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. 2013 Feb 7;111(4):723–730. doi: 10.1093/aob/mct014

Visualization of embolism formation in the xylem of liana stems using neutron radiography

Christian Tötzke 1,2,*, Tatiana Miranda 3, Wilfried Konrad 3, Julien Gout 1, Nikolay Kardjilov 1, Martin Dawson 1,4, Ingo Manke 1, Anita Roth-Nebelsick 3,5
PMCID: PMC3605950  PMID: 23393096

Abstract

Background and Aims

Cold neutron radiography was applied to directly observe embolism in conduits of liana stems with the aim to evaluate the suitability of this method for studying embolism formation and repair. Potential advantages of this method are a principally non-invasive imaging approach with low energy dose compared with synchrotron X-ray radiation, a good spatial and temporal resolution, and the possibility to observe the entire volume of stem portions with a length of several centimetres at one time.

Methods

Complete and cut stems of Adenia lobata, Aristolochia macrophylla and Parthenocissus tricuspidata were radiographed at the neutron imaging facility CONRAD at the Helmholtz-Zentrum Berlin für Materialien und Energie, with each measurement cycle lasting several hours. Low attenuation gas spaces were separated from the high attenuation (water-containing) plant tissue using image processing.

Key results

Severe cuts into the stem were necessary to induce embolism. The formation and temporal course of an embolism event could then be successfully observed in individual conduits. It was found that complete emptying of a vessel with a diameter of 100 µm required a time interval of 4 min. Furthermore, dehydration of the whole stem section could be monitored via decreasing attenuation of the neutrons.

Conclusions

The results suggest that cold neutron radiography represents a useful tool for studying water relations in plant stems that has the potential to complement other non-invasive methods.

Keywords: Xylem, plant water transport, embolism, neutron radiography, Adenia lobata, Aristolochia macrophylla, Parthenocissus tricuspidata.

INTRODUCTION

Plants employ a fascinating mechanism that facilitates water transport against the gravitational potential. According to the cohesion–tension theory, evaporation at the nanoporous walls of mesophyll cells and subsequent transpiration induces capillary forces driving the long-distance water transport without additional metabolic energy input. Water is conducted by the xylem tissue, a transport structure that basically consists of dead capillary-like cells (Tyree and Zimmermann, 2002).While transpiration at the leaves generates suction that pulls the water through the plant, pressure inside the xylem tissue can fall below vapour saturation pressure or may even become negative, i.e. the water is under tension. This thermodynamically metastable state favours the (unlimited) growth of microscopic gas bubbles, leading to embolisms (Tyree and Sperry, 1989). As a consequence, the affected xylem vessels are blocked and lose their capacity for water transport.

Plants have developed various safety strategies to ensure the reliability of their water management even under most unfavourable conditions. A central safety feature is the active control of the transpiration rate by stomata. Specialized guard cells regulate the stomata opening to avoid unnecessary or excessive leaf transpiration that would otherwise lead to a critical drop of xylem pressure (Brodribb et al., 2003). Nonetheless, there are situations, e.g. during periods of persistent drought, where xylem pressure can fall under a critical threshold below which embolism events are promoted. For this reason, the xylem has a dedicated hydraulic architecture to cope with these situations. Individual xylem vessels are interconnected to form a complex transport network with a high degree of flow path redundancy. Hydraulic junctions between the xylem members, so-called pits, act like biologic safety valves. They allow the passage of liquid water but form barriers for the propagation of gas–liquid interfaces, thus preventing the uncontrolled spread of gas emboli (Crombie et al., 1985; Sperry, 2003; Wheeler et al., 2005).

Due to the great redundancy of flow paths represented by the high number of single interconnected conduits, plants can easily compensate the dysfunction of individual vessels. However, with increasing numbers of embolisms the overall conductance of the xylem decreases. Conduits can remain functional for just a few days or for >100 years (Tyree and Zimmermann, 2002), but the first step towards a state of permanent dysfunction probably involves a state of embolism. Repair mechanisms for embolized vessels could, therefore, have an important stabilizing effect on the plant water supply.

There is already theoretical and experimental evidence that embolized xylem vessels can be repaired. Even the refill of empty conduits under negative pressure is possible, as has been shown in various studies (Tyree et al., 1999; Hacke and Sperry, 2003; Salleo et al., 2004). Living tissues in the xylem, rays and xylem parenchyma can play an important role in the refilling process, particularly during the much-debated embolism repair under negative pressure (Vesala et al., 2003; Salleo et al., 2006; Nardini et al., 2011). Most studies on embolism repair apply conventional experimental methods, e.g. the measurement of hydraulic conductivity whose temporal changes demonstrate vessel embolism or refill indirectly (Sperry et al., 1987; Zwieniecki and Holbrook, 1998; Cochard et al., 2001; Vogt, 2001). These methods require destructive sampling and therefore make in vivo measurements impossible.

