Plant, Cell and Environment

(2006)

29

, 1936–1945 doi: 10.1111/j.1365-3040.2006.01569.x

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd

1936

K. A. Nagel

et al

.

Correspondence: Achim Walter. Fax:

+

49 2461 612492; e-mail:a.walter@fz-juelich.de

Dynamics of root growth stimulation in

Nicotiana tabacum

in increasing light intensity

KERSTIN A. NAGEL, ULRICH SCHURR & ACHIM WALTER

Institute of Chemistry and Dynamics of the Geosphere ICG-III: Phytosphere, Research Center Juelich GmbH, 52425 Juelich, Germany

ABSTRACT

Light intensity is crucial for plant growth. In this study, thehypothesis was tested whether a sudden increase in lightintensity leads to an immediate increase of root growth.Seedlings of

Nicotiana tabacum

grown in agar-filled Petridishes were subjected to light intensities of 60 and300

m

mol m

-

2

s

-

1

, respectively. Seedling biomass, sucrose,glucose and fructose concentration as well as primary rootgrowth increased significantly with light intensity. Thedynamics of the increase in root growth were analysed herein more detail. In transition experiments from low to highlight intensities, root growth increased by a factor of fourwithin 4 d, reaching the steady-state level measured inplants that were cultivated in high-light conditions. Thedistribution of relative elemental growth rates along theroot growth zone retained a constant shape throughout thistransition. During the first three hours after light increase,strong growth fluctuations were repeatedly observed withthe velocity of the root tip cycling in a sinusoidal patternbetween 120 and 180

m

m h

-

1

. These dynamic patterns arediscussed in the context of hydraulic and photosyntheticacclimation to the altered conditions. Experiments withexternally applied sucrose and with transgenic plants hav-ing reduced capacities for sucrose synthesis indicatedclearly that increasing light intensity rapidly enhanced rootgrowth by elevating sucrose export from shoot to root.

Key-words

: image processing, electron transport rate, light,relative growth rate, sucrose.

INTRODUCTION

Root growth is closely related to carbon import and hence,to light conditions for the shoot. Carbon gain in roots isrealized predominantly by import from the shoot via thephloem, while the major loss of root carbon occurs viarespiration associated with growth and ion uptake (Farrar& Jones 2000). A number of studies have investigated dif-ferences in root growth between plants acclimated to low-or high-light environments (e.g. Webb 1976; Vincent &

Gregory 1989; Aguirrezabal, Deleens & Tardieu 1994) orbetween plants growing with variable external sucrose sup-ply (Street & McGregor 1952; Freixes

et al

. 2002). It hasbeen proposed that root length is related to cumulativeintercepted radiation (Aresta & Fukai 1984; Vincent &Gregory 1989). Likewise, relations between growth in pri-mary and secondary roots (Bingham & Stevenson 1993) aswell as between root and shoot growth (Thaler & Pages1996) have also been extensively studied in steady-stateexperiments. A key factor directly connecting the irradia-tion of the shoot and the elongation of root tips is the localhexose concentration, which correlates very well withgrowth rates of individual roots of a given species (Scheible

et al

. 1997; Freixes

et al

. 2002). An increase in the sugarcontent of root tissue promotes growth of primary andsecondary roots without affecting branching patterns oroverall root architecture (Bingham, Blackwood & Steven-son 1997), in contrast to increases of other growth sub-strates such as NO

3

−

(Zhang

et al

. 1999) or other mineralnutrients (Forde & Lorenzo 2001), which can lead tomarked alterations of root architecture via localized effectson growth (e.g. PO

43

−

; see Watt & Evans 1999).While a large amount of data on the reaction of overall

root growth to different steady-state light conditions isavailable, much less is known about the differential reac-tions within the growth zone to changing light intensitiesthat are common in natural environments. Muller, Stosser& Tardieu (1998) showed that in maize the length of thegrowth zone decreased with decreasing light intensity, inmuch the same way as with decreasing water availability(Sharp, Hsiao & Silk 1988). The immediate effect of short-term changes in light intensities on the amplitude and dis-tribution of relative elemental growth rates (REGR) withinthe root growth zone has, however, hardly been explored.Recent studies using high-resolution, automated opticalgrowth monitoring methods have shown that alterations inroot growth can take place within less than an hour inreaction to changes in temperature or nutrient availability(Walter

et al

. 2002; Van der Weele

et al

. 2003; Walter, Feil& Schurr 2003). The question arises whether a change inlight environment of the shoot can also induce such fastreactions of root growth. Comparisons of maize rootgrowth in decreasing light intensities indicate that a reduc-tion in root elongation rate takes about 4 d (Muller

et al

.1998).

