Intact plant MRI for the study of cell water relations, membrane permeability, cell-to-cell and long distance water transport

Water content and hydraulic conductivity, including transport within cells, over membranes, cell-to-cell, and long-distance xylem and phloem transport, are strongly affected by plant water stress. By being able to measure these transport processes non-invasely in the intact plant situation in relation to the plant (cell) water balance, it will be possible explicitly or implicitly to examine many aspects of plant function, plant performance, and stress responses. Nuclear magnetic resonance imaging (MRI) techniques are now available that allow studying plant hydraulics on different length scales within intact plants. The information within MRI images can be manipulated in such a way that cell compartment size, water membrane permeability, water cell-to-cell transport, and xylem and phloem flow hydraulics are obtained in addition to anatomical information. These techniques are non-destructive and non-invasive and can be used to study the dynamics of plant water relations and water transport, for example, as a function of environmental (stress) conditions. An overview of NMR and MRI methods to measure such information is presented and hardware solutions for minimal invasive intact plant MRI are discussed.     

Dynamic studies of phloem and xylein flow in fully differentiated plants by fast nuclear-magnetic-resonance microimaging

Summary A fast nuclear-magnetic-resonance imaging method was developed in order to measure simultaneously and quantitatively the water flow velocities in the xylem and the phloem of intact and transpiring plants. Due to technical improvements a temporal resolution of 7 min could be reached and flow measurements could be performed over a time course of 12–30 h. The novel method was applied to the hypocotyl of 35– to 40-day-old, leafy plants ofRicinus communis which were subjected to different light-dark regimes. The results showed that the xylem flow velocities and the xylem volume flow responded immediately to light on-off changes. Upon illumination the flow velocity and the volume flow increased as expected in respect to literature. In contrast, the phloem flow velocity did not change in response to the light-dark regimes. Interestingly, though, the volume flow in the phloem increased during darkness. These findings can be explained by assuming that the conducting area of the phloem becomes enlarged during the dark period due to opening of sieve pores.     

Most Water in the Tomato Truss Is Imported through the Xylem,Not the Phloem: A Nuclear Magnetic Resonance Flow Imaging Study

In this study, we demonstrate nuclear magnetic resonance flow imaging of xylem and phloem transport toward a developing tomato (Solanum lycopersicum) truss. During an 8-week period of growth, we measured phloem and xylem fluxes in the truss stalk, aiming to distinguish the contributions of the two transport tissues and draw up a balance between influx and efflux. It is commonly estimated that about 90% of the water reaches the fruit by the phloem and the remaining 10% by the xylem. The xylem is thought to become dysfunctional at an early stage of fruit development. However, our results do not corroborate these findings. On the contrary, we found that xylem transport into the truss remained functional throughout the 8 weeks of growth. During that time, at least 75% of the net influx into the fruit occurred through the external xylem and about 25% via the perimedullary region, which contains both phloem and xylem. About one-half of the net influx was lost due to evaporation. Halfway through truss development, a xylem backflow appeared. As the truss matured, the percentage of xylem water that circulated into the truss and out again increased in comparison with the net uptake, but no net loss of water from the truss was observed. The circulation of xylem water continued even after the fruits and pedicels were removed. This indicates that neither of them was involved in generating or conducting the circulation of sap. Only when the main axis of the peduncle was cut back did the circulation stop.Fruits are terminal organs that depend completely on long-distance transport to supply them with sugars and water for growth. Water is imported by means of both the xylem and the phloem, whereas sugars are only imported by means of the phloem. Fruits have to compete for water with the rest of the plant, and for that reason, xylem influx is expected to be sensitive to changes in plant water potential. Xylem influx into fruits may thus be lower during the day and higher during the night. When in the apoplast the water potential is especially low, for instance, when the plant is transpiring a lot of water during a hot day, fruits may even experience a xylem efflux and lose water to the vegetative parts of the plant (Johnson et al., 1992; Guichard et al., 2005). It has been suggested that in several species, in order to reduce the sensitivity of fruits to changes in plant water status, during fruit development the xylem connection between fruit and plant is reduced or even severed (Findlay et al., 1987; Lang, 1990; Creasy et al., 1993; Lang and Ryan, 1994; van Ieperen et al., 2003; Drazeta et al., 2004). In contrast to the xylem, the phloem is expected to be relatively insensitive to diurnal changes in water potential (Ehret and Ho, 1986; Ho et al., 1987). For instance, in the main stem of a number of plants, the phloem was found not to respond to diurnal differences in plant water status, whereas the xylem did (Peuke et al., 2001; Windt et al., 2006).The tomato (Solanum lycopersicum) plant has been the subject of many studies dealing with long-distance transport to fruits and has been chosen as a model system in this study as well. It has been estimated that in tomato fruits, about 80% to 90% of the influx of sap takes place by means of the phloem (Ho et al., 1987; Plaut et al., 2004; Guichard et al., 2005). It has been proposed that the low xylem contribution is due to the presence of some form of restriction in the xylem connection between plant and fruit, possibly in the knuckle (Lee, 1989; van Ieperen et al., 2003). Despite the expected low xylem contribution and limited conductivity of the xylem connection between plant and fruit, fruits have been shown to exhibit a diurnal pattern of growth. In most cases, fruits have been observed to grow fastest at night (Lee, 1989; Grange, 1995; van de Sanden and Uittien, 1995; Guichard et al., 2005). The opposite has been found to occur as well (Ehret and Ho, 1986; Pearce et al., 1993), but in these cases, the faster daytime growth was probably caused by a low diurnal stress environment. In a number of studies, even an efflux of xylem sap and fruit shrinkage during the day was reported (Johnson et al., 1992; Leonardi et al., 1999, 2000). It has been proposed that, if the phloem and xylem operate under different diurnal cycles or if their relative contributions can be modified in any way by adjusting the environmental conditions in a greenhouse, it might become possible to control and regulate fruit yield as well as fruit quality and taste.Considering the importance of fruit for the world''s food production, surprisingly little is known about the dynamics of sap flow to fruits. Since the conception of the cohesion tension theory (Dixon and Joly, 1894) and the Munch pressure flow hypothesis (Münch, 1930), there has been a decent theoretical understanding of the basic forces that govern phloem and xylem flow. It has already been attempted to apply this understanding to model fruit growth for a variety of fruits and applications (e.g. Daudet et al., 2002). However, many of the parameters that are needed to model long-distance transport to fruits are currently outside of experimental reach. First, little is known about the pressure and water potential gradients that drive flow to fruits. The xylem and the phloem are extremely sensitive to invasive experimentation and are easily disturbed, and the water potentials in the fruits’ symplast and apoplast are difficult to assess. Second, it is not clear whether xylem and phloem sap only enters the fruit (unidirectional flow), or if return flow is possible as well, and if it is, under which conditions it may occur. As the results of this study show, NMR flow imaging can provide answers to these important questions.

