Order Number 8824647
The roles of polar auxin and calcium transport in the gravitropic response of maize roots
Young, Linda Mull, Ph.D.
The Ohio State University, 1988
Copyright ©1988 by Young, Linda Mull. All rights reserved.
300 N. Zeeb Rd. Ann Arbor, MI 48106 THE ROLES OF POLAR AUXIN AND CALCIUM TRANSPORT
IN THE GRAVITROPIC RESPONSE OF MAIZE ROOTS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of the Ohio State University
By
Linda Mull Young, B.A., M.S.
*****
The Ohio State University
1988
Dissertation Committees Approved by
M. G. Cline
M. L. Evans
G. L. Floyd Advisor F. D. Sack Department of Botany Copyright by
Linda Mull Young
1988 To the Crew of Challenger - may your spirit of adventure live in us always. ACKNOWLEDGMENTS
I wish to acknowledge my advisor, Dr. Michael L. Evans for his encouragement and advice in the preparation of this manuscript and during the performance of the research upon which it is based. I am truly thankful for this opportunity to work with and learn from such an outstanding scientist.
I wish to extend my gratitude to the members of my committee for their valued opinions during the course of this research and their helpful suggestions during the preparation of this dissertation.
Special thanks are extended to Dr. Rainer Hertel whose enthusiasm is contagious. Not only did Rainer suggest many of the experiments described here, but he has also instilled in me some of his scientific curiosity. Rainer's unique approach to research has shown me how satisfying scientific investigation can be.
I wish to thank my friends and co-workers: Mark
Fondren, Karl Hasenstein, John Kiss, June Lee, Chuck
iii Stinemetz, Rosemary White and Rick Yang. Their suggestions, help and support have been invaluable.
Finally, I wish to thank my parents for their encouragement and patience. I also thank and acknowledge my husband, Curtis for his support and understanding during the difficult times. His love and unwavering belief in my abilities helped to make this dissertation possible. VITA
March 31, 1960...... Born - Latrobe, Pennsylvania
1982...... B.A., Wittenberg University Springfield, Ohio Biology and Chemistry
1985...... M.S., The Ohio State University Columbus, Ohio Botany
1985 - Present...... Research and Teaching Associate Department of Botany The Ohio State University Columbus, Ohio
PUBLICATIONS
Mull, Linda L. 1985. An examination of calcium flux patterns in maize root tip protoplasts. Master's Thesis.
Young, LM ML Evans 1987. Correlations between gravitropic curvature and auxin transport across root tips of Zea mays. ASGSB Bulletin 1_: 29.
Young, LM ML Evans 1988. Correlations between gravitropic curvature and auxin transport across root tips of Zea mays. Plant Physiol. 86: 67, suppl.
v Evans, ML, CL Stinemetz, LM Young and WM Fondren 1988. The role of calcium in the response of roots to auxin and gravity. In: Plant Growth Substances 1988. Springer Verlag, ed. R. Pharis.
FIELDS OF STUDY
Major Field: Botany
Studies in Plant Physiology - Michael L. Evans
vi TABLE OF CONTENTS
DEDICATION...... ii
ACKNOWLEDGMENTS...... iii
VITA ......
LIST OF TABLES...... ix
LIST OF FIGURES...... xi
LIST OF ABBREVIATIONS...... xiii
INTRODUCTION...... 1
CHAPTER I. THE NATURE OF AUXIN TRANSPORT IN G RAVI STIMULATED ROOTS OF ZEA MAYS...... 26
Introduction...... 26 Materials and Methods...... 28 Results...... 36 Discussion...... 78
II. CORRELATIONS BETWEEN GRAVITROPIC CURVATURE AND AUXIN TRANSPORT ACROSS GRAVISTIMULATED ROOTS OF ZEA MAYS...... 96
Introduction...... 96 Materials and Methods...... 97 Results...... 100 Discussion...... 112
vii III. POSSIBLE AUXIN TRANSPORT/CALCIUM TRANSPORT INTERACTIONS...... 133
Introduction...... 133 Materials and Methods ...... 134 Results...... 139 Discussion...... 158
SUMMARY...... 176
LIST OF REFERENCES...... 180
viii LIST OF TABLES
TABLE PAGE
1. Polar Auxin Transport Across Caps of Gravistimulated Roots...... 37
2. Time Course of the Development of Downward Auxin Transport Polarity...... 42
3. Auxin Transport Across Tips of Intact and Decapped Roots in Vertical and Horizontal Positions...... 49
4. 3h _i a a Transport Across Isolated Root Caps or Meristems...... 52
5. Effect of KCN on Polarity of ^h - i a a Transport Across the Tips of Gravi stimulated Roots...... 55
6. Transport Across Intact Roots Treated with 10"5 m NPA...... 60
7. 3fj_jAA Transport Across Isolated Root Caps Treated with 10“5 m NPA...... 63
8. Basipetal Movement of 3h - i a a in Gravistimulated Roots Treated with 10"5 M NPA for 60 min...... 65
9. Effect of PBA on Auxin Transport Polarity Across the Caps of Gravistimulated Roots...... 67
10. Effect of PBA on the Basipetal Transport of Auxin in Gravistimulated Roots...... 69
11. 3r -a b a Transport Across Maize Root Tips...... 72
ix 12. Transport of ^h -a b a and ^h - i a a Across Tips of Vertical Maize Roots Following Isotope Loading...... 74
13. Gravicurvature of Control, Prestimulated and Prestimulated/ Rotated Roots...... 110
14. Auxin Transport Across the Caps of Control, Prestimulated and Prestimulated/Rotated Roots...... 113
15. The Effect of Calmidazolium on Gravi- induced Transport of 45ca2+ Across Maize Root Tips...... 140
16. The Effect of Pretreatment of Root Caps with Calmidazolium on Gravi-induced 3h-IAA Redistribution in the Elongation Zone...... 142
17. The Effect of EGTA on ^h -i a a Transport Polarity Across the Caps of Gravi stimulated Roots...... 145
18. Simultaneous Transport of ^h -i a a and 45ca2+ Across the Caps of Gravistimulated Roots...... 148
x LIST OF FIGURES
FIGURE PAGE
1. Auxin Transport in Vertically- and Horizontally-Oriented Roots...... 16
2. The Inositol Trisphosphate Pathway...... 20
3. Time Course of the Development of Auxin Transport Across Caps of Gravistimulated Roots...... 39
4. Comparison of the Rate of Downward Auxin Transport and the Rate of Upward Auxin Transport Across the Caps of Gravi stimulated Roots...... 44
5. Measurement of Auxin Transport Polarity During Discrete Intervals of the Gravitropic Response...... 47
6. Structures of NPA and PBA...... 58
7. Basipetal Transport of 3h -ABA and ^H-IAA in Vertical Roots...... 76
8. The Initial Gravitropic Response of Control Roots vs. Roots Pretreated with Inhibitors of Auxin Transport...... 101
9. Extended Time Course of Gravitropic Curvature in Control Roots vs. Roots Pretreated with Inhibitors of Auxin Transport... 103
xi FIGURE PAGE
10. Short-Term Gravitropic Response of Control, Prestimulated and Prestimulated/ Rotated Roots...... 105
11. Long-Term Gravitropic Response of Control, Prestimulated and Prestimulated/ Rotated Roots...... 107
12. Correlation Between Downward Auxin Transport Polarity and the Rate of Gravicurvature...... 116
13. Auxin Transport Polarity vs. Rate of Gravicurvature...... 119
14. The Time Course of Development of Downward Calcium Transport Polarity Across the Caps of Gravistimulated Roots...... 150
15. A Comparison of Auxin and Calcium Transport Polarities as Determined from Dual Label Experiments...... 153
16. A Comparison of Downward and Upward Calcium Transport in Gravistimulated Roots...... 156
17. A Comparison of the Basipetal Movement of 3H-IAA and 45Ca2+...... 159
18. A Comparison of Auxin and Calcium Transport Rates Across the Caps of Gravistimulated Roots...... 169
xii LIST OF ABBREVIATIONS
CMZ Calmidazolium
DMSO Dimethylsulfoxide
EGTA Ethyleneglycol-bis-(amino ethyl ether) N, N'-tetraacetic acid
3h -IAA 3—(5
NPA Naphthylphthalamic acid
PBA Pyrenoylbenzoic acid INTRODUCTION
A REVIEW OF THE STUDY OF ROOT GRAVITROPISM
Scientists have long been intrigued by the curvature
of plant organs toward or away from the earth's
gravitational pull, a phenomenon called gravitropism. This
ability to sense the field of gravity and to use it for
orienting the direction of growth is advantageous to plant
survival. If a plant is displaced from its original
position with respect to gravity, perception of the earth's
gravitational field by the plant results in the alteration
of root and stem growth patterns. Differential growth
across these organs ultimately results in the re-alignment
of the plant body parallel to the "plane" of the gravitational field. Reorientation of the shoot to an upright position is advantageous since it allows optimal
leaf display for photosynthesis. Also, curvature of the root downward allows anchorage of the plant and acquistion of an adequate water supply.
The phenomenon of gravitropism has been the subject of experimental research since the early 19th century (Knight, 1806). Much of the early work was on gravitropism in
roots, including some of the work of Charles Darwin (1881) with Vicia faba roots. A renewed interest in the response of roots to gravity has occurred with the advent of space exploration. Since it is proposed that future long-term, manned flights should supplement the oxygen supply with oxygen produced by photosynthesizing plants onboard the spacecraft, it becomes increasingly more important to understand the gravitropic response. Once we comprehend the curvature response of a root under normal (earth) conditions, we can better approach problems resulting from the micro-gravity environment of space flight.
To explain the curvature response of a root to the gravity stimulus, we must recognize that any stimulus/response system involves three steps: perception, transduction and response. During the perception event, the gravity stimulus is received by a gravity-sensor within the root. Once perceived, the gravity stimulus must be transduced. Transduction probably involves a complex series or network of events and hence, this step has been the most difficult to elucidate. Although the nature of the transduction phase is still unknown, this step can generally be considered as the conversion of the perceived stimulus into a message that is sent from the gravity sensor to the site of gravicurvature. Downward curvature resulting from differential growth across the elongation zone of the root is the final step in the gravitropism stimulus/response system. This response, or action by the plant, is a direct result of physiological changes caused by the transduction message. By closely examining each of these three steps, we can construct a useful model of root gravitropism.
In order for a plant organ such as a root to curve, the rates of cell elongation across the organ must differ.
Among the possible causes of downward curvature in a gravistimulated root (i.e a horizontally-oriented root) are
1) an increased rate of cell elongation on the upper half of the root, 2) a decreased rate on the lower half and 3) a combination of these two events. To estimate growth patterns in gravistimulated roots, Mulkey and coworkers
(Mulkey and Evans, 1981; Mulkey et al, 1981a, 1981b) took advantage of the fact that normally growing plant cells secrete hydrogen ions (Rayle and Cleland, 1977). They mounted maize seedlings in an agar medium containing the pH indicator dye, bromocrescol purple. The region of the agar/indicator adjacent to the elongation zone turned yellow indicating acidification as expected in the region of rapid cell extension. When seedlings were gravistimulated, the indicator near the elongation zone on the upper half of the gravistimulated roots remained yellow. Agar near the lower half of the elongation zone became purple indicating elevated pH, as expected of root
cells with reduced growth.
Using a more sophisticated system of growth measurement, Nelson and Evans (1986) also showed that growth is suppressed on the lower side of gravistimulated
roots. With a computer-based video digitizer, they monitored the growth and gravicurvature of maize roots.
They found a reduction in the growth rate on both the upper and the lower surfaces of gravistimulated roots. However, the greatest inhibition of growth occurred on the lower half of the root. This differential inhibition of cell elongation is expressed as downward curvature.
Although we now know that differential growth inhibition across the elongation zone causes downward gravicurvature in corn varieties, we lack information on the nature of the mechanism controlling differential growth in this stimulus/response system. How does a root perceive the gravity stimulus and then transduce it, or convert it, into a message that can cause this curvature response?
In 1872, Ciesielski implicated the root cap as the site of gravity perception. He found that decapped roots continued to grow but failed to respond to gravity.
Even though Ciesielski (1872) and Darwin (1880) demonstrated that the root cap contains the gravity-sensing mechanism, they did not determine the nature of this mechanism. As early as 1892, Noll hypothesized that plants perceive gravity in a manner similar to animals, i.e. via a
statocyst system. This would require that plants possess some kind of small, dense particle or body, capable of sedimenting upon reorientation and thus indicating the direction of the earth's gravitational field. Haberlandt
(1900) and Nemec (1900) proposed that the plant statocysts are specific groups of cells (statocytes) located within gravi-responding organs. Inside the statocytes were numerous sedimented statoliths which proved to be amyloplasts, dense starch-filled plastids. Because the presence of these amyloplast-containing statocytes correlated with the graviresponsiveness of the organ,
Haberlandt and Nemec proposed plant gravity perception by means of the starch-statolith hypothesis (Wilkins, 1984).
Since gravity acts uniformly on all of the cells of a plant organ, detection of the gravity stimulus must result in the development of an intracellular asymmetry which is ultimately translated into a transorgan asymmetry.
Sedimentation of the amyloplasts upon gravistimulation would lead to the establishment of such an asymmetry at the cellular level.
In roots, this amyloplast asymmetry occurs in the large, rectangular cells in the core of the root cap, known as columella cells. Since the amyloplasts in these cells sediment to the new lower cell wall upon re-orientation, the columella cells of the root cap appear to serve as statocytes for the root. Hence the starch-statolith hypothesis is consistent with the observed correlation of cap removal with the loss of graviresponsiveness.
Evidence supporting the starch-statolith hypothesis includes the strong correlation between presentation time and the time required for amyloplast sedimentation.
Experimenting with stems of sweet pea, Lathvrus odoratus.
Hawker (1933) noted a correlation between statolith sedimentation rate and temperature. Decreasing temperatures resulted in greater cytoplasm viscosity and hence slower amyloplast sedimentation. Hawker also noted that decreased temperatures resulted in increased presentation times. The excellent correlation between amyloplast sedimentation rate and presentation time supports the starch-statolith hypothesis of graviperception.
Further evidence in support of this hypothesis is the close correlation between the presence of amyloplasts within the columella cells and the graviresponsiveness of the root. After treating L e g i d i i m sativum (cress) roots with high concentrations of 6-furfuryl-aminopurine and gibberellic acid at 30° C for about 35 h, Iversen (1969) found that root caps were not only devoid of amyloplasts, but that the roots were also rendered gravi-insensitive.
About 24 h following termination of the de-starching treatment, Iversen observed a simultaneous return of amyloplasts and graviresponsiveness. Iversen's later work with roots of white and red clover, Trifolium repens L. and
JL«. pratense L, (1974), and with wheat coleoptiles, Triticum durum (1974), and recent work by Song et al. (1988) using
Hordeum vulnare. yielded similar results. Taken together these studies indicate that amyloplasts must be present for graviperception to occur.
Additional studies linking amyloplasts with graviperception have been performed using decapped roots.
Although removal of the cap results in the loss of graviresponsiveness, activity in the root apical meristem leads to the regeneration of the cap, and there is a corresponding restoration of gravisensitivity. Although kinetic studies indicate that gravisensitivity is restored prior to the regeneration of the cap, microscopic examination of the root apex (Grundwag and Barlow, 1973;
Barlow, 1974; Barlow and Grundwag, 1974) reveals the development of starch grains from proplastids in the cells of the quiescent center, immature xylem and the cortex of the root apex (Wilkins, 1984). Therefore, the return of gravisensitivity is correlated with the formation and sedimentation of amyloplasts in the root apex. Thus the apex appears to serve as a "physiological" root cap prior to the regeneration of an anatomically complete cap.
When all of this evidence is considered, it appears that amyloplast sedimentation plays an important role in root gravity perception. But how can gravity perception
via the sedimentation of amyloplasts in columella cells be
transduced into differential growth inhibition across the
elongation zone of gravistimulated roots? One way for this
to occur would be to translate the sedimentation of
amyloplasts into a chemical message that could be
transported to the lower half of the elongation zone
resulting in the inhibition of growth.
There is considerable evidence that the root cap is
the source of a growth inhibitor regulating root extension.
Since the root cap, the site of graviperception, and the
elongation zone, the site of graviresponse, are spatially
separated, there must be communication between the two
sites. Because the curvature response results from a
decreased rate of cell elongation on the lower half of the
root (Nelson and Evans, 1986), it seems likely that this
cap to elongation zone communication occurs via a growth
inhibitor.
Working with roots of Z j y L and EI j l u i i l , Shaw and Wilkins
(197 3) showed that removal of half of the root cap resulted
in strong curvature towards the remaining half-cap
regardless of root orientation. It is doubtful that this
curvature represents a wound response because immediate replacement of the half-cap prevents curvature (Pilet,
1973). To ascertain that the source of the inhibitor was the root cap and not the meristem, Shaw and Wilkins (1973) 9 also removed half of the root apex from which the cap had been previously excised. Since no curvature occurred, it appeared that the cap, not the apical meristem, was the source of the growth inhibitor.
Cholodny and Went (Went and Thimann, 1937) proposed that positive gravicurvature in roots resulted from an inhibition of cell extension on the lower side of the elongation. Consequently, Shaw and Wilkins (1973) proposed that this growth asymmetry results from the lateral transport of an inhibitor downwards across the cap and then back to the elongation zone. This hypothesis is consistent with their findings that stronger gravicurvature occurs in roots with a mica barrier inserted in the upper half of the root just behind the cap, than in roots that had the upper half of the root cap excised. Presumably, more inhibitor is transported downward across the intact cap, than across the lower half-cap as removal of the upper half-cap could correspond to a reduced amount of inhibitor available for downward lateral transport. If the opposite experiment is performed, i.e. a barrier is inserted in the lower half of the root behind the cap and curvature is compared with a root that has had the lower half-cap excised, the former root curves upward less. With an intact cap, downward transport of the inhibitor occurs decreasing the amount of inhibitor that can move basipetally to the elongation zone and cause upward curvature. However, with the lower half 10 of a cap excised/ downward transport cannot occur and thus more inhibitor moves basipetally to the elongation zone causing stronger upward curvature. These experiments support the idea that a root cap inhibitor regulates gravitropic curvature.
Since several plant hormones are known to inhibit the elongation of root cells, a variety of these plant growth regulators have been studied as the possible root cap inhibitor. Because supra-optimal concentrations of indole-
3-acetic acid, IAA, are known to cause inhibition of root cell extension (Went and Thimann, 1937), auxin was among the first of the plant hormones to be investigated for this role. However, Bridges et al. (1973) reported that auxin occurred primarily in the stele of Zea roots, with only minute quantities present in the root apex. Also, Scott and Wilkins (1968) noted strong polar auxin transport toward the root tip in Zea majyL* On the basis of this transport data, they concluded that auxin movement occurs in the wrong direction for it to serve as a mediator of the gravitropic response. In addition, Shaw and Wilkins
(unpublished) were unable to demonstrate lateral auxin transport in gravistimulated Zea ma^s. roots. For these reasons, many investigators abandoned auxin as the possible root cap inhibitor for gravitropism.
During the 1970's, several laboratories provided evidence that the root cap inhibitor may be abscisic acid, 11
ABA. ABA is known to be a general inhibitor of plant growth (Eagles et al., 1973; Dure, 1975), and in 1974,
Kundu and Audus isolated a growth inhibitor from root caps of 2q j l with an Rg similar to ABA. Pilet (1975) asymmetrically applied 10“® M ABA, a concentration claimed to inhibit cell extension, to the elongation zone and observed substantial curvature. Despite recent evidence to the contrary (Mulkey et al., 1983), substitution with 10“8
M IAA, also thought to be an inhibitory concentration, failed to produce curvature.
