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ISSN 0582-9879                                        ACTA BIOCHIMICA et BIOPHYSICA SINICA 2002, 34(6): 685-689                                     CN 31-1300/Q

Structural Analysis of a Gene Cluster Encoding DFR-like Proteins from Rice Chromosome 4

LEI Hai-Yan1,2#, ZHOU Bo1#￥, ZHANG Yu1, HONG Guo-Fan1,2*, HAN Bin1*

( 1 National Center for Gene Research, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences,

Shanghai 200233, China;

2 Institute of Biochemistry and Cell Biology, Shanghai Institutes for  Biological Sciences, the Chinese Academy of Sciences,

Shanghai 200031, China )

Abstract    Sequencing analysis of the 323 kb contig of rice chromosome 4 identified a gene cluster encoding 7 dihydroflavonol-4-reductase (DFR)-like proteins within a 56 kb region. The 7 DFR-like genes were found to be arranged in a tandem array, and all of them comprised 6 exons and 5 introns. Analysis of the predicted amino acid sequences demonstrated that these 7 proteins shared strong similarities with DFR and other enzymes of the phenylpropanoid biosynthesis pathway. RT-PCR revealed the expression pattern of the 7 genes was different in various rice tissues. The structural and functional features of these 7 DFR-like genes and their evolutionary implications are discussed.

Key words    gene cluster; rice; Arabidopsis; dihydroflavonol-4-reductase (DFR); Oryza sativa enzyme of flavonoid biosynthesis (OsEFS)

Flavonoids are secondary metabolites widespread among plants and involved in many plant functions such as UV protection, defense against pathogen attack, legume nodulation and pollen viability[1,2]. Dihydroflavonol-4-reductase (DFR) catalyses the first common step in the flavonoid biosynthetic pathway leading to anthocyanins and proanthocyanidins. The latter compounds are also known as anthocyanogens and condensed tannins. Some of the flavonoids, the anthocyanidins, anthocyanins and tanins are responsible for the red, purple and brown pigmentation of flowers, fruits, seeds and other plant tissues and organs[3]. Since these products are not essential for the viability of the plants, flavonoid biosynthesis represents an excellent model system in which to study the regulation of a complex biosynthetic pathway. Thus the genetic control of flavonoid biosynthesis has been studied in several model plants including maize、snapdragon、petunia and Arabidopsis[4,5]. Most of the genes encoding DFR have been cloned from many plants, such as Z. mays, moring glories, P.hybrida, grape[6-9] and Arabidopsis[5] etc., and their sequences are well conserved among plant species.

In this study, the 323 kb contig of rice chromosome 4 was completely sequenced and analyzed. A large gene cluster consisting of seven predicted DFR-like protein genes in a tandem array was found to be located in the 56 kb region of the contig. The deduced protein sequences of these seven genes all shared significant sequence similarities with DFRs and BANYULS[10] in Arabidopsis. The BANYULS gene encodes a DFR-like protein and is a marker of early seed coat development. Mutations in the BANYULS gene lead to precocius accumulation of anthocyanins in immature seed coat in Arabidopsis. Because DFR and BANYULS both involved in the flavonoid synthesis in plant, we deduced that these seven genes may have similar functions in rice and designated them as OsEFS (Oryza sativa enzyme of flavonoid biosynthesis). Each gene of this cluster was named according to their order in the 56 kb fragment. RT-PCR was performed to elucidate the expression pattern of each gene in this cluster. The structural and functional features of these seven DFR-like genes and their evolutionary implications are discussed.

1  Materials and Methods

1.1  Plant materials and growth conditions

Seeds of rice (Oryza sativa indica Guangluai 4) were germinated at 37 ℃ and the seedlings were grown in the light at 30 ℃ for 3 days for RNA extraction of root and bud. Leaves were collected after 10 days growing. Flowers and young panicles were prepared from 3 month old plants[11].

