Geometric analysis of Arabidopsis root apex.

Emilio Cervantes1* and Angel Tocino2

Corresponding autor (*): Departamento de Produccin Vegetal. IRNASA-CSIC.
Salamanca. Apartado 257. Salamanca. Spain. ecervant@usal.es. Tel.: 34 923
219606. Fax: 34 923 219609

Geometric analysis of Arabidopsis root apex reveals a new aspect of the

ethylene signal transduction pathway in development.
Emilio Cervantes1 and Angel Tocino2
1. Departamento de Produccin Vegetal. IRNASA-CSIC. Salamanca. Apartado
257. Salamanca. Spain. ecervant@usal.es. Tel.: 34 923 219606
2. Departamento de Matemticas. Universidad de Salamanca. Plaza de la
Merced, 1. 37008 Salamanca. Spain. bacon@usal.es.

Summary
Ethylene is structurally the simplest phytohormone and regulates multiple
aspects of plant growth and development. Its effects are mediated by a signal
transduction cascade involving receptors, MAP kinases and transcription
factors. Many morphological effects of ethylene in plant development, including
root size, have been previously described. In this article a combined geometric
and algebraic approach has been used to analyse the shape and the curvature
in the root apex of Arabidopsis seedlings. The process requires the fitting of
Bzier curves that reproduce the root apex shape and the calculation of the
corresponding curvatures. The application of the method has allowed to identify
significant differences in the root curvatures of ethylene insensitive mutants
(ein2-1 and etr1-1) with respect to wild-type Columbia.
Key words: Arabidopsis, Bzier curve, curvature, development, ethylene, root
apex.

Introduction
The Arabidopsis root has been used in recent years as a unique model system
to study cell differentiation during development. In the embryonic root a group of
four central cells in the quiescent center is sourrounded by distinct initial cells.
This group is the origin of the different cell types including the columella rootcap, cortical and endodermal cell files, epidermis, and the steele (pericycle and
vascular tissue) (Schiefelbein and Benfey, 1994; Schiefelbein et al., 1997).
Thus, in a reduced number of cell layers, differentiation goes from the nondividing and non-differentiated status in the quiescent center to cells
differentiated in distinct cell types, passing through dividing non-differentiated
meristematic cells. The molecular basis of cell differentiation involves the
coordinated expression of genes encoding key regulatory proteins such as
scarecrow, a member of the GRAS family of DNA binding proteins (Sabatini et
al., 2003). Hormonal regulatory networks control the expression of these genes
(Fu and Harberd, 2003).
During seed imbibition, embryonic root cells take water and increase their
size leading to germination. After radicle protrusion some cells begin to divide.
Once the developmental program is initiated, root shape will be established by
the coordinated action of their cells in response to hormones and environmental
conditions (Beemster et al., 2003). As it is well known, variables measuring
characteristics (e.g., root length, width or root hair number) which depend on
the additive effect of many low intensity factors follow normal distributions.
Ethylene, the simplest structurally known plant hormone, is produced in
plants in two steps from the amino acid methionine via SAM (S-adenosyl
methionine) and ACC (1-aminocyclopropane-1-carboxylic acid) by the
consecutive action of two enzymes: ACC synthase and ACC oxidase. Ethylene
affects diverse aspects of plant development ranging from germination and
seedling morphology to fruit ripening and senescence (Abeles et al., 1992).
Historically, the effects of ethylene on plant development were first described in
etiolated pea seedlings (Neljubov, 1901). They consist in swelling of the
hypocotyl , growth inhibition in the root and in the hypocotyl and an exaggerated
hook curvature. These characteristics of seedlings grown in the dark in the
presence of ethylene were called the triple response and have been useful in
the identification of mutants in the ethylene biosynthesis and signal transduction
pathways (Guzmn and Ecker, 1990). Other effects of ethylene in development
and in the modulation of several aspects of plant form include the response to
physical stress, leaf abscission, flower development and sex determination (for
a review see Dolan, 1997).
In Arabidopsis, ethylene has a remarkable effect on root development: it
inhibits elongation and promotes radial expansion. Similar effects are obtained
with ACC, the ethylene precursor. Treatments with ethylene and ACC are
effective in inducing root hairs (Masucci and Schiefelbein, 1996; Pitts et al.,
1999; Tanimoto et al., 1995). The response is rapid: changes in cell elongation
and the induction of root hairs can be observed after incubation of minutes (Le
et al., 2001). With longer incubation times, ethylene affects the overall root
shape, resulting in decreased root length and increased width. Thus, ethylene
may strongly influence root shape at early developmental stages. Treatment of
wild-type seedlings with ethylene results in morphological changes that are

