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V. The opaque-2 and floury-2 Mutations in Maize

V. The opaque-2 and floury-2 Mutations in Maize

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GENETIC MODIFICATION OF PROTEIN QUALITY IN PLANTS



181



the first four mutants to be analyzed, the other two being opaque-] and

floury-I. I t had been hypothesized by the author that mutations giving the

opaque phenotype were the most likely to be deficient in zein.

The changes in amino acid composition in seeds or seed components

effected by the mutations have been detailed (Nelson et al., 1965;

Mertz et af.,1966; Nelson, 1968). Table I presents the amino acid comTABLE I

Amino Acid Composition (Grams per 100 g. Protein) for Defatted Whole Kernels

of opaque-2,floury-2, and Normal Maize- 1967 Crop

Amino acid

Lysine

Tryptophan

Histidine

Arginine

Aspartic acid

Threonine

Serine

Glutamic acid

Proline

Glycine

Alanine

Cystine

Valine

Methionine

lsoleucine

Leucine

Tyrosine

Phenylalanine

% Protein



or



+



5.0

1.3

3.5

7.2

8.8

3.8

4.7

17.1

8.4

s.l

6.7

2.0

5.2

2.2

3.4

9.3

4.2

4.4

10.5



3.0

0.7

2.6

4.9

9.2

4. I

5.6

22.6

9.6

4.7

9.2

1.7

s.7

1.3b

4.2

14.6

5.2

5.8

9.0



j?2



4.8



-



2.9

6.3

10.5

4. I

5.2

18.5

8.8

4.7

8.0

I .6

5.7

2.7

4.0

12.0

4.6

5.2

17.0



“ N o analysis available. Previous results show tryptophan values for floury-2 equal to

those for opaque-2.

*Lower than expected. Some may have been destroyed during hydrolysis.



position of proteins from whole seeds of normal maize, and seeds homozygous for either the oy or theJ2 mutation. The contents of lysine, tryptophan, and arginine are higher in the mutant stocks than in normal while

the contents of glutamate (glutamic acid glutamine), alanine, leucine,

tyrosine, and phenylalanine are lower in the mutants than in normal. In

addition, the content of glycine is higher in 0 2 .The content of methionine

in j&is higher than in normal or of. The greater nutritive value of the

mutants compared to normal is largely due to the increased amounts of



+



182



OLIVER E. NELSON



lysine and tryptophan present. The reduced quantity of leucine in the

mutants may also be useful since large quantities of leucine antagonize

the use of isoleucine and valine by rats (De Muelenaere et al., 1967).

It can be shown that the differences of the mutants from normal in

amino acid composition on a whole-seed basis can be attributed entirely

to differences in the storage proteins of the endosperm. Table I1 presents

TABLE I1

Amino Acid Composition (Grams per 100 g. Protein) for Defatted Endosperms of

opaque-2, floury-2, and Normal Maize"

Amino acid



W64A+



W64A02



x



Lysine

Tryptophan

Histidine

Arginine

Aspartic acid

Glutamic acid

Threonine

Serine

Proline

Glycine

Alanine

Valine

Cystine

Methionine

Isoleucine

Leucine

Tyrosine

Phenylalanine

% Protein



1.6

0.3

2.9

3.4

7.0

26.0

3.5

5.6

8.6

3.0

10. I

5.4

1.8

2.0

4.5

18.8

5.3

6.5

12.7



3.7

0.7

3.2

5.2

10.8

19.8

3.7

4.8

8.6

4.7

7.2

5.3

0.9 *

1.8

3.9

11.6

3.9

4.9

11.1



3.3

0.8

2.2

4.5

8. I

19.1

3.3

4.8

8.3

3.7

8.0

5.2

1.8

3.2

4.0

13.3

4.5

5.1

13.6



"Data from Nelson er al. (3965) Science 150, 1469-1470. Copyright 0 1965 by the

American Association for the Advancement of Science.

*Other analysis of opaque-2 stocks have given cystine values equal to or greater than

normal.



the amino acid compositions of nornal and mutant endosperms. The

amino acids whose quantities are changed in the mutants on a whole seed

basis are also found to be altered in amount in the endosperms with the

relative differences between the rnutJds and normal being more pronounced. Table 111 gives the amino acid compositions of embryos from

normal or opaque-2 seeds. There is little indication that the amino acid

composition is different in the normal and the mutant embryos. The amino

acid compositions of leaves and pollen from normal and opaque-2 plants

have also been found to be similar (Nelson, 1968). Clearly, the differences



183



GENETIC MODIFICATION OF PROTEIN QUALITY IN PLANTS



in amino acid content between normal and the opaque2 mutant are confined to the endosperm proteins.

