Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2002 25 Original Article S. Freixes et al.Root architecture and local hexose concentration
Plant, Cell and Environment (2002) 25, 1357–1366
Root elongation and branching is related to local hexose concentration in Arabidopsis thaliana seedlings S. FREIXES,1 M-C. THIBAUD2, F. TARDIEU1 & B. MULLER1 1
Laboratoire d'Ecophysiologie des Plantes sous Stress Environnementaux, UMR 759, INRA/ENSAM, Montpellier, France and 2Laboratoire du Métabolisme carboné, CEA, Cadarache, Saint Paul les Durances, France
ABSTRACT Root architecture can be profoundly affected by the carbon availability in the plant. We hypothesized that this effect could be mediated by the carbon status of root cells involved in elongation and branching processes. Arabidopsis thaliana plants were grown at several photosynthetic photon flux densities (PPFD) and were supplied with various sucrose concentrations in the root medium. Hexose and sucrose concentration was estimated in individual roots in the apical growing region of the primary root and of secondary roots as well as in the zone of primordia development. Local sugar concentration was high in fast-growing and in highly branched roots and robust relationships between root elongation rate or branching and hexose concentration (but not sucrose) were found that were common to all situations experienced. Moreover, these relationships accounted for the plant-to-plant variability within a treatment as well as for the variability among individual secondary roots within a plant. These results support the view that local hexose concentration integrates changes in carbon availability from several sources and acts as a signal to induce at least part of the response of the root architecture to the environment. Key-words: carbon availability; glucose; growing zone; photon irradiance; primordia; sucrose.
INTRODUCTION Consistent indications suggest that root architecture is closely related to the carbon status of roots. Reducing intercepted photosynthetic photon flux densities (PPFD) affects the elongation rate of primary roots and increases the proportion of secondary roots which cease elongation shortly after appearance (Muller, Stosser & Tardieu 1998). In sunflower, Aguirrezabal, Deléens & Tardieu (1994) reported stable relationships between the amount of intercepted PPFD and the elongation rate of the taproot and of secondary roots, with a distribution of responses which is consistent with a source–sink allocation model such as that of Minchin, Thorpe & Farrar (1993). Changing the source-toCorrespondence: Bertrand Muller. Fax: +33 467522116; e-mail:
[email protected] © 2002 Blackwell Publishing Ltd
sink ratio also causes effects that are consistent with this model. Excising all roots but one on a barley plant increases branching density and the length of secondary roots, but has little effect on the length of primary roots (Farrar & Jones 1986). Conversely, defoliating a wheat plant causes a rapid (few hours) decrease in primary and secondary root elongation rate (Bingham, Panico & Stevenson 1996). Changes in elongation rate and/or branching are classically associated with changes in sugar concentration. Fast-growing roots of partly root-pruned plants have high sugar concentration (Farrar & Jones 1986) whereas slow-growing roots of leaf-pruned plants have low sugar concentration (Bingham & Stevenson 1993). In the same way, roots of shaded maize plants, with a reduced elongation rate, have a reduced sugar concentration in the apical zone compared with plants receiving a high incident light (Muller et al. 1998). It has been shown that the impact of sugars on root growth (Bingham & Stevenson 1993) as well on root respiration (Williams & Farrar 1990) is at least in part attributable to a modification of the demand for ATP. This point can be connected with the large body of results that show the ability of sugars to transcriptionnally regulate a large number of enzymes, including those involved in carbon metabolism (Koch 1996; Ho et al. 2001) as well as some enzymes of the cell cycle (Riou-Khamlichi et al. 2000). It is therefore tempting to hypothesize that local sugar concentration could play a crucial role in the determination of root architecture by a signal effect. However, a complication relies on the fact that there is a very large variability in elongation rate of individual secondary roots of a same root system (Pagès 1995). Neighbouring secondary roots can differ by more than 10-fold in elongation rate even when they have similar position in the source–sink system. A possibility might be that these roots also differ in apical sugar concentration, thereby reinforcing the hypotheses of a link between sugar concentration and elongation rate instead of weakening it. The way to test the hypothesis that local sugar concentration plays a crucial role in the determination of root architecture was to carry out a quantitative analysis of the relationship between apical sugar status and elongation rate in roots of plants subjected to a large variability in carbon supply, and to analyse the root-to-root variability in the same way. The same analysis was carried out for the 1357
1358 S. Freixes et al. relationship between branching density and sugar concentration in the subapical zone. Arabidopsis thaliana was an adequate model plant for this analysis, because treatments involving feeding with sucrose can be combined with treatments differing in incident PPFD in plants grown in Petri plates while individual root growth can be followed nondestructively.
