Department of Botany, University of Agricultural Science, Vienna, Austria;
Department of Biochemistry, Max-Planck-Institute for Plant Breeding, Cologne, Germany;
Department of Genetics and Evolution, Max-Planck-Institute for Chemical Ecology, Jena, Germany
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
With the completion of Arabidopsis genomic sequencing and the new 2010 project to determine the function of all Arabidopsis genes, there will be many opportunities to understand gene regulation and its variation within and among species. Large-scale genome annotation is routinely done automatically using database searches and gene prediction programs (Lin et al. 1999
; Mayer et al. 1999
). The latter are usually designed to detect protein-coding regions, leaving the untranslated regulatory sequences of a gene largely unexplored. It is well known that functionally important homologous regions in coding sequences tend to be highly conserved between sibling species. Similarly, it is expected that regulatory elements in untranslated regions might be detected by sequence comparison ("phylogenetic footprinting"; Wasserman et al. 2000
). Here, we test the usefulness of this comparative strategy for the study of gene regulation using two genes, chalcone synthase (Chs) and Apetela3, from 22 cruciferous species.
Recent studies have established a well-supported overview of evolutionary relationships within the Brassicaceae (O'Kane and Al-Shehbaz 1997
; Galloway, Malmberg, and Price 1998
; Koch, Bishop, and Mitchell-Olds 1999
; Koch, Haubold, and Mitchell-Olds 2000, 2001
). Divergence times among various taxa have been estimated using several independent loci and fossil calibrations (Yang et al. 1999
; Koch, Haubold, and Mitchell-Olds 2000
). Consequently, these studies provide a basis for selecting informative species for comparative analysis and provide a key to the understanding of rates and modes of regulatory evolution in the Brassicaceae. Arabidopsis thaliana and many closely related species are diploids with relatively few recent gene duplications. This makes it comparatively easy to identify orthologous loci for comparative studies. However, complications may arise in ancient polyploids such as Brassica (Kowalski et al. 1994
; Lagercrantz 1998
).
To obtain new insights into regulatory evolution, we chose the well-characterized promoter regions of the Chs and Apetala3 genes from selected species of the Brassicaceae. Chalcone synthase participates in plant secondary metabolism and catalyzes the key reaction in flavonoid biosynthesis (Hahlbrock and Scheel 1989
). Although many plant species contain multiple Chs genes (Ryder et al. 1987
; Koes, Spelt, and van der Elzen 1989
; Wingender et al. 1989
; An et al. 1993
; Junghans, Dalkin, and Dixon 1993
; Durbin et al. 1995
; Howles, Arioli, and Weinman 1995
), Chs is single-copy in A. thaliana (Burbulis, Iacobucci, and Shirley 1996
) and most related diploids (Koch, Haubold, and Mitchell-Olds 2000
). The Chs promoter is responsive to UV light, and it has been characterized experimentally in A. thaliana (Hartmann et al. 1998
) and Sinapis alba (Kaiser and Batschauer 1995
; Kaiser et al. 1995
). In addition, detailed information on the cis-acting elements and potential corresponding trans-acting factors of the Chs promoter region are available for many other plant species (Faktor et al. 1997a
; Feldbrügge et al. 1997
; Seki et al. 1997
and references therein).
The floral homeotic gene Apetala3 is a well-known MADS-box regulatory gene responsible for floral organ identity. The function of this gene has been described (e.g., Okamoto et al. 1994
; Yi and Jack 1998
), and its promoter has been analyzed (Hill et al. 1998
). The molecular evolution of the MADS-box genes including Apetala3 has been studied in detail in higher plants (e.g., Purugganan et al. 1995
; Kramer, Dorit, and Irish 1998
; Lawton-Rauh, Alvarez-Buylla, and Purugganan 2000
; Purugganan 2000), and phylogenetic data on Apetala3 from various Brassicaceae are available (Lawton-Rauh, Buckler, and Purugganan 1999
).
In this paper, we address three main questions: (1) Can comparative analysis of promoter sequences among related taxa identify conserved cis-acting regulatory elements? (2) Do conserved promoter elements confer functional patterns of gene regulation? (3) Do patterns of promoter evolution mirror known phylogenetic relationships inferred from nuclear genes, plastidic loci, and noncoding spacer regions of nuclear ribosomal DNA?
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
For each promoter fragment, both strands were cycle-sequenced using the Taq DyeDeoxy Terminator Cycle Sequencing Kit (ABI Applied Biosystem, Inc.). Products of the cycle sequencing reactions were run on an ABI 377XL automated sequencer (ABI Applied Biosystem, Inc.). Cloned PCR products were sequenced using universal t7 forward (5'-gtaacgatttaggtgacactatcg-3') and M13-48 reverse (5'-agcggataacaatttcacacagga-3') primers.
