Glutamine synthetase oligomers and isoforms in sugarbeet (Beta vulgaris L.)

The -amino-N compounds that accumulate in the thickening storage root of sugarbeet (Beta vulgaris L.) were synthesized in the leaves (NO ₃ ^– nutrition) and also in the lateral roots (NH ₄ ⁺ nutrition). Ammonium stimulated glutamine synthetase (GS, EC 6.3.1.2) activity, especially in the lateral roots. With non-denaturing polyacrylamide-gel isoelectric focussing, simultaneously active charge-isomers of GS were separated in both leaves and roots. The leaf isoforms were active in an octameric and also in a tetrameric form. In the root only octameric isoforms were found. The tetramer was more active than the octamer in the leaf blade and vice versa in the leaf stem. Only the tetramer needed -mercaptoethanol for activity stabilization in vitro. A reactivation, however, of an inactive tetramer by the addition of thiol/thioredoxin was not possible. The same isoforms of GS were separated in different organs of sugarbeet but with different patterns of relative activity. The activity pattern depended also on the N-source of the plant. With increasing age of the plant the number of active GS isoforms declined in both leaves and roots although the in-vitro activity remained unchanged (NO ₃ ^– -fed plants) or even increased (NH ₄ ⁺ -fed plants).Abbreviations GS glutamine synthetase (E.C. 6.3.1.2.) - IEF isoelectric focussing - PAGE polyacrylamide gel electrophoresis This work was supported by a grant from Bundesministerium für Forschung und Technologie and by Kleinwanzlebener Saatzucht AG, Einbeck.     

Activity of the tetramer and octamer of glutamine synthetase isoforms during primary leaf ontogeny of sugar beet (Beta vulgaris L.)

In extracts from the primary leaf blade of sugar beet (Beta vulgaris L.) we separated a chloroplastic isoform (GS 2) of glutamine synthetase (GS, EC 6.3.1.2) and one or two (depending on leaf age) cytosolic isoforms (GS 1a and GS 1b). The latter were prominent in the early (GS 1a) and late stages of leaf ontogeny (GS 1a and GS 1b), whereas during leaf maturation GS 2 was the predominantly active GS isoform. The GS 1 isoforms were active exclusively in the octameric state although tetrameric GS 1 protein was detected immunologically. Their activity stayed at a relatively constant level during leaf ontogeny; an increase was observed only in the senescent leaf. The activity of GS 2, however, changed drastically during primary leaf ontogeny and was modulated by changes in the oligomeric state of the active enzyme. In the early and late stages of leaf ontogeny when GS 2 activity was low (lower than that of the GS 1 isoforms), GS 2 was active only in the octameric state. In the maturing leaf, when GS 2 activity had reached its maximum level (much higher than that of the GS 1 isoforms), 80 of total GS 2 activity was due the activity of the tetrameric form of the enzyme and 20 was due to octameric GS 2. Tetrameric GS 2 was a hetero-tetramer and thus not the unspecific dissociation product of homo-octameric GS 2. In addition, GS 2 activity was modulated by an activation/inactivation of the tetrameric GS 2 protein. Due to an activation of the GS 2 tetramer, the activity of tetrameric GS 2 increased during leaf maturation from zero level 23-fold compared with that of GS 1a and 18-fold compared with that of GS 1b. Possible activators of tetrameric GS 2 are thiol-reactive substances. During leaf senescence, GS 2 activity decreased to zero; this decrease was due to an inactivation of the tetrameric GS 2 protein probably caused by oxidation.Abbreviations FLL final lamina length - FPLC fast protein liquid chromatography - GS glutamine synthetase - GHA -glutamyl hydroxamate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase Dr. Roger Wallsgrove's (Rothamsted Experimental Station, Harpenden, UK) generous gift of GS antiserum is greatly appreciated.     

