- Research article
- Open Access
Transcriptional profiling of Arabidopsis root hairs and pollen defines an apical cell growth signature
© Becker et al. 2014
- Received: 11 March 2014
- Accepted: 14 July 2014
- Published: 1 August 2014
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.
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.
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.
- Pollen tube
- Root hair
- Apical growth
- Tip growth
- Apical signature
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 . A direct test of this general principle is facilitated by precise transcriptome analysis using microarrays or RNAseq . 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 -, or more complex ones like roots ,. The large amounts of information in different databases allow formal analysis of the transcriptional profiles of specific cell types or organs, holding 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 . 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, 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 conserved transcriptional signatures that could be used for further genetic or physiological dissection ,. 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 . Modular GRNs are intrinsically robust and quasi-independent complexes of genes, allowing the possibility of disentangling 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 connection elements major targets of adaptive evolution . Plant-microbe interactions have been recently proposed 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 .
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 relatively small volume at the tip, by exocytosis of specific cell wall precursors ,. This form of growth is common among fungi and in some animal cells (neurite outgrowth during the development of the nervous system; see ), 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 - and as they are functionally skewed towards the same objective: perceive the surrounding environment and process this information to direct growth. Previous studies suggested that the molecular and physiological mechanisms employed to direct growth are likely conserved between pollen tubes and root hairs ,. 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 ,-. Developmental definition by specific transcription factors is well described for root hairs (see for example ,) and pollen grains ,. Previous transcriptional profiling of pollen and sperm , 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 complex organ where various hierarchical levels of transcriptional integration are expected . While much is known about root transcriptomics in general, the profile of isolated root hairs is still lacking, limiting the possibility of comparative analysis with pollen tubes, and search for conserved transcriptional network motifs. The advent of more powerful and revealing ways of imaging signal integration in roots (see for example ,) 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 transcriptomic signatures that define functions in similarly growing cells. Root hair transcriptomics was previously approached by a number of studies using FACS of labelled root cell types and nuclei, respectively ,,-, by dataset subtraction from root hair development mutants ,, or by a combination of mutants and FACS . 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.
Isolation of Arabidopsisroot hairs
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%)  as well as ovules (67%) and unpollinated pistils (69%) . In addition, we reanalyzed expression data of single cell types of roots , 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.
Analysis of pollen tube and root hair transcriptomes reveals an apical growth signature
Selectively expressed genes in apical growing cells
Root hair FC
Protein kinase, putative
Protein kinase family protein
Ankyrin protein kinase, putative
Protein kinase family protein
Protein kinase, putative
Protein phosphatase 2C, putative/PP2C, putative
Protein kinase family protein
Protein kinase, putative
Leucine-rich repeat protein kinase, putative
IQD4 (IQ-domain 4); calmodulin binding
AGD11 (ARF-GAP DOMAIN 11)
Calcium-binding EF hand family protein
ATROPGEF11/ROPGEF11 (KINASE PARTNER PROTEIN-LIKE)
ATROPGEF12/MEE64/ROPGEF12 (KINASE PARTNER PROTEIN-LIKE)
AtRABH1d (Arabidopsis Rab GTPase homolog H1d)
Pleckstrin homology (PH) domain-containing protein-related / RhoGAP domain-containing protein
Guanylate-binding family protein
Cell wall proteins
Hydroxyproline-rich glycoprotein family protein
Cell wall Proteins
AGP24 (ARABINOGALACTAN PROTEIN 24)
Cell wall proteins
Proline-rich family protein
Cell wall proteins
Proline-rich family protein
Cell wall proteins
ATCSLD1 (Cellulose synthase-like D1)
Cell wall proteins
pectinesterase family protein
Cell wall proteins
Proline-rich extensin-like family protein
Cell wall proteins
Proline-rich family protein
Transcription initiation factor
Epsin N-terminal homology (ENTH) domain-containing protein / clathrin assembly protein-related
P- and V-ATPases
AHA7 (ARABIDOPSIS H(+)-ATPASE 7)
GDSL-motif lipase/hydrolase family protein
SYP131 (syntaxin 131)
ATEXO70C2 (exocyst subunit EXO70 family protein C2)
ATEXO70C1 (exocyst subunit EXO70 family protein C1)
CAX4 (cation exchanger 4)
Geranylgeranyl pyrophosphate synthase, putative
Kinesin motor family protein
DNAJ chaperone C-terminal domain-containing protein
Inorganic pyrophosphatase, putative (soluble)
NHL repeat-containing protein
Similar to unknown protein (TAIR:AT4G23530.1)
Similar to DTA4 (DOWNSTREAM TARGET OF AGL15-4) (TAIR:AT1G79760.1)
Similar to unknown protein (TAIR:AT5G05840.1)
Similar to unknown protein (TAIR:AT1G07330.1)
Similar to proline-rich family protein (TAIR:AT3G09000.1)
DC1 domain-containing protein
similar to unknown protein (TAIR:AT1G07620.1)
Similar to unknown protein (TAIR:AT1G76270.1)
Dehydration-responsive family protein
Promoters of genes that define the apical growth signature share common cis-elements
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 . 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 bicelullar pollen such as tobacco and petunia -. 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.
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 ,. 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 - or on FACS sorted cells or nuclei ,,-,. 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.  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 ). 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 ,). 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 . 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 . The genes we have identified encode proteins active in a variety of processes, 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 ,). 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.  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 -; reviewed in . It is likely that they are active in aspects of ROS-regulated apical growth in these cell types . 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 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 . 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 , and known to have specific adaptions in root hairs . 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.
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 ), future research will define if the processes regulated by genes of the apical signature are active in other tip growing cells of eukaryotes.
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-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 . 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; ,) were analyzed with dChip 2006 (https://sites.google.com/site/dchipsoft/) as described in  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).
Expression data obtained with dChip were imported into Partek Genomics Suite 6.07 for 3D principal component analysis and hierarchical clustering. For the latter Pearson’s dissimilarity was used to calculate row dissimilarity and Ward’s method for row clustering. Additional CEL files from  were combined with CEL files in this study, analysed with dChip and expression values imported into Chipster 2.12 . Results of PCA analysis were visualized as scatter plots using Origin 9.
Functional annotation tools of DAVID  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.
In order to enhance effectiveness for motif finding, we have delimitated the promoters of apical growth selective genes to -1,000 bp upstream of start codon or predicted transcriptional start sites (TSS), and downstream of adjacent genes if the intergenic regions were less than 1,000 bp. Sequences were obtained from Athena database , and predicted TSSs from PlantPromoterDB (ppdb) . Promoter sequences were analyzed by MUSA  and Promzea , 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  to find correspondence with previously reported elements. Promzea’s output was compared to known promoter motif databases using STAMP .
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 Fundacão 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.
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