Estimating Long-Distance Transport to Fruits

So far, the most important methods to estimate xylem and phloem influx in fruits have been the subtractive method (Lang and Thorpe, 1989) and the mineral accumulation method (Ho et al., 1987). In the subtractive method, the contribution of xylem and phloem are estimated by heat girdling the pedicel (fruit stalk) of a fruit. Heat girdling destroys the sieve tubes, stopping phloem influx, while the xylem is assumed to remain intact and functional. By comparing the growth of nongirdled fruits to that of girdled fruits, the phloem contribution can be estimated. The most critical assumption in this method is that the xylem sap flow is not affected by heat girdling. However, the validity of this assumption is not evident. First, because xylem and phloem flow to fruits are coupled. Xylem influx is driven by a water potential difference between the xylem and the fruit symplast, which is maintained by osmotically active compounds (sugars), which in turn are imported by means of the phloem. Fishman et al. (2001) showed that the coupling between phloem and xylem influx could give rise to significant errors when using the pedicel girdling technique. A second reason is that heat girdling may profoundly affect xylem function. The xylem tissue may apparently escape heat girdling unscathed, as demonstrated by Guichard et al. (2005), but if the surrounding cells are damaged, it is not unlikely that functional damage will occur. For instance, it has been proposed that the cells that surround the xylem protect it against embolisms by preventing the entry of air (Hacke and Sperry, 2001). Van Ieperen et al. (2003) found that in the tomato pedicel, the abscission zone is the site of highest xylem resistance and that only a few xylem conduits traverse it. If an obstruction would occur in these conduits, either by embolisms or by particles of debris, it could significantly affect xylem resistance and have large implications for xylem transport to the fruit.In the second method, the mineral composition of the fruit is used to estimate the relative xylem and phloem contribution. Ho et al. (1987) measured calcium accumulation, net water import, and fruit respiration in tomato fruits. The xylem contribution to fruit growth was then estimated based on a number of assumptions: (1) the calcium content of phloem sap can be neglected compared to that of xylem sap; (2) the calcium content of xylem sap is similar to that measured in root stump exudate; and (3) xylem backflow from fruits does not occur. However, in view of current knowledge, the first and third assumptions are questionable. Calcium is used in signal transduction and as such is known to be present in the phloem. The question is, in what concentration. In phloem sap exudate of castor bean (Ricinus communis) and eucalyptus (Eucalyptus globulus), calcium concentrations have been found that were about 66% and 25% of the concentration in the root stump exudate, respectively (Pate et al., 1998; Peuke et al., 2006). In Banksia prionotes, the calcium concentration in phloem exudate was even found to be 10 times higher than that of the xylem sap (Pate and Jeschke, 1995). It should be noted that in these studies phloem sap was harvested by cutting. This may have elicited a wounding response, causing elevated calcium levels in the phloem (Knoblauch et al., 2001). Still, we argue that these findings illustrate that the calcium concentration in the phloem cannot be assumed to be negligible, especially when the majority of influx of sap is thought to take place via the phloem. The assumption that backflow does not take place also may not hold. In a number of studies, backflow from tomato fruits has already been observed, especially under summer conditions or high vapor deficit (Johnson et al., 1992; Leonardi et al., 1999, 2000; Guichard et al., 2005). The subtractive and the mineral accumulation method thus are likely to be subject to large systematic errors. Better methods to estimate or measure long-distance transport to fruits are needed.

