Os01g44260

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OsDfr is a stress-induced gene that produces dihydroflavonol-4-reductase involved in the flavonoid pathway.[1]

Annotated Information

Introduction

Two well-studied pathways associated with stress responses are the abscisic acid (ABA)-mediated abiotic stress response pathway and the ubiquitous flavonoid biosynthetic pathway.One common element between the ABA mediated pathway and the flavonoid pathway is the role of the MYB and MYC class of transcription activators on target gene expression. In Arabidopsis, the Atmyb2 and rd22BP1 genes regulate rd22 gene expression in the ABA-dependent abiotic stress response pathway. In maize, product of the C1-myb gene regulates expression of the flavonoid pathway genes in cooperation with a MYC family protein encoded by the R gene. The flavonoid biosynthetic pathway genes have been well- characterized and assigned with functions in two major cereal crop plants such as maize and rice. The rice flavonoid pathway is reported to respond to biotic and abiotic stress situations. Flavonoid accumulation in response to UV-B, cold, and drought were reported earilier.[1]

Drought and salinity tolerance in rice are two of the most important agronomic traits, and unraveling the genetic and molecular basis of them is of paramount importance in genetic improvement of this crop for saline and water limited environments. Most importantly, with the availability of the complete genome sequence, saturated genetic maps and good EST coverage, rice serves as a great model crop plant to study stress response mechanisms at the genetic and molecular level. Further, the flavonoid biosynthetic pathway has been one of the most thoroughly investigated pathways in plants and the products have been used extensively as visible markers in genetics and breeding experiments. This pathway is amenable for investigation in rice because of several advantages: availability of cDNA clones for all major genes of the pathway, a wealth of literature on gene expression and regulation, an array of specific mutants, clearly visible phenotypes and well-established extraction and purification protocols. Molecular dissection of the regulatory elements and the mechanisms by which they regulate the expression, temporally and spatially, of a host of genes belonging to diverse but functionally connected pathways will help in elucidation of the genetic basis of abiotic stress response in rice.[1]

Function

Anthocyanin accumulation in response to stress is genotype dependent[1]

Effect of dehydration, high salt and ABA on anthocyanin accumulation in different rice genotypes was analyzed. Ten-day-old seedlings were treated separately with 20%(w/v) PEG, 150mM NaCl and 100uM MABA and shoot samples were harvested 48 h after treatment. Anthocyanins were extracted in acidified methanol and spectrophotometrically quantitated as A530 value. The data revealed an enhanced anthocyanin accumulation in shoot tissues of competent rice genotypes (Fig. 1).

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The homozygous colorless mutant lines, Hamsa and Prasanna, are totally devoid of anthocyanin pigmentation or showed only trace amounts. The fully colored rice genotype, Purpleputtu, showed a significant increase in accumulation of anthocyanins under dehydration, high salt and ABA treatments, whereas the other two colored lines, R27 and G962, and the colorless Nagina 22 showed only a moderate increase. Dehydration stress appears to be more effective in triggering the anthocyanin accumulation among the competent genotypes.

Stress responsive changes in expression of genes of the flavonoid pathway[1]

We have analyzed stress responsive transcript accumulation of four genes of the phenyl propanoid and flavonoid pathways, OsPal (phenylalanine ammonialyase) OsChs (chalcone synthase), OsDfr (dihydroflavanol reductase) and OsAns (anthocyanidin synthase) under stress conditions. Northern analysis show that the transcript levels of the OsPal and OsChs genes were abundant in control seedlings and there were no significant changes upon stress treatment (Fig. 2).

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The OsDfr transcript which was less abundant in control plants, increased dramatically with in 3 h of treatment, reaching a peak by about 6 h, and declining thereafter to the background level by 48 h. Nagina 22 transiently accumulated OsAns transcripts at around 3–6 h post-treatment. The control Nagina 22 plants did not show any detectable levels of OsAns transcripts. The OsDfr and OsAns transcripts accumulation was much higher under dehydration than that of high salt and ABA (Fig. 2). Both the OsDfr and OsAns transcripts reached the maximum by around 6 h post-treatment of dehydration and by about 3 h post-treatment of high salt, followed by a gradual decline to the basal level by 48 h. However, in case of the ABA treatment, the OsDfr transcript level reached to its maximum by 12 h, while that of the OsAns by 6 h after treatment, then declining back to the background level by 48 h.

