Using the Aqueous Phase Produced from Hydrothermal Carbonization Process of Brown Seaweed to Improve the Growth of Phaseolus vulgaris.

Seaweeds are considered a biomass for third-generation biofuel, and hydrothermal carbonization (HTC) is a valuable process for efficiently disposing of the excess of macroalgae biomass for conversion into multiple value-added products. However, the HTC process produces a liquid phase to be disposed of. The present study aims to investigate the effects of seed-priming treatment with three HTC-discarded liquid phases (namely AHL180, AHL240, and AHL300), obtained from different experimental procedures, on seed germination and plant growth and productivity of Phaseolus vulgaris L. To disentangle the osmotic effects from the use of AHL, isotonic solutions of polyethylene glycol (PEG) 6000 have also been tested. Seed germination was not affected by AHL seed-priming treatment. In contrast, PEG-treated samples showed significantly lower seed germination success. AHL-treated samples showed changes in plant biomass: higher shoot biomass was recorded especially in AHL180 samples. Conversely, AHL240 and AHL300 samples showed higher root biomass. The higher plant biomass values recorded in AHL-treated samples were the consequence of higher values of photosynthesis rate and water use efficiency, which, in turn, were related to higher stomatal density. Recorded data strongly support the hypothesis of the AHL solution reuse in agriculture in the framework of resource management and circular green economy.

The optical method based on gas injection overestimates leaf vulnerability to xylem embolism in three woody species.

Plant hydraulic traits related to leaf drought tolerance, like the water potential at turgor loss point (TLP) and the water potential inducing 50% loss of hydraulic conductance (P50), are extremely useful to predict the potential impacts of drought on plants. While novel techniques have allowed the inclusion of TLP in studies targeting a large group of species, fast and reliable protocols to measure leaf P50 are still lacking. Recently, the optical method coupled with the gas injection (GI) technique has been proposed as a possibility to speed up the P50 estimation. Here, we present a comparison of leaf optical vulnerability curves (OVcs) measured in three woody species, namely Acer campestre (Ac), Ostrya carpinifolia (Oc) and Populus nigra (Pn), based on bench dehydration (BD) or GI of detached branches. For Pn, we also compared optical data with direct micro-computed tomography (micro-CT) imaging in both intact saplings and cut shoots subjected to BD. Based on the BD procedure, Ac, Oc and Pn had P50 values of -2.87, -2.47 and -2.11 MPa, respectively, while the GI procedure overestimated the leaf vulnerability (-2.68, -2.04 and -1.54 MPa for Ac, Oc and Pn, respectively). The overestimation was higher for Oc and Pn than for Ac, likely reflecting the species-specific vessel lengths. According to micro-CT observations performed on Pn, the leaf midrib showed none or very few embolized conduits at -1.2 MPa, consistent with the OVcs obtained with the BD procedure but at odds with that derived on the basis of GI. Overall, our data suggest that coupling the optical method with GI might not be a reliable technique to quantify leaf hydraulic vulnerability since it could be affected by the 'open-vessel' artifact. Accurate detection of xylem embolism in the leaf vein network should be based on BD, preferably of intact up-rooted plants.

Critical water contents at leaf, stem and root level leading to irreversible drought-induced damage in two woody and one herbaceous species.

Plant water content is a simple and promising parameter for monitoring drought-driven plant mortality risk. However, critical water content thresholds leading to cell damage and plant failure are still unknown. Moreover, it is unclear whether whole-plant or a specific organ water content is the most reliable indicator of mortality risk. We assessed differences in dehydration thresholds in leaf, stem and root samples, hampering the organ-specific rehydration capacity and increasing the mortality risk. We also tested eventual differences between a fast experimental dehydration of uprooted plants, compared to long-term water stress induced by withholding irrigation in potted plants. We investigated three species with different growth forms and leaf habits i.e., Helianthus annuus (herbaceous), Populus nigra (deciduous tree) and Quercus ilex (evergreen tree). Results obtained by the two dehydration treatments largely overlapped, thus validating bench dehydration as a fast but reliable method to assess species-specific critical water content thresholds. Regardless of the organ considered, a relative water content value of 60% induced significant cell membrane damage and loss of rehydration capacity, thus leading to irreversible plant failure and death.

Delaying drought-driven leaf cell damage may be the key trait of invasive trees ensuring their success in the Mediterranean basin.

