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Xylem sap sampling—new approaches to an old topic
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Xylem sap sampling – new approaches to an old topic Ulrich Schurr Xylem transport has been studied for many years because of the dominant role that it plays in the flow of water through the plant and in nutrient delivery from the root to the shoot. In recent years, increasing awareness about the importance of chemical signals transported in the xylem in several stress responses has added a further dimension to these studies. As a consequence, it is vital to obtain information about the dynamics of the concentrations and fluxes of nutrients and signal molecules in the xylem. The development of methods to study the dynamics of transport with a resolution of minutes rather than days is crucial for understanding the basic mechanisms of nutrient relations both internally and in response to the environment.
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nalysis of blood is a routine procedure in which samples can easily be removed with little disturbance. Information from different cells and organs is integrated in the blood, and thus basic parameters such as composition and pressure provide a wealth of information about the overall health of the organism. By contrast, attempts to develop similar techniques to monitor transport pathways in plants have been problematic. This is partly because of the presence of two distinct long-distance transport systems – xylem and phloem – that operate by two completely different mechanisms and cannot easily be sampled. Phloem exudates can only be obtained in a few species because of the problem of clogging of the sieve tubes. The problems in xylem sap sampling arise because negative pressures are present under normal conditions of transpiration. Thus, incision causes entry of air rather than leakage of the xylem sap. Consequently, the traditional methods to obtain xylem sap involve decapitating the plant and collecting the sap that eventually exudes from the cut stump, or taking detached pieces of a plant, putting them under pressure and collecting the sap that is squeezed out of the cut end. The composition of the sap collected under such traumatic circumstances often differs from that moving in the intact plant (consider the animal analogy). Here, current methods for analyzing xylem sap and recent advances that allow sampling from virtually intact plants are reviewed. Xylem sap sampling
Xylem transport provides the main route of supply of inorganic nutrients to the shoot for maintenance and growth and, together with the phloem, is responsible for transport throughout the plant. In this way, xylem function determines the distribution of ions and compounds derived from them in the plant1 and coordinates nutrient relations at the whole-plant level2. Furthermore, shoot responses to unfavourable conditions in the root zone are often mediated by chemical signals [such as abscisic acid (ABA), cytokinins and 1-amino-cyclopropane-1-carboxylic acid (ACC)] that travel from the root to the shoot in the xylem3–5. In many cases, information about fluxes rather than absolute concentrations is needed2,5 and modelling approaches have been used extensively to analyze net fluxes of nutrients1 and signalling compounds4. However, gross fluxes with high temporal resolution are also needed to understand the impact of dynamic changes of, for example, nutrient uptake, nutrient assimilation6 and variations in the environment that act in hours to minutes. The need for analysis of fluxes is also imperative in the context of root-to-shoot signalling, where it is important to determine the temporal and
spatial coincidence of the proposed change in concentration or flux of signalling compounds and the response of the plant7. Additionally, a high temporal resolution will provide the basis for quantitative treatment of transport by modelling techniques (e.g. time-series analysis). The major difficulty in sampling xylem sap is the negative pressure in the xylem of transpiring plants8. Up to now, it has proved impossible to determine concentrations in the xylem under conditions of negative pressure. The available techniques (Box 1) circumvent the negative-pressure condition in the xylem sap, at least during the sampling itself, either by reduction of the transpirational suction or by compensation of transpirational suction with pressure. As a result, discussions about the reliability of the various sampling techniques always revolve around the problem of whether treatments used to overcome negative pressure in the xylem have any impact on the sample composition. This debate will continue until a method has been developed to probe xylem sap composition directly under negative-pressure conditions.
Box 1. Techniques for sampling xylem sap and analyzing mass flux in the xylem Xylem sap sampling techniques Destructive techniques Sampling from the root • Root exudate • Flux-controlled exudation Sampling from other plant parts • Scholander pressure chamber • Pressure–decompression method • Vacuum extraction • Infiltration and wash-out techniques • Centrifugation techniques Non-destructive techniques • Xylem pressure probe • Xylem-feeding insects • Root pressure chamber • Guttation Mass-flux quantification Sap-flow measurements • Heat-balance techniques • Heat pulses • Thermal dissipation Transpiration measurements • Weighing • Cuvette gas exchange • Remote sensing systems NMR flux measurements
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Box 2. Tracer studies in xylem transport One approach for studying basic mechanisms of transport is to use tracers. Ideal tracers have identical physicochemical properties to the substance of interest and are detectable at low concentrations. Radioactive tracers have often been used because they are easy to detect. However, for some important ion species, radioactive tracers are either not available (e.g. potassium) or very short-lived (e.g. 13N), meaning that experiments are restricted to specialized facilities37. One approach to circumvent this has been to track these nutrients by radioactive isotopes of other elements (e.g. rubidium for potassium), but this can be misleading38,39. In recent years, detection systems for stable isotopes have become more efficient and thus it is more suitable to use these. Xylem transport, in a strict sense, covers only a distinct part of nutrient and signal molecule transport, while tracer studies can be used to analyze substrates involved in wider transport issues (e.g. lateral exchange of nutrients and exchange activities). Combining tracer studies and xylem sap sampling provides an interesting approach for understanding the complex patterns of transport from the root to the shoot. This is especially important with the dynamic variation in xylem sap composition. In steady-state conditions, lateral exchange will have no impact on the net fluxes into different plant parts, but a considerable impact can be expected during dynamic alterations of xylem sap composition. Isotope studies are now even able to localize the isotopes in the tissue40, which will allow the localization of the heterogeneous distribution of tracer during dynamic alterations. This is an important prerequisite for localizing and understanding the mechanisms behind dynamic variations of nutrient fluxes in the xylem.
Quantification of fluxes additionally requires an appropriate technique for measuring the mass flux in the xylem, as this will usually affect the fluxes of all compounds present. Chromatographic transport of ions along ion-exchange sites has been shown by tracer analysis (Box 2). In steady-state situations, when the ion-exchanging groups are stably saturated, these properties would be of minor importance for net fluxes. However, because xylemsap composition varies considerably, these fluxes might play a crucial role in the different mechanisms of nutrient transport (Box 2).
