Hisanori Yamakawa

Three different mechanisms of energy dissipation of a desiccation-tolerant moss serve one common purpose: to protect reaction centres against photo-oxidation

Three different types of non-photochemical de-excitation of absorbed light energy protect photosystem II of the sun- and desiccation-tolerant moss Rhytidium rugosum against photo-oxidation. The first mechanism, which is light-induced in hydrated thalli, is sensitive to inhibition by dithiothreitol. It is controlled by the protonation of a thylakoid protein. Other mechanisms are activated by desiccation. One of them permits exciton migration towards a far-red band in the antenna pigments where fast thermal deactivation takes place. This mechanism appears to be similar to a mechanism detected before in desiccated lichens. A third mechanism is based on the reversible photo-accumulation of a radical that acts as a quencher of excitation energy in reaction centres of photosystem II. On the basis of absorption changes around 800 nm, the quencher is suggested to be an oxidized chlorophyll. The data show that desiccated moss is better protected against photo-oxidative damage than hydrated moss. Slow drying of moss thalli in the light increases photo-protection more than slow drying in darkness.

Two functional sites of phosphatidylglycerol for regulation of reaction of plastoquinone QB in photosystem II

Functional roles of an anionic lipid phosphatidylglycerol (PG) were studied in pgsA-gene-inactivated and cdsA-gene-inactivated/phycobilisome-less mutant cells of a cyanobacterium Synechocystis sp. PCC 6803, which can grow only in PG-supplemented media. 1) A few days of PG depletion suppressed oxygen evolution of mutant cells supported by p-benzoquinone (BQ). The suppression was recovered slowly in a week after PG re-addition. Measurements of fluorescence yield indicated the enhanced sensitivity of Q(B) to the inactivation by BQ. It is assumed that the loss of low-affinity PG (PG(L)) enhances the affinity for BQ that inactivates Q(B). 2) Oxygen evolution without BQ, supported by the endogenous electron acceptors, was slowly suppressed due to the direct inactivation of Q(B) during 10 days of PG depletion, and was recovered rapidly within 10h upon the PG re-addition. It is concluded that the loss of high-affinity PG (PG(H)) displaces Q(B) directly. 3) Electron microscopy images of PG-depleted cells showed the specific suppression of division of mutant cells, which had developed thylakoid membranes attaching phycobilisomes (PBS). 4) Although the PG-depletion for 14 days decreased the chlorophyll/PBS ratio to about 1/4, flourescence spectra/lifetimes were not modified indicating the flexible energy transfer from PBS to different numbers of PSII. Longer PG-depletion enhanced allophycocyanin fluorescence at 683nm with a long 1.2ns lifetime indicating the suppression of energy transfer from PBS to PSII. 5) Action sites of PG(H), PG(L) and other PG molecules on PSII structure are discussed.

Arabitol Provided by Lichenous Fungi Enhances Ability to Dissipate Excess Light Energy in a Symbiotic Green Alga under Desiccation

Lichens are drought-resistant symbiotic organisms of mycobiont fungi and photobiont green algae or cyanobacteria, and have an efficient mechanism to dissipate excess captured light energy into heat in a picosecond time range to avoid photoinhibition. This mechanism can be assessed as drought-induced non-photochemical quenching (d-NPQ) using time-resolved fluorescence spectroscopy. A green alga Trebouxia sp., which lives within a lichen Ramalina yasudae, is one of the most common green algal photobionts. This alga showed very efficient d-NPQ under desiccation within the lichen thallus, whereas it lost d-NPQ ability when isolated from R. yasudae, indicating the importance of the interaction with the mycobiont for d-NPQ ability. We analyzed the water extracts from lichen thalli that enhanced d-NPQ in Trebouxia. Of several sugar compounds identified in the water extracts by nuclear magnetic resonance (NMR), mass spectrometry (MS) and gas chromatography (GC) analyses, only d-arabitol recovered d-NPQ in isolated Trebouxia to a level similar to that detected for R. yasudae thallus. Other sugar compounds did not help the expression of d-NPQ at the same concentrations. Thus, arabitol is essential for the expression of d-NPQ to dissipate excess captured light energy into heat, protecting the photobiont from photoinhibition. The relationship between mycobionts and photobionts is, therefore, not commensalism, but mutualism with each other, as shown by d-NPQ expression.

Molecular assembly of zinc chlorophyll derivatives by using recombinant light-harvesting polypeptides with His-tag and immobilization on a gold electrode.

