Unpacking the point of no return under drought in poplar: insight from stem diameter variation.

A specific, robust threshold for drought-induced tree mortality is needed to improve the prediction of forest dieback. Here, we tested the relevance of continuous measurements of stem diameter variations for identifying such a threshold, their relationship with hydraulic and cellular damage mechanisms, and the influence of growth conditions on these relationships. Poplar saplings were grown under well-watered, water-limited, or light-limited conditions and then submitted to a drought followed by rewatering. Stem diameter was continuously measured to investigate two parameters: the percentage loss of diameter (PLD) and the percentage of diameter recovery (DR) following rewatering. Water potentials, stomatal conductance, embolism, and electrolyte leakage were also measured, and light microscopy allowed investigating cell collapse induced by drought. The water release observed through loss of diameter occurred throughout the drought, regardless of growth conditions. Poplars did not recover from drought when PLD reached a threshold and this differed according to growth conditions but remained linked to cell resistance to damage and collapse. Our findings shed new light on the mechanisms of drought-induced tree mortality and indicate that PLD could be a relevant indicator of drought-induced tree mortality, regardless of the growth conditions.

Detection of acoustic events in lavender for measuring xylem vulnerability to embolism and cellular damage.

Acoustic emission analysis is promising to investigate the physiological events leading to drought-induced injury and mortality. However, their nature and source are not fully understood, making this technique difficult to use as a direct measure of the loss of xylem hydraulic conductance. Acoustic emissions were recorded during severe dehydration in lavender plants (Lavandula angustifolia) and compared with the dynamics of embolism development and cell damage. The timing and characteristics of acoustic signals from two independent recording systems were compared by principal component analysis (PCA). Changes in water potential, branch diameter, loss of hydraulic conductance, and cellular damage were also measured to quantify drought-induced damages. Two distinct phases of acoustic emissions were observed during dehydration: the first one associated with a rapid loss of diameter and a significant increase in loss of xylem conductance (90%), and the second with slower changes in diameter and a significant increase in cellular damage. Based on PCA, a developed algorithm discriminated hydraulic-related acoustic signals from other sources, proposing a reconstruction of hydraulic vulnerability curves. Cellular damage preceded by hydraulic failure seems to lead to a lack of recovery. The second acoustic phase would allow detection of plant mortality.

Into the range: a latitudinal gradient or a center-margins differentiation of ecological strategies in Arabidopsis thaliana?

BACKGROUND AND AIMS: Determining within-species large-scale variation in phenotypic traits is central to elucidate the drivers of species' ranges. Intraspecific comparisons offer the opportunity to understand how trade-offs and biogeographical history constrain adaptation to contrasted environmental conditions. Here we test whether functional traits, ecological strategies from the CSR scheme and phenotypic plasticity in response to abiotic stress vary along a latitudinal or a center- margins gradient within the native range of Arabidopsis thaliana. METHODS: We experimentally examined the phenotypic outcomes of plant adaptation at the center and margins of its geographic range using 30 accessions from southern, central and northern Europe. We characterized the variation of traits related to stress tolerance, resource use, colonization ability, CSR strategy scores, survival and fecundity in response to high temperature (34 °C) or frost (- 6 °C), combined with a water deficit treatment. KEY RESULTS: We found evidence for both a latitudinal and a center-margins differentiation for the traits under scrutiny. Age at maturity, leaf dry matter content, specific leaf area and leaf nitrogen content varied along a latitudinal gradient. Northern accessions presented a greater survival to stress than central and southern accessions. Leaf area, C-scores, R-scores and fruit number followed a center-margins differentiation. Central accessions displayed a higher phenotypic plasticity than northern and southern accessions for most studied traits. CONCLUSIONS: Traits related to an acquisitive/conservative resource-use trade-off followed a latitudinal gradient. Traits associated with a competition/colonization trade-off differentiated along the historic colonization of the distribution range and then followed a center-margins differentiation. Our findings pinpoint the need to consider the joint effect of evolutionary history and environmental factors when examining phenotypic variation across the distribution range of a species.

