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American Society of Plant Biologists Global and Hormone-Induced Gene Expression Changes during Shoot Development in Arabidopsis
a Plant Sciences Institute, Iowa State University, Ames, Iowa 50011 2 To whom correspondence should be addressed. E-mail shh{at}iastate.edu; fax 515-294-5256
A global analysis of gene expression events during shoot development in Arabidopsis was conducted using oligonucleotide array analysis. Shoots can be induced in tissue culture by preincubating root explants on an auxin-rich callus induction medium (CIM) and by transferring explants to a cytokinin-rich shoot induction medium (SIM), during which time explants become committed to shoot formation and ultimately form shoots. Oligonucleotide array data obtained during shoot development from  8000 Arabidopsis genes were subjected to principal component analysis, which demonstrated that the major components of variation in gene expression during shoot development can be represented by groups of genes, each group of which is upregulated at only one developmental stage. Two percent to three percent of the 8000 Arabidopsis genes monitored in this study were upregulated by fourfold or more at any one stage during shoot development. When upregulated and downregulated genes were categorized by function, it was observed that numerous hormone response genes were upregulated during preincubation on CIM. Groups of genes involved in signaling and/or transcription were induced at or before the time of shoot commitment, and genes that encode components of the photosynthetic apparatus were upregulated later in development before shoot emergence. Primary hormone response genes, such as Aux/IAA genes, were upregulated during preincubation on auxin-rich CIM, and cytokinin-responsive response regulator genes were upregulated during incubation on cytokinin-rich SIM. The expression of ARABIDOPSIS RESPONSE REGULATOR5, a type-A response regulator gene, was upregulated at the time of shoot commitment, and its expression was localized to sites of presumptive shoot formation. Two "hybrid" His kinases involved in cytokinin responses, CRE1, which encodes a cytokinin receptor, and CKI1, a gene that is capable of conferring cytokinin-independent shoot development, were upregulated during incubation on SIM.
The power of plant hormones to promote development in tissue culture has amazed and baffled plant scientists for years. In pioneering experiments, Skoog and Miller (1957)  demonstrated that the developmental fate of regenerating tobacco pith cells in tissue culture could be directed by the balance of cytokinin and auxin added to the growth medium: high cytokinin/auxin ratios promoted shoot formation, and low ratios favored the formation of roots. To this day, the findings of Skoog and Miller remain largely unexplained, as do the mechanisms by which cytokinin and auxin promote shoot or root development.
The regeneration of organs from explanted vegetative tissue in culture is a form of organogenesis or adventitious shoot and root development. Regeneration in various species can be classified by whether organogenesis is direct or indirect (Hicks, 1994
The hormone requirements for shoot regeneration in tissue culture have been investigated extensively in various species; however, the developmental events that occur during regeneration and the mechanisms by which the hormones promote shoot development are less well known. Recently, it was found that genes related to those encoding components of the cytokinin signaling pathway influence the formation of shoots (Hwang and Sheen, 2001
CRE1/AHK4/WOL (CRE1) encodes a sensor His kinase that was the first to be reported as a cytokinin receptor (Inoue et al., 2001
The effectors in the cytokinin signaling pathway in Arabidopsis are ARRs. The Arabidopsis genome encodes >30 ARRs or ARR-like proteins (Hwang et al., 2002
Some of the type-B ARRs function as transcriptional activators of cytokinin-induced gene expression, whereas the type-A ARRs may serve as repressors (Hwang and Sheen, 2001
Auxin also plays a critical role in shoot development in tissue culture, serving as the principal hormone in CIM to promote callus formation and, seemingly, as a counterpart to cytokinin in SIM. It is known that auxin activates the expression of three major families of primary response genes: the Aux/IAA (auxin/indoleacetic acid) genes, the GH3 gene family, and the SAUR gene family (Abel and Theologis, 1996
The production of shoots from roots must involve considerable genetic reprogramming. The magnitude of gene expression changes during the regeneration of shoots from roots can be estimated from microarray analyses, such as the study by Ruan et al. (1998) Here, we describe the global program of gene expression during shoot development and the changes in expression of genes involved in cytokinin and auxin signaling. In doing so, we identify some important trends in large-scale gene expression patterns and genes that may be key regulators of this process.
