© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 2735-2744 doi:10.1242/dev.106104
RESEARCH ARTICLE
TECHNIQUES AND RESOURCES
A high-resolution gene expression map of the Arabidopsis shoot
meristem stem cell niche
Ram Kishor Yadav1,2,*, Montreh Tavakkoli1, Mingtang Xie1, Thomas Girke1 and G. Venugopala Reddy1,*
The shoot apical meristem (SAM) acts as a reservoir for stem cells. The
central zone (CZ) harbors stem cells. The stem cell progenitors
differentiate in the adjacent peripheral zone and in the rib meristem
located just beneath the CZ. The SAM is further divided into distinct
clonal layers: the L1 epidermal, L2 sub-epidermal and L3 layers.
Collectively, SAMs are complex structures that consist of cells of
different clonal origins that are organized into functional domains. By
employing fluorescence-activated cell sorting, we have generated gene
expression profiles of ten cell populations that belong to different clonal
layers as well as domains along the central and peripheral axis. Our
work reveals that cells in distinct clonal layers exhibit greater diversity in
gene expression and greater transcriptional complexity than clonally
related cell types in the central and peripheral axis. Assessment of
molecular functions and biological processes reveals that epidermal
cells express genes involved in pathogen defense: the L2 layer
cells express genes involved in DNA repair pathways and telomere
maintenance, and the L3 layers express transcripts involved in ion
balance and salt tolerance besides photosynthesis. Strikingly, the stem
cell-enriched transcriptome comprises very few hormone-responsive
transcripts. In addition to providing insights into the expression profiles
of hundreds of transcripts, the data presented here will act as a resource
for reverse genetic analysis and will be useful in deciphering molecular
pathways involved in cell type specification and their functions.
KEY WORDS: Central zone, CLAVATA, WUSCHEL
INTRODUCTION
A spatiotemporal orchestration of gene expression and cell behaviors
is crucial for cell fate specification (Baurle and Laux, 2003; Reddy,
2008). The stem cells in shoot apical meristems (SAMs) of higher
plants differentiate into various above-ground organs (Steeves and
Sussex, 1989). Traditionally, SAMs have been divided into distinct
domains on the basis of their function and cytological criteria
(Lyndon, 1998; Steeves and Sussex, 1989; Xie et al., 2009). The
central zone (CZ) harbors a set of pluripotent stem cells (Reddy and
Meyerowitz, 2005). The stem cell progeny differentiate into leaves or
flowers within the adjacent peripheral zone (PZ). Upon specification
of leaf or flower primordia, specific cellular behaviors and
differentiation events lead to the formation of boundary regions that
separate developing organs from SAMs, whereas cells in the rib
meristem (RM), situated just below the CZ, differentiate to give rise to
the stem. Collectively, stem cell daughters progress through
1
Department of Botany and Plant Sciences, Center for Plant Cell Biology (CEPCEB),
Institute of Integrative Genome Biology (IIGB), University of California, Riverside,
2
CA 92521, USA. Department of Biological Sciences, Indian Institute of Science
Education and Research, Mohali 140306, India.
*Authors for correspondence ([email protected]; [email protected])
Received 20 November 2013; Accepted 6 May 2014
sequential differentiation programs and acquire distinct cellular
identities (Reddy et al., 2004; Heisler et al., 2005). SAMs of higher
plants are multilayered structures. In Arabidopsis, the tunica consists
of the outer epidermal (L1 layer) and an inner sub-epidermal (L2
layer), while the corpus forms a multilayered structure beneath the L2
layer (Satina and Blackeslee, 1941; Satina et al., 1940; Tilney-Basset,
1986). Thus, the SAM contains cell types that are clonally distinct
and cells that are at different stages of differentiation within the same
cell layer. Both cell type-specific factors and ubiquitously used
signals such as plant hormones have been implicated in SAM
development (Xie et al., 2009; Perales and Reddy, 2012; Barton,
2010; Shani et al., 2006; Yadav et al., 2011). Elucidating gene
networks underlying cell fate specification requires methods that
facilitate the gene expression analysis of individual cell types.
An earlier study describes gene expression profiles of the CZ, the
PZ and the RM (Yadav et al., 2009); however, it does not resolve gene
expression programs associated with cell types in different clonal
layers or with specific specialized cell types. To gain further insights
into transcriptional programs of SAM cell types, we identified novel
promoters that are expressed in discrete clonal layers and in different
cell types of CZ, PZ and RM. Fluorescence-activated cell sorting
(FACS) method was employed to collect seven new cell populations
and Affymetrix GeneChips (ATH1) were used to profile their
transcriptomes. These data were combined with expression data of
three cell populations from an earlier study (Yadav et al., 2009) to
generate a high-resolution gene expression map. Besides the
description of molecular pathways enriched in various cell
populations, our work reveals that cells in the distinct clonal layers
exhibit greater diversity in gene expression and transcriptional
complexity than cell populations within the CZ and the PZ.
RESULTS
Description of cell populations of SAMs
Layered organization
The HIGH MOBILITY GROUP (PHMG; AT1G04880) promoter
was used to label cells of the L1 layer of SAMs (Table 1 and
Fig. 1A; HMG/L1-meristematic). The Arabidopsis thaliana
MERISTEM LAYER1 (AtML1) promoter was used to mark cells in
the L1 layer of both the meristematic region and differentiating
organs (Fig. 1B,F; AtML1/L1-ubiquitous) (Lu et al., 1996). The
promoter of the HOMEODOMAIN GLABROUS4 (PHDG4,
At4g17710) gene was used to label cells in the L2 layer (Table 1;
Fig. 1C,G; HDG2/L2-meristematic) (Yadav et al., 2009). The
microarray data of WUSCHEL-expressing cells (Yadav et al., 2009)
were used to represent cells of the corpus/L3 layers/the RM
(Table 1; WUS/RM/L3/corpus) (Mayer et al., 1998). The phloem
pole pericycle reporter line S17 (At2g22850) was used to label
the phloem (Fig. 1J; see supplementary material Fig. S1B) (Lee
et al., 2006), and the S6/AtHB8 reporter was used to mark
procambial cells (Fig. 1K; see supplementary material Fig. S1A)
(Baima et al., 1995).