Root growth and light intensity

1937

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd,

Plant, Cell and Environment,

29,

1936–1945

If sucrose export from the shoot is among the signalslinking shoot light interception and root growth, transgenicplants with reduced sucrose content (Chen

et al

. 2005)could reveal the degree to which this process is regulatedvia sucrose. Response kinetics of REGR distribution toshoot light conditions may also give some informationabout mechanisms that are involved in the regulation ofthis process. Sugar import into the root growth zone canregulate a vast number of enzymes involved in carbonmetabolism (Koch 1996; Ho

et al

. 2001) or the cell cycle(Riou-Khamlichi

et al

. 2000). Genes regulating root growthhave been recently characterized in

Arabidopsis

(Birn-baum

et al

. 2003) and maize (Bassani, Neumann & Gep-stein 2004).

In this study, the hypothesis was tested whether a suddenincrease in light intensity leads to a rapid increase of rootgrowth. Hence, the effect of light intensity on plant growth,root expansion and sugar concentration both in steady-state and changing light environments was investigated.

MATERIALS AND METHODS

Plant material

Wild-type seedlings of

Nicotiana tabacum

(L.) ecotypeXanthi were surface-sterilized with sodium hypochloritesolution and sown on sterile agar (1%, w/v) in Petri dishes(120

×

120

×

17 mm; five seedlings per dish) that weresealed with fabric tape (Micropore, 3 M Health Care,Neuss, Germany). The medium contained full-strengthIngestad mineral nutrient solution (Ingestad 1982; Walter

et al

. 2003) and in some cases 2% (w/v) sucrose, dependingon the experiment.

Transgenic tobacco plants,

N. tabacum

(L.) ecotypeSamsun SPPi17 with decreased sucrose-6-phosphatephosphatase (SPP) levels, were germinated on an agar con-taining kanamycin. After 10 d, the transgenic plants, whichwere kanamycin-resistant, were selected and transferred toan agar medium without kanamycin.

It is common practice that investigations with seedlingsgrown on agar-filled Petri dishes are performed with shootsgrowing completely inside the sterile Petri dish (see e.g.Freixes

et al

. 2002). Because this is the currently acceptedcultivation system in the scientific community, most exper-iments of this study were performed in such systems as well,but for reasons given below, we also developed cultivationprocedures that allowed for shoot growth outside the Petridish. For shoots grown inside the Petri dish, the dishes werehalf-filled with agar and the seeds were pushed slightly intothe agar. Shoots could then grow in the air volume insidethe dishes. After sowing the seeds, Petri dishes were putalmost horizontal, ensuring that the roots had to growwithin and not at the surface of the agar, thereby improvingthe optical quality of the acquired image sequences sub-stantially. Two days after germination the Petri dishes werepositioned vertically, allowing the roots to grow downwardswithin the agar for weeks.

In the described cultivation system, it is not possible to(1) shade root systems efficiently and (2) ‘handle’ the shoot

to determine photosynthetic activity or treat it mechani-cally. Hence, we established a cultivation procedure thatallowed shoots to grow outside the Petri dish. Here, thePetri dishes were filled completely with agar and sealedwith fabric tape. The seeds were pushed slightly into theagar through five holes (diameter approximately 2 mm) atone side of the closed Petri dishes. The holes were thencovered with laboratory film (Parafilm, Pechiney PlasticPackaging, Menasha, WI, USA) to keep the seeds moistand to keep the agar free of contaminations. After germi-nation the Parafilm was removed. The shoot then grewoutside the Petri dishes, while the roots grew in the agarwhich stayed free of visible fungal or bacterial infectionsthroughout the growth monitoring period.

It has to be noticed that shoots growing outside the Petridish received higher light intensities than those shootsgrowing inside. When light was penetrating the Petri dishperpendicularly, its intensity was only decreased by about5% (data not shown). Yet light that reaches the Petri dishsurface at lower angles is reflected stronger, thus, lightintensity reaching shoots inside the Petri dishes will havebeen lower than light intensity reaching shoots outside thePetri dishes.

After sowing, all Petri dishes were placed at 23

°

C(

±

3

°

C) and 12 h/12 h light/dark cycles. The plants wereilluminated either in steady-state light conditions with 60or 300

µ

mol m

−

2

s

−

1

or for 14 d with 60

µ

mol m

−

2

s

−

1

, andthereafter for 4–5 d with 300

µ

mol m

−

2

s

−

1

. The two lightintensities used in this study corresponded to photon fluxdensities of 2.6 and 13 mol m

−

2

d

−

1

, respectively. Those lightintensities are in the typical range used for laboratory cul-tivations of plants in Petri dishes but are much lower thantypical light intensities under field conditions. The lightintensity was increased by illuminating the plants with coollight (KL 200, Schott, Wiesbaden, Germany) to analyse theeffect of light intensity without increasing the temperature.The root growth and sugar concentrations of the seedlingswere investigated 14–19 d after germination as describedfurther.

