Publications (7)19.08 Total impact
- [Show abstract] [Hide abstract] ABSTRACT: Substantial concentrations of NH4+ are found in the apoplast of the leaves of Brassica napus. Physiological studies on isolated mesophyll protoplasts with 15NH4+ revealed the presence of a high-affinity ammonium transporter that shared physiological similarity to the high-affinity NH4+ transporters in Arabidopsis thaliana (AtAMT1;3). PCR techniques were used to isolate a full-length clone of a B. napus homologue of AMT1 from shoot mRNA which showed 97% similarity to AtAMT1;3. The full-length cDNA when cloned into the yeast expression vector pFL61 was able to complement a yeast mutant unable to grow on media with NH4+ as the sole nitrogen source. Regulatory studies with detached leaves revealed a stimulation of both NH4+ uptake and expression of mRNA when the leaves were supplied with increasing concentrations of NH4+. Withdrawal of NH4+ supply for up to 96 h had little effect on mRNA expression or NH4+ uptake; however, plants grown continuously at high NH4+ levels exhibited decreased mRNA expression. BnAMT1;2 mRNA expression was highest when NH4+ was supplied directly to the leaf and lowest when either glutamine or glutamate was supplied to the leaves, which directly paralleled chloroplastic glutamine synthetase (GS2) activity in the same leaves. These results provide tentative evidence that BnAMT1;2 may be regulated by similar mechanisms to GS2 in leaves.
- [Show abstract] [Hide abstract] ABSTRACT: Translocation of NH4+ was studied in relation to the expression of three glutamine synthetase (GS, EC 188.8.131.52) isogenes and total GS activity in roots and leaves of hydroponically grown oilseed rape (Brassica napus). The concentration of NH4+ in the stem xylem sap of NO3−-fed plants was 0.55–0.70 mM, which was ≈60% higher than that in plants deprived of external nitrogen for 2 days. In NH4+-fed plants, xylem NH4+ concentrations increased linearly both with time of exposure to NH4+ and with increasing external NH4+ concentration. The maximum xylem NH4+ concentration was 8 mM, corresponding to 11% of the nitrogen translocated in the xylem. In the leaf apoplastic solution, the NH4+ concentration increased from 0.03 mM in N-deprived plants to 0.20 mM in N-replete plants. The corresponding values for leaf tissue water were 0.33 and 1.24 mM, respectively. The addition of either NO3− or NH4+ to N-starved plants induced both cytosolic gs isogene expression and GS activity in the roots. In N-replete plants, gs isogene expression and GS activity were repressed, probably due to carbon limitations, thereby protecting the roots against the excessive drainage of photosynthates. Repressed gs isogene expression and GS activity under N-replete conditions caused enhanced NH4+ translocation to the shoots.
- [Show abstract] [Hide abstract] ABSTRACT: Expression and immune-detection studies showed that cytosolic glutamine synthetase (GS1) is the only active GS isozyme in senescing leaves of B. napus L. and that GS1 is a phospho-protein, which is regulated post-translationally by reversible phosphorylation catalysed by protein kinases and microcystin-sensitive serine/threonine protein phosphatases. Dephosphorylated GS1 is much more susceptible to degradation than the phosphorylated form. Phosphorylated GS1 can interact with 14-3-3 proteins. The degree of interaction with 14-3-3-proteins can in vitro be modified by decreasing or increasing the phosphorylation status of GS1 Transgenic oilseed rape plants overexpressing GS1 (2–3 fold higher GS1 activity in senescing leaves compared to wild-type plants) had a more efficient remobilization of N.
- [Show abstract] [Hide abstract] ABSTRACT: Regulation of the cytosolic isozyme of glutamine synthetase (GS(1); EC 184.108.40.206) was studied in leaves of Brassica napus L. Expression and immunodetection studies showed that GS(1) was the only active GS isozyme in senescing leaves. By use of [gamma-(32)P]ATP followed by immunodetection, it was shown that GS(1) is a phospho-protein. GS(1) is regulated post-translationally by reversible phosphorylation catalysed by protein kinases and microcystin-sensitive serine/threonine protein phosphatases. Dephosphorylated GS(1) is much more susceptible to degradation than the phosphorylated form. The phosphorylation status of GS(1) changes during light/dark transitions and depends in vitro on the ATP/AMP ratio. Phosphorylated GS(1) interacts with 14-3-3 proteins as verified by two different methods: a His-tag 14-3-3 protein column affinity method combined with immunodetection, and a far-Western method with overlay of 14-3-3-GFP. The degree of interaction with 14-3-3-proteins could be modified in vitro by decreasing or increasing the phosphorylation status of GS(1). Thus, the results demonstrate that 14-3-3 protein is an activator molecule of cytosolic GS and provide the first evidence of a protein involved in the activation of plant cytosolic GS. The role of post-translational regulation of cytosolic GS and interactions between phosphorylated cytosolic GS and 14-3-3 proteins in senescing leaves is discussed in relation to nitrogen remobilization.
