ALBERT

Notes: The A2 gene family is present in Leishmania donovani, which causes fatal visceral leishmaniasis in human patients, but is not present in Leishmania major, which causes cutaneous leishmaniasis infections. The A2 genes in L. donovani are stage specific and are expressed at high levels in the amastigote stage in the mammalian host, but are not expressed in the promastigote stage in the insect sandfly vector. The A2 genes are tandem repeated with a distinct gene family termed the A2rel genes. In order to characterize the structure and function of the A2–A2rel gene clusters, the 5′ and 3′ DNA sequences flanking the A2–A2rel cluster were isolated, sequenced and used to generate mutants through gene targeting. Although it was possible to generate partial A2–A2rel gene clusters knock-out mutants, it was not possible to delete all the A2–A2rel gene clusters completely from the L. donovani genome, suggesting that, within this cluster, there are genes that are essential for survival in culture. Characterization of these mutants revealed that A2 and A2rel gene expression was compensated by amplifying the remaining intact A2 and A2rel genes, and the proliferation of these mutants in culture and their virulence in BALB/c mice were compromised. In order to explore further the biological role of A2, the L. donovani A2 gene was introduced into L. major. In comparison with the control L. major, the A2-expressing L. major parasites demonstrated an increased ability to survive in the spleen of BALB/c mice. These data suggest that A2 plays a role in the visceralization of infection associated with L. donovani.

Notes: The elongation step of protein synthesis involves binding of aminoacyl-tRNA to the ribosomal A site, formation of a peptide bond and translocation of the newly formed peptidyl-tRNA to the P site. The nucleotide exchange factor EF-1β plays a major role in the regulation of this process by regenerating a GTP-bound EF-1α necessary for each elongation cycle. EF-1β has been shown to be phosphorylated and its phosphorylation is critical for optimal activity. We have previously identified a serine/threonine protein phosphatase 2C (PP2C) from the human malaria parasite Plasmodium falciparum. In the current work, we performed Far-Western analysis to identify PfPP2C substrates. Several components of the translation and transcription machinery were identified, including translation elongation factor 1-beta (PfEF-1β). PfEF-1β is efficiently phosphorylated by protein kinase C and this phosphorylation results in a 400% increase in its nucleotide exchange activity. PKC-phosphorylated PfEF-1β is readily and selectively dephosphorylated by recombinant and native PfPP2C, which downregulates the nucleotide exchange activity to its basal level. The identification of a translation elongation component as substrate for PP2C suggests an important regulatory function for this enzyme and suggests that it may be a good target for drug design in the fight against malaria.

Notes: Lacticin 3147 is a membrane-active, two-component lantibiotic produced by Lactococcus lactis ssp. lactis DPC3147. In this study, the promoters of the lacticin 3147 gene cluster were mapped to the intergenic region between ltnR and ltnA1 (the genes encoding the regulatory protein LtnR and the first structural gene, LtnA1), and Northern analyses revealed that the biosynthetic and immunity genes are divergently transcribed in two operons, ltnA1A2M1TM2D and ltnRIFE respectively. Although the promoter controlling biosynthesis (Pbac) appears to be constitutive, characterization of a downstream β-galactosidase (β-gal) fusion beyond an intragenic stem–loop structure in ltnM1 confirmed that this putative transcriptional attenuator allows limited readthrough to the downstream biosynthetic genes, thus maintaining the correct stoichiometry between structural peptides and biosynthetic machinery. The promoter of the ltnRIFE operon (Pimm) was shown to be regulated by the transcriptional repressor LtnR. A mutant with a truncated ltnR gene exhibited a hyperimmune phenotype, whereas overexpression of ltnR resulted in cells with increased sensitivity to lacticin 3147. Gel mobility shift analysis indicated that LtnR binds to the Pimm promoter region, and fusion of this promoter to the β-gal gene of pAK80 revealed that expression from Pimm is significantly reduced in the presence of LtnR. Thus, we have demonstrated that lacticin 3147 uses a regulatory mechanism not previously identified in lantibiotic systems.

Notes: Erwinia chrysanthemi causes soft-rot disease in a great variety of plants. In addition to the depolymerizing activity of plant cell wall-degrading enzymes, iron acquisition and resistance to oxidative stress contribute greatly to the virulence of this pathogen. Here, we studied the pin10 locus originally thought to encode new virulence factors. The sequence analysis revealed six open reading frames that were homologous to the Escherichia coli sufA, sufB, sufC, sufD, sufS and sufE genes. Sequence similarity searching predicted that (i) SufA, SufB, SufD, SufS and SufE proteins are involved in iron metabolism and possibly in Fe–S cluster assembly; and (ii) SufC is an ATPase of an ABC transporter. The reverse transcription–polymerase chain reaction procedure showed that the sufABCDSE genes constitute an operon. Expression of a sufB::uidA fusion was found to be induced in iron-deficient growth conditions and to be repressed by the iron-sensing Fur repressor. Each of the six suf genes was inactivated by the insertion of a cassette generating a non-polar mutation. The intracellular iron level in the sufA, sufB, sufC, sufS and sufE mutants was higher than in the wild type, as assessed by increased sensitivity to the iron-activated antibiotic streptonigrin. In addition, inactivation of sufC and sufD led to increased sensitivity to paraquat. Virulence tests showed that sufA and sufC mutants exhibited reduced ability to cause maceration of chicory leaves, whereas a functional sufC gene was necessary for the bacteria to cause systemic invasion of Saintpaulia ionantha. The E. coli sufC homologue was inactivated by reverse genetic. This mutation was found to modify the soxR-dependent induction of soxS gene expression. We discuss the possibility that SufC is a versatile ATPase that can associate either with the other Suf proteins to form a Fe–S cluster-assembling machinery or with membrane proteins encoded elsewhere in the chromosome to form an Fe–S ABC exporter. Overall, these results stress the importance of the connection between iron metabolism and oxidative stress during the early steps of infection by E. chrysanthemi.

Notes: The response of Mycobacterium smegmatis to a cold shock was investigated by monitoring changes in both growth and cellular protein composition of the organism. The nature of the cellular response was influenced by the magnitude of the temperature reduction, with the shock from 37°C to 10°C having the most widespread effect on growth, metabolism and protein composition. This 27°C temperature reduction was associated with a lag period of 21–24 h before increases were seen in all the measured cellular activities. The response to cold shock was adaptive, with growth resuming after this period, albeit at a 50-fold slower rate. The synthesis of at least 15 proteins was induced during the lag period. Two distinct patterns of cold-induced synthesis were apparent, namely transient and continuous, indicating the production of both cold-induced and cold-acclimation proteins. One of these cold-shock proteins, CipMa, was identified as the histone-like protein, Hlp, of M. smegmatis, which is also induced during anaerobic-induced dormancy. The corresponding gene demonstrated transient, cold-inducible expression with a five- to sevenfold increase in mRNA occurring 9–12 h after temperature shift. Although bacterial survival was unaffected, CipMa/Hlp knock-out mutants were unable to adapt metabolically to the cold shock and resume growth, thus indicating a key role for CipMa in the cold-shock response.

Notes: An 18 633 bp region containing the replicon from the ≈ 53 kb pBM400 plasmid of Bacillus megaterium QM B1551 has been sequenced and characterized. This region contained a complete rRNA operon plus 10 other potential open reading frames (ORFs). The replicon consisted of an upstream promoter and three contiguous genes (repM400, orfB and orfC) that could encode putative proteins of 428, 251 and 289 amino acids respectively. A 1.6 kb minimal replicon was defined and contained most of repM400. OrfB was shown to be required for stability. Three 12 bp identical tandem repeats were located within the coding region of repM400, and their presence on another plasmid caused incompatibility with their own cognate replicon. Nonsense, frameshift and deletion mutations in repM400 prevented replication, but each mutation could be complemented in trans. RepM400 had no significant similarity to sequences in the GenBank database, whereas five other ORFs had some similarity to gene products from other plasmids and the Bacillus genome. An rRNA operon was located upstream of the replication region and is the first rRNA operon to be sequenced from B. megaterium. Its unusual location on non-essential plasmid DNA has implications for systematics and evolutionary biology.

Notes: Calcineurin is a Ca2+–calmodulin-regulated protein phosphatase that is the target of the immunosuppressive drugs cyclosporin A and FK506. Calcineurin is a heterodimer composed of a catalytic A and a regulatory B subunit. In previous studies, the calcineurin A homologue was identified and shown to be required for growth at 37°C and hence for virulence of the pathogenic fungus Cryptococcus neoformans. Here, we identify the gene encoding the calcineurin B regulatory subunit and demonstrate that calcineurin B is also required for growth at elevated temperature and virulence. We show that the FKR1-1 mutation, which confers dominant FK506 resistance, results from a 6 bp duplication generating a two-amino-acid insertion in the latch region of calcineurin B. This mutation was found to reduce FKBP12–FK506 binding to calcineurin both in vivo and in vitro. Molecular modelling based on the FKBP12–FK506–calcineurin crystal structure illustrates how this mutation perturbs drug interactions with the phosphatase target. In summary, our studies reveal a central role for calcineurin B in virulence and antifungal drug action in the human fungal pathogen C. neoformans.

Notes: Escherichia coli HU protein is a major component of the bacterial nucleoid. HU stabilizes higher order nucleoprotein complexes and belongs to a family of DNA architectural proteins. Here, we report that HU is required for efficient expression of the sigma S subunit of RNA polymerase. This rpoS-encoded alternative σS factor induces a number of genes implicated in cell survival in stationary phase and in multiple stress resistance. By analysis of rpoS–lacZ fusions and by pulse-chase experiments, we show that the efficiency of rpoS translation is reduced in cells lacking HU, whereas neither rpoS transcription nor protein stability is affected by HU. Gel mobility shift assays show that HU is able to bind specifically an RNA fragment containing the translational initiation region of rpoS mRNA 1000-fold more strongly than double-stranded DNA. Together with the in vivo data, this finding strongly suggests that, by binding to rpoS mRNA, HU directly stimulates rpoS translation. We demonstrate here that HU, an abundant DNA-binding, histone-like protein, is able specifically to recognize an RNA molecule and therefore play a role in post-transcriptional regulation.

Notes: The ascomycete Neurospora crassa has the capacity of adapting to a given light quantity, leading to transient blue light responses under continuous light conditions. Here, we present an investigation of this photoadaptation phenomenon. We demonstrated previously that two proteins of the Neurospora blue light signal transduction chain, WC1 and WC2, are subject to light-dependent phosphorylation. WC1 was phosphorylated in parallel with the transient increase in transcript levels of light-regulated genes. Using the light-dependent phosphorylation of WC1 as a marker for an active signalling state of WC1, we show that the transiency of Neurospora blue light responses results from desensitization of the photoreceptor and/or the signalling cascade. Furthermore, a Neurospora mutant was characterized that revealed a specific defect in photoadaptation. In this mutant, the transient expression of light-regulated genes under continuous light, the temporary insensitivity after a light pulse and the capability of differentiating between and adapting to low and high light intensities were abolished. The corresponding protein seems to represent a central component of a negative feedback desensitization mechanism. This negative feedback regulation requires continuous and light-dependent protein de novo biosynthesis.

Notes: The mobilization region of plasmid CloDF13 was localized to a 3.6 kb DNA segment that was analysed by transposon mutagenesis and DNA sequencing. Analysis of the DNA sequence allowed us to identify two mobilization genes and the CloDF13 origin of conjugative transfer (oriT), which was localized to a 661 bp segment at one end of the mobilization (Mob) region. Thus, the overall organization was oriT–mobB–mobC. Plasmid CloDF13 DNA was isolated mainly as a relaxed form that contained a unique strand and site-specific cleavage site (nic). The position of nic was mapped to the sequence 5′-GGGTG/GTCGGG-3′ by primer extension and sequencing reactions. Analysis of Mob− insertion mutants showed that mobC was essential for CloDF13 relaxation in vivo. The sequence of mobC predicts a protein (MobC) of 243 amino acids without significant similarity to previously reported relaxases. In addition to MobC, the product of mobB was also required for CloDF13 mobilization and for oriT relaxation in vivo. mobB codes for a protein (MobB) of 653 amino acids with three predicted transmembrane segments at the N-terminus and the NTP-binding motifs characteristic of the TraG family of conjugative coupling proteins. Membership of the TraG family was confirmed by the fact that CloDF13 mobilization by plasmid R388 was independent of TrwB and only required PILW. However, contrary to the activities found for other coupling proteins, MobB was required for efficient oriT cleavage in vivo, suggesting an additional role for this particular protein during oriT processing for mobilization. Additionally, the cleavage site produced by the joint activities of MobB and MobC was shown to contain unblocked ends, suggesting that no stable covalent intermediates between relaxase and DNA were formed during the nic cleavage reaction. This is the first report of a conjugative transfer system in which niccleavage results in a free nicked-DNA intermediate.

Notes: Pseudomonas putida DOT-T1E grows on a water–toluene double liquid phase. Toluene tolerance in this microorganism is mainly achieved by at least two efflux pumps that belong to the RND family. The TtgDEF efflux pump is induced by toluene, whereas the other efflux pump, called TtgABC, is expressed at a high level in cells not exposed to toluene and at a lower level in cells grown with toluene. The ttgR gene is adjacent to the ttgABC operon and is transcribed divergently from ttgA. The expression level of ttgR was fourfold higher in cells growing in the presence of toluene than in its absence. In a TtgR-deficient background, expression from the ttgA promoter increased about 20-fold, suggesting that TtgR represses expression from the ttgA promoter. In this mutant, background expression of the ttgR gene was also much higher than in the wild-type background; however, its level of expression increased in the presence of toluene. In a ttgR mutant background, expression from the ttgD promoter followed the same pattern of expression as in the wild type. Analysis of a P. putida pTn5cat mutant that exhibited increased sensitivity to a sudden toluene shock, regardless of whether or not it was previously exposed to low toluene concentrations, revealed that pTn5cat had interrupted an lrp-like gene. The ttgR gene was expressed at very high levels in this mutant, with concomitant repression of expression of the ttgABC operon. The second ttgDEF efflux pump was expressed at low levels in this mutant strain, suggesting that the Lrp-like protein is a global regulatory protein involved in the solvent-tolerant response of this strain.

Notes: The endonuclease activity of EcoKI is regulated by the ClpXP-dependent degradation of the subunit that is essential for restriction, but not modification. We monitored proteolysis in mutants blocked at different steps in the restriction pathway. Mutations that prevent DNA translocation render EcoKI refractory to proteolysis, whereas those that permit DNA translocation, but block endonuclease activity, do not. Although proteolysis alleviates restriction in a mutant that lacks modification activity, some restriction activity remains; our evidence indicates residual EcoKI associated with the membrane fraction. ClpXP protects the bacterial chromosome, but little effect was detected on unmodified foreign DNA within the cytoplasm of a restriction-proficient cell. The molecular basis for the distinction between unmodified resident and foreign DNA remains to be determined.

Notes: The yeast HAL1 gene facilitates K+/Na+ selectivity and salt tolerance of cells. Ectopic expression of HAL1 in transgenic tomato (Lycopersicon esculentum Mill.) plants minimized the reduction in fruit production caused by salt stress. Maintenance of fruit production by transgenic plants was correlated with enhanced growth under salt stress of calli derived from the plants. The HAL1 transgene enhanced water and K+ contents in both leaf calli and leaves in the presence of salt, which indicates that HAL1 functions in plants using a similar mechanism to that in yeast, namely by facilitating K+/Na+ selectivity under salt stress.

Notes: The effects of mycorrhizal infection, soil P availability and fruit production on the male function of reproduction were examined in two cultivars of tomato (Lycopersicon esculentum Mill.). Tomato plants were grown in a greenhouse under three treatment combinations: non-mycorrhizal, low P (NMPO); non-mycorrhizal, high P (NMP3); and mycorrhizal, low P (MPO). In addition, all treatment combinations were grown both with and without fruit. Fruit production decreased final leaf biomass, flower production and in vitro pollen tube growth rates, often reducing the beneficial effects of increased P uptake. Thus, fruit production diverted resources from subsequent vegetative growth, flower production and pollen development. As the growing season progressed, mean pollen production per flower and in vitro germination and tube growth decreased. Mycorrhizal infection and high soil P conditions increased final leaf biomass, flower production, mean pollen production per flower (in one cultivar) and in vitro pollen tube growth rates. Thus, mycorrhizal infection and high soil P conditions increased pollen quantity and quality, thereby enhancing fitness through the male function. Similar trends in these treatments suggested that mycorrhizal effects on the male function were largely the result of improved P acquisition.

