The Mg-chelatase complex as a whole affects the ABA-signaling pathway for stomatal movements

Mg‐chelatase H subunit affects ABA signaling in stomatal guard cells, but is not an ABA receptor in Arabidopsis thaliana

by Tsuzuki T., Takahashi K., Inoue S.‐i., Okigaki Y., Tomiyama M., Hossain M. A., Shimazaki K.‐i., Murata Y., Kinoshita T. ( 2011)

Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan.

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In J Plant Res 124: 527– 538 – DOI: 10.1007/s10265-011-0426-x –

https://www.ncbi.nlm.nih.gov/pubmed/21562844?dopt=Abstract

Abstract

Mg-chelatase H subunit (CHLH) is a multifunctional protein involved in chlorophyll synthesis, plastid-to-nucleus retrograde signaling, and ABA perception. However, whether CHLH acts as an actual ABA receptor remains controversial.

Here we present evidence that CHLH affects ABA signaling in stomatal guard cells but is not itself an ABA receptor. We screened ethyl methanesulfonate-treated Arabidopsis thaliana plants with a focus on stomatal aperture-dependent water loss in detached leaves and isolated a rapid transpiration in detached leaves 1 (rtl1) mutant that we identified as a novel missense mutant of CHLH. The rtl1 and CHLH RNAi plants showed phenotypes in which stomatal movements were insensitive to ABA, while the rtl1 phenotype showed normal sensitivity to ABA with respect to seed germination and root growth.

ABA-binding analyses using (3)H-labeled ABA revealed that recombinant CHLH did not bind ABA, but recombinant pyrabactin resistance 1, a reliable ABA receptor used as a control, showed specific binding. Moreover, we found that the rtl1 mutant showed ABA-induced stomatal closure when a high concentration of extracellular Ca(2+) was present and that a knockout mutant of Mg-chelatase I subunit (chli1) showed the same ABA-insensitive phenotype as rtl1.

These results suggest that the Mg-chelatase complex as a whole affects the ABA-signaling pathway for stomatal movements.

BL-induced phosphorylation of the plasma membrane H+-ATPase in stomata

 

Immunohistochemical detection of blue light-induced phosphorylation of the plasma membrane H+-ATPase in stomatal guard cells.

by Hayashi M., Inoue S.-i., Takahashi K.,

Kinoshita T.

 (2011)

in  Plant Cell Physiol 52: 1238–1248. doi: 10.1093/pcp/pcr072 –

PubMed Abstract | CrossRef Full Text | Google Scholar, Abstract/FREE Full Text 

Abstract

Blue light (BL) receptor phototropins activate the plasma membrane H⁺-ATPase in guard cells through phosphorylation of a penultimate threonine and subsequent binding of the 14-3-3 protein to the phosphorylated C-terminus of H⁺-ATPase, mediating stomatal opening.

To date, detection of the phosphorylation level of the guard cell H⁺-ATPase has been performed biochemically using guard cell protoplasts (GCPs). However, preparation of GCPs from Arabidopsis for this purpose requires >5,000 rosette leaves and takes >8 h.

Here, we show that BL-induced phosphorylation of guard cell H⁺-ATPase is detected in the epidermis from a single Arabidopsis rosette leaf via an immunohistochemical method using a specific antibody against the phosphorylated penultimate threonine of H⁺-ATPase.

BL-induced phosphorylation of the H⁺-ATPase was detected immunohistochemically in the wild type, but not in a phot1-5 phot2-1 double mutant.

Moreover, we found that physiological concentrations of the phytohormone ABA completely inhibited BL-induced phosphorylation of guard cell H⁺-ATPase in the epidermis, and that inhibition by ABA in the epidermis is more sensitive than in GCPs.

These results indicate that this immunohistochemical method is very useful for detecting the phosphorylation status of guard cell H⁺-ATPase. Thus, we applied this technique to ABA-insensitive mutants (abi1-1, abi2-1 andost1-2) and found that ABA had no effect on BL-induced phosphorylation in these mutants.

These results indicate that inhibition of BL-induced phosphorylation of guard cell H⁺-ATPase by ABA is regulated by ABI1, ABI2 and OST1, which are known to be early ABA signaling components for a wide range of ABA responses in plants.