MATERIALS AND METHODS Plant material and culture conditions Seeds of Arabidopsis thaliana (ecotype Columbia) were surface sterilized (20 min in 2% w/v Bayrochlore solution in 50% ethanol), rinsed once in ethanol and three times in sterilized water. Seeds were sown in Petri plates (60 seeds per 120 × 120 mm plate) on 40 mL of solidified standard 0·8% agar medium containing micro and macro nutrients (CaSO4 73 mg L−1, KNO3 202 mg L−1, MgCl2 102 mg L−1, KH2PO4 136 mg L−1, NaFeEDTA 18·35 mg L−1, MES 100 mg L−1, NH4Mo 0·04 mg L−1, CuCl2 0·000172 mg L−1, ZnCl2 0·000136 mg L−1, MnCl2 0·00238 mg L−1, H3Bo3 0·0031 mg L−1), and left overnight at 4 °C. Petri plates were then placed vertically in the growth chamber for germination. After day 5, a subset of seedlings was chosen according to the length of their primary root (8–10 mm), and transplanted into new Petri plates (five seedlings/plate) filled with the standard agar medium either containing or not sucrose at two different concentrations (0·5 and 2% w/v). Plates were then left in the growth chamber for 10 d. The temperature was measured inside two Petri plates per treatment with copper–constantan thermocouples inserted in the agar either near the bottom or near the top of plate. PPFD was also measured inside the plate with one miniaturized sensor per treatment, located near the seedlings. PPFD, air temperature and humidity were measured in the air near the plates, and outside them with commercial sensors (LI-190SB; Li-Cor, Lincoln, NE, USA for PPFD; Vaisala HMP 35C, Helsinki, Finland for temperature and humidity). All plants were illuminated 12 h per day at 290 µmol m−2 s−1 (corresponding to 12·5 mol m−2 d−1) before transplanting. After transplanting on day 5, Petri plates were subjected to contrasting PPFDs by placing them either at different distances from the lamp or by shading. The PPFD levels obtained were 460, 290 and 120 µmol m−2 s−1 corresponding to 19·8, 12·5 and 5·2 mol m−2 d−1. In each treatment, a set of three Petri plates (each containing five seedlings) was used for growth and architecture analysis while another set of two plates was used for spatial analysis or for harvests. In each treatment, air temperature of the growth chamber was controlled in such a way that the temperature inside the Petri plates was close to 21 °C night and day (the maximum mean temperature difference observed between two treatments was 1 °C). In addition, vertical temperature gradients in the plates and horizontal temperature gradients between plates were broken by using a fan perpendicular to the gradient. A preliminary experiment
using aluminium foil wrapped around the lower part of the Petri plates showed that root growth was insensitive to the direct exposure of the roots to light.
Growth analysis Numerical images of two to three Petri plates per treatment were taken daily with a scanner at a resolution compatible with the diameter of individual roots (pixel area 56 × 56 µm, root diameter 80–200 µm). They were analysed using an image analysis software (Optimas 6·5; Media Cybernetics, Silver Spring, MD, USA). The first image to be analysed was that corresponding to the last date. On that image, each visible secondary root was given a registration number and the co-ordinates of the insertion into the primary root of each root was recorded. The position of this insertion with respect to the base of the primary root could then be calculated by summing the distances between each individual older root (located closer to the base). The length of the primary root and of each secondary root was recorded on that image and later on images corresponding to earlier dates. The whole process was repeated for 10 plants in each treatment. Some of the treatments were repeated twice. Three main variables were computed from this set of data. The first two, namely the mean (n = 10) elongation rate of the primary root and the elongation rate of each secondary root were computed from successive (1 d interval) measurements of individual root length. Secondary root elongation rate was averaged in each plant on roots longer than 4 mm to avoid roots that were too young and it was then averaged on each treatment (n = 10 plants). The third variable, density of the secondary root was calculated as the mean (n = 10) of the root number in successive 1 cm sectors along the primary root. On day 15, the density of primordia in the region spanning from the first visible primordium to the first visible emerged root was measured on the same 10 plants as those used for growth analysis by using a microscope (Leitz DMRB, Leica, Wetlar, Germany). On day 15 after sowing, the spatial distribution of elongation was analysed along the primary root. Graphite particles were laid on the apical 3 mm of the primary root. An image was taken every hour for 3 h and a set of 10–20 particles that could be found on each image was chosen. The distance between each particle and the root cap interface was measured on each image and the displacement velocity of each particle was assigned to the median position of the particle during the interval. Then, relative elemental growth rate was calculated using the fitting proposed by Morris & Silk (1992).
