NITROGEN METABOLISM IN RED KIDNEY BEAN (PHASEOLUS …...72-22,820 FROTA, Jose Nelson Espindola, 1943-NITROGEN METABOLISM IN RED KIDNEY BEAN (PHASEOLUS VULGARIS L.) UNDER WATER AND

NITROGEN METABOLISM IN RED KIDNEY BEAN (PHASEOLUSVULGARIS L.) UNDER WATER AND SALT STRESS

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72-22,820

FROTA, Jose Nelson Espindola, 1943-NITROGEN METABOLISM IN RED KIDNEY BEAN (PHASEOLUS VULGARIS L.) UNDER WATER AND SSLT STRESS.

The University of Arizona, Ph.D., 1972 Agriculture, soil science

University Microfilms, A XEROX Company, Ann Arbor, Michigan

*- •—.r-r--rrri- T T^ ^ optt^ r v / \ r T T V D ' P f ^ P f ^ r P T )

NITROGEN METABOLISM IN

VULGARIS L.) UNDER

Jose Nelson

RED KIDNEY BEAN (PHASEOLUS

WATER AND SALT STRESS

by

Espindola Frota

A Dissertation Submitted to the Faculty of the

DEPARTMENT OF SOILS, WATER AND ENGINEERING

In Partial Fulfillment of the Requirements For the Degree of

DOCTOR OF PHILOSOPHY WITH A MAJOR IN AGRICULTURAL CHEMISTRY AND SOILS

In the Graduate College

THE UNIVERSITY OF ARIZONA

19 7 2

THE UNIVERSITY OF ARIZONA

GRADUATE COLLEGE

I hereby recommend that this dissertation prepared under my

direction by Jose Nelson Espindola Frota

entitled NITROGEN METABOLISM IN RED KIDNEY BEAN

(PHASEOLUS VULGARIS L.) UNDER WATER AND SALT STRESS

be accepted as fulfilling the dissertation requirement of the

degree of Doctor of Philosophy

jl 6 -J-UL ! 1,1)}7

Dissertation Director Date

After inspection of the final copy of the dissertation, the

following members of the Final Examination Committee concur in

its approval and recommend its acceptance:»

t /jt&k 7~Z~*

This approval and acceptance is contingent on the candidate's

adequate performance and defense of this dissertation at the

final oral examination. The inclusion of this sheet bound into the library copy of the dissertation is evidence of satisfactory

performance at the final examination.

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STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the jnajor department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.

SIGNED

ACKNOWLEDGMENTS

The author expresses his sincere thankfulness and

appreciation to Dr. Thomas C. Tucker for his ideas,

support, and guidance throughout this work.

Appreciation is extended to the members of the

Guidance Committee and the entire staff of the Department

of Soils, Water and Engineering for their contribution to

this study.

I wish to extend my appreciation to the United

States Agency for International Development (USAID) and to

Dr. William G. Matlock, Campus Coordinator of the

University of Arizona/University of Ceara contract for

their cooperation.

Special gratitude is due my wife, Vania, and my

son, Eduardo, whose affection, encouragement, and under

standing made this accomplishment possible.

iii

TABLE OF CONTENTS

Page

LIST OF TABLES vi

LIST OF ILLUSTRATIONS i*

ABSTRACT *

INTRODUCTION 1

REVIEW OF LITERATURE . . 1 3

Water Deficit Affecting Nutrient Uptake .... 3 Water Deficit Affecting Nitrogen

M e t a b o l i s m . . . . . . . . . . . . . . . . . . 6 Effects of Salinity on Nutrient Uptake 8 Effect of Salinity on Nitrogen Metabolism . . . 10

MATERIAL AND METHODS 13

Environmental Conditions 13 Treatment Selection 1^ Experimental Procedures 15

Preliminary Procedures 15 Nitrogen Uptake Experiments 17 Nitrogen Metabolism Experiments 19

Chemical Analyses 20 Solution Samples 20 Total Nitrogen 20 Calculations for Nitrogen Uptake Pattern . . 21 Calculation for Nitrogen Content in

Plant Material Absorbed from Nutrient Solution 22

Ammonium,Nitrate, a-amino Acids, Total Soluble N, and Protein-N 23

Calculation for N Content in Plant in Various N Fractions 2*fc

Statistical Analysis 25

iv

V

TABLE OF CONTENTS—Continued

Page

RESULTS AND DISCUSSION . 26

Influence of NaCl Salinity and Water Stress on Absorption Patterns of Ammonium and Nitrate by Bean Plants 26

Water and Salt Effects on the Distribution of Nitrogen Absorbed as Ammonium and Nitrate, in Bean Shoots and Roots 37

Accumulation of Nitrogenous Substances in Bean Shoots Affected by Salt and Water Stress 40

Nitrate ^0 Ammonium ^3 Free (X-Amino Acids k7 Total Soluble Nitrogen 52 Protein 57 Distribution of Nitrogen in Various

Fractions 63

SUMMARY AND CONCLUSIONS 73

APPENDIX: NITROGEN AND WATER ABSORBED IN 10 HOUR STUDY 77

LITERATURE CITED 8*1

LIST OF TABLES

Table Page

1. Concentrations of carbowax 15^0 and NaCl for different water potential levels (from Riley, 1968) ...... l4

2. Values of t test for slopes of the absorption lines of ammonium and nitrate 29

3 . Total ammonium and nitrate uptake in 1 0 hours by bean plants subjected to salt and water stress 31

4. Water absorption by bean plants under salt and water stress conditions in 10 hours 33

5- Correlation values for water absorption versus ammonium and nitrate uptake by bean plants 3^

6. Hydraulic conductivity (Lp) of the root system of bean plants affected by salt and water stress 35

7* Dry matter, nitrogen percentage, and nitrogen content in total bean plants under salt and water stress 36

15 8. Distribution of N absorbed as ammonium

in bean shoots and roots 3&

15 9. Distribution of N absorbed as nitrate xn

bean shoots and roots 38

15 /• 10. Nitrate- N in bean shoots after 6, 12, 24, and 48 hours in 15no^ solution 42

1 ̂ 11. Ammonium- N in bean shoots after 6, 12,

24, and 48 hours in solution or in 15N0^ solution 46

vi

vii

LIST OF TABLES —Continued

Table Page

15 r 12. a-amino acids- N in bean shoots after b, 12, 24, and 48 hours in soluti on or in I5N0^ solution 51

13. Total soluble-^N in bean shoots after 6, 12, 24, and 48 hours in -^NH^ solution or in solution 55

14. Protein-"*"**N in bean shoots after 6, 12, 24, and 48 hours in -^NH^ solution or in 15NC>3 solution . . 60

1 5 • . 1 5 15. Protein- N to non-protein- N ratio in

bean shoots after 6, 12, 24, and 48 hours in ^NH^ solution or l^NO^ solution 62

15 16. Nitrogenous substances, N labelled, in

bean shoots after 6 hours 64






bean shoots after 48 hours ......... 67

15 20. Distribution of total N in plant shoots

and roots after 6 hours in solution . . 69

15 21. Distribution of total N in plant shoots

and roots after 12 hours in -^N solution . . 70

22. Distribution of total in plant shoots and roots after 24 hours in ^-5n solution . . 71

15 2 3 . Distribution of total N in plant shoots and roots after 48 hours in ^5n solution . . 72

24. Influence of NaCl salinity and water stress (carboivax) on absorption of ammonium by bean plants ..... 78

viii

LIST OF TABLES—Continued

Table Page

25' Effect of NaCl salinity and water stress (carbowax) on nitrate uptake by bean plants 79

1 *5 26. Ammonium- N remained in nutrient solution . . 8 0

27• Nitrate-^^N remained in nutrient solution • . 8l

28. Effect of salt and water stress on water uptake by bean plants--aminonium treatment . 82

29. Effect of salt and water stress on water absorption by bean plants--nitrate treatment 8 3

LIST OF ILLUSTRATIONS

Figure Page

1. Influence of NaCl salinity and water stress (carbowax) on absorption of ammonium by bean plants 27

2. Effect of NaCl salinity and water stress (carbowax) on nitrate uptake by bean plants 28

3. Distribution of nitrogen absorbed as ammonium and nitrate in bean shoots (S) and roots (R) affected by NaCl salinity and water stress (carbowax) .... 39

4. Nitrate-"'"^N in bean shoots aft'er 6, 12, 24, and 48 hours in solution 4l

5 . Ammonium-"*"*^N in bean shoots after 6, 12, 24, and 48 hours in solution 44

15 r 6. Ammonium- N in bean shoots after 6, 12, 24, and 48 hours in -^NO^ solution 45

15 r 7* Ct-amino acids- N in bean shoots after 6, 12, 24, and 48 hours in ^NH^ solution * . 48

15 r 8. a-amino acids- N in bean shoots after 6, 12, 24, and 48 hours in solution ... 49

9. Total soluble-^^N in bean shoots after 6, 12, 24, and 48 hours in •®-5nh/1 solution ... 53

10. Total soluble-^N in bean shoots after 6, 12, 24, and 48 hours in ̂ NO^ solution . . . 54

11. Protein-^N in bean shoots after 6, 12, 24, and 48 hours in solution 58

15 r 12. Protein- N in bean shoots after 0, 12, 24, and 48 hours in 1 ̂NO^ solution 59

ix

ABSTRACT

15 Using inverse isotope dilution techniques with N,

the absorption patterns of ammonium and nitrate by red

kidney bean (Phaseolus vulgaris L". ) were determined under

normal conditions (control), NaCl treatment (salt stress),

and carbowax treatment (water stress). The uptake patterns

of both ammonium and nitrdte were linear functions of time

for all treatments.

Salt and water stress inhibited the absorption of

both ammonium and nitrate. There was no difference between

these two treatments in ammonium uptake, but NaCl promoted

the ujptake of nitrate compared with carbowax. Under NaCl

salinity the plants absorbed the same amount of nitrogen

independent of nitrogen source; however, under water stress

bean plants absorbed more ammonium than nitrate.

Salt and water stress reduced the absorption of

water, but no difference was observed between NaCl and

carbowax. A high positive correlation between nitrogen

and water uptake was obtained. A decrease in root permea

bility was found in plants subjected to stress.

In plants subjected to stress, dry matter produc

tion was decreased and the nitrogen percentage increased,

but the total nitrogen per plant was reduced. The

x

xi

treatments did not affect the distribution in shoots and

1 "5 roots of absorbed as ammonium or nitrate.

15 In another series of experiments, the N in

nitrate, ammonium, free a-amino acids, total soluble

nitrogen, and protein forms in plant shoots was analyzed,

after 6, 12, 24, and 48 hours in plants grown in (NH^J^SO^

and KNO^ solutions.

