Engineered GFP as a vital reporter in plants

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Engineered GFP as a vital reporter in plants Wan-ling Chiu, Yasuo Niwa, Weike Zeng, Takanori Hirano, Hirokazu Kobayashi, Jen Sheen Current Biology Volume 6, Issue 3, Pages 325-330 (March 1996) DOI: 10.1016/S0960-9822(02)00483-9

Figure 1 Engineered GFP gives faster and brighter fluorescent signals. Maize mesophyll protoplasts were electroporated with three plasmids expressing (a,d) the jellyfish GFP (GFP), (b,e) synthetic GFP (sGFP) or (c,f) a mutant S65T sGFP (sGFP(S65T)). The transfected protoplasts were observed after (a–c) 4 h or (d–f) 16 h of incubation. (g) Synthetic GFP gives a high level of protein expression. Untransfected control (C) and transfected maize protoplasts were labeled with 400 μCi ml−1[35S]methionine for 12 h. Total proteins were solublized in protein loading buffer and separated by 12.5 % SDS-PAGE. Current Biology 1996 6, 325-330DOI: (10.1016/S0960-9822(02)00483-9)

Figure 1 Engineered GFP gives faster and brighter fluorescent signals. Maize mesophyll protoplasts were electroporated with three plasmids expressing (a,d) the jellyfish GFP (GFP), (b,e) synthetic GFP (sGFP) or (c,f) a mutant S65T sGFP (sGFP(S65T)). The transfected protoplasts were observed after (a–c) 4 h or (d–f) 16 h of incubation. (g) Synthetic GFP gives a high level of protein expression. Untransfected control (C) and transfected maize protoplasts were labeled with 400 μCi ml−1[35S]methionine for 12 h. Total proteins were solublized in protein loading buffer and separated by 12.5 % SDS-PAGE. Current Biology 1996 6, 325-330DOI: (10.1016/S0960-9822(02)00483-9)

Figure 1 Engineered GFP gives faster and brighter fluorescent signals. Maize mesophyll protoplasts were electroporated with three plasmids expressing (a,d) the jellyfish GFP (GFP), (b,e) synthetic GFP (sGFP) or (c,f) a mutant S65T sGFP (sGFP(S65T)). The transfected protoplasts were observed after (a–c) 4 h or (d–f) 16 h of incubation. (g) Synthetic GFP gives a high level of protein expression. Untransfected control (C) and transfected maize protoplasts were labeled with 400 μCi ml−1[35S]methionine for 12 h. Total proteins were solublized in protein loading buffer and separated by 12.5 % SDS-PAGE. Current Biology 1996 6, 325-330DOI: (10.1016/S0960-9822(02)00483-9)

Figure 1 Engineered GFP gives faster and brighter fluorescent signals. Maize mesophyll protoplasts were electroporated with three plasmids expressing (a,d) the jellyfish GFP (GFP), (b,e) synthetic GFP (sGFP) or (c,f) a mutant S65T sGFP (sGFP(S65T)). The transfected protoplasts were observed after (a–c) 4 h or (d–f) 16 h of incubation. (g) Synthetic GFP gives a high level of protein expression. Untransfected control (C) and transfected maize protoplasts were labeled with 400 μCi ml−1[35S]methionine for 12 h. Total proteins were solublized in protein loading buffer and separated by 12.5 % SDS-PAGE. Current Biology 1996 6, 325-330DOI: (10.1016/S0960-9822(02)00483-9)

Figure 1 Engineered GFP gives faster and brighter fluorescent signals. Maize mesophyll protoplasts were electroporated with three plasmids expressing (a,d) the jellyfish GFP (GFP), (b,e) synthetic GFP (sGFP) or (c,f) a mutant S65T sGFP (sGFP(S65T)). The transfected protoplasts were observed after (a–c) 4 h or (d–f) 16 h of incubation. (g) Synthetic GFP gives a high level of protein expression. Untransfected control (C) and transfected maize protoplasts were labeled with 400 μCi ml−1[35S]methionine for 12 h. Total proteins were solublized in protein loading buffer and separated by 12.5 % SDS-PAGE. Current Biology 1996 6, 325-330DOI: (10.1016/S0960-9822(02)00483-9)

Figure 1 Engineered GFP gives faster and brighter fluorescent signals. Maize mesophyll protoplasts were electroporated with three plasmids expressing (a,d) the jellyfish GFP (GFP), (b,e) synthetic GFP (sGFP) or (c,f) a mutant S65T sGFP (sGFP(S65T)). The transfected protoplasts were observed after (a–c) 4 h or (d–f) 16 h of incubation. (g) Synthetic GFP gives a high level of protein expression. Untransfected control (C) and transfected maize protoplasts were labeled with 400 μCi ml−1[35S]methionine for 12 h. Total proteins were solublized in protein loading buffer and separated by 12.5 % SDS-PAGE. Current Biology 1996 6, 325-330DOI: (10.1016/S0960-9822(02)00483-9)

