ALBERT

Notes: The effect of short-term low temperature treatment on nitrate reductase (NR, EC 1.6.6.1) activity, NR protein and NR transcript levels in excised leaves of winter wheat (Triticum aestivum L. cv. Sadovo-1) was investigated. NR activity, measured in the presence of Mg2+ (NRact), doubled within 2 h at 4°C, whereas NR activity, measured in the presence of EDTA (NRmax), did not respond to the cold treatment. Such an activation of NR occurred only if leaves were exposed to low temperature in the light but not in the dark. It was not affected by feeding cytoplasmic protein synthesis inhibitor, cycloheximide, or protein kinase inhibitor, staurosporin, but was completely prevented by okadaic acid, an inhibitor of protein phosphatases of the type 1 and 2 A. This inhibitory effect decreased gradually when okadaic acid-concentration in the nutrient solution was lowered below 1 µM and tended to disappear when leaves were fed with 10 nM okadaic acid. It was demonstrated that the cold-induced NR activation was dependent neither on cold-triggered calcium influx nor on high endogenous abscisic acid levels. The increased NRact in cold-exposed leaves was found to correlate with a higher level of NR transcript but not with an increased NR protein level. Feeding okadaic acid to these leaves prevented the cold-induced accumulation of NR mRNA. These data point to protein phosphatases of the type 2 A being involved in NR protein dephosphorylation and NR transcript accumulation as targets of activation by low temperature treatment.

Notes: Cutting leaves of Romaine lettuce (Lactuca sativa L. cv. Longifolia) produces a wound signal that induces the synthesis of phenylalanine ammonia lyase (PAL, EC 4.3.1.5) and the accumulation of phenolic compounds in cells up to 2 cm from the site of injury, and tissue browning near the site of injury. The response of leaves within a head of Romaine lettuce to putative chemical wound signals [abscisic acid (ABA), jasmonate (JA) and methyl jasmonate (MeJA)] differed significantly with leaf age. Exposure of harvested heads of lettuce to ABA, JA, MeJA, or salicylic acid (SA) did not induce changes in PAL activity, the concentration of phenolic compounds or browning in mature leaf tissue that was similar to the level induced by wounding. Methyl jasmonate applied as vapour (10, 100 or 1000 µl kg−1 FW), or as an aqueous spray or dip (0.01–100 µM) at 5 or 10°C did not produce an effect on PAL activity or browning that differed significantly from the untreated controls. In contrast, JA, MeJA and SA did induce elevated levels of PAL activity in younger leaves. However, the levels induced were far lower than those induced by wounding. Wound induced phenolic metabolism in mature leaves appears to be induced by different signals than those functioning in young leaves.

Notes: The ATP-dependent Clp protease is one of the newly identified proteolytic systems in plant organelles that incorporate the activity of molecular chaperones to target specific polypeptide substrates and avoid inadvertent degradation of others. We describe new nuclear-encoded ClpC (ClpC1) and ClpP (ClpP3–5) isomers in Arabidopsis thaliana that raise the total number of identified Clp proteins to 19. The extra Clp proteins are localized within the stroma of chloroplasts along with the ClpD, –P1 and –P6 proteins. Potential differential regulation among these Clp proteins was analysed at both the mRNA and protein level. A comparison between different tissues showed increasing amounts of all plastid Clp proteins from roots to stems to leaves suggested the greatest abundance of proteins was in chloroplasts. The increases in protein were mirrored at the mRNA level for most ClpP isomers (ClpP1, −3, −4 and −6) but not for the three Hsp100 proteins (ClpC1, –C2 and –D) and ClpP5, which exhibited little change in transcript levels, suggesting post-transcriptional/translational regulation. Potential stress induction was also tested for all chloroplast Clp proteins by a series of brief and prolonged stress conditions. Short-term moderate and severe stresses (desiccation, high salt, cold, heat, oxidation, wounding and high light) all failed to elicit significant or rapid increases in any chloroplast Clp protein. However, increases in mRNA and protein content for ClpD and several ClpP isomers did occur during long-term high light and cold acclimation of Arabidopsis plants. These results reveal the great complexity of Clp proteins within the stroma of plant chloroplasts, and that these proteins, rather than being rapidly induced stress proteins, are primarily constitutive proteins that may also be involved in plant acclimation to different physiological conditions.

Notes: Peroxidase (POD, EC 1.11.1.7) activity, cellular localization and isozyme patterns were investigated in the seed integument, cotyledon and embryo axis of Brassica oleracea cv. Cappuccio during pregermination and seedling growth. Seeds started to germinate after 24 h of imbibition. POD activity was localized in the pigmented layer of the integument and in procambial strands of the cotyledon and embryo axis in the first 24 h of imbibition. It was localized in the integumental cells of palisade, pigmented and aleurone layers and in epidermal, meristematic, procambial cells and xylem elements of the root and hypocotyl after 48 h of imbibition. POD activity increased during germination and early seedling growth: in the integument, it reached a maximum value after 72 h of imbibition, in the embryo axis and cotyledons, it increased up to 144 h of imbibition. The increase in peroxidase activity was accompanied by the appearance of new isozymes correlated with the development of seedling tissues. The isozyme profile was characterized by nine peroxidases: isoperoxidase of 50 kDa peculiar to integuments, that of 150 kDa to cotyledons and that of 82 kDa to the embryo axis. During pregerminative phase isozymes of 84 kDa were detected in the integument and cotyledons, of 48.5 kDa in the embryo axis. After germination, peroxidase activity and the complexity of the isozyme pattern increased, suggesting that they play a relevant role after rupture of the integument.

Notes: Calli grown from segments of spinach (Spinacia oleracea L.) root in the presence of gibberellic acid (GA3) plus auxin, differentiated to yield somatic embryos after transfer to a medium without growth regulators, while calli formed in the absence of GA3 failed to generate any embryos. We extracted proteins from the two types of callus and analysed them by polyacrylamide gel electrophoresis. Compared with the proteins from calli formed on medium that contained only naphthaleneacetic acid (NAA) as a growth regulator, the proteins from calli grown in the presence of GA3 included appreciably higher levels of a 31-kDa basic protein (pI = 8.8). The protein resembled type I ribosome-inactivating proteins (EC 3.2.2.22) in terms of molecular mass, isoelectric point, sequence of amino-terminal amino acids and extent of glycosylation. The 31-kDa protein was barely detectable in extracts of various tissues from seedlings. Thus, it is possible that an increase in the relative level of this protein might be associated with the expression of embryogenic potential expressed by spinach callus.

Notes: In genotypes of Arabidopsis that exhibit a winter-annual flowering habit, floral induction in response to extended cold exposure (vernalization) is mediated by repression of the flowering-inhibitor gene FLC. We are interested in identifying components of the cold signal transduction pathway leading to FLC repression. We examined the potential involvement of two factors that are known to play roles in plant cold responses: (1) CBF1, a cold-responsive transcription factor that is involved in activating the cold acclimation response, and (2) the phytohormone abscisic acid (ABA), which has traditionally been associated with plant cold responses. We introduced a transgene driving constitutive expression of CBF1 into a winter-annual genotype of Arabidopsis. In transgenic lines expressing CBF1 mRNA to high levels, FLC mRNA expression was not repressed, and flowering was not accelerated relative to control plants. We also introduced mutations that compromise ABA biosynthesis or sensitivity into a winter-annual genotype and found that the vernalization response was not affected. Finally, we found that presumed increases in ABA levels, as a result of direct application of the hormone or severe water stress, were insufficient to substitute for cold to induce flowering. Taken together, these findings indicate that vernalization involves a pathway that is distinct from cold-response mechanisms involving CBF1, cold-regulated genes under CBF1 control, and ABA.

Notes: Five jasmonates, including novel tryptophan conjugates of jasmonic acid and dihydrojasmonic acid, were identified in extracts from spears of Asparagus officinalis L. by electrospray tandem mass spectrometry. Spears were harvested and were held dry or with bases immersed in water. The concentrations of jasmonic acid, dihydrojasmonic acid, their tryptophan conjugates, cucurbic acid and methyl jasmonate, were measured by ELISA in spears in the 10 d following harvest. A transient increase that occurred in all spear tips immediately following harvest in the concentration of jasmonates can be attributed to a wounding response. A second increase in the concentration of jasmonates occurred from 7 d after harvest but only in dry-treated spear tips indicating that jasmonates may have accumulated in response to water stress. Jasmonate levels were also monitored during natural foliar senescence. Increased levels of jasmonates occurred after the onset of senescence, implicating them as a consequence rather than a cause of senescence.

Notes: We have studied photoperiodic control and the effect of phytochrome photoconversion at the end-of-day (EOD) on polyamine (PA) accumulation in petal explants of Araujia sericifera. Petals from immature flowers were cultured under long (LD) and short (SD) days. Light was provided by Gro-lux fluorescent lamps (90–100 µmol m−2 s−1). Red (R), far red (FR), red followed by far-red (R-FR) and far-red followed by red (FR-R) light treatments were applied daily at the end of the photoperiod. The free and bound putrescine (Put), spermidine (Spd) and spermine (Spm) fractions in petal explants were determined 40 days after the beginning of the culture. We also aimed to clarify the involvement of PA changes by using two inhibitors of PA biosynthesis: D-l-α-difluoromethylarginine (DFMA) and methylglyoxal bis(guanylhydrazone) (MGBG). We found PA accumulation to be under photoperiodic control, and the inhibitory effect of DFMA on this accumulation suggests that arginine decarboxylase (ADC) is the major pathway for Put biosynthesis. Polyamine levels were higher under LD, mainly as a result of the accumulation of free and bound Put. FR-EOD treatment, which dramatically reduced the R : FR ratio after LD, increased the accumulation of PA, mainly as free Put and free and bound Spd. Sequential R-FR and FR-R-EOD treatments strongly increased bound Spd. The concentration of MGBG used increased total PA accumulation, mainly as Put. However, all EOD light treatments dramatically reduced Put accumulation in the presence of MGBG. This may be due to a dual role of FR light in PA accumulation: (1) FR per se stimulates PA production, probably via ADC, and (2) in the presence of MGBG, FR inhibits Put accumulation, probably via ethylene production.

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: Rice (Oryza sativa L.) cv. Tulsi is recommended for Eastern India, for upland ecological cultivation systems where a crop experiences natural cycles of water deficit and water sufficiency, depending upon the monsoon rains. In this experiment, this cultivar was subjected to three cycles of water stress of increasing stress intensity. Each stress cycle was terminated by rewatering the plants for a 48-h period. The level of stress was measured by quantification of H2O2. The response of antioxidant metabolites such as ascorbate and glutathione, and enzymes such as superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), glutathione reductase (GR, EC 1.6.4.2) and guaiacol peroxidase (POX, EC 1.11.1.7) was analysed in terms of activity and isozyme pattern for each cycle of stress and recovery. The differential response of the antioxidant enzymes with increasing stress intensity followed by recovery, highlight the different role of each in the drought acclimation process of upland rice. SOD and POX activity in stressed plants was higher than the controls in all the three cycles. The second level of stress saw an increase in all the enzymes with APX and GR showing its maximum activity and there was a better management of H2O2 levels. There was an induction of a new CAT isoform in stressed plants in the third cycle of water stress. The co-ordinated defense helped the plants to recover in terms of growth on rewatering after stress cycles.

Notes: During flooding, when roots are submerged in oxygen-free water, root tissue becomes hypoxic and its metabolism is characterized by fermentation processes and limited respiratory activity. After returning to aerobic conditions (post-hypoxic period) high respiration rates together with symptoms of oxidative stress are observed. Plant mitochondria have two terminal oxidases: a cytochrome oxidase with high affinity to oxygen and an alternative (cyanide resistant) oxidase (AOX) with a relatively low oxygen affinity. We compared mitochondrial respiration and AOX expression immediately after hypoxic and during post-hypoxic period. Four-day-old barley (Hordeum vulgare L. cv Gregor) seedlings were transferred for 5 days to Knop nutrient medium flushed with air (control) or nitrogen (hypoxia) and after returned for 24 h to aerated nutrient medium (post-hypoxia). NADH/NAD+ + NADH and UQH2/UQtotal ratios increased in hypoxia-treated roots. After the hypoxic roots were returned to aerated medium, increases in respiration rate and ATP concentration were observed. Mitochondria isolated from barley roots at the end of hypoxic period exhibited high respiratory rates as compared to mitochondria from control and post-hypoxic roots. Control root mitochondria expressed high AOX capacity in the presence of pyruvate and DTT. Mitochondria isolated at the end of the hypoxic period were highly cyanide sensitive under AOX-activating conditions, however, after 24 h of post-hypoxia, AOX capacity was comparable to that observed in the control. The capacity of AOX was correlated with the amount of AOX protein determined by Western blotting. Faint or no bands were observed immediately after hypoxia with the AOX protein appearing again during the post-hypoxic period. Examination of AOX transcript levels showed no significant differences between control, hypoxic and post-hypoxic roots indicating that the regulation of AOX expression by oxygen availability in barley roots may take place at the translation level.

