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Uridine and the control of phototropism in oat (Avena sativa L.) coleoptiles

Samih M. TamimiDepartment of Biological Sciences, University of Jordan, Amman, Jordan; *e-mail: tamimi@ju.edu.jo

Received 16 July 2003; accepted in revised form 12 May 2004

Key words: Avena sativa, Bruinsma–Hasegawa theory, Cholodny–Went theory, Coleoptile, Phototropism,Uridine

Abstract

The short-term growth response of oat (Avena sativa L.) coleoptiles to exogenously applied uridine wasstudied both in excised apical segments and in the intact seedlings. In both cases growth of coleoptile tissuewas inhibited by uridine. The inhibition of coleoptile growth consistently occurred 20–30 min after uridinetreatment, which is within the lag period of their phototropic response. Asymmetric application of uridineto coleoptiles in the intact seedlings resulted in their bending toward the direction to which uridine wasapplied in the absence of light stimulus. These findings suggest that uridine or its metabolites, plays animportant role in the phototropism of oat coleoptiles and provide support to the Bruinsma–Hasegawatheory as an alternative to the Cholodny–Went theory for explaining phototropism.

Introduction

The phototropic response of higher plant seed-lings results from different rates of elongation bythe two sides of the shoot. The mechanismsinvolved in the establishment of the differentialgrowth during phototropism have been debatedfor more than 60 years but are still largelyunresolved (for reviews, see Firn and Digby 1980;Brigs and Baskin 1988; Iino 1990; Firn 1994).The growth rate changes associated with photo-tropic curvature have been attributed to a lateralmovement of auxin from the lighted to the sha-ded side of stimulated seedlings (Cholodny–Wenttheory) (Went and Thimann 1937). However, thistheory has been challenged because the magni-tude and/or kinetics of auxin asymmetry couldnot account for the differential growth observed(Trewavas 1992). In fact some studies haveshown that the shaded halves of unilaterallyilluminated coleoptiles and hypocotyls did notcontain more auxin than the illuminated ones

(Bruinsma and Hasegawa 1989; Skoda andHasegawa 1989).

Recently, evidence showing that the phototropiccurvature of seedlings is accompanied by a lateralgradient of light-induced growth inhibiting sub-stance(s) have been presented (Hasegawa et al.1986; Bruinsma and Hasegawa 1990; Yamamuraand Hasegawa 2001; Hasegawa et al. 2001;Hasegawa et al. 2002; Yamada et al. 2003) andconstituted the basis of the Bruinsma–Hasegawatheory. This theory suggested that phototropism isregulated by a local gradient of growth inhibi-tor(s), also called phototropism regulating sub-stance(s), induced at the site of illumination. Theaccumulation of these inhibitors at the irradiatedside of the plant causes a unilateral reduction incell elongation and hence the observed curvature.

Results describing the nature of these inhibitorssuggested that different plant species produce dif-ferent and very specific growth inhibitor(s) ascandidate(s) for phototropism regulating sub-stance(s) (Hasegawa et al. 2002). In oat, a plant in

Plant Growth Regulation 43: 173–177, 2004.

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which phototropism have been studied extensively(Denison 1979), the phototropism regulating sub-stance was identified as uridine (Hasegawa et al.2001). Support for the role of uridine in the pho-totropism of oat seedlings comes from two find-ings: first, the concentration of uridine in theilluminated side of stimulated coleoptiles is sig-nificantly higher than those in the shaded side;second, coleoptile growth is inhibited by uridine(Hasegawa et al. 2001). Although the findings thaturidine inhibit coleoptile cell elongation empha-sized its role as the phototropism regulating sub-stance and provided support to the Bruinsma–Hasegawa theory, its effect, however, was dem-onstrated only in excised coleoptile segments.Since intact coleoptiles represent a more naturalsystem than excised segments, data derived fromgrowth experiments conducted on isolated seg-ments may differ from those in the intact plants. Inaddition the inhibitory effect of uridine on cole-optile cell elongation was demonstrated 6 h aftertreatment. Obviously results from such long-termexperiments may not account for the differentialgrowth that causes phototropism. The fact thatchanges in uridine concentration occur during thelag period preceding phototropic bending,approximately 30 min after stimulation (Hasega-wa et al. 2001) makes it important to examine theshort-term growth response of coleoptile tissue toapplied uridine. Such experiments would provideinformation about the lag period preceding uridineaction. Obviously if uridine is to play an importantrole in coleoptile phototropism then its effect oncoleoptile growth should be observed within thelag period of their phototropic response.

