Seasonal changes in carbohydrates and water content predict dynamics of frost hardiness in various temperate tree species.

Predicting tree frost tolerance is critical to select adapted species according to both the current and predicted future climate. The relative change in water to carbohydrate ratio is a relevant trait to predict frost acclimation in branches from many tree species. The objective of this study is to demonstrate the interspecific genericity of this approach across nine tree species. In the studied angiosperm species, frost hardiness dynamics were best correlated to a decrease in water content at the early stage of acclimation (summer and early autumn). Subsequently, frost hardiness dynamics were more tightly correlated to soluble carbohydrate contents until spring growth resumption. Based on different model formalisms, we predicted frost hardiness at different clade levels (angiosperms, family, genus and species) with high to moderate accuracy (1.5-6.0 °C root mean squared error (RMSE)) and robustness (2.8-6.1 °C prediction RMSE). The TOT model, taking all soluble carbohydrate and polyols into account, was more effective and adapted for large scale studies aiming to explore frost hardiness across a wide range of species. The ISC model taking the individual contribution of each soluble carbohydrate molecule into account was more efficient at finer scale such as family or species. The ISC model performance also suggests that the role of solutes cannot be reduced to a 'bulk' osmotic effect as could be computed if all of them were located in a single, common, compartment. This study provides sets of parameters to predict frost hardiness in a wide range of species, and clues for targeting specific carbohydrate molecules to improve frost hardiness.

Can phenological models predict tree phenology accurately in the future? The unrevealed hurdle of endodormancy break.

The onset of the growing season of trees has been earlier by 2.3 days per decade during the last 40 years in temperate Europe because of global warming. The effect of temperature on plant phenology is, however, not linear because temperature has a dual effect on bud development. On one hand, low temperatures are necessary to break bud endodormancy, and, on the other hand, higher temperatures are necessary to promote bud cell growth afterward. Different process-based models have been developed in the last decades to predict the date of budbreak of woody species. They predict that global warming should delay or compromise endodormancy break at the species equatorward range limits leading to a delay or even impossibility to flower or set new leaves. These models are classically parameterized with flowering or budbreak dates only, with no information on the endodormancy break date because this information is very scarce. Here, we evaluated the efficiency of a set of phenological models to accurately predict the endodormancy break dates of three fruit trees. Our results show that models calibrated solely with budbreak dates usually do not accurately predict the endodormancy break date. Providing endodormancy break date for the model parameterization results in much more accurate prediction of this latter, with, however, a higher error than that on budbreak dates. Most importantly, we show that models not calibrated with endodormancy break dates can generate large discrepancies in forecasted budbreak dates when using climate scenarios as compared to models calibrated with endodormancy break dates. This discrepancy increases with mean annual temperature and is therefore the strongest after 2050 in the southernmost regions. Our results claim for the urgent need of massive measurements of endodormancy break dates in forest and fruit trees to yield more robust projections of phenological changes in a near future.

Differentiated dynamics of bud dormancy and growth in temperate fruit trees relating to bud phenology adaptation, the case of apple and almond trees.

Few studies have focused on the characterization of bud dormancy and growth dynamics for temperate fruit species in temperate and mild cropping areas, although this is an appropriate framework to anticipate phenology adaptation facing future warming contexts which would potentially combine chill declines and heat increases. To examine this issue, two experimental approaches and field observations were used for high- and low-chill apple cultivars in temperate climate of southern France and in mild climates of northern Morocco and southern Brazil. Low-chill almond cultivars offered an additional relevant plant material for comparison with apple in northern Morocco. Divergent patterns of dormancy and growth dynamics were clearly found in apple tree between southern France and southern Brazil. Divergences were less pronounced between France and Morocco. A global view outlined main differences in the dormancy chronology and intensity, the transition between endordormancy and ecodormancy and the duration of ecodormancy. A key role of bud rehydration in the transition period was shown. High-chill cultivars would be submitted in mild conditions to heterogeneous rehydration capacities linked to insufficient chill fulfillment and excessive forcing linked to high temperatures. This would favor bud competitions and consequently excessive flowering durations and weak flowering. Low chilling requirements in apple and almond would conversely confer biological capacities to tolerate superficial dormancy and abrupt transition from endordormancy to ecodormancy without important heterogeneous rehydration states within buds. It may also assume that low-chill cultivars can also tolerate high temperatures during ecodormancy as well as extended flowering durations.

Cavitation and water fluxes driven by ice water potential in Juglans regia during freeze-thaw cycles.

