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An Arabidopsis mitochondria-localized RRL protein mediates abscisic acid signal transduction through mitochondrial retrograde regulation involving ABI4.

Yao X, Li J, Liu J, Liu K - J. Exp. Bot. (2015)

Bottom Line: High expression levels of RRL were found in germinating seeds and developing seedlings, as revealed by β-glucuronidase (GUS) staining of ProRRL-GUS transgenic lines.The analyses of the structure and function of mitochondria in the knockout rrl mutant showed that the disruption of RRL causes extensively internally vacuolated mitochondria and reduced ABA-stimulated reactive oxygen species (ROS) production.Furthermore, the results revealed that ABI4 is a downstream regulatory factor in RRL-mediated ABA signalling in seed germination and seedling growth.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China.

No MeSH data available.


Genetic analysis of the double mutant abi4-1 rrl and the hybrid abi4-1 OE-4. (A, B) F2 generation seedlings of crossed plants were subjected to single nucleotide polymorphism (SNP) identification to isolate abi4 mutation loci (A) and semi-quantitative RT-PCR to determine expression of RRL (B). ACTIN2 was used as the internal control. (C) Seeds were sown on MS medium supplemented with 1.0 μM ABA. Photographs were taken at day 5 after stratification. Scale bar=0.5cm. (D) Seeds were sown on MS medium supplemented with the indicated concentration of ABA. Germination rates (%) were analysed at day 5 after stratification. Values are the means ±SE of three independent experiments (n=100). (E) Seedlings grew for 10 d after transfer from MS medium to MS medium supplemented with 10 μM ABA. Seedlings were transferred from ABA-free medium to ABA-containing medium 48h after stratification. Scale bar=0.5cm. (F) Primary root lengths were assayed after seedlings were transferred to MS medium supplemented with 0, 5, 10, 20, or 40 μM ABA for 10 d. Values are the means ±SE of three independent experiments (n=50). (This figure is available in colour at JXB online.)
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Figure 7: Genetic analysis of the double mutant abi4-1 rrl and the hybrid abi4-1 OE-4. (A, B) F2 generation seedlings of crossed plants were subjected to single nucleotide polymorphism (SNP) identification to isolate abi4 mutation loci (A) and semi-quantitative RT-PCR to determine expression of RRL (B). ACTIN2 was used as the internal control. (C) Seeds were sown on MS medium supplemented with 1.0 μM ABA. Photographs were taken at day 5 after stratification. Scale bar=0.5cm. (D) Seeds were sown on MS medium supplemented with the indicated concentration of ABA. Germination rates (%) were analysed at day 5 after stratification. Values are the means ±SE of three independent experiments (n=100). (E) Seedlings grew for 10 d after transfer from MS medium to MS medium supplemented with 10 μM ABA. Seedlings were transferred from ABA-free medium to ABA-containing medium 48h after stratification. Scale bar=0.5cm. (F) Primary root lengths were assayed after seedlings were transferred to MS medium supplemented with 0, 5, 10, 20, or 40 μM ABA for 10 d. Values are the means ±SE of three independent experiments (n=50). (This figure is available in colour at JXB online.)

Mentions: To determine whether ABI4 gene expression is necessary for the RRL-mediated ABA signalling pathway, mutant abi4-1 (maternal) was crossed with rrl (paternal) or the overexpression line OE-4 (paternal). The abi4-1 mutation locus was isolated by a dCAPS marker and RRL expression was analysed by semi-quantitative RT-PCR in the F2 generation seedlings of crossed plants (Fig. 7A, B). In the seed germination assay, the abi4-1 mutant showed an ABA-insensitive phenotype, consistent with a previous study (Finkelstein et al., 1998). The double mutant abi4-1 rrl and hybrid abi4-1 OE-4 showed a similar ABA-insensitive phenotype to abi4 mutants when 1.0 μM ABA was applied (Fig. 7C). Furthermore, germination rates with 0, 0.5, 1.0, 1.5, and 2.0 μM ABA concentrations were assessed by scoring the open green cotyledons at day 5 after stratification. The germination rates of the double mutant abi4-1 rrl and hybrid abi4-1 OE-4 were significantly higher than those of Col-0, which were comparable with those of rrl and abi4-1 mutants. In contrast, the germination rates of the overexpression line OE-4 were drastically reduced compared with Col-0 (Fig. 7D). The length of the primary root of seedling plants grown on MS medium containing the indicated concentration of ABA for 10 d were also measured. The primary root lengths of the double mutant abi4-1 rrl and hybrid abi4-1 OE-4 were also comparable with those of mutant rrl and mutant abi4-1 when the seedlings grew on MS medium containing the indicated concentrations of ABA, whereas the primary root elongation of the overexpression line OE-4 was significantly inhibited by the ABA treatments compared with Col-0 (Fig. 7E, F). Taken together, the data suggest that ABI4 plays a role downstream of RRL in ABA signalling during seed germination and primary root growth.


