Journal of Experimental Botany, Vol. 51, No. 350, pp. 1515-1529,
September 2000
© 2000 Oxford University Press
Leaf development in Ricinus communis during drought stress: dynamics of growth processes, of cellular structure and of sink–source transition
Department of Botany, University of Heidelberg, Im Neuenheimer Feld 360, D-69120 Heidelberg, Germany
Received 11 October 1999; Accepted 8 February 2000
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Abstract |
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Dicot leaf growth is characterized by partly transient tip-to-base gradients of growth processes, structure and function. These gradients develop dynamically and interact with dynamically developing stress conditions like drought. In Ricinus communis plants growing under well-watered and drought conditions growth rates peaked during the late night and minimal values occurred in the late afternoon. During this diurnal course the leaf base always showed much higher rates than the leaf tip. The amplitude of this diurnal course decreased when leaves approached maturity and during drought stress without any significant alteration of the diurnal pattern and it increased during the first days after rewatering. Unique relationships between leaf size and cytological structure were observed. This provided the framework for the analysis of changes in assimilation, transpiration and dark respiration, chlorophyll, protein, carbohydrate, and amino acid concentrations, and of activities of sink–source-related enzymes at the leaf tip and base during leaf development in well-watered and drought-stressed plants. Gas exchange was dominated by physiological rather than by anatomical properties (stomatal density). Tip-to-base gradients in carbohydrate concentrations per dry weight and sink–source-related enzymes were absent, whereas significant gradients were found in amino acid concentrations per dry weight. During drought stress, growing leaves developed source function at smaller leaf size, before specific physiological adaptations to drought occurred. The relevance of the developmental status of individual leaves for the drought-stress response and of the structural changes for the biochemical composition changes is discussed.
Key words: Drought, leaf growth, sink–source transition, diurnal growth patterns, carbohydrate, amino acid, chlorophyll, protein, sucrose phosphate synthase, sucrose synthase.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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Growing tissues are characterized by the simultaneous development of structure and function (Kühn et al., 1996
; Schurr, 1998a; Walter and Schurr, 2000). In many cases the relevance of the interaction of structure and function is straightforward as, for example, in gas exchange where leaves have to develop functional stomata in order to actively control water and CO2 exchange with the atmosphere. In sink–source transition plasmodesmatal and phloem structures have to be adapted to the export function (Oparka and Turgeon, 1999). On the other hand analysis of concentrations of substances (per weight, area or many other reference parameters) require sound information on the structural basis in order to be correctly interpreted: the ratios between tissue layers (Heckenberger et al., 1998; Roggatz et al., 1999), between veins and interveinal areas and between vacuoles and cytoplasm change during development. This may have a direct impact on the composition of a sample, because compartments differ in their principal components. This adds to the general problem of analysing an expanding tissue: the region sampled at a certain point in time changes in size during expansion, and therefore deposition over time (net fluxes into or out of a growing region) can only be calculated on the basis of kinematic growth analysis (Silk, 1992; several other papers in this issue). These simple examples illustrate that structural information is crucial in order to interpret functional data in growing tissues correctly as already pointed out by Körner et al. (Körner et al., 1989).
Structure and function do not build up homogeneously in growing leaves, but develop significant spatial gradients that change dynamically (Maksymowych, 1973; Heckenberger et al., 1998; Granier and Tardieau, 1998; Walter and Schurr, 2000): cell division and elongation cease first in the leaf tip (with a time lapse of several days depending on the species). This allows the tip to mature several days before the leaf base, in its functional relations too (Turgeon, 1989). Strong discrepancies of structure and function develop between the leaf base and tip that are usually transient and disappear when maturity is reached.
Stress can alter leaf structure considerably. Leaves from stressed plants usually reach apparently smaller final sizes and their cytological structure can be altered in comparison to controls (Heckenberger et al., 1998; Granier and Tardieau, 1998). The dynamic of the stress interacts with the dynamic of the development of structure and function in growing tissues, resulting in very different responses to stress in leaves of different developmental situations (Roggatz et al., 1999). Therefore, the parallel analysis of structure and function is of special importance in the analysis of stress responses in growing tissues.
The interaction of structure and function in growing leaves during drought is a complex field. This paper therefore can only highlight some aspects. It intends also to encourage interdisciplinary approaches in this complex field between different areas of botany, as data from different scales have to be combined to understand these complex processes. Additionally, co-operation with other disciplines can supply new techniques and methodology leading to new approaches to this central field of plant physiology.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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Growth of Ricinus communis
Ricinus communis plants were grown as described previously (Heckenberger et al., 1998
). In short, seeds of Ricinus communis L. (var. Carmencita) were germinated in vermiculite and transplanted to special planting pots (4470 cm3) filled with a sandy loam in order to use equivalent conditions for the experiments with the root pressure chamber (Schurr, 1998b) to study the impact of water potential on leaf growth. Plants were cultivated for 3 weeks in a growth cabinet at a day/night cycle of 12/12 h (300 µmol photons m-2 s-1), a constant air temperature of 25 °C and a relative humidity of 60/45% during the light and dark periods, respectively. Plants used in the experiments had developed one pair of green cotyledons, one pair of primary leaves and one fully developed main leaf (Heckenberger et al., 1998). Until the start of the drought experiments, the gravimetric soil water content was kept above 0.20 g g-1 by repeated watering 2 h after the onset of light with modified Hoagland solution (in mol m-3: K+ 3.0, NO-3 4, H2PO-4 1, Ca2+ 1, 
2, Mg2+ 2; in mmol m-3: 
0.12, Fe3+ 18, 
1, Mn2+ 0.09, Zn2+ 0.035, Mo7
0.02).
Drought stress and rewatering treatments
The experiment was designed to study the course of changes occurring in successive growing leaves during drought stress. A detailed description of the drought stress conditions has already been given (Heckenberger et al., 1998). In short, one-half of the plants was subjected to drought stress from day 18 after germination onwards, while control plants were kept well watered. Ricinus plants are highly tolerant to drought stress as even after the complete cessation of watering for up to 20 d leaves did not wilt. Samples from the leaf base and the leaf tip of leaves 2, 3 and 4 were taken from water-stressed and control plants at day 24 (no apparent decline in growth rate), day 26 (significant decline in growth rate), day 29 (cessation of areal growth), and day 35 (growth stopped for 6 d) after germination. Control plants were probed additionally on day 32. In the rewatering experiments water was added to field capacity to drought-stressed plants after 3 d of complete cessation of growth (equivalent to day 32 after germination).
