DOI: 10.25911/608ba2e1921bc
title: Dry season acclimation of leaf and stem traits in the mangrove Sonneratia alba, Daintree River, QLD, 2018, DP180102969

description: This data file contains raw experimental data, summary data and calculation sheets used in analyses. - Leaf pressure volume curves and derived traits. - Minimum cuticular conductance data - Shoot pressure-volume traits - Temp and VPD data logged at site during the experiments. - Diurnal gas exchange and hydraulic conductance measurements. - Stem hydraulic vulnerability curves made using the pneumatic method - Leaf hydraulic vulnerability curves made using the rehydration kinetics method.
description: This data set explores the role of acclimation of leaf and stem traits in mitigating the twin risks of reduced gas exchange and drought-induced hydraulic failure in the mangrove Sonneratia alba Sm. Sonneratia alba is a canopy-forming mangrove tree species wide-spread in the Indo-West Pacific (Duke, Ball & Ellison 1998). S. alba is an ideal system to study acclimation to atmospheric drought in mangroves as it can be found in locations experiencing strong seasonal atmospheric drought with limited changes in salinity at the roots, a coincidence of its preference for mid-estuarine and low intertidal positions (Duke et al. 1998). We hypothesized that leaf and stem water relations traits would differ between the early and late dry season to 1) maintain levels of photosynthesis, and 2) maintain or increase hydraulic safety margins. Specifically, we hypothesized that drought acclimation of leaf traits such as turgor loss point (πTLP), leaf relative water content at turgor loss point (RWCat TLP), leaf osmotic potential at full turgor (πO), elastic modulus (ε), apoplastic fraction (Af), water storage (S) and hydraulic capacitance (C) may enhance gas exchange and sustain photosynthesis. In the mitigation of loss of stem hydraulic function, we hypothesized that seasonal acclimation of water storage and hydraulic capacitance of organs of the shoot may increase hydraulic safety margins, with water retention aided by hydraulic segmentation and/or seasonal acclimation of gmin. Methods: Leaf and shoot water relations: Leaf water relations were determined from pressure-volume (p-v) curves, i.e., plots of leaf water potential versus relative water content (RWC). A p-v curve was constructed for one mature leaf from each of seven trees in the early dry season (n = 7) and six trees in the late dry season (n = 6) using the bench drying method (Tyree & Hammel, 1972). In brief, upon returning to the lab, leaves were cut from shoots underwater and allowed to rehydrate overnight with petioles submerged in a perfusion solution of 1% v/v seawater (~5mM NaCl, -0.02 MPa)(Ball 1988; Stuart, Choat, Martin, Holbrook & Ball 2007). Leaves were rehydrated to greater than -0.2 MPa, with no evident oversaturation or waterlogging observed. After rehydration, excess water was removed with a paper towel and the saturated mass was determined. Leaf areas were measured using a flatbed scanner (LiDE Scan 110, Canon, Tokyo, Japan) and ImageJ (National Institute of Health, Bethesda, USA), and leaves were then allowed to bench dry. Leaf water potentials (Ψleaf) were measured using a Scholander pressure chamber (1050D, PMS Instrument Albany, USA) at intervals of 5-10 mg decline in fresh mass (XP 205 Metter Toledo balance, Mettler � Toledo � Ltd., Griefense, Switzerland) to well below turgor loss point. Relative water content (RWC, %) for each fresh mass measurement was calculated as: RWC= ((fm-dm)/(sm-dm) )100 #(1) where fm is the fresh mass, sm is the saturated mass and dm is the dry mass after oven drying for 72 h at 70�C. Key leaf traits and water relations parameters were determined, including leaf dry mass per area (LMA), saturated water content (SWC), bulk osmotic potential at turgor loss point (πTLP), relative water content at turgor loss point (RWCTLP), osmotic potential at full hydration (πO), leaf bulk modulus of elasticity (ε), symplastic modulus of elasticity (εsymplast), and fractions of leaf water held in the apoplast (Af) and symplast (Sf) were calculated from the p-v curves (Bartlett, Scoffoni & Sack 2012). Capacitance between full turgor and turgor loss (Cleaf), for leaves was calculated as: (C_leaf= (100-〖RWC〗_TLP)/(0-π_TLP ) #(2) Given that variation in capacitance at leaf water potentials below turgor loss is non-linear, C following turgor loss was estimated by plotting RWC as a function of 1/ Ψleaf, fitting a linear function and using the derived slope of RWC for a given