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Enzyme Activity as an Index of Growth
Superiority of Firms clausa var. clausa on Two Soils

RUSSELL MacBAIN BURNS

A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA
1971

ACKNOWLEDGEMENTS

I thank the members of my supervisory committee, particularly
Drs. Spinks, Hortenstine, and Hammond, for their encouragement and
patience. My special thanks go to Dr. William Pritchett, Chairman
of the Committee, for providing guidance during early stages of
research and for his assistance in preparing this manuscript; to
Dr. Robert Stanley, for stimulating my interest in this problem
and for providing a dynamic atmosphere in which to work; to Dr.

Wa^Tie Smith for his counsel and to him and his wife. Midge, for
their warm friendship and hospitality; to Ken Strickland for his
assistance in collecting seeds and plant material.

I appreciate the financial assistance provided by the U. S.
Forest Service and the added encouragement given by Dr. Ray
Brendemuehl .

Lastly, I thank my wife and sons for their love and much needed
patient understanding.

TABLE OF CONTENTS

PAGE

INTRODUCTION 1

OBJECTIVES 3

LITERATURE REVIEW 6

Mineral Nutrition of Sand Pine 7

Photoassimilation of Carbon Dioxide 8

Concentration 8

Light Intensity 9

Exposure -- Length and Temperature 12

Enzymes 14

MATERIALS, EQUIPMENT, AND METHODS 17

Plant Material 17

Photoassimilation Chamber 23

Photoassimilation of ^"^C02 25

Separation of Ethanol-Soluble Components

Measurement of Radioactivity 28

Thin Layer Chromatography 29

Sugars 29

Organic Acids

Amino Acids

Radioautography

Measurement of Enzyme Activity 32

111

TABLE OF CONTENTS

CONTINUED

PAGE

Fructoaldolase 33

Glyceraldehyde- 3- Phosphate Dehydrogenase -NAD
Dependent 33

Phosphoglycerate Kinase 33

Glucose-6-Phosphate Dehydrogenase 33

6-Phosphogluconate Dehydrogenase 35

Acetone Powders -- Preparation and Protein Extraction . 37

Preparation of Polyacrylamide Gels and Electrophoresis 38

Detection of Protein and Isoenzyme Bands 39

Proteins and Dehydrogenase Isoenzymes 39

Glucose-6-Phosphate Dehydrogenase and Malate
Dehydrogenase . 40

Sample Size and Statistical Analysis 41

RESULTS AND DISCUSSION 43

Photoassimilation of ^^CO.2 43

Clarification of Extracts and Quenching 51

Sugars 57

Organic Acids 65

Am.ino Acids 65

Effect of Nutrient Level on Seedling Morphology and ^“^C
Incorporation 67

Color and Weight 67

^'^C Incorporation 70

Biochemical Pathways Involved 75

SUMMr.RY 94

APPENDIX 97

LITER.ATURE CITED 107

LIST OF TABLES

TABLE PAGE

1 Source and concentration of elements used

in the complete nutrient solution 20

2. Chemical and physical properties of soil used

to raise half-sibling seedlings. 21

3 Color and Rf values from chromatograms of

known sugars used to identify unknowns 30

4 Distribution of photoassimilated green

sand pine seedling tissue 56

5 Seedling weight and photoassimilated ^^C

distribution in response to nutrients 71

6 Influence of N and P fertilizer on the distrib-
ution of weight and radioactivity 72

7 Comparisons of the soil x tree interaction showing
the probability of a chance occurrence and the
soil on which the highest values were obtained go

8 Distribution of half-sibling seedlings

possessing 3 to 7 cotyledons 87

9 Protein Rf measurements taken from half-
sibling seedlings grown on Lakeland coarse

sand 98

10 Protein Rf measurements taken from half-

sibling seedlings grown on Paola sand 101

11 Protein Rf measurements taken from parent

trees growing at their original locations 104

LIST OF FIGURES

FIGURE

PAGE

Flow chart of procedures followed in part I

Flow chart of procedures followed in part II

Diagram of the photoassimilation chamber

Reactions for measuring fructoaldolase,
glyceraldehyde-3-phosphate dehydrogenase ,
and 3-phosphoglycerate kinase

Reactions for measuring glucose-6-phosphate
dehydrogenase and 6-phosphogluconate
dehydrogenase

Distribution of radioactivity in seedlings
allowed to photoassimilate 14c02

Distribution of radioactivity in seedling
tissues exposed to 14c02 in the light and
dark

Quenching curves developed to compensate
for loss of counting efficiency

The i4(] incorporated into green tissue
of Finus clausa in response to different
amounts and kinds of acid used to release
14c02 Kh14C03

Comparison of clarifying agents

Chromatogram showing bands of standard
and unknown sugars

Radioautogram of a sugar TLC plate (6
weeks exposure)

Radioautogram of the same TLC plate used
in Figure 12 (2 weeks exposure)

Moribund seedling showing constriction near
ground line

LIST OF FIGURES -- CONTINUED

FIGURE

PAGE

Organic acid TLC plate

Amino acid TLC plate for fertilizer
treatment N2Pj^

Amino acid TLC plate for fertilizer
treatment N2P2

Seed germination from superior and non-
superior sand pine trees at 5 locations
designated A to E

vii

Abstract of Dissertation Presented to the
Graduate Council of the University of Florida in Partial Fulfillment
of the Requirements for the Degree of Doctor of Philosophy

ENZYME ACTIVITY AS AN INDEX OF GROWTH SUPERIORITY
OF PINUS CLAUSA VAR. CLAUSA ON TWO SOILS

Russell MacBain Burns
August, 1971

Chairman: Dr. William L. Pritchett

Co-Chairman: Dr. Robert G. Stanley

Major Department: Soil Science

Tliis study attempted to 1) identify the metabolic pathways

and enzymes involved in the photos>mthetic fixation of carbon

and to determine 2) if metabolic intermediates were altered by

changes in the supply of nitrogen and phosphorus; 3) if activity

level of specific enzymes differed in tissue of superior and

nonsuperior trees and their half-sibling seedling progeny;

4) if the activity was altered by the soils in which seedlings

grew .

Distribution of photoassimilated ^^U02 measured in ethanol-
soluble fractions of green tissue from 3-month-old Ocala sand pines
grown from seed in sand culture and complete nutrient solutions
containing two levels each of nitrogen and phosphorus. Portions of
the metabolic pathways involved in carbon fixation were determined.
Tlie activity and electrophoretic migration rate of malate dehydro-
genase isoenz\nres, glucose-6-phosphate dehydrogenase isoenzymes, and
proteins were compared in green tissue from saw log-size superior

viii

and neighboring, nonsuperior sand pine trees and their half-sibling
progeny grown in pots on Lakeland coarse sand and Paola sand.

Sugars contained 75%, organic acids 19%, and amino acids 6% of
the radioactive carbon in the ethanol -soluble fraction from green
tissue. Radioactivity was highest in fructose, glucose, and galactose.
Nitrogen directly affected chlorophyll formation, seedling growth, and
photosynthetic incorporation of 14c. Phosphorus appeared to be the
principal rate- limiting element in the incorporation of carbon in
sugars and in some organic acid precursors of amino acids. Results
indicated that enzymes of glycolysis, the Calvin cycle, and the
tricarboxylic acid cycle were principally involved in fixation of
carbon in sand pine seedlings allowed to photoassimilate ^^C02
for 10 minutes in a controlled environment.

Measurements of activity and migration of isoenzymes did not
provide an index of growth superiority in parent trees or in half-
sibling seedling progeny. Superior parent trees, but not their
progeny, lacked one or more protein bands found in nonsuperior
trees indicating that a genetic marker exists. However, no rela-
tionship was found between the location of malate dehydrogenase and
glucose-6-phophate dehydrogenase isoenzymes and protein bands.

Activity and Rf values of some malate dehydrogenase iso-
enzyme and protein bands were altered by the soil in which seedlings
grew as well as by genetic factors. Both the isoenzyme and protein
bands and factors influencing them were identifiable.

INTRODUCTION

Superior trees are fast growing and have a desirable morphology.
They are sought and propogated principally to shorten the rotation
age of plantations, i.e., the time needed for trees to reach
merchantable size. Selection of superior trees is based primarily on
a comparison between the candidate and neighboring contemporaries of
the same species. True superiority is adjudged by the ability of
grafted stock and progeny to exhibit the same superior characteristics
as the selected candidate. The approach is sound but very time
consuming.

A more rapid way to screen candidate superior trees may be
by comparing levels of enzymes that catalyze growth processes.

Growth, the primary index of superiority, is cumulative, genetically
controlled (Squillace, 1965), and greater in superior trees than
among others of the general population. Assimilation, the basic
growth process, depends upon the speed of certain biochemical reactions.
Enzymes control the rate of these reactions and, thereby, the rate
at which a tree grows.

Many enzymes are involved in tree growth. This study measures
activity of just a few. The problems are to select for assay those
enzymes that control a biochemical reaction pathway involved in the
synthesis of anabolins, which most differ between superior and
nonsuperior trees, to determine if their activity is influenced by

the soil in which the pine trees grow, and to determine what effect
fertilization with phosphorus (P) and nitrogen (N) has on photosynthetic
fixation of carbon.

Nutrient levels influence rate of growth. Sandy soils in which
sand pines grow are deficient principally in P and N (Brendemuehl ,

1967). Sand pines respond to fertilization with these elements.

Ocala sand pine, Pinus olccusa var. clausa Ward, was studied.

It is native to droughty, infertile sandhill soils in Florida and has
become increasingly important in reforestation of these difficult
sites. Superior tree selections were readily available for seed
collection and tissue sampling.

OBJECTIVES

The study was divided into twj parts, each of which had two
objectives. In part I, experiments were designed to 1) identify
the metabolic pathways and the enzymes involved in the photo-
synthetic fixation of carbon, and to determine 2) how significantly
these pathways might be altered by changes in the supply of
plant nutrients. Figure 1 depicts a flow chart of procedures
used to accomplish these objectives. In part II, objectives were
to determine 3) if the activity level of specific enzymes differs
in tissue of superior and nonsuperior trees and their half-sibling
progeny, and 4) if the activity was altered by the soils in which
seedlings grew. Figure 2 illustrates the procedures followed in
the second part of the study.

I COKPLFTE NUTRIENT

SOLUTION

PLUS; 1

POT

1 SEEDLINGS

ZTTTI

Unr

^^C02

ACHLOROPHYLLOUS

TISSUE

NEEDLES AND
GREEN STEM

ETHANOL

EXTRACTION

SUGARS I r>

ORGANIC

ACIDS

AMINO

ACIDS

U D Q

CHROMATOGRAPHIC

SEPARATION

RADIOAUTOGRAPHIC
IDENTIFICATION OF
LABELED COMPOUNDS

LIQUID

SCINTILLATION
EQUIPMENT TO
MEASURE
1^C02-FIXATI0N

QUALITATIVE AND
QUANTITATIVE
EFFECTS OF N AND P
FERTILIZATION

IDENTIFICATION OF PRINCIPAL
BIOCHEMICAL PATHWAYS
AND ENZYMES

PROTEINS AND ENZYMES
IN FIGURE 2

Figure 1. — Flow chart of procedures followed in part I

+->

!-<

<1)

•t->

1
\

•H

M M

^ H

4 u

S <

§ ^

O O

5h 5

« M

>3 o

P>^ Uh

Z M

a fa

fa z

_g_

O M

LITERATURE REVIEW

Literature pertinent to the development of an enzyme index
for superior trees under the proposed approach encompasses a wide
variety of subjects ranging from nutritional requirements for sand
pine seedling culture to methodology for enzyme assay. To date no
one has published results of an approach with an enzyme assay.

The experiments in this study involve a number of variables, the
effects of which had to be considered in sequence. Factors other
than those to be tested (constants) had to be maintained within
acceptable limits, or at desirable levels, in order to determine
accurately the effect of the variable under consideration. Pertinent
literature is reviewed in those sections dealing with specific
aspects of this research, so that it will be in context.

Mineral Nutrition of Sand Pine

Similar nutrient solutions were employed in sand and water
culture (Hewitt, 1952; Small and Leonard, 1969) and included a
wide range of concentrations of macroelements (Hacskaylo, 1962;

Hoagland and Arnon, 1950) . Tliis suggested that each species
required a particular nutrient regime or that pines exhibited optimum
growth under a diverse range of nutrient conditions. This was
especially true of N and P.

Published (1967) and unpublished work by Brendemuel.l Indicated
that P. clausa growth was enhanced by N but only after the P deficiency
inherent in infertile, acid sand had been corrected. Brendemuehl
(personal communication) recommended using N and P in a ratio of
1 to 2 in acid-washed sand, with N supplied at the rate of 75 ppm.

The form of N used also affects growth and protein synthesis
in conifers. Durzan and Steward (1967) grew white spruce [Piaea
glauca (Moench.) Voss] and jack pine {Pinus banksiana Lamb.)
seedlings for 478 days in sand irrigated with nutrient solutions
containing either ammoniacal nitrogen (NH^-N) or nitrate nitrogen
(NO^-N) . The fresh weight of white spruce seedlings was greater with
NO^-N, but more free, non-protein bound, amino acids were found in
seedlings fed NH^-N, especially in stems and leaves. Jack pine
seedlings supplied with NH^-N were heavier and contained more total
free amino acids than those grov/n with NO^-N. Because free amino
acid reserves were lowest with NO^-N, Durzan and Steward (1967)

concluded tliat more than NH^-N was synthesized into proteins

by conifers, '“.’ork with Southern pines (Barnes and Naylor, 1959;
Barnes, 1962; and Pharis, Bai'nes, and Naylor, 1964) tended to
substantiate this conclusion.

Photoass imi 1 ation of Carbon Dioxide

Concentration

Ihe average concentration of CO2 in the atmosphere is about
300 ppm at sea level. Of this amount only about two-thirds is
available for photosynthesis (Moss, 1962). He found that the ability
of plants to utilize CO^ varied with species. Corn (Zea mays L.),
for example, has a C02-compensation point of less than 10 ppm;

Norway maple {Acer ytatanoides L.), 145 ppm. Furthermore, this
equilibrium between CO^ production and utilization varies with light
intensity and temperature. Moss reported that in a closed system
the average plant reduces CO^ concentration to a level of only
50 to 100 ppm.

At best, only an approximation can be made of CO2 uptake because
photosynthesis and photorespiration occur simultaneously. At most
light intensities much of the CO2 respired is utilized before it can
diffuse to the atmosphere. Zelitch and Day (1968), working with
parent and mutant progeny of tobacco {Niootiana tabacum L.) at
several concentrations of CO2 (including CO^-free air), presented
evidence suggesting that the increased rate of CO2 uptake in hybrid
plants (higher photosynthetic efficiency) may be attributed to
genetic interference with the photorespiratory process, i.e., net
photosynthesis was high because photorespiration was lower in mutant
siblings .

