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: 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: 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.

Notes: In plant tissue, a wound signal is produced at the site of injury and propagates or migrates into adjacent tissue where it induces increased phenylalanine ammonia lyase (PAL, EC 4.3.1.5) activity and phenylpropanoid metabolism. We used excised mid-rib leaf tissue from Romaine lettuce (Lactuca sativa L., Longifolia) as a model system to examine the involvement of components of the phospholipid-signaling pathway in wound-induced phenolic metabolism. Exposure to 1-butanol vapors or solutions inhibited wound-induced increase in PAL activity and phenolic metabolism. Phospholipases D (EC 3.1.4.4), an enzyme involved in the phospholipid-signaling pathway is specifically inhibited by 1-butanol. Re-wounding tissue, in which an effective 1-butanol concentration had declined below active levels by evaporation, did not elicit the normal wound response. It appears the 1-butanol-treated tissue developed resistance to wound-induced increases in phenylpropanoid metabolism that persisted even when active levels of 1-butanol were no longer present. However, a metabolic product of 1-butanol, rather than 1-butanol itself, may be the active compound eliciting persistence resistance. Inhibiting a subsequent enzyme in the phospholipid-signaling pathway, lipoxygenase (LOX; EC 1.13.11.12) with 1-phenyl-3-pyrazolidinone (1P3P) or reducing the product of LOX activity with diethyldithio-carbamic acid (DIECA) also inhibited wound-induced PAL activity and phenolic accumulation. The effectiveness of 1-butanol, DIECA, and 1P3P declined as the beginning of the 1-h immersion period was delayed from 0 to 4 h after excision. This decline in effectiveness is consistent with involvement of the inhibitors in the production or propagation of a wound signal. The wound signal in lettuce moves into adjacent tissue at 0.5 cm h−1, so delaying application would allow the signal to move into and induce the wound response in adjacent tissue before the delayed application inhibited synthesis of the signal. Salicylic acid (SA) inhibits allene oxide synthase (AOS, EC 4.2.1.92), another enzyme in the phospholipid-signaling pathway. Exposure to 1 or 10 mM SA for 60 min reduced wound-induced phenolic accumulation by 26 or 56%, respectively. However, 1 mM SA lost its effectiveness if applied 3 h after excision, while 10 mM SA remained effective even when applied 4 h after excision. At 1 mM, SA may be perturbing the wound signal through inhibition of AOS, while at 10 mM it appears to have some generally inhibitory effect on subsequent phenolic metabolism. These data further implicate the phospholipid-signaling pathway in the generation of a wound signal that induces phenolic metabolism in wounded leaf tissue.

Notes: Many higher plants accumulate free proline (Pro) to counteract osmotic stress. To examine the role of free Pro in salt resistance, we suppressed the tobacco Pro dehydrogenase (NtProDH) gene using a double-stranded RNA interference technique in tobacco Bright Yellow 2 cells. Northern blot analysis showed reduced levels of the NtProDH transcripts in the transgenic line. The free Pro level in transgenic cells was about 1.2- to 3.0-fold, and the Pro dehydrogenase activity was about 4.9–32.2% of those in wild-type (WT) cells. The transgenic cells had an appearance markedly different from that of WT cells. Microscopic analysis revealed that the transgenic tobacco cells were mostly barrel shaped as in a filament, cylindrical and small. In synchronous cultures, transgenic cells showed more active cell division than WT cells. Hypersensitivity to exogenous Pro increased in the transgenic tobacco cells. The transgenic cells showed an increased osmotolerance, perhaps by free Pro accumulation.

Notes: This study examined tobacco (Nicotiana tabacum cv. Petit Havana SR1) leaf respiration in the dark, utilizing both wild-type plants and transgenic plants with increased or decreased levels of alternative oxidase (AOX) protein. AOX represents a non-energy-conserving branch in mitochondrial electron transport. Inhibitor studies showed that the maximum possible flux of electrons to AOX (AOX capacity) correlated with the level of AOX protein present in the different plant lines. A comparison of the plants using online 18O isotope discrimination was done to determine whether AOX protein level would impact the actual steady-state partitioning of electrons to AOX (AOX engagement). Under a range of pretreatment and measurement conditions, there was little if any effect of AOX protein level on the degree of engagement. This suggests that the metabolic conditions inherent to a particular growth condition and/or the biochemical regulatory properties of AOX itself are the critical factors that control partitioning. Interestingly, we found that measurement temperature and water status are parameters that may have some influence over AOX engagement.

Notes: A (1→3),(1→4)-β-glucan synthase catalysing the synthesis of (1→3),(1→4)-β-glucan (mixed-linkage glucan) was investigated using microsomal membranes prepared from developing barley (Hordeum vulgare L. cv. Shikokuhadaka 97) endosperms harvested 21 days after flowering. The microsomal fraction produced (1→3),(1→4)-β-glucan by incorporation of [14C]Glc from UDP-[14C]Glc. The production of (1→3),(1→4)-β-glucan was ascertained by specific enzymatic digestion with endo-(1→3),(1→4)-β-glucanase (lichenase; EC 3.2.1.73) from Bacillus amyloliquefaciens, which released a radiolabelled trisaccharide (3-O-β-cellobiosyl-glucose) and a tetrasaccharide (3-O-β-cellotriosyl-glucose), the diagnostic oligosaccharides for the identification of (1→3),(1→4)-β-glucan. Digestion of the products with exo-(1→3)-β-glucanase (EC 3.2.1.58) from Basidiomycete QM806 released radiolabelled Glc, indicating that not only (1→3),(1→4)-β-glucans but also (1→3)-β-glucans (callose) had been formed due to the presence of (1→3)-β-glucan (callose) synthase (EC 2.4.1.34) in the microsomal fraction. The activity of (1→3),(1→4)-β-glucan synthase was maximal at pH 9.0 and at 25°C and in the presence of at least 2 mM Mg2+. The apparent Km and Vmax values for UDP-Glc were 0.33 mM and 480 pmol min−1 mg protein−1, respectively. Investigating the dependence of enzyme activity on developmental stage (7–35 days after flowering) of the endosperms, we found an increase of activity during the initial development reaching a maximum at 19 days, followed by a gradual decrease as the endosperms matured. The amount of (1→3),(1→4)-β-glucan in the cell walls of the endosperms, however, increased gradually towards maturation, even after 19 days. Analysing the relationship between enzyme activity and (1→3),(1→4)-β-glucan deposition in cell walls of endosperms prepared from 12 different barley varieties harvested 11–22 days after flowering showed that some varieties had both low activity and low glucan content, and in some both were high. But for several other varieties, the availability of donor substrate and other factors seem to influence the production of (1→3),(1→4)-β-glucan as well.

Notes: Red clover, Trifolium pratense L., is the dominant forage legume in Sweden and is usually harvested twice per year, once in June and once in August. Two 15N-based methods –15N isotopic dilution (ID) and 15N natural abundance (NA) – were used to study N2 fixation from spring until first harvest in late June, from first to second harvest in late August, and from second harvest until first frost in autumn in Umeå, Sweden. The material studied comprized three neighbouring fields carrying a first year ley, a second year ley and a third year ley. For the 15N ID method, small amounts of highly enriched 15N-nitrate were added to experimental plots. The non-legumes in the plots, essentially Phleum pratense L. together with Festuca pratensis L., served as reference plants for both the ID and 15N NA measurements. Dry matter, N and 15N were separately analysed in leaves (laminae), stems (including petioles), stubble and roots. The proportion of N derived from air (pNdfa) was then calculated for each plant part and for whole plants. Estimates of the proportion of N derived from N2 fixation (pNdfa) were always very high, usually ≥0.8. Generally, estimates of pNdfa obtained by the ID and NA methods were similar, but the ID method gave higher estimates of pNdfa than the NA method when the highest N2 fixation levels were recorded, at the August harvest. Regression analyses suggest that estimates of pNdfa in leaves could provide useful indications of pNdfa in shoots and whole T. pratense plants, thus avoiding the need for time-consuming root analyses.

Notes: Trehalose exists in most living organisms and functions as a storage carbohydrate and as an osmoprotectant in yeast, fungi, and bacteria. Trace amount of endogenous trehalose was detected in flowering plants, and the trehalose biosynthetic pathway was essential for embryo maturation in Arabidopsis. Conversely, exogenous trehalose was toxic to higher plants and severely curtailed root and shoot growth. In the current study, 30 mM trehalose was added to 2-week-old liquid cultures containing Arabidopsis thaliana (Columbia ecotype) seedlings. Densely stained granular particles were detected in the extracellular space of cotyledons and roots of trehalose-treated seedlings using transmission electron microscopy. Expression levels of 91 transcripts were altered by 1–6 h of trehalose treatment using DNA microarray analysis, and 65 of these encoded either known proteins or putative proteins with known functions. The exogenous trehalose treatment altered transcript levels of transcription factors, cell wall modification, nitrogen metabolism, and stress-related, defense-related, and fatty acid biosynthesis genes. Many of the transcripts altered by exogenous trehalose treatment were associated with the ethylene and methyl jasmonate-signaling pathways. The above findings suggested that trehalose, or metabolites derived from trehalose, are important regulators of plant gene expression in higher plants.

