Evidence that ethylene signalling is not involved in selective root placement by tobacco plants in response to nutrient-rich soil patches

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457

Research

Blackwell Publishing Ltd

Evidence that ethylene signalling is not involved in selective root placement by tobacco plants in response

to nutrient-rich soil patches

Eric J. W. Visser

1

, Gerard M. Bögemann

1

, Maaike Smeets

1

, Susanne de Bruin

1

, Hans de Kroon

1

and Tjeerd J. Bouma

2

1

Department of Experimental Plant Ecology, Institute for Water and Wetland Research, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen,

the Netherlands;

2

NIOO-KNAW Centre for Estuarine and Marine Ecology, Korringaweg 7, 4401 NT Yerseke, the Netherlands

Summary

• Ethylene is a strong controller of root development, and it has been suggested thatit is involved in the increase of lateral root development in nutrient-rich soil patches(selective root placement). Here, this contention was tested by comparing selectiveroot placement of an ethylene-insensitive transgenic tobacco (

Nicotiana tabacum

)genotype (Tetr) with that of wild-type (Wt) plants.• Wt and Tetr plants were grown in pots with locally increased nitrate or phosphateconcentrations, after which the root growth patterns were compared with those ofplants grown in pots with homogeneous nutrient distribution.• Tetr plants responded to nutrient patches in a similar way to Wt plants, by placingtheir roots preferentially at locations with higher nutrient content, and other aspectsof plant morphology were also not greatly influenced by ethylene insensitivity. Theresponse of both Wt and Tetr plants to high-nitrate patches was considerablystronger than to locally high phosphate, suggesting that the two responses are notlinked in any functional or regulatory way.• As the response to nutrient patches was similar in ethylene-sensing and ethylene-insensitive plants, it is concluded that selective root placement in response to nitrateor phosphate is, at least in tobacco, not mediated or modified by ethylene action.

Key words:

ethylene insensitivity,

Nicotiana tabacum

(tobacco), nitrate, nutrientpatches, phosphate, root foraging, selective root placement, soil heterogeneity.

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© The Authors (2007). Journal compilation ©

New Phytologist

(2007)

doi

: 10.1111/j.1469-8137.2007.02256.x

Author for correspondence:

E. J. W. Visser

Tel:

+

31 24 3653382

Fax:

+

31 24 3652409

Email: [email protected]

Received:

17 July 2007

Accepted:

14 August 2007

Introduction

Root system architecture is a crucial trait for plants, as itlargely determines how efficiently water and nutrients will beextracted from the soil. There are almost as many types ofroot systems as there are plant species, but all have in commonthat their architecture is not fixed but can respond toenvironmental stimuli. A well-known but not well-understoodresponse to the environment (De Kroon & Mommer, 2006)is the change in root extension and lateral root development

induced by locally high concentrations of nutrients in otherwiseless rich soils (Hutchings & de Kroon, 1994; Hodge, 2004).This phenomenon is referred to as selective root placement(Fransen

et al

., 1998), and allows newly formed roots tospecifically develop at those sites in the soil where nutrients aremost abundant.

In contrast to the regulation of above-ground morphologicalchanges of plants in response to their environment, relativelylittle information exists on the mechanisms that controlbelow-ground morphological responses. The regulatory

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pathways of selective root placement have received attentionsince Zhang & Forde (1998) showed that expression of theArabidopsis gene

NITRATE REGULATED 1

(

ANR1

), codingfor a MADS-box transcription factor, was essential for selectiveroot placement in response to a high-nitrate patch. Anotherpaper from this group (Zhang

et al

., 1999) indicated a role forauxin via

AXR1

, as might be expected in a response thatcomprises lateral root formation (Benkova

et al

., 2003), but laterwork by Linkohr

et al

. (2002) could not confirm these results.Apart from these few studies, the modes of signal perception,signal transduction and the subsequent developmentalprocesses that underlie the selective root placement responseare largely unknown (Forde, 2002; De Kroon

et al

., 2003;López-Bucio

et al

., 2003; Hodge, 2004). It has, however,repeatedly been suggested that nutrient-induced changes inroot morphology may involve the action of the gaseous planthormone ethylene (Lynch & Brown, 1997; Borch

et al

., 1999;López-Bucio

et al

., 2003). Ethylene can positively affect lateraland adventitious root initiation (Drew

et al

., 1979; Visser

et al

.,1996; Clark

et al

., 1999; Steffens

et al

., 2006) and influencesroot extension rates (e.g. Chadwick & Burg, 1970; Jackson

et al

., 1981; Visser

et al

., 1997; Visser & Pierik, 2007) and rootmeristems (reviewed by Casson & Lindsey, 2003; Souter

et al

.,2004), and may thereby have strong effects on root systemarchitecture. Borch

et al

. (1999) showed that root architecturalresponses of

Phaseolus vulgaris

to low-phosphorus treatmentcould be counteracted by ethylene inhibitors and restored bysimultaneous ethylene supply. A significant role of ethylene inthe signal transduction of nutrient-induced selective rootplacement may therefore be expected, but, to our knowledge,this possibility has never been directly investigated. Here wequantified the importance of ethylene in the growth responseof root systems to soil patches with high nutrient content, bycomparing the responses of transgenic ethylene-insensitiveplants with those of the wild-type genotype.