Drought-Induced Mortality: Branch Diameter Variation Reveals a Point of No Recovery in Lavender Species.

In the context of climate change, determining the physiological mechanisms of drought-induced mortality in woody plants and identifying thresholds of drought survivorship will improve forecasts of forest and agroecosystem die-off. Here, we tested whether continuous measurements of branch diameter variation can be used to identify thresholds of hydraulic failure and physiological recoverability in lavender (Lavandula angustifolia and Lavandula × intermedia) plants exposed to severe drought. Two parameters of branch diameter variation were tested: the percentage loss of diameter and the percentage loss of rehydration capacity. In two greenhouse experiments with different growth conditions, we monitored variation in branch diameter in the two lavender species exposed to a series of drought/rewatering cycles that varied in drought-stress intensity. Water potential, stomatal conductance, loss of xylem hydraulic conductance, and electrolyte leakage were also measured. We observed that plants were not able to recover when percentage loss of diameter reached maximum values of 21.3% ± 0.6% during drought, regardless of species and growth conditions. A percentage loss of rehydration capacity of 100% was defined as the point of no recovery, and was observed with high levels of cellular damage as estimated by electrolyte leakage measured at 75.4% ± 9.3% and occurred beyond 88% loss of xylem hydraulic conductance. Our study demonstrates that lavender plants are not able to recover from severe drought when they have used up their elastic water storage. Additionally, drought-induced mortality in these species was not linked to xylem hydraulic failure but rather to high levels of cell damage.

PtxtPME1 and homogalacturonans influence xylem hydraulic properties in poplar.

While the xylem hydraulic properties, such as vulnerability to cavitation (VC), are of paramount importance in drought resistance, their genetic determinants remain unexplored. There is evidence that pectins and their methylation pattern are involved, but the detail of their involvement and the corresponding genes need to be clarified. We analyzed the hydraulic properties of the 35S::PME1 transgenic aspen that ectopically under- or over-express a xylem-abundant pectin methyl esterase, PtxtPME1. We also produced and analyzed 4CL1::PGII transgenic poplars expressing a fungal polygalacturonase, AnPGII, under the control of the Ptxa4CL1 promoter that is active in the developing xylem after xylem cell expansion. Both the 35S::PME1 under- and over-expressing aspen lines developed xylem with lower-specific hydraulic conductivity and lower VC, while the 4CL1::PGII plants developed xylem with a higher VC. These xylem hydraulic changes were associated with modifications in xylem structure or in intervessel pit structure that can result in changes in mechanical behavior of the pit membrane. This study shows that homogalacturonans and their methylation pattern influence xylem hydraulic properties, through its effect on xylem cell expansion and on intervessel pit properties and it show a role for PtxtPME1 in the xylem hydraulic properties.

Suppression of Dwarf and irregular xylem Phenotypes Generates Low-Acetylated Biomass Lines in Arabidopsis.

eskimo1-5 (esk1-5) is a dwarf Arabidopsis (Arabidopsis thaliana) mutant that has a constitutive drought syndrome and collapsed xylem vessels, along with low acetylation levels in xylan and mannan. ESK1 has xylan O-acetyltransferase activity in vitro. We used a suppressor strategy on esk1-5 to screen for variants with wild-type growth and low acetylation levels, a favorable combination for ethanol production. We found a recessive mutation in the KAKTUS (KAK) gene that suppressed dwarfism and the collapsed xylem character, the cause of decreased hydraulic conductivity in the esk1-5 mutant. Backcrosses between esk1-5 and two independent knockout kak mutants confirmed suppression of the esk1-5 effect. kak single mutants showed larger stem diameters than the wild type. The KAK promoter fused with a reporter gene showed activity in the vascular cambium, phloem, and primary xylem in the stem and hypocotyl. However, suppression of the collapsed xylem phenotype in esk1 kak double mutants was not associated with the recovery of cell wall O-acetylation or any major cell wall modifications. Therefore, our results indicate that, in addition to its described activity as a repressor of endoreduplication, KAK may play a role in vascular development. Furthermore, orthologous esk1 kak double mutants may hold promise for ethanol production in crop plants.

