Targeting mutations to specific genomic loci is invaluable for assessing in vivo the effect of these changes on the biological role of the gene in study. Here, we attempted to introduce a mutation that was previously implicated in an increased heat stability of the mesophilic cyanobacterium Synechocystis sp. PCC6803 via homologous recombination to the psbA gene of Chlamydomonas reinhardtii. For that, we established a strategy for targeted mutagenesis that was derived from the efficient genome wide homologous-recombination-based methodology that was used to target individual genes of Saccharomyces cerevisiae. While the isolated mutants did not show any benefit under elevated temperature conditions, the new strategy proved to be efficient for C. reinhardtii even in the absence of direct positive selection.
Plants experience light intensity over several orders of magnitude. High light is stressful, and plants have several protective feedback mechanisms against this stress. Here we asked how plants respond to sudden rises at low ambient light, far below stressful levels. For this, we studied the fluorescence of excited chlorophyll a of photosystem II in Arabidopsis thaliana plants in response to step increases in light level at different background illuminations. We found a response at low-medium light with characteristics of a sensory system: fold-change detection (FCD), Weber law, and exact adaptation, in which the response depends only on relative, and not absolute, light changes. We tested various FCD circuits and provide evidence for an incoherent feedforward mechanism upstream of known stress response feedback loops. These findings suggest that plant photosynthesis may have a sensory modality for low light background that responds early to small light increases, to prepare for damaging high light levels.
Various species of microalgae have recently emerged as promising host-organisms for use in biotechnology industries due to their unique properties. These include efficient conversion of sunlight into organic compounds, the ability to grow in extreme conditions and the occurrence of numerous post-translational modification pathways. However, the inability to obtain high levels of nuclear heterologous gene expression in microalgae hinders the development of the entire field. To overcome this limitation, we analyzed different sequence optimization algorithms while studying the effect of transcript sequence features on heterologous expression in the model microalga Chlamydomonas reinhardtii, whose genome consists of rare features such as a high GC content. Based on the analysis of genomic data, we created eight unique sequences coding for a synthetic ferredoxin-hydrogenase enzyme, used here as a reporter gene. Following in silico design, these synthetic genes were transformed into the C. reinhardtii nucleus, after which gene expression levels were measured. The empirical data, measured invivo show a discrepancy of up to 65-fold between the different constructs. In this work we demonstrate how the combination of computational methods and our empirical results enable us to learn about the way gene expression is encoded in the C. reinhardtii transcripts. We describe the deleterious effect on overall expression of codons encoding for splicing signals. Subsequently, our analysis shows that utilization of a frequent subset of preferred codons results in elevated transcript levels, and that mRNA folding energy in the vicinity of translation initiation significantly affects gene expression.Significance Statement Significance statementWhile microalgae have emerged as promising host organisms for use in biotechnology industries, the inability to reach high levels of heterologous gene expression in microalgae is hindering progress in this field. In this work we identify conserved sequence elements coded in natural Chlamydomonas reinhardtii genes and show that incorporation of these signals into synthetic genes dramatically affects their overall expression.
The regulatory mechanisms that use signals of low levels of reactive oxygen species (ROS) could be obscured by ROS produced under stress and thus are better investigated under homeostatic conditions. Previous studies showed that the chloroplastic atypical thioredoxin ACHT1 is oxidized by 2-Cys peroxiredoxin (2-Cys Prx) in Arabidopsis plants illuminated with growth light and in turn transmits a disulfide-based signal via yet unknown target proteins in a feedback regulation of photosynthesis. Here, we studied the role of a second chloroplastic paralog, ACHT4, in plants subjected to low light conditions. Likewise, ACHT4 reacted in planta with 2-Cys Prx, indicating that it is oxidized by a similar disulfide exchange reaction. ACHT4 further reacted uniquely with the small subunit (APS1) of ADP-glucose pyrophosphorylase (AGPase), the first committed enzyme of the starch synthesis pathway, suggesting that it transfers the disulfides it receives from 2-Cys Prx to APS1 and turns off AGPase. In accordance, ACHT4 participated in an oxidative signal that quenched AGPase activity during the diurnal transition from day to night, and also in an attenuating oxidative signal of AGPase in a dynamic response to small fluctuations in light intensity during the day. Increasing the level of expressed ACHT4 or of ACHT4DeltaC, a C terminus-deleted form that does not react with APS1, correspondingly decreased or increased the level of reduced APS1 and decreased or increased transitory starch content. These findings imply that oxidative control mechanisms act in concert with reductive signals to fine tune starch synthesis during daily homeostatic conditions.