Notes: Elevated atmospheric CO2 concentration [CO2] and different levels of nitrogen (N) nutrition can influence the amount of excess excitation energy in photosystem (PS) II and related photosynthetic properties. The interactive effect of two [CO2] levels (ambient: 360 µM M−1 and elevated: 720 µM M−1) and two N levels (high: 700 mg N plant−1 and low: 100 mg N plant−1) on these properties was examined in seedlings of Japanese white birch (Betula platyphylla var. japonica) using simultaneous measurements of gas exchange and chlorophyll fluorescence. Photosynthetic acclimation to elevated [CO2], as indicated by a decline in carboxylation efficiency (CE), was observed in plants grown at elevated [CO2] especially under low N. Elevated [CO2] resulted in a decrease in area-based leaf N content (Narea) irrespective of N treatment. The adverse effect of elevated [CO2] and low N on CE may have been exacerbated by a greater accumulation of leaf sugar and starch contents in these plants leading to a lower electron transport rate (ETR). While these plants also showed higher non-photochemical quenching (NqP) that could offset the reduction in energy dissipation through ETR to some extent, they still have a higher risk of photoinhibition from excessive excitation energy in PSII as indicated by a decrease in photochemical quenching (qP). However, chronic photoinhibition was not observed in plant grown at elevated [CO2] and low N because they showed no difference in Fv/Fm (the maximum photochemical efficiency of PSII) from those grown at ambient [CO2] and low N after an overnight dark adaptation. High levels of NqP in plants grown at elevated [CO2] and low N reflect a near saturation of thermal energy dissipation. This impaired capacity of photoprotection would render these plants more vulnerable to photoinhibition in the event of additional environmental stresses such as drought, low or high temperature.

Notes: Carbon isotope ratio of leaf dry matter, δ13C, was measured on species occurring within Baiyin desert community, consisting of valley, slope and ridge microhabitats, and within Shandan desert community, consisting of Gobi desert and seasonal flooded creek microhabitats, in Northwest China. δ13C of C3 species increased with a decrease in soil water availability, suggesting that water-use efficiency (WUE) increased with decreasing soil moisture, whereas for all C4 species, δ13C tended to decrease with decreasing soil water availability, suggesting that WUE also increased with decreasing soil moisture. Above results indicated that water-use pattern was conservative under drought for C4 and C3 plants. In this present study, C4 species' occurrences within different microhabitats were investigated and C4 plants were observed to be absent and/or scarce within relatively lower soil moisture microhabitats, whereas they occurred and/or even had a high abundance within relatively higher soil moisture microhabitats, suggesting limited moisture available was a key factor of limiting C4 distribution in arid region of Northwest China.

Notes: The expression and activity of type 1 NAD(P)H dehydrogenase (NDH-1) were investigated in Synechocystis PCC 6803 cells during different growth phases (i.e. lag, logarithmic, stationary and decline phases). The relative amount of NDH-1, estimated by Western blot analysis using antibodies against NdhH, NdhI and NdhK, increased more than two-fold during growth from the lag to the logarithmic phase and then decreased after the logarithmic phase to reach lowest levels after 15 days (decline phase). The activity of light-dependent NADPH oxidation and cyclic electron flow around photosystem I (PSI) changed nearly in parallel with the amount of NdhH, NdhI and NdhK in cells across the growth phases. In contrast, the activity of photosynthetic O2 evolution and respiratory O2 uptake was not significantly different across phases of growth; the fluctuation of the activity at different phases was within 40%. These results suggested that the activity of light-dependent NADPH oxidation and PSI-cyclic electron flow are restricted by the amount of NDH-1 and that other factor(s) are limiting the rates of photosynthesis and respiration.

Notes: Arabinogalactan proteins (AGPs) are abundant plant cell-surface proteoglycans widely distributed in plant species. Crossed electrophoresis patterns of AGPs isolated from cultured cells before and after protonemata development differed, indicating that AGPs are involved in protonemata differentiation and development. Moreover, the addition of β-glucosyl Yariv reagent (βglcY), which binds specifically to AGPs, inhibits protonemata differentiation in cells of the liverwort Marchantia polymorpha L. cultured in protonemata-inducing medium. Transmission electron microscopic examination revealed that βglcY caused conspicuous disorder at the cell surface and the accumulation of abnormal structures between the plasma membrane and the cell wall. These results suggest that AGPs/βglcY complexes caused disturbances at the cell surface and inhibited cell-wall synthesis required for protonemata differentiation. Our results indicate that AGPs play a significant role in cell-wall synthesis during the protonemata-differentiation process of M. polymorpha.

Notes: When grown at a low P supply, Hakea prostrata R.Br. (Proteaceae) develops dense clusters of determinate branch roots, termed ‘proteoid’ or ‘cluster’ roots and accumulates Mn in its leaves. The aim of this study was to vary the production of cluster roots and assess the relationship between Mn uptake and cluster-root mass. We collected native soil from a location inhabited by H. prostrata and amended this with ‘high’ and ‘low’ amounts of insoluble or soluble P. After 14 months, we measured the impact of the treatments on cluster-root development and the [P], [Mn], [Fe], [Zn] and [Cu] in young (expanding) and mature leaves. Dry mass and leaf area increased with increasing P availability in the soil, but growth decreased at the highest soluble [P], which caused symptoms of P toxicity. The [P] in young leaves (1.3–2.7 mg g−1 DM) exceeded that in older leaves (0.28–0.85 mg g−1 DM), except when plants were grown with soluble P (3.2–21 mg g−1 DM). Cluster-root formation was inhibited when leaf [P] increased; [P] in young leaves, rather than that in old leaves, appeared to be the factor that determined the proportion of the root mass invested in cluster roots. Old leaves of all treatments had [Mn] from 90 to 120 µg g−1 DM, except for plants grown at high levels of soluble P, when [Mn] decreased below 30 µg g−1 DM. The [Mn] and [Zn] in old leaves and the [Cu] in young leaves were positively correlated with the fraction of roots invested in cluster roots. These findings support our hypothesis that cluster roots play a significant role in micronutrient acquisition, and also provide an explanation for Mn accumulation in leaves of H. prostrata, and presumably Proteaceae in general.

Notes: 3-Deoxyanthocyanins provide bright orange-red colours to flowers of some members of the Gesneriaceae, including sinningia (Sinningia cardinalis). We examined 3-deoxyanthocyanin biosynthesis in sinningia, in particular, the expression of key flavonoid biosynthetic genes and the activities of the encoded proteins. Two abundant 3-deoxyanthocyanins, luteolinidin 5-O-glucoside and apigeninidin 5-O-glucoside, three flavone glycosides, luteolin 7-O-glucoside, luteolin 7-O-glucuronide and apigenin 7-O-glucuronide, and the cinnamic acid verbascoside were identified in sinningia petal tissue. Small amounts of a 3-hydroxyanthocyanin were also detected in a limited region of the petal. cDNA clones for three flavonoid enzymes, flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase/flavanone 4-reductase (DFR/FNR) and anthocyanidin synthase (ANS), were isolated from a sinningia cDNA library made from petal RNA and used to measure transcript abundance during petal development. Only very low levels of F3H transcript were detected, while DFR/FNR transcript was highly abundant. ANS transcript levels were intermediate between these two. The F3H cDNA was shown to encode a functional F3H protein by complementation of the phenotype of an Antirrhinum majus F3H mutant. The recombinant DFR/FNR had activity against both flavanone and dihydroflavonol substrates to a comparable extent. The results suggest a mechanism of 3-deoxyflavonoid biosynthesis in sinningia similar to that reported for Zea mays, in which lack of F3H activity allows action of the DFR/FNR on flavanone substrates and production of flavan-4-ols. These are then likely converted to 3-deoxyanthocyanins through the action of the ANS and subsequent glucosylation.

Notes: The responses of photosynthetic gas exchange, chlorophyll fluorescence, activities of antioxidant enzymes and lipid membrane peroxidation of two contrasting Picea asperata Mast. populations to 30% of full sunlight (shade) and full sunlight (sun) were investigated under well-watered and drought conditions. Two contrasting populations were from the wet and dry climate regions in China, respectively. For both populations tested, drought resulted in lower needle relative water content (RWC), CO2 assimilation rate (A), stomatal conductance (gs) and effective PSII quantum yield (Y), and higher non-photochemical quenching (qN), superoxide dismutase (SOD), ascorbate peroxidase (APX) activities as well as malondialdehyde (MDA) levels and electrolyte leakage in sun plants, whereas these changes were not significant in shade plants. For the wet climate population, shade plants showed higher chlorophyll contents (Chla, Chlb and Chla + b) than sun plants under both well-watered and drought conditions. Our study results implied that shade, applied together with drought, ameliorated the detrimental effects of drought. On the other hand, compared with the wet climate population, the dry climate population was more tolerant to drought in the sun treatment, as indicated by less decreases in A and mass-based leaf nitrogen content (Nmass), more responsive stomata, greater capacity for non-radiative dissipation of excitation energy as heat (analysed by qN), and higher level of antioxidant enzyme activities as well as lower MDA content and electrolyte leakage. These results demonstrated that the different physiological strategies were employed by the P. asperata populations from contrasting climate regions when the plants were exposed to drought and shade.

