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Plant CellPhysiol. 41(3): 367-371 (2000) JSPP © 2000 Short Communication Chloroplast Tubules Visualized in Transplastomic Plants Expressing Green Fluorescent Protein Takashi Shiina1, Keiko Hayashi2, Nao Ishii, Kazuya Morikawa and Yoshinori Toyoshima3 Graduate School of Human and Environmental Studies, Kyoto University, Yoshida-nihonmatsu-cho, Sakyo-ku, Kyoto, 606-8501 Japan A fusion between the plastid psbA promoter and the greenfluorescentprotein gene igfp) was introduced into the tobacco chloroplast genome by stable plastid transformation. GFP was synthesized actively and exclusively in the chloroplasts. Tubular projections filled with GFP but containing no chlorophyll were visualized for the first time in chloroplasts of these transplastomic plants. Occasionally, the tubules connect chloroplasts with each other, suggesting the possibility of the exchange of endogenous proteins. However, the fusion of protoplasts between the transplastomic and wild-type plants showed that such chloroplast connections might be rare in mesophyll protoplasts. tween chloroplasts were reported in higher plant cells decades ago (Weier and Thomson 1962, Wildman et al. 1962, Vesk et al. 1965). However, chloroplast interconnections were not recognized in general and neither their function nor their origin has been clarified. Green fluorescent protein (GFP) is a powerful tool to visualize subcellular structures in living cells (Rizzuto et al. 1995, Kohler et al. 1997a, b). Recently, tubular connections between chloroplasts were visualized in transgenic plants in which translational fusion of the chloroplast transit signal and GFP was expressed in cytoplasm and targeted into the stroma of chloroplasts (Kohler et al. 1997b). Similar connections were also observed in etioplasts (Tirlapur et al. 1999). These tubular connections are likely to be involved in the exchange of endogenous proteins between chloroplasts. When GFP was selectively photobleached in one of the two connected chloroplasts, a rapid recovery of GFP fluorescence in the bleached chloroplast was observed, indicating the flow of GFP through the tubules (Kohler et al. 1997b). The tubules may also have an important role as a route for the flow of genomic information between chloroplasts to establish genetic homoplasmicity. However, it is difficult to estimate the exchange rates of macromolecules between chloroplasts by using nuclear transformants. As mentioned above, the chloroplast tubular connections have been so far visualized in nuclear transgenic plants (Kohler et al. 1997b, Tirlapur et al. 1999). In this paper, we visualized chloroplast tubules for the first time in stable transplastomic plants expressing GFP via the plastid genome. The psbA promoter is recognized by eubacterial multi-subunit plastid RNA polymerase (PEP). It comprises the well conserved —35 and —10 elements spaced 18 nucleotides apart and exhibits the highest transcription rates in chloroplasts (Rapp et al. 1992). Previously, we reported that the —35 element was required for transcription initiation from the psbA promoter in younger leaves, but not in developed leaves in wheat (Satoh et al. 1999). In order to examine the role of the —35 element of the psbA promoter in dicots, we constructed tranplastomic tobacco plants with the green fluorescent protein gene (gfp) expressed from psbA promoter derivatives (Hayashi et al., in preparation). By using the transplastomic plant that expresses GFP from the wild-type psbA promoter core Key words: Chloroplast — Chloroplast transformation — Chloroplast tubule — Green fluorescent protein — Nicotiana tobaccum — psbA promoter. Plastids are semiautonomous organelles that contain their own genome and are reproduced by fission. The surface of plastids is bounded by an envelope consisting of two membranes. Most macromolecules, such as proteins and nucleic acids are not able to penetrate the inner membrane of the envelope. Thus, chloroplasts in general have been considered as discrete organelles. On the other hand, fusions and/or connections between chloroplasts have been observed in some algae. It has been reported that Euglena chloroplasts connect to each other by many bridges prior to chloroplast division (Ehara et al. 1990). In Acetabularia, membrane tubules emanating from chloroplasts connect the chloroplasts with each other (Menzel 1994). In addition, similar images of interconnections beAbbreviations: GFP, Green fluorescent protein; MES, 2-(NMorpholino)ethanesulfonic Acid; MS medium, Murashige and Skoog medium; SD signal, Shine-Dalgarno signal. 