Formation of unidentified nitrogen in plants: an implication for a novel nitrogen metabolism

Plants take up inorganic nitrogen and store it unchanged or convert it to organic forms. The nitrogen in such organic compounds is stoichiometrically recoverable by the Kjeldahl method. The sum of inorganic nitrogen and Kjeldahl nitrogen has long been known to equal the total nitrogen in plants. However, in our attempt to study the mechanism of nitrogen dioxide (NO₂) metabolism, we unexpectedly discovered that about one-third of the total nitrogen derived from ¹⁵N-labeled NO₂ taken up by Arabidopsis thaliana (L.) Heynh. plants was converted to neither inorganic nor Kjeldahl nitrogen, but instead to an as yet unknown nitrogen compound(s). We here refer to this nitrogen as unidentified nitrogen (UN). The generality of the formation of UN across species, nitrogen sources and cultivation environments for plants has been shown as follows. Firstly, all of the other 11 plant species studied were found to form the UN in response to fumigation with ¹⁵NO₂. Secondly, tobacco (Nicotiana tabacum L.) plants fed with ¹⁵N-nitrate appeared to form the UN. And lastly, the leaves of naturally fed vegetables, grass and roadside trees were found to possess the UN. In addition, the UN appeared to comprise a substantial proportion of total nitrogen in these plant species. Collectively, all of our present findings imply that there is a novel nitrogen mechanism for the formation of UN in plants. Based on the analyses of the exhaust gas and residue fractions of the Kjeldahl digestion of a plant sample containing the UN, probable candidates for compounds that bear the UN were deduced to be those containing the heat-labile nitrogen–oxygen functions and those recalcitrant to Kjeldahl digestion, including organic nitro and nitroso compounds. We propose UN-bearing compounds may provide a chemical basis for the mechanism of the reactive nitrogen species (RNS), and thus that cross-talk may occur between UN and RNS metabolisms in plants. A mechanism for the formation of UN-bearing compounds, in which RNS are involved as intermediates, is proposed. The important broad impact of this novel nitrogen metabolism, not only on the general physiology of plants, but also on plant substances as human and animal food, and on plants as an integral part of the global environment, is discussed.Abbreviations NO Nitric oxide - NO₂ Nitrogen dioxide - RNS Reactive nitrogen species - UN Unidentified nitrogen - TNNAT, RNNAT, INNAT and UNNAT Total, Kjeldahl, inorganic and unidentified nitrogen in naturally fed plants, respectively - TNNIT, RNNIT, INNIT and UNNIT Total, Kjeldahl, inorganic and unidentified nitrogen derived from nitrate, respectively - TNNO2, RNNO2, INNO2 and UNNO2 Total, Kjeldahl, inorganic and unidentified nitrogen derived from NO₂, respectively     

Alkaline phosphatase activities of anAnabaena sp. from deep-water rice

Anabaena sp. grew with mono- and di-ester phosphate compounds as sources of phosphate, indicating the presence of phosphomonoesterase (PMEase) and phosphodiesterase (PDEase) activities. Cell-bound PMEase and PDEase activities were detected during growth in 0.5 and 10 mg PO₄l^–1 only when the cellular phosphate concentration fell to 0.46% of cell protein and the activities increased as cellular phosphate content decreased. The K_m values for these enzymes were 0.3mm forp-nitrophenyl phosphate and 0.2mm for bis-p-nitrophenyl phosphate, respectively. Only PMEase activity was found extracellularly. The pH optima for PMEase and PDEase were 10.2 and 10.4, respectively, and the temperature optima at pH 10.2 were 37°C and 40°C, respectively. Ca²⁺ increased the enzyme activities while Zn²⁺ caused marked inhibition. The inorganic phosphate repressed the cellular PMEase activity after a lag of 4 h.     

