Role of hydraulic traits in stomatal regulation of transpiration under different vapour pressure deficits across five Mediterranean tree crops.

The differential stomatal regulation of transpiration among plant species in response to water deficit is not fully understood, although several hydraulic traits have been reported to influence it. This knowledge gap is partly due to a lack of direct and concomitant experimental data on transpiration, stomatal conductance, and hydraulic traits. We measured sap flux density (Js), stomatal conductance (gs), and different hydraulic traits in five crop species. Our aim was to contribute to establishing the causal relationship between water consumption and its regulation using a hydraulic trait-based approach. The results showed that the species-specific regulation of Js by gs was overall coordinated with the functional hydraulic traits analysed. Particularly relevant was the negative and significant relationship found between the Huber value (Hv) and its functional analogue ratio between maximum Js and gs (Jsmax/gsmax) which can be understood as a compensation to maintain the hydraulic supply to the leaves. The Hv was also significantly related to the slope of the relationship between gs and Js response to vapour pressure deficit and explained most of its variability, adding up to evidence recognizing Hv as a major trait in plant water relations. Thus, a hydraulic basis for regulation of tree water use should be considered.

On the path from xylem hydraulic failure to downstream cell death.

Xylem hydraulic failure (HF) has been identified as a ubiquitous factor in triggering drought-induced tree mortality through the damage induced by the progressive dehydration of plant living cells. However, fundamental evidence of the mechanistic link connecting xylem HF to cell death has not been identified yet. The main aim of this study was to evaluate, at the leaf level, the relationship between loss of hydraulic function due to cavitation and cell death under drought conditions and discern how this relationship varied across species with contrasting resistances to cavitation. Drought was induced by withholding water from potted seedlings, and their leaves were sampled to measure their relative water content (RWC) and cell mortality. Vulnerability curves to cavitation at the leaf level were constructed for each species. An increment in cavitation events occurrence precedes the onset of cell mortality. A variation in cells tolerance to dehydration was observed along with the resistance to cavitation. Overall, our results indicate that the onset of cellular mortality occurs at lower RWC than the one for cavitation indicating the role of cavitation in triggering cellular death. They also evidenced a critical RWC for cellular death varying across species with different cavitation resistance.

Leaf water potential measurements using the pressure chamber: Synthetic testing of assumptions towards best practices for precision and accuracy.

Leaf water potential (ψleaf ), typically measured using the pressure chamber, is the most important metric of plant water status, providing high theoretical value and information content for multiple applications in quantifying critical physiological processes including drought responses. Pressure chamber measurements of ψleaf (ψleafPC ) are most typical, yet, the practical complexity of the technique and of the underlying theory has led to ambiguous understanding of the conditions to optimize measurements. Consequently, specific techniques and precautions diversified across the global research community, raising questions of reliability and repeatability. Here, we surveyed specific methods of ψleafPC from multiple laboratories, and synthesized experiments testing common assumptions and practices in ψleafPC for diverse species: (i) the need for equilibration of previously transpiring leaves; (ii) leaf storage before measurement; (iii) the equilibration of ψleaf for leaves on bagged branches of a range of dehydration; (iv) the equilibration of ψleaf across the lamina for bagged leaves, and the accuracy of measuring leaves with artificially 'elongated petioles'; (v) the need in ψleaf measurements for bagging leaves and high humidity within the chamber; (vi) the need to avoid liquid water on leaf surfaces; (vii) the use of 'pulse' pressurization versus gradual pressurization; and (viii) variation among experimenters in ψleafPC determination. Based on our findings we provide a best practice protocol to maximise accuracy, and provide recommendations for ongoing species-specific tests of important assumptions in future studies.

Mechanisms of xylem hydraulic recovery after drought in Eucalyptus saligna.

The mechanisms by which woody plants recover xylem hydraulic capacity after drought stress are not well understood, particularly with regard to the role of embolism refilling. We evaluated the recovery of xylem hydraulic capacity in young Eucalyptus saligna plants exposed to cycles of drought stress and rewatering. Plants were exposed to moderate and severe drought stress treatments, with recovery monitored at time intervals from 24 h to 6 months after rewatering. The percentage loss of xylem vessels due to embolism (PLV) was quantified at each time point using microcomputed tomography with stem water potential (Ψx ) and canopy transpiration (Ec ) measured before scans. Plants exposed to severe drought stress suffered high levels of embolism (47.38% ± 10.97% PLV) and almost complete canopy loss. No evidence of embolism refilling was observed at 24 h, 1 week, or 3 weeks after rewatering despite rapid recovery in Ψx . Recovery of hydraulic capacity was achieved over a 6-month period by growth of new xylem tissue, with canopy leaf area and Ec recovering over the same period. These findings indicate that E. saligna recovers slowly from severe drought stress, with potential for embolism to persist in the xylem for many months after rainfall events.

