46

Ethylene in Seed Germination and Early Root

Development
Emilio Cervantes
IRNASA-CSIC, Apartado 257, 37080, Salamanca, Spain
Corresponding author: ecervant@usal.es

Keywords: Arabidopsis, hypoxia, model system, seeds

What is, is uncreated and indestructible, alone, complete, immovable and without end. Nor was it ever, nor will it be.

Poem of Parmenides. On Nature.

ABSTRACT
Seed germination is a model system for the analysis of plant development. Cell elongation and differentiation processes are under the control of
hormones and submitted to the influence of environmental factors. Hormones (abscisic acid, auxin, gibberellic acid, ethylene and others) interact
with each other as well as with diverse components of cell metabolism. Arabidopsis is the model system for the study of developmental
processes in plants and research in this species is now helped by a multiplicity of resources (massive genome sequences available, collections
of mutants and lines expressing GFP, direct observation in confocal microscopy among others). The analysis of seed germination in Arabidopsis
using these tools offers a unique opportunity to dissect the interaction of hormonal pathways during development and its relationship to cellular
processes. Ethylene is one of the hormones whose mechanism of action has been most thoroughly investigated and may serve a pivotal role in
this analysis. An important requirement is the accurate description of phenotypes for the known mutants with altered sensitivity or response to
hormones. This chapter reviews our recent description of root apex curvature as a new phenotype associated with the ethylene signaltransduction pathway mutants. New Arabidopsis sequences induced during germination have been recently isolated. Their expression analysis
may be a useful tool to investigate hormonal interactions during seed germination.

1. INTRODUCTION
1.1. Philosophical introduction: A matter of perception
Parmenides of Ellea is the main figure of a philosophical school from ancient Greece that has been called Monism. Basically this school
defended the thesis that everything is now present, together, single, continuous; that there is no past and no future and everything exists now
and at the same time (or independent of time).
Today, many of us may have a tendency to interpret this as if only is important what exists today, i. e. what it seems to us (we observe) to
exist today; but I believe this is not a correct interpretation. To me, the only correct interpretation is that everything really exists now: in this
moment. To explain it otherwise: The perception of the world as things past, present and future is subjective. It is only a result of our human
condition. Due to our evolution and development as living organisms we have developed senses that inform to us about our environment in this
particular way, but outside us it is possible, as the philosopher suggests that a reality containing everything exists now. Our senses inform to us
on particular aspects of this reality, but our perception is always partial.
This is the beginning to a chapter inside a book on recent advances in Plant Sciences so the reader may ask quite reasonably: What has
what Parmenides said to do with plants?
Plants are complex multi-cellular organisms. They may reproduce by sexual mechanisms that share many features in common with animal
and human reproduction. Plants have also developed mechanisms to sense the changes that occur in environmental variables. The idea that we
have, as human beings, of the world around us is mainly based in our visual perception of radiation (colours), perception of chemical molecules
by specialised sensory systems (smell, taste) and perception of vibrations and mechanical stimuli (hearing, touch). The development of the
nervous system and the brain of mammalians is the result of complex sophistications in the organs of the senses and connections between them
and with the rest of the organism, including the organs of motion that occurred in the course of evolution. On the other hand, plants are
specialized in sensing multiple environmental variables such as radiation, mechanical vibration and chemical compounds. Molecules, and in
particular volatiles, play an important role in the way plants communicate with other organisms and sense the environmental conditions. Plants,
Abbreviations: ABA, abscisic acid; ET, ethylene; GA, gibberellic acid

430

Cervantes

Ethylene in seed germination

in contrast to animals, are static; they do not need to develop a nervous system that directs their motion, but need to possess accurate
mechanisms for sensing environmental variables as well as for communication between cells in the individual to organize growth in a coordinate
fashion. The molecular bases of these mechanisms are now beginning to be understood. Plants and animals perceive different, partial aspects
of reality. Reality is probably more complex: Understanding the way plants perceive it helps us to have new insights of it.
Fig. 1 represents the life cycle of Arabidopsis thaliana, a plant that has been recently adopted by the scientific community and is widely
used as the model plant. This means that Arabidopsis is for the scientific community the best known flowering plant on Earth and that many of
the resources and technologies used in Arabidopsis may be transferred later to other, agriculturally more important plants.
Like humans and animals, plants recall during their life cycles the steps that took place during the evolutionary history of their species. This
is expressed as the principle ontogeny repeats phylogeny. The formation of a living being somehow contains the evolutionary history of all
individuals of its species. This conclusion of modern Biology is somehow a reminder of Parmenides words.

1.2. Introduction (non philosophical)

In plants, like humans and animals, single cells come from the fusion of
gametes, and go through a series of divisions and differentiation
processes to give an embryo. During embryogenesis, both in plants and
animals the cells are heterotrophic, i.e. they depend on the mother for
nutrition and development. An important difference between plant and
F
animal embryos is that plant embryos store reserve molecules, such as
starch and lipids. In addition, before going in further cell differentiation
and organogenesis, plant embryos go through a dessication process that
results in a mature seed able to maintain its metabolism reduced to a
minimum for a variable period of time. Cell differentiation is scarce in
E
plant embryos, and most of the cells remain in a meristematic status
(similar to animal stem cells). For example, there is not a differentiated
vascular tissue in plant embryos (Scheres et al. 1994).
The outlines for the main processes of development are similar for
all plant individuals belonging to thousands of species that cover, in an
important proportion, the surface of earth and nourish its population. For
D
example, in the course of embryogenesis, all seeds go through
desiccation. The capacity to survive after desiccation has played an
important role in the adaptive success of plants during evolution. From a
modern evolutionary point of view, this suggests that the precursors of
actual plants were in the past exposed to the alternation between wet
and dry environments. Ancestors of the actual plants adapted to survive
C
in the absence of water by reducing metabolism to a minimum in special
structures: the seeds. Seeds have mechanisms that allow them to
survive in the absence of water and re-start growth once in the presence
of water. The phytohormone abscisic acid is involved in this process
(Finkelstein et al. 2002, Shinozaki et al. 2003).
During the period of quiescence and arrested growth the embryo
B
remains an ordered structure formed by non-differentiated cells and
Fig. 1 Life cycle of the model plant Arabidopsis thaliana (A). Seeds (B)
enclosed in the seed coat. There, it contains all the information required
germinate (C) in response to appropriate environmental conditions (light,
to develop into a plant. When a seed is in the presence of water and in
temperature, oxygen tension, et.). After germination the seedling continues
appropriate environmental conditions, metabolism is re-initiated, cells
development (D) and all subsequent processes (rosette, shoot development
elongate and the radicle protrudes throughout the seed coat. This
(E), flowering (F)) occur without the possibility of movement.
process is called germination and, when it is completed, some cells enter
into division (Barroco et al. 2005, Bewley and Black 1994). A differentiation program is started by which a new plant will be formed. Thus,
germination is the result of embryonic cell elongation, and cell division occurs only after germination. Seed germination is associated with
important changes: during and around germination the seedling switches from a dehydrated to a hydrated status, from anaerobic to aerobic and
later on, from heterotrophy to autotrophy. Cell differentiation processes occur in the cells and involve the synthesis and re-distribution of subcellular organelles and molecules. Due to the concentration of cell differentiation processes in space (a limited number of cells) and time
(reduced duration), germination is a model process for the study of plant development, the involvement of hormones in gene expression and
cellular differentiation and the study of plant cell responses to the environment.
In this review I will focus on the role of ethylene in the early stages of development that occur during and after seed germination in Arabidopsis.
When appropriate, examples from other plant species will be given to illustrate particular aspects. The effects of ethylene in germination and
early root development will be discussed emphasising recent results from our laboratory.

