bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of arginine decarboxylase by spermine in osmotically‐stressed oat leaves
Authors: Borrell A. , Besford R. T. , Altabella T. , Masgrau C. , Tiburcio A. F.
Published: May 2006

Biosynthesis of polyamines in plants is controlled primarily by the enzymes ornithine decarboxylase (EC 4.1.1.17) and arginine decarboxylase (ADC: EC 4.1.1.19), which are responsible for the production of putrescine, and S‐adenosyl‐L‐methionine (SAM) decarboxylase (EC 4.1.1.50) that is necessary for the formation of spermidine and spermine (Spm). Little is known about the metabolic or molecular mechanisms regulating the synthesis of these enzymes. We have studied the regulation of ADC synthesis by Spm in osmotically‐stressed oat (Avena sativa L. ev. Victory) leaves, using a polyclonal antibody to oat ADC and a cDNA clone encoding oat ADC. Treatment with Spm in combination with osmotic stress resulted in increased steady‐state levels of ADC mRNA, yet the levels of ADC activity decreased. This absence of correlation is explained by the fact that Spm inhibits processing of the ADC proenzyme, which results in increased levels of this inactive ADC form and a consequent decrease in the ADC‐processed form. Spermine treatment leads to delayed loss of chlorophyll in dark‐incubated and osmotically‐treated oat leaves. Thus, post‐translational regulation of ADC synthesis by Spm may be important in explaining its anti‐senescence properties.
Changes in levels of phosphoenolpyruvate carboxylase with induction of Crassulacean acid metabolism (CAM)‐like behavior in the C4 plant Portulaca oleracea
Authors: Mazen A. M. A.
Published: May 2006

Changes in levels of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31, orthophosphate: oxaloacetate carboxy‐lyase, phosphorylating) were followed in leaves and stems of CAM‐expressing and non‐expressing Portulaca oleracea L. plants. CAM expression was induced by growing plants under an 8‐h photoperiod and water stress conditions (SD‐WS). Leaves and stems of these plants (designated CAM) expressed nocturnal acidification with an oscillation pattern and an amplitude characteristic of CAM plants. Generally, PEPC activity increased by ca 3‐fold during the period of CAM induction. Over the day/night cycle. PEPC activity oscillated in a pattern typical of CAM plants. Treatment of the other plant group (designated as non‐CAM) by growth under a 16‐h photoperiod and well‐watered conditions (LD‐WW) did not induce expression of the tested criteria of CAM in plants. In these plants, nocturnal acidification as well as changes in the magnitude of PEPC, activity and fluctuation pattern were undetectable. SDS‐PAGE of leaf extracts of the CAM‐expressing plants and the corresponding densitometric scans show progressive increase in the amount of PEPC subunit protein (ca 95 kDa) during the period of CAM induction. These results show that induction of CAM‐like characteristics in the C4 plant Portulaca oleracea is also accompanied by increased PEPC activity, which may be partly due to an increase in enzyme synthesis.
Changes in non‐structural carbohydrates in olive (Olea europaea) leaves during root zone salinity stress
Authors: Tattini Massimiliano , Gucci Riccardo , Romani Annalisa , Baldi Alessandro , Everard John D.
Published: May 2006

Self‐rooted olive (Olea europaea L.) plants were grown in hydroponics at various NaCl concentrations (from 0 to 200mM) for 28 to 32 days followed by 28 to 30 days of relief from salinity over two growing seasons. Olive leaves accumulated both glucose and mannitol during the period of salinity stress. The concentrations of fructose, myo‐inositol, galactose, galactinol, sucrose, raffinose, and stachyose were not significantly affected by salinity. Starch content was decreased by salinity. The mannitol/glucose and mannitol/soluble carbohydrates ratios increased as the external NaCl concentration was increased, but returned to the control levels during the relief period. The increase in mannitol or glucose molar concentrations, expressed on a leaf tissue water basis, was partially due to a reduction in leaf tissue water content under salinity stress. However, an increase in mannitol concentration was also observed when expressed on a dry weight basis. The accumulation of mannitol in leaf tissue preceded any reduction in leaf area rate or net assimilation rate. The increase in leaf mannitol or glucose concentration was positively correlated with the increasing level of salinity at the root zone, but not with the accumulation of Na+ in the shoot. The role of mannitol. a potential osmoregulator in leaf mesophyll during salinity stress, is discussed in relation to the complex carbohydrate composition of olive leaves.
Storage and remobilisation of sulphur in beech trees (Fagus sylvatica)
Authors: Herschbach Cornelia , Rennenberg Heinz
Published: May 2006

Three‐year‐old beech trees were fed 35S‐sulphate in August 1993 via a flap in a mature leaf of an upper branch. Harvest of beech trees was performed 24 h after feeding 35S‐sulphate, before leaf senescence, after leaf abscission, in early winter (January 1994). in late winter (March 1994). before bud break and after bud break. Twenty‐four h after feeding 35S‐sulphate, 0.7 ± 0.5% of the 35S‐radioactivity taken up was exported out of the fed leaf. When trees were analysed 2 months later, i.e., before leaf senescence, this value had increased to 22 ± 7%. The exported 35S‐radioactivity was located in the branch containing the fed leaf (2.8 ± 13%). in basipetal parts of the trunk (41 ± 77%) and in the main rool (21 ± 6%). Leaves and apical parts of the trunk were no sink organs for the exported sulphur. Along the tree axis the main proportion of the radiolabel was located in the wood, predominantly in the acid soluble fraction. In the bark the greater portion of the radiolabel was found in the acid insoluble fraction. In both tissues the bulk of the 35S of the soluble fraction was sulphate together with small amounts of glutathione. This pattern did not change until bud break. After bud break, basipetal parts of the trunk lost part of its 35S‐radioactivity. Of the 35S‐radioactivity which had been exported out of the fed leaf during the previous autumn, 16 ± 2% remained in the trunk, whereas 47 ± 7% of the 35S was found in branches, mainly in the newly developed leaves. The present results show that sulphur, mainly in the form of sulphate, is stored along the tree axis in both bark and wood of beech trees and is re‐mobilised during leaf development in spring.
Cercospora beticola toxins. IX. Relationship between structure of beticolins, inhibition of plasma membrane H+‐ATPase and partition in lipid membranes
Authors: Gomés Eric , Simon‐Plas Françoise , Milat Marie‐Louise , Gapillout Isabelle , Mikès Vladimir , Pugin Alain , Blein Jean‐Pierre
Published: May 2006