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ABSTRACT
Development (2014) 141, 2735-2744 doi:10.1242/dev.106104
Table 1. Details of reporter lines used in this study
Serial
number
1
2
3
4
5
6
Promoter
reporter
construct
PHMG::
H2B-YFP
PAtML1::
mGFP-ER
PHDG4::
H2B-YFP
PWUS::
mGFP-ER
PCLV3::
mGFP-ER
PLAS::LASGFP
7
PKAN1::
KAN1-GFP
8
PFIL::dsRed-N7
9
PS17s::H2BYFP
PAtHB8::
H2B-YFP
10
Cell types
References
Meristematic L1 layer/
epidermal cell type
Ubiquitous L1 layer/
epidermal cell type
L2 layer/subepidermal
cell type
L3 layer/rib zone cell
type
Central zone/stem
cells
Adaxial organ
boundaries/
peripheral zone cell
type
Abaxial organ
boundaries/
peripheral zone cell
type
Organ primordia/
peripheral zone cell
type
Shoot phloem/
vasculature cell type
Shoot xylem/
vasculature cell type
This study
This study
This study
Yadav et al.,
2009
Yadav et al.,
2009
This study
This study
Yadav et al.,
2009
This study
This study
Central and peripheral organization
The CZ (PCLV3::mGFP5-ER) gene expression dataset was
obtained from an earlier study, referred to as CLV3/CZ (Yadav
et al., 2009). The KANADI1 (KAN1) promoter was used to label
cells in the outer edges of the PZ, referred to as KAN1/outer PZ
(Fig. 1E,I; green fluorescent signal) (Yadav et al., 2013; Kerstetter
et al., 2001). The organ primordia gene expression data set (PFIL::
dsRED-N7) has been described in an earlier study, referred to as
FIL/organ primordia (Table 1; Fig. 1E,I; red fluorescent signal)
(Yadav et al., 2009). The LATERAL SUPPRESSOR (LAS) promoter
was used to isolate cells in the organ boundaries, referred to as
LAS/organ boundaries (Table 1; Fig. 1D,H) (Goldshmidt et al.,
2008; Greb et al., 2003). Seven new fluorescent reporters were
introduced into apetala1;cauliflower1-1 (ap1-1;cal1-1) genetic
background and then we performed protoplasting to disrupt the
SAM, employed FACS to isolate specific cell types and used the
Affymetrix GeneChip ATH1 to perform microarray experiments.
The new data sets were collated with the expression profiles of three
cell populations that have been reported previously (Yadav et al.,
2009) and were analyzed to identify gene expression programs
within the shoot apex.
Identification of cell population elevated gene expressions
without outliers
Based on results from our previous study (Yadav et al., 2009), the
protoplasting method influences the expression of ∼300 genes.
Thus, these genes were excluded from all subsequent analysis steps.
Cell layers in the SAM traverse through the CZ and PZ, whereas
vasculature cell types partially overlap with deeper cell layers of the
RM. Consequently, the reporters used to isolate specific cell
populations have overlapping expressions. To minimize gene
expression redundancies arising from these overlaps in our
analyses of differentially expressed genes (DEGs), we assigned
the ten cell population samples to four non-overlapping comparison
groups. Group A included the HMG/L1-meristematic, HDG4/L2meristematic and WUS/L3/corpus cell populations from the SAM
cell layers. Group B included the CLV3/CZ, FIL/organ primordia,
LAS/organ boundaries and KAN1/outer PZ. Group C included
AtHB8shoot, S17shoot and HDG4/L2-meristematic cell populations,
and is expected to identify genes expressed in vascular cell
populations in comparison with the L2 cell layer. Group D included
AtML1 (L1 ubiquitous), HMG (L1 meristematic) and HDG4 (L2
meristematic) cell populations, and is expected to identify genes
expressed in the L1 layer of meristematic and non-meristematic cells
in comparison with the L2 cell layer. Within those comparison groups
we identified DEGs for all possible sample comparisons using the
LIMMA package (Smyth, 2004). The DEG results were then queried
for genes that showed an elevated expression in one cell type (target)
compared with all other cell types (reference) in a comparison group.
The resulting ‘cell population differentially expressed genes’
(CPEGs) needed to show a ≥1.5-fold higher expression in the target
cell type compared with the reference cell types in a comparison
group. In addition, all fold-changes needed to be supported by an
adjusted P value from LIMMA of ≤0.05. The four groups A, B, C and
D showed 1456, 472, 1031 and 298 CPEGs, respectively (Fig. 2A; see
supplementary material Table S1).
Fig. 1. Expression patterns of reporter lines.
A side view of apetala1;cauliflower1 shoot apex
showing L1 layer marked with PHMG::H2B-YFP
(A). A transverse section of SAM, showing AtML1::
mGFP-ER expression (B). (C-E) Threedimensional reconstructed top views of apetala1;
cauliflower1 SAMs showing L2 layer expression of
PHDG4:H2B-YFP promoter (C), organ boundary
expression of PLAS::LAS-GFP promoter (D), outer
peripheral zone of SAM marked by PKANADI1::
KANADI1-GFP promoter (green) and organ
primordia showing PFIL::ds-Red-N7 expression
(red) (E). (F-I) Reconstructed side views of SAMs
represented in B-E, respectively. Cell outlines
highlighted by FM4-64 dye (red). (J) Confocal
image showing the expression of S17 reporter in
the stem. (K) Confocal image showing the
expression of PAtHB8:H2B-YFP in the stem.