Hexose and sucrose concentration in apical and subapical regions Apical (2·5 mm long), and subapical regions of individual primary roots were harvested at the beginning of the photoperiod on days 10, 13 or 15 after sowing and at one occasion at the end of the photoperiod on day 15. The apical 2·5 mm of some individual randomly chosen second-
© 2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 1357–1366
Root architecture and local hexose concentration 1359
RESULTS Time courses of primary and secondary root elongation rate Examples of time courses of root elongation rate are presented in Fig. 1 for a subset of treatments. Primary root elongation rate increased from day 6–11 after sowing and stabilized afterwards (Fig. 1a). Differences in sugar concentration in the root medium resulted in large differences in elongation rates. The higher the sucrose concentration, the higher the primary root elongation rate. Secondary root elongation rate changed as the root aged and it was also very variable between individual roots. The
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ary roots was also harvested at the beginning of the photoperiod on day 13 or 15. For one of the treatments (PPFD 12·5 mol m−2 d−1, no sucrose in the medium) the 7 mm behind the root tip were divided into 1 mm long sections. In that case only, samples from three plants were pooled to get enough material for the measurements. The unbranched region of the primary root in which primordia are initiated and grow was also harvested. The proximal end was taken at 5 mm from the apex (following observation of most proximal visible primordia 6–10 mm from the apex) whereas the distal end was taken at the site of emergence of the first secondary root at 20–25 mm from the apex depending on the treatment. The length and diameter of all root samples was measured using a binocular lens (Leica) so sugar content could later be divided by segment volume. Each sample was rapidly rinsed in water then placed in 200 µL 80% ethanol at 80 °C for 15 min Extraction was repeated and the two extracts were pooled. The solution was then dried under vacuum (SC110A; Savant Instruments Inc., Farmingdale, NY, USA). The extracts were re-suspended in 150 to 1000 µL of distilled water and the soluble sugars (glucose, fructose and sucrose) were quantified using a highpressure anion exchange chromatography system coupled with pulsed amperometry detection (HPAE-PAD; BioLC; Dionex, Sunnyvale, CA, USA). The separation was performed at room temperature in an anion exchange column (4·6 × 250 mm CarboPac PA-1 and 3 × 25 mm CarboPac PA guard column; Dionex) with NaOH (150 mM) as mobile phase degased with helium and pressurized using a pump (Bio LC; Dionex) at a flow rate of 1 mL min−1. Detection was performed by pulsed amperometry (PAD 2; Dionex) with a gold working electrode. The following pulse potential and duration were used: E1 = 0·05 V (t1 = 240 ms); E2 = 0·75 V (t2 = 180 ms); E3 = −0·2 V (t3 = 360 ms). The signal was recorded using a 30 nA range. The chromatograms were analysed using a software developed by the manufacturer (AI-450; Dionex) and the components (glucose, fructose and sucrose) were quantified using calibration curves performed in the same condition as described above. Quantification was reliable above a threshold of 0·4 ng per peak and this allowed reliable quantification down to 0·06 µg mm−3 and 0·01 µg mm−3 in apical and subapical samples, respectively.