Beticolins are yellow toxins produced by the fungus Cercospora beticola. The effect of one of them, beticolin‐1. has been investigated on corn root plasma membrane H+‐ATPase (EC 3.6.1.35) at different purification levels (plasma membrane fraction, partially, or highly purified enzyme). The results obtained demonstrated that (1) the purified proton pump was inhibited directly by low amounts of the toxin (I50= 1.62 ± 0.18 μM), (2) the biological effects of beticolin‐1 were similar to those of CBT (Cercospora beticola toxin). Furthermore, it was established that the efficiency of the different beticolins was clearly related to their ability to interact with the lipid bilayers, determined by fluorometric studies: the toxins that exhibited the lower I50 (50% inhibitory concentrations) values were the molecules that had the lowest partition coefficient to liposomes.
Molecular expression of genes involved in parthenocarpic fruit set in tomato
Authors: Fos Mariano , Nuez Fernando
Published: May 2006

To characterize the phenomenon of natural parthenocarpy in tomato (Lycopersicon esculentum Mill.) two different approaches have been followed. At a developmental level, the ovary weights of three non‐parthenocarpic lines and three near‐isogenic parthenocarpic (pat‐2) lines were compared. Four developmental stages were considered: flower bud, preanthesis, anthesis and 4 days after anthesis. The parthenocarpic lines displayed ovary weights higher than their respective non‐parthenocarpic lines from preanthesis to 4 days after anthesis. A molecular approach involved comparison of in vitro translation products from flower RNAs taken from the same developmental stages of non‐parthenocarpic and near‐isogenic parthenocarpic (pat‐2 and pat‐3/pat‐4) lines. Analysis by two‐dimensional polyacrylamide gel electrophoresis showed the differential expression of a 30‐kDa product in parthenocarpic materials from preanthesis to anthesis. These results suggest that the physiological and molecular events responsible for parthenocarpy begin at the preanthesis stage, before the flower is completely mature and receptive to pollination. The differential expression of this in vitro translation product in pat‐2 and pat‐3/pat‐4 genotypes also suggests a common or confluent molecular basis in genetically controlled parthenocarpy.
Proteolytic activity in the maize pollen wall
Authors: Radlowski Marek , Kalinowski Andrzej , Adamczyk Jozef , Krolikowski Zygmunt , Bartkowiak Slawomir
Published: May 2006

A new protease from maize (Zea mays L.) pollen is described. It was purified using gel filtration, ion exchange and high performance liquid chromatography. SDS‐PAGE and HPLC showed that the enzyme has a dimeric structure of M, ca 60,000. Inhibitor investigations indicated an aspartic acid residue in its active site. The optimum pH for maize pollen aspartic proteinase activity was 5.6, and the optimum temperature was 45°C. The enzyme is easily eluted from the pollen grains and, as confirmed by enzymoblotting after isoelectric focusing, it is located in the pollen wall. Similar to metallo‐proteinases, its activity is inhibited by Zn2+. The pL value for purified aspartic proteinase, as estimated after IEF, was 5.0. Two‐dimensional electrophoresis analysis of proteins eluted from maize pistils suggests that the enzyme digests the proteins and may be involved in pollen‐tube germination. The properties of serine and aspartic proteinases from maize pollen are compared.
Cytokinins and fruit development in the kiwifruit (Actinidia deliciosa). I. Changes during fruit development
Authors: Lewis David H. , Burge Garry K. , Schmierer David M. , Jameson Paula E.
Published: May 2006

The cytokinin content in fruit tissue of the kiwifruit (Actinidia deliciosa [A. Chev.] C. F. Liang et A. R. Ferguson var. deliciosa cv. Hayward) was monitored during fruit development to identify which cytokinins were present and if they were linked with specific stages of fruit growth. Cytokinins were isolated and purified by column chromatography and high‐performance liquid chromatography and quantified by radioimmunoassay. A novel HPLC step utilising an amine column was successfully introduced as a preparative step in the separation of the O‐ and 9‐glucosides from the free bases and ribosides. The radioimmunoassay results were validated, and the different cytokinins identified, by gas chromatography‐mass spectrometry. Cytokinins detected in fruit included the cytokinin free bases, zeatin and isopentenyladenine, their ribosides, nucleotides and both O‐ and 9‐glucosides. Both qualitative and quantitative changes of the cytokinins occurred during fruit development. A decrease in cytokinin concentration occurred after anthesis (from 342 pmol g−1 fresh weight at anthesis to 41 pmol g−1 fresh weight 27 days after anthesis). A large increase in cytokinin concentration and content per fruit occurred as the fruit reached commercial maturity (to 1900 pmol g−1 fresh weight). Individual cytokinins showed quite different patterns. Zeatin, in particular, showed a peak in concentration (13 pmol g−1 fresh weight) 11 days after anthesis that correlated with the beginning of the cell division phase of fruit growth. The accumulation of cytokinin (mostly zeatin riboside or zeatin nucleotide) in mature fruit may be of significance for the postharvest storage of kiwifruit fruit.
Cytokinins and fruit development in the kiwifruit (Actinidia deliciosa). II. Effects of reduced pollination and CPPU application
Authors: Lewis David H. , Burge Garry K. , Hopping Murray E. , Jameson Paula E.
Published: May 2006

