noriega etal 2008

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Journal of Plant Physiology ](]]]]) ]]]]]]

Ethylene responses in Arabidopsis seedlings include

the reduction of curvature values in the root cap

Arturo Noriegaa, Emilio Cervantesa,, A`ngel Tocinob

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

KEYWORDS

Arabidopsis;Bezier curve;Curvature;Ethylene;Root apex

SummaryRecently, curvature was described as a new trait useful in the analysis of root apexshape. Treating the root profile as a geometric curve revealed that root apexcurvature values are lower in ethylene-insensitive mutants (Cervantes E, Tocino A.Geometric analysis of Arabidopsis root apex reveals a new aspect of the ethylenesignal transduction pathway in development. J Plant Physiol 2005;162:103845). Thisfact suggests that curvature is regulated by ethylene. In this work, we havedetermined the curvature values in embryonic roots of wild-type Columbia as well asin ethylene signal-transduction mutants, and found smaller values in embryos of themutants. We also report on the evolution of root curvature during early development

after seed germination. The line Lt16b that expresses GFP in the cell wall hasallowed us to investigate the evolution of curvature values in three successive celllayers of seedling roots by confocal microscopy. Treatment of seedlings withnorbornadiene resulted in lower curvature values. Our results show detailsillustrating the effect of ethylene in root curvature.&2007 Published by Elsevier GmbH.

Introduction

Plant hormones affect root growth, morphology

and development in a cell-specific manner. Ethy-lene has a demonstrated effect on root growth.Ethylene-insensitive mutants etr1-1 (Bleecker etal.,1988) and ein2-1 (Roman et al., 1995) havelonger and thinner roots than wild-type plants. Onthe other hand, constitutive triple response (ctr1-

1; Kieber et al., 1993) or ethylene over-producermutants (eto1-1;Chae et al., 2003;Woeste et al.,1999) have shorter and thicker roots. Both groupsof mutants represent two diverging patterns ofdevelopment showing characteristics that can bemimicked by adding ethylene or ethylene actioninhibitors to the wild-type seedlings. Adding exo-genous ethylene mimics the effect of the mutantseto1-1 and ctr1-1 and results in short and thickroots with long root hairs. On the contrary, addingethylene action inhibitors mimics the effect of theethylene-insensitive mutants etr1-1 and ein2-1

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0176-1617/$ - see front matter&2007 Published by Elsevier GmbH.doi:10.1016/j.jplph.2007.10.001

Corresponding author. Tel.: +34923219606; fax:+34923219609.

E-mail address:[email protected] (E. Cervantes).

Please cite this article as: Noriega A, et al. Ethylene responses in Arabidopsis seedlings include the reduction of curvature values inthe root.... J Plant Physiol (2007), doi:10.1016/j.jplph.2007.10.001
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resulting in long, thin roots with small root hairsdispersed through the root. The list of easilyscorable macroscopic characteristics that maycontribute to define root morphology is not long:root width, root length, number of root hairs, etc.The definition of a new phenotypic trait can be asignificant contribution in the genetic analysis of

root development.Recently, we defined root curvature as a new

trait in relation with the shape of the root.Basically, root apex curvature is a measure of thedegree of roundness or sharpness in the root apex.The analysis of Arabidopsis roots revealed that apexcurvature values in ethylene-insensitive mutants(etr1-1andein2-1) were smaller than those in rootsof wild-type Columbia (Cervantes and Tocino,2005).

To further investigate the regulation of root apexcurvature and, in particular, the involvement of the

phytohormone ethylene in this process at earlystages of root development, we present theanalysis of curvature values in embryos of wildtype and mutants in the ethylene signal-transduc-tion pathway. The evolution of curvature duringearly root development, after germination is alsoreported. Using the Arabidopsis line Lt16b (Cutleret al., 1999) that expresses GFP in the cell walls,curvature has been analyzed by confocal micro-scopy at early stages of root growth in three celllayers of plants grown in presence of norborna-diene, an ethylene action inhibitor. Our resultsallow us to discuss in detail the role of ethylene in

the regulation of this characteristic.