similar to the phenotypes of ethylene over-producing (eto1-1) (Chae et al.,

2003; Woeste et al., 1999), or constitutive triple response mutants (ctr1-1)
(Kieber et al., 1993). Treatment of wild-type seedlings with ethylene action
inhibitors result in a morphology that resembles ethylene-insensitive mutants
(etr1-1, ein2-1) (Bleecker et al.,1988; Roman et al., 1995). Thus both groups
represent two extreme patterns of development with opposite phenotypes: short
and thick roots with long root hairs in the case of eto1-1 and ctr1-1 ; and long,
thin roots with small root hairs dispersed through the root in the case of etr1-1
and ein2-1.
Ethylene receptors form a protein family (ETR1, ERS1, ETR2, EIN4 and
ERS2) which interacts with other regulatory proteins. For example, ETR1
regulates CTR1, a MAP kinase kinase kinase similar to RAF (Clark et al., 1999;
Huang et al., 2003; Kieber et al., 1993; Ouaked et al., 2003). Constitutive
dominant etr1-1 mutants have a phenotype of ethylene insensitivity, with longer
hypocotyls, longer roots and shorter root hairs than the wild-type in the
presence of ethylene (Bleecker et al., Hall et al., 1999; Masucci and
Schiefelbein, 1996).
In a further attempt to investigate the effects of the ethylene in root
development, we describe in this work a method for the analysis of shape and
curvature in roots of Arabidopsis seedlings. The results show differences
between the ethylene insensitive mutant lines (etr1-1 and ein2-1) and the wildtype Columbia, suggesting that the ethylene signal transduction pathway is
involved in the determination of curvature in the root apex . The method can be
applied in a short time after radicle protrusion in germination, before other
developmental differences are detected.
Materials and Methods
Plant material
Seeds of Arabidopsis thaliana cv Columbia (col) and the mutant genotypes
eto1-1 (eto) (Chae et al., 2003; Woeste et al., 1999), ein2-1 (ein) (Guzmn and
Ecker, 1990) and etr1-1 (etr) (Chang et al., 1993) were imbibed in 1% wateragar plates and stored for three days at 4 oC to allow cold stratification.
Afterwards, the plates were incubated in a growth chamber under a light/dark
cycle (18 hours light, 25 oC; 6 hours dark, 20 oC). After 24 and 48 hours of
incubation, seedlings were carefully collected with forceps and deposited
individually in microscope slides with a drop of water and covered with coverslides for their observation.
Microscopic examination and image analysis.
The slides were observed with transmitted light under an inverted microscope
(Leica DMIRB) with an objective of 20x. Images of the roots were taken and
stored for their analysis.
The program AnalySIS was used to obtain data from the images; the
process is shown schematically in figure 1. A grid of 10 microns width was
drawn over the root image, and with the aid of the grid, a series of
approximately 20 points was regularly marked troughout the root outline. The

coordinates of every point marked were provided by AnalySIS . In this way, for
each image, a data series of the form (xi, yi) were obtained. These data points
were used to create a function representing the curve that approximates the
shape of the root outline.
Curve approximation
Since in this work we focus on the shape of the root, traditional methods forcing
the curve to pass throughout the points are inappropriate. With these fitting
methods, the approximated curve is close to the real shape (in L 2 or L
sense, for example), but oscillates around it (Yang, 2001). So, we have chosen
Bzier curves to approximate the shape of the roots, and curvature as a
measure of shape fidelity.
Bzier curves (Bzier, 1968; Gordon et al., 1974) do not pass throughout
data points (except the first and the last ones), but data points are used as
control points. The multiplicity of data points can be used to improve the fitting.
Bzier curves have been widely used in computer drawing (Rogers and Adams,
1989). They have suitable features for our purposes: 1) Bzier curves have no
great oscillations around the points; 2) they follow the polygonal lines joining the
points and conserve the convexity, i.e. spurious inflection points do not appear;
3) their equations are given by polynomials, and then, are easy to handle (with
a computer code).
From a series of arranged data pi = (xi, yi), i=0,...n, the parametric Bzier
curve is obtained (see e.g. Risler, 1992) as X(t)=(x(t),y(t)) with
n