NMR Flow Imaging

Over the last 10 years, it has been demonstrated that NMR flow imaging can provide an excellent tool to measure xylem and phloem transport (Van As, 2007). NMR flow imaging does not only give information about the average flow velocity, such as heat pulse based methods do, but gives access to all properties of the flowing water, such as the flow conducting area, the distribution of flow velocities, and the volume flow, all on a per pixel basis (Scheenen et al., 2000b). So far, studies have been conducted measuring flow in the stem of a variety of plants, ranging from castor bean seedlings (Köckenberger et al., 1997) to fully developed tomato, castor bean, and tobacco (Nicotiana tabacum) plants, and a small poplar tree (Populus spp.; Windt et al., 2006). The technique has been used to study the diurnal variation in long-distance transport (Peuke et al., 2001; Windt et al., 2006), the effects of cold girdling (Peuke et al., 2006), and xylem embolism repair (Scheenen et al., 2007) and has been used as a reference technique to provide detailed velocity maps for comparison with different heat pulse methods (e.g. Helfter et al., 2007; D. Chavarro, C.W. Windt, M.W. Lubczynski, J. Roy, and H. Van As, unpublished data). These studies have in common that flow was only measured in the main stem of the plant. This is a convenient place to do flow imaging for a variety of reasons. In comparison with other flow-conducting structures in the plant, the stem is large, sturdy, and stable. It conducts the largest fluxes, and the xylem and phloem can be easily distinguished on the basis of their direction of flow. These properties make imaging xylem and phloem transport relatively easy.

Aims and Research Questions

In this study, we used NMR flow imaging to measure long-distance transport to fruits. As a model plant, tomato was chosen. The anatomy of the tomato truss, as well as the dimensions of the magnetic resonance imaging (MRI) device and its components, made it impossible to image the pedicel of a single fruit. The pedicels were too short and too close together to fit them with the radio frequency (RF) coil that is needed for MRI. For this reason, we chose to perform flow imaging on the peduncle of tomato, measuring the transport toward the entire developing truss. After fitting the plant in the imager, it was impossible to remove the plant without damaging it. The plant was therefore left in the imager and allowed to grow there for 8 weeks. In this period, we continuously monitored long-distance transport into the truss, aiming to answer the following questions: (1) can xylem and phloem flow into the truss be visualized and distinguished; (2) what transport tissues conduct sap into the truss during truss development; (3) is phloem and xylem transport into the truss unidirectional, or does backflow occur; and (4) can NMR flow imaging be used to draw up a quantitative balance of xylem and phloem influx into the truss?     

Simultaneous measurement of water flow velocity and solute transport in xylem and phloem of adult plants of Ricinus communis over a daily time course by nuclear magnetic resonance spectrometry

A new method for simultaneously quantifying rates of flow in xylem and phloem using the FLASH imaging capabilities of nuclear magnetic resonance (NMR) spectrometry was applied in this study. The method has a time resolution of up to 4 min (for the xylem) and was used to measure the velocity of flows in phloem and xylem for periods of several hours to days. For the first time, diurnal time course measurements of flow velocities and apparent volume flows in phloem and xylem in the hypocotyl of 40‐d‐old Ricinus communis L were obtained. Additional data on gas exchange and the chemical composition of leaves, xylem and phloem sap were used to assess the role of leaves as sinks for xylem sap and sources for phloem. The velocity in the phloem (0·250 ± 0·004 mm s^?1) was constant over a full day and not notably affected by the light/dark cycle. Sucrose was loaded into the phloem and transported at night, owing to degradation of starch accumulated during the day. Concentrations of solutes in the phloem were generally less during the night than during the day but varied little within either the day or night. In contrast to the phloem, flow velocities in the xylem were about 1·6‐fold higher in the light (0·401 ± 0·004 mm s^?1) than in the dark (0·255 ± 0·003 mm s^?1) and volume flow varied commensurately. Larger delays were observed in changes to xylem flow velocity with variation in light than in gas exchange. The relative rates of solute transport during day and night were estimated on the basis of relative flow and solute concentrations in xylem and phloem. In general, changes in relative flow rates were compensated for by changes in solute concentration during the daily light/dark cycle. However, the major solutes (K⁺, NO₃^?) varied appreciably in relative concentrations. Hence the regulation of loading into transport systems seems to be more important to the overall process of solute transport than do changes in mass flow. Due to transport behaviour, the chemical composition of leaves varied during the day only with regard to starch and soluble carbohydrates.     