Invasive alien species (IAS) threaten the biodiversity richness of the Mediterranean basin, a drought-prone region. However, our knowledge on the adaptive strategies of IAS for facing Mediterranean drought summers is still incomplete. The aim of the present study is to compare the water relations and the critical relative water content (RWC) values leading to loss of cell rehydration capacity of two Mediterranean basin IAS (i.e., Ailanthus altissima (Mill.) Swingle and Robinia pseudoacacia L.) versus two co-occurring native species (i.e., Fraxinus ornus L. and Quercus pubescens Willd.). Study IAS showed higher values of water potential at turgor loss point and osmotic potential at full turgor, lower values of modulus of elasticity and leaf mass area but higher photosynthesis rate, even during the summer, with respect to the Mediterranean native species. These findings supported the hypothesis that IAS are characterized by a resource acquisitive strategy coupled with a safety-efficiency trade-off, compared with Mediterranean native species. However, similar leaf RWC thresholds leading to loss of cell rehydration capacity were recorded in the two groups of species. Moreover, IAS showed higher saturated water content and capacitance values compared with the co-occurring species. Overall, our results suggest that the success of Mediterranean IAS is driven by their ability to delay dehydration damage of mesophyll cells during Mediterranean summer drought, thereby supporting their distinctive high carbon assimilation rate.

When Water Availability Is Low, Two Mediterranean Salvia Species Rely on Root Hydraulics.

Increase in severity and frequency of drought events is altering plant community composition, exposing biomes to a higher risk of biodiversity losses. This is exacerbated in the most fragile areas as Mediterranean biome. Thus, identifying plant traits for forecasting species with a high risk of drought-driven mortality is particularly urgent. In the present study, we investigated the drought resistance strategy of two Mediterranean native species: Salvia ceratophylloides Ard. (Sc) and Salvia officinalis L. (So) by considering the impact of drought-driven water content decline on plant hydraulics. Well-watered samples of Sc displayed higher leaf and stemsaturated water content and lower shoot biomass than So samples, but similar root biomass. In response to drought, Sc showed a conservative water use strategy, as the prompt stomatal closure and leaves shedding suggested. A drought-tolerant mechanism was confirmed in So samples. Nevertheless, Sc and So showed similar drought-driven plant hydraulic conductance (Kplant) recover ability. Root hydraulic traits played a key role to reach this goal. Relative water content as well as loss of cell rehydration capability and membrane damages, especially of stem and root, were good proxies of drought-driven Kplant decline.

Too dry to survive: Leaf hydraulic failure in two Salvia species can be predicted on the basis of water content.

Global warming is exposing plants to increased risks of drought-driven mortality. Recent advances suggest that hydraulic failure is a key process leading to plant death, and the identification of simple and reliable proxies of species-specific risk of irreversible hydraulic damage is urgently required. We assessed the predictive power of leaf water content and shrinkage for monitoring leaf hydraulic failure in two Mediterranean native species, Salvia ceratophylloides (Sc) and S. officinalis (So). The study species showed significant differences in relative water content (RWC) thresholds inducing loss of rehydration capacity, as well as leaf hydraulic conductance (KL) impairment. Sc turned out to be more resistant to drought than So. However, Sc and So showed different leaf saturated water content values, so that different RWC values actually corresponded to similar absolute leaf water content. Our findings suggest that absolute leaf water content and leaf water potential, but not RWC, are reliable parameters for predicting the risk of leaf hydraulic impairment of two Salvia species, and their potential risk of irreversible damage under severe drought. Moreover, the lack of any KL decline until the turgor loss point in Sc, coupled to consistent leaf shrinkage, rejects the hypothesis to use leaf shrinkage as a proxy to predict KL vulnerability, at least in species with high leaf capacitance. Robust linear correlations between KL decline and electrolyte leakage measurements suggested a role of membrane damage in driving leaf hydraulic collapse.

The extra-vascular water pathway regulates dynamic leaf hydraulic decline and recovery in Populus nigra.

