Dynamics of root growth stimulation in Nicotiana tabacum in increasing light intensity

Authors


Achim Walter. Fax: +49 2461 612492; e-mail: a.walter@fz-juelich.de

ABSTRACT

Light intensity is crucial for plant growth. In this study, the hypothesis was tested whether a sudden increase in light intensity leads to an immediate increase of root growth. Seedlings of Nicotiana tabacum grown in agar-filled Petri dishes were subjected to light intensities of 60 and 300 µmol m−2 s−1, respectively. Seedling biomass, sucrose, glucose and fructose concentration as well as primary root growth increased significantly with light intensity. The dynamics of the increase in root growth were analysed here in more detail. In transition experiments from low to high light intensities, root growth increased by a factor of four within 4 d, reaching the steady-state level measured in plants that were cultivated in high-light conditions. The distribution of relative elemental growth rates along the root growth zone retained a constant shape throughout this transition. During the first three hours after light increase, strong growth fluctuations were repeatedly observed with the velocity of the root tip cycling in a sinusoidal pattern between 120 and 180 µm h−1. These dynamic patterns are discussed in the context of hydraulic and photosynthetic acclimation to the altered conditions. Experiments with externally applied sucrose and with transgenic plants having reduced capacities for sucrose synthesis indicated clearly that increasing light intensity rapidly enhanced root growth by elevating sucrose export from shoot to root.

INTRODUCTION

Root growth is closely related to carbon import and hence, to light conditions for the shoot. Carbon gain in roots is realized predominantly by import from the shoot via the phloem, while the major loss of root carbon occurs via respiration associated with growth and ion uptake (Farrar & Jones 2000). A number of studies have investigated differences in root growth between plants acclimated to low- or high-light environments (e.g. Webb 1976; Vincent & Gregory 1989; Aguirrezabal, Deleens & Tardieu 1994) or between plants growing with variable external sucrose supply (Street & McGregor 1952; Freixes et al. 2002). It has been proposed that root length is related to cumulative intercepted radiation (Aresta & Fukai 1984; Vincent & Gregory 1989). Likewise, relations between growth in primary and secondary roots (Bingham & Stevenson 1993) as well as between root and shoot growth (Thaler & Pages 1996) have also been extensively studied in steady-state experiments. A key factor directly connecting the irradiation of the shoot and the elongation of root tips is the local hexose concentration, which correlates very well with growth rates of individual roots of a given species (Scheible et al. 1997; Freixes et al. 2002). An increase in the sugar content of root tissue promotes growth of primary and secondary roots without affecting branching patterns or overall root architecture (Bingham, Blackwood & Stevenson 1997), in contrast to increases of other growth substrates such as NO3- (Zhang et al. 1999) or other mineral nutrients (Forde & Lorenzo 2001), which can lead to marked alterations of root architecture via localized effects on growth (e.g. PO43−; see Watt & Evans 1999).

While a large amount of data on the reaction of overall root growth to different steady-state light conditions is available, much less is known about the differential reactions within the growth zone to changing light intensities that are common in natural environments. Muller, Stosser & Tardieu (1998) showed that in maize the length of the growth zone decreased with decreasing light intensity, in much 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 distribution of relative elemental growth rates (REGR) within the root growth zone has, however, hardly been explored. Recent studies using high-resolution, automated optical growth monitoring methods have shown that alterations in root growth can take place within less than an hour in reaction 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 in light environment of the shoot can also induce such fast reactions of root growth. Comparisons of maize root growth in decreasing light intensities indicate that a reduction in root elongation rate takes about 4 d (Muller et al. 1998).

If sucrose export from the shoot is among the signals linking shoot light interception and root growth, transgenic plants with reduced sucrose content (Chen et al. 2005) could reveal the degree to which this process is regulated via sucrose. Response kinetics of REGR distribution to shoot light conditions may also give some information about mechanisms that are involved in the regulation of this process. Sugar import into the root growth zone can regulate a vast number of enzymes involved in carbon metabolism (Koch 1996; Ho et al. 2001) or the cell cycle (Riou-Khamlichi et al. 2000). Genes regulating root growth have been recently characterized in Arabidopsis (Birnbaum et al. 2003) and maize (Bassani, Neumann & Gepstein 2004).

