The Pennsylvania State University The Graduate School Intercollege Program in Plant Physiology GENETIC, PHYSIOLOGICAL AND ENVIRONMENTAL REGULATION OF ROOT PLAGIOGRAVITROPISM A Thesis in Plant Physiology by Paramita Basu 2006 Paramita Basu Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2006
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The Pennsylvania State University
The Graduate School
Intercollege Program in Plant Physiology
GENETIC, PHYSIOLOGICAL AND ENVIRONMENTAL
REGULATION OF ROOT PLAGIOGRAVITROPISM
A Thesis in
Plant Physiology
by
Paramita Basu
2006 Paramita Basu
Submitted in Partial Fulfillment of the Requirements
for the Degree of
Doctor of Philosophy
August 2006
The thesis of Paramita Basu was reviewed and approved* by the following:
Kathleen M. Brown Professor of Post-Harvest Physiology Thesis Advisor and Chair of Committee
Jonathan P. Lynch Professor of Plant Nutrition
Simon Gilroy Associate Professor of Biology
Paula McSteen Assistant Professor Biology
Teh-hui Kao Professor of Biochemistry and Molecular Biology Program Chair of the Intercollege Program in Plant Physiology
*Signatures are on file in the Graduate School
iii
ABSTRACT
Basal roots together with the primary root forms the scaffolding of the root
system architecture of common bean (Phaseolus vulgaris L.) which responds to gravity
in concert with various environmental cues like phosphorus and hormonal signals such as
ethylene and auxin. Basal roots are a type of secondary roots resembling adventitious
roots, and they arise from tissue with shoot anatomy. They appear in tetrarch pattern like
adventitious and lateral roots. The basal roots emerge from two-three distinct whorls
from a one-centimeter region at the root-shoot interface and exhibit plagiogravitropic
growth which changes over time. Gravitropic growth of roots determines the three-
dimensional root architecture which is essential for efficient acquisition of soil resources.
The growth angle of basal roots is a primary determinant of the roots which impacts
efficient acquisition of limited and immobile nutrients like phosphorus. Genotypes of
common bean vary substantially in the growth angle of basal roots and by altering growth
angles the plants are better adapted to low phosphorus availability. Shallow basal roots
not only aid in topsoil exploration but also reduce intra and interplant competition for
phosphorus.
Since ethylene has been implicated in both gravitropic and edaphic stress, we
studied the role of ethylene and its interaction with phosphorus availability in regulating
growth angles of basal roots. We measured endogenous ethylene production from the
basal roots and also analyzed the response of basal roots to exogenous application of
ethylene in terms of growth angle and root growth. In addition, we developed a new
image analysis program ‘KineRoot’ to study the spatio-temporal patterns of
plagiogravitropic growth of basal roots in response to ethylene and phosphorus
treatments in a reliable semi-automated way, while minimizing user intervention. The
new software allows us to measure the local patterns of basal root growth and
graviresponding zones of basal roots and how these zones are affected by ethylene and
phosphorus availability. Moreover, the software enables us to measure the root diameter
and root midline which was used in calculating root curvature. Since from the available
literature we already know that auxin and ethylene are potential candidates regulating
iv
graviresponse of roots, we studied the possible cross-talk between auxin and ethylene in
modulating graviresponse of basal roots. To test the hypothesis that ethylene modulates
auxin effect on root growth and plagiogravitropic curvature of basal roots, we employed
both parental genotypes and recombinant inbred lines of common bean with contrasting
basal roots traits for this study. Response of basal root angle and root growth to different
doses of auxin was measured. In addition, we examine the effect of application of
ethylene action inhibitor 1-methylcyclopropene (MCP) and ethylene synthesis inhibitor
aminovinylglycine (AVG) on growth angle and root growth in the presence of
phosphorus. Free Indole-3-acetic acid (IAA) content in the basal roots was analyzed and
in a separate experiment the basal roots were treated with tritiated IAA to determine the
transport of 3H-IAA in the basal roots of different whorls.
Our work shows that position of origin i.e. whorl has more influence on growth
angle of basal roots than previously reported effects of genotype and phosphorus
availability. Genotypes of common bean vary in basal root number in each whorl. The
diversity in root architecture is generated partly by variation in basal root number as well
as variation in growth angles of basal roots. Although endogenous ethylene production
from the basal roots did not explain variation in growth angles, tissue sensitivity to
exogenous ethylene application appears to be more important in determining the growth
angle. Our results show that there is a strong correlation between ethylene sensitivity and
growth angle which supports our hypothesis that growth of basal roots may be partially
regulated by ethylene and the difference in ethylene sensitivity might explain variation in
growth angle with whorl, genotype and phosphorus availability. Basal root growth was
also affected by ethylene treatment; however, higher sensitivity for root elongation was
not found consistently in all treatments. Our results indicate that ethylene may be a
modifier of root responses to nutrient availability and ethylene perception may be a
central aspect of root response to low phosphorus availability.
The kinematic analysis shows that the basal roots accelerate growth rate of the
upper whorls at the cost of lower growth rate in lower whorls in response to low
phosphorus. Moreover, study of spatio-temporal patterns of differential growth ratio of
the growing root allows identification and measurement of root bending zones and
v
bending amount. We examine the effects of ethylene and MCP on root curvature and
observe that both of these treatments do not alter local root curvature, but alters the span
and duration of the bending of the root upward or downward and thereby produce
shallow and deep roots respectively.
Our study about the possible interaction of auxin and ethylene supports the
hypothesis that the effect of interaction between auxin and ethylene on regulation of
growth angles is dependent on phosphorus availability. Free IAA analysis in the basal
roots show that lower whorls of basal roots have higher free auxin and are more sensitive
to auxin inhibition of basal root growth compared to upper whorls. However,
radiolabeled IAA treatment to the root-shoot junction just above the basal rooting zone
shows more radiolabeled IAA transported to upper whorls than lower whorls. In addition
while application of AVG or MCP together with IAA increases root growth and reduces
shallowness in phosphorus sufficient conditions, AVG or MCP do not reverse IAA-
inhibition of growth in low phosphorus. These results point to a phosphorus dependent
interaction between ethylene and auxin in regulation of root elongation, but a
phosphorus-independent interaction for control of growth angle.
vi
TABLE OF CONTENTS
LIST OF FIGURES ..................................................................................................... ..x
LIST OF TABLES....................................................................................................... ..xii
ACKNOWLEDGEMENTS......................................................................................... xiii
CHAPTER 1. INTRODUCTION……………………………………………. 1
Gravitropic response of roots……………………………………………………1
Role of auxin in gravitropism…………………………………………………...3
Ethylene as a regulator of root gravitropism…………………………………….6
Role of other hormones in regulating gravitropism……………………………..8
Common bean selected as a model for studying root architecture……………....9
Phosphorus availability and root architecture of common bean………………..10
OVERVIEW OF RESEARCH PROJECTS………………………………………….11
REFERENCES……………………………………………………………………….14
CHAPTER 2. GENETIC, POSITIONAL, AND NUTRITIONAL
REGULATION OF ROOT PLAGIOGRAVITROPISM
MODULATED BY ETHYLENE…………………………………….24
Abstract………………………………………………………………………....25
Introduction……………………………………………………………………..26
Methods………………………………………………………………………....27
Results…………………………………………………………………………..31
1. Morphology of basal root production………………………………....31
2. Basal root angle depends on genotype and position of origin………...31
3. Range of basal root growth angles………………………………….....32
4. Effect of genotype, phosphorus and position of origin
on ethylene …………………………………………………………....33
5. Basal root growth depends only on root position of origin…………...33
Figure 1.1 A macroscopic view of a 2-d old germinated bean seedling showing basal roots emerging from the 3 distinct whorls. Whorls are designated as 1, 2, and 3 from theshoot-side to primary root-side.
CHAPTER 2: GENETIC, POSITIONAL, AND NUTRITIONAL REGULATION
OF ROOT PLAGIOTROPISM MODULATED BY ETHYLENE
Paramita Basu1, Yuan-Ji Zhang2, Jonathan P. Lynch1, 2 and Kathleen M. Brown1, 2
1Intercollege Program in Plant Physiology, The Pennsylvania State University Park, PA
16802, USA
2Department of Horticulture, The Pennsylvania State University Park, PA 16802, USA
25
ABSTRACT
Plagiogravitropic growth of roots strongly affects root architecture and topsoil
exploration, which is important for the acquisition of depth-dependent soil resources such
as phosphorus. Here we show that basal roots of Phaseolus vulgaris L. develop from 2-3
definable whorls at the root-shoot interface and exhibit position-dependent
plagiogravitropic growth. The whorl closest to the shoot produces the shallowest roots,
while lower whorls produce deeper roots. Genotypes vary in both the average growth
angles of roots within whorls and the range of growth angles, i.e. the difference between
the shallowest and deepest basal roots within a root system. Since ethylene has been
implicated in both gravitropic and edaphic stress responses, we studied the role of
ethylene and its interaction with phosphorus availability in regulating growth angles of
genotypes with shallow or deep basal roots. There is only a small correlation between
growth angle and ethylene production in the basal rooting zone, but ethylene sensitivity is
strongly correlated with growth angle. Basal roots emerging from the uppermost whorl
are more responsive to ethylene treatment, displaying shallower angles and inhibition of
growth. Ethylene sensitivity is greater for shallow than for deep genotypes and for plants
grown with low phosphorus compared to those supplied with high phosphorus. Ethylene
exposure increases the range of angles, though deep genotypes grown in low phosphorus
are less affected. Our results show that ethylene mediates regulation of growth angle by
position of origin, genotype, and phosphorus availability.
Figure 2.1. Effect of genotype and position of origin on basal root angle of common bean. Insert shows a close up view of a young seedling (3 d after imbibition) showing three distinct whorls bearing emerging basal roots. All genotypes are from the L88 population. The growth angle of the basal roots was measured after 2 d growth in pouches. The bars show mean growth angles of basal roots emerging from each whorl of 10-12 plants per genotype, with data pooled over phosphorus treatments, ± SE.
45
Figure 2.2. Endogenous ethylene production per gram fresh weight (pooled over both phosphorus treatments together (A) and per basal root (separately for both phosphorus treatments) (B) by the segments of the root-shoot junction bearing basal roots. Segments were harvested 3 d after imbibition. Values shown are means of 8 plants from each of three shallow and three deep genotypes from the L88 population ± SE.
46
Figure 2.3. Growth rate of basal roots measured during the first 24 h growth in pouches. Values shown are means of 8 plants from each of three shallow and three deep genotypes from the L88 population (pooled over both phosphorus treatments together) ± SE. Growth rate is significantly affected only by whorl of origin (P <0.001).
47
Figure 2.4. Effect of MCP and 0.6 µl L-1 ethylene on basal root angle of parent genotypes of L88 populations. The plants were treated with either MCP or ethylene for 24 h immediately after transferring to the pouch. Values shown are means of 10-12 plants per genotype ± SE, with data pooled over both high and low phosphorus treatments.
48
Figure 2.5. Ethylene sensitivity of basal root angles for whorls 1, 2 and 3 of a shallow genotype (TLP19) grown in low phosphorus. The angle was measured for the growth occurring between 24 and 48 h. Values shown are means of basal roots of 5-7 plants per ethylene treatment ± SE.
49
Figure 2.6. Ethylene sensitivity of basal root growth angle as a function of genotype, whorl and phosphorus treatment (low and high P) in three shallow and three deep genotypes from the L88 population. Ethylene sensitivity was measured as the slope of the response functions as illustrated in Fig. 6. Statistical analysis corresponding to these data is shown in Table 3.
50
Figure 2.7. Correlation between ethylene sensitivity and growth angle of basal roots of six L88 genotypes grown in low (low P) and high (high P) phosphorus treatments. Angles on the X-axis are of control plants without ethylene.
51
Figure 2.8. Ethylene sensitivity of growth response of basal roots as a function of genotype, whorl and phosphorus treatment (low and high P) in three shallow and three deep genotypes from the L88 population. Growth was measured between 24 and 48 h. Ethylene sensitivity was calculated as the slope of the response curve (ethylene concentration vs. growth).
52
Figure 2.9. Effect of exogenous ethylene on the range of growth angles of three shallow and three deep genotypes from the L88 population grown in low (low P) or high (high P) phosphorus. Angles were measured for growth occurring between 0 and 48 h. The range of growth angles for each plant was calculated by subtracting the minimum angle from the maximum angle produced by the basal roots of each plant. Values shown are means of the range of growth angles of 4- 7 plants per genotype per ethylene treatment ± SE.
53
Table 2.1. Average number of basal roots per whorl in four parent genotypes. The numbers designate mean numbers of basal roots of 6-8 plants ± SE. The upper whorl is designated as whorl 1, while the lower whorl as whorl 3.
Number of basal roots per whorl
Genotype Whorl 1 Whorl 2 Whorl 3
B98311 2.5±0.2 2.7±0.1 3.5±0.1
TLP19 2.3±0.1 3.2±0.1 3.9±0.1
G19833 3.2±0.2 3.9±0.2 4.1±0.1
DOR364 3.1±0.2 3.9±0.1
Table 2.2 Range of growth angles of basal roots per plant in six genotypes (three deep and three shallow) from the L88 population. The three deep genotypes used for the experiment of growth angle measurement are B98311, RIL7 and RIL76, while the three shallow genotypes are TLP19, RIL15 and RIL57. N = 4-7 plants per genotype.