Assimilation of exogenous and dinitrogen-derived13NH 4 + byAnabaena azollae separated fromAzolla caroliniana Willd

Anabaena azollae was isolated fromAzolla caroliniana by the gentle roller method and differential centrifugation. Incubation of suchAnabaena preparations for 10 min with [¹³N]N₂ resulted in the formation of four radioactive compounds; ammonium, glutamine, glutamate and alanine. Ammonium accounted for 66% of the total radioactivity recovered and 58% of the ammonium was in an extracellular fraction. Since essentially no extracellular¹³N-labeled organic compounds were found, it appears that ammonium is the compound most probably made available toAzolla during dinitrogen-dependent growth of the association.The kinetics of incorporation of exogenous¹³NH ₄ ⁺ into glutamine and glutamate were characteristic of a precursor (glutamine)-product (glutamate) relationship and consistent with assimilation by the glutamine synthetase-glutamate synthase pathway. The results of experiments using the glutamine synthetase inhibitor, methionine sulfoximine, the glutamate synthase inhibitor, diazo-oxonorleucine, and increasing the ammonium concentration to greater than 1 mM, provided evidence for assimilation primarily by the glutamine synthetase-glutamate synthase pathway with little or no contribution from biosynthetic glutamate dehydrogenase.While showing that N₂ fixation and NH ₄ ⁺ assimilation were not tightly coupled metabolic processes in symbioticAnabaena, these results reflect a composite picture and do not indicate the extent to which ammonium assimilatory enzymes might be regulated in filaments associated with specific stages in theAzolla-Anabaena developmental profile.Non-standard abbreviations DON 6-Diazo-5-oxo-l-norleucine - GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase - MSX l-methionine-Dl-sulfoximine     

Microalgae as a source of stable isotopically labeled compounds

Microalgae have the ability to convert inorganic compounds into organic compounds. When they are cultured in the presence of stable (non-radioactive) isotopes (i.e.¹³CO₂,¹⁵NO ₃ ^– ,²H₂O) their biomass becomes labeled with the stable isotopes, and a variety of stable isotopically-labeled compounds can be extracted and purified from that biomass.Two applications for stable isotopically-labeled compounds are as cell culture nutrients and as breath test diagnostics. Bacteria that are cultured with labeled nutrients will produce bacterial products that are labeled with stable isotopes. The presence of these isotopes in the bacterial products, along with recent developments in NMR technology, greatly reduces the time and effort required to determine the three-dimensional structure of macromolecules and the interaction of proteins with ligands. As breath test diagnostics, compounds labeled with¹³C are used to measure the metabolism of particular organs and thus diagnose various disease conditions. These tests are based on the principle that a particular compound is metabolized primarily by a single organ, and when that compound is labeled with¹³C, the appearance of¹³CO₂ in exhaled breath provides information about the metabolic activity of the target organ. Tests of this type are simple to perform, non-invasive, and less expensive than many conventional diagnostic procedures.The commercialization of stable isotopically labeled compounds requires that these compounds be produced in a cost-effective manner. Our approach is to identify microalgal overproducers of the desired compounds, maximize the product content of those organisms, and purify the resulting products.     

Microbial transformation of ginsenoside Rb<Subscript>1</Subscript> by <Emphasis Type="Italic">Acremonium strictum</Emphasis>

Preparative-scale fermentation of ginsenoside Rb₁ (1) with Acremonium strictum AS 3.2058 gave three new compounds, 12β-hydroxydammar-3-one-20 (S)-O-β-d-glucopyranoside (7), 12β, 25-dihydroxydammar-(E)-20(22)-ene-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (8), and 12β, 20 (R), 25-trihydroxydammar-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (9), along with five known compounds, ginsenoside Rd (2), gypenoside XVII (3), ginsenoside Rg₃ (4), ginsenoside F₂ (5), and compound K (6). The structural elucidation of these metabolites was based primarily on one- and two-dimensional nuclear magnetic resonance and high-resolution electron spray ionization mass spectra analyses. Among these compounds, 2–6 are also the metabolites of ginsenoside Rb₁ in mammals. This result demonstrated that microbial culture parallels mammalian metabolism; therefore, A. strictum might be a useful tool for generating mammalian metabolites of related analogs of ginsenosides for complete structural identification and for further use in pharmaceutical research in this series of compounds. In addition, the biotransformation kinetics was also investigated.     