Further evidence that ABA may be the root cap inhibitor was provided by work on cultivars of maize in which the roots require light for normal gravitropism.
Scott and Wilkins (1969) reported that seedling roots of the Giant Horse Tooth cultivar of maize show only a weak gravitropic response when gravistimulated in the dark, but respond strongly when gravistimulated in the light.
Wilkins and Wain (1974) noted that the increase in gravitropic responsiveness in the light correlated with an increase in ABA levels in the cap when exposed to light.
Pilet (1976) transferred the caps of dark-grown roots of Zea mavs var. Anjou, which is graviresponsive in the dark, to the apices of dark-grown decapped roots of the
Kelvedon 33 variety. Although Kelvedon 33 requires light for gravisensitivity, transfer of the Anjou caps resulted in gravicurvature even in the dark. Since Pilet (1975) had 12 previously noted that the gravitropic responsiveness of
Kelvedon 33 in the light correlated with light-induced increases in ABA levels in the cap, he interpreted the data as an indication that cap transfer provided the ABA necessary to confer gravireponsiveness upon the dark-grown
Kelvedon roots.
Although these data support the root cap inhibitor hypothesis and implicate ABA as that inhibitor, other recent experiments force us to question the role of ABA in mediating the transduction of the gravitropic response.
Moore and Smith (1985) observed that roots of maize mutants, that do not synthesize ABA, are graviresponsive.
They also found (1984) that roots of wild type maize seedlings treated with fluridone to prevent ABA synthesis, undergo normal curvature when gravistimulated. Additional evidence against cap-produced ABA as an inhibitor mediating gravitropism, is provided by experiments showing that light does not increase ABA levels in the cap of the light- requiring maize cultivar, Merit. Instead, light induces an increase in the ABA level of the root apical meristem
(Feldman et al., 1985). These findings indicate that ABA does not mediate the transduction phase of the gravitropism stimulus/response system.
A more promising candidate for the gravitropism transduction mediator is the plant hormone auxin. Cholodny and Went (Went and Thimann, 1937) hypothesized that an 13 auxin asymmetry develops across the elongation zone of gravistimulated roots, with the concentration greatest on the lower half. Since roots are very sensitive to auxin
(Thimann, 1937; Aberg, 1957; Gougler and Evans, 1981), an increased auxin concentration in the lower portion of the elongation zone would inhibit cell elongation causing downward curvature. To test the hypothesis that an auxin asymmetry across the elongation zone could lead to curvature, a multitude of experiments have been performed.
One such experiment was performed by Konings (1967) using two-day-old pea roots treated with 14c - i a a .
Following gravistimulation, 67% of the radioactivity was located in the lower portion of the elongation zone.
Konings further noted that no auxin asymmetry or gravicurvature developed if the root cap was removed prior to gravistimulation. Auxin redistribution was also prevented by application of a variety of auxin transport inhibitors to the root cap (1968). Since none of these compounds affected the growth of the roots, Konings concluded that they inhibited the lateral transport of auxin across the root cap.
Other experiments show that artificial establishment of an auxin asymmetry across the elongation zone can cause strong curvature. For example, Lee et al. (1984) applied an auxin-containing agar block to the lower side of a gravistimulated root pretreated with an auxin transport 14
inhibitor. Although the root had been rendered non-
responsive to gravity by pretreatment with the auxin
transport inhibitor, the artificial auxin asymmetry caused
downward curvature by retarding the growth on the lower
half of the elongation zone.
The experiments of Konings and those of Lee et al.,
implicate auxin asymmetry across the elongation zone as the mediator of gravicurvature. In addition, Konings'
experiments with cap-applied auxin transport inhibitors
designates a role for the root cap in the establishment of
this asymmetry. Konings suggested that the columella is
not only the site of graviperception but also the site at which transverse redistribution of auxin is controlled.
His measurements of distribution as a function of distance from the cap, the site of label application, showed that auxin asymmetry extends at least 6 mm behind the apex in gravistimulated roots.
Although Konings' model of gravitropism requires basipetal transport of the hormone, there is controversy over the preferred direction of auxin movement near the root tip. In a study of isolated root segments, Scott and
Wilkins (1968) showed that auxin movement is preferentially toward the tip. However, later studies (Davies el al.,
1972,1976; Tsurumi and Ohwaki, 1978) with intact roots have demonstrated that auxin transport is basipetally polar. This discrepancy appears to arise because the 15 preferential direction of auxin transport depends on whether or not the root cap is present. Since the root cap
is present under normal conditions, data generated from experiments with intact roots are the most relevant.
Evans et al. (1986) have proposed a revised version of
Konings' model to explain the relationship between the cap and the development of auxin asymmetry in the elongation zone. According to this model, auxin is polarly transported through the stele towards the cap (Figure 1).
In vertically-oriented roots, auxin entering the cap is symmetrically redirected toward the periphery of the cap where it enters auxin transport streams moving toward the elongation zone in the cortical and/or epidermal tissues.
This portion of the model is supported by the recent findings of Hasenstein and Evans (1988), that auxin transport in intact vertical roots is strongly polarized in the basipetal direction. The model further proposes that upon horizontal placement, auxin entering the cap is preferentially directed downwards across the cap, resulting in a higher concentration of auxin entering the basipetally moving auxin transport stream on the lower half of the root. The resulting auxin asymmetry in the elongation zone then causes gravicurvature via the Cholodny-Went hypothesis. This portion of the model is supported by
Konings (1968) observations that compounds that interfere 16
Figure 1.
Auxin Transport in Vertically- and Horizontally-Oriented Roots.
A simplified scheme of the auxin transport patterns proposed by Evans et al (1986) in vertically- and horizontally-oriented roots. Gravity
i i
Pleura L 18
with the lateral redistribution of auxin in the cap prevent
gravitropic curvature.
Although this revised model suggests a potential mechanism linking perception in the cap to curvature in the
elongation zone, it does not explain how amyloplast sedimentation, ie. perception, can lead to preferential downward movement of auxin across the root cap. During the
last 25 years, evidence has accumulated implicating a role for calcium movement in the transduction phase root gravitropism. Lee et al (1983b) observed that application of EDTA to the caps of gravistimulated roots prevented gravicurvature. Replacement of the EDTA with calcium restored graviresponsiveness. Shortly after this discovery, Lee et al (1983a) obtained data indicating that gravistimulation induces polar movement of calcium across the root cap toward the lower side (Lee et al., 1983a).
They also found that vertically oriented roots could be induced to curve by artificially establishing a calcium gradient across the root cap (Lee et al, 1983b). These experiments strongly suggest a role for calcium in the gravitropic stimulus/response system.
Can the establishment of a downward calcium gradient across gravistimulated root caps link the falling of the amyloplasts with the development of the downward auxin transport polarity across the caps? Studies indicate that there are high levels of calcium associated with the membranes of the amyloplasts (Chandra et al/ 1982). Also/ there is considerable evidence that calcium is necessary for the movement of auxin from cell to cell (dela Fuente and Leopold/ 1973; deGuzman and dela Fuente/ 1984). This information has led several researchers to speculate about the relationship between amyloplast relocation/ calcium gradients and polar auxin transport across the root cap.
Sievers et al. (1984) proposed that the pressure of amyloplasts on the endoplasmic reticulum (ER) causes a release of calcium from the ER. This would eventually lead to an increased level of calcium along the lower side of the cells and this may in turn lead to net movement of calcium to the lower side of the cap as described below.
One possible mechanism for calcium release from ER in animal cells (Nishizuka, 1986; Berridge and Irvine, 1984) is via inositol 1,4,5-trisphosphate (IP3) functioning as a second messenger. The phosphatidylinositol pathway (Figure
2) is thought to operate as follows. An agonist, e.g. hormone, binding to a surface membrane receptor activates the membrane-bound enzyme, phospholipase C. Phospholipase
C catalyzes the production of IP3 and diacylglycerol (DG) from the membranous pool of phosphatidylinositol 4,5- bisphosphate (PtdIns(4,5)P2)• Since IP3 is water soluble, it can move through the cytosol to the ER where it binds to an IP3 receptor resulting in calcium release. Figure 2.
The Inositol Trisphosphate Pathway. 21 .1*
Tujat
IVS
ct /cm
FltlM S. 22
Could this mechanism function in plant cells? There is good evidence for the existence of phosphatidylinositol in plant membranes as well as evidence of the presence of the kinases and polyphosphorylated inositides (Boss and
Massel, 198 5) necessary for the production of phosphatidylinositol (Morse et al, 1986; Sandelius and
Sommarin, 1986). Irvine et al (1980) reported finding a phospholipase C-like enzyme in celery while Helsper et al
(1985) observed phospholipase C activity in lily pollen.
Since these components of the phosphatidylinositol pathway are found in plant cells, this mechanism of induction of calcium release from the ER may be feasible in root gravitropism if gravistimulation is substituted for the binding of the agonist to the external receptor.
One consequence of elevated intracellular calcium is the promotion of protein phosphorylation. This can occur via direct calcium stimulation of protein kinases or via calcium activation of calmodulin, a small, acidic protein
(Cheung, 1981) capable of activating a variety of enzymes once it has been activated by calcium (Cheung, 1982).
Enzyme activity altered in this manner could play a role in the transduction of the gravity stimulus in roots.
A second consequence of elevating intracellular calcium levels is an efffect on the membrane-bound enzyme protein kinase C (Figure 2). Protein kinase C activity is calcium dependent. However, in the presence of DG, one of 23
the products of phospholipase C degradation of
PtdIns(4,5)P2/ the affinity of protein kinase C for calcium
is enhanced. Since protein kinase C-like activity has been
reported in plants (Shaefer et al, 1985; Ladyzhen et al,
1987) it is quite possible that target proteins
phosphorylated by this enzyme may also be involved in the
transduction phase of root gravitropism.
How could protein phosphorylation by calmodulin or
protein kinase C help mediate the transduction of the
gravitropism stimulus/response system? Recently, Evans et
al (1986) have elaborated upon the hypothesis of Siever's
et al. According to the model of Evan's et al., calcium
escaping from the ER activates calmodulin. They speculate
that activated calmodulin, or a protein activated by
calmodulin, activates calcium pumping enzymes which are on
the plasma membrane. The pumping of calcium from the lower
side of the cells will ultimately lead to the establishment
of the downward calcium gradient.
Calmodulin has been found in maize roots (Kuzmanoff,
1984) and calmodulin activity has been shown to be four
times greater in the root cap than in the elongation zone
(Stinemetz and Evans, 1985; Stinemetz et al, 1987).
Working with a variety of maize in which orthogravitropism
of the primary root is light-dependent, it has been shown
that the level of calmodulin is low in the roots caps of
the dark-grown seedlings. When exposed to light, the roots 24
become graviresponsive and the timing of the development of
gravitropic responsiveness correlates with an increase in
root cap calmodulin (Stinemetz et al, 1987). Also, when
calmodulin antagonists were applied to the caps of
gravistimulated roots, the degree to which gravicurvature
was retarded correlated with the degree to which the
development of the calcium gradient was inhibited
(Stinemetz and Evans, 1986).
Evans et al (1986) speculate that calcium-activated
calmodulin in the columella cells may also activate auxin
pumps in the plasma membranes along the lower side. If
this were true, then much of the auxin entering the cap of
a gravistimulated root would be transported to the lower
half of the cap, rather than being distributed
symmetrically.
Although the model is highly speculative, it can explain the perception, transduction and response steps of
the gravitropism stimulus/response system. The gravity
stimulus is perceived by the displacement of amyloplasts in
the columella cells. The resting of the amyloplasts on the
ER of the lower side of these cells triggers a release of calcium which activates calmodulin. Calmodulin in turn, is proposed to activate both calcium and auxin pumps located
in the plasmalemma on the lower half of the cell resulting
in downward movement of calcium and auxin across the cap.
Accumulated auxin in the lower portion of the cap is 25
loaded, in a calcium-mediated manner, into the basipetal
transport stream. Movement of the hormone to the elongation zone causes decreased cell elongation which ultimately leads to the response, downward gravicurvature.
The purpose of the work presented in this dissertation
is to examine various aspects of this model. Specifically,
I wanted to 1) determine whether or not there is downward auxin transport polarity across the caps of gravistimulated roots; 2) clarify the relationship between this auxin transport polarity and gravicurvature;
3) determine whether or not there is enhanced transport of auxin in the basipetal transport stream on the lower side as predicted by the model; 4) examine the interdependence of calcium accumulation on the lower side of the cap and the downward polarity of auxin movement; and 5) attempt to determine which processess of transduction may be calmodulin dependent. CHAPTER I
THE NATURE OF AUXIN TRANSPORT IN GRAVISTIMULATED
ROOTS OF ZEA MAYS
The Cholodny-Went model of root gravitropism
emphasizes the role of auxin redistribution in the
development of the differential growth inhibition that
results in downward gravicurvature (see Went and Thimann,
1937). Although the differential growth that leads to
curvature occurs in the elongation zone, the root cap is
important to the response. Removal of the cap results in
loss of gravitropic responsiveness (Ciesielski, 1872;
Darwin, 1880) perhaps because the columella tissue of the
cap is the site of graviperception and the site at which
transverse auxin redistribution is controlled (Konings,
1967, 1968). Konings showed that, in gravistimulated
roots, asymmetric auxin redistribution (more on the bottom) extends at least 6 mm back from the apex. Based on measurements of distribution of label from tip-applied
auxin as a function of distance from the tip, he suggested
that auxin asymmetry develops near the tip, probably in the
26 27 cap. However, Konings did not measure auxin transport across the cap or auxin distribution within the cap.
Although Konings's data suggest auxin movement from the cap to the elongation zone, much of the evidence from experiments with excised, decapped root segments indicates that auxin transport is preferentially toward the tip
(Scott and Wilkins, 1968). However, Davies et al (1972,
1976) have shown that auxin moves, possibly in the cortex, from the cap back into the elongation zone in intact roots.
Similar conclusions were drawn by Tsurumi and Ohwaki (1978) from results on auxin movement through root segments from
Vicia faba. Hasenstein and Evans (1988) compared auxin transport in intact vs decapped roots of maize. They found strong tip to base polarity in intact roots and noted that the polarity was reversed when the roots were decapped.
These findings are consistent with Koning's suggestion that asymmetric growth in gravistimulated roots can be controlled by modification of auxin movement from the cap into the elongation zone.
In this context, I examined transverse auxin movement across the caps of gravistimulated roots of maize. My findings support the suggestion that gravi-induced polar auxin movement across the root cap is an important factor in root gravitropism. 28
MATERIALS AND METHODS
Plant Material. Caryopses of maize (Zea mays L. cv
Merit) were soaked in distilled water for 10 h prior to
planting. The grains were placed between wet paper towels
on vertical opaque plastic trays and germinated at room
temperature (20-23°C) under fluorescent laboratory lighting
(intensity 175 uE•m“2«s“2). After 2 days, seedlings with
straight primary roots (approximately 2 cm long) were
selected and mounted vertically using floral tape in Petri dishes lined with moist filter paper. Roots were allowed
to equilibrate for 1 h prior to experimentation.
Application of 3H-IAA or 3H-ABA. Agar (1.5%) sheets
(1.0 cm x 1.0 cm x 1.5 mm) were incubated overnight in distilled water (pH adjusted to 6.5 with NaOH). They were then cut into small blocks (1.5 mm cubes). Receivers were used directly while a stock solution of 3H-IAA (92.5
GBq/mg, Amersham) was applied to blocks to be used as donors. After equilibration with the applied radioactivity, the donor blocks contained 833 Bq 3H-
IAA/block (approximately 50,000 cpm/block).
The same procedure was followed to prepare 3H-ABA donor blocks. A stock solution of 3H-ABA (92.5 GBq/mg,
Amersham) was applied to agar blocks and allowed to equilibrate overnight. As before, the donor blocks contained 50,000 cpm of 3H-ABA/block. 29
Auxin and Abscisic Acid Transport in Intact Roots. To
measure auxin transport across the cap, a donor block and a
receiver block were applied to opposite sides of the root
caps of seedlings oriented vertically within the dishes.
One set of seedlings was held in a vertical position for 90
min to determine the lateral transport of auxin. Total
auxin transported was measured as movement of label
(assumed to be ^h - i a A) across the cap. Following the 90
min transport period, the donor and receiver blocks were
collected and placed into separate scintillation vials for
determination of radioactivity using a Beckman LS7000
scintillation counter. Each data point represents the mean * value of at least 45 roots.
As a test of the potential physiological significance of polar auxin movement to the gravitropic response, I
tested the movement of ^h -a b a across gravistimulated roots.
Abscisic acid, like auxin, is a naturally-occurring
aromatic weak acid. Donor blocks (50,000 cpm of ^h -a b a ) and receiver blocks were placed opposite one another on the
tips of vertically- and horizontally-oriented roots. After
a 90 min transport period, the donors and receivers were
collected to determine upward, downward and lateral ^h -a b a transport across the root tips. This experiment was performed four times with 10 roots/treatment.
To determine the efficiency of IAA vs. ABA uptake, vertically-oriented roots were loaded with labeled hormone 30
for 75 min by applying a donor (50,000 cpm 3H-IAA or 3H-
ABA) to the side of the root. Following the loading
period, a receiver block was applied opposite the donor and
transport monitored for 15 min. The donor blocks, receiver
blocks and excised roots were collected for radioactivity
determination. This experiment was performed four times
with four roots/treatment.
The basipetal transport of 3H-IAA and 3H-ABA was also
compared. Vertically mounted roots were loaded with the
labeled hormones for 90 min by applying a donor block containing either 3H-IAA or 3H-ABA to the side of the root tip. The donor block was collected for radioactivity determination. The roots were then cut into 10, 1 mm sections beginning at the tip. Each section was placed
into a separate scintillation vial and counted. This experiment was performed four times with five roots/ABA treatment and seven times with five roots/IAA treatment.
To determine the time course of the development of downward auxin transport polarity, a set of seedlings was gravistimulated by rotating the Petri dishes 90°. Half of these seedlings were oriented so that the donor block was on the top of the root cap while the other seedlings were oriented with the donor block on the bottom of the root cap. Again, total auxin transported was measured as movement of label across the cap for periods of 15, 30, 45,
60, 75, 90, 105, 120, and 180 min of gravistimulation. 31
Following the transport period, the donor and receiver blocks were collected as before for determination of radioactivity. The transport value for each data point is the mean of at least 45 roots.
Auxin transport across the caps of roots was also monitored at 15 min intervals during a 2 h period of continuous gravistimulation. Application of blocks and orientation of the roots was as described above. However, following 15 min of gravistimulation, the receiver blocks were removed and placed in scintillation vials for determination of radioactivity. Fresh receiver blocks were then applied to the root and this procedure was repeated every 15 min for 120 min. A minimum of 45 roots was used for each data point.
