Biochem. Physiol. Pflanzen 182, 349-358 (1987) VEB Gustav Fischer Verlag Jena

The Effect of Alterations in Nitrogen Supply on Nitrogen Metabolism of Hydroponically Grown Maize Plants, Zea mays L.

J. H. VENE KAMP, F. W. T. PE~NrNG DE VRIES, H. MENSrl\'K, F. VAN MOURIK and J. T. M. KOOT1)

Ccntfl' for Agro biological Rest'anh, Wageningen, The N etherlands

Kcy Term Index: biomass, dry matter, N concentration, ~ content, buffer-soluble proteins, sodium dodecylsulphate (SDS)-soluble proteins, residual proteins, non-protein N, nitrate, redistribution; Zea mays

Summary

:\Iaizc plants hydroponically grown on nutrient solution were used to study the effect of altera­tions in N supply on Neoncentrations in the organs as related to the ability of biomass production.

Omission of nitrate from the nutrient solutiou induced in the vegetative organs a breakdown of nitrogenous compounds which were partly rcdistributed towards the ears. These processes took placc more at the expense of membrane proteins than of the buffer-soluble pro teins and resulted in Neoncentrations lower than the theoretical minimum; then the biomass production stopped.

Re-supply of nitrate to young N-starved plants induccd an increase of Neoncentrations in all plant parts and the rt'sumption of biomass production. The newly produced amount of membrane proteins was larger than that of buffer-soluble proteins. However, the ratio of buffer-soluble to membrane protein eoncrntrations in mature leavcs did not return to the original value. It was eoncluded that re-supply of nitrate resulted in a recuperation of X-starvcd plants to arestricted extent.

Introduction

Nitrogen supply is one of the most important factors for the growth of plants. Altera­tions in N supply during the development of thc plant will induce reactions. According to SPEK (1984a and b) omission of nitrate from the nutrient solution of young maize seedlings 14 d old led to a rapid consumption of accumulated nitrate and a reduction in organic N concelltration by continuing growth, in both shoot and root. Supplying N-starved maize seedlings with nitrate led to a rapid increase in both nitrate-N and organic N contcnts. Also MrLLs and BENTON JONES (1979) found synthesis and break­down of organic N compounds, concurring with changes in N supply. In addition to these studies we wanted to investigate thc following aspects of the cffcct of alterations in ~ supply on N concelltrations in the plant organs:

1. the dependel1ce of this effect on the moment in the plant's development when the N stress starts;

2. the possibility of recuperation of such N-starved plants by re-supply of nitrate;

1) Dedicated to Prof. K. SCHREIBER on the orcasion of his 60th anniversary.

Abbreviations: P.A.R., physiologically active rays; N-ES, early N stress; N-1S, late N stress; SDS, sodium dodecylsulphate

24 349

3. the influence of the original level of nutrient nitrate on the above-mentioned effects.

PENNIKG DE VRIES (1982) stated that growth or mass increase of a plant can occur as long as the ~ eoneentration in the plant is between a maximum and a minimum N level. This led to an additional question:

4. will plant parts resume growth after aperiod of too low or too high N concentra­tions?

Several investigations on consequenees of variations in N supply were restricted to alterations in the eoneentrations of ribulosediphosphate carboxylase (HALL et al. 1977; PHELOUKG and BRADY 1979; and ~OVOA and Lomns 1981) and in concentrations of nitrate reduetase and glutamine synthetase (BARNEIX et al. 1984; and HOFSTRA et al. 1985). For a general description of cOllsequences of alterations in N supply VENEKAlIIP

and KOOT (1984) devised a meaningful grouping of all N compounds. They distinguished 5 eategories: buffer-soluble proteins (particularly eytoplasmic and chloroplast proteins), sodium dodec:'lsulphate (SDS)-soluble pro teins (particularly membrane proteins), resi­dual protpins (particularly eell wall proteins ), non-protein organic N (partieularly nucleic acids and free amino-acids) and nitrate.

This study of the foul' questions mentioned above is a continuation of investigations by VEKEKA~I:P et al. (1985) on the influence of a reduced N nutrition on N metabolism in the organs of maize plants.

Materials and Methods

Plants

Maiz{' plants, Zea mays L. ('v. LG 11, wpr{' grown hydroponieally in gravel after direct sowing, 2 plants per 3 I pot. Thc pots werc plared in six trays (100 X 60 x 20 rlll3 each) in a growth challlber, 14 pots per tray; every 2 h the nutrient solution was eirculated and drained after 15 lllin. The daily rrgilllc was 14 h light of 100 W m- 2 P.A.R. at a temperature of 25 oe and 10 h dark at 18 oe. The lamps wen' 40 ('m from the top of the plants.