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Figure 1. Time course of primary and secondary root elongation rate. Plants were grown at PPFD 5·2 mol d−1 m−2. , Plants grown with 2% sucrose in the medium; grey square, plants grown with 0·5% sucrose in the medium; , plants grown without sucrose. (a) Primary root elongation rate. (b) Secondary root elongation rate. For secondary roots, only roots longer than 4 mm were considered. Error bars illustrate the variability of the mean secondary elongation rate between plants not the variability of secondary root elongation rate within a plant. Insert: elongation rate (ER) of all individual secondary roots in a set of 10 plants as a function of their individual length. Only one treatment is shown for clarity (PPFD 5·2 mol d−1 m−2, 0·5% sucrose). Horizontal bars show when elongation rate was averaged later in the study.
inset in Fig. 1b illustrates how secondary root elongation rate increased rapidly with root length and then tended to stabilize for roots longer than 2–4 mm (only one treatment is shown for clarity but this tendency was very much conserved for all treatments). Taking into account secondary roots longer than 4 mm only, mean secondary root elongation rate was calculated every day and examples of time courses are shown in Fig. 1b for the same three treatments as in Fig. 1a. Mean secondary root elongation rate increased slowly with time and roughly stabilized 5–7 d after the first root was visible in the three treatments presented in Fig. 1b.
© 2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 1357–1366
1360 S. Freixes et al. Mean secondary root elongation rate was twice higher in plants grown in the presence of 2% sucrose than in plants grown at low concentration or in the absence of sucrose.
Root elongation rate was affected by both PPFD and sucrose concentration in the root medium Consistent with time courses presented above, the elongation rate of the primary root was compared between treatments 11–13 d after sowing, while the mean elongation rate of secondary roots was compared in roots longer than 4 mm only, 5–7 d after the first secondary root was visible. In the absence of sucrose in the medium, the elongation rate of primary roots doubled (from 5·2 to 10·7 mm d−1) when daily PPFD increased from 5·2 to 19·8 mol m−2 d−1 (Fig. 2a). At low PPFD, a high primary root elongation rate was restored by the presence of 2% sucrose in the medium. At intermediate PPFD (12·5 mol m−2 d−1), an intermediate sucrose concentration (0·5%) was enough to restore a high elongation rate. A maximum value of root elongation rate
could therefore be reached either under high PPFD without sucrose in the medium or at high concentration of sucrose and low PPFD. At both intermediate and high PPFDs, the elongation rate was insensitive to an increase of sucrose concentration from 0·5 to 2%. At high (2%) sucrose concentration in the medium, elongation rate was no longer responsive to PPFD. The response of the secondary root elongation rate to PPFD and sucrose level in the medium was similar to that of the primary root with the exception that there was no increase of the mean secondary root elongation rate between the low and the intermediate PPFD level (5·2 and 12·5 mol d−1, respectively). A major feature concerning secondary roots was the large variability of individual elongation rate among roots observed in a set of plants in the same treatment (Fig. 2c). In plants grown at low PPFD and in the absence of sucrose, elongation rate of secondary roots varied in the range 1–5 mm d−1 whereas in plants grown at low PPFD and with 2% sucrose, it varied in the range 1– 10 mm d−1. This large distribution of elongation rates was very similar at higher PPFD (not shown).
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rate as a function of PPFD and sucrose concentration in the root medium. (a) Primary root elongation rate (n = 10 ± SD); and (b) mean secondary root elongation rate averaged as in Fig. 1b (n = 10 plants ± SD). Symbols with the same letter are not statistically different (P < 0·05, Newman–Keuls test). , ▲, , plants grown in a 2% sucrose medium; grey square, grey triangle, plants grown in a 0·5% sucrose medium; and , , , plants grown without sucrose. (c) Distribution of elongation rate for all secondary roots longer than 4 mm in a set of 10 plants. Distributions shown are from two treatments at low PPFD (5·2 mol d−1 m−2), with 2% sucrose (filled bars) or without (open bars) sucrose in the root medium.