Notes: Strains of Arabidopsis thaliana that lack a DNA glycosylase to recognize and remove 7,8-dihydro-8-oxoguanine from their DNA are expected to be compromised in their ability to deal with this highly mutagenic base, which is formed in the presence of reactive oxygen species (ROS). We have identified two strains, one containing a Ds insertion in an exon of the gene that codes for oxoguanine glycosylase and one containing a T-DNA insertion in the gene that codes for formamidopyrimidine glycosylase (both EC 3.2.2.23), and have crossed them to produce the double mutant. The homozygous mutant strains showed no phenotypic difference from the wild type in growth, development or reproductive potential under either normal conditions or conditions known to induce the formation of ROS. The lack of phenotype may be ascribed to the redundant nature of the base excision repair pathway in Arabidopsis. Longer multigenerational studies may be needed to determine the quantitative selective advantage of individual DNA glycosylase genes.

Notes: The combined effects of inorganic phosphate (Pi) insufficiency and CO2 enrichment on metabolite levels and carbon partitioning were studied using roots of 9-, 13- and 17-day-old barley seedlings (Hordeum vulgare L. cv. Brant). Plants were grown from seed in controlled environment chambers providing 36 ± 1 Pa (ambient) or 100 ± 2 Pa (elevated) CO2 and either 1.0 mM (Pi sufficient) or 0.05 mM (Pi insufficient) Pi. When values were combined for both Pi treatments, plants grown under enhanced CO2 showed increased root dry matter, adenylates (ATP + ADP), glutamine and non- structural carbohydrates other than starch. In contrast with shoots, enhanced CO2 partially reversed the inhibition of root dry matter formation imposed by Pi insufficiency. The Pi-insufficient treatment also increased sucrose, glucose and fructose levels in barley roots. The Pi and CO2 treatments were additive, so that the highest soluble carbohydrate levels were observed in roots of Pi-insufficient plants from the elevated CO2 treatment. Pi limitation decreased dry matter formation, acid-extractable Pi, nitrate, hexose-phosphates, glutamate, glutamine and acid invertase activity of barley roots in plants grown in both ambient and elevated CO2. Adenylate levels in roots were unaffected by the moderate Pi insufficiency described here. Thus, the reduced hexose-phosphate levels of Pi-insufficient roots were not likely to be the result of low adenylate concentrations. The above results suggest that the capacity of barley roots to utilize carbohydrates from the shoot is inadequate under both Pi-insufficient and CO2-enriched treatments. In addition, the Pi and CO2 treatments used here alter the nitrogen metabolism of barley roots. These findings further emphasize the importance of avoiding nutrient stress during CO2 enrichment experiments.

Notes: The plant hormone auxin plays a crucial role in the upstream regulation of many processes, making the study of its action particularly interesting to understand plant development. In this review we will focus on the effects auxin exerts on cell cycle progression, more specifically, during the initiation of lateral roots. Auxin fulfils a dominant role in the initiation of a new lateral root primordium. How this occurs remains largely unknown. Here we try to integrate the classical auxin signalling mechanisms into recent findings on cell cycle regulation. How both signalling cascades are integrated appears to be complex and is far from understood. As a means to solve this problem we suggest the use of a lateral root-inducible system that allows investigation of the early signalling cascades initiated by auxin and leading to cell cycle activation.

Notes: The sesquiterpenoid hormone abscisic acid (ABA) regulates many aspects of plant growth and development. It has been difficult, however, to understand how this hormone functions in a myriad of events. Genetic analysis, particularly in Arabidopsis, has identified genes that modulate ABA responsiveness, but a molecular framework has not been developed to explain how these genes direct ABA-mediated developmental events. Certainly, some of the diversity of processes influenced by ABA is a result of crosstalk with other signalling pathways. In other cases, the complex development of a multicellular organism with different cell types and growth conditions throughout its life cycle also increases the possible output signals of ABA action. In this article, we touch on some of these issues in the context of ABA signalling during embryogenesis. On a more speculative level, we propose that a developmental and molecular framework of ABA action in the embryo may be gained from two chemically related terpenoid signalling hormones in animals: juvenile hormone (JH) and retinoic acid (RA). Many of the developmental issues with regard to ABA action in plant embryos are mirrored in JH studies from invertebrates, and the molecular action of RA in vertebrates suggests that transcriptional regulation is a direct output of RA addition. Both of these systems may be useful in furthering our developmental and molecular understanding of ABA action in plants.

Notes: Since 1995 the role of fungal hypaphorine in plants has been widely investigated and its IAA-antagonist activity recognized. Evidence of competitive antagonism includes organ development, gene expression or molecule–molecule interaction levels. Based on present knowledge, three sites of hypaphorine/IAA competition and subsequent signalling pathways have been hypothesized: the extracellular signalling pathway, the intracellular signalling pathway, and the transmembrane signalling pathway. Hypaphorine with other active indole alkaloids should be regarded as a new class of IAA antagonist finely regulating specific steps of plant growth or development.

Notes: The plant hormone auxin has long been known to play a crucial role in plant growth and development, but how it affects so many different processes has remained a mystery. Recent evidence from genetic and molecular studies has begun to reveal a possible mechanism for auxin action. In this article we will present an overview with specific emphasis on auxin's role in roots of Arabidopsis thaliana, focusing on cell division, elongation and differentiation.

Notes: Drought stress is the major limitation to crop productivity. However, crops are genetically complex with many loci contributing quantitatively to a given physiological trait. Nonetheless, significant in-roads into the molecular mechanisms of drought-adaptive responses have been made from the use of Arabidopsis thaliana. In this special review, we will discuss results gleaned from reverse and forward genetic studies that revealed the involvement of both ABA-dependent and ABA-independent components. In particular, mutant analyses have highlighted the surprising prevalence of RNA metabolism in many key steps. We will also discuss our recent use of infrared thermography to visualize stomatal closure in response to dehydration as a means to identify novel regulatory genes. This has allowed us to recover mutations belonging to at least eight complementation groups. Analysis of six of these loci revealed that all of their corresponding mutations affect either abscisic acid (ABA) biosynthesis or perception. Hence, in contrast to molecular studies on gene networks which pointed to the clear existence of multiple ABA-independent pathways in the control of dehydration tolerance, our results reinforce ABA-based signalling pathways as the predominant factor in primary or rapid responses. Finally, we will provide some details learned from the molecular analysis of OPEN STOMATA1 (OST1), a gene that encodes an ABA-activated kinase issued from this targeted genetic approach.

Notes: We have studied the stomatal response in relation to the xylem-derived abscisic acid (ABA) accumulation in sunflower leaves. When ABA was introduced into detached leaves of the sunflower through xylem flux, stomatal conductance was regulated, water flux was changed as a result and at the same time the xylem-derived ABA was metabolised in the leaves. We computed the xylem-derived ABA accumulation in the leaves as a function of time by taking into account the variation of ABA flux into the leaves (the product of water flux and ABA concentration) and a continuing ABA metabolism. We found that ABA accumulation was rapid during an initial lag phase, much slowed down during the decreasing phase of stomatal conductance, but still substantial when stomatal conductance reached a new stable state. The results show a poor link between the kinetics of ABA-induced stomatal closure and the xylem-derived ABA accumulation. Xylem-derived ABA was metabolised rapidly in the leaves. Tetcyclacis, as an inhibitor, substantially inhibited this process. Two hours after ABA was fed into a leaf, about 70% of the fed ABA was metabolised, but when tetcyclacis was added into the feeding solution, less than 30% of ABA was metabolised, even after 24 h of incubation. The inhibition of ABA metabolism by tetcyclacis did not lead to more stomatal closure, which was still concentration-dependent. Since the accumulation of xylem-derived ABA was enhanced substantially by the presence of tetcyclacis, these results strongly indicate that stomata mainly respond to the prevailing ABA concentration in the xylem stream, rather than to the accumulated amount of xylem-derived ABA in the leaves.

Notes: Enzymatic digestion of the cell wall of Brassica napus hypocotyls gave a heterogeneous suspension of protoplasts with the cortical microtubules (CMTs) randomly organised or CMTs organised in parallel. The effect of variable g-influences has been tested on CMT organisation. In contrast to the 1 g-protoplasts, which reorganised the CMTs into parallel arrays during the 96 h test period, the frequency of randomly-oriented CMTs in the protoplasts exposed to simulated weightlessness (0 g) on a 2-D clinostat increased significantly during the same period. The opposite effect was obtained when the protoplasts were exposed to hyper-g (7 or 10 g), where the reorganisation of the CMTs into parallel arrays was accelerated compared to the 1 and 0 g-protoplasts. These results indicate that a unidirectional gravity force is a necessity for the reorganisation of CMTs in protoplasts to parallel arrays and that CMTs act as responding elements that are able to sense different levels of gravity. Besides the inability of the protoplasts to reorganise the CMTs into parallel arrays, the quantity of CMTs in the individual protoplast decreased during 4 days of simulated weightlessness, both compared to the CMTs quantity in the protoplasts immediately after isolation and compared to the 1 g- and hyper-g-protoplasts after 24 and 48 h of g-exposure. The size of the protoplasts was also affected by the g-exposure. Protoplasts exposed to simulated 0 g increased significantly after 24 and 48 h, whereas the 1 g- and 10 g-protoplasts maintained the same size during the 48 h test period.

