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Anionic electrolyzers are expected to play a major role in the future massive production of green hydrogen. In this context, anionic membranes are still a bottleneck for their low conductivity and insufficient long-term chemical stability. In this paper we discussed and demonstrated the use of chemically modified, Aquivion®-based anionic membranes for water electrolysis. The membranes were subjected to different ageing treatments up to 3 M at 80 °C and showed better performance and durability than commercial Aemion™. Current density values around 130 mA cm−2 at 2 V were obtained with nickel foam as a benchmark electrode material.

Low temperature alkaline water electrolysis is a well-known technology for hydrogen production and advantageous because less noble metals are needed compared to Proton Exchange Membrane (PEM) electrolysis. However, the two-phase flow behavior and especially the gas bubble distribution and flow during the operation in a zero-gap alkaline electrolysis cell have not been investigated in sufficient detail. Previous studies used e.g. transparent model electrolysis cells or focused on single flow phenomena e.g. gas bubble nucleation, growth or detachment. Recent publications show evidence that the ohmic resistance of zero-gap alkaline electrolysis cells is greater than the ohmic resistance of the used diaphragm, which has been believed the only term contributing to the ohmic resistance in zero-gap cells [1]. This suggests that the evolving gas bubbles might have a relation to the total ohmic resistance of the cell. As possible reasons nano bubbles in the pores of the diaphragm and a possible finite gap between electrodes and diaphragm is mentioned [2]. Haverkort et al. assume that the frontal area of the electrodes might be inactive [3]. To prove the hypothesis and to deepen the general understanding of the two-phase flow inside an alkaline electrolysis cell we conducted operando cell measurements and visualized the electrolyte-gas bubble flow in an alkaline electrolysis cell by using neutron radiography. Thanks to the powerful neutron source at Institute Laue-Langevin in Grenoble, it was possible to observe the whole flow field with all flow channels at once with a sufficient temporal resolution to observe the movement of single gas bubbles, from nucleation to the moment of exiting the channel. The temporal resolution was 50 fps, which makes these measurement, to the best authors’ knowledge, the first to show the transient effects inside of a zero gap alkaline electrolysis cell. For the measurements, the NeXT (Neutron and X-Ray Tomograph) instrument was used which is the imaging station with one of the highest neutron fluxes of the world (1.4 E10 n/cm²/s at the end of the guide) [4]. The neutron beam was used to carry out through-plane measurements of the electrolysis cell. The measurements were conducted at a variety of operation conditions, varying the current density, the electrolyte volume flow and the operation temperature. At different operation conditions Electrical Impedance Spectroscopy (EIS) measurements have been conducted to measure the ohmic resistance of the cell. The used cell is an alkaline single cell out of a Nickel alloy with vertical parallel flow channel and a pocket milled to the backside of the cell in order to increase the transmission through the cell material. From the results of the measurements we observe a relation between current density (forming gas amount) and the ohmic resistance and thus establish a relation to the gas bubble behavior inside the electrolysis cell. Furthermore, we analyzed the velocity of the gas bubbles inside the channel depending on the different operation conditions, as well as the agglomeration behavior of the bubbles and their flow regime. [1] Phillips et al.,Minimising the ohmic resistance of an alkaline electrolysis cell through effective cell design, 2017 [2] de Groot et al., Ohmic resistance in zero gap alkaline electrolysis with a Zirfon diaphragm,2021 [3] Haverkort et al., Voltage losses in zero-gap alkaline water electrolysis,2021 [4] Tengattini et al., NeXT-Grenoble, the Neutron and X-ray tomograph in Grenoble, 2020 Figure 1

Resident adult epithelial stem cells maintain tissue homeostasis by balancing self-renewal and differentiation. The stem cell potential of human epidermal keratinocytes is retained in vitro but lost over time suggesting extrinsic and intrinsic regulation. Transcription factor-controlled regulatory circuitries govern cell identity, are sufficient to induce pluripotency and transdifferentiate cells. We investigate whether transcriptional circuitry also governs phenotypic changes within a given cell type by comparing human primary keratinocytes with intrinsically high versus low stem cell potential. Using integrated chromatin and transcriptional profiling, we implicate IRF2 as antagonistic to stemness and show that it binds and regulates active cis-regulatory elements at interferon response and antigen presentation genes. CRISPR-KD of IRF2 in keratinocytes with low stem cell potential increases self-renewal, migration and epidermis formation. These data demonstrate that transcription factor regulatory circuitries, in addition to maintaining cell identity, control plasticity within cell types and offer potential for therapeutic modulation of cell function., Epidermal homeostasis requires long term stem cell function. Here, the authors apply transcriptional circuitry analysis based on integrated epigenomic profiling of primary human keratinocytes with high and low stem cell function to identify IRF2 as a negative regulator of stemness.

