# Transcriptional profiling of Arabidopsis root hairs and pollen defines an apical cell growth signature

Jörg D. Becker, Seiji Takeda, Filipe Borges, Liam Dolan, José A. Feijó

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Jörg D. Becker, Seiji Takeda, Filipe Borges, Liam Dolan, José A. Feijó. Transcriptional profiling of Arabidopsis root hairs and pollen defines an apical cell growth signature. BMC Plant Biology, 2014, 14, pp.1-14. 10.1186/s12870-014-0197-3. hal-02639633

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# Transcriptional profiling of Arabidopsis root hairs and pollen defines an apical cell growth signature

Jörg D Becker¹*, Seiji Takeda²,⁵, Filipe Borges¹,⁶, Liam Dolan²,³ and José A Feijó¹,⁴

**Abstract**

**Background:** Current views on the control of cell development are anchored on the notion that phenotypes are defined by networks of transcriptional activity. The large amounts of information brought about by transcriptomics should allow the definition of these networks through the analysis of cell-specific transcriptional signatures. Here we test this principle by applying an analogue to comparative anatomy at the cellular level, searching for conserved transcriptional signatures, or conserved small gene-regulatory networks (GRNs) on root hairs (RH) and pollen tubes (PT), two filamentous apical growing cells that are a striking example of conservation of structure and function in plants.

**Results:** We developed a new method for isolation of growing and mature root hair cells, analysed their transcriptome by microarray analysis, and further compared it with pollen and other single cell transcriptomics data. Principal component analysis shows a statistical relation between the datasets of RHs and PTs which is suggestive of a common transcriptional profile pattern for the apical growing cells in a plant, with overlapping profiles and clear similarities at the level of small GTPases, vesicle-mediated transport and various specific metabolic responses. Furthermore, cis-regulatory element analysis of co-regulated genes between RHs and PTs revealed conserved binding sequences that are likely required for the expression of genes comprising the apical signature. This included a significant occurrence of motifs associated to a defined transcriptional response upon anaerobiosis.

**Conclusions:** Our results suggest that maintaining apical growth mechanisms synchronized with energy yielding might require a combinatorial network of transcriptional regulation. We propose that this study should constitute the foundation for further genetic and physiological dissection of the mechanisms underlying apical growth of plant cells.

**Keywords:** Pollen, Pollen tube, Root hair, Transcriptome, Apical growth, Tip growth, Apical signature, Arabidopsis

**Background**

Current views on the control of cell and organ development are anchored on the notion that phenotypes are defined by precise networks of transcriptional activity, acting in a concerted way through a specific combination of transcription factors to specify cell fate [1]. A direct test of this general principle is facilitated by precise transcriptome analysis using microarrays or RNAseq [2]. This approach in combination with Fluorescence Activated Cell Sorting (FACS), has allowed the characterisation of transcriptomic profiles of isolated cells from simple organs, such as pollen [3-5], or more complex ones like roots [6,7]. The large amounts of information

in different databases allow formal analysis of the tran-
scriptional profiles of specific cell types or organs, hold-
ing the promise that subsequently these can be distilled
into specific transcriptional signatures. At the moment
this holy grail of transcriptional regulation is still
unattainable, although the majority of these large scale
biology approaches end up being extremely useful to the
development of smaller scale approaches, focused on a
gene or small group of genes [2]. There are likely to be
multiple reasons for this limitation, including (1) the
limited understanding of additional levels of post-
transcriptional/epigenetic regulation that define the final
phenotype, (2) the absence of a proper understanding at
a formal/mathematical level of network organization and
functioning, or (3) these transcriptional profiles do not
translate into any sort of accessible mechanistic profile,

<footnote>* Correspondence: jbecker@igc.gulbenkian.pt

¹Instituto Gulbenkian de Ciência, 2780-156, Oeiras, Portugal

Full list of author information is available at the end of the article</footnote>
but are an emergent property of the complexity of other
underlying levels of organization based on fundamental
chemical and physical properties of DNA and proteins.
There is no easy way to circumvent these limitations at
our present understanding of biology, but usable clues
could arise from applying an analogue to comparative
anatomy at the cellular level, such as searching for con-
served transcriptional signatures that could be used for
further genetic or physiological dissection [8,9]. Such an
approach can be conceptually rooted into evolutionary
developmental biology (evo-devo), in which specific and
defined small gene-regulatory networks (GRNs) may act
as defined modules that may have been co-opted during
evolution to perform related functions [10]. Modular
GRNs are intrinsically robust and quasi-independent
complexes of genes, allowing the possibility of disentan-
gling evolutionary pathways through comparison with
similar modules from unrelated species or organs. This
architectural feature of the modules, coupled to their
power to generate diversity, makes inter-GRN connec-
tion elements major targets of adaptive evolution [11].
Plant-microbe interactions have been recently pro-
posed to constitute an attractive system to test some of
these concepts, as the communication module seems
to have been both phylogenetically re-deployed and
functionally adapted along co-evolution of both plants
and microbes [12].

Apical growth in filamentous cells is a striking example
of conservation of structure and function in plants. As
opposed to most plant cells, which grow diffusively over
large volumes, these are defined by growing over a rela-
tively small volume at the tip, by exocytosis of specific
cell wall precursors [13,14]. This form of growth is
common among fungi and in some animal cells (neurite
outgrowth during the development of the nervous
system; see [15]), and in flowering plants it occurs only
in root hairs and pollen tubes. Despite differences,
growth and morphogenesis is similar in these two cell
types [16-18] and as they are functionally skewed
towards the same objective: perceive the surrounding en-
vironment and process this information to direct growth.
Previous studies suggested that the molecular and physio-
logical mechanisms employed to direct growth are likely
conserved between pollen tubes and root hairs [19,20].
This conservation is especially well observed at the level
of the cytoskeleton organization, membrane trafficking
and endo/exocytosis and signalling pathways mediated by
calcium, phosphoinositide, ROPs and ROS [18,20-24].
Developmental definition by specific transcription factors
is well described for root hairs (see for example [25,26])
and pollen grains [27,28]. Previous transcriptional profi-
ling of pollen and sperm [3,4] allowed the search of
conserved GRNs that exist in the two different cell types
that compose the male gametophyte. In comparison, root

hairs must be seen in the context of the root, a very com-
plex organ where various hierarchical levels of transcrip-
tional integration are expected [7]. While much is known
about root transcriptomics in general, the profile of isolated
root hairs is still lacking, limiting the possibility of compara-
tive analysis with pollen tubes, and search for conserved
transcriptional network motifs. The advent of more power-
ful and revealing ways of imaging signal integration in roots
(see for example [29,30]) makes it even more obvious the
need of specific transcriptomics of root hairs, one of the
physiologically more important cell types in roots.