force for the upward movement of xylem sap in almost all conditions in the light. Therefore, the flux of water through a detached root system is considerably lower under conditions of root exudation, and the effect of dilution by mass flux will be dramatically diminished in comparison with the situation in an intact plant10,11. It has been argued that the concentration of the sap exuding very early during the sampling procedure should still approximate to the sap formed before decapitation12, but this contradicts the recommendation, based on contamination aspects, that the first droplets of sap should be discarded. Third, root processes seem to be highly dependent on phloem import, at least in small root systems of seedlings that have little storage capacity relative to the activity of the root. Ion uptake13 and membrane potential of root cortex cells14 respond rapidly to girdling or shoot detachment. This can either be because of direct effects such as loss of energy or substrates (e.g. amino acids or potassium) for xylem loading or because of the absence of regulatory signals from the shoot2,15. Transpiration conditions also alter water and ion transport in root systems (e.g. reflection coefficients are altered8,16), which will complicate extrapolation to intact plants. Disruption of phloem import will also interrupt any feedback signals moving from the shoot to the roots, and thus remove a potentially important part of the very regulatory network that the measurements are being carried out to study17. The rate of exudation from the cut stump of the root system can be increased, by application of pressure to the root, to fluxes comparable with those of transpiration (when fluxes at or below the level of transpiration are being considered). As the exudation rate is increased, the concentrations of ions in the sap drop significantly and approach values close to those found under transpiring conditions10 (Fig. 1). This modified root-exudation technique still suffers from the problem that the inflow of sugars, nutrients and signals into the roots via the phloem has been interrupted, disrupting the shoot-to-root signals that regulate root physiology and ion uptake. This slightly more sophisticated approach has shown that alterations of flux rate not only change the dilution of the sap but also the relative levels of different ions in the exudate10. This indicates that either the availability of ions at the site of xylem loading, xylem loading itself or lateral exchange along the xylem is dependent on mass flux rate. These results therefore show that conventional root exudation not only gives misleading information about the absolute concentrations, but also about the relative amounts of different ions moving in the sap.
Destructive sampling techniques
Destructive sampling techniques have two main disadvantages when dynamic changes have to be analyzed: first, because a plant population is not homogenous, a suitable number of samples needs to be taken; second, there is always a high risk of the sampling procedure interfering with basic processes that determine xylem sap composition, such as the flux rate or lateral exchange of compounds9,10. Root exudates
The collection of xylem sap from detached root stumps is based on ‘root pressure’. Active xylem loading increases the concentration of ions inside the xylem relative to the surrounding medium (the apoplast of the stele), resulting in osmotic attraction of water into the xylem vessels. In conditions of little or no transpiration, this causes an increase of pressure inside the xylem vessels above atmospheric pressure and thus an exudation. This approach has the large advantage that it is easy to apply, but also has serious drawbacks. First, because the shoot is usually cut off for sampling, damaged cells can leak their contents into the sample or initiate wounding responses. Second, removing the shoot leads to a cessation of transpirational water movement, which is the driving 294
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Extraction of sap from other parts of the plant
Exudate can be forced out of the xylem of cuttings of plant parts by the application of pneumatic pressure, in a similar way to fluxcontrolled root exudation18. However, there are additional complications to consider in this context. First, in flux-controlled root exudation, exuded water can, in principle, be replenished from soil sources. However, the volume that is lost by exudation (e.g. from a leaf in a Scholander pressure chamber) has to be taken from internal compartments. This limits the volume that can be taken without seriously changing water conditions during sampling. Second, because the water in a transpiring plant is under tension, a release of this tension by cutting will lead to redistribution of water between the adjacent compartments. This would not limit the applicability of these methods as long as there is even remobilization of water from the different compartments during pressurization. However, it is not clear that the exuded water is obtained from those compartments into which xylem water disappears after cutting. For the nutrients themselves, the argument of lateral exchange applies in an even more severe way, because the fluxes present in transpiring leaves cannot even be mimicked in
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Nitrate (mol m–3)
Glutamine (mol m–3)
Protons (mmol m–3)
Abscisic acid (mmol m–3)
detached leaves. This favours redistribution 6.0 of ions between the xylem sap and the neighbouring tissues. 20.0 Other techniques involve sap sampling 4.0 by application of force (vacuum, external 19 pressure, internal gas-bubble formation and centrifugation). The principal idea in 10.0 these techniques is to separate xylem sap 2.0 from other compartments in the plant sample; this can be done because of the lower axial resistance in the xylem vessels, and 0.0 0.0 thus the xylem sap needs less force to be 0 100 200 300 400 500 0 100 200 300 400 500 mobilized than fluid in other compartments. This approach is valid if separable 4.0 2.0 axial conductances are present and if mobilization is uniform over the entire sample. But even if this is the case, sap composition 3.0 1.5 inside the xylem in detached plant parts is likely to differ from the composition in a 2.0 1.0 transpiring plant for reasons of lateral exchange, possible damage of tissue during detachment and application of force. Infil1.0 0.5 tration and volume-replacement techniques have been applied to replenish the extracted 0.0 0.0 volume, but exchange between the xylem 0 100 200 300 400 500 0 100 200 300 400 500 sap and neighbouring compartments deVolume flux rate (mm3 min–1) pends on the concentration gradients, and thus the replenishing fluid should ideally Fig. 1. Relationships between the concentrations of nitrate, glutamine, protons and abscisic have the same composition as the xylem sap, acid and the volume flux rate from a detached root system. Flux rate was increased by an iterative and time-consuming approach. elevating the pneumatic pressure on the root system. Open symbols represent the values Destructive techniques are not well during increasing flux rate and closed symbols indicate values during decreasing flux rate. suited for studying the in vivo composition Modified from Ref. 10. and concentrations in xylem sap, or for monitoring dynamic processes, and extrapolation to intact plants is risky. They are, by default, the methods of choice when nondestructive techniques are not feasible (e.g. in old or large plants The xylem pressure probe and in field studies). However, they should always be tested A minimally invasive method of xylem sap sampling is based on against a range of other techniques20,21, including non-destructive the puncture of individual xylem vessels by means of the xylem ones17,21. Another potential and legitimate application of destruc- pressure probe23. A glass capillary is introduced into the tissue tive techniques, however, is in the disassembly of a complex sys- until negative pressure indicates the positioning of the tip in a tem to study individual aspects such as the dependency of the xylem vessel. Samples are gained by a brief application of an concentration on volume flux through the root system10 or lateral- ‘overpressure’ on the root system to compensate the negative exchange capacities in perfusion systems22. pressure in the individual vessel24. This technique can be used to sample xylem sap from intact plants at very defined locations and Non-destructive techniques even allows between-vessel differences in sap composition to Non-destructive techniques are used to sample xylem sap from be measured (S. Marienfeld, pers. commun.). The possibility of intact, transpiring plants with the aim of maintaining the processes analyzing concentration differences between individual xylem that determine xylem sap composition. However, sampling pro- vessels justifies the high effort and makes it suitable for detailed cedures that gain sap from intact plants are not necessarily any analysis of xylem transport. However, the short-term application closer to the undisturbed plant than destructive techniques – during of pressure might still alter the composition of the sample obtained the procedure, transport processes could be altered by variations of because of differences in the lateral exchange during pressure flux rate, pressure or other important components of xylem trans- increase, even when the extracted volume is small relative to the port. Validation is required to show that the fluxes obtained match vessel volumes. Additionally, the large amount of technical effort the ones determined by modelling approaches1 in fully undisturbed required makes this approach inadequate for determining mean plants; and that tracer distribution and dynamics in the plant xylem sap concentrations – needed, for example, for the calculation (Box 2) remain unaltered, a criterion that shows that the basic trans- of mean ion fluxes – because a large number of such measurements port mechanisms have not been changed. Indeed, this is a general would be needed. strategy for evaluating the suitability of a sampling technique, be it destructive or non-destructive. The effort required to obtain xylem Xylem-feeding insects sap by non-destructive techniques is relatively high for the basic Several types of insect (e.g. cicadas and leaf hoppers) feed on procedure itself, and adaptation to different species is often time- xylem sap. Because of the relatively low concentration of nutrients, consuming. However, these techniques have to be applied to study these animals have to take in large volumes of sap in order to exdynamic processes and as reference methods for destructive ones. tract the compounds they live on (probably amides25). However, August 1998, Vol. 3, No. 8
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Box 3. The root pressure chamber
Potassium Nitrate Concentration (mM)
6 Sensor unit
Controller Sampling sites
4
2
0 0
2
4
6
8
10 12 14 16 18 20 22 24 Time (h)
Nitrogen
Air
Fig. 2. Diurnal change in potassium and nitrate concentration in the xylem sap of Ricinus communis as measured by ion-selective electrodes in the continuous exudation from a root pressure chamber. The dark bar indicates the night period.
