Planta (1992) 187:221-223
P l ~ _ t ~ 9 Springer-Verlag 1992
Synthesis of glutamine and glutamate by intact bundle-sheath cells of maize (Zea mays L.) Estela M . Valle* and H a n s W . Heldt**
Institut ffir Biochemie der Pflanze, Untere Karspfile 2, Universit~it Grttingen, W-3400 Grttingen, Federal Republic of Germany Received 25 September; accepted 30 December 1991
Abstract. Intact bundle-sheath cells with functional plasm o d e s m a t a were isolated f r o m leaves of Z e a m a y s L. cv. Mutin, and the capacity o f these cells to synthesize glutamine and glutamate was determined by simulating physiological substrate concentrations in the bathing medium. The results show that glutamine synthetase can operate at full rate in the presence o f added 8 m M ATP. At lower concentrations of A T P a higher rate o f glutamine synthesis was found in the light than in darkness. Glutamate-synthase activity, on the other hand, was strictly light dependent. It appears that in bundle-sheath cells of maize the nitrate-assimilatory capacities o f glutamine synthetase (located mainly in the cytosol) and o f glutamate synthase (located in the stroma) are high enough to meet the demands o f whole maize leaves. Key words: Bundle sheath cells - G l u t a m a t e synthase G l u t a m i n e synthetase - Nitrate assimilation - Z e a
plasmodesmatal openings are permeable to molecules up to a mass of a b o u t 900 D a (Burnell 1988; Weiner et al. 1988), the bundle-sheath cells are accessible to substrates added to the outside medium. In this way it is possible to determine enzyme activities in intact cells by simulating physiological substrate concentrations in the bathing medium. We have studied the capacity of intact bundlesheath cells of maize leaves to produce amino acids as photosynthetic products. Recently, we reported a study concerning alanine synthesis (Valle and Heldt 1991). In the present publication we demonstrate the capacity of maize bundle-sheath cells to produce the amino acids glutamine (Gin) and glutamate (Glu) under conditions in which malate decarboxylation and the Calvin cycle are operating.
M a t e r i a l and methods Zea mays L., cv "Mutin" (Krrbel, G6ttingen, FRG) was grown in
Introduction
In C4 plants, metabolite transfer between the mesophyll and bundle-sheath cells occurs by diffusion through plasm o d e s m a t a (Hatch 1988). By mechanical treatment it is possible to obtain from C4 plants, such as maize, bundlesheath cells o f high functional integrity and metabolic competence, which have retained the plasmodesmatal openings that originally connected them to the mesophyU cells (Weiner et al. 1988; Valle et al. 1989). As these * Present address: Dipartimento di Biopatologia Umana, Sezione di Biologia Cellulare, Policlinico Umberto 1~ Viale Regina Elena 324, 1-00161 Roma, Italy ** To whom correspondence should be addressed Abbreviations: Gin = glutamine; Glu = glutamate; GOGAT = glutamate synthase; GS = glutamine synthetase; 2-OG = 2-oxoglutarate
soil in a glasshouse under natural illumination supplemented with incandescent and cool-white fluorescent lamps to provide 400-700 ~tmol photons - m -z 9s -1 at 25-30/15-18 ~ C day/night temperature (14 h light, 10 h darkness). The preparation of bundlesheath strands and the determination of chlorophyll were as described by Valle and Heldt (1991). The bundle-sheath strands were virtually devoid of mesophyll cells (Valle and Heldt 1991). For the measurement of amino-acid synthesis by bundle-sheath cells, isolated bundle-sheath strands (30-50 ~tg Chl-ml-1) were suspended in a medium containing 0.3 M sorbitol, 20mM N-[2-hydroxy-l,1bis(hydroxymethyl)ethyl]glycine (Tricine)-KOH (pH 8), 4mM MgC12, 10 mM KC1, and further additions as indicated in the table legends. Amino-acid synthesis was started after 2 min incubation at 30~ by adding the substrates at the concentrations indicated, either in darkness or with illumination by a slide projector giving an incident irradiation of approx. 1200 lamol photons - m -2 9s-1. The reaction times were 10 min. In each experiment a control treatment was carried out with an incubation time of 0.5 min. The incubations were terminated by addition of 3 % HCIO4. The incubation mixture was then frozen at - 80~ C for 1 h, centrifuged and the supernatant neutralized with KOH. In the resulting samples, amino-acid concentrations were determined by high-performance liquid chromatography as described by Riens et al. (1991).
