In situ characterisation of whole-plant stomatal responses to VPD using leaf optical dendrometry.

Vapour pressure deficit (VPD) plays a crucial role in regulating plant carbon and water fluxes due to its influence on stomatal behaviour and transpiration. Yet, characterising stomatal responses of the whole plant to VPD remains challenging due to methodological limitations. Here, we develop a novel method for in situ assessment of whole-plant stomatal responses (gc ) to VPD in the herbaceous plant Tanacetum cinerariifolium. To do this, we examine the relationship between daytime VPD and the corresponding soil-stem water potential gradient (ΔΨ) monitored using the optical dendrometry in well-hydrated plants under nonlimiting light in both glasshouse and field conditions. In glasshouse plants, ΔΨ increased proportionally with the VPD up to a threshold of 1.53 kPa, beyond which the slope decreased, suggesting a two-phase response in gc . This pattern aligned with corresponding gravimetrically measured gc behaviour, which also showed a decline when VPD exceeded a similar threshold. This response was then compared with that of field plants monitored using the optical dendrometry technique over a growing season under naturally variable VPD conditions and nonlimiting light and water supply. Field plants exhibited a similar threshold-type response to VPD but were more sensitive than glasshouse individuals with a VPD threshold of 0.74 kPa. The results showed that whole-plant gc responses to VPD can be characterised optically in T. cinerariifolium, introducing a new tool for the monitoring and characterisation of stomatal behaviour in situ.

Combined heat and water stress leads to local xylem failure and tissue damage in pyrethrum flowers.

Flowers are critical for angiosperm reproduction and the production of food, fiber, and pharmaceuticals, yet for unknown reasons, they appear particularly sensitive to combined heat and drought stress. A possible explanation for this may be the co-occurrence of leaky cuticles in flower petals and a vascular system that has a low capacity to supply water and is prone to failure under water stress. These characteristics may render reproductive structures more susceptible than leaves to runaway cavitation-an uncontrolled feedback cycle between rising water stress and declining water transport efficiency that can rapidly lead to lethal tissue desiccation. We provide modeling and empirical evidence to demonstrate that flower damage in the perennial crop pyrethrum (Tanacetum cinerariifolium), in the form of irreversible desiccation, corresponds with runaway cavitation in the flowering stem after a combination of heat and water stress. We show that tissue damage is linked to greater evaporative demand during high temperatures rather than direct thermal stress. High floral transpiration dramatically reduced the soil water deficit at which runaway cavitation was triggered in pyrethrum flowering stems. Identifying runaway cavitation as a mechanism leading to heat damage and reproductive losses in pyrethrum provides different avenues for process-based modeling to understand the impact of climate change on cultivated and natural plant systems. This framework allows future investigation of the relative susceptibility of diverse plant species to reproductive failure under hot and dry conditions.

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.

Conduit position and connectivity affect the likelihood of xylem embolism during natural drought in evergreen woodland species.

BACKGROUND AND AIMS: Hydraulic failure is considered a main cause of drought-induced forest mortality. Yet, we have a limited understanding of how the varying intensities and long time scales of natural droughts induce and propagate embolism within the xylem. METHODS: X-ray computed tomography (microCT) images were obtained from different aged branch xylem to study the number, size and spatial distribution of in situ embolized conduits among three dominant tree species growing in a woodland community. KEY RESULTS: Among the three studied tree species, those with a higher xylem vulnerability to embolism (higher water potential at 50 % loss of hydraulic conductance; P50) were more embolized than species with lower P50. Within individual stems, the probability of embolism was independent of conduit diameter but associated with conduit position. Rather than the occurrence of random or radial embolism, we observed circumferential clustering of high and low embolism density, suggesting that embolism spreads preferentially among conduits of the same age. Older xylem also appeared more likely to accumulate embolisms than young xylem, but there was no pattern suggesting that branch tips were more vulnerable to cavitation than basal regions. CONCLUSIONS: The spatial analysis of embolism occurrence in field-grown trees suggests that embolism under natural drought probably propagates by air spreading from embolized into neighbouring conduits in a circumferential pattern. This pattern offers the possibility to understand the temporal aspects of embolism occurrence by examining stem cross-sections.

