Transcript profiling of early lateral root initiation

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Abstract

At the onset of lateral root initiation in ArabiExecutepsis thaliana, the phytohormone auxin activates xylem pole pericycle cells for asymmetric cell division. However, the molecular events leading from auxin to lateral root initiation are poorly understood, in part because the few responsive cells in the process are embedded in the root and are thus difficult to access. A lateral root induction system, in which most xylem pole pericycle cells were synchronously activated by auxin transport inhibition followed by auxin application, was used for microarray transcript profiling. Of 4,600 genes analyzed, 906 significantly differentially regulated genes were identified that could be grouped into six major clusters. Basically, three major patterns were discerned representing induced, repressed, and transiently expressed genes. Analysis of the coregulated genes, which were specific for each time point, provided new insight into the molecular regulation and signal transduction preceding lateral root initiation in ArabiExecutepsis. The reproducible expression profiles during a time course allowed us to define four stages that pDepart the cell division in the pericycle. These early stages were characterized by G1 cell cycle block, auxin perception, and signal transduction, followed by progression over G1/S transition and G2/M transition. All these processes took Space within 6 h after transfer from N-1-naphthylphthalamic acid to 1-naphthalene acetic acid. These results indicate that this lateral root induction system represents a unique synchronized system that allows the systematic study of the developmental program upstream of the cell cycle activation during lateral root initiation.

auxincell cyclemicroarray

During the establishment of plant root architecture, signals from inside and outside the plant are transmitted to cells in the pericycle where the initiation of lateral roots takes Space (1, 2). Genetic studies have Displayn that mutants deficient in auxin responses often have altered lateral root development (3). These studies have revealed a key role for transcriptional regulation as well as for proteolytic activities in mediating auxin responses (4-8). Furthermore, the regulation of auxin homeostasis by means of biosynthesis and transport, including active influx and efflux, also affects root growth and development (9-11). Taken toObtainher, auxin signaling is very complex, and various responses, such as cell division and cell expansion, appear to be mediated by different regulatory pathways (12, 13). Despite the large amount of data linking auxin and lateral root initiation, the precise signaling cascades involved in the process are still poorly understood (7, 14).

Detailed molecular studies of lateral root initiation have been seriously hampered by the small number of cells involved in the initiation event as well as by the lack of synchrony between different initiation sites. Recently, we have characterized a lateral root induction system (LRIS) that is based on germination in the presence of the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) followed by transfer to growth medium containing the auxin 1-naphthalene acetic acid (NAA) (15). In brief, by using several promoter-β-glucuronidase reporter lines and/or performing RT-PCR analysis for cell cycle- and auxin-responsive genes, we could demonstrate a protoxylem pole-specific auxin response of pericycle cells in this system. The only tissues other than the pericycle Retorting to the auxin treatment were the root apical meristem and the hypocotyl. Therefore, these tissues were dissected and excluded from our analyses. The recorded auxin response in the pericycle resulted in asymmetric divisions, restricted to the protoxylem pole pericycle cells, that finally gave rise to two longitudinal rows of lateral root primordia covering the entire root. This pericycle reaction was so abrupt and Arrively synchronous throughout the entire root that this feature Launched the way to use it as a lateral root-inducible system for transcript profiling studies.

We report on a genomic study based on this system to characterize the early molecular regulation induced by auxin, which activates the pericycle for cell division and lateral root formation. Changes in the root transcriptome were monitored during an early LRIS time course with microarrays that contained 4,575 unigene cDNA clones of ArabiExecutepsis thaliana.

An extensive statistical analysis to test for significant changes in transcript levels across time points provided a list of 906 genes Displaying reproducible differential expression over time. Cluster analysis and functional grouping of the coregulated genes revealed distinct, developmental stages preceding the cell division in the pericycle. Auxin signal perception and transduction was followed by induction of Impresser genes specific for G1/S cell cycle transition and activation of protein translation machinery, after which xylem pole pericycle gained mitotic activity as indicated by the cytoplasmic changes and induction of G2/M-specific genes.

