Journal of Experimental Botany, Vol. 51, No. 345, pp. 755-768,
April 2000
© 2000 Oxford University Press
Mapping meristem respiration of Prunus persica (L.) Batsch seedlings: potential respiration of the meristems, O2 diffusional constraints and combined effects on root growth
1 INRA, 42 rue Georges Morel, BP 57, 49071, Beaucouzé, France
2 INRA, Unité de Science du Sol, Domaine St-Paul, Site Agroparc, 84914 Avignon Cedex 9, France
3 Unité d'Ecophysiologie et Horticulture, Domaine Saint-Paul, Site Agroparc, 84914 Avignon Cedex 9, France
Received 25 October 1999; Accepted 4 November 1999
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Abstract |
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Root system architecture partially results from meristem activities, which themselves depend on endogenous and environmental factors, such as O2 depletion. In this study, meristem respiration and growth was measured in the root systems of three Prunus persica (L.) Batsch seedlings. The spatial distribution of meristem respiration within the root system was described, and the relationship between the respiration rates and meristem radii was analysed, using a model of radial O2 diffusion and consumption within the root. Histological observations were also used to help interpret the results. Respiration rates were linearly correlated to the root growth rates (
2=0.9). Respiration reached values greater than 3.5x10-13 mol O2s-1 for active meristems. The taproot meristem consumed more O2 than the rest of the entire root system meristems. Similarly, the first order lateral meristems used more O2 than the second order ones. A near hyperbolic relationship between respiration rates and meristem radii was observed. This can be explained by a model of radial O2 diffusion and consumption within the root. Therefore, only one maximum potential respiration rate and one O2 diffusion coefficient was estimated for all the meristems. Key words: O2-microelectrode, meristem respiration, spatial distribution, root system architecture, Prunus persica (L.) Batsch.
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Introduction |
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Hierarchical organization of root system architecture (as described by Atger and Edelin, 1994
), results from meristematic activity, cell elongation, differentiation processes along axes, and the initiation and development of laterals of various orders. The primary meristem plays a major role in plant development. It is often considered that the quiescent centre is involved in generating different numbers of cell ranks and numbers of vascular strands (Torrey, 1957; Feldman and, Torrey, 1975;Torrey and Feldman, 1977; Rost and Jones, 1988). It determines partly the vascularization of the primary structure, which determines nutrient transfer capacities for the axis (Eshel and Waisel, 1996). Therefore, understanding root system formation requires a more precise knowledge of meristem activity.
A number of factors affect meristematic activity, such as competition for carbon assimilates (Gersani and Sachs, 1992; Bingham and Stevenson, 1993), and hormonal relations (Torrey and Feldman, 1977; Torrey, 1986; Wightman and Thimann, 1980; Wightman et al., 1980.). The more strongly the main axis grows, the more it apparently inhibits its laterals (Atzmon et al., 1994a, b). Lateral roots have smaller meristems than a main root axis, while increasing orders of lateral roots have progressively smaller meristems (Cahn et al., 1989; Varney et al., 1991; Varney and McCully, 1991). Changes in the apical diameter, which reflects the size of the meristem, have been linked to the root growth rate (Wilcox, 1962, 1968; Hackett, 1969; Cahn et al., 1989; Pagès, 1995; Thaler and Pagès, 1996a). It was shown that the meristem diameter can vary according to the carbohydrate supply (Pagès, 1995; Thaler and Pagès, 1996a). The size of the meristem may also vary during its period of activity, leading to axes of varying behaviour (Pagès, 1995; Thaler and Pagès, 1996a, b). However, it was reported that the apical diameter of lateral roots of oak was not closely correlated with their growth rate but was still indicative of their potential growth rate (Pagès, 1995).