Genotypes Mean
angle
Standard
deviation
Range of
angles
Min. growth
angle
Max. growth
angle
Deep 41.7 14.0 39.3 21.3 60.6
Shallow 56.4 18.0 54.5 28.5 82.9
54
Table 2.3. ANOVA of growth angle and growth response of basal roots from contrasting genotypes (shallow and deep) of the L88 population as affected by exogenous ethylene treatment. The three deep genotypes used for the experiment of growth angle measurement are B98311, RIL7 and RIL76, while the three shallow genotypes are TLP19, RIL15 and RIL57.
Growth angle Growth rate
Effect DF F-value P-value F-value P-value
Genotype 1 701.9 <0.001 25.29 <0.001
Phosphorus 1 0.178 0.673 4.102 0.046
Ethylene 5 220.0 <0.001 118.1 <0.001
Whorl 2 2218 <0.001 730.5 <0.001
Genotype*Phosphorus 1 2.741 0.098 0.193 0.662
Genotype*Ethylene 5 2.620 0.023 0.642 0.718
Genotype*Whorl 2 64.83 <0.001 0.515 0.584
Phosphorus*Ethylene 5 11.34 <0.001 0.193 0.965
Phosphorus*Whorl 2 3.484 0.031 3.854 0.021
Ethylene*Whorl 10 4.957 <0.001 11.54 <0.001
55
CHAPTER 2 APPENDIX In an initial experiment, we investigated if the genotypic difference in growth
angles of basal roots varies with the basal root lengths. Therefore, we grew contrasting
(shallow and deep) genotypes for 2 d after germination of the seedlings in the growth
pouch containing low or high phosphorus nutrient solution. After 2 d growth in the
pouches, digital images were captured and growth angles of basal roots (BRGA) were
determined at a fixed radius of 2 cm from the base the emerging position of each basal
root (Fig. 10). We observed that even with a fixed root length, the shallow and deep
genotypes significantly (P < 0.001) differ from each other in BRGA.
0
20
40
60
80
100
whorl 1 whorl 2 whorl 3
Bas
al ro
ot a
ngle
(deg
ree
from
ver
tical
)
deep genotypeshallow genotype
Figure 2.10. Genotypic variation in basal root angle for shallow (RIL57) and deep (RIL7) genotypes of common bean genotypes grown in the pouch system for 2 days in low phosphorus. The bars show mean growth angles of basal roots emerging from each whorl of 7 plants per genotype ± SE. The growth angles were measured at a fixed radius of 2 cm from the base of the emerging position of each basal root.
56
Table 2.4. ANOVA of BRGA of two contrasting parent genotypes (TLP19 and B98311) as affected by genotype, phosphorus, ethylene/MCP treatment or whorls. BRGA
Tardieu F, Reymond M, Hamard P, Granier C, Muller B (2000) Spatial distributions of
expansion rate, cell division rate and cell size in maize leaves: a synthesis of the
effects of soil water status, evaporative demand and temperature. Journal of
Experimental Botany 51, 1505-1514.
van der Weele CM, Jiang HS, Palaniappan KK, Ivanov VB, Palaniappan K, Baskin TI
(2003) A new algorithm for computational image analysis of deformable motion
at high spatial and temporal resolution applied to root growth. Roughly uniform
elongation in the meristem and also, after an abrupt acceleration, in the elongation
zone. Plant Physiology 132, 1138-1148.
Walter A, Spies H, Terjung S, Kusters R, Kirchgessner N, Schurr U (2002) Spatio-
temporal dynamics of expansion growth in roots: automatic quantification of
diurnal course and temperature response by digital image sequence processing.
Journal of Experimental Botany 53, 689-698.
86
Figure 3.1. Photo showing the root system of a 2 d old common bean plant (TLP19) in the growth pouch. The plant shows both shallow and deep basal roots growing from the root-shoot interface.
87
Figure 3.2. Photo showing sprinkling of graphite particles on the basal roots of a 1 day old bean seedling using dropper fitted with a pipette tip.
88
Figure 3.3. Photo of the experimental setup showing the position of the camera and two flash units to capture high resolution photos of the basal root of a bean seedling. Bean seedling in the polyethylene pouch was placed inside an air-tight plexiglass box and maintained at temperatures between 25-26°C. Time lapse photography was driven by a laptop computer connected to the camera by a USB cable. Photos were captured using two flashes and light from the flashes was bounced off white papers placed on the top of the plexiglass box.
89
Figure 3.4. Screenshot of the graphical user interface of the image analysis software ‘KineRoot’.
90
Figure 3.5. Schematic showing pattern matching algorithm by finding the highest correlation coefficient between two boxes of pixels. The yellowish projections in the blue background represent the growing root whereas the black spots show the markers due to graphite particles, and (A) shows the reference image and (B) shows the current image. The red circle in (A) is being tracked (B). We choose all pixels within the red square in (A) and correlate it with the cyan boxes in (B). When the dotted cyan box is centered around the green circle in (B), the correlation with (A) is low because of mismatch of the graphite markers, whereas when the solid cyan box placed centered around the red circle, correlation coefficient with the red box in (A) reaches its maximum value identifying the new location of the point in the current image. Note, that there is no requirement for the points to be on a graphite particle for tracking.
A
B
A
B
(x0, y0)
(x*, y*)
91
Figure 3.6. Schematic showing the weights for calculating color-weighted correlation coefficients based on color of the pixel and sampled colors of the root (Rr, Gr, Br) and the background (Rb, Gb, Bb). The red , green and blue lines show the weighting factors for the corresponding colors. If the difference in color intensity between the root and the background is less than 0.2, weighting factor is assigned a value of 1, otherwise weighting factor is w is calculated by linear interpolation for a pixel whose color intensity lies between that of the root and the background. If the color intensity of a pixel is outside this range, a value of 1 or 0 is assigned based on the proximity to the root color or background color respectively.
92
Figure 3.7. Steps of automatic edge detection: (A) two dimensional Gaussian filter, (B) close up image of a basal root, (C) basal root image after noise smoothing by convolution with the Gaussian filter, (D) magnitude of the gradient of the smoothed image showing blurry edges, (E) edge enhanced by non-maxima suppression, (F) detected edges of the root shown by green and yellow lines and the centerline shown by thick white line.
93
Fig. 3.8. (A) Schematic showing projection of tracked points on the root centerline. Distance of the projected tracked points from the root tip Sp is measured along the root centerline. From the detected root edge we also measure the root diameter D as a function of distance from the root tip and time. (B) Schematic showing the spatio-temporal trajectory of the tracked points. The region where the gap between the points increases rapidly with time, identifies the growth zone.
A
Growth zone
Sp
time
B
94
Fig. 3.9. Schematic illustrating the calculation of root growth velocity with respect to the root tip. If a marker point located at Sp distance from the root tip at time t moves to Sp+ δSp distance from the root tip over time interval δt, the growth velocity of the point is given by Up= δSp/δi.
Root tip reference
S2
0
1
2 3
4 5
6
S4 S2 + δS2
S4 + δS4
Time t Time t+δt
95
Fig. 3.10. Schematic showing the growth of a small segment of the root from an initial length l to l+δl over a short period of time δt. Therefore the relative elongation rate is
defined as the fractional change in length per unit time, lrl tδδ
= .
l
l lδ+
time t
time t tδ+
96
Figure 3.11. Montage of 8 images of a basal root at 45 min intervals from a sequence of 72 images originally captured at 5 min intervals. The green and yellow lines show the edges detected by KineRoot and the bold white line shows the root midline. The red dots show the tracked points.
97
Fig. 3.12. Root length map showing the growth of the root by plotting distance of the marker points from the root tip along the root midline at 5 min time intervals.
98
Figure 3.13. (A) Root growth velocity plotted as a function of distance from the root tip. The gray dots show the growth velocity of 25 tracked points in 72 frames. The bold line shows the average growth velocity after grouping the data in bins of 0.5mm. The error bars are standard deviation bars. (B) Mean relative elongation rate plotted against distance from the root tip with standard deviation error bars.
A
B
99
Figure 3.14. Colored isocontours of rate of relative elongation plotted as a function of distance from the root tip and time. Reds, oranges and yellows show high rate of elongation whereas light and dark blues show low/zero rate of elongation.
100
Figure 3.15. Mean root diameter plotted as a function of distance from the root tip. The error bars show standard error.
101
Figure 3.16. (A) Mean root curvature and (B) differential growth ratio between the upper and lower sides of the root plotted as a function of distance from the root tip. Positive curvature and differential growth ratio greater than 1 indicate downward bending and vice-versa. The error bars show standard error.
102
Fig. 3.17. Two images of the root at (A) the initial time and (B) after 6 h showing the spreading of the marker points due to growth.
CHAPTER 4: GROWTH AND CURVATURE OF BASAL ROOTS OF COMMON
BEAN (PHASEOLUS VULGARIS L.) ANALYZED USING
KINEMATIC APPROACH
Paramita Basu1, Anupam Pal2, Jonathan P. Lynch1, 3, Kathleen M. Brown1, 3
1Intercollege Program in Plant Physiology, Penn State University, University Park, PA
16802 USA; 2Department of Mechanical Engineering, Penn State University, University Park, PA
16802 USA; 3Department of Horticulture, Penn State University, University Park, PA 16802 USA.
104
ABSTRACT
Using a newly developed image-analysis program KineRoot, we measured root
growth and curvature of basal roots of common bean using a kinematic approach.
Although computer-assisted kinematic analysis has been applied to primary root growth
of Arabidopsis, it has not been employed for study of plagiogravitropic growth or for
thicker-rooted species. Use of KineRoot permits study of spatio-temporal patterns of
growth of bean basal roots in a reliable, semi-automated way while minimizing user
interventions to allow large scale experiments. We identify and measure the local patterns
of root growth and graviresponding zones of the basal roots, investigate the velocity
profiles within these zones and determine how these zones are affected by low
phosphorus availability and ethylene treatment. We observe that basal roots accelerate
growth rate of the upper whorls at the cost of lower growth rate in lower whorls in
response to low phosphorus availability. Apart from root growth, one of the most
important aspects of this study is to characterize the bending of the basal roots which
leads to graviresponse and reflects shallowness or deepness of basal roots. Root curvature
results from differential growth between upper and lower edges of the root, and the
direction of this curvature varied over time, producing a waving motion. Therefore study
of spatio temporal patterns of differential growth ratio of a growing root allows
identification and measurement of root bending zones and bending amount. Our results
show that ethylene and MCP treatments do not alter local root curvature, but alter the
span and duration of the bending of the root upward or downward which causes altered
response to gravity, thereby producing shallow and deep roots respectively. The results
from this study show new aspects of plagiogravitropic response of basal roots which have
not been observed before.
105
INTRODUCTION
Root architecture, i.e. the three-dimensional spatial configuration of roots, varies
greatly with genotype and environment, influencing plant adaptability and productivity.
In common bean, basal roots emerging from the root-shoot interface, together with the
primary root, form the scaffold of the bean root system. The growth angle of the basal
roots is a major determinant of topsoil exploration, and therefore efficient soil resource
acquisition, especially in environments with phosphorus limitation (Bonser et al. 1996;
Liao et al. 2001). Genotypes vary in basal root growth angle, and some genotypes
respond to phosphorus availability by becoming shallower or deeper (Bonser et al. 1996;
Liao et al. 2001). The position of origin (whorl) is a major determinant of growth angle,
with roots arising from upper whorls growing more shallow than those from lower whorls
(Chapter 2). Growth dynamics responsible for basal root growth and response to gravity
are the subject of this chapter.
Despite intense research on root gravitropism, the detailed mechanism behind the
perception and response to gravity is not yet clearly elucidated. The process of
gravisensing occurs by the perception of gravity stimulus in the root cap statocytes (Sack
1991), followed by graviresponse leading to the growth response in the elongation zones
of the root (Baluska and Hasenstein 1997). The Cholodny-Went theory has been
established as the framework for studying root gravitropism according to which
downward curvature of roots in response to gravity is induced by asymmetric
redistribution of auxin within the elongation zone with accumulation of higher auxin
content along the lower flank of the bending root. Since the optimal concentration of
auxin necessary for root growth is much lower than that for shoot growth (Eliasson et al.
1989), higher auxin content would be inhibitory to root growth i.e. roots are more
sensitive to auxin than shoots. This indicates that increased auxin content in the lower
side of a bending root would result in localized growth inhibition, thereby leading to
downward curvature.
When a vertical root is gravistimulated i.e. the root is reoriented to a certain angle
from the vertical, root curvature is initiated and the root starts to bend until the root tip is
aligned towards the gravitational vector. By using computer-based video digitizing
106
system for tracking marker beads along the surface of maize roots, Ishikawa et al. (1991)
observed that a group of cells termed distal elongation zone (DEZ), lying between the
root apical meristem and the central elongation zone (CEZ), exhibit enhanced elongation
rate in a gravistimulated root. The curvature which initiates in the DEZ is the result of
inhibition of elongation in DEZ and CEZ on the lower flank as well as acceleration of
cell elongation in the DEZ on the upper flank of the gravistimulated root (Mullen et al.