The significance of changes in cytokinin content during early fruit growth was examined in the kiwifruit (Actinidia deliciosa var. deliciosa cv. Hayward). Fruit growth was modified by the reduction of seed number or by the application of the synthetic phenylurea cytokinin N‐(2‐chloro‐4‐pyridyl)‐N‐phenylurea (CPPU). The influence of these treatments on cell division was monitored by flow cytometry and changes in the endogenous cytokinins were measured at days 10 and 20 after anthesis, using high‐performance liquid chromatography and radioimmunoassay. Total cytokinin levels appeared not to be limiting growth since the highest total cytokinin concentration was detected in unpollinated fruit, which abscised by day 25 after anthesis. However, compared with control fruit which had the highest concentration of zeatin (Z) 10 days post anthesis, Z levels were low in unpollinated fruit. It is hypothesised that an increase in Z is the critical change in cytokinin metabolism required for the initiation of cell division and fruit growth. The synthetic cytokinin CPPU promoted fruit development, but there was a decrease in the endogenous cytokinin concentration. Zeatin was not detected in CPPU‐treated fruit. Cell division was reduced in unpollinated fruitlets but there was no significant difference (P > 0.05) between the other treatments. Differences in final fruit size appeared to be due to cell expansion.
Gravitropic responses of the Avena coleoptile in space and on clinostats. IV. The clinostat as a substitute for space experiments
Authors: Brown Allan H. , Johnsson Anders , Chapman David K. , Heathcote David
Published: May 2006

Gravitropic responses of dark grown oat coleoptiles were measured in weightlessness and under clinorotation on earth. The tests in microgravity were conducted in Spacelab during the IML‐1 mission and those on clinostats were conducted in laboratories on earth. The same apparatus was used for both kinds of tests. In both cases autotropism and gravitropic responsiveness were determined. This allowed a quantitative comparison between the plants' responses after receiving the same tropistic stimulations either in weightlessness or on clinostats.

Autotropism was observed with oat coleoptiles responding in weightlessness but it did not occur on clinostats. Gravitropic responsiveness was measured as the ratio between the incremental bending response (degrees curvature) and the corresponding incremental g‐dose (stimulus intensity times duration for which it was applied). Plants were tested at either of two stages of coleoptile development (i.e. different coleoptile lengths). From a total of six different kinds of critical comparisons that could be made from our tests that provided data for clinorotated vs weightless plants, three showed no significant difference between responses in simulated vs authentic weightlessness. Three other comparisons showed highly significant differences. Therefore, the validity of clinorotation as a general substitute for space flight was not supported by these results.
Pyridine nucleotide pool size changes in iron‐deficient Plantago lanceolata roots during reduction of external oxidants
Authors: Schmidt Wolfgang , Schuck Christian
Published: May 2006

The involvement of pyridine nucleotides in the reduction of extracytoplasmatic electron acceptors by iron‐deficient Plantago lanceolata L. roots has been examined by measuring the changes in NAD(P)H and NAD(P)− induced by various external acceptors. Exposure of the plants to FeEDTA, ferricyanide, ferric citrate or hexachloroiri‐date resulted in a transient decrease in NADPH and an increase in NAD−. No major differences in this pattern were observed between acceptors which were assumed to be reduced by different enzymes. The application of the membrane‐permeable oxidant nitro blue tetrazolium led to similar changes in reduced and oxidized pyridine nucleotides and decreased the reduction of external acceptors. The amino acid analog p‐fluorophenylalanine caused a transient decline in both NADPH level and NADPH/ NADP− ratio and a decrease in the ratio of NADH to NAD− without affecting the level of NADH. Exposure of the plants to the translation inhibitor cycloheximide increased both NADH and NADPH concentrations. A comparison of the redox activities and pyridine nucleotide fractions after inhibitor treatment revealed that the constitutive, but not iron stress‐induced redox activity correlates with NADPH levels. These results are interpreted as confirming that the redox systems on the root plasma membrane are separately regulated. Possible metabolic reactions during the reduction processes are discussed.
Polyamine metabolism and gene regulation during the transition of autonomous sugar beet cells in suspension culture from quiescence to division
Authors: Fowler Mark R. , Kirby Melissa J. , Scott Nigel W. , Slater Adrian , Elliott Malcolm C.
Published: May 2006