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RESEARCH ARTICLE
Development (2014) 141, 2735-2744 doi:10.1242/dev.106104
Relaxed CPEGs
Validation of microarray data
Our previously reported experiments involving three cell
populations, CLV3, WUS and FIL, revealed a greater number of
CPEGs in comparison with the current work (Yadav et al., 2009).
This could be due to overlapping expression domains of reporters
used in this study, as seen in the case of FIL and KAN1
(supplementary material Fig. S2). A more relaxed version of the
CPEGs analysis used the same threshold values as above but allowed
one outlier in the target to reference comparisons. For example, a
gene was considered a relaxed CPEG if it met those criteria relative to
at least three out of a total of four reference cell populations: in this
study, the CZ and the PZ domain datasets. In the relaxed CPEG
analysis, the CZ and the PZ domain dataset yielded 2182 genes as
opposed to the 472 detected without an outlier (see supplementary
material Table S1). Many of these newly detected CPEGs were part
of PZ cell types, thereby confirming the utility of the outlier analysis.
To validate the cell population transcriptome, we compared the cell
type-elevated expression of 91 genes in cell populations with RNA
in situ hybridization patterns determined by Yadav et al. (2009) and
other studies (see supplementary material Fig. S3 and Table S8). For
example, 14 L1 CPEGs were found to be expressed in the L1 layer/
epidermis (see supplementary material Table S1). Moreover,
TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1
(AT1G70560) and UNKNOWN PROTEIN (AT5G06270) were
CPEGs in both the L1/HMG (layer-specific analysis data set) and
the CLV3 cell population, which is in agreement with the relatively
higher levels of expression of these genes in the central part of the
L1 layer and to a lesser degree in the L2 sub-epidermal layer (see
supplementary material Fig. S4). Among five L2 layer expressed
genes, four CPEGs were identified (see supplementary material
Table S1). The absence of MEI2 C-TERMINAL RRM LIKE2
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DEVELOPMENT
Fig. 2. Pathways with elevated gene expressions in various cell populations. (A) Layered organization of SAM. (B) A schematic representation of cell types
along the central and peripheral dimension on a 3D reconstructed SAM template showing the expression of PLAS::LAS-GFP. CLV3 and FIL expression domains
are marked green and yellow, respectively. (C) SAM top view showing PKAN1::KAN1-GFP expression in the outer edges of the PZ. (D) GO term enrichment
analysis of DEGs enriched in individual cell layers and cell populations along the central and peripheral zones in SAMs. Gene expression patterns are depicted in
the form of heat maps for genes associated with enriched GO terms.
RESEARCH ARTICLE
The effect of the fold-change cutoffs on CPEG set sizes
To estimate the impact of the fold change cutoffs on the number of
CPEGs identified, we tested four different fold-change cutoffs,
including 0, 1.5, 2 and 4, while using in all cases a constant adjusted
P value cutoff of ≤0.05. As expected, larger fold change cutoffs
result in smaller CPEG sets (supplementary material Table S11).
Examples of genes that were CPEGs at the 1.5-fold but not at 2-fold
cutoff anymore include HDG11, SPT, MCT1, MCT2, CLV1, BAM1,
ARF3, LFY, BOP1, LMI1, PRS, KAN1 and KAN2 (supplementary
material Tables S11 and S12). One reason for this trend is that many
of the weaker expressed genes are unlikely to show a pronounced
fold change in Affymetrix experiments, mainly because of
limitations related to the sensitivity and the signal-to-background
ratio of the technology. Thus, a relatively small fold change of 1.5
can be biologically meaningful, especially for weakly expressed
genes (for example KAN1 and KAN2). This is also supported by the
fact that the CPEG sets for the different fold change cutoffs shared
many statistically enriched Gene Ontology (GO) terms (see below).
Additional reasons could be the relatively broad expression pattern
of candidate genes (e.g. CLV1, BAM1), which does not perfectly
overlap with the expression domains of reporters used for
cell sorting.
CPEGs in SAM cell layers
To analyze the CPEG sets functionally, we performed enrichment
analysis of Gene Ontology (GO) terms (Fig. 2D; see supplementary
material Tables S2 and S12). The GO term enrichment tests were
performed on the CPEG sets generated for each of the four-fold
change cutoffs (see supplementary material Table S12) (see above
for details), while the following discussion is restricted to the GO
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terms enriched (adjusted P≤0.05) in both result sets, the one with
the 1.5- and the 2-fold cutoff.
L1 layer CPEGs
The CPEG datasets from the SAM L1 layer contained several
over-represented GO terms related to L1 layer development
(GO:0032502), defense (GO:0006952) and responses to bacterial
and fungal pathogens (GO:0009607) (Fig. 2D; see supplementary
material Table S2 and Fig. S5). This included: genes involved in wax
biosynthesis, such as 3-KETOACYL-COA SYNTHASE1 (KCS1),
KCS5, KCS6/CUTICULAR1, KCS10/FIDDLEHEAD and WAX2
(Javelle et al., 2011); genes implicated in L1 layer development,
such as HOMEODOMAIN GLABROUS2 and 12, ARABIDOPSIS
CRINKLY4, PROTODERMAL FACTOR 1 and 2 and AtML1 (Abe
et al., 2003); genes that have been shown to function in the
fine-tuning of the basal resistance against Pseudomonas syringaeWRKY11 and WRKY17 (Journot-Catalino et al., 2006); and three
members of the Toll-like/interleukin-1 (TIR-NB) gene family
involved in eliciting responses to fungal pathogens. The cells of
the L1 layer also contained CPEGs annotated as auxin influx carrier
AUX1 and its paralogs, LAX1 and LAX2. Conversely, the CPEGs of
the non-meristematic cell populations of the epidermal layer were
enriched in pathways related to photosynthesis (GO:0015979) and
flavonoid biosynthesis (GO:0009813) (see supplementary material
Table S2) (Zhao et al., 2007).
L2 layer CPEGs
The developmental roles of the L2 layer are not well understood.