An Arabidopsis mitochondria-localized RRL protein mediates abscisic acid signal transduction through mitochondrial retrograde regulation involving ABI4.

Yao X, Li J, Liu J, Liu K - J. Exp. Bot. (2015)

Genetic analysis of the double mutant abi4-1 rrl and the hybrid abi4-1 OE-4. (A, B) F2 generation seedlings of crossed plants were subjected to single nucleotide polymorphism (SNP) identification to isolate abi4 mutation loci (A) and semi-quantitative RT-PCR to determine expression of RRL (B). ACTIN2 was used as the internal control. (C) Seeds were sown on MS medium supplemented with 1.0 μM ABA. Photographs were taken at day 5 after stratification. Scale bar=0.5cm. (D) Seeds were sown on MS medium supplemented with the indicated concentration of ABA. Germination rates (%) were analysed at day 5 after stratification. Values are the means ±SE of three independent experiments (n=100). (E) Seedlings grew for 10 d after transfer from MS medium to MS medium supplemented with 10 μM ABA. Seedlings were transferred from ABA-free medium to ABA-containing medium 48h after stratification. Scale bar=0.5cm. (F) Primary root lengths were assayed after seedlings were transferred to MS medium supplemented with 0, 5, 10, 20, or 40 μM ABA for 10 d. Values are the means ±SE of three independent experiments (n=50). (This figure is available in colour at JXB online.)
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Figure 7: Genetic analysis of the double mutant abi4-1 rrl and the hybrid abi4-1 OE-4. (A, B) F2 generation seedlings of crossed plants were subjected to single nucleotide polymorphism (SNP) identification to isolate abi4 mutation loci (A) and semi-quantitative RT-PCR to determine expression of RRL (B). ACTIN2 was used as the internal control. (C) Seeds were sown on MS medium supplemented with 1.0 μM ABA. Photographs were taken at day 5 after stratification. Scale bar=0.5cm. (D) Seeds were sown on MS medium supplemented with the indicated concentration of ABA. Germination rates (%) were analysed at day 5 after stratification. Values are the means ±SE of three independent experiments (n=100). (E) Seedlings grew for 10 d after transfer from MS medium to MS medium supplemented with 10 μM ABA. Seedlings were transferred from ABA-free medium to ABA-containing medium 48h after stratification. Scale bar=0.5cm. (F) Primary root lengths were assayed after seedlings were transferred to MS medium supplemented with 0, 5, 10, 20, or 40 μM ABA for 10 d. Values are the means ±SE of three independent experiments (n=50). (This figure is available in colour at JXB online.)
Mentions: To determine whether ABI4 gene expression is necessary for the RRL-mediated ABA signalling pathway, mutant abi4-1 (maternal) was crossed with rrl (paternal) or the overexpression line OE-4 (paternal). The abi4-1 mutation locus was isolated by a dCAPS marker and RRL expression was analysed by semi-quantitative RT-PCR in the F2 generation seedlings of crossed plants (Fig. 7A, B). In the seed germination assay, the abi4-1 mutant showed an ABA-insensitive phenotype, consistent with a previous study (Finkelstein et al., 1998). The double mutant abi4-1 rrl and hybrid abi4-1 OE-4 showed a similar ABA-insensitive phenotype to abi4 mutants when 1.0 μM ABA was applied (Fig. 7C). Furthermore, germination rates with 0, 0.5, 1.0, 1.5, and 2.0 μM ABA concentrations were assessed by scoring the open green cotyledons at day 5 after stratification. The germination rates of the double mutant abi4-1 rrl and hybrid abi4-1 OE-4 were significantly higher than those of Col-0, which were comparable with those of rrl and abi4-1 mutants. In contrast, the germination rates of the overexpression line OE-4 were drastically reduced compared with Col-0 (Fig. 7D). The length of the primary root of seedling plants grown on MS medium containing the indicated concentration of ABA for 10 d were also measured. The primary root lengths of the double mutant abi4-1 rrl and hybrid abi4-1 OE-4 were also comparable with those of mutant rrl and mutant abi4-1 when the seedlings grew on MS medium containing the indicated concentrations of ABA, whereas the primary root elongation of the overexpression line OE-4 was significantly inhibited by the ABA treatments compared with Col-0 (Fig. 7E, F). Taken together, the data suggest that ABI4 plays a role downstream of RRL in ABA signalling during seed germination and primary root growth.

Bottom Line: High expression levels of RRL were found in germinating seeds and developing seedlings, as revealed by β-glucuronidase (GUS) staining of ProRRL-GUS transgenic lines.The analyses of the structure and function of mitochondria in the knockout rrl mutant showed that the disruption of RRL causes extensively internally vacuolated mitochondria and reduced ABA-stimulated reactive oxygen species (ROS) production.Furthermore, the results revealed that ABI4 is a downstream regulatory factor in RRL-mediated ABA signalling in seed germination and seedling growth.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China.

No MeSH data available.