Net O2 exchange rates under dark and light conditions across different stem compartments.

Woody plants display some photosynthetic activity in stems, but the biological role of stem photosynthesis and the specific contributions of bark and wood to carbon uptake and oxygen evolution remain poorly understood. We aimed to elucidate the functional characteristics of chloroplasts in stems of different ages in Fraxinus ornus. Our investigation employed diverse experimental approaches, including microsensor technology to assess oxygen production rates in whole stem, bark, and wood separately. Additionally, we utilized fluorescence lifetime imaging microscopy (FLIM) to characterize the relative abundance of photosystems I and II (PSI : PSII chlorophyll ratio) in bark and wood. Our findings revealed light-induced increases in O2 production in whole stem, bark, and wood. We present the radial profile of O2 production in F. ornus stems, demonstrating the capability of stem chloroplasts to perform light-dependent electron transport. Younger stems exhibited higher light-induced O2 production and dark respiration rates than older ones. While bark emerged as the primary contributor to net O2 production under light conditions, our data underscored that wood chloroplasts are also photosynthetically active. The FLIM analysis unveiled a lower PSI abundance in wood than in bark, suggesting stem chloroplasts are not only active but also acclimate to the spectral composition of light reaching inner compartments.

Detecting and Quantifying Xylem Embolism by Synchrotron-Based X-Ray Micro-CT.

The vulnerability to xylem embolism is a key trait underlying species-specific drought tolerance of plants, and hence is critical for screening climate-resilient crops and understanding vegetation responses to drought and heat waves. Yet, accurate determination of embolism in plant's xylem is challenging, because most traditional hydraulic techniques are destructive and prone to artefacts. Hence, direct and in vivo synchrotron-based X-ray micro-CT observation of xylem conduits has emerged as a key reference technique for accurate quantification of vulnerability to xylem embolism. Micro-CT is nowadays a fundamental tool for studies of plant hydraulic architecture, and this chapter describes the fundamentals of acquisition and processing of micro-CT images of plant xylem.

The optical method based on gas injection overestimates leaf vulnerability to xylem embolism in three woody species.

Plant hydraulic traits related to leaf drought tolerance, like the water potential at turgor loss point (TLP) and the water potential inducing 50% loss of hydraulic conductance (P50), are extremely useful to predict the potential impacts of drought on plants. While novel techniques have allowed the inclusion of TLP in studies targeting a large group of species, fast and reliable protocols to measure leaf P50 are still lacking. Recently, the optical method coupled with the gas injection (GI) technique has been proposed as a possibility to speed up the P50 estimation. Here, we present a comparison of leaf optical vulnerability curves (OVcs) measured in three woody species, namely Acer campestre (Ac), Ostrya carpinifolia (Oc) and Populus nigra (Pn), based on bench dehydration (BD) or GI of detached branches. For Pn, we also compared optical data with direct micro-computed tomography (micro-CT) imaging in both intact saplings and cut shoots subjected to BD. Based on the BD procedure, Ac, Oc and Pn had P50 values of -2.87, -2.47 and -2.11 MPa, respectively, while the GI procedure overestimated the leaf vulnerability (-2.68, -2.04 and -1.54 MPa for Ac, Oc and Pn, respectively). The overestimation was higher for Oc and Pn than for Ac, likely reflecting the species-specific vessel lengths. According to micro-CT observations performed on Pn, the leaf midrib showed none or very few embolized conduits at -1.2 MPa, consistent with the OVcs obtained with the BD procedure but at odds with that derived on the basis of GI. Overall, our data suggest that coupling the optical method with GI might not be a reliable technique to quantify leaf hydraulic vulnerability since it could be affected by the 'open-vessel' artifact. Accurate detection of xylem embolism in the leaf vein network should be based on BD, preferably of intact up-rooted plants.

Best Procedures for Leaf and Stem Water Potential Measurements in Grapevine: Cultivar and Water Status Matter.

The pressure chamber is the most used tool for plant water status monitoring. However, species/cultivar and seasonal effects on protocols for reliable water potential determination have not been properly tested. In four grapevine cultivars and two times of the season (early season, Es; late season, Ls, under moderate drought), we assessed the maximum sample storage time before leaf water potential (Ψleaf) measurements and the minimum equilibration time for stem water potential (Ψstem) determination, taking 24 h leaf cover as control. In 'Pinot gris', Ψleaf already decreased after 1 h leaf storage in both campaigns, dropping by 0.4/0.5 MPa after 3 h, while in 'Refosk', it decreased by 0.1 MPa after 1 and 2 h in Es and Ls, respectively. In 'Merlot' and 'Merlot Kanthus', even 3 h storage did not affect Ψleaf. In Es, the minimum Ψstem equilibration was 1 h for 'Refosk' and 10 min for 'Pinot gris' and 'Merlot'. In Ls, 'Merlot Kanthus' required more than 2 h equilibration, while 1 h to 10 min was sufficient for the other cultivars. The observed cultivar and seasonal differences indicate that the proposed tests should be routinely performed prior to experiments to define ad hoc procedures for water status determination.

Structure and function of bark and wood chloroplasts in a drought-tolerant tree (Fraxinus ornus L.).