The CPEG set of this sample was enriched in GO terms related to the
metabolic processes of nucleic acids (GO:0006139) (Fig. 2D; see
supplementary material Table S2). The corresponding genes
include: POLYMERASE DELTA4 (POLD4), a DNA-directed
DNA polymerase; DNA glycosylase family protein (At1g80850);
URACIL DNA GLYCOSYLASE enzymes that are involved in DNA
excision repair; AtMND1, which plays a role in double-strand break
repair during meiotic recombination (Cappadocia et al., 2010); and
genes encoding TELOMERASE REVERSE TRANSCRIPTASE,
which is involved in the maintenance of chromosomal ends
(Ogrocká et al., 2012), and WHIRLY1, which is involved in the
negative regulation of telomere length. Furthermore, the CPEGs
identified for the L2 layer cells include: DOMAINS REARRANGED
METHYLASE1 (DRM1), which is involved in de novo DNA
methylation and the maintenance of asymmetric DNA methylation;
and INCREASE IN BONSAI1 METHYLATION, a jumonji domaincontaining protein involved in H3mK9 demethylation (Stroud
et al., 2013). However, at twofold enrichment cutoff, POLD4, DNA
GLYCOSYLASE and WHIRLY1 were no longer identified as
CPEGs. The preferential enrichment of pathways involved in
maintaining DNA fidelity and telomere length within the L2 layer
suggests that they may provide necessary safeguards for germ cells
that descend from this cell layer (Scott et al., 2004).
The CPEGs identified for the L2 layer sample contained many genes
involved in microtubule organization. They included: AtCDC48,
which encodes ATP binding microtubule motor protein; two genes that
encode P-loop containing triphosphate hydrolase superfamily proteins;
and members of the kinesin family, such as phragmoplast-associated
kinesin-related protein (PAKRP2) and ARABIDOPSIS THALIANA
KINESIN4 (ATK4). PAKRP2, a phragmoplast-localized protein has
been implicated in transport of cargo from the Golgi to the division site
during cell-plate formation (Lee et al., 2001). Whereas ATK4 is a
kinesin-like protein that binds microtubules in ATP-dependent manner
to facilitate microtubule-based movement of vesicles. At the twofold
DEVELOPMENT
(MCT2) (AT5G07930) in the L2 CPEGs may be attributed to the
dynamic expression of MCT2 in the flower meristem in addition to
its enrichment in the L2 layer of SAMs (Aggarwal et al., 2010).
Furthermore, the expression patterns of 12 RM genes were correctly
assigned to the WUS cell population (see supplementary material
Table S1). Collectively, the layer-specific analysis correctly
identified 29 CPEGs out of 30 known candidate genes considered
for analysis.
A similar analysis was performed with datasets of the CZ and the
PZ domains. Of the 15 genes expressed in the CZ, 13 were identified
as CPEGs in CLV3 cell population. One of the genes excluded by
the analysis, TERMINAL EAR LIKE2 (AT1G67770), is expressed in
the CZ and by the PZ of the early stage flower buds (Anderson et al.,
2004) (see supplementary material Table S1). Of the 31 genes
that are expressed in the PZ, our CPEG analysis assigned 24 to the
FIL cell population. Exceptions include the expression patterns
of AUXIN RESPONSE FACTOR3 (AT2G33860) and LATE
MERISTEM IDENTITY2 (AT3G61250), which are expressed
more diffusely in the PZ (see supplementary material Table S1).
Of the seven genes expressed in the outer edge of PZ, six were
assigned to the KAN1 cell population. Similarly, nine out of 11
genes showed elevated expression in boundary regions in the LAS
cell population (see supplementary material Table S1). In summary,
41 of the 46 CPEGs agreed with the RNA in situ hybridization data.
The disagreeing five candidates may have a too dynamic expression
pattern to be detectable by RNA in situ hybridization experiments.
Last, the expression of all ten genes that were considered for the
vascular cell populations were correctly assigned to their respective
cell populations in the CPEG analysis. Taken together, our
microarray experiments correctly identified 94% of the chosen
CPEGs (see supplementary material Tables S1 and S8).
Development (2014) 141, 2735-2744 doi:10.1242/dev.106104
RESEARCH ARTICLE
cutoff, PAKRP2 and ATK4 were not detected as CPEGs. The
enrichment of GO terms related to pathways controlling microtubule
organization and dynamics among cells within the L2 sub-epidermal
layer is intriguing, considering their ubiquitous requirement (Zhong
et al., 2002).
Development (2014) 141, 2735-2744 doi:10.1242/dev.106104
that the L3 cell population responds to the majority of plant
hormones, including jasmonic acid, ethylene, cytokinin, abscisic acid
and gibberellins (Fig. 3; see supplementary material Fig. S5) (Maere
et al., 2005). Collectively, the cells in the L3 layers express genes
implicated in maintaining ionic homeostasis, mediating the water
stress, the salt stress and the response to multiple plant hormones.
L3 layers/corpus CPEGs
CPEGs along the CZ and the PZ domains
CLV3/CZ
CZ cells primarily express genes involved in stem cell maintenance
(GO:0019827), cell differentiation (GO:0030154) and floral organ
development (GO:0048437) (Fig. 2D; see supplementary material
Table S2). This includes CLAVATA3, WUSCHEL, SAPTULA (SPT),
CORONA and PHABULOSA (Barton, 2010). However, at the twofold
enrichment cutoff, SPT was not detected as a CPEG anymore.
FIL/organ primordia
The GO terms related to organ development (GO:0048513),
specification of organ polarity (GO:0010084) and responses to
hormonal stimuli (GO:0009725) were enriched in the CPEG set of
the organ primordia (see supplementary material Table S2). This
CPEG set includes FILAMENTOUS FLOWER, ASYMMETRIC
LEAVES1, GROWTH REGULATING FACTOR7, ROXY1, BLADE
ON PETIOLE2 and MONOPTEROS/ARF5. It also includes the
hormone-responsive genes TONOPLAST INTRINSIC PROTEIN1,
AUXIN RESISTANT2, ARF5, ARABIDOPSIS RESPONSE
REGULATOR11, AtbZIP12, HOMEOBOX-LEUCINE ZIPPER2,
AtMYB13 and BASIC CHITINASE (Barton, 2010).
