Abstract

The expression pattern of genes can identify the cells in which the respective proteins are active during development. As a step towards defining the genetic network that controls the development of roots, a high-throughput method of whole-mount in situ hybridization has been developed that does not require expensive equipment and allows the definition of the expression patterns of 137 transcription factor genes in young developing roots. Of the 137 transcription factors, 81.8% were expressed in the root while 18.2% showed no detectable expression. In all three proximal distal zones (meristem, elongation, and differentiation) of the root, 52.6% were expressed whereas 21.2% were expressed in only two zones. Eight percent of the genes were expressed in a single proximal distal zone. Cell-specific gene expression patterns were also detected. This rapid approach identified potential key regulators of cell differentiation and provides important spatial information for the expression patterns of a large number of transcriptional regulators that function during root development.

Introduction

Methods that precisely define gene expression patterns, such as in situ hybridization and enhancer trapping, are used to identify genes that control precise stages of development in plants and animals (Tomancak et al., 2002; Costa and Dolan, 2003; Imai et al., 2004; Drea et al., 2005a; Kurup et al., 2005; Laplaze et al., 2005). For example, in situ hybridization has been used to identify genes that are expressed in different regions of the developing Drosophila nervous system, and subsequent functional characterization revealed that a subset of these genes were required for neurogenesis (Reeves and Posakony, 2005). Here, a protocol that enables the high-throughput analysis of the expression of a large number of genes in Arabidopsis roots using in situ hybridization is described.

To illustrate the utility of this approach, the distribution of transcripts from genes encoding transcription factors in seedling roots of Arabidopsis was characterized. Transcription factors were chosen as a test set because they are key regulators of development in all domains of life. The spatial control of transcription factor expression is an important component of the formation of the body plan in animals (Mann and Morata, 2000; Hsia and McGinnis, 2003). The sequential spatial-temporal regulation of transcription factors is also critical for the establishment of stem cell populations in plant meristems, and the subsequent tissue patterning that gives rise to the mature organs of the plant (Christensen and Weigel, 1998; Willemsen and Scheres, 2004; Kieffer et al., 2006). Given the important role of spatial regulation in transcription factor function, patterns of transcription factor gene expression is one criterion by which potential regulators of development can be identified. Here, the intention was to test the feasibility of determining the expression patterns of many transcription factors using high-throughput mRNA in situ hybridization to identify transcription factor-encoding genes that are expressed in the different developmental zones of the root, with a view to finding new candidate regulators that control the primary development of Arabidopsis roots.

The Arabidopsis primary root or radicle is laid down during embryogenesis from groups of cells at the basal end of the developing embryo (Scheres et al., 1994). Upon germination, cells in the primary root expand and cell division is initiated in the meristem, resulting in the penetration of the seed coat. The root comprises a distal root cap which ensheaths the root tip. The columella is the central part of the root cap, and the lateral root cap runs along the side of the root. The root cap itself surrounds the dividing cells of the root tip, the meristem, which comprises a number of different groups of cells. Here lie distinct groups of initials, each of which gives rise to a discrete group of cells in the mature root (Dolan et al., 1993). For example, the ground tissue initials give rise to the endodermis and cortex. Initials surround a group of four slowly dividing cells, the central cells of the quiescent centre, which laser ablation experiments have shown to be the source of signals controlling the activity of the adjacent initial cells (Vandenberg et al., 1995, 1997) and clonal analysis has shown can divide to produce cells in any tissue of the root (Kidner et al., 2000). This suggests that the quiescent centre cells may be stem cells for the entire root, while initials are stem cells for distinct tissue types. Cells that are produced by the quiescent centre and initials divide rapidly and constitute the majority of cells in the meristem, and it is through these divisions that the body of the root is built up. After division has ceased these cells continue to grow in the direction of the long axis of the root, before going through a phase of relatively rapid cell expansion. Once elongation stops, cells undergo the final stages of differentiation which completes the primary development of the root (Dolan et al., 1993). Later, after primary development is complete, new meristems are initiated in the pericycle to emerge as lateral roots, and a cambium develops between the xylem and phloem, giving rise to secondary vascular tissue of the root (Dolan and Roberts, 1995).

The expression patterns of 137 genes from six families of transcription factors in roots were examined. The C2C2-Dof (Dofs) family comprises 37 members. Some Dofs are involved in seed development and germination, and light, phytohormone, and defence responses (Yanagisawa, 2002), with a single member implicated in root development (Kang and Singh, 2000). Aux/IAA proteins are transcription factors that repress auxin responses, and plants harbouring gain-of-function mutations in Aux/IAA genes exhibit a range of developmental defects in both root and shoot systems (Liscum and Reed, 2002). HMG (high mobility group) proteins are small (13–20 kDa) chromatin-associated proteins that regulate transcription by controlling the formation and architecture of nucleoprotein complexes (Grasser, 2003). There are 12 HMG genes in Arabidopsis (Grasser, 2003) and no HMG mutant phenotypes have been described in this species. NFY transcription factors form a heteromeric complex that binds to CCAAT box sequences in target promoters that are present in many eukaryotic genes (Johnson and McKnight, 1989; Mitchell and Tjian, 1989). One member, LEAFY COTYLEDON1 (LFY1), is involved in embryo and seed development (Meinke et al., 1994; West et al., 1994; Lotan et al., 1998; Kwong et al., 2003), but the function of the remaining members remains unknown. heat shock factors (HSFs) bind to conserved sequence elements in promoters of heat stress-inducible genes of all eukaryotes and regulate the transcription of genes in response to a variety of stresses, including both heat stress and chemical stress (Bienz and Pelham, 1987; Nover and Scharf, 1997). At least 23 HSF genes are present in Arabidopsis in three classes (Nover et al., 2001) and no developmental role has been described for these genes to date. The squamosa-binding proteins (SBPs) were originally identified in Antirrhinum majus as transcription factors that bind to the promoter of the floral meristem identity gene SQUAMOSA (Klein et al., 1996). There are 16 members of this family in Arabidopsis and these proteins have a variety of roles in plant development, including floral transition (Cardon et al., 1997), plant architecture (Stone et al., 2005), and pollen sac development (Unte et al., 2003).

