Despite the usually high abundance of iron (Fe) in soils, the low solubility of Fe-bearing minerals restricts the available Fe pools in most aerobic soils to levels that are far below those required for microbial or plant growth. To acquire the necessary amounts of Fe from the environment, organisms have evolved mechanisms that enhance the solubility and dissolution rate of Fe(iii) oxyhydroxides prevailing in aerobic soils. Chemically, these mechanisms are based on weakening of the Fe–O bond by reduction, chelation and protonation. Physiologically, two distinct and in all known cases mutually exclusive strategies can be distinguished: the excretion of siderophores capable of solubilizing external ferric Fe and subsequent uptake of the ferric siderophore complex; and reduction of Fe(iii) prior to uptake of the more soluble Fe2+ ion. With the exception of graminaceous species, in which Fe uptake is based on the former mechanism, the latter strategy is found in all cormophytes and certain algae, yeast and bacteria. In higher plants, the increase in their capacity to convert extracellular ferric to ferrous Fe is part of a series of physiological and morphological events that act in concert to achieve appropriate internal levels of Fe. It is this amalgam of features that determines the Fe efficiency of a species or cultivar that in turn affects the yield of economically important plants and the natural distribution of species. Adaptive changes to limited Fe availability have been studied at the molecular, physiological and whole-plant level. This review summarises current knowledge of the components of reduction-based Fe uptake in plants and presents an integrated view of the present understanding of mechanisms that control the rate and extent of Fe absorption by roots.

Biological rhythms are ubiquitous in eukaryotes, and the best understood of these occur with a period of approximately a day – circadian rhythms. Such rhythms persist even when the organism is placed under constant conditions, with a period that is close, but not exactly equal, to 24 h, and are driven by an endogenous timer – one of the many ‘biological clocks’. In plants, research into circadian rhythms has been driven forward by genetic experiments using Arabidopsis. Higher plant genomes include a particularly large number of genes involved in metabolism, and circadian rhythms may well provide the necessary coordination for the control of these – for example, around the diurnal rhythm of photosynthesis – to suit changing developmental or environmental conditions. The endogenous timer must be flexible enough to support these requirements. Current research supports this notion most strongly for the input pathway, in which multiple photoreceptors have been shown to mediate light input to the clock. Both input and output components are now related to putative circadian oscillator mechanisms by sequence homology or by experimental observation. It appears that the pathways linking some domains of the basic clock model may be very short indeed, or the mechanisms of these domains may overlap. Components of the first plant circadian output pathway to be identified unequivocally will help to determine exactly how many output pathways control the various phases of overt rhythms in plants.

Compatible pollination of Brassica napus necessitates pollen hydration, pollen germination and growth of the pollen tube through the loosened walls of stigmatic papillar cells, whereas self-incompatible (SI) pollinations fail at one of these stages. Analyses of the early stages of pollination show that at high (but not low) relative humidities, both compatible and SI pollen hydrates, but SI germination is reduced and the rare pollen tubes generally fail to penetrate the papillar walls, although there is some wall loosening. Inside the papillae, both compatible and SI interactions may induce the formation of callose, but there is no evidence for a major accumulation of cytoplasm or secretory vesicles in the vicinity of the pollen tubes and neither microtubule nor F-actin patterns re-arrange in this zone. These observations indicate that the source of the wall-loosening enzymes is probably the pollen tube or pollen coat, and that the common cellular responses of plants to attempted invasions have become suppressed in the papilla–pollen tube interaction.