Leaf hydraulic conductance (Kleaf ) is highly dynamic and typically responds to changes in water status and irradiance. However, the relative contribution of vascular (Kx ) and extra-vascular (Kox ) water pathways to Kleaf changes in response to water potential decline and recovery in function of light conditions remains poorly investigated. We investigated the dynamic responses of leaf hydraulics in Populus nigra L. by measuring Kleaf , Kx , and Kox changes under drought and upon recovery. Measurements were done at both low and high irradiance (LI and HI, respectively). Kleaf increased and became more vulnerable to dehydration under HI conditions than LI, due to marked changes of Kox . After re-watering, Kleaf recovered in parallel with Kox recovery, but Kleaf response to irradiance remained inhibited. Strong correlations between Kleaf and drought-induced membrane damage demonstrated the relevance of the cell-to-cell water pathway in driving the dynamic responses of Kleaf under drought and recovery. Our findings highlight the importance of coordination between water and light availability in modulating the overall Kleaf response to environmental conditions.

A Leaf Selfie: Using a Smartphone to Quantify Leaf Vulnerability to Hydraulic Dysfunction.

Accurate predictions of species distribution under current and future climate conditions require modeling efforts based on clear mechanistic relationships between climate variables and plant physiological functions. Vulnerability of leaves to xylem embolism is a key mechanistic trait that might be included in these modeling efforts. Here, we propose a simple set-up to measure leaf vulnerability to embolism on the basis of the optical method using a smartphone, a light source, and a notebook. Our data show that this proposed set-up can adequately quantify the vulnerability to xylem embolism of leaf major veins in Populus nigra and Ostrya carpinifolia, producing values consistent with those obtained in temperate tree species with other methods, allowing virtually any laboratory to quantify species-specific drought tolerance on the basis of a sound mechanistic trait.

Non-structural carbohydrate and hydraulic dynamics during drought and recovery in Fraxinus ornus and Ostrya carpinifolia saplings.

The maintenance of hydraulic function during and after a drought event is crucial for tree survival, but the importance of non-structural carbohydrates (NSCs) in the recovery phase is still debated. We tested whether higher NSC availability facilitates post-drought hydraulic recovery, by applying a short-term drought (Sdr) and a long-term drought combined with shading (Ldr+sh) in Fraxinus ornus and Ostrya carpinifolia. Plants were then re-irrigated and recovery was checked 24 h later, by measuring water potential, stem percentage loss of hydraulic conductance (PLC) and NSC content. The relative magnitude of hydraulic and carbon constraints was also assessed in desiccated plants. During drought, PLC increased only in F. ornus, while it was maintained almost constant in O. carpinifolia due to tighter stomatal control of xylem pressure (i.e. more isohydric). In F. ornus, only Sdr plants maintained high NSC contents at the end of drought and, when re-irrigated, recovered PLC to control values. Whereas hydraulic failure was ubiquitous, only F. ornus depleted NSC reserves at mortality. Our results suggest that preserving higher NSC content at the end of a drought can be important for the hydraulic resilience of trees.

Vulnerability to xylem embolism correlates to wood parenchyma fraction in angiosperms but not in gymnosperms.

Understanding which structural and functional traits are linked to species' vulnerability to embolism formation (P50) may provide fundamental knowledge on plant strategies to maintain an efficient water transport. We measured P50, wood density (WD), mean conduit area, conduit density, percentage areas occupied by vessels, parenchyma cells (PATOT) and fibers (FA) on branches of angiosperm and gymnosperm species. Moreover, we compiled a dataset of published hydraulic and anatomical data to be compared with our results. Species more vulnerable to embolism had lower WD. In angiosperms, the variability in WD was better explained by PATOT and FA, which were highly correlated. Angiosperms with a higher P50 (less negative) had a higher amount of PATOT and total amount of nonstructural carbohydrates. Instead, in gymnosperms, P50 vs PATOT was not significant. The correlation between PATOT and P50 might have a biological meaning and also suggests that the causality of the commonly observed relationship of WD vs P50 is indirect and dependent on the parenchyma fraction. Our study suggests that angiosperms have a potential active embolism reversal capacity in which parenchyma has an important role, while in gymnosperms this might not be the case.

Hydraulic recovery from xylem embolism in excised branches of twelve woody species: Relationships with parenchyma cells and non-structural carbohydrates.

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 (P50) 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 ∼50% loss of hydraulic conductivity was recorded (PLC ∼50%). Hydraulic recovery (ΔPLC) and NSC content was, then, assessed after 1h of rehydration. Species showed a different ability to recover their hydraulic conductivity from PLC∼50%. Removing the bark in the species showing hydraulic recovery inhibited the embolism reversal. Strong correlations between the ΔPLC 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.