222
E.M. Valle and H.W. Hetdt: Glutamine and glutamate synthesis by bundle-sheath cells
Results and discussion T o simulate conditions resembling the situation in vivo the rate o f Gin synthesis from Glu and NH3 was determined in the presence o f malate, bicarbonate and 3-phosphoglycerate to achieve a simultaneous and optimal operation of the Calvin cycle (Table 1). The concentration o f Glu in the bundle-sheath cells o f intact maize leaves has been estimated to be in the order o f 50 m M (We• et al. 1991). In experiments not shown here, with isolated bundle-sheath cells, the rate o f Gin synthesis was not altered by changing the Glu concentration from 20 to 80 mM. As shown in Table 1, the rate o f Gin synthesis was not markedly affected by illumination. In the presence o f 8 m M ATP, essentially the same rate was observed in darkness as in the light. This result clearly shows that Gin synthesis can operate at full rate in bundle-sheath cells at the expense o f externally added ATP. Glutamine synthesis, although at a lower rate, was also found in the presence of 1.6 m M ATP, a concentration which may better resemble the in-vivo situation, or with 5 m M A D P which is probably converted to A T P by adenylate kinase activity. In the plants studied so far (spinach, pea), the rate o f ATP transport into the chloroplasts as catalyzed by the A T P / A D P translocator has been found to be very low (Fltigge and Heldt 1991). On the reasonable assumption that this is also the case in maize bundle-sheath chloroplasts, the high rate o f Gin synthesis supported by external ATP indicates that it is catalyzed by glutamine synthetase (GS, EC 6.3.1.2) located in the cytosol. Many plants, including maize and barley, have been shown to contain two different isoenzymes of GS: GS 1 located in the cytosol and GS2 located in the chloroplast stroma (McNally and Hirel 1983). In whole maize leaves, the activity o f GS t was found to be 45% o f the total GS activity (McNally et al. 1983), and in mesophyll and bundle-sheath cells of maize leaves the proportion of the two isoenzymes was found to be very similar (Yamaya and Oaks 1988). In our experiments, in the presence o f ADP or a lower concentration of ATP (1.6 mM), a higher rate o f Gln synthesis was found in the light than in darkness. This may represent a stimulation of chloroplastic GS (GS2), driven by photophosphorylation resulting from a supplementation of the chloroplastic adenine-nucleotide levels by addition of ADP or ATP. Table 1, Rates of Gin synthesis by intact bundle-sheath cells of maize leaves in darkness and in light. The reaction medium contained 20 mM Glu, 1 mM NH4C1, 2 mM Pi, 10 mM malate, 10 mM KHCO3 and 2.5 mM 3-phosphoglycerate. For further details see Material and methods. Mean values of three experiments_+SD Additions (mM) None ATP(I.6) ATP (8) ADP (5)
lamol Gin. (mg Chl)-t . h- 1 Darkness
Light
3-t- 0.5 61_+ 5 135 • 15 46+ 5
4_+ 0.5 80_+ 6 142• 16 74+ 7
Table 2. Glutamate formation by intact bundle-sheath cells of maize in light and in darkness. The reaction medium contained 2.5 mM Gin, 10 mM KHCO3, 2.5 mM 3-phosphoglycerate, 0.5 mM ADP and 2 mM Pi. For further details see Material and methods. Mean values of three experiments • SD Additions (mM)
tlmol Glu. (mg Chl)-x . h 1 Darkness Light
Malate (10) 2-OG (2.5) 2-OG (2.5) malate (10)
2 • 0.3 2+0.4 6 • 0.9
3 • 0.5 34+__6 27 • 3
The capacity of isolated bundle-sheath cells to produce Glu from Gin and 2-oxogtutarate (2-OG) was also investigated (Table 2). It may be noted that for technical reasons the measurements were performed at a Gln concentration of only 2.5 mM, which is much lower than the concentration (20 mM) estimated for the cytosol of maize leaves (Weiner et al. 1991). For this reason, the rates shown in Table 2 are probably lower than those found in vivo. In the absence o f 2 - O G , the rate o f Glu synthesis from Gln was very low, indicating that the activity of glutaminase was negligible. The 2-OG-dependent Glu synthesis appeared to be strictly light dependent. In