Khlestkina EK

Röder MS

Salina EA

English

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 1Flavanone 3-hydroxylase genes in Triticum aestivum L.  Khlestkina EK 1, Röder MS 2, Salina EA1 1 Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Nisibirsk, 630090, Russia, e-mail: khlest@bionet.nsc.ru, 2 Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, D-06466 Gatersleben, Germany  INTRODUCTION An orthologue of the maize gene c1 (which encodes a Myb-like transcriptional factor controlling tissue-specific anthocyanin biosynthesis1) was mapped earlier on each of the short arms of wheat homoeologous group 7 chromosomes2 in positions highly comparable to those of Rc-1 (red coleoptile) genes3 and may represent the same loci. Recognizing elements (MRE) for c1 have 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)4, suggesting that Rc-1 can exert a regulatory role for wheat F3H.   In this paper, we describe the cloning, sequence analysis, mapping and expression of F3H orthologues in bread wheat, and the interaction between F3H and the Rc-1 homoeologues. MATERIALS AND METHODS The bread wheat cultivar ‘Chinese Spring’ (‘CS’) was used for PCR-based cloning. The complete set of ‘CS’ nulli-tetrasomic lines5 and a subset of homoeologous group 2 chromosome deletion lines6 were exploited to establish chromosome bin locations of F3H gene copies. Eight progeny from the cross ‘CS’ (‘Hope’ 7B) x ‘TRI 2732’3 and a set of six homozygous lines each containing a different chromosome 7D segment derived from Ae. tauschii in a ‘CS’ background7 were used to investigate a correlation between the presence of dominant alleles at the Rc-1 and expression of F3H. Quantitative examination of F3H expression was measured in ‘CS’ and ‘Mironovskaya 808’ (‘M808’) and the single chromosome substitution lines ‘CS’ (‘Hope’ 7A) and ‘CS’ (‘Hope’ 7B). RNA was extracted every 24h from two to six day old seedlings grown at 20ºC under a 12h day / 12h night regime using the QIAGEN (http://www1.qiagen.com/) Plant Rneasy Kit. Single-stranded cDNA was synthesized from 1mg total RNA using a (dT)15 primer and the QIAGEN Omniscript Reverse Transcription kit in a 20µl reaction mixture. qRT-PCR was performed using 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. Statistical significance of differences in expression level was assessed by a t-test. The specificity of the qRT-PCR products was confirmed by 2% agarose gel electrophoresis.  RESULTS AND DISCUSSION Four F3H copies were isolated from bread wheat (Genbank accession numbers for copies F3H1, 2, 3 and 4 are EF463100, DQ233636, EU402957 and EU402958, respectively). The length of the coding sequence, which was split into three exons, was 1137bp, and the first intron varied in length among the homoeologues by some hundreds of base pairs. The sequence of the segments of the first intron of the bread wheat copies F3H1, F3H2 and F3H3 not affected by deletions/insertions shared over 80% homology, but the first intron of F3H4 was quite distinct. Gene copy-specific primer pairs were constructed for each of the four F3H copies and used for profiling the nulli-tetrasomic lines of ‘CS’. This analysis showed that F3H1 and 2 are on, respectively, chromosomes 2A and 2D, while 3 and 4 both map to chromosome 2B. A deletion line analysis was then used to define the intra-chromosomal location of F3H1 to the sub-terminal bin (2AL3) of chromosome 2AL, both F3H3 and F3H4 to the terminal bin (2BL6) of chromosome 2BL, and F3H2 to the terminal bin (2DL6) of chromosome 2DL. Additional analyses (data not shown) has revealed that  F3H3 along with F3H1 and F3H2, belong to an F3H homoeoallelic series, whereas F3H4 appears to be a non-homoeologous duplication. Accordingly, the genes were re-designated F3H-A1 (F3H1), F3H-B1 (F3H3), F3H-D1 (F3H2), and F3H-B2 (F3H4). To explore the role of the Rc-1 (red coleoptile) genes as regulators for F3H expression, eight progeny from the cross ‘CS’ (‘Hope’ 7B) x ‘TRI 2732’, along with a set of six different chromosome 7D introgression lines ‘CS’-Ae. tauschii, varying with respect to the dominant allele at either Rc-B1 or Rc-D1, were subjected to RT-PCR analysis from cDNA derived from four day old seedlings. The parental genotypes with pigmented coleoptiles (‘CS’ (‘Hope’ 7B) and ‘CS’ (Ae. tauschii 7D) both showed a high level of F3H expression, whereas those with non-pigmented coleoptiles showed either little (‘TRI 2732’) or none (‘CS’). When this result was compared with the microsatellite-based genotype of the lines3,7, the regulator of F3H expression on chromosome 7B was mapped between Xgwm0263 and Xgwm0573, co-segregating with Rc-B1 (data not shown); similarly, the equivalent locus on chromosome 7D co-segregated with Rc-D1 within the genetic interval Xgwm0044 and Xgwm0111 (Figure 1). RT-PCR was also used to study the contribution of single genes F3H-A1, F3H-B1, F3H-B2 and F3H-D1 to total F3H expression. It was shown  2 that F3H-B2 is not expressed whether or not the coleoptiles are pigmented. In contrast, F3H-A1, F3H-B1 and F3H-D1 were actively expressed in lines with pigmented coleoptiles (‘CS’ (‘Hope’ 7B).                             