Glutamine synthetase from mesophyll and bundle sheath maize cells: isoenzyme complements and different sensitivities to phosphinothricin

 Anion-exchange FPLC has been used to resolve the isoforms of glutamine synthetase (GS, EC 6.3.1.2) from Zea mays mesophyll (MC) and bundle sheath cells (BSC). Two different isoforms were detected in both types of photosynthetic cells. The predominantly active isoform was GS1 (61%) in MC and GS2 (67%) in BSC. The relative contribution of GS1 and GS2 to the overall GS activity in BSC in maize here reported resembles the proportion described for most C3 plants. Differences among these isoforms in terms of their susceptibility to phosphinothricin (PPT), an analogue of glutamate and known inhibitor of GS, were found. The GS1 isoenzyme from MC was the most sensitive form, being inhibited by 50% at approximately 2.0 μM DL-PPT, whereas the GS2 from BSC presented the highest tolerance to the inhibitor (I₅₀=30 μM). The transferase-to-semibiosynthetic activity ratio for the MC isoforms, which was higher than the ratio for the BSC isoforms, and the differences shown by the isoforms in susceptibility to PPT predict important differences in the biochemical properties and regulation of GS isoenzymes. In this regard, the cytoplasmic isoenzymes, and especially the one in MC, due to its relatively high contribution to mesophyll cell GS activity, could play a vital role in nitrogen metabolism in maize. Received: 1 December 1999 / Revised: 7 February 2000 / Accepted: 23 February 2000     

Differential Effects of Nitrate and Light on the Expression of Glutamine Synthetases and Ferredoxin-Dependent Glutamate Synthase in Maize

The effects of nitrate and light on the expression of genesfor glutamine synthetase (GS) isoproteins and ferredoxin-dependentglutamate synthase (Fd-GOGAT) were studied in different organsof maize seedlings by analyzing the levels of the respectivepolypeptides and mRNAs. In roots, the levels of plastidic GSand of a novel, root-specific GS molecule localized in the extraplastidiccompartment were increased markedly by nitrate, whereas Fd-GOGATand cytosolic GS remained at their initial levels. Ammonia wasnot effective in inducing the plastidic GS and Fd-GOGAT butit did induce the novel GS isoprotein. In leaves, cytosolicand plastidic GSs and Fd-GOGAT were present in both mesophyllcells (MC) and bundle sheath cells (BSC). Upon addition of nitrate,the level of plastidic GS increased preferentially in MC, andupon exposure of etiolated seedlings to light, the levels ofplastidic GS and Fd-GOGAT increased in BSC in a coordinatedmanner. The relationship between the expression of genes forGSs and Fd-GOGAT and the physiological role of the GS/GOGATcycle is discussed in terms of the characteristics of nitrogenmetabolism in roots, MC, and BSC. (Received August 11, 1992; Accepted September 21, 1992)     

Inhibition of glutathione synthesis reduces chilling tolerance in maize

 The role of glutathione (GSH) in protecting plants from chilling injury was analyzed in seedlings of a chilling-tolerant maize (Zea mays L.) genotype using buthionine sulfoximine (BSO), a specific inhibitor of γ-glutamylcysteine (γEC) synthetase, the first enzyme of GSH synthesis. At 25 °C, 1 mM BSO significantly increased cysteine and reduced GSH content and GSH reductase (GR: EC 1.6.4.2) activity, but interestingly affected neither fresh weight nor dry weight nor relative injury. Application of BSO up to 1 mM during chilling at 5 °C reduced the fresh and dry weights of shoots and roots and increased relative injury from 10 to almost 40%. Buthionine sulfoximine also induced a decrease in GR activity of 90 and 40% in roots and shoots, respectively. Addition of GSH or γEC together with BSO to the nutrient solution protected the seedlings from the BSO effect by increasing the levels of GSH and GR activity in roots and shoots. During chilling, the level of abscisic acid increased both in controls and BSO-treated seedlings and decreased after chilling in roots and shoots of the controls and in the roots of BSO-treated seedlings, but increased in their shoots. Taken together, our results show that BSO did not reduce chilling tolerance of the maize genotype analyzed by inhibiting abscisic acid accumulation but by establishing a low level of GSH, which also induced a decrease in GR activity. Received: 9 November 1999 / Accepted: 17 February 2000     

