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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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Related in: MedlinePlus

Interactions of CHLH/ABAR with CHLI1 and CHLD. a Test of yeast growth in SD medium lacking Leu, Trp, His, and Ade. AD, activation domain in the prey vector. BD, the DNA binding domain (BD) in the bait vector. ABARn, ABARm and ABARc indicate, respectively, N-terminal peptide (amino acid residues 1–691), median peptide (amino acid residues 347–1038) and C-terminal peptide (amino acid residues 692–1381) of CHLH/ABAR, which are linked, respectively, to BD in the bait vector. CHLI1 (indicated by I) and CHLD (indicated by D) are linked to the AD in the prey vector (indicated by I-AD, D-AD), respectively. The yeast cells were co-transformed with both vectors harboring CHLD (or CHLI1) and ABARs. The yeast cells co-transformed with the I-AD/D-AD of the prey vector and the bait vector carrying BD domain only (empty vector), or with the AD empty vector and BD-vector carrying ABARn, ABARm or ABARc, were taken as negative controls. b Coimmunoprecipitation assay in the same yeast cells as described above in (a). MYC-tagged ABARs are coimmunoprecipitated with HA-tagged CHLI1 or CHLD from yeast total proteins. Total proteins were extracted from the yeast cells transformed, respectively, with construct pairs I-ABARs and D-ABARs. Immunoprecipitation (IP) was performed with anti-HA serum or preimmune serum (p-s, as a negative control), and the immunoprecipitate was blotted (Blot) with the anti-MYC serum. The experiments were repeated three times with the same results. c ABAR and CHLI1 (or CHLD) are coimmunoprecipitated from Arabidopsis total proteins. Immunoprecipitation (IP) was performed with either the anti-CHLI1 (anti-I) or anti-CHLD (anti-D) serum and immunoblotting with the anti-ABAR serum. Immunoprecipitation with preimmune serum (p-s) was taken as a control. KD indicates the molecular mass. The experiments were repeated three times with the same results. d CHLH/ABAR interacts with CHLI1 more tightly than with CHLD. Top panel β-gal activity of the yeast cells harboring both ABARs and CHLI1 (I-ABARs) in comparison with that of the yeast cells expressing ABARs and CHLD (D-ABARs). β-Gal activity is presented as relative units (%), normalized relative to the highest activity of the I-ABARc-transformed cells. 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). Middle panel drop test of yeast growth of the above-mentioned transformed yeast cells. Note that ABA does not affect these bimolecular interactions. ‘+’ indicates (±)ABA (2 μM) treatments, and ‘−’ ethanol solution (for solubilizing ABA) (as a control). Bottom panel levels of the CHLI, CHLD or ABAR protein in the transformed yeast cells. Before drop test, yeast cells of 10 mL with the same OD600 were collected and proteins were extracted for immunoblotting with ABAR/CHLH, CHLI, and CHLD antiserum. Relative band intensities, which are normalized relative to the intensity with the highest activity of the I-ABARc-transformed cells among the I-ABARs interactions, and to that with the highest activity of the D-ABARm-transformed cells among the D-ABARs interactions (indicated by red 100), are indicated by numbers in boxes below the bands. e Firefly Luc complementation imaging to test protein–protein interactions. The tobacco leaves were transformed by infiltration using a needleless syringe with construct pairs ABAR-N-terminal half of Luc (NLuc)/CHLI1-C-terminal half of Luc (CLuc), ABAR-NLuc/CHLD-CLuc, CHLD-NLuc/CHLI1-CLuc or ABAR-NLuc/CLuc (as a negative control, see left panel)
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Fig1: Interactions of CHLH/ABAR with CHLI1 and CHLD. a Test of yeast growth in SD medium lacking Leu, Trp, His, and Ade. AD, activation domain in the prey vector. BD, the DNA binding domain (BD) in the bait vector. ABARn, ABARm and ABARc indicate, respectively, N-terminal peptide (amino acid residues 1–691), median peptide (amino acid residues 347–1038) and C-terminal peptide (amino acid residues 692–1381) of CHLH/ABAR, which are linked, respectively, to BD in the bait vector. CHLI1 (indicated by I) and CHLD (indicated by D) are linked to the AD in the prey vector (indicated by I-AD, D-AD), respectively. The yeast cells were co-transformed with both vectors harboring CHLD (or CHLI1) and ABARs. The yeast cells co-transformed with the I-AD/D-AD of the prey