Notes: The role of mitochondrial respiration in optimizing photosynthesis was assessed in mesophyll protoplasts of pea (Pisum sativum L., cv. Arkel) by using low concentrations of oligomycin (an inhibitor of oxidative phosphorylation), antimycin A (inhibits cytochrome pathway of electron transport) and salicylhydroxamic acid (SHAM, an inhibitor of alternative oxidase). All three compounds decreased the rate of photosynthetic O2 evolution in mesophyll protoplasts, but did not affect chloroplast photosynthesis. The inhibition of photosynthesis by these mitochondrial inhibitors was stronger at optimal CO2 (1.0 mM NaHCO3) than that at limiting CO2 (0.1 mM NaHCO3). We conclude that mitochondrial metabolism through both cytochrome and alternative pathways is essential for optimizing photosynthesis at limiting as well as at optimal CO2. The ratios of ATP to ADP in whole protoplast extracts were hardly affected, despite the marked decrease in their photosynthetic rates by SHAM. Similarly, the decrease in the ATP/ADP ratio by oligomycin or antimycin A was more pronounced at limiting CO2 than at optimal CO2. The mitochondrial oxidative electron transport, through both cytochrome and alternative pathways, therefore akppears to be more important than oxidative phosphorylation in optimizing photosynthesis, particularly at limiting CO2 (when ATP demand is expected to be low). Our results also confirm that the alternative pathway has a significant role in contributing to the cellular ATP, when the cytochrome pathway is limited.

Notes: Recent studies have suggested that Ca2+/calmodulin (CaM) or CaM-like proteins may be involved in blue light (BL)-dependent proton pumping in guard cells. As the increase in cytosolic concentration of Ca2+ is required for the activation of CaM and CaM-like proteins, the origin of the Ca2+ was investigated by measuring BL-dependent proton pumping with various treatments using guard cell protoplasts (GCPs) from Vicia faba. BL-dependent proton pumping was affected neither by Ca2+ channel blockers nor by changes of Ca2+ concentration in the medium used for the GCPs. Addition of Ca2+ ionophores and an agonist to GCPs did not induce proton pumping. However, BL-dependent proton pumping was inhibited by 10 mM caffeine, which releases Ca2+ from the intracellular stores, and by 10 μM 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ) and 10 μM cyclopiazonic acid (CPA), inhibitors of Ca2+-ATPase in the sarcoplasmic and endoplasmic reticulum (ER). By contrast, the inhibitions were not observed by 10 μM thapsigargin, an inhibitor of animal ER-type Ca2+-ATPase. The inhibitions by caffeine and BHQ were reversible. Light-dependent stomatal opening in the epidermis of Vicia was inhibited by caffeine, BHQ, and CPA. From these results, we conclude that the Ca2+ thought to be required for BL-dependent proton pumping may originate from intracellular Ca2+ stores, most likely from ER in guard cells, and that this origin of Ca2+ may generate a stimulus-specific Ca2+ signal for stomatal opening.

Notes: Plant aspartic proteinases have been characterized from seeds, flowers and leaves of a number of different species. The enzymes are generally either monomeric or heterodimeric, containing two peptides processed from the same precursor protein. The plant enzymes, like their mammalian and microbial counterparts, are active at acidic pH and inhibited by a class specific inhibitor pepstatin A. Plant aspartic proteinases are generally either secreted or targeted to the vacuolar/protein storage body compartment. The primary sequences of many of these enzymes have been determined and are very homologous with each other as well as with enzymes from mammalian and microbial origins. Plant aspartic proteinases, however, have a very unique plant specific region, which is not found in mammalian, microbial, or viral aspartic proteinases. The function of this region has not been elucidated. A role for these plant enzymes in protein processing or degradation has been proposed, however, more studies are required to confirm their in vivo functions. Recent intriguing results suggest possible roles for these enzymes in programmed cell-death of tissues and in pathogen resistance.

Notes: In shoots of the garden pea (Pisum sativum L.), the main bioactive gibberellin (GA) is GA1, which is synthesised from GA20 by 3β-hydroxylation. Gibberellin A20 is produced from GA19, as part of the process known as GA 20-oxidation. Because these steps are thought to be negatively regulated by GA1, we compared the metabolism of labelled GA19 and GA20 in mutants deficient in GA1, with that observed in isogenic wild-type (WT) plants. There was a large and specific increase in the 3β-hydroxylation of labelled GA20 in the GA1-deficient (dwarf) mutants, compared with the WT. Metabolism experiments did not provide convincing evidence for feedback regulation of 20-oxidation, possibly because GA19 akppears to be metabolised rapidly, even in WT pea shoots. Both 3β-hydroxylase and 20-oxidase transcript levels were markedly higher in the mutants than in isogenic WT lines. The results sukpport previous suggestions that both biosynthetic steps are feedback-regulated by GA1 in pea.

Notes: An overview is given of electrical events that occur in plant chloroplasts in association with their energization and subsequent photosynthetic performance. Special emphasis is given to the measuring techniques, in particular application of patch-clamp methods, which enable comparison of light-induced photocurrent and -potential kinetics of the thylakoid with kinetics of changes in chlorophyll fluorescence yield.

Notes: Pyrophosphate: d-fructose-6-phosphate 1-phosphotransferase (PFP; EC 2.7.1.90) and ATP: d-fructose-6-phosphate 1-phosphotransferase (PFK; EC 2.7.1.11) activities were determined in sugarcane varieties differing in sucrose content. For this purpose, activities were measured in those internodes where the maximum rate of sucrose accumulation occurs. The specific activity of internodal PFP varied significantly between the sugarcane varieties and was inversely correlated with the sucrose content. There was also a highly significant inverse correlation between PFP and sucrose content in a segregating F1 population. PFK activity was comparable to, or lower than, PFP activity and no relationship was evident between PFK activity and sucrose content. In all tissues investigated, the fructose 2,6-bisphosphate levels were probably sufficient to ensure full activation of PFP. The levels of PFP activity appear to be controlled by the expression of the β-subunit of the protein. The molecular mass of the PFP subunit polypeptide(s) was approximately 63 kDa. There was an inverse correlation between sucrose content and the partitioning of radiolabel into respiration in internodal tissue slices labelled with [U-14C]glucose across 3 sugarcane lines. The estimated flux of carbon into respiration correlated directly with PFP activity.

Notes: Twenty-one durum wheat genotypes originating from different geographic areas were grown during 3 successive years. The trials were characterised by different precipitation regimes. Carbon isotope discrimination (Δ), carbon content (CC) and ash content (ma) were assessed in the flag leaf during anthesis, then in the kernel at full maturity. Differences between the 3 years, due to water availability, were noted for Δ, ma, CC and yield. Genotypic differences were also noted within each year for all the traits studied. Some genotypes from the Middle East exhibited higher flag leaf and kernel Δ than those originating from the West of the Mediterranean basin. The kernel Δ was strongly correlated with grain yield (GY). The leaf Δ correlated with GY only under strong water limitation and with biomass production (BP) in favourable water conditions. For the flag leaf, Δ was correlated with ma and with CC. Silicon content was then assessed in the flag leaf and in the kernel on a subset of 10 genotypes differing in their Δ values. Strong positive correlations were noted between silicon content and Δ and ma for the flag leaf. However, no clear relationship was found between silicon content and GY. The results obtained in this study confirm the validity of kernel Δ as a predictive criterion for GY under water stress and suggest the possible use of kernel ma as an alternative criterion to select genotypes with higher water stress tolerance.

Notes: In order to clarify the effect of abscisic acid (ABA) on anaerobic tolerance in alfalfa (Medicago sativa L.), the seedlings were subjected to anaerobic stress after pretreatment with ABA. At concentrations〉 1 μM, ABA pretreatment increased the root viability of the seedlings to anaerobic stress and the viability increased with increasing ABA doses. At 100 μM ABA, the viability was 2.5-fold greater as compared with that of control seedlings. Roots of the seedlings rapidly lost ATP under the anaerobic stress; however, the decrease in ATP was much slower in the ABA-pretreated seedlings than the control seedlings. At 12 h after onset of the stress, ATP concentrations in the roots of 100 and 10 μM ABA-pretreated seedlings were 2.7- and 2.0-fold that of the control seedlings, respectively. During the period of ABA pretreatment under aerobic condition, ABA increased alcohol dehydrogenase (ADH, EC 1.1.1.1) activity in the roots until 12 h and then leveled off. The maximum ADH activities were 4.3- and 2.8-fold that in the roots of the control seedlings for 100 and 10 μM ABA-pretreated seedlings, respectively. After being subjected to the anaerobic stress, both ADH activities in the roots of the ABA-pretreated and the control seedlings increased but the differences in their activity remained. These results suggest that ABA pretreatment may maintain ATP level due to induction of ADH activity, which may be one of the causes of increasing anaerobic tolerance in the seedlings.

