Possible causes for embolism repair in xylem

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Environmental and Experimental Botany 62 (2008) 139–144

Possible causes for embolism repair in xylem F.Y. Shen a,∗ , R. Guo b , Q. Sun b , R.F. Gao b , Y.B. Shen b , Z.Y. Zhang a a b

College of Science, Beijing Forestry University, Beijing 100083, PR China College of Biology, Beijing Forestry University, Beijing 100083, PR China

Received 18 February 2007; received in revised form 4 July 2007; accepted 30 July 2007

Abstract Wood sections of eight species of angiosperm and gymnosperm were made and observed under microscope. When a dehydrated section was rewet, the air inside its conduits contracted under the force of surface tension for several seconds to form elongated or spherical bubbles. The elongated bubbles in smaller conduits shortened till vanished. In addition, we also discorved that bubbles in larger conduits extended at first, then collapsed and disappeared; the bubbles outside conduits appeared gradualy or popped up in the field of view one after another; for some samples, they originated mainly from the cross sections of the wood rays. The smaller ones also collapsed and the larger ones grew up gradually. We suspected that air might transfer from the bubbles with short radii to those with large radii, both inside and outside conduits. The calculation of the amount of gas in all bubbles in a field of view supported our hypothesis. There are two possible mechanisms to explain the phenomena. First, based on the capillay equation, air can move from a smaller bubble to a larger one. Another reason is that the dissolving air from smaller bubbles can enter into the adjacent bubbles with larger curvature radii. Gas movement should obey the same rules in living plants. Therefore, we suggest that after cavitation events, instead of air moving from xylem into ambient atmosphere, two mechanisms could induce air to transfer from smaller conduits into larger conduits or the regions with lower pressures, leading the embolized conduits in the smaller conduits to repair. Furthermore, the differnce of values of contact angles in conduits might promote the refilling of embolism at lower xylem pressure. © 2007 Elsevier B.V. All rights reserved. Keywords: Bubble; Air movement; Air dissolution; Embolism repair; Xylem pressure

1. Introduction Following daily cavitation the refilling of embolized conduits can occur in both woody and herbaceous plants even when the water in neighboring conduits is under tension (Edwards et al., 1994; Salleo et al., 1996; McCully, 1999; Tyree et al., 1999). Thus, embolized conduits may transport water again. How the ability is recovered at lower xylem pressure is still under question. In this paper, the intention was to give explanations on the mechanism of embolism refilling. Borghetti et al. (1991) concluded that surface tension forces can be accounted for the refilling of embolism of small branch segments in Pinus sylvestris L. However, the refilling of seedling cannot be explained by capillary action. Sperry et al. (1987) suggested three possible mechanisms for the repair of cavitated conduits: condensation of water vapor, dissolution of air, and expulsion of air. Air dissolution from an embolized conduit into the surround-

∗

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0098-8472/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2007.07.015

ing water, which requires positive pressure, may play a major role for embolism refilling in daily life (Tyree and Zimmermann, 2002). Pickard (1989) predicted the process of air dissolution in water. He suggested that, (1) An embolus in a plant should be close to compositional equilibrium with the ambient atmosphere by the end of a day; and (2) If an embolus is compressed to as little as 2% above atmospheric pressure, it should be absorbed in 1000 s or less by sap. Considering the dissolution and movement of air, Tyree and Yang (1992) and Yang and Tyree (1992) conducted an experiment to study the collapse of a large number of emboli in the entire stem and gave theoretical analysis. Lewis et al. (1994) directly observed the formation of the emboli of Thuja occidentalis L. wood as samples dried, as well as the collapse of emboli after the samples were flooded with water under microscope. They suggested that as soon as a sample was rehydrated, the bubbles immediately contracted by surface tension from atmospheric pressure Po (101 kPa) to a little higher pressure (121 kPa). According to Henry’s and Fick’s laws, the gas in the bubbles would diffuse across water into ambient atmosphere. Based on the air dissolution hypothesis, Holbrook and Zwieniecki (1999) and Zwieniecki and Holbrook (2000)

