Monitoring the Electrochemical Failure of Indium Tin Oxide Electrodes via Operando Ellipsometry Complemented by Electron Microscopy and Spectroscopy.

Transparent conductive oxides such as indium tin oxide (ITO) are standards for thin film electrodes, providing a synergy of high optical transparency and electrical conductivity. In an electrolytic environment, the determination of an inert electrochemical potential window is crucial to maintain a stable material performance during device operation. We introduce operando ellipsometry, combining cyclic voltammetry (CV) with spectroscopic ellipsometry, as a versatile tool to monitor the evolution of both complete optical (i.e., complex refractive index) and electrical properties under wet electrochemical operational conditions. In particular, we trace the degradation of ITO electrodes caused by electrochemical reduction in a pH-neutral, water-based electrolyte environment during electrochemical cycling. With the onset of hydrogen evolution at negative bias voltages, indium and tin are irreversibly reduced to the metallic state, causing an advancing darkening, i.e., a gradual loss of transparency, with every CV cycle, while the conductivity is mostly conserved over multiple CV cycles. Post-operando analysis reveals the reductive (loss of oxygen) formation of metallic nanodroplets on the surface. The reductive disruption of the ITO electrode happens at the solid-liquid interface and proceeds gradually from the surface to the bottom of the layer, which is evidenced by cross-sectional transmission electron microscopy imaging and complemented by energy-dispersive X-ray spectroscopy mapping. As long as a continuous part of the ITO layer remains at the bottom, the conductivity is largely retained, allowing repeated CV cycling. We consider operando ellipsometry a sensitive and nondestructive tool to monitor early stage material and property changes, either by tracing failure points, controlling intentional processes, or for sensing purposes, making it suitable for various research fields involving solid-liquid interfaces and electrochemical activity.

N-Acetyl-L-glutamate Kinase of Chlamydomonas reinhardtii: In Vivo Regulation by PII Protein and Beyond.

N-Acetyl-L-glutamate kinase (NAGK) catalyzes the rate-limiting step in the ornithine/arginine biosynthesis pathway in eukaryotic and bacterial oxygenic phototrophs. NAGK is the most highly conserved target of the PII signal transduction protein in Cyanobacteria and Archaeplastida (red algae and Chlorophyta). However, there is still much to be learned about how NAGK is regulated in vivo. The use of unicellular green alga Chlamydomonas reinhardtii as a model system has already been instrumental in identifying several key regulation mechanisms that control nitrogen (N) metabolism. With a combination of molecular-genetic and biochemical approaches, we show the existence of the complex CrNAGK control at the transcriptional level, which is dependent on N source and N availability. In growing cells, CrNAGK requires CrPII to properly sense the feedback inhibitor arginine. Moreover, we provide primary evidence that CrPII is only partly responsible for regulating CrNAGK activity to adapt to changing nutritional conditions. Collectively, our results suggest that in vivo CrNAGK is tuned at the transcriptional and post-translational levels, and CrPII and additional as yet unknown factor(s) are integral parts of this regulation.

Arginine-Dependent Nitric Oxide Generation and S-Nitrosation in the Non-Photosynthetic Unicellular Alga Polytomella parva.

Nitric oxide (NO) acts as a key signaling molecule in higher plants, regulating many physiological processes. Several photosynthetic algae from different lineages are also known to produce NO. However, it remains unclear whether this messenger is produced by non-photosynthetic algae. Among these organisms, the colorless alga Polytomella parva is a special case, as it has lost not only its plastid genome, but also nitrate reductase and nitrite reductase. Up to now, the question of whether NO synthesis occurs in the absence of functional nitrate reductase (NR) and the assimilation of nitrates/nitrites in P. parva has not been elucidated. Using spectrofluorometric assays and confocal microscopy with NO-sensitive fluorescence dye, we demonstrate L-arginine-dependent NO synthesis by P. parva cells. Based on a pharmacological approach, we propose the existence of arginine-dependent NO synthase-like activity in this non-photosynthetic alga. GC-MS analysis provides primary evidence that P. parva synthesizes putrescine, which is not an NO source in this alga. Moreover, the generated NO causes the S-nitrosation of protein cysteine thiol groups. Together, our data argue for NR-independent NO synthesis and its active role in S-nitrosation as an essential post-translational modification in P. parva.