Transcription factor networks shape the gene expression programs responsible for normal cell identity and pathogenic state. Using Core Regulatory Circuitry analysis (CRC), we identify PAX8 as a candidate oncogene in Renal Cell Carcinoma (RCC) cells. Validation of large-scale functional genomic screens confirms that PAX8 silencing leads to decreased proliferation of RCC cell lines. Epigenomic analyses of PAX8-dependent cistrome demonstrate that PAX8 largely occupies active enhancer elements controlling genes involved in various metabolic pathways. We selected the ferroxidase Ceruloplasmin (CP) as an exemplary gene to dissect PAX8 molecular functions. PAX8 recruits histone acetylation activity at bound enhancers looping onto the CP promoter. Importantly, CP expression correlates with sensitivity to PAX8 silencing and identifies a subset of RCC cases with poor survival. Our data identifies PAX8 as a candidate oncogene in RCC and provides a potential biomarker to monitor its activity., Transcription factors are critical regulators of cell identity. Here, the authors use computational and functional genomic approaches to show an oncogenic role of PAX8 in renal cancer. Mechanistic dissection of PAX8 functions reveal its role in activating genes associated with metabolic pathways.

AXIN2 and LGR5 mark intestinal stem cells (ISCs) that require WNT/β-Catenin signaling for constant homeostatic proliferation. In contrast, AXIN2/LGR5+ pericentral hepatocytes show low proliferation rates despite a WNT/β-Catenin activity gradient required for metabolic liver zonation. The mechanisms restricting proliferation in AXIN2+ hepatocytes and metabolic gene expression in AXIN2+ ISCs remained elusive. We now show that restricted chromatin accessibility in ISCs prevents the expression of β-Catenin-regulated metabolic enzymes, whereas fine-tuning of WNT/β-Catenin activity by ZNRF3 and RNF43 restricts proliferation in chromatin-permissive AXIN2+ hepatocytes, while preserving metabolic function. ZNRF3 deletion promotes hepatocyte proliferation, which in turn becomes limited by RNF43 upregulation. Concomitant deletion of RNF43 in ZNRF3 mutant mice results in metabolic reprogramming of periportal hepatocytes and induces clonal expansion in a subset of hepatocytes, ultimately promoting liver tumors. Together, ZNRF3 and RNF43 cooperate to safeguard liver homeostasis by spatially and temporally restricting WNT/β-Catenin activity, balancing metabolic function and hepatocyte proliferation.

Although most acute skin wounds heal rapidly, non-healing skin ulcers represent an increasing and substantial unmet medical need that urgently requires effective therapeutics. Keratinocytes resurface wounds to re-establish the epidermal barrier by transitioning to an activated, migratory state, but this ability is lost in dysfunctional chronic wounds. Small-molecule regulators of keratinocyte plasticity with the potential to reverse keratinocyte malfunction in situ could offer a novel therapeutic approach in skin wound healing. Utilizing high-throughput phenotypic screening of primary keratinocytes, we identify such small molecules, including bromodomain and extra-terminal domain (BET) protein family inhibitors (BETi). BETi induce a sustained activated, migratory state in keratinocytes in vitro, increase activation markers in human epidermis ex vivo and enhance skin wound healing in vivo. Our findings suggest potential clinical utility of BETi in promoting keratinocyte re-epithelialization of skin wounds. Importantly, this novel property of BETi is exclusively observed after transient low-dose exposure, revealing new potential for this compound class.

EKLF/KLF1 is an essential transcription factor that plays a global role in erythroid transcriptional activation. It’s own regulation is of interest, as it displays a highly restricted expression pattern, limited to erythroid cells and its progenitors. Here we use biochemical affinity purification to identify the Ddx5/p68 protein as an activator of KLF1 by virtue of its interaction with the erythroid-specific DNAse hypersensitive site upstream enhancer element (EHS1). We postulate that its range of interactions with other proteins known to interact with this element render it part of the enhanseosome complex critical for optimal expression of KLF1. These individual interactions provide quantitative contributions that, in sum, establish high level activity of the KLF1 promoter and suggest they can be selectively manipulated for clinical benefit.

