JXB Advance Access originally published online on January 30, 2004
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Journal of Experimental Botany, Vol. 55, No. 397, pp. 595-604, March 1, 2004
© 2004 Oxford University Press
Cell and Molecular Biology, Biochemistry and Molecular Physiology |
GFP-labelled Rubisco and aspartate aminotransferase are present in plastid stromules and traffic between plastids
Received 4 September 2003; Accepted 10 November 2003
Department of Molecular Biology and Genetics, 321 Biotechnology Building, Cornell University, Ithaca, NY 14853, USA
* To whom correspondence should be addressed. Fax: +1 607 255 6249. E-mail: mrh5{at}cornell.edu
Abbreviations: AAT, aspartate aminotransferase; AAT3, plastid aspartate aminotransferase; CFP, cyan fluorescent protein; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; LS, large subunit of Rubisco; L8S8, Rubisco holoenzyme; NPC, nuclear pore complex; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; SDS-PAGE, denaturing polyacrylamide gel electrophoresis; SS, small subunit of Rubisco.
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Abstract |
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Plastid stromules are membrane-bound protrusions of the plastid envelope that contain soluble stroma. Stromules are often found connecting plastids within a cell and fluorescence recovery after photobleaching (FRAP) experiments have demonstrated that green fluorescent protein (GFP) can move between plastids via these connections. In this report, the ability of endogenous plastid proteins to travel through stromules was investigated. The motility of GFP-labelled plastid aspartate aminotransferase and the Rubisco small subunit was studied in stromules by FRAP. Both fusion proteins assemble into protein complexes that appear to behave similarly to their endogenous counterparts. In addition, both enzymes are capable of trafficking between plastids via stromules.
Key words: Aspartate aminotransferase, chloroplast, FRAP, photobleaching, plastid, protein trafficking, Rubisco, stromule.
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Introduction |
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Plastid stromules, or stroma-filled tubules, are tubular projections of the plastid envelope membrane. Stromule-like structures were recognized as early as the beginning of the twentieth century, and there have been sporadic observations since then, but a clear understanding of their structure or function has never been achieved (Köhler et al., 1997; Gray et al., 2001). Recently, the use of green fluorescent protein (GFP) targeted to plastids has facilitated the study of stromules in vivo and in non-green tissues. Photobleaching of GFP in plastids connected by stromules has shown that GFP can move between plastids through stromules (Köhler et al., 1997). This discovery of macromolecular movement between plastids has sparked new interest in stromules, which have now been observed in a number of higher plant species by fluorescence, transmitted light, and electron microscopy (Bourett et al., 1999; Tirlapur et al., 1999; Langeveld et al., 2000; Pyke and Howells, 2002).
Stromules are highly variable in appearance. Their length can range from the short projections of 1–2 µm seen in chloroplasts to extensions up to 50 µm long seen in some non-green tissues (Bourett et al., 1999; Arimura et al., 2001). Stromules also show a wide range of diameters, often in the same cell. Widths have been reported ranging from less than 100 nm to greater than 1 µm (Langeveld et al., 2000; Pyke and Howells, 2002). When viewed in vivo, stromules are highly dynamic structures, extending and contracting from plastid bodies and moving through the cytoplasm (Wildman et al., 1962; Kwok and Hanson, 2003). Experiments with inhibitors of the cytoskeleton have demonstrated that microtubules and actin microfilaments both contribute to the morphology of stromules (Kwok and Hanson, 2003). Stromule motility, similar to chloroplast movement, is driven by actin microfilaments, with an inhibitory contribution from microtubules (Gray et al., 2001; Kwok and Hanson, 2003).