MRI of long-distance water transport: a comparison of the phloem and xylem flow characteristics and dynamics in poplar, castor bean, tomato and tobacco

We used dedicated magnetic resonance imaging (MRI) equipment and methods to study phloem and xylem transport in large potted plants. Quantitative flow profiles were obtained on a per-pixel basis, giving parameter maps of velocity, flow-conducting area and volume flow (flux). The diurnal xylem and phloem flow dynamics in poplar, castor bean, tomato and tobacco were compared. In poplar, clear diurnal differences in phloem flow profile were found, but phloem flux remained constant. In tomato, only small diurnal differences in flow profile were observed. In castor bean and tobacco, phloem flow remained unchanged. In all plants, xylem flow profiles showed large diurnal variation. Decreases in xylem flux were accompanied by a decrease in velocity and flow-conducting area. The diurnal changes in flow-conducting area of phloem and xylem could not be explained by pressure-dependent elastic changes in conduit diameter. The phloem to xylem flux ratio reflects what fraction of xylem water is used for phloem transport (Münch's counterflow). This ratio was large at night for poplar (0.19), castor bean (0.37) and tobacco (0.55), but low in tomato (0.04). The differences in phloem flow velocity between the four species, as well as within a diurnal cycle, were remarkably small (0.25-0.40 mm s(-1)). We hypothesize that upper and lower bounds for phloem flow velocity may exist: when phloem flow velocity is too high, parietal organelles may be stripped away from sieve tube walls; when sap flow is too slow or is highly variable, phloem-borne signalling could become unpredictable.     

Non invasive imaging of water flow in plants by NMR microscopy

Summary Water flow in the stems of the horsetailEquisetum hyemale, the flowering plantStachys sylvatica and the mossDendroligotrichum dendroides was observed non-invasively using NMR microscopy. A Pulsed Gradient Spin Echo sequence using a single phase encoding step was used. We demonstrate by this method that water flow inEquisetum occurs in the carinal canals and in the xylem vessels of the vascular tissue ofStachys and in the central water-onducting strand ofDendroligotrichum.Abbreviations CPU central processing unit - ID internal diameter - NMR nuclear magnetic resonance - RF radio frequency - TS transverse section     

Functional imaging of plants by magnetic resonance experiments.

Microimaging based on magnetic resonance is an experimental technique that can provide a unique view of a variety of plant physiological processes. Particularly interesting applications include investigations of water movement and spatially resolved studies of the transport and accumulation of labelled molecules in intact plant tissue. Some of the fundamental principles of nuclear and electron magnetic resonance microimaging are explained here and the potential of these techniques is shown using several representative examples.     

A closed‐form solution for steady‐state coupled phloem/xylem flow using the Lambert‐W function

A closed‐form solution for steady‐state coupled phloem/xylem flow is presented. This incorporates the basic Münch flow model of phloem transport, the cohesion model of xylem flow, and local variation in the xylem water potential and lateral water flow along the transport pathway. Use of the Lambert‐W function allows this solution to be obtained under much more general and realistic conditions than has previously been possible. Variation in phloem resistance (i.e. viscosity) with solute concentration, and deviations from the Van't Hoff expression for osmotic potential are included. It is shown that the model predictions match those of the equilibrium solution of a numerical time‐dependent model based upon the same mechanistic assumptions. The effect of xylem flow upon phloem flow can readily be calculated, which has not been possible in any previous analytical model. It is also shown how this new analytical solution can handle multiple sources and sinks within a complex architecture, and can describe competition between sinks. The model provides new insights into Münch flow by explicitly including interactions with xylem flow and water potential in the closed‐form solution, and is expected to be useful as a component part of larger numerical models of entire plants.     

Nuclear magnetic resonance microscopy ofAncistrocladus heyneanus

Summary The tropical lianaAncistrocladus heyneanus, which is known for its biologically active naphthylisoquinoline alkaloids, has been studied by nuclear magnetic resonance (NMR) microscopy for the first time. The spatial resolution of the cross-sectional NMR images was of the order of 20 m. Quantitative NMR relaxation time images of the root and the shoot show great contrast between different tissue regions. In addition, we observed the regional distribution of chemical compounds inAncistrocladus heyneanus by chemical-shift NMR microscopy. The NMR imaging results were compared with light and fluorescence microscopic images and reveal the excellent tissue characterization using NMR technology.Abbreviations NMR nuclear magnetic resonance - CSI chemical-shift magnetic resonance imaging - FOV field of view - TE echo time - TR repetition time     

Dynamic studies of phloem and xylein flow in fully differentiated plants by fast nuclear-magnetic-resonance microimaging

A fast nuclear-magnetic-resonance imaging method was developed in order to measure simultaneously and quantitatively the water flow velocities in the xylem and the phloem of intact and transpiring plants. Due to technical improvements a temporal resolution of 7 min could be reached and flow measurements could be performed over a time course of 12-30 h. The novel method was applied to the hypocotyl of 35- to 40-day-old, leafy plants of Ricinus communis which were subjected to different light-dark regimes. The results showed that the xylem flow velocities and the xylem volume flow responded immediately to light on-off changes. Upon illumination the flow velocity and the volume flow increased as expected in respect to literature. In contrast, the phloem flow velocity did not change in response to the light-dark regimes. Interestingly, though, the volume flow in the phloem increased during darkness. These findings can be explained by assuming that the conducting area of the phloem becomes enlarged during the dark period due to opening of sieve pores.     