Auxin Transport in Decapped Roots. Since the root cap of a maize seedling is approximately 0.5 mm long
(determined by measuring isolated caps) and the agar blocks used as donors and receivers were 1.5 mm cubes, I was concerned that the transport values obtained may reflect auxin movement across both the cap and the root apical meristem. To test the contribution of the cap to total transport of ^h -i a a across the tips of gravistimulated roots, similar donor/receiver experiments were performed using decapped roots. Prior to mounting the maize seedlings in Petri dishes, they were placed under a dissecting microscope and decapped. This was accomplished 32 by inserting the tip of a scalpel blade into the junction of the root cap and root proper, and gently prying the cap off, leaving the meristem intact. Donor and receiver blocks were then placed on opposite sides of the root apical meristem. Half of the roots remained in a vertical position for a 90 min transport period while the other half was gravistimulated for 90 min with the donors and receivers oriented to determine both upward and downward
3h -IAA transport. In addition to determining the radioactivity in the donor and receiver blocks, the root was excised following transport and the radioactivity within the organ was determined. The transport values obtained represent a mean of at least 40 roots.
Auxin Transport Across Isolated Root Caps. One additional experiment to test the site of ^h - i a a movement across gravistimulated root tips involved measuring auxin transport across isolated root caps. Root caps were removed from seedlings as described above. Four caps were lined up end to end on a microscope slide with 2 donor blocks placed on one side of the caps and 2 receiver blocks placed on the opposite side. A second microscope slide was added as a cover with a capillary tube inserted between the slides acting as a spacer. This double slide apparatus was then reoriented to gravistimulate the isolated caps. As in the previous experiments, transport upward and downward across the caps was measured. As a control for 33
this experiment, four excised root apical meristem
sections, approximately 1 mm long, were also lined up end
to end on the double slide apparatus and ^H-IAA transport
monitored upon gravistimulation of the sections. Following
90 min of gravistimulation, the 2 donor blocks, the 2
receiver blocks and the 4 root caps or 4 meristem sections
were collected for radioactivity determination. This
experiment was repeated 7 times with four isolated root
caps and three times with four meristem sections/treatment.
To test the extent to which 3h _ j a a may move across the
root cap of intact roots by non-physiologically relevant physical means such as diffusion, movement along a surface
film, or movement by some form of capillarity, transport was also measured across roots treated with 1.0 mM KCN as a metabolic inhibitor. Roots were mounted vertically in a plastic basin such that the apical 2 mm were soaking in an aerated 1.0 mM KCN solution. Roots were pretreated for 60 min prior to measuring auxin transport polarity in the previously described manner for a 90 min gravistimulation period. Donors and receivers were collected and counted.
The transport values represent the mean value of 12 roots.
Pretreatment with Auxin Transport Inhibitors. To further test the contribution of polar auxin transport to the total movement of ^h -i a a capillarity across the root cap, transport was measured in roots treated with 10"5 m naphthylphthalamic acid (NPA), an auxin transport inhibitor. Donor and receiver blocks containing 10"5 M NPA
were prepared by equilibrating the blocks overnight with an
aliquot of 10”^ m NPA in 1% dimethylsulfoxide (DMSO). Once
seedlings were mounted in Petri dishes, NPA-containing agar
blocks were placed on each side of the root cap for 60 min.
After this period of incubation, one of the NPA-blocks was
removed and a donor, containing 50,000 cpm of 3H-IAA, was
applied to the opposite side of the cap. Upward, downward
and lateral 3H-IAA transport was measured for a 90 min
period. As before, donors, receivers and excised roots
were collected for determination of radioactivity. The
transport values obtained are the mean of at least 24
roots.
The effect of NPA on the movement of 3H-IAA across
isolated root caps was also determined. Isolated caps were
obtained and mounted as previously described. However, the
receivers contained 10”5 M NPA and were applied for 60 min
prior to the start of the transport period. Once NPA-
containing 3H-IAA donors were added, the caps were
gravistimulated for 90 min. Donors, receivers and the 4
caps were collected for radioactivity determination. This
experiment was repeated 3 times with 4 root caps/treatment.
I also examined the effect of NPA on the basipetal movement of auxin during gravistimulation. Vertically
oriented roots were pretreated for 60 min with 10”3 M NPA
by applying NPA-containing agar blocks. One block was 35 applied to the side of the root tip while the second was placed directly above it in the elongation zone, approximately 3.0-4.5 mm from the root tip. Following pretreatment with the auxin transport inhibitor, the block at the tip was replaced with a donor containing 10"® m NPA and 50,000 cpm of ^H-IAA and the roots were gravistimulated for 90 min. Donors and receivers were collected and radioactivity determined. This experiment was performed four times with 14 roots/treatment.
Since pyrenoylbenzoic acid (PBA) is reported to be a more potent inhibitor of auxin transport than NPA (Katekar,
1981), I also examined the effect of PBA on lateral and basipetal auxin movement in roots. An aliquot of 10"® m
PBA (in 1% ethanol) stock solution was added to agar blocks such that dilution resulted in a 1 0 “ 6 m PBA concentration/block* Two 10"® M PBA-containing blocks were placed opposite one another on the tips of vertically oriented roots for 60 min. After incubation, one of the blocks was replaced with a donor block containing 50,000 cpm of ^H-IAA and the root was oriented horizontally with the donor either on the top or on the bottom. After 90 min donors and receivers were collected for radioactivity determination. This experiment was performed four times with eight roots/treatment.
To test the effect of PBA on the basipetal movement of auxin in gravistimulated roots, vertically-oriented roots 36 were pretreated with 10 " 6 m PBA-containing blocks. One block was placed on the side of the root tip while the second block was placed directly above it in the elongation zone, approximately 4 mm from the root tip. After 60 min, the block at the tip was replaced with a donor containing
1 0 ~ 6 m PBA and 50,000 cpm of ^H-IAA anc^ the root was oriented horizontally with the blocks on the lower side.
After 90 min radioactivity in the donors and receivers was determined. The experiment was performed twice with 12 roots/treatment.
RESULTS
Asymmetric Auxin Movement Across the Caps of
Gravistimulated Roots. Table 1 shows data on the movement of label from ^h -i a a from top to bottom and from bottom to top across the caps of gravistimulated roots.
Radioactivity accumulated in the receivers was measured following 90 min of gravistimulation. A consistent asymmetric movement, or transport polarity (downward movement/upward movement) of about 1.6 was observed.
Time Course of Development of Auxin Transport Polarity
Across the Caps of Gravistimulated Roots. Figure 3 shows results from experiments testing the time course of the development of asymmetric auxin movement as a function of 37
Table 1.
Polar Auxin Transport Across Caps of Gravistimulated Roots.
Donors (50,000 cpm/block of ^H-IAA) an(* receivers were placed on the tips of roots at time zero and transport polarity was measured after 90 min of gravistimulation. The data points represent the mean value + S.E. from at least 48 roots. 38
Table 1.
Polar Auxin Transport Across Caps of Gravistimulated Roots
Duration of Transport Downward Gravistimulation (min) (cpm) Polarity
top-bottom bottom-top
90 2186 + 105 1415 + 78 1.6 39
Figure 3.
Time Course of the Development of Auxin Transport Across Caps of Gravistimulated Roots.
Auxin transport polarity across the root cap was measured over a 3 h gravistimulation period. Each data point represents the mean value of.at least 48 roots. The maximum standard error was 0.1 polarity unit. POLARITY (downward/upward) 0.8 0.9 1 1.2 1.3 1.4 1.0 1.5 1.6 ^ . y 1 >
ui rnpr cosCp Gaitmltd Root Gravistimulated AuxinAcross Cap: Transport
3 6 9 10 5 180 150 120 90 60 30 0 gr 3. Rgure TIME MINUTES IN 41 time after gravistimulation. Each data point is based on total activity accumulated in the receivers during a transport period from zero time (initial horizontal orientation) to the time indicated by the particular data point. No significant auxin transport polarity was detected during the first 30 min of gravistimulation.
However, slight upward movement (polarity < 1) was consistently observed during the first 15 min of gravistimulation. After 45 min of gravistimulation, preferential downward auxin movement (polarity = 1.2) was detected. The magnitude of this preferential downward movement increased with time reaching a maximum (1.6) following 90 min of gravistimulation. Further gravistimulation resulted in a decrease in preferential downward movement, with the downward/upward ratio approaching 1.2 after 105 min of gravistimulation. Table 2 shows the values for upward and downward auxin movement that were used to calculate the auxin transport values of
Figure 3. By plotting downward and upward transport against time, I observed that during the period of development of downward auxin transport polarity, upward auxin transport lagged behind downward auxin transport
(Figure 4a). Using Figure 4a, I determined the rates of downward and upward auxin transport. When auxin transport rates were plotted against time (Figure 4b), it appeared that downward and upward transport rates oscillated in 42
Table 2.
Time Course of the Development of Downward Auxin Transport Polarity.
The downward and upward transport of 3h - i a a across the tips of gravistimulated roots was monitored at various times during a three hour period. Each data point represents total radioactivity accumulating in receivers from zero time to the point measured. These transport values were used to determine the downward auxin transport polarity during gravistimulation. Each data point represents a mean value + S.E. from at least 48 roots. 43
Table 2.
Time Course of the Development of Downward Auxin Transport Polarity
Duration of Transport Downward Gravistimulation (min) (cpm) Polarity
T->B B->T
5 259 + 22 271 + 23 0.9 10 325 + 27 375 + 31 0.9 15 336 + 20 370 + 24 1.0 30 885 + 39 852 + 44 1.0 45 1524 + 78 1242 + 61 1.2 60 2210 + 90 1718 + 81 1.3 75 2216 + 133 1525 + 75 1.5 90 2186 + 105 1415 + 78 1.6 105 2794 + 100 2324 + 118 1.2 120 2653 + 98 2080 + 112 1.3 180 2420 + 68 2227 + 85 1.1 44
Figure 4.
Comparison of the Rate of Downward Auxin Transport and the Rate of Upward Auxin Transport Across the Caps of Gravistimulated Roots.
Figure 4a illustrates the transport of 3H-IAA downward (circles) and upward (triangles) across gravistimulated root caps vs time. These curves were used determine the rates of downward and upward auxin transport shown in Figure 4b. DOWNWARD VS. UPWARD AUXIN TRANSPORT 3000<
O 2500*
2000 “
1 5 0 0 “
30 60 00 120 150 180 TIME M MINUTES Figure 4 . 46 phase with one another throughout the 3 h gravistimulation period. It is noteworthy that the decrease in downward auxin transport polarity after 90 min was the result of the
rate of upward auxin transport increasing faster than the rate of downward auxin transport (Figure 4b).
Measurement of Auxin Transport Polarity During Discrete
Internals of the Gravitropic Response. When auxin transport was measured at intervals by replacement of receiver blocks every 15 min, I detected a period of constant, preferential downward movement (polarity about
1.4) beginning 45 min after gravistimulation and lasting about 60 min (Figure 5). Thereafter, the transport polarity decreased rapidly reaching a value of about 1.2 after 120 min of gravistimulation.
Measurement of Auxin Transport Across the Tips of
Decapped Roots. Table 3 compares the transport of ^h -i a a across the tips of intact and decapped roots oriented either vertically or horizontally. Auxin transport across the tips of intact vertical roots from right to left and from left to right was about equal, yielding a polarity value of 1.0. Gravistimulation resulted in the development of auxin transport polarity (polarity = 1.6 after 90 min).
When roots were decapped and oriented vertically, I again found a transport polarity value of 1.0. However, overall transport of ^H-IAA was greatly reduced in decapped roots.
In intact vertical roots, lateral auxin transport during 90 47
Figure 5.
Measurement of Auxin Transport Polarity During Discrete Intervals of the Gravitropic Response.
The development of auxin transport polarity across the root tip was determined at 15 min intervals by regularly replacing the receivers. Each data point represents the mean value of at least 48 roots. Standard error was 0.1 polarity unit. Transport Polarity (downward/upward) 1.6 1 1 1.0 1 - .3 1 . . . 5 4 2 - - -- 0 Time Course Of Development Of Auxin Transport Polarity Transport AuxinOf Development Of Course Time iue 5. Figure a S E. S. Max 1 nevl ubr(5 min/interval) (15 Number Interval 2 3 4 5 6 7 8 9 00 49
Table 3.
Auxin Transport Across Tips of Intact and Decapped Roots in Vertical and Horizontal Positions.
3h -IAA transport was followed across the tips of vertically- and horizontally oriented intact and decapped roots. The data represent the mean of at least 40 roots/treatment + S.E. Donors contained approximately 50,000 cpm/block. 50
Table 3.
Auxin Transport Across Tips of Intact and Decapped Roots in Vertical and Horizontal Positions
Treatment/Direction Transport Transport of Transport (cpm) Polarity
Intact R->L 1812 + 107 L->R 1845 + 120 1.0
T->B 2049 + 165 B->T 1300 + 120 1.6
Decapped R->L 359 + 35 L->R 340 + 43 1.0
T->B 342 + 38 B->T 318 + 35 1.0 51 min was about 1800 cpm compared with 350 cpm in decapped
roots. Since 12,628 cpm + 1797 of ^H-IAA remained in the decapped roots following transport, uptake was not limiting transport. When the decapped roots were gravistimulated
for 90 min, no polar auxin movement (polarity = 1.0) developed. This lack of asymmetric auxin movement across decapped gravistimulated roots contrasts sharply with the strong (1.6) polarity value obtained using intact roots.
Transport of ^H-IAA Across Isolated Root Caps. To further test the root cap as the site of auxin redistribution during gravitropism, the transport of 3h -i a a was measured across gravistimulated, isolated caps. The data again indicate the development of a downward auxin transport polarity (1.4) across the caps following 90 min of gravistimulation (Table 4). It should be noted that radioactivity transported per cap is about the same in isolated caps (Table 4) as in caps on intact roots (Table
1).
Auxin transport across isolated 1 mm sections of root apical meristem was monitored as a control for transport measurements across isolated root caps. Movement of auxin across the meristem sections was dramatically less than movement across the caps (Table 4). This is consistent with data obtained using intact vs. decapped roots (Table
3). When the transport values of the meristem sections were divided by the number of sections (4), the quotients 52
Table 4.
3H-IAA Transport Across Isolated Root Caps or Meristems.
The transport of 3H-IAA was measured across gravistimulated, isolated caps. The data represent the mean value + S.E. for 7 repetitions using four caps/experiment. Donors contained approximately 50/000 cpm/block. 53
Table 4.
^H-IAA Transport Across Isolated Root Caps
Root Transport Transport Transport ^h - i a a left Region Direction (cpm)3*5 Polarity in Tissue
Cap T->B 8237 + 831 4439 + 797 B->T 6092 + 846 1.4 10047 + 1661
Meristem T->B 1156 + 92 6522 + 600 B->T 1331 + 113 0.9 9861 + 1700
3 90 min transport period. b transport across 4 caps or 4 1-mm meristem sections. 54
were slightly lower than the transport values across
decapped seedlings (Table 3). This may be explained by
recalling that 1.5 mm long blocks were applied to the tips
of the decapped roots and thus auxin movement occurred over
a slightly longer section than in the experiments using
isolated 1 mm long meristem segments.
Although experiments involving intact roots were done
using 1 donor block/seedling and experiments with isolated
caps or meristem sections were done using 1 donor block/2
sections, the amount of available label did not limit
transport. When the amount of ^h -i a a remaining in donor
blocks was compared between these two experiments, I noted
that more label remained in donors used on intact seedlings
(data not shown). Also, since ^H-IAA transport across
individual isolated caps (Table 4) is comparable to
transport across individual intact root tips (Table 1),
there was apparently sufficient ^H-IAA available for optimal auxin transport when experiments were performed using only 1 donor block/2 isolated sections.
The Effect of KCN on the Development of Polar Auxin
Transport in Gravistimulated Roots. In order to determine the extent to which asymmetric auxin movement depends on active processes and to estimate the extent to which passive ^H-IAA movement occurs, 3h -i a a movement was monitored in gravistimulated roots pretreated with KCN
(Table 5). KCN completely eliminated polar auxin transport 55
Table 5.
Effect of KCN on Polarity of ^H-IAA Transport Across the Tips of Gravistimulated Roots.
Root tips were immersed for 60 min in aerated 1 mM KCN. Donor (50/000 cpm of ^H-jAA/block) and receiver blocks were then applied to opposite sides of the root tip. The roots were gravistimulated for 90 min. At the end of this transport period, donor and receiver blocks were placed in individual scintillation vials for determination of radioactivity. Each data point represents the mean value + S.E. of 12 roots. 56
Table 5.
Effect of KCN on Polarity of 3H-IAA Transport Across the Tips of Gravistimulated Roots
Treatment Transport (cpm) Polarity
b ->T
Control 2415 + 285 1265 + 8 6 1.9
+KCN 2851 + 358 2971 + 298 1.0 57 in gravistimulated roots. Thus, it appears that the establishment of the auxin asymmetry across the root tip requires active transport of the hormone.
The Effect of NPA on Auxin Transport in
Gravistimulated Roots. To better understand the nature of auxin transport in gravistimulated roots, intact roots and root caps were pretreated with NPA (Figure 6), an auxin transport inhibitor (Niedergang-Kamien and Leopold, 1957;
Morgan and Soeding, 1958), and lateral and basipetal movement of ^h -i a a was monitored. Table 6 shows the effects of a 60 min pretreatment with 10”5 m NPA on auxin transport across the tips of vertically- and horizontally- oriented roots. NPA treatment resulted in a 42-45% inhibition of lateral auxin transport across the tips of vertically-oriented roots. In horizontally-oriented roots,
NPA inhibited ^h - i a a movement from top to bottom about 45% and movement from bottom to top about 35%. Since the data of Tables 2 and 4 suggest that there is a marked decrease in upward auxin transport across gravistimulated root tips, the lower percent inhibition of upward movement by NPA may reflect the fact that the rate of upward auxin transport is already lagging behind the downward transport rate due to gravistimulation. The net effect of NPA on auxin tip transport was to reduce the auxin asymmetry from 1.6 to 1.4
(Table 6). Figure 6.
Structures of NPA and PBA. 59
HAPHTHYLPHTHALAMIC ACID (HPA)
COOH
C-O-H-H I
PYBBHOYLBBHZOIC ACID (PDA)
COOH
C
0 60
Table 6.
^H-IAA Transport Across Intact Roots Treated with 10"5 m NPA.
Vertically oriented roots were treated by applying two agar blocks with 10“ 5 m npa to opposite sides of the tip for 60 min prior to the examination of auxin transport. The data represent the mean + S.E. of 12 roots. Donors contained approximately 50,000 cpm of ^H-IAA/block. 61
Table 6.
3h -IAA Transport Across Intact Roots Treated with NPA
Treatment/Direction Transport3 Transport % of Transport (cpm) Polarity Inhibition
Control R->L 1812 + 107 — L->R 1845 + 120 1.0 —
T->B 2078 + 149 B->T 1278 + 184 1.6 -
l“5 M NPA R->L 1048 + 166 42 L->R 1020 + 124 1.0 45
T->B 1173 + 154 43 B->T 838 + 89 1.4 34
i0 min transport period. 62
When isolated root caps were pretreated with 10“ 5 m
NPA, I again noted an inhibition of downward transport by about 40% (Table 7). Upward transport decreased only 28% with the overall result of reducing the downward auxin asymmetry from 1.4 to 1.1.
Finally, intact roots were pretreated with NPA and gravistimulated while monitoring basipetal transport of ^h -
IAA. While control roots transported a mean of 1403 cpm
(Table 8) from the root cap to the elongation zone during a
90 min transport period, roots pretreated with NPA transported only 480 cpm. This corresponds to a 66% inhibition of basipetal transport.
