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Journal of Plant Physiology 165 (2008) 911—919

0176-1617/$ - sdoi:10.1016/j.

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Morphological analysis of seed shapein Arabidopsis thaliana reveals altered polarityin mutants of the ethylene signaling pathway

Celine Roberta, Arturo Noriegaa, Angel Tocinob, Emilio Cervantesa,�

aDepartamento de Produccion Vegetal, IRNASA-CSIC, Salamanca, Apartado 257, Salamanca, SpainbDepartamento de Matematicas, Universidad de Salamanca, Plaza de la Merced, 1, 37008 Salamanca, Spain

Received 24 April 2007; received in revised form 3 July 2007; accepted 1 October 2007

KEYWORDSArabidopsis;Curvature;Ethylene;Polarity;Seed

ee front matter & 2007jplph.2007.10.005

ing author. Tel.: +34 9239609.ess: ecervant@usal.es (

SummaryThe shape of Arabidopsis thaliana dry seed is described here as a prolate spheroid.The accuracy of this approximation is discussed. Considering its limitations, it allowsa geometric approximation to the analysis of changes occurring in seed shape duringimbibition prior to seed germination as well as the differences in shape betweengenotypes and their changes during imbibition. The triple mutant ein2-1, ers1-2,etr1-7 presents notable alterations in seed shape. In addition, seeds of this and othermutants in the ethylene signaling pathway (ctr1-1, eto1-1, etr1-1, ein2-1) showdifferent response to imbibition than the wild type. Imbibed seeds of the wild typeincrease their asymmetry compared with the dry seeds. This is detected by therelative changes in the curvature values in both poles. Thus, during imbibition of thewild-type seeds, the reduction in curvature values observed in the basal pole givesthem an ovoid shape. In contrast, in the seeds of the ethylene mutants, reduction incurvature values occurs in both basal and apical poles, and its shape remains as aprolate spheroid. Our data indicate that the ethylene signaling pathway is involved,in general, in the complex regulation of seed shape and, in particular, in theestablishment of polarity in seeds, controlling curvature values in the seed poles.& 2007 Elsevier GmbH. All rights reserved.

Elsevier GmbH. All rights rese

21 9606;

E. Cervantes).

Introduction

Recent analysis by molecular techniques hasrevealed many genes involved in the control oforgan shape as well as general architecture in themodel plant Arabidopsis thaliana. For example,

rved.

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C. Robert et al.912

Dinneny et al. (2006) described genes involved inthe control of shape in stamens and carpels, whileother genes were described that regulate the flatshape of leaves (summarized by Tsukaya, 2005).The shape of organs is the result of the integrationof multiple processes, including cell cycling,cellular elongation and communication and thedirection of cell proliferation, and thus all sets ofgenes active in these processes may also beinvolved in shape determination. The multiplicityof factors that affect organ and plant size andshape indicates complex regulation; thus, globalregulatory mechanisms must also exist that may beable to compensate or buffer sudden environmen-tal changes that, otherwise, would have deleter-ious effects. The cytoskeleton organization andmicrotubules, in particular, are very important incell size and shape determination. Indeed, they areinvolved in the arrangement of the deposition ofthe wall microfibrils (Hepler and Palevitz, 1974). Allthese aspects are known to be under the control ofthe ethylene sensing and signaling pathway. Thus,Apelbaum and Burg (1971) showed that ethylenealtered the orientation of microfibrils in Pisumsativum stems.

Seed shape is also the visible result of processessubmitted to complex regulatory mechanisms.Leon-Kloosterziel et al. (1994) have describedmutations that have an effect in integumentdevelopment with a direct consequence in theArabidopsis seed shape.

In this work, our objective was to investigate indetail the effects that diverse mutants in theethylene signal-transduction exert on seed shape.For this, we first need to adjust the seed shape to ageometric form. After adjusting seed shape to aprolate spheroid and calculating the curvature

Figure 1. Photograph of Arabidopsis thaliana cv. Colu

values in both poles, we described the changesthat occur during imbibition. Further, new pheno-typic traits for mutants in the ethylene signaltransduction pathway are described. Our resultssuggest that the ethylene signaling pathway isinvolved, in general, in the complex regulation ofseed shape and, in particular, in the establishmentof polarity in seeds.

