bluuebunny/crossref_metadata_2025 huggingface dataset:
Can genotypes of soybean (Glycine max) selected for nitrate tolerance provide good “models” for studying the mechanism of nitrate inhibition of nitrogenase activity?
Authors: Neo Hwee Hwee , Hunt Stephen , Layzell David B.
Published: May 2006

In soybeans (Glycine max L. Merr.), high levels of soil nitrate inhibit N2 fixation, and nitrate‐tolerant symbioses have been identified within a chemically mutagenized line of cv. Bragg denoted nts382 and within the line K466, a genotype representative of a number of Korean soybean cultivars. The genotypes nts382 and K466 were examined to see if they could be used as a model system for studying the mechanism responsible for the short‐term (i.e. 3‐day) inhibition of specific nitrogenase activity, especially the mechanism behind the greater O2 limitation of nodule metabolism that is characteristic of nitrate inhibition of N2 fixation in soybean. In nts382, total nitrogenase activity (TNA = H2 production in Ar:O2) was inhibited to a lesser degree (48% of control) relative to Bragg (30% of control), and the nitrate‐treated symbioses showed less of an O2 limitation of nodule metabolism in nts382 than in Bragg. However, the relative proportion of O2 limitation to the total nitrate inhibition was similar (40 and 41%) in nts382 and Bragg, respectively. Therefore, the nts382 symbioses may be useful in elucidating the general mechanism for down‐regulation of nitrogenase activity in soybean, but would not be a useful model system for studying the control of O2‐limited metabolism following nitrate exposure. The effects of nitrate on TNA and on the degree of O2 limitation of nodule metabolism were the same in K466 and a reference cultivar Maple Arrow. Consequently, the tolerance of K466 to nitrate reported previously was attributed to the ability of this symbiosis to maintain nodule biomass in the presence of nitrate, not to any ability to maintain specific nitrogenase activity in the presence of nitrate.
Expression of auxin‐inducible genes in relation to endogenous indoleacetic acid (IAA) levels in wild‐type and IAA‐overproducing transgenic tobacco plants
Authors: Sitbon Folke , Dargeviciute Ausra , Perrot‐Rechenmann Catherine
Published: May 2006

Probes corresponding to the auxin‐inducible genes parA and parB, as well as two aux genes, here denoted Nt‐aux8 and Nt‐aux16, were amplified from tobacco (Nicotiana tabacum L.) cDNA. Transcript levels of these genes were analysed in wild‐type tobacco plants and in the transgenic IAA‐overproducing line C, and related to the endogenous IAA level. In addition, effects of naphtyl‐1‐acetic acid (NAA) treatment on the expression of these genes were determined in both genotypes. Two separate situations were identified. Nt‐aux8 and Nt‐aux16 steady‐state mRNA levels were positively correlated with the IAA content in wild‐type and line C plants. Addition of NAA induced these two genes strongly in wild‐type plants, but only slightly in line C plants. On the other hand, mRNA levels of the parA and parB genes varied more with the organ analysed and its age than with the IAA level, and were induced by NAA treatment to a similar extent in the two genotypes. The results suggest that expression of the two Nt‐aux genes is closely related to the endogenous auxin level, whereas expression of the parA and parB genes to a greater extent is influenced by additional factors.
Endogenous cytokinins in the vascular cambial region of Pinus sylvestris during activity and dormancy
Authors: Moritz Thomas , Sundberg Björn
Published: May 2006

Elucidation of the role of endogenous cytokinins in cambial activity and wood formation requires knowledge of their identity and concentrations in the cambial region. Here, we have used capillary liquid chromatography/frit‐FAB mass spectrometry to identify endogenous cytokinins in the vascular cambial region of mature Pinus sylvestris (L.) trees. Full‐scan mass spectra were obtained for isopentenyladenine, isopentenyladenosine, zeatin riboside, dihydrozeatin and dihydrozeatin riboside. Of these, isopentenyladenine, dihydrozeatin and dihydrozeatin riboside are demonstrated by rigorous physico chemical methods for the first time in a conifer. In addition, an adenine glycoside was found for the first time in a plant. The identified cytokinins were quantified in active and dormant cambial region tissues by isotope dilution techniques using the appropriate deuterated isotope for each cytokinin species. The concentration of the detected cytokinins ranged between 1.3 and 5.5 pmol g‐1 fresh weight, and did not vary greatly between dormant tissues, and in tissues actively dividing and differentiating. This observation indicates that cessation and reactivation of cell division activity in the vascular cambium is controlled by factors other than cytokinin availability.
Cross‐species amplification of glutamine synthetase cDNA by polymerase chain reaction with degenerate primers
Authors: Pérez‐Vicente Rafael , Dorado Gabriel , Maldonado José María
Published: May 2006

We have developed and optimized a consistent polymerase chain reaction (PCR)‐based strategy to quickly obtain specific sequence information on novel plant glutamine synthetase (GS, EC 6.3.1.2) cDNAs. Two sets of degenerate primer pairs were designed to discriminate regions conserved in either any kind of GS messenger or exclusively in those for the chloroplastic GS. Novel GS cDNA sequences were successfully amplified from total RNA obtained from 14 different monocotyledonous and dicotyledonous plants. The procedure, coupled with a further restriction analysis, allowed us to uncover the presence of GS cDNA polymorphism, which most likely stems from the different GS gene family members within a single species. Contrary to previously reported strategies in other systems, GS cDNA oligonucleotide primers were designed keeping the degeneracy level to a minimum, together with a high melting temperature. This approach proved to be particularly effective, generating high yields of the expected products without requiring extra nested amplification steps or time‐consuming optimization steps for each species GS cDNA amplification. Different clones containing sequence information from either the coding or the 3′‐untranslated regions were further sequenced and characterized, confirming the high sequence identity and size uniformity the of GS cDNAs across higher plant species. Therefore, this approach is proposed as a stand‐alone procedure to quickly determine the sequence of unknown GS cDNAs, as well as to speed up and complement classical molecular cloning methodologies.
Pools of non‐structural carbohydrates in soybean root nodules during water stress
Authors: Müller Joachim , Boller Thomas , Wiemken Andres
Published: May 2006