Truncated hemoglobin 2 modulates phosphorus deficiency response by controlling of gene expression in nitric oxide-dependent pathway in Chlamydomonas reinhardtii.

MAIN CONCLUSION: Truncated hemoglobin 2 is involved in fine-tuning of PSR1-regulated gene expression during phosphorus deprivation. Truncated hemoglobins form a large family found in all domains of life. However, a majority of physiological functions of these proteins remain to be elucidated. In the model alga Chlamydomonas reinhardtii, macro-nutritional deprivation is known to elevate truncated hemoglobin 2 (THB2). This study investigated the role of THB2 in the regulation of a subset of phosphorus (P) limitation-responsive genes in cells suffering from P-deficiency. Underexpression of THB2 in amiTHB2 strains resulted in downregulation of a suite of P deprivation-induced genes encoding proteins with different subcellular location and functions (e.g., PHOX, LHCSR3.1, LHCSR3.2, PTB2, and PTB5). Moreover, our results provided primary evidence that the soluble guanylate cyclase 12 gene (CYG12) is a component of the P deprivation regulation. Furthermore, the transcription of PSR1 gene for the most critical regulator in the acclimation process under P restriction was repressed by nitric oxide (NO). Collectively, the results indicated a tight regulatory link between the THB2-controlled NO levels and PSR1-dependent induction of several P deprivation responsive genes with various roles in cells during P-limitation.

Cold Stress Response: An Overview in Chlamydomonas.

Low temperature (or cold) is one of the major environmental factors that limit the growth and development of many plants. Various plant species have evolved complex mechanisms to adjust to decreased temperature. Mesophilic chlorophytes are a widely distributed group of eukaryotic photosynthetic organisms, but there is insufficient information about the key molecular processes of their cold acclimation. The best available model for studying how chlorophytes respond to and cope with variations in temperature is the unicellular green alga Chlamydomonas reinhardtii. Chlamydomonas has been widely used for decades as a model system for studying the fundamental mechanisms of the plant heat stress response. At present, unraveling novel cold-regulated events in Chlamydomonas has attracted increasing research attention. This mini-review summarizes recent progress on low-temperature-dependent processes in the model alga, while information on other photosynthetic organisms (cyanobacteria and land plants) was used to strengthen generalizations or specializations of cold-induced mechanisms in plant evolution. Here, we describe recent advances in our understanding of cold stress response in Chlamydomonas, discuss areas of controversy, and highlight potential future directions in cold acclimation research.

From cyanobacteria to Archaeplastida: new evolutionary insights into PII signalling in the plant kingdom.

The PII superfamily consists of signal transduction proteins found in all domains of life. Canonical PII proteins sense the cellular energy state through the competitive binding of ATP and ADP, and carbon/nitrogen balance through 2-oxoglutarate binding. The ancestor of Archaeplastida inherited its PII signal transduction protein from an ancestral cyanobacterial endosymbiont. Over the course of evolution, plant PII proteins acquired a glutamine-sensing C-terminal extension, subsequently present in all Chloroplastida PII proteins. The PII proteins of various algal strains (red, green and nonphotosynthetic algae) have been systematically investigated with respect to their sensory and regulatory properties. Comparisons of the PII proteins from different phyla of oxygenic phototrophs (cyanobacteria, red algae, Chlorophyta and higher plants) have yielded insights into their evolutionary conservation vs adaptive properties. The highly conserved role of the controlling enzyme of arginine biosynthesis, N-acetyl-l-glutamate kinase (NAGK), as a main PII-interactor has been demonstrated across oxygenic phototrophs of cyanobacteria and Archaeplastida. In addition, the PII signalling system of red algae has been identified as an evolutionary intermediate between that of Cyanobacteria and Chloroplastida. In this review, we consider recent advances in understanding metabolic signalling by PII proteins of the plant kingdom.

Interaction of N-acetyl-l-glutamate kinase with the PII signal transducer in the non-photosynthetic alga Polytomella parva: Co-evolution towards a hetero-oligomeric enzyme.