The effect on root growth of a decrease in meristem activity caused by hypoxia has received little attention. However, hypoxia can be common at ambient [O2], especially for large-diameter meristems (Drew, 1997). Root apical zones, having high local respiration rates (Rroot) and few intercellular air spaces to conduct O2, may experience hypoxia in their centre at temperatures in the range of 298–308 K (Armstrong and Beckett, 1985). In accordance with diffusion-model predictions, several direct [O2] measurements have indicated hypoxia within metabolically active root tissues, such as maize primary meristems and elongating segments (Armstrong, 1994; Armstrong et al., 1994; Ober and Sharp, 1994; Stepniewski et al., 1998). The measurement of other metabolic indicators, such as alanine, ethanol, lactic acid, as well as elevated activities of alcohol dehydrogenase and pyruvate decarboxylase confirmed the occurrence of anaerobiosis for roots exposed to 21% O2 (Crawford, 1982; Saglio and Pradet, 1980; Saglio et al., 1984; Gibbs et al., 1995; Crawford and Braendle, 1996). Despite the role of meristems in morphogenesis, few studies deal with its respiration and oxygenation, because respiration in the meristem alone is difficult to measure. Attempts have been made with sets of equivalent root tips, 3, 5 or 10 mm long, generally introduced into a stirred nutrient solution bathing an O2-Clark macro-electrode (Saglio et al., 1983; Williams and Farrar, 1992; Brouquisse et al., 1992; James, 1994). This procedure did not distinguish between the activities of the meristem and the elongation zone (James, 1994). An analysis of the limiting effects of O2 on meristem activity has never been conducted on meristems of different sizes and growth potentials representative of the variability found in the root system. An approach based on the meristem size-dependent respiration could be proposed if O2 diffusion coefficient and specific respiration rate in O2 non-limiting conditions did not vary between meristems. In this case, meristem bulk respiration would only depend on size and the [O2] at the root surface. Similarly, meristem activities in the same acropetal sequence or on the whole-root-system architecture have not yet been compared. This may be of interest for studying the effect of relative meristem activity on apical dominance processes.
Because respiration in meristematic tissue is primarily related to biosynthesis (Amthor, 1989), meristem respiration was studied to test if it was a good indicator of root growth. Using this meristem activity indicator, an attempt was made to map meristem respiration throughout the seedling root system of Prunus persica, in order to check whether meristem activity depends on morphological and anatomical criteria, as supported by the competition theories for C allocation (Bingham et al., 1996; Thaler and Pagès, 1998). Meristem respiration as a function of meristem diameter and [O2] at the root surface was analysed in order to determine how much O2 diffusion within the root tissues can explain differences in respiration levels.
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Materials and methods |
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Plants and cultivation media
Seeds of Prunus persica L. Batsch GF305 (Nursery Lafond, Valréas, France) were surface-sterilized in a solution of HClO (0.5% for 20 min) and washed with continuously oxygenated deionized water for 2 h. The seeds were then stratified for 3 months in the dark at 277 K in sealed moistened Petri dishes. After this time, the radicle had reached 5–10 mm in length. The germinated seeds were rapidly disinfected and washed again and placed on agar gel.
Plants were grown in agar in order to prevent convective movements of O2, which allowed a diffusion model to be applied to the measurements of O2 concentration around the root. Although the agar was likely to affect root growth, it was considered that the advantages of agar outweighed this disadvantage. Agar gel at a concentration of 6 g l-1 was dissolved in boiling N/2 Hoagland nutrient solution (Hoagland and Arnon, 1950) previously mixed with 2 bactericides (nystatin, 1.0 mg l-1; tetracycline hydrochoride, 1.25 mg l-1, Sigma-Aldrich, Saint Quentin, France) and Rovral fungicide (1.25 g l-1; Rhône-Poulenc, Lyon, France) in order to minimize microbial proliferation. Acidity was controlled by adding 2.0 mM of MES buffer [2-(N-morpholino)ethanesulphonic acid] (Sigma-Aldrich, Saint Quentin, France), following the recommendation which confirmed that this chemical does not disturb root growth (Ewing and Bobson, 1991). Acidification of the rhizosphere was monitored with 0.06% bromocresol purple. In the absence of MES buffer, the colour of agar surrounding the growing roots became bright yellow within 3 d (indicating a pH of about 4.0–4.5), instead of taking more than 2–3 weeks with MES, depending on the roots.