To understand refilling mechanisms thoroughly, direct observations of embolism formation and/or refill are desirable, i.e. in vivo visualization of formation, change and disappearance of gas spaces inside conduits. Recently, the rapid technological progress of imaging techniques has offered novel experimental approaches for studies of embolism formation and refilling. One method to realize in vivo observations of embolism formation is nuclear magnetic resonance imaging (MRI) (Holbrook et al., 2001; Clearwater and Clark, 2003; Windt et al., 2006; Scheenen et al., 2007). The resolution (<100 µm) is sufficiently high to distinguish individual xylem vessels in plants with very wide vessels (D > 100 µm), e.g. some liana species (Ewers et al., 1990). However, the low temporal resolution (17 min per acquisition) hampers the observation of dynamic changes of embolism or refill. Further limitations to MRI studies of xylem transport are given by the physical restrictions on plant samples which must be fitted into the MRI-magnet bore and that represents a laborious and complicated task for larger specimens (Clearwater and Clark, 2003). Moreover, the high magnetic field around the sample makes the use of additional electronic devices during the observation difficult.

Synchrotron X-ray imaging is an alternative technique that was successfully used to visualize embolism formation and refilling of vessels within bamboo and rice leaves and within intact grapevine stems (Lee and Kim, 2008; Brodersen et al., 2010; Kim and Lee, 2010). The advantage of this method is the high spatial (2 µm) and temporal resolution (t = 10 ms) which allows the visualization of small morphological details and dynamic changes at the same time (Banhart, 2008; Banhart et al., 2010), as has also been recently demonstrated by the observation of vessel refill in grapevine stems (Brodersen et al., 2010). A drawback of high resolution X-ray synchrotron imaging for the observation of embolisms in elongated xylem vessels is the small field of view. Depending on the chosen image resolution the field of view dimension is only a few millimetres, whereas elongated xylem vessels have typical lengths of several centimetres.

In this study an alternative method based on cold neutron radiography was applied as a novel approach to study embolism events. Neutrons have unique properties for imaging: they are weakly attenuated by many metals like aluminium but, on the other hand, they are strongly attenuated by H-containing compounds. In contrast to X-rays the attenuation coefficient does not increase proportionally with the atomic number as neutrons do not interact with the electrons in the atomic shell. The high sensitivity for water along with the great penetration potential for metals make neutrons very attractive to investigate water distributions in various systems (Schillinger et al., 2000; Lehmann, 2008; Manke et al., 2009; Banhart et al., 2010; Kardjilov et al., 2011c). Neutron imaging techniques have proven to be a very useful tool to study water distribution in technical systems, e.g. fuel cells (Bellows et al., 1999; Lehmann et al., 2009; Schröder et al., 2009; Hussey et al., 2010; Schröder et al., 2010; Manke et al., 2011; Tötzke et al., 2011). Furthermore, it has also already been successfully applied to study different phenomena of the water transport in plants (Nakanishi and Matsubayashi, 1997; Arif et al., 2006; Oswald et al., 2008; Matsushima et al., 2009a, b; Nakanishi, 2009). The spatial resolution is about 20–50 µm (Boillat et al., 2008, 2010; Hickner et al., 2008; Williams et al., 2012) which is appropriate to study the embolism formation in wider vessels.

MATERIALS AND METHODS

Neutron-imaging technique

The experiments were performed at the CONRAD measuring station located at the end of a curved neutron guide, which faces the cold-neutron source of the BER-II research reactor at the Helmholtz-Zentrum Berlin für Materialien und Energie (HZB). The beam is passed through a collimation system with a circular aperture of 2 cm providing neutrons with wavelengths between about 2 and 12 Å (peaking at 3·1 Å) and a neutron flux of approx. 1·6 × 107 cm−2 s−1 (Kardjilov et al., 2009, 2011b).

A 10 × 10 cm2 lithium fluoride scintillator screen with a thickness of 50 µm was used to convert impinging neutrons into visible light. The absorption image formed on the scintillator screen is projected onto the 16-bit 2048 × 2048 CCD chip of the camera (Andor DW436N-BV) via a mirror and a 105 mm-focus Nikon camera lens (Kardjilov et al., 2011a). Total image acquisition time was 24 s; 20-s exposure plus 4-s readout.