Root pressure chamber
The root pressure chamber can be used for continuous sampling of xylem sap from intact, transpiring plants. Seedlings are planted through a central bore in the top lid of the planting pot. With increasing stem diameter, the central bore is closed and the roots of the plant become sealed. Water and nutrient solutions can be supplied via connectors in the lid, which drain through a grid at the bottom of the pot. The planting pots can than be mounted in the pressure chamber, which is made from stainless steel or pressuretight plastics. A computerized controller adjusts pneumatic pressure and gas composition in the pressure chamber by opening and closing magnetic valves. Oxygen concentration is regulated according to a preset value. Pressure can either be adjusted to a fixed value (constant mode) or controlled by a sensor unit (free-running mode). The sensor unit consists of a glass capillary connected to a cut in a leaf vein and a photo switch that detects the presence or absence of xylem sap in the capillary. Xylem sap exudes into the capillary with the application of pressure at the pressure chamber. In the free-running mode, the controller maintains the pressure in the chamber at ‘compensation pressure’, which is the pressure needed to maintain the xylem sap–air meniscus inside the photoswitch. Xylem sap is continuously sampled from a cut in a leaf vein at a lower leaf position. It is even possible to sample sap from several positions simultaneously.
most of this volume is then released and can be sampled and analyzed. With this entomological background, it can be expected that abundant ions such as calcium will be present in concentrations close to those in native xylem sap (M. Malone, pers. commun.). The frequent release of sap by the animals makes it possible to analyze dynamic changes of sap composition, and diurnal concentration courses have been reported26. A major drawback of this method is the limited availability of the animals because of hibernation and host-specificity. 296
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The root pressure chamber technique resembles the technique of flux-controlled exudation, with the crucial difference that the plant is maintained intact27. The pneumatic pressure applied at the root system is controlled such that the hydraulic pressure in the xylem is equal to the atmospheric pressure at the position at which the sensor is attached. The pressure in the chamber needed to maintain this situation is termed the compensation pressure (Box 3). The application of pressure affects neither gas exchange28 nor – in the long term – the growth of the shoot29, even when the pressure is maintained for several days. Phloem transport, as measured by 11 C-tracer techniques, is transiently impaired during rapid pressure changes, but resembles normal performance within minutes when the new steady-state water potential has been reached (U. Schurr, S. Jahnke and E.D. Schulze, unpublished). Various techniques have been used to sample xylem sap with this apparatus. Small overpressures above the compensation pressure can be used, but this alters water fluxes through the root system and the xylem during sampling. An alternative technique, which does not impair the net water fluxes, employs the fact that compensation pressure is dependent on transpirational suction30,31. Xylem sap can be sampled from intact plants when the pressure in the root vessel is fixed at the compensation pressure present immediately prior to sampling. Exudation from a cut vein is then induced by reduction of transpiration32 and water flux is maintained close to transpirational flux by the applied pressure. Techniques have been used to reduce the transpiration-rate range from putting the leaves in the dark to increasing the air humidity. The volume of water that would have been transpired under normal conditions exudes from the leaf via a cut in a vein. After the end of the sampling procedure, the controlling device readjusts the compensation pressure. Continuous sampling is made possible by separating the site at which the compensation pressure is regulated and the site at which xylem sap is sampled10,30. A cut in the midrib of a leaf that is located lower than the site at which the compensation pressure is regulated results in continuous drainage of xylem sap for as long as several days. Since the same cuts can be used over a long period of time, the initial wounding response and contamination (1–2 h
trends in plant science reviews after cutting) depicted by a variation of sap composition can be avoided. Exudation rates at the cut did not alter the concentration as long as the gross water flux was maintained (U. Schurr, unpublished). It is even possible to sample sap at several sites (Box 3) in order to study the lateral exchange processes between the sampling sites. This mode of sampling allows continuous methods such as ion-selective electrodes to be used to analyze xylem sap composition, and facilitates analysis of dynamic changes in xylem sap composition during the diurnal course (Fig. 2). From concentrations to fluxes
Table 1. Balance of fluxes into and out of a leafa Nutrient
Sucrose Nitrate Sulphate Chloride Phosphate Potassium Calcium Magnesium Amino acids a
Influx via xylem mol leaf ⫺1 12 h⫺1
Efflux via phloem mol leaf ⫺1 12 h⫺1
Ratio influx : efflux
Not determined 462 34 22 17 674 95 36 65
2082 26 15 7 9 307 7 25 484
– 17.5 2.2 3.3 2.0 2.2 13.2 1.4 0.1
2
A leaf (250 cm ) of Ricinus communis was analyzed over a 12 h light period. Import rates were calcuFor many applications, gross fluxes are lated on the basis of continuous determination of the transpiration rate of the leaf and repetitive more important than the concentrations in sampling of xylem sap with the root-pressure chamber. For each sampling period, the transpiration rate the xylem sap. The easiest way to calculate was integrated and multiplied by the concentration in the xylem sap sample determined during that the flux of a certain compound is to multiperiod. Phloem export was calculated on the basis of 11C-tracer determinations of the phloem velocity ply the concentration in the xylem sap by and phloem sap sampling by the incision technique (U. Schurr and S. Jahnke, unpublished). mass flux in the xylem. In a first approximation, the mass flux can be set equivalent to the rate of transpiration, because other processes that compete for water, such as phloem backflow or growth, are <5% (U. Schurr, unpublished). Future work The exception is when transpiration is very low (e.g. in seedlings), A number of techniques are available for sampling xylem sap. Howwhere other pathways make a significant contribution33. When ever, because they interact with the driving forces of transport, it is balances are made from transpiration measurements and xylem crucial to check the relevance of the samples obtained. Even though sap concentrations determined in sap sampled with the root pres- non-destructive techniques cannot be applied in all cases, they prosure chamber, the estimated values (Table 1) are in the same range vide a suitable procedure for validating the results from destructive as those that were obtained from approaches that model the net sampling. In general, all sampling techniques can only be regarded fluxes1, and the ratios of import and export mirror the expected as relevant if they can explain the net fluxes that can be determined flux conditions. in undisturbed plants. Transpiration rates determined with gas-exchange cuvettes are, With recent progress in techniques