Synthesis of p-nitrophenyl sulfated disaccharides with beta-D-(6-sulfo)-GlcNAc units using beta-N-acetylhexosaminidase from Aspergillus oryzae in a transglycosylation reaction

When α-d-GlcNAc-OC₆H₄NO₂ -p and β-d-(6-sulfo)-GlcNAc-OC₆H₄NO₂-p (2) were used as substrates, β-N-acetylhexosaminidase from Aspergillus oryzae transferred the β-d-(6-sulfo)-GlcNAc(unit from 2 to α-d-GlcNAc-OC₆H₄NO₂ -p to afford β-d-(6-sulfo)-GlcNAc-(1→4)-α-d-GlcNAc-OC₆H₄NO₂-p (3) in a yield of 94% based on the amount of donor, 2, added. β-d-(6-sulfo)-GlcNAc-(1→4)-α-d-Glc-OC₆H₄NO₂-p (4) was obtained with α-d-Glc-OC₆H₄NO₂ -p as acceptor in a similar manner. With a reaction mixture of 2 and β-d-GlcNAc-OC₆H₄NO₂-p (1) in a molar ratio of 6:1, the enzyme mediated the transfer of β-d-GlcNAc from 1 to 2, affording disaccharide β-d-GlcNAc-(1→4)-β-(6-sulfo)-d-GlcNAc-OC₆H₄NO₂-p (5) in a yield of 13% based on the amount of 1 added.     

Nutrition of eidamella deflexa V. effect of varying the concentrations of certain inorganic compounds on growth and pigment elaboration

Summary The effect of addition of varying concentrations of certain inorganic compounds in selected media upon growth and pigment elaboration ofEidamella deflexa (Berk.)Benjamin was studied in submerged culture. Preliminary experiments were performed to establish approximate optimum ranges of certain inorganic compounds. Further investigations in a glycine-sucrose basal medium indicated that the following range of concentrations stimulated growth to the greatest extent: 0.2% MgSO₄·7H₂O, 0.2% K₂HPO₄, 0.25 mg% CuSO₄·5H₂O, 0,0% ZnSO₄·7H₂O and 10.0 mg % FeSO₄·7H₂O. The degree of pigment production did not seem to be affected by different concentrations of these compounds.The effects of additional molybdenum and zinc upon growth and pigmentation were studied in the following media: lactose-peptone, lactose-glycine, maltose-peptone and maltose-glycine. Molybdenum increased pigmentation slightly in maltose-glycine and maltose-peptone media, but inhibited the amount of growth. Molybdenum decreased both growth and pigmentation in the two lactose media. Zinc had little affect on either growth or pigment elaboration in the two maltose media. However, in the lactose-glycine medium, zinc decreased both growth and pigmentation while in lactose-peptone media zinc slightly increased growth and pigmentation.     

Growth and H2 production by Synechococcus spp. using organic/inorganic electron donors

Growth with simultaneous photoproduction of H₂ was obtained on various organic and inorganic compounds using axenic cultures of the oxygenic phototrophic bacteria Synechococcus sp. OU 103 and S. cedrorum. Highest H₂ production occurred with resting cells of S. cedrorum on malate (11.8 mmol H₂/vessel), whereas Synechococcus sp. OU 103 preferred sulphide (10.3 mmol H₂/vessel) as electron donor.The authors are with the Microbial Biotechnology Lab. Department of botany, Osmania University, Hyderabad 500 007, India.     

Improved growth conditions for hyphomicrobium sp. B-522 and two additional strains

Growth yields and rates of 3 hyphomicrobia were improved by varying components of or adding compounds to medium 337. Methanol (0.5% v/v), and similarly methylamine·HCl (3.38g/l), were optimal among 22 C-sources tested; increasing the methylamine·HCl concentration to 5.07g/l gave higher Hyphomicrobium B-522 yields but also prolonged lag periods. Ten C-sources (organic acids, alcohols) stimulated growth slightly but significantly, even in subcultures. Sugar compounds were not utilized. Strains B-522 and ZV-580 were stimulated by l-lysine and gluconate, while NQ-521 gr was stimulated by aspartate.N-Sources tested were inorganic (3), organic (3), or complex (3). (NH₄)₂SO₄ (0.5g/l) was optimal for strains ZV-580 and NQ-521 gr, but Hyphomicrobium B-522 grew best with urea-N. With NH ₄ ⁺ , strain B-522 grew as homogeneous suspension, all other N-sources caused clumping and pellicle formation. Inorganic requirements (PO ₄ ^3- , Mg, Ca, Fe, Mn, Mo) of strains B-522 and ZV-580 were optimized. Addition of Ni, Co, or Zn had no effect; metals 44 or Cu, resulted in growth inhibition.Vitamin B₁₂ stimulated Hyphomicrobium B-522; 2.5g/l B₁₂ decreased the doubling time from 9.3–10.8h to 5.4–5.8h. All combined single improvements resulted in a protein increase of 557% (B-522), 141% (NQ-521 gr), or 109% (ZV-580), respectively.     