A chloroplast protein disulfide isomerase (PDI) was previously proposed to regulate translation of the unicellular green alga Chlamydomonas reinhardtii chloroplast psbA mRNA, encoding the D1 protein, in response to light. Here we show that AtPDI6, one of 13 Arabidopsis thalianaPDI genes, also plays a role in the chloroplast. We found that AtPDI6 is targeted and localized to the chloroplast. Interestingly, AtPDI6 knockdown plants displayed higher resistance to photoinhibition than wild-type plants when exposed to a tenfold increase in light intensity. The AtPDI6 knockdown plants also displayed a higher rate of D1 synthesis under a similar light intensity. The increased resistance to photoinhibition may not be rationalized by changes in antenna or non-photochemical quenching. Thus, the increased D1 synthesis rate, which may result in a larger proportion of active D1 under light stress, may led to the decrease in photoinhibition. These results suggest that, although the D1 synthesis rates observed in wild-type plants under high light intensities are elevated, repair can potentially occur faster. The findings implicate AtPDI6 as an attenuator of D1 synthesis, modulating photoinhibition in a light-regulated manner.
The transition from dark to light involves marked changes in the redox reactions of photosynthetic electron transport and in chloroplast stromal enzyme activity even under mild light and growth conditions. Thus, it is not surprising that redox regulation is used to dynamically adjust and coordinate the stromal and thylakoid compartments. While oxidation of regulatory proteins is necessary for the regulation, the identity and the mechanism of action of the oxidizing pathway are still unresolved. Here, we studied the oxidation of a thylakoid-associated atypical thioredoxin-type protein, ACHT1, in the Arabidopsis thaliana chloroplast. We found that after a brief period of net reduction in plants illuminated with moderate light intensity, a significant oxidation reaction of ACHT1 arises and counterbalances its reduction. Interestingly, ACHT1 oxidation is driven by 2-Cys peroxiredoxin (Prx), which in turn eliminates peroxides. The ACHT1 and 2-Cys Prx reaction characteristics in plants further indicated that ACHT1 oxidation is linked with changes in the photosynthetic production of peroxides. Our findings that plants with altered redox poise of the ACHT1 and 2-Cys Prx pathway show higher nonphotochemical quenching and lower photosynthetic electron transport infer a feedback regulatory role for this pathway.
Studies of redox signaling have to take into account the highly reductive intracellular environment and the lability of redox changes in regulatory proteins. Thus, befitting methodology suited to trapping the authentic state in vivo is required. This is particularly relevant in plants where the abundance of redox signaling proteins makes it difficult to discern the cellular function of a specific protein. In this chapter, we present two complementing methods designed first to characterize the redox state in vivo of thioredoxin family proteins and second to capture their in vivo targets. These methods can be used to look at the activity and target proteins of a specific protein under different physiological conditions and in different cellular compartments. Furthermore, as demonstrated here, they can be used to compare the activity of different family members under the same conditions and thus shed light on their general and unique roles.
The reduction and the formation of regulatory disulfide bonds serve as a key signaling element in chloroplasts. Members of the thioredoxin (Trx) superfamily of oxidoreductases play a major role in these processes. We have characterized a small family of plant-specific Trxs in Arabidopsis (Arabidopsis thaliana) that are rich in cysteine and histidine residues and are typified by a variable noncanonical redox active site. We found that the redox midpoint potential of three selected family members is significantly less reducing than that of the classic Trxs. Assays of subcellular localization demonstrated that all proteins are localized to the chloroplast. Selected members showed high activity, contingent on a dithiol electron donor, toward the chloroplast 2-cysteine peroxiredoxin A and poor activity toward the chloroplast NADP-malate dehydrogenase. The expression profile of the family members suggests that they have distinct roles. The intermediate redox midpoint potential value of the atypical Trxs might imply adaptability to function in modulating the redox state of chloroplast proteins with regulatory disulfides.
