A desert Chlorella sp. that thrives at extreme high-light intensities using a unique photoinhibition protection mechanism.

While light is the driving force of photosynthesis, excessive light can be harmful. Photoinhibition is one of the key processes that limit photosynthetic productivity. A well-defined mechanism that protects from photoinhibition has been described. Chlorella ohadii is a green micro-alga, isolated from biological desert soil crusts, which thrives under extreme high light (HL). Here, we show that this alga evolved unique protection mechanisms distinct from those of the green alga Chlamydomonas reinhardtii or plants. When grown under extreme HL, a drastic reduction in the size of light harvesting antennae occurs, resulting in the presence of core photosystem II, devoid of outer and inner antennas. This is accompanied by a massive accumulation of protective carotenoids and proteins that scavenge harmful radicals. At the same time, several elements central to photoinhibition protection in C. reinhardtii, such as psbS, light harvesting complex stress-related, photosystem II protein phosphorylation and state transitions are entirely absent or were barely detected. In addition, a carotenoid biosynthesis-related protein accumulates in the thylakoid membranes of HL cells and may function in sensing HL and protecting the cell from photoinhibition. Taken together, a unique photoinhibition protection mechanism evolved in C. ohadii, enabling the species to thrive under extreme-light intensities where other photosynthetic organisms fail to survive.

The protein phosphorylation landscape in photosystem I of the desert algae Chlorella sp.

The phosphorylation of photosystem II (PSII) and its antenna (LHCII) proteins has been studied, and its involvement in state transitions and PSII repair is known. Yet, little is known about the phosphorylation of photosystem I (PSI) and its antenna (LHCI) proteins. Here, we applied proteomics analysis to generate a map of the phosphorylation sites of the PSI-LHCI proteins in Chlorella ohadii cells that were grown under low or extreme high-light intensities (LL and HL). Furthermore, we analyzed the content of oxidized tryptophans and PSI-LHCI protein degradation products in these cells, to estimate the light-induced damage to PSI-LHCI. Our work revealed the phosphorylation of 17 of 22 PSI-LHCI subunits. The analyses detected the extensive phosphorylation of the LHCI subunits Lhca6 and Lhca7, which is modulated by growth light intensity. Other PSI-LHCI subunits were phosphorylated to a lesser extent, including PsaE, where molecular dynamic simulation proposed that a phosphoserine stabilizes ferredoxin binding. Additionally, we show that HL-grown cells accumulate less oxidative damage and degradation products of PSI-LHCI proteins, compared with LL-grown cells. The significant phosphorylation of Lhca6 and Lhca7 at the interface with other LHCI subunits suggests a physiological role during photosynthesis, possibly by altering light-harvesting characteristics and binding of other subunits.

LHC-like Proteins: The Guardians of Photosynthesis.

The emergence of chlorophyll-containing light-harvesting complexes (LHCs) was a crucial milestone in the evolution of photosynthetic eukaryotic organisms. Light-harvesting chlorophyll-binding proteins form complexes in proximity to the reaction centres of photosystems I and II and serve as an antenna, funnelling the harvested light energy towards the reaction centres, facilitating photochemical quenching, thereby optimizing photosynthesis. It is now generally accepted that the LHC proteins evolved from LHC-like proteins, a diverse family of proteins containing up to four transmembrane helices. Interestingly, LHC-like proteins do not participate in light harvesting to elevate photosynthesis activity under low light. Instead, they protect the photosystems by dissipating excess energy and taking part in non-photochemical quenching processes. Although there is evidence that LHC-like proteins are crucial factors of photoprotection, the roles of only a few of them, mainly the stress-related psbS and lhcSR, are well described. Here, we summarize the knowledge gained regarding the evolution and function of the various LHC-like proteins, with emphasis on those strongly related to photoprotection. We further suggest LHC-like proteins as candidates for improving photosynthesis in significant food crops and discuss future directions in their research.

The desert green algae Chlorella ohadii thrives at excessively high light intensities by exceptionally enhancing the mechanisms that protect photosynthesis from photoinhibition.