In this study, the hypothesis was tested whether a sudden increase in light intensity leads to a rapid increase of root growth. 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.) ecotype Xanthi were surface-sterilized with sodium hypochlorite solution and sown on sterile agar (1%, w/v) in Petri dishes (120 × 120 × 17 mm; five seedlings per dish) that were sealed with fabric tape (Micropore, 3 M Health Care, Neuss, Germany). The medium contained full-strength Ingestad mineral nutrient solution (Ingestad 1982; Walter et al. 2003) and in some cases 2% (w/v) sucrose, depending on the experiment.

Transgenic tobacco plants, N. tabacum (L.) ecotype Samsun SPPi17 with decreased sucrose-6-phosphate phosphatase (SPP) levels, were germinated on an agar containing kanamycin. After 10 d, the transgenic plants, which were kanamycin-resistant, were selected and transferred to an agar medium without kanamycin.

It is common practice that investigations with seedlings grown on agar-filled Petri dishes are performed with shoots growing completely inside the sterile Petri dish (see e.g. Freixes et al. 2002). Because this is the currently accepted cultivation system in the scientific community, most experiments of this study were performed in such systems as well, but for reasons given below, we also developed cultivation procedures that allowed for shoot growth outside the Petri dish. For shoots grown inside the Petri dish, the dishes were half-filled with agar and the seeds were pushed slightly into the agar. Shoots could then grow in the air volume inside the dishes. After sowing the seeds, Petri dishes were put almost horizontal, ensuring that the roots had to grow within and not at the surface of the agar, thereby improving the optical quality of the acquired image sequences substantially. Two days after germination the Petri dishes were positioned vertically, allowing the roots to grow downwards within 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 mechanically. Hence, we established a cultivation procedure that allowed shoots to grow outside the Petri dish. Here, the Petri dishes were filled completely with agar and sealed with fabric tape. The seeds were pushed slightly into the agar through five holes (diameter approximately 2 mm) at one side of the closed Petri dishes. The holes were then covered with laboratory film (Parafilm, Pechiney Plastic Packaging, Menasha, WI, USA) to keep the seeds moist and to keep the agar free of contaminations. After germination the Parafilm was removed. The shoot then grew outside the Petri dishes, while the roots grew in the agar which stayed free of visible fungal or bacterial infections throughout the growth monitoring period.

It has to be noticed that shoots growing outside the Petri dish received higher light intensities than those shoots growing inside. When light was penetrating the Petri dish perpendicularly, its intensity was only decreased by about 5% (data not shown). Yet light that reaches the Petri dish surface at lower angles is reflected stronger, thus, light intensity reaching shoots inside the Petri dishes will have been lower than light intensity reaching shoots outside the Petri dishes.

After sowing, all Petri dishes were placed at 23 °C (± 3 °C) and 12 h/12 h light/dark cycles. The plants were illuminated either in steady-state light conditions with 60 or 300 µmol m−2 s−1 or for 14 d with 60 µmol m−2 s−1, and thereafter for 4–5 d with 300 µmol m−2 s−1. The two light intensities used in this study corresponded to photon flux densities of 2.6 and 13 mol m−2 d−1, respectively. Those light intensities are in the typical range used for laboratory cultivations of plants in Petri dishes but are much lower than typical light intensities under field conditions. The light intensity was increased by illuminating the plants with cool light (KL 200, Schott, Wiesbaden, Germany) to analyse the effect of light intensity without increasing the temperature. The root growth and sugar concentrations of the seedlings were investigated 14–19 d after germination as described further.

Determination of ethanol-soluble sugars

For determination of ethanol-soluble sugars, whole shoots and roots were harvested and immediately frozen in liquid N2 and then extracted with 80% ethanol (v/v) at 80 °C for 20 min according to Arnon (1949). The supernatant was stored 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 coupled enzyme assay (Jones, Outlaw & Lowry 1977) using a multiplate photometer (ht II, Anthos Mikrosysteme GmBH, Krefeld, Germany). Plates were loaded with extract, imidazol buffer, ATP, NADP+ and glucose-6-phosphate dehydrogenase (5.6 U). The enzymes hexokinase (6 U; glucose assay), phosphoglucoisomerase (5 U; fructose assay) and invertase (10 U; sucrose assay) were then sequentially added and the change in absorption at 334 nm was measured.

