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IV. Genetic Control of the Initial Reaction of Nitrogen Metabolism

IV. Genetic Control of the Initial Reaction of Nitrogen Metabolism

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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



A BIOCHEMICAL APPROACH TO CORN BREEDING



65



populations. Schrader and Hageman (1965) determined that Hy2 X

Oh7 (tolerant) had more nitrate reductase activity per plant during the

reproductive (pollination) phase than WF9 C103 (intolerant). Analysis of the whole plant at the end of the season showed that the Hy2 X

Oh7 plants contained more protein but less unreduced nitrate than the

WF9 x C103 plants. Hageman and co-workers (1960a, 1961) found

nitrate reductase activity to decrease, and nitrate content of corn leaf

tissue to increase, in rough proportion to the increase in level of shade

derived from high plant populations or shade structures. Although the

tolerant and intolerant corn hybrids responded in a similar manner to

decreased light intensity, the tolerant hybrids maintained their superiority with respect to nitrate reductase activity throughout the range of

shading. In contrast, “tolerant” hybrids used by Moss and Stinson (1961)

did not differ from “intolerant” types in ability to fix CO, under either

full sun or shade treatment. In addition, they found no difference in

sugar content between tolerant and intolerant hybrids.

The large amounts of nitrogenous fertilizers applied annually and the

high plant densities are most certainly associated with the current record

grain yields (6,000 kg./h.-Illinois state average, 1965). Most of the nitrogen available to plants grown under field conditions is in the form of

nitrate (Virtanen and Rautanen, 1952), because in most soils microorganisms rapidly convert ammoniacal forms to nitrate. Paradoxically,

nitrate must be reduced prior to elaboration into amino acids. An outline

of the reduction sequence, the electron donors, and one mechanism for

the union of ammonia with a keto acid are shown in Fig. 8. The diagram

indicates the interlocking of inorganic nitrogen metabolism with carbohydrate metabolism (Krebs cycle) and that the reduction of nitrite and

“fixed” carbon dioxide have to compete for electrons from ferredodn

( a component of the energy generation system of the chloroplasts)

( Tagawa and Arnon, 1962).

The reduction sequence is initiated by the enzyme nitrate reductase

(NR). NR is a substrate (NO,-) inducible enzyme (Filner, 1966;

Beevers et al., 1965) and in cultured tobacco pith cells is subject to

repressor-type control where amino acids serve as corepressors or derepressors (Filner, 1966). NR is not a stable enzyme as indicated by a

3%-hourhalf-life in excised corn tissue floated in water at 33°C.(unpublished data of L. E. Schrader of our laboratory). These observations

suggest that NR is a major control point for the system that supplies

reduced nitrogen to the plant.

Further support for the concept of NR control is supplied by the

following: Nitrate can and does accumulate to high concentrations

(5,000 pg of NO3- per gram fresh weight) in corn leaf tissue, apparently



x



66



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



without injury to the plant. Nitrite does not accumulate in corn leaf

tissue, at least not in excess of 1 pg. NOz- per gram fresh weight (the

limit of the assay used on thousands of samples). The level of free

ammonia in plant tissue is relatively low (Henderlong and Schmidt,

1966) as it is rapidly assimilated into glutamate or the amides glutamine

From



Krebs cycle



Keto acids



NO;



DPNH ~



Amino acids



_L



D



P



N



Light

Chloroplasts

Fructose I , 6

Diphosphote



-



To starch



FIG. 8. A schematic diagram of inorganic nitrogen metabolism showing its

relationship with carbohydrate metabolism ( a-ketoglutarate from the Krebs cycle)

and with Iight-generated energy ( F D ) . The dotted lines between NOz- and NH,'

indicate that the suggested intermediates, hyponitrous acid and hydroxylamine,

probably do not exist in free form but are bound to the enzyme, nitrite reductase.

Nitrogen metabolism competes with the Krebs cycle for carbohydrate skeletons

(keto acids) and with 3-phosphoglyceric acid (3-PGA) for energy derived froin

light. Evidence for the latter has been published by Moyse (1959).



and asparagine (Fowden, 1965). Other work has shown that increases

in nitrate reductase activity are associated with increased protein formation and decreased nitrate content ( Hageman et al., 1961 ). With

tobacco pith cells cultured on nitrate, growth has been related to NR

activity ( Filner, 1966).

