• TABLE OF CONTENTS
HIDE
 Title Page
 Acknowledgement
 Table of Contents
 List of Tables
 List of Figures
 Abstract
 Introduction
 Review of literature
 Material and methods
 Experiments and results
 Discussion
 Summary and conclusions
 Bibliography
 Biographical sketch














Title: Role of the root tip in development of enhanced rubidium uptake in washed, excised corn root tissue /
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Permanent Link: http://ufdc.ufl.edu/UF00097587/00001
 Material Information
Title: Role of the root tip in development of enhanced rubidium uptake in washed, excised corn root tissue /
Physical Description: 73 leaves : ill. ; 28 cm.
Language: English
Creator: Parrondo, Rolando Teodulo, 1937-
Publication Date: 1973
Copyright Date: 1973
 Subjects
Subject: Roots (Botany)   ( lcsh )
Corn   ( lcsh )
Botany thesis Ph. D
Dissertations, Academic -- Botany -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis (Ph. D.)--University of Florida, 1973.
Bibliography: Includes bibliographical references (leaves 69-72).
Additional Physical Form: Also available on World Wide Web
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by Rolando T. Parrondo.
 Record Information
Bibliographic ID: UF00097587
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000470637
oclc - 37863302
notis - ACN5423

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Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
    Table of Contents
        Page iii
        Page iv
    List of Tables
        Page v
    List of Figures
        Page vi
        Page vii
    Abstract
        Page viii
        Page ix
    Introduction
        Page 1
        Page 2
    Review of literature
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
    Material and methods
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
    Experiments and results
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
        Page 46
        Page 47
        Page 48
        Page 49
        Page 50
        Page 51
        Page 52
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
    Discussion
        Page 58
        Page 59
        Page 60
        Page 61
        Page 62
        Page 63
        Page 64
        Page 65
        Page 66
    Summary and conclusions
        Page 67
        Page 68
    Bibliography
        Page 69
        Page 70
        Page 71
        Page 72
    Biographical sketch
        Page 73
        Page 74
        Page 75
        Page 76
Full Text


















ROLE OF THE ROOT TIP IN DEVELOPMENT OF
ENHANCED RUBIDIUM UPTAKE IN WASHED, EXCISED
CORN ROOT TISSUE






By




ROLANDO T. PARRONDO


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
1973










ACKNOWLEDGEMENTS


The author expresses his sincere thanks to Dr. Richard C.

Smith for his guidance throughout the graduate program and for his

advice and help in the preparation of the manuscript. The help of

Drs. R. H. Biggs, D. S. Anthony, D. G. Griffin, III, and D. B. Ward

as committee members is also acknowledged. Special thanks are due to

Dr. T. E. Humphreys for reading the manuscript and for making many

helpful suggestions. Thanks are also due to the Botany Department

for providing the author with a graduate assistantship while pursuing

graduate studies. Lastly, his deep gratitude to his wife Cynthia for

having endured with patience the privations necessary to bring this

study to fruition.










TABLE OF CONTENTS


Page


ACKNOWLEDGEMENTS . . . . . . . . .

LIST OF TABLES . . . . . . . . .

LIST OF. FIGURES . . . . . . . . . .

ABSTRACT . . . . . . . . . . .

INTRODUCTION . . . . . . . . . .

REVIEW OF LITERATURE . . . . . . . .


Enhancement of Solute Uptake
Storage Tissue . . .
Enhancement of Solute Uptake
Stem and Leaf Tissue .
Enhancement of Solute Uptake
Root Tissue . . . .


by Disks of

by Excised

by Excised


MATERIAL AND METHODS . . . . . . . .

Plant Material . . . . . . . .
Preparation of Samples . . . . . .
Aging Procedure . . . . . . .
Determination of K Content . . . . .
Rubidium Absorption . . . . . .
Assay of Radioactivity . . . . . .

EXPERIMENTS AND RESULTS . . . . . . .


Development of Enhanced Rate of Rubidium
Absorption . . . . . . .
Effects of Environmental Stresses on the
Subsequent Development of Enhanced Rate
of Rubidium Uptake . . . . .


Development of Enhanced Rubidium Absorption Along
the Apical 75 mm of the Primary Root of Corn . 32
Effects of Aging Excised Root Tissue in CaSO4
Solution and in Water Saturated Atmosphere on
the Development of Enhanced Rb Uptake ..... .35
The Effect of Time of Excision of the Root Tip
on the Development of Enhanced Rb Uptake ... . 38


. . vi


vili


. . . 15


. . . 20






Page


Effects of Aging in the Presence of Excised
Root Tips and in Culture Solution . . . ... .43
Effects of Concentrated Growth Solution on
the Development of Enhanced Rb Uptake ...... .48
Induction and Development of Enhanced Rb
Absorption by Tray-grown and Solution-
grown Roots . . . . . . . . . . 51
Rb Uptake and K Efflux from Excised Corn
Root Segments . . . . . . . . . 54
Elongation of Excised Root Segments with
Time in CaSO4 Solution . . . . . . . 54

DISCUSSION . . . . .. .. . . . . . . 58

SUMMARY AND CONCLUSIONS . . . . . . . . . 67

LITERATURE CITED . . . . . .... . . . . 69


BIOGRAPHICAL SKETCH . . . . . . . .


. . 73













LIST OF TABLES


Page


Effects of desiccation on the rate of
absorption of Rb by excised corn root
tissue . . . . . . . . . . .


Table

1.












LIST OF FIGURES


Figure

1. Development of enhanced Rb absorption in
3-centimeter excised, tipless corn root
segments aged in CaSO4 solution . . .

2. Development of enhanced Rb absorption in
corn roots aged intact or aged as
3-centimeter excised, tipless segments

3. Development of enhanced Rb absorption in
3-centimeter excised, tipless corn root
segments aged at 3C as compared to 300C

4. Effect of a 30-minute cold pre-aging
treatment on the subsequent development
of enhanced Rb absorption . . . .


Page


. . 22



. . 24


Effect of osmotic stress during aging on the
development of enhanced Rb absorption . .


6. Effects of a 30-minute period of osmotic
stress on the subsequent development of
enhanced Rb absorption . . . .. . . 31

7. Development of enhanced Rb absorption by
I-centimeter excised segments taken
from three different positions along the
primary root of corn . . . . . . . 34

8. Development of enhanced Rb absorption in humid
air as compared to CaS04 solution . . . . 37

9. Effect of time of excision of root tips on
the development of enhanced Rb absorption ... 40


Effect of time of the upper and the lower
excision of 1-centimeter root segments on
the development of enhanced Rb absorption . ..

Effect of time of the upper and the lower
excision of 1-centimeter root segments on
the long-term development of enhanced Rb
absorption . . . . . . . . .


.






Page


Effect of the presence of root tips,
excised or attached, in the aging solution;
and the effect of old culture solution used
as the aging medium on the development of
enhanced Rb absorption . . . . . .

Effect of two different concentrations of
old culture solution used as the aging
solution on the development of enhanced Rb
absorption . . . . . .

Development of enhanced Rb absorption in
root segments from tray-grown versus
solution-grown seedlings . . . .

Development of enhanced Rb absorption
compared to changes in K content during aging


. 50



. 53


S. 56


Figure






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 Phi-losophy


ROLE OF THE ROOT TIP IN DEVELOPMENT OF
ENHANCED RUBIDIUM UPTAKE IN WASHED, EXCISED
CORN ROOT TISSUE


By

Rolando T. Parrondo

December, 1973


Chairman: Dr. Richard C. Smith
Major Department: Botany


"Low salt" excised corn root segments (DeKalb 805A) were used

throughout this study. Rb was used as a tracer for RbCl. When ex-

cised corn roots in which the apical 5 mm had been removed were washed

in a dilute, well-aerated, CaSO4 solution, the rate of Rb uptake in-

creased rapidly with duration of washing. When root segments were

washed in a water-saturated atmosphere, enhanced ion uptake did not

develop during the first hour, and developed only slightly afterwards.

