AMMONIA ASSIMILATION AND AMINO ACID BIOSYNTHESIS

CHAPTER 14

AMMONIA ASSIMILATION AND AMINO ACID BIOSYNTHESIS P.J. LEA 14.1 Ammonia assimilation 14.1.1 Introduction

CONH 2

COOH

CH2 CH 2

CH2

CHNH 2 COOH

As can be seen from the two previous chapters, ammonia is synthesised either by the reduction of nitrate or nitrogen gas. Ammonia is toxic to all living things: if the internal concentration within a plant cell rises above 1.0 mM all photosynthetic reactions within the chloroplast are switched off. It is therefore essential that the ammonia is assimilated rapidly and efficiently, and this is carried out by the enzyme glutamine synthetase. The enzyme has a very high affinity for ammonia and can remove the compound from solution at levels as low as 1.0 μΜ. In the leaf the majority of glutamine synthetase is present in the chloroplast, but a second different form is also present in the cytoplasm. The ammonia is initially assimilated into the amide position, but this needs to be transferred to the 2 - a m i n o position if it is to become a major constituent of protein amino acids. The enzyme glutamate synthase (sometimes written as GOGAT) carries out the transfer of the amide nitrogen to 2 - oxoglutarate to yield glutamate. The enzyme requires reducing power which is either supplied as reduced ferredoxin or NADH. Thus a two step reaction carries out the process of ammonia assimilation, which is known as the glutamate synthase cycle1 (Figure 14.1). In the leaf, ammonia is assimilated in the chloroplast where the ATP and reduced ferredoxin are generated from light energy. Thus ammonia assimilation can be said to be a true PHOTOSYNTHETIC process.

GLUTAMINE NH;

CH2 C=0

Y

COOH

^2-OXOGLUTARATE _2e_^NAD(P)H ^Ferredoxin

v JA

ATP

GLUTAMATE -

re^

^»-GLUTAMATE

COOH CH2 CH2 CHNH2 COOH

Fig. 14.1. The glutamate synthase cycle. 1. Glutamine synthetase 2. Glutamate synthase (GOGAT). A major source of ammonia that is frequently not mentioned in the text books is that generated in the process of photorespiration. Phosphoglycollate synthesised by the RuBP carboxylase/ oxygenase reaction is metabolised by the pathway shown in Fig. 11.2 in Chapter 11. The C0 2 evolved in photorespiration is released in the conversion of 2 molecules of glycine to one of serine with an equivalent release of ammonia: COOH COOH 2 I | I + H20 CH2NH2 J

Glycine 173

CHNH2 + NH3 + C0 2 | + 2e~ + 2H + CH2OH Serine

174

TECHNIQUES IN BIOPRODUCTIVITY AND PHOTOSYNTHESIS

COOH CH 2 CH 2

+

CHNH 2

N2 -

COOH

- S — ^ 2 GLUTAMATE

NH

N03

^

AMINO

ACIDS

Ί

NH;-^ GLUTAMATE \ *> ASPARAGINE HISTIDINE PURINES NUCLEIC ACIDS

PYRIMIDINES

y PROTEINS

TRYPTOPHAN ARGININE

Fig. 14.2. A summary of the flow of nitrogen into proteins and nucleic acids via glutamine and glutamate. Enzymes: (a) nitrogenase, (b) nitrate reductase, (c) transport of ammonia from outside of organism, (d) glutamine synthetase, (e) glutamate synthase, (f) aminotransferases.

This ammonia is immediately reassimilated by the glutamate synthase cycle (Figure 14.1) at a rate in C3 plants that may be ten times higher than that of nitrate reduction1. In C4 plants where photorespiration rates are lower the requirements for ammonia reassimilation are obviously less. Glutamine and glutamate act as a source of nitrogen for all the protein amino acids as well as nucleic acids, amines and ureides (see Figure 14.2). 14.1.2 Enzyme isolation Unless absolutely essential it is not recommended that any assays are carried out on totally crude extracts. Plant extracts contain a large number of inhibitors (in particular oxidising phenols) which will result in an underestimate (or even a zero determination) of the enzyme activity being measured. It must be remembered that just because an enzyme cannot be detected in an extract under one set of conditions, it does not mean the enzyme is not present. The difficulties in isolating plant enzymes are discussed in more detail by J. Coombs in Chapter 16. It should also be noted that a set of conditions that is optimum for one enzyme is unlikely to be correct for another. Thus it is not always possible to assay a

range of enzymes using the same isolation procedure. It is very important that for each new enzyme and plant material the optimum conditions of extraction and assay are determined on each occasion.

14.1.3 Glutamine synthase E.C. 6.3.1.2. A good extraction buffer is 50 mM Tris-HCl pH 8.0, 1.0 mM EDTA, 1.0 mM dithiothreitol, 10 mM MgCl2, 10% (v/v) glycerol and 2% Polyclar A.T. It may also be necessary to add 1.0 mM mercaptoethanol, 1.0 mM reduced glutathione and 5 mM glutamate, or to substitute ethylene glycol for glycerol. Glutamine synthetase is frequently assayed in the presence of glutamine, hydroxylamine, ADP, arsenate and Mn 2 + . This so-called "transferase" assay is NOT RECOMMENDED as the physiological relevance to glutamine synthetase is not known. The assay is, however, widely used mainly because very high activities may be determined. A more physiological but by no means perfect assay is the "synthetase' ' assay which employs hydroxylamine in place of ammonia.

