Os03g0168600

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Annotated Information

Function

CYP704B2, conserved from moss to angiosperms, is essential for the formation of both cuticle and exine during plant male reproductive and spore development. The recombinant CYP704B2 protein catalyzed the hydroxylation of palmitic acid and unsaturated C18 fatty acids in the position of the carbon chain.

Interestingly, potential ornithology of rice CYP704B2 are found among the EST or genomic databases of bryophytes, pteridophytes, and spermatophytes, suggesting that CYP704B2 is involved in a conserved and ancient pathway for synthesis of cutin and sporopollenin during plant male reproductive and spore development.[1]

Expression

The CYP704B2 transcript is specifically detected in the tapetum and the microspore from stage 8 of anther development to stage 10. Heterologous expression of CYP704B2 in yeast demonstrated that CYP704B2 catalyzes the production of v-hydroxylated fatty acids with 16 and 18 carbon chains. CYP704B2was predicted to be expressed in immature rice panicles. In addition, recent expression profile data from laser microdissection mediated microarray analysis showed that the CYP704B2 gene was expressed in both the tapetum and microspores in the developing anther[3]. Consistently, that CYP704B2 was expressed in the anther from stage 8 to stage 10, but not in the root, stem, leaf palea/lemma, and pistil (Figure 8A). CYP704B2 expression started at stage 8, reached the highest level at stage 10, and then dropped to an undetectable level at stage 13 (Figure 8A). In transgenic plants expressing the CYP704B2pro:GUS fusion (for expression of the -glucuronidase [GUS] marker protein driven by the promoter region of CYP704B2), GUS staining was specifically detected in the anther from stage 8 to stage 10, and no expression was found in the palea, lemma, and pistil (Figures 8B and 8C). When observed by phase-contrast microscopy using cleared mounted anthers[4], only the tapetal layer and microspores showed GUS staining (Figures 8D and 8E). All data therefore converge to indicate a specific function of CYP704B2 in the tapetal cells and microspores essential for anther and pollen development. Huangfengjie1.jpg

Mutation

The cyp704B2 is a rice (Oryza sativa) male sterile mutant, which exhibits a swollen sporophytic tapetal layer, aborted pollen grains without detectable exine, and undeveloped anther cuticle. In addition, chemical composition analysis indicated that cutin monomers were hardly detectable in the cyp704B2 anthers. Like wild-type plants, the cyp704B2 plants developed normal panicles and floral organs, but the mutant anthers appeared smaller and light yellow withoutInsert non-formatted text here pollen grains.Huangfengjie2.jpg

Subsequently, cyp704B2 anthers displayed obvious morphological abnormalities. At stage 9, young microspores were released from the tetrad, tapetal cells became more condensed and deeply stained, and the middle layer appeared degenerated and almost invisible in the wild type (Figure 2C). Although the cyp704B2 microspores appeared normal, the tapetal cells seemed less condensed at this stage (Figure 2H). At early stage 10, vacuolated and round shaped microspores with visible exines were observed in the wild type, and the degradation of the tapetum continued (Figure 2D). By contrast, the tapetal cells in cyp704B2 were swollen and lightly stained, and the microspore became less vacuolated and collapsed without an obvious exine on its outer surface (Figure 2I). At the late stage 10, the wild-type tapetal layer nearly disappeared and it was weakly stained. Vacuolated microspores with abundant exine depositions were visible (Figure 2E). Unlike those of wild-type plants, cyp704B2 microspores were aborted, and only remnants remained at the center of the anther locule (Figure 2J).In addition, the mutant middle layer and tapetum expanded until they occupied the space of locule (Figure 2J).Huangfengjie3.jpg

The cyp704B2 anther epidermis compared with the wild type by scanning electron microscopy examination (Figures 4A and 4B). Moreover, we observed a normal outermost linear-shaped surface of the wild-type anther (Figure 4C), and the pollen developed the exquisite exine pattern and a visible germination pore (Figure 4D). However, a glossy and smooth anther surface (Figure 4E) and shrunken microspores (Figure 4F) in cyp704B2 were observed as a result of the undeveloped cuticle/epicuticular structures and lack of obvious pollen exine.Huangfengjie4.jpg