Although light is the driving force of photosynthesis, excessive light can be harmful. One of the main processes that limits photosynthesis is photoinhibition, the process of light-induced photodamage. When the absorbed light exceeds the amount that is dissipated by photosynthetic electron flow and other processes, damaging radicals are formed that mostly inactivate photosystem II (PSII). Damaged PSII must be replaced by a newly repaired complex in order to preserve full photosynthetic activity. Chlorella ohadii is a green microalga, isolated from biological desert soil crusts, that thrives under extreme high light and is highly resistant to photoinhibition. Therefore, C. ohadii is an ideal model for studying the molecular mechanisms underlying protection against photoinhibition. Comparison of the thylakoids of C. ohadii cells that were grown under low light versus extreme high light intensities found that the alga employs all three known photoinhibition protection mechanisms: (i) massive reduction of the PSII antenna size; (ii) accumulation of protective carotenoids; and (iii) very rapid repair of photodamaged reaction center proteins. This work elucidated the molecular mechanisms of photoinhibition resistance in one of the most light-tolerant photosynthetic organisms, and shows how photoinhibition protection mechanisms evolved to marginal conditions, enabling photosynthesis-dependent life in severe habitats.

The Arabidopsis chloroplast RNase J displays both exo- and robust endonucleolytic activities.

KEY MESSAGE: Arabidopsis chloroplast RNase J displaces both exo- and endo-ribonucleolytic activities and contains a unique GT-1 DNA binding domain. Control of chloroplast gene expression is predominantly at the post-transcriptional level via the coordinated action of nuclear encoded ribonucleases and RNA-binding proteins. The 5' end maturation of mRNAs ascribed to the combined action of 5'→3' exoribonuclease and gene-specific RNA-binding proteins of the pentatricopeptide repeat family and others that impede the progression of this nuclease. The exo- and endoribonuclease RNase J, the only prokaryotic 5'→3' ribonuclease that is commonly present in bacteria, Archaea, as well as in the chloroplasts of higher plants and green algae, has been implicated in this process. Interestingly, in addition to the metalo-ß-lactamase and ß-CASP domains, RNase J of plants contains a conserved GT-1 domain that was previously characterized in transcription factors that function in light and stress responding genes. Here, we show that the Arabidopsis RNase J (AtRNase J), when analyzed in vitro with synthetic RNAs, displays both 5'→3' exonucleolytic activity, as well as robust endonucleolytic activity as compared to its bacterial homolog RNase J1 of Bacillus subtilis. AtRNase J degraded single-stranded RNA and DNA molecules but displays limited activity on double stranded RNA. The addition of three guanosines at the 5' end of the substrate significantly inhibited the degradation activity, indicating that the sequence and structure of the RNA substrate modulate the ribonucleolytic activity. Mutation of three amino acid in the catalytic reaction center significantly inhibited both the endonucleolytic and exonucleolytic degradation activities, while deletion of the carboxyl GT-1 domain that is unique to the plant RNAse J proteins, had a little or no significant effect. The robust endonucleolytic activity of AtRNase J suggests its involvement in the processing and degradation of RNA in the chloroplast.

Identification of LACTB2, a metallo-ß-lactamase protein, as a human mitochondrial endoribonuclease.

Post-transcriptional control of mitochondrial gene expression, including the processing and generation of mature transcripts as well as their degradation, is a key regulatory step in gene expression in human mitochondria. Consequently, identification of the proteins responsible for RNA processing and degradation in this organelle is of great importance. The metallo-ß-lactamase (MBL) is a candidate protein family that includes ribo- and deoxyribonucleases. In this study, we discovered a function for LACTB2, an orphan MBL protein found in mammalian mitochondria. Solving its crystal structure revealed almost perfect alignment of the MBL domain with CPSF73, as well as to other ribonucleases of the MBL superfamily. Recombinant human LACTB2 displayed robust endoribonuclease activity on ssRNA with a preference for cleavage after purine-pyrimidine sequences. Mutational analysis identified an extended RNA-binding site. Knockdown of LACTB2 in cultured cells caused a moderate but significant accumulation of many mitochondrial transcripts, and its overexpression led to the opposite effect. Furthermore, manipulation of LACTB2 expression resulted in cellular morphological deformation and cell death. Together, this study discovered that LACTB2 is an endoribonuclease that is involved in the turnover of mitochondrial RNA, and is essential for mitochondrial function in human cells.

