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0 Research School of Biological Sciences, Australian National University, PO Box 475, Canberra 2601, Australia
Received for publication October 26, 1999. Accepted for publication March 16, 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: cavitation • compensating pressure theory • embolism refilling • leaf veins • protoxylem lacuna • transpiration • xylem vessels • Zea mays
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), and in roots (Buchard, McCully, and Canny, 1999
; McCully, Huang, and Ling, 1998
; McCully, 1999
; Pate and Canny, 1999
; Shane and McCully, 1999
). This pattern of embolism during day and night has been so generally found that it may be distinguished as the typical daily pattern. Also, in these observations the small vessels were less embolized than the large ones. An explanation of these observations was elaborated in terms of fast refilling of embolisms by supplementary water pushed into vessels by tissue-pressure-driven reverse osmosis (Canny, 1998b
). The observations stimulate the question of whether similar changes are occurring in the laminas of leaves, where the tension in the xylem sap is greatest, where the supply of water outside the vessels seems limited, and where the arrangement of tissues and air spaces might be thought inadequate to generate and contain tissue pressure. This paper reports an investigation of this question.
The vein system of leaf laminas comprises two types of vein: large veins with large vessels, which distribute water quickly all over the lamina, and small veins with small vessels, which draw water from the large veins and distribute it locally to the mesophyll from which it evaporates (Canny, 1990, 1993
). The tension in the small veins at the end of the flow path must be greatest and, if both types are equally liable to embolism, might be expected to produce more embolisms there than in the large veins. Embolisms blocking the few small vessels in fine veins would produce patches of leaf under high water stress and might lead to local closures of stomata.
A monocotyledon leaf with parallel veins was selected because of its geometrical simplicity. Any cross section of such a leaf provides right cross sections of the large and small parallel veins, but not, of course, of the transverse veins that connect them. In such a leaf, large veins with large vessels and a protoxylem lacuna are called lateral veins. Small veins with small vessels and no protoxylem lacuna are called either intermediate or small (Kuo, O'Brien and Canny, 1974
). Maize leaves, as used here, have lateral veins and both intermediate and small veins (Fig. 1). Lateral veins are far apart, with many intermediate and small veins between them.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Samples of leaves were taken to measure embolisms from five leaves at each of the following times: 0600, 1000, 1400, 1800, and 2200 (Eastern Summer Time). Each sample was frozen on the plant by clamping a piece of the lamina between the heavy copper jaws of a specially constructed pair of pliers that had been cooled to the temperature of liquid nitrogen (LN2) (McCully, Huang, and Ling, 1998
). The piece was cut from the leaf while still held in the pliers, then released into LN2, trimmed parallel to the veins to strips 
5 mm wide, and placed in a labelled cryo-vial. The vials were stored at LN2 temperature. At each time of sampling measurements of leaf water potential were made on five other (unbagged) leaves with a pressure chamber (Plant water status console model 3000, Soilmoisture Corp., Santa Barbara, California, USA), and the air temperature was recorded.
Embolisms in the vessels of the leaf veins were observed by the method outlined in Canny (1997a)
. Small lengths were cut from the center of the leaf strips under LN2 and mounted in a slot in an aluminium stub with Tissue Tek (Miles Inc., Elkhart, Iowa, USA). The leaf piece was planed in a cryo-microtome at -80°C with its narrow dimension at right angles to the knife, etched in the column of the scanning electron microscope (JSM 6400, JEOL Ltd., Tokyo, Japan) for 1 min at -90°C to reveal cell outlines, coated with 100 nm of aluminium, and observed at 7 kV. Micrographs were made on Kodak TMax-100 120 roll film. The content of the vessels was assessed visually on the screen for each vein in the leaf section. Veins were assigned to one of three classes: lateral, intermediate, or small. Records were made of the number of vessels in each vein class, and of the number of these vessels that contained gas. In lateral veins the large vessels and the protoxylem cavities were scored separately. At each sampling time the counts of vessels and embolisms for all five leaves were pooled to give single totals for each vein class. There are thus no estimates of variance. The extent of embolism at each sampling time was expressed as the percentage of gas-containing vessels observed in each vein class. Because the values for intermediate and small veins were not distinguishable, these two classes were combined.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Sharman, 1942
) and by others to be vessels (e.g., Cheadle, 1942
; Esau, 1943
). Below them is a strand of phloem. Above them is the protoxylem lacuna, sometimes containing remnants of protoxylem vessels. Small and intermediate veins have a single sheath of large, thin-walled bundle sheath cells containing chloroplasts. Their xylem vessels are few and small. Below the vessels lies the phloem. The frozen hydrated leaf specimens showed a surprisingly low proportion of gas space. The surface of a transverse face was mostly frozen liquid (Fig. 2). The proportion of large veins was small, on average one to every 37 of the small and intermediate veins.