1998a; Wolverton 2002). When the bending root comes to a vertical position, the growth
resumes to the symmetrical pattern (Evans and Ishikawa 1997). However, in vertically
growing roots, the maximum rate of cell elongation occurs in the CEZ with a growth rate
three times more than in the DEZ (Ishikawa and Evans 1993).
To analyze the mechanism of root growth and graviresponse, it is important to
identify the regions of the root where growth and bending take place, and also measure
the rate/amount of growth/curvature in space-time coordinates under different treatments.
The method by which this is done is called the ‘kinematic’ analysis. Kinematics is an
aspect of dynamics which involves the study of physical motion (acceleration and growth
velocity) without reference to the forces resulting in the movement (Gandar 1983).
Kinematic analysis has been used by a number of researchers in investigating the growth
zones of an elongating root. This approach has been employed in the study of primary
root growth for a long time (Goodwin and Stepka 1945; Erickson and Sax 1956) and has
become more established in studying both temporal and spatial distribution of a growing
root in the last couple of decades (Erickson and Sax 1956; Goodwin and Avers 1956; Silk
and Erickson 1979; Gandar 1980, 1983; Pahlavanian and Silk 1988; Beemster et al. 1996;
Sacks et al. 1997; Beemster and Baskin 1998; Walter et al. 2002; Ma et al. 2003; van der
Weele et al. 2003). Besides root, the growth profiles of elongating leaf, stem, perianth
etc. have also been analyzed by using kinematic approach (Gould and Lord 1989;
Bernstein et al. 1993; Ben-Haj-Salah and Tardieu 1995; Fiorani et al. 2000; Tardieu et al.
2000; Girousse et al. 2005; Kakanova et al. 2006). Application of the kinematic approach
in such diverse studies shows the utility of the method in understanding the details of
plant growth.
107
To investigate the mechanism behind the longitudinal growth of a root, most
studies depend on the spatial analysis of tissue expansion rate of the elongating zone (Silk
1992) which allows accurate analysis of local events resulting in root elongation. The
expansion growth of a root accelerates and decelerates within a zone of few millimeters
in length. Organ growth is determined by both cell expansion as well as cell division
leading to cell production throughout the growth zone at a given time (Beemster and
Baskin 1998). For quantifying root growth, Walter et al., (2002) applied the tensor
method of image sequence analysis based on intensity gradients where they obtained
velocities at relatively few pixels, resulting in extensive interpolation. Recently the
RootflowRT method (van der Weele et al. 2003) has been developed for measuring the
expansion profile of root elongation at high spatio-temporal resolution by combining the
tensor method with a robust matching algorithm for attaining confident measurements
from more than 50% of pixels. Using these techniques for the quantification of expansion
rates of roots, the growth zone of root can be divided into two distinct zones, an apical
region with steadily increasing velocity and a subapical zone with steeply increasing
velocity with an abrupt transition zone in between (van der Weele et al. 2003). Although
these image sequence analysis methods are elegant in their applications for analysis of
growth in roots, shoots and leaves, they heavily depend on visible natural patterns on the
plant organ. For thicker rooted species like bean where the epidermal cells are not visible
under normal magnification and resolution of microscope, lack of natural patterns on the
root poses a big challenge in estimating local root growth and curvature using these
methods. Although graphite particles add visible markers on the roots, the traditional way
of carefully placing the markers and then tracking them in space-time is very tedious and
does not permit study of a large number of roots. As the roots grow, the graphite particles
also separate in the growth zone, reducing the trackable patterns. Therefore if the tensor
structure method is applied to the kinematic study of bean basal roots sprinkled with
graphite particles, it will be able to calculate root velocity at very few pixels in the
growth zone even when coupled with the robust matching algorithm because of severe
lack of patterns. Therefore a new image analysis method was developed where patterns
from neighboring pixels were used to automatically track marker points resulting in
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velocity measurements even in locations where there are no visible patterns. Chapter 3
describes the image analysis method in detail.
We employed this non-invasive method of kinematic analysis to measure basal
root growth and curvature in response to gravity. Our objectives were to (1) identify the
growth and graviresponding zones of the basal roots, (2) investigate the velocity profiles
within these zones, (3) find the relationship between the growth and the graviresponding
zones, (4) study the time evolution of these zones, and (5) determine how these are
affected by low phosphorus availability and ethylene treatment.
MATERIALS AND METHODS
Plant Culture
Common bean (Phaseolus vulgaris L.) genotype TLP19 developed for tolerance
to low phosphorus at the International Center for Tropical Agriculture (CIAT, Cali,
Colombia) was employed for this study. TLP19 has an indeterminate bush habit i.e. Type
II growth habit. TLP19 produces shallow basal roots and within one plant, we observed
shallow basal roots emerging from the top (closest to the shoot) whorls (whorl 1 and
whorl 2), with progressively deeper basal roots emerging from the lower whorl (whorl 3).
Seeds were surface sterilized with 6% sodium hypochlorite for 5 min, rinsed
thoroughly with distilled water and scarified with a razor blade. Seeds were germinated at
28°C in darkness for 2 d in rolled germination paper (25.5 x 37.5 cm Anchor Paper Co.,
St. Paul, MN, USA) moistened with either low or high phosphorus nutrient solution,
which was composed of (in µM) 3000 KNO3, 2000 Ca(NO3)2, 250 MgSO4, 25 KCl, 12.5
H3BO3, 1 MnSO4, 1 ZnSO4, 0.25 CuSO4, 0.25 (NH4)6Mo7O24, and 25 Fe-Na-EDTA. For
high phosphorus solutions, 1000 µM NH4H2PO4 was added; for low phosphorus, 500 µM
(NH4)2SO4 was added. Germinated seeds with radicals approximately 2-3 cm long were
transferred to a sheet of 30 x 24 cm blue germination paper (Anchor Paper Co., St. Paul,
MN, USA) stiffened by attaching perforated plexiglass sheets to stabilize the root system.
The bottom of the blue paper with plexiglass was placed to allow direct contact with the
nutrient solution containing high (1 mM) or low (0 mM) phosphorus as described above.
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The germination paper containing seedling was suspended in nutrient solution and
covered with aluminum foil to prevent illumination of the roots.
Treatment with ethylene and inhibitors of ethylene action
For ethylene treatment the growth pouch containing the bean seedling in low or
high phosphorus nutrient solution was placed inside a water-sealed plexiglass chamber
(37 L in volume). The seedling was treated with 0. 6 ul L-1 ethylene gas 36 h after the
emergence of the basal roots in the growth pouch and continued for 6-8 h of basal root
growth during which the basal root tends to respond to gravity. It should be noted that to
maintain uniform growth conditions e.g. temperature, humidity etc. the controls were also
placed inside the plexiglass box during time lapse photography.
The ethylene action inhibitor, MCP (EthylBloc, Floralife Inc., Walterboro, SC,
0.43% 1-methylcyclopropene) was used to test the role of ethylene in high or low
phosphorus availability. The plants were kept inside the similar water sealed plexiglass
chamber (37 L in volume). MCP gas was released through the reaction of EthylBloc
powder added to a plastic weighing plate inside the chamber and water added to the plate
by a syringe inserted through a rubber stopper. The ratio of EthylBloc powder to water
was calculated to be 4 mg EthylBloc per 0.08 ml water per liter air space.
Imaging procedure
Graphite particles were sprinkled on the roots carefully without disturbing the
plants and these particles created patterns that could be followed in image analysis on the
otherwise uniformly colored basal root (Chapter 3). During this procedure extra
precaution was taken not to touch the roots and also not to change the orientation of the
seedling with respect to the gravity because touching the root or changing their
orientation with respect to gravity may damage/change their natural behavior. We
checked the effect of adding graphite particles on the length of basal roots and compared
with the controls (Fig. 4.8. in Appendix). Images of root systems were captured at fixed
intervals (5 min) using a high resolution (6 Megapixel) digital single lens reflex camera
(Nikon D70s) fitted with 105 mm Nikkor micro lens. Images were captured for 4-6 h.
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The captured images had a resolution of 10-20 µm/pixel. Figure 3.3 (Chapter 3) shows a
photo of the image capturing setup. The seedling in the growth pouch was placed in an
airtight plexiglass box maintained at 25-26 °C. Photographs of the seedlings were
captured from outside the plexiglass box. The camera was triggered at fixed intervals of
time by a laptop computer through a universal serial bus (USB) cable. Plants were grown
in complete darkness and photos were captured using the camera’s flash to minimize
light exposure of the roots. To avoid shadows from direct flash which interfere with
image analysis, light from the flashes was bounced off the white paper placed on the top
of the plexiglass box. A ruler was attached to the supporting plexiglass sheet for
calibrating pixel dimensions into millimeters.
Measurements
Using a new semi-automated image analysis software, KineRoot (see Chapter 3
for details), marker points are tracked on the root in time. Initially the user selects 10-15
marker points along the body of the root which are then spatially interpolated to generate
a total of 25 marker points. KineRoot automatically tracks the position of these marker
points in all of the images by matching patterns surrounding the marker points. After the
marker points are tracked in all images, the edges of the root are identified by KineRoot
using an edge detection algorithm. From the detected edges of the root, the midline is
calculated by averaging the two edges which is used as the longitudinal axis for
measurement of local root growth velocity. The marker points are projected on the root
midline, and distance of the projected marker points from the root tip along the midline
are measured. Local root velocities relative to the root tip are then calculated as,
( , ) s s s sv s tt t
δ δδ δ
+ −= = (1)
where a marker point at distance s from the root tip moves to s+δs distance from the root
tip over time δt with velocity v(s, t). Spatial derivative of root growth velocity gives the
relative elongation rate of the root
( , ) ve s ts
δδ
= . (2)
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To calculate a meaningful derivative of growth velocity, a smoothed curve was fitted to
the growth velocity data following the overlapping polynomial smoothing procedure used
by Beemster and Baskin (1998) at every time step.
After determining the root midline, the curvature of the root κ is calculated as the
reciprocal of the radius of curvature R of the root midline at point (x, y),
( )
2
2
3/ 22
1
1
d ydx
dydx
Rκ = =
+
. (3)
It should be noted here that (x, y) is the coordinate of a point on the root midline in the
fixed two-dimensional Cartesian frame of reference attached to the plexiglass sheet
holding the germination paper. Therefore (x, y) coordinates of all points lying on the root
midline identify the shape and location of the midline. On the other hand s is the
coordinate of a point on the root midline in the moving one dimensional curvilinear frame
of reference attached to the root tip. Coordinate s of any point on the root gives the
distance from root tip along the root midline for calculation of growth but does not
provide the shape of the root. Therefore s cannot be used to calculate the curvature.
To calculate the root diameter d at distance s from the root tip, a line locally
perpendicular to the root midline is drawn. The distance between the two points of
intersection of the two edges with this perpendicular line is the root diameter at distance s
from the root tip. As a root responds to gravity and bends toward gravity, one side of the
root grows more than the other side. Therefore the ratio of local growth rate along the
two edges of the root can be used to characterize graviresponse of a root. Following Silk
and Erickson (1978), the differential growth ratio of two arcs of length δsu and δsl on the
upper and lower edges of an element of bending root can be calculated as
22
u
l
s ds d
δ κδ κ
+=
−. (4)
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RESULTS
Time history of root growth rate
Figure 4.1 shows the overall basal root growth rate as a function of time in high
and low phosphorus nutrient solutions for control, ethylene and MCP treatments during
the kinematic study. Data from tracking of the root tip were grouped in bins of 1 h, and
averages and standard errors for each bin were calculated, and plotted in Fig. 4.1. During
the time of the study, rate of root growth for most treatments tends to remain nearly
constant for basal roots emerging from whorl 1. However, in phosphorus deficient
nutrient solutions, roots from whorl 1 of controls had a low rate of growth to begin with
and then increased rapidly. Under low P treatment, ethylene inhibited growth rate in
whorl 1 (orange line), whereas MCP enhanced growth rate (magenta line), which then
drops after a certain period. In phosphorus sufficient conditions, control, ethylene and
MCP treatment all show very similar time course of root growth rate for whorl 1. In low
P, growth rate of roots emerging from whorl 3 in controls was less than all other
treatments, whereas application of MCP in low P (magenta line) increased growth rate
compared to other treatments in whorl 3. While MCP treatment increased growth rate in
low P roots from whorl 3, ethylene treatment reduced growth rate under low P treatment.
Root growth velocity
Although overall growth rate of the entire root in Fig. 4.1 indicates the effects of
different treatments on basal root elongation, for understanding the details of root growth
it is important to identify the growth zone and measure the local patterns of growth.
Toward this end Fig. 4.2 shows the velocity profiles of bean basal roots as functions of
distance from the root tip. In the frame of reference fixed with the root tip, any point on
the root moves away from the root tip as the tissue in between grows. Therefore, the
further the point is from the root tip, the higher the velocity. To calculate the average
velocity profiles, data from smoothed velocity profiles of individual pairs of images were
grouped in bins of 0.5 mm. Each of the curves represents data collected from 8-13 roots
obtained from 3-5 plants per treatment for 4-6 hours at 5 min intervals. Although
temporal variations in growth velocity were observed in a few roots, there was no
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consistent temporal pattern in growth velocity that could be extracted. Therefore the
curves in Fig. 4.2 not only show average of multiple roots, but also averages over time.