maize leaves two different isoenzymes of glutamate synthase ( G O G A T ) have been identified, chloroplastic ferredoxin-dependent G O G A T ( F d - G O G A T , EC 1.4.7.1) and an NADH-dependent enzyme ( N A D H G O G A T , EC 1.4.1.14) (Suzuki et al. 1987). We were unable to detect any NADH-dependent G O G A T activity in our bundle-sheath cells (data not shown). It appears from our results that for Glu synthesis by isolated bundle-sheath cells the electron donor is reduced ferredoxin, which is produced in the light by the reaction catalyzed by the chloroplastic F d - G O G A T . Although in bundlesheath cells the activity of PSII is low (Nakano and Edwards 1987), in view of the fact that in the leaves the rate of nitrate assimilation is only about 5% of the rate of carbon assimilation, it seems sufficient to allow G O G A T to operate at a relatively high rate. This conclusion is supported by the finding that the addition o f malate, yielding N A D P H in the stroma, did not increase the rate of Glu formation. When Gin was replaced by ammonia in the incubation medium, the rate o f 2 - O G dependent Glu synthesis was in both light and darkness below 1 l a m o l ' m g Ch1-1" h -~, indicating that glutamate-dehydrogenase activity was negligible under our incubation conditions. Our results indicate that in isolated bundle-sheath cells of maize the high rate o f NH3 fixation involves an unorthodox distribution of enzymes, with GS located in the cytosol and G O G A T in the plastidial stroma (Fig. 1). The overall process o f 2-OG-dependent NH3 fixation by the bundle-sheath cells will therefore require the transport o f 2-OG, Glu and Gin across the chloroplast envelope. Specific transport of these substances has been demonstrated in spinach chloroplasts (Woo et al. 1987; Yu and Woo 1988). It may be noted that, in maize leaves, nitrate reductase and nitrite reductase were found to be primarily located in the mesophyll cells (Harel et al.
E.M. Valle and H.W. Heldt: Glutamine and glutamate synthesis by bundle-sheath cells "~
Chloroplast stroma
Cytosol NH s
Glu
~);~~')i,
Gin
I, Gin
X
ATP ADP+P 2-OG Glu 4
2Fdred 2Fdox Glu
D
2-OG
#
Glu
Fig. 1. Subcellular localization of Gln and Glu synthesis
1977; N e y r a and H a g e m a n 1978). In order to be utilized in the bundle-sheath cells, the NH3 would have to diffuse from the mesophyll to the bundle-sheath cells. Like maize leaves, barley leaves also contain both the cytosolic (GS1) and chloroplastic (GS2) isoenzymes of GS (McNally et al. 1983). Barley mutants totally lacking the chloroplastic isoenzyme GS2, were shown to grow healthily under high CO2 and low 02, conditions which prevent photorespiration (WaUsgrove et al. 1987). It has been concluded f r o m these results that in barley leaves the cytosolic isoenzyme o f GS is sufficient for nitrate assimilation during normal growth. Apparently this also holds for maize leaves. Assuming an N : C assimilation ratio of 1 : 20, at a photosynthesis rate of 200 gmol 9 (mg C h l ) - 1 . h - 1 the rate of nitrate assimilation of a maize leaf would be a b o u t 1 0 g m o l - ( m g Chl) -1 . h -1. The rates o f Gin and Glu synthesis shown in Tables 1 and 2 have been related to chlorophyll contained in the bundlesheath ceils, Since, in maize leaves, the bundle-sheath cells contain only 30 % of the total leaf chlorophyll (Valle and Heldt 1991), the data o f Tables 1 and 2 have to be divided by 3.3 for comparison with the rate of total leaf nitrate assimilation estimated above. Such a comparison shows that in intact bundle-sheath cells the rates of A T P dependent Gin synthesis and the rate of Glu synthesis dependent on added Gin are sufficient for the requirements of nitrate assimilation. Apparently, in bundlesheath cells of maize the capacity of the pathway shown in Fig. 1 is high enough to meet the demands of nitrate assimilation o f whole maize leaves. This work was supported by the Bundesminister ffir Forschung und Technologie (0319296A). We thank Mr. Bernd Raufeisen for the art work of Fig. 1.