The results of the present analysis show that transcripts for the majority of the root-expressed genes are present throughout the root tip but that a smaller yet notable number of genes are expressed in discrete cell type-specific patterns. This protocol can be used to determine the mRNA expression pattern of any number of genes in the root, and the information reported here identifies potential regulators of root development.

Materials and methods

Source of probes and probe preparation

DNA templates for each family of genes were kindly provided by the REGIA (REgulatory Gene Initiative in Arabidopsis) consortium in vectors pGEM, pCR2, or pBluescript. Templates not available from REGIA were obtained from ABRC (Arabidopsis Biological Resource Center) in vectors pUNI, pBluescript, or PZL, where available, and amplified by PCR with the appropriate primers. PCR product purification was done using a QIAquick PCR purification kit (QIAGEN) according to the manufacturer's instructions. In vitro transcription, probe hydrolysis, precipitation, dot-blot analysis, and dilution/storage in hybridization solution were performed as described previously (Drea et al., 2005b).

Plant growth

Seeds of Arabidopsis thaliana L. Heynh, ecotype Columbia-0 (Col-0) were sterilized by immersion in 5% (v/v) Vortex bleach (Procter & Gamble Ltd) (containing 5–15% chlorine-based bleach) for 5 min, and washed three times in sterile distilled water. Seeds were then suspended in sterile distilled water, aspirated into plastic pipettes, and dropped individually onto the surface of the growing medium in horizontal lines at a density of 5–10 seeds per centimetre. Plates were then sealed with Parafilm® laboratory film (Pechiney Plastic Packaging, Menasha, WI, USA). Standard growth medium contained 1× Murashige and Skoog (MS) basal salts (micro- and macro-elements) (Duchefa), 1% (w/v) sucrose, and 0.5% (w/v) Phytagel. These were dissolved in deionized water and the pH adjusted to 5.7 with KOH, followed by autoclaving for 20 min. The medium was cooled to 50–60 °C and ∼20 ml poured into a 9 cm Petri plate (Bibby Sterilin Ltd) and allowed to solidify. Plates were placed in darkness at 4 °C for 48 h to stimulate and synchronize germination. Following cold treatment, plates were transferred to a growth room maintained at 25 °C and incubated in a near vertical position, under fluorescent lamps emitting ∼70 μmol m−2 s−1 in a continuous white light regime.

Whole-mount in situ hybridization

Whole-mount in situ hybridization was performed on 4-d-old seedlings previously fixed in 4% (w/v) paraformaldehyde. Handling of material was aided by collecting seedlings (40–50) into Tissue-Tek® mesh biopsy cassettes (Sakura) followed by brief vacuum infiltration with each change of the following solutions to submerge the cassettes. Material was dehydrated for 1 h each in 30, 65, and 100% (v/v) ethanol, followed by partial rehydration for 1 h each in 65% and 30% (v/v) ethanol. Seedlings were pre-washed at room temperature in 1× PBS for 30 min, 5% (v/v) acetic anhydride in 0.1 M triethanolamine (pH 8) for 30 min, and twice in 1× PBS for 15 min each. Seedlings (10–12) were transferred with a tweezers from cassettes into 1.5 ml microfuge tubes containing 100 μl probe-hybridization solution and incubated at 50 °C for 16 h. Following the hybridization reaction, seedlings were transferred with tweezers into a 48-well mesh-bottom plate (one reaction per well) (Hölle & Hüttner AG, Germany), covered with a lid, and the plate placed into a transparent plastic box, which was 100 mm (w)×200 mm (l)×50 mm (h) with a lid, and containing 100 ml of appropriate washing solution. Material was subjected to three washes in 2× SSC/50% (v/v) formamide and one wash in 1× SSC/50% (v/v) formamide, 30 min each at 50 °C, 1× SSC for 5 min and 1× PBS for 10 min at room temperature. Material was prepared for antibody labelling by washing in Buffer-1 (100 mM TRIS-HCl, 150 mM NaCl, pH 7.4) for 10 min, Buffer-1+0.5% (w/v) blocking reagent (Boehringer Mannheim GmbH, Germany) for 1 h, and Buffer-1+1% (w/v) BSA/0.3% (v/v) Triton X-100 for 1 h. Anti-digoxigenin alkaline phosphatase (Roche) was diluted (1:3000) in the latter buffer and used for seedling incubation at room temperature (1 h) followed by 4 °C for 16 h. Seedlings were given 3× 20 min washes at room temperature in the same buffer with the antibody absent, followed by one wash in Buffer-1 for 20 min, and one wash in Buffer-2 (100 mM TRIS-HCl, 100 mM NaCl, 50 mM MgCl2, pH 9.5) for 10 min. Seedlings were transferred into Nunclon six-well plates (Nalge Nunc International) containing 2 ml of the following solution. Colour detection was carried out by incubating seedlings in Buffer-2 containing NBT (0.15 mg ml−1)/BCIP (0.075 mg ml−1) (Roche) in complete darkness for 2–4 h and then stopped in water. Expression levels were defined by visual inspection broadly separated into four categories; absent (–), weak (+), moderate (++), and strong (+++). The meristematic zone was defined as being up to ∼200 μm from the tip of the root, the elongation zone from ∼200 μm to the point where root hair initiation started (usually ∼700 μm), and the differentiation zone ∼700–1200 μm from the tip of the root. As in a previous analysis in wheat (Drea et al., 2005b), a subset of positive (Fig. 1) and negative (Fig. S7 in Supplementary data available at JXB online) controls was included in each experiment, in addition to a hybridization with no probe.