2. ETHYLENE ACTION: GENERAL ASPECTS

Ethylene (ET) action is closely related with the action of other hormones and free radicals and involves important modifications in gene
regulation and protein degradation. Ethylene acts in central aspects of cell physiology and metabolism and interacts with reactions that may be
active in the absence of the hormone. Thus, it is important to remark that some of the mutants obtained in screenings for altered sensitivity or
Floriculture, Ornamental and Plant Biotechnology Volume I 2006 Global Science Books

431

Cervantes

Ethylene in seed germination

response to ethylene, may be compromised in biochemical reactions of the cell that may be modulated by the hormone, but that in theory, may
occur also independently of the presence of ethylene.
Ethylene has been involved in many aspects of plant development ranging from germination to senescence. The effect of ethylene was
described for the first time more than hundred years ago in pea (Neljubov 1901), and was then called the triple response. As it was then
described in pea, the triple response consists in swelling of the hypocotyl , growth inhibition in the root and in the hypocotyl and an exaggerated
hook curvature in dark-grown seedlings. In many instances ethylene has been involved in cell differentiation processes including differential cell
elongation and cell death. Its effect on cell elongation may be the basis for its role in germination.
Receptors for ethylene sensing and elements in the signal transduction pathway are well known: They cross-interact with central aspects of
cell biology and metabolism (Stepanova and Alonso 2005). The ethylene signal transduction involves ethylene binding to a family of receptors,
whose activity is modulated by the hormone. Ethylene receptors form a family of protein histidine kinases that include: ETR1 (Bleecker et al.
1998, Chang et al. 1993), ETR2 (Sakai et al. 1998), ERS1 (Hall et al. 2000), EIN4 and ERS2 (Hua et al. 1998). These receptors interact with
other regulatory proteins. For example, ETR1 activity regulates CTR1, a MAP kinase kinase kinase similar to RAF, a mammalian protein
involved in processes of cell differentiation and cancer (Clark et al. 1998, Huang et al. 2003, Kieber et al. 1993). 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. 1988, Masucci and Schiefelbein 1996, Hall et al. 1999). Many growth characteristics of ctr1-1 mutants are opposite to
those of ethylene-insensitive mutants (Kieber et al. 1993). Other proteins in the pathway are downstream of CTR1. EIN2 is related to metal
transporters of the NRAMP family (Alonso et al. 1999). The phenotype of ein2-1 mutant is of ethylene insensitivity (Romn et al. 1995), thus
resembling etr1-1 phenotype (Bleecker et al. 1988).
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). The isolation of loss
of function mutants in the ethylene receptors supports this model (Hua and Meyerowitz 1998). According to it, ETR1 protein is active in the
absence of ethylene and its activity is modulated by the phytohormone. Thus, the ethylene receptors posses a biochemical activity
independently of ethylene. This activity is related with cell growth. In the absence of ethylene, a phenotype of defective cell growth in the
hypocotyl has been described for ETR1 loss of function mutants (Hua and Meyerowitz 1998).
It is thus possible to identify other factors, apart from ethylene, that may affect the activity of the ethylene receptors or other regulatory
proteins in the signal transduction cascade. Accordingly, ethylene interacts with other plant hormones and with free radicals. In germination,
ethylene has opposite effects to ABA, and mutants insensitive to ethylene are hypersensitive to ABA (Beaudoin et al. 2002, Ghassemian et al.
2002). Dominant ethylene insensitive etr1-2 mutants have increased levels of ABA and present alterations in other hormones (Chiwocha et al.
2005). The relationship between ethylene and gibberellic acid in Arabidopsis development has been also described (Achard et al. 2003) and, in
many instances, ethylene action has been associated with the response of plants to hypoxia (Jackson 1985, He et al. 1996, Saab and Sachs
1996, Grichko and Glick 2001, Peng et al. 2001) sometimes interacting with free radicals (D'Haeze et al. 2003). Interestingly free radicals have
been recently involved in the action of auxin and in processes of cell elongation (Foreman et al. 2003, Joo et al. 2001, Liszkay et al. 2004,
Rodriguez et al. 2002) as well as in the ABA responses (Meinhard et al. 2002).
When analysing the expression of ADH, Peng et al. (2001) described that ethylene was responsible for the induction of the mRNA for this
gene during hypoxia. This could be confirmed by the use of ethylene inhibitors as well as mutants in the ethylene signal transduction pathway.
But, interestingly, this effect of ethylene occurred after several hours incubation, and the induction began earlier. Thus, some other factor was
responsible for the earlier induction in hypoxia. It may be interesting to investigate in detail how free radicals may regulate the induction of
ethylene-regulated genes and the activity of proteins in the signal-transduction cascade.
Moreover, ethylene signal transduction pathway has been recently implicated in cooperation with auxin in light sensing (Vandenbussche et
al. 2003) as well as related with sugar sensing (Zhou et al. 1998).
The links of plant hormone action with protein degradation in the proteasome have multiplied in recent years. First, it was demonstrated that
auxin action involved proteasome-mediated degradation of Aux/IAA proteins in response to auxin (Gray et al. 1999, Worley et al. 2000). Also the
effect of gibberellic acid consist in the reduction of the levels of inhibitors of the DELLA family of regulatory proteins, thus GA action is regulated
by the ubiquitin-proteasome pathway. The genes SLEEPY1 (SLY1) and SNEEZY (SNE) encode F-box proteins of a Skp1-cullin-F-box (SCF), E3
ubiquitin ligase complex that positively regulates GA signaling trough targeted proteolysis of the DELLA proteins using ubiquitin mediated
destruction (McGinnis et al. 2003, Strader et al. 2004). Ethylene synthesis and action are also directly linked with proteolysis and the activity of
proteasome: The protein ETO1, whose mutants where first identified as ethylene overproducers, is required for appropriate degradation of ACS5,
the protein involved in ethylene biosynthesis (Chae et al. 2003) and the genes RUB1 and 2 regulate both auxin signalling and ethylene
production in Arabidopsis (Bostick et al. 2004). Ethylene action is mediated by the selective degradation of the transcription factor EIN3 by
SCFEBF1/EBF2 (Guo and Ecker 2003, Potuschak et al. 2003). Recently, an auxin receptor has been demonstrated to be the F-box protein TIR1
(Kepinsky and Leyser 2005, Dharmasiri et al. 2005).
Thus ethylene action is linked to other hormones by multiple pathways. It involves protein degradation in the proteasome and is associated
with central aspects of cell metabolism like nutrient and redox sensing. Seed germination offers the opportunity to analyse and dissect the
complex mechanisms of ethylene action in a suitable system.

3. SEED GERMINATION: SENSING THE ENVIRONMENT

Plants present large variation in size and shape and a range of sensitivities to variations in the physical environment that has allowed them to
colonise diverse habitats in earth. Seeds contain, in a latent condition, all the information required for an important part of this variation (Baskin
and Baskin 2001, Matilla et al. 2005). Among all the structures in the plant lifes cycle, the seeds have been responsible for the evolutionary
success of plants, for plant colonization of diverse environments and ultimately for plant diversity.
Developmental processes in plants (Fig. 1), consist in a general schema containing conserved elements with variations in the different
Floriculture, Ornamental and Plant Biotechnology Volume I 2006 Global Science Books

