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II. Heterosis and the Gene-Enzyme Concept

II. Heterosis and the Gene-Enzyme Concept

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extensively by farmers until after 1936. Thereafter its use expanded

dramatically. A t present, hybrid seed is used on a high percentage of the

100 million hectares of the earths surface planted annually to corn.

Despite the extensive use and the wide acclaim given to hybrid corn,

divergent views exist as to the contribution by the hybrids to grain

yields, per se. Whaley (1944) states that the grain yield of the hybrid

may exceed that produced by the original heterozygous parent stock by

as much as 50 percent while Stringfield (1964) estimates a more conservative positive increase of 20 percent. Observations ( Richey, 1950)

also indicate that the individual hybrid plant is not superior in grain

yields to the best individual plant of the original heterozygous openpollinated parental stock. In this view, the superior performance of the

hybrid is due to uniformly high productivity of the individual plant in

contrast to the greater variability among plants of the open-pollinated

variety (Richey, 1950). Overall it is obvious that the better corn hybrids

are usually superior in yield to the best of the open-pollinated varieties,

although exceptions have been noted ( Stringfield, 1964).

Virtually all F, hybrids ( single-crosses) between two inbred lines

will produce grain yields double or more those of the highest-yielding

inbred parents ( Leng, 1954). Yield component analysis reveals (Leng

1954, 1963) that heterotic effects are largely concentrated in the two

“primary components”: kernels per row (ear length), and kernel weight.

Both these components are manifestations of increased vigor and energystoring capacity. Hybrid plants are nearly always taller and have greater

leaf area (Shull, 1952) than their parental inbreds. However, total foliage

leaf number (Mehrotra, 1954) of F, hybrids usually is intermediate between the parental levels. The general phenotypic manifestation of

hybrid vigor, then, is an increase in growth rate and total size of the

plant, with an even greater proportionate increase in kernel number

and total grain weight (Leng, 1954),

Both Richey (1950) and Stringfield (1964) raise the question whether

hybrid corn yield performance has attained a plateau. Both suggest that

there is a need for more efficient breeding methods. Since this question

was raised, average yield levels have increased noticeably. Nevertheless,

the basic question is valid. Richmond (1951) stated that there was a

need to develop more precision in breeding programs in cotton. Precision

was defined as the development of indices that would measure “genetic

potential performance rather than actual end-result behavior.” These

indices were not further defined. Allards discussion (Allard, 1960) of

the evaluation procedure for development of corn inbreds also illustrates

the problem confronting the plant breeder. Specific points are: (1) it

has been estimated that only 60 corn inbreds of over 1001,OOO tested



were good enough to be useful for commercial hybrid production, and

( 2 ) the better inbreds in common commercial use were derived from

a very few open-pollinated varieties including such unimportant varieties

as Lancaster Surecropper. The suggestion most commonly made (Stringfield, 1964; Richey, 1950; Richmond, 1951) for genetic improvement of

agronomic crops is the introduction of new germ plasm to maintain

maximum genetic diversity. The reason for the small percentage of

successful inbreds resides in the inability to evaluate existing and new

germ plasm prior to testing the hybrids. Of course, the development of

more efficient statistical methods for determining usefulness of lines has

promise. Nevertheless, it is clear that most effective breeding techniques

would be those based on understanding and careful evaluation of

physiological efficiency. To date, such techniques have not been put into

practice, and some of the basic data are lacking on which research

workers could proceed to evolve “physiological breeding methods.

As indicated by Whaley (1952) the interacting complex of genes,

metabolism, and environment required for the expression of hybrid

vigor precludes a simple genetic explanation. Yet it is obvious that genes

(in some number) are the underlying basis for the physiological

advantages that lead to hybrid vigor. Because all genes are derived from

the same general parental stock of the species, the phenomenon of heterosis must arise from their proper recombination. Whaley (1952) states

that genetic and physiological studies concerned with the early phases of

development are most likely to lead to the understanding of heterosis.

Although his work suggests guidelines for physiological-genetical investigations of heterosis in higher plants, specific examples especially with

respect to enzymes remained minimal until the mid-1950‘s.