For our study we used tobacco (

Nicotiana tabacum

) plants(Tetr) in which a defect Arabidopsis ethylene receptor (

etr1

)has been introduced (Knoester

et al

., 1998). This genotypehas successfully been used in recent studies of above-groundcompetition of plants, in which ethylene was identified as amodifier of shade avoidance responses (Pierik

et al

., 2004).Ethylene insensitivity resulted in slower responses to thepresence of neighbouring plants, which led to the plantslosing the competition for light (Pierik

et al

., 2003). As single-grown Tetr plants have a shoot phenotype and growth ratesthat are very similar to those of wild-type plants (Pierik

et al

.,2003; Tholen

et al

., 2004), we assumed that they were suitablefor application in selective root placement studies.

Many experiments involving selective root placement haveused nutrient patches with increased concentrations of severalnutrients at the same time, for example through the supply ofpotting soil or other substrates with a high organic content(e.g. Fransen

et al

., 2001; Hodge, 2003). However, it is nowwell documented that responses may differ with the type of

nutrient (Robinson, 2004). Both nitrate and phosphate patchesinduce a higher lateral root weight in the high-nutrient patch,but, depending on the nutrient inducing the response, thisincrease may be accomplished by root elongation or by anincrease in the number of lateral roots (Zhang & Forde, 1998;Linkohr

et al

., 2002). We therefore studied the selective rootplacement of ethylene-insensitive plants in response to nitrateand phosphate patches separately. If ethylene plays any role inthe signalling of one of these nutrients, or in the transductionpathway leading to selective root placement, we would expecta differential response between ethylene-insensitive and wild-type plants to nitrate or phosphate patches, or to both.

Materials and Methods

Plant growth

Seeds of wild-type (Wt) and ethylene-insensitive, transgenic (Tetr)tobacco (

Nicotiana tabacum

L. cv. Samsun NN) were incubatedin vertically positioned Petri dishes on wet filter paper. Growthroom conditions for germination were 22

°

C, with 16 h light(Philips TLD 36 W/840, supplemented with Philips SON-Tplus 600 W; Philips, Eindhoven, the Netherlands) at 175 µmolphotosynthetic photon flux density (PPFD) m

–2

s

–1

and 8 h dark.The Tetr line was produced by Knoester

et al

. (1998), by introduc-ing the mutant

etr1-1

gene of

Arabidopsis thaliana

, coding fora defective ethylene receptor, into Wt plants. These plants displaya strong insensitivity to ethylene (Knoester

et al

., 1998; Pierik

et al

.,2003, 2004), but show no apparent mutant phenotype whengrown individually under optimal conditions (Tholen

et al

., 2004).After 14 d, seedlings were transferred to a climate chamber

with similar conditions but higher light intensity than describedabove (21

°

C; relative humidity 55%; 330 µmol PPFD m

–2

s

–1

)and planted in plastic 104-cell packs filled with a sand–vermiculitemixture (1 : 1; volume/volume (v/v)). The cell packs werethen placed in a shallow container lined with irrigation mats(Brinkman Agro BV, ‘s-Gravenzande, the Netherlands). Thesand–vermiculite mixture was saturated with full-strengthnutrient solution containing 5 m

M

KNO

3

, 0.5 m

M

KH

2

PO

4

,2 m

M

CaCl

2

, 1.2 m

M

MgSO

4

, 30 µ

M

Fe-EDTA, and micro-nutrients. The sand substrate had first been heated at 120

°

Cfor 24 h to prevent infection by soil pathogens, as Tetr plantsare highly susceptible to root rot, even by microorganisms thatare nonpathogenic for Wt plants (Geraats

et al

., 2003; E. J. W.Visser

et al.

, unpublished data). Sufficient water to compensateevapotranspiration was provided every 2–3 d, and nutrientsolution was supplied after 1 wk. When the juvenile plantshad reached a rosette diameter of

c

. 50 mm, which was approx.17 d after being planted in cell packs, a subset of plants selectedfor homogeneity were transplanted to individual free-drainingpots (top diameter 14 cm; bottom diameter 11.5 cm; soil depth13 cm), containing 1.8 l of a 1 : 1 (v/v) sand–vermiculite mixturesaturated with tap water. Immediately after replanting, eachpot received 160 ml of full-strength nutrient solution.


