Andrea Migge

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.

Negative regulation of nitrate reductase gene expression by glutamine or asparagine accumulating in leaves of sulfur-deprived tobacco

Abstract. Tobacco (Nicotiana tabacum L.) plants were subjected to a prolonged period of sulfur-deprivation to characterize molecular and metabolic mechanisms that permit control of primary N-metabolism under these conditions. Prior to the appearance of chlorotic lesions, sulfur-deprived tobacco leaves showed a strong decrease in the sulfate content and changes in foliar enzyme activities, mRNA accumulation and amino-acid pools. The basic amino acids glutamine, asparagine and arginine accumulated in the leaves of sulfur-deprived plants, while the foliar concentrations of aspartate, glutamate, serine or alanine remained fairly unchanged. Maximal extractable nitrate reductase (NR; EC 1.6.6.1) activity decreased strongly in response to sulfur-deprivation. The decrease in maximal extractable NR activity was accompanied by a decline in NR transcripts while the mRNAs of the plastidic glutamine synthetase (EC 6.1.3.2) or the β-subunit of the mitochondrial ATP synthase were much less affected. Nitrate first accumulated in leaves of tobacco during sulfur-deprivation but then declined. An appreciable amount of nitrate was, however, present in severely sulfur-depleted leaves. The repression of NR gene expression is, therefore, not related to the decrease in the leaf nitrate level. However, glutamine- and/or asparagine-mediated repression of NR gene transcription is a possible mechanism of control in situations when glutamine and asparagine accumulate in leaves and provides a feasible explanation for the reduction in NR activity during sulfur-deprivation. The removal of reduced nitrogen from primary metabolism by redirection and storage as arginine, asparagine or glutamine combined with the down-regulation of nitrate reduction via glutamine- and/or asparagine-mediated repression of NR gene transcription may contribute to maintaining a normal N/S balance during sulfur-deprivation and indicate that the co-ordination of N- and S-metabolism is retained under these conditions.

Regulation of nitrate reductase transcript levels by glutamine accumulating in the leaves of a ferredoxin-dependent glutamate synthase-deficient gluS mutant of Arabidopsis thaliana, and by glutamine provided via the roots

Abstract. The regulation by glutamine of the leaf transcript level corresponding to the Arabidopsis thaliana (L.) Heynh. nitrate reductase gene nia2 was examined using a novel approach: we took advantage of the ability of a ferredoxin-dependent glutamate synthase-deficient gluS mutant of A. thaliana to accumulate glutamine in the leaves when illuminated under conditions that favour photorespiration. The accumulation of glutamine in gluS mutant leaves and the concomitant decline in the leaf glutamate pool were not correlated with a reduction in the foliar nia2 transcript level. This result indicates that glutamine may not exert a negative control of the leaf nia2 transcript pool. The pattern of diurnal nia2 mRNA oscillation did not change upon illumination of the gluS mutant in air, although the leaf glutamine level remained high during the diurnal cycle. The amplitude of the diurnal fluctuation in nia2 transcript abundance, therefore, does not seem to depend on the size of the leaf glutamine pool (which normally fluctuates in opposite phase). This result also appears to argue against a role of glutamine as an effective repressor of nia2 transcript accumulation. The application of a solution containing 100 mM glutamine to the roots of A. thaliana resulted in an increase in the leaf glutamine level and in a decrease in the leaf nia2 transcript level. Net CO2 uptake and chlorophyll fluorescence quenching by attached leaves of A. thaliana were determined as a control of the physiological status of the plants and remained unaffected by the glutamine treatment. However, there was a decrease in the foliar nitrate level. The negative effect on the nia2 transcript pool exerted by exogeneous glutamine may, therefore, be explained as a result of the down-regulation of nitrate-uptake permeases in the roots by glutamine.

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.

Photosynthesis and fluorescence quenching, and the mRNA levels of plastidic glutamine synthetase or of mitochondrial serine hydroxymethyltransferase (SHMT) in the leaves of the wild-type and of the SHMT-deficient stm mutant of Arabidopsis thaliana in relation to the rate of photorespiration