Figure 4.2 shows that in whorl 1, growth velocity tends to increase up to about 6 mm
from the root tip, then slows down for next 2.5 mm followed by another increment before
reaching a constant value by about 13.5 mm from the root tip in all but ethylene treatment
under low P. Up to about 4 mm from the root tip, all treatments show similar growth
velocity and then separate into two groups— a higher velocity group comprised of
controls and MCP treatments in low P and a lower velocity group consisting of the rest.
At 9 mm from the root tip, the groups merge and the second velocity increment begins.
This second elongation zone is rather high for controls under low P (blue line) and can
therefore explain the high overall growth rate for controls in low P in whorl 1. Under
ethylene treatment in low P (orange line), root growth velocity is reduced and the second
elongation zone does not exist.
Figure 4.2 also shows growth velocity averages from roots in whorl 3 where the
notable difference with whorl 1 is that this second elongation zone of the root does not
appear in any of the treatments. Furthermore, growth velocity at the end of the elongation
zone (>14 mm) is higher in whorl 3 compared to whorl 1 for all treatments except
controls in low P (blue line). Similar to Fig. 4.1, growth is enhanced in whorl 1 for
controls under low P and this enhanced growth is compensated in whorl 3. Application
of MCP strongly increases growth velocity in low P but only slightly increased it in high
P (Fig. 4.2). Ethylene increases growth velocity in low P but reduces it in high P.
Relative elongation rate
The spatial derivative of root growth velocity with respect to distance from the
root tip in Fig. 4.2 is the relative elongation rate, which is shown in Fig. 4.3 for whorls 1
and 3 under different treatments. The relative elongation rate quantifies the local
expansion rate of tissue along the body of the basal roots. As anticipated from Fig. 4.2,
roots in whorl 1 show a bimodal growth zone in all but ethylene in low P treatments
(orange line). The peak closer to the root tip spans about 6-7 mm with a consistent
relative elongation rate for all treatments, but the second peak is highly sensitive to
114
different treatments, and varies in both magnitude and span. But in whorl 3 except for
controls in high P (red line), none of the other treatments generated a detectable bimodal
relative elongation peak. However, this unimodal relative elongation zone of the roots of
whorl 3 does not make the growth zone shorter compared to whorl 1, rather it joins the
two growth zones. In both whorls 1 and 3, the peak elongation zone is located at about
2.5-3 mm from the root tip. While in whorl 1 rate of relative elongation drops rapidly and
then increases to form the second peak, in whorl 3 rate of relative elongation
monotonically declines.
To understand the bimodal shape of the relative elongation zone in whorl 1, it is
important to examine the data not only as a function of distance from the root tip, but also
as a function of time. To illustrate the spatio-temporal variations in relative elongation
rate, Fig. 4.4 shows, as an example, color isocontours of average relative elongation rate
of the roots of whorl 1 in controls under high P and low P. The reds, oranges and yellows
show relatively high rate of relative elongation whereas the blues and greens show
relatively low values. The relative elongation zone in low P is spread out between 2-13
mm with clearly distinguishable peaks. In high P the peak elongation zone near the root
tip maintains a consistent magnitude before breaking up in to two peaks after 120 min.
But the second peak which is observed between 6-11 mm in Fig. 4.3 does not correspond
to this yellow-orange band between 3.5-6 mm in Fig. 4.4. Rather it is the average of the
high relative elongation rates discontinuously spread between 6 and 11 mm from the root
tip in Fig. 4.4. Furthermore, presence of large amount of yellows and reds in low P
indicate higher relative elongation rates in low P compared to high P (Fig. 4.4). Both
isocontour plots show that relative elongation rate has underlying time dependence, apart
from the variability caused by root to root variations— relative elongation rate is low at
the beginning and then increases over time.
Root curvature and differential growth
Apart from root growth, one of the most important aspects of this study is to
characterize the bending of the basal roots which leads to graviresponse. In many of the
studies we observed that as the root grows, it develops a wavy motion with the root
115
periodically bending upward and downward. Figure 4.5 shows an example of this wave
motion of the root. 16 images of a root from whorl 1 captured at 20 min intervals were
digitally processed by Adobe Photoshop 7.0 TM (Adobe Systems Inc, San Jose, CA) and
superimposed in Fig. 4.5. The trajectory of the root tip is shown by red dots on the tip and
joining the red lines.
When a root bends downward, root growth is higher along the upper edge of the
root compared to the lower edge, and vice versa. Therefore, for downward bending,
differential growth ratio δsu/δsl > 1 and for upward bending δsu/δsl < 1. Measurements of
curvature and root diameter (results not shown) allow calculation of the differential
growth ratio between the upper and lower edges of the roots using equation (4). Two
examples of measured differential growth ratios are shown in Fig. 4.6 where spatio-
temporal changes in differential growth ratio are plotted as color isocontours for two
control roots—one grown in high P and the other in low P. The inserts show three
snapshots of the growing roots. The timings of the images with respect to the isocontour
plots are identified by magenta arrows (Fig. 4.6). In both high and low P treatments, the
left and right side images show that the root is bending downward resulting in differential
growth ratio greater than 1 (red-yellow colors), while the middle images show a subtle
bend upward resulting in differential growth ratio less than 1.0 (blue colors). In this
example, differential growth is nearly uniform along the length of the root in high P as
shown by mostly vertical color bands, whereas the direction of curvature of the root
changes along the body of the root in low P, as colors change in the vertical direction.
Both isocontour plots show periodic temporal changes in differential growth ratio
above and below 1 indicating wavy motion of the root. By applying frequency analysis
using Fourier transform of the differential growth ratio as a function of time at fixed
distances from the root tip, we identified the time periods of the wavy motion of roots
under different treatments which are listed in Table 4.1. Interestingly, application of MCP
tends to reduce the waviness of root growth and no dominant frequency of waviness
could be identified. Ethylene increased the time periods of upward-downward bending
for whorl 1 in both high and low P treatments compared to controls. But in the whorl 3,
compared to controls, ethylene only affected waviness under low P treatment.
116
The spatio-temporal oscillatory changes in differential growth ratio around 1
indicate that averaging differential growth ratio either in space or time will even out the
variations associated with upward or downward bending. Therefore from the isocontour
plots, the spatio-temporal areas where the root is bending upward vs. downward are
identified by setting threshold differential growth ratios of 0.99 for upward bending and
1.01 for downward bending. The solid black lines on the isocontour plots in Fig. 4.6
identify the downward bending regions (δsu/δsl > 1.01) and the areas enclosed by the
dotted black line show the spatio-temporal area of upward bending (δsu/δsl < 0.99).
Differential growth ratio 0.99 ≤ δsu/δsl ≤ 1.01 indicates a straight growing root with
negligible curvature. From each kinematic study of bean basal roots, we measured the
percentage of spatio-temporal areas of upward and downward bending of the roots, and
also calculated the average differential growth ratios during upward and downward
bending. Figure 4.7A shows the percentage of spatio-temporal area for upward and
downward bending of roots emerging from whorl 1 under ethylene and MCP treatments
relative to controls, whereas Fig. 4.7B shows the average differential growth ratios during
upward and downward bending for controls, ethylene and MCP. The dominant bending
pattern of the roots is toward gravity (empty bars) with only 8-10% spatio-temporal areas
occupied by upward bending in controls whereas downward bending is 50% of the
spatio-temporal area (data not shown). Under high phosphorus conditions, the spatio-
temporal area of upward bending increases more than 56% compared to controls,
whereas in low P the increase in upward bending is 25%. On the other hand application
of ethylene causes a reduction in spatio-temporal area for downward bending by 3-5%.
MCP, on the other hand, reduces spatio-temporal area for upward bending by 64% in
high P and 41% for low P while increasing downward bending areas by about 4-5%
compared to controls. Although the spatio-temporal areas of upward vs. downward
bending are influenced by different treatments, the average differential growth ratio
during either upward or downward curvature remain constant irrespective of treatments
as shown in Fig. 4.7B, albeit with high root to root variability.
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DISCUSSION
Using a novel image analysis system, this kinematic study shows local patterns of
root growth and bending of basal roots for common bean. We grew plants under
controlled environment with the specific objective of studying root growth and
graviresponse in different phosphorus treatments and hormonal stimuli like ethylene. Our
earlier studies show that ethylene application influences both basal root growth and
gravitropic angle (Chapter 2). In this kinematic study, the ethylene action inhibitor MCP
was applied to explore the role of ethylene in regulating plagiogravitropic growth locally.
The results from this study show new aspects of plagiogravitropic response of
basal roots which have not been observed before. One of the most interesting
observations from this study is the response of the basal roots in controls under
phosphorus deficient conditions. Figures 4.1 and 4.2 show that in low P treatment, plants
respond by accelerating growth of the roots in upper whorls (blue lines). Our earlier
measurements (Fig. 2.1 in Chapter 2) indicate that basal roots from whorl 1 are
significantly shallower than those of whorl 3. Therefore by enhancing growth of roots
from whorl 1, plants try to acquire limited nutrients like phosphorus more efficiently
from the top soil. This elevated growth rate of the roots in whorl 1 is compensated by a
reduction in growth rate in whorl 3 (Figs. 4.1 and 4.2). This is consistent with the earlier
observations in the field study of increased total basal root length of shallow roots in the
upper soil horizon compared to deep roots (Liao et al. 2001; Liao et al. 2004).
A close look at the rate of relative elongation of roots from whorl 1 in Fig. 4.3
reveals that this additional growth rate of basal roots in low P is a result of enhanced
elongation in the second elongation zone which is apical to the root maturation zone. But
in whorl 3, the second elongation zone does not exist. In addition, the first elongation
zone is reduced compared to other treatments. This phenomenon of accelerated growth of
controls in low P is visualized better when studied in spatio-temporal coordinates using
color isocontour in Fig. 4.4. Clearly, in low P the elongation zone is larger and also the
rate of elongation is higher in whorl 1 which might account for adaptive behavior of basal
roots from different whorls under phosphorus deficient conditions.
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Application of ethylene inhibits root growth in both high and low P conditions
for whorl 1 and whorl 3 (Fig. 4.2) which is consistent with our findings presented in Fig.
2.8 of Chapter 2. The reduction in growth under ethylene treatment in low P is a result of
nonexistence of the second elongation zone even in whorl 1 (Fig. 4.3). In high P
conditions, ethylene causes the second elongation zone to move further from the root tip
and makes this second elongation process very infrequent (data not shown) resulting in
an overall reduction in root growth. Although we notice similar behavior in whorl 3
under ethylene treatment, the deviations from controls due to ethylene are relatively less.
On the other hand, application of the ethylene action inhibitor MCP causes the root
growth rate to increase in low P but root growth rate remains similar to controls in high P
(Fig. 4.1), a result which is contradictory to what we observed in other studies (Fig. 5.17
in Appendix of Chapter 5). This inconsistency could be due the fact that to get good
quality trackable images, imaging had to be started immediately after the application of
MCP with no time for pre-treatment which might have caused delayed activation of MCP
effects.
Together with prior growth angle, root curvature determines shallowness or
deepness of basal roots. Root curvature results from differential growth between upper
and lower edges of the root. Therefore, study of spatio temporal patterns of differential
growth ratio of a growing root allows identification and measurement of root bending
zones and degree of bending. Earlier works have shown that as the root grows it follows a
wavy path (Simmons et al. 1995; Shabala and Newman 1997; Mullen et al. 1998b; Buer
et al. 2000; Buer et al. 2003). Similar observations are made in this study also—an
example is shown in Fig. 4.5. However, not every image clearly displayed this kind of
wavy motion. But measurement of differential growth ratios in kinematic studies show
subtle wavy motions of the roots, even when it may be difficult to identify them directly
on the images, as shown by the changes in the color in isocontour plots of differential
growth ratio in Fig. 4.6. Through systematic frequency analysis using Fourier transforms,
the periodicity of the waviness of root growth was measured for different treatments and
tabulated in Table 4.1. In agreement with Buer et al. (2003), the periodicity
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measurements show that application of ethylene alters the waviness of root motion. But
surprisingly, MCP nearly eliminates waviness of root motion.
The waviness of root growth also makes it difficult to average differential growth
ratios over space or time (Fig. 4.6) for further quantification of root bending, because
alternating high and low values when averaged together flatten the profiles of differential
growth ratio between the upper and lower edges of the root. Therefore from each
isocontour plot, upward and downward bending spatio-temporal areas were identified
(Fig. 4.6). If differential growth was higher than 1%, root growth was identified as a
curved growth; otherwise root growth was categorized as a straight. Therefore,
differential root growth ratio greater than 1.01 was identified as downward bending and
0.99 or lower was identified as upward bending. These thresholds were then used to
calculate the space-time areas of upward and downward bending (Fig. 4.6). Fig. 4.7A
shows a 56% increase in the spatio-temporal area for upward bending due to ethylene
whereas application of MCP reduces upward bending by up to 64% compared to controls.