Effects of cold‐girdling on flows in the transport phloem in Ricinus communis: is mass flow inhibited?

The effects of cold girdling of the transport phloem at the hypocotyl of Ricinus communis on solute and water transport were investigated. Effects on the chemical composition of saps of phloem and xylem as well as of stem tissue were studied by conventional techniques and the water flow in the phloem was investigated by NMR imaging. Cold girdling reduced the concentration of sucrose but not that of inorganic solutes or amino acids in phloem saps. The possibility that cold treatment inhibited the retrieval of sucrose into the phloem, following leaching from the sieve tubes along a chemical gradient is discussed. Leaching of other solutes did not occur, as a result of missing promoting gradients in stem tissue. Following 3 d of cold girdling, sugar concentration increased and starch was synthesized and accumulated in stem tissue above the cold girdling region and along the cold-treated phloem pathway due to leaching of sugars from the phloem. Only in the very first period of cold girdling (<15-30 min) was mass flow inhibited, but recovered in the rest of cold treatment period to values similar to the control period before and the recovery period after the cold treatment. It is concluded that cold treatment affected phloem transport through two independent and reversible processes: (1) a permanent leaching of sucrose from the phloem stem without normal retrieval during cold treatment, and (2) a short-term inhibition of mass flow at the beginning of cold treatment, possibly involving P proteins. Possible further mechanisms for reversible inhibition of water flow are discussed.     

Phloem as Capacitor: Radial Transfer of Water into Xylem of Tree Stems Occurs via Symplastic Transport in Ray Parenchyma

