The C-type hybrid-proline-rich protein (HyPRP) AtCWLP and its homolog AtPRP940 are referred as cell wall (CW)-plasma-membrane (PM) linker proteins, but little is known about their functions. Here we show that N-terminal proline-rich domains of CWLP and PRP940, containing glycosylated hydroxyproline residues, contact the CW, while their C-terminal 8CM domains function as PM-scaffolds. Both proteins are detected in PM nanodomains (PM-ND) and show co-localization and co-immunoprecipitation with aquaporins PIP2;1 and PIP2;7. Inhibition of actin polymerization by latrunculin B promotes CWLP-endosome appearance, while blocking the actomyosin-based transport by a truncated form of myosin XI-K relaxes lateral boundaries of CWLP-PIP2;1 PD-NDs. Mass spectrometry data indicate that CWLP co-purifies with dynamins implicated in fission of endocytic PD-ND invaginations. Lack of co-localization and co-immunoprecipitation with aquaporin-binding flotillin (FLOT2) indicates that CWLP and PRP940 mark a new distinct type of PM-ND. Yeast two-hybrid and co-immunoprecipitation assays demonstrate that CWLP and PRP940 interact with multiple aquaporins and several protein phosphatase PP2A-B’’ regulatory subunits. By preventing irreversible separation of CW and PM, and likely assisting PP2A-mediated dephosphorylation of aquaporins and closure of their water channels, overexpression of CWLP confers tolerance to plasmolysis, dehydration and freezing in Arabidopsis and to water shortage in potato plants. Summary Statement Arabidopsis Hybrid-Proline-Rich Proteins CWLP and PRP940 occur in association with dynamins, recruit PP2A protein phosphatases to aquaporin water channels in plasma-membrane (PM) nanodomains and elevate tolerance to cellular dehydration.

While the prospect of producing hydrogen from photosynthetic microalgae has long been described as one of the promising directions towards achieving a renewable fuel source, current endeavors towards this goal face serious limitations including the inefficient electron supply to hydrogenase and the enzyme's sensitivity to molecular oxygen. In this work we express our ferredoxin‑hydrogenase (fd-hyd) fusion enzyme in a hydrogenase knockout Chlamydomonas reinhardtii mutant and compare its hydrogen production traits to those of wild-type strains. We found that the active enzyme abundance in both systems is in linear correlation with photosynthetic hydrogen production, thus establishing that protein abundance is an additional important bottleneck in the process of hydrogen photo-production. We report here the isolation of two clones with high expression of fd-hyd; OP68 and R2D2, created by either nuclear or chloroplast transformation, respectively. The study of these clones shows that fd-hyd's roughly 4.5-fold higher H2 production efficiency, compared to the native hydrogenase, is maintained in high expression regimes. By comparing a strain's active enzyme pool to its total protein amount, we observed that the engineered clones harbor a large non-mature enzyme pool, indicating that natural maturation of the fd-hyd saturates at a lower level than the native hydrogenase. Subsequently, we measured mRNA levels by quantitative PCR and observed that while the two clones express roughly the same amount of protein, R2D2 has considerably more mRNA. Thus improving transcription rates in nuclear transformants or translation efficiency in chloroplast transformants are both potential routes towards further increasing protein abundance. Finally, we show that these clones are able to continuously produce H2 in sealed bioreactors for five days, significantly outcompeting their parental wild-type strain.

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 in vivo 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.

Despite the impressive progress made in recent years in understanding the early steps in charge separation within the photosynthetic reaction centers, our knowledge of how ferredoxin (Fd) interacts with the acceptor side of photosystem I (PSI) is not as well developed. Fd accepts electrons after transiently docking to a binding site on the acceptor side of PSI. However, the exact location, as well as the stoichiometry, of this binding have been a matter of debate for more than two decades. Here, using Isothermal Titration Calorimetry (ITC) and purified components from wild type and mutant strains of the green algae Chlamydomonas reinhardtii we show that PSI has a single binding site for Fd, and that the association consists of two distinct binding events, each with a specific association constant.