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Table 1 The largest (or average) and smallest lumen diameter of conduits of wood species Wood species (gymnosperm and angiosperm)

Largest (l) or average (a) (␮m)

Platycladus orientalis L. Sabina chinensis (L.) Antoine Sabina chinensis cv. Kaizuca Ginkgo biloba L. Eucommia ulmoides Oliv. Cotinus coggygria Scop. Buxus megistophylla levl Acer truncatum Bunge

13(a) 11(a) 13(a) 17(a) 30(l) 60(l) 16(a) 35(a)

Smallest (␮m)

5 25 5 20

further suggested a mechanism to remove gas bubbles in isolated embolized vessels, which requires non-zero contact angle of water with the vessel wall. Following the work of Zwieniecki and Holbrook (2000), Vesala et al. (2003) proposed a model of water exudation into cylindrical vessels and of gas dissolution with the water pressure in the corner of an embolized conduit equal to the gas pressure under hydraulic isolation. This implies that the contact angle would be of 90◦ in xylem. However, the values of the contact angles of six species measured by Zwieniecki and Holbrook (2000) were 42–55◦ . Thus, the air dissolution hypothesis is not fully satisfied for embolism refilling at lower xylem pressure. Just as Zwieniecki and Holbrook (2000) pointed out that additional studies are needed to determine the hypothesis. Similar to Lewis et al. (1994) study, we conducted experiments using apical shoots of eight species of angiosperm and gymnosperm. Sections of the shoots of the species were made and observed under microscope when dry sections were rehydrated. Similar to Lewis et al. (1994), we observed that the elongated bubbles in smaller conduits contracted till vanished gradually. Besides, we also found that bubbles in larger conduits extended at first, then collapsed and disappeared; the bubbles outside conduits appeared gradualy or popped up in the field of view one after another. In this paper, first, we will describe our experiments in detail. Second, the findings will be illustrated with pictures we took during the experiments and possible explanations will be given. Third, our theory of embolism repair could be generalized to living plants and might be able to give the reasons for the embolism repair at lower xylem pressure. 2. Materials and methods Stem segments were cut from 3–7 year-old apical shoots of eight species of angiosperm and gymnosperm, growing on the campus of Beijing Forestry University. The average diameter and lumen diameter of the conduits of the eight wood species are shown in Table 1. After stem segments were cut and foliage was removed, they were trimmed to an average length of 2–3 cm. In order to soften the segments, we put them in hot water. Then they were sectioned with microtome into tangential sections of 40 or 60 ␮m thick, depending on the diameter of conduit. Since conduits were parallel with a section in longitude, we were able to keep

