Relationship between homoeologous regulatory and structural genes in allopolyploid genome – A case study in bread wheat
© Khlestkina et al; licensee BioMed Central Ltd. 2008
Received: 07 May 2008
Accepted: 13 August 2008
Published: 13 August 2008
The patterns of expression of homoeologous genes in hexaploid bread wheat have been intensively studied in recent years, but the interaction between structural genes and their homoeologous regulatory genes remained unclear. The question was as to whether, in an allopolyploid, this interaction is genome-specific, or whether regulation cuts across genomes. The aim of the present study was cloning, sequence analysis, mapping and expression analysis of F3H (flavanone 3-hydroxylase – one of the key enzymes in the plant flavonoid biosynthesis pathway) homoeologues in bread wheat and study of the interaction between F3H and their regulatory genes homoeologues – Rc (red coleoptiles).
PCR-based cloning of F3H sequences from hexaploid bread wheat (Triticum aestivum L.), a wild tetraploid wheat (T. timopheevii) and their putative diploid progenitors was employed to localize, physically map and analyse the expression of four distinct bread wheat F3H copies. Three of these form a homoeologous set, mapping to the chromosomes of homoeologous group 2; they are highly similar to one another at the structural and functional levels. However, the fourth copy is less homologous, and was not expressed in anthocyanin pigmented coleoptiles. The presence of dominant alleles at the Rc-1 homoeologous loci, which are responsible for anthocyanin pigmentation in the coleoptile, was correlated with F3H expression in pigmented coleoptiles. Each dominant Rc-1 allele affected the expression of the three F3H homoeologues equally, but the level of F3H expression was dependent on the identity of the dominant Rc-1 allele present. Thus, the homoeologous Rc-1 genes contribute more to functional divergence than do the structural F3H genes.
The lack of any genome-specific relationship between F3H-1 and Rc-1 implies an integrative evolutionary process among the three diploid genomes, following the formation of hexaploid wheat. Regulatory genes probably contribute more to the functional divergence between the wheat genomes than do the structural genes themselves. This is in line with the growing consensus which suggests that although heritable morphological traits are determined by the expression of structural genes, it is the regulatory genes which are the prime determinants of allelic identity.
The flavonoid biosynthesis pathway is central to the formation of the phenolic compounds involved in many plant traits, including resistance to abiotic and biotic stresses [1–4]. One branch of the pathway is responsible for the generation of anthocyanin, which is present in various plant organs in most plant species, including the allohexaploid crop species, bread wheat (Triticum aestivum L.). Two major groups of anthocyanin pigmentation genes are present in wheat: the first includes Rc-1, Pc-1, Pan-1, Plb-1 and Pls-1 which encode the pigmentation in, respectively, the coleoptile, culm, anthers, leaf blades and leaf sheaths; while the second consists of Pp and Ra, which are expressed in, respectively, the pericarp and auricle . The former genes are closely linked to one another on each of the short arms of the homoeologous group 7 chromosomes. An orthologue of maize gene c1 (which encodes a Myb-like transcriptional factor controlling tissue-specific anthocyanin biosynthesis ) was mapped earlier on each of the short arms of wheat homoeologous group 7 chromosomes, too  in positions highly comparable to those of Rc-1 (red coleoptile) genes [5, 8]. Furthermore, it was shown that c1, when transferred to wheat, was able to induce anthocyanin pigmentation in non-pigmented wheat coleoptiles . At the same time Rc-1 was shown to upregulate a number of wheat flavonoid biosynthesis pathway genes – DFR (dihydroflavonol-4-reductase), ANS (anthocyanidin synthase) and UFGT (UDPG flavonol 3-0-glucosyl transferase) [10, 11]. Recognizing elements for c1 have also been identified in the promoter sequence of Arabidopsis thaliana F3H gene (flavanone 3-hydroxylase – one of the key enzymes involved in the biosynthesis of flavonoid compounds ), suggesting that Rc-1 can probably exert a regulatory role for wheat F3H, too. F3H orthologues have been isolated in barley and maize [13, 14] as well as in a range of other plant species http://www.ncbi.nlm.nih.gov/Database/, but have yet to be described in wheat.