432

Cervantes

Ethylene in seed germination

species. For example during germination, water uptake, cell elongation, changes in oxygen availability to the cells occur in all species but seeds
from different species respond differently to variations in the environmental variables. This explains the utility of working with a model system:
Today, the analysis of seed germination in Arabidopsis is the first step in the understanding of the process of seed germination in plants. The
comparison of the process in the model with other plant species will then be the next required step.
In the plants life cycle, germination determines the time and place in which all further development will occur. It is thus a process of crucial
importance and as such it must be tightly regulated. The seed has to be equipped with sensing mechanisms that allow it to obtain the information
required to assure that germination will only occur when environmental factors are favourable to complete development. The effect of light on
seed germination in particular species has been thoroughly studied and it was the basis for the discovery of phytochrome (Borthwick et al. 1952).
Temperature constraints are important and very variable for germination of diverse plant species (Bewley and Black 1994). Apart from light and
temperature, other physical factors of the environment may affect seed germination. The effect of metals in Arabidopsis seed germination has
been recently investigated (Li et al. 2005); other factors affecting germination include magnetic fields (Garca Reina et al. 2001), solar radiation
of high intensity, microwave radiation or, even mental energy (Bai et al. 2000). In this respect, physiological analysis of seed germination is
completely dependent of physical advances, discoveries and points of view. When Physics describes a new environmental variable, Plant
Physiology may then study its effects on plant development and in particular in germination. Thus, particular variables still scarcely investigated
may have an effect on germination (for example several types of solar radiation). Also, the possibility exists that variables of the physical world
remain to be identified that may reveal later to be important for germination in general or germination of particular seed types. On the other hand,
the detailed knowledge of mechanisms by which plants perceive the environmental signals may be a useful tool for the chemist and the physicist
in the finding of common aspects between diverse signals or physical processes.
Plants have the possibility to integrate diverse physical inputs forming a net with signal-transduction schemas, and ethylene interacts with
this net (Stepanova and Alonso 2005). For a seed, the result of this complex integrative process will be just one of two possible issues: To
germinate or to remain quiescent. Seed biology tries to explain how the seeds sense the environmental variables, and integrate them in signal
transduction mechanisms to give this final binary result: germination or the lack of it.
In the responses of seeds to environment, there are elements that are species specific and others conserved between species. The
environmental factors that trigger germination are variable with the species, the main events occurring during the process will be the same:
Cellular elongation and differentiation and a change in the metabolism: from hypoxia to normoxia; and, later on, from heterotrophy to autotrophy.
Seed dehydratation was associated with increased cytoplasmic viscosity and consequently with hypoxia (Leprince and Hoekstra 1998). In
legume embryos it has been demonstrated that cells are under hypoxia (Rolletschek et al. 2002). Also mutants defective in the hypoxic/anoxic
induction of ADH (alcohol dehydrogenase) gene in Arabidopsis were defective in their induction during germination, suggesting that at least
some of the regulatory pathways controlling the expression of ADH during seed germination and anoxia/hypoxia are identical (Conley et al.
1999). Once the radicle has emerged, respiration increases and seed metabolism becomes aerobic (Bewley and Black 1994) thus normoxia in
the cells follows cell rehydratation and germination, but most probably, the transition from hypoxia to normoxia is very tightly regulated and
different in the diverse cell types.
During germination and immediately after this process many cell differentiation events occur that include the re-organization, synthesis and
distribution of subcellular components and organelles. The endosymbiotic theory is widely accepted today (Mereschkovsky 1926, Wallin 1927,
Margulis 1970); thus, during embryogenesis, germination and early stages of plant development, the synthesis and sub-cellular distribution of
organelles involves the accurate regulation of genes of different evolutionary origins. Ethylene receptors have its origin in cyanobacteria, thus
share a common origin with plastids (Mount and Chang 2002). Their sub-cellular localisation has been investigated showing that ETR1 protein
resides in the endoplasmic reticulum (Chen et al. 2002). Interestingly ETR1 protein and related ethylene receptors share similarities with more
than forty proteins encoded in the Arabidopsis genome that include the phytochromes, specialized in light sensing, citokinin receptors (Kakimoto
1996, Nishimura et al. 2004) and others. All these proteins share sequence similarities with the two-component regulatory systems of bacteria
that are involved in the perception of a multitude of environmental stimuli including light, nutrients, energy status, pH, osmolarity, etc. up to
approximately eighty different proteins in the E coli genome including the oxygen sensors such as ArcB (Iuchi and Lin 1993, Matsushika and
Mizuno 1998).
Ethylene and cytokinin receptors derive from ancestral two component systems, involved in the coordinate regulation of multiple responses
to environmental factors (including hypoxia) in bacterial species. Of particular interest is the case of Mycobacterium species, in which in the two
component system formed by devR and devS genes is involved in the dormancy associated with hypoxia (Boon and Dick 2002, Mayuri et al.
2002, OToole et al. 2003, Park et al. 2003).
Gibberellic acid is required for seed germination in many plant species including Arabidopsis, but multiple interactions between plant
hormones have been demonstrated, for example between gibberellic acid and ethylene (Achard et al. 2003) or ethylene and ABA (Beaudoin et al.
2002, Ghassemian et al. 2002). Both plant cell growth (Foreman et al. 2003, Liszkay et al. 2004) and hormonal action (Joo et al. 2001, Meinhard
et al. 2002, Mori and Schroeder 2004) are regulated by reactive oxygen species. Oxygen is required for seed germination in many plant species.
The importance of cell elongation and the rapid cell differentiation processes that occur during germination, make of it an interesting system to
investigate the relationships between plant hormones, free radicals and ethylene and their implications in cell elongation and differentiation.

4. ETHYLENE EFFECTS IN SEED GERMINATION

In some species, including Arabidopsis, it has been proven that detectable ethylene production begins with the onset of germination, i.e. with
radicle emergence (Petruzzelli et al. 1994, Thain et al. 2004). On the other hand, many examples through the literature, report that ethylene has
an effect in promoting seed germination (Esashi 1991, Baskin and Baskin 2001, Bewley and Black 1994 and references therein). In Arabidopsis,
constitutive ethylene insensitive mutants, etr1-1, show a delayed germination (Bleecker et al. 1988). The addition of ethylene in the germination
medium to Arabidopsis seeds may result in accelerated germination, and adding norbornadiene, an inhibitor of ethylene action, delays
germination. Nevertheless, these responses in Arabidopsis are variable with the seed lot and germination conditions. In experiments with the
Floriculture, Ornamental and Plant Biotechnology Volume I 2006 Global Science Books

433

Cervantes

Table 1 Root length (mm) in Arabidopsis seedlings grown for 8 days in water-agar,
norbornadiene and ethephon.
Treatment
Water-agar

Mean
6.0326

N
46

Standard dev
3.00629

norbornadiene

8.5217

46

4.02486

ethephon

2.8261

46

1.57486

Total

5.7935

138

3.81828

Ethylene in seed germination

collections of varieties of Medicago truncatula from INRA-Montpellier, we

C
A: norbornadiene
obtained a consistent increased germination rate in all varieties with
ethylene treatments and delayed germination with norbornadiene (de Diego
B: control
et al. 2002).
In seeds of Arabidopsis and other species, ethylene has a
C: ethephon
demonstrated antagonism to ABA, an hormone that inhibits germination.
Mutants with enhanced response to ABA were found to be ethylene
insensitive alleles in known genes of the ethylene pathway. ABA antagonism
may explain, at least partially, ethylenes effects in promoting germination
(Beaudoin et al. 2002, Ghassemian et al. 2002). Promotion of germination
by ethylene may also be explained on the basis of ethylene stimulation of
degradative enzymes as demonstrated in many species (Cervantes et al.
1994, Nascimento et al. 1999, Petruzzelli et al. 1999). Interestingly,
induction of the mRNAs encoding some of these enzymes occurs before
radicle protrusion, whereas ethylene production follows radicle protrusion.
C
B
This indicates that, similarly to what was reported for ADH in hypoxia (Peng A
et al. 2001), a factor, different from ethylene, may be the inductor for these
Fig. 2 Roots of Arabidopsis thaliana line Lt16b (Cutler et al. 2000) that
genes that later on are ethylene-regulated (Cervantes 2001). Free radicals
express GFP in the cell wall. Confocal images of eight days old roots
germinated in water agar (B) or treated with norbornadiene (A) and
interact with ABI proteins, mediators of ABA signalling (Meinhard et al. 2002)
ethephon (C). Top three images, bars = 500 m; bottom three images,
and thus, free radicals are good candidates as regulators of gene
bar = 100 m.
expression before seed germination.
Considering that germination is due to cell elongation, the reported effects of ethylene stimulating germination may seem in contradiction
with the effect inhibiting elongation that has been reported for example in hook formation (Peck et al. 1998) or in root development (see later). It
is important to consider that ethylene may have different effects in different cell types and that both in deep water rice (Kende et al. 1998), where
ethylene promotes cell elongation and in germinating seeds, an important role of ethylene seems to be due to increased elongation growth
associated with decreasing ABA levels or reduced ABA responsiveness (and probably associated with increased GA responsiveness).

