Kinematic Analysis
The kinematic analysis was performed according to Sprangers et al.
(2016). It includes measurements of leaf length, cell length profile
along the leaf axis, calculation of leaf elongation rate and estimation
of the size of the leaf basal meristem. Leaf length was measured daily
with a ruler on the fifth leaf from the moment it was visible among the
older leaves. LER was calculated for the first 3 days of its growth as
the difference in length divided by the time difference between
successive measurements. For meristem measurements, samples harvested 3
days after leaf emergence were analyzed by fluorescence microscopy
(AxioScope A1, Axiocam ICm1, Zeiss, Germany) at 20× magnification. The
size of the meristematic zone of the leaf was estimated by locating the
most distal mitosis in the cell files. Cell length was measured by light
microscopy (AxioScope A1, Axiocam ICm1, Zeiss, Germany) using
differential interference contrast at 40× magnification and the online
measurement module in the Axiovision software (Zeiss, Germany). The leaf
growth zone was split in ten 1cm-long- segments and cell length
measurements were carried out at four locations in each segment: at the
tip (0 cm), at one-third of the segment (0.3 cm), at two-thirds (0.6
cm), and at the end of the segment (0.99 cm). Around 20 cells were
measured at each location. The raw data obtained for individual leaves
were smoothed and interpolated at an interval of 50 mm using the
kernel-smoothing function locpoly of the KernSmooth package (Wand and
Jones, 1995) for the R statistical software environment (R Core Team,
2011; https://www.R-project.org), which allowed averaging between
leaves and comparison between treatments. At least 5 plants were
measured per line/treatment combination.