Cell selection and inheritance of imidazolinone resistance in sugarbeet (Beta vulgaris)

 Sugarbeets are sensitive to imidazolinone herbicide residues applied to rotational crops. Two imidazolinone-resistance (IMI-R) sugarbeet traits were developed by somatic cell selection to overcome rotation restrictions for sugarbeets where imidazolinones have been applied. Sir-13 is an IMI-R/SU-S (sulfonylurea-sensitive) variant selected from an imidazolinone-sensitive (IMI-S) sugarbeet clone, REL-1. A second variant, 93R30B, resistant to imidazolinone as well as to sulfonylurea herbicides (IMI-R/SU-R), was selected from a plant homozygous for a previously described sulfonylurea-specific resistance trait, Sur (IMI-S/SU-R). The IMI-R alleles (Sir-13 and 93R30B) were found to be corresponding allelic variants at the same ALS locus and both were tightly associated with the Sur allele. Each resistant allele is dominant to the sensitive wild-type allele; however, incomplete dominance is shown among resistance alleles. Diploid sugarbeet contains a single ALS gene copy, limiting the ability to stack these resistance traits in the same plant by traditional breeding. Received: 1 May 1997 / Accepted: 30 June 1997     

Native β-glucuronidase activity in sugarbeet (Beta vulgaris)

β-Glucuronidase (EC 3.2.1.31) activity, initially thought absent from plants, has been found in a number of plant families. During an analysis of Agrobacterium -mediated transformation of sugarbeet ( Beta vulgaris L.), significant glucuronidase activity was observed in control (non-transformed) tissues when the fluorogenic substrates 4-methylumbelliferyl-β- d -glucuronic acid, resorufin glucuronic acid and 3-carboxyum-belliferyl-β- d -glucuronic acid were used to quantify β-glucuronidase activity under standard protocol conditions. Similarly, the colorigenic substrate p -nitrophenyl-β- d -glucuronide was hydrolyzed by this sugarbeet-derived glucuronidase. Biochemical and immunological data are presented to indicate significant differences between sugarbeet-derived glucuronidase and that from Escherichia coli (EC 3.2.1.31) encoded by gusA . These differences provide means of distinguishing between the two activities in extracts that contain a mixture of both. Use of X-glue, the substrate utilized in histochemical localizations of glucuronidase activity, gave no reaction product (i.e., indigo precipitate) at pH 7.0. However, at pH 3.0, 4.0 and 5.0 formation of the indigo precipitate was evident within 1 h at 37°C in sugarbeet callus and by 4 h in leaves and petioles. The specific activity of sugarbeet glucuronidase was observed to be strongly pH dependent, with an optimum near pH 4.0. The use of various β-glucuronidase assay techniques as applied to transformation of sugarbeet is discussed.     

Production of transgenetic sugarbeet (Beta vulgaris L.) plants resistant to phosphinothricin

A method of Agrobacterium-mediated genetic transformation of sugarbeet (Beta vulgaris L.) with vacuum infiltration has been developed. Aseptic 3-weeks old etiolated seedlings of two diploid O-type sugarbeet lines (KS3 and KS7) have been used for genetic transformation. Transgenic sugarbeet plants carrying the reporter beta-glucuronidase gene have been selected for their resistance to glufosinate ammonium herbicide. Integration of transgenes into sugarbeet genome was confirmed with GUS assay and PCR using primers for bar and gusA genes.     

Sterol composition of normal and habituated sugarbeet callus (Beta vulgaris L.Altissima)

Summary A detailed qualitative and quantitative analysis of the sterol content of normal (auxin and cytokinin requiring) and habituated (auxin and cytokinin independent) sugarbeet callus (Beta vulgaris L.altissima) was made using mass spectrometry with gas chromatography. The total sterol content of the two lines did not differ significantly. Δ⁷-Sterols were the most important class of sterols in the two sugarbeet callus lines, as in allChenopodiaceae. Elevated levels of Δ⁸-sterols were found in the habituated callus. These sterols are considered to be badly integrated in the membrane of eucaryotic cells. A partial blocking of Δ⁸-Δ⁷-isomerase is hypothesized in the habituated cell line.     

Production of transgenic sugarbeet plants (Beta vulgaris L.) using Agrobacterium rhizogenes

Normal phenotype sugarbeet plants transformed with Agrobacterium rhizogenes were produced using direct regeneration from explants without hairy root phase. Kanamycin resistant plants and Ri-roots carrying the genes of neomycin phosphotransferase II and b-glucuronidase have been obtained. Integration of transgenes into sugarbeet genome was confirmed with GUS-assay and PCR using primers for the introduced genes.     

