Effect of Inorganic Nitrogen Nutrition (Ammonium and Nitrate) on Aerobic and Anaerobic Metabolism in Excised Rice Roots

IDA BRAMBILLA, ALCIDE BERTANI and REMo REGGIANI

Istituto Biosintesi Vegetali CNR - Via Bassini, 15 - 20133 Milano - Italy

Received September 6, 1985 . Accepted November 4, 1985

Summary Excised rice roots grown on ammonium and nitrate media under aerobic and anaerobic con­

ditions were compared. In air, differences between the two culture conditions were observed in the growth of the roots and in the pattern of newly synthesized proteins. Anaerobiosis com­pletely blocked the growth of the roots and cancelled any difference in the polypeptide pattern of newly synthesized proteins. However, in anoxia there were substantial differences in the rate and type of fermentation carried out by the roots supplied with ammonium or nitrate.

Key words: Adenylate energy charge, anaerobiosis, fermentation, polypeptide pattern, respiration.

Introduction

The form of nitrogen applied as fertilizer for rice plants growing in flooded soils is normally ammonium. Fertilizer based on nitrate, in fact, has seldom been used be­cause in waterlogged soils nitrate is absorbed with difficulty by soil particles and often undergoes denitrification (Sasakawa and Yamamoto 1978). Rice plants, how­ever, grow well with nitrate as the sole nitrogen source when cultured on the nutrient culture medium (Malavolta 1954); under such conditions, nitrate is a better nitrogen source than ammonium with respect to plant growth, grain yield, some metabolic activities and cation absorption of the roots (Sutcliffe 1962; Yamasaki and Seino 1965).

Sasakawa and Yamamoto (1978) reported that in rice seedlings, although nitrate and ammonium were assimilated at similar rates under aerobic condition, nitrate was taken up more slowly than ammonium when the culture medium was bubbled with nitrogen gas. Recently we have shown that when rice root tips are incubated under strictly anoxic conditions, the addition of nitrate to the growth medium increases the fermentation activity and AEC, while the reduced pyridine nucleotide content de­creases probably because oxidized in consequence of the reduction of nitrate (Reg­giani et al. 1985 a, b).

Abbreviation list: TCA = trichloroacetic acid; SDS = sodium dodecyl sulfate; PAGE = poly­acrylamide gel electrophoresis; RQ = respiratory quotient; AEC = adenylate energy charge.

J. Plant Physiol. Vol. 123. pp. 419-428 (1986)

420 IDA BRAMBILLA, ALCIDE BERTANI and REMo REGGIANI

Our work has been extended to the investigation of the effect on rice roots of dif­ferent nitrogen sources in both aerobic and anaerobic metabolism. In the presented work, we compared excised rice roots incubated with ammonium or nitrate as re­gards carbohydrate consumption, AEC and protein synthesis.

Materials and Methods Plant materials

Seeds of rice {Oryza sativa cv. Arborio} were surface-sterilized for 2 min with 70% {v/v} ethanol and for 30 min with calcium hypochlorite, and germinated for 3 days on sterile wet paper in Petri dishes at 25°C in the dark according to the method of Reggiani et al. {1985 a}. Apical segments of about 10 mm of seedling roots were excised and incubated in air at 25°C for 1 day on a modified Heller medium {Reggiani et al. 1985 a} at pH 5.1 containing 7 mM NH4CI or KN03. The excised roots were then incubated in 25 ml jars, each containing 25 roots and 5 ml of modified Heller medium. The jars were flushed with nitrogen gas {99.999 % nitrogen} at 3.2 liter h -I through the medium for 20 min to make anoxia {the anoxic grade was checked as described by Reggiani et al. 1985 a}. As to the control, air was flushed into some jars to keep the excised roots under aerobic conditions.

Bacterial contamination was tested by incubating an aliquot of the medium on a agar-nutri­ent medium as described by Reggiani et al. {1985 a}. Data from contaminated samples were dis­carded.

Oxygen, CO2 and ethanol determination

Oxygen uptake and CO2 evolution by aerobic roots was determined by Gilson Differential Respirometer {Gilson, France}. The ethanol produced during anaerobiosis by roots was en­zymatically assayed by the method of Bergmeyer {1974} {see Reggiani et al. 1985 b for details}. CO2 evolution in anoxia was monitored by an Infra Red Gas Analyzer {Leybold-Heraeus, West Germany}.