5. ETHYLENE EFFECTS IN EARLY ROOT DEVELOPMENT

Plants are static organisms, but their seeds are dynamic. Before germination, the seed has possibilities to start development over a range of
options in space and time. After germination, all further development occurs in the same place. Being static organisms implies that the capacity
of the plants to respond to a changing environment consists in changes of shape. In the short term, the root may grow shorter or longer, thinner
or thicker. All the environmental information is integrated in the seedling and cell growth, division and, in consequence, overall morphology are
the result of this integrative process and not of a single factor. In this respect it is very striking to observe the effect of ethylene in root growth. Not
surprisingly, ethylene was one of the first plant hormones to be discovered and its effects to be described. The triple response, as it was
described (Neljubov 1901) indicates the plants sensitivity to ethylene: Swelling of the hypocotyl, growth inhibition in the root and in the hypocotyl
and hook curvature may be broadly modulated in seedlings by altering the concentration of the hormone.
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: decrease 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.
Arabidopsis seedlings grown in the presence of ethylene have shorter and thicker roots (Fig. 2). A similar effect was observed when
inoculating some legumes with particular strains of Rhizobium; in this case it was called a tsr phenotype (for thick short roots); this linked
ethylene with nodulation (van Spronsen et al. 1995). Indeed, ethylene insensitive mutants are hypernodulant (Penmetsa and Cook 1997) .
Although in Arabidopsis the effect of treatments with ethylene and ethylene inhibitors is already detectable one or two days after
germination, notable differences are observed also days later; when the seedlings are treated with ethylene, roots are shorter; when treated with
norbornadiene, an inhibitor of ethylene action, roots are longer (Table 1).
Longer roots may be the result of three different, though not completely independent, processes: 1. Increased cell division; 2. Reduced cell
death; 3. Increased cell expansion.
The effect of ethylene in root growth has not been explained so far by major alterations in rates of cell division or in cell death. Our
preliminary experiments of analysis of a transgenic line expressing GFP associated with Cyclin B (BJ8; Doerner et al. 1996) did not show major
differences in the rate of apical cell divisions between controls in water or treatments with ethylene and norbornadiene (unpublished results).
Floriculture, Ornamental and Plant Biotechnology Volume I 2006 Global Science Books

Cervantes

434

Ethylene in seed germination

Also, our results of staining death cells with propidium iodide did not reveal major
changes in cell death under the ethylene treatments. It is possible that similar
experiments directed to particular cell types, or in specific conditions may reveal an
effect of ethylene in cell division or in cell death, but nevertheless, the main cause of
reduction in root elongation due to ethylene seems to be its effect in cell elongation.
Le et al. (2001) investigated the effect of ethylene on cell elongation by confocal
microscopy and direct observation of roots immediately after ethylene treatments. Their
results show that, in the elongation zone, ethylene down regulates cell elongation in a
concentration dependent manner. This effect was later associated with a re-orientation
of microtubules in cells in the epidermal root (Le et al. 2004). The authors suggest that
primary cause of altered cell elongation may be ethylene altered cell wall metabolism
and not microtubule reorientation. Ethylene could influence the deposition or formation
of wall polysaccharides other than cellulose or influence the secretion or activity of cell
wall proteins, thus affecting cell wall metabolism by other means independent from
microtubule orientation. Both cell wall metabolism and microtubule orientation are
subjected to control by the activity of MAP kinase cascades that regulate response of
cells to changes in their environment. MAPK pathways are well conserved in eucaryonts
and are essential for complex physiological processes (embryonic development,
immune response, cell survival, apoptosis, proliferation, and migration (Robinson and
Cobb 1997, Ip and Davis 1998, Schaeffer and Weber 1999, Widmann et al. 1999).
Ethylene signalling is mediated by a MAP kinase cascade (Kieber et al. 1993, Ecker
2004) thus the cross-talk with other cell signalling pathways is probably complex and
dependent on the cell type.
Fig. 2 contains the images of two day old seedlings treated with norbornadiene and
with ethylene. The plants correspond to the line LT16b that expresses GFP in the cell
wall (Cutler et al. 2000). The effect of ethylene in the elongation zone consists in the
reduction of cell length; In the root cap, this effect is not observed and cells are often
shortened under treatments with norbornadiene, the inhibitor of ethylene action.
The effect of ethylene on vesicle formation and cell wall assembly at the cellular
level is a promising area for future research. Auxin transport is dependent on vesicle
traffic machinery, thus cell elongation is at the crossroads between auxin and ethylene.
Interestingly, a syntaxin protein has been involved in Abscisic acid responses (Zhu et al.

1.5

0.5

0.2

0.4

0.6

0.8

Fig. 3 (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). From: Cervantes
and Tocino (2005) Journal of Plant Physiology 162, 1038-1045
Copyright [2005] Elsevier.

16
15.5
15
14.5
14
13.5
13
2

1.5
1
0.5

0.2

2002).

0.4

0.6

0.8

14
13

6. ETHYLENE EFFECTS IN THE MORPHOLOGY OF THE ROOT APEX

12
11
10
8

10

11

12

13

The observation that the root apex of ethylene insensitive mutants etr1-1 and ein2-1 has
a more rounded shape, led us to develop a geometric analysis of root apex morphology
and curvature. For this, transmitted light images of wild type Col and mutant genotypes
were taken in the root apex of seedlings at 24 and 48 hours after imbibition. Mutants
used were: eto1-1 (Woeste et al. 1999, Chae et al. 2003), ein2-1 (Guzmn and Ecker
1990), etr1-1 (Chang et al. 1993) and ctr1-1 (Kieber et al. 1993).
Using light transmitted photographs of the roots, a series of points were marked
along the root profiles and the program AnalySIS was used to obtain coordinate (x,y)
values for each of the points in the figure. A program was generated with Mathematica
by Dr Angel Tocino that allows to obtain equations (Bezier curves) from the data (x, y)
series. These equations were then used to represent figures (curves) that reproduce the
shape of the apex and to calculate the curvature values along these figures (Cervantes
Fig. 4 Root images (left), adjusted Bzier curves (right,
top) and their corresponding graphics showing curvature
and Tocino 2005; Fig. 3). Curvature is a concept in geometry that indicates the rate of
values (right, bottom) for representative samples of: col
change of the slope of the curves at any given point. The calculations demonstrated that
(above), etr1-1 (middle) and ein2-1 (below) genotypes. Bar
maximum curvature values occur in the root apex and are significantly lower in the
= 100m. From: Cervantes and Tocino (2005) Journal of Plant
ethylene-insensitive mutants (ein2-1, etr1-1) than in the wild-type Columbia (Cervantes
Physiology 162, 1038-1045, Copyright [2005] Elsevier.
and Tocino 2005, Fig. 4). This method allows to treat the root apex profile as a
geometric curve. For this purpose, it is very important to have figures that represent a medial section of the root (perpendicular to the main axis).
This may be the reason why a higher variability was obtained in curvature values for eto1-1 and ctr1-1 mutants as well as with ethylene
treatments: These conditions give thicker roots and it becomes more difficult to obtain a precise perpendicular section through the middle of the
root. Curvature values in these genotypes (eto1-1 and ctr1-1) present the maximum values, but also higher variability (Cervantes and Tocino,
unpublished). We are currently investigating the morphological basis of these reported differences in curvature. Analysis of curvature in the line
LT16b that expresses GFP in the cell wall (Cutler et al. 2000) allows to define curvature values in the successive cell layers of the apex and
under diverse treatments and growth conditions. For example, treatment of seedlings with norbornadiene results in slightly decreased, nonsignificant differences in curvature supporting a role for ethylene in this phenotype. But, when curvature values in roots under norbornadiene
2