Fig. 1.

The Arabidopsis primary root and probe controls: (A) Col-0 wild-type root showing developmental zones; (B) histone H4; (C) CesA3; (D) SCR1; (E) SCR1 transverse section; (F) histone H4 transverse section. Scale bars: A=130 μm, A–D same scale; F=25 μm, E and F same scale. Note: in (D) the roots were washed in ethanol after signal development.

Fig. 1.

The Arabidopsis primary root and probe controls: (A) Col-0 wild-type root showing developmental zones; (B) histone H4; (C) CesA3; (D) SCR1; (E) SCR1 transverse section; (F) histone H4 transverse section. Scale bars: A=130 μm, A–D same scale; F=25 μm, E and F same scale. Note: in (D) the roots were washed in ethanol after signal development.

Imaging of roots

Roots were imaged using a Nikon Coolpix 950 digital camera attached to a Leica WILD M10 binocular microscope. White light from above and white paper underneath the plates improved the signal contrast. Representative images were adjusted using Adobe Photoshop CS to normalize any variation in colour development between experiments.

Embedding, sectioning, and microscopy

Seedlings were embedded in Technovit 7100® (Kulzer GmbH, Germany) resin according to the manufacturer's instructions and 12 μm transverse sections taken from roots. Sections were imaged using a Nikon Coolpix 950 digital camera attached to a Nikon Eclipse E600 microscope in DIC mode.

Data analysis

The 137 genes analysed by whole-mount in situ hybridization were also screened for the presence or absence of transcripts using MPSS (Massively Parallel Signature Sequencing; http://mpss.udel.edu/at/) using nearest comparable tissue, which was 21-d-old roots (ROF/ROS). Microarray data collected by Birnbaum et al. (2003) are available at Genevestigator (https://www.genevestigator.ethz.ch/). This resource was also used to compare gene expression patterns in 3-d-old primary roots by selecting developmental stage 1.00, anatomy 5 (root), which selected 11 chips (22k array). The scale base was set at minimum signal and scale type as linear. Co-expression analysis was performed using PRIMe: Coexpression Gene Search at RIKEN (http://prime.psc.riken.jp/) with the following: Matrix, Tissue and development v.1; Method, union of sets; and Display limit, 20.

Results

The root comprises a proximal–distal axis of developmental zones

The root is organized into proximal–distal zones of development: meristematic, elongation, and differentiation zones (Fig. 1A). Using a number of gene-specific RNA antisense probes for genes that are known to be expressed in different zones of the root, it is shown here that the whole-mount technique reliably determines the diverse patterns of gene expression. For example, histone H4 is transcribed during the S-phase of the cell cycle (Fobert et al., 1996) and is expressed in the meristem and the early (slow) elongation zone where cells are undergoing endoreduplication (Fig. 1B). The CesA3 gene which encodes a cellulose synthase-related protein (Holland et al., 2000) is specifically transcribed in the elongation zone (Fig. 1C).

Because in situ hybridization is being used on whole-mount roots, it is important to demonstrate that the probe can penetrate to the innermost tissues of the organ. To show this, in situ hybridization was carried out using the SCARECROW (SCR) probe (Fig. 1D, E), a transcription factor previously shown to be expressed in the endodermis (DiLaurenzio et al., 1996). Using the in situ protocol, SCR transcript was detected in cells of the quiescent centre and the endodermis, indicating that the probe can access the internal cells of the root and accurately localize transcripts. The expression pattern of histone H4 also demonstrated that the probe could penetrate the innermost regions of the root. A transverse section through a root after in situ hybridization with the histone H4 probe shows that cells at the centre of the root express this gene (Fig. 1F). These results show that this protocol allows detection of gene expression in the different regions of the root and indicates that these patterns are unlikely to be artefacts resulting from differences in probe penetration through the root tissues.

High-throughput in situ hybridization defines proximal–distal patterns of gene expression

To identify transcription factors that are active at the different stages of root development, a whole-mount in situ hybridization experiment was carried out to determine where transcription factor genes were expressed. Using RNA antisense probes and a subset of controls, 137 transcription factor genes comprising almost all members of six families were analysed (Table 1). These include 33 of the C2C2-Dof gene family, 29 Aux/IAAs, 10 HMGs, 31 NFYs, 19 HSFs, and 15 SBPs. Representative images illustrating the major classes of expression patterns that were observed are shown in Fig. 2. HMG At4g23800 expression is confined to the basal part (∼150 μm) of the root tip in a spotty pattern (Fig. 2A). C2C2-Dof At5g62940 is also expressed in this zone but, by contrast to the HMG, is expressed throughout the basal part of the root, with the signal fading ∼300 μm back from the tip into the elongation zone (Fig. 2B). HSF At4g36990 is expressed in the elongation/differentiation zone (Fig. 2C) whereas C2C2-Dof At4g21050 is expressed only in the differentiation zone but in a striped pattern (Fig. 2D). Many of the genes analysed were expressed in all developmental zones of the root tip, such as C2C2-Dof At2g34140 that is strongly expressed in all three zones (Fig. 2E). By contrast, few genes displayed subepidermal expression using this approach, such as C2C2-Dof At5g60200 that is expressed in the meristem and early elongation zone but is specific to the pericycle (Fig. 2F). Whole-mount in situ hybridization of roots therefore allowed a broad range of expression patterns to be detected across the six transcription factor gene families studied. The complete set of in situ hybridization patterns for each gene family and level of expression are described in Tables 2–734567 (see Figs S1S6 in Supplementary data available at JXB online).

Table 1.

