DOI: 10.25911/r4ve-kk98
title: Data and Metadata on leaf carbohydrates and carbon exchange rates - Asao, et al, New Phytologist

description: Sites and sampling
Data were collected from 19 sites across five biomes spanning 68.63° to -43.42° in latitude, and included 382 mature leaf samples belonging to 114 species. Sample sites varied in climate, with mean annual temperatures (MAT) ranging from -11.3 to 25.2 ºC and mean annual precipitation (MAP) ranging from 225 to 4330 mm. Site climate was characterized by 24 bioclimatic variables taken from WorldClim (Fick & Hijmans, 2017), aridity index (AI; annual potential evapotranspiration divided by mean annual precipitation), longitude, and latitude. They were reduced to two principal components that explained 78% of the total variance. The first component represented variables including annual temperature range, precipitation of wettest quarter, and MAT, while the second component represented maximum temperature of warmest month, mean annual potential evaporation, and precipitation of the coldest quarter. The sites used in our study formed part of the global network used in an earlier analysis of global patterns in leaf respiration and associated leaf traits (Atkin et al., 2015). 
 Measurements were made during the summer at each site, or during generally optimal periods (recent rainfall) in a seasonal sites. At most sites (13 of the 19), measurements were made on a single mature leaf from multiple replicate plants per species (n=3-4). Each replicate was analyzed for non-soluble carbohydrates (NSC) concentrations, along with other leaf economic spectrum (LES) traits such as specific leaf area (SLA), nitrogen concentration, and gas exchange variables. In the remaining six sites, we pooled the leaves of replicate plants from each species for analysis of leaf N, P, and NSC concentrations because the leaves were too small to analyze individually. For these six sites, leaf-level values of the measured parameters were therefore averaged into species means (in a total of 43 species). 
Physiological measurements
Gas exchange measurements were made in situ on mature and fully-expanded leaves of wild plants. Leaf gas exchange was typically measured between 9 am and 1 pm on attached and sun-facing leaves in the upper canopy. We measured steady-state light-saturated net CO2 assimilation rates (Asat) using portable photosynthesis systems (Li-6400, Licor, Lincoln, NE, USA). Asat reflects CO2 uptake by Rubisco minus CO2 release by the processes of photorespiration and non-photorespiratory mitochondrial respiration in the light. Cuvette conditions were 1000 - 2000 µmol photons m-2 s-1 of photosynthetically active radiation (as needed to achieve light saturation, depending on the species and site), a reference line [CO2] of 400 ppm, relative humidity of 60-70%, and block temperature set to the prevailing ambient day-time temperature of each site (6-34 °C, depending on site location). Immediately after measuring Asat, the cuvette light was turned off and dark respiration rates (Rdark) were taken after a 30-45 minute dark acclimation period. Both Asat and Rdark were measured at the prevailing ambient temperature at the time of measurement at each site to reflect the rates of photosynthesis and respiration that control sampled leaf NSC concentrations. Flow rates through the leaf cuvette were set to 500 and 300 µmol s-1 for Asat and Rdark measurements, respectively. Using these rates, we estimated daytime cumulative net assimilation (Aday) and nighttime cumulative respiration (Rnight) assuming daylight duration (D) at the summer solstice at each site: Aday = Asat x (D x 0.9), and Rnight = Rdark x (24 – D). Daylight duration was defined as the time from sunrise to sunset at the sampling date for each site and then reduced to 90% to account for low light at dawn and dusk. Aber et al. (1996) have previously reported a value of 76% relating Asat to achieved Aday, so we confirmed our analysis was robust down to a value as low as 20% (see Notes S3). Both Aday and Rnight overestimate leaf C revenue and respiratory cost as Asat was measured at saturating light and Asat and Rdark both were measured at the prevailing temperature during warmer periods of the day. In addition, it is known that R declines during the night period such that it is lower than daytime measurements of Rdark (Bruhn et al., 2022; Bruhn, 2023).
On completion of each set of Asat and Rdark measurements, these and adjacent leaves were harvested typically between mid-morning to mid-afternoon, placed in a moistened plastic bag, and stored in a cool dark location until the measurement of leaf area. Leaves were scanned on a 600 dots/inch flatbed top-illumination optical scanner to quantify leaf area using Image J software (http://imagej.nih.gov/ij/). Scanned leaves were dried at 70 °C for a minimum of 72 h and measured for dry mass. Specific leaf area (SLA, m2 g-1) was then calculated. Most leaves were stored in darkness for 1-5 hours between gas exchange measurements and commencement of oven drying for subsequent analysis. For a majority of sites, both leaf N and P concentrations were determined using Kjeldahl acid digests (Allen et al., 1974) that were analyzed using a LaChat QuikChem 8500 Series 2 Flow Injection Analysis System (Lachat Instruments, Milwaukee, WI, USA). Where only leaf [N] was determined (15 out of 382 samples), ground samples (31–700 Hammer Mill; Glen Creston, Stanmore, UK) were combusted using a Carlo-Erba elemental analyzer NA1500 (Thermo Fisher Scientific, Milan, Italy). These leaves were excluded from data analysis that required both N and P concentrations. 
Dried samples of whole leaves were ground in a mill and analyzed for concentrations of soluble sugars (glucose + sucrose + fructose), starch, and total NSCs (soluble + starch). Soluble sugars were extracted in hot ethanol: 5 mg of ground plant material and 500 uL of 80% (v/v) ethanol were mixed by vortex in an Eppendorf tube, incubated at 80 °C for 20 min while vortex mixing every 10 mins, then centrifuged at 12000 r.p.m for 5 min to separate and collect the supernatant. This process was repeated twice more using 500 uL of 80% (v/v) ethanol, and the collected supernatant was combined. The resultant pellet was used to quantify starch concentration with a Total Starch Assay Kit (Megazyme, Bray, Ireland). The supernatant was used to quantify soluble sugar concentrations (glucose, sucrose, and fructose) with a Fructose Assay Kit (Sigma-Aldrich, Darmstadt, Germany). For glucose, extracts were incubated with hexokinase and glucose-6-phosphate dehydrogenase to enable the reduction of NADP to NADPH which was then followed spectrophotometrically at 340 nm (Dynatech Laboratories MRX, Guernsey, UK). For fructose, extracts were incubated with hexokinase, phosphoglucose isomerase, and glucose-6-phosphate dehydrogenase, the resultant reduction of NADP to NADPH was followed (Scholes et al., 1994) and the glucose concentration was subtracted. For sucrose, extracts were incubated with invertase (Sigma, St Louis, MO, USA) to quantify the concentration of sucrose plus glucose, and glucose concentration was subtracted. Measurements were collected using a micro-titer plate reader (Infinite® M1000 Pro; Tecan). Total NSC concentrations represent the combined concentrations of soluble sugars and starch. All samples were prepared and analyzed within the same laboratory (Quentin et al., 2015; Landhäusser et al., 2018).