Clostridium methylpentosum sp. nov.: a ring-shaped intestinal bacterium that ferments only methylpentoses and pentoses

An intestinal bacterium isolated from a human subject utilized only two methylpentoses (L-rhamnose and L-fucose) and two pentoses (L-lyxose and D-arabinose) as fermentable substrates, among many compounds tested. The isolate was obligately anaerobic and had a distinctive morphology, its cells being rods bent in the shape of rings with the ends slightly overlapping. Single ring-shaped cells and left-handed helical chains of cells were present in cultures. The cells were surrounded by large capsules which appeared as thick, fibrous masses when examined by electron microscopy. Capsules were formed by cells growing in media containing any one of the four fermentable substrates. Terminally located, heat-resistant endospores were formed on plates of an enriched agar medium supplemented with L-rhamnose. End products of L-rhamnose or L-fucose fermentation included acetate, propionate, n-propanol, CO₂, and H₂. The isolate represented a new species of Clostridium for which the name Clostridium methylpentosum (type strain R2. ATCC 43829) is proposed. This organism may participate in intestinal digestive processes by metabolizing rhamnose released via the enzymatic depolymerization of dietary pectin.Abbreviations G+C guanine plus cytosine - OD optical density - TEM transmission electron micrograph     

Comparative effects of salt and alkali stresses on growth, osmotic adjustment and ionic balance of an alkali-resistant halophyte Suaeda glauca (Bge.)

Effects of salt and alkali stresses on growth, osmotic adjustment and ionic balance of Suaeda glauca (Bge.), an alkali-resistant succulent halophyte, were compared. The results showed that alkali stress clearly inhibited the growth of S. glauca. Moreover, the concentrations of Na⁺ and K⁺ both increased with increasing salinity under both stresses, suggesting no competitive inhibition between absorptions of Na⁺ and K⁺. The mechanism underlying osmotic adjustment during salt stress was similar to alkali stress in shoots. The shared essential features were that organic acids, betaine and inorganic ions (dominated by Na⁺) mostly accumulated. On the other hand, the mechanisms governing ionic balance under both stresses were different. Under salt stress, S. glauca accumulated organic acids and inorganic anions to maintain the intracellular ionic equilibrium, but the anion contribution of inorganic ions was greater than that of organic acids. However, the concentrations of inorganic anions under alkali stress were significantly lower than those under salt stress of the same intensity, suggesting that alkali stress might inhibit uptake of anions, such as NO₃ ^– and H₂PO₄ ^–. Under alkali stress, organic acids were the dominant factor in maintaining ionic equilibrium. The contribution of organic acids to anions was 74.1%, while that of inorganic anions was only 25.9%. S. glauca enhanced the synthesis of organic acids, dominated by oxalic acid, to compensate for the shortage of inorganic anions.     

Utility of pentose colorimetric assay for the purification of potato lectin, an arabinose-rich glycoprotein

Potato lectin (Solanum tuberosum agglutinin, STA) is an unusual glycoprotein containing approximately 50% carbohydrates by weight. Of the total carbohydrates, 92% is contributed by L-arabinose, which are O-linked to hydroxyproline residues. The ferric chloride-orcinol assay (Bial’s test), which is specific for pentoses has so far been used only for the determination of free pentoses in biological samples. However, this colorimetric assay has not been used for the detection of pentoses in bound form as it occurs in Solanaceae lectins (potato, tomato, and Datura lectins). Utilizing the pentose colorimetric assay for monitoring the presence of potato lectin, a simpler and shorter procedure for the purification of this lectin from potato tubers has been developed. The yield of potato lectin (1.73 mg per 100 g potato tuber) is twice compared to the yields reported in earlier procedures. Although potato lectin is well known for its specificity to free trimers and tetramers of N-acetyl-D-glucosamine (GlcNAc), it possesses a similar specificity to the core (GlcNAc)₂ of N-linked glycoproteins. The utilization of the pentose assay in the purification of arabinose-rich lectins/agglutinins obviates the necessity for the use of agglutination assay in the various purification steps. The pentose assay appears to be a simple and convenient colorimetric assay for detecting any pentose-rich glycoprotein in plant extracts. The utility of the pentose assay appears to have a significant potential in the detection of hydroxyproline-rich glycoproteins (HRGPs), which are generally O-arabinosylated.     