© 2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 1357–1366
Root architecture and local hexose concentration 1361
Spatial analysis of root elongation, root density and sugar concentration
the primary root, with a tendency to be higher in the first most basal centimetre (Fig. 3b). Both hexose and sucrose concentration were higher in the first 2 mm from the root tip than in the region immediately behind (Fig. 3c). In more distal regions, both concentrations tended to increase slightly with distance from the root tip (Fig. 3c). Hexose and sucrose concentration in the region 6–7 mm from the apex were close to the concentrations found when the whole 5–20 mm region from the root tip was harvested (most right-hand bars on Fig. 3c) suggesting the sugar gradients probably tend to flatten in the region further away from the tip. These data were used to quantify the possible error in estimating the sugar concentration in the growing zone by taking a sample, 2·5 mm in length, irrespective of the length of the growing zone (Fig. 3c inset). In the example shown, the sugar concentration in the first 2 mm (actual length of the growing zone, see Fig. 3a) was 0·36 and 0·2 µg mm−3 for hexose and sucrose, respectively, whereas it was 0·32 and
Examples of spatial analysis of root elongation and branching are presented in Fig. 3a and b. In the examples shown, the elongating zone, identified by kinematic analysis, encompassed the apical 2·0 mm in plant grown at intermediate PPFD without sucrose (Fig. 3a). When 2% sucrose was present in the medium, the elongating zone was increased to about 2·5 mm, maximum strain rate was increased from 0·3 to 00·44 h−1 and the peak shifted 0·2 mm distally. This treatment yielded the fastest roots observed in the present study (11 mm d−1). In all treatments analysed, the elongation zone of the primary root was 1·8– 2·5 mm long (not shown). Primordia were first visible under the microscope (2 cell-layer stage) about 6–10 mm from the apex whatever the treatment. The first emerged roots were observed 20–25 mm from the apex, depending on the treatment. Secondary root density was stable along
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Figure 3. Spatial distribution of (a) relative elongation rate (RER) in the apical region of the primary root; (b) secondary root or primordia density along the primary root; and (c) hexose and sucrose concentration in the apical region of the primary root. Plants were 15 d oldwhen analysed. All data shown concern intermediate PPFD (12·5 mol m−2 d−1) without () or with (▲) 2% sucrose in the medium. In (c) hexose (empty bars) and sucrose (hatched bars) concentration is shown in 1 mm sections of primary roots of plants grown without sucrose. The most right-hand bar show measurements performed in the whole subapical region (5–20 mm from the tip). Data are mean [n = 5 for (a), 10 for (b) and 3 for (c)] ± SD. Inset in (c) shows an estimate of the concentration that would have been measured in samples of various length calculated using data from panel (c) assuming constant concentration along each 1 mm section. (—) hexoses; (- - -) sucrose. Horizontal bar shows the extrema of length of the growing zone in primary roots (1·8–2·5 mm). © 2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 1357–1366
1362 S. Freixes et al. aborted primordia in older (branched) zones of the root (not shown).
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The apical 2·5 mm as well as the subapical region (5–20 mm from the primary root tip) were harvested in a subset of experiments that comprised three PPFD levels with or without 2% sucrose in the root medium and the local hexose and sucrose concentration were measured in individual samples. The apical zone harvested encompassed the growing zone of primary roots (Fig. 3) and very likely encompassed that of secondary roots, which had a lower elongation rate than primary roots. The subapical zone encompassed the region of primordia development. When all samples analysed were considered, glucose, fructose and sucrose concentration was in the range 0·1–1·0,
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0·18 µg mm−3 in a section 2·5 mm long. Therefore, the error introduced (10–12% underestimation) was considered low enough to set the length of the harvested zone to 2·5 mm irrespective of the actual length of the growing zone.
1.0 Fructose concentration (µg mm-3)
sucrose concentration in root medium. , ▲, , plants grown in a 2% sucrose medium; grey square, grey triangle, plants grown in a 0·5% sucrose medium; and , , , plants grown without sucrose. Points are mean (± SD) of 10 plants. Symbols with the same letter are not statistically different (P < 0·05, Newman–Keuls test). Inset: relationship between secondary root and primordia density. The line shows the 1 : 1 relationship.
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Consistent with the spatial analysis presented above, the density of secondary root was compared between treatments in the region distal to 10 mm from the root base. Secondary root density was affected by both PPFD and addition of sucrose in the medium (Fig. 4). Density ranged from 2·2 root cm−1 (low PPFD, no sucrose) to 4·2 root cm−1 (high PPFD and 2% sucrose). It increased with PPFD, from 2·2 root cm−1 at low PPFD to 3 root cm−1 at high PPFD in the absence of sucrose. It increased with sucrose concentration in the medium, from 3 to 4·2 root cm−1 at low PPFD without and with 2% sucrose in the medium, respectively. In contrast to elongation rate, secondary root density was still responsive to PPFD in the presence of both 0·5 and 2% sucrose. In the same way it was still responsive to the presence of 2% sucrose at high PPFD. In each treatment, the density of primordia in the subapical region was very close to the density of secondary roots measured, suggesting a low proportion of primordia abortion in all treatments (Fig. 4 Inset). This was confirmed by the lack of visible
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Glucose concentration (µg mm-3) Figure 5. Relationships between glucose and fructose concentration (a) and between glucose and sucrose concentration (b) in apical zones of primary and secondary roots and in subapical zones of primary roots. , primary roots; secondary roots; grey circle, subapical zones. Each data point corresponds to one single root sample.