Since opaque-2 maize seeds have greatly increased quantities of lysine

and tryptophan, the two limiting amino acids in maize, one would expect

much increased nutritive value for o2 protein as opposed to the protein

of common maize. This expectation has been realized in a number of

TABLE 111

Amino Acid Composition (Grams per 100 g. Protein) for Defatted Embryos of

opaque-2 and Normal Maize"

Amino acid



opaque-2



Lysine

Histidine

Ammonia

Arginine

Aspartic acid

Glutamic acid

Threonine

Serine

Proline

Glycine

Alanine

Valine

Cystine

Methionine

lsoleucine

Leucine

Tyrosine

Phen ylalanine



5.9

2.9

2.1

9.2

9.2

13.9

3.1

5.0

5.3

5.5

5.8

4.4

0.9

I .5

2.5

5.6

2.2

3.6



Normal

6.1



2.9

2.2

9.1

8.2

13.1

3.9

5.5

4.8

5.4

6.0

5.3

I .o

1.7

3. I

6.5

2.9

4. I



"Data from Mertz et al. (1966) Advan. Chem. Ser. 57, 228-242.



experiments with 02 maize. Mertz er at. (1965) reported that weanling

rats on a diet where or maize was the sole source of protein (10 percent)

grew 3.5 times as rapidly as the weanling rats on a diet where common

maize supplied the same level of protein. A third group of rats on a diet

that supplied 10 percent protein as soybean oil meal grew at the same rate

as did the group fed on 0.) maize. Similar estimates of the nutritive superiority of o2 maize in supporting the growth of weanling rats have come

from other laboratories (Mertz, 1966: Piva el al., 1967).

Experiments with weanling pigs have shown that o2 maize supports a

growth rate 3.5 times more rapid than does common maize (Pickett,

1966). Cromwell et a f . ( 1 967) reported that adding lysine to common

maize did not increase the growth rate of weanling pigs: the addition of



184



OLIVER E. NELSON



tryptophan did increase growth rate somewhat. The addition of both

lysine and tryptophan (to the level present in o2maize) increased growth

rate to a rate slightly below that of pigs fed on o2 maize. In a free-choice

preference test between o2 maize and common maize supplemented with

soybean meal to the same protein level as the o2 maize, young pigs ate

about 85 percent o2 maize and 15 percent corn-soy mixture. Finishing

pigs (ca. 130 pounds at the start of the experiment) fed on o2 maize

(1 1.2 percent protein) gained weight approximately as rapidly as those

fed on a maize-soy mixture ( 1 3 percent protein) and significantly more

rapidly than those fed on common corn (8.9 percent protein) or common

maize soy ( 1 1.2 percent protein) (Pickett, 1966).

The nutritional value of o2 maize has been tested in humans. Bressani

( 1966) in nitrogen balance experiments with Guatemalan children demonstrated that o2 maize protein had a nutritive value as high as that of milk

protein when fed a t protein levels of 1.8-2.0 g./kg./day. Children fed at

the same protein level on common maize were in negative nitrogen balance (i.e., were excreting more nitrogen than was supplied in their diets)

indicating the poor quality of common maize protein. Harpstead et al.

( 1 968) and Pradilla et al. ( 1 968) have reported the recovery of children

from kwashiorkor on diets in which part of the nitrogen was supplied by

o2 protein and the remainder by nonessential sources of nitrogen such as

glycine and diammonium citrate. Clark et al. (1967) in tests with adults

found that 5 of 6 subjects tested were in nitrogen balance when consuming

250 to 300 g. of o2 maize daily. For the sixth subject, a large man, 350

g. of o2 corn were needed to establish equilibrium.

The investigations cited have all been carried out with meal made from

the whole seeds of o2 or common maize. When the endosperm meal alone

was used in feeding trials with weanling rats, those fed on o2 endosperm

meal (7.5 percent protein) gained an average of 34.7 g. in 28 days while

those fed on normal endosperm meal at the same protein level gained an

average of 1.4 g. (Wichser, 1966). The much greater difference in nutritive value between the endosperm meals of o2 and common maize than

noted with whole seed meals might have been predicted from Tables I,

11, and 111, which show that all the differences in amino acid composition between o2 and normal maize noted in whole seeds are accounted

for by the differences in endosperm composition. The addition of the

germs tends to bring the composition of the whole seeds of the two

genotypes closer together. Clearly, the use of 0 2 maize will have more

impact and be more valuable relative to common maize in those maize

eating cultures where the maize is degermed before cooking. Discarding

the germs in the preparation of meal markedly reduces the protein quality,

however.



+



GENETIC MODIFICATION O F PROTEIN QUALITY I N PLANTS



185



The nutritive value off12 maize has not been as thoroughly investigated as has that of o2 maize. Veron ( 1967) found that when fed at a level

of 10 percent protein,fle maize was superior to common maize but not

equal to oe maize for weanling rats. However, Cromwell ef al. ( I 968)

reported that in soybean meal-supplemented diets for young chickens

3. was superior to either normal or or maize.