At 0600 and 2200 nearly all the lateral veins had all their tracheary elements filled with liquid, including the protoxylem lacuna (Figs. 3, 9). In Fig. 3, two bands of thick-walled fibers lie above and below the vein, of which only the upper one is visible. The living cells of the mesophyll and vein are filled with white (electron emissive) lines of solute crystallized by freezing. The sap in the tracheary elements has fewer solutes and is less emissive. It can be seen filling the two large metaxylem vessels and, between them, the band of angular small tracheary elements. Above these is a protoxylem element, and beyond that, the protoxylem lacuna, both sap-filled.
At other sampling times one or both of the large vessels usually contained gas (Fig. 4) and sometimes some debris from the planing. Occasionally, in the 1400 samples, the protoxylem lacuna also contained gas (Fig. 5). The small tracheary elements between the vessels were never seen to contain gas.
The vessels of the intermediate (Fig. 6) and small (Fig. 7) veins almost never contained gas. Of 4021 vessels examined only 19 contained gas (Fig. 8), and 17 of these were at the 1000 and 1400 sampling times.
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1.2 MPa) was recorded late in the afternoon, at which time large vessel embolisms had fallen to 20%. The embolism values for the protoxylem lacuna have not been included on the graph. They were zero for all times except 1400, when 25% of the cavities were embolized. Air temperature ranged from 21° to 27°C.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The findings reported here are in full agreement with those reported by many researchers in the older literature, using simpler techniques. Strasburger (1891)
reviewed this literature and concluded: "I myself have never found air bubbles in the ultimate branches of vessels of leaves of many woody plants, which I investigated during daylight hours in summer. However, in the larger veins of the lamina, I confirmed (similar) published statements of Elfving ‘that in the outermost tracheids, air bubbles never occur’. And so also, Scheit has found in the transfusion tissue of conifer leaves, no air, but only water or water vapor." The absence of embolisms in the fine veins of leaf laminas and their concurrent appearance in large vessels of larger veins appear to be very general phenomena.
A similar (and possibly related) distinction between large and small veins was reported by Lo Gullo et al. (1997)
for the acoustic emissions (often considered as evidence of cavitation) from drying holly leaves. Most of the emissions were detected near the midrib. Few emissions were detected from parts of leaves where there were only small veins.
The traditional explanation of the difference in embolisms between large and small vessels would be that vulnerability to cavitation is proportional to diameter (Zimmermann, 1983
). This is less an explanation than a restatement of the fact. A causative link has been surmised: for example, that there are more likely to be nuclei to initiate cavitation in a larger volume or surface or that air seeding would be more likely in a large vessel because the pores in its pit membranes might be larger (Jarbeau, Ewers, and Davis, 1995
). The claimed correlation between vulnerability to cavitation and vessel diameter is contrary to the observations presented here, that the protoxylem lacuna is much less embolized than the large vessels.