To date, research has not revealed any function for stromules in plants. However, fluorescence recovery after photobleaching (FRAP) experiments have shown that GFP residing in plastids can move through stromules between plastids (Köhler et al., 1997). The motility of a foreign protein such as GFP (molecular mass=30 kDa) indicates that other small proteins and metabolites should move freely through stromules as well. Furthermore, the movement of GFP suggests that macromolecules like DNA and RNA might also traffic. Stromules may, therefore, function as pathways for movement of metabolites and macromolecules between plastids in different parts of the cell. Hence, stromules may possibly be considered as organellar analogues of plasmodesmata, the cytoplasmic connections that join plant cells in many tissues.
Macromolecular movement through plasmodesmata is believed to occur via 2.5 nm channels in the cytoplasmic sleeve (Ding et al., 1992). Plasmodesmata are characterized by two forms of trafficking. A non-regulated trafficking, defined by the basal size exclusion limit, allows the diffusion of molecules ranging in size from less than one kDa up to 60 kDa, depending on tissue and developmental state (reviewed in Zambryski and Crawford, 2000). Regulated movement is observed for larger proteins and nucleic acids (Zambryski and Crawford, 2000). Parallels can also be drawn between stromules and the nuclear pore complex (NPC). The NPC is a collection of more than 100 proteins residing in the nuclear envelope that regulates traffic between the nucleus and cytoplasm. The NPC is a cylindrical pore with a diameter of 120 nm and a height of 70 nm (reviewed in Heese-Peck and Raikhel, 1998). NPCs also participate in two forms of trafficking. One system allows the free diffusion of metabolites and small macromolecules of up to 30 kDa, probably by a 9 nm channel (Paine et al., 1975). The second system utilizes a 26 nm regulated channel for energy-dependent import and export of proteins, RNA, and very large ribonucleoproteins of molecular mass up to 10 MDa (Görlich and Kutay, 1999).
The trafficking capacities of plasmodesmata and the NPC have been determined with some precision. By contrast, the nature of molecular movement in stromules is still unknown. As a first step in dissecting protein trafficking in stromules, experiments were conducted to determine if, in addition to GFP, endogenous plastid proteins can traffic via stromules, and if protein trafficking is limited by protein size. Two previous reports describe the presence of endogenous proteins in stromules. Tirlapur et al. (1999) fused two-thirds of the PAC protein to GFP and expressed the fusion in Arabidopsis thaliana. They found that GFP fluorescence was present in stromules and that the GFP could traffic between plastids. Bourett et al. (1999) localized the Rubisco large subunit (LS) to short projections of chloroplasts in Oryza sativa by immunocytochemistry. However, to the authors’ knowledge, no group has reported trafficking of full length endogenous plastid proteins or protein complexes.
In the work described below, GFP was fused to endogenous plastid proteins and their motility in stromules was observed by a photobleaching method. Two proteins were selected for study: the plastid aspartate aminotransferase (AAT3) and the Rubisco small subunit (SS). Both are nuclear-encoded proteins that are translated on cytosolic ribosomes and subsequently imported into plastids. Aspartate aminotransferases (AAT) are highly conserved enzymes found across kingdoms. These 45 kDa proteins are active as 90 kDa homodimers. AAT enzymes catalyse the reversible formation of oxaloacetate and glutamate from aspartate and 
-ketoglutarate using pyridoxal-5'-phosphate as a cofactor (reviewed in Wadsworth, 1997). In plants, AAT is believed to have roles in amino acid synthesis, ammonia assimilation, intercellular carbon shuttling in C4 plants, and interorganellar hydrogen shuttling. Higher plants contain multiple forms of AAT, specialized for function in various compartments in the cell. The plastid-localized enzyme, AAT3, was the focus of attention in this work.
Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) is the major protein constituent of chloroplasts (reviewed in Gutteridge and Gatenby, 1995). This enzyme functions in photosynthetic carbon fixation and photorespiratory oxygen fixation. Rubisco is a 550 kDa enzyme composed of 16 subunits. Eight 14.5 kDa small subunits (SS) combine with eight 55 kDa large subunits (LS) to form the holoenzyme (L8S8).
