Plastids Retained a Functional Prokaryotic-Like Protein Secretion Pathway That Can Export Proteins Synthesized in Chloroplasts into the Cytoplasm

Plastids Retained a Functional Prokaryotic-Like Protein Secretion Pathway That Can Export Proteins Synthesized in Chloroplasts into the Cytoplasm | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Plastids Retained a Functional Prokaryotic-Like Protein Secretion Pathway That Can Export Proteins Synthesized in Chloroplasts into the Cytoplasm Leelavathi Sadhu, Amit Bhardwaj, Krishan Kumar, Saravanan Kumar, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7128036/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Chloroplasts are semiautonomous organelles considered to have evolved from prokaryotic organisms like Cyanobacterium through endosymbiosis. During the course of evolution spanning over a billion years, a part of the organellar genome has migrated to the nucleus, and proteins encoded by such genes are synthesized in the cytoplasm and then imported into chloroplasts for their functions. To import nuclear-encoded and cytoplasm-synthesized proteins, chloroplasts have evolved multiple pathways, and some of them resemble prokaryotic protein export pathways operating in opposite directions. However, it is unknown whether any prokaryotic protein export mechanisms are functionally conserved in present-day land plant chloroplasts, allowing proteins synthesized in the chloroplasts to be exported into the cytoplasm. Results To understand the existence of any functional protein export pathway in chloroplasts, the coding region of a bacterial signal peptide from Bacillus subtilis , involved in the export of a cellulolytic enzyme (BSX), was translationally fused in-frame with GFP at 5’ end to create SP:GFP fusion protein and expressed in chloroplasts. Here we present data providing evidence that shows the existence of a functional protein export mechanism in the chloroplasts, similar to the prokaryotic protein secretion mechanism, which can be exploited for crop improvement and other biotechnological applications. Conclusions Evidence for protein export and prior knowledge about protein import by chloroplasts indicate the presence of unique bidirectional trafficking across the chloroplast envelope. Nonetheless, additional studies are needed to fully understand the molecular components and exact mechanisms underlying protein secretion by chloroplasts. Protein export Bidirectional protein trafficking Chloroplasts Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Protein translocation in both bacteria and plants has been the subject of extensive research. In bacteria, most secreted proteins contain an N-terminal signal sequence that directs their translocation. In Gram-positive bacteria such as Bacillus spp., these proteins typically cross only the plasma membrane to reach the extracellular space. In contrast, in Gram-negative bacteria like Escherichia coli, secreted proteins must traverse both the inner (plasma) membrane to enter the periplasm and subsequently cross the outer membrane. In plants, protein sorting is significantly more complex due to the presence of multiple membrane-bound organelles, each requiring distinct targeting signals and specialized translocation mechanisms. Chloroplasts are considered to have evolved from a photosynthetic Cyanobacterium-like organism through endosymbiosis [1–4]. Through evolutionary processes, a large portion of the chloroplast genome has been transferred to the nuclear genome of the host [5]. Chloroplasts with their own genetic machinery exhibit significant similarities to those of prokaryotes in their DNA replication, transcription, and translation systems [6]. However, proteins encoded by nucleus-migrated genes are synthesized in the cytoplasm and transported into chloroplasts through multiple pathways, most notably via the TOC–TIC system (translocons at the outer and inner chloroplast membranes, respectively). Within the chloroplast, many of these proteins are subsequently directed to the thylakoid membranes, where they are translocated by pathways that have direct bacterial homologs, including the Sec-dependent, SRP-dependent, and spontaneous insertion pathways [7–10]. Although the existence of similar protein secretion mechanisms resembling prokaryotes is reported from plants, they were mainly shown to be involved in the import of nuclear-encoded proteins into plastids and further targeting some of them to thylakoids [11,12]. Several thylakoid signal peptides have been reported that can target proteins for secretion across the plasma membrane of bacteria [13,14]. While mechanisms involved in protein secretion are well-documented in prokaryotes, the functional presence of such mechanisms within contemporary land plant chloroplasts remains ambiguous. Nevertheless, it remains uncertain whether the prokaryotic secretion mechanisms are functionally preserved and capable of exporting proteins expressed in the