1.5

0.5

0.2

0.4

0.6

0.8

9.5

8.5

7.5

6.5

6.5

7.5

8.5

9.5

10

1.5

1.25

0.75

0.5

0.25

0.2

Floriculture, Ornamental and Plant Biotechnology Volume I 2006 Global Science Books

0.4

0.6

0.8

Cervantes

435

Ethylene in seed germination

treatments are compared with the controls (Columbia) and the mutants insensitive to ethylene (etr1-1), it is interesting to observe that the
treatment with norbornadiene is not sufficient to reduce curvature values of the controls to the values obtained in etr1-1, thus suggesting that
etr1-1 phenotype is stronger than just the result of inhibition of ethylene action during seed germination (Noriega, unpublished observation).
Curvature is a characteristic of the root apex that can be observed and quantified. It must be the consequence of a limited number of
metabolic reactions under hormonal control. The root apex of Arabidopsis is an excellent model system to investigate cell wall synthesis and
degradation, cytoskeletal dynamics and their regulation by hormones and free radicals. Our results on curvature analysis support a role for
ethylene in the modulation of reactions that are active in its absence.

7. ETHYLENE MODULATES REACTIONS THAT OCCUR IN THE ABSENCE OF ETHYLENE

Peng et al. (2001) described the effect of ethylene in the induction of ADH gene
in Arabidopsis. Transcripts of this gene were ethylene-regulated at certain
incubation times, but interestingly, at shorter incubation times, activation was due
not to ethylene but to another factors. This reminds the case of genes that are
reported to be induced by ethylene in germination. For example, we identified a
cysteine proteinase mRNA, whose amount detected in northern blots increased
notably in the course of germination of chickpea, from undetected levels in dry
seeds to notable amounts at 24 hours of imbibition and reaching maximum levels
at 96 hours. The expression is ethylene-regulated because transcript levels
decline with ethylene inhibitors (Cervantes et al. 1994); but, because ethylene is
only produced upon germination (with radicle emergence; approximately 22-24
hours after imbibition; Petruzelli et al. 1994), other factors need to be involved in
the initial induction of this gene. In longitudinal sections of the developing radicle,
this gene is expressed very specifically in the procambium cells, precursors of
the vascular bundle in a very precise zone folloing the meristem, below the root
apex (Cervantes et al. 2001). This suggests that the cysteine proteinase
encoded by the gene is involved in the cell death program that results in vascular
Fig. 5 Northern blot showing expression of the mRNA encoding the
chickpea cysteine proteinase cacG1, regulated by ethylene during
differentiation. This cysteine proteinase encoding mRNA was induced in the
germination. The blot shows expression in chickpea cotyledons at
cotyledons as well as in the embryonic radicle during chickpea seed imbibition.
24 hours following seed imbibition in different conditions. Levels of
When the radicle was eliminated from the seed before imbibition, transcript levels
mRNA are sensitive to oxygen concentration and their amounts also
at 48 hours in the cotyledons decreased notably but, interestingly, when the
vary in cotyledons imbibed with or without the testa.
radicle was eliminated and, simultaneously the seminal cover was removed (the
cotyledons peeled), transcript levels increased again reaching almost levels of cotyledons from seedling germinated from intact seeds
(Cervantes et al. 1994). This pattern of mRNA expression (increase during germination, decrease after ablation of the axis, increase again after
simultaneous ablation of the axis and removal of the cover) is the same as for the activity of the enzyme diamine oxydase that was reported to
be oxygen regulated (Hirasawa, 1988). In fact, cysteine proteinase mRNA quantities were responsive to oxygen concentrations in the
germination medium (Fig. 5).
Ethylene was involved in cell death that occurs in aerenchyma formation induced by hypoxia (He et al. 1996) but reactive oxygen
intermediates are known to be universal mediators of cell death not only in animal and in plant systems (Jabs 1999) but also in yeast (Madeo et
al. 1999). Thus, it is a reasonable possibility that ethylene acts as a modulator/mediator in the cell death processes during vascular development
in plants. The cellular response to hypoxia in animals has been thoroughly investigated in recent years and it involves targeting to the
proteasome of HIF mediated by prolyl hydroxylation. Although similar mechanisms have not been as yet described in plants it is interesting to
remark that oxygen sensing may be important in multiple aspects of plant development including vascular formation, and that ethylene may
involved in this process (Cervantes 1998 2002) in a particular way that differentiates plants from animals. Oxygen sensing in plants is still a
developing area that may benefit from more advanced research in animal systems. Although both, plant and animals may share common
features, the pathways involved may be complex and may require to address the issue at a cellular rather than an organism level.

8. NEW ARABIDOPSIS SEQUENCES ARE INDUCED UPON SEED IMBIBITION AND MAY BE USEFUL TOOLS IN THE
ANALYSIS OF HORMONE ACTION
Germination is a complex process. In Arabidopsis, as in many other plant species, seed germination requires GA. Microarray techniques have
been recently applied to investigate the processes under the control of GA during germination. Genes up-regulated during germination include
those encoding proteins for cell elongation and division, transcriptional regulation and hormonal metabolism (Ogawa et al. 2003). Microarray
studies are powerfull and cover a broad range of information, but the results are always limited to genes included previously in the array. A
complementary approach such as cDNA-AFLP may uncover new transcripts involved in germination. Using the technique of cDNA-AFLP we
cloned and analysed thirty five sequences expressed during germination of wild-type and ga1 mutant Arabidopsis seeds. Of them, only seven
corresponded to proteinencoding genes annotated in the Arabidopsis genome; five corresponded to Arabidopsis annotation units in regions not
encoding any known genes; eight were similar to fish DNA in diverse regions of various genes, including microsatellites and transposons; finally,
fourteen sequences did not present any significant similarity to any sequences in the public databases (de Diego et al. 2005). Fig. 6 shows
results of expression analysis (northern blot) for some of these sequences. Interestingly differential expression patterns are observed for diverse
hormone mutants with some of the sequences. For example, sequences 24 (unknown function) and 54a (similar to fish miostatin) present a
Floriculture, Ornamental and Plant Biotechnology Volume I 2006 Global Science Books

436

Cervantes

(A)

Ethylene in seed germination

(B)

Fig. 6 (A) Northern blot showing expression of three clones in dry seeds (0 hours) and at three time points during imbibition (6, 12 and 24 hours) in the wild-type seeds.
Genes encoding ribonucleotide reductase (86.1; left), an unknown sequence (90; middle) and AtHVA22a (54b; right) are shown to be induced during imbibition. (B)
Northern blot showing expression of two clones in dry seeds (0 hours) and at three time points during imbibition (6, 12 and 24 hours) in wild-type as well as in mutant
seeds. Expression of genes encoded in clones 54a and 24 is analysed during seed imbibition in the wild-type seeds, as well as in mutants eto1-1, etr1-1, ga1, and an as
yet uncharacterized T-DNA insertion mutant that presents accelerated germination (named 36, unpublished). Clone 54a contains a sequence homologous to fish DNA
sequences (left). Clone 24 has no similarities to any database sequences (right). These two transcripts are already present in dry seeds, decrease upon imbibition until
12 hours and increase again at 24 hours. Their expression level is reduced in ga1 as well as in etr1-1 and increased in eto1-1 mutant backgrounds. From: De Diego et al.
(2005) Journal of Plant Physiology (in press) Copyright [2005] Elsevier.

reduced expression both in ethylene insensitive mutants (etr1-1) and in ga1 mutants during 24 hours of germination suggesting that seeds of
these mutants may have important differences with the wild-type. A detailed analysis of the expression of these sequences during germination
and its correlation with new phenotypes observable and measurable in germination may broaden our point of view over this fundamental process.

