[Plant Signaling & Behavior 4:5, 1-5; May 2009]; 2009 Landes Bioscience

ib
u

1IRNASA-CSIC;

Salamanca, Spain; 2Department of Mathematics; University of Salamanca; Spain

di

Key words: curvature, ethylene, form, free radicals, genome dynamics, morphology, peroxide, shape

st
r

Emilio Cervantes1,* and ngel Tocino2

.D

no
t

of biology during the XIX and early XX centuries include the

Cell Theory, Bchners experiments demonstrating that enzyme
activities occur in cell-free extracts and the general acceptance of
evolution by natural selection with its extension by neo-darwinism,
a modern unifying paradigm that unifies results of genetics and
biochemistry with the darwinian evolutionary schemes. On this
basis, biology is concentrated in the study and analysis of functions
of the organisms rather than in their forms. This may be explained
by historic reasons.
First, biology is the product of institutionalised science, in
which strong economic interests are involved, thus its objectives
have traditionally been directed towards applied results, based
in the understanding of biological functions. Nevertheless, the
accurate description of the objects in nature requires as much
understanding of forms as of functions.
Second, biology is a specialised science, and as such, it tends
to attribute specific functions to their subjects of study: organs,
tissues, cells, molecules. In this way, the organism becomes traditionally diluted and the main questions concerning global aspects
become inoportune, inadequate and even impossible to answer
under the current perspectives. The Cell Theory provides a way to
see the natural objects as formed by units, but it does not provide
means to address the global mechanisms by which cells communicate to configure and shape an individual. Enzymes work in
cell free extracts allowing the investigation of their mechanism of
action, but there is no guarantee that their action is exactly the
same under the experimental conditions that inside the cells of a
living organism. DNA and RNA molecules contain and transfer
genetic information, but it is becoming increasingly clear that their
modifications by other molecules (methylation, acetylation) may
contribute to essential aspects of this function.
The panorama of a highly institutionalized and specialized
science has important consequences in the way biology confronts
and responds to fundamental questions. For example, to the question: How is the shape of an organism regulated? The answer may
refer to the combination of its genome and the environment. But
this, being correct from an academic point of view, may not provide
the best position to open original ways to further analysis. Among
other reasons, because the environment is something extremely
complex to be defined, and the genome is a plastic entity in

La

nd

es

io
s

ci

en
ce

Treatment with hydrogen peroxide has notable effects in the

morphology of the root apex in Arabidopsis seedlings. The result
was described as consisting in two aspects: first, a reduction in
curvature values in the root profile. Second, alterations in size
and shape of the cells in the root cap. Cells of the root cap were
smaller and had higher circularity index (Fig. 1).1 The results of
peroxide treatment were similar to alterations in the root apex
of ethylene insensitive mutants and wild-type seedlings treated
with ethylene inhibitors. This brings new evidence in favour
of the association between ethylene and hydrogen peroxide
signalling that was recently demonstrated in stomatal cells.2
Notable changes in morphology under peroxide treatment were
previously reported3 in other biological systems (Fig. 2). In
the following paragraphs we make emphasis on the need of an
accurate analysis of morphology. This aspect has not received
the attention required in biology, a discipline dominated by
functional analysis. We suggest that the observed morphological
characteristics in the root apex treated with peroxide may be the
manifestation of global processes of adaptation in the organism.
Alternative forms of roots grown in water or in peroxide are
stable situations representing different global configurations that
may have other (genomic, physiological) traits associated. Each
form represents a different mode of adaptation to environmental
change. The accurate description of morphology in organisms,
with particular emphasis in model systems, and their variations
under stress, is needed to identify and understand the basis of
genomic organization and plasticity.

Introduction: Biology Concentrates More in the Study of

Functions than in the Description of Forms

09

Biology is a young science. Although the word biology was

coined in the XVIII century,4 his extended use in book titles does
not occur until the end of the XIX century. The foundations

20

This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly.

Ethylene, free radicals and the transition between stable states in

plant morphology

te
.

Mini-Review

*Correspondence to: Emilio Cervantes; IRNASA-CSIC; Apartado 257; Salamanca,

Spain; Email: ecervant@usal.es
Submitted: 02/16/09; Accepted: 02/17/09
Previously published online as a Plant Signaling & Behavior E-publication:
http://www.landesbioscience.com/journals/psb/article/8201

Plant Signaling & Behavior

2009; Vol. 4 Issue 5

Ethylene, free radicals and plant morphology

te
.

