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First published online July 10, 2003; 10.1104/pp.103.021436 Plant Physiology 132:1998-2011 (2003) © 2003 American Society of Plant Biologists Expression Profiling of Cytokinin Action in Arabidopsis1,[w]University of North Carolina, Biology Department, CB Number 3280, Chapel Hill, North Carolina 27599–3280
The phytohormone cytokinin is an important regulator of plant growth and development; however, relatively few genes that mediate cytokinin responses have been identified. Genome-wide analyses of Arabidopsis seedlings using the approximately 8,300-element Affymetrix Arabidopsis GeneChips (Affymetrix, Santa Clara, CA) to examine cytokinin-responsive genes were conducted, revealing at least 30 genes whose steady-state level of mRNA was elevated and at least 40 that were down-regulated at multiple time points after application of cytokinin. The cytokinin up-regulated genes include the type-A Arabidopsis response regulators (ARRs), which had been shown previously to be cytokinin primary response genes, cytokinin oxidase, which encodes an enzyme that degrades cytokinins, and several transcription factors. Cytokinin down-regulated genes include several peroxidases and kinases and an E3 ubiquitin ligase. We identified a common sequence motif enriched in the upstream regions of the most consistently cytokinin up-regulated genes. This motif is highly similar to the optimal DNA-binding sites for ARR1/ARR2, type-B ARRs that have been implicated in the transcriptional elevation of the type-A ARRs. Additionally, genome-wide analyses of cytokinin receptor mutants (wol/cre1) revealed large-scale changes in gene expression, including down-regulation of the type-A ARRs and several meristem and cell cycle genes, such as CycD3. Mutations in CRE1 reduced but did not eliminate the effect of cytokinin on gene expression for a subset of cytokinin-responsive genes and had little or no effect on others, suggesting functional redundancy among the cytokinin receptors.
Cytokinins are a group of adenine derivatives that affect multiple aspects of plant growth and development, including cell division, vascular development, sink/source relationships, apical dominance, and leaf senescence (Binns, 1994  ; Mok and Mok,
1994,
2001). A pathway for cytokinin
biosynthesis and metabolism is emerging from molecular and biochemical
studies. This has been highlighted by the recent cloning of several genes
encoding enzymes involved in cytokinin biosynthesis or metabolism, including
ipt (Kakimoto, 2001;
Takei et al., 2001), which
catalyzes the first committed step in cytokinin biosynthesis; cytokinin
oxidase (Houba-Hérin et al.,
1999; Morris et al.,
1999), which cleaves the N6 side chain from cytokinins;
and several enzymes that catalyze the conjugation of sugar moieties to
cytokinins (Martin et al.,
1999a,
1999b,
2001). A model for cytokinin
perception and signal transduction has emerged that is similar to prokaryotic
two-component response pathways (Haberer
and Kieber, 2001; Hutchison
and Kieber, 2002; Lohrmann and
Harter, 2002). A family of genes that are similar to bacterial
two-component response regulators, the type-A Arabidopsis response regulators
(ARRs), was identified as cytokinin primary response genes
(Brandstatter and Kieber, 1998;
Sakakibara et al., 1998;
Taniguchi et al., 1998;
D'Agostino et al., 2000). The
cytokinin receptors (CRE1, AHK2, and AHK3) mediate the induction of these
type-A ARRs and are similar to bacterial two-component His kinases
(Inoue et al., 2001;
Ueguchi et al., 2001;
Yamada et al., 2001). These
receptors act through other two-component elements, including Arabidopsis
homologs of His phosphotransfer proteins (AHPs) and the type-B ARRs, involved
in the up-regulation of type-A ARRs (Hwang
and Sheen, 2001; Sakai et al.,
2001).
CRE1 (also called WOL and AHK4) is the best characterized of the three
cytokinin His kinase receptors. The signal transduction function of this gene
in single-cell systems such as Escherichia coli, yeast
(Saccharomyces cerevisiae), and transient Arabidopsis protoplasts has
been shown to be dependent on the exogenous application of cytokinin.
