Amino Acid Content Related to Gradual Development of Calcium and Boron

Deficiency Symptoms in Tobacco Plants

T. C. Tso and Mary E. Engelhaupt

Crops Research Division, ARS, United States Department of Agriculture Beltsville, Maryland, U.S.A.

The present report is a continued investigation of the changes in o-r­ganic constituents in tobacco plants after growth in various nutrient solutions. In preceding papers (10, 11) changes in plant growth, alka­loids, sugars, and organic acids werereported in relation to the gradualdevelopment of boron and calciumdeficiency symptoms. This paperr pro­vides data on the correspondingchanges in amino acids.

Materials and Methods

The same plant materials used for the sugar and organic acid study (11) were used for the assay ofamino acids. Nicotiana tabacum L.var. Connecticut Broa:dleaf plantswere grown in differrent nutrientsolutions, including complete nutri­ent solution with usual amount ofCa (245 p.p.m.) and B (1 p.p.m.),

solution complete with all nutrients except only 10 % or 1 % of usual amount of Ca or B, and solution without the addition of any Ca or B. Leaf samples were obtained fromthre·e stalk positions, and at foursamplings of the winter crop andthree samplings of the spring crop,based on the gradual development ofCa- and B-defi.ciency symptoms.These samples were extracted with70% ethanol and the tissue: residueswere· hydrolyzed as previously de­scribed (11),

Individual amino acids we·re sepa­rated and semi-quantitatively esti­mated, using Irreverre and Martin's (2) method with modification as de­scribed in an earlieT report (9).

Results are expressed as milli­grams of amino acid per plant. Al­though data are available of the • distribution of amino acid in the top, middle, and bottom leaves for

each harvest, only results of such distribution at the· last harvest are_ reported.

Results

Samples were taken at different stages of gra.dual development of de-; ficiency symptoms as previously de-, scribed (11). Total free amino content in leaves of plants of the: winter crop is shown in Figure L_ The corresponding amino acid con;.;' tent from the hydrolyzate is shoWJ1: in Figure 2. In each figure there it an insert showing the distribution total amino acid content in the to middle, and bottom leaves at the harvest. The free and bound acid contents o.f the spring crop shown in Figure 3 and 4, resp tively.

Most changes in the amino a content in plants given differ treatments occurred in the final W

FIG. I. TOTAL FREE AMINO ACID CONTENT IN LEAVES OF PLANTS AFTER GROWTH IN VARIOUS NUTRIENT SOLUTIONS (winter crop)

FIG.2. TOTAL AMINO ACID CONTENT IN HYDROLYZATE IN LEAVES OF

AFTER GROWTH IN VARIOUS NUTRIENT SOLU170NS

MG

'PER

PLANT

Figure I.

D!S'!f;:lOUTlON '"TOPITl,MIDDLEIMI, a BotTa.,lal LEIIVES OF LAST HARVE'.H

/

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DAYS AFTER TRANSFER TO JJIFFERENT NUTRIENT SOWTJONS

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DISTRIBUTION IN TOP (TJ,MIDDLE(M), 8; 60TTOM(SI LEAVES OF LAST �RVEST

TMO TMB TMB TMB TMB TMB

-�-CONTROL -TOPPED CONTROL

30 ,.,,..,,, ... ,. 10% Ca ,,,,,,. ·Ca

-·-·- i0% B ---•·8

" e "

DAYS AFTER TRANSF�R . TC! DIFfERENT NITTRIENT SCA.IJTIONS

Figure 2.

(Tobacco Science 12)

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, TOTAL FREE AMINO ACID CONTENT IN LEAVES CF PLANTS£R GROWTH IN VARIOUS NUTRIENT SOLUTIONS (sp-ing crop)

FIG.4. TOTAL AMINO ACID CONTENT IN HYDROLYZATE IN LEAVES OF PLANTS AFTER GROWTH IN VARIOUS NUTRIENT SOLUTIONS ( spdog crop)

600 DISTRIBUTION _IN 10P{TJ,MIDDLE(M),!l BOTTOM(B) j LEAVES Of LAST HAfM:ST

J0OO 500

900 400

,o zoo

l

1! 1i:11iTMB TMB TMB TMB TMB TMB

4 --CONTROL --TOPPED CONTRCL

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,,,,,,, -ca ••-•-1 % B

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DISTRIBUTION IN TOP IT),MIDDLE (Ml, a BOTTOM(B) LEAVES OF LAST HARVEST