Growth of plants, and more specifically growth of pine seedlings,
will occur and, in fact, will be enhanced at CO2 concentrations
above 300 ppm. Zelawski and Kinelska (1967) working with Scotch
pine (Firms sylvestris L.) at relatively low light intensities
(maximum approximately 1,000 ft-c), found that the rate of photo-
synthesis was almost directly proportional to the concentration of
CO2 within the range of 200 to 400 ppm. At concentrations above
450 ppm the rate of photosynthesis declined and approached the
point where the response to increased CO2 was negligible irrespective
of light intensity. Similar findings were reported by Hughes and
Cockshull (1969) with China aster (CaZVistephus ohinensis') at
low light intensity. Dry matter production of plants grown in 600 ppm
CO2 was slightly higher than in those grown at 900 ppm, and considerably
higher than in plants grown at 325 ppm. The natural diffusion gradient
for CO2 between leaf and atmosphere is altered at high concentrations
and may cause recycling of re5pired CO2 at higher than normal rates.
Extremely high CO2 concentrations, 2,000 to 4,000 ppm, caused stomata
of some plants to close (Pallas, 1965). He found that dicots were
more tolerant of high CO2 concentrations than monocots.

Light Intensity

Uptake of CO2 is affected by light intensity. Zelawski and
Kinelska (1967) presented a graph showing that the shift in CO2
compensation point with varying light intensity had a parabolic
configuration. At low intensities small changes induced drastic
shifts in the CO2 compensation point. The magnitude of the shifts
diminished in almost exponential fashion as light intensity approached
47.5 and 100 % illumination and, although intermediate intensities were

not tested, assumedly at intervening intensities. Unfortunately,
light saturation was not attained in their experiments.

Light saturation was reached in a study of eastern hemlock
{Tsuga canadensis L.) in Wisconsin (Adams and Loucks, 1971). Hemlock
is tolerant of dense shade and develops well as an understory plant.
Illumination of foliage at midday varied between 25 and 500 ft-c in
the forest. Under controlled conditions the rate of net photosynthesis
increased sharply up to 1,000 ft-c and then began to decrease. At
3,000 and 3,500 ft-c net photosynthesis was approximately the same
indicating that the light compensation point had been reached. The
data suggested that, for this shade-tolerant conifer, changes in
intensity above 1,000 ft-c would not greatly alter CO2 uptake.

Cooper (1957) reported that young sand pines are tolerant of shade.

In this respect the two conifers are alike.

Keller and Koch (1962) examined the influence of mineral
nutrition upon CO2 exchange in poplar {Populus euramerioana
marilandiaa) and found that light saturation occurred at 2,000
ft-c in N-deficient leaves. Light saturation was not reached at
even 4,000 ft-c in "well-fed" leaves. However, at low intensities
of up to 500 ft-c net assimilation was the same in N-deficient and
"well fed" leaves. With regard to the dependence of net photosynthesis
upon CO2 concentration and light intensity, their findings at least
partially substantiated those of Zelawski and Kinelska. At 4,000 ft-c
-the CO2 uptake of poplar leaves was strongly influenced by foliar
N content, N-deficient leaves assimilated only 60% as much CO2
as normal leaves, were proportionately smaller in size, and contained
only 55% as much chlorophyll per unit area. There was a close
correlation between the chlorophyll and N content of poplar leaves.

Light intensity influenced NO^ uptake and the subsequent induction
of NO3 reductase in cereals (Chen and Ries, 1969). Rye seedlings
took up NO3 slowly in dark and rapidly in light. After a 12-hr
exposure, seedlings subjected to about 300 ft-c contained as much NO3-N
as those illuminated with 800 to 1,5C0 ft-c, suggesting that dependence
of NO3 reductase activity on light was satisfied at the lowest
intensity. Presumably NO3 reductase was produced in the dark. Light
and prior uptake of NO3 were essential for the induction of the
enzyme. Once these conditions were met enzyme production continued
to increase for the next 24 hours, even in the dark. Within the
range of 300 to 1,500 ft-c induction of NO3 reductase was proportional
to light intensity.

Sand pine seedlings appeared to be umbraphilic with respect
to light tolerance and juvenile growth (R.M. Burns, unpublished data).
Tests conducted in an experimental nursery support the observation;
seedlings raised in partial shade were taller, larger, and more
verdant than those grown in full sunlight. Results suggested that
chlorophyll-catalyzed photooxidation induced by high light intensities,
and increased respiration in response to high summier temperatures,
as noi'mally encountered on exposed sands, contributed to slower
growth in direct sunlight.

Meidner (1970) , working with sun and shade leaves from a
variety of herbaceous and woody plants, noted that the light compen-
sation point was most closely related to leaf thickness, lliinner
shade leaves had the lowest light compensation point. Kramer and
Kozlowski (1960) made a similar observation with light saturation
of sun and shade leaves of European beech. Differences in saturation

levels exist because higlier intensities are needed to affect
chlorophyll molecules deeply imbedded in thick leaves. Pine
needles are thicker than hardwood leaves and much less efficient
photosynthetically . Bonner and Galston (1952) reported that light
saturation of pine foliage d^d not occur even at intensities approaching
full sunlight, 10,000 to 12,000 ft-c.

Exposure -- Length and Temperature

Uptake of CO2 depends upon the volume involved, the amount of
chlorophyll present, the rate of diffusion inward, and the rate of
photosynthesis, llie latter is temperature, liglit, and CO2
dependent. Small and Leonard (1969) exposed 6-week-old legumes
in plastic bags to a volume of ^^C02 gas with radioactivity of 5
microcuries (pc) . An induction period of 15 min in direct sunlight
was used. No mention was made of how length of exposure for complete
utilization of ^‘^C02 was determined but, in view of the previously
cited work, disposal of residual, labelled gas obviously was necessary.

Paired leaves on intact hybrid and parent Mimulus (Monkey
flower) plants were used by Decker (1959) to measure the effect of
CO2 concentration from 100 to 500 ppm on photosynthesis at temperatures
of 20, 30, and 40 C and 2,000 ft-c. He found that apparent photo-
synthesis increased as CO2 concentration increased and temperature
decreased. Apparent respiration increased with temperature but
decreased with CO2 concentration. The CO2 compensation point increased
almost linearly with temperature. Tlie dependence on temperature
was ascribed to a temperature coefficient larger for respiration than
for photosynthesis, the rate of respiration being more than 3 times
higher in light than in darkness. For Mimulus, at least, to approach

the CO^ compensation point with the shortest possible exposure
time at a fixed' CO^ level necessitates use of a low temperature.

Dr. W. Zelawski (personal communication) suggested evolution of
14

CO2 into a closed system in small increments, then circulating
the gas through the illuminated chamber for 2 hr. This procedure
was used for preliminary work because it provided sufficient time for
translocation of some -labelled products to the roots (Lister et al . ,
1968; Small and Leonard, 1969) and insured biochemical fixation of
a large volume of CO2 containing sufficient quantities of for

detection.

Devlin (1968) noted that an optimum rate of photosynthesis
occurred at 50 C during Snort -term exposure and at 22 C during
long-term e.xposure of Chlovella. Nitssohia closterium reached
an optimum rate at 26 C and. N. palea at 32 C at high light intensities.
Tlie photosynthetic rate of several trees and seedlings was highest
at 20-30 C (Kramer and Kozlowski, 1960). Vose and Spencer (1969)
and Zelawski and Kinelska (1967) reported that temperature in
closed photoassimilation chambers usually is maintained within a
range of 20 to 27 C.

The effect of temperature on photosynthesis and respiration
was measured using sand pine (P. clausa var. imw.ginata') seedlings
from west Florida (Pharis and Woods, 1960). Apparent photosynthesis
(mg. CO2 taken up per hr) was highest at 23 C and actual photosynthesis
(apparent photosynthesis + apparent respiration, as mg. CO.,
exchanged per hr) was equally as high at 23 and 28 C. This was
explained by the fact that apparent respiration increased with tempera-
ture throughout the range tested (18 to 48 C) whereas apparent
photosynthesis peaked at about 23 C.

Fnzymes

Enzymes are proteins that catalyze anabolic or catabolic
reactions along biochemical pathways. The net result is growth and
reproduction. Tlie regulatory mechanisms for protein synthesis and
enzyme activation ai’e not fully understood. One interesting and
widely accepted theory, developed through intensive experimentation with
microorganisms, was advanced by Jacob and Monod (1961). Although
not yet verified in higher plants or animals, it has been used to
explain how specific enzymes are induced and why rate changes occur
during development and maturation of higher plants (Borchert, 1967;
Firenzuoli, et al . , 1968; McClintock, 1961), also as a basis for inter-
preting the genetic implications of evolution in maize (Efron, 1970) and
mutant enzymes (Sch.warcz, 1962), photorespiration in tobacco
(Zelitch and Day, 1968), and induction of NO^ reductase in rye (Chen
and Ries, 1969).

In its simplest form the mechanism involves an operon, composed
of a structural gene and an operator gene, and a regulator gene.

Activity of the structural gene is controlled by the operator gene.

The structural gene dictates the pattern for synthesis of a specific
enzyme, and the operator gene determines the rate and timing of
synthesis. The operon may be activated by presence of exogenous
substrate and external conditions to induce de novo S)oithesis of
specific enz>Tne(s) involved in the sequential metabolism, of the

substrate .

The regulatory gene determines the quantity of enzyme (s)
produced. It produces a specific repressor which, when activated by
metabolites, acts upon the operator gene to block the mechanism
of the operon. Repression may be influenced by a feed-back control
arising from the accumulation of specific catabolic product? in
the cytoplasm.

Level of enzyme activity varies during plant growth (Borchert,
1967), during the advent and end of specific physiological processes
(Chen, Towill, and Loewenberg, 1970), and vvith certain changes in
environment (Chen and Ries, 1969). Exposure of seed to conditions
conducive to germination induces changes in the activity level of
enzymes involved in conversion of stored food to energy and substrate
necessary for assimilation (Firenzuoli et al . , 1968). Activity may
reach a peak in a short time and then decline as the substrate is
depleted or as repressive metabolites are produced as a feed-
back control.

External conditions that influence normal growth processes
also cause change. Sometimes induction of enzyme activity is under
the influence of more than one external factor. Machlis and
Briggs (1965) report that day length as well as temperature may
control breaking of winter dormancy of trees. Premature growth
flushes during unseasonably warm, winter weather may be prevented by
a photo control. Chen and Ries (1969) found that light as vrell
as substrate was needed to induce formation of NO^ reductase. Light
also induced changes in enzyme activity in etiolated bean seedlings
(Filner and Klein, 1968) .

llie use of enzyme activity measurements to predict the
potential for rapid growth in individual plants is not entirely
new. Hybrid vigor (heterosis) in maize is detectable 2 to 6 days
after germination by comparing tlie level of isocitric dehydrogenase
activity of hybridized seedlings with that of progeny of inbred
parent plants (Roos and Sarkissian, 1968). Although hybridization
is known in pine the study material was not hybridized.

llie rapid growth characteristic of superior sand pine results
from a fortunate combination of germ plasm which, through its
sequential control of enzyme synthesis, activity, and repression,
governs the rate of its physiological processes. The hypothesis
under investigation is that the activity of biological catalysts
in rapidly growing, superior trees differs from that in slower
growing, nonsuperior pines.

Because of the great number of enzymes involved in plant growth
and development, some system or method usually is employed to
determine the one(s) involved in particular processes. Roos and
Sarkissian benefited from the works of Gowan (1952), Hageman, Leng,
and Dudley (1966), and others involved in the heterotic behavior
and breeding of corn. In nutritional studies, examination can be
made of enzymes containing the element under investigation. Van
Lear and Smith (1970), for example, examined isoenzymes of poly-
phenoloxidase, peroxidase, and ascorbic acid oxidase in tissue
of pine seedlings grown without copper or without iron because
these enzymes contain copper or iron.

M-XTERIALS, EQUIPMENT, AND METHODS
flant Material

Seeds and foliage uere collected from 30 sand pine trees
(P. clausa var. clausa) on the Ocala National Forest in Marion and
Putnam Counties, Florida. Five of the trees, numbered 77, 82, 120,

193, and 199, were superior selections made jointly by personnel of
the Florida Forest Service, U. S. Forest Service, St. Regis Paper
Company, and the Forest Physiology and Genetics Laboratory of the
University of Florida. Within 100 feet of each superior tree 5 non-
superior trees of similar age were selected as representatives of
the general population growing under conditions similar to the
superior tree.

Cones of the 1967 seed crop were collected from the 30 trees.

Ihey were opened in a force-draft oven at 60-63 C and the extracted
seeds were stored at temperatures below 5 C. Needle samples from
parent trees were collected and used during the summer of 1970. Needles
were placed in plastic bags, quick-frozen in liquid N'2, transported
to the laboratory on dry ice, and stored at -20 C until used.

Seeds composited from nonsuperior pines were used throughout the
first series of experiments. On April 21, 1968, they were surface
sterilized with sodium hypochlorite and planted in S polyethylene-
lined pots, each filled with approximately 27 pounds of dried, acid-
washed sand. Pots were cylindrical, measured 21 cm in diameter and
height, and were made with a 25-mjn diameter hole through the side at

the bottom. A one-hole stopper masked with glass wool was fitted
in the hole to provide for irrigation and drainage without loss of
soil. Fifty seeds were planted in each pot. Distilled water and
subirrigation were used to germinate t’le seeds. They were allowed
to germinate for 30 days and were then thinned to 25 per pot.
lliose that germinated later were discarded.

Seedlings were raised under greenhouse conditions using
complete nutrient solutions patterned after those of Hacskaylo
(1962). Solutions contained either 7.5 ppm (Nj^) or 75.0 ppm (N2)
of N and either 15.0 ppm (Pi) or 150.0 ppm (P2) of P (Table 1).
Seedlings in 2 pots were supplied with one of the 4 solutions
(N^Pj^j ^iP2j '^’2^1’ ^^2^2^ twice weekly throughout the fi 'st series

of experiments.

Seedlings and soil from one pot of each fertilizer treatment
were transferred to tubules on June 19, 1968. Tubules were 15 cm
long, 2.5 cm in diameter, and were constructed from rigid polyvinyl
chloride (PVC) water pipe. ITiey were fitted with a one-hole
neoprene stopper masked with glass wool to permit irrigation and
drainage yet prevent loss of sand. Subirrigation and immediate
drainage were used to feed seedlings and promote soil aeration in
pots and tubules. To prevent accumulation of nutrient salts,
seedling containers were flushed with deionized water at biweekly
intervals .