KAN1/outer PZ
The CPEG set of the KAN1 cell population was enriched in GO terms
related to auxin homeostasis (GO:0010252) (Staswick et al., 2005),
Fig. 3. Inferred functions of CPEGs of selected cell populations of SAMs. GO terms associated with HMG/L1 layer (A), WUS/L3 layer (B), FIL/Organ
primordia (C), AtHB8/Xylem (D) and S17/Phloem (E) were mapped using cytoscape. Different colored nodes represent significantly enriched (P<0.05) GO terms.
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DEVELOPMENT
The CPEG set of the cells of the L3 layer was enriched in GO terms
related to photosynthesis (GO:0015979) (Fig. 2D; see supplementary
material Table S2). Many of these CPEGs encode members of
photosystem I light-harvesting complex A (LHCA1, 3, 4 and 5),
photosystem II components (LHCB3, 4, 4.2 and 5), proteins of the
photosystem I subunits PSAH1, PSAE1, PASF, PSAA and PSAO,
and proteins of photosystem II subunits PSBA and PSBO2 (Friso
et al., 2004).
The GO categories related to responses to ions (GO:0010038) and
salt stress (GO:0009651) were also enriched in the CPEG set of the
L3 cell layer. This includes genes encoding FERRETIN1 (an ironstorage protein); AILP1 (which responds to aluminium ions), CAX1LIKE CATION EXCHANGER 1 and 3, a Ca2+/H+ exchanger;
ANNEXIN1, a Ca2+-dependent protein with peroxidase activity; a
vacuolar Ca2+-binding protein that responds to cadmium ions and salt
stress (Sudre et al., 2013); protein kinases of the SNF1 family
(SNRK2.1, SNRK2.2, SNRK2.6); ABI FIVE BINDING PROTEIN4
(AFP4); and MYB15 (Fujii et al., 2011). At the twofold cutoff,
AILP1 and AFP4 were not identified as CPEGs. In addition, the
CPEG set of the L3 cells contained GO terms associated
with hormonal responses (GO:0009725) that included type B
Arabidopsis response regulators (ARR2, ARR4 and ARR10), which
may explain enhanced cytokinin sensitivity of RM cells observed in
an earlier study (Gordon et al., 2009). In addition, when we applied
the BINGO tool on the L3 layer CPEGs using Cytoscape, we found
wax biosynthesis (GO:0010025), fungal responses (GO:0050832)
and defense responses (GO:0006952) (Fig. 2D; see supplementary
material Table S2) (Consonni et al., 2006). The CPEGs that encode
two auxin-responsive-GH3 family proteins and SLOW MOTION, a
F-box protein implicated in the timing of lateral organ development
were found in the KAN1 cell population (Lohmann et al., 2010).
Organ boundary/LAS
Cells of the organ boundary exhibit unique cell behaviors, including
infrequent cell division and asymmetrical expansion that create a
region of reduced growth (Reddy et al., 2004). GO terms related to
hormonal responses (GO:0009725), including brassinosteroid (BR)
(GO:0009741), were associated with this cell population (Fig. 2B;
see supplementary material Table S2) (Hu et al., 2004). This
included CPEGs that encode CYCLIN D3;1, a D-type CYCLIN,
and Xyloglucan Endotransglycosylase (XET), which mediates cell
wall expansion by hydrolyzing xyloglucan chains. At twofold
enrichment cutoff, CYCLIN D3;1 was not retained as a CPEG.
CPEGs in vascular cells
AtHB8/xylem
The GO terms related to auxin response and signaling pathways
(GO:0009733), and polar auxin transport (GO:0009926) were
enriched in the CPEG set of the xylem cell population (Fig. 2D; see
supplementary material Table S2) (Vernoux et al., 2011). This includes
auxin-induced genes [BODENLOS (BDL), MONOPTEROS, IAA8,
IAA13 and IAA30], genes encoding GH3.3 family proteins [DFL1,
AUX1, AUXIN INDUCED IN ROOT CULTURES3 (AIR3),
AIR12, AXR4, FLAVODOXIN-LIKE QUINONE REDUCTASE1,
PINOID-BINDING PROTEIN1 and BRI1-LIKE2] and genes
involved in polar auxin transport [AtPIN1, AtPIN6, ENHANCER OF
PINOID (ENP), AUX1, ACAULIS5 (ACL5) and MORE AXILLARY
BRANCHES2 (MAX2)]. At the twofold and higher cutoffs, BDL, IAA8,
AXR4, ENP, ACL5 and MAX2 were no longer detected as CPEGs. An
enrichment of the auxin pathway elements in cells that represent the
early stages of xylem differentiation is consistent with the documented
role of auxin in vascular differentiation (Sachs, 1969, 1981).
S17Shoot/phloem
The CPEG set of the phloem cell population contained enriched
GO terms related to glucosinolate biosynthesis (GO:0019761)
(Piotrowski et al., 2004), to gibberellin- and bacterium-induced
responses (GO:0009739; GO:0009617) (Cheng et al., 2004), to
Development (2014) 141, 2735-2744 doi:10.1242/dev.106104
cell wall modification (GO:0042545), and to ion transport
(GO:0006811) (Fig. 2D; see supplementary material Table S2)
(Grotz et al., 1998). Genes involved in cation homeostasis, such
as CYCLIC NUCLEOTIDE-GATED ION CHANNEL 5 and 19
(CNGC5 and CNGC19), K+ UPTAKE PERMEASE9 (AtKUP9),
COPPER TRANSPORTER5 (COPT5), ZINC TRANSPORTER 1
and 5 PRECURSOR (ZIP), iron-regulated transporters IREG1 and
IRT3, nitrate transporters NRT1.5 and NRT1.7, the phosphate
transporter PHO1 and sulfate transporter-SULTR2;1, were
identified as CPEGs in the phloem sample (Lin et al., 2008).
