Elisa Carrayol

Nucleotide sequence of a tobacco cDNA encoding plastidic glutamine synthetase and light inducibility, organ specificity and diurnal rhythmicity in the expression of the corresponding genes of tobacco and tomato

A full-length cDNA encoding glutamine synthetase (GS) was cloned from a λgt10 library of tobacco leaf RNA, and the nucleotide sequence was determined. An open reading frame accounting for a primary translation product consisting of 432 amino acids has been localized on the cDNA. The calculated molecular mass of the encoded protein is 47.2 kDa. The predicted amino acid sequence of this precursor shows higher homology to GS-2 protein sequences from other species than to a leaf GS-1 polypeptide sequence, indicating that the cDNA isolated encodes the chloroplastic isoform (GS-2) of tobacco GS. The presence of C-and N-terminal extensions which are characteristic of GS-2 proteins supports this conclusion. Genomic Southern blot analysis indicated that GS-2 is encoded by a single gene in the diploid genomes of both tomato and Nicotiana sylvestris, while two GS-2 genes are very likely present in the amphidiploid tobacco genome. Western blot analysis indicated that in etiolated and in green tomato cotyledons GS-2 subunits are represented by polypeptides of similar size, while in green tomato leaves an additional GS-2 polypeptide of higher apparent molecular weight is detectable. In contrast, tobacco GS-2 is composed of subunits of identical size in all organs examined. GS-2 transcripts and GS-2 proteins could be detected at high levels in the leaves of both tobacco or tomato. Lower amounts of GS-2 mRNA were detected in stems, corolla, and roots of tomato, but not in non-green organs of tobacco. The GS-2 transcript abundance exhibited a diurnal fluctuation in tomato leaves but not in tobacco leaves. White or red light stimulated the accumulation of GS-2 transcripts and GS-2 protein in etiolated tomato cotyledons. Far-red light cancelled this stimulation. The red light response of the GS-2 gene was reduced in etiolated seedlings of the phytochrome-deficient aurea mutant of tomato. These results indicate a phytochrome-mediated light stimulation of GS-2 gene expression during greening in tomato.

Leaf-specific overexpression of plastidic glutamine synthetase stimulates the growth of transgenic tobacco seedlings

Abstract. The impact of increased plastidic glutamine synthetase (GS-2; EC 6.1.3.2) activity on foliar amino-acid levels and on biomass production was examined in transgenic tobacco. For that, tobacco was transformed via Agrobacterium tumefaciens with a binary vector containing a tobacco GS-2 cDNA downstream of the leaf-specific soybean ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene promotor. Two transgenic tobacco lines with 15- to 18-fold higher foliar GS-2 transcript levels than the wild type were obtained. The GS-2 protein pools and the specific GS-2 activities were, however, only 2- to 2.3-fold higher in the leaves of the transgenic plants than in the leaves of the wild type. This discrepancy may reflect a post-transcriptional control of GS-2 protein accumulation. The increased GS-2 activity was correlated with a decrease in the leaf ammonium pool (3.7-fold) and an increase in the levels of some free amino acids, including glutamate (2.5-fold) and glutamine (2.3-fold). The accumulation of soluble protein per unit fresh weight, however, remained unchanged. This result indicates that a process downstream of the synthesis of the primary organic products of N-assimilation is limiting leaf protein accumulation. Nevertheless, the overexpression of GS-2 stimulated the growth rate of the transgenic tobacco seedlings which, consequently, were larger (20–30% on a fresh-weight basis) than wild-type seedlings grown under identical conditions. This result suggests that GS-2 is the rate-limiting enzyme during biomass production in tobacco seedlings. The requirement for glutamate as the ammonium acceptor in the reaction catalysed by GS-2 may imply that there is co-regulation of GS-2 and ferredoxin dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) gene expression. Increased leaf GS-2 activity had, however, no influence on the foliar Fd-GOGAT protein abundance. This result suggests that in tobacco leaves, more Fd-GOGAT is present than required to meet the demands of primary ammonium assimilation and that there is no strong interdependence between GS-2 and Fd-GOGAT protein expression.

