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VI. Some Recent Developments in Plant Biochemistry Related to Heterosis

VI. Some Recent Developments in Plant Biochemistry Related to Heterosis

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A BIOCHEMICAL APPROACH TO CORN BREEDING



75



curring forms of the enzyme (allodimers and autodimers) specified by

the various other alleles.

I n vitro treatment with glyceraldehyde of various forms of the

esterases derived from the El gene converted all isozymes having varying

net charges, as indicated by band locations on to starch gel, to a single

form with a common net charge, as indicated by a single-band location

on the starch gel (Schwartz, 1962b). All treated isozymes were converted to a more acidic form, and all migrated to the anode (+) at

identical rates. Most ( 6 of 7 ) of the isozymes prior to treatment were

basic in nature and migrated toward the cathode ( - ).

The glyceraldehyde treatment did not cause any appreciable inactivation of the E, esterases. A partially purified esterase preparation incubated with 0.2 M glyceraldehyde at 37°C. for 24 hours retained 96 percent of the activity of a glyceraldehyde-free control as measured by

cleavage of a-naphthyl acetate. Essentially full activity was retained by

the enzyme during the full conversion from a basic to an acidic protein

by the glyceraldehyde treatment. This encompasses many of the charged

forms exhibited by the various isozymes. Does this mean that the minor

changes in charge, which constitute the only known difference among

the various isozymes or hybrid forms, do not cause alteration in

metabolic activity?”

Recently Kitto et al. (1966) using tissue from chickens, demonstrated

multiple forms of mitochondria1 malate dehydrogenase by electrophoretic patterns. Because it was not possible to detect differences in

catalytic properties or in amino acid composition among the various

forms, the term “conformers” was proposed for enzymes with these

characteristics. The work of Fincham (1962) illustrates a divergent

case. The TPN-linked glutamic dehydrogenase of Neurospora crassa is

specified by the am locus. Thirteen different mutations of the am locus

have been observed and the enzymes resulting from these mutations

exhibit great variation in catalytic activities and characteristics including

digerential response to temperature. In spite of these differences, all

the enzymes separate in a similar manner during purification, are

indistinguishable on starch gel electrophoresis, and some of the different

forms exhibit similar “fingerprints” after tryptic digestion. It is obvious

that both qualitative and quantitative measurements are required to

obtain maximum understanding of these complex systems.

Form or type of enzyme becomes important only when it effects a

favorable change (either increasing or decreasing) in rate of substrate

conversion (total enzyme activity) with respect to the metabolic needs

of the organism. Since it is conceivable that one form of enzyme specified

by a homozygous locus might be metabolically adequate only under a



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R. H. HAGEMAN, E. R. LENG, AND J. W. DUDLEY



limited range of environmental conditions, the increased number of

enzyme forms associated with a heterozygous locus could be a causal

factor in hybrid vigor. For corn, this concept must be accepted with

reservations until additional characterizations of the enzymes specified

by heterozygous loci have been obtained.

Even for fungi, Fincham and Day (1965) state that many questions

remain to be answered concerning the observation that “sometimes a

pair of alleles in a heterocaryon is abIe to promote the formation of an

enzyme when neither allele could do so alone.” They point out that the

enzyme formed by interallelic complementation is ( a ) low (25 percent

of normal or less) in activity, and ( b ) usually qualitatively abnormal

with respect to the wild-type form. In addition, an active enzyme can

be formed in certain cases by mixing crude extracts or purified proteins

from separate strains which exhibit complementation, under conditions

not conducive for protein synthesis. From these observations it has

been postulated that the active enzyme is composed of two or more

subunits. The subunits are specified by each allele, and when the alleles

are both wild-type the normal enzyme is formed. If two different mutant

alleles are present, both protein subunits are abnormal and most dimers

(or multiunit enzymes) formed are inactive enzymes; however, a small

number of subunits may unite in such a way that the deficiencies are

masked ( complementation) and an active enzyme results.

Beckman, et at?. (1964a) recently have demonstrated the existence

of four different molecular forms of leucine aminopeptidase in maize

endosperm by starch-gel electrophoresis techniques. Each enzyme

appears to be controlled by a pair of alleles without dominance. In a

similar study with corn endosperm tissue, the same workers found

( Beckman et al., 1964b) three “hybrid catalase enzymes with migration

rates on starch-gel electrophoresis intermediate between their respective

parental enzymes. Their observations suggested that the enzyme might

be a tetramer. Supplemental work by Scandalios (1965) indicated the

occurrence in a single corn inbred of tissue-specific isozymes. Several

different forms (isozymes) were noted for each of the following

enzymes: leucine-amino-peptidase, esterase, catalase, and peroxidase.

