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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 CHLI gene. a Test of the expression levels of CHLI (including CHLI1 and CHLI2) gene in the transgenic lines. Top panel real-time PCR data. Bottom panel immunoblotting data with Actin as a loading control. Col, wild-type Col-0 plants; IR1, IR3, IR4 and IR6, CHLI-RNAi lines 1, 3, 4 and 6, respectively; IOE3, IOE6 and IOE7, CHLI1-overexpression lines 3, 6 and 7, respectively; cs, a T-DNA insertion line of the CHLI1 gene. 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–e, Early seedling growth of the wild-type plants (Col), cch and cs mutants and different transgenic lines [IR3 and IR6, (d); IOE3, IOE6 and IOE7, (e)] as described in (a) in the ABA-free medium (0 μM ABA) and ABA-containing medium [0.8 μM, (d) and (e); 1 μM, (c)] 12 d after stratification. The experiments were repeated independently five times with the same results. f ABA-induced stomatal closure (top panel) and ABA-inhibited stomatal opening (bottom panel) for wild-type Col, ch1-3 and cs mutants and different transgenic lines (IR3, IR6, IOE6 and IOE7) as described in (a) 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. g Whole-plant status in the water loss assays. Intact plants were well watered (Control) or drought stressed (Drought) by withholding water for 15 d for assaying water loss of the two mutants, ch1-3 and cs, and a CHLI-RNAi line IR3 in comparison with wild-type Col. The entire experiment was replicated three times with similar results
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Fig4: Phenotypes of the overexpression and RNAi lines of CHLI gene. a Test of the expression levels of CHLI (including CHLI1 and CHLI2) gene in the transgenic lines. Top panel real-time PCR data. Bottom panel immunoblotting data with Actin as a loading control. Col, wild-type Col-0 plants; IR1, IR3, IR4 and IR6, CHLI-RNAi lines 1, 3, 4 and 6, respectively; IOE3, IOE6 and IOE7, CHLI1-overexpression lines 3, 6 and 7, respectively; cs, a T-DNA insertion line of the CHLI1 gene. 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–e, Early seedling growth of the wild-type plants (Col), cch and cs mutants and different transgenic lines [IR3 and IR6, (d); IOE3, IOE6 and IOE7, (e)] as described in (a) in the ABA-free medium (0 μM ABA) and ABA-containing medium [0.8 μM, (d) and (e); 1 μM, (c)] 12 d after stratification. The experiments were repeated independently five times with the same results. f ABA-induced stomatal closure (top panel) and ABA-inhibited stomatal opening (bottom panel) for wild-type Col, ch1-3 and cs mutants and different transgenic lines (IR3, IR6, IOE6 and IOE7) as described in (a) 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. g Whole-plant status in the water loss assays. Intact plants were well watered (Control) or drought stressed (Drought) by withholding water for 15 d for assaying water loss of the two mutants, ch1-3 and cs, and a CHLI-RNAi line IR3 in comparison with wild-type Col. The entire experiment was replicated three times with similar results

Mentions: Previous report showed that downregulation of CHLI resulted in ABA insensitive phenotype in stomatal movement, but did not affect ABA responses in seed germination and early seedling growth (Tsuzuki et al. 2011). Because the authors used a knockout mutant of CHLI1 (SAIL_230_D11), which exhibited a severe dwarf and pale-green phenotype (Tsuzuki et al. 2011), and therefore may interfere with the investigations related to ABA responses, we verified these observations with a T-DNA insertion knockdown mutant in CHLI1 gene, known as cs (Mochizuki et al. 2001), and transgenic overexpression and RNAi lines of CHLI1 gene. We used the conserved sequences to created CHLI-RNAi lines, which targeted to both CHLI1 and CHLI2. We identified the overexpression and RNAi lines (Fig. 4a) using the antibody against CHLI1, which recognized CHLI1 and CHLI2. The cs mutant and the RNAi lines showed wild-type response to ABA in seed germination and early seedling growth (Fig. 4b–d; Supplementary Fig. 2a, b), but ABA insensitivity in stomatal response and reduced tolerance to dehydration (Fig. 4f, g), which is essentially consistent with previous observations (Tsuzuki et al. 2011). Interestingly, however, the overexpression lines of CHLI1 gene showed significantly hypersensitive to ABA in ABA-induced inhibition of seed germination (Fig. 4b) with wild-type phenotypes in ABA-induced early seedling growth arrest (Fig. 4e; Supplementary Fig. 2c) and in stomatal response to ABA (Fig. 4f). A chlorophyll-deficient mutant ch1-3, which contain lesions in the gene encoding chlorophyll a oxygenase (Espineda et al. 1999), was used as a control and showed wild-type ABA response in stomatal movement and drought tolerance (Fig. 4f, g), indicating that CHLI-mediated stomatal response to ABA is independent of chlorophyll biosynthesis.Fig. 4