N utricnt solutious

.:-I utrient sohltions werr prepared ae('ording to STEIXER (1968). Every two weeks the nutrient sohltions were renewell.

Plants of 3 trays wen' grown on a solution with the normal amollnt of 135 mg 1-1 nitrate-No Plants of the first tray were grown in this solution during the whole experimental p{'riod; they are referred to as 1 ~ -control plants. In the period between 45 and 59 d after planting, nitrate was omitted frolll thl' nutrient solution of thc plants of the sccond tray (an early N stress); these plants are refer­rl'd to as 1 .:-I-ES plants. In the period frOlll the 59th d after planting till the end of the expl'riment on the 73rd d, nitrate was omitted from the nutrient solution of the plants of the third tray (a late N stress); thps(' plants ure referrl't! to as 1 N-LS plants. In both eases nitrate was replaced by sul­phatc.

Another three groups of plants wcre grown on a nutrient solution, containing 45 lllg I-l nitrate-N. Plants of two groups were grown without nitrate during the same, above-Illentioned periods. These three groups of plants are referrl'd to as 1/3 N-control plants, 1/3 N-ES plants, and 1/3 N-LS plants, respectively.

Sampling

The 1 N- and the 1/3 N-control plants were sampled every 3 or 4 d during the period frolll the 42nd d till the 73rd d after planting, the 1 N-ES and the 1/3 N-ES plants frolll the 48th d till the

350 BPP 182 (1987) 5

69th cl (not samplrd on the 73rcl cl) and the 1 X-LS and thc 1/3 N-LS plants from the G2nd d till the 73rd d.

Eaeh sampIe ('onsisted of one pot with two plants whieh ware divided into roots, leaves 1 to G,

leavcs 7 to 9, leaYl's 10 and highl'r, ears, and stems. The sheaths were collcctecl with the leavcs.

Determiilations Thc fr('sh wl'ights of the plant parts \\"('l'(' (h·termim·d; th!'n the parts werc separately cut into

pieces which were mixed thoroughly. Eight-gram sampies of these mixtures werl' frozen at -26 oe and 30-g sampIes were drift! at 70 oe during 2 d.

Thc frozen sam pIes würe Ilsed for detprmination of the concentrations of buffer-soluble, SDS­soltlble and residual proteins aeeording to thc method of VE?U:K"\}!P and KOOT (1984). The dry sampIes wer!' usetl for determination of dry matter, total-N and nitratc-~. From these values total N ('ontents of the plant parts wcrr calclllated. Total-N amI nitrate-~ were mcasured according to methods 7,041 antl 2,rJii8, resp('rtiV!'ly, of "Offirial methods of analysis of the Assoeiation of Official Analytical Chemists", 13th edition, 19tiO, ~Washington D.C.

Results and Discussion

Effect of early or late N stress (question 1)

The levels of .x eoneentration as weIl as the patterns of N redistribution of 1 X­and 1/3 ~-eontrol plants were similar to those described by VENEKA~IP et al. (1985). Omission of nitrate from the nutrient solution induced an immediate breakdown of nitrogenous eompounds and a reduction of N concentration in all vegetative plant parts. Late omission had a smaller effeet than early omission (Fig. 1). In the 1 N-ES and 1/3 N-ES plants, apart of the ~ resulting from breakdown was redistributed in favour of the ears, stern and roots (Table 1). Plants of the 1 ~-LS and 1/3 N-LS groups showed some N redistribution only towards the ears. All these plants, however, lost a consider­able amount of N during the periods of stress. In the 1 ~ plants, this amount did not only originate from the organic nitrogenous compounds, but also from nitrate already prescnt in the plant just before the beginning of the stress period (Table 1).