Sodium chloride and water stress promoted to an

i equal extent the accumulation of nitrate in bean shoots.

An increase in the concentration of ammonium and

free a-amino acids was found in plants subjected to salt

and water stress. There was no difference between NaCl and

carbowax treatments on the accumulation of both ammonium

and free Ot-amino acids. When ammonium was used as the

nitrogen source more ammonium accumulated compared with

nitrate as the source of nitrogen.

Protein synthesis was significantly reduced in bean

shoots when the plants were subjected to NaCl salinity and

carbowax, with either nitrogen source. The inhibition of

protein synthesis was more severe under salt stress. Plants

receiving nitrate as the nitrogen source synthesized more

protein than with ammonium as the source. The protein to

non-protein ratio in control plants was higher than in

stressed plants regardless of nitrogen source. Plants

grown under saline conditions showed a lower protein to

non-protein ratio than water stressed plants.

xii

15 Seventy to eighty per cent of the total N ab

sorbed as ammonium or nitrate in control plants was found

in protein form in and after 12 hours; however, in stressed

15 plants a large portion of the N was in the non-protein

fraction. The percentage of a-amino acids was higher in

plants under stress than in control plants at all times.

INTRODUCTION

Agricultural production in many parts of the world

is severely reduced by water deficit and/or excess of salts.

Approximately 60 per cent of all potentially arable lands

in the world experience six months or more of moisture

deficit each year (Thorne, 1970). The salinity problem is

most conspicuous in arid and semi-arid zones due to lack of

leaching of soluble salts from the rhizosphere.

Water and salt stresses reduce plant growth by

modifying the physiological processes and conditions which

control growth. However, it is not completely understood

how the physiological processes are modified. Even though

plant nitrogen metabolism under normal conditions is well

known, in plants under water and/or salt stress the data

available are incomplete and some controversial. It is

believed that the change in nitrogen metabolism induced by

water deficit and excess of salts has been one of the most

important factors responsible for abnormal plant metabolism

under those conditions. Therefore, understanding the

modifications in nitrogen metabolism in plants cultivated

under water and salt stresses would make it possible to

elucidate the mechanisms by which water deficit and

salinity affect plant metabolism, thereby reducing growth

and development.

1

2

The observed effects on nitrogen metabolism and on

plant growth under water deficit have been related to a

decrease in water uptake caused by osmotic effects. How

ever, under saline conditions the reduction in growth and

changes in nitrogen metabolism can be related to osmotic

effects and to specific or toxic effects of ions due to

accumulation of salts in plants.

The objectives of this investigation were:

1. To study the effect of water and salt stress on

plant absorption of ammonium and nitrate.

2. To study the various steps of nitrogen metabolism

in plants grown under water deficit and salinity

conditions, and to determine which step is more

affected by the stress.

3. To study the osmotic and ionic effect separately in

nitrogen uptake and metabolism.

REVIEW OF LITERATURE

Water Deficit Affecting Nutrient Uptake

The effect of a decrease in available water to

plants on mineral nutrition is difficult to resolve clearly.

The literature dealing with the subject is controversial

and not conclusive. When plant growth is limited by soil

moisture deficit, total mineral nutrient uptake will likely

be limited (Jenne et al. , 1958)* However, the effect of

decreasing water supply is not necessarily of the same

magnitude for growth as for mineral nutrient absorption,

since ion uptake is not directly related to the absorption

of water (Russell and Barber, i960). Wadleigh and Richards

(1951) and Fawcett and Quirk (1962) believe that while the

soil water is in the range available to plants the soil

nutrient availability, per s_e, is not necessarily affected

by water deficit. However, nutrient mobility should be

considered. Fried and Shapiro (1961) think that the mobile

nutrients such as nitrate-nitrogen, can be supplied to

plants by water movement, but immobile nutrients, such as

phosphates, need another type of transport to be adequately

supplied to plants* This theory is supported by several

investigations (Danielson and Russell, 1957; Wiersum, 1958;

Peters and Russell, i960; Mederski and Stackhouse, i960).

3

k

Greenway, Klepper, and Hughes (1968) working with

excised roots of barley and tomato plants observed a de

crease in ion accumulation in roots when submitted to water

deficit. However, they concluded that the decrease was

caused by the increased leakage from cells rather than an

effect on the mechanisms of ion uptake. Metwally and

Pollard (1959) observed that the nutrient uptake by barley

seedlings was always greater at higher moisture levels.

Phosphorus intake by barley shoots was considerably reduced

by low-water treatment and the net assimilation rates of

nitrogen in leaves were depressed (Williams and Shapter,

1955)* Gates (1957) submitted young tomato plants to a

brief period of Abater shortage and the absorption of nitro

gen and phosphorus was significantly reduced. Upon re-

watering, active uptake of both nutrients was resumed.

Meyer and Gingrich (1966) showed a decrease in phosphorus

and nitrogen percentage in wheat plants under osmotic stress

(carbowax treatment) and concluded that the absorption of

both nutrients decreased after application of the stress.

The absorption of phosphorus by 2-day-old corn plants was a

linear function of soil moisture content during 24 hours

(Olsen, Watanabe, and Danielson, 1961). Increasing the

soil moisture from the wilting percentage to saturation

resulted in significant increases in the uptake of nitrogen,

phosphorus, potassium, and calcium by cotton and soybean

plants (Brown, Place, and Pettiet, i960). An increase in

5

potassium uptake was also observed by Mederski and Stack-

house (i960) in corn plants when the soil moisture condi

tions increased. Reichman and Grunes (1966) observed a

decrease in phosporus absorption by barley under high

moisture stress, but the maximum soil suction did not

affect the total nitrogen absorbed by the plants. However,

Fawcett and Quirk (1962) found that the absorption of phos

phorus by young wheat plants was not affected by increasing

soil water stress.

Several investigators (Miller and Duley, 1925?

Abdel Rahman, Shalaby, and El-Monayeri, 19715 Hosner et al.,

1965; Wadleigh and Richards, 1951) reported that a decrease

in soil water availability causes an increase in nitrogen

content in plants. However, as Hosner et al. (1965) pointed

out a dilution effect must be considered. Water stress

affects growth more than nutrient uptake; as a consequence

the relative content of nutrients is higher for stressed

plants, which is evident particularly for calcium and

nitrogen. However, the total nutrient content in the whole

plant is closely correlated with growth. Therefore, in

plants growing under water stress the total content of

nutrients is lower than in plants growing under normal con

ditions , even though stressed plants have a higher concen

tration of the nutrient.

6

Water Deficit Affecting Nitrogen Metabolism

In studies dealing with changes in metabolic pro

cesses of plants grovm under conditions of water deficit,

particular attention has been given to changes in nitrogen

compounds. However, it is not completely clear how the

water stress affects the various aspects of nitrogen metab

olism (Kramer, 19&9).

Gardner and Nieman (1964) reported a marked decrease

in DNA in cotyledonary leaves of radish plants subjected to

water stress. However, this decrease was observed only at

the initial stage of stress development, after which the

level of DNA became relatively constant and independent of

further increase in stress. Shah and Loomis (1965) observed

that RNA synthesis in sugar beets was impaired by stress

and degradation of previously synthesized RNA also occurred.

Similar results were obtained by Zhollcevitch and Koretskaya

(1959) in pumpkin roots. However, Gates and Bonner (1959)

suggested that RNA synthesis continued under stress but a

rapid breakdown of the RNA formed is responsible for the

decrease in total RNA in young tomato leaves. Dove (X967)

also observed an increase in ribonuclease activity in

tomato leaflets caused by water stress. These results

contrast with the report of West (1962) who found an in

crease in RNA content in germinating corn seedlings sub

jected to water stress.

7

According to Petrie and Wood (1938), Mothes (1956),

Todd and Yoo (1964) the protein content in plants decreases

under water stress. Chen, Kessler, and Monselise (1964)

showed that the protein level corresponded differentially

to increasing water stress, increasing at the beginning of

dehydration, decreasing at medium dehydration, and increas

ing again slightly under extreme dehydration conditions.

Ben-Zioni, Itai, and Vaadia (1967) reported that water

stress reduced the ability of tobacco leaves to incorporate

14 1-leucine C into protein. Bermudagrass submitted to water

stress showed an inhibition of protein synthesis with a

consequent decrease in protein level and a continued syn

thesis of amino acid (Barnett and Naylor, 1966). They ob

served an accumulation of free proline, free asparagine,

and valine in stressed plants, but the levels of glutamic

acid and alanine decreased. Changes in the level of free

amino acids and atnides caused by water stress have been

observed also by other investigators (Kudev, 1967;

Prusaltova, i960; Mothes, 1956; Kemble and MacPherson, 195^5

Palfi and Juhasz, 1968). Mothes (1956) thinks that the

accumulation of amides might be the result of incorporation

of free ammonia released by deainination of amino acids

which were in turn released by the breakdown of proteins

induced by water stress. Lahiri and Singh (1968) found an

inhibition of protein synthesis and consequently an increase

8

in amino acids, amides, ammonia, nitrite, and nitrate due

to water deficit in plants.

Todd and Yoo (196*0 showed a decrease in the

activity of several enzymes in detached wheat leaves sub

jected to water stress. Lahiri and Singh (1968) suggested

that the accumulation of nitrite and nitrate is caused by

an effect of water shortage on the enzymes responsible for

nitrate reduction. Younis et al. (1965) presented evidence

that water stress decreases the nitrate reductase activity.

A significant decrease in the activity of both nitrate and

nitrite reductase, caused by water stress in barley plants,

was found also by Huffaker et al. (1971)•

Effects of Salinity on Nutrient Uptake

Excessive accumulation of soluble salts in soil is

responsible for a decrease in osmotic potential of the soil

solution and for an excess of ions in the root zone causing

an unbalanced nutritional medium and specific ion toxicities

The absorption of a given ion is affected by the concentra

tion of that ion and also by the presence and concentration

of other ions in the rhizosphere. The more kinds of ions

present in soil or nutrient solution the more complicated

are the interactions.

Greenway (1962a, 1962b) reported ionic changes in

plants submitted to saline substrates. Hussan et al. (1970)

found a significant decrease in the absorption of phosphorus

9

potassium, calcium, iron, and copper in barley and corn

plants when the. level of salts in the soil were artificially

increased with Na^SO^, MgSO^, and CaCl^. In both species

the uptake of sodium increased significantly. Gauch and

Wadleigh (19^) studied the effect of high concentration of

salts on ionic absorption by bean plants and observed a

decrease in calcium, potassium, and nitrogen content, an

increase in the level of sodium, chloride, and sulfate, and

little effect on phosphate concentration. Several investi

gators have reported a decrease in nitrogen content in

plants, caused by excess of salt in soil and nutrient solu

tion (Kretschmer, Toth, and Bear, 1953? Lunin and Gallatin,

1965; Mattas and Pauli, 1965? Wilson, Haydock, and Robins,

1970; Udovenko, Sinelnikova, and Khazova, 1971)* Palfi

(1963, 1965) reported that Na^SO^ and NaCl in the nutrient

solution inhibited the absorption of nitrogen and phosphate.