The transfer of water from phloem into xylem is thought to mitigate increasing hydraulic tension in the vascular system of trees during the diel cycle of transpiration. Although a putative plant function, to date there is no direct evidence of such water transfer or the contributing pathways. Here, we trace the radial flow of water from the phloem into the xylem and investigate its diel variation. Introducing a fluorescent dye (0.1% [w/w] fluorescein) into the phloem water of the tree species Eucalyptus saligna allowed localization of the dye in phloem and xylem tissues using confocal laser scanning microscopy. Our results show that the majority of water transferred between the two tissues is facilitated via the symplast of horizontal ray parenchyma cells. The method also permitted assessment of the radial transfer of water during the diel cycle, where changes in water potential gradients between phloem and xylem determine the extent and direction of radial transfer. When injected during the morning, when xylem water potential rapidly declined, fluorescein was translocated, on average, farther into mature xylem (447 ± 188 µm) compared with nighttime, when xylem water potential was close to zero (155 ± 42 µm). These findings provide empirical evidence to support theoretical predictions of the role of phloem-xylem water transfer in the hydraulic functioning of plants. This method enables investigation of the role of phloem tissue as a dynamic capacitor for water storage and transfer and its contribution toward the maintenance of the functional integrity of xylem in trees.Physiological and hydraulic functioning of the two long-distance transport systems in trees, xylem and phloem, have intrigued plant researchers for more than a century. Since the pioneering work of Dixon and Joly (1895; cohesion-tension theory for xylem) and Münch (1930; pressure flow hypothesis for phloem), the majority of studies have investigated these systems independently of each other. Although the work of Stout and Hoagland (1939) as well as Biddulph and Markle (1944) laid the foundation for the physiological nexus between xylem and phloem, it is only recently that we have begun to understand the importance of the hydraulic nexus (Hölttä et al., 2006, 2009; Sevanto et al., 2011, 2014). Processes related to both nexus occur in parallel, and here the term physiological nexus covers all metabolite exchange, including the bidirectional flow of amino acids, minerals, and carbohydrates (Wardlaw, 1974; Ferrier et al., 1975; Pfautsch et al., 2009, 2015; De Schepper et al., 2013; for review, see van Bel, 1990, 2003). The term hydraulic nexus refers to the function of phloem as a capacitor, where water stored in phloem moves into xylem vessels to maintain the integrity of the transpiration stream (Zweifel et al., 2000, and refs. therein). Throughout this article, we use the term phloem collectively for cells that make up the transport phloem of woody plants (including sieve element/companion cell complexes, parenchyma cells, etc.), as opposed to collection and release phloem tissue, which differ in structure and function. Transport phloem is characterized by the retention of “high hydrostatic pressure by retrieval of leaked osmotica accompanied by water flux” (Patrick, 2013).According to the cohesion-tension theory, water in xylem vessels is constantly under tension and moves in a metastable state from roots to leaves along a hydrostatic pressure gradient. Depending on both the availability of soil moisture and the vapor pressure deficit of the atmosphere, this tension can exceed the cohesive forces that bind water molecules, resulting in the formation of a gas void that, after expanding, can lead to rupture of the water column inside individual vessels (termed cavitation; Zimmermann, 1983). Once cavitated, vessels become dysfunctional, and the transport of water and nutrients to leaves declines. However, water stored in woody tissues of trees can be mobilized to alleviate the risk of cavitation, and recent theory suggests that both water and carbohydrates from phloem may aid in the reversal of vessel embolism (i.e. air intrusion), although the evidence is indirect (Salleo et al., 2009; Brodersen et al., 2010; Nardini et al., 2011).All parts of plants have a water storage capacity (symplastic and apoplastic), and this capacitance increases with tree size and age (Phillips et al., 2003). The ability to mobilize stored water varies according to the force required to drag it out of storage (Holbrook, 1995). One-half century ago, Reynolds (1965) highlighted the importance of the volume of internally stored water to support the transpiration of trees. Since then, studies have quantified the fraction of stored water in total daily transpiration for a range of tree species. This fraction varies between 2% and 20% (Tyree and Yang, 1990; Čermák et al., 2007, and refs. therein; Barnard et al., 2011; Pfautsch and Adams, 2013) and is generally smaller in angiosperms compared with gymnosperms, where a maximum fraction of 50% was reported for Pinus sylvestris (Waring et al., 1979). Given that the daily water use of large adult trees can easily reach 260 to 380 L (Čermák et al., 2007; Pfautsch et al., 2011), considerable volumes of stored water must be mobilized from and restored back into capacitors on a daily basis. Remobilization of stored water also can prolong stomatal opening and thus increase carbon gain (Goldstein et al., 1998).The volume of stored water released depends on the elasticity of the storage tissue, its connectivity to xylem vessels, and the gradient of water potential (ψ) between the storage tissue and vessels. Cells with elastic walls represent ideal capacitors because they can change their volume as a consequence of small changes in ψ. Thus, phloem, cambium, and juvenile xylem cells are well suited for water storage and release (Yang and Tyree, 1992; Zweifel et al., 2014). The magnitude of release and refill of stored water in trees can be approximated by separately measuring the change in thickness of phloem and xylem during a diel cycle using high-precision dendrometers (Zweifel et al., 2014). Whitehead and Jarvis (1981) have calculated that around 90% of the diurnal change in stem radius can be attributed to changes in the water content of cambial and phloem tissues. To date, it is commonly accepted that tree bark, independent of the wood below, swells during the night and shrinks during the day (Zweifel et al., 2000), reflecting the water flow dynamics that characterize the dynamic exchange of water between phloem and xylem.Phloem and xylem tissues are separated by rows of intermediary cambial cells. However, depending on the species, phloem and xylem are connected through uniseriate or multiseriate strands of radially aligned ray parenchyma cells, commonly termed wood rays. These rays have been shown to be capable of symplastic water transport through plasmodesmata (Höll, 1975). Based on measurements of radial conductance, Sevanto et al. (2011) suggested that aquaporins also might be involved in the radial transfer of water. Both theoretical and experimental approaches have been developed to better understand the dynamic exchange of water between xylem and phloem. Hölttä et al. (2006, 2009) developed a model based on Münch’s hypothesis and included a term that represents the hydraulic connection between the two tissue types. Through incorporating this term, model outputs suggest the occurrence of a constant exchange of water between xylem and phloem along gradients of ψ. However, some authors suggested that changes in ψ of xylem alone might be insufficient to account for the observed diurnal shrinkage and swelling of bark (Sevanto et al., 2003). Along this line of argument, loading and unloading of carbohydrates in phloem tissue has been suggested to further impact the radial transfer of water and associated changes in bark thickness (Mencuccini et al., 2013).Nevertheless, to date, all approaches remain indirect, and the routes of water transfer between phloem and xylem have yet to be determined. Here, we present a technique that enables the visualization of water transfer from phloem to xylem tissues and resolves the apoplastic and symplastic pathways and cell types. The method involves the injection of an aqueous solution that contains fluorescent dye into phloem followed by analyses of woody tissues using confocal laser scanning microscopy. We assess the effectiveness of three different dyes to stain possible transfer pathways. We also introduce dye during different time intervals of the diel transpiration cycle to test the effect of predicted dynamic changes in ψ between phloem and xylem on the transfer processes. We hypothesized that radial transfer of water would be most pronounced during periods where conditions of the hydraulic nexus between phloem and xylem differ the most. These differences are expected during high rates of transpiration that cause a steep decline in xylem ψ, commonly observed during morning hours. We use leaf water potential (ψ_L) and high-precision dendrometer measurements to identify relevant time intervals. The simultaneous assessment of ψ_L and the independent diameter fluctuation of phloem and xylem may provide empirical evidence for the role of phloem as a water storage capacitor that helps mitigate increasing tension in the transpiration stream.     