Materials and methods

Seeds of A. thaliana var. Columbia (col) and its mutantsctr1-1 (Kieber et al., 1993), eto1-1 (Chae et al., 2003;Woeste et al., 1999), etr1-1 (Chang et al., 1993), ein2-1(Guzman and Ecker, 1990), as well as the triple mutantein2-1, ers1-2, etr1-7 (Hall and Bleecker, 2003) were used.

Three different seed treatments were carried out inthe analysis. The first involved dry seeds. For the second,seeds were imbibed for 3 h, and for the third treatment,seeds were imbibed for 24 h in distilled water. For thesecond and third treatments, seeds were previouslysterilized (2min ethanol 70%, 7min 2% sodium hypo-chlorite, 3� washed in distilled water). After steriliza-tion, every seed was placed over a piece of graph paperon a medium containing water–agar (1%).

The visual inspection of seeds of A. thaliana revealed anumber of symmetries and the existence of a longitudinalaxis. In addition, the longitudinal sections resembleellipses and the transversal sections are similar to circles(Figure 1). The seed surface may thus be adjusted by aprolate spheroid. A prolate spheroid is the ellipsoid ofrevolution obtained by rotating an ellipse about its majoraxis (Figure 2). It then has two semi-axes of the samelength as the minor semi-axis of the ellipse (here denotedby b); the third semi-axis of the ellipsoid coincides withthe major semi-axis of the ellipse (here denoted by a).

The seeds were observed, one by one, with a Nikon‘SMZ-2T’ stereo microscope. Considering the seeds as

mbia in longitudinal (left) and polar (right) views.

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Analysis of seed shape in Arabidopsis thaliana: Altered polarity in ethylene mutants 913

ellipsoids of revolution, photographs of the polar andlongitudinal views were taken with a Nikon ‘Coolpix 950’digital camera according to the diagram shown in Figure2 (polar from P1 and longitudinal view from P2). ForColumbia dry seeds, 20 photographs of the longitudinalview and 18 of its transversal view were taken. Regardingother treatments and genotypes, 10 photographs of everyview were used. Photographs were analyzed with thesoftware AnalySISs (Soft Imaging Systems), specialized inimage processing. Long and short diameters were directlyobtained from the photographs. In this process, graphpaper allowed us to convert pixels into mm.

Two independent measures for the areas of every viewwere obtained: one given directly by the image Analy-SISs program and the other calculated using thediameter length in the area formula for circles (polarview), or the axis length for ellipses (longitudinal view).The statistical comparison between both values allows usto accept or reject whether the figures are indeed circlesor ellipses.

In addition, to confirm that the polar view coincideswith a circle, circularity index was calculated (Schwarz,1980). Circularity index, denoted by I, is a measure thatestablishes the degree of roundness of a geometricalfigure in a plane and for a circumference equals to 1. Itwas calculated with the values of area and perimetergiven by the software analysis according to the formula:

I ¼4parea

perimeter2.

To understand changes that occurred in the seedshape, a volume approximation was made. The volumeof an ellipsoid of revolution was calculated from thevalues of the longitudinal view’s area A and the short

Table 1. Statistical analysis (t-test) for the comparison betwlongitudinal and polar views of dry seeds of Arabidopsis cv. C

Longitudinal view

Mean Standard

Calculated area 186,554.59 29,877.4AnalySISs area 193,493.75 31,431.0Probability (0.05) 0.4786

P1

P2

a

b

b

Figure 2. Prolate spheroid (a and b are the long andshort semi-axes, respectively).

semi-axis b obtained from Analysis according to theformula:

V ¼43Ab.

Bezier curves corresponding to the seed poles in theimages were obtained and their curvature valuescalculated according to Cervantes and Tocino (2005).Curvature is expressed in radians per micron (rad/m) andprovides an idea of sharpness or roundness in curves.

