Is xylem of angiosperm leaves less resistant to embolism than branches? Insights from microCT, hydraulics, and anatomy.

According to the hydraulic vulnerability segmentation hypothesis, leaves are more vulnerable to decline of hydraulic conductivity than branches, but whether stem xylem is more embolism resistant than leaves remains unclear. Drought-induced embolism resistance of leaf xylem was investigated based on X-ray microcomputed tomography (microCT) for Betula pendula, Laurus nobilis, and Liriodendron tulipifera, excluding outside-xylem, and compared with hydraulic vulnerability curves for branch xylem. Moreover, bordered pit characters related to embolism resistance were investigated for both organs. Theoretical P50 values (i.e. the xylem pressure corresponding to 50% loss of hydraulic conductance) of leaves were generally within the same range as hydraulic P50 values of branches. P50 values of leaves were similar to branches for L. tulipifera (-2.01 versus -2.10 MPa, respectively), more negative for B. pendula (-2.87 versus -1.80 MPa), and less negative for L. nobilis (-6.4 versus -9.2 MPa). Despite more narrow conduits in leaves than branches, mean interconduit pit membrane thickness was similar in both organs, but significantly higher in leaves of B. pendula than in branches. This case study indicates that xylem shows a largely similar embolism resistance across leaves and branches, although differences both within and across organs may occur, suggesting interspecific variation with regard to the hydraulic vulnerability segmentation hypothesis.

Storage Compartments for Capillary Water Rarely Refill in an Intact Woody Plant.

Water storage is thought to play an integral role in the maintenance of whole-plant water balance. The contribution of both living and dead cells to water storage can be derived from rehydration and water-release curves on excised plant material, but the underlying tissue-specific emptying/refilling dynamics remain unclear. Here, we used x-ray computed microtomography to characterize the refilling of xylem fibers, pith cells, and vessels under both excised and in vivo conditions in Laurus nobilis In excised stems supplied with water, water uptake exhibited a biphasic response curve, and x-ray computed microtomography images showed that high water storage capacitance was associated with fiber and pith refilling as driven by capillary forces: fibers refilled more rapidly than pith cells, while vessel refilling was minimal. In excised stems that were sealed, fiber and pith refilling was associated with vessel emptying, indicating a link between tissue connectivity and water storage. In contrast, refilling of fibers, pith cells, and vessels was negligible in intact saplings over two time scales, 24 h and 3 weeks. However, those compartments did refill slowly when the shoot was covered to prevent transpiration. Collectively, our data (1) provide direct evidence that storage compartments for capillary water refill in excised stems but rarely under in vivo conditions, (2) highlight that estimates of capacitance from excised samples should be interpreted with caution, as certain storage compartments may not be utilized in the intact plant, and (3) question the paradigm that fibers play a substantial role in daily discharge/recharge of stem capacitance in an intact tree.

X-ray microtomography observations of xylem embolism in stems of Laurus nobilis are consistent with hydraulic measurements of percentage loss of conductance.

Drought-induced xylem embolism is a serious threat to plant survival under future climate scenarios. Hence, accurate quantification of species-specific vulnerability to xylem embolism is a key to predict the impact of climate change on vegetation. Low-cost hydraulic measurements of embolism rate have been suggested to be prone to artefacts, thus requiring validation by direct visualization of the functional status of xylem conduits using nondestructive imaging techniques, such as X-ray microtomography (microCT). We measured the percentage loss of conductance (PLC) of excised stems of Laurus nobilis (laurel) dehydrated to different xylem pressures, and compared results with direct observation of gas-filled vs water-filled conduits at a synchrotron-based microCT facility using a phase contrast imaging modality. Theoretical PLC calculated on the basis of microCT observations in stems of laurel dehydrated to different xylem pressures overall were in agreement with hydraulic measurements, revealing that this species suffers a 50% loss of xylem hydraulic conductance at xylem pressures averaging -3.5 MPa. Our data support the validity of estimates of xylem vulnerability to embolism based on classical hydraulic techniques. We discuss possible causes of discrepancies between data gathered in this study and those of recent independent reports on laurel hydraulics.

Excising stem samples underwater at native tension does not induce xylem cavitation.