The patterns of expression of homoeologous genes in wheat have been intensively studied in recent years [15–21], but the interaction between structural genes and their homoeologous regulatory genes is unclear. The question remains as to whether, in an allopolyploid, this interaction is genome-specific, or whether regulation cuts across genomes. The Rc-1 and F3H genes are a suitable model to investigate just this issue, as the expression of Rc-1 generates a clear phenotype, and the latter are well-characterized at the molecular level. In this paper, we describe the cloning, sequence analysis, mapping and expression of F3H orthologues in bread wheat and its relatives, and the interaction between F3H and the Rc-1 homoeologues.
Sequence analysis of F3Hgenes in wheat and its relatives
Length, Genbank accession numbers and chromosome locations for F3H nucleotide sequences determined in the present study.
Length in base pairs (gene segment specification according Figure 1)
Genbank accession number
Identical wheat ESTs*
T. aestivum, F3H1
1852, complete structural part of gene (Segments 1+2+3+4)
T. aestivum, F3H2
1374, complete structural part of gene (Segments 1+4+5)
T. aestivum, F3H3
1626, partial (Segments 2+3+4)
BQ240612 BG262749 CA705431
T. aestivum, F3H4
562, partial (Segment 2)
T. timopheevii, F3H1 t
542, partial (Segment 3)
T. timopheevii, F3H2 t
539, partial (Segment 3)
T. urartu, F3H
542, partial (Segment 3)
Ae. speltoides, F3H
542, partial (Segment 3)
Ae. tauschii, F3H
1326, partial (Segment 5)
Sequence homology and divergence among F3H1 and F3H2 genes.
Part of gene
Length: F3H1/F3H2 (in bp)
Nucleotide sequences homology (%)
There are two major deletion regions, other segments have over 90% homology
Chromosomal assignment and physical mapping of F3Hgenes in hexaploid wheat
Expression analysis of F3Hin lines with and without pigmented coleoptiles
Temporal pattern and the genome specificity of F3Hexpression
T-values for expression levels of different F3H homoeologues in coleoptiles (p = 0.05 for all presented values).
F3H-A1 vs F3H-B1
F3H-A1 vs F3H-D1
F3H-D1 vs F3H-B1
'Chinese Spring' ('Hope' 7A)
'Chinese Spring' ('Hope' 7B)
T-values for F3H expression in different wheat genotypes.
'Chinese Spring' ('Hope' 7A) vs 'Chinese Spring' ('Hope' 7B)
'Chinese Spring' ('Hope' 7A) vs 'Mironovskaya 808'
'Chinese Spring' ('Hope' 7B) vs 'Mironovskaya 808'
Cloning and analysis of F3Hsequences
Previously characterised flavonoid biosynthesis pathway genes in wheat.
Number of cloned copies
Genbank accessions; references
Chromosome location; references
PAL – phenylalanine ammonialyase
Isolation from genomic library
3A, 3B, 3D, 6A, 6B, 6D 
CHS – chalcone synthase
AY286093, AY286095, AY286096, AY286097
1A, 1B, 1D, 2A, 2B, 2D 
CHI – chalcone-flavanone isomerase
5A, 5B, 5D 
F3H – flavanone 3-hydroxylase
F3'5'H – flavonoid 3',5'-hydroxylase
DFR – dihydroflavonol-4-reductase
AB162138, AB162139, AB162140
ANS – anthocyanidin synthase
AB247917, AB247918, AB247919, AB247920, AB247921
6AS (2 copies), 6BS (2 copies), 6DS 
FMT – flavonoid 7-O-methyltransferase
1A, 1B, 1D 
Expression of the three homoeologous F3Hloci in lines with and without pigmented coleoptiles
The patterns of expression of flavanone 3-hydroxylase in lines with and without pigmented coleoptiles indicated that Rc-B1 and Rc-D1 are coincident with the genes regulating its expression (Figure 7). This is in accordance with the suggestion that Rc-1 genes exert a regulatory role for F3H genes, which could be made on the base of combined results obtained earlier [5–9, 12]. The patterns of temporal expression among the F3H homoeologues in the presence of different dominant Rc-1 alleles allowed for an examination as to whether, in an allopolyploid context, there are any genome-specific relationships between the structural and regulatory genes. No such relationship was apparent, since in pigmented coleoptiles, F3H-A1, F3H-B1 and F3H-D1 were all expressed at a similar level (Figure 9). Many sets of wheat homoeologous genes are known to be equally expressed in this way [16, 19, 21], but in others, the expression of one or more members may be either completely [16, 18, 31] or partially [15, 20, 21] suppressed. Generally, when F3H homoeologues are expressed actively (as in pigmented coleoptiles), then they are expressed equally, but where overall F3H transcription level is low, then selective expression of F3H homoeologues could be observed (i.e. F3H-A1 and F3H-B1 were expressed in the green coleoptiles of 'TRI 2732', but F3H-D1 was not; Figure 8). These outcomes are consistent with the activity-selectivity principle  acting at the transcriptional level.