Image processing

The visualization of embolism events by neutron imaging requires appropriate image processing. This includes dark-field and flat-field correction of the neutron absorption images to eliminate the CCD dark current signal and the inhomogeneities of the beam profile, respectively. Moreover, a median filter was applied over five consecutive images to reduce the image noise. The initial image of each sequence served as a reference image for the calculation of quotient images. This approach enhances the sensitivity for the detection of any sample changes occurring during the experiment and allows estimating variations in the sample water content quantitatively. The normalization procedure is based on the Lambert–Beer law which describes the beam attenuation as a function of sample thickness and composition (Banhart, 2008). At time t = n, the intensity In(x, y) of a neutron beam passing the sample with a thickness δn in z-direction is given by

graphic file with name mct014eqn1.jpg (1)

Where μ (x, y, z) denotes the distribution of the local attenuation coefficient in the sample and I0,n (x, y) the initial beam intensity distribution.

To evaluate eqn (1) further, we assume (a) that the plant sample can be partitioned into a water fraction and a ‘dry biomass’ fraction and (b) that the variation of the respective attenuation coefficients μwater and μdry with respect to the x-, y-, z-co-ordinates is negligible. Thus, eqn (1) can be approximated as

graphic file with name mct014eqn2.jpg (2)

δwater (x, y) and δdry (x, y) refer to the hypothetical thickness of water and biomass fraction. Taking the initial image (t = start) as a reference, image normalization of the nth image amounts to

graphic file with name mct014eqn3.jpg (3)

As we further assume δdry and the beam intensity I0 to remain constant during the experiment, eqn (3) simplifies to

graphic file with name mct014eqn4.jpg (4)

Defining Δδwater,n (x, y) = δwater,n (x, y) – δwater,start (x, y) and rearranging eqn (4) one obtains

graphic file with name mct014eqn5.jpg (5)

This expression connects the difference of water thickness in the z-direction between times t = start and t = n at any position (x, y) in the detector plane with the corresponding measured beam intensities. Thus, the normalization procedure transforms the grey value of a pixel at position (x, y) into the thickness variation of water at this position.

Experimental set-up

The aim of these experiments was the detection and visualization of embolism events and potential repair mechanisms in the xylem of liana plants. Some general advantages of this plant type for radiographic experiments are the flexible stems which can be fixed to a supporting structure and positioned into the field of view. Moreover, the xylem tissue contains extraordinarily wide vessels with diameters up to >100 µm which meets the resolution capability of the imaging method. The focus of the experiments was on three different liana species: Adenia lobata, Aristolochia macrophylla and Parthenocissus tricuspidata. The plants were cultivated in flower pots in the Botanical Garden of the Universität Tübingen. At the time of the experiment, the plants were between 6 months and 12 months old. Additionally, stems of outdoor plants of P. tricuspidata were used that climbed the façade of one of the HZB buildings.

To provide basic physiological conditions appropriate to observe embolism formation and subsequent repair we organized the radiographic experiments in the following manner. (a) During the first period the liana plants were subjected to water stress to induce embolism events. This was achieved by stopping watering for 2–4 d prior to the experiments. (b) Additional invasive measures such as partial cutting of xylem tissue were applied to enhance water stress and, thus, the probability of embolism events. (c) Then, plant samples were watered and left in the dark for several hours, thus providing favourable physiological conditions for embolism repair. Neutron radiographic images were taken throughout the whole sequence of steps, except for a short interruption needed for watering.

The general experimental set-up is illustrated in Fig. 1. The liana plants were mounted on an aluminium frame and placed in the neutron beam. The stem sections under investigation were arranged within the field of view and firmly fixed to a perforated aluminium sheet which was positioned 1 cm in front of the scintillator screen. The stems were clamped to the perforated plate to prevent sample movement that would decrease image quality and accuracy of water quantification.

Fig. 1.

Fig. 1.

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General experimental set-up: (A) photograph of a liana plant (Parthenocissus tricuspidata) arranged in the measuring position; (B) detail showing the field of view of the neutron detector, stems are fixed with cable ties to a perforated aluminium sheet; (C) the same detail displayed as a (raw) neutron image.

A high-pressure sodium vapour lamp with an electrical power of 600 W was used to illuminate the plant samples in the measurement room. The average light intensity (PAR) at the leaf surface was about 50 µmol m−2 s−1 and the relative humidity was 30 %. The stomatal conductivity of leaves was measured with a Delta-T porometer of the type AP4. Parthencocissus tricuspidata showed mean values of 22·3 mmol m−2 s−1, Adenia lobata 19·8 mmol m−2 s−1 and Aristolochia macrophylla 23·7 mmol m−2 s−1.