© 2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 1357–1366
Root architecture and local hexose concentration 1363
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Figure 6. Hexose and sucrose concentration in the apical (2·5 mm) region of primary and secondary roots and in the subapical region (5 to 20–25 mm from the apex) of the primary root. Filled bars: plants grown in a 2% sucrose medium; open bars: plants grown without sucrose; Open and filled bars; samples were harvested at the beginning of the photoperiod; Hatched bars: samples were harvested at the end of the photoperiod. Data are means (± SD) of 2–8 samples.
0·1–0·6 and 0–0·4 µg mm−3 in apical zones of primary and secondary roots and in subapical zones of primary roots, respectively (Fig. 5a & b). Moreover, there was a strong relationship between glucose and fructose concentration that was common for all three types of sample (Fig. 5a; [Fru] = 0·73 * [Glc]; r2 = 0·82). Therefore, results are later presented in terms of hexose concentration, which reflects either glucose concentration, fructose concentration, or both. By contrast there was no significant relationship either between glucose and sucrose concentration (Fig. 5b), or between fructose and sucrose concentration (not shown). Despite a large root-to-root variability, primary roots of plants grown at high PPFD or in the presence of sucrose had higher hexose concentration than plants grown without sucrose at low PPFD (Fig. 6a). For instance, the mean hexose concentration was three times higher in roots of plants grown at high PPFD in the presence of sucrose than in roots of plants grown at low PPFD in the absence of sucrose. In secondary roots (Fig. 6b), essentially the same pattern could be observed although with narrower differences. In both primary and secondary roots, there was no consistent effect of PPFD on sucrose concentration whereas the roots of plants grown in the presence of sucrose in the medium had two- to three-fold higher sucrose concentration than roots of plants grown without sucrose. Sugar concentration
was also measured in the subapical region of primary roots, in the zone spanning from 5 mm from the apex to the first emerged secondary root (20–25 mm from the apex). Unfortunately, the treatment with 2% sucrose could not be included in this analysis because of technical problems during measurements. Subapical regions of the primary root of plants grown at high PPFD had higher hexose and sucrose concentration than in plants grown at low PPFD with a twoand three-fold difference between both situations for hexose and sucrose, respectively. For one of the treatments (intermediate PPFD, no sucrose) we checked that there was no diurnal fluctuation of the sugar concentration in the apical region of the primary root by taking samples at the end of the photoperiod (hatched bars in Fig. 6a & d). The pattern identified are therefore probably not substantially altered by the time of harvest of root samples.
Root elongation rate and branching density showed dose–response relationships to the hexose concentration in the apical zone Because a large root-to-root variability of elongation rates and branching density occurred within a treatment, we aimed to analyse the relationship between root architecture and local sugar concentration further by taking into account information at the single root level. Such an analysis was made possible by using a very sensitive detection method that allowed measurements of sugars down to the nanogram range (see Methods). As a result, a relationship between hexose concentration in the root apex and the elongation rate of that root was observed in both primary and secondary roots (Fig. 7a & d). Both relationships included plants grown at various PPFDs, with or without sucrose in the root medium. The relationship was linear for secondary roots whereas a curvilinear model was more adapted for primary roots. Indeed, in the latter case, although the r2 were equivalent between both models, there was a trend of the residual with the hexose concentration when using a linear model. The same relationship accounted for the variability in elongation rate and hexose concentration among roots within a treatment whether they belonged to different plant (primary roots, Fig. 7b) or to the same plant (secondary roots, arrows in Fig. 7e). Finally, the relationship between hexose concentration and elongation rate of the primary root was still valid when root samples were taken at the end of the photoperiod instead of the beginning of the photoperiod (Fig. 7c). Either no or a very weak relationship was observed between primary and secondary root elongation rate and sucrose concentration (data not shown). In the same way, an analysis of the link between branching density and local sugar concentration was carried out at the single root level. (Fig. 8a). As in the case of root elongation (although with a lower r2), a positive relationship was found between root branching density and local hexose (but not sucrose) concentration that also applied to different plants from the same treatment (Fig. 8b).
