bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation of plasma membrane fractions from green wheat leaves by free‐flow electrophoresis or two‐phase partition alone or in combination
Authors: Egger Alfred , Moiré D. James , Sellden Gun , Sandelius Anna Stina
Published: May 2006

Plasma membrane vesicles were isolated from shoots of light‐grown wheat seedlings by preparative free‐flow electrophoresis, aqueous polymer two‐phase partition or both. Plasma membrane vesicles were identified from staining of thin sections prepared for electron microscopy with phosphotungstic acid at low pH. The orientation of the plasma membrane vesicles was determined from latency and trypsin sensitivity of K+ Mg2+ATPase and of glucan synthase II, and concanavalin A‐peroxidase binding and membrane asymmetry visualized by electron microscopy. The K+Mg2+ATPase and of glucan synthase II activities of plasma membrane fractions isolated by two‐phase partition were latent and trypsin resistant. The vesicles bound concanavalin A‐peroxidase strongly and exhibited a cytoplasmic side‐in morphology. These fractions of cytoplasmic side‐in vesicles were less than 10% contaminated by cytoplasmic side‐out vesicles. By free‐flow electrophoresis, two populations of vesicles which stained with phosphotungstic acid at low pH, designated D and E, were obtained. The vesicle population with the lower electrophoretic mobility, fraction E, contained plasma membrane vesicles with properties similar to those of the plasma membrane vesicles obtained after two‐phase partition. The phosphotungstic‐reactive vesicles with greater electrophoretic mobility, fraction D, were concanavalin A unreactive with the cytoplasmic membrane leaflet outwards. Less than 50% of the K+Mg2+‐ATPase activity of this fraction was latent and trypsin sensitive. The vesicles of fraction D appeared to be preferentially cytoplasmic side‐out. The electrophoretic mobilities of cytoplasmic side‐out (non‐latent glucan synthase II activity) and cytoplasmic side‐in (latent glncan synthase II activity) plasma membrane vesicles isolated from a frozen and thawed wheat plasma membrane fraction, corresponded with the mobilities of fraction D and E, respectively, again showing that the plasma membrane vesicles with the lesser electrophoretic mobility were cytoplasmic side‐in. The cytoplasmic side‐in and cytoplasmic side‐out vesicles therefore showed opposite eletrophoretic mobilities compared with a previous free‐flow electrophoretic separation of soybean plasma membranes. The majorities of the plasma membrane vesicles of both fractions D and E entered the upper phase upon two‐phase partition with the phase composition used for purification of wheat plasma membranes. Thus, neither electrophoretic mobility nor phase partitioning characteristics can be used as the only criteria for assignment of vesicle orientation.
In search of the mechanism of nitrate inhibition of nitrogenase activity in legume nodules: Recent developments
Authors: Vessey J. Kevin , Waterer John
Published: May 2006

The mechanisms involved in the inhibition of nitrogenase activity in legume nodules by nitrate is unclear. This paper reviews and evaluates proposed mechanisms of this inhibition. Emphasis is placed on recent developments, which suggest that nitrate causes an O2 limitation of nitrogenase activity. Several mechanisms that involve a nitrate‐induced increase in resistance to O3 diffusion in the nodule cortex are discussed.
Role of xyloglucan breakdown in epidermal cell walls for auxininduced elongation of azuki bean epicotyl segments
Authors: Hoson Takayuki , Sone Yoshiaki , Misaki Akira , Masuda Yoshio
Published: May 2006

Auxin‐induced elongation of epicotyl segments of azuki bean (Vigna angularis Ohwi et Ohashi cv. Takara) was suppressed by a fucose‐binding lectin from Tetragonolobus purpureas Moench and by polyclonal antibodies raised against xyloglucan heptasaccharide (Xyl3Glc4) when the cuticle present in the outer surface of epicotyls was abraded. In contrast, elongation of non‐abraded segments was not influenced by the lectin or the antibodies. Epicotyl segments, from which the epidermal and the outer cortical cells had been removed, elongated rapidly for 2 h and than only slowly. Auxin slightly stimulated elongation of the inner tissue segments in the phase of slow growth. Neither in the presence nor in the absence of auxin did the lectin or the antibodies affect elongation of the inner tissue segments. The split portions of outer surface‐abraded epicotyl segments incubated in buffer extended outward, and auxininhibited this outward bending. The lectin and the antibodies reversed the effect of auxin on bending. The fucose‐binding lectin pretreated with fucose or the immunoglobulin fraction obtained from preimmune serum exhibited little or no inhibitory effect on auxin‐induced elongation of abraded or split segments. These results support the view that a breakdown of xyloglucans in the epidermal cell walls plays an essential role in auxin‐induced elongation in dicotyledons.
Cellular changes in early development of regenerating thin cell layer‐explants of rapeseed analyzed by light and electron microscopy
Authors: Pihakaski‐Maunsbach K. , Nygaard K. Brauner , Jensen K. H. , Rasmussen O.
Published: May 2006

Cellular changes in thin cell layer (TCL) explants of stem origin of Brassica napus L. cv. Vega were studied from 0 to 15 day by light and transmission electron microscopy. Apical and basal ends of the old explants were analysed separately. Quantitative and qualitative analyses showed that during the first culture day the parenchyma cells enlarged significantly as did the cytoplasm/vacuole ratio. The cytoplasm contained increased rough endoplasmic reticulum (RER), polysomes and dictyosomes associated with both coated and uncoated vesicles. The cell enlargement continued during the first 5 days of culture. The structural organization of the cell wall became somewhat loose and inhomogeneous. Parenchyma in the basal end divided frequently, resulting in several centres of division, while cell division in apical cells was less frequent and cells there remained enlarged. Starch accumulation started on the first day and increased until the third day. i. e. until cell divisions became more frequent. The starch content of dividing cells gradually decreased and starch was almost totally lacking in 15‐day‐old explants. Starch grains remained numerous, however, in the large non‐dividing apical cells, except in those cells adjacent to the medium. Cell divisions started close to medium in explants containing vascular tissue, but closer to the epidermis in the explants without vascular tissue.