90 kDa AAT3 and 550 kDa Rubisco therefore provided a convenient size range to assay for the presence in stromules and trafficking between plastids. Protein assembly experiments confirm that the fusion proteins that were generated behave similarly to their endogenous counterparts in transgenic plants. Furthermore, FRAP assays demonstrate that both fusion proteins are capable of moving through stromules.
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Materials and methods |
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Fusion protein constructs
Standard molecular biology techniques were used for all experiments. Unless otherwise noted, all chemicals were obtained from Sigma (St Louis, MO, USA).
Constructs for plant transformation were generated in a pGPTV binary vector (Becker et al., 1992). For the ASP5:GFP construct, a cDNA of the A. thaliana ASP5 gene was used as a template for PCR amplification. The forward primer contained an NcoI restriction site at its 5' end. The reverse primer included sequences encoding a five amino acid linker (GSGGG) at its 3' end to promote proper folding and activity of ASP5 and GFP (Chandler et al., 1998). mGFP5(S65T) was amplified by PCR using a forward primer that overlapped the ASP5 fragment and a reverse primer with an SstI site at the 5' end. These two fragments were joined via recombinant PCR, sequenced, and inserted into the binary vector. For the SS:CFP construct, a cDNA of the Pisum sativum RbcS-3A gene, including the native plastid transit peptide, was used for PCR amplification. The forward primer contained an XbaI restriction site at the 5' end while the reverse primer contained a BamHI site at its 5' end. mCFP was amplified using a forward primer with a BamHI site and sequences encoding the same five amino acid linker used for ASP5:GFP at its 5' end. The reverse primer contained an SstI site at its 5' end. These two fragments were joined by restriction digestion and ligation, sequenced, and inserted into the binary vector. For the TP:CFP construct, the RbcS-3A transit peptide sequence was amplified using a forward primer with an XbaI site at its 5' end and a reverse primer that overlapped with mCFP. mCFP was amplified with a forward primer and a reverse primer identical to the reverse primer used for CFP in the SS:CFP construct. The two fragments were joined by recombinant PCR, sequenced, and inserted into the binary vector.
Agrobacterium-mediated transformation
Leaf strips of Nicotiana tabacum cv. Samsun NN were transformed with Agrobacterium tumefaciens strain LBA4404 as described by Horsch et al. (1985). Transgenic cells were selected on MS104 medium (2.2 g l–1 MS basal medium with Gamborg’s vitamins, 1 mg l–1 6-benzylaminopurine (BA), 0.1 mg l–1 -naphthaleneacetic acid (NAA), 30 g l–1 sucrose, and 8 g l–1 phytagar) supplemented with 300 µg ml–1 kanamycin and 100 µg ml–1 ticarcillin/clavulanic acid (15:1; Duchefa, Haarlem, The Netherlands). Regenerated plants were transferred to soil and screened for fluorescence and GFP protein by western blot (data not shown). Callus cultures were generated from leaf strips on solid NT1 medium (4.33 g l–1 MS basal salt mixture, 100 mg l–1 myo-inositol, 1 mg l–1 thiamine, 0.2 mg l–1 2,4-dichlorophenoxyacetic acid, 180 mg l–1 KH2PO4, and 9 g l–1 phytagar). Callus cells were placed in flasks of liquid NT1 and agitated at 110 rpm to induce suspension cell growth. Suspension cultures were agitated in liquid NT1 at room temperature in the dark and transferred weekly.
Microscopy
Laser scanning confocal microscopy was performed with a Leica TCS-SP2 confocal scanning head mounted on a Leica DMRE-7 (SDK) upright microscope equipped with a 100x HCX PlAPO oil immersion objective (NA=1.40; Leica Microsystems Inc., Bannockburn, IL, USA). GFP was excited with the 488 nm line of a 4-line Argon ion laser and emission was detected by collecting light between 500 and 600 nm. CFP was excited at 458 nm and emission was collected between 500 and 600 nm. Chlorophyll was excited at 633 nm and emission was detected between 660 and 700 nm. DIC images were generated by collecting transmitted light. For each picture, several images were collected at successive focal planes along the z-axis and then reassembled as a maximum projection.