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.

Reproductive water supply is prioritized during drought in tomato.

Reproductive success largely defines the fitness of plant species. Understanding how heat and drought affect plant reproduction is thus key to predicting future plant fitness under rising global temperatures. Recent work suggests reproductive tissues are highly vulnerable to water stress in perennial plants where reproductive sacrifice could preserve plant survival. However, most crop species are annuals where such a strategy would theoretically reduce fitness. We examined the reproductive strategy of tomato (Solanum lycopersicum var. Rheinlands Ruhm) to determine whether water supply to fruits is prioritized above vegetative tissues during drought. Using optical methods, we mapped xylem cavitation and tissue shrinkage in vegetative and reproductive organs during dehydration to determine the priority of water flow under acute water stress. Stems and peduncles of tomato showed significantly greater xylem cavitation resistance than leaves. This maintenance of intact water supply enabled tomato fruit to continue to expand during acute water stress, utilizing xylem water made available by tissue collapse and early cavitation of leaves. Here, tomato plants prioritize water supply to reproductive tissues, maintaining fruit development under drought conditions. These results emphasize the critical role of water transport in shaping life history and suggest a broad relevance of hydraulic prioritization in plant ecology.

Non-invasive imaging reveals convergence in root and stem vulnerability to cavitation across five tree species.

Root vulnerability to cavitation is challenging to measure and under-represented in current datasets. This gap limits the precision of models used to predict plant responses to drought because roots comprise the critical interface between plant and soil. In this study, we measured vulnerability to drought-induced cavitation in woody roots and stems of five tree species (Acacia aneura, Cedrus deodara, Eucalyptus crebra, Eucalytus saligna, and Quercus palustris) with a wide range of xylem anatomies. X-ray microtomography was used to visualize the accumulation of xylem embolism in stems and roots of intact plants that were naturally dehydrated to varying levels of water stress. Vulnerability to cavitation, defined as the water potential causing a 50% loss of hydraulic function (P50), varied broadly among the species (-4.51 MPa to -11.93 MPa in stems and -3.13 MPa to -9.64 MPa in roots). The P50 of roots and stems was significantly related across species, with species that had more vulnerable stems also having more vulnerable roots. While there was strong convergence in root and stem vulnerability to cavitation, the P50 of roots was significantly higher than the P50 of stems in three species. However, the difference in root and stem vulnerability for these species was small; between 1% and 31% of stem P50. Thus, while some differences existed between organs, roots were not dramatically more vulnerable to embolism than stems, and the differences observed were less than those reported in previous studies. Further study is required to evaluate the vulnerability across root orders and to extend these conclusions to a greater number of species and xylem functional types.

Xylem Embolism Spreads by Single-Conduit Events in Three Dry Forest Angiosperm Stems.

Xylem cavitation resulting in air embolism is a major cause of plant death during drought, yet the spread of embolism throughout the plant water transport system is poorly understood. Our study used optical visualization and x-ray microcomputed tomography imaging to capture the spread of emboli in stems of three drought-resistant angiosperm trees: drooping she-oak (Allocasuarina verticillata), black wattle (Acacia mearnsii), and blue gum (Eucalyptus globulus). These species have similar degrees of xylem network connectivity (vessel grouping) with largely solitary vessels. The high temporal resolution of the optical vulnerability technique revealed that in current year branches, >80% of the cavitation events were discrete, temporally separated events in single vessels. This suggests that in xylem networks with low connectivity, embolism spread between conduits leading to multiple conduit cavitation events is uncommon. A. mearnsii showed both the highest number of multivessel cavitation events and the highest degree of vessel connectivity, suggesting a link between vessel arrangement and embolism spread. Knowledge of embolism spread will help us to uncover the links between xylem anatomy, arrangement, and the path of water flow in the xylem in diverse species to ultimately understand the drivers of cavitation and plant vulnerability to drought.