Not surprisingly, the basal roots from whorl 1 respond to gravity and bend mostly toward
gravity and occasionally upward. However even the subtle upward movement of the
basal roots over a long period of time and the longer length of the root can alter root
architecture. In agreement with our earlier results (Figs. 2.4 and 2.6 in Chapter 2), Fig.
4.7A shows that ethylene increases the percentage of time and root length contributing to
upward bending and reduces the spatio-temporal area for downward bending resulting in
shallower roots, while maintaining similar curvatures as controls (Fig. 4.7B). On the
other hand, MCP makes the roots deeper by reducing the time and length of the roots
contributing to upward bending and increasing the time and length of the roots
contributing to downward bending—a result similar to our earlier observations (Fig. 2.4).
Although effects of ethylene or MCP on root architecture in Chapter 2 show the overall
behavior, these new results explain the dynamics associated with graviresponse. In a
seemingly surprising discovery we find that local root curvature remains unaffected by
ethylene or MCP treatments, but the spatio-temporal duration associated with upward vs.
downward motion of the root is affected which causes the root to respond to gravity
differently under different treatments. Since differential growth ratio remains unaltered,
120
local elongation rates along upper and lower edges of the roots under ethylene/MCP
treatments are equally affected in each edge.
To understand the results from this study, it is important to distinguish the
difference between local measurements in space and time with global measurements such
as total root length or angle over a longer period of time. For example, Fig. 4.1 shows
growth rate as a function of time, therefore time integration of each curve in Fig. 4.1, i.e.
the area under each curve, provides total root growth over the study period only,
irrespective of the initial length of the roots. A root can grow fast for a short period of
time and then can slow down resulting in the same overall growth compared to another
root which grows at a steady pace. Therefore, while overall root growth rates presented in
Fig. 4.1 regulate total root length, this is not the only determinant because these results
are based on a relatively short time of 4-6 h, and further changes in growth rate over time
may significantly alter the total growth of the roots.
Basal root growth angle studies in pouch experiments in Chapter 2 show that
there is no statistically significant difference in behavior of basal roots of different whorls
under low and high P. But Figs. 4.1 and 4.2 show that locally root growth rate is higher in
whorl 1 and lower in whorl 3 under low P. This raises the question that if the local pattern
of supposedly plastic behavior of roots can be seen in kinematic studies, then why it is
not observed in overall root growth and growth angle studies in the growth pouch? There
could be several reasons behind this seemingly contradictory behavior. In kinematic
studies root growth was analyzed over a relatively short period of time (4-6 h) during
which the basal roots exhibited a tendency to adapt to low P conditions by enhancing
growth rate of roots from whorl 1. But in the long run this behavior is not visible. The
germination paper which supplies nutrients to the seedling absorbs nutrient solution, and
through capillary action distributes nutrients uniformly to all the roots. So even if there is
phosphorus deficiency in the nutrient solution, all the basal roots, irrespective of
shallowness, get the same amount of phosphorus. But in the field under phosphorus
depleted conditions, more phosphorus is available in the topsoil compared to the subsoil.
This difference between field condition and growth pouch in terms of nutrient gradient
might account for the changed behavior of the basal roots in the long run in laboratory
121
experiments, although initially they show adaptive behavior in kinematic studies. In
addition, the growth pouches are 24 cm wide. Therefore as the basal roots move near the
edge of the paper they are forced to bend resulting in changes in growth angle.
Using detailed kinematic analysis this paper makes surprising discoveries which
could not be studied using other approaches. Firstly, in response to low phosphorus
treatments plants accelerate growth rate of the upper whorls at the cost of lower growth
rate in lower whorls. Ethylene and MCP do not alter local root curvature, but alter the
span and duration of the bending of the root upward or downward and thereby produce
shallow and deep roots respectively.
ACKNOWLEDGEMENT
We would like to thank Dr. Anupam Pal for developing the kinematic program and also
helping in analyzing and discussing the results from kinematic studies.
122
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126
Fig. 4.1. Time course of basal root growth rate from whorl 1 and whorl 3 grown in high (1 mM) and low (0 mM) phosphorus nutrient solutions. The ethylene and MCP treated roots were exposed to 0.6 µl/L ethylene gas and 1 µl/L MCP gas respectively 36 h after the emergence of the basal roots. Data from 8-13 basal roots of 3-5 plant per treatment for 4-6 hours at 5 min intervals were grouped in bins of 0.5 mm. The values shown are means of each of the bins ±SE.
127
Fig. 4.2. Spatial profiles of growth velocity of basal roots as a function of distance from the root tip of whorl 1 and whorl 3 grown on high (1 mM) and low (0 mM) phosphorus nutrient solutions. The ethylene and MCP treated roots were exposed to 0.6 µl/L ethylene gas and 1 µl/L MCP gas respectively 36 h after the emergence of the basal roots. Data from 8-13 basal roots of 3-5 plant per treatment for 4-6 hours at 5 min intervals were grouped in bins of 0.5 mm. The values shown are means of each of the bins ±SE.
128
Fig. 4.3. Spatial profiles of relative elongation rate (or strain rate) versus distance from the root tip for the basal roots of whorl 1 and whorl 3 grown on high (1 mM) and low (0 mM) phosphorus nutrient solutions. The ethylene and MCP treated roots were exposed to 0.6 µl/L ethylene gas and 1 µl/L MCP gas respectively 36 h after the emergence of the basal roots. Data from 8-13 basal roots of 3-5 plant per treatment for 4-6 hours at 5 min intervals were grouped in bins of 0.5 mm. The values shown are means of each of the bins ±SE.
129
Fig. 4.4. Color isocontour plot of relative elongation rate of basal roots emerging from whorl 1 in controls under high P and low P conditions showing the spatio-temporal variation in relative elongation rate. The colors work as a separate axis apart from the horizontal or vertical axes, and show space time locations of high (red, orange, yellow) and low (blue, green) magnitudes of relative elongation rate. The data for each plot were collected from 8 roots of 4 plants, and grouped in space-time bins of 0.5 mm x 5 min and averaged within each space-time bin.
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Fig. 4.5. Superimposed time lapse photos of a growing basal root shot at 20 min intervals showing the wavy motion of the tip (red dots) during a 5 h period. To make all 16 images of the root visible, we only show the edges of the roots after digitally processing each image in Adobe Photoshop 7.0TM (Adobe Systems Inc, San Jose, CA).
131
Fig. 4.6. Examples of spatio-temporal color isocontour plot of differential growth ratio between upper and lower edges of a basal root of whorl 1 in a control plant under low P and high P conditions. The red and yellow colors show differential growth ratio > 1, i.e. bending downward while blue shows differential growth ratio < 1, i.e. bending upward. The inserts show photographs of the root at specific times identified by the dotted magenta arrows. The spatio-temporal regions enclosed by solid black lines identify space-time locations of downward bending roots with differential growth ratio δsu/δsl > 1.01, and the area enclosed within the dotted line identifies upward bending root with differential growth ratio δsu/δsl < 0.99.
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Fig. 4.7. Spatio-temporal comparison of bending of the basal roots emerging from whorl 1. (A) Percentage of spatio-temporal area (see Fig. 4.6) during upward (δsu/δsl < 0.99) and downward (δsu/δsl > 1.01) bending of the roots under different treatments compared to controls. (B) Average differential growth ratio during upward and downward bending of the roots. Data show mean of 8-13 basal roots of 3-5 plant per treatment ±SE.
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Table 4.1. Periodicity of the wavy motion of the bean basal roots for different treatments identified by frequency analysis using Fourier transform of the differential growth ratio as a function of time at fixed distances from the root tip. MCP treatment resulted in elimination of detectable periodicity. Numbers show mean ± SE.
Treatment Whorl 1 Whorl 3
High P 120±11 min 180±3 min
Low P 105±5 min 108±12 min
High P + Ethylene 180±16 min 180±15 min
Low P +Ethylene 210±10 min 140±23 min
High P +MCP - -
Low P +MCP - -
134
CHAPTER 4 APPENDIX
0
1.5
3
4.5
whorl 1 whorl 2 whorl 3
Leng
th o
f bas
al ro
ots
(cm
)
control day 1control day 2before graphite day 1after graphite day 2
Figure 4.8. Graphite does not affect root growth. The graphite particles were sprinkled carefully on the basal roots which were used for the time lapse photography required for kinematic study. N= 4-5 plants ± SE.
CHAPTER 5: HORMONAL REGULATION OF GRAVITROPIC GROWTH OF
BASAL ROOTS – A CROSS-TALK BETWEEN ETHYLENE AND
AUXIN
Paramita Basu1, Jurgen Engelberth2, Jonathan P. Lynch1, 3, Kathleen M. Brown1, 3
1Intercollege Program in Plant Physiology, Penn State University, University Park, PA
16802 USA; 2Department of Entomology, Penn State University, University Park, PA 16802 USA. 3Department of Horticulture, Penn State University, University Park, PA 16802 USA.
136
ABSTRACT
Gravitropic growth of roots determines root architecture, which is essential for
efficient acquisition of soil resources. Auxin and ethylene are potential regulators of the
graviresponse of roots. Basal roots of common bean exhibit plagiogravitropic growth
which varies over time. We hypothesize that ethylene modulates the auxin effect on root
growth and plagiogravitropic curvature of basal roots. Parental genotypes and
recombinant inbred lines of common bean with contrasting basal root traits were
employed for this study. Lower whorls of basal roots had higher free auxin content and
were more sensitive to auxin inhibition of basal root growth compared to upper whorls.
However, transport of auxin from root-shoot junction using 3H-IAA shows more
transport of radiolabeled auxin to upper whorls than lower whorls. Ethylene did not affect
transport of 3H-IAA from the hypocotyls to the roots, but increased free IAA content in
the basal roots. Both ethylene and auxin make the basal roots shallower. Our results show
that auxin concentration in controls is near optimal. Application of aminovinylglycine
(AVG) or 1-methylcyclopropene (MCP) together with exogenous IAA increase root
growth and reduce shallowness in phosphorus sufficient conditions. However, AVG and
MCP do not reverse IAA-inhibition of growth in low phosphorus. These results point to a
phosphorus dependent interaction between ethylene and auxin in regulation of
elongation, but a phosphorus-independent interaction for control of growth angle.
137
INTRODUCTION
Root architecture, the three-dimensional distribution of a root system, is defined
in part by growth angles. Each and every plant organ has distinct and specific response to
gravity, which usually results in plagiogravitropic growth i.e. growth at an angle other
than 0o relative to the gravity vector. This stable angle was referred to as gravitropic set-
point angle (GSA) by Firn and Digby (1997). According to Firn and Digby, the growth of
most plant organs occurs at a stable angle determined by various factors, including
gravity itself. The basal roots of common bean are secondary roots arising from the root-
shoot interface which together with the primary root determine the scaffolding of the
bean root system. They exhibit plagiogravitropic growth which varies over time and also
in response to gravity. Growth angle of basal roots (BRGA) have been associated with
genotypic differences (Chapter 2) in acquisition of limited and immobile nutrients like
phosphorus (P) and adaptation to low-P soils (Bonser et al. 1996; Liao et al. 2001; 2004).
In addition, low phosphorus availability dramatically alters the BRGA in some genotypes
(Bonser et al. 1996; Liao et al. 2001).
Low phosphorus availability is the major constraint for plant productivity in
many terrestrial ecosystems. The growth angle of roots has important implications for
acquisition of soil resources. In common bean shallow-rooted genotypes are better
adapted to low phosphorus availability than deep rooted genotypes (Bonser et al. 1996;
Liao et al. 2001; Liao et al. 2004; Ho et al. 2005). Shallow basal roots not only increase
topsoil exploration but also produce less intraplant and interplant competition for limited
and immobile nutrients like phosphorus (Ge et al. 2000; Lynch and Brown 2001; Rubio
et al. 2001; Rubio et al. 2003).
Our previous work (Chapter 2) has shown that phosphorus stress increases the
ethylene sensitivity of basal roots, making the basal roots shallower. In addition, our
work has also shown that position of emergence of basal roots has important implications
since basal roots from upper whorls explore the upper soil horizon by becoming
shallower, while roots from lower whorls, which are less responsive to ethylene, maintain
a deeper growth angle and would explore different soil domains. Moreover, we have
shown that shallow genotypes produce a greater range of BRGAs than deep genotypes
138
(Chapter 2) in the presence of ethylene, which would enhance topsoil foraging (Lynch
and Brown 2001).
Although variation in growth angles of basal roots has been observed, very little
is known about how the growth angles are regulated. While it seems certain that response
to gravity plays a major role in regulating BRGA, hormonal signals such as ethylene and
auxin most likely play a crucial role in determining genotypic differences. The
involvement of auxin in regulating curvature has been postulated by the Cholodny-Went
theory, which states that laterally redistributed auxin in a gravistimulated organ results in
a differential auxin gradient, promoting differential cell elongation on the opposite flanks
of the stimulated organ and cause downward bending (Blancaflor and Masson 2003). In
addition to its role in regulating gravitropic bending, auxin has another potential role in
controlling root elongation. Although low concentration of auxin applied to the nutrient
solution may stimulate root growth, higher concentration of exogenous auxin reduces
root elongation (Eliasson et al. 1989). Therefore, concentrations of auxin which promote
the growth of shoots inhibit growth of roots i.e. roots are more sensitive to auxin than
shoots.