The results show how rapid (one day) striking changes in the cells take place and suggest that optimal hormone concentration and intertissue relations between epidermis and parenchyma and between parenchyma and vascular tissues as well as intercellular relations among parenchyma cells determine the first cell division sites and planes in the explants. Although the cells change from elongated to spheroid, their original polarity remains as evidenced by the formation of more numerous basal shoot primordia than in apical shoot primordia.
On the interaction of UV‐B radiation (280–315 nm) with water stress in crop plants
Authors: Balakumar T. , Vincent V. Hani Babu , Paliwal Kailash
Published: May 2006

Cowpea (Vigna unguiculata L. Walp.) seedlings (3‐day‐old) were subjected to 4 kinds of experimental treatments: (1) control without exposure to any stress (−D‐UV), (2) moderate water stress with no UV‐B irradiation (+D‐UV), (3) no water stress but to UV‐B radiation (−D+UV). and (4)moderate water stress and exposure to UV‐B (+D+UV). UV‐B and drought stress in the combined form elicited beneficial effects on the morphological and growth characteristics, and a few additive inhibitory effects in some functional processes. An increase in the specific leaf weight (SLW) was observed in the combination of stresses, which could be a defence mechanism against UV‐B. The combination of stresses promoted the synthesis of anthocyanins and phenolic compounds. The responses of plants to the combination of stresses indicate that during simultaneous exposure of plants to multiple stresses, one form of stress could minimize the damage by the other. The enhancement of superoxide dismutase (SOD) and catalase activities appear to serve as acclimation mechanisms to scavenge the toxic, free radicals of oxygen produced under stress conditions. However, the inhibition in nitrate metabolism was greater in the combined stresses than in either of the stresses imposed separately. The results of this study illustrate that the interaction of stresses during simultaneous multiple stress conditions brings out certain beneficial effects.
Antioxidative protection in a drought‐resistant maize strain during leaf senescence
Authors: Pastori G. M. , Trippi V. S.
Published: May 2006

Leaves of 7‐ and 18‐day‐old plants of two maize strains, one resistant (LIZA) and one sensitive (LG11) to water stress, were floated in 1 mM paraquat and 1 mM H2O2 for 12 h in light and in darkness. The aim of this work was to analyse the effects of these substances on the activities of enzymes involved in the scavenging of active oxygen species during senescence. Three senescence parameters; chlorophyll loss, lipid peroxidation and conductivity; showed a general cell damage caused by both oxidative treatments and revealed a higher tolerance of LIZA than LG11 to paraquat and H2O2 both in light and in darkness. Activities of antioxidative enzymes increased by the effect of oxidative treatments in young and senescent leaves of the drought‐resistant maize strain LIZA. These increases were about 3‐to 6‐fold in glutathione reductase. 3‐to 4‐fold in superoxide dismutase and 2‐fold in ascorbate peroxidase activities. The possible correlation between water stress resistance. senescence and the potential of antioxidant enzymes was analysed.
Dimensions and distribution of intercellular spaces in cryo‐planed soybean nodules
Authors: Van Cauwenberghe Owen R. , Newcomb William , Canny Martin J. , Layzell David B.
Published: May 2006

The ability of legume nodules to regulate their permeability to gas diffusion has been attributed to physiological control over the size and distribution of gas‐filed intercellular spaces within the nodule cortex. To examine the size and distribution of intercellular spaces and to determine whether they were filled with gas (high diffusion permeability) or liquid (low diffusion permeability), whole nodules were frozen in liquid nitrogen slush (‐210°C), and then either cryo‐fractured or cryo‐planed before being examined by cold‐stage scanning electron microscopy (SEM). The cryo‐planed tissue was found to have many advantages over cryo‐fractured nodules in providing images which were easier to interpret and quantify. Intercellular spaces throughout the nodule were examined in both tangential and medial planed faces. Since no differences were observed between views in either the size or shape of the open intercellular spaces, it was concluded that the intercellular spaces of nodules were not radially oriented as assumed in many mathematical models of gas diffusion. The inner cortex region in the nodules had the smallest intercellular spaces compared to other zones, and less than 10% of the intercellular spaces were occluded with any type of material in the central zone regions. Vacuum infiltration of nodules with salt solutions and subsequent cryo‐planing for SEM examination showed that open and water‐filled intercellular spaces could be differentiated. The potential is discussed for using this method to study the mechanism of diffusion barrier regulation in legume nodules.
Gibberellin status and responsiveness in shoots of tall and dwarf genotypes of diploid rye (Secale cereale)
Authors: Börner A. , Gale M. D. , Appleford N. E. J. , Lenton J. R.
Published: May 2006