Immunocharacterization of Vitis vinifera L. ferredoxin-dependent glutamate synthase,and its spatial and temporal changes during leaf development

The grapevine (Vitis vinifera L.) partial fragment of cDNA clone pGOGAT1 [Loulakakis and Roubelakis-Angelakis (1997) Physiol Plant 101:220-228], encoding the ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1), was overexpressed in Escherichia coli cells. A hybrid between the Fd-GOGAT fragment and maltose-binding protein was purified and used to raise a polyclonal antibody in a rabbit. The prepared antibody appeared to be specific towards Fd-GOGAT; it recognized a protein band of approximately 160 kDa on nitrocellulose blots after SDS-PAGE of total proteins from leaves, internodes, roots and calluses, and precipitated most of the enzyme activity present in grapevine protein extracts. The quantity of Fd-GOGAT protein was substantially higher in leaves than in other grapevine tissues tested, coincident with a similar distribution of the enzyme specific activity. Intracellular localization studies revealed that both the enzyme activity and the 160-kDa immunoreactive protein were associated with the chloroplastic fraction. Furthermore, the accumulation of Fd-GOGAT, glutamine synthetase (GS) and glutamate dehydrogenase (GDH), at the activity and protein levels, was monitored during leaf development of field-grown plants, from the stage of the newly expanding leaf to the senescing old leaf. Both the specific activity and quantity of the 160-kDa polypeptide of Fd-GOGAT were higher in the mature, full sized leaves and substantially lower in young and senescing leaves. GS specific activity and immunoreactive protein followed the same trend as Fd-GOGAT, while GDH showed opposite developmental patterns of accumulation. The biological significance of the presence of Fd-GOGAT in the various grapevine tissues and its physiological role during early development and natural senescence of the leaves are discussed.     

Isoforms of chalcone synthase in Daucus carota L. and their differential expression in organs from the European wild carrot and in ultraviolet-A-irradiated cell cultures

 Two isoforms of chalcone synthase (CHS) were isolated from cDNA libraries derived from UV-A-irradiated anthocyanin-accumulating (DCb) and non-accumulating (DCs) cell cultures of carrot (Daucus carota L.). The clones designated as DcCHS1, which were present only in the DCb library, had a deduced primary sequence of 389 amino acids and an expected molecular mass of 42.7 kDa, and seem to be alleles of those cloned by Ozeki et al. (1993). The second isoform (DcCHS2) was present in both libraries. It had the highest degree of similarity (97.7%) to parsley CHS over all 397 amino acids. The expected molecular mass of the corresponding protein was 43.6 kDa. Results obtained from Southern blot analysis indicated the existence of at least two CHS genes in carrot. A transient enhancement of the DcCHS1 mRNA level after continuous irradiation with UV-A light could only be observed in anthocyanin-accumulating cultures, whereas an increase in DcCHS2 mRNA was seen in both cell lines. The maximum accumulation of CHS mRNA occurred 48 h after the onset of UV-A irradiation. In the European wild carrot the accumulation of DcCHS1 mRNA was restricted to the red central flowers, whereas the DcCHS2 mRNA was detectable in all red and white petals, as well as leaves, but was absent in stems and roots. The expression of DcCHS1 was restricted to anthocyanin-accumulating cells or organs. The heterologous expression of both cDNAs in Escherichia coli resulted in immunostainable bands of different sizes on the Western blot and high levels of catalytic CHS activity. Received: 2 September 1999 / Accepted: 30 November 1999     

Subcellular and immunocytochemical localization of the enzymes involved in ammonia assimilation in mesophyll and bundle-sheath cells of maize leaves