Polyadenylation and degradation of RNA in the mitochondria.

Mitochondria have their own gene expression machinery and the relative abundance of RNA products in these organelles in animals is mostly dictated by their rate of degradation. The molecular mechanisms regulating the differential accumulation of the transcripts in this organelle remain largely elusive. Here, we summarize the present knowledge of how RNA is degraded in human mitochondria and describe the coexistence of stable poly(A) tails and the nonabundant tails, which have been suggested to play a role in the RNA degradation process.

The Photosystem II D1-K238E mutation enhances electrical current production using cyanobacterial thylakoid membranes in a bio-photoelectrochemical cell.

The conversion of solar energy (SEC) to storable chemical energy by photosynthesis has been performed by photosynthetic organisms, including oxygenic cyanobacteria for over 3 billion years. We have previously shown that crude thylakoid membranes from the cyanobacterium Synechocytis sp. PCC 6803 can reduce the electron transfer (ET) protein cytochrome c even in the presence of the PSII inhibitor DCMU. Mutation of lysine 238 of the Photosystem II D1 protein to glutamic acid increased the cytochrome reduction rates, indicating the possible position of this unknown ET pathway. In this contribution, we show that D1-K238E is rather unique, as other mutations to K238, or to other residues in the same vicinity, are not as successful in cytochrome c reduction. This observation indicates the sensitivity of ET reactions to minor changes. As the next step in obtaining useful SEC from biological material, we describe the use of crude Synechocystis membranes in a bio-photovoltaic cell containing an N-acetyl cysteine-modified gold electrode. We show the production of significant current for prolonged time durations, in the presence of DCMU. Surprisingly, the presence of cytochrome c was not found to be necessary for ET to the bio-voltaic cell.

Dis3-like 1: a novel exoribonuclease associated with the human exosome.

The exosome is an exoribonuclease complex involved in the degradation and maturation of a wide variety of RNAs. The nine-subunit core of the eukaryotic exosome is catalytically inactive and may have an architectural function and mediate substrate binding. In Saccharomyces cerevisiae, the associated Dis3 and Rrp6 provide the exoribonucleolytic activity. The human exosome-associated Rrp6 counterpart contributes to its activity, whereas the human Dis3 protein is not detectably associated with the exosome. Here, a proteomic analysis of immunoaffinity-purified human exosome complexes identified a novel exosome-associated exoribonuclease, human Dis3-like exonuclease 1 (hDis3L1), which was confirmed to associate with the exosome core by co-immunoprecipitation. In contrast to the nuclear localization of Dis3, hDis3L1 exclusively localized to the cytoplasm. The hDis3L1 isolated from transfected cells degraded RNA in an exoribonucleolytic manner, and its RNB domain seemed to mediate this activity. The siRNA-mediated knockdown of hDis3L1 in HeLa cells resulted in elevated levels of poly(A)-tailed 28S rRNA degradation intermediates, indicating the involvement of hDis3L1 in cytoplasmic RNA decay. Taken together, these data indicate that hDis3L1 is a novel exosome-associated exoribonuclease in the cytoplasm of human cells.

RHON1 is a novel ribonucleic acid-binding protein that supports RNase E function in the Arabidopsis chloroplast.