AMMONIA ASSIMILATION AND AMINO ACID BIOSYNTHESIS

glutamate + hydroxylamine + ATP -* glutamyl-hydroxamate + ADP -I- Pj The assay consists of incubating 0.25 ml of enzyme in a final volume of 1.0 ml containing 18 μπιοΐ ATP, 45 μιηοΐ MgS0 4 , 6 μπιοΐ hydroxylamine, 92 ml L-glutamate and 50 μπιοΐ Imidazole-HCl at a final pH of 7.2 for varying times at 30°C. The reaction is stopped by the addition of 1.0 ml of ferric chloride reagent (0.37 M ferric chloride, 0.67 M HC1 and 0.2 M TCA) which forms a brown-coloured complex with any glutamyl-hydroxamate formed and precipitates out the enzyme protein. The reaction tubes are centrifuged to remove the protein, and the absorbance may be read in a spectrophotometer at 540 nm. A standard curve of glutamylhydroxamate may be prepared with up to 3 μπιοΐ per 1.0 ml of assay mixture. The rate of enzyme activity may then be calculated. For very active enzyme preparations it may be necessary to dilute the enzyme or use very short assay times as the production of the hydroxamate is not linear at very high rates. It is not known in crude extracts how close an agreement there is between the true rate of glutamine synthesis and the production of the hydroxamate complex. Two other assays may be used in purer extracts: (1) the determination of the phosphate liberated from ATP, and (2) the determination of ADP based on a spectrophotometric assay involving pyruvate kinase and lactate dehydrogenase. It has recently become apparent that two isoenzymic forms of glutamine synthetase occur in leaves, one present in the chloroplast and one in the cytosol. These forms can be readily separated by ion exchange chromatography (see Chapter 16). However the distribution of the two forms varies considerably between different plants. McNally et al.2 carried out a detailed survey and were able to identify four groups of plants. (1) Parasitic plants containing solely the cytoplasm enzyme (e.g. Orobanche sp.)\ (2) Plants containing 50% each of the cytoplasm and chloroplast enzyme (e.g. Zea, Pennisetum); (3) Plants containing a small proportion of the cytosol enzyme (e.g. barley, wheat, pea); (4) Plants containing solely the chloroplast enzyme (e.g. spinach, tobacco, lupin). The reason for the

175

differences between the plant species is not clear. However it is known that the two isoenzymes, although catalysing the same reaction, may have slight differences in their kinetic constants, molecular weights and stability, and are almost certainly under separate gene control. A frequently used piece of evidence for the operation of the glutamate cycle is that ammonia assimilation in higher plants and algae is blocked by L-methionine sulphoximine (MSO):-

CH3

I

0 = S = NH

I CH2 CH2 CHNH COOH2 The compound acts as an inhibitor by binding to the active site of glutamine synthetase in place of the activated intermediate y-glutamyl phosphate. The following data were obtained at Rothamsted working with glutamine synthetase isolated from pea leaves. In Figure 14.3 the data are presented as the classical Lineweaver-Burke plot at various concentrations of MSO, of the reciprocal of the enzyme activity against the reciprocal of glutamate concentration. The reciprocal of the Vmax of the enzyme at an infinite glutamate concentration is the intercept on the yaxis with a value of approximately 0.8. This value is independent of the amount of inhibitor present indicating that it is a competitive type of inhibition. If the Vmax had been lower with increasing inhibitor concentration then the inhibition would be considered non-competitive. At low concentrations of MSO the plots are linear, but at higher levels an upward curve can be detected, suggesting that there is irreversible binding of MSO to the active site. The slope of the non-inhibited line is equal to:

176

TECHNIQUES IN BIOPRODUCTIVITY AND PHOTOSYNTHESIS

METHIONINE

SULPHOXIMINE

METHIONINE

M50 CONCENTRATION ( μmoles/ml) 0-4

0

01

SULPHOXIMINE

02

03

GLUTAMATE CONCENTRATION ( u moles/ml) 1 2

0-4

MSO CONCENTRATION ( ( j m o l e s / m l ) 0 01 02 03 0-4 05 GLUTAMATE CONCENTRATION (|jmoles/ml)" 1

Fig. 14.3. Inhibition of pea leaf glutamine synthetase by MSO; Lineweaver-Burke plot.

Fig. 14.4. Inhibition of pea leaf glutamine synthetase by MSO; Dixon plot. CH 3 0 - P = 0

I

CH 2 but in the presence of inhibitor the slope is altered to:

I

CH 2 CHNH 2

I

where [I] is the concentration of the inhibitor and Kj is the inhibitor constant. By measuring the slopes of the lines it is possible to calculate the value of Kj. A simpler method of plotting the data is in the Dixon plot of the reciprocal of enzyme activity against inhibitor concentration (Figure 14.4). The lines should all cross at a point that is vertically above the negative Kj value of the inhibitor. Thus in Figure 14.4 the Kj can be read off directly as approximately 0.12 mM. Compounds can be compared for their potency as inhibitors by comparison of their Kj values; the lower the value the stronger the inhibitor. The ability to block ammonia assimilation has formed the basis of a new type of herbicide which utilises a phosphorus derivative of MSO termed phosphinothricine (PPT):

COOH The herbicide has also been patented as the 2 - oxo derivative (PPO) and a dialanyl tripeptide. When these compounds are fed to plants toxic ammonia is liberated and the photorespiratory carbon and nitrogen cycle is disrupted3 (see Figure 11.2, Chapter 11). This disruption can be readily seen in Figure 14.5., where the rate of photosynthetic C 0 2 fixation is inhibited by over 90%, 100 minutes after the addition of the glutamine synthetase inhibitors.

14.1.4 Glutamate synthase Extraction buffer: K phosphate (100 mM, pH 7.2), 5 mM 2 - mercaptoethanol, 1.0 mM phenylmethyl sulphonyl fluoride, 10% glycerol, 5 mM EDTA and 0.1% Triton-X-100.

AMMONIA ASSIMILATION AND AMINO ACID BIOSYNTHESIS

BARLEY LEAVES COMPOUND ADDED

0

40

60

80

40

60

80

100

120 140

Fig. 14.5. Effect of glutamine synthetase inhibitors on the rate of photosynthetic C 0 2 fixation by cut barley leaves.

