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ACHT4-driven oxidation of APS1 attenuates starch synthesis under low light intensity in Arabidopsis plants
Thursday, 2015/10/15 | 07:07:49

Erez Eliyahu, Ido Rog, Dangoor Inbal, and Avihai Danon

Significance

Regulation of protein activity by redox state changes of a disulfide bond between two intrinsic cysteine residues requires both reductive and oxidative signals, yet very little is known of the oxidative pathway. Deciphering the oxidative pathway is further complicated by an increased level of reactive oxygen species when plants are stressed. Here, we show that the chloroplastic atypical thioredoxin (ACHT4) participates under homeostasis conditions in an oxidative signal that diminishes AGPase activity, the first committed enzyme of starch synthesis, during the transition from day to night and attenuates AGPase activity in a dynamic response to small fluctuations in light intensity during the day.

Abstract

The regulatory mechanisms that use signals of low levels of reactive oxygen species (ROS) could be obscured by ROS produced under stress and thus are better investigated under homeostatic conditions. Previous studies showed that the chloroplastic atypical thioredoxin ACHT1 is oxidized by 2-Cys peroxiredoxin (2-Cys Prx) in Arabidopsis plants illuminated with growth light and in turn transmits a disulfide-based signal via yet unknown target proteins in a feedback regulation of photosynthesis. Here, we studied the role of a second chloroplastic paralog, ACHT4, in plants subjected to low light conditions. Likewise, ACHT4 reacted in planta with 2-Cys Prx, indicating that it is oxidized by a similar disulfide exchange reaction. ACHT4 further reacted uniquely with the small subunit (APS1) of ADP-glucose pyrophosphorylase (AGPase), the first committed enzyme of the starch synthesis pathway, suggesting that it transfers the disulfides it receives from 2-Cys Prx to APS1 and turns off AGPase. In accordance, ACHT4 participated in an oxidative signal that quenched AGPase activity during the diurnal transition from day to night, and also in an attenuating oxidative signal of AGPase in a dynamic response to small fluctuations in light intensity during the day. Increasing the level of expressed ACHT4 or of ACHT4ΔC, a C terminus-deleted form that does not react with APS1, correspondingly decreased or increased the level of reduced APS1 and decreased or increased transitory starch content. These findings imply that oxidative control mechanisms act in concert with reductive signals to fine tune starch synthesis during daily homeostatic conditions.

 

See: http://www.pnas.org/content/112/41/12876.abstract.html?etoc

PNAS October 13, 2015; vol. 112 no. 41: 12876–12881

 

Fig. 1.  (A) Immunoblot assay showing the oxidized state of ACHT4 active-site Cys residues captured in plants expressing ACHT4 fused with HA-tag (19) at the end of the night (oxidized) and at 1 min (m), 5 min, 30 min, 1 h, and 2 h after beginning of illumination. Analysis of the purified proteins under reducing conditions (reduced) indicated that the changes in the oxidized level of ACHT4 were not the result of altered protein content. Equal loading was verified by ribulose-1,5-bis-phosphate carboxylase/oxygenase (RBCL) levels. (B) Immunoblot assay showing the ACHT4 intermolecular disulfide complexes, 2-Cys Prx heterotrimeric (Prx-t) and heterodimeric (Prx-d), and a unique additional complex (marked with a star) extracted under nonreducing conditions (NR) from plants expressing either ACHT4MT or ACHT1MT. The conversion of the complexes to the monomer (mono) by chemical reduction (R) indicated the disulfide nature of the complexes. Reciprocal immunoprecipitation identified 2-Cys Prx (C) and APS1 (D) as the intermolecular disulfide partners of ACHT4. Immunoblot assay of proteins immunoprecipitated with anti-HA (ACHT4 IP), anti-2-Cys Prx (Prx IP), and anti-APS1 (APS1 IP) affinity matrixes or with a nonspecific matrix (Cont IP) from plants expressing ACHT4MT. Purified proteins were run under reducing conditions and blotted with antibodies specific to the HA-tagged ACHT4 (αHA), 2-Cys Prx (α2-Cys Prx), or APS1 (αAPS1).

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