Thioredoxin Redox Regulates ATPase Activity of Magnesium Chelatase CHLI Subunit and Modulates Redox-Mediated Signaling in Tetrapyrrole Biosynthesis and Homeostasis of Reactive Oxygen Species in Pea Plants      

Abstract

The chloroplast thioredoxins (TRXs) function as messengers of redox signals from ferredoxin to target enzymes. In this work, we studied the regulatory impact of pea (Pisum sativum) TRX-F on the magnesium (Mg) chelatase CHLI subunit and the enzymatic activation of Mg chelatase in vitro and in vivo. In vitro, reduced TRX-F activated the ATPase activity of pea CHLI and enhanced the activity of Mg chelatase reconstituted from the three recombinant subunits CHLI, CHLD, and CHLH in combination with the regulator protein GENOMES UNCOUPLED4 (GUN4). Yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated that TRX-F physically interacts with CHLI but not with either of the other two subunits or GUN4. In vivo, virus-induced TRX-F gene silencing (VIGS-TRX-F) in pea plants did not result in an altered redox state of CHLI. However, simultaneous silencing of the pea TRX-F and TRX-M genes (VIGS-TRX-F/TRX-M) resulted in partially and fully oxidized CHLI in vivo. VIGS-TRX-F/TRX-M plants demonstrated a significant reduction in Mg chelatase activity and 5-aminolevulinic acid synthesizing capacity as well as reduced pigment content and lower photosynthetic capacity. These results suggest that, in vivo, TRX-M can compensate for a lack of TRX-F and that both TRXs act as important redox regulators of Mg chelatase. Furthermore, the silencing of TRX-F and TRX-M expression also affects gene expression in the tetrapyrrole biosynthesis pathway and leads to the accumulation of reactive oxygen species, which may also serve as an additional signal for the transcriptional regulation of photosynthesis-associated nuclear genes.

Inducible expression of magnesium protoporphyrin chelatase subunit I (CHLI)-amiRNA provides insights into cucumber mosaic virus Y satellite RNA-induced chlorosis symptoms

“…The findings are consistent with the previous observation in CHLI-silenced transgenic tobacco plants [26]. The silencing of Thioredoxin-F and M, which interact with and support the enzymatic activity of CHLI, resulted in the reduced expression of CPRGs and the chlorotic phenotype in pea plants [18]. Although the evidence is indirect in this case, it may support the idea that the downregulation of CHLI gives rise to the reduced expression of CPRGs.…”

Two distinct redox cascades cooperatively regulate chloroplast functions and sustain plant viability

“…Nevertheless, the specific regulatory function of NTRC has remained elusive, because NTRC targets reported to date largely overlap with Trx targets (11). Although there are a few comparative studies addressing the functional difference between NTRC and specific Trx subtypes (16,(32)(33)(34), the results are partly inconsistent and, thereby, insufficient to verify distinct roles for NTRC from those for the Trx family.…”

“…However, given the Trx ability to reduce CHLI in vitro (Fig. 2 E and F) (22) and the physical interaction between Trx-f and CHLI in vivo (33), the FTR/Trx pathway is thought to contribute to CHLI redox regulation possibly in a redundant manner. It is highly plausible that the severely chlorotic phenotype of ftrb ntrc is a consequence of disrupted redox regulation of chlorophyll biosynthesis via both the FTR/Trx and NTRC pathways.…”

Thioredoxin system – a novel therapeutic target

“…Moreover, in vivo experiment by Luo et al demonstrated that one class of chloroplast Trx, named asTrx-M can compensate a lack of different class of chloroplast Trx, named as Trx-F. Both Trxs act as of important redox regulators of Mg-chelatase (Luo et al 2012).…”