Os04g0475600

This gene named dioxygenase for auxin oxidation gene or 2-oxoglutarate-dependent-Fe (II) dioxygenase.Its gene symbol is DAO.This gene code 2-oxoglutarate-dependent-Fe (II) dioxygenase which is essential for anther dehiscence, pollen fertility, and seed initiation in rice.In vitro recombination,the gene DAO make active IAA into no biological activity of 2 - indole 3 acetic oxide. Rice mutant lines lacking a functional DAO display increased levels of free IAA in anthers and ovaries.

Introduction

IAA was found in 1880s and proved very important to plants grown up,the biosynthetic pathway for IAA in plants is still undefined.And this gene can catalyze the irreversible oxidation of IAA to OxIAA and essential for plant reproductive development. The identification of the rice dioxygenase for auxin oxidation (DAO) gene, which encodes a 2-oxoglutarate-dependent-Fe (II) (2OGFe (II))dioxygenase responsible for catalyzing the irreversible oxidation of IAA to OxIAA and essential for plant reproductive development.Sequence and phylogenetic analysis showed that DAO is a single copy gene in rice that is predicted to encode a 2OG-Fe(II) dioxygenase with a dioxygenase domain that is conserved in several classes of dioxygenase, including the GA-2 oxidase(GA2ox), GA-3 oxidase (GA3ox), and GA-20 oxidase (GA20ox). Mutant phynotype:In seedling stage and tillering stage and heading stage,there are no difference in mutant dao and widetype gene dao.flowering period Within the dao of lemma is not open, and there is no pollen from the anther no crack ,male sterility;after the flower Dao ovary and wild type size close to, produce parthenocarpy seeds, seeds are rich in sucrose but no starch accumulation.Mature period, the dao is shrunk seed dry, not germination;Applied auxin IAA or expression synthesis genes OsYUCCA1 consistent with the dao of the mutant phenotype.DAO gene location on the rice chromosome 4 a 52 KB range.Os04g0475600 first exon is a lack of a single nucleotide (25 c).Rt-pcr and protein immunoblot analysis shows that the DAO expressed in root, stem and leaf is low, but expressed in mature anther and development of the ovary is higher, and express the IAA.GUS staining, and early to rise is not observed in flower development, but in the mature anther and the sperm can be seen, moreover, GUS expression in the development of the seed.

Highlight

•Rice dao mutants contain increased levels of free IAA in anthers and ovaries

•dao mutants are defective in anther dehiscence, pollen grains, and seed development

•DAO encodes a 2-oxoglutarate-dependent-Fe (II) dioxygenase

•DAO catalyzes the conversion of active IAA into biologically inactive OxIAA

Expression

•Genetic and Physical Analysis of the Sd-1 Locus. The rice sd-1 dwarfing gene was shown to be tightly linked to restriction fragment length polymorphism (RFLP) markers RG109 and RG220 on the long arm of chromosome 1 in at least three mapping studies. First, in 178 F2 segregants from a cross between Shiokari and a near-isogenic semidwarf line (ID47), the sd-1 gene was flanked by RG109 on the proximal (0.9 cM) and RG220 on the distal side (0.3 cM; ref. 16). Second, the analysis of 2,450 segregating lines derived from a cross between near-isogenic lines Taichung65 and Taichung65 (sd-1) separated sd-1 from RG109 by 0.2 cM (17). Third, in 50 F2 segregants from a cross between Kyeema and Doongara (sd-1), RG109 co-segregated with sd-1, whereas one recombinant was identified between RG220 and sd-1 (≈1 cM) (S. Garland, personal communication). In this study, we positioned RFLP markers RG109 and RG220 on a physical segment of chromosome 1 covered by a BAC contig of approximately 300 kb by using the physical map of Nipponbare available on the RGP database (http://rgp.dna.affrc.go.jp; Fig. ​Fig.1).1). These markers were positioned near the ends of this contig spanning a physical distance of approximately 262 kb. Anchor markers C10419 and S2523, located within this region and separated by 192 kb, were mapped 1.9 cM apart on the high-resolution linkage map (RGP 2000). From this chromosomal segment and linkage map, we estimated that the ratio of physical to genetic distance was approximately 100 kb/cM in this region. When combined with data from the previous mapping studies, these results indicated that the sd-1 gene was located within a 262-kb interval flanked by markers RG109 and RG220.