Diurnal variation of leaf growth
Diurnal variation of leaf expansion of Ricinus was analysed during the development of leaf 3 in control and water-stressed plants by a continuous length detection device based on an angle meter (Schurr, 1998a). In short, the leaf base was fixed by rods in its position. Twine was attached to the leaf tip and led over a roll to a metal rod connected perpendicular to the rotation axis of the angle meter (Novotechnik, Germany). The weight (15 g) on the opposite side kept the leaf midrib in a straight position. Arrays of nylon twines parallel to the midrib enclosed the leaf from top and bottom and fixed the leaf in a plane of the midrib to minimize non-planar movements. The electrical output of the angle meter was digitized every second. For diurnal patterns of expansion hourly means were calculated.
Diurnal variation of leaf growth distribution
Distribution of growth rates at high temporal and spatial resolution was determined in a separate experiment. Image sequences of leaves of a fast and a slow growing Ricinus plant were taken at day/night cycles of 12/12 h. Plants were soil grown at constant temperatures of 25 °C and constant relative humidities of 80%. The fast growing plant was exposed to 500 µmol photons m-2 s-1; the slow growing plant was exposed to 200 µmol photons m-2 s-1. The observed leaves grew with relative growth rates of 17% and 4% d-1, respectively, during observation.
The analysis of distribution of leaf growth was done by image sequence analysis according to Schmundt et al. (Schmundt et al., 1998). In the context of this technique, ‘growth’ means the increase of area. In short, image sequences in the near infrared (>930 nm) were digitized from standard CCD cameras directly. The leaf fixed in the focal plane of the camera system was illuminated day and night by an IR diode array emitting at 950 nm. Motion in the captured sequences was analysed using the structure tensor algorithm (Haussecker and Spies, 1999) in an implementation of the image sequence analysis software HEURISKO (Aeon, Hanau, Germany) previously described in detail (Schmundt et al., 1998). The resulting velocity vector fields were interpolated and local growth rates were calculated from the local divergence in the velocity maps (Schmundt and Schurr, 1999). The growth maps were then used to determine growth within the natural reference system of the leaf, the leaf vein system (Walter and Schurr, 1999), by processing growth rates at defined positions along the midvein of the analysed leaves (Walter and Schurr, 2000).
Analysis of cytological status of Ricinus leaves
The length of the middle lobe of the leaf was used as a measure of leaf size (Heckenberger et al., 1998; Roggatz et al., 1999). Cytological parameters (cell density, cell density in individual cell layers, height of palisade cells, exposed surface area of epidermal cell, stomatal densities) were determined in leaf discs (4 mm diameter) taken from the leaf base and tip of leaves 2, 3 and 4 of well-watered and drought-stressed Ricinus plants. Detailed descriptions of the methods used have been given (Heckenberger et al., 1998). In short, cell density was determined using a Neubauer chamber after complete separation of the cells by enzymatic digestion. The height of palisade cells was determined by microscopic analysis of the isolated palisade cells in the cell suspension. The exposed surface area of epidermal cells and stomatal density was determined from FormVar replicas of the leaf surface and quantified using digital image analysis software (SigmaScan, Jandel Scientific, Erkrath, Germany).
Analysis of gas exchange in growing leaves
Gas exchange (assimilation, transpiration, dark respiration) was analysed by cuvette measurements of Ricinus leaves. For cuvette measurements of the leaf tip and base of growing leaves, a special cuvette was designed which clamps only 2.7 cm2 of the leaf area. This cuvette was attached to the porometer head of a commercial porometer (Walz, Effeltrich, Germany). The air temperature inside the cuvette was maintained at 25 °C and a leaf-to-air vapour pressure difference of 12.7 Pa kPa-1 was provided. CO2-concentrations were at ambient growth conditions. Light measurements were obtained at ambient light conditions in the growth chamber (300 µmol photons m-2 s-1).
Analysis of the composition of growing leaves
For the analysis of the composition of growing leaves it is important to choose a reference unit, which allows suitable interpretation and which is suitable for technical reasons. This is especially relevant in growing tissues, as the cytological structure changes significantly and thereby the relative contribution of different cellular compartments might affect the concentrations measured in the different regions without alteration of the concentration within the individual cellular compartments (cf. Roggatz et al., 1999). Composition data are expressed here on a dry weight basis as calculated from the fresh weight to dry weight ratio determined with equivalent leaf discs.
Leaf samples (3 discs of 1 cm diameter each) were taken from the leaf base and tip of growing leaves of Ricinus plants, respectively. In very small leaves (LML <4 cm) a separation of leaf base and tip was not possible and one sample (3 leaf discs) per leaf was taken. All leaf discs were immediately frozen in liquid nitrogen and stored at -80 °C until extraction. One leaf disc was extracted in ethanol (see below), the other two were used for analysing enzymatic activity (see below).
For ethanol extraction, the frozen leaf disc was transferred into 900 µl of 80 °C ethanol/water (50:50, v/v) for 20 min. The supernatant was stored and the extraction was repeated twice with 200 µl ethanol/water (80 : 20, v/v) until the leaf disc was pale. The supernatants were combined and used for analysis of chlorophyll, glucose, fructose, sucrose, and amino acid concentration. The pale leaf disc was used for starch analysis.
Chlorophyll concentration was analysed photometrically at 652 nm after Arnon (Arnon, 1949). Hexoses and sucrose were analysed after Jones et al. (Jones et al., 1977) by enzymatic analysis. Starch concentration was determined enzymatically as described previously (Matt et al., 1998). Amino acid concentrations and patterns were determined by HPLC as described previously (Gerendas and Schurr, 1999). Proline concentrations were determined separately after Bates et al. (Bates et al., 1973) by toluol-fractionation of ninhydrin derivates.
Enzymatic activities were determined in an extract prepared as follows: Two leaf discs were ground in liquid nitrogen and taken up in 700 µl extraction buffer (50 mM MOPS (pH 7.4), 12 mM MgCl, 5 mM EDTA, 5 mM EGTA, 1 mM benzamidin/ 
-aminocapronic acid, 0.1% Triton X-100, 1 mM DTT, 1 mM PMSF). The extract was centrifuged at 12 000 rpm for 4 min. The supernatant was passed through a G-25 column equilibrated before with 50 mM MOPS (pH 7.4), 12 mM MgCl2 and 1 mM DTT. The eluate was used immediately for the determination of enzymatic activities.
Sucrose phosphate synthase was determined after Reimholz et al. (Reimholz et al., 1994) at non-selective (maximal) and selective conditions. In non-selective conditions the phosphorylated and non-phosphorylated form of SPS was determined. In selective conditions only the activated form of SPS was analysed. Activation state was calculated as the ratio of selective and non-selective activity of SPS in the extracts. Sucrose synthase (SuSy) was determined after Dancer et al. (Dancer et al., 1990) by measuring the formation of UDP-glucose by SuSy in the extracts. Protein concentrations were determined after Bradford (Bradford, 1976) from these extracts.