Two types of auxin transport within roots have been reported: 1) a fast and non-
directional auxin transport in the phloem (Ljung et al. 2001) and 2) a slow, directional
polar transport. Non-polar transport of auxin through phloem occurs in both the basipetal
and the acropetal directions (5-20 cm/hr) coupled with transport of assimilates (sugar)
and inactive auxin conjugates (Baker 2000; Friml and Palme 2002), whereas polar
transport is specific for the cell-to cell movement (5-20 mm/hr) of free active auxin in a
directional manner. In roots, polar auxin transport occurs in two directions: acropetally
from the base of the root to the root apex through the central cylinder and basipetally
away from the root apex through the outermost epidermal and cortical cell layers (Lomax
et al. 1995; Rashotte et al. 2000). Basipetal auxin transport is required for root
gravitropism (Rashotte et al. 2000). In addition to polar transport, lateral auxin movement
across roots is stimulated by a change in gravity and this lateral transport drives
differential gravitropic growth (Muday 2001). Although the two types of transport could
be linked directly or indirectly (Cambridge and Morris 1996), the distinction between the
139
contribution of non-polar and polar auxin transport in generating the auxin pool in
different tissues has not yet been clearly elucidated.
Besides auxin, another potential modulator of root gravitropism is ethylene
(Abeles et al. 1992). Although there is evidence that ethylene mediates gravitropic
responses in roots, shoots and cut-flower stems (Chadwick and Burg 1967; Wheeler and
Salisbury 1980; Lee et al. 1990; Philosoph-Hadas et al. 1996; Kiss et al. 1999; Madlung
et al. 1999; Edelmann 2002; Chang et al. 2004), other research shows that ethylene has
no effect on gravitropic response of plant organs (Harrison and Pickard 1986; Woltering
1991). Evidence suggests that production of an ethylene gradient across a gravistimulated
organ is associated with the manifestation of gravitropic bending (Philosoph-Hadas et al.
1996). However, the role of the ethylene gradient in the signal transduction mechanism
leading to the gravitropic response is still controversial (Madlung et al. 1999; Friedman et
al. 2005; Woltering et al. 2005). Although ethylene applied at low concentrations
promotes gravicurvature, continuous application at higher concentration proved to be
inhibitory in gravistimulated shoot and inflorescence (Madlung et al. 1999; Lu et al.
2002).
The importance of auxin in gravitropism, as well as the close interaction between
ethylene and auxin in various developmental processes including root development, has
already been illustrated by various authors. Extensive studies regarding the physiological
interaction between auxin (IAA) and ethylene have established that at least two kinds of
interactions might exist (Rahman et al. 2001). A well-established auxin-ethylene
interaction is that the application of exogenous auxin stimulates ethylene production
(Chadwick and Burg 1967) and the second potential interaction is that ethylene inhibits
polar and lateral auxin transport (Burg and Burg 1967; Suttle 1988). Ethylene treatment
of pea hypocoyls reduced the amount of auxin transport up to 95% (Burg and Burg 1967;
Ruegger et al. 1997). In roots, by reducing acropetal auxin transport, ethylene could
cause auxin depletion in the root apex, thereby reducing root elongation. A second
possibility is that ethylene retards polar auxin transport from the root tip to the elongation
zone, resulting in an insufficient auxin pool in the elongation zone and reducing root
elongation (Casson and Lindsey 2003). In addition, it has been shown in citrus leaves that
140
ethylene treatment reduces endogenous IAA levels by increasing conjugation of IAA
(Riov et al. 1982) and that the increased auxin conjugation lowers movement of auxin
through the tissue. Increased IAA catabolism is another mechanism by which ethylene
reduces active IAA content (Sagee et al. 1990). Ethylene may affect auxin redistribution
(Lee et al. 1990) or rate of auxin transport (Burg and Burg 1967) or synthesis of auxin in
the root tip (Stepanova et al. 2005). Although the are many reports of ethylene inhibition
of auxin transport, other evidence shows that ethylene stimulates auxin transport (Morgan
and Gausman 1966). Recent work by Madlung et al. (1999) suggested that exogenous
ethylene induces a signal which either stimulates asymmetric redistribution of auxin or
alters auxin sensitivity of the cells of a gravistimulated organ, thereby regulating
graviresponse.
In this report, we investigate the possible role of cross-talk between auxin and
ethylene in regulating growth angle of basal roots (BRGA) as well as root growth. Since
ethylene regulates plagiogravitropic growth of basal roots at an early stage (Chapter 2), it
is possible that ethylene modulates auxin movement, thereby affecting the auxin gradient
needed for graviresponse. However, effects on auxin movement alone might not account
for the complexity of gravity-induced changes in growth rate and curvature patterns
during plagiogravitropic growth. There could be an interaction of auxin redistribution and
time-dependent change in auxin sensitivity as suggested by Ishikawa et al. (1991), if
auxin mediates the gravitropic response as proposed by Cholodny-Went theory.
Therefore we specifically hypothesize that ethylene modulates the auxin response during
root growth and plagiogravitropic curvature of basal roots. This could be possible either
by modification of auxin transport in the growing roots or by altering the sensitivity of
basal roots to auxin. We tested this hypothesis in plagiogravitropic basal roots of
common bean demonstrating genetic and nutrition-induced variation in basal root growth
angles.
141
METHODS
Plant material
Two parent genotypes, B98311 and TLP19, were crossed by Dr. Jim Kelly at
Michigan State University to produce a population of recombinant inbred lines (RILs),
the L88 population consisting of 81 lines. The RILs descending from the cross between
these two parents share a common genetic background, yet segregate for root
architectural traits as well as adaptation to abiotic stress. In addition, they possess
commercial quality of black bean seeds. B98311 is drought-resistant Mesoamerican
genotype from the MSU breeding program and possesses a Type II growth habit and a
deep vigorous primary root (Frahm et al. 2004) and TLP19 was developed for tolerance
to low phosphorus at the International Center for Tropical Agriculture (CIAT, Cali,
Colombia) and also possesses a Type II growth habit. Preliminary experiments showed
that TLP19 produces shallower basal roots both under low and high phosphorus
conditions. In addition to the parent genotypes, we used four contrasting RILs (two
shallow and two deep) in our experiments, selected based on basal root growth angles
assessed in preliminary experiments.
Comparison of growth angle of genotypes
Seeds were surface sterilized with 6% sodium hypochlorite for 5 min, rinsed
thoroughly with distilled water and scarified with a razor blade. Seeds were germinated at
28°C in darkness for 2 d in rolled germination paper (25.5 x 37.5 cm Anchor Paper Co.,
St. Paul, MN, USA) and moistened with either low or high phosphorus nutrient solution,
which was composed of (in µM) 3000 KNO3, 2000 Ca(NO3)2, 250 MgSO4, 25 KCl, 12.5
H3BO3, 1 MnSO4, 1 ZnSO4, 0.25 CuSO4, 0.25 (NH4)6Mo7O24, and 25 Fe-Na-EDTA. For
high phosphorus solutions, 1000 µM NH4H2PO4 was added; for low phosphorus, 500 µM
(NH4)2SO4 was added. Germinated seeds with radicals approximately 2-3 cm long were
transferred to growth pouches consisting of a sheet of 30 x 24 cm blue germination paper
(Anchor Paper Co., St. Paul, MN, USA) inserted into a polyethylene bag of the same size
with evenly spaced (3 cm apart) holes for aeration. Pouches were open at the bottom to
allow direct contact with the nutrient solution containing high (1 mM) or low (0 mM)
142
phosphorus (P) as described above. The pouches were stiffened by attaching perforated
plexiglass sheets to stabilize the root system. The pouches were suspended in nutrient
solution and covered with aluminum foil to prevent illumination of the roots. Root
systems were photographed after 2 d growth in pouches and basal root angles were
determined using Matlab 7.0TM (Mathworks Inc., Natick, MA, USA). Growth angles of
basal roots were measured relative to the vertical, i.e. larger angles indicate shallower
basal roots.
Treatment with auxin and NPA
For experiments with auxin treatment, we conducted preliminary experiments to
determine a suitable method for application of indole-3-acetic acid (IAA) to the
seedlings. Lanolin paste has been the most widely used method for application of IAA,
however, in our case; this method was unsuitable because the preparation and application
procedures were laborious and the lanolin paste did not adhere well to the vertical
seedling. Therefore, we applied IAA in solution directly to the root-shoot interface of the
seedling growing in the pouch without disturbing the seedling. A small plastic ring (cut
from pipette tips) was attached around the root-shoot junction just above the basal root
emergence zone. The bottom of the ring was sealed with a small piece of blue
germination paper to prevent the leakage of solution added to the ring. Experiments were
conducted to determine the effect of IAA concentration on the growth angle and the
growth rate of basal roots. Solutions of IAA (0 – 40 nmol in 20 µl) were applied to the
growing seedlings twice: the first application was done immediately after the transfer of
the seedling to the pouch and the second was done 24 h after the transfer of the seedling
i.e. one day after the basal roots emerged. The roots were photographed after 24 and 48 h
and the basal root growth angles were measured as the angle between the vertical and the
line connecting the root tip positions at 24 h and 48 h using Matlab 7.0TM (Mathworks
Inc., Natick, MA, USA). Root growth (increase in length between 24 and 48 h) was
assessed from the same digital images. The experiments were repeated three times with
2-3 plants per genotype per treatment each time. The slope of the auxin dose-response
curve was estimated by the slope of the linear regression line fitted to BRGA vs. auxin
143
concentration data for each genotype, each whorl position and each P-treatment, and was
defined as the auxin sensitivity.
In a separate experiment, NPA (1-N-naphthylphthalamic acid), an auxin transport
inhibitor, was applied to the seedlings to determine the dose required to examine the
effect of auxin at below-endogenous level on the growth angle and growth rate of basal
roots. Different concentrations of NPA (0 – 20 nmol) were applied in solution in the
similar way as described above.
Measurement of ethylene production
We measured endogenous ethylene production from the basal roots of the auxin
treated (30 nmol) seedlings compared with control plants. For ethylene measurement,
fresh tissue containing the basal roots was harvested 48 h after transfer of the seedlings to
the pouch. The segments were separated into three basal root whorls with a razor blade
and enclosed individually in 9 ml vials capped with septa at 25°. Ethylene was sampled
with a 1-ml syringe from the headspace of the vials 2 h later and quantified by gas
chromatography (HP6890 gas chromatograph) equipped with a flame ionization detector
and an activated alumina column, Hewlett-Packard Company, Wilmington, DE, USA).
Treatment with ethylene inhibitors
In order to assess the possible role of ethylene-auxin interaction on regulation of
growth angle, we treated the seedlings with the inhibitor of ethylene biosynthesis AVG
(aminoethoxyvinylglycine), or the ethylene action inhibitor MCP (EthylBloc, Floralife
Inc., Walterboro, SC 0.43% 1-methylcyclopropene), sometimes in combination with
IAA. For experiments with MCP, seedlings were grown as previously described in either
low or high phosphorus nutrient solution. After transferring to the growth pouches, the
seedlings were treated with 30 nmol IAA and kept inside air-tight growth chambers (118
liter plastic boxes). EthylBloc was added to a plastic weighing plate placed inside the
growth chambers and buffer was added to the plate via a syringe inserted through a
rubber stopper on the top of the chamber. The seedlings were treated for 24 h and 48 h
with MCP released through the reaction of EthylBloc powder with buffer. The basal roots
144
were photographed at 24h and 48 h and the growth angles and root growth were
measured as previously described. The experiment involved 4 plants per genotype per
treatment. The ratio of EthylBloc powder to buffer was 4 mg EthylBloc per 0.08 ml
buffer per liter air space and the actual amount of EthylBloc powder was calculated based
on the volume of the growth chamber, yielding 1 µl L-1 MCP gas inside the chamber. For
AVG experiments, 60 µM AVG and 30 nmol IAA were added to the ring around the
root-shoot junction of the seedlings grown in either low or high phosphorus nutrient
solution.
Quantification of endogenous auxin
In a separate set of experiments, we quantified the amount of endogenous auxin
present in the basal roots. For this experiment, the seedlings of RILs 57 and 7 were
treated with 30 nmol IAA (determined from dose response experiment) or 15 nmol NPA
at the root-shoot interface or with 0.6 ul L-1 ethylene in low phosphorus nutrient solution.
In another set of experiment, the seedlings of RIL57 were treated with 0.6 ul L-1 ethylene
in either low or high phosphorus nutrient solution. The plants were treated with the
specified hormone after the transfer of the seedlings to the pouch and the second
application was done at 24 h after the transfer of the seedlings. The basal roots were
harvested and frozen in liquid nitrogen and then stored in -80°C for analysis of
endogenous free IAA by GC-MS/MS with methanol chemical ionization (Trace GC 2000
attached to a GCQ mass spectrometer, Thermo Finningan, San Jose, CA) as described by
Schmelz et al. (2003) and Engelberth et al. (2004) for analysis of multiple hormones
from a very small amount of tissue. For the analysis of auxin, basal rooting zones were
separated into three basal root whorls with a razor blade and put inside screw-cap vials,
each containing approximately 150 mg – 200 mg basal roots. Since the basal roots of
each plant were very small at the time of harvest, we combined basal roots from of 10-12
plants for each sample to be analyzed. The root samples were transferred to the screw-cap
Philosoph-Hadas S, Friedman H (2005) An auxin-responsive 1-
aminocyclopropane-1-carboxylate synthase is responsible for differential ethylene
161
production in gravistimulated Antirrhinum majus L. flower stems. Planta 220,
403-413.