Immunolabelling of intervessel pits for polysaccharides and lignin helps in understanding their hydraulic properties in Populus tremula × alba.

BACKGROUND AND AIMS: The efficiency and safety functions of xylem hydraulics are strongly dependent on the pits that connect the xylem vessels. However, little is known about their biochemical composition and thus about their hydraulic properties. In this study, the distribution of the epitopes of different wall components (cellulose, hemicelluloses, pectins and lignins) was analysed in intervessel pits of hybrid poplar (Populus tremula × alba). METHODS: Immunogold labelling with transmission electron microscopy was carried out with a set of antibodies raised against different epitopes for each wall polysaccharide type and for lignins. Analyses were performed on both immature and mature vessels. The effect of sap ionic strength on xylem conductance was also tested. KEY RESULTS: In mature vessels, the pit membrane (PM) was composed of crystalline cellulose and lignins. None of the hemicellulose epitopes were found in the PM. Pectin epitopes in mature vessels were highly concentrated in the annulus, a restricted area of the PM, whereas they were initially found in the whole PM in immature vessels. The pit border also showed a specific labelling pattern, with higher cellulose labelling compared with the secondary wall of the vessel. Ion-mediated variation of 24 % was found for hydraulic conductance. CONCLUSIONS: Cellulose microfibrils, lignins and annulus-restricted pectins have different physicochemical properties (rigidity, hydrophobicity, porosity) that have different effects on the hydraulic functions of the PM, and these influence both the hydraulic efficiency and vulnerability to cavitation of the pits, including ion-mediated control of hydraulic conductance. Impregnation of the cellulose microfibrils of the PM with lignins, which have low wettability, may result in lower cavitation pressure for a given pore size and thus help to explain the vulnerability of this species to cavitation.

Modelling the mechanical behaviour of pit membranes in bordered pits with respect to cavitation resistance in angiosperms.

BACKGROUND AND AIMS: Various correlations have been identified between anatomical features of bordered pits in angiosperm xylem and vulnerability to cavitation, suggesting that the mechanical behaviour of the pits may play a role. Theoretical modelling of the membrane behaviour has been undertaken, but it requires input of parameters at the nanoscale level. However, to date, no experimental data have indicated clearly that pit membranes experience strain at high levels during cavitation events. METHODS: Transmission electron microscopy (TEM) was used in order to quantify the pit micromorphology of four tree species that show contrasting differences in vulnerability to cavitation, namely Sorbus aria, Carpinus betulus, Fagus sylvatica and Populus tremula. This allowed anatomical characters to be included in a mechanical model that was based on the Kirchhoff-Love thin plate theory. A mechanistic model was developed that included the geometric features of the pits that could be measured, with the purpose of evaluating the pit membrane strain that results from a pressure difference being applied across the membrane. This approach allowed an assessment to be made of the impact of the geometry of a pit on its mechanical behaviour, and provided an estimate of the impact on air-seeding resistance. KEY RESULTS: The TEM observations showed evidence of residual strains on the pit membranes, thus demonstrating that this membrane may experience a large degree of strain during cavitation. The mechanical modelling revealed the interspecific variability of the strains experienced by the pit membrane, which varied according to the pit geometry and the pressure experienced. The modelling output combined with the TEM observations suggests that cavitation occurs after the pit membrane has been deflected against the pit border. Interspecific variability of the strains experienced was correlated with vulnerability to cavitation. Assuming that air-seeding occurs at a given pit membrane strain, the pressure predicted by the model to achieve this mechanical state corresponds to experimental values of cavitation sensitivity (P50). CONCLUSIONS: The results provide a functional understanding of the importance of pit geometry and pit membrane structure in air-seeding, and thus in vulnerability to cavitation.

Hydrolase treatments help unravel the function of intervessel pits in xylem hydraulics.

