ALBERT

Notes: The redox-state is a critical determinate of cell function, and any major imbalances can cause severe damage or death. The cellular redox status therefore needs to be sensed and modulated before such imbalances occur. Various redox-active components are involved in these processes, including thioredoxins, glutaredoxins and other thiol/disulphide-containing proteins. The cellular reactions for cytoprotection and for signalling are integrated with physiological redox-reactions in photosynthesis, assimilation and respiration. They also determine the developmental fate of the cell and finally decide on proliferation or cell death. An international workshop on redox regulation, organized by the research initiative FOR 387 of the Deutsche Forschungsgemeinschaft, was held in Bielefeld, Germany in 2002. A selection of articles originating from the meeting is printed in this issue of Physiologia Plantarum.

Notes: Ribonucleases (RNases) degrade RNA and exert a major influence on gene expression during development and in response to biotic and abiotic stresses. RNase activity typically increases in response to pathogen attack, wounding and phosphate (Pi) deficiency. Activity also increases during senescence and other programmed cell death processes. The air pollutant ozone (O3) often induces injury and accelerated senescence in many plants, but the biochemical mechanisms involved in these responses remain unclear. The objective of this study was to determine whether RNase activity and isozyme expression was stimulated in wheat (Triticum aestivum L.) flag leaves following treatment with O3. Plants were treated in open-top chambers with charcoal-filtered air (27 nmol O3 mol−1) (control) or non-filtered air plus O3 (90 nmol O3 mol−1) (O3) from seedling to reproductive stage. After exposure for 56 days, RNase activity was 2.1 times higher in flag leaf tissues from an O3-sensitive cultivar in the O3 treatment compared with the control, which generally coincided with foliar injury and lower soluble protein concentration, but not soluble leaf [Pi]. Soluble [Pi] in leaf tissue extracts from the O3 and control treatments was not significantly different. RNase activity gels indicated the presence of three major RNases and two nucleases, and their expression was enhanced by the O3 treatment. Isozymes stimulated in the O3 treatment were also stimulated in naturally senescent flag leaf tissues from plants in the control. However, soluble [Pi] in extracts from naturally senescent flag leaves was 50% lower than that found in green flag leaves in the control treatment. Thus, senescence-like pathological responses induced by O3 were accompanied by increased RNase and nuclease activities that also were observed in naturally senescent leaves. However, [Pi] in the leaf tissue samples suggested that O3-induced injury and accelerated senescence was atypical of normal senescence processes in that Pi export was not observed in O3-treated plants.

Notes: The field of signal transduction has experienced a significant paradigm shift as a result of an increased understanding of the roles of 14-3-3 proteins. There are many cases where signal-induced phosphorylation itself may cause a change in protein function. This simple modification is, in fact, the primary basis of signal transduction events in many systems. There are a large and growing number of cases, however, where simple phosphorylation is not enough to effect a change in protein function. In these cases, the 14-3-3 proteins can be required to complete the change in function. Therefore signal transduction can be either the relatively simple process where phosphorylation alters target activity, or it can be a more complex, multistep process with the 14-3-3 proteins playing the major role of bringing the signal transduction event to completion. This makes 14-3-3-modulated signal transduction a more complicated process with additional avenues for regulation and variety. Adding further complexity to the process is the fact that 14-3-3 proteins are present as multigene families in most organisms (Aitken et al. Trends Biochem Sci 17: 498–501, 1992; Ferl Annu Rev Plant Physiol Plant Molecular Biology 47: 49–73, 1996), with each member of the family being differentially expressed in various tissues and with potentially differential affinity for various target proteins. This review focuses on the 14-3-3 family of Arabidopsis as a model for further developing understanding of the roles of the 14-3-3 proteins as modulators of signal transduction events in plants. The primary approaches to these questions are not unlike the approaches that would be used in the functional dissection of any multigene family, but the interpretation of these data will have wide implications since the 14-3-3 s physically interact with other protein families.

Notes: The metabolic, biochemical and molecular events occurring in the different leaf stages along the main axis of tobacco (Nicotiana tabacum) plants grown either on a nitrogen-rich medium, on a medium containing ammonium as sole nitrogen source or on a nitrogen-depleted medium, are presented. This study shows that the highest induction of cytosolic glutamine synthetase (GS1) protein and transcript occurs when nitrogen remobilization is maximal as the result of nitrogen starvation, whereas both glutamate dehydrogenase (GDH) transcript and activity remain at a very low level. In contrast, GDH is highly induced when plants are grown on ammonium as sole nitrogen source, a physiological situation during which leaf protein nitrogen remobilization is limited. It is therefore concluded that GDH does not play a direct role during the process of nitrogen remobilization but is rather induced following a built up of ammonium provided externally or released as the result of protein hydrolysis during natural leaf senescence.

Notes: Glass microelectrodes were inserted into mesophyll cells of intact leaves from higher plants: Arabidopsis thaliana, Helianthus annuus and Vicia faba var minor, and transient membrane potential changes were recorded in response to a sudden temperature drop. The cold-induced potential changes were unaffected by an anion channel inhibitor (anthracene-9-carboxylic acid) and potassium channel inhibitor (tetraethyl ammonium ion). Verapamil, a calcium channel inhibitor, caused significant suppression of the cold-induced potential changes. In the presence of calmoduline antagonists (trifluoperazine and N-6-aminohexyl-5-chloro-1-naphtalenesulphonamide), their amplitudes decreased and their durations were prolonged. Neomycin, which suppresses phospholipase C, also caused substantial inhibition of the amplitudes of the cold-induced potential changes. It is concluded that cold-evoked membrane potential changes are due to calcium influxes from both the apoplast and internal stores.

Notes: Environmental temperature is a critical factor in the lives of almost all organisms. Plants experience periods of thermal stress related to seasonal patterns of temperature and periodic water deficits. Within the range of non-lethal temperatures, there are a number of thermal effects on metabolism that are a result of the thermal dependence of enzymes. The thermal dependence of enzyme kinetic parameters was used to predict that the efficacy of the herbicide pyrithiobac on Palmer amaranth would be reduced at temperatures outside a 20–34°C thermal application range. This prediction is validated in a controlled environment study described in this paper. Palmer amaranth was grown for 16 days in growth chambers with 34/18°C day/night temperature regime. Pyrithiobac was applied to plants at 18, 27 or 40°C. After 1 h at the application temperatures the plants were returned to the 34/18°C regime for 14 days and post-application biomass accumulation (efficacy) was determined. Dry weight accumulation, as a percentage of untreated controls, was 25, 2.5 and 70% for 18, 27 and 40°C application temperatures. Pyrithiobac efficacy was highest for the application within the thermal application range and significantly reduced at temperatures above and below. The validation of the earlier prediction suggests that temperature-related kinetic limitations on herbicide efficacy may also occur in plants with bioengineered herbicide resistance based on herbicide metabolism. The theoretical aspects of such thermal limitations on herbicide resistance mechanisms are discussed.

Notes: The development of castor bean (Ricinus communis L. var. sanguineus) leaves from bud break to abscission was studied to determine whether senescence of phloem precedes or follows chlorophyll degradation in the course of natural ageing of leaves. The castor bean leaf blade took 20 days for full expansion and its average life span was 60 days. From the day of full expansion on it suffered a substantial loss in N, a small loss in C, K and P and a gain in Ca, Mg and S. The content of soluble sugars increased with time, paralleled by a decrease of photosynthetic activity. Starch accumulated shortly before chlorophyll breakdown. The amino acid level in the leaves decreased steadily together with nitrate reductase and glutamine synthetase activity. Reactive oxygen species increased and oxidation-protecting compounds decreased during the life span of the leaves. Shortly after full leaf expansion an increasing number of sieve plates showed strong callose depositions when visualized by aniline blue method. At day 40 only half of the sieve tubes appeared functional. Chlorophyll breakdown followed these processes with a time lag of approximately 10 days. The sieve tube sap of ageing leaves had the same sucrose concentrations as young leaves, whereas amino acid concentrations decreased. High levels of reduced ascorbic acid and glutathione together with increasing levels of glutaredoxin indicated oxidative strain during senescence. We speculate that the gradual increase of reactive oxygen species during ageing together with the import of calcium ions lead to the stimulation of callose synthesis in plasmodesmata and sieve plates with the consequence of inhibition of phloem transport leading to carbohydrate back-up in the leaf blade. The latter may finally induce chlorophyll breakdown and, at the end, leaf abscission at the petiole base. Thus phloem blockage would precede and may be causal for chlorophyll degradation in leaf senescence.

Notes: In this study, the effect of long-term plum pox virus (PPV) infection on the response of certain antioxidant enzymes at the subcellular level was studied in peach plants (Prunus persica (L.) Batch) (cv. GF305), which are characterized by great susceptibility to the virus. In infected plants, a decrease in the efficiency of excitation energy capture by PSII (Fv′/Fm′) was observed, which was accompanied by a decrease in non-photochemical quenching (NPQ). p-Hydroxy-mercury benzoic acid (pHMB)-insensitive ascorbate peroxidase (APX) activity (class III peroxidase) was detected in both chloroplast and soluble fractions. In soluble fractions from inoculated peaches, a significant increase in pHMB-sensitive APX activity and a significant decrease in superoxide dismutase (SOD) activity were observed. These changes were correlated with the observations in isolated chloroplasts, where an increase in both pHMB-sensitive and pHMB-insensitive APX activities was observed, whereas significant decreases in SOD, monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR) activities were produced. According to these results, as a consequence of PPV infection, an oxidative stress, indicated by an increase in lipid peroxidation and protein oxidation, was produced in peach leaves, which was monitored by the diaminobenzidine (DAB) peroxidase-coupled H2O2 probe. PPV infection produced an alteration in chloroplast ultrastructure, giving rise to dilated thylakoid membranes. PPV-infected peach leaves showed a decreased amount of starch in chloroplasts from palisade parenchyma, as well as an increase in the number and size of plastoglobuli, in relation to control plants. The results suggest that long-term PPV infection produces an oxidative stress, and that an antioxidative metabolism imbalance may be related to the progress of PPV infection and symptoms in peach plants.

Notes: The infection of plants with pathogens results in the induction of defence reactions as well as changes in carbohydrate metabolism. On the one hand, the pathogen attempts to manipulate the carbohydrate metabolism of the plant for its own advantage. On the other, the plant has to reorganize carbon fluxes to ensure fight against the pathogen. In order to further investigate the connection between pathogen infection and carbohydrate metabolism, the effects of two types of pathogen, biotrophic and necrotrophic, on gene expression, endogenous sugar levels and photosynthesis of tomato plants were analysed. Photosynthetic gene expression was downregulated on infection with Pseudomonas syringae and Botrytis cinerea. In contrast, expression of a sink-specific gene encoding a cell wall invertase and of defence genes was induced by both pathogens. These results provide evidence for a co-regulation of defence, sink and photosynthetic gene expression in planta in response to both types of pathogen. The brassinosteroid-containing plant restorative ComCat enhanced resistance against B. cinerea and counter-regulated the repression of photosynthetic gene expression. Endogenous sugar levels decreased and the hexose to sucrose ratio increased on treatment with B. cinerea. The application of chlorophyll fluorescence imaging revealed the spatio-temporal heterogeneity of the pathogen response. At 24 h after infection, inhibition of photosynthetic electron transport was restricted to the direct vicinity of the infection site, which was surrounded by a circle of increased photosynthetic activity. The photosynthesis of the remaining leaf was not affected at this stage. These results show the usefulness of chlorophyll fluorescence imaging for the assessment of the complex spatio-temporal changes and for the definition of the areas relevant for other types of determination, e.g. gene expression.

Notes: The expression of anthocyanin biosynthesis genes during flower colour development in Anthurium andraeanum (anthurium) was studied. A cDNA library was constructed from mRNA from the anthurium spathe, and full-length cDNA clones identified for the flavonoid biosynthetic enzymes chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS). These were used to measure transcript levels in the spathe during flower development, in cultivars with different flower colours, over the diurnal cycle, and in the spadix. CHS, F3H and ANS were expressed at all stages of spathe and spadix development. However, DFR transcript levels varied significantly between stages, and DFR may represent a key point of regulation. A diurnal rhythm of DFR transcript abundance in the spathe was also observed, with transcript levels high at dawn and dusk and low at noon. Control of anthocyanin biosynthesis in anthurium spathe differs from that described for flowers of other species, with DFR a key regulatory point and a complex mix of developmental and environmental control signals.

Notes: The expression of three polyphenol oxidase (PPO; EC 1.10.3.1) genes was investigated in hybrid poplar (Populus trichocarpa × P. deltoides). PtdPPO1 was previously isolated as a wound- and herbivore-inducible PPO (Constabel et al. Plant Physiol 124: 285–295, 2000), whereas PtdPPO2 and PtdPPO3 are two novel hybrid poplar PPO genes isolated from stem and root tissue, respectively. Sequence analysis revealed that the three PPOs were 60–66% identical at the amino acid level. Using gene-specific probes, the expression patterns of the three PPOs was investigated in various organs at different developmental stages. Under normal growing conditions, PtdPPO1 mRNA was absent from all organs tested, while PtdPPO2 was found to be expressed in mid-veins, petioles, stems and roots. PtdPPO3 was expressed only in roots. PtdPPO1 and PtdPPO2 were induced by mechanical wounding and methyl jasmonate, but in different tissues. Overall, the expression patterns suggest that the three PPO genes may have specialized physiological functions in hybrid poplar.

Notes: The possible protective role of endogenous isoprene against oxidative stress caused by singlet oxygen (1O2) was studied in the isoprene-emitting plant Phragmites australis. Leaves emitting isoprene and leaves in which isoprene synthesis was inhibited by fosmidomycin were exposed to increasing concentrations of 1O2 generated by Rose Bengal (RB) sensitizer at different light intensities. In isoprene-emitting leaves, photosynthesis and H2O2 and malonyldialdehyde (MDA) contents were not affected by low to moderate 1O2 concentrations generated at light intensities of 800 and 1240 µmol m−2 s−1, but symptoms of damage and reactive oxygen accumulation started to be observed when high levels of 1O2 were generated by very high light intensity (1810 µmol m−2 s−1). A dramatic decrease in photosynthetic performance and an increase in H2O2 and MDA levels were measured in isoprene-inhibited RB-fed leaves, but photosynthesis was not significantly inhibited in leaves in which the isoprene leaf pool was reconstituted by fumigating exogenous isoprene. The inhibition of photosynthesis in isoprene-inhibited leaves was linearly associated with the light intensity and with the consequently formed 1O2. Hence, physiological levels of endogenous isoprene may supply protection against 1O2. The protection mechanisms may involve a direct reaction of isoprene with 1O2. Moreover, as it is a small lipophilic molecule, it may assist hydrophobic interactions in membranes, resulting in their stabilization. The isoprene-conjugated double bond structure may also quench 1O2 by facilitating energy transfer and heat dissipation. This action is typical of other isoprenoids, but we speculate that isoprene may provide a more dynamic protection mechanism as it is synthesized promptly when high light intensity produces 1O2.