Assembly and asymmetric localization of the photosensory eyespot in the biflagellate, unicellular green alga Chlamydomonas reinhardtii requires coordinated organization of photoreceptors in the plasma membrane and pigment granule/thylakoid membrane layers in the chloroplast. min1 (mini-eyed) mutant cells contain abnormally small, disorganized eyespots in which the chloroplast envelope and plasma membrane are no longer apposed. The MIN1 gene, identified here by phenotypic rescue, encodes a protein with an N-terminal C2 domain and a C-terminal LysM domain separated by a transmembrane sequence. This novel domain architecture led to the hypothesis that MIN1 is in the plasma membrane or the chloroplast envelope, where membrane association of the C2 domain promotes proper eyespot organization. Mutation of conserved C2 domain loop residues disrupted association of the MIN1 C2 domain with the chloroplast envelope in moss cells but did not abolish eyespot assembly in Chlamydomonas. In min1 null cells, channelrhodopsin-1 (ChR1) photoreceptor levels were reduced, indicating a role for MIN1 in ChR1 expression and/or stability. However, ChR1 localization was only minimally disturbed during photoautotrophic growth of min1 cells, conditions under which the pigment granule layers are disorganized. The data are consistent with the hypothesis that neither MIN1 nor proper organization of the plastidic components of the eyespot is essential for localization of ChR1.
Regulatory protein disulfide bonds serve as key signaling elements in chloroplasts in a manner that appears independent of the generally highly reducing intra-organellar conditions. This suggests that both the formation and the reduction reactions of the disulfides are specifically catalyzed. Regulatory disulfides are preferentially reduced by the dithiol reductant, thioredoxin, but their oxidant counterpart is yet to be identified. Regulatory disulfides are found in chloroplast proteins in the dark as well as under low illumination, implying that the source of the oxidative equivalents might not be limited to certain lighting conditions. Several plausible oxidants required for regulatory disulfide formation are discussed herein. By the same token, the recent finding of oxidative protein folding in chloroplasts implies an involvement of an enzymatic system for disulfide formation. Mechanisms for oxidative folding in prokaryotes and eukaryotes share a common design, comprising of a thiol oxidase and an oxidative-type thioredoxin such as protein disulfide isomerase. While the localization of protein disulfide isomerases to chloroplasts seems well established, the identity of a chloroplast thiol oxidase is yet to be determined. The understanding of disulfide formation in chloroplasts should prove key to our understanding of redox signaling in general. (c) 2008 Elsevier Ireland Ltd. All rights reserved.
Biochemical studies have identified two proteins, RB47 and RB60, that are involved in the light-regulated translation of the psbA mRNA in the chloroplast of the unicellular alga Chlamydomonas reinhardtii. RB47, a member of the eukaryotic poly(A)-binding protein family, binds directly to the 5' untranslated region of the mRNA, whereas RB60, a protein disulfide isomerase (PDI), is thought to bind to RB47 and to modulate its activity via redox and phosphorylation events. Our present V studies show that RB47 forms a single disulfide bridge that most probably involves Cys143 and Cys259. We found that RB60 reacts with high selectivity with the disulfide of RB47, suggesting that the redox states of these two redox partners are coupled. Kinetics analysis indicated that RB47 contains two fast reacting cysteines, of which at least one is sensitive to changes in pH conditions. The results support the notion that light controls the redox regulation of RB47 function via the coupling of RB47 and RB60 redox states, and suggest that light-induced changes in stromal pH might contribute to the regulation.
RB60 is an atypical protein disulfide isomerase (PDI) that functions as a member of a redox regulatory protein complex controlling translation in the chloroplast of Chlamydomonas reinhardtii, but also contains a C-terminal endoplasmic reticulum (ER) retention signal, -KDEL. Here, we show by fluorescence microscopy that RB60 resides in the chloroplast but also outside of the chloroplast colocalized with BiP, an ER marker protein. RB60 accumulates in microsomes that exhibit a typical ER magnesium-shift, and cotranslationally translocates into ER microsomes. The first 50-aa leader of R860 is sufficient for both chloroplast and ER targeting. The leader is cleaved upon translocation into the ER, whereas it remains intact after import to the chloroplast. The leader sequence also contains an acidic domain that appears necessary for the protein's association with the thylakoid membranes. Based on these and additional results, we propose that the dual localization of RB60 occurs via the two conserved transport mechanisms, to the chloroplast and to the ER, that the chloroplast RB60 most likely carries an additional function in the ER, and that its mode of transport, including the differential cleavage of its N terminus, plays an important role in its suborganellar localization and organellar-specific function.