Sugar beet cells grown in batch suspension culture have been used to study the regulation of polyamine levels during the transition from a quiescent to a proliferating state. The quiescent state was achieved by maintenance of the phytohormone autonomous cells in the stationary phase of the batch culture cycle. After subculture into fresh medium there was an increase in DNA synthesis which was accompanied by a dramatic increase in cellular polyamine levels. The levels of both free and bound cellular putrescine and spermidine within the cells reached a peak before the onset of the first synchronous division. The levels of putrescine, spermidine and to some extent spermine in the culture medium also increased dramatically shortly after subculture. The increase in polyamines was preceded by a rapid but transient increase in omithine decarboxylase (EC 4.1.1.17) and S‐adenosylmethionine decarboxylase (EC 4.1.1.50). Arginine decarboxylase (EC 4.1.1.19) and S‐adenosylmethionine synthetase (EC 2.5.1.6) activity did not show the same pattern of cell division‐related variation. Inhibition of S‐adenosylmethionine biosynthesis with methylglyoxal bis‐(guanylhydra‐zone) (MGBG) reduced cell division in the suspension culture. Inhibitors of ornithine decarboxylase and arginine decarboxylase individually had little effect on cell division, but in combination led to a reduction in cell division. Addition of polyamines and their precursors to cells in the stationary phase of a batch culture cycle led to the induction of expression of a mitotic cyclin sequence (BvcycII).
Purification and characterization of 1‐SST, the key enzyme initiating fructan biosynthesis in young chicory roots (Cichorium intybus)
Authors: Van den Ende Wim , Van Wonterghem Dominik , Dewil Erna , Verhaert Peter , De Loof Arnold , Van Laere André
Published: May 2006

A genuine 1‐SST (sucrose:sucrose 1‐fructosy] transferase, EC 2.4.1.99) was purified and characterized from young chicory roots (Cichorium intybus L. var. foliosum cv. Flash) by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, anion and cation exchange chromatography. This protocol produced a 63‐fold purification and a specific activity of 4.75 U (mg protein)−1. The mass of the enzyme was 69 kDa as estimated by gel filtration. On SDS‐PAGE apparent molecular masses of 49 kDa (α‐subunit) and 24 kDa (β‐subunit) were found. Further specification was obtained by MALDI‐TOF MS detecting molecular ions at m/z 40109 and 19 896. These two fragments were also found on a western blot using an SDS‐boiled chicory root extract and chicken‐raised polyclonal antibodies against the purified 1‐SST, indicating that the enzyme is a heterodimer in vivo. The N‐terminus of chicory root 1‐SST α‐subunit was shown to be highly homologous with the cDNA‐derived amino acid sequences from barley 6‐SFT and a number of β‐fructosyl hydrolases (in‐vertases and fructan hydrolases). However, chicory root 1‐SST properties could be clearly differentiated from those of chicory root 1‐FFT (EC 2.4.1.100), chicory root acid invertase (EC 3.2.1.26) and yeast invertase. The enzyme mainly produced 1‐kes‐tose and glucose from physiologically relevant sucrose concentrations, indicating that this 1‐SST is the key enzyme initiating fructan biosynthesis in vivo. However, like chicory root 1‐FFT and barley 6‐SFT, the enzyme also showed some β‐fructofuranosi‐dase activity (fructosyl transfer to water) at very low sucrose concentrations. Although sucrose clearly is the best substrate for the enzyme, some transferase and β‐fructofuranosidase activity were also detected using 1‐kestose as the sole substrate.
Abscisic acid both promotes and inhibits photoperiodic flowering of Pharbitis nil
Authors: Takeno Kiyotoshi , Maeda Takahide
Published: May 2006

Abscisic acid (ABA) has been reported to have diverse effects on photoperiodic flowering. Activity of a natural ABA, (+)‐(S)‐abscisic acid (S‐ABA), was recently suggested to be somewhat different from that of racemic ABA, which has been used in previous work. Use of S‐ABA might enable clarification of the role of ABA in flowering. S‐ABA inhibited flowering of the short‐day plant Pharbitis nil (cv. Violet) when given before or 4 h after the start of a 14‐h inductive dark period, and promoted flowering when given 12 h after the start of the dark period or later. The flower‐promoting effect was observed when ABA was applied to the shoot apex. These results indicate that ABA has a dual effect on photoperiodic flowering of P. nil: it may inhibit the time‐measuring process as well as promote some processes that proceed after generation of the flowering stimulus.
CAM variations in the leaf‐succulent Delosperma tradescantioides (Mesembryanthemaceae), native to southern Africa
Authors: Herppich Werner B. , Midgley Guy , von Willert Dieter J. , Veste Maik
Published: May 2006

Drought responses of diurnal gas exchange, malic acid accumulation and water status were examined in Delosperma tradescantioides, a succulent that grows in drought‐prone microenvironments in summer rainfall and all‐year rainfall regions of southern Africa. When well‐watered, this species exhibited Crassulacean acid metabolism (CAM)‐cycling, but its carbon fixation pattern changed during the development of drought, shifting to either low‐level CAM or to CAM‐idling. The rate and pattern of this change depended on environmental conditions, duration of water stress and leaf age. At the onset of drought, diurnal malate fluctuation increased, but was strongly depressed (by ca 70%) as drought continued, and when leaf water content and water potential were low (ca 35 and 50% of the initial levels, respectively). When rewatered, rates of growth and photosynthesis, gas exchange and water status recovered fully to pre‐stressed values within two days. Whole‐shoot carbon uptake rates suggested that leaf growth had continued unabated during a short‐term (≅ one week) drought. This emphasises that CAM‐idling allows the maintenance of active metabolism with negligible gas exchange when soil water is limiting. It is possible that old or senescent leaves may provide water for the expansion of developing leaves during initial periods of drought. Regardless of the water regime and environmental conditions, leaf nocturnal malate accumulation and water content were positively correlated and increased with leaf age. Thus the gradual loss of water from older mature leaves may induce CAM‐idling, which reduces water loss. An important ecological consequence of this combination of CAM modes is the potential to switch rapidly between fast growth via C3 gas exchanges when well‐watered to water‐conserving CAM‐idling during drought.
Relative growth rate, biomass allocation pattern and water use efficiency of three wheat cultivars during early ontogeny as dependent on water availability
Authors: van den Boogaard Riki , de Boer Miriam , Veneklaas Erik J. , Lambers Hans
Published: May 2006

