Molecular heterogeneity of ferredoxin-NADP+ reductase from spinach leaves.

Ferredoxin-NADP+ reductase (NADPH: ferredoxin oxidoreductase, EC 1.6.7.1) from spinach leaves has been purified according to a new procedure. The enzyme shows the presence of five molecular forms as identified by isoelectric focusing, namely a, b, c, d and e with pI values of 6.0, 5.5, 5.2, 5.0 and 4.8, respectively. All the bands are catalytically active and are clearly identifiable after the first steps of the purification procedure. The basic pattern of the ferredoxin-NADP+ reductase forms is the same whether extracted from one or many spinach plants and is not affected by the different purification procedures used. Two distinct classes of molecular weight have been found for the isolated forms b, c and d as measured by sodium dodecyl sulphate electrophoresis, with values of 33 000-34 000 for the first and 36 000-38 000 for the later two forms. Gel electrophoresis in non-denaturing media at different gel concentrations gives the same order of molecular weight values, thus ruling out the possibility that the native enzyme is a dimer, as has been reported by Schneeman, R. and Krogmann, D.W. ((1975) J. Biol. Chem. 250, 4965-4971). No significant kinetic differences were detectable for the isolated forms of ferredoxin-NADP+ reductase.

Physiological evidence for a high-affinity cadmium transporter highly expressed in a Thlaspi caerulescens ecotype.

• Uptake kinetics and translocation characteristics of cadmium and zinc are presented for two contrasting ecotypes of the Cd/Zn hyperaccumulator Thlaspi caerulescens, Ganges (southern France) and Prayon (Belgium). • Experiments using radioactive isotopes were designed to investigate the physiology of Cd and Zn uptake, and a pressure-chamber system was employed to collect xylem sap. • In contrast to similar Zn uptake and translocation, measurements of concentration-dependent influx of Cd revealed marked differences between ecotypes. Ganges alone showed a clear saturable component in the low Cd concentration range; maximum influx Vmax for Cd was fivefold higher in Ganges; and there was a fivefold difference in the Cd concentration in xylem sap. Addition of Zn to the uptake solution at equimolar concentration to Cd did not decrease Cd uptake by Ganges, but caused a 35% decrease in Prayon. • There is strong physiological evidence for a high-affinity, highly expressed Cd transporter in the root cell plasma membranes of the Ganges ecotype of T. caerulescens. This raises evolutionary questions about specific transporters for non-essential metals. The results also show the considerable scope for selecting hyperaccumulator ecotypes to achieve higher phytoextraction efficiencies.

Effects of deuterium oxide on growth, proton extrusion, potassium influx, and in vitro plasma membrane activities in maize root segments.

Elongation of subapical segments of maize (Zea mays) roots was greatly inhibited by (2)H(2)O in the incubation medium. Short-term exposure (30 min) to (2)H(2)O slightly reduced O(2) uptake and significantly increased ATP levels. (2)H(2)O inhibited H(+) extrusion in the presence of both low (0.05 mm) and high (5 mm) external concentrations of K(+) (about 30 and 53%, respectively at 50% [v/v] (2)H(2)O). Experiments on plasma membrane vesicles showed that H(+)-pumping and ATPase activities were greatly inhibited by (2)H(2)O (about 35% at 50% [v/v] (2)H(2)O); NADH-ferricyanide reductase and 1,3-beta-glucan synthase activities were inhibited to a lesser extent (less than 15%). ATPase activities present in both the tonoplast-enriched and submitochondrial particle preparations were not affected by (2)H(2)O. Therefore, the effect of short incubation time and low concentration of (2)H(2)O is not due to a general action on overall cell metabolism but involves a specific inhibition of the plasma membrane H(+) -ATPase. K(+) uptake was inhibited by (2)H(2)O only when K(+) was present at a low (0.05 mm) external concentration where absorption is against its electrochemical potential. The transmembrane electric potential difference (E(m)) was slightly hyperpolarized by (2)H(2)O at low K(+), but was not affected at the higher K(+) concentrations. These results suggest a relationship between H(+) extrusion and K(+) uptake at low K(+) external concentration.

Changes in form of elongation factor 1 during germination of wheat seeds.

Two forms of EF-1 are present in the high-speed supernatant fraction from wheat embryo homogenate. In embryos from dry seeds EF-1H is 55% of the total amount of EF-1, while after 40 h of germination this form completely disappears. When germination and protein synthesis are accelerated by means of 6-benzyladenine, the rate of conversion of EF-1H is increased. On the other hand, the block of germination and of the evolution of protein synthesis by abscisic acid, block this conversion; the block of water uptake, that stops germination and causes a decrease in protein synthesis, reverses the conversion of EF-1H to EF-1L, increasing EF-1H from 15% to 40% of the total.

A low-molecular-mass GTP-binding protein in the cytosol of germinated wheat embryos.

A low-molecular-mass protein able to bind GTP in both native and SDS-denaturating conditions was detected in the cytosol of embryos from wheat (Triticum aestivum L.) seeds germinated for 40 h. The protein fulfilled most of the distinguishing criteria common to eukaryotic small GTP-binding proteins. It retained the ability to bind GTP after SDS/PAGE and nitrocellulose blotting. The protein eluted from Sephadex G-200 gel filtration with a Ve/Vo value corresponding to a molecular mass of 18 kDa, whereas on SDS/PAGE the molecular mass was 20 kDa. The native protein, which showed an intrinsic GTPase activity highly sensitive to NaF, bound the guanine nucleotide with high specificity and with a relatively high affinity (Kd approximately 85 nM). The GTP-binding protein was not detectable in other subcellular fractions; in the microsomal fraction, two other peptides of low molecular mass (23.5 and 21.5 kDa) with GTP-binding activity were detected. These results indicate that in the cytosolic fraction of germinating wheat embryos there is a 20-kDa protein which is biochemically similar to the known small GTP-binding proteins that currently have been detected almost exclusively in the membrane fraction of plant material.