Segments held in cold CaSO4 solution, or in a solution of CaSO4 and

PEG-6000, or mannitol of sufficient strength to lower the osmotic poten-

tial to -8 bars, did not show enhancement. However, once removed from

the stressing environment and placed in CaSO4 solution at 300C, these

segments developed a capacity for a higher rate of ion uptake similar

to controls. A study of the enhancement phenomenon in different por-

tions of the primary root revealed that this response decreased with

Increasing distance from the tip, and at 65 mm from the tip It was

absent. Segments taken 5 to 15 mm behind the tip developed the highest


viii






rate of uptake. Ion uptake in these segments attained a maximum rate

in 2 hours and remained constant thereafter.

When the terminal 5 mm of the root was left attached during aging,

but removed immediately before absorption, the rate of Rb uptake was

only 28 per cent of controls in which the tip was detached before

washing. When decapitated root segments were aged in the culture solu-

tion in which seedlings had grown during the previous 24-hour period,

the rate of uptake was 63 per cent of controls aged in fresh solution.

Agi.ng in concentrated culture solution was even more inhibitory to the

enhancement response. When segments were incubated in fresh solution

containing free-floating, freshly excised tips, the rate of uptake was

20 per cent less than in samples aged in solution containing no free

excised tips.

Tray-grown root segments showed a lag of 20 minutes before any

increase in uptake was apparent. This lag was shorter in solution-

grown roots. The evidence presented in this study shows that the en-

hancement in the rate of Rb uptake with time by excised corn roots is

strongly influenced by the root tip. Moreover, the results also indi-

cate that this action is mediated by an unidentified substance which

is synthesized by the root tip and translocated further back where it

inhibits Rb absorption.












INTRODUCTION


The use of excised root tissue in mineral absorption studies is

a common practice. There are several advantages of using isolated

tissue or organs. The ease of handling the material makes it possible

to establish better control over experimental procedures. Factors

influencing the absorption of substances by the root system of plants

are many. Moreover, the absorption by roots is also influenced by

other processes taking place in the plant. For example, the rate of

transpiration affects the rate at which water is absorbed by the roots.

By using excised roots, the study of absorption is limited to those

processes originating in the root itself, thus avoiding complicating

effects from other organs of the plant.

The advantages of using "low salt" roots in experiments measuring

rates of absorption are evident when one considers that in this tissue

accumulation takes precedence over translocation out of the root.

In using excised roots in uptake studies, it is desirable to reduce

translocation to a minimum. This is further accomplished by short

absorption periods. Epstein et al. (15) listed the advantages of

short-term solute absorption periods by plant tissue. However, it

is realized that difficulties are encountered when trying to relate

results obtained in this manner to processes taking place in intact

plants in nature. Pitman (37) cautioned against assuming that the

uptake by low salt roots is the same as the uptake by plants in

nature. Even though the soil in which a plant is growing may be poor







in nutrients, trace quantities of these elements are present, and

plants are known to "mine" the medium in which they are growing.

Even though it has been known that different parts of the root

respond differently to the accumulation and translocation of minerals,

relatively little attention has been paid to the rate of uptake by

different segments of the root. It is known that the root tip is

morphologically, as well as physiologically, different from the more

mature parts of the root. Moreover, it is known that hormone synthesis

takes place in the root meristem, yet little work has been done to

assess the role of the root tip in physiological processes taking

place distal to the meristem. The root as an organ has been character-

ized as being less complex than the stem. While this may be true

morphologically, physiologically the root is very complex. It was

shown in a recent study by Leonard and Hanson (24) that the capacity

of excised corn root tissue to absorb ions increased with time of in-

cubation in a well-aerated CaSO4 solution. This phenomenon was also

observed in our laboratory in connection with studies dealing with

recovery from physiological drought. In the present study, this in-

crease in the rate of mineral uptake with time which we call the aging

response ("washing" by Leonard and Hanson) was further investigated.

The role of the root tip in the development of this response is speci-

fically examined and its mediation by an endogenous inhibitor is

proposed.












REVIEW OF LITERATURE


Enhancement of Solute Uptake by
Disks of Storage Tissue


Enhanced uptake was first observed in disks of storage tissue

during aging in aerated water, or CaS04 solution (3, 47). This in-

crease in uptake was accompanied by an increase in the rate of respira-

tion (47). Asprey (3) was the first to show clear evidence of enhanced

ion absorption capacity by disks of potato and beets aged in an aerated

solution. Parallel with this increase in ion absorption, there was an

increase in respiration (26, 47). Laties (22) attributed the in-

creased respiration rates in aged disks of storage tissue to the

development of vigorous phosphorylation. Other workers have contended

that the increase in respiration is due to increased protein synthesis

(2). Ellis and MacDonald (11) have reported an increased rate of

leucine incorporation into proteins in beet disks with time in aging

solution. The same workers have reported also an increase in nucleic

acid synthesis. In a later report, MacDonald et al. (29) showed that

puromycin inhibited the development of Cl absorption capacity in aging

disks of storage tissue. There seems to be a general increase in all

aspects of metabolism with time. Anderson (1) stated that the aging

response in storage tissue was due to the breaking of dormancy.

Bryant and Ap Rees (8) summarized the aging phenomenon in slices of

storage tissue as "...a reversal of changes that accompany dormancy."







Enhancement of Solute Uptake by
Excised Stem and Leaf Tissue


More recent studies have shown that other plant tissues are also

capable of increasing their capacity to absorb substances from the

surrounding medium when incubated in well-aerated solutions of vari-

ous salts or water. Bieleski (5) isolated vascular bundle tissue

from celery petioles and aged it in CaSO4 solution for 20 hours.

After 4 to 10 hours of aging, the rate of phosphate uptake rose to

50 times that of fresh tissue. The sucrose accumulation rate was 3

to 8 times that of fresh controls. Bieleski suggested that the low

affinity uptake mechanism of solute absorption was present in fresh

tissue, while the high affinity mechanism developed during aging of

the tissue. Hancock (18, 19) arrived at the same conclusion for 3-o-

methylglucose (a non-metabolizable derivative of glucose) absorption

by squash hypocotyls. The formation of the high affinity mechanism

was not affected by light conditions, shaking, or the absence of

CaSO4 from the solution (49). A typical response curve obtained in

these tests can be described as a lag of 2 hours, then a rapid in-

crease in uptake up to 12 hours followed by a constant rate thereafter.

Palmer and Loughman (34) have reported that incubation of pea

stem segments in potassium maleate buffer caused an increase in the

rate of phosphate absorption. At the same time the rate of respira-

tion decreased. Cotton and sunflower stem segments incubated in the

same medium failed to show any increase in the rate of phosphate ab-

sorption. They showed a doubling in the rate of Rb absorption by pea

stem segments after 18 hours of incubation. In a later study, Palmer

and Blackman (35) reported that the enhancement in phosphate uptake

which developed during washing could be prevented by the addition of





5
2,4-0 to the washing solution. Leonard and Hanson (24) confirmed the

inhibitory effects of 2,4-0 and IAA on the development of enhanced

phosphate uptake. However, they also reported that washing in the

presence of 2,4,6-T, a substance which has little auxin activity, was

even more inhibitory.