The Arabidopsis endonuclease RNase E (RNE) is localized in the chloroplast and is involved in processing of plastid ribonucleic acids (RNAs). By expression of a tandem affinity purification-tagged version of the plastid RNE in the Arabidopsis rne mutant background in combination with mass spectrometry, we identified the novel vascular plant-specific and co-regulated interaction partner of RNE, designated RHON1. RHON1 is essential for photoautotrophic growth and together with RNE forms a distinct ∼800 kDa complex. Additionally, RHON1 is part of various smaller RNA-containing complexes. RIP-chip and other association studies revealed that a helix-extended-helix-structured Rho-N motif at the C-terminus of RHON1 binds to and supports processing of specific plastid RNAs. In all respects, such as plastid RNA precursor accumulation, protein pattern, increased number and decreased size of chloroplasts and defective chloroplast development, the phenotype of rhon1 knockout mutants resembles that of rne lines. This strongly suggests that RHON1 supports RNE functions presumably by conferring sequence specificity to the endonuclease.

Mechanism of RNA stabilization and translational activation by a pentatricopeptide repeat protein.

Pentatricopeptide repeat (PPR) proteins comprise a large family of helical repeat proteins that bind RNA and modulate organellar RNA metabolism. The mechanisms underlying the functions attributed to PPR proteins are unknown. We describe in vitro studies of the maize protein PPR10 that clarify how PPR10 modulates the stability and translation of specific chloroplast mRNAs. We show that recombinant PPR10 bound to its native binding site in the chloroplast atpI-atpH intergenic region (i) blocks both 5'→3' and 3'→ 5 exoribonucleases in vitro; (ii) is sufficient to define the native processed atpH mRNA 5'-terminus in conjunction with a generic 5'→3' exoribonuclease; and (iii) remodels the structure of the atpH ribosome-binding site in a manner that can account for PPR10's ability to enhance atpH translation. In addition, we show that the minimal PPR10-binding site spans 17 nt. We propose that the site-specific barrier and RNA remodeling activities of PPR10 are a consequence of its unusually long, high-affinity interface with single-stranded RNA, that this interface provides a functional mimic to bacterial small RNAs, and that analogous activities underlie many of the biological functions that have been attributed to PPR proteins.

Chloroplast RNase J compensates for inefficient transcription termination by removal of antisense RNA.

Ribonuclease J is an essential enzyme, and the Bacillus subtilis ortholog possesses both endoribonuclease and 5' → 3' exoribonuclease activities. Chloroplasts also contain RNase J, which has been postulated to participate, as both an exo- and endonuclease, in the maturation of polycistronic mRNAs. Here we have examined recombinant Arabidopsis RNase J and found both 5' → 3' exoribonuclease and endonucleolytic activities. Virus-induced gene silencing was used to reduce RNase J expression in Arabidopsis and Nicotiana benthamiana, leading to chlorosis but surprisingly few disruptions in the cleavage of polycistronic rRNA and mRNA precursors. In contrast, antisense RNAs accumulated massively, suggesting that the failure of chloroplast RNA polymerase to terminate effectively leads to extensive symmetric transcription products that are normally eliminated by RNase J. Mung bean nuclease digestion and polysome analysis revealed that this antisense RNA forms duplexes with sense strand transcripts and prevents their translation. We conclude that a major role of chloroplast RNase J is RNA surveillance to prevent overaccumulation of antisense RNA, which would otherwise exert deleterious effects on chloroplast gene expression.

Engineering of an alternative electron transfer path in photosystem II.

The initial steps of oxygenic photosynthetic electron transfer occur within photosystem II, an intricate pigment/protein transmembrane complex. Light-driven electron transfer occurs within a multistep pathway that is efficiently insulated from competing electron transfer pathways. The heart of the electron transfer system, composed of six linearly coupled redox active cofactors that enable electron transfer from water to the secondary quinone acceptor Q(B), is mainly embedded within two proteins called D1 and D2. We have identified a site in silico, poised in the vicinity of the Q(A) intermediate quinone acceptor, which could serve as a potential binding site for redox active proteins. Here we show that modification of Lysine 238 of the D1 protein to glutamic acid (Glu) in the cyanobacterium Synechocystis sp. PCC 6803, results in a strain that grows photautotrophically. The Glu thylakoid membranes are able to perform light-dependent reduction of exogenous cytochrome c with water as the electron donor. Cytochrome c photoreduction by the Glu mutant was also shown to significantly protect the D1 protein from photodamage when isolated thylakoid membranes were illuminated. We have therefore engineered a novel electron transfer pathway from water to a soluble protein electron carrier without harming the normal function of photosystem II.