Image acquisition and processing

The Petri dishes were put in a microrhizotron setup (similar to the rhizotron setup described in Walter et al. 2002; Walter & Schurr 2005), with which images of the root tips were captured by a CCD camera (Sony XC-ST50; Sony, Köln, Germany). The images were taken using infrared illumination (λ = 940 nm) with a frequency of two images per minute. Each image acquired – also designated as a frame – had a resolution of 720 × 560 pixels, corresponding to an area of 3.2 × 2.4 mm. The camera was equipped with a lowpass infrared filter (RG 9, Schott, Mainz, Germany) that blocked visible light. Growing root tips were tracked automatically using three moving stages vertical to each other. The moving stages, which were controlled by an image-based tracking algorithm, allowed one to maintain the root tip in the field of view during the image acquisition. The algorithms which control the root tracker and acquire the image sequences were based on a digital image sequence processing software package (Heurisko, Aeon, Hanau, Germany; Schmundt et al. 1998; Walter et al. 2002, 2003).

The acquired image sequences were used to calculate the velocity of the root tip (vTip) and the REGR along the root growth zone using the structure tensor method (Schmundt et al. 1998; Haußecker & Spies 1999). Motion analysis by structure tensor is based on local grey value structures in the images. The resulting velocity vector fields were interpolated and local growth rates were calculated from local divergence in the velocity maps (Schmundt et al. 1998; Walter et al. 2002; Scharr 2004).

Chlorophyll fluorescence measurements

The chlorophyll a fluorescence measurements were performed with a portable pulse amplitude-modulated photosynthesis yield analyser (Imaging PAM, Heinz Walz GmbH, Effeltrich, Germany). The photosynthetic electron transport rate (ETR) of photosystem (PS) II was obtained as ETR = ΦPSII × PPFD × 0.5 × 0.84, while ΦPSII is the effective quantum yield of PS II. The factor 0.5 assumes that PS II and PS I were equally excited and the so-called empirical ETR correction factor of 0.84 takes into account that only a fraction of incident light (PPFD) is really absorbed by photosystems (Ehleringer 1981).

RESULTS

Effect of increasing light intensity on root growth

Growth-zone length and intensity of growth differed markedly between the two steady-state treatments (60 and 300 µmol m−2 s−1), while the shape of REGR distributions was comparable between treatments (Fig. 1a). The transition in the plants between those two steady states occurred within 4 d, as shown by irradiance transition experiments (Fig. 1b). REGR distributions and total growth intensity (vTip) were analysed in more detail from the data of several replicates. Therefore, four parameters were chosen to characterize the REGR distribution: vTip, maximal REGR, full width at half maximum of the elongation zone (FWHMax.) and length of the entire growth zone (Fig. 2a–d). In steady-state experiments, growth was stable over several days. The vTip, which characterizes the total growth activity of the organ, was four times higher in the high-light treatment compared with the low-light treatment (Fig. 2a), corresponding to the difference in the applied light intensity. Upon transfer of the seedlings from low to high light on day 14, vTip rose in a sigmoidal way for 4 d, reaching the same plateau value of vTip as plants grown under steady-state light. Very similar graphs were obtained for the other three parameters describing the shape of the REGR distributions (Fig. 2b–d), showing quantitatively that the entire root growth zone and hence, each phase of cellular development, was affected very similarly by the increasing light treatment.

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 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. (c) Typical colour-coded REGR distributions along root tips under constant light intensities (60 or 300 µmol m−2 s−1) (mean value ± SE, n = 4).

Figure 2.

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

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

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).

Shoot and root carbohydrate concentrations also differed considerably between low- and high-light treatments (Fig. 3). Sucrose concentrations in leaves from the steady-state experiments differed by a factor of five; glucose and fructose concentrations were about 10 times higher under 300 µmol m−2 s−1, compared with 60 µmol m−2 s−1 on day 14 and day 18. Interestingly, plants transferred to the high-light intensity for 4 d showed higher carbohydrate concentrations in leaves and roots than the plants under the steady-state conditions at 300 µmol m−2 s−1. In roots, sugar concentrations of the transferred plants exceeded 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 the observed results and to provide a solution for ‘handling’ shoots experimentally, plants were also grown in another cultivation system with leaves outside and roots inside the Petri dish. Here, the Petri dish was covered with a plastic sheet to provide dark conditions for roots. With shoots outside the Petri dish, roots grew faster in steady-state low-light conditions compared with the experiments with the conventional cultivation system (Fig. 4a). When shoots were growing outside, plants continued to increase root growth throughout the experimental period. This continuous increase was not observed when plants were exposed to low light around the shoots and high light around the roots. Root growth remained constant throughout some days, indicating that root illumination might interfere with root growth activity. It has been reported that light reaching the root can inhibit root growth (Pilet & Ney 1978; Eliasson & Bollmark 1988), either via stimulated production of ethylene (Eliasson & Bollmark 1988) or flavonols (Hartmann et al. 2005; Schmid et al. 2005). When comparing the two cultivation systems, other factors affecting plant growth potential such as the relative humidity or CO2 -concentration to which the shoot is exposed, can affect root growth strongly. A detailed discussion of such effects is outside the scope of this study.