Agar approximately 3–5 mm thick was solidified by cooling the nutrient solution (at 308 K) in plastic boxes (20x20x1.5 cm). Germinated seeds were fixed in place with mastic (Terostat®) over the gel in which the radicle was settled. A cellophane film was put on the whole preparation to prevent microbial contamination and drying. A hole in this film was made to allow the epicotyl to grow. To prevent the roots from coiling up, the boxes were slightly inclined (angle from vertical: 10%). The plants were raised in the laboratory at 292–293 K. Lighting was low (about 150 W m-2), and large quantities of nutrient solution were poured at daily intervals over the gel surface. Root growth was recorded by tracing on an acetate film. The pH became strongly acid about 10–15 d (pH 
4) after cultivation began.
At the meristem level, Rroot (mol O2 m-3 tissue s-1) was measured on three 15-d-old plants, about 12–15 cm tall with a stem holding 8–12 fully expanded leaves. Plant no. 1 was used to check the reliability of Rroot estimates. Nearly all the meristems on plant no. 2 were analysed for respiration rates. On plant no. 3, measurements were taken 2–3 times per day over 4 d, Rroot being measured both on the taproot meristem and on four early lateral root meristems.
Local estimate of meristem respiration
In order to map Rroot radial O2 profiles were performed around each meristem, using O2-microelectrodes. Rroot was calculated (according to Højberg and Sørensen, 1993; Bidel., 1999):
 | (1) |
[O2]/r)r=rroot the [O2] gradient at the root surface (mol m-4). DO2-gel was measured by the method of Sierra et al. (Sierra et al., 1995): the value was similar to that of O2 diffusion in water. The gradient ([O2]/r)r=rroot was estimated by fitting a model of O2 radial diffusion (Højberg and Sørensen, 1993) within the gel to [O2] experimental data taken in the 0–500 µm region around the root surface, using a non-linear fitting procedure (Bard, 1974). The chosen model accounts for decreasing microbial respiration within the gel with increasing distance from the root and estimates local root respiration rates of Prunus better than other models described in the litterature (Højberg and Sørensen, 1993; Bidel, 1999). In a steady state (i.e. [O2]/t=0 where t is time), the model asserts:
 | (2) |
is a constant (mol O2 m-2 gel s-1) associated with the hyperbolic decrease in microbial respiration /r.
O2-microelectrodes (proposed first by Revsbech and Ward, 1983; Revsbech, 1989), were used to take [O2] profiles around the root. Similar sensors have already been used to describe [O2] distribution within roots (Armstrong et al., 1993, 1994; Ober and Sharp, 1996; Stepniewski et al., 1998). Oxygen is chemically reduced at the cathode surface. In the conditions used in this study, the resulting electrical current was usually between 1 and 200 pA and proportional to [O2] at the tip of the microelectrode. Overall, response time was about 1 s, offset signal (i.e. at 0% O2) was lower than 15 pA, sensitivity was greater than 5 pA per % of [O2] change and tip diameter was about 50 µm. Electrical current was measured using a picoammeter (Keithley 487, Cleveland, Ohio, USA). After calibration of the microelectrode at 0, 20 and 100% [O2], the root system embedded in the agar medium was placed into position (Fig. 1A). The microelectrode was then inserted perpendicular to the root surface at the observed boundary between the cap meristem and the quiescent centre (Fig. 1B). Displacements were made with a motor-driven micromanipulator (Märzhäuser, Steindorf-Wetzlar, Germany), which positioned the electrode tip with an accuracy of 10 µm. The electrode tip and the root were examined with a microscope throughout the experiment. Each measurement was taken within a Faraday cage in a laboratory at 292–293 K. After each [O2] profile, root diameter was measured using a calibrated eyepiece with x25 or x50 magnification. For [O2] profiles, 15–20 points per profile were regularly spaced with 10 points located within the nearest 500 µm zone surrounding the root. The last point of the profile was taken in contact with the root surface.
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Preliminary tests evaluated the bias that may result from the radial O2 diffusion assumption. For these preliminary experiments, [O2] was recorded at every 50 µm during penetration of the microelectrode until the root surface was reached. For plant no. 1, it was verified that the estimates of microbial constant
and root respiration Rroot did not depend on the portion of the oxygen distribution profile around the root used for fitting the model (the fitting area thickness, Fig. 3). For plant no. 2, Rroot was estimated on most meristems of the root system using the same microelectrode over a period of 2 d. Preliminary experiments showed that, in these experimental conditions, the Prunus plants presented no diurnal variations in Rroot and did not have gas flux from shoot to roots (Bidel et al., 1999). It was thus possible to compare Rroot data based on [O2] profiles recorded at different times. Spatial progressions in the meristem were recorded with a calibrated ocular micrometer. Root growth of plants 2 and 3 was estimated from the deplacement of the apex between two dates using capillaries planted in the agar to locate the apex precisely at a particular date.
