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III. Heterosis and Enzyme Activity during Germination

III. Heterosis and Enzyme Activity during Germination

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A BIOCHEMICAL APPROACH TO CORN BREEDING



55



“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-



56



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



way. Another compelling reason was that methods were available to

assay these particular enzymes.

Ungerminated seeds and young (1-to 5-day-old) seedlings of inbreds

WF9, M14, Hy2, and Oh7 and the hybrids WF9 X M14 and Hy2 X Oh7

were used as the experimental material. The seeds were graded in an

attempt to negate gross inequities in initial seed size. To compensate for

Starch



Ir?

Glucose

I - phosphate



Glucose - 6 -phosphate

dehydrogenase



-+

r

G

l(iNl

1



Glucose

6 - phosphate



/-3



TPN



Fructose

6 - phosphate



6 - Phospho gluconate



Ri bulose 5-phosphote



+ co,



TPNH

N 24 kcal



To



Pyruvate



+Krebs

cycle



ZkCi-)

ADP



p&ol



3 - Phosphoglycerote



Fructose

I , 6 - diphosphote



\-



3 - Phospho-



Dihydroxy-



A



ocetone

phosphate



7



glyceroldehyde



N



24 kcol



DL

LY

I , 3 - Diphosphoglycerate



?



Triosephosphate

dehydrogenose



FIG.3. A portion of the metabolic process involved in the conversion of starch

to pyruvate and ribulose 5-phosphate. Only those enzymes that were assayed are

indicated. Note that two enzymes compete for glucose 6-phosphate.



differential plant size, enzymatic activities were expressed as utilization

of substrate or formation of product per unit of time per unit of protein

or plant weight.

Both hybrids exhibited hybrid vigor in seedling growth (scutellum,

root, and shoot only) and endosperm utilization (Fig. 4). “Efficiency”

was estimated by dividing daily increases in dry seedling weight by

daily losses in endosperm weight. No consistent difference in efficiency

could be detected between hybrids and inbreds. Over the 5-day period,

in all experiments, the hybrids were slightly Iess efficient at converting

endosperm reserve into seedling growth than their respective inbred



A BIOCHEMICAL APPROACH TO CORN BREEDING



57



parents. As an example, the conversion efficiencies for WF9, M14, and

WF9 x M14 over the 5-day period were 64, 69, and 63 percent, respectively. In contrast, the hybrids converted their stored reserves into

seedling growth (dry weight) at a faster rate than did their respective

parental inbreds.

Endosperm



UI



n



7



I



I



I



utilization



I



G'.,"

m



iI

P



I

2.



A



1



O'



4



h



b



5 '



2.



0



Days



Growth



-0c



n



2.0-



0



0



WF9

W F 9 x MI4

Mi4



Days



Days



FIG.4. A comparison of the rate of utilization of endosperm and seedling (root,

shoot, and scutellum) growth of corn inbreds WF9, M14, Hy2, and Oh7 and the

hybrids WF9 x M14 and Hy2 x Oh7. Environmental conditions were: dark; aerobic;

29°C.; and 80 to 95 percent relative humidity.



Both hybrids exhibited heterosis with respect to TPD activity in

dormant seeds and in 1- to 5-day-old seedlings (Figs. 5A and 5B).

Aldolase activity was slightly higher in the seedlings (but not in the

dormant seed) of WF9 x M14 than in the two parental inbreds. However, the hybrid Hy2 x Oh7 was essentially intermediate in activity

level throughout, as compared with its parental inbreds (Figs. 5C and

SD).

Attempts to purify and characterize these enzymes were not completely successful. TPD was extracted and partially purified (tenfold)

from ungerminated seeds of WF9, M14, and WF9 X M14. Based on

two preparations and replicate assays, K , values of 2.1, 2.1, and 2.0

were obtained for the WF9, M14, and WF9 X M14 enzymes, respectively. These values are not statistically different. Based on this meager



58



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

TPD octivity

X



I



E



1200-



\



I



I



X



WF9

W F 9 x MI4



0



MI4



I



-



-



240



E



A



0



I



I



1



2



I



I



3

Days



4



\

VI



-



2403.





5



C

0



I



I



I



I



1



2



3



4



5



4



5



Days

Aldolase



activity



600



150



150



3.

E

0



1



2



3

Days



4



5



0



1



2



3

Dcys



FIG. 5. A comparison of amount of enzymatic activity in seeds and whole

seedlings (endosperm, root, shoot, and scutellum) of the two sets of corn inbreds

and hybrids. Enzymes assayed were triosephosphate dehydrogenase ( TPD ) and

aldolase. Material was grown as described in Fig. 1.



evidence, it would appear that the higher level of activity observed for

TPD in WF9 x M14 was due to greater quantity of enzyme rather

than to differences in kind of enzyme fabricated by the hybrid.