The existence of specialized liver stem cell populations, including AXIN2+ pericentral hepatocytes, that safeguard homeostasis and repair has been controversial. Here, using AXIN2 lineage tracing in BAC-transgenic mice, we confirm the regenerative potential of intestinal stem cells (ISCs) but find limited roles for pericentral hepatocytes in liver parenchyma homeostasis. Liver regrowth following partial hepatectomy is enabled by proliferation of hepatocytes throughout the liver, rather than by a pericentral population. Periportal hepatocyte injury triggers local repair as well as auxiliary proliferation in all liver zones. DTA-mediated ablation of AXIN2+ pericentral hepatocytes transiently disrupts this zone, which is reestablished by conversion of pericentral vein-juxtaposed glutamine synthetase (GS)- hepatocytes into GS+ hepatocytes and by compensatory proliferation of hepatocytes across liver zones. These findings show hepatocytes throughout the liver can upregulate AXIN2 and LGR5 after injury and contribute to liver regeneration on demand, without zonal dominance by a putative pericentral stem cell population.

The post-genomic era has seen many advances in our understanding of cancer pathways, yet resistance and tumor heterogeneity necessitate multiple approaches to target even monogenic tumors. Here, we combine phenotypic screening with chemical genetics to identify pre-mRNA endonuclease Cleavage and Polyadenylation Specificity Factor 3 (CPSF3) as the target of JTE-607, a small molecule with previously unknown target. We show that CPSF3 represents a novel synthetic lethal node in a sub-set of acute myeloid leukemia (AML) and Ewing’s sarcoma cancer cell lines. Inhibition of CPSF3 by JTE-607 alters expression of known downstream effectors in AML and Ewing’s sarcoma lines, upregulates apoptosis and causes tumor-selective stasis in mouse xenografts. Mechanistically, it prevents the release of newly synthesized pre-mRNAs, resulting in read-through transcription and the formation of DNA-RNA hybrid R-loop structures. This study implicates pre-mRNA processing, and specifically CPSF3, as a druggable target providing a new avenue to therapeutic intervention in cancer.

Understanding how transcriptional regulators are themselves controlled is important in attaining a complete picture of the intracellular effects that follow signaling cascades during early development and cell-restricted differentiation. We have addressed this issue by focusing on the regulation of EKLF/KLF1, a zinc finger transcription factor that plays a necessary role in the global regulation of erythroid gene expression. Using biochemical affinity purification, we have identified the DEK oncoprotein as a critical factor that interacts with an essential upstream enhancer element of the EKLF promoter and exerts a positive effect on EKLF levels. This element also binds a core set of erythroid transcription factors, suggesting that DEK is part of a tissue-restricted enhanceosome that contains BMP4-dependent and -independent components. Together with local enrichment of properly coded histones and an open chromatin domain, optimal transcriptional activation of the EKLF locus can be established.

Central to the homeostasis of the hematopoietic system is the correct balance of progenitor cell proliferation versus lineage-committed differentiation. Accordingly, impaired differentiation and unrestricted proliferation due to somatic mutations and chromosomal translocations of hematopoietic transcription factors are directly associated with the development of hematopoietic malignancies (37). One of the defining features of the switch from the proliferative to the differentiating state is the exit from the cell cycle. Upon establishment of lineage commitment, cell cycle arrest occurs in the G1 phase as differentiation begins. Cellular proliferation is controlled through the interactions of cyclin-dependent kinases (CDKs) and their inhibitors. In contrast to Ink4 family members such as p16Ink4A and p18Ink4C that inhibit cyclin D-CDK4 complexes and act early in G1, the CIP/KIP family members p21WAF1/CIP1 and p27Kip1 regulate cell cycle transition through inhibition of cyclin E-CDK2 complexes at later stages in G1 (65) that can be p53 independent (40, 43). While p21WAF1/CIP1 expression is required in hematopoietic stem cells to maintain quiescence, its transcriptional upregulation in progenitor cells promotes differentiation (52). Erythropoiesis has served as a useful model to elucidate the mechanisms by which hematopoietic transcription factors regulate the induction of cell cycle inhibition to promote differentiation upon lineage commitment (52, 65). Gata1 (41) and erythroid Kruppel-like factor (EKLF [KLF1]) (31) are highly expressed in the megakaryocytic erythroid progenitor and play directive roles in erythroid versus megakaryocytic commitment and differentiation (reviewed in references 5 and 16). Both are required for terminal erythroid differentiation, as the ablation of either gene leads to an arrest in maturation (10, 58). Analyses of global gene expression profiles in response to either Gata1 or EKLF activity have revealed a plethora of target genes (14, 19, 59), within which a subset is of particular relevance to the balance of proliferation versus differentiation. Gata1-dependent erythroid differentiation correlates with an inhibition of mitogenic signals (32, 47) and cell cycle progression as p18Ink4C and p27Kip1 are upregulated (47). A substantive role for EKLF in this process is suggested by its direct activation of p18Ink4C (54) and E2F2 (42, 53) and by studies showing that EKLF gain of function promotes precocious terminal differentiation (16). Consistent with these molecular effects, EKLF-null fetal livers accumulate progenitor but not Ter119-expressing erythroid cells, showing that erythroid commitment and differentiation cannot proceed in the absence of EKLF expression (19, 42). In the present study, we have used inducible cellular differentiation systems (murine erythroleukemia [MEL] and G1E-ER-Gata1 cells) to further illuminate the role of EKLF in these later stages of the erythroid terminal differentiation process. Using a combination of EKLF gain of function, RNA interference (RNAi)-mediated knockdown, gel shift analyses, cell cycle profiling, luciferase reporter assays, and chromatin immunoprecipitation, we show that p21WAF1/CIP1 activation is highly dependent on cellular EKLF levels and provides another avenue by which EKLF affects terminal differentiation; intriguingly, it performs this in part by use of a novel conserved p21WAF1/CIP1 intronic regulatory region.

