RNase J (RNJ) is a ribonuclease found in bacteria, archaea, and plant chloroplasts, and plays diverse roles in RNA maturation and stability. Chloroplast RNJ is encoded by the nuclear RNJ locus and is essential for embryo maturation. Arabidopsis or tobacco plants depleted for RNJ accumulate massive amounts of double-stranded RNA (dsRNA), which interferes with translation and causes chlorosis. Land plant RNJ uniquely contains a C-terminal GT1 domain, a DNA-binding motif found in transcription factors. Here, we have used complementation of an Arabidopsis rnj mutant with versions of RNJ with a mutated or deleted GT1 domain to investigate its role in RNJ function. We show that in vitro, the recombinant GT1 domain binds both dsRNA and DNA, but not single-stranded nucleic acids, with no sequence specificity. Furthermore, while RNJ lacking GT1 binding complements the rnj mutant, these plants accumulate high levels of dsRNA as detected by immunolocalization and RNA-Seq. GT1 mutations also change RNJ solubility in vivo, suggesting that the GT1 domain is involved in localization within the plastid. Taken together, our results suggest that the GT1 domain plays a key role in dsRNA removal through localizing the enzyme and/or selectively binding the dsRNA substrate.

Non-photochemical quenching (NPQ) mechanisms are crucial for protecting photosynthesis from photoinhibition in plants, algae, and cyanobacteria, and their modulation is a long-standing goal for improving photosynthesis and crop yields. The current work demonstrates that Chlorella ohadii , a green micro-alga that thrives in the desert under high light intensities which are fatal to many photosynthetic organisms, does not perform nor require NPQ to protect photosynthesis under constant high light. Instead of dissipating excess energy, it minimizes its uptake by eliminating the photosynthetic antenna of photosystem II. In addition it accumulates antioxidants that neutralize harmful reactive oxygen species (ROS) and ramps up cyclic electron flow around PSI. These NPQ-independent responses proved efficient in preventing ROS accumulation and reducing oxidative damage to proteins in high-light-grown cells. ### Competing Interest Statement The authors have declared no competing interest.

We describe the importance of researching high-light tolerance in high-light tolerant photosynthetic organisms and how it may contribute to the efforts of improving photosynthesis and enhancing crop yield.

Nonphotochemical quenching (NPQ) mechanisms are crucial for protecting photosynthesis from photoinhibition in plants, algae, and cyanobacteria, and their modulation is a long-standing goal for improving photosynthesis and crop yields. The current work demonstrates that Chlorella ohadii, a green microalga that thrives in the desert under high light intensities that are fatal to many photosynthetic organisms does not perform nor require NPQ to protect photosynthesis under constant high light. Instead of dissipating excess energy, it minimizes its uptake by eliminating the photosynthetic antenna of photosystem II. In addition, it accumulates antioxidants that neutralize harmful reactive oxygen species (ROS) and increases cyclic electron flow around PSI. These NPQ-independent responses proved efficient in preventing ROS accumulation and reducing oxidative damage to proteins in high- light-grown cel

The phosphorylation of photosystem II (PSII) and its antenna (LHCII) proteins has been extensively studied and its involvement in state transitions and PSII repair is well known. Yet, very little is known about the extent and functions of phosphorylation of photosystem I (PSI) and its antenna (LHCI) proteins. Here, two proteomics methods were applied to generate a detailed 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 in these cell types to estimate light-induced oxidative damage to PSI-LHCI.Our work revealed the phosphorylation of 11 out of 22 PSI-LHCI subunits. The analyses detected extensive phosphorylation of the LHCI subunits lhca6 and lhca7. Other PSI-LHCI subunits were phosphorylated to a lesser extent. Additionally, we show the accumulation of oxidatively damaged tryptophans in the psaD subunit of PSI of HL-grown C. ohadii. The significant phosphorylation of lhca6 and lhca7 suggests a physiological role during photosynthesis, possibly by altering light-harvesting characteristics and binding of other LHCI subunits. Moreover, we show that psaD is susceptible to photodamage while LHCI is protected from ROS under HL.

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 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 increasing concern about future environmental disasters as a result of global climate change is driving the efforts to develop novel clean energy technologies that will replace combustible fuels. A promising approach is the utilization of native photosynthesis as an electron source in bio-electrochemical cells. Over the past few years, there have been numerous descriptions of technologies for electrical current production using isolated photosynthetic complexes or membranes, intact microorganisms, or microorganism tissues in different electrochemical setups. In this review, we summarize these methods, and address the key factors that influence photocurrent generation and how can they be improved. Finally, we discuss the potential of photosynthetic-based bioelectricity production to become a real applicative solution that will be able to compete with existing technologies such as solar cells.

Replacing fossil fuels with renewable energy technologies, especially solar energy conversion, are a major pillar in the ongoing efforts to produce useful amounts of energy without significant pollution. Utilization of photosynthetic organisms’ ability to utilize light to produce electrical currents in bio-photoelectrochemical cells (BPECs) are such a source of clean energy. Here, we show that it is possible to harvest photocurrent directly from unprocessed plant tissues from terrestrial, and aquatic environments and use it to produce H2 gas. The source of electrons is shown to originate from the Photosystem II water-oxidation reaction and utilizes exported mediating molecules, especially NADPH. Also, we show that the photocurrent production is dependent on the developmental stage of the leaf. Finally, we show the use of water lilies to generate photocurrent in their native growth environment. 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 establishment of applicative photosynthetic based energy technologies.

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.

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 H 2 . 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 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 / cm 2 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.

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.