Protein analysis
Proteins for AAT activity assay were isolated by grinding plant tissues in an extraction buffer described by Sentoku et al. (2000). Soluble chloroplast proteins were isolated as described by Gruissem et al. (1986). Root tissue was obtained from plants grown on liquid MS medium (2.2 g l–1 MS basal medium with Gamborg’s vitamins, 30 g l–1 sucrose) for 2–3 weeks. Proteins were separated on non-denaturing polyacrylamide gels (PAGE) as described by Schultz and Coruzzi (1995). AAT activity was detected by in-gel assay as described by Wendel and Weeden (1989). Briefly, gels were soaked for 30 min at room temperature with gentle agitation in a solution containing 2.5 mM -ketoglutaric acid, 10 mM L-aspartic acid, 125 µM PVP-40, 1.34 mM Na2EDTA (Fisher Scientific, Fair Lawn, NJ, USA), 100 mM dibasic sodium phosphate (Fisher Scientific), and 1 mg ml–1 Fast Blue BB salt. GFP fluorescence in AAT activity bands was analysed on a Storm 840 phosphorimager using 450 nm excitation (Amersham Biosciences, Piscataway, NJ, USA).
Tissues for immunoblot analysis were frozen in liquid nitrogen and ground in buffer containing 0.1 M TRIS-HCl pH 7.5, 0.1 M NaCl, 5 mM EDTA, 10 mM 2-mercaptoethanol, and 2.5 mM phenylmethylsulphonylfluoride. Proteins in extracts were quantified by Bradford assay and boiled for 5 min in loading buffer (250 mM TRIS-HCl pH 6.8, 20% glycerol, 4% sodium dodecyl sulphate, 2% 2-mercaptoethanol, and a trace of bromophenol blue). Proteins were separated on 12% SDS-PAGE and transferred to nitrocellulose membranes. For the detection of Rubisco, membranes were blocked in TBS-T (8 g l–1 NaCl, 0.2 g l–1 KCl, 3 g l–1 TRIS, and 0.05% Tween-20) containing 5% (w/v) powdered milk. A polyclonal antibody was used to detect Rubisco LS and SS. For detection of GFP and CFP, membranes were blocked in TSW (10 mM TRIS, 9 g l–1 NaCl, 2.5 g l–1 gelatin, 0.1% Triton X-100, 0.2 g l–1 SDS). Fluorescent proteins were detected by a monoclonal antibody that detects both GFP and CFP. Antibody binding was detected by a secondary antibody conjugated to horseradish peroxidase and enzyme activity detected by chemiluminescence.
Proteins for Rubisco assembly analysis were extracted by grinding in non-denaturing buffer (62 mM TRIS-HCl pH 7.0, 0.5% 2-mercaptoethanol). Soluble proteins were separated by PAGE on 4% gels. GFP fluorescence was detected using a phosphorimager as described above.
Fluorescence recovery after photobleaching
FRAP was performed using the Leica confocal microscope described above. For each experiment, a suitable plastid pair was selected and a pre-bleach image acquired at 10% laser power and 4x zoom. Bleaching was accomplished with a single scan by using 100% laser power and zooming into the bleach area at 32x zoom. Recovery images were collected at the same resolution and laser power as the pre-bleach image at 1.6 s intervals for 24 s and then at 10 s intervals for 60 s. 12-bit images were analysed using Leica TCS software. Total fluorescence was calculated in the regions of interest around the plastids in question and expressed as relative fluorescence by dividing by each plastid’s fluorescence in the pre-bleach image.