Addition of poly(A) and poly(A)-rich tails during RNA degradation in the cytoplasm of human cells.

Polyadenylation of RNA is a posttranscriptional modification that can play two somewhat opposite roles: stable polyadenylation of RNA encoded in the nuclear genomes of eukaryote cells contributes to nuclear export, translation initiation, and possibly transcript longevity as well. Conversely, transient polyadenylation targets RNA molecules to rapid exonucleolytic degradation. The latter role has been shown to take place in prokaryotes and organelles, as well as the nucleus of eukaryotic cells. Here we present evidence of hetero- and homopolymeric adenylation of truncated RNA molecules within the cytoplasm of human cells. RNAi-mediated silencing of the major RNA decay machinery of the cell resulted in the accumulation of these polyadenylated RNA fragments, indicating that they are degradation intermediates. Together, these results suggest that a mechanism of RNA decay, involving transient polyadenylation, is present in the cytoplasm of human cells.

Mutational analysis of Arabidopsis chloroplast polynucleotide phosphorylase reveals roles for both RNase PH core domains in polyadenylation, RNA 3'-end maturation and intron degradation.

Polynucleotide phosphorylase (PNPase) catalyzes RNA polymerization and 3'→5' phosphorolysis in vitro, but its roles in plant organelles are poorly understood. Here, we have used in vivo and in vitro mutagenesis to study Arabidopsis chloroplast PNPase (cpPNPase). In mutants lacking cpPNPase activity, unusual RNA patterns were broadly observed, implicating cpPNPase in rRNA and mRNA 3'-end maturation, and RNA degradation. Intron-containing fragments also accumulated in mutants, and cpPNPase appears to be required for a degradation step following endonucleolytic cleavage of the excised lariat. Analysis of poly(A) tails, which destabilize chloroplast RNAs, indicated that PNPase and a poly(A) polymerase share the polymerization role in wild-type plants. We also studied two lines carrying mutations in the first PNPase core domain, which does not harbor the catalytic site. These mutants had gene-dependent and intermediate RNA phenotypes, suggesting that reduced enzyme activity differentially affects chloroplast transcripts. The interpretations of in vivo results were confirmed by in vitro analysis of recombinant enzymes, and showed that the first core domain affects overall catalytic activity. In summary, cpPNPase has a major role in maturing mRNA and rRNA 3'-ends, but also participates in RNA degradation through exonucleolytic digestion and polyadenylation. These functions depend absolutely on the catalytic site within the second duplicated RNase PH domain, and appear to be modulated by the first RNase PH domain.

Harnessing photosynthesis to produce electricity using cyanobacteria, green algae, seaweeds and plants.

The conversion of solar energy into electrical current by photosynthetic organisms has the potential to produce clean energy. Life on earth depends on photosynthesis, the major mechanism for biological conversion of light energy into chemical energy. Indeed, billions of years of evolution and adaptation to extreme environmental habitats have resulted in highly efficient light-harvesting and photochemical systems in the photosynthetic organisms that can be found in almost every ecological habitat of our world. In harnessing photosynthesis to produce green energy, the native photosynthetic system is interfaced with electrodes and electron mediators to yield bio-photoelectrochemical cells (BPECs) that transform light energy into electrical power. BPECs utilizing plants, seaweeds, unicellular photosynthetic microorganisms, thylakoid membranes or purified complexes, have been studied in attempts to construct efficient and non-polluting BPECs to produce electricity or hydrogen for use as green energy. The high efficiency of photosynthetic light-harvesting and energy production in the mostly unpolluting processes that make use of water and CO2 and produce oxygen beckons us to develop this approach. On the other hand, the need to use physiological conditions, the sensitivity to photoinhibition as well as other abiotic stresses, and the requirement to extract electrons from the system are challenging. In this review, we describe the principles and methods of the different kinds of BPECs that use natural photosynthesis, with an emphasis on BPECs containing living oxygenic photosynthetic organisms. We start with a brief summary of BPECs that use purified photosynthetic complexes. This strategy has produced high-efficiency BPECs. However, the lifetimes of operation of these BPECs are limited, and the preparation is laborious and expensive. We then describe the use of thylakoid membranes in BPECs which requires less effort and usually produces high currents but still suffers from the lack of ability to self-repair damage caused by photoinhibition. This obstacle of the utilization of photosynthetic systems can be significantly reduced by using intact living organisms in the BPEC. We thus describe here progress in developing BPECs that make use of cyanobacteria, green algae, seaweeds and higher plants. Finally, we discuss the future challenges of producing high and longtime operating BPECs for practical use.