Seeds from superior and nonsuperior trees were used in the second
series of experim.ents . On December 1, 1969, 50 seeds from each
tree v.-ere planted in each of two polyethylene- lined greenhouse
pots previously described. One pot contained a Lakeland coarse sand
from Calhouii County and the other a Paola sand from Marion County,

Florida. These soils were used because within the limited, natural
range of sand pine they, and their respective hyperthermic and
thermic counterparts, Astatula and Lakewood sands, comprise a
major portion of the sandhill sites upon which the species grows.

Each contained 7.5 cm of topsoil (A^ horizon) and about 12.5 cm of
soil from an underlying horizon (C for Lakeland and A2 for Paola) .

Procedures outlined by Jackson (1958) and by the American
Society of Agronomy and American Society for Testing and Materials
(1965) were used to analyze soils. Soil pH was measured using a
1 to 5 soil-water suspension and a glass electrode. Cation exchange
capacity was determined using normal neutral ammonium acetate.
Available nutrients were extracted from the soil with ammonium
acetate buffered at pH 4.8. The concentration of individual elements
in solution was measured using the equipment or procedures that follow:
Ca and Mg with a Beckman Model DU flame spectrophotometer; K with
a Beckman Model B flame spectrophotometer; P using the ch loros tannous-
reduced molydophosphoric blue color method in a sulfuric acid system
with a Spectronic 20 colorimeter; and A1 with a Perkin-Elmer model
303 atomic absorption spectrophotometer. Organic matter was
determined using the Walkley-Black chromic acid oxidation procedure.

The modified Kjeldahl method was used to determine total N.

Particle size distribution was determined using the hydrometer
method, and sand fractions were separated by dry sieving. Soil
moisture at 15 and 1/3 atmospheres was measured using pressure plate
apparatus. Physical and chemical analyses of these soils are
summarized in Table 2.

Table 1, --Source and concentration

nutrient solution

Nutrient

element

Source

NH4OH and HNO3

H3PO4

KOH

CaCl2* 2H2O

MgSO^ • 7H2O

MgS04 • 7H2O

H3BO3

MnCl2'4H20

ZnCl2

CuCl2‘2H20

H2Mo04-H20

Fe - EDTA

of elements used in the complete

Nutrient Solution*

NiPi

N1P2

N2P1

N2P2

7.5

75.0

15.0

150.0

15.0

150.0

200.0

100.0*

100.0

50.0

65.9

0.4

0.04

0.05

0.02

0.03

5.00

*Solutions were adjusted to pH 5.85 with HCl or NaOH

Table 2. — Chemical and physical properties of soil used to raise half-sibling seedlings

^ 1

2: 1

•

■p

r-*

•H

to Pv CM

O 1

•

I-H

+->

--H 1

•

< 1

»H

75 !

1 —

p 1

i-H

C 1

Q 1

•H

•H 1

P 1

2: e

•

O- Ph O

0) Cl.

t-H 1

rCt 1

•tH

»-H 1

•H 1

cj bO 1

> :s I

< I

C-H

♦H

a \

U I

•H

CN O

to 1

rH 1

U .-1

• 1

ly \

to I

CN 1

ctJ

U •

tXi

i-A

i-H

-4

•

*T3

•

•H

•

•H

t-A

Table 2.-- Continued

•M

f-H

i-H

+->

•

t-H

4->

•

•H

1-H

i-H

t/i

•

•H

I-H

O I-H

•H 03

Tj-

•p p

•

;3 o

^vO H
•H P
?H O
P -H
t/) p
•H O

CTi

P CO
U Ul,

0) 4h >
IM

I-H

•H

to C
cd

O CO CO

I-H tu

•H

4->

. 00

cts in

•

I— (

<r <r

I-H

e apparatus

■M

•

•H

</)

(/)

■)

Seeds were germinated and seedlings grovm using distilled
water and subirrigation. Approximately 45 days after planting,
seedlings were thinned to 25 per pot. On August 25, 1970, needles
and green stems were harvested, quick frozen in liquid N2, then stored
at -20 C until needed.

Photoassimilation Chamber

A chamber for photosynthetic fixation of CO2 in pine seedlings
was designed and built specifically for this project. It was
constructed entirely of plexiglass and consisted of a cubic
chamber, with dimensions of 30.5 cm, surrounded on 5 sides by a
10- cm water jacket. Figure 3. Access to the chamber was provided
through a gas-tight bottom board.

Tne environment of the chamber was controlled. Constant
light intensity of 2,000 ft-c was provided by two overhead, narrow-
spot Sylvania cool-lux lamps (P.4R 300/2NSP) . Temperature was
maintained at 25 ^ 1 C with ice-cooled water piped through the
water jacket. Air and introduced CO^ were circulated through a closed
system at 9 to 10 ft per hr by a sealed pump. Even distribution
of gases within the chamber was assured by a magnetically driven
fan. Gases entered the chamber through the base of the fan
and left via 4 e.xhaust ports in the bottom board. Tests with
smoke showed that circulation within the chamber was good and
that no leakage occurred.

Two CO^ traps and an air drier supplemented the closed
system. The drier (sulfuric acid solution of density 1.40)
maintained a relative humidity of 37% in the air bubbled through
it. CO2 traps containing a 20% solution of KOH were used to

Figure 3. --Diagram of the photoassimilation chamber (A) and components of the closed system:
dehumidifier (B) , 2 CO2 traps (C) , CO- generator (D) , Pump (E) , magnetic-driven
fan (F), magnetic stirrer (G) , removable bottom board (H) , intubuled seedlings (J) ,
heat screen (K) , and adjustable lights (L)

cleanse the system of CO2 and 14c02. Traps were bypassed wlien
was added to the system.

1 A •

CO2 and ■" CO 2 was generated by injection of 10 ml

concentrated H2S0^ into a 5 ml aliquot of XHCO3 and KH^'^CO^.

Ba^^^CO^ with a specifit. ac■^ivity of 39.2 x 10^ pc/mM
was used as the source of 14c02. To provide the 20 yc of
activity and 500 ppm of CO2 needed for each replicated run in
the photoassimilation chamber required 0.10065 mg of Bal^^o^
and 124.6313 mg of BaC03. Because of difficulty in weighing such
minute quantities of Bal^co^, an alternate procedure was devised
to insure accurate replication. CO2 and ^'^C02 were evolved from
sufficient BaCO^ and Bal4c03 for 20 replications (2.0130 mg
Bal^co^ and 2.493 g BaC03) in a closed system using 2 equivalents
of lactic acid. Gases were trapped in a solution containing
an excess of KOH (1.5 g in 100 ml of boiled, glass distilled
water). A 5.0 ml aliquot registered 4.5 x 10^ counts per min
(cprn) on liquid scintillation equipment (101% of the activity
calculated to be in a sample containing 20 yc of activity).

Photoassimilation of 14c02

Seedlings were sealed in their tubules with parafilm and
liquid latex to prevent uptake of by roots (Stemmet,

DeBruyn, and Zeeman, 1962; Fadeel, 1963) and microorganisms in
the soil. Seedlings representing each fertilizer regime were
treated simultaneously. Tliey were preconditioned to the temperature,
humidity, and light intensity of the chamber for 20 min while
atm.ospheric COg was trapped. Preconditioning in light enables

plants "...to generate any reducing agents active in photosynthesis"
or "...to deplete any reducing agents" in darkness (Stutz and
Burris, 1951). After preconditioning, 500 ppm of CO2 containing
20 pc activity v;as released into the circulating air of the
system from Klll^cOg and WICC^, I’sing excess acid. At the conclusion
of the photoassimilaticn period residual was trapped.

Tubules were removed from tlie chamber and plunged into ice
vvater. Seedlings were washed free of sand, blotted dry, and
placed in individually tared and labelled plastic bags. Bags
were sealed after evacuating the air and then plunged into liquid
^2' Frozen seedlings were stored at -20 C until seedlings were
partitioned and weighed.

Low temperatures were maintained during weighing. Bags containing
seedlings were blotted dry of condensation and weighed on a Mettler
balance. Seedlings were removed and divided into: 1) epicotyl

(needles and chlorophyllous stem), 2) nonchlorophyllous stem
(hypocotyl), and 3) roots; then (1) and (2) were weighed;
weight of 3 was obtained by subtraction. Seedling parts were
returned to the bag and stored at -20 C before making ethanol
extracts .

Ethanol -soluble extracts were made of weighed seedling parts.
Needles and green stem, nongreen stem, and roots were individually
ground with a cold mortar and pestle using liquid N2, washed into
Ehrlenmeyer flasks with liquid N2 and 95% ethanol, and boiled for
10 min. Cooled extract was vacuum filtered into scintillation
flasks. Residues were discarded.

Separation of Ethanol -Soluble Comporients

Extracts were evaporated just to dryness in a pan of warm
sand (80-90 C) , cooled, and the residue taken up in 5 ml of 15%
ethanol. Four ml were fractionated into sugars, amino acids,
and organic acids using ion-exchange resins. The remaining 1 ml
was retained to sample radioactivity of the extract.

Procedures for fractionation of ethanol extracts on ion
exchange columns were those of Shiroya et al. (1962, 1966)
modified by Riech (1970) . The extract was pipetted, a drop at
a time, directly onto two seriate resin columns each 5 cm long
X 1 cm diameter. The first contained 50-100 mesh Dowex 1-x 8
resin converted from the Cl" to HCOO" form with formic acid, and
the second contained 200-400 mesh Dowex 50W-x 8 in the H+ form.
The extract was washed through both columns with 150 ml of glass-
distilled, deionized water added at the rate of 30 to 40 drops
per min. The elution contained sugars. Amino acids were
elutriated from the 50W-x 8 resin column with 70 ml of 2N NH4OH,
and organic acids from the 1-x 8 column with 70 ml of 5N formic
acid. Using this procedure in control experiments, Riech reported
98% recovery of glucose-14c in the sugar fraction, 91% recovery
of Leucine- in the amino acid fraction, and 98% recovery of
orotic acid-^'^C in the organic acid fraction.

Eluted fractions were evaporated to dryness (80-90 C)
and taken up in 6 ml of 10% ethanol. Half (3 ml) of each fraction
was reserved for further separation by thin layer chromatography;
the remainder, and the 1 ml sample of ethanol extract, were used
to obtain measurements of radioactivity.

Riech (1970) suspected that heating extracts on a hot
plate to SO or 90 C might cause loss of some of the moi'e volatile
amino and organic acids and modified his technique for concentrating
extracts. The modified technique was not used because plant
tissue was boiled in 95% ethanol to prepare the extract.

Volatile acids do not survive the preparatory steps (Ting and
Dugger, 1965). Vacuum evaporation and lyophilization were used
to concentrate small volumes, but both procedures were excessively
time-consuming and neither proffered special advantages, so the
practice was abandoned.

Organic acids did not rcdissolve well in the diethyl ether
used to remove impurities. Water or ethanol were substituted and
found to give better resolution. Because it evaporated more rapidly
during spotting of plates, 80% ethanol was used.

Measurement of Radioactivity

Samples of ethanol extract, sugars, amino acids, and organic
acids were evaporated to dryness (80-90 C) . The residue,
dissolved in 0.5 ml absolute ethanol, was taken up in 10 ml of
scintillation fluid (BBOT)^, and the radioactivity counted for 5 min.
Counts of radioactivity were measured using Model 3380 Packard

BBOT is 2,5-bis [2- (5-tert-Butylbenoxazolyl) ] thiophene and
the scintillation fluid or "cocktail" consisted of 4 grams of BBOT
dissolved in 1 liter certified grade toluene.

Tri-Caib liquid scintillators, units were normalized, and internal
standards used.

Tliin Layer Cliromatography

Sugars, organic acids, and amino acids were separated into
component parts by thin layer chrv.matography (TLC) . Commercially
available plates were used. For sugars and organic acids
separations were made in one direction on glass plates coated
with silica gel. Two-dimensional separation on cellulose-coated
acetate plates was used for amino acids.

Sugars

Plates were scored vertically so as to contain spots and
convert them to bands (Stahl, 1969). Each was spotted with
unknown sugar solution and with solutions of known sugai's. Tests
showed tliat the clearest bands and best definition were obtained
with 40 to 50 pi of unknown sugar solution per spot.

Sugars were separated by processing the plates twice with a
solvent composed of n-propanol, ethyl acetate, and water (6:3:1).
Plates were completely dried in a fume hood each time the solvent
front reached the upper edge. Bands were detected and colors
developed by gradually heating plates sprayed with a mixture of
anisidine hydrocliloricie, aniline diphenylamine, and phosphoric acid
(5:5:1) from 27 to 36 C in a force-draft oven over a period of
from 5 to 10 min (Lewis and Smith, 1969 as modified by Riech,

1970) . Sugars were identified using a combination of colors and
Rf^ values from samples of known sugars. Table 3.

_ Distance compound moved
Distance solvent moved

Table 3. --Color and Rf-values from chromatograms of knou-n sugars
used to identify unknowns

Sugars

Molecular

Weight

No. of
Carbon
Atoms

Raffinose

594.5

Sucrose

342.3

Galactose

180.2

Glucose

180.2

Fructose

180.2

Mannose

180.2

Arabinose

150.1

. 5

Ribose

150.1

Xylose

150.1

Color

Range of
Rf

Non-Descriptive

.04-. 06

Brov^{nish -yellow

.12-. 15

Brown

.12-. 15

Brown

.15-. 18

Yellow

.16-. 19

Brownish-yellow

.17-. 21

Brownish-blue

.18-. 21

Non-Descriptive

.21-. 25

Non-Descriptive

.24-. 27

Organic Acids

Preliminary tests showed that the quantity of organic acids
in green tissue was low and that separation of the unknowns was
difficult. Spots made from 40 to SO yl of solution required 4
separate processing treatments with water-saturated ethyl 3thfr-
formic acid (7:1) to obtain discernible separation into distinct
bands (Ting and Dugger, 1965). The bands stained yellow against
a blue background when air-dried plates were sprayed with a
tincture of bromcresol green (0.04 g bromcresol green in 100 ml
95% ethanol, then 0.1^ NaOH was added drop by drop until a blue
coloration appeared) (Krebs, Ileusser, and U'immer, 1969).

Amino Acids

Soltanabadi ' s (1966) procedure was used for the two-dimensional
separation of amino acids on cellulose-coated acetate plates.

