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First published online November 5, 2004; 10.1104/pp.104.047233 Plant Physiology 136:4169-4183 (2004) © 2004 American Society of Plant Biologists Analysis of Sequence, Map Position, and Gene Expression Reveals Conserved Essential Genes for Iron Uptake in Arabidopsis and Tomato1,[w]Institute of Plant Genetics and Crop Plant Research, D–06466 Gatersleben, Germany
Arabidopsis (Arabidopsis thaliana) and tomato (Lycopersicon esculentum) show similar physiological responses to iron deficiency, suggesting that homologous genes are involved. Essential gene functions are generally considered to be carried out by orthologs that have remained conserved in sequence and map position in evolutionarily related species. This assumption has not yet been proven for plant genomes that underwent large genome rearrangements. We addressed this question in an attempt to deduce functional gene pairs for iron reduction, iron transport, and iron regulation between Arabidopsis and tomato. Iron uptake processes are essential for plant growth. We investigated iron uptake gene pairs from tomato and Arabidopsis, namely sequence, conserved gene content of the regions containing iron uptake homologs based on conserved orthologous set marker analysis, gene expression patterns, and, in two cases, genetic data. Compared to tomato, the Arabidopsis genome revealed more and larger gene families coding for the iron uptake functions. The number of possible homologous pairs was reduced if functional expression data were taken into account in addition to sequence and map position. We predict novel homologous as well as partially redundant functions of ferric reductase-like and iron-regulated transporter-like genes in Arabidopsis and tomato. Arabidopsis nicotianamine synthase genes encode a partially redundant family. In this study, Arabidopsis gene redundancy generally reflected the presumed genome duplication structure. In some cases, statistical analysis of conserved gene regions between tomato and Arabidopsis suggested a common evolutionary origin. Although involvement of conserved genes in iron uptake was found, these essential genes seem to be of paralogous rather than orthologous origin in tomato and Arabidopsis.
Arabidopsis (Arabidopsis thaliana) serves as the reference for dicot genome analysis regarding gene sequence, gene number, and gene function. In the minimal genome concept, it is assumed that orthologous gene functions transmitted in a lineage-dependent vertical manner in related organisms should be conserved by sequence and function if they are essential (Mushegian, 1999  ). Using systematic gene knockout approaches and genome sequence comparisons, it was calculated that Bacillus subtilis may have about 250 to 300 essential genes (Kobayashi et al., 2003). Ninety-six percent of essential genes are conserved in bacteria and encode proteins with known functions in cell metabolism and energetics, information processing, and cell growth and division (Kobayashi et al., 2003). Yeast (Saccharomyces cerevisiae) may have about 1,000 essential genes (Winzeler et al., 1999; Giaever et al., 2002). In plants, the number of conserved and essential plant genes still remains to be experimentally determined. It is unclear whether searching for conserved sequence homologs in expressed sequence tag (EST) and genomic databases of diverse plant species may provide clues to essential gene functions. A potential obstacle for such homolog searches in plants is the fact that plant genomes underwent large evolutionary genomic rearrangements involving polyploidization, chromosome rearrangement, and partial gene loss (Gaut et al., 2000; Simillion et al., 2002). The analysis of the full genome sequence suggested that the Arabidopsis genome contained duplicated blocks corresponding to ancient and recent genome duplication events (Arabidopsis Genome Initiative, 2000; Blanc et al., 2000, 2003; Vision et al., 2000; Simillion et al., 2002; Bowers et al., 2003; Ermolaeva et al., 2003). These genome duplication events led to horizontal multiplication of homologous genes known as paralogs. The presence of paralogous genes hampers straightforward genome colinearity studies between diverged species of different families.
Full genomic sequence of the five Arabidopsis chromosomes and skeletons of genomic and EST markers mapped onto the chromosomes of other dicot species are currently available for assessing homologous gene functions (Fulton et al., 2002
Since iron is required for many basic enzymatic reactions and biological processes in all organisms, iron uptake is a strictly essential mechanism for growth of any organism. In plants, iron deficiency is caused when iron is not available due to low solubility, as is the case on alkaline and calcareous soils. Plants are able to cope with iron deficiency if they mobilize sufficient iron from their environment (Hell and Stephan, 2003 In this study, the term homologous genes or proteins refers to genes or proteins that have similar sequences that qualify them to share common properties, such as specific transporters or enzymes. The term homologous function specifies that the homologous genes and proteins act in a similar biological context, such as metal transporter in root iron mobilization. We made use of the essential characters of the genes FRO2, IRT1, NRAMP, NAS, and FER to analyze homology between these gene functions in Arabidopsis and tomato. By investigating sequence similarity, map position, and functional expression data, we identified gene pairs that represent the homologous functions in the two species. We discuss conservation of gene function with respect to orthologous and paralogous origin of the genes.
