Embolized conduits of rice (Oryza sativa, Poaceae) refill despite negative xylem pressure

Embolism reversal in rice plants was studied by testing the plant's ability to refill embolized conduits while xylem pressures were substantially negative. Intact, potted plants were water-stressed to a xylem pressure of -1.88 ± 0.1 MPa and a 66.3 ± 3.8% loss of xylem conductivity (PLC) by cavitation. Stressed plants were carefully rewatered, allowing xylem pressure to rise, but not above the theoretical threshold of c. -0.15 MPa for embolism collapse. Despite xylem pressures being more negative than this threshold, the PLC fell significantly (28.5 ± 5.6%), indicating the refilling of vessels. Above c. -1.0 MPa, almost all plants regained their maximum hydraulic conductivity. Dye uptake experiments showed the same pattern of embolism refilling despite negative pressure. Refilling was prevented in plants that were light-starved for 5 d, suggesting the unknown mechanism is dependent on metabolic energy. Results are among the first showing that herbaceous plants can reverse embolism without bulk xylem pressures rising near or above atmospheric.     

Embolism formation during freezing in the wood of Picea abies

Freeze-thaw events can cause embolism in plant xylem. According to classical theory, gas bubbles are formed during freezing and expand during thawing. Conifers have proved to be very resistant to freeze-thaw induced embolism, because bubbles in tracheids are small and redissolve during thawing. In contrast, increasing embolism rates upon consecutive freeze-thaw events were observed that cannot be explained by the classical mechanism. In this study, embolism formation during freeze-thaw events was analyzed via ultrasonic and Cryo-scanning electron microscope techniques. Twigs of Picea abies L. Karst. were subjected to up to 120 freeze-thaw cycles during which ultrasonic acoustic emissions, xylem temperature, and diameter variations were registered. In addition, the extent and cross-sectional pattern of embolism were analyzed with staining experiments and Cryo-scanning electron microscope observations. Embolism increased with the number of freeze-thaw events in twigs previously dehydrated to a water potential of -2.8 MPa. In these twigs, acoustic emissions were registered, while saturated twigs showed low, and totally dehydrated twigs showed no, acoustic activity. Acoustic emissions were detected only during the freezing process. This means that embolism was formed during freezing, which is in contradiction to the classical theory of freeze-thaw induced embolism. The clustered pattern of embolized tracheids in cross sections indicates that air spread from a dysfunctional tracheid to adjacent functional ones. We hypothesize that the low water potential of the growing ice front led to a decrease of the potential in nearby tracheids. This may result in freezing-induced air seeding.     

Water-stress-induced xylem embolism in three species of conifers

Abstract. The mechanism of water-stress-induced xylem embolism was studied in three species of conifers: Abies balsamea (L.) Mill., Picca rubens Sarg, and Juniperus virginiana L. Each species showed a characteristic relationship between xylem tension and the loss of hydraulic conductivity by air embolism. Abics balsamea and Picca rubens began to embolize at tensions between 2 and 3 MPa and were completely non-conducting between 3 and 4 MPa. Juniperus virginiana was least vulnerable, beginning to embolize at 4 and still retaining approximately 10% conductivity at 10 MPa. As with a previous study of the vessel-bearing Accr saccharum Marsh., a brief perfusion of branch segments with an oxalic acid and calcium solution (10 and 0.1 mol m⁻³. respectively) increased the vulnerability of the xylem to embolism; this was especially pronounced in Abies balsamea . In order to test whether embolism was caused by aspiration of air into functional tracheids from neighbouring embolized, ones (the 'air-seeding'hypothesis), hydrated branch segments were injected with air at various pressures and measured for embolism. Results supported the air-seeding hypothesis because the relationship between injection pressure and embolism for both native and oxalic-calcium-treated segments was essentially the same as for embolism induced by xylem tension. Structural and experimental evidence suggested the air seeding occurred through inter-tracheid pit membranes when the thickened torus region of the membrane became displaced from its normal sealing position over the pit aperture. Thus, the embolism-inducing tension may be a function of pit membrane flexibility. This tension is of ecological significance because it reflects to some extent the range of xylem tensions to which a species is adapted.     