Notes: To elucidate how plants adapt to overheating followed by water deficiency, experiments with two cotton (Gossypium hirsutum L.) cultivars (Ok-oltin and INEBR-85) were performed. Preliminary heat-shock (HS) treatment (45°C for 1.5 h) increased resistance of both cultivars to subsequent progressive soil drought [40 days without watering, with soil moisture gradually decreasing from 70 to 20% of field moisture capacity (FMC)]. HS induced accumulation of amino acids and amides and increased their contribution to the osmotic pressure (OP) of the leaf cell sap. HS also enhanced resistance to water deficiency and to overheating of the leaves, especially in cv. INEBR-85, the more drought resistant of the two cultivars. The results suggest the existence of common resistance systems to both stress factors, in particular, accumulation of amino acids and amides (mainly arginine, proline and asparagine) – their concentration in the cell sap increased up to 240-, 160- and 150-fold, respectively.

Notes: Messenger RNA translation is a tightly regulated event in maize axes during germination. To obtain a deeper understanding of this process, the present research was centered on the expression regulation of the translation initiation factor eIF-4E within this period. Two maize eIF-4E isoforms were isolated by mGTP-Sepharose affinity chromatography. The eIF-iso4E protein content remained at a constant level in the axes during the first 24 h of maize germination, whereas the eIF-4E level increased after 12 h of germination as indicated by western blot analysis. Fast in vivo eIF-iso4E, but not eIF-4E, de novo synthesis was found within 6–12 h of germination. Northern blot analysis of total RNA from ungerminated axes with cDNAs encoding either -iso4E or -4E indicated that both transcripts were present in the stored mRNA pool of maize axes. Transcription for any of these mRNAs was not detected before 12 h of germination. Northern blots of polysomal RNAs indicated that the eIF-iso4E stored transcript is selectively recruited into polysomes for translation as early as 6 h of germination, whereas this does not occur for the eIF-4E transcript. The above data demonstrate differential 4E isoform expression regulation during maize germination. Expression of the eIF-iso4E protein appears to be translationally controlled, whereas the expression of the eIF-4E protein might be regulated at the transcriptional level.

Notes: Callus derived from the roots of Inmil® cherry rootstock (Prunus incisa×serrula) proliferates in a hormone-free solid medium. When transferred to a hormone-free liquid medium, such callus forms somatic embryos. On the other hand, leaf-derived callus of P. incisa×serrula and leaf- and root-derived calli of P. domestica require exogenous auxin for sustained growth and never form embryos. Levels of free and esterified indole-3-acetic acid (IAA) were similar in both types of calli grown on a solid medium, whereas the amide-conjugated IAA was higher in the root-derived embryogenic one. Transfer to a liquid medium did not affect the level of both free and conjugated IAA in the nonembryogenic callus, but in the embryogenic callus it decreased the level of amide-conjugated IAA. Isotopic dilution of C-IAA taken up from a medium was faster in the embryoegenic than in the nonembryogenic calli, which shows that the rate of IAA metabolism was higher in embryogenic callus. Besides IAA, indole-3-ethanol and indole-3-acetyl-N-aspartate were detected in nonembryogenic calli and in in vitro-grown shoots of P. domestica, whereas in embryogenic callus and in in vitro-grown shoots of P. incisa×serrula indole-3-acetamide was detected.

Notes: The metabolism of the polyamine precursors arginine and ornithine was studied in maturing and vernalised seeds of Picea abies (L.) Karst. (Norway spruce) in feeding experiments. Incorporation of radioactivity from these C-labelled amino acids into liberated CO2, amino acids, polyamines, proteins and cell wall fractions, as well as polyamine levels were determined in embryos and megagametophytes. Ornithine and especially arginine decarboxylation was more active in the embryo than in the megagametophytic cells, and vernalisation increased arginine metabolism more than it increased ornithine metabolism. Both precursors were metabolised to each other, to other amino acids, and to polyamines. The only polyamine in which radioactivity incorporated was free putrescine, showing either a slow synthesis or a high degradation rate of spermidine and spermine in maturing spruce seeds. The putrescine level was approximately 10 times higher in the embryo than in the megagametophytic tissues, whereas spermidine and spermine levels were almost the same in both tissues. The label from arginine and ornithine was also incorporated into proteins as amino acids and post-translationally as polyamines. Higher radioactivity was seen in the small ≤14-kDa polypeptides. Protein hydrolysates of the embryo and the megagametophytic tissues contained spermidine and spermine and their degradation product 1,3-diaminopropane (DAP), suggesting that polyamines may play a role in the accumulation of seed storage protein and in the maturation of spruce seeds.

Notes: Soybean (Glycine max [L.] Merr. cv. Century) seedlings were germinated in vermiculite, sub-irrigated with a complete nutrient solution, without nitrogen or supplemented with 10 mM KNO3, 5 mM (NH4)2SO4, or 5 mM NH4NO3. After 14 days in the light, or 5 days in the dark, tissues from different organs were harvested separately. Similarly, tissues from different organs from 14- or 21-day-old nodulated or non-nodulated soybean seedlings, maintained in the absence of nitrogen, were harvested. Proteins and total RNA were isolated from the different plant organs and used for immunoblot and RNA blot analyses, respectively. Protein or RNA blots were separately incubated with antisera or hybridized with probes specific for either ferredoxin-dependent glutamate synthase (Fd-GOGAT) or NADH-dependent glutamate synthase (NADH-GOGAT). The specific activity and the abundance of the Fd-GOGAT peptide and transcript increased, approximately 3–10 times, in cotyledons and hypocotyls/stems in plants germinated in the light compared with those germinated in the dark. Fd-GOGAT activity, peptide, and transcript were highest in leaves. Except for increases in the specific activity of samples from roots treated with (NH4)2SO4 or NH4NO3, there were minor or no changes in Fd-GOGAT activity, peptide and transcript among organs of seedlings treated with different nitrogen sources or by nodulation. Low levels of NADH-GOGAT transcript were detected in all organs. NADH-GOGAT activity, peptide, and transcript increased in roots of seedlings treated with different nitrogen sources, but these changes were more apparent on RNA blots versus immunoblots. The highest NADH-GOGAT activity and most abundant amounts of the peptide and transcript were observed in nodules. Despite being induced by different environmental factors, both GOGAT activities are controlled, at least in part, by either gene expression or by RNA stability, because in most instances, both isoenzymes exhibited paralleled changes in specific activity and the abundance of their corresponding peptides and transcripts on immunoblot and RNA blots, respectively. However, there were some exceptions to the parallel increases in specific activities, peptides and transcripts which suggest that post-translational modification may also regulate the activties of the two GOGAT isoforms. Collectively, the results presented here suggest that the two GOGAT isoforms have distinct physiological functions in soybean.

Notes: Kiwifruit (Actinidia deliciosa) is a dioecious vine whose staminate and pistillate flowers nonetheless develop non-functional reproductive structures of the ompposite sex. Ubiquitin is a small, highly conserved protein found in all eucaryotes: a covalent ATP-dependent attachment of ubiquitin marks proteins for degradation. In the present paper, we used immunoblotting to investigate the presence of free ubiquitin and ubiquitin conjugates during pollen development in male (androfertile) and in female (androsterile) genotypes of kiwifruit. In the male, several high molecular mass protein conjugates were present throughout development. On the contrary, such a pattern characterized only early stages of pollen from the female genotype, where conjugates progressively disamppeared, until they were detectable only in trace amounts at anthesis. The highest content of conjugates in the male genotype was observed when microspores were ampproaching the first mitosis. Free ubiquitin increased continuously during development of the male microgametophyte so that mature pollen contained considerable amounts of the ubiquitin monomer at the time of its release from the anther. By contrast, only low levels were detectable in the degenerating microspores in the pistillate flowers. In vitro experiments using labeled ubiquitin indicated that early-uninucleate microspores of the female genotype had a much higher conjugation rate than those of the male genotype at the same stage. However, after feeding α-lactalbumin as exogenous substrate, the rate of ubiquitin conjugation strongly increased and was quite similar in both sexes. Nuclear features of pollen development in both genotypes are also described. The nucleus progressively degenerated in the microspores of the pistillate flowers starting from the early-uninucleate stage, in parallel with the progressive decrease in ubiquitin content and activity. At anthesis, the microspores in the pistillate flowers either had no nucleus or showed only traces of chromatin. Thus, the ubiquitin system seems to play an important role in protein turnover occurring during the normal developmental pathway of the kiwifruit microgametophyte, while it was mainly involved in regressive events related to microspore degeneration in the female genotype.

