Screening of the rice viviparous mutants generated by endogenous retrotransposon Tos17 insertion. Tagging of a zeaxanthin epoxidase gene and a novel ostatc gene

Abstract

The rice (Oryza sativa) retrotransposon Tos17 is one of a few active retrotransposons in plants and its transposition is activated by tissue culture. Here, we present the characterization of viviparous mutants of rice induced by tissue culture to demonstrate the feasibility of the use of retrotransposon Tos17 as an endogenous insertional mutagen and cloning of the tagged gene for forward genetics in unraveling the gene function. Two mutants were shown to be caused by the insertion of Tos17. Osaba1, a strong viviparous mutant with wilty phenotype, displayed low abscisic acid level and almost no further increase in its levels upon drought. The mutant is shown to be impaired in the epoxidation of zeaxanthin. On the other hand, Ostatc, a mutant with weak phenotype, exhibited the pale green phenotype and slight increase in abscisic acid levels upon drought. Deduced amino acids of the causative genes of Osaba1 and Ostatc manifested a significantly high homology with zeaxanthin epoxidase isolated from other plant species and with bacterial Sec-independent translocase TATC protein, respectively. This is the first example of transposon tagging in rice.

Figure 1

A, Water loss assay. Detached leaves of wild type (WT) and mutant (M) were weighed every 10 min until 60 min. Mean and sd of three independent experiments are shown. B, ABA levels in WT and M at 0 h and after 3 h of drought treatment. Mean and sd of three independent experiments are shown. C, Tagging of OsABA1 by Tos17. Cosegregation analysis of XbaI-digested genomic DNA (0.5 μg) was carried out by Southern analyses, and probed with a ³²P-labeled Tos17 (left) or the cloned OsABA1 gene (right). Numbers on the top of each lane represent individual seedling, and the genotype of each seedling is also given on the top of each lane. Lane 1 carries the genomic DNA of Nipponbare and serves as a control for other seedlings. The size of marker (λDNA digested with HindIII) fragments is indicated on the left hand in kilobase pairs.

Figure 2

Structural features of the OsABA1 and position of Tos17 insertion. A, Diagrammatic representation of the OsABA1 ORF showing the initiation codon (ATG) and the stop codon (TGA), and the positions of Tos17 insertion. Nucleotides of OsABA1 flanking the Tos17 insertion or an intron are also given. Hatched lines indicate the putative chloroplast transit peptide. B, Four regions of the tobacco ZEP (ABA2) protein are compared with the OsABA1 protein. The identical amino acid is marked by asterisk. C, Northern analysis of OsABA1 in wild type and mutant (Osaba1). Total RNA (20 μg) was subjected to northern analysis and probed with the ³²P-labeled OsABA1. Equal loading was confirmed by staining with ethidium bromide.

Figure 3

Analysis of carotenoid content in the leaves of wild type and Osaba1 mutant. A, The ABA biosynthetic pathway from zeaxanthin. Adopted from the most recently proposed pathway in plants by Qin and Zeevaart (1999). B, Carotenoid profile in the leaves of wild type and mutant (Osaba1). Presented carotenoid profile is one of three independent measurements.

Figure 4

A, The phenotype of Ostatc mutant (M) and wild type (WT) exhibited by 1-week-old seedlings grown on MS2 medium. B, Tagging of OsTATC by Tos17. Cosegregation analysis of XbaI-digested genomic DNA (0.5 μg) was carried out by Southern analyses, and probed with a ³²P-labeled Tos17 (left) or the cloned OsTATC gene (right).

Figure 5

A, Diagrammatic representation of the OsTATC ORF showing the translation start point (ATG) and the stop codon (TGA), and the position of Tos17 insertion. Nucleotide of OsTATC flanking the Tos17 insertion is given. The putative chloroplast transit peptide (hatched lines), mitochondrial targeting peptide (horizontal lines), and six transmembrane helices (clear areas) are also shown. B, Hydrophilicity analysis of OsTATC protein. Hydrophilicity was analyzed using the Tmpred program. C, Northern analysis of OsTATC in wild type and mutant (Ostatc). Total RNA (20 μg), isolated from leaf and shoot of two individual seedlings, was subjected to northern analysis, and probed with the ³²P-labeled OsTATC. Equal loading was confirmed by staining with methylene blue.

Figure 6

Biochemical and physiological analyses of Ostatc. A, Total chlorophyll (Chl), chlorophyll a, and chlorophyll b contents in the leaves of M and WT are given as nmol g⁻¹ FW. B, Water loss assay. Detached leaves of M and WT were weighed every 15 min until 120 min. Presented data are the mean of three independent experiments. C, ABA levels in M and WT at 0 min (control) and after the drought treatment. Presented data are the mean of three independent experiments.