chloroplasts into the cytoplasm. (Fig. 1 ). Given the above, the present study was aimed at understanding the functional existence of prokaryotic-like protein secretion pathway(s) in present-day land plants. Here we present experimental data based on the expression of Green Fluorescent Protein (GFP) fused with a bacterial signal peptide in the chloroplasts that indicates the existence of a functional protein export mechanism, akin to the protein secretion mechanism observed in prokaryotes. To understand the presence of a functional prokaryotic-like protein secretion mechanism in the chloroplasts of higher plants, a chimeric gene (SP:GFP) coding for Green Fluorescent Protein (GFP) fused at 5’-end with a coding region of a bacterial signal peptide (SP) of a cellulolytic enzyme (xylanase, BSX) [15] from Bacillus subtilis, under the control of a chloroplast gene (psbA) promoter was expressed following transplastomic approach [16]. The same construct having the chimeric gene (SP:GFP) under psbA promoter was also expressed in a gram-negative bacterium, Escherichia coli, as psbA gene promoter was shown to be functional in E. coli , having a double membrane envelope, similar to the chloroplasts and Cyanobacte-rium. Our results showed the presence of GFP in the cytoplasm of transplastomic tobacco cells. Similarly, secretion of GFP was observed in the culture media when the E. coli was transformed with the same construct. These results suggest the presence of an evolutionarily conserved prokaryotic-like protein secretion mechanism in the land plant chloroplasts, which can be exploited for biotechnological applications. Results and Discussion A 51-Amino Acid long Signal Peptide from an Extracellular GH10 Xylanase (BSX) of Bacillus spp. Enables Protein Secretion in Gram-Negative Escherichia coli Previously, we identified and characterized a cellulolytic enzyme, GH10 xylanase (BSX), from Bacillus subtilis NG-27 [15]. BSX is produced as a pre-protein with an N-terminal signal peptide (SP) ( Figure S1 A ) that directs its translocation across the membrane for extracellular secretion ( Figure S1 B ) [17,18]. The previously reported crystal structure of mature, extracellular BSX (PDB id – 2f8q) provided the initial indication that the first 51 amino acids comprise a functional signal peptide [17]. Sequence analysis of the BSX signal peptide (51st amino acids) showed the absence of the conserved twin-arginine motif ([S/T]-R-R-x-F-L-K) required for Tat-pathway-mediated secretion [9], suggesting that BSX could be secreted via some other pathway, such as the Sec-dependent pathway. To assess the ability of SP to secrete BSX when expressed in E. coli , a gram-negative bacterium with a double membrane structure, the BSX was expressed with and without the SP. The results revealed that the BSX enzyme was effectively secreted into the culture medium when co-expressed with the SP ( Figure S1 C and S1D ). The SDS-PAGE analysis of the immunoprecipitated BSX from the membrane fraction using anti-BSX antibodies further confirmed the secretion of BSX when expressed along with SP ( Figure S1 E; Figure S2 ; Table S1 ). These findings imply that the SP derived from B. subtilis is recognized and processed by the secretory pathway(s) of E. coli , leading to the secretion of biologically active BSX into the culture medium. To further validate BSX secretion results in E. coli , the DNA sequence en-coding the SP was translationally fused in-frame to the 5’ end of the GFP coding region [19], generating a SP:GFP fusion construct in the pET vector system and expressed in E, coli (Rosetta (DE3) cells (see Materials and Methods; Fig. 2 A). The subcellular fractionation of E. coli cells expressing SP:GFP revealed the presence of GFP in the culture medium (Fig. 2 B, Lane 1). The GFP protein levels were relatively low in cytoplasmic and periplasmic fractions, whereas a substantial amount of SP:GFP was detected in the membrane-bound fraction, indicative of translocation through the membrane during extracellular secretion (Fig. 2 B, Lane 4). These results suggested that the SP can direct the secretion of recombinant proteins expressed in E. coli , similar to its secretory function in B. subtilis . To determine the cleavage site(s) of the SP by the E. coli secretion machinery, whole-cell lysates from cells overexpressing GFP or SP:GFP were subjected to immunoprecipitation using anti-GFP antibodies, and the resulting bands were analysed via peptide mass fingerprinting. Cells expressing GFP alone exhibited a single band (Fig. 2 C, band 1), while cells expressing SP:GFP displayed multiple bands corresponding to full-length SP:GFP (Fig. 2 C, Band 3) and SP-cleaved GFP (Fig. 2 C, Band 4). This analysis revealed that the SP was cleaved at both the 20th and 51st amino acid positions in E. coli ( Figure S3 ; Table S2 ), suggesting that the bacterial secretion machinery recognizes and processes the SP in a manner analogous to B. subtilis . The cleavage site at the 