for xylem sap sampling and however, often different from the mass fluxes in undisturbed flux determination, it is now feasible to study the dynamics of xylem plants as a significant gas flow through the cuvette is required to transport. Even though it is still not possible to collect xylem sap obtain a dynamic measurement of the gas exchange at the leaf sur- with ease, there are approaches for accessing the long-distance transface. This increased air movement and the resulting decrease in port pathway. In combination with modern tracer techniques, it will boundary-layer resistance will lead to an increase of the transpi- be possible to study the underlying mechanisms of nutrient and ration rate relative to undisturbed plants, and will thus result in an signal transport in whole plants. Hypotheses that have hitherto been apparent overestimation of water flux and hence of the flux of dis- untestable, such as demand-driven control of ion uptake or movesolved compounds. Remote sensing of gas exchange has been ment of hormones in root-to-shoot communication, can be checked. developed on the basis of chlorophyll-fluorescence imaging34. The application of these methods will change our understanding However, extrapolation to water fluxes out of the stomata is rather of nutrient and signal transport from a static to a dynamic view. indirect. Other techniques, such as thermography, have not yet been taken through to a stage that allows a quantitative analysis of Acknowledgements water flow. I thank Arnd Kuhn and Mark Stitt for discussions and critical Alternatively, water loss from the entire shoot can be measured reading of the manuscript and Frank Thürmer, Heike Schneider directly by weighing the plants regularly. This simple but reliable and Mike Malone for providing additional information. Many method has the drawback that it does not provide information thanks also to Uwe Heckenberger and Klaus Herdel for work with about the local rates of water loss. Sap-flow measurements based the root pressure chamber. Funding was provided by the German on heat transport are very commonly made for woody species and Science Foundation (DFG) in SFB 199 TP C1 and Schu 946/1-1. have also been applied in some cases to herbaceous plants35. They allow quantification of sap fluxes in different parts of the plant References without altering the conditions for transpiration. However, the 01 Jeschke, W.D. and Pate, J.S. (1991) Modelling of the partitioning, assimilation spatial resolution is limited, especially in herbaceous plants, and storage of nitrate within the root and shoot organs of castor bean (Ricinus because of the size of the gauge. communis L.), J. Exp. Bot. 42, 1091–1103 Nuclear magnetic resonance (NMR) provides a very refined 02 Marschner, H., Kirkby, E.A. and Cakmak, I. (1996) Effect of mineral measure for quantifying water flux rates in herbaceous plants33,36 nutritional status on shoot–root partitioning of photoassimilates and cycling of and even allows the distribution of flow rates in an intact plant to mineral nutrients, J. Exp. Bot. 47, 1255–1263 be imaged. This approach has also been used in studies of phloem 03 Davies, W.J. and Zhang, J. (1991) Root signals and the regulation of growth transport in intact plants. However, its application is limited and development of plants in drying soil, Annu. Rev. Plant Physiol. Plant Mol. because of the large technical effort required and high costs. Biol. 42, 55–76
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trends in plant science reviews 04 Peuke, A.D., Jechke, W.D. and Hartung, W. (1994) The uptake and flow of C, N and ions between roots and shoots in Ricinus communis L. III. Longdistance transport of abscisic acid depending on nitrogen nutrition and salt stress, J. Exp. Bot. 45, 741–747 05 Jackson, M.B. (1997) Hormones from roots as signals for the shoots of stressed plants, Trends Plant Sci. 2, 22–28 06 Macduff, J.H., Bakken, A.K. and Dhanoa, M.S. (1997) An analysis of the physiological basis of commonality between diurnal patterns of NH4+, NO3⫺ and K+-uptake by Phleum pratense and Festuca pratensis, J. Exp. Bot. 48, 1691–1701 07 Schurr, U. and Gollan, T. (1990) Composition of xylem sap of plants experiencing root water stress – a descriptive study, in Importance of Root to Shoot Communication in the Responses to Environmental Stress (Monograph 21) (Davies, W.J. and Jeffcoat, B., eds), pp. 201–214, British Society for Plant Growth Regulation 08 Schneider, H. et al. (1997) Diurnal variation of radial reflection coefficient of intact maize roots determined with the xylem pressure probe, J. Exp. Bot. 48, 2045–2053 09 Else, M.A. et al. (1994) Concentrations of abscisic acid and other solutes in the xylem sap from root systems of tomato and castor-oil plants are distorted by wounding and variable sap flow rates, J. Exp. Bot. 45, 317–323 10 Schurr, U. and Schulze, E.D. (1995) The concentration of xylem sap constituents in root exudate, and in sap from intact, transpiring castor bean plants (Ricinus communis L.), Plant Cell Environ. 18, 409–420 11 Else, M.A. et al. (1995) Export of abscisic acid, 1-aminocyclopropane-1carboxylic acid, phosphate, and nitrate from roots to shoots of flooded tomato plants. Accounting for effects of xylem sap flow rate on concentration and delivery, Plant Physiol. 107, 377–384 12 Shaner, D.L. and Boyer, J.S. (1976) Nitrate reductase activity in maize (Zea mays L.) leaves, Plant Physiol. 58, 499–504 13 Bloom, A.J. and Caldwell, M.M. (1988) Root excision decreases nutrient absorption and gas fluxes, Plant Physiol. 87, 794–796 14 Graham, R.D. and Bowling, D.J.F. (1977) Effect of the shoot on the transmembrane potential of the root cortical cells of sunflower, J. Exp. Bot. 28, 886–893 15 Pitman, M.G. (1988) Whole Plants, in Solute Transport in Plant Cells and Tissues (Baker, D.A. and Hall, J.L.), pp. 346–391, Longman 16 Zhu, J.J. et al. (1995) Xylem pressure responses in maize roots subjected to osmotic stress: determination of radial reflection coefficients by use of the xylem pressure probe, Plant Cell Environ. 18, 906–912 17 Schurr, U. Dynamics of nutrient transport from the root to the shoot, Prog. Bot. 60 (in press) 18 Berger, A., Oren, R. and Schulze, E.D. (1994) Element concentrations in the xylem sap of Pices abies (L.) Karst. seedlings extracted by various methods under different environmental conditions, Tree Physiol. 14, 111–128 19 Schill, V. et al. (1996) The xylem sap of maple (Acer platanoides) trees – sap obtained by a novel method shows changes with season and height, J. Exp. Bot. 47, 123–133 20 Dodd, I.C., Stikic, R. and Davies, W.J. (1996) Chemical regulation of gas exchange and growth in plants in drying soil in the field, J. Exp. Bot. 47, 1475–1490 21 McDonald, A.J.S. and Davies, W.J. (1996) Keeping in touch: responses of the whole plant to deficits in water and nitrogen supply, Adv. Bot. Res. 22, 229–300 22 Clarkson, D.T. and Hanson, J.B. (1986) Proton fluxes and the activity of a stelar proton pump in onion roots, J. Exp. Bot 37, 1136–1150 23 Balling, A. et al. (1988) Direct measurement of negative pressure in artificialbiological systems, Naturwissenschaften 75, 409–411 24 Zimmermann, G. et al. (1995) Xylem pressure measurements in intact laboratory plants and excised organs: a critical evaluation of methods in the literature and the xylem pressure probe, in Tree Sap (Terazawa, M., McLeoad, C.A. and Tamia, Y., eds), pp. 59–70, Hokkaido University Press 25 Andersen, P.C., Brodbeck, B.V. and Mizell, R.F., III (1992) Feeding by leafhopper, Homalodisca coagulata, in relation to xylem fluid chemistry and tension, J. Insect Physiol. 38, 611–622