vector and the bait vector carrying BD domain only (empty vector), or with the AD empty vector and BD-vector carrying ABARn, ABARm or ABARc, were taken as negative controls. b Coimmunoprecipitation assay in the same yeast cells as described above in (a). MYC-tagged ABARs are coimmunoprecipitated with HA-tagged CHLI1 or CHLD from yeast total proteins. Total proteins were extracted from the yeast cells transformed, respectively, with construct pairs I-ABARs and D-ABARs. Immunoprecipitation (IP) was performed with anti-HA serum or preimmune serum (p-s, as a negative control), and the immunoprecipitate was blotted (Blot) with the anti-MYC serum. The experiments were repeated three times with the same results. c ABAR and CHLI1 (or CHLD) are coimmunoprecipitated from Arabidopsis total proteins. Immunoprecipitation (IP) was performed with either the anti-CHLI1 (anti-I) or anti-CHLD (anti-D) serum and immunoblotting with the anti-ABAR serum. Immunoprecipitation with preimmune serum (p-s) was taken as a control. KD indicates the molecular mass. The experiments were repeated three times with the same results. d CHLH/ABAR interacts with CHLI1 more tightly than with CHLD. Top panel β-gal activity of the yeast cells harboring both ABARs and CHLI1 (I-ABARs) in comparison with that of the yeast cells expressing ABARs and CHLD (D-ABARs). β-Gal activity is presented as relative units (%), normalized relative to the highest activity of the I-ABARc-transformed cells. 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). Middle panel drop test of yeast growth of the above-mentioned transformed yeast cells. Note that ABA does not affect these bimolecular interactions. ‘+’ indicates (±)ABA (2 μM) treatments, and ‘−’ ethanol solution (for solubilizing ABA) (as a control). Bottom panel levels of the CHLI, CHLD or ABAR protein in the transformed yeast cells. Before drop test, yeast cells of 10 mL with the same OD600 were collected and proteins were extracted for immunoblotting with ABAR/CHLH, CHLI, and CHLD antiserum. Relative band intensities, which are normalized relative to the intensity with the highest activity of the I-ABARc-transformed cells among the I-ABARs interactions, and to that with the highest activity of the D-ABARm-transformed cells among the D-ABARs interactions (indicated by red 100), are indicated by numbers in boxes below the bands. e Firefly Luc complementation imaging to test protein–protein interactions. The tobacco leaves were transformed by infiltration using a needleless syringe with construct pairs ABAR-N-terminal half of Luc (NLuc)/CHLI1-C-terminal half of Luc (CLuc), ABAR-NLuc/CHLD-CLuc, CHLD-NLuc/CHLI1-CLuc or ABAR-NLuc/CLuc (as a negative control, see left panel)

Mentions: It is of importance to elucidate clearly the interactions between CHLH and CHLI or CHLD to understand the mechanisms of both Mg-chelatase function and CHLH/ABAR-mediate ABA signaling. Previous reports showed that CHLH is a magnesium- and protoporphyrin IX-binding protein and interacts directly with CHLD (Grafe et al. 1999; Masuda 2008) and GUN4 (Larkin et al. 2003), but whether it interacts directly with CHLI remains unclear. We assayed interactions of different truncated CHLH/ABAR proteins with CHLD and CHLI1. CHLI includes two isoforms in Arabidopsis, CHLI1 (encoded by At4g18480 locus) and CHLI2 (encoded by At5g45930 locus), of which CHLI1 is a major isoform (Huang and Li 2009). The two CHLI isoforms function redundantly (Rissler et al. 2002; Kobayashi et al. 2008; Huang and Li 2009). The assayed truncated CHLH/ABAR proteins include the C-terminal half (ABARc), N-terminal half (ABARn) and middle region (ABARm) of CHLH/ABAR, which correspond to the amino acid residues 692–1381, 1–691, and 347–1038, respectively. In the yeast two-hybrid system, ABARn, ABARm and ABARc are linked, respectively, to the DNA binding domain (BD) in the bait vector; CHLI1 and CHLD are linked to the activation domain (AD) in the prey vector (indicated by I-AD, D-AD), respectively. The yeast cells were co-transformed with both vectors harboring CHLD (or CHLI1) and ABARs. The yeast cells co-transformed with the I-AD/D-AD of the prey vector and the empty bait vector carrying BD domain only, or with the AD empty vector and BD-vector carrying ABARn, ABARm, ABARc or empty BD vector, were taken as negative controls. The results showed that all these truncated CHLH/ABAR proteins interact with both CHLD and CHLI1 in the yeast two-hybrid system (Fig. 