Subcloning of Chs Promoter Fragments and Expression Analysis
Based on the Chs promoter sequences, primers were designed which annealed to the ATG start codon region and a position at about -220 relative to the transcriptional start site. The upstream primer added a HindIII site, and the downstream primer added a NcoI site which was placed on the ATG. PCR fragments representing the various Chs promoter sequences were cut with HindIII and NcoI and cloned into similarly digested pBT10-GUS (Sprenger-Haussels and Weisshaar 2000
). Identities of clones were confirmed by sequencing. Expression analysis was performed as described (Hartmann et al. 1998
; Sprenger-Haussels and Weisshaar 2000
) using a luciferase expression construct as an internal control.
Phylogenetic Analysis
The sequence from S. alba was published by Batschauer, Ehmann, and Schafer (1991)
under accession number X17437. Promoter regions were aligned by hand, and highly conserved regions served as anchor regions for successive alignment. Only those parts of the alignment which we judged to be highly reliable were used for phylogenetic analysis. These parts showed pairwise similarities >50% and were not disrupted by extensive microsatellite sequences. We excluded DNA regions which were used as primer-binding sites for amplification. Phylogenetic distances were computed using Kimura's (1980)
two-parameter model, and the resulting distance matrices were subjected to the neighbor-joining algorithm as implemented in TREECON (version 1b; Van de Peer and De Wachter 1997
), which was also used to perform bootstrap analysis (Felsenstein 1985
) with 1,000 replicates.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
DNA Sequence Variation of Apetala3 Promoter Regions
The sequencing of different subfragments of the Apetala3 promoter region (table 2
) mostly resulted in overlapping fragments of identical sequences. In the case of Rorippa amphibia with the primer combination APET3.for/APET.rev, one additional sequence was found. In Arabis hirsuta, we detected three different genes corresponding to the three primer combinations. However, A. hirsuta is an autotetraploid plant, so we probably isolated alleles from the two duplicated loci.
The alignment of the entire promoter region is shown in figure 2 (supplementary material). The length of the fragment (APET1.for/APET.rev) varied from 486 bp in R. amphibia to 1,036 bp in B. oleracea. The mean G/C content was 34.7% (SD = 1.3%), with a minimum of 32.2% in the C. pyrenaica promoter fragment. The highest G/C content was found in the promoter fragment of A. glabra, with 37.6% (table 1 ). At alignment position 62, we found a 459-bp indel in the B. oleracea promoter.
|
Another H-box-like (or MRE) motif was located at position 397. Database comparisons with PLACE, a collection of promoter elements (http://www.dna.affrc.go.jp/htdocs/PLACE/signalscan.html; Higo et al. 1999
) revealed several other conserved cis-acting regulatory elements with unknown functions in the Chs promoter region. There was an A-box motif (TACGTA) at position 220, related to the G-box or MRE with a core ACGT sequence. However, this ACGT sequence only appeared in the distantly related R. amphibia and Cochlearia excelsa. A CAAT promoter consensus sequence has been found at positions 375 and 407. The sequence is highly conserved and might be responsible for tissue-specific promoter activity (Shirsat et al. 1989
).
A consensus GT-1-binding site (GAAAAA) was found at position 452. This motif is present in many light-regulated genes from numerous species (Villain, Mache, and Zhou 1996
; Zhou 1999
). A related GGTTAAA/TA/T motif has been described by Lawton et al. (1991)
in a silencer region of Chs from the bean. Several other highly conserved regions (>50% invariable nucleotide positions in a fragment a minimum of 15 bp in length) have no known functional role and were labeled as "region 1" and "region 2" (fig. 1
, supplementary material; positions 439555 and 471488, covering the GT-1-binding site). These conserved sequence motifs may represent novel regulatory elements.
Conserved Elements in the Apetala3 Promoter Region
Several MADS-domain-containing proteins bind DNA at a CC(A/T)6GG core consensus binding site which has been referred to as the CArG box (Schwarz-Sommer et al. 1992
; Wynne and Treisman 1992
; Huang, Mizukami, and Ma 1993
; Shiraishi, Okada, and Shimura 1993
; Hill et al. 1998
). The Apetala3 promoter of A. thaliana contains three CArG box motifs (Hill et al. 1998
), which may serve as binding sites for one or more MADS-domain-containing proteins (Irish and Yamamoto 1995
). These boxes are highly conserved among the cruciferous plants analyzed in this study and are identified in figure 2
(supplementary material) at positions 428, 476, and 500.