51st amino acid correlated well with the mature form observed in the BSX crystal structure. Peptide mass fingerprinting also revealed the same cleavage sites (20th and 51st) for SP:BSX, again suggesting that the secretion profile of SP remained similar when fused with two different proteins. These findings indicate that the SP from B. subtilis is recognized and processed by the secretory machinery of E. coli , ultimately leading to the extracellular localization of the fused partner. To identify potential interaction partners of SP:GFP during secretion, we performed proteomic analysis on protein bands uniquely present in the SP:GFP immunoprecipitation sample. One of the unique bands (Fig. 2 C, Band 2) in SP:GFP immunoprecipitation sample was characterized as FtsY protein, a signal-recognition receptor in the bacteria ( Figure S4 A-B , and Table S3 ). The FtsY is known to interact with the SecYEG translocon channel that connects the cytoplasm to the periplasm in gram-negative bacteria and facilitates the translocation of proteins across the cytoplasmic membrane [20], indicating that the secretion of GFP in E. coli might be mediated through the Sec pathway. The mechanisms underlying extracellular protein secretion in Gram-negative bacteria are not fully understood, and multiple pathways may be involved in this process. Once the protein localizes to the periplasmic space, it can cross the outer membrane either via passive diffusion or in a type 2 and type 5 secretion system-dependent manner. It is to be noted here that the presence of homologs of the Sec pathway was reported from plants [21], and the Sec pathway has been implicated in the export of proteins having bacterial secretion signals to thylakoids [22]. A maize mutant defective in the expression of FtsY was found to exhibit pleiotropic defects in the thylakoids [23], suggesting that the Sec pathway is essential for the normal development of plastids. However, export of chloroplast-synthesized proteins to the cytoplasm remains poorly characterized. Bacterial Signal Peptide Facilitates Secretion of Chloroplast-Expressed Recombinant Protein into the Cytoplasm To investigate the presence of a prokaryotic-like secretion mechanism in pre-sent-day land plant chloroplasts, the gene encoding the SP:GFP fusion protein was cloned into the plastid transformation vector pVSR326 [24], driven by the psbA promoter and terminator. The construct was then site-specifically integrated into the tobacco plastid genome via standard chloroplast transformation protocols [25] (see Materials and Methods). As a control, a construct expressing GFP alone was also cloned into pVSR326 under the same regulatory elements, and stable, homotransplastomic lines were generated. Molecular analyses confirmed the transplastomic nature of the transformed plants and verified the efficient transcription and translation of both SP:GFP and GFP genes ( Figures S5–S6 ). Southern blot analysis demonstrated the site-specific integration of the GFP or SP:GFP transgenes, along with the aadA selectable marker, into the tobacco plastome ( Figures S5A–B ). Northern blot analysis confirmed robust transcription of the respective genes ( Figures S5C–D ). Western blot analysis of total leaf protein extracts revealed three distinct bands in all three independent SP:GFP-expressing transplastomic lines (NtVSRSP-GFP1, NtVSRSP-GFP2, and NtVSRSP-GFP3; Fig. 2 D, lanes 1–3). In contrast, the GFP-only transplastomic line (NtVSRGFP1) displayed a single band corresponding in size to the lowest band observed in the SP:GFP-expressing lines (Fig. 2 D, lane 4). These findings suggest that the SP:GFP fusion undergoes processing in chloroplasts. To further validate these results, two-dimensional (2D) gel electrophoresis followed by mass spectrometry was performed to identify GFP isoforms ( Figure S6; Table S4 ). In GFP-only transplastomic plants, a single protein spot was observed within the expected molecular weight and isoelectric point range ( Figure S6A , inset). In contrast, plants expressing SP:GFP exhibited at least two distinct GFP-containing protein spots ( Figure S6B , inset), supporting the hypothesis that the bacterial signal peptide is processed within chloroplasts. These results collectively suggest that the bacterial SP not only remains functional in the chloroplast environment but also facilitates the secretion of recombinant proteins from the chloroplast into the cytoplasm. Subcellular Localization of SP:GFP by Confocal Microscopy Reveals Cytoplasmic Distribution of GFP To determine the subcellular localization of the GFP signal, protoplasts were isolated from the mesophyll tissue of transplastomic tobacco plants and analysed using confocal laser scanning microscopy. In plants expressing GFP alone, green fluorescence was exclusively localized within the chloroplasts, consistent with plastid-targeted expression and retention (Fig. 3 A–B). In contrast, transplastomic plants expressing the SP:GFP fusion