Notes: In root nodules of leguminous plants, such as Vicia faba L., N2 is fixed by rhizobial bacteroids within infected cells. These cells are located in the centre of the nodule, whereas the vascular system serving import and export of solutes is located in the periphery. Within the infected central tissue, metabolites may travel symplastically by using bands of interconnected uninfected cells. Structural evidence, however, speaks against symplastic movement between infected cells themselves. The present work examined the possibility of an apoplastic step in amino acid export from infected cells. Incubation experiments with dissected central tissue demonstrated the release of amino compounds by infected cells. The predominant compound released was asparagine, which is also the major amino acid in xylem sap of legumes forming indeterminate nodules. During incubation of central infected tissue, medium acidification by plasma membrane H+-ATPase quickly turned into slight alkalization, probably caused by the released amino acids. In vivo, this process would lead to an increased apoplastic pH with consequences for processes relying on the proton gradient across the plasma membrane. Uptake of 14C-labelled amino acids by uninfected and infected cells was studied using protoplasts isolated from the central nodule tissue. Uninfected protoplasts accumulated amino acids with low specificity in a ΔpH-dependent, bi-phasic manner, whereas infected protoplasts did not absorb amino acids from the medium. This indicates that uninfected protoplasts not only function in metabolite transport, but also in collection of amino acids from the apoplast. Taken together, both experimental approaches demonstrate the possibility of an apoplastic export step for amino acids in the central tissue of indeterminate legume nodules.

Notes: The putative role of gibberellins in the transition to flowering was investigated in Sinapis alba, a caulescent long-day (LD) plant. It was observed that: (1) physiological doses of exogenous gibberellins (GA1, GA3, GA9) do not cause the floral shift of the meristem when applied to plants grown in short days but have some positive effect on the flowering response to a suboptimal LD; no inhibition was observed in any case; (2) GA-biosynthesis inhibitors (prohexadione-Ca and paclobutrazol) considerably inhibit stem growth but have some negative effect on flowering only when a suboptimal LD is given; and (3) the floral transition induced by one 22-h LD does not correlate with any detectable change in GA content of the apical bud, of the leaves, and of the phloem exudate reaching the apex. Taken together, these results suggest that GAs do not act as a major signal for photoperiodic flower induction in Sinapis.

Notes: The present study examined the response of antioxidant systems to NaCl stress and the relative importance of Na+ and Cl– in NaCl-induced antioxidant systems in roots of rice seedlings. NaCl treatment caused an increase in the activities of ascorbate peroxidase (APX) and glutathione reductase (GR) in roots of rice seedlings, but had no effect on the activities of superoxide dismutase (SOD) and catalase (CAT). There were detectable differences in APX and GR isoenzymes between control and NaCl-treated roots. Levels of activity for SOD and CAT isoenzymes did not change in NaCl-stressed roots compared with the control roots. NaCl treatment produced an increase in H2O2, ascorbate (AsA), dehydro-ascorbate (DHA), reduced glutathione (GSH), and oxidized glutathione (GSSG) levels. Treatment with 50 mM Na-gluconate (whose anion is not permeable to membrane) led to a similar Na+ level in roots to that with 100 mM NaCl. It was found that treatment with 50 mM Na-gluconate affected H2O2, AsA, and DHA levels, APX and GR activities, OsAPX and OsGR mRNA induction in the same way as 100 mM NaCl. These observed changes seem to be mediated by Na+ toxicity and not by Cl– toxicity. On the other hand, it was found that NaCl, but not Na-gluconate and NaNO3, caused an increase in GSH and GSSG levels, indicating that Cl–, rather than Na+, is responsible for the NaCl-increased GSH and GSSG levels in roots of rice seedlings.

Notes: The mechanisms regulating stomatal response following exposure to low (5°C) soil temperature were investigated in aspen (Populus tremuloides Michx.) seedlings. Low soil temperature reduced stomatal conductance within 4 h, but did not alter shoot xylem pressure potential within 24 h. The xylem sap composition was altered and its pH increased from 6.5 to 7.1 within the initial 4 h of the low temperature treatment. However, the increase in abscisic acid (ABA) concentration in xylem sap was observed later, after 8 h of treatment. These changes were accompanied by a reduction in the electrical conductivity and an increase in the osmotic potential of the xylem sap. The timing of physiological responses to low soil temperature suggests that the rapid pH change of the xylem sap and accompanying changes in ion concentration were the initial factors which triggered stomatal closure in low temperature-treated seedlings, and that the role of the more slowly accumulating ABA was likely to reinforce the stomatal closure. When leaf discs were exposed to xylem sap extracted from low soil temperature-treated plants, stomatal aperture was negatively correlated with ABA and positively correlated with K+ concentrations of the xylem sap. The stomatal opening in the leaf discs linearly increased in response to exogenous KCl concentrations when K+ concentrations were in the similar range to those detected in the xylem sap. The lowest concentration of exogenous ABA to induce stomatal closure was several-fold higher compared with the concentration present in the xylem sap.

Notes: The strategy of ‘complementation by functional sufficiency’ was used to isolate XvVHA-c′′1, a vacuolar adenosine triphosphatase (V-ATPase) proteolipid subunit c′′ homologue from Xerophyta viscosa. XvVHA-c′′1 rescued Escherichia coli srl::Tn10 mutants that were subjected to a 1.2 M sorbitol osmotic stress. Bioinformatics analyses conducted on XvVHA-c′′1 revealed all signature characteristics that are common amongst subunit c homologues, which include the four transmembrane domain motifs and a conserved glutamate residue in the fourth transmembrane domain. XvVHA-c′′1 shares 90.96% identity with the Oryza sativa (japonica) subunit c homologue and 86.67% identity with a putative vacuolar ATP synthase proteolipid subunit c′ from Arabidopsis thaliana, at the amino acid level. Southern hybridization analysis conducted on X. viscosa genomic DNA confirmed the presence of XvVHA-c′′1 in the X. viscosa genome. Northern hybridization analysis was conducted on X. viscosa tissue subjected to NaCl stress, dehydration and − 20°C shock, in response to which upregulated transcript levels of XvVHA-c′′1 were seen. XvVHA-c′′1's functional relevance was established through complementation using a Saccharomyces cerevisiae vma3 knockout.

Notes: This study examines how brassinolide (BL) and ethylene interact in the gravitropic response mechanism of maize (Zea mays) primary roots. When applied exogenously, ethylene increases the rate of gravitropic curvature in a dose-dependent manner. This effect of ethylene was confirmed by the fact that AVG, a specific action inhibitor of ACC synthase, reduces the gravitropic curvature in the presence and absence of BL. Since AVG did not inhibit BL-increased gravitropic curvature completely, we investigated the possibility that BL may act on the gravitropic response by ways other than simply through enhanced ethylene production. We show that BL exhibits some of its stimulatory effect in the absence of ethylene. In addition, BL reduces the presentation time and lag period for the gravitropic response, whereas ethylene increases them. One possible mechanism of such action is that BL affects protein kinase activity, since the protein kinase inhibitors, staurosporine and H89, reduce BL-increased gravitropic curvature. In summary, BL is involved in the gravitropic response in maize primary roots via ethylene production, but it acts in a way that differs somewhat from that of ethylene.

Notes: The environmental control of flowering and sex expression has been studied under controlled environment conditions in three populations of the sedge Carex flava L. A dual floral induction requirement was demonstrated in all populations. Low temperature (〈 12°C) was obligatory for, and short photoperiods strongly enhanced, primary induction and inflorescence initiation. Stem elongation and inflorescence development were promoted by long photoperiods, although most plants developed stunted flower stems also under short day (SD) conditions. Growth vigour, abundance of flowering and primary induction requirements varied widely among the populations, with critical exposure times for full flowering varying from less than 9 to about 12 weeks in SD at 9°C, and from about 9 to more than 15 weeks in long days (LD). Sex expression in the normally male terminal spike was shifted towards femaleness by marginal or incomplete primary induction. Primary induction in LD resulted in a complete change to entirely female inflorescences, whereas marginal induction in SD resulted in a similar sex reversal in some plants. The results are discussed in relation to environmental and hormonal factors known to modify sex expression in flowering plants and the significance of the results to Carex systematics and classification.

Notes: Glutamine synthetase (GS) genes, GS1a and GS1b, in pine (Pinus sylvestris L.) are differentially regulated in tissue specificity and during seedling development. To gain insight into the regulatory mechanisms controlling their expression, we have analysed the 5′-flanking sequences of the gene GS1a using a transient expression system in pine protoplasts. Structural analysis of this region revealed the presence of putative regulatory elements including two AT-rich elements and a poly CT consensus sequence. A series of 5′- and 3′-deletions of the untranslated region covering the three putative elements, −800 to −626, −626 to −427 and +118 to +177 were analysed to demonstrate the functional implications of these elements in gene regulation. An electrophoretic mobility-shift assay showed that nuclear proteins prepared from pine cotyledons interact with both AT-rich regions (−800 to −427). Interestingly, no protein binding was detected when the untranslated region (+118 to +177) was included, even if deletion of that region suppressed promoter activity in the transient expression experiments conducted. However, simultaneous deletion of both types of cis elements, A/T and CT, resulted in a recovery of promoter activity of 50%. These results suggest a key regulatory role of the CT box by the interaction with A/T stretches in the distal part of the promoter and possibly with the proximal region (−427 to −1).

Notes: With the characterization of the total genomes of Arabidopsis thaliana and Oryza sativa, several putative plasma membrane components have been identified. However, a lack of knowledge at the protein level, especially for hydrophobic proteins, have hampered analyses of physiological changes. To address whether protein complexes may be present in the native membrane, we subjected plasma membranes isolated from Spinacia oleracea leaves to blue-native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is well established in the separation of functional membrane protein complexes from mitochondria and chloroplasts, but a resolved protein complex pattern from PM of eukaryotic cells has previously not been reported. Using this method, protein complexes from Spinacia oleracea PM could be efficiently solubilized and separated, including the highly hydrophobic aquaporin (apparent molecular mass 230 kDa), a putative tetramer of H+-ATPase, and several less abundant complexes with apparent masses around or above 750 kDa. After denaturation and separation of the complexes into their subunits in a second dimension (SDS-PAGE), several of the complexes were identified as hydrophobic membrane proteins. Large amounts of protein (up to 1 mg) can be resolved in each lane, which suggests that the method could be used to study also low-abundance protein complexes, e.g. under different physiological conditions.

Notes: The common bean root system is composed of several types of root (e.g. tap, basal, and lateral roots), whose physiological functions may be of great difference. However, we do not know if the root system of common bean differs in organic acid secretion and thus aluminium (Al) resistance. In the present study, the tap and basal roots of three common bean genotypes (i.e. G19842, SQ12 and BAT881) from different origins were compared for their citrate secretion and Al resistance. Grown in a simple solution containing 30 µM Al3+ for 24 h, genotype G19842 maintained 75% relative tap root length [RTRL = (tap root length with Al)/(tap root length without Al)], 48% relative basal root length [RBRL = (basal root length with Al)/ (basal root length without Al)], genotype SQ12 maintained 62% RTRL and 57% RBRL, while BAT881 only maintained 31% RTRL and 19% RBRL, indicating differential sensitivity of bean genotypes and root types to Al stress. The amounts of Al-induced citrate secretion by the tap/basal roots were 9.8/5.1, 8.2/5.9 and 5.4/4.1 nmol cm−2 FR (fresh root) [12 h]−1 for G19842, SQ12 and BAT881, respectively, indicating that both bean genotypes and root types differ in organic acid secretion. In G19842, the root surface area was 25% higher in tap root apex than that in basal root apex, and the amounts of citrate secretion per unit surface area and per root apex were 29 and 62% higher in tap root apex than those in basal root apex, respectively, suggesting that the higher citrate secretion in the tap root apex could be attributed to the larger surface area and the higher secretion activity. Stronger inhibition of Al-induced citrate secretion in the basal than tap roots by anthracene-9-carboxylic acid, an inhibitor of anion channel and K-252a, a broad range inhibitor of protein kinase may also imply the differences in the activities of anion channels and K-252a-sensitive protein kinases on the plasma membrane between the tap and basal roots, resulting in differential citrate secretion. We propose that the higher Al resistance in the tap root than in basal roots might be attributed to both greater number and higher activity of the anion channels in the former, thus allowing more citrate secretion in this root type.

Notes: Although mineral N (nitrate and ammonium) is believed to have generally negative effects on nodulation in legume–rhizobia symbioses, previous studies have shown that low, static concentrations of ammonium stimulate nodulation in pea, and that this enhancement may be due to an elevation in cytokinin to auxin levels in roots. Here, the effects of ammonium (0.0, 0.1, 0.5 and 2.5 mM) on nodulation and auxin levels were investigated in wild-type (WT) white clover (Trifolium repens cv. Haifa) and its transformants (lines 38 and 41) which contain the auxin-sensitive reporter gene (GH3:gusA). The effects of exogenous application (10−10, 10−9 and 10−8 M) of the cytokinin 6-benzylaminopurine (BAP) were also assessed. Whole-plant nodulation (nodules plant−1) and dry weight (DW)-specific nodulation (nodules g−1 root DW) were stimulated (up to 49%) in all white clover lines by 0.1 mM NH4+. This represents the first confirmation of an NH4+-induced stimulation of DW-specific nodulation in a species other than pea. At 2.5 mM NH4+, the effect was lost on whole-plant nodulation and was inhibitory on DW-specific nodulation. Rhizobial inoculation resulted in a decline in the expression of GH3:gusA in root tips as expected; however, ammonium treatment did not affect GH3 expression in any root zones. Exogenous application of BAP at 10−9 and 10−8 M stimulated whole-plant and DW-specific nodulation in wild-type white clover to a similar degree as treatment with 0.1 mM NH4+. These results support our previous hypothesis that the stimulation of nodulation by low concentrations of ammonium involves the alteration of the ratio of cytokinin to auxin, specifically by increasing cytokinin.

Notes: We have studied how tree seedlings with differing leaf phenological traits change their nitrogen allocation in seasonally changing light environments. Specifically, we have investigated seasonal changes in maximum leaf photosynthetic rates in situ, and changes in nitrogen, chlorophyll and ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) content. We have also estimated nitrogen allocation characteristics from leaf nitrogen to photosynthetic proteins, light-harvesting protein and other proteins in four successional tree species growing in a larch plantation. The gap phase species, Magnolia hyporeuca Siebold et Zucc., displays higher photosynthetic rates to compensate for the short leafy period and allocates more nitrogen to Rubisco. In contrast, tree species that grow at the forest floor, Prunus ssiori F. Schmidt and Carpinus cordata Blume, use the leafless period of the canopy trees to change their allocation of nitrogen from Rubisco to chlorophyll in line with canopy closure. Quercus mongolica Fischer ex Ledeb. var. crispula (Blume) Ohashi is a mid-successional tree species, and maintains a high photosynthetic rate in September because it allocates more nitrogen to Rubisco. We conclude that phenological differences in tree seedlings are clearly reflected in the nitrogen allocation pattern at gaps and at the forest floor in larch plantations.

