ALBERT

Notes: Wounding, as during excision and preparation of lettuce (Lactuca sativa L.) leaf tissue for salads, induces the synthesis and accumulation of phenolic compounds that participate in subsequent reactions that cause tissue browning. Exposure of excised 5-mm mid-rib segments of romaine lettuce leaf tissue to vapors of mono-carboxylic acids or aqueous solutions of mono-carboxylic acids or their salts inhibited wound-induced phenolic accumulation (WIPA) and subsequent tissue browning. The decline in phenolic content followed a quadratic curve with increasing concentration, reaching a maximum inhibition after 60 min of 74 ± 8% for 50 mM sodium acetate (2 carbons, C2) and 91 ± 4% for 20 mM sodium decanoate (capric acid, C10). Respiration (i.e. carbon dioxide production) was unaffected by concentrations of formic, acetic, or propionic acids that reduced wound-induced phenolic content or that increase ion leakage from the tissue into an isotonic mannitol solution. However, WIPA was suppressed up to 70% at concentrations (20 mM acetate) that did not increase ion leakage over that of water controls. Various acetate salts (i.e. ammonium, calcium, magnesium, and sodium) all produced the same level of inhibition. The effectiveness of the compounds increased with increasing number of carbons in the molecule from 1 to 10, and was unaffected by whether the carbons were a straight chain or branched or whether the treatment was delayed by up to 6 h. The effectiveness of butyrate (C4) in reducing WIPA (27% reduction at 20 mM) was less than that predicted from the response of the two adjacent mono-carboxylates similarly applied: propionate (C3) (62%) and valerate (C5) (73%). It appears that, unlike the n-alcohols, mono-carboxylates are not interfering with the synthesis or propagation of a wound signal but are interfering with subsequent steps in the production and accumulation of wound-induced phenolic compounds.

Notes: Pearl millet, Pennisetum glaucum, is capable of adapting to severely dry environmental conditions. In order to elucidate the mechanism of adaptation to highly dehydrated conditions, we selected both tolerant (IP8210) and susceptible (IP8949) accessions from a total of 15 pearl millet accessions and characterized their morphological and physiological responses to severe drought stress. When these selected accessions were stressed with a severe drought treatment, the leaves of IP8210 exhibited upright folding, a response that effectively reduces the evaporative surface area of the canopy. On the contrary, the leaves of IP8949 exhibited wilting and did not appear to adapt to the drought stress. In comparison with IP8949, the capacity of osmotic adjustment (OA) was greater in both younger leaves and stems of IP8210, while their decrease in relative water content was different. IP8210 accumulated higher concentrations of NO3– than IP8949 in response to drought stress. In addition to inorganic solutes, several organic components such as sucrose, glucose, quaternary ammonium compounds, and amino acids including proline were also accumulated. IP8210 tended to accumulate more amino acids, typically due to the accumulation of asparagine and proline, while IP8949 accumulated more soluble sugars. While it is possible that K+ and NO3– were the major components contributing to osmotic regulations, sugars and amino acids might also function as a cytoprotectant, in addition to their role as osmoprotectants. Collectively, these results demonstrate that the morphological adaptation of leaf folding, OA in both the younger leaves and the stem, and the accumulation of NO3– and amino acids during earlier stress period contribute to superior drought tolerance that was exhibited in IP8210 of pearl millet.

Notes: The C3 halophyte Suaeda salsa L. grown under the high concentration of NaCl (200 mM) was used to investigate the role of the hydrogen peroxide (H2O2)-scavenging system [catalase, ascorbate peroxidase, glutathione reductase (GR), ascorbic acid, and glutathione (GSH)] in removal of reactive oxygen species. The activity of catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), and GR (EC 1.6.4.2) increased significantly after 7 days of NaCl treatment. The isoform patterns of CAT and GR were not affected, but the staining intensities were significantly increased by NaCl treatment. Activities of both the thylakoid-bound APX or GR and stromal APX (S-APX) or GR in the chloroplasts were markedly enhanced under high salinity. Fifty percent of APX in the chloroplasts is thylakoid-bound APX. S-APX and GR activity represented about 74–78 and 64–71% of the total soluble leaf APX and GR activity, respectively. Salt treatment increased the contents of ascorbic acid and GSH. By contrast, a decreased content of H2O2 was found in the leaves of NaCl-treated S. salsa. The level of membrane lipid peroxidation decreased slightly after NaCl treatment. The plants grew well with high rate of net photosynthesis under high salinity. These data suggest that upregulation of the H2O2-scavenging system in plant cells, especially in the chloroplasts, is at least one component of the tolerance adaptations of halophytes to high salinity.

Notes: The photosynthetic performance of the cyanobacterium Synechocystis sp. PCC 6803 exposed to intermittent light was studied by measuring oxygen evolution, respiration and the fluorescence parameters for maximum efficiency of excitation energy capture by photosystem II (PSII) reaction centres (Fv/Fm), PSII quantum yield (ΔF/Fm1) and non-photochemical quenching (NPQ). Cultures were pre-acclimated to constant light conditions. Block and sinusoidal light regimes were tested using four photon-flux densities (PFDs) applied in light/dark intervals of 1:1, 5:5 and 10:10 min. Light use was higher under the sinusoidal light regime compared with the block regime. The accumulated gross photosynthesis of the cyanobacterium was lower under intermittent light conditions compared with predictions from the photosynthesis-irradiance curve (PI curve). The respiration rates were similar for all light/dark intervals tested. However, the respiration slightly increased with increasing oxygen production for both block and sinusoidal light regime. NPQ, ΔF/Fm′ and Fv/Fm depended on the PFD rather than on the duration of the light/dark intervals tested, and there was no detected influence of the two applied light regimes.

Notes: Fructose analog, psicose, and glucose analog, mannose, inhibited root growth of lettuce seedlings. Psicose is phosphorylated by hexokinase and fructokinase (EC 2.7.1.4) to psicose-6-phosphate with no known capacity for further metabolism. Mannose is phosphorylated by hexokinase (EC 2.7.1.1) to mannose-6-phosphate which is further metabolized very slowly. Hexokinase is known to have a sugar-sensing function and possibly triggers a signal cascade resulting in changes of several gene expressions. It was determined, compared with the behaviour of mannose, whether psicose inhibits the root growth through this system. The addition of phosphate into the growth medium of lettuce seedlings did not affect the inhibition by psicose and mannose, and both sugars did not reduce adenosine triphosphate (ATP) level in the roots, suggesting that the inhibition is not due to phosphate starvation and ATP depletion. The inhibiting effects of psicose and mannose were overcome by adding sucrose into the medium, which suggests that the inhibition is not caused by accumulation of psicose-6-phosphate or mannose-6-phosphate in the seedlings. Mannoheptulose, a specific competitive inhibitor of hexokinase, defeated the mannose-induced inhibiting but was not able to relieve the psicose-induced inhibition. Thus, the phosphorylation of mannose by hexokinase may trigger a signal cascade resulting in the growth inhibition of lettuce roots, which is consistent with the hypothesis established in Arabidopsis. However, psicose cannot inhibit the growth of lettuce roots via a hexokinase-mediated pathway, and the phosphorylation of psicose by fructokinase might trigger a hexokinase-independent signal cascade resulting in the growth inhibition.

