Group Title: Geotropism and transport of indoleacetic acid in normal and ageotropic Zea Mays L. /
Title: Geotropism and transport of indoleacetic acid in normal and ageotropic Zea Mays L.
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 Material Information
Title: Geotropism and transport of indoleacetic acid in normal and ageotropic Zea Mays L.
Physical Description: ix, 137 leaves : ; 28 cm.
Language: English
Creator: Holmsen, Theodore Waage, 1930-
Publication Date: 1961
Copyright Date: 1961
Subject: Geotropism   ( lcsh )
Corn   ( lcsh )
Soil Science thesis Ph. D
Dissertations, Academic -- Soil Science -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
Thesis: Thesis (Ph. D.)--University of Florida, 1961.
Bibliography: Includes bibliographical references (leaves 126-135).
Additional Physical Form: Also available on World Wide Web
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by Theodore Waage Holmsen.
 Record Information
Bibliographic ID: UF00097984
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 - 000469626
oclc - 36814411
notis - ACN4355


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nl' l .1*.r i....:I)R OF PHILOSOPHY

I.r.- 1961


The author expresses appreciation to Dr. H. J. Teas

for guidance in early phases of his graduate study; to Dr.

A. L. Koch for assuming direction of this research in its

final phases; to Dr. G. R. Noggle for providing facilities

in the Department of Botany for the research; to Dr. P. H.

Senn for facilitating graduate study in the Department of

Agronomy; to Dr. M. J. Soule for effective criticism of this

manuscript; to Drs. W. O. Ash and A. E. Brandt for advice on

design and analysis of the experiments; to Dr. Poul Larsen

for permission to cite from his unpublished manuscripts; to

Dr. E. Homer for corn seed and facilities for field experi-

ments; to the General Biological Supply House, Inc. for a

scholarship: and to the United States Atomic Energy Commis-

sion for financial support of the research.

My wife Claire Holmsen, not only assisted measurably

in many of the experiments and in preparing this manuscript

but also efficiently assumed most of the responsibilities of

raising our family during the writer's graduate study.

Without her help and confidence this study would never have

been completed.


4a p/?


,'CKI OL.E'DCEIIE T . . . . . .

LIST OF TABLES . . . . . .

LI T OF FIGUFE . . . . .

IIFTRODUCTI . . . . . .


Th.; G--o ropic F.eaction . . . .

Chemical Inhibition of G'otropi.m .

Auxin Transport . . . . .


Active Transport vs. Fereaion .

Dispo.siton of ,Auzin in Tissue . .

Lazy Corn--.n Aqgeotropic ut ant .


;.latcrials and I1,cthods . . . .

Flant production

breathing strength and growth of stem?
Geotropic rcactior of la:y and normal corn

Result ts . . . . . . . . .

Brealzinq Ettienath and gro'.:th of sr.EcrTi
Geotropic reaction of lazy and normal corn
Grow'.th habit of lazy corn plants


F aq:'
. . . . ii

. . . . iiL

. . . 4


. . . . 19

. . . . 21

. . . . 23

. . . . 24
-/ -



Discussion . . . . ... . . . . .. 44


Methods . . . . . . . . . . 47

Results . . . . . . . . . . 49

Discussion . . . . ... . . . . . 57

TRANSPORT OF IAA . . . . . . . . ... 60

Materials and Methods . . . . . . .. 60

Results . . . . . . . . . . . 64

Losses of radioactivity from tissue blocks
Characteristics of lateral transport
Effect of gravity on lateral transport
Effect of gravity on polar transport
Characteristics of polar transport

Discussion . . . . . . . . . . 90


Methods . . . . . . . . ... . . 94

Results . . . . . . . . 95

Effect of gravity and NP
Effect of gravity and temperature
Upper half vs. lower half

Discussion . . . ... . . . . . . 116

SUMMARY . . . . . . . . ... . . 121

BIBLIOGRAPHY . . . . . . . . ... .. . 125

APPENDIX . . . . . . . . .. . . . 136



Table Fage
1. Compounds -'riich Inhibit Straight Growtch and.or
Geotropic Bending . . . . . . 14

2. Breaking Strength of First Generation Inbred Corn
Segregating for the Laz: Character ..... 39

3. Height of First Generation Inbred Corn Segre-
gating for the Lazy Character . . . ... 41

4. The Effect of Various Reagents on the Angle of
Bend of nodal Sections of Second Inbred
Generation Corn . . .... . ...... 503

5. The Effect of lP on the ..ngle of Bend per uode
of Stem Seg.ments from Sibling normal and Lazy
Corn Plants . ... . . . . ...... 53

6. The Effect of Iu.. on the Angle of Bending per
[lode of StTem Segqmients from Normal Corn Plants .. 5:3

7. The Effect of Time on the VAmount of Radioactiv.ity
Femaining in Tissue Blocks from Corn Stms after
Infiltration with IAA 2-C14 . . . . . . 65

8. Lateral Transport of Radioactive IAA through
Horizontally Placed Corn Stear Blocks during an
Eight-Hour Period ...... . . . . 67

9. Calculation of the Diffusion Constant, D. of IlA
Based on Lateral Transport through Corn Stem
Tissue Blocks . . . . . . . . . 69

10. Lateral Transport of Padioactvi., I.AA in Blocks of
Corn Sten Tissue in the Horizontal-Above Configu-
ration as a Function of rlodal rozition . . 73

11. Effect of Tissue Orientation before Transport on
Lateral Transport of Radioacti.-c IAA . . . 74


Table Page
12. The Effect of Gravity on Lateral Transport of
Radioactive IAA in Tissue Blocks from Stems of
Normal and Lazy Corn . . . . . .. 76

13. Lateral Transport of Radioactive IAA in Tissue
Blocks of Normal and Lazy Corn Stems as Affected
by Gravity and as Compared to Polar Transport 7

14. Lateral Transport of IAA 2-C14 in Horizontal
Tissue Blocks from the Periphery of Corn Stem
Nodes . . . . .... .. . . . . 80

15. Transport of IAA in the Polar and Lateral Di-
rections in Blocks of Normal and Lazy Corn Stem
Tissue as Affected by Gravity . . . ... 83

16. Influence of Gravity on Polar Transport of IAA
in Blocks of Normal Corn Stem Tissue . . . 86

17. The Influence of Gravity on Polar Transport of
IAA in Corn Stem Tissue Blocks Infiltrated with
DNP, NP, Buffer, or Air . . . . ... 88

18. Comparison of Polar and Anti-Polar Transport in
Tissue Blocks from Normal and Lazy Corn Stem
Nodes . . . . . . . . ... .. . 89

19. Total Radioactivity in Tissue Blocks from Normal
and Lazy Corn Stems after Three Hours Transport 99

20. Parameters of E = 100 Rekrt Se-kst Obtained
from the Data Shown in Figures 11 to 14 . . 103

21. The Effect of Gravity on the Form of Radioactive
IAA in Tissue Blocks from Normal and Lazy Corn
Stems Infiltrated with NP and Buffer . . .. 106

22. Parameters of the Equation E = 100 Re-krt -
Se-kst Obtained from the Data of the Experiment
Summarized in Table 23 . . . . . ... 108

LIST OF T/-BLES---Continued

Tabl,- P3age
2). The- tff-ctL of Grav'.ty and T-'r- pe-rature on the
Fo-rm of Rad ioacti'.'ve I;AA in Tissuc- Blocks fr om
[Iorm3al Corn t . . . . . . 110

2-. r.el-as- of Cloi- CElated radrjio;ati': I.A from
Corn tlodc Ti -sue Blocks into 5 : 1,-4 LIA at
25 and IL-.A-free Soluti.--ns at L'- C . . . 113

25. Peleaase .of Radioact. '. I;AA from !Iodal Tissue
Blo;kc from Normnal and Lazy Corn St'ams into
Deionized after r 10- ,M r4. 10 ;i EP. and
5 x 10 plus 4 1 [ HP aft.-,r 20 Hours
Caution in Wjater . . . . . . ... 117

'.' 1


Figure Page
1. Third generation sibling lazy and normal corn
plants grown in the greenhouse . . . .. 26

2. The geotropic reaction in normal corn stems re-
sults from growth in an area just distal to the
nodes . . . . .. . . . . . 30

3. Crossing plan and utilization of inbred lines
segregating for the lazy character . . .. 34

4. The uninhibited growth habit of lazy corn plants 45

-5. Corn node segments in plastic foam 96 hours
after horizontal placement of the segments . 51

6. Response of nodal stem segments of lazy corn to
various concentrations of NP . . . ... 56

J. Response of nodal stem segments of normal corn
to various concentrations of IAA . . . .. 56

8. Configurations of blocks of corn stem tissue and
agar to study transport of IAA through the
tissue . . . . . . . . ... .. . 63

9. Time-course of lateral transport of radioactive
IAA in horizontally placed corn stem tissue
blocks . . . . . . . . ... .. . 68

10. Time-course of release of radioactivity from
tissue blocks of corn stem nodes into water or
10-4 M IAA . . . . . . . . ... 97

11. Time-course of elution of radioactive IAA from
tissue blocks of normal corn stems infiltrated
with buffer . . . . . . . . . 100

v i

LIST OF FICUiRE.--Contlnnucd

Figure Page
12. TLmn--cour:c of Jlution of radioactivt'. IAA from
tissue block of nor-Tal corn stemn irnfiltrated
'.'Ith .IF' .......... ......... 100

13. Ti~re-co'urse of elution of radioact.i-:.e I;,i from
ti.ue bllocks of lazy corn steI-nI infiltrated
'..'i h bu f .r . . . . . . . . 101

14. T f'.---courrse of *lut on of radioactive : I.-A from
tiLru- blocl.s of lazyi corn rt, ~-: Infiltratc.d
'with lIF' . . . .. . . . ... . 101

15. Tlme -cou r-e of re;lea.: of raJio'actl .'e I;' at
250 in 5 10-4 11 LVA and at 10 C in '-*actr or
buffer from corn node tLisue blocV.z pre'.iou:1'"
Esoa:.- 13 hour . . . . . . . 115

16. TLm e-course of rel-aze of radji.,activ. IAA from
tissue blc.-K from normal and lazy corn stems-
after 20 hour soaJ:inq in watsr . .. ... 119


Living organisms respond in a variety of ways to

stimuli in their environment. Gravity, for example, has

three general types of effects on plants: geotonic effect,

effect of gravity on the growth rate of certain plant or-

gans, e.g., internodes of grasses (92); geomorphic effect,

effect of gravity on morphological differentiation, e.g.,

root formation in sugar cane cuttings (33) or flower for-

mation in pineapple (93); and geotropic effect, effect of

gravity on the orientation of plant organs. Geotropism was

defined by Frank (21) as "active movement induced by gravity

and oriented in a direction determined by the angle between

the direction of the force of gravity and either 1) the axis

of the plant part (curvature) or 2) the plane of symmetry of

a bilaterally symmetrical (or dorsiventral) plant part

(torsion). The direction of the movement may be that of the

force of gravity (positive geotropism) or the opposite di-

rection (negative geotropism) or the movement may take place

in a plane at an angle to the direction of the force of

gravity (lateral geotropism)."

Geotropic curvatures are classified according to the


liminal direction of a plant organ. The liminal geotropic

direction is defined as the orientation (with respect to the

plumb line) which can be maintained by a plant organ for

prolonged periods of time without t the organ carrying out

gross geotropic reactions (51). Comron types of geotrropi-m

(51, 63) arc:

1. Lateral geotropisn (horizontal geotropiEri)---the

cur'vatures produced are in a plane at right or

oblique angles to the plumb line.