BACKGROUND:Hydrogen photo-production in green algae, catalyzed by the enzyme [FeFe]-hydrogenase (HydA), is considered a promising source of renewable clean energy. Yet, a significant increase in hydrogen production efficiency is necessary for industrial scale-up. We have previously shown that a major challenge to be resolved is the inferior competitiveness of HydA with NADPH production, catalyzed by ferredoxin-NADP(+)-reductase (FNR). In this work, we explored the in vivo hydrogen production efficiency of Fd-HydA, where the electron donor ferredoxin (Fd) is fused to HydA and expressed in the model organism Chlamydomonas reinhardtii.RESULTS:We show that once the Fd-HydA fusion gene is expressed in micro-algal cells of C. reinhardtii, the fusion enzyme is able to intercept photosynthetic electrons and use them for efficient hydrogen production, thus supporting the previous observations made in vitro. We found that Fd-HydA has a ~4.5-fold greater photosynthetic hydrogen production rate standardized for hydrogenase amount (PHPRH) than that of the native HydA in vivo. Furthermore, we provide evidence suggesting that the fusion protein is more resistant to oxygen than the native HydA.CONCLUSIONS:The in vivo photosynthetic activity of the Fd-HydA enzyme surpasses that of the native HydA and shows higher oxygen tolerance. Therefore, our results provide a solid platform for further engineering efforts towards efficient hydrogen production in microalgae through the expression of synthetic enzymes.

Photosynthetic hydrogen production in the microalga Chlamydomonas reinhardtii is catalyzed by two [FeFe]-hydrogenase isoforms, HydA1 and HydA2, both irreversibly inactivated upon a few seconds exposure to atmospheric oxygen. Until recently, it was thought that hydrogenase is not active in air-grown microalgal cells. In contrast, we show that the entire pool of cellular [FeFe]-hydrogenase remains active in air-grown cells due to efficient scavenging of oxygen. Using membrane inlet mass spectrometry, (18)O2 isotope, and various inhibitors, we were able to dissect the various oxygen uptake mechanisms. We found that both chlororespiration, catalyzed by plastid terminal oxidase, and Mehler reactions, catalyzed by photosystem I and Flavodiiron proteins, significantly contribute to oxygen uptake rate. This rate is considerably enhanced with increasing light, thus forming local anaerobic niches at the proximity of the stromal face of the thylakoid membrane. Furthermore, we found that in transition to high light, the hydrogen production rate is significantly enhanced for a short duration (100 s), thus indicating that [FeFe]-hydrogenase functions as an immediate sink for surplus electrons in aerobic as well as in anaerobic environments. In summary, we show that an anaerobic locality in the chloroplast preserves [FeFe]-hydrogenase activity and supports continuous hydrogen production in air-grown microalgal cells.

Proline-rich proteins (PRP) are cell wall and plasma membrane-anchored factors involved in cell wall maintenance and its stress-induced fortification. Here we compare the synthesis of P5C as the proline (Pro) precursor in the cytosol and chloroplast by an introduced alien system and evaluate correlation between PRP synthesis and free Pro accumulation in plants. We developed a Pro over-producing system by generating transgenic tobacco plants overexpressing E. coli P5C biosynthetic enzymes; Pro-indifferent gamma-glutamyl kinase 74 (GK74) and gamma-glutamylphosphate reductase (GPR), as well as antisensing proline dehydrogenase (ProDH) transcription. GK74 and GPR enzymes were targeted either to the cytosol or plastids. Molecular analyses indicated that the two bacterial enzymes are efficiently expressed in plant cells, correctly targeted to the cytosol or chloroplasts, and processed to active enzymatic complexes in the two compartments. Maximal Pro increase is obtained when GK74 and GPR are active in chloroplasts, and ProDH mRNA level is reduced by anti-sense silencing, resulting in more than 50-fold higher Pro content compared to that of wild type tobacco plants. The Pro over-producing system efficiently works in tobacco and Arabidopsis. The elevation of Pro levels promotes accumulation of ectopically expressed Cell Wall Linker Protein (AtCWLP), a membrane protein with an external Pro-rich domain. These results suggest that the Pro-generating system can support endogenous or alien PRP production in plants.