162
Figure 5.1. Effect of genotype and position of origin on basal root growth angle of common bean genotypes of the L88 population. The growth angle of the basal roots was measured after 2 d growth in pouches. The bars show mean growth angles of basal roots emerging from each whorl of 10-12 plants per genotype, with data pooled over phosphorus treatments, ± SE. Inset shows a close up view of a young seedling (1 d after transplanting to the growth pouch) showing distinct whorls with emerging basal roots.
163
Figure 5.2. Auxin sensitivity of growth angles and growth rate of basal roots for whorls 1, 2, 3 of a deep (B98311) and a shallow (TLP19) genotype grown in low phosphorus. The angle and growth were measured for the root growth occurring between 24 and 48 h. Values shown are means of 4-5 plants per auxin treatment ± SE.
164
Figure 5.3. Auxin sensitivity of growth angle of basal roots as a function of genotype and whorl position in three shallow (TLP19, RIL57 and RIL15) and three deep (B98311, RIL7 and RIL76) genotypes (pooled over both phosphorus treatments). Auxin sensitivity was calculated as the slope of the response curve (auxin concentration vs. growth angle of basal roots).
165
high Py = 0.0686x + 0.2292
R2 = 0.5239
low Py = 0.0751x - 0.162
R2 = 0.8577
0
1.5
3
4.5
6
20 35 50 65 80
Growth angle (without auxin)
Aux
in s
ensi
tivity
Figure 5.4. Correlation between auxin sensitivity and growth angle of basal roots of six L88 genotypes (deep genotypes B98311, RIL7, RIL76 and shallow genotypes TLP19, RIL57, RIL15) grown in low P and high P. The symbols show values for each genotype and for each whorl position. Growth angles on X-axis designate control plants without auxin treatment.
166
Figure 5.5. Auxin sensitivity of growth response of basal roots as a function of genotype and whorl in three shallow (TLP19, RIL57 and RIL15) and three deep (B98311, RIL7 and RIL76) genotypes (pooled over both phosphorus treatments together). Root growth was measured between 24 and 48 h. Auxin sensitivity was calculated as the slope of the response curve (auxin concentration vs. growth).
167
0
25
50
75
100
whorl 1 whorl 2 whorl 3 whorl 1 whorl 2 whorl 3
shallow genotype deep genotype
Ethy
lene
pro
duct
ion
(nl/h
/g F
W) control Low P
IAA Low Pcontrol High PIAA High P
Figure 5.6. Endogenous ethylene production per gram fresh weight by the segments of the root-shoot junction bearing basal roots of a deep (RIL7) and a shallow (RIL57) genotype treated with 30 nmol IAA in either low P or high P nutrient solution. Segments were harvested 48 h after transplanting. Values shown are means of 4-7 plants per genotype per hormone treatment and phosphorus treatment. Bars indicate standard errors.
168
0
25
50
75
100
whorl 1 whorl 2 whorl 3 whorl 1 whorl 2 whorl 3
Deep genotypes Shallow genotypesBas
al ro
ot a
ngle
(deg
rees
from
ver
tical
) control IAA AVG+IAA
Figure 5.7. Combined effect of AVG (60 µM) and IAA (30 nmol) on the growth angle of basal roots of deep (B98311 and RIL7) and shallow (TLP19 and RIL57) genotypes (pooled over both phosphorus treatments). AVG prevents the increase in root shallowness caused by IAA (P <0.001). Values shown are means of the growth angles of 4 plants per genotype per hormone treatment and phosphorus treatment. Bars indicate standard errors.
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Figure 5.8. Combined effect of AVG (60 uM) and IAA (30 nmol) on the basal root growth of deep (B98311 and RIL7) and shallow (TLP19 and RIL57) genotypes grown in low P or high P nutrient solution. AVG reverses the IAA-inhibition of growth only for plants grown with high phosphorus (P < 0.001). Values shown are means of the growth angles of 4 plants per genotype per hormone treatment per phosphorus treatment. Bars indicate standard errors.
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0
25
50
75
100
whorl 1 whorl 2 whorl 3 whorl 1 whorl 2 whorl 3
Deep genotypes Shallow genotypesBas
al ro
ot a
ngle
(deg
rees
fro
m v
ertic
al)
control IAA MCP +IAA
Figure 5.9. Combined effect of MCP and IAA on the growth angle of basal roots of deep (B98311 and RIL7) and shallow (TLP19 and RIL57) genotypes (pooled over both phosphorus treatments). MCP prevents the increase in root shallowness caused by IAA (P <0.001). Values shown are means of the growth angles of 4 plants per genotype per hormone treatment and phosphorus treatment. Bars indicate standard errors.
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Figure 5.10. Combined effect of MCP and IAA on the basal root growth of deep (B98311 and RIL7) and shallow (TLP19 and RIL57) genotypes grown in low P or high P nutrient solution. MCP reverses the IAA-inhibition of growth only for plants grown with high phosphorus (P < 0.001). Values shown are means of the growth angles of 4 plants per genotype per hormone treatment per phosphorus treatment. Bars indicate standard errors.
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Figure 5.11. Free IAA in common bean basal roots of seedlings of a deep genotype (RIL7) and a shallow genotype (RIL57) grown in low phosphorus nutrient solution for exogenous ethylene (A), exogenous IAA (B), and exogenous NPA (C) applications. Values shown are the mean of three samples each containing 12 to 20 basal roots ± SE.
0.0
0.2
0.3
0.5
0.6
whorl 1 whorl 3 whorl 1 whorl 3
Deep genotype Shallow gentoypeEndo
geno
us fr
ee IA
A c
onte
nt (n
g/ba
sal r
oot)
control ethyleneA
0.0
0.2
0.3
0.5
0.6
whorl 1 whorl 3 whorl 1 whorl 3
Deep genotype Shallow gentoypeEndo
geno
us fr
ee IA
A c
onte
nt (n
g/ba
sal r
oot)
control NPAC
0.0
0.2
0.3
0.5
0.6
whorl 1 whorl 3 whorl 1 whorl 3
Deep genotype Shallow gentoypeEndo
geno
us fr
ee IA
A c
onte
nt (n
g/ba
sal r
oot)
control IAAB
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0.0
0.1
0.2
0.3
0.4
0.5
whorl 1 whorl 3 whorl 1 whorl 3
Low P High P
Endo
geno
us fr
ee IA
A c
onte
nt (n
g/ba
sal r
oot)
control
ethylene
Figure 5.12. Free IAA in common bean basal roots of seedlings of a shallow genotype (RIL57) grown in low (low P) and high (high P) phosphorus nutrient solution. Values shown are the mean of three samples each containing 12 to 22 basal roots ± SE.
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Figure 5.13. Basal root growth rate vs. free IAA content per basal root plotted for whorls 1 and 3 of a shallow (RIL57) and a deep (RIL7) genotype. For each of the four line segments the left most symbols were for NPA treated basal roots, the middle points were for controls and the right most were for IAA treated basal roots.
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0
15000
30000
45000
60000
75000
hypocotyl appliedzone
whorl 1 whorl 3 primaryroot
3-H
IAA
tran
spor
t (cp
m)
IAAethylene + IAA
Figure 5.14. Auxin transport activity in roots of common bean seedlings of a shallow genotype (RIL57). Amount of radioactive IAA transported to the basal roots of different whorls, primary roots, hypocotyls and applied zone( root-shoot junction) after application of 3H-IAA for 24 h. Individual segments were immersed in scintillation cocktail for 48 h. Values show means ± SE of 4 seedlings per treatment.
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Figure 5.15. Anatomical sections of basal root emergence zone of a parent genotype TLP19. Figures A-C show the zone from where basal roots emerge. Figures D-E show the longitudinal view of the root-shoot interface from where the basal roots develop from 3 distinct whorls (Fig. D) and 1 whorl (Fig. E). Figure F shows the transverse section of the region just below the basal root emergence zone.
Table 5.1. ANOVA of growth angle and growth response of basal roots from contrasting genotypes –shallow (TLP19, RIL57, RIL15) and deep (B98311, RIL7, RIL76) as affected by exogenous auxin treatment.
Growth angle Growth rate
Effect DF F-value P-value F-value P-value
Genotype 1 1752.39 <0.001 4.23 0.039
Phosphorus 1 23.51 <0.001 0.15 0.696
Auxin 4 260.82 <0.001 340.27 <0.001
Whorl 2 3756.14 <0.001 722.75 <0.001
Genotype*Phosphorus 1 0.59 0.442 0.06 0.796
Genotype*Auxin 4 5.88 <0.001 20.75 <0.001
Genotype*Whorl 2 434.87 <0.001 5.58 0.003
Phosphorus*Auxin 4 0.05 0.994 0.11 0.977
Phosphorus*Whorl 2 0.07 0.924 0.08 0.919
Auxin*Whorl 8 10.63 <0.001 20.99 <0.001
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Table 5.2. Effect of NPA treatment on the basal root growth angle (BRGA) and growth rate of two shallow (TLP19 and RIL57) and two deep (B98311 and RIL7) genotypes (pooled over both phosphorus treatments). Values shown are means of the growth angles of 4 plants per genotype per phosphorus treatment ± SE.
Figure 5.16. Correlation between endogenous ethylene production and growth angle of a deep genotype – RIL7 (A) and a shallow genotype-RIL57 (B) treated with 30 nmol exogenous IAA concentration in both low P and high P treatments.
Low Py = 0.7128x + 23.267
R2 = 0.3729, P = 0.011
High Py = 0.2549x + 35.727
R2 = 0.2102, P = 0.085
0
25
50
75
100
125
30 45 60 75 90Growth angle (degrees from vertical)
Ethy
lene
pro
duct
ion
(ng/
h/g
FW) A
High Py = 0.2233x + 49.846
R2 = 0.3694, P = 0.003
Low Py = 0.3951x + 35.849
R2 = 0.3948, P = 0.016
0
25
50
75
100
125
30 45 60 75 90 105Growth angle (degrees from vertical)
Ethy
lene
pro
duct
ion
(ng/
h/g
FW)
B
180
0
1
2
3
4
whorl 1 whorl 2 whorl 3 whorl 1 whorl 2 whorl 3
Deep genotype Shallow genotype
Gro
wth
rate
(cm
/day
)
low P low P + MCPhigh P high P+MCP
Figure 5.17. Effect of MCP on growth of basal roots of a deep genotype (B98311) and a shallow genotype (TLP19) in both low P and high P treatments for 24 h immediately after transferring to the growth pouch. Each bar shows mean of basal roots of 5-7 plants ±SE.
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Table 5.3. ANOVA of endogenous ethylene production as affected by genotype, phosphorus, IAA treatment or whorls.
Table 5.6. Free IAA in common bean basal roots of seedlings of a deep genotype (RIL7) and a shallow genotype (RIL57) grown in low phosphorus nutrient solution. The reported values are the mean and S.E. of three samples each containing 117 to 280 mg of basal root tissue.
Table 5.7. Free IAA in common bean basal roots of seedlings of a shallow genotype (RIL57) grown in low (Low P) and high (High P) phosphorus nutrient solution. The reported values are the mean and S.E. of three samples each containing 117 to 240 mg of basal root tissue.
Table 5.8. ANOVA of endogenous free IAA from a shallow genotype (RIL57) as affected by ethylene treatment, phosphorus or whorls (1 and 3).
Free IAA (ng/g FW) Free IAA (ng/basal root)
Effect DF F-value P-value F-value P-value
Phosphorus 1 0.009 0.926 0.172 0.684
Ethylene 1 1.229 0.284 20.519 <0.001
Whorl 1 1.523 0.235 24.765 <0.001
Phosphorus*Hormone 1 0.058 0.812 0.370 0.552
Phosphorus*Whorl 1 0.003 0.960 0.183 0.674
Hormone*Whorl 1 0.033 0.858 0.033 0.858
CHAPTER 6: SUMMARY OF THE WORK
The overall objective of the research work presented here was to study the root
architecture of common bean plants, with special focus on the basal root growth angle in
response to gravity in concert with various environmental cues like phosphorus and
endogenous hormonal signals such as ethylene and auxin. Basal roots of common bean
together with the primary root form the scaffolding of the entire root system. Basal roots
have been considered as the specialized lateral roots (Zobel 1991) developing from root-
shoot interface. However, we found that the basal roots emerge from root-shoot junction,
the anatomy of which displays typical shoot anatomy (Figs. 5.15 A-C), while the region
just below the basal root emergence zone displays root anatomy (Figs. 5.15 D-F).
Therefore, basal roots resemble adventitious roots although they appear in 4 xylem files
(tetrarch) like lateral roots.