Rains (41) noted an increased absorption of K by bean stem slices

aged for 20 hours in CaSO4 solution at 300C. He showed that this

enhancement in K absorption was prevented if the tissue was incubated

at 40C. Addition of benzyladenine (an analog of kinetin) to the ab-

sorption medium inhibited the enhancement of K absorption. The same

effects of benzyladenine have also been shown in aging disks of

tobacco leaves (44). In a later report, Rains and Floyd (42) reported

that the increased K absorption rate in aged bean stem slices was

promoted by Ca. Aging of bean stem slices in the presence of cyclo-

heximide prevented the enhancement of K uptake, thus indicating that

inhibition of protein synthesis also prevented the increase in K up-

take developed during agirg. The process controlling the increased

ion uptake in bean stem tissue appeared to be different from the

process in storate tissue in as far as there was no increase in the

rate of oxygen uptake. On the contrary, respiration decreased during

the first 4 hours of aging (16).

Macklon and Higinbotham (30) reported a considerable increase in

K and NO3 content in excised segments of pea epicotyls immersed in

complete nutrient solution for 72 hours, as compared to intact, etio-

lated tissue. During this time, there was an increase in cell electro-

potential differences. Although the increase in ion uptake showed a

lag of 6 -to 8 hours, the difference in cell potential increased




6

rapidly. They considered the increase in potential difference a pre-

requisite for the rapid ion accumulation. Incubation of sufficient

duration in water, prior to placing in solutions containing K, elimi-

nated the lag in K uptake. Sacher (43) reported an increase in up-

take of orotic acid, glucose, and phenylalanine from 5-to 50-fold in

bean endocarp after aging the tissue in water for 24 hours. Auxin

(NAA) largely prevented this enhancement in substrate uptake.


Enhancement of Solute Uptake by Excised Root Tissue


Enhanced solute uptake has also been reported for non-dormant,

excised root tissue. Tanada (48) using segments of mung bean roots

showed an enhancement in Rb uptake with time by samples incubated in

solutions containing Ca. No increase in uptake was shown when Ca

was absent. In the presence of Ca, the uptake of Rb was not immedi-

ately stimulated. There was a lag period of about 10 minutes before

the rate of absorption gradually increased and then proceeded linearly

at a rate much faster than the initial rate. In mung bean, the enhance-

ment of Rb uptake was most pronounced in the 2.5 to 5.0 mm zone from

the root tip. Tanada attributed the enhanced Rb capacity to effects

of Ca on the availability of binding sites in the membrane-bound

carrier. The role of Ca in ion transport by plant tissue has been

emphasized by Epstein (13).

Cereals, particularly barley, have been favorite plants for

mineral uptake studies. Pitman (39) reported that when excised barley

roots were kept in 0.5 mM CaSOq solution for 2 to 4 hours, this tissue

took up more K than either intact plants or freshly excised roots.

He attributed the net increase in K uptake to an increased selectivity







of K over Na, and to increased K efflux from the tissue. However,

no difference in tracer uptake was reported. In a recent study

Pitman et al. (40) reported on the changes in electropotential dif-

ference between excised barley roots and the dilute CaS04 solution

in which they were aging. After 6 to 8 hours of aging, the potential

difference increased from 65 millivolts to 185 millivolts. As pre-

viously reported, there was no difference in tracer uptake between

fresh and aged tissue. They suggested 2 possible hypotheses to

explain this effect of aging on potential difference: (1) that cutting

exposed plasmadesmata which are leaky initially, but which seal in

time, and (2) that some internal factor, such as hormones, have a

regulatory effect on cell potential; an influence which dissipates

with time after excision. Leonard and Hanson (24) reported a 150 per

cent increase in the rate of phosphate absorption by barley root

segments after 3 hours of washing in aerated 0.2 mM CaCl2 solution,

and a 200 per cent increase in phosphate absorption by oat root tissue

incubated for the same period of time under the same conditions.

There are several reports of increased rate of mineral absorption

in excised corn root tissue. Brown and Cartwright (7), using apical

segments of corn roots in which cells were not fully elongated, showed

that increased Rb uptake was due to higher protein content in expand-

ing cells. Handley et al. (20) using segments of corn root tissue

taken 1.8 to 3.8 mm from the root tip,showed that aging increased the

rate of Rb uptake over freshly excised tissue. When pre-incubated for

2 hours in CaCl2, the rate of absorption remained constant. They

attributed this phenomenon to a dual effect of Ca on Rb uptake: an

initial inhibitory effect, followed by a stimulation with time. A







similar effect of Ca on the rate of K uptake in corn root tissue,

but not in barley, was reported by Elzam and Hodges (12).

Laties and Budd (23) reported a 20-fold increase in Cl uptake by

aged corn root steles over fresh steles, while the increase in aged

cortex tissue was only 2.5 times that of freshly separated cortex.

They attributed this difference in absorption between fresh and aged

steles to a leaky condition of freshly isolated steles. That some

stelar cells are normally leaky was proposed earlier by Crafts and

Broyer (10). In a later study, Luttge and Laties (27) reported a

10-fold increase in K absorption by aged corn root steles. They con-

cluded that this increase in uptake in aged steles was due to develop-

ment of a high affinity mechanism of solute uptake (14). Yu and

Kramer (49,50 ) reported a higher content of Rb in excised corn root

tissue than in intact roots after 23 hours of absorption. However,

these workers were not able to confirm the early findings of Laties

and co-workers on the increased capacity of uptake by aged, isolated

steles over the cortex. In a recent study, Hall et al. (17) reported

a greater increase in the rate of Cl uptake by isolated steles than

by cortex of corn roots, after 24 hours of aging. However, the in-

crease in uptake by isolated steles was much less when the seedlings

were grown under sterile conditions.

Leonard and Hanson (24) have reported a 280 per cent increase in

the rate of phosphate absorption by excised corn root tissue that had

been aged in 0.2 mM CaCI2 solution for 3 hours before absorption.

This enhanced rate of uptake was prevented by anaerobiosis, low

temperatures, metabolic inhibitors, and several plant hormones. An

enhanced uptake capacity appeared to be general, since the rate of





9
absorption of several substances was also increased during 2 hours of

washing in dilute CaC12 solution. The presence of Ca in the washing

solution was not necessary since incubation in distilled water gave

similar results. After a lag of 30 minutes, the tissue showed a

constant rate of increase which continued for 4 hours before leveling

off. Analysis of membrane-bound ATPase showed an increase with time

in washing (25). Electron microscopy of subcellular structures of

fresh and washed tissue showed no detectable changes due to washing.

Since efflux of ions has been reported to take place readily

(21, 31, 36, 38), some workers have attributed the increase in absorp-

tion to the loss of minerals during aging (39). However, Leonard and

Hanson (24) reported net accumulation of K after the tissue was washed

at 300C, but not in freshly excised tissue. Handley et al. (20)

showed no change in K content in corn root tissue after 6 hours of

aging in solution.

The influence of microorganisms on the development of enhanced

solute uptake by excised tissue has been reported by several workers.

Palmer (33) and MacDonald (28) have shown that in storage tissue, non-

sterile conditions reduced considerably the rate of uptake. However,

Bowen and Rovira (6) reported that uptake of 32P by tomato and clover

tissue was higher when plants were grown in non-sterile conditions.

Barber and Frankenburg (4) reported rates of uptake of phosphate and

Rb by excised barley roots to be higher in plants grown in non-sterile

conditions than in plants grown under sterile conditions. Leonard

and Hanson (24) aged corn root segments in the presence of chloram-

phenicol and found no difference in the rate of phosphate uptake be-

tween control and treated samples during 8 hours of incubation.