223
References
Burnell, J.N. (1988) An enzymic method for measuring the molecular weight exclusion limit of plasmodesmata of bundle sheath ceils of C4 plants. J. Exp. Bot. 39, 1575-1580 Fliigge, U.-I., Heldt, H.W. (1991) Metabolite translocators of the chloroplast envelope. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, 129-144 Harel, E., Lea, P.J., Miflin, B.J. (1977) The localisation of enzymes of nitrogen assimilation in maize leaves and their activities during greening. Planta 134, 195-200 Hatch, M.D. (1988) Ca photosynthesis: a unique blend of modified biochemistry, anatomy and ultrastructure. Biochim. Biophys. Acta 895, 81-106 McNally, S., Hirel, B. (1983) Glutamine synthetase isoforms in higher plants. Physiol. Veg. 21, 761-774 McNally, S., Hirel, B., Gadal, P., Mann, A.F., Stewart, G.R. (1983) Glutamine synthetases of higher plants. Evidence for a specific isoform content related to their possible physiological role and their compartmentation within the leaf. Plant Physiol. 72, 22-25 Nakano, Y., Edwards, G.E. (1987) Hill reaction, hydrogen peroxide scavenging, and ascorbate peroxidase activity of mesophyll and bundle-sheath chloroplasts of NADP-malic enzyme type C4 species. Plant Physiol. 85, 294-298 Neyra, C.A., Hageman, R.H. (1978) Pathway for nitrate assimilation in corn (Zea mays L.) leaves. Plant Physiol. 62, 618-621 Riens, B., Lohaus, G., Heineke, D., Heldt, H.W. (1991) Amino acid and sucrose content determined in the cytosolic, chloroplastic and vacuolar compartments and in the phloem sap of spinach leaves. Plant Physiol. 97, 227-233 Suzuki, A., Andet, C., Oaks, A. (1987) Influence of light on the ferredoxin-dependent glutamate synthase in maize leaves. Plant Physiol. 84, 578-581 Valle, E.M., Craig, S., Hatch, M.D., Heldt, H.W. (1989) Permeability and ultrastructure of bundle-sheath cells isolated from C4 plants: structure-function studies and the role of plasmodesmata. Bot. Acta 102, 276-282 Valle, E.M., Heldt, H.W. (1991) Alanine synthesis by bundle sheath cells of maize. Plant Physiol. 95, 839-845 Wallsgrove, R.M., Turner, J.C., Hall, N.P., Kendall, A.C., Bright, S.W.J. (1987) Barley mutants lacking chloroplast glutamine synthetase. Biochemical and genetic analysis. Plant Physiol. 83, 155-158 Weiner, H., Burnell, J.N., Woodrow, J.E., Heldt, H.W., Hatch, M.D. (1988) Metabolite diffusion into bundle-sheath cells from C4 plants: relation to C4 photosynthesis and plasmodesmatal function. Plant Physiol. 88, 815-822 Weiner, H., Blechschmidt-Schneider, S., Mohme, H., Eschrich, W., Heldt, H.W. (1991) Phloem transport of amino acids. Comparison of amino acids contents of maize leaves and of the sieve tube exudate. Plant Physiol. Biochem. 29, 1-5 Woo, K.C., Fliigge, U.-I., Heldt, H.W: (1987) A two-translocator model for the transport of 2-oxoglutarate and glutamate in chloroplasts during ammonia assimilation in the light. Plant Physiol. 84, 624-632 Yamaya, T., Oaks, A. (1988) Distribution of two isoforms of glutamine synthetase in bundle sheath and mesophyll cells of corn leaves. Physiol. Plant 72, 23-28 Yu, J., Woo, K.C. (1988) Glutamine transport and the role of the glutamine translocator in chloroplasts. Plant Physiol. 88, 1048-1054
P l ~ _ t ~ 9 Springer-Verlag 1992
Synthesis of glutamine and glutamate by intact bundle-sheath cells of maize (Zea mays L.) Estela M . Valle* and H a n s W . Heldt**
Institut ffir Biochemie der Pflanze, Untere Karspfile 2, Universit~it Grttingen, W-3400 Grttingen, Federal Republic of Germany Received 25 September; accepted 30 December 1991