Notes: The pools of photoprotective molecules respond to changes in the environmental conditions and sometimes to leaf ageing. We asked to what extent both factors contribute to the contents of α-tocopherol and xanthophyll cycle [V + A + Z (VAZ)] pigments. To address this question, we used boxtree (Buxus sempervirens) as model species because its leaves are long-lived and evergreen and are subjected to a succession of different stress conditions during their lifespan. In three age classes of sun and shade leaves of this species, seasonal changes in photoprotective compounds were followed during 15 months and a leaf age interval of 40 months was covered. As could be expected, VAZ and α-tocopherol pools increased in parallel during stress periods (summer and winter), but only VAZ recovered to the initial pools once stress disappeared. As a result, the basal α-tocopherol level increased linearly in a time-dependent manner that was also higher in sun leaves of this species when compared with shade leaves, and in fact, the rate of tocopherol increase was directly proportional to irradiance in another evergreen (Laurus nobilis). To study whether light dependency of tocopherol accumulation is observed in other species, we performed a literature survey that revealed that this age-dependent tocopherol increase was significant in sun leaves from 65% of the species for which age-dependent tocopherol changes have been reported, and it was on average 2.2-fold higher in sun leaves as compared with shade leaves. We conclude that there are two components in the α-tocopherol pool, one dynamic that responds to environmental changes and one age-related which increases linearly with time in a light-dependent manner. The physiological meaning of the latter remains obscure.

Notes: B-function genes determine the identity of petals and stamens in the flowers of model plants such as Arabidopsis and Antirrhinum. Here, we show that a putative B-function gene BpMADS2, a birch homolog for PISTILLATA, is expressed in stamens and carpels of birch inflorescences. We also present a novel birch gene BpMADS8, a homolog for APETALA3/DEFICIENS, which is expressed in stamens. Promoter-GUS analysis revealed that BpMADS2 promoter is active in the receptacle of Arabidopsis flower buds while BpMADS8 promoter is highly specific in mature stamens. BpMADS2 promoter::BARNASE construct prevented floral organ development in Arabidopsis and tobacco. In birch, inflorescences with degenerated stamens and carpels were obtained. BpMADS8::BARNASE resulted in degeneration of stamens in Arabidopsis and birch causing male sterility. In tobacco, only sepals were developed instead of normal flowers. The results show that the BpMADS2::BARNASE construct can be used to specifically disrupt floral organ development in phylogenetically distant plant species. The stamen-specific promoter of BpMADS8 is a promising tool for biotechnological applications in inducing male sterility or targeting gene expression in the late stamen development.

Notes: We have used yeast two-hybrid screens and biochemical methods to identify glycolytic enzymes that interact with subcellular structures in hypoxic maize seedlings. As binding domain-bait fusion constructs, we have cloned actin, cytosolic aldolase, the three sucrose synthase (SUS) isoforms SUS1, SUS3, and SH1 as well as the SNF1-related protein kinase into yeast and identified cytosolic isoforms of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), enolase, tubulin, and mitochondrial porin voltage-dependent anion channel protein (VDAC) as well as protein kinases and proteins involved in ubiquitinylation and proteasome-linked degradation as interacting activation domain-prey clones. The results were further confirmed using overlay blots (VDAC) as well as co-polymerization and co-precipitation assays (tubulin and actin). Some results were obtained that support the idea of metabolite and modification effects on the association, namely guanosine triphosphate (GTP)/MgCl2 was necessary for the binding of enolase to actin. GAPDH is inactivated upon association with tubulin but then serves to stabilize the microtubules. The findings support the idea of the dynamic formation of locally associated complexes of enzymes involved in sucrose breakdown and glycolysis in plant cells depending on their metabolic state.

Notes: Acid invertases play a key role in sugar metabolism, and the plant hormone abscisic acid (ABA) enhances sugar accumulation in crop sink organs, but information about the relationship between ABA and acid invertases has been limited. The present experiments were done with both in vivo pre-incubation of the grape (Vitis vinifera × V. labrusca L.) berry tissues in ABA-containing medium and in vivo infiltration of ABA into the intact berries. The results show that ABA activates both the soluble and cell wall-bound acid invertases during fruit development by enhancing their activities and amounts as assessed by immunoblotting or enzyme-linked immunosorbent assay. This activation was pH, time course and ABA dose dependent. The serine/threonine protein kinase inhibitors K252a, staurosporine and H7 and acid phosphatase increased the activation of ABA-induced acid invertase, but the tyrosine protein kinase inhibitor quercetin strongly suppressed the ABA-induced effects, suggesting that a complex reversible protein phosphorylation is involved in the ABA-induced activation of acid invertases. The effects of the protein kinase inhibitors were dependent on the in vivo state of the tissues but independent of the expression of acid invertases. Two ABA analogues, (–)-ABA and trans-ABA, had no effect on acid invertases, showing that the ABA-induced activation of acid invertases is specific to the physiologically active form of ABA. These data suggest that ABA may be involved in fruit development by activating acid invertases.

Notes: Onions were grown in environmentally controlled growth chambers for 85 days to investigate the effect of relatively low light intensity (350 µmol m−2 s−1) at two different total irradiance periods (12-h and 24-h photoperiods) on growth and photosynthetic performance. To test whether photosynthetic downregulation occurred due to carbohydrate feedback, we used onions that differed in bulb-forming capacity. Allium fistulosum (L. cv. ‘Kinka’) is a non-bulbing onion, with potentially limited carbohydrate storage capacity, while Allium cepa (L. cv. ‘Cal 296’) is a bulb-forming onion with possibly greater carbohydrate storage capacity. In A. fistulosum, photosynthetic downregulation was observed in 24-h plants as indicated by reductions in the light- and CO2-saturated photosynthetic capacity (Asat and Amax, respectively) by 26%, reduced maximum rate of carboxylation (Vcmax) by ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) by 33%, reduced maximum rate of electron transport (Jmax) by 27% and 3-fold higher foliar sugar concentration. In contrast, the photosynthetic and biochemical capacity of A. cepa was not affected by exposure to 24-h photoperiod, presumably because substantial amounts of foliar carbohydrates were re-allocated to bulbs. In 24-h A. cepa, up to 84% of total plant mass was allocated to bulbs, while in 12-h plants, more mass was allocated to leaves. Production of greater leaf area in 12-h plants compared with 24-h plants compensated for lower total daily irradiance such that 12-h and 24-h plants of both species exhibited similar daily total leaf net CO2 exchange and plant mass at the end of the experiment.