1.2  Cloning and sequencing of BAC clones

A BAC (bacterial artificial chromosome) contig, which was anchored on the region from 110.0 cM (centimorgan) to 111.5 cM of chromosome 4, was constructed by using colony hybridization and chromosome walking. The contig consisted of 20 BAC clones which came from two BAC libraries of Oryza sativa Guangluai 4, and the genetic markers used as probes were provided by rice genome program (RGP) in Japan[12,13]. Six tiled BAC clones (BAC H0410G08, H0315F07, H0613A10, B0808H03, H0105C05, H0323C08) with minimum overlaps were sequenced and analyzed. The BAC DNA was purified by cesium chloride gradient centrifugation, and subcloned into pBluescript II plasmid vector (Stratagene) after sonication. Subclones were sequenced at both ends using the DYEnamicTM ET dye terminator kit (Amersham Pharmacia) and analyzed on Megabase100 (Amersham Pharmacia). The sequence data were assembled using PHRED/PHRAP software. Homology searches were performed using the Blast program[14]. GENSCAN program was used to predict possible genes in this contig[15].

The nucleotide sequences of the BACs H0410G08 and B0808H03 have been submitted into the EMBL database under the accession numbers AL512546 and AL512545.

1.3  Oligonucleotides

All oligonucleotides used in this study were synthesized by Sangon company, China; except for Oligo dT-adaptor primer which was provided by RNA RCR Kit. The sequences of all oligunucleotides were shown in Table 1.

1.4  RT-PCR

Total RNA of roots，buds，leaves，flowers and young panicles of rice were extracted following the manufacture's instruction (Qiagen RNeasy Plant mini kit). For RT-PCR, 1 μg of DNase-treated total RNA was reverse-transcribed with gene-specific antisense primers and AMV reverse transcriptase using an RNA PCR kit (TaKaRa, Japan), and the entire reaction mixture was used as a template in the subsequent PCR. Each PCR cycle consisted of 94 ℃ denaturation for 30 s, 60 ℃ annealing for 30 s, 72 ℃ extension for 2 min, for 30 cycles.

2  Results

2.1  OsEFS gene cluster was revealed in the 56 kb fragment

Six overlapping BACs representing the 323 kb region of rice chromosome 4 were sequenced and analyzed by using the gene identification software GENSCAN to predict the location of the genes in this contig. The prediction result showed that seven DFR-like genes were clustered in a 56 kb fragment, and the predicted protein sequences of these 7 genes all had high similarities to DFR of rice and BANYULS of Arabidopsis. The similarities between 7 OsEFS proteins and BANYULS of Arabidopsis are 55.52%-60.18%, and the similarities to DFR in rice are 48.19%-55.56%. One mutator-like transposon was found to be located between OsEFS3 and OsEFS4. These 7 genes all had the same transcription direction and contained 6 exons and 5 introns. Though the length of each gene in genomic level is different from each other, the lengths of their coding regions are almost same. The average G+C content of each gene's coding and noncoding region is similar to other plant genes (Table 2). The predicted seven OsEFS protein sequences had high similarities with each other (Table 3). The relevant features of each deduced protein were shown in Table 4. It is interesting to find that other 6 OsEFS proteins are all acidic proteins except that the isoelectric point of OsEFS4 is 7.27. Though the amino acid compositions of 7 proteins are similar, the basic amins acid Arg is higher in OsEFS4 than in other six proteins. The number of basic amino acid Arg in OsEFS1-3, 5-7 protein is 17, 11, 12, 17, 13, 12, respectively, however the number in OsEFS4 is 25.

2.2  Transcript levels of OsEFS gene cluster

he expression of the OsEFS gene cluster in different tissues of rice was examined by RT-PCR. Total RNAs were extracted from roots, buds, leaves, flowers and young panicles. These RNA were all treated with DNase to remove any contaminated genomic DNA. As a control, amplification by RT-PCR was performed using two primers (actin-P1/actin-P2) specific for the rice actin 1 gene (ACT1). The gene-specific primers (OsEFSs 5′/ OsEFSs 3′, see Table 1) were designed according to the GENSCAN prediction result. Each gene's corresponding PCR fragment was sequenced and confirmed that they all came from the transcript of OsEFS. The amplification fragments of seven genes were transferred to membranes and hybridized with corresponding genomic DNA.

Results of RT-PCR and Southern blot revealed that seven OsEFSs transcribed at different levels in five rice tissues [Fig.1 (A), (B)]. We found that seven genes all had strong expressions in flowers and young panicles and they had different transcription in other three tissues. The expression pattern of seven genes may be due to that these genes involved in the biosynthesis of anthocyanins.

