Ziyadin Ramazanov

The induction of the CO2-concentrating mechanism is correlated with the formation of the starch sheath around the pyrenoid of Chlamydomonas reinhardtii

The pyrenoid is a prominent proteinaceous structure found in the stroma of the chloroplast in unicellular eukaryotic algae, most multicellular algae, and some hornworts. The pyrenoid contains the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase and is sometimes surrounded by a carbohydrate sheath. We have observed in the unicellular green alga Chlamydomonas reinhardtii Dangeard that the pyrenoid starch sheath is formed rapidly in response to a decrease in the CO2 concentration in the environment. This formation of the starch sheath occurs coincidentally with the induction of the CO2-concentrating mechanism. Pyrenoid starch-sheath formation is partly inhibited by the presence of acetate in the growth medium under light and low-CO2 conditions. These growth conditions also partly inhibit the induction of the CO2-concentrating mechanism. When cells are grown with acetate in the dark, the CO2-concentrating mechanism is not induced and the pyrenoid starch sheath is not formed even though there is a large accumulation of starch in the chloroplast stroma. These observations indicate that pyrenoid starch-sheath formation correlates with induction of the CO2-concentrating mechanism under low-CO2 conditions. We suggest that this ultrastructural reorganization under lowCO2 conditions plays a role in the CO2-concentrating mechanism C. reinhardtii as well as in other eukaryotic algae.

The Low CO2-Inducible 36-Kilodalton Protein Is Localized to the Chloroplast Envelope of Chlamydomonas reinhardtii

The localization of the 36-kD polypeptide of Chlamydomonas reinhardtii induced by photoautotrophic growth on low CO2 concentrations (0.03% in air [v/v], low CO2-grown cells) has been investigated. This polypeptide was specifically localized to the chloroplast envelope membranes isolated from low CO2-grown cells and was not present in the chloroplast envelopes isolated from high (5% CO2 in air [v/v]) CO2-grown cells. The 36-kD protein does not show carbonic anhydrase activity and was not present on the plasma membranes isolated from low CO2-grown cells. This protein may, in part, account for the different inorganic carbon uptake characteristics observed in chloroplasts isolated from high and low CO2-grown cells of C. reinhardtii.

Effect of vanadate on photosynthesis and the ATP/ADP ratio in low-CO2-adapted Chlamydomonas reinhardtii cells

We have assessed the effect of vanadate as an inhibitor of plasma-membrane ATPase on photosynthesis and the ATP/ADP ratio in Chlamydomonas reinhardtii CW-92 (a mutant strain lacking a cell wall). This effect was compared in low-CO2-adapted cells grown in media bubbled with air containing 400 or 70 μL · L−1 CO2. Evidence is presented indicating that cells grown at 70 μL · L−1 CO2 have a higher rate of photosynthetic O2 evolution than cells grown at 400 μL · L−1 CO2, at limiting carbon concentrations. Extracellular and intracellular carbonic-anhydrase activities were, however, similar in cells grown in both of the low-carbon conditions. Vanadate inhibited, to a different extent, the HCO3−-dependent O2 evolution in cells grown at 400 and 70 μL · L−1 CO2. At 400 μM vanadate, inhibition reached 70–75% in cells grown at 400 μL · L−1 but only 50% in those grown at 70 μL · L−1 CO2. The ATP/ADP ratios determined with and without vanadate at limiting concentrations of dissolved inorganic carbon indicated that more ATP was hydrolysed in algae grown at 70 μL · L−1 than in those grown at 400 μL · L−1 CO2. We conclude that the maximal capacity to accumulate dissolved inorganic carbon is inversely related to the CO2 concentration in the medium. Activation and — or synthesis of vanadate-sensitive ATPase may be the major explanation for the higher capacity for HCO3−-dependent O2 evolution in cells grown under limited CO2 concentrations.

Regulation of the low-CO2-inducible polypeptides in Chlamydomonas reinhardtii

Polypeptides of 21, 36 and 37 kDa are induced in the unicellular green alga Chlamydomonas reinhardtii Dang. when cells are transferred from high (2%) to low (0.03%) CO2 concentrations. The synthesis of these polypeptides is correlated with the induction of the CO2-concentrating mechanism. In this work we studied the effect of the growth conditions on the synthesis of these polypeptides with the aim of clarifying whether the induction of all three of these low-CO2-inducible polypeptides requires the same environmental factor. Our results showed that induction of the 21- and 36-kDa polypeptides under low-CO2 conditions occurred only in the light, while the 37-kDa periplasmic carbonic anhydrase (EC 4.2.1.1) was induced in light, darkness, and in both synchronous and asynchronous cultures. In addition, induction of these polypeptides appeared to be determined more by the O2/CO2 ratio than by the CO2 concentrations. None of these polypeptides could be induced in either of two different mutants of C. reinhardtii, one lacking ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) and the other with inactive enzyme. Our results indicate that the 21- and 36-kDa polypeptides are regulated by a mechanism different from that controlling the 37-kDa polypeptide.

Low-temperature-induced β-carotene and fatty acid synthesis, and ultrastructural reorganization of the chloroplast in Dunaliella salina (Chlorophyta)

The effect of suboptimal growth temperature on β-carotene and fatty acid biosynthesis, and on the ultrastructural reorganization of the chloroplast, in the green unicellular alga Dunaliella salina has been studied. A decrease from the optimal temperature for growth (30°C) to suboptimal (18°C) temperatures induced β-carotene synthesis and increased lipid content in D. salina cells, thereby promoting the formation of lipid-carotene globules in the chloroplast periphery. The content of polyunsaturated fatty acids was higher in cells cultured at low temperature. Results suggest that the induction of carotenogenesis and accumulation of polyunsaturated fatty acids are mechanisms of acclimation to unfavourable environmental conditions for growth.