Relations between vulnerability to xylem embolism and xylem conduit dimensions in young trees of Quercus corris

Measurements of xylem conduit length and width and the distribution of xylem conduit ends were made in inter-nodes (I), nodes (N) and twig junctions (J) of 1-, 2- and 3-year-old twigs of plants of Quercus cerris L. Parallel measurements were also made of the loss of hydraulic conductivity of twigs subjected to pressure differentials across conduit pit membranes, equalling the leaf water potential at the turgor loss point. The loss of theoretical hydraulic conductivity was calculated as the ratio of i esivr⁴ (where r is the conduit radius) of the non-conducting conduits to that of all the conduits in the outermost wood ring of I, N and J. Stem zones such as 1-year-old nodes and junctions were localized with narrower and shorter xylem conduits and with higher percentages of conduit ends than internodes. Such ‘constricted zonesrsquo; were less vulnerable to embolism than internodes. Latewood conduits were consistently narrower, shorter and less vulnerable to embolism than earlywood ones. A positive relation therefore existed between conduit diameter and length and vulnerability to embolism. The overall vulnerability to embolism of Q. cerris plants is discussed in terms of xylem conduit width and length and of the distribution of conduit ends.     

A method for inducing xylem emboli in situ: experiments with a field-grown tree

A pressure collar, assembled around 25cm sections of 4-year-old willow twigs, was used to examine cavitation events under field conditions. When the air pressure inside the collar was raised to between 1–8 and 2–8MPa, ultrasound acoustic emission signals were triggered which indicated the breaking of water columns in the xylem. The hydraulic conductivity of the twig portion inside the chamber decreased markedly. As a result, water potentials and conductances in leaves at the end of the twig decreased. Similar changes were induced at comparable pressures in detached twigs. The equipment used is described in detail, and evidence is presented that the mechanism of this artificial production of emboli follows the air-seeding principle hypothesized for natural cavitation events.     

元宝枫苗木的水力结构特征

在温室条件下,控制不同干旱梯度,用改良的冲洗法测定了4年生元宝枫苗木的水力结构参数．研究表明,随着小枝水势的降低,水力结构各参数随茎段功能木质部直径的变化可以用不同的方程来模拟;导水率的大小受茎段所在区域的影响,限速区的导水率明显低于非限速区,限速区的存在对苗木个体的生存竞争有利．导水率、比导率和叶比导率都和功能木质部直径和小枝水势呈明显的正相关．较粗茎段的叶比导率远高于多次分枝的未端细小分枝,有利于苗木在干旱时保存那些光合积累较大的器官．在落叶之前,相同直径枝条的胡伯尔值随小枝水势的变化很小,说明苗木水分胁迫主要源于木质部空穴和栓塞．     

Relationship of Xylem Embolism to Xylem Pressure Potential, Stomatal Closure, and Shoot Morphology in the Palm Rhapis excelsa

Xylem failure via gas embolism (cavitation) induced by water stress was investigated in the palm Rhapis excelsa (Thumb.) Henry. Xylem embolism in excised stems and petioles was detected using measurements of xylem flow resistance: a decrease in resistance after the removal of flow-impeding embolisms by a pressure treatment indicated their previous presence in the axis. Results supported the validity of the method because increased resistance in an axis corresponded with: (a) induction of embolism by dehydration, (b) increased numbers of cavitations as detected by acoustic means, (c) presence of bubbles in xylem vessels. The method was used to determine how Rhapis accommodates embolism; results suggested four ways. (a) Embolism was relatively rare because pressure potentials reach the embolism-inducing value of about −2.90 megapascals only during prolonged drought. (b) When embolism did occur in nature, it was confined to the relatively expendable leaf xylem; the stem xylem, which is critical for shoot survival, remained fully functional. (c) Even during prolonged drought, the extent of embolism is limited by complete stomatal closure, which occurred at the xylem pressure potential of −3.20 ± 0.18 megapascals. (d) Embolism is potentially reversible during prolonged rains, since embolisms dissolved within 5 h at a pressure potential of 0.00 megapascals (atmospheric), and xylem sap can approach this pressure during rain.     