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Results and discussion |
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Temporal and spatial scales relevant for growth analysis in well-watered and water-stressed leaves
Before individual parameters of growth can be analysed it is necessary to recall the importance of spatial and temporal aspects of growing leaves especially in their interaction with dynamically developing stress conditions such as drought.
Growing leaves are characterized by dynamic gradients of growth processes and functional development. Different temporal scales interact in a complex manner. In well-watered Ricinus leaves, expansion rates varied by a factor of more than 6 during the day with a distinct maximum during the second half of the dark period (Fig. 1A, B). In young leaves, expansion was detected throughout 24 h, periods of zero expansion were observed only in almost mature leaves. Most of the expansion took place during the few hours around the day–night transition. A distinct smaller peak of expansion was consistently detected at the beginning of the night. This peak was still present even in almost mature leaves. A local minimum of expansion of similar amplitude was observed at the beginning of the day. These minor patterns were closely associated with the closure and the opening of stomata (data not shown).
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In many species diurnal variation of leaf expansion has been reported (for an overview see Walter and Schurr, 2000
). It is beyond the scope of this paper to analyse the mechanisms underlying this diurnal course, however, despite the common increase in water potential and sometimes turgor (Boyer, 1968; Bunce, 1977) it seems unlikely that increased water status is responsible for the higher growth activity during the night hours (Schurr, 1998a), especially as maintaining the shoot water potential at maximum by means the root pressure chamber did not alter the diurnal course of leaf growth (Fig. 1C
). Expansion rates are more likely to be controlled by the expansibility of the cell wall, as the spatial distribution of turgor does not fit the pattern of expansion (Tomos and Pritchard, 1994) and the transient increase in the expansion rate after an increase in turgor by the short-term application of pneumatic pressure is due to unphysiological spatial patterns of expansion and is only transient despite higher turgor pressures being maintained (Schmundt et al., 1998). In contrast, the spatial patterns of cell wall properties along growth zones fit well with their potential role in growth regulation (overview in Schurr, 1998a; Wu and Cosgrove, 2000). Little is known about the diurnal patterns of cell wall properties, but more data on this topic have to be sampled in order to elucidate the mechanism behind the spatio-temporal patterns of leaf expansion in the diurnal course.
The transient peaks at the beginning of the dark and the light phases have been linked to turgor changes due to stomatal activity and a transient imbalance of turgor, as the height of these peaks can be affected by the vapour pressure deficit (Christ, 1978; Cutler et al., 1980) and transient alterations of expansion rate can be induced by alterations of light and air humidity (Shakel et al., 1987; McDonald and Davies, 1996; Clifton-Brown and Jones, 1999). However, even when water potential in the leaves was held constantly high by controlled application of pneumatic pressure to the root system, short-term changes in growth rate occurred during the light–dark transition (Fig. 1C).
Drought stress mainly affected the amplitude of the diurnal growth peak with little influence on the overall diurnal pattern and the number of days with significant growth activity (Fig. 1B). Zero growth was first detected temporally at day 4 after the onset of the analysis, but the peak activity of expansion rate was still significant (0.48 mm h-1). Even at severe drought stress, when almost no expansion was detected any more (Fig. 1B, days 5 and 6), the small expansion peak at the beginning of the night was detected. Thus, from the diurnal pattern of expansion rate, leaves from drought-stressed plants mimicked more mature leaves of control plants at smaller leaf size.
Rewatering (at the beginning of day 7, Fig. 1B) immediately increased the growth rate, showing that there was still the potential to grow in these leaves 2 d after the complete cessation of expansion. Significant expansion was regained within 30–40 min after rewatering (data not shown), but expansion rates comparable to well-watered rates were only detected a day later and maintained for one additional day (Fig. 1B). Thereafter leaves reduced their expansion rate again for developmental reasons.
The restoration of expansion rates comparable to well-watered controls of the actual leaf size was fastest in the smallest leaves (data not shown). Final leaf size in rehydrated plants was reduced most in leaves that were small at the onset of drought. However when the length of the middle lobe of a leaf (LML) was above 12 cm at the onset of drought, rehydration within 2 d after complete growth stop allowed full recovery of the final leaf size. This leaf size compares well with the one at which cell division stopped entirely (Heckenberger et al., 1998). This result hints to a limitation of cell division as the cause of a reduced final leaf size in these cases. Cell division has already been identified to be of central importance for the cytological stress response of growing leaves (Granier and Tardieau, 1998), as has been found previously in roots (Sacks et al., 1997) and under nitrogen limitation (Roggatz et al., 1999).
Spatial heterogeneity is another essential feature characteristic for different classes of growing tissues (Schurr, 1998a). Dicot leaves are characterized by their tip-to-base gradients in growth rates which occur transiently during development (Maksymowych, 1973; Taylor et al., 1994) and respond to drought stress (Granier and Tardieau, 1998). Biochemical and molecular control of expansion processes acts in the range of minutes to hours (Hsiao and Xu, this issue) rather than in the range of days. Therefore, the temporal resolution that is relevant to analyse spatio-temporal relationships of the biochemical and molecular mechanisms responsible for, for example, variation of cell wall properties, was not available until recent technical developments based on image sequence analysis (Schmundt et al., 1998; Schmundt and Schurr, 1999; Walter and Schurr, 2000; for some details see Materials and methods). Previous approaches were limited to monocot leaves (Schnyder et al., 1988) with the problem that the growth zone in monocots is enclosed in the leaf sheath and is thus not optically accessible for the analysis of growth distribution prior to sampling. In dicots, the spatial and temporal resolution of the growth analysis was much too low to do such an analysis (Dale, 1988). The new imaging techniques have not yet been applied to drought-stressed plants, but distinct differences in the spatio-temporal patterns of relative growth rate between leaves growing at high and low growth rates due to light conditions have been observed (Fig. 2). Essentially, the diurnal variation reflects the pattern observed by continuous length detection systems (see above).It is clear from these first data in Ricinus that the tip-to-base gradients change significantly. During the diurnal course in fast-growing leaves, areas that grow with rates of 5% h-1 at peak activity, completely cease growth in the middle of the day (Fig. 2A). Even in slow-growing Ricinus leaves distinct patterns of diurnal growth distribution were observed. The diurnal peak activity was still observed in the late night and entirely located at the leaf base (Fig. 2B).
