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.

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.

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.

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.

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.

The aim of this work was to examine the correspondence between apoplastic/symplastic antioxidant status and previously reported plant age-related shifts in the ozone (O3) resistance of Plantago major L. Seed-grown plants were fumigated in duplicate controlled environment chambers with charcoal/Purafil®-filtered air (CFA) or CFA plus 70 nmol mol−1 O3 for 7 h d−1 over a 42 d period. Measurements of stomatal conductance and antioxidants were made after 14, 28 and 42 d fumigation, on leaves at an equivalent stage of development (youngest fully expanded leaf, measured c. 9 d after emergence). Ozone exposure resulted in a similar decline in stomatal conductance across plant ages, indicating that increases in O3 resistance with plant age were mediated through changes in the tolerance of leaf tissue rather than enhanced pollutant exclusion. Leaf apoplastic washing fluid was found to contain ‘unspecific’ peroxidase, ascorbate peroxidase, superoxide dismutase and ascorbate, but not glutathione and the enzymes required to facilitate the regeneration of ascorbate from its oxidized forms. A weak induction in the activity of certain symplastic antioxidants was found after 14 d O3 fumigation, despite a lack of visible symptoms of injury, but shifts in symplastic antioxidant enzyme activity were not consistent with previously observed increases in resistance to O3 with plant age. By contrast, changes in ‘unspecific’ peroxidase activity and in the small pool of ascorbate in the leaf apoplast were found to accompany age-related shifts in O3 resistance. It is concluded that constituents of the leaf apoplast may constitute a potentially important front line defence against O3.

We report a survey of four viruses (beet western yellows luteovirus (BWYV), cauliflower mosaic caulimovirus (CaMV), turnip mosaic potyvirus (TuMV), turnip yellow mosaic tymovirus (TYMV)) in five natural populations of Brassica oleracea in Dorset (UK). All four viruses were common; 43% of plants were infected with BWYV, 60% with CaMV, 43% with TuMV and 18% with TYMV. For each virus there were significant differences in the proportion of infected plants among populations, which were not completely explained by differences in the age of plants. Multiple virus infections were prevalent, with 54% of plants having two or more virus types. There were statistically significant associations between pairs of viruses. The CaMV was positively associated with the other three viruses, and BWYV was also positively associated with TuMV. There was no detectable association between BWYV and TYMV, whereas TuMV and TYMV were negatively associated. We suggest these associations result from BWYV, CaMV and TuMV having aphid vectors in common, as aphids are attracted to plants that already have a virus infection. Infected plants were distributed randomly or were very weakly aggregated within populations. The implications of widespread multiple virus infections in natural plant populations are discussed with respect to the release of transgenic plants expressing virus-derived genes.

Native upland species, Nardus stricta, Eriophorum vaginatum, Erica cinerea and Vaccinium vitis-idaea were given 3 or 60 kg N ha−1 yr−1, over 2 yr, applied as a mist (NH4NO3). The high N treatment increased above-ground biomass in all four species, but only significantly in E. cinerea, E. vaginatum and N. stricta. Biomass increases in E. vaginatum and N. stricta resulted from enhanced tiller production rather than shoot elongation. Root growth increased in N. stricta, so that root[ratio ]shoot ratio in this species was unchanged by N. Root growth in E. vaginatum, E. cinerea and V. vitis-idaea did not respond to N and their root[ratio ]shoot ratios decreased. Tissue N concentrations increased in both shoots and roots of all species in response to N. The accumulated foliar N did not increase the proportion of N allocated to Rubisco and the photosynthetic capacities of N. stricta, E. vaginatum and V. vitis-idaea were unchanged. Thus growth responses to N were due to altered allocation rather than increased rate of photosynthesis per unit leaf area. The high N treatment increased flower production significantly in E. cinerea but not in the other species. Although in this experiment dwarf shrubs were more responsive than graminoids to N, in the field at current N inputs the enhanced tillering of the graminoids may be more competitively advantageous, especially where gaps develop in the canopy. Thus increasing N deposition may lead to increased grassiness of upland heath, and in particular, a spread of N. stricta.

