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Roles of the different components of magnesium chelatase in abscisic acid signal transduction.

Du SY, Zhang XF, Lu Z, Xin Q, Wu Z, Jiang T, Lu Y, Wang XF, Zhang DP - Plant Mol. Biol. (2012)

Bottom Line: The overexpression lines of the CHLD gene showed wild-type ABA sensitivity in Arabidopsis.Both the GUN4-RNA interference and overexpression lines of Arabidopsis showed wild-type phenotypes in the major ABA responses.These findings provide clear evidence that the Mg-chelatase-catalyzed Mg-ProtoIX production is distinct from ABA signaling, giving information to understand the mechanism by which the two cellular processes differs at the molecular level.

View Article: PubMed Central - PubMed

Affiliation: MOE Systems Biology and Bioinformatics Laboratory, School of Life Sciences, Tsinghua University, Beijing, China.

ABSTRACT
The H subunit of Mg-chelatase (CHLH) was shown to regulate abscisic acid (ABA) signaling and the I subunit (CHLI) was also reported to modulate ABA signaling in guard cells. However, it remains essentially unknown whether and how the Mg-chelatase-catalyzed Mg-protoporphyrin IX-production differs from ABA signaling. Using a newly-developed surface plasmon resonance system, we showed that ABA binds to CHLH, but not to the other Mg-chelatase components/subunits CHLI, CHLD (D subunit) and GUN4. A new rtl1 mutant allele of the CHLH gene in Arabidopsis thaliana showed ABA-insensitive phenotypes in both stomatal movement and seed germination. Upregulation of CHLI1 resulted in ABA hypersensitivity in seed germination, while downregulation of CHLI conferred ABA insensitivity in stomatal response in Arabidopsis. We showed that CHLH and CHLI, but not CHLD, regulate stomatal sensitivity to ABA in tobacco (Nicotiana benthamiana). The overexpression lines of the CHLD gene showed wild-type ABA sensitivity in Arabidopsis. Both the GUN4-RNA interference and overexpression lines of Arabidopsis showed wild-type phenotypes in the major ABA responses. These findings provide clear evidence that the Mg-chelatase-catalyzed Mg-ProtoIX production is distinct from ABA signaling, giving information to understand the mechanism by which the two cellular processes differs at the molecular level.