New evidence for a role of vessel-associated cells and phloem in the rapid xylem refilling of cavitated stems of Laurus nobilis L.

Xylem recovery from embolism was studied in Laurus nobilis L. stems that were induced to cavitate by combining negative xylem pressure potentials (P_X = ?1.1 MPa) with positive air pressures (P_C) applied using a pressure collar. Xylem refilling was measured by recording the percentage loss of hydraulic conductance (PLC) with respect to the maximum 2 min, 20 min and 15 h after pressure release. Sodium orthovanadate (an inhibitor of many ATP‐ases) strongly inhibited xylem refilling while fusicoccin (a stimulator of the plasma membrane H⁺‐ATPase) promoted complete embolism reversal. So, the refilling process was interpreted to result from energy‐dependent mechanisms. Stem girdling induced progressively larger inhibition to refilling the nearer to the embolized stem segment phloem was removed. The starch content of wood parenchyma was estimated as percentages of ray and vasicentric cells with high starch content with respect to the total, before and after stem embolism was induced. A closely linear positive relationship was found to exist between recovery from PLC and starch hydrolysis. This, was especially evident in vasicentric cells. A mechanism for xylem refilling based upon starch to sugar conversion and transport into embolized conduits, assisted by phloem pressure‐driven radial mass flow is proposed.     

Interpretation of seasonal changes of xylem embolism and plant hydraulic resistance in Fagus sylvatica

The annual course of xylem embolism in twigs of adult beech trees was monitored, and compared to concurrent changes of tree water status and hydraulic resistances. Xylem embolism was quantified in 1-year-old apical twigs by the hydraulic conductivity as a percentage of the maximum measured after removal of air emboli. Tree and root hydraulic resistances were estimated from water potential differences and sap flux measurements. The considerable degree of twig embolism found in winter (up to 90% loss of hydraulic conductivity) may be attributed to the effect of freeze-thaw cycles in the xylem. A partial recovery from winter embolism occurred in spring, probably because of the production of new functional xylem. Xylem embolism fluctuated around 50% throughout the summer, without significant changes. Almost complete refilling of apical twigs was observed early in autumn. A significant negative correlation was found between xylem embolism and precipitation; thus, an active role of rainfall in embolism reversion is hypothesized. Tree and root hydraulic resistances were found to change throughout the growing period. A marked decrease of hydraulic resistance preceded the refilling of apical twigs in the autumn. Most of the decrease in total tree resistance was estimated to be located in the root compartment.     

Field measurements of xylem cavitation: are acoustic emissions useful?

Cavitation of water columns within the xylem is deleteriousfor plant water relations as it results in embolism, so reducinghydraulic conductivity. This cavitation can be detected as itis accompanied by the production of acoustic emissions, whichcan be detected ultrasonically and logged automatically overperiods of days. The acoustic emission technique is useful todetermine the threshold water potential at which damage to thewater-conducting system of the plant is initiated. It can revealwhich environmental variables are deleterious to the plant waterrelations, and which parts of the plant are most sensitive tocavitation. Species comparisons, and comparisons of the samespecies in different environments can be made, to obtain indicationsof drought tolerance. However, acoustic emissions have onlya limited use in determining the proportion of embolism in aconducting stem, and other methods are needed to find the percentagereduction in hydraulic conductivity. Key words: Embolism, hydraulic conductivity, draught stress, vulnerability curves     

Cavitation fatigue. Embolism and refilling cycles can weaken the cavitation resistance of xylem

