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Embolism repair ability has been documented in numerous species. Although the actual mechanism driving this phenomenon is still debated, experimental findings suggest that non-structural carbohydrates (NSC) stored in wood parenchyma would provide the osmotic forces to drive the refilling of embolized conduits. We selected 12 broadleaved species differing in vulnerability to xylem embolism (P-50) and amount of wood parenchyma in order to check direct evidence about the possible link(s) between parenchyma cells abundance, NSC availability and species-specific capacity to reverse xylem embolism. Branches were dehydrated until similar to 50% loss of hydraulic conductivity was recorded (PLC similar to 50%). Hydraulic recovery (Delta PLC) and NSC content was, then, assessed after 111 of rehydration. Species showed a different ability to recover their hydraulic conductivity from PLC similar to 50%. Removing the bark in the species showing hydraulic recovery inhibited the embolism reversal. Strong correlations between the Delta PLC and: a) the amount of parenchyma cells (mainly driven by the pith area), b) the consumption of soluble NSC have been recorded. Our results support the hypothesis that refilling of embolized vessels is mediated by the mobilization of soluble NSC and it is mainly recorded in species with a higher percentage of parenchyma cells that may be important in the hydraulic recovery mechanism as a source of carbohydrates and/or as a source of water.

Diurnal changes in percentage loss of hydraulic conductivity (PLC), with recorded values being higher at midday than on the following morning, have been interpreted as evidence for the occurrence of cycles of xylem conduits' embolism and repair. Recent reports have suggested that diurnal PLC changes might arise as a consequence of an experimental artefact, that is, air entry into xylem conduits upon cutting stems, even if under water, while under substantial tension generated by transpiration. Rehydration procedures prior to hydraulic measurements have been recommended to avoid this artefact. In the present study, we show that xylem rehydration prior to hydraulic measurements might favour xylem refilling and embolism repair, thus leading to PLC values erroneously lower than those actually experienced by transpiring plants. When xylem tension relaxation procedures were performed on stems where refilling mechanisms had been previously inhibited by mechanical (girdling) or chemical (orthovanadate) treatment, PLC values measured in stems cut under native tension were the same as those measured after sample rehydration/relaxation. Our data call for renewed attention to the procedures of sample collection in the field and transport to the laboratory, and suggest that girdling might be a recommendable treatment prior to sample collection for PLC measurements.

Drought-induced tree decline is a complex event, and recent hypotheses suggest that hydraulic failure and carbon starvation are co-responsible for this process. We tested the possible role of non-structural carbohydrates (NSC) content on post-drought hydraulic recovery, to verify the hypothesis that embolism reversal represents a mechanistic link between carbon starvation and stem hydraulics. Measurements were performed in laurel plants subjected to similar water stress levels either over short or long term, to induce comparable embolism levels. Plants subjected to mild and prolonged water shortage (S) showed reduced growth, adjustment of turgor loss point driven by changes in both osmotic potential at full turgor and bulk modulus of elasticity, a lower content of soluble NSC and a higher content of starch with respect to control (C) plants. Moreover, S plants showed a lower ability to recover from xylem embolism than C plants, even after irrigation. Our data suggest that plant carbon status might indirectly influence plant performance during and after drought via effects on xylem hydraulic functioning, supporting the view of a possible mechanistic link between the two processes.

Drought stress can impair leaf hydraulic conductance (Kleaf), but the relative contribution of changes in the efficiency of the vein xylem water pathway and in the mesophyll route outside the xylem in driving the decline of Kleaf is still debated. We report direct measurements of dehydration-induced changes in the hydraulic resistance (R=1/K) of whole leaf (Rleaf), as well as of the leaf xylem (Rx) and extra-vascular pathways (Rox) in four Angiosperm species. Rleaf, Rx, and Rox were measured using the vacuum chamber method (VCM). Rleaf values during progressive leaf dehydration were also validated with measurements performed using the rehydration kinetic method (RKM). We analysed correlations between changes in Rx or Rox and Rleaf, as well as between morpho-anatomical traits (including dehydration-induced leaf shrinkage), vulnerability to embolism, and leaf water relation parameters. Measurements revealed that the relative contribution of vascular and extra-vascular hydraulic properties in driving Kleaf decline during dehydration is species-specific. Whilst in two study species the progressive impairment of both vascular and extra-vascular pathways contributed to leaf hydraulic vulnerability, in the other two species the vascular pathway remained substantially unaltered during leaf dehydration, and Kleaf decline was apparently caused only by changes in the hydraulic properties of the extra-vascular compartment.