Notes: In citrus, the majority of fine roots are distributed near the soil surface – a region where conditions are frequently dry and temperatures fluctuate considerably. To develop a better understanding of the relationship between changes in soil conditions and a plant’s below-ground respiratory costs, the effects of temperature and soil drying on citrus root respiration were quantified in controlled greenhouse experiments. Chambers designed for measuring the respiration of individual roots were used. Under moist soil conditions, root respiration in citrus increased exponentially with changes in soil temperature (Q10 = 1·8–2·0), provided that the changes in temperature were short-term. However, when temperatures were held constant, root respiration did not increase exponentially with increasing temperatures. Instead, the roots acclimated to controlled temperatures above 23 °C, thereby reducing their metabolism in warmer soils. Under drying soil conditions, root respiration decreased gradually beginning at 6% soil water content and reached a minimum at 〈2% soil water content in sandy soil. A model was constructed from greenhouse data to predict diurnal patterns of fine root respiration based on temperature and soil water content. The model was then validated in the field using data obtained by CO2 trapping on root systems of mature citrus trees. The trees were grown at a site where the soil temperature and water content were manipulated. Respiration predicted by the model was in general agreement with observed rates, which indicates the model may be used to estimate entire root system respiration for citrus.

Notes: Changes in specific leaf area (SLA, projected leaf area per unit leaf dry mass) and nitrogen partitioning between proteins within leaves occur during the acclimation of plants to their growth irradiance. In this paper, the relative importance of both of these changes in maximizing carbon gain is quantified. Photosynthesis, SLA and nitrogen partitioning within leaves was determined from 10 dicotyledonous C3 species grown in photon irradiances of 200 and 1000 µmol m−2 s−1. Photosynthetic rate per unit leaf area measured under the growth irradiance was, on average, three times higher for high-light-grown plants than for those grown under low light, and two times higher when measured near light saturation. However, light-saturated photosynthetic rate per unit leaf dry mass was unaltered by growth irradiance because low-light plants had double the SLA. Nitrogen concentrations per unit leaf mass were constant between the two light treatments, but plants grown in low light partitioned a larger fraction of leaf nitrogen into light harvesting. Leaf absorptance was curvilinearly related to chlorophyll content and independent of SLA. Daily photosynthesis per unit leaf dry mass under low-light conditions was much more responsive to changes in SLA than to nitrogen partitioning. Under high light, sensitivity to nitrogen partitioning increased, but changes in SLA were still more important.

Notes: Diurnal patterns of photosynthesis were studied in July and April populations of Cladophora glomerata (L.) Kütz. from open and from shaded sites. Summer samples exposed to full sunlight showed decreased efficiency of open photosystem II at noon, and only slight differences were found between samples that had grown at open or at shaded sites. Electron transport rate was limited at highest fluence rates in shade plants, and non-photochemical quenching (NPQ) revealed faster regulation in samples from open sites. Daily course of de-epoxidation was not linearly correlated with the course of NPQ. The comparison of samples from open and from shaded sites revealed a higher capacity of thermal energy dissipation and an increase in the total amount of xanthophyll-cycle pigments (21%) in samples from open sites. In April, down-regulation of the efficiency of open photosystem II was related to lower water temperature, and hence, increased excitation pressure. In April the pool size of xanthophyll-cycle pigments was increased by 21% in comparison with summer and suggested higher levels of thermal energy dissipation via de-epoxidized xanthophylls. In both, summer and spring the amount of xanthophyll-cycle pigments was 20% higher in samples from open sites. Acclimation of C. glomerata to growth light conditions was further shown by experimental induction of NPQ, indicating NPQ increases of 23%, and increases of 77% in the reversible component of NPQ in open site samples. The effect of temperature on photosynthetic rate was non-linear, and different optimum temperatures of electron transport rate and oxygen evolution were exhibited.

Notes: A detailed histochemical analysis of the expression of the soybean small heat shock protein gene promoter, GmHSP 17·5E, fused to the GUS reporter gene, has been made in all organs and tissues of the flower as a function of stage of development and heat stress. This promoter is not uniformly expressed after a heat shock in all floral tissues and organs. Expression is seen at all stages of development in the sepals but not in the petals. The expression pattern in the pistil and in anthers is complex. Heat stress-induced GUS staining is seen in the style and upper portion of the ovary, but not in the stigmatic papillae or in the lower part of the ovary or in ovules. In stamens the heat shock response is seen in the filament and in the extension of the vascular tissue from the filament into the anther. No induction is seen in other tissues of the anther or in microspores or pollen at any stage of development. Vegetative organs in contrast are more uniform in the heat shock inducibility of GUS activity. Based on evidence from transient assays after microprojectile particle bombardment of the GmHSP 17·5E/GUS construct into pollen, it is likely that the gene is transcriptionally in an inactive configuration in pollen nuclei in stably transformed transgenic plants. These results are discussed with reference to other information in the literature.

Notes: The development of beech leaves (Fagus sylvatica L.) was characterized by determination of the pigment and electrolyte concentrations as well as the accumulation of dry mass and specific leaf mass from bud break to senescence. To test the hypothesis that stress tolerance and responsiveness of defences show developmental and/or seasonal changes, leaf discs were either incubated in the absence (control) or presence of paraquat to induce oxidative stress. Controls displayed developmental changes in stress susceptibility ranging from less than 15% of maximum electrolyte leakage in mature leaves to more than 20% leakage in senescent and 36–46% in immature leaves. Paraquat concentrations were chosen to result in about 95% of maximum electrolyte conductivity within 24 h in all developmental stages. Paraquat accumulation was about two-fold lower in senescent as compared with immature leaves, whereas stress susceptibility, as characterized by the kinetics of the increase in relative leakage, was similar in these developmental stages with 50% of maximum electrolyte conductivity (EC50) = 6·5 h in immature and 7·5 h in senescent leaves. In mature leaves with intermediate paraquat accumulation rates, two classes of stress-sensitivity were distinguished, namely stress-resistant and stress-susceptible leaves with EC50= 9·5 and 5·2 h, respectively. Stress-resistance of mature leaves was accompanied by a rapid, approximately two-fold induction of superoxide dismutase activity. Stress-sensitive mature leaves initially contained high superoxide dismutase activities but showed a rapid, more than six- fold loss in activity in 24 h. Correlation of meteorological data with leakage rates suggested that high air temperatures and low precipitation might have been predisposing for loss of resistance against oxidative stress in beech leaves.

Notes: Chilling (4 °C) induced a prolonged high level of intracellular Ca2+ (Ca2+ overload) and lipid peroxidation in maize (Zea mays L. cv Black Mexican Sweet) cultured cells. However, such Ca2+ overload and enhanced lipid peroxidation were not seen in abscisic acid (ABA)-treated cells, which had an improved chilling tolerance. A Ca2+ ionophore, A23187, caused Ca2+ overload in both ABA-treated maize cells and the untreated control, whereas an enhanced lipid peroxidation was detected only in the control. The high level of active oxygen species (AOS) in the control during chilling at 4 °C could be reduced by the presence of lanthanum (La3+), a Ca2+ channel blocker, in the medium. Moreover, both the A23187-induced lipid peroxidation and AOS production in the control could be reduced by extracellular EGTA, a Ca2+ chelator. Laser-scanning confocal microscopy revealed that mitochondria were one of the major AOS sources under chilling and during A23187 treatment. In vitro assays showed that superoxide production in isolated maize mitochondria was enhanced by the presence of Ca2+. Findings suggest that chilling-induced Ca2+ influx in the control triggers a marked generation of AOS, which in turn results in the enhanced lipid peroxidation. The ability of ABA-treated cells to avoid the chilling-induced Ca2+ influx may serve as a mechanism that prevents the chilling-induced oxidative stress and thus results in less chilling injury.

Notes: Cellular responses of 1-, 2- and 4-d-old Fucus spiralis embryos subjected to a single dose of elevated photosynthetically active photon flux density (PPFD), with or without ultraviolet (UV) radiation, were investigated by measuring the effects on the effective quantum yield of photosystem II (ΔF / Fm′) and intracellular production of active oxygen species (AOS). Production of AOS was determined by the in vivo conversion of 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-DCFH2-DA) to the fluorescent compound dichlorofluorescein (DCF) using confocal laser scan microscopy (CLSM) and image analysis. The role of xanthophyll cycle pigments in photoprotection was also assessed. A rapid decline in ΔF / Fm′ was observed under all elevated light conditions. A correlation was found between non-photochemical quenching and the de-epoxidation ratio zeaxanthin/(zeaxanthin + violoxanthin). Active oxygen formation increased with PPFD and was higher in older embryos and when UVB was present. Two photoinhibition responses were recognized: (i) a rapid decline of the PSII yield due to the violoxanthin–zeaxanthin cycle (photoprotection), and (ii) a slower second-phase decline, correlated with active oxygen production. Electron transport rate (ETR) increased with embryo age, and was correlated with AOS production. As a result of enhanced AOS production, there was a slow recovery of the PSII yield, in particular with increased effective UV dose. In general, embryos were able to recover from the imposed light conditions, but UVB had a more damaging effect. Overall, our data suggest that under natural conditions, embryos of F. spiralis are susceptible to elevated light levels, and that UVB radiation is an important stress factor.

Notes: Time series of net CO2 exchange (JCO2) and leaf conductivity for water vapour (gH2O) were measured and subsequently analysed mathematically in the Crassulacean acid metabolism (CAM) plant Kalanchoe daigremontiana (Hamet et Perrier de la Bâthie) under constant environmental conditions and under imposed external rhythms of lower and higher light intensity. The time series were analysed by Fourier methods and a correlation analysis considering the first time derivatives of JCO2, gH2Oand photosynthetically active photon flux density (PPFD). The ratio of internal to external CO2 (ci/ca) was also considered in the analysis, leading to a discussion of the interaction of stomata and carbon assimilation under periodic stimulation. It is suggested that for stimulation with frequencies close to the endogeneous circadian period, stomatal conductance and carbon assimilation oscillate synchronously, guard-cell movements trailing behind changes in internal CO2 with a delay of 10–15 min. For stimulation frequencies far shorter than the endogeneous period, this synchrony can be disturbed due to independent responses of stomata and assimilation to light pulses, leading to an arrhythmic gas exchange pattern. These results are discussed in the context of understanding circadian oscillations as the output of a multioscillator, multisignalling pathway system on the organismic and metabolic level.

Notes: The two most common oak species in western Europe, Quercus robur and Quercus petraea, display different ecological behaviours, particularly with respect to their responses to drought. The ecophysiological basis of this niche difference is not understood well. Here we test the hypothesis that these two species present distinct water use efficiencies (WUEs), using the carbon isotope discrimination approach. Leaves and 13 dated ring sequences were sampled in 10 pairs of adult trees growing side by side. Carbon isotope composition was measured on cellulose extracts. In addition, relationships between carbon isotope discrimination and wood anatomy were assessed at the tree level. Quercus robur displayed a 1·0‰ larger isotopic discrimination than Q. petraea, and therefore a lower intrinsic WUE (−13%). This interspecific difference of isotopic discrimination was quite stable with time and independent of tree radial growth and climate fluctuations. A strong positive correlation was observed between average tree values of earlywood vessel surface area and 13C isotopic discrimination. This correlation was even higher with 13C of the 1976 dry year (r = 0·86). These observations led to the hypothesis that hydraulic properties of xylem could exert a constraint on leaf gas exchange, resulting in a larger WUE for individuals with smaller vessel cross-section area.

Notes: Vulnerability to cavitation of leaf minor veins and stems of Laurus nobilis L. was quantified together with that of leaflets, rachides and stems of Ceratonia siliqua L. during air-dehydration of 3-year-old branches. Embolism was estimated by counting ultrasound acoustic emissions (UAE) and relating them to leaf water potential (ΨL). The threshold ΨL for cavitation was less negative in L. nobilis than in C. siliqua according to the known higher drought resistance of the latter species. Leaf minor vein cavitation was also quantified by infiltrating leaves with fluorescein at different dehydration levels and observing them under microscope. Distinct decreases in the functional integrity of minor veins were observed during leaf dehydration, with high correlation between the two variables. The relationship between leaf conductance to water vapour (gL) and ΨL showed that stomata of L. nobilis closed in response to stem and not to leaf cavitation. However, in C. siliqua, gL decreased in coincidence to the leaf cavitation threshold, which was, nevertheless, very close to that of the stem. The hypothesis that stem cavitation acts as a signal for stomatal closure was confirmed, while the same role for leaf cavitation remains an open problem.

Notes: We characterized the response of root hair density to phosphorus (P) availability in Arabidopsis thaliana. Arabidopsis plants were grown aseptically in growth media with varied phosphorus concentrations, ranging from 1 mmol m−3 to 2000 mmol m−3 phosphorus. Root hair density (number of root hairs per mm of root length) was analysed starting at 7 d of growth. Root hair density was highly regulated by phosphorus availability, increasing significantly in roots exposed to low-phosphorus availability. The initial root hairs produced by the radicle were not sensitive to phosphorus availability, but began to respond after 9 d of growth. Root hair density was about five times greater in low phosphorus (1 mmol m−3) than in high phosphorus (1000 mmol m−3) media. Root hair density decreased logarithmically in response to increasing phosphorus concentrations within that range. Root hair density also increased in response to deficiencies of several other nutrients, but not as strongly as to low phosphorus. Indoleacetic acid (IAA), the auxin transport inhibitor 2-(p-chlorophenoxy)-2-methylpropionic acid (CMPA), the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), and the ethylene synthesis inhibitor amino-oxyacetic acid (AOA) all increased root hair density under high phosphorus but had very little effect under low phosphorus. Low phosphorus significantly changed root anatomy, causing a 9% increase in root diameter, a 31% decrease in the cross-sectional area of individual trichoblasts, a 40% decrease in the cross-sectional area of individual atrichoblasts, and 45% more cortical cells in cross-section. The larger number of cortical cells and smaller epidermal cell size in low phosphorus roots increased the number of trichoblast files from eight to 12. Two-thirds of increased root hair density in low phosphorus roots was caused by increased likelihood of trichoblasts to form hairs, and 33% of the increase was accounted for by changes in low phosphorus root anatomy resulting in an increased number of trichoblast files. These results show that phosphorus availability can fundamentally alter root anatomy, leading to changes in root hair density, which are presumably important for phosphorus acquisition.

Notes: Nitric oxide (NO) is a bioactive molecule that exerts a number of diverse activities in phylogenetically distant species, as well as opposing effects in related biological systems. It was firstly described in mammals as a major messenger in the cardiovascular, immune and nervous system, in which it plays regulatory, signalling, cytoprotective and cytotoxic effects (Ignarro, Annual Review of Pharmacology and Toxicology 30, 535–560, 1990; Anbar, Experientia 51, 545–550, 1995). This versatility is mainly achieved through interactions with targets via either a redox or an additive chemistry (Stamler, Cell 78, 931–936, 1994). For this reason, metal- and thiol-containing proteins serve as major target sites for NO: these include signalling proteins, receptors, enzymes, transcription factors and DNA, among others. Furthermore, NO is a small, highly diffusible molecule. It rapidly crosses biological membranes and triggers various different processes in a short period of time. In this context, NO can co-ordinate and regulate cellular functions of microsomes and organelles such as mitochondria. The ubiquity of NO reactions, as well as the finding that the biochemical and molecular mechanisms underlying many physiological processes are well conserved between diverse species, have opened the exploration of NO chemistry in different organisms. Among these, plants were not the exception. The research in plants has been focused on three main fields: (i) the search for NO or any source of NO generation; (ii) the examination of the effects of NO upon exogenous treatments; and (iii) the search for the same molecules involved in NO-sensitive transduction pathways as in animals (e.g. cGMP, Ca2+, calmodulin). As it is evident from this review, recent progress on NO functionality in plants has been impressive. With the use of biochemistry, molecular genetics and structural biology, together with classical physiological approaches, an explosion of new discoveries will surely begin. It is certainly a good time for plant biologists.