Notes: A growing body of evidence indicates that phenylpropanoid and flavonoid metabolism is catalyzed, not by free-floating ‘soluble’ enzymes, but via one or more membrane-associated multienzyme complexes. This type of macromolecular organization has important implications for the overall efficiency, specificity, and regulation of these pathways. Classical biochemical studies of phenylpropanoid and flavonoid metabolism have laid a solid foundation for this model, providing evidence of the channeling of intermediates between enzyme active sites and co-localization of enzymes in cell membranes. This work is now being extended using transgenic plants to determine how the partitioning of metabolites within these pathways is controlled, as well as applying sensitive methods to define specific interactions among the individual enzymes. Information from these studies promises to provide new insights into the structuring of biosynthetic pathways within cells, which should lead to more effective means for engineering the production of plant metabolites with nutritional and agronomic importance.

Notes: Tomato plants (Lycopersicon esculentum Mill. cv. Pera) were transformed via Agrobacterium tumefaciens with the binary vector pKYLX71 containing a tomato basic peroxidase (EC 1.11.1.7) gene, tpx1, under the control of the cauliflower mosaic virus (CaMV35S) promoter. Transgenic plants showed a 2–5-fold increase in the activity of the peroxidase ionically bound to the cell wall, whereas soluble peroxidase activity remained similar or even lower than wild-type plants. Isoelectric focusing showed the presence of a new isoperoxidase of pI ca 9 in the ionically bound extract. Western blot also showed the presence of a new band at 41 kDa that was absent in the wild-type extract. A 40–220% increment of lignin content of the leaf was found in transgenic plants. Shoot phenotype of transgenic plants was similar to wild type, although under stress, the plants appeared wilted and the new leaves had a reduced area and were thicker than wild-type or older transgenic leaves. The root system was underdeveloped in transgenic plants, but the rooting ability of the stem was not affected by the overexpression of peroxidase. Finally, the morphogenetic response of cotyledon and hypocotyl explants from transgenic plants was evaluated. In the case of cotyledons, the percentage of explants with shoot was not different from wild-type plants. For hypocotyl, one of the transgenic lines showed a 30% reduction in the percentage of shoot organogenesis. The results are discussed in relation to the role of tpx1 in lignin synthesis.

Notes: Levels of three major dehydrins of 65, 60, and 14 kDa have been observed to increase in blueberry (Vaccinium smpp.) floral buds during chill unit accumulation and cold acclimation and decrease during deacclimation and resumption of growth. Indeed, levels of the 65-, 60-, and 14-kDa dehydrins increase such that they become the most predominant proteins visible on sodium dodecyl sulfate (SDS)-polyacrylamide gels. The peptide sequence information from the 65- and 60-kDa dehydrins was used to synthesize degenerate DNA primers for amplification of a part of the gene(s) encoding the dehydrins. One pair of primers amplified a 174-bp fragment. The 174-bp fragment was used to screen a cDNA library (prepared from RNA from cold-acclimated blueberry floral buds) and resulted in the isolation of a clone with a 2.0-kb insert. The cDNA was sequenced and found to be a full-length clone encoding a K5-type dehydrin (5 K boxes). Five high-confidence peptide sequences, ranging from 9 to 25 amino acids long, obtained from the 60-kDa dehydrin exactly matched sequences encoded within the cDNA clone. Furthermore, amino acid composition of the 60-kDa dehydrin agreed well with the expected amino acid composition based on the cDNA sequence. However, the DNA sequence and coupled in vitro transcription/translation reactions of the cDNA clone indicated that it encodes a dehydrin with a native molecular mass of ∼40 kDa instead of 60 kDa. Experiments to determine if the dehydrins undergo post-translational modifications revealed that the 65- and 60-kDa dehydrins are glycosylated. Thus, our results indicate that the 2.0-kb dehydrin cDNA encodes the native version of the 60-kDa dehydrin. The dehydrin cDNA hybridized on RNA blots to two chilling/cold-responsive messages of 2.0 and 0.5 kb. Both the 2.0- and 0.5-kb messages increased to higher levels more quickly in the cold-hardy cultivar Bluecrop than in the less hardy cultivar Tifblue. In addition, the 0.5-kb message remained at a higher level longer in Bluecrop than in Tifblue.

Notes: Winter annual and perennial crop species grown in the northern boreal ecosystem must survive periods of protracted snow cover and low temperatures during the winter. In deep snow regions, plants are susceptible to winter stresses caused by both snow molds and low temperatures. Therefore, high levels of tolerance to freezing and snow molds are requisite for crops adapted to these regions. Accumulation of soluble carbohydrates in winter wheat during the autumn is linked to both hardening and resistance to attack by snow molds. Snow mold-resistant cultivars accumulate higher levels of carbohydrate and metabolize them at slower rates than susceptible cultivars. The quantity and quality of carbohydrates, particularly fructans, remaining in the spring after snow mold attack appear important for survival of winter wheat. However, the total accumulation of carbohydrates is dependent on the stage of development of the winter cereal plant at the beginning of the winter. Recent research findings have shown that sugars are pivotal metabolic activators of the sugar-sensing enzyme, hexokinase, which initiates signal transduction and activation of numerous metabolic genes including host defense genes. Thus, an understanding of the metabolism of soluble carbohydrates, particularly fructans, during plant growth, hardening, and snow mold infection, is essential to the elucidation of survival mechanisms in plants subjected to these winter stresses.

Notes: The involvement of the ascorbate-glutathione cycle in the defence against Cu-induced oxidative stress was studied in the roots of Phaseolus vulgaris L. cv. Limburgse vroege. All the enzymes of this cycle [ascorbate peroxidase (APOD), EC 1.11.1.11; monodehydroascorbate reductase (MDHAR), EC 1.6.5.4; dehydroascorbate reductase (DHAR), EC 1.8.5.1; glutathione reductase (GR), EC 1.6.4.2] were increased, and the total ascorbate and glutathione pools rose after a 15 μM root Cu treatment. In the first hours after the start of the experiment, the accumulation of dehydroascorbate (DHA), formed as a result of a Cu-mediated direct oxidation of ascorbate (AA), was limited by a non-enzymatic reduction using glutathione (GSH) as the reductant. At 24 h, the enzyme capacities of both DHAR and GR were increased to maintain the redox status of the AA and GSH pools. After 72 h of Cu application, the DHAR capacity was inhibited and MDHAR was responsible for maintaining the AA pool in its reduced form. Although the GR capacity was enhanced after 72 h in the treated plants, the GSSG/GSH ratio was increased. This could be due to direct participation of GSH in the detoxification of Cu through reduction and complexation.

Notes: In an effort to study regulation of starch synthesis in the source and sink tissues, a cDNA clone for starch granule-bound starch synthase (GBSSI), which encodes a 67-kDa protein, was isolated from a cDNA library prepared from tuberous roots of sweet potato (Ipomoea batatas Lam. cv. Tainong 62). This GBSSI protein contains a signal peptide of 77 amino acids, and the mature protein has a molecular mass of about 59 kDa. The mature protein shares 75–85% sequence identity with GBSSIs of dicotyledonous plants and about 70% identity with those of monocotyledons. The sequence of the signal peptide, on the other hand, differs substantially from those of the dicotyledons and monocotyledons studied, although their hydropathic profiles are all similar. This GBSSI gene was well expressed in tuberous roots, leaves, and stems, but not in roots. However, mechanisms involved in regulating the expression of this gene were different between tuberous roots and leaves. In tuberous roots, the synthesis of GBSSI transcript increased coordinately with tuberous root expansion; nevertheless, accumulation rates of GBSSI protein in starch granules remained constant regardless of tuberous root sizes, suggesting an involvement of post-transcriptional regulation for the synthesis of this protein. The levels of GBSSI transcript were investigated in photosynthetic tissues during diurnal cycles, and the results suggest that the transcription of the GBSSI gene in leaves is controlled by the endogenous circadian rhythm.

Notes: The early cellular events in leaf explants of Medicago sativa L. cultured on somatic embryogenesis- and callogenesis-inducing media (EIM and CIM, respectively) were correlated with the endogenous contents of polyamines (PAs) and aromatic monoamines (AMs). On the second day of culture, replication of DNA occurred in epidermal and subepidermal cells on the edges of explants on EIM and was a prerequisite for proembryonal mass and, later, globular proembryo formation. In explants cultured on CIM, replication occurred at least one day later and in fewer cells, which were randomly spread all over the explant. Transition of leaf explant cells to rapidly dividing meristematic-like cells on EIM or to enlarged, highly vacuolated cells on CIM was observed. The increase in total PA levels in cultured explants was primarily a consequence of increases in putrescine (Put) and spermidine (Spd) contents and was much more pronounced on EIM than on CIM. High Spd levels were characteristic of meristematic cells and might be essential for the development of globular structures. The higher amount of insoluble PA conjugates was determined in explant cells on EIM, as compared with CIM. Proembryogenic cell mass formation was positively correlated with free tyramine and negatively correlated with free phenylethylamine contents.

