Chlorophyll (Chl) and nitrogen (N) content measurements
Leaf Chl content and Chl a /b ratio were determined as
reported previously (Takagi et al., 2019). In brief, leaf segments were
incubated in N, N -dimethylformamide at 4 °C overnight and the
absorbance values of the aliquot were measured at 750 nm, 663.8 nm, and
646.8 nm to calculate the chlorophyll content (Porra et al., 1989). The
total leaf nitrogen content was determined by using the Nessler’s
reagent in a digestion solution after the addition of potassium sodium
tartrate (KNaC4H4O6) and
then measuring the absorbance at 420 nm (Takagi et al., 2017).