1a, b, d). The different negative controls showed no interaction signal (Fig. 1a), indicating that these detected bimolecular interactions are specific and reliable. It is noteworthy that CHLH/ABAR is a trans-chloroplast-membrane protein (Shang et al. 2010), and thus the truncated CHLH/ABAR could not move into the yeast nucleus to interact with CHLD and CHLI1 if the truncated CHLH/ABAR are associated with yeast membranes in this GAL4-based two hybrid system that requires interactions in the nucleus. One possible explanation for the interaction of the truncated CHLH/ABAR with CHLD and CHLI1 in the yeast cells is that CHLH/ABAR has a low hydrophobicity (Shang et al. 2010) and may likely not linked to the membranes of yeast cells that lacks plastids, and another possibility is that the truncation-caused mutations prevent association of the truncated proteins into yeast membranes. Nevertheless, we further tested these bimolecular interactions in plant systems. Coimmunoprecipitation assay in plant total protein and an in vivo luciferase complementation imaging assay (LCI) confirmed that CHLH interacts with both CHLD and CHLI1 (Fig. 1c, e). Interestingly, we found that CHLH/ABAR interacts more strongly with CHLI1 than with CHLD, and that the C-terminal half of CHLH/ABAR protein (ABARc) interacts most strongly with CHLI1 in comparison with the ABARn and ABARm truncated proteins, as evidenced by both β-galactosidase activity and drop test of yeast growth in the yeast two-hybrid system (Fig. 1d). We carefully performed these yeast-two hybrid assays and showed that the differences in the detected bimolecular-interaction intensities were not caused by the differences in the expression levels of the related proteins, in which the CHLH protein showed substantially no differences in their amounts among different treatments, and the quantities of CHLI and CHLD proteins were also carefully controlled in order that their amounts in the stronger bimolecular interactions were not higher than those in the weaker interactions (Fig. 1d), indicating that the estimations of the bimolecular-interaction intensities are reliable. None of the interactions between ABARs and CHLI1 or CHLD was affected by ABA treatment in the yeast two-hybrid system (Fig. 1d).Fig. 1


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)

Interactions of CHLH/ABAR with CHLI1 and CHLD. a Test of yeast growth in SD medium lacking Leu, Trp, His, and Ade. AD, activation domain in the prey vector. BD, the DNA binding domain (BD) in the bait vector. ABARn, ABARm and ABARc indicate, respectively, N-terminal peptide (amino acid residues 1–691), median peptide (amino acid residues 347–1038) and C-terminal peptide (amino acid residues 692–1381) of CHLH/ABAR, which are linked, respectively, to BD in the bait vector. CHLI1 (indicated by I) and CHLD (indicated by D) are linked to the AD in the prey vector (indicated by I-AD, D-AD), respectively. The yeast cells were co-transformed with both vectors harboring CHLD (or CHLI1) and ABARs. The yeast cells co-transformed with the I-AD/D-AD of the prey vector and the bait vector carrying BD domain only (empty vector), or with the AD empty vector and BD-vector carrying ABARn, ABARm or ABARc, were taken as negative controls. b Coimmunoprecipitation assay in the same yeast cells as described above in (a). MYC-tagged ABARs are coimmunoprecipitated with HA-tagged CHLI1 or CHLD from yeast total proteins. Total proteins were extracted from the yeast cells transformed, respectively, with construct pairs I-ABARs and D-ABARs. Immunoprecipitation (IP) was performed with anti-HA serum or preimmune serum (p-s, as a negative control), and the immunoprecipitate was blotted (Blot) with the anti-MYC serum. The experiments were repeated three times with the same results. c ABAR and CHLI1 (or CHLD) are coimmunoprecipitated from Arabidopsis total proteins. Immunoprecipitation (IP) was performed with either the anti-CHLI1 (anti-I) or anti-CHLD (anti-D) serum and immunoblotting with the anti-ABAR serum. Immunoprecipitation with preimmune serum (p-s) was taken as a control. KD indicates the molecular mass. The experiments were repeated three times with the same results. d CHLH/ABAR interacts with CHLI1 more tightly than with CHLD. Top panel β-gal activity of the yeast cells harboring both ABARs and CHLI1 (I-ABARs) in comparison with that of the yeast cells expressing ABARs and CHLD (D-ABARs). β-Gal activity is presented as relative units (%), normalized relative to the highest activity of the I-ABARc-transformed cells. 