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New phenotypes of rtl1, a new mutant allele of CHLH gene. a The rtl1 mutant seeds showed ABA insensitivity in germination like cch mutant. Seed germination rate of the wild-type plants (Col) and two mutant alleles cch and rtl1 of CHLH gene in the ABA-free medium (0 μM ABA) and ABA-containing medium (0.5, 1 and 3 μM) from 60 to 72 h after stratification. Each value is the mean ± SE of five independent biological determinations and different letters indicate significant differences at P < 0.05 (Duncan’s multiple range test) when comparing values within the same ABA concentration. b and c The rtl1 mutant seedlings showed wild-type ABA sensitivity, while the cch mutant seedlings showed ABA insensitivity in early growth. The wild-type plants (Col) and cch and rtl1 mutant seeds were directly planted in the ABA-free medium (0 μM ABA) and ABA-containing medium [3 μM in (b) and 0.8 μM in (c)], and seedling growth was recorded 12 d, 16 d or 24 d after stratification as indicated. d Both cch and rtl1 mutants showed ABA insensitivity in ABA-induced stomatal closure. Each value is the mean ± SE of five independent biological determinations and different letters indicate significant differences at P < 0.05 (Duncan’s multiple range test) when comparing values within the same group (within the same ABA concentration applied for assaying stomatal aperture); n = 60 apertures per experiment. e Comparison of ABA-induced inhibition of seed germination among different genotypes. Seed germination rate of the wild-type plants (Col), cch, rtl1, cs, abi4 (abi4-1), abi5 (abi5-1), a CHLI-RNAi (IR) and a CHLI-overexpression lines in the ABA-free medium (0 μM ABA) and ABA-containing medium (0.5, 1 and 3 μM) from 60 to 72 h after stratification. Each value is the mean ± SE of five independent biological determinations and different letters indicate significant differences at P < 0.05 (Duncan’s multiple range test) when comparing values within the same ABA concentration
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Fig3: New phenotypes of rtl1, a new mutant allele of CHLH gene. a The rtl1 mutant seeds showed ABA insensitivity in germination like cch mutant. Seed germination rate of the wild-type plants (Col) and two mutant alleles cch and rtl1 of CHLH gene in the ABA-free medium (0 μM ABA) and ABA-containing medium (0.5, 1 and 3 μM) from 60 to 72 h after stratification. Each value is the mean ± SE of five independent biological determinations and different letters indicate significant differences at P < 0.05 (Duncan’s multiple range test) when comparing values within the same ABA concentration. b and c The rtl1 mutant seedlings showed wild-type ABA sensitivity, while the cch mutant seedlings showed ABA insensitivity in early growth. The wild-type plants (Col) and cch and rtl1 mutant seeds were directly planted in the ABA-free medium (0 μM ABA) and ABA-containing medium [3 μM in (b) and 0.8 μM in (c)], and seedling growth was recorded 12 d, 16 d or 24 d after stratification as indicated. d Both cch and rtl1 mutants showed ABA insensitivity in ABA-induced stomatal closure. Each value is the mean ± SE of five independent biological determinations and different letters indicate significant differences at P < 0.05 (Duncan’s multiple range test) when comparing values within the same group (within the same ABA concentration applied for assaying stomatal aperture); n = 60 apertures per experiment. e Comparison of ABA-induced inhibition of seed germination among different genotypes. Seed germination rate of the wild-type plants (Col), cch, rtl1, cs, abi4 (abi4-1), abi5 (abi5-1), a CHLI-RNAi (IR) and a CHLI-overexpression lines in the ABA-free medium (0 μM ABA) and ABA-containing medium (0.5, 1 and 3 μM) from 60 to 72 h after stratification. Each value is the mean ± SE of five independent biological determinations and different letters indicate significant differences at P < 0.05 (Duncan’s multiple range test) when comparing values within the same ABA concentration

Mentions: A previous report identified a new mutant allele of CHLH gene rtl1 (rapid transcription of leaves1) with a single nucleotide substitution, resulting in a single amino acid mutation Leu690 → Phe (see Supplementary Fig. 1), which is susceptible to dehydration with defect in stomatal response to ABA, but the mutant was shown to have wild-type response in ABA-induced inhibition of seed germination and early developmental arrest (Tsuzuki et al. 2011). We assayed the ABA responses of this mutant in our experimental conditions, and observed that the seed germination of the rtl1 mutant was significantly insensitive to ABA with a weaker phenotype than cch mutant (Fig. 3a), which is a mutant harbors a single nucleotide substitution at a different site of CHLH gene, resulting in a single amino acid mutation Pro642 → Leu (Mochizuki et al. 2001; see Supplementary Fig. 1) and leading to ABA-insensitive phenotypes in seed germination, early seedling growth and stomatal movement (Shen et al. 2006; Wu et al. 2009). This observation of the defect of the rtl1 mutant in ABA response of seed germination is inconsistent with the previous report (Tsuzuki et al. 2011). However, we observed that the rtl1 mutant has wild-type response to ABA in early seedling growth (Fig. 3b, c), which is consistent with the previous observation (Tsuzuki et al. 2011), while cch mutant showed significant ABA-insensitive phenotype in early seedling growth (Fig. 3b, c) as we previously reported (Shen et al. 2006; Wu et al. 2009). Also, we confirmed the previous observation (Tsuzuki et al. 2011) that the rtl1 mutant is insensitive to ABA in stomatal movement, and found that the ABA-insensitive phenotype of this mutant in stomatal movement was significantly weaker that the cch mutant (Fig. 3d).Fig. 3


Roles of the different components of magnesium chelatase in abscisic acid signal transduction.