Intervessel pits are structures that play a key role in the efficiency and safety functions of xylem hydraulics. However, little is known about the components of the pit membrane (PM) and their role in hydraulic functions, especially in resistance to cavitation. We tested the effect of commercial chemicals including a cellulase, a hemicellulase, a pectolyase, a proteinase and DTT on xylem hydraulic properties: vulnerability to cavitation (VC) and conductance. The effects were tested on branch segments from Fagus sylvatica (where the effects on pit structure were analyzed using TEM) and Populus tremula. Cellulose hydrolysis resulted in a sharp increase in VC and a significant increase in conductance, related to complete breakdown of the PM. Pectin hydrolysis also induced a sharp increase in VC but with no effect on conductance or pit structure observable by TEM. The other treatments with hemicellulase, proteinase or DTT showed no effect. This study brings evidence that cellulose and pectins are critical components underpinning VC, and that PM components may play distinct roles in the xylem hydraulic safety and efficiency.

Methods for measuring plant vulnerability to cavitation: a critical review.

Xylem cavitation resistance has profound implications for plant physiology and ecology. This process is characterized by a 'vulnerability curve' (VC) showing the variation of the percentage of cavitation as a function of xylem pressure potential. The shape of this VC varies from 'sigmoidal' to 'exponential'. This review provides a panorama of the techniques that have been used to generate such a curve. The techniques differ by (i) the way cavitation is induced (e.g. bench dehydration, centrifugation, or air injection), and (ii) the way cavitation is measured (e.g. percentage loss of conductivity (PLC) or acoustic emission), and a nomenclature is proposed based on these two methods. A survey of the literature of more than 1200 VCs was used to draw statistics on the usage of these methods and on their reliability and validity. Four methods accounted for more than 96% of all curves produced so far: bench dehydration-PLC, centrifugation-PLC, pressure sleeve-PLC, and Cavitron. How the shape of VCs varies across techniques and species xylem anatomy was also analysed. Strikingly, it was found that the vast majority of curves obtained with the reference bench dehydration-PLC method are 'sigmoidal'. 'Exponential' curves were more typical of the three other methods and were remarkably frequent for species having large xylem conduits (ring-porous), leading to a substantial overestimation of the vulnerability of cavitation for this functional group. We suspect that 'exponential' curves may reflect an open-vessel artefact and call for more precautions with the usage of the pressure sleeve and centrifugation techniques.

Arabidopsis thaliana as a model species for xylem hydraulics: does size matter?

While Arabidopsis thaliana has been proposed as a model species for wood development, the potential of this tiny herb for studying xylem hydraulics remains unexplored and anticipated by scepticism. Inflorescence stems of A. thaliana were used to measure hydraulic conductivity and cavitation resistance, whereas light and electron microscopy allowed observations of vessels. In wild-type plants, measured and theoretical conductivity showed a significant correlation (R (2) = 0.80, P < 0.01). Moreover, scaling of vessel dimensions and intervessel pit structure of A. thaliana were consistent with structure-function relationships of woody plants. The reliability and resolution of the hydraulic methods applied to measure vulnerability to cavitation were addressed by comparing plants grown under different photoperiods or different mutant lines. Sigmoid vulnerability curves of A. thaliana indicated a pressure corresponding to 50% loss of hydraulic conductance (P 50) between -3 and -2.5MPa for short-day and long-day plants, respectively. Polygalacturonase mutants showed a higher P 50 value (-2.25MPa), suggesting a role for pectins in vulnerability to cavitation. The application of A. thaliana as a model species for xylem hydraulics provides exciting possibilities for (1) exploring the molecular basis of xylem anatomical features and (2) understanding genetic mechanisms behind xylem functional traits such as cavitation resistance. Compared to perennial woody species, however, the lesser amount of xylem in A. thaliana has its limitations.

Water stress-induced xylem hydraulic failure is a causal factor of tree mortality in beech and poplar.