During evolution, several algae and plants became heterotrophic and lost photosynthesis; however, in most cases, a nonphotosynthetic plastid was maintained. Among these organisms, the colourless alga Polytomella parva is a special case, as its plastid is devoid of any DNA, but is maintained for specific metabolic tasks carried out by nuclear encoded enzymes. This makes P. parva attractive to study molecular events underlying the transition from autotrophic to heterotrophic lifestyle. Here we characterize metabolic adaptation strategies of P. parva in comparison to the closely related photosynthetic alga Chlamydomonas reinhardtii with a focus on the role of plastid-localized PII signalling protein. Polytomella parva accumulates significantly higher amounts of most TCA cycle intermediates as well as glutamate, aspartate and arginine, the latter being specific for the colourless plastid. Correlating with the altered metabolite status, the carbon/nitrogen sensory PII signalling protein and its regulatory target N-acetyl-l-glutamate-kinase (NAGK; the controlling enzyme of arginine biosynthesis) show unique features: They have co-evolved into a stable hetero-oligomeric complex, irrespective of effector molecules. The PII signalling protein, so far known as a transiently interacting signalling protein, appears as a permanent subunit of the enzyme NAGK. NAGK requires PII to properly sense the feedback inhibitor arginine, and moreover, PII tunes arginine-inhibition in response to glutamine. No other PII effector molecules interfere, indicating that the PII-NAGK system in P. parva has lost the ability to estimate the cellular energy and carbon status but has specialized to provide an entirely glutamine-dependent arginine feedback control, highlighting the evolutionary plasticity of PII signalling system.

Truncated Hemoglobins 1 and 2 Are Implicated in the Modulation of Phosphorus Deficiency-Induced Nitric Oxide Levels in Chlamydomonas.

Truncated hemoglobins (trHbs) form a widely distributed family of proteins found in archaea, bacteria, and eukaryotes. Accumulating evidence suggests that trHbs may be implicated in functions other than oxygen delivery, but these roles are largely unknown. Characterization of the conditions that affect trHb expression and investigation of their regulatory mechanisms will provide a framework for elucidating the functions of these globins. Here, the transcription of Chlamydomonas trHb genes (THB1-12) under conditions of phosphorus (P) deprivation was analyzed. Three THB genes, THB1, THB2, and THB12 were expressed at the highest level. For the first time, we demonstrate the synthesis of nitric oxide (NO) under P-limiting conditions and the production of NO by cells via a nitrate reductase-independent pathway. To clarify the functions of THB1 and THB2, we generated and analyzed strains in which these THBs were strongly under-expressed by using an artificial microRNA approach. Similar to THB1 knockdown, the depletion of THB2 led to a decrease in cell size and chlorophyll levels. We provide evidence that the knockdown of THB1 or THB2 enhanced NO production under P deprivation. Overall, these results demonstrate that THB1 and THB2 are likely to contribute, at least in part, to acclimation responses in P-deprived Chlamydomonas.

Effects of arginine on Polytomella parva growth, PII protein levels and lipid body formation.

MAIN CONCLUSION: L-Arginine supports growth and resulted in increased PII signaling protein levels and lipid droplet accumulation in the colorless green alga Polytomella parva. Polytomella parva, a model system for nonphotosynthetic green algae, utilizes ammonium and several carbon sources, including ethanol and acetate. We previously reported that P. parva accumulates high amounts of arginine with the key enzyme of the ornithine/arginine biosynthesis pathway, N-acetyl-L-glutamate kinase, exhibiting high activity. Here we demonstrate that L-arginine can be used by this alga as a nitrogen source. Externally supplied arginine directly influenced the levels of PII signaling protein and formation of triacylglycerol (TAG)-filled lipid bodies (LBs). Our results suggest that the nitrogen source, but not nitrogen starvation, may be critical for the accumulation of LBs in a PII-independent manner in P. parva.

Dual positive and negative control of Chlamydomonas PII signal transduction protein expression by nitrate/nitrite and NO via the components of nitric oxide cycle.