The aim of this study was to examine how the pools of non‐structural carbohydrates in soybean nodules are affected under water stress conditions depending on the nature of the symbiont strains with particular emphasis on the plant‐borne carbohydrates sucrose and pinitol, and on trehalose, a compatible solute synthesized by the bacteroids. Soybean (Glycine max [L.] Merr. cv. Maple Arrow) plants were inoculated with the nitrogen‐fixing strains Bradyrhizobium japonicum 61‐A‐101 or USDA 110 spc4 and cultivated axenically under conditions in which nodules formed in an upper soil compartment while roots for water supply grew into a compartment filled with nutrient solution. When the nodules were well established (1 month post inoculation), 10% (w/v) PEG 6000 was added to the nutrient solution. This led to a slowly progressing, moderate water stress, as determined by measuring the decrease of transpiration, and to a decrease in nitrogen fixation. The pool sizes of the major non‐structural nodule carbohydrates changed during progression of water stress. Sucrose, the major soluble carbohydrate in nodules of unstressed plants (2 and 4%, respectively of nodule dry weight depending on symbiont strain), strongly increased in nodules of stressed plants, reaching nearly 10% of dry weight. The activities of two major sucrose‐consuming enzymes, sucrose synthase and alkaline invertase, decreased markedly in nodules of stressed plants. Starch decreased only transiently upon water stress. Pinitol, a cyclitol serving as compatible solute in many plants, increased more than 4 times, reaching about 1% of nodule dry weight during the stress. Trehalose, the major soluble carbohydrate synthesized by the bacteroids, increased in nodules colonized by USDA 110 spc4 from about 0.2 to 0.8% of nodule dry weight, while in nodules colonized by 61‐A‐101 it amounted to more than 1.5% of dry weight both with and without stress.
Effects of fusicoccin and indole‐3‐acetic acid on the levels of ascorbic acid and dehydroascorbic acid in the apoplast during elongation of epicotyl segments of Vigna angularis
Authors: Takahama Umeo
Published: May 2006

The levels of ascorbic acid (AA) and dehydroascorbic acid (DHA) in the apoplast of epicotyl segments from Vigna angularis L. cv. Erimoshouzu decreased to nearly zero and about 35%, respectively, of their initial levels, 3 h after the preparation of the epicotyl segments. The decreased level was kept nearly constant between 3 and 7 h. Fusicoccin (FC) and indole‐3‐acetic acid (IAA) slightly amplified the initial decrease in the level of AA, but suppressed the initial decrease in the level of DHA while enhancing elongation growth. During incubation for 3 and 7 h, FC then increased the levels of both AA and DHA, whereas IAA did so only with DHA. By the addition of FC 4 h after the start of incubation, the levels of both AA and DHA were also increased. The uncoupler carbonylcyanide m‐chlorophenyl hydrazone increased the levels of both AA and DHA in the apoplast inhibiting elongation growth. These results suggest that the electrochemical proton gradient across the plasma membrane is one of the factors that control the apoplastic levels of AA and DHA.
Capsidiol: Its role in the resistance of Capsicum annuum to Phytophthora capsici
Authors: Egea Catalina , Alcázar M. Dolores , Candela M. Emilia
Published: May 2006

Inoculation of the stems of three Capsicum annuum L. cultivars showing different degrees of sensitivity to the fungal pathogen Phytophthora capsici, resulted in a hypersensitive reaction being expressed along the stems. One of the peppers (cv. Smith‐5) showed resistance by total inhibition of fungal growth. Capsidiol, a phytoalexin, which accumulates in the area of necrosis appears to be involved in this resistance. Capsidiol accumulation was analyzed by gas chromatography and was correlated with the restricted growth of P. capsici, in vivo and in vitro, confirming the former's fungistatic and fungitoxic properties. The capacity to inhibit pathogenic growth was evident only when capsidiol production exceeded 1 204 μg ml‐1, a level reached in the resistant variety after 6 days of incubation. Experiments on induced resistance showed that a second inoculation of the stems of the three cultivars also resulted in necrosis and in an accumulation of capsidiol, although to a lesser extent than in the first inoculation. The greater accumulation of capsidiol in the stems of cv. Smith‐5 is in accordance with the resistance shown by this cultivar to P. capsici, and confirms the implication of capsidiol in the disease resistance of this cultivar to fungal pathogens. Capsidiol has a fungistatic character at a mean concentration of 3.75 mM, and is fungitoxic at levels above 5 mM. This level must be exceeded and all the growing hyphae must be affected for capsidiol to qualify from being fungistatic to being fungitoxic.
Changes in cell cycle status and expression of p34cdc2 kinase during potato tuber meristem dormancy
Authors: Campbell Michael A. , Suttle Jeffrey C. , Sell Thomas W.
Published: May 2006