Nepenthes spp. are carnivorous plants that have developed insect capturing traps, evolved by specific modification of the leaf tips, and are able to utilize insect degradation products as nutritional precursors. A chitin-induced antifungal ability, based on the production and secretion to the trap liquid of droserone and 5-O-methyldroserone, is described here. Such specific secretion uniquely occurred when chitin injection was used as the eliciting agent and probably reflects a certain kind of defence mechanism that has been evolved for protecting the carnivory-based provision of nutritional precursors. The pitcher liquid containing droserone and 5-O-methyldroserone at 3:1 or 4:1 molar ratio, as well as the purified naphthoquinones, exerted an antifungal effect on a wide range of plant and human fungal pathogens. When tested against Candida and Aspergillus spp., the concentrations required for achieving inhibitory and fungicidal effects were significantly lower than those causing cytotoxicity in cells of the human embryonic kidney cell line, 293T. These naturally secreted 1,4-naphthoquinone derivatives, that are assumed to act via semiquinone enhancement of free radical production, may offer a new lead to develop alternative antifungal drugs with reduced selectable pressure for potentially evolved resistance.

The Cry1C group of Bacillus thuringiensis delta-endotoxins contains 10 highly homologous members of the Cry1Ca toxin sub-group and additional three members of the Cry1Cb sub-group that differ in domain III sequence. The Cry1Ca bioinsecticidal spectrum encompasses lepidopteran insects that are completely or partially tolerant to the current commercially used Bt crops. Plant-expressed Cry1Ca proteins successfully control specific lepidopteran pests, however, Bt crops expressing Cry1Ca have not been commercialized. This review summarizes the accumulating data in Cry1C research. Multiple sequence alignments of closely related Cry1Ca homologues show that the N-terminal half of the protein, comprising the “active toxin”, is less conserved than the C-terminal part, which is involved in the assembly of the toxin-containing crystalline structure during the bacterial sporulation stage. Bioinformatics analyses predict high evolutionary diversity of amino acid residues in the regions identified as toxin–membrane interaction sites. All the three structural domains of Cry1Ca “active toxin” interact in vitro with membrane vesicles produced from epithelial cells of the larval gut. This multi-site-interaction depends on the normal assembly of membrane lipid raft domains, which is disturbed during cell division, when transient Cry1Ca insensitivity is observed. Cry1Ca interaction with the gut epithelial cells involves specific aminopeptidase-N receptors that differ from those described for other Cry1 toxins. The involvement of other membrane components in the interaction remains to be studied. Cry1A-resistant insect pests, such as the Cry1Ac-tolerant mutants of diamondback moth, are sensitive to Cry1Ca, due to the involvement of different genetic loci. Hence pyramiding expression of Cry1Ca and other Cry toxins can broaden the bioinsecticidal spectrum of Bt crops and simultaneously delay the evolution of Cry-resistant insect populations.