Two Holocene lake sequences from Lac de Creno, Corsica were analysed on the basis of 119 pollen spectra and with the support of 13 14C-calibrated dates. The lower part of one of these sequences, corresponding to the late- glacial period, has been published previously. The first third of the Post-glacial is characterized by very particular forest dynamics, namely the absence of a role for deciduous Quercus and Corylus, the presence of mesophilous vegetation types dominated by Taxus, and the major forest role of Erica arborea at lower and mean altitude. At about 7440 cal BP, the occurrence of a major anthropogenic action brought about significant changes in the vegetation, notably an increase of deciduous Quercus and the expansion of Quercus ilex. Later, three major human-induced events are identified: the first, at about 2290 cal BP, is the cause of a short local expansion of Abies; the second, at about 1150 cal BP, is the degradation of deciduous forests to the benefit of Fagus; the third, at about 310 cal BP, corresponds to the disappearance of Fagus and its replacement by Pinus. Pollen data indicate that Q. ilex, Abies and Fagus are not indigenous in Corsica but spread there during the Postglacial ; this probably took place at about 6980 cal BP for Q. ilex.

Among citrus rootstocks, higher specific root length (SRL, root length/d. wt) has been linked to several specific morphological and physiological traits, including smaller average root diameter, higher root hydraulic conductivity and higher rates of root proliferation. In this study, thickness of the outer tangential exodermal (hypodermal) wall and its suberin layer, number of passage cells, presence of epidermis, and stelar anatomy were examined and related to variation in root diameter of field roots of known maximum age. We also compared root morphology and anatomy of young roots in the field with those of potted rootstock seedlings in the glasshouse. Fibrous roots were measured separately from pioneer (framework) roots. Among the fibrous roots, only the first-order (root links having a root tip) and second-order (root links bearing first-order roots) laterals were examined. Among first-order field roots, larger root diameter was caused by larger rather than more numerous cells in the cortex. Root diameter of first-order roots was positively correlated with both number of passage cells in the exodermis and thickness of the secondary walls of the exodermis in both field and potted plants.

Exodermal walls were about 80% thicker in field- than pot-grown roots. In the field, in more than 50% of the first-order roots examined less than 30% of the root surface was still covered by epidermis, with few differences among rootstocks. In contrast, in roots of 19-wk-old glasshouse plants generally 70–100% of the epidermis was still intact. There was no evidence of secondary xylem development in second-order fibrous roots in the field; in seedling, pot-grown rootsystems, 75–97% of second-order roots had formed secondary xylem despite their small diameter (<0.8 mm).

It is argued that there can be suites of physiological, morphological and anatomical traits in roots that co-vary with specific root length. Investigations of how root morphology and anatomy are linked to root function, moreover, need to recognize trait variability and the potentially important differences between field- and pot- grown (seedling) roots.

Several DNA-based techniques, developed for identifying and differentiating fungi in the Gaeumannomyces–Phialophora complex associated with take-all diseases of cereals and grasses, were used to compare fungi from maize. Maize isolates obtained as G. graminis (Sacc.) Arx & H Olivier var. tritici Walker, from the UK, having been identified by ascospore morphology and in pathogenicity tests on wheat, were indistinguishable from isolates of the same variety obtained from wheat. Isolates of G. graminis (Sacc.) Arx & H Olivier var. maydis Yao et al., recently described as the maize take-all fungus from China, were identical in DNA tests to the anamorphic fungus Phialophora radicicola Cain and almost identical to Phialophora zeicola Deacon & Scott, whose description was originally based on isolates from South Africa and France. These species appear to represent the holomorph of the same fungus. The late wilt pathogen of maize, from India and Egypt, commonly known as Cephalosporium maydis Samra et al., but suggested as being the Phialophora anamorph of a Gaeumannomyces species, was closely related to other Gaeumannomyces species included in the tests.