2. Orthogeotropism (parallelogeotropisml--thc liminal

geotropic direction is parallel to th, pluwib line.

3. Plagiogeotropi _m--the liminal geotropic direction

is at an angle to the plumb line.

a. Diageotropism--liminal qeotropic direction is

at 90 dejree3 to th.e pLurmb line.

b. Klinogcotropismr--lminal geotropic direction

ot-her than parallel or perpendicular to the

plLumb line.

The study reported here is limited to the negative

orthogeotropic reaction of Zea mays L. (corn). This plant

was chosen as experimental material because of the availa-

bility of a single gene ageotropic mutant which would be ex-

pected to possess a single primary physiological deficiency.


Radioactive indole acetic acid (IAA) and recently discovered

selective inhibitors of the geotropic reaction were employed

in conjunction with the methods of physiological genetics to

examine this important biological reaction. The radio-

activity was used to measure transport and binding of the

hormone; and the inhibitors used to block selectively the

geotropic reaction without inhibiting growth.

The results of this study indicate that reorientation

of a corn stem causes a redistribution in the physical-

chemical associations of IAA within the stem. The stems of

ageotropic mutant corn and stems of normal corn infiltrated

with a selective inhibitor of geotropism have patterns of

redistribution different from normal corn. Horizontal

placement of a corn stem results in an inhibition of polar

transport of IAA but has no detectable effect on lateral

transport of IAA.


The Geotropic Reaction

Dodart (19) in 1703 referred to the propensity of

plant stems to grow upward and roots to grow downward. This

was apparently the first written mention of the geotropic

reaction. Frank (21) coined the term geotropismm" for the

"peculiar active force" liberated in plants by gravity.

Many extensive reviews of geotropism, such as those by

Rawitscher (68), Schrank (79), Brauner (9), and Larsen (51,

52) have been published in the past 25 years.

The geotropic reaction may be divided into three

phases from an operational point of view: presentation,

lag, and differential growth. According to Hawker (30), the

presentation phase is the period of time plant organs must

be maintained in a horizontal orientation such that upon

subsequent vertical placement 75 percent of these organs de-

velop at least 5 degrees curvature. Larsen (51) listed

other definitions which have been used and presented a

criticism of them. Hawker (30) examined the presentation

time of a number of species of plants representing the

Gymnospermae, Dicotyledonea, and Monocotyledoneae and


found times varying from 3 minutes for seedling stems of

Asparagus officinalis to 24 hours for those of Phoenix

dactylifera. The lag or latent phase is that period of time

from the completion of the presentation phase until the ob-

servation of the first visible response (30). Hawker ob-

served lag times of from 35 to 240 minutes. Hawker also

found that presentation and lag times varied considerably

with the height (age) of the seedlings tested. Prankerd

(66) observed diurnal and Brain (7) seasonal variability in

the duration of these phases.

The geotropic reaction may also be considered to occur

in several phases from a mechanistic point of view: stimu-

lation, transmission, and reaction (51, 68). The stimula-

tion phase includes a physiological phase, perception or re-

ception, and at least one physical phase, susception. Per-

ception produces an excitation which initiates an unknown

number of physiological transmission steps which culminate

in the final reaction.

Two hypotheses regarding the mode of susception of

geotropic stimulation have been proposed. The first and

most generally accepted one states that plant organs are

sensitive to differences between the liminal and actual di-

rection of their axis (51). A more recent hypothesis

proposes that gravitational susception consists in the mo-

tion of the plant organ in moving from its normal position

to a new one (6).

Regardless of the mode of susccption, the effect of

gravitc is apparently limited to the acceleration of mass.

The miiagnitude of this acceleration on the earth varies

slightly with location but has an appro::;xmate value- of 980

cm per sec2 (g). Acceleration required to initiate the geo-

tropic response is much less than this value. Chance and

Smith (13) employing a large centrifuge determined that a

reFultant force between 0.019 and 0.025 g was required to

elicit curvature (7.16 degrees) in seedling ster s of

Fagopyrum esculentun. Lyon (54), employlinq vibrating wires

attached to an horizontal clinostat, determined that about

0.000045 g would cause bending of corn seedling roots in the

dark. Experiments of Haines (27) indicated that the primary,

effect of gravity on plant? is a redistribution of "rela-

tively solid" particles in the protoplasts.

About 1900, Haberlandtand rlemec (26) postulated the

statolith-starch theory to explain the perception of gravity

by plants. According to this theory, gravity produces a

displacement of starch granules which in turn excite the

neighboring protoplasm. .'ork of many investigators, notably

Hawker (30, 31), has lent support to the theory. There are,

however, many examples of plants which respond to gravity

but which do not contain any starch granules (68). The

existence of statoliths in plants must still be assumed in

order to understand the geotropic reaction, but the morpho-

logical identity of these statoliths is unknown.

Larsen (50, 52) proposed a new model to explain geo-

tropic stimulation as the result of extensive experiments

with young roots of Artemisia absinthium (47, 49). The ob-

served geotropic behavior of these roots fit the model of a

statolith as an electrically charged pendulum with oscil-

lations damped by a constant longitudinal force. Larsen

proposed that displacement of a plant organ from its liminal

direction causes a displacement of the statoliths resulting

in a transverse potential which initiates the physiological

processes culminating in geotropic bending. Larsen (52)

developed this model mathematically and found good agreement

between the model and observed geotropic behavior.

Bunning and Glatzle (11) found that presentation in

two interrupted periods elicited a greater bending response

than continuous presentation of the same total time. An op-

timum time interval of interruption was also observed. From

these observations Bunning and Glatzle proposed that there

is an absolute refractory state and a relative one after

geotropic irritation. According to this hypothesis, the

time between successive presentations allows statoliths

which were not stimulated during the first period to re-

orient in a position favorable to stimulation, whereas

stimulated statoliths are maintained in a temporary state

of irreversible stimulation. The second period of presen-

tation therefore allows greater stimulation, more cells are

irritated, and the reaction is stronger.

Larsen (51) reviewed experiments which demonstrate the

localization of cells capable of perceiving gravity. In

these experiments plant organs were mounted above the axis

of a centrifuge such that an extension of the axis inter-

sected the organ at various distances from its apex. In

this manner it was demonstrated that the tips of roots were

far more perceptive than the region of elongation, whereas

in shoots the perceptive area was more diffuse, extending

into the region of elongation. These observations were con-

firmed by experiments in which root or shoot tips were re-

placed either by auxin (2) or by geotropically stimulated

or nonstimulated root or shoot tips (29, 44). Further con-

firmation of these observations was obtained from experi-

ments in which mica was inserted along the median plane of

horizontally placed roots for various distances from the

apex before the geotropic reactivity of the roots was

measured (43). These results indicate that the morphologi-

cal tip of an organ, although sensitive, is not necessary

for perception of gravity but functions chiefly as a source

of auxin.

De Wit (18) concluded that auxin was necessary for the

perception of gravity by deseeded Avena coleoptiles. This

conclusion was based, among other things, on the fact that

decapitated coleoptiles placed horizontally in water and

then vertically in IAA solutions did not bend, whereas

similar coleoptiles placed horizontally in IAA would bend

when placed vertically.

It is clear, regardless of the mechanism of stimula-

tion, that a physiological polarization has been developed

at the end of geotropic presentation which leads to local

differences in rates of growth. Factors examined in an at-

tempt to characterize the nature of this polarization are:

osmotic pressure, viscosity, pH, hydrolyzable sugar, reduc-

ing sugars, catalase activity, and respiration (52, 68). As

Larsen (52) pointed out most of the observed changes must be

regarded as a prerequisite or consequence of changes in

growth rate.

Shrank (75) found that horizontal placement of an

Avena coleoptile caused the lo..,er surface to become about 10

my more positive than the upper surface. This polarity was

e::pres.ed "long before" bending or difference:. in au:-:in

could be demonstrated (76) The presence of the coleoptile

tip was not necessary for production of the polarity al-

though it ias nece:-sar, for blending to occur (77). If the

coleoptile was filled with an elezctrolyte both the geotropic

bending and establishment of a potential cere inhibited

roughly as a fun-tion of the conductivity of the solution

(79 80). The -ffect of el ctroly ts was not osmotic 1 73)

The *establishment of this potential is the first observ.ablc:

effect of gravity.

Central to any explanation of the geotropic reaction

is the Cholodny-Went theory. According to this theory

"growth curvatures . are due to an unequal distribution

of auxin between the two sides of the curving organ. In the

tropisms induced by light and gravity the unequal distribu-

tion is brought about by a transverse polarization of the

cells, which results in a lateral transport of the auxin"

(95). The fact that auxin is required for the geotropic re-

action is well established (2, 95). It is also well es-

tablished that the lower half of a horizontally placed organ

contains more diffusible auxin than the upper half (18).

Furthermore, Went (94) observed that the course of produc-

tion of auxin obtained by diffusion into agar followed the

course of recovery of geotropic reactivity.

However, direct confirmation of gravity-induced

lateral transport has not been obtained.

In fact, recent experiments with C14 labeled IAA sug-

gest that an alternative explanation is required. Bunning

and co-workers (12) applied IAA 2-C14 to decapitated coleop-

tile stumps either in agar blocks or by dripping IAA solu-

tion on them. These coleoptiles were then illuminated uni-

laterally. After phototropic curvatures had developed, the

coleoptiles were bisected. Determinations of radioactivity

in the "light" and "dark" halves of over 1,000 coleoptiles

failed to demonstrate any transverse transport of radio-

activity. These authors also state that preliminary experi-

ments of the same nature with the geotropic reaction failed

to support the hypothesis of transverse distribution. The

geotropic reaction observed could not have resulted from

native auxin alone since decapitated coleoptiles require

exogenous IAA for reaction (2).

Reisener (70) immersed 1.2-1.5 cm coleoptile tips ver-

tically in solutions of 1 mg per liter radioactive IAA. The

coleoptile_ were bisected three hours- after horizontal

placement. Again, the radioactivity in each half or the

geotropically bent coleoptiles was equivalent: i.e., no evi-

dence of lateral transport of the radioactivity was OD-


Ching and Fang (14) applied carboi:yl labeled IAA-Cl4

to pea, lima bean, and corn roots and shoots and then de-

termined the radioactivit.' in the upper and lo'-er halve. of

horizontally placed organs. Geotropic b-nding was observed

in some of the organs assayed since samples were taken at

intervals of 20 to 180 minutes after horizontal placement.

And again, no unequal distribution of radioactivity was ob-

serv'.d. Five to 10 percent of the recoverable radioac-

tivity ,.as found chromatographically identical with IA...

An alternative to lateral redistribution of aaxin has

been proposed in the case of certain plant organs. The geo-

tropic behavior of rhizomes of Aegopodium podagrarla (5) and

of roots of Pisu sativum seedlings ( ., 4) suggested that

the geotropic reaction results from the de no-.o production

of an inhibitor rather than the redistribution of au.:in.

The final geotropic reaction results from unequal

growth of the upper and lower halves of a horizontally

placed organ. It is well established for many species that


cell elongation is intimately involved in this differential

growth (51). Brandes and McGuire (8) found that cell di-

vision as well as cell elongation contributed to the geo-

tropic bending of sugar cane stems.