We have investigated the water use efficiency of whole plants and selected leaves and allocation patterns of three wheat cultivars (Mexipak, Nesser and Katya) to explore how variation in these traits can contribute to the ability to grow in dry environments. The cultivars exhibited considerable differences in biomass allocation and water use efficiency. Cultivars with higher growth rates of roots and higher proportions of biomass in roots (Nesser and Katya) also had higher leaf growth rates, higher proportions of their biomass as leaves and higher leaf area ratios. These same cultivars had lower rates of transpiration per unit leaf area or unit root weight and higher biomass production per unit water use. They also had higher ratios of photosynthesis to transpiration, and lower ratios of intercellular to external CO2 partial pressure. The latter resulted from large differences in stomatal conductance associated with relatively small differences in rates of photosynthesis. There was little variation between cultivars in response to drought, and differences in allocation pattern and plant water use efficiency between cultivars as found under well‐watered conditions persisted under dry conditions. At the end of the non‐watered treatment, relative growth rates and transpiration rates decreased to similar values for all cultivars. High ratios of photosynthesis to transpiration, and accordingly high biomass production per unit of transpiration, is regarded as a favourable trait for dry environments, since more efficient use of water postpones the decrease in plant water status.
Role of purine metabolism in thidiazuron‐induced somatic embryogenesis of geranium (Pelargonium×hortorum) hypocotyl cultures
Authors: Hutchinson Margaret J. , Saxena Praveen K.
Published: May 2006

Somatic embryogenesis in geranium (Pelargonium×hortorum Bailey cv. Scarlet Orbit Improved) was achieved by culturing hypocotyl sections on Murashige and Skoog (1962) (MS) medium containing 10 μM thidiazuron (TDZ) (induction medium) for 3 days and subsequently transferring the sections onto a basal medium lacking any plant growth regulators (expression medium). Addition of the purine analogue 2.6‐diaminopurine (DAP) to the somatic embryo induction medium completely inhibited the embryogenic response as well as chlorophyll accumulation without affecting enlargement of the treated tissues. Addition of 20 μM adenine sulphate to the expression medium, i.e during embryo growth and development phase, completely reversed the DAP‐induced inhibition of the embryogenic response while addition during the induction phase caused only a 50% reversal of the inhibition. Analysis of endogenous levels of plant growth substances indicated that TDZ alone elevated the levels of auxins, cy‐tokinins and abscisic acid while the presence of DAP during the induction phase caused a further increase in the levels of adenine and adenosine. These findings indicate a possible critical role for purines in embryogenesis from geranium hypocotyl tissues. However, the conversion of cytokinin bases to their corresponding nucleotide forms was not evident as the levels of isopentenyl adenine and zeatin increased during the second day of culture.
Divergence in properties of two closely related α‐amylase inhibitors of barley
Authors: García‐Casado Gloria , Sánchez‐Monge Rosa , Puente Xose S. , Salcedo Gabriel
Published: May 2006

The only inhibitor of human salivary α‐amylase identified so far in Hordeum has been isolated from barley cv. Bomi endosperm. This protein has the same N‐terminal sequence (23 residues), molecular mass, and isoelectric point as one of the subunits of the barley tetrameric inhibitor previously characterized. However, enzymatic cleavage of both proteins with endoproteinase Glu‐C revealed that they are products of different genes. The two isoforms have diverged in their aggregative and inhibitory properties. Thus, the subunit previously characterized forms, along with two other subunits, a tetramer active towards insect but not human salivary α‐amylase, while the isoform reported here behaves as a homodimer effective against the human enzyme. These results are discussed in the context of the evolution of the cereal α‐amylase inhibitor family.
Regulation of nitrogenase activity in Bradyrhizobium japonicum/soybean symbiosis by plant N status as determined by shoot C:N ratio
Authors: Bacanamwo Méthode , Harper James E.
Published: May 2006

Regulation of nitrogenase is not sufficiently understood to engineer symbioses that achieve a high N2 fixation rate under high levels of soil N. In the present hydroponic growth chamber study we evaluated the hypothesis that nitrogenase activity and the extent of its inhibition by NO3− may be related to both N and carbohydrate levels in plant tissues. A wide range of C:N ratios in various plant tissues (8.5 to 41.0, 1.9 to 3.7, and 0.8 to 1.8, respectively, in shoots, roots, and nodules) was generated through a combination of light and CO2 levels, using two soybean genotypes differing in C and N acquisition rates. For both genotypes, N concentration in shoots was negatively correlated to nitrogenase activity and positively correlated to the extent of nitrogenase inhibition by NO3−. Furthermore, nitrogenase activity was positively correlated to total nonstructural carbohydrates (TNC) and C:N ratio in shoot and nodules for both genotypes. Nitrogenase inhibition by NO3− was negatively correlated to TNC and C:N ratio in shoots, but not in nodules for both genotypes. At the onset of nitrogenase inhibition by NO3−, C:N ratio declined in shoots but not in nodules. These results indicate that both C and N levels in plant tissues are involved in regulation of nitrogenase activity. We suggest that the level of nitrogenase activity may be determined by (1) N needs (as determined by shoot C:N) and (2) availability of carbohydrates in nodules. Modulation of the nitrogenase activity may occur through sensing changes in plant N, i.e. changes in shoot C:N ratio, possibly through some phloem translocatable compound(s).
Immunodetection and immunolocalization of globulin storage proteins during zygotic and somatic embryo development in Zea mays
Authors: Thijssen Marja H. , Spoelstra Patrick , Emons Anne Mie C.
Published: May 2006