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26 Andersen, P.C., Brodbeck, B.V. and Mizell, R.F., III (1993) Diurnal variations of amino acids and organic acids in xylem fluids from Lagerstroemia indica: an endogenous circadian rhythm, Physiol. Plant. 89, 783–790 27 Passioura, J.B. and Tanner, C.B. (1985) Oscillations in apparent hydraulic conductance of cotton plants, Aust. J. Plant Physiol. 12, 455–461 28 Gollan, T., Passioura, J.B. and Munns, R. (1987) Soil water status affects the stomatal conductance of fully turgid wheat and sunflower leaves, Aust. J. Plant Physiol. 13, 459–464 29 Passioura, J.B. and Gardner, P.A. (1990) Control of leaf expansion in wheat seedlings growing in dry soil, Aust. J. Plant Physiol. 17, 149–157 30 Schurr, U. and Schulze, E.D. (1996) Effect of drought on nutrient transport and ABA transport in Ricinus communis, Plant Cell Environ. 19, 665–674 31 Stirzaker, R.J., Hayman, P.T. and Sutton, B.G. (1997) Misting of tomato plants improves leaf water status but not leaf growth, Aust. J. Plant Physiol. 24, 9–16 32 Gollan, T., Schurr, U. and Schulze, E.D. (1992) Stomatal response to drying soil in relation to changes in the xylem sap composition of Helianthus annuus. I. The concentration of cations, anions, amino acids and the pH of the xylem sap, Plant Cell Environ. 15, 551–559 33 Köckenberger, W. et al. (1997) A non-invasive measurement of phloem and xylem water flow in castor bean seedlings by nuclear magnetic resonance microimaging, Planta 201, 53–63 34 Siebke, K. and Weis, E. (1995) Assimilation images of leaves from Gleochoma hederacea. Analysis of non-synchronous stomata related oscillations, Planta 196, 148–165 35 Smith, D.M. and Allen, S.J. (1996) Measurement of sap flow in plant stems, J. Exp. Bot. 47, 1833–1844 36 Kuchenbrod, E. et al. (1996) Measurement of water flow in the xylem vessels of intact maize plants using flow-sensitive NMR-imaging, Bot. Acta 109, 184–186 37 Clarkson, D.T. et al. (1996) Nitrate and ammonium influxes in soybean (Glycine max) roots: direct comparison of 13N and 15N tracing, Plant Cell Environ. 19, 859–868 38 Marschner, H. and Schimansky, C. (1968) Unterschiedliche Aufnahme von Kalium und Rubidium durch Gerste, Naturwissenschaften 55, 499 39 Jeschke, W.D. (1970) Über die Verwendung von 86Rb als Indikator für Kalium, Untersuchungen am lichtgeförderten 42K/K- und 86Rb/Rb-Influx bei Elodea densa, Z. Naturforsch. 25, 624–630 40 Kuhn, A.J., Bauch, J. and Schröder, W.H. (1995) Monitoring uptake and contents of Mg, Ca and K in Norway spruce as influenced by pH and Al, using microprobe analysis and stable isotope labelling, Plant Soil 168, 135–150
Ulrich Schurr is at the Institute of Botany, University of Heidelberg, Im Neuenheimer Feld 360, D-69120 Heidelberg, Germany (tel +49 6221 545334; fax +49 6221 545859; e-mail [email protected]).
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Xylem sap sampling – new approaches to an old topic Ulrich Schurr Xylem transport has been studied for many years because of the dominant role that it plays in the flow of water through the plant and in nutrient delivery from the root to the shoot. In recent years, increasing awareness about the importance of chemical signals transported in the xylem in several stress responses has added a further dimension to these studies. As a consequence, it is vital to obtain information about the dynamics of the concentrations and fluxes of nutrients and signal molecules in the xylem. The development of methods to study the dynamics of transport with a resolution of minutes rather than days is crucial for understanding the basic mechanisms of nutrient relations both internally and in response to the environment.
A
nalysis of blood is a routine procedure in which samples can easily be removed with little disturbance. Information from different cells and organs is integrated in the blood, and thus basic parameters such as composition and pressure provide a wealth of information about the overall health of the organism. By contrast, attempts to develop similar techniques to monitor transport pathways in plants have been problematic. This is partly because of the presence of two distinct long-distance transport systems – xylem and phloem – that operate by two completely different mechanisms and cannot easily be sampled. Phloem exudates can only be obtained in a few species because of the problem of clogging of the sieve tubes. The problems in xylem sap sampling arise because negative pressures are present under normal conditions of transpiration. Thus, incision causes entry of air rather than leakage of the xylem sap. Consequently, the traditional methods to obtain xylem sap involve decapitating the plant and collecting the sap that eventually exudes from the cut stump, or taking detached pieces of a plant, putting them under pressure and collecting the sap that is squeezed out of the cut end. The composition of the sap collected under such traumatic circumstances often differs from that moving in the intact plant (consider the animal analogy). Here, current methods for analyzing xylem sap and recent advances that allow sampling from virtually intact plants are reviewed. Xylem sap sampling
Xylem transport provides the main route of supply of inorganic nutrients to the shoot for maintenance and growth and, together with the phloem, is responsible for transport throughout the plant. In this way, xylem function determines the distribution of ions and compounds derived from them in the plant1 and coordinates nutrient relations at the whole-plant level2. Furthermore, shoot responses to unfavourable conditions in the root zone are often mediated by chemical signals [such as abscisic acid (ABA), cytokinins and 1-amino-cyclopropane-1-carboxylic acid (ACC)] that travel from the root to the shoot in the xylem3–5. In many cases, information about fluxes rather than absolute concentrations is needed2,5 and modelling approaches have been used extensively to analyze net fluxes of nutrients1 and signalling compounds4. However, gross fluxes with high temporal resolution are also needed to understand the impact of dynamic changes of, for example, nutrient uptake, nutrient assimilation6 and variations in the environment that act in hours to minutes. The need for analysis of fluxes is also imperative in the context of root-to-shoot signalling, where it is important to determine the temporal and
spatial coincidence of the proposed change in concentration or flux of signalling compounds and the response of the plant7. Additionally, a high temporal resolution will provide the basis for quantitative treatment of transport by modelling techniques (e.g. time-series analysis). The major difficulty in sampling xylem sap is the negative pressure in the xylem of transpiring plants8. Up to now, it has proved impossible to determine concentrations in the xylem under conditions of negative pressure. The available techniques (Box 1) circumvent the negative-pressure condition in the xylem sap, at least during the sampling itself, either by reduction of the transpirational suction or by compensation of transpirational suction with pressure. As a result, discussions about the reliability of the various sampling techniques always revolve around the problem of whether treatments used to overcome negative pressure in the xylem have any impact on the sample composition. This debate will continue until a method has been developed to probe xylem sap composition directly under negative-pressure conditions.