Subset of transcription factor genes in Arabidopsis thaliana

Gene family Total no. of genes No. of genes in the REGIA collection No. of genes whose cDNA was amplified by PCR No. of genes analysed in the present study No. of genes not analysed in the present study 
C2C2-Dof 37a 29 33 
Aux/IAA 30b 26 29 
HMG 12c 10 
NFY 34d 30 31 
HSF 23e 11 19 
SBP 16f 15 
Total 152 107 30 137 15 
Gene family Total no. of genes No. of genes in the REGIA collection No. of genes whose cDNA was amplified by PCR No. of genes analysed in the present study No. of genes not analysed in the present study 
C2C2-Dof 37a 29 33 
Aux/IAA 30b 26 29 
HMG 12c 10 
NFY 34d 30 31 
HSF 23e 11 19 
SBP 16f 15 
Total 152 107 30 137 15 
Table 2.

C2C2-Dof gene expression patterns

Gene Meristematic zone Elongation zone Differentiation zone 
At1g07640 ++ 
At1g21340 –/+ 
At1g26790 +/++ 
At1g28310 +/++ – 
At1g47655 –/+ – 
At1g51700 – –/+ – 
At1g64620 ++ ++/+++ ++ 
At1g69570 – – – 
At2g28510 ++/+++ +++ ++/+++ 
At2g28810 +/++ +/++ +/++ 
At2g34140 +++ +++ +++ 
At2g37590 ++ ++ – 
At2g46590 – ++/+++st ++/+++st 
At3g21270 – ++ ++ 
At3g45610 +/++pe +/++pe –/+ 
At3g47500 
At3g50410 +/++ –/+ 
At3g52440 +/++ 
At3g55370 –/+ +/++ –/+ 
At3g61850 – ++ – 
At4g00940 ++ –/+ 
At4g21040 –/+ 
At4g21050 – – ++/+++st 
At4g21080 – – 
At4g24060 – 
At4g38000 ++ +++ ++ 
At5g02460 +/++ ++ –/+ 
At5g39660 ++ 
At5g60200 ++/+++pe ++/+++pe – 
At5g60850 – – – 
At5g62430 –/+ +/++ –/+ 
At5g62940 ++/+++ ++/+++fa – 
At5g65590 – 
Gene Meristematic zone Elongation zone Differentiation zone 
At1g07640 ++ 
At1g21340 –/+ 
At1g26790 +/++ 
At1g28310 +/++ – 
At1g47655 –/+ – 
At1g51700 – –/+ – 
At1g64620 ++ ++/+++ ++ 
At1g69570 – – – 
At2g28510 ++/+++ +++ ++/+++ 
At2g28810 +/++ +/++ +/++ 
At2g34140 +++ +++ +++ 
At2g37590 ++ ++ – 
At2g46590 – ++/+++st ++/+++st 
At3g21270 – ++ ++ 
At3g45610 +/++pe +/++pe –/+ 
At3g47500 
At3g50410 +/++ –/+ 
At3g52440 +/++ 
At3g55370 –/+ +/++ –/+ 
At3g61850 – ++ – 
At4g00940 ++ –/+ 
At4g21040 –/+ 
At4g21050 – – ++/+++st 
At4g21080 – – 
At4g24060 – 
At4g38000 ++ +++ ++ 
At5g02460 +/++ ++ –/+ 
At5g39660 ++ 
At5g60200 ++/+++pe ++/+++pe – 
At5g60850 – – – 
At5g62430 –/+ +/++ –/+ 
At5g62940 ++/+++ ++/+++fa – 
At5g65590 – 

–, Absent; +, weak; ++, moderate; +++, strong; fa, fading in strength across zone; pe, pericycle layer; st, striped pattern.

Table 3.

Aux/IAA gene expression patterns

Gene Meristematic zone Elongation zone Differentiation zone 
At1g04100 – – – 
At1g04240 +/++ 
At1g04250 –/+ +/++ –/+ 
At1g04550 – ++/+++ ++/+++ 
At1g15050 – ++ ++ 
At1g15580 – – – 
At1g51950 – +/++ +/++ 
At1g52830 – – – 
At1g80390 – +/++ +/++ 
At2g01200 – 
At2g22670 – – – 
At2g33310 – – – 
At2g46990 – 
At3g04730 – – – 
At3g15540 – ++ ++ 
At3g16500 ++ ++/+++ +/++ 
At3g17600 – – – 
At3g23030 –/+ –/+ 
At3g23050 – – – 
At3g62100 –/+ –/+ – 
At4g14550 – – – 
At4g14560 ++ ++ 
At4g28640 – – – 
At4g29080 – ++ ++ 
At4g32280 – – – 
At5g25890 +++ +++ ++ 
At5g43700 – – – 
At5g62000 – – – 
At5g65670 +/++ +/++ –/+ 
Gene Meristematic zone Elongation zone Differentiation zone 
At1g04100 – – – 
At1g04240 +/++ 
At1g04250 –/+ +/++ –/+ 
At1g04550 – ++/+++ ++/+++ 
At1g15050 – ++ ++ 
At1g15580 – – – 
At1g51950 – +/++ +/++ 
At1g52830 – – – 
At1g80390 – +/++ +/++ 
At2g01200 – 
At2g22670 – – – 
At2g33310 – – – 
At2g46990 – 
At3g04730 – – – 
At3g15540 – ++ ++ 
At3g16500 ++ ++/+++ +/++ 
At3g17600 – – – 
At3g23030 –/+ –/+ 
At3g23050 – – – 
At3g62100 –/+ –/+ – 
At4g14550 – – – 
At4g14560 ++ ++ 
At4g28640 – – – 
At4g29080 – ++ ++ 
At4g32280 – – – 
At5g25890 +++ +++ ++ 
At5g43700 – – – 
At5g62000 – – – 
At5g65670 +/++ +/++ –/+ 

–, Absent; +, weak; ++, moderate; +++, strong.

