Coaction of blue/ultraviolet-A light and light absorbed by phytochrome in controlling the appearance of ferredoxin-dependent glutamate synthase in the Scots pine (Pinus sylvestris L.) seedling

The appearance of NADH- and ferredoxin (Fd)-dependent glutamate synthases (GOGATs) was investigated in the major organs (roots, hypocotyl and cotyledonary whorl) of the Scots pine seedling. It was found that cytosolic NADH-GOGAT (EC 1.4.1.14) dropped to a low level during the experimental period (from 4 to 12 d after sowing) and was not significantly affected by light. On the other hand, plastidic Fd-GOGAT (EC 1.4.7.1) increased strongly in response to light. Whereas similar amounts of NADH-GOGAT were found in the different organs, Fd-GOGAT was mainly found in the cotyledons even in the presence of nitrate. Protein chromatography revealed only a single Fd-GOGAT peak. No isoforms were detected. Experiments to investigate regulation of the appearance of Fd-GOGAT in the cotyledonary whorl yielded the following results: (i) In darkness, neither nitrate (15 mM KNO₃) nor ammonium (15 mM NH₄Cl) had an effect on the appearance of Fd-GOGAT. In the light, nitrate stimulated Fd-GOGAT activity by 30% whereas ammonium had no effect. The major controlling factor is light. (ii) The action of long-term white light (100 W · m^–2) could be replaced quantitatively by blue light (B, 10 W · m^–2). Since the action of long-term far-red light was very weak, operation of the High Irradiance Reaction of phytochrome is excluded. On the other hand, light-pulse experiments with dark-grown seedlings showed the involvement of phytochrome. (iii) Red light, operating via phytochrome, could fully replace B, but only up to 10 d after sowing. Thereafter, there was an absolute requirement for B for a further increase in the enzyme level. It appears that the operation of phytochrome was replaced by the operation of cryptochrome (B/UV-A photoreceptor). (iv) However, dichromatic experiments (simultaneous treatment of the seedlings with two light beams to vary the level of the far-red-absorbing form of phytochrome (Pfr) in blue light) showed that B does not affect enzyme appearance if the Pfr level is low. It is concluded that B is required to maintain responsiveness of Fd-GOGAT synthesis to phytochrome (Pfr) beyond 10 d after sowing.Abbreviations and Symbols B blue light - c continuous - D darkness - Fd-GOGAT ferredoxin-dependent glutamate synthase (EC 1.4.7.1) - FR far-red light - HIR high-irradiance reaction of phytochrome - NADH-GOGAT nicotinamide-dinucleotide-dependent glutamate synthase (EC 1.4.1.14) - R red light - RG9 long-wavelength far-red light defined by the properties of the Schott glass filter (_RG9<0.01) - Pfr/Ptot far-red-absorbing form of phytochrome/total phytochrome, wavelength-dependent photoequilibrium of the phytochrome system Research supported by Deutsche Forschungsgemeinschaft (SFB 46 und Schwerpunkt Physiologie der Bäume). We thank E. Fernbach for his help with the dichromatic experiments.     

Salt stress increases ferredoxin-dependent glutamate synthase activity and protein level in the leaves of tomato

Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) catalyzes an essential step in the pathway of glutamate biosynthesis. Exposing detached tomato ( Lycopersicon esculentum ) leaves for 6 h to 12 g l⁻¹ NaCl resulted in a significant two-fold increase in the activity of ferredoxin-dependent glutamate synthase extracted from the leaves. Western blot studies demonstrated that salt treatment also increased the ferredoxin-dependent glutamate synthase content of the leaves. A similar effect of salt on the concentration of this enzyme was found in the leaves of hydroponically-grown tomato plants. The induction of ferredoxin-dependent glutamate synthase under salt stress may provide the glutamate required for the proline synthesis which is a common response to salt stress.     

Response of transgenic poplar overexpressing cytosolic glutamine synthetase to phosphinothricin

Glutamine synthetase (GS) is the main enzyme involved in ammonia assimilation in plants and is the target of phosphinothricin (PPT), an herbicide commonly used for weed control in agriculture. As a result of the inhibition of GS, PPT also blocks photorespiration, resulting in the depletion of leaf amino acid pools leading to the plant death. Hybrid transgenic poplar (Populus tremula x P. alba INRA clone 7171-B4) overexpressing cytosolic GS is characterized by enhanced vegetative growth [Gallardo, F., Fu, J., Cantón, F.R., García-Gutiérrez, A., Cánovas, F.M., Kirby, E.G., 1999. Expression of a conifer glutamine synthetase gene in transgenic poplar. Planta 210, 19-26; Fu, J., Sampalo, R., Gallardo, F., Cánovas, F.M., Kirby, E.G., 2003. Assembly of a cytosolic pine glutamine synthetase holoenzyme in leaves of transgenic poplar leads to enhanced vegetative growth in young plants. Plant Cell Environ. 26, 411-418; Jing, Z.P., Gallardo, F., Pascual, M.B., Sampalo, R., Romero, J., Torres de Navarra, A., Cánovas, F.M., 2004. Improved growth in a field trial of transgenic hybrid poplar overexpressing glutamine synthetase. New Phytol. 164, 137-145], increased photosynthetic and photorespiratory capacities [El-Khatib, R.T., Hamerlynck, E.P., Gallardo, F., Kirby, E.G., 2004. Transgenic poplar characterized by ectopic expression of a pine cytosolic glutamine synthetase gene exhibits enhanced tolerance to water stress. Tree Physiol. 24, 729-736], enhanced tolerance to water stress (El-Khatib et al., 2004), and enhanced nitrogen use efficiency [Man, H.-M., Boriel, R., El-Khatib, R.T., Kirby, E.G., 2005. Characterization of transgenic poplar with ectopic expression of pine cytosolic glutamine synthetase under conditions of varying nitrogen availability. New Phytol. 167, 31-39]. In vitro plantlets of GS transgenic poplar exhibited enhanced resistance to PPT when compared with non-transgenic controls. After 30 days exposure to PPT at an equivalent dose of 275 g ha(-1), growth of GS transgenic poplar plantlets was 5-fold greater than controls. The response of young leaves to PPT treatment depends on physiological state as indicated by GS and Rubisco (LSU) levels. Young leaves from control plants, typically in a low differentiation state, respond to the herbicide showing up-regulation of GS and LSU. In contrast, young leaves from transgenic lines, with higher initial GS and LSU levels compared to control, display up-regulation of NADP(+)-isocitrate dehydrogenase. Differences between control and GS transgenics in their response to PPT are discussed in relation to their differences in photosynthetic and photorespiratory capacities (El-Khatib et al., 2004).