Freeze-thaw cycles induce major hydraulic changes due to liquid-to-ice transition within tree stems. The very low water potential at the ice-liquid interface is crucial as it may cause lysis of living cells as well as water fluxes and embolism in sap conduits, which impacts whole tree-water relations. We investigated water fluxes induced by ice formation during freeze-thaw cycles in Juglans regia L. stems using four non-invasive and complementary approaches: a microdendrometer, magnetic resonance imaging, X-ray microtomography, and ultrasonic acoustic emissions analysis. When the temperature dropped, ice nucleation occurred, probably in the cambium or pith areas, inducing high water potential gradients within the stem. The water was therefore redistributed within the stem toward the ice front. We could thus observe dehydration of the bark's living cells leading to drastic shrinkage of this tissue, as well as high tension within wood conduits reaching the cavitation threshold in sap vessels. Ultrasonic emissions, which were strictly emitted only during freezing, indicated cavitation events (i.e. bubble formation) following ice formation in the xylem sap. However, embolism formation (i.e. bubble expansion) in stems was observed only on thawing via X-ray microtomography for the first time on the same sample. Ultrasonic emissions were detected during freezing and were not directly related to embolism formation. These results provide new insights into the complex process and dynamics of water movements and ice formation during freeze-thaw cycles in tree stems.

Differentiated Responses of Apple Tree Floral Phenology to Global Warming in Contrasting Climatic Regions.

The responses of flowering phenology to temperature increases in temperate fruit trees have rarely been investigated in contrasting climatic regions. This is an appropriate framework for highlighting varying responses to diverse warming contexts, which would potentially combine chill accumulation (CA) declines and heat accumulation (HA) increases. To examine this issue, a data set was constituted in apple tree from flowering dates collected for two phenological stages of three cultivars in seven climate-contrasting temperate regions of Western Europe and in three mild regions, one in Northern Morocco and two in Southern Brazil. Multiple change-point models were applied to flowering date series, as well as to corresponding series of mean temperature during two successive periods, respectively determining for the fulfillment of chill and heat requirements. A new overview in space and time of flowering date changes was provided in apple tree highlighting not only flowering date advances as in previous studies but also stationary flowering date series. At global scale, differentiated flowering time patterns result from varying interactions between contrasting thermal determinisms of flowering dates and contrasting warming contexts. This may explain flowering date advances in most of European regions and in Morocco vs. stationary flowering date series in the Brazilian regions. A notable exception in Europe was found in the French Mediterranean region where the flowering date series was stationary. While the flowering duration series were stationary whatever the region, the flowering durations were far longer in mild regions compared to temperate regions. Our findings suggest a new warming vulnerability in temperate Mediterranean regions, which could shift toward responding more to chill decline and consequently experience late and extended flowering under future warming scenarios.

Non-structural carbohydrates in woody plants compared among laboratories.

Non-structural carbohydrates (NSC) in plant tissue are frequently quantified to make inferences about plant responses to environmental conditions. Laboratories publishing estimates of NSC of woody plants use many different methods to evaluate NSC. We asked whether NSC estimates in the recent literature could be quantitatively compared among studies. We also asked whether any differences among laboratories were related to the extraction and quantification methods used to determine starch and sugar concentrations. These questions were addressed by sending sub-samples collected from five woody plant tissues, which varied in NSC content and chemical composition, to 29 laboratories. Each laboratory analyzed the samples with their laboratory-specific protocols, based on recent publications, to determine concentrations of soluble sugars, starch and their sum, total NSC. Laboratory estimates differed substantially for all samples. For example, estimates for Eucalyptus globulus leaves (EGL) varied from 23 to 116 (mean = 56) mg g(-1) for soluble sugars, 6-533 (mean = 94) mg g(-1) for starch and 53-649 (mean = 153) mg g(-1) for total NSC. Mixed model analysis of variance showed that much of the variability among laboratories was unrelated to the categories we used for extraction and quantification methods (method category R(2) = 0.05-0.12 for soluble sugars, 0.10-0.33 for starch and 0.01-0.09 for total NSC). For EGL, the difference between the highest and lowest least squares means for categories in the mixed model analysis was 33 mg g(-1) for total NSC, compared with the range of laboratory estimates of 596 mg g(-1). Laboratories were reasonably consistent in their ranks of estimates among tissues for starch (r = 0.41-0.91), but less so for total NSC (r = 0.45-0.84) and soluble sugars (r = 0.11-0.83). Our results show that NSC estimates for woody plant tissues cannot be compared among laboratories. The relative changes in NSC between treatments measured within a laboratory may be comparable within and between laboratories, especially for starch. To obtain comparable NSC estimates, we suggest that users can either adopt the reference method given in this publication, or report estimates for a portion of samples using the reference method, and report estimates for a standard reference material. Researchers interested in NSC estimates should work to identify and adopt standard methods.