Identification and Sequence Comparison of Tomato-Arabidopsis Iron Uptake Homologs To identify homologs of iron uptake proteins from tomato and Arabidopsis, we screened the databases using the amino acid sequences of AtIRT1, LeIRT1, LeNRAMP1, LeNRAMP3, AtFRO2, LeNAS, and LeFER ("Materials and Methods;" Table I). We only retained those sequences for further analysis that were most related according to E-values ("Materials and Methods"). Partial sequences were named according to their transcript unit number in the database (e.g. LeFRO-TC129233). The available peptide sequences were aligned and represented in phylogenetic trees (Fig. 1, shown for IRT and NAS sequences).
LeIRT1 and LeIRT2 showed highest sequence similarity with each other (Fig. 1A). Among the 15 Arabidopsis zinc and iron-regulated (ZIP) transporter sequences (Mäser et al., 2001 ), five of them could be grouped together with LeIRT1 and LeIRT2 (Fig. 1A). One of these, AtIRT1, was shown to be essential for iron uptake, whereas AtIRT2 was iron regulated but not essential for iron uptake (Vert et al. 2001, 2002; Varotto et al., 2002). In the sequence comparisons, AtIRT1 and AtIRT2 did not appear to be the closest homologs. In fact, AtIRT1 was most similar to AtZIP8, and LeIRT1 and LeIRT2 were most similar to AtZIP10. AtZIP7 was distantly related to IRT proteins. All other ZIP sequences from the database were even more distant to IRT than AtZIP7.
LeNRAMP1, LeNRAMP3, AtNRAMP1, AtNRAMP3, and AtNRAMP4 are encoded by iron-regulated genes (Curie et al., 2000
AtFRO2 is required for iron reduction upon low iron supply (Robinson et al., 1999
NAS is essential for iron homeostasis in tomato. Four different Arabidopsis NAS sequences were in the database (see also Suzuki et al., 2001
A single Arabidopsis protein that we named FER-like regulator of iron uptake (AtFRU;At2g28160, bHLH029; Heim et al., 2003 In summary, it was possible to predict a unique homologous Arabidopsis-tomato gene pair in only a single case, namely that of LeFER-AtFRU. For the other four gene functions Arabidopsis had more and larger gene families encoding these functions than tomato.
A further criterion for gene homology between two species is location in a colinear region of the two genomes. For map position comparison, it was necessary first to map the tomato genes.
LeNAS and LeFER map positions were known to be on tomato chromosomes 1 and 6, respectively (Ling et al., 1999
Analysis of Conserved Gene Content in Arabidopsis and Tomato Chromosomal Regions Containing Iron Uptake Genes
Chromosomal regions harboring iron uptake genes of Arabidopsis and tomato genomes were analyzed for their level of conserved genes. For this analysis, we selected COS markers that were mapped by Fulton et al. (2002) We found that 8 tomato COS markers out of 24 were located in the region of LeIRT1/LeIRT2 and recognized at least one Arabidopsis region containing either AtIRT1/AtIRT2, AtZIP8, or AtZIP10 (Fig. 3A). Two of these tomato COS markers were homologous to multiple Arabidopsis COS markers that were located in two and three IRT/ZIP regions, respectively. Therefore, we could identify three Arabidopsis regions harboring IRT/ZIP genes that shared several conserved gene sequences with the LeIRT1/LeIRT2 region in tomato. Although only 10 COS markers were available for the LeNRAMP1 region, we could identify 2 that were located near AtNRAMP1 (Fig. 3B). Single LeNRAMP1-neighboring COS markers identified regions of AtNRAMP4, AtNRAMP5, and AtNRAMP6. Three COS markers were located near AtNRAMP3. The LeNRAMP3 region showed similarity to the region of AtNRAMP3 (3 COS markers out of 26), and, to a lesser extent, to that of AtNRAMP4 (two COS markers) and AtNRAMP5 (one COS marker; Fig. 3C). The tomato region of LeFRO-TC124302 showed similar gene sequences to the Arabidopsis regions around the FRO-like homologs At5g23990/At5g23980 (4 COS markers out of 35), AtFRO2/AtFRO1 (one COS marker), and At5g50160/At5g49730/At5g49740 (seven COS markers; Fig. 3D). The LeFRO1/LeFRO-TC129233 region showed similarity to the regions of AtFRO2/AtFRO1 (3 COS markers out of 21) as well as At5g23980/At5g23990 (one COS marker; Fig. 3E). No indication for conserved genes was found for the region of Arabidopsis FRO3. For the LeNAS regions we identified four corresponding regions in Arabidopsis (twice 2 and twice 3 COS markers out of 29; Fig. 3F), indicating that all four Arabidopsis NAS regions were related. Only a single COS marker out of 21 recognized the LeFER and AtFRU regions (Fig. 3G). With the exception of the LeFER/AtFRU regions, we could thus determine that 20% to 50% of the analyzed tomato COS markers recognized Arabidopsis genes located in clusters in