Spots were separated in the first dimension using a solvent
composed of 2-propanol, formic acid, and water (40:2:10), and,
after drying, were separated in the second dimension with
tertbutanol, methyl ethyl ketone, 3% NH4OH, and water (50:30:10:10).
Spots were developed on thoroughly air-dried plates with ninhydrin
spray (0.5 g ninhydrin in 100 ml acetone) and heat (65 C for 30
min) .

Radioautography

Counts of radioactivity using liquid scintillation equipment
provided a quantitative measure of photoassimi lated in
ethanol soluble fractions of plant parts, i.e., sugars, organic
acids, and amino acids. Radioautography was used to obtain a
comparative measure of radioactivity in individual compounds

separated and identified by TLC. Films were exposed to the TLC

plates for 2 weeks or 6 weeks; exposure for 6 weeks recorded the

relative concentration of in all compounds. At 2 weeks only

the most radioactive compounds exposed the film. ' Although methods

are available for more quantitative i.ieasurements of photoassimilatcd
14

C, radioautography served the purpose of this study, i.e., to
identify those compounds incorporating the most ^‘^C.

Measurement of Enzyme Activity

Needles and green stems were homogenized in a Sorvall Omni-
mixer with a 2-fold weight of extracting solution (10 mM 2-
mercaptoethanol , 2 mM F.DTA, 100 itlM phosphate buffer, pH 7.2) for
1 min at -15 C. Tlie brei wa‘= extracted for 60 min at 0 C with
magnetic stirring. Tlie insoluble fraction was sedimented by
centrifugation at 37,000 x g for 10 min at 4 C, then the supernatant
was centrifuged at 100,000 x g for 30 min at 0 C. Clear supernatant
was used for detei'mination of enzyme activity.

Tne activity of three enzymes of glycolysis: fructose

diphosphate aldolase (EC 4.1.2.13), glyceraldehyde-3-phosphate
dehydrogenase-NAD dependent (EC 1.2.1.12), and 3-phosphoglycerate
kinase (2. 7. 2. 3), and two enzymes of the pentose phosphate shunt:
glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate
dehydrogenase (EC 1.1.1.44) were measured using optical tests
based on the extinction coefficient of pyridine nucleotide
coenzymes at -340 my and a Beckman model DB spectrophotometer with
a Sargent recorder. Procedures were those employed by Firenzuoli
et al. (1967) but were modified for use on the Beckman spectro-
photometer. Extinction was measured at a wavelength of 340
my instead of 366 my.

Fructoaldolase

Measurement of fructoaldolase activity involved a coupled
reaction (Figure 4) with two accessory enzymes (triose isoraerase
and glycerol-3-phosphate dehydrogenase). Ihe reaction mixture
contained: 50 mM triethanolamine hydrochloride (TRA-HCl) buffer,

5 mM EDTA, 0.15 mM nicotinamide adenine dinucleotide-reduced
form (NADH), 4 mM fructose-1, 6-diphosphate, 0.72 lU per ml glycerol-
3-phosphate deliydrogenase, 0.72 lU per ml triosephosphate isomerase,
and green tissue extract. Tlie product of the reaction was
glycerophosphate .

Glyceraldehydc-3- Phosphate Dehydrogenase-NAD Dependent

One accessory enzyme (phosphoglycerate kinase) was used in
the coupled reaction, depicted in Figure 4. The reaction mixture
included: 50 imM TFL\-HC1, 5 mM EDTA, 0.15 mM NADH, 1.5 mM adenosine

triphosphate (.^TP) , 3.3 mM MgS04, 10 mM cysteine-hydrochloride,

7 mM 3-phosphoglycerate, 1.8 iU per ml phosphoglycerate kinase, and
extract. Tlie product of the reaction was glyceraldehyde-3-phosphate ,
Phosphog lycerate Ki nase

This is the same reaction as that used for glyceraldehyde-3-
phosphate dehydrogenase reversed. The reaction mixture was identical
except that 0.9 IU per ml glyceraldehyde-3-pliosphate dehydrogenase
substituted for 3-phosphoglycerate kinase. The product was 3-
phosphoglycerate .

Glucose- 6 -Phosphate Dehydrogenase

Tlie reaction mixture contained 50 mM 1R,'\-HC1 (pH 7.6),

5 mM EDTA, 0.5 mM NADP, 1.8 mM glucose-6-phosphate, and extract.

The product was gluconoiactcne-5-phosphate (Conn and Stiimpf,

1964). In aqueous solution containing the obligatory metal ion

FRUCTOALDOLASE

'Ph

t/^

I— <

-p

0
p

! ^ ^

X 0
P
O 03
U rd
'U P
X CO

rC O

0
x:
p
•H
'O

o p

O I
XCO

r-H

C/2

I— (

c_)

(/)

cq)

'T“

E-

‘i:

+->

«0

r-*

i-H

r-H

§ ^ P

^ p

0
rC

CO
I

X
o
"0

0
p
rt
U
0
o
X 0

r— < CO
bO ci
o c
x: -H

P.rl^

:2:

y >•

r-H

0
X
P-

•H

Figure 4. Reactions for measuring fructoaldolase, glyceraldehyde-3-phosphate dehydrogenase, and
3-phosphoglycerate kinase

cofactor Mg++ the reaction may proceed to form 6-phosphogluconate
(Figure 5) ^ the substrate for phosphogluconate dehydrongenase .
6-Phosphogluconate Dehydrogenase

'Die reaction mixture contained 50 miM iRA-HCl (pH 7.6),

5 miM CDJA, 0.5 mM .N'.A.DP, 6.6 inM MgSO^j, 1 mM 6-phosphogluconate, end
extract. Tlie product was ribulose-5-phosphate .

Using the spectrophotometric assays, only very low and short-
lived enzyme activity was detected by these procedures. On several
occasions the reaction reversed after starting. Use of internal
standards and commercially prepared enz>Tnes showed that procedures
were valid. Polyphenols, resins, terpenes (Firenzuoli, Vanni , and
Mastronuzzi, 1969; Anderson, Lowe, and Vaughn, 1969), quinones,
and tannins formed after cells are ruptured by extraction procedures
(Anderson and Rowan, 1967) and other endogenous substances in
tissue extracts interfere with biochemical reactions a^id inhibit
enzyme activity. Attempts to prevent oxidation of phenols and
quinones with reducing agents (Stokes, Anderson, and Rowan, 1968;
Wildes et al . , 1969), to remove or nullify them with polyvinylpyrolli-
done (Jones, Hulme, and Wooltorton, 1965; Walker and Hulme, 1965;
Loomis and Battaile, 1966), and to extract sufficient proteins for
analysis from acetone powders failed, as did efforts to concentrate
enzyme extracts .

An alternate approach proved more successful and was used
in this study. Acetone powders were made from green tissue, and
the enzymes and proteins were extracted from the powders. The
quantity extracted was too small for spectrophotometric use but
was ample for electrophoretic detection and colorimetric measurements

GLUCOSE -6 -PHOSPHATE DEHYDROGENASE AND 6-PHOSPHOGLUCONATE DEHYDROGENASE

0
p.

ir>

ff)

rO
♦ H

■M

0
ft

vO -

0 (1)

ft 3

1 C
vO (1)

O T3
O X
3 x:
*— 1 0^
bo *3

0
Ph

i-H

I'-

(N bO
O ^ tD
U

+->

Figure 5. Reactions for measuring glucose-6-phosphate dehydrogenase and 6-phosphogluconate
dehydrogenase

of enzyme activity. Tne technique is not as accurate as the optical
test, but it has been used successfully (De.Jong, Jansen, and Olson,
1967; Efron and Schwartz, 1968). Considering the comparative use
made of these data, it provided an acceptable alternative.

7 cet one Powders --Preparati on an.d Protein Ext r a c t i o n

To prevent denaturation of enzymes all operations were
carried out at temperatures below 5 C with cold I'eagents, tissue,
and glassware (Hare, 1970).

Twenty grams of frozen tissue and approximately 125 ml of 80%
acetone were "grated" then "liquified" in an Oster Blender. Tlie
brei was poured through a No. 60 soil sieve. Fibers were discarded
and the suspension sedimented at 37,000 x g for 10 min. T]ie
supernatant was discarded and the pellet was mixed into fresh 80%
acetone. The suspension was . sedimented, as before, and the pellet
blended into 100% acetone using a glass homogenizer and teflon
pestle. After vacuum filtration through a plastic filter-funnel,
the residue was washed with 100% acetone until white. Tlie residue,
air-dried by vacuum filti'ation, was sifted through No. 35 and No. 100
soil sieves. These fines were stored at -20 C.

Proteins were extracted from acetone powders with a solution
containing: 2.5 M urea, 22.5 niM potassium metabisulphite, 56.8

mM ascorbic acid, 6.5 mM dithiothreitol (Clelands reagent),

"Tween 20", tris buffer [tris (hydroxymethyl) amino methane], and
water to give. a product containing 0.004% "Tween 20" at pH 8.5
(Hare, 1970). Protein extracts were made by mixing acetone powder
and extracting solution 200:3, extraction for 1 hr, and centrifugation
at 37,000 X g for 10 min. Tlie supernatant, extracted with a
syringe, contained dissolved proteins. The pellet was discarded.

Preparation of Polyacrylamide Gels and Electrophore sis

Preliminary tests were made comparing polyacrylamide gel
formulations and layering techniques proposed by Bakay and Nyhan
C1969), Dar^is (1964), and Hare (1970). Use of a large-pore
sample gel as an anticonvection medium proved inconvenient and
unnecessary; the high concentration of urea in Hare's extracting
solution served as well when layering was done by syringe under
the upper buffer of the electrophoresis chamber. Separation of
glucose-6-phosphate dehydrogenase isoenzymes was enhanced with a
large-pore, spacer gel, but the clearest definition of malate
dehydrogenase isoenzymes was obtained without one, i.e., by layering
directly on the small-pore, running gel. Davis' large- and small-
pore gels gave the best resolution of proteins.

Buchler Polyanalist disc electroplioresis apparatus was used.
Buffers were those suggested by Hare: tris-glycine (pH 8.9)

containing bromphenol blue dye in the upper chamber and tris-HCl
(pH 8.1) in the lower chamber. Twelve tubes were processed
simultaneously using 50 ma of current.

Gels were precharged, i.e., processed without a layer of
protein extract, to remove contaminants. Protein extract was
layered on gels under the buffer using a syringe calibrated in
pi. The urea in the extract prevented dispersion into tlie upper
buffer. Gels developed for glucose-6-phosphate dehydrogenase
isoenzymes and for protein bands were layered with 40 pi of protein
extract on the spacer gel; 20 pi of extract was layered directly
onto the small pore, running gel used for development of malate
dehydrogenase isoenzymes and for protein bands. Electrophoresis was
complete when the colored front reached the lower end of the gel.

Detection of Protein and Isoenzyme Bands

Gels vv'ere removed from the electrophoresis tubes with a fine
jet of water then placed in 100 x 12 mm test tubes.

Proteins and Dehydrogenase Isoenzymes

Gels were covered with lO-i trichloroacetic acid then agitated in
a reciprocal shaker for 30 min. Three drops of 0.2% coomassie blue
were mixed with the acid, and the immersed gels were stored for 48 hr
in the dark. Stained gels were rinsed and stored in 8% acetic acid in
the dark at 4 C until scanned with a Gilford Model 2000 gel scanner
(densitometer) .

Existing techniques for preparing substrates to color isoenzymes
of a specific enzyme in polyacrylamide gels were used (Johnson,

Brannaman, and Zscheile, 1966; Macko, Honold, and Stalimann, 1967; Roggen,
1967).

The staining procedure involves action of the enzyme on its
substrate in a coupled reaction that results in the reduction of
nitroblue tetrazolium (NBT) to a colored product (Goldberg, 1963).

Bands stain blue. The intensity of the stain is related to the pH
of the solution, temperature, length of incubation, and the activity
of the enzyme (Gabriel and Wang, 1969) .

Dietz and Lubrano (1967) recommended 90-min incubation for
optimum development of lactate dehydrogenase isoenzymes and cautioned
against using less than 45 or more than 120 min for quantitative work.

'Ihe upper limit was set because substrate in the vicinity of the most
active isoenzymes is exhausted while that near less active isoenzymes
allows staining to continue. Ihis was true with malate dehydrogenase

for which a 45-5iiin incubation period \vas used. However, comparisons of
staining intensity with glucose-6-phosphate dehydrogenase subst7:ate
showed that the most active isoenzymes continued to darken even after
6 hr, indicating a low level of enzyme activity and an adequate supply
of sibstrate. Background staining, attributed to protein sislfliydryl
groups (Dietz and Lubrano, 1967), occurred witli long periods of
incubation, so development of isoenzymes of glucose-6-phosphate de-
hydrogenase was reduced to 3 hr or less.

Hie basic incubation media was prepared using two solutions to
which specific substrate and pyridine nucleotide coenzyraes were added.
Both were made with 0.05 M tris-HCl buffer pH 7.5. Solution I contained
30 mg NBT (dissolved in 95% ethanol) and 100 mg MgS04'7H20 dissolved
in 50 ml buffer. Solution II was composed of 150 mg EUTA dissolved
in 40 ml hot buffer to which. 3 rag phenazine methosulfate (PMS) was
added when cool, liie solution w^as brought to 50 ml volume with buffer.

Both NBT and PMS solutions are light-sensitive, and required storage
in brown bottles and use in subdued light.

'Phe incubation mixtures for developing glucose-6-phosphate
dehydrogenase and malate dehydrogenase bands were made up in quantities
to develop 21 gels.

Glucose-6-Phosphate Dehydrogenase and Malate Dehydrogenase

Gels were inundated in an incubation mixture containing 10 mg
NADP, 84 mg giucose-6-phosphate , 14 ml solution I, and 14 ml solution
II, then incubated at 37 C in the dark.

I'lie incubation mixture consisted of 188 mg malic acid, 14 ml
solution I, and 14 ml solution II (adjusted to pH 7.5 with NaOH)
to which 20 mg NAD was added. Gels were covered with the incubation

mixture and incubated at 37 C in the dark. At the end of the
incubation period S% acetic acid was substituted for the incubation
mixtures. Gels were stored at 4 C in the dark until scanned on
the densitometer.

Reaction mixtures for staining isoenzymes that involve use of
one or more accessory enzymes are difficult to formulate. Experi-
ments aimed at developing or modifying existing procedures (Bergmeyer,
1965) to stain fructose-1 6-diphosphate aldolase, glyceraldehyde-3-
phosphate dehydrogenase, and phosphoglycerate kinase isoenzyme bands
were not entirely successful. Bands were obtained but results
were not consistently reproducible.