The various isozymes were detected and described only by starch-gel

electrophoresis and staining techniques. The function of these four

enzymes in differentiation and development of the corn plant is not

known.

It is the authors’ opinion that the observations of Schwartz,

Scandalios, Beckman, and Brewbaker are of great interest and importance. Work of this type presently provides the best approach to the

biochemical understanding of interallelic and intergenic complementa-



A BIOCHEMICAL APPROACH TO CORN BREEDING



77



tion. The proposition that many enzymes are composed of aggregates

of subunits, and that these protein subunits are specified by the individual

alleles, permits a logical explanation of variation in metabolic activity

of corn inbreds and their hybrids. It is also obvious from the work of

Kitto et al. (1966) and Fincham (1962) that the qualitative separation

of enzymes needs to be supplemented by extensive quantitative evaluations. With respect to agronomic production, the authors strongly believe

that such qualitative and quantitative evaluations need to be continued

throughout the life of the plant. The interaction of genotype with

environment is dynamic, and a valid conclusion reached in growth

chamber studies may not be true under field conditions.

The complexities of metabolism preclude a single factor from being

the universal underlying cause of hybrid vigor. Yet in certain cases a

single enzyme, hormone, vitamin, or growth factor could be solely

responsible for the enhanced growth rate of a hybrid. Where such cases

have been found, the experiments usually have been of short duration

and have not extended over the life cycle of the plant. The work reported

by Robbins (1940, 1941b) serves to illustrate this single-factor concept.

In these experiments, separate extracts from equal weights of grain from

two corn inbreds and their two progenies were added to a standard

minimal medium used for culturing Phycomyces blakesleeaniu Burgess.

When added to the media, extracts from the hybrid seeds essentially

doubled the mycelial growth over that obtained with extracts from the

inbred seeds. Extracts from seeds of the two corn inbreds did not

enhance mycelial growth over that of the basal medium. The growthstimulating factor in the hybrid seeds was never fully identified.

In a similar experiment two decades later, Matskov and Manzyuk

(1961) reported that extracts of hybrid seed, seedlings of leaf tissue

stimulated the growth of yeast more than comparable extracts from the

parental material. Mixtures of extracts from appropriate inbred material

invoked the same growth effect on the yeast as did the extracts of

hybrid progeny. In some instances (four of eighteen) no differences were

observed between inbreds and their progeny. The causal factor was

reported to be a mixture of B vitamins. No difference was observed in

auxin content of the inbreds and hybrids tested. These results are in

general agreement with the nutrient supplement work of Robbins

( 1941a), who used excised hybrid and inbred tomato root cultures. The

addition of pyridoxine to the minimal basal nutrient stimulated the root

growth of one of the parental inbreds, whereas nicotinamide had no

effect. The converse was true for the second inbred. The root growth

of the hybrid was superior in all cultures.

Sinkovics (1963) analyzed various plant parts of two corn inbreds



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R. H. HAGEMAN, E. R. LENG, AND J. W. DUDLEY



and their F, progeny for vitamins of the B group (biotin, thiamine,

pyridoxine, pantothenic acid, and nicotinic acid). Except for thiamine,

the hybrid contained a much higher quantity of these B vitamins.

Hormones are single components that could exert manifold effects

on metabolic systems and metabolism, Key (1964) reported that 2,4-D

( a synthetic auxin) enhanced the rate of cell elongation of excised

soybean hypocotyl tissue. Cell elongation in turn is dependent upon

nucleic acid and protein synthesis. Apparently 2,4-D enhances the

formation of specific nucleic acids. Varner (1964) and Varner and

Chandra, 1964) have shown that gibberellic acid causes the de w v o

synthesis of a-amylase in the aleurone layer of half barley seeds.

