Cervantes 2006. Floriculture, Ornamental and Plant Biotechnology Volume I ©2006 Global Science...

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Abbreviations: ABA, abscisic acid; ET, ethylene; GA, gibberellic acid

46

Ethylene in Seed Germination and Early Root

Development

Emilio Cervantes

IRNASA-CSIC, Apartado 257, 37080, Salamanca, Spain

Corresponding author : [email protected]

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 tohormones. This chapter reviews our recent description of root apex curvature as a new phenotype associated with the ethylene signal-

transduction 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) toexist 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 themand 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,



Cervantes Ethylene in seed germination

A

B

C

D

E

F

Fig. 1 Life cycle of the model plant Arabidopsis thaliana (A). Seeds (B)

germinate (C) in response to appropriate environmental conditions (light,

temperature, oxygen tension, et.). After germination the seedling continues

development (D) and all subsequent processes (rosette, shoot development

(E), flowering (F)) occur without the possibility of movement.

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

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 thatresults in a mature seed able to maintain its metabolism reduced to a

minimum for a variable period of time. Cell differentiation is scarce in

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

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 wetand dry environments. Ancestors of the actual plants adapted to survive

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

remains an ordered structure formed by non-differentiated cells and

enclosed in the seed coat. There, it contains all the information required

to develop into a plant. When a seed is in the presence of water and in

appropriate environmental conditions, metabolism is re-initiated, cells

elongate and the radicle protrudes throughout the seed coat. Thisprocess 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 sub-

cellular 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

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




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 (Román 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 theabsence 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 ofproteasome: 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 life’s 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

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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 plant’s 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 (García 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 signaltransduction 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, O’Toole 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

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A B

CA: norbornadiene

B: control

C: ethephon

A B C

Fig. 2 Roots of Arabidopsis thaliana line Lt16b (Cutler et al. 2000) that

express GFP in the cell wall. Confocal images of eight days old roots

germinated in water agar (B) or treated with norbornadiene (A) and

ethephon (C). Top three images, bars = 500 µm; bottom three images,

bar = 100 m.

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

obtained a consistent increased germination rate in all varieties with

ethylene treatments and delayed germination with norbornadiene (de Diego

et al . 2002).

In seeds of Arabidopsis and other species, ethylene has a

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, ethylene’s 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.

This indicates that, similarly to what was reported for ADH in hypoxia (Peng

et al . 2001), a factor, different from ethylene, may be the inductor for these

genes that later on are ethylene-regulated (Cervantes 2001). Free radicals

interact with ABI proteins, mediators of ABA signalling (Meinhard et al . 2002)

and thus, free radicals are good candidates as regulators of gene

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 plant’s 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).

Table 1 Root length (mm) in Arabidopsis seedlings grown for 8 days in water-agar,

norbornadiene and ethephon.

Treatment Mean N Standard dev

Water-agar 6.0326 46 3.00629

norbornadiene 8.5217 46 4.02486

ethephon 2.8261 46 1.57486

Total 5.7935 138 3.81828

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0.2 0.4 0.6 0.8 1

0.5

1

1.5

2

Fig. 3 (A) Adjusted Bézier curve taking nineteen pointsthroughout 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.

2 3 4 5 6

13

13.5

14

14.5

15

15.5

16

0.2 0.4 0.6 0.8 1

0.5

1

1.5

2

8 9 10 11 12 13

10

11

12

13

14

0.2 0.4 0.6 0.8 1

0.5

1

1.5

2

0.2 0.4 0.6 0.8 1

0.25

0.5

0.75

1

1.25

1.5

6.5 7 7.5 8 8.5 9 9.5 10

6

6.5

7

7.5

8

8.5

9

9.5

Fig. 4 Root images (left), adjusted Bézier curves (right,

top) and their corresponding graphics showing curvature

values (right, bottom) for representative samples of: col

(above), etr1-1 (middle) and ein2-1 (below) genotypes. Bar

= 100µm. From: Cervantes and Tocino (2005) Journal of Plant

Physiology 162, 1038-1045, Copyright [2005] Elsevier.

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 .

2002).

6. ETHYLENE EFFECTS IN THE MORPHOLOGY OF THE ROOT APEX

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 (Guzmán 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

and Tocino 2005; Fig. 3). Curvature is a concept in geometry that indicates the rate of

change of the slope of the curves at any given point. The calculations demonstrated that

maximum curvature values occur in the root apex and are significantly lower in the

ethylene-insensitive mutants (ein2-1, etr1-1) than in the wild-type Columbia (Cervantes

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, non-

significant differences in curvature supporting a role for ethylene in this phenotype. But, when curvature values in roots under norbornadiene

434





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 22-24hours 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

differentiation. This cysteine proteinase encoding mRNA was induced in the

cotyledons as well as in the embryonic radicle during chickpea seed imbibition.

When the radicle was eliminated from the seed before imbibition, transcript levels

at 48 hours in the cotyledons decreased notably but, interestingly, when the

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 aftersimultaneous 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 protein–encoding 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

Fig. 5 Northern blot showing expression of the mRNA encoding the

chickpea cysteine proteinase cacG1, regulated by ethylene during

germination. The blot shows expression in chickpea cotyledons at

24 hours following seed imbibition in different conditions. Levels of

mRNA are sensitive to oxygen concentration and their amounts also

vary in cotyledons imbibed with or without the testa.

435





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.

(A) (B)

reduced expression both in ethylene insensitive mutants (etr1-1) and in ga1 mutants during 24 hours of germination suggesting that seeds ofthese 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 don’t 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, dependingon 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 Gutiérrez de Diego, F David Rodríguez, Sabrine Humbert and JL Rodríguez Lorenzo (Department of Biochemistry; Salamanca University); Angel

Tocino (Department of Mathematics; Salamanca University) and José Javier Martín Gómez 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.

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