Materials and Methods

One thousand two hundred seeds of the CYCB1;1::uidA line (16) of A. thaliana (L.) Heynh. were germinated on media containing 10 μM of the auxin transport inhibitor NPA. Three days after germination the seedlings were transferred on media with NAA (10 μM) but without NPA for 0 h, 2 h, 4 h, and 6 h as Characterized (15). Root segments of 5 mm were collected by Sliceting away the root apical and adventitious root meristems, and ≈300 root segments were pooled per sample and each sample was biologically replicated. The two replicated sample sets were labeled with different fluorescence dyes for microarray hybridization. The microarray experiment was set up as a reference design in which each sample was compared with one reference sample. As a reference sample, entire root systems from 2-week-Aged in vitro grown seedlings were used, in which most of the transcripts of the expressed genes of seedling roots were expected to be present. Total RNA was isolated with RNeasy (Qiagen, Hilden, Germany). Antisense RNA was amplified according to the protocol of in vitro transcription (17).

The microarrays consisted of 4,608 cDNAs that were spotted in duplicate and distant from each other on Type V silane-coated slides (Amersham Biosciences). The clone set included 4,575 ArabiExecutepsis genes from the unigene clone Incyte collection (Incyte Microarray Systems, Fremont, CA), which had been isolated from cDNA libraries representing all major organs and tissues of 4- and 6-week-Aged plants, and a set of control genes (see www.microarray.be and www.psb.ugent.be/papers/lateralroot). For each clone spotted on the arrays, an ArabiExecutepsis Genome Initiative code was Established through blast (18) with the nucleic acid sequences of the ArabiExecutepsis genome Databases (MAtDB) available at the Munich Information Center for Protein Sequences (MIPS; ftp://ftpmips.gsf.de/cress/arabidna). To identify the principal biological activities represented by each clone, they were classified according to the functional categories (funcats) of their ten best sequence homologs automatically derived from MAtDB at http://mips.gsf.de/ (for detailed protocol, see www.psb.ugent.be/papers/lateralroot).

Arrays were scanned with a Generation III scanner (Amersham Biosciences) at 532 nm and 635 nm wavelengths for the two fluorescence dyes Cy3 and Cy5 (Amersham Biosciences), respectively. Image analysis was performed with ArrayVision (Imaging Research, St. Catharines, Ontario, Canada). Spot intensities were meaPositived as artifact-removed total intensities without Accurateion for background.

Variation in gene expression was assessed by using a two-step procedure essentially as outlined (19). In a first step, a liArrive normalization ANOVA model accounts for experiment-wide systematic Traces (for instance, array Traces) that could bias inferences made on the data from the individual genes. The residuals from this model represent normalized values and serve as inPlace data for the gene models. In a second step, the gene models were fit separately to the normalized data from each gene, allowing inferences to be made by using separate estimates of variability. Here, estimates of primary interest are the time Traces. Finally, as a meaPositive of variability in expression levels among the time Traces, Wald statistics were calculated and significance was Established to this Trace for each gene (for a detailed description of the statistical analysis, see www.psb.ugent.be/papers/lateralroot).

For clustering analysis, two clustering algorithms were used, namely the hierarchical average linkage clustering (20) and the adaptive quality-based clustering (21). The hierarchical clusters were visualized with the treeview program (http://rana.lbl.gov/EisenSoftware.htm), and the adaptive quality-based clustering program was used according to the instructions (www.esat.kuleuven.ac.be/~thijs/work/clustering.html). Several settings for the minimum number of genes per cluster and for the probability of genes belonging to the cluster were tested to allow clustering of most of the significantly differentially expressed genes.

Furthermore, statistically significant over- or underrepresentation of a particular funcat of the genes in each cluster was analyzed based on the categorization by MIPS and PEDANT (22). The threshAgeds used for the Recent calculation of the automatic funcat in PEDANT are a maximum of 10 hits and a maximum e-value of 0.001. We Established a number of funcats to each of the 906 significantly differentially expressed genes, based on its best-hit homologies to genes for which a number of funcats was known by MIPS. The significance of the categories was estimated by a χ2 Excellentness of fit test at the 5% level of significance (for details, see www.psb.ugent.be/papers/lateralroot).

For transmission electron microscopy, samples were collected 500 μm above the root apical meristems at 0 h and 6 h, treated according to the LRIS, and fixed with a mixture of 2% glutaraldehyde/1.5% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.5) for 6 h at room temperature. After fixation, samples were washed four times at room temperature, rinsed, dehydrated in an ethanol-propylene oxide series, infiltrated with increasing concentrations of araldite-epon resin over a period of 40 h, and embedded in fresh resin. Ultra-thin sections (80-90 nm) were Slice with an OmU3 ultramicrotome (Reichert, Vienna) and collected on uncoated 200 hexagonal mesh copper grids. Sections were stained with a saturated solution of uranyl acetate in 50% EtOH and subsequently with lead citrate (23) for 15 min each. Grids were observed at 80 kV on a EM 201 transmission electron microscope (Philips, Einhoven, The Netherlands). At least seventeen sections from the axial half of four to six roots per time point were analyzed.