Global Gene Expression Patterns The developmental events involved in shoot formation can be studied during regeneration of shoots from root explants in Arabidopsis tissue culture. To do so, root explants were preincubated on CIM for 4 days and then transferred to SIM for 14 or 15 days. Under these conditions, explants acquire competence to respond to shoot formation signals during preincubation on CIM (Cary et al., 2001 ). On transfer to SIM, explants become committed to shoot formation after 6 days on SIM, and shoots begin to emerge 6 to 8 days later (12 to 14 days on SIM; Cary et al., 2002). To describe the program of gene expression that underlies shoot development, a global analysis of gene expression was undertaken using oligonucleotide array analysis. Root segments were explanted in bulk from Arabidopsis seedlings and harvested for RNA extraction at seven different time points (time 0, 2 and 4 days on CIM, and 3, 6, 10, and 15 days on SIM). Total RNA was used to generate cDNA probes and hybridized to Affymetrix Arabidopsis GeneChips. Gene expression changes at different time points in shoot development were compared using scatterplots. The error variation from chip to chip was assessed in scatterplots by hybridizing the time-0 probe to two different chips (Figure 1A) . The variation resulting from experimental error from chip to chip increased with decreasing signal intensity. We eliminated from consideration gene expression profiles with maximum signal intensities of <500 (Figure 1A, dotted lines), because signals below that threshold frequently varied by more than twofold (Figure 1A, solid gray lines.) Thus, by focusing on genes with a greater than fourfold change in gene expression and with a maximum signal intensity of >500, we eliminated most false-positive results.
Error variation arising from tissue culture procedures was reduced by replicating and pooling samples within a time point. Root segments were explanted onto 10 to 20 Petri plates. (More plates and root segments were required for earlier time points to obtain 1 g fresh weight of material.) The root segments on each plate were maintained as separate groups during transfer from CIM to SIM plates and were pooled with samples from other plates before RNA extraction. Error variation arising from time point to time point was estimated by comparing the expression of ubiquitin genes at different time points. For ubiquitin 4 (At5g20620), the mean signal intensity and standard error was 6378 ± 338 (±5.2% of the mean). For ubiquitin 11 (At4g05320), there are three sets of oligonucleotides on the Affymetrix 8000 GeneChips. The standard errors for the mean of determinations at various time points ranged from ±3.3 to ±4.9% of the mean. Because we chose to study genes that are upregulated or downregulated by fourfold or more (Figure 1A, dashed lines), the error variation from extraction to extraction appears to lie well within the experimental variation for most genes of interest with signal intensities of >500. Error variation from experiment to experiment was estimated by conducting another independent experiment (several months later) and comparing one of the time points (3 days on SIM) between the two experiments. (This time point was chosen because, as described below, there are interesting changes in the expression of genes that encode signaling components and transcription factors at this stage.) Scatterplots comparing the data at the single time point between the two experiments (Figure 1B) show no greater error variation than the chip-to-chip variation described above (Figure 1A). Scatterplots were used to compare gene expression levels (signal intensity) at the first time point, 2 days on CIM (Figure 1C), and the last time point, 15 days on SIM (Figure 1D), with those at time 0 (time of explant). The scatterplots demonstrated that many genes were upregulated and downregulated (with respect to the time-0 control) at both the earliest and latest time points. After 2 days of incubation on CIM, 189 genes (2.3% of the total) were upregulated by more than fourfold (and had signal intensities of >500) compared with time 0. At 15 days of incubation on SIM, 257 genes (3.1% of the total) were upregulated by more than fourfold (and had signal intensities of >500) compared with time 0. It was surprising that more genes were not upregulated as shoot development progressed. One might expect that if shoot development was the result of a gene cascade stimulated by hormonal signals, more genes might be upregulated with time. Some of this was seen during incubation on SIM. At 3 days on SIM, 141 genes (1.7% of the total) were upregulated by more than fourfold compared with time 0. The number of genes upregulated by more than fourfold approximately doubled at 15 days of incubation on SIM. However, one might expect greater numbers of genes to be upregulated in a gene cascade.