Results

Geometric description of seed shape in dryseeds of Arabidopsis thaliana cv. Columbia

The visual inspection of seed images suggeststheir adjustment with an ellipsoid of revolution. Toconfirm this, two tests were developed. One wasbased on comparison of areas, the other utilizedcurvature analysis in both poles.

The values given by the program analysis for thearea of the figures corresponding to longitudinaland polar views of seeds were compared with thosecorresponding to an ellipse (longitudinal view) or toa circle (for the polar view). No differences werefound between the calculated and the observedvalues (Table 1).

Circularity indices for the images observed inpolar views of wild-type Columbia dry seeds yieldedan average value of 0.88 (Table 2).

The comparison of curvature values between thetwo seed poles provides information about whetherthere are differences in shape between them. Inthe case of dry seeds, there was no differencebetween maximum curvature values obtained forboth poles (data not shown).

These results led us to accept the idea that theshape of the dry seed is approximately an ellipsoidof revolution.

Change in the shape of wild-type Columbiaseeds following imbibition

Figure 3 represents graphically the increase inmajor parameters at 3 h and 24 h of imbibition of

een calculated areas and software given areas values forolumbia

Polar view

error Mean Standard error

113 142,716.233 24,534.4761973 148,642.588 25,811.4664

0.4976

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Table 2. Mean and standard error of circularity index for the polar views of dry seeds in the different genotypes understudy

col ctr1-1 ein2-1 eto1-1 etr1-1 triple

Mean 0.87817394 0.85808874 0.85197882 0.86379887 0.85877989 0.84418956Std. error 0.0065203 0.01867804 0.02369746 0.0377162 0.02287831 0.03137029

0.00%

5.00%

10.00%

15.00%

20.00%

25.00%

30.00%

35.00%

long diameter

short diameter

areaperim

eter

volume

% o

f d

ry s

ee

d

3h imbibition

24h imbibition

Figure 3. Diagram showing percent of increase in thedimensions of Arabidopsis thaliana wild-type after 3 and24 h of imbibition.

C. Robert et al.914

Arabidopsis thaliana wild-type Columbia seeds. Theshort diameter presented a 10% increase after 3 hof imbibition showing no further significativechange up to 24 h after imbibition. The longdiameter also increased in the first 3 h, though toa lesser extent (5%), and kept growing until 24 h.Area, perimeter and volume values correspondingto the figures of longitudinal views of seedsincreased proportionally after 3 h of imbibition,showing very little increase between 3 and 24 h ofimbibition.

In the course of imbibition, maximum curvaturevalues decreased in the basal pole but not in theapical pole (Table 3). This suggests that, whiledry seeds are similar to ellipsoids of revolution,after 24 h imbibition, wild-type Columbia seedsresemble the shape of an ovoid (roughly speaking,an egg). The term ovoid refers to a three-dimensional figure obtained by the revolution of acurve (also called ovoid) similar to an ellipse butonly with one axis of symmetry. Although ovoidsand ellipsoids are similar figures and have manycharacteristics in common, there exists an impor-tant difference between them: the lack of sym-metry of ovoids across one equatorial plane. Thisproperty is a reflection of the polarity observed inseed shape.

The shape of dry seeds in mutants in theethylene signaling pathway

Seed volume was larger in the wild-type Colum-bia and in the triple mutant (Table 4) than in themutants eto1-1 and ctr1-1; these genotypes pre-sented shorter lengths in both axes.

In general, the shape of the wild-type Columbiaseeds is more homogeneous than the shape of themutants. This is confirmed by the comparison ofstandard error of the mean values of circularityindex for the polar view (Table 2).

Seeds of the mutant genotypes deviated in anumber of aspects from the wild type. Forexample, eto1-1 and the triple mutant genotypeshave more elongated seeds. This is supportedby the ratios long/short axis length as shown inTable 5. Seeds of etr1-1 and the triple mutant havevery irregular morphologies (Figure 4).

A comparison of changes occurring in theshape of wild-type and mutant seeds afterimbibition

As a consequence of imbibition, a general sizeincrease is expected. The increase was notablygreater in all mutant genotypes than in wild-typeseeds (Table 6).