Furthermore, loss-of-function mutants of CRE1 result in reduced cytokinin
sensitivity (Inoue et al.,
2001
Several researchers have examined gene expression in response to
application of exogenous cytokinin (Crowell
and Amasino, 1994 In this study, we sought to identify genes regulated by cytokinin to better understand the mode of action of this hormone. Additionally, we examined the role of the cytokinin receptor CRE1 in mediating the effect of cytokinin on gene expression in Arabidopsis roots.
Regulation of Gene Expression by Cytokinin Light-grown Arabidopsis seedlings were treated with exogenous cytokinin for various times over a 24-h period, and gene expression was analyzed using the approximately 8,300-element Affymetrix Arabidopsis GeneChips (Affymetrix, Santa Clara, CA). Ten cytokinin treatments were conducted and normalized to control samples prepared and treated identically with dimethyl sulfoxide (DMSO; the solvent used for the cytokinin). For our analyses, we chose a 2-fold change in expression level compared with the control as a minimum for a gene to be called altered in response to cytokinin and a minimum raw value cutoff of 175 to 500, depending on the particular chip hybridization. These raw level cutoffs were assigned based on scatter plot analyses of a treated sample versus its control and used because expression levels of genes below these cutoff thresholds frequently showed variability greater than 2-fold and often had Affymetrix flag calls of Absent (data not shown). In addition, only the cytokinin-treated samples with an Affymetrix flag call of Present and the minimum raw cutoff level were used to identify up-regulated genes; likewise, only the control samples with the same restrictions were used to identify down-regulated genes. By using these restrictions, we sought to distinguish authentic changes in expression from background noise and false positives. Using these parameters, the steady-state levels of expression of more than 1,000 genes were altered by cytokinin alone in at least one treatment; however, the majority of these were affected at only a single treatment and generally not in replicate samples (see Supplemental Tables I and II at http://www.plantphysiol.org).
Thirty genes were found to be up-regulated at five or more of the 10
cytokinin time course treatments, and 17 were up-regulated in at least six
independent treatments (Fig.
1A). The observation that these genes were reproducibly
up-regulated by cytokinin suggests that they are cytokinin responsive. A
Welch's t test confirmed that all of the top 17 genes showed
significant changes in expression levels with cytokinin treatment. We chose to
include other genes that fit the criteria described above for cytokinin
regulation but that showed a P value
Approximately 40 genes were identified that showed greater than a 2-fold decrease in their expression level at five or more of the 10 treatments after cytokinin application (Fig. 1B). One group of genes that showed reductions in their steady-state mRNA levels was a set of genes involved with oxidation, including four peroxidases and two oxidases. Another group of down-regulated genes was three kinases, including an Ste-20 and SNF1-related kinase. Other reproducibly identified cytokinin down-regulated genes include an ubiquitin ligase SCF complex subunit, three transcription factors, and the leaf development protein Argonaute (Fig. 1B). In comparison with the set of induced genes, which contains only two unknowns, the cytokinin-repressed genes include more than one-quarter (11 genes) for which there is no known function (Fig. 1A,Fig. 1B). A number of distinct induction profiles can be identified from this time course (Fig. 1A,Fig. 1B). There is a set of genes that is elevated or reduced at all time points examined. This includes most of the type-A ARRs, the cytokinin oxidase, the AP2s, and the P450 from the up-regulated list, and the respiratory burst oxidase and the three kinases from the down-regulated list. There is only a small set of genes that are induced rapidly (15, 45, and 120 min), but transiently (return to baseline at 480 and 1,440 min), such as the nodulin-26-like gene NIP1;1, whereas no down-regulated genes appear to fit this pattern. There is a larger set of genes that are up- or down-regulated only upon prolonged cytokinin treatment (>120 min). For the up-regulated genes, these include a glutaredoxin, a disease resistance response protein, and an unknown protein. For the down-regulated genes, these include a zinc finger protein and three of the four down-regulated peroxidases. Another class of genes displays alterations in gene expression at specific time points. Most of these genes are specifically induced or repressed at a single time point, such as a putative peroxidase that is primarily repressed at 120 min by both BA and