Basic cellular processes such as electron transport in photosynthesis and respiration require the precise control of iron homeostasis. To mobilize iron, plants have evolved at least two different strategies. The nonproteinogenous amino acid nicotianamine which is synthesized from three molecules of S-adenosyl-l-methionine, is an essential component of both pathways. This compound is missing in the tomato mutant chloronerva, which exhibits severe defects in the regulation of iron metabolism. We report the purification and partial characterization of the nicotianamine synthase from barley roots as well as the cloning of two corresponding gene sequences. The function of the gene sequence has been verified by overexpression in Escherichia coli. Further confirmation comes from reduction of the nicotianamine content and the exhibition of a chloronerva-like phenotype due to the expression of heterologous antisense constructs in transgenic tobacco plants. The native enzyme with an apparent M r of < 105 000 probably represents a trimer of S-adenosyl-l-methionine-binding subunits. A comparison with the recently cloned chloronerva gene of tomato reveals striking sequence homology, providing support for the suggestion that the destruction of the nicotianamine synthase encoding gene is the molecular basis of the tomato mutation.Keywords: antisense constructs; chloronerva mutation; gene isolation; Hordeum vulgare; iron metabolism.Iron is essential for fundamental cellular processes such as electron transfer in photosynthesis, respiration, nitrogen fixation as well as DNA synthesis [1]. Excessive accumulation causes severe damage to cellular components due to the formation of highly reactive hydroxyl radicals by the Fenton reaction [2]. Thus, the precise control of iron homeostasis is a basic prerequisite for cellular function. According to WHO data the health of more than three billion people worldwide is affected by iron deficient diet. Crop plants with a higher iron content, for example in the endosperm of cereals, could contribute to the improvement of this situation. In soil iron is mainly found as stable Fe(III) compounds with low solubility at neutral pH [1,3]. Therefore, plants have evolved special mechanisms of iron acquisition, classified into two strategies [4]. Strategy I plants, including dicots and nongraminaceous monocots, facilitate iron uptake mainly by increased acidification of the rhizosphere due to enhanced proton extrusion and the reduction of Fe(III) to Fe(II) by an inducible plasma membrane-bound reductase. In contrast, graminaceous monocots (strategy II plants) release phytosiderophores of the mugineic acid family into the rhizosphere. These compounds act as chelators of ferric ions and are taken up by root cells as Fe(III)-phytosiderophore complexes.The nonproteinogenous amino acid nicotianamine (NA) is found in all multicellular plants [5] and is considered to be a key component for both strategies of iron acquisition (Fig.1). In strategy I plants NA might function as a chelator of iron in symplastic...

Scenedesmus cells grown on high CO2, when adapted to air levels of CO2 for 4 to 6 hours in the light, formed two concentrating processes for dissolved inorganic carbon: one for utilizing CO2 from medium of pH 5 to 8 and one for bicarbonate accumulation from medium of pH 7 to 11. Similar results were obtained with assays by photosynthetic 02 evolution or by accumulation of dissolved inorganic carbon inside the cells. The CO2 pump with Ko.5 for 02 evolution of less than 5 micromolar CO2 was similar to that previously studied with other green algae such as Chlamydomonas and was accompanied by plasmalemma carbonic anhydrase formation. The HC03-concentrating process between pH 8 to 10 lowered the Ko.5 (DIC) from 7300 micromolar HC03-in high CO2 grown Scenedesmus to 10 micromolar in air-adapted cells.The HC03-pump was inhibited by vanadate (K, of 150 micromolar), as if it involved an ATPase linked HC03-transporter. The CO2 pump was formed on low CO2 by high-CO2 grown cells in growth medium within 4 to 6 hours in the light. The alkaline HC03-pump was partially activated on low CO2 within 2 hours in the light or after 8 hours in the dark. Full activation of the HC03-pump at pH 9 had requirements similar to the activation of the CO2 pump. Air-grown or air-adapted cells at pH 7.2 or 9 accumulated in one minute 1 to 2 millimolar inorganic carbon in the light or 0.44 millimolar in the dark from 150 micromolar in the media, whereas C02-grown cells did not accumulate inorganic carbon. A general scheme for concentrating dissolved inorganic carbon by unicellular green algae utilizes a vanadate-sensitive transporter at the chloroplast envelope for the CO2 pump and in some algae an additional vanadate-sensitive plasmalemma HC03-transporter for a HC03-pump.