Solution culture studies have demonstrated that apoplasmic iron (Fe) deposited in the roots of dicotyledonous and graminaceous plants can be mobilized to improve plant iron status in conditions of limited Fe supply. The present study investigated the formation of the apoplasmic Fe pool in dicotyledonous (soybean and cucumber) and graminaceous (wheat) plants in a pot experiment. The pots had three compartments such that plants could take up Fe and other nutrients from two calcareous soils treated with different Fe forms without their roots touching with soil directly. In this way overestimating Fe accumulation in root apoplasm was avoided. The results showed that while the root d.wt of wheat did not vary when soils were supplied with different Fe resources, the root d. wt of soybean and cucumber supplied with FeEDTA decreased compared with the control (without Fe treatment). Supplying FeEDTA in the side compartment increased shoot d. wt and Fe concentration in shoots of all species. However, supplying Fe(OH)3 had no effect on shoot d. wt or Fe concentration in the shoots of any species. Soybean and cucumber accumulated little or no Fe in the root apoplasm in controls or in Fe(OH)3 treatments. By contrast, a large amount of Fe was deposited in the root apoplasm of wheat grown in similar conditions. Remarkably, when FeEDTA was supplied in the soils, large apoplasmic iron pools were formed in the roots of all three species. Therefore, in dicotyledonous plants grown on calcareous soils, little or no apoplasmic iron pool forms, because there is not enough available Fe in the soil solution and the plants have little ability to mobilize Fe3+ in the soil. By contrast, a larger apoplasmic iron pool could form in graminaceous plants at lower concentrations of available soil-Fe possibly by enhancing the release of phytosiderophores which could mobilize Fe3+ in the soil and then transfer the Fe3+-complexes to the root apoplasm.

The post-cytokinetic guard cells of Asplenium nidus display a prominent perinuclear microtubule system and a few microtubules under the periclinal walls. Afterwards, microtubules appear on the whole surface of the ventral wall, whereas those below the periclinal walls proliferate and tend to become parallel with the ventral wall. The perinuclear microtubules gradually diminish but persist in later stages of guard cell differentiation. In post-cytokinetic guard cells, actin is found in the perinuclear cytoplasm and in the cortical cytoplasm lining all the walls. In differentiating guard cells, the following cortical microtubules and actin filament ‘systems’ appear in succession: (a) radial microtubule and actin filament arrays beneath the periclinal walls converging on the stomatal pore region, (b) anticlinal microtubule bundles, which are co-localized with actin filaments, along the ventral wall outlining the region of the stomatal pore, (c) periclinal microtubules and actin filaments on the polar ventral wall ends. These cytoskeletal systems, except for the radial actin filaments, persist in advanced stages of guard cell differentiation. Instead of the radial actin filaments, a prominent actin filament reticulum is organized under the margins of the developing wall thickenings of the stomatal pore. In addition, an extensive endoplasmic actin filament reticulum develops around the plastids. It seems likely that the successive microtubule systems in guard cells are formed by putative microtubule organizing centres operating in a definite spatial and temporal succession. Guard cell morphogenesis is the outcome of a definite process, in which the cortical microtubule cytoskeleton plays the primary role, implicated in the deposition of cellulose microfibrils and probably of the local wall thickenings. Callose or a callose-like glucan is deposited on the whole surface of the nascent ventral wall and in the wall regions where thickenings are deposited. Finally, the guard cells of Asplenium assume a kidney shape and display polar hypostomatic swellings. Particular structural features established in guard cell mother cells affect guard cell morphogenesis.

When freshly cut segments of naturally decorticated steles of horizontal roots of the Australian grass tree (Xanthorrhea preissii) are subjected to gentle suction, bubbles of gas sometimes appear, as well as liquid, in capillaries attached to the aspirated ends of the steles. We tested the hypothesis that this gas comes from vessels embolized in the intact xylem stream, and that the gas volume extruded is therefore an effective measure of the extent of this embolism. To do this, twin samples were taken from individual roots of X. preissii in the field, one was fast-frozen intact for subsequent estimation of vessel embolisms in the cryo-scanning electron microscope (CSEM), the other 15-cm segment rapidly assessed for volume of gas aspirated into a standard micropipette tube. The two measures mutually confirmed one another by showing a strong positive correlation between numbers of embolized vessels and extracted gas volume. Similar gas volumes were obtained from replicate root segments excised directly from a root when the ends of the segment were frozen before excision, and aspiration conducted after subsequent thawing of the ends under water. The pattern of changes in embolisms during unstressed conditions in early summer, shown by both CSEM and aspiration, indicated almost no embolisms before dawn, followed by a rapid rise to a peak in mid morning, than a progressive loss of embolisms in late afternoon. It was also shown that the amount of embolism did not change with time after excision of the roots up to at least 30 min. A comparison of changes in leaf transpiration with gas volumes in steles during a 24-h cycle at peak transpiration stress in mid summer showed rapid rates of transpiration in early morning and late afternoon, with an intervening period of low water loss during the rest of the day. Numbers of embolisms rose to an early morning peak, followed by apparent repair of these before noon. There was a second spate of embolisms in late afternoon, followed by complete refilling of all xylem with liquid by an hour or so after dusk. All vessels then remained fully recharged until the following dawn. We believe that aspiration is a direct and reliable technique, which offers a simple, inexpensive means of assessing the relative extent of embolism of vessels in xylem, and a means to test earlier findings by the other direct method of the CSEM. In a broad context, the technique should provide new opportunities for evaluating water relations of the xylem of whole plants.