The genus Nepenthes represents carnivorous plants with pitcher traps capable of efficient prey capture and digestion. The possible involvement of plant chitinases in this process was studied in Nepenthes khasiana. Two different types of endochitinases were identified in the liquid of closed traps exhibiting substrate specificity for either long chitin polymers or N-acetylglucosamine (GlcNAc) oligomers. Injection of chitin into such closed sterile pitchers induced the appearance of additional endochitinase isoenzymes, with substrate specificity only for long chitin polymers. No significant exochitinase (N-acetyl-beta-glucosaminidase) or chitobiosidase activity could be detected in the non-induced or induced trap liquid. Four genes representing two subgroups of basic chitinases, denoted as Nkchit1b and Nkchit2b, were isolated from the secretory region of N. khasiana pitchers. The main differences between the two subgroups are the presence of a proline-rich hinge region only in NkCHIT1b and a C-terminal putative vacuole targeting extension only in NkCHIT2b, indicating different compartmentalization of the two enzymes. Reverse transcription-polymerase chain reaction (RT-PCR) evaluation of mRNA levels showed that the Nkchit2b genes are constitutively expressed in the secretory cells while transcription of Nkchit1b genes is induced by chitin injection. These results show for the first time the involvement of genes encoding chitinases in prey-trap interaction and their differential expression and activity during prey trapping.

Cis-acting regulatory elements of the wheat acetyl-CoA carboxylase (ACC) gene family were identified by comparing the promoter activity of 5' end gene fragments fused to a reporter gene in two transient expression systems: wheat protoplasts and epidermal cells of mature embryos. Expression of the plastid and the cytosolic ACC genes is each driven by two nested promoters responsible for the synthesis of two transcript types. The internal promoter is located in an intron removed from transcripts originating at the first promoter. These complex promoters, which are different for the cytosolic and plastid ACC genes, control tissue-specific expression of the enzymatic activity supplying cytosolic, plastid, and mitochondrial pools of malonyl-CoA. The activity of one such complex promoter, driving expression of one of the cytosolic ACC genes, was studied throughout development of transgenic wheat plants carrying a full-length promoter-reporter gene fusion. High activity of the promoter was detected in the coleoptile, in the upper sheath section of the leaf, on the top surface of the ovary, in some sections of the main veins in the lemma and glume, and in abaxial epidermis hair cells of the lemma, glume, and rachis. The findings are consistent with the developmental and environmental requirements for very-long-chain fatty acids and flavonoids, whose synthesis begins with the ACC reaction in the cytosol of these specific cell types.

Rubisco (EC 4.1.1.39) catalyzes photosynthetic COz fixation and has a key role in plant growth and productivity. The holoenzyme is composed of eight chloroplast-encoded LSUs and eight nuclear-encoded SSUs (Andrews and Lorimer, 1987). In plants the LSU gene, rbcL, is present in a single copy per chloroplast genome, whereas the SSUs are encoded by a nuclear multigene family (rbcS) (Dean et al., 1989). We have previously characterized a unique system in which two distinct types of Rubisco, differing in carboxylation activity, appear in fem gametophytes when exposed to either red or blue light (Eilenberg et al., 1991). In addition to having different V,,, values for carboxylation, these two enzymes appear to consist of SSUs that differ in antigenicity. We are currently verifying the molecular differences between the two types of SSUs that could affect enzyme activity. We report here the isolation and analysis of an rbcS cDNA clone isolated from fem gametophytes grown under red light. The fem gametophyte cDNA library was screened by in situ hybridization using as probe a homologous cDNA clone corresponding to amino acids 10 to 117 of the mature SSU. Two independent rbcS cDNA clones, which differ significantly at their 3' noncoding region, were isolated. The longer clone, designated FSRZ, was sequenced, and its amino acid sequence was deduced (Table I). The clone contains a single open reading frame of 162 amino acids consisting of the mature SSU protein of 122 amino acids preceded by a partial transit peptide of 40 amino acids. Comparison of the fem mature SSU to severa1 higher plant and cyanobacterial SSUs reveals that the fern polypeptide has 61 and 59% identity with Pinus thunbergii (Yamamoto et al., 1988) and Solanum tuberosum (Wolter et al., 1988), respectively, and has 45 and 44% identity with Synechococcus sp. PCC 6301 (Shinozaki and Sugiura, 1985) and Anabaena sp. PCC 7120 (Nierzwicki-Bauer et al., 1984), respectively. Both the fem mature SSU and the pine SSU have an identical substitution of Thr6' to Va16' within the highly conserved region. Furthermore, there is an amino acid deletion at position 46, similar to that found in monocots such as wheat (Broglie et al., 1983) and Lemna gibba (Stiekema et al., 1983).