water potential (ΔRWC / ΔΨleaf) for p-v curves of each season (Figure S1). To assess the water storage capacity of stem tissues and leaf tissues in shoots (including stem and leaves), shoot pressure-volume curves were constructed in the early and late dry season using a modified bench drying method (Gleason, Blackman, Cook, Laws & Westoby 2014). One shoot, &gt;60cm in length, was cut from each of four trees in the early dry season and five trees in the late dry season in the late afternoon, each with several dozen leaves. Upon return to the lab, these shoots were recut under perfusion solution of 1 % seawater (~0.02 MPa) and rehydrated overnight. Upon rehydration to &gt; -0.5 MPa, terminal shoots were re-cut, weighed, and Ψshoot was determined as the average Ψleaf of two leaves, measured using a pressure chamber. Shoots were then allowed to bench dry, permitting sufficient time for Ψshoot to decline ~0.5 MPa, after which the shoots were incubated in black plastic bags for 40-90 min, to allow equilibration. Shoots were then re-weighed before and after measurement of Ψshoot as above. This sequence was repeated and records were kept to account for the decline in fresh mass due to repeated removal of leaves for determination of Ψshoot after each drying interval, and determining the dry mass of removed leaves, and of the remaining stems and leaves and stems after oven drying for &gt;72h at 70� C. From these data, the shoot water content (WC; gwater g-1dry mass) and RWC was determined throughout the dehydration, yielding a shoot p-v curve. The leaf water fraction of shoot water was determined from leaf capacitance (gwater released g-1dry mass MPa-1) derived from the leaf p-v curves based on the ∆Ψ for each dehydration interval. Leaf water content was then subtracted from total shoot water content to determine the stem water content during shoot dehydration, yielding a stem p-v curve. For shoots and stems, C was then calculated from the linear slope describing decline in water content with decrease in shoot water potential during the initial linear part of the slope. Diurnal hydraulic function and gas exchange characteristics Diurnal changes in stem and leaf water status, gas exchange and leaf hydraulic conductance were monitored in five co-occurring trees in both the early dry season and late dry season. Diurnal measurements of Ψleaf and Ψstem were measured over three clear sunny days (with no leaf wetting on the preceding evening) in the early dry season (August 19-21) at 6:00 (predawn; ΨPD), 9:00 (morning), 12:00 (midday), and 15:00 h (afternoon), and on one clear sunny day in the late dry season (November 22) at 5:00 (predawn; ΨPD), 8:00 (morning), 11:00 (midday), 14:00 (afternoon) and 17:00 h. Sampling times were shifted forward 1 h between seasons to standardize comparisons of morning, midday and afternoon measurements based on hours from sunrise, which differed between seasons. Sunrise occurred at 6:35am and 5:38am for early and late dry season respectively. On the evening preceding measurements, on each tree, a pair of healthy sun leaves were selected to be sampled at each time point. For each pair, one leaf was left exposed (i.e., allowed to transpire normally), while the other leaf was wrapped in plastic film and aluminium foil, to equilibrate with the stem and thus enable measurement of stem water potential (Melcher et al., 1998). The first round of measurements on the subsequent day assessed predawn Ψleaf, Ψstem and estuarine salinity. At subsequent time points, gas exchange measurements were made on the exposed leaf (using a portable photosynthesis system, LI-COR 6400XT, LI-COR Biosciences, Nebraska, USA). Photosynthetically active radiation was standardized at 1000 �mol photons m-2 s-1 and the flow rate was set to 500 �mol s-1, and all other parameters (CO2, temperature, humidity) were left at ambient levels. After attaching the gas analyser head, measurements were logged after photosynthetic rate and stomatal conductance stabilized (usually 2-3 min). The time elapsed between first tree and last tree during a measurement cycle was &lt; 45 min. After gas exchange measurements, the exposed and the wrapped leaves were harvested, placed into zip-lock bags and their water potentials measured in the lab within 90 min. Leaf hydraulic conductance (Kleaf) was calculated from diurnal gas exchange and water potential measurements on an area basis: (K_(leaf )=E/((Ψ_stem-Ψ_leaf ) ) #(3) where E is the transpiration rate and Ψstem - Ψleaf represents the water potential gradient between the transpiring leaf (Ψleaf) and stem (Ψstem). Similarly, whole-plant conductance (Kplant) was calculated on a leaf area basis as: (K_(plant )=E/((Ψ_est-Ψ_leaf ) )#(4) ) where Ψest is the estuarine water potential. To assess seasonal changes in the hydraulic resistance in the roots and stem due to drought, the sum of stem and root resistance was obtained by subtracting leaf from whole plant resistance as in Tsuda and Tyree (1997): (R_(roots and stem)=1/K_plant - 1/K_leaf #(5) Minimum leaf conductance (gmin), was determined for leaves from each of nine trees in the early dry season and five in the late dry season. Petioles of hydrated leaves were sealed with petroleum jelly, and leaves were then suspended to dry for 5 � 6 h in 1.8 L chambers containing 50 g silica gel. Chamber air was circulated using a 40 mm, 9V DC fan (YX2503, Sirocco Industrial Co. Taiwan). Leaves were weighed at 45 min intervals. By plotting water loss versus time, cuticular transpiration was determined for the linear region (r2 &gt; 0.995, from a minimum of 6 points per leaf) after initial stomatal closure. Temperature and relative humidity (RH) of chambers were recorded at 10 min intervals and were averaged from two data loggers (ibutton DS1923, Maxim integrated products, San Jose, USA) per chamber. Mean temperature and RH were 25 � 2 �C and 12.5 � 4 % respectively. Minimal leaf conductance, gmin, was determined by methods previously described (Kerstiens, 1996; Sack et al., 2003) as the cuticular transpiration, per two sided area, per mole fraction difference in water vapor between leaf and air, where air inside the leaf was assumed saturated with water vapour (Pearcy et al., 2000). Hydraulic vulnerability The relationship between Kleaf (leaf hydraulic conductance via the petiole) and declining leaf water status (Ψleaf) was determined using the rehydration kinetics method (Brodribb & Holbrook, 2003). Large shoots were obtained from six trees at the study site in the early dry season only. From each branch, branchlets containing &gt; 6 leaves were cut and were dehydrated to differing degrees, then allowed to equilibrate in black plastic bags for &gt;30 min. Initial water potential was measured from a proxy leaf on the equilibrated branchlets. Petioles of the remaining leaves were then cut under a perfusion solution of 5 mM NaCl (0.02 MPa) and then leaves were allowed to rehydrate via the petiole for periods of 30, 60, 120, 240 and 300 seconds. Post rehydration, leaves were held in the dark in zip-lock bags for &gt;10 min to allow equilibration before water potential was re-measured. Leaf area and leaf dry mass were then determined as previously described. Kleaf was calculated as: (K_leaf=(C_leaf ln(Ψ_(l,0)/Ψ_(l,t) ))/t #(6) where Cleaf is the leaf area normalized instantaneous capacitance derived from leaf pressure-volume curves as described above, t is the duration of rehydration, Ψl,0 is the water potential of the proxy leaf before hydration treatments, and Ψl,t is the water potential of a leaf at time t of rehydration. Each branchlet yielded one Kleaf measurement, averaged from five leaves, each with increasing rehydration periods. The response of stomatal conductance and Ψleaf was plotted using paired measurements of gas exchange and Ψleaf made in the late dry season. Early dry season measurements were not included in gs response curves as seasonal differences in modes of diurnal gas exchange were observed between early and late dry season. In the early dry season stomatal conductance was moderate throughout the day, whereas in the late dry season stomatal conductance was characterized by high values in the morning followed by rapid decline with declining water status in the afternoon. Loss of stem hydraulic conductivity with declining water potential (Ψstem), was estimated in the early dry season using the pneumatic method (Pereira et al. 2016; Zhang et al. 2018). Maximum vessel length was determined for shoots from five trees by supplying the basal end of a cut branch with air under low positive pressure, and making progressive cuts at the distal, submerged end of the branch until air was discharged from the distal end, indicating the longest vessel had been cut. The length of the remaining branch was determined with a measuring tape (Ewers & Fisher, 1989). Maximum vessel length was 47 � 1 cm; subsequently, pneumatic curves were constructed for shoots &gt; 60cm in length, from each of six trees. Shoots were harvested late afternoon, kept in black plastic bags during transit and re-cut in the lab under a perfusion solution of 1% seawater (~0.02 MPa) and