Notes: The genus Brassica includes species with two levels of polyploidy: diploids that have replicated genomes and appear to be ancient polyploids, and allopolyploids that were recently derived from hybridization of the diploid species. Research on these species has provided evidence that polyploidy contributes to phenotypic variation through several mechanisms. Polyploidy increases the potential variation of dosage-regulated gene expression, and this mechanism appears to affect flowering time variation through the effects of replicated copies of the flowering time gene FLC. Homoeologous chromosome transpositions occur in allopolyploids that alter allele composition, and this has created novel flowering time variation in newly formed Brassica allopolyploids. New allopolyploids also may have epigenetic changes or altered regulatory interactions that affect gene expression and phenotypic variation. Continued research on Brassica and other species should provide insight into the relative importance of these mechanisms for generating novel variation in polyploids.

Notes: Parameters for the evaluation of the effects of photoinhibition on photosynthetic carbon gain were studied in Chenopodium album leaves. The light-response curve of photosynthetic rate was determined at 36 Pa CO2 partial pressure and fitted by a non-rectangular hyperbola. Both the initial slope of the curve and the light-saturated rate decreased in photoinhibited leaves, although the decrease in the latter was small. The convexity of the curve was also smaller in photoinhibited leaves. The capacities of ribulose-1,5-bisphosphate carboxylation (Vcmax) and electron transport (Jmax) were estimated from the CO2-response curves. Vcmax and Jmax decreased similarly with increasing photoinhibition. Energy partitioning in photosystem II (PSII) was estimated using chlorophyll fluorescence parameters. The fraction of energy that was consumed by photochemistry decreased with increasing photoinhibition. However, an increase in inactive PSII, decreasing energy partitioning to active PSII, relaxed the excitation pressure in PSII, and led to a reduction in the fraction of excess energy that was neither consumed by photochemistry nor dissipated as heat.

Notes: The pigments of etiolated leaves of barley (Hordeum vulgare L.) were analysed during dark periods after flash illumination, and the results were compared with in vivo spectroscopy of the leaves. Pretreatment of the leaves with kinetin slightly stimulated and pretreatment with NaF and anaerobiosis inhibited the esterification of chlorophyllide a (Chlide) at 10–40 min after the flash, whereas the rapid esterification within 30 s after the flash remained unchanged. Irrespective of pretreatment, the amount of esterified pigment was, at any time, identical with the amount of pigment that had shifted its absorption from 684 to 672 nm (Shibata shift). Cycloheximide (CHI) had only a small inhibitory effect on esterification, but drastically inhibited the hydrogenation of geranylgeraniol to phytol, bound to Chlide. The regeneration of long-wavelength protochlorophyllide a (Pchlide650) was stimulated by kinetin and inhibited by CHI and NaF. During the rapid phase (0–30 s after the flash), the esterification was faster than the regeneration of Pchlide650, and this, in turn, was faster than the formation of photoactive Pchlide. The kinetics changed after pretreatment with 5-aminolaevulinic acid: regeneration of Pchlide650 was the fastest reaction and the Shibata shift preceded the esterification of Chlide. The results are discussed as pigment exchange reactions at NADPH:protochlorophyllide oxidoreductase (POR; EC 1.6.99.1).

Notes: The investigation and improvement of nitrogen efficiency in oilseed rape (Brassica napus L.) are important issues in rapeseed breeding. The objective of this study was to modify ammonium assimilation in transgenic rapeseed plants through the expression of the Escherichia coli asparagine synthetase (AsnA, E.C. 6.3.1.1) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and to study its influence on amino acid composition in leaves and on seed traits related to nitrogen efficiency. In regenerated transgenic plants, the 37 kDa AsnA protein was detected by Western blot analysis, but was lacking in untransformed control plants of cv. Drakkar. In the transformants, in vitro asparagine synthetase activities ranged from 105 to 185 nmol asparagine mg−1 protein h−1, whereas, in untransformed control plants, only negligible asparagine synthetase activities of up to 5 nmol asparagine mg−1 protein h−1 were found. Despite these significant activities, no changes in the amino acid composition in the leaves or in the phloem of transgenic plants were detectable. In a pot experiment, two transgenic lines expressing the prokaryotic asparagine synthetase clearly performed inferiorly to control plants at limiting nitrogen (N) fertilizer supply. Although the seed N content was increased, the seed yield and the seed N yield were reduced, which was interpreted as an increased nitrate assimilation leading, at limiting N supply, to a reduced seed yield and seed N yield. At high N fertilizer supply, the differences were less pronounced for one transgenic line, whereas the other showed a higher seed N yield and an improved nitrogen harvest index. The results show that the expression of the E. coli asnA gene in oilseed rape could be of advantage at high N supply, but not at limiting N fertilizer supply.

Notes: GALACTINOL SYNTHASE (GOLS) acts on UDP-galactose and myo-inositol to form galactinol. The production of galactinol is thought to be the committed step in the biosynthesis of the raffinose family oligosaccharides (RFOs), a group of carbohydrates hypothesized to play a number of roles in plant development. Three cDNAs for maize (Zea mays L. inbred line B73) GOLS (ZmGOLS) were identified from the Pioneer Hi-Bred expressed sequence database. The recombinant proteins from these cDNAs synthesize galactinol. Transcript amounts in developing and germinating seeds were determined using northern blot, ribonuclease protection assays, and multiplex RT-PCR. ZmGOLS1 mRNA was not detected in any of the tissues examined. ZmGOLS3 transcripts accumulated late during seed development and were present in mature seeds but declined quickly to undetectable amounts following imbibition. Experiments in which germination was interrupted by desiccation, but not by cold, heat, or salt stress, detected ZmGOLS2 transcripts. Soluble sugar amounts and GALACTINOL SYNTHASE activity were determined for developing maize seeds as well as for mature seeds during germination and upon stress during germination. Neither enzyme activity nor galactinol or raffinose quantities could be related to ZmGOLS transcript abundance. Galactinol amounts were most closely related to myo-inositol quantities during development and early germination. Maturation desiccation appears to be a switch delineating a realm of expression for ZmGOLS3, predominant during seed development, from that of ZmGOLS2, principally associated with post-imbibition dehydration stress.

Notes: Accumulation of transcript from three maize (Zea mays L. inbred line B73) GALACTINOL SYNTHASE (ZmGOLS) genes in abscised developing seeds and callus before and after stress was examined. Previous work showed that transcript from ZmGOLS3 predominates during seed development, whereas ZmGOLS2 transcript accumulates when triggered by certain stresses following imbibition. Abscission stimulated accumulation of ZmGOLS transcript in immature kernels. This accumulation was enhanced by increasing durations of dehydration stress. In callus tissue, neither ZmGOLS1 nor ZmGOLS3 was detected regardless of media composition or the nature or duration of the applied stress. Accumulation of ZmGOLS2 transcript in heat- or dehydration-stressed callus was induced by exogenously supplied myo-inositol in low amounts but not by sucrose. However, abundance of transcript was a poor indicator of activity of GOLS or amounts of galactinol/raffinose regardless of type of tissue, composition of media, or imposition of stress. Neither were amounts of galactinol/raffinose tightly associated with activity of GOLS nor amounts of myo-inositol, despite the ability of exogenously supplied raffinose to maintain the growth of dehydration-stressed callus. The abundance of ZmGOLS transcript did not increase in response to exogenously supplied abscisic acid.

Notes: A new stress-responsive gene BoRS1 (GenBank accession No. 〈accessionId ref="info:ddbj-embl-genbank/AY373021"〉AY373021) was isolated from Brassica oleracea var. acephala by rapid amplification of cDNA ends (RACE). The full-length cDNA of BoRS1 was 2076 bp and contained a 1851 bp open reading frame (ORF) encoding 617 amino acids. Sequence analysis indicated that the deduced BoRS1 shared some identities with 〈accessionId ref="info:ddbj-embl-genbank/LTI65"〉LTI65, RD29A, RD29B and 〈accessionId ref="info:ddbj-embl-genbank/COR78"〉COR78 from Arabidopsis thaliana. Southern blot analysis of genomic DNA indicated that other related genes existed and there were two copies of BoRS1 in the genome of B. oleracea. Northern blot analysis revealed that BoRS1 was up-regulated by cold, mannitol, NaCl and abscisic acid (ABA). Expressional fluctuation of time course with ABA implied a two-step induction process. Tissue-specific expression analysis indicated that BoRS1 was expressed in all the tested plant tissues including leaves, stems and roots. Our studies imply that BoRS1 is a new gene that is responsive to environmental stresses such as low temperature, salinity and osmotic stress.

Notes: This paper describes the first measurement of enzyme activities in cluster roots under –Fe stress, at different stages of cluster root development and function. In Lupinus albus L., Cluster roots are produced both under iron- and phosphorus-deficient conditions. In both cases the structure is similar, but the level of exudation is much greater in iron-deficient plants. Much work has been done on the enzyme kinetics of P-deficient cluster roots, but none on enzyme activities of Fe-deficient cluster roots. The enzymes investigated were citrate synthase (EC 4.1.3.7), aconitase (EC 4.2.1.3), isocitrate dehydrogenase [IDH(NAD) (EC 1.1.1.41) and IDH (NADP) (EC 1.1.1.42)] and lactate dehydrogenase (LDH) (EC 1.1.1.27). In cluster roots, citrate synthase activity was initially lower than in lateral roots but, after 5 days, recovered to the lateral root level. Cluster root aconitase levels initially increased, but fell sharply on day 3, and no activity was detected after day 5. IDH (NAD) levels were much lower in cluster roots than in laterals, dropping to a low on day 3, and then rising throughout development. IDH (NADP) levels were always higher in cluster roots than in lateral roots, increasing throughout development. LDH levels in cluster roots fell throughout development. Internal tissue concentrations of citrate were markedly higher in –Fe laterals than in +Fe lateral roots and in cluster roots. Cluster root levels of citrate increased dramatically after day 3. Results are discussed within the context of previous work on enzyme kinetics under –P, and the importance of a block in aconitase activity is highlighted.

Notes: The relationship between active oxygen species (AOS) and membrane damage, and between antioxidant enzyme activity and chilling tolerance has been documented, but the mechanisms responsible for perennial forage grass to survive winter with temperatures at −30°C in temperate alpine regions is not well understood. In this study, the seasonal pattern of enzymatic antioxidant systems superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), peroxidase (EC 1.11.1.7), ascorbate peroxidase (EC 1.11.1.11) and lipid peroxidation in roots and leaves of alpine perennial grasses grown in their natural environment were investigated to understand the role of the enzymatic antioxidant system in freezing tolerance of perennial grasses. Four grasses, Poa sphyondylodes Trine., Bromus inermis Leyss., Bromus sinensis Keng. and Elymus nutans Griseb., were established in alpine conditions in 1993. The grasses were sampled at approximately semi-monthly intervals in the autumn of 1995 and spring of 1996. The results showed that leaves were dead in the autumn and membrane damage seems to play a key role in the decline of this organ. Antioxidant enzyme activities of the roots strongly changed with declining temperature in the autumn and winter or increasing temperature in the spring. With the decrease in temperature in the autumn the antioxidant enzyme activities increased rapidly, reaching maximum values in early November and then slowly declining during the following winter period, although they were still higher than in September In the spring, antioxidant enzymes activities increased again in the roots with the rise of temperature from mid April to early May when the shoots began re-growth. In contrast, thiobarbituric acid-reactive substances content in the roots increased markedly in the autumn, reaching maximum values in early October and remaining constant with little fluctuation during the following winter. In the autumn when the roots experienced winter acclimation, the formation of freezing tolerance in the roots was correlated with the activities of the antioxidant enzyme, indicating that antioxidant activity systems in the roots played an important role in limiting the production of free radicals to protect membrane integrity. Freezing tolerance in alpine grasses correlated with an increased capacity to scavenge or detoxify activated AOS by the antioxidant enzymatic system. AOS accumulated with decreasing temperature in early cold acclimation may be an inducer in activating the antioxidant enzyme defence system for the formation of freezing tolerance in roots.

Notes: Somatic embryogenesis (SE) of Pinus strobus L. has been greatly improved over the last few years with respect to both the initiation frequencies from a number of seed families and production of mature somatic embryos that readily convert to plants. However, there are no data on biochemical characterization of somatic embryos in relation to zygotic embryos of eastern white pine and on the optimal duration of the maturation stage. It is believed that somatic embryos closely resembling zygotic embryos not only morphologically but biochemically would display more vigorous growth. Hence, in this study the accumulation pattern of the most abundant seed storage proteins in zygotic and somatic embryos were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and identified by amino acid sequencing and tandem mass spectrometry (MS/MS). This showed that somatic embryos accumulated storage proteins in a similar manner to zygotic embryos and that the most abundant were the buffer-insoluble 11S- globulins MW 59.6 kDa, which dissociated under reduced conditions to 38.2–40.0 and 22.5–23.5 kDa range polypeptides, and buffer-soluble 7S vicilin-like proteins MW 46.0–49.0 kDa, which did not separate under reduced conditions. Other relatively abundant soluble proteins were in the ranges of 25–27 and 27–29 kDa. The only group of proteins that showed different migration profiles in the presence of β-mercaptoethanol (ME) were the low molecular mass proteins of 14.6–16.5 kDa. Somatic embryos that matured for 9 weeks on medium with 6% sucrose accumulated more storage proteins than those matured on medium with 3% sucrose and the extension of the maturation period to 12 weeks resulted in significant reduction of the storage proteins on both media. As expected, somatic embryos matured on medium with 6% sucrose had lower water potential (Ψ) than those from medium with 3% sucrose. Nonetheless, the somatic embryos matured under the best of tested conditions (6% sucrose for 9 weeks) had slightly higher water content; 1.35 ± 0.28 g H2O g−1 DM (mean ± sd) than the mature non-dried zygotic embryos; (1.16 ± 0.09 g H2O g−1 DM), and accumulated less storage proteins, whose amounts were either similar to (7S-vicilins) or below (11S-globulins) those found in the immature zygotic embryos collected 2 weeks prior to the usual cone collection. The implications of these results for further research and development of viable artificial seed is discussed.

Notes: Seeds gradually lose their viability during dry storage. The damage that occurs at the biochemical level can alter the seed physiological status and is affected by the storage conditions of the seeds. Although these environmental conditions controlling loss of viability have been investigated frequently, little information is available on the genetics of seed longevity. Using Arabidopsis mutants in defined developmental or biochemical pathways such as those affected in seed coat composition, seed dormancy, hormone function and control of oxidative stress, we tried to gain insight into the genes and mechanisms controlling viability of stored seeds. Mutations like abscisic acid insensitive3 (abi3) as well as abscisic acid deficient1 (aba1) show reduced longevity, which may be partially related to the seed dormancy phenotype of these mutants. Mutants with seed coat alterations, especially aberrant tests shape (ats), showed a stronger reduction in germination percentage after storage, indicating the importance of a ‘functional’ seed coat for seed longevity. A specific emphasis was placed on mutants affected in dealing with Reactive Oxygen Species (ROS). Because several pathways are involved in protection against ROS and because gene redundancy is a common feature in Arabidopsis, ‘double’ mutants were generated. These ‘double’ mutants and the corresponding single mutants were subjected to a controlled deterioration test (CDT) and a germination assay on hydrogen peroxide (H2O2) after prolonged storage at two relative humidities. CDT and germination on H2O2 affected all genotypes, although it appears that other effects like genetic background are more important than the deficiencies in the ROS scavenging pathway. Explanations for this limited effect of mutations affecting ROS scavenging are discussed.