Seasonal dynamics of phloem and xylem formation in silver fir and Norway spruce as affected by drought

The dynamics of phloem growth ring formation in silver fir (Abies alba Mill.) and Norway spruce (Picea abies Karst.) at different sites in Slovenia during the droughty growing season of 2003 was studied. We also determined the timing of cambial activity, xylem and phloem formation, and counted the number of cells in the completed phloem and xylem growth rings. Light microscopy of cross-sections revealed that cambial activity started on the phloem and xylem side simultaneously at all three plots. However, prior to this, 1–2 layers of phloem derivatives near the cambium were differentiated without previous divisions. The structure of the early phloem was similar in silver fir and Norway spruce. Differences in the number of late phloem cells were found among sites. Phloem growth rings were the widest in Norway spruce growing at the lowland site. In all investigated trees, the cambium produced 5–12 times more xylem cells than phloem ones. In addition, the variability in the number of cells in the 2003 growth ring around the stem circumference of the same tree and among different trees was higher on the xylem side than on the phloem side. Phloem formation is presumably less dependent on environmental factors but is more internally driven than xylem formation.     

Transpiration rate affects the mobility of foliar-applied boron in Ricinus communis L. cv. Impala

In plant species not containing polyols, boron (B) is regarded as practically phloem immobile. This has been explained by the high membrane permeability of boric acid (BA) resulting in a rapid efflux out of the phloem and re-transport into the leaf in the xylem. The present study investigated how the xylem flow rate affects the phloem mobility of foliar-applied BA in Ricinus communis L. cv. Impala. Xylem flow rates were varied by exposure of the canopy to different levels of relative humidity (RH). In seedlings with severed hypocotyls, i.e. without xylem flow, B was highly mobile. In intact seedlings and plants, the degree of mobility and the within-plant distribution of B were strongly RH-dependent. At RH of 70% or above, up to 16–24% of the B was translocated to other plant parts, whereas at lower RH no significant movement of B was detected. Only at an intermediate RH (70–80%), did leaf-applied B accumulate in roots. At 100% RH, B transport in the xylem was significantly increased, suggesting that the build up of root pressure induced the recycling of phloem delivered B from roots to shoots. These results indicate that in R. communis phloem B mobility is not constant, but strongly affected by transpiration rates.     

Developmental changes in the diurnal water budget of the grape berry exposed to water deficits

The diurnal water budget of developing grape (Vitis vinifera L.) berries was evaluated before and after the onset of fruit ripening (veraison). The diameter of individual berries of potted ‘Zinfandel’ and ‘Cabernet Sauvignon’ grapevines was measured continuously with electronic displacement transducers over 24 h periods under controlled environmental conditions, and leaf water status was determined by the pressure chamber technique. For well-watered vines, daytime contraction was much less during ripening (after veraison) than before ripening. Daytime contraction was reduced by restricting berry or shoot transpiration, with the larger effect being shoot transpiration pre-veraison and berry transpiration post-veraison. The contributions of the pedicel xylem and phloem as well as berry transpiration to the net diurnal water budget of the fruit were estimated by eliminating phloem or phloem and xylem pathways. Berry transpiration was significant and comprised the bulk of water outflow for the berry both before and after veraison. A nearly exclusive role for the xylem in water transport into the berry was evident during pre-veraison development, but the phloem was clearly dominant in the post-veraison water budget. Daytime contraction was very sensitive to plant water status before veraison but was remarkably insensitive to changes in plant water status after veraison. This transition is attributed to an increased phloem inflow and a partial discontinuity in berry xylem during ripening.     

Evidence supporting a non-phloem source of water for export of solutes in the xylem of soybean root nodules

The vascular anatomy of soybean nodules [Glycine max (L.) Merr.] suggests that export of solutes in the xylem should be dependent on influx of water in the phloem. However, after severing of stem xylem and phloem by shoot decapitation, export of ureides from nodules continued at an approximately linear rate for 5h. This result was obtained with decapitated roots remaining in the sand medium, but when roots were disturbed by removal from the rooting medium prior to shoot decapitation, export of ureides from nodules was greatly reduced. Stem exudate could not be collected from disturbed roots, indicating that flow in the root xylem had ceased. Thus, ureide export from nodules appeared to be dependent on a continuation of flow in the root xylem. When seedlings were fed a mixture of ³H₂O and ¹⁴C-inulin for periods of 14–21 min, nodules had higher ³H/¹⁴C ratios than roots from which they were detached. The combined results are not consistent with the proposal that export of nitrogenous compounds from nodules is dependent on import of water via the phloem. The results do support the view that a portion of the water required for xylem export from soybean nodules is supplied via a symplastic route from root cortex to nodule cortex to the nodule vascular apoplast.     