Materials and methods

Plant material

Seeds of Arabidopsis thaliana cvs. Columbia (col) andWassilewskja (ws) and line Lt16b (col) expressing GFP inthe cell wall (Cutler et al., 1999) were imbibed in 1%wateragar plates and stored for 3 days at 4 1C to allow

cold stratification. Then, the plates were incubated in agrowth chamber under a light/dark cycle (18h light,25 1C; 6h dark, 20 1C). Norbornadiene (norbornadiene issynonymous of bi-cycle-hepta-diene) from Aldrich wasused at 30 mM for the treatments. After 12, 24 and 48 h ofincubation, seedlings were carefully collected withforceps and deposited individually on microscope slideswith a drop of water and covered with coverslips for theirobservation. For the analysis of curvature in embryonicroots, seeds were imbibed in water and the embryosisolated after putting a drop of water containing theseeds between a slide and a coverslip and applying gentlepressure.

Microscopic examination and image analysis

The slides were observed with transmitted light underan inverted microscope (Leica DMIRB) or in confocalmicroscopy (Leica TCS-SPII) with an objective of 60 .Images of the roots were taken and stored for analysis.

The program AnalySISs was used to obtain data fromthe images. A series of 21 points were marked along theroot apex profile. The series began at one side of the rootsurface close to the quiescent center, and then continuedby marking 10 points upwards to the root tip and 10points downwards to the opposite side of the quiescentcenter in the root profile. The program gives for eachpoint a pair of coordinates (x, y).

The same procedure was employed to obtain curvesreproducing the shapes corresponding to the cell walls inthree successive layers of the root apex. For these,confocal images corresponding to the medial section ofthe root in line Lt16b were obtained (Figure 1).

Curve approximation and geometric analysis

With each 21-point data set, the Be zier curve (Bezier,1968) was obtained to adjust the root profile (fortransmitted light images) or the layer shape (for confocalimages), seeFigures 1 and 4.

The curvatures of these Bezier curves were calculatedfrom their parametric Eqs. (x(t), y(t)) by means of theformula

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Figure 1. Confocal microscopy image of the medialsection of a plant of the line Lt16b, which expressesGFP in the cell wall, showing the three successive layersused in the determination of curvature values. The pointsused for the definition of the Bezier curves correspondingto the three cell layers and the resultant curves havebeen added.

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kt jx0ty00t x00ty0tj

x0t2 y0t23=2 ; t2 0; 1.

Curvature represents the rate at which the unittangent vector is changing with respect to arc length.For our adjusted Bezier curves the values of thecurvature near the extremes of the interval are low,since the curve is similar to a line in their proximities.

When the parameter moves from one extreme to thecenter of the interval, curvature increases, reaching itsmaximum value at a point corresponding to the root tip.For each adjusted curve, the maximum value of thecurvature was calculated and variables containing thesemaxima for each layer were created. Curvature valuesare given here in radians/micron.

A Mathematicas code which, from a set of input datapoints, gives the Be zier curve, its graphical representa-tion, the graphic of a curve representing the curvatures(Figure 4) and the maximum value of the curvature hasbeen written out and is available upon request at theauthors e-mail addresses. For more details, see Cer-vantes and Tocino (2005).

Statistical analysis

To test hypotheses about the equality of curvaturemean values for different populations, data weresubjected to analysis of variance (ANOVA) followed byTukey and Duncan multiple comparison tests. Thestatements made in the text are based on differencesthat were statistically significant at po0.05. Theseprocedures were carried out using SPSS. All experimentsin this study were repeated at least once with similarresults.

Results

Genotypic differences in curvature values

Mean root apex curvature values in embryos werecompared in four different genotypes. Theseinclude two varieties: Columbia and Wassilewskja,and two mutant genotypes in the genetic back-

ground of Columbia: eto1-1 and etr1-1 (Figures 2and 3). The same genotypes were analyzed at 24and 48 h (Figure 3).

Curvature values are smaller in embryonic roots

of ethylene-insensitive etr1-1 mutants

Curvature values in embryonic roots of etr1-1

mutants are significatively smaller than values foreto1-1 and Col.

