A rapid and simple method for the determination of a ferric iron pool in the free space of roots is described. Formation of this pool depended on the source of iron in the nutrient solution. During growth in water culture at pH 5 to 6 with Fe-ethylenediaminetetraacetate, a free space pool of 500 to 1000 nanomoles Fe per gram fresh weight was formed in the roots of bean (Phaselus vullgaris L. var. Prelude), maize (Zea mays L. var. Capella), and chlorophytum (Chlorophytum comosum IThunb. Jacques). No si nt pool (less than 100 nanomoles per gram fresh weight) was formed with ferrioxamine. Upon impending Fe deficiency, bean and chlorophytum were able to mobilize this pool. Fe-deficient bean plants mobilized iron from the free space iron pool of another plant in the same vessel.Iron uptake by plants is fastest when iron is present in the ferrous form (10). In anaerobic soils, high concentrations of ferrous ions may lead to iron toxicity by excessive iron uptake. Plants may limit iron uptake under those conditions by oxidation of ferrous ions with oxygen which is transported from the shoot via aerenchyma (12). Iron in aerobic soils is mainly present as the ferric ion in precipitates (14) or in soluble chelates (22). Dicotyledonous plants may enhance their capacity for iron uptake in response to a developing deficiency, by increasing their ability to reduce ferric chelates at the root surface (10). The uptake of iron by roots is therefore a result of several processes which may occur simultaneously: (a) reduction of ferric chelates in the rhizosphere by excreted reducing compounds (20) Crude Cell Wall Preparations. Root systems were washed in 0.5 mM CaSO4 for 10 to 15 min, frozen in liquid N2, and milled with an iron-free mortar and pestle. The resulting powder was suspended in 0.5 mM CaSO4, and the material precipitating at 500g was collected. This washing procedure was repeated at least three times, after which the final precipitate was suspended in 0.5 mM CaSO4, 10 mM Mes (pH 5.5).Determination of Apoplasmic Iron. A plant was transferred from nutrient solution to a beaker with 0.5 mm CaSO4 under vigorous aeration. After 10 to 15 min, the plant was placed with its root system in a wide 40-ml tube with 21 ml 10 mm Mes, 0.5 mM Ca(NO3)2, 1.5 mM 2,2'-bipyridyl (pH 5.5) at 25°C. Nitrogen was bubbled through the solution and the tube was covered with a cotton plug to prevent entry of oxygen by turbulence. After 5 min under N2, 1 ml 250 mm Na2S204 was added from a syringe.The A520 of the solution (A520 of 1 mM Fe[bipyridyl]3 = 8.650) was followed on 2-ml samples. The first sample was taken just before addition ofdithionite. The reaction was stopped, routinely after 10 min, by transferring the plant to a beaker with 0.5 L vigorously aerated 0.5 mM CaSO4. The plant could then be used for other determinations, or be put back on nutrient solution for further culture.Reducible iron in crude cell wall preparations was determined in the same way as described for intact roots, but samples taken from the incubation suspens...