MATERIAL AND METHODS


Plant Material


Solution-grown Roots

The tissue was obtained from the primary root of 4-day-old seed-

lings of Zea mays (DeKalb 805A) grown according to the method de-

scribed by Smith et al. (46), but slightly modified. One hundred

grams of dry seeds were placed in a 2-liter Erlenmeyer flask contain-

ing 1 liter of distilled water, shaken and the water drained. This

washing procedure was repeated twice. The seeds were surface ster-

ilized in 200 ml of 15 per cent Clorox (5 per cent sodium hypochlorite)

to which a few drops of liquid detergent were added. After 5 minutes

of gentle shaking, the detergent-Clorox solution was poured off and

the seeds rinsed with distilled water 10 times. In the last 3 rinses

the flask was allowed to overflow. The flask containing the seeds was

filled to the 1800 ml mark with distilled water and the seeds were

soaked 18 to 20 hours with vigorous aeration. Then the seeds were

rinsed 3 times with distilled water, and the flask was filled to the

1800 ml mark with distilled water and placed in the incubator. After

2 to 3 more hours of soaking, the water was decanted, and the seeds

rinsed with distilled water. At this stage most of the seeds showed

the coleorhiza protruding through the seed coat. Seeds not showing

the coleorhiza at the end of the soaking period were discarded. The

remaining seeds were planted between 2 layers of boiled, coarse-grade








cheesecloth. The cheesecloth was supported by a stainless steel

mesh and hung at the top of a 4-liter beaker filled with 0.2 mM CaSO4

solution. The seeds were distributed among 4 beakers. A sintered

glass aeration tube was placed inside a glass jacket and this assembly

placed in each beaker for aeration purposes. The corners of the

cheesecloth were placed to wick the solution, but the seeds were held

a few mm above the aerated solution. The beakers were covered with

a watch glass and placed in the incubator. After 24 hours the top

cheesecloth was removed and discarded and the seeds washed thoroughly

with distilled water. At this stage most of the roots were about 2

centimeters long and were extending into the solution. The seeds

were placed over fresh 0.2 mM CaS04 solution in a clean beaker, the

watch glass replaced, and the beakers placed back in the incubator.

The third day the same procedure was followed, except that the watch

glass was not replaced because of the length of the coleoptiles. The

roots were harvested the fourth day. At this stage the roots were

usually 12 to 15 cm long.


Tray-grown Roots

The seeds were sterilized as described above. After the tenth

rinse with distilled water, the seeds were planted in Pyrex trays,

with the embryo side in contact with several layers of white paper

towel saturated with 0.2 mM CaS04 solution. The trays were covered

with transparent food wrap in which holes were punched to allow gas

exchange. The trays were placed in the incubator at 280C and left

undisturbed until harvest time. The roots were harvested the fourth

day. At this stage the roots were usually 8 to 10 cm long.







Preparation of Samples


Root segments of either 3 cm or 1 cm, taken 5 mm from the root

tip, were the experimental material. Immediately after excision,

the segments were placed in a small fiberglass basket in a 2-liter

beaker containing 0.5 mM CaS04. They were then agitated for 1 minute

to remove material from damaged cells, removed, gently blotted,

weighed to 0.1 mg, and placed in fiberglass bags for handling.

Each bag of 10 or 15 root segments was prepared separately and as

rapidly as possible, requiring 6 to 8 minutes.


Aging Procedure


The aging solution consisted of 4 liters of 0.5 mM CaSO4 solution

prepared in 4-liter beakers. The temperature of the aging solution

was maintained at 300C in a water bath. The solution was vigorously

aerated throughout the aging process. Hereafter these are referred

to as standard aging conditions. Seven or eight samples, each con-

taining 10 or 15 root segments were aged in each beaker before dis-

carding the solution.


Determination of K Content


Samples used for K content determination were rinsed 3 times in

distilled water after aging, placed in crucibles and ashed at 5000C.

The ashes were dissolved in distilled water and diluted to exactly

25 ml in a volumetric flask. The K content was determined by using

a Flame Emission Spectrophotometer (Beckman B and DU).







Rubidium Absorption


Preparation of the Solution

The absorption solution consisted of 0.1 mM RbCl and 0.5 mM

CaCI2, prepared in a 2-liter volumetric flask. Enough 8Rb was added

to give a counting rate of approximately 10,000 cpm per pmole of

RbCI. Four-hundred ml volumes were poured in 500 ml wide mouth

Erlenmeyer flasks and placed in a water bath at 300C. The flask con-

tents were aerated vigorously throughout the absorption period. At

the end of 10 minutes of absorption, the concentration of Rb in the

solution had decreased less than 1 per cent.


Uptake Procedure

Thirty seconds before absorption started, each sample was removed

from the aging solution, and swung around to remove excess water from

the bag. At exactly zero time the sample was dropped in the absorp-

tion solution. After 10 minutes, the absorption of Rb was stopped by

dropping the sample in cold (30C) exchange solution, containing 5.0 mM

KCI and 0.5 mM CaCl2. After 3 rinses, the samples were submerged for

30 minutes in 4 liters of aerated exchange solution kept at 30C. At

the end of exchange the samples were rinsed 3 times in distilled water

and the tissue placed in stainless steel planchets for determination

of radioactivity.


Assay of Radioactivity


The planchets containing the samples were ashed at 5000C. After

cooling, the ashes were dissolved in water, and a drop of detergent

solution added to break the surface tension. The samples were evap-




14

orated to dryness under low heat on a hot plate.. The activity of the

samples was determined by a low-background counting system in which

the sample detector was a G-M tube with window thickness of 150
2
pg/cm2. The background averaged less than 2 cpm. Each sample was

counted at least 2 times to 10,000 counts, or for 10 minutes, which-

ever came first. Each experiment was repeated at least 2 times with

similar results. The data shown, however, are from single experiments.













EXPERIMENTS AND RESULTS


Development of Enhanced Rate of Rubidium Absorption


When 3-cm segments of corn root, in which the terminal 5 mm of

the tip had been removed, were aged in well-aerated 0.5 mM CaSO4

solution, the rate of Rb absorption increased rapidly (Figure 1) for

several hours until it reached a maximum and then approached a con-

stant value. In some experiments, however, after reaching a maximum,

the rate of uptake declined. This was attributed to a depletion of

food in the excised tissue. Figure 1 shows that after 8 hours of

incubation, the rate of absorption of Rb was 13 times that of freshly

excised tissue.

To test whether the development of the enhanced rate of absorp-

tion was due to excision, an experiment was designed in which one

group of roots were excised and aged under standard conditions. In

another group, intact roots were incubated in 0.5 mM CaSO4 at 300C,

and then segments excised before determining the rate of absorption.

At different periods of time after aging (incubation) started, a

sample from each treatment was taken out and its rate of Rb uptake

determined. The results of this experiment are shown in Figure 2.

In both groups the capacity to absorb Rb was increased. However, the

rate of absorption was greater in roots which had been excised.

Excision amplified this response, but was not the cause of it. Six

hours after transfer to fresh CaSO4 solution, segments from roots














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20
which had been incubated intact were absorbing Rb at a rate 6 times

that of samples which had been given zero aging time. After 6 hours

of aging, excised segments were absorbing at a rate 9.5 times that

of excised roots at zero aging time.


Effects of Environmental Stresses on the Subsequent
Development of Enhanced Rate of Rubidium Uptake


Two of the most common environmental stresses encountered by

plants in nature are low temperatures and drought.