protein exhibited green fluorescence in both chloroplasts and the cytoplasm (Fig. 3 C–F), suggesting that the bacterial signal peptide facilitated the export of GFP from the chloroplasts into the cytoplasm. The cytoplasmic distribution of GFP fluorescence was particularly pronounced in cells containing differentiating plastids (Fig. 3 C–D), while mesophyll cells with fully developed and mature chloroplasts showed relatively weaker cytoplasmic fluorescence (Fig. 3 E). This differential localization pattern indicates that the efficiency of GFP export may be influenced by the plastid developmental stage. Additionally, GFP fluorescence was observed in the cytoplasm of epidermal trichome cells (Fig. 3 F), where it appeared more prominent, likely due to the lower abundance of plastids in these cells, thereby reducing signal overlap and masking effects ( Figure S7 ). Molecular and Proteomic Analyses of Chloroplast-Expressed SP:GFP Reveal Conserved Protein Secretion Mechanisms Shared Between Bacteria and Chloroplasts To investigate the conserved nature of prokaryotic-type protein secretion mechanisms in plant chloroplasts, mass spectrometry-based analysis was performed. Given the observed cleavage of the BSX signal peptide (SP) after the 20th and 51st amino acids in both Bacillus subtilis and Escherichia coli, it was hypothesized that the SP would undergo similar processing in chloroplasts if analogous mechanisms were present. Mass spectrometry-based identification of the N-terminal region of SP:GFP expressed in chloroplasts revealed cleavage after the 20th and 50th amino acids (corresponding to the lower bands in Fig. 2 D, lanes 1–3; Figure S8 ), closely resembling the cleavage pattern observed in B. subtilis and E. coli ( Table S5; Figures S8 - S11 ). The cytoplasmic localization of GFP fluorescence further supports the existence of a functionally conserved, prokaryotic-like protein secretion pathway in modern land plant chloroplasts (Fig. 4 ). The secretion of BSX and GFP into the extracellular medium when expressed as fusion proteins with the BSX SP in E. coli , along with the identification of FtsY among co-immunoprecipitated proteins using anti-GFP antibodies, suggests that the Sec pathway is likely involved in this export process in E. coli . The Sec pathway is also known to facilitate the targeting of a subset of nuclear-encoded proteins, imported into chloroplasts, to the thylakoid membrane [26]. Interestingly, the inner envelope membrane (IEM) and the thylakoid membrane share similar lipid compositions and protein components involved in protein translocation [27, 28]. For a chloroplast-expressed SP:GFP fusion protein to reach the cytoplasm, as observed in this study, it must first cross the IEM to enter the intermembrane space, followed by translocation across the outer envelope membrane via an unidentified mechanism. The fact that the SP is cleaved at the same sites in both E. coli and chloroplasts raises the possibility that the chloroplast Sec pathway, which usually mediates thylakoid protein targeting, may also facilitate SP:GFP targeting to the IEM, allowing subsequent export into the intermembrane space. Following this, GFP may either be actively exported into the cytoplasm through a novel pathway or passively diffuse. Alternatively, a thylakoid-independent export mechanism might exist, though its identity remains to be elucidated. The discovery of a functional protein export mechanism in chloroplasts, as suggested by our findings, combined with the well-established capacity of chloroplasts to produce foreign proteins at high levels, opens up exciting possibilities for crop improvement and various biotechnological applications [29, 30]. However, further work is needed to understand the exact mechanism(s) involved in the secretion of proteins by chloroplasts. Conclusions In the present study, we have provided evidence that a signal peptide derived from a single membrane containing gram-positive bacteria, Bacillus subtilis , can secrete recombinant proteins by a double membrane containing gram-negative E. coli . Molecular and proteomic analysis suggested that the secretion of the recombinant proteins might be taking place through the SecY pathway. Similar secretion of recombinant proteins was observed when the SP-GFP fusion protein was expressed in the chloroplasts. Proteomic analysis revealed that the processing of signal peptides in E. coli and chloroplasts is very similar, suggesting an evolutionarily conserved prokaryotic-like protein secretion mechanism operating in the opposite direction to the well-known protein import mechanisms in chloroplasts. Our results indicate the presence of a unique bidirectional protein trafficking across the double membrane envelope of the plastids. Nonetheless, additional studies are needed to fully understand the molecular components and exact mechanisms underlying protein secretion by chloroplasts. Materials and Methods Cloning and Expression of SP:BSX