Adenine nucleotide determination

HCI04-soluble extracts prepared frol? the excised roots as described previously {Reggiani et al. 1985 b} were assayed for A TP, ADP, and AMP. A TP was determined by the luciferin-Iuci­ferase assay method with the ATP bioluminescence HS Kit Boehringer Mannheim, West Ger­many. The emission of light in the luciferin-Iuciferase assay was monitored by Biolumat LB 9500 apparatus (Berthold company, West Germany). ADP and AMP were estimated after their conversion to ATP by the method described by Carver and Walker (1983). AEC was defined as by Pradet and Raymond (1983).

In vivo labelling

The excised roots were incubated in Heller medium containing 35S-methionine 1.8 MBq ml- I

for 3 h under aerobic and anaerobic conditions. The roots were then rinsed with ice cold water and homogenized with 0.1 M Tris-HCI buffer, pH 7.4 (1 ml per 25 roots) in a cold mortar. After centrifugation (at 100,000 xg for 30 min), the pellet was again extracted with the buffer (0.2ml per 25 roots) and centrifuged. The supernatants were combined and an aliquot was mixed with an Insta-gelliquid scintillation cocktail (Packard, U.S.A.) and assayed for radioactiv­ity with a Packard-Tri-Carb liquid scintillation spectrometer. The radioactivity of polypeptides in the supernatant was determined by precipitating the proteins with 10 % TCA. The insoluble materials were filtered on nitrocellulose filters and counted.

J. Plant Physiol. Vol. 123. pp. 419-428 (1986)

Nitrogen nutrition and root metabolism 421

SDS·PA GE and jluorography

SDS·PAGE was carried out as described earlier {Bertani et al. 1981}. The acrylamide concen­tration in the running gel was 15 %. Samples with approximately 50,000 cpm each were loaded on the geL To locate radioactive polypeptides, fluorography of the gel was carried out as by Bertani et aL (1981).

Results and Discussion

Root growth

The growth in length and in fresh weight of excised rice roots in air and after 24 h of anaerobic treatment is shown in Table 1. The growth in length and in fresh weight of the roots grown for 1 day in aerobic conditions was greater when nitrate instead of ammonium was present in the culture medium. After this growth, the imposition of anaerobiosis completely blocked the growth of the roots both in length and in fresh weight irrespective of the nitrogen source.

Table 1: Effect of nitrogen source on growth of excised rice roots. The means and standard er­rors of three different experiments with 25 roots each are reported. At the beginning of the ex­periments, the apical parts excised from germinated rice seedlings were 11.70 ± 0.80 mm long with a fresh weight of 2.80±0.10 mg.

Treatments Aerobic Aerobic growth + growth 24 h of anaerobiosis

+NH4+ length (mm) 14.61±0.91 14.60±0.94 fresh wt (mg root-I) 3.18±0.20 3.16±0.25

+N03 -length (mm) 17.41±0.90 17.39±0.90 fresh wt (mg rooC I) 3.84±0.18 3.80±0.21

Carbohydrate consumption

During aerobic growth, a significant difference of RQ between the two cultural conditions was observed: values of 1.00 and 1.13 for roots fed with ammonium and nitrate respectively were estimated. This difference of RQ as attributable not to the CO2 evolution, which was fairly similar (40.2 x 10- 6 molg- I fresh wt), but to a differ­ent oxygen uptake (40.2 x 10-6 and 36.3 x 10- 6 mol g-I fresh wt in ammonium and nitrate respectively). The influence of the nitrogen source on RQ was similar to that reported with regard to excised barley roots by Willis and Yemm (1955) and suggested by Lee (1980), who interpreted the lower oxygen uptake by roots grown on nitrate as a partial re-oxidation of pyridine nucleotides through nitrate reductase rather than through the respiratory chain. The similar CO2 evolution in air, on the other hand, indicates that the equivalents of glucose consumed aerobically are similar in roots fed with ammonium or nitrate (Fig. 1).

The production of CO2 and ethanol during 24 h of anaerobic treatment is shown in Figs.2 and 3. Higher evolution of CO2 and ethanol in anaerobically treated roots

]. Plant Physiol. Vol. 123. pp. 419-428 (1986)

422 IDA BRAMBILLA, ALCIDE BERTANI and REMo REGGIANI

12

10

I/) .... i 8 z w

-= ..J

N°i ~ 5~ o 01

6 w

w .. ~ I/) 0 (J

4 ::t ~ ..J CI

co I

S2 2

o 3 8 24

HOURS OF ANAEROBIOSIS

Fig. 1: Glucose equivalents consumed by excised rice roots grown in air and in anaerobiosis in the presence of 7 rnM ammonium or nitrate. The data were obtained making use of the produc­tion of CO2 in air and the production of CO2 and ethanol at 3, 8, and 24 h of anaerobiosis. Glu­cose equivalents were reckoned by dividing the CO2 produced by 6 and the ethanol by 3.