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Mathematical analysis of O2 depletion within the root
Under steady-state conditions, radial O2 diffusion within the root can be described by the following equation (Armstrong, 1979):
 | (3) |
, 1987). As differentiation has not already occurred at the apex (i.e. in the meristem and the subsequent elongating zone), root tissues were assumed to behave as a radially homogeneous porous medium. It was characterized by an average oxygen diffusivity that takes into account both transfer in the liquid phase and transfer in the gaseous phase within intercellular spaces filled with gas. As no aerenchyma was observed in the cortex, connectivity of air spaces for longitudinal transfer were supposed not to be of significance. It was assumed that DO2-root does not vary with root thickness and MRroot remains constant as long as O2 is locally available (Van Noordwijk and de Willigen 1984; De Willigen and van Noordwijk, 1989).
In order to compare Rroot between meristems of different sizes, a normalization procedure was used, which enabled us to compare Rroot when O2 concentrations at the root surface (i.e. [O2]s, mol m-3) are different. Equation (3) can thus be transformed:
 | (4) |
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 | (5) |
There is no anaerobiosis within the root (i.e. ||r0||=0) as long as the normalized root radius ||rroot|| is lower than a critical normalized radius ||rc||. When it becomes higher, however, anaerobiosis may appear:
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):
 | (7) |
If the MRroot/DO2-root ratio is identical in different locations, checking for a unique relationship between the local root respiration measured at various points in a root system and the normalized local root radii ||rroot|| may be a means of investigating whether Rroot variability results from limited O2 diffusion or not. This is illustrated in Fig. 2, where the relationship between Rroot and ||rroot|| radius for contrasted MRroot and DO2-root values was plotted, corresponding to young (e.g. meristem and expanding zones) and mature segments zones (e.g. primary and woody zones) having (i) high respiration and low O2 diffusion coefficient and (ii) low respiration and high O2 diffusion coefficient, respectively. Rroot at the meristem level was analysed as a function of the nomalized radius ||r|| for plants 2 and 3. In this analysis, the results of Bidel (Bidel, 1999) on Rroot in root segments along the taproot of another 45-d-old Prunus (plant 4) were included.
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Meristem dimensions and volumetric growth rate
Apical diameter was measured at the boundary between cap and quiescent centre. The length hmer and the apical diameter dmer were measured with a graduated eyepiece micrometer (magnification x50). The meristem volume Vmer was estimated according to Barlow and Rathfelder, assuming its shape to be one half of a spheroid (Barlow and Rathfelder, 1984):
 | (8) |
V) was assumed to be equal to the difference between the meristem volumes at these two times. However, the volume due to root elongation l (m) was also added. This last term was estimated assuming conical root segments of radii r1 and r2 at their two ends:
 | (9) |
Meristem histological treatments
The apical 10 mm long root segments were excised, washed in a phosphate buffer and immediately fixed in glutaraldehyde. They were then dehydrated and embedded in metachrylate resin Technovit 500 (Heraeus Kulzer GmbH, Philipp-Reis-S trasse 8/13 D-61273 Wehrheim /Ts) as described previously (Bidel et al., 1999). Microtome sections (0.5–3.0 µm) were stained using the periodic acid–Schiff's (PAS) reaction followed by toluidine blue-O-(TOB) (Varney and McCully, 1991). They were mounted on slides covered with Histolaque (Labo-Moderne, 75015, Paris). Photomicrography used Kodak 160 T film and an Olympus Vanox microscope. The mean length of the meristematic zone was estimated as the distance between the cap junction and the nearest cell mitosis in the central cortex (Barlow et al., 1991; Barlow, 1992).