Notes: A versatile two-step cultivation procedure for Arabidopsis thaliana is described for the production of large quantities of leaf material suitable for biochemical and biophysical analysis. The first step comprises a miniature greenhouse made out of a plastic pipette box to grow the seedlings to the six-leaf stage. For continued growth, the seedlings are transferred to hydroponic cultivation using an opaque container covered by a styrofoam lid. Transfer of the small seedlings to hydroponic culture is facilitated by growth in separate pipette tips, which protects vulnerable roots from damage. The hydroponic cultivation system is easy to scale-up and produces large amounts of relatively large leaves and roots. This hydroponic system produces enough plant material to make Arabidopsis a feasible model for biochemical and biophysical experiments, which can be combined with the available genetic information to address various aspects of plant functional genomics.

Notes: Phytochelatins (PCs) have been detected in a large range of plant species, but their role in heavy-metal tolerance is unclear. Various clones of Salix viminalis are differently tolerant to heavy metals, and the aim of this work was to investigate whether PCs were differently expressed in tolerant compared with sensitive clones. In a long-term study, five clones with high or low metal tolerance were cultivated 21 days without or with Cd (1 or 10 µM), Cu (0.3 or 7 µM), Ni (15 µM), Pb (7 µM) or Zn (10 or 100 µM). Controls were further used in a short-term study where Cd (1 µM) was added and samples were collected 0, 15 and 30 min, 1, 3 and 24 h after start of treatment. PCs were analysed on high performance liquid chromatography (HPLC) using two different methods: post-column derivatization using Ellmans reagent and pre-derivatization with monobromobimane. Thlaspi caerulescens treated with Cd was used as internal PC standard. No PCs could be detected in Salix with either of the two methods in any of the treatments: different clones, metals, concentrations, plant parts or treatment time. The 16 thiol peaks shown were the same in both control and treated plants. Both HPLC methods showed PC peaks when Thlaspi was used but these peaks could not be associated with any of the 16 peaks. The amino acid composition of the 16 peaks was not the expected composition of that of PCs. Thus, Salix viminalis have no detectable levels of PCs, which in turn are not involved in heavy metal tolerance in Salix.

Notes: The contents of protochlorophyllide, protochlorophyll and chlorophyll together with the native arrangements of the pigments and the plastid ultrastructure were studied in different leaf layers of white cabbage (Brassica oleracea cv. capitata) using absorption, 77 K fluorescence spectroscopy and transmission electron microscopy. The developmental stage of the leaves was determined using the differentiation of the stoma complexes as seen by scanning electron microscopy and light microscopy. The pigment content showed a gradual decrease from the outer leaf layer towards the central leaves. The innermost leaves were in a primordial stage in many aspects; they were large but had typical proplastids with few simple inner membranes, and contained protochlorophyllide and its esters in a 2 : 1 ratio and no chlorophyll. Short-wavelength, not flash-photoactive protochlorophyllide and/or protochlorophyll forms emitting at 629 and 636 nm were dominant in the innermost leaves. These leaves also had small amounts of the 644 and 654 nm emitting, flash-photoactive protochlorophyllide forms. Rarely prolamellar bodies were observed in this layer. The outermost leaves had the usual characteristics of fully developed green leaves. The intermediary layers contained chlorophyll a and chlorophyll b besides the protochlorophyll(ide) pigments and had various intermediary developmental stages. Spectroscopically two types of intermediary leaves could be distinguished: one with only a 680 nm emitting chlorophyll a form and a second with bands at 685, 695 and 730 nm, corresponding to chlorophyll–protein complexes of green leaves. In these leaves, a large variety of chloroplasts were found. The data of this work show that etioplasts, etio-chloroplasts or chloro-etioplasts as well as etiolated leaves do exist in the nature and not only under laboratory conditions. The specificity of cabbage leaves compared with those of dark-grown seedlings is the retained primordial or intermediary developmental stage of leaves in the inner layers for very long (even for a few month) period. This opens new developmental routes leading to formation of specially developed plastids in the various cabbage leaf layers. The study of these plastids provided new information for a better understanding of the plastid differentiation and the greening process.

Notes: The impact of phosphorous nutrition on plant growth, symbiotic N2 fixation, ammonium assimilation, carbohydrate and amino-acid accumulation, as well as on nitrogen, phosphorus and ATP content in tissues in common bean (Phaseolus vulgaris) plants was investigated. Plants inoculated with Rhizobium tropici CIAT899 were grown in Leonard jars under controlled conditions, with P-deficient (0 and 0.1 mM), P-medium (0.5, 1 and 1.5 mM) and P-high (2 mM) conditions in a N-free nutrient solution. The P application, increased leaf area, whole plant DW (67%), nodule biomass (4-fold), and shoot and root P content (4- and 6-fold, respectively) in plant harvested at the onset of flowering (28-days-old). However, P treatments decreased the total soluble sugar and amino acid content in vegetative organs (leaf, root and nodules). The root growth proved less sensitive to P deficiency than did shoot growth, and the leaf area was significantly reduced at low P-application. The absence of a relationship between shoot N content, and P levels in the growth medium could indicate that nitrogen fixation requires more P than does plant growth. The optimal amount for the P. vulgaris–R. tropici CIAT899 symbiosis was 1.5 mM P, this treatment augmented nodule-ARA 20-fold, and ARA per plant 70-fold compared with plants without P application.

Notes: A dwarf mutant of broad bean (Vicia faba L.), the variety Rinrei, has been created by γ-ray irradiation. Rinrei is characterized by dark green leaves and by reduced plant length, internode and petiole length, shoot weight, and number of branches. Genetic analysis of hybrids between Rinrei and two wild-type lines indicated that these characteristics are controlled by a single recessive gene. The phenotype of Rinrei was restored to that of the wild type by application of brassinolide, but not by GA3. Qualitative and quantitative analysis by gas chromatography–mass spectrometry indicated that 24-methylenecholesterol and isofucosterol accumulated in Rinrei to levels more than 30 times higher than in the wild type. In contrast, Rinrei had lower than wild-type levels of campesterol, sitosterol and brassinosteroids. Therefore, Rinrei is a brassinosteroid-deficient mutant defective in sterol C-24 reduction. The gene was tentatively designated as brassinosteroid deficient dwarf 1, bdd1, which seems to be a homologue of Arabidopsis dwf1 (dim, cbb1) and pea lkb.

Notes: Fruit starch reserves can be an important contributor to the sugar content of some ripe fruit, and despite the relatively high financial premiums (compared to other fruit) commanded by ripe strawberries, neither their starch or sugar biochemistry has been examined in detail. This study assessed the rate of starch biosynthesis and breakdown in developing strawberry and sought to determine the temporal changes in the activities of selected enzymes known to be involved in sucrose-starch interconversions. Scanning electron microscopy revealed that starch levels appeared greatest in immature strawberry (Fragaria×ananassa, cv. Elsanta) at 7 days postanthesis, as evidenced by a decrease in the number of cells containing starch granules as ripening progressed. Levels of key enzymes of starch and sugar metabolism estimated using Western blotting and enzyme activity analysis showed that activities did not correlate with antigen levels. In particular, enzyme activity recovery experiments indicated that losses were due to non-proteinaceous inhibitors, and in particular protein binding: highlighting the potential for misinterpretation of enzyme activity data gathered from ripening (strawberry) fruit tissue extracts. Consequently, in vitro experiments using [U-14C] glucose revealed that incorporation to starch is low (11%) at the earliest developmental stages when starch content is greatest. Starch synthesis rate then declines to non-detectable levels as fruit expand and ripen. These results show that starch accumulates extremely early in the fruit formation process and that starch degradation predominates during fruit growth and development. We estimate that breakdown of transient starch can contribute up to 3% of the sugar accumulated in ripe fruit.

Notes: The effects of three ecdysteroids, 20-hydroxyecdysone (20E), 2-deoxy-20-hydroxyecdysone (2d20E) and 20-hydroxyecdysone 22-acetate (20E22Ac), on growth and the levels of cellular components in Chlorella vulgaris Beijerinck (Trebouxiophyceae) are reported and compared with data previously reported for ecdysone (E; Bajguz A and Koronka A, Plant Physiol Biochem 39: 707–715, 2001). All three 20-hydroxyecdysteroids stimulate growth of C. vulgaris cells over a wide concentration range (10−16 to 10−7 M). Optimal stimulation is observed at 10−9 M with each ecdysteroid. High concentrations (〉10−6 M) are cytotoxic. The potency ranking of the ecdysteroids is 20E 〉 20E22Ac 〉 2d20E 〉 E. Levels per cell of DNA, RNA, protein, sugars, organic and inorganic phosphorus, chlorophylls a and b and phaeophytins a and b are all stimulated by ecdysteroid treatment when compared with the untreated control cells. Possible modes of action of ecdysteroids on C. vulgaris cells are discussed.

Notes: A comparison of the effects of a rapid and a slowly imposed water deficit on photosynthesis was performed in Setaria sphacelata var. splendida (Stapf) Clayton, a C4 NADP-ME grass. Gas exchange was measured in rapidly and slowly dehydrated adult leaves either under atmospheric CO2 partial pressure with an infrared gas analyser or under saturating CO2 partial pressure with a leaf disc oxygen electrode. These measurements were used to calculate stomatal and non-stomatal limitations to photosynthesis. These were further investigated using modulated chlorophyll a fluorescence measurements and photosynthetic pigment quantification. The decrease of net photosynthesis, leaf conductance and water use efficiency was more pronounced under rapid stress than in slow stress. However, photosynthesis is always mainly limited by stomata in both types of stress, albeit the contribution of non-stomatal limitations increases at severe water deficits in slow stress experiments. The substomatal CO2 partial pressure significantly increased in both types of stress, suggesting an increased resistance due to an internal barrier to CO2 diffusion. Physical alterations in the structure of the intercellular spaces due to leaf shrinkage may account for these results. The maximal photochemical efficiency of photosystem II (PSII) was remarkably resistant to stress, as the Fv/Fm ratio decreased only at severe water deficit. On the contrary, the effective photochemical efficiency of PSII (ΔF/F′m) measured under high actinic light decreased linearly in both types of stress, although in a more pronounced way under rapid stress. A similar variation in photochemical quenching suggests that the decrease of ΔF/F′m is mainly due to the closure of PSII reaction centres. The non-photochemical quenching did not change significantly except under severe dehydration indicating that the energization state of thylakoids remained stable under stress. The decrease observed in photosynthetic pigments may be an adaptation to stress rather than a limiting factor to photosynthesis. Results suggests that, although intrinsic mesophyll metabolic inhibitions occur, stomatal limitation to CO2 diffusion is the main reason for the decrease in photosynthesis.

Notes: A complete cDNA and a genomic DNA fragment coding for urate oxidase (urate: oxygen oxidoreductase, EC 1.7.3.3) from chickpea (Cicer arietinum L.) were isolated and characterized. The 1032 bp cDNA (CAUR1) contains a complete open reading frame that encodes a 308 amino acid protein with a predicted size of 34.06 kDa and a pI of 9.38. This protein shows a strong similarity with other uricases present in the databases. Heterologous expression in Escherichia coli showed urate oxidase activity in crude extracts from induced cells, demonstrating that CAUR1 encodes a complete and functional uricase enzyme. Kinetic properties of the recombinant enzyme were similar to those of the native uricase purified from chickpea leaves. The genomic organization of the chickpea uricase gene (CAUR1) remarkably resembles that of the soybean (Glycine max L.) uricase gene, presenting eight exons and seven introns. Within the 5′-flanking region of the chickpea gene, nucleotide sequences matching consensus motifs of soybean nodule NAT2-nuclear factors and common bean (Phaseolus vulgaris L.) proteins (PNF1) are present. It demonstrates that a single copy of the uricase gene is present in the chickpea genome and that the gene is expressed not only in nodules, but also in leaves and roots.

Notes: Phenylalanine ammonia-lyase (PAL) catalyses the first step controlling the rate of phenylpropanoid metabolism. Wounding is a ubiquitous stress in nature and in the harvesting and preparation of fruits and vegetables that induces an increase in PAL activity, an accumulation of phenolic compounds and subsequent tissue browning. A wound-inducible PAL gene (LsPAL1) was isolated from Romaine lettuce by RT-PCR. The putative protein encoded by LsPAL1 is similar to predictive polypeptides sequences for other PALs. The kinetics of PAL mRNA accumulation is similar to those of induced PAL enzyme activity, with enzyme activity following mRNA accumulation by 12 h. Wound-induced PAL transcripts accumulated in cells close to the wound sites. Tissue printing showed that PAL mRNA was associated with tissue next to the epidermis and vascular bundles. A heterologous PAL protein was expressed in E. coli and was found to show significant PAL activity.

Notes: A Pantoea agglomerans isolate from barley seeds, selected for inducing growth promotion in tomato test plants, was found to excrete several hormones of the cytokinin class into its culture medium. In addition to isopentenyladenine, isopentenyladenosine and the 2-methylthiol derivates of these, an unknown compound with affinity to anticytokinin antibodies was also isolated. Mass spectroscopy indicated the structure of this to be a deoxyisopentenyladenosine. The structure of 9-(5′-deoxy-β-d-ribofuranosyl)-6-(3-methyl-2-butenylamino)purine was verified after synthesis of standards and analysis with GC–MS. The synthesized 5′-deoxyisopentenyl-adenosine showed activity in the Amaranthus bioassay, specific for cytokinins. To our knowledge this is the first report of a naturally occurring cytokinin containing 5′-deoxyribose.

Notes: In order to continuously supply horticultural products for long periods, it is essential to store them after harvest in low temperatures. However, many tropical and subtropical fruits and vegetables, such as citrus, are sensitive to chilling. In previous studies, the authors have shown that a short hot water rinsing treatment (at 62°C for 20 s) increased chilling tolerance in grapefruit. In order to gain more insight into the molecular mechanisms involved in heat-induced chilling tolerance, PCR cDNA subtraction analysis was performed which isolated four different PCR fragments whose expression was enhanced 24 h after the heat treatment, and that showed high sequence homology with various plant HSP18-I, HSP18-II, HSP22 and HSP70 genes. It was found that the short hot water treatment given at 62°C for 20 s, but not at lower temperatures of 20 or 53°C, increased the expression of the various HSP cDNAs in grapefruit peel tissue. However, when the fruits were kept at ambient temperatures, the increases in HSP mRNA levels following the hot water treatment were temporary and lasted only between 6 and 48 h. Similar temporary increases in the HSP mRNA levels were detected following exposure of the fruit to a hot air treatment at 40°C for 2 h. Nevertheless, when the fruits were treated with hot water but afterwards stored at chilling temperatures of 2°C, the mRNA levels of the various HSP18-I, HSP18-II, HSP22 and HSP70 cDNAs increased and remained high and stable during the entire 8-week cold-storage period, suggesting their possible involvement in heat-induced chilling-tolerance responses. The chilling treatment by itself increased the expression of the HSP18-I cDNA, but had no effect on the mRNA levels of any of the other HSP cDNAs. Exposure of fruit to other stresses, such as wounding, UV irradiation, anaerobic conditions and exposure to ethylene, had no effect on the expression of the various HSPs. Overall, the study explored the correlation between the expression and persistence of various HSP cDNAs in grapefruit peel tissue during cold storage, on the one hand, and the acquisition of chilling tolerance, on the other hand, and the results suggest that HSPs may play a general role in protecting plant cells under both high- and low-temperature stresses.