Box 1. Techniques for sampling xylem sap and analyzing mass flux in the xylem Xylem sap sampling techniques Destructive techniques Sampling from the root • Root exudate • Flux-controlled exudation Sampling from other plant parts • Scholander pressure chamber • Pressure–decompression method • Vacuum extraction • Infiltration and wash-out techniques • Centrifugation techniques Non-destructive techniques • Xylem pressure probe • Xylem-feeding insects • Root pressure chamber • Guttation Mass-flux quantification Sap-flow measurements • Heat-balance techniques • Heat pulses • Thermal dissipation Transpiration measurements • Weighing • Cuvette gas exchange • Remote sensing systems NMR flux measurements
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Box 2. Tracer studies in xylem transport One approach for studying basic mechanisms of transport is to use tracers. Ideal tracers have identical physicochemical properties to the substance of interest and are detectable at low concentrations. Radioactive tracers have often been used because they are easy to detect. However, for some important ion species, radioactive tracers are either not available (e.g. potassium) or very short-lived (e.g. 13N), meaning that experiments are restricted to specialized facilities37. One approach to circumvent this has been to track these nutrients by radioactive isotopes of other elements (e.g. rubidium for potassium), but this can be misleading38,39. In recent years, detection systems for stable isotopes have become more efficient and thus it is more suitable to use these. Xylem transport, in a strict sense, covers only a distinct part of nutrient and signal molecule transport, while tracer studies can be used to analyze substrates involved in wider transport issues (e.g. lateral exchange of nutrients and exchange activities). Combining tracer studies and xylem sap sampling provides an interesting approach for understanding the complex patterns of transport from the root to the shoot. This is especially important with the dynamic variation in xylem sap composition. In steady-state conditions, lateral exchange will have no impact on the net fluxes into different plant parts, but a considerable impact can be expected during dynamic alterations of xylem sap composition. Isotope studies are now even able to localize the isotopes in the tissue40, which will allow the localization of the heterogeneous distribution of tracer during dynamic alterations. This is an important prerequisite for localizing and understanding the mechanisms behind dynamic variations of nutrient fluxes in the xylem.
Quantification of fluxes additionally requires an appropriate technique for measuring the mass flux in the xylem, as this will usually affect the fluxes of all compounds present. Chromatographic transport of ions along ion-exchange sites has been shown by tracer analysis (Box 2). In steady-state situations, when the ion-exchanging groups are stably saturated, these properties would be of minor importance for net fluxes. However, because xylemsap composition varies considerably, these fluxes might play a crucial role in the different mechanisms of nutrient transport (Box 2).
force for the upward movement of xylem sap in almost all conditions in the light. Therefore, the flux of water through a detached root system is considerably lower under conditions of root exudation, and the effect of dilution by mass flux will be dramatically diminished in comparison with the situation in an intact plant10,11. It has been argued that the concentration of the sap exuding very early during the sampling procedure should still approximate to the sap formed before decapitation12, but this contradicts the recommendation, based on contamination aspects, that the first droplets of sap should be discarded. Third, root processes seem to be highly dependent on phloem import, at least in small root systems of seedlings that have little storage capacity relative to the activity of the root. Ion uptake13 and membrane potential of root cortex cells14 respond rapidly to girdling or shoot detachment. This can either be because of direct effects such as loss of energy or substrates (e.g. amino acids or potassium) for xylem loading or because of the absence of regulatory signals from the shoot2,15. Transpiration conditions also alter water and ion transport in root systems (e.g. reflection coefficients are altered8,16), which will complicate extrapolation to intact plants. Disruption of phloem import will also interrupt any feedback signals moving from the shoot to the roots, and thus remove a potentially important part of the very regulatory network that the measurements are being carried out to study17. The rate of exudation from the cut stump of the root system can be increased, by application of pressure to the root, to fluxes comparable with those of transpiration (when fluxes at or below the level of transpiration are being considered). As the exudation rate is increased, the concentrations of ions in the sap drop significantly and approach values close to those found under transpiring conditions10 (Fig. 1). This modified root-exudation technique still suffers from the problem that the inflow of sugars, nutrients and signals into the roots via the phloem has been interrupted, disrupting the shoot-to-root signals that regulate root physiology and ion uptake. This slightly more sophisticated approach has shown that alterations of flux rate not only change the dilution of the sap but also the relative levels of different ions in the exudate10. This indicates that either the availability of ions at the site of xylem loading, xylem loading itself or lateral exchange along the xylem is dependent on mass flux rate. These results therefore show that conventional root exudation not only gives misleading information about the absolute concentrations, but also about the relative amounts of different ions moving in the sap.