Notes: Gibberellin (GA) and cytokinin (CK) were exogenously supplied at different periods of the vegetative development in Sorghum bicolor. Growth response to these hormonal treatments differed according to the developmental stage. This reveals the existence of discrete phenophases, each one characterized by a specific sensitivity to plant growth regulations (PGRs). Developmental changes in sensitivity were less accentuated in plants grown in optimal conditions than in plants exposed to 150 mM NaCl. Variations in organ connectance (the level of coordination in growth of the shoot, adventitious roots and seminal root) were analysed during vegetative growth of salt-treated plants. This analysis shows a temporary decrease in connectance during the transition period between phenophases. From the effect of hormonal treatments on connectance, it was concluded that (i) the transition period coincides with a partial dismantling of the initial regulatory network followed by the emergence of a new network coordinating growth of the different organs; (ii) GA is involved in the process of emergence of a transition period; and (iii) duration of the transition period is considerably enlarged for plants exposed to NaCl stress. This dynamics of alternance of phenophase and transition periods enables the integration of the two modes of action of the PGRs (dose–response and change in sensitivity) within a unified framework.

Notes: Abscisic acid (ABA) integrates the water status of a plant and causes stomatal closure. Physiological mechanisms remain poorly understood, however, because guard cells flanking stomata are small and contain only attomol quantities of ABA. Here, pooled extracts of dissected guard cells of Vicia faba L. were immunoassayed for ABA at sub-fmol sensitivity. A pulse of water stress was imposed by submerging the roots in a solution of PEG. The water potentials of root and leaf declined during 20 min of water stress but recovered after stress relief. During stress, the ABA concentration in the root apoplast increased, but that in the leaf apoplast remained low. The ABA concentration in the guard-cell apoplast increased during stress, providing evidence for intra-leaf ABA redistribution and leaf apoplastic heterogeneity. Subsequently, the ABA concentration of the leaf apoplast increased, consistent with ABA import via the xylem. Throughout, the ABA contents of the guard-cell apoplast, but not the guard-cell symplast, were convincingly correlated with stomatal aperture size, identifying an external locus for ABA perception under these conditions. Apparently, ABA accumulates in the guard-cell apoplast by evaporation from the guard-cell wall, so the ABA signal in the xylem is amplified maximally at high transpiration rates. Thus, stomata will display apparently higher sensitivity to leaf apoplastic ABA if stomata are widely open in a relatively dry atmosphere.

Notes: Alfalfa (Medicago sativa L.) N-sufficient plants were fed 1·5 mM N in the form of NO3−, NH4+ or NO3− in conjunction with NH4+, or were N-deprived for 2 weeks. The specific activity of phosphoenolpyruvate carboxylase (PEPC) from the non-nodulated roots of N-sufficient plants was increased in comparison with that of N-deprived plants. The PEPC value was highest with NO3− nutrition, lowest with NH4+ and intermediate in plants that were fed mixed salts. The protein was more abundant in NO3−-fed plants than in either NH4+- or N mixed-fed plants. Nitrogen starvation decreased the level of PEPC mRNA, and nitrate was the N form that most stimulated PEPC gene expression. The malate content was significantly lower in NO3−-deprived than in NO3−-sufficient plants. Root malate accumulation was high in NO3−-fed plants, but decreased significantly in plants that were fed with NH4+. The effect of malate on the desalted enzyme was also investigated. Root PEPC was not very sensitive to malate and PEPC activity was inhibited only by very high concentrations of malate. Asparagine and glutamine enhanced PEPC activity markedly in NO3−-fed plants, but failed to affect plants that were either treated with other N types or N starved. Glutamate and citrate inhibited PEPC activity only at optimal pH. N-nutrition also influenced root nitrate and ammonium accumulation. Nitrate accumulated in the roots of NO3−- and (NO3− + NH4+)-fed plants, but was undetectable in those administered NH4+. Both the nitrate and the ammonium contents were significantly reduced in NO3−- and (NO3− + NH4+)-starved plants. Root accumulation of free amino acids was strongly influenced by the type of N administered. It was highest in NH4+-fed plants and the most abundant amides were asparagine and glutamine. It was concluded that root PEPC from alfalfa plants is N regulated and that nitrate exerts a strong influence on the PEPC enzyme by enhancing both PEPC gene expression and activity.

Notes: Sugars are not only metabolic substrates: they also act as signals that regulate the metabolism of plants. Previously, we found that glycolysis is induced in transgenic tubers expressing a yeast invertase in the cytosol but not in those expressing invertase in the apoplast. This suggests that either the low level of sucrose, the increased formation of cytosolic glucose or the increased levels of metabolites downstream of the sucrose cleavage is responsible for the induction of glycolysis in storage organs. In order to discriminate between these possibilities, we cloned and expressed a bacterial sucrose phosphorylase gene from Pseudomonas saccharophila in potato tubers. Due to the phosphorolytic cleavage of sucrose, formation of glucose was circumvented, thus allowing assessment of the importance of cytosolic glucose – and, by implication, flux through hexokinase – in glycolytic induction. Expression of sucrose phosphorylase led to: (i) a decrease in sucrose content, but no decrease in glucose or fructose; (ii) a decrease in both starch accumulation and tuber yield; (iii) increased levels of glycolytic metabolites; (iv) an induction of the activities of key enzymes of glycolysis; and (v) increased respiratory activity. We conclude that the induction of glycolysis in heterotrophic tissues such as potato tubers occurs via a glucose-independent mechanism.

Notes: Extracts of the central infected zone and the surrounding cortex of nodules from Lupinus angustifolius L., Vigna unguiculata L. (Walp), Pisum sativum L., Phaseolus vulgaris L., Vicia faba L. and Medicago sativa L. contained significant activities of carbonic anhydrase (CA). Immunoassay of extracts using antisera to a putative nodule CA (Msca1) cloned from M. sativa also indicated expression in both tissue types. Quantitative confocal microscopy using laser scanning imaging and a fluorescent CA-specific probe (5-dimethylaminonaphthalene-1-sulfonamide [DNSA]) localized expression to the infected cells in the central zone tissue and a narrow band of 2–3 files of cells in the cortical tissue that corresponded to the inner cortex. In the infected cells, the enzyme activity was distributed evenly in the cytosol, but in the inner cortical cells, it was restricted to the periphery – possibly to the plasma membrane or cell wall. The functions of CA in these two tissues are considered in relation to the carbon metabolism of nodules and the participation of the inner cortex in the regulation of gaseous diffusive resistance.

Notes: Leaf gas exchange parameters and the content of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the leaves of two 2-year-old aspen (Populus tremuloides Michx.) clones (no. 216, ozone tolerant and no. 259, ozone sensitive) were determined to estimate the relative stomatal and mesophyll limitations to photosynthesis and to determine how these limitations were altered by exposure to elevated CO2 and/or O3. The plants were exposed either to ambient air (control), elevated CO2 (560 p.p.m.) elevated O3 (55 p.p.b.) or a mixture of elevated CO2 and O3 in a free air CO2 enrichment (FACE) facility located near Rhinelander, Wisconsin, USA. Light-saturated photosynthesis and stomatal conductance were measured in all leaves of the current terminal and of two lateral branches (one from the upper and one from the lower canopy) to detect possible age-related variation in relative stomatal limitation (leaf age is described as a function of leaf plastochron index). Photosynthesis was increased by elevated CO2 and decreased by O3 at both control and elevated CO2. The relative stomatal limitation to photosynthesis (ls) was in both clones about 10% under control and elevated O3. Exposure to elevated CO2 + O3 in both clones and to elevated CO2 in clone 259, decreased ls even further – to about 5%. The corresponding changes in Rubisco content and the stability of Ci/Ca ratio suggest that the changes in photosynthesis in response to elevated CO2 and O3 were primarily triggered by altered mesophyll processes in the two aspen clones of contrasting O3 tolerance. The changes in stomatal conductance seem to be a secondary response, maintaining stable Ci under the given treatment, that indicates close coupling between stomatal and mesophyll processes.

Notes: Studies of uptake of ionic sources of N by two hydroponically grown rice (Oryza sativa L.) cultivars (paddy-field-adapted Koshihikari and dryland-adapted Kanto 168) showed that the magnitude of the nitrogen isotope fractionation (ɛ) for uptake of NH4+ depended on the concentrations of NH4+ and cultivar (averaging –6·1‰ for Koshihikari and –12·0‰ for Kanto 168 at concentrations from 40 to 200 mmol m−3 and, respectively, –13·4 and –28·9‰ for the two cultivars at concentrations from 0·5 to 4 mol m−3). In contrast, the ɛ for uptake of NO3− in similar experiments was almost insensitive to the N concentration, falling within a much narrower range (+3·2‰ to –0·9‰ for Koshihikari and –0·9‰ to –5·1‰ for Kanto 168 over NO3− concentrations from 0·04 to 2 mol m−3). From longer term experiments in which Norin 8 and its nitrate-reductase deficient mutant M819 were grown with 2 or 8 mol m−3 NO3− for 30 d, it was concluded that the small concentration-independent isotopic fractionation during absorption of this ion was not related to nitrate reductase activity.

Notes: The consequences of manipulating abscisic acid (ABA) biosynthesis rates on stomatal response to drought were analysed in wild-type, a full-deficient mutant and four under-producing transgenic lines of N. plumbaginifolia. The roles of ABA, xylem sap pH and leaf water potential were investigated under four experimental conditions: feeding detached leaves with varying ABA concentration; injecting exogenous ABA into well-watered plants; and withholding irrigation on pot-grown plants, either intact or grafted onto tobacco. Changes in ABA synthesis abilities among lines did not affect stomatal sensitivity to ABA concentration in the leaf xylem sap ([ABA]xyl), as evidenced with exogenous ABA supplies and natural increases of [ABA]xyl in grafted plants subjected to drought. The ABA-deficient mutant, which is uncultivable under normal evaporative demand, was grafted onto tobacco stock and then presented the same stomatal response to [ABA]xyl as wild-type and other lines. This reinforces the dominant role of ABA in controlling stomatal response to drought in N. plumbaginifolia whereas roles of leaf water potential and xylem sap pH were excluded under all studied conditions. However, when plants were submitted to soil drying onto their own roots, stomatal response to [ABA]xyl slightly differed among lines. It is suggested, consistently with all the results, that an additional root signal of soil drying modulates stomatal response to [ABA]xyl.

Notes: The effects of the treatment of different tissues of three bean cultivars (Pinto, Vilmorin and Arroz) with ultra-violet (UV) UV-B and UV-C radiation and red light on the activity, quantity and RNA levels of NADP-malic enzyme (NADP-ME) were determined. Exposure to UV-B radiation for 8 h caused a marked increase of NADP-ME from leaves, stems and roots in the three cultivars studied. A similar induction was observed in the leaves and stems after 8 h of exposure under UV-C, but not in the roots, suggesting that a different signal might be acting to induce the expression of NADP-ME after UV-B and UV-C exposure. In contrast, red light was ineffective in inducing NADP-ME in either tissue, so the regulation of the expression of this enzyme is phytocrome-independent. The activity of superoxide dismutase, ascorbate peroxidase, catalase and peroxidase was also different in plants treated with UV-B, UV-C and photosynthetically active radiation, suggesting that various pathways may be acting in the regulation of these enzymes by UV-B and UV-C. Reactive oxygen species (ROS) were also required for UV-B induction of NADP-ME, as the addition of ascorbic acid before UV-B treatment prevented NADP-ME induction, whereas salicylic acid was not effective in inducing the enzyme, showing that NADP-ME induction by UV-B is ROS dependent but salicylic acid independent.

Notes: The variations of δ13C in leaf metabolites (lipids, organic acids, starch and soluble sugars), leaf organic matter and CO2 respired in the dark from leaves of Nicotiana sylvestris and Helianthus annuus were investigated during a progressive drought. Under well-watered conditions, CO2 respired in the dark was 13C-enriched compared to sucrose by about 4‰ in N. sylvestris and by about 3‰ and 6‰ in two different sets of experiments in H. annuus plants. In a previous work on cotyledonary leaves of Phaseolus vulgaris, we observed a constant 13C-enrichment by about 6‰ in respired CO2 compared to sucrose, suggesting a constant fractionation during dark respiration, whatever the leaf age and relative water content. In contrast, the 13C-enrichment in respired CO2 increased in dehydrated N. sylvestris and decreased in dehydrated H. annuus in comparison with control plants. We conclude that (i) carbon isotope fractionation during dark respiration is a widespread phenomenon occurring in C3 plants, but that (ii) this fractionation is not constant and varies among species and (iii) it also varies with environmental conditions (water deficit in the present work) but differently among species. We also conclude that (iv) a discrimination during dark respiration processes occurred, releasing CO2 enriched in 13C compared to several major leaf reserves (carbohydrates, lipids and organic acids) and whole leaf organic matter.

Notes: The interactive effects of elevated atmospheric CO2 and temperature on seasonal patterns of photosynthesis in Douglas fir (Psuedotsuga menziesii (Mirb.) Franco) seedlings were examined. Seedlings were grown in sunlit chambers controlled to track either ambient (~400 p.p.m.) CO2 or ambient +200 p.p.m. CO2, and either ambient temperature or ambient +4 °C. Light-saturated net photosynthetic rates were measured approximately monthly over a 21 month period. Elevated CO2 increased net photosynthetic rates by an average of 21% across temperature treatments during both the 1996 hydrologic year, the third year of exposure, and the 1997 hydrologic year. Elevated mean annual temperature increased net photosynthetic rates by an average of 33% across CO2 treatments during both years. Seasonal temperature changes also affected net photosynthetic rates. Across treatments, net photosynthetic rates were highest in the spring and autumn, and lowest in July, August and December–January. Seasonal increases in temperature were not correlated with increases in the relative photosynthetic response to elevated CO2. Seasonal shifts in the photosynthetic temperature optimum reduced temperature effects on the relative response to elevated CO2. These results suggest that the effects of elevated CO2 on net photosynthetic rates in Douglas fir are largely independent of temperature.

Notes: Relationships between growth, nitrogen and concentration of unique biont components were investigated for the tripartite lichens Nephroma arcticum (L.) Torss. and Peltigera aphthosa (L.) Willd. Nitrogen availability was manipulated during 4 summer months by removing cephalodia and their associated N2 fixation activity, or by weekly irrigation with NH4NO3. Chlorophyll and ribulose 1·5-biphosphate carboxylase/oxygenase (Rubisco), and chitin and ergosterol were used as photobiont and mycobiont markers, respectively. Nitrogen concentrations were similar in older and newer parts of the same thallus, varying between 2 and 5 g m−2, with P. aphthosa having higher concentrations than N. arcticum. Both chlorophyll (Chl a) and chitin were linearly correlated with thallus N, but N. arcticum invested more in fungal biomass and had lower Chl a concentrations in comparison with P. aphthosa at equal thallus N. During the 4 months, control and N-fertilized thalli of N. arcticum increased in area by 0·2 m2 m−2 and P. aphthosa by 0·4 m2 m−2. Thallus expansion was significantly inhibited in samples without cephalodia, but there was no effect on lichen weight gain. Mean relative growth rate (RGR; mg g−1 d−1) was 3·8 for N. arcticum and 8·4 for P. aphthosa, when time (d) reflected the lichen wet periods. RGR was 2–3 times lower when based on the whole time, i.e. when including dry periods. The efficiency (e) of converting incident irradiance into lichen biomass was positively and linearly correlated with thallus Chl a concentration to the same extent in both species. The slower growth rates of N. arcticum, in comparison with P. aphthosa, could then be explained by their lower nitrogen and Chl a concentrations and a subsequently lower light energy conversion efficiency. Functional and dynamic aspects of resource allocation patterns of the two lichens are discussed in relation to the above findings.

