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Plant Physiology Preview. Published on November 5, 2012, as DOI:10.1104/pp.112.206441 Running head: iRootHair: root hair genomics database Correspon...
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Plant Physiology Preview. Published on November 5, 2012, as DOI:10.1104/pp.112.206441

Running head: iRootHair: root hair genomics database

Corresponding author: Miroslaw Kwasniewski Department of Genetics, University of Silesia, Jagiellonska 28, 40-032 Katowice, Poland Telephone: +48 (32) 200-94-57, Fax: +48 (32) 200-93-96 E-mail: [email protected]

Research area: Genetics, Genomics, and Molecular Evolution

Copyright 2012 by the American Society of Plant Biologists

Title of article: iRootHair: a comprehensive root hair genomics database

Miroslaw Kwasniewski Urszula Nowakowska Jakub Szumera Karolina Chwialkowska Iwona Szarejko

Department of Genetics, University of Silesia, Jagiellonska 28, 40-032 Katowice, Poland

Footnotes: This work was supported by the EU-FP7 project "EURoot: Enhancing resource Uptake from Roots under stress in cereal crops", 2012-2015, project number 289300; www.euroot.eu.

Corresponding author: Miroslaw Kwasniewski, e-mail: [email protected], fax: +48 (32) 200-93-96

ABSTRACT The specialized root epidermis cells of higher plants produce long, tubular outgrowths called root hairs. Root hairs play an important role in nutrient and water uptake and they serve as a valuable model in studies of plant cell morphogenesis. More than 1,300 articles that describe the biological processes of these unique cells have been published to date. As new fields of root hair research are emerging, the number of new papers published each year and the volumes of new relevant data are continuously increasing. Therefore, there is a general need to facilitate studies on root hair biology by collecting, presenting and sharing the available information in a systematic, curated manner. Consequently, in this paper we present the comprehensive database of root hair genomics, iRootHair, which is accessible as a Web-based service. The current version of the database includes information about 153 root hair-related genes that have been identified to date in dicots and monocots along with their putative orthologs in higher plants with sequenced genomes. In order to facilitate the use of the iRootHair database, it is subdivided into interrelated, searchable sections that describe: Genes, Processes of root hair formation, root hair Mutants and available References. The database integrates bioinformatics Tools with a focus on sequence identification and annotation. iRootHair is a unique resource for root hair research that integrates the large volume of data related to root hair genomics in a single, curated and expandable database that is freely available at: http://www.iroothair.org.

INTRODUCTION Root hairs are long, tubular outgrowths of specialized epidermal cells that through the enlargement of the root surface area play an important role in the absorption of water and nutrients and provide plant anchorage during germination and growth (Cutter, 1978; Gahoonia et al., 1997). Additionally, root hairs function as sites of interactions with fungal and bacterial soil microorganisms (Mylona et al., 1995). Despite their normally important role, under controlled laboratory conditions root hairs are not necessary for plants to survive. This makes them an important model for genetic and consequently, phenotypic manipulations in fundamental studies of higher plant cell growth and development. The first scientific publication about root hairs, which was recorded in the PubMed database, is dated on 1893, where T. Jamieson reviewed the current knowledge on the role of root hairs in the absorption of nutrients from the soil. At present, a PubMed search with combined basic keywords: reveals 1,340 publications concerning broad aspects of root hair biology that have been published to date. Significant progress in publishing in the field of root hair research has been observed since 1990. After that, the number of new papers published yearly has increased regularly showing linear trend (Fig. 1). A stepwise increase in publishing was noticed in 2000 when the Arabidopsis thaliana genome was published. In fact, the broad range of genomics tools that is accessible for this species, including a large collection of T-DNA mutants, straightforward methods of genetic transformation, a small, sequenced genome with highly saturated genetic maps have made Arabidopsis the most important model plant for root hair studies (Schiefelbein and Somerville, 1990; Parker et al. 2000; Schiefelbein, 2000). Nevertheless, new, high throughput methodologies, such as global transcriptome or proteome analyses have also opened new fields in root hair research in non-model species and crop plants. It has been demonstrated that global transcriptome profiling in a wild-type/mutant system results in the rapid identification of new genes that are involved in root hair development in Arabidopsis (Jones et al., 2006) and allows the identification of new candidate genes that are presumably associated with this process in the crop plant barley (Kwasniewski et al., 2010). Similar approaches, applied on a proteome level, allowed the identification of new candidate proteins which can be involved in the initiation of root hair formation in barley (Janiak et al., 2012) and were used to establish a protein reference map for soybean root hair cells (Brechenmacher et al., 2009) and to identify the conserved and unique protein features of root hairs in maize (Nestler et al., 2011). All of these studies delivered a large amount of new data that enabled