Notes: Few studies have investigated the interaction of ultraviolet (UV)-B radiation and CO2 concentration on plants. We studied the combined effects of UV-B radiation and CO2 concentration on canola (Brassica napus cv. 46A65) under four growth conditions – ambient CO2 with UV-B (control), elevated CO2 with UV-B, ambient CO2 without UV-B, and elevated CO2 without UV-B – to determine whether the adverse effects of UV-B are mitigated by elevated CO2. Elevated CO2 significantly increased plant height and seed yield, whereas UV-B decreased them. Elevated CO2 ameliorated the adverse effects of UV-B in plant height. UV-B did not affect the physical characteristics of leaf but CO2 did. Certain flower and fruit characteristics were affected negatively by UV-B and positively by CO2. UV-B did not affect net photosynthesis, transpiration and stomatal conductance but decreased water use efficiency (WUE). Elevated CO2 significantly increased net photosynthesis and WUE. Neither UV-B nor CO2 affected chlorophyll (Chl) fluorescence. UV-B significantly decreased Chl b and increased the ratio of Chl a/b. Elevated CO2 decreased only the ratio of Chl a/b. UV-B significantly increased UV-absorbing compounds while CO2 had no effect on them. Both UV-B and CO2 significantly increased epicuticular wax content. Many significant relationships were found between morphological, physiological, and chemical parameters. This study showed that elevated CO2 can partially ameliorate some of the adverse effects of UV-B radiation in B. napus.

Notes: Xyloglucan endotransglucosylase/hydrolases (XTHs) are thought to be involved in various aspects of plant development by modifying the structure of xyloglucan cross-links. To address one of the roles of XTHs in plant growth, we identified an XTH, VrXTH1, in the mungbean through a differential reverse transcriptase-polymerase chain reaction. The deduced amino acid sequence of VrXTH1 shows high similarity to other XTHs. In addition, a signal peptide consisting of 17 amino acids is found at the N-terminus. The gene expression of VrXTH1 was differentially regulated in tissues and was higher in hypocotyls and stems than it was in other tissues. The steady state level of VrXTH1 transcripts was closely related to the elongation regions of hypocotyls. Notably, in the elongation region of hypocotyls, most VrXTH1 mRNAs were limited to the epidermis and to some layers of the cortex that act as growth-limiting tissue. Growth-promoting hormones, such as auxin and brassinolide, strongly enhanced mRNA accumulation of VrXTH1. However, abscisic acid, a hormone which is antagonistic to auxin, acted as a downregulator. Overall, VrXTH1 seems to play a role in plant growth at the gene level and, thus, by possibly altering cell wall morphogenesis in mungbean hypocotyls.

Notes: The light-induced de-epoxidation of xanthophylls is an important photoprotective mechanism in plants and algae. Exposure to ultraviolet radiation (UVR, 280–400 nm) can change the extent of xanthophyll de-epoxidation, but different types of responses have been reported. The de-epoxidation of violaxanthin (V) to zeaxanthin (Z), via the intermediate antheraxanthin, during exposure to UVR and photosynthetically active radiation (PAR, 400–700 nm) was studied in the marine picoplankter Nannochloropsis gaditana (Eustigmatophyceae) Lubián. Exposures used a filtered xenon lamp, which gives PAR and UVR similar to natural proportions. Exposure to UVR plus PAR increased de-epoxidation compared with under PAR alone. In addition, de-epoxidation increased with the irradiance and with the inclusion of shorter wavelengths in the spectrum. The spectral dependence of light-induced de-epoxidation under UVR and PAR exposure was well described by a model of epoxidation state (EPS) employing a biological weighting function (BWF). This model fit measured EPS in eight spectral treatments using Schott long pass filters, with six intensities for each filter, with a R2 = 0.90. The model predicts that 56% of violaxanthin is de-epoxidated, of which UVR can induce as much as 24%. The BWF for EPS was similar in shape to the BWF for UVR inhibition of photosynthetic carbon assimilation in N. gaditana but with about 22-fold lower effectiveness. These results demonstrate a connection between the presence of de-epoxidated Z and the inhibition under UVR exposures in N. gaditana. Nevertheless, they also indicate that de-epoxidation is insufficient to prevent UVR inhibition in this species.

Notes: SULTR2;1 is a low-affinity sulfate transporter expressed in the vascular tissues of roots and leaves for interorgan transport of sulfate in Arabidopsis thaliana. Transgenic Arabidopsis carrying a fusion gene construct of SULTR2;1 5′-promoter region and β-glucuronidase coding sequence (GUS) demonstrated that within the reproductive tissues, SULTR2;1 is specifically expressed in the bases and veins of siliques and in the funiculus, which connects the seeds and the silique. The antisense suppression of SULTR2;1 mRNA caused decrease of sulfate contents in seeds and of thiol contents both in seeds and leaves, as compared with the wildtype (WT). The effect of antisense suppression of SULTR2;1 on seed sulfur status was determined by introducing a sulfur-indicator construct, p35S::βSRx3:GUS, which drives the expression of GUS reporter under a chimeric cauliflower mosaic virus 35S promoter containing a triplicate repeat of sulfur-responsive promoter region of soybean β-conglycinin β subunit (βSRx3). The mature seeds of F1 plants carrying both the SULTR2;1 antisense and p35S::βSRx3:GUS constructs exhibited significant accumulation of GUS activities on sulfur deficiency, as compared with those carrying only the p35S::βSRx3:GUS construct in the WT background. These results suggested that SULTR2;1 is involved in controlling translocation of sulfate into developing siliques and may modulate the sulfur status of seeds in A. thaliana.

Notes: The expression of totipotency in plant protoplasts is a complex developmental phenomenon and is affected by genetic and physiological factors. Polyamines (PAs) are known to be involved in a variety of growth and developmental processes in higher plants, as well as in adaptation to stresses. In this study, we present the homeostatic characteristics of the endogenous PA putrescine (Put), spermidine (Spd), and spermine (Spm) in totipotent (T) and non-totipotent (NT) tobacco protoplasts and in recalcitrant (R) grapevine protoplasts. T-tobacco protoplasts, with high division rates, have the highest level of endogenous PAs. In these protoplasts, the soluble-hydrolyzed fraction predominates and increases, and the insoluble-hydrolyzed fraction also increases, whereas soluble (S) PAs decrease rapidly during culture. The isolation process contributes to the increased Put levels, which are higher in freshly isolated NT-tobacco protoplasts than in T-protoplasts. During culture, total Put predominates over Spd and Spm, and the highest accumulation is found in T-protoplasts. Ornithine decarboxylase and arginase activities both increase in T-protoplasts, whereas arginine decarboxylase activity causes Put accumulation in NT-tobacco protoplasts. R-grapevine protoplasts show a different PA profile, mostly due to the lower PA content, the higher S-fraction, and the higher ratio of Spm to total PAs. The data suggest that the levels and metabolism of the intracellular PAs could be related to the expression of totipotency of plant protoplasts.