As important and exciting as these results are, it is still extremely

difficult to know how to extrapolate and adapt these findings to the

selection of genetic material with optimum hormonal balance throughout

the life of the plant. The complexities and difficulties involved are

indicated by Wright ( 1961, 1966). His data demonstrate that growth

responses to exogenous gibberellic acid and kinetin were greatest in

young excised wheat coleoptiles (18 to 78 hours after sowing). In

contrast, exogenous indolyl-3-acetic acid exerted little effect on growth

until the coleoptiles were much older (30 to 42 hours ) . Gibberellic acid

and kinetin effects could be separated by alteration in temperature. It

is also quite probable that at certain developmental stages hormones

will exert synergistic effects and at other stages antagonistic action (Van

Overbeek, 1962).

McDaniel and Sarkissian (1966) reported that at 1:1 mixture of

mitochondrial preparations from seedlings or scutella of two corn inbreds

as well as the mitochondrial preparations from the hybrid (WF9 x

C103) showed heterosis with respect to respiration and oxidative

phosphorylation. In a second case, using the “hybrid Oh43 x Oh45

which does not show heterosis with respect to growth, no heterosis was

observed with the hybrid mitochondrial preparation or from mixing

preparations from the two respective inbreds. Interpretation of these

comparisons is complicated by the fact that the “hybrid Oh43 x Oh45

actually is a cross between two very closely related inbred lines, and in

fact is essentially similar to an inbred. It is not known whether the

heterotic response with the WF9 C103 and WF9 x C103 material is

attributable to a single chemical compound or to a complementation of

enzymes. The latter alternative would appear to be quite complex.

Sarkissian and Huffaker (1962) reported that a barley hybrid (Kk)

derived from essentially isogenic parents, exhibited heterosis in seedling

growth (weight) and ability to fix CO, via the ribulose-diphosphatecarboxylase pathway. The greater fixation of CO, could be correlated



+



A BIOCHEMICAL APPROACH TO CORN BREEDING



79



with heterosis. In contrast there was no difference among these same

three genotypes with respect to ability to fix CO, by the phosphoenolpyruvic carboxylase system. Thus, hybrid vigor was not found to be

associated with a general stimulation of all enzymes involved in

metabolism.

The ability of the hybrid to fix CO, by the ribulose diphosphate

pathway was greater (0.37 DS 0.26 pmoles of CO, fixed per gram fresh

weight per hour) prior to and during the first 10 hours of illumination

than either parental line. The hybrid apparently reached a maximum

rate of CO, fixation after 10 hours. During the subsequent 14 hours of

illumination, the difference in fixation rate between hybrid and parents

gradually decreased, although the rate of CO, fixation by the hybrid

was still greater than that of either parent at the end of the experiment.

This advantage of the hybrid is stressed because it suggests that the

hybrid has the ability to shift its metabolism more rapidly in response to

changes in environment, and to regulate the output of specific systems

to the needs of the whole system. Perhaps the parental lines ( K K and

kk) in time would have attained or even exceeded the maximum rate of

CO, fixation exhibited by the hybrid, even though they could not compensate for their initial disadvantage.

This work (Sarkissian and Huffaker, 1962) suggests that studies of

changes in enzyme activity or enzyme synthesis in response to changes

in environment would permit an evaluation of the efficiency of the

complex system of protein synthesis among an array of inbreds and

their progeny and varieties. Under field conditions, corn hybrids customarily appear able to adapt readily to changes in environment. A

change in environment would undoubtedly alter the enzyme complement, which then would alter metabolism. Rapidity of response to an

environmental change, as reflected by the rate of synthesis of the needed

enzyme, should be a highly desirable attribute.

For precise studies, the initial level of enzyme (or other component)

of inbreds and hybrids should be equal, and should be expressed on some

unit of measure other than plant size. In the work with barley (Sarkissian

and Huffaker, 196Z), the initial rate of CO, fixation (cpm/O.l ml) just

prior to illumination was twice as high for the hybrid as for the parental

lines. Based on the initial values, the rate of increase of CO, fixation

was the same for inbreds and hybrids during the first 10 hours, and

greater increases were exhibited by the inbreds in the subsequent

14-hour period.

Some attempts have been made in our laboratories to utilize the

induction and synthesis of nitrate reductase as a tool for evaluation of

corn inbreds and hybrids. Corn seedlings grown on a medium devoid



80



R. H. HAGEMAN, E. R. LENG, AND J. W. DUDLEY



of nitrate contain negligible amounts of nitrate reductase. The enzyme

can then be induced in detectable amounts in the shoot tissue within a

few ( 2 to 4) hours (Beevers et al., 1965). Initially, light was thought to

be involved in the induction process, but subsequent work indicated

that light was more directly involved in uptake and movement of nitrate

to the site of induction (Beevers et al., 1965). Whole-plant induction

studies measure not only the ability of the inbred or hybrid to synthesize nitrate reductase, but also its ability to absorb and transport nitrate.