Recovery of hydraulic conductivity after the induction of embolisms was studied in woody stems of laurel (Laurus nobilis). Previous experiments confirming the recovery of hydraulic conductivity when xylem pressure potential was less than −1 MPa were repeated, and new experiments were done to investigate the changes in solute composition in xylem vessels during refilling. Xylem sap collected by perfusion of excised stem segments showed elevated levels of several ions during refilling. Stem segments were frozen in liquid N2 to view refilling vessels using cryoscanning electron microscopy. Vessels could be found in all three states of presumed refilling: (a) mostly water with a little air, (b) mostly air with a little water, or (c) water droplets extruding from vessel pits adjacent to living cells. Radiographic probe microanalysis of refilling vessels revealed nondetectable levels of dissolved solutes. Results are discussed in terms of proposed mechanisms of refilling in vessels while surrounding vessels were at a xylem pressure potential of less than −1 MPa. We have concluded that none of the existing paradigms explains the results.

Quasi-steady-state measurements of root hydraulic conductance (KR) of Olea oleaster Hoffmgg. et Link potted seedlings were performed using a pressure chamber with the aim of: (a) measuring the impact of different water-stress levels on a KR; (b) measuring the kinetics of KR recovery several days after soil rewetting; (c) relating changes in KR to changes in root anatomy and morphology. Increasing water-stress was applied in terms of ratio of leaf water potential (ΨL) measured at midday to that at zero turgor (ΨTLP), i.e. ΨL/ΨTLP=0·5, 1·0, 1·2, 1·6; KR was measured initially and at 24, 48, 72, 96 h after irrigation. Values of KR in seedlings stressed to ΨL/ΨTLP=1·2 increased for 48 h after irrigation from 0·23 to 0·97×10−5 kg s−1 m−2 MPa−1 i.e. from 16% to 66% of that measured in unstressed seedlings. A marked shift of the x-axis intercept of the straight line relating flow to pressure (zero flow at non-zero pressure) was recorded initially after irrigation and persisted up to 48 h. Recovery of KR occurred within 24 h after irrigation in seedlings at ΨL/ΨTLP=0·5 and 48 h later in those at ΨL/ΨTLP=1·0. Severe drought stress (ΨL/ΨTLP=1·6) caused anatomical changes to roots which formed a two-layered exodermis with thicker suberized walls and a three- to four-layered endodermis with completely suberized tangential walls. Recovery of KR in these roots required resumed growth of root tips and emergence of new lateral roots.

This work reports on experimental evidence for the role of ion-mediated changes of xylem hydraulic conductivity in the functional response of Solanum lycopersicum L. cv. Naomi to moderate salinity levels. Measurements were performed in fully developed 12-week-old plants grown in half-strength Hoagland solution (control, C-plants) or in the same solution added with 35 mM NaCl (NaCl-plants). NaCl-plants produced a significantly less but heavier leaves and fruits but had similar gas-exchange rates as control plants. Moreover, NaCl-plants showed higher vessel multiple fraction (FVM) than control plants. Xylem sap potassium and sodium concentrations were significantly higher in NaCl-plants than in control plants. When stems were perfused with 10 mM NaCl or KCl, the hydraulic conductance of NaCl plants was nearly 1.5 times higher than in control plants. Accordingly, stem hydraulic conductance measured in planta was higher in NaCl- than in control plants. Our data suggest that tomato plants grown under moderate salinity upregulate xylem sap [Na+] and [K+], as well as sensitivity of xylem hydraulics to sap ionic content, thus, increasing water transport capacity.

Xylem cavitation is a common occurrence in drought-stressed plants. Cavitation-induced embolism reduces xylem hydraulic conductivity (kxylem) and may lead to stomatal closure and reduction of photosynthetic rates. Recent studies have suggested that plants may compensate for kxylem loss through ion-mediated enhancement of the residual water transport capacity of functioning conduits. To test this hypothesis, field-grown laurel (Laurus nobilis L.) plants were subjected to mild drought stress by suspending irrigation. Drought treatment induced a significant increase in xylem embolism compared with control (well watered) plants. Xylem sap potassium concentration ([K+]) increased during the day both in control and water stressed plants. Midday values of sap [K+] were significantly higher in water stressed plants. The recorded increase in sap potassium concentration induced significant enhancement of residual kxylem when solutions with different [K+] were perfused through excised stems sampled in the field and measured in the laboratory. In planta measurements of stem hydraulic conductance revealed no change between water stressed plants and controls. Our data suggest that ion-mediated enhancement of residual kxylem buffered the actual loss of hydraulic conductance suffered by plants during the warmest hours of the day as well as under mild drought stress conditions.

