The frozen leaf sample was homogenized using a chilled mortar
and pestle with cooled extraction buffer containing 50 mM Tris–HCl
(pH 7.5), 1 mM ethylene diamine tetraacetic acid (EDTA), 1 mM
MgCl2, 12.5% (v/v) glycerin, 10% poly vinyl pyrrolidone (PVP), and
10 mM -mercaptoethanol. The homogenate was centrifuged at
15,000
×
g for 15 min at 4 ◦C.