Contrasting stem water uptake and storage dynamics of water-saver and water-spender species during drought and recovery.

Drought is projected to occur more frequently and intensely in the coming decades, and the extent to which it will affect forest functioning will depend on species-specific responses to water stress. Aiming to understand the hydraulic traits and water dynamics behind water-saver and water-spender strategies in response to drought and recovery, we conducted a pot experiment with two species with contrasting physiological strategies, Scots pine (Pinus sylvestris L.) and Portuguese oak (Quercus faginea L.). We applied two cycles of soil drying and recovery and irrigated with isotopically different water to track fast changes in soil and stem water pools, while continuously measuring physiological status and xylem water content from twigs. Our results provide evidence for a tight link between the leaf-level response and the water uptake and storage patterns in the stem. The water-saver strategy of pines prevented stem dehydration by rapidly closing stomata which limited their water uptake during the early stages of drought and recovery. Conversely, oaks showed a less conservative strategy, maintaining transpiration and physiological activity under dry soil conditions, and consequently becoming more dehydrated at the stem level. We interpreted this dehydration as the release of water from elastic storage tissues as no major loss of hydraulic conductance occurred for this species. After soil rewetting, pines recovered pre-drought leaf water potential rapidly, but it took longer to replace the water from conductive tissues (slower labeling speed). In contrast, water-spender oaks were able to quickly replace xylem water during recovery (fast labeling speed), but it took longer to refill stem storage tissues, and hence to recover pre-drought leaf water potential. These different patterns in sap flow rates, speed and duration of the labeling reflected a combination of water-use and storage traits, linked to the leaf-level strategies in response to drought and recovery.

Do 2 H and 18 O in leaf water reflect environmental drivers differently?

We compiled hydrogen and oxygen stable isotope compositions (δ2 H and δ18 O) of leaf water from multiple biomes to examine variations with environmental drivers. Leaf water δ2 H was more closely correlated with δ2 H of xylem water or atmospheric vapour, whereas leaf water δ18 O was more closely correlated with air relative humidity. This resulted from the larger proportional range for δ2 H of meteoric waters relative to the extent of leaf water evaporative enrichment compared with δ18 O. We next expressed leaf water as isotopic enrichment above xylem water (Δ2 H and Δ18 O) to remove the impact of xylem water isotopic variation. For Δ2 H, leaf water still correlated with atmospheric vapour, whereas Δ18 O showed no such correlation. This was explained by covariance between air relative humidity and the Δ18 O of atmospheric vapour. This is consistent with a previously observed diurnal correlation between air relative humidity and the deuterium excess of atmospheric vapour across a range of ecosystems. We conclude that 2 H and 18 O in leaf water do indeed reflect the balance of environmental drivers differently; our results have implications for understanding isotopic effects associated with water cycling in terrestrial ecosystems and for inferring environmental change from isotopic biomarkers that act as proxies for leaf water.

Evidence for distinct isotopic compositions of sap and tissue water in tree stems: consequences for plant water source identification.

The long-standing hypothesis that the isotopic composition of plant stem water reflects that of source water is being challenged by studies reporting bulk water from woody stems with an isotopic composition that cannot be attributed to any potential water source. The mechanism behind such source-stem water isotopic offsets is still poorly understood. Using a novel technique to extract selectively sap water from xylem conduits, we show that, in cut stems and potted plants, the isotopic composition of sap water reflects that of irrigation water, demonstrating unambiguously that no isotopic fractionation occurs during root water uptake or sap water extraction. By contrast, water in nonconductive xylem tissues is always depleted in deuterium compared with sap water, irrespective of wood anatomy. Previous studies have shown that isotopic heterogeneity also exists in soils at the pore scale in which water adsorbed onto soil particles is more depleted in deuterium than unbound water. Data collected at a riparian forest indicated that sap water matches best unbound soil water from depth below -70 cm, while bulk stem and soil water differ markedly. We conclude that source-stem isotopic offsets can be explained by micrometre-scale heterogeneity in the isotope ratios of water within woody stems and soil micro-pores.

Contrasting ecophysiological strategies related to drought: the case of a mixed stand of Scots pine (Pinus sylvestris) and a submediterranean oak (Quercus subpyrenaica).

Submediterranean forests are considered an ecotone between Mediterranean and Eurosiberian ecosystems, and are very sensitive to global change. A decline of Scots pine (Pinus sylvestris L.) and a related expansion of oak species (Quercus spp.) have been reported in the Spanish Pre-Pyrenees. Although this has been associated with increasing drought stress, the underlying mechanisms are not fully understood, and suitable monitoring protocols are lacking. The aim of this study is to bring insight into the physiological mechanisms anticipating selective decline of the pines, with particular focus on carbon and water relations. For this purpose, we performed a sampling campaign covering two growing seasons in a mixed stand of P. sylvestris and Quercus subpyrenaica E.H del Villar. We sampled seasonally twig xylem and soil for water isotope composition (δ18O and δ2H), leaves for carbon isotope composition (δ13C) and stems to quantify non-structural carbohydrates (NSC) concentration, and measured water potential and leaf gas exchange. The first summer drought was severe for both species, reaching low predawn water potential (-2.2 MPa), very low stomatal conductance (12 ± 1.0 mmol m-2 s-1) and near-zero or even negative net photosynthesis, particularly in P. sylvestris (-0.6 ± 0.34 µmol m-2 s-1 in oaks, -1.3 ± 0.16 µmol m-2 s-1 in pines). Hence, the tighter stomatal control and more isohydric strategy of P. sylvestris resulted in larger limitations on carbon assimilation, and this was also reflected in carbon storage, showing twofold larger total NSC concentration in oaks than in pines (7.8 ± 2.4% and 4.0 ± 1.3%, respectively). We observed a faster recovery of predawn water potential after summer drought in Q. subpyrenaica than in P. sylvestris (-0.8 MPa and -1.1 MPa, respectively). As supported by the isotopic data, this was probably associated with a deeper and more reliable water supply in Q. subpyrenaica. In line with these short-term observations, we found a more pronounced negative effect of steadily increasing drought stress on long-term growth in pines compared with oaks. All these observations confer evidence of early warning of P. sylvestris decline and indicate the adaptive advantage of Q. subpyrenaica in the area.