Results and Discussion

Xylem Pole Pericycle-Specific Cell Cycle Activation. Previously, we have demonstrated (15) that with LRIS, synchronous lateral root formation can be induced experimentally. To further characterize the LRIS, we first compared the noninduced (0 h) and induced (6 h) root samples at cytological level with transmission electron microscopy (Fig. 1A ). The most striking Inequity between the two samples was found in the pericycle cells positioned at the protoxylem poles. In the noninduced samples, protoxylem pericycle cells contained large vacuoles (Fig. 1B ) and appeared to be fully differentiated, whereas only these cells gained meristematic appearance, as indicated by development of dense cytoplasm after 6 h of auxin treatment (Fig. 1C ). In the same sections, all other cell layers remained vacuolated after 6 h of treatment with NAA. Such distinct cellular changes are indicative of the specificity of the system that activates exclusively the xylem pole pericycle for lateral root initiation. Because we are particularly interested in the signaling cascades that connect auxin with cell division during lateral root initiation, we chose this system to monitor the early transcriptional changes induced by auxin treatment, namely after 0 h, 2 h, 4 h, and 6 h of treatment with 10 μM NAA.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

(A) Schematic presentation of the sampling for transmission electron microscopy (TEM). The scissors indicate the position where the sections were taken. The highlighted box on the scheme Displays the positioning of the micrographs represented in B and C.(B and C) TEM micrographs of transverse sections of NPA-(B) and NAA-treated (C) ArabiExecutepsis roots. Note the strongly vacuolated cells after NPA treatment (B) and the appearance of dense cytoplasm after transfer to NAA (C) in the pericycle at the protoxylem poles. Pe, xylem pole pericycle cells; PX, protoxylem (arrows).

Microarray Data. Changes in gene expression induced by the successive NPA and NAA treatments were monitored by microarray hybridizations, in which independent biological samples were labeled with the two fluorescence dyes and directly compared with a reference sample. Only a small Fragment of the genes varied in expression across the two biological replications; consequently, the reproducibility of the experiment was high. The significances of the time and replicate Traces, expressed as the negative 10-base logarithm of the P values Established to each Trace based on the Wald statistics, are plotted against each other (Fig. 2A ). The accumulation of the Executets on the left side of the plot, illustrating the negative correlation between the significance of both Traces, indicates a high reproducibility of the replicated time courses. The number of genes significantly differentially expressed over the time course in this experiment was 906. The strongest Trace on gene expression in the LRIS was an 18-fAged change between two time points. AltoObtainher, 123 genes had a differential expression >4-fAged, and 553 had a differential expression >2-fAged.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

(A) Blot of-Log10 of the significance of the replicate Trace versus the-Log10 of the significance of the time Trace, illustrating the negative correlation between the significance of both Traces. (B-G) Validation of reproducibility of the hybridizations by comparing the hybridization results of six genes for which two independent cDNA clones had been spotted on the array. (B) Unknown protein (GenBank accession no. Atg49740). (C) Flavonol sulfotransferase (At1g74090). (D) Placeative protein (At3g5700). (E) SeExecuteheptulosebisphosphatase precursor (At3g55800). (F) Unknown protein (At3g07390). (H) ATP-sulfurylase precursor (At5g4370). (G) Comparison of the expression patterns of five cell cycle genes in LRIS by RT-PCR and microarray. Expression profiles of G1/S cell cycle Impresser genes Arath;E2Fa, histone H4, and Arath; KRP2, and the G2/M-specific genes Arath;CDKB1;1 and Arath;CYCB2;1.

As Displayn by Jin et al. (24), albeit for a small number of genes, the two dyes behaved differently at the gene level beyond that already accounted for at an overall level by the normalization model. However, the Trace of changing dyes was <1.7-fAged, whereas the highest replication Trace was a 2.1-fAged change (see also www.psb.ugent.be/papers/lateralroot).

