterisks
indicate significant differences between stu1 and wild type (P<0.05, by Student’s ttest). (B)GUS expression in root tips of 10day-old Dbox CYCB1;1-GUS and stu-1
Dbox CYCB1;1-GUS seedlings mock treated
(MOCK) or treated with PAC or GA for 10
hours. Scale bar: 50m. (C)Quantitative
analysis of GUS activity in root tips of Dbox
CYCB1;1-GUS plants treated as shown in B.
Values representing the mean ± s.d. were
scored from at least 30 plants of each
genotype. Asterisks indicate significant
difference between GA or PAC treatment
and mock treatment (P<0.05, by Student’s
t-test). (D)Comparison of 3-week-old ga13, ga1-3 35S:STU and ga1-3 stu-1 plants.
Scale bar: 1 cm. (E)Comparison of the
maximum rosette radius of ga1-3, ga1-3
35S:STU and ga1-3 stu-1 from day 10 to 35
after germination. Maximum rosette radius
was measured on the longest rosette leaf of
each plant. Values representing the mean ±
s.d. were scored from at least 15 plants of
each genotype. (F)Relative expression of
SIM and SMR1 in 8-day-old seedlings with
various genetic backgrounds mock treated
or treated with 10M GA for 10 hours.
Transcript levels were determined by
quantitative real-time PCR analyses. Relative
gene expression levels were normalized
against the expression of TUB2. Asterisks
indicate significant changes in gene
expression between mock and GA
treatment (P<0.05, by Student’s t-test).
Error bars indicate s.d.
1574 RESEARCH ARTICLE
Development 139 (9)
background and found increased plant stature in 35S:STU lines
(Ler background) as those in the Col background (supplementary
material Fig. S5C). One representative 35S:STU line was selected
for genetic crossing with ga1-3 (Ler background). As expected,
35S:STU was able to partially rescue the reduced stature of ga1-3
(Fig. 5D,E). We further created ga1-3 stu-1 double mutants, in
which stu-1 (Col) had been backcrossed to Ler background for
three times. ga1-3 stu-1 showed comparable stature to ga1-3,
suggesting that STU acts downstream of the GA biosynthetic
pathway (Fig. 5D,E). Taken together, these results substantiate that
STU acts in the GA pathway to control plant stature. Interestingly,
GA dose-response assays of the maximum rosette radius and time
to bolting and flowering showed that although stu-1 always grew
slower than wild-type plants, stu-1 still responded to GA treatment
(supplementary material Fig. S10). This implies that GA also
affects plant stature in an STU-independent manner.
DISCUSSION
STU encodes an RLCK that functions in
maintaining normal plant stature
In this study, we characterized the biological function of STU, an
RLCK VI family protein, in Arabidopsis. Although STU is
ubiquitously detectable in all the tissues examined, it is highly
expressed in shoot apices and roots, which contain actively dividing
cells, indicating that STU activity is relevant to active cell
Fig. 6. sim-1 is epistatic to stu-1. (A)Comparison of wild-type, stu-1,
sim-1 and sim-1 stu-1 plants at 30 days after germination.
(B)Comparison of leaf blade area, average area of a cell and total cell
number of the first true leaf pair of wild-type, stu-1, sim-1 and sim-1
stu-1 plants at 22 days after sowing. Asterisks indicate significant
differences between stu-1 and the other plants. (P<0.05, by Student’s
t-test). (C)Ploidy level distribution of the first true leaves of 3-week-old
wild-type, stu-1, sim-1 and sim-1 stu-1 plants as measured by flow
cytometry. Values representing mean ± s.d. shown in the diagram (left)
were scored from at least eight plants of each genotype. The mean
values are also shown in the table (right). Asterisks indicate significant
differences between stu-1 and the other plants (P<0.05, by Student’s
t-test).
proliferation. stu-1 mutants exhibit retarded growth in many aspects
of plant development during vegetative and reproductive stages. stu1 seedlings develop smaller leaves and shorter roots compared with
wild-type seedlings at the vegetative phase, while at the reproductive
phase, stu-1 exhibits delayed floral transition and lower fertility, and
produces deformed pollen grains. A detailed analysis of cell division
and expansion in leaves has revealed that the reduced stature of stu1 at least partly results from a reduction in cell proliferation (Fig. 5).
These observations suggest that STU functions in the control of cell
proliferation to maintain normal plant stature.