Functional difference between homoeologous Rc-1genes
Whereas each dominant Rc-1 allele affects the expression of each of the three F3H homoeologues equally, overall F3H expression was dependent on the identity of which dominant Rc-1 allele was present (Figure 9). This difference was observed not only at specific time points, but also from the total amounts of F3H mRNA produced over the period of coleoptile pigmentation. The delayed start of expression and the lesser level of transcript present in 'Chinese Spring' ('Hope' 7B) compared to 'Chinese Spring' ('Hope' 7A) was consistent with the observed accumulation of pigmentation in the coleoptile, both in the present experiments and in those reported earlier . In order to test for background effects on F3H expression or variability within transcriptional factors encoded by dominant Rc-1 alleles in other genotypes, it would be of interest to investigate the extent to which the profiles of F3H expression of 'Chinese Spring' ('Hope' 7A), 'Chinese Spring' ('Hope' 7B) and 'Mironovskaya 808' are typical, i.e. for instance to compare profile of 'Mironovskaya 808' to those of some other varieties carrying the same dominant allele (Rc-D1).
There are at least four flavanone 3-hydroxylase gene copies in the hexaploid genome of bread wheat, three of which are the homoeologues on chromosomes 2AL, 2BL and 2DL, highly similar at structural and functional level, while the fourth one represents a distinct non-homoeologous copy on chromosome 2BL with suppressed expression in red coleoptiles.
Expression of the F3H homoeologues (F3H-1) in wheat coleoptiles is determined by the presence of dominant alleles in Rc-1 (red coleoptiles) loci. Rc-1 and F3H-1 genes represent a suitable model to investigate relationship between homoeologous regulatory and homoeologous structural genes in allopolyploid wheat genome (which have never been studied before). The lack of any genome-specific relationship between F3H-1 and Rc-1 observed in the present study implies an integrative evolutionary process among the three diploid genomes, following the formation of hexaploid wheat.
Furthermore, based on F3H expression analysis it was observed for the first time that activity-selectivity principle  acts at the transcriptional level.
Our general conclusion is that regulatory genes probably contribute more to the functional divergence between the wheat genomes than do the structural genes themselves. This is in line with the growing consensus which suggests that although heritable morphological traits are determined by the expression of structural genes, it is the regulatory genes which are the prime determinants of allelic identity.
Plant materials and RNA extraction
The bread wheat cultivars 'Chinese Spring', 'Opata', 'Flair', 'Prinz', 'Golubka', 'Novosibirskaya 67', the synthetic hexaploid wheat 'W7984', tetraploid T. timopheevii k-38555 (AAGG) and the diploids T. urartu TMU06 (AA), Aegilops speltoides TS01 (SS) and Ae. tauschii TQ17 (DD) were used for PCR-based cloning. The complete set of 'Chinese Spring' nulli-tetrasomic lines , a subset of homoeologous group 2 chromosome deletion lines , introgression line 842 derived from the cross T. aestivum cv. 'Saratovskaya 29' × T. timopheevii  were exploited to establish chromosome bin locations. Eight progeny from the cross 'Chinese Spring' ('Hope' 7B) × 'TRI 2732'  and a set of six homozygous lines each containing a different chromosome 7D segment derived from Ae. tauschii in a 'Chinese Spring' background  were used for RT-PCR. Quantitative examination of F3H expression was measured in 'Chinese Spring' and 'Mironovskaya 808' and the single chromosome substitution lines 'Chinese Spring' ('Hope' 7A) and 'Chinese Spring' ('Hope' 7B). DNA was extracted from seven day old seedlings following the procedure described earlier . RNA was extracted from seedlings grown at 20°C under a 12 h day/12 h night regime using the QIAGEN http://www1.qiagen.com/ Plant Rneasy Kit, followed by DNAse treatment. For RT-PCR, RNA was extracted on the fourth day after germination. For quantitative RT-PCR, RNA was extracted every 24 h from two to six day old seedlings.