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Results |
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Reliability of meristem respiration estimates
Even for meristems with low Rroot, the decrease in [O2] between the agar surface and the root surface corresponded to an equivalent decrease in the O2 fraction in air greater than 4%: [O2] at the meristem surface corresponded to O2 fractions in air between 17.6% and 3.8%. The lower values corresponded generally to high microbial respiration around the root (Table 1
). The electrode signal drift was never greater than 2%. Simulating numerical experiments by adding noise to a perfect signal showed that a random noise of 3% on the microelectrode signal yields an estimate of Rroot to the nearest 10% (data not shown).
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At first sight, the estimated Rroot and the constant
measuring microbial respiration did not depend on the fitting-area thickness, i.e. the maximum cylinder volume of gel used for model fitting (Fig. 3
). Microbial activities in the agar gel markedly varied between meristems, with ranging between 2.1x10-11 and 2.3x10-7 mol O2 m-2 s-1.
Map of meristem respiration in the 15-d-old plant
Meristems of plant no. 2 grown in agar differed both in size and respiration rate (Table 1; Fig. 4). The volume of the taproot meristem was about 0.680 mm3, while meristems of first-order laterals ranged between 0.012 and 0.143 mm3. The volumes of the second-order lateral meristems and the meristems from the distal short-time growing laterals occupied between 0.0002 and 0.013 mm3. The taproot meristem (M1) was 3644 times larger than the smallest second-order lateral meristem (M23), but it only consumed O2 340 times more rapidly. Rroot was thus approximately ten times lower for larger meristems. Similarly, the early first-order lateral meristem (M17) was 33 times larger than the second-order lateral meristem (M13), but it only consumed O2 11 times faster. Second-order lateral meristems were larger and consumed more O2 (M10, M7) than some first-order laterals ones. The largest second-order lateral meristem (M15) exhibited an Rroot similar to first-order lateral meristems of comparable size. Some first- and second-order lateral meristems had very low Rroot (M24, M25, M26). Unfortunately, [O2] profile measurements were not taken on the eight first order lateral meristems. Nevertheless, morphometric measurements made it possible to estimate that their respiration rate would hardly reach 15x10-14 mol O2 s-1. This estimate was calculated by multiplying the measured meristem volumes by the highest Rroot estimate for first-order lateral meristems. Consequently, at 15 d, the taproot meristem respired more O2 than the sum of all the other meristems in the whole root system.
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Correlation between respiration rate and volumic root growth
Meristem progression in agar is the result of both the process of cell elongation in the expanding zone and cell division in the meristem. The growth rate was roughly correlated with the meristem Rroot (Fig. 5
). These data made it possible to estimate a maintenance respiration of 5x10-14 mol O2 s-1 (i.e. the limit of Rroot when the meristem progression tended to zero). This value is less than 1/8 of the maximum respiration rate at the meristem level. According to the linear regression calculated between the volumetric elongation rate and Rroot shown in Fig. 5, O2 efficiency was 8.62x10-4 mol O2 g-1 dry matter. This allowed calculation of the construction cost, 1/Yg (ratio of the total weight of substrate consumed to the weight of tissue produced). In agreement with the estimate of 1.02 for the whole tomato root system, (Gary et al., 1998) 1/Yg was calculated here to be 1.045 g equivalent glucose g-1 dry matter.
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Relationship between the respiration rates and the meristem radii: normalization procedure
Since Rroot appeared related to the size of the meristem, respiration estimates made on plants 2 and 3 were plotted as a function of the meristem radius at the measurement level (Fig. 6A). Results in Rroot measured on segments and the taproot meristem of a 45-d-old Prunus persica, (Bidel, 1999), were also included in this figure and referred to as plant 4. The general trend is a hyperbolic curve. To account for [O2] at the root surface, which may differ between meristems, the normalization procedure, previously described in the Materials and methods section, was applied to this data set (Fig. 6B). By using the normalization procedure, (i) a distinction was made between segments and meristems, because of the lower [O2] at the segment root surface (Bidel, 1999) and, possibly, (ii) the residual variability of Rroot was reduced close to the overall trend (i.e. the hypothetical hyperbolic curve).