Fertilizer nitrogen budgets of 15N-labelled sugarbeet (Beta vulgaris) tops and Na 15NO3 dressings split-applied to winter wheat (Triticum aestivum) in microplots on a loam soil

The fate of N from sugarbeet (Beta vulgaris L.) tops returned to the soil (50 T ha^-1) in autumn 1986 before sowing winter wheat (Triticum aestivum L.), and from NaNO₃ split-applied in 3 equal dressings (at tillering, stem elongation and flag leaf stages) was studied using isotopically labelled ¹⁵N in open stainless-steel cylinders pressed into the soil.At harvest, the percentage utilization (PU) of N from sugarbeet was very low (6.66%) and negatively influenced by fertilizer N (5.59%), while that of fertilizer N was rather high (69.64%) and unchanged by addition of tops. Residual N in soil represented 25.9% of the amount applied in tops and ranged from 33% for the tillering application to 21% for the flag leaf application. N losses (mainly denitrification) from sugar beet tops amounted to 67% and were very low for mineral fertilizer (less than 5%).     

Production of transgenic sugarbeet (Beta vulgaris L.) plants of O-type using Agrobacterium tumefaciens

A method of Agrobacterium-mediated genetic transformation of sugarbeet (Beta vulgaris L.) by vacuum infiltration has been developed. Transgenic sugarbeet plants of Ukraine breeding were selected for their resistance either to the antibiotic kanamycin or to the herbicide glufosinate ammonium. Integration of transgenes was confirmed by PCR and GUS-assay.     

Relation of light and nitrogen source to growth, nitrate reductase and glutamine synthetase activity of jack pine seedlings

Two-month-old jack pine ( Pinus banksiana Lamb.) seedlings were placed in a greenhouse where both nitrogen source and light level were varied. After 4 months, whole seedling biomass, leaf biomass and relative growth rate were greatest in seedlings grown with NH⁺₄/NO/NO⁻₃-N and full light (FL) and least in seedlings grown with NO ⁻₃-N and low light (LL). NO ⁻₃-seedlings grown under full light and NH⁺₄/NO⁻₃-seedlings grown under low light were approximately equal. This indicates that the extra carbon costs of assimilating only NO⁻₃-N were similar to the reduction of carbon fixation resulting from a 50% decrease in photon flux density. Percentage and total nitrogen content of needles were greater in seedlings grown under low light independent of nitrogen fertilization. Percentage and total nitrogen content of roots were higher under low light and lower when fertilized with NO⁻₃.
Nitrate reductase (NR) activity was higher in roots than in needles, while glutamine synthetase (GS) activity was higher in needles than in roots. Low light resulted in decreased NR activity (mg N)⁻¹ in needles, but not in roots. However, no nitrate was detected in the needles in any treatment. GS activity, on the other hand, was greater under low light in both needles and roots. GS activity in needles is most likely involved with the reassimilation rather than the initial assimilation of ammonium. Some implications of these shifts in enzymatic activity for ecological phenomena in forests are discussed.     

Nodule-specific glutamine synthetase is expressed before the onset of nitrogen fixation in Phaseolus vulgaris L.

Glutamine synthetase expression was studied in developing root-nodules of common bean with regard to the time-course of specific activity, antigen accumulation, polypeptide composition and in vitro translation products. This analysis shows that the nodule-specific GS polypeptide (GS-gamma) is detected prior to the nitrogenase acetylene-reducing activity, and that its accumulation together with that of the GS-alpha and GS-beta polypeptides vary with nodule age. GS-gamma is present in ineffective nodules, although in a lower ratio to GS-beta than in wild-type nodules. Comparisons of in vitro translated and in vivo synthesized GS polypeptides suggest no post-translational modifications. The possible factors and mechanisms involved in the regulation of expression of GS polypeptides are discussed.     