•Identification of a Putative GA 20-Oxidase in the sd-1 Region. Because the sd-1 phenotype is consistent with the dwarfism that results from a deficiency in bioactive GA1, we investigated whether GA biosynthetic genes were present near the Sd-1 locus. The Rice Genome Automated Annotation system (RiceGAAS, RGP) was used to identify predicted coding regions within the interval flanked by RFLP markers RG109 and RG220, as well as within 500 kb of sequence on either side of these markers. Most of the 28 predicted coding regions located between RG109 and RG220 were related to hypothetical proteins of unknown function or retrotransposable elements. However, one predicted ORF (between positions 136550–139292 on BAC clone AP003561, Fig. ​Fig.1)1) encoded a protein of 389 amino acids that was closely related to GA 20-oxidases previously isolated from Arabidopsis (GA5, 47% identity), pea (50% identity), rice (51% identity; refs. 18–20), and related GA 20-oxidase sequences from cereals such as wheat (Fig. ​(Fig.2).2). There was no other predicted coding region identified that contained significant amino acid sequence relatedness to proteins involved in GA biosynthesis in the regions either proximal or distal to the BAC contig. Genetic analysis has linked a mutant GA 20-oxidase gene to a semidwarf phenotype in Arabidopsis (18). Therefore, we investigated the possibility that the sd-1 allele is associated with a mutation in the putative GA 20-oxidase gene.

•Characterization of Mutant GA 20-Oxidase Genes in sd-1 Lines. The putative GA 20-oxidase gene (Os20ox2) was amplified by PCR by using genomic DNA of cvs Kyeema (tall) and Doongara (sd-1) as template and primers developed from the Nipponbare genomic sequence (http://rgp.dna.affrc.go.jp). Sequence analysis of these PCR products demonstrated that the predicted ORF isolated from Kyeema was identical in sequence to Nipponbare. Intron positions (557–558 and 879–880 in Fig. ​Fig.3)3) were confirmed by RT-PCR (see Materials and Methods) and were conserved in relation to GA 20-oxidase genes isolated from Arabidopsis and pea (18, 19). The corresponding sequence amplified from the semidwarf cultivar Doongara contained a 280-bp deletion within the coding region. This deletion spanned parts of exon 1 and exon 2 and was predicted to result in a truncated, nonfunctional polypeptide containing the first 99 amino acid residues of the GA 20-oxidase, followed by an additional 3 amino acids that were in the wrong reading frame before a termination codon (Fig. ​(Fig.3).3). Hybridization of a DNA probe derived from exon 2 to digested genomic DNA of Kyeema (tall), Doongara (sd-1), and IR36 (sd-1) resulted in an RFLP pattern which was consistent with a deletion in the putative GA 20-oxidase gene (Fig. ​(Fig.4).4). The RFLP analysis identified two gene members in the rice genome: a strong band (4.6 kb) of the predicted size corresponding to a single member at the sd-1 locus, and a weaker band (≈8 kb) probably identifying a related GA 20-oxidase gene (Os20ox1) located on chromosome 3 (GenBank U50333; ref. 20).

We investigated whether an independent semidwarf mutant, allelic to sd-1, also contained an altered Os20ox2 sequence by amplifying sequences from Calrose (tall) and a semidwarf Calrose76 (11). The predicted amino acid sequence of Calrose was identical to the sequence of Nipponbare and Kyeema. The DNA sequence of Calrose76 was identical to Calrose except for a C to T transition at position 798 that resulted in a change of the predicted amino acid leucine (Leu-266) in Calrose to phenylalanine in Calrose76 (Fig. ​(Fig.3).3). This change represents a conservative substitution between hydrophobic amino acids, and although the side chain of phenylalanine is larger than that of leucine, such a substitution may not necessarily affect enzyme activity. However, a comparison of dioxygenase gene sequences from public databases revealed that Leu-266 was conserved in all GA 20-oxidase sequences. Furthermore, this residue was still conserved when 20-oxidase sequences were compared with more distantly related dioxygenases such as GA 3-oxidase and 2-oxidase sequences (refs. 7, 21; Fig. ​Fig.22).

The deduced amino acid sequence of Os20ox2 was submitted to the SWISS-MODEL protein server (22), and a portion of 94 aa (starting at amino acid residue 244 in Fig. ​Fig.2)2) was selected with a very close match to the crystal structure of a region of another dioxygenase, isopenicillin N synthase, from Aspergillus nidulans (results not shown). The α-carbon atoms of this region closely match those of the crystal structure, with an rmsd of 0.6–0.7 Å. This region includes the Leu-266 residue that is altered in the sd-1 Calrose76 mutant, and the corresponding residue in isopenicillin N synthase is also a leucine (Leu-231) that has been shown to interact by van der Waals contact with the substrate of the enzyme (23). The extremely high degree of conservation of this leucine residue, and its interaction with substrate in at least one case, suggest that any substitution will be likely to reduce or abolish enzyme activity.