Determination of ethanol-soluble sugars

For determination of ethanol-soluble sugars, whole shootsand roots were harvested and immediately frozen in liquidN

2

and then extracted with 80% ethanol (v/v) at 80

°

C for20 min according to Arnon (1949). The supernatant wasstored at 4

°

C and the extraction was repeated with 50%,and then again with 80% ethanol (v/v).

Glucose, fructose and sucrose were analysed with a cou-pled enzyme assay (Jones, Outlaw & Lowry 1977) using amultiplate photometer (ht II, Anthos MikrosystemeGmBH, Krefeld, Germany). Plates were loaded withextract, imidazol buffer, ATP, NADP

+

and glucose-6-phosphate dehydrogenase (5.6 U). The enzymes hexoki-nase (6 U; glucose assay), phosphoglucoisomerase (5 U;fructose assay) and invertase (10 U; sucrose assay) werethen sequentially added and the change in absorption at334 nm was measured.

1938

K. A. Nagel

et al

.

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd,

Plant, Cell and Environment,

29,

1936–1945

Image acquisition and processing

The Petri dishes were put in a microrhizotron setup (similarto the rhizotron setup described in Walter

et al

. 2002;Walter & Schurr 2005), with which images of the root tipswere captured by a CCD camera (Sony XC-ST50; Sony,Köln, Germany). The images were taken using infraredillumination (

λ

=

940 nm) with a frequency of two imagesper minute. Each image acquired – also designated as aframe – had a resolution of 720

×

560 pixels, correspondingto an area of 3.2

×

2.4 mm. The camera was equipped witha lowpass infrared filter (RG 9, Schott, Mainz, Germany)that blocked visible light. Growing root tips were trackedautomatically using three moving stages vertical to eachother. The moving stages, which were controlled by animage-based tracking algorithm, allowed one to maintainthe root tip in the field of view during the image acquisition.The algorithms which control the root tracker and acquirethe image sequences were based on a digital imagesequence processing software package (Heurisko, Aeon,Hanau, Germany; Schmundt

et al

. 1998; Walter

et al

. 2002,2003).

The acquired image sequences were used to calculate thevelocity of the root tip (

v

Tip

) and the REGR along the rootgrowth zone using the structure tensor method (Schmundt

et al

. 1998; Haußecker & Spies 1999). Motion analysis bystructure tensor is based on local grey value structures inthe images. The resulting velocity vector fields were inter-polated and local growth rates were calculated from localdivergence in the velocity maps (Schmundt

et al

. 1998;Walter

et al

. 2002; Scharr 2004).

Chlorophyll fluorescence measurements

The chlorophyll

a

fluorescence measurements were per-formed with a portable pulse amplitude-modulated photo-synthesis yield analyser (Imaging PAM, Heinz Walz GmbH,Effeltrich, Germany). The photosynthetic electron trans-port rate (

ETR

) of photosystem (PS) II was obtained as

ETR

=

Φ

PSII

×

PPFD

×

0.5

×

0.84, while

Φ

PSII

is the effectivequantum yield of PS II. The factor 0.5 assumes that PS IIand PS I were equally excited and the so-called empirical

ETR

correction factor of 0.84 takes into account that onlya fraction of incident light (PPFD) is really absorbed byphotosystems (Ehleringer 1981).

RESULTS

Effect of increasing light intensity on root growth

Growth-zone length and intensity of growth differed mark-edly between the two steady-state treatments (60 and300

µ

mol m

−

2

s

−

1

), while the shape of REGR distributionswas comparable between treatments (Fig. 1a). The transi-tion in the plants between those two steady states occurredwithin 4 d, as shown by irradiance transition experiments(Fig. 1b). REGR distributions and total growth intensity(

v

Tip

) were analysed in more detail from the data of several

replicates. Therefore, four parameters were chosen tocharacterize the REGR distribution:

v

Tip

, maximal REGR,full width at half maximum of the elongation zone (FWH-Max.) and length of the entire growth zone (Fig. 2a–d). Insteady-state experiments, growth was stable over severaldays. The

v

Tip

, which characterizes the total growth activityof the organ, was four times higher in the high-light treat-ment compared with the low-light treatment (Fig. 2a), cor-responding to the difference in the applied light intensity.Upon transfer of the seedlings from low to high light on

Figure 1.

Distributions of relative elemental growth rate (REGR) along the root growth zone (shoots inside Petri dishes). (a) Plants were exposed for 18 d to constant light intensities (60 or 300

µ

mol m

−

2

s

−

1

) or (b) for 14 d to 60

µ

mol m

−

2

s

−

1

and thereafter for 4 d to 300

µ

mol m

−

2

s

−

1

. (c) Typical colour-coded REGR distributions along root tips under constant light intensities (60 or 300

µ

mol m

−

2

s

−

1

) (mean value

±

SE,

n

=

4).