the vicinity of the respective iron uptake genes. We realized that clusters of conserved COS markers could also be found between regions that did not appear related by the presence of homologous iron uptake genes. To analyze this point further, we searched for matches between COS markers of 6 unrelated iron uptake regions (42 comparisons of nonhomologous regions and the 7 comparisons of homologous regions as control; Table II). We found that the number of matching COS markers was highest when comparing Arabidopsis and tomato regions with corresponding iron uptake genes (three to five conserved genes; only exception was LeFER/AtFRU). Only in the case of LeFER was the highest level of gene conservation observed with a different region. Only 4 out of 42 comparisons of regions with nonhomologous iron uptake genes showed similar levels of conserved gene content as the regions with corresponding iron uptake genes (three and four conserved COS markers), namely Lefro-TC124302-AtIRT1/AtIRT2, LeFRO-TC124302-At1g09240 (AtNAS3), LeFER-At5g23990 (FRO-like), and LeNRAMP1-AtFRU. In 19 comparisons with nonhomologous iron uptake gene regions, no matching COS marker was detected. In 12 cases, a single matching COS marker was found. In six cases, two matching COS markers were found. These observations indicate that the level of conserved gene sequences between homologous iron uptake gene regions generally tends to be higher between homologous iron uptake gene regions than between regions with unrelated iron uptake genes.
To analyze the significance of these findings, we determined the probability of the occurrence of tomato-Arabidopsis homolog clusters at random within 2-Mb intervals in the Arabidopsis genome. Similar results were obtained for 4-Mb (±2 Mb) windows (data not shown). We calculated P-values for the occurrence of clustering of the selected Arabidopsis gene homologs within ±1-Mb windows by taking into account the number of neighbors recognizing tomato homologs in the iron uptake region, the number of homologous genes of the same tomato region in the entire Arabidopsis genome, and the number of all genes in the Arabidopsis genome ("Materials and Methods"). Small P-values (P < 0.05) indicate that the number of homologous Arabidopsis neighbors recognized by the corresponding tomato region exceeded significantly the number of neighbors according to random clustering within the ±1-Mb window. We found that 7 out of 31 Arabidopsis iron uptake genes analyzed (22.6%) had P-values less than 0.05, and 43 out of 279 noniron uptake genes analyzed (15%) showed clustering with P-values less than 0.05 (Supplemental Table II). The cumulative distribution of P-values for iron uptake genes indicated that overall iron uptake genes were significantly more clustered than noniron uptake genes (data not shown). Three Arabidopsis homologs of LeIRT1/LeIRT2 had P-values less than 0.015, namely At1g31260, At5g45105, and At4g19690/At4g19680 (Table III). Moreover, 9 out of 11 COS homologs that we suspected to be located in these regions (compare Supplemental Table I and Fig. 3) had P-values less than 0.02 (Table III). The 2 out of 11 remaining markers had P-values of about 0.06, just slightly above the cutoff value of 0.05 below which we considered results to be statistically significant (Table III). Thus, LeIRT1 maps together with four other tomato markers from the ±10-cM region surrounding the LeIRT1 locus to a single ±1-Mb window on the Arabidopsis genome region containing At1g31260 together with 419 genes. This means that in this Arabidopsis region the homologs of tomato markers of the LeIRT1/LeIRT2 region are highly enriched, which is reflected by the average density of about one of these markers every 83 genes (5/419). We would expect only about one of these markers every 500 genes (52/26,404) if the genes were randomly distributed. The significantly similar gene content of the IRT/ZIP regions of tomato and Arabidopsis suggests a common evolutionary origin. An equally significant conservation of clustered genes was found for LeFRO-TC124302/At5g47930/At5g49740/At5g50160 (Table III). Significant clustering was obtained for LeFRO1/LeFRO-TC129233/At1g01580/At1g01590 as well as LeNAS/At1g09240 (Table III). P-values between 0.05 and 0.075 were found for the regions of LeNRAMP1/At2g23150 and LeFRO-TC124302/At1g01580/At1g01590 (Table III). All other comparisons of iron uptake regions between tomato and Arabidopsis showed no significant clustering, with P-values higher than 0.1 (Table III). However, we speculate that, for some of the NRAMP, FRO, and NAS gene regions, P-values would decrease if more COS markers were available for the analysis of these regions.