BACKGROUND: The PII proteins constitute a large superfamily, present in all domains of life. Until now, PII proteins research in Chloroplastida (green algae and land plants) has mainly focused on post-translation regulation of these signal transductors. Emerging evidence suggests that PII level is tightly controlled with regard to the nitrogen source and the physiological state of cells. RESULT: Here we identify that a balance of positive (nitrate and nitrite) and negative (nitric oxide) signals regulates Chlamydomonas GLB1. We found that PII expression is downregulated by ammonium through a nitric oxide (NO)-dependent mechanism. We show that nitrate reductase (NR) and its partner, truncated hemoglobin 1 (THB1), participate in a signaling pathway for dual control of GLB1 expression. Moreover, NO dependent guanilate cyclase appeared to be involved in the negative control of GLB1 transcription. CONCLUSION: This study has revealed the existence of the complex GLB1 control at transcription level, which is dependent on nitrogen source. Importantly, we found that GLB1 gene expression pattern is very similar to that observed for nitrate assimilation genes, suggesting interconnecting/coordinating PII-dependent and nitrate assimilation pathways.

Regulation of alternative oxidase 1 in Chlamydomonas reinhardtii during sulfur starvation.

The mitochondrial respiratory chain in plants, some protists and many fungi consists of the ATP-coupling cyanide-sensitive cytochrome pathway and the cyanide-resistant alternative respiratory pathway. The alternative pathway is mediated by alternative oxidase (AOX). Although AOX has been proposed to play essential roles in nutrient stress tolerance of plants and protists, the effects of sulfur (S) deprivation, on AOX are largely unknown. The unicellular green alga Chlamydomonas reinhardtii reacts to S limitation conditions with the induced expression of many genes. In this work, we demonstrated that exposure of C. reinhardtii to S deprivation results in the up-regulation of AOX1 expression and an increased AOX1 protein. Furthermore, S-deprived C. reinhardtii cells display the enhanced AOX1 capacity. Moreover, nitrate assimilation regulatory protein (NIT2) is involved in the control of the AOX1 gene expression in the absence of S. Together, the results clearly indicate that AOX1 relates to S limitation stress responses and is regulated in a NIT2-dependent manner, probably together with yet-unknown regulatory factor(s).

The PII signaling protein from red algae represents an evolutionary link between cyanobacterial and Chloroplastida PII proteins.

PII superfamily consists of widespread signal transduction proteins found in all domains of life. Whereas they are well-studied in Archaea, Bacteria and Chloroplastida, no PII homolog has been analyzed in Rhodophyta (red algae), where PII is encoded by a chloroplast localized glnB gene. Here, we characterized relevant sensory properties of PII from the red alga Porphyra purpurea (PpPII) in comparison to PII proteins from different phyla of oxygenic phototrophs (cyanobacteria, Chlamydomonas and Physcomitrella) to assess evolutionary conservation versus adaptive properties. Like its cyanobacterial counterparts, PpPII binds ATP/ADP and 2-oxoglutarate in synergy with ATP. However, green algae and land plant PII proteins lost the ability to bind ADP. In contrast to PII proteins from green algae and land plants, PpPII enhances the activity of N-acetyl-L-glutamate kinase (NAGK) and relieves it from feedback inhibition by arginine in a glutamine-independent manner. Like PII from Chloroplastida, PpPII is not able to interact with the cyanobacterial transcriptional co-activator PipX. These data emphasize the conserved role of NAGK as a major PII-interactor throughout the evolution of oxygenic phototrophs, and confirms the specific role of PipX for cyanobacteria. Our results highlight the PII signaling system in red algae as an evolutionary intermediate between Cyanobacteria and Chlorophyta.

Regulation of sulfur deprivation-induced expression of the ferredoxin-encoding FDX5 gene Chlamydomonas reinhardtii in aerobic conditions.

The unicellular green alga Chlamydomonas reinhardtii reacts to sulfur (S) starvation with the increased expression of numerous genes. One gene which is induced in illuminated anaerobic S-deprived cells is the ferredoxin-5 gene (FDX5). To test FDX5 transcriptional regulation in aerobic cultures, we used a real-time PCR analysis and an artificial microRNA approach. We demonstrated that FDX5 gene is controlled by S deprivation independently of anoxia-treatment. The Ser/Thr kinase SNRK2.1 is necessary for expression of FDX5 during deprivation to S. Copper response regulator 1 (CRR1) is not involved in FDX5 up-regulation in S-deficient cells under aerobic conditions. Furthermore, expression of FDX5 is negatively regulated by nitric oxide (NO). Moreover, truncated hemoglobin 1 (THB1) underexpression resulted in the decrease in FDX5 transcript abundance in S-deficient cells under aerobic conditions. Together, our results imply that the FDX5 gene is controlled by NO in THB1-dependent pathway under conditions of depleted S supply.

