The GUN4 protein plays a regulatory role in tetrapyrrole biosynthesis and chloroplast‐to‐nucleus signalling in Chlamydomonas reinhardtii

Abstract

SUMMARYThe GENOMES UNCOUPLED 4 (GUN4) protein is found only in aerobic photosynthetic organisms. We investigated the role of GUN4 in metabolic activities of the Mg branch of the tetrapyrrole biosynthesis pathway and the plastid signal-mediated changes of nuclear gene expression in Chlamydomonas reinhardtii. In light, gun4 accumulates only 40% of the wild-type chlorophyll level. Light-or dark-grown gun4 mutant accumulates high levels of protoporphyrin IX (Proto), and displays increased sensitivity to moderate light intensities. Despite the photooxidative stress, gun4 fails to downregulate mRNA levels of the tetrapyrrole biosynthesis and the photosynthesis-associated nuclear genes (PhANGs). In contrast, upon illumination, the Proto-accumulating and light-sensitive chlD-1 mutant displays the expected downregulation of the same nuclear genes. Although chlD-1 and the wild type have similar GUN4 transcript levels, the GUN4 protein in chlD-1 is hardly detectable. Overexpression of GUN4 in chlD-1 modifies the downregulation of nuclear gene expression, but also increases light tolerance. Therefore, GUN4 is proposed to function in 'shielding' Proto, and most likely MgProto, by reducing reactivity with O 2 . Furthermore, GUN4 seems to be involved in sensing elevated levels of these photoreactive tetrapyrrole intermediates, and contributing to 1 O 2 -mediated retrograde signalling, originating from chlorophyll biosynthesis.

Porphyrin Binding to Gun4 Protein, Facilitated by a Flexible Loop, Controls Metabolite Flow through the Chlorophyll Biosynthetic Pathway

“…However, the Gun4 mutations are generally more pronounced with increasing light intensity (14,19,38). The sensitivity of Gun4-less mutants to light and changes in ALA synthesizing capacity led to a hypothesis that Gun4 binds excessive porphyrins, which serves as a signal to down-regulate ALA formation (19,20). Although this model can explain reduced ALA synthesis in Gun4 or MgCh mutants (40), it is not consistent with the massive accumulation of P IX in ferrochelatase mutants (8,41).…”

“…Overexpression of Gun4 in plants causes an aberrant accumulation of Chl, accompanied by elevated steady state levels of P IX and MgP porphyrins (19). In the green alga Chlamydomonas reinhardtii, overexpression of Gun4 also increases Chl content per cell (38), whereas a C. reinhardtii strain possessing a low Gun4 content has reduced both Chl and P IX levels (15,20). Only after the complete elimination of Gun4 do cyanobacteria, algae, and plants accumulate aberrant concentrations of P IX (14,20,39).…”

“…According to the available data, it appears that there is crosstalk between the activity of the MgCh-Gun4 complex and regulation of metabolite flux through the whole tetrapyrrole pathway, keeping the P IX pool small (19,20,40,43). As already discussed, other enzymes in the tetrapyrrole pathway, including ferrochelatase, could be a part of the identical membrane domain (5), so it is not difficult to imagine that the deactivation of MgCh allows ferrochelatase to attenuate ALA formation, e.g.…”

“…Inactivation of the gun4 gene perturbs Chl biosynthesis and accumulation of Chl-binding proteins in cyanobacteria (14), green algae (15), and plants (11,19). However, phenotypes of available Gun4 mutants are difficult to explain simply by a defect in MgCh activity, and the in vivo activities of Gun4 appear to be complex (15,19,20).…”

“…Although it is widely accepted that the role of Gun4 depends on tight porphyrin binding (20,21), there is no conclusive model of how the Gun4-porphyrin complex is implicated in the control of tetrapyrrole biosynthesis. Here, we employed in silico modeling, which allows us to identify the MgP-binding pocket and provides an atomic level description of the MgP-binding mechanism for cyanobacterial Gun4.…”