Direct somatic embryogenesis and plant regeneration from mature sugarbeet (Beta vulgaris L.) zygotic cotyledons

An in vitro protocol has been developed for direct somatic embryogenesis of zygotic cotyledons from mature sugarbeet (Beta vulgaris L.) embryos. Explants were sequentially cultured on modified Murashige and Skoog (MS) medium supplemented with different combinations of 2,4-D, NAA, BAP and TIBA. Somatic embryogenesis was induced within 4 weeks of culture on embryogenesis induction medium which contained MS medium supplemented with BAP and TIBA. Proliferation of somatic embryos was observed on embryo proliferation medium, which contained MS medium supplemented with BAP and NAA within 4 weeks of culture. Plants were regenerated on hormone free half; strength MS medium containing a low sucrose concentration. With some sugarbeet lines, high frequencies of plant regeneration in excess of 90percnt; were observed. The incorporation of TIBA in the media was essential for successful regeneration.     

Biochemical and genetic analysis of a Chlamydomonas reinhardtii mutant devoid of chloroplastic glutamine synthetase activity

A spontaneous double mutant of Chlamydomonas reinhardtii, designated ARF3, was resistant to L-methionine-S-sulfoximine (MSX), lacked chloroplastic glutamine synthetase (GS2) activity, and grew very poorly in all media tested. In segregants obtained after genetic crosses, the poor-growth phenotype was always linked to the lack of GS2 and to a diminished rate of consumption of ammonium, even under conditions where photorespiration was minimized. The ammonium permeases in mutant ARF3, however, were not altered. This indicates that, unlike in higher plants, GS2 contributes substantially to the primary assimilation of ammonia in this alga, and that its function cannot be replaced by the cytosolic glutamine synthetase. In genetic crosses, the MSX resistance and the lack of GS2 segregated independently, indicating that resistance was not due to an altered form of GS2. Received: 5 June 1998 / Accepted: 10 September 1998     

甜菜胞质型谷氨酰胺合成酶基因组DNA的克隆

以甜菜叶片为材料,用CTAB法提取基因组DNA.以分段PCR法扩增得到了完整的甜菜胞质型谷氨酰胺合成酶(GS1)基因组DNA.采用RT-PCR法扩增此GS1基因(GS1)的cDNA序列应用于对照.获得了长度为9 606bp的完整的GS1 DNA序列和长度为1 068 bp的GSI cDNA序列.分析GS1基因组DNA序列表明,它包含13个外显子,被12个内含子分隔开.其外显子区与已公布的GS1 mRNA序列的相似性达99.5%.RT-PCR法获得的cDNA序列与已知的GS1 mRNA序列相似性达99.6%.而2次实验中GS1基因组DNA外显子区与GS1 cDNA序列的相似性达99.9%.GenBank登录号为EU370974.     

Osmotic stress enhances the competence of Beta vulgaris vacuoles to respond to inositol 1,4,5-trisphosphate

Isolated vacuoles from Beta vulgaris storage roots respond to the intracellular signalling molecule inositol 1,4,5-trisphosphate (InsP₃). Whole vacuole patch clamp enables measurement of an inward current (cytosol-directed) induced by cytosolic InsP₃ which is fully reversible upon removal of InsP₃. The reversal potentials of the InsP₃-induced whole vacuolar currents indicate a permeability ratio (P,Ca:P,K) of 200:1. Competence of vacuoles to respond to InsP₃ is dependent upon the root tissue undergoing hyperosmotic stress before vacuole isolation. The magnitude of the hyperosmotic stress and the density of InsP₃-induced current per unit membrane area are exponentially related. A standing osmotic gradient across the vacuolar membrane further enhances the InsP₃-induced current, the current being larger when there is net water flux from the cytosol to the vacuolar lumen. InsP₃-induced currents are not affected by the cytosolic free Ca²⁺ concentration. The conductance of InsP₃-induced single channel currents varied greatly between individual outside-out patches, but all showed a non-linear increase in single channel current at physiological potentials. The reversal potentials of these currents indicated a P_Ca:P_K of between 100:1 and 800:1. The significance of these findings is discussed in relation to technical aspects of monitoring InsP₃-induced currents in plant vacuoles and in the context of the physiological roles of InsP₃ and its receptor in cell water relations.     