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Results |
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GFP fusion proteins for monitoring protein trafficking
To study the movement of plastid proteins in stromules, two nuclear-encoded plastid proteins of different sizes were selected for fusion to fluorescent proteins: the major plastid-localized aspartate aminotransferase and the Rubisco small subunit. In A. thaliana, the ASP5 gene encodes AAT3 activity (Wilkie et al., 1996; Schultz et al., 1998). Rubisco SS is coded in the nucleus by a small gene family in most plants, including N. tabacum (Jamet et al., 1991).
Transgenic N. tabacum plants were generated using a pGPTV binary vector that carries a kanamycin resistance marker for selection in plants (Becker et al., 1992). Coding regions for fusion proteins were inserted downstream of a double cauliflower mosaic virus 35S promoter and alfalfa mosaic virus translation enhancer (Datla et al., 1993). The coding regions were followed by a nopaline synthase 3' untranslated region (Becker et al., 1992). Constructs were introduced into N. tabacum cv. Samsun NN via Agrobacterium-mediated transformation of leaf strips.
Constructs for plant transformation are shown in Fig. 1. The aspartate aminotransferase construct consisted of the A. thaliana ASP5 coding region (Wilkie et al., 1995) followed by mGFP5(S65T) (Siemering et al., 1996). This construct was designated ASP5:GFP. GFP was fused to the carboxy-terminus of ASP5 to preserve the cleavage site between the ASP5 transit peptide and mature protein, thus ensuring proper plastid import and processing. A five amino acid linker peptide (GSGGG) was inserted between ASP5 and GFP to promote proper folding and activity of both proteins. The Rubisco small subunit construct consisted of the rbcS-3A coding region of P. sativum (Fluhr et al., 1986) followed by cyan fluorescent protein (mCFP) (Haseloff, 1999). This construct was designated SS:CFP. The rbcS-3A coding region includes a plastid transit peptide sequence. As with ASP5:GFP, a five amino acid linker peptide was used to join SS and CFP. A control construct in which CFP alone was targeted to plastids via the rbcS-3A transit peptide was also made. This construct was designated TP:CFP.
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Expression of fusion proteins in transgenic plants
Stable transgenic lines that showed both high and low levels of fluorescence were regenerated for each construct. None of the plants showed any obvious changes in growth or morphology relative to the wild type when grown on soil or in culture. In all transgenic lines, fluorescence was detected specifically in chloroplasts and non-green plastids (Fig. 2). Epifluorescence and confocal fluorescence microscopy also revealed fluorescence in plastid stromules in all tissues and organs known to have high frequencies of stromules, namely: hypocotyl epidermis, cultured suspension cells, flower petals, and root epidermis and hair cells (Fig. 2). Thus ASP5:GFP and SS:CFP are present in stromules of N. tabacum. In most transgenic lines, an even fluorescence signal was observed in all tissues and organs. The single exception was found in roots of SS:CFP plants. SS:CFP fluorescence in roots was extremely low relative to the fluorescence signal seen in other parts of the same transgenic plant (Fig. 2D). This difference in fluorescence may be due to post-translational regulation of Rubisco subunits (see Discussion).
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Before testing the fusion proteins for stromule trafficking, it was necessary to assess whether the fusion proteins behaved as their endogenous counterparts and could therefore serve as indicators of native protein activity in stromules. To this end, wild-type expression of the two proteins in N. tabacum tissues and organs that are rich in stromules was examined. Fusion proteins were also tested for their ability to assemble into the expected higher order molecular complexes.
Endogenous AAT3 expression and assembly of ASP5:GFP
Aspartate aminotransferase activity assays were performed on untransformed N. tabacum to determine the distribution of AAT3 expression (Fig. 3). In A. thaliana, AAT activity resolves into three distinct bands on native PAGE (Schultz and Coruzzi, 1995). The plastid isozyme, AAT3, migrates fastest on these gels, with mitochondrial and cytosolic AAT running significantly slower (Schultz and Coruzzi, 1995). A similar pattern was observed in N. tabacum (Fig. 3). Although it was not always possible to resolve the mitochondrial from the cytosolic bands, a fast-migrating band specific to chloroplasts was visible. Analysis of extracts from various organs and tissues confirmed that N. tabacum plastid AAT is present in all tissues that carry high numbers of stromules (Fig. 3).