© 2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 1357–1366
1364 S. Freixes et al.
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Figure 7. Relationship between local hexose concentration in the growing zone of the root and elongation rate. Sugar concentration was quantified in primary (a, b and c) and in secondary (d and e) root apical (0–2·5 mm from the root tip) zones. Each data point corresponds to individual roots whose elongation rate was measured over the 24–48 h period before harvest. (a) and (d) show all data points collected. All samples were collected at the beginning of the photoperiod except those indicated by open circles () that were collected at the end of the photoperiod. Open and filled symbols correspond to roots of plants grown without and with 2% sucrose, respectively. , , plants grown at low PPFD (5·2 molm−2 d−1); , , ▲, plants grown at medium PPFD (12·5 mol m−2 d−1); , , plants grown at high PPFD (19·8 mol m−2 d−1). (b) and (e) show data from one treatment only (plants grown at high PPFD without sucrose in the medium). In (e), arrows show data from one single plant. (c) show data from samples collected at the end of the photoperiod only. Line shows curvilinear (a) and linear (d) model that best fit the data. Their respective r2 is indicated in the panel.
DISCUSSION The variability of root elongation rate is related to the local hexose concentration in the growing region of the root The fact that fast-growing roots under high carbon availability have higher sugar concentration than slow-growing roots under low carbon availability has been illustrated by others (Farrar & Jones 1986; Bingham & Stevenson 1993; Bingham, Blackwood & Stevenson 1998). Here, we report the occurrence of a robust relationship between local hexose concentration in the growing zone and root elongation rate. Indeed, both the increase in incident PPFD and in
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Hexose concentration (µg mm-3) Figure 8. Relationship between hexose concentration in the subapical region of the primary root and secondary root density. Hexose concentration was quantified in the region spanning from 5 mm from the apex to the first emerged secondary root (20– 25 mm). (a) Shows all data points collected at three PPFD levels without sucrose in the medium. , plants grown at low PPFD (5·2 mol m−2 d−1), , plants grown at medium PPFD (12·5 mol m−2 d−1); , plants grown at high PPFD (19·8 mol m−2 d−1). All samples were collected at the beginning of the photoperiod. (b) shows data originating from one treatment only (low PPFD).
external sugar concentration caused an increase in hexose and sucrose concentrations in the growing region of the root. These changes in carbon supply also generated a variability in root elongation rate. As a result, a relationship was found between both variables and was common to several experiments (Fig. 7a & d). The variability in hexose concentration in the apical region accounted for differences in primary root elongation rate between plants within a treatment (Fig. 7b), as well as for differences in secondary root elongation rate within a single plant (Fig. 7e). Finally, the relationship was conserved when the primary root apex was harvested either at the beginning or at the end of the photoperiod (Fig. 7a & e).
© 2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 1357–1366
Root architecture and local hexose concentration 1365 Large hexose and sucrose gradients are commonly observed in the apical region of the root. Sucrose concentration is high in the meristematic region whereas hexose concentration is low in the meristem and high in regions with expansion only (Sharp, Hsiao & Silk 1990; Scheible et al. 1997; Muller et al. 1998). In the present study, the spatial analysis (Fig. 3c) concentrated on the difference between the growing zone (about 2 mm) and the region located just further away. In the case examined, the hexose concentration fell by 50–60% after the first 2 mm (Fig. 3c). This gradient generated an error of about 10% on the sugar concentration of the sampled growing zone when the error in sample length was 0·5 mm. This error is lower than the scatter of points in the relationship on Fig. 7a & d. We therefore believe that the sampling procedure did not induce a major bias in the analysis. A striking results is that the relationship of Fig. 7 accounted for the variability among secondary roots within a single plant. A large variability in individual elongation rate among secondary roots is a classic feature in both monocots (Cahn, Zobel & Bouldin 1989) and dicots (Pagès 1995). Our data suggest that the processes which cause this variability are associated with the local hexose concentration. The mean by which local hexose concentration differ among secondary roots in a root system is not known. A possibility is that slow-growing secondary roots would be those which are connected with the phloem sieve tubes with the lowest section area and drive the lowest amount of assimilates (Varney et al. 1991). Due to the rapid turnover of the sugar pool in roots, changes in sugar flux would rapidly induce changes in local concentration (Farrar & Jones 2000).