Abstract.The regulation by photorespiration of the transcript level corresponding to plastidic glutamine synthetase (GS-2) was investigated in the leaves of Arabidopsis thaliana (L.) Heynh.. Photorespiration was suppressed by growing the plants in an atmosphere containing 300 Pa CO2. Suppression of photorespiration was demonstrated by the ability of the conditionally lethal serine hydroxymethyltransferase (SHMT)-deficient stm mutant of A. thaliana to grow normally under these conditions. In contrast to previous studies with bean or pea that were performed at very high CO2 partial pressure (2–4 kPa; Edwards and Coruzzi, 1989, Plant Cell 1: 241–248; Cock et al., 1991, Plant Mol Biol 17: 761–771), suppression of photorespiration during growth of A. thaliana in an atmosphere with 300 Pa CO2 had no effect on the leaf GS-2 transcript level. In the short term, neither suppression of photorespiration induced by the transfer of air-grown A. thaliana plants into a CO2-enriched atmosphere, nor an increase in the rate of photorespiration achieved by the transfer of high-CO2-grown A. thaliana plants into air resulted in a change in the GS-2 mRNA level. The absence of photorespiratory ammonium release in leaves of the stm mutant had no effect on the GS-2 transcript level. Overall, our data argue against a control by photorespiration of the A. thaliana leaf GS-2 mRNA pool. In contrast, regulation of the leaf SHMT mRNA level may involve a negative feedback effect of at least one metabolite derived from the glycine/serine conversion during photorespiration, as indicated by the overexpression of SHMT transcripts in the leaves of the stm mutant.

Greenhouse-grown conditionally lethal tobacco plants obtained by expression of plastidic glutamine synthetase antisense RNA may contribute to biological safety.

A cDNA corresponding to plastidic glutamine synthetase (GS-2), an enzyme involved in photorespiration, was expressed in antisense orientation under the control of a leaf-specific soybean ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene promotor in transgenic tobacco to yield conditionally lethal plants. Three transgenic tobacco lines with decreased (at most 64%) foliar GS-2 activity were obtained. These plants grew normally when maintained in an atmosphere with a CO(2) partial pressure sufficiently high (300 Pa CO(2)) to suppress photorespiration. However, when photorespiration was initiated by the transfer of the plants to air (35 Pa CO(2)), ammonium accumulated in the leaves. With time, the transgenic plants exhibited severe chlorotic lesions and, eventually, the plants died. A stable atmosphere containing at least 300 Pa CO(2) can be established easily in the greenhouse but is unlikely to occur in a natural environment. Therefore, the transgenic tobacco plants with decreased leaf GS-2 activity may contribute to biological safety for production of desired proteins.

Prolonged exposure of tobacco to a low oxygen atmosphere to suppress photorespiration decreases net photosynthesis and results in changes in plant morphology and chloroplast structure

Air-grown tobacco (Nicotiana tabacum L.) plants were transferred for one week into a low oxygen atmosphere (2 kPa O2, LO) to study both immediate and long-term effects of the suppression of photorespiration on net photosynthetic rate (PN), plant morphology, and chloroplast ultrastructure. The PN and the leaf conductance for CO2 increased upon exposure of attached tobacco leaves to LO. These results may suggest that under LO, external CO2 is used to consume the radiant energy normally utilized in photorespiration by net CO2 assimilation at the expense of an increased rate of transpiration. The increase in the coefficient of nonphotochemical fluorescence quenching indicates that under LO, (surplus) radiant energy is also dissipated as heat. Prolonged LO-treatment of tobacco resulted in a decrease in the PN (measured in air) and in a reduction in the number of starch grains in the chloroplasts. Concomitantly, large lipid globuli appeared in the chloroplasts and the distance between the thylakoids forming the grana decreased. These changes in the ultrastructure of chloroplasts may have contributed to the decline in the PN. The LO-treated plants were considerably smaller than the control plants maintained in air. This appears to have resulted from a reduction in the rate of leaf area expansion at the expense of an increase in the specific mass of the leaves. This long-term response to LO-treatment may allow the plants to conserve water.

Coaction of light and the nitrogen substrate in controlling the expression of the tomato genes encoding nitrite reductase and nitrate reductase

Summary The co-action of light and the nitrogen source in the regulation of the expression of the genes encoding nitrite reductase (NiR; EC 1.7.7.1) or nitrate reductase (NR; EC 1.6.6.1) was investigated in the cotyledons of dark- and white light-grown tomato ( Lycopersicon esculentuni ) seedlings. Light acting at red/far-red or at blue regions of the spectrum stimulated both the NiR transcript and the NiR protein level in dark-grown tomato seedling cotyledons, and resulted in an increase in NiR activity. The light-stimulation of tomato NiR gene expression does not depend on the availability of nitrate. Thus, the mode of co-action between light and nitrate in the regulation of NiR gene expression in tomato is different than in all other plant species examined so far. In the cotyledons of tomato seedlings grown during a 16-h photoperiod in white light, nitrate is required for maximum NiR gene expression. Glutamine seems to repress NiR protein expression in green cotyledons. Taken together, our results suggest that the mode of regulation by light and nitrate of the NiR protein level in white light-grown tomato seedling cotyledons is different than during the illumination of etiolated tomato cotyledons. In etiolated and in green tomato seedling cotyledons, both light and nitrate were required for the induction of NR transcripts, NR protein and N R activity. This result confirms a synergistic coaction of both factors in the regulation of NR gene expression.

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.

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.