However, at the twofold and higher cutoffs, CNGC5, AtKUP9 and
ZIP1 were no longer detected as CPEGs.
Similarities among cell types based on expression profiles
We performed the sample-wise hierarchical clustering of
transcriptome to determine similarities in the gene expression
profiles among cell types. Data from the ten SAM cell populations
as well as from 16 cell populations of the Arabidopsis root were
considered (Birnbaum et al., 2003; Brady et al., 2007). The distance
matrix required for this step was generated from the distancetransformed Spearman correlation coefficients obtained from all pairwise sample comparisons using the average among their normalized
replicates (see supplementary material Table S6). The resulting
clustering dendrogram (Fig. 4C) indicates that the expression profiles
of adjacent cell types in the CZ and the PZ are more similar than those
that are spatially distant. For example, FIL cell population share
greater similarities with KAN1 cell population compared with CLV3
(CZ) cell population (Fig. 4C). Similarly, the two vascular cell types,
the xylem and phloem, clustered together; however, they showed a
lower degree of relatedness to stem cell population of the CZ.
Notably, the extent of this similarity was not always related to the
spatial proximity of cell types within the SAMs. For example, the
clonally distinct but adjacent L1-meristematic (HMG) and L2meristematic (HDG4) cells showed a lesser degree of relatedness in
their gene expression profiles. Similarly, cell populations of the root
system and the SAM, including vascular cells, formed separate
clusters (Fig. 4D). Clonal separation of SAM cell layers at early stages
of embryonic development might have contributed to the greater
diversity of gene expression profiles.
The estimation of the amount of expressed (detected) genes in the
ten cell populations of the SAM presented in this study and in root
samples (S4, J2501, xylem, S17, RM1000, JO121, SUC2, phloem,
S32, cortex, COBL9, src5, J0571, wol, LRC, gl2) (Brady et al.,
Fig. 4. A comparison of genes expressed in shoot and root cell type. (A) Venn diagram showing the intersection of genes detected in shoot and root cell
populations. (B) Summary of GO term enrichment results highlighting some of the most dominant classifications. (C) Sample-wise hierarchical clustering of shoot
microarray replicates. (D) A comparison of shoot- and root-derived microarray replicates to assess their relative closeness in terms of their transcriptome profile.
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2007) (see Materials and methods for details) revealed 958 and
2187 genes that are expressed exclusively in the shoot and the root,
respectively (Fig. 4A; see supplementary material Table S3). GO
terms associated with flower development (GO:0009908), stomata
development (GO:0010103) and chlorophyll metabolic processes
(GO:0015994) were overrepresented in the genes expressed in shoot
cell populations (Fig. 4B; see supplementary material Table S4). On
the contrary, the gene set of the root cell-types contained GO terms
associated with responses to toxins (GO:0009404) (Fig. 4B) and
hormones (GO:0042446), which included genes involved in auxin
biosynthesis (YUCCA3 and YUCCA5), cytokinin biosynthesis
[ISOPENTENYL TRANSFERASE (IPT) 1, 3, 5, 7 and 8] (Zhao,
2010) and ethylene biosynthesis (1-AMINOCYCLOPROPANE-1CARBOXYLATE SYNTHASE 7, 8 and 11) (Tsuchisaka et al., 2009).
The IPT class genes were not expressed in SAM cell populations,
which suggests that cytokinin precursor-N6-(Δ2-isopentenyl)
adenine (iP) may be transported into meristematic cells where it is
converted into active forms of cytokinin through the action of
locally synthesized LONELYGUY class of proteins (Kurakawa
et al., 2007; Kuroha et al., 2009).
Orchestration of transcriptional programs
To understand the orchestration of the gene regulatory networks
(GRNs) mediated by transcription factors (TFs), we mined our data
sets for the distribution expression patterns of TFs (Jiao and
Meyerowitz, 2010). Of the 1270 TF genes considered for this
analysis, 1225 were detected in SAM cell populations. Among
these, only 296 TFs were CPEGs (see supplementary material
Table S1). About 39% CPEGs encoding for TFs were found in
clonally distinct cell layers of the SAMs, whereas only 22% CPEGs
encoding for TFs were present in distinct cell populations located in
the CZ and the PZ. Thirty-five percent of CPEGs encoding for TFs
were present in vasculature cell populations. The greater diversity of
TF representation among the distinct layers of SAMs may explain
lesser similarity in the transcriptome among cell layers.
AP2_EREBP, C2H2, MADS, MY and WRKY family TFs are
Development (2014) 141, 2735-2744 doi:10.1242/dev.106104
highly expressed in the meristematic L1 layer; C2C2_DOF
members are predominant in the L2 meristematic layer; and
bHLH, GRAS, HB, NAC, SBP and ZF_HD are enriched in L3
layer (see supplementary material Fig. S6). Furthermore, members
of the AP2_EBERP family, which are involved in mediating stress
responses, and members of the WRKY family, which are involved
in defense responses, are expressed in the L1 epidermal layer. The
CPEGs of the CLV3/stem cell population contained genes encoding
HOMEOBOX and MADS TFs, whereas the FIL/differentiating cell
population contained genes that code for C2C2_YABBY, MYB and
SBP TFs (see supplementary material Fig. S6 and Table S1). Last,
the genes that encode AP2_EREBP, AUX_IAA and WKRY TFs
were among xylem cell type CEPEGs, whereas genes that encode
members of the bHLH, C2C2_DOF, C2H2, GRAS, MYB, NAC
and SBP TF families were found in phloem CPEGs. The analysis of
the spatial expression of TFs and identification of cis-motifs to
which they bind may provide clues to the GRNs that underlie
specification of different cell types. We identified the TF-binding
motifs by studying the enrichment of upstream regulatory elements
within the promoter regions of CPEGs identified in the L1, L2, L3
cell layers, and the CZ and the PZ domains (Fig. 5A,B; see
supplementary material Table S7 and see Materials and Methods).