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.

Xylem cavitation isolates leaky flowers during water stress in pyrethrum.

Flowers underpin plant evolution, genetic legacy and global food supply. They are exposed to similar evaporative conditions as leaves, yet floral physiology is a product of different selective forces. We used Tanacetum cinerariifolium, a perennial daisy, to examine the response of flowers to whole-plant water stress, determining if flowers constitute a liability during drought, and how this species has adapted to minimize risk associated with reproduction. We determined the relative transpiration cost of flowers and leaves and confirmed that flowers in this species are xylem-hydrated. The relative water stress tolerance of leaves and flowers then was compared using xylem vulnerability measurements linked with observed tissue damage during an acute drought treatment. Flowers were a major source of water loss during drought but the xylem supplying them was much more vulnerable to cavitation than leaves. This xylem vulnerability segmentation was confirmed by observations that most flowers died whereas leaves were minimally affected during drought. Early cavitation and hydraulic isolation of flowers during drought benefits the plant by slowing the dehydration of perennial vegetative organs and delaying systemic xylem damage. Our results highlight the need to understand flower xylem vulnerability as a means of predicting plant reproductive failure under future drought.

Genetic differentiation in functional traits among European sessile oak populations.

The vulnerability of forest species and tree populations to climate change is related to the exposure of the ecosystem to extreme climatic conditions and to the adaptive capacity of the population to cope with those conditions. Adaptive capacity is a relatively under-researched topic within the forest science community, and there is an urgent need to understand to what extent particular combinations of traits have been shaped by natural selection under climatic gradients, potentially resulting in adaptive multi-trait associations. Thus, our aim was to quantify genetic variation in several leaf and woody traits that may contribute to multi-trait associations in which intra-specific variation could represent a source for species adaptation to climate change. A multi-trait approach was performed using nine Quercus petraea provenances originating from different locations that cover most of the species' distribution range over Europe and that were grown in a common garden. Multiple adaptive differences were observed between oak provenances but also some evolutionary stasis. In addition, our results revealed higher genetic differentiation in traits related to phenology and growth than in those related to xylem anatomy, physiology and hydraulics, for which no genetic differentiation was observed. The multiple associations between those traits and climate variables resulting from multivariate and path analyses suggest a multi-trait association largely involving phenological and growth traits for Q. petraea.

Non-invasive imaging shows no evidence of embolism repair after drought in tree species of two genera.

Drought stress can result in significant impairment of the plant hydraulic system via blockage of xylem conduits by gas emboli. Recovery after drought stress is an essential component of plant survival but is still a poorly understood process. In this study, we examined the capacity of woody species from two genera (Eucalyptus and Quercus) to refill embolized xylem vessels during a cycle of drought and recovery. Observations were made on intact plants of Eucalyptus calmudulensis, E. grandis, E. saligna and Quercus palustris using X-ray microtomography. We found no evidence of an effective xylem refilling mechanism in any of the plant species. Despite rehydration and recovery of plant water potential to near pre-drought levels, embolized vessels were not refilled up to 72 h after rewatering. In E. saligna, water droplets accumulated in previously air-filled vessels for a very small percentage of vessels. However, no instances of complete refilling that would restore embolized vessels to hydraulic function were observed. Our observations suggest that rapid refilling of embolized vessels after drought may not be a wide spread mechanism in woody plants and that embolism formed during drought represents long term cost to the plant hydraulic system.

Similar geometric rules govern the distribution of veins and stomata in petals, sepals and leaves.