lots of complete vessels or tracheids in a sample. The sections were put into clear water, which were changed everyday to prevent mildewed sections. Before experiments they were trimmed again with razor blade into 10 mm in width and 20 mm in length. When the sections were kept in air for several minutes, hours, and even overnight, they would dehydrate and air would be sucked into their conduits. To make a sample a dry section was placed on a glass slide and covered with a coverslip of 22 mm × 22 mm. Two methods were used to rewet the dehydrated sections. First, a dehydrated sample was placed under a microscope 400× or a stereomicroscope, and was rewet by injecting water, which was held in air for several days to guarantee the eventual equilibrium of the air in atmosphere and the gas in the water. Second, a dehydrated section with slide and coverslip was fully submerged into a culture dish of water. A stereomicroscope at 144× was used to observe the refilling process. The room temperature ranged from 20 ◦ C in winter to 33 ◦ C in summer and the ambient RH ranged from 34% in winter to 78% in summer. The process was supposed to be isothermal. Videotapes and pictures were taken during the refilling process in order to help us make detailed observations. 3. Results The following descriptions were based on samples of Eucommia ulmoides Oliver. Similar observations were found with other species. First, when water was injected into a dehydrated section, some bubbles flashed in the field of view and the air in its vessels collapsed at once under the force of surface tension in 2 s to form elongated or spherical bubbles (Fig. 1) (for other species, the processes were all within several seconds). This was similar to the observation described in Lewis et al. (1994) study. Then the section became relatively stable. At the moment, we observed that some vessels became translucent and elongated bubbles (emboli) appeared in some large vessels. The length of the elongated bubbles was different. The longest bubble took up to 2–3 vessel elements and the smaller ones were spheres with radii less than their vessels’ radii. Extending a radius to be infinitesimal, we suspected that there might be tiny bubbles within some small vessels changing into translucent appearance. There were also many long dim thin bands in the frames, which represented the elongated bubbles in small vessels. We also observed several white bands, which might be damaged large vessels. When wood parenchyma rays were perpendicular to the surface of coverslip, we also saw clusters of their cross sections. Some of samples were among this type of wood rays perpendicular to surface. Afterward, the elongated bubbles in small vessels contracted till vanished gradually (the process was similar to Lewis et al. (1994) study). In addition, we had some new, interesting findings. The elongated bubbles in some large vessels extented at first, and then they began to collapse till disappear. For some samples with wood rays perpendicular to the surface of coverslip there were some round bubbles appearing or poping up in the

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Fig. 1. Evolution of the bubbles of a section of Eucommia ulmoides Oliver. The elongated bubbles in some large vessel elements extended at first, and then they collapsed till disappeared. There were some round bubbles appearing. They originated mainly from the cross section of the wood rays. The smaller ones collapsed, and the larger ones grew up gradually. The smaller vessels are too small to be shown clearly. From the microscope at 400×. Scale: 50 ␮m.

field of view one after another. They originated mainly from the cross sections of wood rays and a few of them came out from the joints of two large vessel elements or the damaged points of conduits. The smaller ones collapsed, and the larger ones grew up gradually (Fig. 1). As the diameter of the round bubbles became larger than the adjacent vessels, they were outside the vessels and between the coverslip and slide. Taken together, we concluded that the bubbles with short radii would collapse and those with long radii would extend both inside and outside conduits. The size of bubbles changed (increase or decrease) with different rate. Second, when a section with coverslip and slide was fully submerged into water, fewer gas bubbles floated off essentially, indicating that little gas escaped from the section into ambient atmosphere upon rewetting and almost all air remained within the section. For a while spherical bubbles appeared and became larger in quick succession. They were numerous and close to

each other under the coverslip. The larger ones expanded continually while the smaller ones contracted in size till vanished (Fig. 2). From the above observations, it is natural to conclude that air might transfer from the bubbles with shorter radii to those with larger radii, both inside and outside conduits. To prove our hypothesis, we calculated the amount of air in all bubbles in a field of view of a section during the rewetting process. We took Platycladus orientalis L. as an example. There was sufficient water between the slide and the coverslip so that the round bubbles outside conduits appeared as spheres. Elongated bubbles appeared as a cylinder with two semi spheres at both ends. According to the Ideal Gas Law PV = nRT and Eq. (1) (see next), the amount of gas (in molar) in both the elongated bubbles and the spherical bubbles within a field of view were calculated at different stage of rewetting (e.g., early,

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Fig. 2. Spherical bubbles outside conduits in a section of Platycladus orientalis L. They were close and numerous. The larger ones expanded continually while the smaller ones contracted in size till vanished. From the microscope at 32×. Scale: 0.3 mm. Table 2 The amount of air in bubbles in a field of view of Platycladus orientalis L. at different stage Dataset