RESULTS AND DISCUSSION

Testing the imaging method

In this study neutron radiography was applied as a novel method to visualize embolism formation and potential refilling. The suitability of the experimental set-up was tested in a preliminary experiment. The water ascent in cut liana stems was visualized using heavy water (D2O) as a contrast agent. Plant samples were arranged to fit into the field of view and fixed to a perforated aluminium sheet as described above. The stems were cut and submerged into a D2O reservoir. Upon illumination, the plant began to transpire water from its leaves, replacing increasing amounts of xylem water with heavy water. As D2O attenuates neutrons much less than H2O, the ascending D2O front in the xylem tissue could be tracked as a contrast change in the neutron images. The image sequence of Fig. 2 visualizes the D2O transport in cut stems of Aristolochia macrophylla and Parthenocissus tricuspidata. In fact, water and D2O are transported simultaneously. The water column inside the stem xylem is moving upwards and pulls the D2O column into the xylem vessels. The consecutive images are normalized with respect to the initial image of the sequence. The quotient pictures reveal the exchange of water for D2O over a 3-h period, illustrating the ascending water. Note the stems of both samples are cut in half to study the effect of severed vessels on the water balance of the tissue located above. For both samples, the image sequences prove that the D2O uptake takes place in both the intact and the injured parts of the stem. Apparently, lateral interchange, e.g. due to interconnectivity and tortuosity of vessels and diffusive transport, can compensate the loss of transport capacity in the transected vessels.

Fig. 2.

Fig. 2.

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Normalized neutron images showing D2O ascent in cut stems of Aristlochia macrophylla (left) and Parthenocissus tricuspidata (right). The displayed stem section starts approx. 10 cm above the submerged stem ends. t denotes the time elapsed after D2O watering. Note: stems of both samples are cut in half at the positions outlined by dashes in (B).

Transpiration rates inside the facility were always quite low, and amounted to about 20–40 mmol m−2 s−1. This was very probably due to the environmental conditions. Low transpiration rates were also observed elsewhere on study plants under similar conditions (Clearwater and Clark, 2003).

Observation of embolism formation

Preliminary neutron radiographic experiments showed that water stress caused by substrate desiccation, i.e. no watering of the pots over several days, failed to trigger embolism events in the considered plant species. Therefore, various methods were tested to intensify the water stress and to promote embolism, for instance, partial and complete stem cutting as well as exposure to ultrasonic waves. Stem cutting, however, turned out to be the most effective method. We will focus on selected results obtained by partial and complete stem transection.

A potted Adenia lobata plant was subjected to water stress by interrupting the daily watering procedure for 2 d. Before starting the radiographic measurement approximately half of the xylem tissue was cut in such a way that a complete wedge was removed from the stem exposing the cut area to the air.

Figure 3A displays a (flatfield- and darkfield-corrected) radiograph of the sample showing the general shape and structure of the stem. In Fig. 3B a detail of the stem section around the cut is displayed as a normalized image revealing changes of the stem water content occurring within 10 min after notching the stem. The bright rhomboid area surrounding the lesion turns up after cutting, intensifies and expands for 10 min before its size and intensity stabilizes. Obviously, the contrast change is the result of evaporative water loss of the plant tissue adjacent to the lesion that was exposed to air. The evolution of the bright shape is probably linked to the process of wound closure which counteracts evaporative loss of injured tissue. Wound closure seems to be completed after 10 min since no further significant contrast change is observed. However, no other changes were observed that could be attributed to embolism events. This is remarkable since the disconnection of a significant portion of the conducting tissue was expected to affect the water system substantially, thereby, leading to embolism. However, no embolism could be detected during several hours after the manipulation. Figure 3 demonstrates the robustness of the water transport despite stem injury. It proves that only a small stem region around the cut is affected by water loss, suggesting that wound closure quickly counteracts evaporative water loss of stem tissue.

Fig. 3.

Fig. 3.

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Radiograph of an Adenia lobata stem 10 min after cutting: (A) flatfield- and darkfield-corrected radiograph of the sample showing general shape and structure of the stem; (B) normalized detail revealing changes in stem water content; the grey scale displays the change in effective water thickness Δδwater in micrometres.

The overall difficulty to provoke embolism may be attributed to the conditions in the experimental chamber, particularly the low-light conditions affecting stomatal conductance. Embolism tends to be markedly reduced under these conditions. However, there are also other reports that show that it can be difficult to promote embolism during non-invasive imaging. Kim and Lee (2010) dehydrated plants of Oryza sativa until severe wilting occurred to observe embolism via X-ray imaging methods. However, no embolism event could be detected unless the leaves were cut to generate air/water interfaces inside the conduits. Also no embolism could be provoked in intact Ripogonum scandens plants during MRI imaging and it was necessary to place the cut end of a severed stem into a PEG solution with high osmotic potential (Clearwater and Clark, 2003). The conditions under which embolism events set in is strongly species dependent and various taxa appear to avoid embolism (Vogt, 2001). Remarkable in our studies is the circumstance that none of the liana species considered showed embolism with intact stems, even if the stem was severely damaged by a wedge cut with a large area of stem tissue being exposed to air.