The rice regulatory gene, OsC1-myb, of the flavonoid pathway is responsive to abiotic stress[1]

We examined the effect of dehydration, high salt and ABA treatment on expression of the OsC1-myb homologues in rice seedlings. The OsC1-myb transcript which was not detectable in control plants, increased significantly up to 6 h and dropped down to background level by 48 h (Fig. 2). The pattern of expression of transcripts of the OsDfr, OsAns and OsC1-myb was similar in all the three treatment regimes. For each of the genes examined in these studies, the transcript levels remained constant in unstressed plants throughout the treatment period (data not shown).

Promoter region of the OsDfr and the OsAns genes harbor multiple putative stress responsive elements[1]

To understand the correlated stress responsive expression of the OsDfr and the OsAns genes, the 5' upstream region of these two genes were analyzed in detail. Plant cis acting elements databases, PLACE and PlantCARE were searched using the OsDfr and the OsAns promoter sequences as a query to identify putative stress responsive motifs present in these two promoters. Visual inspection of the sequences was used to further characterize the domain organization in comparison with well-characterized and confirmed stress responsive regulatory domains. Putative sites of significance in stress response, identified in promoter region of the OsDfr and the OsAns, are listed in Table 2.

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The C1-MYB binding sequences that resemble the consensus site, A(C/A)C(T/A)A(C/A)C, present in most of the anthocyanin gene promoters studied, were identified on both the OsDfr and the OsAns promoters (Fig. 3A).

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A MYB binding domain (TAACAAA) with a proximal pyramidine rich domain resembling the gibberellic acid responsive complex (GARC) of the rice and barley alpha-amylase gene promoter was found in 5' upstream region of the OsDfr gene (Fig. 3B). Also present on the OsDfr and the OsAns promoters is a domain sharing homology with the Arabidopsis Atmyb2 binding region (CAGTTA on the OsDfr and TGGTTAG on the OsAns) that is present in several drought stress responsive genes. Several putative ABRE/G-Box like elements with the central ACGT core are present in the promoter region of the OsAns gene (Table 2, Fig. 3C). A low temperature responsive element (LTRE) with a consensus sequence CCGACC flanked by ATGC like regions in the OsAns promoter was similar to that of the multiple stress responsive Arabidopsis cor 15a and the cold responsive Brassica BN115 genes promoter (Fig. 3C). Another interesting feature found in the OsAns promoter is the presence of a sph element, CACATGCATGCAC, (Fig. 3D).

Putative myb responsive domains on OsDfr and OsAns promoter show differential binding affinities, in vitro, for the OsC1-MYB protein[1]

To examine whether OsC1-MYB protein binds to different putative myb responsive domains identified in the OsDfr and the OsAns promoters, we used a recombinant 6His-OsC1-MYB fusion protein expressed in E. coli for protein–DNA binding studies. Gel mobility shift assay was used to study interaction between affinity purified recombinant OsC1-MYB protein and the PCR amplified oligonucleotide probes carrying different putative MYB binding domains.

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The results (Fig. 4A) show that the recombinant OsC1-MYB fusion protein bound to different oligonucleotide probes carrying putative MYB binding domains leading to the formation of a lower mobility complex (lanes 4, 6, 8, 10 and 12). However, the recombinant OsC1-MYB protein failed to bind to the putative ZmPMyb binding domain identified on the OsDfr promoter (Fig. 4A, lane 2). To assess the relative affinity of each of these domains to the OsC1-MYB protein, each oligonucleotide probe was competed with an excess of cold oligonucleotide carrying a different MYB responsive domain. Among all the tested oligonucleotides probes, the putative GAMYB binding domain showed the highest affinity to the recombinant OsC1-MYB protein, which was effectively competed out only by the addition of other cold competitors in excess of nearly 100 times. (Fig. 4B, lanes 1–8). The recombinant OsC1-MYB protein bound to the putative AtMYB2 and C1-MYB binding domains present on both the OsDfr and the OsAns promoters, respectively, with almost same affinity where merely 10 times excess of cold probes could significantly reduce the extent of binding (Fig. 4B, lanes 9–40).

Expression

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Evolution

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Structured Information

Gene Identification[2]

Gene Product Name: dihydroflavonol-4-reductase, putative, expressed

Locus Name: LOC_Os01g44260.1

Gene Attributes[2]

Chromosome: Chr1

CDS Coordinates (5'-3'): 25382698 - 25384678

Nucleotide length: 855

Predicted protein length: 285

Predicted molecular weight: 31218.5

Predicted pI: 4.8564

Gene Structure[2]

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References

  1. 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 Nagabhushana Ithal, Arjula R. Reddy. (2004) Rice flavonoid pathway genes, OsDfr and OsAns, are induced by dehydration, high salt and ABA, and contain stress responsive promoter elements that interact with the transcription activator, OsC1-MYB. Plant Science 166(6): 1505-1513.
  2. 2.0 2.1 2.2 [[1]]