Xylem resistance to water stress-induced cavitation is an important trait that is associated with drought tolerance of plants. The level of xylem cavitation experienced by a plant is often assessed as the percentage loss in conductivity (PLC) at different water potentials. Such measurements are constructed with samples that are excised underwater at native tensions. However, a recent study concluded that cutting conduits under significant tension induced cavitation, even when samples were held underwater during cutting. This resulted in artificially increased PLC because of what we have termed a 'tension-cutting artefact'. We tested the hypothesized tension-cutting artefact on five species by measuring PLC at native tension compared with after xylem tensions had been relaxed. Our results did not support the tension-cutting artefact hypothesis, as no differences were observed between native and relaxed samples in four of five species. In a fifth species (Laurus nobilis), differences between native and relaxed samples appear to be due to vessel refilling rather than a tension-cutting effect. We avoided the tension-cutting artefact by cutting samples to slightly longer than their measurement length and subsequent trimming of at least 0.5 cm of sample ends prior to measurement.

X-ray microtomography (micro-CT): a reference technology for high-resolution quantification of xylem embolism in trees.

As current methods for measuring xylem embolism in trees are indirect and prone to artefacts, there is an ongoing controversy over the capacity of trees to resist or recover from embolism. The debate will not end until we get direct visualization of the vessel content. Here, we propose desktop X-ray microtomography (micro-CT) as a reference direct technique to quantify xylem embolism and thus validate more widespread measurements based upon either hydraulic or acoustic methods. We used desktop micro-CT to measure embolism levels in dehydrated or centrifuged shoots of laurel - a long-vesseled species thought to display daily cycles of embolism formation and refilling. Our direct observations demonstrate that this Mediterranean species is highly resistant to embolism and is not vulnerable to drought-induced embolism in a normal range of xylem tensions. We therefore recommend that embolism studies in long-vesseled species should be validated by direct methods such as micro-CT to clear up any misunderstandings on their physiology.

Interactive ion-mediated sap flow regulation in olive and laurel stems: physicochemical characteristics of water transport via the pit structure.

Sap water is distributed and utilized through xylem conduits, which are vascular networks of inert pipes important for plant survival. Interestingly, plants can actively regulate water transport using ion-mediated responses and adapt to environmental changes. However, ionic effects on active water transport in vascular plants remain unclear. In this report, the interactive ionic effects on sap transport were systematically investigated for the first time by visualizing the uptake process of ionic solutions of different ion compositions (K+/Ca2+) using synchrotron X-ray and neutron imaging techniques. Ionic solutions with lower K+/Ca2+ ratios induced an increased sap flow rate in stems of Olea europaea L. and Laurus nobilis L. The different ascent rates of ionic solutions depending on K+/Ca2+ ratios at a fixed total concentration increases our understanding of ion-responsiveness in plants from a physicochemical standpoint. Based on these results, effective structural changes in the pit membrane were observed using varying ionic ratios of K+/Ca2+. The formation of electrostatically induced hydrodynamic layers and the ion-responsiveness of hydrogel structures based on Hofmeister series increase our understanding of the mechanism of ion-mediated sap flow control in plants.

Relax and refill: xylem rehydration prior to hydraulic measurements favours embolism repair in stems and generates artificially low PLC values.

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.

Does short-term potassium fertilization improve recovery from drought stress in laurel?

Xylem hydraulic conductance varies in response to changes in sap solute content, and in particular of potassium (K(+)) ion concentration. This phenomenon, known as the 'ionic effect', is enhanced in embolized stems, where it can compensate for cavitation-induced loss of hydraulic conductance. Previous studies have shown that in well-watered laurel plants (Laurus nobilis L.), potassium concentration of the xylem sap and plant hydraulic conductance increased 24 h after fertilization with KCl. The aim of this work was to test whether water-stressed laurel plants, grown under low potassium availability, could recover earlier from stress when irrigated with a KCl solution instead of potassium-free water. Two-year-old potted laurel seedlings were subjected to water stress by suspending irrigation until leaf conductance to water vapour (g(L)) dropped to ∼30% of its initial value and leaf water potential (ψ(L)) reached the turgor loss point (ψ(TLP)). Plants were then irrigated either with water or with 25 mM KCl and monitored for water status, gas exchange and plant hydraulics recovery at 3, 6 and 24 h after irrigation. No significant differences were found between the two experimental groups in terms of ψ(L), g(L), plant transpiration, plant hydraulic conductance or leaf-specific shoot hydraulic conductivity. Analysis of xylem sap potassium concentration showed that there were no significant differences between treatments, and potassium levels were similar to those of potassium-starved but well-watered plants. In conclusion, potassium uptake from the soil solution and/or potassium release to the xylem appeared to be impaired in water-stressed plants, at least up to 24 h after relief from water stress, so that fertilization after the onset of stress did not result in any short-term advantage for recovery from drought.

