Plant Science

Brown algae belong to a phylogenetic lineage distantly related to green plants and animals, and are found predominantly, but not exclusively, in the intertidal zone, a harsh and frequently changing environment. Because of their unique evolutionary history and of their habitat, brown algae feature several peculiarities in their metabolism. One of these is the mannitol cycle, which plays a central role in their physiology, as mannitol acts as carbon storage, osmoprotectant, and antioxidant. This polyol is derived directly from the photoassimilate fructose-6-phosphate via the action of a mannitol-1-phosphate dehydrogenase (M1PDH, EC 1.1.1.17) and a mannitol-1-phosphatase (M1Pase, EC 3.1.3.22). This protocol describes the biochemical characterization of the recombinant catalytic domain of one of the three M1PDHs identified in Ectocarpus sp. This recombinant catalytic domain, named hereafter M1PDHcat, catalyzes the reversible conversion of fructose-6-phosphate (F6P) to mannitol-1-phosphate (M1P) using NAD(H) as a cofactor. M1PDHcat activity was assayed in both directions i.e., F6P reduction and M1P oxidation (Figure 1).


Figure 1. Reversible reaction of mannitol-1-phosphate dehydrogenase

Brown algae belong to a phylogenetic lineage distantly related to green plants and animals, and are found predominantly, but not exclusively, in the intertidal zone, a harsh and frequently changing environment. Because of their unique evolutionary history and of their habitat, brown algae feature several peculiarities in their metabolism. One of these is the mannitol cycle, which plays a central role in their physiology, as mannitol acts as carbon storage, osmoprotectant, and antioxidant. This polyol is derived directly from the photoassimilate fructose-6-phosphate via the action of a mannitol-1-phosphate dehydrogenase (M1PDH, EC 1.1.1.17) and a mannitol-1-phosphatase (M1Pase, EC 3.1.3.22). This protocol describes the biochemical characterization of a recombinant M1Pase of Ectocarpus sp. The M1Pase enzyme catalyzes the conversion of mannitol-1-phosphate to mannitol (Figure 1).


Figure 1. Reaction catalyzed by a mannitol-1-phosphatase

Chlamydomonas reinhardtii is a model organism for chloroplast studies. Besides other convenient features, the feasibility of chloroplast genome transformation distinguishes this unicellular alga as ideal for the manipulation of chloroplastic gene expression aiming biotechnological goals, such as improved biofuel and biomass production. Ribulose 1, 5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39, Rubisco) is the photosynthetic carbon-fixing enzyme which is considered crucial for biomass accumulation in algal cultures. Purification of wild type and site-directed mutants of Rubisco in C. reinhardtii is usually performed to study its catalytic properties and assess the carbon-fixing potential of the strains. In this protocol Rubisco is extracted through sonication of cell pellets, and purified by ammonium sulfate precipitation, sucrose gradient centrifugation (Goldwaithe and Bogorad, 1975) and anion exchange chromatography.

The performance of the carbon-fixing enzyme, ribulose 1, 5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39, Rubisco), controls biomass accumulation in green plants, algae and most autotrophic bacteria. In particular, the carboxylase activity of Rubisco incorporates carbon from CO₂ to ribulose 1, 5-bisphosphate (RuBP) producing two molecules of 3-phosphoglycerate. Here a detailed protocol is given for the assay of the carboxylase activity of Rubisco from Chlamydomonas reinhardtii, a model organism for chloroplast studies and a fitting host for biotechnologically oriented genetic manipulation of the enzyme. Rubisco has to be pre-incubated with Mg²⁺ ions and bicarbonate to induce the catalytically competent active center (Laing and Christeller, 1976). Once Rubisco is activated, the assay of its carboxylase activity described here is based on the fixation of ¹⁴C-carbon dioxide/bicarbonate into acid-resistant radioactivity (Lorimer et al., 1977). Although a spectrophotometric assay is also available (Lilley and Walker, 1974), the method based on fixation of a radioactive substrate is irreplaceable when processing a large number of samples, and it is still the technique most often used for the determination of Rubisco activity.

Here we describe the activity measurements of heterologous expressed pyruvate:ferredoxin oxidoreductase (Noth et al., 2013) (Noth et al.,2013) from Chlamydomonas reinhardtii. This enzyme catalyzes the reversible reaction (I) from pyruvate to acetyl CoA and CO₂ generating low potential electrons which are in vivo transferred to ferredoxin.



In this assay we use methyl viologen as artificial electron acceptor which turns into dark violet (ε₆₀₄ = 13.6 mM^-1 cm^-1) (Mayhew, 1978) in its reduced state (Figure 1).

This protocol describes the heterologous expression and purification of proteins related to anoxic hydrogen production of Chlamydomonas reinhardtii (Noth et al., 2013). For this, the bacterial expression hosts Escherichia coli BL21 (DE3) ΔiscR (Akhtar MK et al., 2008) and Clostridium acetobutylicum ATCC 824 are used, which are grown either aerobic or anaerobic with glucose. Two standard chromatographic methods for purification were applied using His- and StrepII-tagged proteins (Figure 1). All procedures have been performed in an anaerobic tent to avoid the access of oxygen.

Western blotting allows for the specific detection of proteins by an antibody of interest. This protocol utilizes isolation of total proteins protocol for Chlorella vulgaris prior to gel electrophoresis. After electrophoresis, the selected antibodies are used to detect and quantify levels of chloroplast HSP70B.

The method of immunoelectron microscopy is intended for localization of proteins inside the cells of Chlamydomonas reinhardtii or other microalgae and cyanobacteria. This protocol was used to study localization of carbonic anhydrase Cah3 with antibodies raised in rabbit, though it can be used to localize any other abundant protein. Primary rabbit antibodies are recommended because they react quickly and specifically with proteins of C. reinhardtii. If primary antibodies other than rabbit are used, the blocking procedure and time of incubation with primary and secondary antibodies should be adjusted.