zeatin cytokinin treatments, as seen in Figure 1B. Our experimental design using both BA and zeatin treatments allowed us to examine the effect of these different classes of cytokinins on gene expression. At 120 min, seedlings were treated with either BA, a cytokinin with an aromatic-type side chain attached to the N6 position of the adenine ring, or trans-zeatin, a naturally occurring cytokinin with an isoprenoid-type side chain. Almost every gene found to be up-regulated by BA at both 120-min treatments was also up-regulated in at least one of the two zeatin treatments and vice versa. In contrast, there are many fewer genes down-regulated by zeatin as compared with BA, which suggests that some of these genes may be specifically down-regulated by BA or that they are not actually regulated by cytokinin. It is important to note that for the genes that have altered expression levels in fewer numbers of cytokinin treatments, there is a decreased confidence that any particular gene is truly regulated by cytokinin. This is reflected in the statistical analysis of the data, which indicates that some but not all of the genes that we identified as cytokinin regulated were found to have significant P values. The genes identified as altered in both replicate treatments for a single time point are potentially transiently cytokinin-regulated genes and are more likely to be truly altered by cytokinin than those affected at only two distinct time points, but not in the replicates. Thus, we have included genes affected in both replicates of any single time point above a 2.5-fold change in expression in Figure 1A,Figure 1B. Nevertheless, it is possible that some of the genes identified as cytokinin regulated (Fig. 1A) are false positives, and it is likely that we have excluded genes that are weakly induced for a narrow window of time along with genes whose expression levels are relatively low. In summary, the set of genes presented in Figure 1A,Figure 1B represents the list of genes found in this study to most likely be regulated by cytokinin, with a decreasing confidence in this conclusion as a gene is identified as altered in fewer cytokinin-treated samples.
We used GeneSpring 5.0 software (Silicon Genetics, Redwood City, CA) to
search for common motifs enriched within 1 kb upstream of the translational
start site of the 17 genes that are consistently up-regulated by cytokinin
(those genes induced in six or more of the cytokinin treatments as in
Fig. 1A). The sequence GATCTT
was identified as a core sequence motif found significantly enriched in the
upstream regions of this set of genes. The relative positions of this motif
upstream of the type-A ARRs and the other cytokinin-regulated genes are shown
in Figure 2. White boxes
represent this common upstream sequence motif GATCTT on the forward strand,
and black boxes represent the complement of the motif AAGATC. Upstream regions
of the cytokinin-regulated type-A ARRs showed the highest concentration of
these motifs, generally clustering in the 500 bp proximal to the coding
region. The sequence of this common upstream sequence motif closely matches
(G/A)GAT(T/C), the optimal DNA-binding sequence identified in vitro for ARR1
and ARR2, two type-B ARRs (Sakai et al.,
2000
The frequency of this common upstream sequence motif in the upstream regions of the type-A ARRs is positively correlated with the level of their induction by cytokinin. The most highly cytokinin-induced genes, ARR5, ARR7, and ARR16, have multiple copies of this motif. Additionally, genes that are either only weakly induced by cytokinin or not expressed at detectable levels, ARR3, ARR4, and ARR17, have one or no motifs (Fig. 2; data not shown). ARR6, which is moderately induced by cytokinin, does not fit this pattern because it has no consensus motif in its upstream region, although two such motifs are found within the fourth intron of this gene. The common upstream sequence motif (GATCTT) should randomly occur approximately 0.32 times per kilobase pair, taking into account the GC content observed in intergenic regions of Arabidopsis (approximately 33%). Our search of all genes present on the Affymetrix GeneChip that are expressed with a raw of at least 500 but not induced by cytokinin (950 genes) revealed that the sequence motif GATCTT is present a total of 358 times in the 1 kb upstream of the translational start site, or approximately 0.38 times per kilobase pair. In contrast, this core sequence motif is present 14 times among the upstream regions of the top 17 cytokinin up-regulated genes and only six times among the upstream regions of the 21 cytokinin down-regulated genes. This enrichment, coupled with the similarity to the previously defined ARR1/ARR2-binding sequence, suggests that this motif may play a role in the transcriptional regulation of these genes by cytokinin.
