Exchange of protein molecules through connections between higher plant plastids

Science

Volumn 276, Issue 5321, 1997, Pages 2039-2042

  Köhler, Rainer H ^a   Cao, Jun ^a   Zipfel, Warren R ^b   Webb, Watt W ^b   Hanson, Maureen R ^a  

^a Department of Obstetrics Gynecology   (United States)

^b Cornell University   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

VEGETABLE PROTEIN;

ARTICLE; FLUORESCENCE; NONHUMAN; PLANT; PLASTID; PRIORITY JOURNAL; PROTEIN ANALYSIS; TRANSGENE;

AMINO ACID SEQUENCE; BASE SEQUENCE; CHLOROPLASTS; CYTOPLASM; GREEN FLUORESCENT PROTEINS; LUMINESCENT PROTEINS; MICROSCOPY; MICROSCOPY, FLUORESCENCE; MOLECULAR SEQUENCE DATA; PLANT LEAVES; PLANTS, GENETICALLY MODIFIED; PLANTS, TOXIC; RECOMBINANT FUSION PROTEINS; TOBACCO;

EID: 0038620661     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.276.5321.2039     Document Type: Article

References (29)

1. Reviewed in J. K. Hoober, Chloroplasts (Plenum, New York, 1984), and J. T. O. Kirk and R. A. E. Tilney-Bassett, The Plastids: Their Chemistry, Structure, Growth and Inheritance (Elsevier/North-Holland, Amsterdam, ed. 2, 1978).
2. Reviewed in J. K. Hoober, Chloroplasts (Plenum, New York, 1984), and J. T. O. Kirk and R. A. E. Tilney-Bassett, The Plastids: Their Chemistry, Structure, Growth and Inheritance (Elsevier/North-Holland, Amsterdam, ed. 2, 1978).
3. Reviewed in R. Cline and R. Henry, Annu. Rev. Cell Dev. Biol. 12, 1 (1996).
4. R. H. Köhler, W. R. Zipfel, W. W. Webb, M. R. Hanson, Plant J. 11, 613 (1997).
5. The modified GFP4 coding region [J. Haseloff and B. Amos, Trends Genet. 111, 328 (1995)], which was further altered (3) by a Ser-to-Thr mutation at codon 65 (S65TmGFP4), was fused in frame with the Arabidopsis recA transit peptide (CT) [H. Cerutti, M. Osman, P. Grandoni, A. T. Jagendorf, Proc. Natl. Acad. Sci. U.S.A. 89, 8068 (1992)].
6. The modified GFP4 coding region [J. Haseloff and B. Amos, Trends Genet. 111, 328 (1995)], which was further altered (3) by a Ser-to-Thr mutation at codon 65 (S65TmGFP4), was fused in frame with the Arabidopsis recA transit peptide (CT) [H. Cerutti, M. Osman, P. Grandoni, A. T. Jagendorf, Proc. Natl. Acad. Sci. U.S.A. 89, 8068 (1992)].
7. The CT-GFP fusion was cloned into the Bam HI and Sst I sites of pBIN19-based binary vector pCT37 [C. M. Tobias and J. B. Nasrallah, Plant J. 10, 523 (1996)], introduced into A. tumefaciens pCIB542/ A136 [E. E. Hood, G. L. Helmer, R. T. Fraley, M. D. Chilton, J. Bacteriol. 168, 1291 (1986)], and used to transform plants [R. B. Horsch et al., Science 227, 1229 (1985)].
8. The CT-GFP fusion was cloned into the Bam HI and Sst I sites of pBIN19-based binary vector pCT37 [C. M. Tobias and J. B. Nasrallah, Plant J. 10, 523 (1996)], introduced into A. tumefaciens pCIB542/ A136 [E. E. Hood, G. L. Helmer, R. T. Fraley, M. D. Chilton, J. Bacteriol. 168, 1291 (1986)], and used to transform plants [R. B. Horsch et al., Science 227, 1229 (1985)].
9. The CT-GFP fusion was cloned into the Bam HI and Sst I sites of pBIN19-based binary vector pCT37 [C. M. Tobias and J. B. Nasrallah, Plant J. 10, 523 (1996)], introduced into A. tumefaciens pCIB542/ A136 [E. E. Hood, G. L. Helmer, R. T. Fraley, M. D. Chilton, J. Bacteriol. 168, 1291 (1986)], and used to transform plants [R. B. Horsch et al., Science 227, 1229 (1985)].
10. We prepared the chloroplasts by breaking tobacco leaf cells with a mortar and pestle, followed by differential centrifugation and separation on a Percoll gradient according to J. Huang, E. Hack, R. W. Thornburg, and A. M. Meyer [Plant Cell 2, 1249 (1990)], except that a 15%/40%/80% Percoll gradient was used.
11. A BioRad MRC-600 CLSM with a standard K1/K2 filter set was used. Pseudocolor similar to the color observed by eye with the Olympus microscope was added to the images by the import of data collected in the green and red channels from the BioRad microscope into Adobe Photoshop. Optical sections were taken along the optical axis and projected into one image with the COMOS software (BioRad).
12. Purified chloroplasts were lysed for 10 min at 4°C in 50 mM Hepes at pH 7.5 and 1 mM phenymethylsulfonyl fluoride and separated by 15 min of centrifugation at 4°C into a soluble and a membrane fraction. The membrane fraction was washed twice in lysis buffer. Protein was quantified by the Bradford protein assay (Bio-Rad) and separated by SDS-polyacrylamide gel electrophoresis on a 12% gel, transferred to nitrocellulose, and probed with 1:1000 dilutions of the different antibodies as described in (3).
13. J. Zhao and R. L. Last, J. Biol. Chem. 270, 6081 (1995).