One reason why more genes are not upregulated by 15 days on SIM is that many genes are expressed transiently (i.e., their expression is stage specific). This can be seen in a hierarchical cluster of expression profiles grouped by pattern similarity (Figure 2A)
. Although not all
Principal Component Analysis of Gene Expression Patterns Because the data sets in the scatterplots and cluster analysis were large, we attempted to reduce their dimensionality to seven or eight components using principal component analysis. Principal component analysis showed that the most significant component contributing to the variation in gene expression profiles was a pattern representing "no change" in gene expression (Figure 2B). The second most significant component was one that peaked at two time points, 2 and 4 days on CIM. However, the next most significant components were profiles that peaked at single time points. To understand the gene expression changes that gave rise to these principal components, expression profiles were sorted into groups of genes with stage-specific patterns that peaked at different time points. The groups were defined by selecting genes with prototypic expression profiles peaking at each time point and using the profiles of these genes to search for other genes with similar profiles. The identification of some of the highly upregulated genes in each group gave a snapshot of the changes in the gene expression program at different developmental stages.
The gene AIR1A (At4g12550), which encodes the wall–plasma membrane disconnecting protein, was selected as the most highly upregulated gene with a profile peaking at 2 days on CIM (Table 1). This is a critical time because it is when root explants acquire nearly full competence to respond to shoot induction signals (Cary et al., 2001
The gene with a prototypical expression profile peaking at 4 days on CIM was PDF1 (At2g42840), which encodes protodermal factor 1 (Table 1). Four days on CIM also is a key time point because explants acquire full competence for shoot formation at this time and are poised to respond to shoot development signals when transferred to SIM (Cary et al., 2001 ). The high performers at this time point included two genes that encode nonspecific lipid transfer protein isoforms (At2g38540 and At2g38530) (Table 1). Among the putative transcription factors and signaling proteins with similar profiles were those encoding a putative AP2 domain transcription factor (At4g23750), a putative heat shock transcription factor (At2g26150), and a homeodomain-containing protein (At4g36740). A gene that encodes a cold- and abscisic acid–inducible protein (At5g15960) was chosen as a highly expressed gene with a stage-specific pattern peaking at 3 days on SIM (Table 1). Curiously, other high performers with similar expression profiles were genes, such as KIN1 (At5g15960) and KIN2 (At5g15970), that also encode cold-regulated proteins (Table 1). Genes with similar expression profiles that encode putative transcription factors included the genes encoding transcription factor BBFa (At3g61850), a myb-like transcription factor (At5g60890), and a PERIANTHIA transcription factor (At1g68640).
A prototypic gene selected with an expression profile peaking at 6 days on SIM was an expansin gene (At2g40610) (Table 1). A profile similarity search revealed another expansin gene (At1g69530) as a highly upregulated gene at this stage, along with an aquaporin/MIP-like protein (At3g54820), a putative pectate lyase (At4g247880), and a type-A response regulator, ARR5 (At3g48100) (Table 1). (ARR5 will be discussed further below.) CUP-SHAPED COTYLEDON2 (At5g53950), a gene encoding a NAC domain–containing protein required for shoot meristem formation (Aida et al., 1999 A gene that encodes a hypothetical protein (At4g27730) was chosen as the gene with a prototypical profile peaking at 10 days on SIM (Table 1). This gene was used to cull others with similar profiles, and among the top performers were two genes that encode putative peroxidases (At4g36430 and At2g18150) and one that encodes a metallothionein (At5g02380) (Table 1). Interesting upregulated genes that encode transcription factors and signaling components included a putative protein kinase (At2g32800) and myb-related transcription factors (At5g57620 and At5g23000). The final time point in our study was 15 days on SIM, a time when shoots emerge. The prototypic gene upregulated at this stage was one that encodes a chlorophyll a/b binding protein (At5g01530) (Table 1). The most highly induced genes with expression profiles similar to the cab gene included a gene encoding a small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (At5g38410) and one encoding a Gly-rich protein (At2g05520) (Table 1). Genes that encode a putative RING-H2 zinc finger protein (At2g15580) and two putative transcription factors, a homeodomain (At2g35940) and a CCAAT binding factor (At4g14540), also were upregulated at 15 days on SIM. These and other gene expression profiles during shoot development can be seen at http://www.bioinformatics.iastate.edu/howell/.