Chemical Inhibition of Geotropism

Many growth regulators have been found to inhibit the

geotropic response. Some of these growth regulators inhibit

both straight growth and geotropic bending, others inhibit

geotropic bending but not straight growth, and still others

inhibit straight growth but not geotropic bending (Table 1).

The existence of these three classes of compounds indicates

that the mechanism of the differential growth phase of the

geotropic reaction is not identical with the mechanism of

straight growth. For example, compounds which inhibit

straight growth but not differential growth must act on loci

of the straight growth process which do not exist in the

differential growth process.

The effects of two of the compounds, 2,3,6-trichloro-

benzoic acid (TCBA) and N-l-naphthylphthalamic acid (NP),

have been studied extensively. Vander Beek (87) found that

TCBA inhibited the geotropic response of seedling shoots of

oat, barley, and cucumber. Jones and co-workers (40) re-

ported that TCBA inhibited both geotropic and phototropic


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responses of rye-grass roots without inhibiting straight

growth. Recently, Schrank (82) showed that concentrations

of 10-5 to 10-3 M TCBA stimulated the growth of both 5 mm

subapical and 15 mm apical coleoptile sections. This stimu-

lation was obtained only in the absence of IAA. At concen-

trations of 10-4 and 10-3 M TCBA inhibited both geotropic

and phototropic bending of 15 mm apical coleoptile segments.

Growth of these segments at 40 C was neither stimulated nor

inhibited by 10-4 M TCBA, whereas the geotropic reaction was

measurably inhibited. Schrank interpreted these results as

indicating that TCBA in some way inhibits the geotropic per-

ception mechanism.

NP has been observed to inhibit the geotropic reaction

of radicles of Lens esculenta, Pisum sativum, several of the

Cruciferae and Compositae (57), inflorescence stems of

Antirrhinum majus (86) and roots and stems of many other

monocotyledonous and dicotyledonous species (59). Jones,

et al (40) found that low concentrations of NP inhibited the

geotropic response of rape and rye-grass roots without in-

hibiting root growth. Ching, et al (15) observed that NP

inhibited the geotropic response of Avena coleoptiles,

coleoptiles and roots of corn, and roots and shoots of Pisum

sativum. Certain concentrations of NP inhibited the

geotropic rcactLon of these tr-st organs ith only a slight

inhibition of straight growth.

.n extenisiv.e study of chc- r. lationship of chemical

-tructure to activity in inhibiting thi- geotropsc- reaction

was made bd Mlntzer and co-workers (56). T'enty-fiv'. com-

pounds with structures rc-latrd to UP Fwer- assay;ed for ac-

tt"i.ty in inhibiting the geotropic response of Lens

e-:cuienta seedlLna roots. These authors deduced the chemi-

cal structure necessary for activity (Diagram 1). .,- tivity

I J'

h ,' ,,C *

was cnhanc,-a d: (1) if the ring (I) was multiple. (2) if the

carbon-carbon bond (II) was unsaturated. or (") if the car-

bon-carbon bond (II) was associated with a ring system.

Jones. t al (40) crTpioying r,'--grass and rape seedling

roots found that the p-:Fptlde linkage was not necessar-; for

activt.ty in inhibLting the gyotropic response (e.g. 2-

carboxybiphFnyiam in) .

Jones and co-workers (40) also found that iP was nri-

thrr an antagonist nor syrnergist of IAA in the split pea

test and concluded that th. antigeotropic activity of I1P re-

sults neither from auxin nor anci-awxin actciit;. Ching, ct

al (15) could not "unambiguously" clasitfy [jP either as a


growth-promoting substance or a competitive inhibitor of IAA

on the basis of Avena section tests. In contrast to these

results, Morgan and Soding (58) found that 1 to 100 mg per

liter solutions of NP promoted growth of 3 mm Avena sections

floating on the solutions. Growth promotion by NP occurred

both in the absence and presence of exogenous IAA (0.1 to

1.0 mg per liter). However, in assay methods requiring

polar transport NP inhibited growth both in the presence and

absence of IAA. Morgan and Soding (58) concluded that al-

though NP stimulates growth it is an inhibitor of polar

transport of auxin.

Auxin Transport

The role of auxin transport in the geotropic reaction

is implicit in the Cholodny-Went theory. Furthermore, the

physical separation of regions of geotropic perception and

reaction suggests the involvement of transport processes.

Recent investigations of auxin transport have been re-

viewed by Leopold (53) and Van Overbeek (91); earlier

studies on polar, i.e., basipetal, transport are summarized

by Went and Thimann (95). Polar transport is a metabolic

process having a Q10 of about three. The rate of polar

transport is greater than accounted for by diffusion, being

about 1 to 1.5 cm per hour. At 00 C the polarity of the

process is maintained, but the rate of transport approaches

that of diffusion. In low concentrations of ether vapor the

polarity of the process is reversibly suspended, and the

rate of transport approaches that of diffusion. Wickson and

Thimann (96) found that older stem segments transported less

auxin than younger ones and that both light and kinetin re-

duced the rate of polar transport. The rate of polar trans-

port is also proportional to oxygen tension in the range of

0 to 5 percent oxygen (25).

The transport process in Avena coleoptiles is strictly

polar, that is, no acropetal transport is observed (95).

More recent studies indicate that in other plant tissues

this strict polarity does not hold (53). For example,

Wickson and Thimann (96) were able to demonstrate measurable

acropetal transport of IAA-C14 in pea stem sections.

Polar transport of IAA is affected by a number of com-

pounds which also inhibit geotropic bending to a greater ex-

tent than straight growth. Among these are: TCBA (46);

NP (58); 2,3,5-triiodobenzoic acid (60, 62); 2,4,6-trichloro-

phenoxyacetic acid (61); 2,4-dichlorophenoxyacetic acid

(61); and 2,6-dichlorobenzoic acid (46). Niedergang-Kamien

and Leopold (61) pointed out the similarity between the ef-

fects of eleven chlorinated phenoxyacetic acids on IAA

transport and their adsorption onto charcoal. This fact, in

addition to others, suggested to them that the inhibition of

polar transport by these compounds might result from

interference at some transport site of attachment.

The literature contains little information on the ef-

fect of gravity on polar transport. Vander Weij (88) re-

ported that polar transport in inverted Avena coleoptile

sections was slightly inhibited. In a subsequent study

Pfaeltzer (64) found no effect of gravity, acting along the

longitudinal axis of the plant, on polar transport of auxin

in Avena coleoptiles.

Direct studies on lateral transport of auxin in plants

have likewise been neglected. However, since unilateral ap-

plication of auxin to plants produces bending, the rate of

lateral transport must be low. Regarding lateral transport

in general, Zimmermann (98) states that "lateral transport

in the phloem is known to be very slight." In support of

this statement he cites several examples in which unilateral

defoliation produces asymmetric growth of stems, flowers,

and fruit.

Auxin Uptake

Studies of auxin uptake not only provide information

on transport processes, but also on physical-chemical asso-

ciations of auxin in cells. Reinhold (69) was able to dis-

tinguish two phases of uptake of IAA by pea epicotyl seg-

ments and carrot root disks; a metabolic phase and a

physical phase. Uptaxe by the metabolic process was in-

hibited by cyanide, lodoacetate, and arsenite and depressed

by diethyldithiocarbamate and 2,4-dichlorophenoxyacetic

acid (2,4-D). The IAA taken into the tissue by the meta-

bolic process was not recoverable but was either bound, con-

verted, or destroyed. Uptake by the physical process re-

sembled adsorption rather than diffusion. Uptake by this

process accounted for about 50 percent of total uptake. The

IAA taken up by the physical process was essentially re-

coverable from the tissue.

Johnson and Bonner (37) were able to distinguish three

kinds of uptake of 2,4-D into Avena coleoptile sections:

metabolic uptake, diffusion, and exchangeable binding. The

phase of metabolic uptake had the same properties as found

for IAA (69). The diffusion phase was complete in 30 min-

utes and the 2,4-D taken up was free to diffuse out again

into water. The inward diffusion was not influenced by

1,000-fold excesses of IAA. Exchangeable binding within the

tissue was also complete within 30 minutes. The 2.4-D taken

up by exchange could not be recovered into water but was re-

leased into either 2,4-D or IAA solutions. Exchangeable

binding also differed from the diffusion process in that e::-

cess IAA suppressed exchangeable binding.

Recently Andreae and Ysselstein (1) found that pea

roots accumulated IAA to a much greater extent than did

epicotyls. During the first two to four hours of uptake IAA

was recoverable from the tissue in the free form. After

this period IAA was rapidly conjugated to indoleacetylas-

partic acid. No other conjugated form of IAA was recover-

able from the tissue during the 24-hour period of their ex-

periments. They also found evidence that degradation of IAA

occurred in only a small area of the tissue, probably the

epidermis and root cap.

Active Transport vs. Permeation

Collander (16) defined "permeation" as the transfer

process "in which the protoplast plays the passive role of a

mere resistance to be overcome by the substance as it leaves

or enters the cell." He listed six criteria to distinguish

between permeation and active transport: (1) Generally the

rate of a permeation process is proportional to concentra-

tion, whereas in active transport this proportionality is

not found. (2) Chemically similar substances rarely compete

with one another in simple permeation, but often mutually

depress the uptake of one another by active transport. (3)

The permeation power of substances is correlated with their

molecular size and lipid solubility. If the uptake of two

substances of similar molecular jize and lipid solubilitie-

La markedly diffeLreont then the uptake of at least one of

th.2em is probably not due to permeation alone. (4) Permea-

tion is little affect-d by the absence of ox-:ygen v'*herea.

anaerobiois, in aarooic organlsns, -i thor depresses ocL

prev'ents .active transport. (5) The effects of narcotics on

permeation are complex:, whlic active transport mray be- re-

.ersibly reduced by, narco-is. (6) 'Substancres. kno'-'n to in-

hibit certain enzyvmes, such as h;ydrocyanic acid. carbon

mono::ide, sodium azide. iinit rophenol. iodoacetate. and

fluoride have Deen success fully used to show. that particu-

lar enzye: ar- involved. directly or indi-ctly,. in certain

absorptn process. proce prionounc--d effect of eLnzYm' inhi-

bitors on perieationr processes. although conceivable, is- not

'ery probable' (16).

Collander (16) pointed out that accumulation of a _=,b-

.tarncC withinin a cell does not necessarily, iLrrply an active

metabolic transport of that substance. For e:.ample, many

weak bases enter c.?ll= by permeation process, but accumulate

within the cells as a result of binding. Similarly, dif-

fusible ions may accumulate within cells as the result of

Donnan equilibrium.

In the case of simple diffuslo-n Fick's la' states that

dQ dC
= -Da ;
dt dx

where dQ is the quantity of a substance which in time, dt,

passes across an area, a, in which dC/dx is the concentra-

tion gradient. The constant of proportionality, D, is the

diffusion coefficient with the dimensions of area divided by

time, e.g., square centimeters per second (34). Larsen (48)

tabulated the results of several determinations of the dif-

fusion coefficient of IAA; D was found to be in the range of

0.596 to 0.677 cm2 per day or an average value of about

7.45 x 10-6 cm2 per second at 250 C.

If the cell membrane is a barrier to diffusion then

obviously Fick's law does not hold for the permeation

process. In this case, Fick's law may be modified to define

a permeation constant instead of a diffusion constant (16).