Notes: In the southeast of the Qinghai-Tibetan Plateau of China, sea buckthorn (Hippophae rhamnoides L.), which is a thorny nitrogen-fixing deciduously perennial shrub, has been widely used in forest restoration as the pioneer species. In our study, two contrasting populations from the low and high altitudinal regions were employed to investigate the effects of drought, ultraviolet-B (UV-B) and their combination on sea buckthorn. The experimental design included two watering regimes (well watered and drought stressed) and two levels of UV-B (with and without UV-B supplementation). Drought significantly decreased total biomass, total leaf area and specific leaf area (SLA), and increased root/shoot ratio, fine root/coarse root ratio and abscisic acid content (ABA) in both populations. However, the high altitudinal population was more responsive to drought than the low altitudinal population. On the other hand, elevated UV-B induced increase in anthocyanins in both populations, whereas the accumulation of UV-absorbing compounds occurred only in the low altitudinal population. The drought-induced enhancement of ABA in the high altitudinal population was significantly suppressed in the combination of drought and elevated UV-B. Moreover, significant drought × UV-B interaction was detected on total biomass in both populations, total leaf area and fine root/coarse root in the low altitudinal population, and SLA in the high altitudinal population. These results demonstrated that there were different adaptive responses between two contrasting populations, the high altitudinal population exhibited higher tolerance to drought and UV-B than the low altitudinal population.

Notes: Deoxyhypusine synthase (DHS; EC 2.5.1.46) mediates the first of two enzymatic reactions that convert inactive eukaryotic translation initiation factor-5A (eIF-5A) to an activated form, thought to facilitate translation. A full-length cDNA clone encoding canola (Brassica napus cv. Westar) DHS was isolated from a cDNA-expression library prepared from senescing leaves. Transgenic canola lines with suppressed DHS expression were obtained by introducing a transgene expressing antisense 3′-UTR canola DHS cDNA under the regulation of the constitutive cauliflower mosaic virus 35S (CaMV-35S) promoter. Transformed seed was obtained by vacuum infiltration of canola inflorescences using the protocol developed for Arabidopsis with modifications. The resultant transgenic plants had reduced levels of leaf DHS protein and exhibited delayed natural leaf senescence. Suppression of DHS also increased leaf size by 1.5- to two-fold and resulted in increases in seed yield of up to 65%. Moreover, the enhanced performance of transgenic plants reflected increased tolerance to chronic sublethal stress. When wild-type and transgenic plants were grown in 6-inch pots, the increase in seed yield accruing from suppression of DHS was approximately 4.5-fold greater than when the plants were grown in 12-inch pots. Thus, suppression of DHS appears to ameliorate the effects of sublethal stress engendered by growth in small containers.

Notes: In higher plants, the xylem vessels functionally connect the roots with the above-ground organs. The xylem sap transports various organic compounds, such as proteins and amino acids. We examined drought and rewatering-inducible changes in the amino acid composition of root xylem sap collected from Cucurbita maxima roots. The major free amino acids in C. maxima root xylem sap were methylglycine (MeGly; sarcosine) and glutamine (Gln), but MeGly was not detected in the xylem sap of cucumber. MeGly is an intermediate compound in the metabolism of trimethylglycine (TMG; betaine), but its physiological effects in plants are unknown. Drought and rewatering treatment resulted in an increase in the concentration of MeGly in root xylem sap to 2.5 mM. After flowering, the MeGly concentration in the xylem sap dropped significantly, whereas the concentration of Gln decreased only after fruit ripening. One milli molar MeGly inhibited the formation of adventitious roots and their elongation in C. maxima, but glycine, dimethylglycine, or TMG had no effect. Similar effects and the inhibition of stem elongation were observed in shoot cuttings of cucumber and Phaseolus angularis. These observations seem to imply a possible involvement of xylem sap MeGly in the physiological responses of C. maxima plants to drought stress.

Notes: In this work we present the first study of the behaviour of tobacco plants, under saline conditions, grafted to salinity-resistant rootstocks of tomato cultivars. To test the viability and efficiency of this grafting technique in tobacco plants subjected to salinity, we analyse the production of foliar biomass and different quality parameters in this crop. With this aim, Nicotiana tabacum cv. Sevilla (scion) was grafted to two cultivars of Lycopersicum esculentum (rootstocks): cv. Jaguar (Sevilla/Jaguar) and cv. Brillante (Sevilla/Brillante). Furthermore, as controls, tobacco plants of cv. Sevilla were used grafted to themselves (Sevilla/Sevilla) and non-grafted plants of cv. Sevilla. Plants were grafted by needle graft following the procedure described by Rivero RM, Ruiz JM, Romero L (2002) Role of grafting in horticultural plants, pp 229–254. In the present work, we demonstrate that the graft of tobacco scions with tomato rootstocks is an effective agricultural approach to improve production and quality in tobacco leaves under conditions of saline stress. Our results show that the rootstock of the cv. Brillante best induced salt resistance in tobacco cv. Sevilla, registering the lowest foliar concentrations of Na+ and Cl–, the lowest lipid peroxidation and the highest proline and sugar concentrations. Overall, this is reflected in better biomass production of the aerial part of the plant. Finally, it is noteworthy that grafting in tobacco plants to tomato rootstocks essentially eliminates foliar nicotine levels (reduced to 1%). These results are of great importance, as this technique implies a rapid, efficient and natural alternative in increasing tobacco-leaf quality and thus reducing harmful effects of this alkaloid on the health of smokers.

Notes: Cucumber seedling radicles decrease in chilling tolerance as they increase in length or decrease in vigor. The protein content of the apical 5 mm of the radicle decreased with decreases in chilling tolerance (R2 = 0.92). This general reduction in protein content was reflected in a decrease of six dehydrin-like proteins with apparent molecular weights of 13.0, 15.0, 16.8, 23.0, 26.8, and 33.5 kDa. The disappearance of naturally occurring dehydrin-like proteins in cucumber seedling radicles as they elongate or lose vigor was correlated with a loss of chilling tolerance. Exposure to an osmotic (0.6 M mannitol) or heat (2 min at 45°C) stress enhanced chilling tolerance. The osmotic-shock treatment induced both chilling tolerance and the appearance or strengthening of dehydrin-like proteins previously present in radicles. The heat-shock treatment also induced high levels of chilling tolerance and protein(s) that reacted with a 23 and 70 kDa antibody. However, these heat-shock protein (HSPs) did not cross react with the probe for dehydrin-like proteins. When organized into high, medium, and low chilling tolerance groups, radicle that were chilling tolerant contained either the 13.0 and 16.8 kDa dehydrin-like proteins, or the 15.0 and 23.0 kDa dehydrin-like proteins, or the 23 or 70 kDa HSP.