Effects of Low Temperature

Three-centimeter excised root segments were aged for different

periods of time in aging solution at either 30C or 30 C before Rb

absorption. Both solutions were well aerated. Figure 3 shows the

results of this experiment. When excised roots were aged at 30C, the

enhancement of Rb uptake was largely prevented, and at the end of 4

hours of aging it was still only 17 per cent of-samples aged at 300C

for the same period of time. This is consistent with the concept

that mineral absorption by roots is metabolically dependent since low

temperature is known to reduce metabolism. Figure 4 shows the results

of an experiment in which root segments were pre-treated in cold

CaSO4 solution for 30 minutes before aging under standard conditions.

Thirty minutes of cold treatment at 30C did not prevent the subsequent

development of enhanced Rb uptake. Cold-treated samples showed a

slightly lower rate of uptake than control samples. This has been

interpreted as being due to chilling injury.
















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Effects of Water Stress

Drought conditions were simulated in the laboratory in two dif-

ferent ways: by desiccation and by incubation in solutions of low

osmotic potential.

Excised root segments were suspended over 500 ml of 5.0 M NaCl

in 2-liter flasks where they remained for various time periods.

Control samples were suspended over distilled water. The flasks had

earlier been sealed with Parafilm, and placed in a water bath at 300C

for 24 hours before the experiment started to allow water vapor equil-

ibration between air and solution. For treatment, the samples were

suspended just above the solution. The time lapse between opening

and re-sealing the flask was no more than a few seconds, thus minimiz-

ing changes in humidity inside the flask. The results are presented

in Table 1. Roots held over 5.0 M NaCI showed a lower rate of Rb

absorption than controls held over distilled water. Samples held over

NaCI solution lost as much as 52.9 per cent of their fresh weight in

6 hours, while control samples lost less than 5 per cent of their

fresh weight. A lower rate of Rb uptake by control samples after I

hour over distilled water was frequently observed, but the cause of

it could not be assessed. Two significant results are shown in this

experiment: (1) desiccation reduced the rate of Rb absorption, and

(2) control samples over distilled water did not show the rapid in-

crease in uptake (aging response) shown by roots incubated in aerated

CaS04 solution. This shows that the "aging" response is not elicited

by time alone. The loss of weight by control samples was not con-

sidered to be stressing to the tissue.

Two different osmotica were used to simulate drought: Polyethelene












Table 1. Effects of desiccation on the rate of absorption of Rb
by excised corn root tissue. (Absorption by freshly
excised root segments was 0.108 pmoles/g./10 min.)



Treatment
Distilled water 5M NaCI

Rb Rb
Time % loss Absorption % loss of Absorption
(hours) of wt. (pmol./g. 10 min.) wt. (pmol./g 10 min.)


1 2.4 0.050 11.9 0.023

2 0 0.161 22.5 0.038

4 3.1 0.141 40.0 0.041

6 4.7 0.119 52.9 0.024







glycol, with an average molecular weight of 6000 (PEG-6000), and

mannitol. PEG-6000 does not plasmolyse the tissue, while mannitol

is known to cause plasmolysis (32). Excised root segments, 3 cm

long, were aged for various time periods in 0.5 mM CaSO4, or in

CaSOq containing 212 g/1 of PEG-6000, or 55 g/1 mannitol. The con-

centration of the osmoticum was adjusted so that the osmotic potential

of the solution was approximately -8 bars. The results with both

osmotica are similar, but only the results with PEG-6000 are shown in

Figure 5. Incubation of root segments in a solution of low osmotic

potential prevented the increase in rate of Rb uptake, while samples

aged in CaSOq solution without osmoticum showed a rapid increase in

the rate of Rb uptake with time.

In the next experiment, samples were incubated for 30 minutes in

0.5 mM CaSO4, or CaSO plus mannitol or PEG-6000 as described in the

previous experiment. After this stressing period, the samples were

rinsed 3 times with 0.5 mM CaS04 to remove the osmoticum, and were

transferred to the aging solution under standard conditions for dif-

ferent periods of time before determining the rate of Rb uptake. The

results are presented in Figure 6. The most significant feature is

the fact that water stress did not prevent the subsequent development

of enhanced Rb uptake. At the end of 2 hours of aging, the Rb uptake

by mannitol-treated segments was 3 times the initial rate of stressed

samples. However, this rate was only about 50 per cent of that of the

controls. PEG-treated samples showed a greater enhancement than mannitol-

treated samples. This difference Is attributed to damage due to plas-

molysis by mannitol. The difference in uptake by PEG-treated samples and

control Indicates that there was a certain amount of tissue damage

























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32

by this osmoticum. It is not known whether this damage was due to

direct toxicity or due to damage to membranes or other subcellular

structures.


Development of Enhanced Rubidium Absorption along the
Apical 75 mm of the Primary Root of Corn


This experiment was conducted to determine to what extent the

enhanced capacity of Rb uptake developed at different distances from

the apex of the primary root of corn. Due to the presence of lateral

root primordia near the base of the root, this study was limited to

the apical 75 mm of the root. One cm segments were taken 5-15,

35-45, and 65-75 mm behind the root tip. Each sample contained 15

root segments with an average weight of 120 mg. These segments were

aged in 0.5 mM CaSO4 for periods up to 9 hours before measuring the

rate of Rb uptake. The results of this experimentare presented in

Figure 7. Segments taken 5 to 15 mm behind the root tip showed the

highest increase in the capacity to absorb Rb. The maximum rate was

reached in 2 hours, and remained constant thereafter. The basal

segments had the highest initial rate of absorption (0.31 pmoles/g-

10 min.), and this rate was not affected by the aging treatment. The

middle segments, which initially absorbed at the same rate as the

apical segments, increased their rate of uptake to the level of the

basal segments, but did not go any higher. This experiment was re-

peated 3 times, with similar results being obtained. On the basis

of these results, all subsequent experiments utilized only the apical

segment, 5 to 15 mm behind the root apex.


















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Effects of Aqing Excised Root Tissue in CaSOS1 Solution
and in Water Saturated Atmosphere on the Development
of Enhanced Rb Uptake


The results of Table 1 show that segments placed over distilled

water developed only a small increase in their capacity for Rb uptake

with time. This was further investigated. One-centimeter segments

were aged in a water saturated environment or in aerated 0.5 mM CaSO4

under standard conditions. Experimental samples were placed in

2-liter Erlenmeyer flasks containing 500 ml of distilled water. The

flasks were sealed with Parafilm and placed in a water bath at 300C,

24 hours before the experiment started, as described before. One

sample was placed in each flask. To insure that the tissue did not

suffer any significant loss of water, the fiberglass bag containing

the tissue was covered with a piece of damp cheesecloth. In the flask,

the edges of the cheesecloth touched the water, but the bag containing

the roots remained several centimeters above the water. After hanging

the sample inside, the mouth of the flask was sealed immediately with

a new piece of Parafilm. The oxygen in the flask was calculated to be

300 times that consumed by respiration of the tissue during a 4-hour

period. The results of this experiment are shown in Figure 8. Samples

aged in CaSO4 solution showed the usual enhancement of Rb uptake with

time, reaching the maximum rate in 2 hours. However, little enhance-

ment of the absorption capacity occurred in experimental samples held

over distilled water. After 1 hour, the rate of uptake had not changed.

After 2 hours, the rate of uptake was only 15 per cent of controls.

A sample taken out after 2 hours of aging over distilled water and

immersed in aerated CaSO4 solution for 1 hour showed an increase in

the rate of absorption similar to controls aged in CaSO4 solution















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throughout the aging period. Changes in weight in experimental

samples were less than 1 per cent and in some cases the tissue

gained weight.


The Effect of Time of Excision of the Root Tip
on the Development of Enhanced Rb Uptake


Apical root segments were aged with and without the root tip

attached, but the root tips were excised in all cases before Rb ab-

sorption. Figure 9 shows the results of this experiment. The presence

of the root tip during aging largely prevented the enhancement of Rb

absorption. Segments aged without the tip rapidly increased their

rate of uptake, most of it taking place within the first hour of aging.