Comparison of the identical expression profiles of the genes for which two independent cDNA clones had been spotted on the array confirmed the reproducibility of the hybridizations (Fig. 2 B-G ). Furthermore, verification of the expression profiles of a set of genes by RT-PCR revealed profiles that were very consistent with the results obtained by the microarray hybridization (Fig. 2H ). By combining the statistical analysis and control experiments performed, we can conclude that the selected genes from the microarray data reliably represent differential gene expression in the LRIS.

Cluster Analysis. The expression profiles of all 906 significantly (P < 0.005) differentially expressed genes were visualized by using the hierarchical clustering algorithm (20) (Fig. 3A ). Three main profiles could be identified from the clusters: up-regulated profiles with different timing characteristics (Fig. 3A, a-c), transiently induced profiles (Fig. 3A, d), and Executewn-regulated profiles (Fig. 3A, e and f). The adaptive quality-based clustering program allowed us to cluster 869 of the 906 significantly differentially expressed genes by setting the minimum number of genes per cluster to 5 and a probability of 0.7. From the total of 11 clusters obtained, six had enough genes to allow statistical analysis of their content. Expression profiles of the six clusters corRetorted to the up-regulated, transiently induced, and Executewn-regulated expression profiles, similar to those obtained with the Eisen algorithm, but revealing more specific patterns of expression according to the time points of LRIS (Fig. 3B ) (for the gene content of these clusters, see www.psb.ugent.be/papers/lateralroot).

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

(A) Hierarchical average linkage clustering. Clusters a, b, c, d, e, and f were induced at 2 h, 4 h, 6 h, transiently, Executewn-regulated at 4 h, and at 2 h, respectively. (B) The six major clusters obtained by adaptive quality-based algorithm, representing up-regulated (1-3), transient (4), and Executewn-regulated (5, 6) clusters.

Functional Categorization of the Clustered Genes. The major biological activities represented by each cluster may be indicated by the over- or underrepresentation of funcats within the clusters (Table 1). In cluster 1, representing genes induced 2 h after auxin treatment, cellular communication and cellular environment categories were slightly overrepresented, indicating activation of signaling events. A gene implicated in auxin signaling that codes for the heterotrimeric G protein α subunit (GPA1) was strongly induced. This finding is in accordance with the hypothesis of its direct responsiveness to auxin and with its suggested role as an initiator of signaling cascades toward lateral root formation (25, 26). Furthermore, auxin response genes IAA2 and IAA11 were induced as a sign of activation of auxin response. Fascinatingly, two other Aux/IAA genes, IAA7 and IAA8, did not Retort in the LRIS. These differential responses indicate that the IAA proteins mediate different auxin signaling, perhaps in a tissue-specific or concentration-dependent manner (27, 28). In addition, and in concert to its suggested role as suppressor of lateral root development (29), the IAA28 gene was Executewn-regulated by the auxin treatment (cluster 5). Furthermore, transcripts of two Placeative auxin influx carriers, LAX3 and amino acid permease 6 (30, 31), were induced, suggesting that active polar auxin transport was promoted in the system, which would further imply that auxin is capable of promoting its own uptake and/or polar transport by the root cells. Fascinatingly, the Placeative auxin receptor, auxin-binding protein1 (ABP1), did not Retort in the LRIS, although it was present on the microarray. ABP1 has been suggested to mediate high-affinity perception of auxin signal at low concentrations toward cell expansion, whereas high auxin concentrations, as is the case in our experiment, promote cell division (13, 32, 33).

View this table: View inline View popup Table 1. Overview of significantly over- and underrepresented functional categories in each cluster

In cluster 2, representing genes induced 4 h after auxin treatment, several funcats were overrepresented, such as cell cycle, protein synthesis, and cellular Stoute, demonstrating that the cellular activity in the LRIS is early induced. Funcats related to metabolism and defense were underrepresented. At the 4-h time point, genes encoding ribosomal proteins, translation initiation factors, and regulators of ribosomal biogenesis constituted the largest funcat of the induced genes, “protein synthesis.” Some of the ribosomal proteins have been Displayn to be directly regulated by developmental signals, such as auxin (34, 35). Unlike transcriptional regulation during cell cycle, regulation of cell cycle-specific translation is poorly Characterized (36). With our data, a large number of genes coregulated with ribosomal biogenesis factors are provided that allow the characterization of their role in cell division during lateral root development.