According to him the large amounts of sodium absorbed im

paired the uptake of ammonium ions by rice plants, and

sulfate and chloride ions were antagonistic to phosphate

uptake. Inhibition of nitrate uptake by chloride, bromide,

and iodide was observed by Lontai, Cseh, and Boszormenyi

(1967)* However, Reifenberg and Rosousky (19^7) and

Hernando, Jimeno, and Cadahia (1967) found that nitrate up

take by plants was not influenced by the presence of sodium

chloride. Langdale and Thomas (1973-) and Khalil, Ainer, and

Elgabaly (1967) also found that soil salinity did not

10

inhibit the absorption of nitrogen by plants. No marked

changes in nitrogen, phosphorus, iron, copper, and zinc

content in crops in presence of choride or sulfate was

observed by Dilley et al. (1958)* The presence of chloride

and sulfate sodium had little effect on total nitrogen and

phosphorus concentration in barley plants according to

Gauch and Eaton (l9*t2), but in this case the level of cal

cium and potassium was reduced.

In contrast some authors have reported an increase

in nitrogen content in plants growing in saline substrate

(Bernstein and Pearson, 1956; Wadleigh and Ayres, 19^5;

Bhardwaj and Rao, 1962; Shimose, 1963; Strogonov, 1964;

Lunin, Gallatin, and Batchelder, 1964). As was already

mentioned for water stress, these different results were

probably due to a dilution effect. In other words, the

plant growth was impaired more severely by salt than was

the nitrogen uptake.

Effect of Salinity on Nitrogen Metabolism

The specific conditions prevailing in saline soils

markedly influence plant metabolism. Schimper as cited by

Prisco (1971) proposed that the deleterious effects of

salinity were due to decreased osmotic potential in the

root medium and consequently the plants wex*e exposed to

physiological drought. This theory was accepted for many

years. In the early 1960's Bernstein (1961, 1963) proposed

11

that the inhibition in metabolism might be due to effects

of osmotic concentration on various enzyme systems. In

fact excess salts in the root medium has been shown to

affect several enzyme activities (Kessler et al•, 1964;

Weimberg, 1970; Livne and Levin, 1967; Porath and Polja-

koff-Mayber, 1964, 1970» Mattas and Pauli, 1965)*

According to Rauser and Hanson (1966) the DNA and

RNA synthesis in soybean roots exposed to saline media was

inhibited. A strong suppression on DNA and RNA accumula

tion was also observed by Kessler et al. (1964).

Sarin (1961) working with wheat seedlings observed

that excess of Na^SO^ in nutrient solution disturbed the

cation balance and protein metabolism. Kahane and Polja-

koff-Mayber (1968a, 1968b) reported a decrease in incor

poration of amino acids into pea root protein. Several

other investigators showed a depressing effect of salts on

protein synthesis (Nightingale and Farnham, 1936; Lapina,

1966; El-Shourbagy, 1964; Prisco and O'Leary, 1970, 1971)•

Palfi (1963, 1965) and Palfi and Juhasz (1968) found a

great increase in total amino acids and amides in plants

exposed to salt stress. Udovenko, Sinelnikova, and

Khazova (1970) obtained an accumulation of ammonia in

barley, wheat, peas, and beans under saline conditions.

Accumulation of amino acids, amides, and ammonia

has also been reported by Strogonov (1964). This author

emphasized the poisoning effect of accumulated toxic

12

intermediaries of metabolism, among them intermediaries of

nitrogen metabolism, in plant cells submitted to salinity.

MATERIAL AND METHODS

Red kidney bean (Phaseolus vulgaris L.) plants were

used in this work. This plant was selected because i.t is

relatively salt sensitive (Richards, 195^) and has been

studied extensively as far as the effect of salinity on its

growth and metabolism is concerned (Gauch and Wadleigh,

Lagerwerff and Eagle, 196la; Bernstein, 1 9 6 1 , 19&3;

Riley, 1968; O'Leary and Prisco, 1970; Prisco and O'Leary,

1970, 1971; Prisco, 1971).

Environmental Conditions

All experiments were conducted in a walk-in growth

chamber. The light intensity at the upper leaf surface

was approximately 2,000 ft-c., provided by 12, 100-watt

tungsten bulbs and 28 fluorescent bulbs, type F 96T12

CW/VHO.

Day length consisted of a 12 hour photoperiod. The

temperature was 25 C in the light period and 20 C during

the dark period, and the relative humidity was 45-50 and

60-65 per cent at light and dark periods, respectively.

Both temperature and relative humidity were recorded on a

hygrothermograph.

13

14

Treatment Selection

To distinguish between osmotic and specific ion

effects on plant growth and metabolism a polyethylene

glycol (carbowax), average molecular weight 15^0 (Matheson,

Coleman, & Bell) was used. This chemical lowers the water

potential of the nutrient solution and is not absorbed by

plants (Riley, 1 9 6 8 ) . Table 1 shows the amount of carbowax

used to obtain the desired decrease in water potential.

T

Table 1. Concentrations of carbowax 1J540 and NaCl for different water potential levels (from Riley, 1968).

Water potential (bars)

Carbowax 15^0 (g in 1 liter H^O)

NaCl (meq/liter)

-1 47.5 24.0

-2 75-0 48.0

-3 97-5 72.0

-k 119.0 9 6.O

The salinity effect was achieved by adding NaCl to

the nutrient solution (Table l). This salt was chosen for

this work because it contains the most common ions, Na and

Cl , found in saline soils (Mulwani and Pollard, 1939)*

The water potential of the nutrient solution in all

15

experiments was -4 bars based on pi-evious studies (Prisco,

1969, 1971j 0'Leary and Prisco, 1970)*

Since nitrogen is absorbed mainly in ammonium and

nitrate foi-m by plants and since these two ions probably

are absorbed by different mechanisms, both were used as

nitrogen sources in the nutrient solution. By using

nitrate it was also possible to study the reduction of

nitrate in plants grown under water and salt stress.

Experimental Procedures

15 The nitrogen sources used were N-enriched

(NH^SO^ (95 A % 15N) and KNO^ (96.6 A % 15N). An inverse

isotope dilution technique (Frota and Tucker, 1972) was

used to measure the uptake of nitrogen by plants, and the

absorption rate and patterns of both ammonium and nitrate

nitrogen were determined. The various nitrogenous sub

stances (nitrate, ammonium, free a-amino acids, total

15 soluble nitrogen, and protein nitrogen) labelled with N

were determined at different time intervals (6, 12, 24, andtb

48 hours).

Preliminary Procedures

Red kidney bean seeds (W. Atlee Burpee Company,

Riverside, California, Lot p3l) were germinated on brown

paper (Kimberly-Clark Corporation) and after a period of

7 days the seedlings were transplanted into complete

Hoagland and Arnon (1950) nutrient solution No. 1. Two

l6

plants were used in each two liter brown polyethylene

container covered to avoid evaporation. The cover contained

two holes where the plants were fitted by wrapping a piece

of plastic foam around the hypocotyl.

The source of iron was iron chelate at a concentra

tion of 1 ppm Pe in the nutrient solution. The pH of the

solution was regulated to about 6.0 by adding 0.1 N NaOH

solution.

Two days after the plants had been transplanted,

the water potential of the nutrient solution was gradually

decreased (to provide osmotic adjustment) by adding carbo-

wax, corresponding to water stress treatment, and NaCl,

salt treatment, one bar every two days, until the desired

level of water potential (-k bars) was reached. The level

of the nutrient solution in the containers was maintained

during the experiments by adding deionized water.

During the 13-day growth period in these containers

the nutrient solution was changed twice. After the growth

period the plants were transferred to other similar con

tainers containing 1/10 Hoagland solution without nitrogen,

and the treatments with the desired amount of carbowax and

NaCl for 3 days.

The air used for aeration of the nutrient solution

was cleaned, by passing it through 6 N H^SO^ and then

through distilled water into a thick wall rubber tubing.

The flow of air from the rubber tubing was regulated by

17

hypodermic needles inserted into the main air tube and

connected to short sections of glass tubing immersed in the

nutrient solution.

From germination through the end of the experiments

the plants were kept in a growth chamber with the environ

mental conditions described above.

Nitrogen Uptake Experiments

Pots containing two liters of 1/10 Hoagland

nutrient solution (control), 1/10 nutrient solution plus

238.O g of carbowax (water stress), and 1/10 nutrient

solution plus 96 meq/liter of NaCl (salt stress), all

without nitrogen were used. Nitrogen solutions containing

15 approximately Ik mg of N as (NH^J^SO^ or KNO^ were added

to each pot. The nutrient solution was agitated during

the experiment with a magnetic stirrer in each pot.

Initial two ml solution samples were taken from

each pot, and two plants were transferred to each pot. Two

ml samples were taken hourly for 10 hours. All solution

samples were added to vials containing 3 ml of (NH^J^SO^

solution (ammonium treatment) or KNO^ solution (nitrate

treatment) corresponding to 1.000 mg of normal nitrogen.

The solution level in the pots was maintained at

two liters by adding deionized water. The water added was

measured, recorded, and called water absorbed.

18

At the end of the 10 hour study the plants were

removed from the nutrient solution and root permeability

determined. The root systems were immersed in distilled

water and enclosed in a pressure chamber (O'Leary, 1965)

and a constant pressure of two bars was applied. The

exudate from the stumps of the root systems was collected

at 30 minute intervals for two hours. The root systems

then were removed from the pressure chamber and weighed.

The surface area of the root systems was determined on the

basis of the relationship between root fresh weight and

surface area (O'Leary, 1971)• By the use of a regression

equation relating electric conductivity in mmhos/cm to

osmotic pressure (Riley, 1968), the osmotic pressure of the

root exudate was measured. Root permeability expressed as

hydraulic conductivity of the root surface was determined

by using the following equation:

J = L (AY) w p

where 3 -2 J = water flow across the root (cm . cm . sec )

w

Lp = hydraulic conductivity of the root surface'

(cm. sec . bar "*")

AT = the water potential gradient from the external

solution to the interior of the root (bars)

The plant material, roots, and shoots was dried in

an oven at 60 C and" dry weights were determined. Three

replications were used, each at a different time.