The possible role of water expelled from cavitated xylem conduits in the rehydration of water-stressed leaves has been studied in one-year-old twigs of populus deltoides Bartr. Twigs were dehydrated in air. At desired values of leaf water potential (Ψl) (between near full turgor and -1.62 MPa), twigs were placed in black plastic bags for 1–2h. Leaf water content was measured every 3–5 min before bagging and every 10 min in the dark. Hydraulic conductivity and xylem cavitation were measured both in the open and in the dark. Cavitation was monitored as ultrasound acoustic emissions (AE). A critical Ψl value of -0.96 MPa was found, at which AE increased significantly while the leaf water deficit decreased by gain of water. Since the twigs were no longer attached to roots, it was concluded that water expelled from cavitated xylem conduits was transported to the leaves, thus contributing to their rehydration. Xylem cavitation is discussed in terms of a ‘leaf water deficit buffer mechanism’, under not very severe water stress conditions.

The ability of stems of Laurus nobilis (L.) to refill embolised xylem conduits was studied in plants both at optimal water supply (W) and under conditions of soil drought inducing xylem pressures (Px) of –1.54 (S1) and –2.35 MPa (S2). Starch depolymerisation in wood parenchyma was measured as percentage of cells ‘with high starch content’ (HSC-cells) counted under a microscope. HSC-cells decreased during embolism and increased again in refilled stems. A direct relationship was found between percentage of HSC-cells and Px, with HSC-cells between 65 and 75% of the total at Px ≥ –0.6 MPa, at which recovery from PLC was recorded. At low transpiration, starch re-appeared in wood parenchyma cells but only in plants that showed diurnal stomatal opening (W- and S1-plants). In S2-plants showing diurnal stomatal closure and nocturnal opening with Px between –1.2 to –2.4 MPa, HSC-cells were only 25% and plants did not recover from PLC. This finding suggests that (i) the Px threshold for embolism repair was ≥ –0.6 MPa, and (ii) impeded phloem loading limits starch content in wood parenchyma and embolism repair. We conclude that starch depolymerisation acts as a signal to phloem unloading sugars to embolised conduits thus generating the necessary osmotic gradients driving refilling.

Recent studies have shown that, in some species, xylem hydraulic conductivity (K(h)) increases with increasing cation concentration of xylem sap. Evidence indicates that K(h) increases as a result of the de-swelling of pit membrane pectins caused by cation neutralization of polygalacturonanes. We tested whether this ionic effect partly compensates for the embolism-induced loss of stem hydraulic conductivity (PLC) by increasing K(h) of functioning conduits. We report changes in PLC, leaf water status and potassium concentration ([K(+)]) of xylem sap measured in April and July in two evergreens (Ceratonia siliqua L. and Phytolacca dioica L.) and one deciduous tree (Platanus orientalis L.) growing in the field in Sicily. In summer, Ceratonia siliqua and Phytolacca dioica showed similar native embolism (PLC = 30-40%) and [K(+)] of xylem sap (14 to 17 mM), and K(h) of stems perfused with 10 to 25 mM KCl increased by 15 to 18% compared with K(h) of stems perfused with a low concentration of a multi-ionic solution. In contrast, native [K(+)] of sap of Platanus orientalis was 50% of that in the two evergreens in summer, with a parallel lack of detectable changes in PLC that was below 10% in both spring and summer. The ionic effect was PLC-dependent: the enhancement of K(h) induced by 10 to 25 mM KCl changed from 15% for fully hydrated stems to 50-75% for stems with PLC = 50%. In Ceratonia siliqua, PLC was less than 10% in spring and about 40% in summer; concurrently, xylem sap [K(+)] increased from 3 to about 15 mM. This [K(+)] at the recorded PLC would cause an increase in residual K(h) of about 30%. Hence, the actual reduction in water transport capacity of Ceratonia siliqua stems in summer is about 20%. Similar calculations for Phytolacca dioica suggest that the actual loss of hydraulic conductivity in stems of this species in summer would be only about 10%, and not 30% as suggested by hydraulic measurements performed in the laboratory. We conclude that an increase in [K(+)] of xylem sap might be involved in the up-regulation of residual K(h), thus substantially alleviating the embolism-induced reduction in leaf water supply.

