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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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Phenotypes of the overexpression and RNAi lines of GUN4 gene. a Test of the expression levels of GUN4 gene in the transgenic lines by real-time PCR analysis. Col, wild-type Col-0 plants; GOE3 and GOE9, GUN4-overexpression lines 3 and 9, respectively; RG4, RG10, RG12, RG13 and RG15, GUN4-RNAi lines 4, 10, 12, 13 and 15, respectively. b Seed germination rate of the wild-type plants (Col) and different transgenic lines as described in (a) in the ABA-free medium (0 μM ABA) and ABA-containing medium (0.5, 1 and 2 μM) 60 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. c and d, Early seedling growth of the wild-type plants (Col), different GUN4-RNAi lines (c) and two GUN4-overexpression lines (d) in the ABA-free medium (0 μM ABA) and ABA-containing medium (0.8 μM) 12 d after stratification. The experiments were repeated independently five times with the same results. (e) and (f), ABA-induced stomatal closure (top panel) and ABA-inhibited stomatal opening (bottom panel) for wild-type Col and different GUN4-RNAi lines (e) and two GUN4-overexpression lines (f) in the ABA-free medium (0 μM ABA) and ABA-containing medium (20 μM). Values are the mean ± SE from three independent experiments and different letters indicate significant differences at P < 0.05 (Duncan’s multiple range test) when comparing values within the same ABA concentration. n = 60 apertures per experiment
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Fig7: Phenotypes of the overexpression and RNAi lines of GUN4 gene. a Test of the expression levels of GUN4 gene in the transgenic lines by real-time PCR analysis. Col, wild-type Col-0 plants; GOE3 and GOE9, GUN4-overexpression lines 3 and 9, respectively; RG4, RG10, RG12, RG13 and RG15, GUN4-RNAi lines 4, 10, 12, 13 and 15, respectively. b Seed germination rate of the wild-type plants (Col) and different transgenic lines as described in (a) in the ABA-free medium (0 μM ABA) and ABA-containing medium (0.5, 1 and 2 μM) 60 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. c and d, Early seedling growth of the wild-type plants (Col), different GUN4-RNAi lines (c) and two GUN4-overexpression lines (d) in the ABA-free medium (0 μM ABA) and ABA-containing medium (0.8 μM) 12 d after stratification. The experiments were repeated independently five times with the same results. (e) and (f), ABA-induced stomatal closure (top panel) and ABA-inhibited stomatal opening (bottom panel) for wild-type Col and different GUN4-RNAi lines (e) and two GUN4-overexpression lines (f) in the ABA-free medium (0 μM ABA) and ABA-containing medium (20 μM). Values are the mean ± SE from three independent experiments and different letters indicate significant differences at P < 0.05 (Duncan’s multiple range test) when comparing values within the same ABA concentration. n = 60 apertures per experiment

Mentions: We created the GUN4-RNAi and overexpression lines (Fig. 7a), and observed that all these transgenic RNAi and overexpression lines showed wild-type phenotypes in ABA-induced inhibition of seed germination (Fig. 7b) and early seedling growth arrest (Fig. 7c, d; Supplementary Fig. 2e, f), and ABA-induced stomatal closure and inhibition of stomatal opening (Fig. 7e, f). These data demonstrate that GUN4 is not involved in ABA signaling.Fig. 7


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)

Phenotypes of the overexpression and RNAi lines of GUN4 gene. a Test of the expression levels of GUN4 gene in the transgenic lines by real-time PCR analysis. Col, wild-type Col-0 plants; GOE3 and GOE9, GUN4-overexpression lines 3 and 9, respectively; RG4, RG10, RG12, RG13 and RG15, GUN4-RNAi lines 4, 10, 12, 13 and 15, respectively. b Seed germination rate of the wild-type plants (Col) and different transgenic lines as described in (a) in the ABA-free medium (0 μM ABA) and ABA-containing medium (0.5, 1 and 2 μM) 60 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. c and d, Early seedling growth of the wild-type plants (Col), different GUN4-RNAi lines (c) and two GUN4-overexpression lines (d) in the ABA-free medium (0 μM ABA) and ABA-containing medium (0.8 μM) 12 d after stratification. The experiments were repeated independently five times with the same results. (e) and (f), ABA-induced stomatal closure (top panel) and ABA-inhibited stomatal opening (bottom panel) for wild-type Col and different GUN4-RNAi lines (e) and two GUN4-overexpression lines (f) in the ABA-free medium (0 μM ABA) and ABA-containing medium (20 μM). Values are the mean ± SE from three independent experiments and different letters indicate significant differences at P < 0.05 (Duncan’s multiple range test) when comparing values within the same ABA concentration. n = 60 apertures per experiment
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Fig7: Phenotypes of the overexpression and RNAi lines of GUN4 gene. a Test of the expression levels of GUN4 gene in the transgenic lines by real-time PCR analysis. Col, wild-type Col-0 plants; GOE3 and GOE9, GUN4-overexpression lines 3 and 9, respectively; RG4, RG10, RG12, RG13 and RG15, GUN4-RNAi lines 4, 10, 12, 13 and 15, respectively. b Seed germination rate of the wild-type plants (Col) and different transgenic lines as described in (a) in the ABA-free medium (0 μM ABA) and ABA-containing medium (0.5, 1 and 2 μM) 60 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. c and d, Early seedling growth of the wild-type plants (Col), different GUN4-RNAi lines (c) and two GUN4-overexpression lines (d) in the ABA-free medium (0 μM ABA) and ABA-containing medium (0.8 μM) 12 d after stratification. The experiments were repeated independently five times with the same results. (e) and (f), ABA-induced stomatal closure (top panel) and ABA-inhibited stomatal opening (bottom panel) for wild-type Col and different GUN4-RNAi lines (e) and two GUN4-overexpression lines (f) in the ABA-free medium (0 μM ABA) and ABA-containing medium (20 μM). Values are the mean ± SE from three independent experiments and different letters indicate significant differences at P < 0.05 (Duncan’s multiple range test) when comparing values within the same ABA concentration. n = 60 apertures per experiment
Mentions: We created the GUN4-RNAi and overexpression lines (Fig. 7a), and observed that all these transgenic RNAi and overexpression lines showed wild-type phenotypes in ABA-induced inhibition of seed germination (Fig. 7b) and early seedling growth arrest (Fig. 7c, d; Supplementary Fig. 2e, f), and ABA-induced stomatal closure and inhibition of stomatal opening (Fig. 7e, f). These data demonstrate that GUN4 is not involved in ABA signaling.Fig. 7

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