Chilling and heat requirements for leaf unfolding in European beech and sessile oak populations at the southern limit of their distribution range.

With global warming, an advance in spring leaf phenology has been reported worldwide. However, it is difficult to forecast phenology for a given species, due to a lack of knowledge about chilling requirements. We quantified chilling and heat requirements for leaf unfolding in two European tree species and investigated their relative contributions to phenological variations between and within populations. We used an extensive database containing information about the leaf phenology of 14 oak and 10 beech populations monitored over elevation gradients since 2005. In parallel, we studied the various bud dormancy phases, in controlled conditions, by regularly sampling low- and high-elevation populations during fall and winter. Oak was 2.3 times more sensitive to temperature for leaf unfolding over the elevation gradient and had a lower chilling requirement for dormancy release than beech. We found that chilling is currently insufficient for the full release of dormancy, for both species, at the lowest elevations in the area studied. Genetic variation in leaf unfolding timing between and within oak populations was probably due to differences in heat requirement rather than differences in chilling requirement. Our results demonstrate the importance of chilling for leaf unfolding in forest trees and indicate that the advance in leaf unfolding phenology with increasing temperature will probably be less pronounced than forecasted. This highlights the urgent need to determine experimentally the interactions between chilling and heat requirements in forest tree species, to improve our understanding and modeling of changes in phenological timing under global warming.

Frost hardiness in walnut trees (Juglans regia L.): how to link physiology and modelling?

In the literature, frost hardiness (FH) studies in trees have often been restricted to one organ (buds, leaves, needles or twigs). To extend our knowledge and gain a unified view, FH differences between organs and tissues or throughout the life of the tree have to be characterized in relation to physiological changes. In this study, different organs and tissues of young potted and mature orchard walnut trees (Juglans regia L.) were compared for seasonal changes in FH during different years. FH was assessed using the electrolyte leakage method. Physiological parameters were concomitantly monitored focusing on two significant traits: water content (WC) and carbohydrate content (glucose + fructose + sucrose, GFS). No seasonal variation in FH was observed in the root system, but acclimation and deacclimation were observed aboveground. Among organs and tissues, cold sensitivity levels were different in deep winter, with buds most sensitive and bark most resistant, but acclimation/deacclimation dynamics followed similar patterns. Physiological variation was also similar among organs: FH increased when WC decreased and/or soluble carbohydrates increased. Based on these results, relations between soluble carbohydrate content, WC and FH were calculated independently or in interaction. The key results were that: (i) the relationship between FH and physiological parameters (GFS and WC), which had previously been shown for branches only, could be generalized to all aboveground organs; (ii) lower WC increased the cryoprotective effect of GFS, showing a synergic effect of the two factors; (iii) the best fit was a non-linear function of WC and GFS, yielding a predictive model with an root mean square error of 5.07 °C on an independent dataset and 2.59 °C for the most sensitive stages; and (iv) the same parameters used for all organs yielded a unified model of FH depending on physiology, although the variability of GFS or WC was wide. The model should be of value for predicting FH in walnut independently of previous growing conditions (i.e., after sublethal stress accumulation).

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.

Modulation of bud survival in Populus nigra sprouts in response to water stress-induced embolism.

Understanding drought tolerance mechanisms requires knowledge about the induced weakness that leads to tree death. Bud survival is vital to sustain tree growth across seasons. We hypothesized that the hydraulic connection of the bud to stem xylem structures was critical for its survival. During an artificial drastic water stress, we carried out a census of bud metabolic activity of young Populus nigra L. trees by microcalorimetry. We monitored transcript expression of aquaporins (AQPs; plasma membrane intrinsic proteins (PIPs), X intrinsic proteins (XIPs) and tonoplast membrane intrinsic proteins (TIPs)) and measured local water status within the bud and tissues in the bearer shoot node by nuclear magnetic resonance (NMR) imaging. We found that the bud respiration rate was closely correlated with its water content and decreased concomitantly in buds and their surrounding bearer tissues. At the molecular level, we observed a modulation of AQP pattern expressions (PIP, TIP and XIP subfamilies) linked to water movements in living cells. However, AQP functions remain to be investigated. Both the bud and tree died beyond a threshold water content and respiration rate. Nuclear magnetic resonance images provided relevant local information about the various water reservoirs of the stem, their dynamics and their interconnections. Comparison of pith, xylem and cambium tissues revealed that the hydraulic connection between the bud and saturated parenchyma cells around the pith allowed bud desiccation to be delayed. At the tree death date, NMR images showed that the cambium tissues remained largely hydrated. Overall, the respiration rate (Rco2) and a few AQP isoforms were found to be two suitable, complementary criteria to assess the bud metabolic activity and the ability to survive a severe drought spell. Bud moisture content could be a key factor in determining the capacity of poplar to recover from water stress.