x(t) = xi Bi ,n (t ) ,
where t [0,1] and

i =0

y(t) = y i Bi ,n (t )
i =0

n
Bi ,n (t ) = (1 t ) n i t i
i = 0,...n,
i
represent the n+1 Bernstein polynomials (Lorentz, 1986). So, x(t) and y(t) are
polynomials of degree n and the parameter t, which varies in the interval [0,1],
expresses the percentage of the total curve length traversed. Notice that X (0)
is the initial point (x0, y0) and X (1) is the final point (xn, yn).
Geometric analysis
The curvatures of the adjusted Bzier curves were calculated from their
parametric equations (x(t), y(t)) by means of formula (DoCarmo, 1976) :
x' (t ) y ' ' (t ) x' ' (t ) y ' (t )
,
t [0,1] .
(t) =
( x' (t ) 2 + y ' (t ) 2 ) 3 / 2
Curvature measures the rate at which the unit tangent vector is changing with
respect to arc length. So, all graphical representations of the curvatures of
Bzier curves approximating root outlines have similar bell-shaped form (see
Figure 2b). The values of the curvature near to the extremes of the interval are
low, since the curve is similar to a line in their proximities. When the parameter
goes up to the root tip, curvature increases. The maximum value of the
curvature is attained on the apex vertex. Bzier curves fitting pointed roots lead

to tall and narrow bells, whereas those corresponding to blunt roots give
flattened bells.
Once calculated the maximum value of the curvature function for each root, a
variable called APEXCUR containing these values was created. For each root
this variable measures curvature value in the root tip.
A Mathematica code which, from a set of input data points, gives the Bzier
curve, its graphical representation (figure 1a), the graphic of a curve
representing the curvatures (figure 1b), and the maximum value of the curvature
has been written out and is available upon request at the authors e-mail
addresses.
Statistical analysis
We are interested in the homogeneity of populations col, ein and etr with
respect to variable APEXCUR. Thus, a sample of six root images for each
population (24 hours after imbibition) was analysed and, following the process
described above, their APEXCUR values were obtained. Main statistics of
variable APEXCUR were calculated.
ANOVA procedure (Fisher, 1935; Bliss, 1967) was used to test the significance
of the difference between the three populations. The null hypothesis H0 was that
they represent samples from the same normal distributed population, i.e.
populations col, ein and etr are normally distributed and their parameters (mean
and variance) coincide. We tested H0 in a level of significance = 0.01 .
To study if data have significant difference in dispersion, i.e. to test the variance
homogeneity, a Levene test was applied (Levene, 1960). In this case, the null
hypothesis H1 was that the samples come from normal distributed populations
with the same variance (level of significance = 0.01 ).
If H0 is rejected after ANOVA test and H1 is accepted in Levene test, then we
must conclude that populations col, ein and etr differ in their means but not in
their variances with respect to variable APEXCUR. In this case, to find which
pair or pairs of populations (col-ein, col-etr, ein-etr) give this heterogeneity,
many multiple comparison tests are available. In our case, a Scheff multiple
comparison test (Scheff, 1953) was carried out.
The analysis was repeated with an equal size sample of seedlings taken at 48
hours after imbibition.
Results
For each image, a Bzier curve fitting the root outline and the corresponding
curvature function, were obtained. Figure 3, which contains one example of
each genotype, illustrates this process. Afterwards, APEXCUR values of this
sample were calculated. Main statistics of variable APEXCUR are summarized
in Table 1.
The significance of the difference between root tip curvatures of groups col, ein
and etr was tested by analysis of variance. From the F value obtained in Levene
test (Table 2), there is insufficient evidence to reject the hypothesis that the
variances are equal. So we accept that our three samples represent normal
populations with the same variance.
The obtained value F=17.586 in ANOVA test (Table 3) corresponds to a
probability lower than P=0.001. On this basis, it was concluded that the groups