The tremendous recent advances in knowledge and understanding of

the relationship between genes, metabolism, and growth are presented

in current texts and reviews (Bonner and Varner, 1965; Fincham and

Day, 1965; Frisch, 1961, 1963; Pollard, 1965; Crick, 1964; Watson, 1965;

Herskowitz, 1962; Sager and Ryan, 1961). Because of the complexity of

the processes involved, it is understandable that most studies have been

conducted with microorganisms. Even with these simpler organisms,

final resolution of the complexities of genetics has not been achieved.

However, there is no reason that these findings and research approaches

cannot be applied in a general sense to the study of heterosis and breeding of agronomic crops.

In brief, current concepts can be outlined as follows. The genetic

(chromosomal) material is capable of precise replication, except for

mutations, generation after generation (Fig. 1). In microorganisms which

have been studied intensively, the single chromosome is a double-



stranded, helix-wound, continuous loop of polymerized deoxyribomonophosphate nucleotides ( deoxyribonucleic acid-DNA ) , Each successive

nucleotide is coupled by a 3’-Y-phosphodiester bond. The molecular

weight of this circular chromosome is estimated at 2 billion, and each

strand contains an estimated 2.5 to 3.0 million individual nucleotides.

The chromosomal (DNA) strand is composed primarily of four deoxyribonucleotides: deoxyadenosine monophosphate ( d-AMP), deoxyguanosine monophosphate ( d-GMP), deoxycytidine monophosphate ( d-CMP ),

and thymidine monophosphate ( d-TMP ) ,

.. .. .. .. .. .. .. .. .. .. ..



. . . . . . . . . . .







. .. .. .. .. .. .. .. .. .. ..

FIG. 1. Schematic diagram showing the two parental strands of deoxyribonucleic

acid (DNA), and the replication process that involves strand separation and strand

duplication. The latter process is catalyzed by the enzyme DNA polymerase. Key to

the symbols: solid line connecting circles represents the 3’-5’-phosphodiester bond that

couples the nucleotides forming the DNA strand; the two dots represent the hydrogen

bond between the compIementary nucleotide pair; dA, dG, dC, and d T represent the

four deoxyribonucleotides; deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxycytidine monophosphate, and thymidine monophosphate, respectively. dA always pairs with dT, and dG with dC.

The chromosome strands are held together by hydrogen bonds between the nucleotides, and d-AMP is always H-bonded to d-TMP and

d-CMP is always H-bonded t o d-GMP. This type of bonding establishes

complementary structures, i.e., the nucleotide in one strand specifies the

( complementary) nucleotide in the other chromosome strand. In replication the strands separate, and each serves as a template to make a new

complementary strand, thus precisely duplicating the genetic material,

A second primary function of the chromosome (DNA) strand is to

specify, albeit indirectly, the kind (identical over time periods) and

amount as well as time of synthesis of protein (enzymes) needed for the

growth, development, and function of the cell. This process is shown

schematically in Fig. 2.



The coding procedure which specifies the kind of protein to be

synthesized resides in the sequential order of the nucleotides along a

single strand of DNA. Specifically three nucleotides in sequence are

required to specify the insertion of one amino acid in a protein. DNA

does not serve as a direct template for the synthesis of protein but does

serve as a template for the synthesis from ribonucleotides, of a complementary single strand polymer of “message” ribonucleic acid (mRNA ) .











FIG.2. Schematic diagram of the DNA strands; the unwinding and strand separation that precedes transcription or formation of the messenger ri!3onucleic acid

(mRNA) [the coded information for the synthesis of a specific protein (enzyme)];

and translation, the process of forming the protein specified by the mRNA. Transcription is catalyzed by the enzyme RNA-polymerase, and translation is a very

complex process that involves ribosomes ( ribonucleic acid-protein-organelles ) and

soluble or transfer ribonucleic acids. Key to the symbols: the double bars between

DNA and mRNA indicate hydrogen bonds between complementary base pairs; A,

G, C, and U represent the ribomonophosphate nucleotides found in RNA, adenosine,

guanosine, cytidine, and uridine ( rib0 ) monophosphates, respectively; the three sets

of nucleotide triplets (code) on the mRNA, UUU, CAC, and GAA, specify the

protein phenylalanine ( p ) , histidine ( h ) and glutamic acid ( g), respectively. The

dotted line between the amino acids represents peptide bonds. Other symbols are

the same as in Fig. 1.