Pyridine Nucleotide-dependent Enzyme E.C.I. 4.1.13.: The enzyme in roots, root nodules and developing fruits may be assayed by measuring the decrease in absorbance of NADH at 340 nm in a recording spectrophotometer. The assay medium should contain 12.5 μιτιοΐ 2-oxoglutarate, 200 nmol NADH and 125 μιτιοΐ Tricine-KOH pH 7.5 in 2.5 ml. Divalent cations are not required for the reaction, and it is normal to have EDTA present in the reaction medium to remove any other ions. The rate of reaction is normally relatively low and care should be taken to avoid artifacts due to the presence (particularly in crude extracts) of contaminating NADH oxidases and glutamate dehydrogenase. Activity is determined by measuring the difference between the rate of NADH oxidation in the presence and absence of highly purified glutamine. The actual rate of the enzyme reaction can be calculated as it is known that a solution of NADH of 1 μπιοΐ ml - 1 (1 mM) has an absorbance at 340 nm of 6.2. Ferredoxin-dependent Enzyme E.C.I.4.7.1.: The enzyme in leaf tissue is almost totally ferredoxin-dependent but the enzyme from other sources is apparently able to use NADH or reduced ferredoxin. It is probable that two separate enzymes are responsible for the two reactions.

177

The assay mixture contains 10 mM 2-oxoglutarate, lOmM glutamine, 100 μg of methyl viologen and up to 100 μΐ of enzyme in a final volume of 0.5 ml. For assaying crude extracts 10 mM aminooxyacetate may be added to inhibit any transaminase activity. The reaction is started by the addition of 100 μΐ of a solution containing 16 mg ml - 1 sodium dithionite and 16 mg ml" 1 sodium bicarbonate (prepared immediately before use). After incubation at 30°C for 5 - 1 5 minutes the reaction is stopped by the addition of 1.0 ml of ethanol followed by vigorous shaking to oxidise the methyl viologen and dithionite. A blank without dithionite is always run for each reaction. Methyl viologen is used as a substitute for ferredoxin as it is much cheaper, and is very stable on storage. It also has the added advantage that it forms a blue colour when reduced, and its oxidation to a colourless form may be followed in the assay tube. Ferredoxin may be used in place of methyl viologen in the assay mixtures and reaction rates are in general faster although care has to be taken to ensure that the ferredoxin is in a fully reduced state. After centrifugation 200 μΐ of the reaction mixture is spotted on to Whatman No. 4 chromatography paper. The separation of glutamate may be carried out by chromatography in 75% (w/w) phenol in the presence of ammonia vapour. The papers should be dried thoroughly after use and sprayed with a freshly prepared specific ninhydrin reagent suggested by Atfield and Morris 4 . The reagent comprises 0.05 g cadmium acetate, 1.0 ml acetic acid and 5.0 ml of H 2 0 in 50 ml acetone containing 0.5 g ninhydrin. The papers should be left in the dark in an enclosed container for a period of 12 - 18 hours in the presence of a small beaker of concentrated sulphuric acid. The glutamate spots may then be cut out and the dark-red colour eluted with 8 ml of a reagent containing 600 ml ethyl acetate, 600 ml water, 600 ml methanol, 18 g glacial acetic acid and 18 g cadmium acetate. The absorbance of the coloured solution may be determined at 500 nm. A standard curve of glutamate concentrations should be made, and an internal standard of 20 μΐ of a 1.0 mg ml - 1 solution should always be run on EACH chromatography paper, to allow for variations in relative humidity and temperature during development. There is a linear relationship

178

TECHNIQUES IN BIOPRODUCTIVITY AND PHOTOSYNTHESIS

between the absorbance at 500 nm and the glutamate applied over a range 0 - 5 0 μg. Glutamate may also be separated from glutamine using Dowex-1 columns. The Dowex-1 chloride form may be converted to the acetate form by thorough washing with 10% Na 2 C0 3 , 2 M acetic acid followed by excess distilled H 2 0 . Small columns (0.5 x 5.0 cm) of Dowex-1-acetate may be prepared in glass pasteur pipettes plugged at the tapered end with glass wool; 0.5 ml of the assay medium may be loaded on the columns and glutamine is eluted with 4 ml of distilled H 2 0 . Glutamate may then be eluted with 4 ml of 0.2 M acetic acid. The glutamate content of the eluate may be determined by any ninhydrin method provided that a standard glutamate curve is first constructed. 14.1.5 Glutamate dehydrogenase E.C.I.4.1.2 The enzyme was originally considered the one primarily used for ammonia assimilation. The activity of the enzyme can be readily measured in extracts of all plant tissues, although its role in plant metabolism is not clear. Extraction buffer: Tris-acetate (50 mM, pH 8.2), 10% glycerol, 0.5 mM EDTA, 5 mM mercaptoethanol. The reaction mixture contains in a final volume of 2.5 ml: 370 μπιοΐ of ammonium acetate, 200 nmol NADH, 31 μπιοΐ 2 - oxoglutarate, 2 μπιοΐ CaCl2 and 125 μπιοΐ Tris/acetate buffer pH 8.2. All solutions are adjusted to pH 8.2 with Tris. Activity is determined by measuring the difference between the rate of NADH oxidation in the presence and absence of ammonium acetate in a recording spectrophotometer. Two important points to note are: (1) The concentration of ammonium ion must be high to ensure that the enzyme is fully saturated; (2) There must be excess of a divalent cation to activate the enzyme completely. It is not possible to make an accurate determination of glutamate dehydrogenase activity in the presence of EDTA alone, as the majority of the enzyme activity would be inhibited.