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).

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

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

In two additional experiments, it was tested whether sucrose concentration is the key factor mediating root growth response to increased light interception. In the first experiment, shoots were excised and growth in isolated roots was monitored with or without addition of sucrose to the medium (Fig. 5). Without the addition of sucrose, elongation velocity decreased rapidly and plants from both low and high light showed similar kinetics of growth decay (Fig. 5a). When sucrose was present in the external medium, root growth decreased much more slowly, indicating that externally applied sucrose can maintain root growth substantially. Interestingly, in all three populations investigated the length of the growth zone decreased very slowly (Fig. 5b). This implies that developmental processes of cells can proceed in a relatively orderly manner as long as the root system still has some growth potential. Maximal REGR (Fig. 5c) was affected by the lack of sucrose import from the shoot much more severely than growth-zone length. This suggests that a sustained supply of sucrose is of special importance for that region within the growth zone, in which cell walls and macromolecules are assembled most rapidly.

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).

In a second experiment, transgenic plants that show reduced sucrose synthesis were investigated for their root growth reactions in response to alterations in light intensity. SPP, which catalyses the final step in the sucrose synthesis pathway, is strongly reduced in these plants via RNA interference, resulting in a decrease of SPP activity to less than 10% of wild-type SPP activity (Chen et al. 2005). At low light intensity, the root growth of the transgenic plants was somewhat 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: while wild-type plants increased vTip by 300%, sucrose synthesis-deficient plants could increase vTip by only 50%. Comparison of REGR distributions revealed that (1) both plant lines increased growth mainly by extension of the growth-zone length and (2) the transgenic plants could not achieve maximal REGR values of more than 30% h−1, while wild-type plants reached almost 40% h−1 (Fig. 6b).

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].

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

A closer analysis of the first few hours of light transition indicated that there were two phases in rapid responses to light acclimation: the first 30 minutes and the following two hours after light transition (Fig. 7). During the first 30 minutes in low-light-acclimated plants transferred to higher light, there was a transient decrease in vTip of 15–20% in both wild-type and transgenic plants (Fig. 7a,b). When light intensity was decreased for high-light-acclimated plants, the opposite response was observed: growth increased transiently by about 20% of the steady-state value and decreased thereafter (Fig. 7c). During the subsequent 2.5 h the wild-type plants showed a very characteristic, sinusoidal growth pattern with a period length of about 2 h when transferred from low to high light (Fig. 7a; Supplementary Video Clip S1). In contrast to this, a relatively constant vTip was recorded when plants were transferred from high to low light (Fig. 7c). In total, 3 h after the transition of light intensity, growth was increased by about 15% in the light-increase experiment (vTip increased from 140 to 160 µm h−1) and decreased by about 20% in the light-decrease experiment (vTip decreased from 400 to 320 µm h−1), respectively. The increase in growth of 15% during 3 h corresponds nicely to the ‘longer-term’ increase of 400% within 4 d (Fig. 2). In the transgenic plants the light increase also enhanced root growth by about 20% within 3 h, but did not show the characteristic, sinusoidal growth pattern (Fig. 7b; Supplementary Video Clip S2).

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).

To test the hypothesis if this sinusoidal growth oscillation could be caused by the influx of photosynthates into the roots, we measured the ETR of PS II in the shoot after changing the light conditions. During the first hour after light transition ETR increased by more than a factor of two in the light-increase experiment (Fig. 7a), and decreased to about one-third of the initial steady-state value in the light-decrease experiment (Fig. 7c), respectively. During the following two hours, ETR responded to light increase by changing sigmoidally to a level clearly above the initial dark-acclimated value. The ETR values declined to low-light values upon switching back light intensity to low light again (Fig. 7a). In the light decrease experiment ETR remained constant and returned to the initial steady-state value within minutes after increasing the light intensity again.