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Considering the limited number of measurements and the contrast between apex and segments for both the porosity and Rroot, the normalized model, equations (5), (6) and (7), was fitted separately to meristems and segments data thus yielding two experimental relationships between Rroot and normalized root radius (Fig. 6B
). The model fitted the data well and no positive or negative biases were observed. DO2-root and MRroot were estimated to be equal to 9.2x10-12 m2 s-1 and 5.7x10-2 mol O2 m-3 of tissue s-1 for meristems, and 2.36x 10-7 m2 s-1, and 2.53x10-3 mol O2 m-3 of tissue s-1 for segments. DO2-root was expressed for O2 diffusing in a liquid phase: [O2]s at the root surface equalled actual O2 concentration within the agar gel, and [O2] within the root was expressed assuming that O2 diffuses in water. If O2 diffusion happened in the air-filled intercellular spaces, the corresponding O2 diffusion coefficient could be estimated by multiplying the previous values by the coefficient of O2 solubility in water (approximately 0.034 at 293 K), because the previous [O2] gradient would be divided by this value to obtain [O2] gradient in a gas phase. Therefore, the diffusion coefficients would then be 3.1x10-13 and 8.0x10-9 m2 s-1 for meristems and segments, respectively.
Histology
Variability in the length of the meristematic area is greater than in the diameter (Fig. 7A–E). Some meristems lost a part of their cap embedded in agar gel during sampling (Fig. 7C, D). Smaller meristems with higher Rroot were less stained by meristematic indicators, such as PAS-TBO or methyl green (Gahan, 1984). These presented cap cells without amyloplasts. Longitudinal histological sections revealed that it finally reconstituted a new root cap that was not always typical in size and staining.
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Respiration estimates
The model of radial O2 diffusion within the agar gel apparently fitted well with measurements, even when [O2] measurements were taken close to the root tip, as long as the fitting area thickness did not exceed 500 µm (and sometimes more as in Fig. 3
). The resulting estimates of Rroot and constant that characterized the microbial respiration did not depend on the fitting area thickness in this domain (Fig. 3). Furthermore, the lack of variation in the parameter with changes in the fitting area thickness would indicate that microbial respiration in the agar gel can be modelled by a function proportional to the (1/r) ratio, as already suggested (Højberg and Sørensen, 1993; Bidel, 1999). Additional assumptions were made to estimate total O2 consumption in each meristem. Their shape was assumed to be spheroid, and all their tissues were assumed to have a specific respiration rate equal to the estimated Rroot value. There was generally a good agreement between the observed length of the translucent zone of the meristem and the length of the meristematic area on histological sections. However, there was not good agreement for the two largest meristems (the taproot meristems of plants no. 2 and no. 3) out of the 27 meristems for which Rroot was measured. It was only possible to obtain 18 histological sections and these were not always in the axial plane. For these, volume estimations based on microscopic observation were made. Therefore, the O2 consumption of the taproot meristem was probably underestimated by about 10–15%. Despite these uncertainties, the results of this study are indicative of the relative activity of the set of meristems in an entire root system.
Rroot of the taproot meristems of plants 1, 2 and 3 were equal to 5.3, 13.8, and 7–8.9 nmol O2 m-1 s-1. These values were in fairly good agreement with measurements taken on excised root tips placed into stirred nutrient solutions (Table 2). For these root tips, however, meristem respiration was not discernible from that of the elongation zone with the ‘excised root’ experimental procedure. No results were found in the literature about respiration rate of second and third order meristems.
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The conditions of low light and very high humidity probably caused Rroot meristem values to be lower than those expected for roots growing in a rhizotron under standard climatic conditions (Pagès, 1995
). Moreover, for the same aerial climate, plants grown in the agar layer had achieved only one-half to one-third of the growth rate of plants cultivated in a rhizotron or on nylon mesh (L Pagès, unpublished results). As an indirect confirmation of this effect of agar gel, meristems that emerged from the agar layer thereafter progressed quicker than meristems remaining in the gel. These two facts suggested the occurrence of limiting factors for root growth in the experimental design, such as O2 depletion. Other adverse factors such as a progressive dehydration, acidification and mineral depletion of the agar around older root segments may also have reduced the meristem growth.
Respiration rate as an indicator of meristem activity
Since meristem respiration rate appears significantly correlated to root elongation rate (r2=0.90), it may be considered as a growth indicator. However, not enough points were plotted to ensure that the relationship was strictly linear. The residual variability may partially result from measurement errors on meristem diameter and length, and biased shape assumption. The taproot meristem of the 15-d-old plant consumed O2 at a rate more than ten times higher than that of its nearest laterals (Table 1), suggesting that it could deprive these laterals of carbohydrates and reduce their activity. The total active tissue in the root tip, including both the elongation zone and the meristem, would probably have increased the magnitude of competition. The length of the elongation zone of roots with small meristems was very small compared with roots with larger meristems, which is visible when Fig. 7A–E are compared.