In summary, Arabidopsis and tomato genome regions with homologous iron uptake genes were generally characterized by the presence of multiple conserved genes. Since the Arabidopsis genome frequently contained more than one region with conserved gene content for any of the studied tomato iron uptake regions, it did not appear to be sufficient to determine a functional homology based on map position and sequence alone.
Within gene families, specific biological functions of gene family members are conferred by their specific expression patterns. Iron uptake genes should be expressed in the root and/or induced by iron deficiency, indicating a function in iron mobilization of external or internal iron. Here, we investigated which of the iron uptake homologs fulfilled these expression pattern criteria in tomato and Arabidopsis. Gene expression was surveyed by analyzing EST expression data available at http://www.tigr.org for tomato genes (Supplemental Table III), as well as experimental gene expression studies in tomato and Arabidopsis ("Materials and Methods;" Figs. 4 and 5).
First, we analyzed expression of IRT/ZIP genes (Fig. 4A). LeIRT1 and LeIRT2 were previously shown to be expressed in the root, whereby LeIRT1 was iron regulated (Bereczky et al., 2003 ). LeIRT2 EST sequences were available in the EST database, but no data were available for LeIRT1. LeIRT2 EST sequences were all derived from root tissue (data not shown). In Arabidopsis (Fig. 4A), AtIRT1 and AtIRT2 were mainly expressed in roots and expression was induced upon iron deficiency, as expected (Eide et al., 1996; Vert et al., 2001). AtIRT2 appeared to be induced at a higher level than AtIRT1. The AtZIP8 expression pattern was similar to that of AtIRT2. AtZIP10 transcripts were detected in roots upon sufficient iron supply. AtZIP7 was mainly expressed in leaves and induced upon low iron supply.
Expression of NRAMP genes was not further analyzed here. LeNRAMP1 and AtNRAMP1 were previously found to be root specific and iron regulated (Curie et al., 2000
EST sequences for LeFRO-TC124302 were found in roots and callus (data not shown). Experimental expression analysis showed that expression of LeFRO-TC124302 was not only root specific, but also slightly iron regulated and dependent on a functional LeFER gene in tomato (Fig. 5). On the other hand, transcripts for LeFRO-TC129233 were not detected experimentally in leaves and roots (data not shown). All EST sequences were derived from flower libraries, indicating that LeFRO-TC129233 was not involved in root iron uptake. As analyzed previously, LeFRO1 was expressed in roots and leaves, up-regulated by iron deficiency, and dependent on a functional LeFER gene (Li et al., 2004
We found expression of Arabidopsis NAS genes in leaves and roots upon sufficient and low iron supply (Fig. 4C), supporting the tomato data that NAS activity was indeed required constantly (Ling et al., 1999
LeFER is expressed in the root but not in the leaves and cotyledons (Ling et al., 2002 Overall, expression data contributed to assigning homologous functions. In general, we found that gene family members from tomato differed more significantly in their expression patterns than did the homologous Arabidopsis gene family members, which tended to retain similar expression patterns.
In this study, we assigned functionally and structurally homologous gene functions involved in iron uptake between tomato and Arabidopsis. For this purpose, we based our studies not only on sequence comparisons but also took into account map position and functional expression data. Analyzing, in addition, levels of conserved gene content adjacent to the genes of interest allowed us to predict whether gene family members were redundant genes resulting from internal genome duplication events or nonredundant single-acting genes with distinct biological functions. We found that it was easier to determine the functional homologs in tomato using the Arabidopsis information than vice versa. Due to the lower gene complexity in tomato, we hypothesize that it might be generally convenient to utilize Arabidopsis genome information to predict tomato homologs.