Morphological changes and transversal growth kinetics along the apical meristem in the pericycle cell types of the onion adventitious root

Summary In the apical meristem of the adventitious root ofAllium cepa, all pericycle cells show a marked increase in cross-sectional area between 400 and 800 m behind the tip, this transversal growth ceasing in the 1,200–1,400 m interval. However, different pericycle cell types (opposite xylem, intervening and opposite phloem) show different transversal growth kinetics. Along the meristem, the opposite xylem cells are narrower than both the intervening and opposite phloem cells, and these latter are similar in cross-sectional area. Another relevant difference is in the polarity of the transversal expansion, which in turn gives rise to changes in cell shape. In fact, in apical most portions of the meristem, the opposite phloem cells mainly expand tangentially, while the intervening cells do so radially, and the opposite xylem cells undergo a similar tangential and radial expansion. By contrast, in basal most portions of the meristem, radial expansion continues in the opposite phloem cells when it has ceased in the intervening cells. These latter expand tangentially once again when tangential expansion has ceased in the opposite phloem cells. As a consequence of this transversal growth, the opposite xylem cells, which can initiate lateral root primordia, retain their isodiametric transversal shape along the meristem, whereas the transversal shape of the opposite phloem and intervening cells initially changes from isodiametric to markedly enlarged tangentially (opposite phloem) or radially (intervening), after which both cell types tend to become more rounded in shape.     

Modeling xylem and phloem water flows in trees according to cohesion theory and Münch hypothesis

Water and solute flows in the coupled system of xylem and phloem were modeled together with predictions for xylem and whole stem diameter changes. With the model we could produce water circulation between xylem and phloem as presented by the Münch hypothesis. Viscosity was modeled as an explicit function of solute concentration and this was found to vary the resistance of the phloem sap flow by many orders of magnitude in the possible physiological range of sap concentrations. Also, the sensitivity of the predicted phloem translocation to changes in the boundary conditions and parameters such as sugar loading, transpiration, and hydraulic conductivity were studied. The system was found to be quite sensitive to the sugar-loading rate, as too high sugar concentration, (approximately 7 MPa) would cause phloem translocation to be irreversibly hindered and soon totally blocked due to accumulation of sugar at the top of the phloem and the consequent rise in the viscosity of the phloem sap. Too low sugar loading rate, on the other hand, would not induce a sufficient axial water pressure gradient. The model also revealed the existence of Münch “counter flow”, i.e., xylem water flow in the absence of transpiration resulting from water circulation between the xylem and phloem. Modeled diameter changes of the stem were found to be compatible with actual stem diameter measurements from earlier studies. The diurnal diameter variation of the whole stem was approximately 0.1 mm of which the xylem constituted approximately one-third.     

Detection and characterization of acidic compartments (vacuoles) in Chlorella vulgaris 11h cells by 31P-in vivo NMR spectroscopy and cytochemical techniques

Acidic inorganic phosphate (Pi) pool (pH around 6) was detected besides the cytoplasmic pool in intact cells of Chlorella vulgaris 11h by ³¹P-in vivo nuclear magnetic resonance (NMR) spectroscopy. It was characterized as acidic compartments (vacuoles) in combination with the cytochemical technique; staining the cells with neutral red and chloroquine which are known as basic reagents specifically accumulated in acidic compartments. Under various conditions, the results obtained with the cytochemical methods were well correlated with those obtained from in vivo NMR spectra; the vacuoles were well developed in the cells at the stationary growth phase where the acidic Pi signal was detected. In contrast, cells at the logarithmic phase in which no acidic Pi signal was detected contained only smaller vesicles that accumulated these basic reagents. No acidic compartment was detected by both cytochemical technique and ³¹P-NMR spectroscopy when the cells were treated with NH₄OH. The vacuolar pH was lowered by the anaerobic treatment of the cells in the presence of glucose, while it was not affected by the external pH during the preincubation ranging from 3 to 10. Possible vacuolar functions in unicellular algae especially with respect to intracellular pH regulation are discussed.Non-standard abbreviations EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MDP methylene diphosphonic acid - NMR nuelear magnetic resonance - PCA perchloric acid - PCV packed cell volume - Pi inorganic phosphate - Pic sytoplasmic inorganic phosphate - Piv vacuolar inorganic phosphate - ppm parts per million - SP sugar phosphates - TCA trichloroacetic acid     

Xylem ingestion by winged aphids

When aphids and their host plant are incorporated in a DC electrical circuit, phloem and xylem ingestion register as separate waveforms of the electrical penetration graph (EPG) signal. Aphids are primarily phloem feeders; xylem ingestion is seldom reported but can be induced experimentally by fasting the insects in desiccating conditions. In experiments with the black bean aphid, Aphis fabae Scop., young winged (alate) and unwinged (apterous) virginoparous adults were collected from their natal host plants (broad bean, Vicia faba L.) and allowed 3-h continuous EPG-recorded access to V. faba seedlings. Several aphids (47% of both morphs) showed ingestion from phloem sieve elements. Alate aphids also showed frequent xylem ingestion (60% of individuals), but no apterous aphids exhibited this activity. The EPG technique involves attachment of a fine gold wire electrode to each insect, a process that may affect normal behaviour at the plant surface. However, when the technique was modified to monitor the stylet activities of freely-settled aphids, high levels of xylem ingestion by alates were also recorded. The results suggest that the developmental physiology of winged aphids somehow predisposes them to xylem ingestion, possibly as a result of dehydration during the teneral period. Alate aphids may reduce their weight by fasting before take-off, giving aerodynamic benefits, but making rehydration, via xylem uptake, a priority following plant contact.