Notes: During illumination of dark-grown plants protochlorophyllide (Pchlide) is continuously transformed to chlorophyllide (Chlide). Different dark-grown plants, maize (Zea mays cv. Sundance), wheat (Triticum aestivum cv. Kosack), pea (Pisum sativum cv. Kelwedon wonder), the lip1 mutant of pea, and the aurea mutant of tomato (Solanum lycopersicum), have various ratios of spectral Pchlide forms in darkness. When the plants were illuminated and then returned to darkness Pchlide re-accumulated. The proportions of different Pchlide forms within the pool of re-accumulated Pchlide were followed by low temperature fluorescence emission and excitation spectra in green and greening leaves. After 1 h of illumination the spectral characteristics of regenerated Pchlide forms mirrored those of Pchlide in dark-grown plants and were thus species dependent. After a prolonged illumination period (24 h) as well as in fully green leaves energy transfer to chlorophyll (Chl) masked the presence of long-wavelength Pchlide in the fluorescence emission spectra. However, excitation spectra showed Pchlide absorption around 650 nm and its flash-induced disappearance confirmed its nature of phototransformable Pchlide. In fact the excitation spectra showed that the proportions of different Pchlide forms in green leaves highly resembled the proportions of Pchlide forms in dark-grown leaves and were specific for the plant variety. Thus Chl formation in both dark-grown and light-grown leaves can occur in a similar way through the main photoactive long-wavelength form of Pchlide.

Notes: Water stress-induced spikelet sterility limits rice production under upland conditions. The causes of spikelet sterility under drought stress are poorly understood. In this study the role of antioxidant defence management in drought-induced spikelet sterility was investigated in two rice (Oryza sativa) genotypes differing in drought resistance. Drought-resistant N22 genotype showed less water stress-induced spikelet sterility when compared to the susceptible N118 genotype under upland conditions. The N22 panicles maintained higher RWC and turgor potential and lower H2O2 levels across the developmental stages under water stress than that of N118 panicles. Drought-induced enhancement in superoxide dismutase (SOD, EC 1.15.1.1) activity coupled with higher ascorbate (AsA), glutathione (GSH) content and enhanced ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) activities resulted in lower H2O2 levels in N22 panicles. In contrast, insufficient enhancement in SOD, APX and GR activities resulted in relatively higher H2O2 levels under water stress in N118 panicles. The N22 panicles exhibited a higher number of SOD and APX isozymes in comparison with N118 panicles that might provide better reactive oxygen species scavenging. Hence it is concluded that well-equipped antioxidant defence plays an important role in minimizing water stress-induced spikelet sterility in upland rice.

Notes: The involvement of cortical microtubules in the control of plant cell expansion was studied in the Arabidopsis root epidermis. In the zone of fast elongation microtubules were transverse to the root axis in all epidermal cells. However when cells entered the differentiation zone cell type-specific microtubule reorientation took place. In the trichoblasts that were then approximately 130 µm long and formed the root hair bulge, the microtubules switched to a random distribution. In the adjoining atrichoblasts microtubules adopted a slightly oblique orientation. In more proximal parts of the differentiation zone atrichoblast microtubules were found in a more oblique and finally in a longitudinal orientation. Upon exposure to ethylene or 1-aminocyclopropane-1-carboxylic acid (ACC – the precursor of ethylene) at a saturating dose, cell elongation abruptly stopped. From then on trichoblast cells reached only a length of about 35 µm, and developed root hairs. Cortical microtubules changed orientation within 10 min. In trichoblasts they adopted the typical random orientation, in atrichoblasts however, they took up a longitudinal orientation. Microtubule reorientation was complete within 60 min. The possible role of microtubules in the control of cell elongation is discussed.

Notes: To investigate the relationship between growth, biomass partitioning and lignification we used tobacco (Nicotiana tabacum) in which O-methyl transferase (OMT) activity, an enzyme involved in the pathway of sinapyl alcohol formation for lignin synthesis, was suppressed by antisense transformation. To modulate growth, controls and transformed tobacco plants were grown under ambient (approximately 380 p.p.m) or elevated CO2 (700 p.p.m), respectively. Lignin concentrations and composition were determined with spectrophotometric methods (thioglycolate and acetyl bromide) and Fourier transform infrared (FTIR) spectroscopy, respectively. A comparison of the thioglycolate and acetylbromide method suggested that the thioglycolate method was sensitive to changes in the syringyl/guaiacyl (S/G)-ratio in lignin and therefore not suitable for quantification in tissues with altered lignin composition. Growth under elevated CO2 increased leaf and stem biomass of both genotypes by 40 and 20%, respectively, compared with ambient CO2 and had no effect on root biomass. OMT suppression did not affect lignin concentrations in the leaves but caused a shift in biomass partitioning from the structural to the non-structural fraction. Elevated CO2 caused a shift towards production of structural compounds resulting in decreased foliar lignin concentrations and indicating that the lignin/structural mass ratio is flexible in leaves. By contrast, the lignin concentrations of stems were unaffected by elevated CO2 or OMT suppression and increased about three-fold from the apex to the base. Antisense-OMT plants produced more stem biomass than controls but showed no changes of the relative partitioning of biomass to the different pools. This indicates that the metabolic control of carbon fluxes to the production of structural versus non-structural fractions is tighter in stems than in leaves. FTIR spectroscopy indicated a relative increase in guaiacyl- as compared with syringyl-units in antisense-OMT tobacco, which was more pronounced under elevated as compared with ambient CO2. Since there was no evidence for decreased lignin concentrations in the antisense-OMT plants but increased biomass formation we suggest that less methylated lignins are ‘cheaper’ in biosynthetic carbon and energy demand and, thus, may enable plants to allocate increased resources to growth.

Notes: The protein fraction extracted with a high ionic strength buffer from the cell wall preparation of oat (Avena sativa L.) coleoptiles and first leaves contained starch-degrading (amylase) activity. The activity of apoplastic amylase in the coleoptiles and first leaves continued to increase in parallel with organ growth. One of the apoplastic amylases recovered from shoot cell wall preparations was purified by sequential ion exchange and gel filtration chromatography, and the catalytic properties of the enzyme were analysed. The purified enzyme gave a single 25 kDa protein band on SDS-PAGE. The enzyme exhibited maximum activity at pH 5.0 against maltooligosaccharides. The purified enzyme hydrolysed soluble starch and maltooligosaccharides larger than tetraose at maltose unit, but did not hydrolyse β-limit dextrin or p-nitrophenyl-α-d-glucopyranoside. These results as well as the findings that the molecular size and the catalytic properties of the purified enzyme are different from those of known amylases obtained from Gramineae caryopses suggest that this enzyme is a novel type of β-amylase present in cell walls of vigorously elongating Gramineae shoot organs.

Notes: An NADPH-dependent oxidoreductase (NADPH-DO) with an isoelectric point of 5.2 and a molecular mass of 35 kDa was isolated from suspension-cultured rice cells. NADPH-DO was inducible by auxin and gibberellin. NADPH-DO mRNA was expressed in roots and leaf sheaths of rice seedlings, but not in leaf blades. The levels of NADPH-DO mRNA and protein in suspension-cultured cells were increased by auxin; they were further raised by the application of zinc. In contrast, NADPH-DO mRNA and protein accumulation in intact roots was stimulated by auxin, but in this case the stimulation was inhibited by zinc. Auxin-induced callus growth and root formation in intact rice plants were further enhanced by zinc. These results indicate that high levels of NADPH-DO are necessary for auxin- and zinc-induced callus formation, and imply that zinc plays a role in the regulation of NADPH-DO induction by auxin.

Notes: In a previous report from the present authors, it was shown that the 1-aminocyclopropane-1-carboxylate (ACC) oxidation may play a crucial role during zygotic embryogenesis of turnip tops seeds. The present study was performed to elucidate the contribution of the silique-wall and seeds in ethylene production during this developmental process. ACC content in the silique wall is only higher than in seeds during the middle phases of zygotic embryogenesis. The ACC-oxidase (ACO) activity peaks in the silique-wall and seeds during the onset of embryogenesis, declining gradually afterwards, being undetectable during desiccation period. Using reverse transcriptase-polymerase chain reaction, one cDNA clone coding for an ACO and called BrACO1, was isolated. The deduced protein for BrACO1 has a molecular weight of 36.8 kDa and a high homology with other crucifer ACOs. The heterologous expression of this cDNA confirmed that BrACO1 is an ACO. The expression of this gene was high during the first phases of silique-wall development, low during the middle phases and undetectable during desiccation. By contrast, BrACO1 transcript was accumulated only in the earliest phases of seed embryogenesis and may participate in the highest ACO activity and ethylene production by seeds at the beginning of embryogenesis. Finally, in this work a correlation between the heterogeneity of Brassica rapa L. cv. Rapa seeds and the ability to oxidize the ACC to ethylene has been demonstrated.

Notes: The rind of fruits of muskmelon (Cucumis melo L. var. reticulatus) contains a network of suberized tissue referred to as the ‘netting’, and peroxidase (EC 1.11.1.7) activity is necessary to the polymerization of the aromatic domain of suberin. Peroxidase activity increased dramatically during the early stages of melon fruit netting, and in fruits exhibiting incomplete netting, peroxidase activity was significantly higher in netted than in non-netted regions of the same fruit. Moreover, analysis of peroxidase activity in three varieties of smooth-rind melons (Cucumis melo var. inodorous) indicated lower levels of the activity in rind samples, taken throughout fruit development, than in rinds of netted varieties. Netting-associated anionic peroxidase (NAPOD) was isolated from the melon rind at an early stage of netting development, partially purified, microsequenced and its cDNA was cloned. It was found to be a single-copy gene within the genome of netted and smooth melon varieties, and highly homologous to other Cucurbitaceous anionic peroxidases. A high transcript level was only detected in the rind of the netted variety. Monitoring the gene expression of netting-associated anionic peroxidase, together with other enzymes involved in the netting will shed light on the molecular control of the suberization processes in the melon rind and in plants in general.

Notes: Salt-extractable proteins from the cell walls of immature and ripe strawberry (Fragaria × ananassa Duch. cv. Elsanta) fruit were separated using two-dimensional polyacrylamide gel electrophoresis. Seven polypeptides (enzymes) were characterized from their N-terminal sequences: (1) glyceraldhyde-3-phosphate dehydrogenase (EC 1.2.1.12); (2) triose phosphate isomerase (TPI; EC 5.3.1.1); (3) mitochondrial malate dehydrogenase (mMDH; EC 1.1.1.37); (4) NADH glutamate dehydrogenase (EC 1.4.1.3); (5) chalcone synthase (ChS; EC 2.3.1.74); (6) mitochondrial citrate synthase (mCS; EC 4.1.3.7); and (7) UDP glucose:flavonoid 3-O-glucosyltransferase (UDPG:FGT; EC 2.4.1.91). The sequenced polypeptides identified only cytosolic proteins, two of which (ChS and UDPG:FGT) had already been identified as being up-regulated in ripening (strawberry) fruit and important contributors to ripe fruit character. Our focus was therefore diverted to the enzymes mMDH and mCS for further molecular characterization as potentially important determinants of fruit flavour via regulation of the sugar : acid balance. Citrate synthase (CS) and malate dehydrogenase (MDH) enzyme activities increased substantially during ripening, as did citrate and malate contents. The increase in CS activity is supported by western blot analysis. One strawberry mCS (Fa-mCS-I) and two mMDH (Fa-mMDH-I and -II) cDNAs were cloned that were 77, 82 and 53% identical (respectively) to sequences from other plant sources. Northern analysis showed that CS and MDH expression did not correlate with enzyme activities and these findings are discussed.

Notes: The development of flowers is regulated by a complex network of transcriptional activators and repressors, many of which belong to the MADS box gene family. In this study, we describe two MADS box genes of silver birch (Betula pendula Roth), BpMADS1 and BpMADS6, which are similar to SEPALLATA3 and AGAMOUS in Arabidopsis thaliana, respectively. In situ hybridization showed that BpMADS1 was expressed in the inflorescence meristem at a very early stage, but not later. Both genes were expressed in developing carpels, ovules and stamens but not in tepals or scales. Ectopic expression of BpMADS1 in Arabidopsis resulted in a reduced number of floral organs or whole whorls and in petaloid or carpelloid sepals, a phenotype reminiscent of that of fil mutants. 35S::BpMADS6 caused very early flowering in Arabidopsis. In tobacco, both 35S::BpMADS1 and 35S::BpMADS6 accelerated flowering and, in addition, 35S::BpMADS6 caused changes in sepals and petals. In some transgenic birch plants, 35S::BpMADS1 antisense resulted in the development of both male and female organs in the axil of a single bract and in a change of some inflorescences into vegetative shoots. In two plants, either 35S::BpMADS6 sense or antisense constructs resulted in an increase in the number of tepals and in complete lack of stamens in some male inflorescences. These results suggest that BpMADS1 participates both in inflorescence and in flower formation and BpMADS6 participates in flower formation and that they are functional homologues to SEPALLATA3 and AGAMOUS, respectively.

Notes: Molecular studies were conducted on Metrosideros excelsa to determine if the current genetic models for flowering with regard to inflorescence and floral meristem identity genes in annual plants were applicable to a woody perennial. MEL, MESAP1 and METFL1, the fragments of LEAFY (LFY), APETALA1 (AP1) and TERMINAL FLOWER1 (TFL1) equivalents, respectively, were isolated from M. excelsa. Temporal expression patterns showed that MEL and MESAP1 exhibited a bimodal pattern of expression. Expression exhibited during early floral initiation in autumn was followed by down-regulation during winter, and up-regulation in spring as floral organogenesis occurred. Spatial expression patterns of MEL showed that it had greater similarity to FLORICAULA (FLO) than to LFY, whereas MESAP1 was more similar to AP1 than SQUAMOSA. The interaction between MEL and METFL1 was more similar to the interaction between FLO and CENTRORADIALIS than that between LFY and TFL1. Consequently, the three genes from M. excelsa fit a broader herbaceous model encompassing Antirrhinum as well as Arabidopsis, but with differences, such as the bimodal pattern of expression seen with MEL and MESAP1. In mid-winter, at the time when both MEL and MESAP1 were down-regulated, GA1 was below the level of detection in M. excelsa buds. Even though application of gibberellin inhibits flowering in members of the Myrtaceae, MEL was responsive to gibberellin with expression in juvenile plants up-regulated by GA3. However, MESAP1 was not up-regulated indicating that meristem competence was also probably required to promote flowering in M. excelsa.