The permeation constant of IAA has not been determined, how-

ever, since IAA may be taken up or bound in several ways.

Disposition of Auxin in Tissue

It is apparent that the auxin available for transport,

diffusible auxin, is only a fraction of the total auxin con-

tent. The term diffusiblee" is not necessarily descriptive

of the mechanism of the transport process itself. The auxin

remaining in the tissue after diffusion into agar blocks is


called "bound" au:-in. Roughly 50 percent of the au::in in a

plant is bound (53). The identLty of the molecules to which

auw in is bound and the nature of the binjinq is not cer-tain.

Se.'eral kinds of au:-:in-protein comple'.e- been found

(24 84 97) The possibility' r-.rains. howe'.ver, that these

corfll:I :.2s rcsuitcd as an artifact of preparation (23). P.e-

centil'. Galston and Kaur k22) found ,e'.'den:e of au::in bind-

ing to protein in the supernatant of homogeniized-centrifuged

pea stemi sections but could find no awu:in associated with

ceil particulate They al'o found an au::in-induced de-

crease in the heat _oagulability of cytoplasmic protein. In

a review, Galston and Furv'e. (23) Cite the work of Mi. Bach

and also of '.'. Freed which also showed awu-:in-induced changes

in the heat coagulability of cytoplamnic protein. '.'. Freed

also found altered infrared spectra for enzyT;me-au1:1n com-

Lazy Co-n--An Ageotropic Mutant

The literature contains a number of references to,.

plant part: which normally change their racponse to gravity

during some phase of their 11if, cycle. Striking e:.arnples of

such plant parts ace the stamens of Hosta caeruil.a (65) and

the inflorescence3 of v.ater hyacinth t63). Of equal physio-

logical interest are genetic mutants which fail to respond

to gravity. Such ageotropic mutants are known in rice

(38, 67), Cajanus cajan (17), Pisum sativum (74), and in


The ageotropic mutant of corn, discovered in 1923, was

named "lazy" by Jenkins and Gerhardt (36). In field plant-

ings lazy corn usually cannot be distinguished from normal

corn until the plants enter the phase of rapid elongation

just prior to tasseling. At this time the stems of lazy

plants gradually bend until the stalk above the fifth or

sixth node above the prop roots rests on the ground (Figure

1). After becoming prostrate the lazy plants continue to

grow above the ground. Analysis of the breeding behavior of

over 4,000 plants in F2, F3, and backcross generations indi-

cated that the lazy character is inherited as a simple Men-

delian recessive (36).

Jenkins and Gerhardt (36) undertook a detailed com-

parison of the characteristics of lazy and normal corn in an

attempt to elucidate the action of the lazy gene. In this

comparison they employed sibling plants from a backcross of

homozygous lazy (la la) with heterozygous normal (La la) Fl

plants. They found that: (1) Lazy and normal plants had

similar morphological structure. (2) The cell walls of lazy

plant stems were thinner than those of normal plant stems.

Fig. 1.--Third gnrrration sibling laz-' (Left) and normal
(right) corn plants grown in the greenhouse.


(3) The breaking strength of mature green lazy plant stems

was about 50 percent of the breaking strength of normal

plants. (4) Lazy plant stems were lower in cellulose,

lignin, and pentosans than were normal stems. (5) The ex-

pressed sap of lazy plant stems contained less ash, total

solids, and ionizable constituents and had lower osmotic

pressure than the sap of normal stems. Jenkins and Gerhardt

(36) concluded that the lazy habit of growth resulted from a

structural weakness in the stem of lazy corn.

Van Overbeek (89) found that five-to six-day-old lazy

corn seedlings were negatively geotropic in the dark. How-

ever, ten-day-old seedlings in the greenhouse appeared ageo-

tropic since they continued to grow in the direction in

which they were pointed for some weeks. Furthermore, both

lazy and normal plants grew parallel to the axis of a hori-

zontal clinostat (one-half rpm) for one month. These re-

sults suggested to Van Overbeek (89) that the lazy habit of

growth resulted from a deficiency in the geotropic reaction

rather than a structural deficiency.

Van Overbeek (90) compared the auxin content of normal

and lazy corn plants segregating from a backcross of homo-

zygous lazy (la la) with heterozygous normal (La la) F1

plants. Auxin was obtained by short-term cold ether

extraction and determined b" A"e'na bioassay. Van O-erbeek

determined that the "nodes' (one-fourth inch on either side

of the point of leaf insertion) of horizontally placed

norm-al corn ste.'m contained less auxin than the nodes of

lazy corn stcrs: 0.19710.054 mniroqrams IPA equival.ents per

kilogram fre-san '.'eight for normal as opposed to 0. 32210.042

for lazy. Both type. of plants had equivalent azTiountf of

au:in in Lnternodal tissue.

The relative distribution of 3u:in in the upper and

lo'.--er halves of normal corn stlan nodes and internodes was in

agr.2 ment with previous findings with other plants (95). If

the upper half of the stemi is taken to have 100 parts of

auxin, then the iow.'er half of nodal tissue of normal corn

stLn,-m was found to contain an ave.rag;e of 121 parts (range

84-144) and internodal tissue 107 parts (range '35-114' In

contrast, the lower half of lav'.- corn :stmi nodes contain-d

an average of 90 parts aux.:in (rang? 67-109) and the lower

half of internodal tissue 37 parts au:i;n (range 44-114).

From these data Van (90) concluded that the lazy

habit of growth resulted from an impairment of gravity-in-

duced lateral transport of auxin.

Van O.'erbeek (90) observed three classes of lazy

plant- in field plantings: plants lying flat with tips

"more or less" curved up, plants lying flat and "entirely

straight," and plants "more or less curved downward." If

plants of this last class were rotated 180 degrees along

their longitudinal axis, the tip of the stem bent down again

after ten days.

Shafer (83) determined that the growth of both normal

and lazy corn stems occurred in regions one to four mm above

the leaf insertion points of the leaves. In agreement with

previous work (95) he found that horizontal placement of

normal corn stems stimulated growth at nodes which would not

ordinarily have grown more. Geotropic bending of normal

corn stems was accompanied by the formation of a visible

wedge of tissue in the region of the growth ring; the inter-

nodal regions remained straight (Figure 2). Horizontal

placement of lazy corn stems elicited neither growth nor

geotropic responses. Similarly the application of 0.2 per-

cent heteroauxin in lanolin produced no growth response.

Shafer (83) compared auxin production in normal and

lazy sibling plants from a backcross, i.e., no inbreeding

was employed. Shafer obtained auxin from coleoptile tips of

both lazy and normal seedlings (classified by subsequent

growth) and then assayed the auxin by the standard Avena

test. He could find no difference in auxin production

.1 ,- TI

3 C 'C'
,-J .C '


C .C C7
cr Z* r"

--1 3 ^:

* C" C''


i3J1 -
> J


4 -' c


between normal and lazy coleoptile tips. Shafer stated that

this result was expected since young lazy seedlings are

negatively geotropic.

Shafer (83) also studied auxin transport in segments

of the growing regions of normal and lazy stems. One cm

segments of stem were clamped in a horizontal position and

the morphological base of the segment divided into two equal

parts by the horizontal insertion of a piece of razor blade.

Plain agar platelets were placed in contact with the end of

the segment, above and below the razor blade and an agar

platelet containing auxin placed on the morphologically

upper end of the segment. The concentration of the auxin

was "varied in the direction that seemed to promise the best

transport results." Shafer did not specify either the con-

centration of auxin used or the time interval for transport.

Further, it is not clear what auxinn" Shafer was talking


In normal stem segments more auxin was found in the

lower agar platelet on six independent trials, more in the

upper twice, and essentially the same amount in each plate-

let nine times. The ratio of auxin in the lower platelets

to that in the upper was three to two. In lazy stem seg-

ments more auxin was found in the lower agar platelet on

fi'.e independent trials, more in the upper one seen times,

and the ainme amount in each platelet five times. The ratio

of au-in in the lo' platelet to that in the upper was

about four to five. On th average, lazy ste.m segmcnnt-

tranzporte-d more au:in than nonnal. The results ,ere quite

,ariable and tne basis for comparison of normal and lazy is

not clear. Shafer (8:.) stated that the: lazy and normal

plants used were "nearly al\wayS from seeds from the saire

*ear; but since this secd wa= not inbred the planr.t must hav.,.

varied much in spite of -are used to s-elect similar ones."

Nonetheless Shafer interpreted these results to ugqq.est that

lazy corn is posztivel:- geotropi.. Shafer also observed

that lazy plant- become aphototropic at about Ut-e sajne tLmw

as they become ageotropic.


Materials and Methods

Plant production

The corn plants used in this study were derived from

Maize Genetics Cooperative stock 50-409-1/-2 which had the

genotype (M14/la su g13) sibling. A crossing program

(Figure 3) was initiated to produce inbred lines adapted to

Florida growing conditions which would segregate for the

lazy character. A secondary objective of the program was to

obtain these lines free of the sugary (su) character which

is linked with lazy (39). First generation inbred seeds

were used for field experiments; second and third generation

inbred seeds were used as a source of plants for laboratory


Plants for laboratory experiments were raised in the

greenhouse under a minimum photoperiod of twelve hours.

Night temperatures were maintained at 650 F; day tempera-

tures never exceeded 950 F. The plants were raised in six-

inch clay pots filled with fumigated soil. Four seeds from

a single line were planted per pot. During early stages of

growth the plants in each pot were irrigated twice weekly


r.1 I4 '-
ia Su Qi,

P fI I
7 I ., t F 4 n I F ] E

FIELD C 0-5 9 60-60 60 6
E.,PE I',MErITS ., IB ,1, a

second in red. gp rerar ion for e-,perrrir.:

6".-. l2 6(-103 6'-,'-I.4 6 ,- 105 600-06

Irrd g.- rer er.j (Cor B ,per P r, nts

* numiiber.

**(F44 :x F6) is one of the parents of Dixic 18, 3
hybrid widely grown in the South.

Fig. 3.--Crossing plan and utilization of inbred lines
segregating for the lazy character.

with about 150 cc of Hoagland's solution. The plants were

irrigated with this nutrient solution daily, after the onset

of rapid growth just prior to tasseling. No visible defi-

ciency symptoms were observed.

Immediately after the onset of rapid growth the geo-

tropic reactivity of the plants was determined by placing

the pot and plants horizontally. Those plants which ex-

hibited a bending response within 48 hours were tentatively

classified as normal and those which did not respond clas-

sified as lazy. At this time two of the four plants in the

pot were removed. Whenever possible, a normal plant and a

lazy plant were left in the pot. Presumptive lazy plants

were tied to bamboo stakes placed vertically in the pots.

Reorientation of the pot vertically gave an additional check

on the geotropic reactivity of the presumed normal plants.

Plants were classified as normal if they responded to

gravity after these two reorientations and if they subse-

quently maintained a vertical orientation. Growth of a lazy

plant above the point at which it was tied to the bamboo

stake was accompanied by a curvature of this new growth away

from the vertical. A presumptive lazy plant was classified

as lazy if it had to be tied frequently to the bamboo stake

to maintain it in a vertical orientation. The latter


critcrion was also used for classification of lazy plants in

field plantings.

Plants were taken for laboratory experiments after

they had tasseled. The plants .ere kept in the dark at

25110 C overnight in the laboratory before each e::periment

to minimize both the erffct of seasonal differences in the

qrenrhouse environments and any disturbing effect incurred in

transporting th. plants.

