effect of nitrogen nutrition on nitrate and nitrite reductase, glutamine synthetase, glutamate...

Plant Science, 84 (1992) 145-152 145 Elsevier Scientific Publishers Ireland Ltd.

Effect of nitrogen nutrition on nitrate and nitrite reductase, glutamine synthetase, glutamate synthase and glutamate

dehydrogenase in the CAM plant Kalanch6e lateritia Engl.

Isabel Santos and R. Salema

Institute of Botany and Centre of Experimental Cytology (INIC), University of Porto, Rua do Campo Alegre, 823, P-4100 Porto (Portugal)

(Received November 29th, 1991; revision received April 10th, 1992; accepted April 10th, 1992)

The activity of the enzymes involved in nitrogen metabolism NR, NiR, GS, GOGAT and GDH was studied in the CAM Kalanch6e lateritia using nitrogen starved plants later grown under three different nitrogen nutritional conditions (N, N/5, N/10 plant groups). After l day of the nitrogen resupply the NR activity increased similarly in all groups; the rise was higher after 7 days and significantly different from one group to the other. After 1 day the NiR activity increased in all plants with close values in N and N/5 and the lowest one in the N/10 group and with ongoing treatment an increase of activity was only observed in N plants. GS and Fd-GOGAT behaved similarly to each other, their activities increase with nitrogen increase; the NADH-GOGAT behaved in the opposite way and moreover its activity fluctuates along the day. NADH- and NADPH-GDH also fluctuate however the maximum activity found for the former isoenzyme was not affected by nitrogen level whereas the activity of the latter was lowest in N/10 plants. The data showed that in this CAM plant the activity of all enzymes was affected by nitrogen nutrition with exception of the mitochondrial NADH-GDH.

Key words: nitrate reductase; nitrite reductase; glutamine synthetase; glutamate synthase; glutamate dehydrogenase; CAM Kalanch6e lateritia

Introduction

Ni t rogen is the minera l nut r ien t tha t most l imits p lant g rowth and under no rma l field condi t ions is

mainly avai lable in the fo rm of ni t rate . The up take o f this ion is med ia ted by t ranspor te rs , some o f them induced by the ion i tself [1,2] and the enzymes ca ta lyz ing its reduc t ion to a m m o n i a (ni t rate

and nitr i te reductase) are also n i t ra te inducible , processes demand ing complex mechanisms to regulate the expression o f the genes involved (Refs. 3, 4 and references therein). The n i t rogen

ass imi la tory pa thway involves var ious enzymes

Correspondence to: Isabel Santos, Centre of Experimental Cytology (INIC), University of Porto, Rua do Campo Alegre 823, P-4100 Porto, Portugal.

0168-9452/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Printed and Published in Ireland

with different cel lular local izat ion. Ni t r a t e reductase loca ted in the cy top lasm accord ing to most evidence [5,6] conver ts n i t ra te to nitrite; ni tr i te reductase local ized in the p las t ids then reduces the ni tr i te to ammonia . Isoforrns o f g lu tamine synthetase were ident if ied by ion-exchange ch roma- tog raphy and des ignated as GSl and GS2 [7] and by tissue f rac t iona t ion studies were shown tha t GS1 is local ized in the cytosol while GS2 is ch loroplas t id ia l [7,8]. This loca l iza t ion was fur ther conf i rmed by immunogo ld a n t i b o d y label l ing [9]. This enzyme catalyzes the convers ion o f g lu tamate to g lu tamine th rough i nco rpo ra t i on o f free ammonia . Coup led with g lu tamine synthetase the g lu tamate synthase ( G O G A T ) media tes the t rans- ference o f the amide N o f g lutamine, fo rmed dur ing ass imi la t ion o f NH4 +, to oxog lu ta ra te

Ltd.

146

producing two molecules of glutamate. The reactions mediated by these two enzymes (GS and GOGAT) are considered the most important route for the incorporation of ammonia into the organic compounds (Ref. 10 and references therein); glutamine synthetase is then considered to be the key enzyme in ammonia assimilation in higher plants. Also the role of the glutamate dehydrogenase (GDH) in the NH4 + assimilation has been reconsidered based on presence of high levels of this enzyme in plants under certain environmental or nutritional conditions [11,12]. However such in- volvement was questioned when taking together enzymological, isotopic labelling and genetic data [10]. In addition to that using deeply searching techniques it was recently considered that GDH was not involved in the ammonia assimilation but active in the catabolism of glutamate instead [13]. There are two GDH distinct isoenzymes, one mitochondrial and generally referred to as NADH-linked and the other chloroplastidial and referred to NADPH-linked [14].