The two nitrogen mobilisation- and senescence-associated GS1 and GDH genes are controlled by C and N metabolites

In tobacco, the two enzymes of nitrogen metabolism, cytosolic glutamine synthetase (GS1; E.C.6.3.1.2) and glutamate dehydrogenase (GDH; E.C.1.4.1.2), are induced during leaf senescence, whereas the chloroplastic glutamine synthetase (GS2; E.C.6.3.1.2) and nitrate reductase (NR; E.C.1.6.1.1) are repressed in the course of ageing. In this report, we showed in discs of fully expanded Nicotiana tabacum L. cv. Xanthi leaves that sucrose (Suc) and amino acids were involved in the regulation of the expression of GS1 and GDH genes. Suc induced the expression of GS1 and repressed that of GDH. Therefore, we concluded that in response to Suc, GS1 behaved as an “early” Senescence Associated Gene (SAG), whereas GDH behaved as a “late” SAG. Moreover, amino acids induced the expression of both genes. Among the amino acids tested as signal molecules, proline (Pro) and glutamate (Glu) were major inducers of GDH and GS1 expression, respectively. Interestingly, an opposite regulation of GS1 and GS2 by Pro and Glu was shown. The contrary effect of Suc on NIA (NR encoding gene) and GDH mRNA accumulation was also emphasized.

Glutamine synthetase and glutamate dehydrogenase isoforms in maize leaves : localization, relative proportion and their role in ammonium assimilation or nitrogen transport

Abstract. Mesophyll cells (MCs) and bundle-sheath cells (BSCs) of leaves of the C4 plant maize (Zea mays L.) were separated by cellulase digestion to determine the relative proportion of the glutamine synthetase (GS; EC 6.3.1.2) or the NADH-glutamate dehydrogenase (GDH; EC 1.4.1.2) isoforms in each cell type. The degree of cross-contamination between our MC and BSC preparations was checked by the analysis of marker proteins in each fraction. Nitrate reductase (EC 1.6.6.1) proteins (110 kDa) were found only in the MC fraction. In contrast, ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) proteins (160 kDa) were almost exclusively present in the BSC fraction. These results are consistent with the known intercellular distribution of nitrate reductase and Fd-GOGAT proteins in maize leaves and show that the cross-contamination between our MC and BSC fractions was very low. Proteins corresponding to cytosolic GS (GS-1) or plastidic GS (GS-2) were found in both the MC and BSC fractions. While equal levels of GS-1 (40 kDa) and GS-2 (44 kDa) polypeptides were present in the BSC fraction, the GS-1 protein level in the MC fraction was 1.8-fold higher than the GS-2 protein pool. Following separation of the GS isoforms by anion-exchange chromatography of MC or BSC soluble protein extracts, the relative GS-1 activity in the MC fraction was found to be higher than the relative GS-2 activity. In the BSC fraction, the relative GS-1 activity was very similar to the relative GS-2 activity. Two isoforms of GDH with apparent molecular weights of 41 kDa and 42 kDa, respectively, were detected in the BSC fraction of maize leaves. Both GDH isoenzymes appear to be absent from the MC fraction. In the BSCs, the level of the 42-kDa GDH isoform was 1.7-fold higher than the level of the 41-kDa GDH isoform. A possible role for GS-1 and GDH co-acting in the synthesis of glutamine for the transport of nitrogen is discussed.

Purification and properties of tobacco ferredoxin-dependent glutamate synthase, and isolation of corresponding cDNA clones : Light-inducibility and organ-specificity of gene transcription and protein expression.

Ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) was purified to electrophoretic homogeneity from leaves of tobacco (Nicotiana tabacum L.). The holoenzyme is a monomeric flavoprotein with a molecular weight of 164 kDa. Polyclonal rabbit antibodies against the purified enzyme were used to isolate a 450-bp Fd-GOGAT cDNA clone (C16) from a tobacco λgt11 expression library. A longer Fd-GOGAT cDNA clone (C35) encoding about 70% of the amino acids of tobacco Fd-GOGAT was isolated from a tobacco λgt10 cDNA library using C16 as the probe. The amino-acid sequence of the protein encoded by the Fd-GOGAT cDNA clone C35 was delineated. It is very likely that Fd-GOGAT is encoded by two genes in the amphidiploid genome of tobacco while only a single Fd-GOGAT gene appears to be present in the diploid genome of Nicotiana sylvestris. Two Fd-GOGAT isoenzymes could be distinguished in extracts of tobacco leaf protein. In contrast, a single Fd-GOGAT protein species was detected in leaves of Nicotiana sylvestris speg. et Comes. In tobacco leaves, the 6-kb Fd-GOGAT mRNA is about 50-fold less abundant than chloroplastic glutamine synthetase (EC 6.3.1.2) mRNA. Both Fd-GOGAT mRNA and Fd-GOGAT protein accumulated during greening of etiolated tobacco leaves, and a concomitant increase in Fd-GOGAT activity was observed. These results indicate that tobacco Fd-GOGAT gene expression is light-inducible. Levels of Fd-GOGAT mRNA in tobacco organs other than leaves were below the detection limit of our Northern-blot analysis. Polypeptides of Fd-GOGAT were present in tobacco leaves and, to a lesser extent, in pistils and anthers, but not in corollas, stems and roots. These results support organ specificity in tobacco Fd-GOGAT gene expression.

Regulation of the subunit composition of tomato plastidic glutamine synthetase by light and the nitrogen source

The co-action of light and the N-source in the regulation of the expression of the single-copy gene encoding plastidic glutamine synthetase (GS-2) and of the multigene family encoding cytosolic glutamine synthetase (GS-1) was investigated in the cotyledons of tomato (Lycopersicon esculentum L.). Light, acting at red/far red or at blue regions of the spectrum increased the abundance of the GS-2 gene product and induced a modification of GS-2 subunits, resulting in the appearance of two GS-2 proteins exhibiting different molecular weights. The magnitude of the light stimulation of GS-2 gene expression was independent of the nitrogen source. However, following red- or far-red-light treatment of etiolated tomato cotyledons, two GS-2 proteins were found when nitrate was the N-source, while only one GS-2 protein was present with ammonium as the sole nitrogen source. Thus, light of specific wavelengths and N-substrates seem to act in concert to regulate GS-2 subunit composition. Tomato GS-1 gene expression was unaffected by light. Ammonium provided externally increased the level of the tomato GS-1 protein. Irrespective of the N-source or the light quality, the GS-1 subunits were represented by polypeptides of similar molecular weight in tomato cotyledons. However, phosphinothricin-induced inhibition of GS activity resulted in the appearance of at least one additional GS-1 polypeptide in etiolated or in green tomato cotyledons. In addition, impairment of GS activity in green tomato cotyledons by phosphinothricin was correlated with an increased level of the GS-1 transcript. Taken together, our data suggest a metabolic control of GS-1 gene expression in green tomato cotyledons.

Ammonium Alleviates Redox State in Solanum Seedlings under Cadmium Stress Conditions

Cadmium effects on growth and oxidative stress were investigated in 21-day-old tomato seedlings (Solanum) grown in hydroponics media containing 5 mM of KNO3 or (NH4)2SO4 and three Cd levels as CdCl2 (0, 5 and 25 μM) for 14 d. Cadmium was more accumulated in nitrate-fed tomato compared to ammonium-fed ones. Dry weight, Chla, Chlb and carotenoides contents were reduced in NO3--fed tomato. But in NH4 +-fed plants the parameters were increased. Cadmium induced an increase in the H2O2 and MDA levels which was more pronounced in nitrate-fed tomato. Antioxidant enzyme activities such as superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase(APX) were induced with Cd. But, the enhancement degree of these enzymes activities were higher in ammonium-fed tomato compared to those grown with nitrate. These data suggested that antioxidative activity developed by tomato leaves is more induced by cadmium when ammonium was added in nutrient solution as nitrogen source. This can be related to the ability of cadmium to induce an accumulation of reactive oxygen species (ROS) less pronounced in presence of ammonium regime. The beneficial effect of NH4+ on Cd toxicity was confirmed by a significant decrease in MDA level and accumulation of photosynthetic pigments.

Regulation of the subunit composition of plastidic glutamine synthetase of the wild-type and of the phytochrome-deficient aurea mutant of tomato by blue/UV-A- or by UV-B-light.