The yeast and human mitochondrial sulfhydryl oxidases of the Erv1/Alr family have been shown to be essential for the biogenesis of mitochondria and the cytosolic iron sulfur cluster assembly. In this study we identified a likely candidate for the first mitochondrial flavin-linked sulfhydryl oxidase of the Erv1-type from a photosynthetic organism. The central core of the plant enzyme (AtErv1) exhibits all of the characteristic features of the Erv1/Alr protein family, including a redox-active YPCXXC motif, noncovalently bound FAD, and sulfhydryl oxidase activity. Transient expression of fusion proteins of AtErv1 and the green fluorescence protein in plant protoplasts showed that the plant enzyme preferentially localizes to the mitochondria. Yet AtErv1 has several unique features, such as the presence of a CXXXXC motif in its carboxyl-terminal domain and the absence of an amino-terminally localized cysteine pair common to yeast and human Erv1/Alr proteins. In addition, the dimerization of AtErv1 is not mediated by its amino terminus but by its unique CXXXXC motif. In vitro assays with purified protein and artificial substrates demonstrate a preference of AtErv1 for dithiols with a defined space between the thiol groups, suggesting a thioredoxin-like substrate.
The 5'-leader and 3'-tail of chloroplast mRNAs have been suggested to play a role in posttranscriptional regulation of expression of the message. The regulation is thought to be mediated, at least in part, by regulatory proteins that are encoded by the nuclear genome and targeted to the chloroplast where they interact with chloroplast mRNAs. Previous studies identified high affinity binding of the 5'-untranslated region (UTR) of the chloroplast psbA mRNA by Chlamydomonas reinhardtit proteins. Here we tested whether the 3'-UTR of psbA mRNA alone or linked in cis with the 5'-UTR of the mRNA affects the high affinity binding of the message in vitro. We did not detect high affinity binding that is unique to the 3'-UTR. However, we show that the cis-linked 3'-UTR increases the stability of the 5'-UTR binding complex. This effect could provide a means for translational discrimination against mRNAs that are incorrectly processed.
Plant, genomes typically contain several sequences homologous to protein disulfide isomerase (PDI). PDI was first identified as an abundant enzyme in the endoplasmic reticulum, where it catalyzes the formation, reduction, and isomerization of disulfide bonds during protein folding. PDI-like proteins have also been implicated in a variety of other functions, such as the regulation of cell adhesion, and may act as elicitors of the autoimmune response in mammals. A PDI-Iike protein (RB60) was recently shown to be imported into chloroplasts in the unicellular green alga Chlamydomonas reinhardtii and a higher plant, Pisum sativum, where it associates with thylakoid membranes. This suggests that the different PDI-like proteins in plant and animals may have diverse biological roles. To begin to elucidate the roles of PDI-like proteins, we have cloned, characterized, and generated knock-out mutants for three PDI-like genes that have high, medium, and low levels of expression, respectively, in the moss Physcomitrella patens. Phylogenetic analysis indicates that the three PDI-like proteins cluster with RB60 and four proteins from Arabidopsis thaliana. They are typified by an N-terminal domain rich in negatively charged residues. The knock-out mutants, which are the first knockouts available for PDI-Iike proteins in a multicellular organism, were found to be viable, indicating that the function of each single gene is dispensable, and suggesting that they may be functionally complementary.
Signaling by redox state regulates the transcriptional and post-transcriptional events that control gene expression. To elucidate redox signaling in vivo, the effects of the reductive intracellular redox environment on regulatory redox events must be taken into account. This article focuses on proteins that contain regulatory disulfides, considering whether regulatory proteins can be oxidized and how the redox state of regulatory proteins can be uniquely controlled to allow redox signaling via specific pathways. It is possible that the favored kinetics of the redox reactions of regulatory proteins are important for attaining specificity in redox signaling.
The 5 ' untranslated region (5 ' UTR) of the psbA mRNA (pshA encodes the PSII reaction center protein, DI) is a key site for FNA-protein interactions in the post-transcriptional regulation of gene expression. In this study, we mapped the major pshA mRNA 5 ' -terminus at -77 nt, and two minor termini clusters centered at -48 and -64 nt, upstream from the psbA translational start codon of Arabidopsis thaliana. RNA mobility shift, RNase protection and UV-crosslinking assays were used to characterize the interaction of chloroplast proteins with the RNA 5 ' UTR. RNA-protein interactions depended upon a thermolabile secondary structure and specific sequences in a 35 nt region of the 5 ' UTR, which were 80% conserved with the psbA 5 ' UTRs from five other plants. Major and minor proteins of 43- and 30-kDa, respectively, were detected by UV-crosslinking to RNA. Oxidizing conditions abolished the association of the proteins with the 5 ' UTR, while RNA-binding activity was recovered upon incubation with a reductant. Based on these findings, we hypothesize that post-transcriptional regulation of psbA gene expression in chloroplasts of vascular plants involves redox-dependent interactions between specific sequences in the 5 ' UTR and 43- and 30-kDa RNA-binding proteins.