19

Nitrogen Metabolism Experiments

A similar basic procedure was followed in prepara

tion for and during the conduct of the nitrogen metabolism

experiment. Nitrogen solutions containing approximately

15 10, 15, 20, and 30 mg of N in ammonium sulfate or

potassium nitrate form were added to individual pots. The

10, 15, 20, and 30 mg of. corresponded to 6, 12, 24, and

48 hours study, respectively.

Initial two ml solution samples were taken and

added to the vials containing 1.000 mg of normal nitrogen

and mixed. Two plants were then transferred to each pot.

After 6 hours the first set of plants (control, carbowax,

and NaCl treatments) were harvested. After 12, 24, and 48

hours from the initial time the other sets of plants were

harvested. Immediately before harvesting the plants, two

ml solution samples were taken from each pot and added to

the vials containing 1.000 mg of normal nitrogen. These

and the initial solution samples were used to determine

the total nitrogen absorbed.

After harvesting, the plants were separated into

shoots and roots and frozen in dry ice. The material was

lyophylized for three days at 0.05 mm Hg pressure, weighed,

ground to pass a 40-mesh screen in a Willey mill, and

stored in a refrigerator until analyzed for ammonium,

nitrate, free oc-aniino acids, total soluble nitrogen, and

20

15 protein nitrogen. The distribution of N among the

various plant nitrogen fractions was determined.

Chemical Analyses

Solution Samples

15 The atom per cent N in solution samples was cal

culated from the 28 to 29 mass ratio determined by a mass

spectrometer (Consolidated Electrodynamics Corporation,

Model 21-621) following the methods described by Bremner

(1965a)• The nitrate samples were first reduced to ammonium

15 by Devardas Alloy, and then analyzed for N according to

procedure described for ammonium.

Total Nitrogen

The total nitrogen in the shoots and roots from the

uptake experiments and roots from the metabolism study was

analyzed by micro-Kjeldahl determination. Samples of 100

mg of plant material were digested using four ml of con

centrated HgSOj^ and 1.5 g of the mixture 100:10:1 (K^SO^,

CuSO^, Se) until a yellowish-green color appeared. After

the flask was cooled, about 20 ml of deionized water was

added to dilute the HgSO^. The nitrate samples were

reduced to ammonium by Devardas Alloy. Approximately 20 ml

k0% NaOH was added and the flask connected to steam distil

lation apparatus. The ammonia liberated was collected in

5 ml boric acid indicator (li0 g boric acid in 9500 ml of

21

CO -free deionized water plus 50 ml of 0*1% bromocresol 2

green and 0.1% methyl red in a 5:1 ratio). After titration

with 0.01 N KH(I0^)2 the samples were redistilled by adding

10 ml li0% NaOH solution. The ammonia recovered in 1 ml

15 0.5 N Hg, 0^ was saved for atom per cent N determination.

15 The determination of atom per cent N followed the same

procedure as for solution samples.

Calculations for Nitrogen Uptake Pattern

15 Prom the atom per cent N solution samples, nitro

gen absorption at different time intervals was calculated

from the equations below:

At. = ztn - zt. a. 0 l

V x Jlg^N x A% Ex. 15N Z =

1 ̂ S x A% N enrichment in source v

where

Z = total nitrogen remaining in solution, jig

V = volume of root solution, 2000 ml

jXg lZtN = 1000 [lg N as (NH^SO^ or KNO^

15 15 A % Ex. N = atom per cent excess N in solution

samples (J^N A % - natural abundance A

= samples volume, 2 ml

The total nitrogen absorbed at a given time, A , v • X

is equal to the initial nitrogen in solution, Z, , less 0

the amount remaining at a given time, .

22

Calculation for Nitrogen Content in Plant Material Absorbed from Nutrient Solution

15 From the atom per cent N and total nitrogen in *

15 shoots and roots the N can be calculated:

mg N titrated = N KH(I0 ) x (volume titration j £

- volume blank) x l*t

^ x P x A% Ex. 15N S w ip i ^

^ A% enrichment

where

T JJ = total nitrogen recovered from plant due to

absorption, |ig

= N in plant sample by micro Kjedahl digestion, mg

•S = plant sample weight for analysis, mg

P = total plant part weight, p,g

15 15 Ex. N = atom per cent excess

15 o/„ _ enrichment per cent

T/ . % R = - ( t + r )

T n sl

where

R = per cent recovery of N in plant due to absorption

from solution

15 T = total g N lost from solution due to absorption

15 T(£ + ̂ = total N in plant shoot and root

23

Ammonium, Nitrate, a-amino Acids, Total Soluble N, and Protein-N

The plant shoots of the metabolism experiment were

analyzed for nitrate, ammonium, a-amino acids N, total

soluble-N, and protein-N. Approximately 2.5 g of ground

material was placed in a Soxhlet extractor and extracted

with 75 ml of Q0% ethanol for 24 hours. The alcoholic

extract was transfered to a 100 ml volumetric flask, the

volume completed, and stored in a refrigerator until

analyzed for nitrate-N, ammoniutn-N, free a-amino acids-N,

and total soluble-N (Bremner, 1965b). The extracted resi-*

due, called total protein, was lyophilized for 48 hours at

0.05 nun Hg and stored in a refrigerator for protein-N

analysis.

Ammonium-N. Thirty ml aliquot of the extract was

distilled by steam distillation with MgO. The amount of

ammonia liberated was collected in 5 nil boric acid-

indicator. After titration with 0.01 N KHtlO^)^, 10 ml

k0% NaOH was added and the samples redistilled. The

ammonia was collected in 1 ml 0.5 N H^SO^ and saved for

15 15 atom per cent N determination. Before N determination

the alcohol was evaporated in a vacuum-oven at 60 C.

Ammonium + Nitrate-N. Thirty ml aliquot of the

extract was distilled by steam distillation with MgO plus

2k

Devairdas Alloy. From this point the same procedure used

for ammonium was followed.

Free cx-amino Acids-N. Twenty ml aliquot of the

extract plus 1 ml 0.5 N, NaOH was placed in the distillation

flask and heated in boiling water until the volume was

reduced to 2-3 ml (about ^5 minutes). The flask was allowed

to cool, and 500 mg of citric acid and 100 mg ninhydrin was

added. The flask was returned to boiling water for 10

minutes. When the flask was cooled, 10 ml of phosphate

borate buffer and 1 ml of 5 N. NaOH was added. By steam

15 distillation the nitrogen was determined and saved for N

analysis after redistillation.

Total Soluble-N. Twenty ml aliquot of the extract

was acidified with concentrated H^SO^ and heated to reduce

the volume. The nitrogen was determined by the same pro

cedure used for total nitrogen. The nitrate samples were

reduced by Devardas Alloy.

Protein-N. The nitrogen in the total protein

residue was analyzed by the same procedure used for total

nitrogen.

Calculation for N Content in Plant in Various N Fractions

For alcoholic extract:

mg N = (mg N titrated)(100/A)(D/T)

25

•where

A = ml of aliquot taken

D = dry weight of shoots

T = dry weight of sample extract.

For alcoholic residue:

mg N = (mg N titrate)(D/C)

where

C = weight of digested sample.

For N-15:

15» „ A% Ex. 15N jig N = jigN

A% enrichment

where

p,N = N determined by the equations above

A% Ex.^^N = atom per cent excess

k% = enrichment.

Statistical Analysis

Linear regression, correlation values, and analysis

of variance were obtained from the data according to

methods outlined by Steel and Torrie (i960). The Duncan

New Multiple Range Test was used to compare the means.

RESULTS AND DISCUSSION

Influence of NaCl Salinity and Water Stress on Absorption Patterns of Ammonium and Nitrate by Bean Plants

The mean values for ammonium absorption by bean

plants in 10 hours under normal Hoagland solution (con

trol) , Hoagland solution plus 96 meq NaCl/liter (salt

stress), and Hoagland solution plus 119 g of carbowax/liter

(water stress) are presented in Figure 1. Figure 2 shows

the nitrate uptake under the. same conditions. The indi

vidual points used to draw the curves in Figures 1 and 2

are presented in Tables 24 and 25 in the Appendix. Linear

regression analyses were performed on these data and the

slope (b) , intercept (a), and correlation coefficient (r)

between ammonium and nitrate uptake and time are also

given.

A high degree of association between ammonium and

nitrate absorption by bean plants and time at all treat

ments was characterized by the high correlation coefficient,

r, obtained. The statistical analyses for comparing the

slopes of the absorption lines are shown in Table 2. These

results suggest that the uptake rate of ammonium and

nitrate is reduced when bean plants are subjected to salt

or water stress. A higher uptake rate was observed for

nitrate than ammonium in the controls, and a higher

26

2.0

1.5

U i)

*-» d E

2? 1.0 "O CT1

z in O) E

.5

•o CONTROL

Na CI

A A CARBOWAX

/I _ * w

1 0

T I M E ( h o u r s )

Figure 1» Influence of NaCl salinity and water stress (carbowax) on absorption of ammonium by bean plants.

2.0

O C O N T R O L

• Na CI

C A R B O W A X

1.5

u V

a E

u 1.0 -a

cn

z in

cn E

5 10


Figure 2. Effect of NaC.l salinity and water stress (carbowax) on nitrate uptake by bean plants.

29

Table 2. Values of t test for lines of ammonium and

slopes of the nitrate.

abs orption

Treatment t values

Ammonium vs. nitrate—control 2.79*

Ammonium vs. nitrate—NaCl 1.6l n.s.

Ammoniuin vs. nitrate-~carbowax 3.37*

Ammonium---NaCl vs. carbowax 1*3^ n.s.

Nitrate— -NaCl vs. carbowax 2.90*

*Significant at .05 level.

30

ammonium absorption rate in water stress treatment (carbo-

wax). However, no difference was observed between the

absorption rate of ammonium and nitrate in the salinity

treatment. The absorption rate of ammonium was the same

for salt and water stressed plants, but the nitrate

uptake rate was higher in NaCl salinity than in water

stress. It was not possible to detect the ion effect on

total nitrate uptake in 10 hours by using the standard

error based on all observation (Table 3)« This table shows

a significant decrease in uptake of both ammonium and

nitrate, by intact bean plants when submitted to salt or

water stress. A decrease in nitrogen absorption by plants

under osmotic stress was also observed by Gates (1957) and

Meyer and Gingrich (1966), In contrast some authors

(Miller and Duley, 19255 Abdel Rahman et al., 19715 Hosner

et al. , 1965; Wadleigh and Richards, 1951) reported an

increase in nitrogen content in plants caused by soil

.'water deficit. However, these investigators did not

measure the real absorption since their results are

nitrogen concentration rather than total uptake. -

A decrease in nitrogen uptake by plants growing in

salinity substrate is supported by the works of Kretschmer

et al. (1953), Lunin and Gallatin (19&5), Mattas and Pauli

(1965), Wilson et al. (1970), and Udovenko et al. (1971)*

However, Reifenberg and Rosousky (19^7) and Hernando et al.