Expression Changes of Genes Grouped by Function Some interesting trends were observed during the time course of development among the upregulated genes. In particular, the number of genes that were involved in hormone responses increased dramatically at 2 days on CIM and decreased rapidly during shoot development (Figure 3A) . Most of the hormone response genes in this group were Aux/IAA genes, and these genes will be described more fully below. Another interesting trend late in shoot development was the dramatic increase in genes that encode components of the photosynthetic apparatus (Figure 3A). As stated above, this was not unexpected because explants green and shoots emerge later during incubation on SIM. Another trend was the increase in the numbers of upregulated genes that encode transcription factors and signaling components during the transition from CIM (4 days on CIM) to SIM (3 days on SIM) (Figure 3A). This appears to be a time during shoot development when the gene regulatory machinery undergoes a major transition.
Unlike the upregulated genes, there were almost no significant trends during shoot development among the downregulated genes (Figure 3B). In comparing the overall patterns of upregulated to downregulated genes, it is apparent that the downregulated genes are dominated by fewer categories of genes, particularly by the hypothetical/unknown genes and the genes encoding cell wall components or enzymes that are involved in cell wall metabolism. Cell wall metabolism genes are both upregulated and downregulated in greater numbers than most other categories of genes during shoot development (Figures 3A and 3B). It is of interest that the most highly downregulated genes were large numbers of genes encoding peroxidases.
Cytokinin-Related Gene Expression Patterns
Cytokinins rapidly activate the expression of type-A ARRs (Brandstatter and Kieber, 1998
Because ARR5 is highly responsive to cytokinin and is induced during shoot development, we attempted to localize ARR5 expression by generating an ARR5 promoter:reporter construct (pARR5:GUS). Several transgenic lines containing the construct showed high-level induction of  -glucuronidase (GUS) in seedlings exposed to cytokinin (20 µM 6-benzyladenine) (data not shown). In root explants, we were interested in whether ARR5 expression, which peaked during incubation on SIM, marked presumptive sites of shoot emergence. We found that pARR5:GUS generally was expressed in the proliferating pericycle/callus tissue along the length of the root segment during preincubation on CIM and early during incubation on SIM (Figure 4C). GUS expression peaked at 6 days on SIM; after that, it diminished except at sites of callus outgrowth, from which shoots tended to emerge. Thus, later during incubation on SIM, pARR5:GUS expression was concentrated in regions of callus protuberances and may mark presumptive sites of shoot formation. Emerging shoots showed very little ARR5 expression. In general, ARR5 expression levels tended to increase with the production of undifferentiated callus tissue. ARR5 expression levels remained high in callus destined to give rise to green callus and shoots, but the gene was not expressed in differentiated tissues of the emerging shoot, except in the newly formed shoot meristem.
Type-B ARR genes are thought to be genetic activators in the cytokinin signaling pathway (Hutchison and Kieber, 2002
We also examined the expression profiles of genes that encode "hybrid" His kinases involved in cytokinin perception and signaling. CRE1/AHK4/WOL (At2g01830; hereafter referred to as CRE1) was the first hybrid His kinase shown to function as a cytokinin receptor and to be capable of conferring cytokinin responsiveness in a heterologous (yeast) system (Inoue et al., 2001
Auxin-Related Gene Expression Patterns Auxin is a key hormone present in CIM and one that is critical to maintain in balance with cytokinin during shoot development. A number of hormone response genes are upregulated during incubation on CIM, and many of these encode Aux/IAA proteins. The Affymetrix Arabidopsis 8000 GeneChip contains 14 of the 30 known Aux/IAA genes. Of those, eight were upregulated on CIM; IAA5 (At1g15580) and IAA1 (At4g14560) were upregulated by >20-fold and IAA19 (At3g15540) was upregulated by >10-fold (Figure 6A)
. All but one, IAA8 (At2g22670), was downregulated after transfer to SIM. Abel et al. (1995) observed that IAA8 is not highly regulated by auxin, so its pattern of expression may reflect the relative nonresponsiveness of that gene to the high levels of auxin in CIM. The six other Aux/IAA genes have different expression profiles that do not relate strictly to the presence of auxin in CIM (Figure 6B). For example, levels of the IAA17/AXR3 (At1g04250) transcripts increased during incubation on SIM and peaked at 6 days on SIM, declining thereafter. Many of the Aux/IAA genes that were upregulated on CIM were identified originally by their responsiveness to auxin, particularly IAA1 to IAA14 (At4g14550), except for IAA8 (Abel et al., 1995), whereas the others were recognized as members of the Aux/IAA gene family by sequence similarity (Liscum and Reed, 2002).