Breaking strength and growth of stemn

ji-nJlin and Gerhardt (36) that the lazy habit

of qro'th resulted from the stru.-tural weakness of the stem.

Before examining the geotropic behavior of the lazy mutant

sto.-k on hand it was necessary to test this hypothesis. A

field experiment to examine breaking strength and growth of

normal and lazy siblings employed a randomized blocK design

with six replication. Each block was four rows wide with

ten plants per row. A row was planted with seed from on.

of the four selected first generation selfcd plants. The

expected sgrc-gation in these lines is one lazy plant to

three normal plants. As a consequence of this segregation,

the experimental results were evaluated by covariance

analysis to adjust for unequal numbers of the two plant

types (S5).

The height of the plants was estimated immediately

after pollen was shed and stem elongation had essentially

stopped. Height was measured from the ground level to the

first node below the tassel. The breaking strength of the

stems was estimated immediately thereafter by a modification

of the method employed by Rogers (72). One end of a piece

of strong cord was looped around the center of the second

internode above the ground. The other end of the cord was

attached to a 100 pound capacity spring balance. The break-

ing strength was taken to be the equivalent of the force in

pounds required to break the stem when the balance was

pulled slowly and steadily in a horizontal direction.

Geotropic reaction of lazy and normal corn

One hundred of the first generation selfed seed were

sown in two-inch wooden plant bands filled with soil and

then grown for two weeks before being transplanted to the

field. The geotropic reaction of these seedlings was de-

termined while they were in the coleoptile stage by placing

the wooden bands on their sides. The bands remained in this

orientation for two days during which time the coleoptiles

of the plants ruptured, and the first two leaves expanded.

The bands were then reoriented vertically and the geotropic

reaction of the seedlings again measured. The seedlings


*..ere then planted in the field where they were subsequentlv

claSifieJ as lazy or normal.

Pesul ts

Breaking strength an3 growth of

Measurements of the breaking strerngtri of stems of

normal and lazy zorn segregating after one- generation of in-

breeding ar.- given in Table 2. The mean breaking strength

of the 60 lazy plants 22.1 pounds, of the 151 normal

plants -4.6 pounds. Th- adjusted means were -1.4 pounds for

lazy plants and A4.5 pounds for normal plants. Covariance

analysis indicated that there '.as no significant difference

between Lti. breaking strengths of normal and laz. plant

3 ten 3.

Growth meaEureme.nts of these normal and lazy plants

are gien n in Tabl-e The a'.erage height of the 60 lazy

plants wa-. 43.9 inches, of the 151 normal plants 47.3

inches. The adjusted means were 44.1 inches for lazy and

48.5 inches for normal. There was no significant difference

in height between normal and lazy plants. There was, how-

-ever. a significant difference in the heights of plants be-

twe.en these first generation inbred lines. The adjusted

mean heights per plant for these lines were 46.2. 51.4

45.9, and 41.3 inches.

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a, 9 --4 o 10 mI mA a% Ou 0 it LAN
O N HH d NN N Hm


E a o0 0 00 m0 (U N mo

644 4 z 4 4

0 H N C LA o
I ~ '- 4 l lO nlO t t O f

C d L 0 '0 CO 0 -<
SC.,0 r0 ui r'- Is. L',
-I c0 --- r rj0 -0 ,

C..C' l N L; A
*I* -I **J r3 -4

S. L'

N" 0 N ,. 0 0. 'i %D ,
a. N0J N 0 '. cm c0 l

T, .V r- 0 -4 l --4 i *r 0 aD

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cP in -- T 00 f- n 4i L

,0 N' r' Jj \ r- i
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-P L" T CO u -E .-

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Ca 'r r N- u l 0 N ,

C C D O *1
- '4 r C , u-J rre 7 I '
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.-a -- r- ".-- J "-) Q rM -0 "- C

v 0 J (, O fl, C4
? 1' > ,z o

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o o i D L if" 0 V) G r *-0 %
*. i i rll to -i ( n rM 'n in C

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r C C t -4
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I L) ..ri 0 /c i ^ tr 3

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Geotropic reaction of lazy and normal corn

In the coleoptile stage the seedlings which were

proven by subsequent testing to be lazy had bent an average

of 88.3 degrees four hours after reorientation; normal

plants had bent an average of 87.6 degrees in the same

period. After the coleoptile had ruptured, lazy plants bent

an average of only 3.4 degrees in 48 hours after reorienta-

tion, whereas normal plants bent an average of 40.3 degrees.

Five days after reorientation normal plants were vertical

but lazy plants still averaged only about three degrees of


The geotropic reactivity of normal corn plants was

also examined in more mature plants. Five days after hori-

zontal placement, 50 six-week-old potted plants exhibited an

average curvature of only 39.7 degrees resulting from bend-

ing at only one node. Young plants continued to respond in

this manner up to the period of rapid elongation which com-

mences approximately 60 days after planting. At this time

plants become more responsive to gravity and remain so for

most of their subsequent life. Average bending of 90 de-

grees is usually achieved in about 48 hours with the bending

distributed over four or five nodes (Figure 2). As the

plants mature, the nodes which respond are located further

up the stem, presumably since lower nodes have lost their

potential for elongation. For example, when silks appear on

the ear, the ear node has largely lost its potential to re-

spond to gravity.

Growth habit of lazy corn plants

In the course of growing corn for the crossing program

pits were dug next to several lazy plants after they had

started to bend over. As anticipated, the lazy corn stems

continued to bend until the stems were vertical with the

tassel pointing downward (Figure 4). This inverted growth

habit is taken to be the typical habit of growth of mature

lazy plants.


Both Van Overbeek (89) and Shafer (83) observed that

young lazy corn seedlings responded to gravity. These ob-

servations were confirmed in the present study with the

qualification that only coleoptiles of lazy seedlings are

capable of responding to gravity. Young seedling stems are

not responsive. The expression of the lazy gene is ap-

parently manifested in the stem but not in the coleoptile of

lazy plants. The expression of this gene, in the genetic

stock examined, was not associated with significant effects


Fig. 4.--The uninhibited growth habit of lazy corn plants.
The pit into which this plant is growing was dug
after the plant had started to bend.


on eitherr the growth or the breakLng strength of stems of

lazy plants.

The coleoptiles of normal corn seedlings respond to

gravity at a much faster rate than young seedling stems. As

the stem elongates, however, it becomes progressively more

responsive to gravity. The response is maximal and involves

four or five nodes subsequent to the period of rapid elonga-

tion of the stem. The experiments reported in subsequent

sections utilized plants in this growth period when there

was a maximum amount of geotropically responsive tissue.



A convenient assay was needed to test the effect of

various compounds on the geotropic reaction of corn stems.

The assay was particularly needed to test the effect of IAA

since it was planned to use IAA in transport experiments.

Corn stems were defoliated one internode at a time,

and the stem cut into segments one cm below each node. The

morphological base of the stem segment was placed immedi-

ately in an appropriate solution one cm deep. The upper end

of the segment was inserted into tight-fitting tubing at-

tached to an aspirator and a vacuum applied for one minute.

In preliminary tests with safranin, the dye appeared at the

upper end of the segment 20 to 30 seconds after the applica-

tion of vacuum. Subsequent dissection of the stem showed

red dye associated with the vascular strands.

After infiltration, the section was trimmed two cm be-

low the node and five cm above, and the distal end of the

segment covered with parafilm. The segment was then ori-

ented horizontally by inserting its base into a block of

water-saturated plastic foam (Oasis brand). The angle of


the segment from the horizontal was determined. If the

angle deviated more than five degrees, the segment was re-

inserted. The block of plastic foam with the stem segments

was placed in a chamber in the dark at a temperature of

25t10 C and a relative humidity of 90 to 95 percent. The

angle of bend of a segment from the horizontal was deter-

mined after 96 hours.

The chemicals employed were all dissolved in 0.02 M

pH 6.0 potassium phosphate buffer. The buffering capacity

of the phosphate was adequate for all compounds used. IAA

was first dissolved in an equimolar amount of 0.10 N sodium

hydroxide (5 mg IAA equivalent to 0.286 ml of base) with a

magnetic stirrer before being diluted with phosphate. Lar-

sen (48) stated that a five-fold excess of base is required

to affect solution of IAA. One lot of IAA (Nutritional

Biochemicals Corporation), however, dissolved in less than

10 minutes; another lot of IAA (Fisher Lot 794428) did not

dissolve in two hours. These lots of IAA have identical

melting point ranges which agree with literature values and

equivalent biological activity in the standard Avena test,

and in reversing radiation inhibition of the geotropic re-

action.1 Both lots produce only one spot upon chromatography

H. J. Teas and T. W. Holmsen, unpublished experiments.

with two solvents and three color reagents. Apparently

these lots of IAA differ only in their physical form. The

Nutritional Biochemicals Corporation product is supplied in

the form of platelets, whereas the Fisher product is granu-

lar. Because of the difference in solubility, only the Nu-

tritional Biochemicals Corporation IAA was used.


In preliminary tests with the assay, the eight apical

nodal sections of 87-day-old second inbred generation plants

were all treated with the same compound (Table 4). The type

of response observed is shown in Figure 5. (The plant ma-

terial for these photographs came from a later experiment.)

NP and 2,5-dinitrophenol (DNP) inhibited geotropic reac-

tivity of normal corn, as reported by Larsen (51) and others

for a number of plants. The apparent stimulation by de-

ionized water and 10-4 M NaCN might have been an artifact.

In further preliminary screening, this apparent stimulation

was not observed, nor did 10-4 M 2,3,5-triiodobenzoic acid

and 10-4 M chlorogenic acid have an effect. However, 10-3 M

2,3,5-triiodobenzoic acid markedly inhibited the geotropic

reaction as in the case of other plants (71, 87).

The average angle of bend for the reacting nodes ex-

hibited a maximum at the fourth node from the apex


Normal Plants

Progeny Treatment**

1 2 3


Deionized water
10-5 M IAA
10-4 M IAA
10-5 M NP
10-4 M NP
10-4 M NaCN
10 M NaCN
10-4 M DNP
10-3 M DNP
10 M DNP

Avg.-Nodes of Reacting

0 10
.2 21
5 8
3 5
0 5
0 0
0 32
0 16
0 3
0 0

Node Number*

4 5 6
17 8 5
30 30 0
2 10 0
13 7 8
12 22 18
0 0 0
36 18 7
19 0 0
20 17 0
0 0 0

7 8 sum

2 12 17 19 14 5 4 1

Lazy Plants

Progeny Treatment**


Deionized water
10-5 M IAA
10-4 M IAA
10-5 M NP
10-4 M NP
10-4 M NaCN
10-3 M NaCN
10-4 M DNP
10-3 M DNP
*Node number 1 is

Node Number*
1 2 3 4 5 6 7 8 Sum

0 0 6
0 0 0
0 0 0
6 0 0
0 0 0
5 31 11
0 0 0
0 0 0
0 0 0
0 0 0
apical node.

10 0
0 8
0 0
7 4
0 0

**The concentration listed for a compound refers to
the concentration infiltrated and not to the concentration
in the tissue.

0 0 0

v v "




Fig. 5.--Corn node segments in plastic foam 96 hours after
horizontal placement of the segments. The segments
on the left in the upper view correspond to the
segments in front in the lower view. In the lower
view, the fifth segment from the right is the ear
node. Note that segments proximal to the ear node
are relatively unreactive, whereas distal nodes
exhibit a marked response to gravi y.