. The fern Pteris vittata L. belongs to the evolutionarily highest group of vascular plants that still maintains a free-living gametophytic stage. The two-dimensional gametophytes developed under blue light exhibit higher CO 2 fixation efficiency and different ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit (SSU) composition when compared to the red-induced filamentous gametophytes (H. Eilenberg et al., 1991, Plant Physiol 95: 298–304). To unravel the correlation between SSU structural differences and light regulation, two rbc S genes and two additional partial cDNAs were characterized. Fern rbc S genes resemble those of higher plants in their promoter light-regulatory elements (LREs) and intron number and positions. However, the primary structure of the fern mature SSUs displays much higher divergency within the gene family. This structural variability was correlated with differential steady-state mRNA levels under red and blue light. Genes rbc S-1 and -4 show 4- to 6-fold higher transcript levels in red light while rbc S-2 and -3 contribute relatively more to the blue rbc S mRNA levels. Five of the 12 amino acids that differ between rbc S-2 and -4 affect hydrophobicity and might play a crucial role in determining the efficiency of CO 2 fixation. Dendrograms of Rubisco SSUs and LSUs indicate early divergence of the fern types from the rest of the vascular plants. However, prominent higher-plant-like Rubisco features such as high carboxylation efficiency, promoter LREs and exon-intron structure, suggest that molecular specialization of the higher-plant Rubisco prototype occurred earlier than the emergence of ferns.

Rubisco (EC 4.1.1.39) catalyzes photosynthetic COz fixation and has a key role in plant growth and productivity. The holoenzyme is composed of eight chloroplast-encoded LSUs and eight nuclear-encoded SSUs (Andrews and Lorimer, 1987). In plants the LSU gene, rbcL, is present in a single copy per chloroplast genome, whereas the SSUs are encoded by a nuclear multigene family (rbcS) (Dean et al., 1989). We have previously characterized a unique system in which two distinct types of Rubisco, differing in carboxylation activity, appear in fem gametophytes when exposed to either red or blue light (Eilenberg et al., 1991). In addition to having different V,,, values for carboxylation, these two enzymes appear to consist of SSUs that differ in antigenicity. We are currently verifying the molecular differences between the two types of SSUs that could affect enzyme activity. We report here the isolation and analysis of an rbcS cDNA clone isolated from fem gametophytes grown under red light. The fem gametophyte cDNA library was screened by in situ hybridization using as probe a homologous cDNA clone corresponding to amino acids 10 to 117 of the mature SSU. Two independent rbcS cDNA clones, which differ significantly at their 3' noncoding region, were isolated. The longer clone, designated FSRZ, was sequenced, and its amino acid sequence was deduced (Table I). The clone contains a single open reading frame of 162 amino acids consisting of the mature SSU protein of 122 amino acids preceded by a partial transit peptide of 40 amino acids. Comparison of the fem mature SSU to severa1 higher plant and cyanobacterial SSUs reveals that the fern polypeptide has 61 and 59% identity with Pinus thunbergii (Yamamoto et al., 1988) and Solanum tuberosum (Wolter et al., 1988), respectively, and has 45 and 44% identity with Synechococcus sp. PCC 6301 (Shinozaki and Sugiura, 1985) and Anabaena sp. PCC 7120 (Nierzwicki-Bauer et al., 1984), respectively. Both the fem mature SSU and the pine SSU have an identical substitution of Thr6' to Va16' within the highly conserved region. Furthermore, there is an amino acid deletion at position 46, similar to that found in monocots such as wheat (Broglie et al., 1983) and Lemna gibba (Stiekema et al., 1983).