PCR-based cloning and sequence analysis
Primers designed to amplify wheat F3H for cloning, chromosomal localization and for expression analysis.
Gene segment specification (according Figure 1) or former gene name
DNA/cDNA-derived PCR product length (bp)
Chromosomal assignment and
T. aestivum F3H1
T. aestivum F3H3
T. aestivum F3H4
T. aestivum F3H2
T. timopheevii F3H2 t
T. aestivum F3H1
T. aestivum F3H3
T. aestivum F3H4
T. aestivum F3H2
Chromosomal assignment and physical mapping of F3H
Specific primer pairs were designed to amplify each wheat F3H copy (Table 6). To obtain a unique amplification product, the 3' end of at least one of the two primers matched the copy-specific sequence. A touchdown PCR protocol was used to amplify from templates of the 'Chinese Spring' nulli-tetrasomic and deletion lines and the T. aestivum × T. timopheevii introgression line 842 in 20 μl reactions by applying a denaturing step of 94°C/2 min, 13 cycles of 94°C/15 s, 65°C/30 s (decreasing by 0.7°C/cycle), 72°C/45 s, 24 cycles of 94°C/15 s, 56/30 s, 72°C/45 s; and a final extension of 72°C/5 min. The specificity of the amplifications was confirmed by cloning and sequencing of the PCR product from 'Chinese Spring'. The microsatellite analysis of the 'Chinese Spring' deletion lines was performed using procedures described earlier . The microsatellite genotypic data of the T. aestivum × T. timopheevii introgression line 842 have been published .
RT-PCR and qRT-PCR
Single-stranded cDNA was synthesized from 1 mg total RNA using a (dT)15 primer and the QIAGEN Omniscript Reverse Transcription kit in a 20 μl reaction mixture. RT-PCR was performed with F3H primers published earlier  or with F3H gene copy-specific primers (Table 6). The standardization of cDNA template was performed using ubiquitin (UBC) primers . PCR products were separated by 2% agarose gel electrophoresis. F3H gene copy-specific primers were also applied for qRT-PCR, which used a QIAGEN QuantiTect SYBR Green kit. UBC and GAPDH primers were used to standardize the cDNA template. The amplifications were performed in an Applied Biosystems 7900 HT fast real time PCR system. Pre-determined amounts of cloned cDNA were used to generate standard curves. Each sample was run in three replicates. The specificity of the qRT-PCR products was confirmed by 2% agarose gel electrophoresis. Statistical significance of differences in F3H expression level either between F3H homoeologues or between different genotypes was assessed by Student's t-test for matched pairs. When F3H homoeologues were compared, T-values were calculated for each pair (F3H-A1 vs F3H-B1, etc.) in each genotype (Table 3), and 'matched pairs' were represented by expression level values obtained for respective pair of F3H homoeologues at the same day in the same genotype. When comparison was made between genotypes, T-values were calculated for each pair of genotypes ('Chinese Spring' ('Hope' 7A) vs 'Chinese Spring' ('Hope' 7B), etc.; Table 4), and 'matched pairs' were represented by expression level values obtained in respective pair of genotypes at the same day for the same F3H gene copy.
Accession numbers for sequence data
GenBank: EF463100, EU402957, EU402958, DQ233636, EU402959, EU402960, EU402961, EU402963, DQ233637.
We thank Drs. A. Börner and B.S. Gill for seed of the wheat cultivars and lines, Drs. I. Leonova and E. Pestsova for providing the microsatellite genotyping data for, respectively, T. aestivum x. timopheevii and T. aestivum/Ae. tauschii introgression lines. We also thank Dr. R. Koebner for fruitful discussion and Stefanie Lück for valuable suggestions regarding qRT-PCR. This study was supported by the Russian Foundation for Basic Research (08-04-00368-a), INTAS (04-83-3786), the program "Biodiversity and Dynamics of Gene Pools" of the Presidium of the Russian Academy of Sciences, SB RAS (Lavrentjev grant and Integration Project 5.8), the Russian Science Support Foundation, Timofeeff-Ressovsky Scientific Society "Biosphere and Mankind", and a grant from the President of the Russian Federation (MK-566.2007.4). We also thank www.smartenglish.co.uk for linguistic advice in the preparation of this manuscript.
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