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). Middle panel drop test of yeast growth of the above-mentioned transformed yeast cells. Note that ABA does not affect these bimolecular interactions. ‘+’ indicates (±)ABA (2 μM) treatments, and ‘−’ ethanol solution (for solubilizing ABA) (as a control). Bottom panel levels of the CHLI, CHLD or ABAR protein in the transformed yeast cells. Before drop test, yeast cells of 10 mL with the same OD600 were collected and proteins were extracted for immunoblotting with ABAR/CHLH, CHLI, and CHLD antiserum. Relative band intensities, which are normalized relative to the intensity with the highest activity of the I-ABARc-transformed cells among the I-ABARs interactions, and to that with the highest activity of the D-ABARm-transformed cells among the D-ABARs interactions (indicated by red 100), are indicated by numbers in boxes below the bands. e Firefly Luc complementation imaging to test protein–protein interactions. The tobacco leaves were transformed by infiltration using a needleless syringe with construct pairs ABAR-N-terminal half of Luc (NLuc)/CHLI1-C-terminal half of Luc (CLuc), ABAR-NLuc/CHLD-CLuc, CHLD-NLuc/CHLI1-CLuc or ABAR-NLuc/CLuc (as a negative control, see left panel)
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3472068&req=5

Fig1: Interactions of CHLH/ABAR with CHLI1 and CHLD. a Test of yeast growth in SD medium lacking Leu, Trp, His, and Ade. AD, activation domain in the prey vector. BD, the DNA binding domain (BD) in the bait vector. ABARn, ABARm and ABARc indicate, respectively, N-terminal peptide (amino acid residues 1–691), median peptide (amino acid residues 347–1038) and C-terminal peptide (amino acid residues 692–1381) of CHLH/ABAR, which are linked, respectively, to BD in the bait vector. CHLI1 (indicated by I) and CHLD (indicated by D) are linked to the AD in the prey vector (indicated by I-AD, D-AD), respectively. The yeast cells were co-transformed with both vectors harboring CHLD (or CHLI1) and ABARs. The yeast cells co-transformed with the I-AD/D-AD of the prey vector and the bait vector carrying BD domain only (empty vector), or with the AD empty vector and BD-vector carrying ABARn, ABARm or ABARc, were taken as negative controls. b Coimmunoprecipitation assay in the same yeast cells as described above in (a). MYC-tagged ABARs are coimmunoprecipitated with HA-tagged CHLI1 or CHLD from yeast total proteins. Total proteins were extracted from the yeast cells transformed, respectively, with construct pairs I-ABARs and D-ABARs. Immunoprecipitation (IP) was performed with anti-HA serum or preimmune serum (p-s, as a negative control), and the immunoprecipitate was blotted (Blot) with the anti-MYC serum. The experiments were repeated three times with the same results. c ABAR and CHLI1 (or CHLD) are coimmunoprecipitated from Arabidopsis total proteins. Immunoprecipitation (IP) was performed with either the anti-CHLI1 (anti-I) or anti-CHLD (anti-D) serum and immunoblotting with the anti-ABAR serum. Immunoprecipitation with preimmune serum (p-s) was taken as a control. KD indicates the molecular mass. The experiments were repeated three times with the same results. d CHLH/ABAR interacts with CHLI1 more tightly than with CHLD. Top panel β-gal activity of the yeast cells harboring both ABARs and CHLI1 (I-ABARs) in comparison with that of the yeast cells expressing ABARs and CHLD (D-ABARs). β-Gal activity is presented as relative units (%), normalized relative to the highest activity of the I-ABARc-transformed cells. 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). Middle panel drop test of yeast growth of the above-mentioned transformed yeast cells. Note that ABA does not affect these bimolecular interactions. ‘+’ indicates (±)ABA (2 μM) treatments, and ‘−’ ethanol solution (for solubilizing ABA) (as a control). Bottom panel levels of the CHLI, CHLD or ABAR protein in the transformed yeast cells. Before drop test, yeast cells of 10 mL with the same OD600 were collected and proteins were extracted for immunoblotting with ABAR/CHLH, CHLI, and CHLD antiserum. Relative band intensities, which are normalized relative to the intensity with the highest activity of the I-ABARc-transformed cells among the I-ABARs interactions, and to that with the highest activity of the D-ABARm-transformed cells among the D-ABARs interactions (indicated by red 100), are indicated by numbers in boxes below the bands. e Firefly Luc complementation imaging to test protein–protein interactions. The tobacco leaves were transformed by infiltration using a needleless syringe with construct pairs ABAR-N-terminal half of Luc (NLuc)/CHLI1-C-terminal half of Luc (CLuc), ABAR-NLuc/CHLD-CLuc, CHLD-NLuc/CHLI1-CLuc or ABAR-NLuc/CLuc (as a negative control, see left panel)