Notes: Despite the major impacts of fire on plants, responses to fire damage have not been closely studied on the level of gene expression. Here, we present analyses of novel transcripts from tomato (Lycopersicon esculentum cv. Heinz), which are systemically upregulated in leaves after a distant leaf is wounded by flame. Nine cDNA fragments were isolated from a subtractive cDNA library of leaf tissue 1 h after flaming. Using data mining and polymerase chain reaction (PCR), full-length open-reading frames were predicted, amplified, and then sequenced. Real-time (RT)-PCR using leaf RNA after flaming confirmed the systemic accumulation of 4 and 7 transcripts within 30 and 60 min, respectively, before returning to basal levels within 3 h. During this same time course, proteinase inhibitor I levels gradually increased over 30-fold in 6 h. Expression analyses also showed that eight of the transcripts are present in unwounded leaf, stem, and root tissues. The predicted proteins include an acyl carrier, adenylyl sulfate reductase, PS II oxygen-evolving complex protein 3, anion : sodium symporter, chloroplast-specific ribosomal protein, a histidine triad family protein, and an unknown wound/stress-related protein. Homologs of several of these proteins have been associated with other types of wound and stress responses. It appears that, within an hour after being damaged by fire, plants systemically upregulate a variety of genes involved with basic cell metabolism and upkeep, in addition to classic defense genes such as proteinase inhibitors.

Notes: Among the longest cell types known in plants, cotton fibers are economically important seed trichomes that provide a unique single-celled model system for studying fundamental biological processes. Functional genomic approaches have served to characterize dynamic changes to the cotton fiber transcriptome in response to developmental signals that control fiber morphogenesis at the level of a single cell. The genetic complexity of the fiber transcriptome is very high and accounts for as much as 45–50% of the genes in the cotton genome. In addition to a large diverse group of constitutively expressed genes, expression profiling of the transcriptome revealed two developmentally regulated stage-specific expression patterns that define rapid cell elongation during primary cell wall (PCW) synthesis relative to secondary cell wall biogenesis. In developing cotton fibers, many fiber genes involved in PCW synthesis and turgor-driven cell expansion are differentially expressed in a manner that parallels the growth rate. Characterization of the cotton fiber transcriptome has immediate applications in agricultural biotechnology and molecular breeding programs geared toward the genetic improvement of yield and fiber quality.

Notes: Methyl salicylate (MeSA) is thought to have a major role in biotic and abiotic stresses by acting as a signal to trigger the oxidative burst, which is needed to activate gene expression in plant stress responses. To assess the potential effects of sustained foliar accumulation of MeSA on plant stress tolerance, the extent of photo- and antioxidant protection, lipid peroxidation and visual leaf area damage were evaluated in MeSA-treated (c. 60 nl l−1 in air) and control holm oak (Quercus ilex L.) plants exposed to heat stress. Control plants showed an increase in foliar MeSA levels up to 1.8 nmol [gDW]−1 as temperature increased and they displayed tolerance to temperatures as high as 45°C, which might be attributed, at least in part, to enhanced xanthophyll de-epoxidation and increases in ascorbate and α-tocopherol. MeSA-treated plants showed a sustained foliar accumulation of this compound, with values ranging from 10 to 23 nmol [gDW]−1 throughout the experiment. These plants showed lower ascorbate and tocopherol levels and higher oxidative damage at 50°C than controls, as indicated by enhanced malondialdehyde levels and leaf area damage and lower maximum efficiency of PSII photochemistry (Fv/Fm ratio). These results demonstrate that a sustained foliar accumulation of MeSA is detrimental to plant function and that it can reduce thermotolerance in holm oak by altering antioxidant defences.

Notes: The virescent character is a genetic variant in pigmentation characterized by a delay in greening. Seedlings of the virescent mutants v1, v2, v3, v4, v13, v16, v18, v19 and v26 of maize exhibit chlorosis when grown at low temperature. Chlorotic leaves contain plastids that appear to have been arrested at an early stage of development. The results indicated that V16, V2, V3 and V4 loci control early stages of chloroplast development while V1, V13 and V19 may play a role at the end of development. The mutations in the V18 and V26 loci may control an intermediate step. At the pigment level, the virescent mutants of maize differ widely from analogous mutations existing in other plants. The mutations were characterized by a reduced amount of chlorophyll a and b (up to 100 times in v16) and chlorophyll a/b ratio above normal (up to 13.7 in v16). Lutein content was reduced in all mutants (less than 3% in v16 compared to wild type) but v13, while pigments of the xanthophyll cycle were found at higher levels in v1 and v13 (more than 10 and 90%, respectively). The v2, v3, v4, v16 and v18 mutants that are most depleted in β-carotene (36 times less in average than wild type) are also deprived in D1 and D2 polypeptides. Moreover, the v2, v3, v4, v16 and v18 mutants characterized by a lower accumulation in lutein are most depleted of light-harvesting complex II. All mutants possess a functioning, fully reversible, non-photochemical quenching mechanism. This is most developed in the v13 and v19 mutants (φn = 0.48 and 0.44, respectively). These two mutants also have a relatively high primary photochemical yield for photosystem II and a functioning photosystem I (φp = 0.23 and 0.39, respectively). The most interesting mutant is v13 that shows severe chlorosis and possesses the most effective non-photochemical quenching mechanism(s), which is thought to provide protection against excess photon absorption by photosystem II.

Notes: A UV-B exclusion-experiment was conducted in the high Arctic Zackenberg, NE Greenland, in which Salix arctica leaves during most of the growing season were fixed perpendicular to the solar zenith angle, thereby receiving maximal solar radiation. Covered with Teflon and Mylar foil, the leaves received approximately 90 and 40% of the ambient UV-B irradiance, respectively. The effects were examined through recordings of chlorophyll a fluorescence transients, determination of biomass and analysis of total carbon and nitrogen content and amount of soluble flavonoids in the leaves. The processing of light was analysed by means of the chlorophyll a fluorescence transient, using the so-called JIP test, as evolved by Reto J. Strasser and his coworkers. Reduction of the UV-B irradiance caused a rise in many of the fluorescence parameters during July, but not in August (late season). Thus increases in the efficiency that an absorbed photon will be trapped by the PSII reaction centre with the resultant reduction of QA to QA– (ET0/ABS = FV/FM) and the efficiency that an electron residing on QA– will enter the intersystem electron transport chain (ET0/TR0) were observed in reduced UV-B. Moreover, estimated per cross-section of leaf sample, the number of active PSII reaction centres (RC/CSM) and electron transport rate (ETM/CSM) and all performance indexes (PIABS, PICSo and PICSm) were increased in reduced UV-B. The total soluble flavonoid content was highest in ambient UV-B. The treatment effects on fluorescence parameters that were directly measured (e.g. F0 and FM) and those that were derived (e.g. quantum efficiencies, parameters per PSII reaction centres and per cross-section of leaf sample) are discussed in relation to one another, in relation to daily and seasonal variation, and from the perspective of evaluating the relative importance of UV-B of donor and acceptor side capacity in Photosystem II. In conclusion, the experimental set-up and non-invasive measurements proved to be a sensitive method to screen for effects of UV-B stress.