The observation that the taproot meristem has the greatest O2 consumption suggests that it is also the strongest carbohydrate sink. This is consistent with previous reports in the literature, although such data are rarely quantitative (Webb, 1977; Daie, 1985; Schulz, 1994). For many species, the more developed the main axis is, the more it seems to inhibit growth of its laterals (Atzmon et al., 1994). Furthermore, the removal of the root tips is known to stimulate the growth of the youngest and nearest laterals greatly (Wightman et al., 1980; Atzmon et al., 1994a, b).
Oxygen diffusion within root tissue as a factor limiting meristem respiration
The theoretical model based on O2 diffusion and consumption within root tissue is quantitatively in fairly good agreement with the experimental results, especially in view of the experimental difficulties and the theoretical approximations made for obtaining these values. Two independent reasons suggested that [O2] diffusion and consumption within the root was really the main factor involved in Rroot variations between the meristems. (1) No discrepancy was observed between experimental and simulated data (Fig. 6B). A discrepancy would be expected if carbohydrate supply was selectively limiting respiration rate in aerobic tissues as a function of meristem size. (2) The estimated O2 diffusion coefficients within the root tissue were consistent with the real structure seen in histological sections.
The O2 diffusion coefficient in pure water is about 2.09x10-9 m2 s-1 at 293 K and decreases in saline water. This value is 4 orders of magnitude lower than the coefficient in air. Given that the calculated O2 diffusion coefficient at the meristem level is about 9.2x 10-12 m2 s-1, O2 must have moved there as a solute in a liquid phase. The root:water O2 diffusion coefficient ratio was about 0.004, indicating that the cell walls and plasma membrane greatly reduced O2 diffusion. It was assumed by other authors that the diffusion coefficient of the cell walls was about 6.3x10-10 m2 s-1 (Armstrong et al., 1994); these results suggest an even greater resistance of the cell walls and membranes. In the meristem, it seems unrealistic to consider O2 diffusion in the intercellular pore space, because it is difficult to observe such a space in histological sections. Conversely, it may be considered that only O2 moves in the air-filled intercellular pore space at the segment level, because O2 diffusion in pure water is lower than the estimated O2 diffusion coefficient within the root. Considering the O2 diffusion coefficient in air (2.01x10-5 m2 s-1 from Jaynes and Rogowski, 1983), the root segment:air O2 diffusion coefficient ratio would be equal to 0.0004. Using Buckingham's model (Buckingham, 1904) to relate this ratio to the air-filled porosity of porous media, it was estimated that the air-filled intercellular pore space actually involved in O2 diffusion corresponded to approximately 2% of the root volume. This is only a rough estimate, because the porous media:air O2 diffusion coefficient ratio greatly depends on the actual geometry of the intercellular space (Cousin et al., 1999). The estimate of 2% is in the range of published values from various methods, such as picnometer measurements: values of 2, 3 and 4–9% for Festuca, barley, and tomato, respectively, have been reported (Glinski and Stepniewski, 1990); and values between 1% and 4% for bean have been reported (De Willigen and Van Noordwijk, 1989).
Assuming a constant DO2-root for all the meristems of the root system, the present diffusion model suggests the existence of a unique maximum respiration rate value MRroot. This corresponds to a potential for Rroot in the meristem tissues. An unique potential Rroot has still to be confirmed by different measurements on other plants. In theory, this would depend on plant species and environmental conditions, such as temperature. For the 15-d-old plants, a unique potential Rroot would also indicate that the activities in all meristematic zones in aerobic conditions were not limited by the carbohydrate supply. This could have resulted from the presence of cotyledons and the slow development of the plants in agar gel.