The opaque phenotype characteristic of the or andflz mutants is a

definite asset in breeding programs since it eliminates considerable

chemical analysis. The transfer of either mutation into a desired background can be followed by the opaque phenotype with the assurance that

improved nutritive value is also being transferred.

In genotypes conditioning the production of maize with a higher percentage of protein, the or mutation still effects its characteristic changes

in amino acid composition (Nelson, 1966). This is most important since

o2 maize with 10 percent protein does not support optimal growth for

children or young pigs, but o2 maize with 14 or 15 percent protein should

do so. Such protein levels have been attained. If such selections are

equal in yield to o2 maize of lower protein content, they will be most

valuable since no protein supplementation would be needed. The Jz,

mutation is especially interesting in this connection since the mutation

itself results in higher protein production (Nelson, 1968). It may be possible to attain the desired protein levels in many f12 stocks without

additional selection.

The effects of the or andj& mutations on the production of the endosperm proteins has been investigated by Jimenez ( 1966, I968), Mosse

( I966), and Mosse et al. ( 1966). The results from the two laboratories are

are not directly comparable since Mosse and his collaborators extracted

their protein fractions from entire seeds. Jimenez extracted the proteins

from the endosperms alone. Therefore, Mosse et al. ( 1966) found a larger

amount of albumins plus globulins in all genotypes than Jimenez owing to

the inclusion of the germ (embryo and scutellum) proteins which largely

fall into these classes. This accounts also for the differences found in the

amino acid compositions of the water- and salt-soluble fractions by the

two groups. They agree on the pronounced reduction in the amount of

alcohol-soluble proteins synthesized by the two mutants. Additionally,

Jimenez found a large increase in the amount of the endosperm albumins

and globulins synthesized by the mutants even though these classes of

proteins make up a relatively small proportion of the total endosperm

protein.

In view of the great reduction in zein synthesis, it is interesting that

no difference exists in the amino acid compositions in the zeins from

normal and o2 maize. Starch gel urea electrophoresis of the zeins does not



186



OLIVER E. NELSON



reveal any bands in o2 that are not present in normal (Mosse, 1966;

Jimenez, 1968). Jimenez found that 3 of the 13 bands found in normal

were absent in o2 zein preparations. The starch gel urea electrophoresis

patterns of normal and onalbumin and globulin fractions were identical as

were the amino acid compositions of these fractions. Both groups find a

slightly different amino acid composition for the glutelins from the mutant

stocks as compared to normal with higher lysine contents in the mutant

preparations. It is the conclusion of both Mosse and Jimenez that the

the change in the relative proportions of the solubility fractions (less

alcohol-soluble, more water-, salt-, and alkali-soluble proteins) is the

factor contributing most significantly to the change in amino acid composition of the mutant seeds.

The changes effected by the f l n mutation are qualitative as well as

quantitative. Jimenez (1968) finds one band of the 22 present in the

globulin fraction and one of the 13 present in the zein fraction to be

altered in electrophoretic mobility on starch gel urea electrophoresis,

although the possibility that these differences may be conditioned by

different genetic backgrounds and not by the flz mutation cannot be

rigorously excluded.

In our initial investigations, we were searching for mutations that would

block the ability of the endosperm to synthesize the alcohol-soluble

fraction. The two mutations, opaque-2 and floury-2, that were identified

as affecting the synthesis of this fraction, do not completely block synthesis. In the mutant endosperms, some zein is still synthesized, and this

zein corresponds to that synthesized by normal endosperms on the basis

of amino acid composition and electrophoretic mobility (excepting one

band inJlz). It is a reasonable assumption that the normal allele at the

on locus is concerned with the regulation of zein synthesis. Apparently

no proteins are altered, but the proteins of the prolamine fraction (apart

from the 3 missing bands) are present in much reduced quantities. The

increase in synthesis of the other fractions would be secondary to the

reduction in zein synthesis which is suggested as the primary effect of the

o2mutation. The increase in the components of the other solubility classes

is not uniform. Jimenez ( 1968) has shown, for example, that the increase

in the albumin fraction of 02 is largely an increase in one protein.

There is no definitive evidence that the primary effect of the o2mutation

is on zein synthesis. It is possible but less likely that the enhancement of

the other fractions is primary, and the reduction in zein synthesis is

secondary. The observations that the total increase in the other fractions

does not equal the decrease in zein and that the quantity of free amino

acids is higher in o2 suggests that the reduction in zein synthesis is pri-



GENETIC MODIFICATION OF PROTEIN QUALITY IN PLANTS



187



mary. The embryos in 0 2 seeds are larger than in normal controls and in

some lines may contain more protein (Bauman, unpublished data).