Are budburst dates, dormancy and cold acclimation in walnut trees (Juglans regia L.) under mainly genotypic or environmental control?

As observed for most stresses, tree frost resistance can be split into two main processes: avoidance and tolerance. Avoidance of freezing is achieved by introducing species only in the climatic context in which the probability of freezing events is very low for the sensitive stages of buds or stems; i.e., when good synchronism exists between the annual cycle and the critical climatic periods. Buds become able to grow only after chilling requirements have been satisfied (endodormancy released) during winter; they subsequently break after heat requirements have been completed (end of ecodormancy) in early spring. Actually, this period is often subject to more or less severe freezing events. Trees are also able to adjust their freezing tolerance by increasing their capacity of extracellular freezing and decreasing the possibility of intracellular freezing through the process of frost acclimation. Both freezing resistance processes (avoidance and tolerance) are environmentally driven (by photoperiod and temperature), but there are also genotypic effects among species or cultivars. Here, we evaluated the degree to which differences in dormancy release and frost acclimation were related to environmental and genetic influences by comparing trees growing in common garden conditions. This investigation was carried out for two winters in lowland and mountain locations on different walnut genotypes differing significantly for budburst dates. Chilling requirement for endodormancy release and heat requirement during ecodormancy were evaluated in all situations. In addition, frost acclimation was assessed by the electrolyte leakage method on stems from the same trees before leaf fall through budburst. No significant differences were observed in chilling requirements among genotypes. Moreover, frost acclimation dynamics were similar between genotypes or locations when expressed depending on chilling units accumulated since 15 September as a time basis instead of Julian day. The only exception was for maximal frost hardiness observed during winter with the timber-oriented being significantly more resistant than fruit-oriented genotypes. Heat requirement was significantly different among genotypes. Thus, growth was significantly faster in fruit-oriented than in wood-oriented genotypes. Furthermore, among wood-oriented genotypes, differences in growth rate were observed only at cold temperatures. Frost acclimation changes differed significantly between fruit- and wood- walnuts from January through budburst. In conclusion, from September through January, the acclimation dynamic was driven mainly by environmental factors whereas from January through budburst a significant genotype effect was identified in both frost tolerance and avoidance processes.

Carbohydrate uptake from xylem vessels and its distribution among stem tissues and buds in walnut (Juglans regia L.).

Bud break pattern is a key determinant of tree architecture. The mechanisms leading to the precedence of certain buds over the others are not yet fully explained, but the availability of soluble sugars may play a significant role, especially those in the xylem sap at the onset of the growing period. Here, we measured carbon availability in the different tissues (bud, xylem and bark). To assess the capacity of buds to use the xylem sap carbohydrates, the fluxes between xylem vessels and parenchyma cells, bark and buds of walnut (Juglans regia cv 'Franquette') were measured during the rest period until bud break. This uptake capacity varies according to the temperature, the sugar and the position on the branch of the fragment studied. Between December and March, in xylem tissues, the active component of sucrose uptake was predominant compared with diffusion (90% of the total uptake), whereas the active component accounted for more moderate amounts in buds (50% of the uptake). The active uptake of hexoses took place belatedly (April) in xylem. The flow rates between xylem vessels and buds increased 1 month before bud break and reached 2000 microg sucrose h(-)(1) g DW(-)(1). Fluxes seemed to depend on bud position on the branch. However, this study strongly suggests that they were mainly dependent on the sink strength of the buds and on the sink competition between bud, xylem parenchyma and bark.

Sucrose (JrSUT1) and hexose (JrHT1 and JrHT2) transporters in walnut xylem parenchyma cells: their potential role in early events of growth resumption.