Distance from root tip (mm)

0.0 0.5 1.0 1.5 2.0R

EG

R (

% h

–1)

0

10

20

30

RE

GR

(%

h –1

)

0

10

20

30

40

60

300(a)

day 18day 17

day 16day 15

day 14

(b)

Root growth and light intensity

1939

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd,

Plant, Cell and Environment,

29,

1936–1945

day 14,

v

Tip

rose in a sigmoidal way for 4 d, reaching thesame plateau value of

v

Tip

as plants grown under steady-state light. Very similar graphs were obtained for the otherthree parameters describing the shape of the REGR distri-butions (Fig. 2b–d), showing quantitatively that the entireroot growth zone and hence, each phase of cellular devel-opment, was affected very similarly by the increasing lighttreatment.

Fresh weight (FW) differed by a factor of five betweenplants from the two steady-state treatments (Fig. 3). Whilethe sum of root and shoot FW was 6 mg per individual plantfrom the low-light treatment on day 14, the sum of root andshoot FW was 28 mg per individual plant from the high-light treatment. Plants from the transition experimentexhibited intermediate values; they did not reach the sizeof the high-light-grown plants during 4 d of high-lighttreatment.

Shoot and root carbohydrate concentrations alsodiffered considerably between low- and high-light treat-ments (Fig. 3). Sucrose concentrations in leaves from the

steady-state experiments differed by a factor of five; glu-cose and fructose concentrations were about 10 timeshigher under 300

µ

mol m

−

2

s

−

1

, compared with 60

µ

molm

−

2

s

−

1

on day 14 and day 18. Interestingly, plants trans-ferred to the high-light intensity for 4 d showed highercarbohydrate concentrations in leaves and roots than theplants under the steady-state conditions at 300

µ

mol m

−

2

s

−

1

. In roots, sugar concentrations of the transferred plantsexceeded those of the high-light plants by a factor of two(sucrose) to three (glucose and fructose).

To test whether the illumination of roots affects theobserved results and to provide a solution for ‘handling’shoots experimentally, plants were also grown in anothercultivation system with leaves outside and roots inside thePetri dish. Here, the Petri dish was covered with a plasticsheet to provide dark conditions for roots. With shootsoutside the Petri dish, roots grew faster in steady-state low-light conditions compared with the experiments with theconventional cultivation system (Fig. 4a). When shoots weregrowing outside, plants continued to increase root growth

Figure 2.

Changes in the parameters characterising the root growth zone (shoots inside Petri dishes): (a) Growth velocity of root tip (

v

Tip

), (b) maximal relative elemental growth rate (REGR), (c) full width at half maximum (FWHMax.) of elongation zone and (d) length of growth zone. Plants were exposed for 18 d to constant light intensities (60 or 300

µ

mol m−2 s−1) or for 14 d to 60 µmol m−2 s−1 and thereafter for 5 d to 300 µmol m−2 s−1 (60–300) (mean value ± SE, n = 4).Days after germination

14 15 16 17 18 19

Length (mm

)

0.0

0.5

1.0

1.5

Days after germination

14 15 16 17 18 19

FW

HM

ax.

(m

m)

0.0

0.2

0.4

0.6

0.8

v Tip (mm

h –1

)

0

50

100

150

200

250

300

RE

GR

max. (%

h–1)

5

10

15

20

25

30

35

6060 – 300300

(a) (b)

(c) (d)

Figure 3. Fresh weight (FW) and sugar concentrations (C; glucose, fructose and sucrose) of leaves and roots harvested on day 14 or 18, respectively (shoots inside Petri dishes). Plants were exposed for 14 or 18 d to constant light intensities (60 or 300 µmol m−2 s−1) or for 14 d to 60 µmol m−2 s−1 and thereafter for 4 d to 300 µmol m−2 s−1 (60–300) (mean value ± SE, n = 5).

C (m

mol g

–1 F

W)

0

2

4

6

8

10

12

60 300 6060–300 300

C (m

mol g

–1 F

W)

0.0

0.5

1.0

1.5

60 300 6060–300

300

C (mm

ol g

–1 F

W)

0

2

4

6

8

10

Glucose

Fructose Sucrose

day 14 day 14day 18 day 18

FW

(m

g)

0

10

20

30

40

50LeafRoot

FW

1940 K. A. Nagel et al.

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd, Plant, Cell and Environment, 29, 1936–1945

throughout the experimental period. This continuousincrease was not observed when plants were exposed to lowlight around the shoots and high light around the roots.Root growth remained constant throughout some days, indi-cating that root illumination might interfere with rootgrowth activity. It has been reported that light reaching theroot can inhibit root growth (Pilet & Ney 1978; Eliasson &Bollmark 1988), either via stimulated production of ethyl-ene (Eliasson & Bollmark 1988) or flavonols (Hartmannet al. 2005; Schmid et al. 2005). When comparing the twocultivation systems, other factors affecting plant growthpotential such as the relative humidity or CO2-concentrationto which the shoot is exposed, can affect root growthstrongly. A detailed discussion of such effects is outside thescope of this study.