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This technique will be used in the future to elucidate different mechanism of stress response in dicot leaves. In roots, it has already been shown that the decrease of overall growth rate in response to stress is due either to variation in amplitude or to the spatial distribution of expansion rate (roots: Silk, 1992
; Sharp et al., 2000; monocot leaves: Durand et al., 1995). With the strong spatio-temporal patterns present, growth in dicot leaves needs to be studied on a diurnal scale in order to link the underlying biochemical (and molecular) mechanisms acting in that time scale. Additionally, quantitative analysis of deposition rates of substances in growing tissues (kinematic growth analysis, Silk, 1992; Granier and Tardieu, 1998) require techniques which analyse the spatio-temporal patterns of growth, as the presented data show that the assumption of steady growth in dicot leaves throughout the day is wrong. A relevant analysis of growth in stressful conditions needs to take into account that (i) processes directly involved in growth or in its control must be functional at the correct time and space and (ii) that development and stress vary within the same time scale.
The cytological status as a framework for functional analysis
The dynamic nature of leaf growth requires a thorough analysis of potential reference systems, as common reference units like area, weight or chlorophyll change dynamically in growing tissues. A number of possible reference systems have been proposed (Erickson and Michelini, 1957; Poethig and Sussex, 1985; Hejnowicz and Karaczewski, 1993; Granier and Tardieu, 1998; Walter and Schurr, 1999; Tardieu et al., 2000), but a single one matching all requirements may be impossible to find (Erickson and Michelini, 1957).
Cytological status is attractive as a framework for functional analysis as it circumvents problems with variation in cell size and relative share of cells to the tissue samples (see Introduction), which is especially evident for the biochemical analysis of plant material taken from different cytological states due to the stress of other treatments (Schurr, 1998a; Walter and Schurr, 2000). However, a complete analysis of the cellular status during development with spatial and temporal resolution is complex and tedious (Taylor et al., 1994; Granier and Tardieau, 1998; Heckenberger et al., 1998; Roggatz et al., 1998).
Leaf size, as expressed by the length of the middle lobe of the Ricinus leaf (LML), has been proposed as a potential reference system for the analysis of interaction of structure and function due to its links to cellular parameters (Heckenberger et al., 1998; Roggatz et al., 1999). In drought-stressed plants subjected to the indicated conditions, unique relationships were found in leaf 3 between the cytological parameters, as well as their tip-to-base gradients to leaf size in water-stressed and well-watered plants (Heckenberger et al., 1998). Very similar relationships between leaf size and all the cytological parameters analysed were found for successive leaves (Fig. 3). Even the relative share of tissue layers (palisade and spongy parenchyma, upper and lower epidermis) was identical at a given leaf size (data not shown). This makes ‘leaf size’ an especially good reference system for structure–function relationships, as leaves of identical size can be compared for ‘functional analysis’ without the need for a complex analysis of the cytological structure just on the basis of simple leaf size measurements. This may not be the case in all drought stress treatments or under other stress conditions (e.g. nutrient stress: Roggatz et al., 1999), especially when cell division is affected. However, it opens direct approaches to study the interaction of structure and function.
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Sink–source transition in growing leaves
In parallel to cytological changes, sink–source transition alters physiological processes in the developing leaf (Turgeon, 1989) ranging from gas exchange to composition (concentration and relative) and to the enzymatic apparatus of the leaf regions. The following will mainly concentrate on carbon metabolism but, additionally, simultaneously and strongly interlinked, other aspects of primary metabolism (e.g. nitrogen metabolism) and secondary metabolism change fundamentally during sink–source transition.
Gas exchange
Mature stomata are required for a functional gas exchange (Kebede et al., 1994) and thus for sink–source transition. In Heckenberger et al. it has been shown that the production of stomatal complexes and the ‘dispersal’ of these by the expanding epidermal cells (Fig. 3) interact to produce a characteristic spatio-temporal pattern of stomatal density in growing leaves (Heckenberger et al., 1998). Mature stomata were present at a leaf size of approximately 5 cm (LML) and 8 cm (LML) at the leaf tip and base, respectively. This corresponds to a developmental delay of 2–3 d at the base relative to the leaf tip. Stomatal density decreased with increase in leaf size after the maximum due to the increase of the functional ratio between leaf area expansion and formation of stomatal complexes. Stomatal density in the leaf base peaked again approximately 2 d later (LML 11 cm). This pattern of development is consistent within successive leaves (Fig. 3D).
Drought stress (as described inHeckenberger et al., 1998) did not significantly alter the relationship between stomatal density and leaf size in leaf 3 from which gas exchange parameters were analysed (Fig. 3). Therefore, leaves of similar size were equipped with the same structural apparatus at a given leaf size, irrespective if they were well-watered or water-stressed (Heckenberger et al., 1998). The delay in development caused leaves from water-stressed plants at a given size to have higher stomatal densities than leaves from control plants at a given point in time. Variations in stomatal density and distribution in response to environmental stresses need therefore to be interpreted on the basis of the acceleration or deceleration of development (Taylor et al., 1994).
In control plants, transpiration rates per area increased with leaf size and barely differed between the leaf base and the leaf tip and (Fig. 5). In contrast, assimilation rates were significantly higher at the leaf tip than at the leaf base in small leaves (Fig. 4A). Assimilation rate became positive at a LML of 5 cm and 10 cm at the leaf tip and base, respectively. When the leaf at the base just started to gain carbon on its own photosynthetic activity, the assimilation rate of the leaf tip was already half the mature rate (Fig. 4A). Assimilation rate was highly and linearly correlated with cell density, with the leaf base and the leaf tip showing the same relationship (Fig. 4B). Dark respiration per area dropped significantly with increase in leaf size (Fig. 4C), with the leaf base declining at larger leaf sizes than the leaf tip. At maturity no gradients of dark respiration between tip and base were present any more. The gradients were predominantly due to differences in cell density between leaf base and leaf tip (Fig. 4D), as dark respiration per cell at the leaf base and leaf tip were similar throughout the development of the leaf.
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Assimilation and transpiration rate did not follow stomatal density. Therefore physiological parameters dominated gas exchange at the stomatal level. This is in contrast to findings by Kebede et al. (Kebede et al., 1994
). The gas exchange gradients studied here were rather stable, in contrast to short-term fluctuations of stomatal conductance and assimilation rate as reported for mature leaves (Lawson and Weyers, 1999). However, in contrast to their approach, temperature and humidity conditions in these experiments were maintained as constant as possible during the measurements. Mapping transpiration rates by thermography (Kümmerlen et al., 1999) will be a valuable tool to analyse the developmental aspects of gas exchange in growing leaves further. The close relationship between assimilation rate and cell density and the transient maximum of dark respiration per cell in mid-sized leaves underline the importance of cellular development for the photosynthetic capacity in growing leaves.