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AAT3, like all other plant aspartate aminotransferases, assembles into homodimers in vivo (Wilkie et al., 1996). Monomeric AAT has been demonstrated to be non-functional in vitro (Arrio-Dupont and Coulet, 1979). To investigate the assembly of ASP5:GFP fusion proteins, protein extracts of transgenic plants were assayed for AAT activity and GFP fluorescence (Fig. 4). Non-denaturing PAGE gels stained for AAT activity indicate that ASP5:GFP is able to dimerize with itself and also forms heterodimers with the endogenous N. tabacum AAT3 (Fig. 4A). Extracts from transgenic plants contain two additional AAT activity bands that migrate between the endogenous plastid and cytosolic/mitochondrial bands. The presence of two additional activity bands indicates the fusion protein is able to form dimers with itself (giving rise to the slower moving band) and also with the endogenous AAT3 protein (giving rise to the faster moving band) to give the functional enzyme. Comparison of activity staining in the wild type (Fig. 3) and ASP5:GFP (Fig. 4) shows a relative loss of endogenous plastid AAT activity with respect to cytosolic and mitochondrial AAT activity in the ASP5:GFP plants. This is due to the diversion of some of the endogenous plastid AAT into the formation of heterodimers with ASP5:GFP fusions. Fluorescence analysis of the activity gels confirmed the presence of GFP in the bands containing fusion proteins (Fig. 4B). ASP5:GFP homodimers and heterodimers were found in all stromule-bearing organs and tissues. Thus A. thaliana ASP5, with GFP fused to its carboxy-terminus, can interact with itself and with the N. tabacum AAT3 to form enzymes that are functional in vitro.
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Endogenous SS expression and assembly of SS:CFP
High level expression of Rubisco SS is normally limited to chloroplasts in green tissue, as expected for a protein involved in photosynthesis (Tobin and Silverthorne, 1985). Experiments on N. tabacum indicate that both LS and SS accumulate to low levels in flower petals and dark-grown seedlings (Fig. 5). Protein extracts of wild-type N. tabacum were separated by SDS-PAGE and blotted to nitrocellulose. Immunodetection with a polyclonal antibody directed against Rubisco detected both LS and SS in leaf, petal, and dark-grown hypocotyls. Rubisco was undetectable in light-grown roots and suspension cells (Fig. 5).
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SS normally forms a 550 kDa holoenzyme with plastid-encoded LS. In SS:CFP transgenic plants, the SS:CFP fusion assembles into the 16-subunit holoenzyme in leaves and dark-grown seedlings of transgenic plants (Fig. 6). Extracts of leaf proteins were separated by non-denaturing PAGE, preserving the holoenzyme assembly of Rubisco (Fig. 6A). Transgenic protein extracts from leaves and dark-grown seedlings contain a single slow-migrating protein band. SS:CFP transgenic petals contain a similarly slow-migrating band as well as a significantly faster migrating fluorescent band. Leaf extracts of TP:CFP plants contain a single fast-migrating protein band. The similar mobility of the TP:CFP band and the fast-migrating band of SS:CFP petals suggests this SS:CFP petal protein is unassembled SS:CFP fusion protein. The fact that SS:CFP petal extracts contained both assembled SS:CFP and an unassembled SS:CFP protein made it unsuitable for further analysis, since the unassembled protein would not accurately represent endogenous Rubisco. Wild-type leaf extracts showed no fluorescent protein bands, as expected.