A greater degree of diversity was observed among the TF-binding
motifs present in the promoters of distinct cell layers CPEGs relative
to the CZ and the PZ domain CPEGs, suggesting that a highly
complex GRN specifies cell types in the SAM layers.
Hormonal pathways
An earlier study has shown that 1638 genes respond to abscisic
acid, indole-3 acetic acid, gibberellin, cytokinin, jasmonate,
brassinosteroid and ethylene treatments (Nemhauser et al., 2006).
Of the 1638 hormone-responsive genes, 1298 were detected in
SAM cell populations, out of which 469 were identified as CPEGs.
A majority of these genes were CPEGs in one of the three cell layers
of SAMs (see supplementary material Table S1). Notably, stem
cells or CZ CPEGs contained very few hormone-responsive genes
Fig. 5. Identification of cis-element and hormone-responsive transcripts in SAM cell populations. (A,B) Enrichment of cis elements in cell layers (A) and in
the central and peripheral zones (B). (C) Number of hormone-responsive CPEGs plotted as a percentage against the total number of CPEGs for each cell
population. (D) Relative contributions of hormone-responsive CPEGs depicted as a percentage against the number of all hormone-responsive CPEGs.
2741
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RESEARCH ARTICLE
DISCUSSION
The SAM is a complex developmental field consisting of cell types
that form clonal layers within the CZ and the PZ that are closely
related and clonal layers that are distantly related. Our analysis
indicates cell types in clonal layers that are distantly related by birth
show a lesser degree of relatedness in gene expression profiles than
those that are closely related. A greater diversity of TF classes and
cis-elements detected in promoters of genes that are differentially
expressed in cell layers suggests that a highly complex GRN may
underlie cell type specification in the SAM cell layers relative to the
GRNs that specify cell types in the CZ and the PZ. Furthermore,
small fractions of the total number of genes expressed (∼13% in the
root system and ∼5.7% in the SAM) are organ specific, suggesting
that the network topology of interacting components within a given
cell type may play a crucial role in cell type specification.
Our work shows that CPEGs of the L1 cell populations are
implicated in pathways related to biosynthesis and secretion of
epicuticular wax. Genetic studies have implicated two functionally
redundant HD-ZIP IV classes of TFs (AtML1 and PROTODERMAL
FACTOR2) in epidermal cell differentiation (Lu et al., 1996;
Abe et al., 2003). Upon differentiation, the L1 epidermal cells
asymmetrically secrete a hydrophobic lipid layer consisting of cutin
and waxes that forms a protective film (Jeffree, 2006). Furthermore,
the AP2/EREBP and MYB TFs have been shown to regulate the L1
layer expression of genes involved in cuticle biosynthesis (Javelle
et al., 2011). Analysis presented here may be used in future studies to
decipher the mechanisms by which developmental TFs, such as
AtML1 and PDF2, function in tandem with the AP2/EREBP and
MYB family TFs in controlling epidermal cell type development and
its function. Besides protective function, the L1 layer has been shown
to play a role in stem cell maintenance (Knauer et al., 2013), regulation
of cell division patterns in sub-epidermal layers (Reinhardt et al.,
2003a,b; Savaldi-Goldstein et al., 2007), lateral organ primordia
specification (Reinhardt et al., 2005, 2003a,b) and establishment of
lateral organ polarity (Reinhardt et al., 2005). The L1 layer
transcriptome presented here should facilitate functional analysis of
these genes, leading to better molecular insights into the involvement
of epidermal layer in plant development.
Until recently, it was widely believed that the SAM is
heterotrophic and contains only proplastids that lack thylakoids
and chlorophyll-binding proteins (Fleming, 2006). However, recent
work has revealed that plastids in SAMs contain PSII-associated
chlorophyll and thylakoid membranes (Charuvi et al., 2012). Our
data show elevated expression of genes encoding photosystem I and
II light-harvesting complexes, along with other accessory proteins
of the photosynthetic machinery, in the L3 cell population; this
provides molecular support for the presence of the functional
chloroplast machinery in SAMs. Several developmental processes
in SAMs, including leaf initiation and positioning, depend on light
(Yoshida et al., 2011). Given that chlorophyll biosynthesis in higher
plants is light dependent, the light-dependent developmental
regulation may be intricately linked to the chloroplast maturation
and metabolic status of cells of SAMs.
Earlier studies have shown that a higher level of auxin signaling is
required for specification and growth of the lateral organ primordia
2742
(Reinhardt et al., 2003a,b). Furthermore, GA has been implicated in
promoting cell differentiation, and members of the class I KNOX
family have been shown to repress GA biosynthesis in the meristem
center (Sakamoto et al., 2001; Bolduc and Hake, 2009). In agreement
with earlier studies, the genome level analysis presented here shows
poor representation of auxin and GA-responsive genes in the CZ. The
cell population transcriptome presented here can be used to
reconstruct cellular responsiveness to various plant hormones, as
shown for stem cell promoting TF-WUS (Yadav et al., 2013), which
may lead to new insights into the hormonal regulation of stem cell
maintenance and organ differentiation.
MATERIALS AND METHODS
DNA constructs and transgenic lines
PAtML1::mGFP5-ER was generated by fusing ∼3.3 kb promoter fragment
to mGFP5-ER. A ∼2 kb upstream sequence including the first exon of HMG
(locus At1g04880) was fused to H2B-YFP to generate PHMG::H2B-YFP. A
3 kb HDG4 promoter was fused with H2B-YFP to generate PHDG4::H2BYFP. A ∼2 kb AtHB8 promoter region was fused with H2B-YFP to generate
PAtHB8::H2B-YFP. PLAS::LAS-mGFP5 (Goldshmidt et al., 2008),
PKAN1::KAN1-mGFP5 (Yadav et al., 2013), and PCLV3::mGFP-ER,
PWUS::mGFP-ER and PFIL::ds-Red (Yadav et al., 2009) have been
described previously. The oligonucleotides used to amplify promoter
fragments of PHMG, PHDG4 and PAtHB8 are in supplementary material
Table S10. All transgenic lines were generated in ap1-1;cal1-1 genetic
background using the floral dip method (Clough and Bent, 1998).