In companion articles, we identified developmental events, such as the "acquisition of competence" and "shoot commitment," that occur during shoot formation (Cary et al., 2001 , 2002). Explants acquire the competence to respond to shoot formation signals during preincubation on CIM (Figure 7)
. Explants do not form shoots on SIM unless they are preincubated on CIM. Explants also become committed to producing roots during CIM preincubation, so that if explants are transferred to basal medium, they continue to develop roots. Explants rapidly lose their commitment to form roots when transferred to SIM (Cary et al., 2002). After that, they become committed to the formation of shoots (6 to 8 days on SIM). Green foci (centers of green callus) begin to form at approximately that time, and shoots emerge.
The general patterns in gene expression during shoot development provide insights into the mechanisms that may underlie this developmental process. First, a number of hormone response genes, largely Aux/IAA genes, were upregulated during preincubation on CIM. Second, many genes that encode signaling and/or transcription components were highly induced before shoot emergence at approximately the time of shoot commitment. This finding suggests that there may be substantial changes in signaling and gene regulatory activities at these stages. Third, as shoots emerged, genes that encode products of differentiated cells were the most highly induced—mostly genes that encode components of the photosynthetic apparatus. We found little evidence in the overall phenomenon of gene expression changes for a simple cascade of events during shoot development. Based on such a model, one might expect exponentially increasing numbers of genes to be upregulated with time. Instead, many genes were upregulated throughout the time course of shoot development, and most genes that were upregulated subsequently were downregulated. Principal component analysis demonstrated that the major components that contribute to the variation in the overall pattern of gene expression represent groups of genes, each group of which is upregulated at a single developmental stage. However, the total picture may miss many important subpatterns that take the form of gene cascades. One concern that we have had about this analysis is that early events leading to shoot development might be obscured by the presence of other tissues in the root explants that do not participate in the organization of the shoot primordium. We have tried to minimize this problem by using root explants from seedlings of the Columbia ecotype, which are fairly robust in forming shoots, usually producing one or two shoots per explant. However, that leaves much of the explant uninvolved in shoot formation. If the early events in shoot formation engage only a few progenitor cells, it is unlikely that one would see gene expression changes associated with a minority of cells. On the other hand, it is possible that much tissue in the root explants engage in the early events of shoot formation and that cells progressively drop out of the process as development proceeds. One gains that impression by comparing green callus formation with shoot development. Shoots generally develop from broader areas of green callus, usually from protuberances in the green callus patches (green foci) that develop on explants. Not all green calli and protuberances give rise to shoots. However, if green callus formation is a step in the production of shoots, then shoot development may be a selective process whereby fewer and fewer cells reach later milestones in the shoot formation process as development proceeds.