Abstract. Intact bundle-sheath cells with functional plasm o d e s m a t a were isolated f r o m leaves of Z e a m a y s L. cv. Mutin, and the capacity o f these cells to synthesize glutamine and glutamate was determined by simulating physiological substrate concentrations in the bathing medium. The results show that glutamine synthetase can operate at full rate in the presence o f added 8 m M ATP. At lower concentrations of A T P a higher rate o f glutamine synthesis was found in the light than in darkness. Glutamate-synthase activity, on the other hand, was strictly light dependent. It appears that in bundle-sheath cells of maize the nitrate-assimilatory capacities o f glutamine synthetase (located mainly in the cytosol) and o f glutamate synthase (located in the stroma) are high enough to meet the demands o f whole maize leaves. Key words: Bundle sheath cells - G l u t a m a t e synthase G l u t a m i n e synthetase - Nitrate assimilation - Z e a
plasmodesmatal openings are permeable to molecules up to a mass of a b o u t 900 D a (Burnell 1988; Weiner et al. 1988), the bundle-sheath cells are accessible to substrates added to the outside medium. In this way it is possible to determine enzyme activities in intact cells by simulating physiological substrate concentrations in the bathing medium. We have studied the capacity of intact bundlesheath cells of maize leaves to produce amino acids as photosynthetic products. Recently, we reported a study concerning alanine synthesis (Valle and Heldt 1991). In the present publication we demonstrate the capacity of maize bundle-sheath cells to produce the amino acids glutamine (Gin) and glutamate (Glu) under conditions in which malate decarboxylation and the Calvin cycle are operating.
M a t e r i a l and methods Zea mays L., cv "Mutin" (Krrbel, G6ttingen, FRG) was grown in
Introduction
In C4 plants, metabolite transfer between the mesophyll and bundle-sheath cells occurs by diffusion through plasm o d e s m a t a (Hatch 1988). By mechanical treatment it is possible to obtain from C4 plants, such as maize, bundlesheath cells o f high functional integrity and metabolic competence, which have retained the plasmodesmatal openings that originally connected them to the mesophyU cells (Weiner et al. 1988; Valle et al. 1989). As these * Present address: Dipartimento di Biopatologia Umana, Sezione di Biologia Cellulare, Policlinico Umberto 1~ Viale Regina Elena 324, 1-00161 Roma, Italy ** To whom correspondence should be addressed Abbreviations: Gin = glutamine; Glu = glutamate; GOGAT = glutamate synthase; GS = glutamine synthetase; 2-OG = 2-oxoglutarate
soil in a glasshouse under natural illumination supplemented with incandescent and cool-white fluorescent lamps to provide 400-700 ~tmol photons - m -z 9s -1 at 25-30/15-18 ~ C day/night temperature (14 h light, 10 h darkness). The preparation of bundlesheath strands and the determination of chlorophyll were as described by Valle and Heldt (1991). The bundle-sheath strands were virtually devoid of mesophyll cells (Valle and Heldt 1991). For the measurement of amino-acid synthesis by bundle-sheath cells, isolated bundle-sheath strands (30-50 ~tg Chl-ml-1) were suspended in a medium containing 0.3 M sorbitol, 20mM N-[2-hydroxy-l,1bis(hydroxymethyl)ethyl]glycine (Tricine)-KOH (pH 8), 4mM MgC12, 10 mM KC1, and further additions as indicated in the table legends. Amino-acid synthesis was started after 2 min incubation at 30~ by adding the substrates at the concentrations indicated, either in darkness or with illumination by a slide projector giving an incident irradiation of approx. 1200 lamol photons - m -2 9s-1. The reaction times were 10 min. In each experiment a control treatment was carried out with an incubation time of 0.5 min. The incubations were terminated by addition of 3 % HCIO4. The incubation mixture was then frozen at - 80~ C for 1 h, centrifuged and the supernatant neutralized with KOH. In the resulting samples, amino-acid concentrations were determined by high-performance liquid chromatography as described by Riens et al. (1991).