The Effect of PBA on Auxin Transport in Gravistimulated
Roots. PBA is reported to be a more potent inhibitor of auxin transport than NPA (Katekar, 1981). Although PBA eliminated gravi-induced auxin transport polarity (Table
9), I was surprised to find that total movement of auxin downward across the root tip was not reduced. A mean value of 2398 cpm of ^H-IAA moved downward across control root tips while 2808 cpm moved downward across tips of roots pretreated with 10“ 6 M PBA. This indicates that abolishment of asymmetric auxin movement across the tip resulted primarily from increased movement of label from bottom to top across tips of PBA-treated roots.
PBA pretreatment reduced basipetal auxin movement in gravistimulated roots by 84% (Table 10). 63
Table 7.
^H-IAA Transport Across Isolated Root Caps Treated with 10~ 5 M NPA.
Isolated caps were incubated by applying two agar blocks with 10“5 m NPA to opposite sides for 60 min prior to the measurement of ^H-IAA transport. Upward and downward auxin transport were measured across the caps following 90 min of gravistimulation. The data represent mean transport values + S.E. of at least 7 repetitions using 4 caps/treatment. Donors contained approximately 50,OOOcpm/block. 64
Table 7.
3h -IAA Transport Across Isolated Root Caps Treated with 10” 5 M NPA
Treatment/ Transport3 Transport % Inhib Transport (cpm) Polarity ition
Control T->B 8237 + 831 B->T 6092 + 846 1.4
+10“5 M NPA T->B 4311 + 651 47 B->T 3522 + 849 1.1 42
3 90 min transport period, 4 caps per treatment. 65
Table 8.
Basipetal Movement of ^h -i a a in Gravistimulated Roots Treated with 10“5 m NPA for 60 minT
Vertically oriented roots were treated with NPA by placing an agar block containing 10”5 m NPA on one side of the root tip and another on the same side in the elongation zone, about 4 mm from the tip. Following 60 min of pretreatment with NPA, the block at the tip was replaced with a donor containing NPA and 50,000 cpm of ^h -IAA. The roots were gravistimulated for 90 min. Donor and receiver blocks were collected for radioactivity determination. Each data point represents the mean + S.E. for 54 roots. 66
Table 8.
Basipetal Movement of 3H-IAA in Gravistimulated Roots Pretreated with NPA
Treatment Basipetal Transport (cpm) % Inhibition
Control 1403 + 112 +NPA 480 + 3 5 66 67
Table 9.
Effect of PBA on Auxin Transport Polarity Across the Caps of Gravistimulated Roots.
Agar blocks containing 10"® M PBA were placed opposite one another on the tips of vertically oriented roots for 1 h. Following pretreatment with the auxin transport inhibitor, one block was replaced with a donor block containing 10"® M PBA plus 50,000 cpm ^h -IAA. The roots were gravistimulated for 90 min before donor and receiver blocks were collected and counted# Each data point represents the mean value + S.E. of from least 30 roots. 68
Table 9.
Effect of PBA on Auxin Transport Polarity Across Caps of Gravistimulated Roots
Treatment Transport (cpm) Polarity T->B B->T
Control 2398 + 132 1549 + 91 1.6 +PBA 2808 + 256 2703 + 206 1.0 69
Table 10.
Effect of PBA on the Basipetal Transport of Auxin in Gravistimulated Roots.
One agar block containing 10”® M PBA was applied to the side of the tip of a vertically oriented root while a second block containing the auxin transport inhibitor was placed on the same side of the elongation zone about 4 mm from the tip. Following 60 min of pretreatment, the block at the tip was replaced with a donor block containing both PBA and 50,000 cpm ^h -i a a . The roots were gravistimulated for 90 min. Each data point represents the mean value + S.E. from 24 roots. 70
Table 10.
Effect of PBA on the Basipetal Transport of Auxin in Gravistimulated Roots
Treatment Transport (cpm) % Inhibition
Control 1372 + 118 +PBA 218 + 34 84 71
An Examination of 3jj_a b a Transport in Gravistimulated
Roots. As a further test of the potential physiological relevance of gravistimulated polar auxin movement, I examined the influence of gravity on the transport of ^h -
ABA, an aromatic acidic plant hormone also considered a candidate for growth regulation during root gravitropism
(Wilkins, 1984). As with ^h -Ia a (Table 3), -^h -a BA transport from right to left was equal to transport from left to right in vertically-oriented roots (Table 11).
However, upon gravistimulation, ^H-ABA transport across the root tip remained non-polar.
Although 3h _a b a transport was non-polar, the overall transport of ^h -a b a (Table 11) across the root tip was considerably greater than the transport of ^h -IAA (Table
3). To determine if this difference was due to differences in uptake, vertically-oriented roots were loaded with labeled hormone for 75 min prior to a 15 min transport period. Table 12 shows that more ^h - i a a than ^h -a b a entered and remained within the root.
I also compared the accumulation of cap-applied ^H-IAA and ^H-ABA in 10, 1 mm segments cut from the tip through the elongation zone (Figure 7). ^h -a b a accumulation was maximal in the first mm of the root, i.e. the site of application, and decreased 63% in the second segment. The
^h -ABA content decreased sharply with distance from the site of application, indicating little movement of ABA back 72
Table 11.
3h -ABA Transport Across Maize Root Tips.
Donor (50,000 cpm 3h -ABA) and receiver blocks were applied to opposite sides of the root tip and the roots were oriented either vertically or horizontally for 90 min. Following the transport period, the blocks were placed in scintillation vials and the root was excised at the base of the caryopsis and placed in a scintillation vial for determination of radioactivity remaining in the tissue. All data points represent the mean + S.E. of at least 40 roots. 73
Table 11.
^H-ABA Transport Across Maize Root Tips
Direction of Transport Transport Transport (cpm) Polarity
R->L 4418 + 333 L->R 4440 + 287 1.00
T->B 4396 + 253 B->T 4145 + 284 1.06 74
Table 12.
Transport of ^H-ABA and ^H-IAA Across Tips of Vertical Maize Roots Following Isotope Loading.
A donor (50,000 cpm ^H-ABA or ^H-IAA) was applied to one side of the tip of a vertically oriented root for 75 min. Following this isotope loading period, a receiver block was applied to the opposite side of the root tip. After 15 min of lateral transport, both donor and receiver blocks were removed for determination of radioactivity. The roots were excised just below the caryopses and also placed in scintillation vials to determine the amount of isotope remaining in the tissue following lateral transport. All data points represent the mean + S.E. of at least 16 roots. 75
Table 12.
Transport of 3H-ABA and 3 H-IAA Across Tips of Vertical Maize Roots Following Isotope Loading.
Treatment Isotope Transport (cpm) Uptake (cpm) __ __
3 H-ABA 1024 + 154 939 + 104 2238 + 293 3 H-IAA 512 + 48 564 + 33 11007 + 650 76
Figure 7.
Basipetal Transport of 3 H-ABA and 3H-IAA in Vertical Roots.
A donor (50,000 cpm 3 H-ABA or 3 H-IAA) was applied to one side of the cap of a vertically-oriented root for 90 min. Following this treatment, the donor block was removed and placed in a scintillation via for determination of radioactivity. The root was then cut into 10, 1 mm sections beginning at the tip. The sections were placed into individual scintillation vials for determination of radioactivity. The ABA data points (open bars) represent the mean value of 20 roots while the IAA data points (striped bars) represent the mean value of 35 roots. RADIOACTIVITY TRANSPORT (CPM) 1000 1000 - 0 0 2 1 1400- 1800 1600- 400 -I 0 0 8 600 -I 0 0 2 0
iue . 7 Figure BASIPETAL HORMONE TRANSPORT IN VERTICALROOTS IN BASIPETALTRANSPORT HORMONE i i \ \ \ \ DISTANCE FROM ROOT TIP (MM) ROOTDISTANCE FROMTIP A JQ * JDl 6 nl nfl nl 8 10
11 78
from the root cap. Conversely, 3H-IAA accumulated
maximally in the second, third and fourth mm segments,
those sections corresponding to the elongation zone.
Although 3H-IAA accumulation was less in the fifth segment,
the decrease in 3H-IAA accumulation with distance from the
site of application was gradual with 2.4 times more 3 H-IAA
in the tenth segment than 3 H-ABA.
DISCUSSION
A Metabolically-Dependent Downward Auxin Transport
Polarity Develops Across the Caps of Gravistimulated Maize
Roots. The data obtained are consistent with the
suggestion (e.g. Konings, 1967) that the cap is the site of
auxin redistribution in gravistimulated roots. These data
are also consistent with the major points of the model
proposed by Evans et al. (1986). According to this model,
auxin moves into the root cap through the stele and then moves back from the cap toward the elongation zone through
cortical cells. In vertically oriented roots, this flow is
proposed to be radially symmetric while in gravistimulated
roots it is proposed that auxin entering the cap moves preferentially toward the lower side of the cap. It then enters the transport stream moving auxin back into the elongation zone where the resulting increase in 79 concentration of auxin causes the differential growth inhibition leading to positive gravicurvature.
If the cap is the site of auxin redistribution, then one should be able to detect auxin transport polarity across the caps of gravistimulated roots using exogenously applied ^H-IAA. When auxin transport across the tip was monitored as ^h -i a a redistribution, I noted significant auxin transport polarity across root tips following 90 min of gravistimulation (Table 1). To determine the approximate onset of this asymmetric auxin movement, tip transport was measured after various periods of gravistimulation, beginning with 5 min. The time course of auxin transport polarity revealed transient polar auxin movement upward (Table 2) which, with further gravistimulation, reversed direction resulting in significant downward polarity after 45 min of gravistimulation. This downward auxin transport polarity increased with the duration of gravistimulation reaching a peak polarity of about 1.6 by 90 min (Figure 3). Prolonged gravistimulation (> 105 min) led to a decline in transport polarity (Figures 3 and 5). The decrease in auxin transport polarity after 90 min of gravistimulation appears to reflect changes in the rate of downward and upward auxin transport across the root cap. Figure 4 shows that the rate of upward auxin transport initially lags behind the rate of downward auxin movement. This difference between 80
downward and upward transport rates appears responsible for
establishing the gravitropic downward auxin transport polarity. However, since the rate of upward auxin
transport eventually increases more than the downward rate
of transport, polarity is reduced. It seems likely that
the rapid decrease in auxin transport polarity via changing hormone transport rates may reflect a reduction in the strength of the gravity stimulus as gravitropic curvature brings the root apex back toward vertical orientation.
It is noteworthy that the auxin transport rates do not simply continue to increase with the duration of gravistimulation. Rather, downward and upward rates increase and decrease in a periodic fashion, as though the roots were constantly adjusting hormone transport. This is consistent with the hypothesis that the root is responding to changing stimulus strength due to gravicurvature or perhaps to changes in tissue sensitivity due to elevated auxin levels.
When asymmetric auxin movement was examined at discrete intervals following gravistimulation, I again observed that significant transport polarity had developed after 45 of gravistimulation. However, further gravistimulation did not increase this polarity (Figure 5).
Instead, the level of auxin transport polarity observed at
45 min was maintained at a nearly constant value for the next 60 min of gravistimulation. Prolonged 81
gravistimulation then resulted in a decrease in auxin
transport polarity. Again, the decrease in polarity is
consistent with the idea that gravicurvature reduced the
stimulus strength. This suggests that a graviresponding
root establishes and sustains a certain minimal downward
auxin transport polarity (1.4) when the gravity sensor is optimally stimulated.
Apparent downward auxin transport polarity (1.6) was greater after 90 min of continuous transport (see Figure 3)
than the steady asymmetry (1.4) measured by periodic replacement of receiver blocks (see Figure 5). Since the model of Evans et al. (1986) proposes that auxin entering the cap of a gravistimulated root is polarly redistributed and then basipetally transported, this higher than expected polarity may indicate that basipetal auxin movement occurs more slowly than the asymmetrical redistibution of auxin across the cap. If this is the case, then one would expect to see enhanced auxin accumulation on the lower side of the root cap during the early phase of gravistimulation.
Since the agar blocks used in these experiments were longer than the length of a root cap this method measures the movement of hormone across both the cap and the apical meristem. To determine the relative contribution of the root cap to this asymmetric auxin movement, 3{j_ x a a transport across vertically- and horizontally-oriented decapped roots was measured. There was approximately five 82
times more auxin transported across vertically-oriented
intact roots than across vertically-oriented decapped roots
(Table 3) indicating that lateral auxin movement requires
the root cap. Horizontal orientation of decapped roots had no effect on auxin distribution (polarity = 1 .0 ) while gravistimulation of controls led to the expected preferentially downward movement of auxin (polarity = 1 .6 ).
These data indicate that the root cap may also be required for the development of downward auxin transport polarity.
This hypothesis is further supported by the development of downward auxin transport polarity (polarity
= 1.4) across gravistimulated, isolated root caps (Table
4). Since the absolute amount of ^h -i a a transported across the isolated caps was the same as the amount of ^h - i a a transported across the caps of intact roots, it was not surprising that the auxin polarity value obtained using isolated caps is identical to the minimum polarity maintained by graviresponding roots (Figure 5). However, without basipetal transport out of the cap into the root proper, one would expect more auxin to accumulate in the cap and hence in the receiver contacting the cap.
Several possible explanations exist for the observation that radioactivity entering the receiver from isolated caps was about the same as from caps on intact roots despite the absence of alternative sinks with isolated caps. First, despite efforts to minimize injury 83
during cap removal and to maintain conditions of high
humidity, the isolated caps used in this study may have
been damaged resulting in sub-optimal downward auxin
transport. Another possible explanation considers that
chemical communication may occur between the cap and the
root proper under normal conditions. Decapping the root
and preventing this communication may halt further downward
auxin transport following development of the minimum auxin
asymmetry that results from stimulation of the gravisensor.
This hypothesis could be verified by examining ^h -i a a
transport polarity across isolated caps after 45 min of
gravistimulation, the time required to establish this same
constant downward auxin transport polarity across the caps of intact roots. If a mean polarity of 1.4 has already been established during this shorter transport period, it would indicate that downward auxin transport ceases in
isolated caps following the establishment of a minimum auxin asymmetry. This would indicate that communication with the root proper may be necessary for auxin accumulation in the lower half of the cap prior to the onset of basipetal auxin transport.
During the experiment with isolated caps, it was noted that considerably more auxin remained in caps used to monitor upward auxin transport (Table 4). This observation plus the data shown in Figure 4 suggest that an important component in the development of a downward auxin transport 84
polarity may be that the rate of upward auxin transport
initially lags behind the rate of downward auxin transport
in gravistimulated caps.
As a control for experiments performed with isolated caps, 3h-iaa transport across isolated 1 mm apical meristem sections was examined. This study provided further evidence that initially the downward auxin transport polarity develops only across the cap since auxin movement across meristem sections was slight and non-polar.
Although these studies indicate that gravistimulation results in preferential downward movement of auxin across the root cap, I was concerned that a portion of the observed asymmetry may be an artifact of the experimental design. To determine the extent to which passive auxin movement contributes to total movement across the cap, I treated root tips with 1 mM KCN. By eliminating the active transport of auxin with a metabolic inhibitor, I would able to separate the physiologically important transport of the hormone during gravistimulation from passive movement via diffusion or capillarity. The results of these experiments indicate that establishment of downward auxin asymmetry in gravistimulated roots requires active transport (Table 5).
Although pretreatment of the root tips with KCN abolished auxin asymmetry, it did not decrease the overall movement of label within the cap. In fact, downward movement of ^H-IAA was enhanced 15% in KCN-treated roots. 85
Since this apparent increase in downward movement of 3H-IAA
in KCN-treated roots was within the limits of the standard
errors, it may represent normal experimental variation.
Alternatively, it could reflect cyanide-induced membrane
damage resulting in increased permeability to auxin,
especially the anionic form (IAA“ ). The passive
permeability of healthy membranes to IAA- is low
(Goldsmith, 1977; Goldsmith and Goldsmith, 1981).
Perhaps even more plausible, is the hypothesis that
KCN indirectly effects auxin transport by acting on the H+-
ATPase that normally generates the pH gradient necessary
for chemiosmotic auxin uptake by the cell. Mulkey et al
(1981a, 1981b) showed that growing regions of the root,
i.e. the elongation zone, secreted H+ . This is consistent
with the chemiosmotic model of auxin uptake (Goldsmith,
1981). According to the model, a plasma membrane H+-ATP
pumps H+ from the cell out, maintaining a neutral
intracellular pH and acidifying the free space. This
enhances the uptake of IAA. Once inside the cell, the
higher pH (about 7) causes the deprotonation of IAAH+ to
IAA“ . Since IAA- is highly impermeable to the membrane, it
is removed from the cell primarily by auxin efflux
carriers.
In these same experiments, Mulkey et al (1981a, 1981b) also noted that the root cap was basic, i.e. the H+-ATPase pumped H+ from the free space into the cell. By increasing 86
the pH of the free space/ the contribution of auxin uptake
via the chemiosmotic hypothesis would be minimal. However,
it is known that an auxin influx carrier exists (Jacobs and
Hertel, 1978) and thus auxin uptake would still occur in root cap cells, but at a reduced level.
If KCN treatment of the root tip inhibits the H+-
ATPase, then the pH in the free space would decrease. This would lead to enhanced auxin uptake via the chemiosmotic hypothesis. If we assume that gravistimulation causes an
increase in the auxin affinity of the auxin efflux carriers on the lower half of the cells, then the KCN-enhanced auxin uptake should result in increased downward auxin transport, as auxin availability would no longer limit pumping. This hypothesis accounts for the 15% increase in downward auxin transport observed following KCN-treatment.
However, the data also show that the elimination of transport polarity in KCN-treated roots results from an increase in the upward movement of ^H-IAA. This was most surprising. I can explain the KCN-enhanced downward auxin transport as described above, but how could KCN-treatment increase upward auxin movement? Since I assume that gravistimulation has increased the affinity of the auxin efflux carriers on the lower side of the cells, these carriers would normally compete more effectively for the available auxin than the auxin efflux carriers on the upper side. This is illustrated by the upward auxin transport 87
lagging behind the downward auxin transport during the
development of downward auxin transort polarity (Figure 4).
However, if KCN enhances auxin uptake, even the lower
affinity auxin efflux carriers on the upper side of the
cell could operate at maximum levels. Consequently, I would predict an even greater relative increase in the upward transport of auxin across KCN-treated root tips which would ultimately lead to the elimination of downward auxin transport polarity in the presence of both increased downward and upward auxin movement.
This is precisely what the data indicate (Table 5).
This experiment adds to the evidence that the almost constant rate of upward auxin movement during a period of increasing rate of downward auxin movement in gravistiraulated root caps, may be crucial to the development of downward auxin transport polarity. It also suggests that this may result from the more effective competition of the lower efflux carriers, due to a higher auxin affinity, for the available auxin. Since H+-ATPases are involved in the operation of a variety of other cellular functions, i.e. the maintenance of a membrane potential, the suggestion of a reverse, KCN-sensitive H+-
ATPase in the plasmalemma of root cap cells may at first appear to be a drastic change to propose. However, in direct data are consistent with this hypothesis indicating that it warrants further study. The Nature of Auxin Transport in Gravistimulated Roots as Determined by Investigations with Auxin Transport
Inhibitors. When intact roots were pretreated with 10~5 m
NPA/ lateral auxin movement across the caps of vertical roots and downward auxin movement across the caps of horizontal roots were inhibited 42-45% while upward movement was inhibited only 34% (Table 6 ). The overall effect of NPA treatment was to decrease downward auxin transport polarity from 1.6 to 1.4. The decrease in polarity is the result of greater NPA-induced inhibition of downward auxin transport than upward transport. One potential explanation of this observation is that gravistimulation leads to maximal activation of the auxin efflux carriers on the lower side of the cells via enhancement of their auxin affinity while the upper carriers are operating at a lower level. If this is the case, an equal inhibition of both sets of carriers would result in a relatively greater effect on the more active carriers. Consequently, I observed a greater inhibition of downward than upward auxin transport, resulting in decreased auxin transport polarity across the root cap.