Notes: Immunofluorescent and immunogold labeling was used to study the localization of cytokinins in developing somatic and zygotic embryos of Tilia cordata Miller. Broad-specificity polyclonal antibodies active against dihydrozeatin riboside (DHZR)/zeatin riboside (ZR)-type and isopentenyladenosine (iPR)-type cytokinins were used for immunolabeling. Immunofluorescent microscopy showed that these cytokinins were concentrated in highly cytoplasmic cells showing meristematic character. Cotyledon initials and primary meristems of heart-stage somatic embryos, as well as heart-stage zygotic embryos, were labeled. During elongation of embryos, cytokinin immunoreactive material was concentrated to areas having meristematic character. Root apex, shoot meristem, and cotyledon cells of somatic and zygotic cotyledonary embryos, as well as epidermal and subepidermal cell layers of the hypocotyl, showed the strongest immunoreaction. The nucleoli, especially, had a very strong signal. Results at the ultrastructural level with gold-conjugated protein A supported these conclusions. Gold particles were distributed in the nuclei, especially in the nucleoli and throughout the ground cytoplasm. They were occasionally associated with plastids and mitochondria, but seldom with other organelles.

Notes: Leaf discs of Capsicum annuum L. were illuminated in air enriched with 1% CO2 in the absence or presence of lincomycin, an inhibitor of chloroplast-encoded protein synthesis. The loss of functional photosystem (PS) II complexes with increase in cumulative light dose (photon exposure), assessed by the O2 yield per single-turnover flash, was greater in leaves of plants grown in low light than those in high light; it was also exacerbated in the presence of lincomycin. A single exponential decay can describe the relationship between the loss of functional PSII and increase in cumulative photon exposure. From this relationship we obtained both the maximum quantum yield of photoinactivation of PSII at limiting photon exposures and the coefficient k, interpreted as the probability of photoinactivation of PSII per unit photon exposure. Parallel measurements of chlorophyll fluorescence after light treatment showed that 1/Fo−1/Fm was linearly correlated with the functionality of PSII, where Fo and Fm are the chlorophyll fluorescence yields corresponding to open and closed PSII reaction centers, respectively. Using 1/Fo−1/Fm as a convenient indicator of PSII functionality, it was found that PSII is present in excess; only after the loss of about 40% functional PSII complexes did PSII begin to limit photosynthetic capacity in capsicum leaves.

Notes: Characteristics of the cell cycle in cortical regions (0–0.6 mm from the root-cap junction) of the primary root of lentil (Lens culinaris L.) during germination in the vertical position on earth were determined by iododeoxyuridine labelling and image analysis. All cells were in the G1 phase at the beginning of germination and the duration of the first cell cycle was about 25 h. At 29 h, around 14% of the cortical nuclei were still in the G2 or M phases of the first cell cycle, whereas 53 and 33% of the nuclei were respectively in the G1 or S phase of the second cell cycle. In parallel, the cell cycle was analysed in root tips of lentil seedlings grown in space during the IML 2 mission (1994), (1) on the 1-g centrifuge for 29 h, (2) on the 1-g centrifuge for 25 h and placed in microgravity for 4 h, (3) in microgravity for 29 h, (4) in microgravity for 25 h and placed on the 1-g centrifuge for 4 h. The densitometric analysis of nuclear DNA content showed that in microgravity there were less cells in DNA synthesis and more cells in G1 than in the controls on the 1-g centrifuge (flight and ground). The comparison of the sample grown continuously on the 1-g centrifuge in space and of the sample grown first in 1-g and then in microgravity indicated that 4 h of microgravity modified cell cycle, increasing the percentage of cells in the G1 phase. On the contrary, the transfer from microgravity to the 1-g centrifuge (for 4 h) did not provoke any significant change in the distribution of the nuclear DNA content. Thus the effect of microgravity could not be reversed by a 4 h centrifugation. As the duration of the first cell cycle in the lentil root meristem is about 25 h, the results obtained are in agreement with the hypothesis that the first cell cycle and/or the second G1 phase was lengthened in absence of gravity. The difference observed in the distribution of the nuclear DNA content in the two controls could be due to the fact that the 1g control on board was subjected to a period of 15 min of microgravity for photography 25 h after the hydration of the seeds, which indicated an effect of short exposure to weightlessness. The mitotic index of cortical cells was greater on the 1-g centrifuge in space than in any other sample (flight and ground) which could show an effect of the centrifugation on the mitosis.

Notes: Olive trees are often subjected to low temperatures during winter. To quantify the effects of low temperatures on the water relations of olive trees, we studied the responses to low soil temperatures on winter days of variable evaporative demand (ET0) in 1-year-old potted olive (Oleo europaea L. cv. Picual) trees in 1996 and 1997. Low night (2.5 and 5.2°C) but ambient day soil temperatures (above 10°C) did not affect stomatal conductance (gs), leaf (Ψleaf) and stem (Ψstem) water potentials. Soil temperature levels inducing water stress in olive trees were determined for winter days with ET0 typical for southern Spain (ET0= 1.5 ± 0.3 mm day−1). Leaf and stem water potential decreased and root hydraulic resistance (rroot) increased when trees were exposed to night and day soil temperatures below 10°C. Stomatal conductance was not affected at soil temperatures between 6.4 and 10°C, but decreased at temperatures below 6.4°C. The soil temperature levels affecting the water uptake of olive trees remained relatively constant over the range of ET0 of 1-2 mm day−1 during winter and early spring months. However, the soil temperature influencing gs appeared to be more variable and was affected by ET0. Olive tree recovery from low soil temperature stress depended on stress duration and severity and interacted with ET0. Recovery of ψ started already during the stress period, probably induced by stomatal closure and high rroot, thus allowing tree rehydration overnight. Root hydraulic resistance contributed the major part of whole-tree hydraulic resistance in response to cold stress, accounting for 76 and 89% at 6.4 and 4.6°C, respectively; which indicates that rroot is the primary control of the water status in olive trees under low temperatures.

Notes: The effects of the ratio of Rubisco activase to Rubisco (activase/Rubisco ratio) on light dependent activation of CO2 assimilation were investigated during leaf aging of rice. Changes of photosynthetic CO2 gas exchange rates in relation to step increases of light intensity from two photon flux densities of 60 µmol m−2 s−1 (low initial PFD) and 500 µmol m−2 s−1 (high initial PFD) to saturated PFD of 1 800 µmol m−2 s−1 were measured. These photosynthetic activation processes were considered to be limited by the Rubisco activation rate when analyzed by the relaxation method. The relaxation time of low initial PFD gradually declined from 3 to 33 days after leaf emergence and showed high and negative correlation to the activase/Rubisco ratio. The initial rate of Rubisco activation under low initial PFD linearly correlated to the amounts of Rubisco activase, whereas these were almost constant from 3 to 23 days after leaf emergence. But these correlations could not be recognized in the case of high initial PFD. Moreover, the relaxation times were more sensitive to intercellular CO2 concentration (Ci) under high initial PFD than under low initial PFD, especially, at Ci below 300 µl l−1. These results suggest the involvement of the activase/Rubisco ratio in the photosynthetic activation under relatively low initial PFD, and the limitation of photosynthetic activation under relatively high initial PFD by Rubisco carbamylation during leaf aging of rice.

Notes: Peroxidase (EC 1.11.1.7) activity from homogenized tissue or in apoplastic fluid was analyzed along the developmental gradient of expanding B73 maize (Zea mays L.) leaf blades. Soluble plus ionically bound peroxidase activity from homogenized tissue was present in high levels at the leaf base, which includes the region of cell division, and decreased as tissue was displaced away from the base by growth. A different pattern of change in peroxidase activity was seen in apoplastic fluid extracted from segments of intact tissue, where an increase in peroxidase activity preceded a rapid decrease in leaf elongation rate. Similar patterns in peroxidase activity from homogenized and intact tissue have been found in leaf blades of tall fescue (Festuca arundinacea Schreb.), suggesting a common phenomenon. At the location within the elongation zone where the increase in apoplastic peroxidase activity occurred, the activities of neutral and acidic (pl 4.6) peroxidase isoforms were also elevated in both the homogenate and in apoplastic fluid. The coincidence of these isoforms with the decline in leaf elongation rate suggests they may contribute to cessation of growth. At the distal end of the elongation zone, the activities of other acidic peroxidases (pI 5.6 and 5.7) increased in the homogenate and in apoplastic fluid, and remained elevated as tissue was displaced into the maturation region. The location of their appearance and their relatively high activity in the maturation region suggest the involvement of these isoforms in lignification.