col, ein and etr have different means with a degree of reliability higher than
99.9%.
Finally, Scheffes multiple comparison test gives that a pair of populations
have unequal means if their sample means i , j fulfill the inequality
i j 0.0193 F1 ; 2,12 .
With = 0.01 , the value of the term on the right is 0.366 and we conclude that,
with this level of significance, col population differs from ein and etr in their
curvatures. Obviously, there is no evidence to reject the hypothesis that the
populations ein and etr have the same mean.
Figure 4 contains the twelve curvature functions corresponding to col and etr
samples (green, col functions; red, etr). It shows the differences between the
curvature functions for both populations and, in particular, the lack of
overlapping between their APEXCUR values.
The analysis was repeated taking different sets of points with similar results.
Also similar conclusions were obtained when the analysis was repeated for a
sample consisting of six seedlings for each genotype at 48 hours after
imbibition.
On the other hand, seedlings of the mutant genotype eto1-1 were included in a
similar analysis. The mean curvature value for eto1-1 was 1.965. The values
were included between 1.65 and 2.47 and the standard deviation for this
genotype was 0.29. Scheffs test revealed that curvature values for eto1-1 are
not statistically different from the values corresponding to the other genotypes
under study.
Discussion
Plant shape results from the coordinated growth of many thousands of cells.
From fertilization and early embryonic development, very efficient comunication
mechanisms are established involving the different organs of a plant. During
plant development, a multiplicity of mechanisms exert their effects both in short
(i.e. between neighbour cells) and long distance (e.g. hormonal signals
transmitted throughout the plant). Recently, root length has been shown to be
partly controlled by shoot produced phytohormones and regulated by the
coordinated action of gibberellins, auxins and ethylene (Achard et al., 2003;
Cervantes, 2003; Fu and Harberd, 2003).
In a recent article, Rolland-Lagan et al. (2003) proposed that growth is
governed by three factors: growth rate, anisotropy and direction. In root growth
the main axis determines the direction and, after germination, growth is
predominantly driven by the gravitropic stimulus. Major variations may concern
rate of growth and anisotropy. Changes in anisotropy are related to variability in
radial expansion. Ethylene is known to inhibit growth and to promote radial
expansion in roots (Dolan and Davies, 2004). Thus, this phytohormone
regulates crucial aspects in root morphogenesis allowing the adaptation of root
growth to rapid responses in environmental changes (Le et al., 2001).
From a practical point of view it may be convenient to analyse
developmental aspects that: 1) may be observed at early developmental stages.
2) can respond to known signals or environmental factors, and 3) can be
expressed in mathematical terms. Growth characteristics observed at early
stages of development are the consequence of interactions among a lower