Figure 1 Engineered GFP gives faster and brighter fluorescent signals. Maize mesophyll protoplasts were electroporated with three plasmids expressing (a,d) the jellyfish GFP (GFP), (b,e) synthetic GFP (sGFP) or (c,f) a mutant S65T sGFP (sGFP(S65T)). The transfected protoplasts were observed after (a–c) 4 h or (d–f) 16 h of incubation. (g) Synthetic GFP gives a high level of protein expression. Untransfected control (C) and transfected maize protoplasts were labeled with 400 μCi ml−1[35S]methionine for 12 h. Total proteins were solublized in protein loading buffer and separated by 12.5 % SDS-PAGE. Current Biology 1996 6, 325-330DOI: (10.1016/S0960-9822(02)00483-9)

Figure 2 The expression of AtCAB2-sGFP(S65T) is regulated by light in maize leaf protoplasts. (a) Untransfected control protoplasts. (b,c) Transfected protoplasts incubated (b) in the dark or (c) under light for 20 h. Current Biology 1996 6, 325-330DOI: (10.1016/S0960-9822(02)00483-9)

Figure 2 The expression of AtCAB2-sGFP(S65T) is regulated by light in maize leaf protoplasts. (a) Untransfected control protoplasts. (b,c) Transfected protoplasts incubated (b) in the dark or (c) under light for 20 h. Current Biology 1996 6, 325-330DOI: (10.1016/S0960-9822(02)00483-9)

Figure 2 The expression of AtCAB2-sGFP(S65T) is regulated by light in maize leaf protoplasts. (a) Untransfected control protoplasts. (b,c) Transfected protoplasts incubated (b) in the dark or (c) under light for 20 h. Current Biology 1996 6, 325-330DOI: (10.1016/S0960-9822(02)00483-9)

Figure 3 The expression of 35SC4PPDK-sGFP(S65T) in tobacco mesophyll protoplasts. (a) Untransfected control and (b,c) transfected tobacco protoplasts after 20 h incubation. In (c)the red autofluorescence of chlorophyll was blocked using a interference filter. Current Biology 1996 6, 325-330DOI: (10.1016/S0960-9822(02)00483-9)

Figure 3 The expression of 35SC4PPDK-sGFP(S65T) in tobacco mesophyll protoplasts. (a) Untransfected control and (b,c) transfected tobacco protoplasts after 20 h incubation. In (c)the red autofluorescence of chlorophyll was blocked using a interference filter. Current Biology 1996 6, 325-330DOI: (10.1016/S0960-9822(02)00483-9)

Figure 3 The expression of 35SC4PPDK-sGFP(S65T) in tobacco mesophyll protoplasts. (a) Untransfected control and (b,c) transfected tobacco protoplasts after 20 h incubation. In (c)the red autofluorescence of chlorophyll was blocked using a interference filter. Current Biology 1996 6, 325-330DOI: (10.1016/S0960-9822(02)00483-9)

Figure 4 Organelle targeting in Arabidopsis. Constructs carrying (a,d)35SΩ-sGFP(S65T), (b,e)35SΩ-NLS-sGFP(S65T) or (c,f)35SΩ-TP-sGFP(S65T) were bombarded into Arabidopsis (a–c) leaves or (d–f) roots. The expression and localization of sGFP(S65T) was observed after 24 h of incubation. Current Biology 1996 6, 325-330DOI: (10.1016/S0960-9822(02)00483-9)

Figure 4 Organelle targeting in Arabidopsis. Constructs carrying (a,d)35SΩ-sGFP(S65T), (b,e)35SΩ-NLS-sGFP(S65T) or (c,f)35SΩ-TP-sGFP(S65T) were bombarded into Arabidopsis (a–c) leaves or (d–f) roots. The expression and localization of sGFP(S65T) was observed after 24 h of incubation. Current Biology 1996 6, 325-330DOI: (10.1016/S0960-9822(02)00483-9)

Figure 4 Organelle targeting in Arabidopsis. Constructs carrying (a,d)35SΩ-sGFP(S65T), (b,e)35SΩ-NLS-sGFP(S65T) or (c,f)35SΩ-TP-sGFP(S65T) were bombarded into Arabidopsis (a–c) leaves or (d–f) roots. The expression and localization of sGFP(S65T) was observed after 24 h of incubation. Current Biology 1996 6, 325-330DOI: (10.1016/S0960-9822(02)00483-9)