Since the partial transection of the xylem was not sufficient to trigger embolisms in either A. lobata or in other species (data not shown), the procedure was changed and in another experiment a liana stem (Parthenocissus tricuspidata) was completely cut through and subsequently radiographed for 1·5 h. While leaves were illuminated to initiate transpiration and the build-up of water stress, no water was supplied to the cut end of the stem. The sequence of normalized images in Fig. 4 documents the development of the stem water status. Within the first 36 min the intensity was quite stable for the whole stem section except for the stem edges which brighten (Fig. 4A). This effect continues throughout the whole experiment and can be explained by the contraction of elastic portions of the xylem tissue. This contraction is caused by the decreasing water potential and leads to a slight shrinkage of the liana stem. A coupling between stem water content and stem diameter was also recently demonstrated by using MRI (De Schepper et al., 2012). However, after 38 min a fibre-like bright structure appears along the stem axis (indicated by the arrow in Fig. 4B). In the subsequent images (Fig. 4C and D, t = 40 and 42 min) the shape and intensity of the bright structure further increases and stays almost constant for the rest of the experiment. The shape of the affected region as well as its rapid development suggests that xylem embolism has occurred.

Fig. 4.

Fig. 4.

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Normalized image sequence showing the embolism formation in P. tricuspidata. The same stem section is displayed at different times after cutting: (A) after 36 min prior to embolism formation, bright stem edges indicate stem shrinking (dashed arrows); (B) at 38 min, a bright fibre-like structure appears (solid arrows) and intensifies for 4 min (see C and D: after 40 min and 42 min) indicating embolism formation. Note: images are shown as negatives to allow for easier identification of contrast changes.

As described in the Methods, the normalization procedure allows a quantitative estimation of the change of water thickness which is transmitted by the neutron beam. The grey value of each pixel denotes the reduction in water thickness in the z-direction at the detector position (in μm). In Fig. 5 the variation of the mean water thickness during the experiment was evaluated for a stem section that was obviously affected by an embolism (area marked red in Fig. 5B) and, for comparison, a section located next to the embolized vessel (area marked white in Fig. 5B).

Fig. 5.

Fig. 5.

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(A) Plots of the mean water thickness change for the stem regions marked red and white in (B). Data points 1, 2, 3 and 4 correspond to the labels in Fig. 4A, B, C and D, respectively. (C) Schematic representation of δwater at t = 0, t = 36 min and t = 42 min, due to stem shrinkage and embolism. Note that the diagram is not drawn to scale and that the position of the embolized vessel is speculative with respect to the z-direction.

According to Fig. 5A, the evolution of the water thickness in the stem section marked red (see Fig. 5B) is characterized by two phases (t = 0 min … 38 min and t = 42 min … 85 min) of continuous and approximately linear thickness reduction interrupted by a short intermediate phase of rapid water loss between t = 38 min and t = 42 min (see also data points 1, 2, 3 in Fig. 5). The early and late phases of moderate thickness reduction can be explained by the contraction of elastic tissue caused by the falling plant water potential (equivalent to increasing water stress). This amounts to shrinkage of the stem diameter at a rate of about 1·7 µm min−1, which corresponds to the increasingly bright stem edges observed in Fig. 4. Between data points 1 and 3 the water thickness reduction rate (about 25 µm min−1) is one order of magnitude higher than before and afterwards. During these 4 min, the water thickness is reduced by about 100 µm. Since typical vessel diameter of this species range between 50 and 100 µm, it is most likely that an embolism was formed in a xylem vessel. This interpretation is corroborated by the sudden emergence of the fibre-like structure observed in the normalized images of Fig. 4. For comparison, the evolution of the water thickness is also plotted for a stem area next to the embolized vessel (see area marked white in Fig. 5B). While in the first 36 min the effective water thickness decreases in both regions simultaneously at the same rate, the rapid water loss between t = 38 and t = 42 min is only detected in the stem section affected by the embolism. In contrast, in the stem section next to it the approximately linear trend of water thickness reduction occurring continuously throughout the measured period is caused by the contraction of elastic tissue due to increasing water stress.

Behaviour after watering

In the second part of the experiment it was attempted to observe rehydration and, ideally, refilling processes in the stem of P. tricuspidata. To provide appropriate rehydration conditions the plant was watered by submerging the end of the stem into a water reservoir after cutting. Furthermore, the plant illumination was switched off to minimize leaf transpiration. Radiographs were taken for another 2·5 h; the initial image of this series served as a reference image for the normalization procedure.

It is to be expected that rehydration leads to an increase in the thickness of the xylem tissue and, therefore, the water column within it. The evolution of the liana stem is documented in Fig. 6 by a sequence of normalized images including the evaluation of the mean change in water thickness for two selected stem regions. The sequence of images shows that with elapsing time the whole stem area brightens, i.e. the stem tissue does not rehydrate but continues to lose water. Moreover, several additional embolisms form in the xylem which is reflected by the emergence of bright fibre-like structures, e.g. at t = 12 min and t = 122 min after watering (Fig. 6B, D). The embolism events correlate with a sudden reduction in the water thickness in the affected stem areas (cf. Fig. 6).