With the shoots outside, plants transferred from low tohigh light seemed to reach an increased steady-state levelof root growth faster than those with the shoots inside (3 dversus 4 d; Fig. 4b).

Is sucrose a key regulatory element in root growth response to increased light?

In two additional experiments, it was tested whethersucrose concentration is the key factor mediating root

growth response to increased light interception. In the firstexperiment, shoots were excised and growth in isolatedroots was monitored with or without addition of sucrose tothe medium (Fig. 5). Without the addition of sucrose, elon-gation velocity decreased rapidly and plants from both lowand high light showed similar kinetics of growth decay(Fig. 5a). When sucrose was present in the externalmedium, root growth decreased much more slowly, indicat-ing that externally applied sucrose can maintain rootgrowth substantially. Interestingly, in all three populationsinvestigated the length of the growth zone decreased veryslowly (Fig. 5b). This implies that developmental processesof cells can proceed in a relatively orderly manner as longas the root system still has some growth potential. MaximalREGR (Fig. 5c) was affected by the lack of sucrose import

Figure 4. Growth velocity of root tip (vTip) with shoots growing inside or outside Petri dishes, respectively. (a) Plants were exposed for 14 d to constant light intensity (60 µmol m−2 s−1) or (b) for 14 d to 60 µmol m−2 s−1, and thereafter for 4 d to 300 µmol m−2 s−1. Roots were grown either for 18 d in the dark (60 outside/root 0) or for 14 d in the dark and thereafter for 4 d at 300 µmol m−2 s−1 (60 outside/root 0–300) (mean value ± SE, n = 4).

Days after germination

14 15 16 17 18 19

0

100

200

300

400

60–300 inside60–300 outside

0

100

200

300

400

50060 inside60 outside/root 0 60 outside/root 0–300

(b)

(a)

v Tip (mm

h–1

)v T

ip (mm

h–1

)

Figure 5. Root growth after removing the shoot (shoots outside Petri dishes). Plants were exposed to constant light intensities (60 or 300 µmol m−2 s−1) or grown at 60 µmol m−2 s−1 with 2% sucrose in the agar medium (Suc). Normalized values of (a) growth velocity of root tip (vTip), (b) length of the growth zone and (c) maximal relative elemental growth rate (REGRmax). Shoots were excised at time 0. Values prior to removing the shoot were averaged for each replicate and treatment and set to 100% (mean value ± SE, n = 3).

Time after removing the shoot (h)

0 10 20 30 40 6050

RE

GR

max

,nor

m (

%)

0

20

40

60

80

100

120

140

Leng

thno

rm (

%)

0

20

40

60

80

100

120

140

v Tip

,nor

m (

%)

0

20

40

60

80

100

120

14060300Suc

(a)

(b)

(c)

Root growth and light intensity 1941

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd, Plant, Cell and Environment, 29, 1936–1945

from the shoot much more severely than growth-zonelength. This suggests that a sustained supply of sucrose is ofspecial importance for that region within the growth zone,in which cell walls and macromolecules are assembled mostrapidly.

In a second experiment, transgenic plants that showreduced sucrose synthesis were investigated for their rootgrowth reactions in response to alterations in light intensity.SPP, which catalyses the final step in the sucrose synthesispathway, is strongly reduced in these plants via RNA inter-ference, resulting in a decrease of SPP activity to less than10% of wild-type SPP activity (Chen et al. 2005). At lowlight intensity, the root growth of the transgenic plants wassomewhat lower than that of wild-type plants (Fig. 6a, day

14). During four consecutive days of high-light exposure,the root growth of the two lines diverged significantly: whilewild-type plants increased vTip by 300%, sucrose synthesis-deficient plants could increase vTip by only 50%. Compari-son of REGR distributions revealed that (1) both plantlines increased growth mainly by extension of the growth-zone length and (2) the transgenic plants could not achievemaximal REGR values of more than 30% h−1, while wild-type plants reached almost 40% h−1 (Fig. 6b).

Reaction dynamics of growth and photosynthesis during the first hours after light transition

A closer analysis of the first few hours of light transitionindicated that there were two phases in rapid responses tolight acclimation: the first 30 minutes and the followingtwo hours after light transition (Fig. 7). During the first 30

Figure 6. Root growth of wild-type (WT) and transgenic (SPP) plants (shoots outside Petri dishes): (a) Growth velocity of root tip vTip and (b) distributions of relative elemental growth rate (REGR) along the root growth zone. Plants were exposed for 14 d to 60 µmol m−2 s−1, and thereafter for 4 d to 300 µmol m−2 s−1 [mean value ± SE, n = 4; (b) SE for every 10th data point are shown].