Figure 4 Organelle targeting in Arabidopsis. Constructs carrying (a,d)35SΩ-sGFP(S65T), (b,e)35SΩ-NLS-sGFP(S65T) or (c,f)35SΩ-TP-sGFP(S65T) were bombarded into Arabidopsis (a–c) leaves or (d–f) roots. The expression and localization of sGFP(S65T) was observed after 24 h of incubation. Current Biology 1996 6, 325-330DOI: (10.1016/S0960-9822(02)00483-9)

Figure 4 Organelle targeting in Arabidopsis. Constructs carrying (a,d)35SΩ-sGFP(S65T), (b,e)35SΩ-NLS-sGFP(S65T) or (c,f)35SΩ-TP-sGFP(S65T) were bombarded into Arabidopsis (a–c) leaves or (d–f) roots. The expression and localization of sGFP(S65T) was observed after 24 h of incubation. Current Biology 1996 6, 325-330DOI: (10.1016/S0960-9822(02)00483-9)

Figure 4 Organelle targeting in Arabidopsis. Constructs carrying (a,d)35SΩ-sGFP(S65T), (b,e)35SΩ-NLS-sGFP(S65T) or (c,f)35SΩ-TP-sGFP(S65T) were bombarded into Arabidopsis (a–c) leaves or (d–f) roots. The expression and localization of sGFP(S65T) was observed after 24 h of incubation. Current Biology 1996 6, 325-330DOI: (10.1016/S0960-9822(02)00483-9)

Figure 5 Nuclear localization of sGFP(S65T) in onion cells. Constructs carrying (a)35SΩ-sGFP(S65T) or (b)35SΩ-NLS-sGFP(S65T) were bombarded into onion skin epidermal cells. The expression and localization of sGFP(S65T) was observed after 24 hours incubation. Current Biology 1996 6, 325-330DOI: (10.1016/S0960-9822(02)00483-9)

Figure 5 Nuclear localization of sGFP(S65T) in onion cells. Constructs carrying (a)35SΩ-sGFP(S65T) or (b)35SΩ-NLS-sGFP(S65T) were bombarded into onion skin epidermal cells. The expression and localization of sGFP(S65T) was observed after 24 hours incubation. Current Biology 1996 6, 325-330DOI: (10.1016/S0960-9822(02)00483-9)

Figure 6 sGFP(S65T) as a vital marker in transgenic tobacco plants. (a,d) Untransformed control and (b,c,e,f) transgenic tobacco plant (a–c) leaves and (d–f) protoplasts were observed using a fluorescence microscope with a FITC filter set without or (c,f) with an interference filter. (g–i) Drought-inducible expression of sGFP(S65T) in transgenic tobacco plants. Transgenic plants carrying the AtRD29A-sGFP(S65T) construct were allowed to wilt and the expression of sGFP(S65T) on leaf surface was observed (g) without or (h,i) with an interference filter. Bright green fluorescence was most striking in the nucleus of trichome and guard cells as indicated by arrows. Current Biology 1996 6, 325-330DOI: (10.1016/S0960-9822(02)00483-9)

Figure 6 sGFP(S65T) as a vital marker in transgenic tobacco plants. (a,d) Untransformed control and (b,c,e,f) transgenic tobacco plant (a–c) leaves and (d–f) protoplasts were observed using a fluorescence microscope with a FITC filter set without or (c,f) with an interference filter. (g–i) Drought-inducible expression of sGFP(S65T) in transgenic tobacco plants. Transgenic plants carrying the AtRD29A-sGFP(S65T) construct were allowed to wilt and the expression of sGFP(S65T) on leaf surface was observed (g) without or (h,i) with an interference filter. Bright green fluorescence was most striking in the nucleus of trichome and guard cells as indicated by arrows. Current Biology 1996 6, 325-330DOI: (10.1016/S0960-9822(02)00483-9)

Figure 6 sGFP(S65T) as a vital marker in transgenic tobacco plants. (a,d) Untransformed control and (b,c,e,f) transgenic tobacco plant (a–c) leaves and (d–f) protoplasts were observed using a fluorescence microscope with a FITC filter set without or (c,f) with an interference filter. (g–i) Drought-inducible expression of sGFP(S65T) in transgenic tobacco plants. Transgenic plants carrying the AtRD29A-sGFP(S65T) construct were allowed to wilt and the expression of sGFP(S65T) on leaf surface was observed (g) without or (h,i) with an interference filter. Bright green fluorescence was most striking in the nucleus of trichome and guard cells as indicated by arrows. Current Biology 1996 6, 325-330DOI: (10.1016/S0960-9822(02)00483-9)