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. 2016 Sep 7;28(9):2147–2160. doi: 10.1105/tpc.16.00428

Figure 2.

Figure 2.

Mg-Dechelating Activity and Substrate Specificity of Recombinant SGR1 and SGRL.

(A) Pigment analysis after incubation of chlorophyll derivatives with SGR. Chlorophyll a and chlorophyllide a were incubated with recombinant GFP, SGR1 with a FLAG-tag (SGR1-FLAG) for 60 min, or with recombinant SGRL with a FLAG-tag (SGRL-FLAG) for 15 min. Recombinant proteins were prepared with a wheat germ protein expression system and diluted 3-fold with the reaction buffer without purification. GFP was used as a negative control because it has a similar molecular weight as SGR. Chlorophyll b was incubated with recombinant GFP, SGR1-FLAG, and SGRL-FLAG for 60 min. The concentration of substrates was 6 µM. After incubation, pigments were analyzed using HPLC. Pigments were detected at 410 nm for chlorophyll a derivatives or 435 nm for chlorophyll b derivatives.

(B) An increase in chlorophyll derivatives by SGR activity. The levels of pheophytin a and pheophorbide a were determined after incubation of recombinant GFP and SGR1 with a FLAG-tag (SGR1-FLAG and SGRL-FLAG) with 6 µM of chlorophyll a and chlorophyllide a (n = 3 ±sd). The incubation times of SGR1-FLAG and SGRL-FLAG were 60 and 15 min, respectively. Recombinant proteins were prepared with a wheat germ protein expression system and diluted with the reaction buffer without purification. GFP was used as a negative control because it has similar molecular weight as SGR.