Most comparative genome programs are based on finding orthologs by using the criterion sequence similarity (e.g. http://www.tigr.org/tdb/tgi/lgi/GO.html). In this study, sequence similarity alone was not a sufficient criterion for determining functional homologs for iron uptake between Arabidopsis and tomato. For a given gene, we frequently identified more than one related gene in the other species. The Arabidopsis genome in particular had a higher number of homologs compared to tomato. Extrapolating from our data would suggest that Arabidopsis may have more and larger gene families than tomato. Taking the number of most related FER-like, NAS, IRT, NRAMP, and FRO genes together would indicate that Arabidopsis had 21 genes (1+4+5+6+5), whereas tomato had only 10 genes for these functions (1+1+2+3+3). Van der Hoeven et al. (2002)
The comparative mapping studies presented here showed that 20% to 50% of analyzed COS markers were located in the vicinity of corresponding iron uptake genes in tomato and in Arabidopsis (except for those of the LeFER region). In contrast, only 0% to 14% of these COS markers matched with Arabidopsis regions containing nonhomologous iron uptake genes. The clustering of conserved COS marker positions in between tomato and Arabidopsis was calculated to be significant for several of them, and so it can be excluded that all clusters occurred at random. For some of the compared Arabidopsis-tomato regions with P-values greater than 0.05, we speculate that an increase of the number of analyzed markers would decrease the P-values and make the clustering statistically significant. We avoid utilizing the term colinearity for these observations since the actual gene order was not conserved in all cases. Similar gene content suggests a common evolutionary origin of the corresponding Arabidopsis and tomato genome regions. The analysis also showed internally duplicated regions in the Arabidopsis genome. It seems likely that the reason for higher multigene family copy numbers in Arabidopsis was the genome structure. Some duplications uncovered here have been found in previous studies as internally duplicated regions in the Arabidopsis genome; for example, the duplication events involving chromosome 1, chromosome 4, and chromosome 5 containing IRT/ZIP gene regions as well as the chromosome 1 duplication involving NAS genes (Blanc et al., 2003
This study dealt mainly with essential gene functions required in the root and/or for iron regulation. We found that expression data greatly contributed to assigning appropriate and unique homologous gene pairs. For IRT and FRO sequences, it was indispensable to consider sequence, map position, and gene expression aspects together. For example, among the 15 Arabidopsis ZIP genes (Mäser et al., 2001
The most drastic example of why functional gene analysis data are needed for investigating homologous gene functions was provided by the FRO genes. Among the eight Arabidopsis FRO-like genes, AtFRO2 was unequivocally identified as the FRO gene in Arabidopsis due to genetic experiments (Robinson et al., 1999
The chloronerva (nas) tomato mutant is characterized by a distinct leaf chlorosis and root phenotype upon low iron supply (Scholz et al., 1992
The Arabidopsis genome contains 162 predicted BHLH genes (Bailey et al., 2003
Arabidopsis and tomato show similar physiological responses to iron deficiency (strategy I), so that most likely iron regulation and iron uptake are conserved processes in these two species that involve conserved gene functions. If these gene functions were not performed by orthologs, at least they were expected to be compensated by paralogs. We predict from our analysis that essential homologous gene functions of iron uptake are indeed involved and conserved between Arabidopsis and tomato. However, detailed sequence and map position analysis indicated that these conserved genes are most likely of paralogous origin rather than of orthologous origin. Despite the similarities of genes and proteins involved in iron mobilization, tomato induces root morphological alterations, root hair proliferation, and transfer cell development as a response to iron deficiency (Schmidt, 1999
Sequence Analysis and Database Searches
Amino acid sequences of AtFRO2 (Robinson et al., 1999
Genomic tomato DNA fragments were mapped by restriction fragment length polymorphism analysis using 43 F2 individuals of an L. esculentum/Lycopersicon pennellii mapping population according to Tanksley et al. (1992) Tomato COS markers that mapped within a distance of ±10 cM of iron uptake genes were selected for analysis of conserved gene regions at http://www.sgn.cornell.edu/maps/tomato_Arabidopsis/synteny_map.html. The encoded amino acid sequences of the selected COS markers were used to BLAST for corresponding Arabidopsis amino acid sequences at http://www.Arabidopsis.org/Blast. Arabidopsis genes were used as COS markers if their encoded peptides gave expected values below e-20 in alignments with encoded peptides of tomato COS markers (Supplemental Table I). Arabidopsis COS markers located up to 400 genes from iron uptake genes (approximately ±2 Mb) were considered to be in that same chromosomal region. For example, genes in the region of At4g19690 would be expected to have gene locus numbers between At4g15690 and At4g23690.