Individual bands were identified by tlieir position relative
to the dye front (Rf value). Activity of identified isoenzymes
measured by staining intensity was quantified using heights of
densitometer scanning peaks. Gabriel and Wang (1969) report that
quantification of isoenzyme activity using reduced tetrazolium
is accurate for comparative purposes. Comparisons using the
number, Rf, and activity of dehydrogenase isoenzymes were made
between the superior tree and each of its 5 nonsuperior, neighboring
trees, and between the half-sibling progeny of the superior tree
and that of each of its neighbors growing on two soils. .Similar
comparisons were made using the number, Rf, and staining intensity
of protein bands.

Sample Size and Statistical .^alysis
Use of two electrophoresis chambers permitted processing 24
gels simultaneously and replication of samples from a single location

and soil at least 3 times. Measurements v;ere obtained from scanned
gels representing the average of each tree x soil x protein combination.

A randomized complete block design was used for the experiment
involving measurem.ents of isoenzjcnes and proteins. Tlie number of
oands, Rf values, and quantified staining intensity data froni densitometer
scannings were subjected to analysis of variance.

Location of the 5 superior trees and their associated nonsuperior
contemporaries, from which seeds and tissues were collected, served
as blocks. Comparisons were made among soils on vdiich parent trees
grew (original), and those on which seedlings were grown (Lakeland
sand and Paola sand), and among trees from each location. Orthogonal
partitioning of sums of squares was used to make the contrasts specified
under soils and trees. Dunnett's procedure (Steel and Torrie, 1960)
was used to compare the superior tree with each neighbor. Selected
comparisons also were made (at a single degree of freedom) by partitioning
the tree x soil interaction sums of squares.

RESULTS AND DISCUSSION

Photoassimilation of

To obviate the need for processing all seedling parts, time-
course studies were used to estimate an optim.uin period for photo-
assimilation of ^'^C02. Tlie purpose was to limit time for translocation
of labelled compounds into the nonchlorophyllous portions of the seedling,
yet permit incorporation of easily measurable quantities in green
tissue. Radioactivity of the ethcinol-soluble fraction of needles and
green stem, stem, and roots was measured for seedlings allowed to
photoassimilate ^'^C02 for 15, 30, 60 and 120 min.

Results summarized in Figure 6 show that, with minor
exceptions, there was a orogressive increase in radioactivity in
stem and root tissue with the length of the photoassimilation
period, and the proportion of activity in green tissue decreased as
the length of photoassimilation increased. It appears that even
with photoassimilation as short as 15 min, ample time is provided
for some translocation of labelled compounds to the roots. The
proportionately high concent cation of radioactivity in the stem,
compared to root tissue for all lengths of exposui’e suggests that
the stem of young sand pine seedlings serves more as storage
tissue than primarily as an avenue for translocation. Tliis does not
discount possibilities that some C02-fixation may occur in jion-green
tissue or that a high rate of metabolism in the roots may catabolize
and respire some labelled compounds. Extrapolation of the data suggested
a 10 min photoassimilation period as near optimum.

LEVEL OF ACTIVITY IN CLOSED SYSTEM = 44,382,000 CPM

C/D

SONVSnOHl NI IHDIHM HSHHd WVHD yScJ WdD

Figure 6. Distribution of radioactivity in seedlings allowed to photoassimilate ^“^CO,

Results of the tiir.e-course experiment raised two questions:

1) would assimilation of ^^C02 and CO2 continue during the post-illumination
period while the chamber was being cleansed of radioactive gas and
seedlings were being processed for freezing? 2) 'V.liy was such a small
proportion of the 44 million cpm released into the closed system utilized
even after a 2 hr photoassimilation period? Experiments were conducted
to answer these questions.

Tlie first was designed to answer question 1 and to test the
conclusion that a 10 min photoassimilation period was near optimum
for fixing measureable amounts of labelled carbon in green tissue
without allowing appreciable amounts to translocate to the stem and
roots, i.e., to limit radioactivity primarily to green tissue.

Working at night to reduce the likelihood of contamination by
incident daylight, com.parison was made of the radioactivity in
ethanol extracts of green tissue, stem, and roots between seedlings
subjected to a) preconditioning and exposure to ^‘^C02 for 10 min
in darkness or b) preconditioning and pliotoassimilation for 10
min using 2,000 ft-c light. Post-induction periods in both treatments
were in darkness, and seedlings were processed for preliminary
freezing in subdued light (just enough to ascertain that all sand
was washed from roots) .

Results presented in Figure 7 support the previous contention
that pliotoassimilation for less than 15 min confines metabolites
with 14c to g.reen tissue without permitting appreciable amounts to
translocate to the stem and roots. Reducing the photoassimilation
period from 15 to 10 min increased the proportion of radioactivity
in green tissue by about 3% compared to activity in stem and
roots. It also reduced radioactivity by 70% in

light dark

Figure 7. Distribution of radioactivity in seedling tissues exposed to
^C02 in the light and dark

green tissue, 75% in stems, and 87% in roots. Shorter photoassimilation
periods might have increased the proportion of radioactivity in green
tissue and greatly limited translocation to the stem and roots, but
extrapolation of 15 and 10 min data suggests that radioactivity in
green tissue also might be lowered to a level of questionable utility.
For purposes of this study a 10 min photoassimilation period appeared
near optimum.

In the dark radioactivity was recorded in the stem, but none in
the roots or green tissue of all 4 seedling replicates. Results,
while not analyzed statistically, emphasize the need to extinguish
light at the conclusion of the photoassimilation period and to process
seedlings in subdued light.

The large discrepancy between the radioactivity of labelled
carbon in the system and the amount photoassimilated has at least
5 possible explanations:

1. Uptake of CO2 was limited by some unexplained physiological
condition (s) .

2. Preferential uptake of CO2 over ^“^002 .

3. An excess of C02was used.

4. Activity was lost during preparation of ethanol extracts or
was masked by chlorophyll and other extraneous material.

5. Not all the radioactive carbon was released from KHI4CO3
by the lactic acid.

The system was gas tight. Photo- and dark-respiration do not account
for the large discrepancy. Possibilities 1 and 2 are beyond the
scope of this investigation, and 3 was intentional. Experiments were
undertaken to test 4 and 5.

Aliquots of green tissue extract prepared from 5 sand pine
seedlings were pipetted into scintillation vials to form an arith-
metic series from 0.5 through 5.0 ml in 0.5 ml increments. A
blank of 95% ethanol was used. To each vial 10 ]jl ^^C-sucroso was
added. All w’ere evaporated to dryness, tlien tlie residue was taken
up in 0.5 ml absolute alcohol and diluted with 10 ml BBOT scintillation
cocktail. One additional vial containing 2.5 ml green tissue extract
was similarly prepared and processed except that the extract,
including ^^C-sucrose, was clarified with charcoal before evaporating
the liquid. Counting efficiency and cpm were recorded on identical
liquid scintillators at two cooperating laboratories (Agronomy and
Pesticides).

Chlorophyll in the scintillation fluid lowered both counting efficiency
and cpm. High concentrations of chlorophyll, such as those found in
tissue from high nitrogen fertilizer treatments, were most seriously
affected. Effectiveness of the scintillators was lowered in direct
proportion to the amount of chlorophyll present. Clarification with
charcoal improved counting efficiency by 800% and increased cpm by
250% in the vial containing 2.5 ml of chlorophyll-containing extract.

Some loss of radioactivity occurred during clarification but, considering
gains experienced, it was considered tolerable. At best, however,
the counting efficiency was only 0.70 in scintillation fluid containing
no green tissue extract, indicating that the cocktail or absolute
ethanol lowered counting efficiency. Ihese possibilities were tested.

Results of the tests showed that the cocktail and the absolute
ethanol used with scintillation equipment in the Forest Physiology
and Genetics Laboratory (F.P.G.L.) caused quenching in scintillator

counters at cooperating laboratories. The cooperators equipment had
been standardized against cocktail other than BBOT. To compensate
for quenching caused by chlorophyll and ethanol and for differences
in cocktail, curves were drawn to equate all measurements of radioactivity
at 100% counting efficiency regardless of cooperator equipment used.

Figure 8. BBOT alone was responsible for a reduction in counting

of more than 20-s. Absolute ethanol caused a further reduc-
tion of about 11%. Increasing the proportion of BBOT to absolute
ethanol improved efficiency by about 2% but reduced cpm by more than
14%. Linear regressions of the form Logg AES Ratio = a+b Logg
cpm were fitted to a plot of the data. Test of the regression by
analysis of variance showed that the probability of obtaining an
F value larger than that obtained for regression was less than 0.0001
for cooperator equipment. No meaningful loss of accuracy occurred
from fitting straight line functions to the data. The slope of each
curve appears to be unique for each scintillator.

Seedlings appear to utilize only a minute amount of the ^‘^C02
released into the photoassimilation chamber. Possibly not all of the
20 yc of activity (45 million dpm) was evolved from Kh1^C03 by
lactic acid even though two equivalents of acid were used to one of
the bicarbonate.

To test the effectiveness of the acid, a comparison was made of
radioactive carbon photoassimilated by seedlings when 10 ml of
concentrated lactic, hydrochloric, or sulfuric acid were used.
Stoichiometrically, this provides twice the amount of lactic and
hydrochloric and 4 times the ajnount of sulfuric acid needed to
evolve 500 ppm of CO2 containing 20 yc of activity from the potassium

EFFICIENCY

Figure 8. Quenching curves developed to compensate for loss of
counting efficiency

bicarbonate. To verify results of a previous experiment, which
indicated that postil luinination processing of seedlings should be
done in darkness, one additional treatment was included. Two
equivalents of lactic acid w’ere used to evolve > but liglits v/ere
left on for an additional 5 m-’ n following the 10 min photoa ;siii ilation
period (while CO2 in the chamber was trapped). 'Ilien, seedlings
were processed in fluorescent rather than subdued light. Adjustments
were made for quenching, and cpm were expressed per gram of fresh
tissue weight.

Inorganic acids more effectively evolved CO^ from KHCO3 than
lactic acid. Figure 9. On an equivalent basis HCl was as effective
as H2S0^, but in this experiment, only two equivalents of HCl were
used as compared to four of H2S0^.

Photoassimilation of continued during 5 additional min
of light while the chamber was cleansed of ^"*002 . Tlie exact amount
could not be determined because seedlings subjected to the additional
photoassimilation period also were processed in fluorescent rather
than subdued light. Some loss of have occurred to the atmos-

phere due to photorespiration.

Clarification o f Ex tracts and Quen ching

Extraneous material in ethanol extracts caused quenching, i.e.,
loss of counting efficiency and a reduction in recorded cpm, in the
liquid scintillator. Dark green extracts from seedlings supplied
high rates of N proved particularly troublesome. Attempts to
rcm.cve the chlorophyll by filtration or centrifugation failed.

Powdered, activated charcoal was lased routinely in previous
experiments as a clarifying agent; and even though some loss of

4 Equiv.

J—J

2 Equiv.

LACTIC HCL H2SO4

LACTIC

PROCESSED : IN DARKNESS

IN LIGHT

Figure 9. The C incorporated into green tissue of Pinus Clausa in
response to different founts and kinds of acid used to
release ^‘^C02 from Kh14c03

radioactivity occurred, gains in counting efficiency moi'e than
compensated for losses. Powdered, activated alumina was compared
with powdered, activated cliarcoal to provide a more efficient
clarifying agent.

Ethanol extracts from 2 s 2ts of seedlings (each represented by
the 4 fertilizer treatments) were prepared from seedlings that had
photoassimilated Color of the extracts, ranged from light to

dark green (no color charts for foliage were available).

NjPj NjP^ NjPj

Dark green Light green

One set of extracts was clarified with activated alumina
(80-200 mesh size chromatogranhic grade) using 3 additions of
approximately 0.5 g each plus 3 min spinning at maximum speed on a
clinical centrifuge after each addition. Com.parison with untreated
extiact showed no color change although tlie alumina had attained

a light yellowish-green color.

Tne other set of extracts was clarified with powdered activated
charcoal using 3 additions of approximately 0.3 g each, as above. All
clarified extracts were light yel low'ish-green and only a slight
difference in color remained between extracts of N2P^ and NjP^ fertilized
seedlings. Because alumina failed to clarify the extracts, they
were treated with charcoal. Meaningful differences in counting
efficiency or cpm between charcoal clarified and alumina plus charcoal
clarified extracts were attributed to alumina.

Figure 10 shows that when both counting efficiency and retention
of radioactivity are considered, clarification with charcoal was as
good or superior to clarification with both alumina and charcoal.

Alumina by itself was not an efficient clarifying agent.

CPM PER GRAM FRESH WEIGHT IN THOUSANDS COUNTING EFFICIENCY

230

210

190

170

150

130

110

Figure 10. Comparison of clarifying agents

Objectives of another series of experinents were: to identify

metabolic pathways most responsible for fixation of atmospheric
carbon in green sand pine seedling tissue; to determine the affect
of each of the 4 levels of fertilizer on the seedlings, on radio-
activity in the ethanol-soluble fraction of green tissue, and on
the relative amounts of labelled sugars, organic acids, and amino
acids produced.

Ethanol extracts made from green tissue of fertilized, 3-
month-old sand pine seedlings, after HCO2 was photoassimilated for
10 min at 2,000 ft-c and 25 1 C, were fractionated on ion exchange

columns. Radioactive counts were made of sugar, organic acid, and
amino acid fractions. Sugars and acids were separatee, i ico
constituent compounds chromatographical ly, for identification, and
radioautograms were made of TLC plates to permit comparison of
radioactivity among constituents.

Table 4 shows the actual and proportional distribution of
in the ethanol soluble fractions, llie insoluble fraction, composed
primarily of cellulose, hemicellulose, and lignin, was not assayed.
Shiroya et al. (1962, 1966j working with white and red pine seedlings
and Balatinecz, Forward, and Bidwell (1966) working with 8-month-
old jack pine found no more than 10% of photoassimilated
in the ethanol insoluble fraction. Extrapolation of their data
suggests that virtually all the photoassimilated in 10 min
was contained in ethanol-soluble compounds. In 'O-raonth-old sand
pines Riech (1970) reported very little translocation of labelled,
ethanol-soluble compound from needles supplied by photoassimilated
^^C02, even after 8 hours.

Table 4 . --Distribution of photoassimilated 14C in green sand pine
seedling tissue

Ethanol Soluble Fractions

Organic Acid Amino Acid Total

lo^/g fresh tissue

55.7 13.8 4.8 74.3

. Percent of fraction

75.0 18.6 6.4 100.0

Of the ethanol-soluble fraction, sugars contained 75%, organic acid
19-6, and amino acids 6% of the labelled carbon in green tissue.