This process is known as transcription, and the polymer formed

(mRNA) directs the synthesis of the protein. Three of the four common

nucleotides of mRNA are the same as the nucleotides found in DNA,

except that the sugar portion of each molecule is ribose instead of

deoxyribose. The fourth nucleotide, uridine (ribose ) monophosphate,

is substituted for thymidine monophosphate. Again, as in DNA, the

nucleotides are linked together by 3’-5’-phosphodiester bonds.

The transcription operation is initiated by the unwinding of the

double-stranded chromosome (DNA), which permits the “reading out”



of the mRNA. Complementation of the nucleotide bases is observed

between the DNA and the mRNA. For example, if d-TMP, d-CMP,

d-GMP, d-AMP, d-AMP, d-AMP and d-AMP occur in sequence along

the DNA strand, the mRNA reads in sequence AMP, GMP, CMP, UMP,

UMP, UMP, and UMP, respectively. It is currently visualized that a

gene, defined as a segment of a single strand of chromosome (DNA)

within which a chiasma cannot be formed without modification of the

organism, is the template for the individual mRNA formed. The mRNA

in turn serves as the template for the synthesis of the protein (enzyme

or subunit of an enzyme). Since there are four nucleotides and a combination of three nucleotides is required to specify one amino acid, there

are sixty-four possible codes of nucleotide triplets available to specify

the position of the twenty common amino acids found in proteins. As an

example, a triplet nucleotide sequence along a segment of mRNA of

UMP, UMP, and UMP when read out (translated from mRNA to

protein) would specify the insertion of the amino acid phenylalanine.

Since there are sixty-four possible code triplets and only twenty amino

acids, some amino acids can be specified by more than one code. The

reasons for and implications of this multicoding are not fully established

at present.

Enzymes (proteins) are linear polymers of amino acids. Each amino

acid in the polymer is joined to each of the two adjacent amino acids by

a peptide bond. Thus its carboxyl group is linked with the amino group

of one adjacent amino acid while its amino group is linked to the second

amino acid.

Enzymes range in molecular weight from 10,000 to several million

and accordingly would contain from 100 to several thousand amino acids.

In function, enzymes are biological catalysts, They are considered to

operate like other catalysts, providing a surface upon which reacting

components can bind while undergoing changes in orbital electron

configuration. Thus reacting metabolites are bonded to the enzyme for

periods of time ranging from microseconds to days. It is obvious that

enzymes must be very specific as to reactions that they catalyze if they

control the metabolism of an organism. This specificity is achieved by

the sequential order of amino acids along the linear protein polymer

and by the conformation (secondary and tertiary structure) the enzyme

assumes in its active state. This specificity is not absolute, otherwise the

competitive inhibition of enzymes would be impossible. In fact, compounds of related structure and charge can also bond to a given enzyme.

One other important facet of enzyme structure is that only certain

segments (active centers) of the molecule can bind the metabolite

(substrate). Thus portions of the enzyme can be deleted (by isolation



techniques or genetic mutations) without completely destroying the

catalytic efficiency of the enzyme. Usually, a deletion of part of the

enzyme structure is associated with decreased enzymatic function. The

bonding of metabolite (substrate) and enzyme is partially described by

the Michaelis-Menten kinetics and constant ( K , ) , K, is defined as that

concentration of metabolite in moles per liter where reaction velocity is

one-half maximal. At this concentration of substrate, the binding site of

the enzyme can be assumed to be half saturated whereas maximum

reaction rates are achieved only when the binding sites are fully


Because any change in active centers, charge, structure, or form of

an enzyme, produced by genetic mutation or environmental conditions,

could greatly affect the bonding of substrate and enzyme, the rate of

individual reactions, and in turn, metabolism as a whole, could readily

be altered. Changes in the sequential order of amino acids in the enzyme

reflect changes (mutations) in the gene. Currently the best way to

characterize a gene is by characterization of the enzyme product specified by the gene.