Unicellular green algae have a mechanism for concentrating dissolved inorganic carbon (DIC) only when grown in low CO2. To find proposed transporter protein(s) for DIC, we isolated intact chloroplasts from Dunaliel/a tertiolecta cells, separated the chloroplast envelopes by isopyknic centrifugation, and separated their polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two peptides of apparent molecular masses of 45 and 47 kD were constituents of the inner chloroplast envelope only if the cells had been adapted to low CO2 in the light or grown in low CO2. These two low C02-induced peptides appear to be part of the algal DIC pump.Cells and isolated chloroplasts from unicellular green algae, such as Dunaliella (9) and Chlamydomonas (17), concentrate DIC3 to increase photosynthetic efficiency when growing with air levels of CO2. This DIC pump is suppressed by elevated CO2 concentrations (1-5% CO2 in air [v/v]) and is formed in these 'high-CO2 cells" within 4 to 6 h in the light when cultures receive only air levels of CO2 ("low-CO2 cells'). During the induction of the DIC pump in Chlamydomonas, a number of proteins are formed preferentially (1,4,7,13,14,21,22), one of which is a periplasmic carbonic anhydrase (3, 16). Working models for the DIC-concentrating mechanism in green algae suggest that active DIC transporters should be membrane-bound proteins, which may be located at the plasmalemma and/or at the chloroplast envelope (23). In this communication, we report two peptides in the inner chloroplast envelope from Dunaliella, which are formed as the DIC pump is activated during adaptation to low CO2 in the light. MATERIALS AND METHODSThe marine, cell wall less phytoflagellate Dunaliella tertiolecta (Commonwealth Scientific and Industrial Research Organization, Australia strain) was grown photoautotrophically in minimal medium containing 0.17 M NaCl at 26 ± 20C with continuous shaking and bubbling with 5% CO2 in air (v/v (8), and then the chloroplast envelopes were isolated as described for spinach chloroplasts (10). The main steps for the envelope isolation consisted of gentle lysis of the chloroplasts by two freeze-thaw cycles under hyperosmotic conditions, differential centrifugations to remove most of the thylakoids (heavy membranes), and a floating density centrifugation of the light membrane fraction on a discontinuous sucrose gradient (Fig. 1). Before polypeptide separation by SDS-PAGE, membranes were washed with a buffer (1 mm EGTA, 10% glycerol, pH adjusted to 7.5 with Tris base) and concentrated by ultracentrifugation; soluble proteins were concentrated by ultrafiltration (Centricon-10, Amicon). Samples were resuspended in denaturing sample buffer containing 1% SDS and boiled for 1 min at 1000C. Aliquots of 40 to 100 jig of protein were subjected to electrophoresis on a linear 7.5 to 15% gradient acrylamide gel (0.8% Bis) of 0.75 mm thickness. The gels were run with a discontinuous buffer system (11). Polypeptides were stained with Coomassie brilliant blue R-250. The mole...

Photosynthetic adaptation of the unicellular green alga Scenedemus obliquus to different light conditions was investigated with respect to chlorophyll synthesis. Cultures were grown under white light (20 W · m(-2)) from fluorescent lamps and were then transferred and subjected to the actual adaptation regime which consisted of a 24-h irradiation by different fluence rates and wavelengths. Fluence rate-response curves for chlorophyll synthesis were measured between 4 · 10(-2) and 1 · 10(2) W · m(-2). In white light from incandescent lamps, in blue and red light the fluence rate-response curves for chlorophyll (Chl) a and also for Chl b were bell-shaped. In red light the threshold was about the same as under blue light. The maximal amounts of Chl a and b were about twofold increased under blue light relative to the values obtained with red light. Action spectra for the stimulation of chlorophyll synthesis (Chl a + Chl b) as well as those for the separate chlorophylls showed two maxima near 450 and 500 nm. However, the action spectrum for Chl b synthesis demonstrated a considerably higher value in the 450-nm peak. Experiments with the photosynthesis inhibitor 3-(3,4-dichlorphenyl)-1,1-dimethylurea (DCMU) indicated that photosynthetic energy supply supported the photostimulation of chlorophyll synthesis. The action spectra indicate the cooperation of two photoreceptors. The 460-nm peak is attributed to the "typical" blue-light receptor, being more active in Chl b formation. The peak at 500 nm may represent carotenoproteins acting as an accessory pigment system.

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