Notes: A bioactivity-guided chemical study of Iostephane heterophylla (Asteraceae) led to the isolation of xanthorrhizol (1) as the compound that causes inhibition of ATP synthesis, H+-uptake and electron flow from water to methylviologen (basal, phosphorylating and uncoupled) in freshly lysed spinach chloroplasts, thus acting as an inhibitor of the Hill reaction. Acetyl (2), dihydro (3) and acetyl-dihydro (4) derivatives were synthesized. It was found that 4 was less active than 1 and 2 in ATP synthesis, whereas 3 was the most potent inhibitor of the Hill reaction and was also an inhibitor of H+-ATPase. Studies of the photosynthetic partial redox reactions from PQ to MV indicated that 1 partially inhibited the PQ pool, but that 3 did not. However, both inhibited the uncoupled electron transport in PSII from water to DCBQ. Uncoupled electron flow from water to silicomolybdate was completely inhibited by 3 and partially by 1. The reaction from DPC to DCPIP was inhibited by both 1 and 3. These results indicate that the inhibition site is located within PSII for 1 and 3 as was corroborated by fluorescence decay data.

Notes: Glutathione transferases (GSTs) are ubiquitous, multifunctional proteins encoded by large gene families. In different plant species this gene family is comprised of 25–60 members, that can be grouped into six classes on the basis of sequence identity, gene organization and active site residues in the protein. The Phi and Tau classes are the most represented and are plant specific, while Zeta and Theta GSTs are found also in animals. Despite pronounced sequence and functional diversification, GSTs have maintained a highly conserved three-dimensional structure through evolution. Most GSTs are cytosolic and active as dimers, performing diverse catalytic as well as non-catalytic roles in detoxification of xenobiotics, prevention of oxidative damage and endogenous metabolism. Among their catalytic activities are the conjugation of electrophilic substrates to glutathione, glutathione-dependent isomerizations and reductions of toxic organic hydroperoxides. Their main non-catalytic role is as hormone and flavonoid ligandins. GST genes are predominantly organized in clusters non-randomly distributed in the genome. Phylogenetic studies indicate that plant GSTs have mainly evolved after the divergence of plants, the two prevalent Phi and Tau classes being the result of recent, multiple duplication events.

Notes: The carbon assimilation efficiency and the internal composition of the chlorophyte Dunaliella viridis have been studied under conditions of current (0.035%) and enriched (1%) levels of CO2, with and without N limitation (supplied as nitrate). Results show that both photosynthesis and growth rates are enhanced by high CO2, but the strategy of acclimation also involves the light harvesting machinery and the nutritional metabolism in an N supply dependent manner. D. viridis carried out a qualitative rather than a quantitative acclimation of the light harvesting system leading to increased PSII quantum yields. Total internal C decreased as a consequence of either active growth or organic carbon release to the external medium. The latter process allowed photosynthetic electron transport to proceed at higher rates than under normal CO2 conditions, and maintained the internal C:N balance in a narrow range (under N sufficiency). N limitation generally prevented the effects of high CO2, with some exceptions such as the photosynthetic O2 evolution rate.

Notes: Fusicoccin (FC) is a well known toxin acting as a 14-3-3 protein-mediated activator of the plasma membrane H+-ATPase and it has been widely used to study the regulatory mechanism and the physiological role of this enzyme's activity. Recently, FC has been shown to induce other responses similar to those occurring under a stress condition, perhaps not strictly dependent on the activation of proton extrusion. In this paper we report that in cultured sycamore (Acer pseudoplatanus L.) cells FC induces H2O2 overproduction as well as other novel, presumably related responses, such as the activation of the alternative oxidase and the leakage of cytochrome c from the mitochondria, accompanied by a decrease of the cytochrome pathway capacity. The relationship between H2O2 production and other phenomena has also been studied by means of exogenously added H2O2.

Notes: It is currently accepted that, along with nutrients, arbuscular mycorrhizal (AM) fungi also transport water to their host plant. However, the quantity of water supplied and its significance for plant water relations remain controversial. The objective of this work was to evaluate and compare the ability of six AM fungi to alter rates of root water uptake under drought stress conditions. Soil drying rates of uninoculated control plants of comparable size and nutritional status and mycorrhizal plants were recorded daily. Lactuca sativa plants colonized by Glomus coronatum, G. intraradices, G. claroideum and G. mosseae depleted soil water to a higher extent than comparably sized uninoculated control plants or plants colonized by G. constrictum or G. geosporum. The differences ranged from 0.6% volumetric soil moisture for G. mosseae-colonized plants to 0.95% volumetric soil moisture for G. intraradices-colonized plants. These differences in soil moisture were equivalent to 3–4.75 ml plant−1 day−1, respectively, and could not be ascribed to differences in plant size, but to the activity of AM fungi. The AM fungi tested in this study differed in their effectiveness to enhance plant water uptake from soil. This ability seems to be related to the amount of external mycelium produced by each AM fungus and to the frequency of root colonization in terms of live and active fungal structures.

Notes: Ear photosynthesis may be an important source of C for grain growth in water-stressed plants of cereals. The main objectives of this work were to determine the stability of the photosynthetic apparatus and the photochemical efficiency of ears in plants subjected to post-anthesis drought. Plants of wheat (Triticum aestivum L. cv. Granero INTA) were grown in pots under a rain shelter and subjected to water stress (soil water potential around −0.6 to −0.8 MPa) starting 4  days after anthesis. Post-anthesis drought substantially accelerated the loss of chlorophyll, Rubisco and the light-harvesting complex of photosystem II (LHCII) in the flag leaf, but the degradation of these photosynthetic components was much less affected by water deficit in awns and ear bracts. Quantum yield of PSII (ΦPSII) decreased in leaves of water-stressed plants. In contrast, ear bracts had a higher ΦPSII than leaves, and ΦPSII of ear bracts did not decrease at all in response to drought. Removing the grains immediately before fluorescence measurements (less than 30 min) slightly reduced ΦPSII, indicating that CO2 supplied by grain respiration may contribute to the high photochemical efficiency of ears in droughted plants. However, other factors may be involved in maintaining high ΦPSII, since even in the absence of grains ΦPSII remained much higher in ear bracts than in the flag leaf. The relative stability of ear photosynthetic components and their relatively high photochemical efficiency may help to maintain ear photosynthesis during the grain filling period in droughted plants.

Notes: Circumnutation in Helianthus annuus L. was investigated by measurements lasting 4–7 weeks using a picture analysis system. The rhythmicity of circumnutation vigour (intensity) with regard to the trajectory length and period of individual circumnutations were examined. Three photoperiod conditions were applied [light/dark (LD), continuous light (LL) and LD followed by LL]. Data were processed by the Fourier analysis. Statistical analysis included the examination of circumnutation mean frequencies and correlation tests. Both parameters, trajectory length and period, revealed a daily (24 h) modulation in LD with a weak correlation between them, whereas in LL no daily modulation of the parameters was observed. After LD–LL transition, the parameters were gradually losing their daily modulation. Despite a very strong modulation of the trajectory length in LD, the period was quite stable in all groups tested, but only in LD were there no statistical differences in the number of circumnutations per 24 h among the plants studied. LD was concluded to be the strong synchronizer, making the plants circumnutate regularly. Regardless of the presence or absence of daily modulation, the infradian (several and more days long) harmonics of the trajectory length were the same in each group. These findings strongly support the view that circumnutation in sunflower, widely known as an ultradian rhythm, also possesses daily and infradian modulations of its intensity. To the authors' knowledge, this is the first report of circumnutation that was obtained by a picture analysis system in such a large timescale.

Notes: Two hypotheses, namely the occurrence of post-thaw oxidative stress or imbibitional damage, were tested to explain the high sensitivity of coffee seeds to liquid nitrogen (LN) exposure. Oxidative stress was studied by measuring primary and secondary products of lipid peroxidation in seeds during the desiccation and rehydration periods. The 4-hydroxynonenal (4-HNE) content of seeds remained constant throughout the desiccation step. No significant difference was observed between desiccated seeds and seeds desiccated and exposed to LN for the evolution of their 4-HNE and hydroperoxide contents during rehydration. In both cases, an increase in 4-HNE and hydroperoxide contents of seeds was observed during the first hours of culture under germination conditions, followed by a progressive decrease down to values comparable to those observed in desiccated seeds. The hydroperoxide composition of frozen seeds was not significantly different from that of control seeds. The (S)/(R) enantiomeric ratios of 9- and 13-hydroxy-octadecadienoic acid extracted from rehydrating seeds were chiral, suggesting that they originated from lipoxygenase activity. These results suggest that the high sensitivity of coffee seeds to LN exposure is not directly associated with the occurrence of an oxidative stress during post-thaw rehydration. The effect on seed viability of different rehydration procedures previously identified to reduce membrane imbibitional injury was studied after desiccation and LN exposure. Desiccation tolerance increased with, by increasing order, seed osmoconditioning, pre-heating and pre-humidifying prior to their culture under germination conditions. Among the four combinations of pre-humidification durations (24 or 48 h) and temperatures (25 or 37°C) tested, pre-humidification for 24 h at 37°C gave the highest level of desiccation tolerance. This rehydration procedure also dramatically increased seed viability after LN exposure. Seed desiccation sensitivity modelling in combination with the calculation of the decrease in seed water activity during cooling facilitated the explanation of the beneficial effect of controlled rehydration after desiccation and LN exposure. These results support the hypothesis that imbibitional membrane damage is involved in the sensitivity of coffee seeds to LN exposure.

Notes: Plant growth and adaptation to cold and freezing temperatures in a CO2-enriched atmosphere have received little attention despite their predicted effects on plant distribution and productivity. In this study we looked at the interaction between elevated CO2 and development of freezing tolerance in Norway spruce (Picea abies (L.) Karst.). First-year seedlings were grown under controlled conditions in an atmosphere enriched in CO2 (70 Pa) for one simulated growth season. We measured shoot growth, registered the timing of growth cessation and bud set, measured needle net photosynthetic rate, and determined needle carbohydrate concentration (fructose+pinitol, glucose, sucrose, inositol, raffinose and starch). Freezing tolerance (LT50) was determined after exposing whole seedlings to temperatures ranging from −6.5 to −36.0°C and scoring for visual needle browning. Elevated CO2 did not affect height growth or the timing of growth cessation and bud set. The only statistically significant effects of CO2 treatment were on seedling dry weight, percent dry matter and starch content. During the three weeks after growth cessation and bud set, freezing tolerance increased from −10 to −35°C, and there was a marked increase in all soluble sugars except inositol. However, neither freezing tolerance nor the concentration of soluble sugars was significantly influenced by elevated CO2.

Notes: The effect of Zn on growth, chlorophyll a fluorescence, net photosynthetic rate, gas exchange, water content and mineral concentrations (Zn, Mn and Mg) in ryegrass infected with or free from Neotyphodium lolii was studied by addition of ZnSO4 (0–20 mM) to the nutrient solution. Zn induced a decrease in growth of plants at 1, 5 and 10 mM and cessation of growth at 20 mM ZnSO4. From 1 to 10 mM, the decrease was less pronounced in the presence of the endophytic fungus than in its absence. The growth limitation was due to an accumulation of Zn in leaves. From 8 to 15 days, the presence of the fungus in the plant led to a limitation of the Zn concentration in the leaves (24–32% lower with N. lolii than without). This restriction of Zn concentrations in leaves also had a beneficial effect on photosystem II (PSII) activities, net photosynthetic rate and internal CO2 concentration. Particularly at 1 and 5 mM, the quantum yield of electron flow throughout PSII was greater in the presence of the fungus than in its absence and at 5 and 10 mM, the internal CO2 concentration was maintained at a normal level. Compared with the endophyte-free ryegrass, the symbiotic plants showed higher values of total dry weight and tiller number, indicating a tolerance to environmental Zn stress.

Notes: The aim of this work was to study the effect of ultraviolet-B (UV-B) exposure on oxidative status in chloroplasts isolated from soybean (Glycine max cv. Hood). Chloroplasts were isolated from soybean leaves excised from either control seedlings or those exposed to 30 and 60 kJ m−2 day−1 of UV-B radiation for 4 days. Chloroplastic oxidative conditions were assessed as carbon-centered radical, carbonyl groups and ascorbyl radical content. Treatment with UV-B increased the carbon-centered radical-dependent EPR signal significantly by 55 and 100% in chloroplasts from leaves exposed to 30 and 60 kJ m−2 day−1 UV-B, respectively, compared to radical content in chloroplasts from control leaves. The content of carbonyl groups increased by 37 and 62% in chloroplasts isolated from soybean leaves irradiated for 4 days with 30 and 60 kJ m−2 day−1 UV-B, respectively. The content of soluble metabolites in isolated chloroplasts should not be taken as absolute in vivo values; however, these data are valuable for comparative studies. UV-B exposure did not significantly affect ascorbyl radical content compared to controls. The content of ascorbic acid and thiols in chloroplasts isolated from leaves exposed to 60 kJ m−2 day−1 UV-B was increased by 117 and 20.8%, respectively, compared to controls. Neither the content of total carotene nor that of β-carotene or α-tocopherol was affected by the irradiation. The results presented here suggest that the increased content of lipid radicals and oxidized proteins in the chloroplasts isolated from leaves exposed to UV-B could be ascribed to both the lack of antioxidant response in the lipid soluble fraction and the modest increase in the soluble antioxidant content.