number of cells and organs involving, in general, simpler signal-response

networks. The development of mathematic formulae is essential in the accurate
description of developmental phenotypes.
Recently, the application of image analysis resulted in the precise
description of growth in the root apical zone including the meristem and
elongation zone (van der Weele et al, 2003).
To analyse the shape of the root in the apex we have used root images
from transmitted light microscopy and obtained adjusting curves for them. Their
curvature is a descriptive measure of root shape.
The method has been applied to compare etr1-1, ein2-1, eto1-1 and wildtype Columbia genotypes. Differences in root morphology among these
genotypes were described previously and are related to root length and root
hair size and shape (Bleecker et al., 1988; Guzmn and Ecker, 1990; Roman et
al., 1995; Hall et al., 1999; Masucci and Schiefelbein, 1996).
The application of the method has allowed to identify differences in the
root curvatures of ethylene insensitive mutants (ein2-1 and etr1-1) with respect
to wild-type Columbia that are statistically significant at a p=0,01 confidence
level. The study of curvatures here presented was done on seedlings at 24 and
48 hours after germination and can be applied at earlier times after germination,
or in embryos before other major differences in growth parameters, e.g. root
length, are visible.
The activity of ETR1 protein as ethylene receptor has been demonstrated
(Shiu and Bleecker, 2001). The current model of ethylene action admits that the
wild-type ETR1 protein activity is negatively modulated by the binding of
ethylene, whereas the mutant allele etr1-1 encodes a protein unable to bind
ethylene, and thus constitutively activated (Kende, 2001). EIN2 is related to
metal transporters of the NRAMP family (Hirayama and Alonso, 2000). EIN2 is
downstream of ETR1 and the phenotype of ein2-1 mutant is of ethylene
insensitivity (Romn et al., 1995), thus resembling etr1-1 phenotype (Bleecker
et al., 1988). ETR1 protein is active in the absence of ethylene and its activity is
modulated by the phytohormone. Thus, in theory, aspects not controlled by
ethylene can be different between wild-type Columbia and etr1-1 genotypes.
This situation may occur when ETR1 is active in a developmental stage in
which the hormone is absent or the receptor is unable to bind the hormone (e.g.
due to physical isolation).
In this article, we have defined and expressed mathematically the
curvature of the root apex, a developmental characteristic detectable early upon
seed imbibition or even in embryonic roots. Statistical analysis has shown that
root tip curvature values are smaller in ethylene-insensitive mutants (etr1-1 and
ein2-1) when compared with wild-type seedlings. Curvature is, thus, a new
phenotypic feature that may be useful to study the cellular basis of root growth.
Different curvature values may result from the effect of mutations in hormone
sensing pathways (as it is shown here) or in response to environmental factors.
We have demonstrated that curvature is dependent on the activities of
ETR1 and EIN2 proteins in the ethylene signal transduction pathway. This does
not necessarily imply that curvature is ethylene dependent. Our results with
eto1-1 suggest that, at least at early root development stages, curvature does
not respond to increased ethylene levels.

Although ethylene-insensitive mutants have been obtained in screening

experiments designed to disrupt the ethylene signalling pathway, the pathway
may be active in absence of the phytohormone and may regulate important
processes related with cell growth and structure. The analysis of curvature
values of Columbia seedlings treated with ethylene inhibitors can give more
data to illustrate whether this characteristic is controlled by ethylene or is
regulated by ETR1 protein activity, independently of this phytohormone.
It would be interesting to observe if ethylene insensitive mutants have
lower curvature values at earlier times after germination, or in their embryos.
Another question is if low curvature is a specific feature for ethylene insensitive
lines or if it is a shared feature with other hormone response mutants. Curvature
analysis in roots of mutants in different signal transduction pathways and in
wild-type seedlings grown under different treatments may give new lights on the
molecular basis of development and hormone signal transduction mechanisms.
These possibilities are currently under investigation. Finally, the tools developed
in this work may be applied to analyse whether curvature values in the root tip
are associated with size and distribution of cells in the root apex. The methods
will be extended and completed by confocal imaging into the third dimension
and the analysis of available Arabidopsis lines tagged with GFP in the cell wall.

Acknowledgements
We thank Jos Javier Martn Gmez for his help in the elaboration of figures.
References
Abeles F, Morgan P, Saltveit M (1992). Ethylene in Plant Biology, San Diego:
Academic press.
Achard P, Vriezen WH, Van Der Straeten D, Harberd NP (2003). Ethylene
regulates Arabidopsis development via the modulation of DELLA protein growth
repressor function. Plant Cell 15 (12), 2816-25.
Beemster, GTS, Fiorani F, Inz D (2003). Cell cycle: the key to plant growth
control? Trends in Plant Science, 8(4), 154-158.
Bzier, PE (1968). How Renault uses numerical control for car body design and
tooling. SAE Congress, paper 680010, Detroit.
Bleecker, A B, Estelle, MA, Somerville C, Kende H (1988). Insensitivity to
ethylene conferred by a dominant mutation in Arabidopsis thaliana. Science
241, 1086-1089.
Bliss, CI (1967). Statistics in Biology (vol. I), McGraw Hill, New York.