(1967) found that nitrate uptake by plants was not

31

Table 3* Total ammonium and nitrate uptake in 10 hours by bean plants subjected to salt and water stress.

Treatments

g "^N/g dry matter

Treatments Ammonium Nitrate

Control 1.899 a 1.717 a

NaCl O.883 be 0.7^5 be

Carbowax 1.122 b 0.517 c

s- = O.165.

All values followed by the same letter are not significantly different at the .05 level.

influenced by excess of NaCl in the root zone. Langdale

and Thomas (1971) and Khalil et al* (1967) also observed

that soil salinity did not inhibit the absorption of

nitrogen by plants. The conclusions of these investigators

are questionable since the absorption process per se was

not determined, but the conclusions were based on nitrogen

concentration in plants.

According to Palfi (1963, 1965) the uptake of

ammonium was impaired by Na ions when this ion was in

excess in the nutrient solution. Inhibition of nitrate

absorption by Cl ions was observed by Lontai et al.

(1967). S ince the absorption of ammonium by plants

submitted to NaCl salinity and water stress was not

32

different (Table 3) "the observed decrease in uptake

compared with the controls was due to osmotic effects

+ — only, that is, the Na and Cl ions did not affect the

absorption of ammonium by bean plants, but they affected

the absorption rate of nitrate. Gauch and Eaton (19^2)

also observed no influence of NaCl on nitrogen absorption.

Ammonium and nitrate were absorbed equally in

NaCl treatment; however, under water stress bean plants

absorbed more ammonium than nitrate.

Even though the absorption rate of nitrate was

higher than the ammonium uptake rate in control plants,

there was no difference in the total absorption of both

ions in 10 hours. This can be explained by the low level

of nitrate in nutrient solution after six hours. When the

nitrate content in the nutrient solution reached a level

of approximately 2 ppm (six hours) the absorption rate

diminished, suggesting that at this level the concentra

tion of nitrate in the nutrient solution was a limiting

factor. The concentration of ammonium in the pots was

approximately 6 ppm at the end of the 10 hour study

(Tables 26 and 27 i*i the Appendix) .

The total water absorbed in 10 hours is presented

in Table 4. The plants subjected to salt and water stress

absorbed significantly less water than the controls. No

ionic effect was observed in the uptake of water, since

there was no difference in the absorption of water between

33

Table h. Water absorption by bean plants under salt and water stress conditions in 10 hours.

Ml of water/pot

Treatments Ammonium Nitrate

Control 164.3 a 175*3 a

NaCl 60.0 b 61.3 b

Carbowax 72 .Ob 59.6 b

s- = 5.5. x


NaCl and carbowax treatments. Tables 28 and 29 in the

Appendix show the amount of water absorbed hourly during

10 hours. These values are probably associated with the

relative loss of water transpired by the plants.

A high positive correlation between ion and water

uptake was observed (Table 5) in all treatments. Since

NaCl and carbowax stressed plants absorbed significantly

less water than the controls, the decrease in water

absorption might be at least one factor responsible for

the decrease in ammonium and nitrate uptake by stressed

plants. Data in the literature are controversial as far

as the relationship between water and nutrient uptake is

concerned. Whereas some authors think that there is a

34

Table 5* Correlation values for water absorption versus ammonium and nitrate uptake by bean plants.

Treatments

Ammonium Nitrate

Carbo- Carbo-Control NaCl wax Control NaCl wax

Correlation (r) .985** .907** .674** .805** .909** .978**

Slope (b) .0112 .0134 .0124 .0145 .0120 .0096

sb .00037 .00118 .00257 .00268 . oo io4 .00039

* ""Significant at .01 level.

quantitative relation between the two processes (Epstein,

1956; Hylmo, 1953; Kylin and Hylmo, 1957; Kramer, 1956)

others showed evidence that the ion and water absorption

are independent to each other (Lagert^erff and Eagle, 196lb;

Hoagland and Broyer, 1942; Brower, 1956; Honert, Hooymans,

and Vollcers, 1955)-

The root permeability, expressed as the hydraulic

conductivity of the root system is presented in Table 6.

The root permeability was significantly decreased by NaCl

and carbowax, but there was no difference between these two

treatments, suggesting no ionic effect on the hydraulic

conductivity of the roots. The decrease in root

35

Table 6. Hydraulic conductivity (Lp) of the root system of bean plants affected by salt and water stress.

L (cm.sec. har ^ x 10 ) P „

Treatment Ammonium Nitrate

Control 1.64 a I.85 a

NaCl 0.27 b 0.25 b

Carbowax . 0.?6 b 0.83b

s- - 0.2k. x


permeability might be another factor affecting the observed

decrease in nutrient uptake.

Table 7 shows the dry matter production, per

centage of nitrogen, and total nitrogen in plants. The

plants under stress had their dry matter production

reduced. Although NaCl and carbowax inhibited the absorp

tion of ammonium and nitrate, in both cases the plants, had

a higher percentage of nitrogen than the controls. How

ever, the total nitrogen per plant is quite higher in

control plants.

The fact that plants grown in NaCl salinity and

carbowax, even absorbing less nitrogen, had a higher

percentage of this nutrient than the controls might be

Table 7• Dry matter, nitrogen percentage, and nitrogen content in total bean plants under salt and water stress.

Treatment

Dry matter Nitrogen percentage Total nitrogen

Treatment Ammonium Nitrate Ammonium Nitrate Ammonium Nitrate

Gram °A ms/plant

Control 5-32 5.75 3.30 3.08 175-5 176.1

NaCl 3.03 3.59 3.57 3.37 108.2 121.0

Carbowax 2.03 2.89 3.82 3.60 77.5 92.0

ON

37

related to the low dry matter production. Therefore, the

yield of dry matter was more severely affected by the I

treatments than was the nitrogen uptake, consequently the

reduction in nutrient absorption was not responsible for

the inhibition in growth when plants were submitted to

water and salt stress.

Water and Salt Effects on the Distribution of Nitrogen Absorbed as Ammonium and Nitrate.

in Bean Shoots and Roots

Data in Tables 8 and 9 show the influence of NaCl

15 salinity and water deficit on the distribution of N,

absorbed as ammonium and nitrate, in bean plants. The per

15 cent of the total N absorbed that was found in shoots and

roots of bean plants is graphically presented in Figure 3«

These data indicate an absolute higher amount of

15 ^N in total plant, shoots, and roots of control plants

than in stressed plants, for both nitrogen sources. How

ever, expressing the results in relative terms there was

15 no difference in the distribution of the total N,

absorbed as ammonium and nitrate, in shoots and roots for

all treatments, expressed in terms of percentage of the

total. Therefore, the difference in absorption can not be

attributed to effects on translocation.

38

15 Table 8. Distribution of N absorbed as ammonium in bean

shoots and roots.

Treatment Total Shoots Roots Shoots Roots Recovery

Mff N/planta % of total °A Control 8 . 6 8 5.45 3.23 62.79 37.21 8 5 . 7 9

NaCl 2.30 1. 36 0.94 59.13 40.87 8 6 . 3 6

Carbowax 1. 6 5 1.05 O. 6 5 63.64 3 6 . 3 6 7 3 . 2 1

a.

plants. Values expressed on total shoots , roots , or total

15 Table 9* Distribution of N absorbed as nitrate in bean shoots and roots.


Mp; N/planta % of total °A Control 8.4o 5.14 3.26 61.19 3 8 . 8 1 84.30

NaCl 2.05 1.26 0.79 61.46 38.54 77.03

Carbowax H

•

to

O

0.76 0.44 63.33 3 6 . 6 7 8 1 . 2 6

Bk

plants. Values expressed on total shoots , roots, or total

39

100

yfy N source - N H4

1 j N sourcc - NOg

a *-»

o 50

o JC

o o

1 1 I 1 1

R I i 1

s S

! ! CO N TROL Na CI C A R B O W A X

Figure 3 . Distribution of nitrogen absorbed as ammonium and nitrate in bean shoots (S) and roots (R) affected by NaCl salinity and water stress (carbowax).

40

Accumulation of Nitrogenous Substances in Bean Shoots Affected by Salt and Water Stress

Nitrate

Values for the mean nitrate-nitrogen content in

bean shoot plants grown in normal conditions (Hoagland

solution), and salt and water stress are found in Figure 4.

15 The N was supplied in KNO^ form, and the plants harvested

at 6, 12, 24, and 48 hours after the labelled nitrogen had

been applied. There was an increase in nitrate-nitrogen

content with time in all treatments.

Table 10 reports the statistical analyses for the

mean values at different times and treatments. Nitrate

accumulated in bean shoots when the plants were subjected

to NaCl salinity and to water stress compared with the

control. No significant difference at any time was observed

between NaCl and carbowax treatments, suggesting that the

nitrate accumulation was due to osmotic effects only.

An accumulation of nitrate in plants under water

.deficit conditions was reported by Lahiri and Singh ( 1 9 6 8 ) .

They suggested that this accumulation was caused by an

effect of water shortage on the enzymes responsible for

nitrate reduction. A significant decrease in the activity

of both nitrite and nitrate reductase in plants was found

by Huffaker et al. (1971) caused by water stress.

Strogonov (1964) and Udovenlco et al. (1971) showed an

increase in nitrate content in plants salinized with NaCl

'il

4.0

N a C

C A R B O W A X

i. o

n( e >»

£ 2.0 O)

z i n cn

48 0 12 24 6


Figure 4. Nitrate-^N in bean shoots after 6, 12, 24, and 48 hours in 15N0^ solution.

42

Table 10. Nitrate-^^N in bean shoots after 6, 12, 24:, and 48 hours in ^-5no<j solution.

15 fig N/g dry matter

Time • Hours Control NaCl Carbowax

6 0.23 a O . 3 6 b 0.40 b

12 0.50 a 0.73 b 0.70 b

24 1.06 a 1.43 b 1.66 b

48 2.06 a 3.33 b 3.46 b

s- = 0.033, 0.056, 0.090, 0.056 at 6, 12, 24, and 48 hours, respectively.

All values within a row, followed by the same letter are not significantly different at the .05 level.

43

and NagSOj^. Therefore, a reduction in the nitrate

reductase activity might be one factor responsible for the

accumulation of nitrate in bean shoots under salt and

water stress.

Ammonium

The effect of NaCl salinity and water stress on the

accumulation of ammonium in bean shoots at different times

(6, 12, 24, and 48 hours) is shown in Figures 5 and 6.