Although cavitation and refilling cycles could be common in plants, it is unknown whether these cycles weaken the cavitation resistance of xylem. Stem or petiole segments were tested for cavitation resistance before and after a controlled cavitation-refilling cycle. Cavitation was induced by centrifugation, air drying of shoots, or soil drought. Except for droughted plants, material was not significantly water stressed prior to collection. Cavitation resistance was determined from "vulnerability curves" showing the percentage loss of conductivity versus xylem pressure. Two responses were observed. "Resilient" xylem (Acer negundo and Alnus incana stems) showed no change in cavitation resistance after a cavitation-refilling cycle. In contrast, "weakened" xylem (Populus angustifolia, P. tremuloides, Helianthus annuus stems, and Aesculus hippocastanum petioles) showed considerable reduction in cavitation resistance. Weakening was observed whether cavitation was induced by centrifugation, air dehydration, or soil drought. Observations from H. annuus showed that weakening was proportional to the embolism induced by stress. Air injection experiments indicated that the weakened response was a result of an increase in the leakiness of the vascular system to air seeding. The increased air permeability in weakened xylem could result from rupture or loosening of the cellulosic mesh of interconduit pit membranes during the water stress and cavitation treatment.     

New evidence for large negative xylem pressures and their measurement by the pressure chamber method

Pressure probe measurements have been interpreted as showing that xylem pressures below c. –0.4 MPa do not exist and that pressure chamber measurements of lower negative pressures are invalid. We present new evidence supporting the pressure chamber technique and the existence of xylem pressures well below –0.4 MPa. We deduced xylem pressures in water-stressed stem xylem from the following experiment: (1) loss of hydraulic conductivity in hydrated stem xylem (xylem pressure = atmospheric pressure) was induced by forcing compressed air into intact xylem conduits; (2) loss of hydraulic conductivity from cavitation and embolism in dehydrating stems was measured, and (3) the xylem pressure in dehydrated stems was deduced as being equal and opposite to the air pressure causing the same loss of hydraulic conductivity in hydrated stems. Pressures determined in this way are only valid if cavitation was caused by air entering the xylem conduits (air-seeding). Deduced xylem pressure showed a one-to-one correspondence with pressure chamber measurements for 12 species (woody angiosperms and gymnosperms); data extended to c. –10 MPa. The same correspondence was obtained under field conditions in Betula occidentalis Hook., where pressure differences between air- and water-filled conduits were induced by a combination of in situ xylem water pressure and applied positive air pressure. It is difficult to explain these results if xylem pressures were above –0.4 MPa, if the pressure chamber was inaccurate, and if cavitation occurred by some mechanism other than air-seeding. A probable reason why the pressure probe does not register large negative pressures is that, just as cavitation within the probe limits its calibration to pressures above c. –0.5 MPa, cavitation limits its measurement range in situ.     

How do water transport and water storage differ in coniferous earlywood and latewood?

The goal of this research project was to determine the water transport behaviour of earlywood versus latewood in the trunk of 21-year-old Douglas-fir [Pseudostuga menziesii (Mirb.) Franco] trees. Specific conductivity (k(s)) and the vulnerability of xylem to embolism were measured on a single growth ring and in a subset of earlywood and latewood samples within the same ring. Earlywood/latewood ratio, trunk water potential (Psi) and relative water content (RWC) were used to predict differences in conductivities and vulnerability to embolism. Earlywood has about 11 times the k(s) of latewood, and up to 90% of the total flow occurred through the earlywood. Earlywood's vulnerability to embolism followed the same trend as that of the whole wood, with 50% loss of conductivity at -2.2 MPa (P(50)). Latewood was more vulnerable to embolism than earlywood at high Psi, but as Psi decreased, the latewood showed very little further embolism, with a P(50) <-5.0 MPa. The lowest trunk Psi estimated in the field was about -1.4 MPa, indicating that latewood and earlywood in the field experienced about 42% and 16% loss of k(s), respectively. The higher vulnerability to embolism in latewood than in earlywood at field Psi was associated with higher water storage capacity (21.8% RWC MPa(-1) versus 4.1% RWC MPa(-1), latewood and earlywood, respectively). The shape of the vulnerability curve suggests that air seeding through latewood may occur directly through pores in the margo and seal off at lower pressure than earlywood pores.     