Notes: Chilling whole cucumber seedlings that had 10-mm long radicles for 4 days at 2.5°C significantly inhibited subsequent radicle growth both by increasing the time it took the seedlings to recover from chilling and attain a linear rate of radicle growth, and by decreasing the subsequent rate of linear growth. Exposing cucumber seedlings to 45°C for up to 20 min had no effect on subsequent radicle growth, while longer exposures produced reductions in growth. A heat shock at 45°C for 10 min induced the optimal protection to 4 days of chilling at 2.5°C by reducing chilling inhibition from 60 to 42%. Two hours after being chilled, heat shocked or heat shocked and then chilled, there was no difference in protein content of the apical 1 cm of the seedling radicle among these treatments and the non-heat shocked, non-chilled control. Two days after treatment, the protein content was still similar in tissue that had been heat shocked or heat shocked and chilled, while it was significantly reduced in tissue that had been chilled. In general, 2 h after treatment, the activity of the 5 antioxidant enzymes examined in this study [superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11), guaiacol peroxidase (GPX; EC 1.11.1.7) and glutathione reductase (GR; EC 1.6.4.2)] were reduced by chilling and unaffected or increased by heat shock. When heat shock was followed by chilling, there was a consistent effect of the heat shock treatment on preventing the loss of enzyme activity following chilling. This protective effect of the heat shock treatment was even more pronounced after 2 days of recovery at 25°C for SOD, CAT and APX. In contrast, the activity of GR and GPX was substantially higher in chilled tissue than in tissue that had been heat shocked before being chilled. Elevated levels of GR and GPX therefore appear to be correlated with the development of chilling injury, while elevated levels of SOD, CAT and APX appear to be correlated with the development of heat shock-induced chilling tolerance.

Notes: The Ingestad approach to the culture of higher terrestrial plants for physiological studies is discussed in relation to a number of resources, organisms and growth situations that were not part of the original design and rationale of Ingestad's methodology. The additional resource considered is photosynthetically active radiation, and difficulties of applying the Ingestad approach to this resource as well as to atmospheric CO2 are considered. The relationship of the Ingestad approach to reductionist studies based on enzyme kinetic studies is then briefly considered. The organisms considered next are aquatic plants, including both micro- and macrophytes. The consideration of photosynthetic microorganisms leads to a comparison of the Ingestad approach with growth in batch, and in continuous (chemostat and turbidostat) cultures, and with studies on growth in synchronous cultures in which cyclic changes in cell composition in the cell growth and division cycle can be identified. The natural environmental conditions for these organisms are a natural extension of the light/dark synchronization of laboratory cultures, and the bloom (batch culture equivalent to new production) and of grazing and parasitism removing biomass and recycling nutrients (chemostat or turbidostat culture equivalent to recycled production) situations for phytoplankton. The overall conclusion is that, while the Ingestad approach is a useful mirror in which to examine other concepts of plant resource acquisition and manipulation, the Ingestad methodology seems to make assumptions about the intrinsic growth rate and composition of plants that cannot be independently verified.

Notes: Total soluble phenols, soluble flavanols, (+)-catechin, ferulic acid and 1-O-feruloyl-β-d-glucose were analyzed during the development of a strawberry (Fragaria×ananassa, cv. Chandler) callus culture. The time-course changes of the different phenols assayed were well correlated with callus growth and morphology. The changes in polyphenol oxidase (EC 1.10.3.1-2) and β-glucosidase (EC 3.2.1.21) activities in the callus were also examined. The total phenol, soluble flavanols and (+)-catechin contents were high during the preexponential and exponential phases of growth. The subsequent decrease in (+)-catechin concentration coincided with high levels of polyphenol oxidase activity. The 1-O-feruloyl-β-d-glucose content was highest as callus growth ceased, and its subsequent decrease was accompanied by the increased production of ferulic acid. This increase in ferulic acid was accompanied by an increase in β-glucosidase activity. The ferulic acid content decreased at the end of culture, when callus growth had stopped and showed clear symptoms of senescence. This decrease in the ferulic acid concentration was accompanied by an increase in the levels of ferulic acid bound to cell wall components.

Notes: The carbohydrate pool within the bulbs of Lachenalia minima W.F. Barker (Hyacinthaceae) consists of similar amounts of fructans and starch. This study was conducted to examine the changes within the pool of non-structural carbohydrates that occur during sprouting under field conditions. The bulbs were watered over a period of 23 days to simulate the onset of the rainy season. Even though there was no significant change of the total fructan content, the distribution and the composition of the fructan fraction within the different leaf scales of the bulbs altered during sprouting. The major changes occurred in the innermost scales, the total fructan content increased from 300 (day 0) to 607 (day 23) g kg−1 dry mass and high-performance anion-exchange chromatography analysis revealed a significant increase of fructans with low degree of polymerization (DP). With respect to starch, the most pronounced difference accompanying the transition to growth was also in the innermost scales. In contrast to fructans, starch content decreased from 241 (day 0) to 60 (day 14) g kg−1 dry mass. These results demonstrate that starch, and not fructan, is used as the carbon and energy source for sprouting. The water content data suggest the involvement of fructans in water relations. The preferential accumulation of low DP fructans and sucrose within the innermost scales directs the water flow to where it is most needed for growth. Similar changes were obtained for bulbs in the dry soil, but transformation rates were much slower and occurred to a lesser extent, indicating that these reactions were not triggered but were accelerated by water.

Notes: Previous studies on the tomato (Lycopersicon esculentum Mill.) peroxidase TPX1, including the development of transgenic tomato over-expressing this gene, supported an involvement of this peroxidase in the synthesis of lignin and suberin. The transgenic plants showed a wilty phenotype at flowering, but the relationship between this role in ligno-suberization and this phenotype was not clear. In the present study a histological approach and the measurement of water-related parameters have been performed in order to obtain an insight into the origin of this phenotype. Clear differences between transgenic and non-transgenic roots were observed in the cross-sections of the basal root zones where secondary growth was evident. The diameter of the xylem vessel was diminished in the transgenic plants. Total area corresponding to xylem in the basal cross-sections decreased 3.9 fold in the transgenic roots. In addition, the radial and outer tangential walls of the exodermis cells were more ligno-suberized in transgenic than in non-transgenic plants. After fruit set, predawn and midday water potentials were lower in transgenic than in-non-transgenic plants. At midday, the stomatal conductance was also lower in the transgenic plants, 494±69 versus 594±60 mmol m−2 s−1. Root hydraulic conductances of the transgenic and non-transgenic plants were 1.4±0.38 and 3.47±0.19 g water min−1 MPa−1, respectively. The results obtained support that the phenotype is caused by the anatomical differences found in the transgenic roots. These differences would be the cause of a increased resistance to water flow in the roots that would negatively affect the water supply to the shoot and, as a consequence, resulted in a decreased water potential in the leaves.

Notes: The differential display technique was used to generate cDNA probes in order to identify mRNAs that are up-regulated during senescence of Arabidopsis leaves. Three mRNAs were examined that had not previously been associated with senescence. The steady-state levels of these mRNAs are detectable in small amounts in mature green leaves, but increase considerably as chlorophyll levels begin to decline. This relationship to senescence occurs under natural circumstances as well as when senescence is accelerated by leaf detachment in the dark or by addition of 1-aminocyclopropane-1-carboxylic acid (ACC). Retardation of senescence by benzyladenine slows the increase of the mRNAs. One of these mRNAs appears to code for a protein (Sec 13) that may be involved in vesicle formation at the endoplasmic reticulum. Another mRNA codes for a protein with WD-repeat motif whose function is as yet unidentified, and the third codes for a putative calcium-dependent protein kinase. A fourth cDNA has also been cloned by subtractive hybridization from senescing Arabidopsis leaves that encodes vacuolar-processing enzyme (γVPE). Incubation of detached leaves in darkness also caused an abrupt elevation in the steady-state levels of the γVPE, similar to that of the senescing attached leaves. The possible functions of the gene products and their involvement in cellular and biochemical processes during senescence are discussed.

Notes: The metabolism and intracellular localization of salicylic acid (SA) was investigated in soybean (Glycine max[L.] cv Williams 82) cell suspension cultures. [7–14C]SA was added to the cell cultures, the metabolites were extracted from the cells at various time points and analysed by TLC and HPLC. The [7–14C]SA was taken up rapidly from the culture media and converted primarily to SA 2-O-β-d-glucose (SAG). Lower levels of glucosylated 2,5-dihydroxbenzoic acid (gentisic acid) and methyl salicylate 2-O-β-d-glucose were also formed. Examination of the intracellular localization of the glucose conjugates revealed that all of the conjugates associated with the protoplasts were found in the vacuoles. An SA glucosyltransferase (SAGT) that could catalyse the formation of SAG from SA and UDP-glucose could be extracted from soybean cells and assayed in vitro. Increasing concentrations of SA added to the culture media induced the SAGT activity. The highest levels of SAGT activity were observed in cells treated with 0.5 mM SA. The SAGT activity in these cells was 88-fold greater than the SAGT activity in the untreated cells. The intracellular localization of the SAGT activity was also examined and it was determined that the majority of the SAGT activity in the protoplasts was located outside the vacuole. Therefore, it appears as if SAG is formed from SA outside the vacuole, presumably in the cytoplasm, and then subsequently transported into the vacuole where it accumulates.

Notes: The cDNA for a β-glucosidase (EC3.2.1.21) was isolated from rye (Secale cereale, cv Motto) and the sequence corresponding to the mature protein cloned into pET21a expression vector and used for transformation of Escherichia coli. The recombinant β-glucosidase expressed in E. coli was recognized by antibodies to maize β-glucosidase and exhibited the same kinetic properties on the endogenous substrates hydroxamic acid glucosides and artificial substrates as the native enzyme purified from rye. The enzyme monomer had an apparent molecular weight of about 67 kDa. The isolated cDNA was analysed with web-based chloroplast targeting prediction programs. The programs predicted a chloroplast targeting peptide with a cleavage site between amino acid 49 and 50. Sequence alignment of the plastidic rye β-glucosidase showed that the putative sites for substrate specificity of maize Glu1, W378 and F198 (F197) are conserved in the rye enzyme, whereas F205, F466 and A467 of maize Glu1 are exchanged for histidine, glycine and serine, respectively, in rye. The plastidic β-glucosidase is expressed in all plant parts and the highest levels were found in the coleoptile and mesocotyl.

Notes: Kinetic fluorescence imaging was used to set a new detection limit for plant exposure to low levels of destruxins – phytotoxins of Alternaria brassicae. A general experimental algorithm is presented that can be used to identify the combination of fluorescence parameters providing the highest contrast between the affected and unaffected plants or plant segments. Leaves of canola (Brassica napus) and white mustard (Sinapis alba) were exposed to various concentrations of destruxins and images of key fluorescence signals (F0, FM, FP, and of FS) were captured in a single kinetic experiment. Contrast was quantified within these images between the leaf areas exposed to destruxins and the untreated areas. The highest contrast was found in the image constructed by pixel-to-pixel division of images F0 by FP and F0 by FM. Using the F0/FM ratio image, we were able to detect exposure to destruxin concentration as low as approximately 0.05 mg l−1 applied to canola leaf and approximately 10 mg l−1 when applied to mustard. The detection limits were significantly lower than those obtained by optical microscopy indicating that kinetic chlorophyll fluorescence imaging can be used as a diagnostic tool in screening for varieties with an enhanced resistance to destruxins of Alternaria brassicae.

Notes: In the gravity-perceiving cells (statocytes), located in the centre of the root cap, polarity is expressed in the arrangement of the organelles since, in most genera, the nucleus and the endoplasmic reticulum are maintained at the opposite ends of each cell by actin. Polarity is also evident in the distribution of plasmodesmata, which are more numerous in the transverse walls than in the longitudinal walls. The centre of each statocyte is depleted of microtubules (they are only located at the periphery) but is occupied by numerous amyloplasts (statoliths), denser than the cytoplasm. The amyloplasts do not contribute to the inherent structural polarity since their position is dependent upon the gravity vector. This article focuses on new microscopic analyses and on data obtained from experiments performed in microgravity, which have contributed to our better understanding of the architecture of the actin web implicated in the perception of gravity. Depending upon the plant, the actin network seems to be formed of single filaments arranged in various ways, or, of thin bundles of actin filaments. The amyloplasts are enmeshed in this web of actin and their envelopes are associated with it, but they can have autonomous movement via myosin in the absence of gravity. From calculations of the value of the force necessary to move one amyloplast in the lentil root, and from videomicroscopy performed with living statocytes of maize roots, it is hypothesized that actin microfilaments could be orientated in an overall diagonal direction in the statocyte. These observations could help in understanding how slight amyloplast movements may trigger and transmit the gravitropic signal.

Notes: Nodulation in pea (Pisum sativum L.) grown in hydroponic and sand culture systems is stimulated by low concentrations (〈1.0 mM) of ammonium, but the physiological mechanisms underlying this stimulation are unknown. The current study involves a series of experiments, which investigate if the ammonium-induced stimulation of nodulation involves changes in endogenous hormone (auxin and cytokinin) levels. P. sativum L. cv. Express was grown in growth pouches for 1 week with mineral N (0.5 and 2.0 mM NH4+ or NO3–) or for 3 weeks exposed to exogenous indole-3-acetic acid (IAA) or 6-benzylaminopurine (BAP) at a range of concentrations (10-9−10-5 M). Ammonium enhanced nodulation on the basis of both early whole plant (nodules plant−1) and specific nodulation (nodules g−1 root DW), especially in 0.5 mM treatment in which nodulation was approximately 4-fold of the mineral-N-free control 1 week after inoculation. Correspondingly, the roots treated with ammonium contained much higher levels of t-zeatin (Z) and lower t-zeatin riboside (ZR) than that the control or nitrate-treated plants. There was no significant difference in IAA levels between the control and ammonium treatments. Exogenous application of BAP for 3 weeks at concentrations of 10-7−10-5 M strongly inhibited nodulation. However, 10−9 M BAP, but not IAA, significantly enhanced nodulation. These data support the theory that a relatively high ratio of cytokinin:auxin in roots is favourable for nodule initiation, but that an excessively high level of cytokinin inhibits nodulation. Based on these results we propose that stimulation of nodulation by low concentrations of ammonium may be mediated through increasing Z level in roots, which alters the balance of cytokinin and auxin, which in turn induces cortical cell divisions leading to nodule initiation.