.............E l

(Table 4). The reactivity of nodes distal to the fourth

node fell off gradually; that of proximal nodes was more

abrupt. This abrupt reduction occurred at the ear node

(see Figure 5).

It is significant to note that nodal sections from

lazy plants did not exhibit a positive geotropic reaction

(Table 4). In fact, after infiltration with either buffer

or deionized water an occasional node exhibited a negative

geotropic reaction. The results suggest that NP may cause

nodal sections of lazy plants to become negatively geotropic.

This compound was selected for more intensive study.

The results of an experiment employing five levels of

NP are presented in Table 5. A Latin square design (85) was

employed with a pot containing both sibling lazy and normal

plants as columns and nodal sections as rows. The corre-

sponding nodes of normal and lazy plants growing in the same

pot received the same treatment. This design permitted a

comparison of the reactions of lazy and normal plants to the

compound. The experimental plants were 87-day-old third

generation siblings. NP clearly reduced the geotropic re-

action of normal nodal segments. However, there was no sig-

nificant trend in the effect of NP with respect to concen-

tration. As in the screening experiments (Table 4), nodal



Treatment Normal Plants Lazy Plants
degrees degrees
Buffer 29.0 7.5
10-5 M NP 6.8 4.3
5x10-5 M NP 2.3 6.5
10-4 M NP 14.8 -0.5
5x10-4 M NP 9.5 6.3
10-3 M NP 4.3 2.5

Analyses of Variance


Source of Variation d.f. Mean Square

Pots 5 112.368
Nodes 5 77.258
Buffer vs. NP 1 1,575.025**
Remainder 4 41.807
Error (a) 20 111.247

Normal vs. lazy (C) 1 806.681*
Pots x C 5 154.780
Nodes x C 5 144.547
Treatment x C
Buffer vs. NP 1 789.136*
Remainder 4 151.817
Error (b) 20 181.240

Table 5--Continued

Source of Variation

Normal Corn
Buffer vs. NP
Error (a)

Lazy Corn
Error (b)
Comparison (+ vs.

0.05 level.


Mean Square





at the 0.01 level; *significant at the


segments from lazy plants exhibited a negative geotropic re-

action although significantly less than that of normal

plants. On the average, NP treatment did not differ sig-

nificantly from buffer treatment of lazy plants. There was

a significant quartic trend in the effect of NP, however, on

the geotropic reaction of nodal stem segments of lazy plants

(Figure 6).

The results of this experiment were confirmed in a

similar subsequent experiment; that is, the geotropic re-

action of nodal stem segments from normal plants was reduced

by NP but the effect did not exhibit significant maxima,

minima, or trend in the concentration range 10-5 M to 10-3 M

NP. Nodal stem segments from lazy plants exhibited a nega-

tive geotropic reaction which was significantly less than

that of normal segments. The quartic trend of reactivity

of nodal stem segments from lazy plants with concentration

of NP was again observed (Figure 6).

In three experiments with a total of 108 nodal stem

segments from lazy plants, no effect of IAA in the concen-

tration range of 10-5 to 10-3 M was observed. This result

was expected since Van Overbeek (90) found that lazy plants

contain a slightly greater auxin content than normal plants.

The effect of IAA on geotropic bending of nodal stem

0 91- day-old plants
.. .J6-day-old plants

0 / /

zw / /




0 10-5 5x5 10-4 5x10-4 10-3

Fig. 6.--Response of nodal stem segments of lazy corn to
various concentrations of NP. Each bar represents
the average of six observations.

Lj 92-day-old plants
gr 87-day-old plants
w 40
C I :I .. . . .... / / . . .. .. .

CD 30

j 20/ / /

0 10-5 5x10-5 10-4 5xl0-3 10-3

Fig. 7.--Response of nodal stem segments of normal corn to
various concentration- of TPA. Each bar represents
the average of siy: observations. Dotted line
represents level of response of control.

segments from normal plants was examined in a Latin square

design (85) with plants as columns and nodal segments as

rows. Bending was increased when nodal stem segments were

infiltrated with 10-4 M IAA (Figure 7). However, a signifi-

cant effect of IAA treatment on the geotropic response of

these segments could not be demonstrated (Table 6). Concen-

trations of 10-4 M and 5 x 10-4 M IAA also stimulated bend-

ing in a subsequent experiment (Figure 7). A significant

effect of IAA treatment, however, could not be demonstrated.


The fact that isolated nodal segments of corn stems

respond to gravity indicates that these segments contain the

entire geotropic apparatus. Reduction in geotropic reac-

tivity resulting from NP, DNP, and 2,3,5-triiodobenzoic

acid, therefore, can not be attributed to any one phase of

the geotropic reaction on the basis of this assay.

IAA is known to exist in large quantities (105,000

microgram IAA equivalents per kg fresh weight) in endosperm

of corn seed (53). Housley and co-workers (35), however,

could find no chromatographically identifiable IAA in ex-

tracts of four-day-old corn seedlings. Apparently there is

no record of attempts to identify the auxin(s) of mature

corn plants.





10-5 M IAA

5x10-5 M IAA

10-4 M IAA

5x10-4 M IAA

10-3 M IAA








Analysis of Variance

Source of Variation








Mean Square





Treatment effect not significant at the 0.05 level.

The effect of IAA concentration on geotropic bending

of corn node segments (Figure 7) follows a typical IAA re-

sponse curve in going through an optimum (53). It is not

surprising that a significant effect of IAA was not observed

since horizontal placement of corn stems results in an in-

creased production of auxin (83). Brandes and McGuire (8)

observed an effect of IAA on the geotropic response of sugar

cane stems only after treating the stems for 20 minutes at

520 C. A similar depletion of auxin from corn nodal seg-

ments should permit detection of a significant effect of IAA

on geotropic bending of the segments.

Nodal stem segments from lazy corn plants exhibit a

slight, but real, negative geotropic reaction. The exist-

ence of this reactivity is confirmed by the observation that

NP inhibits geotropic bending of the segments. The facts at

hand do not permit identification of the deficiency in the

geotropic reaction inherent in lazy corn. It is, however,

capable of perception of the stimulus of gravity, at least

to a limited degree. It is also significant that nodal stem

segments from lazy plants do not exhibit a positive geo-

tropic reaction.


Materials and Methods

As a logical extension of the Cholodny-Went theory one

might expect to observe an increase in lateral transport of

exogenous auxin from the upper to the lower surface of a

horizontally placed stem as compared to transport from the

lower surface to the upper surface. IAA transport in corn

stems was examined in isolated blocks of internal tissue

(ground parenchyma and vascular tissue [20]) taken from

growth rings of corn stems. The blocks were cut from the

morphological center of the stem by means of two parallel

single edge ejector-type razor blades mounted with their

long axis perpendicular to the jaws of a jewelers vise. The

size of the blocks varied from experiment to experiment.

Two lots of IAA 2-C14 were used in these experiments.

The first lot (Nuclear-Chicago) had a specific activity of

21.7 microcuries per mg, and the second (Orlando Research)

6.58 microcuries per mg. Both lots were examined by as-

cending filter paper strip chromatography in two solvents

(water-saturated n-hexane and butanol:acetic acid:water,

10:l:l,v:v:v) and with three indole color reagents:

p-dimethylaminobenzaldehyde (53), p-dimethylaminocinnamal-

dehyde (28), and FeC13-HCIO4 (53). In all cases IAA was the

only compound discovered on the chromatograms. After color

development the filter paper strips were cut perpendicularly

to the direction of solvent flow into one cm segments and

the radioactivity in the segments determined by counting

them directly. Determinations of radioactivity were made

with a gas flow counter with a "micromil" window and operat-

ing in the Geiger region. In all cases the peak of radio-

activity coincided with the color spot on the chromatogram.

The methods outlined by Larsen (48) were adopted for

making solutions of IAA and IAA agar. The radioactive IAA

was dissolved in redistilled ether and stored until used in

cork-stoppered brown glass bottles at 50 C. A given amount

of the ether solution was evaporated in a test tube in a

water bath at 500 C by passing CaC12 dried air at 1.5 p.s.i.

into the tube. As soon as the ether was evaporated, equal

quantities of 0.02 M pH 6 citrate buffer and 0.004 M CaCl2

were added to the tube which was then stoppered and set

aside at room temperature for 30 minutes. Filtered-auto-

claved 2.5 percent agar which had been melted and then

cooled to 500 C was then added to the tube. The final

concentrations of the various components were 1.4 x 10-4 M

IAA, 0.01 M pH 6 citrate buffer, 0.002 M CaCI2, and 1.25

percent agar. The warm agar was pipetted into 2.0 mm thick

stainless steel forms to solidify. The agar blocks were

trimmed to the thickness of the form and then cut into

platelets with the same cross-sectional area as the tissue

block to which they were subsequently applied. Recipient

agar platelets were made in the same manner with the omis-

sion of radioactive IAA.

The agar platelets were applied to freshly cut tissue

blocks; a platelet containing IAA 2-C14 being applied to one

side of the block and recipient agar containing no IAA

placed on the opposite side. The various combinations of

tissue configuration and orientation are shown in Figure 8.

The tissue and agar platelets on shallow aluminum planchets

were placed in a humid chamber for the period of transport.

Transport was carried out in diffuse light (2-3 foot can-

dles) at a temperature of 2510 C and a relative humidity

of 90-95 percent. At the termination of the time for trans-

port the tissue block was removed and the agar dried di-

rectly on the planchet under infrared light. The radioac-

tivity of the dried agar platelet was then determined.






Fig. 8.--Configurations of blocks of corn stem tissue and
agar to study transport of IAA through the tissue.
Lines on the tissue indicate orientation of vascu-
lar tissue. Heavy double line indicates morpho-
logically upper end of block. Stippled area repre-
sents donor agar containing 2-C14 IAA.




Losses of radioactivity' from tissue blocks

It was necessary to learn whether IAA 2-C14 was me-

tabolized to CO2 by corn tissue blocks and whether tissue

orientation had an effect. Tissue blocks were infiltrated

with IAA 2-C14 in a vertical orientation from donor agar

pl3te-lts at both the pro:-lmial and distal ends of the block.

Three hours after placement of the agar the tissue blocks

were removed from the agar and bisected vertically. One-

fourth of the bisected blocks were put immediately into

formalin-acetic acid-alcohol (FAA); one-fourth remained ver-

tical; one-fourth were placed horizontally so that both

halves became upper halves; and one-fourth placed horizon-

tally so that both halves became lower halves. After 24

hours in the dark at 25110 C and 92-95 percent relative hu-

midity the last three groups of blocks were also placed in

FAA. The radioactivity removed from these blocks by three

extractions with FAA is tabulated in Table 7. There was no

evidence for catabolism of the added IAA 2-C14 to CO2 during

the 24-hour period following administration of the isotope.

In transport e:-:periuents, then, no correction need be made

for losses of radioactivity during the experimental period.