The fact that the protoxylem lacuna, whether in Equisetum or in the monocotyledon vascular bundles, is filled with liquid and is a pathway for water transport parallel with the vessels of the bundle has been known since the investigation of Westermaier (1884)
. Strasburger (1891)
ascribes water conduction as the main function of the lacuna. The conduction of water by the lacuna was confirmed with dye tracers by Buchholz (1921)
for a wide range of monocotyledons, Equisetum and several water plants, by Bierhorst (1958)
for Equisetum, and by Dong, McCully, and Canny (1997)
for sugarcane.
The conducting protoxylem lacuna does not conform to the traditional view (confirmed here for vessels of maize leaf veins) that large vessels are more vulnerable to cavitation than small ones. The discovery that the lacuna is much less subject to embolism than the two vessels flanking it, although it is of comparable diameter, requires explanation. It seems most unlikely that the lacuna would be less susceptible to air seeding than the vessels, with their stout walls and small pits.
Possible explanations of both the relation of embolism to vessel diameter, and of the anomalous behavior of the protoxylem lacuna, follow from the observed refilling of embolized vessels as outlined by Canny (1998b)
. He proposes a small flux of water (supplementary water) into vessels from the surrounding tissues, driven by tissue pressure, and expressing water by reverse osmosis from reservoir cells at lower pressure. In Appendix 1 of that paper some calculations were attempted about the flux of water and the necessary pressure difference. The time to refill is proportional to vessel diameter, and this could explain why small vessels are found to have less embolism than large vessels. Even with a common rate of cavitation, small vessels would spend less time empty than large ones. The objections to such a mechanism put forward by Tyree et al. (1999)
show that he has not understood the proposal. He does not refer to the statement of it in Canny (1998b).
To explain the low embolism of the wide protoxylem lacuna requires a further step. In the calculations of Appendix 1 of Canny (1998b)
the whole surface of the vessel was assumed to be permeable to water. That this is an oversimplification is shown by the published images of water entering embolized vessels through pits (e.g., fig. 2 of Canny, 1998b
; fig. 10 of Canny, 1998a
; fig. 4 j, l of McCully, Huang, and Ling, 1998
). The calculations should include a proportionality factor for the pit area as a fraction of the total wall area. With this revision, the special property of the protoxylem lacuna is that it has no impermeable walls, but is surrounded by thin-walled parenchyma cells. If the pits occupy say 10% of the walls of the large vessels, then the protoxylem lacuna would fill ten times faster than the vessels and would spend a tenth of the time that they do empty. Looked at in this way, the protoxylem lacuna would form part of that water continuum that maintains minimum embolism (along with the small tracheary elements of the lateral veins and the small vessels of the intermediate and small veins). It would be a vital part of the water supply network at times of high water demand. In justice, it must be stated that Westermaier (1884)
was fully aware of the special permeability of the walls around the lacuna to water.
The existence of sufficient reservoirs of water to supply the refilling process is less easy to imagine in leaves than in other organs. The large proportion of ice in the frozen transverse leaf face in Fig. 2 is in contrast to the comparable views of frozen hydrated dicotyledon leaves, where air spaces make up a large part of the surface (e.g., Jeffree et al., 1987
). Maize leaves do have a low volume fraction of gas space. Byott (1976)
showed that the air space volume of C4 plants was less than that of C3 plants, and for C4 monocotyledons ranged from 10.5% in Setaria to 2.8% in Saccharum. Zea was at the higher end of the range, with 10%. Dicotyledon leaves could have percentages of air space ranging up to 52% in Nicotiana. For maize leaves, the volume of water in the lamina available for refilling appears substantial. It seems possible that the higher water use efficiency of C4 leaves may derive, in part, from their having a larger volume of reservoir water for refilling embolisms.
