Planta 9 by Springer-Verlag 1980

Planta 150, 299-302 (1980)

Effect of Chemical Inhibitors of Photorespiration on Nitrogenase Activity in Nodulated Alfalfa Plants Eulogio J. Bedmar and Jos60livares Departamento de Microbiologia, Estacidn Experimentaldel Zaidin. C.S.I.C., Prof. Albareda, 1, Granada, Spain

Abstract. Nitrogen fixation (measured as acetylene

reduction) by whole nodulated alfalfa plants was stimulated when the plants were treated with isonicotinic acid hydracide (INH) and glyoxylate, both inhibitors of the glycolate pathway of carbohydrate metabolism, at concentrations of 300 and 100 mM, respectively. Reducing energetic loses caused by photorespiration results in an increase in the symbiotic nitrogen fixation. Key words. Glyoxylate Isonicotinic acid hydracide M e d i c a g o - Nitrogen fixation Photorespiration Rhizobium.

Introduction

Recent studies have identified photosynthate availability to the nodules root legumes as a major limitation of symbiotic nitrogen fixation (Hardy and Havelka 1975, 1976; Quebedeaux et al. 1975; Hardy et al. 1977; Ogren 1977; Bethlenfalvay and Phillips 1977 a, b). This process requires a continued supply of photosynthate for nitrogenase activity that is to a high degree energy dependent. In addition, photosynthate must also be provided with reductant power and carbon skeletons to incorporate the ammonia produced. Net CO2 reduction during photosynthesis is diminished by the rapid synthesis of glycolate. Photorespiration involves the oxidation of this glycolate to glyoxylate which is further converted either to formic acid and CO2 or to glycine to be metabolized to serine with the release of NH3 and CO2 (Tolbert 1971; Zelitch 1971, 1975a, 1976). In most crops, at least half the CO2 assimilated during photosynthesis is lost by the photorespiratory Abbreviation." INH =isonicotinic acid hydracide

C O 2 (Zelitch 1975 b; Kelly et al. 1976). Photorespiration can be reduced by increasing the CO2 concentration in the atmosphere (Hardy and Havelka 1976) and by eventually using chemical inhibitors of the glycolate pathway. Between them, glycidate, glyoxylate, and I N H have been widely studied. Glycidate (2,3 epoxy propionate) has been used to inhibit the conversion of glyoxilic acid to glycine in illuminated tobacco leaf discs (Zelitch 1974) and in isolated spinach chloroplasts (Chollet 1976). Glyoxylate, an intermediate in the glycolate pathway, normally formed in leaves from the action of glycolateoxidase on glycolate has been shown to inhibit glycolate synthesis and photorespiration in tobacco leaf discs (Oliver and Zelitch 1977; Oliver 1978a, b) and in soybean leaves (Hunt and Ogren 1979). I N H prevents the formation of serine from glycine, the reaction which releases photorespiratory CO2 in C h l o r e l l a (Pritchard et al. 1962, 1963) and in leaves of higher plants (Asada et al. 1965; Mifflin et al. 1966; Zelitch 1973; Servaites and Ogren 1977; Oliver 1979). The aim of this study has been to learn the effect of these chemicals which inhibit some reaction involved in the glycolate pathway on the nitrogen fixation in whole nodulated alfalfa plants.

Material and Methods Plant Culture. Culture Conditions. Seedlings of two-day-old alfalfa plants (Medieago sativa L.) (from seeds surface-sterilized with HgCI2, germinated on filter paper in Petri dishes) were put on filter paper strips in 20.200 mm glass tubes (5 plants per tube) containing 10 ml nitrogen-freemineralsolution (Evans et al. 1972). The plants were maintained in a controlled environment chamber (day/night temperature 25/19~ C; photoperiod 16/8h light/dark). Sylvania Grolux tubes were used as the light source. Seven-day-old plants were inoculated with 1 ml of a bacterial suspension (approximately 109 cells ml-1) of Rhizobium meliloti wild strain Rm 11 (Bedmar and Olivares 1979).