Notes: The past few years have provided many advances in the role and biosynthesis of L-ascorbic acid (AsA) in plants. There is an increasing body of evidence confirming that AsA plays an important role in the detoxification of reactive oxygen species. The role of AsA in photoprotection has been confirmed in vivo with the use of Arabidopsis mutants. A player in the defence against reactive oxygen species, AsA peroxidase, has been extensively studied at the molecular level, and regulation of this key enzymatic activity appears to occur at several levels. As a cofactor in the hydroxylation of prolyl and lysl-residues by peptidyl-prolyl and -lysyl hydroxylases, AsA plays a part in cell wall synthesis, defence, and possibly cell division. The maintenance of reduced levels of AsA appears to be highly regulated, involving the interplay of both monodehydroascorbate and dehydroascorbate reductases and possibly auxin. A major breakthrough in plant AsA biosynthesis has been made recently, and strong biochemical and genetic evidence suggest that GDP-mannose and L-galactose are key substrates. In addition, evidence for an alternative AsA biosynthetic pathway(s) exists and awaits additional scrutiny. Finally, newly described Arabidopsis mutants deficient in AsA will further increase our understanding of AsA biosynthesis

Notes: The addition of gibberellin A3 (GA3) to culture media induced a dramatic elongation of the first internode in Hong Mang Mai, a wheat variety tolerant to deep-seeding conditions. The length of the first internode in the GA3-treated seedlings reached approximately 45 cm, which is twice as long as that of the control, whereas first internodes of other varieties were 3 to 12 cm long and hardly affected by GA3. The level of endogenous gibberellins (GA) in Hong Mang Mai was not greater than that of other wheat varieties. The expression of GAMyb, a transcription factor, was abundant in the first internode and substantially increased by GA3 application in Hong Mang Mai, compared with other wheat varieties. These results suggest that the first internode of Hong Mang Mai is more sensitive to GA in inducing strong elongation. The presence of potassium in the culture media was indispensable for the first internode elongation. Application of GA3 enhanced the uptake of potassium in Hong Mang Mai. Higher sensitivity of the first internode to GA could cause strong elongation by increasing the amount of osmotic solute, which plays an important role in the tolerance mechanism of Hong Mang Mai to deep-seeding conditions.

Notes: This is the first report on using green fluorescent protein (GFP) as a pH reporter in plants. Proton fluxes and pH regulation play important roles in plant cellular activity and therefore, it would be extremely helpful to have a plant gene reporter system for rapid, non-invasive visualization of intracellular pH changes. In order to develop such a system, we constructed three vectors for transient and stable transformation of plant cells with a pH-sensitive derivative of green fluorescent protein. Using these vectors, transgenic Arabidopsis thaliana and tobacco plants were produced. Here the application of pH-sensitive GFP technology in plants is described and, for the first time, the visualization of pH gradients between different developmental compartments in intact whole-root tissues of A. thaliana is reported. The utility of pH-sensitive GFP in revealing rapid, environmentally induced changes in cytoplasmic pH in roots is also demonstrated.

Notes: Leaf photosynthesis (Ps), nitrogen (N) and light environment were measured on Populus tremuloides trees in a developing canopy under free-air CO2 enrichment in Wisconsin, USA. After 2 years of growth, the trees averaged 1·5 and 1·6 m tall under ambient and elevated CO2, respectively, at the beginning of the study period in 1999. They grew to 2·6 and 2·9 m, respectively, by the end of the 1999 growing season. Daily integrated photon flux from cloud-free days (PPFDday,sat) around the lowermost branches was 16·8 ± 0·8 and 8·7 ± 0·2% of values at the top for the ambient and elevated CO2 canopies, respectively. Elevated CO2 significantly decreased leaf N on a mass, but not on an area, basis. N per unit leaf area was related linearly to PPFDday,sat throughout the canopies, and elevated CO2 did not affect that relationship. Leaf Ps light-response curves responded differently to elevated CO2, depending upon canopy position. Elevated CO2 increased Pssat only in the upper (unshaded) canopy, whereas characteristics that would favour photosynthesis in shade were unaffected by elevated CO2. Consequently, estimated daily integrated Ps on cloud-free days (Psday,sat) was stimulated by elevated CO2 only in the upper canopy. Psday,sat of the lowermost branches was actually lower with elevated CO2 because of the darker light environment. The lack of CO2 stimulation at the mid- and lower canopy was probably related to significant down-regulation of photosynthetic capacity; there was no down-regulation of Ps in the upper canopy. The relationship between Psday,sat and leaf N indicated that N was not optimally allocated within the canopy in a manner that would maximize whole-canopy Ps or photosynthetic N use efficiency. Elevated CO2 had no effect on the optimization of canopy N allocation.

Notes: Previous studies indicate that methylated cyclitols are potentially important osmolytes in plants. In a search for genetic diversity for pinitol (D-3-O-methyl-chiro-inositol) accumulation in soybean (Glycine max (L.) Merr.), two- to three-fold differences in pinitol accumulation in leaf blades were found among Chinese plant introductions. Furthermore, it was found that genotypes that accumulated high concentrations of pinitol, when grown under well-watered conditions, had been selected for performance in regions of China having low rainfall. Among the carbohydrates analysed, pinitol accumulation was uniquely associated with adaptation to dry areas of China. A detailed study of pinitol accumulation in the soybean plant showed two- to three-fold gradients in pinitol concentration from the bottom to the top of the plant. The gradient shifted during plant development, with consistently higher concentrations of pinitol in the uppermost leaves. Pinitol accumulation was not correlated with activity of the key biosynthetic enzyme, inositol methyl transferase. This result and other lines of evidence indicated that shifting patterns of pinitol accumulation were due to translocation of the cyclitol from lower to upper nodes. Pinitol, proline, and sugars accumulated in leaf blades on soybean plants subjected to drought, but the molar concentration of pinitol in stressed plants was greater than the concentrations of proline or sugars. Although the mechanism by which pinitol participates in drought tolerance is not fully known, our results provide additional correlative evidence linking pinitol and drought tolerance in soybean.

Notes: Plant culture in oxygen concentrations below ambient is known to stimulate vegetative growth, but apart from reports on increased leaf number and weight, little is known about development at subambient oxygen concentrations. Arabidopsis thaliana (L.) Heynh. (cv. Columbia) plants were grown full term in pre-mixed atmospheres with oxygen partial pressures of 2·5, 5·1, 10·1, 16·2, and 21·3 kPa O2, 0·035 kPa CO2 and the balance nitrogen under continuous light. Fully expanded leaves were harvested and processed for light and transmission electron microscopy or for starch quantification. Growth in subambient oxygen concentrations caused changes in leaf anatomy (increased thickness, stomatal density and starch content) that have also been described for plants grown under carbon dioxide enrichment. However, at the lowest oxygen treatment (2·5 kPa), developmental changes occurred that could not be explained by changes in carbon budget caused by suppressed photorespiration, resulting in very thick leaves and a dwarf morphology. This study establishes the leaf parameters that change during growth under low O2, and identifies the lower concentration at which O2 limitation on transport and biosynthetic pathways detrimentally affects leaf development.

Notes: The impact of inoculation with Paxillus involutus on the utilization of organic carbon compounds by birch roots was studied by feeding [14C]Glu or [14C]malate to the partners of the symbiosis, separately or in association, and by monitoring the subsequent distribution of 14C. Inoculation increased [14C]Glu and [14C]malate absorption capacities by up to eight and 17 times, respectively. Six- and 15-d-old mycorrhizal roots showed about four-fold higher [14C]Glu and [14C]malate absorption capacities compared with 60-d-old mycorrhizal roots, suggesting that the early stages of mycorrhiza formation induced higher requirements for C skeletons. Moreover, the results demonstrated that inoculation strongly modified the fate of [14C]Glu and [14C]malate. It was demonstrated that exogenously supplied Glu and malate might serve as C skeletons for amino acid synthesis in mycorrhizal birch roots and in the free-living fungus. Gln was the major 14C-sink in mycorrhizal roots and in the free-living P. involutus. In contrast, citrulline and insoluble compounds were the major 14C sinks in non-mycorrhizal roots, whatever the 14C source. It was concluded that mycorrhiza formation leads to a profound alteration of the metabolic fate of exogenously supplied C compounds. The ecological significance of amino acid and organic acid utilization by mycorrhizal plants is further discussed.

Notes: Changes in the amount of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4·1·1·39) synthesized and degraded and the levels of rbcL and rbcS mRNAs were examined in the eighth leaf blades of rice from emergence to senescence. Synthesis of Rubisco was very active during leaf expansion, became quite low at the time of full expansion and then declined further during senescence. The changes in the levels of rbcL and rbcS mRNAs co-ordinated approximately with those in the amount of Rubisco synthesized. Thus, it is suggested that the amount of Rubisco synthesized is determined primarily by the levels of rbcL and rbcS mRNAs during the life span of the leaves. Degradation of Rubisco started just before the time of full expansion and became far more active than its synthesis during senescence. Since the synthesis of Rubisco during senescence scarcely contributed to its amount, it can be concluded that the degradation of Rubisco is the major determinant for the amount of Rubisco in senescent leaves. The decline in the level of rbcL mRNA occurred much earlier in the developmental stage and proceeded at a much faster rate than that of rbcL DNA, indicating that the level of rbcL DNA is not a major determinant for the level of rbcL mRNA in senescent leaves of rice.

Notes: Aerenchyma formation in roots of maize (Zea mays L.) involves programmed death of cortical cells that is promoted by exogenous ethylene (1 µL L−1) or by endogenous ethylene produced in response to external oxygen shortage (3%, v/v). In this study, evidence that degeneration of the cell wall accompanies apoptotic-like changes previously observed in the cytoplasm and nucleus (Gunawardena et al. Planta 212, 205–214, 2001), has been sought by examining de-esterified pectins (revealed by monoclonal antibody JIM 5), and esterified pectins (revealed by monoclonal antibody JIM 7). In controls, de-esterified wall pectins were found at the vertices of triangular junctions between cortical cells (untreated roots). Esterified pectins in control roots were present in the three walls bounding triangular cell-to-cell junctions. After treatment with 3% oxygen or 1 µL L−1 ethylene, this pattern was lost but walls surrounding aerenchyma gas spaces became strongly stained. The results showed that cell wall changes commenced within 0·5 d and evidently were initiated by ethylene in parallel with cytoplasmic and nucleoplasmic events associated with classic intracellular processes of programmed cell death.

Notes: It emerged recently that there is an inter-relationship between drought and ultraviolet-B (UV-B) radiation in plant responses, in that both stresses provoke an oxidative burst. The purpose of this investigation was to compare the effects and interaction of drought and UV-B in wheat and pea. The absence of changes in relative leaf water content (RWC) after UV-B treatments indicate that changes in water content were not involved. RWC was the main factor resulting in reduced growth in response to drought. Increases in anthocyanin and phenols were detected after exposure to UV-B. The increases do not appear to be of sufficient magnitude to act as a UV-B screen. UV-B application caused greater membrane damage than drought stress, as assessed by lipid peroxidation as well as osmolyte leakage. An increase in the specific activities of antioxidant enzymes was measured after UV-B alone as well as after application to droughted plants. Proline increased primarily in drought-stressed pea or wheat. Proline may be the drought-induced factor which has a protective role in response to UV-B. The physiological and biochemical parameters measured indicate the UV-B light has stronger stress effectors than drought on the growth of seedlings of both species. The two environmental stresses acted synergistically to induce protective mechanisms in that pre-application of either stress reduced the damage caused by subsequent application of the other stress.

Notes: Etiolated seedlings of wild-type wheat and a transgenic line overexpressing an oat PHYA gene were investigated by the use of in situ low-temperature fluorescence spectroscopy. The red-absorbing phytochrome form, Pr, was characterized by (1) fluorescence emission spectrum; (2) total phytochrome content, and (3) by the extent of the Pr → lumi-R photoconversion at low temperature (γ1), and of the Pr → Pfr photoconversion at ambient temperature (γ2) as derived from emission data. All the characteristics were shown to be variable and to depend on (1) organ and tissue used; (2) seedling age; (3) transgenic wheat modification, and (4) continuous far-red irradiation of seedlings during their growth. These variations were interpreted in terms of the existence in wheat seedlings of the two phenomenological Pr types: (a), Pr′– major longer wavelength (687/673 nm, emission/absorption maxima) variable and light-labile with γ1 ≈ 0·5; and (b), Pr′′– minor, shorter wavelength (682/668 nm), relatively constant with its concentration not changing significantly with the increase of total phytochrome content in tissues and light-stable with γ1 ≤ 0·05–0·1. Overexpression of oat phyA increases primarily the content of Pr′ suggesting that it is comprised of phyA (phyA′) whereas Pr′′ is believed to consist of the minor phyA fraction (phyA′′) and phyB. The transgenic wheat line has been demonstrated to have a modified phenotype – the appearance of the far-red high irradiance reaction (FR-HIR) (Shlumukov et al. Plant, Cell and Environment 24, 703–712). The increased content of phyA′ in the transgenic line, whereas the total [phyA′′ + phyB] remains the same as in the wild type, indicates that the phyA′ pool is primarily responsible for the observed modification of the phenotype and suggests that even in wild-type plants the phyA′ component of the phyA pool may mediate the FR-HIR.

Notes: A canopy photosynthesis model was modified to assess the effect of photoinhibition on whole-plant carbon gain. Photoinhibitory changes in maximum quantum yield of photosystem II (Fv/Fm) could be explained solely from a parameter (Lflux) calculated from the light micro-environment of the leaves. This relationship between Fv/Fm and the intercepted cumulative light dose, integrated and equally weighted over several hours was incorporated into the model. The effect of photoinhibition on net photosynthesis was described through relationships between photoinhibition and the shaping parameters of the photosynthetic light-response curve (quantum use efficiency, convexity, and maximum capacity). This new aspect of the model was then validated by comparing measured field data (diurnal courses of Fv/Fm) with simulation results. Sensitivity analyses revealed that the extent of photoinhibitory reduction of whole-plant photosynthesis was strongly dependent on the structural parameters (LAI and leaf angle). Simulations for a Mediterranean evergreen oak, Quercus coccifera, under climatic conditions which cause mild photoinhibition revealed a daily loss of 7·5–8·5% of potential carbon gain in the upper sunlit canopy layers, a 3% loss in the bottom canopy, and an overall loss of 6·1%. Thus, this canopy photoinhibition model (CANO-PI) allows the quantitative evaluation of photoinhibition effects on primary production.

Notes: Pending cell death was visualized by thermographic imaging in bacterio-opsin transgenic tobacco plants. Cell death in these plants was characterized by a complex lesion phenotype. Isolated cell death lesions were preceded by a colocalized thermal effect, as previously observed at sites infected by tobacco mosaic virus (TMV) (Chaerle et al. 1999Nature Biotechnology 17, 813–816). However, in most cases, a coherent front of higher temperature, trailed by cell death, initiated at the leaf base and expanded over the leaf lamina. In contrast to the homogenous thermal front, cell death was first visible close to the veins, and subsequently appeared as discrete spots on the interveinal tissue, as cell death spread along the veins. Regions with visible cell death had a lower temperature because of water evaporation from damaged cells. In analogy with previous observations on the localized tobacco–TMV interaction (Chaerle et al. 1999), the kinetics of thermographic and continuous gas exchange measurements indicated that stomatal closure preceded tissue collapse. Localized spontaneous cell death could also be presymptomatically visualized in the Arabidopsis lsd2 mutant.