Xylem embolism repair is preceded by starch depolymerisation in vessel-associated cells (VAC) of Laurus nobilis L. (laurel) twigs, but the primary signal triggering such a process is still unknown. We tested the hypothesis that conduit wall vibrations during cavitation may be sensed by VAC inducing starch-to-sugar conversion. Twigs of laurel from watered or stressed plants were exposed to ultrasound for 60 min to simulate acoustic waves emitted by cavitating conduits. Preliminary tests showed that ultrasound caused no damage to cell membrane integrity nor did they cause xylem embolism. The number of VAC with high starch content (HSC-cells) was estimated microscopically by counting the cells with more than 50% of their lumen filled with starch granules. Sonication had no effect on HSC-cells in twigs from watered plants while it induced a drop in the percentage HSC-cells from 80 to 40% in twigs from stressed plants, at the ultrasound source location. No effect was recorded in these twigs 20 mm from the ultrasound source. Sonication was a good simulator of cavitation in inducing starch depolymerisation which suggests a possible bio- physical nature for the signal initiating embolism repair.

The physiological significance of ion-mediated enhancement of xylem hydraulic conductivity (K h ) in planta has recently been questioned. The phenomenon has been suggested to be an artefact caused by the use of deionized water as a reference fluid during measurements of the impact of different ions on K h . In the present study, ion-mediated changes in K h were measured in twigs of five woody species during perfusion with 25 mM KCl compared with different reference fluids like deionized water, a commercial mineral water containing different ions (including 0.5 mM Ca 2+ ), and a 1 mM CaCl 2 solution. Both fully hydrated twigs and twigs with about 50% loss of hydraulic conductivity due to cavitation-induced embolism were tested. Adding 25 mM KCl to the three reference fluids caused K h to increase by about 20%. The KCl-mediated increase of K h was even larger (up to 100%) in embolized twigs. The presence of Ca 2+ in the reference solution decreased, but not suppressed, the KCl-mediated enhancement of K h in fully hydrated twigs of three species, but not in the other two species tested. Ca 2+ did not affect the K h response to KCl in embolized twigs. These data suggest that the recently reported suppression of the ‘ionic effect’ by the presence of calcium in the xylem sap is not a general phenomenon and that ion-mediated changes of K h may play a role in planta partially to compensate for cavitation-induced loss of xylem hydraulic conductivity.

Xylem recovery from embolism was studied in stems of Laurus nobilis L. that were induced to cavitate by combining negative xylem pressures with positive air pressures applied with a pressure collar. Xylem refilling was measured 2 and 20 min and 15 h after air pressure release in January, March and June when increasing percentages of wood parenchyma cells with high starch content (HSC-VAC) were counted (from 0% in January to 87.3% in June). In January, no xylem repair was measured. In June, stems refilled by 75% of previous conductivity loss with a parallel decrease of HSC-VAC. Xylem refilling was tested for stems with phloem either intact or excised by 20 and 50% and with phloem inactivated by girdling stems at both sides of the embolised segment. Stems with 50% of the cortex removed showed some recovery 15 h after embolism. Girdled stems did not recover from embolism and no starch depolymerisation was measured. Girdled stems where a radial mechanical pressure was applied for 20 min after embolism refilled in the same way as stems with intact phloem. Our conclusion is that phloem may export some signal for starch depolymerisation and this, in turn, would drive sugar efflux into embolised conduits with consequent osmotic water flows and refilling.

Measurements of Leaf Specific Conductivity (LSC) of the vertical water potential gradients and of vessels diameters in 1-year-old twigs of young olive trees have been made. LSC prooves to vary along the twigs differently from other species we have already studied in that it is significantly lower in the proximal twig's segment. Consequently, the water potential gradients are in this region higher so indicating that a higher resistance to flow exists. The frequency distribution of the vessels diameters shows that changes in the xylem hydraulic conductivity are mainly due to analogous changes in the relative number of large-diameter vessels. Also the nodes appear to behave as constriction zones of the xylem system. The functional significance of such constrictions in terms of a safety system against cavitation is discussed.