The effect of the& mutation may be more complex than that of 0 2 .The

synthesis of zein is suppressed almost as much as with og In addition,

several proteins with altered mobilities are produced. The mode of inheritance is semidominant and each successive dose o f j & in the endosperm adds an additional increment of lysine (Bates, 1966).

VI.



The Prospects of Improvements in Other Plants



Since the amino acid composition of maize can be drastically improved

by mutations that act to suppress the synthesis of the nutritionally poor

prolamine fraction, the prospect of improving other cereal grains by the

detection of homologous mutations exists if the grain contains a sizable

prolamine fraction. Only rice and oats among commonly grown cereals

do not. These two species rank at the top of the cereal grains with respect

to lysine content expressed as grams per 100 g. of protein. For these two

species, mutations homologous to o2 or fr, would have relatively little

effect on amino acid composition. For the other cereal grains, such mutations would be expected to effect considerable improvement. In polyploid species such as the hexaploid Triticum vulgare, one of the genomes

could contain a recessive gene homologous to o2 without any effect on

amino acid composition. The difficulty of obtaining such a mutation in all

the genomes is formidable. On the other hand, the presence of a semidominant mutation such asJ2 in one of the genomes would effect a change

in amino acid composition and should be identifiable. Such genotypes

would then constitute the starting point for a second cycle of nutritional

improvement.

With barley and sorghum, mutations leading to larger quantities of the

limiting amino acid should be detectable. Recent reports of lines containing larger quantities of lysine have been made for each species. Virupaksha and Sastry (1968) have reported a wide range of lysine content in

the whole seeds of various accessions of sorghum. One accession,

Cernum 160, had a lysine content of 3.14 g. per 100 g. of protein, which

is high for sorghum. This variety had a low pro1amine:glutelin ratio, so

the change in composition here is brought about by a change in the relative proportions of the storage proteins. Only one sample of the variety

was analyzed, and there is no report of inheritance studies with this

variety. Hagberg and Karlsson ( 1968) have reported a barley accession

that contains 4.6 g. of lysine per 100 g. of protein, which is above the

usual levels for barley - 3.6 g. per 100 g. of protein (Mosse, 1968; Eggum,

1968). Again, this represents the analysis of a single sample, and it is not



188



OLIVER E. NELSON



yet known whether the accession grown in another year would again

have a high lysine content.

Assuming that one could detect mutations that suppress prolamine

synthesis and significantly change the prolamine: glutelin ratio in any of

the cereals rich in alcohol-soluble proteins, the results might not be as

striking as with the o1 and j12 mutations in maize as Mertz (1969) has

pointed out. The prolamines of cereals can be divided into 3 groups on

the basis of their amino acid composition: the first group contains the

gliadin of wheat, hordein of barley, and secalin of rye; the second contains the zein of maize, panicin of millet; the third contains the avenin of

oats (Mosse, 1968). The prolamines of the second group contain very low

amounts of lysine. To this group can be added kafirin of sorghum with a

lysine content of less than 0.2 g. per I00 g. of protein. The consequence of

suppressing prolamine synthesis with consequent compensatory synthesis of other protein fractions would be expected to be greater in terms

of lysine content for maize, millet, and sorghum than for cereals of the

first group, where the lysine content of the prolamine fraction is ca. 1 g.

per 100 g. of protein.

The possibility of raising the methionine content in the proteins of the

legumes deserves careful study since methionine is the limiting amino

acid for all legumes. For obvious reasons, the proteins of the soybean

seed have been intensively investigated. As in the other leguminous

seeds, the bulk of the seed protein is globulin in nature and was once

thought to be a homogeneous protein (glycinin). Ultracentrifugal studies

have revealed that 4 components (2, 7, 1 1 , and 15 S) are present (Naismith, 1955). Wolf and Sly ( 1 967) have shown that other methods of

fractionation will separate the components. Roberts and Briggs ( 1 965)

reported that the 7 S component that comprises 30 percent of the total

protein has an extremely low methionine content-0.19 g. per 100 g. of

protein. For comparison, the entire globulin fraction has a methionine

content of 1.4 g. per 100 g. of protein. The 7 S fraction also differs appreciably from the total in its content of threonine and glycine. If the

synthesis of the 7 S fraction could be blocked or suppressed genetically,

and compensatory synthesis of the other fractions resulted, the methionine content would be raised substantially. Wolf et al. (1961) have reported that the relative proportions of the 7 S and 1 1 S fractions were

quite different in Clark soybeans grown in Illinois and Hakuhou No. 1

soybeans grown in Japan. In this instance, it is not clear whether variety,

location, or both are responsible. The possibility that lines with a low

quantity of the 7 S fraction exist, or could be induced, should be investigated.

In this review, the principal concern has been the enhancement of



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