14.1.6 Aminotransferases Aminotransferases catalyse the transfer of the amino group of an amino acid to a 2 - oxo acid to yield a new amino acid and 2 - o x o acid respectively5 e.g. glutamate-oxaloacetate aminotransferase E.C.2.6.1.1 :Glutamate + oxaloacetate -* 2 - oxoglutarate ■f aspartate Extraction buffer: Tris-HCl (pH 7.5, 40 mM), EDTA 0.25 mM, glutathione 2 mM. All aminotransferases are reversible; therefore the direction chosen may depend upon the substrates available. The enzymes are pyridoxal phosphate requiring, although it is now believed that in plants the coenzyme is bound tightly to the enzyme. The requirement for pyridoxal phosphate for the enzyme under test should be checked. Probably the simplest method of testing for aminotransferase is to incubate the enzyme with 5 mM 2 - oxo acid and 5 mM amino acid in 50 mM Tris-HCl buffer pH 7.5 for varying times and stopping the reaction with an equal volume of ethanol. After centrifuging the protein, the extract may be chromatographed on paper or TLC plates in a solvent that gives a good separation of the initial end product amino acid (e.g. butanol: acetic acid: water in proportions 90:10:29 by volume gives a good separation of the amino acids aspartate and alanine). The rate of synthesis of the product amino acid may be determined by the method of Atfield and Morris described in the previous section. A second method is to incubate the amino acid with a very small amount of 2 - o x o acid and determine the product 2 - oxo acid by formation of a dinitrophenylhydrazone. Alanine amino­ transferase may be assayed by incubating the enzyme with 100 mM alanine and 2 mM 2 - oxoglutarate in 0.1 M Tris-HCl buffer pH 7.4. The pyruvate formed may be determined by reaction with 2,4-dinitrophenylhydrazine and measuring the colour at 546 nm. A standard curve of varying pyruvate concentrations must, of course, be constructed. A third more refined but expensive method is to couple the 2 - o x o acid produced to NADH

AMMONIA ASSIMILATION AND AMINO ACID BIOSYNTHESIS

oxidation by an added enzyme. A standard reaction mixture may be set up containing 25 μΐ 10 mM 2-oxoglutarate, 20 μΐ 10 mM EDTA, 10 μΐ 10 mM NADH and 40 μΐ enzyme extract. If aspartate aminotransferase is to be measured the product is oxaloacetate which is rapidly converted to malate by malic dehydrogenase with the subsequent oxidation of NADH, which may be measured on a spectrophotometer at 340 nm in a similar manner to glutamate dehydrogenase. The final reaction medium in the spectrophotometer cell should also include 25 μΐ 10 mM aspartate, 0.5 ml 0.1 M HEPES buffer pH 8.0, 0.1 ml of 100-fold diluted commercial malate dehydrogenase and 0.28 ml H 2 0 . In crude extracts of plants there is often sufficient malate dehydrogenase already present to drive the reaction without further addition of the enzyme. If alanine aminotransferase is to be measured, the product is pyruvate, which may be converted to lactate by lactate dehydrogenase. In this case the final reaction medium should also include 0.1 ml 0.1 M alanine, 0.5 ml 0.1 HEPES buffer pH 7.5, 0.1 ml of 100-fold diluted commercially available lactate dehydrogenase and 0.2 ml H 2 0 . The activity of the enzyme may be calculated in both cases using a blank reaction cuvette containing no 2-oxoglutarate.

v v

O

NH 2 O

c

I1

CH 2 1

If ammonia is formed in the roots it has to be transported to the leaves and developing fruits in a non-toxic form. Nitrate, on the other hand may be transported directly in the xylem as the unmetabolised ion. Plants can apparently be divided into two groups on the basis of their nitrogen transport compounds. Cereals and temperate legumes (e.g. Pisum and Vicia) utilise the amides asparagine and glutamine and to a lesser extent arginine.

1

1

CH 2

CHNH 2

(CH2)3 11 CHNH 2 COOH

1

COOH

1

CHNH 2 11 COOH Asparagine

NH 2

C 1 1 NH

1

1

Glutamine

Arginine

Tropical legumes (e.g. Glycine, Phaseolus and Vigna) utilise the ureides allantoin and allantoic acid: O NH 2 O = C

C — NH

/ \ CH C = O \ / N N H H Allantoin Οκ \

O = C

14.2.1 Compounds utilised

\/

NH

C 11 CH 2

NH 2

14.2 Transport of nitrogenous compounds

NH 2

179

OH / C

NH 2 = O

H NH

NH

Allantoic acid

All the compounds above are characterised by a high N:C ratio. It is not clear yet why tropical legumes are able to synthesise large amounts of the ureides with a very high nitrogen content. Organic nitrogen may be transported in either the phloem or the xylem. Techniques for determining the content of the phloem are somewhat difficult. The composition of the xylem sap can be readily determined, particularly in young plants grown in well-watered soil. The only essential requirements are a very sharp razor blade and a piece of clear plastic tubing 3 - 4 cm long, with an internal diameter the same as the thickness of the stem. The shoot is cut immediately above the surface of

180

TECHNIQUES IN BIOPRODUCTIVITY AND PHOTOSYNTHESIS

the soil and the tubing placed rapidly over the cut end. Sap can be readily seen to collect in the tubing after about 30 minutes and may be collected with a syringe or pipette. However, frequently under field conditions with older plants it is not possible to obtain xylem sap. In this case stem apices or the shoot plus petiole should be dried and extracted in 1:1 mixture of ethanol and 0.1 M K-phosphate buffer pH 7.0. Most authors recommend heating at 80°C for a few minutes, although this will cause some breakdown of glutamine. 14.2.2 Assay of transport compounds The amino acid content of the extracts may be determined by spotting 1 0 - 2 0 μΐ onto a TLC plate or paper chromatogram as described in Chapter 11. Amino acids can be readily identified by spraying with ninhydrin, and quantitative determinations can be made by the method of Atfield and Morris (see 14.1.4). The ureide content of the extracts may be qualitatively determined by spraying TLC plates with Ehrlich's reagent ( 1 % solution of 4 - dimethylaminobenzaldehyde in 96% ethanol). Allantoin and allantoic acid give brown colours when the plates are exposed to HCl vapour. The best quantitative method of ureide analysis involves measuring the amount of glyoxylate produced after hydrolysis. The sap should be heated in 0.05 M HCl for 2 minutes at 100°C and immediately cooled in ice. The hydrolysed samples should then be incubated with phenylhydrazine hydrochloride at a concentration of 0.66 mg ml" 1 at 30°C for 15 minutes. The mixture should be cooled in a salt-ice bath and made up to 3 M HCl and 3.3 mg ml - 1 potassium ferricyanide. After allowing colour development the absorbance of the solutions can be read at 520 nm. Standard curves of allantoic acid must be prepared at least in duplicate before any determination can be made. The above test is not given by allantoin itself only allantoic acid. However, it is possible to hydrolyse allantoin to the free acid in alkali prior to the assay. An easier method has recently been published by Patterson et al.1. The method involves the use

