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
/
"
DAYS AFTER TRANSFER TO JJIFFERENT NUTRIENT SOWTJONS
'"
""
PLANT
,0
mo
ec
=
w
me
,co
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
'"'""'""'I %C�
,,,,,,, -ca ••-•-1 % B
2 •--•- B ·
7 15
1000
"" aoo
em ,oo
PLANT
,oo
,oo
22 Figure 4,
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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oo:iot-t-o:i o <Nl0"1400t-= o C\l,....,,....,,....,,...., 00
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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-
I ments,
TheJ in the Ca defi
protein
1. Ga1
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9. TsoJr.,ganplanPhy
10. Tso,andficieingrovlatecCaPl. j
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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).
.. Ill GI
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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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