1 Present address: Faculty of Human Environment, Kyoto Prefectural University Hangi-cho, Shimogamo, Sakyo-ku, Kyoto, 606-8522 Japan. 2 Present address: Hokuriku National Agricultural Experiment Station, Inada 1-2-1, Joetu, Niigata, 943-0193 Japan. 3 Corresponding author: Fax: +81-75-753-6577, E-mail: ytoyoshi @ip. media. kyoto-u. ac .j p. 367 Tubular connections between chloroplasts 368 (KH6), we could visualize tubular projections emanating from chloroplasts. This transgenic line offers an opportunity for estimating rates of protein transport among chloroplasts via tubular connections by examining the distribution of GFP among chloroplasts following protoplast fusion. In wheat chloroplasts, the psbA promoter core is responsible for maintaining the high transcription rate (Satoh et al. 1999). Thus, the tobacco psbA promoter core was cloned in upstream of the gfp cassette consisting of a SD signal; gfp and Trpsl6 (Fig. 1). The gfp gene was modified by introducing mutations into a chromophore region (Phe^Ser65 to Leu^Thr65). The chimeric gfp gene was cloned into a pKH5 plastid transformation vector designed following pRV112A (Zoubenko et al. 1994). The pKH5 contains the aadA cassette (P16SrDNA::aadA:: TpsbA) excised from a plasmid pCT08 (Shikanai et al. 1998). Details of the plasmid construction will be described elsewhere (Hayashi et al., in preparation). Transformation of tobacco (Nicotiana tabacum cv. Xanthi) plastids was carried out using a Biorad PDSlOOOHe Biolistic gun at 1100 PSI following the method described by Svab and Maliga (1993). Transgenic shoots were selected on agarsolidified MS medium (Murashige and Skoog 1962) containing sucrose (3%), 1-naphthaleneacetic acid (0.1 mg liter"1), A^-benzyladenine (1 mg liter" 1 ), thiamine (1 mg liter" 1 ), inositol (100 mg liter"1) and spectinomycin dihydrochloride (500 mg liter"1) as described by Svab et al. (1990). Transgenic plants rooted on MS agar medium containing sucrose (3%) or soil were used for observation with microscopes. Confocal imaging of cells was performed using a confocal laser-scanning microscope (CLSM) (MRC600 Z or /^Radiance BioRad U.S.A.). The GFP was excited with the 488 nm of blue line and the chlorophyll was excited with the 568 nm line. These fluorescence images were col- TpsbA I 16SrDNA | |"| tmV lected in green or red channels, respectively. Serial optical sections were obtained at 0.5 //m intervals and processed using NIH image and PhotoShop (Adobe Systems Inc). GFP fluorescence was detected in transplastomic plants, while control wild-type plants did not show GFP fluorescence (data not shown). Fig. 2 shows fluorescence images of mesophyll and guard cells in a transplastomic tobacco plant. As expected, GFP fluorescence was localized in chloroplasts, indicating that GFP was synthesized and accumulated in chloroplasts and did not move to other organelles. Thin tubular projections emanating from chloroplasts were detected, but their avandance varied from cell to cell. The diameter of tubules was around 0.5 /um and their length was up to 10 /um. Sometimes a branching of tubules could also be observed. No chlorophyll autofluorescence was detected from the tubules, indicating that thylakoid membranes do not extend into them. The shape Prm PpsbA Trps16 iiinili 70B rps7/12 —*• 1 2kb 5bp Fig. 1 A gfp cassette driven by psbA promoter. PpsbA indicates the position of the psbA promoter; gfp encodes the modified green fluorescent protein; Trpsl6 refers to the 3-UTR of the rpsl6 ribosomal protein gene; Prrn indicates the position of the 16SrRNA promoter; aadA encodes aminoglycocide 3 "-adenylyltransferase; TpsbA refers to the 3'-UTR of the psbA gene. The psbA promoter core was connected with a gfp gene via a linker containing a synthesized SD signal. Fig. 2 Images of GFP (a, d) and chlorophyll (c, f) fluorescence taken with a laser confocal microscope (CLSM). (a-c) are projections of 85 optical sections of the same mesophyll cells; (d-f) are projections of 34 optical sections of a guard cell. Panels