The recessive dwarfing alleles of rye (Secale cereale L.), ct1 and ct2, caused a 35–55% reduction in the length of leaf 2 compared with corresponding tall lines grown at both 10°C and 20°C. The dwarf lines were 45–50% as responsive to applied GA3 as the tall lines at 20°C but the absolute GA‐responsiveness of the dwarfs was greater at 10°C than at 20°C. There was no significant difference in the contents of GA19, GA20, GA29, GA1, GA3 and GA8 in the leaf extension zone of tall and dwarf seedlings grown at 20°C. It was concluded that the mechanism whereby GA homeostasis is maintained is functional in both tall and dwarf lines despite marked differences in leaf extension rate. The recessive rye mutations may cause loss of function late in the GA‐cell elongation pathway or, alternatively, indirectly affect GA‐responsiveness in vegetative tissues. The genetic and physiological evidence indicates that ct1 and ct2 are unrelated to the GA‐insensitive Rht genes in hexaploid bread wheat.
Determination of floral organ type in cultured Silene shoot apices
Authors: Donnison Iain Simon , Francis Dennis
Published: May 2006

Shoot apices of the long day plant, Silene coeli‐rosa, were cultured on a basal medium (+3% sucrose) in non‐inductive short days (SD) following their excision from plants which had been exposed to long day (LD) treatments in order to examine the period for determination of each floral whorl. In response to the inductive LD treatments, the pattern of whorl formation in vitro reflected their normal appearance in Silene: sepals, stamens 1–5, petals, stamens 6–10 and carpels, although the number of apices initiating each whorl was lower in vitro compared with apices in vivo. However, supplementing the medium with 7 instead of 3% sucrose corrected this deficiency and, for the first time, resulted in apices initiating floral whorls in SD. The interval between the shortest treatment to result in whorl initiation in vitro, 4 LD (which also resulted in 50% flowering in vivo), and the treatment which gave 50% initiation of the corresponding whorl in vitro, was taken to be the period for determination of that whorl. The determination times on the 3% medium were: sepals (2 days), stamens 1–5 (3 days), petals (3 days), stamens 6–10 (4 days) and carpels (4 days); all of these periods shortened to about 1 day on the 7% medium. Tissue culture did not perturb the pattern of initiation of each whorl since apices excised and cultured from plants which had received 7 LD + 2 SD, exhibited each whorl over the same time scale as those of intact plants which received the same treatment. The data are consistent with a sequential determination and initiation of each whorl in the order that they appear normally in Silene. Synchronisation of cell division, as represented by peaks of the mitotic index and G2/G1 ratios on day 8 (7 LD + 2 SD), did not occur in vitro but the mitotic index did not descend to zero, further emphasising that tissue culture did not perturb the Silene apex.
Inhibition of mugineic acid‐ferric complex uptake in barley by copper, zinc and cobalt
Authors: Ma Jian Feng , Nomoto Kyosuke
Published: May 2006

The interfering effects of copper, zinc, and cobalt on the uptake of mugineic acid‐ferric complex were studied in barley (Hordeum vulgare, cv. Minorimugi) grown in nutrient solution. Short‐term uptake experiments of 3 h were performed utilizing both ionic and mugineic acid‐complex forms of each metal at two different concentrations. Copper was most effective in decreasing iron uptake when added in an ionic form at either concentration. The inhibition order at higher concentrations followed Cu(II) > Zn(II) ≥ Co(II), Co(III), which is consistent with the stability constants of these metal complexes with mugineic acid. The displacement of iron from its mugineic acid complex by these metals is suggested as a probable explanation for the decreased iron uptake. The inhibitory effect of metal complexes with mugineic acid on iron uptake was only found in cases with higher concentrations of Cu(II) and Zn(II) complexes. Deformation of the specific iron transport system in the plasma membrane due to their adsorption may be responsible for this effect.
Metabolism of cell wall polysaccharides from persimmon fruit. Pectin solubilization during fruit ripening occurs in apparent absence of polygalacturonase activity
Authors: Cutillas‐Iturralde Antonio , Zarra Ignacio , Lorences Ester P.
Published: May 2006

Pectins from persimmon (Diospyros kaki L.) fruit pericarp were sequentially extracted with 0. 05 M trans‐1,2‐diaminocyclohexane‐N,N, N′, N′‐tetraacetic acid (CDTA), 0. 05 M Na2CO3 (1°C) and Na2CO3 (20°C) and the carbohydrate composition and metabolism during development determined. Young persimmon fruits contained a large proportion of pectins, 46% by dry weight, that decreased to 20% with ripening. This decrease occurred in the CDTA and Na2CO3 (1°C) fractions, mainly composed of uronic acids, and represents a net loss of uronic acids, arabinose and galactose. The amount of non‐cellulosic neutral sugars was especially high in the Na2CO3 (20°C) fraction. The loss of pectins was also accompanied by a depolymerisation of the polysaccharides extracted in the three pectic fractions. However, none of these changes can be attributed to the action of polygalacturonase activity. Proteins were extracted from the pericarp tissue, but endopolygalacturonase (EC 3. 2. 1. 15) activity, determined as a decrease in viscosity of polygalacturonic acid, was not observed in the extract. Determination of exopolygalacturonase (EC 3. 2. 1. 67) activity by measuring the release of reducing groups from polygalacturonic acid was also negative. The results presented indicate that polygalacturonase is not responsible for the metabolism of pectins during persimmon fruit ripening.
Comparison of seasonal growth and polyamine content in shoots of orchard‐grown standard and genetic dwarf apple tress
Authors: Wang Shiow Y. , Faust Miklos
Published: May 2006