Short-term dynamics of evaporative enrichment of xylem water in woody stems: implications for ecohydrology.

In ecohydrology, it is generally assumed that xylem water reflects the water source used by plants. Several studies have reported isotopic enrichment within woody tissues, particularly during dormancy periods or after long periods of inactivity. However, little is known about the short-term dynamics of this process. Here we assessed the magnitude and dynamics of xylem isotopic enrichment in suberized twigs of pines and oaks. We performed a series of laboratory experiments, in which we monitored hourly changes in water content and isotopic composition under two contrasting scenarios of sap flow restriction. First, we simulated the effect of extreme hydraulic failure by excising twigs to restrict sap flow, while sealing the wounds to ensure that water loss took place only through the leaves or bark, as would be the case for evaporation in attached stems. Second, we studied the effect of reduced leaf transpiration by darkening with aluminium foil all the leaves of healthy, well-watered saplings growing in pot conditions. We found evidence of fast evaporative enrichment in metabolically active stems, as a consequence of a temporal decline in sap flow rates, and not necessarily linked to a traceable decline in stem water content. The excision experiments showed significant isotopic changes (~+1‰ in oxygen) appearing in <1 h. Similarly, the pot experiment showed a progressive increase in isotope composition (up to +8‰ in oxygen in a 3-day cycle) when the leaves were covered, and a rapid recovery to initial values when sap flow rates were re-established. We conclude that evaporative enrichment of xylem water in stems is a highly dynamic process that may have significant effects even during short periods of restricted water flow. This has important implications for the study of plant water uptake, as well as for ecosystem- and global-scale hydrological models.

Circadian rhythms have significant effects on leaf-to-canopy scale gas exchange under field conditions.

BACKGROUND: Molecular clocks drive oscillations in leaf photosynthesis, stomatal conductance, and other cell and leaf-level processes over ~24 h under controlled laboratory conditions. The influence of such circadian regulation over whole-canopy fluxes remains uncertain; diurnal CO2 and H2O vapor flux dynamics in the field are currently interpreted as resulting almost exclusively from direct physiological responses to variations in light, temperature and other environmental factors. We tested whether circadian regulation would affect plant and canopy gas exchange at the Montpellier European Ecotron. Canopy and leaf-level fluxes were constantly monitored under field-like environmental conditions, and under constant environmental conditions (no variation in temperature, radiation, or other environmental cues). RESULTS: We show direct experimental evidence at canopy scales of the circadian regulation of daytime gas exchange: 20-79 % of the daily variation range in CO2 and H2O fluxes occurred under circadian entrainment in canopies of an annual herb (bean) and of a perennial shrub (cotton). We also observed that considering circadian regulation improved performance by 8-17 % in commonly used stomatal conductance models. CONCLUSIONS: Our results show that circadian controls affect diurnal CO2 and H2O flux patterns in entire canopies in field-like conditions, and its consideration significantly improves model performance. Circadian controls act as a 'memory' of the past conditions experienced by the plant, which synchronizes metabolism across entire plant canopies.

Isotope-ratio infrared spectroscopy: a reliable tool for the investigation of plant-water sources?

Stable isotopes are extensively used as tracers for the study of plant-water sources. Isotope-ratio infrared spectroscopy (IRIS) offers a cheaper alternative to isotope-ratio mass spectroscopy (IRMS), but its use in studying plant and soil water is limited by the spectral interference caused by organic contaminants. Here, we examine two approaches to cope with contaminated samples in IRIS: on-line oxidation of organic compounds (MCM) and post-processing correction. We assessed these methods compared to IRMS across 136 samples of xylem and soil water, and a set of ethanol- and methanol-water mixtures. A post-processing correction significantly improved IRIS accuracy in both natural samples and alcohol dilutions, being effective with concentrations up to 8% of ethanol and 0.4% of methanol. MCM outperformed the post-processing correction in removing methanol interference, but did not effectively remove interference for high concentrations of ethanol. By using both approaches, IRIS can overcome with reasonable accuracy the analytical uncertainties associated with most organic contaminants found in soil and xylem water. We recommend the post-processing correction as the first choice for analysis of samples of unknown contamination. Nevertheless, MCM can be more effective for evaluating samples containing contaminants responsible for strong spectral interferences at low concentrations, such as methanol.