Leaves are the most important photosynthetic organs in most woody plants, but chloroplasts are also found in organs optimized for other functions. However, the actual photosynthetic efficiency of these chloroplasts is still unclear. We analyzed bark and wood chloroplasts of Fraxinus ornus L. saplings. Optical and spectroscopic methods were applied to stem samples and compared with leaves. A sharp light gradient was detected along the stem radial direction, with blue light mainly absorbed by the outer bark, and far-red-enriched light reaching the underlying xylem and pith. Chlorophylls were evident in the xylem rays and the pith and showed an increasing concentration gradient toward the bark. The stem photosynthetic apparatus showed features typical of acclimation to a low-light environment, such as larger grana stacks, lower chlorophyll a/b and photosystem I/II ratios compared with leaves. Despite likely receiving very few photons, wood chloroplasts were photosynthetically active and fully capable of generating a light-dependent electron transport. Our data provide a comprehensive scenario of the functional features of bark and wood chloroplasts in a woody species and suggest that stem photosynthesis is coherently optimized to the prevailing micro-environmental conditions at the bark and wood level.

No Evidence for Light-Induced Embolism Repair in Cut Stems of Drought-Resistant Mediterranean Species under Soaking.

(1) Recent studies suggested that stem photosynthesis could favor bark water uptake and embolism recovery when stem segments are soaked in water under light conditions, but evidence for this phenomenon in drought-resistant Mediterranean species with photosynthetic stems is missing. (2) Embolism recovery upon immersion in water for 2 h-4 h under light was assessed (i) via a classical hydraulic method in leafless Fraxinus ornus and Olea europaea branch segments stressed to xylem water potentials (Yxyl) inducing ca. 50% loss of hydraulic conductivity (PLC) and (ii) via X-ray micro-CT imaging of the stem segments of drought-stressed potted F. ornus saplings. Hydraulic recovery was also assessed in vivo in intact drought-stressed F. ornus saplings upon soil re-irrigation. (3) Intact F. ornus plants recovered hydraulic function through root water uptake. Conversely, the soaked stem segments of both species did not refill embolized conduits, although Yxyl recovered to pre-stress levels (between -0.5 MPa and -0.2 MPa). (4) We hypothesize that xylem embolism recovery through bark water uptake, even in light conditions, may not be a common phenomenon in woody plants and/or that wounds caused by cutting short stem segments might inhibit the refilling process upon soaking.

Shade-induced reduction of stem nonstructural carbohydrates increases xylem vulnerability to embolism and impedes hydraulic recovery in Populus nigra.

Nonstructural carbohydrates (NSCs) have been suggested to affect xylem transport under fluctuating water availability, but conclusive evidence is still lacking. We tested the effect of shade-induced NSC depletion on xylem vulnerability to embolism and hydraulic recovery on Populus nigra saplings. Vulnerability was assessed in light-exposed (L) and shaded (S) plants with the hydraulic method, and in vivo with the optical method and X-ray micro-computed tomography. Plants were stressed to 80% loss of hydraulic conductance (PLC) and re-irrigated to check for possible recovery. We measured PLC, bark and wood NSC content, as well as xylem sap pH, surface tension (γsap ) and sugar concentration, before, during and after drought. Shading induced depletion of stem NSC (mainly starch) reserves. All methods converged in indicating higher xylem vulnerability in S than in L plants. This difference was not explained by xylem vessel and pit anatomy or by γsap . Shading impeded sap acidification and sugar accumulation during drought in S plants and prevented hydraulic recovery, which was observed in L plants. Our results highlight the importance of stem NSCs to sustain xylem hydraulic functioning during drought and suggest that light and/or adequate stem NSC thresholds are required to trigger xylem sap chemical changes involved in embolism recovery.

Water 'on the rocks': a summer drink for thirsty trees?

Drought-induced tree mortality frequently occurs in patches with different spatial and temporal distributions, which is only partly explained by inter- and intraspecific variation in drought tolerance. We investigated whether bedrock properties, with special reference to rock water storage capacity, affects tree water status and drought response in a rock-dominated landscape. We measured primary porosity and available water content of breccia (B) and dolostone (D) rocks. Saplings of Fraxinus ornus were grown in pots filled with soil or soil mixed with B and D rocks, and subjected to an experimental drought. Finally, we measured seasonal changes in water status of trees in field sites overlying B or D bedrock. B rocks were more porous and stored more available water than D rocks. Potted saplings grown with D rocks had less biomass and suffered more severe water stress than those with B rocks. Trees in sites with B bedrock had more favourable water status than those on D bedrock which also suffered drought-induced canopy dieback. Bedrock represents an important water source for plants under drought. Different bedrock features translate into contrasting below-ground water availability, leading to landscape-level heterogeneity of the impact of drought on tree water status and dieback.

A Leaf Selfie: Using a Smartphone to Quantify Leaf Vulnerability to Hydraulic Dysfunction.

Accurate predictions of species distribution under current and future climate conditions require modeling efforts based on clear mechanistic relationships between climate variables and plant physiological functions. Vulnerability of leaves to xylem embolism is a key mechanistic trait that might be included in these modeling efforts. Here, we propose a simple set-up to measure leaf vulnerability to embolism on the basis of the optical method using a smartphone, a light source, and a notebook. Our data show that this proposed set-up can adequately quantify the vulnerability to xylem embolism of leaf major veins in Populus nigra and Ostrya carpinifolia, producing values consistent with those obtained in temperate tree species with other methods, allowing virtually any laboratory to quantify species-specific drought tolerance on the basis of a sound mechanistic trait.