Influence of nitrogen source and growth status on glutamine synthetase and glutamate synthase activity in Mycobacterium avium

An investigation was made of the activity of glutamine synthetase and glutamate synthase from batch-cultured cells of Mycobacterium avium. The bacteria were grown in medium with ammonium chloride concentrations of 0, 0.1, 0.25, 1, 5, or 25 mumol/mL or with glutamine at 0.1 or 1 mumol/mL. The specific activity of the two enzymes was determined at 0, 22, 45, and 70 h of incubation. Regardless of the ammonia concentration in the medium, glutamate synthase specific activity was two to five times higher in extracts from elongating cells, incubated 22 h, than in those from shortened cells, incubated 45 or 70 h. In contrast, there was no apparent difference in glutamine synthetase specific activity with regard to culture age; however, glutamine synthetase specific activity varied inversely with the concentration of ammonium chloride in the medium. Cells grown in glutamine had high activity of glutamine synthetase.     

Soil carbon fractions in grasslands respond differently to various levels of nitrogen enrichments

Background and Aims

Soil contains many different C fractions which have diverse physical and chemical compositions. Examining these differential soil C fractions in response to N enrichment is helpful for better understanding soil C changes under the predominantly increasing N deposition. In this study, we used a field N addition experiment in a grassland to explore the effects of various N enrichment levels on soil C fractions.

Methods

We conducted a field manipulative experiment which used a Latin square design with six N addition levels of 0, 2, 4, 8, 16 and 32 g N m^?2 year^?1 since 2003 in a semiarid grassland in northern China. Soil samples were collected in August (when plants have the greatest biomass), 2011. We measured C and N concentrations in soil light fraction, microbial biomass, extractable organic matter, heavy fraction, and total soil C and N.

Results

The results showed that total soil C and N, and heavy fraction C and N were not significantly affected by N addition after 9 years of treatments. In contrast, different N enrichment levels changed soil light fraction C and N, ranging from 4.3 to 27.7 % and 3.3–30.0 %, respectively. Moreover, both light fraction C and N had a nonlinear relationship with N addition rates, and the threshold for N-induced change in light fraction C and N was near 16 g N m^?2 year^?1 in this semiarid grassland. Increases of soil light fraction C and N primarily resulted from changes in biotic (N-stimulated aboveground biomass) and abiotic (soil temperature, moisture and pH) factors under N enrichment. Soil microbial biomass exponentially declined with increasing N, but extractable organic C showed a positive linear response to N enrichment rates. Changes in microbial biomass C and extractable organic C were primarily due to the reduced soil pH under N addition.

Conclusions

Our findings suggest that various soil C fractions differentially respond to elevated N, because different sets of biotic and abiotic factors regulate those fractions under N enrichment.     

Photosynthesis on individual leaves of sugar beet (Beta vulgaris) during the ontogeny at variable water regimes

It is well known that the extent of yield reduction depends not only on the severity of water stress but also on the stage of plant development. Assessing photosynthetic response of individual leaves to water deficit during the ontogeny may, therefore, offer a clue to better understand the whole plant behaviour. This research aimed at investigating the influence of early and late water stress on net photosynthesis (P_n), carbon‐isotope discrimination and other related traits on individual leaves during ontogeny. Sugar beet plants were grown in rain‐sheltered soil columns of relevant volume (300 L), subdivided into well‐watered (WW); early (S1) and late (S2) stress. In general, water stress significantly reduced leaf lifespan and P_n. Relieving the stress at about one‐third and two‐thirds of potential leaf life substantially restored P_n at the levels of WW. Stressing a previously WW leaf brought about a comparatively heavier loss than stressing a leaf since the beginning. As for leaves at different phenological times, the early leaves had higher initial photosynthetic peaks but steeper falls during their lives. An insight into the relationships between P_n and substomatal CO₂ concentration (C_i) shows that in mature leaves the photosynthetic restoration following stress relief did not entail a full recovery of the electron transport rate, the parameter most severely affected by the stress. The partial reversibility of the effects of water deficiency, associated to the anticipated leaf senescence and to the natural slow‐down of net assimilation during leaf life, may be seen as a key factor in predicting to what extent the plant can tolerate drought and the damages caused by water stress.