A similar inhibition of auxin transport (Table 7) occurred when isolated caps were pretreated with NPA. The maximum polarity across control caps was 1.4 compared with a polarity of 1.2 across NPA-treated caps. As with intact roots downward auxin transport was inhibited by about 45%. 89
Although NPA consistently reduced downward auxin
transport across the root tip by about 45%, it inhibited
basipetal auxin movement even more (6 6 %, see Table 8 ).
Equal amounts of NPA were applied for identical pretreatment and transport periods in experiments examining
lateral and basipetal 3H-IAA transport. Since these precautions were taken to ensure similar levels of NPA uptake during the two experiments, the difference in NPA- induced auxin transport inhibition cannot be attributed to a difference in NPA uptake.
These data may indicate that the mechanism for auxin transport across the root tip differs from the mechanism for basipetal auxin transport. This conclusion is based on the observation that treatment with the same coumpound results in different levels of transport inhibition when applied to the two systems.
Conversely, the data may represent different diffusional contributions to total auxin movement in these two transport systems. It appears that both lateral and basipetal auxin transport are NPA-sensitive. If the same mechanism of auxin transport, and hence NPA-inhibition, is involved in both systems, I would expect less apparent NPA- inhibition of lateral auxin transport since the diffusional contribution would be greater over the shorter distance.
Since the distance for basipetal auxin transport was considerably greater (about 4 mm), the diffusional contribution to total auxin movement would be much smaller.
Diffusion of IAA in roots and shoots is approximately 1 mm/h (Salisbury and Ross, 1985). Since I monitored basipetal ^h -i a a transport for 90 min, I would expect the diffusional ^h -i a A front to be only 1.5 mm from the root tip. Consequently, the ^h - i a a collected in the receiver blocks should represent primarily transported hormone.
Therefore, although basipetal auxin transport appears to be more strongly inhibited by NPA than lateral auxin transport, the data may simply reflect different diffusional contributions to total auxin movement in similar auxin transport systems.
Since PBA is an excellent phytotropin and is reported to abolish root gravicurvature at 10“^ M (Katekar, 1981), I expected pretreatment with 10”6 M PBA to eliminate downward auxin transport polarity in gravistimulated roots.
Although treatment did eliminate downward auxin transport polarity as expected (Table 9), I was surprised to find that this resulted from an increase in upward auxin transport with no decrease in downward movement of auxin.
These data are similar to the data obtained by pretreatment of roots with 1 mM KCN. In both experiments, the inhibitors increased upward auxin movement and actually increased downward auxin transport slightly (about 15%).
Once again, this data can be explained via the chemiosmotic hypothesis for auxin uptake. Since PBA- 91 treatment produced results similar to KCN-treatment, PBA may also be effecting the H+-ATPases of the root cap cells.
As before, this would lead to an acidification of the free space, an increase in chemiosmotic auxin uptake and enhanced downward and upward auxin transport.
Although PBA did not inhibit the lateral movement of auxin across the tips of gravistimulated roots, it strongly
(84%) inhibited basipetal transport of auxin. These results would be predicted by the chemiosmotic hypothesis of auxin uptake. In the elongation zone, the H+-ATPase pumps H+ out acidifying the free space and promoting auxin uptake. However, if PBA affects the H+-ATPase, the pH of the free space will increase, reducing the chemiosmotic uptake of ^h -i a a . With less ^h -i a a uptake, I would expect decreased levels of auxin transport via the efflux carriers. This explanation is consistent with the 84% PBA- induced inhibition of basipetal ^h - i a a .
PBA was more effective than NPA in inhibition of basipetal auxin transport. These results, along with the fact that PBA decreases basipetal auxin transport but not auxin transport across the tip while NPA decreases auxin transport in both systems, further suggest that NPA and PBA affect auxin transport via different mechanisms.
A Comparison of IAA and ABA Transport Patterns in
Gravistimulated Maize Roots. Positive gravicurvature in roots results from an inhibition of cell extension on the 92
lower side of the elongation zone (Nelson and Evans, 1986).
This inhibition may result from an increase in auxin
concentration in that region (Went and Thimann, 1937).
Since the initial auxin asymmetry develops across the caps
of gravistimulated roots (Table 4) and since 3h-IAA is
basipetally transported (Table 8 and 10), my auxin
transport data are consistent with the model of Evans et
al. (1986) for auxin movement during root gravitropism.
Although my findings strongly indicate a role for auxin transport in the transduction phase of root gravitropism, I was concerned that the transport of other
inhibitory plant hormones may be involved, specifically
ABA. Consequently a series of experiments was performed comparing the transport of auxin and ABA in gravistimulated roots.
An examination of ^h -a b a transport across the tip showed equal amounts of label transported laterally in vertically- as well as horizontally-oriented roots, i.e.
3H-ABA transport across gravistimulated root caps is non polar (Table 11). These data suggest that the development of a downward auxin asymmetry across the cap may be specifically related to gravitropism since no asymmetry develops with a related aromatic, weak acid growth regulator.
Despite the non-polar nature of 3jj-aba movement, there was more overall 3h _aija than 3h - i a a movement across the root (Table 3 vs. Table 11). I considered the possibility
that the greater 3h _a b a transport may result from greater
^H-ABA uptake. However, an examination of radioactivity
remaining in donor blocks following the transport studies
indicated that more ^h -i a a than ^H-ABA was taken up (data
not shown). There are a number of alternative explanations
for the observation that IAA, once taken up by root cap cells, moves less readily into receivers than absorbed ABA.
Among these possible explanations are: 1) more active ABA than IAA efflux pumps; 2) more rapid conversion of IAA to conjugated, non-mobile forms; 3) more rapid metabolism of
IAA to non-mobile products; and 4) more IAA transported basipetally out of the cap and therefore unavailable to move into a receiver block.
To compare rates of efflux into receivers, vertically-
oriented roots were loaded with ^H-IAA or ^H-ABA f°r 75 min prior to a 15 min lateral transport period. Table 12 shows that despite five times more 3h -i a a uptake, twice as much 3h -a b a moved out of the root tips into a receiver.
Although these data may indicate that ABA efflux pumps are more efficient than IAA efflux pumps, they are also consistent with the other three interpretations.
I examined basipetal transport patterns for IAA and
ABA in order to determine whether differences in the transport of these two hormones in this system might be responsible for the apparent difference in their lateral 94
transport across the cap. Roots preloaded by application
of ^H-IAA or ^H-ABA to the cap, were cut into 1 mm segments
(beginning at the tip) following a transport period of 90
min. I found that ^H-ABA was confined primarily to the
first mm, i.e. the site of application while 3h -i a a
accumulated 2-4 mm behind the root tip (Figure 7).
Although the amount of labeled IAA decreased with distance
from the site of application, the drop was gradual compared
with the abrupt drop in the amount of ^H-ABA behind the
first mm of the root. These results are consistent with
the suggestion that the greater efflux of preloaded ^H-ABA than 3H-IAA from the root tip may simply be due to greater basipetal transport of auxin reducing the amount of 3h-IAA
in the vicinity of the receiver block. If this
interpretation is correct, I would predict that pretreatment of vertically oriented roots with PBA would eliminate the difference between efflux of pre-absorbed ^H-
ABA and 3h -i a a into receivers. This experiment was not done.
Conclusions on the Nature of Auxin Transport in
Gravistimulated Maize Roots. The experiments outlined in this chapter lead to the following conclusions: 1 ) downward auxin transport polarity develops across the tips of gravistimulated roots; 2 ) this transport polarity begins after 45 min of gravistimulation, reaches a value of
1.6 at 90 min, and decreases with further 95
gravistimulation? 3) once transport polarity is
established, it continues at a constant rate for about 60 min? 4) polarity occurs only across the root cap? 5)
root gravitropism appears to require the development of a certain minimal auxin asymmetry across the cap? 6 ) the development of auxin transport polarity is metabolically- dependent? 7) downward auxin transport polarity and basipetal auxin transport are sensitive to NPA and PBA, auxin transport inhibitors? 8 ) ABA is neither polarly transported across gravistimulated root caps nor basipetally transported from cap to elongation zone. CHAPTER II
CORRELATIONS BETWEEN GRAVITROPIC CURVATURE AND AUXIN
TRANSPORT ACROSS GRAVISTIMULATED ROOTS OF ZEA MAYS
According to the Cholodny-Went model of root gravitropism (Went and Thimann, 1937), gravicurvature results from the inhibition of cell extension in the lower half of the elongation zone. This is due to an increased concentration of auxin in this region following gravistimulation. The model proposed by Evans et al (1986) incorporated this hypothesis into its explanation of auxin movement within gravistimulated roots. My observations are consistent with this current model as I have noted both preferential downward auxin asymmetry across the caps of gravistimulated roots and the basipetal transport of auxin during gravistimulation.
Chapter I discussed the establishment of downward auxin transport polarity across the caps of gravistimulated roots with respect to time. Since this pattern of auxin movement consistently developed upon gravistimulation, it may be related to the curvature-inducing auxin asymmetry across the elongation zone described by the Cholodny-Went
(1937) hypothesis. If auxin is indeed responsible for
96 97 mediating positive gravicurvature, then one would predict a correlation between the development of specific auxin transport patterns and the kinetics of curvature. In view of the increasing evidence that the cap may be the initial site of auxin redistribution during the gravitropic response of roots, I compared the time course of transverse auxin movement across the caps of gravistimulated maize roots with the time course of gravicurvature. My observations show an excellent correlation between gravi- induced polar auxin movement across the root cap and the development of gravicurvature, indicating that polar auxin movement across the cap is an important component of the gravitropic transduction phase.
MATERIALS AND METHODS
For a detailed description of the plant material and the application of ^h -i a a , see the "Materials and Methods" section in Chapter I.
Determination of the Rate of Gravicurvature.
Approximately 10 seedlings were mounted in a humid chamber, oriented 90° for gravistimulation and photographed at 5 min intervals for 180 min. The degree of root curvature was measured using a protractor and rate of curvature determined by plotting curvature vs. time. This experiment was repeated 6 times. 98
Application of Inhibitors of Downward Auxin Transport
Polarity. The tips of vertically oriented roots were soaked for 60 min in an aerated 1 mM KCN solution.
Following treatment with KCN, these roots and control roots were mounted in a humid chamber and gravicurvature was monitored as before. This experiment was performed twice with 5 roots/treatment.
Gravicurvature was measured in roots pretreated with auxin transport inhibitors by applying 2 agar blocks containing either 10“ 5 M NPA or 10” 6 m PBA to opposite sides of the caps of vertically oriented roots. After 60 min, the roots were gravistimulated and curvature and rate of curvature determined in the usual fashion. This experiment was performed twice with 5 roots/treatment.
Prestimulation of Roots. Roots mounted in Petri dishes were rotated 90° and gravistimulated for 2 min. The roots were then reoriented 180° and again gravistimulated for 2 min. This alternate gravistimulation on opposite flanks was repeated every 2 min for 1 h. Roots pretreated by alternate gravistimulation in this fashion are referred to here as prestimulated roots. Following prestimulation, the roots were given long-term gravistimulation. Curvature and rate of curvature were determined as previously described. This experiment was repeated four times with 10 roots/treatment. 99
To control for effects that may arise from the mechanical manipulation of the roots during prestimulation,
one group of roots was prestimulated every 2 min for 1 h in
the usual manner but then rotated 90° out of the plane of prestimulation to expose a non-stimulated surface to the gravity stimulus. These roots are referred to here as prestimulated/rotated roots. Curvature and rate of
curvature were measured for the prestimulated/rotated roots
as described earlier. This experiment was performed four
times using 10 roots/treatment.
Measurement of Downward Auxin Transport Polarity in
Prestimulated Roots. Following 60 min of prestimulation, downward auxin transport polarity was monitored by applying a donor block (50,000 cpm of 3 H-IAA) and a receiver block to opposite sides of the root caps. The prestimulated seedlings were then gravistimulated with half of the seedlings oriented so that the donor block was on top of the cap and half oriented with the donor block on the bottom of the root cap. After a 30 min transport period, donor and receiver blocks were collected for determination of radioactivity. As a control, the same procedure was followed using prestimulated/rotated roots. Ten roots were used for each treatment and the treatments were repeated four times. 100
RESULTS
Time Course of Gravitropic Curvature. Figure 8 shows
the time course of development of gravitropic curvature in
primary roots of Merit under the conditions used for
transport experiments. Notice that there is an initial
slight upward curvature phase. This initial "backward"
curvature is characteristic of roots of Merit. During the
first 20 min of gravistimulation, the roots demonstrated a
mean upward curvature (-2°) but returned to a horizontal
position by 30 min. Continued gravistimulation resulted in
normal downward curvature reaching a maximum angle of
approximately 67° (Figure 9).
The response of control roots to gravistimulation
differs greatly from the response of roots in which the
tips were pretreated with 1 mM KCN, 10"5 m NPA or 10"6 M
PBA, treatments that abolish preferential downward auxin movement across the cap. Roots pretreated with any of
these inhibitors showed no response to gravistimulation
(Figure 8 ). Even with prolonged gravistimulation (Figure
9), the pretreated roots remained unresponsive to the gravity stimulus.
Data from experiments comparing gravitropic curvature
in control roots vs. prestimulated roots are shown in
Figures 10 and 11. When prestimulated roots were 101
Figure 8 .
The Initial Gravitropic Response of Control Roots vs. Roots Pretreated with Inhibitors of Auxin Transport.
The gravitropic curvature of control roots (circles) and roots in which the tips were pretreated with inhibitors of auxin transport (triangles) was monitored during the first 60 min of gravistimulation. Note the initial upward curvature by the control roots. The inhibitors tested were 1 mM KCN, 10-5 M NPA and 10-6 m PBA. The control data points represent the mean values of 60 roots while the pretreated data points are means of 10 roots. Standard error bars were omitted since error values were extremely small (maximum error = + 3°). CURVATURE (DEGREES) -I 5 - 25 ------iue 8. Figure NTA RVTOI RSOS FMIE ROOTSMAIZE OF GRAVITROPICINITIAL RESPONSE 0 0 0 0 0 60 50 40 30 - 20 10 1 ------1 ------IE (MINUTES) TIME 1 ------h 103
Figure 9.
Extended Time Course of Gravitropic Curvature in Control Roots vs. Roots Pretreated with Inhibitors of Auxin Transport.
The gravicurvature of control roots (circles) and roots in which the tips were pretreated with auxin transport inhibitors (triangles) was monitored over a 3 h gravistimulation period. The Inhibitors tested were 1 mM KCN, 10“ 5 m NPA and 10“6 M PBA. Each control data point represents the mean value of 60 roots. A mean value of 10 roots is shown for roots pretreated to eliminate the downward auxin tip asymmetry. Standard error bars were not included as error values were very small (maximum error = + 3°). CURVATURE (DEGREES) 80 EXTENDED GRAVICURVATURE TIME COURSE FOR MAIZE ROOTS iue 9. Figure 0 0 0 10 4 160 140 120 100 80 60 IE N MINUTES INTIME 180
104 105
Figure 10.
Short-Term Gravitropic Response of Control/ Prestimulated and Prestimulated/Rotated Roots.
The gravicurvature of control (circles), prestimulated (triangles) and prestimulated/rotated (squares) was monitored during the first 60 min of gravistimulation. The data points represent the mean values of 60 roots. Standard error bars were omitted since error values were quite low (maximum error = + 3°). CURVATURE (DEGREES) -i 0 2 - Q RVTOI RSOS FMIE ROOTSMAIZE OF GRAVITROPIC RESPONSE 3Q 20 ' 0 + + + 1 2 3 4 5 60 50 40 30 20 10 0 ------\ iue 10. Figure 4 1 ------a - - a 1 ------• / * ' - IE N MINUTES IN TIME 1 ------H
~~r m / 106 107
Figure 11.
Long-Term Gravitropic Response of Control/ Prestimulated and Prestimulated/Rotated Roots.
The curvature of primary roots of Merit was monitored over a three hour gravistimulation period. Curvature of control (circles), prestimulated (triangles), and prestimulated/rotated (squares) roots was compared. Each data point represents the mean value of 60 roots. Standard error bars were not included as error values were extremely small (maximum error = + 4°). CURVATURE (DEGREES) iue 11. Figure RVTOI RSOS O MIE ROOTS MAIZE OF GRAVITROPIC RESPONSE 0 0 0 120 100 80 60 IE N MINUTES IN TIME 0 8 1
801 ' 109
gravistimulated they began downward curvature almost
immediately (Figure 10)/ and attained a maximum curvature
of approximately 92° (Figure 11). Prestimulated/rotated
roots initially responded with negative curvature (Figure
10). The backward curvature of these prestimulated/rotated
roots was slightly exaggerated during the first hour of
gravistimulation/ but later the roots behaved similarly to
the controls with prolonged gravistimulation resulting in
positive curvature with a maximum angle of 68° (Table 13).
Comparison of Curvature and Auxin Transport Polarity during Gravistimulation: Roots of Prestimulated vs. Non- prestimulated Seedlings. Studies of the kinetics of root gravicurvature using the Bear hybrid (WF9 x 38) cultivar of maize have shown that they can be divided into two classes on the basis of their graviresponse (Evans, 1985). Class I
roots begin downward curvature within 15 min of gravistimulation. They overshoot 90° (70-100 min) and eventually return to a curvature value less than 90°.
Class II roots exhibit a transient upward curvature and then return to a horizontal position. After about 20 min, they curve downward eventually reaching a maximum curvature of about 80°. I have observed that primary roots of most seedlings of the Merit cultivar are of the Class II type
(Figures 10 and 11).
Although Merit roots typically exhibit Class II curvature kinetics when gravistimulated, prestimulation 110
Table 13.
Gravicurvature of Control/ Prestimulated and Prestimulated/Rotated Roots.
The data represent the mean value of 60 roots. Standard error was calculated for each 30 min interval. Ill
Table 13.
Gravicurvature of Prestimulated and Prestimulated/Rotated Roots
Duration of Curvature (degrees) Gravistimulation (min)
Prestimulated/ Control Prestimulated Rotated
30 3 + 1 12+1 -14 + 3 60 20+2 27+2 4 + 3 90 39+2 57+3 19 + 3 120 52+3 75+3 35 + 4 150 61+3 84+1 50 + 5 180 67+3 87+2 63 + 4 112
appears to convert them to Class I curvature kinetics.
Thus, after 30 min of gravistimulation, control roots show
essentially no net curvature having just returned to the
horizontal position from their initial backward curvature
phase (Table 13). By contrast, prestimulated roots develop
12° downward curvature during this same period since they
don't show the initial wrong-way curvature. Auxin
transport polarity was also determined after 30 min of
gravistimulation in roots that had been prestimulated or
prestimulated/rotated. Table 14 shows that within 30 min
of gravistimulation, a strong (1.7) downward auxin
transport polarity had developed in the prestimulated roots while there was essentially no auxin polarity (polarity =
1) in control roots or prestimulated/rotated roots.