Figure 1. Confocal section of the root of Arabidopsis

seedlings of the line LT16b expressing GFP in the cell
wall (Cuttler et al. PNAS 97, 371823, 2000,38). Left,
control. Right, seedlings treated with 10 mM hydrogen
peroxide.

st
r
di
no
t
o

20

09

La

nd

es

io
s

ci

en
ce

Textbooks and articles on morphology throughout the XX

century represent a small proportion of the total of publications in
biology. The treatise entitled On growth and form,5 by dArcy
Wentworth Thompson is a classical one, and, as such, it appears
quoted in many publications in the field. This book contains the
description of fundamental principles of the discipline such as the
biological importance and scope of the relation surface/volume,
or the application of coordinate-axis modifications in the representation of morphological relations in organisms. In addition, it
includes multiple examples and discussions on diverse topics with
abundant quotations of classical authors (even long un-translated
quotes in greek and latin). The existence of such a classical treatise
is important to establish the bases of the discipline, but paradoxically, this could have had also a negative effect, inhibiting further
developments on biological morphology.
Other books in this area are often the result of advanced
mathematic approaches that, if in some instances may include
original and interesting developments, in others present highly
sophisticated models taking the subject far away from the point
of view that natural objects are observed by the biologist. As an
example of the former, simulation programs for particular aspects
of plant growth were presented by Prusinkewitz and Lindenmayer
(1990).6
In his book, first published in French in 1977, Ren Thom
(1989)7 presents sophisticated models more in the context of
advanced mathematics than in the form of useful resources for
the biologist. In this line, the application of complex mathematics

.D

Classical Approaches to the Analysis of Form in Biology:

Contributions and Drawbacks

ib
u

constant interaction with many undefined environmental factors.

A change is required to compensate this unbalanced situation that tends to favour the study of
function over form in living organisms. In the
following sections we present a brief and subjective
outline of classical and modern approximations to
the study of form in biological objects. Although
the general impression is that morphological analysis needs more attention in biology, it is important
to have at least an overview of the main characteristics, milestones and drawbacks in the study of forms in a discipline
that has been dominated by functional objectives. The accurate
description of forms may be an important way to open new views
on fundamental biological problems.

Figure 2. Erythrocytes. Top, control. Bottom, echinocytes produced by peroxide treatment. Figure 7.1, page 486 from Free radicals in biology and
medicine by Barry Halliwell and John MC Gutteridge edited by Oxford
Science Publications (2001). By permission of Oxford University Press.
www.landesbioscience.com

Plant Signaling & Behavior

Ethylene, free radicals and plant morphology

1370

2438

19

(magnifolia)

Flower
(magniflora)

(magnipetala)

129

52

(rotundifolia)

Flower
(rotundiflora)

(rotundipetala)

1203

11

Adjective: Magni (large) (190)

Applied to (organ)
Leaf

Nr of species

Adjective: Rotundi (round) (1219)

Applied to (organ)
Leaf

Nr of species

Petal

Petal

Petal

te
.

Effects of Ethylene in Cell Morphology and Differentiation

are Related with Free Radicals

io
s

ci

en
ce

in biology has often resulted in exhibitory works more in the line

of demonstrating the high mathematical capacities of the authors
than providing useful methods to approach plant form.
Thus, we are in a rather curious and interesting situation in
which the study of forms in nature, an aspect considered fundamental by naturalists until the XIX century, has become today,
in the time of biology (XX and XXI centuries), often secondary,
sometimes ignored or the subject of specialised and profound
speculation of abstract minds operating far from the way biologists look at the objects of nature. The solution may require new
approximations including original descriptions of the forms of
biological objects and their approximations to geometric shapes
supported by statistical analysis, and the description and application of new useful magnitudes.

ib
u

(parvipetala)

st
r

Flower
(parviflora)

di

Nr of species

(parvifolia)

no
t

Adjective: Parvi (small) (3827)

Applied to (organ)
Leaf

offer new avenues for the investigation of plant genomics, for

example relating genome composition and dynamics with variations in shape in related species, or under different environmental
conditions. The identification of forms resembling geometric
objects and their adequate (reproducible) description and measurement may be a first step. The description of plant morphology
may include increased comparison with geometric objects as well
as the use of magnitudes for their measurement. Variations in the
forms of plants may be associated with global changes representing
the transition between different stable stages corresponding with
physiological and genomic properties.
In addition to length, width, number or proportion of particular organs and structures, new magnitudes can be defined and
measured. Curvature was described as a new magnitude useful in
the description of the root apex morphology.8,9 Maximum curvature values occur in the root apex and are reduced in ethylene
insensitive mutants or in wild-type roots treated with ethylene
inhibitors.8-10 Similarly, a reduction in curvature was observed
as the result of a treatment with 10 mM hydrogen peroxide,1
thus supporting the interaction between hydrogen peroxide and
the ethylene signal transduction pathway that was demonstrated
in stomatal cells,2 and the idea that alterations in free radicals
and ethylene may be able to switch plant development between
different morphological patterns.