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To identify the proteins contained in the slow-migrating fluorescent bands, the fluorescent bands from leaf extracts of SS:CFP, and TP:CFP, as well as the gel region in the wild-type sample at the same position as the SS:CFP band were cut from the non-denaturing gels (white boxes in Fig. 6A) and introduced into the lanes of SDS-PAGE gels (Fig. 6B). Denatured and separated proteins from the gel slices were probed with antibodies for Rubisco (Fig. 6B). Wild-type samples contained both LS and SS, confirming that the gel slices excised from native gels in Fig. 6A contained the Rubisco holoenzyme and indicating that SS:CFP fusion protein forms a complex that migrates identically to the endogenous Rubisco holoenzyme. This was confirmed by the presence of LS and SS in the SS:CFP sample. The SS:CFP sample also contained an additional band migrating slightly faster than LS that corresponds to the predicted size of the SS:CFP fusion protein. Thus the fluorescence in SS:CFP plants is due to SS:CFP fusion protein that is assembled with endogenous SS and LS to form the Rubisco holoenzyme. The TP:CFP sample contained no Rubisco protein, confirming that CFP by itself does not interact with Rubisco SS or LS.
Trafficking of fusion proteins
The above experiments confirmed that ASP5:GFP and SS:CFP assembled into their respective complexes and could accumulate in stromules. It was next determined whether these fusion proteins could also traffic between plastids via stromules, as had been demonstrated for unfused GFP. Fluorescence recovery after photobleaching was used to monitor the movement of the fusion proteins between plastids in dark-grown hypocotyls. FRAP was conducted on dark-grown hypocotyls because stromules are numerous in these organs and often connect pairs of plastids. In addition, both AAT3 and Rubisco are normally expressed in these cells and the fusion proteins had been shown to assemble correctly here. When plastids containing SS:CFP or ASP5:GFP were bleached of fluorescence, fluorescence gradually recovered, at the expense of fluorescence in a connected plastid (Fig. 7). Recovery was generally complete within the 2 min observation period. Control experiments in which a single plastid without stromules was bleached and then monitored over time showed no significant recovery of fluorescence (data not shown), confirming that bleaching was due to the irreversible destruction of the GFP or CFP fluorophore (Kwok and Hanson, 2003). Likewise, unbleached plastids that were monitored over time showed no significant loss of fluorescence, confirming that fluorescence is not lost by photobleaching under these experimental conditions. The observation of fluorescence recovery for the fusions in hypocotyl epidermal cells demonstrates that SS:CFP and ASP5:GFP can traffic between plastids via stromules.
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Discussion |
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FRAP experiments (Fig. 7) showed that fusion proteins of fluorescent proteins and the endogenous plastid proteins aspartate aminotransferase and Rubisco small subunit are able to traffic through stromules. The fusion proteins introduced into N. tabacum plastids behaved identically to their endogenous counterparts in terms of assembly into multimeric complexes. The trafficking data from fusion proteins can therefore be extended to the endogenous N. tabacum proteins. Thus, 90 kDa plastid AAT dimers and 550 kDa Rubisco holoenzymes are motile in plastid stromules.
Plastid aspartate aminotransferase is believed to have a number of metabolic activities in the cell and is therefore expressed throughout the plant body (Fig. 3). The ASP5:GFP fusion protein was able to assemble with itself and with N. tabacum AAT3 in vivo and both of these dimers were functional in vitro (Fig. 4). Therefore, the fusion protein provides an accurate tool for studying endogenous AAT3 motility. In fact, because the fusions are larger than endogenous AAT3 because of the extra 30 kDa associated with the fused GFP, the FRAP experiments underestimate the motility of endogenous AAT3.
