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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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SPR assays: CHLH, but not CHLI1, CHLD or GUN4, binds ABA. The sample proteins [CHLH (a) and (b); CHLI (c) and (d); CHLD (e) and (f); GUN4 (g) and (h)] were immobilized to a chip by an amino-coupling process, and (+)ABA binding to these proteins was tested by recording the response data. Left panels (a, c, e, g) show the row data of a representative response record, and right panels (b, d, f, h) show the corresponding saturation curves of ABA binding to each of the proteins where the colour circles indicate the data presented in the corresponding left panels, while the filled circles and open circles represent the data, respectively, from other two independent repetitions. The experiments were repeated independently five times with the similar results
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Fig2: SPR assays: CHLH, but not CHLI1, CHLD or GUN4, binds ABA. The sample proteins [CHLH (a) and (b); CHLI (c) and (d); CHLD (e) and (f); GUN4 (g) and (h)] were immobilized to a chip by an amino-coupling process, and (+)ABA binding to these proteins was tested by recording the response data. Left panels (a, c, e, g) show the row data of a representative response record, and right panels (b, d, f, h) show the corresponding saturation curves of ABA binding to each of the proteins where the colour circles indicate the data presented in the corresponding left panels, while the filled circles and open circles represent the data, respectively, from other two independent repetitions. The experiments were repeated independently five times with the similar results

Mentions: It is essential to investigate ABA-binding abilities of all the four components of Mg-chelatase to understand their possible roles in ABA signaling. We newly adopted the surface plasmon resonance (SPR) technique for assaying ABA binding for these Mg-chelatase proteins. We observed that only CHLH binds ABA with a saturation curve typical for receptor-ligand binding (Fig. 2a, b). However, it should be noted that, in this SPR system, the detected ABA-binding affinity of CHLH was low (equilibrium dissociation constant Kd = 20 μM), which may be due to the technical limitations of this technique for testing interaction of this huge, more or less hydrophobic CHLH protein (about 150 kDa) with a small ligand.Fig. 2


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)

SPR assays: CHLH, but not CHLI1, CHLD or GUN4, binds ABA. The sample proteins [CHLH (a) and (b); CHLI (c) and (d); CHLD (e) and (f); GUN4 (g) and (h)] were immobilized to a chip by an amino-coupling process, and (+)ABA binding to these proteins was tested by recording the response data. Left panels (a, c, e, g) show the row data of a representative response record, and right panels (b, d, f, h) show the corresponding saturation curves of ABA binding to each of the proteins where the colour circles indicate the data presented in the corresponding left panels, while the filled circles and open circles represent the data, respectively, from other two independent repetitions. The experiments were repeated independently five times with the similar results
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3472068&req=5

Fig2: SPR assays: CHLH, but not CHLI1, CHLD or GUN4, binds ABA. The sample proteins [CHLH (a) and (b); CHLI (c) and (d); CHLD (e) and (f); GUN4 (g) and (h)] were immobilized to a chip by an amino-coupling process, and (+)ABA binding to these proteins was tested by recording the response data. Left panels (a, c, e, g) show the row data of a representative response record, and right panels (b, d, f, h) show the corresponding saturation curves of ABA binding to each of the proteins where the colour circles indicate the data presented in the corresponding left panels, while the filled circles and open circles represent the data, respectively, from other two independent repetitions. The experiments were repeated independently five times with the similar results
Mentions: It is essential to investigate ABA-binding abilities of all the four components of Mg-chelatase to understand their possible roles in ABA signaling. We newly adopted the surface plasmon resonance (SPR) technique for assaying ABA binding for these Mg-chelatase proteins. We observed that only CHLH binds ABA with a saturation curve typical for receptor-ligand binding (Fig. 2a, b). However, it should be noted that, in this SPR system, the detected ABA-binding affinity of CHLH was low (equilibrium dissociation constant Kd = 20 μM), which may be due to the technical limitations of this technique for testing interaction of this huge, more or less hydrophobic CHLH protein (about 150 kDa) with a small ligand.Fig. 2

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