Several other highly conserved regions were located exclusively upstream of the CArG1 motif, and none of them have been characterized experimentally. (As for the analyzed Chs-promoter region, we defined regions as "highly conserved" if more than 50% of a DNA sequence with a minimum length of 15 bp was invariable.) However, strong evidence for functional relevance of promoter regions upstream of the CArG1 motif is provided by experimental analysis of the Apetala3 promoter in A. thaliana (Hill et al. 1998
).
The Apetala3 alignment (fig. 2
, supplementary material) shows four conserved regions, which we designated region 1 (alignment positions 73133), region 2 (alignment positions 216230), region 4 (alignment positions 365381), and duplicated region 3a (alignment positions 328347) and region 3b (alignment positions 383402). In each of regions 1, 2, and 4, there was one MYB-binding site (with a consensus motif CNGTTR; Urao et al. 1993
). Only in the highly conserved regions 3a and 3b (90% invariable sites) were we unable to identify known regulatory motifs.
Evidence for additional motifs is ambiguous. At position 78 there was a second putative MYBhomolog-binding site, previously recognized in maize (consensus motif CCWACC; Grotewold et al. 1994
). Overlapping with this myb element was a sequence similar to a region necessary for circadian expression of the tomato Lhc (light harvesting complex) gene (CAANNNNATC; Piechulla, Merforth, and Rudolph 1998), and overlapping with the 3' end of this putative myb element was a G-box-like CACCTG motif. At positions 73 and 243 there were also core sites (AAAG) required for binding of Dof proteins (DNA-binding proteins) in maize, with presumably only one zinc finger (Yanagisawa and Schmidt 1999
). Interestingly, a few base pairs upstream of this AAAG recognition sequence, there was an ACTTTA motif (alignment position 53), which has been shown to act as a Dofprotein-binding site (Baumann et al. 1999
). We also found the same motif at alignment position 421 close to the CArG1 box.
Phylogenetic Analysis
With the exception of Lepidium campestre and M. incana, the evolutionary relationships of these species have been considered in previous studies (Koch, Haubold, and Mitchell-Olds 2000, 2001
). A schematic phylogenetic network based on matK and Chs coding regions considering only taxa analyzed herein is redrawn from Koch, Haubold, and Mitchell-Olds (2001)
in figure 2
for further comparisons. The phylogenetic tree estimated from Chs promoter sequence divergence values (fig. 3
) differs from the combined matK/Chs tree (fig. 2
) only in the relative positions of L. campestre and M. incana. However, as outlined above, there is little statistical support (bootstrap values and decay indices in Koch, Haubold, and Mitchell-Olds 2001
) for the relative positions of Lepidium and Matthiola, and therefore the conflict among these differing topologies is statistically insignificant. This means that Chs promoter sequence evolution corresponds closely with Chs gene coding sequences as well as several other genes and DNA regions. This holds at least for the selected portions of the sequences for which a reliable alignment was found. However, distance analysis of the total alignment resulted in the same tree topology with some extremely long branches. Because it is not possible to calculate simple synonymous or nonsynonymous mutation rates for pairwise comparisons, we used the Kimura (1980)
two-parameter distances to compare Chs promoter regions (alignable regions as indicated in fig. 1
, supplementary material) with the corresponding Chs coding regions (Koch, Haubold, and Mitchell-Olds 2000
). These data showed that within the selected promoter regions the mean substitution rate in the Chs promoter region is 0.58 times as low (SD = 0.18) as the mean synonymous substitution rate in the corresponding coding Chs region.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hartmann et al. (1998)
showed that a light regulation unit (LRU) containing the ACE and the MRE was sufficient for UV/blue light-regulated expression of Chs. The corresponding LRU from bean Chs15 has been intensively studied (Arias, Dixon, and Lamb 1993
), and it has been shown that the G-box (ACE) and the H-box (MRE) make major contributions to the transcription of the Chs15 promoter in vivo. Combinatorial specificity of these elements has also been found in promoters of genes encoding phenylpropanoid biosynthetic enzymes (Lois et al. 1989
; Leyva et al. 1992
). The corresponding LRU regions in Chs promoters of the French bean (Faktor et al. 1996
) and the petunia (van der Meer et al. 1990
) are also responsible for tissue-specific activity. Finally, additional H-boxes binding MYB classes of transcription factors are present upstream of the LRU unit of bean Chs15 (Arias, Dixon, and Lamb 1993
). This corresponds to our observation of a highly conserved H-box-like sequence at alignment position 396 (fig. 1
, supplementary material).