Notes: Seedlings of Arabidopsis thaliana (L.) Heynh. fail to become green when germinated and grown on media containing high concentrations of glucose (Glc). Although previous studies have shown that sugar concentration affects chlorophyll levels and photosynthetic gene expression, the possibility that sugar concentration might affect actual chloroplast biogenesis has received little attention. Therefore, experiments were conducted to determine whether germination and growth on Glc impairs development of mature chloroplasts from the proplastids found in plant embryos. To monitor chloroplast biogenesis, the levels of a chloroplast-specific fatty acid, hexadecatrienoic (16:3) fatty acid, were measured in Arabidopsis seedlings grown on media containing different concentrations of Glc. These experiments indicate that moderate concentrations of Glc delay accumulation of 16:3. The effects of Glc on 16:3 levels are not solely due to osmotic stress, as equi-molar and even twice equi-molar concentrations of sorbitol do not exert comparable effects. Seedlings grown on concentrations of Glc high enough to prevent greening accumulate almost no 16:3, even after 22 days of growth under continuous light conditions. The lack of 16:3, a major structural component of chloroplast membranes, suggests that seedlings do not develop mature chloroplasts when grown in the presence of high concentrations of exogenous Glc. Further support for this hypothesis is provided by electron microscopy studies revealing that seedlings grown on high concentrations of Glc lack identifiable chloroplasts. Although Glc has been reported to inhibit chloroplast development in unicellular organisms, similar studies on intact higher plants have been lacking.

Notes: A full-length cDNA encoding sucrose synthase was isolated from the tropical epiphytic orchid Oncidium Goldiana. The cDNA is 2829 bp in length containing an open reading frame of 2447 bp encoding 816 amino acids with a predicted molecular mass of 93.1 kDa. The deduced amino acid sequence of O. Goldiana sucrose synthase (Osus) shares more than 80% identity with those from other monocotyledonous plants. The sucrose synthase gene was demonstrated to encode a functional sucrose synthase protein by expression as recombinant protein in Escherichia coli. The Osus mRNA is present in all the tissues analysed, with the highest levels in strong sinks such as developing inflorescence and root tips. Incubation with sucrose or glucose resulted in a significant increase in the steady-state Osus mRNA levels in root tips and mature leaves in a similar pattern to maize Sus1. Expression of the Osus mRNA in mature leaves was markedly enhanced by anaerobic conditions and elevated CO2. The expression pattern and regulation of the gene suggest that the sucrose synthase plays an important role in the growth and development of the tropical epiphytic orchid O. Goldiana.

Notes: The generation of reactive oxygen species (ROS) is a central component of the elicitor-induced defence reactions in cultured cells as well as the resistance responses of plants to pathogen challenge. We show that N-acetylchitooligosaccharide elicitor induces rapid and transient activation of phosphatidylinositol-specific phospholipase C (PI-PLC) and that phosphatidylcholine-specific phospholipase D (PC-PLD) in suspension-cultured rice cells and their products, phosphatidic acid (PA) and diacylglycerol (DG), especially the former, play an important role in the elicitor-induced ROS generation based on the following observations: (1) the amount of PA and DG in rice cells was rapidly increased by the elicitor treatment. (2) Elicitor-induced activation of PI-PLC and PC-PLD in the membrane fraction was confirmed by the analysis of enzymatic products from radio-labelled phospholipids as well as by 1-butanol (1-ButOH)-specific formation of phosphatidylbutanol (PtdBut) (for PC-PLD). Inhibitors of these phospholipases at least partly inhibited the elicitor-induced ROS generation. (3) Exogenously applied PA and DG could induce ROS generation in the rice cells in the absence of the elicitor. (4) PA phosphohydrolase (PAPH) and diacylglycerol kinase (DGK) activities, which catalyse the conversion of PA and DG with each other, are present in the rice cells and the inhibitors of these enzymes inhibited/stimulated the elicitor-induced ROS generation depending on the direction of the PA accumulation. These results indicate the important role of PI-PLC/PC-PLD and their products, especially PA, in the signal transduction cascade downstream of the N-acetylchitooligosaccharide receptor.

Notes: The induction of cyclobutane pyrimidine dimers (CPDs) by ultraviolet-B radiation (UV-B, 280–315 nm) and repair mechanisms were studied in the lichen Cladonia arbuscula ssp. mitis exposed to different temperatures and water status conditions. In addition, the development and repair of CPDs were studied in relation to the different developmental stages of the lichen thallus podetial branches. Air-dried lichen thalli exposed to UV-B radiation combined with relatively high visible light (HL, 800 μmol m−2 s−1; 400–700 nm) for 7 days showed a progressive increase of CPDs with no substantial repair, although HL was present during and after irradiation with UV-B. Fully hydrated lichen thalli, that had not been previously exposed to UV-B radiation for 7 days, were given short-term UV-B radiation treatment at 25°C, and accumulated DNA lesions in the form of CPDs, with repair occurring when they were exposed to photoreactivating conditions (2 h of 300 μmol m−2 s−1, 400–700 nm). A different pattern was observed when fully hydrated thalli were exposed to short-term UV-B radiation at 2°C, in comparison with exposure at 25°C. High levels of CPDs were induced at 2°C under UV-B irradiation, without significant repair under subsequent photoreactivating light. Likewise, when PAR (300 μmol m−2 s−1) and UV-B radiation were given simultaneously, the CPD levels were not lowered. Throughout all experiments the youngest, less differentiated parts of the lichen thallus – namely ‘tips’, according to our arbitrary subdivision – were the parts showing the highest levels of CPD accumulation and the lowest levels of repair in comparison with the older thallus tissue (‘stems’). Thus the experiments showed that Cladonia arbuscula ssp. mitis is sensitive to UV-B irradiation in the air-dried state and is not able to completely repair the damage caused by the radiation. Furthermore, temperature plays a role in the DNA damage repairing capacity of this lichen, since even when fully hydrated, C. arbuscula ssp. mitis did not repair DNA damage at the low temperatures.

Notes: Pringlea antiscorbutica R. Br., an endemic crucifer from the Kerguelen Archipelago in the subantarctic, has been previously shown to be unable to acclimatize to 25°C when transferred after several months cultivation under cold conditions. Furthermore, the polyamine composition was greatly modified in such high-temperature-treated plants. The development of seedlings of this species was investigated under a regime mimicking the subantarctic summer thermoperiod (5/10°C night/day) and a regime with high temperatures (22/25°C night/day). In parallel, the associated changes in polyamine composition that occurred during the first 6 days of seedling life were determined. Marked acceleration of seedling growth and intense cotyledon greening were observed at day 4 in 5/10°C-grown seedlings but not in 22/25°C-grown seedlings. Seedlings grown at high temperature accumulated agmatine and putrescine, whereas cold-cultivated seedlings maintained high levels of spermidine. Cold-cultivated seedlings accumulated the uncommon long-chain polyamines norspermidine and homospermidine. These seedlings also accumulated free 1,3-diaminopropane, cadaverine, N1-acetylspermidine, N1-acetylspermine and bound polyamines, whereas seedlings under high temperature accumulated N1-acetylputrescine. Aromatic amine metabolism also appeared to be very responsive to temperature: seedlings under a cold regime accumulated free dopamine and bound phenylethylamine and tyramine, whereas seedlings grown at high temperature accumulated free tyramine. The possible relationships between the observed amine patterns and seedling growth under low and high temperature are discussed.

Notes: The aim of this study was to evaluate how physiological processes of potted Pinus halepensis plants, grown under controlled conditions, were affected by ozone (O3) and/or water stress, integrating the gas exchange and biochemical data with fluorescence OJIP polyphasic transient data. Plants submitted to only water stress (T1) and with ozone (T3) showed a strong decrease in stomatal conductance and gas exchange, coinciding with a reduction of maximum yield of photochemistry (ϕpo) and very negative values of leaf water potential. Simultaneously, a great increase of both PSII antenna size, indicated by absorption per reaction centre, and electron transport per reaction centre were found. The reduction of photosynthesis in the O3-treated plants (T2) by a slowing down of the Calvin cycle was supported by the increase of related fluorescence parameters such as relative variable fluorescence, heat de-excitation constant, energy de-excitation by spillover, and the decrease of ϕpo. We suggest an antagonistic effect between the two stresses to explain the delayed ozone-induced decrease of stomatal conductance values for T3 with respect to T1 plants, by an alteration of the physiological mechanisms of stomatal opening, which involve the increase of intra-cellular free-calcium induced by ABA under co-occurring water shortage. We emphasise the importance of considering the intensity of the individual stress factor in studies concerning the interaction of stresses.

Notes: A simple new method, competitive hybridization, for identification of differentially regulated genes was used to isolate novel genes induced by ethylene in citrus (Citrus sinensis [L.] Osbeck cv. Shamouti) leaves. One of the isolated genes, an ethylene-induced esterase gene (EIE), was further characterized. The deduced protein sequence of this gene shows a similarity to those of several plant α/β hydrolase gene family members, which are known to be involved in secondary metabolism. Northern blot analysis demonstrated that EIE mRNA was induced by ethylene within 4 h and accumulated to a very high level 24 h after the initiation of ethylene treatment. Induction of EIE by ethylene could be counteracted by 1-methylcyclopropene, a potent ethylene perception inhibitor, indicating that the expression of EIE is ethylene-dependent. The bacterially expressed protein of EIE was recognized by antiserum against Pir7b, a naphthol AS esterase induced in rice by the non-host pathogen, Pseudomonas syringae pv. syringae. The EIE protein was identified in ethylene-treated leaves using anti-Pir7b antibodies. An α-naphthyl acetate esterase accumulated concomitantly with the increase in EIE protein in ethylene-treated citrus leaves. An enzyme activity assay followed by western analysis confirmed that the esterase was EIE.

Notes: In young cucumber seedlings, the peg is a polar outgrowth of tissue that functions by snagging the seed coat, thereby freeing the cotyledons. The development of the peg is thought to be gravity-dependent and has become a model system for plant-gravity response. Peg development requires rapid cell expansion, a process thought to be catalyzed by α-expansins, and thus was a good system to identify expansins that were regulated by gravity. This study identified 7 new α-expansin cDNAs from cucumber seedlings (Cucumis sativus L. cv Burpee Hybrid II) and examined their expression patterns. Two α-expansins (CsExp3 and CsExp4) were more highly expressed in the peg and the root. Earlier reports stated that pegs tend not to form in the absence of gravity, so the expression levels were compared in the pegs of seedlings grown in space (STS-95), on a clinostat, and on earth (1 g). Pegs were observed to form at high frequency on clinostat and space-grown seedlings, yet on clinostats there was more than a 4-fold reduction in the expression of CsExp3 in the pegs of seedlings grown on clinostats vs. those grown at 1 g, while the CsExp4 gene appeared to be turned off (below detection limits). There were no detectable differences in expansin gene expression levels for the pegs of seedlings grown in space or in the orbiter environmental simulator (OES) (1 g) at NASA. The microgravity environment did not affect the expression of CsExp3 or CsExp4, and the clinostat did not simulate the microgravity environment well.

Notes: Natural leaf senescence proceeds through an orderly program of events referred to, generally, as the ‘senescence syndrome’. Leaf senescence consists of primarily, but not exclusively, a set of degradative and remobilization activities that salvage valuable nutrients by reallocation to the seeds or other viable parts of the plant. The program requires changes in gene expression and eventually culminates in death of the leaf or whole plant. Leaf/whole plant senescence has now been scrutinized extensively using molecular genetic approaches and a clearer picture of the events that comprise the developmental program is beginning to emerge. However, while understandings of the phenomenological aspects of the program have become apparent, the mechanistic aspects, particularly with regard to the processes required for induction and regulation of the program, are still far from clear. Molecular evidence suggests the process is complex in terms of the wide array of genes and activities expressed, and in terms of the overall regulation of progression of the events of the syndrome. This article attempts to review our current understanding of leaf senescence and includes a brief discussion of aspects of the process that require clarification if we are to more fully understand this complex developmental program.

Notes: Recent research has shown that nodule nitrogen fixation is limited under a wide range of environmental constraints by lowered carbon flux within the nodule due to down-regulation of sucrose synthase activity. The aim of this work was to elucidate whether an increase in both carbon flux and activity of enzymes of carbon metabolism in nodules may lead to an increased nitrogen fixation. We report the effects caused by a continuous exposure to atmospheric CO2 enrichment in nodulated pea plants. CO2 enrichment led to an enhanced whole-plant growth and increased nodule biomass. Moreover, nodules of plants grown at increased CO2 showed a higher sugar content as well as enhancement of some activities related to nodule carbon metabolism, such as sucrose synthase, UDP glucose pyrophosphorylase and phosphoenolpyruvate carboxylase. Indeed, acetylene reduction activity, measured by the classical technique, was increased more than four times. However, when specific nitrogen fixation was determined as hydrogen evolution, no significant differences were detected, consistent with the lack of changes of enzymes involved in nitrogen metabolism such as glutamate synthase and aspartate aminotransferase. These results are discussed in the context of the regulation of nitrogen fixation and nodule metabolism.