Notes: The effect of NaCl on the plasma membrane and tonoplast ATPases measured as the hydrolytic and H+-pumping activity was studied. Treatment of cucumber seedlings with salt increased the membrane-bound ATPases of the plasma membrane as well as the tonoplast. In both types of membranes the stimulation of ATP-hydrolysis was much higher than the stimulation of H+-transport suggesting that the salt- treatment of plants partially uncoupled the membrane proton pumps. It was shown that the soluble fraction obtained from the unstressed or NaCl-stressed roots stimulated the ATPase activities in both membranes isolated from unstressed plants. A stimulatory effect of the soluble fraction on the proton pump activities was considerably enhanced in the salt conditions indicating the presence of a salt-inducible factor (s) in the soluble fraction, which could rapidly modulate the membrane-bound ATPases. Staurosporine, a specific protein kinase inhibitor, totally abolished the stimulatory action of the soluble fractions on the membrane proton pumps, whereas okadaic acid, a phosphatase inhibitor, had no effect. Inclusion of calcium in the mixture of membranes and the soluble fraction from unstressed roots elevated the ATPase activities to the levels determined with the soluble fraction isolated from NaCl-stressed roots. Cation chelators (EGTA), as well as calmodulin antagonist (W7) cancelled the stimulatory effect of calcium ions. The above results strongly suggest the involvement of specific calcium–calmodulin-dependent protein kinases in the activation of the membrane ATPases under salt-stress conditions.

Notes: Polyamines have been suggested to play an important role in stress protection. However, attempts to determine the function of polyamines have been complicated by the fact that, dependent on the conditions, polyamine contents increase or decrease during stress. To determine the importance of polyamine formation during salt stress, we analysed polyamine contents and salt tolerance in two Arabidopsis thaliana mutants, spe1-1 and spe2-1 (Watson et al. Plant J 13: 231–239, 1998), with reduced activity of arginine decarboxylase (EC 4.1.1.19), an important enzyme in polyamine synthesis. Polyamines accumulated in wild-type plants (Col-0 and Ler-0) that were pre-treated with 100 mM NaCl before transfer to 125 mM NaCl, but not in plants that were directly transferred to 125 mM NaCl without prior treatment with 100 mM NaCl. This shows that polyamine accumulation depends on acclimation to salinity. The salt treatment that induced polyamine accumulation in wild-type plants did not lead to polyamine accumulation in the spe1-1 and spe2-1 mutants. Decreased fresh weight, chlorophyll content and photosynthetic efficiency indicated that the spe1-1 mutant was more severely affected by salt stress than its wild type, Col-0. In the spe2-1 mutant decreased salt tolerance compared to its wild type, Ler-0, became apparent as bleaching under severe salt stress. The present results demonstrate that decreased polyamine formation due to lower arginine decarboxylase activity leads to reduced salt tolerance.

Notes: Reversibly glycosylated polypeptides (RGPs) belong to a family of self-glycosylating proteins believed to be involved in plant polysaccharide synthesis. The precise function of these enzymes remains to be elucidated. Our results showed that the RGP 38-kDa subunit is phosphorylated in potato extracts (Solanum tuberosum L.). An increase in the self-glycosylation of Solanum tuberosum RGP (StRGP) 38-kDa subunit was observed after alkaline phosphatase (AP) treatment. Our results suggest that phosphorylation of StRGP appears to regulate its self-glycosylation. It was determined that when the StRGP reaction was carried out in the presence of UDP-[14C]Glc as the sugar donor and then 1 mM UDP was added in a chase-out experiment, radioactive UDP-Glc was obtained indicating that StRGP reaction seems to be reversible. The anomeric configuration of transferred sugars to StRGP protein was also studied.

Notes: Embryogenesis and plant regeneration have been obtained from isolated immature pollen of two poplar hybrids (Populus nigra L. × hybrid ‘Aue1’ and ‘Aue2’). In total, 1487 calli or embryos, respectively, larger than 1 mm were generated in a 2-year study. By using a cytokinin containing induction medium, on average 19 calli per responsive immature catkin were formed. Additional supplementation with auxin in 2002 increased the frequency to 72 calli per catkin. Microsatellite marker analyses confirmed haploid origin in most regenerants studied. So far six out of eight obtained regenerative callus lines have maintained their haploid level up to 24 months of development. A number of haploid and doubled haploid plants of different lines are available and have been transferred to soil.

Notes: The N-terminal amino-acid sequence was determined for a major rice phloem protein with a molecular mass of 31 kDa, named RPP31. The corresponding full-length rice EST-clone was cloned based on the amino acid sequence. The predicted total amino-acid sequence of RPP31 shared high similarity with plant glutathione S-transferases (GSTs). Recombinant RPP31 produced in Escherichia coli and rice phloem sap showed GST activity. Immunocytological analysis indicated that RPP31 is localized in the phloem region of leaves. In mature leaves, the signal was restricted to sieve element–companion cell complexes, and was stronger in sieve elements than in companion cells. Although some plant GSTs are known to be induced by xenobiotics, the amount of RPP31 was not affected by treatments with an herbicide, pretilachlor, and/or its safener, fenclorim. These results suggest that RPP31 is an active GST restricted to the phloem region of normal rice leaves.

Notes: Within its wide range across Canada, jack pine is exposed to salinity from both natural and anthropogenic sources. To compare the effects of Cl and SO4 on salt injury, sand and solution-culture grown jack pine (Pinus banksiana Lamb.) seedlings were treated with nutrient solutions containing 60 or 120 mM NaCl, 60 mM Na2SO4, or a mixture of 60 mM NaCl and 30 mM Na2SO4. After 5 weeks of salt treatments, concentrations of Cl, K, Na, and SO4 were determined in roots, stem and needles of the current and previous years growth, and in necrotic needles. To determine the role of water uptake in the absorption and translocation of salts in plants, total transpiration was measured as the loss of water from a sealed system and related to total plant uptake of Cl, Na, and SO4. Sodium uptake and root-to-shoot transport rates were greater in treatments containing Cl. A delay in root-to-shoot transport of both Na and Cl indicates retention of these ions in the roots. Electrolyte leakage of needles was more closely related to treatment Cl concentrations than treatment Na concentrations. The transport of Na ions to the shoot was related to the presence of Cl, but was not related to transpiration rate.

Notes: Coupling growth of Lolium perenne L. in sterile solution culture with steady-state 13CO2 labelling allowed quantification of the contribution of C, assimilated either before or after a specific time point, both to plant compartments and root exudates. Plants were grown for 27 days in atmospheres containing CO2 with δ13C signatures of either −13.5 or −36.1‰. Air supplies to plants were then reciprocally switched to the opposing signature (day 0), plants were destructively harvested and root exudates collected over the next 8 days. Following the switch, C assimilated after day 0 and transported to the roots initially only appeared in root tips, later appearing in both tip and non-tip material. The δ13C signature of the root exudate changed exponentially with time. Assimilation pre- and post-day 0 contributed equally to exudate C at 4.5 days. Beyond day 8, assimilation pre-day 0 still contributed 41.7% of exudate C. Over all 8 days, a linear relationship existed between the δ13C signatures of root tips and exudate, suggesting that all newly assimilated C in the exudate was from root tips. Results imply pulse-labelling approaches to study root exudates are discriminative in the sources of exudates labelled and in the sites from which exudation occurs.

Notes: Although the catalytic activity of Rubisco increases with temperature, the low affinity of the enzyme for CO2 and its dual nature as an oxygenase limit the possible increase in net photosynthesis with temperature. For cotton, comparisons of measured rates of net photosynthesis with predicted rates that take into account limitations imposed by the kinetic properties of Rubisco indicate that direct inhibition of photosynthesis occurs at temperatures higher than about 30°C. Inhibition of photosynthesis by moderate heat stress (i.e. 30–42°C) is generally attributed to reduced rates of RuBP regeneration caused by disruption of electron transport activity, and specifically inactivation of the oxygen evolving enzymes of photosystem II. However, measurements of chlorophyll fluorescence and metabolite levels at air-levels of CO2 indicate that electron transport activity is not limiting at temperatures that inhibit CO2 fixation. Instead, recent evidence shows that inhibition of net photosynthesis correlates with a decrease in the activation state of Rubisco in both C3 and C4 plants and that this decrease in the amount of active Rubisco can fully account for the temperature response of net photosynthesis. Biochemically, the decrease in Rubisco activation can be attributed to: (1) more rapid de-activation of Rubisco caused by a faster rate of dead-end product formation; and (2) slower re-activation of Rubisco by activase. The net result is that as temperature increases activase becomes less effective in keeping Rubisco catalytically competent. In this opinionated review, we discuss how these processes limit photosynthetic performance under moderate heat stress.

Notes: Some gibberellin (GA) analogues, especially with C-16,17 modifications of GA5, can inhibit growth of plants apparently by acting as competitors with the endogenous substrate of GA biosynthetic enzymes. Here, we directly confirm the competitive action of GA derivatives but also show that some analogues may retain significant bioactivity. A recombinant 3-oxidase from pea, which converts GA20 to bioactive GA1, was inhibited by GA5, and 16,17-dihydro-GA5 derivatives, especially if the C-17 alkyl chain length was increased by up to three carbons or if the C-13 hydroxyl was acetylated. Genetic confirmation that GA5 analogues target 3-oxidases in vivo was provided by comparing the growth response of a WT (LE) pea with a 3-oxidase mutant (le-1). Two pea 2-oxidases that inactivate bioactive GAs, were inhibited by GA1 and GA3 but were generally insensitive to GA5 analogues. α-Amylase production by barley half-seeds in response to GA analogues provided a method to study their action when effects on GA biosynthesis were excluded. This bioactivity assay showed that 16,17-dihydro GA5 analogues have some inherent activity but mostly less than for GA5 (5–50-fold), which in turn was 100-fold less active than GA1 and GA3. However, although C-17 alkyl derivatives with one or two added carbons showed little bioactivity and were purely 3-oxidase inhibitors, adding a third carbon (the 17-n-propyl-16,17-dihydro GA5 analogue) restored bioactivity to that of GA5. Furthermore, this analogue has lost its capacity to inhibit stem elongation of Lolium temulentum (Mander et al., Phytochemistry 49:1509–1515, 1998a), although it strongly inhibits the 3-oxidase. Thus, the effectiveness of a GA derivative as a growth retardant will reflect the balance between its bioactivity and its capacity to inhibit the terminal enzyme of GA biosynthesis. The weaker growth inhibition in dicots including pea (approximately 10%) than in monocots such as L. temulentum (〉35%) is suggestive of taxonomic differences in the bioactivity of GAs and/or their effects on GA biosynthesis.

Notes: Resurrection plants have the unique capacity to revive from an air-dried state. In order to tolerate desiccation they have to overcome a number of stresses, mechanical stress being one. In leaves of the Craterostigma species, an extensive shrinkage occurs during drying as well as a considerable cell wall folding. Our previous microscopically analysis using immunocytochemistry on the resurrection plant Craterostigma wilmsii, has shown an increase in labelling of xyloglucan and unesterified pectins in the cell wall during drying. In this study, we have undertaken a biochemical approach to separate, quantify and characterize major cell wall polysaccharides in fully hydrated and dry leaves of C. wilmsii. Our results show that the overall cell wall composition of C. wilmsii leaves was similar to that of other dicotyledonous plants with respect to the pectin content. However, the structure of the hemicellulosic polysaccharide xyloglucan was characterized to be XXGG-type. The data also demonstrate marked changes in the hemicellulosic wall fraction from dry plants compared to hydrated ones. The most conspicuous change was a decrease in glucose content in the hemicellulosic fraction of dry plants. In addition, xyloglucan from the cell wall of dry leaves was relatively more substituted with galactose than in hydrated walls. Together these findings show that dehydration induces significant alteration of polysaccharide content and structure in the cell wall of C. wilmsii, which in turn might be involved in the modulation of the mechanical properties of the wall during dehydration.

Notes: Arabinogalactan-proteins (AGPs) are a class of highly glycosylated, hydroxyproline-rich glycoproteins that function in plant growth and development. Tomato LeAGP-1 represents a major AGP expressed in cultured cells and plants. Based on cDNA and amino acid sequence analyses along with carbohydrate and other biochemical analyses, tomato LeAGP-1 is hypothesized to be a classical AGP localized to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. Here, this hypothesis was tested and supported with the following experiments. First, tomato (Lycopersicon esculentum, cv. UC82B) cotyledon protoplasts were isolated following cell wall digestion with cellulase and pectinase, and LeAGP-1 was immunolocalized to the plasma membrane with a LeAGP-1 antibody. Second, LeAGP-1 was shown to be a major AGP component in plasma membrane vesicles from tomato cv. Bonnie Best suspension-cultured cells by Western blot analysis with the LeAGP-1 antibody. Third, fluorescence microscopy of plasmolysed, transgenic tobacco (Nicotiana tabacum BY-2) suspension-cultured cells expressing a green fluorescent protein (GFP)-LeAGP-1 fusion product demonstrated localization to the plasma membrane and Hechtian threads. Fourth, the GFP-LeAGP-1 fusion protein was present in plasma membrane preparations from these transgenic tobacco cells by Western blot analysis with a GFP antibody. Fifth, GFP-LeAGP-1 secreted into the culture media contained ethanolamine, presumably attached to the C-terminal amino acid residue, consistent with its processing and release from the plasma membrane. Thus, these data support the hypothesis that LeAGP-1 is localized to the plasma membrane via a GPI anchor and suggest possible roles for LeAGP-1 in cellular signalling and matrix remodelling.

Notes: We examined the influence of short-term exposure of different UV wavebands on the fine-scale kinetics of hypocotyl growth of dim red light-grown cucumbers (Cucumis sativus L.) and other selected dicotyledonous seedlings to evaluate: (1) whether responses induced by UV-B radiation (280–320 nm) are qualitatively different from those induced by UV-A (320–400 nm) radiation, and (2) whether different wavebands within the UV-B elicit different responses. Responses to brief (30 min) irradiations with 3 different UV wavebands all included transient inhibition of elongation during irradiation followed by wavelength specific responses. Irradiations with proportionally greater short wavelength UV-B (37% of UV-B between 280 and 300 nm) induced inhibition of hypocotyl elongation within 20 min of onset of irradiation, while UV-B including only wavelengths longer than 290 nm (and only 8% of UV-B between 290 and 300 nm) induced inhibition of hypocotyl elongation with a lag of 1–2 h. The response to short wavelength UV-B was persistent for at least 24 h, while the response to long wavelength UV-B lasted only 2–3 h. The UV-A treatment induced reductions in elongation rates of approximately 6–9 h following exposure followed by a continued decline in rates for the following 15–18 h. Short wavelength UV-B also induced positive phototropic curvature in both cucumber and Arabidopsis seedlings, and this response was present in nph-1 mutant Arabidopsis seedlings defective in normal blue light phototropism. Reciprocity was not found for the response to short wavelength UV-B. The short wavelength and long wavelength UV-B responses differed in dose–response relationships and both short wavelength responses (phototropic curvature and elongation inhibition) increased sharply at wavelengths below 300 nm. These results indicate that different photosensory processes are involved in mediating growth and morphological responses to short wavelength UV-B (280–300 nm), long wavelength UV-B (essentially 300–320 nm) and UV-A. The existence of two separate types of hypocotyl inhibition responses to UV-B, with one that depends on the intensity of the light source, provides alternate interpretations to findings in other studies of UV-B induced photomorphogenesis and may explain inconsistencies between action spectra for inhibition of stem growth.