The cellular localization of the enzymes involved in primary nitrogen assimilation was investigated following separation of mesophyll protoplasts and bundle-sheath cells of maize (Zea mays L.) leaves. Determination of the enzymatic activities in the two types of cell revealed that nitrate and nitrite reductase are principally located in the mesophyll cells whereas glutamine synthetase (GS) and ferredoxin-dependent glutamate synthase (Fd-GOGAT) are present in both tissues with a preferential location in the bundle-sheath strands. In order to confirm the results obtained by this conventional biochemical method we have used an in-situ immunofluorescence technique to unambiguously localize GS and Fd-GOGAT at the cellular level. Thin-sectioned maize leaves treated with specific GS and Fd-GOGAT antisera followed by conjugation with fluorescein-isothiocyanate-labelled sheep anti-rabbit immunoglobulins clearly show that GS is equally distributed within the leaf whereas Fd-GOGAT is mostly present in the chloroplasts of the bundle-sheath cells. The cellular localization of nitrate reductase, nitrite reductase, GS-2 and Fd-GOGAT in maize leaf cell types strongly indicates that primary nitrogen assimilation functions in the mesophyll cells while photorespiratory nitrogen recycling is restricted to the bundle-sheath cells.     

Application of two-dimensional electrophoresis for monitoring gastrointestinal digestion of milk

Summary. Two-dimensional electrophoresis (2-DE) was used for tracing in vivo gastrointestinal digestion of milk proteins in a rapid model system with rats. Contents of stomach and small intestine from digestion trials with rats given a single dose of milk powder were recovered after 1 hour. They were then subjected to 2-DE (IEF and SDS-PAGE). 2-DE showed undigested proteins in a MW range 13.0–66.0 kDa in stomach and 13.0–25.0 kDa in the small intestine, thus indicating that milk proteins are slowly digested. This approach may shed light on pattern of protein digestion and mechanism of amino acid and peptide assimilation.     

Nectin-like molecule 1 is a high abundance protein in cerebellar neurons

Summary. Nectins and Nectin-like molecules belong to the Ca-independent immunoglobulin superfamily of cell adhesion molecules and are mandatory for various cellular functions such as morphogenesis, differentiation and proliferation. Among them, Nectin-like molecule 1 (Necl-1) is unique for its exclusive expression in the brain where it is localized at the contact sites among axon terminals and glia cell processes, cooperatively forming synapses. We hereby aimed to unambiguously characterize Necl-1 at the protein level in rat brain. Rat cerebellar neurons were lysed, proteins extracted and run on two-dimensional gel electrophoresis with subsequent in-gel digestion and mass spectrometrical (MS/MS) analysis of protein spots. One spot at pI 5.96 with an observed molecular weight of 26 kDa was identified as Nectin-like molecule 1. MS/MS analyses of three matching peptides warranted unambiguous identification for the first time. Additionally, we verified the result by immunoblotting and detected two bands at about 48 kDa and 60 kDa. The proposed roles of Necl-1 in cerebellar morphogenesis as well as plasticity of synapses challenge further research on its function in more detail and we hereby provide a fair analytical tool for the unequivocal determination of Necl-1, independent of antibody availability and specificity.     

Response of Glutamine Synthetase and Glutamate Synthase Isoforms to Nitrogen Sources in Rice Cell Cultures

As a model system with no photorespiration and no long distancetransport, rice cell cultures (Oryza saliva L. cv Sasanishiki)were used to investigate the effect of nitrogen sources on thelevels of isoforms of glutamine synthetase (GS) and glutamatesynthase (GOGAT). Isoforms of GS and GOGAT were analyzed byimmunoblotting methods and their activities in early growthphase of the cells. Cytosolic type GS (41 kDa subunit) and NADH-GOGATwere the major isoforms in the rice cells grown in normal R-2medium. However, contents of plastid type GS (44 kDa subunit)and Fd-GOGAT increased in response to NO_–³ supply. NADH-GOGATactivity also increased following the supply of NO_–³.In vitro translated products from poly(A)⁺RNA prepared fromthe cells showed that the precursor of plastid type GS (49 kDa)was detected at 48 h after the inoculation. Supply of NH₊⁴ resultedin an increase in NADH-GOGAT activity but had no effect on thelevels of Fd-GOGAT, of polypeptides of the plastid type GS orof the corresponding mRNAs. (Received May 30, 1990; Accepted August 23, 1990)     