Detoxification of SO2 in conifers differing in SO2-tolerance

Contents of organic sulfur, sulfate and the inorganic cations K⁺, Ca²⁺, Mg²⁺, Mn²⁺ and Na⁺ were compared in needles of three conifer species differing in tolerance to chronic SO₂ immissions. Sulfate and organic sulfur compounds were also measured in bark and wood. Field material was collected from Norway Spruce (Picea abies (L.) Karst.), Colorado Spruce (Picea pungens Engelm.) and Scots Pine (Pinus sylvestris L.) at sites where the SO₂ concentration in air was high, and at another site where it was low. In general, sulfate contents were higher but cation contents lower at the sites where SO₂ concentrations were high than where they were low. Up to 114mmol · (kg DW)^–1 sulfate was measured in fouryear-old needles of Norway Spruce from the Erzgebirge (annual mean of SO₂ in air 32 nl · 1^–1). Sulfate accumulation in this SO₂-sensitive conifer increased with SO₂ concentration in ambient air and with needle age, indicating that the main part of the sulfate resulted from the oxidative detoxification of SO₂. Loss of inorganic cations from ageing needles was reduced, or cation levels even increased, with increasing needle age, while sulfate accumulated. Apparently, cations served as counter-ions for sulfate, which is sequestered in the vacuoles. Individual trees differed in regard to the nature of cations which accumulated with sulfate. Calcium, potassium and magnesium were the dominating cations. Sodium levels were very low. Needles of the SO₂-tolerant conifers Colorado Spruce and Scots Pine growing next to Norway Spruce in the Erzgebirge did not accumulate, or accumulated less, sulfate with increasing needle age as compared to needles of Norway Spruce. However, somewhat more sulfate was found in the bark of the SO₂-tolerant species than in the bark of Norway Spruce. Scots Pine contained distinctly more sulfate in the wood than the other conifers. Since accumulation of organic sulfur compounds could not be observed with increasing needle age, or in bark and wood, reduction does not appear to play a major role in the detoxification of SO₂ by the investigated species. Physiological mechanisms permitting Colorado Spruce and Scots Pine to avoid the sulfate accumulation in the needles and the accompanying sequestration of cations that are observed in neighbouring Norway Spruce are discussed on the basis of the obtained data.Abbreviations S_org organic sulfur compounds Died June 10, 1991, aged 29, in a traffic accident. He initiated this work.This work was supported by the Sonderforschungsbereich 251 of the University of Würzburg and by the Projektgruppe Bayern zur Erforschung der Wirkung von Umweltschadstoffen (PBWU). The authors with to thank Prof. Dr. W Kaiser and Prof. Dr. W. Urbach (both Julius-von-Sachs-Institut, University of Würzburg, Germany) for HPLC-analysis and ICP-analysis.     

Effects of organic sulfur compounds on extraction and determination of inorganic sulfate

The common methods for determining inorganic soil sulfate may be affected by the extraction of sulfate from organic sulfur compounds such as ester sulfates. In order to test this, various synthetic organic sulfur compounds (ranging from ester sulfates to sulfonates and C-bonded sulfur) were extracted with deionized water or with two common sulfate extractors (0.5 M NaHCO₃ and 0.02 M NaH₂PO₄). Similar amounts of dissolved sulfate were detected in all extracts of the aromatic ester sulfate hydroxyquinoline sulfate. Sulfate was not released from aliphatic ester sulfates or C-bonded sulfur. Ion chromatography was compared to a turbidimetric method for the determination of sulfate. The latter method, based on BaSO₄-precipitation, appeared to be unsuitable for determining sulfate in organically influenced solutions. Barium precipitated sulfate as well as ester sulfates. Furthermore, the photometry of BaSO₄ was influenced by specific absorption of dissolved organic compounds, leading to a misinterpretation of the sulfate concentration in the solution.     