In CAM plants there have been very few studies concerning ammonia assimilation. The presence of enzymes necessary to assimilate ammonia either via GS/GOGAT or via the glutamate dehydrogenase in leaves of K. fedtschenkoi led the authors [15] to admit that in this plant the ammonia could be assimilated by both pathways, that is, GDH and GS/GOGAT. In another CAM plant it was demonstrated that ammonia assimilation proceeds via the GS/GOGAT pathway [16].

In spite of the studies referred to above concerning the nitrogen assimilatory pathway in CAM plants there remain many aspects to be clarified, one of which is how the enzymes involved are affected by different nitrogen levels matter that has been addressed in C3 and C4 plants. Work previously done on CAM activity in K. lateritia showed that the highest expression of this photosynthetic pathway appeared only when plants grew under a certain nitrogen level [17]. These results pointed out the interest in deeping the study of nitrogen assimilatory pathway in this plant, since photosynthetic efficiency and the assimilation of nitrogen are metabolically interdependent.

The aim of the present work was to study in the CAM plant K. lateritia the influence of the three

nitrogen nutritional levels, used in the previous study [17], on the activity of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH), enzymes considered involved in nitrogen metabolism.

Material and Methods

Plant material Plants of K. lateritia Engl. (K. zimbabwensis

Rendle) potted in vermiculite were grown in a growth cabinet (Heraeus-Votsch 'Ecophyt Plant Growth Chamber') under a short photoperiod (8 h light) of a photon flux density of 700 ~zmol. m -2 s -l and 27°C day/17°C night temperature with relative air humidity held at 55% day, 65% night. For ten days plants were watered three times with Hoagland's solution [18] modified to omit nitrogen ions producing N starved plants. After this starving treatment, plants were divided into three sets; one of them was henceforth watered with full strength Hoagland nutrient solution (N plants), another with a similar solution but with nitrogen ions reduced to one-fifth the normal formulation (NO 3- -- 2.984 mM and NH4 + = 0.208 mM; N/5 plants) and the third group received solution with one-tenth the normal nitrogen ions (NO3- = 1.447 mM and NH4 ÷ = 0.104 mM; N/10 plants),

Enzyme extraction and assays Third leaf pair from the apex were used to study

enzyme activities and material was collected after the preconditioning treatment (N starved plants) and after 1 and 7 days of watering them with the different nutrient solutions except whenever differently stated in the figure legend. Material used for the study of the activity of the NAD(P)H- dependent enzymes was collected at 10:00 h, 13:00 h and 16:00 h. At least, six assays were run for each enzyme from each plant group and the results presented as means -4- S.E. (standard error of the mean attached to 95% confidence limits).

Nitrate reductase (EC 1.6.6.1, NR). Assayed in vivo following the method of Hageman and Reed [19] slightly modified.The reaction assay was stopped by transferring the tubes to boiling water and after cooling they were centrifuged at 2000 x g

for 10 min and to the supernatant was added phenazine methosulfate (55 /~M) to oxidize the residual NADH. After 20-min aliquots were removed for colorimetric determination of NO2-.

Nitrite reduetase (EC 1.7. 7.1, NiR). Studied in vitro through dithionite assay as reported by Vega et al. [20].

Glutamine synthetase (EC 6.3.1.2, GS). To study the activity of this enzyme, leaf tissue was homogenized in the extraction buffer described by Kanamori and Matsumoto [21] to which was added 0.1 M cysteine and PVP (1%). Crude extract was centrifuged at 14 000 × g for 20 min; GS protein was concentrated in 40-90% ammonium sulfate fraction which was collected after centrifugation and then dissolved in 2 cm 3 of grin- ding buffer and passed through a Sephadex G-25 column previously equilibrated with a solution (pH 7.5) containing Tris (0.01 M), 2-mercaptoethanol (1 mM) and NaEDTA (2 mM). All steps were run at 4°C. The eluate was used for quantification of GS activity according to Kanamori and Matsumoto [21].