Previous studies have implicated Arabidopsis homologs of two-component
signaling elements in the cytokinin response pathway. We analyzed the
expression pattern of these genes under our experimental conditions
(Fig. 3; see Supplemental Table
III). As noted above, the five type-A ARR genes present on the chip were
up-regulated at most of the cytokinin treatments. The three AHP genes on the
chip are not affected by cytokinin, nor are the five type-B ARRs, consistent
with previous results suggesting that the steady-state mRNA level of these
classes of two-component genes are unaffected by cytokinin treatment
(Imamura et al., 1998
We examined the effects that loss-of-function mutations of the cytokinin
receptor CRE1 had on the pattern of gene expression by analyzing RNA isolated
from the roots of two different cre1 mutant alleles, wol and
cre1-1. Root tissue was examined because these mutations have been
reported to principally affect root development
(Scheres et al., 1995
Of the genes that are down-regulated in both wol and
cre1-1, one group of particular interest is those involved in
regulation of cell division and meristem function, consistent with the primary
defect in root development of this mutant. Interestingly, the cell cycle
regulators CycD1 and CycD3 are down-regulated in both
wol and cre1-1 relative to wild-type roots, independent of
cytokinin treatment, and the cdc2 gene is down-regulated specifically
in wol (see Supplemental Table IV). As in the shoots, we do not
observe an induction of either of these genes in response to cytokinin in
contrast to previous reports
(Riou-Khamlichi et al., 1999
The type-A ARR genes downstream of the cytokinin receptor CRE1 were still
induced by cytokinin in the wol and cre1-1 mutants, although
the fold increase relative to untreated mutant roots was not always to the
same level observed in wild-type roots
(Fig. 3). Basal expression
levels of the type-A ARR and several others of the top cytokinin-inducible
genes were also lower in these cytokinin receptor mutants
(Table II), as previously
observed in the cre1-1 mutant for two type-A genes, ARR15
and ARR16 (Kiba et al.,
2002
Generally, genes that are involved in the primary response to a signal are still induced by that signal when protein synthesis is blocked. Furthermore, many primary response genes are elevated upon inhibition of protein synthesis, an effect that has been attributed to the rapid degradation of a negative regulator of transcription or the involvement of a short-lived RNase in the degradation of those transcripts. The effect of inhibition of protein synthesis on cytokinin up-regulated gene expression was examined in seedlings pretreated with cycloheximide, in the presence or absence of cytokinin (Table III).
A large number (236) of genes was found to be elevated greater than 3-fold
in response to cycloheximide (data not shown), including many genes previously
shown to be up-regulated by cycloheximide treatment such as indole-3-acetic
acids (IAAs; Koshiba et al.,
1995
We have analyzed the effect of exogenous cytokinin on gene expression in Arabidopsis seedlings to identify novel cytokinin-regulated genes and to better understand the role of cytokinin in plant developmental processes. Our analysis revealed over 70 genes whose expression is altered by application of exogenous cytokinin in at least one-half of the treatments examined. The genes that are affected by cytokinin and their kinetics of induction provide some clues as to how seedlings respond to elevated cytokinin levels. One reaction to elevated cytokinin at the transcriptional level appears to be a reduction of both the level of active hormone and the sensitivity of the response pathway. The cytokinin signaling response is desensitized by up-regulation of type-A ARRs, which act as negative regulators of the cytokinin response pathway (J. To, G. Haberer, and J. Kieber, unpublished data). The level of cytokinin is potentially reduced by increased expression of a cytokinin-degrading enzyme, cytokinin oxidase, consistent with previous studies demonstrating that cytokinin oxidase enzyme activity was elevated in response to exogenous cytokinin (Terrine and Laloue, 1980 ; Palmer and
Palni, 1987) and by the induction of a gene encoding a potential
cytokinin-conjugating enzyme, a putative glucosyltransferase (At2g16890;
Fig. 1A). These feedback
responses may explain why we do not observe large secondary transcriptional
responses after prolonged exogenous cytokinin treatment. In contrast,
expression of the cytokinin receptor, CRE1, is up-regulated over time in
response to the hormone, which is similar to the induction of ethylene
receptors by ethylene. This slight increase may reflect a potential negative
regulator role of CRE1 in cytokinin response because His kinases in other
two-component systems can act as both His kinases and phosphatases
(Stock et al., 2000).