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 CHLI gene. a Test of the expression levels of CHLI (including CHLI1 and CHLI2) gene in the transgenic lines. Top panel real-time PCR data. Bottom panel immunoblotting data with Actin as a loading control. Col, wild-type Col-0 plants; IR1, IR3, IR4 and IR6, CHLI-RNAi lines 1, 3, 4 and 6, respectively; IOE3, IOE6 and IOE7, CHLI1-overexpression lines 3, 6 and 7, respectively; cs, a T-DNA insertion line of the CHLI1 gene. 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–e, Early seedling growth of the wild-type plants (Col), cch and cs mutants and different transgenic lines [IR3 and IR6, (d); IOE3, IOE6 and IOE7, (e)] as described in (a) in the ABA-free medium (0 μM ABA) and ABA-containing medium [0.8 μM, (d) and (e); 1 μM, (c)] 12 d after stratification. The experiments were repeated independently five times with the same results. f ABA-induced stomatal closure (top panel) and ABA-inhibited stomatal opening (bottom panel) for wild-type Col, ch1-3 and cs mutants and different transgenic lines (IR3, IR6, IOE6 and IOE7) as described in (a) 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. g Whole-plant status in the water loss assays. Intact plants were well watered (Control) or drought stressed (Drought) by withholding water for 15 d for assaying water loss of the two mutants, ch1-3 and cs, and a CHLI-RNAi line IR3 in comparison with wild-type Col. The entire experiment was replicated three times with similar results
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Fig4: Phenotypes of the overexpression and RNAi lines of CHLI gene. a Test of the expression levels of CHLI (including CHLI1 and CHLI2) gene in the transgenic lines. Top panel real-time PCR data. Bottom panel immunoblotting data with Actin as a loading control. Col, wild-type Col-0 plants; IR1, IR3, IR4 and IR6, CHLI-RNAi lines 1, 3, 4 and 6, respectively; IOE3, IOE6 and IOE7, CHLI1-overexpression lines 3, 6 and 7, respectively; cs, a T-DNA insertion line of the CHLI1 gene. 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–e, Early seedling growth of the wild-type plants (Col), cch and cs mutants and different transgenic lines [IR3 and IR6, (d); IOE3, IOE6 and IOE7, (e)] as described in (a) in the ABA-free medium (0 μM ABA) and ABA-containing medium [0.8 μM, (d) and (e); 1 μM, (c)] 12 d after stratification. The experiments were repeated independently five times with the same results. f ABA-induced stomatal closure (top panel) and ABA-inhibited stomatal opening (bottom panel) for wild-type Col, ch1-3 and cs mutants and different transgenic lines (IR3, IR6, IOE6 and IOE7) as described in (a) 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. g Whole-plant status in the water loss assays. Intact plants were well watered (Control) or drought stressed (Drought) by withholding water for 15 d for assaying water loss of the two mutants, ch1-3 and cs, and a CHLI-RNAi line IR3 in comparison with wild-type Col. The entire experiment was replicated three times with similar results
Mentions: Previous report showed that downregulation of CHLI resulted in ABA insensitive phenotype in stomatal movement, but did not affect ABA responses in seed germination and early seedling growth (Tsuzuki et al. 2011). Because the authors used a knockout mutant of CHLI1 (SAIL_230_D11), which exhibited a severe dwarf and pale-green phenotype (Tsuzuki et al. 2011), and therefore may interfere with the investigations related to ABA responses, we verified these observations with a T-DNA insertion knockdown mutant in CHLI1 gene, known as cs (Mochizuki et al. 2001), and transgenic overexpression and RNAi lines of CHLI1 gene. We used the conserved sequences to created CHLI-RNAi lines, which targeted to both CHLI1 and CHLI2. We identified the overexpression and RNAi lines (Fig. 4a) using the antibody against CHLI1, which recognized CHLI1 and CHLI2. The cs mutant and the RNAi lines showed wild-type response to ABA in seed germination and early seedling growth (Fig. 4b–d; Supplementary Fig. 2a, b), but ABA insensitivity in stomatal response and reduced tolerance to dehydration (Fig. 4f, g), which is essentially consistent with previous observations (Tsuzuki et al. 2011). Interestingly, however, the overexpression lines of CHLI1 gene showed significantly hypersensitive to ABA in ABA-induced inhibition of seed germination (Fig. 4b) with wild-type phenotypes in ABA-induced early seedling growth arrest (Fig. 4e; Supplementary Fig. 2c) and in stomatal response to ABA (Fig. 4f). A chlorophyll-deficient mutant ch1-3, which contain lesions in the gene encoding chlorophyll a oxygenase (Espineda et al. 1999), was used as a control and showed wild-type ABA response in stomatal movement and drought tolerance (Fig. 4f, g), indicating that CHLI-mediated stomatal response to ABA is independent of chlorophyll biosynthesis.Fig. 4

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