About 40-6 of the activity was lost during fractionation of the
ethanol extract on ion exchange columns. Although this loss is
approximately 2 1/2 times as great as that reported by Riech using
the identical procedure, the proportionate distribution of he
reported in 6-month-old sand pines wa.s almost identical i.e.,

76% sugars, 17% organic acids, and 7% amino acids.

Sugars

Glucose, fructose, and galactose were the 3 most common sugars identified
for seedlings raised at all levels of fertilization. Rf values
for glucose and frucLOse overlapped (fable 3) but were differentiated
by color. UTien sprayed with developer and heated, glucose stained
brown, and fructose yellow. Rf values for glucose and fructose
differed enough from galactose to avoid confusion, but Rf's for
galactose and sucrose were similar. Here, too, color was used to
differentiate oetween them; galactose was brown, and sucrose was
brownish-yellow. One other band developed primarily in the extract
from seedlings raised at low levels of nitrogen. Its Rf identified
it as a pentose, but it bracketed the ranges of ribose, arabinose,
and mannose. The brownish-blue color indicated it was arabinose.

Tlie chromatogram illustrated in Figure 11 shows other bands at
relatively high Rf values, but they were either faint or not present
at all levels of fertilization. They were not specifically identified;
yet, since Rf values in sugars are inversely proportional to molecular
weight and number of hydroxyl groups (Lewis and Smith, 1969) or
the number of carbons, this suggests they were probably tetroses or

Figure 12. Radioautogram of a sugar TLC plate (6 weeks exposure)

Figure 13. Radioautogram of the same TLC plate used in Figure 12 (2 weeks exposure)

trioses. llie bands were for the most part in extracts from seedlings
raised at high levels of N and P.

Radioautograms of TLC plates show that most of the 14^ ‘

contained in glucose and fructose, less in galactose, virtually
none in high Rf, low molecular weight sugars, and none in pentoses
(Figures 12 and 13). No difference in radioactivity between glucose
and fructose was apparent here even though the green tissue
included active apical, leaf, and stem meristem.

Rangnekar and Forward (1969) reported differential fixation of
^4c in fructose and glucose following 6 days of 14^2 assimilation.
From 2.7 to 8.4 times as much radioactivity was found in fructose
as in glucose in the stem, root, and bud of red pine seedlings.

The pattern was reversed in needles: 1.4 times as much 14^

recorded in glucose. Tlie high proportions in active meristematic
tissue was attributed to metabolism of the glucose moiety of
translocated sucrose and the presumed sequestration of fructose
within a cell compartment or in the individual cell. This explanation
is in agreement with the theory for higher plants. Sucrose acts as
a protective derivative and source of glucose, the primary metabolite
of all living organisms (Arnold, 1968).

Some labelled carbon remained at and near the origin. The
concentration was particularly high for seedlings raised at the
high N level. Their low mobility with respect to sucrose
and raffinose suggested that they were oligosaccharides, possibly
verbascose, composed of sucrose plus 3 galactose units. Presence
of oligosaccharides containing sucrose might serve to explain
absence of sucrose on the chromatograms. A body of evidence exists

to show that sucrose is a primary translocate in higher plants
(Clauss, Mortimer, and Gorham, 1964; Gordon and Larson, 1968;

Shiroya et al . , 1962; IVillenbrink, 1966; Hofstra and Nelson, 1969),
including conifers. Hida, Sacko, and ILnrada (1962), however,
found that the sucrose content in pine needles appeared lower than
in foliage of other conifers. In some plants raffinose, (Pridham,
Walter, and Worth, 1969) stachyose, and verbascose (Webb and Burley,
1964, Trip, Nelson, and Krotkov, 1968) were found among translocates
(sucrose plus 1, 2, and 3 galactose units, respectively). It
seems apparent that oligosaccharides wore present. Tliey may serve
as translocates in young sand pine seedlings.

Tlie extract from one seedling fertilized at the high level of
N and low level of P remained at the origin of the TLC plate.

Extract from all other seedlings raised under this fertilizer
regime migrated upward during separation. iTie seedling is believed
to have been moribund. Seedlings raised at low P levels (P]^)
suffered comparatively high mortality in both pots and tubules.
Mortality am.ong the faster growing N2PJ seedlings was higher than
among slower growing N^Pj^ seedlings. Dead and dying seedlings were
characterized by a constriction of the stem at groundline reminiscent
of damping off disease (Figure 14) . 'Hie constriction and area
immediately adjacent to it was black. It appeared charred. Dr.

R. Schmidt, Forest Pathologist at F.P.G.L., identified a species
of Verticillium in one of several tissue sample cultures. Alexopoulos
(1952) recognizes Va'x^'t’VQ'Ll^'L'tw^ as a cause of wilt disease in many
plants and as a fungus associated with damping-off.

Figure 14. Moribund seedling showing constriction near groundline

Another possible cause of mortality among seedlings raised
at the low level of P might have been chemical burn. HCl was used
to adjust the acidity of nutrient solutions to pH 5.85 + 0.05.

Prior to adjustment solutions containing the low level of P were
pH 9.8 and pH 10.5, whereas those containing the high level were
pH 3.5 and pH 5.8. Accumulation of Cl“ during the 2 weeks between
flushings of plant containers with deionized water could have
resulted in a Cl build-up and bui'n.

Regardless of the cause of death, seedlings supplied the NCP

^ 1

nutrient solution had a comparatively high rate of mortality. The
possibility exists that one healthy-looking but moribund seedling
was used in these experiments. If this were a fact, assimilation
processes may have all but ceased thereby causing a low level of
C fixation in sugars. More likely, however, is the possibility
that the stem was girdled causing accumulation of translocation
products in the stem and green tissue above the girdle. If these
products were high molecular weight oligosaccharides, such as
stachyose or verbascose, their movement from the origin would be
slight. Idle seedlings' moribund condition would permit progressively
less assimilation of in comparatively low caibon sugars such as
fructose, glucose, and galactose and reduced respiratory loss of
labelled compounds. Subsequent coupling with previously formed
sucrose might result in an accumulation of translocated ^Re-
labelled high carbon sugars, such as stachyose and verbascose, at
the origin of the TLC plate. Examination of the radioautogram
shows this to have been the case.

Organic Acids

Incorporation of 14c in the organic acid fraction amounted
to only about one-fourth that in sugars, (Table 4). Separation
by TLC required several treatments with solvent to obtain
separation for identification of some components. Figure 15.
Exposure of plates to x-ray film for two weeks yielded no clearly
discernible bands of radioactivity; 6 weeks exposure was required
to obtain faint bands. The intensity of the bands attests to
the low activity obtained on liquid scintillation equipment.

Several bands appeared on the radioautograms that did not
correspond to those obtained visually on TLC plates. These were
labelled "hot" on the chromatogram. "Hot" bands were found only
in syrupy extract. The Rf of one "hot" band coincided with that
of malic acid standards, another with glutamic acid. Glutamic
acid is a dicarboxylic amino acid formed from the organic acid
c^-ketoglutarate and NH^ . An intermediate is “^-iminoglutarate which
requires NADH + H+ and glutamic dehydrogenase to catalyze the
second reaction (Devlin, 1968) . It is conceivable that oc_
iminoglutarate or the acidic glutamic acid was absorbed on the
50w X 8, H+-form resin to become a contaminant contributing to
the syrupy consistency of the extract.

Malic acid was the only organic acid identified containing
detectable amounts of ^4q_

Amino Acids

This fraction of ethanol-soluble, green tissue extract
contained the least radioactivity, about one-twelfth of that in
sugars. Activity was so low that spots could barely be detected

Hatched bands are unknowns

Organic acid standards are labelled: glutamic acid (G) ,

oxalic acid (0) , ascorbic acid (A) , and malic acid (M)

Radioactive bands (HOT) appeared in syrupy (S) extract

on x-ray film after 6 weeks of exposure. Prints could not
be made.

Only extract from trees fed the higji level of nitrogen
contained radioactive spots. Figures 16 and 17.' They v/ere
identified by using Soltanabedi' s (1966) amino acid map as;
glutamic acid, aspartic acid, and, because of the close proximity
of spots, either lysine or arginine, most probably the latter.

Labelled malic acid, glutamic acid, and aspartic acid suggest
activity of the tricarboxylic acid cycle, amination of
ketoglutarate to glutamic acid, and possibly transamination to form
aspartate leading to arginine in the urea cycle (IVilson, King,
and Burris, 1954). Because of the extremely low incorporation
Or C in amino acids, no further work was done with amino acids
and the enzymes involved in their synthesis.

Effect of Nutrient Level on Seedling
Morphology and Incorporation

Distribution of weight and in seedlings supplied 7.5

ppm (%) or 75.0 ppm (N2) N, and 15.0 ppm (Pi) or 150.0 ppm

Pj is summarized in Table 5. The proportional distribution
of weight and radioactivity as influenced by N and P nutritional
levels is shown in Table 6.

Color and Weight

Foliage was darker green in N2 than Nj treatments and
darkest in the N2P1 treatment. P, by itself, liad no noticeable
Influence on color. (See re-^ults of experiment testing clarifying
agents on page 53.)

N more than P influenced seedling weight and size. The entire
seedling and each of its component parts was larger and heavier •

YELLOW

glutamic acid

aspartic

acid

arginine

lysine

ORIGIN

Figure 16, Amino acid TLC plate for fertilizer treatment N2PJ

Radioactive spots are labelled

glutamic acid

aspartic

acid

^^^^arginine
or

lysine

ORIGIN

Figure 17. Amino acid TLC plate for fertilizer treatment N2P2

Radioactive spots are labelled *

One * spot (dotted outline) did not appear on the TLC plate

in N2 than Nj treatments. The effect of P was not as pronounced
and appeared to have been centered in green tissue and roots.

P2 treatments contained the heaviest seedlings. The N.,Po
fertilizer regime produced the largest and heaviest seedlings.

Ratios between green and nongreen tissue weight were
virtually unaffected by P but strongly influenced by N. The
ratio was lowest for seedlings which suggests that, like the
more common top-root ratio, a favorable balance existed between
green and nongreen tissue. This was expected because N2 treat-
ments produced seedlings with the most green tissue.

Fertilizer combinations most conducive to green tissue
production contained the high level of N. N2^ produced seedlings
with heavy foliage, stem, and roots. N2P2 produced proportionately
less root and stem tissue and, therefore, seedlings with the
highest green-nongreen tissue ratio. Seedlings grown in N^Pj
were smallest but the weight distribution between green and nongreen
tissue was most equally balanced.

Incorporation

Foliage of N2-fed seedlings was heavier and darker green
than those fed Nj . It contained more chlorophyll and photo-
assimilated more ^^C02, consequently, green tissue extracts from
N2 -supplied seedlings were more radioactive. The affect of N
and P on C incorporation becomes apparent only when radioactivity
is adjusted for differences in tissue weight, i.e., when expressed
on a weight of green tissue basis.

Both P and N influenced the level of radioactivity in
ethanol extract and most of its component fractions. Highest

Table 5. --Seedling weight and photoassimilated distribution
in response to nutrients

NUTRIENT

REGIME

ITEM

NjPl

N1P2

N2P1

N2P2

Weight in grams

Entire seedling

0.375

0.425

0.929

1.196

Needle + green stem

.146

.168

.542

0.675

Stem

.024

.042

.048

Roots

.205

.233

.345

.473

Ratio

Green/nongreen wt.

Adj . cpm/g green tissue

.594

X 10^

.675

1.367

1.264

Ethanol extract

55.2

69.0

41.1

74.7

Sugars

38.9

58.0

54.9

71.2

Organic acids

10.9

22.0

10.0

12.5

Amino acids

5.5

3.0 -

5.9

4.6

Nutrient treatments were: 7.5 ppm (Nj) or 75.0 ppm (N^,) of

N and 15.0 ppm (Pj) or 150.0 ppm (P2) of P

Table 6. -Influence of N and P fertilizer on the distribution of
weight and radioactivity

NUTRIENT PJEGIME

NITROGEN

LEVELS

PHOSPHORUS

LEVELS

ITEM (7

.5 ppm)

(75 ppm)

(15 ppm)

(150 ppm)

of seedlings-

Heaviest seedling

5.6

94.4

44.4

55.6

Heaviest green tissue

0.0

100.0

50.0

Heaviest stem

11.1

88.9

50.0

Heaviest root

5 .6

94.4

44.4

55.6

Lowest green-nongreen
tissue wt. ratio

100.0

0.0

50.0

Highest adj . cpm per
gram green tissue

Ethanol extract

55 . 6

44.4

55.6

Sugars

37.5

62.5

12.5

87.5

Organic acids

50.0

Amino acids

12.5

87.5

62.5

37.5

C incorporation in the extract was recorded in P2 and
treatments. Treatment combinations with the highest radioactivity
were N2r*2 and Njp2'

Sugars contained 75% of the photoassimilate -*-4c and, in
this fraction, tlie level of activity was influenced almost
exclusively by P. Seedlings supplied NU were more radioactive than
those supplied Nj , but, on the average, P contributed more to
incorporation than N. Seedlings grown in N2P^ contained the

most

Interpretation of the affects of N and P on fixation of
in organic and amino acids is confounded by presence of, what
appears to be, glutamic acid in both acid fractions. To facili-
tate interpretation, the presumed contaminant in the organic acid
fraction was considered to be “-iminoglutarate, the oxidized •
precuisor of glutamic acid, lliis was possibly true as no
“-iminoglutarate was used as a standard for identification.

Organic acids from seedlings supplied P2 and contained the
most c. Here, as in ethanol extract and sugars, incorporation
of 14c appears to be influenced more by P than N. Highest
radioactivity was recorded in the N^Pg treatment combination.

In amino acids, as in the other fractions, 14^; fixation
was influenced more by the level of P than N. However, the
difference is much less pronounced in amino acids. The apparent
inciease in importance of N on carbon fixation is not inter-
pieted as indicating an extraordinary relationship between
incorporation and .N’ but rather an expression of the requirement
for N in amiino acid synthesis via am.ination of ketoacids

utilizing carbon derivatives. Radioactivity was highest in
and 1^2 treatrnents and in tlic ^^2^1 ^urtilizer regime,

P appeals to be a rate-limiting element in pliotosyntlietic
incorporation of carbon in sand pine grown on acid-washed sand,
it had its most pronounced affects on the fixation of carbon
in sugars, the principal assimilate and translocate in sand pine,
and presumably, in some organic acid precursors of amino acid
synthesis .