Drought stress affected the development of assimilation rate later than transpiration rate (Fig. 5). While transpiration rates at the leaf base and tip were significantly lower than controls 8 d after withholding water, with no difference between leaf base and tip (Fig. 5A), assimilation rates of leaves from drought-stressed plants increased in a similar way to the controls due to ongoing maturation for at least two more days (Fig. 5B). At day 11 of withholding water, assimilation rates at the leaf tip declined strongly and reached values similar to the ones of the less mature leaf base. In the leaf base the developmental increase of assimilation rates was only stopped by drought after more than 11 d of withholding water. Even at the strongest drought stress assimilation rate was still positive and no net respiration was observed during the light period. Upon rewatering the assimilation rate again recovered faster than transpiration rate and both reached values similar to controls after 2 d (data not shown). Dark respiration was strongly reduced during drought stress at the leaf base and tip in comparison to control plants (Fig. 6). However, rates of dark respiration in mature leaves were similar in well-watered and water-stressed plants.
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The higher sensitivity of transpiration to drought in comparison to assimilation rate has often been observed in mature leaves (for an overview see Schulze and Hall, 1982
). Chlorophyll fluorescence analysis showed that non-photochemical quenching processes increased in Ricinus during drought stress to dissipate excess energy (U Heckenberger and U Schurr, unpublished results). In stress conditions the physiological control again strongly overrode the structural pattern, as gas exchange declined irrespective of the higher stomatal density in drought-stressed plants (Heckenberger et al., 1998). In contrast to assimilation and transpiration rates, dark respiration was closely associated with the earlier maturation of stressed leaves.
Determination of biochemical composition in growing and stressed tissues
Due to the dynamic situation in growing tissues, it is difficult to find a single reference unit, which allows easy interpretation (for a more detailed analysis see Walter and Schurr, 2000). Under the conditions of these experiments, ratios of dry weight over fresh weight were not statistically different between leaf positions and between water-stressed and well-watered leaves (well-watered: leaf 2, 0.24±0.06; leaf 3, 0.23±0.06; leaf 4, 0.229±0.04; water stressed: leaf 2, 0.27±0.05; leaf 3, 0.28±0.07; leaf 4, 0.3113±0.07). The maximal difference obtained between values on the fresh weight and the dry weight basis was in leaf 4 between well-watered and water-stressed plants resulting in a maximal change by 30%. Nevertheless, concentrations were expressed on a dry weight basis in order to circumvent any problems of interpretation in the drought-stress treatments.
Chlorophyll and protein concentrations
Alterations in chlorophyll and protein concentrations are essential steps in sink–source transition. Chlorophyll concentration per unit dry weight increased almost linearly with the increase in leaf size and stayed constant after the final leaf size was reached (Fig. 7). The difference in chlorophyll concentration between the leaf base and the leaf tip was small, with the leaf tip having a slightly higher concentration than the leaf base in mid-sized leaves. As the leaf tip and base had different cell densities (Fig. 3) the leaf tip had an approximately 1/3 higher amount of chlorophyll per cell than the leaf base throughout most of the development of the leaf. This increase in chlorophyll concentration is typical during the development of dicot leaves (Harn et al., 1993; Merlo and Passera, 1991). Protein concentration per dry weight increased slightly with leaf size (Fig. 7B). No significant differences were present between the leaf base and the leaf tip despite significant differences in cell density (Fig. 3).
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During drought stress, chlorophyll concentrations per dry weight increased in leaves 2, 3 and 4 of water-stressed plants at smaller leaf sizes than in well-watered plants (Fig. 8A
). However, concentrations at the smaller final size of the leaves under stress were not significantly different from those of mature leaves of well-watered plants. Protein concentration per dry weight of leaves from drought-stressed plants were not significantly different from controls at a given leaf size.
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The rewatering experiments (Fig. 9
) supported these findings for water-stressed plants, with chlorophyll concentrations in drought-stressed plants being similar to the concentrations at maturity of well-watered controls, but at a smaller leaf size. Leaves of rewatered plants increased their leaf size, but maintained their chlorophyll concentration at mature levels. This indicates the induction of chlorophyll biosynthesis to counteract the dilution due to regained leaf expansion. Protein concentrations were not analysed in the rewatering experiment.
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Chlorophyll and protein content per dry weight at the final leaf size were similar in leaves of well-watered and drought-stressed plants. As leaf size and cellular parameters were closely associated, the protein and chlorophyll content per cell increased during drought. The functional development of the leaf continued during stress and similar ‘mature’ concentrations per dry weight of major constituents equivalent to the mature control leaves were reached. A fixed protein and chlorophyll concentration per dry weight was characteristic for a functional mature leaf. Even during the rewatering experiments, when leaf size increased considerably, the ‘mature’ concentration of chlorophyll was maintained. These findings support the hypothesis that the content of these constituents per dry weight is closely controlled in developing leaves with an apparent upper limit.
In other experiments chlorophyll concentrations have been reported to decline during drought stress, which might be related to membrane disintegration due to oxidative stress in less drought-tolerant species (Moran et al., 1994). Additionally, the nitrogen form used by the plant can cause considerable differences in the response of chlorophyll concentration during drought stress (Mihailovic et al., 1992).
Carbohydrate concentrations
Glucose (Fig. 10A) and fructose (Fig. 10B) content per dry weight increased in parallel with leaf size by a factor of 7, but no tip-to-base gradients were observed. In mature leaves, hexose concentrations tended to decline with time after the final size had been reached. Sucrose concentrations were significantly higher than glucose and fructose concentrations in small leaves, but were similar to hexose concentrations in mature leaves (Fig. 10C). Sucrose concentration increased by only a factor of 2 during leaf development. No tip-to-base gradients were present in sucrose concentration, even though assimilation rates were significantly higher at the tip (Fig. 4). Sucrose concentrations remained constant after the cessation of growth. Starch was hardly quantifiable in small leaves and increased to a mean value of 300 µmol hexose equivalents g-1 dry weight in mature leaves. After the cessation of growth, variation between leaves increased significantly in all measured carbohydrates, even though samples were taken at fixed times of day. The increase of sucrose-to-hexose ratios is common during leaf development and due to the interaction of sucrose-phosphate synthase, sucrose synthase and invertase activities during sink–source transition (Weber et al., 1996).
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During drought stress, glucose and fructose concentrations increased at a smaller leaf size up to values equivalent to or slightly above those of mature leaves of well-watered controls in leaves 2, 3 and 4 (Fig. 11A
, B). When these mature values were reached, glucose and fructose concentrations decreased slightly in leaf 2, but declined 3-fold in leaf 3. Leaf 4 did not reach mature concentrations during the experiment. Sucrose concentrations also reached mature concentrations (Fig. 11C), but concentrations increased thereafter, equivalent to the decline of hexoses. Starch concentrations increased slightly in leaves of water-stressed plants (Fig. 11D).