- [Show abstract] [Hide abstract] ABSTRACT: Plants have a compensation point for NH3 which ranges from 0.1 to 20 nmol mol-1, and may be several-fold higher or lower than naturally occurring atmospheric NH3 concentrations. This implies that NH3 fluxes over vegetated surfaces are bi-directional and that ammonia exchange with the atmosphere in many cases contributes significantly to the nitrogen economy of vegetation. Physiological regulation of plant–atmosphere NH3 fluxes is mediated via processes involved in nitrogen uptake, transport and metabolism. A rapid turnover of NH3 + in plant leaves leads to the establishment of a finite NH3 + concentration in the leaf apoplastic solution. This concentration determines, together with that of H+, the size of the NH3 compensation point. Barley and oilseed rape plants with access to NH3 + in the root medium have higher apoplastic NH3 + concentrations than plants absorbing NO3 -. Furthermore, the apoplastic NH3 + concentration increases with the external NH3 + concentration. Inhibition of GS leads to a rapid and substantial increase in apoplastic NH3 + and barley mutants with reduced GS activity have higher apoplastic NH3 + than wild-type plants. Increasing rates of photorespiration do not affect the steady-state NH3 + or H+ concentration in tissue or apoplast of oilseed rape, indicating that the NH3 + produced is assimilated efficiently. Nevertheless, NH3 emission increases due to a temperature-mediated displacement of the chemical equilibrium between gaseous and aqueous NH3 in the apoplast. Sugarbeet plants grown with NO3 - seem to be temporarily C-limited in the light due to a repression of respiration. As a consequence, the activity of chloroplastic GS declines during the day causing a major part of NH3 + liberated in photorespiration to be assimilated during darkness when 2-oxoglutarate is supplied in high rates by respiration.
- [Show abstract] [Hide abstract] ABSTRACT: Ammonium is constantly generated from a variety of processes in plant nitrogen metabolism. Re-fixation of the liberated ammonium is catalysed by the enzyme glutamine synthetase (GS). Cytosolic GS isoforms dominate in the roots, while a chloroplastic GS isoform is the quantitatively most important in green leaves. Despite the central role of NH 4+ as an intermediate in plant nitrogen metabolism very little is known about the processes regulation NH 4+ distribution between cell organelles and plant organs. The extent of NH 4+ translocation from roots to shoots in oil-seed rape plants (Brassica napus L.) depended on the external N supply and the activity of GS isoforms in the root. Addition of either NO 3− or NH 4+ to N-starved induced both cytosolic GS isogene expression and GS activity in the roots. In N-replete plants, root GS isogene expression and activity were repressed causing enhanced NH 4+ translocation to the shoots. Xylem NH 4+ concentrations increased linearly both with time of exposure to NH 4+ and with increasing external NH 4+ concentration. The maximum xylem NH 4+ concentration in NH 4+-fed plants was 8 mM, corresponding to 11% of the nitrogen translocated in the xylem, while in NO 3−-fed plants the xylem NH 4+ concentration was around 0.6 mM, constituting 2% of the nitrogen translocated in the xylem. Regulation of apoplastic NH 4+ concentration in leaves of oil-seed rape was studied using a vacuum infiltration technique allowing controlled manipulations of the apoplastic solution. The apoplastic solution constituted a highly dynamic NH 4+ pool. Ammonium was constantly added to this pool via NH3 efflux from the mesophyll cells. The NH 4+ was retrieved by a system involving a transporter with channel-like properties which was able to respond very rapidly to perturbations in apoplastic NH 4+ concentration, thereby maintaining apoplastic NH 4+ homeostasis More detailed investigations into 15NH 4+ uptake into B. napus leaf protoplasts revealed the presence of an additional high-affinity NH 4+ uptake system, similar in kinetics to the AMT1 transporter observed in Arabidopsis, tomato and rice. Primers based on conserved regions of the AMT1 transporter were used in RT-PCR reactions to identify and subsequently clone a homologue in B. napus (BnAMT1). The Bnamt1 gene was found to be strongly expressed under all N conditions but regulated by N supply.
- [Show abstract] [Hide abstract] ABSTRACT: Short and long term effects of variations in external N supply on the content of soluble N compounds and glutamine synthetase (GS) activity were investigated in leaves of vegetatively growing oilseed rape (Brassica napus L., cv. Global). Only the chloroplastic isoform of GS had a detectable activity. Plants growing continuously for 5 weeks at elevated nitrogen levels had 3-fold higher levels of NH4+ and soluble amides compared with plants growing at low levels of N supply. In contrast, GS activities were similar at the different levels of N supply. Addition of a pulse of nitrogen to plants of medium N status caused a rapid and large increase in NH4+ and soluble amides within the leaves. Pulse addition of nitrogen also triggered a temporary increase in GS activity, thereby maintaining the concentration of NH4+ below 14 μmol NH4+·g−1 leaf fresh weight (corresponding to 21 mM NH4+ ammonium on tissue water basis). This level of NH4+ was equal to the maximum level observed in leaves of plants growing permanently at elevated N supplies in spite of the fact that the latter plants received a higher total amount of nitrogen. It is concluded that tissue concentrations of NH4+ and soluble amides in B. napus may vary considerably with external N supply, although accumulation of exceedingly high NH4+ concentrations can be avoided by stimulation of GS activity.