Seasonal variations in growth rate, and polyamine and arginine content were determined in shoots of standard and genetic dwarf apples (Malus domestica Borkh.) in orchard conditions. Putrescine, spermidine, spermine and arginine exhibited the same seasonal variations. Polyamine titer and arginine content were correlated. Putrescine, spermidine, spermine and arginine levels increased during March and April, reaching the maximum at the end of April. From the onset of May, they decreased gradually until they reached a steady level at the end of July and beginning August. Then the level increased until September when growth stopped. Shoots of both standard and dwarf trees started to grow in April, elongated slowly during the summer and ceased growth in September. Polyamine and arginine content of the shoots of standard apple trees were higher compared to the shoots of genetic dwarfs, but the concentration of polyamine and arginine were not correlated with the growth rate.
Preparation and polypeptide composition of plasma membrane and other subcellular fractions from wild oat (Avena fatua) aleurone
Authors: Walker Robert P. , Waterworth Wanda M. , Hooley Richard
Published: May 2006

Plasma membranes can be isolated from a variety of plant tissues by first preparing a post‐mitochondrial membrane fraction enriched in plasma membranes, by differential centrifugation, and partitioning this on a dextran‐polyethylene glycol two‐phase system. With wild oat aleurone, however, we observed that differential centrifugation could not be used to produce a microsomal fraction enriched in plasma membrane. Approximately 70% of the plasma membrane in aleurone homogenates was pelleted by sequential centrifugation at 100 g× 10 min and 1000 g× 10 min. The remainder sedimented at 112 000 g× 1 h. All the material that was pelletable by centrifugation was, therefore, subjected to dextran‐polyethylene glycol two‐phase partitioning. The plasma membrane marker enzymes glucan synthase II (GSII, EC 2. 4. 1. 34) and UDP‐glucose:sterol glucosyltransferase (SGT, EC 2. 4. 1.) were enriched in the upper phase, whereas cytochrome c oxidase activity (EC 1. 9. 3. 1), a mitochondrial marker enzyme, was depleted. The presence of endoplasmic reticulum (ER) and protein body membranes in the phase system was assessed by probing western blots, of SDS‐PAGE separated proteins, with polyclonal antiserum either to binding protein (BiP, an ER marker) or to tonoplast intrinsic protein (TIP, a protein body membrane marker). BiP and TIP were present in the lower phase, but were not detected in the upper phase. In addition, the polypeptide patterns of material in the upper and lower phases were very different. These observations suggested that high purity aleurone plasma membrane had been isolated. Although the procedure for isolating plasma membranes was applicable to both aleurone protoplasts and layers, the polypeptide patterns of plasma membranes prepared from these sources were very different. The major protein components of wild oat aleurone were 7 S and 12 S storage globulins. These proteins were present in the lower phase, but not in the plasma membrane enriched upper phase, after aqueous two‐phase partitioning. Differential centrifugation studies showed that it was necessary to homogenise aleurone in a buffer of pH 6. 0 or less if a soluble protein fraction, essentially devoid of storage globulins, was to be obtained. The use of these fractionation techniques is discussed in relation to photoaffinity labelling of gibberellin (GA)‐binding proteins in aleurone.
Induction of chitinase and β‐1,3‐glucanase in Parthenocissus quinquefolia cells cultured in vitro
Authors: Flach Jean , Jollés Pierre , Pilet Paul‐Emile
Published: May 2006

Chitin, chitosan and peptidoglycan induced chitinase (EC 3. 2. 1. 14) activity in Parthenocissus quinquefolia cells cultured in vitro, while cellulose did not. The real inducers seemed to be oligomers released from the large size polymers by hydrolytic enzymes secreted into the medium during the cell growth and division. This effect was mimicked by the addition to the medium of a partially purified Parthenocissus chitinase/lysozyme (EC 3. 2. 1. 17), which was also able to hydrolyse chitosan. Oligomers of chitin and of chitosan induced the activity to the same level and with the same time course, while peptidoglycan oligomers induced less activity. Oligomers also induced β‐1,3‐glucanase (EC 3. 2. 1. 6) activities. The changes with time of both activities and the relative effects of the three kinds of polymers suggested that the induction of both enzymes involves a common element early in the signal pathway.
Inhibition of the activity of aminoacyl‐tRNA synthetases in pollen grains of maize
Authors: Radlowski Marek , Bartkowiak Slawomir , Krolikowski Zygmunt
Published: May 2006

Extracts from pollen grains of maize (Zea mays L.) show a low activity of aminoacyltRNA synthetases (EC 6. 1. 1). They also contain a specific factor inhibiting the activity of these enzymes. The molecular mass of this factor, which may be a short peptide, is about 3000 Da as determined by column chromatography on Sephadex G‐25 Fine. The Michaelis constant (Km), determined for the amino acid in the presence of this factor, suggests its allosteric influence on the affinity of the enzyme. Short‐term incubation of the factor with pronase R resulted in conversion of the inhibiting action into a stimulating one. Kinetics of aminoacylation reactions confirm inhibitory and stimulative influences of the effector on the enzyme activity. High performance liquid chromatography shows that inhibition of the activity of aminoacyl‐tRNA synthetases is affected by a group of compounds of similar molecular masses.
A comparison of the influence of sterols on the specific activity of the H+‐ATPases in isolated plasma membrane vesicles from oat, rye and rice shoots
Authors: Cooke D. T. , Ros R. , Burden R. S. , James C. S.
Published: May 2006