15 Figure 5 shows the content of ammonium when the N was

15 applied in (NH^J^SO^ form and in Figure 6 the source of N

was KNO^. In both cases there was an increase in ammonium

content in bean shoots with time for all treatments*

The mean values for the ammonium content at dif

ferent times and treatments are recorded in Table 11. The

control plants accumulated significantly less ammonium in

shoots than the plants treated with NaCl and carbowax at 6,

12, 24, and 48 hours, when the nitrogen source was either

(NH^JgSO^ or KNO^. In the (NH^J^SO^ treatment there was no

difference in ammonium accumulation between NaCl and

carbowax at 6, 12, and 24 hours. However, the ionic

effect of NaCl resulted in a higher ammonium content at

48 hours. When the nitrogen source was KNO^ there was no

ionic effect on the accumulation of ammonium, as indicated

by the same content of ammonium at each given time for the

NaCl and carbowax treatments.

kk

30 C O N T R O L

N a C

C A R B O W A X

o> //

/ / / /

cn

,-y

0 6 12 24 48

T I M E C h o u r s )

15 Figure 5* Ammonium- N in bean shoots after 6, 12, 24, and

48 hours in solution.

45

25.0

S A

CAR BOWAX

en

O G 12 24 48


gure 6. Ammonium"'''in bean shoots after 6, 12, 24, and 48 hours in solution.

Table 11. Ammonium-^"**N in bean shoots after 6, 12, 24, and 48 hours in "^NH^ solution or in -^NO^ solution.

Ammonium solution Nitrate solution Time • • Hours Control NaCl Carbowax Control NaCl Carbowax

UK "^N/g dm

6 1 . 9 6 a 3.43 b 3.60 b I. 8 3 a 2.63 c 3.06 be

1 2 4.46 ab 6.70 cd 7.86 d 3.53 a 4.70 ab 5.73 be

24 9.20 a 16.13 be 17.66 c 9.86 a 13.20 b 13.83 b

48 1 8 . 6 3 a 2 8 . 6 6 c 23.96 b 17.46 a 22.46 b 23.53 b

s- = 0.167, 0.437, 0.958, 1.180 at 6, 12, 24, and 48 hours, respectively* DC


47

The control plants contained the same amount of

ammonium in shoots for both sources of nitrogen at all

times. However, the plants salinized with NaCl

15 accumulated more ammonium when the N was applxed as

(NH^J^SO^ than KNO^ except at 2k hours. At 6 and 48 hours

the ammonium content in water stressed plants was equal

for both nitrogen sources, but at 12 and 2k hours more

ammonium accumulated when the source was (NH^^SO^.

Several authors (Udovenlco et al. , 1970; Strogonov,

1964; Lapina, 19^6) have reported an accumulation of

ammonium in plants under saline conditions, and water

deficit (Mothes, 195^; Lahiri and Singh, 1968).

The accumulation of ammonium found in this experi-«

ment might have contributed to the increase in nitrate

discussed previously. Thus, possibly both pehnomena, the

decrease in nitrate reductase activity and accumulation of

ammonium, might be responsible for an increase in nitrate

in plants under salt and water stress.

Free a-Amino Acids

The content of a-amino acids in shoots of bean

plants affected by NaCl salinity and water stress at dif

ferent times is shown graphically in Figures 7 and 8.

Figure 7 presents the a-amino acids content in shoots of

bean plants that received (NH^J^SO^ in the nutrient

solution, and in Figure 8 KNO^ was applied as nitrogen

350|—

L.

O E

> u XI

cn •—

i n

cn

175

• o C O N T R O L

• — — N q C I

A * C A R B O W A X

I

12 24


48

Figure ?. a-amino acids-1^N in bean shoots after 6, 12, 24, and '±8 hours in solution.

4 9

350

f-i)

a £

>>

« 175g-o>

z in

cn a.

• o C O N T R O L

- • N q C I

• A C A R B O W A X

I

12 24


48

Figure 8. 15 a-amino acids- in. bean shoots after 6, 12, 24, and 48 hours in solution.

50

15 source. The incorporation of N in a-ainino acids

increased in all treatments with time.

15 When the N source was ammonium more a-amino acids

were found in salt and water stress than in control, at 6,

12, and 48 hours, and no difference was observed between

the two treatments (Table 12). However, at 24 hours the

control and salt plants had the same amount of a-amino

acids and significantly less than carbowax treated plants.

The plants treated with KNO^ accumulated less a-amino acids

in control at 6 and 48 hours than plants submitted to salt

and water stress, but no significant difference was observed

at 12 and 24 hours.

By comparing the a-amino acids found in plants

under NaCl salinity and water stress it can be seen that

the only significant increase in a-ainino acids content was

obtained at 12 hours in plants submitted to water stress.

Plants salinized with NaCl accumulated equal

amounts of a-amino acids independent of the nitrogen source

at 6, 24, and 48 hours. At 12 hours more amino acids was

observed in plants receiving (NH^),,S0^ as the nitrogen

source. The water stressed plants had their amino acid

content increased at 6 and 24 hours when the nitrogen was

applied in ammonium form, but at 12 and 48 hours no differ

ence was obtained.

The observed accumulation of free a-amino acids in

plants subjected to salt and water stress, even in the

IS IS Table 12. Ot-amino acids- N in bean shoots after 6, 12, 24, and 48 hours in ^NH4

solution or ih solution.

Ammonium solution Nitrate solution Time 1

Hours Control NaCl Carbowax Control NaCl Carbowax

15 U-g N/g dm

6 23-73 a 3 8 . 3 0 be 40.63 c 24.96 a 37- 6 0 be 33-93 b

12 53.03 a 73. 9 6 d 6 9 . 6 6 cd 6 6.20 bed 5 6 . 9 6 abc 80.73 d

24 115.73 a l4l.00 a 175-03 b 125-43 a 120.13 a 135-70 a

48 2 6 3 . 3 0 a 3 3 0 . 6 0 b 347.20 b 239*46 a 3 2 5 . 2 0 b 339-46 b

s- = I. 7 6 , 4.44, 9-12, 1 1 . 7 2 at 6 , 1 2 , 24, and 48 hours, respectively.


ui H

52

presence of nitrate and ammonium accumulation, indicated

that further steps of nitrogen metabolism (incorporation of

amino acids into protein) was somehow impaired by the

treatments. Additional support for this assumption is

presented in Figures 11 and 12 and in Table l4. Barnett

and Naylor (1966), Kudev (1967), Prusaltova (i960), Mothes

(1956), and Kemble and MacPherson (195^) reported increases

in the level of free amino acids caused by water deficit.

A decreased incorporation 'of amino acids into proteins was

observed by Kahane and Poljakoff-Mayber (1968a, 1968b) and

Prisco and 0'Leary (1970) in plants subjected to salinity

treatment. Palfi ( 1 9 6 3 , 1965), Palfi and Juhasz ( 1 9 6 8 ) , .

and Lapina (1966) also found a great increase in total

amino acids in plants exposed to salt stress.

Total Soluble Nitrogen

The total alcohol soluble nitrogen fraction (mainly

nitrate, ammonium, free amino acids, amides, and chloro

phyll) in shoots of the control, NaCl, and carbowax plants

at different times is presented in Figures 9, as

nitrogen source, and 10, nitrogen in KNO^ form. The

content of soluble nitrogenous substances in all treatments

increased with time.

Data in Table 13 show the statistical comparison

for the mean values of total soluble nitrogen at different

1,000 o C O N T R O L

C A R B O W A X

cn

O)

200 48 24


gure 9* Total soluble-^N in bean shoots after 6, 12, 24, and '*8 hours in solution.

1,200

C A R B O W A X

•o 700

200 0 48 12 24 6


Figure 10. 15 Total soluble- N in bean shoots after 6, 2^ , and ^8 hours in solution.

12,

Table 13- Total soluble-^^N in bean shoots after 6, 12, 24, and 48 hours in solution or in -^NO^ solution*

Ammonium solution Nitrate solution Time 1

Hours Control NaCl Carbowax Control NaCl Carbowax

15 US N/jr dm

6

S 1 2

246.40 ab 286.75 c 249.76 ab 236.90 a 273.13 be 271.08 abc 6

S 1 2 3 2 1 . 2 6 a 3 8 1 . 3 6 abc 403.56 c 337-23 ab 39.9.83 be 391.70 be

24 435.16 a 487.50 ab 625.36 b 489.23 ab 602.86 ab 623.10 b

48 747.50 a 860.30 ab 995.36 be 805.90 ab 935*43 abc 1092.03 c

s- = 10.75, 19*05, 51*58, 59*19 at 6, 12, 24, and 48 hours, respectively.


VI VI

56

treatments and times. Nitrogen source did not affect sig

nificantly the content of the soluble fraction in any

treatment.

When (NH^JgSO^ was the nitrogen source the control

had less soluble nitrogen than water stressed plants but

amounts equal to NaCl plants, at 12, 24, and 48 hours. At

6 hours the control and carbowax treatments were equal,

but the NaCl plants had a higher soluble nitrogen content.

No difference was observed between NaCl and carbowax

treatments at 12, 24, and 48 hours.

No significant difference was obtained among the

15 treatments at 12 and 24 hours when the N was supplied as

KNOj. At 6 hours the content of soluble nitrogen in the

control was lower than in NaCl salinized plants but it was

not possible to detect a difference in carbowax treated

plants. At 48 hours the NaCl and carbowax treated plants

showed no difference in soluble nitrogen, and the control

plants had the same content of this fraction as NaCl but

less than carbowax.

Plants treated with NaCl and carbowax accumulated

more nitrogen in soluble form than the control. The

inconsistency of the results found within treatments might i

be a consequence of fractions of soluble nitrogen other

than nitrate, ammonium, and free amino acids. The behavior

of such compounds as chlorophyll in salt and water stressed

plants is different from the other soluble forms. While

57

those forms increase in stressed plants the chlorophyll

content is significantly decreased (Prisco, 1971)*

Protein

Figures 11 and 12 show the content of protein in

shoots of bean plants affected by NaCl salinity and

15 carbowax at 6, 12, 24, and 48 hours. The N absorbed as

ammonium and incorporated into protein is presented in

Figure 11 and Figure 12 reports the content of protein when

the nitrogen source was nitrate. In both cases there was

15 an increase in N labelled protein with time xn all treat

ments .

The statistical analyses for the mean values at

different times and treatments are presented in Table l4.

There was a significant decrease in the protein content in

bean shoots of plants treated with NaCl and carbowax,

compared with the control, at all times with both nitrogen

sources except at 24 hours in the ammonium treatment when

the content of protein was equal in the control and water

stressed plants.