NADH-glutamate synthase (EC 1.4.1.14, NADH-GOGAT). Leaf samples were homogenized in 100 mM Tris-HC1 buffer (pH 8.0) containing 2 mM NaEDTA, 1 mM dithiothreitol and 0.1% (w/v) Triton X-100. The homogenate was centrifuged at 20 000 × g for 15 min and the supernatant was used for the enzyme assays; all steps run at 2-4°C. The reaction was started by adding the extract and followed at 30°C by observing the decrease in absorbance at 340 nm. The assay mixture contained NADH (0.1 mM), glutamine (25 mM), oxoglutarate (20 mM), Tris (100 mM; pH 7.5) and 0.2 cm 3 extract.

Ferredoxin-glutamate synthase (EC 1.4. 7.1, Fd- GOGAT). The activity of this enzyme was determined through the measurement of glutamate produced in the reaction between oxoglutarate and glutamine with reduced methylviologen as electron donor. Leaf samples were homogenized using a buffer containing KH2PO4/KOH (pH 7.5), 0.5 mM NaEDTA, 100 mM KC1, 0.1% Triton X-100 and 0.2% (v/v) mercaptoethanol. The homogenate was centrifuged at 11 000 x g for 20 min and the supernatant used for the enzyme assays. The reaction mixture, in a final volume of 1.5 cm 3 contain-

147

ed 50 mM KH2PO4/KOH (pH 7.5), 10 mM glutamine, 15 mM 2-oxoglutarate, 15 mM methylviologen, 1.5 mg Na-dithionite and the extract (0.4 cm3). Also added to the assay solution was l0 mM aminooxyacetate, a specific inhibitor of trans- aminase reactions. The acidic 2-oxoglutarate solution was neutralized with KOH before use and the reaction was started by addition of dithionite. After 20 min incubation at 30°C the reaction was stopped by the addition of 0.1 cm 3 5 M acetic acid. Blanks were incubated without dithionite. The glutamate produced was isolated from the assay mixture using a Dowex-acetate column (1 x 8, 200-400 mesh, acetate form). Glutamine was eluated from the column using 15 cm 3 of distilled water followed by glutamate with 6 cm 3 3 M acetic acid eluation which was completed by centrifugation for 2 min at 600 x g. Glutamate concentration was determined adding 1 cm 3 of the ninhydrin reagent [22] to 0.5 cm 3 of the eluate. After 10 min incubation at 80°C in a water bath, the samples were cooled and absorbance measured at 506 nm.

Glutamate dehydrogenase (NADH-GDH, EC 1.4.1.2; NADPH-GDH, EC 1.4.1.4). The activity of both isoenzymes was assayed according to the method described by Joy [23].

Results

The activity of the enzymes studied was affected by the different nitrogen levels of the nutritional conditions under which plants were grown. When the nitrogen supply was increased the activity of the enzymes mediating the reduction of nitrate to ammonia as well as the activity of GS, Fd- GOGAT and N A D P H - G D H increased whereas that of N A D H -G O G A T decreased and appeared unchanged for NADH-GDH.

In starved plants a very low value (0.022 nkat g-l fresh wt.) for the NR activity was found. Starved plants were then transferred to three different nitrogen nutritional conditions and, in the material collected after 1 day, a significant increase of the activity was observed in all of them with fairly close values (Fig. 1A). After 7 days a further increase in activity was quantified but differently in the 3 groups of 'plants. The highest increase

148

2 .0-

A 12

• N [ ] N/5 I0

~" [ ] N/10 ) 1.5 ~ 8

"5= 6 1.0 ~ _ .~

"~ "~ 4

"~ 0.5 z 2 ~ .-T-. _

1 7 Days 0.0

B

• N

[ ] N/5

[ ] N/10

T

1 7 Days

Fig. l. (A) NR activity in K. lateritia grown under N, N/5 and N/10 nitrogen nutritional conditions. Significantly different values appeared only after 7 days of treatment, lowering with nitrogen decrease. (B) Activity of NiR under identical conditions as in A; note that only N plants showed an increase of NiR activity from I to 7 days making all 3 values significantly different by this time.

observed in N plants, followed by N/5 plants and the lowest value appeared in N/10 material, this latter one represents a decrease o f about 39% in relation to N group. The values found at this time were significantly different among themselves and also in relation to the previous quantification (Fig. 1A)

The NiR activity in nitrogen starved plants was 4.02 + 0.39 nkat g- i fresh wt. After 1 day of the nutrient resupply an increase in the activity was observed in all plants irrespective o f the nitrogen concentrat ion used (Fig. 1B). The value found in either N and N/5 plants was very close and higher than the one observed in N/10 material. A further

'~ 3

2

Fig. 2.