the identification of known and new root hair genes and thus provided new insights into the molecular mechanisms of the complex processes of root hair formation. Consequently, a wide-range of available information on the molecular bases of root hair morphogenesis revealed the potential of root hairs as models for plant cell systems biology (Libault et al., 2010.) Recently, a cutting-edge, genome-wide transcriptome differentiation approach using root epidermal cell fate mutants and fluorescence-activated cell-sorting has been applied to define and organize a large set of root hair development-related genes into a transcriptional regulatory network. The integration of genetic, genomic and computational methods allowed for preliminary studies of the composition, architecture and logic of the root epidermal transcriptional network in Arabidopsis (Bruex et al., 2012). Nevertheless, regardless of the methodologies used, all the studies on the molecular mechanisms of root hair growth and development, either single-gene analyses or large scale systems-level studies relay and use the information about genes, which function in root hair formation has been undoubtedly confirmed. The above-mentioned evidences demonstrate that the processes of root hair formation have been described in dispersed literature; in each study the information about root hair-related genes has to be reviewed, BLAST searches need to be performed against primary databases, the subsequent description of gene function must be studied etc. Thus, we believe that there is a serious need to facilitate studies on root hair biology by collecting, presenting and sharing the available information in a systematic, curated manner. Consequently, in this paper we present the comprehensive database of root hair genomics information, iRootHair, to assist in the study of root hair development and system biology.

RESULTS Root Hair Genomics Information A PubMed database search identifies more than 1,300 root hair research-related publications. The records that were found were systematically examined for papers that refer to information about genes that have a documented function in root hair development. The main criterion used during the reviewing process was a clear root hair phenotype resulting from gene mutation, overexpression or silencing. In some cases, even if the root hair phenotype was not precisely recognized, additional aspects, such as a specific root hairassociated expression pattern or well-defined interactions with root hair genes were

considered. The genes described in the literature that were based solely on global transcriptome or proteome analyses were not selected if their function was not confirmed in subsequent studies. A comprehensive literature search resulted in the identification of 139 genes with a described function in root hair formation in Arabidopsis thaliana. Similarly, 5 genes that play a role in root hair formation were found in Solanum lycopersicum and another 9 root hair genes were found in monocotyledonous species, including Oryza sativa (six genes), Zea mays (two genes) and Hordeum vulgare (one gene). The genes that were found in that way were later called primary root hair genes (Supplementary Table 1). For all of the genes found, any associated significant information was collected. This comprised mainly: the full gene name, its aliases and abbreviations, its predicted protein function, a comprehensive description of the function of the gene in root hair development, mutant and/or overexpression/silencing phenotype and the gene expression pattern. Moreover, the genomic sequence of the gene, its mRNA, promoter (1000 bp upstream of the transcription start site, TSS) and encoded protein were downloaded from the GenBank database. All genes were categorized according to their function in root hair development and the phenotype of mutant or overexpression/silencing. Three main categories related to the gene function in root hair development were selected: 1) cell differentiation, 2) root hair initiation and 3) tip growth. In addition, the category unknown was proposed for genes whose function has not yet been well documented. All of the genes were classified into six categories according to the root hair phenotype of mutants caused by mutations in the gene: 1) hairless roots, 2) changes in root hair position, 3) changes in root hair shape, 4) changes in root hair number, 5) changes in root hair length and 6) branched root hairs. The category unknown was created for genes whose phenotype could not be determined or was not clearly visible (Supplementary Table 1). In addition, all bibliographic references were collected during the literature searches. Protein sequences encoded by the primary root hair genes were used for the identification of their putative orthologs in higher plant species that have sequenced genomes. The reciprocal best BLASTP hits method resulted in defining orthologous groups within the following species: Arabidopsis thaliana, Arabidopsis lyrata, Populus trichoarpa, Vitis vinifera, Ricinus communis, Sorghum bicolor, Oryza sativa, Zea mays, Brachypodium distachyon and Hordeum vulgare. For most of the primary root hair genes, the identification of orthologs in all of the above-mentioned species was possible. However, the identification of putative orthologs was more problematic for some genes and thus the orthologous group is not complete (Supplementary Table 2). The genomic sequences of each identified orthologous