In the present investigation further experimentson the role of uridine in oat coleoptile phototro-pism were carried out. The short-term growthresponse of coleptile tissue to applied uridine wasstudied both in excised segments and the intactplants to determine the lag period of uridine actionon coleoptile cell elongation. The response ofintact seedling to asymmetrical application of uri-dine was also investigated. It was argued that ifuridine has an important role in phototropism thenits inhibitory effect on coleoptile elongation shouldbe demonstrated not only in excised segment butalso in intact plants and should occur within the lagperiod that precedes their phototropic curvature.Furthermore, if the lateral gradient of endogenousuridine reported to exist across the two flanks of

stimulated coleoptiles is responsible for theirbending then an asymmetric application of uridineto intact coleoptiles in darkness should causebending response.

Materials and methods

Plant material

Oat (Avena sativa L.) seeds were germinated onmoist filter paper in covered plexiglass boxes at25 �C under dim red light (1 lmol/m�2 s�1) for24 h. The germinated seeds in boxes were allowedto grow in the dark at 25 �C for 3 days. Forobtaining individual seedlings, germinated seedsobtained as described above were transplantedindividually in small plastic pots containing moistvermiculite and were grown in the dark at 25 �Cfor 3 days. Only uniform seedlings with straightcoleoptiles were used for experiments and allexperimental manipulation were carried out underdim green light.

Growth experiments

Long-term growth experiments were initially con-ducted on isolated coleoptile segments to establishthe dose–response relationship of uridine action oncloeoptile elongation. Twenty apical coleoptilesegments, 5 mm in length, were treated with dif-ferent concentrations of uridine in small petridishes. Uridine (Sigma) was dissolved in 5 mM K-succinate buffer (pH 6) to give a final concentra-tion ranging from 10�3 to 10�6 M. Control seg-ments were treated with 5 mM K-succinate buffer(pH 6). Segments in Petri dishes were incubated indarkness at 25 �C for 6 h and the final length ofthe segments was measured. The concentrations ofuridine that inhibited coleoptile elongation by50% relative to control was selected for the short-term growth experiments.

The short-term growth response of coleoptiletissue to uridine treatment was studied using bothisolated segments and intact seedlings. Thesegrowth experiments were designed to determinewhether coleoptile growth in the intact plant isalso inhibited by uridine treatment and to find outthe lag time required for uridine action in bothsystems. For experiments with isolated segments, a

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5 mm apical segment was placed in the uprightposition in an aerated perspex reservoir thermo-stated at 25 �C. The reservoir was filled with5 mM K-succinate buffer (pH 6) and the growthrate of the segments was monitored. The coun-terbalanced core of a linear displacement trans-ducer (Sangmo D2/5) was rested on the tip of thesegment with the help of a small part of a dis-posable pipette tip. The voltage output from thetransducer was linked to a microcomputer and thegrowth rate of the segments was computed using2nd degree five point differentiation formula(Erikson 1976). The effect of uridine on coleoptilegrowth was examined by replacing the K-succinatebuffer in the reservoir with 10�4 M uridine solu-tion. For experiments with intact plants, thecounterbalanced linear displacement transducerwas rested on the coleoptile tip of an intact seed-ling and its growth rate was monitored as de-scribed above. When steady growth rate wasreached, 10 ll of 10�3 M uridine were appliedsymmetrically as small droplets to the apical re-gion of the coleoptiles (two 2.5 ll droplets at eachflank). All growth measurements were carried outin darkness and results presented are the meanfrom 5 replicates.

Effect of exogenous uridine on coleoptile bending

The effect of different concentrations of uridine oncoleoptile bending was studied in intact seedlingsby the application of five droplets (2 ll each) ofuridine solution at the desired concentration toone side of the upper 10 mm of the coleoptile. Foreach concentration 15 seedlings were used and allseedlings were placed in darkness at 25 �C for 3 h.The bending response of the seedlings was thenestimated by measuring the angle of coleoptilecurvature. All experimental manipulations wereconducted under dim green light and each experi-ment was repeated at least 5 times.