Potato (Solanum tuberosum L. cv. Russet Burbank) tubers undergo a period of endodormancy that is characterized by cell division arrest. At the time of harvest, the tubers used in this study were completely endodormant (i.e. 0% sprouting). After 120 days of storage at 3°C, tubers transferred to 20°C had begun to exit endodormancy and exhibited ca 50% sprouting. After 223 days of 3°C storage, tubers transferred to 20°C were completely nondormant and exhibited 100% sprouting. Based on flow cytometry, about 70% of nuclei isolated from endodormant meristems are arrested in the G1/G0 phase of the cell cycle. Storage of tubers at 3°C did not alter the cell cycle position nor did transfer of tubers from 3 to 20°C for 7 days prior to analysis unless tubers had been stored for at least 223 days. After 223 days of cold (3°C) storage, tubers transferred to 20°C for 7 days showed sprout growth in excess of 5 mm and an increase in the percentage of nuclei in the G2 phase of the cell cycle. Uptake and incorporation of 3H‐thymidine into DNA was low in all tubers up until 120 days postharvest. After that time, only tubers incubated at 20°C for 7 days prior to analysis exhibited an increase in 3H‐thymidine incorporation. This increase coincided with visible sprout growth, demonstrating that cell cycle shifts in tuber meristems relate directly to sprout growth and not the breakage of the endodormancy per se. Using degenerate primers, a portion of a p34cdc2 homolog was amplified from RNA isolated from logphase potato suspension culture cells by polymerase chain reaction. Northern analysis with this probe demonstrated that mRNA levels for two p34cdc2 homologs were present throughout the endodormant period. Immunoblot analysis demonstrated that levels of at least four proteins containing a PSTAIRE epitope (i.e. cdc2‐like) were present at reduced levels in endodormant meristems and increased in reactivated tuber meristems that showed a shift in cell cycle kinetics based on flow cytometry and increased 3H‐thymidine incorporation. These results indicate that the temporal shift in competence for cell division in potato meristems induced by dormancy is not accompanied by alterations in the level of mRNA for p34cdc2 homologues but is correlated with a change in the level of PSTAIRE‐containing proteins. This suggests that during endodormancy cell division in potato tuber meristems is regulated indirectly by posttranscriptional regulation of genes controlling the cell cycle.
Purification and characterization of UDP‐D‐galactose 4‐epimerase from the red alga Galdieria sulphuraria
Authors: Prosselkov Pavel V. , Gross Wolfgang , Igamberdiev Abir U. , Schnarrenberger Claus
Published: May 2006

UDP‐D‐galactose 4‐epimerase of the unicellular red alga Galdieria sulphuraria has been purified to apparent electrophoretic homogeneity by chromatography on DEAE‐Fractogel, hydroxylapatite and by affinity chromatography on Dyematrex Orange. The holoenzyme is a homodimer with an apparent molecular mass of 83 and 76 kDa as determined by gelfiltration and by sucrose gradient centrifugation, respectively. The size of the subunits was 42 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 4‐epimerase from G. sulphuraria does not require external NAD for activity, unlike the enzyme from some other organisms, and inhibition by NADH was not observed. The apparent Km for UDP‐D‐galactose was 64 μM. The pH optimum was at 8 and the apparent equilibrium constant for UDP‐Glc/UDP‐Gal was 3.5. The enzyme in crude as well as in purified samples was unusually stable and was not inactivated even on incubation at 46°C for several hours.
Rooting and acclimatization of micropropagated cuttings of Pinus pinaster and Pinus sylvestris are enhanced by the ectomycorrhizal fungus Hebeloma cylindrosporum
Authors: Normand Laurence , Bärtschi Henry , Debaud Jean‐Claude , Gay Gilles
Published: May 2006

The effect of different strains of the ectomycorrhizal fungus Hebeloma cylindrosporum on rooting in vitro and acclimatization of micropropagated cuttings of Pinus pinaster and Pinus sylvestris was studied. Two clones of P. pinaster and one of P. sylvestris were unable to root in the absence of auxin, but were induced to root on a medium devoid of auxin by all the fungal strains. Wild‐type and indoleacetic acid (IAA)‐overproducing mutant strains of the fungus stimulated rooting of clones showing a good reactivity to auxin to the same extent. In contrast, with a clone of P. sylvestris that showed low reactivity to auxin, IAA‐overproduction by the fungus was advantageous for the induction of rooting of cuttings. Adventitious roots formed in the presence of a fungal strain were completely surrounded by a loosely packed network of hyphae which formed mycorrhizas as soon as roots grew outside the agar medium. During acclimatization, fungal inoculation improved the survival of rooted cuttings. At the end of acclimatization, fungal mycelia could be easily detected in the culture substrate of cuttings inoculated with dikaryotic strains and most of the pines' short roots were mycorrhizal. Monokaryotic mycelia, which have a lower growth rate and a lower infectivity, displayed poor ability to colonize the substrate and to form mycorrhizas. Two months after the end of acclimatization, fungal inoculation frequently depressed the growth of acclimatized cuttings of the clone J of P. pinaster. No depressive effect was observed with clone 78 and growth stimulation could even be observed with the infective dikaryon D1 which formed numerous mycorrhizas. From these studies, it was concluded that ectomycorrhizal fungi could be a suitable tool for improving rooting in vitro and survival at acclimatization of micropropagated conifer cuttings.
Auxin induces exocytosis of acid phosphatase in coleoptiles from Zea Mays
Authors: Pfeiffer Wolfgang
Published: May 2006