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Localized nutrient supply

Two separate experiments were performed, in which nitrate(‘nitrate experiment’) and phosphate (‘phosphate experiment’)distributions were either homogeneous or heterogeneous.Starting 2 d after the plants had been placed in single pots,nutrient solution was provided three times a week, creatinghomogeneous and heterogeneous nutrient distributionsvia a dripping system (Fig. 1). This method was first designedby Campbell & Grime (1989) and later modified by Jansen

et al

. (2006). In brief, four 60-ml syringes (with the plungerremoved) were positioned vertically above a pot, and 40 mlof nutrient solution was poured into each syringe. Silicontubing, connected to a small section of nylon tubing(internal diameter 0.5 mm), led the solution from a syringe tothe surface of a specific, marked quadrant of the pot, at a rateof 25 ml min

–1

. Pots with homogeneous nutrient distributionreceived full-strength nutrient solution (see previous section;5 m

M

nitrate; 0.5 m

M

phosphate) in all quadrants. In contrast,

pots with heterogeneous nutrient distribution in the nitrateexperiment received full-strength nutrient solution with extra7.5 m

M

Ca(NO

3

)

2

in one quadrant (referred to as the ‘rich’ orhigh-nitrate quadrant; final concentration 20 m

M

nitrate),whereas the other three (‘poor’ or low-nitrate) quadrants receivednitrate-deficient nutrient solution (0.3 m

M

), in which most ofthe KNO

3

had been replaced by K

2

SO

4

(resulting in equimolarK

+

concentrations). In the phosphate experiment, heterogeneouspots received extra 1.5 m

M

NaH

2

PO

4

in the ‘rich’ (high-phosphate) quadrant (final concentration 2 m

M

phosphate),whereas K

2

SO

4

partially replaced KH

2

PO

4

(equimolar K

+

)in the ‘poor’ (low-phosphate) quadrants, resulting in a finallocal concentration of 0.03 m

M

phosphate. Special care wastaken to let the four outlet tubes drip at exactly the same flowrate, as any deviation might lead to imprecise positioning ofthe nutrient patch (i.e. ‘rich’ nutrient solution infiltrating intothe ‘poor’ quadrants of the pot). The total amount of 160 mlnutrient solution per pot was sufficient to bring the pots tofield capacity. Excess nutrient solution drained freely via holesin the bottom of the pots. The resulting nutrient concentrationsin the pore water were checked by drawing samples via porouscups (Rhizon SMS-10 cm; Eijkelkamp Agrisearch Equipment,Giesbeek, the Netherlands) and analysing phosphate and nitrateconcentrations colorimetrically with Technicon AA II systems(Technicon Industrial Systems, Tarrytown, NY, USA), usingammonium molybdate- and hydrazine sulphate-based assays,respectively (Lamers

et al

., 1998). Differences in concentrationsbetween the ‘rich’ quadrant and the opposite ‘poor’ quadrantin the planted heterogeneous pots 2 d after nutrient solutionwas provided (i.e. immediately before the next application ofnutrients) were on average

>

80-fold for nitrate and

>

50-foldfor phosphate towards the end of the experiment (Fig. 2). Eventhe two quadrants adjacent to the ‘rich’ quadrants showedonly very low nitrate and phosphate concentrations (Fig. 2),indicating minimal diffusion of nitrate and phosphate betweenquadrants. This concurs with previous measurements indiscontinuous dripping systems (Johnson & Biondini, 2001;Jansen

et al

., 2006), and also with the maximum rates ofdiffusion calculated for nitrate and phosphate in such systemsby Johnson & Biondini (2001), which were estimated toapproximate only 0.66 and 0.02 cm d

–1

, respectively.

Final harvest

The diameter of the rosette was measured throughout theexperiment, and plants were harvested when they reached (onaverage) a rosette diameter of 230 mm (i.e. 14 d after theonset of treatments).

First, the shoot was cut at the base with a razor blade, afterwhich the fresh weight was determined. Then, the soil columnof each pot, including the roots, was separated into four quartersaccording to the quadrants described in the previous section,using steel blades that were driven into the soil column fromthe top surface. These four parts of the soil column were gently

Fig. 1 Experimental set-up for the formation of high-nitrate or high-phosphate patches in pots filled with sand–vermiculite mixture. Nutrient solution was supplied three times a week through four syringes that simultaneously dripped 40 ml of nutrient solution each at a fixed quadrant of the pot. Homogeneous pots received the same concentrations of nitrate (5 mM) or phosphate (0.5 mM) in each quadrant; in heterogeneous pots one quadrant received a high nitrate (20 mM) or phosphate (2 mM) concentration, whereas the other three quadrants were supplied with low nitrate (0.3 mM) or phosphate (0.03 mM).

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washed out on a sieve with tap water in order to retrieve theroots, after which the lateral roots were separated from the taproots. Lateral roots from the nutrient-rich quadrant (or arandomly assigned quadrant in the case of the homogeneoustreatments; quadrant 1) and those from the opposite quadrant(quadrant 3) were stored in glass flasks containing 100 mg l–1

HgCl2 (to prevent microbial decomposition) in tap water at4°C for root length and diameter determination.

Root length and root diameter were determined using aflat-bed scanner system, essentially following the protocoldescribed in Bouma et al. (2000). In short, lateral roots werefirst stained with neutral red (0.5 g l–1 in phosphate buffer;pH 6.5) to increase optical contrast, and then spread out inwater in a transparent plastic tray. The roots were thenscanned at 400 dpi, using a flatbed Epson 10000XL scannerwith two-sided lighting system, and root length and averagediameter were analysed using image analysis software(WINRHIZO; Régent Instruments Inc., Quebec, Canada).