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The rewatering experiment supported the findings of the drought-stress experiment as, during drought, hexose and sucrose concentrations increased to mature levels (Fig. 9B
) in leaves 3 and 4, while they stayed in the same range as in controls in the almost mature leaf 2. The decline in hexose concentrations in leaf 3 was also observed, but was coincident with rewatering the plants. The rewatering response of soluble carbohydrates was clearly different to the drought response: upon rewatering both hexose and sucrose concentrations declined to the concentrations of leaves equivalent to leaves of that size in controls. Again, these findings support the hypothesis that the leaf finishes sink–source transition at a smaller leaf size during drought stress. It is clear from the results presented here that the impact of drought on the speed of development has to be taken into account in growing leaves for understanding the changes of biochemical composition. Rewatering allowed the leaf to expand and to regain the ability to expand its source function.
Amino acids
Amino acid concentrations per dry weight varied strongly in growing leaves (range 50–350 µmol g-1 DW). In small leaves, tips had significantly higher amino acid concentrations than the leaf base, but decreased consistently with leaf size (Fig. 12A). During this decline, the amount of amino acids per cell remained constant, indicating that the decline was mainly due to cell expansion. At a leaf size (LML) of 16 cm the tip-to-base gradient had vanished and amino acid concentration fell to values of 30 µmol g-1 DW, which were maintained constant in the mature tissues (data not shown). During this decline the content of amino acids per cell decreased. This was mainly due to falling concentrations in glutamine and arginine, which were the two major amino acids in growing leaves (Fig. 13B, C).
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standard error, n=3, except for tip of leaf 4 and age class 24: n=2). In leaf 4 a differentiation between leaf tip and base was not possible for age 24 and 26 d after germination. The closed lines indicate the relation obtained for leaf 3 in well-watered plants during their development, ‘ww’ indicates well-watered and ‘ws’ water-stressed plants.The developmental change in amino acid composition is a common phenomenon associated with the transition of the growing leaf from a sink to a source for amino acids. In particular, the decline in glutamine as the major transport (and storage) form is characteristic. Amino acids showed the most prominent tip-to-base gradients in all measured leaf constituents.
During drought stress, amino acid concentrations decreased at smaller leaf sizes until final leaf area was reached (Fig. 13A). Strong tip-to-base gradients were observed. This decline was similar to the maturation in control plants, as (i) the concentration at final leaf size was 50–100 µmol g-1 DW, (ii) it was mainly due to falling concentrations of glutamine and arginine (Fig. 13B, D) and (iii) transient tip-to-base gradients were present in leaf 3 (Fig. 13B, D). However, when the final leaf size was reached, amino acid concentrations increased again (Fig. 13A). This increase was mainly (40% of the entire increase) due to arginine (Fig. 13B), which increased its concentration more than 4-fold compared to the concentration at the day when growth ceased first. Proline concentration was low and almost constant in leaves of well-watered plants during development (Fig. 13C solid line). During drought stress it increased strongly by factors of 10, 3 and 4 in leaves 4, 3 and 2, respectively. By far the most dramatic increase took place in the youngest leaves (leaf 4), where proline concentrations reached values close to the concentrations of glutamine.
The decline of amino acid again suggests that sink–source transition occurs at smaller leaf size (see above). This hypothesis is supported by the transient yet strong tip-to-base gradients in amino acid concentrations, which also occur at a smaller leaf size during drought stress. The increase after reaching the final leaf size is clearly a different physiological stage, similar to the changes in carbohydrates after cessation of growth. The strong increase in proline concentration has often been reported in drought-stressed plants (Heuer, 1994) and has been interpreted as osmotic adjustment with proline being a compatible solute or as increased storage of unused nitrogen (Heuer, 1994; Lawler and Leech, 1985).
Sink–source transition: enzymatic level
The biochemical (and molecular) basis of sink–source transition is the alteration of the enzymatic apparatus of the leaf. The activities of sucrose-phosphate synthase and sucrose synthase activity in leaves are reported. Analysis of invertases (acid and alkaline) in the same material (data not shown) supported the general conclusion (see below). Again it has to be emphasized that leaves of a given size in this experiment had a similar cytological structure in well-watered and drought-stressed plants and thus a change in activity of an enzyme in response to drought cannot be related simply to a change in the number of cells.
Sucrose phosphate synthase (SPS) is predominantly involved in source activity, providing sucrose for export (Weber et al., 1994; Harn et al., 1993). Maximal SPS activity, correlated to SPS protein (Walker and Huber, 1989), increased during the second half (in relation to leaf size) of the leaf development in well-watered control plants (Fig. 14A). Tip-to-base gradients in SPS activity were not present despite the considerable difference in assimilation rate. Due to the different cell density in the leaf base and the leaf tip, SPS activity per cell was higher in the leaf tip than in the leaf base throughout most of the development of the leaf (data not shown). SPS-activity declined to levels of activity similar to those in small leaves soon after final leaf size was reached. SPS is activated by phosphorylation and its activation state can be measured in vitro (Huber et al., 1989; Reimholz et al., 1994). SPS activation increased with leaf size by a factor of 3 (Fig. 14B) and, in contrast to the maximal activity, was maintained constant in mature leaves. Therefore, the increase in SPS function in mature source tissues in Ricinus was predominantly due to an increased activation state. However, no tip-to-base gradients were present in SPS-activation that would relate to sink–source gradients in the growing tissues. This is the same on a per cell basis, as the activation is calculated from the ratio of the maximal and the selective activity of SPS.
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Drought stress caused an increase of SPS activity at smaller leaf size in leaf 3, while leaf 4 did not reach mature SPS activities even at the end of the experiments (Fig. 15A
). The effect of drought on the SPS activation state was more pronounced and again caused an increase of activation at smaller leaf size (Fig. 15B). Hence SPS activity reached mature levels at a smaller leaf size indicating a sink–source transition at smaller leaf size. It is clear from these data that SPS is more related to sink–source transition than to growth and that the deduction of a central role for SPS in growth from the close correlation with elongation (Seneweera et al., 1995) is an oversimplification. This analysis of SPS during development and drought did not take into account that different isoforms of SPS have been reported to be expressed during the different developmental stages of the leaf and during stress response (Reimholz et al., 1997).
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Sucrose synthase (SuSy) is central for the activation of sucrose imported from the phloem into sink leaf (Weber et al., 1994) and is generally found markedly up-regulated in developing leaves (Sturm et al., 1995
). Sucrose synthase activity declined with leaf size (Fig. 14C). The decline with leaf size was predominantly due to the expansion of the cells and thus ‘dilution’ of the activity, as the SuSy activity per cell decreased only by a factor of 2 with development (data not shown). Again no tip-to-base gradients in enzyme activity were found on a dry weight basis, making the SuSy activity per cell in the tip higher than at the base. Drought stress did not significantly alter the relationship between SuSy activity and leaf size, again emphasizing the role of cell expansion (dilution effect) in the decline in SuSy activity. Sucrose synthase has been reported to increase during drought (Keller and Ludlow, 1993), but in those experiments sucrose concentrations decreased and hexose concentrations increased during the stress.
