Anatomical analysis
For measurements of partial volume of total vasculature, vascular
bundles (both including and excluding the bundle sheath), and xylem,
cross sections were made on leaf 5 at the point of maximum lamina width.
The freehand transverse sections were immersed in water and imaged under
UV light filter (with excitation wavelength range 350-520nm) using cell
wall autofluorescence to maximize the contrast between the bundle sheath
cell wall and the surrounding mesophyll cells. Images were captured
using Leitz Epi-Fluorscent Microscope with Leica DFC425C Camera at 100x
for the leaf blade and 250x for the midrib. Additional images were taken
at 40x magnification for the leaf blade and 25x for the midrib to
calculate cross sectional leaf area. The section area of all vascular
bundles (including and excluding the bundle sheath) was measured. For
xylem area, diameter of each xylem element was measured twice
perpendicular to each other, and mean diameter was used to calculate
xylem area as the area of a circle. All measurements were made in ImageJ
(Schneider 2012). The total vasculature
was calculated as area of the vascular bundles in the leaf blade plus
the entire midrib. Partial volume of each was calculated as the total
cross-sectional area of each respective anatomical feature relative to
the total leaf cross-sectional area (blade area + midrib area).
The surface of three fresh leaves per genotype per treatment was
analyzed under the low vacuum mode of FEI Scanning Electron Microscope
(SEM) Quanta 200F (FEI Company, Field Emission Instruments, Hillsboro,
OR, USA). The leaves were the same or similar to those used for gas
exchange measurements and anatomical analyses. Images of the adaxial and
abaxial epidermal surfaces were randomly captured across the entire leaf
blade avoiding the central vein (min. 10 images per leaf) to determine
the number of stomata per mm2 of leaf surface
(stomatal density) and axis length of stomatal pores.