gene, its mRNA, promoter region (1000 bp upstream of TSS) and encoded protein were downloaded from the GenBank or MIPS databases. In total, more than 3,450 unique sequences for all primary root hair genes and their putative orthologs were collected.

iRootHair Database In order to store all of the information collected for the primary root hair genes and their respective putative orthologs, a MySQL (Structured Query Language; MySQL 5.1 Reference Manual, http://dev.mysql.com/doc/refman/5.1/en/index.html) relational database was created. The database resources are offered externally as the Web-based service, iRootHair, which is freely accessible at www.iroothair.org. The iRootHair database hosts a large volume of available information about the genomics of root hair morphogenesis. In order to facilitate its use, the iRootHair is subdivided into interrelated, searchable main sections that describe: Genes, Processes of root hair formation, root hair Mutants and available References. The database also integrates bioinformatic Tools with a focus on sequence identification and annotation as well as useful external Links. The primary root hair genes are the pivotal objects of the iRootHair database and are accessible through the main section Genes (Fig. 2). Each primary root hair gene is described in detail (Fig. 3) and presented in the following subsections: -

Summary – basic information about the gene, including the gene identifier (Locus Tag), gene name(s) and abbreviation(s), a description of the encoded protein function, the category of its function in root hair development and a detailed description of its function in root hair development;

-

Sequences – with paragraphs: Primary source (hyperlink to NCBI/GenBank) and View sequence with links to the genomic, mRNA, protein and promoter sequences of the gene;

-

Mutants – a description of its mutant and/or overexpression/silencing phenotypes, supported by original photographs of root phenotypes or by graphical models; additionally, the paragraph Mutant resources offers a direct link to the SALK T-DNA mutant lines via the TAIR database (The Arabidopsis Information Resource; www.tair.org);

-

Expression – a description of the expression pattern of the gene and original photographs with visualized expression based on reporter genes, if available; the paragraph Expression profile also offers a direct link to the eFP Browser Tool, accessible via The Bio-Array Resource for Plant Biology (bar.utoronto.ca);

-

Orthologs – direct links to the putative orthologs of the primary root hair genes;

-

References – a list of bibliographic references that describe the function of a gene, subdivided into paragraphs of Original and Review articles; each reference is formatted according to the Plant Physiology journal style and linked directly to the PubMed database for easy access to an abstract and original papers. The second main section of the iRootHair database, Processes of root hair formation is

divided into three subsections that allow a search for and selection of genes specifically involved in the cell differentiation, root hair initiation and tip growth processes. The genes are briefly described here with an emphasis on the function of the encoded protein and its particular role in the subsequent phases of root hair development. A direct link to the primary root hair gene of interest in the section Genes allows quick access to a detailed description and additional information. The section Mutants allows a search for and subsequent assessment of genes whose mutations lead to the particular, main root hair phenotype: hairless roots, changes in root hair position, changes in shape, changes in root hair number and length or to a phenotype with branched root hairs. Although the function of a particular gene is described only briefly, a link to the detailed description in the Genes section is provided. More emphasis was given to the description of the mutant phenotype and to the direct access to the mutant’s resources in this section, particularly to the SALK mutant lines and to the bibliographic references. The References section is a collection of the available root hair development-related articles that were used as sources of information for the iRootHair database. In order to facilitate searching, this section has been divided into two subsections: the original and review articles. All references are formatted according to the Plant Physiology journal style and are linked to the PubMed database for quick access to the abstracts and full articles. The database also integrates bioinformatics Tools with a focus on sequence identification and annotation. A BLAST tool allows all DNA and/or protein sequences accessible in the database to be searched. Additionally, the iRootHair database offers a batch or individual sequence Download tool and a Search tool that can be used to identify the particular gene of interest or a broader set of information about root hair genomics. At the end of the current version of the iRootHair database, useful external Links have been provided, including hyperlinks to the Root Hair Gene Expression Database (www.roothairs.org; Bruex et al., 2012), The Arabidopsis Information Resource – TAIR (www.arabidopsis.org), the Plant Biology section at the Genevestigator platform (www.genevestigator.com/gv/plant.jsp; Zimmermann et al., 2004) and the Bio-Array Resource for Plant Biology (bar.utoronto.ca; Toufighi et al., 2005).