Notes: Plant sterols differ from cholesterol in having an alkyl group at Δ-24, and, in the case of stigmasterol, also a Δ-22 double bond. The effects of 10 mol% of three plant sterols (campesterol, β-sitosterol, stigmasterol) and cholesterol on the molecular dynamics and phase behavior in multilamellar liposomes made from different phosphatidylcholine (PC) molecular species have been compared, utilizing the fluorescent probe Laurdan (2-dimethyl-amino-6-laurylnaphthalene). Laurdan reports the molecular mobility in the hydrophilic/hydrophobic interface of the membrane by determining the rate of dipolar relaxation of water molecules close to the glycerol backbone of PC. Our results showed that the Δ-24 alkyl group of plant sterols did not affect their ability to reduce molecular mobility in this region of the PC membranes. However, the plant sterols had a decreased capacity compared to cholesterol to inhibit formation of co-existing domains of gel and liquid-crystalline phases in membranes composed of equimolar dilauroyl-PC and dipalmitoyl-PC. The Δ-22 double bond present in stigmasterol decreased the ability of this sterol, compared to the other phytosterols, to reduce the molecular mobility at the hydrophobic/hydrophilic interface in membranes made of a saturated PC molecular species. However, in membranes made from 16:0/18:2-PC, a lipid species common in plant plasma membranes, stigmasterol was as efficient as other sterols in affecting the polarity and molecular mobility at the hydrophilic/hydrophobic interface of the membrane at 25°C, but was, in contrast to the other sterols, without effect at 0°C. Our results thus confirm as well as contradict the results of previous studies of the interactions between saturated PC and sterols, where other membrane regions were probed. The physiological relevance of the findings is discussed.

Notes: The leaves of trees emit significant amounts of acetaldehyde which is synthesized there by the oxidation of ethanol. In the present study, we examined plant internal and environmental factors controlling the emission of acetaldehyde by the leaves of young poplar (Populus tremula×P. alba) trees. The enzymes possibly involved in the oxidation of ethanol in the leaves of trees are catalase (CAT; EC 1.11.1.6) and alcohol dehydrogenase (ADH; EC 1.1.1.1), both expressed constitutively in the leaves of poplars. Inhibition of ADH in excised leaves caused a significant decrease of acetaldehyde emission accompanied by an increased ethanol emission. Since inhibition of CAT by aminotriazole did not affect acetaldehyde and ethanol emission, it is concluded that the oxidation of ethanol in the leaves is mediated by ADH rather than by CAT. Further studies indicated that aldehyde dehydrogenase (ALDH; EC 1.2.1.5) seems to be responsible for the oxidation of acetaldehyde. The present results demonstrate that acetaldehyde emission is clearly dependent on its production in the leaves as controlled by the delivery of ethanol to the leaves via the transpiration stream. Environmental factors that control stomatal conductance seem to be of less importance for acetaldehyde emission by the leaves.

Notes: The potential contribution of polyamines (PAs) in the regulation of physiologically induced fruitlet abscission was investigated in cuttings from two cultivars of Vitis vinifera L., Pinot noir (PN) and Merlot (MRT). Abscission was higher in MRT than in PN and was preceded by a decrease in free PA levels. This decline was more pronounced in inflorescences than in leaves of the sensitive cultivar. Soluble conjugated PA showed an opposite trend in both cultivars. This suggests a cause-effect relationship between free and/or conjugated PA levels in floral organs and susceptibility to abscission. Spermidine (Spd), but not putrescine (Put) or diaminopropane, supplied at 0.5–1 mM to the nutritive medium prior to the anthesis, increased free and conjugated PA levels in the inflorescences and markedly inhibited abscission. α-Difluoromethylarginine, an inhibitor of arginine decarboxylase, but not α-difluoromethylornithine, an inhibitor of ornithine decarboxylase, lowered PA levels and increased abscission. Treatment with cyclohexylamine or β-hydroxyethylhydrazine as potent inhibitors of Spd synthase and PA oxidases, respectively, reduced the Spd and/or spermine levels and enhanced free Put in the inflorescences, inducing an increased abscission of floral organs shortly after anthesis. These data suggest that PAs, particularly Spd, could be involved in the regulation of grapevine fruitlet physiological abscission.

Notes: We investigated the role of non-photochemical energy quenching (NPQ) in cold acclimation in potato. We first analyzed the expression of the PsbS gene, which encodes a PSII subunit involved in NPQ, during chilling treatment in two potato species, a cold-tolerant Solanum sogarandinum and a cold-sensitive Solanum tuberosum (cv. Cisa). In in vitro plantlets, a transient transcript accumulation was observed after 1 h in the light at room temperature in both species, and this light-induced PsbS transcript accumulation was strongly amplified at 4°C. Nuclear run-off transcription experiments indicated that this increase likely originates from a higher transcriptional activity of PsbS gene. In phytotron-grown plants, chilling treatment was shown also to result in a substantial increase in PsbS mRNA level. However, no change in protein abundance was noticed in either Solanum species. PSII photochemistry and photosynthetic electron transport were severely decreased in S. tuberosum plants at low temperature, while both activities were only slightly affected in S. sogarandinum. NPQ was substantially reduced in both species during chilling stress. These results indicate that neither PsbS nor NPQ are involved in acclimation of S. sogarandinum to low temperature. In contrast, the level of two other plastid proteins, one related to thioredoxins, CDSP32, and the other homologous to plastid lipid-associated proteins, CDSP34 (for chloroplastic drought-induced stress proteins of 32 and 34 kDa, respectively), was higher at low temperature in the cold-tolerant species. This result is discussed in relation to the potential roles of CDSPs in the protection of photosynthetic structures.

Notes: Glycine-rich proteins (GRPs) are members of a family of proteins with unique repeats of glycine-containing motifs. This feature facilitates their association with plant structural components such as cell wall and vascular tissues. A transcript encoding a new member (LeGRP1) of the GRP family was identified from roots of tomato by the method of differential display of mRNA species. The cDNA is 1.15 kb in size and has an open reading frame encoding a peptide of 284 amino acids. Glycine residues make up 55% of the deduced peptide and the sequence is characterized by repeating (Gly-X)n domains. The presence of a hydrophobic N-terminus with a putative signal peptide indicates a probable association of the protein with the cell wall. Southern analysis shows that several LeGRP1 homologues are present in the genome. The LeGRP1 transcript accumulates predominantly in the roots, stem and petioles of tomato. A relatively lower level of LeGRP1 message was observed in the leaf mid-rib, while the message is undetectable in the leaf lamina. Abiotic stresses like cold, heat, salt, desiccation and phosphate starvation did not significantly affect the expression of LeGRP1, whereas wounding resulted in a significant repression of the gene expression. Temporal regulation of LeGRP1 was observed during fruit development.

Notes: Two cDNAs, designated MWPL1 and MWPL2, encoding putative pectate lyases (Pel; EC 4.2.2.2), which catalyze the cleavage by β-elimination of α-1→4-linked galacturonosyl residues of pectins found mostly in middle lamella and primary cell wall in plants, were isolated from ripening fruit of banana (Musa acuminata) and their expressions in fruit during ripening and in response to ethylene were investigated. MWPL1 and MWPL2 encode a single polypeptide of 407 and 454 amino acid residues, respectively. The two cDNAs shared an overall identity of 75% in both nucleotide and deduced amino acid sequences. Sequence comparison of MWPL1 and other plant Pels revealed the homology ranging from 76% with zinnia to 48% with ragweed. Southern analysis indicated that MWPL1 might be present as a single copy gene, and there might be up to two copies of MWPL2 in the banana genome. The two cDNAs were expressed differentially and/or spatially in various banana organs, with female flower and fruit tissues showing accumulation of the MWPL2 transcript, which was not detected in root, pseudostem, leaf, male flower and ovary, whereas the MWPL1 transcript was not detectable in all organs tested. In fruit tissue during ripening, although transcripts of both members were not detectable in unripe preclimacteric fruits, they began to accumulate as ripening progressed and the level remained high thereafter in overripe fruits. However, the magnitude of transcript accumulation differed between the two Pel members, with substantially more abundant MWPL2 than MWPL1 in ripening fruit. Similar differential transcript accumulation was also observed between peel and pulp, where the former was markedly higher than the latter. Expression of both Pel members was also affected by exogenous ethylene, whose presence at 5–100 ppm stimulated accumulation of MWPL1 and MWPL2 transcripts in preclimacteric fruit, suggesting that ethylene may play an important regulatory role in regulating Pel expression during fruit ripening of the banana.

Notes: We studied the effect of three factors on the induction of flowering in Arabidopsis thaliana, i.e. vernalization, day length and DNA demethylation. Seven natural late flowering genotypes and 13 late flowering mutants were used in the experiments. The effect of the vernalization and the short day (SD) was uniform in all genotypes used, resulting in shortening (vernalization) or extension of the period before the appearance of the first flower primordia. On the other hand, the effect of the demethylating agent (5-azacytidine [5-azaC]) was not uniform in the genotypes used. In all natural late genotypes (except Lu-1), the shortening of the flowering time (FT) after 5-azaC treatment was observed. On the contrary, only five mutants –dl, pm, M63, M73 and fca-1– showed a shortening of the FT, while in the majority of the late flowering mutants, no significant response (earlier flowering) was found. The different response to the vernalization and demethylation treatment in late flowering mutants shows the possibility of two different pathways leading to the flowering, both of which are regulated by DNA demethylation. The different response of natural and induced late flowering genotypes after 5-azaC treatment shows that genes that play a role in flower development are of a different nature.

Notes: In vivo leaf characteristics were examined to describe longitudinal gradients of UV-absorbing screening pigments in barley. Chlorophyll fluorescence properties and in vivo absorption spectra (210–750 nm) of leaves were measured from the base to the tip. Barley leaves showed strong longitudinal gradients of chlorophyll, where chlorophyll concentration increased within the first 5–8 cm from the leaf base, and did not significantly change for the remaining part of the leaf. Fluorescence microscopy was used to localize cell wall bound screening pigments different from flavonoids, since flavonoids lack a blue-green fluorescence emission (Lichtenthaler and Schweiger 1998). Measurements of in vivo chlorophyll fluorescence indicated that the ratio of UV-absorbing screening pigments per leaf area increases from the leaf base to the tip. These gradients were confirmed by in vivo absorption spectra. It is demonstrated that leaves in the early stage of development are less protected against UV-radiation than fully developed mature leaf regions. The experiments show that measurements of in vivo chlorophyll fluorescence are ideally suited as a fast non-invasive tool to estimate the epidermal UV-transmittance in different leaf sections.

Notes: Monoterpene synthase activities were measured in current year and 1-year-old leaves of holm oak (Quercus ilex L.). The monoterpene synthase activities of the leaves strongly changed with leaf development and leaf age. Enzyme activities increased rapidly in spring after leaf emergence, reaching maximum values in summer, which declined during the following winter period. In the next spring monoterpene synthase activities recovered in the old leaves to about one-third of values in the previous years and showed a similar seasonal variation as in young leaves. In both leaf age classes the pattern of enzymatic monoterpene formation was stable with α-pinene (33%), β-pinene (28%), and myrcene (26%) as prominent compounds followed by minor fractions of sabinene (10%) and limonene (3%). Monoterpene emission correlated with the activity of the synthetizing enzymes, indicating that monoterpene synthase activities in Q. ilex reflect the seasonal monoterpene emission potential of the leaves.

Notes: Galactan: galactan galactosyltransferase (GGT), an enzyme involved in the biosynthesis of the long-chain raffinose family of oligosaccharides (RFOs) in Ajuga reptans, catalyses the transfer of an α-galactosyl residue from one molecule of RFO to another one resulting in the next higher RFO oligomer. This novel galactinol (α-galactosyl-myo-inositol)-independent α-galactosyltransferase is responsible for the accumulation of long-chain RFOs in vivo. Warm treatment (20°C) of excised leaves resulted in a 34-fold increase of RFO concentration and a 200-fold increase of GGT activity after 28 days. Cold treatment (10°C/3°C day/night) resulted in a 26- and 130-fold increase, respectively. These data support the role of GGT as a key enzyme in the synthesis and accumulation of long-chain RFOs. GGT was purified from leaves in a 4-step procedure which involved fractionated precipitation with ammonium sulphate as well as lectin affinity, anion exchange, and size-exclusion chromatography and resulted in a 200-fold purification. Purified GGT had an isoelectric point of 4.7, a pH optimum around 5, and its transferase reaction displayed saturable concentration dependence for both raffinose (Km = 42 mM) and stachyose (Km = 58 mM). GGT is a glycoprotein with a 10% glycan portion. The native molecular mass was 212 kDa as determined by size-exclusion chromatography. Purified GGT showed one single active band after native PAGE or IEF separation, respectively, which separated into three bands on SDS-PAGE at 48 kDa, 66 kDa, and 60 kDa. The amino acid sequence of four tryptic peptides obtained from the major 48-kDa band showed a high homology to plant α-galactosidase (EC 3.2.1.22) sequences. GGT differed, however, in its substrate specificity from α-galactosidases; it neither hydrolysed nor transferred α-galactosyl-groups from melibiose, galactinol, UDP-galactose, manninotriose, and manninotetrose. Galactinol, sucrose, and galactose inhibited the GGT reaction considerably at 10–50 mM.