Notes: Photosynthetic capacity and leaf properties of sun and shade leaves of overstorey sweetgum trees (Liquidambar styraciflua L.) were compared over the first 3 years of growth in ambient or ambient + 200 μL L−1 CO2 at the Duke Forest Free Air CO2 Enrichment (FACE) experiment. We were interested in whether photosynthetic down-regulation to CO2 occurred in sweetgum trees growing in a forest ecosystem, whether shade leaves down-regulated to a greater extent than sun leaves, and if there was a seasonal component to photosynthetic down-regulation. During June and September of each year, we measured net photosynthesis (A) versus the calculated intercellular CO2 concentration (Ci) in situ and analysed these response curves using a biochemical model that described the limitations imposed by the amount and activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Vcmax) and by the rate of ribulose-1,5-bisphosphate (RuBP) regeneration mediated by electron transport (Jmax). There was no evidence of photosynthetic down-regulation to CO2 in either sun or shade leaves of sweetgum trees over the 3 years of measurements. Elevated CO2 did not significantly affect Vcmax or Jmax. The ratio of Vcmax to Jmax was relatively constant, averaging 2·12, and was not affected by CO2 treatment, position in the canopy, or measurement period. Furthermore, CO2 enrichment did not affect leaf nitrogen per unit leaf area (Na), chlorophyll or total non-structural carbohydrates of sun or shade leaves. We did, however, find a strong relationship between Na and the modelled components of photosynthetic capacity, Vcmax and Jmax. Our data over the first 3 years of this experiment corroborate observations that trees rooted in the ground may not exhibit symptoms of photosynthetic down-regulation as quickly as tree seedlings growing in pots. There was a strong sustained enhancement of photosynthesis by CO2 enrichment whereby light-saturated net photosynthesis of sun leaves was stimulated by 63% and light-saturated net photosynthesis of shade leaves was stimulated by 48% when averaged over the 3 years. This study suggests that this CO2 enhancement of photosynthesis will be sustained in the Duke Forest FACE experiment as long as soil N availability keeps pace with photosynthetic and growth processes.

Notes: Acclimation of respiration to the light environments is important for a plant’s carbon balance. Respiratory rates of mature leaves of Alocasia odora, a typical shade-tolerant species, were measured during the night for 14 d after reciprocal transfers between high- (330 µ mol m−2 s−1) and low-light (20 µ mol m−2 s−1) environments. Following the transfer, both the rate of CO2 efflux and that of O2 uptake of A. odora leaves adjusted to the new light environments. The O2-uptake rates changed more slowly than the CO2-efflux rates under the new environments. Leaf mass per area also changed after the transfer. We analysed whether substrate availability or ATP-consumption rates influence the respiratory acclimation. Since the addition of sucrose to leaf segments did not influence the O2-uptake rates, the change of respiratory substrate availability was not responsible for the respiratory acclimation. The addition of an uncoupler induced increases in the O2-uptake rates, and the degree of enhancement significantly decreased after the transfer from low to high irradiance. Thus, the change in ATP-consumption rates was responsible for the changes in respiratory rates in the plants transferred from low to high light. Potential rates of O2 uptake, as measured in the presence of both the substrate and the uncoupler, changed after the transfer, and strongly correlated with the O2-uptake rates, irrespective of the directions of transfer (r = 0·961). There was a strong correlation between maximal activities of NAD-isocitrate dehydrogenase and the potential rates of O2 uptake (r = 0·933), but a weaker correlation between those of cytochrome c oxidase and the potential rates (r = 0·689). These data indicate that the changes of light environments altered the respiratory rates via the change of the respiratory ATP demand, and that the altered rates of respiration will induce the changes of the respiratory capacities.

Notes: Humidity in a small area of a Vicia faba L. leaf was perturbed with a flow of dry air from an 80 µm (inside diameter) needle, while the remainder of the leaf was maintained at high and constant humidity. The influence of the needle flow on the humidity at the leaf surface was quantified by using a spatially explicit dewpoint hygrometer to observe condensation patterns. When the dry air from a needle was applied to the leaf, stomata within the influence of the needle opened within the first few minutes of the perturbation, and local epidermal turgor pressure declined within the same time frame. When the needle flow was removed from the leaf, these responses were reversed, but with more variable kinetics. Stomata and epidermal cells outside the influence of the needle flow, which were exposed to a constant and high humidity, showed similar, but smaller, responses when the needle flow was applied to the leaf. Since the opening of these stomata should have had only a small effect on transpiration (because of the high humidity), it is likely that the reduction in epidermal turgor was the cause (rather than the result) of the stomatal opening. The magnitude of the turgor response was only loosely related to the distance from the needle flow up to distances of almost 400 µm. The data support the idea that neighbouring stomata can interact through the influence of transpiration on epidermal turgor.

Notes: Temporal trends in photosynthetic capacity are a critical factorin determining the seasonality and magnitude of ecosystem carbonfluxes. At a mixed deciduous forest in the south-eastern United States (Walker Branch Watershed, Oak Ridge, TN, USA), we independently measured seasonal trends in photosynthetic capacity (using single-leaf gas exchange techniques) and the whole-canopycarbon flux (using the eddy covariance method). Soil respiration was also measured using chambers and an eddy covariance system beneath the canopy. These independent chamber and eddy covariance measurements, along with a biophysical model (CANOAK), areused to examine how leaf age affects the seasonal pattern of carbon uptake during the growing season. When the measured seasonality in photosynthetic capacity is representedin the CANOAK simulations, there is good agreement with the eddy covariance data on the seasonal trends in carbon uptake. Removing the temporal trends in the simulations by using the early season maximum value of photosynthetic capacity over the entire growing season over estimates the annual carbon uptake by about 300 g C m−2 year−1– halfthe total estimated annual net ecosystem exchange. Alternatively, use of the mean value of photosynthetic capacity incorrectly simulates the seasonality in carbon uptake by the forest. In addition to changes related to leaf development and senescence, photosynthetic capacitydecreased in the middle and late summer, even when leaf nitrogenwas essentially constant. When only these middle and late summer reductions were neglected in the model simulations, CANOAK still overestimated the carbon uptake by an amount comparable to 25% ofthe total annual net ecosystem exchange.

Notes: The water relations and hydraulic architecture of growing grass tillers (Festuca arundinacea Schreb.) are reported. Evaporative flux density, E (mmol s−1 m−2), of individual leaf blades was measured gravimetrically by covering or excision of entire leaf blades. Values of E were similar for mature and elongating leaf blades, averaging 2·4 mmol s−1 m−2. Measured axial hydraulic conductivity, Kh (mmol s−1 mm MPa−1), of excised leaf segments was three times lower than theoretical hydraulic conductivity (Kt) calculated using the Poiseuille equation and measurements of vessel number and diameter. Kt was corrected (Kt*) to account for the discrepancy between Kh and Kt and for immature xylem in the basal expanding region of elongating leaves. From base to tip of mature leaves the pattern of Kt* was bell-shaped with a maximum near the sheath–blade joint (≈ 19 mmol s−1 mm MPa−1). In elongating leaves, immature xylem in the basal growing region led to a much lower Kt*. As the first metaxylem matured, Kt* increased by 10-fold. The hydraulic conductances of the whole root system, 〈inlineGraphic alt="inline image" href="urn:x-wiley:01407791:PCE657:PCE_657_mu1" location="equation/PCE_657_mu1.gif"/〉 (mmol s−1 MPa−1) and leaf blades, 〈inlineGraphic alt="inline image" href="urn:x-wiley:01407791:PCE657:PCE_657_mu2" location="equation/PCE_657_mu2.gif"/〉 (mmol s−1 MPa−1) were measured by a vacuum induced water flow technique. 〈inlineGraphic alt="inline image" href="urn:x-wiley:01407791:PCE657:PCE_657_mu1" location="equation/PCE_657_mu1.gif"/〉 and 〈inlineGraphic alt="inline image" href="urn:x-wiley:01407791:PCE657:PCE_657_mu2" location="equation/PCE_657_mu2.gif"/〉 were linearly related to the leaf area downstream. Approximately 65% of the resistance to water flow within the plant resided in the leaf blade. An electric-analogue computer model was used to calculate the leaf blade area-specific radial hydraulic conductivity, 〈inlineGraphic alt="inline image" href="urn:x-wiley:01407791:PCE657:PCE_657_mu3" location="equation/PCE_657_mu3.gif"/〉 (mmol s−1 m−2 MPa−1), using 〈inlineGraphic alt="inline image" href="urn:x-wiley:01407791:PCE657:PCE_657_mu2" location="equation/PCE_657_mu2.gif"/〉, Kt* and water flux values. 〈inlineGraphic alt="inline image" href="urn:x-wiley:01407791:PCE657:PCE_657_mu3" location="equation/PCE_657_mu3.gif"/〉 values decreased with leaf age, from 21·2 mmol s−1 m−2 MPa−1 in rapidly elongating leaf to 7·2 mmol s−1 m−2 MPa−1 in mature leaf. Comparison of 〈inlineGraphic alt="inline image" href="urn:x-wiley:01407791:PCE657:PCE_657_mu2" location="equation/PCE_657_mu2.gif"/〉 and 〈inlineGraphic alt="inline image" href="urn:x-wiley:01407791:PCE657:PCE_657_mu3" location="equation/PCE_657_mu3.gif"/〉 values showed that ≈ 90% of the resistance to water flow within the blades resided in the liquid extra-vascular path. The same algorithm was then used to compute the xylem and extravascular water potential drop along the liquid water path in the plant under steady state conditions. Predicted and measured water potentials matched well. The hydraulic design of the mature leaf resulted in low and quite constant xylem water potential gradient (≈ 0·3 MPa m−1) throughout the plant. Much of the water potential drop within mature leaves occurred within a tenth of millimetre in the blade, between the xylem vessels and the site of water evaporation within the mesophyll. In elongating leaves, the low Kt* in the basal growth zone dramatically increased the local xylem water potential gradient (≈ 2·0 MPa m−1) there. In the leaf elongation zone the growth-induced water potential difference was ≈ 0·2 MPa.

Notes: Recent work has shown that stomatal conductance (gs) and assimilation (A) are responsive to changes in the hydraulic conductance of the soil to leaf pathway (KL), but no study has quantitatively described this relationship under controlled conditions where steady-state flow is promoted. Under steady-state conditions, the relationship between gs, water potential (Ψ) and KL can be assumed to follow the Ohm's law analogy for fluid flow. When boundary layer conductance is large relative to gs, the Ohm's law analogy leads to gs = KL (Ψsoil−Ψleaf)/D, where D is the vapour pressure deficit. Consequently, if stomata regulate Ψleaf and limit A, a reduction in KL will cause gs and A to decline. We evaluated the regulation of Ψleaf and A in response to changes in KL in well-watered ponderosa pine seedlings (Pinus ponderosa). To vary KL, we systematically reduced stem hydraulic conductivity (k) using an air injection technique to induce cavitation while simultaneously measuring Ψleaf and canopy gas exchange in the laboratory under constant light and D. Short-statured seedlings (〈 1 m tall) and hour-long equilibration times promoted steady-state flow conditions. We found that Ψleaf remained constant near − 1·5 MPa except at the extreme 99% reduction of k when Ψleaf fell to − 2·1 MPa. Transpiration, gs, A and KL all declined with decreasing k (P 〈 0·001). As a result of the near homeostasis in bulk Ψleaf, gs and A were directly proportional to KL (R2 〉 0·90), indicating that changes in KL may affect plant carbon gain.

Notes: The transport of mineral ions into and out of tissues and cells is central to the life of plants. Ion transport and the plasma membrane transporters themselves have been studied using a variety of techniques. In the last 15 years, measurement of specific ion fluxes has contributed to the characterization of transport systems. Progress in molecular genetics is allowing gene identification and controlled expression of transporter molecules. However the molecular expression of transporter gene products must be characterized at the functional level. The ion-selective microelectrode technique to measure specific ion fluxes non-invasively is ideally suited to this purpose. This technique, its theory, its links with others and its application and prospects in plant science, are discussed. Ions studied include hydrogen, potassium, sodium, ammonium, calcium, chloride and nitrate. Applications discussed include: solute ion uptake by roots; gravitropism and other processes in the root cap, meristematic and elongation zones; Nod factor effect on root hairs; osmotic and salt stresses; oscillations; the effects of light and temperature. Studies have included intact roots, leaf mesophyll and other tissues, protoplasts and bacterial biofilms. A multi-ion capability of the technique will greatly assist functional genomics, particularly when coupled with imaging techniques, patch clamping and the use of suitable mutants.

Notes: The regulation of ribulose-1,5-biphosphate carboxlase/oxygenase (Rubisco) and Rubisco activase was followed for 3 months in an experiment studying the effects of ozone and water stress on Aleppo pine. Rubisco activity was shown to be reduced by 30% in the presence of ozone, whereas no significant effect of water stress was noticed. The effect of combined stresses on Rubisco activity was similar to the effect of ozone. The changes in protein quantity of Rubisco large subunit (LSU) and Rubisco activase (RCA), compared with control plants, were similar to that of the Rubisco activity. Using homologous probes obtained by reverse transcription (RT)-polymerase chain reaction (PCR), rbcL and rca transcript quantities were quantified during the course of the experiment. RbcL and rca mRNA quantities decreased in ozone and after drought. Changes in rbcL transcript quantity in needles subjected to the combination of ozone and drought were similar to the ones detected when drought was applied alone. On the contrary, the pattern of rca changes under the combination of the two stresses was similar to that of ozone applied alone. A positive correlation existed between the effects of ozone on Rubisco activase and Rubisco LSU protein quantities, which was not so obvious by comparing transcript quantities. This could suggest a potential post-transcriptional coordinated regulation of the two proteins under stress-imposed conditions.

Notes: Acclimation to periodic high-light stress was studied in tree seedlings from a neotropical forest. Seedlings of several pioneer and late-succession species were cultivated under simulated tree-fall gap conditions; they were placed under frames covered with shade cloth with apertures of different widths that permitted defined periods of daily leaf exposure to direct sunlight. During direct sun exposure, all plants exhibited a marked reversible decline in potential photosystem II (PSII) efficiency, determined by means of the ratio of variable to maximum Chl a fluorescence (Fv/Fm). The decline in Fv/Fm under full sunlight was much stronger in late-succession than in pioneer species. For each gap size, all species exhibited a similar degree of de-epoxidation of violaxanthin in direct sunlight and similar pool sizes of xanthophyll cycle pigments. Pool sizes increased with increasing gap size. Pioneer plants possessed high levels of β-carotene that also increased with gap size, whereas α-carotene decreased. In contrast to late-succession plants, pioneer plants were capable of adjusting their Chl a/b ratio to a high value in wide gaps. The content of extractable UV-B-absorbing compounds was highest in the plants acclimated to large gaps and did not depend on the successional status of the plants. The results demonstrate a better performance of pioneer species under high-light conditions as compared with late-succession plants, manifested by reduced photoinhibition of PSII in pioneer species. This was not related to increased pool size and turnover of xanthophyll cycle pigments, nor to higher contents of UV-B-absorbing substances. High β-carotene levels and increased Chl a/b ratios, i.e. reduced size of the Chl a and b binding antennae, may contribute to photoprotection in pioneer species.

Notes: We show here that both salinity and osmotic stress trigger transient increases in intracellular free Ca2+ concentration ([Ca2+]i) in cells of the nitrogen-fixing filamentous cyanobacterium Anabaena sp. PCC7120, which constitutively expresses apoaequorin. Isoosmolar concentrations of salt (NaCl) and osmoticum (sucrose) induced calcium transients of similar magnitude and shape, suggesting that cells sense, via Ca2+ signalling, mostly osmotic stress. The Ca2+ transients induced by NaCl and sucrose were completely blocked by the calcium chelator ethylene glycol-bis(b-aminoethylether)N,N,N¢,N¢-tetraacetic acid (EGTA) and were partially inhibited by the calcium channel blocker verapamil. Increased external Ca2+ and the Ca2+ ionophore calcimycin (compound A23187) enhanced Ca2+ influx further, suggesting the involvement of extracellular Ca2+ in the observed response to salinity and osmotic stress. However, the plant hormone abscisic acid (ABA) did not provoke any effect on the Ca2+ transients induced by both stresses, indicating that it may not be acting upstream of Ca2+ in the signalling of salinity and/or osmotic stress in Anabaena sp. PCC7120.