Conditions of the Experiment

Plants: Third inbred generation normal (102-11 and 102-7);
87 days old; shedding pollen
Internodes: 4-7 from apex
Tissue blocks: 4 x 4 x 4 mm
IAA: 5 x 10-4 M; about 10,100 cpm per donor platelet
Design: Two 4 x 4 Latin squares with plants as squares, in-
ternodes as columns, and position on internode as

Treatment Radioactivity
per Block
Radioactivity determined immediately 7,546
Radioactivity determined 24 hours after:
horizontal placement-lower halves 7,443
horizontal placement-upper halves 7,770
vertical placement 7,913

Analysis of Variance

Source of Variation d.f. Mean Square

Plants 1 90,979,933
Internodes within plants 6 2,192,672
Position in internode
within plants 6 1,629,151
Treatments 3 361,864
Remainder 3 1,210,573
Combined error 12 970,623

Treatment effect not significant at the 0.05 level.

Characteristics of lateral transport

The time-course of lateral transport waz e::amlned for

both the horizontal-above and horizontal-below configura-

tions. Diagrams of the appropriate configurations are shown

in Figure 8 and Tables 8 and following. In this experiment,

a randomized block design (85) with plants as replications,

tissue blocks were cut only from nodes of the stem. The

same tissue blocks were used throughout the eight-hour

transport period, but the recipient agar platelet was re-

placed one, two, and four hours after commencement of the

experiment. In the eight hour transport period, there was

no significant difference in the amount transported between

the two orientations (Table 8).

The time-course for transport is shown in Figure 9.

After an initial lag period of about 1.14 hours, lateral

transport in horizontally placed stems was linear with time.

If the concentration gradient in the tissue block is linear,

an apparent diffusion coefficient, D, for lateral transport

may be calculated (Table 9). The table includes estimates

from a subsequent experiment with the horizontal-above con-

figuration ("Horizontal-above-2").

The diffusion coefficient, D25, estimated from the re-

sults of these experiments ranged from 1.18 x 10-7 to


Conditions of the Experiment

Plants: Second inbred generation normal (60-8, 60-5); 61
days old; pre-tassel stage
Nodes: 8 and 9 from apex
Tissue blocks: 7.5 x 7.5 x 4 mm; transport through 4 mm
Time: 8 hours
IAA: 1.15 x 10-4 M, about 38,900 cpm per donor platelet


Average Radioactivity in Recipient Agar


Analysis of Variance

Source of Variation d.f. Mean Square

Plants 9 846.44

Orientation 1 346.11

Error 9 120.82



horizontal- below

ww 30

0 horizontal above

O O'
0 C

0 2 4 6 8

Fig. 9.--Time-course of lateral transport of radioactive
IAA in horizontally placed corn stem tissue


Corn Tissue Blocks




(1) Activity in donor

(2) Volume of donor

(3) IAA concentration

(4) Rate of transport





(5) dQ=(2)(3)(4) moles
dt 3600(1) sec 8.77x0-15

(6) a-cm2

(7) A x-cm

(8) D25-cm2/sec







1.15x10-4 5.0x10-4









5.41x10-7 4.60x10-7 1.18x10-7

dQ -Da dC
dt dx



5.41 x: 10-7 cm2 per sec. These values for lateral transport

of LAA through corn stem sections are considerably lower
than the value of 7.45 :: 10-6 2 pr c or th ffuion
.. per sec for the diffusion

of IAA through 1.5 percent agar calculated by Larsen (48).

In one of the subsequent transport e>-perrLient (Table

14) the average distribution of radioactivity at the end of

a six-hour transport period was 3,735 cpm in the donor agar

platelet, 6,380 cpm in the tissue, and 35 cpm in the re-

cipient agar platelet. The amount of radioactivity in the

tissue was determined by difference from the total amount

added, 10,150 cpm (average of 10 determinations). IAA dif-

fusion was carried out in a stack of two mm agar platelets

concurrently with the transport experiment. At the end of

three hours, the distribution of radioactivity in these

platelets was 3,953 cpm in the donor agar platelet, 3,042

cpm in the next lower agar platelet, 1,862 cmp in the next

lower agar platelet, and 1,389 in the lowest agar platelet.

Radioactivity in a parallel stack of platelets reached equi-

librium at the end of six hours. It should be noted that

the stack of agar platelets was one mm thicker than the

stack of agar and tissue blocj. combined.

If IAA were moving through the tissue block by a

process of diffusion and were not accumulated by the tissue,


then a linear concentration gradient would exist in the tis-

sue block. Under these conditions, the tissue block would

contain (3,700)(1.5/2) or about 2,670 cpm. Clearly, then,

IAA accumulates in the tissue.

The effect of DNP on lateral transport of IAA was

examined in an experiment employing two nodal sections from

each of four normal plants. The concentration of DNP used,

10-3 M, was found to inhibit the geotropic response of corn

nodal sections (Table 4). Nodal sections were infiltrated

with either DNP or buffer (0.02 M potassium phosphate,

pH 6.0) and after one hour 5 x 5 x 3 mm tissue blocks were

cut from the nodes of the sections. Transport was carried

out in the horizontal-above configuration for three hours

with 5 x 10-5 M IAA 2-C14 in the donor agar. Radioactivity

in the recipient agar platelets of buffer-treated tissue

blocks was 11.2t1.5 cpm. The radioactivity of recipient

agar platelets of DNP-treated tissue blocks was 11.22.4

cmp. DNP at a concentration which inhibits the geotropic

reaction of nodal stem segments did not inhibit the lateral

transport of IAA 2-C14 through tissue blocks from these seg-


Since there is a gradient of geotropic responsiveness

along the successive nodes of a corn stem (Table 4, Figure


5), lateral transport was examined with respect to nodal po-

sition on the stem. The results of an experiment measuring

transport in the horizontal-above configuration (Table 10)

failed to indicate any such gradient. The slope of the re-

gression line (0.0176) of lateral transport compared to node

position did not differ significantly from zero (t=0.0157,

32 d.f.). Although there was considerable variation between

plants, there was no significant deviation from parallelism

between the regression lines for the individual plants (85).

Effect of gravity on lateral transport

As shown in Table 8, transport in the horizontal-below

configuration was slightly greater than transport in the

horizontal-above configuration, but the difference was not

significant. Transport in these configurations was examined

in several additional experiments. In the first of these,

an evaluation was made of the effect of the orientation of

the stem prior to the transport measurements. One group of

plants was placed horizontally 12 hours before tissue blocks

were taken from them; a comparable group of plants remained

vertical during this time. As shown in Table 11, there was

no significant difference in transport between the horizon-

tal-above and horizontal-below configurations regardless of

the orientation of the stem prior to measurement of



Conditions of the Experiment

Plants: Second inbred generation normal (59-1, 59-18, 60-8,
and 61-11); 63 days old; pre-tassel stage
Nodes: 6-10 from apex
Tissue blocks: 7.5 x 7.5 x 4 mm; transport through 4 mm
Time: 2 hours
IAA: 1.15 x 10- M, about 38,900 cpm per donor platelet

Node Transport of IAA
6 13.9
7 15.4
8 13.6
9 14.7
10 16.9

Analysis of Variance

Source of Variation d.f. Mean Square

Plants 7 308.26

Joint Regression 1 1.26

Parallelism 7 .37

Error 18 28.54


Conditions of the Experiment

Plants: Second inbred generation normal (60-6 and 60-10);
56 days old; pre-tassel stage
Nodes: 9-11 from apex
Tissue blocks: 7.5 x 7.5 x 4 mm; transport through 4 mm
Time: 1 hour
IAA: 1.15 x 10- M, about 38,900 cpm per donor platelet

Average Radioactivity in Recipient Agar
Orientation Stems Vertical Stems Horizontal
before 12 Hours before
Experiment Experiment
cpm cpm
4.91 4.49

6.11 4.08

Analyses of Variance

Stems Vertical Stems Horizontal
before 12 Hours before
Experiment Experiment
Source of
Variation d.f. Mean Square Mean Square

Plants 7 17.09 4.73
Orientation 1 5.76 0.68
Error 7 12.64 11.44


transport. This and a subsequent experiment (Tables 11, 12)

originally included a comparison of the horizontal transport

configurations with the vertical-lateral orientation (Figure

8). An artifact resulted, hence the data are not included

in Tables 11 and 12.

A comparison was made of lateral transport in lazy as

well as normal plants. The experimental design employed was

a randomized block (85) with a pot containing a normal and

lazy plant taken as a block. The analysis of the experiment

followed procedures outlined previously (Table 5) modified

to a randomized block design.

The results of the experiment (Table 12) again indi-

cated no significant difference in lateral transport in the

horizontal-above and horizontal-below configurations. No

effect of gravity on lateral transport could be demonstrated

in either normal or lazy tissue blocks. Lateral transport

in tissue blocks from lazy stems was less than lateral

transport in comparable tissue from normal stems but the

difference fell short of significance (F=5.23; F05=6.61).

It should be noted that the normal and lazy plants compared

were not siblings but were from sister lines which differ by

one generation of independent segregation.

A method which permitted evaluation of lateral


Conditions of the Experiment

Plants: Second inbred generation normal (61-16) and lazy
(61-2) growing in the same pot; 75 days old; shed-
ding pollen
Nodes: 7-9 from apex
Tissue block: 7.5 x 7.5 x 3 mm; transport through 3 mm
Time: 2 hours
IAA: 1.15 x 10-4 M, about 38,900 cpm per donor platelet

Configuration Average Radioactivity in Recipient Agar
Normal Plants Lazy Plants
cpm cpm
17.2 7.9

16.6 13.9

Analysis of Variance

Source of Variation d.f. Mean Square

Pots 5 45.14
Orientation 1 63.69
Error (a) 5 81.49

Lazy vs. Normal (C) 1 217.80
Pots x C 5 80.51
Orientation x C 1 45.10
Error (b) 5 41.61

transport in the vertical-lateral orientation consisted of

placing the tissue block on the edge of a glass microscope

slide held firmly in a horizontal staining dish. The place-

ment of the tissue block was such that neither donor nor re-

cipient agar platelet came in contact with the glass slide,

thus preventing the artifact obtained in previous trials.

An experiment to compare the three configurations of

lateral transport with polar transport (Figure 8) again

failed to demonstrate any influence of gravity on lateral

transport in either normal or lazy plants (Table 13). The

results indicated that polar transport was about three times

as fast as lateral transport. This difference was signifi-

cant. As in the previous experiment, tissue blocks from

lazy plants transported less than tissue blocks from normal

plants. This difference was significant in the present ex-

periment, which had a higher sensitivity than the previous

one. It should be noted again that this comparison of nor-

mal and lazy plants was made between sister lines and not

between sibling plants.

In all of the foregoing transport experiments, tissue

blocks were taken from median sections of nodal tissue, that

is, all sections included the morphological center of the

stem. Measurements of lateral transport in such horizontally


Conditions of the Experiment

Plants: Second generation inbred normal (61-16) and lazy
(61-2) growing in the same pot; 77 days old; shed-
ding pollen
Nodes: 6-9 from apex
Tissue blocks: 7.5 x 7.5 x 3 mm; transport through 3 mm
Time: 3 hours
IAA: 1.15 x 10-4 M, about 38,900 cpm per donor platelet

Orientation Average Radioactivity in Recipient Agar
Normal Plants Lazy Plants
cpm cpm
14.3 5.0

9.2 6.5

E__ 9.3 5.2

S32.0 17.1

Analysis of Variance
Source of Variation d.f. Mean Square
Pots 7 34.56
Polar vs. Lateral 1 3,176.88**
Remainder 2 24.41
Error (a) 21 114.98
Lazy vs. Normal (C) 1 956.36**
Pots x C 7 110.43
Transport x C 3 124.11
Error (b) 21 89.51

**Indicates significance at the 0.01 level.


placed tissue blocks estimate the net effect of transport in

the upper and lower halves of the block. If the effect of

gravity were to induce an increase in lateral transport in

one half of a horizontally placed stem and a proportionate

decrease in lateral transport in the other half, then the

methods employed would not detect this change.