Auxin‐stimulated elongation growth of maize coleoptiles has been suggested to be associated with enhanced exocytotic activity. However, the problem in plants is one of finding a soluble parameter, which can be used as a direct measure of exocytosis (H. D. Blackbourn and N. H. Battey [1993]. Physiol. Plant. 89: 27–32). In yeast, acid phosphatase (EC 3.1.3.2) is used as a marker for secretory activity (E. Harsay and A. Bretscher [1995]. J. Cell Biol. 131: 297–310). Therefore, extracellular acid phosphatase activities in maize tissues were investigated. Coleoptile (7.36 nkat mg‐1) and mesocotyl (8.9) showed higher specific extracellular acid phosphatase activities than primary leaf (6.0), root (4.9) and root tip (2.7). In coleoptiles extracellular acid phosphatase activity was 6.7% of total homogenate activity (mesocotyls 10.6%). Auxin (30 μM IAA) increased the extracellular acid phosphatase activity of coleoptiles (146% of control). This effect was tissue‐specific; extracellular acid phosphatase activity of mesocotyls was not enhanced by IAA. The stimulating effect of auxin on extracellular acid phosphatase activity in coleoptiles was reversed by the protonophore nigericin (0.3 μM). Furthermore, localization of an acid phosphatase activity in Golgi vesicles was shown by co‐migration of the Golgi marker latent IDPase (EC 3.6.1.6) and acid phosphatase activity (65% of total microsomal activity) on isopycnic continuous sucrose density gradients. Tonoplast‐enriched membrane frctions (24% of microsomal acid phosphatase) and plasma membrane‐enriched fractions (11%) contained lower amounts of acid phosphatase. The data presented suggest that acid phosphatase activity is a useful marker for hormone‐induced secretory activity in plant cells.
Photosynthetic characteristics of leaves of male‐sterile and hermaphrodite sex types of Plantago lanceolata grown under conditions of contrasting nitrogen and light availabilities
Authors: Poot Pieter , Pilon Jorn , Pons Thijs L.
Published: May 2006

Plantago lanceolata is a gynodioecious species: In natural populations male steriles (MS) coexist with hermaphrodites (H). Since male steriles have a reproductive disadvantage, without any compensation for their loss in male function by an increase in female function, they are expected to disappear from the population. In this study we investigated the possibility that differences in ecologically important photosynthetic characteristics, between MS and H lines of P. lanceolata. play a role in maintaining gynodioecy. One MS line and two H lines were grown under conditions of high N and light availability, as well as under either N limitation or light limitation, to investigate whether the sex types respond differently to environmental constraints. Photosynthetic light‐response and CO2‐response curves were made, together with leaf organic N and chlorophyll determinations. There were only few small differences between the lines and since the MS line did not differ in any of the determined photosynthetic characteristics from either H line, it is unlikely that these differences are involved in maintaining male sterility in populations of P. lanceolata. The low‐light‐grown plants showed a high degree of acclimation as shown by a two‐fold higher leaf area to leaf weight ratio (SLA), a two‐fold higher investment of N in light harvesting, and higher net photosynthetic rates under low‐light conditions, as compared to the high‐light‐grown plants. The low‐N‐grown plants used their organic N more efficiently in photosynthesis compared to plants grown at an optimal N supply. This was mainly due to the N‐limited plants having leaves with a lower organic N content and thus lower photosynthetic capacities. To a lesser extent it was due to the higher value for the curvature factor of the light‐response curves of the N‐limited plants, to their decreased rates of photorespiration and possibly to their relatively higher allocation of organic N to photosynthetic functions.
The induction of the CO2 concentrating mechanism in a starch‐less mutant of Chlamydomonas reinhardtii
Authors: Villarejo Arsenio , Martinez Flor , del Pino Plumed Maria , Ramazanov Ziyadin
Published: May 2006

In Chlamydomonas reinhardtii the formation of a starch sheath surrounding the pyrenoid is observed when cells grown under high CO2 (5% CO2 in air) are transferred to low CO2 (0.03%) conditions. Formation of the starch sheath occurs coincidentally with induction of the CO2 concentrating mechanism and with de novo synthesis of 5 polypeptides with molecular masses of 21, 36, 37, 42–44 kDa. We studied the effect of CO2 concentrations on photosynthesis, ultrastructure and protein synthesis in Chlamydomonas reinhardtii cw‐15 (wild phenotype for photosynthesis) and in the starch‐less mutant BAFJ‐6, with the aim to clarify the role of the pyrenoid starch sheath in the operation of the CO2 concentrating mechanism and whether these low CO2‐inducible polypeptides are involved in the formation of starch sheath. When wild type and starch‐less mutant cells were transferred from high to low CO2, the CO2 requirement for half‐maximal rates of photosynthesis decreased from 40 μM to 2 μM CO2. 35SO42‐ labeling analyses showed that the starch‐less mutant induced the 5 low CO2‐inducible polypeptides. These observations suggest that the starch‐less mutant was able to induce a fully active CO2 concentrating mechanism. Since the starch‐less mutant did not form a pyrenoid starch sheath, we suggest that the starch sheath is not involved in the operation of the CO2 concentrating mechanism and that none of these 5 low CO2‐inducible proteins is involved in the formation of the starch sheath in Chlamydomonas.
Accumulation of a 50‐kDa protein during leaf senescence of alstroemeria cut flowering stems
Authors: Jordi W. , Stoopen G. M. , Argiroudi I. , in ‘t Veld E. , Heinen P. , van Tol H.
Published: May 2006