Notes: Arsenate [As (V)] is taken up by phosphate [P (V)] transporters in the plasma membrane of roots cells, but the translocation of As from roots to shoots is not well understood. Two mutants of Arabidopsis thaliana (L.) [(pho1, P deficient) and (pho2, P accumulator)], with defects in the regulation and translocation of P (V) from roots to shoots, were therefore used in this study to investigate uptake, translocation and speciation of As in roots and shoots of plants grown in soil or nutrient solution. The shoots of the pho2 mutant contained higher P concentrations, but similar or slightly higher As concentrations, in comparison with the wild type. In the pho1 mutant, the P concentration in the shoots was lower, and the As concentration was higher, in comparison with the wild type. Both pho2 and the wild type contained mainly As (III) in roots and shoot (67–90% of total As). Arsenic was likely to be translocated by a different pathway to P (V) in the pho2 and pho1 mutants. Therefore, it is suggested that As (III) is the main As species translocated from roots to shoots in Arabidopsis thaliana.

Notes: Fluridone and norflurazon, two carotenoid-biosynthesis inhibitors, shortened the conditioning period required by seeds of Orobanche minor in order to respond to the germination stimulant strigol. Neither fluridone nor norflurazon alone induced seed germination of O. minor, they promoted strigol-induced germination. In addition, these compounds restored the conditioning and germination of seeds at a supraoptimal temperature (30°C) as well as in the light. Gibberellic acid (GA3) showed similar promotive and protective effects on the conditioning and germination of O. minor seeds. Although fluridone and norflurazon are known to prevent abscisic acid (ABA)-biosynthesis, and stresses such as supraoptimal temperatures have been reported to induce ABA accumulation in plants, the amount of ABA in the seeds or that released from the seeds into the conditioning media was not affected by the fluridone treatment and by exposure to the supraoptimal temperature. These results indicate that the promotive and protective effects of fluridone and norflurazon on the conditioning and germination of O. minor seeds would be attributed to other perturbations rather than the inhibition of ABA-biosynthesis.

Notes: Dehydrins (DHNs; late embryogenesis abundant D-11) are a family of plant proteins induced in response to environmental stresses such as water stress, salinity and freezing or which occur during the late stages of embryogenesis. Previously, it was reported that citrus contains a small gene family encoding a unique class of dehydrins that differs from most other plant dehydrins in various respects, such as having an unusual K-segment similar to that of gymnosperms. In the present study, we identified by cDNA differential display analysis a ‘Navel’ orange 202-bp polymerase chain reaction (PCR) fragment, which encoded the typical plant angiosperm-type K-segment consensus sequence, and of which the expression was down-regulated by exposure to low oxygen levels. The full-length cDNA sequence of the orange DHN, designated csDHN (for Citrus sinensis DHN), was further isolated by 5′-and 3′-RACE; it had a total length of 933 bp and encoded a predicted polypeptide of 235 amino acids. In addition, the same 202-bp ‘Navel’ dehydrin PCR fragment was used to screen a ‘Star Ruby’ grapefruit flavedo cDNA library, and its full-length grapefruit homologue, designated cpDHN (for C. paradisi DHN) was isolated and found to have a total length of 1024 bp and to encode a predicted polypeptide of 234 amino acids. The defined orange and grapefruit DHN proteins were completely identical in the 196 amino acids of their N-terminus but differed in their C-terminus region. Overall, the csDHN and cpDHN proteins share 84% identity and contain the conserved dehydrin serine cluster (S-segment) and a putative nuclear localization signal, but csDHN has one conserved dehydrin K-segment consensus sequence, whereas cpDHN contains two dehydrin K-segments. Both csDHN and cpDHN represent single copy genes, in ‘Navel’ orange and ‘Star Ruby’ grapefruit genomes, respectively. We found that the cpDHN gene was consistently expressed in the fruit peel tissue at harvest, but that its message levels dramatically decreased during storage at either ambient or low temperatures. However, a pre-storage hot water treatment, given to enhance fruit-chilling tolerance, increased cpDHN mRNA levels during the first 3 weeks of cold storage at 2°C, and enabled the message levels to be retained for up to a further 8 weeks of cold storage at 2°C. The hot water treatment by itself had no inductive effect on cpDHN gene expression when the fruits were held at non-chilling temperatures. Other stresses applied to the fruit, such as wounding, UV irradiation, water stress, low oxygen and exposure to the stress hormone ethylene decreased DHN mRNA levels, whereas abscisic acid had no effect at all.

Notes: An increasingly important area of research is based on sulphydryl chemistry, since the oxidation of -SH groups is one of the earliest observable events during oxidant-mediated damage and -SH groups play a critical role in the function of many macromolecular structures including enzymes, transcription factors and membrane proteins. Glutaredoxins and thioredoxins are small heat-stable oxidoreductases, conserved throughout evolution, which play key roles in maintaining the cellular redox balance. Much progress has been made in analysing these systems in the yeast Saccharomyces cerevisiae which is a very useful model eukaryote due to its ease of genetic manipulation, its compact genome, the availability of the entire genome sequence, and the current rate of progress in gene function research. Yeast, like all eukaryotes, contains a number of glutaredoxin and thioredoxin isoenzymes located in both the cytoplasm and the mitochondria. This review describes recent findings made in yeast that are leading to a better understanding of the regulation and role of redox homeostasis in eukaryotic cell metabolism.

Notes: The nature of the active site and the substrate specificity of poplar type II peroxiredoxin, an enzyme which preferentially uses glutaredoxin as an electron donor, were investigated in this study. The type II peroxiredoxin is able to use phospholipid hydroperoxide nearly as efficiently as hydrogen peroxide. Two of the hyper-conserved amino acid residues in peroxiredoxins have been altered, by site-directed mutagenesis, generating the mutants T48V and R129Q. The two mutant proteins are inactive with hydrogen peroxide or tertiary butyl hydroperoxide as substrates. On the other hand, the mutant enzymes catalyse the degradation of cumene hydroperoxide with low efficiency. This suggests that the thiol-dependent regeneration process of the catalytic cysteine is not affected by the mutations and that all substrates are not accommodated identically in the active site.

Notes: In terms of gene expression and carbohydrate metabolism, the response of wheat seedlings to hypoxia is dramatically different from the anoxic response. Total carbohydrate content of roots increased 4-fold during 6 days of hypoxia, with a 17-fold increase in fructans. In contrast, anoxically treated roots depleted all soluble carbohydrates and died within 72 h. Gas exchange measurements (CO2 release vs. O2 uptake) demonstrate that hypoxia establishes a new balance between fermentation and aerobic respiration in the roots without altering the flux of carbon through glycolysis. Furthermore, the respiratory component of this new balance is 55% higher in roots that have been hypoxically pretreated compared to non-hypoxically pretreated roots. The establishment of this new homeostasis under hypoxia involves the induction of glycolytic (aldolase and enolase) and fermentative enzymes (pyruvate decarboxylase, alcohol dehydrogenase, and lactate dehydrogenase). Enzyme induction is generally complete within 24 h with mRNA induction occurring primarily during Period I (0–6 h of hypoxia), and maximal enzymes activities attained during Period II (6–24 h of hypoxia). Accumulation rates of Suc, hexoses, and fructans also change during Periods I and II. By the start of Period III (24–144 h of hypoxia), the metabolic adjustments are complete and fructans are the major carbohydrate accumulated. In anoxia, the pattern of enzyme induction was dramatically different: aldolase was not induced and declined throughout the treatment. Alcohol dehydrogenase, pyruvate decarboxylase, and lactate dehydrogenase were induced as in hypoxia, but rapidly declined within 72 h of anoxia. Only enolase exhibited a similar expression pattern in both anoxia and hypoxia.

Notes: It is increasingly evident that transcription control might be conserved among organisms. For this reason, genome sequencing and gene expression profiling methods, which have yielded a plethora of data in different organisms, may be applied in species where genomic sequence is limited to mostly expression array and EST data. The identification of transcription factors and promoters associated with gene expression profiles and ESTs could therefore contribute to elucidate and predict complex regulatory events in plants.

Notes: The changes in osmotic potential and the concentration of osmotic solutes in the cell sap of the root tips exposed to Al were examined in two cultivars of wheat (Triticum aestivum) differing in Al resistance. Root elongation was less influenced by an 8-h exposure to 20 μM or 50 μM Al in Al-resistant cv. Atlas 66 than in Al-sensitive cv. Scout 66. After Al treatment the osmotic potential of the root cells was decreased in Atlas 66 but increased in Scout 66 indicating that the Al treatment osmotically stimulated the driving force for water uptake in Atlas 66 but suppressed it in Scout 66. Al increased the concentration of soluble sugars, the major osmotic solute in the root cells in Atlas 66, but decreased it in Scout 66. Al at both low (5 μM) and high (50 μM) concentrations, also increased the concentration of soluble sugars in the Al-resistant genotype ET8 but a high Al concentration decreased it in Al-sensitive genotype ES8. Enzymatic analyses and thin-layer chromatography revealed that soluble sugars in the root cells of both Atlas 66 and Scout 66 mainly consisted of monosaccharides such as glucose, fructose and a small amount of sucrose. These results suggest that the accumulation of soluble sugars in Al-resistant wheat Atlas 66 keeps the osmotic potential in the root cells low and thus, enables the root cells to take up water and to elongate against the pressure produced by cell wall rigidification under Al stress.

Notes: Phytohormones are well-known regulators of the symbiotic Rhizobium–legume association in the plant host. The enod40 nodulin gene is associated with the earliest phases of the nodule organogenesis programme in the legume host and modifying its expression resulted in perturbations of nodule development in Medicago truncatula. Therefore in our pursuit to mimic the initial signal transduction steps of legume nodulation in the alien physiological set-up of a rice plant, we have expressed the Mtenod40 gene in rice. Molecular data confirm the stable integration, inheritance and transcription of the foreign gene in this non-legume. We have compared the phytohormonal responses of Mtenod40-overexpressing and control plants in a homologous legume background (M. truncatula) and in the non-legume rice. An enod40-mediated root growth response, induced by inhibition of ethylene biosynthesis, was observed in both plants. On the other hand, a significant differential effect of cytokinins was observed only in rice plants. This suggests that ethylene inhibits enod40 action both in legumes and non-legumes and reinforces that some of the early signal transduction steps of the nodule developmental programme may function in rice.

Notes: In Cantharanthus roseus transformed roots, the application of methylglyoxal bis(guanylhydrazone) (MGBG), an inhibitor of S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50), inhibited the root growth in a dose-dependent manner with a DL50 of about 300 µm. Spermidine and spermine (Spm) levels and SAMDC and phospholipase C (PLC; EC 3.1.4.3) activities were reduced in the presence of the inhibitor. The inhibition was reversed by the addition of Spm. Radioactivity from [14C]Spm was detected in an immunoprecipitated fraction with an antibody anti-PLC-δ. To our knowledge, this is the first direct evidence that demonstrates an interaction of Spm with the signal transduction cascade phosphoinositide-Ca2+.

Notes: This study presents the effects of methyl jasmonate (MeJA) on growth, N uptake, N partitioning, and N storage in taproots of non-nodulated alfalfa (cv. Lodi). When compared to untreated plants, addition of 100 µM MeJA to the nutrient solution for 14 days reduced total growth and modified biomass partitioning between shoots and roots in favour of taproots and lateral roots. MeJA decreased N uptake (after 7 days) and increased N partitioning towards roots after 14 days. This preferential N partitioning to roots was accompanied by increased N storage in taproots as soluble proteins. Compared to total soluble proteins, VSP accumulation occurred earlier (7 days), and was greater (2-fold increase) in plants treated with 100 µM MeJA. Steady-state transcript levels for two VSPs (32 and 57 kDa) also increased markedly (about 4-fold) in roots of plants treated with 100 µM MeJA. This suggests that MeJA could act directly (transcriptional regulation) or indirectly (via the changes of N partitioning among alfalfa organs) on N storage as soluble proteins and in particular, VSPs. Because the deduced amino acid sequence of the 32 kDa VSP clone reveals high homology with Class III chitinases, we propose that the 32 kDa VSP may have a role in pathogen defense, in addition to its function as a storage protein.

Notes: The lack of knowledge about the tissue and subcellular distribution of polyamines (PAs) and the enzymes involved in their metabolism remains one of the main obstacles in our understanding of the biological role of PAs in plants. Arginine decarboxylase (ADC; EC 4.1.1.9) is a key enzyme in polyamine biosynthesis in plants. We have characterized a cDNA coding for ADC from Nicotiana tabacum L. cv. Petit Havana SR1. The deduced ADC polypeptide had 721 amino acids and a molecular mass of 77 kDa. The ADC cDNA was overexpressed in Escherichia coli, and the ADC fusion protein obtained was used to produce polyclonal antibodies. Using immunological methods, we demonstrate the presence of the ADC protein in all plant organs analysed: flowers, seeds, stems, leaves and roots. Moreover, depending on the tissue, the protein is localized in two different subcellular compartments, the nucleus and the chloroplast. In photosynthetic tissues, ADC is located mainly in chloroplasts, whereas in non-photosynthetic tissues the protein appears to be located in nuclei. The different compartmentation of ADC may be related to distinct functions of the protein in different cell types.

Notes: Variegation mutants offer excellent opportunities to study interactions between the nucleus-cytoplasm, the chloroplast, and the mitochondrion. Variegation in the immutans (im) mutant of Arabidopsis is induced by a nuclear recessive gene and the extent of variegation can be modulated by light and temperature. Whereas the green sectors have morphologically normal chloroplasts, the white sectors are devoid of pigments and accumulate a colourless carotenoid, phytoene. The green sectors are hypothesized to arise from cells that have avoided irreversible photooxidative damage whereas the white sectors originate from cells that are photooxidized. Cloning of the IMMUTANS (IM) gene has revealed that IMMUTANS (IM) is a plastid homologue of the mitochondrial alternative oxidase. This finding suggested a model in which IM functions as a redox component of the phytoene desaturation pathway, which requires phytoene desaturase activity. Consistent with this idea, IM has quinol oxidase activity in vitro. Recent studies have revealed that IM plays a more global role in plastid metabolism. For example, it appears to be the elusive terminal oxidase of chlororespiration and also functions as a light stress protein.

