Two chloroplast-localized MORF proteins act as chaperones to maintain tetrapyrrole biosynthesis.

Tetrapyrroles have essential functions as pigments and cofactors during plant growth and development, and the tetrapyrrole biosynthesis pathway is tightly controlled. Multiple organellar RNA editing factors (MORFs) are required for editing of a wide variety of RNA sites in chloroplasts and mitochondria, but their biochemical properties remain elusive. Here, we uncovered the roles of chloroplast-localized MORF2 and MORF9 in modulating tetrapyrrole biosynthesis and embryogenesis in Arabidopsis thaliana. The lack or reduced transcripts of MORF2 or MORF9 significantly affected biosynthesis of the tetrapyrrole precursor 5-aminolevulinic acid and accumulation of Chl and other tetrapyrrole intermediates. MORF2 directly interacts with multiple tetrapyrrole biosynthesis enzymes and regulators, including NADPH:PROTOCHLOROPHYLLIDE OXIDOREDUCTASE B (PORB) and GENOMES UNCOUPLED4 (GUN4). Strikingly, MORF2 and MORF9 display holdase chaperone activity, alleviate the aggregation of PORB in vitro, and are essential for POR accumulation in vivo. Moreover, both MORF2 and MORF9 significantly stimulate magnesium chelatase activity. Our findings reveal a previously unknown biochemical property of MORF proteins as chaperones and point to a new layer of post-translational control of the tightly regulated tetrapyrrole biosynthesis in plants.

GUN4 Affects the Circadian Clock and Seedlings Adaptation to Changing Light Conditions.

The chloroplast is a key organelle for photosynthesis and perceiving environmental information. GENOME UNCOUPLED 4 (GUN4) has been shown to be required for the regulation of both chlorophyll synthesis, reactive oxygen species (ROS) homeostasis and plastid retrograde signaling. In this study, we found that growth of the gun4 mutant was significantly improved under medium strong light (200 µmol photons m-2s-1) compared to normal light (100 µmol photons m-2s-1), in marked contrast to wild-type (WT). Further analysis revealed that GUN4 interacts with SIGNAL RECOGNITION PARTICLE 54 KDA SUBUNIT (SRP43) and SRP54. RNA-seq analysis indicated that the expression of genes for light signaling and the circadian clock is altered in gun4 compared with (WT). qPCR analysis confirmed that the expression of the clock genes CLOCK-RELATED 1 (CCA1), LATE ELONGATION HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1 (TOC1) and PSEUDO RESPONSE REGULATOR 7 (PRR7) is significantly changed in the gun4 and srp54 mutants under normal and medium strong light conditions. These results suggest that GUN4 may coordinate the adaptation of plants to changing light conditions by regulating the biological clock, although it is not clear whether the effect is direct or indirect.

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

In oxygenic phototrophs, chlorophylls, hemes, and bilins are synthesized by a common branched pathway. Given the phototoxic nature of tetrapyrroles, this pathway must be tightly regulated, and an important regulatory role is attributed to magnesium chelatase enzyme at the branching between the heme and chlorophyll pathway. Gun4 is a porphyrin-binding protein known to stimulate in vitro the magnesium chelatase activity, but how the Gun4-porphyrin complex acts in the cell was unknown. To address this issue, we first performed simulations to determine the porphyrin-docking mechanism to the cyanobacterial Gun4 structure. After correcting crystallographic loop contacts, we determined the binding site for magnesium protoporphyrin IX. Molecular modeling revealed that the orientation of α6/α7 loop is critical for the binding, and the magnesium ion held within the porphyrin is coordinated by Asn-211 residue. We also identified the basis for stronger binding in the Gun4-1 variant and for weaker binding in the W192A mutant. The W192A-Gun4 was further characterized in magnesium chelatase assay showing that tight porphyrin binding in Gun4 facilitates its interaction with the magnesium chelatase ChlH subunit. Finally, we introduced the W192A mutation into cells and show that the Gun4-porphyrin complex is important for the accumulation of ChlH and for channeling metabolites into the chlorophyll biosynthetic pathway.

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

The 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 O2 . Furthermore, GUN4 seems to be involved in sensing elevated levels of these photoreactive tetrapyrrole intermediates, and contributing to (1) O2 -mediated retrograde signalling, originating from chlorophyll biosynthesis.

Impact of Porphyrin Binding to GENOMES UNCOUPLED 4 on Tetrapyrrole Biosynthesis in planta.