LH1-α and -β polypeptides, which make up the light-harvesting 1 (LH1) complex of purple photosynthetic bacteria, along with bacteriochlorophylls, have unique binding properties even for various porphyrin analogs. Herein, we used the porphyrin analogs, Zn-Chlorin and the Zn-Chlorin dimer, and examined their binding behaviors to the LH1-α variant, which has a His-tag at the C-terminus (MBP-rubα-YH). Zn-Chlorin and the Zn-Chlorin dimer could bind to MBP-rubα-YH and form a subunit-type assembly, similar to that from the native LH1 complex. These complexes could be immobilized onto Ni-nitrilotriacetic acid-modified Au electrodes, and the cathodic photocurrent was successfully observed by photoirradiation. Since Zn-Chlorins in this complex are too far for direct electron transfer from the electrode, a contribution of polypeptide backbone for efficient electron transfer was implied. These findings not only show interesting properties of LH1-α polypeptides but also suggest a clue to construct artificial photosynthesis systems using these peptide materials.

A novel ″oxygen-induced″ greening process in a cyanobacterial mutant lacking the transcriptional activator ChlR involved in low-oxygen adaptation of tetrapyrrole biosynthesis

Background: ChlR activates the transcription of genes encoding low-oxygen-type enzymes in response to hypoxia in cyanobacteria. Results: The chlR-lacking mutant showed a novel “oxygen-induced” greening process upon exposure to air. Conclusion: The contents of photosystems were correlated well with the chlorophyll contents in the greening process. Significance: Oxygen-induced greening provides a promising alternative system to investigate the biogenesis of photosystems. ChlR activates the transcription of the chlAII-ho2-hemN operon in response to low-oxygen conditions in the cyanobacterium Synechocystis sp. PCC 6803. Three genes in the operon encode low-oxygen-type enzymes to bypass three oxygen-dependent reactions in tetrapyrrole biosynthesis. A chlR-lacking mutant, ΔchlR, shows poor photoautotrophic growth due to low chlorophyll (Chl) content under low-oxygen conditions, which is caused by no induction of the operon. Here, we characterized the processes of etiolation of ΔchlR cells in low-oxygen conditions and the subsequent regreening of the etiolated cells upon exposure to oxygen, by HPLC, Western blotting, and low-temperature fluorescence spectra. The Chl content of the etiolated ΔchlR cells incubated under low-oxygen conditions for 7 days was only 10% of that of the wild-type with accumulation of almost all intermediates of the magnesium branch of Chl biosynthesis. Both photosystem I (PSI) and photosystem II (PSII) were significantly decreased, accompanied by a preferential decrease of antenna Chl in PSI. Upon exposure to oxygen, the etiolated ΔchlR cells resumed to produce Chl after a short lag (∼2 h), and the level at 72 h was 80% of that of the wild-type. During this novel “oxygen-induced” greening process, the PSI and PSII contents were largely increased in parallel with the increase in Chl contents. After 72 h, the PSI content reached ∼50% of the wild-type level in contrast to the full recovery of PSII. ΔchlR provides a promising alternative system to investigate the biogenesis of PSI and PSII.

Dissipation of Excess Excitation Energy by Drought-Induced Nonphotochemical Quenching in Two Species of Drought-Tolerant Moss: Desiccation-Induced Acceleration of Photosystem II Fluorescence Decay

Drought-tolerant mosses survive with their green color intact even after long periods of dehydration that would kill ordinary plants. The mechanism of dissipation of excitation energy under drought stress was studied in two species of drought-tolerant moss, Rhytidium rugosum and Ceratodon purpureus. They showed severe quenching of photosystem II chlorophyll fluorescence (PSII) after being dehydrated in the dark. Quenching was induced by the acceleration of the fluorescence decay rate. This drought-induced nonphotochemical quenching (designated d-NPQ) was fully reversed by rehydration. Global analysis of fluorescence decay at 77 K indicated rapid 46 ps transfer of excitation energy from the 680-690 nm PSII bands to a 710 nm band, and to 740-760 nm bands. The latter bands decayed to the ground state with the same time constant showing the rapid dissipation of excitation energy into heat. The quenching by d-NPQ in dry moss was stronger than that by PSII charge separation or nonphotochemical quenching (NPQ), which operates under hydrating conditions. Drought-tolerant mosses, thus, dissipate excess excitation energy into heat. The d-NPQ mechanism in moss resembles that reported in lichens, suggesting their common origin.