Notes: Stepwise selection was carried out with increasing glyphosate concentrations to produce suspension cultures of Medicago sativa L. (alfalfa), Glycine max L. (Merr.) (soybean) and Nicotiana tabacum L. (tobacco) (two lines) that were at least 100-fold more resistant than the original culture as measured by the I50. The selection process required from 8 to 11 transfers to fresh medium over a total period from 161 to 312 days. The alfalfa and soybean lines contained 62- and 21-fold higher activity levels of the glyphosate target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), respectively. The tobacco lines had EPSPS enzyme activity levels more than 800-times higher than the original cultures. The EPSPS gene copy number and mRNA were increased in all of the lines as measured by southern and northern hybridization, respectively. Thus, as has been found before with most glyphosate-resistant suspension cultures, the resistance is caused by high EPSPS enzyme activity due to EPSPS gene amplification. Alfalfa and soybean EPSPS gene amplification and the very high EPSPS enzyme activity increases found in the tobacco cultures have not been reported before. These studies show that EPSPS gene amplification can occur in many plant species to confer glyphosate tolerance.

Notes: In order to better understand control of the mitochondrial pyruvate dehydrogenase complex (PDC), total catalytic activity was determined during development of the primary leaves of pea (Pisum sativum L.) seedlings, as well as in each leaf pair of 21-day-old plants. Activity of the PDC in clarified homogenates was highest in the youngest organs and then dropped dramatically as the leaves matured and became photosynthetically competent. As leaves began to senesce, total PDC activity dropped to zero. Steady-state mRNA levels were determined using E1 and E3 cDNA probes. The overall pattern of transcript abundance matched the pattern observed for total PDC activity; transcript levels for E1α and E1β approached zero during senescence. Levels of the E1α, E1β, E2 and E3 subunits of the PDC were analyzed in the same samples, using specific antibodies. Quantitation of the immunoblotting results throughout this developmental series showed a pattern in parallel with that of catalytic activity and mRNA levels, although the relative changes in subunit protein levels were not as extreme as the changes in activity. The exception to the global pattern was that of the E3 subunit: lipoamide dehydrogenase. Expression of this enzyme was highest in mature, fully expanded leaves, which were active in photosynthesis and photorespiration, reflecting the additional role of E3 as a component of glycine decarboxylase.

Notes: The present work examines the requirement for RGD-binding sites, such as those in the plasma membrane protein integrin during somatic embryogenesis in carrot (Daucus carota L. cv. Scarlet nantes). It is possible to assay for this requirement by competitively inhibiting binding of the site to the natural RGD-containing ligand by adding an excess of synthetic RGDS. We found that treatment of carrot callus cultures with RGDS (2.7 mM) inhibited the development of a normal shoot pole in carrot somatic embryos. The structures that formed contained separate zones of highly cytoplasmic and vacuolate cells and no evidence of embryonic organization, although occasionally a root-like structure was observed. If the aspartic acid residue in the peptide was replaced by a chemically similar amino acid (glutamic acid), the resultant somatic embryos were indistinguishable from those developing in untreated cultures. These effects are similar to those reported in animal systems where the protein receptor involved has been identified as integrin. Our results are thus consistent with the conclusion that a binding site for a motif similar to the integrin-binding site in a variety of animal extracellular matrix proteins exists in plants and appears to be important in somatic embryo development in carrot.

Notes: Oil bodies are lipid storage organelles which have been analyzed biochemically due to the economic importance of oil seeds. Although oil bodies are structurally simple, the mechanisms involved in their formation and degradation remain controversial. At present, only two proteins associated with oil bodies have been described, oleosin and caleosin. Oleosin is thought to be important for oil body stabilization in the cytosol, although neither the structure nor the function of oleosin has been fully elucidated. Even less is known about caleosin, which has only recently been described [Chen et al. (1999) Plant Cell Physiol 40: 1079–1086; Næsted et al. (2000) Plant Mol Biol 44: 463–476]. Caleosin and caleosin-like proteins are not unique to oil bodies and are associated with an endoplasmatic reticulum subdomain in some cell types. Here we review the synthesis and degradation of oil bodies as they relate to structural and functional aspects of oleosin and caleosin.

Notes: When grown in darkness the photomorphogenic lip1 mutant of pea (Pisum sativum L.) has a slender stem, expanded leaves, prolamellar body (PLB) lacking plastids with the size of chloroplasts and a low level of phytochrome A. The lack of PLBs in a dark-grown material (lip1) created a possibility to further study the regulation of their formation in relation to plant development. Inclusion of a cytokinin, 2-isopentenyladenine (2iP), in a medium supporting growth of the pea seedlings in darkness was found to reduce epicotyl length in the wild type. In lip1 the formation of a slender stem was inhibited and a short epicotyl developed. Furthermore, leaf expansion was inhibited, the plastid size reduced and the formation of PLBs induced. The PLB formation in lip1 was not accompanied by an increase in the amount of protochlorophyllide (Pchlide) or Pchilde oxidoreductase (POR). In the presence of 2iP the level of phytochrome A protein was increased in lip1 and the POR mRNA levels decreased in both lip1 and wild-type plants. The chloroplast characteristic trans-3-hexadecenoate acyl group of phosphatidylglycerol, present in the plastids of dark-grown lip1, was not influenced by 2iP. Thus, not all photomorphogenic processes reacted similarly in the lip1 mutant, but leaf expansion and plastid differentiation, including PLB formation, seemed to be regulated by the same signal transduction chain. Exogenously applied brassinolide could rescue neither dark- nor light-grown defects of the lip1 mutant. Thus, cytokinins but not brassinolides seem to be involved in the regulation of certain characteristic traits of skotomorphogenesis in pea, including plastid development and PLB formation.

Notes: Changes in the protein level and phosphorylation state of sucrose synthase (SS) were studied throughout the development of Japanese pear fruit. The level of SS protein was high at the young stage, dropped with fruit enlargement and increased again with fruit maturation. Antibody against phospho-Ser reacted with SS from young fruit, but did not react with SS that had been dephosphorylated by alkaline phosphatase (AP). The activities of SS isozymes were separated by ion-exchange chromatography. It was found that the fluctuation in SS activity was caused by two SS isozymes (SSI and SSII); (SSI reacted with antibody against phospho-Ser, while SSII did not. Phosphorylation of SS affected its kinetic parameters, that is, the affinity of phosphorylated SS for UDP was higher than that of dephosphorylated SS, while it was the contrary for UDP-glucose. The reaction of dephosphorylated SS was inclined toward sucrose synthesis more than that of phosphorylated SS. Phosphorylated SS protein was most abundant in young fruit, but decreased with fruit development, while non-phosphorylated SS protein increased in mature fruit. These results suggest that SS isoforms may be affected by post-translational modifications such as phosphorylation, and that the regulation of phosphorylation may potentially control the properties and functions of SS throughout the development of Japanese pear fruit.

Notes: Exposure of Galega orientalis plants to diamines putrescine (Put) and cadaverine (Cad) at concentrations from 0.01 to 2.0 mM significantly altered carbon and nitrogen metabolism in their root nodules. Correlative studies of bacteroid poly-β-hydroxybutyrate (PHB) content and acetylene-reduction capacity of the nodules revealed a negative relationship between these parameters. Utilisation of PHB deposits by bacteroids and high acetylene reduction activity was observed when applying low diamine concentrations. The increase in PHB accumulation in response to high diamine levels was accompanied by a considerable decline in nodule nitrogenase activity. Supplying isolated Galega bacteroids with various diamine concentrations significantly modified bacteroid oxygen consumption, which might be associated with alterations in carbon flux to the bacteroids. Finally, modulation of the bacteroid content upon Put and Cad treatment was examined. The results are discussed in terms of possible causes of the diamine-induced changes in nodule metabolism.

Notes: Glutathione is an important component of the ascorbate-glutathione cycle, which is involved in the regulation of hydrogen peroxide (H2O2) concentrations in plants. During chilling and cold acclimation, i.e. exposure to temperatures between 0 and 15°C, H2O2 may accumulate. Excess electrons from the photosynthetic and respiratory electron transport chains can be used for the reduction of oxygen, thus producing superoxide radicals (O2⋅−); these are subsequently transformed to H2O2 via superoxide dismutase (SOD; EC 1.15.1.1). During the removal of excess H2O2, reduced glutathione (GSH) is converted to its oxidised form (GSSG), and GSH is regenerated by the activity of NADPH-dependent glutathione reductase (GR; EC 1.6.4.2). At low non-freezing temperatures, high GSH content and GR activity were detected in several plant species, indicating a possible contribution to chilling tolerance and cold acclimation. Changes in H2O2 concentration and GSH/GSSG ratio alter the redox state of the cells and may activate special defence mechanisms through a redox signalling chain. The finding that several defence genes have antioxidant responsive elements or GSSG binding sites in their regulatory regions supports the idea that redox signalling is involved in regulating gene expression in response to low temperature.

Notes: A genomic clone encoding a thiohydroximate S-glucosyltransferase (S-GT) was isolated from Brassica napus by library screening with probes generated by PCR using degenerated primers. Its corresponding cDNA was amplified by rapid amplification of cDNA ends (RACE) PCR and also cloned by cDNA library screening. The genomic clone was 5 896 bp long and contained a 173-bp intron. At least two copies of the S-GT gene were present in B. napus. The full-length cDNA clone was 1.5 kb long and contained an open reading frame encoding a 51-kDa polypeptide. The deduced amino acid sequence shared a significant degree of homology with other glucosyltransferases characterized in other species, including a highly conserved motif within this family of enzymes corresponding to the glucose-binding domain. The recombinant protein was expressed in Escherichia coli, and the enzyme activity was tested by a biochemical assay based on the measure of glucose incorporation. The high thiohydroximate S-GT activity detected from the recombinant protein confirmed that this clone was indeed a S-glucosyltransferase.

Notes: The monophenolase activity of Terfezia claveryi tyrosinase (EC 1.14.18.1) is described for the first time. This enzyme is fully latent and can only be detected if SDS is present in the reaction medium. Monophenolase activity was localized within the ascocarp using histochemical techniques. A detailed kinetic study of the parameters affecting this activity has been carried out. Both the characteristic lag period and the steady-state rate are affected by pH and the enzyme and substrate concentrations. The presence of catalytic concentrations of o-diphenols affected the lag period but not the steady-state rate. By increasing the concentration of o-diphenols, it was possible to evaluate the enzyme activation constant, Kact, which showed a value of 7.2 μM. The experimental results are compatible with the mechanism previously described for tyrosinases from other sources.

Notes: We studied copper uptake in inside-out plasma membrane vesicles derived from roots of copper-sensitive, moderately copper-tolerant and highly copper-tolerant populations of Silene vulgaris (Amsterdam, Marsberg and Imsbach, respectively). Plasma membrane vesicles were isolated using the two-phase partitioning method and copper efflux was measured using direct filtration experiments. Vesicles derived from Imsbach plants accumulated two and three times more copper than those derived from Marsberg and Amsterdam plants, respectively. This accumulation was ATP-dependent. Also, 9-amino-6-chloro-2-methoxyacridine fluorescence quenching rates upon copper addition decreased in the order Imsbach〉Marsberg〉Amsterdam. Our results support the hypothesis that efflux of copper across the root plasma membrane plays a role in the copper tolerance mechanism in S. vulgaris.

Notes: When rooted cuttings of Corylus maxima Mill. cv. Purpurea are moved from the wet and humid conditions of the rooting environment, the leaves frequently shrivel and die. Since the newly formed adventitious root system has been shown to be functional in supplying water to the shoot, stomatal behaviour in C. maxima was investigated in relation to the failure to prevent desiccation. Stomatal conductance (gs) in expanding leaves (L3) of cuttings increased almost 10-fold over the first 14 days in the rooting environment (fog), from 70 to 650 mmol m−2 s−1. In contrast, gs of expanded leaves (L1) changed little and was in the region of 300 mmol m−2 s−1. Midday leaf water potential was much higher in cuttings than in leaves on the mother stock-plant (−0.5 versus −1.2 MPa) even before any roots were visible. Despite this, leaf expansion of L3 was inhibited by 〉50% in cuttings and stomata showed a gradual reduction in their ability to close in response to abscisic acid (ABA). To determine whether the loss of stomatal function in cuttings was due to severance or to unnaturally low vapour pressure deficit and wetting in fog, intact plants were placed alongside cuttings in the rooting environment. The intact plants displayed reductions in leaf expansion and in the ability of stomata to close in response to dark, desiccation and ABA. However, in cuttings, the additional effect of severance resulted in smaller leaves than in intact plants and more severe reduction in stomatal closure, which was associated with a 2.5-fold increase in stomatal density and distinctively rounded stomatal pores. The similarities between stomatal dysfunction in C. maxima and that observed in many species propagated in vitro are discussed, as is the possible mechanism of dysfunction.