The hierarchical progression of stem and progenitor cells to their more-committed progeny is mediated through cell-to-cell signaling pathways and intracellular transcription factor activity. However, the mechanisms that govern the genetic networks underlying lineage fate decisions and differentiation programs remain poorly understood. Here we show how integration of Bmp4 signaling and Gata factor activity controls the progression of hematopoiesis, as exemplified by the regulation of Eklf during establishment of the erythroid lineage. Utilizing transgenic reporter assays in differentiating mouse embryonic stem cells as well as in the murine fetal liver, we demonstrate that Eklf expression is initiated prior to erythroid commitment during hematopoiesis. Applying phylogenetic footprinting and in vivo binding studies in combination with newly developed loss-of-function technology in embryoid bodies, we find that Gata2 and Smad5 cooperate to induce Eklf in a progenitor population,followed by a switch to Gata1-controlled regulation of Eklftranscription upon erythroid commitment. This stage- and lineage-dependent control of Eklf expression defines a novel role for Eklf as a regulator of lineage fate decisions during hematopoiesis.

International audience; Symmetrical dimethylation on arginine-3 of histone H4 (H4R3me2s) has been reported to occur at several repressed genes, but its specific regulation and genomic distribution remained unclear. Here, we show that the type-II protein arginine methyltransferase PRMT5 controls H4R3me2s in mouse embryonic fibroblasts (MEFs). In these differentiated cells, we find that the genome-wide pattern of H4R3me2s is highly similar to that in embryonic stem cells. In both the cell types, H4R3me2s peaks are detected predominantly at G + C-rich regions. Promoters are consistently marked by H4R3me2s, independently of transcriptional activity. Remarkably, H4R3me2s is mono-allelic at imprinting control regions (ICRs), at which it marks the same parental allele as H3K9me3, H4K20me3 and DNA methylation. These repressive chromatin modifications are regulated independently, however, since PRMT5-depletion in MEFs resulted in loss of H4R3me2s, without affecting H3K9me3, H4K20me3 or DNA methylation. Conversely, depletion of ESET (KMT1E) or SUV420H1/H2 (KMT5B/C) affected H3K9me3 and H4K20me3, respectively, without altering H4R3me2s at ICRs. Combined, our data indicate that PRMT5-mediated H4R3me2s uniquely marks the mammalian genome, mostly at G + C-rich regions, and independently from transcriptional activity or chromatin repression. Furthermore, comparative bioinformatics analyses suggest a putative role of PRMT5-mediated H4R3me2s in chromatin configuration in the nucleus.