The Effect of Two Amino acid Residue Substitutions via RNA Editing on Dark-operative Protochlorophyllide Oxidoreductase in the Black Pine Chloroplasts

Dark-operative protochlorophyllide oxidoreductase (DPOR) is a key enzyme to produce chlorophyll in the dark. Among photosynthetic eukaryotes, all three subunits chlL, chlN, and chlB are encoded by plastid genomes. In some gymnosperms, two codons of chlB mRNA are changed by RNA editing to codons encoding evolutionarily conserved amino acid residues. However, the effect of these substitutions on DPOR activity remains unknown. We first prepared cyanobacterial ChlB variants with amino acid substitution(s) to mimic ChlB translated from pre-edited mRNA. Their activities were evaluated by measuring chlorophyll content of dark-grown transformants of a chlB-lacking mutant of the cyanobacterium Leptolyngbya boryana that was complemented with pre-edited mimic chlB variants. The chlorophyll content of the transformant cells expressing the ChlB variant from the fully pre-edited mRNA was only one-fourth of the control cells. Co-purification experiments of ChlB with Strep-ChlN suggested that a stable complex with ChlN is greatly impaired in the substituted ChlB variant. We then confirmed that RNA editing efficiency was markedly greater in the dark than in the light in cotyledons of the black pine Pinus thunbergii. These results indicate that RNA editing on chlB mRNA is important to maintain appropriate DPOR activity in black pine chloroplasts.

Biochemistry of Chlorophyll Biosynthesis in Photosynthetic Prokaryotes

Chlorophylls (Chls) are tetrapyrrole pigments that are essential for photosynthesis, which supports almost all organisms on the planet. Thus, elucidation of the molecular mechanisms of Chl biosynthesis is a major biological challenge. Nearly 100 different Chls with differing ring structures and substituents are represented by Chls a, b, c, d, and f and bacteriochlorophylls a, b, c, d, e, and g. Phototrophic prokaryotes perform photosynthesis using specific sets of Chls that capture available light under the conditions of their natural habitats. For example, cyanobacteria grow photosynthetically in the top layers of water columns using Chl a, whereas purple bacteria perform anoxygenic photosynthesis using bacteriochlorophyll a in the deeper layers of the water columns. Extensive gene searches have been performed in photosynthetic prokaryotes since the 1990s, and the largely complete scheme of Chl biosynthetic pathways includes a core pathway that is conserved among all photosynthetic organisms and comprises diverse reactions for the production of a variety of Chls. With this framework of biosynthetic pathways, further studies of Chl biosynthesis are directed at understanding the physiological and biochemical aspects. The physiological aspects include elucidation of regulatory networks that are integrated with other cellular processes, and the biochemical aspects include elucidation of three-dimensional structures of Chl biosynthetic enzymes to understand molecular mechanisms. In this chapter, we describe the current investigations of molecular mechanisms of enzymes in the Mg branch focusing on the latter aspects.

Functional expression of an oxygen-labile nitrogenase in an oxygenic photosynthetic organism

Transfer of nitrogen fixation ability to plants, especially crops, is a promising approach to mitigate dependence on chemical nitrogen fertilizer and alleviate environmental pollution caused by nitrogen fertilizer run-off. However, the need to transfer a large number of nitrogen fixation (nif) genes and the extreme vulnerability of nitrogenase to oxygen constitute major obstacles for transfer of nitrogen-fixing ability to plants. Here we demonstrate functional expression of a cyanobacterial nitrogenase in the non-diazotrophic cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803). A 20.8-kb chromosomal fragment containing 25 nif and nif-related genes of the diazotrophic cyanobacterium Leptolyngbya boryana was integrated into a neutral genome site of Synechocystis 6803 by five-step homologous recombination together with the cnfR gene encoding the transcriptional activator of the nif genes to isolate CN1. In addition, two other transformants CN2 and CN3 carrying additional one and four genes, respectively, were isolated from CN1. Low but significant nitrogenase activity was detected in all transformants. This is the first example of nitrogenase activity detected in non-diazotrophic photosynthetic organisms. These strains provide valuable platforms to investigate unknown factors that enable nitrogen-fixing growth of non-diazotrophic photosynthetic organisms, including plants.

In vivo transposon tagging in the nonheterocystous nitrogen‐fixing cyanobacterium Leptolyngbya boryana

Nitrogenase is an oxygen‐vulnerable metalloenzyme that catalyzes nitrogen fixation. It largely remains unknown how nitrogenase coexists with oxygenic photosynthesis in nonheterocystous cyanobacteria, since there have been no appropriate model cyanobacteria so far. Here, we demonstrate in vivo transposon tagging in the nonheterocystous cyanobacterium Leptolyngbya boryana as a forward genetics approach. By conjugative transfer, a mini‐Tn5‐derived vector, pKUT‐Tn5‐Sm/Sp, was transferred from Escherichia coli to L. boryana cells. Of 1839 streptomycin‐resistant colonies, we isolated three mutants showing aberrant diazotrophic growth. Genome resequencing identified the insertion sites of the transposon in the mutants. This in vivo transposon tagging mutagenesis of L. boryana provides a promising system to investigate molecular mechanisms to resolve the Oxygen Paradox between nitrogen fixation and oxygenic photosynthesis in cyanobacteria.