At the end of the first hour of aging the rate of uptake by experi-

mental samples was only 10 per cent of controls, and at the end of

8 hours of aging the rate was still only 24 per cent of controls.

In order to determine further the role of the root tip in prevent-

ing the enhancement of Rb uptake, another experiment was designed in

which the roots were aged under 4 different experimental conditions.

(1) Roots excised and aged with the terminal 5 mm of the root intact

during aging but removed immediately before absorption. (2) Roots

treated as in 1, except that the terminal 5 mm of the root tip was

removed before aging. (3) Root tip (5 mm) removed,the seedling

incubated in 0.5 mM CaS04, and root segments excised just prior to Rb

absorption. (4) Intact seedlings incubated in a culture solution in

which seedlings had grown for 24 hours before the experiment, and

samples excised before Rb absorption. At different time periods a

sample from each group was taken out and the rate of Rb uptake deter-

mined. The results are shown in Figure 10. As before, excised segments














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Figure 10. Effect of time of the upper and the lower excision of
1-centimeter root segments on the development of enhanced
Rb absorption. The root segments were-taken 5 to 15 mm
from the tip. In cases where the tissue was aged with the
tip attached, the tip was removed after the aging period.













-c- Excised, tip removed
-o- Intact, tip removed
-- Excised, with tip attached
--,- Control, no aging


I I I


AGING TIME (Hours)


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43

aged with the tip showed lower rates of absorption than segments whose

tip was excised before aging. Rates of uptake into segments from

seedlings incubated without the root tip were close to those of

excised, tipless segments. Moreover, in identical experiments in

which the aging time was extended over a 24-hour period, segments

from seedlings aged without the root tip showed a higher rate of Rb

uptake than excised, tipless segments 12 hours after aging started.

The enhancement of the rate of Rb uptake with time in aging solu-

tion under standard conditions was followed over a 24-hour period in

intact roots, excised roots with intact tip, and in excised, tipless

roots. The purpose of this experiment was to determine whether the

rate of Rb uptake in intact seedlings declined with time after reach-

ing a maximum. At different times during the aging period, a sample

from each treatment was taken and the rate of Rb absorption determined.

Figure 11 shows the results of this experiment. Root segments from

intact seedlings showed an enhancement in uptake, but after 12 hours

the rate declined. Excised roots did not show this decline. Samples

in which the root tip was left attached, whether the root remained

attached to the seedling during aging or not, showed a lower enhance-

ment than excised, tipless roots.


Effects of Aging in the Presence of Excised Root
Tips and in Culture Solution


One-centimeter segments were aged in 400 ml volumes of test solu-

tion for 2 hours before determining the rate of Rb uptake. The roots

were treated in 5 different ways as described in Figure 12. Each

sample was duplicated and the average of the 2 is given. Variation

between replicate samples is indicated by vertical lines. Only a
















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48

slight enhancement of uptake was obtained with segments aged with the

tip intact: about one fourth of that obtained with samples aged

without tips. Aging of tipless root segments in growth solution was

also inhibitory to the enhanced Rb uptake. Their rate of uptake was

two-thirds of controls without the tip aged in fresh CaSO4 solution.

The enhancement of Rb uptake was also less pronounced in samples aged

with root tips floating in the aging solution.


Effects of Concentrated Growth Solution on
the Development of Enhanced Rb Uptake


in the previous experiment it was shown that the solution in

which seedlings had been growing for 24 hours was inhibitory to the

development of enhanced rate of Rb uptake. In the following experiment,

this growth solution was concentrated by reduction to one-half of its

original volume by lyophilization, to determine if concentrating this

medium would permit even less enhancement of Rb uptake by excised,

tipless corn root segments. Since the concentration of CaS04 in the

growth solution was 0.2 mM, and that of the aging solution was 0.5 mM,

a reduction in the volume of the growth solution by one-half would not

increase the CaSO4 concentration above that of the aging solution.

The samples were aged for 2 hours before determining the rate of Rb

uptake. One sample was aged in growth solution, a second sample was

aged in concentrated growth solution. A third sample was aged in

fresh 0.5 mM CaS04. A fourth sample was used to determine uptake by

freshly excised tissue. Each sample was duplicated, and the average

of the 2 is given in Figure 13. Variation between replicate samples

is indicated by vertical lines. The enhancement in uptake by root

tissue incubated in growth solution was again less than in control
















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51

samples aged in fresh CaSO4 solution. The prevention of the enhance-

ment of Rb uptake, however, was more pronounced in,tissue aged in

concentrated growth solution than in tissue aged in normal strength

growth solution.


Induction and Development of Enhanced Rb Absorption
by Tray-grown and Solution-grown Roots


The effects of aging on Rb uptake in one-centimeter root segments

from tray-grown versus solution-grown seedlings are shown in Figure 14.

Freshly excised root tissue of both solution-grown and tray-grown

seedlings showed initially the same rate of absorption (0.020 moles/

g .10 min.). Tray-grown roots showed a 20-minute lag period before an

increase in the rate of absorption became apparent. An even longer

lag has been reported for phosphate absorption (24). Solution-grown

roots always showed an increase in the rate of uptake within 10 minutes

after aging began. However, this increased rate of Rb uptake after

10 minutes was not always as pronounced as that shown in Figure 14.

After 3 hours of aging, tray-grown tissue was absorbing at a rate

13 times that of freshly excised tissue, whereas solution-grown tissue

was absorbing at a rate 23 times that of freshly excised tissue. The

difference in absorption between tray and solution grown tissue with

time in aging, presented in Figure 14, was not always as great as

shown here, however, solution-grown roots consistently showed a

higher rate of absorption with time in aging solution than tray-grown

tissue.



















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Rb Uptake and K Efflux from Excised
Corn Root Segments


The following experiment was carried out to determine the extent

of K loss from corn root segments and the degree to which this loss

is influenced by aging. Excised root segments were divided into 2

groups. Both groups were aged under standard conditions. Two samples

were taken out at each sampling during aging. One sample was used

to determine the rate of Rb absorption, and the other sample was used

to determine the K content. The K in the roots would necessarily

have to come from the seed since K was not supplied at any time during

the growth of the seedlings. The results are presented in Figure 15.

The data show that the K content of the tissue remained constant

during 8 hours of aging. The enhanced Rb uptake with time was not

accompanied by an increased efflux of K from the tissue. Although K

efflux might be significant in tissue with high salt content, tissue

with low salt content such as that used in these experiments did not

lose significant quantities of K during aging in CaSO4 solution.


Elongation of Excised Root Segments with
Time in CaSO, Solution


To determine whether further elongation occurred after excision

and during aging, the change in length of I-cm root segments, taken

5 to 15 mm behind the root tip was checked. These segments were accu-

rately cut with the aid of a stereoscopic microscope. After 4 hours

of aging in CaSO4 solution under standard conditions, the increase in

length was less than 1 mm. Thus It can be assumed that most of the

cells in the segments had completed their elongation, or conditions

for further cell elongation were lacking. Therefore, the enhancement






















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63


- I Ir





57
in the rate of Rb uptake of such proportions as shown in this study

could not be attributed to cell growth.













DISCUSSION


There have been scattered reports in the literature (20, 24, 48)

of a time-dependent enhanced capacity for mineral uptake by excised

roots. Many of these reports have been limited to isolated observa-

tions made in the course of other studies relating'to mineral absorp-

tion. Leonard and Hanson were the first to publish the results of

experiments aimed at determining the causes of this increase in uptake.

At the same time this phenomenon was being observed in our laboratory.