9. CONCLUSION
After the precedent chapters we may turn back to the more broad and philosophical subjects with which we were dealing in the introduction. The
perception that we may have of all biological phenomena is partial and biased. It depends on many social constraints that often dont receive the
adequate attention. When we observe seed germination from the classical point of view of a naturalist equipped with a low magnifying
microscope, our impression may be that of a simple process. Briefly, the root may emerge from the seed or not; their cells may elongate or not.
But the approaches of biochemistry and molecular biology, reveal inside germination a series of processes of great importance and complexity.
In Arabidopsis, germination becomes now a process at the interface between cellular and organismal levels. Important changes in metabolism
and in gene expression, a switch from anaerobic to aerobic metabolism and the consequent changes in the redox state of the cells occur in a
limited number of cells. These processes may be investigated in the early stages that precede and surround seed germination and, depending
on the points of view and technologies used, each single process may reveal again an unexpected complexity. Germination processes are under
the control of hormonal action, and we have began to observe how the hormones interact with each other, and with free radicals and the
proteolytic machinery of the cell. Details of their interaction as well as with other central aspects of cell metabolism are being discovered.
Important regulatory processes are well conserved in plants, thus the study of development and hormone action in Arabidopsis may be of
broad application to understanding plant processes. Seed germination and early seedling development are processes adequate for the study of
hormonal interaction.

ACKNOWLEDGEMENTS
I wish to thank Juana Gutirrez de Diego, F David Rodrguez, Sabrine Humbert and JL Rodrguez Lorenzo (Department of Biochemistry; Salamanca University); Angel
Tocino (Department of Mathematics; Salamanca University) and Jos Javier Martn Gmez and Arturo Noriega (IRNASA-CSIC) for their continued collaboration and
contribution to diverse aspects of the research mentioned in this work. I would like to thank Peter Doerner for providing us with clone BJ8. Research in our laboratories has
been supported by CICYT (Spanish Ministry of Science and Technology), INIA (Spanish Ministry of Agriculture), Junta de Castilla y Leon and EEC (FEDER programs).
Special thanks to Jaime Teixeira da Silva for his invitation to participate in this book and continuous help with aspects of the edition of this chapter.

REFERENCES
Achard P, Vriezen WH, Van Der Straeten D, Harberd NP (2003) Ethylene regulates Arabidopsis development via the modulation of DELLA protein growth repressor function. The
Plant Cell 15, 2816-28
Alonso JM, Hirayama T, Roman G, Nourizadeh S, Ecker JR (1999) EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis. Science 284, 2148-2152
Bai F, Sun C, Shen J, Shen Y, Ge R, Bei C, Zhang J, Shi X, Liu Y, Liu X (2000) Seeds induced to germinate rapidly by mentally projected Qi energy are apparently genetically
altered. American Journal of Chinese Medicine 28, 3-8
Barroco RM, van Poucke C, Bergevoet JHW, de Veylder L. Groot SPC, Inz D, Engler G (2005) The role of the cell cycle machinery in resumption of postembryonic development.
Plant Physiology 137, 127-140
Baskin CC, Baskin JM (2001) Seeds: Ecology, Biogeography and Evolution of Dormancy and Germination, Academic Press, London
Beaudoin N, Serizet C, Gosti F, Giraudat J (2002) Interactions between abscisic acid and ethylene signaling cascades. The Plant Cell 12, 1003-1115
Bewley JD, Black M (1994) Seeds: Physiology of Development and Germination (2nd ed), Plenum Press, NY, 444 pp
Bleecker A, Estelle MA, Somerville C, Kende H (1988) Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana. Science 241, 1086-1089
Boon C, Dick T (2002) Mycobacterium bovis BCG response regulator essential for hypoxic dormancy. Journal of Bacteriology 184, 6760-6767
Borthwick HA, Hendricks SB, Parker MW, Toole EH, Toole VK (1952) A reversible photoreaction controlling seed germination. Proceedings of the National Academy of Science
USA 38, 662-666
Bostick M, Lochhead SR, Honda A, Palmer S, Callis J (2004) Related to ubiquitin 1 and 2 are redundant and essential and regulate vegetative growth, auxin signaling, and ethylene
production in Arabidopsis. The Plant Cell 16, 2418-2432
Cervantes E, Rodriguez A, Nicolas G (1994) Ethylene regulates the expression of a cysteine proteinase gene during germination of chickpea (Cicer arietinum L). Plant Molecular
Biology 25, 207-215
Cervantes E (1998) Oxygen sensing in plants. In: Lopez-Barneo J, Weir K (eds) Oxygen Regulation of Ion Channels and Gene Expression, Futura Publishing, NY, pp 57-66
Cervantes E (2001) A distinctive voice for ethylene signalling in hypoxia. Trends in Plant Science 6, 450

Floriculture, Ornamental and Plant Biotechnology Volume I 2006 Global Science Books