Dry weights of shoots, tap roots and lateral roots weremeasured after drying at 70°C for at least 48 h.

From the dry weights of lateral roots in the ‘rich’ quadrantand the opposite ‘poor’ quadrant (or, in homogeneous pots,a randomly chosen quadrant and the opposite quadrant), therelative root placement (RRP) was calculated as follows (cf.Jansen et al., 2005):

(DW, dry weight.) The values of this parameter range from–1 (all roots in quadrant 3), via 0 (same amounts of roots

in the two quadrants), to 1 (all roots in quadrant 1). Forinstance, if 70% of the total root biomass in quadrants 1 and3 is positioned in the rich quadrant (quadrant 1), thenRRP = (0.7 – 0.3)/(0.7 + 0.3) = 0.4.

Statistical analyses

Significant differences between treatments and genotypeswere tested with one-way ANOVA for comparisons between pots.Contrasts in root lengths and diameters within individualpots (opposite quadrants; ‘rich’ vs ‘poor’ in the heterogeneoustreatment) were analysed with a repeated measures t-test, withthe different quadrants as pairs. Data were ln-transformedif homogeneity of variances differed significantly betweengroups. A probability of P < 0.05 indicates a significantdifference. All analyses were performed with SPSS statisticalsoftware (SPSS Inc., Chicago, IL, USA).

Results

Growth of Tetr plants

Before we could compare the responses of Wt and transformed,ethylene-insensitive Tetr plants to nutrient patches, we neededto establish if the two genotypes showed comparable growthcharacteristics. Wt and Tetr plants reached a similar rosettesize after 14 d of treatment (6.5 wk after sowing; data notshown), and although Wt plants tended to have higher dryweights of shoots and lateral roots than Tetr plants (Table 1),these differences were relatively small and only significantin the nitrate experiment. Tap roots were poorly developed

Fig. 2 Pore water nitrate and phosphate concentrations in pots with either a homogeneous or a heterogeneous distribution of nitrate or phosphate. Each circle represents a pot, divided into quadrants from which pore water samples were taken along an approx. 100-mm vertical length in the centre of the quadrants, 2 d before the final harvest and immediately before application of fresh nutrient solution. Heterogeneous pots were sampled in each quadrant (‘rich’ quadrants indicated in black); homogeneous pots were sampled in two randomly chosen opposite quadrants. Concentrations of nitrate (first two rows of pots in the diagram) and phosphate (last two rows of pots in the diagram) are given in mM. Four of the eight replicate pots in the experiment were sampled.

RRP DW DW

DW DWquadrant1 quadrant3

quadrant1 quadrant3

=−+

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in these young plants, explaining the larger variationamong individuals in this parameter (Table 1), and mostlateral roots originated within some centimetres of theshoot base. We conclude that the slight differences inbiomass characteristics would not appreciably affect thecapacity of Tetr plants to respond to locally high nutrientconcentrations.

Selective root placement

Wild-type plants in the heterogeneous nitrate treatmentshowed a RRP exceeding 0.4 (Fig. 3a), meaning that approx.

70% of the combined root weight in the ‘rich’ quadrant andthe opposite ‘poor’ quadrant of the pot was positioned in thehigh-nitrate quadrant. This indicates that Wt tobacco plantsshowed a considerable preference for growing roots in the patchwith high nitrate concentrations. Interestingly, no significantdifferences in RRP were found between ethylene-insensitiveTetr plants and Wt plants (Fig. 3a); selective root placementof Tetr plants in response to nitrate was as favourable as thatof Wt plants. Plants growing in homogeneous pots showed,as expected, an RRP that did not significantly differ from 0,which means that root biomass was divided equally over thetwo opposite quadrants.

Table 1 Effects of either homogeneous or heterogeneous distribution of nitrate and phosphate on dry weights (DWs) of wild-type (Wt) Nicotiana tabacum plants and the ethylene-insensitive transgenic genotype Tetr

Parameter

Wt Tetr

Homogeneous Heterogeneous Homogeneous Heterogeneous

Nitrate experimentShoot DW (g) 1.18 ± 0.04a 1.09 ± 0.05a 1.01 ± 0.06ab 0.88 ± 0.06b

Tap root DW (mg) 14.4 ± 2.8a 13.7 ± 2.0a 8.1 ± 1.7a 6.5 ± 1.9a

Lateral root DW (mg) 221 ± 12a 202 ± 10ab 165 ± 12bc 146 ± 15c

Phosphate experimentShoot DW (g) 1.21 ± 0.09a 1.16 ± 0.07a 1.03 ± 0.15a 1.09 ± 0.10a

Tap root DW (mg) 9.9 ± 2.3a 12.8 ± 3.4a 6.9 ± 2.9a 6.5 ± 2.2a

Lateral root DW (mg) 234 ± 19a 238 ± 28a 192 ± 28a 228 ± 21a

Nutrient solution was supplied for 14 d via a dripping system. For nitrate treatments, all quadrants of each pot in the homogeneous treatment received 5 mM nitrate, and three quadrants in the heterogeneous treatment received 0.3 mM nitrate and one quadrant received 20 mM nitrate. For phosphate treatments, all quadrants of each pot in the homogeneous treatment received 0.5 mM phosphate, and three quadrants in the heterogeneous treatment received 0.03 mM phosphate and one quadrant in the heterogeneous treatment received 2 mM phosphate. Values are means ± SE (n = 8); different letters indicate statistically significant differences between treatments and genotypes (P < 0.05).