Gravitropism does not necessarily mean vertical upward growth of shoots and
vertical downward growth of roots. While various reports exist on the root gravitropism,
they focus mainly on primary roots of Arabidopsis, maize, rice etc. Basal roots of
common bean exhibit plagiogravitropic growth i.e. grow at a predetermined set-point
angle other than 0° or 180° with respect to gravity. However, this angle of growth
changes with time. The growth angle of basal roots is a primary determinant of the roots
with soil depth which impacts phosphorus acquisition efficiency (Bonser et al. 1996; Ge
et al. 2000; Liao et al. 2001). Common bean genotypes vary substantially in the growth
angle of basal roots (Liao et al. 2004) and by altering their growth angles, the plants are
better adapted to nutrient limited environment like low phosphorus availability (Bonser et
al. 1996; Liao et al. 2001; Ho et al. 2005). Shallow basal roots not only increase topsoil
exploration, but also produce less intraplant and interplant competition for phosphorus
which are beneficial under conditions of non-uniform availability of phosphorus in soil
(Ge et al. 2000; Lynch and Brown 2001).
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The overall theme of this thesis research is to address the regulation of
plagiogravitropic growth of basal roots by genotypic, physiological and environmental
variations. A detailed study of the genetic and physiological basis of the basal root traits
which account for efficient phosphorus acquisition would increase the scope for selection
and breeding of crops with improved adaptation to low phosphorus availability (Lynch
1998). Although it may seem that the shallowness of the basal roots is correlated with
inhibition of basal root growth, we observed that even with fixed root length there is a
significant difference in the BRGA of shallow and deep genotypes (Appendix Fig. 2.10).
With a view to attain our objective, we first investigated the role of ethylene in
modulating the growth angle in interaction with phosphorus availability. Secondly, we
focused on the more detailed study of localized growth and curvature of basal roots by
kinematic approach using semi-automated image analysis software. We also measured
root growth velocity and diameter as functions of distance from the root tip and time. In
addition, the kinematic method was employed in investigating how the elongation and
curvature of basal roots are affected by phosphorus treatments, and application of
exogenous ethylene and ethylene action inhibitor, 1-methylcyclopropene (MCP). The
final project presented in this thesis is based on the subject of exploring the cross-talk
between auxin and ethylene in regulation of plagiogravitropic growth in response to low
phosphorus availability.
The secondary roots of other plant species are gravitropic (Yamashita et al. 1997;
Kiss et al. 2002; Mullen and Hangarter 2003). Similarly basal roots of common bean
genotypes are also gravitropic (Walk 2005) and the genotypes differ in the
graviresponsiveness. Moreover, phosphorus and ethylene were hypothesized to regulate
growth angle of basal roots leading to the production of shallow root system (Bonser et
al. 1996; Zhang 2002). The work presented in chapter 2 is the continuation of our
hypothesis that ethylene might play a role in regulating genetic, positional and nutrient
induced variation of growth angle of basal roots. The results of this project indicate that
ethylene might act as a modulator of root responses to nutrient availability. In addition,
ethylene perception may be an important factor in the response of basal roots to low
phosphorus availability (Lynch and Brown 1997). Moreover our study shows that
187
position of emergence of basal roots from root-shoot junction plays a key role in
determining the non-vertical orientation of basal roots. This study has important
implications where basal roots from upper whorls would explore upper soil horizon by
becoming shallower, while roots from lower whorls less responsive to ethylene maintain
deeper growth angle and would explore different soil domains. This dimorphic root
architecture would be beneficial in acquisition of limited nutrients like phosphorus and
water from soil minimizing the competition among the roots of an individual plant.
Chapter 3 of my dissertation work describes the kinematic approach using semi-
automated computer-aided image analysis program, KineRoot, used for measuring
localized growth of basal roots and curvature, while chapter 4 focuses on the results and
conclusions made from the experimental work using that technique. We developed a
semi-automated way to study the spatio-temporal patterns of root growth of bean in a
reliable way while reducing user interventions to allow large scale experiments. In this
project we studied the plagiogravitropic growth of thicker rooted species like common
bean. The primary difficulty in studying bean is that epidermal cells are invisible
resulting in images of roots devoid of any trackable patterns. Basal roots were sprinkled
with graphite particles randomly, while the KineRoot program was used to track the
displacement of the patterns of the graphite particles over space and time using a number
of algorithms from the digital images taken by time lapse photography over a period of 4-
6 h. The tracking algorithm also took advantage of the color difference between the root
and the background for higher accuracy and reliability. The new software enables us to
measure the local root growth, diameter, and root midline which was used in calculating
root curvature. In addition, the program was able to produce growth velocity data with a
high degree of accuracy and consistency. Spatio-temporal study of root growth is
beneficial for characterizing the root growth accurately.
Chapter 4 of the thesis aims to study the growth and curvature of basal roots of
common bean using the image-analysis program KineRoot. We identified and measured
the local patterns of root growth and graviresponding zones of the basal roots,
investigated the velocity profiles within these zones and determine how these zones are
affected by low phosphorus availability and ethylene treatment. We observed that basal
188
roots accelerate growth rate of the upper whorls at the cost of lower growth rate in lower
whorls in response to low phosphorus availability. Apart from root growth, one of the
most important aspects of this study was to characterize the bending of the basal roots
which leads to graviresponse and reflects shallowness or deepness of basal roots. Root
curvature results from differential growth between upper and lower edges of the root.
Therefore study of spatio temporal patterns of differential growth ratio of a growing root
allows identification and measurement of root bending zones and bending amount. Our
results show that ethylene and MCP treatments do not alter local root curvature, but alters
the span and duration of the bending of the root upward or downward which causes the
root to respond to gravity differently under different treatments and thereby produce
shallow and deep roots respectively. The results from this study show new aspects of
plagiogravitropic response of basal roots which has not been observed before.
Chapter 5 of the thesis focuses on the potential interaction of auxin and ethylene
in regulating the plagiogravitropic curvature and growth of basal roots. Our results
support the hypothesis that auxin-ethylene interaction regulates growth angles which are
also dependent on phosphorus availability. For this project we measured free auxin
content endogenously present in the basal roots and observed both higher auxin content
and higher sensitivity to auxin for root growth in basal roots of lower whorls than upper
whorls. In addition, we showed that more radio-labeled IAA transported to upper whorls
compared to lower whorls. Measurement of growth angles show that both ethylene and
auxin make the basal roots shallower. Our results show that auxin concentration in
controls is near optimal. Application of aminoethoxyvinylglycine (AVG) or MCP
together with exogenous IAA increases root growth and reduces shallowness in
phosphorus sufficient conditions. However, AVG and MCP do not reverse IAA-
inhibition of growth in low phosphorus. These results point to a phosphorus dependent
interaction between ethylene and auxin in regulation of elongation, but a phosphorus-
independent interaction for control of growth angle. In addition, our results show that
under low P treatment, ethylene inhibitors like AVG or MCP may have more effects on
ethylene rather than auxin resulting in root growth inhibition, whereas in high P treatment
ethylene inhibitors might affect the free IAA content resulting in increased root growth.
189
However, we did not check the second possibility. It would be worthwhile if this
possibility can be checked to have better understanding of the role of ethylene-auxin
interaction in controlling root elongation under different phosphorus availability.
However, there are several areas of future work which can be pursued to have a
more detailed study of the effects of auxin-ethylene interaction on plagiogravitropic
growth of basal roots. First of all, longitudinal sectioning of basal roots might be done to
analyze the auxin redistribution in the graviresponding basal roots which would be
beneficial for verifying the role of auxin gradient in regulating the graviresponse of basal
roots. Moreover, analysis of conjugated auxin in addition to free IAA content could be
carried out to estimate total IAA content which is contributed by both free IAA and
conjugated auxin inside the basal roots. While this report focuses on the hormonal effect
on growth and BRGA of basal roots at a very early stage, future study could explore the
effects of auxin and ethylene at a later stage of root growth and curvature. Greenhouse
experiments would be essential for the study of older bean basal roots since the basal
roots grown in pouch always become deeper after 5-6 d due to lack of space in the pouch,
resulting in the absence of difference of BRGA between shallow and deep genotypes.
My thesis research on plagiogravitropic growth of basal roots of common bean
identifies a new aspect of basal root growth in terms of position of origin i.e. whorls. It
shows that apart from all other factors such as genotypic variations, hormones,
phosphorus availability etc., the whorl from which basal roots emerge is one of the most
important determinants of root depth. Roots also vary in their responses to gravity based
on whorl position. Root depth is strongly responsive to exogenous ethylene, but weakly
correlated with endogenous ethylene production. Furthermore our kinematic study
indicates adaptive behavior of basal roots under phosphorus stress. This is the first time
kinematics has been used to analyze plagiogravitropic growth. We show that the change
in basal root depth is not a result of changes in root curvature; rather it is a result of
changes in time and span of upward vs. downward bending of the roots. We also show
that auxin makes basal roots shallower but auxin response is regulated by ethylene which
changes auxin sensitivity and auxin content of the basal roots.
190
REFERENCES
Bonser AM, Lynch J, Snapp S (1996) Effect of phosphorus deficiency on growth angle of
basal roots in Phaseolus vulgaris. New Phytologist 132, 281-288.
Ge Z, Rubio G, Lynch JP (2000) The importance of root gravitropism for inter-root
competition and phosphorus acquisition efficiency: results from a geometric
simulation model. Plant & Soil 218, 159-171.
Ho MD, Rosas JC, Brown KM, Lynch JP (2005) Root architectural tradeoffs for water
and phosphorus acquisition. Functional Plant Biology 32, 737-748.
Kiss JZ, Miller KM, Ogden LA, Roth KK (2002) Phototropism and gravitropism in
lateral roots of Arabidopsis. Plant and Cell Physiology 43, 35-43.
Liao H, Rubio G, Yan XL, Cao AQ, Brown KM, Lynch JP (2001) Effect of phosphorus
availability on basal root shallowness in common bean. Plant and Soil 232, 69-79.
Liao H, Yan XL, Rubio G, Beebe SE, Blair MW, Lynch JP (2004) Genetic mapping of
basal root gravitropism and phosphorus acquisition efficiency in common bean.
Functional Plant Biology 31, 959-970.
Lynch J (1998) The role of nutrient efficient crops in modern agriculture. Journal of
Crop Production 1, 241-264.
Lynch J, Brown KM (1997) Ethylene and plant responses to nutritional stress.
Physiologia Plantarum 100, 613-619.
Lynch JP, Brown KM (2001) Topsoil foraging - an architectural adaptation of plants to
low phosphorus availability. Plant and Soil 237, 225-237.
Mullen JL, Hangarter RP (2003) Genetic analysis of the gravitropic set-point angle in
lateral roots of Arabidopsis. Space Life Sciences: Gravity-Related Processes in
Plants 31, 2229-2236.
Walk TC (2005) 'Variation in root architecture of common bean and effects on
phosphorus acquisition.' PhD thesis, (Pennsylvania state University, PA).
Yamashita M, Takyu T, Saba T (1997) Gravitropic reaction in the growth of tea roots.
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Zhang YJ (2002) 'Ethylene and phosphorus responses in plants.' PhD thesis,
(Pennsylvania State University, PA).
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Zobel R (1991) Root growth and development. In 'The Rhizosphere and Plant Growth.'
(Eds Keister DCregan P) pp. 61-71. (Kluwer: Dordrecht, The Netherlands).
VITA
PARAMITA BASU
EDUCATION
• PhD Plant Physiology, Pennsylvania State University, 2006 • M.Sc. Botany, Calcutta University, Kolkata, India, 1997 • B.Sc Botany, Calcutta University, Kolkata, India, 1994
AWARDS AND HONORS
• Thomas Walter Memorial Scholarship, Penn State University 2002-2005. • 3rd place in Twentieth Annual Graduate Exhibition, Penn State University 2005,
University Park, PA, USA. • Graduate Student Travel awards, College of Agricultural Sciences, Penn State
University, 2005, University Park, PA, USA. TEACHING EXPERIENCE
• Lecturer in Vidyasagar College for Women, Calcutta University, India 1997-1999. • Teaching Assistant for Conservation Biology, Penn State University, Fall 2001. • Teaching Assistant for Plant Ecology, Penn State University, Fall 2002. • Teaching Assistant for Plant Nutrition Lab, Penn State University, Spring 2004. • Teaching Assistant for Post Harvest Physiology, Penn State University, Spring 2005 SELECTED JORUNAL PUBLICATIONS • Paramita Basu, Yuan-Ji Zhang, Jonathan P. Lynch, and Kathleen M. Brown.
Genetic, positional and nutritional regulation of root plagiogravitropism modulated by ethylene. To be submitted to Functional Plant Biology.
• Paramita Basu, Anupam Pal, Jonathan P. Lynch, and Kathleen M. Brown. Kinematic analysis of root growth and gravitropism using semi-automated image analysis. In Preparation.
• Paramita Basu, Anupam Pal, Jonathan P. Lynch, and Kathleen M. Brown. Growth and curvature of basal roots of common bean (Phaseolus vulgaris L.) analyzed using kinematic approach. In Preparation.
• Paramita Basu, Jurgen Engelberth, Jonathan P. Lynch, and Kathleen M. Brown. Hormonal regulation gravitropic growth of basal roots – a cross-talk between ethylene and auxin. In Preparation.