Distance from root tip (mm)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

RE

GR

(%

h–1

)

0

10

20

30

40

50

day 14day 15

day 16day 17

day 18

Days after germination13 14 15 16 17 18 19

0

100

200

300

400

500WT 60–300SPP 60–300

RE

GR

(%

h–1

)

0

10

20

30

40

50

day 14day 15

day 16 day 17 day 18WT

SPP

(a)

(b)

v Tip (mm

h–1

)

Figure 7. Growth velocity of root tip (vTip) and electron transport rate (ETR) of photosystem II during the first three hours (0–3 h) after light transition (shoots outside Petri dishes). (a) Wild-type (WT, light increase; mean value ± SE, n = 4) and (b) transgenic plants (SPP, light increase; mean value ± SE, n = 4) were acclimated to constant low light intensity (60 µmol m−2 s−1). At time 0, light intensity was increased to 300 µmol m−2 s−1. (c) Wild-type (WT, light decrease) plants were acclimated to constant high light intensity (300 µmol m−2 s−1). At time 0 light intensity was decreased to 60 µmol m−2 s−1 (mean value ± SE, n = 4).

100

120

140

160

180

200

5

10

15

20

25

30

35

40

vTip

ETR

60 60300WT(a)

(c)

Time (h)–3 –2 –1 0 1 2 3 4 5 6

100

200

300

400

500 ET

R (m

mol m

–2 s

–1) E

TR

(mm

ol m–2

s–1)

0

10

20

30

40

50

60300 30060

WT

light increase

light decrease

70

80

90

100

110

120

13060 60300

SPP(b)

light increase

v Tip (mm

h–1

)v T

ip (mm

h–1

)v T

ip (mm

h–1

)

1942 K. A. Nagel et al.

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd, Plant, Cell and Environment, 29, 1936–1945

minutes in low-light-acclimated plants transferred to higherlight, there was a transient decrease in vTip of 15–20% inboth wild-type and transgenic plants (Fig. 7a,b). When lightintensity was decreased for high-light-acclimated plants, theopposite response was observed: growth increased tran-siently by about 20% of the steady-state value anddecreased thereafter (Fig. 7c). During the subsequent 2.5 hthe wild-type plants showed a very characteristic, sinusoidalgrowth pattern with a period length of about 2 h whentransferred from low to high light (Fig. 7a; SupplementaryVideo Clip S1). In contrast to this, a relatively constant vTip

was recorded when plants were transferred from high tolow light (Fig. 7c). In total, 3 h after the transition of lightintensity, growth was increased by about 15% in the light-increase experiment (vTip increased from 140 to 160 µmh−1) and decreased by about 20% in the light-decreaseexperiment (vTip decreased from 400 to 320 µm h−1), respec-tively. The increase in growth of 15% during 3 h corre-sponds nicely to the ‘longer-term’ increase of 400% within4 d (Fig. 2). In the transgenic plants the light increase alsoenhanced root growth by about 20% within 3 h, but did notshow the characteristic, sinusoidal growth pattern (Fig. 7b;Supplementary Video Clip S2).

To test the hypothesis if this sinusoidal growth oscillationcould be caused by the influx of photosynthates into theroots, we measured the ETR of PS II in the shoot afterchanging the light conditions. During the first hour afterlight transition ETR increased by more than a factor of twoin the light-increase experiment (Fig. 7a), and decreased toabout one-third of the initial steady-state value in the light-decrease experiment (Fig. 7c), respectively. During the fol-lowing two hours, ETR responded to light increase bychanging sigmoidally to a level clearly above the initialdark-acclimated value. The ETR values declined to low-light values upon switching back light intensity to low lightagain (Fig. 7a). In the light decrease experiment ETRremained constant and returned to the initial steady-statevalue within minutes after increasing the light intensityagain.

DISCUSSION

Changes in the light environment are translated via sucrose into root growth acclimation

The results presented here show clearly that root growthacclimates rapidly to light changes. Several aspects of thedynamics of root growth acclimation indicate an importantrole of sucrose here. Clearest evidence for this wasobtained from the root growth data of SPP-reduced trans-genic plants during the transition from low to high lightintensity (Fig. 6). In those plants that show inhibition ofphotosynthesis and reduced levels of sucrose comparedwith wild-type plants (Chen et al. 2005), root growthscarcely increased in response to elevated light intensityand the growth reaction during the first hours after lighttransition differed strongly from that of wild-type plants(Fig. 7). The remaining amount of sucrose, or other forms

of long-range transport sugars such as raffinose or stachy-ose, might maintain carbohydrate delivery to the root sys-tem of the transgenic plants. However the amounts ofsugars available in the root growth zone were clearly notenough in these plants to realize the full growth potentialof N. tabacum in the given environment.