Here we compare the transcriptional profile of isolated
root hairs and pollen with other cell and organ types to
test the hypothesis that there are conserved transcrip-
tomic signatures that define functions in similarly grow-
ing cells. Root hair transcriptomics was previously
approached by a number of studies using FACS of la-
belled root cell types and nuclei, respectively [6,7,31-33],
by dataset subtraction from root hair development mu-
tants [34,35], or by a combination of mutants and FACS
[36]. Here we developed a new way of isolating mRNA
directly from mechanically purified frozen wild type root
hairs. We conclude that root hairs and pollen have highly
overlapping transcriptional profiles, with clear similarities
at the level of small GTPases, vesicle-mediated transport
and various specific metabolic responses, likely defining
the unique regulatory processes that occur in these cell
types. We propose that this study should constitute the
foundation for further genetic and physiological dissection
of the mechanisms underlying apical growth of plant cells.

Results

Isolation of *Arabidopsis* root hairs

The purity of total RNA isolated from root hairs was
important for this study, because the slightest contami-
nation would have obscured a potential apical growth
signature. Therefore, we established a method using an
aluminum tower partially immersed in liquid nitrogen
and a brush to isolate root hairs from Arabidopsis seed-
lings (Figure 1, see Methods). To determine the quality
of the total RNA isolated from root hairs, several genes
expressed in specific cell types in roots were investigated
by RT-PCR (Figure 2). *SCARECROW* (*SCR*) expressed in
cortex, *SHORT ROOT* (*SHR*) in stele, and *PLETHORA1*
(*PLT1*) in stem cells, were amplified from root cDNA
but not from root hair cDNA [37-39], whereas *Arabi-
dopsis thaliana* *EXPANSIN7* (*AtEXP7*), which has been
shown to be expressed in root hair cell files [40], was de-
tected both in root and root hair cDNA. *ACTIN8*
(*ACT8*), expressed throughout the plant including the
root hairs [41] was used as a positive control. *GLABRA2*
(*GL2*) is preferentially expressed in non-hair cells of the
root epidermis but is also expressed in low levels in
some root hair cells [42,43], and was detected in our
<figure><img src="image_2.png" /></figure>

root hair sample. Moreover, *ENHANCER OF TRY AND CPC1 (ETC1)* and *MYB23*, both of which are non-hair cell markers [44,45], were called “absent” in our microarray data. Together, our data indicated that the extracted RNA was rich in root hair specific transcripts.

Root hairs and pollen overlap significantly in their
transcriptional programs

We obtained the transcriptional profile of the root hairs
using Affymetrix Arabidopsis ATH1 arrays. 11,696 genes
were detected as expressed, corresponding to 51% of the
transcripts represented on the array (mean percentage of
Present calls). The expression profile of root hairs was
compared with those of cell sorted hydrated pollen
grains (29% of Present calls), leaves (62%), seedlings
(68%), siliques (69%), flowers (68%) [5] as well as ovules
(67%) and unpollinated pistils (69%) [46]. In addition, we
reanalyzed expression data of single cell types of roots
[6,47] resulting in 58% of Present calls for stele, 62% for
endodermis plus quiescent center, 66% for cortex and
53% for epidermal atrichoblasts. Thus, the number of
genes expressed in root hairs is significantly higher than
in pollen, but smaller than in other vegetative tissues and
even in a number of root cell types. It is however similar
in root hairs and epidermal atrichoblasts.

When the expression data derived from our data sets
is subjected to principal component analysis and hie-
rarchical clustering, closely related or overlapping tissues
like seedling and leaves, pistils and ovules and siliques

and flowers form sub-clusters (Figure 3A). Interestingly
root hairs form a sub-cluster with pollen and not with
any of the tissues. Principal component analysis shows a
similar picture with root hairs and pollen being clearly
separated from the other tissues in the first principal
component (Figure 3B). Cell types with apical growth
type (root hairs and pollen) are conclusively separated
from tissues containing cells only with diffuse growth
type (pistils, ovules, siliques and leaves) or even a mix-
ture of diffuse and apical growth cell types as found in
flowers containing pollen and seedlings containing root
hairs. This result statistically shows a relation between
the datasets which is suggestive of a common transcrip-
tional profile pattern for the apical growing cells in a
plant. Importantly, other root cell types [33] do not
cluster together with pollen and root hair samples
(Additional file 1: Figure S1 and Additional file 2:
Table S1 for PCA loadings). This is an indication that
the separation observed is not solely based on green
versus non-green tissue features, although one has to
keep in mind that comparison with the root cell type
datasets might be confounded by protoplasting and
FACS effects. 1814 genes show enriched expression in
root hairs in relation to expression levels in leaf, pistil,
ovule and silique samples. When compared with “root
hair genes” as defined in other studies [7,31,32,34,36]
the highest overlap (125 genes out of 153) is achieved
with the “core set hair genes” identified by Bruex et al.
[36] (Additional file 3: Table S2).
<figure><img src="image_3.png" /></figure>

Analysis of pollen tube and root hair transcriptomes
reveals an apical growth signature

We hypothesized that the differences observed in the tran-
scriptional profiles would predominantly derive from tran-
scripts that show enriched or selective expression in root
hairs and pollen when compared with tissues containing
solely cells with diffuse growth type. Of the 4989 genes
expressed in both pollen and root hairs our comparative
analysis identified 277 genes as showing enriched expres-
sion in these apical growing cells (Additional file 4: Table
S3). Based on comparison with our restricted data set of 4
tissues with cell types showing diffuse growth, 105 genes
are selectively expressed in apical growing cells (Figure 4).
However, extending this comparison by including other
Arabidopsis tissue types and developmental stages (Schmid
et al. 2005) strictly containing only cell types with diffuse
growth type, reduces this list of selectively expressed genes

to 49 (Table 1). Transcriptome analysis of growing pollen
tubes of Arabidopsis has shown that there is a moderate in-
crease in transcript diversity and abundance when compar-
ing growing pollen tubes with hydrated pollen grains [48].
To assess if we are missing potential apical growth signa-
ture genes we crossed our list of 1814 root hair enriched
transcripts with the list of genes up-regulated during pollen
tube growth [48] and our 4989 genes common to mature
hydrated pollen and root hairs (Additional file 3: Table S2).
34 of the 41 genes identified as being enriched in root hairs,
up-regulated in growing pollen tubes and not in our apical
growth list were called Absent in our pollen data and
would thus potentially have to be added to our list of 277
apical growth enriched genes, if not being expressed at
higher levels in the sporophytic tissues analyzed. Further-
more, in a recent study 104 genes were identified as poten-
tial polar cell expansion genes by crossing tobacco pollen
tube with Arabidopsis trichoblast transcriptomic data [49].
We found 48 of those genes to be expressed in Arabidopsis
pollen and root hairs, three showing enriched expression
and none being selective (Additional file 4: Table S3).