A case study To demonstrate the usefulness of the iRootHair service, we carried out a simple analysis based on our earlier study. We show that a global root transcriptome differentiation analysis of the root-hairless barley mutant rhl1.a and a wild-type parent variety ‘Karat’ using Affymetrix Arrays allowed the identification of 10 genes that are presumably involved in the initiation of root hair formation in barley (Kwasniewski et al., 2010). A homology analysis in a barley-rice-Arabidopsis comparison using GreenPhyl software revealed that two out of the 10 identified genes are putative orthologs of SCN1 (SUPERCENTIPEDE 1) and COW1 (CAN OF WORMS 1), respectively; these are genes that are involved in root hair morphogenesis in Arabidopsis (Böhme et al., 2004; Carol et al., 2005). Moreover, two additional genes that encoded peroxidase proteins were identified (Kwasniewski et al., 2010). In this study, we repeated the homology analysis using the same 10 genes that were identified in our previous study. A simple BLASTX analysis using barley mRNA/EST sequences against sequences within the iRootHair database (Tools section of the iRootHair service) allowed the identification of four orthologous groups representing genes/proteins related to four primary root hair genes in Arabidopsis: CAN OF WORMS 1 (COW1), SUPERCENTIPEDE 1 (SCN1), ROOT HAIR SPECIFIC 19 (RHS19) and ROOT HAIR SPECIFIC 18 (RHS18) (Table 1). These results confirmed our previous analysis with respect to COW1 and SCN1. Moreover, we have now demonstrated that two peroxidase genes identified previously are putative orthologs of RHS18 and RHS19 – two primary root hair genes encoding peroxidase proteins that were identified earlier by Won et al. (2009). Their expression in the Arabidopsis root is root hair cell-specific; both contain Root Hair-specific cis-Elements (RHEs) in their proximal promoters and the overexpression of RHS18 reduces root hair elongation by 20% compared with the control (Won et al., 2009).

DISCUSSION The presented iRootHair database is the first, unique resource for root hair research that integrates the large amount of data related to root hair genomics in a single, curated, easily accessible Web-based service. It was intended to constitute a helpful tool for organizing and sharing information about the molecular basis of root hair biology. The iRootHair database can be used as a reference database when a search for root hair-related genes is needed. In order to facilitate its use, we have categorized genes according to their function in

root hair development and the phenotype of the mutant or its overexpression/silencing lines. Therefore, the database was subdivided into main searchable sections that describe: Genes, Processes of root hair formation, root hair Mutants and available References. Moreover, a comprehensive bioinformatics analyses allowed the identification of the putative orthologs of the primary root hair genes. The genomic, mRNA or protein sequences of primary root hair genes and their putative orthologs can be used as records for BLASTbased analyses. We demonstrated that a simple BLAST search against the database records with genes found in microarray experiments on the non-model crop species, Hordeum vulgare (Kwasniewski et al., 2010), allowed the rapid identification of the orthologous groups of primary root hair genes in other species. The orthologous groups provided for the primary root hair genes in the iRootHair database helped to determine the evolutionary association of two peroxidase genes that were identified in our previous study with the root hair peroxidase genes RHS18 and RHS19, which were identified in Arabidopsis by Won et al. (2009). The expression of both root hair-related peroxidase genes identified in Arabidopsis is root hairspecific and both contain Root Hair-specific cis-Elements (RHEs) in their proximal promoters that drive the root-hair specific expression of many root hair formation-related genes (Kim et al., 2006; Won et al., 2009). In our recent study, we demonstrated that the expression of both peroxidase genes identified in barley – as shown here the putative orthologs of RHS18 and RHS19 – is correlated with the initiation of root hair formation and is root hair-specific, and that both contain RHEs conserved within their orthologs in monocotyledonous species in their promoters (Kwasniewski et al., 2012, in press). Therefore, the iRootHair resources can be used not only for the identification of primary root hair genes and their orthologous groups, but also for secondary studies like comparative promoter analyses for the identification of conserved cis-acting elements that can act as a putative functional regulatory sequences (Balazadeh et al., 2011). A similar BLAST-based approach can be very useful in other large-scale transcriptome or proteome analyses related to root hairs. In the studies of Brechenmacher et al. (2009), Nestler et al. (2011) and Janiak et al. (2012), a global analysis of root hair proteome was applied in order to identify the proteins/genes which are presumably involved in root hair formation, or to characterize protein features which are conserved and typical among different plant species. The iRootHair database, with the implemented BLAST tool, can be used in analogous studies as a reference database for screening for known root hair formation-related proteins/genes. All records returned from a BLAST search are linked with the main pages of the primary root hair genes; therefore, a simple BLAST search against