This work was pursued and additional data were obtained with

plant material grown under two different environments-aerobic and

semiaerobic (Fig. 6A-F). There are six distinctive features of data

presented in Fig. 6: ( a ) The hybrids did not exhibit “heterotic” levels

of enzyme activity under the aerobic or semiaerobic conditions. In fact

the activities of all three enzymes in the hybrid seedlings tended to be

intermediate as compared with those of the parental inbreds. ( b ) The

semiaerobic environment enhanced the activities of ALD and TPD in

all material, as compared with material grown under aerobic conditions. ( c ) In Hy2 x Oh7 and its parental inbreds and in M14 seedlings

grown under the semiaerobic environment, ALD activity continued to

increase over the 5-day period. In contrast, hybrid WF9 x M14 and

inbred WF9 showed sharp decreases in activity on day 4. ( d ) TPD



59



A BIOCHEMICAL APPROACH TO CORN BREEDING



Triosephosphate dehydrogenoze

Avg. values

Aerobic

600



1000 -0 WF



c

._

0

a



,



Semi- 1;

aerobic,

785 / \



0 Hy2



Aerobic Semiaerobic

675

695



800 -



I



0



1



2



I



I



I



3 4 5

0 1 2 3

Seedling age in days



I



4



5



0



1



2



3 4 5

0 1 2

Seedling age in duys



Aldolase

Avg values



3



4



5



11,050



Aerobic Semiaerobic



0



1



2



3 4 5 0 1 2 3

Seedling age in days



Aerobic

0

X



WF9

WF9 x MI4



11.0

14.0



4



5



0



1



2



Glucose 6 - P Dehydrogenose

Semiaerobic

Avg. VaIUeS

11.0

14.0



Seeding age in days



-



3 4 5

0 1 2 3 4 - 5

Seedling age in days



Aerobic Semiaerobic

o Hy2

16.5

14.0

x H y 2 x Oh7 16.0

13.0



Seeding age in days



FIG. 6. A comparison of amounts of enzymatic activity in seeds and whole

seedlings (endosperm, root, shoot, and scutellum) of two sets of corn inbreds and

hybrids. Environment was as described in Fig. 1 except that semiaerobic conditions

were achieved by resealing the plant culture flat with a clear plastic film after

opening for watering. Other details of plant culture are published (Hageman and

Flesher, 1960b) ,



60



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



activities of the Hy2 x Oh7 set were enhanced by the semiaerobic

environment only to a small degree (5 percent). In contrast, TPD

activities of the WF9 X MI4 set of material were increased by 25 percent under similar treatment. ( e ) Activity of G-6-PD was decreased in

both sets of material under the semiaerobic environment. This observation is not inconsistent with the current concepts of TPNH reoxidation

under semiaerobic conditions. ( f ) The level of G-6-PD activity in M14

was consistently higher and in WF9 was consistently lower than in WF9

Aerobic

Reduced

Substrates

(organic

l a ( l ,acid)

, N r Dc



Oxidized

Substrates



+



( c l glutamate



(bl NO;

H P gyenr+,



cop



(ATP)



(a1 0 2

(b) NO;

( c I (I- ketoglutaric + NH;

( d l Other similar reductions



Ana e r o b i c



I , 3 - Diphospho-



I ,3-Diphosphoglycerote



p-



Acetaldehyde



CO,



Pyruvate



FIG. 7 . A comparison of the aerobic and anaerobic conditions with respect to

the systems that recycle (reduce and oxidize) diphosphopyridine nucleotide ( DPN ) .

Competition for the energy of DPNH is illustrated in the aerobic phase.



x M14. In the authors’ opinion, these differences in G-SPD activity

are due to genetic differences. This suggests that these two inbreds

should be useful as divergent sources of germ plasm for this enzyme.

The influence of the semiaerobic environment on the expression of

activity of TPD and ALD (Fig. 6) can be explained. This explanation

also affords an opportunity to relate environment, genetic composition,

and phenotypic expression. With corn seedlings under aerobic conditions, the pyruvate formed (Fig. 3 ) is the natural metabolite to be

supplied to the Krebs cycle where the oxidation of di- and tricarboxylic

acids generate energy (DPNH). Much of this DPNH is reoxidized by

oxygen (Fig. 7 ) , with the subsequent generation of ATP. Under conditions favorable to growth, other reactions proceed which also reoxidize



A BIOCHEMICAL APPROACH TO CORN BREEDING



61



DPNH (Fig. 7 ) . As indicated in the figure, the recycling of DPN+

DPNH is also a mass-action type of control mechanism; i.e., energy

(ATP) cannot be generated if DPNH is not available. An aerobic

environment thus leads to conditions favoring a reasonably high level

of ATP, which in turn can act to control the rate of flow of metabolites

through the glycolytic pathway (Fig. 3 ) . Specifically, high levels of

ATP inhibit the enzyme ( phosphofructokinase) that leads to the production of fructose 176-diphosphate ( Bonner and Varner, 1965; Passonneau and Lowry, 1962; Lowry and Passonneau, 1964). This regulation of

carbohydrate catabolism by ATP offers one plausible explanation for

the Pasteur effect, i.e., the addition of oxygen to a fermenting system

slows down the rate of carbohydrate utilization.