Recently, Seki et al. (1996)
presented an overview of these elicitor-responsive cis elements and clearly showed similarities among Chs and PAL gene promoters from different species. Interestingly, only a few additional regions have been subjected to functional dissection analysis. Nuclear factors (SBF-1) have been identified in P. vulgaris that bind to SBF-1 boxes in the 5' region of the bean Chs15 gene promoter regulating tissue-specific expression (Hotter et al. 1995
). These SBF-boxes contain a consensus motif identical to the GT-1 recognition motif. We found a highly conserved GT1-like motif (fig. 1
, supplementary material) at alignment position 452 which might have a similar function.
Perspective
We analyzed interspecific patterns of sequence conservation in promoter regions of two genes, Chs and Apetala3. Our results show that known functionally important regulatory elements are conserved among crucifer relatives of Arabidopsis (figs. 1 and 2 , supplementary material). Other, previously unknown, elements were identified which are even more highly conserved than the known motifs which have been identified by functional studies. In addition, we showed that Chs promoter sequences from a broad sample of related species are sufficient for light-regulated gene expression in transient expression assays in Arabidopsis.
Comparative analyses of putative regulatory regions are increasingly important in genomics. Sequences of CFTR, the gene responsible for cystic fibrosis in humans, have been compared in humans, mice, and Fugu (Davidson et al. 2000
; Ellsworth et al. 2000
). Human-mouse comparisons found many intergenic and intron segments with high levels of conservation, providing little ability to identify which regions were functionally important in gene regulation. With greater evolutionary divergence, genomic sequence 5' of CFTR is highly divergent between pufferfish and mammals, except for conserved putative CRE and CAAT box elements. Likewise, human-mouse comparison of the SNCA gene, which may influence development of Alzheimer's and Parkinson's diseases, identified a novel 5' element that regulates normal expression in transient assays (Touchman et al. 2001
). Wasserman et al. (2000)
studied conserved promoter regions from 28 human-mouse pairs of skeletally expressed genes and identified a number of known and inferred enhancer elements.
Comparative analysis of promoter sequences from related species is a powerful tool to identify conserved and putatively functional elements. Rapidly accumulating knowledge about the function and regulation of genes coming from the model plant A. thaliana can be easily transferred to its closest relatives. Wild relatives provide a broad spectrum of naturally occurring genotypes and phenotypes, which are presumably adapted to a range of environments and natural histories. This will permit hypothesis testing about the evolutionary significance of character change and suites of traits. Well-supported phylogenies of numerous Brassicaceae and estimates of divergence times provide an important resource for these studies (Koch, Haubold, and Mitchell-Olds 2000, 2001
). These can be used to compare rates of evolution. In our analysis, the greatest evolutionary divergence was about 45 Myr (for the Chs promoter of A. thaliana compared with A. grandiflora).
Bioinformatic analyses have used three approaches to identify putative regulatory regions within promoters: pattern recognition, phylogenetic footprinting among several species, and within-species comparison of promoters from coregulated genes (Fickett and Wasserman 2000
; Wasserman et al. 2000
). Phylogenetic footprinting is particularly important, because it reduces the search space for subsequent computational analysis. In addition, comparison of promoter sequences from several species at different levels of evolutionary divergence may provide improved identification of putative functionally important sites (e.g., Davidson et al. 2000
). Likewise, expression profiling can identify groups of coregulated genes which may display above-average frequencies of particular regulatory motifs (Maleck et al. 2000
). All three approaches will be useful for understanding gene regulation in A. thaliana.
Despite successful comparisons in our experiments and in other studies, regulatory function may be maintained even in the absence of sequence conservation. For example, the Drosophila melanogaster and Drosophila pseudoobscura promoters of even-skipped mediate conserved patterns of stripe 2 expression during embryo development. In contrast, the functionally important stripe 2 enhancer shows considerable divergence between these two species. Chimeric promoters (containing both combinations of the 5' and 3' halves of the D. melanogaster and D. pseudoobscura upstream regions) are no longer expressed in the wild-type pattern (Ludwig et al. 2000
). This decoupling of conservation and function will complicate attempts to identify functionally important regulatory motifs by comparative genomics.