222
E.M. Valle and H.W. Hetdt: Glutamine and glutamate synthesis by bundle-sheath cells
Results and discussion T o simulate conditions resembling the situation in vivo the rate o f Gin synthesis from Glu and NH3 was determined in the presence o f malate, bicarbonate and 3-phosphoglycerate to achieve a simultaneous and optimal operation of the Calvin cycle (Table 1). The concentration o f Glu in the bundle-sheath cells o f intact maize leaves has been estimated to be in the order o f 50 m M (We• et al. 1991). In experiments not shown here, with isolated bundle-sheath cells, the rate o f Gin synthesis was not altered by changing the Glu concentration from 20 to 80 mM. As shown in Table 1, the rate o f Gin synthesis was not markedly affected by illumination. In the presence o f 8 m M ATP, essentially the same rate was observed in darkness as in the light. This result clearly shows that Gin synthesis can operate at full rate in bundle-sheath cells at the expense o f externally added ATP. Glutamine synthesis, although at a lower rate, was also found in the presence of 1.6 m M ATP, a concentration which may better resemble the in-vivo situation, or with 5 m M A D P which is probably converted to A T P by adenylate kinase activity. In the plants studied so far (spinach, pea), the rate o f ATP transport into the chloroplasts as catalyzed by the A T P / A D P translocator has been found to be very low (Fltigge and Heldt 1991). On the reasonable assumption that this is also the case in maize bundle-sheath chloroplasts, the high rate o f Gin synthesis supported by external ATP indicates that it is catalyzed by glutamine synthetase (GS, EC 6.3.1.2) located in the cytosol. Many plants, including maize and barley, have been shown to contain two different isoenzymes of GS: GS 1 located in the cytosol and GS2 located in the chloroplast stroma (McNally and Hirel 1983). In whole maize leaves, the activity o f GS t was found to be 45% o f the total GS activity (McNally et al. 1983), and in mesophyll and bundle-sheath cells of maize leaves the proportion of the two isoenzymes was found to be very similar (Yamaya and Oaks 1988). In our experiments, in the presence o f ADP or a lower concentration of ATP (1.6 mM), a higher rate o f Gln synthesis was found in the light than in darkness. This may represent a stimulation of chloroplastic GS (GS2), driven by photophosphorylation resulting from a supplementation of the chloroplastic adenine-nucleotide levels by addition of ADP or ATP. Table 1, Rates of Gin synthesis by intact bundle-sheath cells of maize leaves in darkness and in light. The reaction medium contained 20 mM Glu, 1 mM NH4C1, 2 mM Pi, 10 mM malate, 10 mM KHCO3 and 2.5 mM 3-phosphoglycerate. For further details see Material and methods. Mean values of three experiments_+SD Additions (mM) None ATP(I.6) ATP (8) ADP (5)
lamol Gin. (mg Chl)-t . h- 1 Darkness
Light
3-t- 0.5 61_+ 5 135 • 15 46+ 5
4_+ 0.5 80_+ 6 142• 16 74+ 7
Table 2. Glutamate formation by intact bundle-sheath cells of maize in light and in darkness. The reaction medium contained 2.5 mM Gin, 10 mM KHCO3, 2.5 mM 3-phosphoglycerate, 0.5 mM ADP and 2 mM Pi. For further details see Material and methods. Mean values of three experiments • SD Additions (mM)
tlmol Glu. (mg Chl)-x . h 1 Darkness Light
Malate (10) 2-OG (2.5) 2-OG (2.5) malate (10)
2 • 0.3 2+0.4 6 • 0.9
3 • 0.5 34+__6 27 • 3
The capacity of isolated bundle-sheath cells to produce Glu from Gin and 2-oxogtutarate (2-OG) was also investigated (Table 2). It may be noted that for technical reasons the measurements were performed at a Gln concentration of only 2.5 mM, which is much lower than the concentration (20 mM) estimated for the cytosol of maize leaves (Weiner et al. 1991). For this reason, the rates shown in Table 2 are probably lower than those found in vivo. In the absence o f 2 - O G , the rate o f Glu synthesis from Gln was very low, indicating that the activity of glutaminase was negligible. The 2-OG-dependent Glu synthesis appeared to be strictly light dependent. In maize leaves two different