An initial survey with 2- to 3-week-old seedlings established that the

three corn inbreds C103, Oh7, and Hy2 contained about the same level

of NR and were approximately twice as high in activity as WF9

(Zieserl and Hageman, 1962). Based on the importance of NR implicit

in this concept, studies were undertaken to determine the range of

genetic diversity for NR in Corn Belt inbred lines and hybrids. Although

these preliminary results indicated that lines differed in NR, the data

were obtained from seedlings grown in the greenhouse. Critical evalua-



A BIOCHEMICAL APPR0.4CH TO CORN BREEDING



67



tion of the importance of NR in determining heterotic response depends

on demonstration of differences between lines per se and their hybrids

grown under field conditions.

In 1960 field trials were begun when 47 inbreds were evaluated for

NR throughout the growing season. The highest and lowest inbreds in

NR activity differed by fivefold in seasonal mean activity (Zieserl and

Hageman, 1962; Zieserl et al., 1963) (Table I). Lines also differed in

TABLE I

Specific Activity for Nitrate Reductase (Seasonal Mean Values)

of 12 Inbred Lines of Corn in 1960 and 1961

Specific activity“



a



Inbred



1960



1961



Oh43

R177

H49

WF9

A545

HY2

R181

R168

CI2lE

Oh7

M14

R151



0.16

0.21

0.23

0.26

0.35

0.35

0.37

0.42

0.48

0.51

0.51

0.58



0.28

0.34

0.34

0.41

0.61

0.70

0.94

0.55

0.82

0.70

0.78

0.83



Expressed as micromoles of KNOz per milligram of protein per hour.



seasonal distribution of NR, although all lines were low in activity near

the end of the season.

Twelve inbred lines representing a wide range of NR activity, as

observed in 1960, were evaluated for NR in 1961. Samples were taken

at weekly intervals from June 28 to August 24. In general, the inbred

rankings based on seasonal mean activities were the same as in 1960

even though the general mean level of activity was higher in 1961

(Table I). Thus certain inbred lines were found to differ consistently in

NR activity, indicating that activity of this enzyme is under genetic

control.

A more important question was to determine the NR activity of hybrids between lines of known activity levels, since evidence on this point

could be of prime importance in assessing the role of NR in heterotic

responses. Therefore, inbreds classified as “high” and “low” in NR were

crossed to produce “high X “high,” “low” X “low,” and “high” x ‘‘low’’

hybrids (Schrader et al., 1966). The hybrids and inbreds studied and



68



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



their seasonal mean values for activity per gram fresh weight are shown

in Fig. 9. Seasonal means of “high x “high hybrids were significantly

lower than the mid-parental level in three cases but not significantly

different from the mid-parent in three other cases. No “heterotic” levels

of NR activity were observed in any of the “high” x “high hybrids.

The cross Hy2 x Oh7 grown in 1963 was the only one of seven

comparisons of “high” x ‘‘low’’ hybrids in which the hybrid differed

significantly from the mid-parent. In general, crosses of “high” x “low”

lines gave hybrids intermediate between their parents in activity. Again,

no cases of “heterosis”in NR activity were found.

One “low” x “low” hybrid, B14 x Oh43, was significantly higher

in NR activity than either parent in both 1962 and 1963 (unpublished

data in our laboratory verified this observation in 19f34, 1965, and 1966).

A similar result was obtained for Hy2 X B14. Thus, heterosis for NR

was indicated in some “low” x “low” crosses. However, other ‘‘low’’ x

“low” hybrids showed no heterosis for NR (Fig. 9). Even though heterosis for NR was observed, the activity of the “heterotic” hybrids really

was in the intermediate range, since it did not approach the activity of

certain “high inbreds. Yet all hybrids studied, regardless of level of

NR, showed heterosis for grain yield and vegetative growth. This clearly

indicates that heterotic levels of NR are not necessary for “agronomic

heterosis,” though it is not likely that NR activity is unimportant to vigor

and yielding ability.

Some attention has been given to the question “Are the differences in

levels of nitrate reductase activity among the corn inbreds and hybrids

due to quantitative or qualitative differences in the enzyme?” The major

difficulty in determining qualitative differences is that nitrate reductase

is an extremely unstable enzyme and all attempts to obtain a pure

“homogeneous” enzyme have been unsuccessful.