Table 4.

HMG gene expression patterns

Gene Meristematic zone Elongation zone Differentiation zone 
At1g14900 – – – 
At1g20693 – –/+ – 
At1g20696 –/+ –/+ – 
At2g17560 +/++ +/++ –/+ 
At2g34450 +/++ ++/+++ +/++ 
At3g51880 ++ – 
At4g11080 – – – 
At4g23800 +++sp – – 
At4g35570 – – – 
At5g23405 ++ 
Gene Meristematic zone Elongation zone Differentiation zone 
At1g14900 – – – 
At1g20693 – –/+ – 
At1g20696 –/+ –/+ – 
At2g17560 +/++ +/++ –/+ 
At2g34450 +/++ ++/+++ +/++ 
At3g51880 ++ – 
At4g11080 – – – 
At4g23800 +++sp – – 
At4g35570 – – – 
At5g23405 ++ 

–, Absent; +, weak; ++, moderate; +++, strong; sp, spotty pattern.

Table 5.

NFY gene expression patterns

Gene Meristematic zone Elongation zone Differentiation zone 
At1g08970 ++/+++ ++/+++ ++/+++ 
At1g09030 –/+ ++ ++ 
At1g17590 ++ +++ ++ 
At1g21970 ++ – 
At1g30500 ++ ++ +/++ 
At1g45830 +/++ ++/+++ +/++ 
At1g50220 –/+ +/++ –/+ 
At1g54160 ++/+++ +++ ++/+++ 
At1g56170 +++ +++ +++ 
At1g72830 ++ ++/+++ ++ 
At2g13570 –/+ ++/+++ ++/+++ 
At2g34720 +++ +++ ++/+++ 
At2g37060 +/++ ++/+++ +/++ 
At2g38880 ++ ++/+++ +/++ 
At2g47810 ++ +++ ++ 
At3g05690 – –/+ – 
At3g12480 –/+ –/+ – 
At3g14020 – ++sp/st ++sp/st 
At3g20910 +++ +++ ++/+++ 
At3g48590 – +++ – 
At3g53340 +++ +++ ++ 
At4g14540 +++ +++ ++fa 
At5g06510 –/+ ++/+++ 
At5g12840 ++ ++/+++ +/++ 
At5g27910 –/+ ++ –/+ 
At5g47640 +++ +++ ++fa 
At5g47670 –/+ ++ +/++ 
At5g50470 ++/+++ ++/+++ ++ 
At5g50480 +/++ ++/+++ ++/+++ 
At5g50490 +/++ ++/+++ –/+ 
At5g63470 ++/+++ ++/+++ +/++ 
Gene Meristematic zone Elongation zone Differentiation zone 
At1g08970 ++/+++ ++/+++ ++/+++ 
At1g09030 –/+ ++ ++ 
At1g17590 ++ +++ ++ 
At1g21970 ++ – 
At1g30500 ++ ++ +/++ 
At1g45830 +/++ ++/+++ +/++ 
At1g50220 –/+ +/++ –/+ 
At1g54160 ++/+++ +++ ++/+++ 
At1g56170 +++ +++ +++ 
At1g72830 ++ ++/+++ ++ 
At2g13570 –/+ ++/+++ ++/+++ 
At2g34720 +++ +++ ++/+++ 
At2g37060 +/++ ++/+++ +/++ 
At2g38880 ++ ++/+++ +/++ 
At2g47810 ++ +++ ++ 
At3g05690 – –/+ – 
At3g12480 –/+ –/+ – 
At3g14020 – ++sp/st ++sp/st 
At3g20910 +++ +++ ++/+++ 
At3g48590 – +++ – 
At3g53340 +++ +++ ++ 
At4g14540 +++ +++ ++fa 
At5g06510 –/+ ++/+++ 
At5g12840 ++ ++/+++ +/++ 
At5g27910 –/+ ++ –/+ 
At5g47640 +++ +++ ++fa 
At5g47670 –/+ ++ +/++ 
At5g50470 ++/+++ ++/+++ ++ 
At5g50480 +/++ ++/+++ ++/+++ 
At5g50490 +/++ ++/+++ –/+ 
At5g63470 ++/+++ ++/+++ +/++ 

–, Absent; +, weak; ++, moderate; +++, strong; sp/st, spots in a striped pattern; fa, fading in strength across zone.

Table 6.

HSF gene expression patterns

Gene Meristematic zone Elongation zone Differentiation zone 
At1g32330 – –/+ – 
At1g77570 +/++ +++ +/++ 
At2g26150 +/++ ++/+++ ++/+++ 
At2g41690 +/++ ++/+++ +/++ 
At3g02990 –/+ –/+ – 
At3g22830 –/+ –/+ – 
At3g24520 – – – 
At3g51910 – – – 
At4g11660 ++ 
At4g13980 +/++ ++/++ +/++ 
At4g17750 ++ ++/+++ ++/+++ 
At4g18880 – – – 
At4g19630 – ++ ++ 
At4g36990 – +/++ –/+ 
At5g03720 –/+ ++/+++ ++/+++ 
At5g16820 +/++ +/++ +/++ 
At5g45710 – ++/+++ – 
At5g54070 –/+ –/+ – 
At5g62020 ++ +++ ++ 
Gene Meristematic zone Elongation zone Differentiation zone 
At1g32330 – –/+ – 
At1g77570 +/++ +++ +/++ 
At2g26150 +/++ ++/+++ ++/+++ 
At2g41690 +/++ ++/+++ +/++ 
At3g02990 –/+ –/+ – 
At3g22830 –/+ –/+ – 
At3g24520 – – – 
At3g51910 – – – 
At4g11660 ++ 
At4g13980 +/++ ++/++ +/++ 
At4g17750 ++ ++/+++ ++/+++ 
At4g18880 – – – 
At4g19630 – ++ ++ 
At4g36990 – +/++ –/+ 
At5g03720 –/+ ++/+++ ++/+++ 
At5g16820 +/++ +/++ +/++ 
At5g45710 – ++/+++ – 
At5g54070 –/+ –/+ – 
At5g62020 ++ +++ ++ 

–, Absent; +, weak; ++, moderate; +++, strong.