The plants under NaCl salinity had a lower content

of protein than plants treated with carbowax at all times

"t* in ammonium treatment, suggesting that the excess of Na

and/or Cl~ ions inhibited protein synthesis. However, when

nitrate was the nitrogen source the ionic effect appeared

only at 48 hours.

58

3,000

C A R B O W A X

1.500 —

cn

48 12 24 0 6


15 Figure 11. Protein- in. bean shoots after 6, 12, 2,k, and


59

3,500

O CONTROL

-A C ARBOWAX

CD

0 6 12 24 48


15 Figure 12. Protein- N in bean shoots after 6, 12, 24, and


Table 14. Protein-"'""*N in bean shoots after 6, 12, 24, and 48 hours in solution or in solution.

Ammonium solution Nitrate solution Time • • -•• • - — • Hours Control NaCl Carbowax Control NaCl Carbowax

15 U.c: ^N/g dm

6 352.76 a 66.53 c 110.83 b 3 2 2 . 2 0 a 6 3 . 2 0 c 73.66 c

1 2 9 0 8 . 5 3 a 158.73 c 541.76 b 1135.56 a 117.70 c 1 1 2 . 8 0 c

24 1 1 9 8 . 2 3 b 417.63 c 975.96 b 1993.46 a 291.96 c 399.93 c

48 2 8 3 0 . 0 0 b 8 7 7 . 1 6 e 1424.0 6 d 3439.90 a 1 2 8 3 . 0 0 d 2179.23 c

s- = 10.22, 75*89, 151*03, 115«89 at 6, 12, 24, and 48 hours, respectively.


C\ O

6l

At 6, 12, and 24 hours the protein content in

shoots of* NaCl treated plants was the same for both sources

of" nitrogen, but a higher amount of protein was found at

48 hours with the nitrate source. Water stressed plants

synthesized more proteins when the nitrogen source was

nitrate at 48 hours but less at all other times. A higher

protein content was found in control plants at 24 and 48

hours when "^N was supplied in nitrate form.

Table 15 shows the protein to non-protein ratio in

bean shoots after 6, 12, 24, and 48 hours in ammonium and

nitrate solution. The statistical analyses performed on

the data show that the protein to non-protein ratio in

control plants was significantly higher than in NaCl and

carbowax plants at all times either in ammonium or nitrate

solution. Plants subjected to NaCl salinity presented a

lower protein to non-protein ratio than water stressed

plants except at 24 hours when ammonium was the nitrogen

source. However, this difference.was observed only at 48

hours with nitrate nutrition.

No difference in the protein to non-protein ratio

was observed in NaCl stressed plants between the two

sources of nitrogen at 6, 12, and 24 hours, but at 48 hours

a higher ratio was obtained in plants receiving nitrate.

The plants submitted to water stress presented a higher

protein to non-protein ratio when the nitrogen source was

ammonium at 6 and 12 hours. At 24 hours no difference was

15 15 Table 15. Protein- N to non-protein- N ratio in bean shoots after 6, 12, 24,

and 48 hours in 15NH^ solution or solution.

Ammonium solution Nitrate solution Time Hours Control NaCl Carbowax Control NaCl Carbowax

UK 15N/K dm

6 1.43 a O

• to

c 0.44 b 1.36 a 0.23 c

r-01 •

0 c

12 2 . 8 3 a 0.42 c 1.34 b 3-37 a 0.29 c

O

0-\

•

0 c

24 2.75 b 0.86 c 1.56 c 4.07 a 0.48 c 0.64 c

48 3 . 7 8 b 1 . 0 2 e 1.43 d 4.27 a 1-37 d 1.99 c

s- = 0.044, 0.207, 0.358, 0.110 at 6, 12, 24, and 48 hours respectively.

All values within a row, followed by the same letter sire not significantly different at the .05 level.

ON E0

63

detected, but a higher ratio was obtained when nitrate was

used as nitrogen source at 48 hours.

The above results provide evidence that both salt

and water stress impaired protein synthesis in bean shoots,

•j* and that the presence of excess Na and CI ions also

affected the content of protein. Therefore, the inhibition

of protein synthesis in bean shoots was accomplished by

osmotic and ionic effects. Several investigators (Petrie

and Wood, 19385 Mothes, 1956; Todd and Yoo, 1964) reported

a decrease in protein content in plants under water stress.

Lapina (1966) and Nightingale and Farnham (1936) showed a

depressing effect of salts on protein synthesis.

Distribution of Nitrogen in Various Fractions

The protein, non-protein (total soluble), a-amino

15 acids, ammonium, and nitrate expressed as jig N/g dry

15 matter and as per cent of the total N found in bean

shoots after 6, 12, 24, and 48 hours from ammonium and

nitrate solutions are reported in Tables 1 6, 17, 18, and

15 19, respectively. The data expressed as jig N/g of dry

matter have been already discussed but are included in

these tables for direct comparisons. In terms of per cent

15 of the total most of the N in control plants was found

in the protein fraction, principally after 6 hours. How

ever, in stressed plants a high percentage of nitrogen was

Table l6. Nitrogenous substances, labelled, in bean shoots after 6 hours

Forms of N

Control NaCl Carbovax

Forms of N |lg15N/ga % total Iig1^N/g' a % total jig1^N/ga % total

Ammonium source

Total 599-2 100.00 353.3 100.00 360.6 100.00 Protein ^ 352.8 58.87 66.51 18.83 110.8 30.74 Non-protein 246.4 4i.i4 286 .8 81.67 249 .8 69 .26 a-amino acids 23.71 3.96 38.3 10.84 40.6 11.27 Ammonium 2.0 0.33 3.4 0.97 3.6 1.00

Nitrate source

Total 559-1 100.00 336.3 100.00 344.7 100.00 Protein ^ 322.2 57.63 63.2 18.79 73.7 21.37 Non-prot ein 236.9 42.37 273.1 81.21 271.0 78.63 a-amino acids 24.96 4.46 37.6 11.18- 33.9 9.84 Ammonium 1.8 0.33 2.6 0.78 3.1 0.89 Nitrate 0.2 o.o4 0.4 0.11 0.4 0.12

aValues expressed on the dry weight of total shoots.

^Non-protein includes a-amino acids, ammonium, nitrate, and other soluble nitrogenous compounds.

15 Table 17• Nitrogenous substances, N labelled, in bean shoots after 12 hours.

Forms of N

Control NaCl Carbowax

Forms of N lAS15N/ga % total jig15N/ga % total [ig15N/ga % total

Ammonium source

Total " 1229-8 100.00 540.1 100.00 945.3 100.0 Protein 908.5 73.88 158.7 29.39 5^1.7 57.31 Non-protein 321.3 26.12 381.4 70.61 403.6 42.69 a-amino acids 53.0 4.31 73-9 13.69 69.6 7.37 Ammonia 4.4 0.36 6.7 1.24 7-9 0.83

Nitrate source

Total 1472.8 100.00 517.5 100.00 504.5 100.00 Protein ^ 1135.6 77.10 117.7 22.74 112.8 22.36 Non-protein 337.2 22.90 399.8 77.26 391-7 77.64 a-amino acids 66.2 4.49 56.9 11.00 80.7 16.00 Ammonia 3.5 0.24 4.7 0.91 5.7 l.l4 Nitrate 0.5 0.03 0.7 0.14 0.7 o.i4

aValues expressed on the dry weight of total shoots.

Non-protein includes a-amino acids, ammonium, nitrate, and other soluble nitrogenous compounds.

a\ \ji

Table 18. Nitrogenous substances, labelled, in bean shoots after 24 hours.

Forms of N

Control NaCl Carbowax

Forms of N HS15N/ga % total (J-g^N/g3 % total }Ig15N/ga % total

Ammonium source

Total 1633.4 100.00 905.1 100.00 1601.3 100.00 Protein ^ 1 1 9 8 . 2 7 3 . 3 6 417.6 46.14 975-9 6 0 . 9 5 Non-protein 435.2 . 26.64 487.5 5 3 . 8 6 625.4 3 9 . 0 5 OC-amino acids 115.7 7 . 0 8 l4l.00 1 5 . 5 8 175.0 1 0 . 9 3 Ammonia 9-2 0 . 5 6 1 6 . 1 3 1 . 7 8 17.7 1 . 1 0

Nitrate source

Total 2482.7 1 0 0 . 0 0 894.8 1 0 0 . 0 0 1023.0 1 0 0 . 0 0 Protein ^ 1993-5 8 0 . 2 9 292.0 3 2 . 6 3 399.9 3 9 . 0 9 Non-protein 489.2 19.71 602.8 67.37 623.1 6 0 . 9 1 CX-amino acids 125.4 5 . 0 5 120.13 13.43 135.7 1 3 . 2 6 Ammonia 9 . 8 0.40 13.2 1.48 13.8 1 . 3 5 Nitrate l.l 0.04 1.4 0.16 1.7 0 . 1 6

£ Values expressed on the dry weight of total shoots.

Non-protein includes a-amino acids, ammonium, nitrate, and other soluble nitrogenous compounds.

1 c Table 19* Nitrogenous substances, N labelled, in bean shoots after 48 hours.

V

Forms of N

Control

^S15N/ga

NaCl

% total Iig^^N/g3 % total

Carbowax

Jig15N/ga % total

Ammonium source

Total 3577-5 100.00 1737-5 100.00 2419.1 100.00 Protein 2830.0 79-10 877-2 50.49 1424.1 5 8 . 8 6 Non-protei/ 747-5 20.90 8 6 0 . 3 49.51 995-0 4l.l4 a-amino acids 2 6 3 . 3 7-36 330. 6 19-03 347-2 14-35 Ammonia 1 8 . 6 O. 5 2 2 8 . 7 I. 6 5 24.0 0.99

Nitrate source

Total Protein ^ Non-protein a-amino acids Ammonia Nitrate

4245.8 3439-9 805-9 239-5 17-5 2.1

100.00 81.02 18.98 5.64 o.4i 0 . 0 5

2 2 1 8 . 4 1 2 8 3 . 0 935-4 325-2

2 2 . 5 3-3

1 0 0 . 0 0 57.83 42 . 1 7 14.66

1 . 0 1 0 . 1 5

3271.3 2179-2 1092.0 339.5 23.5 3-5

1 0 0 . 0 0 6 6 . 6 2 33.38 1 0 . 3 8

0 . 7 2 0 . 1 1

Values expressed on the dry weight of total shoots.

Non-protein includes oc-amino acids, ammonium, nitrate, and other soluble nitrogenous compounds.

68

found in non-protein form. The percentage of a-amino acids

was higher in plants under stress than in control at all

times.