of Dowex HCR-S, a strongly acidic cationic exchange resin which may be obtained from Sigma Chemical Company Ltd. The resin must first be soaked in 2 vol 0.5 M NaOH and washed with enough distilled H 2 0 until neutral and then transferred to 2 vol 1 M HCl and washed again with water until neutral. The process can be carried out on a sintered glass funnel. After a final wash in 50% ethanol the resin can be stored in a dried state. Approximately 2 g of the resin is added to the plant extract and shaken for 30 minutes. The decanted extract is then placed in a 18 x 150 mm test tube and adjusted to 1 ml. 1 ml of potassium phthalate (0.2 M, pH 4.0) and 0.5 ml of commercial household bleach (3:7 v/v with water) are added and the mixture shaken vigorously. After 5 minutes at room temperature, 2.0 ml phenol reagent* is added and again vigorously mixed. After 10 minutes, 5.5 ml of water is added and the absorbance determined at 625 nm. A standard amount of allantoin or allantoic acid should have been previously prepared. The method is designed to measure the ammonia liberated on the oxidation of the ureides. The Dowex resin removes interfering amino acids. * Phenol reagent: Phenol saturated with water is mixed with methanol (1:1, v/v) and stored in a dark bottle. Just before use it is mixed (1:4 v/v) with 20% NaOH. 14.2.3 Nitrogen fixation Such is the dependence by tropical legumes on using ureides as transport compounds, that the ureide content can be used as an indirect measurement of nitrogen fixation. There is a positive correlation between ureide content of the xylem sap and the rate of nitrogen fixation8. This has been further refined to the term: Ureide-N Ureide N + Nitrate N and it is now possible to use the content of the stem plus petiole (but not leaves) to avoid any problems with obtaining bleeding xylem sap.

AMMONIA ASSIMILATION AND AMINO ACID BIOSYNTHESIS

Thus it is possible to take small samples of a plant in the field (the plant can be left to grow for breeding purposes), take them back to the laboratory (samples may be stored for up to 24 hours without any serious effect), and carry out a cheap and easy assay for nitrogen fixation that does not require sophisticated and expensive GLC apparatus to be taken out in the field. Clearly it would be advisable to carry out a series of acetylene reduction assays on a set of plants to standardise the ureide content.

14.3 The biosynthesis of amino acids It is still generally accepted that only twenty amino acids are incorporated into protein. There may, however, be considerable post-translation modification (e.g. the formation of Nmethylysine, 4 - hydroxyproline and 4-carboxyglutamate). Other amino acids (e.g. homoserine, ornithine and diaminopimelic acid) are formed during metabolic interconversions. However at least two hundred other "non-protein" amino acids occur in plants, some of which have a restricted taxonomic distribution. The protein amino acids can be divided into separate "families" depending on the precursor compound common to their synthesis. ASP ART ATE:

Asparagine, lysine, isoleucine, methionine and threonine. GLUTAMATE: Glutamine, proline and arginine. PHOSPHOENOL The aromatic amino acids PYRUVATE: phenylalanine, tyrosine and tryptophan. 3-PHOSPHOGLYCERATE: Serine, glycine and cysteine. PYRUVATE: Alanine, leucine and valine. Histidine is somewhat unusual in that it is derived from ribose-5-phosphate. Plants and bacteria are able to synthesise all twenty protein amino acids9. Animals however, are not able to do so, and they require nine

181

"essential" amino acids (marked in italics) in their diet. Technically tyrosine and cysteine are also essential, but they may be synthesised from phenylalanine and methionine respectively, provided they are available in adequate quantities. Recent data have now confirmed an original suggestion that those amino acids that animals require in their diet are synthesised solely inside the chloroplast in higher plants 1 . 14.3.1 The aspartate family A pathway that has been extensively studied at Rothamsted is the synthesis of lysine, threonine and methionine from aspartate (Figure 14.6). The amino acids are frequently those that limit the quality of plant foodstuffs; in particular, cereal seeds are deficient in lysine and legume seeds deficient in methionine. The ability of chloroplasts to synthesise these amino acids can be readily demonstrated using intact chloroplasts prepared by the method described in Chapter 9. Chloroplasts containing 100 μg chlorophyll should be resuspended in 0.5 ml buffer containing sorbitol (300 mM), EPPS (pH 8.3) and 30 mM KC1, with 2 μ ο of 14C-aspartate for 20 minutes in a bright light (controls should be run in complete darkness). The reaction is stopped by the addition of an equal volume of ice-cold 10% trichloroacetic acid. The reaction products can be separated by two-dimensional chromatography on TLC plates in a similar method to that described in Chapter 11. Amino acids can be identified by cochromatography with authentic standards. Radioactivity in each amino acid can be determined by scraping each spot into a scintillation vial. A full description of this technique is given in Mills et al10. Pea leaf chloroplasts readily convert aspartate to lysine, threonine and homoserine. Initial experiments suggested that chloroplasts could also synthesise methionine, but later work showed that homocysteine was the chloroplast product and the last step of the methionine synthesis took place in the cytoplasm (Figure 14.6.) It is interesting that the requirement for methionine in animals can be met by homocysteine, and that animals can also carry out the final methylation step.