Protoplasting and microarray hybridization
Protoplasting, RNA extraction and ATH1 gene chip hybridization have been
described previously (Yadav et al., 2009). All microarray data generated in
this study are deposited in GEO under accession number GSE28109.
Analysis of microarray data
Raw data analysis and quality assessment
Microarray data analyses were performed in the statistical environment R
using Bioconductor packages (Gentleman et al., 2005; R Development Core
Team, 2011). The probe set to locus mappings for the ATH1 chip were
obtained from TAIR (V-2010-12-20). Controls and probe sets matching no
or several loci in the Arabidopsis genome were ignored in the downstream
analysis steps. In addition, redundant probe sets that represent the same
locus several times were counted only once. The quality of the Affymetrix
GeneChips was assessed with analysis routines provided by the affyPLM
and arrayQualityMetrics packages (Bolstad et al., 2005; Kauffmann et al.,
2009). The normalization of the raw data Cel files was performed with the
MAS5 algorithm using the default settings of the corresponding R function
of the Affymetrix package (Gautier et al., 2004).
Sample clustering
Sample-wise hierarchical clustering was used to evaluate global expression
similarities among cell types. The distance matrix required for this step was
generated from the distance-transformed Spearman correlation coefficients
obtained from all pair-wise comparisons of the sample expression vectors
using the average among their normalized replicates. The actual clustering
was performed with the hclust function in R, by choosing complete linkage
as cluster joining rule.
DEG analysis
Analysis of differentially expressed genes was performed with the LIMMA
package using the normalized expression values as input. The Benjamini
and Hochberg method was selected to adjust P values (P) for multiple
testing and controlling false discovery (FDR) rates (Benjamini and
Hochberg, 1995). As confidence thresholds, we used an adjusted P≤0.05
and a minimum fold change of 1.5. A gene was considered to be a DEG if it
met both threshold criteria in at least one of all possible sample comparisons
(contrasts) among the ten cell types. Cell population expressed genes
(CPEGs) were identified by filtering the DEG results for candidate genes
DEVELOPMENT
when compared with the CPEGs in the PZ and the RM (Fig. 5D; see
supplementary material Table S9). In addition, we observed
ethylene-responsive genes in the L1-meristematic layer, the L3
layer and vascular cell population CPEGs (Fig. 5C; see
supplementary material Table S1; Fig. 3A,B,D,E) which suggests
that ethylene may be broadly perceived in SAMs.
Development (2014) 141, 2735-2744 doi:10.1242/dev.106104
with an N-fold higher expression in the target cell type compared to all of the
chosen reference cell types, where N (fold change cutoff ) was set to values
of ≤0, ≤1.5, ≤2 and ≤4. At the same time, all fold changes needed to be
supported by an adjusted P value of ≤0.05 from LIMMA (see columns N-R
in supplementary material Table S1). A more relaxed version of the CPEG
analysis allowed one outlier. For example, if a gene was higher expressed in
cell type ‘A’ compared with at least three out of a total of four reference cell
types (e.g. ‘BCDE’), then it was considered to be a relaxed CPEG of cell
type ‘A’ (see columns S-U in supplementary material Table S1). To estimate
the amount of expressed (detected) and unexpressed genes in the SAM and
root samples (S4, J2501, xylem, S17, RM1000, JO121, SUC2, phloem,
S32, cortex, COBL9, src5, J0571, wol, LRC, gl2) (Brady et al., 2007), the
present call information of the non-parametric Wilcoxon signed rank test
was computed. Genes that received present calls in all replicates of a given
cell type were considered to be expressed (see supplementary material
Tables S5 and S6) (McClintick and Edenberg, 2006).
Gene ontology analysis
Enrichments analysis of GO terms was performed as described in an earlier
study (Horan et al., 2008) (see supplementary material Tables S2 and S12).
The CPEG sets from each cell population was imported into Cytoscape 2.8.1
plug-in BINGO 2.4.4 (Maere et al., 2005; Smoot et al., 2011). The network of
enriched categories was organized using first the force directed layout, and
subsequently the hierarchical layout (see supplementary material Fig. S5).
Identification of regulatory motifs
The sequence regions 1000 bp upstream of each gene model were
considered. For the motif finding, 471 cis-regulatory elements available in
the Agris database were downloaded (Yilmaz et al., 2011) and mapped to
the upstream sequences of each gene set while allowing one mismatch per
ten nucleotides (see supplementary material Table S7). The hypergeometric
distribution was used to compute P values for estimating the significance of
this enrichment and the Bonferroni method to adjust the P values for
multiple testing (Sinha et al., 2006).
Acknowledgements
FACS, microarray and imaging were carried out at Institute for Integrative Genome
Biology and Center for Plant Cell Biology, UCR. We thank Yuval Eshed for sharing
reporters and K. S. Sandhu for help with data analysis.
Competing interests
Authors declare that they have no competing interests.
Author contributions
R.K.Y. and G.V.R. conceived research and designed experiments. R.K.Y. and M.T.
performed the experiments. M.X. contributed H2B:YFP pGreen plasmid. T.G. performed
microarray analysis. R.K.Y., T.G. and G.V.R. analyzed the data and wrote the paper.
Funding
This work was supported by NSF 2010 grant (IOS-0820842) to G.V.R. R.K.Y. is
Ramalingaswami fellow and recipient of Innovative Young Biotechnologist Award
from the Department of Biotechnology, Govt. of India and acknowledges DBT for
current funding.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.106104/-/DC1
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