There are 14 members in the subfamily of class VI RLCKs in
Arabidopsis (Jurca et al., 2008). A few of them have been shown
to interact with and be specifically activated by Rop GTPases
(Molendijk et al., 2008), implying that RLCKs may function as
effectors of Rop GTPases, which are plant-specific Rho small
GTPases involved in signaling pathways and act as molecular
switches to control the transmission of extracellular signals in
plants (Zheng and Yang, 2000; Li et al., 2001; Yang, 2002;
Agrawal et al., 2003). Although the diverse tissue expression of
STU and ubiquitous subcellular localization of the protein imply a
possible role of STU as an effector of Rop in cellular signaling,
whether STU affects plant stature through its interaction with Rop
GTPases needs to be further investigated.
RGA negatively regulates STU in the GA pathway
The phytohormone GA plays an important role in controlling
many aspects of plant development throughout the plant life
cycle. DELLA proteins serve as the major repressors of GA-
DEVELOPMENT
STU affects cell division partly through SMR1 and
SIM
A recent study has found that DELLA restrain of cell proliferation
results from the accumulation of transcript levels of cyclin-dependent
kinase inhibitors, a group of proteins that bind to cyclin/cyclindependent kinase complexes (CYC/CDK) and inhibit cell cycle
(Achard et al., 2009). Hence, we further examined the link between
the expression of several cyclin-dependent kinase inhibitor genes and
STU activity in the GA pathway. Quantitative real-time PCR revealed
that two of the cyclin-dependent kinase inhibitor genes, SMR1 and
SIM, were regulated through STU in the GA signaling pathway (Fig.
5F). In Ler wild-type plants, GA treatment reduced the expression of
SMR1 and SIM, which is consistent with the previously published
data (Achard et al., 2009), indicating that both genes are regulated in
the GA pathway. Without GA treatment, both SMR1 and SIM were
downregulated in 35S:STU, but upregulated in stu-1, indicating that
STU inhibits the expression of SMR1 and SIM. As GA treatment
triggers the degradation of RGA and increases STU levels, 35S:STU
seems to mimic the effect of GA treatment on cell proliferation, thus
inhibiting the expression of SMR1 and SIM. Furthermore, GA
treatment of 35S:STU and stu-1 (Ler) had less pronounced effects on
reducing the expression of SMR1 and SIM than GA treatment of
wild-type plants (Fig. 5F). The similar attenuated effect of GA
treatment on regulating the expression of SMR1 and SIM was also
observed in ga1-3 35S:STU and ga1-3 stu-1 when compared with
ga1-3 (Fig. 5F). These results corroborate that STU acts in the GA
pathway to regulate SMR1 and SIM expression. Consistently, loss of
SIM function in sim-1 significantly suppressed the defects in stu-1,
including the stunted stature (Fig. 6A), reduced cell number (Fig.
6B) and higher ploidy levels (Fig. 6C), suggesting that SIM indeed
acts downstream of STU in the control of cell division.
In contrast to SMR1 and SIM, the expression of other two cyclindependent kinase inhibitor genes, SMR2 and Kip-related protein 2
(KRP2), was not similarly affected by STU activity and GA
treatment (supplementary material Fig. S11), implying that they
might be differentially regulated.
responsive growth and null mutations of different combinations
of DELLA proteins rescue different aspects of GA-deficient
phenotypes (Dill and Sun, 2001; Cheng et al., 2004; Yu et al.,
2004b; Cao et al., 2006). Although several microarray analyses
have been performed to identify genes regulated by DELLA
proteins or GA in Arabidopsis, detailed analyses on the effectors
that function downstream of DELLA proteins and bring about
GA-responsive growth and development remain largely
unknown. Our studies have shown that STU expression is
repressed by RGA activity and is an immediate target of RGA
regulation, because induced RGA activity represses STU
expression within 4 hours and independently of protein
synthesis. On the contrary, GA treatment stimulates the
degradation of RGA, and thus promotes STU expression. In
particular, STU expression in shoot apices and roots where cell
division actively occurs is specifically mediated by RGA activity
and GA signaling. These observations strongly suggest that STU
is a downstream effector of the GA signaling pathway, and that
GA promotes STU expression through degradation of RGA,
which might be relevant to the regulation of cell proliferation.