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Conclusion |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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In this paper, data from the whole leaf to the biochemical level have been summarized to illustrate the complexity and interdependency of structure and function in growing leaves and its interaction with drought stress. The results indicate that (i) on a macroscopic scale, growth rate and its distribution vary strongly in a diurnal manner, (ii) cytological structure alters strongly during development and this provides a framework for the analysis of function in growing leaves in combination with leaf size as a reference system, (iii) gas exchange properties of a developing leaf are dominated by the physiological properties with the stomatal development providing the basic structures, (iv) tip-to-base gradients in carbohydrate concentrations and activities of enzymes linked to sink–source transition were not strongly developed, in contrast to gradients of amino acids, (v) drought stress (and rewatering) caused a maturation of leaves at a smaller leaf size with a complete syndrome in growth dynamics, carbohydrate and amino acid concentrations as well as sink–source related enzymes. Finally, these data sets show the complexity of the interaction of structural and functional development in a dynamically changing environment.
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Notes |
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1 To whom correspondence should be addressed. Fax: +49 6 221 545 859. E-mail: uschurr{at}botanik1.bot.uni\|[hyphen]\|heidelberg.de
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Abbreviations |
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LML, length of the middle lobe of the Ricinus leaf; MOPS, 3-(N-morpholino)propane sulphonic acid; EDTA, ethylene diamine tetraacetic acid; EGTA, ethyleneglycol-bis-(2-aminoethyl-)-tetraacetic acid; DTT, dithiothreitol; PMSF, phenylmethylsulphonylfluoride..
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References |
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Arnon DI.1949. Copper enzymes in isolated chloroplasts. Polyphenol-oxidase in Beta vulgaris. Plant Physiology 24, 1–15.
Bates LS, Waldren RP, Teare ID.1973. Rapid determination of proline. Plant and Soil 39, 205–207.[Web of Science]
Boyer JS.1968. Relationship of water potential and growth of leaves. Plant Physiology 43, 1056–1062.
Bunce JA.1977. Leaf elongation in relation to leaf water potential in soybean. Journal of Experimental Botany 28, 156–161.
Bradford MM.1976. A rapid and sensitive method for quantification of microgram quantities of protein using the principle of protein–dye binding. Analytical Biochemistry 72, 248–254.[Web of Science][Medline]
Christ RA.1978. The elongation of wheat leaves. I. Elongation rates during day and night. Journal of Experimental Botany 29, 603–610.
Clifton-Brown JC, Jones MB.1999. Alteration of transpiration rate, by changing air vapour pressure deficit, influences leaf extension rate transiently in Miscanthus. Journal of Experimental Botany 50, 1393–1401.
Cutler JM, Steponkus PI, Wach MJ, Shahan K.1980. Dynamic aspects and enhancements of leaf elongation in rice. Plant Physiology 80, 147–152.
Dale JE.1988. The control of leaf expansion. Annual Reviews of Plant Physiology 37, 377–405.
Dancer JE, Hatzfeld W-D, Stitt MN.1990. Cytosolic cycles regulate the turnover of sucrose in heterotrophic cell-suspension cultures of Chenopodium rubrum L. Planta 182, 223–231.
Durand J-L, Onillion B, Schnyder H, Rademacher I.1995. Drought effects on cellular and spatial parameters of leaf growth in tall fescue. Journal of Experimental Botany 46, 1147–1155.
Erickson RO, Michelini FJ.1957. The Plastochron Index. American Journal of Botany 44, 297–305.[Web of Science]
Gerendas J, Schurr U.1999. Physicochemical aspects of ion relations and pH regulation in plants: a quantitative approach. Journal of Experimental Botany 50, 1101–1114.
Granier C, Tardieau F.1998. Spatial and temporal analysis of expansion and cell cycle in sunflower leaves. Plant Physiology 116, 991–1001.
Harn C, Khayat E, Daie J.1993. Expression dynamics of genes encoding key carbon metabolism enzymes during sink to source transition in developing leaves. Plant and Cell Physiology 34, 1045–1053.
Haussecker H, Spies H.1999. Motion. In: Jähne B, Haussecker H, Geissler P, eds. Handbook of computer vision and applications. Vol. 2. Signal processing and pattern recognition. New York: Academic Press, 310–396.
Heckenberger U, Roggatz U, Schurr U.1998. Effect of drought stress on the cytological status in Ricinus communis. Journal of Experimental Botany 49, 181–189.
Hejnowicz Z, Karaczewski J.1993. Modelling of meristematic growth in root apices in a natural co-ordinate system. American Journal of Botany 80, 309–315.[Web of Science]
Heuer B.1994. Osmoregulatory role of proline in water- and salt-stressed plants. In: Pessarakli M, ed. Handbook of plant and crop stress. New York: Marcel Dekker, 363–481.
Huber JLA, Huber SC, Nielsen TH.1989. Protein phosporylation as a mechanism for regulation of spinach leaf sucrose phosphate synthase. Archives of Biochemistry and Biophysics 270, 681–690.[Web of Science][Medline]
Jones MGK, Outlaw WH, Lowry OH.1977. Enzymatic assay of 10-7 to 10-14 moles of sucrose in plant tissues. Plant Physiology 60, 379–383.
Kebede H, Martin B, Nienhuis J, King G.1994. Leaf anatomy of two Lycopersicon species with contrasting gas exchange properties. Crop Science 34, 108–113.
Keller F, Ludlow MM.1993. Carbohydrate metabolism in drought-stressed leaves of pigeonpea (Cajanus cajan). Journal of Experimental Botany 44, 1351–1359.
Körner Ch, Pelaez Menendez-Riedl S, Hohn PCL.1989. Why are Bonsai plants small? A consideration of leaf size. Australian Journal of Plant Physiology 16, 443–448.
Kühn C, Quick WP, Schulz A, Riesmeier JW, Sonnewald U, Frommer WB.1990. Companion cell-specific inhibition of the potato sucrose transporter SUT1. Plant, Cell and Environment 19, 1115–1123.
Kümmerlen B, Dauwe S, Schmundt D, Schurr U.1999. Thermography to measure water relations of plant leaves. In: Jähne B, Haussecker H, Geissler P, eds. Handbook of computer vision and applications, Vol. 3. Systems and applications. New York: Academic Press, 763–781.
Lawlor DW, Leach JE.1985. Leaf growth and water deficits: biochemistry in relation to biophysics. In: Baker NR, Davies WJ, Ong CK, eds. Control of leaf growth. Cambridge: Cambridge University Press, 267–294.
