Effects of phosphinotricin treatment on glutamine synthetase isoforms in Scots pine seedlings

The occurrence of GS isoenzymes has been investigated in Scots pine (Pinus sylvestris) seedlings. A transient increase of glutamine synthetase (GS, EC 6.3.1.2) activity was observed in the cotyledon whorl of plants treated with the herbicide phosphinotricin (PPT). The increase in GS activity was accompanied by a parallel accumulation of GS1 protein, which remained at high levels throughout the PPT treatment. Two-dimensional SDS-PAGE western analysis showed that pine extracts contained two GS1 polypeptides which differ in their corresponding isoelectric points. Analysis of crude extracts by ion-exchange chromatography led to the separation of two GS isoforms. The first peak (GS1-a) eluted from the columns at a low ionic strength (0.15-0.18 M KCl), whereas the second one (GS1-b) was detected at 0.5 M KCl. A detailed molecular study of both GS holoenzymes confirmed that their subunits were similar in size (about 41 kDa) but different in charge. All these data clearly demonstrate the presence of two GS1 forms in Scots pine cotyledons. Moreover, a comparison of isolated GS isoproteins with the recombinantly expressed Scots pine cytosolic subunit suggests that GS1-a corresponds to the previously characterized cDNA (pGSP114) whereas GS1-b is a minor GS isoenzyme with increased relative abundance in phosphinotricin treated plants.     

Functional expression of two pine glutamine synthetase genes in bacteria reveals that they encode cytosolic holoenzymes with different molecular and catalytic properties

Two glutamine synthetase isogenes, GS1a and GS1b, isolated from pine have been functionally expressed in E. coli and the characteristics of individual gene products compared. When bacteria were grown at 37 degrees C most pine GS1 protein was found in the insoluble fraction but lowering of the expression temperature increased yield of both GS1 polypeptide and activity in the soluble fraction. High levels of functionally active GS1a (309 + or - 35 nkat mg(-1)) and GS1b (1,166 + or - 65 nkat mg(-1)) enzymes were obtained by decreasing the expression temperature to 10 degrees C. Purification and characterization of recombinant products showed that pine GS1 polypeptides are assembled in octameric GS holoenzymes showing structural and kinetic differences. The results are discussed with regard to the specific localization of GS1a and GS1b in different cell types of pine seedlings. The isoform GS1a may control the assimilation of the high levels of ammonium released in photosynthetic tissues, whereas GS1b enzyme could mitigate oscillations in glutamate availability providing a constant flux of glutamine for nitrogen transport in vascular cells.     

Molecular characterization of a cDNA clone encoding glutamine synthetase from a gymnosperm,Pinus sylvestris

A full-length cDNA clone (pGSP114) encoding glutamine synthetase was isolated from a gt11 library of the gymnosperm Pinus sylvestris. Nucleotide sequence analysis showed that pGSP114 contains an open reading frame encoding a protein of 357 amino acid residues with a calculated molecular mass of 39.5 kDa. The derived amino acid sequence was more homologous to cytosolic (GS1) (78–82%) than to chloroplastic (GS2) (71–75%) glutamine synthetase in angiosperms. The lack of N-terminal presequence and C-terminal extension which define the primary structure of GS2, also supports that the isolated cDNA encodes cytosolic GS. Southern blot analysis of genomic DNA from P. sylvestris and P. pinaster suggests that GS may be encoded by a small gene family in pine. GS mRNA was more abundant in cotyledons and stems than in roots of both Scots and maritime pines. Western blot analysis in P. sylvestris seedlings showed that only one GS polypeptide, similar in size to GS1 in P. pinaster, could be detected in several different tissues. Our results suggest that cytosolic GS is mainly responsible for glutamine biosynthesis in pine seedlings.This paper is dedicated to the memory of Dr. Jesús S. Olavarría.     

Sucrose Represses the Developmentally Controlled Transient Activation of the Plastocyanin Gene in Arabidopsis thaliana Seedlings

The plastocyanin (PC) gene of Arabidopsis thaliana is activated independently of light during early seedling development. In etiolated seedlings, PC mRNA levels increase transiently and a maximum dark level is reached after 2 d of growth in darkness. In etiolated transgenic seedlings carrying a chimeric PC-promoter: luciferase fusion gene, luciferase activity is similarly increased after 2 d of growth. The transient increase in PC mRNA and luciferase activity levels can be repressed by sucrose. Nonmetabolizable sugars and polyethylene glycol do not have a major effect on PC gene expression. Also, light-grown seedlings show a similar transient and sucrose-sensitive increase in PC mRNA levels and luciferase activity, as in dark-grown seedlings, but here expression levels are 15- fold higher. These findings suggest the presence of a sucrose-sensitive, developmentally controlled expression mechanism that operates independently of light.     

