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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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ABA-induced stomatal closure of the RNAi lines for CHLH, CHLI and CHLD genes in tobacco plants. a Immunoblotting analysis for CHLI (including CHLI1 and CHLI2, top) in the CHLI-VIGS lines VI-1 and VI-2, CHLD (middle) in the CHLD-VIGS lines VD-1 and VD-2, and CHLH proteins (bottom) in the CHLH-VIGS lines VH-1 and VH-2. The immunoblotting signals in the wild-type non-transgenic lines (WT) were serviced as controls, and Actin was used as a loading control. b Plant status of the wild-type tobacco (WT) and the VI-1, VD-1 and VH-1 transgenic lines with a VIGS line for PDS (phytoene desaturase) gene (VPDS) as a control. Bottom panel shows the chlorophyll concentrations in the corresponding lines. c ABA-induced stomatal closure for the wild-type plants (WT) and different transgenic lines (VPDS, VI-1, VD-1 and VH-1) as described in (a) and (b) in the ABA-free medium (0 μM ABA) and ABA-containing medium (30 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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Fig5: ABA-induced stomatal closure of the RNAi lines for CHLH, CHLI and CHLD genes in tobacco plants. a Immunoblotting analysis for CHLI (including CHLI1 and CHLI2, top) in the CHLI-VIGS lines VI-1 and VI-2, CHLD (middle) in the CHLD-VIGS lines VD-1 and VD-2, and CHLH proteins (bottom) in the CHLH-VIGS lines VH-1 and VH-2. The immunoblotting signals in the wild-type non-transgenic lines (WT) were serviced as controls, and Actin was used as a loading control. b Plant status of the wild-type tobacco (WT) and the VI-1, VD-1 and VH-1 transgenic lines with a VIGS line for PDS (phytoene desaturase) gene (VPDS) as a control. Bottom panel shows the chlorophyll concentrations in the corresponding lines. c ABA-induced stomatal closure for the wild-type plants (WT) and different transgenic lines (VPDS, VI-1, VD-1 and VH-1) as described in (a) and (b) in the ABA-free medium (0 μM ABA) and ABA-containing medium (30 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 failed to obtain the CHLD-RNAi lines through the transgenic manipulations as described in the Materials and Methods section because all the transgenic lines were pale and not able to establish growth. The T-DNA insertion knockout mutants of the CHLD gene available in the public resources such as Arabidopsis Biological Resource Center (ABRC) are lethal (Strand et al. 2003; Tsuzuki et al. 2011). Given that the CHLH, CHLI and CHLD are highly conserved in both their sequences and functions from bacteria to high plants (Gibson et al. 1996; Willows et al. 1996; Walker and Willows 1997; Guo et al. 1998; Papenbrock et al. 2000; for some plant CHLH, CHLI and CHLD, see Supplementary Figs. 3-6), we designed RNAi-constructs according to the ArabidopsisCHLH, CHLI and CHLD genes, and used a tobacco rattle virus (TRV) based virus induced gene silencing (VIGS) system (Liu et al. 2002) to down-regulate the expression of CHLD in tobacco (N. benthamiana). The gene silencing efficiency of the VIGS system was assessed by suppressing the expression of the phytoene desaturase (PDS) gene in tobacco, in which silencing of PDS leads to the inhibition of carotenoid synthesis, causing the plants to a photo-bleached phenotype (Fig. 5b, VPDS) as described previously (Liu et al. 2002; Kumagai et al. 1995). We successfully obtained more than ten lines that expressed the CHLD gene at relatively low levels, had slightly yellow leaves and survived well (Fig. 5a, b). Also, we created the VIGS lines for CHLH and CHLI genes both to serve as controls and to verify the functions of these two genes in tobacco. We observed that the transgenic tobacco leaves displayed pale or even blanched phenotypes when the amount of any of the three subunit proteins was downregulated to a very low level, verifying their indispensible role for chlorophyll biosynthesis as functional Mg-chelatase subunits (data not shown). We showed that the VIGS lines of CHLD gene showed wild-type stomatal response to ABA, while the VIGS lines of both the CHLH and CHLI genes showed ABA insensitive phenotype in ABA-induced stomatal closure (Fig. 5c; Supplementary Fig. 7). These data indicate that CHLH and CHLI regulate ABA response in stomatal movement in both Arabidopsis and tobacco plants, and that CHLD does not function in this stomatal response to ABA.Fig. 5


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)