The photomorphogenetic aurea mutant of tomato severely deficient in spectrophotometrically active phytochromes was used to study the light-regulation of the single-copy nuclear gene encoding plastidic glutamine synthetase (GS-2; EC 6.1.3.2). The de-etiolation of dark-grown aurea mutant seedling cotyledons showed an obligatory dependency on blue light. A limited red light-responsiveness of etiolated aurea cotyledons is, however, retained as seen by the stimulation of both the GS-2 transcript and protein level in the cotyledons of aurea seedlings during growth in red light. The subunits of the octameric GS-2 enzyme were represented by polypeptides with similar electrophoretic mobilities (polypeptides a) in etiolated wild-type or aurea mutant cotyledons. GS-2 proteins with similar apparent molecular masses were also seen in the cotyledons of red light-grown aurea mutant seedlings. In contrast, GS-2 polypeptides with different apparent molecular masses (polypeptides a and b) were detected in the cotyledons of wild-type seedlings grown in red light. This difference indicates that the (post-translational) modification of tomato GS-2 subunit composition is mediated by the photoreceptor phytochrome. The illumination of etiolated wild-type or aurea cotyledons with UV-A- or UV-B-light light resulted in an increase in both the GS-2 transcript and protein level. Following illumination of etiolated wild-type seedlings with UV-A-light, the relative proportion of the GS-2 polypeptides a and b was similar than upon irradiation with blue light but different than after exposure to UV-B- or red light. This result suggests the involvement of a blue/ UV-A-light-specific photoreceptor in the regulation of tomato GS-2 subunit composition.

Physiological and biochemical effects of cadmium toxicity in enzymes involved in nitrogen and amino—acid metabolism in tomato plants

Abstract The activities of several enzymes related to amino—acid metabolism were investigated in tomato plants (Lycopersicon esculentum Mill. 63/5F1) in control and treated leaves and roots. Glutamine synthetase and glutamate synthase ferredoxine dependent activities rapidly declined in stress condition. In presence of cadmium, the decline of glutamine synthetase activity was retarded, while the activity of glutamate synthase significantly and more rapid declined. The activity of NADH—dependent glutamate dehydrogenase was markedly increased in presence of cadmium. Western blots analysis from cadmium treated plants, showed a decrease in chloroplastic GS (GS2) protein and an increase in cytosolic GS (GS1) and glutamate dehydrogenase (GDH) proteins. The activities of phosphoenol pyruvate carboxylase and NADP—dependent isocitrate dehydrogenase also increased in both organs of plantes treated. The activity of NAD and NADP—dependent malate dehydrogenase were decreased especially in leaves.

Two nitrite reductase isoforms are present in tomato cotyledons and are regulated differently by UV-A or UV-B light and during plant development

Abstract. The regulation by UV-A or UV-B light of the nuclear gene(s) encoding the plastidic enzyme nitrite reductase (NiR; EC 1.7.7.1) was examined in the cotyledons of tomato (Lycopersicon esculentum L.). Two NiR isoforms designated NiR1 and NiR2 with apparent molecular masses of 63 kDa and 62 kDa, respectively, were detected by immunoblot analysis in total soluble protein extracts derived from tomato seedling cotyledons. Genomic Southern blot analysis indicated the presence of two NiR genes per haploid tomato genome. In etiolated tomato cotyledons, the total NiR protein pool was almost exclusively constituted by NiR1. In contrast, NiR2 was the predominant NiR isoform in the cotyledons of tomato seedlings grown in white light. Illumination of etiolated tomato cotyledons with UV-A or UV-B light resulted in an increase in both the total NiR transcript level and the NiR2 protein abundance. Blue light stimulated the NiR2 protein pool above the level obtained with red light of equal photon fluence rate. These results show that NiR2 protein expression is light-inducible and that the light-stimulation of NiR2 protein accumulation involves the action of both phytochrome and a specific blue-light photoreceptor. The NiR1 protein level remained virtually unaffected by the light treatments. The change in the relative proportion of the NiR isoforms during greening of etiolated tomato cotyledons is, therefore, due to the different light-responsiveness of the genes corresponding to NiR1 or NiR2. The physiological significance of the presence of NiR isoforms that are regulated differently by light in tomato cotyledons is discussed.