In addition to X distribution over the plant organs, the partitioning of N between the nitrogenous compounds was investigated. These were for the greater part proteins. The average total protein-N as a percentage of the average organic N in the leaves was 71 ± 16 %. The residual proteil1-~ in the leaves was only 1 % of the total protein­N; these residual pro teins hardly played a role in the proeesses involved in N stress (VENEKX)IP et al. 1985). The course of the protein composition is refleeted by the ratio of buffer-soluble to SDS-soluble proteins (Table 2). Early as weIl as late withholding of nitrate redueed the amount of SDS-soluble proteins more than that of buffer-soluble proteins, and the ratio between these proteins in leaves of both groups of plants was high er than that of the control plants (Table 2). Apparently ~ was redistributed more at the expense of the SDS-soluble pro teins than of the buffer-soluble proteins. Redis­tribution of N was relatively most distinct in the oldest leaves and we found the highest ratios there. YIoreover, during the period of late stress a larger amount of SDS-soluble proteins was brokcn down than during early stress (Table 1). Under the present condi­tions the buffer-soluble pro teins were probably more important for the maintenance of the physiologieal activities of the plants than the SDS-soluble proteins.

24* BPP 182 (1987) 5 351

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Table 1. Alteratiolls in the arnouuts of nitrogenous substances in the organs of maize plants during early and la te stress periods, 45-59 d anel 59-7'1 d after planting, respectively.

Plant Period, Organic Huffer- SDS- Non- Nitrate-N

Group Organ days ~ soluble soluble protein-N after protein-X protein-N planting mg mD"

" mg mg lUg

1N ears 45-59 + ,38 0 leaves 10-tops - 20 -6 -12 -2 -4 lcaves 7-9 - 18 0 -10 -8 -6 leaves 1-6 - 46 -18 -20 -8 -32 stern + 16 -12 roots + 16 -8

total -14 -62

ears ;)9-73 + 16 0 leavt's 10-top - 70 -12 -16 -42 -10 leaves 7-9 - 68 -16 -28 -24 -6 leav~s 1-6 8 -2 -3 -3 -2 stern 0 -23 roots 8 -8

total -138 -49

1/3 N pars 45-ö9 + 27 0 leaves 10-top - 1G -4 -6 -G 0 leaves 7-9 - 40 -14 -16 -10 0 leavcs 1-G - 18 -ö -5 -7 -1 stern + 19 0 roots + 1 0

total - 27 -1

ears ö9-73 .l 1 0 I

leaves 10-top - 30 -8 -12 -10 -4 leavcs 7-9 -M., -22 -24 -12 0 leaves 1-6 3 -1 -1 -1 0 stern 0 0 roots 0 0

total - 90 -4

Fig. 1. Neoncentrations in the organs of ll1aize plants, grown under different conditions of -y supply. Control plants were grown on a complete nutrient solution, 1; plants oi early stress were grown on the same nutrient solution, but nitrate was omitted in the period between 45 and 59 d after plant­ing, 2; plants of late stress were grown on the same nutrient solution, but nitrate was omitted in the period between 59 ami 73 d after planting, 3. The maximum level of N concentration, above which a plant cannot grow, 4; the minimum level oi Neoncentration, below which a plant tannot grow, 5. a: Nutrient solution eontained 13ö mg I-I nitrate-N; b: nutricnt solution containe1l4ö mg I-I nitrate-No

BPP 182 (1987) 5 353

Table 2. Influcnce of earl!} or late stress Oll ihe ratios of buffer-soluble and SDS-soluble protein contenls il1 Ihe lea~'es of maize pllllds. Data are giwn as averagl's for eac'h prriod, with their standard devia­tions.

Plant Lcaf Ratio of buHer-soluble and SDS-soluble protrin contents in leaves of group nUffibl'f Control plants Plants during Plants aftn Plants during

dlLrin" wholl' "

carly stress early stress late stress dl'Vclopment

IX 1~6 1.24 ± 0.08 1.69 ± 0.22 2.08 ± 0.25 2.72 ± 0.35 7~9 1.17 ± 0.11 1.G7 ± 0.27 1.42 ± 0.15 1.32 ± 0.43 10~top l.()9 ± 0.06 1.24 ± 0.12 0.95 ± 0.11 1.22 ± 0.22

1/3 N l~G 1.18 ± 0.08 1.44 ± 0.12 1.37 ± 0.12 1.64 ± 0.07 7~9 1.24 ± 0.14 1.41±0.11 1.44 ± 0.19 1.37 ± 0.52 10~top 0.97 ± 0.09 1.3G ± 0.09 0.79 ± 0.13 1.03 ± 0.38

Table 3. AUeratiolis ill Ihe all10unts of nitrogenous substallces in the organs of maize plants aftcr the per iod of earl!} stress: diffrrences of the amounts on cl 78 from those at the end of the stress period, d 59.