Expression of NADH-dependent glutamate synthase protein in the epidermis and exodermis of rice roots in response to the supply of ammonium ions

The mRNA and protein for NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) in root tips of rice (Oryza sativa L. cv. Sasanishiki) plants increases dramatically within 12 h of supplying a␣low concentration (>0.05 mM) of ammonium ions (T.␣Yamaya et al., 1995, Plant Cell Physiol 36: 1197–1204). To identify the specific cells which are responsible for this rapid increase, the cellular localization of NADH-GOGAT protein was investigated immunocytologically with an affinity-purified anti-NADH-GOGAT immunoglobulin G. When root tips (>1 mm) of rice seedlings which had been grown for 26 d in water were immuno-stained, signals for the NADH-GOGAT protein were detected in the central cylinder, in the apical meristem, and in the primordia of the secondary roots. Signals for ferredoxin-dependent GOGAT (Fd-GOGAT; EC 1.4.7.1) protein were also seen in the same three areas. When the roots were supplied with 1 mM ammonium ions for 24 h, there were strong signals for the NADH-GOGAT protein in two cell layers of the root surface, i.e. epidermis and exodermis, in addition to the cells giving signals in the absence of ammonium ions. The supply of ammonium ions was less effective on the profile of signals for Fd-GOGAT. Although the supply of ammonium ions had less effect on the expression of cytosolic glutamine synthetase (GS; EC 6.3.1.2), this enzyme was also found to be located in the epidermis and exodermis, as well as in the central cylinder and cortex. The results indicate that NADH-GOGAT, coupled to the cytosolic GS reaction, is probably important for the assimilation of ammonium ions in the two cell layers of the root surface. Received: 21 June 1997 / Accepted: 11 September 1997     

Evidence for secretion of vacuolar α-mannosidase, class I chitinase, and class I β-1,3-glucanase in suspension cultures of tobacco cells

We have investigated the possibility that vacuolar proteins can be secreted into the medium of cultured cells of Nicotiana tabacum L. Time-course and balance-sheet experiments showed that a large fraction, up to ca. 19%, of vacuolar α-mannosidase (EC 3.2.1.24) and vacuolar class I chitinase (EC 3.2.1.14) in suspension cultures accumulated in the medium within one week after subculturing. This effect was most pronounced in media containing 2,4-dichlorophenoxyacetic acid (2,4-D). Under comparable conditions only a small fraction, 1.8–5.1% of the total protein and ca. 1% of malate dehydrogenase (EC 1.1.1.37), which is localized primarily in the mitochondria and cytoplasm, accumulated in the medium. Pulse-chase experiments showed that newly synthesized vacuolar class I isoforms of chitinase and β-1,3-glucanase (EC 3.2.1.39) were released into the medium. Post-translational processing, but not the release of these proteins, was delayed by the secretion inhibitor brefeldin A. Only forms of the proteins present in the vacuole, i.e. mature chitinase and pro-β-1,3-glucanase and mature β-1,3-glucanase, were chased into the medium of tobacco cell-suspension cultures. Our results provide strong evidence that vacuolar α-mannosidase, chitinase and β-1,3-glucanase can be secreted into the medium. They also suggest that secretion of chitinase and β-1,3-glucanase might be via a novel pathway in which the proteins pass through the vacuolar compartment. Received: 3 September 1997 / Accepted: 30 October 1997     

Affinity for inorganic carbon of Gracilaria tenuistipitata cultured at low and high irradiance

