Ancestral function of the phytochelatin synthase C-terminal domain in inhibition of heavy metal-mediated enzyme overactivation

Abstract

Phytochelatin synthases (PCSs) play essential roles in detoxification of a broad range of heavy metals in plants and other organisms. Until now, however, no PCS gene from liverworts, the earliest branch of land plants and possibly the first one to acquire a PCS with a C-terminal domain, has been characterized. In this study, we isolated and functionally characterized the first PCS gene from a liverwort, Marchantia polymorpha (MpPCS). MpPCS is constitutively expressed in all organs examined, with stronger expression in thallus midrib. The gene expression is repressed by Cd2+ and Zn2+. The ability of MpPCS to increase heavy metal resistance in yeast and to complement cad1-3 (the null mutant of the Arabidopsis ortholog AtPCS1) proves its function as the only PCS from M. polymorpha. Site-directed mutagenesis of the most conserved cysteines of the C-terminus of the enzyme further uncovered that two twin-cysteine motifs repress, to different extents, enzyme activation by heavy metal exposure. These results highlight an ancestral function of the PCS elusive C-terminus as a regulatory domain inhibiting enzyme overactivation by essential and non-essential heavy metals. The latter finding may be relevant for obtaining crops with decreased root to shoot mobility of cadmium, thus preventing its accumulation in the food chain.

Responses to Cadmium in Early-Diverging Streptophytes (Charophytes and Bryophytes): Current Views and Potential Applications

“…Although it was thought for long time that bryophytes do not synthesize PCn under metal stress [ 55 , 57 , 100 , 101 , 102 ], Petraglia et al [ 103 ] demonstrated instead that constitutively expressed and functional PCSs are present in a number of bryophytes, as well as in other early-diverging streptophytes, arguing that the ability to synthesize PCn, as well as the presence of active PCSs, are ancestral (plesiomorphic) traits of early-diverging plants [ 103 ]. This study was followed by others, supporting the idea that PCn synthesis is an important mechanism of intracellular Cd detoxification, both in bryophytes, such as in L. riparium [ 62 ], L. cruciata [ 75 , 104 ], M. polymorpha [ 105 , 106 ], and in charophytes, such as N. mucronata [ 77 ]. In addition to the ability to synthesize PCn, vacuolar compartmentalization plays a key role in metal detoxification in bryophytes and charophytes, as well as in higher plants, as supported by the intravacuolar electron-dense Cd deposit detected in L. cruciata [ 75 ].…”

Overexpression of BnPCS1, a Novel Phytochelatin Synthase Gene From Ramie (Boehmeria nivea), Enhanced Cd Tolerance, Accumulation, and Translocation in Arabidopsis thaliana

“…For PCS, being rich in Cys is one of its important features. The C 358 C 359 XXXC 363 XXC 366 motif is a heavy metal sensor, which is capable of transferring heavy metal ions to the N-terminal catalytic region when they are detected (Vestergaard et al, 2008;Li et al, 2020 1A), which is identical to the motif in PcPCS1 and similar to that in LjPCS1. These results reveal that BnPCS1 has similar characteristics to the PCS homologs from the other diverse plant species, and may perform similar functions.…”

“…For PCS, being rich in Cys is one of its important features. The C 358 C 359 XXXC 363 XXC 366 motif is a heavy metal sensor, which is capable of transferring heavy metal ions to the N-terminal catalytic region when they are detected ( Vestergaard et al, 2008 ; Li et al, 2020 ). This motif appears as C 368 C 369 RETC 373 MKC 376 in LjPCS1 and C 369 C 370 QETC 374 VKC 377 in PcPCS1, while this motif is present as C 371 C 372 QETC 376 VKC 379 in BnPCS1 ( Figure 1A ), which is identical to the motif in PcPCS1 and similar to that in LjPCS1.…”

Development of affinity beads-based in vitro metal-ligand binding assay reveals dominant cadmium affinity of thiol-rich small peptides phytochelatins beyond glutathione

“…Such metal-selectivity of PCSs raises a significant question of how PCSs distinguish the activating ions and others. Recent studies from our group and others suggest that the different regions of the C-terminal regulatory domain of PCSs are involved in the metal(loid)-specific activation [16,[49][50][51][52].…”

“…Such metal-(loid) PCS activation mechanism has been a significant question. Recent studies suggest that the different regions of the C-terminal regulatory domain of PCSs are required for the metal(loid)-specific activation [17, [50][51][52][53],…”