Number of bubbles at early stage

Amount of gas within all bubbles at different stages (10−12 mol) Early stage

Midway

End

1

Elongated: 15 Spherical: 4 Total: 19

21.1 48.5 69.6

3.5 61.7 65.2

0 65.7 65.7

2

Elongated: 13 Spherical: 2 Total: 15

24.5 47.7 72.1

0.1 64.7 64.9

0 54.2 54.2

3

Elongated: 11 Spherical: 6 Total: 17

15.9 23.7 39.6

2.3 37.5 39.8

0 39.6 39.6

4

Elongated: 13 Spherical: 1 Total: 14

29.0 26.9 55.9

9.9 41.9 51.8

0 51.5 51.5

5

Elongated: 16 Spherical: 4 Total: 20

18.2 25.8 43.9

2.2 42.9 45.1

0 49.1 49.1

midway, and end). As can be seen from Table 2, the total amount of air in the elongated and spherical bubbles was similar across three stages. Therefore, we concluded that, (1) air transferred from bubbles in small conduits into bubbles in large conduits; and (2) air also transferred from bubbles in small conduits into wood rays at the cross section of which spherical bubbles outside conduits appeared or popped up. As a result, large bubbles expanded while small bubbles collapsed till vanished.

4. Disscusion 4.1. Explanations of gas movement Gas movement from one bubble to another could be explained in two ways. First, when two air bubbles in liquid connect to each other, air will move from the smaller bubble to the larger one. This is because when an air bubble with curvature radius of r is sub-

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merged in water with pressure of Pl , the gas pressure (Pg ) in the bubble is calculated as:

living plant the air pressure in an embolized conduit should be lower than the atmospheric pressure.

Pg = Pl + 2σ/r

4.3. Mechanisms for embolism repair of living plants

(1)

Here σ is surface tension. From the equation, the air pressure is always higher in a small bubble than in a large one. Therefore, air directly moves from the high pressure bubble to the low pressure bubble. Second, according to stastistical theory, Ward et al. (1986) first predicted that when there are two separated bubbles with different radii in water in a closed system, air in the smaller bubble can dissolve into the surrounding water, and then diffuses into the adjacent larger bubble. The theory has been supported by experiment. Both theories predicted that smaller bubble would collapse and larger bubble would expand. Our experiments should obey the same rules. In our experiments, the radii of wood rays of Eucommia ulmoides Oliver were longer than the radii of adjacent small vessels. Thus, the gas in the small conduits moved into the adjacent wood rays through ray-conduit pitting, causing the spherical bubbles at the cross-sections of wood rays to appear and enlarge continually till all elongated bubbles in small vessels vanished. The gas in smaller conduits also entered larger vessels, inducing the elongated bubbles in some large vessels to extend at first. Afterward, due to the pressure difference between large conduits and the spherical bubbles outside conduits, the gas moved from some larger vessels into the spherical bubbles outside conduits. Since the pressure differences among bubbles were not the same, the speeds of air transferring were different. That is, the size of bubbles changed in different rate. Taken together, two mechanisms caused gas movement observed in our experiments, which underlined the process of emboli repair in the conduits of our sections. 4.2. Pressures in cavitated conduits For embolism repair in living plants we should consider the pressure in a cavitated conduit. In our experiment sections were kept in air for several minutes, hours, and even overnight before rewetting. Air could be sucked into the cavitated conduits. However, upon rewetting little gas escaped from the sections. This implied that the amount of air in the conduits was nearly the same before and after the rewetting. Based on the Ideal Gas Law, if air under atmospheric pressure was fully sucked into a dehydrated conduit, surface tension would force an elongated air bubble in a conduit to take up 83% of its initial length after rewetting. From our videotapes we observed that there were bubbles in the conduits shorter than 83% of their initial length. Therefore, the air pressure in cavitated conduits should be lower than atmospheric pressure. The results are commensurate with Lewis et al. (1994) conclusion. In a living plant, the xylem is surrounded by phloem, cortex, and epidermis. It is more difficult for air to be sucked into its conduits than into the cavitated conduits of the sections in our experiments, which were only of 40 or 60 ␮m thick. Thus, in a