Fig. 3 Relative root placement (RRP) of wild-type (Wt) Nicotiana tabacum plants and the ethylene-insensitive transgenic genotype Tetr in response to either a homogeneous (open bars) or a heterogeneous (hatched bars) distribution of (a) nitrate or (b) phosphate in the soil. Nutrient solution was supplied via a dripping system, by which the homogeneous treatment received 5 mM nitrate or 0.5 mM phosphate in all quadrants of the pot, whereas the heterogeneous treatment received 0.3 mM nitrate or 0.03 mM phosphate in the three ‘poor’ quadrants and 20 mM nitrate or 2 mM phosphate in the ‘rich’ quadrant. RRP was calculated as the difference in lateral root dry weight between a ‘rich’ quadrant of a heterogeneous pot and its opposite ‘poor’ quadrant, divided by the sum of these root weights. For homogeneous pots, root dry weights of two opposite, randomly selected quadrants were taken for the calculation. RRP = 1 indicates that all roots were positioned in the high-nutrient quadrant, and RRP = 0 indicates an even distribution over the high- and low-nutrient quadrants. Values are means (n = 8). Error bars indicate SE, and the different letters indicate statistically significant differences between treatments and genotypes (P < 0.05).


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Surprisingly, no clear selective root placement took place inthe phosphate treatment (Fig. 3b). Only Tetr plants showed asignificant, but still small, increase of their RRP in heterogeneouspots, but the response was far less obvious than in the case ofthe nitrate patch (Fig. 3a). The RRP in the heterogeneousphosphate treatment reached an average of just 0.16,corresponding to 58 and 42% of the total root weightgrowing in the phosphate ‘rich’ and ‘poor’ quadrants of thepots, respectively.

The distributions of lateral root lengths over the ‘rich’ and‘poor’ quadrants in both the nitrate and the phosphate exper-iments showed similar patterns, as might be expected from theRRP data, but it is interesting to note that the combined rootmass in the two quadrants did not differ significantly betweenhomogeneous and heterogeneous treatments (Fig. 4a,b). Inother words, root length in the nitrate experiment was allocatedfrom the low-nutrient to the high-nutrient quadrant, throughan increase in root formation in one quadrant and, at the sametime, decreased root development in the other. Although theresponse of root length to local high nitrate concentrationswas as strong as the response based on root weight (Fig. 3a),the small stimulation of root mass found in the high-nutrientpatch in the phosphate treatment (Fig. 3b) could not bedetected in the lateral root length measurements (Fig. 4b).This discrepancy could potentially be explained by differencesin root diameters within one root system, with thickerroots in the ‘poor’ quadrant, resulting in higher biomass perroot length. However, root diameters did not significantlydiffer between homogeneous and heterogeneous treatments,and within the heterogeneous treatment they did not differbetween the ‘rich’ and the ‘poor’ quadrants, neither with nitratenor with phosphate patches (Fig. 5a,b).

Discussion

In the last decade, the view has evolved that few plant processesare controlled by just a single plant hormone, and networks ofinteracting hormones are more the rule than the exception(Gazzarrini & McCourt, 2003). Ethylene is therefore involvedin many processes regulating the growth of plants (Stepanova& Alonso, 2003). Pierik et al. (2006) recently reviewed theimportance of ethylene in a variety of processes in severalplant species, and found that, during vegetative growth,ethylene mostly plays a role in the response of plants toadverse conditions, rather than affecting intrinsic growth anddevelopment. Consistent with this, Lynch & Brown (2001)suggested that ethylene appears to be involved in many ofthe responses of root systems to phosphate availability. Forinstance, low phosphate induced increased ethylene productionin tomato (Lycopersicon esculentum) plants, which could inits turn induce adventitious and lateral root formation. Inexperiments with maize (Zea mays) the opposite was found:phosphate deficiency (and also nitrate deficiency) on the onehand decreased ethylene production rates (Drew et al., 1989),but on the other hand increased ethylene sensitivity so muchthat ethylene-induced formation of aerenchyma was stimulated(He et al., 1992). Particularly relevant to our experiments isthe interaction of ethylene and low-phosphate treatments intheir effects on main root length and lateral root density ofcommon bean (Phaseolus vulgaris) plants, through which theyaffect root system architecture (Borch et al., 1999). Blockingof ethylene production with an inhibitor decreased lateralroot density in phosphate-sufficient plants, but increased it inphosphate-deficient plants. At the same time, ethylene at alow concentration stimulated main root lengths when the