BACKGROUND AND AIMS: Extreme water stress episodes induce tree mortality, but the physiological mechanisms causing tree death are still poorly understood. This study tests the hypothesis that a potted tree's ability to survive extreme monotonic water stress is determined by the cavitation resistance of its xylem tissue. METHODS: Two species were selected with contrasting cavitation resistance (beech and poplar), and potted juvenile trees were exposed to a range of water stresses, causing up to 100 % plant death. KEY RESULTS: The lethal dose of water stress, defined as the xylem pressure inducing 50 % mortality, differed sharply across species (1·75 and 4·5 MPa in poplar and beech, respectively). However, the relationships between tree mortality and the degree of cavitation in the stems were similar, with mortality occurring suddenly when >90 % cavitation had occurred. CONCLUSIONS: Overall, the results suggest that cavitation resistance is a causal factor of tree mortality under extreme drought conditions.

Hydraulic failure and tree mortality: from correlation to causation.

Xylem hydraulic failure has been recognized as a pervasive factor in the triggering of drought-induced tree mortality. However, foundational evidence of the mechanistic link connecting hydraulic failure with living cell damage and tree death has not been identified yet, compromising our ability to predict mortality events. Meristematic cells are involved in the recovery of trees from drought, and focusing on their vitality and functionality after a drought event could provide novel information on the mechanistic link between hydraulic failure and drought-induced tree mortality. In this Opinion, we focus on the cell's critical hydration status for tree recovery from drought and how it links with the membrane integrity of the meristems.

Calcium is a major determinant of xylem vulnerability to cavitation.

Xylem vulnerability to cavitation is a key parameter in the drought tolerance of trees, but little is known about the control mechanisms involved. Cavitation is thought to occur when an air bubble penetrates through a pit wall, and would hence be influenced by the wall's porosity. We first tested the role of wall-bound calcium in vulnerability to cavitation in Fagus sylvatica. Stems perfused with solutions of oxalic acid, EGTA, or sodium phosphate (NaPO(4)) were found to be more vulnerable to cavitation. The NaPO(4)-induced increase in vulnerability to cavitation was linked to calcium removal from the wall. In contrast, xylem hydraulic conductance was unaffected by the chemical treatments, demonstrating that the mechanisms controlling vulnerability to cavitation and hydraulic resistance are uncoupled. The NaPO(4) solution was then perfused into stems from 13 tree species possessing highly contrasted vulnerability to cavitation. Calcium was found to be a major determinant of between-species differences in vulnerability to cavitation. This was evidenced in angiosperms as well as conifer species, thus supporting the hypothesis of a common mechanism in drought-induced cavitation.

Influence of Rootstock Genotype and Ploidy Level on Common Clementine (Citrus clementina Hort. ex Tan) Tolerance to Nutrient Deficiency.

Nutrient deficiency, in particular when this involves a major macronutrient (N, P, and K), is a limiting factor on the performance of plants in their natural habitat and agricultural environment. In the citrus industry, one of the eco-friendliest techniques for improving tolerance to biotic and abiotic stress is based on the grafting of a rootstock and a scion of economic interest. Scion tolerance may be improved by a tetraploid rootstock. The purpose of this study was to highlight if tolerance of a common clementine scion (C) (Citrus clementina Hort. ex Tan) to nutrient deficiency could be improved by several diploid (2×) and their tetraploid (4×) counterparts citrus genotypes commonly used as rootstocks: Trifoliate orange × Cleopatra mandarin (C/PMC2x and C/PMC4x), Carrizo citrange (C/CC2x and C/CC4x), Citrumelo 4475 (C/CM2x and C/CM4x). The allotetraploid FlhorAG1 (C/FL4x) was also included in the experimental design. The impact of nutrient deficiency on these seven scion/rootstock combinations was evaluated at root and leaf levels by investigating anatomical parameters, photosynthetic properties and oxidative and antioxidant metabolism. Nutrient deficiency affects foliar tissues, physiological parameters and oxidative metabolism in leaves and roots in different ways depending on the rootstock genotype and ploidy level. The best known nutrient deficiency-tolerant common clementine scions were grafted with the doubled diploid Citrumelo 4475 (C/CM4x) and the allotetraploid FlhorAG1 (C/FL4x). These combinations were found to have less foliar damage, fewer changes of photosynthetic processes [leaf net photosynthetic rate (P net ), stomatal conductance (g s ), transpiration (E), maximum quantum efficiency of PSII (F v /F m ), electron transport rate (ETR), ETR/P net ], and effective quantum yield of PSII [Y(II)], less malondialdehyde accumulation in leaves and better functional enzymatic and non-enzymatic antioxidant systems. Common clementine scions grafted on other 4× rootstocks did not show better tolerance than those grafted on their 2× counterparts. Chromosome doubling of rootstocks did not systematically improve the tolerance of the common clementine scion to nutrient deficiency.