Figure 1. F3H RT-PCR analysis from four day old seedlings of the substitution ‘CS’ (Ae. tauschii 7D) (1), ‘Chinese Spring’ (2) and the ‘CS’-Ae. tauschii 7D introgression lines (3-8). (+), (-) indicate presence/absence of anthocyanin pigmentation in the coleoptiles. The status of chromosome 7D of each line is indicated in the lower part of the panel.   To investigate the possibility of more subtle differences between expression levels of the F3H homoeologues in the presence of particular alleles of Rc-1, quantitative RT-PCR was applied to a set of cDNAs sampled from two to six day old seedlings (Figure 2). The test genotypes were ‘CS’ (‘Hope’ 7A) [Rc-A1b], ‘CS’ (‘Hope’ 7B) [Rc-B1b] and ‘M808’ [Rc-D1b], along with the control ‘CS’ which carries the non-pigmented alleles at all three Rc-1 loci. In the latter, none of the F3H copies was expressed at any time during the sampling period. F3H-B2 was not expressed in any of three test line seedlings, but F3H-A1, F3H-B1 and F3H-D1 were all expressed in these lines. No within genotype significant difference (p=0.05) in the expression level of the three homoeologues could be detected at any of the sampling times. Many sets of wheat homoeologous genes are known to be equally expressed in this way8-10, but in others, the expression of one or more members may be either completely8,11-12 or partially10,13-14 suppressed.   The overall level of F3H expression differed very significantly between each pair of lines (Figure 2) - ‘CS’ (‘Hope’ 7A) vs ‘CS’ (‘Hope’ 7B) p>0.999, ‘CS’ (‘Hope’ 7A) vs ‘M808’ p>0.999, and ‘CS’ (‘Hope’ 7B) vs ‘M808’ p>0.95).  Figure 2. Quantitative RT-PCR analysis with respect to the various copies of F3H (a - F3H-B2, b - F3H-A1, c - F3H-B1, d - F3H-D1) in ‘CS’, ‘CS’ (‘Hope’ 7A), ‘CS’ (‘Hope’ 7B) and ‘M808’  (each genotyped is characterized with 5 rows, corresponding to 2, 3, 4, 5 and 6-day old seedlings, respectively).  Overall, therefore, each Rc-1 gene appeared to regulate the expression of the three F3H homoeologues equally, but the level of expression was governed by which of the Rc-1 genes was present as a dominant allele. 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. 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. ACKNOWLEDGEMENTS This study was supported by the Russian Foundation for Basic Research (08-04-00368-a), INTAS (04-83-3786), SB RAS (Lavrentjev grant), the Russian Science Support Foundation, and a grant from the President of the Russian Federation (MK-566.2007.4). d c b aCS CS/H7A CS/H7B M808  1      2      3      4      5       6       7     8      +       -      +       -      -     +       -      -         Xgwm1187 Xgdm0130 Xgwm1672 Xgwm1002 Xgwm0044 Xgwm0437 Xgwm1242 Xgdm0046 Xgwm1168 Xgdm0067 Xgwm0428 Xgwm0111 Xgwm1335 Rc-D1 3REFERENCES 1 Paz-Ares et al. (1987) The regulatory c1 locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators. EMBO J., 6, 3553-3558.  2  Li et al. (1999) Genomic mapping of defense response genes in wheat. Theor. Appl. Genet., 98, 226-233. 3  Khlestkina et al. (2002) Molecular mapping, phenotypic expression and geographical distribution of genes determining anthocyanin pigmentation of coleoptiles in wheat (Triticum aestivum L.). Theor. Appl. Genet., 104, 632-637. 4  Hartmann et al. (2005) Differential combinatorial interactions of cis-acting elements recognized by R2R3-MYB, BZIP, and BHLH factors control light-responsive and tissue-specific activation of phenylpropanoid biosynthesis genes. Plant Mol. Biol., 57, 155-171. 5  Sears, E.R. (1953) Nullisomic analysis in common wheat. Amer. Nat., 87, 245-252. 6  Endo, T.R. and Gill, B.S. (1996) The deletion stocks of common wheat. J. Hered., 87, 295-307. 7  Pestsova et al. (2006) Development and QTL assessment of Triticum aestivum-Aegilops tauschii introgression lines. Theor. Appl. Genet., 112, 634-647. 8  Bottley et al.  (2006) Homoeologous gene silencing in hexaploid wheat. Plant J., 47, 897-906.  9  Morimoto et al. (2005) Intragenic diversity and functional conservation of the three homoeologous loci of the KN1-type homeobox gene Wknox1 in common wheat. Plant Mol. Biol., 57, 907-924.  10  Shitsukawa et al. (2007) Genetic and epigenetic alteration among three homoeologous genes of a class E MADS box gene in hexaploid wheat. Plant Cell, 19, 1723-1737.  11  Kashkush et al. (2002) Gene loss, silencing and activation in a newly synthesized wheat allotetraploid. Genetics, 160, 1651-1659.  12 Kawaura et al. (2005) Expression profile of two storage-protein gene families in hexaploid wheat revealed by large-scale analysis of expressed sequence tags. Plant Physiol., 139, 1870-1880.  13  Appleford et al. (2006) Function and transcript analysis of gibberellin-biosynthetic enzymes in wheat. Planta, 223, 568-582.   14  Nomura et al. (2005) Three genomes differentially contribute to the biosynthesis of benzoxazinones in hexaploid wheat. Proc. Natl Acad. Sci. USA, 102, 16490-16495.   

Flavanone 3-hydroxylase genes in Triticum aestivum L.

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Flavanone 3-hydroxylase genes in Triticum aestivum L.

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