Fig. 4 Lateral root lengths of wild-type (Wt) Nicotiana tabacum plants and the ethylene-insensitive transgenic genotype Tetr in quadrants of pots with either homogeneous or heterogeneous distribution of (a) nitrate or (b) phosphate in the soil. Columns represent roots from two opposite quadrants of the pot, which were randomly chosen for homogeneous treatments (Hom; light and darker grey bars), and ‘rich’ (black bars) and ‘poor’ (open bars) in (a) nitrate or (b) phosphate, respectively, for heterogeneous treatments (Het). Nutrient solution was supplied via a dripping system, by which the homogeneous treatment received 5 mM nitrate or 0.5 mM phosphate in all quadrants of the pot, whereas the heterogeneous treatment received 0.3 mM nitrate or 0.03 mM phosphate in the three ‘poor’ quadrants and 20 mM nitrate or 2 mM phosphate in the ‘rich’ quadrant. Values are means (n = 7–8). Error bars indicate SE. ns, no significance difference (P > 0.05); ***, significant difference between pot quadrants (P < 0.001).


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supply of phosphate was low and inhibited root elongationwhen phosphate was sufficient. This suggests that ethyleneperception can change with phosphate (and probably alsonitrate) availability and thereby alter the architecture of theroots. We therefore anticipated that ethylene could well playa role in directing selective root placement in response tohigh-nitrate and/or high-phosphate patches.

Although ethylene-mediated responses have successfullybeen studied by application of inhibitors of ethylene productionand/or perception (e.g. He et al., 1992; Visser et al., 1996;Borch et al., 1999), toxic or other aspecific effects of thesechemicals may easily occur, particularly when a continuedsupply of the inhibitor is needed to suppress ethylene responsesin new roots that develop during the experiment. The use oftransgenic plants that lack perception of ethylene facilitatesthe experimentation, and transgenic ethylene-insensitivetobacco plants have been used in previous studies to success-fully elucidate the role of ethylene in shade avoidance (e.g.Pierik et al., 2003, 2004), shoot and root growth (Tholen et al.,2004), adventitious root formation (McDonald & Visser,2003), and disease resistance (Knoester et al., 1998; Geraatset al., 2003). The Tetr genotype used in these experimentsshows an extreme lack of response to endogenous andexternally applied ethylene, even with regard to processes thatare commonly regarded as very sensitive to ethylene, such asroot elongation (McDonald & Visser, 2003).

In our experiments there were only small effects of thegenotype (ethylene-responsive vs ethylene-insensitive) ongrowth (Table 1), but, importantly and in contrast to ourexpectation, we did not observe a decreased ability of Tetrplants to respond to nutrient patches. In the nitrate experiment,where selective root placement by Wt plants was most

pronounced, ethylene-insensitive plants developed an equallyhigh RRP (Fig. 3a), caused by increased proliferation of lateralroots in the high-nitrate patch (Fig. 4a). This local increase inroot growth resulted from allocation of root length from otherparts of the pot, as the combined root length of opposingquadrants with high and low nitrate contents was similar tothat of opposed quadrants in the homogeneous treatment(Fig. 4a). These data suggest that there may be two separateregulatory events at work at the same time: one that stimulatesroot proliferation in the patch with high nitrate, and one thatdown-regulates root formation and/or elongation in theparts of the pot that are low in nitrate. Alternatively, the localstimulation of root proliferation by nitrate could simply leadto a shift in internal plant source–sink relations, decreasingavailable resources for the remainder of the root system.Recent findings that the costs of new root formation may berelatively small ( Jansen et al., 2006) suggest that the formeroption is more likely.

Surprisingly, phosphate patches caused a much moremodest shift in root placement in both Wt and Tetr plants(only detectable in Wt as DW (Fig. 3b) and not as root length(Fig. 4b)). In earlier studies, the responses of root systems tonitrate and phosphate patches showed a superficial resemblance,as in both conditions more root biomass was produced in thenutrient patch (Drew, 1975; Linkohr et al., 2002). However,given the small response of selective root placement tophosphate in our current experiments, this is not the casefor tobacco. The high sensitivity of the species to local nitrateconcentrations did not lead to a similarly strong responseto phosphate (even though the contrast between patches andbackground soil was similar for the two nutrients; Fig. 2), whichmeans either that either the signal transduction pathway is

Fig. 5 Lateral root diameters of wild-type (Wt) Nicotiana tabacum plants and the ethylene-insensitive transgenic genotype Tetr in response to either homogeneous or heterogeneous distribution of (a) nitrate or (b) phosphate in the soil. Columns represent roots from two opposite quadrants of the pot, which were randomly chosen for homogeneous treatments (Hom; light and darker grey bars), and ‘rich’ (black bars) and ‘poor’ (open bars) in (a) nitrate or (b) phosphate, respectively, for heterogeneous treatments (Het). Nutrient solution was supplied via a dripping system, by which the homogeneous treatment received 5 mM nitrate or 0.5 mM phosphate in all quadrants of the pot, whereas the heterogeneous treatment received 0.3 mM nitrate or 0.03 mM phosphate in the three ‘poor’ quadrants and 20 mM nitrate or 2 mM phosphate in the ‘rich’ quadrant. Values are means (n = 8). Error bars indicate SE. ns, no significance difference (P > 0.05); *, significant difference between pot quadrants (P < 0.05).