The role of external Ca2+ in the homeostasis of intracellular pH (pHi) of Anabaena sp. strain PCC7120 in response to a decrease in the external pH (pHex) has been studied in cell suspensions. Increase in cytoplasmic pH after acid shock is dependent on the presence of Ca2+ in the medium. The observed Ca2+-mediated alkalization of the cytoplasm depends on the extent of the shift in external pH. Acid pH shifts resulted in an increased permeability of the cytoplasmic membrane to protons, which could be reversed by increasing the concentration of Ca2+ in the medium. Thus, the ability of Ca2+ to increase cytoplasmic pH might be correlated with an inhibition of net proton uptake by increasing concentrations of external Ca2+ under these conditions. This combined response resulted in the generation and maintenance of a larger pH gradient (ΔpH) at acid external pH values. All Ca2+ channel blockers tested, such as verapamil and LaCl3, inhibited the observed Ca2+-mediated response. On the other hand, the Ca ionophore calcimycin (compound A23187) was agonistic, and stimulated both cytoplasmic alkalization and inhibition of net proton uptake. The protonophorous uncoupler carbonylcyanide m-chlorophenyl hydrazone, inhibited this Ca2+-mediated response, whereas monensin, an inhibitor of the Na+/H+ antiporter, had no significant effect. The results of the present study suggest that an influx of Ca2+ from the extracellular space is required for the regulation of cytoplasmic pH in Anabaena sp. strain PCC7120 exposed to low external pH values.

This study aims to characterize the translocation of photosynthates within and from developing tall fescue (Festuca arundinacea) leaves at the time of transition from sink to source. The developing leaf contains a source, the exposed tip, and a sink, the growing basal portion. When the exposed tip of the developing blade is labelled with 14CO2, it exports photosynthates exclusively to sinks within the developing blade until the blade reaches 80% of its final length, when photosynthates begin to be exported from the blade and pass through the collar to reach the growing sheath and the next expanding leaf. Concurrently, the previous mature leaves reduce their level of photosynthate export to the developing blade; export stops as soon as the sheath of the developing leaf elongates beyond 10 mm. Export from the mature leaves to the growing sheath and to the next expanding leaf blade increases rapidly. Thus, in a developing tall fescue leaf blade photosynthate importation and exportation are exclusive events: the expanding blade imports photosynthate from mature leaves until it reaches 80% of its final length, then exportation begins and importation ceases.

The treatment of wheat roots with 2,4-dichlorophenoxyacetic acid (2,4-D) led to the formation of tumour-like structures, para-nodules, which were readily colonized by cyanobacteria. The amount of cyanobacteria found on 2,4-D treated roots was 3.6 times higher than that found on untreated roots. Cyanobacteria penetrated the para- nodules by migrating in between loosely arranged cells that covered their surfaces or by penetrating the space at the junction of root and para-nodule. In plants treated with 2,4-D and co-cultivated with cyanobacteria in medium without combined nitrogen, the rate of acetylene reduction was three times that seen in untreated but colonized roots. Addition of 2,4-D itself did not change the rate of acetylene reduction in free-living culture of Nostoc sp. strain 2S9B. In plants treated with 2,4-D and co-cultivated with Nostoc sp. strain 2S9B in medium without combined nitrogen the nitrogen content of roots but not shoots was significantly increased.