Further support for the key role of sucrose in the growthacclimation of roots towards changing light intensity wasobtained in the experiments of isolated root systems withand without externally applied sucrose (Fig. 5). Becauseexternal sucrose can be taken up by isolated root systems(Chin, Haas & Still 1981), it can be concluded that a con-tinuous sucrose import into the growing root could havebeen sustaining growth. Other factors such as phytohor-mones exported from the shoot are also necessary for uti-lizing the full potential of root growth (Robbins & Hervey1978). However, sucrose is likely to be the most criticalfactor as root growth decreased practically to zero within10 h upon shoot excision (Fig. 5a). When sucrose was sup-plied, growth was still at 50% of its initial value after 10 h.For tomato root systems, it has been shown that growth ofthe isolated root system could be sustained for 8 d whensucrose was applied externally (Street & McGregor 1952).Carbohydrates accumulated in the root cannot sustain rootgrowth for a long time, while the carbohydrate depot ofsource leaves is a critical factor determining the extent ofroot growth (Eliasson 1968). This indicates that rootsrequire a constant influx of sucrose that cannot easily bereplaced by other carbohydrate pools to sustain growthactivity. Sucrose reaching the root growth zone is cleavedinto glucose and fructose by invertase that has its maximalactivity in the region of cell elongation (Toko et al. 1987),leading to balanced relations between sucrose, glucose andfructose. Our results on carbohydrate concentrations inleaves and roots showed that elevated concentrations ofglucose, fructose and sucrose in the shoot coincided withelevated concentrations of these carbohydrates as well aselevated growth activity in the roots (Fig. 3). Four daysafter transition of plants from low to high light, sugar con-centrations were higher than in plants from steady-statehigh light. This indicates an accumulation of sugars andhighlights that in future studies it will be important to quan-tify the exact correlation between the temporal dynamicsof sugar fluxes and concentrations and changes in rootgrowth rate to come to a mechanistic understanding. Alinear correlation between hexose concentrations in rootsand root growth activity has already been demonstrated byFreixes et al. (2002). In the same study, it has also beenpointed out that externally applied sucrose amelioratesroot growth (Freixes et al. 2002).

Apart from their role as material growth substrates, car-bohydrates affect growth by playing an important role assignal molecules in feedback mechanisms of gene regula-tion. Sucrose acts as signal molecule in source–sink rela-tions throughout all stages of plant development (Roitsch1999; Smeekens 2000) and can modulate the expression ofa large number of genes (Koch 1996; Smeekens 1998).Sucrose and glucose can up-regulate growth-related genes

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and down-regulate stress-related genes (Ho et al. 2001),demonstrating clearly their key function as signalling mol-ecules for light-acclimation processes. Cell division and cellcycle may be controlled by sucrose at the transitionbetween G1-S and between G2-M phases (Van’t Hof 1968;Smeekens 2000). Moreover, cell division is affected in Ara-bidopsis by sugar availability, which regulates the activityof cyclins (Riou-Khamlichi et al. 2000). Our finding thatacclimation to shoot-intercepted light intensity does notinvolve alterations in the distribution of REGR along theroot growth zone indicates that the relation between tissue,which is located in the meristematic zone, and tissue, whichis located in the elongation zone, remains constant (Figs 1,2 & 6). This is in agreement with the findings of Muller et al.(1998) that the flux of cells through the root growth zoneis affected by light intensity mainly through the durationduring which cells remain competent to divide, but notthrough the duration of the cell cycle itself.

Temporal dynamics of root growth acclimation

Within 4 d after switching the light intensity from 60 to300 µmol m−2 s−1, root growth reached the level observed insteady-state high-light-grown plants (Figs 1 & 2). This cor-responds well to the kinetics observed in a light-decreasetransition experiment (from 400 to 80 µmol m−2 s−1) per-formed with maize plants (Muller et al. 1998). In thesemaize plants, the distribution of REGR was followed dur-ing the transition period by measuring distances betweenink marks applied on the root surface. By this ‘classical’method Muller et al. (1998) demonstrated that the REGRdistribution within the root growth zone is mainly scaledbetween the two steady states (80 and 400 µmol m−2 s−1) andthe light-decrease transition does not cause a marked shiftin the proportions of the meristematic zone – characterizedby low growth rates at the root apex – and the zone of cellelongation which includes the maximal REGR. Our resultsobtained in a different species and with a higher precisionconfirmed the findings by Muller et al. (1998). For steady-state growth conditions at different light intensities, con-nections between root growth, root biomass and light inten-sity have been investigated extensively (Barney 1950;Vincent & Gregory 1989; Aguirrezabal et al. 1994; Mont-gomery & Chazdon 2002). These results generally show anincrease in root growth under high light intensities, whichis mediated by increased photosynthesis (Webb 1976; Hanet al. 1999).