A second potential pattern that emerges from cytokinin-regulated genes is
an effect on redox state. Four peroxidases and two oxidases are down-regulated
by cytokinin in multiple treatments, and both a glutathione
S-transferase and a glutaredoxin are up-regulated. The down-regulated
peroxidase genes are phylogenetically dispersed within the Arabidopsis
peroxidase gene family, which includes 73 members
(Tognolli et al., 2002
A number of transcription factors are rapidly up-regulated by cytokinin,
including two AP2/ERF family members and a WRKY DNA-binding protein. The two
AP2 genes cluster within the same subgroup of the large Arabidopsis
ERF/AP2 gene family, and this subgroup as of now has no members with
assigned functions (Sakuma et al.,
2002
Other genes that we have identified as cytokinin up-regulated reflect
previous findings on the effects of cytokinin. Consistent with our
identification of a cytokinin up-regulated expansin, cytokinin has been
implicated in regulating cell expansion in certain tissues, most notably
cotyledons, and a cytokinin up-regulated
One group of genes (cell cycle and meristem genes) was conspicuously absent
from our set of induced genes (Fig.
1A; see Supplemental Table V). The cdc2 protein kinase,
CycD3 cyclin, and the STM1 homeobox transcription factor
have all been reported previously to be elevated in response to cytokinin
(Hemerly et al., 1993 Our examination of the temporal expression patterns of genes over our time course of treatments revealed two major patterns. The first is a constant pattern of induction or repression from the first 15-min time point to the 24-h time point, including several of the type-A ARRs, cytokinin oxidase and two AP2s that are constantly induced, and a respiratory burst oxidase and several kinases that are constantly repressed. The second major pattern represented by a small set of genes is one of alteration only after prolonged exposure to cytokinin. This occurs both for induced genes such as a putative disease resistance protein and a glutaredoxin, which are up-regulated after 45 min, and for repressed genes, such as the four peroxidases that do not show down-regulation until after 120 min. While other expression patterns can be identified, they are represented by relatively few genes. Although most rapidly induced genes remain altered at later time points, in many cases, a decrease in the magnitude of the response is observed. This suggests that there is a partial desensitization of the response pathway after prolonged exposure to cytokinin.
We were also able to identify a common upstream sequence motif, GATCTT. The
similarity of this motif to the previously identified type-B ARR-binding site
suggests that type-B ARR genes may mediate induction of genes in addition to
type-A ARRs (Sakai et al.,
2000 To better understand the role of the cytokinin receptor CRE1 in mediating cytokinin response, we examined expression profiles of cytokinin treatment in the receptor mutant alleles wol and cre1-1. We found lower baseline expression levels of the type-A ARRs, suggesting that the response to endogenous cytokinin is affected by the loss of this receptor. However, the general trend for gene expression in the presence of cytokinin was a reduction, but not elimination, of the alteration in transcript levels in both receptor mutants. This suggests a redundancy of cytokinin receptor function, probably by the other CHASE domain-containing His kinase homologs, AHK2 and AHK3. There is also another group of genes that show a lack of induction by cytokinin in the receptor mutants or even in some cases a repression in the mutants relative to the wild type. Although some of these genes may be explained by general variation in expression levels, it is possible that for some, the cytokinin signal acts solely through CRE1. Additional analysis of lines disrupted for CRE1 and other cytokinin receptors may shed further light on this. In conclusion, we have identified through expression profiling a set of cytokinin-regulated genes. Some of these correspond to previously identified cytokinin-regulated genes, and others are novel cytokinin-regulated genes that provide new insight into the mechanism of cytokinin function. More detailed analyses of the function of these novel genes are in progress to better elucidate their role in cytokinin response. Our examination of CRE1 cytokinin receptor mutants suggests that there is redundancy at the level of cytokinin perception, although levels of downstream genes in the absence of exogenous cytokinin are reduced in these receptor mutants. Further analyses of gene expression with full genome arrays and with additional cytokinin mutants should provide additional insight into the function of this hormone.