In the absence of oxygen, normal utilization of pyruvate, oxidation

of DPNH, and most ATP production ceases. In this environment, corn

seedlings initiate alternate or fermentative pathways that permit recycling of DPNH (anaerobic system-Fig. 7). This results in the

utilization of large amounts of the endosperm starch and the production

of an excess of ethanol that is excreted into the external medium

( Hageman and Flesher, 196Qb).This production of ethanol is wasteful,

since it appears that corn seedlings cannot utilize exogenous ethanol

even under aerobic conditions. However, without the production of

ethanol, DPNH reoxidation and the concurrent meager (4 moles ATP

per mole of glucose us. 38 moles ATP per mole of glucose under aerobic

conditions) production of energy cease. When corn seedlings are placed

in an anaerobic environment, growth (fresh weight) and net increase

in protein stop within a few hours. However, some metabolic changes

do occur. It has been shown that under anaerobic conditions, corn

seedlings show a marked increase in activity of some enzymes, e.g.,

alcohol dehydrogenase, and a decrease in activity of others, e.g.,

cytochrome oxidase (Hageman and Flesher, 196Ob). It can also be

shown that increased amounts of alcohol dehydrogenase could be induced

in corn seedlings by the addition of exogenous pyruvate or acetaldehyde

under aerobic conditions. Thus alteration of environment or level of

substrate (any of the vast array of metabolites found in the plant) may

increase or decrease the level of activity of the various enzymes.

The semiaerobic environment (Fig. 6 ) was sufficiently depleted of

oxygen for part of each day to permit limited ethanol production, but

provided enough oxygen the rest of the time to permit respiration,

generation of energy (ATP), and synthesis of protein. Therefore, the

increased activity of TPD and ALD observed (Fig. 6A, B, and E ) was

anticipated. Although the reason for the relative lack of increase in TPD

activity in the Hy2 X Oh7 set of material (Fig. 6D) is not known,



62



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



genetic control is one possible explanation. From these data (Fig. 6A,

B, D, and E ) it is obvious that the phenotypic expression of enzyme

activity varies with environment and with genotype. The consequence

of these divergencies in enzyme activity upon the subsequent go&

and development of the plant are not known. However, it can be

visualized that such major variations in metabolism would exert significant influences on the final characteristics of the plant.

It is more difficult to explain the decrease in level of activity of

G-6-PD (Fig, 6C and F) found in the seedlings grown under the semiaerobic conditions. One possible explanation is that under these conditions the reoxidation of TPNH is limited, The lack of TI" would curtail

the utilization of glucose 6-phosphate, and this in turn might lead to the

formation of a corepressor for G-6-PD. Inhibition of the enzyme activity

by product formation provides another explanation. Since the semiaerobic environment should diminish the utilization of the products of

this pathway, the accumulating products could contribute to control.

The relatively high level of G-6-PD activity in M14 plants grown under

semiaerobic conditions implies a genetic system less subject to control.

No matter which control system is operative, the phenotypic expression

of enzymatic activity obviously is significantly different when environment or genotype are altered.

Based on the data in Figs. 5 and 6 it is concluded that with the

possible exception of the low level of G-6-PD in WF9 there was no real

difference in enzyme activity of the material tested. This statement

appears paradoxical, since many of the differences observed (Fig. 5 )

were found to be significant and consistent results were noted from

experiment to experiment. However, these differences were noted only

as long as seed source, environment, etc. remained constant. This is

verified by the data of Fig. 6. It is suggested that until experimental

techniques for assaying and purifying enzymes improve and general

knowIedge in this area is expanded, work of this type should be initiated

by surveying a large number of corn inbreds for divergent expression of

activity of the enzyme selected. In other words, it is important to

establish maximum genetic variability. It is also evident that changes

in environment drastically alter the level of enzymatic activity and that

there are dramatic environment-genotype interactions.

It is difficult to envisage the precise consequences of the interaction

of genotype with environment since so little is known about the genetic

control of metabolic systems in plants. Because there is competition for

energy and metabolites (Moyse, 1959), the level of activity of any given

enzymes established by the genotype could alter metabolism and ultimately the final phenotypic expression of yield.