Notes: Hexanal and cis-3-hexenal are principal flavor volatiles in ripe tomato fruit, but whether they accumulate during ripening or are formed upon maceration of the tissue has not been clarified. This has been addressed by measuring levels of these aldehydes in green and ripe fruit with discrimination between intrinsic aldehyde content and aldehyde generation following tissue disruption. Volatile sampling of tomato fruit homogenates was accomplished by purge/trapping, followed by thermal desorption on a gas chromatograph equipped with a mass selective detector. Incubation of some samples with alcohol dehydrogenase to convert the aldehydes to their respective alcohols permitted positive identification of the isomeric form of hexenal as cis-3-hexenal. Red and green tomato fruit homogenized in buffer with saturated CaCl2 contained low (0.1-0.8 µg g−1 fresh weight) levels of hexanal and cis-3-hexenal; thus there is minimal endogenous volatile content in intact fruit. Volatile levels increased rapidly, up to 10-fold, following homogenization of ripe tomato fruit in the absence of CaCl2, and more modestly in corresponding green tomato fruit homogenates. Incubation with the appropriate lipoxygenase/hydroperoxide lyase substrate (linoleic acid for hexanal, linolenic acid for cis-3-hexenal) doubled the amount of volatile compound produced. Hexanal generation was suppressed in the presence of linolenic acid, suggesting that the enzyme complex has greater affinity for this substrate. As well, levels of cis-3-hexenal, but not hexanal, tended to decline within 30 min of homogenization, possibly reflecting a specific degradative process. The results collectively indicate that the contribution of six-carbon aldehydes to tomato fruit flavor is attributable to metabolism invoked following tissue disruption rather than within the intact fruit.

Notes: A protein kinase (PK-II), phosphorylating casein, was purified from ripening mango, Mangifera indica L., fruit tissue. The purification procedure consisted of ammonium sulphate fractionation and sequential anion exchange-, dye-ligand, and gel filtration chromatography. The enzyme was purified over 500-fold to near homogeneity with a recovery of 4%. The purified enzyme had a specific activity of ca 1 µmol mg−1 protein min−1 with ATP as phosphoryl donor. SDS-PAGE results indicated a monomeric enzyme with molecular mass of 35 kDa. The protein kinase phosphorylated the acidic substrates casein and phosvitin, but had a very low activity with histones and protamine sulphate. The optimum pH and temperature for catalysis were determined to be 9.6 and 35°C, respectively. Mn2+ could not substitute for the Mg2+ needed for activity and Ca2+ had a slight stimulatory effect. Phospholipids, cAMP, calmodulin and the calmodulin inhibitor, calmidazolium, did not have any significant effect on activity, but the enzyme was inhibited by heparin and the specific inhibitor, CKI-7, (N-[2-aminoethyl]-5-chloroisoquinoline-8-sulphonamide). Autoradiographic studies revealed the ability of the protein kinase to autophosphorylate as well as the presence of endogenous protein substrates in the crude extract. Initial velocity studies with casein as substrate and product inhibition studies with ADP indicated a Km (ATP) and Km (casein) of 14 µ M and 0.18 mg ml−1, respectively, with a Ki (ADP) of 3.2 µM. The enzyme can be classified as a casein kinase I type of protein kinase (EC 2.7.10).

Notes: Stem elongation can be suppressed by a temperature drop at the onset of the photoperiod (DROP) or with a cooler day than night temperature (DT and NT, respectively), commonly described as DIF (DT - NT). To test our hypothesis that phytochrome A (phyA) mediated the reduction of stem elongation caused by −DIF and DROP, we conducted experiments with photomorphogenic mutants of tomato (Solarium lycopersicon L.) and transgenic potato (Solarium tuberosum L.). The plants studied were tomato mutants fri1 (deficient in phyA) and tri3 (deficient in phytochrome B1 [phyBl]) and their isogenic wild-type (WT) cv. Moneymaker, nontransformed potato, and two lines each of antisense phyA (15-9 and 15-11) and overexpressed phyA (PS-2 and PS-4). Plants were placed in three temperature regimens with a daily mean of 20°C: a constant 20°C (0 DIF), an 8°C DROP for 3 h, and a - 8°C DIF. For all tomato genotypes, −DIF and DROP reduced intemode length by ≥ 21% and stem elongation by 30% compared to that of plants at 0 DIF. Interactions between temperature treatment and genotype were nonsignificant. For potato, −DIF, but not DROP, significantly reduced intemode length of WT (by 39%) and both antisense lines (by 36 or 48%) but only one of the two lines of overexpressed phyA plants (by 18%). The −DIF significantly reduced stem length for only antisense phyA (by 36 or 48%) and WT (by 35%) plants. Thus, at least for tomato and potato, it appears that phyA does not control stem extension in relation to cool-temperature treatments.

Notes: Purified preparations of NAD(H)-glutamate dehydrogenase (GDH, EC 1.4.1.2.) were assayed to determine the effects of mono- and divalent cations, nucleotides and select carbon compounds on NAD(H)-dependent GDH activity. The amination reaction was stimulated 2- to 17-fold by divalent cations (Ca2+ 〉 Cd2+ 〉 Co2+ 〉 Mg2+ 〉 Mn2+ 〉 Zn2+ between 1 and 1000 µM), but the reaction was unaffected by monovalent cations (Na + and K +). The amination reaction was most responsive to changes in Ca2+ in a NADH-dependent manner. The addition of EDTA or EGTA nullified the stimulatory effects of Ca2+. Calmodulin alone or in combination with calmodulin antagonists did not affect the amination reaction. Divalent cations (at 1 mM) inhibited the rate of the deamination reaction by 15 to 25%, while monovalent cations had no effect. ATP inhibited the amination reaction by 10 to 60%, while ADP had little or no effect. ATP or ADP decreased the rate of the deamination reaction 23 to 60 or 20 to 38%, respectively. Many tricarboxylic acid cycle intermediates inhibited the amination reaction, 20 to 50% of the inhibition could be attributed to the chelating capacity of intermediates. Conversely, most of the carbon sources tested did not affect the deamination reaction, the only appreciable differences were increases in activity with sucrose (21%) and glucose (41%) and a decrease in activity with pyruvate (34%). Inhibitors of sulfhydryl groups were used to examine the importance of reduced thiol groups in the amination or deamination reactions. The amination was not dependent on reduced thiol groups, whereas the deamination reaction was dependent on reduced thiol groups.

Notes: The mechanism of cobalt uptake was investigated using cells of the giant alga Chara corallina in which it is possible to resolve separately uptake by the cell wall and actual influx across the cell membrane. The absorption of 60Co by Chara cells appeared to saturate within 2 h, but this was mainly due to rapid uptake into the cell wall which accounted for 87–92% of the total activity. Even after prolonged desorption most of the cell-associated 60Co was found on the cell wall. The intracellular distribution of absorbed 60Co was investigated by fractionating the cell into cytoplasm and vacuole. It was shown that 60Co influx to the vacuole occurs simultaneously with influx to the cytoplasm. The transported species appears to be Co2+ rather than the less charged Co(OH)+ or Co(OH)2. 60Co influx is pH dependent (optimum pH 7–9), and is sensitive to some other divalent metals. Influx from solutions containing 1 µM60Co was inhibited by 5 µM Cd2+, Cu2+, and Zn2+, but Mn2+ and Ni2+ had no significant effect. The sensitivity of Co uptake to N-ethyl maleimide (NEM) and cysteine suggests that the transport system involves direct binding of CO2+ to -SH groups.

Notes: Experiments were carried out to evaluate the effects of exposure to nitric oxide on the ability by NADPH-dependent microsomal electron transfer to generate oxygen radicals. Such interactions could play a role in the potential antioxidant action of nitric oxide (NO). Isolated microsomes from soybean (Glycine max [L.] Merr. cv. Hood) embryonic axes were exposed to an exogenously added source of nitric oxide (NO) (S-nitrosoglutathione + dithiothreitol). The O2− generation rate by microsomes exposed to NO decreased significantly as compared to the rate measured in microsomes incubated in the absence of NO. The exposure of the microsomes to the NO donor did not alter the microsomal rate of hydroxyl radical generation. Preincubation of the microsomes with the NO donor affected neither iron reduction rate nor activity of cytochrome c reductase. However, cytochrome P450 activity was significantly inhibited after exposure to NO. This inhibition was completely prevented by hemoglobin. The data are consistent with the hypothesis that NO exhibits a potential antioxidant role in the plant cell by decreasing the rate of generation of superoxide anion. Since endogenous NO was detected in homogenates of soybean embryonic axes by EPR studies, this interaction between NO and cytochrome P450 in soybean embryonic axes could be a factor of relevance for the control of oxidative stress in vivo.

Notes: Three soluble invertase (EC 3.2.1.26) isoforms from Easter lily (Lilium longiflorum Thunb. cv. Nellie White) flower buds were purified to apparent homogeneity. Non-denaturing PAGE showed one band for all three invertases that corresponded to the invertase activity. SDS-PAGE of purified invertase I gave a single band at 78 kDa, whereas invertases II and III gave three bands at 54, 52 and 24 kDa. Antibodies against tomato fruit acid invertase and Urtica dioica leaf acid invertase recognized all three invertase isoforms, whereas antibodies against wheat coleoptile acid invertase recognized only 56- and 54-kDa bands of invertases II and III. Antibodies against wheat coleoptile invertase recognized the 54- and 52-kDa proteins from crude extracts of all flower organs, and a 72-kDa protein in both leaf and bulb scale extracts. All three invertases bound to Con-A peroxidase. Deglycosylation of invertase I with glycopeptidase F was complete and resulted in a peptide of 75 kDa. Invertases II and III were deglycosylated partially by glycopeptidase F and resulted in proteins of 53, 51, 50 and 22 kDa. Invertase I was localized only in anther and filament, whereas the other two isoforms were present in all flower organs.