To recapitulate, the operations synthesizing an enzyme (assumed to

be monomeric in this case) containing 150 amino acids with molecular

weight of 15,000, would require mRNA containing approximately 150

triplet codes, or 450 nucleotides. If the DNA undergoes no variation in

sequence of nucleotides along the chromosome chain, if the transcription

of DNA to mRNA proceeds without error, and if no mistakes are made

in translation of mRNA into protein, enzyme molecules identical in

structure will be produced.

Since at present it is believed that a different mRNA must be synthesized for each enzyme, and since the number of enzymes in an organism

is probably at least 2,000 to 3,000, the system obviously is extremely

complex. The “lifetime” (stability) of mRNA also is variable, ranging

from a half-life of minutes to several hours in the few examples which

have been carefully studied. This is important because the number of

individual enzyme molecules which can be translated from one mRNA

varies both with the rate at which the translation can be carried out and

with the “lifetime” of the mRNA. The precise number of enzyme molecules produced by any given mRNA is not known. This turnover of

mRNA is obviously an important metabolic control mechanism, since

stability (“lifetime”) of enzymes also varies,

Jacob and Monod (1961) have developed a theory of gene action

which postulates two categories of genes: (1) structural genes; ( 2 )

regulatory genes. It is considered that “structural genes” specify the kind

of protein (enzyme) to be produced, and “regulatory” genes determine



whether and to what extent a given protein would be synthesized under

a given environment. “Regulatory” genes would not alter the kind of

protein specified by the “structural” genes.

Environmental changes could affect enzyme content of cells in

several different ways. One likely possibility is by enzyme induction,

where a metabolite could invoke the synthesis of the particular enzyme

needed for its utilization. A second is enzyme repression, the situation

in which synthesis of a given enzyme is suppressed by a certain metabolite, It appears likely (Jacob and Monod, 1961) that induction and

repression are basicaIly controlled by the same mechanism. It has been

shown in some cases that induction is the release of repression and that

different metabolites can activate the same repression. Exactly how the

activation or repression of the “regulatory” gene occurs is not known.

Neither are there clear indications of the nature of the control products

(repressors) formed by “regulatory” genes, or of the sites of activity of

the repressor materials. Currently, it is assumed that the “repressed

state (“structural” genes nonfunctional) is normal. However, since the

control of state is genetic, it is obvious that gene mutations could occur

which would specify continuous function, intermittent function, or no

function of a given “structural” gene. Finally, one “regulatory” gene

could control the action of one or several “structural” genes. Such functional groups of genes have been termed “operons” (Jacob and Monod,

1961).Little evidence to support the “operon theory” has been obtained

with nonbacterial organisms (Fincham and Day, 1965). With respect to

agronomic plants, Filner (1966) has reported the induction of nitrate

reductase in cultured tobacco pith cells by nitrate and the repression of

this induction with certain amino acids. Although nitrate reductase can

be induced by nitrate in corn seedlings, repression is not affected by

amino acids (unpublished data of our laboratory).

To summarize, the function of enzymes is to catalyze the chemical

reactions which make up metabolism and which are expressed in the

growth, development, and maturation of cells, tissues, and the entire

organism. Since both intensity and direction of metabolic processes vary

with time, it is essential that the enzyme complement or its functional

effectiveness vary, both within the cell and from cell to cell, during

different stages of development. Thus, as stated by Glass (1958), “genes

must function differently in different cells.” Also they may function

differently in the same cell at different times. From this it may be concluded that sequential development results from differential gene action,

in point of time. If the ‘parsimony of nature” principle is valid for

metabolic systems, it is more likely that control is exerted over the fabrication of enzymes, than that there is a constant rate of synthesis of a11



enzymes with control being achieved by preferential degradation or


From the above, it is clear that a “biochemical-genetic” approach to

the problem of heterosis and its effective utilization in plant breeding

must be based on the fundamentaI facts of enzyme activity and its

genetic control. From this, it follows that a direct attack on the problems

involved in major metabolic processes is indicated. The following sections

of this paper will treat recent experimental evidence relating to several

such problems.


