Frank Buhr

Chlorophyll biosynthesis: spotlight on protochlorophyllide reduction

Photosynthetic organisms require chlorophyll or bacteriochlorophyll for their light trapping and energy transduction activities. The biosynthetic pathways of chlorophyll and bacteriochlorophyll are similar in most of their early steps, except for the reduction of protochlorophyllide (Pchlide) to chlorophyllide. Whereas angiosperms make use of a light-dependent enzyme, cyanobacteria, algae, bryophytes, pteridophytes and gymnosperms contain an additional, light-independent enzyme dubbed dark-operative Pchlide oxidoreductase (DPOR). Anoxygenic photosynthetic bacteria such as Rhodobacter capsulatus and Rhodobacter sphaeroides rely solely on DPOR. Recent atomic resolution of reductase and catalytic components of DPOR from R. sphaeroides and R. capsulatus, respectively, have revealed their similarity to nitrogenase components. In this review, we discuss the two fundamentally different mechanisms of Pchlide reduction in photosynthetic organisms.

Photoprotective role of NADPH:protochlorophyllide oxidoreductase A

A homology model of NADPH:protochlorophyllide (Pchlide) oxidoreductase A (POR; E.C. 1.3.33.1) of barley is developed and verified by site-directed mutagenesis. PORA is considered a globular protein consisting of nine α-helices and seven β-strands. The model predicts the presence of two functionally distinctive Pchlide binding sites where the pigment is coordinated by cystein residues. The pigment bound to the first, high-affinity Pchlide binding site is used for the formation of the photoactive state of the enzyme. The pigment bound to the second, low-affinity Pchlide binding site is involved in the PORA:PORB interaction, allowing for resonance energy transfer between the neighboring PORs in the complex. In the in vitro reconstituted light-harvesting POR:Pchlide complex (LHPP), light absorbed by PORA-bound Pchlide b is transferred to PORB-bound Pchlide a. That induces the conversion of Pchlide a to chlorophyllide (Chlide) a. This energy transfer eliminates the possibility of Pchlide b photoreduction and prevents that excited triplet states of either Pchlides a or b accumulate and provoke singlet oxygen production. Together, our results provide a photoprotective role of PORA during greening.

A plant porphyria related to defects in plastid import of protochlorophyllide oxidoreductase A

The plastid envelope of higher plant chloroplasts is a focal point of plant metabolism. It is involved in numerous pathways, including tetrapyrrole biosynthesis and protein translocation. Chloroplasts need to import a large number of proteins from the cytosol because most are encoded in the nucleus. Here we report that a loss-of-function mutation in the outer plastid envelope 16-kDa protein (oep16) gene causes a conditional seedling lethal phenotype related to defects in import and assembly of NADPH:protochlorophyllide (Pchlide) oxidoreductase A. In the isolated knockout mutant of Arabidopsis thaliana, excess Pchlide accumulated in the dark operated as photosensitizer and provoked cell death during greening. Our results highlight the essential role of the substrate-dependent plastid import pathway of precursor Pchlide oxidoreductase A for seedling survival and the avoidance of developmentally programmed porphyria in higher plants.

Singlet oxygen-dependent translational control in the tigrina-d.12 mutant of barley

The tigrina (tig)-d.12 mutant of barley is impaired in the negative control limiting excess protochlorophyllide (Pchlide) accumulation in the dark. Upon illumination, Pchlide operates as photosensitizer and triggers singlet oxygen production and cell death. Here, we show that both Pchlide and singlet oxygen operate as signals that control gene expression and metabolite accumulation in tig-d.12 plants. In vivo labeling, Northern blotting, polysome profiling, and protein gel blot analyses revealed a selective suppression of synthesis of the small and large subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (RBCSs and RBCLs), the major light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCB2), as well as other chlorophyll-binding proteins, in response to singlet oxygen. In part, these effects were caused by an arrest in translation initiation of photosynthetic transcripts at 80S cytoplasmic ribosomes. The observed changes in translation correlated with a decline in the phosphorylation level of ribosomal protein S6. At later stages, ribosome dissociation occurred. Together, our results identify translation as a major target of singlet oxygen-dependent growth control and cell death in higher plants.

The outer chloroplast envelope protein OEP16-1 for plastid import of NADPH:protochlorophyllide oxidoreductase A in Arabidopsis thaliana

The outer plastid envelope protein OEP16-1 was previously identified as an amino acid-selective channel protein and translocation pore for NADPH:protochlorophyllide oxidoreductase A (PORA). Reverse genetic approaches used to dissect these mutually not exclusive functions of OEP16-1 in planta have led to descriptions of different phenotypes resulting from the presence of several mutant lines in the SALK_024018 seed stock. In addition to the T-DNA insertion in the AtOEP16-1 gene, lines were purified that contain two additional T-DNA insertions and as yet unidentified point mutations. In a first attempt to resolve the genetic basis of four different lines in the SALK_024018 seed stock, we used genetic transformation with the OEP16-1 cDNA and segregation analyses after crossing out presumed point mutations. We show that AtOEP16-1 is involved in PORA precursor import and by virtue of this activity confers photoprotection onto etiolated seedlings during greening.