.D

Table 1A total of 5,236 plant species bear in their

names adjectives indicative of three simple
morphological characteristics (small, large,
round) applied to one of three organs (leaf,
flower, petal)

Geometrical Shapes: Magnitudes in their Description

20

09

La

nd

es

During centuries, plant species were named and classified

according to morphological characteristics. Thus, the names of many
plant families are related to their forms (Magnoliaceae, Asteraceae,
Campanulaceae, Crucipherae,). Morphological properties are also
used to name the plant species below the family level. Searches in the
IPNI database (International Plant Names Index; http://www.ipni.
org/index.html) with adjectives indicating simple morphological
attributes show how often this occurs. For example, Table 1 reveals
a total of 5,236 plant species whose names contain adjectives related
with only three simple morphological characteristics (small, large,
round) applied to one of three organs (leaf, flower, petal).
Today, when the number of available nucleotide sequences
corresponding to the genomes of plant species has grown exponentially, it is striking to remark how little our knowledge about plant
forms has improved. This paradoxal situation indicates an important drawback: the lack of attention or insufficient description of
the visible aspects (forms) may be limiting the comprehension of
the relationship between plant forms and their genomic composition. Increased attention to the description of plant forms may
3

Ethylene has well-known effects on the size and shape of plant

organs.11 The triple response of legume seedlings to ethylene was
one of the first hormonal effects described in plants.12 The triple
response consists in stem swelling, inhibition of stem elongation,
and exaggerated hook curvature indicating the effects of ethylene
on cell morphology and differential elongation. Ethylene treatments
have an effect on the dimensions of diverse types of plant cells,13,14
as well as on the rate of cell divisions in the quiescent centre of
Arabidopsis root.15 In the root apex of Arabidopsis seedlings, curvature values and cell morphology are altered in ethylene insensitive
mutants in a way that resembles treatments with ethylene inhibitors or with peroxide (Fig. 1).8-10 Thus, ethylene is involved in cell
division, cell shape and differential elongation processes in diverse
types of plant cells.
The ethylene signal transduction pathway is one of the best
known hormonal sensing mechanisms in plants.16 Genes and
proteins involved where termed including the word ethylene in
their names, such as EIN17 (ethylene insensitive) ERS18 (ethylene
response sensor), ETR19 (ethylene resistant). The phenotypes of
their mutants resemble morphologically the effects of growing
the wild-type plants in presence of ethylene or ethylene inhibitors. Interestingly, the ethylene signal transduction pathway may
not be exclusively involved in mediating the response to ethylene.
The ethylene receptors have a GAF domain involved in energy
sensing20 and it has been demonstrated recently that the ETR1
activity is regulated by hydrogen peroxide.2 In addition, ETR2 was
found to be activated during low oxygen stress in Arabidopsis root
cultures,21 a complex physiological response that involves changes

Plant Signaling & Behavior

2009; Vol. 4 Issue 5

Ethylene, free radicals and plant morphology

Acknowledgements

te
.

Supported by Junta de Castilla y Len under the Project

SA071A07. Figure 2 is a reproduction of Figure 7.1, page 486
from Free radicals in biology and medicine by Barry Halliwell
and John MC Gutteridge edited by Oxford Science Publications
(2001). By permission of Oxford University Press.