With respect to mean curvature values at thepoles in dry seeds, no differences were detectedamong genotypes (Figure 5). In the basal pole,curvature values oscillated between 0.0076 for coland 0.0093 for eto, and in the apical pole, between0.0085 for col and 0.0122 for eto. For eachgenotype there were no differences betweencurvature values in both poles in the dry seeds.

After 24h of imbibition, curvature values (Figure 5)decreased in both poles for all genotypes, exceptin the apical pole of the wild-type Columbia seeds.The greatest change in curvature was in thetriple mutant, and was more pronounced in thebasal pole.

Thus, differences in curvature between poleswere detected only in the wild type after 24 h ofimbibition. These seeds had smaller curvaturevalues in the basal pole and larger curvature values

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Table 4a. Main parameters (obtained from Analysis, except volume) corresponding to seeds from different genotypesof Arabidopsis thaliana

Long diam (mm) Short diam (mm) Area (mm2) Perimeter (mm) Volume (mm3)

col 599.85 394.34 1.93�105 1519.33 5.15�107

49.58 36.45 3.14� 104 140.09 1.33� 107

ctr1-1 535.73 340.61 1.48�105 1517.79 3.46�107

38.12 54.77 3.06� 104 148.78 1.26� 107

ein2-1 591.85 370.52 1.78�105 1660.24 4.45�107

40.93 42.40 2.94� 104 129.74 1.26� 107

eto1-1 533.98 309.99 1.30�105 1448.17 2.73�107

36.46 31.29 2.25� 104 115.87 7.35� 106

etr1-1 544.90 365.42 1.66�105 1604.92 4.09�107

60.24 33.99 2.77� 104 134.81 1.01� 107

ein2-1, ers1-2, etr1-7 633.76 375.78 1.88�105 1741.16 4.74�107

73.53 31.45 2.44� 104 130.08 8.47� 106

In bold types, mean values. In normal types (below each mean), standard deviations. Obtained from a sample of 20 seeds for Columbiaand 10 seeds for other genotypes.

Table 3. t-test to compare curvature values in the basal and apical poles. Curvature values were obtained for bothpoles from images of dry seeds and seeds after 24 hour of imbibition of the wild-type Arabidopsis thaliana var. Columbia

Treatments Dry seeds 24 hr imbibed seeds t-student (po0,05)

Apical pole 0,0085 0,0077 0,230Basal pole 0,0076 0,0066 0,001

Table 4b. ANOVA

Genotypes N Alfa ¼ 0.05

1 2 3

eto1-1 10 2.73� 107

ctr1-1 10 3.46� 107 3.46� 107

etr1-1 10 4.09� 107 4.09� 107

ein2-1 10 4.45� 107 4.45� 107

triple 10 4.74� 107

col 20 5.15� 107

Sig. 0.13891324 0.05750903 0.05012817

Comparison of seed volumes among genotypes.

Table 5. Mean values and standard deviations for theratio between long and short diameters in the dry seeds

col ctr1-1 ein2-1 eto1-1 etr1-1 triple

Mean 1.52 1.60 1.61 1.73 1.50 1.70Std. dev. 0.12 0.19 0.12 0.15 0.17 0.24

Analysis of seed shape in Arabidopsis thaliana: Altered polarity in ethylene mutants 915

in the apical pole. Only in this case can we concludethat the approximation of the seed shape to theellipsoid of revolution was not accurate. Wild-typeseeds after 24 h of imbibition look more like ovoids

than like ellipsoids of revolution. For the dry seedsof all genotypes, the approximation by ellipsoids ofrevolution is correct. After 24 h of imbibition, theshape of seeds of all other genotypes, except forthe wild type, is modeled to an ellipsoid ofrevolution.