Table 7.

SBP gene expression patterns

Gene Meristematic zone Elongation zone Differentiation zone 
At1g02065 –/+ ++/+++ ++/+++ 
At1g20980 ++ 
At1g27370 ++ 
At1g53160 – – – 
At1g69170 – – – 
At1g76580 – – – 
At2g33810 +/++ ++/+++ +/++ 
At2g42200 – –/+ – 
At2g47070 ++ +/++ 
At3g15270 – 
At3g57920 +/++ 
At3g60030 –/+ +/++ –/+ 
At5g18830 –/+ 
At5g43270 ++ 
At5g50570 – – – 
Gene Meristematic zone Elongation zone Differentiation zone 
At1g02065 –/+ ++/+++ ++/+++ 
At1g20980 ++ 
At1g27370 ++ 
At1g53160 – – – 
At1g69170 – – – 
At1g76580 – – – 
At2g33810 +/++ ++/+++ +/++ 
At2g42200 – –/+ – 
At2g47070 ++ +/++ 
At3g15270 – 
At3g57920 +/++ 
At3g60030 –/+ +/++ –/+ 
At5g18830 –/+ 
At5g43270 ++ 
At5g50570 – – – 

–, Absent; +, weak; ++, moderate; +++, strong.

Fig. 2.

Selection of expression patterns in primary root: (A) ‘spotty’ meristem in HMG At4g23800; (B) meristematic/(fading) elongation expression in C2C2-Dof At5g62940; (C) elongation/differentiation-specific in HSF At4g36990; (D) striped pattern in C2C2-Dof At4g21050; (E) all zones in C2C2-Dof At2g34140; (F) pericycle-specific in C2C2-Dof At5g60200. Scale bar in A=130 μm, all figures to same scale.

Fig. 2.

Selection of expression patterns in primary root: (A) ‘spotty’ meristem in HMG At4g23800; (B) meristematic/(fading) elongation expression in C2C2-Dof At5g62940; (C) elongation/differentiation-specific in HSF At4g36990; (D) striped pattern in C2C2-Dof At4g21050; (E) all zones in C2C2-Dof At2g34140; (F) pericycle-specific in C2C2-Dof At5g60200. Scale bar in A=130 μm, all figures to same scale.

Gene expression patterns vary between each family

The proportion of genes expressed in one or more zones of the root and the patterns displayed varies between each of the six families studied. These patterns are summarized in Fig. 3 (see Table S1 in Supplementary data available at JXB online). The most diverse patterns within a family are in the C2C2-Dofs (Table 2; see Fig. S1 in Supplementary data at JXB online) in which most of the genes studied (94%) are expressed in the root. Of the genes expressed in this family, >50% (18) are present in all three developmental zones of the root (Fig. 3), one of which (At3g45610) is pericycle-specific (see Fig. S1, panel B5, in Supplementary data at JXB online). Seven of the C2C2-Dofs are expressed in both the meristem and elongation zones, one of which (At5g60200) is also pericycle-specific (shown previously in Fig. 2F). Only two members are expressed in the elongation and differentiation zones, one of which (At2g46590) displays a striped pattern characteristic of epidermal trichoblasts or atrichoblasts (see Fig S1, panel B3, in Supplementary data at JXB online). Four members of this family are specifically expressed in one zone: three in the elongation zone and one in the differentiation zone, the latter again showing a characteristic striped pattern (shown previously in Fig. 2D).

Fig. 3.

Venn diagram for each family showing the number of genes expressed in each zone of the root: meristematic, red; elongation, green; differentiation, blue.

Fig. 3.

Venn diagram for each family showing the number of genes expressed in each zone of the root: meristematic, red; elongation, green; differentiation, blue.

By contrast to the C2C2-Dof family, none of the Aux/IAA genes is specifically expressed in a single zone, with only 16 (55%) of the 29 genes studied are expressed in the root. Half of these are expressed in both the elongation and differentiation zones and one is confined to the meristem and elongation zone (Table 3, see Fig. S2 in Supplementary data available at JXB online). However, similar to the C2C2-Dof family, almost half of the Aux/IAA genes are expressed in all three zones of the root.

This pattern is repeated in the HMG family, where nearly half of the genes expressed are present in all developmental zones (Table 4, see Fig. S3 in Supplementary data available at JXB online). Seven (70%) of the 10 HMG genes studied are expressed in the root tip. Two members of the HMG family are expressed in only one zone, one in the meristem with a spotty pattern characteristic of cell cycle-regulated genes (shown previously in Fig. 2A), and one in the elongation zone (see Fig. S3, panel A2, in Supplementary data at JXB online).

Of the six families studied, only the NFY family showed expression for all members (31) in the root. The majority (83%) of the NFY genes are expressed in all developmental zones (Table 5; see Fig. S4 in Supplementary data available at JXB online). Expression of two NFY genes (At4g14540 and At5g47640) fades in the differentiation zone (see Fig. S4, panels C2 and C6, respectively, in Supplementary data at JXB online). Two genes are expressed in the meristem and elongation zone, and two are specific to the elongation zone. The most interesting pattern was produced by NFY At3g14020, which was expressed only in the elongation and differentiation zone, showing a discontinuous striped pattern (see Fig. S4, panel B8, in Supplementary data at JXB online).