Coping with drought-induced xylem cavitation: coordination of embolism repair and ionic effects in three Mediterranean evergreens.

Embolism repair and ionic effects on xylem hydraulic conductance have been documented in different tree species. However, the diurnal and seasonal patterns of both phenomena and their actual role in plants' responses to drought-induced xylem cavitation have not been thoroughly investigated. This study provides experimental evidence of the ability of three Mediterranean species to maintain hydraulic function under drought stress by coordinating the refilling of xylem conduits and ion-mediated enhancement of stem hydraulic conductance (K stem). Vessel grouping indices and starch content in vessel-associated parenchyma cells were quantified to verify eventual correlations with ionic effects and refilling, respectively. Experiments were performed on stems of Ceratonia siliqua L., Olea europaea L. and Laurus nobilis L. Seasonal, ion-mediated changes in K stem (ΔK stem) and diurnal and/or seasonal embolism repair were recorded for all three species, although with different temporal patterns. Field measurements of leaf specific stem hydraulic conductivity showed that it remained quite constant during the year, despite changes in the levels of embolism. Starch content in vessel-associated parenchyma cells changed on diurnal and seasonal scales in L. nobilis and O. europaea but not in C. siliqua. Values of ΔK stem were significantly correlated with vessel multiple fraction values (the ratio of grouped vessels to total number of vessels). Our data suggest that the regulation of xylem water transport in Mediterranean plants relies on a close integration between xylem refilling and ionic effects. These functional traits apparently play important roles in plants' responses to drought-induced xylem cavitation.

Changes of xylem sap ionic content and stem hydraulics in response to irradiance in Laurus nobilis.

This study reports experimental evidence for light-mediated changes of stem hydraulic conductance (K(stem)) in field-grown laurel plants. Field measurements based on the evaporative flux method revealed that sun-exposed branches had 60% higher K(stem) with respect to shade-exposed branches. Xylem sap potassium concentration was approximately 3 mM as recorded in shaded branches and up to 12 mM in illuminated ones. Such a difference in [K(+)] proved to induce significant increase in xylem hydraulic conductance of excised twigs when artificially perfused with various solutions, as a likely consequence of the interaction of cations with the pectic matrix of pit membranes (the so-called 'ionic effect'). We propose that this mechanism provides plants with a large potential for fine regulation of water flow towards different parts of the canopy exposed to different environmental conditions with the result of optimizing light and water utilization.

Gas exchange characteristics of a Canarian laurel forest tree species (Laurus azorica) in relation to environmental conditions and leaf canopy position.

Diurnal courses of gas exchange were measured over a 1-year period in fully expanded current-year leaves in the upper (sun-exposed, 18 m above ground) and the lower (shaded, 12 m above ground) canopy of Laurus azorica (Seub.) Franco, a major canopy species of the Canarian laurel forest in Tenerife, Canary Islands, Spain. Laurus azorica exhibited high leaf plasticity in gas exchange characteristics, with a maximum carbon assimilation rate (Amax) of shade leaves about 50% that of sun leaves. This difference reflects the high leaf area index (LAI) of the stand and the correspondingly sharp light attenuation with increasing canopy depth. In sun leaves, Amax peaked at about 11 micromol m-2 s-1 and maximum transpiration (E) was about 8 mmol m-2 s-1, which corresponded with a maximum stomatal conductance (gs) of about 650 mmol m-2 s-1. Mean maximum instantaneous water-use efficiency (WUE) was 1.5 mmol mol-1 and the mean maximum A/gs was 20-35 micromol mol-1. Mean minimum internal CO2 concentration (Ci) was 225 micromol mol-1. Although high air vapor pressure deficit (VPD) caused a small decrease in gs, it remained high enough to maintain relatively high A and E. These gas exchange characteristics indicate a non-conservative use of water, which is appropriate for a species subject to droughts that are mild or of short duration. In this respect, Laurus azorica differs from its congener, L. nobilis L., of the Mediterranean region and other shrubs growing in Mediterranean-type climates in California and Chile that have to withstand more severe or more prolonged droughts.