A new method for vulnerability analysis of small xylem areas reveals that compression wood of Norway spruce has lower hydraulic safety than opposite wood

Compression wood is formed at the underside of conifer twigs to keep branches at their equilibrium position. It differs from opposite wood anatomically and subsequently in its mechanical and hydraulic properties. The specific hydraulic conductivity (k_s) and vulnerability to drought‐induced embolism (loss of conductivity versus water potential ψ) in twigs of Norway spruce [Picea abies (L.) Karst.] were studied via cryo‐scanning electron microscope observations, dye experiments and a newly developed ‘Micro‐Sperry’ apparatus. This new technique enabled conductivity measurements in small xylem areas by insertion of syringe cannulas into twig samples. The hydraulic properties were related to anatomical parameters (tracheid diameter, wall thickness). Compression wood exhibited 79% lower k_s than opposite wood corresponding to smaller tracheid diameters. Vulnerability was higher in compression wood despite its narrower tracheids and thicker cell walls. The P₅₀ (ψ at 50% loss of conductivity) was ?3.6 MPa in opposite but only ?3.2 MPa in compression wood. Low hydraulic efficiency and low hydraulic safety indicate that compression wood has primarily a mechanical function.     

Pit Membrane Degradation and Air-Embolism Formation in Ageing Xylem Vessels of Populus tremuloides Michx

Air-embolism formation in xylem vessels of Populus tremuloidesMichx. was quantified by its reduction of hydraulic conductivityin branch segments. Embolism was induced by increasing xylemtension in drying stems, or by inserting one end of a hydratedstem in a pressure bomb and increasing air pressure in the bomb.Both treatments produced the same response suggesting that embolismby water stress was caused by air entering water-filled vessels,presumably through inter-vessel pits. In rapidly-growing P.tremuloides branches, the vessels of the outer growth ring werefunctional whereas vessels in older xylem were mostly embolized.This selective embolizing of older vessels was associated witha marked increase in permeability of their inter-vessel pitsto air, relative to pits of younger vessels. Air-injection pressuresless than 1·0 MPa completely embolized older vesselsthat had been re-filled in the laboratory, whereas pressuresover 4·0 MPa were required to embolize young vessels.Greater permeability of old vessels was due to degradation oftheir pit membranes as seen in the scanning electron microscope;large openings were present that were not seen in pit membranesof young vessels. These holes would allow air to penetrate vesselends at low pressure differences causing embolism. Degradationof pit membranes causing the selective dysfunction of oldersapwood may be a general phenomenon initiating heartwood formationin many species. Key words: Xylem embolism, hydraulic conductivity, heartwood formation, cavitation, Populus tremuloides, Michx     

Pit membrane porosity and water stress-induced cavitation in four co-existing dry rainforest tree species

Aspects of xylem anatomy and vulnerability to water stress-induced embolism were examined in stems of two drought-deciduous species, Brachychiton australis (Schott and Endl.) A. Terracc. and Cochlospermum gillivraei Benth., and two evergreen species, Alphitonia excelsa (Fenzal) Benth. and Austromyrtus bidwillii (Benth.) Burret., growing in a seasonally dry rainforest. The deciduous species were more vulnerable to water stress-induced xylem embolism. B. australis and C. gillivraei reached a 50% loss of hydraulic conductivity at -3.17 MPa and -1.44 MPa, respectively; a 50% loss of hydraulic conductivity occurred at -5.56 MPa in A. excelsa and -5.12 MPa in A. bidwillii. To determine whether pit membrane porosity was responsible for greater vulnerability to embolism (air seeding hypothesis), pit membrane structure was examined. Expected pore sizes were calculated from vulnerability curves; however, the predicted inter-specific variation in pore sizes was not detected using scanning electron microscopy (pores were not visible to a resolution of 20 nm). Suspensions of colloidal gold particles were then perfused through branch sections. These experiments indicated that pit membrane pores were between 5 and 20 nm in diameter in all four species. The results may be explained by three possibilities: (a) the pores of the expected size range were not present, (b) larger pores, within the size range to cause air seeding, were present but were rare enough to avoid detection, or (c) pore sizes in the expected range only develop while the membrane is under mechanical stress (during air seeding) due to stretching/flexing.     