was always observed when exogenous nitrate was present. This difference is much more evident if the data of CO2 and ethanol are expressed as equivalent of glucose consumed (Fig.1). Under both cultural conditions, ethanol and CO2 were not pro­duced in equimolar ratio as expected of a pure alcoholic fermentation: the amount of CO2 produced was always greater than that of ethanol (Table2). In consequence of this behaviour, it is necessary to hypothesize the functioning during anoxia of meta­bolic pathways other than alcoholic fermentation. In nitrate fed roots, however, the ethanol/C02 ratio was lower than that of ammonium fed roots (Table2). This clearly indicates that the exogenous nitrate permitted a greater carbohydrate con­sumption through metabolic pathways other than alcoholic fermentation. The na­ture of these pathways remains rather obscure, even if one of them, the pentose phosphate pathway, has been demonstrated to function in plant tissues subjected to anaerobiosis (Lee 1979; Rumpho and Kennedy 1983). In a recent work (Reggiani et al. 1985 b) of comparison of the fermentative activity of excised rice roots anaerobically grown in the presence or absence of nitrate, it was shown that nitrate stimulated carbohydrate breakdown. On the other hand, the ethanol/C02 ratio during 24 h of

J. Plant Physiol. Vol. 123. pp. 419-428 (1986)

Nitrogen nutrition and root metabolism 423

600

500

N°i

400

w i NHt

Q

~ x 300 0 is "j

en

Z '0 ~ E 200 II: co

I <I: ~ u

100

o 3 8 24

HOURS OF ANAEROBIOSIS

Fig. 2: The effect of the different source of nitrogen in the external medium on the production of carbon dioxide by excised rice roots. Means of three replicates. Standard error under 10%.

anaerobiosis was similar in roots incubated in the presence or absence of nitrate. In the light of this evidence, the higher values of ethanol/C02 ratio here reported in roots grown in the presence of ammonium could indicate that ammonium partially inhibits carbohydrate consumption through metabolic pathways other than alco­holic fermentation.

Adenylate energy charge

The levels of adenine nucleotides and AEC of the roots are shown in Table3. Dur­ing aerobic growth the AEC was similar in roots incubated with ammonium or ni­trate. On transfer of the roots from air to nitrogen gas the level of the total adenine nucleotides and AEC dropped to a lower level in the case of both the nitrogen sources. The drop in AEC was mainly attributed in both conditions to a decrease of the ATP content and to the build-up in AMP level. Between the 3rd and 24th hour the level of AEC decreased slowly. A slightly higher level was, however, observed in roots grown on nitrate. We think that this slight higher value can be related to the greater carbohydrate breakdown reported above.

J. Plant Physiol. Vol. 123. pp. 419-428 (1986)

424 InA BRAMBILLA, ALCIDE BERTANI and Ram REGGIANI

300

200

i ..J ~ ... 0 'i z c Ol ::t I- "0 w E

Ul 100 I

~

o 3 8 24

HOURS OF ANAEROBIOSIS

Fig. 3: The effect of the presence of ammonium or nitrate in the culture medium of excised rice roots on the production of ethanol. Means of three experiments. Standard error under 10 %.

Table 2: Effect of the nitrogen source on ethanol/C02 ratio. The means and standard errors of three different experiments are reported. Use was made of excised rice roots grown on a modi­fied Heller medium (7mM ammonium or nitrate) for 24h in air and then subjected to anaero­biosis in the presence of ammonium or nitrate.

Hours of anaerobiosis

3 8

24

Protein synthesis

Treatments

ammomum

0.74±O.02 O.69±O.03 O.64±O.Ol

nitrate

O.SS±O.OS 0.56±O.03 0.57±O.02

During aerobic growth, the uptake and incorporation of 35S-methionine were slightly greater when nitrate rather than ammonium was present in the culture me­dium, whilst the efficiency of label incorporation [(total cpm into proteins)· (total cpm uptake)-l . 100] was similar in the two conditions (Table 4).

The imposition of anaerobiosis drastically reduced the incorporation of 35S_

methionine into proteins in both ammonium and nitrate fed roots (Table4). The

f. Plant Physiol. Vol. 123. pp. 419-428 (1986)

Nitrogen nutrition and root metabolism 425

Table 3: Levels of adenine nucleotides and adenylate energy charge. All values are calculated from three different experiments. The coefficient of variation was less than ±10%.