A BIOCHEMICAL APPROACH TO CORN BREEDING



63



The following concept can be deduced from this work: The genetic

code is expressed in the phenotype through the mechanism of an

enzymatically catalyzed metabolic system, and through the interaction

of this system with the environment. Logically, then, hybrid vigor must

be the result of a properly balanced and catalyzed metabolic system for

a given environment. This implies that the amount of activity of each

enzyme arising from one or more loci-regardless of the allelic composition-must be in balance with the system as a whole. Either too high or

too low a level of enzymatic activity could be detrimental to the system

as a whole.

The work on energy-transfer systems, summarized above, was not

pursued far enough to elucidate or establish the “balanced metabolic

system” concept as it may apply specifically to heterosis and early vigor.

However, this work made it clear that hybrid seedlings-despite their

marked superiority (in vigor) to their inbred parents-do not exhibit

heterotic levels of activity of these three major enzymes involved in

important metabolic processes related to early growth. Clear differences

were observed between inbred lines only with respect to G-6-PD-also

not necessarily related to growth-rate characteristics. The essentially

intermediate condition found in F, hybrids indicates that some explanation other than “heterotic” levels of enzymes must be found if hybrid

vigor is to be satisfactorily explained.

IV.



Genetic Control of the initial Reaction of Nitrogen Metabolism



Because of new developments in experimental procedures ( Hageman

and Waygood, 1959; Miflin and Hageman, 1963), it became possible to

investigate enzymes and enzyme systems of green corn leaves throughout the growing season. This type of investigation is deemed most important because it permits a better evaluation of genotype under the

environment in which it is normally grown. It also provides more insight

into the complex interaction of genotype-environment-metabolism stage

of physiological development. Although heterosis is exhibited in early

seedling growth, this early season vigor in no way assures maximum

grain production per hectare ( McIlrath, 1964). Since grain production

also exhibits heterosis, studies of the synthetic systems that fabricate

“building blocks” needed for protein and carbohydrate synthesis in the

grain should be of value. Because the corn plant has a high light requirement (Hesketh and Musgrave, 1962), it is much easier to achieve “normal” grain production under natural light than in the currently available

environmental growth chambers. For these reasons investigations were

shifted to metabolic systems strongly affected by light and more directly

related to grain production.



64



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



Interest in this complex problem developed from the results of

shading experiments with corn at Illinois (Earley et al., 1966) and other

stations. Initially, this work was not directly related to the heterosis

problem, but it soon became clear that field and laboratory techniques

were available which would enable a study of the enzyme systems

involved in nitrogen metabolism, and also of chloroplast activity.

The enzyme nitrate-reductase ( N R ) was chosen for study because

it is a major control-point in nitrogen metabolism, which in turn was

considered to be of prime importance to yielding ability of corn grown

under management systems involving high levels of nitrogen supply

and high plant populations. Evidence bearing on these points will be

developed in the following paragraphs.

Certain corn hybrids have been clearly shown to be tolerant of high

plant populations in respect to grain yield (Lang et al., 1956; Stinson

and Moss, 1960). Since the “tolerant” hybrids produced more grain

under shade treatment where light was presumably the only variable, it

was concluded that light was the causal factor for reduced grain production (Earley et al., 1966). Duncan (1958) has shown that as rate of

planting increases the log values of grain yield per plant decreases in a

linear manner, regardless of fertility level. It is difficult to assess the

effects of competition for light, nutrients, and moisture on grain yields

under high plant populations (Brenchley, 1920), however the linear

decrease in yield per plant, with increased plant population under

various environments, implicates light as a major factor.

Although competitive (inter- and intra-plant ) and artificial shading

of plants causes both reduction in CO, fixation (Moss and Stinson, 1961;

Hesketh and Musgrave, 1962; Moss et al., 1961; Thomas, 1956) and

accumulation of nitrate (Schimper, 1888; Knipmeyer et al., 1962), it

could be that the lack of carbohydrate and carbohydrate derivatives

constitutes the first limiting factor in grain production under high plant

populations. Thus when shade experiments failed to show that nitrate

accumulation in plants was due to a lack of carbohydrate and carbohydrate derivatives ( Knipmeyer et al., 1962), other explanations were

needed. These experiments indicated that nitrogen metabolism was

more adversely affected by the decreased light intensity than was carbohydrate metabolism. Other experiments provided additional support for

this view. Candela et al. (1957) observed that nitrate reductase activity

disappeared from cauliflower plants placed in the dark. Zieserl et al.

(1963) compared two corn hybrids rated as “tolerant” with respect to

grain production at high planting rates with an “intolerant” hybrid. The

“tolerant” types contained more nitrate reductase per gram of fresh leaf

tissue or per milligram of protein than the hybrid intolerant of high plant



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III. Heterosis and Enzyme Activity during Germination

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