Two mechanisms described above can be extended to intact plants. Since gas movement is caused partially by air pressure difference, we should consider where gas goes during the refilling process. From the vulnerable curves (Tyree and Sperry, 1989; Vogt, 2003) it is known that the initial conductivity of each stem segment is below its maximum conductivity. This means that in a living plant some conduits are embolized by gas even at early morning. In the above text we have pointed out that it is very difficult for atmosphere to be sucked into conduits in living plants. Therefore, the pressures in all embolized conduits may be at lower than atmospheric pressure. After a cavitation event in a conduit, the gas inside the conduit should be in mechanical equilibrium with the surrounding water. From Eq. (1), we know that the gas pressure in the conduit depends on the curvature radius of water/gas meniscus. If the contact angles are the same within all conduits and the water potentials of xylem increase to reach a high value, like our experiments, the gas pressures in all embolized conduits only depend on the radii of the conduits. Thus, gas will shift from the bubbles in small conduits (directly or through wood rays) to the conduits with long curvature radii at the same height, inducing the embolism to be refilled with water in the bubbles of short radii and leaving some larger conduits to be filled with air. When water stress is severe and the tension of water in a plant is high, less water in the corners of a conduit forms water/gas menisci after a cavitation event in the conduit. If the stereoscope angles of the corners are equal the curvature radii of the menisci depend on contact angles of water with the conduit wall. If all contact angles are equal, the gas pressure in all conduits would be the same. Then the gas in embolized conduits cannot move. If the contact angles are of different values, and the radii of menisci are not the same, the water/gas system in a plant is not at equilibrium. The gas at the same height might move from the bubbles in the conduits with small contact angles into the bubbles in the conduits with large contact angles. The water entering the cavitated conduits may be supplied by the adjacent parenchyma and conduits. Since the gas in living plants always losses balance gas movement may proceed all the time during transpiration. The work of Zwieniecki and Holbrook (2000) has shown that the contact angles measured in the lumens of six species of trees are in the range of 42–55◦ , meaning that contact angle is not zero as it was known before. The contact angles in the conduits of the sections in our experiments did not seem to be the same either. The value of a contact angle depends on the materials, which are lignin and cellulose, lining on the wall of conduits. The distribution of the materials in plants changes with regions and seasons. However, we are not sure if the contact angle indeed plays a role in embolism refilling at low xylem pressure. Future study should be conducted to examine our hypothesis.

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In summary, our experiments showed that when a dehydrated section was rewetted, the elongated bubbles in smaller conduits shortened till vanished. In addition, the bubbles in larger conduits grew up at first, then collapsed and disappeared. What’s more, the bubbles outside conduits appeared or popped up one after another; they originated mainly from the cross sections of wood rays. The smaller ones also collapsed, the larger ones grew up gradually. Two mechanisms can explain these phenomena. First, air would move directively from one bubble to another, instead of being pushed out of a section. Second, air would diffuse through the surounding water into the adjacent bubbles with long curvature radii. The experiments also showed that the initial gas pressure in a cavitated conduit was lower than the atmospheric pressure. Therefore, we further suggest that for living plants there might be some larger conduits with gas at lowerthan-atmospheric-pressure and two mechanisms could induce air to move from conduits with higher pressure to those with lower pressure, leading the conduits to repair and leaving some regions to be filled with air. For the refilling of embolism at lower xylem pressure the values of contact angle might play an important role. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (#30270343). The authors also thank Dr. M.T. Tyree for his encouragement. References Borghetti, M., Edwards, W.R.N., Grace, J., Jarvis, P.G., Raschi, A., 1991. The refilling of embolized xylem in Pinus sylvestris L. Plant Cell Environ. 14, 357–369. Edwards, W.R.N., Jarvis, P.G., Grace, J., Moncrieff, J.B., 1994. Reversing cavitation in tracheids of Pinus sylvestris L. under negative potential. Plant Cell Environ. 17, 389–397.

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