iRootHair records allows quick access to comprehensive information about the identified primary root hair genes. This, among other things, includes the description of mutant and/or overexpression or gene silencing phenotypes and is supported by original photographs of root phenotypes or by graphical models. Additionally, the paragraph Mutant resources provides a direct link to the TAIR database and later to the SALK T-DNA mutant lines, where suitable seeds can be ordered. Also, in the Mutants section, we provided an appropriate reference that should simplify the eventual contact with the research group that was, or is still, working on the presented mutant. Similarly, whenever possible, in the Expression section, we provided pictures and references for the plant lines which were used for the analysis of the gene expression profile. The number of bibliographic references for each gene is significantly extended in the Reference section, which is subdivided into paragraphs of Original and Review articles. This should greatly simplify the process of literature reviewing and collecting information about the gene of interest. The iRootHair database can be useful in basic studies on root hair morphogenesis and root hair system biology as a comprehensive source of curated information and sequences. Moreover, recent trends in plant science that look forward to translational genomics suggest the potential usefulness of root hairs as a tool for crop improvement. The presented work was supported by the EU-FP7 initiative "EURoot: Enhancing resource Uptake from Roots under stress in cereal crops"; we are convinced that the iRootHair database can be useful in studies that explore the role of root hairs in the field and link basic information about root hair genomics, plant physiology, viability and yield (Brown et al., 2012).

MATERIALS AND METHODS Database content All of the available information about genes involved in root hair morphogenesis was collected through a comprehensive literature search. The PubMed database was searched for relevant publications using combination of two sets of keywords: "root hair"[All Fields] or "root hairs"[All Fields]. The literature was reviewed in order to specifically find information about genes with a described function in root hair development. The main criteria were the mutant phenotype and/or root hair-related expression pattern. When the specific gene was found, associated information about the gene name, its aliases, protein function, the function of the gene in root hair development, mutant phenotype and expression pattern were collected.

Additionally, the genomic sequence of the gene, its mRNA, promoter (1000 bp upstream of the Transcription Start Site, TSS) and encoded protein were downloaded from the NCBI (National Center for Biotechnology Information) GenBank database (www.ncbi.nlm.nih.gov). The genes found in this way were later called the primary root hair genes. In order to identify orthologs of the primary root hair genes in the sequenced genomes of higher plants, the reciprocal best BLASTP hits method was applied. As a source of protein sequences, the NCBI GenBank and the MIPS (Munich Information Center for Protein Sequence Database; http://mips.helmholtz-muenchen.de/plant/genomes.jsp) databases were used. Additionally, as a cross-check reference and support for the analysis, the information about putative orthologs described in the NCBI UniGene database was used. The analysis resulted in the identification of orthologous groups of the primary root hair genes in the following species: Arabidopsis thaliana, Arabidopsis lyrata, Populus trichoarpa, Vitis Vinifera, Ricinus communis, Sorghum bicolor, Oryza sativa, Zea mays, Brachypodium distachyon and Hordeum vulgare. The genomic sequences of each orthologous gene, its mRNA, promoter (1000 bp upstream of TSS) and encoded protein were downloaded from the GenBank or MIPS databases.

