Measurement of structural and anatomical traits associated
with gm and Kleaf
Light and electron microscopy techniques were used to measure important
structural and anatomical traits listed in Table 1. The details of
sample preparation for light and electron microscopy and measurements
were presented in Pathare et al ., (2020). Light microscopy images
of leaf cross sections were used to measure average leaf thickness
(calculated as average of maximum and minimum leaf thickness) ,
interveinal distance (IVD), vein-to-adaxial epidermis distance
(VEDada), vein-to-abaxial epidermis distance
(VEDaba), average VED (calculated as
(VEDada + VEDaba) /2) and length of
mesophyll cell walls exposed to intercellular air spaces (IAS) using
10-15 different fields of view for each leaf (n = 3 per species)
taken at x 50 and x 100 magnifications. Portion of BS cell walls exposed
to IAS (BSias) was calculated as a percentage from the
total BS cell wall length. BS area ratio was calculated as a percentage
using BS area and mesophyll area (BS area/ [BS area + Mesophyll
area]) for each species (Griffiths et al ., 2013). The mesophyll
surface area exposed to IAS per unit leaf area
(S mes) was calculated from measurements of total
length of mesophyll cell walls exposed to IAS and width of section
analyzed using equation from Evans et al., (1994) with curvature
correction factor (F) of 1.34. Mesophyll and BS cell wall thickness
(MCW and BSCW) was measured from TEM
micrographs using at least 15 images for each leaf. Total leaf vein
length per unit leaf area (VLA) was calculated from the total number of
veins per section length and expressed per mm2considering parallel venation in grasses.
Images of the adaxial and abaxial epidermal surfaces, captured on four
leaves (each from a different replicate; n = 4) per species under
the low-vacuum mode with a FEI Scanning Electron Microscope Quanta 200F
(FEI Co., Field Emission Instruments), were used to measure the stomatal
number on each surface of leaf and expressed per mm2as adaxial stomatal density (SDada) and abaxial stomatal
density (SDaba). The SR was calculated as ratio of the
SD on the adaxial and abaxial surfaces. The stomatal size for adaxial
(SSada) and abaxial (SSaba) stomata was
calculated as guard cell length x Ʃ guard cell widths,
µm2.