Destructive sampling techniques
Destructive sampling techniques have two main disadvantages when dynamic changes have to be analyzed: first, because a plant population is not homogenous, a suitable number of samples needs to be taken; second, there is always a high risk of the sampling procedure interfering with basic processes that determine xylem sap composition, such as the flux rate or lateral exchange of compounds9,10. Root exudates
The collection of xylem sap from detached root stumps is based on ‘root pressure’. Active xylem loading increases the concentration of ions inside the xylem relative to the surrounding medium (the apoplast of the stele), resulting in osmotic attraction of water into the xylem vessels. In conditions of little or no transpiration, this causes an increase of pressure inside the xylem vessels above atmospheric pressure and thus an exudation. This approach has the large advantage that it is easy to apply, but also has serious drawbacks. First, because the shoot is usually cut off for sampling, damaged cells can leak their contents into the sample or initiate wounding responses. Second, removing the shoot leads to a cessation of transpirational water movement, which is the driving 294
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Extraction of sap from other parts of the plant
Exudate can be forced out of the xylem of cuttings of plant parts by the application of pneumatic pressure, in a similar way to fluxcontrolled root exudation18. However, there are additional complications to consider in this context. First, in flux-controlled root exudation, exuded water can, in principle, be replenished from soil sources. However, the volume that is lost by exudation (e.g. from a leaf in a Scholander pressure chamber) has to be taken from internal compartments. This limits the volume that can be taken without seriously changing water conditions during sampling. Second, because the water in a transpiring plant is under tension, a release of this tension by cutting will lead to redistribution of water between the adjacent compartments. This would not limit the applicability of these methods as long as there is even remobilization of water from the different compartments during pressurization. However, it is not clear that the exuded water is obtained from those compartments into which xylem water disappears after cutting. For the nutrients themselves, the argument of lateral exchange applies in an even more severe way, because the fluxes present in transpiring leaves cannot even be mimicked in
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Nitrate (mol m–3)
Glutamine (mol m–3)
Protons (mmol m–3)
Abscisic acid (mmol m–3)
detached leaves. This favours redistribution 6.0 of ions between the xylem sap and the neighbouring tissues. 20.0 Other techniques involve sap sampling 4.0 by application of force (vacuum, external 19 pressure, internal gas-bubble formation and centrifugation). The principal idea in 10.0 these techniques is to separate xylem sap 2.0 from other compartments in the plant sample; this can be done because of the lower axial resistance in the xylem vessels, and 0.0 0.0 thus the xylem sap needs less force to be 0 100 200 300 400 500 0 100 200 300 400 500 mobilized than fluid in other compartments. This approach is valid if separable 4.0 2.0 axial conductances are present and if mobilization is uniform over the entire sample. But even if this is the case, sap composition 3.0 1.5 inside the xylem in detached plant parts is likely to differ from the composition in a 2.0 1.0 transpiring plant for reasons of lateral exchange, possible damage of tissue during detachment and application of force. Infil1.0 0.5 tration and volume-replacement techniques have been applied to replenish the extracted 0.0 0.0 volume, but exchange between the xylem 0 100 200 300 400 500 0 100 200 300 400 500 sap and neighbouring compartments deVolume flux rate (mm3 min–1) pends on the concentration gradients, and thus the replenishing fluid should ideally Fig. 1. Relationships between the concentrations of nitrate, glutamine, protons and abscisic have the same composition as the xylem sap, acid and the volume flux rate from a detached root system. Flux rate was increased by an iterative and time-consuming approach. elevating the pneumatic pressure on the root system. Open symbols represent the values Destructive techniques are not well during increasing flux rate and closed symbols indicate values during decreasing flux rate. suited for studying the in vivo composition Modified from Ref. 10. and concentrations in xylem sap, or for monitoring dynamic processes, and extrapolation to intact plants is risky. They are, by default, the methods of choice when nondestructive techniques are not feasible (e.g. in old or large plants The xylem pressure probe and in field studies). However, they should always be tested A minimally invasive method of xylem sap sampling is based on against a range of other techniques20,21, including non-destructive the puncture of individual xylem vessels by means of the xylem ones17,21. Another potential and legitimate application of destruc- pressure probe23. A glass capillary is introduced into the tissue tive techniques, however, is in the disassembly of a complex sys- until negative pressure indicates the positioning of the tip in a tem to study individual aspects such as the dependency of the xylem vessel. Samples are gained by a brief application of an concentration on volume flux through the root system10 or lateral- ‘overpressure’ on the root system to compensate the negative exchange capacities in perfusion systems22. pressure in the individual vessel24. This technique can be used to sample xylem sap from intact plants at very defined locations and Non-destructive techniques even allows between-vessel differences in sap composition to Non-destructive techniques are used to sample xylem sap from be measured (S. Marienfeld, pers. commun.). The possibility of intact, transpiring plants with the aim of maintaining the processes analyzing concentration differences between individual xylem that determine xylem sap composition. However, sampling pro- vessels justifies the high effort and makes it suitable for detailed cedures that gain sap from intact plants are not necessarily any analysis of xylem transport. However, the short-term application closer to the undisturbed plant than destructive techniques – during of pressure might still alter the composition of the sample obtained the procedure, transport processes could be altered by variations of because of differences in the lateral exchange during pressure flux rate, pressure or other important components of xylem trans- increase, even when the extracted volume is small relative to the port. Validation is required to show that the fluxes obtained match vessel volumes. Additionally, the large amount of technical effort the ones determined by modelling approaches1 in fully undisturbed required makes this approach inadequate for determining mean plants; and that tracer distribution and dynamics in the plant xylem sap concentrations – needed, for example, for the calculation (Box 2) remain unaltered, a criterion that shows that the basic trans- of mean ion fluxes – because a large number of such measurements port mechanisms have not been changed. Indeed, this is a general would be needed. strategy for evaluating the suitability of a sampling technique, be it destructive or non-destructive. The effort required to obtain xylem Xylem-feeding insects sap by non-destructive techniques is relatively high for the basic Several types of insect (e.g. cicadas and leaf hoppers) feed on procedure itself, and adaptation to different species is often time- xylem sap. Because of the relatively low concentration of nutrients, consuming. However, these techniques have to be applied to study these animals have to take in large volumes of sap in order to exdynamic processes and as reference methods for destructive ones. tract the compounds they live on (probably amides25). However, August 1998, Vol. 3, No. 8
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Box 3. The root pressure chamber
Potassium Nitrate Concentration (mM)
6 Sensor unit
Controller Sampling sites
4
2
0 0
2
4
6
8
10 12 14 16 18 20 22 24 Time (h)
Nitrogen
Air
Fig. 2. Diurnal change in potassium and nitrate concentration in the xylem sap of Ricinus communis as measured by ion-selective electrodes in the continuous exudation from a root pressure chamber. The dark bar indicates the night period.
Root pressure chamber
The root pressure chamber can be used for continuous sampling of xylem sap from intact, transpiring plants. Seedlings are planted through a central bore in the top lid of the planting pot. With increasing stem diameter, the central bore is closed and the roots of the plant become sealed. Water and nutrient solutions can be supplied via connectors in the lid, which drain through a grid at the bottom of the pot. The planting pots can than be mounted in the pressure chamber, which is made from stainless steel or pressuretight plastics. A computerized controller adjusts pneumatic pressure and gas composition in the pressure chamber by opening and closing magnetic valves. Oxygen concentration is regulated according to a preset value. Pressure can either be adjusted to a fixed value (constant mode) or controlled by a sensor unit (free-running mode). The sensor unit consists of a glass capillary connected to a cut in a leaf vein and a photo switch that detects the presence or absence of xylem sap in the capillary. Xylem sap exudes into the capillary with the application of pressure at the pressure chamber. In the free-running mode, the controller maintains the pressure in the chamber at ‘compensation pressure’, which is the pressure needed to maintain the xylem sap–air meniscus inside the photoswitch. Xylem sap is continuously sampled from a cut in a leaf vein at a lower leaf position. It is even possible to sample sap from several positions simultaneously.