Nitric oxide upregulates expression of alternative oxidase 1 in Chlamydomonas reinhardtii.

The mitochondrial respiratory chain in plants, many fungi and some protists consists of the ATP-coupling cyanide-sensitive cytochrome pathway and the cyanide-resistant alternative respiratory pathway. The alternative pathway is mediated by alternative oxidase (AOX). In unicellular algae, AOXs are monomeric fungi-type proteins. Studies performed in the model plant Chlamydomonas reinhardtii showed that a range of stress factors lead to induction of its AOX1. However, signaling molecules that trigger upregulation of AOX1 have not yet been identified. Here, we were able to discriminate between two alternative oxidases of the alga. In this work, we demonstrated that exposure of C. reinhardtii to nitric oxide (NO) resulted in the up-regulation of AOX1 expression and an increased AOX1 protein. Furthermore, NO-treated C. reinhardtii cells displayed the enhanced AOX1 capacity. We also clearly demonstrated that AOX1 can function in C. reinhardtii when the cytochrome oxidase became inhibited by NO. Although the pathway(s) that leads to increased AOX1 levels and activity upon NO treatment is yet unknown, it is now clear that NO serves as the signal to trigger this regulatory process in C. reinhardtii.

Truncated hemoglobin 1 is a new player in Chlamydomonas reinhardtii acclimation to sulfur deprivation.

Truncated hemoglobins constitute a large family, present in bacteria, in archaea and in eukaryotes. However, a majority of physiological functions of these proteins remains to be elucidated. Identification and characterization of a novel role of truncated hemoglobins in the model alga provides a framework for a more complete understanding of their biological functions. Here, we use quantitative RT-PCR to show that three truncated hemoglobins of Chlamydomonas reinhardtii, THB1, THB2 and THB12, are induced under conditions of depleted sulfur (S) supply. THB1 underexpression results in the decrease in cell size, as well in levels of proteins, chlorophylls and mRNA of several S-responsive genes under S starvation. We provide evidence that knock-down of THB1 enhances NO production under S deprivation. In S-deprived cells, a subset of S limitation-responsive genes is controlled by NO in THB1-dependent pathway. Moreover, we demonstrate that deficiency for S represses the nitrate reduction and that THB1 is involved in this control. Thus, our data support the idea that in S-deprived cells THB1 plays a dual role in NO detoxification and in coordinating sulfate limitation with nitrate assimilation. This study uncovers a new function for the Chlamydomonas reinhardtii THB1 in the control of proper response to S deprivation.

New insights into AOX2 transcriptional regulation in Chlamydomonas reinhardtii.

A feature of the mitochondrial electron transport chain in plants, some protists and many fungi is the presence of two terminal oxidases, the energy-conserving cytochrome oxidase and another termed alternative oxidase (AOX). AOX branches from the main respiratory chain, directly coupling the oxidation of ubiquinol with reduction of oxygen to water. The AOX genes can be divided into two discrete subfamilies, AOX1 and AOX2. Although AOX has been proposed to play essential roles in stress tolerance of organisms, the role of subfamily AOX2 is largely unknown. In the model green alga Chlamydomonas reinhardtii, two genes have been identified that encode for AOX, AOX1 and AOX2. To test AOX2 transcriptional regulation in this alga, we used a real-time PCR analysis and an artificial microRNA approach. The C. reinhardtii AOX2 gene is up-regulated by oxygen or copper deprivation. Moreover, in dark-adapted unstressed cells, AOX2 is induced. Together, our results imply that the AOX2 gene is a stress-inducible and is regulated by the copper response regulator 1 (CRR1), probably together with yet-unknown regulatory factor(s).

Responses triggered in chloroplast of Chlorella variabilis NC64A by long-term association with Paramecium bursaria.