Investment in leaf veins (supplying xylem water) is balanced by stomatal abundance, such that sufficient water transport is provided for stomata to remain open when soil water is abundant. This coordination is mediated by a common dependence of vein and stomatal densities on cell size. Flowers may not conform to this same developmental pattern if they depend on water supplied by the phloem or have high rates of nonstomatal transpiration. We examined the relationships between veins, stomata and epidermal cells in leaves, sepals and petals of 27 angiosperms to determine whether common spacing rules applied to all tissues. Regression analysis found no evidence for different relationships within organ types. Both vein and stomatal densities were strongly associated with epidermal cell size within organs, but, for a given epidermal cell size, petals had fewer veins and stomata than sepals, which had fewer than leaves. Although our data support the concept of common scaling between veins and stomata in leaves and flowers, the large diversity in petal vein density suggests that, in some species, petal veins may be engaged in additional functions, such as the supply of water for high cuticular transpiration or for phloem delivery of water or carbohydrates.

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.

Ferns are less dependent on passive dilution by cell expansion to coordinate leaf vein and stomatal spacing than angiosperms.

Producing leaves with closely spaced veins is a key innovation linked to high rates of photosynthesis in angiosperms. A close geometric link between veins and stomata in angiosperms ensures that investment in enhanced venous water transport provides the strongest net carbon return to the plant. This link is underpinned by "passive dilution" via expansion of surrounding cells. However, it is not known whether this 'passive dilution' mechanism is present in plant lineages other than angiosperms and is another key feature of the angiosperms' evolutionary success. Consequently, we sought to determine whether the 'passive dilution' mechanism is; (i) exclusive to the angiosperms, (ii) a conserved mechanism that evolved in the common ancestor of ferns and angiosperms, or (iii) has evolved continuously over time. To do this we first we assessed the plasticity of vein and stomatal density and epidermal cell size in ferns in response to light environment. We then compared the relationships between these traits found among ferns with modelled relationships that assume vein and stomatal density respond passively to epidermal cell expansion, and with those previously observed in angiosperms. Vein density, stomatal density and epidermal cell size were linked in ferns with remarkably similar relationships to those observed in angiosperms, except that fern leaves had fewer veins per stomata. However, plasticity was limited in ferns and stomatal spacing was dependent on active stomatal differentiation as well as passive cell expansion. Thus, ferns (like angiosperms) appear to coordinate vein and stomatal density with epidermal cell expansion to some extent to maintain a constant ratio between veins and stomata in the leaf. The different general relationships between vein density and stomatal density in ferns and angiosperms suggests the groups have different optimum balances between the production of vein tissue dedicated to water supply and stomatal tissue for gas exchange.

Vein density is independent of epidermal cell size in Arabidopsis mutants.

Densities of leaf minor veins and stomata are co-ordinated within and across vascular plants. This maximises the benefit-to-cost ratio of leaf construction by ensuring stomata receive the minimum amount of water required to maintain optimal aperture. A 'passive dilution' mechanism in which densities of veins and stomata are co-regulated by epidermal cell size is thought to facilitate this co-ordination. However, unlike stomata, veins are spatially isolated from the epidermis and thus may not be directly regulated by epidermal cell expansion. Here, we use mutant genotypes of Arabidopsis thaliana (L.) Heynh. with altered stomatal and epidermal cell development to test this mechanism. To do this we compared observed relationships between vein density and epidermal cell size with modelled relationships that assume veins and stomata are passively diluted by epidermal cell expansion. Data from wild-type plants were consistent with the 'passive dilution' mechanism, but in mutant genotypes vein density was independent of epidermal cell size. Hence, vein density is not causally linked to epidermal cell expansion. This suggests that adaptation favours synchronised changes to the cell size of different leaf tissues to coordinate veins and stomata, and thus balance water supply with transpirational demand.

Xylem and stomata, coordinated through time and space.

Land plants exhibit a degree of homeostasis in leaf water content to protect against damage to photosynthetic and xylem tissues, and to maintain an efficient allocation of resources. This is achieved by a strong coordination between the systems regulating water delivery (xylem) and water loss (stomata). This review discusses evolution in xylem and stomatal function, specifically focussing on the interactions between them.