In subsequent work the effects of light and the root absorption and

translocation process were eliminated by floating the cotyledon or

excised young corn seedling in a medium containing nitrate. Since

nitrate reductase is substrate inducible (Tang and Wu, 1957; Hewitt

and Afridi, 1959), and since the rate of nitrate reductase synthesized is

initially dependent upon the amount of nitrate present (Beevers et al.,

1965)) the amount of enzyme formed in a given time per milligram of

tissue or protein should also be expressed as a function of the nitrate

present in the tissue.

Preliminary data obtained with the hybrid Hy2 X Oh7 and its

parental inbreds have not shown a consistent advantage for either inbred

or progeny in induction of enzyme or uptake of nitrate. Currently, plans

are underway to expand this study to an array of inbreds and hybrids.

Since the rate of induction of an enzyme measures the apparent ability

of the plant to mobilize and direct the complex system involved in

protein synthesis, this measurement of induction rates may be useful

for genetic selection.

VII.



A Concept for the Future



From a practical agronomic viewpoint, hybrid vigor in corn and

other crop plants is expressed phenotypically as the production of greater

amounts of pIant tissue, grain, or derived products. Obviously, then, the

hybrid forms exhibit greater growth than their inbred parents. Since

differences in “initial capital” do not explain the heterotic effects

observed, these effects must result primarily from a higher rate of

growth. In turn, since growth is the product of metabolism (biochemical

reactions), heterosis must be manifested either in increased rates of

metabolic reactions or in a more efficiently organized metabolic system,

Increased reaction rates are most likely to be achieved by enchanced

enzyme activity, since nearly all important metabolic reactions are

catalyzed by enzymes. Enhanced activity must result from either ( a )

the presence of more enzyme at the reaction site, or ( b ) more efficient

(qualitatively different) enzymes or enzyme systems.

Although there are some exceptions, most of the data now available



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A BIOCHEMICAL APPROACH TO CORN BREEDING



do not support the concept of either ( a ) more enzyme, or ( b ) more

efficient systems. This suggested that the enzymes and enzyme systems

are more efficiently organized as a whole in the hybrid.

FENDERS



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10

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DOORS



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10

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DOORS



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ID

LEFT

FENDERS



10

RIGHT

FENDERS



L

m

J

14

LEFT

DOORS



6

RIGHT



10

RIGHT

FENDERS



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L n J

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LOUT L,,YS

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v



Production / hr.

4 Cars

ASSUME EACH LINE HAS THE SAME AMOUNT OF STEEL. AND THAT EQUAL

AMOUNTSOFSTEELARE USEDFOR DOORS AND FENDERS.



FIG. 11. Diagrammatic analogy to illustrate the need for balance of systems

and integrated control of these systems. (Note: The word “fender” is defined as

that part of the body that covers the wheels.)



In this connection it may be useful to visualize how closely a plant

and its metabolic system resemble a modern, completely automated

industrial factory (Fig. 11). Comparable parts or systems could be

considered as:

Factory



Plant



Automated machines tf Enzymes or enzyme systems

Competition for doors and fenders tf Competition for energy and metabolites

Assembly lines tf Metabolic pathways

Computerized control c-f Environmental and hormonal control



In a factory, the size of the building may, but need not, indicate the

rate or total of production. The rate and total production depends on

the efficiency of the individual machines, and on the properly coordinated rates of operation of all machines in all assembly lines. Flow lines

1 and 2 (left to right) of Fig. 11 could, given time, reconvert components

and produce 5 cars, but not within the prescribed time.

As with the factory, the initial size of a plant or its meristem cannot

be the basic cause of hybrid vigor. Rather, this cause must lie in the

efficient and timely operation of the individual enzymes and in the

proper balance and integration of the enzymatically catalyzed metabolic

systems. To be sure, a larger plant, tissue, or cell may confer certain

advantages to the metabolic system through providing greater amounts

of needed “raw products.” Without greater efficiency of metabolism,



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VI. Some Recent Developments in Plant Biochemistry Related to Heterosis

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