Database and the iRootHair service construction All information collected for the primary root hair genes and their respective putative orthologs have been entered into the database and offered externally as the iRootHair service. The iRootHair website (www.iroothair.org) was constructed using the Hypertext Markup Language (HTML), Hypertext Preprocessor (http://php.net/) for retrieving database entries, jQuery

(http://jquery.com/)

for

dynamic

styling

and

navigation

and

C++

(http://www.cplusplus.com/) for BLAST output parsing and locusTag linking. The database content is served through the Apache 2.2.17 server. The MySQL 5.0 was used for the database engine (http://dev.mysql.com/). The entire service runs on a server managed by the Fedora operating system.

ACKNOWLEDGEMENTS We thank Agnieszka Janiak for the revision of the database and helpful comments and Paweł Sega who helped to create the graphical models of the root hair mutants. We are grateful to the root hair scientific community for providing their unpublished pictures of root hair mutants, gene expression profiles and helpful comments. We would especially like to

thank: Tore Brembu, Atle M. Bones, Hyung-Taeg Cho, Tokitaka Oyama, Takashi Hirayama, Christoph Ringli, John Schiefelbein, Lukas Synek and Viktor Žárský.

LITERATURE CITED Balazadeh S, Kwasniewski M, Caldana C, Mehrnia M, Zanor MI, Xue GP, MuellerRoeber B (2011) ORS1, an H₂O₂-responsive NAC transcription factor, controls senescence in Arabidopsis thaliana. Mol Plant 4: 346-60 Böhme K, Li Y, Charlot F, Grierson C, Marrocco K, Okada K, Laloue M, Nogué F (2004) The Arabidopsis COW1 gene encodes a phosphatidylinositol transfer protein essential for root hair tip growth. Plant J 40: 686-98 Brechenmacher L, Lee J, Sachdev S, Song Z, Nguyen TH, Joshi T, Oehrle N, Libault M, Mooney B, Xu D, Cooper B, Stacey G (2009) Establishment of a protein reference map for soybean root hair cells. Plant Physiol 149: 670–682 Brown LK, George TS, Thompson JA, Wright G, Lyon J, Dupuy L, Hubbard SF, White PJ (2012) What are the implications of variation in root hair length on tolerance to phosphorus deficiency in combination with water stress in barley (Hordeum vulgare)? Ann Bot 110: 319-28 Bruex A, Kainkaryam RM, Wieckowski Y, Kang YH, Bernhardt C, Xia Y, Zheng X, Wang JY, Lee MM, Benfey P, Woolf PJ, Schiefelbein J (2012) A gene regulatory network for root epidermis cell differentiation in Arabidopsis. PLoS Genet 8: e1002446 Carol RJ, Takeda S, Linstead P, Durrant MC, Kakesova H, Derbyshire P, Drea S, Zarsky V, Dolan L (2005) A RhoGDP dissociation inhibitor spatially regulates growth in root hair cells. Nature 438: 1013-6 Cutter EG. The epidermis. In Plant Anatomy. London: Clowes & Sons, 1978. Gahoonia TS, Care D, Nielsen NE (1997) Root hairs and phosphorus acquisition of wheat and barley cultivars. Plant and Soil 191: 181-188 Jamieson T (1893) On root hairs. Science 22: 354-6 Janiak A, Piórko S, Matros A, Mock H-P, Kwasniewski M, Chwialkowska K, Chmielewska B, Szarejko I (2012) A comparative analysis of proteins that accumulate during the initial stage of root hair development in barley root hair mutants and their parent varieties. Journal of Applied Genetics DOI: 10.1007/s13353-012-0105-1