To validate our microarray results, we performed RT-
PCR analysis for eleven of these apical growth selective
transcripts. Ten were detected in both pollen and root hair
samples, while *At5g04960* could not be amplified from our
pollen cDNA sample (Figure 5), possibly reflecting its low
signal value of 67 on the pollen arrays. RT-PCR analyses
have additionally shown that even if a transcript is called
Absent on a Genechip experiment, it might still be detected
by RT-PCR. This holds true for *At2g29620*, *At5g01280* and
*At1g63930*, which were detected also in ovules, seedlings
and siliques, respectively (Figure 5), although the latter two
are likely to be root hair- and pollen-derived, respectively.
Thus it seems that ten out of eleven apical growth genes
are mainly expressed in root hairs and pollen, which is a
significantly positive result to allow downstream analyses
based on the array data. In addition, comparing detection
levels for pollen and root hair samples confirms a signifi-
cant correlation between microarray data and the semi
quantitative RT-PCR performed.

Next we asked if genes expressed in both pollen and
root hairs are functionally skewed towards biological
process classes known or expected to be involved in
apical cell growth. Our comparative Gene Ontology
analysis showed that genes involved in membrane
lipid metabolism and vesicle-mediated transport are
over-represented in apical growing cells (Figure 6 and
Additional file 5: Table S4). In addition energy metabo-
lism, represented by the classes oxidative phosphorylation,
mitochondrial transport and coenzyme metabolism, as
well as signal transduction, comprising the classes re-
sponse to reactive oxygen species, small GTPase signaling
and biopolymer modification, are over-represented func-
tions in these cell types. Most but not all of these classes
<figure><img src="image_4.png" /></figure>

**Figure 3** Principal component analysis and hierarchical clustering of Arabidopsis transcriptome data. (A) Principal component analysis is an exploratory technique used to describe the structure of high dimensional data, e.g. derived from microarrays, by reducing its dimensionality. Here, expression values for 22.800 genes in 8 tissue/cell types are projected onto the first three principal components. The first principal component separates pollen and root hairs from the other tissues, while the second and third principal components show a further, though less significant, separation of the samples. (B) Hierarchical clustering is used to group similar objects into “clusters”, producing a tree (called dendrogram) that shows the hierarchy of the clusters. The dendrogram shows a clear separation of a pollen and root hair cluster from a cluster including the other sample types.

are statistically significantly enriched even when the
complete set of genes in the root hair and pollen tran-
scriptome, respectively, are analyzed separately (Figure 6).

The MapMan tool [50] was used to map differential gene
expression in apical versus diffuse growing cell types on
the most relevant gene families (Figure 7 and Additional
file 6: Table S5). This detailed gene family and pathway
analysis facilitates the identification of primary targets for
reverse genetics confirmation of a possible role for respec-
tive gene products in apical cell growth.

Promoters of genes that define the apical growth
signature share common cis-elements

The identification of conserved cis-regulatory elements
is important to understand regulatory networks and

combinatorial gene expression. To identify conserved
motifs associated with the apical growth gene expression
signature, we analysed the promoter regions of apical
growth selective genes. In order to overcome recognized
limitations of most motif discovery tools available, from
which different motifs are obtained after each run, we
performed promoter sequence analysis using two diffe-
rent tools, and compared the results based on sequence
consensus alignment and annotation to different plant
promoter databases. As expected, different motifs were
detected by Musa [51] and Promzea [52] as overrepre-
sented in the promoters of apical growth genes (Figure 8).
While we were not able to find correspondence to many
of the motifs identified by Musa within the publicly avail-
able plant promoter database PLACE [53], it was possible
<figure><img src="image_5.png" /><figcaption>**Figure 4** Venn Diagram depicting the number of expressed genes (as defined by Present calls) in apical growing and diffuse cell types, and their respective overlaps. Flowers and seedlings were excluded from this analysis, since they contain pollen and root hairs, respectively.</figcaption></figure>

to identify the most statistically significant consensus sequences detected by Promzea using STAMP [54]. We found common elements such as the TATA box and pyrimidine patch (Y Patch) elements [55-57] that generally appear near the transcriptional start site (TSS). This might be the case for the TCTTCT and TTCTCT motifs (Figure 8), which probably form part of the higher plant-specific core promoter element Y Patch. Musa was able to detect the AGAAA motif, which is a *cis*-regulatory element of the *Lat52* promoter that is preferentially active in the vegetative cell during pollen maturation [58].

Interestingly, the only motif detected by both tools was AAAACAAA, a *cis*-element that was previously detected in the promoters of genes whose expression is induced anaerobically [59]. It is likely that both pollen tube and root hairs growth might sometimes suffer hypoxia, owing to submergence either inside sporophyte tissues or by water flooding, respectively. In fact, an alternative to mitochondrial respiration has been previously characterized in species with bicellular pollen such as tobacco and petunia [60-63]. Oxygen availability was never a limiting factor for pollen germination in vitro, while ethanol fermentation either involving alcohol dehydrogenase (ADH) and pyruvate carboxylase (PDC) pathways were demonstrated to be essential for pollen tube growth and fertilization. Taken together, our results

suggest that maintaining apical growth mechanisms synchronized with energy yielding might require a combinatorial network of transcriptional regulation.

Discussion

Cell growth takes place at a restricted area at the cell apex in pollen tubes and root hairs, a process called tip or apical growth [13,14]. While many components of the mechanism required for growth of these extremely polarised cells also occur in other cell types that grow by diffuse growth, our analysis of the root hair and pollen transcriptome demonstrates that tip growing cells are defined by a common set of proteins that carry out activities required for tip-growth. We propose that the core set of genes that comprise this apical signature encode proteins that are active in a variety of cellular activities that are required for this mode of cell elongation.

