PMC

Time course of chlorophyllide a formation during pigment extraction. Chlorophyll was extracted by immersing leaves in pure acetone as described in the “Time-course experiments” subsection of the Methods section. A. Levels of chlorophyll a and chlorophyllide a per gram fresh weight of leaves. B. Chlorophyllide a levels in sample extracts expressed as the ratio of chlorophyllide a to the sum of chlorophyll a and chlorophyllide a. Chl a, chlorophyll a. Chlide a, chlorophyllide a. Error bars indicate standard deviations. Sample size, n = 3. n.d. = not detected.

Comparison of extraction methods for chlorophyllide formation with three different species (Glebionis coronaria, Pisum sativum L. and Prunus sargentii Rehd.). Leaves were homogenized in acetone at room temperatures (“room temperatures”) or sub-zero temperatures (“sub-zero temperatures”), respectively, by grinding leaves with steel stainless beads. Alternatively, leaves were immersed in pure acetone at 4°C (“acetone 4°C”) or in pure DMF at 4°C (“DMF 4°C”), respectively, without the grinding procedure. In the last pair of experiments, leaves were boiled for 5 (“boiling 5 sec”) or 10 sec (“boiling 10 sec”), and were subsequently immersed in acetone at 4°C for chlorophyll extraction. Details of the methods is described in the Method section. A, C and E. Levels of chlorophyll a and chlorophyllide a per gram FW of leaves. B, D and F. Chlorophyllide a levels in sample extracts expressed as the ratio of chlorophyllide a to the sum of chlorophyll a and chlorophyllide a. Chl a, chlorophyll a. Chlide a, chlorophyllide a. Error bars indicate standard deviations. Sample size, n = 3.

Formation of chlorophyllide a by the extraction of chlorophyll with pure or 80% buffered acetone. Frozen leaves were immersed in pure acetone or 80% acetone containing 20% (v/v) Tris–HCl, pH 8 and then incubated in these solvents for 12 h at 4°C in the dark. Pigments were subsequently extracted by grinding with stainless steel beads as described in the Methods section. A. Levels of chlorophyll a and chlorophyllide a per gram fresh weight of leaves. B. Chlorophyllide a levels in sample extracts expressed as the ratio of cholorophyllide a to the sum of chlorophyll a and chlorophyllide a. Chl a, chlorophyll a. Chlide a, chlorophyllide a. Error bars indicate standard deviations. Sample size, n = 3.

Effect of quick boiling on the formation of chlorophyllide a during extraction. Mature leaves (7th to 9th leaves counting from the bottom of the plant) were collected from 4-week-old wild-type plants that had been sprayed with 100 μM MeJA and kept for 3 days at the same growth conditions. The collected leaves were immersed in pure acetone at room temperature directly or were boiled for 5 and 10 sec respectively before they were immersed in pure acetone, then they were ground with stainless steel beads by vigorous shaking (A and B). Alternatively, pigments were extracted by immersing leaves in pure acetone at 4°C for 12 hours (C and D). A and C, Levels of chlorophyll a and chlorophyllide a per gram fresh weight of leaves. B and D, Chlorophyllide a levels in sample extracts expressed as the ratio of chlorophyllide a to the sum of chlorophyll a and chlorophyllide a. Chl a, chlorophyll a. Chlide a, chlorophyllide a. Error bars indicate standard deviations. Sample size, n = 3.