![]() |
Supplementary Material |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
1 Keywords: Apetala3
Chs, comparative genomics
phylogeny
promoter function
regulatory evolution
2 Address for correspondence and reprints: Marcus A. Koch, Department of Botany, University of Agricultural Sciences, Gregor-Mendel-Strasse 33, A-1180 Vienna, Austria. koch{at}edv1.boku.ac.at
.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
An C., Y. Ichinose, T. Yamada, Y. Tanaka, T. Shiraishi, H. Oku, 1993 Structure and organization of the genes encoding chalcone synthase in Pisum sativum Plant Mol. Biol 21:789-803[ISI][Medline]
Arias J. A., R. A. Dixon, C. J. Lamb, 1993 Dissection of the functional architecture of a plant defense gene promoter using a homologous in vitro transcription initiation system Plant Cell 5:485-496
Batschauer A., B. Ehmann, E. Schafer, 1991 Cloning and characterization of a chalcone synthase gene from mustard and its light-dependent expression Plant Mol. Biol 16:175-185[ISI][Medline]
Baumann K., A. De Paolis, P. Costantino, G. Gualberti, 1999 The DNA binding site of the Dof protein NtBBF1 is essential for tissue-specific and auxin-regulated expression of the rolB oncogene in plants Plant Cell 11:323-333
Block A., J. L. Dangl, K. Hahlbrock, P. Schulze-Lefert, 1990 Functional borders, genetic fine structure, and distance requirements of cis elements mediating light responsiveness of the parsley chalcone synthase promoter Proc. Natl. Acad. Sci. USA 87:5387-5391[Abstract]
Burbulis I., M. Iacobucci, B. W. Shirley, 1996 A null mutation in the first enzyme of flavonoid biosynthesis does not effect male fertility in Arabidopsis Plant Cell 8:1013-1025
Davidson H., M. S. Taylor, A. Doherty, A. C. Boyd, D. J. Porteous, 2000 Genomic sequence analysis of Fugu rubripes CFTR and flanking genes in a 60 kb region conserving synteny with 800 kb of human chromosome 7 Genome Res 10:1194-1203
Durbin M. L., G. H. Learn, G. A. Huttley, M. T. Clegg, 1995 Evolution of the chalcone synthase gene family in the genus Ipomoea Proc. Natl. Acad. Sci. USA 92:3338-3342[Abstract]
Ellsworth R. E., D. C. Jamison, J. W. Touchman, et al. (31 co-authors) 2000 Comparative genomic sequence analysis of the human and mouse cystic fibrosis transmembrane conductance regulator genes Proc. Natl. Acad. Sci. USA 97:1172-1177
Faktor O., J. M. Kooter, R. A. Dixon, C. J. Lamb, 1996 Functional dissection of a bean chalcone synthase gene promoter in transgenic tobacco plants reveals sequence motifs essential for floral expression Plant Mol. Biol 32:849-859[ISI][Medline]
Faktor O., J. M. Kooter, G. J. Loake, R. A. Dixon, C. J. Lamb, 1997a Differential utilization of regulatory cis-elements for stress-induced and tissue-specific activity of a French bean chalcone synthase promoter Plant Sci 124:175-182[ISI]
Faktor O., G. Loake, R. A. Dixon, C. J. Lamb, 1997b The G-box and H-box in a 39 bp region of a French bean chalcone synthase promoter constitute a tissue-specific regulatory element Plant J 11:1105-1113[ISI]
Feldbrügge M., M. Sprenger, K. Hahlbrock, B. Weisshaar, 1997 PcMYB1, a novel plant protein containing a DNA-binding domain with one MYB repeat, interacts in vivo with a light-regulatory promoter unit Plant J 11:1079-1093[ISI][Medline]
Felsenstein J., 1985 Confidence limits on phylogenies: an approach using the bootstrap Evolution 39:783-791[ISI]
Fickett J. W., W. W. Wasserman, 2000 Discovery and modeling of transcriptional regulatory regions Curr. Opin. Biotechnol 11:19-24[ISI][Medline]
Galloway G. L., R. L. Malmberg, R. A. Price, 1998 Phylogenetic utility of the nuclear gene arginine decarboxylase: an example from Brassicaceae Mol. Biol. Evol 15:1312-1320
Grotewold E., B. J. Drummond, B. Bowen, T. Peterson, 1994 The myb-homologous P gene controls phlobaphene pigmentation in maize floral organs by directly activating a flavonoid biosynthetic gene subset Cell 76:543-553[ISI][Medline]
Hahlbrock K., D. Scheel, 1989 Physiology and molecular biology of phenylpropanoid metabolism Annu. Rev. Plant Physiol. Plant Mol. Biol 40:347-369[ISI]