0032-0935/80/0150/0299/$01.00

300

E.J. Bedmar and J. Olivares: Nitrogenase Activity and Photorespiration

Experimental Procedures. Nitrogenase activity was determined every three days from the appearance of the first visible nodule (approximately 7 days after inoculation) until the plants were 50 days old. To test the chemicals, the plants were taken out of the tubes and homogeneously sprayed with 100, 200, 300, and 400 mM INH (Sigma) or 25, 50, 100, and 200 mM glyoxylate (Sigma) solutions in distilled water with added smfactant (Bayer). Afterwards, the plants were again put into 15.150 mm glass tubes and stoppered with serum-bottle caps and again put under adequate light and temperature, In all experiments, 10 tubes per treatement were used. Ten test tubes remained as the control after spraying plants with distilled water supplemented with surfactant. Nitrogenase Activity Determination. Ten per cent of the internal atmosphere was removed from the corresponding tubes and replaced with the same volume of acetylene. Gas samples of 0.2 ml were removed at 5, 30, 60, 90, 120, 150, and 180 rain and assayed for ethylene by gas chromatography (Perkin-Elmer F-33 ; 80-mesh Poropak N; 50-cm column; 100 ~ nitrogen as carrier gas at

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,'o Results

Figure 1 shows the profile of the nitrogenase activity curve of nodulated alfalfa plants for a period of 50 days under the experimental conditions used. A maximum of nitrogenase activity appears when the culture is becoming stabilized. This evolution has been followed to find the optimal moment at which plants give a good response on being treated with the chemicals to be tested. There are two clear phases concerning nitrogenase activity. At the beginning it increases up to 30-35 days to fall later during the last half of the growth cycle. The nodule number and dry weight of these plants are also shown in Fig. 1. The data presented in Table 1 indicate that plants 30-35 days old respond better to the treatment with chemicals than younger ones. It could be thought that the appropiate time seems to correspond to the decrease in nitrogenase activity, possibly because the photosynthate provision decreases in this phase and there is not enough carbohydrate to maintain the enzyme activity rate. This Table also shows that when plants to be sprayed come directly from the dark, the effect of the chemicals is greater than when plants have previously been kept in the light for several hours. After a period of darkness, photosynthate is exhausted and the photosynthate formed at the beginning of the light period is not sufficient to support plant requeriments and a high nitrogenase activity. According to the data in this Table, plants 30 35 days old coming directly from the dark have been used for all experiments carried out to test different chemicals and concentrations.

Treatments with INH, Glyoxylate and Glycidate. Relative nitrogenase activity values (taking controls as

io

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time(d)

Fig. 1. Specific nitrogenase activity (e), nodule number (o) and dry weight ( x ) of nodulated alfalfa plants found under conditions described in the text

Table 1. Specific nitrogenase activity of nodulated alfalfa plants treated with INH and glyoxylate under different experimental conditions (values_+ average error in ten tubes)

Conditions

Plants coming

Specific nitrogenase activity (nmol C2H4"tube- 1 .h- 1) INH

Glyoxylate

Controls

706.5+31.3

987.7_+34.4

454.5_+21.3

from dark Plants coming 532.5_+22.5 669.0+_26.2 451.3 -+16.8 from light Plants younger 113.5-+12.1 109.4+7.6 100.0+9.3 than 30-35 days Plants o l d e r 706.5+_31.3 987.7_+34.4 454.5_+21.3 than 30-35 days

100) found in plants treated with I N H and glyoxylate at different times and concentrations are presented in Table 2. It can be deduced that 100 mM glyoxylate is the optimal concentration for spraying plants to recieve the maximum increase in enzyme activity as compared to the controls. The absolute values for one of the concentrations of this chemical applied and of the control have been graphically represented in Fig. 2, to give a better idea of the differences found in nitrogenase activity. It can also be seen that the effect performed by the chemical does not last for a long time. The specific

E.J. Bedmar and J. OIivares: Nitrogenase Activity and Photorespiration

Table 2. Relative nitrogenase activity of nodulated alfalfa plants treated with INH and glyoxylate at different times and concentrations, Values are expressed as percentage of nitrogenase activity referred to controls. They are mean of ten tubes Time (rain) 5

30

60

90

I20

301

nitrogenase activity found are lower than those obtained with 100 mM glyoxylate. Preliminary experiments carried out using glycidate (kindly provided by R. Chollet) have shown that this chemical acts at a lower concentration than glyoxylate. Unfortunately the amount of glycidate available was not sufficient to complete the study.

I N H (mM) 100 200 300 400

-3.24 -2.28 1.33 6.05

-3.14 26.87 35.87 10.38

-3.19 41.95 57.02 16.58

-3.17 22.35 27.43 13.97

-3.04 20.67 33.09 9.06

-5.71 2.85 17.14 0.00

-10.29 10.75 39.24 4.65

39.11 25.78 51.23 26.72

27.79 27.11 87.36 25.90

10.10 26.03 62.61 24.17

Glyoxylate (raM) 25 50 100 200

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5

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30

60

90

120

150

180

Time(m) Fig. 2. Nitrogenase activity in plants 35 days old. After being sprayed with 100 m M glyoxylate solution (e) and controls (o).