A N-P ratio of 1:2 produced the heaviest seedlings, a low
green-nongreen tissue ratio, and the highest level of photoassimi lated
C in ethanol extract and sugars. Except for the green-nongreen
tissue ratio, the most beneficial combination of N and P included
the high level of N (75.0 ppm) and the high level of P (150.0 ppm),
i.e., the N^p^ nutrient regime recommended by R. H. Brendemuehl
(personal communication) . The green-nongreen tissue ratio was
most nearly balanced using the same ratio of N to P. Tlie amount of
ladioactivity in the amino acid fraction of green tissue extract
was slightly higher for the N2P2 than the N^Pj regime. In organic
acids, N^P^ was second best to N2^P2-

lliese data suggest that, of the treatments used, the ratio of
one part N to 2 parts P is best for sand pine seedlings on infertile
sands. Furthermore, they suggest that procedures involving
assimilation of labelled compounds can be used for rapid deter-
mination of optimum nutrient regimes involving more than just two

elements .

Biochemical Pathways Involved

Tlie highest proportion of photoassimilated was contained
in the sugar fraction of the ethanol extract from green tissue.
Fructose, glucose, and to a lesser extent galactose, contained

i/irtually all tlie labelled caibon in sugars. Organic acids contained

some C principally as malic acid. Virtually no activity was
contained in the amino acid fraction, but the little there was,
was identified as components leading to, and involved in, the urea
cycle .

Sugars constitute the major photosynthates leading to forma-
tion of organic and amino acids and eventually to protein synthesis.
'Ihese data suggested that the glycolytic pathway, Calvin cycle, and
the tricarboxylic acid cycle were m.echanisms for interconversion
of these compounds. Furthermore, they suggested that activity of
key enzymes along these biochemical pathways might provide an
index of superior sand pine tree growth.

Objectives of the final phase of the study were to determine ;

1) whether differences exist in the isoenzymes of glucose-6-
phosphate dehydrogenase and malate dehydrogenase and in proteins
of superior and nonsuperior sand pine trees and their half-sibling,
seedling progeny, and 2) if the soils on which seedlings were
grov\'n altered their morphology or the migration rate and activity
of isoenzymes and protein bands.

Seeds collected from parent superior and nonsuperior sand
pines were planted in Lakeland and Paola sands. Germination,
num.ber of cotyledons, and foliar color were recorded.

The protein extract from green tissue of parent plants and
half-siblings was separated by gel electrophoresis then stained

to develop isoenzymes of malate dehydrogenase, glucose-6-phosphate
dehydrogenase and protein bands. Stained gels were scanned and
measurements were made of the number and Rf value of bands and the
intensity of densitometer tracings.

Measurement of glucose-6-phosphate dehydrogenase isoenzymes
was limited to the 3 bands near the center of the gels. This
limitation was caused by degredation products (Bakay and Nyhan,
1969) and "nothing" dehydrogenase that produced false bands; and,
protein sulfhydryl groups (Dietz and Lubrano, 1967) and lightly
stained, unresolved proteins (Hall et al., 1969) that caused a
foggy, colored background at both extremes of the gels. Bakay
and Nyhan interpreted the 3 bands as subresolved glucose-6-
phosphate dehydrogenase isoenzyme and identified them, by rate of
migration, as fast, medium, and slow components. They were
treated as 3 distinct isoenzymes.

Quantified data were subjected to analysis of variance;
selected orthogonal and single-degree-of-freedom comparisons
were made of soil and tree treatment main effects and the soil x
tree interaction.

Isoenzymes and proteins bands are separate entities. Any
one can act as a marker indicating genetic variation between
superior and nonsuperior trees. The Rf and activity of three
glucose-6-phosphate dehydrogenase and six malate dehydrogenase
isoenzymes and the Rf and staining intensity of 17 protein bands
were examined. To obviate repeated qualification to differentiate
between isoenzyme activity and protein band staining intensity,
the term "activity" is used hereafter with both isoenzymes and
protein bands .

To be meaningful, consistent differences have to exist in
tissue of superior and nonsuperior trees growing on the same
soil. Two individual comparisons differed statistically, but
examination of the data showed that neither was consistent for all
nonsuperior trees. For example, average Rf's for glucose-6-phosphate
dehydrogenase isoenzymes 1, 2, and 3, were significantly higher in
superior trees than in nonsuperior trees labelled D, but not in
nonsuperior trees A, .6, C, and E. Statistically, no meaningful
differences were found among parent trees or among their progeny for
these isoenzymes. None of the isoenzymes studied here provided an
index of superiority.

llie only indication of a possible genetic marker was
discovered in the raw data (Appendix Tables 9, 10, and 11). No
protein band existed between Rf 0.29 and 0.39 in superior parent
trees; other parent trees contained at least one band within this
range. The marker was absent among siblings.

The Rf and activity of some isoenzymes and protein bands
are affected by the soil in which trees grow as well as by genetic
factors. In some instances, both the band and the controlling
influence can be identified. Two orthogonal comparisons were made
among soils irrespective of superiority of tissue source. 'Fhe
first compared results obtained from Lakeland (L) with that from
Paola (P) sand. Half-sibling seedling tissue w.as used. Since
tissues were similar, the comparison was between soils developed under
thermic (L) and hyperthermic (P) conditions. Tlie natural range
of Ocala sand pine is limited almost exclusively to hyperthermic soils.

In this comparison the significant response obtained can be
attributed to soil.

Hie second comparison among soils measured the response from
half-sibling seedlings grouai on thermic and hypertliermic sand
(L+P) with that from parent trees growing i?i situ (0, for
original soils) on h)q5erthermic sands. The significant response
obtained can be attributed to genetic factors, half-sibling
vs parent, or to age, seedling vs grown tree, as well as to the
soil. Differences in age were discounted, however, because only
green tissue fi'om the current year's growth was used and because
the tissue served an identical function involving the same
enzymes and proteins in both trees and seedlings.

The Rf of malate dehydrogenase isoenzymes 2, 3, and 4 and of
protein bands 12 and 16 were significantly higher for seedlings
grown in L and P than for parents (0). Rf's in the L vs P
comparison did not differ significantly suggesting that differences
in the rate of migration of isoenzymes 2, 3, and 4 and of protein
bands 12 and 16 v/as influenced by a combination of soil and genetic
factors .

.Activity of malate dehydrogenase isoenzymes 2 and 3 was
significantly higher in seedlings grown in L than P, but activity
in the L+P vs 0 comparison did not differ significantly. This
suggests’ that their activity was influenced by the soil more than
by genetic factors or else the parent-half sibling comparison
would also have been significant. Activity of protein bands 7,

12, 14, 16, and 17 was significantly higher in L than P. Two
of these bands, 12 and 16, plus three additional bands, 2, 9, and

15 were significantly higher in the L+P vs 0 comparison. This
implies that activity of some protein bands, o.g., 12 and 16, was
influenced by soil and genetic factors, some by soil, e.g., 7,

14, and 17, and still others by genetic factors, e.g., 2, 9,
and 15.

Factors believed to have influenced the Rf or activity of
specific isoenzpie and protein bands are entered under "Remarks"
in Table 7 for future discussion.

The implication that the soil on which trees grew strongly
influenced the Rf and activity of isoenzymes and proteins was
substantiated by comparisons made of the soil x tree interaction.
Interpretation of results summarized in Table 7 is facilitated
by considering separately comparisons made under columns headed:
Lakeland vs Paola, Paola vs Original, and L vs P+0 (Lakeland vs
Paola plus Original) . Isoenzymes and protein bands ^re listed
numerically in the column to the left of the comparisons according
to migration rate; 1 was fastest. Consistency among superior and
among nonsuperior trees was essential to identify genetic markers
and to provide an index of superiority. It was also essential
to substantiate differences attributable to soils, genetic factors,
and soil plus genetic factors suggested by the comparisons made
among soils.

The original groupings for comparisons in the soil x tree
interaction were made to determine if anticipated differences in
isoenzymes and proteins between superior and nonsuperior tissue
could be detected in siblings grown on soils other than those on
which the parents grew. Here they serve to test the validity of
inferences suggested in the soils com.parison.

Table 7. --Comparisons of the soil x tree interaction showing the probability of a chance
occurrence and the soil on which the highest values were obtained

•H

•rH

<1>

WcD*

o£T

1-H

•H

1-3 3 +

> 3-

V— '

^ PI

O 3 <

< > 2

Cu i-i

V— ^

N— /

E-

•p

3 ^

,£) <D

t-i 3

I-H CO
<

'3 •

C O
rt 2:
aa

■X

/ \ / \ / N

O- 3-

V / \ /

XXX

CM bO rt m vO

P) PI l-J

\ f ^ '

XXX

V /

X X
X X

PI PI PI PI

X X X X X
X X X X X

PI ,p PI PI

X X X X X
X XXX

PI PI PI

X X X X
XXX

^ J

PI IPI PI

XXX
X X

PI PI ^ PI

V ' \ f '' — / V /

X X X X
XXX

PI PI PI

^ ^ \ — f \ — '

XXX

rH CM CO LO \0

•H

PI
' — /
X

r— I

CL,

•H

e>\°

•H

•H •

3 -g
bO 3

•H 3
CO 3

o\®

probability

(/)

•H

•P

<1>

•H

0
u

r-H

iJ to +

> a.

►J kJ

owe

< > 2

O. I-H

f-H

V !

3 U
,o (U
CM
y-^ 3
i-H to
<

CJl

cq|

01
D

2
W
C3

VO
■ 1
OJ
CO

CX, O

V ' \ ‘

* *

o
\ /

O O O D-

V — / c*-' \ /

■* * * 4!

* * *

2
* *

/■ — \
•-J

V — /
*

^ Cu J

V— ^ s-_> V / V /

' -J< ♦ -K -ic

•K *

1-3

V—/

\ /

Ph CU (X

* -K *

♦ -Jc

■K

a a

V ' N /

•J< -k
•k

•k

V /

•-3

'w'

•k

i-^CMtO’^lOsOr^COOCMtO^LOvO

Soil § Genetics

0
u

CIS

iJ w +

tn <C
> 2
)— I

I— (

Pl,

►J

V -

•rH

4->

+-)

ctS

to ID

•H

•rH

tJiJ-

•rH CaD*

4->

+->

(D rH r-H

• rH

•fH

•rH

C •H 'H

o o o

CJ3 CO CO

/ — \

r-^

/— N

r — N <• — N

/ s

h4 1-4

1-4 1-4

h4 »-4 h4

N— / V—/

V— '

N— ✓

v— ^ '

V— ' ^

4c 4e

4c 4c

4c 4c 4c

4c 4c

f —

/ — N

t — \

/■ — \

O- pH

Cl.

r-H

<. /

N— '

V /

N— /

4c 4c

+->

CQ (D

1-1 5^
r-» CO
<

pjI

c o
05 Z
CQ

cqI

hJ -3

'w/ '

■»« ♦

•Jc ^

*-J«^c-}e-lc4e^c^c-jc

J h4

♦

•jc

■):

1-3

'w/

•jc

V—/

•je

V /

♦

hJ J

V / V /

•Je •)«

•Jc

•Jt

•-4

V y

h4 1-3 h4 m4

♦ -Je -Jc 4:

4c ■)« 4c

^ J kJ

4c 4t * *

4c 4c 4c

*-3 iJ

4c 4c
4c

►J hJ

4c 4c
4c 4c

1-3

1-4 k4

V— /

4c 4c

(N^LOvOt^OOC^Oi-nCMtO^LONOr*^

+->

.-3

V /

*t3

4-1

Paola vs Original comparisons were made on sandy soils from
the sarr.e general, area. For this reason no consistent and signifi-
cant response should be expected in the Rf or activity of isoenzymes
or protein bands influenced primarily by soil. A response
attributable to .genetic factors might be anticipated in appropriately
labelled bands because the comparison is primarily between parent
vs half-sibling progeny.

Table 7 shews that the labels are correct. In siblings, Rf
of isoenzymes 2,, 3, and 4, and of protein bands 12 and 16, and
the activity of protein bands 2, 4, 8, 12, and 15, and, in parents,
activity of protein band 6 were highest by a statistically signifi-
cant degree. Prxotein bands 4, 6, and 8 were not labelled, but
all the rest bare a genetic or a genetic and soil designation.
Conversely, no significance was recorded for the activity of
malate dehydrogemase isoenzyme 2 and 3 or protein bands 7,

14, and 17, influenced by soil, or of band 16, labelled soil and
genetics. The only exception was in protein band 9 which bore a
genetic label but exliibited no significant oi' consistent response.

A comparison of Lakeland vs Paola plus Original was made to
examine differences in Rf and activity of isoenzymes and proteins
attributable to "native vs non-native" soils in superior trees.
Combining the data from Paola and Original soils also introduced
the element of genetic makeup, lialf-sibling vs parent, into the
comparison; thus it is not surprising that mixed results were
obtained. Tlie Rf and activity of many labelled and unlabelled
isoenzymes and proteins exhibited a significant response. In

every instance the Rf and activity were highest on Lakeland
sand .

Comparisons of Lakeland vs Paola examine the effect of a
native ;,md non-native soil on the Rf and activity of isoenzymes
and protein bands in progeny from superior and nonsuperior trees.
Individual comparisons were made for 1) superior trees, 2) non-
superior trees collectively, and 3) nonsuperior trees bearing a
common, identifying letter. Comparison 3 provides a statistical
breakdown of comparison 2 but is meaningless by itself.

The Rf and activity of all significantly affected isoenzymes
and most significantly affected protein bands in the 3 compari-
sons were higher on Lakeland than Paola sand for seedling from
superior and nonsuperior parents alike. With respect to soil,
genetic factors, and soil plus genetic factors, the data defy
interpretation. Had the effect of soil, genetic factors, and the
combined effect of soil and genetics on isoenzymes and protein
bands been anticipated, provision could have been made to clarify
results by use of backcrossed or control-pollinated stock or
possibly rooted cuttings from parents.

It is tempting and sometimes convenient to identify protein
bands with isoenzymes that I'eact similarly to a given stimulus by
direct comparison of gels. Both are protein. Gabriel and Wang
(1969) successfully accomplished staining for both protein and
enzyme on paired gels using triphenyltetrazolium. With nitroblue
tetrazolium the practice does not appear to be reliable. Malate
dehydrogenase isoenzymes 2 and 3 reacted similarly to protein
bands 12 and 16 in the L vs P and L+P vs 0 soils comparisons,

yet the Rf's of the isoenzymes averaged 48. 7 and 37.4 ivhile those
of the protein bands averaged 40.3 and 24.8. Possible application
of this method was further tested by developing ])rotein bands in
gels previously stained for isoenzymes and then comparing these
with gels stained either for isoenzymes or protein bands.