We report on the role of as-grown and plasma treated multi-walled carbon nanotubes (MWCNTs) as cathode components in solid acid fuel cells (SAFCs). SAFCs combine a true solid state, proton conducting electrolyte, CsH2PO4 with good conductivity at mild temperatures slightly above 228 °C, with fuel flexibility and a high resistance to catalyst poisoning.1,2 The widespread application of solid acid fuel cells has been partly hindered by the necessity of the precious metal catalyst platinum in the electrodes.3,4 A general new approach in electrode catalysis bearing immense potential for electrochemical technologies is the prospect of precious metal free electrodes based on carbon nanomaterials.5,6 MWCNTs were synthesised by chemical vapour deposition using Ni nanoparticles as the growth catalyst on carbon paper current collectors. Oxygen plasma treatment was applied on selected samples with the dual effect of removing remaining amorphous carbon and increasing the concentration of oxygen functional groups on the surface. Symmetric electrochemical cells (MWCNTs|CsH2PO4|MWCNTs) were fabricated by uniaxial cold-pressing for impedance spectroscopy in a humidified oxygen environment at 240 °C. A good long term stability over a 19 day period and electrode impedances as low as 22 Ω cm2were measured, Figure 1. The oxygen plasma treated samples showed significantly lower impedances compared to as-synthesized. However, the application of a 500 mV DC bias voltage caused a dramatic drop in the electrode impedance when using as-synthesised MWCNTs. The impedance data suggests the electrochemical activity towards oxygen reduction and water splitting reaction after an initial reversible activation step. However, carbon corrosion is shown to have a non-negligible but small contribution to the measured currents. Scanning electron microscopy, Raman and XPS spectroscopy before and after the electrochemical measurements show increased oxygen concentration, increased graphitisation and slight morphological changes in all electrodes. Caption Figure 1: (a) Scanning electron micrograph of representative MWCNTs on carbon paper fiber. (b) Electrode impedance of symmetric electrochemical cells (MWCNTs|CsH2PO4|MWCNTs) measured in humidied oxygen at 240 °C as a function of oxygen concentration of MWCNTs. References [1] S. M. Haile, D. A. Boysen, C. R. I. Chisholm and R. B. Merle, Nature, 2001, 410, 910-913. [2] D. A. Boysen, T. Uda, C. R. I. Chisholm and S. M. Haile, Science, 2004, 303, 68-70. [3] A. B. Papandrew, C. R. I. Chisholm, R. A. Elgammal, M. M. Ozer and S. K. Zecevic, Chemistry of Materials, 2011, 23, 1659-1667. [4] M. W. Louie, A. Hightower and S. M. Haile, ACS Nano, 2010, 4, 2811-2821. [5] S. Zhang, Y. Y. Shao, G. P. Yin and Y. H. Lin, Journal of Materials Chemistry A, 2013, 1, 4631-4641. [6] K. N. Wood, R. O'Hayre and S. Pylypenko, Energy & Environmental Science, 2014, 7, 1212-1249 Figure 1

Micro- and nanometer sized carbon materials play an important role as components in low and intermediate temperature fuel cell electrodes. Vulcan carbon, active carbon, etc. have been used as catalyst substrates and electronic interconnect materials. As such, they reduce agglomeration of nanoparticulate catalyst and thus increase long-term stability. The catalyst utilization can be also increased significantly by improving the geometrical distribution of the catalyst. Recently, the drive towards ever smaller electrode structures has shifted attention towards carbon nanotubes (CNTs) and graphene. Their excellent electronic conductivity, extremely high specific surface area, and relatively good stability makes them suitable candidates as fuel cell electrode components. Both the catalyst distribution and utilization have been successfully improved, lowering costs and bringing the technology closer to market application. Importantly, numerous studies have recently shown functionalized CNTs and graphene, without precious metals, to be an effective catalyst for the oxygen reduction reaction. Especially nitrogen doped CNTs and graphene flakes show tremendous potential.1 Solid acid fuel cells based on CsH2PO4 as the solid state electrolyte operate at ca. 240°C and are currently performance limited at the cathode. Here, vulcan carbon has been mainly used as an electronic interconnect and catalyst support.2 Recently, carbon nanotubes have been employed, increasing the mass normalized activity of the Pt catalyst up to 61 S/mgPt.3,4 Here we combine the benefits of recent findings and present new fabrication routes based on spraydrying, metal-organic chemical vapor deposition to create highly active, nanostructured composite electrode materials, based on Pt decorated CNTs and graphene. In addition, we evaluate the suitability of surface functionalized carbon nanomaterials as precious metal free catalyst in solid acid fuel cell cathodes. Functionalization includes plasma treatment and electron beam irradiation under controlled atmospheres. Raman and XPS analysis is used before and after electrochemical measurements to evaluate structural and chemical changes. Data from impedance spectroscopy of symmetric electrochemical cells suggest effective electrochemical activity of CNTs and graphene in solid acid fuel cell cathodes. (1) Gong, K.; Du, F.; Xia, Z.; Durstock, M.; Dai, L. Science 2009, 323, 760. (2) Chisholm, C. R. I.; Boysen, D. A.; Papandrew, A. B.; Zecevic, S. K.; Cha, S.; Sasaki, K. A.; Varga, Á.; Giapis, K. P.; Haile, S. M. Interface Magazine 2009, 18, 53. (3) Varga, Á.; Pfohl, M.; Brunelli, N. A.; Schreier, M.; Giapis, K. P.; Haile, S. M. Physical chemistry chemical physics : PCCP 2013, 15, 15470. (4) Thoi, V. S.; Usiskin, R. E.; Haile, S. M. Chem. Sci. 2015, 6, 1570.