Notes: Nine species from the tribe Triticeae – three crop, three pasture and three ‘wild’ wetland species – were evaluated for tolerance to growth in stagnant deoxygenated nutrient solution and also for traits that enhance longitudinal O2 movement within the roots. Critesion marinum (syn. Hordeum marinum) was the only species evaluated that had a strong barrier to radial O2 loss (ROL) in the basal regions of its adventitious roots. Barriers to ROL have previously been documented in roots of several wetland species, although not in any close relatives of dryland crop species. Moreover, the porosity in adventitious roots of C. marinum was relatively high: 14% and 25% in plants grown in aerated and stagnant solutions, respectively. The porosity of C. marinum roots in the aerated solution was 1·8–5·4-fold greater, and in the stagnant solution 1·2–2·8-fold greater, than in the eight other species when grown under the same conditions. These traits presumably contributed to C. marinum having a 1·4–3 times greater adventitious root length than the other species when grown in deoxygenated stagnant nutrient solution or in waterlogged soil. The length of the adventitious roots and ROL profiles of C. marinum grown in waterlogged soil were comparable to those of the extremely waterlogging-tolerant species Echinochloa crus-galli L. (P. Beauv.). The superior tolerance of C. marinum, as compared to Hordeum vulgare (the closest cultivated relative), was confirmed in pots of soil waterlogged for 21 d; H. vulgare suffered severe reductions in shoot and adventitious root dry mass (81% and 67%, respectively), whereas C. marinum shoot mass was only reduced by 38% and adventitious root mass was not affected.

Notes: Physiological properties of photosynthesis were determined in the marine diatom, Phaeodactylum tricornutum UTEX640, during acclimation from 5% CO2 to air and related to H2CO3 dissociation kinetics and equilibria in artificial seawater. The concentration of dissolved inorganic carbon at half maximum rate of photosynthesis (K0·5[DIC]) value in high CO2-grown cells was 1009 mmol m−3 but was reduced three-fold by the addition of bovine carbonic anhydrase (CA), whereas in air-grown cells K0·5[DIC] was 71 mmol m−3, irrespective of the presence of CA. The maximum rate of photosynthesis (Pmax) values varied between 300 and 500 μmol O2 mg Chl−1 h−1 regardless of growth pCO2. Bicarbonate dehydration kinetics in artificial seawater were re-examined to evaluate the direct HCO3− uptake as a substrate for photosynthesis. The uncatalysed CO2 formation rate in artificial seawater of 31·65°/oo of salinity at pH 8·2 and 25 °C was found to be 0·6 mmol m−3 min−1 at 100 mmol m−3 DIC, which is 53·5 and 7·3 times slower than the rates of photosynthesis exhibited in air- and high CO2-grown cells, respectively. These data indicate that even high CO2-grown cells of P. tricornutum can take up both CO2 and HCO3− as substrates for photosynthesis and HCO3− use improves dramatically when the cells are grown in air. Detailed time courses were obtained of changes in affinity for DIC during the acclimation of high CO2-grown cells to air. The development of high-affinity photosynthesis started after a 2–5 h lag period, followed by a steady increase over the next 15 h. This acclimation time course is the slowest to be described so far. High CO2-grown cells were transferred to controlled DIC conditions, at which the concentrations of each DIC species could be defined, and were allowed to acclimate for more than 36 h. The K0·5[DIC] values in acclimated cells appeared to be correlated only with [CO2(aq)] in the medium but not to HCO3−, CO32−, total [DIC] or the pH of the medium and indicate that the critical signal regulating the affinity of cells for DIC in the marine diatom, P. tricornutum, is [CO2(aq)] in the medium.

Notes: Plants respond to various biotic and abiotic stresses by the specific induction of a certain set of genes. This study involved a search for transcripts induced in alfalfa (Medicago sativa L. bv Verko) upon infection with dodder (Cuscuta trifolii Bab. et Gibs). A 570 bp cDNA, designated PPRG1, was isolated by differential display. Southern blot analysis indicates that PPRG1 is a member of a new small gene family in alfalfa. PPRG1 encodes a putative 19·2 kDa calmodulin-related protein with 59% amino acid homology to the touch-inducible TCH2 gene isolated from Arabidopsis. The level of PPRG1 transcript shows a rapid transient increase to mechanical stimuli such as rain, touch and wounding, to osmotic and salt stresses and to external treatment with the plant hormone abscisic acid. Constantly elevated levels of PPRG1 mRNA can be observed during cold stress, whereas heat treatment does not induce PPRG1 expression. These results implicate roles for Ca2+ and PPRG1 in the transduction of signals from the environment.

Notes: The light–nitrogen hypothesis suggests canopy photosynthesis is maximized when there is a positive relationship between irradiance received by foliage, its nitrogen content (per unit area Narea), and maximum rate of photosynthesis (Amax). Relationships among relative irradiance and Narea, allocation of nitrogen within the photosynthetic apparatus to Rubisco and chlorophyll, and Amax were examined in Pinus pinaster Ait. needles up to 6 years of age. Measurements were made before bud break in August 1998, and in May 1999 after the first ‘winter’ rains. In August, Narea in P. pinaster needles decreased from 5·1 to 5·7 g m−2 in sunlit 1-year-old needles to 2·3 g m−2 in shaded 6-year-old needles. In May, Narea was 5–40% less but spatial trends were the same. At both sampling dates, Amax was less in old shaded needles compared with young sunlit needles, and was thus consistent with the light–nitrogen hypothesis. Relationships between Narea and Amax were positive at both dates yet varied in strength and form. Allocation of nitrogen within the photosynthetic apparatus was qualitatively consistent with acclimation to light (i.e. Rubisco/Chl decreased with shading), but quantitatively suboptimal with respect to photosynthesis owing to consistent over-investment in Rubisco. This over-investment increased with height in the canopy and was greater in May than in August.

Notes: In order to study convective heat transfer of small leaves, the steady-state and transient heat flux of small leaf-shaped model structures (area of one side = 1730 mm2) were studied under zero and low (= 100 mm s−1) wind velocities by using a computer simulation method. The results show that: (1) distinct temperature gradients of several degrees develop over the surface of the model objects during free and mixed convection; and (2) the shape of the objects and onset of low wind velocities has a considerable effect on the resulting temperature pattern and on the time constant τ. Small leaves can thus show a temperature distribution which is far from uniform under zero and low wind conditions. The approach leads, however, to higher leaf temperatures than would be attained by ‘real’ leaves under identical conditions, because heat transfer by transpiration is neglected. The results demonstrate the fundamental importance of a completely controlled environment when measuring heat dissipation by free convection. As slight air breezes alter the temperature of leaves significantly, the existence of purely free convection appears to be questionable in the case of outdoor conditions. Contrary to the prognoses yielded by standard approximations, no quantitative effect of buoyancy on heat transfer under the considered conditions could be detected for small-sized leaf shapes.

Notes: This field study with the C3 bromeliad Vriesea sanguinolenta (Cogn. & Marchal 1874) was initiated to explore the importance of size-related ecophysiological changes in vascular epiphytes in a natural tropical setting. In this species, a step change from atmospheric to tank-forming life form occurs during early ontogeny, followed by a continuous size increase of individuals with water-impounding tanks. Although our study focused on the water-impounding phase, this growth pattern also allowed us to compare ecophysiological consequences of a step change in life form with those associated with size increments among plants of identical life form. The shift in life form was accompanied by relatively minor changes, for example in leaf morphology (decrease in leaf thickness and trichome density) and leaf physiology (decrease in photosynthetic capacity), while there were more substantial changes during the tank-forming phase. A major trend was a decreasing dependence of larger plants on internally stored water due to a more efficient tank. We suggest that the resulting, more reliable water supply in larger plants may be the proximate cause for the observed size-related differences in leaf anatomy (relative reduction of water storage tissue, and relative and absolute increase in chlorenchyma thickness), leaf morphology (increase in stomatal density, decrease in trichome density), and leaf physiology (increase in net rates of CO2 uptake, more conservative stomatal behaviour, higher residual transpiration). The results are compared with previous studies on heteroblasty in bromeliads, but are also discussed in the context of a gradual shift from a drought-tolerance to a drought-avoidance strategy.

Notes: Native scrub-oak communities in Florida were exposed for three seasons in open top chambers to present atmospheric [CO2] (approx. 350 μmol mol−1) and to high [CO2] (increased by 350 μmol mol−1). Stomatal and photosynthetic acclimation to high [CO2] of the dominant species Quercus myrtifolia was examined by leaf gas exchange of excised shoots. Stomatal conductance (gs) was approximately 40% lower in the high- compared to low-[CO2]-grown plants when measured at their respective growth concentrations. Reciprocal measurements of gs in both high- and low-[CO2]-grown plants showed that there was negative acclimation in the high-[CO2]-grown plants (9–16% reduction in gs when measured at 700 μmol mol−1), but these were small compared to those for net CO2 assimilation rate (A, 21–36%). Stomatal acclimation was more clearly evident in the curve of stomatal response to intercellular [CO2] (ci) which showed a reduction in stomatal sensitivity at low ci in the high-[CO2]-grown plants. Stomatal density showed no change in response to growth in high growth [CO2]. Long-term stomatal and photosynthetic acclimation to growth in high [CO2] did not markedly change the 2·5- to 3-fold increase in gas-exchange-derived water use efficiency caused by high [CO2].

Notes: The distribution of hydraulic resistances in xylem throughout the pathway leading to the tomato fruit was investigated. Previous work had indicated that there were large resistances within the supporting sections of this pathway (the peduncle and pedicel), perhaps associated with interruptions in the xylem. These high resistances are believed to impede calcium flux into the fruit and thus impair fruit development. It is shown here that fruit on intact plants do not shrink detectably during drought, even when the drought is sufficient to cause marked shrinkage of leaves and visible wilting of the shoot. In explants, it is possible to induce back-flow from the fruit into the stem (probably via the xylem) but this flow is small and very slow. These observations support the view that there is a large hydraulic resistance in the pathway between fruit and stem. When pulses of water were made available within explants, by scorching of one leaflet, there was a rapid swelling of leaves and sepals. Such rapid fluxes indicate the presence of strong hydraulic (xylem) connections throughout the pathway between leaf and calyx. This shows that there are no significant hydraulic constrictions in the xylem proximal to the calyx. This finding is contrary to some previous conclusions but it is supported by experiments with dyes which showed continuous, functional xylem throughout the peduncle and pedicel. Calculations show that over 90% of the hydraulic resistance between stem and fruit must reside within the fruit pericarp. Implications for calcium nutrition are discussed.

Notes: This paper discusses the essentials of the oil-filled pressure probe technique in the measurement of negative xylem pressures, focusing in particular on the technique and physics underlying our recent, successful experiment which has rekindled the debate on the validity of the Cohesion–Tension theory. We illustrate a number of general problems associated with the cell pressure probe and xylem pressure probe techniques, and propose appropriate criteria for micropipette construction. We enumerate factors dealing with the cavitation problem and suggest methods for eliminating air seeds in the system. We introduce reliable criteria for the successful measurement of xylem pressure, and emphasize the importance of the probe pressure relaxation test. Several problems regarding the controversy over the Cohesion–Tension theory are also discussed. We discuss the correlation between xylem pressure and the transpiration rate, the existence of absolute negative xylem pressure in intact plants, the most negative values of xylem pressure measured by the pressure probe, the agreement between the pressure probe and pressure bomb techniques, and the vulnerability to cavitation (tensile strength) of pressure probes.

Notes: Anatomy and some physiological characteristics of the leaves in Polygonum cuspidatum Sieb. et Zucc., a dioecious clonal herb, were compared between two populations, one from a lowland in Shizuoka City (10 m above sea level), and another from a highland on Mt. Fuji (2500 m above sea level). Leaf mass per area (LMA) of the highland plants was about twice that of the lowland plants. The greater leaf thickness, thicker mesophyll cell walls and higher mesophyll cell density in the highland leaves contributed to the larger LMA. Although mesophyll area exposed to intercellular airspaces was greater in the highland leaves than in the lowland leaves by 30%, the surface area of chloroplasts facing intercellular airspaces was similar between these leaves. CO2 transfer conductance inside the leaf (gi) of the highland leaves (0·75 μmol m−2 s−1 Pa−1) is the lowest recorded for herbaceous plants and was only 40% of that in the lowland leaves. On the other hand, the difference in stomatal conductance was small. δ13C values in the leaf dry matter were greater in the highland leaves by 4‰. These data and the estimation of CO2 partial pressures in the intercellular air spaces and in the chloroplast suggested that the greater dry matter δ13C in the highland leaves, indicative of lower long-term ratio of the chloroplast stroma to the ambient CO2 partial pressures, would be mainly attributed to their lower gi.

Notes: In this study the question whether the alternative respiratory pathway acts as an electron bypass for the cytochrome pathway under conditions of growth on limited phosphorus in leaves of bean (Phaseolus vulgaris L.), tobacco (Nicotiana tabacum L.) and Gliricidia sepium Walp was investigated. The oxygen isotope fractionation technique was used to assess the in vivo activities of the cytochrome and alternative respiratory pathways in the absence of added inhibitors. The response of respiration to low phosphorus supply varied among species. Growth at low phosphorus reduced cytochrome pathway activity in bean and tobacco. Alternative pathway activity increased only in bean leaves in response to low phosphorus and not in tobacco. In the case of G. sepium, cytochrome pathway activity remained unchanged whereas the alternative pathway activity increased with low nutritional phosphorus. At low phosphorus, alternative oxidase protein levels increased in the leaves of bean and G. sepium but not in tobacco, suggesting a dependence of alternative pathway activity on protein level. Alternative pathway activity was also not correlated with soluble carbohydrate concentration in bean or tobacco at any phosphorus level. These results show that the alternative pathway does not always act as an electron bypass in response to the downstream restriction of the cytochrome pathway imposed by low phosphorus supply. These results suggest that factors in addition to cellular carbohydrate level and adenylate control can act to regulate alternative pathway activity.

Notes: We investigated the role that manganese superoxide dismutase (MnSOD, EC 1·15·1·1), an important enzyme of the antioxidant pathway, may play in aluminium (Al) toxicity/resistance. A wheat (Triticum aestivum) cDNA (WMnSOD) was cloned and up-regulation of the transcript was observed in root tips after 24 h exposure to 100 µm Al. The WMnSOD1 under the control of the CaMV35S promoter was expressed in canola (Brassica napus). Transgenic plants were phenotypically normal. Northern analyses showed enhanced levels of the WMnSOD1 and WMnSOD1-GUS transcripts and total SOD activity was 1·5- to 2·5-fold greater in transgenic plants than in wild type (WT) plants. Transgenic (T1) leaf discs showed increased retention of chlorophyll and reduced electrolyte leakage when exposed to methyl viologen (MV) as compared with WT leaf discs, suggesting that transgenic plants were more resistant to oxidative stress. When WT canola plants were exposed to aluminium (0–200 µm), inhibition of root growth, higher SOD activity and increased levels of malondialdehyde (MDA, an indicator of lipid peroxidation) were observed in roots. Aluminium-induced inhibition of root growth and accumulation of MDA was lower in homozygous transgenic plants (T2) compared with WT plants. Transgenic lines also showed lower synthesis of 1,3-β-glucans (callose, a sensitive marker for Al injury) as compared with WT. These data suggest that resistance to Al toxicity can be improved by overexpressing WMnSOD1.

Notes: Recent soil pressurization experiments have shown that stomatal closure in response to high leaf–air humidity gradients can be explained by direct feedback from leaf water potential. The more complex temperature-by-humidity interactive effects on stomatal conductance have not yet been explained fully. Measurements of the change in shoot conductance with temperature were made on Phaseolus vulgaris (common bean) to test whether temperature-induced changes in the liquid-phase transport capacity could explain these temperature- by-humidity effects. In addition, shoot hydraulic resistances were partitioned within the stem and leaves to determine whether or not leaves exhibit a greater resistance. Changes in hydraulic conductance were calculated based on an Ohm’s law analogy. Whole-plant gas exchange was used to determine steady- state transpiration rates. A combination of in situ psychrometer measurements, Scholander pressure chamber measurements and psychrometric measurements of leaf punches was used to determine water potential differences within the shoot. Hydraulic conductance for each portion of the pathway was estimated as the total flow divided by the water potential difference. Temperature-induced changes in stomatal conductance were correlated linearly with temperature-induced changes in hydraulic conductance. The magnitude of the temperature-induced changes in whole-plant hydraulic conductance was sufficient to account for the interactive effects of temperature and humidity on stomatal conductance.