Jones MA, Raymond MJ, Smirnoff N (2006) Analysis of the root-hair morphogenesis transcriptome reveals the molecular identity of six genes with roles in root-hair development in Arabidopsis. Plant J 45: 83-100 Kim DW, Lee SH, Choi SB, Won SK, Heo YK, Cho M, Park YI, Cho HT (2006) Functional conservation of a root hair cell-specific cis-element in angiosperms with different root hair distribution patterns. Plant Cell 18: 2958-70 Kwasniewski M, Chwialkowska K, Kwasniewska J, Kusak J, Siwinski K, Szarejko I (2012) Accumulation of peroxidase-related reactive oxygen species in trichoblasts correlates with root hair initiation in barley. J Plant Physiol: in press Kwasniewski M, Janiak A, Mueller-Roeber B, Szarejko I (2010) Global analysis of the root hair morphogenesis transcriptome reveals new candidate genes involved in root hair formation in barley. J Plant Physiol 167: 1076-1083 Libault M, Brechenmacher L, Cheng J, Xu D, Stacey G (2010) Root hair systems biology. Trends Plant Sci 15: 641-50 Mylona P, Pawlowski K, Bisseling T (1995) Symbiotic Nitrogen Fixation. Plant Cell 7: 869885 Nestler J, Schütz W, Hochholdinger F (2011) Conserved and unique features of the maize (Zea mays L.) root hair proteome. J Proteome Res 10: 2525-37 Parker JS, Cavell AC, Dolan L, Roberts K, Grierson CS (2000) Genetic interactions during root hair morphogenesis in Arabidopsis. Plant Cell 12: 1961-74 Schiefelbein JW (2000) Constructing a plant cell. The genetic control of root hair development. Plant Physiology 124: 1525-1531 Schiefelbein JW, Somerville C (1990) Genetic control of root hair development in Arabidopsis thaliana. The Plant Cell 2: 235-243 Won SK, Lee YJ, Lee HY, Heo YK, Cho M, Cho HT (2009) Cis-element- and transcriptome-based screening of root hair-specific genes and their functional characterization in Arabidopsis. Plant Physiol 150: 1459-73

FIGURE LEGENDS Fig. 1. The number of new root hair-related articles published yearly since 1990. The best-fit linear trend is indicated as a dashed line. Fig. 2. The schematic structure of the iRootHair database along with the content of main sections. Fig. 3. A print screen of an example of a gene description (here ROP4; AT1G75840) in the iRootHair database. The subsections of the detailed gene description are divided by green bars.

TABLES Table 1. The results of a BLASTX analysis of four genes previously identified by global transcriptome differentiation of the root hairless mutant rhl1.a vs. wild-type Karat variety in barley (Kwasniewski et al., 2010) against the iRootHair database.

Contig12866_at

JQ649323 NIASHv2125H19

NIASHv1138L11

NIASHv2031A18

Barley mRNA

HR01N23u_at

Contig13621_s_at

HW09I06u_at

Barley AffyChip ProbeID

Species

LocusTag

Description

Score

E-value

B. distachyon O. sativa S. bicolor Z. mays R. communis P. trichocarpa V. vinifera A. lyrata A. thaliana O. sativa S. bicolor B. distachyon Z. mays R. communis P. trichocarpa A. lyrata V. vinifera A. thaliana O. sativa S. bicolor S. bicolor V. vinifera B. distachyon A. lyrata Z. mays P. trichocarpa R. communis O. sativa A. thaliana O. sativa S. bicolor O. sativa R. communis A. lyrata P. trichocarpa V. vinifera A. lyrata A. thaliana

BRADI3G11140 OS10G0122600 SORBIDRAFT_04G021100 LOC100274086 RCOM_0534520 POPTRDRAFT_201228 LOC100245225 ARALYDRAFT_656539 AT4G34580 OS02G0719000 SORBIDRAFT_04G029740 BRADI3G53540 LOC100285962 RCOM_0549620 POPTRDRAFT_246928 ARALYDRAFT_896959 LOC100262024 AT3G07880 OS03G0121300 SORBIDRAFT_03G004380 SORBIDRAFT_01G007230 LOC100266458 BRADI1G07780 ARALYDRAFT_489097 LOC100382459 POPTRDRAFT_553951 RCOM_0578390 OS07G0115300 AT5G67400 OS03G0121300 SORBIDRAFT_03G004380 OS07G0115300 RCOM_1502930 ARALYDRAFT_919965 POPTRDRAFT_901246 LOC100266458 ARALYDRAFT_489097 AT5G22410