Globulins (GLB) are storage proteins that accumulate to high levels during zygotic embryo development of Zea mays L. We visualized the distribution of GLB during zygotic embryo development by immunolabelling of polyethylene glycol sections with a GLB‐specific antiserum and a fluorescent secondary antibody. In sections of embryos at 10 days after pollimation (DAP), GLB were detected in the scutellar node only. Sections of embryos of 17 DAP showed, besides the presence of GLB in the scutellar node, the presence of a low amount of GLB in the coleoptile and the leaf primordia. In 30‐DAP embryos GLB were localized in the root, the coleorhiza, the leaf primordia, the coleoptile and in all cells of the scutellum with the exception of the epidermis and the pro‐vascular tissues. The subcellular location of GLB was visualized by immunolabelling of ultrathin sections with anti‐GLB and a gold‐conjugated secondary antibody. Scutellum cells and root cortex cells of 30‐DAP embryos were packed with protein storage vacuoles (PSV), which differed in electron density. GLB were either evenly distributed throughout the PSV or were localized in electron‐dense inclusions within the PSV. SDS‐PAGE and immunoblot analysis of total protein extracts indicated the presence of a low amount of the GLB1 processing intermediate proGLB1′ in globular as well as mature somatic embryos. After maturation on an ABA‐containing medium, somatic embryos showed the additional presence of the next GLB1 processing intermediate GLB1′. By immuno‐electron microscopy it was possible to localize GLB in globular deposits in PSV in scutellum cells of these somatic embryos.
Photoperiodic control of cytokinin transport and metabolism in Chenopodium rubrum
Authors: Macháčková Ivana , Eder Josef , Motyka Václav , Hanuš Jan , Krekule Jan
Published: May 2006

Cytokinin (CK) levels in the short‐day plant Chenopodium rubrum L. are known to fluctuate diurnally. The aim of this work was to investigate if the diurnal changes are brought about by changes in transport and/or metabolism of CKs. The effect of photo‐period on cytokinin transport was studied by analysing CK concentrations in root, leaf and apical exudates, respectively, under constant light (CL), a 12‐h photoperiod (DL) inductive for flowering, DL in which darkness was interrupted at the end of hour 6 by 15 min red light (R), or by 15 min R followed by 30 min far‐red irradiation (R/FR). The concentrations of cytokinins (zeatin, zeatin riboside, isopentenyladenine, isopen‐tenyladenosine) in all three types of exudates were significantly higher in the first 12‐h period after the end of 12 h darkness than in CL. The R break almost fully negated the effect of darkness and its effect was reversed by FR, showing the involvement of phytochrome in the regulation of CK transport. In the next 12‐h interval, i.e. 12–24 h after the end of darkness, the CK level remained high in the leaf exudate only, but to a much lower extent than in the previous 12 h. The highest CK concentration (increase by 108%) was observed in apical exudates during inductive darkness. A comparison of the CKs present in the individual exudates indicates that those arriving at the apical part are derived mostly from leaves with varying contributions by the xylem. The metabolism of applied [3H]‐zeatin riboside (ZR) was studied using HPLC separation of the metabolites. Metabolism was found to be very rapid and different glucosides, adenine and adenosine were the main metabolites after 12 h incubation with labelled ZR in all regimes tested. The only metabolite that seems to be under photoperiodic control is ZR‐5′‐monophosphate. It is as yet not clear if photoperiod controls the phosphorylation or dephosphorylation reaction. The activity of the main cytokinin degradative enzyme, cytokinin oxidase, did not change during the photoperiodic regimes tested.
Nitrogen allocation in response to partial shading of a plant: Possible mechanisms
Authors: Pons Thijs L. , Bergkotte Marc
Published: May 2006

The significance of photosynthetic and transpiration rates for the perception by plants of light gradients in leaf canopies has been investigated with regard to nitrogen allocation and re‐allocation. A gradient of photon flux density (PFD) over a plant's foliage was simulated by shading one leaf of a pair of primary leaves of bean (Phaseolus vulgaris L. cv. Rentegever). Photosynthetic rate was manipulated independently of PFD and, to some extent, also of transpiration, by subjecting the leaf to different CO2 concentrations. Transpiration rate was changed independently of PFD and photosynthetic rate by subjecting the leaf to different vapour pressure differences (VPD). A reduced partial pressure of CO2 reduced specific leaf mass (SLM) as did a decreased PFD, but did not change leaf N per unit area (NLA) and light saturated rate of photosynthesis (Amax). A reduced VPD caused several effects consistent with the effect of PFD. It decreased NLA and Amax and increased the chlorophyll to N ratio in old and young leaves. Furthermore, it decreased the chlorophyll a to b ratio and inhibited leaf growth in young leaves. The transpiration stream is partitioned among the leaves of a plant according to their transpiration rates. The results suggest that relative rates of import of xylem sap into leaves of a plant play an important role in the perception of partial shading of a plant, a situation normally found in dense vegetations. The possible role of cytokinin influx into leaves as controlled by transpiration rate, is discussed.
A comparison of the kinetics of aluminum (Al) uptake and distribution in roots of wheat (Triticum aestivum) using different aluminum sources. A revision of the operational definition of symplastic Al
Authors: Archambault Daniel J. , Zhang Guichang , Taylor Gregory J.
Published: May 2006