Data presented in Tables 20, 21, 22, and 23 show

15 the distribution of total N in plant shoots and roots

after 6, 12, 24, and 48 hours, respectively. Approximately

15 65 to 80 per cent of the total N absorbed as ammonium or

nitrate was found in shoots from the control and salinity

treatments. In water stressed plants only 48 to 6 3 per

15 cent of the N was found in the shoots and in two cases

15 considerably less N was in the shoots from the nitrate

source.

Table 20. Distribution of total in plant shoots and roots after 6 hours in solution.


Me « N/plant3 % of total °A NH. 2.15 1.47 0.68 68.44 31.56 73.2

Control NO3 2.94 1.67 1.27 55-93 44.07 71.0

NH. 1.06 0.7^ 0.32 69.95 30.05 68.5 NaCl

NO3 0.94 0.69 0.25 7 2 . 8 5 27.15 84.6

NH. 0.97 0.49 0.48 50.88 49.12 8 9 . 6 Carbowax

NO3 1.19 0.66 0.53 55.35 44.65 88.4

aValues expressed on total shoots, roots, and total plants.

cr. vO

15 Table 21. Distribution of total N in plant shoots and roots after 12 hours in

solution.


Mr ^ N/planta % of total °A NH. 4.36 3.51 0.85 80.65 19.35 7 9 . 3

Control NO3 6.75 4.61 2.14 68.33 31.67 7 5 . 2

NH, 1.42 1.07 1.35 75.60 24.40 7 0 . 3 NaCI

NO3 1.36 1.03 0.33 76.05 23.95 8 1 . 7

*

NH. 1.79 1.12 0.67 62.71 37.29 8 8 . 9 Carbowax

NO 1.86 0.89 0.97 47.71 52.29 8 9 . 5


*vl O

15 "Table 22. Distribution of total N in plant shoots and roots after 24 hours in solution.


MR 15 N/planta % of total H NH. 7-31 5.09 2.22 69.60 30.40 76.0

Control TC

NO3 10.46 7.79 2.67 74.53 25.47 81.4

NH. 2.36 1.63 0.73 68.94 31.06 72.2 NaCl

NO3 3.02 2.20 0.82 73-00 27.00 76.5

NH. 3.93 2.70 1.23 68.60 31.40 74.8 Carbowax

Jt

NO3 2.83 1.36 1.47 47.99 52.01 73.4


-n] H

Table 23- Distribution of total "^N in plant shoots and roots after 48 hours in solution.


m !5 M.c: N/planta % of total

NH, 19.88 • 12.79 7.09 64.34 35.66 80.8 Control

NO3 23 .26 18.97 4.29 8 1 . 5 6 18.44 83.7

NH, 7.21 4.69 2.52 65.09 34.91 79.8 NaCl -

NO3 7.81 5-42 2.39 69.34 30.66 8 0 . 3

NHj, 9.22 5.18 4.04 56.22 43.78 78.9 Carbowax

*±

NO3 11.62

CO

• 4.78 58.83 41.17 8 1 . 5


•Sj to

SUMMARY AND CONCLUSIONS

The ammonium and nitrate uptake patterns of bean

plants grown under normal conditions (control), NaCl treat

ment (salt stress), and carbowax treatment (water stress)

were linear functions of time in all treatments.

The absorption of both nitrate and ammonium was

inhibited by the presence of NaCl and carbowax in the

nutrient solution. The inhibition of absorption due to

NaCl was similar to that under water deficit for ammonium,

but the effect of Na and Cl promoted an increase in the

uptake of nitrate compared with carbowax. The plants

salinized with NaCl absorbed the same amount of nitrogen in

10 hours independent of the nitrogen source. Under water

stress, however, bean plants absorbed more ammonium than

nitrate.

Significantly less water was absorbed by plants sub

jected to salt and water stress, but no difference in water

uptake occurred between NaCl and carbowax treatmen'Cs.

Since a high positive correlation between ion and water

uptake was obtained, the decrease in nutrient uptake by

plants under NaCl salinity and water deficit was correlated

to the decrease in water absorption. Another factor that

might have affected the mineral uptake was the reduction

73

in the root permeability observed in plants grown in

salinity and carbowax substrate.

In the bean plants subjected to stress, growth (dry

matter production) was inhibited, percentage of nitrogen

increased, but total nitrogen per plant was reduced. Even

though the nitrogen uptake was inhibited by stress over

short periods, the reduction in the nutrient absorption was

not responsible for the inhibition in growth since dry

matter production was more severely affected by the treat

ments than was nitrogen uptake.

There was an accumulation of nitrate in bean shoots

when the plants were submitted to stress, without regard to

absorption time. Carbowax and NaCl affected the accumula

tion of nitrate similarly, so that the observed increase in

nitrate content was due only to osmotic effects. This

accumulation of nitrate might be the result of a decrease

in nitrate reductase activity and/or ammonium accumulation.

An increase in ammonium and free a-amino acids was

observed in the plants subjected to salt and water stress.

No difference between NaCl and carbowax treatments was

obtained on ammonium and free a-amino acids accumulation.

In general there was a higher content of ammonium in bean

shoots when (NH^) SO^ was used in the nutrient solution.

However, the accumulation of free Ot-amino acids was in

dependent of the form of nitrogen used. The higher amount

of ammonium and a-amino acids found in stressed plants

75

suggests that salinity and water deficit caused a depres

sing effect on the incorporation of ammonium into amino

acids, and impaired the utilization of amino acids in the

subsequent steps of nitrogen metabolism.

The effect of salt and water stress on the content

of total soluble nitrogen in bean shoots was not as clearly

identified as it was on other nitrogen fractions.

The protein synthesis in bean shoots was inhibited

by NaCl and carbowax treatments at all times when the

nitrogen source was either ammonium or nitrate. The Na+

15 and CI ions affected the incorporation of N into protein

at all times with the ammonium source, but the ionic effect

was observed only at *18 hours in the case of nitrate. The

protein to non-protein ratio followed the same patterns as

15 the protein content. Therefore, the incorporation of N

into protein was highly reduced by salt and water stress,

and in addition to the osmotic effects, the excess of Na

and CI caused a substantial decrease in the protein content

of bean plants.

15 Seventy to eighty per cent of the total N in

control plants was found in protein form in or after 12

hours absorption: however, in stressed plants a high per-

15 centage of N was in non-protein form. The percentage of

free (X-amino aci.ds was higher in plants under stress than

in the control at all times.

76

A modification in nitrogen metabolism was shown for

bean plants exposed to the effect of salt and water defi

cit. Nitrogen uptake was reduced, accumulation of nitrate,

ammonium, and free Ot-amino acids was observed. A signifi

cant reduction in protein content was found in stressed

plants. The ionic effect of Na+ and Cl~ under saline

conditions was relevant only in depressing protein syn

thesis. The excess of these ions altered the metabolism

probably by interfering with the enzyme activity involved

in the biochemical process.

APPENDIX

NITROGEN AND WATER ABSORBED IN 10 HOUR STUDY

77

78

Table 24. Influence of NaCl salinity and water stress (carbowax) on absorption of ammonium by bean plants.

Uptake tittie % Mg N/ga

Hours Control. NaCl Carbowax

1 0.282 0.132 0.124

2 0.458 0.164 0.319

3 0.628 0.277 0.372

4 0.810 0. 342 0.450

5 0.961 0.442 0.506

6 1.132 0.538 0.648

7 1.341 0.601 0.888

8 1.542 0.721 0.925

9 1.716 ,f '

0.802 0.993

10 1.899 O.883 1.122

a O.O87 0.016 0.034

b 0.180 0.086 0.108

r 0.962 0.964 0.794

Absorption expressed as mg N/g dry weight of total plants. Average of three replications.

79

Table 25» Effect of NaCl salinity and water stress (carbowax) on nitrate uptake by bean plants.

Uptake time Mg N/ga

Hours Control NaCl Carbowax

1 0.199 0.087 0.028

2 0.450 0.148 0.048

3 0.738 0.239 0.135

4 1.052 O.349 0.193

5 1.353 0.407 0. 241

6 1.596 0.437 0.268

7 1.681 0.542 0.389

8 1.686 0.6l4 0.408

9 1.713 0.662 0.449

10 1.717 0.745 0.517

a -0.103 0.018 -0.039

b 0.286 0.074 0.055

r 0.889 0.914 0.991

aAbsorption expressed as mg N/g of dry weight of total plants. Average of three replications.

80

1 e Table 26. Ammonium- N remained in nutrient solution.

Time Mg "*"^N/2 liters


0 15.658 15.587 16.224

1 14.163 15.189 15.917

2 13.248 15.085 15.466

3 12.345 14.747 15•3^7

4 11.451 14.598 15.204

5 10.648 14.248 15 .058

6 9.787 13.952 14.777

7 8.716 13.763 14.187

8 7.691 13.4oi 14.086

9 6.785 13.156 13.932

10 5.873 12.905 13.685

81

15 Table 27- Nitrate- N remained in nutrient solution.

Time Mg "^N/2 liters


0 13.449 13.583 13. 8 6 5

1 12.317 13.227 13.782

2 10.945 13 .212 13.726

3 9.332 12.673 13.473

4 7.603 12.284 13.319

5 5.908 12.066 13.168

6 4.552 11.945 13.038

7 3.992 11.570 12.750

8 3-971 11.311 12.695

9 3.833 11.125 12.530

10 3.789 10.823 12.369

82

Table 28. Effect of salt and water stress on water uptake by bean plants—ammonium treatment.

Time ml of water/pot


1 15-7 6 . 0 6.7

2 31.3 1 1 . 6 14.3

3 44.0 1 6 .3 2 0 . 7

4 6 2 . 7 2 2 . 6 24.0

5 77-3 25-7 3 6 . 0

6 8 9 . 7 32.7 42.0

7 1 0 5 . 7 3 8 . 0 49. 0

8 1 2 6 . 3 45.7 57.0

9 1^5.3 5 2 . 6 6 3 . 6

1 0 16^.3 6 0 . 0 7 2 . 0

83

Table 29* Effect of salt and water stress on water absorption by bean plants--nitrate treatment.

Time ml of water/pot

Hours Control NaCl Car bowax

1 1 7 . 0 4.7 7-3

2 32 .7 1 1 . 3 13.3

3 50 .3 17 .3 1 8 . 6

4 69.0 24.0 23-7

5 85 .7 3 0 . 0 29.7

6 io4 .o 3 5 . 0 34.0

7 1 2 6 . 7 4i.3 4i . 0

8 i4o .o 48.0 46 . 7

9 1 6 0 . 0 52.7 51-3'

10 175.3 6 1 . 3 59.6

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