It is of particular interest since so much work has been published on

the subject of mineral absorption using excised roots of grasses. A

clear understanding of this phenomenon is needed to relate uptake

by excised tissue to intact plants. In this study an attempt has been

made to determine the nature of this enhancement, as well as the extent

of its development along the primary root. An effort has also been

made to determine the cause or causes which induce this enhancement

and the locus in the plant where it originates.

The mechanism of increased mineral uptake in corn root tissue

does not seem to be the same as that operating in slices of storage

tissue, the time dependence curves are different. In storage tissue,

a lag time of 24 hours has been reported before an increase in uptake

was apparent (47). In excised, solution-grown corn roots an increase

in uptake was evident after 10 minutes of aging. In tray-grown roots,

a lag of 20 minutes was required before increased uptake could be

detected. Leonard and Hanson reported a 30-minute lag in excised corn







roots (24). In corn root tissue the increase in uptake reached a

maximum in 2 to 4 hours, while in potato tuber slices the rate of

phosphate uptake was still increasing after 30 hours of aging (26).

The increase in uptake by storage tissue is accompanied by an increase

in respiration, typical of tissue breaking dormancy (26). In corn

root tissue the rate of respiration remains constant (24).

The enhanced rate of Rb uptake was not considered to be due to

microbial contamination. Even though no attempt was made at determin-

ing the bacterial count in the aging solution, there is evidence which

indicates that microorganisms were not responsible for the enhancement.

If microorganisms were responsible for increased uptake, the rate would

continue to rise as the bacterial population continues to increase.

However, the rate of Rb uptake reached a maximum in 2 hours and remained

constant thereafter. Further evidence published by others (24) showed

that incubation of corn roots in the presence of chloramphenicol at

bacteriostatic concentrations did not have any effect on the develop-

ment of enhanced mineral uptake capacity.

The response of Rb uptake to aging could not be attributed to

increased K efflux from the tissue. The K content of excised roots

remained constant during 8 hours of aging in CaSO4 solution under

standard conditions. Similar findings have been reported for phosphate

uptake by Leonard and Hanson (24), and for Rb uptake by Handley et al.

(20).

Whatever the cause, or causes, it appears that the increase in

uptake is dependent on the metabolism of the cell. This is indicated

by the fact that when excised roots were aged at low temperature at

which metabolism is minimal, the enhancement of Rb uptake was reduced





60

(Figure 3), or prevented (24). However, when the tissue was returned

to 300C the enhancement proceeded rapidly (Figure 4). Further evidence

which indicates that this process is metabolically dependent is given

by results from water-stressed tissue. In tissue under water stress,

the enhancement of Rb uptake was prevented. Excision does not appear

to be the cause of this phenomenon since roots from intact seedlings

when transferred to fresh CaS04 solution showed a similar enhancement,

although not quite as high as when roots were excised before aging.

Leonard and Hanson did not observe any difference in the rate of up-

take by samples in which the roots were cut into several segments from

samples in which the root was not subdivided.

The potential for enhancement was not manifested along the entire

length of the primary root of corn, but rather it was limited to the

portion of the root near the root apex (Figure 7). Canning and Kramer

(9) have shown that in corn root the ability to accumulate minerals is

highest at a distance of 20 to 25 mm from the apex and decreases to-

ward the base of the root. Smith (45) has shown that exudation volume

as well as Rb output was higher in apical segments than in basal seg-

ments. It is of no surprise then that enhancement of Rb uptake also

varies along the length of the root. There are certain peculiarities

in the way in which different segments of the corn root respond to the

aging effect that are worthy of comment. Freshly excised root segments

taken 5 to 15 mm behind the tip had a rate of uptake of only 25 per

cent of freshly excised segments taken 65 to 75 mm from the tip. At

the end of 2 hours of incubation, however, the apical segments had

increased their uptake dramatically, while basal segments continued

to absorb at the Initial rate. Intermediate segments taken 35 to 45 mm





61

from the tip initially absorbed at the same rate as apical segments,

but their highest rate of uptake after 5 hours of aging was essen-

tially the same as that of basal segments. It appears that the

ability to respond to aging decreased with distance from the apex.

Moreover, the rate of Rb absorption appears to be permanently set at

a lower level once the tissue has reached some particular stage of

development. This difference in response could not be attributed to

cell elongation after excision since elongation of the segments was

less than 10 per cent during the time that the rate of Rb uptake

reached a maximum.

The increase in uptake by segments from intact seedlings when

transferred to fresh CaSO4 solution suggested that the enhanced rate

of Rb uptake may be due to the leaching of an inhibitor which builds

up in the culture solution with time. It was possible to test this

hypothesis by aging root segments in an atmosphere in which leaching

could not take place. The results shown in Figure 9 lend support to

this hypothesis. Root segments aged above water showed a much lower

rate of Rb absorption than segments aged in CaSO4 solution. However,

when tissue held in the air was immersed in CaS04 solution for 1 hour,

enhancement similar to that of samples aged in CaSO4 solution occurred.

The lower rate of absorption by samples aged in air can hardly be

attributed to desiccation since the tissue did not lose more than 5

per cent of the fresh weight. The oxygen supply was calculated to be

300 times that required by the tissue for the longest period of time

that a sample was kept in the flask.

If enhancement of the rate of Rb absorption'by corn roots is due

to leaching out of an inhibitor, it should be possible to identify




62

the source of the inhibitor. It would have to be synthesized in situ

or it must come from the shoot or root tip. If the inhibitor is

synthesized in the region where it exerts its regulatory influence,

tipless segments and tipless seedlings would not have increased their

uptake with time in the aging solution, since the synthesis of this

inhibitor presumably should continue after excision of the roots.

Also the fact that the response to aging decreased with distance from

the root apex suggests that the control of enhanced absorption may

come from the root tip (support for this hypothesis is shown in

Figure 10). When root segments were aged with the tip intact, but

removed before absorption, the enhancement of Rb uptake was much less

than when the tips were removed prior to aging. Further evidence was

shown in Figure 11. When the terminal 5 mm of the primary root of

intact corn seedlings was removed and the decapitated roots of the

otherwise intact plants were incubated in CaSOq solution, the uptake

was higher than when excised roots were aged with tips intact.

If an endogenous inhibitor leaks from the root tip the rate of Rb

uptake by root segments from intact seedlings, as well as by excised

roots with intact tips, should decrease with time, if left in the

aging solution long enough for the concentration of the inhibitor to

build up. The results in Figure 12 confirm this expectation for in-

tact seedlings, but not for excised roots with intact tips. The

decrease in uptake by root segments from intact seedlings after 12

hours of incubation in CaS04 solution could hardly be attributed to

the lack of food in the tissue since the roots were not excised until

after the aging period. Moreover, roots from seedlings left in the

solution where they had grown for 24 hours before the experiment





63

showed no enhancement in the rate of Rb uptake (Figure 10). Tipless

root segments aged for 2 hours in fresh CaS04 solution absorbed less

Rb in 10 minutes when the tips were present in the solution than

when the tips were absent (Figure 13). However, if the tips remained

attached during aging, the enhanced rate of Rb uptake was much less

than when the tips were floating in the solution. This difference

probably resulted from dilution of the inhibitor or discontinuity of

the supply when the tip was severed. Low penetration of the membrane

by the inhibitor may have also contributed to this difference. In

intact tissue, the inhibitor probably is translocated from the tip to

other parts of the root in the symplastic continuum.