Discussion

The shape of organs is a property integrated inthe architecture of organisms. Because these resultfrom adaptation to environmental conditionsthrough generations, both are programmed in thegenome and must be accurately regulated. Changesin shape are not the result of the action of one, nora single group of genes or proteins, but need to bethoroughly regulated by the interaction of globalpathways able to integrate signals from differentenvironmental stimuli. The ethylene signal trans-duction pathway is one such global integrativepathway regulating plant architecture (Dolan,1997), and as such it is well described in the modelplant Arabidopsis thaliana (Stepanova and Alonso,2005). Our objective in this work was to investigatewhether mutations in diverse genes encodingproteins of the ethylene transduction pathway

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Genotype Longitudinal view Polar view Genotype Longitudinal view Polar viewcol 2005 eto1-1 2000

ctr1-12005

etr1-1 2005

ein2-12005

triple 2004 (ein2-1, ers1-2, etr1-7)

Figure 4. Photographs of Arabidopsis thaliana cv Columbia in longitudinal and polar views corresponding to dry seedsof all the mutants used in this study.

Table 6. Increase of size (percent) of the six genotypes under study after 24 h of imbibition

col (%) ctr1-1 (%) ein2-1(%) eto1-1 (%) etr1-1(%) triple (%)

Long diameter 7.90 22.26 13.11 14.30 25.82 17.78Short diameter 10.80 45.52 29.63 46.11 34.94 52.45Area (long. view) 20.06 77.56 49.43 67.67 63.00 82.66Perimeter (long. view) 8.84 28.85 19.50 23.88 24.79 28.34Volume 32.24 153.76 96.40 142.67 118.87 180.66

0.0000

0.0020

0.0040

0.0060

0.0080

0.0100

0.0120

0.0140

0.0160

0.0180

col eto1-1 ctr1-1 etr ein2-1 ein2-1etr1-

7ers1-2

Genotypes

Cu

rva

ture

po

les

apical pole dry seed basal pole dry seedapical pole 24hr in water basal pole 24hr in water

Figure 5. Representation of the change in curvature values (rad/micron) in both poles, basal and apical, for allgenotypes. Bars represent curvature values for dry seeds and after 24 h of imbibition.

C. Robert et al.916

could affect seed shape, and to investigatehow. Previous results from our laboratory showedthat ethylene-insensitive mutants had reduced

curvature values in their root apex during earlyroot development (Cervantes and Tocino, 2005) andalso in the embryonic root (Noriega et al., 2007).

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Analysis of seed shape in Arabidopsis thaliana: Altered polarity in ethylene mutants 917

Because the seed contains the embryo, of whichthe radicle is an important part, and also becausecurvature in the poles of the seed is an importantparameter defining seed shape, we reasoned thatthe ethylene mutants could also present eitheralterations in the curvature values of the seedin the poles, or in their polarity. To analyzesuch differences in polarity, or in general variationsin shape, we first adjusted seed shape to ageometric form.

The shape of A. thaliana seeds has been definedhere as prolate spheroid. A prolate spheroid is theellipsoid of revolution obtained by rotating anellipse about its major axis. In a general sense,polarity refers to differences between the poles,which, in the seed, may be due to the asymmetricaldistribution of the embryo, endosperm and othercell types, tissues or structures inside the seedcover. But in the geometric analysis presentedhere, we prefer to use the term polarity in a strictsense, i.e., as the sign of differences in curvaturevalues between the two poles. In this strict,geometrical sense, the A. thaliana var. Columbiawild-type seeds acquired their polarity in thecourse of imbibition. Only after 24 h of imbibitionwas a difference between curvature values in bothpoles observed in the wild-type seeds. Duringimbibition, swelling of the cells leads, in general,to increased roundness through the surface of theseed, but in the wild-type seeds, swelling isrestricted to the basal pole (BP), and does notoccur in the apical pole (AP). Curvature values inthe BP decrease (i.e. the extremes became morerounded), whereas in the AP, curvature maintainsthe values corresponding to the dry seed (Figure 5).Thus, the wild-type Columbia dry seeds areellipsoids of revolution that, as a consequence ofthe increase in polarity that occurs in the courseof imbibition, become ovoids. In contrast, allmutant seeds analyzed in this work are ellipsoidsof revolution that increase their volumes andremain being ellipsoids of revolution after imbibi-tion. Polarity is due to the differential swellingof both poles; it occurs only in the wild typeafter 24 h imbibition and not in any of theethylene signal transduction mutants tested.Thus, the regulation of polarity in seeds is underthe control of the ethylene signal transductionpathway.