b and e show merged images of both GFP and chlorophyll fluorescence. All optical sections were taken at 0.5 ^m intervals by CLSM. Chloroplast tubules are indicated by arrows. Tubules connecting chloroplasts are indicated by red arrows. Scale bars, 10 ^m. Tubular connections between chloroplasts and length of the tubules changed dynamically in the living cell. They wave, shrink and extend continuously. In some cells, tubules filled with GFP connect two or more chloroplasts (Fig. 2b). It is expected that such connections between chloroplasts would contribute to keep the concentration of inorganic ions, metabolic intermediates, proteins and enzymes uniform among several chloroplasts in the same cell. Transplastomic plants in which GFP was exclusively synthesized in chloroplasts enable us to estimate the rate of GFP transfer among chloroplasts by fusioning protoplasts between the transplastomic and wild-type plants. We isolated protoplasts from mesophyll cells of both plants by treatment with a solution containing 0.5 M mannitol, 2.5 mM MES (pH 5.7), 1% Cellulase Onozuka and 0.5% Macerozyme at 25°C in the dark for I d . Wild-type protoplasts were mixed with protoplasts from the transplastomic plants and polyethylene glycol-induced fusion was carried out. The fused cells were incubated at 25°C in the light. At first, the two types of chloroplast with and without GFP fluorescence coexisted at the ratio of one to one in most of the fused cells. A rapid flow of GFP through the tubular connections between chloroplasts has been demonstrated by selective photobleaching of individual chloroplasts (Kohler et al. 1997b, Tirlapur et al. 1999). If tubular connections between chloroplasts were so frequent that all chloroplasts connected with each other via tubules, GFP fluorescence would distribute in all fused cell chloroplasts. Contrary to this, we found that the fraction of chloroplasts exhibiting GFP fluorescence in the fused cells did not increase significantly up to 3 d (Fig. 3). This strongly suggests that chloroplast connections are rare in most mesophyll protoplasts and exchange of endogenous proteins between chloroplasts does not occur frequently. One may assume that the tubular connections observed in mesophyll cells of leaves disappeared during protoplast Green channel 369 Merged image Red channel Fig. 3 Fluorescent image of fused protoplasts with a conventional fluorescent microscope, (a) GFP fluorescence; (c) chlorophyll fluorescence; (b) merged image of both GFP and chlorophyll fluorescence. Images were taken 2 d after the fusion of protoplasts. preparation due to a loss of turgor pressure or change of light or hormone conditions. However, the following observations provided further support to the above conclusion. When transient expression of GFP in chloroplasts was examined two days after the introduction of the gfp gene by bombardment, GFP fluorescence was usually detected in only a single chloroplast per cell (data not shown). The similar observation has been reported by Hibberd et al. (1998). GFP did not distribute to all chloroplasts in a few days following microinjection of the gfp gene into a single chloroplast in a living mesophyll cell (Knoblauch et al. 1999). Furthermore, Khan and Maliga (1999) reported that some plastids express GFP whereas others do not in the same cell in chimeric transplastomic sectors. From these observations, it may be concluded that exchange of endogenous proteins between chloroplasts is a rare event in most leaf cells. However, it may occur actively at some specific cell stages or in some special cells. In facts, the tubules were more frequently observed in mesophyll cells of the matured leaves than those of the etiolated cotyledons in tobacco (Fig. 4). Furthermore, we could observe well-developed Fig. 4 Images of GFP fluorescence of mesophyll cells of the matured leaves (a) and those of the etiolated cotyledons (b) taken with CLSM. All images are single optical sections taken at 0.5 ^m intervals by CLSM and merged images of both GFP and chlorophyll fluorescence are shown. In etioplasts, GFP fluorescence overwhelms red fluorescence of chlorophyll. Chloroplast tubules are indicated by arrowheads. Scale bar, 10 fxm. Tubular connections between chloroplasts 370 Green channel Red channel Fig. 5 Images of GFPfluorescenceof