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Byott, G. S. 1976 Leaf air space systems in C3 and C4 species. New Phytologist 76: 295–299[CrossRef][ISI]
Buchard, C., M. McCully, and M. Canny. 1999 Daily embolism and refilling of root xylem vessels in three dicotyledonous crop plants. Agronomie 19: 97–106[CrossRef][ISI]
Buchholz, M. 1921 Über die Wasserleitungsbahnen in den interkalaren Wachstumszonen monokotyler Sprosse. Flora 114: 119–186
Canny, M. J. 1990 What becomes of the transpiration stream? New Phytologist 114: 341–368[CrossRef][ISI]
———. 1993 The transpiration stream in the leaf apoplast: water and solutes. Philosophical Transactions of the Royal Society (London) B 341: 87–100
———. 1997a Vessel contents after excision—a test of Scholander's assumption. American Journal of Botany 84: 1217–1222[Abstract]
———. 1997b Vessel contents during transpiration—embolisms and refilling. American Journal of Botany 84: 1223–1230[Abstract]
———. 1998a Transporting water in plants. American Scientist 86: 152–159[CrossRef]
———. 1998b Applications of the compensating pressure theory of water transport. American Journal of Botany 85: 897–909[Abstract]
Cheadle, V. I. 1942 The occurrence and types of vessels in the various organs of the plant in the Monocotyledonae. American Journal of Botany 29: 441–450[CrossRef][ISI]
Dong, Z., M. E. McCully, and M. J. Canny. 1997 Does Acetobacter diazotrophicus live and move in the xylem of sugarcane stems? Some anatomical and physiological data. Annals of Botany 80: 147–158
Esau, K. 1943 Ontogeny of the vascular bundle in Zea mays. Hilgardia 15: 327–356
Hayward, E. H. 1938 The Structure of Economic Plants. Macmillan, New York
Jarbeau, J. A., F. W. Ewers, and S. D. Davis. 1995 The mechanism of water-stress induced embolism in two species of chapparal shrubs. Plant Cell and Environment 18: 189–196
Jeffree, C. E., N. D. Read, J. A. C. Smith, and J. E. Dale. 1987 Water droplets and ice deposits in leaf intercellular spaces: redistribution of water during cryofixation for scanning electron microscopy. Planta 172: 20–37[CrossRef][ISI]
Kuo, J., T. P. O'Brien and M. J. Canny. 1974 Pit-field distribution, plasmodesmatal frequency and assimilate flux in the mestome sheath cells of wheat leaves. Planta 121: 97–118[CrossRef][ISI]
Lo Gullo, M. A., A. Nardini, H. Richter, and S. Salleo. 1997 Ultrasound acoustic emissions from dehydrating leaves of deciduous evergreen leaves. Plant, Cell and Environment 20: 1381–1390[CrossRef]
McCully, M. E. 1999 Root xylem embolisms and refilling. Relation to water potentials of soil, roots, and leaves and osmotic potentials of root xylem sap. Plant Physiology 119: 1001–1008
———, C. X. Huang, and L. E. C. Ling. 1998 Daily embolism and refilling of xylem vessels in the roots of field-grown maize. New Phytologist 138: 327–342[CrossRef][ISI]
Pate, J. S., and M. J. Canny. 1999 Quantification of embolisms by direct observation: a comparison of two methods. New Phytologist 141: 33–44[CrossRef][ISI]
Shane, M., and M. E. McCully. 1999 Root xylem embolisms: implications for water flow to the shoot in large, field-grown maize plants with only one root. Australian Journal of Plant Physiology 26: 107–114
Sharman, B. C. 1942 Developmental anatomy of the shoot of Zea mays L. Annals of Botany NS 6: 245–262
Strasburger, E. 1891 Über den Bau und die Verrichtungen der Leitungsbahnen in den Pflanzen. Histologischer Beiträge 3. Jena
Tyree, M. T., S. Salleo, A. Nardini, M. A. Lo Gullo, and R. Mosca. 1999 Refilling embolized vessels in young stems of laurel. Do we need a new paradigm? Plant Physiology 120: 11–21
Westermaier, M. 1884 Untersuchungen über die Bedeutung todter Röhren und lebender Zellen für die Wasserbewegung in der Pflanze. Sitzungsberichte der deutschen Akademie der Wissenschaft zu Berlin 1105–1117
Zimmermann, M. H. 1983 Xylem structure and the ascent of sap. Springer-Verlag, New York, New York, USA
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