Plant Growth All plants, except for those in the wol/cre1-1 experiments, were planted at a density of approximately 500 seeds per plate (150 mm) using 0.8% (w/v) top agar (0.8% [w/v] low-melt agarose in 1x Murashige and Skoog salts) on filter paper (150 mm, Whatman no. 3, Whatman, Clifton, NJ) placed on 1% (w/v) agar containing 1x Murashige and Skoog salts + 1% (w/v) Suc buffered to pH 5.7 with MES (Murashige and Skoog media). Seedlings were stratified at 4°C for 4 d, then grown horizontally under constant light (90 µM m–2 s–1) at 22°C for 10 d. The seedlings were then gently scraped off the filter paper into flasks containing 500 mL of liquid Murashige and Skoog media and shaken lightly for 2 h in the light before treatment. At time 0, the indicated concentration of cytokinin (1 or 5 µM BA or 1 µM trans-zeatin) was added, or an equal volume of DMSO was added as a vehicle control to the seedlings, with 0.1% (v/v) DMSO in a final volume of Murashige and Skoog. The treatment of seedlings with cycloheximide was conducted in the same manner as with the cytokinin treatments using either a cytokinin (5 µM BA) or DMSO treatment for 45 min, except that seedlings were treated with 50 µM cycloheximide for 30 min before and during the 45-min cytokinin or DMSO treatment. After the appropriate time in all gene chip treatments, seedlings were harvested and frozen at –80°C until RNA was extracted. For all experiments, RNA from two independent biological replicates was pooled before hybridizing to the chip, except for the wol/cre1-1, for which only one replicate was made. For the time course, there was a gene chip for each of the following: 5 µM BA treatment at 15, 45, and 120 min; two treatments at 480 and 1,440 min; a 1 µM BA treatment at 120 min; and two treatments with 1 µM zeatin at 120 min, as shown in Figure 1A,Figure 1B, along with DMSO control gene chips in parallel with each of the above cytokinin gene chip times. Plants in the wol/cre1-1 experiments were treated in a similar manner as above except that these seedlings were grown vertically on agar plates without filter paper from a line of seeds such that the roots could be harvested easily. After growth at the above conditions for 10 d, roots were harvested and immediately moved to flasks of liquid Murashige and Skoog, where they were treated in a similar manner as the seedlings above. wol- and cre1-treated root samples were normalized to two independent biological wild-type root DMSO-treated gene chip samples. Seedlings used in these experiments treated with 5 µM BA and its DMSO control were of the Wassilewskija ecotype, with 1 µM BA and zeatin treatments, and their DMSO control was of the Columbia ecotype. In the wol/cre1-1 experiment, the Ler ecotype was used as both the wol and the cre1-1 mutants were in the Ler background.
Total RNA was extracted from frozen plant tissue using TRIzol reagent following the manufacturer's instructions (Introgen, Grand Island, NY).Seven micrograms of total RNA was used to synthesize cDNA. A custom cDNA kit (Life Technologies/Gibco-BRL, Gaithersburg, MD) was used with a T7-(dT)24 primer for this reaction. Biotinylated cRNA was then generated from the cDNA reaction using the BioArray High Yield RNA Transcript Kit (Life Technologies/Gibco-BcL, Gaithersburg, MD). The cRNA was then fragmented in fragmentation buffer (5x fragmentation buffer: 200 mM Tris-acetate [pH 8.1], 500 mM KOAc, and 150 mM MgOAc) at 94°C for 35 min before the chip hybridization. Fifteen micrograms of fragmented cRNA was then added to a hybridization cocktail (0.05 µg µL–1 fragmented cRNA; 50 pM control oligonucleotide B2; BioB, BioC, BioD, and cre1-1 hybridization controls; 0.1 mg mL–1 herring sperm DNA; 0.5 mg mL–1 acetylated bovine serum albumin; 100 mM MES; 1 M NaCl; 20 mM EDTA; and 0.01% [v/v] Tween 20), and 10 µg of cRNA was used for each hybridization. Arrays were hybridized for 16 h at 45°C in a GeneChip Hybridization Oven 640. The arrays were washed and stained with R-phycoerythrin streptavidin in the GeneChip Fluidics Station 400. The arrays were subsequently scanned with the Hewlett-Packard GeneArray Scanner (Hewlett-Packard, Palo Alto, CA). Affymetrix GeneChip Microarray Suite 5.0 software was used for washing, scanning, and basic analysis. Sample quality was assessed by examination of 3' to 5' intensity ratios of certain genes.