The lack of information about the movement of aluminum (Al) across the plasma membrane presents a significant barrier to the elucidation of resistance mechanisms which may involve exclusion of Al from the symplast. An understanding of mechanistic aspects of exclusion requires the estimation of symplastic Al levels. Such measurements may be achievable through the use of a kinetic approach. A kinetic protocol was developed to characterize the accumulation and distribution of Al in various cellular compartments in roots of wheat (Triticum aestivum L.). The kinetics of uptake and desorption were similar when Al was supplied as AIK(SO4)2 or as AlCl3. When both salts were supplied at low concentration (50 μM), Al bound to a purified cell wall fraction could be reduced to less than 10–20% of non‐exchangeable Al, if roots were washed for 30 min in citric acid following exposure. In contrast, when AlK(SO4)2 was supplied at a high concentration (200 μM), a strong linear phase of uptake into cell wall material was observed, which accounted for approximately 48% of non‐exchangeable Al in roots. These results suggest that the use of low concentrations of Al in simple salt solutions is required to minimize accumulation of non‐exchangeable Al in the apoplasm. A series of multiple‐desorption experiments confirmed that citric acid was effective in removing Al from the cell wall compartment of roots exposed to Al for short periods (3 h). However, long exposures (48 h) appeared to create conditions conducive to the accumulation of non‐exchangeable Al in the cell wall. In experiments where uptake from solutions containing 50 μM AlCl3 was followed by desorption in citric acid, non‐exchangeable Al in microsomal membrane fractions represented less than 4% of total non‐exchangeable Al. Thus, we can exclude the plasma membrane and cell wall as major sites for accumulation of non‐exchangeable Al in short exposure studies. Although we cannot provide unequivocal evidence for the localization of Al within the symplast, use of simple salt solutions followed by desorption in citric acid provides the best kinetic technique currently available for the quantitation of Al in the symplasm.
Reduction of root iron in Plantago lanceolata during recovery from Fe deficiency
Authors: Schmidt Wolfgang , Boomgaarden Bettina , Ahrens Vera
Published: May 2006

Changes in root‐associated Fe(III) reductase activity and Fe concentration during recovery from temporary iron starvation were investigated in hydroponically grown Plantago lanceolata L. Within two days, interruption of the Fe supply resulted in enhanced rates of reduction by intact plant roots. Transfer of iron deficient plants to a solution containing various amounts of chelated Fe caused a transient increase in reduction activity before the rates declined to a level determined by the amount of Fe added. Repression of root‐associated redox activity was independent of the Fe concentration in the preculture. When iron deficient plants were submitted to a supply of Fe localized to a part of the root system (split‐root plants), the decrease in reduction rates was much more pronounced in the Fe‐deprived portion of the roots than in the Fe‐supplied one. No correlation was observed between root Fe concentration and Fe(III) reductase activity. Continued growth of split‐root plants in the +Fe/‐Fe regime increased the reduction rates of the +Fe‐grown portion of the root system over the rates in iron sufficient plants with non‐divided roots. The results are discussed in relation to putative factors mediating intra‐ and interorgan regulation of iron nutrition.
Transmembrane movements of artificial redox mediators in relation to electron transport and ionic currents in chloroplasts
Authors: Bulychev Alexander A. , van Voorthuysen Tijmen , Vredenberg Wim J.
Published: May 2006

Electrochemical data obtained with TMPD+‐sensitive electrodes indicate that ammonium‐uncoupled chloroplasts retain TMPD (N,N,N′,N′‐tetramethyl‐p‐phenylenediamine) mainly in the reduced form during illumination, whereas uncoupled DCMU‐treated chloroplasts accumulate TMPD in the oxidized form (TMPD+). This observation indicates that the reduced plastoquinol is the preferred electron donor for photosystem I (PSI) and TMPD can only compete efficiently when plastoquinone reduction is blocked. After adding DCMU the formation of a transmembrane gradient for TMPD+ is reflected by a slow‐down of the electrogenic electron transport and by the emerging of the overshoot of the membrane current in the light‐off response. A light‐dependent increase in photoelectric current generated by chloroplasts in the presence of NH4Cl and TMPD is observed and considered to be caused by a reversible release of current limitation in the interfacial conductance barriers in the lumen.
Evidence that cytokinin controls bud size and branch form in Norway spruce
Authors: Chen Hao‐Jie , Bollmark Marie , Eliasson Lennart
Published: May 2006

Shoot elongation in many coniferous species is predetermined during bud formation the year before the shoot extends. This implies that formation of the primordial shoot within the bud is the primary event in annual shoot growth. Hormonal factors regulating bud formation are consequently of utmost importance. We followed the levels of the endogenous cytokinins zeatin riboside (ZR) and isopentenyladenosine (iPA) in terminal buds, whorl buds and lower lateral buds of the uppermost current‐year whorl shoots of 15‐ to 20‐year‐old trees of Norway spruce [Picea abies (L.) Karst.] from June to September. Cytokinins were isolated with affinity chromatography columns, purified by high performance liquid chromatography, and quantified by ELISA. The level of ZR was low in June but increased gradually in all buds until September. Throughout the measurement period, the ZR level was highest in terminal buds and lowest in the scattered lateral, buds, with the whorl buds intermediate. The level of iPA peaked in July and decreased later without any consistent differences among the three classes of buds. The development of different kinds of buds was followed by scanning electron microscopy. We found that bud growth was greatest during August and September. The final size of primordial shoots within the buds varied considerably and the weight of the terminal bud was three times that of the whorl buds and more than five times that of the other lateral buds.