Do observed relationships tell something about signalling properties of sugars on root elongation? Four arguments can be put forward to hypothesize that local hexoses concentration per se act as a signal that regulates root elongation. (1) Root growth is known to tightly depend on carbon nutrition. Shading plants reduces root elongation (e.g. Muller et al. 1998) whereas feeding roots with sugar increases elongation rate (Bingham et al. 1998). (2) It is unlikely that a phloem translocated molecule different from sugars but known to alter root growth such as potassium (Walker, Black & Miller 1998) or amino acids (e.g. Zhang et al. 1999) was responsible for observed responses. The relationship found in the present study (Fig. 7a & d) accounted for responses to both high incident light and feeding sucrose to the roots although the flow of phloem sap was probably increased in the former and decreased in the latter case, respectively (e.g. Bingham et al. 1998). Concentrations in elements other than sugars were probably affected in a different way in the two treatments. (3) The observed relationship was robust enough to account for various sources of variability including within and between plants variability (Fig. 7b & e). Moreover, it was still valid when root samples were harvested at the end of the photo-
period, consistent with the lack of diurnal variations of sugar content in primary root tip (Fig. 7c). (4) In roots of shaded maize plants, the decrease in local sugar concentration precedes the decrease in root elongation rate suggesting the slowdown of root elongation was the consequence, not the cause of carbon shortage (Muller et al. 1998). In a different context, a positive relationship between root growth rate and root carbohydrate status was found by Scheible et al. (1997) in wild-type and nitrate reductasedeficient tobacco plants grown on a large range of nitrogen treatments. The authors argued that the inhibition of root growth in plants grown on high N concentration could be at least partly mediated by a reduction in carbon allocation to the roots. By contrast, there are some obvious cases in which sugar accumulates in association with a slow root growth rate. This is the case when root growth is impeded by low temperature (Pollock & Eagles 1988) or by compacted soils (Atwell 1990). In these cases, increase in concentrations are due to an inbalance between sugar flux and the ability of the root to utilize the sugar to grow.
Branching density can be related to carbon availability via the local hexose concentration in the subapical region of the root The increase in incident PPFD caused an increase in hexose concentration in the subapical region of the primary root where primordia development takes place (Dubrovsky et al. 2001). These changes in hexose concentration were associated with a variability in branching density, with a relationship between both variables which was common to three different experiments (Fig. 8a). Differences in branching density between plants within a treatment (Fig. 8b) were accounted for by the variability in hexose concentration in their apical region with a similar relationship as that found when all treatments were considered. Branching density is believed to be a less plastic architectural feature of the root than the root elongation rate since it changes little in response to several environmental conditions such as low light (Aguirrezabal & Tardieu 1996), high N (Zhang et al. 1999), or low P (Mollier & Pellerin 1999). Nevertheless, when changes in carbon availability are drastic, for instance when roots are supplied with sucrose (Street & McGregor 1952) or when all the roots but one are pruned (Bingham, Blackwood & Stevenson 1997), root and primordia density can be significantly altered. In wheat, primordia density was increased when a portion of the root system was fed with glucose (Bingham et al. 1998). This increase occurred while the carbon flux to that root portion was decreased; a result which led the author to propose that root branching is controlled by the local sugar concentration (and not the flux). This conclusion is very close to that drawn by Farrar & Jones (2000) that the internal sugar pool is ‘a time-integrated sensor of the balance between production and consumption of sugars’. Our study would suggest that these sugar pools are located in the regions where elongation rate and branching intensity are determined.
© 2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 1357–1366
1366 S. Freixes et al.
CONCLUSION Our results demonstrate the existence of robust relationships between local hexose concentration and root elongation and branching. The robustness of these relationships under the range of conditions experienced suggest they are not fortuitous but rather support the view that local sugar concentration (1) integrates changes in carbon availability from several sources of variation; and (2) acts as a signal to induce the architectural response of the root through yet unknown mechanisms.
ACKNOWLEDGMENTS This work was partly funded by INRA ‘Ecogène’ action. Christine Granier is acknowledged for critical reading of an earlier draft of the manuscript.
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