As part of this study we have developed a novel method to isolate growing and mature root hairs directly from seedlings. It circumvents problems associated with methods used in other studies aiming at identifying root hair-rich expression, e.g. by relying on mutants with decreased or increased abundance of root hairs [34-36] or on FACS sorted cells or nuclei [6,7,31-33,36]. Altered transcriptional profiles due to the mutations or due to the extensive manipulations needed before FACS in combination with the limitation in purity for the FACS approaches might explain the limited overlap of our root hair enriched gene list with comparable lists from these studies. Further confounding factors are technical differences like the platforms used (RNAseq or different microarrays) and the tissue types used to identify enriched or selective expression. Given these restrictions the 82% overlap with the 153 "core set hair genes" identified by Bruex et al. [36] is remarkable and validates our approach.

It is long known that the growth in both pollen tubes and root hairs is accompanied by similar physiological processes (reviewed by [20]). Probably the best characterised is the formation of a tip-high gradient of cytoplasmic calcium in both cell types and that is required for growth (reviewed by [17,64]). This local elevation in cytoplasmic calcium concentration is believed to be formed as a result of the activity of channels that transport calcium ions from the outside of the cell to the cytoplasm in the apical region of the cell [65]. It is likely that other physiological processes that are specific to tip growing cells exist and remain to be identified. Our analysis of the pollen and root hair transcriptome has identified sets of genes that are common to elongating pollen tubes and root hairs and may thus define such a suite of apical growth-specific processes. This increases significantly a previously defined list of 104 potential polar cell expansion genes [49]. The genes we have identified encode proteins active in a variety of processes,
<table><caption>Table 1 Selectively expressed genes in apical growing cells</caption><thead><tr><th>Function</th><th>Probe set</th><th>AGI ID</th><th>Gene</th><th>Pollen</th><th>Root hair</th><th>Enriched</th><th>FC</th><th>Pollen FC</th><th>Root hair FC</th></tr></thead><tbody><tr><td>Protein modifications</td><td>264284_at</td><td>At1g61860</td><td>Protein kinase, putative</td><td>13390</td><td>360</td><td>X</td><td>72.4</td><td>141.2</td><td>3.5</td></tr><tr><td>Protein modifications</td><td>249950_at</td><td>At5g18910</td><td>Protein kinase family protein</td><td>8337</td><td>261</td><td>X</td><td>56.5</td><td>110.0</td><td>3.0</td></tr><tr><td>Protein modifications</td><td>251433_at</td><td>At3g59830</td><td>Ankyrin protein kinase, putative</td><td>2322</td><td>279</td><td>X</td><td>19.1</td><td>34.2</td><td>3.9</td></tr><tr><td>Protein modifications</td><td>258832_at</td><td>At3g07070</td><td>Protein kinase family protein</td><td>795</td><td>2708</td><td>X</td><td>19.0</td><td>8.3</td><td>29.7</td></tr><tr><td>Protein modifications</td><td>267582_at</td><td>At2g41970</td><td>Protein kinase, putative</td><td>2680</td><td>2854</td><td>X</td><td>12.5</td><td>12.2</td><td>12.8</td></tr><tr><td>Protein modifications</td><td>263378_at</td><td>At2g40180</td><td>Protein phosphatase 2C, putative/PP2C, putative</td><td>1481</td><td>197</td><td>X</td><td>10.8</td><td>19.2</td><td>2.4</td></tr><tr><td>Protein modifications</td><td>248909_at</td><td>At5g45810</td><td>CIPK19</td><td>2006</td><td>560</td><td>X</td><td>6.4</td><td>10.2</td><td>2.6</td></tr><tr><td>Protein modifications</td><td>265178_at</td><td>At1g23540</td><td>Protein kinase family protein</td><td>620</td><td>133</td><td>X</td><td>6.3</td><td>10.6</td><td>2.0</td></tr><tr><td>Protein modifications</td><td>264127_at</td><td>At1g79250</td><td>Protein kinase, putative</td><td>347</td><td>452</td><td>X</td><td>4.3</td><td>3.8</td><td>4.7</td></tr><tr><td>Protein modifications</td><td>253718_at</td><td>At4g29450</td><td>Leucine-rich repeat protein kinase, putative</td><td>233</td><td>176</td><td></td><td></td><td>2.9</td><td></td></tr><tr><td>Calcium signalling</td><td>245036_at</td><td>At2g26410</td><td>IQD4 (IQ-domain 4); calmodulin binding</td><td>12177</td><td>1885</td><td>X</td><td>59.5</td><td>105.5</td><td>13.6</td></tr><tr><td>Calcium signalling</td><td>259064_at</td><td>At3g07490</td><td>AGD11 (ARF-GAP DOMAIN 11)</td><td>4146</td><td>1254</td><td>X</td><td>17.5</td><td>27.3</td><td>7.6</td></tr><tr><td>Calcium signalling</td><td>254774_at</td><td>At4g13440</td><td>Calcium-binding EF hand family protein</td><td>90</td><td>173</td><td></td><td></td><td></td><td>1.8</td></tr><tr><td>G-protein signalling</td><td>259836_at</td><td>At1g52240</td><td>ATROPGEF11/ROPGEF11 (KINASE PARTNER PROTEIN-LIKE)</td><td>3003</td><td>282</td><td>X</td><td>51.8</td><td>95.5</td><td>8.1</td></tr><tr><td>G-protein signalling</td><td>260161_at</td><td>At1g79860</td><td>ATROPGEF12/MEE64/ROPGEF12 (KINASE PARTNER PROTEIN-LIKE)</td><td>3436</td><td>661</td><td>X</td><td>44.0</td><td>75.0</td><td>13.1</td></tr><tr><td>G-protein signalling</td><td>263458_at</td><td>At2g22290</td><td>AtRABH1d (Arabidopsis Rab GTPase homolog H1d)</td><td>458</td><td>674</td><td>X</td><td>12.2</td><td>9.4</td><td>14.9</td></tr><tr><td>G-protein signalling</td><td>254173_at</td><td>At4g24580</td><td>Pleckstrin homology (PH) domain-containing protein-related / RhoGAP domain-containing protein</td><td>782</td><td>164</td><td>X</td><td>4.8</td><td>8.0</td><td>1.6</td></tr><tr><td>G-protein signalling</td><td>266190_at</td><td>At2g38840</td><td>Guanylate-binding family protein</td><td>1383</td><td>1343</td><td>X</td><td>2.3</td><td>2.5</td><td>2.2</td></tr><tr><td>Cell wall proteins</td><td>263453_at</td><td>At2g22180</td><td>Hydroxyproline-rich glycoprotein family protein</td><td>5725</td><td>233</td><td>X</td><td>37.8</td><td>73.0</td><td>2.7</td></tr><tr><td>Cell