Leaf senescence of alstroemeria cut flowering stems in the dark was accompanied by a decrease in the soluble protein and chlorophyll content. Two‐dimensional (2D) electrophoresis revealed that a polypeptide with an apparent molecular mass of 50 (± 2) kDa and isoelectric point of 5.0 (± 0.1) accumulated during the senescence process. Treatments which delayed leaf senescence (light and gibberellic acid) diminished the accumulation of this polypeptide. This polypeptide was purified and one of the peptides, resulting from digestion with trypsin, was sequenced. The se quence shows a high degree of homology to that of 3‐isopropylmalate dehydrogenase. This enzyme plays a role in the biosynthesis of leucine. Possibly, leucine plays a role in carbon partitioning between sources and sinks in this plant.
Physical and kinetic properties of acetohydroxyacid synthase from wheat leaves
Authors: Southan Michael D. , Copeland Les
Published: May 2006

Acetohydroxyacid synthase (AHAS, EC 4.1.3.18; also known as acetolactate synthase), which catalyses the first reaction common to the biosynthesis of the branched‐chain amino acids, L‐valine, L‐leucine and L‐isoleucine, and is the target of several classes of herbicides, has been studied in hydroponically‐grown seedlings of wheat (Triticum aestivum L. cv. Vulcan). Enzyme activity was greater in leaves than roots, reaching a maximum between 4 and 6 days after germination. AHAS was associated with the chloroplasts after centrifugation in a density gradient. A preparation of the enzyme was obtained from wheat leaves which gave a single band after electrophoresis in native gels but was resolved by denaturing sodium dodecyl sulphate‐polyacrylamide gel electrophoresis into three polypeptide bands of molecular mass 58, 57 and 15 kDa. The native molecular mass was approximately 128 kDa. AHAS had optimum activity at pH 7 and did not require the addition of flavin adenine dinucleotide (FAD), thiamine pyrophosphate (TPP) and MgCl2 for activity. The enzyme did not display typical hyperbolic kinetics, in that the double reciprocal plot of activity against pyruvate concentration was non‐linear. The concentration of pyruvate that gave half of the maximum activity was 4 mM. Sulfonylurea and imidazolinone herbicides were potent inhibitors of wheat leaf AHAS, with 50% inhibition being observed at concentrations of 0.6 and 0.3 μM for chlorsulfuron and metsulfuron methyl, respectively, and at 2.5, 5 and 10 μM for imazaquin, imazethapyr and imazapyr. Inhibition by both classes of compounds was reversed by removal of the inhibitor. Progress curves of product formation against time in the presence of the herbicides were non‐linear, and based on the assumption that inhibition by the sulfonylureas was of the slow, tight‐binding type, estimates of 0.17 and 0.1 nM were obtained for the dissociation constants of chlorsulfuron and metsulfuron methyl, respectively, from the steady‐state enzyme‐inhibitor complex.
5‐Aminoimidazole‐4‐carboxamide riboside activates nitrate reductase in darkened spinach and pea leaves
Authors: Huber Steven C. , Kaiser Werner M.
Published: May 2006

Nitrate reductase (NR) activity is modulated in vivo by phosphorylation (inactivation)/dephosphorylation (activation) in response to light/dark signals. The dephosphorylation of phospho‐NR in vitro, catalyzed by endogenous protein phosphatases, is known to be stimulated by 5′‐AMP suggesting that this metabolite may be an important regulator of the activity of NR, e.g. under anoxia. To determine whether 5′‐AMP might be a regulatory metabolite in vivo, excised spinach (Spinacia oleracea) and pea (Pisum sativum) leaves were provided 5‐aminoimidazole‐4‐carboxamide riboside (AICAR) via the transpiration stream, and the apparent phosphorylation status of NR was assessed by assay of activity in the presence of free Mg2+. NR was activated in darkened spinach leaves in a time‐ and concentration‐dependent manner when leaves were fed AICAR; there was also an accumulation of nitrite in treated leaves in the dark. The activation by AICAR could be blocked by several type 2A protein phosphatase inhibitors (microcystin‐LR, okadaic acid and cantharidin), and was not the result of a reduction of kinase activity by lack of ATP because cellular adenylates were unaffected. It was confirmed that AICAR‐P, but not AICAR, mimicked 5′‐AMP in the activation of phospho‐NR in vitro. Our results are consistent with the notion that AICAR is converted to the monophosphorylated derivative, which accumulates in cells and acts as a structural analog of 5′‐AMP. Our results suggest that a rise in cytosolic [5′‐AMP] may be sufficient to activate NR in vivo. AICAR should be a useful compound for identifying AMP‐regulated processes in plant systems.
Growth and growth substrate levels in spinach under non‐steady state conditions of nitrogen nutrition and light
Authors: Buysse Jan , Van den Broeck Hilde , Merckx Roel
Published: May 2006

Spinach plants (Spinacia oleracea L. cv. Subito) were grown in a complete nutrient solution under ample light intensity (14 h day−1 at 660 μmol m−2 s−1) before being transferred either to a minus‐N solution (experiment 1), or to limiting light conditions (6 h day−1 at 220 μmol m−2 s−1; experiment 2). Shoot growth in experiment 1 decreased significantly from 0.24 day−1 to 0.07 day−1 after the fourth day of transfer. Root relative growth rate increased after 1 day from 0.25 to 0.31 day−1, but decreased on the fifth day after transfer to 0.11 day−1. Shoot growth in experiment 2 decreased significantly from 0.25 to 0.17 day−1 after the fourth day of transfer, while root growth decreased to half of its original level (0.25 day−1) already on the second day. Growth substrate levels in the plants (free sugars, free amino acids) and starch levels depended on the plant age, the moment in the diurnal cycle, and the imposed treatment. Fluctuations in shoot growth or root growth resulting from the light or N limitation could not be explained by a correspondent increase or decrease in the levels of growth substrates. The hypotheses underlying the functional equilibrium theory, assuming shoot and root growth to be controlled by N‐ and C‐containing substrates respectively, and several other growth and partitioning models are therefore questioned. A neglect of the osmotic role of the free sugars in these models might be the explanation for this.
Inside or outside? Localization of the receptor relevant to auxin‐induced growth
Authors: Claussen Maike , Lüthen Hartwig , Böttger Michael
Published: May 2006