The changes in pigment composition, photosynthesis and PSII photochemistry were investigated in cells of Spirulina platensis adapted to salt stress (<0.75 M NaCl). A decrease in the phycocyanine/chlorophyll and no significant change in the carotenoid/chlorophyll ratio were observed in salt-adapted cells. Salt stress inhibited the apparent quantum efficiency of photosynthesis and PSII activity while stimulating PSI activity and dark respiration significantly. Salt stress also resulted in a decrease in overall activity of the electron transport chain, which could not be restored by diphenylcarbazide, an artificial electron donor to the reaction centres of PSII. Measurements of the polyphasic fluorescence rise in fluorescence transients including phases O, J, I and P showed that salt stress had no effect on the fluorescence yield at phase O but decreased the fluorescence yield at phases J, I and P. Analyses of the JIP test developed from the polyphasic rise of fluorescence transients showed that salt stress led to a decrease in both the maximum quantum efficiency of PSII photochemistry and the maximum quantum efficiency of electron transport beyond the primary quinone electron acceptor. However, salt stress induced no significant changes in the probability of transporting an electron beyond QA, the trapping flux per PSII reaction centre, or the electron transport flux per PSII reaction centre. A theoretical analysis of fluorescence parameters indicated a decrease in the rate constant of excitation energy trapping by PSII reaction centres. In addition, salt stress induced an increase in the complementary area above the fluorescence induction curve in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, suggesting an increase in the proportion of closed PSII reaction centres in salt-adapted cells. Based on these results, it is suggested that modifications in PSII photochemistry in salt-adapted Spirulina cells maintained a high conversion efficiency of excitation energy, such that no significant change was observed in either the trapping flux or the electron transport flux per PSII reaction centre.

The presence and variation of activity of the type 1 ribosome-inactivating protein saporin has been evaluated in cultured roots of the soapwort Saponaria officinalis. Results from western analysis and in vitro protein synthesis inhibition indicate that saporin production is increased in senescing cultures, reaching a maximum value during the late stationary phase. Accordingly, cultures treated with the senescence-related hormone abscisic acid show a significant increase in saporin activity, independently from the culture growth phase. Stress conditions, such as the presence of hydrogen peroxide in the culture medium, had no effect on the modulation of enzymatic activity. The putative regulation of saporin production by abscisic acid and its possible role in accomplishing the ageing programme is discussed.

Elimination of calcium ions from the medium of undifferentiated cell cultures of Digitalis thapsi increased cardenolide production and induced extracellular H2O2 accumulation, as measured by the quenching of pyranine fluorescence. The addition of catalase reduced the response and the inclusion of superoxide dismutase enhanced the loss of fluorescence. This suggested that, besides H2O2, the superoxide anion was also formed before dismutating to H2O2. Additionally, exogenous H2O2 or superoxide dismutase stimulated cardenolide production whereas the addition of catalase markedly reduced it. These results point to a connection between H2O2 and cardenolide formation. The absence of calcium did not alter the levels of lipid peroxidation products; however, changes in the antioxidant system of D. thapsi cells were observed. Catalase activity was extremely low in control cultures and remained unaltered upon calcium elimination. Ascorbate peroxidase activity was not modified in calcium-free cultures. By contrast, calcium deprivation stimulated superoxide dismutase activity and strongly inhibited glutathione reductase activity. Also, a significant decrease in reduced glutathione was observed. These responses were emulated by treatment of the cultures with the glutathione biosynthesis inhibitor buthionine sulfoximine and by ethyleneglycol-bis-β-aminoethyl ether and LaCl3. All these results indicate that the depletion of extracellular calcium induces changes in the redox state of cells and suggest that this alteration stimulates cardenolide formation in D. thapsi cultures.