In the course of imbibition, the seeds swell,resulting in an increase in volume. This increase involume is greater in the ethylene mutants, andin particular in the triple mutant ein2-1, ers1-2,etr1-7 (Table 4) and is in agreement with higherwater uptake rates during seed imbibition in themutants (unpublished results). The relationship

between water distribution in seeds and ethylenehas been reported previously (Fountain et al.,1998).

Water uptake during imbibition is triphasic, andthe first phase is mainly the consequence of matricforces (Bewley and Black, 1994). Thus, our resultssuggest that matric forces are restricted in thewild-type seeds when compared with mutants.Seed swelling during imbibition is due primarily toan increase in the length of the short axis that isassociated with increased seed volume very earlyupon imbibition, and followed by a slight elonga-tion (increase in the long axis; Figure 3). Thenumber of cells remains constant before seedgermination and divisions occur only after radicleprotrusion (Barroco et al., 2005). Thus, theincrease in seed volume may be attributed to arapid hydration of intercellular spaces as well asswelling of the cells. As a consequence, the seedbecomes more rounded, and the curvature in thebasal pole decreases. The increase in volume is alsorelated to the circularity index in the polar view.The lowest circularity index in the polar viewoccurs in mutant seeds, and in particular in thetriple mutant. This is associated with a greatervariation (i.e. higher standard error values,Table 2) and, thus, with increased irregularity inseed shape that may allow a greater increase involume than in the case of more regular seeds, withhigher circularity index, as in the wild-type seeds.

Water uptake through the seed is more uniform inseeds of mutants in the ethylene signal transduc-tion pathway (in particular the triple mutant) thanin wild-type seeds. In the mutants, there is areduction in curvature in both poles, whereas in thewild type, there is not such a reduction in curvaturein the apical pole. During imbibition, the wild-typeseeds must have a mechanism that creates polarity,i.e. that maintains the initial curvature values inthe apical pole and generates differences incurvature values between both poles with in-creased values (curves more pronounced) in theapical pole. This may be related with the reducedincrease in volume observed in the wild type, and,in general, with the control of seed shape. In theethylene signal transduction pathway mutants, theprocess of establishment of polarity is disrupted,and in the triple mutant, the shape of the seeds isvery irregular and distorted. Changes in polarcurvature values during imbibition are maximal inthis genotype. Thus, our results show the relationbetween the creation of polarity in the seeds(altered in all the ethylene mutants) and theoverall maintenance of a regular shape in the seeds(altered in the triple mutant with high variation inthe shape and aberrant seeds).

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C. Robert et al.918

Two signaling mechanisms, depending on auxinsand cell wall components, respectively, are im-portant in the establishment and maintenance ofpolarity in plant cells (Souter and Lindsey, 2001).Ethylene signal pathways interact closely withauxin action (Rahman et al., 2002; Stepanovaet al., 2005), but also it has been long known thatethylene may have an effect in the cytoskeleton(Hepler and Palevitz, 1974). Auxins also interactwith the cytoskeleton (Sun et al., 2004). Thus, cellshape and cell wall structure may be affected bythe direct action of ethylene signaling pathway oncell wall components, or indirectly, via changes inregulatory proteins, alterations in the sensibility tohormones, calcium or other secondary messengersor affecting general physiological aspects like theredox status.

Geometric polarity is the result of anatomicalpolarity and may be accurately measured bycurvature values in both apical and basal poles. Inthe course of seed imbibition in the wild-typeseeds, geometric polarity, i.e. the differencesbetween maximum curvature values in both poles,increase. Our results show that an intact ethylenesignal transduction pathway is required for theestablishment of polarity upon seed imbibition.Wild-type seeds show this polarity at 24 h ofimbibition, which is apparent as differences be-tween curvature values in both poles. Mutants inany of the genes encoding proteins of the ethylenesignal transduction pathway resulted in a lack ofpolarity after 24 h of imbibition.