Notes: Gravicurvature in water- and auxin (IAA)-incubated coleoptiles of rye (Secale cereale L.) is similar, despite a general strongly enhancing effect of exogenous IAA on the overall (cell) elongation of these organs. Longitudinally split coleoptiles or isolated longitudinally halved coleoptiles (horizontally positioned as upper or lower halves) respond gravitropically in the same way as water-incubated intact coleoptiles, irrespective of whether the halves are incubated in distilled water or IAA. A new model for the principal mechanism of regulation of gravitropic growth is proposed which depends on, yet does not involve, the redistribution of IAA as the means for gravistimulated differential growth, as postulated by the Cholodny-Went hypothesis (CWH). It is based on a gravimediated temporarily restrained infiltration of IAA-induced wall-loosening factors into the growth-limiting outer epidermal walls of the concave organ flank.

Notes: The effects of root application of brassinolide (BL) on the growth and development of Arabidopsis plants (Arabidopsis thaliana ecotype Columbia [L.] Heynh) were evaluated. Initially, all leaves were evaluated on plants 18, 22, 26 and 29 days old. The younger leaves were found to exhibit maximal petiole elongation and upward leaf bending in response to BL treatment. Therefore, based on these results leaves 6, 7 and 8 on 22–24-day-old plants were selected for all subsequent studies. Elongation along the length of the petiole in response to BL treatment was uniform with the exception of an approximately 4 mm region next to the leaf where upward curvature was observed. Both BL and 24-epibrassinolide (24-epiBL) were evaluated, with BL being more effective at lower concentrations than 24-epiBL. The exaggerated growth induced by 0.1 μM BL was not observed in plants treated with 1 000-fold higher concentrations of GA3, IAA, NAA or 2,4-D (100 μM). In addition, no exaggerated growth effects were observed when plants were treated with 200 ppm ethylene or 1 mM ACC. All treatments with BL, NAA, 2,4-D, IAA or ACC promoted ethylene and ACC production in wild type Arabidopsis plants, but only BL triggered exaggerated plant growth. BL also promoted exaggerated growth and elevated levels of ACC and ethylene in the ethylene insensitive mutant etr1-3, showing that the effect of BR on growth is independent of ethylene. This work provides evidence that BR-induced exaggerated growth of Arabidopsis plants is independent of gibberellins, auxins and ethylene.

Notes: To elicit the roles of cytokinins in the regulation of maturation of Pinus radiata D. Don, the spectrum of endogenous cytokinins and their concentration in the mature buds were analysed using double-solvent extraction, column complex purification and separation, a novel immunoaffinity purification method, normal and reverse phase high-pressure liquid chromatography, enzymatic treatment, radioimmunoassay and electrospray MS/MS spectrometry. We have isolated two novel cytokinin glycosides whose proposed structures are isopentenyladenine-9-(glucopyranosyl riboside), dihydrozeatin-9-(glucopyranosyl riboside) and confirmed the presence of zeatin-9-(glucopyranosyl riboside). We have also found the presence of novel phosphorylated forms of these 3 cytokinin ribosyl-linked glycosides. Quantitative analyses revealed that the cytokinin ribosyl-linked glycosides predominate in P. radiata mature buds. Although cytokinin free base, riboside and nucleotide forms are also present, we could find no evidence of the traditional cytokinin O- or N-glucosides in the conifer buds. Thus, cytokinin metabolism in mature buds of P. radiata is very different from other species previously examined.

Notes: Graft compatibility has been studied in vitro using callus tissues of apricot (Prunus armeniaca) and different Prunus rootstocks to form scion/rootstock combinations with different degrees of graft compatibility. In these species, incompatibility is manifested by a breakdown of the trees at the union area that can occur some years after grafting. Here, the possibility of obtaining an early detection method to determine graft incompatibility is explored by callus fusion in vitro. The adhesion of the two callus partners, the development of the cells at the contact surface (cell arrangement, intensity of cell-wall staining), and the presence of lipid and phenolic compounds have been studied during the first 3 weeks after grafting in both compatible and incompatible combinations. Differences were observed at the second and the third week of callus co-culture in most of the characters determined, although these differences were present as early as the first week in the case of phenolic compounds. The behaviour of the grafts grown in vitro was correlated to that of the same combinations in the field, suggesting that callus fusion in vitro could be a possible and reliable method for an early detection of graft incompatibility in different Prunus combinations.

Notes: To clarify the causal factors for ploidy variation in plant cell culture, we attempted to alter ploidy distribution in cell cultures of a tetraploid cultivar of Doritaenopsis by changing the plant growth regulators (PGRs) in the culture medium. The original suspension cultured cells, which had been maintained in medium containing 0.1 mg l−1 1-naphthaleneacetic acid and 1 mg l−1 benzyladenine, were transferred onto various gellan gum solidified media with a single application of PGRs, and the ploidy distributions of the cells were examined using flow cytometry analysis during 3 weeks of culture. Among the PGRs tested, 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid caused a drastic reduction in the 4C-cell proportion in cell cultures with an increased cell proportion of 8C or higher C-values. In the case of 2,4-D application, a reduction of cell viability was observed. A decreasing proportion was also observed in the 8C-cell population accumulated by 2,4-D treatment, following transfer back to the medium containing the standard PGR composition. These results suggest that the exogenous application of 2,4-D arrested the cell cycle at G2 phase in the Doritaenopsis cells, and the removal of 2,4-D might induce further endoreduplication or recover the mitotic cycle of the G2-arrested cells.

Notes: Research on plant nitrogen (N) uptake and metabolism has more or less exclusively concerned inorganic N, particularly nitrate. Nevertheless, recent as well as older studies indicate that plants may have access to organic N sources. Laboratory studies have shown that ectomycorrhizal and ericoid mycorrhizal plants can degrade polymeric N and absorb the resulting products. Recent studies have also shown that some non-mycorrhizal plants are able to absorb amino acids. Moreover, amino acid transporters have been shown to be present in both plant roots and in mycorrhizal hyphae. Although both mycorrhizal and non-mycorrhizal plants appear to have a capacity for absorbing a range of organic N compounds, is this capacity realized in the field? Several lines of evidence show that plants are outcompeted by microorganisms for organic N sources. Such studies, however, have not addressed the issue of spatial and temporal separation between plants and microorganisms. Moreover, competition studies have not been able to separate uptake by symbiotic and non-symbiotic microorganisms. Qualitative assessment of organic N uptake by plants has been performed with dual-labelled glycine in several studies. These studies arrive at different conclusions: some indicate that plants do not absorb this organic N source when competing with other organisms in soil, while others conclude that significant fractions of amino acid N are absorbed as intact amino acid. These variable results may reflect species differences in the ability to absorb glycine as well as differences in experimental conditions and analytical techniques. Although theoretical calculations indicate that organic N might add significant amounts of N to plant N uptake, direct quantitative assessment of the fraction of plant N derived from uptake by organic N sources is a challenge for future research.

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: Application experiments have suggested that short-day-induced cessation of elongation growth in trees is caused by photoperiodic regulation of the conversion of gibberellin GA19 to GA20. In the present study we examined further the photoperiodic control of GA metabolism in trees with focus on the conversion of GA19 in Salix pentandra, hybrid aspen (Populus tremula × tremuloides) and silver birch (Betula pendula) using [17,17-2H2]-GA19 or unlabelled GAs in application studies. GA20 and GA1 were able to restore growth also in hybrid aspen and silver birch under short days (SD), whereas GA19 had no or only a very small activity. Contrary to hybrid aspen and S. pentandra, the activity of GA20 in silver birch was significantly lower than that of GA1. Gas chromatography-mass spectrometry (GC-MS) analysis revealed a smaller turnover of [2H2]-GA19 in SD than in long days (LD) in hybrid aspen. No such difference in turnover of [2H2]-GA19 was observed in photoperiod-insensitive hybrid aspen overexpressing PHYA. Application of unlabelled GAs to seedlings of S. pentandra, hybrid aspen and silver birch under SD followed by quantification of metabolites by GC-MS analysis, showed that applied GA19 was not readily converted to GA20 and GA1. Although the sensitivity to GAs is also known to decrease under SD, the present data are in favour of a photoperiodic regulation of the metabolism of GA19in vivo in the woody species S. pentandra, hybrid aspen and silver birch. The data might also suggest that silver birch differs from S. pentandra and hybrid aspen by exhibiting a possible photoperiodic control also of the conversion of GA20 to GA1.

Notes: Inhibition of nitrogenase (EC 1.18.6.1) activity by O2 has been suggested to be an early response to disturbance in carbon supply to root nodules in the Frankia-Alnus incana symbiosis. Intact nodulated root systems of plants kept in prolonged darkness of 22 h were used to test responses to O2 and short-term N2 deprivation (1 h in Ar:O2). By using a Frankia lacking uptake hydrogenase it was possible to follow nitrogenase activity over time as H2 evolution in a gas exchange system. Respiration was simultaneously recorded as CO2 evolution. Dark-treated plants had lower initial nitrogenase activity in N2:O2 (68% of controls), which declined further during a 1-h period in the assay system in N2:O2 at 21 and 17% O2, but not at 13% O2. When dark-treated plants were deprived of N2 at 21 and 17% O2 nitrogenase activity declined rapidly to 61 and 74%, respectively, after 20 min, compared with control plants continuously kept in their normal light regime. In contrast, there was no decline in dark-treated plants at 13% O2, and only a smaller and temporary decline in control plants at 21% O2. When dark-treated plants were kept at 21% O2 during 45 min prior to N2 deprivation at 17% O2 the decline was abolished. This supports the idea that the decline in nitrogenase activity observed in N2:O2 at 21% O2 and during N2 deprivation was caused by O2, which affected a sensitive nodule fraction. Nodule contents of the amino acids Gln and Cit decreased during N2 deprivation, suggesting decreased assimilation of NH4+. Contents of ATP and ADP in nodules were not affected by short-term N2 deprivation. ATP/ADP ratios were about 5 indicating a highly aerobic metabolism in the root nodule. We conclude that nitrogenase activity of Alnus plants exposed to prolonged darkness becomes more sensitive to inactivation by O2. It seemed that dark-treated plants could not adjust their nodule metabolism at higher perceived pO2 and during cessation of NH4+ production.

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: Chlorophyll (Chl) and total soluble protein decreased and proteolytic activity increased over a 12-day period during dark-induced senescence in detached leaves of Tara, a yellowing cultivar (Y) of Dendranthema grandiflora. In Boaldi, a non-yellowing cultivar (NY), Chl and soluble protein remained near initial levels and little change in proteolytic activity was observed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of soluble proteins showed no major differences in banding patterns between the two cultivars at day 0; however, all of the resolved proteins were diminished in Tara by day 12. On the other hand, in NY Boaldi, the intensity of the protein bands did not change over the 12-day period. Attached and detached leaves exhibited similar senescence patterns for each cultivar. Ethylene (100 μl l−1) accelerated the rate of Chl loss in detached leaves of Tara, but had no effect on Boaldi. These observations suggest that Boaldi is a stay-green genotype, possibly a functional type. The results are discussed in relation to the role of ethylene in chrysanthemum leaf senescence.

Notes: Production of transgenic plants started more than a decade ago, but it is still a time-consuming operation. One of the critical points is the selection procedure used for the recovery of transgenic shoots after transformation. Moreover, as more transgenic traits are to be incorporated into crops that already have been transformed, it is clear that there is a need for new methods with higher efficiencies. In this article, recently developed selection systems are reviewed. They differ from conventional selection techniques as they are based on supplementing the transgenic cells with a metabolic advantage rather than killing the non-transgenic cells. In many cases, these new selection systems have been found to be superior to conventional methods.

Notes: The role of gibberellins (GAs) in the induction of parthenocarpic fruit-set and growth by the pat-3/pat-4 genetic system in tomato (Lycopersicon esculentum Mill.) was investigated using wild type (WT; Cuarenteno) and a near-isogenic line derived from the German line RP75/59 (the source of pat-3/pat-4 parthenocarpy). Unpollinated WT ovaries degenerated but GA3 application induced parthenocarpic fruit growth. On the contrary, parthenocarpic growth of pat-3/pat-4 fruits, which occurs in the absence of pollination and hormone treatment, was not affected by applied GA3. Unpollinated pat-3/pat-4 fruit growth was negated by paclobutrazol, an inhibitor of ent-kaurene oxidase, and this inhibitory effect was negated by GA3. The quantification of the main GAs of the early 13-hydroxylation pathway (GA1, GA8, GA19, GA20, GA29 and GA44) in unpollinated ovaries at 3 developmental stages (flower bud, FB; pre-anthesis, PR; and anthesis, AN), by gas chromatography-selected ion monitoring, showed that the concentration of most of them was higher in pat-3/pat-4 than in WT ovaries at PR and AN stages. The concentration of GA1, suggested previously to be the active GA in tomate, was 2–4 times higher. Unpollinated pat-3/pat-4 ovaries at FB, PR and AN stages also contained relatively high amounts (5–12 ng g−1) of GA3, a GA found at less than 0.5 ng g−1 in WT ovaries. It is concluded that the mutations pat-3/pat-4 may induce natural facultative parthenocarpy capacity in tomato by increasing the concentration of GA1 and GA3 in the ovaries before pollination.

Notes: It has been suggested that the function of the chloroplast-localized small heat shock protein (sHsp) is to protect photosystem II (PSII) from heat inactivation. This paper reports that addition of purified sHsp protein to isolated thylakoid membranes gave no protection of PSII and questions that there is any direct effect of the sHsp on PSII. The opinion is forwarded that the primary role for the chloroplast-localized sHsp may not even be protection of PSII.