Notes: The basidiomycete Piriformospora indica interacts with Arabidopsis roots and mimics an arbuscular mycorrhiza. A MATH [meprin and TRAF (tumour necrosis factor receptor-associated factor) homology] domain-containing (MATH) protein at the plasma membrane of Arabidopsis roots is one of the first components to respond to the presence of this fungus. MATH proteins are involved in nodule formation in Medicago and protein degradation in the Arabidopsis cytosol. They exhibit sequence similarities to meprins, extracellular peptidases which cleave (signal) peptides, and to TRAFs, intracellular proteins which interact with receptor kinases at the plasma membrane. Fifty-nine genes for MATH proteins are present in the Arabidopsis genome. Members of this protein family are predicted to be found in the ER–plasma membrane–extracellular space continuum, in the nucleus–cytosol compartment and in organelles. In this article, we describe this novel class of plant genes. We also use MS-MS analyses to identify the subcellular localization of individual members of the MATH protein family in Arabidopsis thaliana.

Notes: Maize (Zea mays L., line F2) plants were grown in the field under high or low fertilization input to monitor the metabolic, biochemical and molecular events occurring in young vegetative leaves and in the different leaf stages along the main axis in plants harvested 15 days after silking. This study shows that in maize which possess large sinks represented by the seeds, nitrogen (N) management is different compared with tobacco in which sink strength is much lower and mostly limited to young developing leaves. Although in young leaves nitrate assimilation predominates in both species, ammonium assimilation exhibits some species-specific differences with respect to inorganic and organic N metabolite accumulation during leaf ageing. These differences are likely to be related to the high sink strength of the ear in maize, which continuously imports carbon and N assimilates during grain filling. Consequently, a number of cytosolic glutamine synthetase isoenzymes are expressed during leaf ageing to maintain a constant flux of reduced N necessary for the synthesis of organic N molecules used either for leaf protein synthesis or directly translocated to the grain. This situation contrasts with that found in tobacco for which leaf ammonium assimilation in the plastids is shifted to the cytosol during the transition from sink leaves to source leaves. These species-specific differences for N assimilation and recycling are discussed in relation to the evolution of leaf photosynthetic activity and leaf senescence, which both seem to be largely dependent on the different sink strength in each species.

Notes: Effect of low temperature on anthocyanin accumulation in seedlings of Alternanthera bettzickiana and activity changes of calmodulin (CaM) and Ca2+-ATPase under low temperature were studied. Results indicate that the increase of anthocyanin content was obviously paralleled not only by the activity of CaM but also by the activity of Ca2+-ATPase. In addition, seedlings were pretreated with CaM antagonist [chlorpromazine (CPZ)] before low-temperature treatment in order to further investigate whether CaM plays a role in anthocyanin accumulation. CPZ pretreatment inhibited the activity of CaM and Ca2+-ATPase and caused a reduction in anthocyanin levels. Hence, it is concluded that CaM and Ca2+-ATPase were directly correlated with anthocyanin accumulation under low temperature, Ca2 ± CaM may be involved in low-temperature signal transduction leading anthocyanin synthesis.

Notes: Arabinogalactan-proteins (AGPs) are a class of large hydroxyproline-rich glycoproteins (HGRPs) found in almost all plant species, and have been implicated in various plant growth and developmental processes including xylogenesis. A total of six AGP-like genes or gene families have been cloned from differentiating pine xylem. In this study, seven different members of the ptaAGP5 gene family with between 54% and 73% similarity at the amino acid level were newly identified. Gene-specific primers were designed and relative transcript levels of 11 loblolly pine AGP and AGP-like genes were examined using real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. Expression was examined in different tissues: earlywood and latewood; xylem from two populations; drought-stressed and well-watered roots; compression, opposite and vertical wood; and in vitro cultured cells induced for lignification. The different loblolly pine AGP and AGP-like genes showed varying expression patterns under the different conditions, suggesting different functions for each loblolly pine AGP. The results from this study also suggest that some AGPs are associated with xylogenesis, but not with lignification, and that different xylem AGPs probably have different functions.

Notes: The effects of soil drying on the activity of nitrate reductase (NR; EC 1.6.6.6) were studied in Helianthus annuus L. and non-nodulated Lupinus albus L. plants growing under two nutrient supply regimes. NR activity was assessed in leaf and root extracts by measuring the activity of the unphosphorylated active form (NRact), the maximal extractable activity (NRmax) and the activation state. To obtain an insight into potential signalling compounds, nitrate, free amino acids and soluble sugars were also quantified. In both species, foliar NRact and NRmax were negatively affected by soil drying and a decreased supply of nutrients, the observed changes in NR activity being linearly correlated with the depletion of nitrate. Similar results were obtained in the roots of sunflower. Conversely, in white lupin roots, NRmax was found to be independent of tissue nitrate concentration. Regardless of the species and organ, the activation state of the enzyme was unaffected by the nutrient supply regime. In well-watered sunflower roots, only about 50% of the existing NR was unphosphorylated, but the activation state increased significantly in response to drought. In contrast, lupin roots always exhibited NR activation state values close to 80%, or even higher. At the leaf level, the NR activation state was hardly changed in response to soil drying. The observed changes in the concentrations of soluble sugars and free amino acids are discussed in terms of their possible contribution to the variations in NR activity.

Notes: The phototransformation of protochlorophyllide forms was studied in epicotyls of dark-germinated pea (Pisum sativum L. cv. Zsuzsi) seedlings. Middle segments were illuminated with white or 632.8 nm laser flash or continuous light at room temperature and at −15°C. At low light intensities, photoreduction could be distinguished from bleaching. 77 K fluorescence emission spectra were measured, difference spectra of illuminated and non-illuminated samples were calculated and/or the spectra were deconvoluted into Gaussian components. The 629 nm-emitting protochlorophyllide form, P629 (Pxxx where xxx is the fluorescence emission maximum), was inactive. For short-period (2–100 ms) and/or low-intensity (0.75–1.5 µmol m−2 s−1) illumination, particularly with laser light, the transformation of P636 into the 678 nm-emitting chlorophyllide form, C678 (Cxxx where xxx is the fluorescence emission maximum), was characteristic. This process was also found when the samples were cooled to −15°C. The transformation of P644 into C684 usually proceeded in parallel with the process above as a result of the strong overlap of the excitation bands of P636 and P644. The Shibata shift of C684 into a short-wavelength form, C675–676, was observed. Long-period (20–600 s) and/or high-intensity (above 10 µmol m−2 s−1) illumination resulted in the parallel transformation of P655 into C692. These results demonstrate that three flash-photoactive protochlorophyllide forms function in pea epicotyls. As a part of P636 is flash photoactive, its protochlorophyllide molecule must be bound to the active site of a monomer protein unit [Böddi B, Kis-Petik K, Kaposi AD, Fidy J, Sundqvist C (1998) The two short wavelength protochlorophyllide forms in pea epicotyls are both monomeric. Biochim Biophys Acta 1365: 531–540] of the NADPH:protochlorophyllide oxidoreductase (EC 1.3.1.33). Dynamic interconversions of the protochlorophyllide forms into each other, and their regeneration, were also found, which are summarized in a scheme.