DISCUSSION
The data obtained in this study are consistent with
the suggestion made by Konings (1968) that the cap is the site of initial auxin redistribution in gravistimulated
roots. These data are also consistent with the model of
auxin transport patterns within gravistimulated roots proposed by Evans et al (1986). According to this model auxin entering the cap moves preferentially to the lower side of the cap in gravistimulated roots. This pattern of auxin movement corresponds to my observation that there is 113
Table 14.
Auxin Transport Across the Caps of Control, Prestimulated and Prestimulated/Rotated Roots.
Auxin transport polarity was measured following 30 min of gravistimulation. The data represent the mean of at least 24 roots/treatment with a maximum standard error of + 0.1 polarity units. Donors contained approximately 50,000 cpm/block. Table 14. 114
Auxin Transport Across the Caps of Control Rootsj Prestimulated Roots and Prestimulated/Rotated Roots.
Treatment Polarity (downward/upward)
Control 1.0 Prestimulated 1.7 Prestimulated/Rotated 1.0
30 min transport time. 115 downward auxin transport polarity across the caps of gravistimulated maize roots. According to the model, the auxin accumulating in the lower half of gravistimulated root caps is transported to the lower half of the elongation zone where the resulting increase in the concentration of auxin causes the differential growth inhibition leading to positive gravicurvature (Evans et al., 1986). I have observed basipetal movement of auxin from the cap to the elongation zone in experiments monitoring the transport of tip applied 3h -IAA (Figure 7).
If this model is correct and the cap is the site of initial auxin redistribution, then one would predict a close correlation between the kinetics of gravitropic curvature and the kinetics of the development of asymmetric auxin movement across the cap. This was found to be the case.
The time course of the development of downward auxin transport polarity (Figure 3) paralleled the time course for the rate of gravitropic curvature (Figure 12). The magnitude of preferential downward auxin movement increased with the duration of gravistimulation, as did the rate of gravicurvature. Both auxin transport polarity and rate of curvature peaked between 75 and 90 min of gravistimulation, and subsequently decreased with additional stimulation.
The close relationship between transport polarity and rate of curvature (Figure 12) indicates that auxin asymmetry may 116
Figure 12.
Correlation Between Downward Auxin Transport Polarity and the Rate of Gravicurvature.
Auxin transport polarity is represented by circles and rate of gravicurvature by triangles. Standard error of transport polarity was +0.1 polarity unit. The rate of gravicurvature was determined by plotting curvature vs. time and taking tangents to the curve at the indicated times. Correlation Between Polarity and Rate of Curvature cn v i (o ut I I I RATE OF CURVATURE (degrees/mRATE in) o • oo • o o • o o o o • o o * o o • o • c j - * ootoo-^rooiAuicnvi
o 15 30 45 60 75 90 105 120 135 150 165 180 TIME IN MINUTES (downward/upward) POLARITY TRANSPORT 118
cause the growth asymmetry responsible for gravicurvature
as originally proposed by Cholodny and Went. The correlation between auxin transport polarity and rate of
curvature is displayed more directly in Figure 13 where transport polarity is plotted directly against rate of curvature.
Although the general correlation between curvature rate and auxin movement was strong, I could detect asymmetric auxin transport only after the initiation of gravicurvature. This is contrary to the suggestion that asymmetric auxin movement across the cap establishes the hormone gradient causing curvature. However, the lag in measurable auxin transport polarity behind curvature may be attributed to the methods used to monitor asymmetric auxin movement. Auxin movement was determined by collecting 3h -
IAA in agar blocks applied to the surface of the root cap.
Using this method, one would expect an internal auxin transport polarity to develop prior to asymmetric accumulation in applied agar blocks.
Another potential explanation for this observation considers the effect of the basipetal auxin transport streams on the development of polarity. As ^h - i a a from the donor moves into the cap, some of the label is transported across the columella cells and into the receiver. However,
Figure 7 indicates that a large portion of the ^H-IAA entering the cap is loaded into the basipetal auxin 119
Figure 13.
Auxin Transport Polarity vs. Rate of Gravicurvature.
Data from Figure 12 are replotted to show more directly the relationship between transport polarity and rate of curvature. TRANSPORT POLARITY (down/up) 1.00 1.10 1.20 1.30 1.40 1.50 1.60 + 1.60 1.70 0. 0. . 03 . 0.7 0.5 0.3 0.1 .1 -0 .3 -0 + + + + + + iue 13. Figure TRANSPORTPOLARITY RATEvs OF RATECURVATUREOF (dcgrees/min)
F R U T A V R U C 120 121
transport streams and moved into the root proper. The
constant removal of 3h - i a A from the cap via this mechanism
would lead to a delay in the appearance of a downward auxin
transport polarity.
A second apparent discrepancy in the correlation
between auxin polarity and rate of gravicurvature is the
finding that the rate of gravicurvature begins to decrease
75-90 min after the beginning of gravistimulation even
though asymmetric auxin movement reaches a maximum at 90 min (Figure 10). I have considered the possibility that
the decrease in curvature rate despite high auxin transport asymmetry may reflect a decrease in tissue sensitivity to auxin resulting from extended exposure to elevated concentrations of the hormone. The ability of roots to adapt to inhibitory levels of auxin and resume normal growth is well documented (List, 1969). Gougler and Evans
(1981) found that primary roots of maize immersed in 0.01 uM IAA begin to recover from growth inhibition within 30 min.
Prolonged gravistimulation led to a decline in the rate of gravicurvature as well as a drop in the asymmetry of auxin movement. It seems likely that these changes reflect a reduction in the strength of the gravity stimulus as gravitropic curvature brings the root apex back toward vertical orientation. Decreased asymmetry of auxin movment across the cap would result in decreased auxin delivery to 122 the elongation zone on the lower side. Thus, in addition to a possible decrease in tissue sensitivity to auxin, less auxin would be transported to the elongation zone. This combination of events may account for the shape of the curvature rate vs. time curve for gravistimulated roots.
The suggestion that there is a link between auxin transport polarity and rate of curvature is strengthened by experiments in which gravicurvature was measured in roots pretreated to eliminate asymmetric auxin movement. When the tips of roots were treated with 1 mM KCN (Table 5), 10"
5 M NPA (Table 6) or 10"6 M PBA (Table 9) prior to gravistimulation, no downward auxin transport polarity developed across the caps. Since Figure 13 indicates a strong correlation between the development of asymmetric auxin movement in the cap and the rate of curvature, one would predict that pretreatments interfering with polar auxin movement across the cap would affect gravicurvature. Figure 9 shows this to be true, as roots pretreated with inhibitors that prevent the development of polar auxin transport exhibited no gravicurvature even during a 3 h gravistimulation period.
Figure 12 indicates that transport polarity and rate of gravicurvature correlate even during the initial negative curvature phase. Although the initial upward gravicurvature in gravistimulated roots of Merit appears to correlate with upward auxin movement, I am unable to 123
explain why this temporary reverse in preferred direction
of auxin movement occurs. Hild and Hertel (1972) reported
a similar phenomenon in gravistimulated coleoptiles of maize. Initially the coleoptiles responded to gravity with
slight downward curvature. The coleoptiles then returned
to the horizontal and began normal upward curvature. Hild
and Hertel noted that the initial downward gravicurvature
correlated with auxin transport to the upper side. When similar experiments were performed using amylomaize, a cultivar with small, slowly-sedimenting amyloplasts, the
initial phase of downward curvature did not occur. Hild and Hertel interpreted these results as an indication that the initial wrong way curvature of the wild type results from an overstimulation of the gravity-sensing mechanism.
They proposed that prestimulation via the alternate gravistimulation method may allow for adaptation of the sensory mechanism and permit a response to subsequent gravistimulation without an initial period of wrong-way curvature.
Based on the work reported here, it appears that the initial upward curvature of gravistimulated Merit roots may also reflect overstimulation of the gravity-sensing mechanism. Prestimulated roots curved downward with no initial wrong-way curvature (Figure 8). These roots also exhibited earlier and stronger preferential downward auxin movement than control roots (Table 14). The data are 124
consistent with the hypothesis that prestimulation allows
adaptation of the gravity-sensing mechanism preventing
overstimulation and wrong-way curvature upon
gravistimulation.
To control for effects that may arise from the mechanical manipulation of the roots during prestimulation,
a group of roots was prestimulated and then rotated 90° out of the plane of prestimulation, exposing a non-adapted flank. Since the gravity-sensor would not be adapted in this plane, one expects curvature and auxin transport similar to control values. I found this to be the case.
Prestimulated/rotated roots demonstrated Class II curvature kinetics typical of control roots (Figure 11). Also, following 30 min of gravistimulation, prestimulated/rotated roots demonstrated a mean auxin transport polarity value of
1.0, as did the control roots (Table 14). These data support the hypothesis that initial wrong-way curvature may reflect an overstimulation of the gravity-sensing device and that the gravi-sensor can be adapted via an alternate gravistimulation treatment prior to gravistimulation. In addition, the data underscore the correlation between gravitropic curvature and auxin transport polarity across the cap.
Although auxin transport polarity and the rate of gravicurvature correlate in control, prestimulated and prestimulated/rotated roots, I noted an exaggerated 125
negative curvature response in the prestimulated/rotated
roots. Table 13 indicates that following 60 min of
gravistimulation control roots have achieved a downward
curvature of 20° while prestimulated/rotated roots have
just begun their downward curvature phase (4°). Not only
does the wrong-way curvature persist for a longer period of
gravistimulaton in prestimulated/rotated roots, the extent
of curvature is also greater. These roots demonstrate a mean maximum negative curvature of -17° in 20 min while the mean curvature of control roots at this time is only -2°.
To better understand this exaggerated negative curvature,
auxin transport polarity should be measured across the caps
of prestimulated/rotated roots following 15 min of
gravistimulation. Because of the excellent polarity/curvature rate correlation, I would predict a
strong upward auxin transport polarity, especially since slight upward curvature in control roots after 10 min of gravistimulation (Figure 10) corresponded to a slight upward auxin transport polarity (Table 2).
Although auxin transport polarity is 1.0 in prestimulated/rotated roots after 30 min of gravistimulation (Table 14), the roots are -14° from the horizontal. This appears contrary to the consistent polarity/curvature rate correlation. However, one should note that 30 min is the inflection point in the gravicurvature time course for these roots. Thus, the 126
polarity value of 1.0 may be a reflection of the reversal
in the preferential direction of auxin movement at this
time. This hypothesis is supported by the observation that
control roots show -2° curvature and a transport polarity
of 1.0 after 15 min of gravistimulation, i.e. at the
inflection point in the curvature time course. In fact,
significant downward auxin transport polarity in control
roots was detectable only after 45 min of gravistimulation
(Figure 3), by which time the rate of downward
gravicurvature was substantial. As previously discussed,
the lag in the appearance of auxin transport polarity may
be an artifact of the experimental design and/or the result
of continuous siphoning of 3H-IAA from the cap into the
root proper via the basipetal auxin transport streams. In
view of these observations with control roots, I would predict that significant downward 3H-IAA polarity could
also be measured 30 min after the inflection point (60 min
gravistimulation) in the prestimulated/rotated curvature
time course. I suggest that auxin transport polarity
across the caps of prestimulated/rotated roots be measured after 15 and 60 min of gravistimulation to confirm that despite an exaggerated gravicurvature response, the polarity/curvature rate correlation still holds for prestimulated/rotated roots. If these experiments continue to support this correlation, as I suspect they will, then the exaggeration of upward curvature in these roots may 127
reflect an even more pronounced overstimulation of the
gravi-sensor perhaps due to adaptation of the sensing
mechanism in a plane different from the final plane of
stimulation.
Although the data presented in this chapter are
consistent with the idea that auxin moves into the caps of
gravistimulated roots and is then redistributed resulting
in gravicurvature (Konings, 1968; Evans et al., 1986;
Hasenstein and Evans, 1988), one cannot eliminate the
possibility that the auxin controlling gravitropic
curvature is synthesized in the cap. However, Feldman
(1981) reported that auxin biosynthesis in roots of this
cultivar of maize is strongest in the proximal meristem
with no synthesis detectable in the cap. Also, Hasenstein
and Evans (1988) showed that auxin movement in maize roots
is basipetal only when the root cap is intact. In the
absence of the cap the net direction of auxin transport is
toward the tip. This implies that the cap is required for
strong basipetal auxin movement but may not serve as a
source of auxin.
The experiments presented in this chapter provide strong support for a correlation between auxin transport polarity and rate of curvature. In fact, the correlation between these two events is so strong that it becomes difficult to explain gravicurvature via the model proposed by Evans et al (198 6). Because the model proposes that the 128
auxin redistributed in the cap of the gravistimulated root
is ultimately responsible for curvature, I would predict a
lag between the development of auxin transport polarity in
the cap and the beginning of gravitropic curvature. A lag
would seem to be required for the redistributed auxin in
the cap to be transported to the lower half of the
elongation zone and cause inhibition of cell extension.
Since the suspected rate of basipetal auxin transport is
approximately 1-5 mm/h (Salisbury and Ross, 1985), I would
expect a 30 min lag between the development of downward
auxin transport polarity across the cap and the development
of gravicurvature. However, Figure 13 indicates that no
such lag occurs.
There are at least three possible explanations that
would fit the correlation between transport polarity and
curvature to the current model. First, experiments in
Chapter 1 indicate that polar auxin movement across the cap
and basipetal auxin movement toward the elongation zone may occur via two different mechanisms. If this is the case,
then it may be that the redistributed auxin is loaded into a fast-moving basipetal transport stream. If auxin moves / rapidly from the lower half of the cap to the lower half of
the elongation zone, the correlation between curvature and auxin transport polarity across the cap would be consistent with the model. However, a comparison of data on auxin
transport across the caps obtained by allowing continuous 129 accumulation in a single receiver (Figure 3) with transport measured at intervals, i.e. periodic replacement of receivers (Figure 5), suggests that there may be a short
lag in the initiation of basipetal transport. Since continuous transport results in a higher polarity (1.6) than transport measured at intervals by receiver replacement (1.4), it appears the increased polarity during continuous transport may reflect auxin accumulation due to a lag in the initiation of basipetal transport. This auxin accumulation in the lower half of the cap resulting from an initial lag in basipetal transport would not be observed during interval transport measurements since receivers are replaced periodically.
A second possible explanation for the observed results is that auxin asymmetry across the cap triggers the very rapid movement of some other messenger from the tip to the elongation zone resulting in gravicurvature.
Finally, although it does appear that gravistimulation results in downward auxin transport polarity across the root cap and that basipetal auxin transport also occurs in gravistimulated roots, the auxin mediating curvature may not be the same auxin that is redistributed in the cap.
Although basipetal transport would ultimately move the auxin from the cap to the elongation zone, the auxin responsible for initiating gravicurvature may come from a different source. I propose that establishment of auxin 130
asymmetry leads somehow to rapid release of auxin, perhaps
from the stele, in the region of the elongation zone, and
that the auxin redistributed in the cap arrives in the
elongation zone only after a substantial lag and thus
contributes to but does not initiate gravicurvature.
Several qualitative observations support this
hypothesis. I have noted that asymmetric application of
low concentrations of auxin (5 x 10“® M) to the root caps of vertically oriented roots results in curvature toward
the hormone within 30 min while application directly to the elongation zone has no effect. However, asymmetric application of 10”5 m IAA to the cap causes curvature toward the hormone only after 2 or more hours. Conversely,
10“5 m IAA applied to one side of the elongation zone results in the initiation of curvature within 30 min.
This experiment has been repeated by C. Stinemetz (personal communication) using auxin concentrations of 10“® M and
10”5 m and similars experiments were performed by Davies et al (1976). These observations suggest that the auxin redistributed in the cap may not be directly responsible for the curvature in the elongation zone. Rather, development of a small auxin asymmetry across the cap may trigger a larger auxin asymmetry across the elongation zone from auxin originating in the elongation zone.
Considering these qualitative experiments along with the quantitative studies of auxin transport polarity and 131 gravicurvature, I propose the following modification to the model of Evans et al (1986): downward auxin transport polarity across gravistimulated root caps leads to a subtle asymmetry of auxin movement out of the caps. This acts as a signal to trigger rapid release of auxin in the lower half of the elongation zone, possibly from the stele
(personal communication with C.L. Stinemetz). It is this stele-associated auxin which initiates gravicurvature while basipetally transported auxin sustains or contributes to the response. This hypothesis is supported by experiments on the movement of pulse application of ^h -i a a in roots
(Stinemetz, unpublished). Following the loading of the stele at the basal end of an intact apical root segment with 3h_ i a A, stinemetz observed high levels of ^h -i a a in the stele, the apical meristem and the lower half of the elongation zone of gravistimulated roots. However, there were only trace levels of the ^h -IAA in the root cap, again suggesting that the growth-inhibiting levels of auxin necessary for gravicurvature do not come from the auxin asymmetry across the cap but rather from the stele.
Conclusions on the Correlation between Gravitropic
Curvature and Auxin Transport Polarity Across the Caps of
Gravistimulated Roots. The experiments described in this chapter have led to the following conclusions: 1) There is an excellent correlation between the development of downward auxin transport polarity and the kinetics of 132 gravicurvature; 2) The initial wrong-way curvature of
Merit roots may reflect an overstimulation of the gravi- sensor and this initial upward curvature phase can be by passed by pre-adaptation of the gravi-sensor; 3) The auxin asymmetry across the caps of gravistimulated roots may indirectly cause gravicurvature by triggering the release of large amounts of auxin within the elongation zone rather than by basipetal transport of the redistributed auxin itself. CHAPTER III
POSSIBLE AUXIN TRANSPORT/CALCIUM TRANSPORT INTERACTIONS
The data presented in Chapters I and II suggest that
development of auxin asymmetry across the elongation zone
may be triggered by the establishment of downward auxin
transport polarity across the root cap. Recent evidence
indicates that calcium movement may also be involved in
mediating the gravitropic response. Lee et al (1983b)
observed that application of EDTA to the root cap had no
effect on root growth but rendered the root unresponsive to
gravity. Removal of EDTA and replacement with calcium
restored graviresponsiveness. These experiments support a
role for mobile calcium in the mediation of root
gravitropism.
Lee et al (1983a) also noted that gravistimulation
resulted in the development of a preferential downward movement of calcium across the root cap, much as I observed
a preferential downward movement of auxin. This
observation coupled with the fact that auxin transport
inhibitors reduce both downward auxin transport polarity
(Tables 6 and 9) and downward calcium movement (Lee et al,
133 134
1984) indicate that gravi-induced transport of auxin and
calcium may be related. Evans et al (1986) propose that a
common component in gravi-induced downward auxin and
calcium transport may be the activation and/or regulation
of trans-membrane auxin and calcium pumps via calmodulin.
To explore possible auxin/calcium interactions in the mediation of root gravitropism, a series of transport studies was performed. I tested the involvement of calmodulin in the establishment of the downward auxin and calcium asymmetries using calmidazolium, a calmodulin antagonistic drug; I evaluated the effect of calcium depletion on the development of gravi-induced auxin asymmetry across the cap; and I examined simultaneous auxin and calcium movement in dual label transport studies.
MATERIALS AND METHODS
For a description of the plant material and the application of ^h - i a a , see the "Materials and Methods" section in Chapter I.