Two distinct ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit (SSU) populations were observed in Pteris vittata gametophytes grown under different illumination conditions. Exposure of the fern gametophytes to continuous red light (R) resulted in Rubisco SSUs that were not recognized by polyclonal antibodies raised against SSUs from spinach. Unlike the R-induced SSUs, blue light (B) induced SSUs were well recognized. This difference in SSU composition also reflected in Rubisco activity. In vitro, B-induced Rubisco exhibits a significantly higher carboxylation activity as compared to the R-induced Rubisco. Approximately a two- to threefold increase in the V(max) value of the B-induced carboxylase as compared to the R-induced one was measured. It thus seems very likely that certain domains in the SSU molecule affect enzyme activity.

Rubisco is a bifunctional enzyme, catalyzing carboxylation and oxygenation of ribulose 1,5-bisphosphate (RuBP) at the same catalytic site. It is a chloroplast stromal enzyme comprising of 8 chloroplast encoded large submits (LSU), and 8 nuclear encoded small subunits (SSU) (1,2). The LSUs contain binding sites for the three substrates: CO2, O2 and RuBP and other features required for assembly, activation and catalytic activity (3,4). The LSU chloroplast gene, rbcL, is present in a single copy per genome and the amino acid sequences around the active sites are conserved in most photosynthetic organisms (5).

The activity of rat liver microsomal squalene epoxidase is inhibited effectively by digitonin. Concentrations of 0.8 to 1.2 mg/ml of digitonin cause total inhibition of microsomal (0.75 mg protein/ml) squalene epoxidase either in microsomes that were pretreated with digitonin and subsequently washed and subjected to epoxidase assay or when digitonin was added directly to the assay. The inhibition of squalene epoxidase by digitonin is concentration-dependent and takes place rapidly within 5 min of exposure of the microsomes to digitonin. Octylglucoside, dimethylsulfoxide, CHAPS, as well as cholesterol or total microsomal lipid extract were ineffective in restoring the digitonin-inhibited squalene epoxidase activity. Epoxidase activity in digitonin-treated microsomes was fully restored by Triton X-100. The reactivation by Triton X-100 displays a concentration optimum with maximal reactivation of the epoxidase (0.7 mg protein/ml) occurring at 0.2% Triton X-100. Microsomal 2,3-oxidosqualene-lanosterol cyclase is also inhibited by digitonin. Higher concentrations of digitonin are required to obtain full inhibition of the cyclase activity and only 40% inhibition of cyclase activity is observed at 1 mg/ml of digitonin. Solubilized (subunit size 55 to 66 kDa) and microsomal (subunit size 97 kDa) 3-hydroxy-3-methylglutaryl CoA reductase are totally unaffected by the same concentration of digitonin. Squalene synthetase, another microsomal enzyme in the biosynthetic pathway of cholesterol, is activated by digitonin. A 2.2-fold activation of squalene synthetase is observed at 0.8 mg/ml of digitonin. The results agree with a model in which squalene, and to a lesser degree 2,3-oxidosqualene, are segregated by digitonin into separate intramembranal pools.(ABSTRACT TRUNCATED AT 250 WORDS)

Squalene epoxidase activity has been studied in cell-free preparations of Chinese hamster ovary (CHO) cells and rat liver. In contrast to rat liver microsomal squalene epoxidase, the enzyme of CHO cells is only slightly activated by the autologous cytosolic fraction, whereas phosphatidylglycerol or rat liver cytosolic preparations are potent stimulators of this enzyme. Triton X-100, a known stimulator of the hepatic squalene epoxidase, has no activating effect on the enzyme of CHO cells. The squalene epoxidase activity of both rat liver and CHO cells varies significantly according to the lipid content of the growth medium or diet. The changes in enzyme activity are shown to be entirely due to altered microsomal enzyme per se and not to changes in the activating properties of the soluble fraction. These results further support the proposed regulatory role of squalene epoxidase in cholesterogenesis.