Plant

Group

IN

Organ

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total

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rng rng rng mg rng

+ 50 0 +104 +4G +52 + G + 4

+ 28 ,-11 +13 + 4 0

+ 8 + 3 + 1 + 4 0

+ 5ß +15 + 42 +27 +2Hii +46

+ EJ 0

+ 50 -[-H; +28 + (j 0

+ 48 +23 +21 + 4 0

+ ii + 2 + 1 + 2 0

+ 28 0

+ 27 0

+lGH 0

Effect of re-supply of nitrate (question 2)

Re-supply of nitrate to the N-starved plants immediately stopped the N redistri­bution and the ~ concentration in all plant parts increased (Fig. 1).

Re-supply can induce a de novo synthesis of several buffer-soluble enzymes, such as ribulose bisphosphate earboxylase in rice leaves (MAKINO et al. 1984), nitrate reductase in maize (QL'ETZ et al. 1982) and nitrate reductase, nitrite reductase and glutamine synthetase in barley (BARKE IX et al. 1984). Re-addition of nitrate to the nutrient solu-

354 HPP 182 (1987) [)

tion of our plants, however, resulted in a synthesis of buffer-soluble as weIl as SDS­soluble proteins in the leaves (Table 3). Because in the older leaves both protein types increased almost equaIly, the value of the ratio after the stress period did not differ significantly from the value during early stress (Table 2). Young 1 ~-ES leaves on the other hand showed a decreased ratio of buffer-soluble to SDS-soluble proteins after re-supply of nitrate. The newly produced amount of SDS-soluble proteins was larger than that of buffer-soluble proteins ; this larger amount would be necessary for the synthesis of the latter. This may be iIIustrated by investigations of ELLrs (1979) and BRADY (1981). They reported the need of chloroplast SDS-soluble proteins for the syn­thesis of the buffer-soluble ribulose bisphosphate carboxylase. With respect to the ratio between the buffer-solublr and SDS-soluble protein contents (Table 2) it may finaIly be concluded that all 1 X-ES lraves rrmained different from the corresponding control leaves even after re-supply of nitratr. Apparently thrse lraves were not able to produce more buffN-soluble proteills for causing the ratio to reach the original value. Recupera­tion of these plants was therefore incomplete.

Re-supply of nitrate had a small effect on the amounts of non-protein N in the leaves. The increase in nitrate-K was largely restricted to the stern and roots of 1 N-ES plants (Table 3).

Effect of original nitrate level on plant reactions (question 3)

With respect to this qurstion, 1/3 K plants were compared with 1 N plants, growing on a nutrit'nt solution with 45 and 135 mg 1-1 nitrate-N, respectively. Withholding of nitrate from the nutriellt solution early or late in the development of the plant reduced the N concentration of 1/3 X plant parts relatively less than that of 1 N plant parts (compare Fig. 1b with Fig.1a). This reduction was smaller in 1/3 ~-LS parts than in 1/3 N-ES plant parts. A similar differellce was found in corresponding 1 N plant parts.

During both periods of stress, synthesis and breakdown of nitrogenous compounds were of more importancr in the organs of 1 N plants than in the 1/3 N plant parts (Table 1). Re-supply of nitrate had relatively more effect on synthetic processes in the 1/3 N-ES plants than on th08e in the 1 N-ES plants (Table 3). Omission of nitrate from the nutrient solution resuIted in the ratios of buffer-soluble to S nS-soluble proteins having the highest valurs in the oldest leaves, irrespective of the stage of plant developmen (Table 2).

Consequences of too low and too high N concentrations (question 4)

In Figs. 1 a and 1 b the theoretical maximum and minimum N concentrations of each vegetative organ are given as expected from the data of VENEKAMP et al. (1985). Some N concentrations in the 1 N-control plant parts exceeded the maximum (Fig. 1a), while many in the 1/3 N-control plant parts fell below the minimum (Fig. 1 b). Early nitrate omission from the nutrient solution resulted in N concentrations lower than the minimum, not only in all vegetative parts of the 1/3 N-ES plant parts but also in th08e of 1 N-ES plants. After re-supply of nitrate, the N concentration8 increased to 01' became high er than the theoretical minimum. In the 1 N-ES root8 they even rose to the theoretical maximum. During the period of late stress, the N concentrations

BPP 182 (1987) [) 355

Table 4. The dependence of biomass production on the difference between actual and theoretical mini-mum N cOlleentrations in fhe organs of maize plants. Data dcrived from mcasurements during the second week of the stress period.