ib
u

References

.D

no
t

di

st
r

1. Noriega A, Tocino A, Cervantes E. Hydrogen peroxide treatment results in reduced

curvature values in the Arabidopis root apex. J Plant Physiol 2008; In press.
2. Desikan R, Hancock JT, Bright J, Harrison J, Weir I, Hooley R, Neill SJ. A role for
ETR1 in hydrogen peroxide signaling in stomatal guard cells. Plant Physiol 2005;
137:831-4.
3. Halliwell B, Gutteridge JMC. Free radicals in biology and medicine. Oxford University
Press, 3rd ed 1999; 936.
4. McLaughlin P. Naming biology. J Hist Biol 2002; 35:1-4.
5. DArcy Wentworth Thompson. On Growth and form. Cambridge University Press
1917; (1961).
6. Prusinkiewicz P, Lindenmayer A. The algorithmic beauty of plants New York: Springer,
cop. 1990; (imp. 1996) 228.
7. Thom R. Structural stability and Morphogenesis: an outline of a general theory of models.
Addison-Wesley, Redding, Massachusetts 1989; 348.
8. Cervantes E, Tocino A. Geometric analysis of Arabidopsis root apex reveals a new aspect
of the ethylene signal transduction pathway in development. J Plant Physiol 2005;
162:1038-45.
9. Tocino A, Cervantes E. Curvature analysis reveals new functions for the ethylene signaling pathway in the determination of the shape of seeds and roots. Plant Signal Behav
2008; 3:362-6.
10. Noriega A, Cervantes E, Tocino A. Ethylene responses in Arabidopsis seedlings include
the reduction of curvature values in the root cap. J Plant Physiol 2008; 165:960-6.
11. Dolan L. The role of ethylene in the development of plant form. J Exp Bot 1997;
48:201-10.
12. Neljubow D. Ueber die horizontale Nutation der Stengel von Pisum sativum und einiger
anderer. Pflanzen Beith Bot Zentralbl 1901; 10:128-39.
13. Le J, Vandenbussche F, Van Der Straeten D, Verbelen JP. In the early response of
Arabidopsis roots to ethylene, cell elongation is up and downregulated and uncoupled
from differentiation. Plant Physiol 2001; 125:519-22.
14. Le J, Vandenbussche GF, Van der Straaten D, Verbelen JP. Position and cell typedependent microtubule reorientation characterizes the early response of the Arabidopsis
root epidermis to ethylene. Physiol Plant 2004; 121:513-22.
15. Ortega-Martnez O, Pernas M, Carol RJ, Dolan L. Ethylene modulates stem cell division
in the Arabidopsis thaliana root. Science 2007; 317:507-10.
16. Ecker JR. The ethylene signal transduction pathway in plants. Science 1995;
268:609-772.
17. Hall AE, Bleecker AB. Analysis of combinatorial loss-of-function mutants in the
Arabidopsis ethylene receptors reveals that the ers1;etr1 double mutant has severe developmental defects that are EIN2 dependent. Plant Cell 2003; 15:2032-41.
18. Hua J, Chang C, Sun Q, Meyerowitz EM. Ethylene insensitivity conferred by
Arabidopsis ERS gene. Science 1995; 269:1712-4.
19. Bleecker AB, Estelle MA, Somerville C, Kende H. lnsensitivity to ethylene conferred by
a dominant mutation in Arabidopsis thaliana. Science 1988; 241:1086-9.
20. Aravind L, Ponting CP. The GAF domain: an evolutionary link between diverse phototransducing proteins. Trends Biochem Sci 1997; 22:458-9.
21. Klok EJ, Wilson IW, Wilson D, Chapman SC, Ewing RM, Somervillee SC, et al.
Expression profile analysis of the low-oxygen response in Arabidopsis root cultures. Plant
Cell 2002; 14:2481-94.
22. Jones MA, Raymond MJ, Yang Z, Smirnoff N. NADPH oxidase-dependent reactive
oxygen species formation required for root hair growth depends on ROP GTPase. J Exp
Bot 2007; 58:1261-70.
23. Fath A, Bethke PC, Jones RL. Enzymes that metabolize reactive oxygen species are downregulated prior to GA-induced programmed cell death in barley aleurone. Plant Physiol
2001; 126:156-66.
24. Joo JH, Bae YS, Lee JS. Role of auxin-induced reactive oxygen species in root gravitropism. Plant Physiol 2001; 126:1055-60.
25. Cervantes E. ROS in root gravitropism: The auxin messengers? Trends Plant Sci 2001;
6:556.
26. Hung KT, Kao CH. Hydrogen peroxide is necessary for abscisic acid-induced senescence
of rice leaves. J Plant Physiol 2004; 161:1347-57.
27. De Cnodder T, Vissenberg K, Van Der Straeten D, Verbelen JP. Regulation of cell length
in the Arabidopsis thaliana root by the ethylene precursor 1-aminocyclopropane-1carboxylic acid: a matter of apoplastic reactions. New Phytol 2005; 168:541-50.
28. Peng H-P, Chan C-S, Shih M-C, Yang SF. Signaling events in the hypoxic induction of
alcohol dehydrogenase gene in Arabidopsis. Plant Physiol 2001; 126:742-9.

io
s

ci

en
ce

in mitochondrial activity and in the balance of free radicals.