Notes: A novel membrane lipoxygenase (LOX; EC 1.13.11.12) from eggplant (Solanum melongena L. cv. Belleza negra) fruit chloroplasts has been purified 20-fold to a specific activity of 207 enzymatic units per mg of protein with a yield of 72%. The purification was carried out by sonicating the chloroplastic membranes in the presence of Triton X-114 followed by phase partitioning and anion exchange chromatography. The purified membrane LOX preparation consisted of a single major band with an apparent molecular mass of 97 kDa after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results obtained using intact chloroplasts indicate that the enzyme is not localized in the stroma. When the enzyme reacts with linoleic acid, it produces a single peak, which comigrates with standard 9-hydroperoxy-octadecadienoic acid. A physiological role for this chloroplastic LOX is proposed.

Notes: Pyruvate kinase (PK, EC 2.7.1.40) was partially purified from the plant cytosolic fraction of N2-fixing soybean (Glycine max [L.] Merr.) root nodules. The partially purified PK preparation was completely free of contamination by phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), the other major phosphoenolpyruvate (PEP)-utilizing enzyme in legume root nodules. Latency experiments with sonicated nodule extracts showed that Bradyrhizobium japonicum bacteroids do not express either PK or PEPC activity in symbiosis. In contrast, free-living B. japonicum bacteria expressed PK activity, but not PEPC activity. Antibodies specific for the cytosolic isoform of PK from castor bean endosperm cross-reacted with a 52-kDa polypeptide in the partially purified PK preparation. At the optimal assay pH (pH 8.0 for PEPC and pH 6.9 for PK) and in the absence of malate, PEPC activity in crude nodule extracts was 2.6 times the corresponding PK activity. This would tend to favour PEP metabolism by PEPC over PEP metabolism by PK. However, at pH 7.0 in the presence of 5 mM malate, PEPC activity was strongly inhibited, but PK activity was unaffected. Thus, we propose that PK and PEPC activity in legume root nodules may be coordinately regulated by fluctuations in malate concentration in the plant cytosolic fraction of the bacteroid-containing cells. Reduced uptake of malate by the bacteroids, as a result of reduced rates of N2 fixation, may favour PEP metabolism by PK over PEP metabolism by PEPC.

Notes: Feeding 5-aminoimidazole-4-carboxiamide ribonucleoside (AICAR) through the petiole of detached young barley leaves rapidly increased activities of NADH-nitrate reductase (NR) and glutamine synthetase (GS) in leaf extracts and at least partly prevented the usual slow decrease of these enzyme activities during prolonged illumination. Further, AICAR caused drastic changes in amino acid levels: glutamine and serine levels were increased whereas glutamate and glycine were decreased, probably indicating a higher GS activity and more rapid conversion of glycine into serine. The latter may be responsible for the higher ammonium contents found in AICAR treated leaves. We tentatively suggest that GS (located in the chloroplast) and glycine decarboxylase (located in the mitochondria) are regulated in a manner similar to NR. This is discussed in the light of recent reports that 14-3-3 isoforms exist in chloroplasts and that GS binds to 14-3-3s in vitro.

Notes: The leaves of necrotic hybrid of wheat (Triticum aestivum L.) exhibited high superoxide content associated with increased lipid peroxidation and membrane damage in earlier studies (Khanna-Chopra et al. 1998, Biochem Biophys Res Commun 248: 712–715; Dalal and Khanna-Chopra 1999, Biochem Biophys Res Commun 262: 109–112). In the present study, we investigated the activities of the antioxidant enzymes in the leaves of necrotic wheat hybrids, Kalyansona×C306 (K×C) and WL711×C306 (WL×C) and their parents at different developmental stages. The K×C hybrid exhibited more severe necrosis than WL×C. In K×C, superoxide dismutase (SOD) activity showed no increase over the parents, while WL×C showed an early increase, but it was possibly insufficient to scavenge increased superoxide. Activities of guaiacol peroxidase, ascorbate peroxidase and glutathione reductase were enhanced, while catalase exhibited a decrease in activity, with the appearance of visible necrosis in both the hybrids. The isozyme profile of the antioxidant enzymes was similar in the hybrids and their parents. One existing isoform of guaiacol peroxidase showed an early appearance in the hybrid and increased in intensity with the progression of necrosis. The results reveal a differential response of antioxidant enzymes in necrotic wheat hybrids as compared to their parents. The response differed in magnitude at developmental stages of the leaves, which might be related to the intensity of necrosis expressed by the hybrids.

Notes: The effects of 700 μmol mol−1 CO2 and 200 nmol mol−1 ozone on photosynthesis in Pinus halepensis seedlings and on N translocation from its mycorrhizal symbiont, Paxillus involutus, were studied under nutrient-poor conditions. After 79 days of exposure, ozone reduced and elevated CO2 increased net assimilation rate. However, the effect was dependent on daily accumulated exposure. No statistically significant differences in total plant mass accumulation were observed, although ozone-treated plants tended to be smaller. Changes in atmospheric gas concentrations induced changes in allocation of resources: under elevated ozone, shoots showed high priority over roots and had significantly elevated N concentrations. As a result of different shoot N concentration and net carbon assimilation rates, photosynthetic N use efficiency was significantly increased under elevated CO2 and decreased under ozone. The differences in photosynthesis were mirrored in the growth of the fungus in symbiosis with the pine seedlings. However, exposure to CO2 and ozone both reduced the symbiosis-mediated N uptake. The results suggest an increased carbon cost of symbiosis-mediated N uptake under elevated CO2, while under ozone, plant N acquisition is preferentially shifted towards increased root uptake.

Notes: Stimulation of photosynthesis in response to elevated carbon dioxide concentration [CO2] in the short-term (min) should be highly temperature dependent at high photon flux. However, it is unclear if long-term (days, weeks) adaptation to a given growth temperature alters the temperature-dependent stimulation of photosynthesis to [CO2]. In velveltleaf (Albutilon theophrasti), the response of photosynthesis, determined as CO2 assimilation, was measured over a range of internal CO2 concentrations at 7 short-term measurement (12, 16, 20, 24, 28, 32, 36°C) temperatures for each of 4 long-term growth (16, 20, 28 and 32°C) temperatures. In vivo estimates of VCmax, the maximum RuBP saturated rate of carboxylation, and Jmax, the light-saturated rate of potential electron transport, were determined from gas exchange measurements for each temperature combination. Overall, previous exposure to a given growth temperature adjusted the optimal temperatures of Jmax and VCmax with subsequently greater enhancement of photosynthesis at elevated [CO2] (i.e., a greater enhancement of photosynthesis at elevated [CO2] was observed at low measurement temperatures for A. theophrasti grown at low growth temperatures compared with higher growth temperatures, and vice versa for plants grown and measured at high temperatures). Previous biochemical based models used to predict the interaction between rising [CO2] and temperature on photosynthesis have generally assumed no growth temperature effect on carboxylation kinetics or no limitation by Jmax. In the current study, these models over predicted the temperature dependence of the photosynthetic response to elevated [CO2] at temperatures above 24°C. If these models are modified to include long-term adjustments of Jmax and VCmax to growth temperature, then greater agreement between observed and predicted values was obtained.

Notes: Recent evidence has indicated that activated oxygen species (AOS) may function as molecular signals in the induction of defence genes. 
In the present work, the response of antioxidative enzymes to the plum pox virus (PPV) was examined in two apricot (Prunus armeniaca L.) cultivars, which behaved differently against PPV infection. In the inoculated resistant cultivar (Goldrich), a decrease in catalase (CAT) as well as an increase in total superoxide dismutase (SOD) and dehydroascorbate reductase (DHAR) activities were observed. Ascorbate peroxidase (APX), glutathione reductase (GR) and monodehydroascorbate reductase (MDHAR) did not change significantly in relation to non-inoculated (control) plants. In the susceptible cultivar (Real Fino), inoculation with PPV brought about a decrease in CAT, SOD and GR, whereas a rise in APX, MDHAR and DHAR activities was found in comparison to non-inoculated (control) plants. 
Apricot leaves contain only CuZn-SOD isozymes, which responded differently to PPV depending on the cultivar. Goldrich leaves contained 6 SODs and both SOD 1 and SOD 2 increased in the inoculated plants. In leaves from Real Fino, 5 SODs were detected and only SOD 5 was increased in inoculated plants. 
The different behaviour of SODs (H2O2-generating enzymes) and APX (an H2O2-remover enzyme) in both cultivars suggests an important role for H2O2 in the response to PPV of the resistant cultivar, in which no change in APX activity was observed. This result also points to further studies in order to determine if an alternative H2O2-scavenging mechanism takes place in the resistant apricot cultivar exposed to PPV. On the other hand, the ability of the inoculated resistant cultivar to induce SOD 1 and SOD 2 as well as the important increase of DHAR seems to suggest a relationship between these activities and resistance to PPV. 
This is the first report about the effect of PPV infection on the antioxidative enzymes of apricot plants. It opens the way for the further studies, which are necessary for a better understanding of the role of antioxidative processes in viral infection by PPV in apricot plants.

Notes: The recessive gene af produces a modification of Pisum sativum L. leaf morphology, where leaflets are replaced by tendrils. Previous reports have suggested that tendrils may contribute to plant growth in a similar way to flat leaf structures, but these reports have been restricted to carbon metabolism. In this work, we investigate the nitrate reduction (nitrate reductase activity) capacity of tendrils of a semi-leafless variety, Solara, in relation to other leaf structures. Maximum nitrate reductase activity (EC 1.6.6.1), expressed on a protein basis, was significantly lower in tendrils compared to flat structures. However, the activation state of nitrate reductase was significantly higher in tendrils, reaching 70%, compared to flat leaf structures. According to these results, tendrils contributed up to 25% of the overall plant nitrate reduction in the semi-leafless variety. This figure was even higher when nitrate reduction was calculated from in vivo measurements. The results are discussed in relation to nitrate, magnesium, carbohydrates, amino acids and adenylate levels of tendrils.

Notes: The characterization and expression of PP-ACO1 and PP-ACO2, two members of the peach 1-aminocyclopropane-1-carboxylate (ACC) oxidase (ACO) gene family, are reported. PP-ACO1 is organized in 4 exons interrupted by 3 introns, whereas PP-ACO2 has only 2 of the 3 introns present in PP-ACO1. Comparison of the deduced amino acid sequences of PP-ACO1 and PP-ACO2 reveals a 77.7% identity. PP-ACO1 and PP-ACO2 show highest degree of similarity with petunia (PH-ACO3; 84.1%) and apple (85.4%) ACO genes, respectively. PP-ACO1 is expressed in flowers, fruitlet abscission zones, mesocarp and in young fully expanded leaves. PP-ACO1 transcript accumulation strongly increases during fruitlet abscission, in ripe mesocarp and senescing leaves, and is enhanced by propylene. PP-ACO2 mRNA accumulation is detected in fruits only during early development and is unaffected by propylene treatment. Both ACO genes are expressed in epicotyl and roots of growing seedlings, although a stronger accumulation of PP-ACO2 mRNA is observed.

Notes: We have isolated 5 cDNA clones (din2,din6, din9, din10 and din11) corresponding to genes, the transcripts of which accumulated in leaves of Arabidopsisthaliana kept in the dark. These cDNA clones encode proteins similar to β-glucosidase (EC 3.2.1.21, din2), asparagine synthetase (EC 6.3.5.4, din6), phosphomannose isomerase (EC 5.3.1.8, din9), seed imbibition protein (din10) and 2-oxoacid-dependent dioxygenases (din11). Accumulation of the transcripts from din6 and din10 occurred within 3 h after plants were transferred to darkness. The transcripts from din2,din9 and din11 were only detected after 24 h of dark treatment. We also observed the accumulation of the din transcripts in senescing leaves. Application of a photosynthesis inhibitor, 3-(3,4-dichlorophenyl)-1-1-dimethyl-urea, induced the expression of the din genes under illumination. Application of sucrose to detached leaves suppressed the accumulation of the din transcripts in the dark. These results indicate that expression of these genes partly depends on cellular sugar level. The sugar-modulated expression of the din genes suggests that dark-induced expression of these genes might be related to sugar starvation occurring in leaf cells in the dark, when the photosynthesis is hindered.

Notes: In cereals, a progressively increasing root cortical cell death (RCD) occurs from the root tip and upwards when measured with vital staining methods. In this study, nuclear DNA fragmentation was studied in seminal root segments of wheat and barley in order to investigate if the cell death resembled apoptosis. The fraction of cells with TUNEL-positive nuclei increased gradually with increasing root age in both the cortex and the stele. Southern analysis showed a typical ladder pattern, indicating nucleosomal fragmentation already in 2-day-old root segments, and this became more pronounced in older root segments. DNA fragmentation appeared to be more extensive in wheat than in barley roots. These results confirm earlier studies, where RCD has been found to be earlier initiated and to proceed at a faster rate in wheat. The characteristic DNA fragmentation found in the roots indicates programmed cell death with mechanistic similarities to apoptosis. Ultrastructural examination of nuclei in cortex cells with transmission electron microscopy revealed an increased chromatin condensation in older roots, particularly in wheat.In addition, we found nucleosomal DNA ladders in young leaf tissue from wheat but not from barley.

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.