(Values_+average error in 10 tubes) enzyme activity decreases in plants treated two hours after application to become the same as that of the control ones. Similar results have been found using INH. In this case the optimal concentration appears higher than that for glyoxylate. In spite of this, a higher concentration is required for INH, and the increments of

Discussion

Since photorespiration reduces the efficiency of photosynthesis with no demonstrable profit, some authors have considered it to be an unnecesary and wasteful process (Chollet and Ogren 1975; Ogren 1975; Ogren 1976; Zelitch 1975 a). Thus, the elimination of photorespiration by genetic or chemical means is thought to be a promising approach for increasing the productivity of plants. In the case of legumes, the inhibition of photorespiration might lead to an increase in the nitrogen fixation rate since this process is closely related to photosynthesis. A considerable proportion of fixed CO2 travels from the leaves to the roots to cover nodule requirements (Pate 1976). Any action directed to increase net photosynthesis will act positively on root nodule development and functions. The importance of photosynthate provision is especially marked during pod-filling, when the necessities for carbohydrate are higher in comparison to nitrogen fixation. Although in the study presented plants never reached the flowering state, conditions derived from the nature of the experiments caused plant growth to become constant and nitrogenase activity in nodules to decrease (approximately 30 days after sowing). In this phase, the response of this enzyme activity is considerably higher than when plants can supply the photosynthate requirements. On the other hand, the effect is more evident when plants are treated at the beginning of the light period; during the dark period they have almost exhausted their nodule carbohydrate reserves. In any case, the effect shown by the use of chemicals is short. It does not last for more than two hours. After this time enzyme activity declines and control and treated plants show similar specific activity. Although the composition of the atmosphere around the plants varies when tubes are sealed for nitrogenase activity determinations the same modification occurs in the control tubes too. So an effect derived from an enrichment of CO2 (Hardy and Havelka 1976) is not the cause of the results obtained. From the data it can be deduced that the more active the chemical inhibitor of photorespiration is the higher is the degree of increasing nitrogenase ac-

302

E.J. Bedmar and J. Olivares: Nitrogenase Activity and Photorespiration

tivity found. In experiments carried out using INH (Zelitch 1973), glyoxylate (Oliver and Zelitch 1977) and glycidate (Chollet 1976) on leave pieces, the optimal concentrations reported have been 10 mM, 525 mM, and 3 raM, respectively. Referring to the effect on nitrogenase activity, the optimal concentrations found have been for INH and glyoxylate 300 and 100 mM, and we assume that glycidate, according to previous data, would be more active. This study was supported by a grant of the Fundaci6n Ram6n Areces (1978).

References Asada, K., Saito, K., Kitoh, S., Kasai, Z. (1965) Photosynthesis of glycine and serine in green plants. Plant Cell Physiol. 6, 47-59 Bedmar, EJ., Olivares, J. (1979) Nitrogen fixation (acetylene reduction) by free-living Rhizobium melitoti. Curr. Microbiol. 2, 11 13 Bethlenfalvay, G.J., Phillips, D.A. (1977 a) Ontogenetic interaction between photosynthesis and symbiotic nitrogen fixation in legumes. Plant Physiol. 60, 419-421 Bethlenfalvay, G.J., Phillips, D.A. (1977b) Photosynthesis and nitrogen fixation in Phaseolus vulgaris, L. In : Genetic engineering for nitrogen fixation, pp. 401-408. Plenum, N.Y. Basic Life Sci. Chollet, R. (1976) Effect of glycidate on glycolate formation and photosynthesis in isolated spinach chloroplasts. Plant Physiol. 57, 239-240 Chollet, R., Ogren, W.L. (1975) Regulation of photorespiration in C3 and C4 species. Bot. Rev. 41, 137-179 Evans, H.J., Koch, B., Klucas, R. (1972) Preparation of nitrogenase from nodules and separation into components. In: Methods in enzymology, vol XXIV, pt B, pp. 470477 Hardy, R.W.F., Havelka, U.D. (1975) Nitrogen fixation research: a key to world food? Science 188, 633-643 Hardy, R.W.F., Havelka, U.D. (1976) Photosynthate as a major factor limiting nitrogen fixation by field-grown legumes with emphasis on soybeans. In: SymbiotiC nitrogen fixation in plants, vol VII, pp. 421-439, Nutman P.S., ed. Int. Biol. Programme Series. Cambridge University Press, London Hardy, R.W.F., Havelka, U.D., Quebedeaux, B. (1977) Increasing crop productivity: the problem, strategies, approach and selected rate-limitations related to photosynthesis. In: Proc. Fourth Int. Congress on Photosynthesis, pp. 695-719, Hall, D.O., Coombs, J., Goodwin, T.W., eds. The Biochemical Society Hunt, L.D., Ogren, W.L. (1979) Glyoxylate stimulation of photosynthesis in isolated soybean leaf mesophyll-cells. Plant Physiol. (Suppl.) 63, 38 Kelly, G.J., Latzko, E., Gibbs, M. (1976) Regulatory aspects of photosynthetic carbon metabolism. Annu. Rev. Plant Physiol. 27, 181 205 Mifflin, B.J., Marker, A.F.H., Whittingham, C.P. (1966) The metabolism of glycine and glycolate by pea leaves in relation to photosynthesis. Biochim. Biophys. Acta 120, 266~73