•Analysis of GA Contents in Elongating Stems. Most of the variation in final plant height between tall and semidwarf lines result from differences in stem length of the first (subtending panicle) and second (subtending flag leaf) stem internodes (24). We initially compared the GA contents of Kyeema (tall) and Doongara (sd-1) by using the uppermost stem internodes at equivalent stages of growth (about 50% of final length). The results (Table ​(Table1)1) showed a higher (about three-fold) content of GA53 in the semidwarf compared with the tall line, and lower (about two-fold) contents of GA20 and GA1, and possibly GA17, which is derived from GA19 (Table ​(Table1).1). There was no significant difference between semidwarf and tall lines in the contents of GA44 and GA19. This analysis also revealed that early 13-hydroxylated GAs predominated in the stem segments, and the peaks identified for GA9 and GA34 were just above the detection limits. The elevated content of GA53 in the semidwarf, and the reduced amount of GA20 (and GA1) were consistent with impaired GA 20-oxidase activity (Fig. ​(Fig.5)5) and dwarfing caused by a deficiency of GA1. Because sd-1 rice still contained considerable amounts of GA44, GA19, GA20, and GA1, yet is proposed to have a nonfunctional Os20ox2 gene, we conclude that there must be another functional GA 20-oxidase isozyme whose activity provides biosynthetic precursors to GA1 in the stem segment.

We also compared GA contents in equivalent stem segments of Calrose and Calrose76. The results (Table ​(Table1)1) were similar to those above, in that there was an accumulation of GA53 and a deficiency of GA20 in the semidwarf variety, consistent with reduced activity of a GA 20 oxidase. The extent of these changes was slightly less than that observed for the Doongara/Kyeema pair, as was the degree of reduction in GA1 content of Calrose76 compared with Calrose (nonsignificant at P < 0.05). Calrose76 also had lower contents of GA44 and of GA19 than Calrose. These differences might be because of (i) other genetic differences between the Doongara/Kyeema pair, (ii) slight activity of the mutant GA20ox2 gene product in Calrose76, or (iii) experimental variation.

Discussion

The data from the public rice genome sequence was combined with previous mapping studies to locate a putative GA 20-oxidase gene (Os20ox2) at the expected position of sd-1 on chromosome 1. Os20ox2 was the only predicted ORF identified within a target interval of 262 kb that contained significant amino acid sequence relatedness to proteins involved in GA biosynthesis. Two independent semidwarf, allelic mutants contained alterations within Os20ox2: a deletion within Os20ox2 was predicted to encode a nonfunctional protein in Doongara (DGWG source of sd-1), whereas a change in an amino acid residue (Leu-266), which was highly conserved among dioxygenase sequences, could explain the loss of function of Os20ox2 in the semidwarf mutant Calrose76. The quantification of GAs in elongating stems revealed that the initial substrate of GA 20-oxidase (GA53) accumulated, whereas the content of the major product (GA20) was reduced in semidwarf compared with tall lines. These results were consistent with impaired GA 20-oxidase activity and dwarfing caused by a deficiency of GA1. Therefore, we propose that the semidwarf (sd-1) phenotype of rice is the result of reduced GA 20-oxidase activity, and that the defective GA 20-oxidase gene defines the sd-1 locus.

The determination of GA contents in elongating stems revealed very high levels of GA19, as observed previously in rice seedlings (20). In both semidwarfs, there was a lowered content of GA20, presumably a consequence of reduced 20-oxidase activity. In turn, the amounts of GA1, a major growth active GA, were only 65% (Doongara/Kyeema) or 80% (Calrose76/Calrose) of the corresponding tall values, although in the latter case the difference was not significant. This modest reduction in GA1 content is consistent with the relatively small reduction (approximately 25%) in the height of semidwarf vs. tall lines. The mutant GA 20-oxidase in Doongara should be a nonfunctional polypeptide, so our results imply that rice contains additional 20-oxidase activity that provided precursors contributing to approximately half of the GA1 content found in the stem internodes. A GA 20-oxidase mapping to chromosome 3 could account for the GA20 detected in stems (20). Similarly in Arabidopsis, the GA-responsive semidwarf mutant ga5 resulted from a defective GA 20-oxidase gene, and other gene members were predicted to supplement GA 20-oxidase activity resulting in a semidwarf phenotype (18).