rehydrated overnight. After rehydration to &gt; 0.3 MPa shoots were recut, and the cut end sheathed in 1cm diameter rubber tubing. Tubing was fastened around the branch with two cable ties, and sealed with silicon sealant at the branch-tubing interface (Aquarium SikaSeal, Sika Australia Pty Ltd, NSW, Australia), and the tubing was then connected to a fitting (Luer-lock, Cole-Palmer, Vernon Hills, IL, United States). Air discharge was measured by exposing the cut end of a branch, via a three-way stop-cock to a 3.9mL vacuum reservoir with an absolute pressure of ~40 kPa attached to a pressure transducer (PX140 Series, Omega, CT). Pressure was logged every second (using a data logger shield, Arduino Uno, Arduino.cc), and room temperature (ibutton data logger DS1923, Maxim Integrated Products, San Jose, USA). Initial pressure (Pi) and pressure after 150 seconds (Pf) were recorded. Air discharged (AD) from the stem into the vacuum reservoir was calculated using the ideal gas law as: AD=(V (P_f-P_i ))/RT #(7) where V was the volume of the vacuum reservoir (0.0039 L), R the gas constant (8.314 kPa L mol-1 K-1) and T the room temperature (K). The percentage of air discharged (AD, %), was calculated for each branch as: (AD %=((〖AD〗_x-〖AD〗_min ))/((〖AD〗_max-〖AD〗_min ) )#(8) ) where ADx was the volume of air discharged during each measurement, ADmin the minimum volume of air discharged when the branch was maximally hydrated and ADmax the maximum volume of air discharged. The initial air discharged (ADmin) accounts for the non-conduit air volume from the pith, intercellular spaces and outside air. Increases in air discharge as the branch dehydrates are understood to come from air-seeding into conduits (Pereira et al., 2016). Shoots were allowed to bench dry from fully hydrated to -10 MPa with measurements made at ~1 MPa intervals. Kstem, Kleaf and gs values, were plotted against Ψ values for measurements and fitted with a re-parameterized Weibull model (Ogle et al. (2009): (K= K_max�(1/2)^(〖50/P_50 〗^(((P50*Sx)/((Ψ-100)*log⁡(1/2) )) ) )#(9)) where Kmax was the average maximum observed conductance, P50 was the water potential corresponding to 50% loss of conductance, Sx was the slope of the curve at P50 and Ψ was the water potential of the leaf or stem. For comparison of the relative responsiveness of stomatal conductance, leaf hydraulic conductance and percent stem air discharge, percent loss of conductance (PLC) was calculated for each as: (PLC=100 � (1-K/K_max )#(10) ) where K was measured conductance and Kmax was maximum observed conductance. Leaf or stem water potentials corresponding to 12% (P12), 50% (P50) and 88% (P88) loss of conductance were estimated and, where sample size permitted, 95% bootstrap confidence intervals were generated with 1000 resamples using the fitplc package for R (cran.r-project.org/package=fitplc). Measurements from all shoots were pooled and random effects associated with individual shoots were factored into estimates and confidence intervals using the random effects argument of the fitplc package in R (Version 3.6.1, R Development Core Team, 2019). Strength of modelled fits were assessed by plotting curve predictions of conductance against observed conductance, and significance of correlations were assessed with linear models. Hydraulic vulnerability segmentation was assessed using hydraulic safety margins HSM calculated for leaves and stems, as in Choat et al. (2012), of the late dry season as the difference between the minimum water potential (Ψmin) and the water potential associated with 50% and 88% loss of conductance (P50 and P88, respectively) as: (〖HSM〗_50= Ψ_min-P_50 #(11) ) (〖HSM〗_88= Ψ_min-P_88 #(12) The minimum leaf water potential was determined as the lowest diurnal Ψleaf measurements made during the late dry season. The minimum stem water potential was determined similarly, but in leaves which were covered the previous day with plastic film and foil to prevent transpiration and allow equilibration with the stem (Melcher et al., 1998). Seasonal differences in the capacity of leaf water to buffer exposure to critical stem water potentials were assessed through the integration of the lowest midday water potential measurements, stem hydraulic vulnerability curves, and leaf pressure-volume curves. Hydraulic safety margins between leaf midday RWC and leaf RWC corresponding to stem P50 were determined by calculating the RWC margin between the water potentials associated with ΨMD and stem P50 as: (〖HSM〗_(50 stem)= SWC�LMA (〖RWC〗_(leaf at ΨMD)-〖RWC〗_(leaf at stem P50) )#(13)