Beevers et al. (1964) used partially purified nitrate reductase from

Zea mays L. (Corn belt inbreds and hybrids and Peruvian corn) and

teosinte-Zea mexicana Reeves and Mangelsdorf ( Euchleana mexicanu

Schrad.) to determine apparent K , values for nitrate. K , values (magnitude

M.) of 1.7, 1.6, 1.4, 1.4, 1.5, and 2.0 were determined for

Hy2 X Oh7, WF9 X C103, R151, R177, Peruvian corn, and teosinte,

respectively. The variation among the values are in the range of experimental error. Based on these K , values for nitrate, the enzymes from

these different materials are considered to be qualitatively the same.

Other attempts to purify and characterize nitrate reductase from B14,

Oh43, and B14 X Oh43 were conducted by Dr. G. Ritenour (unpublished results from our laboratory). The I(, values for B14, Oh43, and

B14 X Oh43 were: for DPNH (

M.) 3.9, 3.2, and 3.4 and for nitrate



A BIOCHEMICAL APPROACH TO CORN BREEDING



69



(

M.) 1.6, 1.4, and 1.2, respectively. These data indicate no qualitative differences in the enzymes.

Over the years other evaluations (pH optima, change with purification, temperature sensitivity, etc. ) with this and other enzymes have

tended to support the view that the difference in level of enzyme activity

is quantitative rather than qualitative. This conclusion is restricted by

High x low category



High x high category

1962



RBI



Rl8l

MI4 a Oh43

Oh43aM14



W F 9 aOh43



W F 9 I 814



Cl03



814 xOh43

Oh43

814 rOh43

oh43



FIG.9. Comparison of seasonal mean values of nitrate reductase of 15 F1maize

hybrids with their respective parental inbreds. Details of seasonal patterns have been

published ( Schrader et al., 1966)

I



the lack of success in obtaining highly purified enzymes from corn tissue.

From the evidence previously cited, genetic control of NR activity

has been established for maize, and some information is available from

which activity levels of hybrids between lines of known NR activity can

be predicted. However, the number of genes involved, the mode of

action of these genes, and the way in which they affect activity are still

to be determined.



70



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



Some indication of the complexity of the genetic system controlling

NR may be inferred from work with microorganisms. However, in these

organisms NO,- is taken directly into the cell in which it is utilized. Also,

the photosynthetic process is not involved, the organisms are grown

under rigidly controlled environments, and the character being determined is not level of activity, but the presence or absence of the enzyme.

In a corn plant with its elaborate transport mechanism for moving ions

from the soil through the root and stalk to the leaf and its long growing

season with varying periods of rapid and slow growth, opportunity for

expression of genes which affect NR indirectly or which mask the direct

effects of genes for NR activity is many times that in microorganisms.

The complement of genes affecting NR in corn therefore must be much

more complex than that in microorganisms. Even in microorganisms,

several Ioci have been found which can bIock production of NR. Cove

and Pateman (1963) found mutants at six different loci, in at least four

different linkage groups, which blocked the production of NR in

Aspergillus nidulans (Eidam ) Winter. Sorger and Giles ( 1965) found

mutants at four different loci which blocked production of NR in

Neurospora crassu (Shear.) Dodge. Only a few mutants, each of which

could utilize NO,- as a source of nitrogen, were evaluated. In both

organisms, mutants which required NH,” in the medium were also found.

The complete absence of NR in actively growing corn plants has not

been demonstrated. This reflects the fact that NO3- is the major source

of nitrogen available to plants when grown in soil. In addition, the corn

material studied has been selected for its ability to produce well under

a high NO3- regime. Differences in the ability of lines to grow on media

having NH,” as the sole source of nitrogen have been demonstrated

( Harvey, 1939). Recent unpublished work in this laboratory indicates

that certain corn mutant genotypes show more growth when nitrogen is

supplied as NH,’ than when supplied as NO,-. At present, it is not

possible to use the simple techniques available to the microbial geneticist

to study genetics of NR in corn. However, further evaluation of divergent germ plasms under carefuliy controlled environmental conditions

might make such studies feasible.