Fig. 6.

Fig. 6.

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Plot of the mean water thickness change after watering for the stem regions marked in images (B) and (D), respectively. Note: images are shown as negatives to allow an easier identification of contrast changes.

The appearance of additional embolism events indicates that no rehydration via the cut surface occurred. Possibly the stem region above the cut was already too dehydrated for water uptake. Consequently, the drought stress in the plant increased further, entailing additional contraction of elastic tissue. The shrinking rate of about 0·9 µm min−1 is much lower compared with that observed before watering (1·7 µm min−1). Probably, the turning-off of the illumination leads to stomatal closure, which reduced leaf transpiration significantly. Unaffected by the lower shrinking rate due to contraction of living stem tissue, embolism events show the same dynamic behaviour, i.e. reducing the water thickness by 50–100 µm within a few minutes.

Conclusions

The observations made within this study show that neutron radiography is a suitable tool for direct monitoring of gas spaces within the xylem by detecting changes in transmitted water thickness in whole plant stems. The temporal resolution of the observed embolism events allowed for quite exact determination of the discharge time of a single vessel; this was completed in our study in about 4 min. To our knowledge, this was the first attempt to use neutron imaging for the visualization of embolism formation in plants. One of the advantages of neutron radiography is that a considerable portion of a stem can be observed with a quite acceptable temporal resolution. The results of this study demonstrate the great potential of neutron imaging for biological studies of water transport phenomena in plants. The approach should principally allow observation of gas spaces in the xylem of intact plants under suitable conditions. Further work is necessary to address the conditions and circumstances under which embolism formation and repair occur. It is suggested that this method represents a very promising tool for gaining more information on the in vivo processes occurring during embolism and vessel refill.

ACKNOWLEDGMENTS

The research activities were partly funded by the German Federal Ministry for Education and Science (BMBF) under grant number 01RB0713A (BIONA).