Regulation of mesophyll photosynthesis in intact wheat leaves by cytoplasmic phosphate concentrations

The effect of D-(+)-mannose, inorganic phosphate (Pi) and mannose-6-phosphate on net mesophyll CO₂ assimilation rate (A) and stomatal conductance (g_s) of wheat (Triticum aestivum L.) leaves was studied. The compounds were supplied through the transpiration stream of detached leaves from plants grown in sand in growth cabinets or glasshouses, with different concentrations of Pi (0.25, 1.0 and 4.0 mM) supplied during growth. In all cases, 10 mM D-(+)mannose caused 40–60% reduction of A within 30 min, though the time courses differed for flag leaves and the sixth leaf on the mainstem of glasshouse- and cabinet-grown plants. D-(+)Mannose had a similar effect on A in leaves having a fourfold range in total phosphate content. Effects of D-(+)mannose in reducing g_s were always slower than on A. When the CO₂ concentration in the leaf chamber was adjusted to maintain intercellular CO₂ concentration (C_i) constant as A declined after mannose supply, g_s still declined indicating that stomatal closure was not caused by changing C_i. Supplying mannose-6-phosphate at 10 and 1 mM and Pi at 5 and 10 mM concentrations caused rapid reductions in g_s and also direct reductions in A. The observed effects of mannose and Pi on assimilation are consistent with the proposed regulatory role of cytoplasmic Pi in determining mesophyll carbon assimilation that has been derived previously using leaf discs, protoplasts and chloroplasts.Abbreviations and symbols A net mesophyll CO₂-assimilation rate - C_a, C_i external (assimilation-chamber) and intercellular CO₂ concentration, respectively - g_s stomatal conductance - Man6P mannose-6-phosphate - Pi orthophosphate     

Long-term effects of SO2 on plants,SO2 metabolism and regulation of intracellular pH

The impact of SO₂ on the ionic balance of plants and its implications for intracellular pH regulation was studied to find explanations for long-term effects of SO₂. When sulphur, taken up as SO₂ by the shoots of plants, is not assimilated in organic compounds, but stored as sulphate, an equivalent amount of H⁺ is produced. These H⁺ ions are not buffered chemically, but removed by metabolic processes.On the basis of knowledge on metabolic buffering mechanisms a conceptual model is proposed for the removal of shoot-generated H⁺ by (i) OH^- ions, produced in the leaves when sulphate and nitrate are assimilated in organic compounds and/or by (ii) OH^- ions produced by decarboxylation of organic anions (a biochemical pH stat mechanism). The form in which nitrogen is supplied largely determines the potential of the plant to neutralize H⁺ in the leaves during SO₂ uptake by the proposed mechanisms.In field experiments with N₂ fixing Vicia faba L. crops, the increase of sulphate in the shoots of SO₂-exposed plants was equivalent in charge to the decrease of organic anion content, calculated as the difference between inorganic cation content (C) and inorganic anion content (A), indicating that H⁺ ions produced in the leaves following SO₂ uptake were partly removed by OH^- from sulphate reduction and partly by decarboxylation of organic anions.The appearance of chronic SO₂ injury (leaf damage) in the field experiment at the end of the growing period is discussed in relation to the impact of SO₂ on the processes involved in regulation of intracellular pH. It is proposed that the metabolic buffering capacity of leaf cells is related to the rates of sulphate and nitrate reduction and the import rate of organic anions, rather than to the organic anion content in the vacuoles of the leaf cells.     

Land plants of amphibious Littorella uniflora (L.) Aschers. maintain utilization of CO2 from the sediment

Summary Submerged macrophytes of the isoetid life form derive the majority of their CO₂ for photosynthesis from the sediment. The experiments described here were designed to test the hypothesis that root uptake of CO₂ is important also in the terrestrial form of Littorella uniflora. The results of ¹⁴CO₂ experiments showed that sediment CO₂ contributed 56% of the total fixation at 0.1mm CO₂ in the rhizosphere, 83% at 0.5mm and 96% at 2.5mm. Sediment CO₂ in emergent Littorella stands ranged from 0.1 to 1.0mm and averaged 0.5mm. Measurements of the net CO₂ exchange over the leaves showed an even higher dependence of the sediment as CO₂ source. Littorella leaves had no stomata at the base and densities (ca. 100 mm^–2) typical of terrestrial plants at the tip, allowing sediment-derived CO₂ to be supplied along the length of the leaf. The stomata permit supply of CO₂ from the air during periods of reduced sediment CO₂ concentrations (e.g. if the sediment dries up) and regulate transpiration.     