Hours of ATP*) ADP*) AMP*) Total AEC anaerobiosis nucleotides*)

+NH4+ 0 154.9 52.7 5.6 213.2 0.85 3 39.7 44.6 54.6 138.9 0.45 8 28.3 20.1 55.3 98.7 0.39

24 8.7 22.0 53.5 90.3 0.34

+N03~ 0 177.1 46.8 12.5 236.4 0.85 3 51.9 43.6 59.1 154.6 0.48 8 34.2 17.7 52.9 104.9 0.41

24 13.7 19.9 63.9 97.5 0.37

*) 1O~9 mol g~ 1 fresh wt.

Table 4: Effect of nitrogen source on uptake and incorporation into proteins of 35S-methionine. The data of uptake and incorporation into proteins are expressed as 103 cpm root ~ 1 and are the mean of two different experiments. The excised rice roots were incubated for 3 h at 25°C in the presence of 35S-methionine at the concentration of 1.8 MBq per ml of Heller medium.

Treatments Aerobic Anaerobic treatment between: treatment 3-6th hour 20 - 23rd hour

+NH4+ Uptake 753 149 135 Incorporation 315 14 11 into proteins Efficiency of 41.8 9.4 8.1 incorporation*)

+N03~ Uptake 925 90 100 Incorporation 441 19 16 into proteins Efficiency of 47.7 21.1 16.0 incorporation*)

*) Efficiency of incorporation: (cpm into proteins)· (total cpm uptake)~ 1 ·100.

uptake of labelled methionine in was, however, less affected by anaerobiosis in the ammonium medium than in the nitrate medium. As a consequence of this behaviour, the anaerobic efficiency of label incorporation into proteins in the presence of ammonium was half that of roots incubated in the presence of nitrate (Table4). We have not so far found a clear explanation of this behaviour. Considering, however, that the efficiency of protein synthesis was lower in ammonium fed roots, we can suppose that the rate of anaerobic protein synthesis was not greatly affected by the different source of nitrogen.

Polypeptide pattern of synthesized proteins

The qualitative analysis of synthesized proteins carried out by SDS-PAGE and fluorography is shown in Fig. 4. Several differences were observed in the polypeptide

J Plant Physiol. Vol. 123. pp. 419-428 (1986)

426 InA BRAMBIILA, ALCIDE BERTANI and Ralo REG GIANI

1_

2--. 3_

4-5-6_ 7_

8-

a b c d

-92.5

_18.4

mol. wt. x103

Fig.4: Fluorography of rice polypeptides separated by 5D5-Polyacrylamide gel electropho­resis. Use was made of excised roots grown for 24 h in aerated Heller medium in the presence of 7 mM ammonium or nitrate, and then subjected to aerobic or anaerobic treatment in the same growth media. After 3 h of treatment, the roots were incubated for 3 h in the presence of 355_ methionine at 1.8 MBq per ml of Heller medium. a) aerobic condition in the presence of ammonium; b) aerobic condition in the presence of nitrate; c) anaerobic condition in the pres­ence of ammonium; d) anaerobic condition in the presence of nitrate.

patterns of aerobic proteins synthesized in the presence of ammonium or nitrate (Fig. 4 lanes a and b, bands marked by number from 1 to 8). These differences con­cerning a large number of polypeptides, suggest that, besides proteins linked to the as­similation pathways of ammonium and nitrate (e.g. glutamate dehydrogenase, nitrate reductase), other proteins are induced by the different forms of inorganic nitrogen nutrition.

The absence of oxygen utterly cancelled the differences between roots supplied with ammonium and nitrate observed during the aerobic growth. Anaerobiosis, in fact, resulted in simplification and similarity of the polypeptide patterns of newly synthesized proteins in both cultural conditions (Fig. 3 lanes c and d). This indicates

J. Plant Physiol. Vol. 123. pp. 419-428 (1986)

Nitrogen nutrition and root metabolism 427

that the few and essential proteins synthesized in anoxia by excised rice roots do not include proteins detectable by our analytical system that are associated with the pres­ence of ammonium or nitrate in the growth medium.

The cancelling of differences in the polypeptide patterns observed in anaerobic con­ditions as a result of the lack of oxygen has been reported also by Okimoto et al. (1980), who, working with maize seedlings, found that the aerobic protein synthesis profiles of root, endosperm, scutellum, and anther wall were very different, while their patterns of protein synthesis during anaerobiosis were remarkably similar.

On the whole, these observations seem to indicate that, in comparison with the aerobic condition, the polypeptide pattern expressed during anoxia is not only drastically changed and simplified, but is also less influenced by the different condi­tions of growth.