Regulation by irradiance level of the mechanism for dissolved inorganic carbon (DIC) acquisition was examined in the red macroalga Gracilaria tenuistipitata Zhang et Xia. For this purpose, affinity for external DIC, carbonic anhydrase (CA; EC 4.2.1.1) activity and content of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) were determined in thalli grown at 45 and 500 μmol photons m⁻² s⁻¹. Oxygen evolution rates declined by 50% when the medium pH was changed from 8.1 to 8.7, and the pH compensation point attained was ca. 9.2. These characteristics were unaffected by the light treatments. In contrast, photosynthetic conductance for DIC at pH 8.7 was doubled in thalli grown at high irradiance compared with those grown at low irradiance (to 0.74 × 10⁻⁶ from 0.33 × 10⁻⁶ m s⁻¹). Photosynthetic rates at saturating DIC concentration were also higher by 60% in thalli grown at high irradiance. These differences could not be attributed to changes in the use of external DIC, since external CA activity did not vary. Although the irradiance level did not modify the pool size of Rubisco, Rubisco content expressed on a chlorophyll a basis was almost doubled at high irradiance. These results likely indicate that the internal transport of DIC towards the active-site of Rubisco, rather than the external use of DIC, is enhanced in the thalli grown at high irradiance. Received: 7 June 1999 / Accepted: 16 October 1999     

Isolation and characterization of a jacalin-related mannose-binding lectin from salt-stressed rice (Oryza sativa) plants

A novel plant lectin was isolated from salt-stressed rice (Oryzasativa L.) plants and partially characterized. The lectin occurs as a natural mixture of two closely related isoforms consisting of two identical non-covalently linked subunits of 15 kDa. Both isoforms are best inhibited by mannose and exhibit potent mitogenic activity towards T-lymphocytes. Biochemical analyses and sequence comparisons further revealed that the rice lectins belong to the subgroup of mannose-binding jacalin-related lectins. In addition, it could be demonstrated that the lectins described here correspond to the protein products of previously described salt-stress-induced genes. Our results not only identify the rice lectin as a stress protein but also highlight the possible importance of protein-carbohydrate interactions in stress responses in plants. Received: 27 July 1999 / Accepted: 11 November 1999     

Symplastic communication between the central cell and the egg apparatus cells in the embryo sac of Torenia fournieri Lind. before and during fertilization

 Various membrane-impermeable, water-soluble fluorescent tracers with different molecular weights were microinjected into the central cell of the embryo sac of Torenia fournieri Lind. before and during fertilization. Before anthesis, there was high symplastic permeability between the central cell and the egg apparatus cells. In this stage, fluorescent tracers up to 10 kDa could pass from the central cell into the egg apparatus cells, whereas those with larger molecular weight remained in the central cell. As the embryo sac matured, symplastic permeability decreased such that 2 d after anthesis only tracers less than 3 kDa could spread from the central cell into the egg cell. There appeared to be no symplastic permeability between the primary endosperm and zygote after fertilization, since tracers as small as 521 Da could not pass into the zygote in about half of the microinjected embryo sacs. This is the first report of a change in cell-to-cell communication among the cells of the female germ unit before and after fertilization. Received: 16 December 1999 / Accepted: 4 February 2000     

Intraprotoplasmic and wall-localised formation of arabinoxylan-bound diferulates and larger ferulate coupling-products in maize cell-suspension cultures