wall Proteins</td><td>249375_at</td><td>At5g40730</td><td>AGP24 (ARABINOGALACTAN PROTEIN 24)</td><td>19904</td><td>13284</td><td>X</td><td>16.3</td><td>19.7</td><td>12.9</td></tr><tr><td>Cell wall proteins</td><td>259720_at</td><td>At1g61080</td><td>Proline-rich family protein</td><td>533</td><td>482</td><td>X</td><td>11.0</td><td>11.6</td><td>10.3</td></tr><tr><td>Cell wall proteins</td><td>246872_at</td><td>At5g26080</td><td>Proline-rich family protein</td><td>128</td><td>152</td><td>X</td><td>1.5</td><td>1.4</td><td>1.6</td></tr><tr><td>Cell wall proteins</td><td>245159_at</td><td>At2g33100</td><td>ATCSLD1 (Cellulose synthase-like D1)</td><td>11021</td><td>55</td><td></td><td></td><td>132.6</td><td></td></tr><tr><td>Cell wall proteins</td><td>250801_at</td><td>At5g04960</td><td>pectinesterase family protein</td><td>67</td><td>8360</td><td></td><td></td><td></td><td>114.1</td></tr><tr><td>Cell wall proteins</td><td>251842_at</td><td>At3g54580</td><td>Proline-rich extensin-like family protein</td><td>447</td><td>150</td><td></td><td></td><td>4.2</td><td></td></tr><tr><td>Cell wall proteins</td><td>265275_at</td><td>At2g28440</td><td>Proline-rich family protein</td><td>488</td><td>136</td><td></td><td></td><td>6.2</td><td></td></tr><tr><td>Transcription</td><td>261643_at</td><td>At1g27720</td><td>Transcription initiation factor</td><td>540</td><td>196</td><td>X</td><td>3.1</td><td>4.7</td><td>1.6</td></tr><tr><td>ENTH</td><td>247941_at</td><td>At5g57200</td><td>Epsin N-terminal homology (ENTH) domain-containing protein / clathrin assembly protein-related</td><td>717</td><td>108</td><td>X</td><td>9.3</td><td>16.3</td><td>2.3</td></tr><tr><td>P- and V-ATPases</td><td>251405_at</td><td>At3g60330</td><td>AHA7 (ARABIDOPSIS H(+)-ATPASE 7)</td><td>654</td><td>2829</td><td>X</td><td>22.7</td><td>8.2</td><td>37.2</td></tr><tr><td>Lipid degradation</td><td>267439_at</td><td>At2g19060</td><td>GDSL-motif lipase/hydrolase family protein</td><td>181</td><td>396</td><td>X</td><td>3.4</td><td>2.6</td><td>4.2</td></tr></tbody></table>
<table><caption>Table 1 Selectively expressed genes in apical growing cells (Continued)</caption><tr><td>Vesicle transport</td><td>259338_at</td><td>At3g03800</td><td>SYP131 (syntaxin 131)</td><td>13646</td><td>603</td><td>X</td><td>45.0</td><td>86.6</td><td>3.3</td></tr><tr><td>Exocyst</td><td>250204_at</td><td>At5g13990</td><td>ATEXO70C2 (exocyst subunit EXO70 family protein C2)</td><td>1428</td><td>695</td><td>X</td><td>16.4</td><td>21.1</td><td>11.7</td></tr><tr><td>Exocyst</td><td>245979_at</td><td>At5g13150</td><td>ATEXO70C1 (exocyst subunit EXO70 family protein C1)</td><td>3238</td><td>1780</td><td>X</td><td>15.8</td><td>20.5</td><td>11.1</td></tr><tr><td>Aging</td><td>249868_at</td><td>At5g23030</td><td>TET12 (TETRASPANIN12)</td><td>97</td><td>1167</td><td></td><td></td><td></td><td>19.5</td></tr><tr><td>Cytoskeleton organisation</td><td>266697_at</td><td>At2g19770</td><td>PRF5 (PROFILIN5)</td><td>5226</td><td>165</td><td>X</td><td>30.4</td><td>59.1</td><td>1.8</td></tr><tr><td>Ion transport</td><td>251053_at</td><td>At5g01490</td><td>CAX4 (cation exchanger 4)</td><td>495</td><td>342</td><td></td><td></td><td>1.8</td><td></td></tr><tr><td>Isoprenoid biosynthesis</td><td>257274_at</td><td>At3g14510</td><td>Geranylgeranyl pyrophosphate synthase, putative</td><td>58</td><td>112</td><td></td><td></td><td></td><td>1.8</td></tr><tr><td>Microtubule-based movement</td><td>254205_at</td><td>At4g24170</td><td>Kinesin motor family protein</td><td>1216</td><td>143</td><td>X</td><td>13.0</td><td>23.5</td><td>2.5</td></tr><tr><td>Protein Folding</td><td>260478_at</td><td>At1g11040</td><td>DNAJ chaperone C-terminal domain-containing protein</td><td>1136</td><td>112</td><td></td><td></td><td>17.5</td><td></td></tr><tr><td>Pyrophosphatase activity</td><td>266765_at</td><td>At2g46860</td><td>Inorganic pyrophosphatase, putative (soluble)</td><td>1024</td><td>598</td><td>X</td><td>10.8</td><td>14.2</td><td>7.4</td></tr><tr><td>Unknown</td><td>246592_at</td><td>At5g14890</td><td>NHL repeat-containing protein</td><td>7155</td><td>144</td><td>X</td><td>48.7</td><td>95.6</td><td>1.8</td></tr><tr><td>Unknown</td><td>260320_at</td><td>At1g63930</td><td>Similar to unknown protein (TAIR:AT4G23530.1)</td><td>1169</td><td>1495</td><td>X</td><td>18.0</td><td>15.8</td><td>20.1</td></tr><tr><td>Unknown</td><td>267051_at</td><td>At2g38500</td><td>Similar to DTA4 (DOWNSTREAM TARGET OF AGL15-4) (TAIR:AT1G79760.1)</td><td>4282</td><td>546</td><td>X</td><td>13.6</td><td>24.3</td><td>2.8</td></tr><tr><td>Unknown</td><td>256506_at</td><td>At1g75160</td><td>Similar to unknown protein (TAIR:AT5G05840.1)</td><td>958</td><td>310</td><td>X</td><td>12.8</td><td>19.9</td><td>5.6</td></tr><tr><td>Unknown</td><td>266674_at</td><td>At2g29620</td><td>Similar to unknown protein (TAIR:AT1G07330.1)</td><td>337</td><td>839</td><td>X</td><td>9.7</td><td>5.6</td><td>13.8</td></tr><tr><td>Unknown</td><td>251135_at</td><td>At5g01280</td><td>Similar to proline-rich family protein (TAIR:AT3G09000.1)</td><td>134</td><td>1001</td><td>X</td><td>7.8</td><td>1.6</td><td>14.0</td></tr><tr><td>Unknown</td><td>249185_at</td><td>At5g43030</td><td>DC1 domain-containing protein</td><td>346</td><td>487</td><td>X</td><td>4.8</td><td>4.0</td><td>5.6</td></tr><tr><td>Unknown</td><td>251047_at</td><td>At5g02390</td><td>similar to unknown protein (TAIR:AT1G07620.1)</td><td>388</td><td>321</td><td>X</td><td>4.6</td><td>5.2</td><td>4.0</td></tr><tr><td>Unknown</td><td>252987_at</td><td>At4g38390</td><td>Similar to unknown protein (TAIR:AT1G76270.1)</td><td>106</td><td>423</td><td></td><td></td><td></td><td>5.5</td></tr><tr><td>Unknown</td><td>254972_at</td><td>At4g10440</td><td>Dehydration-responsive family protein</td><td>135</td><td>144</td><td></td><td></td><td></td><td></td></tr><tr><td>Unknown</td><td>260195_at</td><td>At1g67540</td><td>Unknown protein</td><td>540</td><td>51</td><td></td><td></td><td>6.5</td><td></td></tr></table>