Plant Days Control plants ~-starvcd plants

Group Organ after Biomass Actual- Biomass Actual-planting In(')'rase minimum Increase minimum

N-ronren- N-rollC'pn-tration tration

g/(l-! mg N/g dry g/d-1 mg X/g dry matter-1 matter-1

IN ears 5:2-09 +0.7 +0.1 leaves 1O-top +0.7 + 0 ° -6 leaves 7-9 +0.3 + '8 ° -1 leaves 1-6 ° + 1 ° -7 stern +1.0 + 2 ° -4 roots ° 8 ° -2

ears 66-73 + 1.3 ° leaves 1O-top -0.4 , 9 -0.1 ° T

leaves 7-9 -0.1 + 7 +0.3 +4 leavcs 1-6 ° -L 2 ° +1 stem +1.2 + 7 +1.6 ° roots +0.2 +10 ° +1

1/3 N ears 52-09 +0.2 +0.1 leaves 1O-top +0.5 ° -0.1 -9 lcaves 7-9 ° -2 -0.2 -8 leaves 1-6 -0.4 -7 ° -9 stem +0.8 () ° -4 roots ,0.:2 + 6 ° -2

cars 66-73 +0.2 ()

leaves 10-top -0.2 0 -7 leaves 7-9 ° -3 ° -8 leaves 1-6 -0.1 -7 () -7 stem +1.3 ° ° ° root8 +0.3 + 1 ° -2

in the 1 N-LS plant parts did not fall below the minimum (Fig. la). Apparently meta­bolie eonversiol1s are slow in old plal1ts.

The dependence oi biomass production on the level oi N eoncentration is given in Table 4. This production was mostly positive when the aetual N eoncel1tration was higher thall the theoretical minimum. In some eases the productiol1 was negative becltuse oi breakdown of biomass, partly as a consequence oi redistribution processes. An N concentration below the minimum did not permit synthesis oi biomass at all. However, such too low N concentrations did not prevent an incrcase oi the N concen­tration after re-supply of nitrate. Then we might conclude that N concentration limits have only a restricted meaning in relation to inerease of biomass. From the retarded development of the kerneIs during the periods of stress it is concluded that the N

356 BPP 182 (1987) 5

concentrations in the vegetative organs should be weIl above their minimum values to permit normal seed formation.

In some cases the N concentration was above thc theoretical maximum, mostly in the bcginning of the observation period and after renewal of the nutrient solution, on thc 45th and 59th d after planting. Excccding the maximum Neoncentration did not harm thc plant, but did not result in an excessive production of biomass either.

Acknowledgements

The authors arr greatly inuebteu to the late Prof. Dr. R. BROUWER, Department of Biology, State Univcrsity of Utrecht, Utreeht, Thc Nctherlanus for his valuable auvice and eneouragement in these investigations. Our grateful acknowledgement is maue to Dr. H. C. ~L DE STIGTER (Centre for Agro­biologie al Research, Wageningen, The ~ethrrlands) and to Dr. G. W. :\hLLER (Department of Biology, Utah State University, Logan, lJtah, V.S.A.) for helpful rriticism of the manuscript and eorrecting the English text.

Analyses arcording to "Official methods of analysis of the Association of Offirial Analytieal Chemists" were done at thl' Dcpartmrnt of Chemistry of the Centre for Agrobiologi('al Research.

References

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SPEK, L. Y.: Respousl' of plants to nitrogen nutrition. I. Effects of interruption of thc nitrate supply and aeration of thc nlltricnt solution on absorption and distribution of nitrogen in maize plants. Prol'o K. Ncd. Akad. Wet. (C). 87, 319-i32G (1984a).

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BPP 182 (1987) 3 357

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VEXEKAMP, J. H., PE;';XL\'G DE VWES, F. W. T., and KOOT, J. T. :JI.: Influ('m(' of different levels of nitrogen on nitrogen nlltrition and metabolism of maizr plants, Zea mll!Js L. Z. Acker- und Pflanzenbau U;', 217-22G (198ö).

Reeeired July 29, 1986; rerised for1ll {(ccfpted l\Iareh 04, 1987

Author's addrcss: Dr. J. H. VE"EK.\}]P, C('ntre for Agrobiological Research (CABO), Bornsesteeg G5, Postbus 14, 6700 AA Wageningen, The Nethcrlands.

358 BPP 182 (1987) 5