Although all the gels had been processed simultaneously in the
same electrophoresis chamber, the position of the bands did not
coincide. Development of isoenzymes and proteins in paired
halves of gels split lengthwise was not tried.

A clue to deciphering the consistently higli response to
Lakeland soil in Lakeland vs Paola plus Original and Lakeland vs
Paola comparisons was sought among measurements made of seedlings
prior to harvest. Seed germination vv’as consistently higher on
Paola than Lakeland sand (Figure 18). 'Hie difference might be
attributed to a response to origin of the soils; it seems more
probable that it reflects the influence of soil texture on moisture
available for imbibition by seeds planted at the surface. Texture
of Lakeland sand was coarse while that of Paola sand was medium.

No genetic variance was expected because seeds were from the
sam.e trees, and samples for planting were selected at random.

A comparison of cotyledon numbers (Table 8) showed no meaningful
differences in the proportional distribution between soils or
between superior and nonsuperior progeny. There was no apparent
difference in size of cotyledons.

Lakeland Sand

+->

•H

o w

•H

P
P
pH

to
•H P
•M T)
P

P W

6 O
P *H
P +J
bO P
o

•3 O
P fH
P

CO LO

N0I1VNIWH3D lN3D83d

•H

A to

Table 8 . --Distribution of half-sibling seedlings possessing 3 to 7
cotyledons

PARENT
TREE LO-

CATION

AVERAGE

NONSUPERIOR TREES

SUPERIOR

TREE

LAKELAND

SAND

120

193

199

PAOLA SAND

120

193

199

Color of the foliage '.vas fairly uniform on each soil, but it
differed markedly between soils. Color differences increased
with age indicating that possibly soil aeration and moisture in
Lakeland coarse vs Paola medium sand, but most probably the availability
of nutrients v;as primarily re:,ponsible . A comparison of colors
was made shortly before harvest against standards in liunsell
Color Charts for Plant Tissue. It showed that the foliage of
seedlings grown on Lakeland sand was dark greenish-yellow, approxi-
miately 7.5 GY 4/6 to 7.5 GY 5/6, while foliage on Paola sand
had a lighter hue, 2.5 GY 8/10 to 5 GY 7/8. No other differences
in the foliage were observed. Some fascicled needles developed
on all seedlings, and the needles were of about the same size.

Ihese data raise several questions: 1) What characteristics

of the soil differ sufficiently to alter color of seedling
foliage? 2) Are these differences in any way related to the
consistently high Rf and activity of isoenzymes and proteins
observed in seedlings raised on Lakeland soil?

Photosynthetic rate can be as much as 3 times higher in
dark green than light green leaves (Kramer and Kozlowski, 1960).

A corollary that serves to explain the consistently higher
activity of enzymes and proteins on Lakeland than Paola sand is
that the darker leaves produced more substrate for protein
synthesis because they contained more chlorophyll. Since seedlings
of genetically similar origin were raised under identical conditions
except for the soils, differences in chlorophyll content are
ascribed to available nutrients in the two soils. Hie possibility
exists that chlorophyll formation also could have been affected

by a deficiency or an excess of water in the soils, but,

considering the slight difference in texture between the

sands, and the care with which seedlings were raised, tlie probability

that either factor played a meaningful role seems extremely

remote .

Table 2 showed rhac Lakeland sand contained twice as much
total N, considerably more Mg, and more K than Paola sand. N
and Mg are constituents, of chlorophyll; a deficiency of either
causes chlorosis. The amount of N in soil is a major determinant
of leaf protein (Kynd and Noggle, 1945). In the plant, it is
an element essential for amino acid and protein synthesis.
Conceivably, the divergence in N between Lakeland and Pacla
sand could have modified the amino acid and enzyme components
of sand pine seedlings growing on them in a manner similar to
that reported in slash pine seedling tissue following addition
of N (Stanley and Smith, 1970), i.e., by altering isoenzyme
patterns. In this respect differences in available Mg could
also have had an effect. The Mg ion is also a cofactor in many
biochemical reactions including conversion of glucose-6-
phosphate to 3-lactone catalyzed by glucose-6-phosphate dehydrogenase
(Conn and Stumpf , 1964) , and influences the reactivity of at
least one glucose-6-phosphate dehydrogenase isoenzyme in some
animals (Hori and Matsui, 1967; Hori, Tsutom.u, and Matsui, 1967).
Aside from the difference in foliar color, no apparent syiTiptoms
of N or Mg deficiency were observed.

The K requirement of most plants is liigh. Bollard (1955)
'reported that dried leaves of healthy plants contain about 15,000

ppm. Brendemuehl (unpublished data) found that dried needles of
Choctawhatchee sand pine seedlings grovm in a Lakeland sand with
14 ppm available K contained 6,560 ppm K. So, although K accumulates
in needles of sand pine seedlings against a concentration gradient,
the relatively low level of ; vailable K in the Lakeland and Paola
sands of this study suggests that the sand pine seedlings on both
soils grew at suboptimum, but not necessarily deficient, levels
of K. Deficiency symptoms such as those characterized for
foliage of white pine (Hacskaylo, 1962), loblolly pine and
V'irginia pine (Sucoff, 1961) seedlings were not apparent.

Lakeland sand contained slightly less available Ca, 1/4 as
much available P, and 10 times the A1 of Paola sand. Sucoff
reported no deficiency symptoms in loblolly pines supplied with
0.8 ppm Ca nor in Virginia pines raised with as little as
0.2 ppm Ca. It seems unlikely, therefore, that the supply of Ca
was limiting in either Paola (44 ppm) or Lakeland (30 ppm) sand.

No deficiency symptoms were evident.

As in most acid sandhill soils, P availability was low. Tliis
was especially true in the Lakeland sand, pH 5.6, where the
presence of a comparatively high concentration of Al, 50.5 ppm,
suggests that P was fixed as the insoluble hydroxy-phosphate
of Al . Although the threshold concentration for P deficiency in
Ocala sand pine seedlings is unknowni for sandy soils, the data
suggest that it may lie between concentrations found on Lakeland
and Paola sand. Seedlings on Lakeland sand, 0.35 ppm P, evidenced
the dark green color characteristic of a P deficiency (Bonner
and Galston, 1952) while those on Paola sand, 1.45 ppm P, did not.

Tile relatively dai'k green color of seedlings grown on
Lakeland vs Paola soil may be attributed to a P deficiency and
to a greater availability of N and Mg. Because seedlings were
destroyed during preparation of acetone powders for protein
extraction, foliar analyses could not be made to corroborate
suppositions based upon foliar color and the soils' available
nutrient content. If plants were deficient in P, apparent
conflict exists.

P is essential in plant metabolism. Its high-energy bonds
provide a mechanism for stoi'age and energy transfer (Arnon,

1953). How, then, can plants growing in soil containing only
0.35 ppm of available P and signalling a P deficiency symptom
exhibit high protein and enzyme activity levels? Lacking cori'obora-
tive plant tissue analyses, the answer can only be conjectural.

Two possible e.xplanations follow.

Tl;e rate of P absorption reaches its maximum early in the
growth cycle; at a. time when the amount absorbed per unit of growth
is higlier than at later stages of growth (Dean and Fried, 1953).

In this study young seedlings were used. Tliey could have absorbed
enough P during their first month or so of existence to sustain
a normal metabolic rate for about a month. In the interim,
natural recycling of P and translocation from relatively old to
meristematic tissue could sustain sufficient sources of high
energy compounds to provide tlie increased protein synthesis
ascribed to comparatively high levels of available N and Mg found

in Lakeland sand.

Plants react differently to a P deficiency. Eaton (1949,

1950, and 1952) raised sunflowers, soybeans, and black mustard
plants in sand-solution culture with and without P. Analyses of
stem tissue extracts showed an accumulation of carbohydrates in
ill plants but a significant increase in water-soluble N,
nitrate, ammonia, amino, and amide, only occurred in soybean.
Accumulation of cai'bohydrates and nitrogen is symptomatic of
P deficiency and was explained by interference with a) protein
synthesis at the nitrate-reduction stage or b) protein synthesis
at the amide stage.

Sunflowers were grown for 64 days and, according to Eaton,
would have eventually matured under P-deficiency conditions,
presumably because they were able to utilize P of complex
organic compounds, 'fhe sand pines in this study seemed to
react similarly to sunflowers. Under field conditions both
varieties of sand pine grow to maturity on P-deficient soils.

If protein synthesis was not blocked by a P deficiency in the
period of growth studied, then differences in levels of N and Mg
could account for the consistently higher protein and enzyme
activity levels found in seedlings on Lakeland than on Paola sand.

A solution to the apparent dilemma offers opportunity for
further investigation into the differenctial uptake of available
nutrients under stress conditions imposed by deficiency of one
or more nutrients, as related to the affect of soil and plant
nutrients on protein production and enzyme activity. Hie continued
quest for a genetic marker and index of superior growth using
isoenzymes affords opportunities for future investigations.

TTie physiological response of superior tree selection to soils
and soil nutreint levels other than those found in the parent
habitat also offers opportunities for future research.

SUMMARY

'ITiis was a study of sand pine growth on sandhill soils. It
included rneasuren'ents of the affect of nutrient levels on
fixation of photoassiniilated and comparisons of isoenzyme
and protein migration rates and activity between superior and
nonsuperior trees and between their half-sibling, seedling
progeny grown on Lakeland and Paola sands.

1. Photoassimilation of ^^C02 in a closed system and at
steady-state conditions is directly related to length of
exposure .

2. Translocation of ^^C-labelled photos>nithate from green
seedling tissue was negligible for photoassimilation
periods of 10 min or less, lliereafter, the proportion

of labelled compounds remaining in green tissue decreased
in relation to nongreen tissue.

3. Young Ocala sand pine seedlings did not incorporate ^'^C02
in the dark,

4. Chlorophyll in ethanol extracts decreased the efficiency
a)id tile accuracy of liquid scintillation measurements.

Both were improved by use of quenching curves and
clarification of the extract with powdered activated
charcoal. Quenching curves were unique for each scintilla-
tion counter,

5. Ihe ethanol-soluble fraction of green tissue contained
most of the photoassimilated within 10 min. Sugars

contained 75%, organic acids 19%, and amino acids 6% of
the in this fraction.

6. N treatments seemed to affect chlorophyll formation, seedling
growth, and the photosyntlietic incoi'poration of ^'^C.

7. P appeared to be a rate- limiting element in the photos>Tithetic
incorporation of C in sugars and in some organic acid
precursors of amino acids.

8. A N-P ratio of 1:2 in complete nutrient solution
produced seedlings with a high ratio of nongreen to green
tissue and resulted in a high level of incorporation in
sugars .

9. The activity and Rf values of malate dehydrogenase and
glucose-6-phosphate dehydrogenase isoenzymes did not
provide an index for rapid growth in superior Ocala
sand pine tree selections or in their seedling progeny.

10. Absence of protein bands between Rf 0.29 and 0.39 in
superior trees was the only indication of a genetic
difference between superior and nonsuperior parent
trees. This genetic marker was not found among half-
sibling, seedling progeny.

11. Tlie activity and Rf of som.e malate dehydrogenase iso-
enzyme and protein bands were affected by the soil

on which seedlings were grown as well as by genetic
factors . Both the bands and the factors influencing

them were identified.

12. Procedures developed in this study may be applicable to
research seeking to detect genetic aberrations in trees
exhibiting a superior growth rate and to the development
of optimum levels of tree nutrition.

APPENDIX

" ->

!-■ 's^'l

'•■■ ?

Table 9. --Protein Rf measurements taken from half-sibling seedlings
grown on Lakeland coarse sand

TREE

Band

Super

Rf X

100 --

74 .

"'O

Table 9. --Continued

TREE

Band

Super .

Rf X

100---

. 00

100

Table 9. --Continued

TREE

Band

Super

Rf X

100

101

Table 10. --Protein Rf measurer^ents taken from half-sibling seedlings grown
on Paola sand

TREE

Super .

Rf X

100- -

. 87

102

Table 10 . --Continued

TREE

Band

1 on

Super

.60

11 «

103

Table 10. --Continued

TREE

Band

1 nn

Super

, 44

104

Table 11. --Protein Rf measurements taken from parent trees growing at their
original locations.

TRF.E

Band

Super

Rf X

100---

—

91.

105

Table 11 . --Continued

TREE

Band

1 nn

Super

■ 62

106

Table 11 . --Continued

TREE

Band

Super

RF X

100

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BIOGRAPHICAL SKETCH

Russell MacBain Burns was born August 25, 1926, at New
York, New York. In June, 1944, he was graduated from Haaren
High School. From 1944 until 1946 he served in the Infantry of
the United States Army in Europe. Following his discharge from
the Army, he attended the Associated Colleges of Upper New
York and Michigan State University and, in 1950, received his
degree of Bachelor of Science with a major in Forest Management.

He worked for the Southern Forest Experiment Station of the U.S.
Forest Service throughout the South, and while stationed at
Oxford, Mississippi, attended the University of Mississippi.

In 1959, he received the degree of Master of Science with a
major in Biology and a minor in Mycology. In 1966 he was selected
by the Forest Service for advanced training under the Government
Employees Training Act at the University of Florida. The Ph.D.
degree was received in August 1971, with a major in Soils and a
minor in Forest Physiology.

Russell MacBain Burns is married to the former Mildred Ann
Nastasia'and is the father of three children, Stephen, John, and
Russell. Memberships are held in Sigma Xi, The American Society
of Plant Physiologists, Soil Conservation Society of America, Society
of American Foresters, and the Florida Academy of Science.

116

I certify tliat I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.

August, 1971

Chai]

Professor, Forest Soils

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.

August, 1971

Professor, Forest Physiologist

I certify that 1 have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.

Robert G. Staley, Co-jGHairman

August, 1971

Professor, Soil Physicist

August, 1971 (‘4\

Charles C. Hortenstine
Associate Professor, Soil Chemist

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly presentation
and is fully adequate, in scope and quality, as a dissertation
for the degree of Doctor of Philosophy.

Wayne H. Smith

Associate Professor, Forester

August, 1971

August, 1971 /^/

Daniel 0. Spinl^

Assistant Dean, So:

August, 1971

Rayji^ond H . Brendemuehl
Principal Silviculturist, U.S,
Forest Service

This dissertation was submitted to the Dean of the

College of Agriculture and to the Graduate Council, and was
accepted as partial fulfillment of the requirements for the
degree of Doctor of Philosophy.

August, 1971

Dean, Graduate School

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