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different, or that putative phosphate receptors are far lesssensitive than the respective nitrate receptors in this species.In any case, for both nitrate and phosphate responses,ethylene does not interfere with the signal transductionpathway leading to increased root proliferation in thenutrient-rich patch.

Conclusion

Ethylene has been shown to affect root growth and developmentin various ways (Visser et al., 1997; Borch et al., 1999; Clarket al., 1999; Steffens et al., 2006), and seemed to be a goodcandidate to play a role in the selective root placement thatplants exhibit in response to high-nitrate and -phosphatepatches. However, ethylene-insensitive Tetr tobacco plantsdid not differ from Wt in selective root placement when theirroot system was grown in poor soil that contained a patch withhigh nitrate or phosphate. Both Tetr and Wt plants showedstrong selective root placement in nitrate patches, and far lessroot proliferation in phosphate patches. Apparently, ethyleneis not an essential component of the signal transductioncascade leading to selective root placement in tobacco.

References

Benkova E, Michniewicz M, Sauer M, Teichmann T, Seifertová D, Jürgens G, Friml J. 2003. Local, efflux-dependent auxin gradients as a common module for plant organ formation. Cell 115: 591–602.

Borch K, Bouma TJ, Lynch JP, Brown KM. 1999. Ethylene: a regulator of root architectural responses to soil phosphorus availability. Plant, Cell & Environment 22: 425–431.

Bouma TJ, Nielsen KL, Koutstaal B. 2000. Sample preparation and scanning protocol for computerised analysis of root length and diameter. Plant and Soil 218: 185–196.

Campbell BD, Grime JP. 1989. A new method of exposing developing root systems to controlled patchiness in mineral nutrient supply. Annals of Botany 63: 395–400.

Casson SA, Lindsey K. 2003. Genes and signalling in root development. New Phytologist 158: 11–38.

Chadwick AV, Burg SP. 1970. Regulation of root growth by auxin–ethylene interaction. Plant Physiology 45: 192–200.

Clark DG, Gubruim EK, Barrett JE, Nell TA, Klee HJ. 1999. Root formation in ethylene-insensitive plants. Plant Physiology 121: 53–59.

De Kroon H, Mommer L. 2006. Root foraging theory put to the test. Trends in Ecology and Evolution 21: 113–116.

De Kroon H, Mommer L, Nishiwaki A. 2003. Root competition: towards a mechanistic understanding. In: De Kroon H, Visser EJW, eds. Root ecology. Heidelberg, Germany: Springer-Verlag, 215–234.

Drew MC. 1975. Comparison of the effects of a localized supply of phosphate, nitrate, ammonium and potassium on the growth of the seminal root system, and the shoot, in barley. New Phytologist 75: 479–490.

Drew MC, He C-J, Morgan PW. 1989. Decreased ethylene biosynthesis, and induction of aerenchyma, by nitrogen- or phosphate-starvation in adventitious roots of Zea mays L. Plant Physiology 91: 266–271.

Drew MC, Jackson MB, Giffard S. 1979. Ethylene-promoted adventitious rooting and development of cortical air spaces (aerenchyma) in roots may be adaptive responses to flooding in Zea mays L. Planta 147: 83–88.

Forde BG. 2002. Local and long-range signaling pathways regulating plant responses to nitrate. Annual Review of Plant Biology 53: 203–224.

Fransen B, de Kroon H, Berendse F. 1998. Root morphological plasticity and nutrient acquisition of perennial grass species from habitats of different nutrient availability. Oecologia 115: 351–358.

Fransen B, de Kroon H, Berendse F. 2001. Soil nutrient heterogeneity alters competition between two perennial grass species. Ecology 82: 2534–2546.

Gazzarrini S, McCourt P. 2003. Cross-talk in plant hormone signalling: What Arabidopsis mutants are telling us. Annals of Botany 91: 605–612.

Geraats BPJ, Bakker PAHM, Lawrence CB, Achuo EA, Hofte M, van Loon LC. 2003. Ethylene-insensitive tobacco shows differentially altered susceptibility to different pathogens. Phytopathology 93: 813–821.

He C-J, Morgan PW, Drew MC. 1992. Enhanced sensitivity to ethylene in nitrogen- or phosphate-starved roots of Zea mays L. during aerenchyma formation. Plant Physiology 98: 137–142.

Hodge A. 2003. Plant nitrogen capture from organic matter as affected by spatial dispersion, interspecific competition and mycorrhizal colonization. New Phytologist 157: 303–314.

Hodge A. 2004. The plastic plant: root responses to heterogeneous supplies of nutrients. New Phytologist 162: 9–24.

Hutchings MJ, de Kroon H. 1994. Foraging in plants – the role of morphological plasticity in resource acquisition. Advances in Ecological Research 25: 159–238.