[] []

[] []

GS

F d - G O G A T

N A D H - G O G A T

N A D H - G D H

N A D P H - G D H

N N/5 N/10 Activity of the enzymes GS, GOGAT and GDH after 7 days of treatment; with the exception of NADH-GDH all others

are affected by N, N/5 and N/10 nutrition. Discounting NADH-GDH (unchanged) and NADH-GOGAT (increased) all other enzymes showed activities decreasing when lowering nitrogen.

149

increase in NiR activity was observed after 7 days of growth but only in N plants. The values of activity of NiR at this time were significantly different among the three plant groups (Fig. 1B).

The activity of the enzymes mediating ammonia assimilation, GS, GOGAT and GDH was also affected by nitrogen nutrition. After 7 days the values found for GS activity decreased significantly from N group (2.86 ± 0.26 nkat g-J fresh wt.) to N/5 (2.25 ± 0.20 nkat g-~ fresh wt.) to N/10 plants (1.46 ± 0.10 nkat g-J fresh wt.) the value in the latter reduced about 49% relative to the former (Fig. 2)

Both isoforms of GOGAT were affected by nitrogen nutrition although in opposite ways. Fd- GOGAT activity showed a similar behaviour to what was observed for GS albeit with much higher susceptibility to nitrogen level. An 80% reduction of nitrogen (N/5 plants) provoked a marked decline of activity (from 1.65 ± 0.141 nkat g-l fresh wt. to 0.78 4- 0.081 nkat g-l fresh wt.) and a decrease of nitrogen to 10% (N/10) led to a 74% reduction in activity (0.428 ± 0.039 nkat g-a fresh wt.; Fig. 2). NADH-GOGAT showed a different behaviour; as can be seen in Fig. 2 its activity increased with decreased nitrogen level. Besides that, it was also observed fluctuation of the activity during the day and in both N and N/10 plants the highest value was found in the morning whereas in N/5 plants the activity was highest on afternoon (16:00 h). Considering the sum of the activity of both NADH- and Fd-GOGAT the N/5 plants showed a reduction of about only 33% instead of 52% and the N/10 only 40% instead of 74% in relation to the N group (Fig. 2).

The activity of both isoforms of the GDH was also studied and showed a different behaviour; the highest activity of NADH-GDH reached in each group had the same value notwithstanding the fact they were attained at different time of the day (at 10:00 h in N, at 16:00 h in N/5 and at 13:00 h in N/10 plants; Fig. 2). The activity of the NADPH- GDH also fluctuates and was significantly affected by the nutritional growth conditions. N plants had the highest activity whereas the lowest was found in N/10 plants and in all groups the maximum activity was attained at the same hour of the day (13:00 h; Fig.2).

Discussion

The present study shows that in K. lateritia, nitrogen nutrition interferes with the activity of the enzymes mediating reduction of nitrate to ammonia and the subsequent assimilation of nitrogen.

Plants were initially nitrogen-starved to create conditions for enhanced nitrate uptake [24-26] and to decrease the inducible NR and NiR which showed very low activity in accordance with results on nitrogen starvation studies [26,27]. After 1 day of nitrogen resupply NR increased but similarly in the three groups possibly due to the time required to induction of nitrate transporters [1] and its 'de novo' synthesis [24]. After 7 days, the activity of NR was significantly different in the three groups, with values in the same range as those reported for K. fedtschenkoi [15] and also consistent with the thought that induction of NR is dependent on external supply and substrate concentration, as observed in maize suspension cultures [3].

The NiR activity also increased in all groups, after 1 day, with the lowest value in N/10 and significantly different from the other two; this increase agrees well with results reported [27-29]. After 7 days the enzyme activity increased only in N plants, showing that a certain level of nitrogen is needed to promote high activity, whereas values below that are only sufficient to maintain the level attained after 24 h of the nitrogen resupply.