Phialocephala fortinii is among the few identified hyphomycetes belonging to the Mycelium radicis atrovirens complex. This ‘dark-septate endophyte’ has a global distribution and colonizes a wide variety of host plants. In this study, the spatial distribution of discrete genets of P. fortinii on the forefront of a receding glacier was analysed using the randomly amplified polymorphic DNA (RAPD) technique to determine plants colonized and patterns of colonization. In two consecutive years of sampling, a total of 74 isolates of P. fortinii were obtained from nine plant species, typically ectomycorrhizal, ericoid mycorrhizal or non-mycorrhizal. The isolates showed substantial variation, sharing on average approx. half the RAPD markers. In the first year, three isolates belonging to a single genet were obtained from two plants separated by a distance of nearly 1.5 m. The continuity of this genet was assessed by a sampling the following year. No isolates similar to that, or any of the genets collected the year before were observed. Consequently, the identical isolates from the previous year were concluded to represent discontinuous ramets. Two additional large genets were observed during the second year of sampling, which inhabited roots of several plants representing three different species. These data suggest that the sharing of P. fortinii genets among plant species might play a fundamental role in adaptation and interaction within the whole plant community in a system undergoing primary succession.

The time course of initiation and development of root nodules was investigated in the South American actinorhizal shrub Discaria trinervis (Rhamnaceae). A local strain of Frankia (BCU110501) which was isolated from D. trinervis nodules, was used as inoculum. Inoculated seedlings were periodically studied under the light microscope after clearing with aqueous NaClO. In parallel, semithin and ultrathin sections were analysed by light and electron microscopy. Infection by Frankia BCU110501 involved intercellular penetration among epidermal and cortical root cells. Nodule primordia were detected from 6 d after inoculation, while bacteria were progressing through intercellular spaces of the outer layers of cortical cells. Invasion of host cells by the symbiont occurred 7–9 d after inoculation, and hypertrophy of the primordium cells was associated with Frankia penetration. Root hairs were not deformed during the early events of nodule formation. From 13 to 16 d after inoculation, the proximal cellular zone of the primordia behaved differently from the other tissues after NaClO treatment and remained darkly pigmented. At the same time, differentiation of Frankia vesicles started to occur inside already infected cells. By 16 d after inoculation, spherical vesicles of BCU110501 were homogeneously distributed in the host cells. These vesicles were septate and surrounded by void space. Frankia spores or sporangia were not observed in the nodule tissue. This study has clarified the mode of Frankia penetration in D. trinervis, one of the Rhamnaceae which also includes Ceanothus. The events involved in infection, nodule induction, host-cell infection and vesicle differentiation have been characterized and identified as time-segregated developmental processes in the ontogeny of D. trinervis root nodules.

Endophytes of the genus Epichloë (Clavicipitaceae, Ascomycota) are systemic symbionts of cool-season grasses. Their interactions with grass hosts may vary between mutualistic and pathogenic depending on the mode of endophyte reproduction. Sexual strains prevent flowering and seed set (choke disease) of the host and can be horizontally transmitted by ascospores, while asexual strains remain asymptomatic and are vertically transmitted through seeds. In Switzerland nearly all plants of Brachypodium sylvaticum (Huds.) P.B. are infected by Epichloë sylvatica Leuchtmann & Schardl, but choke symptoms are formed very rarely, and are restricted to particular locations and to a minority of plants at those locations. Earlier research has revealed that E. sylvatica is genetically differentiated into sexual and asexual subpopulations. Given the high level of infection and assuming horizontal transmission of sexual strains, multiple host infections have been predicted. In this study, 25 plants out of 63 examined by isozyme analysis were found to be infected by two or three different endophyte genotypes. In most cases endophyte genotypes appeared to be correlated with the symptom type of a particular tiller, suggesting that the fungal genome controls choke formation and that the sexual and asexual subpopulations are separated at the ramet (tiller) level rather than at the genet (plant) level. These conclusions were further supported by analyses with log-linear models of the population structure of E. sylvatica at four locations where choke symptoms were present. These analyses also revealed a geographic structure in the asexual subpopulation but not in the sexual subpopulation which could be caused by the different dispersal ranges of their propagules. The rare occurrence of sexually reproducing strains and the dominance of a single genotype in asymptomatic plant populations may be explained by the colonization history of B. sylvaticum and its endophyte in Switzerland.