Visual and hydraulic techniques produce similar estimates of cavitation resistance in woody species.

Hydraulic failure of the plant vascular system is a principal cause of forest die-off under drought. Accurate quantification of this process is essential to our understanding of the physiological mechanisms underpinning plant mortality. Imaging techniques increasingly are applied to estimate xylem cavitation resistance. These techniques allow for in situ measurement of embolism formation in real time, although the benefits and trade-offs associated with different techniques have not been evaluated in detail. Here we compare two imaging methods, microcomputed tomography (microCT) and optical vulnerability (OV), to standard hydraulic methods for measurement of cavitation resistance in seven woody species representing a diversity of major phylogenetic and xylem anatomical groups. Across the seven species, there was strong agreement between cavitation resistance values (P50 ) estimated from visualization techniques (microCT and OV) and between visual techniques and hydraulic techniques. The results indicate that visual techniques provide accurate estimates of cavitation resistance and the degree to which xylem hydraulic function is impacted by embolism. Results are discussed in the context of trade-offs associated with each technique and possible causes of discrepancy between estimates of cavitation resistance provided by visual and hydraulic techniques.

Declining root water transport drives stomatal closure in olive under moderate water stress.

Efficient water transport from soil to leaves sustains stomatal opening and steady-state photosynthesis. The aboveground portion of this pathway is well-described, yet the roots and their connection with the soil are still poorly understood due to technical limitations. Here we used a novel rehydration technique to investigate changes in the hydraulic pathway between roots and soil and within the plant body as individual olive plants were subjected to a range of water stresses. Whole root hydraulic resistance (including the radial pathway from xylem to the soil-root interface) constituted 81% of the whole-plant resistance in unstressed plants, increasing to > 95% under a moderate level of water stress. The decline in this whole root hydraulic conductance occurred in parallel with stomatal closure and contributed significantly to the reduction in canopy conductance according to a hydraulic model. Our results demonstrate that losses in root hydraulic conductance, mainly due to a disconnection from the soil during moderate water stress in olive plants, are profound and sufficient to induce stomatal closure before cavitation occurs. Future studies will determine whether this core regulatory role of root hydraulics exists more generally among diverse plant species.

Hydraulic Traits Emerge as Relevant Determinants of Growth Patterns in Wild Olive Genotypes Under Water Stress.

The hydraulic traits of plants, or the efficiency of water transport throughout the plant hydraulic system, could help to anticipate the impact of climate change and improve crop productivity. However, the mechanisms explaining the role of hydraulic traits on plant photosynthesis and thus, plant growth and yield, are just beginning to emerge. We conducted an experiment to identify differences in growth patterns at leaf, root and whole plant level among four wild olive genotypes and to determine whether hydraulic traits may help to explain such differences through their effect on photosynthesis. We estimated the relative growth rate (RGR), and its components, leaf gas exchange and hydraulic traits both at the leaf and whole-plant level in the olive genotypes over a full year. Photosynthetic capacity parameters were also measured. We observed different responses to water stress in the RGRs of the genotypes studied being best explained by changes in the net CO2 assimilation rate (NAR). Further, net photosynthesis, closely related to NAR, was mainly determined by hydraulic traits, both at leaf and whole-plant levels. This was mediated through the effects of hydraulic traits on stomatal conductance. We observed a decrease in leaf area: sapwood area and leaf area: root area ratios in water-stressed plants, which was more evident in the olive genotype Olea europaea subsp. guanchica (GUA8), whose RGR was less affected by water deficit than the other olive genotypes. In addition, at the leaf level, GUA8 water-stressed plants presented a better photosynthetic capacity due to a higher mesophyll conductance to CO2 and a higher foliar N. We conclude that hydraulic allometry adjustments of whole plant and leaf physiological response were well coordinated, buffering the water stress experienced by GUA8 plants. In turn, this explained their higher relative growth rates compared to the rest of the genotypes under water-stress conditions.

Mapping xylem failure in disparate organs of whole plants reveals extreme resistance in olive roots.