Dynamics of freeze-thaw embolism in Smilax rotundifolia (Smilacaceae)

Freeze-thaw cycles pose a major physiological challenge for all temperate perennial plants, but monocotyledonous vines face a still greater risk because their few large vessels are especially susceptible to embolism and are not replaced by secondary growth. The genus Smilax is particularly remarkable because it is widespread in the tropics but includes species that survive the hard frosts of New England winters. Smilax rotundifolia was monitored for a year for evidence of stem xylem freeze-thaw cavitation and refilling. Embolism of metaxylem was complete by late November and was completely reversed by late April, when root pressures rose as high as 100 kPa. Protoxylem remained full of sap throughout the year in cryogenic scanning electron micrographs. Three methods were used to quantify embolism: percent loss conductivity (PLC), gravimetric air fraction (GAF: mass of water in stem xylem relative to capacity), and cryogenic scanning electron microscopy (cryo-SEM). The three methods corroborated one another well and gave quantitatively similar results. Osmolality of xylem sap extracted from exuding stems was 64 mol/kg (±7.0, N = 8), consistent with the root pressures observed. Strong root pressure can account for Smilax's survival in temperate regions with severe frosts, where few monocots with persistent aboveground organs are found.     

The Pneumatron: An automated pneumatic apparatus for estimating xylem vulnerability to embolism at high temporal resolution

Xylem vulnerability to embolism represents an important trait to determine species distribution patterns and drought resistance. However, estimating embolism resistance frequently requires time-consuming and ambiguous hydraulic lab measurements. Based on a recently developed pneumatic method, we present and test the “Pneumatron”, a device that generates high time-resolution and fully automated vulnerability curves. Embolism resistance is estimated by applying a partial vacuum to extract air from an excised xylem sample, while monitoring the pressure change over time. Although the amount of gas extracted is strongly correlated with the percentage loss of xylem conductivity, validation of the Pneumatron was performed by comparison with the optical method for Eucalyptus camaldulensis leaves. The Pneumatron improved the precision of the pneumatic method considerably, facilitating the detection of small differences in the (percentage of air discharged [PAD] < 0.47%). Hence, the Pneumatron can directly measure the 50% PAD without any fitting of vulnerability curves. PAD and embolism frequency based on the optical method were strongly correlated (r² = 0.93) for E. camaldulensis. By providing an open source platform, the Pneumatron represents an easy, low-cost, and powerful tool for field measurements, which can significantly improve our understanding of plant–water relations and the mechanisms behind embolism.     

Direct observation and modelling of embolism spread between xylem conduits: a case study in Scots pine

Xylem embolism is one of the main processes involved in drought‐related plant mortality. Although its consequences for plant physiology are already well described, embolism formation and spread are poorly evaluated and modelled, especially for tracheid‐based species. The aim of this study was to assess the embolism formation and spread in Pinus sylvestris as a case study using X‐ray microtomography and hydraulics methods. We also evaluated the potential effects of cavitation fatigue on vulnerability to embolism and the micro‐morphology of the bordered pits using scanning electron microscopy (SEM) to test for possible links between xylem anatomy and embolism spread. Finally, a novel model was developed to simulate the spread of embolism in a 2D anisotropic cellular structure. Results showed a large variability in the formation and spread of embolism within a ring despite no differences being observed in intertracheid pit membrane anatomical traits. Simulations from the model showed a highly anisotropic tracheid‐to‐tracheid embolism spreading pattern, which confirms the major role of tracheid‐to‐tracheid air seeding to explain how embolism spreads in Scots pine. The results also showed that prior embolism removal from the samples reduced the resistance to embolism of the xylem and could result in overestimates of vulnerability to embolism.