Notes: The growth dynamics of vessel formation and the effect of auxin transport inhibition on vessel differentiation were investigated in the pedicel of developing apple (Malus domestica Borkh.). Quantitative microscopic analysis showed that a majority of vessels were differentiated pre-bloom with the commencement of pedicel extension but that the full conducting capacity of the xylem was attained shortly after bloom. The effect of a post-bloom application of N-(1-Napthyl)phthalamic acid showed that an auxin-like signal emanating from the young fruit not only stimulated vessel differentiation in the pedicel but also controlled fruit abscission and the development of the seed and fruit.

Notes: This communication presents a short outline of the current knowledge on the molecular basis of PII signal transduction in unicellular cyanobacteria with respect to the perception of environmental stimuli. First, the general characteristics of the PII signalling system in unicellular cyanobacteria are presented, the hallmark of which is modification by serine-phosphorylation, as compared to the paradigmatic PII signal transduction system in proteobacteria, which is based on tyrosyl-uridylylation. Then, the focus is turned on the signals controlling PII phosphorylation state. Recently, the cellular phosphatase (termed PphA), which specifically dephosphorylates phosphorylated PII (PII-P) was identified in Synechocystis sp. strain PCC 6803. With the availability of a PphA-deficient mutant and the purified components for in vitro assay of PphA mediated PII-P dephosphorylation, novel insights into the signals, to which PII-P dephosphorylation responds, can be obtained. Here we present an investigation of the response of PII-P dephosphorylation towards treatments that affect the redox-balance of the cells. Furthermore, a possible role of varying ATP/ADP ratios on PII-P dephosphorylation was examined. From these studies, together with previous investigations, we conclude that PII-P dephosphorylation specifically responds to changes in the levels of central metabolites of carbon metabolism, in particular 2-oxoglutarate.

Notes: The physiology of hyperhydricity in relation to oxidative stress, mineral nutrients, antioxidant enzymes and ethylene has been studied in three micropropagated carnation cultivars under experimentally induced hyperhydricity. A marked increase in Fe content in comparison with normal tissues was observed in the hyperhydric tissues from the three cultivars. The levels of ethylene, solute leakage and malondialdehyde content were also significantly higher in the hyperhydric tissues. In relation to the time course of H2O2 production measured by fluorescence quenching, a similar trend could be observed for the three cultivars, with a clear increase in the generation of hydrogen peroxide in hyperhydric tissues. The activities of all the antioxidative enzymes studied, except lipoxygenase, were higher in the hyperhydric shoots. Phenylalanine ammonia-lyase (PAL) showed a significant decrease in activity in the hyperhydric tissues in comparison with the controls for the three cultivars. Soluble guaiacol peroxidase had a strong increase in activity in hyperhydric shoots of the three cultivars. These results provide, for the first time, direct evidence of H2O2 generation in hyperhydric tissues, characterize the response of the antioxidant system to an oxidative stress during hyperhydricity in carnation leaves and point to the accumulation of toxic forms of oxygen as the inducer of some of the abnormalities observed.

Notes: A large genetic variability was observed in the shoot NO3– content of recombinant inbred lines (RILs) of Lotus japonicus. To determine the cause of this variability, we have studied some aspects of nitrate uptake and assimilation in the two parental ecotypes (Gifu and Funakura) and four representatives of the RILs population differing both in their shoot biomass and shoot NO3– content. Higher shoot NO3–content was mainly due to an increase in the uptake of the ion regardless of the plant biomass production. The positive correlation observed between the shoot NO3– content and the steady state level of mRNA encoding high affinity NO3– transporters suggests that the higher NO3– influx is due to enhanced expression of the transporters. In contrast, neither the level of nitrate reductase mRNA, nor the potential enzyme activity in vivo in the different lines was correlated with the shoot NO3– content. This indicates that NO3– transport in Lotus is one of the main checkpoints controlling shoot NO3– accumulation. In addition, this study shows that at least in Lotus, it is possible, through breeding strategies, to lower the NO3– content without affecting biomass production.

Notes: Expression profiles of Synechocystis sp. PCC 6803 genes in response to growth in iron-deficient versus iron-sufficient media and after 30 min treatment with H2O2 were determined using a full-genome microarray. We used an anova model that accounted for slide and replicate (random) effects as well as dye (a fixed) effects to identify statistically significant, differentially expressed genes that changed by 1.25-fold or greater during each of these experiments. We utilized this microarray data to identify gene clusters that were regulated under these stresses, because we are interested in cellular redox control and the way in which the cell responds to oxidative stresses. We are particularly interested in using differential expression to help determine the function of genes involved in redox control and cluster analysis aids this process. We concentrated on four gene clusters, two of which were similarly affected by both stresses, and two that were only differentially regulated by one of the stresses. We also analysed the regulatory genes that responded to these oxidative stresses and discussed the changes in transcription of the RNA polymerase sigma factors and the other regulatory proteins, many of which represent two-component regulatory systems.

Notes: In green parts of the plant, during illumination ATP and NAD(P)H act as energy sources that are generated mainly in photosynthesis and respiration, whereas in darkness, glycolysis, respiration and the oxidative pentose-phosphate pathway (OPP) generate the required energy forms. In non-green parts, sugar oxidation in glycolysis, respiration and OPP are the only means of producing energy. For energy-consuming reactions, the delivery of NADPH, NADH, reduced ferredoxin and ATP has to take place at the required rates and in the specific compartments, since the pool sizes of these energy carriers are rather limited and, in general, they are not directly transported across biomembranes. Indirect transport of reducing equivalents can be achieved by malateoxaloacetate shuttles, involving malate dehydrogenase (MDH) for the interconversion. Isoenzymes of MDH are present in each cellular compartment. Chloroplasts contain the redox-controlled NADP-MDH that is only active in the light. In addition, a plastid NAD-MDH that is permanently active and is present in all plastid types has been found. Export of excess NAD(P)H through the malate valves will allow for the continued production of ATP (1) in photosynthesis, and (2) in oxidative phosphorylation. In the latter case, the coupled production of NADH is catalysed by the bispecific NAD(P)-GAPDH (GapAB) in chloroplasts that is active with NAD even in darkness, or by the specific plastid NAD-GAPDH (GapCp) in non-green tissues. When plants are subjected to conditions such as high light, high CO2, NH4+ nutrition, cold stress, which require changed activities of the enzymes of the malate valves, changed expression levels of the MDH isoforms can be observed. In nodules, the induction of a nodule-specific plastid NAD-MDH indicates the changed requirements for energy supply during N2 fixation. Furthermore, the induction of glucose 6-phosphate dehydrogenase isoforms by ammonium and of ferredoxin and ferredoxin-NADP reductase by nitrate has been described. All these findings are in line with the assumption that a changed redox state caused by metabolic variability leads to the induction of enzymes involved in redox poise.

Notes: Abstract –  Technological advances have lead to the production of micro radio-transmitters capable of being implanted in fish as small as c. 5 g. Although the actual tags are small, transmitters are equipped with long antennas that can increase drag and tangle in debris. We examined the effects of radio-transmitter antenna length on the swimming performance of juvenile rainbow trout, Oncorhynchus mykiss, (N = 156, mean mass = 34 g, mean fork length = 148 mm). Although we tested a variety of different antenna lengths up to a maximum of 300 mm, only the longest antenna significantly impaired swimming performance relative to control fish (P 〈 0.001). There was no difference in swimming performance between the sham (surgery, but no transmitter) and the control fish (handled, but no surgery), suggesting that the surgical procedure itself did not negatively affect the fish. Regression analysis, however, indicated that there was a significant decrease in swimming performance associated with increased antenna length (R2 = 0.11, P 〈 0.001). In addition, when held in laboratory tanks, fish with the three longest antennas (150, 225 and 300 mm) frequently became entangled with the standpipe. We suggest that researchers, under the guidance of the tag manufacturer, trim antennas to the shortest possible length required to detect fish in their specific study area. Antenna length is clearly an important issue for small fish, especially for species that inhabit complex habitats where antennas may become entangled, and where fish must attain speeds near limits of their swimming capacity.

Notes: Abstract –  Previous studies have offered suggestive but sometimes conflicting glimpses at the range and patterns of seasonal and interannual variation in female darter life-history traits. The present study examined clutch-size and egg-size variation from a single Mississippi population of brighteye darters (Etheostoma lynceum), collected at regular intervals over four sequential spawning seasons. Both life-history traits were significantly correlated with body size, and they both showed significant variation within and among breeding seasons. The overall intraseasonal trends in these traits involved an increase in clutch size (CS) and a decrease in egg size from early to late periods of the spawning season. Clutch size and egg size showed a weak but significant negative correlation, which was indicative of a trade-off between the two clutch parameters. Seasonal changes in temperature appear to be related to differing patterns of seasonal variation in egg size reported in the literature for darters. Inverse seasonal shifts in egg size and CS in the brighteye darter may represent adaptive phenotypic plasticity that allows females to produce larger, competitively superior offspring early in the reproductive season when there is low supply of food for them (parental investment hypothesis) or to produce larger eggs early in the reproductive season to ensure that each egg is adequately provisioned in the face of uncertainty (bet-hedging hypothesis) if the food available for the young is unpredictable early in the season. Consistent with theoretical predictions, egg size showed less phenotypic variability than CS.

Notes: Abstract –  Residency and movement of bullheads (Cottus gobio) were assessed by mark-recapture from November 2001 to June 2002 in two Flemish rivers [Steenputbeek (SPB), Laarse Beek (LB)]. Although the majority of the recaptured bullheads (SPB: 66%; LB: 55%) was found in their initial tagging site before the spawning season, some fish had covered distances between 10–100 m (mean: 17 ± 2 m) and 10–70 m (mean: 18 ± 7 m), respectively. During the spawning season, the proportion of fish that moved (SPB: 58%; LB: 75%) and the distances travelled (SPB: between 10 and 90 m, mean 26 ± 3 m; LB: between 30 and 260 m, mean 133 ± 3 m) were significantly larger. In addition, analysis of individual movement behaviour of multiple recaptured bullheads showed that some fish were either always resident or mobile, while others switched between both behaviours. In general, our results suggest that not all bullheads exhibit sedentary behaviour but that the populations under study consist of both stationary and mobile individuals.

Notes: Abstract –  Movement and habitat use of river blackfish (Gadopsis marmoratus R.) was studied in a highly modified central Victorian stream (Birch Creek), Australia, using radio telemetry (N = 10) and mark–recapture (N = 113) between October 2001 to May 2002, and November to December 2002 respectively. Blackfish had a small home range of 10–26 m with strong affinity to a pool. However, small movements within a pool were common which resulted in relatively large cumulative movements. At a diel scale there were no significant differences in blackfish movement between day and night (Kruskal–Wallis test, P = 0.95). Six of seven blackfish translocated 40 m upstream or downstream of their original positions returned within 48 h, suggesting strong affinity not only to a pool, but also to a small region within a pool. Blackfish utilised slow flowing (0–20 cm s−1) and deep waters (40–60 cm). Blackfish were strongly associated with the instream cover habitats of undercut banks and boulders. Significant differences were recorded in instream cover and water velocity used by blackfish between day and night (Kruskal–Wallis test, P = 〈0.01). At night blackfish used open areas with high water velocities whereas during the day blackfish used complex undercut banks with low water velocities.

Notes: Abstract –  Induced morphological defences, where individuals develop morphological structures as a response to signals from sympatric predators, are common among invertebrates. Such defences have also been reported in a freshwater fish, the crucian carp Carassius carassius. In crucian carp, the presence of piscivorous fish presumably induces the development of a deep body as a defence mechanism. We tested how the growth rate (in body depth, length and mass) of individually tagged crucian carp responds to pike Esox lucius odours and control water. In general, crucian carp exposed to pike odour grew better (both in length and mass) than control fish. The treatment crucian carp also developed deeper bodies than control crucian carp. Further, there was a strong positive relationship between individual growth rate and the rate of change in body depth and this relationship did not differ between control and treatment fish.

Notes: Abstract –  The different reproductive roles of the sexes can predict the direction and magnitude of sexual dimorphism of external and internal morphology. Males should have enlarged structures that enhance the acquisition of mating opportunities, whereas females are predicted to have enlarged organs that are associated with the production of eggs. We tested these predictions in male and female lake whitefish, a species in which both sexes have similar overall body size and shape. After controlling for body size, male lake whitefish had significantly longer jaws and pectoral and pelvic fins, larger hearts, and more muscle than females. Sexual dimorphism in relative muscle mass may be one of the most fundamental morphological differences between males and females. Females had relatively heavier livers than males. Because the liver is important for the breakdown of fats and vitellogenesis, selection should favour an enlarged liver in females for the processing of energy and the production of large numbers of eggs.

Notes: Abstract –  Anthropogenic barriers that may interfere or prevent fish migration are commonly found in streams throughout the distribution of salmonids. Construction of fish passages in streams is a common solution to this problem. However, the goal with fish passages is often, at least in Scandinavia, to allow Atlantic salmon (Salmo salar L.) and migratory brown trout (S. trutta L.) to get access to spawning areas above these barriers. Hence, the fish passages may often only be open during the spawning migration of salmonids (late summer to autumn). We present data, on wild brown trout migration, from two trapping systems in two Scandinavian streams showing that intra- and interstream migrations are common throughout the summer and autumn. Moreover, differences in size were found between trap-caught trout and electrofished trout where trapped trout were generally larger than electrofished trout. We suggest that the current regime with fish passages only open parts of the year can have negative effects on populations by depriving trout from the possibility to perform migrations throughout the year.

Notes: Abstract –  We examined the site fidelity of spawning adult sockeye salmon (Oncorhynchus nerka) by tagging and releasing fish in the same stream reach (controls) and displacing them among different but nearby sites (c. 50 m away). Three sites – two above a stream junction (‘upper’ reach and ‘pond’) and one below (‘lower’ reach) – allowed us to compare the behavior of salmon in the presence and absence of olfactory cues and habitat similarity. Most controls of both sexes (90%) remained in the immediate vicinity of the tagging and release site. When displaced downstream, where the odors of both the upper reach and the pond were detectable, most salmon returned to their former site (65%). Displaced sockeye were more likely to return to the pond from the lower reach than from the upper one (P = 0.05), consistent with olfactory orientation and the hypothesis that salmon prefer certain habitats. Salmon displaced from the upper to the lower reach were much more likely to return than those displaced to the pond (P 〈 0.01), consistent with the role of odors in orientation and inconsistent with the habitat choice hypothesis.

Notes: Abstract –  We examined whether mortality and growth during the migration phase (the lacustrine phase after smolting) differ between sexes in migratory (lake-run) masu salmon (Oncorhynchus masou) in a lacustrine population, northern Hokkaido, Japan. The sex ratio of 1+ smolts was more skewed to females than that of the returning adults in three annual cohorts examined, indicating that females have a higher mortality rate during the migration phase. Although there was no difference in smolt length between sexes, female adults were larger in body length than males of the same age: female-biased sexual size dimorphism (SSD) occurs. We suppose that breeding selection favouring a larger body size for females than for males in this population probably requires that females undertake more intensive foraging behaviour with its greater inherent risks but faster growth rates during the migration phase. It therefore seems to be likely that the selection is the ultimate cause of the female-biased mortality rate and SSD.