Nodal tissue was cut longitudinally into thirds and

the center third discarded in an experiment to measure

lateral transport separately in upper and lower halves of

the stems. Tissue blocks cut from the peripheral thirds

were made "upper halves" by placing the edge formed by the

original longitudinal cut downward. Similarly, tissue

blocks were made "lower halves" by placing the edge formed

by the original longitudinal cut upward. Two nodes were

used per plant. The two tissue blocks from one node were

made either "upper" or "lower." One of the blocks was used

to measure lateral transport of IAA from the lower surface

of the block to the upper surface. The other block was used

to measure lateral transport from the upper surface to the

lower surface of the block. A split-plot design was em-

ployed (85). No differences were observed in either the

overall capacity to transport or the capacity to transport

laterally in either direction (Table 14).


Conditions of the Experiment

Plants: Third generation inbred normal (102-2, 102-11, and
106-4); 84 days old; shedding pollen
Nodes: 7 and 8 from apex
Tissue blocks: 4 x 4 x 3 mm; transport in 3 mm direction
Time: 6 hours
IAA: 5 x 10-4 M, about 10,100 cpm per donor platelet

Orientation Direction of Average Radioactivity
Transport in Recipient Agar
Upper From Above 34.3
Upper From Below 39.7
Lower From Above 29.3
Lower From Below 35.1

Analysis of Variance

Source of Variation d.f. Mean Square

Main plots
Plants 4 283.60
Orientation: Upper vs.
Lower (0) 1 114.24
Error (a) 4 1,412.79

Transport: Above vs.
Below (T) 1 155.68
OxT 1 270.05
Error (b) 8 443.89

Effect of gravity on polar transport

In all lateral transport experiments, the radioactive

IAA was supplied to the tissue block chiefly through the

parenchyma of the stem and perhaps occasionally through one

or two vascular bundles exposed in cutting the block. If

auxin is normally supplied to the stem through vascular

tissue, then failure to demonstrate the influence of gravity

on lateral transport may result from the mode of application

of the radioactive IAA. The diagonal configurations (Figure

8) were adopted to examine this possibility. In these con-

figurations, the tissue block is supplied with IAA from both

the polar and lateral directions. They provide a measure of

net lateral and polar transport in one half of a tissue

block as limited by lateral transport from the other half of

the block.

Experiments on lateral transport utilized tissue

blocks only from the growth rings (nodes) of the stems since

geotropic bending is manifested in this area. This differ-

ence in response between the growth ring and the remainder

of the internode may result from the difference in growth

potential and not from intrinsic differences in the capa-

bility to transport auxin. In preliminary experiments to

work out techniques for examining transport in the diagonal

configurations, the average transport for eight tissue

blocks cut from the growth ring was 18.1 cpm and was 20.4

cpm for similar tissue blocks cut from the adjacent inter-

nodal segments.

As a result of these observations, the experimental

design previously employed was modified. A pot containing a

normal and lazy plant was taken as the basic unit for a

4 x 4 Latin square with internodes as columns and position

on the internode as rows. Tissue blocks and agar in the

diagonal configuration were placed on planchets held at an

angle of 45 degrees to the horizontal. The tissue blocks in

the polar-horizontal configuration (Figure 8) rested on the

edge of a glass microscope slide in a manner similar to that

described for the vertical-lateral orientation. As in all

previous experiments, there was no evidence to support the

hypothesis that gravity influences lateral transport of ex-

ogenously applied IAA (Table 15). The experimental results

did show, however, that polar transport was significantly

greater than transport in the polar-horizontal configura-

tion, that is, gravity reduced polar transport in horizon-

tally placed tissue blocks. Furthermore, these results con-

firmed the observation made in the previous experiment that

polar transport of IAA was significantly greater than


Conditions of the Experiment

Plants: Second inbred generation segregating normal and
lazy (58-12); 106 days old
Internodes: 2-5 from apex
Tissue blocks: 3 x 3 x 3 mm
Time: 3 hours
IAA: 10-4 M, about 6,200 cpm per donor platelet

Orientation Average Radioactivity in Recipient Agar
Normal Plants Lazy Plants
cpm cpm
44.9 35.3

D 23.1 29.3

17.9 25.3

20.0 22.3

Table 15.--Continued

Analysis of Variance

Source of Variation

d.f. Mean Square

Internodes in plants
Position on internode in plants
Polar vs. Diagonal
Polar vs. Polar-Horizontal
Diagonal vs. Diagonal-Horizontal
Error (a)

Normal vs. Lazy (C)
Plants x C
Internodes in plants x C
Position on internode in plant x C
Orientation x C
Error (b)





**Indicates significance at the 0.01 level; *0.05

lateral transport. There was also no significant differ-

ence in IAA transport between sibling lazy and normal


The influence of gravity on polar transport was ex-

amined in another experiment with hybrid normal plants

(Table 16). Gravity again reduced the polar transport of

IAA in horizontally placed tissue blocks. The influence of

gravity in these two experiments (Tables 15 and 16) was

relatively the same, causing a reduction in polar transport

of about 45 to 48 percent.

Characteristics of polar transport

NP and DNP both inhibit the geotropic reaction of corn

stems (Tables 4 and 5) and are known to reduce polar trans-

port in other species (58, 60). Therefore, the effect of

these compounds on polar transport of IAA in corn stem tis-

sue blocks was examined. Nodal stem segments were infil-

trated with the reagents as in the geotropic assay of iso-

lated corn node segments. Each stem segment was divided

laterally into three sections and two tissue blocks cut from

each section. One tissue block was placed in the polar

orientation, the other block in the polar-horizontal orien-

tation. A split-plot design was employed for the experi-

ment (85).


Conditions of the Experiment

Plants: Hybrid; 89 days old; had shed pollen
Nodes: 4 and 5 from apex
Tissue blocks: 7.5 x 7.5 x 3 mm; transport through 3 mm
Time: 3 hours
IAA: 10-4 M, about 6,200 cpm per donor platelet

Orientation Average Radioactivity in Recipient Agar


Analysis of Variance

Source of Variation d.f. Mean Square

Internodes 5 224.04
Orientation 1 851.93**
Error 5 47.05

**Indicates significance at the 0.01 level.

The extent of participation of xylem and adjacent

lacunae in polar transport was also evaluated. If air is

pulled through a stem section, transport through these ele-

ments is prevented. In the present experiment, nodal stem

segments were infiltrated with buffer (0.02 M potassium

phosphate, pH 6.0), air was pulled through one-half of these

infiltrated segments, and then tissue blocks cut from them.

NP and DNP greatly reduced polar transport in corn

stems (Table 17) as in other species of plants (58, 60).

The action of these compounds was such that no further re-

duction of transport was observed in horizontally placed

tissue blocks. Transport observed in these tissue blocks

might be attributed to diffusion of IAA through the xylem

and lacunae. To test such a suggestion, however, one must

know how long it takes for these reagents to reach a locus

of action so that subsequent air treatment will not remove


The experimental results also showed that about 40

percent of polar transport as previously measured is attri-

butable to transport through xylem and lacunae. The portion

of the IAA thus transported was not influenced by gravity.

There was, however, a significant influence of gravity on

that portion of the IAA transported by non-lacunar tissue.


Conditions of the Experiment

Plants: Second inbred generation normal (58-8); 84 days
old; shedding pollen
Internodes: 3-6 from apex
Tissue blocks: 3 x 3 x 3 mm
Time: 3 hours
IAA: 1 x 10-4 M; about 34,800 cpm per donor platelet

Treatment Average Radioactivity in Recipient Agar

cpm cpm
10-3 M DNP 5.9 5.8
10-4 M NP 6.3 6.0
Air 11.9 3.9
Buffer 20.0 11.7

Analysis of Variance

Source of Variation d.f. Mean Square

Main plot
Plants 1 0.91
NP vs. DNP (A) 1 0.58
Buffer vs. air (B) 1 381.60**
Buffer+air vs. NP+DNP (C) 1 418.90**
Error (a) 19 3.48
Polar vs. Polar-Horizontal (0) 1 210.84**
Ax O 1 0.20
Bx O 1 0.48
C x O 1 190.32**
Error (b) 20 10.46

**Indicates significance at the 0.01



This component of polar transport was reduced about 60 per-

cent by gravity. Transport in the polar and anti-polar

(Figure 8) configurations was measured as part of another

experiment. Four tissue blocks were used for each configu-

ration for both normal and lazy plants. The tissue blocks

were 7.5 x 7.5 x 3 mm, and transport was carried out for two

hours. Anti-polar (acropetal) transport occurred in corn

stem tissue, but to a lesser extent than polar transport

(Table 18). Lazy and normal plants apparently have equiva-

lent capacities for these two kinds of transport. The re-

sults of the previous experiment (Table 17) suggest that a

considerable portion of transport in the acropetal direction

may have resulted from diffusion through lacunae in the



Average Radioactivity in Recipient Agar

Plant Type ___
cpm cpm

Normal 26.4112.8* 14.611.2

Lazy 25.112.8 17.8111.3

+ standard error.

Polar transport in tissue blocks from sibling normal

and lazy corn was again compared. Under comparable experi-

mental conditions with 24 nodal tissue blocks from each

plant type, polar transport in normal tissue blocks resulted

in 9.112.8 cpm in the recipient agar. and polar transport in

lazy corn resulted in 9.4+4.7 cpm. Thus, there are three

independent observations (Tables 15 and 18) that polar

transport in lazy plants does not differ from polar trans-

port in normal plants.


The rate of lateral transport of IAA in corn stems is

only about 5 percent of the rate of diffusion of IAA

through agar (Table 10). During transport the tissue blocks:

were observed to contain more radioactive IAA than could be

accounted for by a linear concentration gradient (Page 71).

As shown in calculations in the Appendix, cells with a uni-

form capacity on all surfaces to pump a particular compound

into them are incapable of accumulating that compound.

Since parenchyma cells appear to be physiologically uniform

in all directions, they would be unable to accumulate IAA

even Lf th-ey possessed the capacity to pump it into the

cells. The high concentration of IAA in the cells and the

limited rate of lateral transport through them may,

therefore, result from binding but not accumulation of the

IAA in the cells.

DNP decreases both polar transport (Table 18) and

geotropic bending (Table 5) but has no effect on lateral

transport. These facts suggest that lateral transport is a

permeation process rather than an active transport process.

The results of six independent experiments (Tables 9

and 12 to 15) involving 50 pairs of tissue blocks failed to

demonstrate any effect of gravity on lateral transport.

Furthermore, the gradient of geotropic reactivity with nodal

position (Table 5) is not associated with a gradient in

capacity for lateral transport (Table 11). About 60 percent

of the diffusible auxin of corn stems is obtained from the

lower half of the stem. If lateral transport were respon-

sible for this unequal distribution then the ratio of lat-

eral transport from the lower to the upper half of the stem

to that from the upper to the lower half would be about

0.67. The weighted mean ratio observed in these experiments

was 0.989. These experiments were more sensitive to changes

in lateral transport than previous investigations (12, 15,

70) since the IAA flux instead of accumulation in the tissue

was measured.

These data suggest, therefore, that processes other

than lateral transport are responsible for the unequal

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