Curvature values at 24 h are smaller in

Wassilewskja than in Columbia

In agreement with the above statement forembryos and with previous results (Cervantes andTocino, 2005), curvature values at 24 and 48 h weresmaller in etr1-1 than in wild-type Columbia or ineto1-1genotypes. Curvature values for Wassilewsk-ja did not present significant difference with any ofthe other genotypes both in embryos and at 48 h,

but when the values at 24 h were compared, twohomogeneous groups appeared: one comprisingeto1-1 and wild-type Columbia, and the other oneincluding genotypes Wassilewskja and etr1-1.

Curvature values in the root surface increaseduring the first days of root growth

The evolution of curvature values in the course ofthe first 2 days of root development followinggermination was investigated in the varietiesColumbia and Wassilewskja, and genotypes eto1-1

andetr1-1 (Figure 3).Curvature values in roots after 48 h growth weresignificantly greater than in embryos for allgenotypes, whereas increases in curvature valuesbetween embryos and 24 h were only observed inCol and eto1-1.

Multivariate ANOVA leads us to conclude thatthere exists significant interaction between vari-ables genotype and age. Further analysis revealsthat the significance of the interaction may be

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Figure 2. Representative transmitted light images of wild-type Columbia (a) and etr1-1 (b) embryos, used in thedetermination of curvature values. Bar equals 100 mm.

Ethylene response in root apex curvature 3



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explained by the different rate of evolution ofcurvature values in the two varieties, Columbia andWassilewskja (Figure 3).

Correlation between curvature values in thesuccessive cell layers of the Lt16b line

Figure 1contains a representative image of theroot apex of a seedling of the line Lt16b showingthe three cell layers in which curvature values weredetermined at 12, 24 and 48h. A significant positivePearson correlation was found between curvaturevalues of layers 1, 2 and 3 (Table 1).

Curvature values increase with time in thecell layers of root apex in the Lt16b line

In layers 1 and 2, curvature increased from 12 to24 h, as well as from 24 to 48 h. With respect tolayer 3, curvature values were higher at 48 h thanat 12 or 24 h, but there was no evidence to affirmthat curvature changed between 12 and 24 h.

Effects of norbornadiene, an inhibitor ofethylene action, in curvature of three celllayers of the Lt16b line

Norbornadiene alters the evolution of curvature

in the outer cell layers

Curvature values in layer 3 of seedlings treatedwith norbornadiene followed the same pattern ofevolution as that of the controls, i.e., at 48 h theywere higher than at 12 or 24 h, and there was nochange between 12 and 24 h. In contrast, in rootstreated with norbornadiene, there was no differ-

ence in curvature between layers 1 and 2 (Figure5).

Norbornadiene causes a significant reduction in

curvature values

Curvature values between the controls andnorbornadiene-treated seedlings were comparedfor the three cell layers (Figures 4 and 5).

Norbornadiene affected in a different way thedifferent layers and its effect was dependent onthe time of exposure. Thus, significant differencesbetween control and norbornadiene treatmentswere observed in the outer layer (L1) at 24 and48 h after seed imbibition as well as in theintermediate layer (L2) at 48 h. Norbornadiene

treatment had no significant effect on any of thethree layers at 12 h. Norbornadiene treatment hadno significant effect on the inner layer for the threetime points considered, nor in the intermediatelayer at 24 h.

Discussion

The distal part of the root is formed by the rootcap that has a function in the penetration through

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Figure 3. Evolution of root apex curvature values for the root surface curves of col, ws, eto and etr genotypes inembryos, as well as 24 and 48 h after transfer to light.

Table 1. Correlations between curvature values inlayers

Layer 1 Layer 2 Layer 3

Layer 1 1 0.701** 0.612**Layer 2 1 0.593**Layer 3 1

**Significant level po0.01.

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Figure 4. Analysis of root apex curvature in seedlings of the line Lt16b germinated in wateragar (control; left) or30mM norbornadiene (right). Top: confocal images. Bottom: graphic representation of curves corresponding to each ofthe three layers (L1, L2, L3; from top to bottom) and their corresponding curvatures.

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Figure 5. Evolution of curvature values in the three layers of control and norbornadiene-treated roots in seedlings at12, 24 and 48 h after transfer to light.