The sd-1 gene in rice and Rht genes in wheat have played similar roles in height reduction associated with significant yield increases. The molecular bases for semidwarf stature seem to be different, with sd-1 resulting from a mutant GA 20-oxidase gene causing a deficiency of bioactive GA in elongating stems, whereas Rht genes are negative regulators of GA signaling that in the mutant are no longer modulated by GA (4). Unlike Rht semidwarf wheat, sd-1 semidwarf rice retains the ability to respond to applications of bioactive gibberellins. Most of the dwarf mutants identified in rice (d1 to d60), including those resulting from mutations in GA biosynthetic genes (dx and d18), are not used in crop improvement because they are associated with severe dwarfism, floret sterility, or abnormal plant and grain development (25). The agronomic success of sd-1 may be intimately linked to the developmental timing of its moderate dwarfing in relation to stem growth and panicle development. It will be of considerable interest to determine the detailed expression patterns of the two 20-oxidase isozymes in different parts of the rice plant, because this determination will underlie physiological interpretations of semidwarf productivity.

During the final stages of the review process of this manuscript, Monna et al. (26) and Sasaki et al. (27) described independent mutations in the GA20-oxidase gene located at the sd-1 locus, two of which confirmed the sequence alterations reported here.

Evolution

Indole-3-acetic acid (IAA), the natural auxin in plants, regulates many aspects of plant growth and development. Extensive analyses have elucidated the components of auxin biosynthesis, transport, and signaling, but the physiological roles and molecular mechanisms of auxin degradation remain elusive. Here, we demonstrate that the dioxygenase for auxin oxidation (DAO) gene, encoding a putative 2-oxoglutarate-dependent-Fe (II) dioxygenase, is essential for anther dehiscence, pollen fertility, and seed initiation in rice. Rice mutant lines lacking a functional DAO display increased levels of free IAA in anthers and ovaries. Furthermore, exogenous application of IAA or overexpression of the auxin biosynthesis gene OsYUCCA1 phenocopies the dao mutants. We show that recombinant DAO converts the active IAA into biologically inactive 2-oxoindole-3-acetic acid (OxIAA) in vitro. Collectively, these data support a key role of DAO in auxin catabolism and maintenance of auxin homeostasis central to plant reproductive development.

This gene is belong to oryza sativa and it is one of the world's oldest crop specise.In a heavy huge scale study,US researchers have thought oryza sativa orginated in China.As soon as 800 years ago appear in the Yangtze basin China.This gene is on the fourth chromsome in the rice. DAO is important in keep auxin metabolism and the balance of auxin.The most important is take action in plant reproductive development.It is indispensible on rice seeds of anther dehiscence and pollen development and morphogenesis.

Labs working on this gene

Shenyang Institute of Aeronautical Engineering, Feng-t’ien, Liaoning, China

Climate Stress Laboratory Janet P. Slovin

Horticultural Crops Quality Laboratory Jerry D. Cohen

United States Department of Agriculture, Beltsville, Maryland

References

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[2]Normanly, J., Slovin, J.P., and Cohen, J.D. (1995). Rethinking auxin biosynthesis and metabolism. Plant Physiol. 107, 323–329.

[3]Zhigang Zhao, Yunhui Zhang, Xi Liu.ect(2013). A Role for a Dioxygenase in Auxin Metabolism and Reproductive Development in Rice. Developmental Cell. 14 October 2013, 113–122.

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[11]Koga J, Adachi T, Hidaka H. Molecular cloning of the gene for indolepyruvate decarboxylase from Enterobacter cloacae. Mol Gen Genet. 1991 Apr;226(1-2):10–16.

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[13]Normanly J, Cohen JD, Fink GR. Arabidopsis thaliana auxotrophs reveal a tryptophan-independent biosynthetic pathway for indole-3-acetic acid. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):10355–10359.

[14]Szerszen JB, Szczyglowski K, Bandurski RS. iaglu, a gene from Zea mays involved in conjugation of growth hormone indole-3-acetic acid. Science. 1994 Sep 16;265(5179):1699–1701.

[15]Tam YY, Slovin JP, Cohen JD. Selection and Characterization of [alpha]-Methyltryptophan-Resistant Lines of Lemna gibba Showing a Rapid Rate of Indole-3-Acetic Acid Turnover. Plant Physiol. 1995 Jan;107(1):77–85.