DISCUSSION

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

The results presented here show clearly that root growth acclimates rapidly to light changes. Several aspects of the dynamics of root growth acclimation indicate an important role of sucrose here. Clearest evidence for this was obtained from the root growth data of SPP-reduced transgenic plants during the transition from low to high light intensity (Fig. 6). In those plants that show inhibition of photosynthesis and reduced levels of sucrose compared with wild-type plants (Chen et al. 2005), root growth scarcely increased in response to elevated light intensity and the growth reaction during the first hours after light transition 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 stachyose, might maintain carbohydrate delivery to the root system of the transgenic plants. However the amounts of sugars available in the root growth zone were clearly not enough in these plants to realize the full growth potential of N. tabacum in the given environment.

Further support for the key role of sucrose in the growth acclimation of roots towards changing light intensity was obtained in the experiments of isolated root systems with and without externally applied sucrose (Fig. 5). Because external sucrose can be taken up by isolated root systems (Chin, Haas & Still 1981), it can be concluded that a continuous sucrose import into the growing root could have been sustaining growth. Other factors such as phytohormones exported from the shoot are also necessary for utilizing the full potential of root growth (Robbins & Hervey 1978). However, sucrose is likely to be the most critical factor as root growth decreased practically to zero within 10 h upon shoot excision (Fig. 5a). When sucrose was supplied, growth was still at 50% of its initial value after 10 h. For tomato root systems, it has been shown that growth of the isolated root system could be sustained for 8 d when sucrose was applied externally (Street & McGregor 1952). Carbohydrates accumulated in the root cannot sustain root growth for a long time, while the carbohydrate depot of source leaves is a critical factor determining the extent of root growth (Eliasson 1968). This indicates that roots require a constant influx of sucrose that cannot easily be replaced by other carbohydrate pools to sustain growth activity. Sucrose reaching the root growth zone is cleaved into glucose and fructose by invertase that has its maximal activity in the region of cell elongation (Toko et al. 1987), leading to balanced relations between sucrose, glucose and fructose. Our results on carbohydrate concentrations in leaves and roots showed that elevated concentrations of glucose, fructose and sucrose in the shoot coincided with elevated concentrations of these carbohydrates as well as elevated growth activity in the roots (Fig. 3). Four days after transition of plants from low to high light, sugar concentrations were higher than in plants from steady-state high light. This indicates an accumulation of sugars and highlights that in future studies it will be important to quantify the exact correlation between the temporal dynamics of sugar fluxes and concentrations and changes in root growth rate to come to a mechanistic understanding. A linear correlation between hexose concentrations in roots and root growth activity has already been demonstrated by Freixes et al. (2002). In the same study, it has also been pointed out that externally applied sucrose ameliorates root growth (Freixes et al. 2002).

Apart from their role as material growth substrates, carbohydrates affect growth by playing an important role as signal molecules in feedback mechanisms of gene regulation. Sucrose acts as signal molecule in source–sink relations throughout all stages of plant development (Roitsch 1999; Smeekens 2000) and can modulate the expression of a large number of genes (Koch 1996; Smeekens 1998). Sucrose and glucose can up-regulate growth-related genes and down-regulate stress-related genes (Ho et al. 2001), demonstrating clearly their key function as signalling molecules for light-acclimation processes. Cell division and cell cycle may be controlled by sucrose at the transition between G1-S and between G2-M phases (Van’t Hof 1968; Smeekens 2000). Moreover, cell division is affected in Arabidopsis by sugar availability, which regulates the activity of cyclins (Riou-Khamlichi et al. 2000). Our finding that acclimation to shoot-intercepted light intensity does not involve alterations in the distribution of REGR along the root growth zone indicates that the relation between tissue, which is located in the meristematic zone, and tissue, which is 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 zone is affected by light intensity mainly through the duration during which cells remain competent to divide, but not through the duration of the cell cycle itself.

Temporal dynamics of root growth acclimation

Within 4 d after switching the light intensity from 60 to 300 µmol m−2 s−1, root growth reached the level observed in steady-state high-light-grown plants (Figs 1 & 2). This corresponds well to the kinetics observed in a light-decrease transition experiment (from 400 to 80 µmol m−2 s−1) performed with maize plants (Muller et al. 1998). In these maize plants, the distribution of REGR was followed during the transition period by measuring distances between ink marks applied on the root surface. By this ‘classical’ method Muller et al. (1998) demonstrated that the REGR distribution within the root growth zone is mainly scaled between the two steady states (80 and 400 µmol m−2 s−1) and the light-decrease transition does not cause a marked shift in the proportions of the meristematic zone – characterized by low growth rates at the root apex – and the zone of cell elongation which includes the maximal REGR. Our results obtained in a different species and with a higher precision confirmed the findings by Muller et al. (1998). For steady-state growth conditions at different light intensities, connections between root growth, root biomass and light intensity have been investigated extensively (Barney 1950; Vincent & Gregory 1989; Aguirrezabal et al. 1994; Montgomery & Chazdon 2002). These results generally show an increase in root growth under high light intensities, which is mediated by increased photosynthesis (Webb 1976; Han et al. 1999).