We conclude that the increase in ZR level during the period of active bud development is indicative of the importance of cytokinin for this process. Furthermore, the positive correlation between the level of ZR and bud growth during the period of predetermination of next year's branch growth suggests that this hormone indirectly controls the form of single branches in the spruce tree.
A reassessment of the intervention of calmodulin in the regulation of stomatal movement
Authors: Cotelle Valérie , Forestier Cyrille , Vavasseur Alain
Published: May 2006

Commelina cammunisL., a monocotyledonous plant whose stomata are highly sensitive to calcium ions, was used to study calmodulin (CaM) involvement in stomatal movements. CaM was detected and quantified in guard cell and mesophyll cell protoplasts by western blot and by45Ca2+‐overlays. CaM was found to be 3‐ to 7‐fold more abundant on a per protein basis in guard cell than in mesophyll cell protoplasts. Numerous guard cell proteins that bind CaM in a Ca2+‐dependent manner were detected by gold‐labelled CaM overlays. Using bioassays with epidermal strips, different CaM‐antagonists were found to induce a net stimulation of stomatal opening in darkness or under illumination (trifluoperazine > compound 48/80 ≅ fluphenazine > W7 > W5). As CaM is frequently involved in the regulation of phosphorylation processes, the effects of different inhibitors of protein kinases on stomatal movements were studied. In red plus blue light, a promotion of the stomatal aperture was observed in the nanomolar range with K252a and KT5926 and in the micromolar range with KT5720 ≫ ML7 ≅ ML9 ≫ H7 > KN62. Only the inhibitors with a high specificity for Ca2+‐CaM dependent protein kinases (K252a, KT5926, ML7, ML9) triggered a stomatal opening in darkness and increased stomatal aperture in red plus blue light. Taken together, these data strongly suggest that a Ca2+‐ or a Ca2+‐CaM‐dependent protein kinase plays a central role in the calcium transduction pathway leading to the maintaining of stomatal closure.
Caffeine biosynthesis in young leaves of Camellia sinensis: In vitro studies on N‐methyltransferase activity involved in the conversion of xanthosine to caffeine
Authors: Kato Misako , Kanehara Tomomi , Shimizu Hisayo , Suzuki Takeo , Gillies Fiona M. , Crozier Alan , Ashihara Hiroshi
Published: May 2006

The aim of this study was to investigate the S‐adenosylmethionine dependent N‐methyltransferase(s) (NMT) associated with the three methylation steps in the caffeine biosynthesis pathway in tea (Camellia sinensis L.). NMT activity in cell‐free preparations from young leaves was purified by anion‐exchange and gel‐filtration column chromatography. In both systems, a single zone of NMT activity, with broad substrate specificity was detected. The N‐3 position of dimethylxanthine and monomethylxanthines was methylated more readily than N‐1 while comparatively little substitution occurred at the N‐7 locus. When xanthosine was used as a substrate only the N‐7 position was methylated. These results indicate that a single NMT may participate in the conversion of xanthosine to caffeine. The apparent Mr of the NMT, estimated by gel filtration chromatography, was 61 000. The substrate specificity of the NMT is compatible with the operation of a xanthosine → 7‐methylxanthosine → 7‐methylxanthine → theobromine → caffeine pathway as the main biosynthetic route to caffeine in young tea leaves. The data also indicate that the conversion of 7‐methylxanthine → paraxanthine → caffeine may function as one of a number of minor pathways that also contribute to the production of caffeine.
Protease activity, organisation of vegetative primordia and effect of putrescine in tobacco thin layers
Authors: Torrigiani P. , Scaramagli S. , Franceschetti M. , Capitani F. , Altamura M. M. , Bagni N.
Published: May 2006

Since in tobacco thin layers exogenous putrescine alters the physiological and mor‐phogenic responses induced by IAA (indole‐3‐acetic acid) and/or BA (benzylade‐nine), the effect of this polyamine on protease activity and on the formation of meristemoids and vegetative primordia was studied during morphogenesis. Superficial thin layer explants, excised from the stem of tobacco (Nicotiana tabacum L. cv. Samsun) plants in the vegetative stage, were cultured under various hormonal conditions (IAA, IAA+BA, BA) and in a hormone‐free medium, in the presence or absence of 100 μM putrescine. Histological analysis showed that no meristemoids were formed on the control medium or with putrescine alone and only a few were formed on IAA‐treated explants with or without putrescine. An increasing number of meristemoids was observed in IAA+BA and BA treatments during culture; in both cases this number was enhanced by the presence of exogenous putrescine. Protease activity was evaluated spectrophotometrically using two synthetic substrates, azocasein and N‐benzoyl‐DL‐arginine‐p‐nitroanilide (BAPNA). In the former, maximum protease activity was observed in IAA+BA‐ and BA‐treated explants on days 10 and 15, respectively, while with IAA activity was lowest, the maximum occurring on days 5–10. In this case exogenous putrescine enhanced protease activity in the presence of IAA alone or with BA, while it decreased it in the presence of BA. BAPNA‐mediated proteolytic activity (serine‐proteases) was highest in IAA+BA‐treated explants, intermediate in BA‐ and not different from controls in IAA‐treated explants. Putrescine only affected proteolytic activity in IAA+BA treatments. The use of specific inhibitors of protease activities indicated that these enzymes belong to two main classes of proteases, that is serine‐ and thiol‐proteases. The pattern of proteolytic activities during culture appeared to be related to the differentiation of meristemoids into vegetative primordia. The effect of exogenous putrescine on protease activity was different depending on different synthetic substrates, developmental patterns, pH and ionic strength.