Effects of prolonged drought on stem non-structural carbohydrates content and post-drought hydraulic recovery in Laurus nobilis L.: The possible link between carbon starvation and hydraulic failure.

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.

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.

The contribution of vascular and extra-vascular water pathways to drought-induced decline of leaf hydraulic conductance.

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.

Species-specific reversal of stem xylem embolism after a prolonged drought correlates to endpoint concentration of soluble sugars.

Recent reports on tree mortality associated with anomalous drought and heat have raised interest into processes underlying tree resistance/resilience to water stress. Hydraulic failure and carbon starvation have been proposed as main causes of tree decline, with recent theories treating water and carbon metabolism as interconnected processes. We subjected young plants of two native (Quercus pubescens [Qp] and Prunus mahaleb [Pm]) and two invasive (Robinia pseudoacacia [Rp] and Ailanthus altissima [Aa]) woody angiosperms to a prolonged drought leading to stomatal closure and xylem embolism, to induce carbon starvation and hydraulic failure. At the end of the treatment, plants were measured for embolism rates and NSC content, and re-irrigated to monitor recovery of xylem hydraulics. Data highlight different hydraulic strategies in native vs invasive species under water stress, and provide physiological explanations for species-specific impacts of recent severe droughts. Drought-sensitive species (Qp and Rp) suffered high embolism rates and were unable to completely refill xylem conduits upon restoration of water availability. Species that better survived recent droughts were able to limit embolism build-up (Pm) or efficiently restored hydraulic functionality after irrigation (Aa). Species-specific capacity to reverse xylem embolism correlated to stem-level concentration of soluble carbohydrates, but not to starch content.

Diurnal changes in embolism rate in nine dry forest trees: relationships with species-specific xylem vulnerability, hydraulic strategy and wood traits.

Recent studies have reported correlations between stem sapwood capacitance (C(wood)) and xylem vulnerability to embolism, but it is unclear how C(wood) relates to the eventual ability of plants to reverse embolism. We investigated possible functional links between embolism reversal efficiency, C(wood), wood density (WD), vulnerability to xylem embolism and hydraulic safety margins in nine woody species native to dry sclerophyllous forests with different degrees of iso versus anisohydry. Substantial inter-specific differences in terms of seasonal/diurnal changes of xylem and leaf water potential, maximum diurnal values of transpiration rate and xylem vulnerability to embolism formation were recorded. Significant diurnal changes in percentage loss of hydraulic conductivity (PLC) were recorded for five species. Significant correlations were recorded between diurnal PLC changes and P50 and P88 values (i.e., xylem pressure inducing 50 and 88% PLC, respectively) as well as between diurnal PLC changes and safety margins referenced to P50 and P88. WD was linearly correlated with minimum diurnal leaf water potential, diurnal PLC changes and wood capacitance across all species. In contrast, significant relationships between P50, safety margin values referenced to P50 and WD were recorded only for the isohydric species. Functional links between diurnal changes in PLC, hydraulic strategies and WD and C(wood) are discussed.

Plant performance on Mediterranean green roofs: interaction of species-specific hydraulic strategies and substrate water relations.

Recent studies have highlighted the ecological, economic and social benefits assured by green roof technology to urban areas. However, green roofs are very hostile environments for plant growth because of shallow substrate depths, high temperatures and irradiance and wind exposure. This study provides experimental evidence for the importance of accurate selection of plant species and substrates for implementing green roofs in hot and arid regions, like the Mediterranean area. Experiments were performed on two shrub species (Arbutus unedo L. and Salvia officinalis L.) grown in green roof experimental modules with two substrates slightly differing in their water retention properties, as derived from moisture release curves. Physiological measurements were performed on both well-watered and drought-stressed plants. Gas exchange, leaf and xylem water potential and also plant hydraulic conductance were measured at different time intervals following the last irrigation. The substrate type significantly affected water status. Arbutus unedo and S. officinalis showed different hydraulic responses to drought stress, with the former species being substantially isohydric and the latter one anisohydric. Both A. unedo and S. officinalis were found to be suitable species for green roofs in the Mediterranean area. However, our data suggest that appropriate choice of substrate is key to the success of green roof installations in arid environments, especially if anisohydric species are employed.

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.

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.