The localization of the auxin receptor relevant to the control of elongation growth is still a matter of controversy. Auxin‐induced elongation of maize coleoptile segments was measured by means of a high resolution auxanometer. When indole‐3‐acetic acid (IAA) was removed from the bathing solution, a rapid cessation of auxin‐induced elongation was detected. This decline was delayed when the auxin efflux carrier was blocked by the phytotropins naphthylphthalamic acid (NPA) and pyrenoylbenzoic acid (PBA) or by triiodobenzoic acid (TIBA). The IAA concentration in NPA‐pretreated segments was 2–3 times higher than in NPA‐free controls 35 min after the removal of IAA in the bathing medium.

A similar rapid drop of growth after removal of auxin was observed for the rapidly‐transported synthetic auxin, naphthaleneacetic acid (NAA). When the auxin efflux was blocked, growth induced by NAA was sustained much longer than IAA‐stimulated elongation.

In comparison with NAA, the synthetic auxin 2,4‐dichlorophenoxyacetic acid (2,4‐D) is known to be excreted very slowly by the efflux carrier. 2,4‐D‐induced growth remained at a stimulated level when the auxin was washed off, even in the absence of any auxin efflux inhibitor. We conclude from these results that the presence of intracellular auxin is a necessary and sufficient condition for sustained auxin‐induced elongation growth, at least for the phases during the 2 h after its application. Consequently, we postulate the existence of an intracellular auxin receptor relevant to the control of growth.
Barley scutellum protoplasts: Isolation, culture and plant regeneration
Authors: Nobre J. , Davey M. R. , Lazzeri P. A.
Published: May 2006

Many applications of cereal protoplast culture systems are still limited by the difficulties of regeneration from suspension cells which are the usual protoplast source. The objective of the present study therefore was to investigate the conditions for the development of a culture system for protoplasts capable of plant regeneration isolated directly from immmature scutella of barley. The procedure developed involves a two‐stage pre‐culture of scutellar tissue, followed by vacuum infiltration with cell wall degrading enzymes and the culture of alginate‐embedded protoplasts. The pre‐culture of the scutella and the co‐cultivation of protoplasts with nurse cells were the most important factors for the success of the culture system, but several other parameters affecting protoplast yield, viability and sustained division were identified, including the developmental stage of the embryo, the use of cold conditioning periods during pre‐culture, the composition of the pre‐culture and protoplast culture medium, and the embedding matrix. Protoplasts isolated from scutellar tissues of barley cvs Dissa, Clipper, Derkado and Puffin were capable of sustained division in culture. Macroscopic protoplast‐derived tissues were obtained in all cultivars, except ev. Puffin, and fertile plants were regenerated from cvs Dissa and Clipper 3–4 months after protoplast isolation. The procedure described provides a novel approach for the isolation of totipotent protoplasts in barley which avoids the need for suspension cultures.
Physiological and growth responses of two African species, Acacia karroo and Themeda triandra, to combined increases in CO2 and UV‐B radiation
Authors: Wand Stephanie J. E. , Midgley Guy F. , Musil Charles F.
Published: May 2006

The interactive effects of increased carbon dioxide (CO2) concentration and ultraviolet‐B (UV‐B, 280–320 nm) radiation on Acacia karroo Hayne, a C3 tree, and Themeda triandra Forsk., a C4 grass, were investigated. We tested the hypothesis that A. karroo would show greater CO2‐induced growth stimulation than T. triandra, which would partially explain current encroachment of A. karroo into C4 grasslands, but that increased UV‐B could mitigate this advantage. Seedlings were grown in open‐top chambers in a greenhouse in ambient (360 μmol mol‐1) and elevated (650 μmol mol‐1) CO2, combined with ambient (1.56 to 8.66 kJ m‐2 day‐1) or increased (2.22 to 11.93 kJ m‐2 day‐1) biologically effective (weighted) UV‐B irradiances. After 30 weeks, elevated CO2 had no effect on biomass of A. karroo, despite increased net CO2 assimilation rates. Interaction between UV‐B and CO2 on stomatal conductance was found, with conductances decreasing only where elevated CO2 and UV‐B were supplied separately. Increases in water use efficiencies, foliar starch concentrations, root nodule numbers and total nodule mass were measured in elevated CO2. Elevated UV‐B caused only an increase in foliar carbon concentrations. In T. triandra, net CO2 assimilation rates were unaffected in elevated CO2, but stomatal conductances and foliar nitrogen concentrations decreased, and water use efficiencies increased. Biomass of all vegetative fractions, particularly leaf sheaths, was increased in elevated CO2. and was accompanied by increased leaf blade lengths and individual leaf and leaf sheath masses. However, tiller numbers were reduced in elevated CO2. Significantly moderating effects of elevated UV‐B were apparent only in individual masses of leaf blades and sheaths, and in total sheath and shoot biomass. The direct CO2‐induced growth responses of the species therefore do not support the hypothesis of CO2‐driven woody encroachment of C4 grasslands. Rather, differential changes in resource use efficiency between grass and woody species, or morphological responses of grass species, could alter the competitive balance. Increased UV‐B radiation is unlikely to substantially alter the CO2 response of these species.
Hydroxynitrile lyases: Functions and properties
Authors: Hickel Andrea , Hasslacher Meinhard , Griengl Herfried
Published: May 2006

Plant hydroxynitrile lyases (Hnl) have attracted the attention of bioorganic scientists for more than 90 years. However, the most important increase in knowledge of this class of enzymes has only arisen in the recent decade. The industrial application of these enzymes as biocatalysts for the synthesis of enantiomerically pure α‐cyanohydrins may be responsible for the growing interest in this area.