Light controls the translation of several mRNAs in fully developed chloroplasts via at least two regulatory pathways. In the first, the light signal is transduced as a thiol-mediated signal that modulates translation in parallel to light intensity. The second light-controlled pathway, termed priming, is a prerequisite to the thiol-mediated regulatory pathway. Light regulation is rapid and requires intra-chloroplast photoreceptor(s). To delineate the signaling pathways controlling each of these regulatory events, we assayed the effect of photosynthetic inhibitors and electron donors on the translation of chloroplastic psbA mRNA. We show that the thiol-mediated signal is generated by photosystem I and transduced by vicinal dithiol-containing proteins. We also found that the priming signal probably initiates on reduction of plastoquinone. These findings suggest that translation of chloroplast psbA mRNA is controlled by both linear photosynthetic electron transport, exerted by the reduction of the ferredoxin-thioredoxin system, and the relative activities of photosystems I and II, signaled by the redox state of the plastoquinone pool. These data underscore the function of the light-capturing reactions of photosynthesis as chloroplast photoreceptors.
Translation of psbA mRNA in Chlamydomonas reinhardtii chloroplasts is regulated by a redox signal(s), RB60 is a member of a protein complex that binds with high affinity to the 5'-untranslated region of psbA mRNA. RB60 has been suggested to act as a redox-sensor subunit of the protein complex regulating translation of chloroplast psbA mRNA, Surprisingly, cloning of RB60 identified high homology to the endoplasmic reticulum-localized protein disulfide isomerase, including an endoplasmic reticulum-retention signal at its carboxyl terminus. Here we show, by in vitro import studies, that the recombinant RB60 is imported into isolated chloroplasts of C, reinhardtii and pea in a transit peptide-dependent manner. Subfractionation of C, reinhardtii chloroplasts revealed that the native RB60 is partitioned between the stroma and the thylakoids, The nature of association of native RB60, and imported recombinant RB60, with thylakoids is similar and suggests that RB60 is tightly bound to thylakoids, The targeting characteristics of RB60 and the potential implications of the association of RB60 with thylakoids are discussed.
Light has been proposed to stimulate the translation of Chlamydomonas reinhardtii chloroplast psbA mRNA by activating a protein complex associated with the 5' untranslated region of this mRNA. The protein complex contains a redox-active regulatory site responsive to thioredoxin. We identified RB60, a protein disulfide isomerase-like member of the protein complex, as carrying the redox-active regulatory site composed of vicinal dithiol. We assayed in parallel the redox state of RB60 and translation of psbA mRNA in intact chloroplasts. Light activated the specific oxidation of RB60, on the one hand, and reduced RB60, probably via the ferredoxin-thioredoxin system, on the other. Higher light intensities increased the pool of reduced RB60 and the rate of psbA mRNA translation, suggesting that a counterbalanced action of reducing and oxidizing activities modulates the translation of psbA mRNA in parallel with fluctuating light intensities. In the dark, chemical reduction of the vicinal dithiol site did not activate translation. These results suggest a mechanism by which light primes redox-regulated translation by an unknown mechanism and then the rate of translation is determined by the reduction-oxidation of a sensor protein located in a complex bound to the 5' untranslated region of the chloroplast mRNA.
Recombinant interferon alpha-C (rIFN-alpha-C, Interpharm), is a new type of alpha-interferon that has a specific activity of 1-2 x 10(9) units/mg protein. The pharmacokinetics of rIFN-alpha-C were studied in 11 patients with metastatic renal-cell carcinoma. A total of 10 million units IFN-alpha-C were injected intramuscularly and the serum level of IFN was evaluated up to 72 h post-administration. Measurable IFN concentrations appeared in the serum as early as 0.5 h, and levels peaked at 4-6 h (C(max) = 53.2 +/- 4.6 units/ml). Relatively high levels persisted for 24 h and declined thereafter with an apparent half-life of 3-4 h. The mean area under the serum-concentration curve (AUC) was 1,259 +/- 145 units h ml-1, indicating good bioavailability of the preparation from the intramuscular injection.