Rubisco, with its 550 kDa size and its critical role in photosynthesis is a compelling target for study. However, the fact that it is not expressed to a high degree in non-green tissues, where stromules are most frequently found, limited its analysis. Expression analysis showed that Rubisco accumulated to significant levels only in leaves, dark-grown seedlings, and flower petals, with no protein accumulating in roots or suspension cells (Fig. 5). Coruzzi et al. (1984) reported similar findings in P. sativum: the Rubisco small subunit protein accumulated to high levels in leaves and petals. However, these authors found that SS protein accumulation in dark-grown P. sativum seedlings was less than 1% of leaf levels, though protein was detected at a high level in seeds, and rbcS mRNA was detected in dark-grown seedlings (Coruzzi et al., 1984).
Expression studies of Rubisco in wild-type N. tabacum showed no protein accumulation in suspension cells or roots (Fig. 5). However, fluorescence was detected in suspension cells and roots of transgenic plants (Fig. 2), suggesting the SS:CFP fusion was stable in the absence of other Rubisco subunits. This is unexpected, as the accumulation of SS and LS is tightly regulated to maintain stoichiometric amounts of both proteins in the plastid stroma (Rodermel, 1999). In particular, Schmidt and Mishkind (1983) studied SS accumulation in Chlamydomonas reinhardtii in which LS was not expressed because of inhibition of plastid translation. They found that SS protein was translated and imported into chloroplasts, but was then rapidly degraded. These data predict that SS:CFP should be rapidly degraded in root cells, where no LS is expressed. However, it should be noted that fluorescence in roots of SS:CFP plants was extremely low relative to all other tissues of the plant, suggesting post-import degradation did occur to some extent. Suspension cells also showed high levels of fluorescence, suggesting a lack of regulation in these cells. Similarly, in flower petals, unassembled SS:CFP was detected in addition to Rubisco holoenzyme (Fig. 6). This is another instance where the regular subunit control is violated in SS:CFP transgenic plants. Dark-grown hypocotyls were the only organ outside of leaves where endogenous Rubisco was expressed and where SS:CFP fusions assembled correctly. FRAP experiments on these cells showed trafficking of the fusion proteins.
Enzymatic activity of hybrid Rubisco enzymes formed by the combination of endogenous LS and a mixture of endogenous SS and SS:CFP was not determined in the transgenic plants. However, recent studies involving the creation of hybrid forms of Rubisco suggest that Rubisco subunits can be exchanged and retain function. Kanevski et al. (1999) replaced N. tabacum LS with LS from Helianthus annuus (sunflower). They found the hybrid protein to have reduced, but detectable activity. Getzoff et al. (1998) overexpressed P. sativum rbcS-3a in A. thaliana and found only a slight reduction in Rubisco activity. It is therefore possible that the P. sativum RbcS-3a, N. tabacum LS hybrids in this study also retained some level of activity.
In conclusion, stromules represent a significant avenue of protein movement within the plant cell. The above experiments indicate that protein complexes as large as 550 kDa can move via stromules. Stromules are thus another component in the protein trafficking machinery of plant cells, with the ability to move macromolecules much like plasmodesmata and nuclear pore complexes. Currently, the function of stromules in plants is unknown. The capacity of stromules for macromolecular trafficking suggests stromules may serve as conduits for mixing the contents of plastids in different parts of the cell. The presence of enzymes in stromules indicates that stromules participate in plastid-biochemical activity. The high surface area:volume ratio of stromules may enhance the exchange of reactants and products between plastids and the cytoplasm. Future characterization of the molecular contents of stromules and the mechanism of stromule transport will shed more light on stromule function.
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Acknowledgements |
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The authors would like to express their gratitude for the material contributions of several colleagues. The ASP5 coding region of A. thaliana was provided by Sue Wilkie and Martin J Warren (University College, London). The rbcS-3a coding region from P. sativum was contributed by Danny Schnell (University of Massachusetts, Amherst). Coding regions for mCFP and mGFP5(S65T) were provided by Jim Haseloff (University of Cambridge). Hans Bohnert (University of Illinois, Urbana-Champaign) contributed the antibody directed against Rubisco. This work was supported by Department of Energy grant DE FG02-89 ER14030.
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References |
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