The effect of Glc6P uptake and its subsequent oxidation within pea root plastids on nitrite reduction and glutamate synthesis.

In roots, nitrate assimilation is dependent upon a supply of reductant that is initially generated by oxidative metabolism including the pentose phosphate pathway (OPPP). The uptake of nitrite into the plastids and its subsequent reduction by nitrite reductase (NiR) and glutamate synthase (GOGAT) are potentially important control points that may affect nitrate assimilation. To support the operation of the OPPP there is a need for glucose 6-phosphate (Glc6P) to be imported into the plastids by the glucose phosphate translocator (GPT). Competitive inhibitors of Glc6P uptake had little impact on the rate of Glc6P-dependent nitrite reduction. Nitrite uptake into plastids, using (13)N labelled nitrite, was shown to be by passive diffusion. Flux through the OPPP during nitrite reduction and glutamate synthesis in purified plastids was followed by monitoring the release of (14)CO(2) from [1-(14)C]-Glc6P. The results suggest that the flux through the OPPP is maximal when NiR operates at maximal capacity and could not respond further to the increased demand for reductant caused by the concurrent operation of NiR and GOGAT. Simultaneous nitrite reduction and glutamate synthesis resulted in decreased rates of both enzymatic reactions. The enzyme activity of glucose 6-phosphate dehydrogenase (G6PDH), the enzyme supporting the first step of the OPPP, was induced by external nitrate supply. The maximum catalytic activity of G6PDH was determined to be more than sufficient to support the reductant requirements of both NiR and GOGAT. These data are discussed in terms of competition between NiR and GOGAT for the provision of reductant generated by the OPPP.

Nitrate assimilation in the forage legume Lotus japonicus L.

Nitrate assimilation in the model legume, Lotus japonicus, has been investigated using a variety of approaches. A gene encoding a nitrate-inducible nitrate reductase (NR) has been cloned and appears to be the only NR gene present in the genome. Most of the nitrate reductase activity (NRA) is found in the roots and the plant assimilates the bulk of its nitrogen in that tissue. We calculate that the observed rates of nitrate reduction are compatible with the growth requirement for reduced nitrogen. The NR mRNA, NRA and the nitrate content do not show a strong diurnal rhythm in the roots and assimilation continues during the dark period although export of assimilated N to the shoot is lower during this time. In shoots, the previous low NR activity may be further inactivated during the dark either by a phosphorylation mechanism or due to reduced nitrate flux coincident with a decreased delivery through the transpiration stream. From nitrate-sufficient conditions, the removal of nitrate from the external medium causes a rapid drop in hydraulic conductivity and a decline in nitrate and reduced-N export. Root nitrate content, NR and nitrate transporter (NRT2) mRNA decline over a period of 2 days to barely detectable levels. On resupply, a coordinated increase of NR and NRT2 mRNA, and NRA is seen within hours.

In vivo activation of plasma membrane H(+)-ATPase hydrolytic activity by complex lipid-bound unsaturated fatty acids in Ustilago maydis.

As an adaptation process to the growth retardation provoked by the presence of nonlethal concentrations of ergosterol biosynthesis inhibitors, Ustilago maydis alters the ratio of linoleic to oleic acid bound to plasma membrane complex lipids [Hernández, A., Cooke, D.T., Lewis, M. & Clarkson, D.T. (1997) Microbiology 143, 3165-3174]. This alteration increases plasma membrane H(+)-ATPase hydrolytic activity. Activation of H(+)-ATPase by the linoleic/oleic acid proportion is noncompetitive, nonessential and only involves changes in the maximum velocity of the pump. Optimum pH, affinity to MgATP and constants for the inhibition by vanadate and erythrosin B remain unchanged. This all indicates that activation of plasma membrane H(+)-ATPase by unsaturated fatty acids differs clearly from glucose-induced activation observed in yeast. Also, it is a physiologically relevant event similar to other, as yet uncharacterized, changes in plasma membrane H(+)-ATPase hydrolytic activity observed in plants and fungi, as part of an adaptation process to different stress conditions.

Fungicides and sterol-deficient mutants of Ustilago maydis: plasma membrane physico-chemical characteristics do not explain growth inhibition.

Plasma membrane vesicles from erg11 and erg2 sterol-deficient mutants and from wild-type Ustilago maydis sporidia treated with and without inhibitors of sterol 14α-demethylase or sterol ∆8-∆7 isomerase (triadimenol and fenpropimorph fungicides, respectively) were purified by aqueous two-phase partitioning. Changes in plasma membrane lipid composition were mostly restricted to sterols and complex lipid-bound fatty acids (CLB fatty acids). There was a greater accumulation of abnormal sterols (14α-methyl-or ∆ 8-unsaturated sterols) in plasma membranes from sterol-deficient mutants than from those treated with their fungicide counterparts. However, greater growth inhibition was observed on fungicide-treated wild-type than on mutants. Changes in CLB fatty acids were restricted to alterations in the relative proportion of linoleic acid (18:2) with respect to oleic acid (18:1). The 18:2 to 18:1 ratio found in CLB fatty acids in plasma membranes could be correlated to rates of sporidial growth but not to accumulation of a particular abnormal sterol or to the extent of sterol replacement. Plasma membrane permeability to protons was increased moderately in the mutants only. No changes were observed in plasma membrane fluidity. Plasma membrane H+-ATPase activity was increased up to twofold in those cases with lower growth rate. It was concluded that fungicide-induced growth inhibition in U. maydis was not due to accumulation of abnormal sterols in plasma membranes but probably due to intracellular ATP depletion by the H+-ATPase and that changes in 18:2 to 18:1 ratio in CLB fatty acids were not directly dependent on the plasma membrane physical state or lipid composition but were possibly part of a stress adaptation mechanism.