Heterosis and Enzyme Activity during Germination

From the discussion above, it is clear that an experimental approach

to a biochemical solution of the heterosis problem must focus on

metabolic systems of major importance for growth, development, and

maturation. A prime requirement of such studies is that the system to be

investigated be one which is not only important to the organism, but

capable of reasonably exact investigation under environmental conditions

which will produce normal growth and development. Even today, this

latter requirement presents major difficulties. When research was initiated

at Illinois on this problem in 1954, far less biochemical-genetic information (particularly pertaining to higher plants) was available. Initially

the work had to be restricted to etiolated corn seedlings because many

enzymes could not be extracted in active form from green corn leaf

tissue by standard and established procedures (Hageman and Waygood,


The first experimental approach was to examine the energy transfer

system in young corn seedlings. The basic rationale of this study was

that extreme manifestations of heterosis were nearly always apparent in

seedling vigor, i.e., rate and amount of early growth. Ashby (1930,

1932) postulated that this early vigor resulted from an increased “initial

capital” of embryo tissue in the hybrid, as compared with its inbred

parents. Groszmann and Sprague ( 1948), however, clearly showed that

“initial capital” differences have little if any basic influence on heterotic

early vigor. Therefore, it was clear that the explanation for this phenomenon must be sought elsewhere.

The work begun in 19% at Illinois was planned to determine whether

enhanced early vigor in corn hybrids could be associated with greater

amounts or more efficient operation of major enzymes involved both

directly and indirectly in energy transfer. The basic premise was that

since the hybrid grows at a faster rate than its inbred parents, its cells

must convert stored food reserves to energy, and ultimately to the



“building blocks” of new tissue, at a faster rate, Alternatively, the hybrid

simply operates more efficiently than the inbred. I n either case, differences in enzyme activity should occur, since most if not all major metabolic reactions are catalyzed by enzymes. As is true for other catalysts,

enzymes affect the rate (velocity) of chemical reactions but do not alter

the reaction equilibrium, nor do they appear as products of the catalyzed

reaction. The conclusion should be obvious that a hybrid which is growing more rapidly than its inbred parents should show either ( a ) more

efficient (qualitatively different) enzymes, or ( b ) greater amount of

enzymes (quantitatively different), for the enzymes involved in the

major growth reactions. Logically, it would be expected that quantitative

differences would be most likely, since increases in quantity of catalyst

(enzyme) normally are associated with increases in the amount of

reaction products per unit time of reaction.

Germination studies, conducted in dark constant environment growth

chambers with corn seedlings, offered an experimental approach to test

the postulate that hybrid vigor was due to qualitative or quantitative

differences in enzymes, The growth of etiolated seedlings for the initial

5 days is almost exclusively dependent (85to 90 percent) upon the stored

endosperm starch for its energy source (Ingle et al., 1964). In addition

to energy, starch is a major reserve for carbon compounds needed in

growth and development. The enzyme glucose-6-phosphate dehydrogenase (Fig. 3 ) initiates a pathway that leads to the production of ribulose

diphosphate used in the photosynthetic fixation of CO, and ribose-a

constituent of nucleotides. Other metabolites of the glycolytic pathway

and Krebs cycle are useful and necessary for growth and development.

Enzymes concerned with the release of energy and conversion of starch

to other compounds are of major importance to the growth and development of the seedling.

The selection of enzymes for study posed problems because of the

large number of enzymes available for testing (Bonner and Varner,

1965). The enzymes, triosephosphate dehydrogenase (TPD ), aldolase

( ALD ), and glucose-6-phosphate dehydrogenase ( G-6-PD ) were selected for study. ALD and TPD were chosen because they are two of the

ten enzymes of the glycolytic pathway (Fig. 3 ) which couples the

stored endosperm starch to the Krebs cycle. Although about 80 percent

of the energy derived from starch is converted in the Krebs cycle into

forms [adenosine triphosphate (ATP) and reduced di- and triphosphopyridine nucleotides ( DPNH and TPNH), respectively] useful to the

seedling for growth and development, the enzymes TPD and G-6-PD

are the sites of first energy conversion. The functioning of the Krebs

cycle is also dependent upon pyruvate supplied by the glycolytic path-

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II. Heterosis and the Gene-Enzyme Concept

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