Self-Enclosed Bio-Photoelectrochemical Cell in Succulent Plants.

Harvesting an electrical current from biological photosynthetic systems (live cells or isolated complexes) is typically achieved by immersion of the system into an electrolyte solution. In this study, we show that the aqueous solution found in the tissues of succulent plants can be used directly as a natural bio-photo electrochemical cell. Here, the thick water-preserving outer cuticle of the succulent Corpuscularia lehmannii serves as the electrochemical container, the inner water content as the electrolyte into which an iron anode and platinum cathode are introduced. We produce up to 20 µA/cm2 bias-free photocurrent. When 0.5 V bias is added to the iron anode, the current density increases ∼10-fold, and evolved hydrogen gas can be collected with a Faradaic efficiency of 2.1 and 3.5% in dark or light, respectively. The addition of the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibits the photocurrent, indicating that water oxidation is the primary source of electrons in the light. Two-dimensional fluorescence measurements show that NADH and NADPH serve as the major mediating electron transfer molecules, functionally connecting photosynthesis to metal electrodes. This work presents a method to simultaneously absorb CO2 while producing an electrical current with minimal engineering requirements.

Trichodesmium erythraeum produces a higher photocurrent than other cyanobacterial species in bio-photo electrochemical cells.

The increase in world energy consumption, and the worries from potential future disasters that may derive from climate change have stimulated the development of renewable energy technologies. One promising method is the utilization of whole photosynthetic cyanobacterial cells to produce photocurrent in a bio-photo electrochemical cell (BPEC). The photocurrent can be derived from either the respiratory or photosynthetic pathways, via the redox couple NADP+/NADPH mediating cyclic electron transport between photosystem I inside the cells, and the anode. In the past, most studies have utilized the fresh-water cyanobacterium Synechocystis sp. PCC 6803 (Syn). Here, we show that the globally important marine cyanobacterium Trichodesmium erythraeum flourishing in the subtropical oceans can provide improved currents as compared to Syn. We applied 2D-fluorescence measurements to detect the secretion of NADPH and show that the resulting photocurrent production is enhanced by increasing the electrolyte salinity, Further enhancement of the photocurrent can be obtained by the addition of electron mediators such as NAD+, NADP+, cytochrome C, vitamin B1, or potassium ferricyanide. Finally, we produce photocurrent from additional cyanobacterial species: Synechocystis sp. PCC6803, Synechococcus elongatus PCC7942, Acaryochloris marina MBIC 11017, and Spirulina, using their cultivation media as electrolytes for the BPEC.

Production of photocurrent and hydrogen gas from intact plant leaves.

Here, we show that it is possible to harvest photocurrent directly from unprocessed plant tissues from terrestrial or aquatic environments in bio-photoelectrochemical cells (BPECs) and use the current to produce molecular H2. The source of electrons is shown to originate from the Photosystem II water-oxidation reaction and utilizes exported mediating molecules, especially NADPH. The photocurrent production is dependent on the concentration of the photosynthetic complexes, as an increase in total chlorophyll and oxygen evolution rates in the leaves lead to increased photocurrent rates. The permeability of the outer leaf surface is another important factor in photocurrent harvesting. Different tissues produce photocurrent densities in the range of ∼1-10 mA/cm2 which is significantly higher than microorganism-based BPECs. The relatively high photocurrent and the simplicity of the plants BPEC may pave the way toward the development of future applicative photosynthetic based energy technologies.