182

TECHNIQUES IN BIOPRODUCTIVITY AND PHOTOSYNTHESIS

Asportate CHLOROPLAST

III· Asp

ASA

Homoserine

®

Pyruvate

^_

ASA

->

DHDP

HS®

Jl·

> DAP

L

-» Lysine

-> Threonine

Homoserine Cysteine -

Cystathionine

-> Homocysteine Homocysteine

Methylthioribose S-Adenosyl Ethylene / ~~ methionine Polyammes

Methyl donation reactions CYTOPLASM

Fig. 14.6. The localisation of the enzymes of aspartate pathway in leaf cells. Enzymes: 1. aspartate kinase; 2. ASA dehydrogenase; 3. DHDP synthase (EC 4.2.1.52); 4. DAP decarboxylase (EC 4.1.1.20). 5. homoserine dehydrogenase; 6. homoserine kinase; 7. threonine synthase; 8. cystathionineß-synthase; 9. cystathionine-y-lyase; 10. methionine synthase; 11. methionine adenosyltransf erase. Abbreviations: Asp P = aspartyl phosphate; ASA = asparticsemialdehyde; DHDP = dihydrodipicolinate; DAP = diaminopimelate; HsP = phosphohomoserine.

The complex pathway to lysine, threonine, methionine, and further on to isoleucine, leucine and valine requires a considerable amount of energy input in the form of NADPH and ATP. If these reactions are carried out in the chloroplast the energy is derived directly from light. The light dependence of the reactions can be demonstrated by keeping the reaction tubes in the dark, when little conversion of aspartate to other amino acids takes place. As these reactions utilize NADPH and ATP directly from the electron transport chain of the light process, they are true photosynthesis in the same manner as C0 2 fixation. If amino acid synthesis is carried out in the root or maturing seed then the energy required for the synthesis must be derived from previously fixed carbon via oxidation in the mitochondria. It is possible to calculate the amount of glucose required to synthesise 1.0 g of methionine in the

root from nitrate and sulphate as 2.13 g. If the reaction had gone on in the chloroplast using light energy then the cost would only be 0.51 g. Thus there is a considerable saving in energy if plants carry out their synthetic reactions in the chloroplast, and there could be a considerable increase in total yield if plants were selected which carried out more of their metabolism in the leaf rather than in the root or seed. The metabolic interconversions in the different pathways are subject to strict feedback regulatory control by the endproduct amino acids. Lysine almost totally inhibits its own synthesis at levels above 1.0 mM, and also that of homoserine and threonine. Threonine, however, only inhibits its own synthesis and that of homoserine. The feedback inhibition can be readily explained by the known properties of the enzymes involved in the pathway. Aspartate kinase which catalyses the

AMMONIA ASSIMILATION AND AMINO ACID BIOSYNTHESIS

ASPARTIC ACID

Fig. 14.7. The regulation of the biosynthesis of lysine, threonine, methionine and isoleucine. ( - ) Feedback inhibition ( + ) Feedback stimulation. first step, the formation of aspartyl phosphate, is inhibited strongly by lysine and to a lesser extent by threonine. The inhibition by lysine and threonine is additive suggesting the presence of at least two separate enzymes. Lysine is also able to inhibit the first enzyme unique to its own synthesis, dihydrodipicolinate synthase, and in a similar manner threonine inhibits homoserine dehydrogenase (see Figure 14.7). The plant is thus able to carefully regulate the flow of metabolites along the complex pathways of amino acid synthesis. High levels of amino acids do not build up, and the rate of synthesis is then determined by the rate of incorporation into protein. A similar system operates in bacteria, where the regulation of metabolite flow is even more important. Bacteria are also able to regulate amino acid synthesis by "repressing'' the amount of a particular enzyme synthesised within the cell. In most cases such a system does not operate in higher plants. However, recent evidence now suggests that the first enzyme unique to methionine synthesis (which is not feedback regulated), cystathionine-y-synthase, is repressed by methionine. 14.3.2 Selection of amino acid metabolism mutants In Figure 14.7 it can be seen that lysine and threonine are able to inhibit the passage of carbon from aspartate to homoserine by inhibiting both aspartate kinase and homoserine dehydrogenase.

183

If they are added together to a growing plant they are able to kill the plant by preventing the formation of methionine due to the prevention of homoserine synthesis. Under normal conditions it is not possible to add lysine and threonine to growing plants: (1) due to the large cost of the amino acids required to feed to plants growing in a pot or in the field; (2) due to the stimulation of bacterial and fungal growth which will remove the amino acids and interfere with the natural growth of the plants. This problem was initially overcome by growing plants in tissue culture, but it was not always possible to regenerate healthy plants after the tests had been carried out. Studies at Rothamsted over the last few years by Dr. S.W.J. Bright have developed a technique for growing young barley plants in sterile culture, in such a way that the action of various compounds can be tested, and viable plants can be obtained at the end of the experiment. Method: Barley seeds should be dehusked in 50% v/v H 2 S0 4 for 3 hours, washed in tap water and three changes of distilled water before being soaked overnight at 5°C. A wash in saturated CaC0 3 solution for 20 minutes after the acid treatment is also recommended. The embryos may then be dissected by hand from the seeds and allowed to dry on filter paper at 25°C for at least 24 hours. Petri dishes containing agar (6 g Γ 1 ), Murashige and Skoog's medium12 without hormones or casein hydrolysate and sucrose 30 g Γ 1 which has been previously autoclaved, should be prepared. If amino acids are to be tested they should be sterile filtered and added to the mixture whilst it is still liquid; 20 ml of medium may be added to a standard 9 cm plastic petri dish. Dry embryos should be sterilised by shaking for 15 minutes at 25°C in the supernatant of 70 g Γ 1 calcium hypochlorite with a small amount of Teepol added as a wetting agent. The embryos are then rinsed in sterile distilled water, washed for 5 minutes in sterile 0.01 M HC1 and finally rinsed with 400 ml sterile distilled water. Embryos should be placed on the agar medium under sterile conditions so that the scutellum faces downwards. The plates should be incubated for 7 days at 25 °C under white lights with a 16-hour day. The