Notes: Expression of selected genes in relation to phosphate (Pi) starvation and sugar sensing was studied in leaves of Arabidopsis. Excised leaf segments with different P status were supplied with combinations of Pi and sugars. Sugar-inducible genes, encoding β-amylase (β-AMY) and chalcone synthase (CHS), were also induced by P deficiency, and were more strongly regulated by sugars when leaf segments originated from P-starved plants. Furthermore, transcript levels of the P-starvation-inducible genes ACP5 (encoding an acid phosphatase), RNS1 (encoding a ribonuclease), and IPS1 (unknown function) increased in response to exogenously applied sugars. Supply of Pi to the leaf segments reversed both P-starvation-induced and sugar-induced gene expression. These interactions reveal a close relationship between P and sugar sensing. To differentiate between hexokinase-dependent and hexokinase-independent sugar sensing the effect of the glucose analogue 2-deoxyglucose and gene expression in the hexokinase-1 deficient mutant, gin2-1, were studied. Both β-AMY and CHS were induced by supplying sucrose to excised leaves but not by 2-deoxyglucose, confirming that these genes are regulated by hexokinase-independent sugar sensing. In the gin2-1 mutant both β-AMY and CHS responded clearly to P starvation excluding that hexokinase-1 mediates the response to P. Similarly, the P-responding genes, IPS1 and RNS1 were repressed by addition of Pi also in the gin2-1 mutant. In conclusion, several phosphate starvation-induced genes are also sugar-induced and hexokinase-independent sugar sensing in Arabidopsis is strongly intensified by phosphate starvation.

Notes: S-Adenosylmethionine decarboxylase (SAMDC, E.C. 4.1.4.50) is a key enzyme involved in the polyamine (PA) biosynthetic pathway. An understanding of how SAMDC genes are regulated is important for elucidating the molecular basis of PA biosynthesis and the role of PAs in plant growth and development. However, information regarding transcriptional regulation of SAMDC has been limited. In an attempt to address this question, we isolated four cDNAs from mustard (Brassica juncea), designated BJSAMDC1, BJSAMDC2, BJSAMDC3 and BJSAMDC4, encoding predicted SAMDC. A comparison of deduced amino acid sequence revealed that they are highly homologous to other plant SAMDCs. These proenzymes also possess the conserved cleavage domain and putative PEST sequence for SAMDC. Northern analysis showed that the SAMDC transcripts were most abundant in reproductive organs and roots but that the level was low in young leaves and petioles. Meanwhile, SAMDC expression in the leaf was up-regulated differentially in response to stress such as chilling and exogenous ACC. The effect of exogenous PAs on SAMDC expression appears to be divergent. While putrescine up-regulated the expression of BJSAMDC1, spermidine and spermine down-regulated its expression. Furthermore, mannitol was also shown to up-regulate SAMDC expression in a gene-specific manner, in which the BJSAMDC1 transcript increases but other SAMDC transcripts are not affected.

Notes: FtsH is a membrane-bound ATP-dependent metalloprotease complex found in prokaryotes and organelles of eukaryotic cells. It consists of one or two trans-membrane helices at its amino-terminus, a highly conserved ATPase domain, which relates it to the AAA protein family, and a zinc-binding domain towards its carboxy-terminus that serves as the proteolytic site. Most bacteria contain a single FtsH gene, but the cyanobacterium Synechocystis has four. The Arabidopsis thaliana genome contains 12 genes encoding FtsH proteins, nine of them can be targeted to chloroplasts, whereas the other three are mitochondrial. Chloroplast FtsH protease is located in the thylakoid membrane, where it forms complexes, most likely hexamers, whose ATPase and proteolytic domains are exposed to the stroma. It is involved in the degradation of the D1 protein of photosystem II reaction centre during its repair from photoinhibition, as well as in the degradation of unassembled proteins in the thylakoid and the stroma. In Arabidopsis, FtsH2 is the most abundant isomer, followed by FtsH5, 8 and 1. This hierarchy is well reflected in the severity of the variegated phenotype of mutants in these genes.

Notes: Lon-, Clp- and FtsH-like proteases, members of three families of ATP-dependent proteases derived from bacterial ancestors, have been identified in plant mitochondria. Classifications of mitochondrial-specific paralogues of plant ATP-dependent proteases, based on targeting prediction programs and different experimental methods, are compared. Accumulating evidence points to similarities in the structure and the mechanisms of action used by various ATP-dependent proteases. Therefore, before focusing on plant mitochondrial ATP-dependent proteases, the paper discusses general features of ATP-dependent proteases. To date, information about structure and function of plant mitochondrial Lon-like, Clp-like and FtsH-like proteases is rather scarce, but indicates that these enzymes, like their bacterial and eukaryotic homologues, combine proteolytic and chaperone-like activities to form mitochondrial protein quantity and quality control system in plants.

Notes: Several studies have shown that protease inhibitors can suppress programmed cell death in various plant species and plant tissues. This is especially true of caspase inhibitors that can block programmed cell death and its marker DNA laddering. There are up to six different caspase-like activities that can be measured in plant extracts, the most prominent being caspase1-like and caspase3-like. These activities can be located in vacuoles and also in the nucleus or the cytoplasm. This represents a striking apparent similarity with animal programmed cell death. Because there are no caspase orthologue in plant genomes, a major challenge is to identify these proteases. Recently two proteases with caspase-like activities have been recognized as belonging to two different protease families that are not closely related to animal caspases. Various other protease families have been implicated and this suggests that complex protease networks have been recruited for the plant cell demise.

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: Soil salinity is a major factor affecting crop productivity worldwide. This study explores mechanisms that contribute to salt tolerance in rice (Oryza sativa L.). Hydroponically grown, 2-week-old salt tolerant and sensitive indica rice varieties, Pokkali and Jaya, respectively, were exposed to a 48-h stress period with NaCl (0–250 mM). When exposed to 200 mM NaCl, micromolar levels of external Ca2+ elevated survival of both varieties. The Ca2+ levels required were lower for Pokkali than for Jaya, but resulted in significantly higher survival. Estimates of Na+ and K+ in root and shoot compartments were made by flame photometry, while X-ray microanalysis was used to localize Na+ in the extracellular matrix of the shoot. Transpirational bypass flow was estimated using the apoplastic tracer, 8-hydroxypyrene-1,3,6-trisulphonic acid, trisodium salt. Our data demonstrate a Ca2+-dependent reduction in Na+ transport to shoots, which correlated with a decline in bypass flow and of Na+ in the transpirational stream. In addition, the Na+ that enters the shoot is partitioned among several distinct compartments. Survival is inversely correlated with Na+ levels in the shoot apoplastic fluid, which surrounds the cell and influences cytosolic composition. Pokkali maintained lower Na+ in its apoplast compared with the salt sensitive Jaya at the same total shoot Na+. Na+ in the apoplast appears to be regulated by sequestration into intracellular compartments. This sink supplements the primary response of reducing Na+ influx into the shoot and effectively buffers the apoplastic fluid in Pokkali. All of these mechanisms are operational in Jaya as well but are deployed less effectively.