Tetraploid Citrumelo 4475 rootstocks improve diploid common clementine tolerance to long-term nutrient deficiency.

Nutrient deficiency alters growth and the production of high-quality nutritious food. In Citrus crops, rootstock technologies have become a key tool for enhancing tolerance to abiotic stress. The use of doubled diploid rootstocks can improve adaptation to lower nutrient inputs. This study investigated leaf structure and ultrastructure and physiological and biochemical parameters of diploid common clementine scions (C) grafted on diploid (2x) and doubled diploid (4x) Carrizo citrange (C/CC2x and C/CC4x) and Citrumelo 4475 (C/CM2x and C/CM4x) rootstocks under optimal fertigation and after 7 months of nutrient deficiency. Rootstock ploidy level had no impact on structure but induced changes in the number and/or size of cells and some cell components of 2x common clementine leaves under optimal nutrition. Rootstock ploidy level did not modify gas exchanges in Carrizo citrange but induced a reduction in the leaf net photosynthetic rate in Citrumelo 4475. By assessing foliar damage, changes in photosynthetic processes and malondialdehyde accumulation, we found that C/CM4x were less affected by nutrient deficiency than the other scion/rootstock combinations. Their greater tolerance to nutrient deficiency was probably due to the better performance of the enzyme-based antioxidant system. Nutrient deficiency had similar impacts on C/CC2x and C/CC4x. Tolerance to nutrient deficiency can therefore be improved by rootstock polyploidy but remains dependent on the rootstock genotype.

Acclimation of hydraulic and morphological traits to water deficit delays hydraulic failure during simulated drought in poplar.

The capacity of trees to tolerate and survive increasing drought conditions in situ will depend in part on their ability to acclimate (via phenotypic plasticity) key hydraulic and morphological traits that increase drought tolerance and delay the onset of drought-induced hydraulic failure. However, the effect of water-deficit acclimation in key traits that determine time to hydraulic failure (THF) during extreme drought remains largely untested. We measured key hydraulic and morphological traits in saplings of a hybrid poplar grown under well-watered and water-limited conditions. The time for plants to dry-down to critical levels of water stress (90% loss of stem hydraulic conductance), as well as the relative contribution of drought acclimation in each trait to THF, was simulated using a soil-plant hydraulic model (SurEau). Compared with controls, water-limited plants exhibited significantly lower stem hydraulic vulnerability (P50stem), stomatal conductance and total canopy leaf area (LA). Taken together, adjustments in these and other traits resulted in longer modelled THF in water-limited (~160 h) compared with well-watered plants (~50 h), representing an increase of more than 200%. Sensitivity analysis revealed that adjustment in P50stem and LA contributed the most to longer THF in water-limited plants. We observed a high degree of trait plasticity in poplar saplings in response to water-deficit growth conditions, with decreases in stem hydraulic vulnerability and leaf area playing a key role in delaying the onset of hydraulic failure during a simulated drought event. These findings suggest that understanding the capacity of plants to acclimate to antecedent growth conditions will enable better predictions of plant survivorship during future drought.

Plasticity of the xylem vulnerability to embolism in Populus tremula x alba relies on pit quantity properties rather than on pit structure.