Cervantes

437

Ethylene in seed germination

Cervantes E, De Diego JG, Gmez MD, De Las Rivas J, Igual JM, Velzquez E, Grappin P, Cercs M, Carbonell J (2001) Expression of a cysteine proteinase in chickpea (Cicer
arietinum L.) is localized to provascular cells in the developing root. Journal of Plant Physiology 158, 1463-1469
Cervantes E, Tocino A (2005) Geometric analysis of Arabidopsis root apex reveals a new aspect of the ethylene signal transduction pathway in development. Journal of Plant
Physiology 162, 1038-1045
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. Plant Cell 15, 545-559
Chang C, Kwok SF, Bleecker AB, Meyerowitz EM (1993) Arabidopsis ethylene-response gene ETR1: similarity of product to two-component regulators. Science 262, 539-544
Chen YF, Randlett MD, Findell JL, Schaller GE (2002) Localization of the ethylene receptor ETR1 to the endoplasmic reticulum of Arabidopsis. Journal of Biological Chemistry 277,
19861-19866
Chiwocha SD, Cutler AJ, Abrams SR, Ambrose SJ, Yang J, Ross AR, Kermode AR (2005) The etr1-2 mutation in Arabidopsis thaliana affects the abscisic acid, auxin, cytokinin
and gibberellin metabolic pathways during maintenance of seed dormancy, moist-chilling and germination. Plant Journal 42, 35-48
Clark KL, Larsen PB, Wang X, Chang C (1998) Association of the Arabidopsis CTR1 Raf-like kinase with the ETR1 and ERS ethylene receptors. Proceedings of the National
Academy of Science USA 95, 5401-5406
Conley TR, Peng H-P, Shih M-C (1999) Mutations affecting induction of glycolytic and fermentative genes during germination and environmental stresses in Arabidopsis. Plant
Physiology 119, 599-608
Cutler SR, Ehrhardt DW, Griffitts JS, Somerville CR (2000) Random GFP::cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency.
Proceedings of the National Academy of Science USA 97, 3718-3723
Dharmasiri N, Dharmasiri S, Estelle M (2005) The F-box protein TIR1 is an auxin receptor. Nature 435, 441-445
de Diego JG, Rodrguez FD, Cervantes E (2002) The control of germination in model plants: Ethylene, cell elongation and cell divisin cycle. In: Vendrell M (ed) Biology and
Biotechnology of the Plant Hormone Ethylene III., IOS Press, pp 246-247
de Diego JG, Rodrguez FD, Rodrguez-Lorenzo JL, Grappin P, Cervantes E (2005) cDNA-AFLP analysis of seed germination in Arabidopsis thaliana identifies transposons and
new genomic sequences. Journal of Plant Physiology In press
Doerner P, Jorgensen J-E, You R, Steppuhn J, Lamb C (1996) Control of root growth and development by cyclin expression. Nature 380, 520-523
Ecker JR (2004) Reentry of the Ethylene MPK6 Module. The Plant Cell 16, 3169-3173
Esashi Y (1991) Ethylene and seed germination. In: Mattoo AK, Suttle JC (eds) The Plant Hormone Ethylene, CRC Press, Boca Raton, pp 133-157
Finkelstein RR, Gampala SS, Rock CD (2002) Abscisic Acid signaling in seeds and seedlings. Plant Cell 14S, 15-45
Foreman J, Demidchik V, Bothwell JHF, Mylona P, Miedema H, Torres MA, Linstead P, Costa S, Brownlee C, Jones JDG, et al (2003) Reactive oxygen species produced by
NADPH oxidase regulate plant cell growth. Nature 422, 442-446
Garcia RL, Arza PL, Almarza FI (2001) Influence of a stationary magnetic field on water relations in lettuce seeds. Part II: Experimental results. Bioelectromagnetics 22, 596-602
Ghassemian M, Nambara E, Cutler S, Kawaide H, Kamiya Y, McCourt P (2002) Regulation of abscisic acid signaling by the ethylene response pathway in arabidopsis. The Plant
Cell 12, 11117-111126
Gray WM, del Pozo JC, Walker L, Hobbie L, Risseeuw E, Banks T, Crosby WL, Yang M, Ma H, Estelle M (1999) Identification of an SCF ubiquitin-ligase complex required for auxin
response in Arabidopsis thaliana. Genes and Development 13, 1678-1691
Grichko VP, Glick BR (2001) Ethylene and flooding stress in plants. Plant Physiology and Biochemistry 39, 1-9
Guo H, Ecker JR (2003) Plant responses to ethylene gas are mediated by SCFEBF1/EBF2-dependent proteolysis of EIN3 transcription factor. Cell 115, 667-677
Guzman P, Ecker JR (1990) Exploiting the triple response of Arabidopsis to identify ethylene-related mutants. Plant Cell 2, 513-523
D'Haeze W, De Rycke R, Mathis R, Goormachtig S, Pagnotta S, Verplancke C, Capoen W, Holsters M (2003) Reactive oxygen species and ethylene play a positive role in lateral
root base nodulation of a semiaquatic legume. Proceedings of the National Academy of Science USA 100, 11789-11794
Hall AE, Chen QG, Findell JL, Schaller GE, Bleecker AB (1999) The relationship between ethylene binding and dominant insensitivity conferred by mutant forms of the ETR1
ethylene receptor. Plant Physiology 121, 291-300
Hall AE, Findell JL, Schaller GE, Sisler E, Bleecker AB (2000) Ethylene perception by the ERS1 protein in Arabidopsis. Plant Physiology 123, 1449-1457
He C-J, Morgan PW, Drew MC (1996) Transduction of an ethylene signal is required for cell death and lysis in the root cortex of maize during aerenchyma formation induced by
hypoxia. Plant Physiology 112, 463-572
Hirasawa E (1988) Amine oxidase in cotyledons of Pisum sativum develops as a result of the supply of oxygen through the embryonic axis during germination. Plant Physiology 88,
441-443
Hua J, Meyerowitz EM (1998) Ethylene responses are regulated by a receptor gene family in Arabidopsis thaliana. Cell 94, 261-271
Hua J, Sakai H, Nourizadeh S, Chen QG, Bleecker A, Ecker B, Meyerowitz EM (1998) EIN4 and ERS2 are members of the putative ethylene receptor gene family in Arabidopsis.
Plant Cell 10, 1321-1332
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 Journal 33, 221-233
Ip YT, Davis RJ (1998) Signal tansduction by the c-Jun NH2-terminal kinase (JNK)-from inflammation to development. Current Opinion in Cell Biology 10, 205-219
Iuchi S, Lin ECC (1993) Adaptation of Escherichia coli to redox environments by regulation of gene expression. Cell 66, 5-7
Jabs T (1999) Reactive oxygen intermediates as mediators of programmed cell death in plants and animals. Biochemical Pharmacology 57, 231-245
Jackson MB (1985) Ethylene and responses of plants to soil waterlogging and submergence. Annual Review of Plant Physiology 36, 145-174
Joo JH, Bae YS, Lee JS (2001) Role of auxin-induced reactive oxygen species in root gravitropism. Plant Physiol 126, 1055-1060
Kakimoto T (1996) CKI1, a histidine kinase homolog implicated in cytokinin signal transduction. Science 274, 982-985
Kende H (2001) Hormone response mutants. A plethora of surprises. Plant Physiology 125, 81-84
Kende H, van der Knaap E, Cho H-T (1998) Deepwater rice: A model plant to study stem elongation. Plant Physiology 118, 1105-1110
Kepinski S, Leyser O (2005) The Arabidopsis F-box protein TIR1 is an auxin receptor. Nature 435, 446-451
Kieber JJ, Rothenberg M, Roman G, Feldman 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, 427-441
Le J, Vandenbussche GF, Van der Straaten D, Verbelen JP (2001) In the early response of Arabidopsis roots to ethylene, cell elongation is up- and down-regulated and uncoupled
from differentiation. Plant Physiology 125, 519-522
Le J, Vandenbussche GF, Van der Straaten D, Verbelen JP (2004) Position and cell type-dependent microtubule reorientation characterizes the early response of the Arabidopsis
root epidermis to ethylene. Physiologia Plantarum 121, 513-522
Leprince O, Hoekstra FA (1998) The responses of cytochrome redox state and energy metabolism to dehydration support a role for cytoplasmic viscosity in desiccation tolerance.
Plant Physiology 118, 1253-1264
Li W, Khan MA, Yamaguchi S, Kamiya Y (2005) Effects of heavy metals on seed germination and early seedling growth of Arabidopsis thaliana. Plant Growth Regulation 46, 45-51
Liszkay A, van der Zalm E, Schopfer P (2004) Production of reactive oxygen intermediates (O2, H2O2, and OH) by maize roots and their role in wall loosening and elongation
growth. Plant Physiology 136, 3114-3123
Madeo F, Frlich E, Ligr M, Grey M, Sigrist S, Wolf D, Frlich KW (1999) Oxygen stress: A regulator of apoptosis in yeast. The Journal of Cell Biology 145, 757-767
Margulis L (1970) Origin of Eukaryotic Cells, Yale University Press, New Haven
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
Matilla A, Gallardo M, Puga-Hermida MI (2005) Structural, physiological and molecular aspects of heterogeneity in seeds: A review. Seed Science Research 15, 63-76
Matsushika A, Mizuno T (1998) A dual-signaling mechanism mediated by the ArcB hybrid sensor kinase containing the histidine-containing phosphotransfer domain in Escherichia coli.
Journal of Bacteriology 180, 3973-3977
Mayuri BG, Das TK, Tyagi JS (2002) Molecular analysis of the dormancy response in Mycobacterium smegmatis: expression analysis of genes encoding the DevR-DevS twocomponent system, Rv3134c and chaperone alpha-crystallin homologues. FEMS Microbiology Letters 211, 231-237
McGinnis KM, Thomas SG, Soule JD, Strader LC, Zale JM, Sun TP, Steber CM (2003) The Arabidopsis SLEEPY1 gene encodes a putative F-box subunit of an SCF E3 ubiquitin
ligase. Plant Cell 15, 1120-1130
Meinhard M, Rodriguez PL, Grill E (2002) The sensitivity of ABI2 to hydrogen peroxide links the abscisic acid-response regulator to redox signalling. Planta 214, 775-782
Mereschkovsky KS (1926) Symbiogenesis and the Origin of Species
Mori IC, Schroeder JI (2004) Reactive oxygen species activation of plant Ca2+ channels. A signaling mechanism in polar growth, hormone transduction, stress signaling, and
hypothetically mechanotransduction. Plant Physiology 135, 702-708
Mount SM, Chang C (2002) Evidence for a plastid origin of plant ethylene receptor genes. Plant Physiology 130, 10-14
Nascimento WM, Cantlife DJ, Huber DJ (1999) Lettuce seed germination and endo-b-mannanase activity at high temperature is stimulated by ethylene. HortScience 34, 513-519
Neljubov DN (1901) Uber die horizontale nutation der stengel von Pisum sativum und einiger anderen. Pflanzen Beitrage und Botanik Zentralblatt 10, 128-139