The Hnls are involved in the catabolic degradation of cyanogenic glycosides, releasing HCN which serves as defense agent against herbivores and microbial attack, or as a nitrogen source. Hydroxynitrile lyases from various plant families appear to represent a new example of enzymes that originated from the convergent evolution of different precursor proteins. The enzymes have been classified into non‐FAD‐ and FAD‐containing proteins. FAD‐containing enzymes have been isolated exclusively from the Rosaceae, whereas the FAD‐independent Hnls, which are more heterogenous in structure, have been characterized from various plant families (Poaceae, Euphorbiaceae, Linaceae, Olacaceae. Filitaceae). The aim of this review is to present a general survey of the natural function and localization of this class of enzymes and a comprehensive summary of the biochemical and genetic data of the isolated proteins.
Life in a changing world: TCH gene regulation of expression and responses to environmental signals
Authors: Braam Janet , Sistrunk Melissa L. , Polisensky Diana H. , Xu Wei , Purugganan Mary M. , Antosiewicz Danuta M. , Campbell Paul , Johnson Keith A.
Published: May 2006

The Arabidopsis TCH genes were discovered as a consequence of their marked upregulation of expression in response to seemingly innocuous stimuli, such as touch. Further analyses have indicated that these genes are upregulated by a variety of diverse stimuli. Understanding the mechanism(s) and factors that control TCH gene regulation will shed light on the signalling pathways that enable plants to respond to changing environmental conditions. The TCH proteins include calmodulin, calmodulin‐related proteins and a xyloglucan endotransglycosylase. Expression analyses and localization of protein accumulation indicate that the potential sites of TCH protein function include expanding cells and tissues under mechanical strain. We hypothesize that the TCH proteins may collaborate in cell wall biogenesis.
Light regulation of photosynthetic genes
Authors: Kloppstech Klaus
Published: May 2006

Light influences plant development at various levels of gene expression. Preferentially photosynthetic genes are light controlled. In the nuclear‐cytosolic compartment this regulation occurs primarily at the level of transcription. To date, the light regulation of chloroplast genes has been observed mainly at the level of translation. However, mainly fully mature chloroplasts have been analyzed and the situation might be different in young chloroplasts, as has been found earlier for nuclear transcription. Finally, regulation also occurs by posttranslational modification. The involvement of the circadian clock in the regulation of gene expression is described in detail. During these studies it has been found that the circadian oscillator influences gene expression at various levels, probably resulting in the coordination of processes which might be harmful to the plant. The synthesis of chlorophylls as well as of their corresponding apoproteins are co‐regulated and reduce dangerous radical formation, especially at the higher light intensities. The maxima of chlorophyll and binding apoproteins occur during the first half of the light phase while other, presumably light‐insensitive processes, are shifted to the dark phase. Under light stress, e.g. full sunlight at 5°C, a different set of genes is activated; these include proteins such as catalases, peroxidases, superoxide dismutase or ELIPs, which are upregulated while the well known ‘light‐in‐ducible genes’ are down‐regulated under light stress conditions.
Pigment ligation to proteins of the photosynthetic apparatus in higher plants
Authors: Paulsen Harald
Published: May 2006

Ligation of pigments to proteins of the thylakoid membrane is a central step in the assembly of the photosynthetic apparatus in higher plants. Because of the potentially damaging photooxidative activity of chlorophylls, it is likely that between their biosynthesis and final assembly, chlorophylls will always be bound to protein complexes in which photooxidation is prevented by quenchers such as carotenoids. Such complexes may include chlorophyll carriers and/or membrane receptors involved in protein insertion into the membrane. Many if not all pigment‐protein complexes of the thylakoid are stabilised towards protease attack by bound pigments. The major light‐harvesting chlorophyll a/b protein (Lhebl,2) folds into its native structure in vitro only when it binds pigments. Pigment‐induced folding may also be a general feature of chlorophyll‐carotenoid proteins of the photosynthetic apparatus.
Novel aspects of chlorophyll a/b‐binding proteins
Authors: Bassi Roberto , Sandonà Dorianna , Croce Roberta
Published: May 2006