15 22

DAYS AFTER TRANSFER TO DIFFERENT NUTRIENT SOWTlOIIS

DAYS AFTER TRANSFER TO DIFFERENT NUTRIENT SOLUTICXIIS

re the last harvest, the same 'od in which changes in sugars

organic acids occurred (11). ·ng this final week, leaves in con­plants reached maturity and

in plants given -Ca and -B ents showed severe deficiency

ptoms. !ants grown in 10% Ca, 1 % Ca, -Ca nutrient solutions showed

nite increases in free amino acid tent over the controls. Boron de­ncy and 1 % B treatments also eased free amino acids but not uch as -Ca. Ten per cent B ap­ed to have little effect on free

'no acids. However, the results of nd amino acid content due to

treatments were not:so- consist-

�'enty-nine different amino- acids � detected in thes�, samples, in­mg a-alanine, fl-alanine, a­nobutyric acid, y-aminobutyric , arginine, asparagine, aspartic : citrulline, cysteic acid, cysteine,:ne, glutamic acid, glutamine,

. me, histidine, isoleucine, leucine, e, methionine, norleucine, orni­' phenylalal).ine, praline, se,rine; nme, tryptophan, tyramine, ty­e, and valine. The six amino

s of greatest abundance in each tment at last harvest of winter spring crops are, shown in les 1 and 2, respectively, with

1 amount of each amino acid found eaves of the whole plant, and the ,aective percentage of total amino

· These top six amino acidsounted to 81 to 90% of the totalt"lnino acids and 70 to 84% of

0und amino acids. The remain-

ing twenty or more amino acids were present in minute quantities.

Discussion

The direct toxic action of excessive accumulation of metabolites was at­tributed by Steinberg et al .. (7) asthe primary cause of chloroses due to mineral deficiencies. However, Steinberg et al. (7) found sharp free amino acid increases in Ca-deficient plants, but did not find definite chemical differences in plant tissues in B"deficient plants.

In this study, both Ca- and B­deficient plants showed a higher free amino acid content than the control, particularly in the spring crop wherein plants grew vigorously and deficiency symptoms developed rapid­ly. In the hydrolyzate fraction, changes were very inconsistent at the last week of growth, but a lesser amount of bound amino acid oc­curred in Ca- or B-deficient plants than in the control.

As the deficiency symptoms gradu­ally advanced, so did the, differences in amino acids among various treat­ments. The general composition of the amino acids found in ethanol extracts and in hydrolyzate· fractions was rather similar, but the amounts of individual amino acids were markedly affected by the treatments.

Both Ca and B are known to have an important role in nitrogen metab­olism (1, 9). Many reports (1) stated that among B-deficient plants there are increases in amino acids and decreases in protein, in amina­tion of carbohydrate derivatives, in nitrate reduction, and in absorption

(Tobacco Science 13)

or accumulation of nitrate. Since nitrogen metabolism is associated ·with carbohydrates and the latter inplants are affected by boron (11), itis apparent that boron would affectnitrogen metabolism. It was also re­ported ( 4) that absorption and as­similation of nitrates failed to takeplace in Ca-deficient plants.

It appea.rs that both Ca and B participant actively in the· formation and breakdown of amino acids, pro­teins, and intermediate products. However, no information is ava.ilable in the literature whether the specific effect of Ca or B deficiency is on the inhibition of protein synthesis or on the· breakdown of protein. Richards and Templeman (5) reported that the accumulation of free amino acids was due to, breakdown of protein in potash-deficient barley and to an inhibition of protein synthesis in phosphorous-deficient barley.

In studying nitrogen metabolism in tobacco plants, Vickery (12) ad­ministered N15 ammonium salt to a young, rapidly growing tobacco plant and observed a significantly greater enrichment of NH in aspartic and glutamic acids than the other amino acids from the leaf protein. In dis­cussing the formation of amides, as­paragine and glutamine in plants, Vickery (13) supported the hypo­thesis that amide metabolism was connected with respiration invoJving Krebs citric acid cycle.

As shown in Tables 1 and 2, as­partic or glutamic acid was found to occupy the top position in hydroly­zates in all plants. In free amino acid fraction, either one of the two

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(Tobacco Science 14)

amides, glutamine and asparagine, or proline, which is interrelated with glutamic acid (3), was the most abundant.

In discussing the contracts in the behavior of glutamine and aspara­gine in plants, Steward and Thomp­son (8) stated that glutamine is often more closely connected with protein synthesis than is asparagine, which is to be regarded more as a reserve substance and a store of the nitrogen which accrues from protein breakdown. In the winter crop in which the plant growth and symptom development we,re slow, more gluta­mine than asparagine was found in all plants. In the spring crop in which the plant growth and symptom development were fast, however, more asparagine than glutamine was found in 1 % B and -B plants, but more glutamine than asparagine was found in 1 % Ca, - Ca, and control plants. These differences in the amounts of the amides appear to suggest that the accumulation of free amino acids was due possibly to protein breakdown in B-deficient plants, and to an inhibition of pro­tein synthesis in Ca-deficient plants.