Du SY, Zhang XF, Lu Z, Xin Q, Wu Z, Jiang T, Lu Y, Wang XF, Zhang DP - Plant Mol. Biol. (2012)

New phenotypes of rtl1, a new mutant allele of CHLH gene. a The rtl1 mutant seeds showed ABA insensitivity in germination like cch mutant. Seed germination rate of the wild-type plants (Col) and two mutant alleles cch and rtl1 of CHLH gene in the ABA-free medium (0 μM ABA) and ABA-containing medium (0.5, 1 and 3 μM) from 60 to 72 h after stratification. Each value is the mean ± SE of five independent biological determinations and different letters indicate significant differences at P < 0.05 (Duncan’s multiple range test) when comparing values within the same ABA concentration. b and c The rtl1 mutant seedlings showed wild-type ABA sensitivity, while the cch mutant seedlings showed ABA insensitivity in early growth. The wild-type plants (Col) and cch and rtl1 mutant seeds were directly planted in the ABA-free medium (0 μM ABA) and ABA-containing medium [3 μM in (b) and 0.8 μM in (c)], and seedling growth was recorded 12 d, 16 d or 24 d after stratification as indicated. d Both cch and rtl1 mutants showed ABA insensitivity in ABA-induced stomatal closure. Each value is the mean ± SE of five independent biological determinations and different letters indicate significant differences at P < 0.05 (Duncan’s multiple range test) when comparing values within the same group (within the same ABA concentration applied for assaying stomatal aperture); n = 60 apertures per experiment. e Comparison of ABA-induced inhibition of seed germination among different genotypes. Seed germination rate of the wild-type plants (Col), cch, rtl1, cs, abi4 (abi4-1), abi5 (abi5-1), a CHLI-RNAi (IR) and a CHLI-overexpression lines in the ABA-free medium (0 μM ABA) and ABA-containing medium (0.5, 1 and 3 μM) from 60 to 72 h after stratification. Each value is the mean ± SE of five independent biological determinations and different letters indicate significant differences at P < 0.05 (Duncan’s multiple range test) when comparing values within the same ABA concentration
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Fig3: New phenotypes of rtl1, a new mutant allele of CHLH gene. a The rtl1 mutant seeds showed ABA insensitivity in germination like cch mutant. Seed germination rate of the wild-type plants (Col) and two mutant alleles cch and rtl1 of CHLH gene in the ABA-free medium (0 μM ABA) and ABA-containing medium (0.5, 1 and 3 μM) from 60 to 72 h after stratification. Each value is the mean ± SE of five independent biological determinations and different letters indicate significant differences at P < 0.05 (Duncan’s multiple range test) when comparing values within the same ABA concentration. b and c The rtl1 mutant seedlings showed wild-type ABA sensitivity, while the cch mutant seedlings showed ABA insensitivity in early growth. The wild-type plants (Col) and cch and rtl1 mutant seeds were directly planted in the ABA-free medium (0 μM ABA) and ABA-containing medium [3 μM in (b) and 0.8 μM in (c)], and seedling growth was recorded 12 d, 16 d or 24 d after stratification as indicated. d Both cch and rtl1 mutants showed ABA insensitivity in ABA-induced stomatal closure. Each value is the mean ± SE of five independent biological determinations and different letters indicate significant differences at P < 0.05 (Duncan’s multiple range test) when comparing values within the same group (within the same ABA concentration applied for assaying stomatal aperture); n = 60 apertures per experiment. e Comparison of ABA-induced inhibition of seed germination among different genotypes. Seed germination rate of the wild-type plants (Col), cch, rtl1, cs, abi4 (abi4-1), abi5 (abi5-1), a CHLI-RNAi (IR) and a CHLI-overexpression lines in the ABA-free medium (0 μM ABA) and ABA-containing medium (0.5, 1 and 3 μM) from 60 to 72 h after stratification. Each value is the mean ± SE of five independent biological determinations and different letters indicate significant differences at P < 0.05 (Duncan’s multiple range test) when comparing values within the same ABA concentration
Mentions: A previous report identified a new mutant allele of CHLH gene rtl1 (rapid transcription of leaves1) with a single nucleotide substitution, resulting in a single amino acid mutation Leu690 → Phe (see Supplementary Fig. 1), which is susceptible to dehydration with defect in stomatal response to ABA, but the mutant was shown to have wild-type response in ABA-induced inhibition of seed germination and early developmental arrest (Tsuzuki et al. 2011). We assayed the ABA responses of this mutant in our experimental conditions, and observed that the seed germination of the rtl1 mutant was significantly insensitive to ABA with a weaker phenotype than cch mutant (Fig. 3a), which is a mutant harbors a single nucleotide substitution at a different site of CHLH gene, resulting in a single amino acid mutation Pro642 → Leu (Mochizuki et al. 2001; see Supplementary Fig. 1) and leading to ABA-insensitive phenotypes in seed germination, early seedling growth and stomatal movement (Shen et al. 2006; Wu et al. 2009). This observation of the defect of the rtl1 mutant in ABA response of seed germination is inconsistent with the previous report (Tsuzuki et al. 2011). However, we observed that the rtl1 mutant has wild-type response to ABA in early seedling growth (Fig. 3b, c), which is consistent with the previous observation (Tsuzuki et al. 2011), while cch mutant showed significant ABA-insensitive phenotype in early seedling growth (Fig. 3b, c) as we previously reported (Shen et al. 2006; Wu et al. 2009). Also, we confirmed the previous observation (Tsuzuki et al. 2011) that the rtl1 mutant is insensitive to ABA in stomatal movement, and found that the ABA-insensitive phenotype of this mutant in stomatal movement was significantly weaker that the cch mutant (Fig. 3d).Fig. 3