Notes: To assess how diurnal changes of nitrate reductase (NIA) expression in leaves interact with upstream and downstream processes during nitrate utilization, nitrate uptake, and nitrate and ammonium metabolism were investigated at several times during the diurnal cycle in wild-type tobacco. Plants were grown hydroponically on 2 mM nitrate to exclude possible complications due to changes in the external availability of nitrate, and to allow nitrate uptake to be measured in the growth conditions. (a) In leaves, the NIA transcript decreases during the day and recovers at night, and NIA activity increases three-fold during the first part and declines during the second part of the light period. Nitrate decreases during the day and recovers at night, ammonium, glutamine, glycine and serine increase during the day and decrease at night, and 2-oxoglutarate increases three-fold after illumination and decreases during the last part of the light period. The amplitudes of the diurnal changes are similar to or larger than in tobacco grown on high nitrate in sand. The transcript for plastid glutamine synthetase (GLN2) is low at the end of the night and increases during the day, and glutamine synthetase activity increases to a peak at the end of the day and decreases at night. (b) In the roots, transcript levels for the high affinity nitrate transporter (NRT2) increase in the day and decrease at night. Nitrate uptake is about 40% higher during the day than at night. (c) Comparison of the diurnal changes of the leaf metabolite pools with the rate of nitrate uptake allows diurnal changes in fluxes to be estimated. During the first part of the light, the rate of nitrate assimilation is about two-fold higher than the rate of nitrate uptake, and also exceeds the rate at which reduced nitrogen is metabolized in the GOGAT pathway. The resulting decrease of leaf nitrate and accumulation of nitrogen in intermediates of ammonium metabolism and photorespiration represent about 40 and 15%, respectively, of the total nitrate that enters the plant in 24 h. Later in the diurnal cycle as NIA expression and activity decline, this imbalance is reversed. NRT2 expression and nitrate uptake remain relatively high, and nitrate taken up during the night is used to replenish the leaf nitrate pool. Increased GLN2 expression in leaves during the second part of the light period allows continued assimilation of ammonium released during photorespiration and remobilization of the reduced nitrogen that accumulated earlier in the diurnal cycle.

Notes: Using the patch-clamp technique, we investigated the transport properties of vacuolar ion channels from the roots of water hyacinth, Eichhornia crassipes (Mart. Solms, Pontederiacae). Eichhornia crassipes vacuoles displayed large voltage-dependent rectifying slow-vacuolar (SV) currents, which activated in a few seconds at positive potentials and deactivated at negative voltages in a few hundreds of millseconds. Similarly to SV channel previously identified in the tonoplast of terrestrial plants, SV currents in E. crassipes were activated by micromolar concentrations of Ca2+ and current slightly increased (25%) on addition (10 mm) of the reducing agent dithiothreitol (DTT). Eichhornia crassipes SV channels were equally permeable to K+ and Na+. The permeability sequence derived from current values is: K+ ≈ Na+ 〉 Rb+ 〉 NH4+ ≈ Cs+ 〉〉 TEA+. Excised membrane patches displayed single channel transitions typical of SV-type single channel openings with a conductance of (83·0 ± 5·6) pS; a smaller channel with a conductance of (31·0 ± 2·7) pS was also identified. Metals such as Ni2+ and Zn2+ decreased the vacuolar current in a reversible manner. However, although Zn2+ inhibition is comparable to that induced by the same metal in vacuoles from the main root of sugar beet (Beta vulgaris L.), the inhibition of the SV currents by Ni2+ is not as substantial in E. crassipes as in sugar beet. To our knowledge, this is the first electrophysiological characterization of ionic transport in E. crassipes, a pervasive troublesome aquatic weed, which has exceptional absorption properties of several water contaminants such as heavy metals, pesticides and phenols.

Notes: The model RATP (radiation absorption, transpiration and photosynthesis) is presented. The model was designed to simulate the spatial distribution of radiation and leaf-gas exchanges within vegetation canopies as a function of canopy structure, canopy microclimate within the canopy and physical and physiological leaf properties. The model uses a three-dimensional (3D) representation of the canopy (i.e. an array of 3D cells, each characterized by a leaf area density). Radiation transfer is computed by a turbid medium analogy, transpiration by the leaf energy budget approach, and photosynthesis by the Farquhar model, each applied for sunlit and shaded leaves at the individual 3D cell-scale. The model typically operates at a 20–30 min time step. The RATP model was applied to an isolated, 20-year-old walnut tree grown in the field. The spatial distribution of wind speed, stomatal response to environmental variables, and light acclimation of leaf photosynthetic properties were taken into account. Model outputs were compared with data acquired in the field. The model was shown to simulate satisfactorily the intracrown distribution of radiation regime, transpiration and photosynthetic rates, at shoot or branch scales.

Notes: Sun and shade leaves of several plant species from a neotropical forest were exposed to excessive light to evaluate the responses of photosystem I in comparison to those of photosystem II. Potential photosystem I activity was determined by means of the maximum P700 absorbance change around 810 nm (ΔA810max) in saturating far-red light. Leaf absorbance changes in dependence of increasing far-red light fluence rates were used to calculate a ‘saturation constant’, Ks, representing the far-red irradiance at which half of the maximal absorbance change (ΔA810max/2) was reached in the steady state. Photosystem II efficiency was assessed by measuring the ratio of variable to maximum chlorophyll fluorescence, Fv/Fm, in dark-adapted leaf samples. Strong illumination caused a high degree of photo-inhibition of photosystem II in all leaves, particularly in shade leaves. Exposure to 1800–2000 μmol photons m−2 s−1 for 75 min did not substantially affect the potential activity of photosystem I in all species tested, but caused a more than 40-fold increase of Ks in shade leaves, and a three-fold increase of Ks in sun leaves. The increase in Ks was reversible during recovery under low light, and the recovery process was much faster in sun than in shade leaves. The novel effect of high-light stress on the light saturation of P700 oxidation described here may represent a complex reversible mechanism within photosystem I that regulates light-energy dissipation and thus protects photosystem I from photo-oxidative damage. Moreover, we show that under high-light stress a high proportion of P700 accumulates in the oxidized state, P700+. Presumably, conversion of excitation energy to heat by this cation radical may efficiently contribute to photoprotection.

Notes: Comprehensive studies on the processes involved in photosynthetic acclimation after a sudden change in light regime are scarce, particularly for trees. We tested (i) the ability of photosynthetic acclimation in the foliage of walnut trees growing outdoors after low-to-high and high-to-low light transfers made early or late in the vegetation cycle, and (ii) the relative importance of changes in total leaf nitrogen versus changes in the partitioning of leaf nitrogen between the different photosynthetic functions during a 2 month period after transfer. Changes in maximum carboxylation rate, light-saturated electron transport rate, respiration rate, total leaf nitrogen, ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) and total chlorophylls were surveyed before and after the change in light regime. Respiration rate acclimated fully within 1 week of transfer, and full acclimation was observed 1 month after transfer for the amount of Rubisco. In contrast, total nitrogen and photosynthetic capacity acclimated only partially during the 2 month period. Changes in photosynthetic capacity were driven by changes in both total leaf nitrogen and leaf nitrogen partitioning. The extent of acclimation also depended strongly on leaf age at the time of the change in light regime.

Notes: Diurnal courses of nutrient transport in the xylem and their response to external availability of nutrients were studied. In soil culture, maximal concentrations in all analysed substances were observed during night-time. Over experimental periods of up to 20 d, concentrations of some ions increased, most by accumulation in the soil. Stringent nutrient conditions were established in a novel pressure chamber. An aeroponic nutrient delivery system inside allows the sampling of xylem sap from intact plants under full control of the nutrient conditions at the root. Analysis of xylem transport under these highly defined conditions established that (1) diurnal variations in concentrations and fluxes in the xylem are dominated by plant-internal processes; (2) concentrations of nutrients in the xylem sap are highly but specifically correlated with each other; (3) nitrate uptake and nitrate flux to the shoot are largely uncoupled; and (4) in continuous light, diurnal variations of xylem sap concentrations vanish. Step changes in nitrate concentrations of the nutrient solution established that (5) the concomitant increase in nitrate concentration and flux in the xylem is delayed by 2–3 h and is only transient. Diurnal variations of xylem sap composition and use of the new technique to elucidate xylem-transport mechanisms are discussed.

Notes: We investigated the physiological and growth responses of native (Populus fremontii S. Wats. and Salix gooddingii Ball) and exotic (Tamarix chinensis Lour.) riparian trees to ground water availability at the free-flowing Hassayampa River, Arizona, during dry (1997) and wet (1998) years. In the drier year, all species experienced considerable water stress, as evidenced by low shoot water potentials, low leaf gas exchange rates and large amounts of canopy dieback. These parameters were significantly related to depth of ground water (DGW) in the native species, but not in T. chinensis, in 1997. Canopy dieback was greater in the native species than in T. chinensis when ground water was deep in 1997, and dieback increased rapidly at DGW 〉 2·5–3·0 m for the native species. Analysis of combined data from wet and dry years for T. chinensis tentatively suggests a similar physiological sensitivity to water availability and a similar DGW threshold for canopy dieback. In 1998, shoot water potential and leaf gas exchange rates were higher and canopy dieback was lower for all species because of increased water availability. However, T. chinensis showed a much larger increase in leaf gas exchange rates in the wet year than the native species. High leaf gas exchange rates, growth when water is abundant, drought tolerance and the maintenance of a viable canopy under dry conditions are characteristics that help explain the ability of T. chinensis to thrive in riparian ecosystems in the south-western United States.

Notes: Changes in net photosynthetic rate on a leaf area basis and anatomical properties during leaf development were studied in an evergreen broad-leaved tree, Castanopsis sieboldii and an annual herb, Phaseolus vulgaris. In C. sieboldii, surface area of mesophyll cells facing the intercellular air spaces on a leaf area basis (Smes) was already considerable at the time of full leaf area expansion (FLE). However, surface area of chloroplasts facing the intercellular air spaces on a leaf area basis (Sc), and chlorophyll and Rubisco contents on a leaf area basis increased to attain their maximal values 15–40 d after FLE. In contrast, in P. vulgaris, chloroplast number on a leaf area basis, Sc and Smes at 10 d before FLE were two to three times greater than the steady-state levels attained at around FLE. In C. sieboldii, the internal CO2 transfer conductance (gi) slightly increased for 10 d after FLE but then decreased toward the later stages. Limitation of photosynthesis by gi was only about 10% at FLE, but then increased to about 30% at around 40 d after FLE. The large limitation after FLE by gi was probably due to the decrease in CO2 concentration in the chloroplast caused by the increases in thickness of mesophyll cell walls and in Rubisco content per chloroplast surface area. These results clearly showed that: (1) in C. sieboldii, chloroplast development proceeded more slowly than mesophyll cell expansion and continued well after FLE, whereas in P. vulgaris these processes proceeded synchronously and were completed by FLE; (2) after FLE, photosynthesis in leaves of C. sieboldii was markedly limited by gi. From these results, it is suggested that, in the evergreen broad-leaved trees, mechanical protection of mesophyll cells has priority over the efficient CO2 transfer and quick construction of the chloroplasts.

Notes: The involvement of nitrogenous substances in the transition to flowering was investigated in Sinapis alba and Arabidopsis thaliana (Columbia). Both species grown in short days (SD) are induced to flower by one long day (LD). In S. alba, the phloem sap (leaf and apical exudates) and the xylem sap (root exudate) were analysed in LD versus SD. In A. thaliana, only the leaf exudate could be analysed but an alternative system for inducing flowering without day-length extension was used: the displaced SD (DSD). Significant results are: (i) in both species, the leaf exudate was enriched in Gln during the inductive LD, at a time compatible with export of the floral stimulus; (ii) in S. alba, the root export of amino acids decreased in LD, whereas the nitrate remained unchanged – thus the extra-Gln found in the leaf exudate should originate from the leaves; (iii) extra-Gln was also found very early in the apical exudate of S. alba in LD, together with more Glu; (iv) in A. thaliana induced by one DSD, the leaf export of Asn increased sharply, instead of Gln in LD. This agrees with Asn prevalence in C-limited plants. The putative role of amino acids in the transition to flowering is discussed.

Notes: The effects of free-air CO2 enrichment (FACE) on leaf growth in Populus, was studied. For the first time in field conditions, both the production and expansion of leaf cells were shown to be sensitive to atmospheric carbon dioxide. Leaf area expansion rate and final leaf size were stimulated under FACE for three species (Populus x euramericana (I-214), P. nigra (Jean Pourtet) and P. alba (2AS-11), with the largest effect observed for P. x euramericana (61%). In this species and in P. nigra, both epidermal cell size and cell number were increased, whereas for P. alba, only cell production was increased in FACE. Two findings suggest that changes in the cell wall may be important in explaining larger leaf cells in FACE: (i) Leaf cell wall extensibility of rapidly growing leaves increased in all species in FACE; and (ii) an increase in xyloglucan endotransglycosylase activity, a cell wall-loosening enzyme, was increased in FACE and associated with leaf growth rate. The results suggest that the mechanisms by which FACE promotes leaf growth differ, depending on species. Despite this, increases in final leaf size provide an important component driving increased biomass accumulation in POPFACE, during this first year of rapid growth, prior to canopy closure. The question as to whether these effects are the result of a direct response to CO2, or are driven indirectly through substrate availability remains unresolved, although evidence from the literature suggests that the latter mechanism is most likely.

Notes: The Polylepis tarapacana forests found in Bolivia are unique with respect to their altitudinal distribution (4200–5200 m). Given the extreme environmental conditions that characterize these altitudes, this species has to rely on distinct mechanisms to survive stressful temperatures. The purpose of this study was to determine low-temperature resistance mechanisms in P. tarapacana. Tissue was sampled for carbohydrate and proline contents and micro-climatic measurements were made at two altitudes, 4300 and 4850 m, during both the dry cold and wet warm seasons. Supercooling capacity (−3 to −6 °C for the cold dry and −7 to −9 °C for the wet warm season) and injury temperatures (−18 to −23 °C for both seasons), determined in the laboratory, indicate that P. tarapacana is a frost-tolerant species. On the other hand, an increase in supercooling capacity, as the result of significant increase in total soluble sugar and proline contents, occurs during the wet warm season as a consequence of higher metabolic activity. Hence, P. tarapacana, a frost-tolerant species during the colder unfavourable season, is able to avoid freezing during the more favourable season when minimum night-time temperatures are not as extreme.

Notes: Single, double, triple and quadruple mutants of phyA, phyB, cry1 and cry2 were exposed to different sunlight irradiances and photoperiods to investigate the roll played by phytochrome A, phytochrome B, cryptochrome 1 and cryptochrome 2 during de-etiolation of Arabidopsis thaliana seedlings under natural radiation. Even the quadruple mutant retained some hypocotyl-growth inhibition by sunlight. Hypocotyl length was strongly affected by interactions among photoreceptors. Double phyA phyB, phyA cry1, and cry1 cry2 mutants were taller than expected from the additive action of single mutations. Some of these redundant interactions required the presence of phytochromes A and/or B. Interactions among photoreceptors resulted in a 44% reduction of the response to irradiance and a 70% reduction of the response to photoperiod. The complex network of interactions among photoreceptors is proposed to buffer de-etiolation against changes in irradiance and photoperiod, i.e light fluctuations not related to the positions of the shoot above or below soil level

Notes: A model is presented which quantifies a possible role for the carbonic anhydrase in the mitochondrial matrix of Chlamydomonas reinhardtii which incorporates the observation that the expression of this enzyme is increased under growth conditions in which the expression of the carbon dioxide-concentrating mechanism is increased. It is assumed that the inorganic carbon enters the cytosol from the medium, and leaves the cytosol to the plastids, as HCO3− and that there is negligible carbonic anhydrase activity in the cytosol. The role of the mitochondrial carbonic anhydrase is suggested to be the conversion to HCO3– of the CO2 produced in the mitochondria in the light from tricarboxylic acid cycle activity and from decarboxylation of glycine in any photorespiratory carbon oxidation cycle activity which is not suppressed by the carbon concentrating mechanism. If there is a HCO3− channel in the inner mitochondrial membrane then almost all of the inorganic carbon leaves the mitochondria as HCO3−, thus limiting the potential for CO2 leakage through the plasmalemma. This mechanism could increase inorganic C supply to ribulose bisphosphate carboxylase-oxygenase by some 10% at the energetic expense of less than 1% of the total ATP generation by plastids plus mitochondria.