most of this volume is then released and can be sampled and analyzed. With this entomological background, it can be expected that abundant ions such as calcium will be present in concentrations close to those in native xylem sap (M. Malone, pers. commun.). The frequent release of sap by the animals makes it possible to analyze dynamic changes of sap composition, and diurnal concentration courses have been reported26. A major drawback of this method is the limited availability of the animals because of hibernation and host-specificity. 296
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The root pressure chamber technique resembles the technique of flux-controlled exudation, with the crucial difference that the plant is maintained intact27. The pneumatic pressure applied at the root system is controlled such that the hydraulic pressure in the xylem is equal to the atmospheric pressure at the position at which the sensor is attached. The pressure in the chamber needed to maintain this situation is termed the compensation pressure (Box 3). The application of pressure affects neither gas exchange28 nor – in the long term – the growth of the shoot29, even when the pressure is maintained for several days. Phloem transport, as measured by 11 C-tracer techniques, is transiently impaired during rapid pressure changes, but resembles normal performance within minutes when the new steady-state water potential has been reached (U. Schurr, S. Jahnke and E.D. Schulze, unpublished). Various techniques have been used to sample xylem sap with this apparatus. Small overpressures above the compensation pressure can be used, but this alters water fluxes through the root system and the xylem during sampling. An alternative technique, which does not impair the net water fluxes, employs the fact that compensation pressure is dependent on transpirational suction30,31. Xylem sap can be sampled from intact plants when the pressure in the root vessel is fixed at the compensation pressure present immediately prior to sampling. Exudation from a cut vein is then induced by reduction of transpiration32 and water flux is maintained close to transpirational flux by the applied pressure. Techniques have been used to reduce the transpiration-rate range from putting the leaves in the dark to increasing the air humidity. The volume of water that would have been transpired under normal conditions exudes from the leaf via a cut in a vein. After the end of the sampling procedure, the controlling device readjusts the compensation pressure. Continuous sampling is made possible by separating the site at which the compensation pressure is regulated and the site at which xylem sap is sampled10,30. A cut in the midrib of a leaf that is located lower than the site at which the compensation pressure is regulated results in continuous drainage of xylem sap for as long as several days. Since the same cuts can be used over a long period of time, the initial wounding response and contamination (1–2 h
trends in plant science reviews after cutting) depicted by a variation of sap composition can be avoided. Exudation rates at the cut did not alter the concentration as long as the gross water flux was maintained (U. Schurr, unpublished). It is even possible to sample sap at several sites (Box 3) in order to study the lateral exchange processes between the sampling sites. This mode of sampling allows continuous methods such as ion-selective electrodes to be used to analyze xylem sap composition, and facilitates analysis of dynamic changes in xylem sap composition during the diurnal course (Fig. 2). From concentrations to fluxes
Table 1. Balance of fluxes into and out of a leafa Nutrient
Sucrose Nitrate Sulphate Chloride Phosphate Potassium Calcium Magnesium Amino acids a
Influx via xylem mol leaf ⫺1 12 h⫺1
Efflux via phloem mol leaf ⫺1 12 h⫺1
Ratio influx : efflux
Not determined 462 34 22 17 674 95 36 65
2082 26 15 7 9 307 7 25 484
– 17.5 2.2 3.3 2.0 2.2 13.2 1.4 0.1
2
A leaf (250 cm ) of Ricinus communis was analyzed over a 12 h light period. Import rates were calcuFor many applications, gross fluxes are lated on the basis of continuous determination of the transpiration rate of the leaf and repetitive more important than the concentrations in sampling of xylem sap with the root-pressure chamber. For each sampling period, the transpiration rate the xylem sap. The easiest way to calculate was integrated and multiplied by the concentration in the xylem sap sample determined during that the flux of a certain compound is to multiperiod. Phloem export was calculated on the basis of 11C-tracer determinations of the phloem velocity ply the concentration in the xylem sap by and phloem sap sampling by the incision technique (U. Schurr and S. Jahnke, unpublished). mass flux in the xylem. In a first approximation, the mass flux can be set equivalent to the rate of transpiration, because other processes that compete for water, such as phloem backflow or growth, are <5% (U. Schurr, unpublished). Future work The exception is when transpiration is very low (e.g. in seedlings), A number of techniques are available for sampling xylem sap. Howwhere other pathways make a significant contribution33. When ever, because they interact with the driving forces of transport, it is balances are made from transpiration measurements and xylem crucial to check the relevance of the samples obtained. Even though sap concentrations determined in sap sampled with the root pres- non-destructive techniques cannot be applied in all cases, they prosure chamber, the estimated values (Table 1) are in the same range vide a suitable procedure for validating the results from destructive as those that were obtained from approaches that model the net sampling. In general, all sampling techniques can only be regarded fluxes1, and the ratios of import and export mirror the expected as relevant if they can explain the net fluxes that can be determined flux conditions. in undisturbed plants. Transpiration rates determined with gas-exchange cuvettes are, With recent progress in techniques for xylem sap sampling and however, often different from the mass fluxes in undisturbed flux determination, it is now feasible to study the dynamics of xylem plants as a significant gas flow through the cuvette is required to transport. Even though it is still not possible to collect xylem sap obtain a dynamic measurement of the gas exchange at the leaf sur- with ease, there are approaches for accessing the long-distance transface. This increased air movement and the resulting decrease in port pathway. In combination with modern tracer techniques, it will boundary-layer resistance will lead to an increase of the transpi- be possible to study the underlying mechanisms of nutrient and ration rate relative to undisturbed plants, and will thus result in an signal transport in whole plants. Hypotheses that have hitherto been apparent overestimation of water flux and hence of the flux of dis- untestable, such as demand-driven control of ion uptake or movesolved compounds. Remote sensing of gas exchange has been ment of hormones in root-to-shoot communication, can be checked. developed on the basis of chlorophyll-fluorescence imaging34. The application of these methods will change our understanding However, extrapolation to water fluxes out of the stomata is rather of nutrient and signal transport from a static to a dynamic view. indirect. Other techniques, such as thermography, have not yet been taken through to a stage that allows a quantitative analysis of Acknowledgements water flow. I thank Arnd Kuhn and Mark Stitt for discussions and critical Alternatively, water loss from the entire shoot can be measured reading of the manuscript and Frank Thürmer, Heike Schneider directly by weighing the plants regularly. This simple but reliable and Mike Malone for providing additional information. Many method has the drawback that it does not provide information thanks also to Uwe Heckenberger and Klaus Herdel for work with about the local rates of water loss. Sap-flow measurements based the root pressure chamber. Funding was provided by the German on heat transport are very commonly made for woody species and Science Foundation (DFG) in SFB 199 TP C1 and Schu 946/1-1. have also been applied in some cases to herbaceous plants35. They allow quantification of sap fluxes in different parts of the plant References without altering the conditions for transpiration. However, the 01 Jeschke, W.D. and Pate, J.S. (1991) Modelling of the partitioning, assimilation spatial resolution is limited, especially in herbaceous plants, and storage of nitrate within the root and shoot organs of castor bean (Ricinus because of the size of the gauge. communis L.), J. Exp. Bot. 42, 1091–1103 Nuclear magnetic resonance (NMR) provides a very refined 02 Marschner, H., Kirkby, E.A. and Cakmak, I. (1996) Effect of mineral measure for quantifying water flux rates in herbaceous plants33,36 nutritional status on shoot–root partitioning of photoassimilates and cycling of and even allows the distribution of flow rates in an intact plant to mineral nutrients, J. Exp. Bot. 47, 1255–1263 be imaged. This approach has also been used in studies of phloem 03 Davies, W.J. and Zhang, J. (1991) Root signals and the regulation of growth transport in intact plants. However, its application is limited and development of plants in drying soil, Annu. Rev. Plant Physiol. Plant Mol. because of the large technical effort required and high costs. Biol. 42, 55–76
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Ulrich Schurr is at the Institute of Botany, University of Heidelberg, Im Neuenheimer Feld 360, D-69120 Heidelberg, Germany (tel +49 6221 545334; fax +49 6221 545859; e-mail [email protected]).
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