Plant tetrapyrrole biosynthesis (TPS) provides the indispensable chlorophyll (Chl) and heme molecules in photosynthetic organisms. Post-translational mechanisms control the enzymes to ensure a balanced flow of intermediates in the pathway and synthesis of appropriate amounts of both endproducts. One of the critical regulators of TPS is GENOMES UNCOUPLED 4 (GUN4). GUN4 interacts with magnesium chelatase (MgCh), and its binding of the catalytic substrate and product of the MgCh reaction stimulates the insertion of Mg2+ into protoporphyrin IX. Despite numerous in vitro studies, knowledge about the in vivo function of the GUN4:porphyrin interaction for the whole TPS pathway, particularly in plants, is still limited. To address this, we focused on two highly conserved amino acids crucial for porphyrin-binding to GUN4 and analyzed GUN4-F191A, R211A, and R211E substitution mutants in vitro and in vivo. Our analysis confirmed the importance of these amino acids for porphyrin-binding and the stimulation of plant MgCh by GUN4 in vitro. Expression of porphyrin-binding deficient F191A, R211A, and R211E in the Arabidopsis gun4-2 knockout mutant background revealed that, unlike in cyanobacteria and green algae, GUN4:porphyrin interactions did not affect the stability of GUN4 or other Arabidopsis TPS pathway enzymes in vivo. In addition, although they shared diminished porphyrin-binding and MgCh activation in vitro, expression of the different GUN4 mutants in gun4-2 had divergent effects on the TPS and the accumulation of Chl and Chl-binding proteins. For instance, expression of R211E, but not R211A, induced a substantial decrease of ALA synthesis rate, lower TPS intermediate and Chl level, and strongly impaired accumulation of photosynthetic complexes compared to wild-type plants. Furthermore, the presence of R211E led to significant growth retardation and paler leaves compared to GUN4 knockdown mutants, indicating that the exchange of R211 to glutamate compromised TPS and Chl accumulation more substantially than the almost complete lack of GUN4. Extensive in vivo analysis of GUN4 point mutants suggested that F191 and R211 might also play a role beyond porphyrin-binding.

Mutations of the Genomes Uncoupled 4 Gene Cause ROS Accumulation and Repress Expression of Peroxidase Genes in Rice.

The Genomes Uncoupled 4 (GUN4) is one of the retrograde signaling genes in Arabidopsis and its orthologs have been identified in oxygenic phototrophic organisms from cyanobacterium to higher plants. GUN4 is involved in tetrapyrrole biosynthesis and its mutation often causes chlorophyll-deficient phenotypes with increased levels of reactive oxygen species (ROS), hence it has been speculated that GUN4 may also play a role in photoprotection. However, the biological mechanism leading to the increased ROS accumulation in gun4 mutants remains largely unknown. In our previous studies, we generated an epi-mutant allele of OsGUN4 (gun4 epi ), which downregulated its expression to ∼0.5% that of its wild-type (WT), and a complete knockout allele gun4-1 due to abolishment of its translation start site. In the present study, three types of F2 plant derived from a gun4-1/gun4 epi cross, i.e., gun4-1/gun4-1, gun4-1/gun4 epi and gun4 epi /gun4 epi were developed and used for further investigation by growing them under photoperiodic condition (16 h/8 h light/dark) with low light (LL, 100 µmol photons m-2 s-1) or high light (HL, 1000 µmol photons m-2 s-1). The expression of OsGUN4 was light responsive and had two peaks in the daytime. gun4-1/gun4-1-F2 seeds showed defective germination and died within 7 days. Significantly higher levels of ROS accumulated in all types of OsGUN4 mutants than in WT plants under both the LL and HL conditions. A comparative RNA-seq analysis of WT variety LTB and its gun4 epi mutant HYB led to the identification of eight peroxidase (PRX)-encoding genes that were significantly downregulated in HYB. The transcription of these eight PRX genes was restored in transgenic HYB protoplasts overexpressing OsGUN4, while their expression was repressed in LTB protoplasts transformed with an OsGUN4 silencing vector. We conclude that OsGUN4 is indispensable for rice, its expression is light- and oxidative-stress responsive, and it plays a role in ROS accumulation via its involvement in the transcriptional regulation of PRX genes.

The catalytic power of magnesium chelatase: a benchmark for the AAA(+) ATPases.

In the first committed reaction of chlorophyll biosynthesis, magnesium chelatase couples ATP hydrolysis to the thermodynamically unfavorable Mg(2+) insertion into protoporphyrin IX (ΔG°' of circa 25-33 kJ·mol(-1) ). We explored the thermodynamic constraints on magnesium chelatase and demonstrate the effect of nucleotide hydrolysis on both the reaction kinetics and thermodynamics. The enzyme produces a significant rate enhancement (kcat /kuncat of 400 × 10(6) m) and a catalytic rate enhancement, kcat/KmDIXK0.5Mgkuncat, of 30 × 10(15) m(-1) , increasing to 300 × 10(15) m(-1) with the activator protein Gun4. This is the first demonstration of the thermodynamic benefit of ATP hydrolysis in the AAA(+) family.