Hartmann U., W. J. Valentine, J. M. Christie, J. Hays, G. I. Jenkins, B. Weisshaar, 1998 Identification of UV/blue light-response elements in the Arabidopsis thaliana chalcone synthase promoter using a homologous protoplast transient expression system Plant Mol. Biol 36:741-754[ISI][Medline]
Higo K., Y. Ugawa, M. Iwamoto, T. Korenaga, 1999 Plant cis-acting regulatory DNA elements (PLACE) database Nucleic Acids Res 27:297-300
Hill T. A., C. D. Day, S. C. Zondlo, A. G. Thackeray, V. F. Irish, 1998 Discrete spatial and temporal cis-acting elements regulate transcription of the Arabidopsis floral homeotic gene Apetala3 Development 125:1711-1721
Horikawa Y., N. Oda, N. J. Cox, et al. (25 co-authors) 2000 Genetic variation in the gene encoding calpain-10 is associated with type 2 diabetes mellitus Nat. Genet 26:163-175[ISI][Medline]
Hotter G. S., J. Kooter, I. A. Dubery, C. J. Lamb, R. A. Dixon, M. J. Harrison, 1995 Cis elements and potential trans-acting factors for the developmental regulation of the Phaseolus vulgaris CHS15 promoter Plant Mol. Biol 28:967-981[ISI][Medline]
Howles P. A., T. Arioli, J. J. Weinman, 1995 Nucleotide sequence of additional members of the gene family encoding chalcone synthase in Trifolium subterraneum Plant Physiol 107:1035-1036
Huang H., Y. Mizukami, H. Ma, 1993 Isolation and characterization of the binding sequence for the product of the Arabidopsis floral homeotic gene AGAMOUS Nucleic Acids Res 21:4769-4776[Abstract]
Irish V. F., Y. T. Yamamoto, 1995 Conservation of floral homeotic gene function between Arabidopsis and Antirrhinum Plant Cell 7:1635-1644
Junghans H., K. Dalkin, R. A. Dixon, 1993 Stress response in alfalfa (Medicago sativa L.) 15. Characterization and expression patterns of members of a subset of the chalcone synthase multigene family Plant Mol. Biol 22:239-253[ISI][Medline]
Kaiser T., A. Batschauer, 1995 Cis-acting elements of the CHS1 gene from white mustard controlling promoter activity and spatial patterns of expression Plant Mol. Biol 28:231-243[ISI][Medline]
Kaiser T., K. Emmler, T. Kretsch, B. Weisshaar, E. Schäfer, A. Batschauer, 1995 Promoter elements of the mustard CHS1 gene are sufficient for light regulation in transgenic plants Plant Mol. Biol 28:219-229[ISI][Medline]
Kimura M., 1980 A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences J. Mol. Evol 16:111-120[ISI][Medline]
Kobayashi H., Y. Oikawa, H. Koiwa, S. Yamamura, 1998 Flower-specific gene expression directed by the promoter of a chalcone synthase gene from Gentiana triflora in Petunia hybrida Plant Sci 131:173-180[ISI]
Koch M., J. Bishop, T. Mitchell-Olds, 1999 Molecular systematics and evolution of Arabidopsis and Arabis Plant Biol 1:529-537[ISI]
Koch M., B. Haubold, T. Mitchell-Olds, 2000 Comparative evolutionary analysis of chalcone synthase and alcohol dehydrogenase loci in Arabidopsis, Arabis and related genera (Brassicaceae) Mol. Biol. Evol 17:1483-1498
. 2001 Molecular systematics of the Brassicaceae: evidence from coding plastidic matK and nuclear CHS sequences Am. J. Bot 88:534-544
Koes R. R., C. E. Spelt, P. J. M. van der Elzen, 1989 Cloning and molecular characterization of the chalcone synthase multigene family of Petunia hybrida Gene 81:245-257[ISI][Medline]
Kopp A., I. Duncan, S. B. Carroll, 2000 Genetic control and evolution of sexually dimorphic characters in Drosophila Nature 408:553-559[ISI][Medline]
Kowalski S. P., T.-H. Lan, K. A. Feldmann, A. H. Paterson, 1994 Comparative mapping of Arabidopsis thaliana and Brassica oleracea chromosomes reveals islands of conserved organization Genetics 138:499-510
Kramer E. M., R. L. Dorit, V. F. Irish, 1998 Molecular evolution of genes controlling petal and stamen development: duplication and divergence within the Apetala3 and PISTILLATA MADS-box gene lineages Genetics 149:765-783
Lagercrantz U., 1998 Comparative mapping between Arabidopsis thaliana and Brassica nigra indicates that Brassica genomes have evolved through extensive genome replication accompanied by chromosome fusions and frequent rearrangements Genetics 150:1217-1228
