Haviva Eilenberg

Elevation of free proline and proline-rich protein levels by simultaneous manipulations of proline biosynthesis and degradation in plants

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.

Induced production of antifungal naphthoquinones in the pitchers of the carnivorous plant Nepenthes khasiana

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 dual effect of a ferredoxin-hydrogenase fusion protein in vivo: successful divergence of the photosynthetic electron flux towards hydrogen production and elevated oxygen tolerance

BackgroundHydrogen 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.ResultsWe 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.ConclusionsThe 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.

A possible regulatory role of squalene epoxidase in chinese hamster ovary cells

Growth of Chinese Hamster Ovary (CHO) cells in the presence of 20% lipid depleted serum (LDS) for only 2 hr results in an increase in the synthesis of [14C] sterols from [14C] mevalonate and from [14C] squalene compared with cells grown under normal growth conditions in the presence of 10% fetal calf serum (FCS). This enhanced sterol synthesis increases with time of exposure of the cells to LDS. However, exposing these cells for time periods up to 42.5 hr to a growth medium containing 20% LDS did not result in enhanced [14C] sterol synthesis from [14C] 2,3-oxidosqualene. Incubation of these cells with [14C] mevalonate resulted in the accumulation of [14C] squalene regardless of the presence of either LDS or FCS. These results suggest that squalene epoxidase is a regulatory enzyme in the cholesterol biosynthetic pathway in CHO.

Microoxic Niches within the Thylakoid Stroma of Air-Grown Chlamydomonas reinhardtii Protect [FeFe]-Hydrogenase and Support Hydrogen Production under Fully Aerobic Environment

Hydrogen production catalyzed by the extremely anaerobic enzyme [FeFe]-hydrogenase in air-grown microalgae reports on microoxic niches within the thylakoid stroma. 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, 18O2 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.

Characterization of rbcS genes in the fern Pteris vittata and their photoregulation

Abstract. 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 CO2 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 rbcS genes and two additional partial cDNAs were characterized. Fern rbcS 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 rbcS-1 and -4 show 4- to 6-fold higher transcript levels in red light while rbcS-2 and -3 contribute relatively more to the blue rbcS mRNA levels. Five of the 12 amino acids that differ between rbcS-2 and -4 affect hydrophobicity and might play a crucial role in determining the efficiency of CO2 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.

Red or Blue Light Induced Different Rubisco Small Subunits in Fern Gametophytes Affecting Carboxylation Rates

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).

Binding of ferredoxin to algal photosystem I involves a single binding site and is composed of two thermodynamically distinct events

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.