184

TECHNIQUES IN BIOPRODUCTIVITY AND PHOTOSYNTHESIS

Fig. 14.8. Different stages of barley embryo development grown in sterile culture.

embryos develop roots and leaves, and examples of the plants at different stages of growth can be seen in Figure 14.8. After the leaves reach 6 cm long the plants may be successfully transplanted to a small pot of compost and, provided they are maintained in a humid atmosphere for a further period until the roots establish, they will grow into healthy mature plants. If the plants are grown in the presence of 2 mM lysine and 2 mM threonine then there is an 80% reduction in growth. The inhibition in growth can be prevented by the addition of 0.5 mM methionine to the medium, suggesting that the action of lysine + threonine is to prevent methionine synthesis (see Figure 14.7). If normal embryos are grown in the presence of lysine and threonine then none of them will show any significant leaf development. However, if the seeds are first treated with a mutagen then there is a large increase in the probability of obtaining a mutant plant that is in some way resistant to the toxic action of lysine + threonine. In Fig. 14.9. such a plant can be seen, after azide treatment. A number of these mutants in barley have now been obtained at Rothamsted and their enzymology examined. The majority of mutants examined have alteration in the feedback regulation of the enzyme aspartate kinase (E.C.2.7.24)13 . The enzyme which converts aspartate into aspartyl phosphate can be separated into three isoenzymic forms in barley (Secton 14.3.4), one sensitive to inhibition by threonine and two sensitive to

lysine14. Mutations were found in one or other of the lysine inhibited isoenzymes such that the enzyme activity had a greatly reduced sensitivity to lysine. In Figure 14.10. the sensitivity of aspartate kinase II (cv. Borni) is shown; the enzyme is about 80% inhibited by 1 mM lysine. However the enzyme from the homozygous mutant line is totally insensitive to lysine, whilst the hetero­ zygous line shows intermediate sensitivity. In Table 14.1 the soluble levels of lysine, threonine and methionine in the mutants analysed so far at Rothamsted are shown13. It is clear that an alteration to the feedback sensitivity of aspartate kinase can cause a build up in the seed of the levels of threonine and to a lesser extent lysine. The quantities are sufficient to eliminate the second limiting amino acid status of the threonine in the barley grain and to make a significant contribution to reducing the requirement for lysine supplementation. 14.3.3 Mutagen treatment technique The treatment described, although used on barley, can probably be used for most dry seeds. A simple test as to whether a mutagen has acted is the appearance of a high proportion of ''chlorophyll-less'' or albino plants, when the seeds are germinated ( 5 - 10%). Seeds should be soaked overnight in water at 4°C, and then incubated in 1 mM sodium azide in 0.1 M phosphate buffer pH 3.0 for 2 hours. The seeds should then be washed in two volumes of

AMMONIA ASSIMILATION AND AMINO ACID BIOSYNTHESIS

1

**V X

oR^Il

185

fc; "rJ^

ß _s *

1 S „

'-.

%

ψ* |

Fig. 14.9. The growth of barley embryos in the presence of 2 mM lysine and 2 mM threonine. The top left plate also contains 0.5 mM methionine. Note in the bottom centre plate, the presence of one plant that is growing normally.

distilled water and under running tap water in the cold for 30 minutes. The seeds should be dried and then may be planted in the field or in pots. The seeds that have been harvested after the growth of these plants (M2 generation) should be used for the embryo growth experiments discussed above.

2

3 A LYSINE (mM)

5

10

10 + 0 e Ado Met

Fig. 14.10. The effect of lysine on the barley aspartate kinase isoenzyme AKII. (A) homozygous mutant plant R2501, (D) heterozygous mutant plant; (o) Wild type plant cv. Borni.

Warning: Azide is a volatile mutagen and a respiratory poison, coupled with which it may form an explosive mixture if poured down the sink. All handling of azide must be carried out in a fume cupboard, the original solution and the first two washings must be treated with a 15% solution of ammonium eerie nitrate before they are poured down the sink; after this treatment the azide is decomposed. The method is perfectly safe as long as reasonable precautions are taken.

186

TECHNIQUES IN BIOPRODUCTIVITY AND PHOTOSYNTHESIS

Table 14.1. Threonine, lysine and methionine content of the free amino acid pools of mature grains from four barley mutants resistant to lysine plus threonine. Amino acid content nmol g 1 dry weight Plant

Threonine

Lysine

Methionine

Total

Borni R2501 R2506

118 6129 9032

84 1266 1620

24 149 72

10400 28200 20400

Borni R3004 R3202 Double mutant R3004 x R3202

127 2376 125

79 107 109

19 23 19

6900 12400 11700

689

129

21

10100

14.3.4 Isolation and characterisation of aspartate kinase A suitable extraction buffer is 25 mM K phosphate, pH 7.5; 2 mM MgCl2; 2 mM EDTA; 15% (v/v) glycerol and 0.2% (v/v) 2-mercaptoethanol. Prior to assay the extract should first be precipitated with 65% saturation ammonium sulphate and passed through Sephadex G.25. The assay is similar to that used for glutamine synthetase. The mixture should contain 75 mM Tr­ aspariate, 15 mM MgCl2, 30 mM ATP and 225 mM hydroxylamine hydrochloride at pH 7.4; zero time and minus aspartate blanks should be used. The formation of aspartyl-hydroxamate should then be assayed in a similar manner to that described for the glutamyl derivative (Section 14.1.3). A more refined assay using 14C-aspartate is also available14. In order to separate the three isoenzymes of aspartate kinase the enzyme should be subjected to DEAE - Sephacel chromatography. The extract should be loaded in the presence of 50 mM KCl on to a 1 x 11 cm column and washed with 30 ml of extraction buffer plus 75 mM KCl; this will elute the threonine sensitive isoenzyme AKI. The first lysine sensitive isoenzyme, AKII can then be eluted with 15 ml of extraction buffer plus 120 mM KCl. The second lysine sensitive isoenzyme, AKIII requires 20 ml of extraction buffer plus 220 mM KCl for complete elution14. The feedback sensitivities of the different fractions can then be