Lawson T, Weyers J.1999. Spatial and temporal variation in gas exchange over the lower surface of Phaseolus vulgaris L. primary leaves. Journal of Experimental Botany 50, 1381–1391.
Maksymowych R.1973. Analysis of leaf development. Cambridge: Cambridge University Press.
Matt P, Schurr U, Klein D, Krapp A, Stitt MN.1998. Growth of tobacco in short-day conditions leads to high starch, low sugars, altered diurnal changes in Nia transcript and low nitrate reductase activity, and inhibition of amino acid synthesis. Planta 207, 27–41.[Web of Science][Medline]
McDonald AJS, Davies WJ.1996. Keeping in touch: responses of whole plant to deficits in water and nutrient supply. Advances in Botanical Research 22, 229–300.
Merlo L, Passera C.1991. Changes in carbohydrate and enzyme levels during development of leaves of Prunus persica a sorbitol synthesizing species. Physiologia Plantarum 83, 621–626.
Mihailovic N, Jelic G, Filipovic R, Djurdjevic M, Dzeletovic Z.1992. Effect of nitrogen form on maize response to drought stress. Plant and Soil 144, 191–197.
Moran J, Becana M, Iturbe-Oraetxe I, Frechilla S, Klucas RV, Aparicio-Tejo P.1994. Drought induces oxidative stress in pea plants. Planta 194, 346–352.[Web of Science]
Oparka KJ, Turgeon R.1999. Sieve elements and companion cells: traffic control centers of the phloem. The Plant Cell 11, 739–750.
Poethig RS, Sussex IM.1985. The cellular parameters of leaf development in tobacco. Planta 165, 170–184.[Web of Science]
Reimholz R, Geigenberger P, Stitt MN.1994. Sucrose-phosphate-synthase is regulated via metabolites and protein phosphorylation in potato tubers, in a manner analogous to the enzyme in the leaves. Planta 192, 480–488.
Reimholz R, Geiger M, Haake V, Deiting U, Krause K-P, Sonnewald U, Stitt MN.1997. Potato plants contain multiple forms of sucrose phosphate synthase, which differ in their tissue distributions, their levels during development, and their responses to low temperature. Plant, Cell and Environment 20, 291–305.
Roggatz U, McDonald AJS, Stadenberg I, Schurr U.1999. Effect of nitrogen deprivation on cell division and expansion of Ricinus communis L. Plant, Cell and Environment 22, 81–90.
Sacks MM, Silk W, Borman P.1997. Effects of water stress on cortical cell division rates within the apical meristem of primary roots of maize. Plant Physiology 114, 519–527.[Abstract]
Schmundt D, Schurr U.1999. Plant-leaf growth studied by image sequence analysis. In: Jähne B, Haussecker H, Geissler P, eds. Handbook of computer vision and applications. Vol. 3. Systems and applications. New York: Academic Press, 719–735.
Schmundt D, Stitt M, Jähne B, Schurr U.1998. Quantitative analysis of the local rates of growth of dicot leaves at a high temporal and spatial resolution, using image sequence analysis. The Plant Journal 16, 505–514.[Web of Science]
Schnyder H, Nelson CJ, Spollen WG.1988. Diurnal growth of tall fescue leaf blades. Plant Physiology 86, 1077–1083.
Schulze E-D, Hall AE.1982. Stomatal responses, water loss and CO2 assimilation rates of plants in contrasting environments. In: Lange OL, Nobel PS, Osmond CB, Ziegler H, eds. Enzyclopedia of plant physiology, New Series, Vol. 12B. Berlin: Springer.
Schurr U.1998a. Growth physiology: approaches to a spatially and temporally varying problem. Progress in Botany 59, 355–373.
Schurr U.1998b. Xylem sap sampling—new approaches to an old topic. Trends in Plant Sciences 3, 293–298.
Seneweera S, Basra AS, Barlow EW, Conroy J.1995. Diurnal regulation of leaf blade elongation in rice by CO2: Is it related to sucrose-phosphate synthase activity. Plant Physiology 108, 1471–1477.[Abstract]
Shakel KA, Matthews MA, Morrison JC.1987. Dynamic relation between expansion and cellular turgor in growing grape (Vitis vinifera L.) leaves. Plant Physiology 84, 1166–1171.
Sharp RE, LeNoble ME, Else MA, Thorne ET, Gherardi F.2000. Endogenous ABA maintains shoot growth in tomato independently of effects on plant water balance: evidence for an interaction with ethylene. Journal of Experimental Botany 51, 1575–1584.
Silk WK.1992. Steady form from changing cells. International Journal of Plant Sciences 153, S49–S58.
Sturm A., Sebkova V, Lorenz K, Hardegger M, Lienhard S, Unger C.1995. Development- and organ-specific expression of the genes for sucrose synthase and three isoenzymes of acid beta-fructofuranosidase in carrot. Planta 195, 601–610.
Tardieu F, Reymond M, Hamard P, Granier C, Muller B.2000. Spatial distributions of expansion rate, cell division and cell size in maize leaves: a synthesis of the effects of soil water status, evaporative demand and temperature. Journal of Experimental Botany 51, 1505–1514.
Taylor G, Ranashinghe S, Bosac C, Gardner SDL, Ferris R.1994. Elevated CO2 and plant growth: cellular mechanisms and responses of whole plants. Journal of Experimental Botany 45, 1761–1774.
Tomos AD, Pritchard J.1994. Biophysical and biochemical control of cell expansion in roots and leaves. Journal of Experimental Botany 45, 1721–1731.[Web of Science]
Turgeon R.1989. The sink–source transition in leaves. Annual Review of Plant Physiology 40, 119–138.[Web of Science]
Walker JL, Huber SC.1989. Regulation of sucrose-phosphate-synthase activity in spinach leaves by protein level and covalent modification. Planta 177, 116–120.
Walter A, Schurr U.1999. The modular character of growth in Nicotiana tabacum plants under steady-state nutrition. Journal of Experimental Botany 50, 1169–1177.
Walter A, Schurr U.2000. Spatio-temporal variation of leaf growth and function. In: Marshall B, Roberts J, eds. Leaf development and canopy growth. Sheffield: Sheffield Academic Press.
Weber H, Buchner P, Borisjuk L, Wobus U.1996. Sucrose metabolism during cotyledon development of Vicia faba L. is controlled by the concerted action of both sucrose-phosphate synthase and sucrose synthase: expression patterns, metabolic regulation and implications for seed development. The Plant Journal 9, 841–850.[Web of Science][Medline]
Wu Y, Cosgrove D.2000. Adaptations of roots to low water potentials by changes in cell wall extensibility and cell wall proteins. Journal of Experimental Botany 51, 1543–1553.
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