Addition of 2 mM nitrite or ammonium to aerobically incubated cultures of Gloeothece rapidly inhibited N2 fixation (measured as acetylene reduction). In contrast, 2 mM nitrate inhibited N2 fixation less rapidly and less extensively, and often temporarily stimulated nitrogenase activity. The inhibitory effects of both nitrate and ammonium could be prevented by addition of 3 mM L-methionine-DL-sulphoximine, suggesting that the true inhibitor of N2 fixation was an assimilatory product of ammonium rather than either ammonium or nitrate itself. The inhibition of N2 fixation by nitrite could not, however, be prevented by addition of L-methionine-DL- sulphoximine. On the other hand, nitrite (unlike nitrate and ammonium) did not inhibit N2 fixation in cultures incubated under a gas phase lacking oxygen. These findings suggest that the mechanism whereby nitrite inhibits N2 fixation in Gloeothece differs from that of either nitrate or ammonium. The inhibitory effect of nitrite on N2 fixation did not involve reduction of nitrite to nitric oxide, though nitric oxide was a potent inhibitor of nitrogenase activity in Gloeothece. Nitrate and nitrite inhibited the synthesis of nitrogenase in Gloeothece, while ammonium not only inhibited nitrogenase synthesis but also stimulated degradation of the enzyme. In addition, all three compounds favoured the appearance of the Fe-protein of nitrogenase in its larger, presumed inactive, form.

Uptake of the lipophilic cation, tetraphenylphosphonium (TPP+), which is often used as a membrane potential probe, was studied in the cyanobacterium Synechococcus R-2 (PCC 7942) to determine if the technique was valid for these cells. Lipophilic cation probes are notorious for giving inconsistent estimates of membrane potential (ΔΨ) using the Nernst equation (ETPP+=ΔΨ). It is necessary to correct for the large amounts of TPP+ bound to the surface of the cells. The rate of equilibration of intracellular TPP+ is very fast (t½≈1–3 min), hence the permeability of the plasmalemma to TPP+ (PTPP+≈10–50 nm s−1) is very high. There appears to be an active uptake of TPP+ with a pH optimum at about pHo 7.5. TPP+ does not readily uncouple Synechococcus cells. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and dinitrophenol depolarized ETPP+, but monensin made ETPP+ more negative. Accumulation of TPP+ is not affected by [K+] in the medium in a way consistent with it acting as a reporter of the membrane potential. Addition of K+, NH4+ and HCO3− altered the uptake rate of TPP+ in a way consistent with electrogenic uptake of these ions but the effects were smaller than those found using the 86Rb+/valinomycin and 201TI+ probes. A comparison has been made with ΔΨ determinations using other membrane potential probes. ETPP+ was often close to the value of the membrane potential obtained using other methods under control conditions but effects of experimental treatments are sometimes inconsistent with those found using other ΔΨ probes. The TPP+ method is therefore unreliable and could be seriously misleading.

A pea (Pisum sativum cv. Puget) cultivar was grown on a medium containing different NaCl concentrations (0–160 mol m−3) in order to study the effects of salt stress on leaf water relations and on the activity of antioxidant enzymes. NaCl stress caused a rapid decline in chlorophyll content. Both leaf water (ψl) and osmotic potentials (ψs) decreased progressively with the severity of the stress (from 90–160 mol m−3 NaCl) whereas leaf turgor pressure (ψp) increased in treated plants. Pea leaves contained an iron-containing superoxide dismutase (Fe-SOD) isozyme in chloroplasts alongside a copper-zinc-containing (CuZn-SOD) form (CuZn-SOD II). The lowest NaCl concentration (70 mol m−3) had no effect on the activity of these antioxidant enzymes while higher concentrations (110–130 mol m−3) enhanced the activity of cytosolic CuZn-SOD I and chloroplastic CuZn-SOD II as well as that of mitochondrial and/or peroxisomal manganese-containing superoxide dismutase (Mn-SOD). These inductions were matched by increases in the activity of ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR). The increased activities coincided with decreased stomatal conductance and were unaffected by the severity of stress except in the case of CuZn-SOD II which fell to control values under the highest stress conditions (140–160 mol m−3 NaCl), when a concomitant increase in chloroplastic Fe-SOD activity was observed. Glutathione reductase (GR) and dehydroascorbate reductase (DHAR) activities were only induced under severe NaCl stress (130–160 mol m−3) and were accompanied by losses in the ascorbate and glutathione pools, lower ASC/DHA and GSH/GSSG ratios and increases in GSSG. Electron microscopy showed that the thylakoidal structure of the chloroplasts became disorganized and their starch content decreased in plants treated with 160 mol m−3 NaCl. Overall, the results suggest that salt stress is accompanied by oxidative stress, perhaps at the cell compartment level. The capacity of Puget cultivar to ensure cell turgor and to enhance the activity of enzymes involved in the defence against oxidative stress seems to be important in determining adaptation to moderate NaCl stress conditions. In plants exposed to severe NaCl stress (130–160 mol m−3) it seems that such resistance to oxidative stress is overcome, which might contribute to the deleterious effects of salt and significant growth reduction in these conditions.