The first column shows the functional classification of the gene (see also Figure 7). The second column depicts the Affymetrix probe set, followed by TAIR locus (AGI ID) assigned to this probe set and gene annotation in the third and fourth column. In columns five and six the expression values for pollen and root hairs, respectively, are given. The following three columns (7 to 8) depict, if a gene is selectively and/or enriched in root hairs and pollen, followed by the average of the lower confidence bound of the fold change (FC) for apical growing cells. The last two columns give the average FC of pollen and root hairs, respectively.

including signalling, cell wall modification, oxidative phosphorylation, mitochondrial transport and coenzyme metabolism. We therefore propose that the apical-growth gene expression signature defines a suite of cellular activities that, like the tip high calcium gradient, are required for the extension of tip growing cells.

Among the processes that are defined by the apical transcriptome are genes involved in signalling processes that control growth. GTPases are key regulators of signalling cascades in cells that play important roles in the co-ordination of cellular activities during growth (reviewed in [66,67]). The Rab GTPase homolog H1d (At2g22290) for example is a selectively expressed component of our apical growth signature and has

been identified by Lan et al. [32] as potential key component of a Rho-signaling network in root-hair differentiation. Reactive oxygen species play important roles in signaling and cell wall modification during growth of pollen tubes and root hairs and genes that are induced in response to reactive oxygen species are components of the apical-growth signature [68-70]; reviewed in [17]. It is likely that they are active in aspects of ROS-regulated apical growth in these cell types [71]. We propose that these different sets of signalling modules are central components of the apical growth mechanism.

The coordinated expression of genes in pollen tubes and root hairs likely involves a common set of regulatory elements. Cis-regulatory elements in the DNA sequence
<figure><img src="image_6.png" /><figcaption>**Figure 5 RT-PCR analysis.** Gel figures for ten genes whose expression was detected only in pollen and root hair samples but not in vegetative tissues (ovule, silique and seedling) by microarray. *TUB4* - tubulin β-4 chain (*At5g04180*) was used as positive control.</figcaption></figure>

surrounding a gene play important roles in the control of gene expression. Different *cis*-regulatory elements are required for the induction of gene expression in different cell types or in response to changes in environmental conditions. For example short WHHDTGNNN(N)KCACGWH elements occur in the promoters of genes that are expressed in the root hair of Arabidopsis [35]. Our analysis demonstrates that there are conserved *cis*-regulatory elements in the promoters of genes that are expressed in pollen tubes and root hairs. We found the AAAACAAA *cis*-regulatory element that is found in genes whose transcription is induced in anaerobic conditions. This is consistent with the hypothesis that tip growing cells suffer anoxia, an hypothesis long set forth for pollen tubes [72], and known to have specific adaptions in root hairs [73]. These conserved *cis*-regulatory elements are likely required for the expression of genes of the apical signature, but given the divergent results of the two prediction tools experimental validation will be needed.

## Conclusions

Together our analyses of the pollen tube and root hair transcriptome indicate that there is a core of 277 genes whose expression is higher in these cell types when

compared to others in the plant. We propose that the proteins that are encoded by these genes define activities that are common to both cell types. We predict that like the tip-high calcium gradient and the apical production of reactive oxygen species that are required for growth in these cells, these activities will define cellular processes that are required for the growth of tip-growing cells in land plants. Given that the tip-high calcium gradient also occurs in other organisms such as fungi (see for example [74]), future research will define if the processes regulated by genes of the apical signature are active in other tip growing cells of eukaryotes.

## Methods

### Plant growth conditions

Seeds for root hair isolation were sterilized in 5% sodium hypochlorite, washed by water and sown on half strength Murashige and Skoog (Duchefa, Haarlem, The Netherlands) medium (pH 5.8) containing 1% sucrose and 0.8% phytagel.

### Root hair RNA isolation and RT-PCR

The scheme of isolating root hairs is shown in Figure 1. Four to five surface-sterilized seeds of *Arabidopsis thaliana* Columbia (Col-0) were sowed on a 3 cm-diameter cellophane disc of type 325P (AA packaging Ltd, Preston, UK), placed on growth media and incubated horizontally under continuous light for 4 to 5 days. The discs on which plants grew were frozen for 1-2 seconds on an aluminium tower (20 cm height) half-sunk in liquid nitrogen (Figure 1). A small flat paint brush was used to carefully remove the leaves, hypocotyls and roots from the frozen plant tissue, except for root hairs that were retained on the discs. These hairs were collected in RNA extraction buffer. Contaminating root tips were removed under a stereomicroscope.

Total RNA from root hairs was isolated by RNeasy Mini extraction kit (Qiagen, Hilden, Germany) and integrity was confirmed using an Agilent 2100 Bioanalyzer with a RNA 6000 Nano Assay (Agilent Technologies, Palo Alto, CA). Total RNA was reverse-transcribed by Superscript II reverse transcriptase (Invitrogen, Paisley, UK) and used for RT-PCR.