Statistical analysis and calculation of P-values for random clustering of homologous genes between Arabidopsis and tomato were performed as follows: For each of the Arabidopsis genes of interest, an interval of ±1 Mb was considered in which the gene of interest was in the center. If K was the total number of Arabidopsis genes in the ±1-Mb interval, L was the number of Arabidopsis genes located in this interval and homologous to genes from the corresponding ±10-cM tomato region minus tandem duplicates, M was the total number of genes in the Arabidopsis genome, and N was the number of genes in the Arabidopsis genome homologous to genes from the ±10-cM tomato region minus tandem duplicates, then the probability pL of finding L genes by chance given K, M, and N was computed by Fisher's exact test as the right tail of the hypergeometric distribution:
When determining K, L, M, and N, the gene in the center of the ±1-Mb interval was not counted. The statistical significance was computed as the probability that L or more than L genes could occur by chance in the ±1-Mb region if the numbers K, M, and N were fixed. The P-values were assigned to the genes in the center of the ±1-Mb intervals, and P-values less than 0.05 were considered significant for nonrandom clustering. For example, At1g31260 maps at position 11,175,540 bp on Arabidopsis chromosome 1. The ±1-Mb interval centered at position 11,175,540 contains 420 genes, including At1g31260 (K = 419). Out of those 419 genes, five are homologs of COS markers from the ±10-cM tomato region containing LeIRT1 (L = 5). In total, there are 26,405 Arabidopsis genes, including At1g31260 (M = 26,404). Out of those 26,404 genes, 51 are homologs of COS markers from the ±10-cM region of LeIRT1 (n = 51). The density of 5 genes out of 419 is 6-fold higher than the density of 51 genes out of 26,404, and the P-value is 0.001.
Tomato plants used in RNA expression analysis were derived from the lines L. esculentum T3238fer (fer mutant phenotype) and L. esculentum Moneymaker (wild type). Homozygous plants of the first-generation introgression lines from L. pennellii (LA 716) in the genetic background of the processing tomato variety M82 were propagated and utilized in this study (Eshed et al., 1992
For tomato iron uptake studies, 12-d-old plants were grown in a hydroponic Hoagland medium containing 0.1 (low iron) or 10 µM (sufficient iron) FeNaEDTA for 1 week according to Bereczky et al. (2003) For expression studies in Arabidopsis, 2-week-old Arabidopsis Columbia plants grown on solid Hoagland medium in the presence of 10 µM FeNaEDTA were transferred to Hoagland medium containing 10 µM FeNaEDTA (sufficient iron) or no iron and 200 µM bathophenanthroline disulfonic acid (low iron) for 5 d. nas1, nas2, and nas4 T-DNA insertion lines were identified by database searches at the SALK Institute Web site (http://signal.salk.edu) and ordered from the Arabidopsis Biological Resource Center.
Semiquantitative reverse transcription (RT)-PCR analysis was performed according to Bereczky et al. (2003)
Tomato mapping filters were provided by Dr. M. Ganal. Help with the MAPMAKER program by Dr. X. Huang and Dr. M. Röder is greatly acknowledged. Received June 2, 2004; returned for revision July 7, 2004; accepted July 7, 2004.
1 This work was supported by Deutsche Forschungsgemeinschaft grants in the Emmy Noether program (Ba1610/3–1 to P.B.) and in the Arabidopsis Functional Genomics Network program (Ba1610/4–1 to P.B. and R.H.), and by the Bundesministerium für Bildung und Forschung to the Bioinformatics Center Gatersleben-Halle (grant to I.G.). 
2 Present address: Heidelberg Institute for Plant Sciences, University of Heidelberg, Im Neuenheimer Feld 360, D–69120 Heidelberg, Germany.
[w] The on-line version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.047233. * Corresponding author; e-mail bauer{at}ipk-gatersleben.de; fax 49–39482–5139.
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(At5g19510) served as constitutive control.