Cell expansion not cell differentiation predominantly co-ordinates veins and stomata within and among herbs and woody angiosperms grown under sun and shade.

BACKGROUND AND AIMS: It has been proposed that modification of leaf size, driven by epidermal cell size, balances leaf water supply (determined by veins) with transpirational demand (generated by stomata) during acclimation to local irradiance. We aimed to determine whether this is a general pattern among plant species with contrasting growth habits. METHODS: We compared observed relationships between leaf minor vein density, stomatal density, epidermal cell size and leaf size in four pairs of herbs and woody species from the same families grown under sun and shade conditions with modelled relationships assuming vein and stomatal densities respond passively to epidermal cell expansion. Leaf lignin content was also quantified to assess whether construction costs of herbaceous leaf veins differ from those of woody plants and the leaf mass fraction invested in veins. KEY RESULTS: Modelled relationships accurately described observed relationships, indicating that in all species, co-ordinated changes to the density of minor veins and stomata were mediated by a common relationship between epidermal cell size, vein density and stomatal density, with little or no impact from stomatal index. This co-ordination was independent of changes in leaf size and is likely to be an adaptive process driven by the significant proportion of biomass invested in veins (13·1 % of sun leaf dry weight and 21·7 % of shade leaf dry weight). Relative costs of venation increased in the shade, intensifying selective pressure towards economizing investment in vein density. CONCLUSIONS: Modulation of epidermal cell size appears to be a general mechanism among our experimental species to maintain a constant ratio between leaf anatomical traits that control leaf water fluxes independently of habit. We propose that this process may co-ordinate plasticity in hydraulic supply and demand in the majority of eudicot angiosperms.

Acclimation to humidity modifies the link between leaf size and the density of veins and stomata.

The coordination of veins and stomata during leaf acclimation to sun and shade can be facilitated by differential epidermal cell expansion so large leaves with low vein and stomatal densities grow in shade, effectively balancing liquid- and vapour-phase conductances. As the difference in vapour pressure between leaf and atmosphere (VPD) determines transpiration at any given stomatal density, we predict that plants grown under high VPD will modify the balance between veins and stomata to accommodate greater maximum transpiration. Thus, we examined the developmental responses of these traits to contrasting VPD in a woody angiosperm (Toona ciliata M. Roem.) and tested whether the relationship between them was altered. High VPD leaves were one-third the size of low VPD leaves with only marginally greater vein and stomatal density. Transpirational homeostasis was thus maintained by reducing stomatal conductance. VPD acclimation changed leaf size by modifying cell number. Hence, plasticity in vein and stomatal density appears to be generated by plasticity in cell size rather than cell number. Thus, VPD affects cell number and leaf size without changing the relationship between liquid- and vapour-phase conductances. This results in inefficient acclimation to VPD as stomata remain partially closed under high VPD.

Differential leaf expansion can enable hydraulic acclimation to sun and shade.

Although leaf size is one of the most responsive plant traits to environmental change, the functional benefits of large versus small leaves remain unclear. We hypothesized that modification of leaf size within species resulting from differences in irradiance can allow leaves to acclimate to different photosynthetic or evaporative conditions while maintaining an efficient balance between hydraulic supply (vein density) and evaporative demand. To test this, we compared the function and anatomy of leaf hydraulic systems in the leaves of a woody angiosperm (Toona ciliata M. Roem.) grown under high and low irradiance in controlled conditions. Our results confirm that in this species, differential leaf expansion regulates the density of veins and stomata such that leaf hydraulic conductance and stomatal conductance remain proportional. A broader sample of field-grown tree species suggested that differences in leaf venation and stomatal traits induced by sun and shade were not regulated by leaf size in all cases. Our results, however, suggest that leaf size plasticity can provide an efficient way for plants to acclimate hydraulic and stomatal conductances to the contrasting evaporative conditions of sun and shade.