ABA-induced stomatal closure of the RNAi lines for CHLH, CHLI and CHLD genes in tobacco plants. a Immunoblotting analysis for CHLI (including CHLI1 and CHLI2, top) in the CHLI-VIGS lines VI-1 and VI-2, CHLD (middle) in the CHLD-VIGS lines VD-1 and VD-2, and CHLH proteins (bottom) in the CHLH-VIGS lines VH-1 and VH-2. The immunoblotting signals in the wild-type non-transgenic lines (WT) were serviced as controls, and Actin was used as a loading control. b Plant status of the wild-type tobacco (WT) and the VI-1, VD-1 and VH-1 transgenic lines with a VIGS line for PDS (phytoene desaturase) gene (VPDS) as a control. Bottom panel shows the chlorophyll concentrations in the corresponding lines. c ABA-induced stomatal closure for the wild-type plants (WT) and different transgenic lines (VPDS, VI-1, VD-1 and VH-1) as described in (a) and (b) in the ABA-free medium (0 μM ABA) and ABA-containing medium (30 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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Related In: Results  -  Collection

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Fig5: ABA-induced stomatal closure of the RNAi lines for CHLH, CHLI and CHLD genes in tobacco plants. a Immunoblotting analysis for CHLI (including CHLI1 and CHLI2, top) in the CHLI-VIGS lines VI-1 and VI-2, CHLD (middle) in the CHLD-VIGS lines VD-1 and VD-2, and CHLH proteins (bottom) in the CHLH-VIGS lines VH-1 and VH-2. The immunoblotting signals in the wild-type non-transgenic lines (WT) were serviced as controls, and Actin was used as a loading control. b Plant status of the wild-type tobacco (WT) and the VI-1, VD-1 and VH-1 transgenic lines with a VIGS line for PDS (phytoene desaturase) gene (VPDS) as a control. Bottom panel shows the chlorophyll concentrations in the corresponding lines. c ABA-induced stomatal closure for the wild-type plants (WT) and different transgenic lines (VPDS, VI-1, VD-1 and VH-1) as described in (a) and (b) in the ABA-free medium (0 μM ABA) and ABA-containing medium (30 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 failed to obtain the CHLD-RNAi lines through the transgenic manipulations as described in the Materials and Methods section because all the transgenic lines were pale and not able to establish growth. The T-DNA insertion knockout mutants of the CHLD gene available in the public resources such as Arabidopsis Biological Resource Center (ABRC) are lethal (Strand et al. 2003; Tsuzuki et al. 2011). Given that the CHLH, CHLI and CHLD are highly conserved in both their sequences and functions from bacteria to high plants (Gibson et al. 1996; Willows et al. 1996; Walker and Willows 1997; Guo et al. 1998; Papenbrock et al. 2000; for some plant CHLH, CHLI and CHLD, see Supplementary Figs. 3-6), we designed RNAi-constructs according to the ArabidopsisCHLH, CHLI and CHLD genes, and used a tobacco rattle virus (TRV) based virus induced gene silencing (VIGS) system (Liu et al. 2002) to down-regulate the expression of CHLD in tobacco (N. benthamiana). The gene silencing efficiency of the VIGS system was assessed by suppressing the expression of the phytoene desaturase (PDS) gene in tobacco, in which silencing of PDS leads to the inhibition of carotenoid synthesis, causing the plants to a photo-bleached phenotype (Fig. 5b, VPDS) as described previously (Liu et al. 2002; Kumagai et al. 1995). We successfully obtained more than ten lines that expressed the CHLD gene at relatively low levels, had slightly yellow leaves and survived well (Fig. 5a, b). Also, we created the VIGS lines for CHLH and CHLI genes both to serve as controls and to verify the functions of these two genes in tobacco. We observed that the transgenic tobacco leaves displayed pale or even blanched phenotypes when the amount of any of the three subunit proteins was downregulated to a very low level, verifying their indispensible role for chlorophyll biosynthesis as functional Mg-chelatase subunits (data not shown). We showed that the VIGS lines of CHLD gene showed wild-type stomatal response to ABA, while the VIGS lines of both the CHLH and CHLI genes showed ABA insensitive phenotype in ABA-induced stomatal closure (Fig. 5c; Supplementary Fig. 7). These data indicate that CHLH and CHLI regulate ABA response in stomatal movement in both Arabidopsis and tobacco plants, and that CHLD does not function in this stomatal response to ABA.Fig. 5

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