During the first three hours after light transition, the growth reaction exhibited characteristic features, clearly demonstrating the capacity of plants to respond to dynamic fluctuations of ambient light intensity (Fig. 7). In all three experiments (wild-type and transgenic plants in increasing light and wild-type plants in decreasing light), plants showed a transient change in growth intensity of about 15–20% during the first 30 minutes. When light intensity increased, growth decreased and vice versa. This effect has been 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 intensity affect cell wall pH (Mühling et al. 1995) and stomatal conductance (Mott & Buckley 2000) and are, hence, likely to affect cell wall extensibility and turgor. Here, it is shown for the first time that roots react in the same way as has been described for leaves before. This could imply that altered hydraulic properties of the plant (such as stomatal closure upon a sudden decrease of light intensity) or altered apoplastic ion relations (such as pH or membrane potential) are propagated within the entire plant almost instantaneously and hence, might trigger growth reactions in roots.

Within the first hour after changing the light intensity, ETR also changed in parallel with light intensity. In the light-increase experiment, ETR decreased sigmoidally 1–3 h after light change (Fig. 7a). In parallel with this, growth rose to a maximum first, decreased sharply thereafter and eventually reached a steady state. We hypothesize that this sinusoidal growth oscillation is caused by the influx of sucrose reaching the root. Increased photosynthesis in the shoot would promote sucrose export and increased sucrose fluxes into the root would then lead to an overshooting reaction of growth. Again, our results obtained with transgenic plants indicate that sucrose, and not for example a hormonal signal, might be the trigger of this reaction chain (Fig. 7b): while the sucrose-deficient plants showed the transient, ‘hydraulic-induced’ growth depression 30 min after light increase, they did not show the subsequent sinusoidal growth pattern after light change. Instead, growth increased in these plants rather continuously. In future studies, the aforementioned hypotheses could be tested by monitoring the influx of labelled sucrose from the shoot.

Non-destructive high-resolution methods of growth analysis such as the one used in the present study can provide information on the dynamic responses of plant growth to short-term fluctuations of light intensity, which occur naturally on cloudy days, in sunflecks, in gaps of forest stands or in other heterogeneous natural growth settings. The potential of primary root growth to acclimate rapidly to altered environmental conditions such as temperature (Walter et al. 2002) and nutrient availability (Walter et al. 2003) has been demonstrated with high-resolution growth monitoring methods before. Concerning the mechanistic regulation of root growth acclimation to altered or fluctuating light intensity, it will be important to look into the rapid reaction of the root during those first hours after alteration of external conditions. As gene expression and signalling cascades are often regulated within very short time frames, it is important to learn more about the dynamics of plant acclimation in time frames of hours to minutes.

CONCLUSION

While it is well known from steady-state experiments that increased light levels lead to increased root growth via higher export of carbohydrates from the shoot, we demonstrated in this study that characteristic root growth reactions already occur during the first hours after light transitions. We propose that an initial phase of growth acclimation may result from changes in cell wall extensibility or turgor that can be quickly propagated within the whole plant. The subsequent phase – lasting only a few hours – manifests how the root growth reaction is influenced by sucrose import and hence, is critical for understanding the mechanistic processes of growth acclimation to fluctuating light intensity.

ACKNOWLEDGMENTS

We are indebted to Shizue Matsubara for her help during the chlorophyll fluorescence measurements and during the preparation of this manuscript. We thank Andres Chavarria-Krauser, Hanno Scharr, Sabine Briem and Kathrin Cloos for help with the analysis of the digital image sequences and Maja M. Christ and Vicky Temperton for their comments on the manuscript. We are grateful for the provision of transgenic seeds by Uwe Sonnewald. K.A.N. acknowledges the support for her PhD thesis at the Heinrich-Heine-Universität Düsseldorf by the German Science Foundation (DFG; Grant no. Schu 946 2-2).

Ancillary