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: An experiment was performed to elucidate interspecific differences in survival time of grass species subjected to an extreme climatic event. We exposed eight grass species to a simulated heat wave in the field (‘free air’ temperature increase at 11°C above ambient) combined with drought. We determined whether interspecific differences in survival time were related to the responses of the species to the imposed stress or could be explained by their ecophysiological or morphological characteristics in unstressed conditions. Surprisingly, there was no effect of specific leaf area, but species with a higher total leaf area survived longer. This may arise from a greater water reserve in the plant as a whole, which could delay the desiccation of the meristem, or from reduced evaporation due to a higher leaf area index. Species in which the decrease in light-saturated stomatal conductance (gs) and photosynthetic CO2 uptake rate (Amax) was strongly related to the decrease in soil water availability (measured as soil relative water content and stress duration) survived longer than species in which gs and Amax likewise declined but responded more to daily fluctuations in irradiance, temperature, and vapor pressure deficit during the heat wave. We, therefore, hypothesize that interspecific differences in stress survival time might be related to the extent to which stomata react to changes in soil water conditions relatively to changes in other environmental and physiological factors. The results suggest that resistance to extremes is governed by other mechanisms than resistance to moderate drought.

Notes: Rice (Oryza sativa L.) seeds were soaked for 18 h in distilled water in the absence (–PBZ) or presence (+PBZ, a triazole) of 100 mg l−1 paclobutrazol and then air dried. These air-dried seeds were germinated in the dark and then cultivated in a Phytotron. Twelve-day-old –PBZ and +PBZ seedlings were treated or not with CdCl2. Cd toxicity was judged by the decrease in biomass production, decrease in chlorophyll and protein content, increase in NH4+ content and induction of oxidative stress. The results indicated that PBZ applied to seeds was able to protect rice seedlings from Cd toxicity. On treatment with CdCl2, the abscisic acid (ABA) content increased in +PBZ leaves, but not in –PBZ leaves. The decrease in the transpiration rate of –PBZ seedlings by CdCl2 was less than that of +PBZ seedlings. Exogenous application of the ABA biosynthesis inhibitor, fluridone (Flu), reduced ABA accumulation, increased the transpiration rate and Cd content, and decreased the Cd tolerance of +PBZ seedlings. The effects of Flu on the Cd toxicity, transpiration rate and Cd content were reversed by the application of ABA. It seems that the PBZ-induced Cd tolerance of rice seedlings is mediated through an accumulation of ABA.

Notes: A comparison was performed of the tetrapyrrole transformations that occur upon irradiation of epicotyl or leaves of dark-grown Pisum sativum L. (var. Zsuzsi, Hungary). High performance liquid chromatography analysis after continuous or flash-irradiation showed that the biosynthetic pathway from protochlorophyllide (Pchlide) to chlorophyll (Chl) a was markedly slowed down at the step of the reduction of geranylgeranyl(gg)-Chl to dihydrogeranylgeranyl (dhgg)-Chl in epicotyls, whereas phytyl-Chl was synthesized in leaves subjected to the same light treatments. Quantitative pigment analysis during continuous irradiations of different intensities also showed that significant Pchlide photodestruction occurred in epicotyls even under weak light. When both Pchlide and chlorophyllide and/or chlorophylls were present in epicotyls, Pchlide photodestruction was faster under 630-nm light than under 670-nm light, which indicates that this process is most efficiently promoted by Pchlide excitation. Pre-incubation of epicotyl segments with 10 mM ascorbate partly alleviated pigment photodestruction in white light. It is concluded that formation of photoactive Pchlide–Pchlide oxidoreductase complexes is important to prevent fast pigment photooxidation after Pchlide accumulation in the dark.

Notes: Ethylene is produced by plants in response to a wide variety of environmental signals and mediates several developmental processes in higher plants. We investigated whether ethylene has a regulatory function in nodulation in the actinorhizal symbiosis between Discaria trinervis and Frankia BCU110501. Roots of axenic D. trinervis seedlings showed aberrant growth and reduced elongation rate in the presence of ethylene donors [i.e. 2-aminocyclopropane carboxylic acid (ACC) and 2-chloroethylphosphonic acid (CEPA)] in growth pouches. By contrast, inhibitors of ethylene synthesis (aminoethoxyvinylglycine, AVG) or perception (Ag+) did not modify root growth. This indicates that the development of D. trinervis roots is sensitive to elevated ethylene levels in the absence of symbiotic Frankia. The drastic response to higher ethylene levels did not result in a systemic impairment of root nodule development. Nodulation occurred in seedlings inoculated with Frankia BCU110501 in the presence of ethylene donors or inhibitors. Overall, the ability of the seedlings to shut down nodule formation in the younger portions of the root (i.e. to autoregulate nodulation) was not significantly impaired by a modification of endogenous ethylene levels. In contrast, we detected subtle changes in the nodulation pattern of the taproots. As a result of exposing the roots to CEPA, less nodules developed in older portions of the taproot. In line with this observation, AVG or Ag+ caused the opposite effect, i.e. a slight increase in nodulation of the mature regions of the taproot. These results suggest that ethylene is involved in modulating the susceptibility for nodulation of the basal portion of D. trinervis seedling roots.

Notes: Feedback de-excitation (FDE) is a process that protects photosystem II from damage during short periods of overexcitation. Arabidopsis thaliana mutants lacking this mechanism have reduced fitness in environments with variable light intensities. We have assayed the physiological consequences of mutations resulting in the lack of FDE and analysed the differences between field-grown plants and plants grown under fluctuating light in the laboratory. We show that FDE is an important mechanism in short-term responses to fluctuating light. Anthocyanin and carbohydrate levels indicated that the mutant plants were stressed to a higher degree than wild-type (WT) plants. Field-grown mutants were photo-inactivated to a greater degree than WT, whereas mutant plants in the fluctuating light environment in the laboratory seemed to downregulate the photosynthetic quantum yield, thereby avoiding photo-damage but resulting in impaired growth in the case of one mutant. Finally, we provide evidence that FDE is most important under conditions when photosynthesis limits plant growth, for example during flower and seed development.