In the classical treatise entitled Free radicals in biology and
medicine, Halliwell and Gutteridge3 present striking examples of
morphological alterations in mammalian cells treated with oxidants
or peroxide (see the Fig. 4.4 in p. 255 of this book for the effect on
hepatocytes, and the Fig. 7.1, p. 486 shown here as the Fig. 2 for
erythrocytes). In this last case, the authors attribute the alteration
of shape to global changes including increased membrane rigidity,
lipid peroxidation and damage to membrane proteins.
Free radicals have been involved in particular developmental
processes (root hair formation,22 cell death,23 differential cell
elongation in the gravitropic response24,25) as well as in global
changes affecting the organism, related to organ senescence and
aging.26 In many of these instances, ethylene interacts with free
radicals. For example, treatment of Arabidopsis seedlings with
the ethylene precursor 1-aminocyclopropane-1-carboxylic acid
resulted in reduced cell elongation characterized by accumulation
of free radicals and oxidative crosslinking of hydroxyproline-rich
glycoproteins and callose deposition in the cell wall.27
During the hypoxic response, ethylene modulates the physiological response (gene expression) to alterations in free radical
balance,28,29 and a similar effect may occur during germination,
when the seedling begins a new developmental program based
in the onset of respiration and aerobic metabolism. In chickpea,
the expression zone of an mRNA encoding a cysteine proteinase
upregulated by ethylene during germination,30 was identified by in
situ hybridization in the provascular tissue,31 thus suggesting that
ethylene regulation of germination is related with vascular formation and cell death, processes interconnected and regulated both
by free radicals and ethylene.23,32 Ethylene is also involved in the
process of programmed cell death during aerenchyma formation
in maize roots.33

Conclusion: Genome Dynamics and Morphology

20

09

La

nd

es

The sequencing of plant genomes has been one of the main

objectives of recent research in plant biology. The accurate description of the changes in shape in organisms may contribute to
understanding the functions of the elements found in the genomes
(repeated sequences, transposons, microRNAs,).
The morphology of cells and organisms can be interpreted as
the result of two factors: genome composition and the physiological
status in response to environmental conditions. Changes in shape
may be either the result of developmental programs in a regular
environment or the response to changes (stress) in environmental
conditions. Genome changes in response to stress were described in
the work of Barbara McClintock34 and have been the subject of recent
investigation, suggesting the role of free radicals in the generation of
systemic signals involved in genome dynamics.35 In Drosophila, the
morphological alterations in response to hypoxia include chromosomal abnormalities36 and in maize roots the genomic responses to
hypoxia include the activation of particular microRNAs.37
The accurate morphological description of plant organs may
contribute to define different metabolic situations associated with
genomic activity. This is an important research objective for future
work in model systems, in particular Arabidopsis.

www.landesbioscience.com

Plant Signaling & Behavior

Ethylene, free radicals and plant morphology

20

09

La

nd

es

io
s

ci

en
ce

.D

no
t

di

st
r

ib
u

te
.

29. Cervantes E. A distinctive voice for ethylene signalling in hypoxia. Trends Plant Sci
2001; 6:450.
30. Cervantes E, Rodriguez A, Nicolas G. Ethylene regulates the expression of a cysteine
proteinase gene during germination of chickpea (Cicer arietinum L). Plant Mol Biol
1994; 25:207-15.
31. Cervantes E, De Diego JG, Gmez MD, De Las Rivas J, Igual JM, Velzquez E, et al.
Expression of a cysteine proteinase in chickpea (Cicer arietinum L.) is localized to provascular cells in the developing root. J Plant Physiol 2001; 158:1463-9.
32. Courtois-Moreau CL, Pesquet E, Sjdin A, Muiz L, Bollhner B, Kaneda M, et al. A
unique program for cell death in xylem fibers of Populus stem. Plant J 2009; In press.
33. Drew MC, He CJ, Morgan PW. Programmed cell death and aerenchyma formation in
roots. Trends in Plant Science 2000; 5:123-7.
34. McClintock B. The significance of responses of the genome to challenge. Science 1984;
226:792-801.
35. Filkowski J, Yeoman A, Kovalchuk O, Kovalchuk I. Systemic plant signal triggers
genome instability. Plant J 2004; 38:1-11.
36. Foe VE, Alberts BM. Reversible chromosome condensation induced in Drosophila
embryos by anoxia: visualization of interphase nuclear organization. J Cell Biol 1985;
100:1623-36.
37. Zhang Z, Wei L, Zou X, Tao Y, Liu Z, Zheng Y. Submergence-responsive microRNAs
are potentially involved in the regulation of morphological and metabolic adaptations in
maize root cells. Ann Bot 2008; 102:509-19.
38. Cuttler SR, Ehrhardt Dw, Griffitts JS, Sommerville CR. Random GFP::cDNA fusions
enable visualization of subcellular structures at a high frequency, Proc Natl Acad Sci USA
2000; 97:3718-23.

Plant Signaling & Behavior

2009; Vol. 4 Issue 5