Lawton M. A., S. M. Dean, M. Dron, J. M. Kooter, K. M. Kragh, M. J. Harrison, L. Yu, L. Tanguay, R. A. Dixon, C. J. Lamb, 1991 Silencer region of a chalcone synthase promoter contains multiple binding sites for a factor, SBF-1, closely related to GT-1 Plant Mol. Biol 16:235-249[ISI][Medline]
Lawton-Rauh A. L., E. R. Alvarez-Buylla, M. D. Purugganan, 2000 Molecular evolution of flower development Tree 15:144-149[Medline]
Lawton-Rauh A. L., E. S. Buckler, M. D. Purugganan, 1999 Patterns of molecular evolution among paralogous floral homeotic genes Mol. Biol. Evol 16:1037-1045[Abstract]
Leyva A., X. Liang, J. A. Pintor-Toro, R. A. Dixon, C. J. Lamb, 1992 Cis-element combinations determine phenylalanine ammonia-lyase gene tissue-specific expression patterns Plant Cell 4:263-271
Lin X. Y., S. S. Kaul, S. Rounsley, et al. (26 co-authors) 1999 Sequence and analysis of chromosome 2 of the plant Arabidopsis thaliana Nature 402:761-768[ISI][Medline]
Loake G. J., O. Faktor, C. J. Lamb, R. A. Dixon, 1992 Combination of H-box [CCTACC(N)7CT] and G-box (CACGTG) cis elements is necessary for feed-forward stimulation of a chalcone synthase promoter by the phenylpropanoid-pathway intermediate p-coumaric acid Proc. Natl. Acad. Sci. USA 89:9230-9234[Abstract]
Logemann E., M. Parniske, K. Hahlbrock, 1995 Modes of expression and common structural features of the complete phenylalanine ammonia-lyase gene family in parsley Proc. Natl. Acad. Sci. USA 92:5905-5909
Lois R., A. Dietrich, K. Hahlbrock, W. Schulz, 1989 A phenylalanine ammonia-lyase gene from parsley: structure, regulation and identification of elicitor and light-responsive cis-acting elements EMBO J 8:1641-1648[Abstract]
Ludwig M. Z., C. Bergman, N. H. Patel, M. Kreitman, 2000 Evidence for stabilizing selection in a eukaryotic enhancer element Nature 403:564-567[ISI][Medline]
Maleck K., A. Levine, T. Eulgem, A. Morgan, J. Schmid, K. A. Lawton, J. L. Dangl, R. A. Dietrich, 2000 The transcriptome of Arabidopsis thaliana during systemic acquired resistance Nat. Genet 26:403-410[ISI][Medline]
Mayer K., C. Schuller, R. Wambutt, et al. (100 co-authors) 1999 Sequence and analysis of chromosome 4 of the plant Arabidopsis thaliana Nature 402:769-777[ISI][Medline]
Mummenhoff K., M. Koch, 1994 Chloroplast DNA restriction site variation and phylogenetic relationships in the genus Thlaspi sensu lato (Brassicaceae) Syst. Bot 19:73-88[ISI]
Okamoto H., A. Yano, H. Shiraishi, K. Okada, Y. Shimura, 1994 Genetic complementation of a floral homeotic mutation, apetala3, with an Arabidopsis thaliana gene homologous to DEFICIENS of Antirrhinum majus Plant Mol. Biol 26:465-472[ISI][Medline]
O'Kane S. L., I. A. Al-Shehbaz, 1997 A synopsis of Arabidopsis (Brassicaceae) Novon 7:323-327[ISI]
Piechulla B., N. Merforth, B. Rudolph, 1998 Identification of tomato LHC promoter regions necessary for circaelian expression Plant Mol. Biol 38:655-662[ISI][Medline]
Purugganan M. D., 1997 The MADS-box floral homeotic gene lineages predate the origin of seed plants: phylogenetic and molecular clock estimates J. Mol. Evol 45:392-396[ISI][Medline]
. 2000 The molecular population genetics of regulatory genes Mol. Ecol 9:1451-1461[ISI][Medline]
Purugganan M. D., S. D. Rounsley, R. J. Schmidt, M. F. Yanofsky, 1995 Molecular evolution of flower development: diversification of the plant MADS-box regulatory gene family Genetics 140:345-356
Ryder T. B., S. A. Hedrick, J. N. Bell, X. Liang, S. D. Clouse, C. J. Lamb, 1987 Organization and differential activation of a gene family encoding the plant defense enzyme chalcone synthase in Phaseolus vulgaris Mol. Gen. Genet 210:219-233[ISI][Medline]
Schulze-Lefert P., M. Becker-Andre, W. Schulz, K. Hahlbrock, J. L. Dangl, 1989a Functional architecture of the light-responsive chalcone synthase promoter from parsley Plant Cell 1:707-714