Plasma membrane vesicles were extracted from the shoots of 10‐day‐old oat, rye and rice plants and incubated with either cholesterol, stigmasterol or a mixture of sitosterol + campesterol (60:40). After ascertaining that the sterol composition of the vesicles had been altered by this treatment, the specific hydrolytic activity of the membrane‐bound H+‐ATPase (EC 3. 6. 1. 35) was measured. The results indicated that, although all sterols were taken up, cholesterol was best integrated into the plasma membrane of the species tested. After treatment, ATPase activity was altered in oat and rice, but not in rye. The results are discussed in the context of sterol/lipid and sterol/protein interactions in the plasma membrane.
Growth patterns and endogenous indole‐3‐acetic acid concentrations in current‐year coppice shoots and seedlings of two Betula species
Authors: Rinne Päivi , Tuominen Hannele , Sundberg Björn
Published: May 2006

Apical dominance, internode elongation, radial growth and xylem cell size in coppice and apical shoots of Betula pubescens B. Pendula were determined and related to endogenous indole‐3‐acetic acid (IAA) levels, measured by gas chromatography‐selected ion monitoring in the apical bud and at three positions along the stem. The effects of defoliation and debudding on morphological and anatomical characters and endogenous IAA levels were also investigated. The coppice shoots displayed superior stem elongation and increased branching during the initial phase of growth, after which their growth pattern was similar to that of the seedlings; however, their radial growth was greater throughout the experiment. Both plant types produced smaller‐sized xylem cells at the top of the shoot than at the bottom with coppice shoots tending to form larger tracheids and smaller vessels than the seedlings. There was no consistent difference in IAA concentration between the coppice shoots and the seedlings. Defoliation and debudding reduced the IAA level in the stem within 36 h and it was still low after 25 days. Although the extent of the IAA decrease was similar in both coppice shoots and seedlings, the treatments affected the morphological and anatomical characters differently in the two plant types. The results suggest that the observed differences between seedlings and coppice shoots were not mediated through a drastic change in IAA level.
Unusual ethylene‐related behavior in senescing flowers of the carnation Sandrosa
Authors: Mayak Shimon , Tirosh Tsipora
Published: May 2006

The time course of ethylene production by senescing carnation (Dianthus caryophyllus L. cv. Sandrosa) flowers was studied. These flowers are unusual in that they do not exhibit an autocatalytic increase in ethylene production nor do they develop petal in‐rolling. Exposure of the flowers to exogenous ethylene resulted in a rise in their ethylene‐forming enzyme (EFE) activity and ethylene production, and at the same time a marked decline in their fresh weight. Natural senescence was also accompanied by a rise in EFE activity, but with no concomitant rise in 1‐amino cyclopropane carboxylic acid synthase activity nor in ethylene production. A shift in responsiveness to ethylene was observed, with young flowers more responsive to exogenous ethylene than older flowers. The results are discussed in terms of a proposed mechanism allowing for the decline in competence of this cultivar to respond to ethylene during senescence.
Embryology in Norway spruce (Picea abies). Immunochemical studies on transport of a seed storage protein
Authors: Hakman Inger
Published: May 2006

One of the main seed storage proteins of Norway spruce (Picea abies), is a salt‐soluble protein with an average molecular mass of 42 kDa. This protein was localized by immunocytochemical methods in ultrathin sections of megagametophytes active in storage protein synthesis, as analyzed by SDS‐PAGE. The megagametophyte in spruce starts accumulating storage materials, proteins and lipids, as the young embryo grows into the gametophytic tissue. It then continues to accumulate these storage products throughout seed development (Hakman 1993). Megagametophytes at an early stage of storage protein accumulation were chosen in this study for analysing the likely transport pathway of the proteins, since only a small amount of lipid had yet accumulated in the cells, and cell organelles were still easy to distinguish. An antibody against the 42 kDa storage protein showed very good reactivity with the 42 kDa protein in immunoblot experiments with total protein extracts from megagametophytes and embryos. In ultrathin sections of the megagametophyte, the antibodies were preferentially localized in the lumen of Golgi cisterna, in Golgi‐associated vesicles, protein deposits close to the vacuolar membrane and in protein storage vacuoles (protein bodies). These observations indicate that the transport is mediated by the Golgi apparatus.

Also, proteins present in storage vacuoles in mature zygotic and somatic embryos showed intense labelling with these antibodies in ultrathin sections.
De novo shoot morphogenesis and plant growth of mustard (Brassica juncea) in vitro in relation to ethylene
Authors: Pua E.‐C. , Chi G.‐L.
Published: May 2006

Role of ethylene in de novo shoot morphogenesis from explants and plant growth of mustard (Brassica juncea cv. India Mustard) in vitro was investigated, by culturing explants or plants in the presence of the ethylene inhibitors aminoethoxyvinylglycine (AVG) and AgNO3. The presence of 20 μM AgNO3 or 5 μM AVG in culture medium containing 5 μM naphthaleneacetic acid and 10 μM benzyladenine were equally effective in promoting shoot regeneration from leaf disc and petiole explants. However, AgNO3 greatly enhanced ethylene production which reached a maximum after 14 days, whereas ethylene levels in the presence of AVG remained low during 3 weeks of culture. The promotive effect of AVG on shoot regeneration was overcome by exogenous application of 25 μM 2‐chloroethylphosphonic acid (CEPA), but AgNO3‐induced regeneration was less affected by CEPA. For whole plant culture, AVG did not affect plant growth, although it decreased ethylene production by 80% and both endogenous levels of 1‐aminocyclopropane‐1‐carboxylate (ACC) synthase and ACC by 70–80%. In contrast, AgNO3 stimulated all 3 parameters of ethylene synthesis. Both AgNO3 and CEPA were inhibitory to plant growth, with more severe inhibition occuring in AgNO3. Leaf discs derived from plants grown with AVG or AgNO3 were highly regenerative on shoot regeneration medium without ethylene inhibitor, but the presence of AgNO3 in the medium was inhibitory to regeneration of those derived from plants grown with AgNO3.
Acetolactate synthase from Brassica napus: Immunological characterization and quaternary structure of the native enzyme
Authors: Bekkaoui Faouzi , Schorr Peter , Crosby William L.
Published: May 2006