The current model representing the ethylenesignal transduction pathway has been built onthe basis of diverse experimental evidence fromdifferent sources that include the analysis ofmutant genotypes, genetic analysis of epistasis,yeast transformation, two-hybrid interaction, etc.(Stepanova and Alonso, 2005). The genetic analysisis based generally in an all or nothing, lineal effectof mutants, where all mutations tend to be groupedinto two classes: first, those turning the mechanismoff, and second, those maintaining the system in anactive status resulting in the transcriptional activa-tion of ethylene-regulated transcription factors andgenes. In the first group, phenotypes resemblewild-type plants treated with ethylene inhibitors(either of action or synthesis). The second group ofmutants are characterized by phenotypes thatresemble wild-type plants treated with ethylene.In this model, it is difficult to find new phenotypesfor elements redundant in the model. For example,etr1-1 mutant has a dominant constitutive effect.Its phenotype is, in many aspects, similar to that ofthe mutant ein2-1. Both mutants yield plants thatresemble wild-type plants treated with ethylene

inhibitors. It has been much more difficult to findphenotypes for null mutants in the etr1 gene suchas etr1-7, because as predicted in the model, theeffect of this mutant will be complemented by theaction of genes encoding homologous proteins suchas etr2, ers1, ers2 or ein4 (Qu et al., 2007).Similarly, if all the receptor’s actions converge inEIN2, as predicted in the model, then it would beimpossible to find an effect for the triple mutantein2-1, ers1-2, etr1-7 presenting additional traitsother than those described for the null mutantein2-1. In fact, developmental defects found in thistriple mutant, including altered cell size, werefound to be ein2-dependent (Hall and Bleecker,2003). Nevertheless, further analysis demonstratedthat the triple mutant also presents growthresponses that are not present in ein2-1 nullmutants (Binder et al., 2004), thus demonstratingthat not all the activities of the ethylene receptorsare mediated through the action of EIN2 protein. Inthis work, we have reported developmental altera-tions in the triple mutant. They are (1) irregula-rities in seed shape, (2) abnormal increase in seedvolume during germination and (3) abnormalreduction of curvature values in both poles of theseed and lack of polarity. Of these traits, 1 isexclusive in the triple mutant and is absent in ein2-1 mutants, whereas 2 and 3 are more notable in thetriple mutant but are also present in ein2-1. Thisresult indicates that null mutants in both receptorsETR1 and ERS1 have additional effects other thanthose mediated via EIN2 protein, and presentnew traits suitable for further analysis of thephysiological action of the proteins. Interestingly,the role for ETR1 receptor protein in hydrogenperoxide signaling in stomatal guard cells hasbeen recently shown (Desikan et al., 2005),suggesting that the ethylene receptors may beinvolved not only in ethylene binding and signaltransduction but also as transducers of hydrogenperoxide. Ethylene receptors may be sensors of theredox state in the cells, involved in generalregulatory circuits beyond ethylene sensing. Thishighlights an important aspect of broad interestin genetics concerning nomenclature. When weaccept the name of a gene, we must be aware thatthe name indicates one function, but never theonly function. Ethylene mutants are altered notonly in ethylene perception or signaling but also inother processes.

A common effect reported here for all theethylene mutants consists in the alteration ofpolarity. This indicates that polarity may be viewedas the result of a balanced situation in which,alterations in any of two opposite directions wouldresult in a reduced curvature resulting in the lack

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Analysis of seed shape in Arabidopsis thaliana: Altered polarity in ethylene mutants 919

of polarity. This is in contrast with the knownphenotypes in ethylene signaling pathways, result-ing in all or nothing responses where two contrast-ing effects are always observed similar to thepresence or absence of the phytohormone. On thecontrary, the response consisting in the lack ofpolarity resembles more a non-linear process, inwhich polarity is observed when the conditionsreach an equilibrium but disappears in case theequilibrium is disrupted. Further disruption resultsin severe shape alterations. It may be interesting toinvestigate the specific effects that redox statusand free radicals may have in polarity.

Acknowledgments

We thank the late Dr. Anthony Bleecker and Dr.Brad Binder for kindly providing us with seeds of thetriple mutant ein2-1, ers1-2, etr1-7. This work hasbeen partly supported by Junta de Castilla y Leon,under the project SA071A07.

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