Computational protocol: Importance of leaf anatomy in determining mesophyll diffusion conductance to CO2 across species: quantitative limitations and scaling up by models

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[…] After the gas-exchange measurements, 1×1mm pieces were cut between the main veins from the same leaves for anatomical measurements. Leaf material was quickly fixed under vacuum with 4% glutaraldehyde and 2% paraformaldehyde in 0.1M phosphate buffer (pH 7.2). Afterwards, the samples were fixed in 1% osmium tetroxide for 1h and dehydrated in a graded ethanol series, followed by washing three times in propylene oxide. The dehydrated segments were embedded in Spurr’s resin (Monocomp Instrumentación, Madrid, Spain) and cured in an oven at 60 ºC for 48h. Semi-thin (0.8 µm) and ultrathin (90nm) cross-sections were cut with an ultramicrotome (Reichert & Jung model Ultracut E). Semi-thin cross-sections were stained with 1% toluidine blue and viewed under an Olympus BX60light microscope. Photos were taken at 200× and 500× magnification with a digital camera (U-TVO.5XC; Olympus) to measure the leaf thickness and thickness of the palisade and spongy tissue layers (Supplementary Fig. S1A–C). Ultrathin cross-sections for transmission electron microscopy (TEM H600; Hitachi) were contrasted with uranyl acetate and lead citrate. Photos were taken at 2000× magnification (Supplementary Fig. S1D–F) to measure the length of mesophyll cells and chloroplasts adjacent to intercellular air spaces and chloroplast width and thickness, and the volume fraction of intercellular air space calculated as:where ΣS s is the total cross-sectional area of mesophyll cells, W is the width of the section, and t mes is the mesophyll thickness between the two epidermises. Mesophyll (S m/S) and chloroplast (S c/S) surface area exposed to intercellular air spaces per leaf area were calculated separately for spongy and palisade tissues as described by and . The curvature correction factor was measured and calculated for each species according to ) for palisade and spongy cells by measuring their width and height and calculating an average width/height ratio. The curvature factor correction ranged from 1.16 to 1.4 for spongy cells and from 1.4 to 1.5 for palisade cells. All parameters were analysed at least in four different fields of view and at three different sections. Weighted averages based on tissue volume fractions were calculated for S m/S and S c/S. T cw and cytoplasm thickness (T cyt) were measured at 20 000–40 000× magnification depending on the species (Supplementary Fig. S1G–I). Three different sections per species and four to six different fields of view were measured for each anatomical characteristic. Micrographs were selected randomly in each section and T cw was measured for two to three cells per micrograph. Ten measurements for spongy tissue and ten for palisade parenchyma cells were made for each anatomical trait, and weighted averages based on tissue volume fractions were calculated. All images were analysed with Image analysis software (ImageJ; Wayne Rasband/NIH, Bethesda, MD, USA). [...] Regression and correlation analyses were conducted using the Sigma Plot 10.0 software package (SPSS; Chicago, IL, USA). Univariate analysis of variance was performed to reveal differences between species in the studied characteristics. Differences between means were revealed by Tukey analyses (P <0.05). These analyses were performed with the IBM SPSS statistics 19.0 software package (SPSS). […]

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