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.