Pyridine nucleotides were determined in lateral roots of iron-deficient and iron-sufficient Phaseolus vulgaris L. cv Prelude. In iron-deficient plants, total NADP per gram fresh weight and the NADPH/NADPI ratio were twice the values found in iron-sufficient plants. The NADPH/ NADP ratio in iron-deficient plants was considerably lowered after a 2 minute incubation in I millimolar ferricyanide. Total NAD was not influenced by growth conditions and was mainly present in oxidized form.These results indicate that NADPH is the electron donor for the high Fe"' reduction activity found in iron-deficient roots, a process that is part of the Fe-uptake mechanism.The reduction of Fe"' to Fe" is an essential step in the uptake of iron by roots of dicotyledonous plants (6). Evidence is accumulating that this reduction takes place at the plasma membrane of root cells by an enzymic process (1,3,4,13,14). The enzyme involved should be capable ofelectron transfer across the plasma membrane. We demonstrated that cytosolic reduced pyridine nucleotides are the direct electron donors for this enzyme (14,15).Plants suffering from iron deficiency show a considerable increase in their Fe"' reduction capacity at the root surface (6, 12). We established that the potential supply of reduced pyridine nucleotides is greatly enhanced under iron deficiency and that the level of cytosolic NAD(P)H is strongly lowered when irondeficient roots are exposed to extracellular Fe"' salts (14). We set out to determine more precisely the electron donor for extracellular Fe"' reduction. Through direct measurements of pyridine nucleotides in root extracts, we demonstrate that NADPH is the source of electrons for the membrane-bound enzyme system, of which the activity is stimulated by iron deficiency. (17), the Mn concentration in the iron-free nutrient solution was lowered from 9 to 0.9 uM. The solutions were replaced with freshly prepared nutrient solution on the 3rd, 6th, and 8th d after transfer and the plants were used for experiments on the 8th or 9th d. The plants were grown in a growth chamber (16 h light, 22C) under 26 w m 2 (Philips TL 33 fluorescent) and 65% RH. MATERIALS AND METHODSIsolation and Pretreatment of Lateral Roots. For each experiment, about 20 g (fresh weight) lateral roots of 2 to 5 cm length were isolated from 30 plants. Isolated lateral roots were kept in aerated iron-free nutrient solution at room temperature until all roots were harvested (about 2 h). The nutrient solution was decanted and the roots were gently blotted with tissue paper and weighed. They were then quickly divided into four equal portions (on fresh weight basis) and returned to aerated iron-free nutrient solution for 10 min. After this preincubation, two portions were incubated in 1 mM K3Fe(CN)6 for 2 min at room temperature. The nutrient solutions of all four portions were then decanted and the roots were immediately frozen in liquid N2 and homogenized with mortar and pestle. To one untreated and one ferricyanide-treated portion, a fixed amount of the p...

Chlamydomonas eugametos gametes of opposite mating type make cell-cell contact via their flagellar surfaces. This contact triggers an increase in the intracellular level of cyclic AMP (cAMP) and several cellular responses which are necessary for cell fusion. Here, we show that wheat-germ agglutinin, which binds to the flagellar surface and induces all mating responses, also increased the intracellular cAMP level. Dibutyryl-cAMP added to non-mating gametes induced flagellar twitching, cell-wall lysis, mating-structure activation, flagellartip activation and an increase in agglutinability. It did not induce agglutinin transport to the flagellar tip (tipping) and may not be the direct cause of flagellar twitching and flagellar-tip activation. In non-illuminated cells, dibutyryl-cAMP was far more effective in evoking mating reactions than in illuminated cells. Light induced a 50% decrease in the cAMP level within 1 min. Adenylate cyclase was found to be associated with cell membranes but only 8% of the total was present in the gamete flagella.

During sexual reproduction in the heterothallic, biflagellate, green alga Chlamydomonas, gametes adhere together via their agglutinins, sex-specific glycoproteins extrinsically bound to the flagellar membrane. Using an antibody specific for a C. eugametos agglutinin, we illustrate that agglutinins engaged in adhesion are transported to the flagellar tips. This tipping phenomenon, together with a particular orientation of the flagella, forms part of the mechanism by which gametes position themselves properly for fusion in pairs.Sexual cell fusion; Membrane receptor transport; (Chlamydomonas eugametos)

Evidence is presented that French-bean (Phaseolus vulgaris) seed ferritin is composed of one type of subunit with an apparent Mr of 26500. In normal and iron-loaded leaf tissues it is detected immunologically with an antiserum raised against purified bean seed ferritin and migrates in SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis with the same mobility as the bean seed ferritin subunit. The biosynthetic pathway of ferritin in normal and iron-loaded leaves was investigated. RNA was extracted, fractionated into polyadenylated RNA and translated in a cell-free rabbit reticulocyte lysate and a wheat-germ-extract system. The products were identified by SDS/polyacrylamide-gel electrophoresis after indirect immunoprecipitation. In all cases the ferritin product had an Mr 5000 higher than that of the native subunit. Uptake and processing of the precursor form of ferritin from iron-loaded leaves by intact chloroplasts was demonstrated. This indicates that, in iron-loaded leaves, ferritin acts as a chloroplast protein. We propose that the ferritin precursor in normal leaves follows the same biosynthetic pathway. This suggests that the iron-buffering function of ferritin in plants takes place in the chloroplast and that non-functional cellular iron will accumulate in this cell organelle.