In aerobic photosynthetic organisms, GUN4 binds the chlorophyll intermediates protoporphyrin and Mg protoporphyrin, stimulates Mg chelatase activity, and is implicated in plastidic retrograde signaling. GUN4 expression is most abundant in young and greening tissues and parallels the activity of 5-aminolevulinic acid (ALA) ALA and Mg porphyrin biosynthesis during photoperiodic growth. We explored function and mode of action of GUN4 using GUN4-deficient and overexpressing plants. GUN4 overexpression leads to a general activation of the enzymes of chlorophyll biosynthesis. During photoperiodic growth GUN4 deficiency prevents ALA synthesis and chlorophyll accumulation. All these metabolic changes do not correlate with altered gene expression or changes of protein abundance in tetrapyrrole biosynthesis. While ALA feeding fails to compensate GUN4 deficiency during light-dark growth, this approach results in chlorophyll accumulation under continuous dim light. A new model defines the involvement of GUN4 in posttranslational regulation of ALA and Mg porphyrin synthesis, to sustain chlorophyll synthesis, namely under varying environmental conditions.

The NADPH-dependent thioredoxin reductase C (NTRC) is involved in redox-related regulatory processes in chloroplasts and nonphotosynthetic active plastids. Together with 2-cysteine peroxiredoxin, it forms a two-component peroxide-detoxifying system that acts as a reductant under stress conditions. NTRC stimulates in vitro activity of magnesium protoporphyrin IX monomethylester (MgPMME) cyclase, most likely by scavenging peroxides. Reexamination of tetrapyrrole intermediate levels of the Arabidopsis (Arabidopsis thaliana) knockout ntrc reveals lower magnesium protoporphyrin IX (MgP) and MgPMME steadystate levels, the substrate and the product of MgP methyltransferase (CHLM) preceding MgPMME cyclase, while MgP strongly accumulates in mutant leaves after 5-aminolevulinic acid feeding. The ntrc mutant has a reduced capacity to synthesize 5-aminolevulinic acid and reduced CHLM activity compared with the wild type. Although transcript levels of genes involved in chlorophyll biosynthesis are not significantly altered in 2-week-old ntrc seedlings, the contents of glutamyl-transfer RNA reductase1 (GluTR1) and CHLM are reduced. Bimolecular fluorescence complementation assay confirms a physical interaction of NTRC with GluTR1 and CHLM. While ntrc contains partly oxidized CHLM, the wild type has only reduced CHLM. As NTRC also stimulates CHLM activity in vitro, it is proposed that NTRC has a regulatory impact on the redox status of conserved cysteine residues of CHLM. It is hypothesized that a deficiency of NTRC leads to a lower capacity to reduce cysteine residues of GluTR1 and CHLM, affecting the stability and, thereby, altering the activity in the entire tetrapyrrole synthesis pathway.During the last decades, almost all enzymes of tetrapyrrole biosynthesis and their complex network of transcriptional regulation have been comprehensively studied (Tanaka et al., 2011). These studies revealed a complex control of the expression of genes encoding enzymes in the light-regulated chlorophyll (Chl)-synthesizing branch of tetrapyrrole metabolism. In brief, 5-aminolevulinic acid (ALA) is synthesized in a transfer RNA (tRNA) GLU -mediated pathway, and eight molecules of ALA are ultimately converted in a series of enzymatic steps to protoporphyrin IX. The polymeric magnesium (Mg) chelatase complex consisting of the three different subunits CHLH, CHLI, and CHLD directs protoporphyrin IX into the Mg branch of tetrapyrrole biosynthesis. Methylation of magnesium protoporphyrin (MgP) by MgP methyltransferase (CHLM) at the C13 of pyrrole ring C initiates the formation of the typical fifth ring. The product of this step, magnesium protoporphyrin monomethylester (MgPMME), is then converted to divinyl protochlorophyllide (PChlide) by an oxidative cyclase complex. NADPH:protochlorophyllide oxidoreductase (POR) synthesizes chlorophyllide (Chlide). PChlide and Chlide are most likely the main substrates of a divinyl reductase that reduces the C7-C8 double bond, forming a monovinyl product. The two final steps of Chl a and b synthesis are likely ...

The Rhizoclosmatium globosum genome encodes three rhodopsin-guanylyl cyclases (RGCs), which are predicted to facilitate visual orientation of the fungal zoospores. Here, we show that RGC1 and RGC2 function as light-activated cyclases only upon heterodimerization with RGC3 (NeoR). RGC1/2 utilize conventional green or blue-light-sensitive rhodopsins (λmax = 550 and 480 nm, respectively), with short-lived signaling states, responsible for light-activation of the enzyme. The bistable NeoR is photoswitchable between a near-infrared-sensitive (NIR, λmax = 690 nm) highly fluorescent state (QF = 0.2) and a UV-sensitive non-fluorescent state, thereby modulating the activity by NIR pre-illumination. No other rhodopsin has been reported so far to be functional as a heterooligomer, or as having such a long wavelength absorption or high fluorescence yield. Site-specific mutagenesis and hybrid quantum mechanics/molecular mechanics simulations support the idea that the unusual photochemical properties result from the rigidity of the retinal chromophore and a unique counterion triad composed of two glutamic and one aspartic acids. These findings substantially expand our understanding of the natural potential and limitations of spectral tuning in rhodopsin photoreceptors.

Gun4 is a porphyrin-binding protein that activates magnesium chelatase, a multimeric enzyme catalyzing the first committed step in chlorophyll biosynthesis. In plants, GUN4 has been implicated in plastid-to-nucleus retrograde signaling processes that coordinate both photosystem II and photosystem I nuclear gene expression with chloroplast function. In this work we present the functional analysis of Gun4 from the cyanobacterium Synechocystis sp. PCC 6803. Affinity co-purification of the FLAG-tagged Gun4 with the ChlH subunit of the magnesium chelatase confirmed the association of Gun4 with the enzyme in cyanobacteria. Inactivation of the gun4 gene abolished photoautotrophic growth of the resulting gun4 mutant strain that exhibited a decreased activity of magnesium chelatase. Consequently, the cellular content of chlorophyll-binding proteins was highly inadequate, especially that of proteins of photosystem II. Immunoblot analyses, blue native polyacrylamide gel electrophoresis, and radiolabeling of the membrane protein complexes suggested that the availability of the photosystem II antenna protein CP47 is a limiting factor for the photosystem II assembly in the gun4 mutant.

At the last step of the chlorophyll biosynthetic pathway chlorophyll synthase (CHLG) esterifies chlorophyllide a and b with phytyl or geranyl-geranyl pyrophosphate in chloroplasts. Transgenic tobacco plants expressing CHLG RNA in sense and antisense orientation were examined for the effects of excessive and reduced ectopic CHLG expression, respectively, on the chlorophyll biosynthetic pathway and the expression of chlorophyll-binding proteins. Reduced chlorophyll synthase activity does not result in accumulation of chlorophyllide and caused reduced ALA formation and Mg and ferrochelatase activity, while CHLG overexpression correlated with enhanced ALA synthesizing capacity and more chelatase activities. The transcript levels of genes expressing proteins of chlorophyll biosynthesis and chlorophyll-binding proteins were down-regulated in response to reduced CHLG expression. Thus, reduced expression and activity of chlorophyll synthase caused a feedback-controlled inactivation of the initial and rate limiting step of the pathway leading to down regulation of the metabolic flow, while overexpression can mediate a stimulation of the pathway. Chlorophyll synthase is proposed to be important for the co-regulation of the entire pathway and the coordination of synthesis of chlorophyll and the chlorophyll-binding proteins.