Over 80% of the HSF genes analysed are expressed in the root (Table 6; see Fig. S5 in Supplementary data available at JXB online). Nine out of 16 (56%) of these genes were expressed in all zones. Three are expressed in both the meristem and elongation zone, and two in both the elongation and differentiation zone. Only two genes are expressed in one zone, the elongation zone.

Finally, 11 (73%) of the 15 SBPs studied are expressed in the root (Table 7, see Fig. S6 in Supplementary data available at JXB online). Most of these (82%) are expressed in all developmental zones, a similar proportion to that seen in the NFY family. One is expressed only in the meristem and elongation zone, and one is specific to the elongation zone.

Global overview of gene expression patterns

Of the 137 genes studied, 112 (82%) are expressed in the root and 25 (18%) were not detectable by in situ hybridization. Most of the 112 genes are expressed in more than one zone and are not restricted to any one region of the root. Of these, 72 genes (almost 65%) are expressed in all three zones (Fig. 3; see Table S1 in Supplementary data available at JXB online). For example, C2C2-Dof At2g34140 (Fig. 2E) is expressed in all zones of the root, from meristem to differentiation zone. Only 11 (10%) of the 112 genes are expressed in a single zone. One of these, HMG At4g23800 (Fig. 2A), was the only gene identified that is expressed in the meristem alone. In this zone, its expression is patchy and resembles the pattern of histone H4 (Fig. 1B, F), suggesting that its expression varies throughout the cell cycle. Nine genes were expressed specifically in the elongation zone, for example HSF At5g45710 (Table 6, see Fig. S5, panel B7, in Supplementary data at JXB online), and only one gene, C2C2-Dof At4g21050, displays specific expression in the differentiation zone (Fig. 2D). Sixteen (14%) of the 112 genes were expressed in the meristem and elongation zone but not in the differentiation zone; for example, C2C2-Dof At5g62940 (Fig. 2B). Similarly, 13 (12%) genes were expressed in both the elongation and differentiation zone, but not in the meristem; for example, C2C2-Dof At2g46590 (Table 2; see Fig. S1, panel B3, in Supplementary data at JXB online). None of the genes studied was expressed in only the meristem together with the differentiation zone.

A small proportion of genes (five) are expressed in single cell types. For example, C2C2-Dof At5g60200 (Fig. 2F) is expressed in the pericycle layers of the meristematic zone and early elongation zone. C2C2-Dof At4g21050 (Fig. 2D) is only expressed in the epidermis of the differentiation zone, and within the epidermis it is expressed in a striped pattern. This indicates that it is located in either the trichoblasts or the atrichoblasts but not both. These are candidate genes that may control the process of either pericycle or epidermal development, respectively.

Discussion

Here, a high-throughput in situ hybridization protocol is reported for use in Arabidopsis roots. To illustrate how this protocol could be used, the expression patterns of 137 transcription factor genes in young roots are described. Of the 137 transcription factor genes, 112 are expressed in the 4-d-old primary root in one or more developmental zones. More than half are expressed throughout the three developmental zones while ∼30% are expressed in two zones. Only 10% of the genes are specific to a single developmental zone. One such gene (At4g23800) was found to be meristem specific, and its patchy pattern indicates that its expression is likely to be controlled by cell cycle regulation of its transcription. Similarly, only one gene (At4g21050) was found to be specific to the differentiation zone whereas nine elongation zone-specific genes were identified.

Eleven of the genes studied here have previously been found to mutate to defective root development phenotypes (see Table S2 in Supplementary data available at JXB online). In nine of these 11 genes, expression in the primary root was detected, highlighting the usefulness of this approach to screen for key players in root development. In most cases, previous studies have not determined mRNA localization of these transcription factors in the primary root. However, two Aux/IAA genes, At1g04240 (SHY2/IAA3) and At1g04250 (AXR3/IAA17), have been implicated in root development (Leyser et al., 1996; Rouse et al., 1998; Tian and Reed, 1999), and the expression patterns described here for these genes are similar to those previously published (Knox et al., 2003), showing the accuracy of the method used in this study. Aux/IAAs are short-lived auxin-responsive transcription factors whose levels are degraded in response to auxin. Root growth and root hair growth are controlled by the levels of two antagonistically acting proteins SHY2/IAA3 and AXR3/IAA17 (Knox et al., 2003). For example, dominant stabilizing mutations in AXR3/IAA17 block root hair initiation and elongation, while plants carrying stabilizing mutations in SHY2/IAA3 precociously develop hairs which grow longer than in the wild type because they elongate for a greater duration. The expression patterns of other Aux/IAA family members that are shown here (Table 3; see Fig. S2 in Supplementary data available at JXB online) indicate that other members are also likely to be involved in processes such as root elongation and differentiation. Auxin responses are known to be modulated by the relative abundance of different Aux/IAA proteins.

Several databases are now publicly available, allowing a gene's expression profile to be correlated with that of other genes during particular stages of development or stress conditions. Such information is useful in identifying downstream targets of transcription factors. Using the Coexpression Gene Search facility at RIKEN, a search was made for potential targets of some the transcription factor genes described in this study. For the C2C2-Dof (At2g46590) expressed in a striped pattern in the elongation and differentiation zones of the root, other potential target or co-regulated genes include an arabinogalactan protein (AGP25) that is also present in the growing tips of pollen tubes (Pereira et al., 2006), a process analogous to root hair tip growth; and a β-galactosidase involved in cell wall xyloglucan metabolism and cell growth (Iglesias et al., 2006). Similarly, a plethora of cell-cycle-regulated genes are strongly co-expressed with the meristem-specific HMG (At4g23800) described previously. Most genes may be independently co-regulated in the two examples given here; however, some may be direct targets of transcriptional regulation. The information presented here will complement other methods of gene expression studies such as microarrays, providing a basis for further comparative genomic analysis and mutant screens. Moreover, mRNA localization at cell- and tissue-specific resolution will potentially identify key transcriptional regulators that are involved in various aspects of root development.