Putative ortholog of AT4G34580 CAN OF WORMS1 (COW1) Putative ortholog of AT4G34580 CAN OF WORMS1 (COW1) Putative ortholog of AT4G34580 CAN OF WORMS1 (COW1) Putative ortholog of AT4G34580 CAN OF WORMS1 (COW1) Putative ortholog of AT4G34580 CAN OF WORMS1 (COW1) Putative ortholog of AT4G34580 CAN OF WORMS1 (COW1) Putative ortholog of AT4G34580 CAN OF WORMS1 (COW1) Putative ortholog of AT4G34580 CAN OF WORMS1 (COW1) CAN OF WORMS1 (COW1) Putative ortholog of AT3G07880 SUPERCENTIPEDE1 (SCN1) Putative ortholog of AT3G07880 SUPERCENTIPEDE1 (SCN1) Putative ortholog of AT3G07880 SUPERCENTIPEDE1 (SCN1) Putative ortholog of AT3G07880 SUPERCENTIPEDE1 (SCN1) Putative ortholog of AT3G07880 SUPERCENTIPEDE1 (SCN1) Putative ortholog of AT3G07880 SUPERCENTIPEDE1 (SCN1) Putative ortholog of AT3G07880 SUPERCENTIPEDE1 (SCN1) Putative ortholog of AT3G07880 SUPERCENTIPEDE1 (SCN1) SUPERCENTIPEDE1 (SCN1) Putative ortholog of AT5G22410 ROOT HAIR SPECIFIC 18 (RHS18) Putative ortholog of AT5G22410 ROOT HAIR SPECIFIC 18 (RHS18) Putative ortholog of AT5G67400 ROOT HAIR SPECIFIC 19 (RHS19) Putative ortholog of AT5G67400 ROOT HAIR SPECIFIC 19 (RHS19) Putative ortholog of AT5G67400 ROOT HAIR SPECIFIC 19 (RHS19) Putative ortholog of AT5G22410 ROOT HAIR SPECIFIC 18 (RHS18) Putative ortholog of AT5G67400 ROOT HAIR SPECIFIC 19 (RHS19) Putative ortholog of AT5G67400 ROOT HAIR SPECIFIC 19 (RHS19) Putative ortholog of AT5G67400 ROOT HAIR SPECIFIC 19 (RHS19) Putative ortholog of AT5G67400 ROOT HAIR SPECIFIC 19 (RHS19) ROOT HAIR SPECIFIC 19 (RHS19) Putative ortholog of AT5G22410 ROOT HAIR SPECIFIC 18 (RHS18) Putative ortholog of AT5G22410 ROOT HAIR SPECIFIC 18 (RHS18) Putative ortholog of AT5G67400 ROOT HAIR SPECIFIC 19 (RHS19) Putative ortholog of AT5G22410 ROOT HAIR SPECIFIC 18 (RHS18) Putative ortholog of AT5G67400 ROOT HAIR SPECIFIC 19 (RHS19) Putative ortholog of AT5G22410 ROOT HAIR SPECIFIC 18 (RHS18) Putative ortholog of AT5G67400 ROOT HAIR SPECIFIC 19 (RHS19) Putative ortholog of AT5G22410 ROOT HAIR SPECIFIC 18 (RHS18) ROOT HAIR SPECIFIC 18 (RHS18)

907 850 822 812 551 540 539 513 511 347 347 344 343 291 290 289 288 286 243 190 188 182 180 179 176 174 173 169 167 193 191 177 174 172 170 169 168 167

0.0 0.0 0.0 0.0 4e-158 1e-154 2e-154 9e-147 3e-146 6e-97 6e-97 4e-96 8e-96 4e-80 8e-80 2e-79 3e-79 1e-78 2e-65 2e-49 5e-49 3e-47 1e-46 3e-46 3e-45 1e-44 2e-44 2e-43 1e-42 1e-50 6e-50 8e-46 7e-45 4e-44 1e-43 2e-43 5e-43 7e-43

SUPPLEMENTAL DATA FILES Supplementary Table 1. The list of the primary root hair genes described in the iRootHair database. Categories of the function in root hair development and mutant phenotypes are indicated instead of full descriptions. Also, only a single, main bibliographic reference is indicated.

Supplementary Table. 2.1. Primary root hair genes in Arabidopsis thaliana and their putative orthologs in sequenced plant species. Supplementary Table. 2.2. Primary root hair genes in Oryza sativa and their putative orthologs in sequenced plant species Supplementary Table. 2.3. Primary root hair genes in Zea mays and their putative orthologs in sequenced plant species. Supplementary Table. 2.4. Primary root hair gene in Hordeum vulgare and its putative orthologs in sequenced plant species. Supplementary Table. 2.5. Primary root hair gene in Solanum lycopersicum and its putative orthologs in sequenced plant species.