10

Cervantes, E (2003). DELLA proteins, keys in plant hormone interaction.

http://update.bmn.com/rsearch/section/record?uid=UPDATE.Cervantes0912200
3104
Chae, HS, Faure, F, Kieber JJ (2003). The eto1, eto2, and eto3 Mutations and
Cytokinin Treatment Increase Ethylene Biosynthesis in Arabidopsis by
Increasing the Stability of ACS Protein. The Plant Cell 15, 545-564.
Chang, C, Kwok, S F, Bleecker, A B, Meyerowitz, E M, (1993). Arabidopsis
ethylene-response gene ETR1: similarity of product to two-component
regulators . Science 262, 539-41.
Clark, K L, Larsen, P B, Wang, X, Chang, C (1998). Association of the
Arabidopsis CTR1 Raf-like kinase with the ETR1 and ERS ethylene receptors.
Proc. Nat. Acad. Sci. USA, 95, 5401-5406.
DoCarmo, MP (1976). Differential Geometry of Curves and Surfaces. Prentice
Hall, New Jersey.
Dolan, L (1997). The role of ethylene in the development of plant form. Journal
of Experimental Botany, 48 (307), 201-210.
Dolan, L, Davies J (2004). Cell expansion in roots. Curr Opin Plant Biol. 7(1),
33-9.
Fisher, RA (1935). The Design of Experiments, Oliver and Boyd, London.
Fu, X, Harberd, N (2003). Auxin promotes Arabidopsis root growth by
modulating gibberellin response. Nature 421, 740-743.
Gordon, WJ, Riesenfeld, RF (1974). Bernstein-Bzier methods for the computer
aided design of free-form curves and surfaces, Journal of the Association of
Computing Machinery, 21, 293-310.
Guzmn P, Ecker JR (1990). Exploiting the triple response of Arabidopsis to
identify ethylene-related mutants. The Plant Cell 2,513-523.
Hall A E, Chen QG, Findell J L, Schaller G E, Bleecker A B (1999). The
Relationship between Ethylene Binding and Dominant Insensitivity Conferred by
Mutant Forms of the ETR1 Ethylene Receptor1 Plant Physiol. 121, 291-300
Hirayama T, Alonso, JM (2000). Ethylene captures a metal! Metal ions are
involved in ethylene perception and signal transduction. Plant Cell Physiol.
41,548-55.
Huang Y, Li H, Hutchison, CE, Laskey J, Kieber JJ (2003). Biochemical and
functional analysis of CTR1, a protein kinase that negatively regulates ethylene
signaling in Arabidopsis. Plant J 33(2) 221-33.

11

Kende, H (2001). Hormone Response Mutants. A Plethora of Surprises. Plant

Physiol 125, 81-84.
Kieber JJ, Rothenberg M, Roman G, Feldmann KA, Ecker JR (1993). CTR1, a
negative regulator of the ethylene response pathway in Arabidopsis, encodes a
member of the raf family of protein kinases. Cell 72(3) 427-41
Le J, Vandenbussche F, Van Der Straeten D, Verbelen J-P (2001). In the Early
Response of Arabidopsis Roots to Ethylene, Cell Elongation Is Up- and DownRegulated and Uncoupled from Differentiation. Plant Physiol. 125, 519-522.
Levene H (1960). Robust tests for equality of variances. Contributions on
Probability and Statistics, ed. I. Olkin, pp. 278-92, Standford University Press,
California.
Lorentz GG (1986). Bernstein Polynomials, Chelsea, New York.
Masucci JD, Schiefelbein JW (1996). Hormones act downstream of TTG and
GL2 to promote root hair outgrowth during epidermis development in the
Arabidopsis root. Plant Cell 8, 1505 1517.
Neljubov D N (1901). Uber die horizontale nutation der stengel von Pisum
sativum und einiger anderen. Pflanzen Beitrage und Botanik Zentralblatt
10,128-139.
Ouaked F, Rozhon W, Lecourieux D, Hirt, H (2003). A MAPK pathway mediates
ethylene signaling in plants. The EMBO Journal 22 (6), 1282-1288.
Pitts RJ, Cernac A, Estelle M (1999). Auxin and ethylene promote root hair
elongation in Arabidopsis. Plant J 16, 553 560.
Risler JJ (1992). Mathematical Methods for CAD. Cambridge University Press,
Cambridge.
Rogers DF, Adams, JA (1989). Mathematical Elements for Computer Graphics,
McGraw Hill, New York.
Rolland-Lagan A-G, Bangham JA, Coen E (2003). Growth dynamics underlying
petal shape and asymmetry. Nature 422, 163-165.
Roman G, Lubarsky B, Kieber JJ, Rothenberg M, Ecker JR (1995). Genetic
analysis of ethylene signal transduction in Arabidopsis thaliana: five novel
mutant loci integrated into a stress response pathway. Genetics 139, 1393-1409
Scheff H (1953), A method for judging all contrasts in the analysis of variance,
Biometrika, 87-104.
Sabatini S, Heidstra R, Wildwater M, Scheres B (2003). SCARECROW is
involved in positioning the stem cell niche in the Arabidopsis root meristem.
Genes & Development 17, 354-358.