LITERATURE CITED

  1. Arif M, Jacobson DL, Hussey DS. Neutron imaging study of the water transport in operating fuel cells. FY 2006 Annual Progress Report. 2006:875–877. [Google Scholar]
  2. Banhart J. Advanced tomographic methods in materials research and engineering. Oxford: Oxford University Press; 2008. [Google Scholar]
  3. Banhart J, Borbely A, Dzieciol K, et al. X-ray and neutron imaging: complementary techniques for materials science and engineering. International Journal of Materials Research. 2010;101:1069–1079. [Google Scholar]
  4. Bellows RJ, Lin MY, Arif M, Thompson AK, Jacobson D. Neutron imaging technique for in situ measurement of water transport gradients within nafion in polymer electrolyte fuel cells. Journal of the Electrochemical Society. 1999;146:1099–1103. [Google Scholar]
  5. Boillat P, Kramer D, Seyfang BC, Frei G, et al. In situ observation of the water distribution across a PEFC using high resolution neutron radiography. Electrochemistry Communications. 2008;10:546–550. [Google Scholar]
  6. Boillat P, Frei G, Lehmann EH, Scherer GG, Wokaun A. Neutron imaging resolution improvements optimized for fuel cell applications. Electrochemical and Solid-State Letters. 2010;13:B25–B27. [Google Scholar]
  7. Brodersen CR, McElrone AJ, Choat B, Matthews MA, Shackel KA. The dynamics of embolism repair in xylem: in vivo visualizations using high-resolution computed tomography. Plant Physiology. 2010;154:1088–1095. doi: 10.1104/pp.110.162396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brodribb TJ, Holbrook NM, Edwards EJ, Gutiérrez MV. Relations between stomatal closure, leaf turgor and xylem vulnerability in eight tropical dry forest trees. Plant, Cell & Environment. 2003;26:443–450. [Google Scholar]
  9. Clearwater MJ, Clark CJ. In vivo magnetic resonance imaging of xylem vessel contents in woody lianas. Plant, Cell & Environment. 2003;26:1205–1214. [Google Scholar]
  10. Cochard H, Lemoine D, Améglio T, Granier A. Mechanisms of xylem recovery from winter embolism in Fagus sylvatica. Tree Physiology. 2001;21:27–33. doi: 10.1093/treephys/21.1.27. [DOI] [PubMed] [Google Scholar]
  11. Crombie DS, Hipkins MF, Milburn JA. Gas penetration of pit membranes in the xylem of Rhododendron as the cause of acoustically detectable sap cavitation. Australian Journal of Plant Physiology. 1985;12:445–453. [Google Scholar]
  12. De Schepper V, van Dusschoten D, Copini P, Jahnke S, Steppe K. MRI links stem water content to stem diameter variations in transpiring trees. Journal of Experimental Botany. 2012;63:2645–2653. doi: 10.1093/jxb/err445. [DOI] [PubMed] [Google Scholar]
  13. Ewers FW, Fisher JB, Chiu ST. A survey of vessel dimensions in stems of tropical lianas and other growth forms. Oecologia. 1990;84:544–552. doi: 10.1007/BF00328172. [DOI] [PubMed] [Google Scholar]
  14. Hacke UG, Sperry JS. Limits to xylem refilling under negative pressure in Laurus nobilis and Acer negundo. Plant, Cell & Environment. 2003;26:303–311. [Google Scholar]
  15. Hickner MA, Siegel NP, Chen KS, Hussey DS, Jacobson DL, Arif M. In situ high-resolution neutron radiography of cross-sectional liquid water profiles in proton exchange membrane fuel cells. Journal of The Electrochemical Society. 2008;155:B427–B434. [Google Scholar]
  16. Holbrook NM, Ahrens ET, Burns MJ, Zwieniecki MA. In vivo observation of cavitation and embolism repair using magnetic resonance imaging. Plant Physiology. 2001;126:27–31. doi: 10.1104/pp.126.1.27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hussey DS, Jacobson DL, Arif M, Coakley KJ, Vecchia DF. In situ fuel cell water metrology at the NIST Neutron Imaging Facility. Journal of Fuel Cell Science and Technology. 2010;7(021024) http://dx.doi.org/10.1115/1.3007898 . [Google Scholar]
  18. Kardjilov N, Hilger A, Manke I, Strobl M, Dawson M, Banhart J. New trends in neutron imaging. Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment. 2009;605:13–15. [Google Scholar]
  19. Kardjilov N, Dawson M, Hilger A, et al. A highly adaptive detector system for high resolution neutron imaging. Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment. 2011a;651:95–99. [Google Scholar]
  20. Kardjilov N, Hilger A, Manke I, Strobl M, Dawson M, Williams S, Banhart J. Neutron tomography instrument CONRAD at HZB. Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment. 2011b;651:47–52. [Google Scholar]
  21. Kardjilov N, Manke I, Hilger A, Strobl M, Banhart J. Neutron imaging in materials science. Materials Today. 2011c;14:248–256. [Google Scholar]
  22. Kim HK, Lee SJ. Synchroton X-ray imaging for nondestructive monitoring of sap flow dynamics through xylem vessel elements in rice leaves. New Phytologist. 2010;188:1085–1098. doi: 10.1111/j.1469-8137.2010.03424.x. [DOI] [PubMed] [Google Scholar]
  23. Lee S-J, Kim Y. In vivo visualization of the water-refilling process in xylem vessels using X-ray micro-imaging. Annals of Botany. 2008;101:595–602. doi: 10.1093/aob/mcm312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lehmann E. Recent improvements in the methodology of neutron imaging. Pramana Journal of Physics. 2008;71:653–661. [Google Scholar]
  25. Lehmann EH, Boillat P, Scherrer G, Frei G. Fuel cell studies with neutrons at the PSI's neutron imaging facilities. Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment. 2009;605:123–126. [Google Scholar]
  26. Manke I, Strobl M, Kardjilov N, et al. Investigation of soot sediments in particulate filters and engine components. Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment. 2009;610:622–626. [Google Scholar]