In vitro-mutagenesis of NADPH:protochlorophyllide oxidoreductase B: two distinctive protochlorophyllide binding sites participate in enzyme catalysis and assembly

NADPH:protochlorophyllide oxidoreductase (POR) B is a key enzyme for the light-induced greening of etiolated angiosperm plants. It is nucleus-encoded, imported into the plastids posttranslationally, and assembled into larger light-harvesting POR:protochlorophyllide complexes termed LHPP (Reinbothe et al., Nature 397:80–84, 1999). An in vitro-mutagenesis approach was taken to study the role of the evolutionarily conserved Cys residues in pigment binding. Four Cys residues are present in the PORB of which two, Cys276 and Cys303, established distinct pigment binding sites, as shown by biochemical tests, protein import studies, and in vitro-reconstitution experiments. While Cys276 constituted the Pchlide binding site in the active site of the enzyme, Cys303 established a second, low affinity pigment binding site that was involved in the assembly and stabilization of imported PORB enzyme inside etioplasts.

JIP60-mediated, jasmonate- and senescence-induced molecular switch in translation toward stress and defense protein synthesis

Significance Quantitative trait loci (QTLs) are major targets for plant breeders. Despite intensive efforts undertaken over the last decades, still little is known how QTLs affect plant resistance to biotic and abiotic stresses and what exact molecular markers (genes) are involved. Here we identified a gene in barley that maps to previously identified QTLs for boron sensitivity, plant height, lodging, stem breaking, days to heading, yield, seed weight, days to maturity, as well as powdery mildew and spot blotch resistance. This gene is identical to a previously described jasmonate-induced protein designated JIP60 that by virtue of its unique structure and processing is capable of reprogramming protein translation for increased stress tolerance and controlled senescence. Two closely related genes encoding the jasmonate-induced protein 60 (JIP60) were identified in the barley genome. The gene on chromosome arm 4HL encodes the previously identified protein encoded by the cDNA X66376.1. This JIP60 protein is characterized here and shown to consist of two domains: an NH2-terminal domain related to ribosome-inactivating proteins and a COOH-terminal domain, which displays similarity to eukaryotic translation initiation factor 4E (eIF4E). JIP60 undergoes processing in vivo, as a result of which JIP60’s COOH-terminal eIF4E domain is released and functions in recruiting a subset of cellular messengers for translation. This effect was observed for both MeJA-treated and naturally senescing plants. Because the JIP60 gene is in close proximity to several quantitative trait loci for both biotic and abiotic stress resistance, our results identify a unique target for future breeding programs.

Implication of the oep16-1 mutation in a flu-independent, singlet oxygen-regulated cell death pathway in Arabidopsis thaliana.

Singlet oxygen is a prominent form of reactive oxygen species in higher plants. It is easily formed from molecular oxygen by triplet-triplet interchange with excited porphyrin species. Evidence has been obtained from studies on the flu mutant of Arabidopsis thaliana of a genetically determined cell death pathway that involves differential changes at the transcriptome level. Here we report on a different cell death pathway that can be deduced from the analysis of oep16 mutants of A. thaliana. Pure lines of four independent OEP16-deficient mutants with different cell death properties were isolated. Two of the mutants overproduced free protochlorophyllide (Pchlide) in the dark because of defects in import of NADPH:Pchlide oxidoreductase A (pPORA) and died after illumination. The other two mutants avoided excess Pchlide accumulation. Using pulse labeling and polysome profiling studies we show that translation is a major site of cell death regulation in flu and oep16 plants. flu plants respond to photooxidative stress triggered by singlet oxygen by reprogramming their translation toward synthesis of key enzymes involved in jasmonic acid synthesis and stress proteins. In contrast, those oep16 mutants that were prone to photooxidative damage were unable to respond in this way. Together, our results show that translation is differentially affected in the flu and oep16 mutants in response to singlet oxygen.

NADPH:protochlorophyllide oxidoreductase B (PORB) action in Arabidopsis thaliana revisited through transgenic expression of engineered barley PORB mutant proteins

NADPH:protochlorophyllide oxidoreductase (POR) is a key enzyme for the light-induced greening of etiolated angiosperm plants. It belongs to the ‘RED’ family of reductases, epimerases and dehydrogenases. All POR proteins characterized so far contain evolutionarily conserved cysteine residues implicated in protochlorophyllide (Pchlide)-binding and catalysis. cDNAs were constructed by site-directed mutagenesis that encode PORB mutant proteins with defined Cys→Ala exchanges. These cDNAs were expressed in transgenic plants of a PORB-deficient knock-out mutant (porB) of Arabidopsis thaliana. Results show that porB plants expressing PORB mutant proteins with Ala substitutions of Cys276 or Cys303 are hypersensitive to high-light conditions during greening. Hereby, failure to assemble higher molecular weight complexes of PORB with its twin isoenzyme, PORA, as encountered with (Cys303→Ala)-PORB plants, caused more severe effects than replacing Cys276 by an Ala residue in the active site of the enzyme, as encountered in (Cys276→Ala)-PORB plants. Our results are consistent with the presence of two distinct pigment binding sites in PORB, with Cys276 establishing the active site of the enzyme and Cys303 providing a second, low affinity pigment binding site that is essential for the assembly of higher molecular mass light-harvesting PORB::PORA complexes and photoprotection of etiolated seedlings. Failure to assemble such complexes provoked photodynamic damage through the generation of singlet oxygen. Together, our data highlight the importance of PORB for Pchlide homoeostasis and greening in Arabidopsis.