The light‐harvesting proteins (LHC) constitute a multigene family including, in higher plants, at least 12 members whose location, within the photosynthetic membrane, relative abundance and putative function appear to be very different. The major light‐harvesting complex of photosystem II (LHCII) is the most abundant membrane protein in the biosphere and fulfil a constitutive light‐harvesting function for photosystem II while the early light‐induced proteins (ELIPs) are expressed in low amounts under stress conditions. Primary sequence analysis suggests that all these proteins share a common structure which was resolved at 3.7 Å resolution by electron crystallography in the case of the major LHCII complex: Three transmembrane helices connected by hydrophilic loops coordinate seven chlorophyll a and five chlorophyll b molecules by histidine, glutamine, asparagine lateral chains as well as by charge compensated ionic pairs of glutamic acid and arginine residues; moreover, at least two xantophyll molecules are located at the centre of the structure in close contact with seven porphyrins, tentatively identified as chlorophyll a. The antenna system is also involved in the regulation of excitation energy transfer to reaction centre II. This function has been attributed to three members of the protein family, namely CP29, CP26 and CP24 (also called minor chlorophyll proteins) which have been recently characterised and shown to bind most of the xantophyll cycle carotenoids, thus suggesting that the non‐photochemical quenching mechanism is acting in these proteins. Further support to this assignment comes from the recent identification of protonation sites in CP29 and CP26 by covalent dicyclohexhylcarbodiimide binding suggesting that these respond to low lumenal pH. In addition, CP29 is reversibly phosphorylated under light and cold stress conditions, undergoing conformational change, supporting the hypothesis that these subunits, present in low amounts in photosystem II, have a major regulatory role in the light‐harvesting function and are thus important in environmental stress resistance.
Proteolytic activities and proteases of plant chloroplasts
Authors: Andersson Bertil , Aro Eva‐Mari
Published: May 2006

A concise overview on the current knowledge of the proteolytic activities in chloroplasts is presented, with an emphasis on the proteolytic events associated with thylakoid membranes. The Dl reaction centre protein of photosystem II undergoes rapid light‐dependent turnover and chlorophylla/b‐binding proteins are effectively degraded upon acclimation of plants to higher irradiances. Insights into the partially characterized proteolytic systems in each case will be presented, but the proteases involved still remain unknown. It can be envisaged, however, that the proteolysis is probably an as highly regulated phenomenon as the various steps during biosynthesis of the photosynthetic multiprotein complexes. From the protease point of view, more progress has recently been made in characterization of processing proteases involved in protein import into chloroplasts and in C‐terminal processing of the Dl protein. Moreover, there are an increasing number of proteases in chloroplasts which have been discovered and identified as bacterial homologues. These include a Clp‐type protease, a homologue of the bacterial protease FtsH and the cyanobacterial PcrA protease, all of which have a specific location in the chloroplast but their definite physiological substrates are still missing. Attempts are made to bring together the recent progress in the identification of proteases and characterisation of proteolytic events in chloroplasts.
ELIPs – Light‐induced stress proteins
Authors: Adamska Iwona
Published: May 2006

Exposure of plants to light intensities higher than those required to saturate photosynthesis leads to a reduction in photosynthetic capacity. This effect is known as photoinhibition. Photoinhibition is followed by destruction of carotenoids, bleaching of chlorophylls and increased lipid peroxidation due to damage by oxygen‐derivatives. The oxygen concentration in chloroplasts in the light is high because of oxygen production by photosystem II (PSII). This can result in the release of reactive intermediates of reduced dioxygen such as superoxide radicals, hydroxyl radicals, hydrogen peroxide or singlet oxygen. In order to maintain their normal function under light stress conditions, chloroplasts have developed multiple repair and protection systems. The induction of specific light stress proteins, the ELIPs (for early light‐induced proteins) can be considered to be part of these protective responses. The accumulation of ELIPs under light stress conditions is correlated with the photoinactivation of PSII, degradation of the Dl‐protein of PSII reaction centre and changes in the level of pigments. Futhermore, the accumulation of ELIPs in the thylakoids is strictly controlled by the pigment content, especially by chlorophylls. Isolation of ELIPs in a native form and analysis of pigments bound to these proteins revealed that ELIPs can bind chlorophyll a and lutein. These data indicate that ELIPs might represent unique chlorophyll‐binding proteins which have a transient function(s) during light stress. A transient ‘pigment‐carrier’ function is postulated for ELIPs.
The xanthophyll cycle, its regulation and components
Authors: Eskling Marie , Arvidsson Per‐Ola , Åkerlund Hans‐Erik
Published: May 2006

During the last few years much interest has been focused on the photoprotective role of zeaxanthin. In excessive light zeaxanthin is rapidly formed in the xanthophyll cycle from violaxanthin, via the intermediate antheraxanthin, a reaction reversed in the dark. The role of zeaxanthin and the xanthophyll cycle in photoprotection, is based on fluorescence quenching measurements, and in many studies a good correlation to the amount of zeaxanthin (and antheraxanthin) has been found. Other suggested roles for the xanthophylls involve, protection against oxidative stress of lipids, participation in the blue light response, modulation of the membrane fluidity and regulation of abscisic acid synthesis. The enzyme violaxanthin de‐epoxidase has recently been purified from spinach and lettuce as a 43‐kDa protein. It was found as 1 molecule per 20–100 electron‐transport chains. The gene has been cloned and sequenced from Lactuca sativa, Nicotiana tabacum and Arabidopsis thaliana. The transit peptide was characteristic of nuclear‐encoded and lumen‐localized proteins. The activity of violaxanthin de‐epoxidase is controlled by the lumen pH. Thus, below pH 6.6 the enzyme binds to the thylakoid membrane. In addition ascorbate becomes protonated to ascorbic acid (pKa= 4.2) the true substrate (Km= 0.1 mM) for the violaxanthin de‐epoxidase. We present arguments for an ascorbate transporter in the thylakoid membrane. The enzyme zeaxanthin epoxidase requires FAD as a cofactor and appears to use ferredoxin rather than NADPH as a reductant. The zeaxanthin epoxidase has not been isolated but the gene has been sequenced and a functional protein of 72.5 kDa has been expressed. The xanthophyll cycle pigments are almost evenly distributed in the thylakoid membrane and at least part of the pigments appears to be free in the lipid matrix where we conclude that the conversion by violaxanthin de‐epoxidase occurs.