Acetolactate synthase (ALS, AHAS; EC 4. 1. 3. 18) from Brassica napus has been partially purified and characterized using polyclonal antibodies. Following denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis and western blot analysis, 65 and 66 kDa ALS subunit polypeptides were immunologically detected, along with a novel 36 kDa polypeptide which cross‐reacted with the anti‐ALS antibody. Partial peptide sequencing of the 36 kDa peptide revealed significant similarity to plant aldolase proteins. ALS activity from stromal extracts fractionated by gel filtration chromatography as a single species of estimated molecular mass of 124 kDa, while comparative sedimentation coefficient in glycerol gradients indicated a corresponding molecular mass of 132 kDa. The results suggest that the native enzyme is a dimer of 65 and/or 66 kDa subunits. Anion exchange chromatography resolved two classes of ALS activity of equal native molecular weight, but which exhibited different properties with respect to subunit structure, sensitivity to inhibition by chlorsulfuron and feedback inhibition by branched chain amino acids.
Nicotianamine: mediator of transport of iron and heavy metals in the phloem?
Authors: Stephan Udo W. , Scholz Günter
Published: May 2006

Recent work has demonstrated that minerals in plants are circulated between root and shoot. This occurs during the whole life time and renders possible response to changing environmental conditions. This mineral circulation occurs through intensive solute exchange between xylem and phloem in roots, stems, and leaves. The transport form of heavy metals such as iron, manganes, zinc and copper in the phloem, whether ionic or chelated, is unclear in most cases.

The unusual amino acid nicotianamine (NA) is ubiquitous throughout the plant kingdom. It is a chelator of several divalent transition metals. Its physiological role was investigated with the tomato mutant chloronerva, the only known NA‐free multicellular plant. The mutant also exhibits disturbances of its iron metabolism and that of other heavy metals. This leads, among others, to a typical intercostal chlorosis and progressive iron accumulation in the leaves. From the heavy metal chelating properties of NA and from the phenotype of the mutant chloronerva it is concluded that NA is needed for normal distribution of heavy metals in young growing tissues fed via the phloem. This function could be fulfilled by mediating phloem loading or unloading of heavy metals as well as by preventing their precipitation in the alkaline phloem sap. An attempt is made to explain the chloronerva phenotype in the light of the phloem transport hypothesis of chelated iron.
Abscisic acid and assimilate partitioning to developing seeds. II. Does abscisic acid influence the sink strength of Arabidopsis seeds?
Authors: de Bruijn S. M. , Vreugdenhil D.
Published: May 2006

The role of abscisic acid (ABA) in the regulation of transport of assimilates to seeds was investigated with the aid of Arabidopsis thaliana mutants that were ABA‐deficient and/or insensitive to ABA. Subsequent flowers of mutant mother plants were alternately pollinated with pollen from either wild‐type or mutant plants, and the transport of radiolabelled photoassimilates to the genetically different seeds was studied. The experiments were performed under conditions of reduced availability of source material, achieved either by reduced light quantity or by combining the ABA‐deficient mutant with a starchless mutant. No effect of the genotype on the import rate of assimilates was detected, indicating that endogenous ABA does not influence the sink strength of Arabidopsis seeds. Reports describing contrary results are discussed.
Effect of culture conditions on the isocitrate dehydrogenase and isocitrate lyase activities in Chlamydomonas reinhardtii
Authors: Martínez‐Rivas José M. , Vega José M.
Published: May 2006

In the green alga Chlamydomonas reinhardtii, nitrogen staravation induced a reversible increase (2‐fold) in NAD‐isocitrate dehydrogenase (NAD‐IDH; EC 1.1.1.41) and NADP‐isocitrate dehydrogenase (NADP‐IDH; EC 1.1.1.42) activities. Both enzymes were not affected by the concentration of CO2, the dark or the nature of the nitrogen source (nitrate, nitrite, or ammonium). When cells growing autotrophically were transferred to heterotrophic conditions, a 40% reduction of the NAD‐IDH activity was detected, a 2‐fold increase of NADP‐IDH was observed and isocitrate lyase (ICL; EC 4.1.3.1) activity was induced. The replacement of autotrophic conditions led to the initial activity levels. NAD‐ and NADP‐IDH activities showed markedly different patterns of increase in synchronous cultures of this alga obtained by 12 h light/12 h dark transitions. While NAD‐IDH increased in the last 4 h of the dark period, NADP‐IDH increased during the last 4 h of the light period, remaining constant for the rest of the cycle.
Electroporation of rapeseed protoplasts – transient and stable transformation
Authors: Bergman Per , Glimelius Kristina
Published: May 2006