isoenzymes of glutamate synthase ( G O G A T ) have been identified, chloroplastic ferredoxin-dependent G O G A T ( F d - G O G A T , EC 1.4.7.1) and an NADH-dependent enzyme ( N A D H G O G A T , EC 1.4.1.14) (Suzuki et al. 1987). We were unable to detect any NADH-dependent G O G A T activity in our bundle-sheath cells (data not shown). It appears from our results that for Glu synthesis by isolated bundle-sheath cells the electron donor is reduced ferredoxin, which is produced in the light by the reaction catalyzed by the chloroplastic F d - G O G A T . Although in bundlesheath cells the activity of PSII is low (Nakano and Edwards 1987), in view of the fact that in the leaves the rate of nitrate assimilation is only about 5% of the rate of carbon assimilation, it seems sufficient to allow G O G A T to operate at a relatively high rate. This conclusion is supported by the finding that the addition o f malate, yielding N A D P H in the stroma, did not increase the rate of Glu formation. When Gin was replaced by ammonia in the incubation medium, the rate o f 2 - O G dependent Glu synthesis was in both light and darkness below 1 l a m o l ' m g Ch1-1" h -~, indicating that glutamate-dehydrogenase activity was negligible under our incubation conditions. Our results indicate that in isolated bundle-sheath cells of maize the high rate o f NH3 fixation involves an unorthodox distribution of enzymes, with GS located in the cytosol and G O G A T in the plastidial stroma (Fig. 1). The overall process o f 2-OG-dependent NH3 fixation by the bundle-sheath cells will therefore require the transport o f 2-OG, Glu and Gin across the chloroplast envelope. Specific transport of these substances has been demonstrated in spinach chloroplasts (Woo et al. 1987; Yu and Woo 1988). It may be noted that, in maize leaves, nitrate reductase and nitrite reductase were found to be primarily located in the mesophyll cells (Harel et al.
E.M. Valle and H.W. Heldt: Glutamine and glutamate synthesis by bundle-sheath cells "~
Chloroplast stroma
Cytosol NH s
Glu
~);~~')i,
Gin
I, Gin
X
ATP ADP+P 2-OG Glu 4
2Fdred 2Fdox Glu
D
2-OG
#
Glu
Fig. 1. Subcellular localization of Gln and Glu synthesis
1977; N e y r a and H a g e m a n 1978). In order to be utilized in the bundle-sheath cells, the NH3 would have to diffuse from the mesophyll to the bundle-sheath cells. Like maize leaves, barley leaves also contain both the cytosolic (GS1) and chloroplastic (GS2) isoenzymes of GS (McNally et al. 1983). Barley mutants totally lacking the chloroplastic isoenzyme GS2, were shown to grow healthily under high CO2 and low 02, conditions which prevent photorespiration (WaUsgrove et al. 1987). It has been concluded f r o m these results that in barley leaves the cytosolic isoenzyme o f GS is sufficient for nitrate assimilation during normal growth. Apparently this also holds for maize leaves. Assuming an N : C assimilation ratio of 1 : 20, at a photosynthesis rate of 200 gmol 9 (mg C h l ) - 1 . h - 1 the rate of nitrate assimilation of a maize leaf would be a b o u t 1 0 g m o l - ( m g Chl) -1 . h -1. The rates o f Gin and Glu synthesis shown in Tables 1 and 2 have been related to chlorophyll contained in the bundlesheath ceils, Since, in maize leaves, the bundle-sheath cells contain only 30 % of the total leaf chlorophyll (Valle and Heldt 1991), the data o f Tables 1 and 2 have to be divided by 3.3 for comparison with the rate of total leaf nitrate assimilation estimated above. Such a comparison shows that in intact bundle-sheath cells the rates of A T P dependent Gin synthesis and the rate of Glu synthesis dependent on added Gin are sufficient for the requirements of nitrate assimilation. Apparently, in bundlesheath cells of maize the capacity of the pathway shown in Fig. 1 is high enough to meet the demands of nitrate assimilation o f whole maize leaves. This work was supported by the Bundesminister ffir Forschung und Technologie (0319296A). We thank Mr. Bernd Raufeisen for the art work of Fig. 1.
223
References
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