Many of the transcription factor genes controlling cell differentiation in the root are expressed in cell-specific patterns. These include SCR which is expressed in the quiescent centre and endodermis (Fig. 1D, E). In this study, two additional cell-specific gene expression patterns were observed. One pattern was specific to the pericycle in two C2C2-Dof genes, At3g45610 and At5g60200, where expression continues from the meristem into the elongation zone, suggesting that these genes may be involved in pericycle development, sites of lateral root hair formation, or in regulating pericycle-specific transcription. Three genes, C2C2-Dof At2g46590, C2C2-Dof At4g21050, and NFY At3g14020, are expressed in subsets of cells in the developing epidermis, indicating that they may be involved in cell-specific differentiation of the epidermis and in root hair formation. Higher magnification images of these tissue-specifically expressed genes are shown in Fig. S8 in Supplementary data available at JXB online. This approach therefore provides a sensitive method by which cell-specific patterns of transcription factor gene expression in the root can be detected, providing another criterion for the identification of potential regulators of root development.

The information available from this analysis complements other approaches to determine the spatial patterns of gene expression. At the lowest level of resolution, MPSS provides information about genes that are expressed in roots without tissue or cell resolution. Of the 112 root-expressed transcription factors described in this study, 83 (74%) were also shown to be expressed in the root by MPSS. However, MPSS did not detect the other 29 (26%) genes that were shown to be expressed in the root. Therefore, differences in the sensitivity of transcript analysis and baselines employed by the different methods may account for the quarter of the genes that were not identified by MPSS. Similarly, the expression of 25 of 137 transcription factor genes was not detected in this study. Of these 25 genes, five (20%) were also not detectable by MPSS, suggesting that these five genes may not be expressed in the root. Therefore, 20 genes were shown to be expressed in the root by MPSS but were not detected in the present survey. This discrepancy is not unexpected, since the present analysis only identified genes that are expressed in the 1 mm region around the root tip which contains the meristematic, elongation, and differentiation zones. By contrast, the MPSS study isolated mRNA from complete 21-d-old roots which would include not only the 1 mm root tip but also cambium, its derivatives, and the earliest stages of lateral root formation.

While MPSS provides organ-specific gene expression information, increased spatial resolution of whole genome expression analysis has been achieved using array-based approaches that utilize fluorescent cell sorting to purify cell populations from living meristems as a source of mRNA (Birnbaum et al., 2003). In this analysis, roots of plants expressing cell-specific marker genes were separated into three developmental zones, which broadly reflect the meristem (and root cap), elongation, and differentiation zones. Specific cells were isolated from the roots and protoplasts prepared for fluorescence cell sorting. This allowed the identification of gene expression profiles in tissues for which there are fluorescence cell markers available. Genevestigator was used to analyse these microarray data to look for the presence or absence of transcripts in each of the three developmental zones of the root. Of the 112 transcription factor genes that were found to be expressed in the root, 32 (29%) show patterns that are consistent with the proximal–distal patterns described by Genevestigator/Birnbaum et al. (2003), 35 (31%) show different patterns, 36 (32%) were not detected, and nine (8%) were not available in the microarray analysis. Thus, of the genes (103) available for pair-wise comparison of expression in the basal 1 mm of the primary root 65% agree; however, only half of these genes have the same expression pattern across the three developmental zones. Of the 25 genes not detected by whole-mount in situ hybridization, three (12%) were also not detected by Genevestigator/Birnbaum et al. (2003). Twenty-two were shown to be expressed, suggesting that microarray-based experiments may be more sensitive at detecting expression than the conventional mRNA localization methods used.

The whole-mount in situ approach provides further spatial resolution to that provided by complementary methods. Most importantly it defines genes that may be involved in the regulation of root development at cell-level resolution. This is best exemplified by the HMG At4g23800 gene (Fig. 2A). The fluorescence cell-sorting approach indicates that this gene is expressed in the meristem but cannot reveal the characteristic patchy cell-cycle-regulated pattern. Also, the availability of cell-specific GFP lines and their variable expression can be limiting for the fluorescent cell-sorting approach. For example, pericycle and striped pattern genes could not have been identified as pericycle- or tricoblast/atrichoblast-specific, since no cell populations were generated for these cell types in the Birnbaum study (Birnbaum et al., 2003). Nevertheless, it is only a matter of time before genes will be used to fluorescently label specifically more cell types to aid their purification, allowing the expression of the entire genome to be examined at a greater level of resolution.

Characterization of the role of the genes identified in this study in combination with further detailed high-throughput in situ hybridization studies will further define the complex network of interactions that regulate the development of the Arabidopsis root. Extension of this approach to other classes of genes involved in signal transduction events will provide more details of the regulatory network underpinning the development of plants.

Supplementary data

Figure S1 C2C2-Dof gene expression patterns.

Figure S2 Aux/IAA gene expression patterns.

Figure S3 HMG gene expression patterns.

Figure S4 NFY gene expression patterns.

Figure S5 HSF gene expression patterns.

Figure S6 SBP gene expression patterns.

Figure S7 Subset of control probes.

Figure S8 Tissue-specific gene expression patterns.

Table S1 Summary of gene expression patterns.

Table S2 Root phenotypes of mutations in transcription factor genes described in this study and ISH localisation.

PD was funded by Syngenta, SD by a BBSRC responsive mode grant, and PJS, JHD, and LD by a grant-in-aid to the John Innes Centre from BBSRC. We acknowledge the EU-REGIA consortium for cDNA clones.

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Author notes

*
Present address Department of Molecular, Cellular and Developmental Biology, PO Box 208104, Yale University, 266 Whitney Ave, New Haven, CT 06520-8104, USA.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

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