Knowledge on variations of drought resistance traits are needed to predict the potential of trees to acclimate to coming severe drought events. Xylem vulnerability to embolism is a key parameter related to such droughts, and its phenotypic variability relies mainly on environmental plasticity. We investigated the structural determinants controlling the plasticity of vulnerability to embolism, focusing on the key elements involved in the air bubble entry in vessels, especially the intervessel pits. Poplar saplings (Populus tremula x alba (Aiton) Sm., 1804) grown in contrasted water availability or light exposure exhibited differences in the vulnerability to embolism (P50) in a range of 0.76 MPa. We then characterized the structural changes in features related to pit quantity and pit structure, from the pit ultrastructure to the organization of xylem vessels, using different microscopy techniques (transmission electron microscopy, scanning electron microscopy, light microscopy). A multispectral combination of X-ray microtomography and light microscopy analysis allowed measuring the vulnerability of each single vessel and testing some of the relationships between structural traits and vulnerability to embolism inside the xylem. The pit ultrastructure did not change, whereas the vessel dimensions increased with the vulnerability to embolism and the grouping index and fraction of intervessel cell wall both decreased with the vulnerability to embolism. These findings hold when comparing between trees or between the vessels inside the xylem of an individual tree. These results evidenced that plasticity of vulnerability to embolism in hybrid poplar occurs through changes in the pit quantity properties such as pit area and vessel grouping rather than changes on the pit structure.

Improved response of triploid citrus varieties to water deficit is related to anatomical and cytological properties.

Polyploidy plays a major role in citrus plant breeding to improve the adaptation of polyploid rootstocks as well as scions to adverse conditions and to enhance agronomic characteristics. In Citrus breeding programs, triploidy could be a useful tool to react to environmental issues and consumer demands because the produced fruits are seedless. In this study, we compared the physiological, biochemical, morphological, and ultrastructural responses to water deficit of triploid and diploid citrus varieties obtained from 'Fortune' mandarin and 'Ellendale' tangor hybridization. One diploid clementine tree was included and used as a reference. All studied scions were grafted on C-35 citrange rootstock. Triploidy decreased stomatal density and increased stomata size. The number of chloroplasts increased in 3x varieties. These cytological properties may explain the greater photosynthetic capacity (Pnet, gs, Fv/Fm) and enhanced water-holding capacity (RWC, proline). In addition, reduced degradation of ultrastructural organelles (chloroplasts and mitochondria) and thylakoids accompanied by less photosynthetic activity and low oxidative damages were found in 3x varieties. Triploid varieties, especially T40-3x, had a better ability to limit water loss and dissipate excess energy (NPQ) to protect photosystems. Higher starch reserves in 3x varieties suggest a better carbon and energy supply and increases in plastoglobuli size suggest less oxidative damage (H2O2, MDA), especially in T40-3x, and preservation of photosynthetic apparatus. Taken together, our results suggest that desirable cytological and ultrastructural traits induced by triploidy improve water stress response and could be a useful stress marker during environmental constraints.

Does sample length influence the shape of xylem embolism vulnerability curves? A test with the Cavitron spinning technique.

The Cavitron spinning technique is used to construct xylem embolism vulnerability curves (VCs), but its reliability has been questioned for species with long vessels. This technique generates two types of VC: sigmoid 's'-shaped and exponential, levelling-off 'r'-shaped curves. We tested the hypothesis that 'r'-shaped VCs were anomalous and caused by the presence of vessels cut open during sample preparation. A Cavitron apparatus was used to construct VCs from samples of different lengths in species with contrasting vessel lengths. The results were compared with VCs obtained using other independent techniques. When vessel length exceeded sample length, VCs were 'r'-shaped and anomalous. Filling vessels cut open at both ends with air before measurement produced more typical 's'-shaped VCs. We also found that exposing segments of 11 woody species in a Cavitron at the pressure measured in planta before sampling considerably increased the degree of embolism above the native state level for species with long vessels. We concluded that open vessels were abnormally more vulnerable to cavitation than intact vessels. We recommend restricting this technique to species with short conduits. The relevance of our conclusions for other spinning techniques is discussed.