This paper reports general patterns of relative growth rate and related traits in response to elevated atmospheric CO2 in eight woody species ranging widely in life form, leaf habit, taxonomy and ecology. Young plants of these species, all of comparable ontogenetic phases, were grown simultaneously in large containers with favourable nutrient and water availability in transparent outdoor chambers at 350 and 700 μl l−1 CO2 for one growing season. We found the following consistent responses. (1) All species grew faster at elevated CO2, whereas the following leaf and allocation traits were consistently lower in CO2-enriched environments: specific leaf area (quotient of leaf area and leaf weight), leaf area ratio (quotient of total leaf area and plant weight), weight-based foliar N concentration and, to a smaller extent, leaf weight fraction (quotient of leaf weight and plant weight). (2) There was important interspecific variation in the magnitude of the response of relative growth rate to CO2. Specific leaf area at ambient CO2 explained 88% of the variation in relative growth rate response to CO2 among the eight species. At ambient CO2, relative growth rate itself, was significantly correlated with the relative growth rate response to CO2 only if the leafless species Ulex gallii was excluded from analysis. (3) The four deciduous species had a significantly stronger relative growth rate response to CO2 than the four evergreens. This corresponded with their generally higher specific leaf area. (4) Specific leaf area and leaf habit might be useful for scaling up exercises, as easy-to-measure substitutes for growth responses of (woody) vegetation to elevated CO2. However, the usefulness of such traits in this context needs to be tested in realistic, longer-term manipulative experiments in real ecosystems.

The physiological potential for acquisition of atmospheric ammonia (NH3) was investigated in three European meadow grasses (Arrhenatherum elatius, Bromus erectus and Brachypodium pinnatum) competing in chalk grasslands. Experiments were carried out with plants cultivated for about three months on a soil–sand mixture at high root nitrogen supply or in nutrient solutions at low root nitrogen supply. Two different root nitrogen regimes were applied to the solution-grown plants: 130 μmol NO3 plant−1 wk−1 (approx. 50 kg nitrogen ha−1 in three months); or 130 μmol NO3− plus 130 μmol NH4+ plant−1 wk−1. Each regime was combined with two levels of NH3 fumigation (0 and 70 nmol mol−1 air for 24 d). Uptake of gaseous NH3 in the shoots was investigated under controlled environmental conditions including NH3 concentrations ranging from 0 to 30 nmol mol−1 air. Concurrently, photosynthesis, glutamine synthetase activity, nitrogen allocation, biomass allocation and apoplastic cation composition were measured. For A. elatius, the influence of photorespiration on NH3 acquisition was also assessed. Independently of plant nitrogen status, ammonia compensation points in A. elatius and B. erectus plants were <0.5 nmol mol−1. The total leaf conductance to NH3 absorption remained constant at increasing NH3 concentrations, showing that the capacity for assimilation was unaltered. Whereas internal factors in the leaves did not cause differences in the potential for NH3 acquisition between the species, other factors of NH3 acquisition were quite different: B. erectus had higher stomatal conductance and, thus, higher NH3 uptake rates per unit leaf area compared to A. elatius and B. pinnatum; higher stomatal conductances of B. erectus were to a large extent offset by a lower leaf area per plant, resulting from a lower growth rate and thicker leaves than in the two other species. The rate of photorespiration in Arrhenatherum constituted at least 15% of the net photosynthetic rate. Surprisingly, suppression of photorespiration indicated that NH3 uptake was supported by photorespiration. Bromus responded to fumigation with 70 nmol NH3 mol−1 air for 24 d by lowering the root[ratio ]shoot ratio and increasing the nitrogen concentration in the stem dry matter. The total leaf conductance to NH3 uptake decreased in all three species upon exposure to NH3, while the stomatal conductance was unaffected. The NH3 exposure caused lower apoplastic concentrations of H+, Mg2+ and Ca2+ in A. elatius and B. erectus.