Schulze-Lefert P., J. L. Dangl, M. Becker-Andre, K. Hahlbrock, W. Schulz, 1989b Inducible in vivo DNA footprints define sequences necessary for UV light activation of the parley chalcone synthase gene EMBO J 8:651-656[Abstract]
Schwarz-Sommer Z., I. Hue, P. Huijser, P. J. Flor, R. Hansen, F. Tetens, W.-E. Lönning, H. Saedler, H. Sommer, 1992 Characterization of the Antirrhinum floral homeotic MADS-box gene deficiens: evidence for DNA binding and autoregulation of its persistent expression throughout flower development EMBO J 11:251-262[Abstract]
Seki H., Y. Ichinose, M. Ito, T. Shiraishi, T. Yamada, 1997 Combined effects of multiple cis-acting elements in elicitor-mediated activation of PSCHS1 gene Plant Cell Physiol 38:96-100[ISI]
Seki H., Y. Ichinose, H. Kato, K. Hisaharu, T. Shiraishi, T. Yamada, 1996 Analysis of cis-regulatory elements involved in the activation of a member of chalcone synthase gene family (PsChs1) in pea Plant Mol. Biol 31:479-491[ISI][Medline]
Shiraishi H., K. Okada, Y. Shimura, 1993 Nucleotide sequences recognized by the AGAMOUS MADS domain of Arabidopsis thaliana in vitro Plant J 4:385-398[ISI][Medline]
Shirsat A., N. Wilford, R. Croy, D. Boulter, 1989 Sequences responsible for the tissue specific promoter activity of a pea legumin gene in tobacco Mol. Gen. Genet 215:326-331[ISI][Medline]
Sprenger-Haussels M., B. Weisshaar, 2000 Transactivation properties of parsley proline-rich bZIP transcription factors Plant J 22:1-8[ISI][Medline]
Ting C., S. Tsaur, M. Wu, C. Wu, 1998 A rapidly evolving homeobox at the site of a hybrid sterility gene Science 282:1501-1504
Touchman J. W., A. Dehejia, O. Chiba-Falek, D. E. Cabin, J. R. Schwartz, B. M. Orrison, M. H. Polymeropoulos, R. L. Nussbaum, 2001 Human and mouse alpha-synuclein genes: comparative genomic sequence analysis and identification of a novel gene regulatory element Genome Res 11:78-86
Urao T., K. Yamaguchi-Shinozaki, S. Urao, K. Shinozaki, 1993 An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence Plant Cell 5:1529-1539
Van de Peer Y., R. De Wachter, 1997 Construction of evolutionary distance trees with TREECON for Windows: accounting for variation in nucleotide substitution rate among sites Comput. Appl. Biosci 13:227-230[Abstract]
Van der Meer I. M., C. E. Spelt, J. N. M. Mol, A. R. Stuitje, 1990 Promoter analysis of the chalcone synthase (chsA) gene of Petunia hybrida: a 67 bp promoter region directs flower-specific expression Plant Mol. Biol 15:95-109[ISI][Medline]
Villain P., R. Mache, D. X. Zhou, 1996 The mechanism of GT element-mediated cell type specific transcription control J. Biol. Chem 271:32593-32598
Wang R. L., A. Stec, J. Hey, L. Lukens, J. Doebley, 1999 The limits of selection during maize domestication Nature 398:236-239[ISI][Medline]
Wasserman W. W., M. Palumbo, W. Thompson, J. W. Fickett, C. E. Lawrence, 2000 Human-mouse genome comparisons to locate regulatory sites Nat. Genet 26:225-228[ISI][Medline]
Wingender R., H. Röhrig, C. Höricke, D. Wing, J. Schell, 1989 Differential regulation of soybean chalcone synthase genes in plant defense, symbiosis and upon environmental stimuli Mol. Gen. Genet 218:315-322[ISI][Medline]
Wynne J., R. Treisman, 1992 SRF and MCM1 have related but distinct DNA binding specificities Nucleic Acids Res 20:3297-3303[Abstract]
Yanagisawa S., R. J. Schmidt, 1999 Diversity and similarity among recognition sequences of Dof transcription factors Plant J 17:209-214[ISI][Medline]
Yang Y.-W., K. N. Lai, P.-Y. Tai, W.-H. Li, 1999 Rates of nucleotide substitution in angiosperm mitochondrial DNA sequences and dates of divergence between Brassica and other angiosperm lineages J. Mol. Evol 48:597-604[ISI][Medline]
Yi Y., T. Jack, 1998 An intragenic suppressor of the Arabidopsis floral organ identity mutant apetala31 functions by suppressing defects in splicing Plant Cell 10:1465-1477
Zhou D. X., 1999 Regulatory mechanism of plant gene transcription by GT-elements and GT-factors Trends Plant Sci 4:210-214[ISI][Medline]
Zhu T., X. Wang, 2000 Large-scale profiling of the Arabidopsis transcriptome Plant Physiol 124:1472-1476