Taking into consideration that the values ob- tained for the nitrate inducible NR and NiR were only significantly different after 7 days of nitrogen resupply as well as the known interference of nitrogen nutrition on the chloroplast size [17] the other enzymes involved in ammonia metabolism were studied only after this period of growth. The reference to daily fluctuation of the activity of the piridine nucleotide-dependent enzymes in another CAM plant [16] led us to determine the activity of these enzymes also at different time of the day. The effects of nitrate and ammonia on GS activity are conflicting [references in 30] and also there are only few reports on the influence of these ions on GOGAT activity [references in 31]. In K. lateritia when the nitrogen amount was reduced to 20%, a

150

small decrease of GS activity was observed, whereas a nitrogen reduction to 10% (N/10 plants) provoked a sharp decrease of about 49% lower than in N plants. This behaviour seems to indicate that in this CAM plant the activity of GS attains a fairly high value with N/5 conditions and that in- creasing nitrogen concentration five-fold had a relatively small effect on the activity of the enzyme and lowering it just to half leads to a considerable loss of activity. Our results are in disagreement with some reports [32,33] and in accordance with some others [30,34,35]. In K. lateritia, a plant with the CAM pathway, the activity of both NiR and GS had a parallel pattern of response to nitrogen, as observed in Sinapis alba [34]. Previous work in K. lateritia, under the same conditions used in the present study, showed that nitrate accumulated in leaves was highest in N plants and lowest in N/10 ones [17], results that led us to admit that the availability of nitrate induces synthesis of NiR and GS, as in S. alba.

The activity of Fd-GOGAT was higher in N plants and lower in N/10 plants, results which deviate from what was reported by Hecht et al. [31]. The decline of Fd-GOGAT in both N/5 and N/10 plants, seems to be circumvented through an increase of NADH-GOGAT which showed higher values in leaves with lower Fd-GOGAT activity. The Fd-GOGAT/NADH GOGAT ratio declined from the N to N/10 plants in agreement with other report which ascribe this transition from NADH- to Fd-GOGAT to developmental changes of chloroplasts [36], a dependence that very likely also influenced the two enzymes in our material. The behaviour of NADH-GOGAT contrasts with what was found in maize seedlings [37] and in mustard seedlings [31]. In general, it was seen that the activity of both the GS and GOGAT lowered with decreased nitrogen supply. It is known that in CAM plants photorespiration is negligible occurr- ing mostly during a short period of the phase II and phase IV of the CAM plants [38,39], thus practically excluding the apport of photorespiration-derived ammonia as substrate for the GS/GOGAT cycle. The dependence of GS from nitrogen nutrition is very likely due to its behaviour as inducible enzyme [34,40]; the different values of Fd-GOGAT activity can be ascribed to the interference of nitrogen with the development

of chloroplasts, which occurs in the plant studied [17]. In K. lateritia it was observed a diurnal fluctuation of the NADH-GOGAT activity in accordance with what was reported in another CAM plant [16]. The activity of the two GDH isoenzymes also fluctuates although their behaviour was different from each other. Maximum activity of NADH-GDH was found at different time of the day, albeit with no significant dif- ferences from one plant group to the others in- dicating that the level of the nitrogen used interfered with the time of its maximum activity. This difference is possibly related to the different level of CAM that was found for the plants of these three groups. The activity of NADPH-GDH was different for each group decreasing with the reduction of nitrogen supply although the maximum was reached at the same time of the day. Curiously, the pattern followed by the chloroplastidial GDH activity paralleled that observed for NiR, GS and Fd-GOGAT which reinforce the referred to above correlation between the chloroplast size and the amount of the enzymes.

Previous work done in K. lateritia showed that the level of CAM activity was dependent on the nitrogen level [17] and altogether the results presented here show clearly that in this C A M plant the activity of the enzymes NR, NiR, GS, GOGAT and NADPH-GDH was strongly affected by the level of nitrogen nutrition, whereas NADH-GDH was not. The high sensitivity of the former enzymes to the level of nitrogen, most of them chloroplast-related, might well be a reflex of the known dependence that these organelles have for that nutrient and such behaviour influence the level of CAM photosynthesis since the assimilation of nitrogen and photosynthetic CO2, fixation are interwoven processes.

Acknowledgments

The skillful contributions of Mrs Andrea Costa and Isabel Guimar~,es are gratefully acknow- ledged.

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