Dynamic regulation of histone methylation by methyltransferases and demethylases plays a central role in regulating the fate of embryonic stem (ES) cells. The histone H3K9 methyltransferase KMT1E, formerly known as ESET or Setdb1, is essential to embryonic development as the ablation of the Setdb1 gene results in peri-implantation lethality and prevents the propagation of ES cells. However, Setdb1-null blastocysts do not display global changes in H3K9 methylation or DNA methylation, arguing against a genome-wide defect. Here we show that conditional deletion of the Setdb1 gene in ES cells results in the upregulation of lineage differentiation markers, especially trophectoderm-specific factors, similar to effects observed upon loss of Oct3/4 expression in ES cells. We demonstrate that KMT1E deficiency in ES cells leads to a decrease in histone H3K9 methylation at and derepression of trophoblast-associated genes such as Cdx2. Furthermore, we find genes that are derepressed upon Setdb1 deletion to overlap with known targets of polycomb mediated repression, suggesting that KMT1E mediated H3K9 methylation acts in concert with polycomb controlled H3K27 methylation. Our studies thus demonstrate an essential role for KMT1E in the control of developmentally regulated gene expression programs in ES cells.

Megakaryocytes and erythroid cells are thought to derive from a common progenitor during hematopoietic differentiation. Although a number of transcriptional regulators are important for this process, they do not explain the bipotential result. We now show by gain- and loss-of-function studies that erythroid Krüppel-like factor (EKLF), a transcription factor whose role in erythroid gene regulation is well established, plays an unexpected directive role in the megakaryocyte lineage. EKLF inhibits the formation of megakaryocytes while at the same time stimulating erythroid differentiation. Quantitative examination of expression during hematopoiesis shows that, unlike genes whose presence is required for establishment of both lineages, EKLF is uniquely down-regulated in megakaryocytes after formation of the megakaryocyte-erythroid progenitor. Expression profiling and molecular analyses support these observations and suggest that megakaryocytic inhibition is achieved, at least in part, by EKLF repression of Fli-1 message levels.

Even though light is the driving force in photosynthesis, it also can be harmful to plants. The water-splitting photosystem II is the main target for this light stress, leading to inactivation of photosynthetic electron transport and photooxidative damage to its reaction center. The plant survives through an intricate repair mechanism involving proteolytic degradation and replacement of the photodamaged reaction center D1 protein. Based on experiments with isolated chloroplast thylakoid membranes and photosystem II core complexes, we report several aspects concerning the rapid turnover of the D1 protein. ( i ) The primary cleavage step is a GTP-dependent process, leading to accumulation of a 23-kDa N-terminal fragment. ( ii ) Proteolysis of the D1 protein is inhibited below basal levels by nonhydrolyzable GTP analogues and apyrase treatment, indicating the existence of endogenous GTP tightly bound to the thylakoid membrane. This possibility was corroborated by binding studies. ( iii ) The proteolysis of the 23-kDa primary degradation fragment (but not of the D1 protein) is an ATP- and zinc-dependent process. ( iv ) D1 protein degradation is a multienzyme event involving a strategic (primary) protease and a cleaning-up (secondary) protease. ( v ) The chloroplast FtsH protease is likely to be involved in the secondary degradation steps. Apart from its significance for understanding the repair of photoinhibition, the discovery of tightly bound GTP should have general implications for other regulatory reactions and signal transduction pathways associated with the photosynthetic membrane.

Light stress is an essential factor limiting plant growth by inhibition of the overall photosynthetic process. The photochemistry of photosystem II (PSII) is the primary target for photoinhibition [1]. As a consequence, the D1 protein is degraded, generating 23 kDa N-terminal and 10 kDa C-terminal primary fragments, which are subjected to secondary proteolysis to yield smaller peptides, and eventually amino acids [1–3].