Seasonal patterns of NR vary with genotype (Fig. 10).Two lines may

differ widely in NR at certain times during the active growing season

whereas at other times there may be no difference in activity. Lack of

differences in activity late in the season may be attributed to the beginning of senescence in the plant; i.e., the grain or product is formed and

the “factory” is turned off. Differences between the same lines early in

the season may be amenable to genetic interpretation. A realistic understanding of the genetic mechanisms controlling NR will require study



71



A BIOCHEMICAL APPROACH TO CORN BREEDING



of segregating populations at a particular time in the life cycle of the

plant, and perhaps at several different times.

The consistent “heterotic” level of NR activity in the hybrid B14 X

Oh43 indicates that the two inbreds showing similar levels of NR may

differ in genes responsible for this activity. Unpublished results from

our laboratory demonstrate that genetic segregation occurs in the F,

and backcross populations from B14 x Oh43. F, populations studied



5 g O c I l

+.

e

n



I



I



1



F



1



1



1



1



1



Miscellaneous



,L-L-2 5 30

June



I



1



10

July



I



I



20



30



1



I



4



L

9



U

13



18 24



August



FIG.10. Seasonal patterns of nitrate reductase activity of groups of corn inbreds.

WF9 and related inbreds exhibited similar patterns while the inbreds of divergent

origin exhibited unrelated patterns ( Zieserl and Hageman, 1962 ).



showed a range in NR from the level of the F, down to the level of the

lower parent. These data are not yet adequate to determine the mode

of inheritance of NR, but some evidence suggests that very few loci

(perhaps two to three) may be involved in the differences-at least at

certain developmental stages.

Though the details of the genetic mechanism controlling NR in corn

are unclear, enough is known to put together hybrids of known levels of

NR if this is desirable. As pointed out previously, growth (or yield) is

the end result of a complex series of chemical reactions any one of which



72



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



may be limiting, The data presented make it clear that NR activity level

can be controlled by genetic manipulation to the extent that NR need

not be a limiting factor in nitrogen metabolism in corn.



V. Specific Chloroplast



Activity



The importance of light as a major environmental control on nitrate

reductase activity, and consequently on nitrogen metabolism, has been

stressed in the preceding section. Nitrate reduction, like the reduction of

"fixed" CO, is indirectly related to light in that both reactions utilize

chemical energy produced by the chloroplasts from light energy. Since

light is the sole primary source of energy for plants, a comparison of the

light-converting enzyme system of the chloroplasts ( Arnon et ul., 1958)

from corn inbreds and their progeny should be of signal importance.

Because standard techniques (Arnon et al., 1954; Jagendorf and

Avron, 1958) would not permit the isolation of chloroplasts active in

photophosphorylation (chemical energy generation) from corn leaf tissue, it was necessary to develop special isolation procedures (Miflin and

Hageman, 1963).

Three assays, Hill reaction, noncyclic and cyclic photophosphorylation, were used to estimate the conversion of light energy to chemical

energy by the corn chloroplasts. Comparable evaluations of chloroplast

activity were obtained by each method. Therefore the simplest assay

(cyclic photophosphorylation ) was used to evaluate the chloroplast

activities of five corn inbred lines (WF9, R151, R177, R181, and Oh43)

and seven F, hybrids-counting reciprocals-among these lines ( Miflin

and Hageman, 1966). Objectives were ( 1 ) to determine whether chloroplasts isolated from the different inbreds and hybrids differed in activity,

and ( 2 ) to determine whether heterosis was manifested in chloroplast

activity.

The essential data from studies of the inbred lines are summarized in

Table 11. It is apparent that the lines differed markedly in ability to

convert light energy into chemical energy, per unit of chlorophyll. One

line, R151, had a significantly higher level of cyclic photophosphorylative

activity than the other four. Another inbred, WF9, was consistently the

lowest in activity. The other three lines showed intermediate levels.

These findings are of particular interest because the same inbreds had

been evaluated for nitrate reductase activity, also shown in Table 11. As

may readily be seen, R151 was the only inbred ranking similarly (high)

for both activities. WF9, with the lowest chloroplast activity, was moderately low in nitrate reductase activity. Two of the other three lines were

low in nitrate reductase activity and medium for cyclic photophosphorylation activity. R181, also intermediate for cyclic photophosphoryla-



A BIOCHEMICAL APPROACH TO CORN BREEDING



73



tion, had a high level of nitrate reductase activity. This comparison

clearly illustrates the expected situation, i.e., a given inbred line is

likely to be high in activity of some enzymes, low in others, and intermediate in yet others.

