Sobre los estudia ntes eternos

which contained dithionite. Therefore, it is uncertain whether the low levels of measured activity in minus-nitrate grown or dark-grown plants represents a true measure of the activity present, due to the low levels of nitrite reductase protein detectable in these plants, or is an overstatement of the activity, caused by an inherent bias from the design of the controls used.

6.3.6. Environmental control of nitrate assim ilation

Although nitrite reductase is influenced by environmental factors such as nitrate availability, light intensity and the abundance of the end products of nitrate assimilation it is clear that nitrate reductase is the logical point to effect regulation of input of reduced nitrogen as it is the first and normally rate limiting enzyme in the assimilation of nitrate into amino-N (Beevers and Hageman, 1980).

Nitrate reductase activity is high in plants grown on nitrate in the light (Beevers and Hageman, 1969: Gupta and Beevers, 1983). The increases observed in nitrate reductase activity after nitrate treatment is due to de novo synthesis of nitrate reductase molecules (Somers et aL, 1983). Thus, both nitrate reductase and nitrite reductase exhibit the same form of regulation in response to nitrate and light, by controlling the amount of enzyme molecules synthesised within the plant tissues. This co-ordinated regulation clearly conserves the plant resources of amino-N when nitrate availability is low by preventing the futile production of nitrate and nitrite reductase protein molecules.

The reduced levels of leaf nitrate and nitrite reductase activity in plants treated with equimolar amounts of nitrate and either ammonium ions or glutamine may occur through either the control of the de novo synthesis of enzyme molecules or through some form of inactivation of existing enzyme molecules. Since both nitrate and the end products of nitrate assimilation are available to these plants it may be that the observed decrease in the level of nitrate and nitrite reductase activities constitute some form of fine control to balance the plants requirements for further nitrate assimilation and prevent over production of ammonium ions and/or glutamine.

Chapter 7

91

7,1. I n tr o d u c tio n

7.1.1. In vivo location of nitrite reductase

Nitrite reductase is widely accepted to be located within the chloroplasts in higher plant leaf tissue with fractionation studies generally demonstrating the coincidental sedimentation of nitrite reductase with chloroplast marker enzymes and chlorophyll (Ritenour et al., 1967; Dalling et al., 1972a; Miflin, 1974). Physiological studies also support the chloroplastic location of nitrite reductase with intact chloroplasts from a variety of species capable of photoreduction of nitrite to ammonium ions (Paneque et al., 1963) and thence to a-NH^ nitrogen (Anderson and Done, 1978) with reduced ferredoxin as the physiological electron donor.

More recently molecular studies have demonstrated that in vitro translation of polyA^ RNA and immuno-precipitation of products with specific nitrite reductase antiserum reveals the synthesis of a peptide of higher molecular weight in both wheat (Small and Gray, 1984) and pea (Gupta and Beevers, 1985). In pea, this in vitro synthesised peptide can be cleaved, in a two-step process, to a peptide of the same size as that of the native enzyme by a proteinaceous extract from chloroplasts (Gupta and Beevers, 1987), suggesting that nitrite reductase mRNA is translated in the cytoplasm as a larger precursor protein which is transported into the chloroplast where the modified protein is functional in vivo. The existence of a precursor form of the protein has been confirmed by the molecular cloning of spinach nitrite reductase cDNA species (Back et al., 1988). These workers showed that the precursor protein for nitrite reductase, with a molecular weight of about 66,000, has a 32 amino acid extension at the N-terminal end which probably serves as the transit peptide. 7.1.2. Im munolocalisation and electron microscopy

The direct in situ morphological localisation of nitrite reductase may be achieved by immunogold labelling of ultrathin tissue sections viewed under the electron microscope. This technique involves the incubation of ultrathin tissue sections with specific antiserum to allow antigen-antibody binding followed by labelling of the specifically bound primary antibody with either protein A or a secondary anti-IgG antiserum conjugated with gold colloids. Thus, the intracellular localisation of the antigen may be visualised, on the ultrathin sections under the electron microscope, due to the presence of the electron dense gold particles (Roth, 1984).

Electron dense particles are particularly useful for immunolabelling ultrathin sections since alternatives such as radiolabels combined with autoradiography or enzyme markers give a less precise antigen localisation and require more complicated labelling procedures which are not compatible with good preservation of ultrastructural detail. One of the first electron dense immunomarkers was the iron storage protein ferritin (Singer, 1959). However, more recently colloidal gold (Faulk and Taylor, 1971) has been used extensively in immunolocalisation studies because of its high electron density, the availability of many different particle sizes and the simple procedure by which they may be coupled to macromolecules.