The unicellular green alga Chlorella variabilis NC64A is an endosymbiont of the ciliate Paramecium bursaria. The host's control, including the transfer of biochemical substrates from P. bursaria to C. variabilis, is involved in symbiotic relationships. C. variabilis NC64A that had been re-infected to P. bursaria for more than 1 year and isolated from the host showed higher chlorophyll levels compared to those in free-living cells. Unlike the host, the expression of C. variabilis NC64A heat shock 70 kDa protein was independent of establishment of endosymbiosis. In symbiotic cells, the levels of PII signal transduction protein (CvPII) that coordinate the central C/N anabolic metabolism were slightly higher than those in free-living cells. Furthermore, the environmental cues (light and host food bacteria availability) affected the abundance of CvPII, suggesting that synthesis of the protein was influenced by the host. Moreover, arginine concentrations in the symbiotic algae of P. bursaria were also controlled by the host's nutritional conditions. Together, our results imply that signal substrates and/or products of metabolism in host cells might act as messengers mediating the regulation of key events in symbiont cells.

The HSP70 chaperone machines of Chlamydomonas are induced by cold stress.

The responses of Chlamydomonas reinhardtii cells to low temperatures have not been extensively studied compared with other stresses. Like other organisms, this green alga has heat shock protein 70s (HSP70s) that are located in chloroplast, mitochondrion and cytosol. To test whether temperature downshifts affected HSP70s synthesis, we used real-time PCR and protein gel blot analysis. C. reinhardtii cells exposed to cold stress show increased HSP70s mRNA levels. Genes encoding other components of HSP70 chaperone machines (e.g. CGE1, CDJ1, HSP90C and HSP90A) are also up-regulated in response to decreased temperature. We demonstrated that the accumulation of all analyzed mRNA occur more slowly and with reduced amplitude in cells exposed to cold than in cells treated with heat. Furthermore, C. reinhardtii cells display the splicing of the CGE1 transcript that was dependent on low temperature. Finally, the transcription regulator of C. reinhardtii HSF1 is also cold-responsive, suggesting its role in the transcriptional regulation of HSP genes at low temperature.

The Chlamydomonas reinhardtii alternative oxidase 1 is regulated by heat stress.

The alternative oxidase (AOX) is a non-energy conserving terminal oxidase that has emerged as an important mitochondrial component of the cell stress responses. Although the most studied abiotic condition in relation to Chlamydomonas reinhardtii is high temperature, changes in AOX capacity of the alga were studied only under oxidative stress and cold. To examine whether elevated temperatures affected AOX1 expression, we applied quantitative real-time PCR and pharmaceutical approaches. In this work, we demonstrated a sharp increase in AOX1 transcript and protein abundance under heat stress. Furthermore, C. reinhardtii cells displayed a large increase in alternative respiration in response to high temperature. Feeding with the protein kinase inhibitor staurosporine strongly retarded the AOX1 transcription. Finally, the addition of the calcium chelator EGTA prevented heat-induced AOX1 expression. Together, our results imply that heat-inducible Ca(2+) influx and protein kinase(s) may mediate AOX1 expression at elevated temperatures. Characterization of heat-induced AOX1 regulation in the green alga C. reinhardtii provides a framework for a more complete understanding of the function of this conserved protein.

Reduction of PII signaling protein enhances lipid body production in Chlamydomonas reinhardtii.

In all examined organisms that have the PII signal transduction machinery, PII coordinates the central C/N anabolic metabolism. In green algae and land plants, PII is localized in the chloroplast and controls the L-arginine biosynthetic pathway pathway. To elucidate additional functions of PII in the model photosynthetic organism Chlamydomonas reinhardtii (CrPII), we generated and analyzed four strains, in which PII was strongly under-expressed by artificial microRNA (GLB1-amiRNA strains). In response to nitrogen deficiency, Chlamydomonas produces triacylglycerols (TAGs) that are accumulated in lipid bodies (LB). Quantification of LBs by confocal microscopy in four GLB1-amiRNA strains showed that reduced PII levels resulted in over-accumulation of LBs compared to their parental strains. Moreover, knock-down of PII caused also an increase in the total TAG level. We propose that the larger yields of TAG-filled LBs in N-starved GLB1-amiRNA cells can be attributed to the strain's depleted PII level and their inability to properly control acetyl-CoA carboxylase activity (ACCase). Together, our results imply that PII in Chlamydomonas negatively controls TAG accumulation in LBs during acclimation to nitrogen starvation of the alga.