For confirmation of selective expression of apical growth genes we used cRNA amplified from pollen, root hair, ovule, silique and seedling samples to prepare double-stranded cDNA. Five nanograms of each template cDNA were subsequently used in reactions of 35 PCR cycles. The primer sequences for all RT-PCRs are shown in Additional file 7: Table S6.

## Target synthesis and hybridization to Affymetrix GeneChips

The GeneChip experiment was performed with biological duplicates. Root hair total RNA was processed for use on Affymetrix (Santa Clara, CA, USA) Arabidopsis ATH1 genome arrays, according to the manufacturer's Two-
<figure><img src="image_7.png" /><figcaption>**Figure 6** Functional enrichment analysis of genes expressed in root hairs, in pollen and in both (apical growth) based on Gene Ontology biological process terms. An asterisk denotes classes that are not statistically significantly enriched in the particular cell type. See Additional file 5: Table S4 for a list of the genes comprising the classes in apical growth.</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>**Figure 7** Gene family analysis of apical versus diffuse growing cell types. Gene expression data from root hairs and pollen relative to siliques, pistils, ovules and leaves are shown on a scheme depicting shank and tip of an apical growing cell. Genes are symbolized by color-encoded squares (red, down-regulation; blue, up-regulation; white, present call in root hairs and pollen, but no concordant change; grey, Absent call in pollen and/or root hairs; X, selective expression in root hairs and pollen). Abbreviation: ENTH, Epsin N-Terminal Homology domain-containing protein; Prec., Precursor; z.f., zinc finger; S., Signalling.</figcaption></figure>
<table><thead><tr><th>Motifs detected by Musa</th><th>Quorum</th><th>p-value</th><th>PLACE Db</th><th>Motifs detected by Promzea</th><th>Sequence consensus</th><th>p-value (STAMP)</th></tr></thead><tbody><tr><td>Motif1</td><td>AAAA (14) TTCT</td><td>36 of 49</td><td>1.38e-17</td><td>Motif1</td><td>AATCTATCTA</td><td>3AF1BOXPSRBCS3 (AAATAGATAAATAAAAAACATT)</td></tr><tr><td>Motif2</td><td>ATTTT (16) AAAC</td><td>22 of 49</td><td>2.83e-13</td><td>Motif2</td><td>AGAGAAGAAA</td><td>RGATAOS (CAGAAGATA)</td></tr><tr><td>Motif3</td><td>TCTTCT</td><td>44 of 49</td><td>1.78e-11</td><td>Motif3</td><td>TAACGTGCAA</td><td>T_GBOXATPIN2 (AACGTG)</td></tr><tr><td>Motif4</td><td>AAAACAAA</td><td>30 of 49</td><td>4.09e-11</td><td>ANAERO1CONSENSUS (AAACAAA)</td><td>ACGTGCTTAA</td><td>bHLH66_M01054 (CACGTGC)</td></tr><tr><td>Motif5</td><td>TTCTCT</td><td>41 of 49</td><td>3.71e-10</td><td>Motif5</td><td>AAAAACAAA</td><td>ANAERO1CONSENSUS (AAACAAA)</td></tr><tr><td>Motif6</td><td>AACAGA</td><td>38 of 49</td><td>9.91e-10</td><td>Motif6</td><td>GACGTGCA</td><td>RYREPEATBNNAPA (CATGCA)</td></tr><tr><td>Motif7</td><td>ACCAC</td><td>39 of 49</td><td>1.72e-09</td><td>Motif7</td><td>ACTTATTCAA</td><td>minus300ELEMENT (TGNAARK)</td></tr><tr><td>Motif8</td><td>AAGAAAA</td><td>39 of 49</td><td>8.76e-09</td><td>POLLEN1LELAT52 (AGAAA)</td><td>GAAGAAGAAG</td><td>TL1ATSAR (CTGAAGAGAA)</td></tr><tr><td>Motif9</td><td>CAACG</td><td>37 of 49</td><td>1.17e-08</td><td>Motif9</td><td>GAAAGAGAGA</td><td>CTRMCAMV35S (TCTCTCTCT)</td></tr><tr><td>Motif10</td><td>AAGAAG</td><td>37 of 49</td><td>4.94e-08</td><td>Motif10</td><td>GGCGCGCA</td><td>ZF5_M00333</td></tr><tr><td>Motif11</td><td>CAAAAC</td><td>38 of 49</td><td>5.36e-08</td><td>Motif11</td><td>AACAAAAACA</td><td>STE11_M01005</td></tr><tr><td>Motif12</td><td>AGAGG</td><td>38 of 49</td><td>6.09e-08</td><td>Motif12</td><td>AAGAAGAAGA</td><td>TCA1MOTIF (TCATCTTCTT)</td></tr></tbody></table>

**Figure 8** Motifs reported by MUSA [51] and Promzea [52] for 49 promoter sequences of apical growth selective genes. Motifs detected by MUSA are ranked by p-value, highlighting correspondence to cis-elements summarized in PLACE database [53]. The quorum value shows the number of query sequences in which a certain motif stands. The sequence consensus for each motif detected by Promzea was compared to known plant promoter database by STAMP [54], and the results were ranked by p-value. Only the most significant result is shown.

### Cycle Target Labeling Assay.
Briefly, 100 ng of total RNA containing spiked in Poly-A RNA controls (GeneChip Expression GeneChip Eukaryotic Poly-A RNA Control Kit; Affymetrix) was used in a reverse transcription reaction (Two-Cycle DNA synthesis kit; Affymetrix) to generate first-strand cDNA. After second-strand synthesis, double-stranded cDNA was used in an *in vitro* transcription (IVT) reaction to generate cRNA (MEGAscript T7 kit; Ambion, Austin, TX). 600 ng of the cRNA obtained was used for a second round of cDNA and cRNA synthesis, resulting in biotinylated cRNA (GeneChip Expression 3'-Amplification Reagents for IVT-Labeling; Affymetrix). Size distribution of the cRNA and fragmented cRNA, respectively, was assessed using an Agilent 2100 Bioanalyzer with a RNA 6000 Nano Assay.

15 µg of fragmented cRNA was used in a 300-µl hybridization containing added hybridization controls. 200 µl of mixture was hybridized on arrays for 16 h at 45°C. Standard post hybridization wash and double-stain protocols (EukGE-WS2v5_450) were used on an Affymetrix GeneChip Fluidics Station 450. Arrays were scanned on an Affymetrix GeneChip scanner 3000.