Conversion of solar energy into electrical current by photosynthetic organisms has the potential to produce clean energy. Previously reported bio-photoelectrochemical cells (BPECs) have utilized unicellular photosynthetic microorganisms. In this study, we describe for the first time BPECs that utilize intact live marine macroalgae (seaweeds) in natural seawater or saline buffer or natural seawater. The BPECs produce electrical currents from of >50mA/cm2, from both light-dependent (photosynthesis) and light independent processes. These values are significantly greater than the current densities that have been reported for single-cell microorganisms. The photocurrent is inhibited by the Photosystem II inhibitor DCMU, indicating that the source of light-driven electrons is from water oxidation via NADPH and other reduced molecules. We show here that intact seaweed cultures can be used in a large-scale BPEC containing seawater that produces bias-free photocurrent. The ability to produce bioelectricity from intact seaweeds may pave the way to future development of a low-cost energy technology using BPECs.

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.

While light is the driving force of photosynthesis, excessive light can be harmful. Photoinhibition, or light-induced photo-damage, is one of the key processes limiting photosynthesis. When the absorbed light exceeds the amount that can be dissipated by photosynthetic electron flow and other processes, damaging radicals are formed that mostly inactivate photosystem II (PSII). A well-defined mechanism that protects the photosynthetic apparatus from photoinhibition has been described in the model green alga Chlamydomonas reinhardtii and plants. Chlorella oha-dii is a green micro-alga, isolated from biological desert soil crusts, that thrives under extreme high light (HL) in which other organisms do not survive. Here, we show that this alga evolved unique protection mechanisms distinct from those of C. reinhardtii and plants. When grown under extreme HL, significant structural changes were noted in the C. ohadii thylakoids, including a drastic reduction in the antennae and the formation of stripped core PSII, lacking its outer and inner antennae. 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, the stress-related light harvesting complex, PSII protein phosphorylation and state-transitions are entirely absent or were barely detected in C. ohadii . Taken together, a unique photoinhibition protection mechanism evolved in C. ohadii , enabling the species to thrive under extreme-light intensities where other photo-synthetic organisms fail to survive.

Photosynthesis in deserts is challenging since it requires fast adaptation to rapid night-to-day changes, that is, from dawn's low light (LL) to extreme high light (HL) intensities during the daytime. To understand these adaptation mechanisms, we purified photosystem I (PSI) from Chlorella ohadii, a green alga that was isolated from a desert soil crust, and identified the essential functional and structural changes that enable the photosystem to perform photosynthesis under extreme high light conditions. The cryo-electron microscopy structures of PSI from cells grown under low light (PSILL) and high light (PSIHL), obtained at 2.70 and 2.71 Å, respectively, show that part of light-harvesting antenna complex I (LHCI) and the core complex subunit (PsaO) are eliminated from PSIHL to minimize the photodamage. An additional change is in the pigment composition and their number in LHCIHL; about 50% of chlorophyll b is replaced by chlorophyll a. This leads to higher electron transfer rates in PSIHL and might enable C. ohadii PSI to act as a natural photosynthesiser in photobiocatalytic systems. PSIHL or PSILL were attached to an electrode and their induced photocurrent was determined. To obtain photocurrents comparable with PSIHL, 25 times the amount of PSILL was required, demonstrating the high efficiency of PSIHL. Hence, we suggest that C. ohadii PSIHL is an ideal candidate for the design of desert artificial photobiocatalytic systems.

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.

In recent years, finding alternatives for fossil fuels has become a major concern. One promising solution is microorganism-based bio-photo electrochemical cells (BPECs) that utilize photosynthetic solar energy conversion as an energy source while absorbing CO2 from the atmosphere. It was previously reported that in cyanobacterial-based BPECs, the major endogenous electron mediator that can transfer electrons from the thylakoid membrane photosynthetic complexes and external anodes is NADPH. However, the question of whether the same electron transfer mechanism is also valid for live eukaryotic microalgae, in which NADPH must cross both the chloroplast outer membrane and the cell wall to be secreted from the cell has remained elusive. In this work, we show that NADPH is also the major endogenous electron mediator in the microalgae Dunalliela salina (Ds). We show that the ability of Ds to tolerate high salinity enables the production of a photocurrent that is 5–6 times greater than previously reported for freshwater cyanobacterial-based BPECs in the presence or absence of exogenous electron mediators. Additionally, we show that the electron mediator Vitamin B1 can also function as an electron mediator enhancing photocurrent production. Finally, we show that the addition of both FeCN and NADP+ to Ds has a synergistic effect enhancing the photocurrent beyond the effect of adding each mediator separately.

RNA quality control is an indispensable but poorly understood process that enables organisms to distinguish functional RNAs from nonfunctional or inhibitory ones. In chloroplasts, whose gene expression activities are required for photosynthesis, retrograde signaling, and plant development, RNA quality control is of paramount importance, as transcription is relatively unregulated. The functional RNA population is distilled from this initial transcriptome by a combination of RNA-binding proteins and ribonucleases. One of the key enzymes is RNase J, a 5'→3' exoribonuclease and an endoribonuclease that has been shown to trim 5' RNA termini and eliminate deleterious antisense RNA. In the absence of RNase J, embryo development cannot be completed. Land plant RNase J contains a highly conserved C-terminal domain that is found in GT-1 DNA-binding transcription factors and is not present in its bacterial, archaeal, and algal counterparts. The GT-1 domain may confer specificity through DNA and/or RNA binding and/or protein-protein interactions and thus be an element in the mechanisms that identify target transcripts among diverse RNA populations. Further understanding of chloroplast RNA quality control relies on discovering how RNase J is regulated and how its specificity is imparted.