The relationships between inbred lines and their F, hybrids for cyclic

photophosphorylation were much like those observed in the nitrate

reductase work. No “heterotic effects” on chloroplast activity were

observed. In the three hybrids studied, no significant differences between

reciprocals were found. Cyclic photophosphorylation activity of the

hybrids was essentially intermediate between the parental values. In

TABLE I1

A Comparison of Five Corn Inbreds with Respect to Chloroplast

(Cyclic-Photophosphorylation) and Nitrate Reductase Activity

Inbred



Chloroplast activity’



Nitrate reductase activityb



345

317

240

220

168



0.71

0.28

0.22

0.66

0.34



~



R151

R177

Oh43

R181

WF9

~~



~~



~



Micromoles of phosphate esterified per milligram of chlorophyll per hour.

b Micromoles of nitrite produced per milligram of protein per hour. Data presented

are the average of several assays.



WF9 X R151 (“low” X “high) and WF9 X R181 (“low” X “intermediate”) the cyclic photophosphorylation of the hybrid was not significantly

different from the mid-parent values. In R151 X R177 ( ‘ I i g h X

“medium-high”), the F, value was significantly lower than the midparent, and in fact was slightly lower than that of R177, the lower of the

two parents ( Miflin and Hageman, 1966).

From studies of the three major metabolic systems (i.e., energy transfer in seedling growth, nitrate reductase and nitrogen metabolism, and

energy generation by chloroplasts) it is apparent that genetic variability

is indicated by divergent levels of enzymatic activity. From the comparisons of inbreds and F, hybrids for activities of the various enzymes,

it is obvious that heterotic activity levels are the exception-not the rule.

In addition, preliminary characterization of the enzymes studied in two

of the three systems failed to show qualitative differences between

enzymes of the parents and hybrids. Other recent investigations have

reported the occurrence of qualitative differences between enzymes of

corn inbreds and hybrids. The implications of these findings are presented in Section VI.



74



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



VI.



Some Recent Developments in Plant Biochemistry

Related to Heterosis



Schwartz ( 1960) reported the occurrence of qualitatively different

esterases in developing corn seeds and seedlings. Starch-gel electrophoresis and stain techniques were used to separate and identify three

forms (isozymes) of a basic protein with esterase activity. The substrate

was a-naphthyl acetate, a nonspecific, nonmetabolic ester. According to

Schwartz, three allelic genes, EF, EN, and ES, specified a single esterase

in homozygous plant material, The three esterases were identifiable on

the starch gel (pH 8.6) by their rate of migration toward the cathode,

and were designated F, N, and S, respectively. Crosses made between

homozygous plants gave heterozygotes that exhibited both parental

enzyme types und a new “hybrid”enzyme. The hybrid enzyme migrated

on the starch gel at a rate intermediate between those of the two parental

types. It was suggested that the new enzyme was a dimer which was

formed by random association of the monomers specified by each allele.

In Schwartz’s (1960) initial paper, it was postulated that these

hybrid esterases would be more active in metabolic transformation than

the enzyme types of the parental lines. The formation of hybrid enzymes

in general then would be a factor in hybrid vigor. While this concept is

attractive, it is not necessary for the enzymes in the hybrid plant to

exhibit a higher level of activity than their inbred parents for yield

heterosis to occur.

In a subsequent paper, (Schwartz, 1964), it was reported that the

new (hybrid) enzyme found in maize plants heterozygous for the two

allelic E, genes was synthesized as a dimer. Separation of the dimer into

its monomeric units was not reported. Further, the hybrid enzyme did not

appear to be formed by random association of two monomers. Schwartz

( 1962a) hypothesized a random interaction between messenger-RNA

molecules specified by the two alleles, to permit the synthesis of the

hybrid enzyme on the ribosome.

Subsequent examination of the esterase band patterns on the starch

gel after electrophoresis of extracts from selected and crossed corn and

teosinte plants has permitted Schwartz et al. (1965) to conclude that

there are seven alleles of the El gene. When tissue containing homozygous El alleles was treated in vivo with sodium borohydride, multiple

forms of the esterase were detectable in the extract (Schwartz, 1964).

Based on band positions, the new forms of the esterase were more

negative in charge than the esterase from comparable untreated tissue.

These new forms of the esterase, resulting from the borohydride treatment, were similar in charge (migration rate) to other naturally oc-



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