Application of 45ca2+. a s in the previously described
^H-IAA and ^H-ABA transport experiments, agar (1.5%) sheets were incubated overnight in distilled water (pH adjusted to
6.5 with NaOH). Once cut into small blocks (1.5 mm cubes), a stock solution of 45caCl2+ (92.5 GBq/mg, Amersham) was 135
applied to incorporate 833 Bq 4 5 Ca2+/clonor block
(approximately 50,000 cpm/block). *
To measure calcium movement (as 45ca2+ transport)
across the root cap, donor and receiver blocks were placed
opposite one another on the caps of vertically-oriented
roots mounted in Petri dishes and the roots were
gravistimulated by rotating the Petri dishes 90°. The
roots were oriented such that the donor block was on the
upper side of the root cap allowing the measurement of downward 4^ca2+ movement. Following transport periods of
30, 60, 90, and 120 min, donor and receiver blocks were
collected and placed in separate scintillation vials for
the determination of radioactivity. This experiment was performed four times with eight roots/transport period.
Pretreatment of Roots with Calmidazolium. To determine whether or not calmodulin may be involved in mediating the development of the downward calcium gradient across the caps of gravistimulated roots, the effects of calmidazolium (CMZ), a calmodulin antagonistic drug, were examined. A stock solution of 10"4 M CMZ (in 1% ethanol) was diluted to make 10"6 M and 10"7 M solutions. Since CMZ binds to glass (personal communication, R. David Johnson), care was taken to prepare all solutions with plastic labware. These solutions were added to three plastic Petri dishes, each containing agar sheets. The agar sheets were incubated in CMZ overnight. To prevent the loss of CMZ 136
incorporated into the sheets via glass-binding, the agar was cut into 1.5 mm cubes on a small plastic base rather than on a glass microscope slide as in previous experiments. Receiver blocks were used directly while donor blocks were incubated overnight following application of 45ca2+ stock solution. Vertically-oriented roots were pretreated by applying CMZ-containing blocks to opposite sides of the root cap for 30 min. Following the
30 min pretreatment, one of the blocks was replaced with a
CMZ-containing ^5ca2+ donor block. The roots were gravistimulated for 30, 60, 90, and 120 min to monitor the development of the downward 45Ca2+ gradient. After the designated period of transport, donor and receiver blocks were collected for determination of radioactivity. This time course was performed four times with eight roots for each concentration of CMZ.
To discern the possible relationship between calcium movement and auxin movement within gravistimulated roots and the possible mediation of this transport by calmodulin, the accumulation of 3h - i a a in the elongation zone was monitored following pretreatment of the root cap with 10"5
M, 10-6 M or 10“7 M CMZ. As before, CMZ-containing blocks were applied to opposite sides of the caps of vertically- oriented roots. Following 30 min of pretreatment, a plain agar block and one containing 50,000 cpm were applied opposite each other across the elongation zone, 137
approximately 4 mm behind the root apex. The roots were
then gravistimulated 30 or 120 min with the donor on the
upper side of the elongation zone The four blocks were
then collected and placed into separate scintillation vials
for the determination of radioactivity. This time course was performed once using eight roots for each concentration
of CMZ.
Pretreatment of Root Tips with EGTA. In order to determine the effect of calcium depletion on auxin
transport polarity, 32 seedlings were pretreated (apical 2 mm) with aerated 2 mM EGTA for 30 min. Half of the seedlings were then soaked in aerated distilled water for
30 min. The remaining roots were soaked for 30 min in aerated 5 mM CaCl2 » As controls, a third group of roots was soaked for 30 min in distilled water and a fourth group
in 2 mM EGTA for 30 min with no additional treatment.
Following these pretreatments, the roots were mounted in
Petri dishes and prepared for auxin transport by applying
^H-IAA donor blocks and gravistimulating for 90 min as described in Chapter 1. This experiment was performed three times using 8 roots/treatment. EGTA solutions were prepared by stirring overnight on low heat.
Simultaneous Monitoring of Downward Auxin Transport
Polarity and Downward Calcium Transport Polarity Across the
Caps of Gravistimulated Roots. To better define the relationship between the transport of IAA and Ca2+, donor 138 blocks containing both ^h -IAA and 4f>ca2+ were prepared
(50,000 cpm ^h - i a a and 50,000 cpm ^^Ca2+/donor block).
Donor and receiver blocks were placed on opposite sides of the caps of vertically-oriented roots. The roots were then rotated 90° and gravistimulated for 5, 10,15, 30, 45, 60,
75, 90, 105, 120, and 180 min to generate time courses for the simultaneous transport of ^h -i a A and 45ca2+ across the root cap. Following the prescribed transport period, donor and receiver blocks were collected for radioactivity determination using a dual label counting mode. This experiment was performed three times with eight roots/transport period.
Simultaneous Monitoring of and *5ca2+ Basipetal
Transport. The basipetal movement of auxin and calcium was followed by applying 3H-iAA/45ca2+ donor blocks to one side of the caps of vertically-oriented roots for 90 min.
Following this loading period, the roots were cut into 1 mm sections beginning at the tip and each section was placed into a separate scintillation vial for determination of radioactivity using a dual label counting mode. This experiment was performed four times with eight roots/experiment. 139
RESULTS
The Effects of Calmidazolium on Calcium and Auxin
Transport in Gravistimulated Roots. Table 15 shows the
relationship between the concentration of calmidazolium
(CMZ) used for pretreatment and the development of the downward movement of calcium. Pretreatment of the root tip with 10-7 m CMZ had no effect on the development of the gravi-induced downward calcium asymmetry over a 2 h transport period. However, pretreatment with 10“6 M CMZ resulted in a 43% inhibition of downward ^5ca2+ transport after only 30 min of gravistimulation. Calcium transport inhibition increased to 63% following 60 min of gravistimulation and reversed somewhat (46% inhibition) after 120 min.
Gravi-induced redistribution of ^H-IAA applied to the elongation zone was examined following pretreatment of the root cap with 10“ 5 M, 10“® M or 10“7 m CMZ. 3h -IAA was applied to the upper half of the elongation zone (about 4 mm from the tip). Receiver blocks were placed on the upper and lower sides of the cap (positions 1 and 2 respectively) and on the lower side of the elongation zone directly beneath the donor block (position 3). Table 16 indicates variable results for the accumulation of ^H-IAA at positions 1 and 2 following 30 or 120 min of gravistimulation. However, after 30 min of Table 15.
The Effect of Calmidazolium on Gravi-induced Transport of Across Maize Root Tips.
Root tips were pretreated with 10“® M or 10“7 M calmidazolium (CMZ) for 30 min prior to gravistimulation for periods of 30, 60, 90 and 120 min. Donors contained 50,000 cpm of ^^Ca^+/block, All data points represent the mean value + S.E. of at least 32 roots. 141
Table 15.
The Effect of Calmidazolium on Gravi-induced Transport of 45q 32+ Across Maize Root Tips.
Concentration Transport Time
30 60 90 120
cpm
0 1538 + 115 2790 + 396 3680 + 297 4687 + 315 0.1 1507 + 192 2624 + 283 3739 + 308 4593 + 352 1.0 872 + 80 974 + 119 1746 + 225 2529 + 268 142
Table 16.
The Effect of Pretreatment of Root Caps with Calmidazolium on Gravi-induced ^H-IAA Redistribution in the Elongation Zone.
Roots were pretreated with 10”5 M, 10“ 6 M or 10”7 M CMZ for 30 min prior to gravistimulation. Following transport periods of 30 or 120 min, donors (50,000 cpm 3h - IAA) and the three receivers were collected from each root and placed into separate scintillation vials for determination of radioactivity. Position 1 refers to the receiver block located on the upper side of the root cap; position 2 refers to the block on the lower side of the cap; and position 3 refers to the receiver located approximately 4 mm from the root tip on the lower side of the elongation zone. The data points represent the mean + S.E. of only 8 roots. 143
Table 16.
The Effect of Pretreatment of Root Caps with Calmidazolium on Gravi-induced ^H-IAA Redistribution in the Elongation Zone.
CMZ (uM) 30 min Transport Period
Position 1 2 3
0 130 + 7 91 + 11 321 + 75 0.1 131 + 9 96 + 13 259 + 25 1.0 140 + 10 142 + 22 101 + 18
120 min Transport Period Position 1 2 3
0 157 + 15 183 + 36 354 + 26 0.1 141 + 14 187 + 27 384 + 24 1.0 168 + 27 140 + 19 357 + 39 144 gravistimulation, ®H-IAA accumulation at position 3 was reduced 19%, 35% and 69% for roots pretreated with 10"? m ,
10"® M and 10"® M CMZ, respectively. Surprisingly, the inhibitory effect of CMZ on auxin accumulation in the lower part of the elongation zone seen after 30 min of transport was not expressed after 120 min of transport.
The Effects of EGTA on the Development of the Downward
Auxin Transport Polarity. Pretreatment of root tips with calcium promotes downward auxin transport in gravistimulated roots (Karl Hasenstein, personal communication). Since Hasenstein's data indicate that auxin transport may be calcium-dependent, I hypothesized that calcium depletion would abolish gravi-induced auxin asymmetry. To test this hypothesis, I pretreated root tips with 2 mM EGTA for 30 min. Gravistimulated control roots developed strong auxin transport polarity (polarity = 1.6) while EGTA-treated roots did not (polarity = 1.1) (Table
17). Since I could abolish the gravi-induced auxin asymmetry by pretreating with 2 mM EGTA , I was curious to see if this effect could be reversed by calcium application. Table 17 shows that pretreatment for 30 min with 2 mM EGTA followed by treatment with 5 mM CaCl2 for an additional 30 min, not only reversed the EGTA effect but actually enhanced downward auxin transport polarity
(polarity = 2.0) relative to the untreated control. 145
Table 17.
The Effect of EGTA on ^h -i a A Transport Polarity Across the Caps of Gravistimulated Roots.
Root tips were soaked 30 min in aerated 2 mM EGTA. One group of EGTA-treated roots was then soaked in aerated distilled water for 30 min while a second group was soaked in 5 mM calcium for 30 min. Control root tips were soaked in aerated distilled water for 30 min. The roots were then used to monitor ^h -i a a transport polarity upon gravistimulation. This experiment was performed four times with eight roots/treatment. The data points represent the mean value + S.E. of at least 32 roots/treatment. 146
Table 17.
The Effect of EGTA on ^H-IAA Transport Across the Caps of Gravistimulated Roots.
Treatment Transport Direction Polarity T->B B->T
cpm
Control 2381 + 136 1501 + 88 1.6 + 2mM EGTA 2305 + 47 2111 + 112 1.1 + 2mM EGTA/H20 2067 + 158 1604 +149 1.3 + 2mM EGTA/Ca2+ 2870 + 144 1443 + 104 2.0 147
As an additional control for this experiment, a group of roots was pretreated with 2 mM EGTA for 30 min then treated an additional 30 min by soaking the tips in aerated distilled water. These roots developed a downward auxin transport polarity of 1.3 (Table 17).
The Simultaneous Monitoring of Downward Auxin
Transport Polarity and Downward Calcium Transport Polarity
Across the Caps of Gravistimulated Roots. Donor blocks containing 50,000 cpm ^H-IAA a n & 50,000 cpm 45ca2+ were used to monitor simultaneously the development of downward auxin transport polarity and downward calcium transport polarity. The time course for the development of these two transport polarities is illustrated in Table 18. Note that the auxin transport values and the auxin asymmetry values measured in the presence of 45ca2+ (Table 18) essentially correspond to the previous data in which auxin transport and polarity were monitored alone (Table 2).
Downward calcium transport polarity was determined in the same manner as auxin transport polarity, i.e. by dividing downward calcium transport by upward calcium transport. When downward calcium transport polarity was plotted against time (Figure 14), I noted an initial upward polarity (polarity = 0.9). Additional gravistimulation resulted in a reversal of this upward polarity as significant downward calcium transport polarity was established after 45 min (polarity - 1.2). The downward 148
Table 18.
Simultaneous Transport of ^H-IAA and 45ga2+ Across the Caps of Gravistimulated Roots.
Donors (50,000 cpm ^H-IAA, 50,000 cpm 45ca2+) and receivers were placed opposite one another on the caps of gravistimulated roots. Following the designated transport period, both were placed in separate scintillation vials for the determination of radioactivity. Each data point represents the mean + S.E. of 24 roots/transport period. 149
Table 18.
Simultaneous Transport of ^H-IAA and 45ca 2+ Across the Caps of Gravistimulated Roots.
Time (min) ^H-IAA Tranport (cpm) 3h -i a a Polarity
T->B B->T
5 445 + 34 435 + 38 1.02 10 476 + 47 539 + 66 0.88 15 589 + 43 621 + 65 0.95 30 931 + 60 919 + 82 1.01 45 1498 + 98 1165 + 102 1.29 60 1783 + 155 1317 + 101 1.35 75 2300 + 81 1543 + 112 1.49 90 2531 + 116 1528 + 53 1.66 105 2479 + 217 2090 + 147 1.19 120 2908 + 205 2527 + 184 1.15 180 3127 + 189 2767 + 205 1.13
45ca2+ Transport (cpm) 45ca2+ polarity
T->B B->T
5 643 + 59 638 + 62 1.01 10 858 + 67 931 + 121 0.92 15 859 + 46 842 + 82 1.02 30 949 + 55 966 + 74 0.98 45 1704 + 128 1471 + 130 1.16 60 1940 + 228 1555 + 178 1.25 75 2100 + 192 1666 + 121 1.26 90 3024 + 286 2638 + 243 1.15 105 3793 + 84 3322 + 321 1.14 120 4898 T 250 4509 + 200 1.09 180 6172 + 492 5398 + 405 1.14 150
Figure 14.
The Time Course of Development of Downward Calcium Transport Polarity Across the Caps of Gravistimulated Roots.
Donors and receivers were placed opposite one another on the caps of gravistimulated roots. Following the designated transport period, both were collected and placed into separate scintillation vials for the determination of radioactivity. The data points represent the mean value of 24 roots/transport period. The maximum standard error is 0.1 polarity unit. TRANSPORT POLARITY (down/up) 9- .9 0 0.8 1.0 1 - .3 1 1.4 . 2 AA A -A A— - 0 CALCIUM TRANSPORT ACROSS POLARITY ROOT TIPS iue 14. Figure 30 IE N MINUTES INTIME 90 120 150
18060 151 152
calcium transport asymmetry peaked at 1.3 following 60-75
min of gravistimulation. Continued gravistimulation
resulted in the decrease of calcium polarity to 1.1 after
105 min where it remained during the next 75 min of
gravistimulation.
A comparison of downward auxin and calcium transport polarities reveals several similarities (Figure 15).
During the first 10 min of gravistimulation, both auxin and
calcium demonstrate preferential upward movement, but both polarities are 1.0 following 30 min of gravistimulation.
Although the auxin asymmetry is stronger than the calcium
asymmetry, both develop significant polarities after 45 of gravistimulation, reach maximum polarities with additional stimulation and return to a polarity of 1.1 after 180 min.
Figure 15 also illustrates several important differences between the development of downward auxin and calcium transport polarities. Calcium transport polarity peaks after 60 min of gravistimulation while the auxin polarity continues to increase until 90 min. Once the downward calcium asymmetry has achieved its maximum value, it remains high for about 15 min before gradually decreasing to 1.2 (at 90 min). Conversely, auxin transport polarity peaks (polarity = 1.7 at 90 min) and decreases sharply within the next 15 min (polarity = 1.2). Finally, downward calcium transport polarity never exceeds 1.3 in contrast to the peak downward auxin asymmetry of 1.7. 153
Figure 15.
A Comparison of Auxin and Calcium Transport Polarities as Determined from Dual Label Experiments.
Auxin (striped bars) and calcium (open bars) transport were measured simultaneously in this dual label experiment. Maximum standard error is 0.1 polarity unit and each polarity bar represents the mean of 24 roots. TRANSPORT POLARITY (down/up) * "°*9 n o.8 -LL 1 1.1 1.3- 1 1.4- 1.5- 1 1*71 . . . 0 2 iue 15. Figure 6 - - - - o 30 U I V. CALCIUMAUXIN VS. TRANSPORT POLARITY 0 0 120 90 60 IE N MINUTES IN TIME 150 180 155
To better understand the possible relationship between downward auxin and calcium transport polarities/ the calcium transport data from Table 18 was plotted against time. Figure 16 shows that upward calcium transport lags behind downward calcium transport for the duration of gravistimulation. When the rates of downward and upward calcium transport were determined by measuring the slopes of tangents to the curves in Figure 16af and then plotted against the time of gravistimulation (Figure 16b)/ I noted that the development of calcium transport polarity was the result of the downward transport rate increasing more than the upward rate. As polarity peaked (60-75 min)/ both downward and upward transport rates dropped sharply with the upward rate decreasing more than the downward rate.
The decline in downward calcium transport asymmetry at 90 min was reflected by the upward transport rate increasing slightly more than the downward rate. The constant calcium polarity maintained (1.1) for the remainder of the gravistimulation period appeared to result from synchronous changes in both downward and upward transport rates (Figure
16b).
The Simultaneous Basipetal Transport of ^h -i a a and
45ca2+ in Vertically-Oriented Roots. When the basipetal movement of 3h - i a a and 45ca2+ was monitored simultaneously/ striking differences in transport were observed. As expected (see Figure 7)f 3H-IAA was basipetally transported 156
Figure 16.
A Comparison of Downward and Upward Calcium Transport in Gravistimulated Roots.
Downward (circles) and upward (triangles) calcium transport were plotted against time (upper graph). These curves were then used to determine the rates of downward and upward calcium transport by measuring the slopes of tangents to these curves. The data points represent the mean value of 24 roots/transport period. CALCIUM TRANSPORT RATE (CPM/MlN) CALCIUM TRANSPORT (CPM ) 3000 2000 4000 5000 6000 1000 DOWNWARD VS. UPWARD CALCIUM TRANSPORT CALCIUM UPWARD DOWNWARD VS. 80 40 $0 20 20 0 0 Figure Figure 0 2 10 180 190 120 0 0 60 0 3 0 18 . IEI MINUTES IN TIME 158 with significant radioactivity (621 cpm) moving at least 6 mm back from the root tip (Figure 17). In contrastf 45Ca2+ movement was restricted to the first 2 mm of the root tip,
i.e. the site of application. The accumulation of 43ca2+ dropped from 8184 cpm in the first mm to 628 cpm in the third mm, indicating no significant basipetal transport of calcium in vertically-oriented roots. It is noteworthy that three times more 45Ca2+ than 3H-IAA accumulated in the root cap and apical meristem. This would suggest that
45ca2+ uptake did not limit its basipetal movement.
DISCUSSION
A Possible Role for Calmodulin in the Transduction Phase of
Root Gravitropism. As researchers began to correlate changes in available calcium levels (Lee et al, 1983b) and calcium transport (Lee et al, 1984) with the gravicurvature of shoots and roots, many gravitropism models incorporated a role for calcium in the mediation of the response.
Recently, Evans et al (1986) proposed that gravistimulation leads to elevation of free calcium levels in the cytoplasm along the lower side of columella cells. They proposed that the increase in free calcium is sufficient to cause localized activation of calmodulin. According to Evans et al (1986), asymmetric activation of calmodulin would lead to asymmetric activation of CaM-dependent calcium pumps 159
Figure 17.