Effect of nitrogen supply on photosynthesis and carbonic anhydrase activity in the green seaweed Ulva rigida (Chlorophyta)

Photosynthesis rate and carbonic anhydrase (CA) activity have been studied in the green seaweed Ulva rigida C. Agardh (Chlorophyta) grown in seawater (SW) and SW supplemented with 40 μM NH4Cl (N-SW). Higher growth and maximal O2 evolution rates were observed in N-SW- than in SW-grown sea-weeds. Western blot analysis of the total homogenates probed with antibodies raised against small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) showed crossreaction with a 15 kdalton polypeptide in both SW- and N-SW-grown plants, although the band was more intense in N-SW-grown plants. Carbonic anhydrase activities in the total homogenate and in the soluble protein fraction were higher in N-SW-grown plants. Although the pellets from both plants showed a considerable CA activity, the activity of CA in the thylakoid membranes was undetectable. The low nitrogen concentration is a major environmental factor that affects the level of RuBisCO and CA, and therefore CO2 assimilation in U. rigida.

Low-CO2-inducible protein synthesis in the green alga Dunaliella tertiolecta

In the green marine alga Dunaliella tertiolecta, a CO2-concentrating mechanism is induced when the cells are grown under low-CO2 conditions (0.03% CO2). To identify proteins induced under low-CO2 conditions the cells were labelled with 35SO42−, and seven polypeptides with molecular weights of 45, 47, 49, 55, 60, 68 and 100 kDa were detected. The induction of these polypeptides was observed when cells grown in high CO2 (5% CO2 in air) were switched to low CO2, but only while the cultures were growing in light. Immunoblot analysis of total cell protein against pea chloroplastic carbonic anhydrase polyclonal antibodies showed immunoreactive 30-kDa bands in both high- and low-CO2-grown cells and an aditional 49-kDa band exclusively in low-CO2-grown cells. The 30-kDa protein was shown to be located in the chloroplast. Western blot analysis of the plasmamembrane fraction against corn plasma-membrane AT-Pase polyclonal antibodies showed 60-kDa bands in both high- and low-CO2 cell types as well as an immunoreactive 100-kDa band occurring only in low-CO2-grown cells. These results suggest that there are two distinct forms of both carbonic anhydrase and plasma-membrane ATPase, and that one form of each of them can be regulated by the CO2 concentration.

Dark induction of nitrate reductase in the halophilic alga Dunaliella salina

The effect of nitrogen starvation on the NO3-dependent induction of nitrate reductase (NR) and nitrite reductases (NIR) has been investigated in the halophilic alga Dunaliella salina. When D. salina cells previously grown in a medium with NH4+as the only nitrogen source (NH4+-cells) were transferred into NO3−medium, NR was induced in the light. In contrast, when cells previously grown in N-free medium were transferred into a medium containing NO3−, NR was induced in light or in darkness. Nitrate-dependent NR induction, in darkness, in D. salina cells previously grown at a photon flux density of 500 umol · m−2 s−1 was observed after 4 h preculture in N-free medium, whilst in cells grown at 100 umol · m−2 s−1 NR induction was observed after 7–8 h. An inhibitor of mRNA synthesis (6-methylpurine) did not inhibit NO3−-induced NR synthesis when the cells, previously grown in NH4+medium, were transferred into NO3−medium (at time 0 h) after 4-h-N starvation. However, when 6-methylpurine was added simultaneously with the transfer of the cells from NH4+to NO3−medium (at time 0 h), NO3−induced NR synthesis was completely inhibited. The activity of NIR decreased in N-starved cells and the addition of NO3−to those cells greatly stimulated NIR activity in the light. The ability to induce NR in darkness was observed when glutamine synthetase activity reached its maximal level during N starvation. Although cells grown in NO3−medium exhibited high NR activity, only 0.33% of the total NR was found in intact chloroplasts. We suggest that the ability, to induce NR in darkness is dependent on the level of N starvation, and that NR in D. salina is located in the cytosol. Light seems to play an indirect regulatory role on NO3−uptake and NR induction due to the expression of NR and NO3−-transporter mRNAs.

Effect of external CO2 concentrations on protein synthesis in the green algae Scenedesmus obliquus (Turp.) Kütz and Chlorella vulgaris (Kosikov)

Unicellular algae grown under low-CO2 conditions (0.03% CO2) have developed a means of concentrating CO2 at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase. Cells with the CO2-concentrating mechanism (CCM) acquire the ability to accumulate inorganic carbon to a level higher than that obtained by simple diffusion. To identify proteins which are involved in the organization of the CCM, cells of Scenedesumus obliquus and Chlorella vulgaris grown in high CO2 (5% CO2 in air) were transferred to low-CO2 (0.03%) conditions in the presence of 35SOinf4sup2−and, thereafter, polypeptides labeled with 35S were detected. Under low-CO2 conditions the inducton of 36-, 39-, 94- and 110- to 116kDa polypeptides were particularly observed in S. obliquus and 16-, 19-, 27-, 36-, 38- and 45-kDa polypeptides were induced in C. vulgaris. Western blots with antibodies raised against 37-kDa subunits of the periplasmic carbonic anhydrase (CA) of Chlamydomonas reinhardtii showed immunoreactive bands with the 39-kDa polypeptide in the whole-cell homogenates from S. obliquus and with 36 and 38-kDa polypeptides in both high- and low-CO2grown cells of C. vulgaris. Anti-pea-chloroplast CA antibodies cross-reacted with a single polypeptide of 30 kDa in the whole-cell homogenates but not with thylakoid membranes. The CA activity was associated with soluble and membrane-bound fractions, except thylakoid membranes.