  27. Manke I, Markotter H, Totzke C, et al. Investigation of energy-relevant materials with synchrotron X-rays and neutrons. Advanced Engineering Materials. 2011;13:712–729. [Google Scholar]
  28. Matsushima U, Herppich WB, Kardjilov N, Graf W, Hilger A, Manke I. Estimation of water flow velocity in small plants using cold neutron imaging with D2O tracer. Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment. 2009a;605:146–149. [Google Scholar]
  29. Matsushima U, Kardjilov N, Hilger A, Manke I, Shono H, Herppich WB. Visualization of water usage and photosynthetic activity of street trees exposed to 2 ppm of SO2: a combined evaluation by cold neutron and chlorophyll fluorescence imaging. Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment. 2009b;605:185–187. [Google Scholar]
  30. Nakanishi TM. Neutron imaging applied to plant physiology: neutron imaging and applications. In: Bilheux HZ, McGreevy R, Anderson IS, editors. New York, NY: Springer; 2009. pp. 305–317. [Google Scholar]
  31. Nakanishi TM, Matsubayashi M. Nondestructive water imaging by neutron beam analysis in living plants. Journal of Plant Physiology. 1997;151:442–445. [Google Scholar]
  32. Nardini A, Lo Gullo MA, Salleo S. Refilling embolized xylem conduits: is it a matter of phloem unloading? Plant Science. 2011;180:604–611. doi: 10.1016/j.plantsci.2010.12.011. [DOI] [PubMed] [Google Scholar]
  33. Oswald SE, Menon M, Carminati A, Vontobel P, Lehmann E, Schulin R. Quantitative imaging of infiltration, root growth, and root water uptake via neutron radiography. Vadose Zone Journal. 2008;7:1035–1047. [Google Scholar]
  34. Salleo S, Lo Gullo MA, Trifiló P, Nardini A. New evidence for a role of vessel-associated cells and phloem in the rapid xylem-refilling of cavitated stems of Laurus nobilis L. Plant, Cell & Environment. 2004;27:1065–1076. [Google Scholar]
  35. Salleo S, Trifiló P, Lo Gullo MA. Phloem as a possible major determinant of rapid cavitation reversal in stems of Laurus nobilis (laurel) Functional Plant Biology. 2006;33:1063–1074. doi: 10.1071/FP06149. [DOI] [PubMed] [Google Scholar]
  36. Scheenen TWJ, Vergeldt FJ, Heemskerk AM, Van As H. Intact plant magnetic resonance imaging to study dynamics in long-distance sap flow and flow-conducting surface area. Plant Physiology. 2007;144:1157–1165. doi: 10.1104/pp.106.089250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schillinger B, Lehmann E, Vontobel P. 3D neutron computed tomography: requirements and applications. Physica B: Condensed Matter. 2000;276–278:59–62. [Google Scholar]
  38. Schröder A, Wippermann K, Mergel J, et al. Combined local current distribution measurements and high resolution neutron radiography of operating Direct Methanol Fuel Cells. Electrochemistry Communications. 2009;11:1606–1609. [Google Scholar]
  39. Schröder A, Wippermann K, Lehnert W, et al. The influence of gas diffusion layer wettability on direct methanol fuel cell performance: a combined local current distribution and high resolution neutron radiography study. Journal of Power Sources. 2010;195:4765–4771. [Google Scholar]
  40. Sperry JS. Evolution of water transport and xylem structure. International Journal of Plant Science. 2003;164:S115–S127. [Google Scholar]
  41. Sperry JS, Holbrook NM, Zimmermann MH, Tyree MT. Spring filling of xylem vessels in wild grapevine. Plant Physiology. 1987;83:414–417. doi: 10.1104/pp.83.2.414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tötzke C, Manke I, Arlt T, et al. High resolution large area neutron imaging detector for fuel cell research. Journal of Power Sources. 2011;196:4631–4637. [Google Scholar]
  43. Tyree MT, Sperry JS. Vulnerability of xylem to cavitation and embolisms. Annual Review of Plant Physiology. 1989;40:19–38. [Google Scholar]
  44. Tyree MT, Zimmermann MH. Xylem structure and the ascent of sap. Berlin: Springer-Verlag; 2002. [Google Scholar]
  45. Tyree MT, Salleo S, Nardini A, Lo Gullo MA, Mosca R. Refilling of embolized vessels in young stems of laurel: do we need a new paradigm? Plant Physiology. 1999;120:11–21. doi: 10.1104/pp.120.1.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Vesala T, Hölttä T, Perämäki M, Nikinmaa E. Refilling of a hydraulically isolated xylem vessel: model calculations. Annals of Botany. 2003;91:419–428. doi: 10.1093/aob/mcg022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Vogt UK. Hydraulic vulnerability, vessel refilling, and seasonal courses of stem water potential of Sorbus aucuparia L. and Sambucus nigra L. Journal of Experimental Botany. 2001;52:1527–1536. doi: 10.1093/jexbot/52.360.1527. [DOI] [PubMed] [Google Scholar]
  48. Wheeler JK, Sperry JS, Hacke UG, Hoang N. Intervessel pitting and cavitation in woody Rosaceae and other vesselled plants: a basis for a safety versus efficiency trade-off in xylem transport. Plant, Cell & Environment. 2005;28:800–812. [Google Scholar]
  49. Williams SH, Hilger A, Kardjilov N, et al. Detection system for microimaging with neutron. Journal of Instrumentation. 2012;7:P02014. http://dx.doi.org/10.1088/1748-0221/7/02/P02014 . [Google Scholar]
  50. Windt CW, Vergeldt FJ, De Jager PA, Van As H. MRI of long-distance water transport: a comparison of the phloem and xylem flow characteristics and dynamics in poplar, castor bean, tomato and tobacco. Plant, Cell & Environment. 2006;29:1715–1729. doi: 10.1111/j.1365-3040.2006.01544.x. [DOI] [PubMed] [Google Scholar]
  51. Zwieniecki MA, Holbrook NM. Diurnal variation in xylem hydraulic conductivity in white ash (Fraxinus americana L.), red maple (Acer rubrum L.) and red spruce (Picea rubens Sarg.) Plant, Cell & Environment. 1998;21:1173–1180. [Google Scholar]

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