Os10g0567400

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This gene locus named after OsCAO1 of Oryza sativa Japonica Group, whose length is 2983 bps and 541 amino acids ,plays a major role in chlorophyll b biosynthesis.

Annotated Information

Function

OsCAO1 plays a major role in chlorophyll b biosynthesis.Knockout mutant lines tagged by T-DNA or Tos17 have pale green leaves, indicating chlorophyll b deficiency.And the manifested phenotype was one of pale green leaves and retarded growth,besides those particular plants produced fewer tillers, usually 3–5 compared with 13–15 in the wild types[1].

Figure 1.Phenotype comparisons between OsCAO1 KO and WT plants at 30 DAG. T/T, homozygous mutant plant; W/W, wild-type segregant from tagged line; W/T, heterozygous plant.


Mutant

The schematic diagrams of the rice CAO genes and insertion positions of T-DNA and Tos17.

Figure 2.Schematic diagrams of the rice CAO genes and insertion positions of T-DNA and Tos17

  • Line 1C-039-43, in which T-DNA insertion occurred in the first intron of OsCAO1; the direction of the gus reporter gene was the same.Genotyping our 30

T2 progeny resulted in the isolation of 4 homozygous(Lanes 7 , 12, 18 and 27), 17 heterozygous(1, 3, 5, 6, 10, 11, 14, 15, 17, 19, 20, 21 23, 24, 25, 26, and 28, and 9), wild-type (Lanes 2, 4, 8, 9, 13, 16, 22, 29, and 30) segregating plants[1].

Figure 3.Genotyping and GUS activity in progeny of Line 1C-039-43. Thirty plants were genotyped with primers a and b (upper), or primers a and RB (lower). The 0.6-kb genomicDNAbands are PCR product using primers a and b, if no T-DNA insertion; 0.4-kb bands are PCR products using primers a and RB, if T-DNA was inserted in first intron

  • Line 1B-044-10, in which the Tos17 was inserted into the fourth exon of OsCAO1.In genotyping its T2 progeny, we isolated three homozygous plants, all of which showed the phenotype of pale green leaves, stunted growth, and less tillering[1].

Figure 4.(B), ND1064 (C) and 1B-025-43 (D). LTR2 is the 3' region-specific primer of Tos17.

Expression

OsCAO1 is induced by light and is preferentially expressed in photosynthetic tissues.OsCAO1 was expressed strongly in shoots and leaves[1].

The expression pattern of OsCAO1 was light-dependent[1]

During the reproductive development stages, its expression level was higher in mature than in immature panicles. Figure 5.PCR was performed with cDNA prepared from various vegetative and reproductive organs: 1, calli; 2 and 3, shoots and roots of 7-day-old seedlings; 4, 5, and 6, leaves, stems and roots from 30-day-old plants; 7, fully expanded mature leaves at the flowering stage; 8, panicles <5 cm long; 9, panicles between 10 and 20 cm; 10, panicles between 20 and 30 cm; 11, immature seeds 2 to 5 d after pollination. Rice actin1 OsAct1 transcript was amplified as control.

When seedlings were grown under continuous light, they accumulated higher levels of OsCAO1 transcript than were measured in darkgrown plants. Figure 6.Expression levels of OsCAO1 and OsCAO2 in seedlings 4, 5, and 6 d after germination; DAG grown under continuous light L or darkness D.

When transferred dark-grown seedlings to light conditions,Within the first half hour after transfer, OsCAO1 transcript levels started to increase rapidly, reaching a maximum level in 2 h; when light-grown plants were transferred to dark conditions, OsCAO1 transcript levels decreased. Figure 7.(A) Expression levels after etiolated 4 DAG seedlings were exposed to illumination.(B) Expression patterns of 4 DAG light-grown seedlings after transfer to dark conditions.

Transcript levels of OsCAO1 followed a circadian rhythm[1]

Because the expression pattern of OsCAO1 was light-dependent, we examined whether its transcript level followed a circadian rhythm.Transcript levels of OsCAO1 were higher during the daytime, but much lower in the late afternoon and at night.During the nighttime,OsCAO1 transcript levels were high.the change in expression for OsCAO1 was gradual, reaching a maximum level at 2 PM; its accumulation began in the dark.

Figure 8.Plants were grown in greenhouse 14 h/10 h light/dark, and then transferred to continuous darkness. Flag leaves of two or three plants were harvested for RNA extraction.

OsCAO1 was inducible by all light treatments, but most significantly under blue light.

Figure 9.Expression levels were determined in 4-day-old dark-grown wild-type seedlings after exposure to various light sources. D, darkness; Fr, far-red light; R, red light; B, blue light; W, white light.

Primer Forward primer Reverse primer
RT-PCR 5'-tcaaccattggcatctcaaa-3' 5'-cgtgatgctgtcgctagtgt-3'
DNA pool screening 5'-tacagatcatgggattcaaaattggac-3' 5'-ttacccactgctcctcaaaacaatcta-3'

Evolution

This class of proteins (chlorophyll a oxygenase (CAO) protein) they encode contain two conserved functional motifs – the Rieske Fe–sulfur coordinating center and a non-heme mononuclear Fe-binding site[1].

Figure 10.Phylogeneic trees of the proteins containing the conserved Rieske [2Fe–2S] binding site from rice and Arabidopsis

Labs working on this gene

  • National Research Laboratory of Plant Functional Genomics, Division of Molecular and Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Korea
  • Department of Molecular Biology, Pusan National University, Keumjung-ku, Pusan 609-735, Korea
  • Department of Molecular Genetics, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan

References

[1] Sichul Lee,et.al,Differential regulation of chlorophyll a oxygenase genes in rice,Plant Molecular Biology (2005),57:805–818

Structured Information

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