Protoplasts of Brassica napus hypocotyls were transfected using electroporation. Parameters such as discharge potential, protoplast density and buffer constituents were tested to determine the most suitable conditions for gene transfer. To monitor the introduction of DNA into protoplasts a plasmid containing the β‐glucuronidase (EC 3.2.1.31), and the neomycin phospotransferase (EC 2.7.1.95) genes was used. By using this construct, expression of a screenable marker gene for transient expression analysis as well as an antibiotic resistance marker gene for selection of stable transformants were obtained. Refined electroporation conditions resulted in a frequency of 0.1% transiently transformed protoplasts. Microcalluses were cultured under selective conditions in a bead‐type culture system. Resistant callus, with an absolute transformation frequency of 4.9 × 10−5 and a relative transformation frequency of 0.3% could be achieved. X‐ray irradiation of newly electroporated protoplasts did not enhance absolute transformation frequencies. From some of the resistant calluses, transgenic plants could be regenerated which were characterized by molecular analysis.
Exothermic responses of dormant Salix stems during exposure to subzero temperatures
Authors: von Fircks Heinrich A.
Published: May 2006

Differential thermal analysis (DTA) was used to determine the exothermic responses in dormant stems and excised lengths of stem ofSalix dasyclados Wimmer subjected to artificial freezing treatments.

The presence of ice on the surfaces of intact stems restricted the mechanism of freezing avoidance to temperatures above –4°C. In contrast, excised lengths of stem started to freeze as soon as the ambient temperature fell below –2°C, demonstrating that extracellular ice formation takes place earlier if cut surfaces are present. Exposure of dormant excised lengths of stem to subfreezing temperatures for more than 8 weeks did not alter their nucleation temperature not their exothermic differential responses. Early extracellular crystallisation of freezable cellular water provides conditions that allow dormant Salix dasyclados stems or excised lengths of stem to survive extreme freezing stress.

Crystallisation of extracellular and cellular water took place in the cortex, and did not result in visual damage or reduced survival. This nucleation of extracellular water took place over the same temperature range whether the excised dormant lengths of stem were partly (bark only) or completely thawed. Exposure of dormant tissue to 20°C for up to 24 h did not alter the level of freezing tolerance, nor did it increase the susceptibility of excised lengths of stem to damage by extreme temperature fluctuations.
Relationship between evolution of CO2 and growth of plum shoot tips cultured in vitro under different light/dark regimes
Authors: Morini Stefano , Muleo Rosario , Sciutti Renato , Fortuna Paola
Published: May 2006

The application of 4 h light followed by 2 h darkness (4/2 h), compared to 16 h light‐8 h darkness (16/8 h). induced very different patterns of CO2 evolution from proliferating shoots of Prunus cerasifera in vitro. Under the former light/dark regime, fresh and dry weights of shoot clutures and the number of newly formed shoots were increased. Cultures under 4/2 h showed a higher photosynthetic capacity at an earlier stage of growth but this did not appear to be a factor in the enhanced growth. It is suggested that phytochrome may be involved in determining a different pattern of growth.
Comparative study of metabolism and forms of transport of phosphate between Ascophyllum nodosum and Polysiphonia lanosa
Authors: Penot Marthe , Hourmant Annick , Penot Michel
Published: May 2006

Polysiphonia lanosa (L.) Tandy is a marine red alga that usually grows epiphytically on the fucale Ascophyllum nodosum (L.) Le Jolis. The present work was conducted in order to obtain more information on the relationships between these two algae, especially as regards the metabolism and long‐distance transport of phosphorus. Three types of experiments were carried out using labelled phosphorus. (1) Comparative study of the metabolism of 32P1 absorbed by the tissues of each species. By means of two‐dimensional chromatography and autoradiography, it was shown that 32P1 was rapidly incorporated into organic soluble compounds (adenosine triphosphate, hexose monophosphate, uridine diphosphoglucose, phosphoenolpyruvate + phosphoglyceric acid). Although the two algae belong to different phylae the phosphorylated compounds were not very different. The energy charges (0. 72 for both species) were in the usual range for aerobic plant tissues. On the other hand the incorporation of 32P1 into the insoluble P0 fraction was doubled in P. lanosa compared to in A. nodosum (ca 80 and 40%, respectively). At the source level, the air bladder of A. nodosum. the same soluble compounds (inorganic phosphate, P1 adenosine triphosphate, hexose monophosphate. etc.) represented the likely forms transported. A part of the soluble P0 fraction may return to the P1 fraction. (2) In translocation experiments conducted in situ, 32P1 locally injected into an air bladder moved over long distances not only through the thallus of A. nodosum but also into P. lanosa. The reciprocal transfer remained unsuccessful. (3) The 32P1 represented the predominant compound identified in the two species: this argues in favour of P1 as the translocated form of phosphorus. Our results support the hypothesis of a parasitic rather than a simple epiphytic relationship between the two algae.
The presence of a short redox chain in the membrane of intact potato tuber peroxisomes and the association of malate dehydrogenase with the peroxisomal membrane
Authors: Struglics André , Fredlund Kenneth M. , Rasmusson Allan G. , Møller Ian M.
Published: May 2006

Peroxisomes and mitochondria were purified from potato tubers (Solanum tuberosum L. cv. Bintje) by differential centrifugation followed by separation on a continuous Percoll gradient containing 0.3 M sucrose in the lower half and 0.3 M mannitol in the upper half. The peroxisomes band at the bottom and the mitochondria in the middle of this type of gradient. Mitochondrial contamination of the peroxisomes was only 2% [as judged by cytochrome c oxidase (EC 1.3.9.1) activity]. Contamination by amyloplasts, plasma membrane and endoplasmic reticulum was also minimal. The peroxisomes were 80% intact as judged by malate dehydrogenase (MDH, NAD−‐dependent; EC 1.1.1.37) latency.