Conclusions

During aerobic growth, the different form of nitrogen nutrition (NH4 +, N03 -)

influenced the growth, RQ, and polypeptide pattern of newly synthesized proteins. The imposition of anaerobiosis cancelled difference in growth and in the poly­

peptide pattern, but induced qualitative and quantitative changes in carbohydrate consumption. This last aspect, together with the AEC, could play a central role in root survival in waterlogged soils in which energy supply can be a limiting factor. Al­leviation of flooding injury in barley and wheat plants by means of nitrate has in fact been reported (Drew et al. 1979; Trought and Drew 1981). However, to clarify the role of the different source of nitrogen with regard to root survival in the absence of oxygen, further investigations are necessary.

Acknowledgements

We thank Dr. F. Mazzini, Ente Nazionale Risi - Mortara, for the generous supply of seeds. Research work supported by C.N.R. Italy. Special grant I.P.R.A. - Sub-project 1. paper no. 756.

References

BERGMEYER, H. U.: Methods of enzymatic analysis. Vol. 4, pp. 1499-1501. Verlag Chemie Weinheim-Academic Press, Inc. New York and London Second edition (1974).

BERTANI, A., F. MENEGUS, and R. BOLLINI: Some effect of anaerobiosis on protein metabolism in rice roots. Z. Pflanzenphysiol. 103, 37 -43 (1981).

CARVER, K. A. and D. A. WALKER: The effect of adenylate kinase activity in a reconstituted chlo­roplast system; determination of ATP, ADP and AMP levels with luciferin-Iuciferase. Sci. Tools 30, 1-5(1983).

DREW, M. c., E. J. SISWORO, and L. R. SAKER: Alleviation of waterlogging damage to young barley plants by application of nitrate and a synthetic cytokinin, and a comparison between the effects of waterlogging, nitrogen deficiency and root excision. New Phytol. 82, 315-329 (1979).

LEE, R. B.: The release of nitrite from barley roots in response to metabolic inhibitors, un­coupling agents, and anoxia. J. Exp. Botany 114, 119-133 (1979).

J. Plant Physiol. Vol. 123. pp. 419-428 (1986)

428 loA BRAMBILLA, hCIDE BERTANI and REMo REGGIANI

LEE, R. B.: Source of reductant for nitrate assimilation in non-photosynthetic tissue: a review. Plant Cell Environ. 3,65-90 (1980).

MAuVOLTA, E.: Study on the nitrogenous nutrition of rice. Plant Physiol. 29, 98-99 (1954). OKIMOTO, R., M. M. SACHS, E. K. PORTER, and M. FREELING: Patterns of polypeptide synthesis

in various maize organs under anaerobiosis. Planta 150, 89-94 (1980). PUDET, A and P. RAYMOND: Adenine nucleotide ratios and adenylate energy charge in energy

metabolism. Ann. Rev. Plant Physiol. 34, 199-224 (1983). REGGIANI, R., I. BRAMBILLA, and A. BERTANI: Effect of exogenous nitrate on anaerobic metabo­

lism in excised rice roots. I. Nitrate reduction and pyridine nucleotide pools. J. Exp. Botany 36,1193-1199{1985a).

REGGIANI, R., 1. BRAMBILLA, and A. BERTANI: Effect of exogenous nitrate on anaerobic metabo­lism in excised rice roots. II. Fermentative activity and adenylic energy charge. J. Exp. Botany 36, 1698-1704 (1985 b).

RUMPHO, M. E. and R. A. KENNEDY: Activity of the pentose phosphate and glycolytic pathways during anaerobic germination of Echinocloa crus-galli (Barnyard grass) seeds. J. Exp. Botany 144,893-902 (1983).

SASAKAWA, H. and Y. YAMAMOTO: Comparison of the uptake of nitrate and ammonium by rice seedlings. Plant Physiol. 62, 665-669 (1978).

SUTCUFFE, J. F.: Mineral salts absorption in plants. Pergamon Press, New York (1962). TROUGHT, M. C. T. and M. C. DREW: Alleviation of injury to young wheat plants in anaerobic

solution cultures in relation to the supply of nitrate and other inorganic nutrients. J. Exp. Botany 32,509-522 (1981).

WILUS, A. J. and E. W. YEMM: The respiration of barley plants, VIII. Nitrogen assimilation and the respiration of the root system. New Phytol. 54, 163 -181 (1955).

YAMASAKI, T. and K. SEINO: Use of nitrate fertilizers for the cultivation of paddy rice (part 1). About the physiological character of rice seedlings supplied with nitrate as the source of ni­trogen. J. Sci. Soil Manure Japan 36, 153-158 (1965).

,. Plant Physiol. Vol. 123. pp. 419-428 (1986)