Floriculture, Ornamental and Plant Biotechnology Volume I 2006 Global Science Books

Cervantes

438

Ethylene in seed germination

Nishimura C, Ohashi Y, Sato S, Kato T, Tabata S, Ueguchi C (2004) Histidine kinase homologs that act as cytokinin receptors possess overlapping functions in the regulation of
shoot and root growth in Arabidopsis. Plant Cell 16, 1365-7137
O'Toole R, Smeulders MJ, Blokpoel MC, Kay EJ, Lougheed K, Williams HD (2003) A two-component regulator of universal stress protein expression and adaptation to oxygen
starvation in Mycobacterium smegmatis. Journal of Bacteriology 185, 1543-1554
Ogawa M, Hanada A, Yamauchi Y, Kuwahara A, Kamiya Y, Yamaguchi S (2003) Gibberellin biosntesis and responses during Arabidopsis seed germination. Plant Cell 15, 15911604
Park HD, Guinn KM, Harrell MI, Liao R, Voskuil MI, Tompa M, Schoolnik GK Sherman DR (2003) Rv3133c/dosR is a transcription factor that mediates the hypoxic response of
Mycobacterium tuberculosis. Molecular Microbiology 48, 833-843
Peck SC, Pawlowski K, Kende H (1998) Asymmetric responsiveness to ethylene mediates cell elongation in the apical hook of peas. Plant Cell 10, 713-720
Peng HP, Chan CS, Shih MC, Yang SF (2001) Signaling events in the hypoxic induction of ADH in Arabidopsis. Plant Physiology 126, 742-749
Penmetsa RV, Cook D (1997) A legume ethylene-insensitive mutant hyperinfected by its rhizobial symbiont. Science 275, 527-530
Petruzzelli L, Harren F, Reuss J (1994) Patterns of C2H2 production during germination and seedling growth of pea and wheat as indicated by a laser-driven photoacoustic system.
Environmental and Experimental Botany 34, 55-61
Petruzzelli L, Kunz S, Waldvogel R, Meins F, Leubner-Metzger G (1999) Distinct ethylene and tissue-specific regulation of B-1,3-glucanases and chitinases during pea seed
germination. Planta 290, 195-201
Pitts RJ, Cernac A, Estelle M (1999) Auxin and ethylene promote root hair elongation in Arabidopsis. Plant Journal 16, 553-560
Potuschak T, Lechner E, Parmentier Y, Yanagisawa S, Grava S, Koncz C, Genschik P (2003) EIN3-dependent regulation of plant ethylene hormone signaling by two Arabidopsis F
box proteins: EBF1 and EBF2. Cell 115, 679-689
Robinson MJ, Cobb MH (1997) Mitogen-activated protein kinase pathways. Current Opinion in Cell Biology 9, 180-186
Rodrguez AA, Grunberg KA, Taleisnik EL (2002) Reactive oxygen species in the elongation zone of maize leaves are necessary for leaf extension. Plant Physiology 129, 16271632
Rolletschek H, Borisjuk L Koschorreck M, Wobus U, Weber H (2002) Legume embryos develop in a hypoxic environment. Journal of Experimental Botany 53, 1099-1107
Romn 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
Saab X, Sachs Y (1996) A flooding-induced xyloglucan endo-transglycosylase homolog in maize is responsive to ethylene and associated with aerenchyma. Plant Physiology 112,
385-391
Sakai H, Hua J, Chen QG, Chang C, Medrano LJ, Bleecker AB, Meyerowitz EM (1998) ETR2 is an ETR1-like gene involved in ethylene signaling in Arabidopsis. Proceedings of the
National Academy of Science USA 95, 5812-5817
Schaeffer HJ, Weber MJ (1999) Mitogen-activated protein kinases: specific messages from ubiquitous messengers. Molecular and Cellular Biology 19, 2435-2444
Scheres B, Wolkenfelt H, Willemsen V, Terlow M, Lawson E, Dean C, Weisbeek P (1994) Embryonic origin of the Arabidopsis primary root and root meristem initials. Development
120, 2475-2487
Shinozaki K, Yamaguchi-Shinozaki K, Seki M (2003) Regulatory network of gene expression in the drought and cold stress responses. Current Opinion in Plant Biology 6, 410-417
van Spronsen PC, van Brussel AA, Kijne JW (1995) Nod factors produced by Rhizobium leguminosarum biovar viciae induce ethylene-related changes in root cortical cells of Vicia
sativa ssp. nigra. European Journal of Cell Biology 68, 463-469
Stepanova A, Alonso JM (2005) Ethylene signalling and response pathway: A unique signalling cascade with a multitude of inputs and outputs. Physiologia Plantarum 123, 195-206
Strader LC, Ritchie S, Soule JD, McGinnis KM, Steber CM (2004) Recessive-interfering mutations in the gibberellin signaling gene SLEEPY1 are rescued by overexpression of its
homologue, SNEEZY. Proceedings of the National Academy of Science USA 101, 12771-12776
Tanimoto M, Roberts K, Dolan L (1995) Ethylene is a positive regulator of root hair development in Arabidopsis thaliana. Plant Journal 8, 943-948
Thain SC, Vandenbussche F, Laarhoven LJJ, Dowson-Day MJ, Wang Z-Y, Tobin EM, Harren FJM, Millar AJ, Van der Straeten D (2004) Circadian rythms of ethylene emission in
Arabidopsis. Plant Physiology 136, 3751-3761
Vandenbussche F, Vriezen WH, Smalle J, Laarhoven LJJ, Harren FJM, Van Der Straeten D (2003) Ethylene and auxin control the Arabidopsis response to decreased light intensity.
Plant Physiology 133, 517-527
Wallin I (1927) Symbionticism and the Origins of Species, Williams and Wilkins, Baltimore
Widmann C, Gibson S, Jarpe MB, Johnson GL (1999) Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human. Physiology Reviews 79, 143180
Woeste KE, Ye C, Kieber JJ (1999) Two Arabidopsis mutants that overproduce ethylene are affected in the posttranscriptional regulation of 1-1-aminocyclopropane-1-carboxylic acid
synthase. Plant Physiology 119, 521-528
Worley CK, Zenser N, Ramos J, Rouse D, Leyser O, Theologis A, Callis J (2000) Degradation of Aux/IAA proteins is essential for normal auxin signalling. Plant Journal 21, 553-562
Zhou L, Jang J-Ch, Jones TL, Sheen J (1998) Glucose and ethylene signal transduction crosstalk revealed by an Arabidopsis glucose-insensitive mutant. Proceedings of the
National Academy of Science USA 95, 10294-10299
Zhu J, Gong Z, Zhang C, Song C-P, Damsz B, Inan G, Koiwa H, Zhu J-K, Hasegawa PM, Bressan RA (2002) OSM1/SYP61: A syntaxin protein in Arabidopsis controls abscisic
acid-mediated and non-abscisic acid-mediated responses to abiotic stress. The Plant Cell 14, 3009-3028

Floriculture, Ornamental and Plant Biotechnology Volume I 2006 Global Science Books