Fig.1  RT-PCR analysis of OsEFS transcripts in various tissues

(A) RT-PCR products detected by ethidium bromide staining. (B) RT-PCR products detected by Southern blot hybridization. From left to right is root, immature shoot, mature leaf,  panical and immature seed, respectively. The products of the transcripts of 7 genes were confirmed by sequencing.

3  Discussion

We found a large gene cluster encoding seven OsEFS genes in the 56 kb fragment of chromosome 4 of Oryza sativa ssp. indica cv. Guangluai 4. Protein sequence analysis demonstrated that OsEFS belongs to a superfamily of NADP(H) binding oxido-reductases as defined by Baker et al.[16]. Comparison with various databases revealed similarities to known genes encoding the dihydroflavonol reductase (DFR) of rice and many other plant species and to BANYULS of Arabidopsis. A phylogenetic tree was constructed using the CLUSTALX program and is presented in Fig.2. The tree summarizes the theoretical evolutionary distances among the different NADPH-dependent oxido-reductase superfamily members and BANYULS and OsEFS. The DFR proteins from several plant species represent a separate cluster from which OsEFSs and BANYULS are excluded. The motif of 13 amino acid residues common to DFRs and thought to define their substrate specificity are not found in BANYULS and seven OsEFS[10]. However, these 13 conserved amino acids were found in rice DFR[17]. DFR genes have been cloned from rice[17] 、sorghum[18] , barley[19] , maize[6], Arabidopsis[5] etc.. Though the copy numbers of DFR gene in each species are different, they mostly locate in a single locus. BANYULS[10] has been cloned from Arabidopsis recently and it encodes a DFR-like portein. The BANYULS locates in the chromosome 4 of Arabidopsis and the DFR gene of Arabidopsis locates in the chromosome 1. Devic et al.[10] thought  that BANYULS is not another copy of DFR of Arabidopsis, and it may encode the leucoanthocyanidin synthesis. OsEFSs have a closer evolution relation with BANYULS than that of rice DFR. From all these thoughts, we can conclude that OsEFS gene cluster were not a second DFR gene in rice and they may have a closer function with BANYULS.

Fig.2  Phylogenetic tree of the members of the superfamily of NADPH-dependent oxido-reductase including 7 OsEFS and BANYULS

The tree was constructed by the program CLUSTALX 1.0.

The predicted protein sequences of seven OsEFS genes showed high similarities to each other. From the phylogenetic tree analysis, we found that seven OsEFS genes formed four evolution branchs and the branchs were consistent with their localization order in the gene cluster. Members of gene families were often found to be located in the gene cluster in genome. Five perioxicase genes were found in rice chromosome 4[20]. Three nit genes were located in the 13.8 kb region in Arabidopsis genome[21]. Gene duplication and subsequent divergence are thought to play important roles in evolution of genes[22]. The putative evolution mechanism of the seven OsEFS genes by duplication was shown in Fig.3. At least, three gene duplication processes must be involved in generating seven tandem copies from a single copy, and a transposon insertion might occur prior to the second duplication. The insertion of this transposon led to the phenomenon that previous three genes had higher similarities than other four genes. Transposons were often found in the intergenic regions of gene clusters in the genome[23].

Fig.3  Schematic reprensentation of the putative evolution mechanism of the 7 OsEFS genes by duplication in rice

In summary, we found a large gene cluster encoding seven DFR-like protein in the 56 kb fragment of rice chromosome 4. RT-PCR analysis revealed the expression pattern of these seven genes. We discussed the structural and functional features of these seven genes. The putative evolutionary mechanism of this gene cluster was also discussed in this text.

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Received: March 27, 2002    Accepted: May 21, 2002

This work was supported by grants from the Ministry of Science and Technology, the Chinese Academy of Sciences and the Shanghai Municipal Commission of Sciences and Technology

# These authours contribute equally

￥Present address: Department of Plant Pathology, Ohio State University, Columbus, Ohio, 45210, USA

*Corresponding author: HAN Bin: Tel, 86-21-64825260; Fax, 86-21-64825775; e-mail, [email protected]. HONG Guo-Fan: Tel, 86-21-64822885; Fax, 86-21-64825775; e-mail, [email protected].