### GeneChip data analysis

Scanned arrays were first analyzed with Affymetrix GCOS 1.4 software to obtain Absent/Present calls using the MAS5 detection algorithm. Based on a non-parametric statistical test (Wilcoxon signed rank test) it determines whether significantly more perfect matches show more hybridization signal than their corresponding mismatches, leading to a detection call (Absent (A), Present (P) or Marginal (M)) for each probe set [75]. Transcripts were considered as expressed, if their detection call was “Present” in at least one of the two replicates. Subsequently the 16 arrays used in this study (root hairs; [5,46]) were analyzed with dChip 2006 (https://sites.google.com/site/dchipsoft/) as described in [5] with the only difference that no filter for high variation within the replicates was applied. Annotations were obtained from the NetAffx database (www.affymetrix.com) as of July 2007. The raw data is available at Gene Expression Omnibus under the series number GSE38486 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE38486).

CEL files containing raw expression data of single cell types from roots [6,47] were obtained from the AREX
database (www.arexdb.org) and detection calls analyzed
as described above.

Expression data obtained with dChip were imported
into Partek Genomics Suite 6.07 for 3D principal com-
ponent analysis and hierarchical clustering. For the latter
Pearson's dissimilarity was used to calculate row dissimi-
larity and Ward's method for row clustering. Additional
CEL files from [33] were combined with CEL files in this
study, analysed with dChip and expression values imported
into Chipster 2.12 [76]. Results of PCA analysis were
visualized as scatter plots using Origin 9.

Functional annotation tools of DAVID [77] were employed for enrichment analysis of Gene Ontology (GO) terms (biological process; GO level 5) with the following thresholds: Count ≥2; EASE (modified Fisher Exact P-value) ≤0.05; Benjamini-Hochberg ≤0.05, False Discovery Rate ≤10%. Subsequently genes comprising enriched GO terms were subjected to functional annotation clustering followed by manual analysis to identify GO terms with gene lists showing more than 50% overlaps. For GO terms, for which such high redundancy was identified, only the most representative GO terms were retained.

Promoter analysis

In order to enhance effectiveness for motif finding, we have
delimited the promoters of apical growth selective genes
to -1,000 bp upstream of start codon or predicted tran-
scriptional start sites (TSS), and downstream of adjacent
genes if the intergenic regions were less than 1,000 bp.
Sequences were obtained from Athena database [78], and
predicted TSSs from PlantPromoterDB (ppdb) [79]. Pro-
moter sequences were analyzed by MUSA [51] and Prom-
zea [52], using default values for each parameter. MUSA's
output has shown the distribution of motifs detected
through each uploaded sequence (Quorum), ranked
by p-value. Detected sequences were queried against
PLACE database [53] to find correspondence with previ-
ously reported elements. Promzea's output was compared
to known promoter motif databases using STAMP [54].

Additional files

**Additional file 1: Figure S1.** Principal component analysis of Arabidopsis transcriptome data. Expression values for 22.800 genes are projected onto the first two principal components (PC1 and PC2). (A) The same 8 tissue/cell types as in Figure 1, showing a clear separation of pollen and root hairs from the other tissues. (B) Samples as in a, but adding root cell types from Dinneny et al. [33]: A, columella root cap; B, cortex; C, endodermis and quiescent center; D, epidermis and lateral root cap; E, protophloem; F, stele.

**Additional file 2: Table S1.** Data underlying PCAs in Additional file 1: Figure S1, including variance and loadings.

**Additional file 3: Table S2.** List of 1814 genes showing enriched expression in root hairs and its overlap with "root hair genes" or pollen tube up-regulated genes as defined in other studies.

**Additional file 4: Table S3.** Detailed expression date for 4989 transcripts expressed both in root hairs and pollen. In addition information is provided

whether a gene is selectively expressed in growing cells, enriched, or
depleted. The average fold change (FC\*) is given as the lower confidence
bound fold change of all relevant comparisons. Transcript that are
selectively expressed based on our data set, but not if compared with
publicly available ATH1 datasets are denoted with "(X)\#".

**Additional file 5: Table S4.** Genes used for comparative Gene Ontology analysis, sorted in their respective functional classes.

**Additional file 6: Table S5.** Selectively expressed and enriched genes in apical growing cells. The first column shows the functional classification of the gene (see also Figure 7). The second column depicts the Affymetrix probe set, followed by TAIR locus (AGI ID) assigned to this probe set and gene annotation in the third and fourth column. In columns five and six the expression values for pollen and root hairs, respectively, are given. The following three columns (7 to 8) depict, if a gene is selectively and /or enriched expressed in root hairs and pollen, followed by the average of the lower confidence bound of the fold change (FC) for apical growing cells. The last two columns give the average FC of pollen and root hairs, respectively.

**Additional file 7: Table S6.** Primer sequences used for RT-PCR on root hair samples and for confirmation of selectively expressed apical growth transcripts.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

JB, ST and FS did experiments and performed the bioinformatic analysis of the data. JB, LD and JF conceived the project, participated in the design of the study and wrote the manuscript. All authors read, corrected and approved the final manuscript.

Acknowledgments

We would like to thank Christopher Liseron-Monfils for help with motif discovery. JAF acknowledges grants PTDC/BEX-BCM/0376/2012 and PTDC/BIA-PLA/4018/ 2012 and JDB grants PTDC/AGR-GPL/103778/2008 and PTDC/BIA-BCM/103787/ 2008 (all from Fundação para a Ciência e a Tecnologia-FCT, Portugal). FB acknowledges FCT PhD fellowship SFRH/BD/48761/2008. ST acknowledges Marie Curie Incoming fellowship (MCIIF - 0A272J05C) from the European Union and a grant from the Biotechnology and Biological Research Council (BBS/B/ 04498) of the United Kingdom to LD.

Author details

¹Instituto Gulbenkian de Ciência, 2780-156, Oeiras, Portugal. ²Department of Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, UK.

³Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK. ⁴Department of Cell Biology and Molecular Genetics, University of Maryland, 0118 BioScience Research Bldg, College Park, MD 20742-5815, USA. ⁵Present address: Cell and Genome Biology, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kitaina-Yazuma Oji 74, Seika-cho, Soraku-gun, Kyoto 619-0244, Japan.

⁶Present address: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.

Received: 11 March 2014 Accepted: 14 July 2014

Published: 1 August 2014

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doi:10.1186/s12870-014-0197-3

**Cite this article as:** Becker et al.: Transcriptional profiling of Arabidopsis root hairs and pollen defines an apical cell growth signature. BMC Plant Biology 2014 **14**:197.

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