These reasons cannot explain, however, the differences in growth rate between meristems, resulting mainly from their size. It may be assumed that differences in growth rates are due to competition for carbohydrates: the supply of carbohydrate to a meristem would influence its own diameter. On a short time-scale, both the anatomy and the O2 tissue diffusion properties of the meristem define a Rroot that could be satisfied if carbohydrate availability made it possible. When the meristem is supplied by lower carbohydrate availability caused by competition with other organs, a lower respiration rate and a lower mitotic activity could be expected. Consequently, a reduced number of cell ranks could be generated as reported for meristems in vitro (Feldman and Torrey, 1975; Barlow and Adams, 1989). That may explain daily variation in size of the apical diameter in parallel to photosynthesis activity in vivo for Hevea brasiliensis Müell. Arg. seedlings plant in the phytotron at constant temperature (Thaler and Pagès, 1996a). This would lead to a kinetic adjustment of the meristem dimensions that minimizes carbohydrate limitation for aerobic metabolism in a larger time-scale.
The taproot meristem usually increases its size during the establishment stage on nylon mesh culture with similar [O2] and temperature. The proposed cylindrical model of respiration simulates an increase of total carbohydrate consumption by aerobic metabolism with the increase of the meristem diameter, although the meristem anoxic fraction increases. The size of excised meristems cultivated in vitro was positively correlated with hexose supply, which also governed the complexity of the vascular pattern of the formed axes (Feldman and Torrey, 1975; Scadeng and MacLeod, 1976).
When temperature increases, thinner lateral roots grow faster than the main roots (MacDuff et al., 1986; Gregory, 1986). Growth rate in soybean (Glycine max [L.] Merr.) taproots decreases as temperature increases, whereas lateral growth rate remains steady (McMichael and Quisenberry, 1993). Based on the results presented here, it is suggested that these different responses of root types to temperature may result from the effect of O2 depletion on meristematic activity. A 10 K increase in temperature slightly affects O2 diffusion (Renault and Stengel, 1994), whereas root respiration increases exponentially with a Q10 between 1.5 and 3.0 (Lambers et al., 1996; Sprugel et al., 1994; Amthor, 1989). Consequently, hypoxia within the root may appear and/or increase as temperature increases (Glinski and Stepniewski, 1985). The thinner lateral roots would be expected to be less affected by hypoxia than the wider taproot as temperature increased, and therefore have a greater temperature optimum for elongation.
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsAppendixReferences |
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As far as is known, this is the first work that describes a map of meristem O2 consumption for a root system architecture. The experimental procedure and the related device appeared to be adequate for investigating root activity with low disturbing effect for seedling, although it may affect meristem respiration because of [O2] limitation at the root surface. The proposed normalization of the data (i.e. the definition of a normalized root radius equal to the actual radius:square root of the surface [O2] ratio) made it possible to discuss the data, regardless of the actual effect of the agar gel on root respiration. Diffusion of O2 within the root tissues appeared to be the main limiting factor for meristem respiration. The proposed model of O2 radial diffusion and consumption within the root tissues was in fairly good quantitative agreement with the experimental data. It enabled an estimatation of a unique O2 diffusion coefficient and a unique maximum specific respiration rate for all the apical meristems of the root systems studied. Due to the uniqueness of these parameters, using this theoretical approach would also make possible the definition of the minimum [O2] at the root surface to avoid O2 limitation, and the estimation of the meristem respiration rates for roots growing in aerated conditions (i.e. 20% for the O2 fraction). The uniqueness of these parameters would also suggest that the activity of the central volume of root tissue in anaerobic condition is not limited by the carbohydrate supply. This might be caused by kinetic adjustment of the meristem dimensions to the local carbohydrate supply.
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List of symbols
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Acknowledgments |
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This work was carried out in the Soil Science Unit at INRA, Avignon (France). We thank NP Revsbech (University of Aarhus, Denmark) and the members of his Laboratory for training one of us in the construction of O2 microelectrodes. We also thank M Dever of the Language Service, INRA, for reviewing the English version of the manuscript, S Parry for helping to construct O2 microelectrodes and V Serra for young peach trees cultivation. The authors wish to thank JL Poessel and V Restier (INRA, Avignon) for their advice and help with logistic assistance in preparing the micro-sections, and M Chevalier (INRA, Angers) for microscopy.
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4 To whom correspondence should be addressed. Fax: +33 4 32 72 22 12. E-mail:Pierre.Renault{at}avignon.inra.fr
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