a trichome cell taken with CLSM. (a) GFPfluorescence;(b) chlorophyllfluorescence.All images are projections of 6 optical sections taken at 0.5 /xm intervals by CLSM. The cell margin is indicated by dot lines. Scale bar, chloroplast tubules in mesophyll cells adjacent to the epidermal cells in some leaves (Fig. 4a). These observations suggest that chloroplast tubules may be regulated developmentally and/or tissue-specifically and have functions related to photosynthesis and the intracellular signal transduction. Previously, the presence of tubular connections between etioplasts has been demonstrated in stomatal guard cells of Arabidopsis (Tirlapur et al. 1999). The chloroplast tubules may be regulated in a different manner during chloroplast development between tobacco and Arabisopsis. Recently, Kohler and Hanson (2000) reported that the appearance of the plastid tubules (stromules) are abundant in root cells. However, we could not observe them due to the low level GFP expression from the leafspecific psbA promoter in root cells. Further study is required to understand the developmental regulation of chloroplast tubules. In addition, we found that chloroplast tubules were most abundant in trichome cells of tobacco leaves (Fig. 5). Several tubules emanate from single chloroplasts and many of them end at a near cell membrane. They might be involved in transducing membrane signals to the chloroplasts or acquiring molecules which are localized in some special compartments in the cell (Tilapur et al. 1999). Further study is needed to characterize the protein transport through the tubular connections between chloroplasts. It would be interesting to know whether chloroplast genome or genetic information is exchangeable through the tubules. There are more than 10 plastids in a leaf cell. As described above, in transient expression of GFP in chloroplasts, GFP fluorescence was usually detected in only a single chloroplast in a cell after bombardment. However, in stable plastid transformation all the plastid genome copies are uniformly altered. Transformed and non-transformed plastids may sort out during cultivation of the cells on a selective medium. Alternatively, it is expected that the introduced gfp gene may pass to other plastids through chloroplast tubules during this process. In addition, intergenomic recombination between chloroplast genomes has also been reported in some somatic hybrids in higher plants (Medgyesy et al. 1985, Fejes et al. 1990, Baldev et al. 1998). The chloroplast tubules may have an important role in recombination of the chloroplast genome and establishing homoplasmicity. The chloroplast tubules are reminiscent of bacterial pilli. It is interesting to note that the appearance of the tubular connections is coordinated with a cycle of plastid division in Euglena (Ehara 1990). Interconnections of chloroplasts have been observed in Euglena (Ehara 1990), Acetabularia (Menzel 1994) and several higher plants (Koler et al. 1997b, Tirlapur et al. 1999). The evolutional diversity of the chloroplast tubules also supports the physiological importance of the interconnection between chloroplasts. We thank Dr. T. Shikanai (Nara Institute of Science and Technology, Japan) for providing the plasmid pCT08, and Drs. S. Tsukita and A. Nagafuchi (Graduate School of Medicine, Kyoto University, Japan) for access to a confocal laser microscope. Thanks are also due to Dr. E.O. Hideg (Biological Reserach Center of the Hungarian Academy of Sciences, Hungary) for critical reading of the manuscript. This work was supported partly by a Grant-in-Aid for Scientific Research in Priority Areas (no. 11151218) to YT and Grants-in-Aid for Scientific research (no. 10640629 and 10309008) to TS from the Ministry of Education, Science and Culture of Japan; by a grant from the Research for the Future program, Japan Society for the Promotion of Science (JSPS-RFTF96L00604) to TS. KM is a Research Fellow of the Japan Society for the promotion of Science. References Baldev, A., Gaikwad. K., Kirti, P.B., Mohapatra, T., Prakash, S. and Chopra, V.L. (1998) Recombination between chloroplast genomes of Trachystoma ballii and Brassica juncea following protoplast fusion. Mol. Gen. Genet. 260: 357-361. Ehara, T., Osafune, T. and Hase, E. 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