In these investigations the amount of tryptophan was not positively re­lated to levels of boron as it has been reported for several other crops ( 6) .

Summary

This report relates to amino acid content with a gradual development of Ca- and B-deficiency symptoms in Nicotiana tabacum L. var. Connecti­cut Broadleaf. Most changes in amino acid content occurred during the last week to ten days of plaPt growth before final sampling, par­ticularly when Ca- or B-deficiency symptoms were severe.

Plants grown in nutrient solutions with decreased levels (10% or 1 % ) of Ca or - Ca showed marked in­creases in free amino acids. One per cent boron or - B showed a similar but lesser increase. In the hydroly­zate fraction, absence of Ca re­sulted in a decreased amount of

amino acids. Absence of B, 10% or 1 % B showed a similar but less pro­nounced decrease.

Twenty-nine amino acids were de­tected in these samples. The princi­pal free amino acids were glutamine, asparagine, proline, glutamic acid, aspartic acid, serine, and y-amino­butyric acid; and the principal bound amino acids were glutamic acid, as­partic acid, glycine, arginine, a­alanine, proline, serine, and leucine. Changes found in the,se principal amino acids due to various treat-

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ments were mainly quantitative. The possible effect of B deficiency

in the breakdown of protein and of Ca deficiency in the inhibition of protein synthesis is discussed.

Literature Cited

1. Gauch, H. G. and W. M. Dugger,Jr., "The physiological action ofboron in higher plants: A re­view and interpretation." Univ.of Md. Agr. Exp. Sta. Tech. Bu!.A-80 (1954).

2. Irreverre, F. and W. Martin,"Versatile technique of paperchromatography." Anal. Chem.26: 257-260 (1954).

3. McElroy, W. D. and B. Glass, "Asymposium on amino acid me­tabolism." Contribution 105 ofthe McCollum-Pratt Institute.The Johns Hopkins Press. Balti­more, Md. (1954).

4. Nightingale, G. T., R. M. Ad­doms, W. R. Robbins and L. G.Schermerhorn, "Effects of Cadeficiency on nitrate absorptionand on metabolism in tomato.';Plant Phy.'{iol. 6: 603-630 (1931).

5. Richards, P. J. and W. G. Temple­man, "Physiological studies innlant nutrition. IV. Nitrogen,u��abolism in relation to nutri­ent deficiency and age in leavesof barley." Ann. Bot .. 50: 367-402 (1936).

6. Sheldon, V. L., W. G. Blue andW. A. Albrecht, "Biosynthesisof amino acid8 according to soilfertility. I. Tryptophane inforage crops." Plant and Soil 3:33-40 (1951).

7. Steinberg. R. A., J. D. Bowlingand J. E. McMurtrey, Jr., "Ac­cumulation of free animo acidsas a chemical basis for morpho­logical symptoms in tobacco man­ifesting frenching and mineraldeficiency sympt oms." PlantPhysiol. 25: 279-288 (1950).

8. Steward, F. C. and J. F. Thomp­son, "Properties and physiologi­cal role of asparagine and gluta­mine, with a new interpretationof the structure of asparagine.''Nature 169: 739-742 (1952).

9. Tso, T. C. and J. E. McMurtrey,Jr., "Mineral deficiency and or­ganic constituents in tobaccoplants. II. Amino acids." Pl.Physiol. 35: 865-870 (1960).

10. Tso, T. C., J. E. McMurtrey, Jr.,and R. N. Jeffrey, "Mineral de­ficiency and organic constituentsin tobacco plants. III. Plantgrowth and alkaloid content re­lated to gradual development ofCa- and B-deficiency symptoms."Pl. Physiol. 37: 804-808 (1962).

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(Tobacco Science 15)

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11. Tso, T. C. and Tamara Sorokin,"Sugars and organic acid con­tent related to gradual develop­ment of Ca- and B-deficiencysymptoms in tobacco plants."

Tobacco Science 7: 7-11 (1963). 12. Vickery, H. B., "End products of

nitrogen metabolism in plants."Biol. Symposia 5: 3-19 (1941).

13. Vickery, H. B. and G. W. Pucher,

(To/;acco Science 16)

"The metabolism of amides in green plants. III. The mechan­ism of amide synthesis." Jour.

Biol. Chem.128: 703-713 (1939).

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