In temperate woody species, the vegetative growth period is characterized by active physiological events (e.g., bud break), which require an adequate supply of soluble sugars imported in the xylem sap stream. One-year-old shoots of walnut (Juglans regia L. cv. 'Franquette') trees, which have an acrotonic branching pattern (only apical and distal vegetative buds burst), were used to study the regulation of xylem sugar transporters in relation to bud break. At the end of April (beginning of bud break), a higher xylem sap sucrose concentration and a higher active sucrose uptake by xylem parenchyma cells were found in the apical portion (bearing buds able to burst) than in the basal portion (bearing buds unable to burst) of the sample shoots. At the same time, xylem parenchyma cells of the apical portion of the shoots exhibited greater amounts of both transcripts and proteins of JrSUT1 (Juglans regia putative sucrose transporter 1) than those of the basal stem segment. Conversely, no pronounced difference was found for putative hexose transporters JrHT1 and JrHT2 (Juglans regia hexose transporters 1 and 2). These findings demonstrate the high capacity of bursting vegetative buds to import sucrose. Immunological analysis revealed that sucrose transporters were localized in all parenchyma cells of the xylem, including vessel-associated cells, which are highly specialized in nutrient exchange. Taken together, our results indicate that xylem parenchyma sucrose transporters make a greater contribution than hexose transporters to the imported carbon supply of bursting vegetative buds.

Spatial activity and expression of plasma membrane H+-ATPase in stem xylem of walnut during dormancy and growth resumption.

Plasma membrane H+-ATPase (PM H+-ATPase) plays a key role in nutrient transport, stress responses and growth. To evaluate proton motive force differences between apical and basal parts of acrotonic 1-year-old shoots of walnut (Juglans regia L. cv 'Franquette') trees, spatial and seasonal changes in PM H+-ATPase were studied in mature xylem tissues. During both the dormancy and growth resumption periods, and in both the apical and basal parts of the stem, PM H+-ATPase activity showed positive correlations with the amount of immunodetectable protein. In spring, at the time of growth resumption, higher activities and immunoreactivities of PM H+-ATPase were found in the apical part of the stem than in the basal part of the stem. In spring, the decrease in xylem sugar concentration reflected the high sugar uptake rate. Our data suggest that PM H+-ATPase plays a major role in the uptake of carbohydrates from xylem vessels during growth resumption. These results are discussed in the context of the acrotonic tendency of walnut shoots.

Trophic control of bud break in peach (Prunus persica) trees: a possible role of hexoses.

Vegetative buds of peach (Prunus persica L. Batsch.) trees act as strong sinks and their bud break capacity can be profoundly affected by carbohydrate availability during the rest period (November-February). Analysis of xylem sap revealed seasonal changes in concentrations of sorbitol and hexoses (glucose and fructose). Sorbitol concentrations decreased and hexose concentrations increased with increasing bud break capacity. Sucrose concentration in xylem sap increased significantly but remained low. To clarify their respective roles in the early events of bud break, carbohydrate concentrations and uptake rates, and activities of NAD-dependent sorbitol dehydrogenase (SDH), sorbitol oxidase (SOX) and cell wall invertase (CWI) were determined in meristematic tissues, cushion tissues and stem segments. Only CWI activity increased in meristematic tissues shortly before bud break. In buds displaying high bud break capacity (during January and February), concentrations of sorbitol and sucrose in meristematic tissues were almost unchanged, paralleling their low rates of uptake and utilization by meristematic tissues, and indicating that sorbitol and sucrose play a negligible role in the bud break process. Hexose concentrations in meristematic tissues and glucose imported by meristematic tissues correlated positively with bud break capacity, suggesting that hexoses are involved in the early events of bud break. These findings were confirmed by data for buds that were unable to break because they had been collected from trees deprived of cold. We therefore conclude that hexoses are of greater importance than sorbitol or sucrose in the early events of bud break in peach trees.

Sorbitol uptake is regulated by glucose through the hexokinase pathway in vegetative peach-tree buds.

In peach trees (Prunus persica L. Batsch cv. Redhaven), sorbitol is a primary photosynthetic product and may play an important role in the budbreak process. Surprisingly, before budbreak (from January to early March), the concentration of sorbitol in the xylem sap decreases, while that of hexoses (glucose and fructose) increases. The aim of this work was to study the control of sorbitol uptake into vegetative buds by hexoses. Sorbitol uptake was selectively inhibited by hexoses at low and physiological concentrations and this effect was both reversible and concentration-dependent. In addition, the active uptake of sorbitol significantly declined in the plasma membrane vesicles-enriched fraction purified from glucose-treated vegetative buds, suggesting that the inhibitory action of glucose was at the membrane level. Finally, among several glucose analogues tested, only hexokinase substrates (2-deoxyglucose and mannose) were able to mimic the glucose effect, which was completely blocked by the hexokinase inhibitor mannoheptulose. These results represent the first steps towards a better understanding of polyol transport control in plants.