12

Schiefelbein JW, Benfey P (1994). Root Development, in: Meyerowitz E.,

Sommerville, C. (Eds), Arabidopsis, Chapter 13, pp. 335-353, Cold Spring
Harbor Laboratory Press.
Schiefelbein JW, Masucci JD, Wang, H (1997). Building a root: The control of
patterning and morphogenesis during root development. The Plant Cell 9, 19891098.
Shiu S-H, Bleecker AB (2001). Receptor-like kinases from Arabidopsis form a
monophyletic gene family related to animal receptor kinases. Proc. Nat. Acad.
Sci. USA, 98, 10763-10768.
Tanimoto M, Roberts K, Dolan L (1995). Ethylene is a positive regulator of root
hair development in Arabidopsis thaliana. Plant J, 8, 943948.
van der Weele CM, Jiang HS, Palaniappan KK, Ivanov V, Palaniappan K,
Baskin T (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 Physiol 132, 1138-1148.
Wang W, Hall AE, O'Malley R, Bleecker AB (2003). Canonical histidine kinase
activity of the transmitter domain of the ETR1 ethylene receptor from
Arabidopsis is not required for signal transmission. Proc. Nat. Acad. Sci. USA,
100, 352-357.
Woeste KE,Ye, C.Kieber J J
(1999). Two Arabidopsis mutants that
overproduce ethylene are affected in the posttranscriptional regulation of 1-1aminocyclopropane-1-carboxylic acid synthase. Plant Physiol, 119, 521-528.
Yang X. (2001). Planar point set fairing and fitting by arc splines. ComputerAided Design, 33, 35-43.
Figure legends
Figure 1. Process of data acquisition. The images of the root apex were
visualized with AnalySIS and points were marked along their outline with the
help of a grid. The program gives the coordinates of the points.
Figure 2. a) Adjusted Bzier curve taking nineteen points throughout the profile
of the root surface. b) Graphical representation of corresponding curvature
values through the length of the curve represented in a).
Figure 3. Root images (left, a), adjusted Bzier curves (top right, b) and their
corresponding graphics showing curvature values (bottom right, c) for
representative samples of: col (above), etr (middle) and ein (below) genotypes.
Bar represents 100 micrometers.
Figure 4. Curvature functions corresponding to Bzier curves for six root
samples of col (green) and etr (yellow) genotypes.

13

Figures

Figure 1

1.5

0.5

0.2

a)

0.4

0.6

0.8

b)

Figure 2

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15.5
15
14.5
14
13.5
13

b)

2
1.5
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c)

0.2

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a)
14
13
12
11
10

b)

10

11

12

13

2
1.5
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c)

0.4

0.6

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a)
9.5
9
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7.5
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6.5
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6.5

b)

7.5

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10

1.5
1.25
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0.75
0.5
0.25

c)

0.2

0.4

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a)
Figure 3

15

3
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0.3

0.4

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0.9

Figure 4

16

Tables
TYPE
col

Mean
2.17450

Cases
6

St. D.
.225846

Var.
.051006

Min.
1.921

Max.
2.509

ein

1.66496

.129154

.016680

1.509

1.848

etr

1.67350

.139406

.019434

1.432

1.847

Total

1.83766

18

.329275

.108422

1.432

2.509

Table 1. Summary statistics for variable APEXCUR

Levene
1.969

DF1
2

DF2
15

Sig.
.174

Table 2. Levene test for homogeneity of variances

ANOVA Table

SS
APEXCUR
* TYPE

Between groups
Within groups
Total

DF

MS

1.021

.511

.436

15

.029

1.457

17

F
17.586

Sig.
.000

Table 3. Results of ANOVA test comparing the means of groups col, ein and etr for variable
APEXCUR.

17