Jackson MB, Drew MC, Giffard S. 1981. Effects of applying ethylene to the root system of Zea mays on growth and nutrient concentration in relation to flooding tolerance. Physiologia Plantarum 52: 23–28.

Jansen C, van de Steeg HM, de Kroon H. 2005. Investigating a trade-off in root morphological responses to a heterogeneous nutrient supply and to flooding. Functional Ecology 19: 952–960.

Jansen C, van Kempen MML, Bögemann GM, Bouma TJ, de Kroon H. 2006. Limited costs of wrong root placement in Rumex palustris in heterogeneous soils. New Phytologist 171: 117–126.

Johnson HA, Biondini ME. 2001. Root morphological plasticity and nitrogen uptake of 59 plant species from the Great Plains grasslands, USA. Basic and Applied Ecology 2: 127–143.

Knoester M, Van Loon LC, Van den Heuvel J, Hennig J, Bol JF, Linthorst HJM. 1998. Ethylene-insensitive tobacco lacks nonhost resistance against soil-borne fungi. Proceedings of the National Academy of Sciences, USA 95: 1933–1937.

Lamers LPM, Tomassen HBM, Roelofs JGM. 1998. Sulfate-induced eutrophication and phytotoxicity in freshwater wetlands. Environmental Science and Technology 32: 199–205.

Linkohr BI, Williamson LC, Fitter AH, Ottoline Leyser HM. 2002. Nitrate and phosphate availability and distribution have different effects on root system architecture of Arabidopsis. Plant Journal 29: 751–760.

López-Bucio J, Cruz-Ramírez A, Herrera-Estrella L. 2003. The role of nutrient availability in regulating root architecture. Current Opinions in Plant Biology 6: 280–287.

Lynch J, Brown KM. 1997. Ethylene and plant responses to nutritional stress. Physiologia Plantarum 100: 613–619.

Lynch JP, Brown KM. 2001. Topsoil foraging – an architectural adaptation of plants to low phosphorus availability. Plant and Soil 237: 225–237.

McDonald MP, Visser EJW. 2003. A study of the interaction between auxin and ethylene in wild type and transgenic ethylene-insensitive tobacco during adventitious root formation induced by stagnant root zone conditions. Plant Biology 5: 550–556.

Pierik P, Tholen D, Poorter H, Visser EJW, Voesenek LACJ. 2006. The Janus face of ethylene: growth inhibition and stimulation. Trends in Plant Science 11: 176–183.

Pierik R, Visser EJW, De Kroon H, Voesenek LACJ. 2003. Ethylene is required in tobacco to successfully compete with proximate neighbours. Plant, Cell & Environment 26: 1229–1234.

Pierik R, Whitelam GC, Voesenek LACJ, De Kroon H, Visser EJW. 2004. Canopy studies on ethylene-insensitive tobacco identify ethylene as a novel element in blue light and plant–plant signalling. Plant Journal 38: 310–319.


Research 465

Robinson D. 2004. The responses of plants to non-uniform supplies of nutrients. New Phytologist 127: 635–674.

Souter MA, Pullen ML, Topping JF, Zhang X, Lindsey K. 2004. Rescue of defective auxin mediated gene expression and root meristem function by inhibition of ethylene signalling in sterol biosynthesis mutants of Arabidopsis. Planta 219: 773–783.

Steffens B, Wang J, Sauter M. 2006. Interactions between ethylene, gibberellin and abscisic acid regulate emergence and growth rate of adventitious roots in deepwater rice. Planta 223: 604–612.

Stepanova AN, Alonso JM. 2003. Ethylene signalling and response pathway: a unique signalling cascade with a multitude of inputs and outputs. Physiologia Plantarum 123: 195–206.

Tholen D, Voesenek LACJ, Poorter H. 2004. Ethylene insensitivity does not increase leaf area or relative growth rate in Arabidopsis, Nicotiana tabacum, and Petunia × hybrida. Plant Physiology 134: 1803–1812.

Visser EJW, Cohen JD, Barendse GWM, Blom CWPM, Voesenek LACJ. 1996. An ethylene-mediated increase in sensitivity to auxin induces adventitious root formation in flooded Rumex palustris Sm. Plant Physiology 112: 1687–1692.

Visser EJW, Nabben RHM, Blom CWPM, Voesenek LACJ. 1997. Elongation by primary lateral roots and adventitious roots during conditions of hypoxia and high ethylene concentrations. Plant, Cell & Environment 20: 647–653.

Visser EJW, Pierik R. 2007. Inhibition of root elongation by ethylene in wetland and non-wetland plant species and the impact of longitudinal ventilation. Plant, Cell & Environment 30: 31–38.

Zhang H, Forde BG. 1998. An Arabidopsis MADS box gene that controls nutrient-induced changes in root architecture. Science 279: 407–409.

Zhang H, Jennings A, Barlow PW, Forde BG. 1999. Dual pathways for regulation of root branching by nitrate. Proceedings of the National Academy of Sciences, USA 96: 6529–6534.

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Documents

Evidence that ethylene signalling is not involved in selective root placement by tobacco plants in response to nutrient-rich soil patches