Evolution

Each plant species has a large number of P450 members with a diversity of biological functions in biosynthetic and catabolic pathways[2]. A total of 39 putative ESTs and annotated protein sequences that are related to riceCYP704B2 were obtained from 19 different species. Along with the six and two members of the CYP704A subfamily encoded by the genome of rice and Arabidopsis, respectively (Figure 7), these P450s belonged to four families all from the CYP86 clan: CYP86, CYP94, CYP704, and CYP96.Huangfengjie5.jpg

Labs working on this gene

School of Life Science and Biotechnology, Shanghai Jiaotong University, Shanghai, China

Institut de Biologie Mole ´ culaire des Plantes, Centre National de la Recherche Scientifique, Unite ´ Propre de Recherche Universite de Strasbourg, Strasbourg Cedex, France

Institute of Cellular and Molecular Botany, University of Bonn, Bonn, Germany

Bio-X Research Center, Key Laboratory of Genetics and Development and Neuropsychiatric Diseases, Ministry of Education,Shanghai Jiaotong University, Shanghai , China

References

[1] Hui Li, Franck Pinot, Vincent Sauveplane, Danie ` le Werck-Reichhart, Patrik Diehl, Lukas Schreiber, Rochus Franke, Ping Zhang, Liang Chen, Yawei Gao, Wanqi Liang,and Dabing Zhang(2010).Cytochrome P450 Family Member CYP704B2 Catalyzes the w-Hydroxylation of Fatty Acids and Is Required for Anther Cutin Biosynthesis and Pollen Exine Formation in Rice. The Plant Cell, Vol. 22: 173–190.

[2] Schuler, M.A., and Werck-Reichhart, D. (2003). Functional genomics of P450s. Annu. Rev. Plant Biol. 54: 629–667.

[3] Hobo, T., et al. (2008). Various spatiotemporal expression profiles of anther-expressed genes in rice. Plant Cell Physiol. 49: 1417–1428.

[4] Willemsen, V., Wolkenfelt, H., de Vrieze, G., Weisbeek, P., and Scheres, B. (1998). The HOBBIT gene is required for formation of the root meristem in the Arabidopsis embryo. Development 125: 521–531.

Structured Information

Based on primary mapping, we mapped this gene to a genetic distance of 16.8 and 17.9 centimorgans with CL6-4 and SJ301 markers on chromosome 3 (Figure 6A). To localize the mutant gene more precisely, 2700 individuals of F2 mapping population were analyzed using a set of primer pairs. Finally, this mutant gene was located between two markers SJ622 and LH301, defining a DNA region of 116 kb, and these two markers were located in the BAC clones OSJNBa0026H19 and OSJNBa0091P11, respectively. No recombinant was found using another marker CL8-1 (Figure 6A). By PCR amplification using the forward primer 3F and reverse primer 2R (Figure 6B) and sequencing analysis, we found in the mutant a DNA fragment deletion of 3102 bp, which included 258 bp of the 39 coding region of Os03g07260, 1732 bp of the genic region of Os03g07250, and 1112 bp of the Inter genic region between the two genes (Figure 6B).

To determine which gene is responsible for the main defects of anther development in cyp704B2, two constructs were generated by PCR amplification from the BAC clone (OSJN- Ba0091P11): one carrying a 3.8-kb wild-type genomic fragment for Os03g07250/CYP704B2 using the primer pair 1F and 2R, and another one containing the fragment of 4 kb for Os03g07260 from the primer pair 4F and 3R (Figure 6B). Among the transgenic plants obtained from transformed calli induced from the flower of the cyp704B2 mutant, only the construct including Os03g07250 was able to restore pollen fertility and normal anther epidermal surface in the homozygous mutant plants (see Supplemental Figure 4 online). These results confirmed that the deletion of Os03g07250 is responsible for the developmental defects found in this mutant.

To verify the gene structure of the CYP704B2 gene, we determined exon-intron junctions by sequencing the cDNA frag- ment amplified by RT-PCR. Comparison with the genomic sequence indicated thatCYP704B2 contained four exons and three introns (Figure 6B). CYP704B2 putatively encodes a cytochrome P450 protein with 545 amino acids containing a transmembrane segment of 23 amino acids (VTSFFPVAGIHKLIAIFLVVLSW) in the N-terminal region (Figure 6C).Huangfengjie6.jpg