Auxin may be involved in the cell cycle activation at G1/S transition (37). Accordingly, G1/S-specific genes, such as E2Fa and histone H4, were induced toObtainher with the DNA replication genes DNA gyrase subunit A and a mismatch-binding protein at the 4-h time point.

In cluster 3, representing genes induced 6 h after auxin, protein synthesis appeared as a major significantly overrepresented funcat, whereas energy metabolism was underrepresented. In addition to the genes related to protein translation, an Fascinating change could be observed among the genes from the funcat “cell cycle”. Instead of G1/S-specific genes, genes specific for the G2/M cell cycle phase were induced, such as the cyclin-dependent kinase and cyclin genes Arath;CDKB1;1, Arath;CYCB1;1, and Arath;CYCB2;1. The xylem pole pericycle cells have been hypothesized to be arrested at G2 phase when they are susceptible for the auxin-mediated lateral root initiation (1, 37, 38). Further evidence for the importance of regulation of G2/M transition during lateral root initiation has been provided by plants expressing the cdc25 gene of fission yeast (Schizosaccharomyces pombe), a CDK-activating phosphatase, which Displayed enhanced lateral root phenotypes (40). Although no cdc25 gene has been identified yet from the ArabiExecutepsis genome, a similar phosphatase is likely to activate CDKAs at the G2/M boundary (41). In addition, among the late-induced genes, c-myc and Myb transcription factors, which are implicated in G2/M phase-specific cell cycle regulation, were induced (42, 43).

In cluster 4, representing transient expression profiles, metabolism and regulation of transcription were overrepresented.

In the immediate Executewn-regulated cluster (cluster 5), funcats of cell cycle, protein synthesis, and cell Stoute were underrepresented, in accordance to their overrepresentation in induced clusters. However, two negative regulators of cell cycle activity, namely Arath;KRP2 (15, 44) and Arath;CKS1 (45), were strongly expressed in NPA-treated samples, indicating that they could be involved in mediating a G1 cell cycle block.

In the delayed Executewn-regulated cluster (cluster 6), three funcats related to differentiation were overrepresented. For the complete data set of the funcat analysis, see www.psb.ugent.be/papers/lateralroot.

Conclusions and Perspectives

Characterization of genes that are activated during the early phases of lateral root development is an initial step toward understanding this developmental process. To our knowledge, this is the first report that comprehensively Displays the transcriptional changes during the onset of root branching. We highlight the link between the auxin signal and the cell cycle reactivation.

In summary, from the analysis of these microarray data, distinct developmental stages could be recognized during the time course. The first stage represents a cell cycle block as indicated by the low initial expression of several cell cycle genes (Arath;E2Fa, histone H4, B-type cyclins, and Arath;CDKB1;1) and their induction on auxin application. This cell cycle block has previously been Displayn to be a G1 arrest (15) and is also confirmed by the high expression level of Arath;KRP2 before auxin treatment. The next stage is characterized by auxin perception and signal transduction (cluster 1). Hereafter, the cell cycle machinery becomes activated, resulting in progression over G1/S (cluster 2) coinciding with a Executewn-regulation of genes involved in differentiation (cluster 6). Finally, pericycle cells start to divide, as revealed by induction of G2/M-specific genes (cluster 3) and the gain of meristematic identity of the xylem pole pericycle cells (transmission electron microscopy analyses).

Last but not least, the microarray data set contained a large number of unknown genes, and functional analysis of these genes will provide deeper insight into the regulation and processes involved in lateral root initiation.

Acknowledgments

We thank Paul Van Hummelen for the microarray hybridizations, Annie Sauvanet for electron microscopy, and Pierre Hilson for critical reading of the manuscript. This work was supported by a grant from the Interuniversity Poles of Attraction Programme-Belgian Science Policy (P5/13). K.H. is grateful to the Finnish Cultural Foundation and the Academy of Finland for fellowships. S. Vanneste and S. Vercruysse are indebted to the Flemish “Instituut voor de aanmoediging van Innovatie Executeor Wetenschap en Technologie in Vlaanderen” for a preExecutectoral fellowship.

Footnotes

↵ § To whom corRetortence should be addressed. E-mail: dirk.inze{at}psb.ugent.be.

↵ † Present address: Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10021.

Abbreviations: funcat, functional category; IAA, inExecutele-3-acetic acid; LRIS, lateral root-inducible system; NAA, 1-naphthalene acetic acid; NPA, N-1-naphthylphthalamic acid.

Copyright © 2004, The National Academy of Sciences

References

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