Different effects of supplied ammonium on glutamine synthetase activity in mustard (Sinapis alba) and pine (Pinus sylvestris) seedlings

The activity of glutamine synthetase (GS) in mustard ( Sinapis alba L.) and Scots pine ( Pinus sylvestris L.) seedlings was used as an index to evaluate the capacity to cope with excessive ammonium supply. In these 2 species GS activity was differently affected by the application of nitrogen compounds (NH₄⁺ or NO₃⁻). Mustard seedlings older than 5 days showed a considerable increase in GS activity after NH₄⁺ or NO₃⁻ application. This response was independent of the energy flux, but GS activity in general was positively affected by light. Endogenous NH₄⁺ did not accumulate greatly after nitrogen supply. In contrast, seedlings of Scots pine accumulated NH₄⁺ in cotyledons and roots and showed no stimulation of GS activity after the application of ammonium. In addition, root growth was drastically reduced. Thus, the pine seedlings seem to have insufficient capacity to assimilate exogenously supplied ammonium. NO₃⁻, however, did not lead to any harmful effects.     

Two different modes of early development and nitrogen assimilation in gymnosperm seedlings†

Light-independent chloroplast development and expression of genes encoding chloroplast proteins occur in many but not all species of gymnosperms. Early development in maritime pine (Pinus pinaster) seedlings was strongly light-independent, whereas Ginkgo biloba seedlings exhibited a typical angiosperm-like morphogenesis with differentiated patterns in light and dark. In pine, chloroplast polypeptides were undetectable in the seed embryo and accumulated in cotyledons of both light- and dark-grown plants in good correlation with light-independent chlorophyll synthesis. In contrast, chlorophyll and chloroplast proteins were only detected in light-grown ginkgo. Pine cytosolic glutamine synthetase (GS) and ferredoxin glutamate synthase (Fd-GOGAT) were present at low levels in the seeds and accumulated at comparable amounts in light- and dark-grown seedlings. Fd-GOGAT was also barely detectable in the seeds of ginkgo and only accumulated in green plants with mature chloroplasts. In G. biloba seeds and etiolated plants only cytosolic GS was identified, while in light-grown seedlings this molecular form was present at low abundance and choroplastic GS was the predominant isoenzyme. The above results have been confirmed by immunolocalization of GS protein in pine and ginkgo plantlets. In pine, GS was present in the peripheral cytoplasm of mesophyll cells and also in the phloem region of the vascular bundle. Immunocytochemical analysis showed that the labelling of mesophyll and phloem cells was only cytoplasmic. In developing ginkgo, GS antigens were present in the chloroplasts of mesophyll parenchyma cells of leaflets and green cotyledons. In contrast, a weak labelling of GS was observed in the parenchyma and phloem cells of non-green cotyledons enclosed in the seed coat. Taking all this into account, our data indicate the existence of two different modes of GS and GOGAT regulation in gymnosperms in close correlation with the differential response of plants to light. Furthermore, the results suggest that glutamine and glutamate biosynthesis is confined to the chloroplast of mesophyll cells in species with light-dependent chloroplast, development whereas compartmentation would be required in species with light-independent plastid development.     

Expression of ferredoxin-dependent glutamate synthase in dark-grown pine seedlings

Pine seedlings are able to accumulate chlorophylls and develop green plastids in a light-independent manner. In this work, we have characterized ferredoxin-dependent glutamate synthase (EC 1.4.7.1; Fd-GOGAT), a key enzyme in nitrogen interconversion during this process. Fd-GOGAT has been purified about 170-fold from cotyledons of maritime pine (Pinus pinaster). As occurs in angiosperms, the native enzyme is a single polypeptide with an apparent molecular mass of 163–168 kDa that is confined to the chloroplast stroma. Polyclonal antibodies generated against the purified enzyme were used to immunoscreen a gt11 expression library from Scots pine (Pinus sylvestris) seedlings and partial cDNA clones were isolated and characterized. The clone with the longest cDNA insert (pGOP44) contained the codification for the C-terminal (550 amino acids) of the pine Fd-GOGAT polypeptide. Immunological cross-reactivity and comparative amino sequence analysis revealed that Fd-GOGAT is a well conserved protein in higher plants. Western blot analyses showed that protein was expressed in chloroplast-containing pine tissues and this expression pattern was not affected by exogenously supplied nitrogen. Fd-GOGAT mRNA, polypeptide and enzyme activity accumulated in substantial amounts in dark-grown pine seedlings. The presence of a functional Fd-GOGAT may be important to provide the required glutamate for the biosynthesis of nitrogen compounds during chloroplast biogenesis in the dark.     

Role of Light in the Appearance of Glutamine Synthetase in Leaves of Pennisetum glaucum

Leaves of Pennisetum [Pennisetum glaucum (L) HHB 67] seedlings contained two isozymes of glutamine synthetase (GS, EC 6.3.1.2): cytosolic GS1 and chloroplastic GS2. Leaves of seedlings grown in light for seven days contained about twofold higher GS activity than etiolated leaves. In both light and dark grown seedlings, total GS, GS1 and GS2 activity declined with plant age with more pronounced effect in leaves of etiolated seedlings, and GS2 declined at a much faster rate than GS1. Exposure of etiolated seedlings to light markedly enhanced GS1 and GS2 activity. This increase in activity was not affected by cycloheximide, precluding light dependent de novo synthesis of the enzyme. Treatment of etiolated seedlings with photosynthetic inhibitor, dichlorophenyl dimethyl urea (DCMU) inhibited light dependent appearance of GS. Exogenous supply of sucrose to dark grown seedlings greatly increased the GS activity in dark. These results suggest that light-mediated stimulation in activity of GS in Pennisetum leaves is dependent on photosynthetic reaction.