14. L. A. Payan and K. Cline, J. Cell Biol. 112, 603 (1991).
15. R. H. Köhler, J. Cao, W. R. Zipfel, W. W. Webb, M. R. Hanson, data not shown.
16. W. Denk, J. H. Strickler, W. W. Webb, Science 248, 73 (1990); R. M. Williams, D. W. Piston, W. W. Webb, FASEB J. 8, 804 (1994). The images were obtained with a two-photon microscope consisting of a Zeiss IM-35 microscope, a modified BioRad MRC-600, and a Spectra-Physics Tsunami Ti:S laser. GFP fluorescence was collected in non-descanned mode through a 500 dichroic long pass mirror and a 520DF35 emission filter; excitation was at 800 nm (with a 100-fs pulse width and an 80-MHz repetition rate). A Zeiss 40/1.2 water immersion objective lens was used. Localized photobleaching was carried out by repetitive rapid line scanning of the targeted subcellular structure.
17. W. Denk, J. H. Strickler, W. W. Webb, Science 248, 73 (1990); R. M. Williams, D. W. Piston, W. W. Webb, FASEB J. 8, 804 (1994). The images were obtained with a two-photon microscope consisting of a Zeiss IM-35 microscope, a modified BioRad MRC-600, and a Spectra-Physics Tsunami Ti:S laser. GFP fluorescence was collected in non-descanned mode through a 500 dichroic long pass mirror and a 520DF35 emission filter; excitation was at 800 nm (with a 100-fs pulse width and an 80-MHz repetition rate). A Zeiss 40/1.2 water immersion objective lens was used. Localized photobleaching was carried out by repetitive rapid line scanning of the targeted subcellular structure.
18. S. G. Wildman, T. Hongladarom, S. I. Honda, Science 138, 434 (1962); S. G. Wildman, in Biochemistry of Chloroplasts, T. W. Goodwin, Ed. (Academic, Press, New York, 1967), vol. 2, pp. 295-319; T. Hongladarom, I. Shigeru, S. I. Honda, S. G. Wildman, "Organelles in Living Plant Cells," color sound film produced by P. Kahana and Y. Kahana (Extension Media Center, University of California, Berkeley, 1965); D. Spencer and S. G. Wildman, Aust. J. Biol. Sci. 15, 599 (1962).
19. S. G. Wildman, T. Hongladarom, S. I. Honda, Science 138, 434 (1962); S. G. Wildman, in Biochemistry of Chloroplasts, T. W. Goodwin, Ed. (Academic, Press, New York, 1967), vol. 2, pp. 295-319; T. Hongladarom, I. Shigeru, S. I. Honda, S. G. Wildman, "Organelles in Living Plant Cells," color sound film produced by P. Kahana and Y. Kahana (Extension Media Center, University of California, Berkeley, 1965); D. Spencer and S. G. Wildman, Aust. J. Biol. Sci. 15, 599 (1962).
20. S. G. Wildman, T. Hongladarom, S. I. Honda, Science 138, 434 (1962); S. G. Wildman, in Biochemistry of Chloroplasts, T. W. Goodwin, Ed. (Academic, Press, New York, 1967), vol. 2, pp. 295-319; T. Hongladarom, I. Shigeru, S. I. Honda, S. G. Wildman, "Organelles in Living Plant Cells," color sound film produced by P. Kahana and Y. Kahana (Extension Media Center, University of California, Berkeley, 1965); D. Spencer and S. G. Wildman, Aust. J. Biol. Sci. 15, 599 (1962).
21. S. G. Wildman, T. Hongladarom, S. I. Honda, Science 138, 434 (1962); S. G. Wildman, in Biochemistry of Chloroplasts, T. W. Goodwin, Ed. (Academic, Press, New York, 1967), vol. 2, pp. 295-319; T. Hongladarom, I. Shigeru, S. I. Honda, S. G. Wildman, "Organelles in Living Plant Cells," color sound film produced by P. Kahana and Y. Kahana (Extension Media Center, University of California, Berkeley, 1965); D. Spencer and S. G. Wildman, Aust. J. Biol. Sci. 15, 599 (1962).
22. M. Vesk, F. V. Mercer, J. V. Possingham, Aust. J. Bot. 13, 161 (1965); T. E. Weier and W. W. Thomson, Am. J. Bot. 49, 807 (1962).
23. M. Vesk, F. V. Mercer, J. V. Possingham, Aust. J. Bot. 13, 161 (1965); T. E. Weier and W. W. Thomson, Am. J. Bot. 49, 807 (1962).
24. D. Menzel, Protoplasma 179, 166 (1994).
25. T. Osafune, T. Ehara, E. Hase, J. A. Schiff, J. Electron Microsc. 42, 201 (1993); T. Ehara, T. Osafune, E. Hase, Exp. Cell Res. 190, 104 (1990).
26. T. Osafune, T. Ehara, E. Hase, J. A. Schiff, J. Electron Microsc. 42, 201 (1993); T. Ehara, T. Osafune, E. Hase, Exp. Cell Res. 190, 104 (1990).
27. M. W. Gray, Int. Rev. Cytol. 141, 233 (1992).
28. Differential interference contrast microscopic images were collected with an Olympus BX50 microscope equipped with a cooled charge-coupled device camera [Optronics (Chelmsford, MA) DEI-750T].
29. We thank J. Haseloff for the mGFP4 gene and T. Horiagon, R. Last, S. Lawrence, and K. Cline for antibodies to GFP, AS, and LHCP. Supported by DOE Energy Biosciences Program grant DE-FG02-89ER14030 to M.R.H. and grants to W.W.W. and Developmental Resources for Biophysical Imaging and Opto-electronics by NSF (DIR8800278) and NIH (RR04224 and R07719).