Affymetrix gene chip data files were imported into GeneSpring 5.0 software and normalized as recommended by the GeneSpring manual for Affymetrix gene chips. Using 50% median normalization, each treatment chip was specifically normalized to a control chip treated with DMSO for a similar time to the actual treatment. Once the data were normalized within GeneSpring, there were analyzed as a ratio to signal in a variety of ways. Data for all experimental treatments are from a single gene chip compared with its respective DMSO time point control. Data for tables and figures were restricted such that values were considered only if genes had positive expression levels for both treated and control microarrays and if the raw value for a gene on a treated microarray was above a raw expression level of 500 or 175 in the DMSO or treated sample when examining down- and up-regulated genes, respectively. A raw level cutoff of 500 was assigned to all gene chip experiments conducted on seedlings with 5 µM BA or the corresponding DMSO control, whereas a raw cutoff of 175 was assigned to all other gene chips experiments. These raw level cutoffs were assigned because expression levels of genes below these cutoff thresholds frequently showed variability greater than 2-fold and often had Affymetrix flag calls of Absent (data not shown). Statistical analyses were performed on Affymetrix gene chip treatments normalized as described above and examined as a log ratio to have a normally distributed population for parametric analysis. Welch's t tests were performed using the Statistical Group Comparison tool in GeneSpring 5.0 software using the GeneSpring Cross-Gene Error model variances. Comparisons were made between treatments of cytokinin or DMSO either for individual time point treatments or across all time points after a Welch's ANOVA determined that there was no significant effect of the time treatment of samples. A P value cutoff of 0.05 was selected for all tests. In addition, the Benjamini and Hochberg False Discovery Rate Multiple Testing Correction option was applied to the examination of the cytokinin up-regulated genes to eliminate against false positives in the larger number of significant genes identified there. The search for potential cis-acting regulatory sequence motifs in the cytokinin up-regulated genes was conducted using the Find Potential Regulatory Sequence tool in GeneSpring 5.0 software. We searched using Find New Sequences to look for oligonucleotides 5 to 13 bp in length that were enriched in the open reading frames from 0 to 1,000 bp upstream of the top 17 cytokinin up-regulated genes from Figure 1A compared with other genes in the Arabidopsis genome. The sequences GATCTT, GATCTTA, AGATCTTA, AAGATC, and AAGATCTT were identified as having single P values below 0.05, observed rates 2 to 3 times the expected rate for those sequences. The only sequences also identified using the above criteria were AAAAAGA and AAAGAAAA, which are likely to simply reflect the A/T-rich value of the non-coding region in the Arabidopsis genome.
We would like to thank Mike Vernon and the University of North Carolina Affymetrix chip facility for their help in labeling and reading the Arabidopsis Affymetrix GeneChips. We also thank the members of the Kieber lab for their helpful comments and suggestions. Received February 4, 2003; returned for revision February 25, 2003; accepted May 12, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.021436.
1 This work was funded by the National Science Foundation (grant no.
DBI–0077503) and by the National Institutes of Health (grant no.
GM4421–01).
[w] The online version of this article contains Web-only data. The supplemental
material is available at
http://www.plantphysiol.org. * Corresponding author; e-mail jkieber{at}unc.edu; fax 919–962–1625.
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ABSTRACT
RESULTS
-expansin
(Crowell et al., 1990
0.05 because these are
still potential cytokinin-regulated genes. These results are further supported
by the fact that four of the top 17 genes, ARR4, ARR5, ARR7, and
ARR16, have been identified previously as cytokinin-responsive type-A
ARRs (D'Agostino et al., 2000