Bottom Line: The overexpression lines of the CHLD gene showed wild-type ABA sensitivity in Arabidopsis.Both the GUN4-RNA interference and overexpression lines of Arabidopsis showed wild-type phenotypes in the major ABA responses.These findings provide clear evidence that the Mg-chelatase-catalyzed Mg-ProtoIX production is distinct from ABA signaling, giving information to understand the mechanism by which the two cellular processes differs at the molecular level.

View Article: PubMed Central - PubMed

Affiliation: MOE Systems Biology and Bioinformatics Laboratory, School of Life Sciences, Tsinghua University, Beijing, China.

ABSTRACT
The H subunit of Mg-chelatase (CHLH) was shown to regulate abscisic acid (ABA) signaling and the I subunit (CHLI) was also reported to modulate ABA signaling in guard cells. However, it remains essentially unknown whether and how the Mg-chelatase-catalyzed Mg-protoporphyrin IX-production differs from ABA signaling. Using a newly-developed surface plasmon resonance system, we showed that ABA binds to CHLH, but not to the other Mg-chelatase components/subunits CHLI, CHLD (D subunit) and GUN4. A new rtl1 mutant allele of the CHLH gene in Arabidopsis thaliana showed ABA-insensitive phenotypes in both stomatal movement and seed germination. Upregulation of CHLI1 resulted in ABA hypersensitivity in seed germination, while downregulation of CHLI conferred ABA insensitivity in stomatal response in Arabidopsis. We showed that CHLH and CHLI, but not CHLD, regulate stomatal sensitivity to ABA in tobacco (Nicotiana benthamiana). The overexpression lines of the CHLD gene showed wild-type ABA sensitivity in Arabidopsis. Both the GUN4-RNA interference and overexpression lines of Arabidopsis showed wild-type phenotypes in the major ABA responses. These findings provide clear evidence that the Mg-chelatase-catalyzed Mg-ProtoIX production is distinct from ABA signaling, giving information to understand the mechanism by which the two cellular processes differs at the molecular level.

Show MeSH
Related in: MedlinePlus