The Magnesium-Chelatase H Subunit Binds Abscisic Acid and Functions in Abscisic Acid Signaling: New Evidence in Arabidopsis    

Abstract

Using a newly developed abscisic acid (ABA)-affinity chromatography technique, we showed that the magnesium-chelatase H subunit ABAR/CHLH (for putative abscisic acid receptor/chelatase H subunit) specifically binds ABA through the C-terminal half but not the N-terminal half. A set of potential agonists/antagonists to ABA, including 2-trans,4-trans-ABA, gibberellin, cytokinin-like regulator 6-benzylaminopurine, auxin indole-3-acetic acid, auxin-like substance naphthalene acetic acid, and jasmonic acid methyl ester, did not bind ABAR/CHLH. A C-terminal C370 truncated ABAR with 369 amino acid residues (631-999) was shown to bind ABA, which may be a core of the ABA-binding domain in the C-terminal half. Consistently, expression of the ABAR/CHLH C-terminal half truncated proteins fused with green fluorescent protein (GFP) in wild-type plants conferred ABA hypersensitivity in all major ABA responses, including seed germination, postgermination growth, and stomatal movement, and the expression of the same truncated proteins fused with GFP in an ABA-insensitive cch mutant of the ABAR/CHLH gene restored the ABA sensitivity of the mutant in all of the ABA responses. However, the effect of expression of the ABAR N-terminal half fused with GFP in the wild-type plants was limited to seedling growth, and the restoring effect of the ABA sensitivity of the cch mutant was limited to seed germination. In addition, we identified two new mutant alleles of ABAR/CHLH from the mutant pool in the Arabidopsis Biological Resource Center via Arabidopsis (Arabidopsis thaliana) Targeting-Induced Local Lesions in Genomes. The abar-2 mutant has a point mutation resulting in the N-terminal Leu-348/ Phe, and the abar-3 mutant has a point mutation resulting in the N-terminal Ser-183/Phe. The two mutants show altered ABA-related phenotypes in seed germination and postgermination growth but not in stomatal movement. These findings support the idea that ABAR/CHLH is an ABA receptor and reveal that the C-terminal half of ABAR/CHLH plays a central role in ABA signaling, which is consistent with its ABA-binding ability, but the N-terminal half is also functionally required, likely through a regulatory action on the C-terminal half.

ABI3 controls embryo degreening through Mendel'sIlocus

“…Whether the grs/abi3-1 mutant accumulates phototoxic chl catabolites that affect seed longevity or the mechanism behind how grs mutation enhances abi3-1 phenotype are not known. The chloroplast localized, ABA binding, Mg-chelatase H subunit, CHLH, has been shown to participate in ABA responses independent of chlorophyll biosynthesis (24). Overexpression of CHLH resulted in increased sensitivity to ABA, without altering chlorophyll levels (24), whereas the chl-deficient chlh (cch) mutant was insensitive to ABA (24).…”

“…The chloroplast localized, ABA binding, Mg-chelatase H subunit, CHLH, has been shown to participate in ABA responses independent of chlorophyll biosynthesis (24). Overexpression of CHLH resulted in increased sensitivity to ABA, without altering chlorophyll levels (24), whereas the chl-deficient chlh (cch) mutant was insensitive to ABA (24). Thus, proteins involved in chl biosynthesis (CHLH) or degradation (SGR) could also influence ABA responses, although the insensitivity displayed is quite weak compared with strong ABA insensitive mutants such as abi3-6.…”

The Nuclear Interactor PYL8/RCAR3 ofFagus sylvaticaFsPP2C1 Is a Positive Regulator of Abscisic Acid Signaling in Seeds and Stress

“…Most recently, an ABA-specific-binding protein, ABAR/CHLH (for putative abscisic acid receptor/ Mg-chelatase H subunit), that mediates ABA signaling as a positive regulator in Arabidopsis, has been found to specifically bind ABA using a newly developed ABA-affinity chromatography technique and a [ 3 H]ABA-binding assay, supporting that ABAR/ CHLH may act as an intracellular ABA receptor (Wu et al, 2009).…”

Structural insights into ABA perception and signalling: structure of ABA receptor PYR1

“…CHLH, in addition to its central role in producing Mg-protoporphyrin-IX (Walker and Willows, 1997), it was also known to play a key role in mediating plastid-to-nucleus signalling (Mochizuki et al, 2001). To further corroborate that CHLH was binding ABA, Shen et al (2006) performed a direct binding assay with [ (Shang et al, 2009), which will be further discussed in the General discussion.…”

“…Isolation of the ABA-binding protein from broad bean, and corroboration of CHLH/ABAR binding to ABA, was carried out using an ABA affinity matrix. This matrix was constructed by coupling ABA to a shepharose affinitychromatography column through its carboxylate group Wu et al, 2009). This avenue was raising the first question mark of this ABA receptor.…”