In another study (Cary et al., 2002
Response Regulators
By contrast, type-B ARR genes such as ARR1 and ARR2 were not regulated demonstrably during shoot development in our system, despite the fact that these genes have extraordinary powers in shoot development. For example, ARR2 overexpression can drive cytokinin-independent shoot formation (Hwang and Sheen, 2001
His Kinases
Because CKI1 overexpression promotes cytokinin-independent shoot development (Kakimoto, 1996
Aux/IAA Genes
Shoot Development Conditions Arabidopsis thaliana ecotype Columbia seedlings were grown to 7 days of age on plant nutrient solution medium [5 mM KNO3, 2.5 mM KH2PO4, 2 mM MgSO4, 2 mM Ca(NO3)2, 0.05 mM Fe/EDTA, 70 µM H3BO3, 14 µM MnSO4, 0.5 µM CuSO4, 1.0 µM ZnSO4, 0.2 µM Na2MoO4, 10 µM NaCl, 0.01 µM CoCl2, 5 g/L Suc, pH 5.5, and 0.7% Bacto-agar]. Root segments (5 mm) were cut and transferred to callus induction medium (CIM), which consists of Gamborg's B5 medium (Gamborg et al., 1968 ) with 5 g/L Mes, 2.2 µM 2,4-D, 0.2 µM kinetin, and 0.8% agarose. Explants were incubated on CIM for 4 days under constant light conditions and then transferred to shoot induction medium (SIM). SIM is prepared as CIM except that SIM contains the hormones isopentenyladenine (5.0 µM) and 3-indoleacetic acid (0.9 µM). Root reduction medium contains 0.9 µM 3-indoleacetic acid. After transfer to SIM, explants were incubated under constant-light conditions to induce shoot formation. Shoot development was recorded with a Nikon CoolPix 990 digital camera (Tokyo, Japan).
RNA Procedures RNA samples (20 µg), isolated from various hormone- and inhibitor-treated tissues, were denatured at 95°C and subjected to electrophoresis in 1% (w/v) agarose and 1 x Mops [0.2 M 3-(N-morpholino)-propanesulfonic acid, 0.5 M sodium acetate, and 0.01 M EDTA] on 13.3% formaldehyde gels. RNA was blotted to Hybond-N membranes (Amersham Pharmacia). Probes specific for ARR5 and UBQ5 were generated by PCR and labeled by random-hexamer labeling with a DNA-bead labeling kit, as described by the manufacturer (Amersham Life Sciences). The primers used for UBQ5 amplification were UBQ5F (5'-CTTGAAGACGGCCGTACCCTC-3') and UBQ5R (5'-CGCTGAACCTTTCAAGATCCATCG-3'). ARR5-specific primers were IBC6F (5'-CTGAGGTTTTGCGTCCCGAGATG-3') and IBC6R (5'-GCGCGTTTTAGCTGCGAGTAGATATC-3'). Probes were hybridized to the membranes overnight in Church buffer (7% SDS, 1% BSA, 1 mM EDTA, and 250 mM NaH2PO4). The membranes were washed and imaged using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Signals were quantified with ImageQuant software (Molecular Dynamics).
Quantitative Reverse Transcription PCR
Probe Synthesis and Hybridization to Affymetrix GeneChips RNA samples were submitted to the DNA facility at the University of Iowa (http://dna-9.int-med.uiowa.edu/microarrays.htm) and were handled as follows. An aliquot of copy RNA (cRNA) control mixture was added to 10 µg of purified total RNA. The cRNA control mixture is composed of a set of four in vitro transcripts generated by T3 RNA polymerase, encoding the Bacillus subtilis genes thr, trp, phe, and lys. Probes corresponding to these bacterial transcripts are represented on all GeneChips (including test chips) from Affymetrix (Santa Clara, CA). Each synthetic transcript is quantified and represented at copy numbers of 5 x 106 to 5 x 108, corresponding approximately to the expected dynamic range of detection for the GeneChip system. This set of control transcripts allows for the monitoring of cDNA synthesis and the in vitro transcription of biotinylated targets and also provides a reference sample for normalizing between experiments. RNA was converted to double-stranded cDNA using the SuperScript Choice system (Life Technologies) according to the supplier's protocol, except that an HPLC-purified T7-dT24 primer (Genosys) was used for first-strand synthesis. After synthesis, double-stranded cDNA was purified by phenol/chloroform extraction and ethanol precipitation. The purified cDNA was used to generate biotinylated cRNA target using a Bioarray High-Yield RNA Transcript-Labeling Kit (Enzo, Farmingdale, NY) according to the supplier's protocol. The labeled cRNA then was purified using the RNeasy total RNA cleanup protocol (Qiagen) and quantified by absorbance at 260 nm. cRNA (20 µg) was fragmented by heating at 94°C for 35 min in fragmentation buffer (40 mM Tris-acetate, pH 8.1, 125 mM KOAc, and 30 mM MgOAc). An aliquot of fragmented and unfragmented cRNA was analyzed by formaldehyde/agarose gel