Results and discussion

The physiological importance of nucleotides andtheir metabolism in regulating various aspects ofplant growth and development is only recentlybeginning to emerge (for a review see Stasolla et al.2003). Uridine has been reported to play an

important role in the control of phototropism inoat coleoptiles and is described as the phototro-pism regulating substance of oats (Hasegawa et al.2001). In this work further support to the roleplayed by uridine in the control of oat coleoptilephototropism is provided. Data presented inFigure 1 show the long-term growth response ofapical coleoptile segments from young oat seed-lings to applied uridine. Treatment of coleoptilesegments with 10�6 M uridine for 6 h reduced theirgrowth by 18% relative to untreated segments andthe degree of growth inhibition increased with theincrease in the concentration of uridine. A 50%inhibition in the growth of segments was observedat 10�4 M uridine and this concentration was se-lected for the short-term growth experiments.

Figure 2 shows that significant reduction in thegrowth rate of apical coleoptile segments occured30 min after uridine treatment. Growth rates ofcoleoptile segments in 5 mM K-succinate buffer(pH 6) showed little variations and ranged from 4to 4.5 lm/min. Thirty minutes after the replace-ment of the K-succinate buffer with a solution of10�4 M uridine the growth rate of the segmentsdropped to 0.5–1 lm/min and remained at this lowrate for as long as growth was measured.

-6 -5 -4 -30

20

40

60

80

Gro

wth

inhi

bitio

n (P

erce

nt)

Log uridine concentration (Molar)

Figure 1. Effect of different concentrations of uridine on the

long-term growth of apical segments of oat coleoptiles. Results

were recorded 6 h after uridine treatment and presented as %

growth inhibition relative to untreated control. Each value is

the mean from 5 replicates of 10 segments � SE.

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Growth of intact coleoptiles tissue is alsostrongly inhibited by uridine (Figure 3). Treat-ment of intact coleoptiles with 10 ll of 10�3 M

uridine, applied symmetrically to the two flanks ofthe coleoptile, reduced their growth from anaverage of approximately 12 lm/min (growth ratebefore treatment) to an average of 3–4 lm/min.While uridine caused a similar inhibition of growthin coleoptiles tissue both in the intact seedlings andisolated segments, its effect, however, was morerapid in the intact plant and consistently occured20 min after uridine treatment. It is possible thatfaster uptake/transport of uridine by coleoptiletissue of intact seedlings was responsible for thedifference in their responses. Nevertheless theinhibitory action of uridine on the growth of oatcoleoptiles appears to occur within the lag periodof their phototropic curvature. Taken together,these results and the findings of Haesegawa et al.(2001), which demonstrated the establishment of aconcentration gradient of endogenous uridineacross the two sides of oat coleoptiles within thefirst 30 min from light stimulation suggest thaturidine either directly or indirectly (possiblythrough its metabolites) plays an important role inthe phototropic response of oats. Additional sup-port to the role of uridine in the phototropism ofoat coleoptiles comes from the findings thatcoleoptiles treated asymmetrically with different

Figure 2. The short-term growth response of apical coleoptile

segments to uridine. Apical segments, 5 mm in length, were

initially grown in 5 mM K-succinate buffer (pH 6) until steady

growth rate was attained. Arrow indicates the time at which the

buffer was replaced with a solution of 10�4 M uridine. Each

data point is the mean from 5 replicates � SE.

Figure 3. The short-term growth response of intact coleoptiles

to uridine treatment. Arrow indicates the time of uridine

treatment. Treatment consisted of four 2.5 ll droplets of

10�3 M uridine symmetrically applied (2 droplets at each flank)

to the upper 5 mm of coleoptiles. Each data point is the mean

from 5 replicates � SE.

-6 -5 -4 -30

10

20

30

40

Ang

le o

f cur

vatu

re (

degr

ees

)

Log uridine concentration (Molar)

Figure 4. The bending response of intact coleoptiles to asym-

metrically applied uridine. Uridine was applied as small drop-

lets to one flank of coleoptiles. Following treatment, seedlings

were kept in darkness for 3 h and the angle of coleoptile cur-

vature was then recorded. Each data point is the mean from 5

replicates (with 15 seedlings per treatment/replicate) � SE.

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concentrations of uridine bend toward the direc-tion of the side to which uridine was applied in theabsence of light stimulus (Figure 4). Althoughrelatively high concentrations of uridine had to beused in order to obtain an appreciable bendingresponse, low uptake of uridine and/or the possi-bility of applied uridine being quickly metabolizedby the coleoptile tissue may account for therequirement of these high concentrations. In con-clusion the results of this study support the find-ings of Haesegawa et al. (2001) that uridine plays arole in the phototropism of oat coleoptiles andsuggest that the Bruinsma–Hasegawa theory couldprovide an alternative to the Chlodny–Went the-ory as an explanation to coleoptile phototropismin oats.

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