The specific activity of NADH‐ferricyanide reductase and NADH‐Cyt c reductase was 0.22 and 0.051 μmol (mg protein)−1 min−1 in freshly isolated peroxisomes, respectively. The active site of the reductase appeared to be on the inner surface of the membrane. The peroxisomes also contained a b‐type cytochrome. Frozen peroxisomes were subfractionated by osmotic rupture followed by centrifugation to separate the soluble proteins from the peroxisomal membrane. About half the MDH and 30% of the NADH‐ferricyanide reductase activity was associated with the membrane but only 6% of the catalase (EC 1.11.1.6) activity. A further wash removed 75% of the residual catalase with only a small loss of MDH or NADH‐ferricyanide reductase activity. MDH appears to be closely associated with the peroxisomal membrane.

When the purified peroxisomal membrane was analyzed by SDS‐PAGE followed by silver staining, prominent bands at 22, 40, 41, 48, 53 and 74 kDa were observed. After immunoblotting the purified peroxisomal membrane, a band at 53 kDa showed strong cross‐reactivity with antibodies raised against NADH‐ferricyanide reductase. Since the NADH‐ferricyanide reductase activity in the peroxisomal membrane could be shown to be specific for the β‐hydrogen of NADH, the activity could not be due to contamination by endoplasmic reticulum where the reductase is α‐specific. We conclude that the peroxisomal membrane contains a short redox chain, consisting of a NADH‐ferricyanide reductase and a b‐type cytochrome, similar to that of e.g. the plasma membrane. The role of this redox chain has yet to be elucidated.
Selection in vitro for UV‐tolerant sugar beet (Beta vulgaris) somaclones
Authors: Levall Mats W. , Bornman Janet F.
Published: May 2006

With a reduced stratospheric ozone concentration, the generation of UV‐tolerant plants may be of particular importance. Among different crop plants there is large variation in sensitivity to UV‐B radiation. This study was undertaken to investigate the possibilities of using somaclonal variation and selection in vitro for improving UV‐B tolerance in sugar beet (Beta vulgaris L.).

Sugar beet callus was exposed to UV radiation (280–320 nm, 0.863–5.28 kJ m‐2 day‐1, unweighted) and resultant shoots were selected from surviving cells. After establishment of the plants, they were grown under either visible radiation (114 μmol m‐2 s‐1 PAR) or with the addition of UV radiation (6.3 kJ m‐2 day‐1 biologically effective UV‐B). Screening of regenerants in vivo for tolerance to UV radiation was undertaken 10 months after termination of the UV selection pressure. Screening was done visually and by using a number of physiological parameters, including chlorophyll fluorescence induction, ultraweak luminescence, pigment analysis and total content of UV‐screening pigments. A clear difference between the unselected and the UV‐selected somaclones was observed when visually studying the UV damage and other leaf injury. The observations were supported by the ultraweak luminescence measurements. Unselected plants showed significantly greater damage when subjected to subsequent UV radiation as compared to the selected plants. The clones subjected to UV selection pressure displayed a significantly higher concentration of UV‐screening pigments under subsequent UV radiation. The unselected plants under subsequent UV treatment showed a lower carotenoid concentration when compared to selected plants. However, no significant difference between treatments was found for chlorophyll a/b, or F/Fmax, a measure of photosynthetic quantum yield.
Physiological responses of Prunus cerasus to whole‐plant source manipulation. Leaf gas exchange, chlorophyll fluorescence, water relations and carbohydrate concentrations.
Authors: Layne Desmond R. , Flore James A.
Published: May 2006

The source‐sink ratio of 1‐year‐old, potted sour cherry trees (Prunus cerasus L.) was altered by whold‐plant partial defoliation or continuous illumination to determine if trees were primarily sink limited and to elucidate the means whereby photosynthetic enhancement or inhibition occurs. Leaf xylem water potential was not affected by either treatment. Although stomatal conductance was reduced by 1 to 3 days of continuous illumination, internal CO2 concentration was not significantly affected indicating that the enhanced physical limitation imposed by the stomata was of no physiological significance. Net CO2 assimilation (A) was significantly higher 4 days after partial defoliation and lower from 1 to 4 days following continuious illumination. The increase in A in partially defoliated plants was associated with reduced leaf starch and increased surose and sorbitol concentrations. The decrease in A in continuously illuminated plants was associated with a decrease in variable fluorescence, photochemical efficiency of photosystem II (PSII) and an increase in instantaneous fluorescence, indicating that leaves were photoinhibited and that irreversible damage had occurred to PSII. In addition, leaves of continuously illuminated plants had 80% more starch and significantly less sucrose and sorbitol. These altered leaf carbohydrate concentrations indicate that the existing sink limitation may have been aggravated by continuous illumination leading to an insufficient utilization of sucrose from the leaf. Whether the altered photochemical and biochemical events occurred simultaneously and/or to the same degree to lead to the observed responses remains equivocal.
Aneuploidy and polyploidization in haploid tissue cultures of Larix decidua
Authors: von Aderkas Patrick , Anderson Parker
Published: May 2006

Haploid embryogenic lines of Larix decidua which had been maintained on medium free of plant growth regulatiors were screened for chromosomal abnormalities. Chromosome squashes were prepared from fast‐growing suspension culture. alternatively, a method using burst protoplasts was devised for improving chromosome spreads. One line showed polyploidization with chromosome mumbers ranging from haploid to tetraploid. This line is presently predominantly diploid. Aneuploidization also occurred. The most effective method for counting chromosomes was from burst protoplasts. It is recommended that long‐term cultures of conifer tissues be checked for genetic stability.