electrophoresis to ensure appropriate size distribution (average size, 700 bp of unfragmented cRNA and 100 bp after fragmentation). Fragmented cRNA (15 µg) was added to a hybridization "cocktail" containing 50 pM of control oligonucleotide B2 (Genosys, The Woodlands, TX), 1 x control cRNA mixture, 0.1 mg/mL herring sperm DNA, and 1 x Mes hybridization buffer in a volume of 300 µL. Control oligonucleotide B2 (5'-biotin:GTCGTCAAGATGCTACCG-TTCAGGA-3') was designed to hybridize to structural features on the chip to allow for proper scanning and grid alignment. The control cRNA mixture was composed of a second set of four biotinylated in vitro antisense transcripts of cDNAs encoding the Escherichia coli biotin synthesis genes bioB, bioC, and bioD and the P1 bacteriophage cre recombinase gene. Probes corresponding to these bacterial transcripts also are represented on all Affymetrix GeneChips (including test chips). Each synthetic transcript was quantified and represented at copy numbers of 2 x 108 to 2 x 1010, corresponding approximately to the expected dynamic range of detection for the GeneChip. This set of control cRNAs allows for the monitoring of hybridization, washing, and staining conditions and also provides a second set of reference samples for normalizing between experiments. The hybridization cocktail was heated to 95°C and then cooled to 45°C, and then 100 µL was applied to a Test3 GeneChip array. The remainder of the cocktail was stored at -20°C, and the test chip was hybridized at 45°C for 16 h. After hybridization, the hybridization cocktail was removed from the chip and stored at -70°C. The chip was placed immediately in the Affymetrix GeneChip Fluidics Station 400 and washed using the preprogrammed step "mini_euk2." This wash program involves a low-stringency wash, a high-stringency wash, a steptavidin/phycoerythrin stain, a low-stringency wash, an anti-streptavidin antibody stain, a second steptavidin/phycoerythrin stain, and a final low-stringency wash. After washing and staining, the test chip was placed in the Affymetrix GeneChip array scanner, and image data were captured and converted to numerical output using Microarray Analysis Suite version 5.0 (Scanalytics, Fairfax, VA). For the test chip, the levels of bioB, bioC, bioD, cre, phe, trp, lys, and thr were assessed. When the labeled cRNA target passed all metrics of the Test3 chip, it was again heated to 95°C, cooled to 45°C, and hybridized to the GeneChip of interest. Hybridization, washing, staining, and scanning were repeated as described above except that the automated fluids program "euk2" was used. Experimental data acquired from the chip were analyzed using the GeneChip Microarray Analysis Suite version 5.0. For comparison between two chips, the "baseline chip" intensity values were measured and normalized to the average signal intensity. Intensity values of the "experimental chip" then were compared with baseline chip values and a "difference change" was calculated. Data were analyzed using GeneSpring version 4.1.5 (Silicon Genetics, Redwood City, CA).
ARR5 Promoter/Reporter Gene Plasmid Construction
Plant Transformation and Culture Conditions Seeds from the dipped plants were surface-sterilized with 50% bleach and 0.02% Triton X-100. Seeds were resuspended in 0.1% sterile agar and plated on kanamycin–plant nutrient solution selection plates with 50 µg/mL kanamycin. Transformants were identified and transferred to plant nutrient solution plates to recover for 7 days in the growth room and then planted in soil. Transformants were grown for an additional 4 to 6 weeks and allowed to self-pollinate, and seeds representing the T1 generation were collected. Upon request, all novel materials described in this article will be made available in a timely manner for noncommercial research purposes. No restrictions or conditions will be placed on the use of any materials described in this article that would limit their use for noncommercial research purposes.
D.J.G. and S.H.H. acknowledge support from the National Science Foundation/Cornell Plant Cell and Molecular Biology Program and from the Boyce Thompson Institute. This work was supported in part by the Plant Sciences Institute at Iowa State University.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.006668.
1 Current address: Department of Horticulture, University of Wisconsin, Madison, WI 53706. Received August 12, 2002; accepted August 19, 2002.
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Abstract
INTRODUCTION