tested by carrying out the aspartate kinase assays in increasing concentrations of lysine as shown in Figure 14.10. 14.3.5 Proline and drought stress The majority of plants when subject to a reduction in the water potential of tissues or cells accumulate the cyclic imino acid proline (see Paleg and Aspinall15 for review articles). The progressive accumulation of proline is accompanied by a fall in tissue water potential with time. Examples of estimates of the lower limit to the threshold water potential for the response are barley ( - 0 . 7 MPa), cotton ( - 1 . 2 MPa) and sorghum ( - 2 . 4 MPa). The concentration of proline may rise to 10% of the total leaf dry weight although most values are in the region of 2 0 - 3 0 mg g - 1 dry weight. Most studies have examined the proline concentration in shoot or leaves but the amino acid will also accumulate in the root and other organs although it occurs both later and less extensively. Proline accumulation may also be stimulated by high or low temperatures, salinity and abscisic acid. A number of workers have attempted to establish a correlation between the ability of a plant to accumulate proline and drought resistance. The subject has caused some controversy and it is difficult to assess the importance of proline accumulation in the complex of factors which determine crop resistance to water deficit in the field.

AMMONIA ASSIMILATION AND AMINO ACID BIOSYNTHESIS

Assay for proline: Approximately 200 mg of tissue should be extracted successively with 0.5 ml 3% w/v 5 - sulphosalicylic acid four times in a glass pestle and mortar. The pooled homogenate was centrifuged at 500 xg for 10 minutes and reacted with 2 ml glacial acetic acid and 2 ml acid ninhydrin (1.25 g ninhydrin warmed in 30 ml glacial acetic acid plus 20 ml 6 M phosphoric acid; this mixture is stable at 4°C for 24 hours) for 1 hour at 100°C in a boiling tube. The mixture was then cooled in an ice bath and 4 ml of toluene added. After vigorous mixing the two layers separate out and the pink-red colour at the top may be removed with a Pasteur pipette. The top colour-containing layer should be allowed to warm up to room temperature and the absorbance read at 520 nm using toluene as a blank in a spectrophotometer. A standard curve should always be obtained at the same time. The absorbance is linear up to 40 μg of proline per 2 ml sample. Note: It is important for this assay that either fresh tissue or that which has been rapidly frozen (e.g. in liquid nitrogen) is used. If tissue is allowed to dry after it has been removed from the plant (even for a very short period), proline will accumulate and give spurious results. References 1. Wallsgrove, R.M., A.J. Keys, P.J. Lea and B.J. Miflin (1983) Photosynthesis, photorespiration and nitrogen metabolism. Plant Cell Environment 6, 301-309. 2. McNally, S.F., B. Hirel, P. Gadal, A.F. Mann and G.R. Stewart (1983) Glutamine synthetase of higher plants. Plant Physiol. 72, 22-25. 3. Lea, P.J., K.W. Joy, J.L. Ramos and M.G. Guerrero (1984) The action of 2-amino-4(methylphosphinyl)-butanoic acid (phosphoro-

187

thricine) and its 2-oxo derivative on the metabolism of cyanobacteria and higher plants. Phytochemistry 23, 1-6. 4. Atfield, G.N. and C.J.O.R. Morris (1961) Analytical separation by high voltage electrophoresis of amino acids in protein hydrosylates. Biochem J. 81, 606- 13. 5. Givan, C.V. (1980) Aminotransferases in higher plants. In: The Biochemistry of Plants, Vol 5. (B.J. Miflin, ed.) pp. 329-357. Academic Press, New York. 6. Pate, J.S. (1980) Transport and partitioning of nitrogenous solutes. Ann. Rev. Plant Physiol. 31, 313-340. 7. Patterson, T.G., R. Glenister and T.A. La Rue (1982) Simple estimate of ureides in soybean tissue. Anal. Biochem. 119, 90-95. 8. Herridge, D.F. (1982) Relative abundance of ureides and nitrate in plant tissues of soybean as a quantitative assay of nitrogen fixation. Plant Physiol. 70, 1-6. 9. Miflin, B.J. and P.J. Lea (1982) Ammonia assimilation and amino acid metabolism. In: Encyclopaedia of Plant Physiology, Vol. 14A. (D. Boulter and B. Parthier, eds.) pp. 3-64. SpringerVerlag, Berlin. 10. Mills, W.R., P.J. Lea and B.J. Miflin (1980) Photosynthetic formation of the aspartate family of amino acids in isolated chloroplasts. Plant Physiol. 65, 1166-1171. 11. Thompson, G.A., A.H. Datto, S.H. Mudd and J.G. Giovanelli (1982) Methionine synthesis in Lemna. Plant Physiol. 69, 1077-1083. 12. Murashige, T. and F. Skoog (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 15, 473-497. 13. Bright, S.W.J., P.J. Lea, P. Arruda, N.P. Hall, A.C. Kendall, A.J. Keys, J.S.H. Kueh, M.L. Parker, S.E. Rognes, J.C. Turner, R.M. Wallsgrove and B.J. Miflin (1984) In: The Genetic Manipulation of Plants and its Application to Agriculture (P.J. Lea, G.R. Stewart, eds.), pp. 141-169. Oxford University Press. 14. Rognes, S.E., S.W.J. Bright and B.J. Miflin (1983) Feedback insensitive aspartate kinase isoenzymes in barley mutants resistant to lysine plus threonine. Planta 157, 32-38. 15. Paleg, L.G. and D. Aspinall (1981) The Physiology and biochemistry of drought resistance in plants. Academic Press, Sydney.