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the soil, opening the way and indicating the placesfor further root development. In the center of theroot cap, the columella is formed by a group of cellsthat have a role in gravity sensing (Ottenschlager etal., 2003). The columella initials are a reducedgroup of meristematic cells located between thecolumella and the quiescent center, that, by

successive divisions, give rise to the columellaand the root cap cells. The root cap has itsembryonic origin in the first cellular division andderives from the cell remaining under the plane ofcell division, whereas all the other cells andstructures of the plant originate from the cell thatremains, after this first division, located over thehorizontal plane. Gravitropic sensing and soilpenetration are known functions of the root cap.Its form is an adaptation to these purposes: the rootends in a conic shape that facilitates penetrationthrough the soil.

In Arabidopsis, the region comprising the colu-mella and root cap is formed by three to five celllayers (Figure 1), which are being constantlyrecycled. Penetration through the soil causes aconstant deterioration of the cells in the outerlayer that need to be replaced by new cellsproduced in the meristem.

Ethylene is involved in the regulation of root sizeand shape (Stepanova et al., 2005), as well as in thegravity-sensing process (Chang et al., 1993). Thus,it has an effect on multiple aspects of root shape aswell as on growth rate. We demonstrated recentlythat root apex curvature was reduced in ethylene-

insensitive mutants (Cervantes and Tocino, 2005),opening the possibility that ethylene regulates rootapex curvature. In this work, we have shown thatembryos of ethylene-insensitive mutants etr1-1also have smaller root apex curvature values thanembryos of the wild-type Columbia. It remains tobe investigated whether this difference is due toethylene action during embryogenesis or to othereffects of the etr1-1 mutation that may bepleiotropic and independent of ethylene.

We also report here data on the evolution of rootapex curvature during the first days of root growth.

From the onset of germination, root apex curvaturevalues tend to increase in all genotypes andvarieties tested. This is associated with the cellelongation process that occurs prior to, and is anessential part of, germination. Curvature values inall experiments were significantly smaller in etr1-1than in the wild type. At 24h, curvature valueswere smaller in Wassileskja than in Columbia.Curvature values increased from embryos to 48 hin etr1-1, but in this genotype there was noevidence of change between embryos and 24h.This indicates a role for ethylene in the increase of

the curvature values that occurs during andimmediately after germination. This conclusion isalso supported by our results with the ethyleneaction inhibitor norbornadiene (see later). Ourresults also point towards a reduced ethylenesensitivity early upon germination in roots of thevariety Wassileskja that may deserve further

investigation.A comparison between curvature values in the

three layers of the root cap shows that curvatureincreases in the successive layers from the centerto the periphery, this being in agreement withincreased values in the outer layer with time,because in both cases it reflects smaller curvaturevalues in layers formed by younger cells. Cells inthe outer layer of younger roots are younger thanthose in the outer layer of older roots, whereas inthe same root, cells in the inner layer are youngerthan those in the outer. Thus, larger curvature

values are associated with differential cell elonga-tion that occurs concomitantly with progress in rootcell maturity in early developmental stages. Ethy-lene has been involved in processes of cell elonga-tion in other cell types (Le et al., 2001, 2004;Lehman et al., 1996; Rahman et al., 2002). Itinteracts with other hormones in the regulation ofthese processes, in particular with auxin (Stepano-va et al., 2005) and with gibberellin (Achard et al.,2003).

The experiments reported above indicated aneffect of ethylene in the development of rootcurvature. To verify this possibility, the seedlings

were germinated and allowed to grow in thepresence of the ethylene action inhibitor norbor-nadiene. Differences in curvature values betweenroots of seedlings grown in norbornadiene and thecontrols were detected in the two outer layers ofcells after 48h, with smaller values in thenorbornadiene-treated roots (Figures 4 and 5).

The reported effect of norbornadiene in rootapex curvature is thus rapid, and points towardsthe involvement of ethylene in the development ofroot curvature in early root development aftergermination. Ethylene has been involved in micro-

tubule organization as well as in cell wall poly-saccharide synthesis (Le et al., 2004), bothprocesses that may contribute to the accurateshaping of the cells in this part of the root. Thus, itis not surprising to find that inhibition of ethyleneaction by norbornadiene has a rapid effect on thecellular growth processes underlying root curva-ture. It may be interesting to investigate rootcurvature in mutants with particular aspects ofcytoskeleton or cell wall synthesis altered and toanalyze other factors involved in this new pheno-

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type as well as to explore in detail the associationbetween curvature and differential cell growth.

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