Ogren, W.L. (1975) Control of photorespiration in soybean and maize. In: Environmental and biological control of photosynthesis, pp. 45-52, R. Marcelle, W. Junk, eds. The Hague Ogren, W.L. (1976) Search for higher plants with modifications of the reductive pentose phosphate pathway of CO2 assimilation. In: CO 2 metabolism and plant productivity, pp. 19 29, Burris, R.H., Black, C.C., eds. University Park Press, Baltimore Ogren, W.L. (1977) Increasing carbon fixation by crop plant. In: Proc. Fourth Int. Congress on Photosynthesis pp. 721-733, Hall, D.O., Coombs, J., Goodwin, T.W., eds. The Biochemical Society Oliver, D.J. (1978a) Effect of glyoxylate on the sensitivity of net photosynthesis to oxygen (The Warburg effect) in tobacco. Plant Physiol. 62, 938-940 Oliver, D.J. (1978b) Decrease in inhibition of net photosynthesis by 02 in presence of glyoxylate. Plant Physiol. (Suppl.) 61, 7 Oliver, D.J. (1979) Photorespiratory glycolate and glycine metabolism by isolated soybean cells in the dark. Plant Physiol. (Suppl.) 63, 154 Oliver, D.J., Zelitch, I. (1977) Increasing photosynthesis by inhibiting photorespiration with glyoxylate. Science 196, 1450-1451 Pate, J.S. (1976) Physiology of the reaction of nodulated legumes to environment. In: Symbiotic nitrogen fixation in plants, vol VII, pp. 335-360, Nutman, P.S. ed. Int. Biol. Programme Series, Cambridge University Press, London Pritchard, G., Griffin, W., Whittimgham, C.P. (1962) The effect of CO2 concentration, light intensity and isonicotinyl hydracide on the photosynthetic production of glycolate acid by Chlorella. J. Exp. Bot. 13, 176-184 Pritchard, G., Whittimgham, C.P., Griffin, W. (1963) The effect of isonicotinyl hydracide on the photosynthetic incorporation of radioactive COz into ethanol-soluble compounds of ChlorefIa. J. Exp. Bot. 14, 281~89 Quebedeaux, B., Havelka, U.D., Livak, K.L., Hardy, R.W.F. (1975) Effect of altered pO2 in the aerial part of soybean on symbiotic nitrogen fixation. Plant Physiol. 56, 761-764 Servaites, J.C., Ogren, W.L. (1977) Chemical inhibition of the glycolate pathway in soybean leaf cells. Plant Physiol. 60, 461466 Tolbert, N.E. (1971) Microbodies-peroxisomes and glyoxysomes. Annu. Rev. Plant Physiol. 22, 45 69 Zelitch, I. (1971)Photosynthesis, Photorespiration and Plant Productivity. Academic Press, New York Zelitch, I. (1973) Alternate pathway of glycolate synthesis in tobacco and maize leaves in relation to rates of photorespiration. Plant Physiol. gl, 299-305 Zelitch, I. (1974) The effect of glycidate, an inhibitor of glycolate synthesis on photorespiration and net photosynthesis. Arch. Biochem. Biophys. 163, 367-377 Zelitch, I. (1975a) Improving the efficiency of photosynthesis. Science 188, 626-633 Zelitch, I. (1975b) Pathway of carbon fixation in green plants. Annn. Rev. Biochem. 44, 123-145 Zelitch, I. (1976) Biochemical and genetic control of photorespiration. In: CO 2 metabolism and plant productivity, pp. 343 358, Burris, R.H., Black, C.C., eds. University Park Press, Baltimore

Received 11 April; accepted 25 June 1980