The capacity of plant species to resist xylem cavitation is an important determinant of resistance to drought, mortality thresholds, geographic distribution and productivity. Unravelling the role of xylem cavitation vulnerability in plant evolution and adaptation requires a clear understanding of how this key trait varies between the tissues of individuals and between individuals of species. Here, we examine questions of variation within individuals by measuring how cavitation moves between organs of individual plants. Using multiple cameras placed simultaneously on roots, stems and leaves, we were able to record systemic xylem cavitation during drying of individual olive plants. Unlike previous studies, we found a consistent pattern of root > stem > leaf in terms of xylem resistance to cavitation. The substantial variation in vulnerability to cavitation, evident among individuals, within individuals and within tissues of olive seedlings, was coordinated such that plants with more resistant roots also had more resistant leaves. Preservation of root integrity means that roots can continue to supply water for the regeneration of drought-damaged aerial tissues after post-drought rain. Furthermore, coordinated variation in vulnerability between leaf, stem and root in olive plants suggests a strong selective pressure to maintain a fixed order of cavitation during drought.

Most stomatal closure in woody species under moderate drought can be explained by stomatal responses to leaf turgor.

Reduced stomatal conductance (gs ) during soil drought in angiosperms may result from effects of leaf turgor on stomata and/or factors that do not directly depend on leaf turgor, including root-derived abscisic acid (ABA) signals. To quantify the roles of leaf turgor-mediated and leaf turgor-independent mechanisms in gs decline during drought, we measured drought responses of gs and water relations in three woody species (almond, grapevine and olive) under a range of conditions designed to generate independent variation in leaf and root turgor, including diurnal variation in evaporative demand and changes in plant hydraulic conductance and leaf osmotic pressure. We then applied these data to a process-based gs model and used a novel method to partition observed declines in gs during drought into contributions from each parameter in the model. Soil drought reduced gs by 63-84% across species, and the model reproduced these changes well (r(2) = 0.91, P < 0.0001, n = 44) despite having only a single fitted parameter. Our analysis concluded that responses mediated by leaf turgor could explain over 87% of the observed decline in gs across species, adding to a growing body of evidence that challenges the root ABA-centric model of stomatal responses to drought.

Role of leaf hydraulic conductance in the regulation of stomatal conductance in almond and olive in response to water stress.

The decrease of stomatal conductance (gs) is one of the prime responses to water shortage and the main determinant of yield limitation in fruit trees. Understanding the mechanisms related to stomatal closure in response to imposed water stress is crucial for correct irrigation management. The loss of leaf hydraulic functioning is considered as one of the major factors triggering stomatal closure. Thus, we conducted an experiment to quantify the dehydration response of leaf hydraulic conductance (Kleaf) and its impact on gs in two Mediterranean fruit tree species, one deciduous (almond) and one evergreen (olive). Our hypothesis was that a higher Kleaf would be associated with a higher gs and that the reduction in Kleaf would predict the reduction in gs in both species. We measured Kleaf in olive and almond during a cycle of irrigation withholding. We also compared the results of two methods to measure Kleaf: dynamic rehydration kinetics and evaporative flux methods. In addition, determined gs, leaf water potential (Ψleaf), vein density, photosynthetic capacity and turgor loss point. Results showed that gs was higher in almond than in olive and so was Kleaf (Kmax = 4.70 and 3.42 mmol s(-1) MPa(-1) m(-2), in almond and olive, respectively) for Ψleaf > -1.2 MPa. At greater water stress levels than -1.2 MPa, however, Kleaf decreased exponentially, being similar for both species, while gs was still higher in almond than in olive. We conclude that although the Kleaf decrease with increasing water stress does not drive unequivocally the gs response to water stress, Kleaf is the variable most strongly related to the gs response to water stress, especially in olive. Other variables such as the increase in abscisic acid (ABA) may be playing an important role in gs regulation, although in our study the gs-ABA relationship did not show a clear pattern.

Vulnerability to cavitation in Olea europaea current-year shoots: further evidence of an open-vessel artifact associated with centrifuge and air-injection techniques.

Different methods have been devised to analyze vulnerability to cavitation of plants. Although a good agreement between them is usually found, some discrepancies have been reported when measuring samples from long-vesseled species. The aim of this study was to evaluate possible artifacts derived from different methods and sample sizes. Current-year shoot segments of mature olive trees (Olea europaea), a long-vesseled species, were used to generate vulnerability curves (VCs) by bench dehydration, pressure collar and both static- and flow-centrifuge methods. For the latter, two different rotors were used to test possible effects of the rotor design on the curves. Indeed, high-resolution computed tomography (HRCT) images were used to evaluate the functional status of xylem at different water potentials. Measurements of native embolism were used to validate the methods used. The pressure collar and the two centrifugal methods showed greater vulnerability to cavitation than the dehydration method. The shift in vulnerability thresholds in centrifuge methods was more pronounced in shorter samples, supporting the open-vessel artifact hypothesis as a higher proportion of vessels were open in short samples. The two different rotor designs used for the flow-centrifuge method revealed similar vulnerability to cavitation. Only the bench dehydration or HRCT methods produced VCs that agreed with native levels of embolism and water potential values measured in the field.