Preparation of tissue for electron microscopy requires the chemical fixation, organic solvent dehydration and embedding of tissue in resin. It is widely recognised that these processes adversely affect antigenicity and appropriate conditions have to be devised for each particular antigen.

Tissue is usually fixed with various concentrations of glutaraldehyde (0.5 to 4 per cent) or mixtures of paraformaldehyde (2 to 4 per cent) and glutaraldehyde (0.1 to 2 per cent). Dehydration is performed with either ethanol or acetone before the tissue is embedded in resin.

Development of resins such as L.R. White for immunolabelling studies (Goodchild, 1985; Van den Bosch and Newcomb, 1985; Vaughn, 1987) allows immunochemicals to permeate the supporting resin, due to its hydrophilic nature, and react with tissue antigens, if they have been preserved in the tissue. Resins such as L.R. White must be thermally cured during the polymerisation step (Van den Bosch and Newcomb, 1986) which may also affect antigenicity. However, low temperature embedding procedures with specialised resins such as Lowicryl^ K4M photopolymerised by ultraviolet light (C3oodchild et aL, 1985; Kamachi et aL, 1987) allows the embedding of tissue containing thermally susceptible antigens.

Therefore, the technique of immunogold labelling may allow the in situ localisation of proteins that up to now may only have been inferred from biochemical, physiological and molecular studies. However, the procedure demands a "trial and error" approach in order to determine the optimum conditions for fixation and labelling of tissue most suited to the preservation of antigenic sites.

93

7,1,3. M u ta n t a n a ly s is

The technique may also be of use in the characterisation of mutants defective in nitrite reduction. As stated earlier many types of mutation may be deduced a priori from a previous knowledge of the pathway. Clearly some types of mutation affecting nitrite reduction would be characterised by an abnormal distribution of the enzyme within leaf tissue. Mutations may result in defects in chloroplast recognition, processing of the precursor protein, transport into the chloroplast and even localisation within the chloroplast itself. In such cases immunogold labelling may be the only method to provide definitive evidence for the biochemical basis of the mutation responsible for the defect in nitrite reduction.

7J2. R e su lts

7.2.1, Leaf tissue fixation

A range of fixation protocols, 1 to 4 per cent glutaraldehyde and mixtures of glutaraldehyde and paraformaldehyde (Table 7.1) were employed to determine the optimal conditions for barley leaf tissue fixation. Tissue integrity was maintained with a fixative concentration of at least 2.5 per cent glutaraldehyde with the cellular ultrastructure well defined (Fig 7.1) in tissue post-fixed stained with osmium tetroxide.

In vitro nitrite reductase activity in leaf extracts from 7 day old plants to be fixed (treated with 25mM KNOg for 3 days) was almost double that found in leaf extracts from 7 day old plants treated with 25mM KNOg for 24 hours on a fresh weight basis (130|imoles nitrite reduced/gm fresh weight/hour (Table 7.1) compared to 67|0.moles nitrite reduced/gm fresh weight/hour) whereas the specific activity increases on average by only just over 40 per cent (10.5 jxmoles nitrite reduced/mg protein/hour (Table 7.1) compared to 7.9pmoles nitrite reduced/mg protein/hour (section 6.2.2.)). Thus, the increase in nitrite reductase activity is due to induction by nitrate and an overall increase in protein content in the seedlings.

7.2.2. Immunogold localisation

Nitrite reductase antiserum was used at various dilutions (neat to 1:2,000) to determine the optimum dilution of antiserum for immunogold labelling of sections of barley leaf tissue for nitrite reductase (Table 7.2). Dilutions below 1:100 resulted in a high density of label covering the entire section (Table 7.2) while dilutions greater than 1:100 resulted in a reduction in total label unless the sections were incubated for longer periods of time (Table 7.2). Thus, a primary antiserum dilution of 1:100 was chosen as the optimum for immunogold labelling which was finely tuned by adjustments made to the basic procedure involving buffer composition and method for washing grids as well as an increase in the dilution and incubation time for GAR G15 to give the final procedure described in the materials and methods (section 2.5.5.).

Using this procedure sections of barley leaf, from nitrate-grown plants, demonstrate preferential labelling of the chloroplasts after treatment with nitrite reductase antiserum (Fig 7.2). Label is not present on control sections incubated with pre-immune antiserum or when the primary antiserum was omitted (Fig 7.3).