STU mediates the control of cell division by GA
GA has been shown to modulate growth by promoting cell
expansion (Yang et al., 1996; Cowling and Harberd, 1999;
Cheng et al., 2004; Ubeda-Tomas et al., 2008) and cell division
(Achard et al., 2009; Ubeda-Tomas et al., 2009). Our study has
provided several pieces of evidence that supports a role for STU
in mediating the control of cell division by GA. First, stu-1 lossof-function mutants exhibit a decrease in cell proliferation in
leaves, whereas cell expansion is unaffected (Fig. 5A). This
result, together with the observation that the expression of STU
transcripts is mainly in the tissues where active cell division
takes place (Fig. 2), suggests that STU is involved in the control
of cell division. Second, staining and quantitative analysis of
Dbox CYCB1;1-GUS in root tips (Fig. 5B,C) and gene
expression analyses of CYCB1;1 and KNOLLE in seedlings
(supplementary material Fig. S9A) have shown that mitotic
activity is obviously reduced in stu-1 when compared with wildtype plants. This further substantiates that STU controls cell
division. Third, in wild-type roots, GA treatment promotes cell
proliferation, whereas PAC treatment reduces cell proliferation
(Fig. 5B,C). However, these effects are impaired or almost
completely abolished in stu-1 roots, demonstrating that, in the
absence of STU, the effect of GA signaling on cell proliferation
is compromised. Last, increased STU activity is able to rescue
the reduced plant stature in ga1-3, whereas loss of STU function
in ga1-3 mimics the phenotypes of ga1-3 (Fig. 5D,E). These
results all support that STU serves as an important regulator that
mediates the control of cell proliferation by GA.
It has been found that DELLA proteins restrain cell production
in leaves and root meristems by enhancing the transcript levels of
the cyclin-dependent kinase inhibitors KRP2, SIM, SMR1 and
SMR2 (Achard et al., 2009). These inhibitors prevent cell cycle
progression by interacting with D-type CYC and A-type CDK
subunits (Inze, 2005). Our results suggest that two of the cyclindependent kinase inhibitor genes, SMR1 and SIM, are regulated
through STU in the GA signaling pathway (Fig. 5F). Further
genetic and phenotypic analyses have shown that loss of SIM
function rescues stu-1 mutant phenotypes (Fig. 6). These results
propose a role for STU in conveying the signal for growth from GA
through RGA to the repression of cyclin-dependent kinase
inhibitors, which in turn mediate cell division.
RESEARCH ARTICLE 1575
Fig. 7. STU mediates the control of Arabidopsis plant stature by
GA. GA binds to its receptor GID, which enhances the interaction with
RGA. This inactivates RGA activity partially through SLY1-mediated
degradation of RGA by the 26S proteasome. Inactivation of RGA
upregulates STU expression, which in turn decreases the expression of
two cyclin-dependent kinase inhibitors, SIM and SMR1.
Downregulation of these inhibitors drives cell proliferation and normal
plant growth.
An updated model for GA-mediated control of
plant stature
Our results have led to an updated model that includes a new
player, STU, in GA-mediated control of plant stature (Fig. 7). In
this model, the presence of GA is perceived by the GA receptor
GID1. The GA-GID1 complex interacts with RGA and inactivates
RGA activity partially through SLY1-mediated degradation of
RGA by the 26S proteasome. STU expression is repressed by RGA
in the default state and inactivation of RGA causes an increase in
STU transcript levels. STU in turn decreases the levels of SIM and
SMR1, thus driving cell proliferation and promoting GA-responsive
growth and development.
The exact mode of action of STU is an interesting topic for
future studies as it will reveal how plant growth is executed in its
entirety, from hormone perception to cell proliferation. Several
recent publications have shown that members of RLCK VI family
in Arabidopsis and Medicago truncatula can interact with and be
specifically activated by Rop GTPases (Molendijk et al., 2008;
Dorjgotov et al., 2009). Although we have not found any
interaction between STU and Rop GTPases by yeast two-hybrid
analyses, it is still possible that STU participates in cellular
signaling through its interaction with other families of GTPases,
such as ARF, RAN and RAB. Further elucidation of the regulatory
cascade and substrate(s) of STU will greatly advance our
understanding of the intricate details of GA-responsive growth.
Acknowledgements
We thank P. Doerner for providing the pCYCB1;1:Dbox-GUS line, the
Arabidopsis Biological Resource Centre for the seeds of stu-1 (Salk_039301)
and sim-1 (CS23884), and Lisha Shen and Chang Liu for critical reading of the
manuscript.
Funding
This work was supported by the Academic Research Fund MOE2011-T2-1-018
from the Ministry of Education, Singapore, and by the intramural research
funds from Temasek Life Sciences Laboratory.
DEVELOPMENT
STU mediates cell proliferation
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.079426/-/DC1
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