More evidence in support of the presence of an inhibitor is shown

in Figures 13 and 14. Incubation of excised, tipless root segments in

the culture solution in which the seedlings had been grown for 24

hours prior to the experiment was inhibitory to the development of

enhanced Rb uptake (Figure 13). Root tissue aged in this solution

for 2 hours absorbed only 66 per cent as much as samples aged for the

same period of time in fresh solution. Concentrating the growth

solution further inhibited the rate of Rb uptake by excised, tipless

root tissue (Figure 14).

The mechanism by which this inhibitor regulates the rate of mineral

uptake is not yet known. Elucidation of the mechanism will have to

await future research, possibly until the identity of the inhibitor

is known. Several possibilities are suggested here. First, it may

be that the increase in uptake is brought about by an increase in the

membrane-bound carrier, the synthesis of which is held in check by

the Inhibitor. Another possibility IS that the Inhibitor itself




64

combines with the carrier in the membrane, rendering it inoperative.

The inhibitor-carrier association may be weak so that leaching of the

inhibitor takes place readily when the roots are immersed in fresh

solution. The first possibility is supported by the findings of

Leonard and Hanson, who reported that an increase in protein content

accompanied the enhancement of phosphate uptake in corn root tissue.

This mechanism would require a lag period before the increase in the

rate of uptake is apparent. Leonard and Hanson have reported a lag

period of 30 minutes before the rate of phosphate uptake increased.

A 20-minute lag is reported here for Rb absorption by tray-grown roots

(Figure 15), but not by solution-grown roots. It may be significant

that most of the tissue used by Leonard and Hanson was tray-grown.

In our laboratory, solution-grown roots showed an increase in uptake

within 10 minutes after aging started. It would seem that this short

period of time would be adequate for leaching out of a water-soluble

compound (the hypothetical inhibitor) whereas it might not be long

enough for appreciable protein synthesis. One difficulty in interpret-

ing these data is that an increase in total protein content may not

necessarily mean an increase in the carrier. This will be difficult

to show since the identity of the carrier is not yet known. The

second suggestion receives some support from the experiments with

solution-grown tissue by the fact that the lag period was 10 minutes

or less. As soon as the inhibitor-carrier association is broken, the

carrier is free to bind with a Rb ion, thus an immediate increase in

the rate of uptake. Still another possibility Is that the inhibitor

may act by interfering with the delivery of energy required for active

transport. Leonard and Hanson have shown an increase in membrane-

bound ATPase activity with aging In corn root tissue.





65

Leonard and Hanson attributed the increase in phosphate uptake

to an increase in protein synthesis brought about by submersion of

the root tissue. As evidence for this they showed that freshly

excised solution-grown roots absorbed at a much higher rate than tray-

grown roots. They showed this difference in the absorption of several

ions, including Rb. However, this difference in freshly excised

tissue between tray and solution grown roots could not be verified in

our laboratory for Rb absorption. It could very well be that this

difference was due to differences in handling the tissue, or perhaps

to the genetic differences in the corn strains. The difference in Rb

uptake with time in CaSO4 solution between tray-grown and solution-

grown tissue (Figure 15) may have been due to the fact that roots

grown in trays had been under the influence of higher concentrations

of the inhibitor during the growth period. In solution-grown roots,

the CaSO4 solution was changed every 24 hours, and the seedlings were

rinsed with distilled water each time that the solution was changed.

Tray-grown roots were left undisturbed during the entire growth period.

Tray-grown roots may not be able to overcome the effects of the in-

hibitor to the extent that solution-grown roots do. Conditions in the

tray, however, resemble more closely the natural environment than

growing the seedlings in solution.

It is not known whether both mechanisms of ion uptake reported by

Epstein et al. (14) are influenced by this inhibitor. The Rb con-

centration of the absorption solution used in these experiments (0.1 mM)

was in the range of the high affinity mechanism. Thus it appears that

at least the high affinity mechanism is involved in the aging effect.

The significance of this response of corn roots is viewed in





66

regard to its possible survival value to the plant. A mechanism of

this kind, whereby the rate of mineral uptake is increased rapidly

during short periods of high water content in the soil when the

maximum amount of dissolved nutrients are available, would be of a

definite advantage to the plant.













SUMMARY AND CONCLUSIONS


The rate of mineral uptake by excised corn root tissue has been

shown to increase following aging in a solution of CaSO4. This in-

creased capacity for solute uptake was prevented by conditions which

lowered the metabolism of the tissue. However, once the tissue was

returned to normal metabolic conditions, the enhancement of Rb uptake

was restored. Excision does not appear to cause this enhancement,

since roots from intact seedlings when transferred to fresh CaSO4

solution showed a similar response, although somewhat smaller. How-

ever, the rate of Rb uptake began declining in tissue from intact

seedlings 10 hours after being transferred to fresh CaSO4 solution.

It appears that the Rb absorption capacity in corn root tissue is

limited by the presence of an inhibitor, and the increased rate of

uptake is caused by the leaching of this substance when the tissue is

immersed in aerated CaSO4 solution. This conclusion is strengthened

by the fact that enhanced capacity for Rb absorption is prevented or

greatly reduced when excised roots were aged in humid air. However,

when roots aged in air were subsequently submerged in aerated CaSO4

solution, the capacity for Rb uptake developed similar to that developed

in samples aged in solution without pre-aging in the air. The idea

that a substance inhibitory to Rb uptake leaches from the roots is

also supported by the fact that the CaSO4 solution In which roots had

been growing partially prevented the enhancement of Rb uptake during

aging. Concentrating the solution where seedlings had grown further

reduced the response to aging.





68

The experimental data obtained in this investigation have been

interpreted to indicate that some water-soluble substancess, orig-

inating in the root tip prevents the enhancement response of tissues

farther back in the root. This hypothesis is specifically supported

by the following: (1) The aging response decreased as the distance

from the tip increased; (2) at a distance of 65 to 75 mm from the tip

there was no response at all; (3) aging the segments with the tip

attached largely prevented'the response; (4) aging tipless roots

which had been left attached to the plant showed a response similar

to that of excised, tipless roots.

The response to aging could not be attributed to Rb absorption

by microorganisms, or to increased efflux of K from the tissue. The

data presented in this investigation strongly suggest that the enhanced

rate of Rb absorption with time by excised corn root tissue was not

due to submersion itself, but to the leaching out of a water-soluble

endogenous inhibitor which occurs when the tissue is submerged.













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BIOGRAPHICAL SKETCH


Rolando T. Parrondo was born in Camaguey, Cuba, on December 23,

1937.. He attended high school at Colegio Pinson, in Cuba. In 1961,

he came to the United States to attend Andrew Junior College, in

Cuthbert, Georgia. In 1962, he attended Auburn University, in Auburn,

Alabama, and later transferred to George Peabody College for Teachers,

in Nashville, Tennessee, where he received a B.S. degree in Biology.

Before returning to the University for graduate studies, he taught

biology and chemistry at Englewood High School in Jacksonville, Florida.

In 1968, he entered graduate school at the University of Florida. In

1969, he received an M.S. degree in Botany. From 1969, to the present

time, he has pursued work toward the degree of Doctor of Philosophy.

While a graduate student he was a teaching assistant in general botany,

general biology, and plant physiology. He is a member of the Botanical

Society of America, and the American Society of Plant Physiologists.

Rolando T. Parrondo is married to the former Cynthia Cora Melton.







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.




Richard C. Smith, Chairman
Associate Professor of Botany


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.




Robert H. Biggs
Professor of Fruit Crops


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.




Da a G. Griffin, III /
Associate Professors f Botany


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.




David S. Anthony
Professor of Botany






This dissertation was submitted to the Dean of the College of Agricul-
ture and to the Graduate Council, and was accepted as partial fulfill-
ment of the requirements for the degree of Doctor of Philosophy.

December, 1973


Dean, Graduate School




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