During the first three hours after light transition, thegrowth reaction exhibited characteristic features, clearlydemonstrating the capacity of plants to respond to dynamicfluctuations of ambient light intensity (Fig. 7). In all threeexperiments (wild-type and transgenic plants in increasinglight and wild-type plants in decreasing light), plantsshowed a transient change in growth intensity of about 15–20% during the first 30 minutes. When light intensityincreased, growth decreased and vice versa. This effect hasbeen reported for leaf growth in numerous studies (Hsiao,Acevedo & Henderson 1970; Christ 1978; Walter & Schurr

2005). It has been shown that step changes in light intensityaffect cell wall pH (Mühling et al. 1995) and stomatal con-ductance (Mott & Buckley 2000) and are, hence, likely toaffect cell wall extensibility and turgor. Here, it is shownfor the first time that roots react in the same way as hasbeen described for leaves before. This could imply thataltered hydraulic properties of the plant (such as stomatalclosure upon a sudden decrease of light intensity) or alteredapoplastic ion relations (such as pH or membrane poten-tial) are propagated within the entire plant almost instan-taneously and hence, might trigger growth reactions inroots.

Within the first hour after changing the light intensity,ETR also changed in parallel with light intensity. In thelight-increase experiment, ETR decreased sigmoidally1–3 h after light change (Fig. 7a). In parallel with this,growth rose to a maximum first, decreased sharply thereaf-ter and eventually reached a steady state. We hypothesizethat this sinusoidal growth oscillation is caused by the influxof sucrose reaching the root. Increased photosynthesis inthe shoot would promote sucrose export and increasedsucrose fluxes into the root would then lead to an over-shooting reaction of growth. Again, our results obtainedwith transgenic plants indicate that sucrose, and not forexample a hormonal signal, might be the trigger of thisreaction chain (Fig. 7b): while the sucrose-deficient plantsshowed the transient, ‘hydraulic-induced’ growth depres-sion 30 min after light increase, they did not show the sub-sequent sinusoidal growth pattern after light change.Instead, growth increased in these plants rather continu-ously. In future studies, the aforementioned hypothesescould be tested by monitoring the influx of labelled sucrosefrom the shoot.

Non-destructive high-resolution methods of growth anal-ysis such as the one used in the present study can provideinformation on the dynamic responses of plant growth toshort-term fluctuations of light intensity, which occur natu-rally on cloudy days, in sunflecks, in gaps of forest standsor in other heterogeneous natural growth settings. Thepotential of primary root growth to acclimate rapidly toaltered environmental conditions such as temperature(Walter et al. 2002) and nutrient availability (Walter et al.2003) has been demonstrated with high-resolution growthmonitoring methods before. Concerning the mechanisticregulation of root growth acclimation to altered or fluctu-ating light intensity, it will be important to look into therapid reaction of the root during those first hours afteralteration of external conditions. As gene expression andsignalling cascades are often regulated within very shorttime frames, it is important to learn more about the dynam-ics of plant acclimation in time frames of hours to minutes.

CONCLUSION

While it is well known from steady-state experiments thatincreased light levels lead to increased root growth viahigher export of carbohydrates from the shoot, we demon-strated in this study that characteristic root growth

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reactions already occur during the first hours after lighttransitions. We propose that an initial phase of growth accli-mation may result from changes in cell wall extensibility orturgor that can be quickly propagated within the wholeplant. The subsequent phase – lasting only a few hours –manifests how the root growth reaction is influenced bysucrose import and hence, is critical for understanding themechanistic processes of growth acclimation to fluctuatinglight intensity.

ACKNOWLEDGMENTS

We are indebted to Shizue Matsubara for her help duringthe chlorophyll fluorescence measurements and during thepreparation of this manuscript. We thank Andres Chavar-ria-Krauser, Hanno Scharr, Sabine Briem and KathrinCloos for help with the analysis of the digital imagesequences and Maja M. Christ and Vicky Temperton fortheir comments on the manuscript. We are grateful for theprovision of transgenic seeds by Uwe Sonnewald. K.A.N.acknowledges the support for her PhD thesis at theHeinrich-Heine-Universität Düsseldorf by the GermanScience Foundation (DFG; Grant no. Schu 946 2-2).

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Received 22 December 2005; received in revised form 17 March2006; accepted for publication 12 June 2006

SUPPLEMENTARY MATERIAL

The following supplementary material is available for thisarticle:

Video Clip S1. Time-lapse movie of relative elementalgrowth rate (REGR) along the root growth zone (shootoutside Petri dish) of a wild-type plant. The video wasacquired throughout the first three hours after increasinglight intensity from 60 to 300 µmol m−2 s−1 (movie displayed770 times faster than original sequence). Root diameter:300 µm, colour-code as displayed in Fig. 1, but extending toa maximum of 35% h−1.Video Clip S2. Time-lapse movie of relative elementalgrowth rate (REGR) along the root growth zone (shootoutside Petri dish) of a transgenic plant (SPP). The videowas acquired throughout the first three hours after increas-ing light intensity from 60 to 300 µmol m−2 s−1 (movie dis-played 770 times faster than original sequence). Rootdiameter: 300 µm, colour-code as displayed in Fig. 1.

This material is available as part of the online articlefrom: http://www.blackwell-synergy.com/doi.abs/10.1111/j.1365-3040.2006.01569.x(This link will take you to the article abstract)

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