Determination of photosynthetic and nonphotosynthetic pigment
contents
The procedure for the determination of chlorophyll (Chl) was as follows.
Three 2-cm-long stem segments or 10-mm-wide leaf discs were homogenized
in 4 mL of 80% acetone using a mortar and pestle and then centrifuged
at 8000 g and 4°C for 10 min. The contents of Chl a , Chl band the total Chl in the supernatant were determined and calculated
according to the methods of Wellburn (1994) and expressed as micrograms
per unit area (cm2). Since Chl was mainly distributed
on the surface layer of stems, the surface area was used in the
calculation of Chl contents.
Anthocyanins in the stems and leaves were extracted with methanol-HCl
(99:1, v/v) (Craker et al., 1971). Three 1-cm-long stem segments or 1-cm
leaf discs were submerged in 4 mL of methanol-HCl overnight in the dark.
The Chl in the extract was removed by the addition of 4 mL of chloroform
and 1.5 mL of deionized water. The absorbance of the anthocyanin extract
at 530 nm was measured. Anthocyanin concentrations were calculated by
using a standard curve constructed by cyanidin-3-O-glucoside (5–200 μM)
and ultimately expressed as micrograms per unit surface area
(cm2).