Maize (Zea mays L.) cell cultures incorporated radioactivity from [¹⁴C]cinnamate into hydroxycinnamoyl-CoA derivatives and then into polysaccharide-bound feruloyl residues. Within 5–20 min, the CoA pool had lost its ¹⁴C by turnover and little or no further incorporation into polysaccharides then occurred. The system was thus effectively a pulse–chase experiment. Kinetics of radiolabelling of diferulates (also known as dehydrodiferulates) varied with culture age. In young (1–3 d) cultures, polysaccharide-bound [¹⁴C]feruloyl- and [¹⁴C]diferuloyl residues were both detectable within 1 min of [¹⁴C]cinnamate feeding. Thus, feruloyl residues were dimerised <1 min after their attachment to polysaccharides. For at least the first 2.3 h after [¹⁴C]cinnamate feeding, polysaccharide-bound [¹⁴C]diferuloyl residues remained almost constant at ≈7% of the total polysaccharide-bound [¹⁴C]ferulate derivatives. Since feruloyl residues are attached to polysaccharides <1 min after the biosynthesis of the latter, and >10 min before secretion, the data show that extensive feruloyl coupling occurred intra-protoplasmically. Exogenous H₂O₂ (1 mM) caused little additional feruloyl coupling; therefore, wall-localised coupling may have been peroxidase-limited. In older (e.g. 4 d) cultures, less intraprotoplasmic coupling occurred: during the first 2.5 h, polysaccharide-bound [¹⁴C]diferuloyl residues were a steady 1.4% of the total polysaccharide-bound [¹⁴C]ferulate derivatives. In contrast to the situation in younger cultures, exogenous H₂O₂ induced a rapid 4- to 6-fold increase in all coupling products, indicating that coupling in the walls was H₂O₂-limited. In both 2- and 4-d-old cultures, polysaccharide-bound ¹⁴C-trimers and larger coupling products exceeded [¹⁴C]diferulates 3- to 4-fold, but followed similar kinetics. Thus, although all known dimers of ferulate can now be individually quantified, it appears to be trimers and larger products that make the major contribution to cross-linking of wall polysaccharides in cultured maize cells. We argue that feruloyl arabinoxylans that are cross-linked before and after secretion are likely to loosen and tighten the cell wall, respectively. The consequences for the control of cell expansion and for the response of cell walls to an oxidative burst are discussed. Received: 19 January 2000 / Accepted: 13 April 2000     

Changes in the levels of enzymes involved in ammonia assimilation during the development of Phaseolus vulgaris seedlings.Effects of exogenous ammonia

Seeds of Phaseolus vulgaris L. cv. White Kidney were germinated and grown either in a nitrogen-free or in an ammonia-supplied medium. The changes in the soluble protein concentration and in the levels of glutamine synthetase (GS, EC 6.3.1.2), NADH–glutamate synthase (NADH-GOGAT, EC 1.4.1.14), ferredoxin-glutamate synthase (Fd-GOGAT, EC 1.4.7.1) and glutamate dehydrogenase (GDH, EC 1.4.1.2), both NADH- and NAD⁺-dependent, were examined in cotyledons and roots during the first 10 days after sowing. Soluble protein declined rapidly in the cotyledons and increased slightly in the roots. GS activity was initially high both in cotyledons and roots but subsequently decreased during seedling growth. Exogenous ammonia hardly affected GS activity. High levels of NADH-GOGAT were present both in cotyledons and roots during the first days of germination. The activity then gradually declined in both organs. In contrast, Fd-GOGAT in cotyledons was initially low and progressively increased with seedling development. In roots, the levels of Fd-GOGAT were higher in young than in old seedlings. Supply of ammonia to the seedlings increased the levels of NADH-GOGAT and Fd-GOGAT both in cotyledons and roots. NADH-GDH (aminating) activity gradually increased during germination. In contrast, the levels of NAD⁺-GDH (deaminating) activity were highest during the first days of germination. Exogenous ammonia did not significantly affect the activities of GDH.     

O-Acetylation of plant cell wall polysaccharides: identification and partial characterization of a rhamnogalacturonan O-acetyl-transferase from potato suspension-cultured cells

 A microsomal preparation from suspension-cultured potato stem cells (Solanum tuberosum L. cv. AZY) was incubated with [¹⁴C]acetyl-CoA resulting in a precipitable radiolabeled product. Analysis of the product revealed that it consisted mostly of acetylated proteins and cell wall polysaccharides, including xyloglucan, homogalacturonan and rhamnogalacturonan I. Thus, acetyl-CoA is a donor-substrate for the O-acetylation of wall polysaccharides. A rhamnogalacturonan acetylesterase was used to develop an assay to measure and characterize rhamnogalacturonan O-acetyl transferase activity in the microsomal preparation. Using this assay, it was shown that the transferase activity was highest during the linear growth phase of the cells, had a pH-optimum at pH 7.0, a temperature optimum at 30 °C, an apparent K _m of 35 μM and an apparent V _max of 0.9 pkat per mg protein. Further analysis of the radiolabeled acetylated product revealed that it had a molecular mass >500 kDa. Received: 3 July 1999; Accepted: 27 September 1999