Notes: Heavy metal hyperaccumulation in plants is an intriguing and poorly understood phenomenon. Transmembrane metal transporters are assumed to play a key role in this process. We describe the cloning and isolation of three zinc transporter cDNAs from the Zn hyperaccumulator Thlaspi caerulescens. The ZTP1 gene is highly similar to the Arabidopsis ZAT gene. Of the other two, one is most probably an allele of the recently cloned ZNT1 gene from T. caerulescens (Pence et al; Proceedings of the National Academy of Science USA 97, 4956–4960, 2000). The second, called ZNT2, is a close homologue of ZNT1. All three zinc transporter genes show increased expression in T. caerulescens compared with the non-hyperaccumulator congener T. arvense, suggesting an important role in heavy metal hyperaccumulation. ZNT1 and ZNT2 are predominantly expressed in roots and ZTP1 is mainly expressed in leaves but also in roots. In T. arvense, ZNT1 and ZNT2 are exclusively expressed under conditions of Zn deficiency. Their expression in T. caerulescens is barely Zn-responsive, suggesting that Zn hyperaccumulation might rely on a decreased Zn-induced transcriptional downregulation of these genes. ZTP1 expression was higher in plants from calamine soil than in plants from serpentine or normal soil. The calamine plants were also the most Zn tolerant, suggesting that high ZTP1 expression might contribute to Zn tolerance.

Notes: Nitrate assimilation in leaves requires synthesis of malate to counteract alkalinization, and synthesis of 2-oxoglutarate to act as an acceptor in the GOGAT pathway. We have investigated whether malate or 2-oxoglutarate regulate nitrate reductase (NIA, EC 1·6.6·1) expression. (i) Diurnal changes of NIA expression and organic acid levels were compared in tobacco leaves. The NIA transcript rose during the night and decreased during the day, and NIA activity rose to a maximum during the first 4 h of the light period and fell during the second part of the light period. Malate accumulated to high levels during the light period and decreased during the night. The 2-oxoglutarate increased by 40% at the beginning and decreased towards the end of the light period. The glutamine : 2-oxoglutarate ratio was steady during the first part of the light period and increased markedly during the second part of the light period. The diurnal changes of the NIA transcript level were inversely correlated to the diurnal changes of malate, and unrelated to the changes of 2-oxoglutarate or the glutamine : 2-oxoglutarate ratio. The decrease of NIA activity in the second part of the light period correlated with an increase of the glutamine : 2-oxoglutarate ratio. (ii) Leaves were detached 4 h into the light period and supplied with malate or 2-oxoglutarate via the petiole, to investigate their impact on the gradual decrease of the NIA transcript and NIA activity during the second part of the light period. Physiologically relevant changes of malate led to a further decrease of the NIA transcript level and a 27–60% decrease of NIA activity. A large increase of 2-oxoglutarate stabilized the NIA transcript level but had only slight effects on NIA activity. (iii) Plants were darkened for 16–24 h to reduce the NIA transcript level and NIA activity to low levels, and leaves were then detached and supplied with malate or 2-oxoglutarate for 4 h in the light to investigate their impact on the light-induction of NIA. The increase of the NIA transcript and NIA activity was antagonized by malate, and slightly accelerated by 2-oxoglutarate. (iv) Plants were placed in the dark for 60 h to reduce NIA activity to the limit of detection, and leaf discs were then incubated in the dark on sucrose to achieve a photosynthesis-independent increase of NIA activity. This was strongly inhibited by malate. (v) It is concluded that malate inhibits NIA expression, affecting both the NIA transcript level and NIA activity. Although the results are consistent with a role for 2-oxoglutarate in the regulation of NIA expression, the impact is less marked and no endogenous changes of 2-oxoglutarate were found that are likely to have a significant effect on NIA expression.

Notes: Two experiments, a split-root experiment and a root pressurizing experiment, were performed to test whether hydraulic signalling of soil drying plays a dominant role in controlling stomatal closure in herbaceous bell pepper plants. In the split-root experiment, when both root parts were dried, synchronous decreases in stomatal conductance (gs), leaf water potential (LWP) and stem sap flow (SFstem) were observed. The value of gs was found to be closely related to soil water potential (SWP) in both compartments. Tight relationships were observed between gs and stem sap flow under all conditions of water stress, indicating a complete stomatal adjustment of transpiration. When the half-root system has been dried to the extent that its water uptake dropped to almost zero, declines in gs of less than 20% were observed without obvious changes in LWP. The reduced plant hydraulic conductance resulting from decreased sap flow and unchanged LWP may be a hydraulic signal controlling stomatal closure; the results of root pressurizing supported this hypothesis. Both LWP and gs in water-stressed plants recovered completely within 25 min of the application of root pressurizing, and decreased significantly within 40 min after pressure release, indicating the hydraulic control of stomatal closure. Our results are in contrast to those of other studies on other herbaceous species, which suggested that chemical messengers from the roots bring about stomatal closure when plants are in water stress.

Notes: Predicting the environmental responses of leaf photosynthesis is central to many models of changes in the future global carbon cycle and terrestrial biosphere. The steady-state biochemical model of C3 photosynthesis of Farquhar et al. (Planta 149, 78–90, 1980) provides a basis for these larger scale predictions; but a weakness in the application of the model as currently parameterized is the inability to accurately predict carbon assimilation at the range of temperatures over which significant photosynthesis occurs in the natural environment. The temperature functions used in this model have been based on in vitro measurements made over a limited temperature range and require several assumptions of in vivo conditions. Since photosynthetic rates are often Rubisco-limited (ribulose, 1-5 bisphosphate carboxylase/oxygenase) under natural steady-state conditions, inaccuracies in the functions predicting Rubisco kinetic properties at different temperatures may cause significant error. In this study, transgenic tobacco containing only 10% normal levels of Rubisco were used to measure Rubisco-limited photosynthesis over a large range of CO2 concentrations. From the responses of the rate of CO2 assimilation at a wide range of temperatures, and CO2 and O2 concentrations, the temperature functions of Rubisco kinetic properties were estimated in vivo. These differed substantially from previously published functions. These new functions were then used to predict photosynthesis in lemon and found to faithfully mimic the observed pattern of temperature response. There was also a close correspondence with published C3 photosynthesis temperature responses. The results represent an improved ability to model leaf photosynthesis over a wide range of temperatures (10–40 °C) necessary for predicting carbon uptake by terrestrial C3 systems.

Notes: Inhibition of root growth and accumulation of putrescine caused by exogenous spermidine in roots of maize seedlings (Zea mays L., cv Samodek) were partially prevented by a concomitant treatment with dimethylthiourea (DMTU), that traps H2O2 produced from spermidine by the activity of polyamine oxidase (PAO) in the apoplast. Treatment with spermidine caused a strong increase of ascorbate peroxidase (APX) gene expression, that was induced to a lesser extent by removing spermidine-generated H2O2 by DMTU. Over-expression of APX was associated with increased APX activity in spermidine-treated seedlings whereas the addition of DMTU to spermidine completely prevented spermidine-induced increase of APX activity. Thus, DMTU permitted the demonstration that exogenous spermidine supplied to maize seedlings causes an oxidative stress and induces APX, a key enzyme of the antioxidant defence mechanism, through H2O2, a spermidine catabolic product.

Notes: Carbonic anhydrases catalyse the reversible hydration of CO2, increasing the interconversion between CO2 and HCO3− + H+ in living organisms. The three evolutionarily unrelated families of carbonic anhydrases are designated α-, β-and γ-CA. Animals have only the α-carbonic anhydrase type of carbonic anhydrase, but they contain multiple isoforms of this carbonic anhydrase. In contrast, higher plants, algae and cyanobacteria may contain members of all three CA families. Analysis of the Arabidopsis database reveals at least 14 genes potentially encoding carbonic anhydrases. The database also contains expressed sequence tags (ESTs) with homology to most of these genes. Clearly the number of carbonic anhydrases in plants is much greater than previously thought. Chlamydomonas, a unicellular green alga, is not far behind with five carbonic anhydrases already identified and another in the EST database. In algae, carbonic anhydrases have been found in the mitochondria, the chloroplast thylakoid, the cytoplasm and the periplasmic space. In C3 dicots, only two carbonic anhydrases have been localized, one to the chloroplast stroma and one to the cytoplasm. A challenge for plant scientists is to identify the number, location and physiological roles of the carbonic anhydrases.

Notes: The role that the constituents of the ascorbate–glutathione cycle play in the mechanism of contrasting ozone sensitivities was examined in mature and old tobacco leaves after acute ozone-fumigation (150 p.p.b., 5 h). Levels of the enzyme activities associated with the detoxifying system were lower in ozone-sensitive Bel W3 control plants than in unfumigated ozone-tolerant Bel B plants. In particular, the endogenous activities of ascorbate peroxidase (APX) and glutathione reductase (GR), and the metabolites ascorbic acid (AA) and reduced glutathione (GSH) were more abundant in Bel B than Bel W3 control plants. These results suggest that the higher tolerance of Bel B to O3 is associated with a greater initial content of the antioxidant enzymes or metabolites. Only in the mature leaves of the ozone-tolerant Bel B cv. did fumigation trigger activation of APX and, weakly, of dehydroascorbate reductase (DHAR). The activity of these enzymes was significantly lower after ozone treatment in both mature and old leaves of Bel W3 than in control plants. Fumigation had little effect on the ascorbate content. Its main effects on the glutathione pool were that it boosted the oxidized form and lowered the reduced form, particularly in mature Bel W3 leaves. Extractable GR activity remained unchanged in both Bel B and Bel W3 immediately after fumigation, but increased slightly 24 h later, particularly in mature leaves of Bel W3. Exposure to O3 caused a sharp decline in chloroplastic GR mRNA levels in both cultivars. However, as Western blot analysis failed to detect any major changes in GR protein content at this time, the protein must be highly stable. There is therefore a good correlation between tolerance to O3 and high endogenous levels of antioxidant metabolites such as AA and GSH in tobacco. In addition, the degree of inducibility of the system discriminates the two cultivars investigated.

Notes: A new method for simultaneously quantifying rates of flow in xylem and phloem using the FLASH imaging capabilities of nuclear magnetic resonance (NMR) spectrometry was applied in this study. The method has a time resolution of up to 4 min (for the xylem) and was used to measure the velocity of flows in phloem and xylem for periods of several hours to days. For the first time, diurnal time course measurements of flow velocities and apparent volume flows in phloem and xylem in the hypocotyl of 40-d-old Ricinus communis L were obtained. Additional data on gas exchange and the chemical composition of leaves, xylem and phloem sap were used to assess the role of leaves as sinks for xylem sap and sources for phloem. The velocity in the phloem (0·250 ± 0·004 mm s−1) was constant over a full day and not notably affected by the light/dark cycle. Sucrose was loaded into the phloem and transported at night, owing to degradation of starch accumulated during the day. Concentrations of solutes in the phloem were generally less during the night than during the day but varied little within either the day or night. In contrast to the phloem, flow velocities in the xylem were about 1·6-fold higher in the light (0·401 ± 0·004 mm s−1) than in the dark (0·255 ± 0·003 mm s−1) and volume flow varied commensurately. Larger delays were observed in changes to xylem flow velocity with variation in light than in gas exchange. The relative rates of solute transport during day and night were estimated on the basis of relative flow and solute concentrations in xylem and phloem. In general, changes in relative flow rates were compensated for by changes in solute concentration during the daily light/dark cycle. However, the major solutes (K+, NO3−) varied appreciably in relative concentrations. Hence the regulation of loading into transport systems seems to be more important to the overall process of solute transport than do changes in mass flow. Due to transport behaviour, the chemical composition of leaves varied during the day only with regard to starch and soluble carbohydrates.

Notes: The effect of defoliation on the deposition of carbon (C) and nitrogen (N) and the contribution of reserves and current assimilates to the use of C and N in expanding leaf tissue of severely defoliated perennial ryegrass (Lolium perenne L.) was assessed with a new material element approach. This included 13C/12C-and 15N/14N-steady-state labelling of all post-defoliation assimilated C and N, analysis of tissue expansion and displacement in the growth zone, and investigation of the spatial and temporal changes in substrate and label incorporation in the expanding elements prior to and after defoliation. The relationship between elemental expansion and C deposition was not altered by defoliation, but total C deposition in the growth zone was decreased due to decreased expansion of tissue at advanced developmental stages and a shortening of the growth zone. The N deposition per unit expansion was increased following defoliation, suggesting that N supply did not limit expansion. Transition from reserve- to current assimilation-derived growth was rapid (〈1 d for carbohydrates and approximately 2 d for N), more rapid than suggested by label incorporation in growth zone biomass. The N deposition was highest near the leaf base, where cell division rates are greatest, whereas carbohydrate deposition was highest near the location of most active cell expansion. The contribution of reserve-derived relative to current assimilation-derived carbohydrates (or N) to deposition was very similar for elements at different stages of expansion

Notes: We studied the effects of phosphorus (P) and light on the physiological and morphological components of growth of young tomato plants (Lycopersicon esculentum Mill. cv. Capita). The importance of dry-mass partitioning and starch accumulation in explaining the effects of P limitation on growth was examined more closely. Plants were grown at a wide range of exponential P supply rates (between 70 and 320 mg g−1 d−1) and one free-access treatment (1 mm). Two light levels (70 and 300 µmol m−2 s−1) were applied. Growth response coefficients (GRCs) were calculated to address the importance of different growth parameters in explaining relative growth rate (RGR). At both light levels, net assimilation rate (NAR) was more important than leaf area ratio (LAR) in explaining the effects of P on growth as indicated by GRCs. At less severe P limitation, LAR became more important and NAR less important. Dry-mass partitioning to both roots and leaves played a minor role in determining the effects of P limitation on growth as indicated by low GRCs. The increase in starch at mild P limitation showed that the assimilate supply was not limiting. At severe P limitation, the rate of photosynthesis was decreased, as suggested by the decrease in starch accumulation.

Notes: Over a large part of the photoperiod, light energy absorbed by upper canopy leaves saturates photosynthesis and exceeds the energetic requirements for light-saturated linear electron flow through photosystem II (JPSII), so that photoinhibition results. From a theoretical consideration of the response of light-saturated photosynthesis to elevated atmospheric CO2 partial pressure (pCO2) it may be predicted that, where light-saturated photosynthesis is Rubisco-limited, an increase in pCO2 will stimulate JPSII. Therefore, the proportion of absorbed quanta dissipated photochemically will increase and the potential for photoinhibition of photosynthesis will decrease. This was tested by measuring modulated chlorophyll a fluorescence from Quercus myrtifolia Willd. growing in the field in open-top chambers, at either current ambient or elevated (ambient + 35 Pa) pCO2 on Merritt Island, Florida, USA. During spring and summer, light-saturated photosynthesis at current ambient pCO2 was Rubisco-limited. Consistent with theoretical prediction, JPSII was increased and photoinhibition decreased by elevated pCO2 in spring. In the summer, when growth had largely ceased, an acclimatory decrease in the maximum Ribulose 1,5 bisphosphate saturated carboxylation capacity (Vc max) removed the stimulation of JPSII seen in the spring, and photoinhibition was increased in elevated pCO2. It is concluded that, for Q. myrtifolia growing in the field, the effects of elevated pCO2 on JPSII and photoinhibition will reflect seasonal differences in photosynthetic acclimation to elevated pCO2 in a predictable manner.