Mentions: It is of importance to elucidate clearly the interactions between CHLH and CHLI or CHLD to understand the mechanisms of both Mg-chelatase function and CHLH/ABAR-mediate ABA signaling. Previous reports showed that CHLH is a magnesium- and protoporphyrin IX-binding protein and interacts directly with CHLD (Grafe et al. 1999; Masuda 2008) and GUN4 (Larkin et al. 2003), but whether it interacts directly with CHLI remains unclear. We assayed interactions of different truncated CHLH/ABAR proteins with CHLD and CHLI1. CHLI includes two isoforms in Arabidopsis, CHLI1 (encoded by At4g18480 locus) and CHLI2 (encoded by At5g45930 locus), of which CHLI1 is a major isoform (Huang and Li 2009). The two CHLI isoforms function redundantly (Rissler et al. 2002; Kobayashi et al. 2008; Huang and Li 2009). The assayed truncated CHLH/ABAR proteins include the C-terminal half (ABARc), N-terminal half (ABARn) and middle region (ABARm) of CHLH/ABAR, which correspond to the amino acid residues 692–1381, 1–691, and 347–1038, respectively. In the yeast two-hybrid system, ABARn, ABARm and ABARc are linked, respectively, to the DNA binding domain (BD) in the bait vector; CHLI1 and CHLD are linked to the activation domain (AD) in the prey vector (indicated by I-AD, D-AD), respectively. The yeast cells were co-transformed with both vectors harboring CHLD (or CHLI1) and ABARs. The yeast cells co-transformed with the I-AD/D-AD of the prey vector and the empty bait vector carrying BD domain only, or with the AD empty vector and BD-vector carrying ABARn, ABARm, ABARc or empty BD vector, were taken as negative controls. The results showed that all these truncated CHLH/ABAR proteins interact with both CHLD and CHLI1 in the yeast two-hybrid system (Fig. 1a, b, d). The different negative controls showed no interaction signal (Fig. 1a), indicating that these detected bimolecular interactions are specific and reliable. It is noteworthy that CHLH/ABAR is a trans-chloroplast-membrane protein (Shang et al. 2010), and thus the truncated CHLH/ABAR could not move into the yeast nucleus to interact with CHLD and CHLI1 if the truncated CHLH/ABAR are associated with yeast membranes in this GAL4-based two hybrid system that requires interactions in the nucleus. One possible explanation for the interaction of the truncated CHLH/ABAR with CHLD and CHLI1 in the yeast cells is that CHLH/ABAR has a low hydrophobicity (Shang et al. 2010) and may likely not linked to the membranes of yeast cells that lacks plastids, and another possibility is that the truncation-caused mutations prevent association of the truncated proteins into yeast membranes. Nevertheless, we further tested these bimolecular interactions in plant systems. Coimmunoprecipitation assay in plant total protein and an in vivo luciferase complementation imaging assay (LCI) confirmed that CHLH interacts with both CHLD and CHLI1 (Fig. 1c, e). Interestingly, we found that CHLH/ABAR interacts more strongly with CHLI1 than with CHLD, and that the C-terminal half of CHLH/ABAR protein (ABARc) interacts most strongly with CHLI1 in comparison with the ABARn and ABARm truncated proteins, as evidenced by both β-galactosidase activity and drop test of yeast growth in the yeast two-hybrid system (Fig. 1d). We carefully performed these yeast-two hybrid assays and showed that the differences in the detected bimolecular-interaction intensities were not caused by the differences in the expression levels of the related proteins, in which the CHLH protein showed substantially no differences in their amounts among different treatments, and the quantities of CHLI and CHLD proteins were also carefully controlled in order that their amounts in the stronger bimolecular interactions were not higher than those in the weaker interactions (Fig. 1d), indicating that the estimations of the bimolecular-interaction intensities are reliable. None of the interactions between ABARs and CHLI1 or CHLD was affected by ABA treatment in the yeast two-hybrid system (Fig. 1d).Fig. 1

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