A^Comgarison of the Basipetal Movement of ^H-IAA and
Donors (50/000 cpm ^H-IAA, 50,000 cpm 45ca2+) were placed on one side of the caps of vertically-oriented roots. After 90 min, the roots were cut into 1 mm sections beginning at the tip. Basipetal auxin movement is shown by striped bars and basipetal calcium movement is represented by open bars. Each section was placed into a separate scintillation vial for the determination of radioactivity. Each data bar represents the mean value of 32 roots. ISOTOPE TRANSPORT(CPM) 8000 4000 H 5000 7000 3000-1 2000 6000 1000-1 0 IUTNOS ACU N UI BASIPETALSIMULTANEOUS CALCIUMTRANSPORTAUXIN AND
iue 17. Figure I 234567 ITNE RM OTTP (MM) ROOT DISTANCE FROMTIP nl B « a . -B 6 8 7 6 5 9 8 JSL 10
11 160 161 leading in turn to the establishment of downward calcium transport polarity across the root cap. It may be significant that the levels of calmodulin are four times higher in the root cap than in the subjacent 3 mm of the root (Stinemetz and Evans, 1986).
To determine whether or not calmodulin may be involved in mediating the gravitropic response, I monitored the development of polar calcium movement in roots pretreated with the calmodulin antagonist, calmidazolium (CMZ). When applied at 10” 7 m , CMZ had no effect on the downward movement of calcium across the root cap (Table 15).
However, a ten-fold higher concentration (10“® M) inhibited calcium transport by 43% after only 30 min of gravistimulation. Continued gravistimulation (60 min) resulted in increased inhibition of the downward movement of calcium, reaching a maximum inhibition of 65%. During the next hour of gravistimulation, the inhibition of downward calcium movement gradually decreased to 46%. This partial recovery of calcium transport may indicate adaptation of the roots to lower levels of activated calmodulin, metabolism of the calmidazolium, or synthesis of additional calmodulin.
The inhibition of downward calcium movement by CMZ correlated with a reduction in gravicurvature (Stinemetz, unpublished data). As expected, control roots and roots pretreated with lO”? M CMZ demonstrated normal 162 gravicurvature. Stinemetz also determined the effects of
CMZ on root growth. He observed no inhibition of growth at concentrations of 10"6 m or less. This indicates that the
reduction of gravicurvature following 10"® M CMZ-treatment
is not caused by a reduction in root growth.
The correlated inhibition of calcium transport and gravicurvature by CMZ suggests that calmodulin may be
involved in activating the membrane-bound calcium pumps as proposed by Evans et al (1986). However/ the use of calmodulin antagonistic drugs, including CMZ, is often criticized as these compounds are generally quite lipophilic and may have non-specific effects on membrane properties (Rahwan and Witiak, 1982).
The model of Evans et al (1986) also proposes that calmodulin activates auxin pumps which indirectly results in the accumulation of auxin in the lower half of the elongation zone. I therefore examined the movement of 3H-
IAA in gravistimulated roots pretreated with three different concentrations of CMZ. These preliminary data
(Table 16) indicate that pretreatment with 10"^ m CMZ, a concentration that had no effect on calcium transport, gravicurvature or growth, may slightly inhibit (19%) the accumulation of 3H-IAA in the lower half of the elongation zone within 30 min. However, since this experiment was only performed once with eight roots, the difference in 3H-
IAA accumulation may reflect experimental variation as the 163 transport values are within the limits of the standard errors. A more convincing inhibition of ^H-IAA accumulation (35%) occurred when roots were treated with
10"® M CMZ, a concentration known to reduce calcium transport and gravicurvature. Also, as expected, 10"® M
CMZ, a growth inhibiting concentration of the antagonist, resulted in drastic reduction (69%) of ®H-IAA accumulation.
Since auxin in the elongation zone has been strongly implicated in causing the growth asymmetry of the gravitropic response, these results were predicted by
Stinmetz's gravicurvature data. Any compound that reduced the levels of auxin in the lower half of the elongation zone would necessarily retard gravicurvature.
Suprisingly, after 120 min of gravistimulation, roots pretreated with the various concentrations of CMZ showed no significant differences from non-treated roots in the accumulation of ®H-IAA in the elongation zone. Once again, this apparent recovery from CMZ-treatment may reflect several possible mechanisms. As before, the roots may have adapted to the lower level of activated calmodulin; the antagonist may have been metabolized; or additional calmodulin may have been synthesized. Alternatively, the data may reflect some metabolism-controlled maximum in auxin asymmetry. Also, it should be remembered that these data are preliminary. The experiment should be repeated including sampling times of 60 and 90 min. 164
In addition to applying receiver blocks on the lower
half of the elongation zone, receivers were placed on the
upper and lower sides of the root cap. I expected that
CMZ-inhibition of 3h -i a a accumulation in the elongation
zone would be accompanied by an increase in the amount of
auxin in these two receivers. However, Table 16 shows
variable results for roots gravistimulated for 30 or 120
min. These experiments need repeating.
The Role of Free Calcium in the Establishment of
Downward Auxin Transport Polarity. Unpublished experiments
performed by Karl Hasenstein indicate that calcium
pretreatment of root tips enhances the development of downward auxin asymmetry upon gravistimulation. This led me to hypothesize that auxin transport polarity could be
reduced or eliminated by lowering the level of free calcium within the root cap.
To test this hypothesis, I pretreated roots with 2 mM
EGTA. This reduced the auxin asymmetry in gravistimulated
roots to 1.1 (Table 17) and eliminated gravicurvature measured at 90 min.
Since I could eliminate auxin asymmetry across the cap and gravicurvature with EGTA, I suspected that subsequent addition of calcium would reverse these effects.
Table 17 shows that roots pretreated with 2 mM EGTA for 30 min followed by a 30 min treatment with 5 mM CaCl2 / showed enhanced downward auxin transport polarity (2.0). These 165
roots also showed stronger gravicurvature than control
roots (polarity = 1.6). It should be noted that the
abolishment of gravi-induced downward auxin transport
polarity in roots pretreated with EGTA occurred via an
increase in the upward transport of auxin rather than
inhibition of downward transport (Table 17). This also
occurred in roots pretreated with KCN or PBA (Tables 5 and
9). Suprisingly, the recovery of auxin transport polarity
after CaCl2 was added to EGTA-pretreated roots resulted
from enhancement of downward transport of auxin rather than
from re-establishment of a low rate of upward transport
(Table 17).
These data are easily explained by the following model. Calcium-dependent auxin movement has been demonstrated by Dela Puente (1984). The contribution of upward auxin transport in gravistimulated roots is reduced as this calcium-dependent auxin movement into the cells, enhanced by the high levels of free calcium (10"3 m ) in the apoplasm, serves to counteract upward auxin transport.
Downward auxin transport may also occur in a calcium- dependent fashion according to the levels of available cytoplasmic calcium. The addition of 2 mM EGTA decreases the levels of apoplastic calcium which leads to a reduction in the calcium-dependent movement of auxin into the cell, and hence to a reduction in its ability to counteract upward movement of auxin. Consequently, I observed a reduction in downward auxin transport polarity via an
increase in upward auxin movement (Table 17). Since EGTA does not enter the cell# intracellular calcium levels remained unchanged and hence calcium-dependent downward auxin transport remained the same. Subsequent treatment with 5 mM CaCl^ would restore high apoplastic calcium levels causing enhanced calcium-dependent auxin movement into the cells. This calcium-mediated auxin transport would once again counteract the upward movement of auxin, resulting in the decreased upward auxin transport that I observed. Also, since calcium can enter the cell, increased intracellular calcium levels should correspond to enhanced calcium-dependent downward auxin transport. This is precisely what I observed as downward auxin transport increased 21% over control roots corresponding to an increased polarity of 2.0.
A Possible Relationship Between Downward Auxin and
Calcium Transport Polarities. The data suggest that the movement of free calcium during gravistimulation is related to the development of downward auxin transport polarity across the cap (Table 17). They also indicate that calmodulin may indirectly regulate both the movement of calcium and the accumulation of auxin in the elongation zone (Table 16). All of these data support the hypothesis that gravi-induced downward auxin and calcium asymmetries are related and perhaps even inter-dependent. To evaluate 167
this possible relationship, auxin and calcium transport patterns were examined simultaneously in gravistimulated roots.
When the pattern of auxin transport was compared to the pattern of calcium movement, I observed several similarities. Both showed an intial upward asymmetry which reversed with continued gravistimulation and resulted in the development of significant downward polarity within 45 min (Figure 14). Not only are the initial polarity values for auxin and calcium transport identical (polarities = 0.9 at 10 min and 1.0 at 30 min), both become downwardly polar within 45 min of gravistimulation (Figure 15). Further gravistimulation leads to a peak calcium polarity of 1.3 at
60 min which was maintained for 15 min prior to the gradual decrease in calcium polarity to 1.1 after 105 min (Figure
14). This calcium polarity was maintained during extended gravistimultion. Downward auxin transport polarity also increased to a maximum polarity with additional gravistimulation and eventually returned to a polarity of
1.1 after 180 min.
In addition to these similarities, I found that, like the auxin asymmetry (Figure 4), the establishment of downward calcium asymmetry correlated with subtle changes in the downward and upward calcium transport rates. The development of downward calcium polarity at 45 min corresponded to a greater increase in the downward than 168
upward rate of calcium transport (Figure 16b) just as with
the development of the auxin asymmetry. As calcium polarity peaked/ the downward transport rate was maintained
at a a higher level than the upward rate. The gradual decline in calcium polarity and its subsequent maintenance at a constant lower level (1.1) reflected a slight increase
in the upward rate at 90 min followed by almost identical changes in both transport rates thereafter.
As with auxin transport, downward and upward calcium transport rates fluctuated in a cyclic manner. Figure 18a compares the cyclic nature of downward auxin and calcium transport rates while Figure 18b illustrates the cycling of upward transport rates. During the first 45 min of gravistimulation, downward auxin and calcium transport rates cycle together, as do the upward transport rates.
However, further gravistimulation leads to a shifting of these rate curves such that the transport rates of auxin and calcium cycle out of phase. Again, this periodic alteration of transport rates suggests the involvement of a dynamic system, including control by feedback inhibition or adaptation resulting in oscillation toward a new steady state.
Although there are many similarities between gravi- induced auxin and calcium transport, Figure 15 illustrates several important differences. For example, calcium transport polarity peaked after only 60 min of 169
Figure 18.
A Comparison of Auxin and Calcium Transport Rates Across the Caps of Gravistimulated Roots.
The downward transport rates of auxin (circles) and calcium (triangles) are plotted against time in the upper graph while the lower graph illustrates upward auxin (circles) and calcium (triangles) transport rates plotted against time. Rates were determined by measuring the slopes of tangents to the transport vs. time curves shown in Figures 4 and 16. UPWARD TRANSPORT RATE (C P M /M IN ) DOWNWARD TRANSPORT RATE (C P M /M IN )
-j o 171 gravistimulation while auxin polarity continued to increase/ peaking 30 min later. Also, once the maximum calcium asymmetry had been established/ it was inaintained for approximately 15 min before gradually decreasing. In contrast/ the achievement of peak auxin transport polarity was immediately followed by a dramatic decrease in polarity. Finally, maximum downward calcium polarity never exceeded 1.3 while the peak polarity was 1.7.
These differences in polarity become more apparent when the various transport rates are compared. For example, once downward auxin and calcium polarities have been established (Figure 18), they peak at different times because the downward auxin transport rate remained above the upward rate for the next 45 min (Figure 4b) while the downward calcium transport rate remains significantly above the upward rate for only 15 min (Figure 16b). In addition to causing peak polarities to occur at different periods of gravistimulation, this also shifts the cycling of auxin and calcium transport rates out of phase (Figure 18).
The maintenance of the downward calcium asymmetry vs. the sharp drop in auxin transport polarity can also be explained by examining transport rates. Figure 16b shows that the rate of upward calcium transport remains constant over the period of peak polarity and then increases slightly more than the downward rate as the calcium asymmetry gradually diminishes. In contrast, the upward 172 auxin transport rate increased dramatically following the development of peak polarity (Figure 4b).
Figure 18 reveals a difference in auxin and calcium transport patterns not evident simply by examining polarity values. Following 3 h of gravistimulation, downward and upward calcium transport rates were considerably higher than auxin transport rates. This resulted in the accumulation of high levels of calcium in the lower half of the root cap (Figure 16a). In contrast, auxin transport rates had been reduced to 0 cpm/min (Figure 4b) resulting in a relatively constantly level of auxin within the cap
(Figure 4a).
Although there are some similarities in auxi/i and calcium transport patterns across gravistimulated root caps, there are sufficient differences to indicate that they are not inter-dependent and are probably regulated by different mechanisms.
The Possible Relationship Between Auxin and Calcium
Basipetal Transport. In Chapter I, I presented evidence that 3h - i a a transported from the root cap to the elongation zone (Figure 7). Because of the many similarities between downward auxin and calcium transport polarities, I was curious to see if 45ca2+ might also move basipetally when applied to the root cap. This was not the case. Figure 17 shows that ^h - i a a moves at least 6 mm back from the tip. However, there was little or no significant basipetal
transport of 43Ca2+ .
The fact that both auxin and calcium were
asymmetrically transported downward across the root caps,
but only auxin was basipetally transported, indicates that
there is no obligatory coupling between auxin and calcium
transport patterns in gravistimulated roots. However,
according to the model of Evans et al (1986), the elevated
calcium levels in the lower half of the cap serve to enhance the loading of the accumulated 3H-IAA into the basipetal transport stream. They propose that this auxin
is then carried back toward the lower side of the elongation zone where it inhibits growth and causes gravicurvature. If this is the case, then the basipetal transport of 43Ca2+ would not be necessary to obtain a graviresponse.
The model of Evans et al (1986) conflicts with the hypothesis presented in Chapter II that the auxin redistributed across the cap is not the auxin that mediates curvature in the elongation zone. As discussed in Chapter
II, I proposed that the establishment of a slight downward auxin asymmetry across the root cap triggers the release of large amounts of auxin in the region of the elongation zone, perhaps from the stele. It is this auxin that is responsible for the rapid initiation of gravicurvature. 174
This hypothesis can be modified to include the data on
the development of a downward calcium transport polarity.
Since calmodulin antagonists reduce both polar calcium movement and gravicurvature, the data are consistent with the model of Evans et al (1986) that the elevated calcium levels enhance the loading of auxin into the basipetal stream. However, since there is virtually no lag between the development of auxin asymmetry across the cap and gravicurvature (Figure 13), the establishment of this auxin asymmetry cannot itself cause curvature. I propose that a slight asymmetry in auxin flux from the cap triggers asymmetric release of auxin from the stele into the elongation zone and that it is this larger asymmetry that initiates gravicurvature.
This hypothesis is supported by the experiments using
CMZ. The decreased gravicurvature of CMZ-treated roots correlated with a decreased downward calcium asymmetry and hence less efficient loading of auxin into the basipetal stream. If in fact the loading of redistributed auxin initiates the release of curvature-inducing auxin in the elongation zone, treatment with CMZ or any other compound which would directly or indirectly reduce auxin loading, i.e. KCN, NPA, PBA and EGTA, would be expected to decrease the graviresponse.
Conclusions on the Relationship Between Auxin and
Calcium Transport Patterns in Gravistimulated Roots. The 175 experiments with calmodulin antagonists, Stinemetz's unpublished ^h -i a a transport data and the simultaneous monitoring of 2H-IAA an^ ^5Ca2+ movement during gravistimulation have led to the following conclusions: 1) the downward transport of calcium across the caps of gravistimulated roots may be mediated by calmodulin; 2) the development of normal downward auxin transport polarity requires free calcium; 3) both downward auxin and calcium transport polarities are induced by gravity; 4) the timing of the development of auxin and calcium transport asymmetries indicates that they are related but probably occur by different mechanisms; 5) 45ca2+ applied to the root tip is not basipetally transported; and 6) asymmetric basipetal movement of auxin out of the cap may trigger the asymmetric release of auxin from the stele into the elongation zone at levels sufficient to account for gravicurvature. SUMMARY
Gravistimulation of maize roots results in the development of downward auxin transport polarity across the tips within 45 min. Continued gravistimulation leads to the achievement of maximal polarity after 90 min with an abrupt decline in hormone asymmetry thereafter.
When auxin transport was monitored across the tips of decapped, horizontally-oriented roots, I observed no hormone asymmetry and much less overall auxin transport than across intact roots, indicating that the root cap was the site of polar auxin movement. This conclusion is supported by experiments showing preferential downward movement of auxin across isolated, gravistimulated root caps.
An examination of the kinetics of establishment of auxin asymmetry indicated that its development is regulated by cyclic changes in the downward and upward auxin transport rates. Also, downward asymmetric auxin movement requires metabolic energy and is sensitive to the auxin transport inhibitors, NPA and PBA.
There was an excellent correlation between the establishment of downward auxin transport polarity and the development of gravicurvature. Compounds (such as KCN,
176 177
NPA, PBA and EGTA) that inhibited downward auxin transport
polarity, also inhibited gravicurvature. Also, the initial
upward auxin transport polarity correlated with the initial
wrong-way curvature response characteristic of most
gravistimulated Merit roots.
Considering the hypothesis proposed by Hild and Hertel
(1972) that initial wrong-way gravicurvature results from
overstimulation of the gravi-sensing mechanism, I
prestimulated, or "pre-adapted", roots using alternate
brief gravistimulation on opposite flanks and then examined gravicurvature. The pre-adapted roots by-passed their
initial upward curvature phase and began immediate downward
curvature and this correleated with direct establishment of downward auxin transport polarity. These experiments reinforce the proposal that auxin mediates gravitropic curvature since there was a consistent correlation between curvature and auxin transport polarity.
I found that downward calcium movement across gravistimulated root caps was inhibited by the calmodulin antagonist, CMZ. Stinemetz (unpublished data) noted that this concentration of CMZ inhibits gravicurvature without reducing growth.
I investigated the possible correlation between the establishment of downward auxin and calcium transport polarities by examining the effects of the calcium- chelating agent, EGTA, on gravi-induced polar auxin 178
movement. I found that EGTA sharply decreased the polarity
of auxin movement in gravistimulated roots and that this
effect was reversible by adding calcium. These experiments
indicate that calcium is required for the gravi-induced
development of downward auxin transport polarity.
This relationship was further studied by monitoring
the simultaneous movement of ^H-IAA and 45ca2+ across
gravistimulated root caps. The data show that downward
transport asymmetries develop for both auxin and calcium.
Early transport kinetics were identical and calcium
transport polarity also appeared to be continually
regulated by cyclic changes in the downward and upward
calcium transport rates as was the case for auxin transport polarity. However, I noted that: 1) calcium transport polarity peaked 30 min before auxin transport polarity; 2) maximum calcium transport polarity was maintained for a short time while auxin transport polarity dropped almost
immediately after reaching its maximum; 3) maximum calcium transport polarity was significantly less than maximum auxin transport polarity; 4) and auxin but not calcium is basipetally transported from the cap to the elongation zone. These experiments indicate that the relationship between downward auxin and calcium transport asymmetries may be indirect.
Based on these findings, I propose a modification of the root gravitropism model proposed by Evans et al (1986). According to the model of Evans et al (1986), in vertically-oriented roots, auxin moves acropetally through the stele into the root cap where it is radially redirected to the sides of the cap and then basipetally transported, possibly through the cortical and/or epidermal tissues, to the elongation zone. Upon gravistimulation, more auxin is redirected downward to the lower side of the cap and hence more auxin moves basipetally to the lower half of the elongation zone ultimately causing gravicurvature.
I propose that the auxin redirected across the gravistimulated root cap, though it may indirectly initiate gravicurvature, does not itself cause the differential growth inhibition of the gravitropic response. Instead, I propose that the initial wave of auxin asymmetry moving back from the cap triggers rapid asymmetric release of larger amounts of auxin in the elongation zone from the stele, or perhaps from sequestered pools of auxin in the cells of the elongation zone itself. LIST OF REFERENCES
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