Synthesis ofN-Acetylglucosamine derivatives as probes for specificity of chicken hepatic lectin

Syntheses of the following compounds are described: 6-(Trifluoroacetylamino)hexyl 2-acetamido-2,6-dideoxy--d-glucopyranoside and 2-acetamido-2-deoxy--d-xylopyranoside, two allyl 2-acetamido-2-deoxy--d-glucopyranosiduronic acid derivatives, and several allyl 2-acylamido-2-deoxy--d-glucopyranosides having different acyl groups. These and other compounds were used as inhibitors in the binding assay for the chicken hepatic lectin specific forN-acetylglucosamine. We found that: 1) The inhibitory potency ofN-acylglucosamine derivatives decreased progressively with increase in the size of acyl group, 2) absence of either 3-or 4-OH group ofN-acetylglucosamine lowered the binding affinity more than 100-fold, and 3) the presence of a negatively charged group (carboxylic acid) at the C-6 position did not lower the affinity. The first two items are similar to the mammalian hepatic galactose/N-acetylgalactosamine lectins, but the last item is in a strong contrast to the mammalian lectins.Abbreviations XyLNAc N-acetyl-d-xylosamine - BSA bovine serum albumin - NeuAc N-acetylneuraminic acid - GlcNAc34-BSA amidino-type neoglycoprotein [6] containing on the average 34N-acetylglucosaminyl residues per BSA molecule     

Isolation and characterization of free glycans of the oligomannoside type from the extracellular medium of a plant cell suspension

The oligosaccharides Man₅GlcNAc and Man₃(Xyl)GlcNAc(Fuc)GlcNAc presumed to originate fromN-glycosyl proteins have been purified from an extracellular medium (concentration: 2–5 mg/l of 14 day cultures) of white campion (Silene alba) suspension culture. Their primary structures have been determined by¹H-400-MHz NMR spectroscopy and FAB-MS spectrometry. They are probably the result of an autophagic process including protein catabolism due to sucrose starvation. Additional identification of digalactosylglycerol (galactolipid breakdown) argues for this hypothesis.Abbreviations Fuc l-fucose - Man d-mannose - Xyl d-xylose - GlcNAc N-acetyl-d-glucosamine - Gal d-galactose - Glc d-glucose - FAB-MS fast atom bombardment mass spectrometry - NMR nuclear magnetic resonance     

Solubilisation of tomato fruit pectins by ascorbate: a possible non-enzymic mechanism of fruit softening

The aim of this work was to test the hypothesis that endogenous ascorbate, released into the apoplast by membrane permeabilisation early in fruit ripening, could promote the solubilisation and depolymerisation of polysaccharides, and thus contribute to fruit softening. In vitro, ascorbate (1 mM), especially in the presence of traces of either Cu²⁺ or H₂O₂, solubilised up to 40% of the total pectin from the alcohol-insoluble residue of mature-green tomato (Lycopersicon esculentum Mill.) fruit. Solubilisation was due to the action of ascorbate-generated hydroxyl radicals (^·OH), which can cause non-enzymic scission of polysaccharides. The pectins solubilised by ascorbate in vitro were polydisperse (4–1,000 kDa), partially esterified and galactose-rich. Excised pieces of living tomato fruit released ascorbate into the medium (apoplast); the ability of different tissues to do this increased in the order pericarp < placenta < locule. In all three tissues, but especially in the locule, the ability to release ascorbate increased during ripening. The Cu content of each tissue also increased during ripening, whereas neither Fe nor Mn showed a similar trend. We suggest that progressively increasing levels of Cu and ascorbate in the fruit apoplast would lead to elevated ^·OH production there and thus to non-enzymic scission of pectins during ripening. Such scission could contribute to the natural softening of the fruit. De-esterified citrus pectin was more susceptible to ascorbate-induced scission in vitro than methylesterified pectin, suggesting a possible new significance for pectin methylesterase activity in fruit ripening. In conclusion, non-enzymic mechanisms of fruit softening should be considered alongside the probable roles of hydrolases, xyloglucan endotransglucosylases and expansins.Abbreviations AIR alcohol-insoluble residue - Ara l-arabinose - DMSO dimethylsulphoxide - endo-PG endo-polygalacturonase - Gal d-galactose - GalA d-galacturonic acid - Glc d-glucose - k_·OH rate constant for reaction with the hydroxyl radical - K_av elution from Sepharose column relative to the void volume (K_av=0.0) and totally included volume (K_av=1.0) - MG mature-green - PME pectin methylesterase - Rha l-rhamnose - RR red-ripe