and SP:GFP in E. coli Full-length BSX gene along with signal peptide (SP:BSX) was polymerase Chain Reaction (PCR) amplified using genomic DNA of Bacillus sp. NG-27 [15] as a template using xylaFL5 and xyla3-10 primers containing NcoI and BamHI sites, respectively, and cloned into pET14b plasmid, creating the pET14b SP:BSX vector. The BSX signal peptide was also fused with GFP using a PCR-based method and SP:GFP gene was cloned at the same NcoI and BamHI sites of the pET14b plasmid, creating the pET14b SP:GFP vector. To compare the effect of the signal peptide on protein secretion, the GFP gene was cloned into the pET14b vector at the NcoI and BamHI sites, creating a pET14bGFP vector. The cloning of pET14bxyla5-14, carrying the mature part of BSX gene (excluding the Signal peptide) has already been described previously [17]. All four constructs were transformed into Rosetta (DE3) cells (Novagen) for recombinant expression. In case of SP:BSX and SP:GFP, cells were grown at 28oC, and overnight induction was performed by adding IPTG (1mM final concentration). In the case of BSX and GFP, cells were grown at 37oC and induced for 4 hours by adding IPTG (1mM). Cloning and Expression of SP:GFP and GFP in Tobacco Chloroplast To express SP:GFP fusion protein in chloroplasts, the SP:GFP gene was placed under a chloroplast gene (psbA) promoter and terminator sequences. For a better comparison, transplastomic plants (transgenic plants transformed with foreign genes into the plastid genome) were also generated using a pVSRGFP vector having the GFP gene alone without the SP [31]. The GFP or SP:GFP gene cassettes are expected to get integrated into the tobacco plastome in a site-specific manner at the intergenic spacer region between rbcL and accD genes through two homologous recombination (as shown below in Fig. 5 ). Stable and site-specific integration of GFP, SP:GFP, and the selectable aadA gene into tobacco plastome was confirmed by Southern hybridization ( Figure S5A-B ). A 14.3 kb and 14.45 band for GFP and SP:GFP, respectively, provided evidence for stable and site-specific integration of transgenes into tobacco plastome (Figure S5A-B). On the other hand, the presence of an 11.45 kb band in the control untransformed plant and the absence of the same band in the transplastomic plants is proof of the homoplasmy for the transplastome in all three transformed plants analyzed ( Figure S5B and 5 ). Similarly, Southern hybridization analysis also confirmed the stable and site-specific integration of the GFP gene when tobacco plants were transformed with the pVSRGFP vector (data not shown). Northern blot analysis was used to identify efficient transcription of GFP (Figure S5C) and SP:GFP ( Figure S5D ). An expected size transcript of 0.85 kb was observed in the transplastomic plants transformed with SP:GFP gene ( Figure S5D , lanes 1 and 2), and no signal was found in the control untransformed plant (Figure S5D, lane 3). Similarly, an expected size transcript (0.7 kb) was observed in GFP-expressing plants ( Figure S5C , lane 2). Western Blotting Analysis Induced cultures of SP:BSX, SP:GFP, BSX, and GFP were sonicated in 1X PBS containing protease inhibitors (Roche). The supernatant fraction was loaded and subjected to SDS-PAGE analysis [32]. Total protein from tobacco untransformed/control and transplastomic plants (such as NtVSRSP-GFP1, NtVSRSP-GFP2, NtVSRSP-GFP3, and NtVSRGFP1) leaf was extracted as described in our earlier study [16]. Western blotting was performed using the wet transfer method, and in-house developed primary polyclonal antibodies raised against BSX and GFP proteins in rabbits were used to hybridize the PVDF membrane to detect the expression of recombinant proteins following standard procedures [16, 32]. Immunoprecipitation Induced culture of SP:BSX, SP:GFP, BSX, and GFP were sonicated in equal volumes of ice-cold 1X TNET buffer (50mM Tris-Cl pH 8.0, 400mM NaCl, 5mM EDTA, and 2% Triton X-100) in the presence of protease inhibitors cocktail (Roche Diagnostics). The clarified extract was incubated with 100ul of Protein-A beads (GE Healthcare) coupled with anti-BSX or anti-GFP antibodies overnight at 4oC. Beads were washed with TNET buffer alone and subsequently with high-salt TNET buffer (600 mM NaCl) and low-salt TNET buffer (50 mM NaCl + 0.1% Triton X-100). Finally, proteins were extracted from the Protein-A conjugated beads by boiling at 100°C with 2× reducing dye for 5 minutes and subjected to 1D-SDS-PAGE analysis. Protein bands were excised from the Coomassie-stained gel and subjected to MALDI-TOF/TOF (Bruker Daltonics, Germany) analysis as mentioned below. Two-Dimensional Gel Electrophoresis Analysis (2D-PAGE) An equal amount of protein extracted from the same-age leaf tissue expressing GFP and SP:GFP was taken and resolved on a 7cm IpG strip (PH range 4–7) during first-dimensional isoelectric focusing (IEF) using Ettan IPGphor II IEF system, followed by second dimension SDS-PAGE as per manufacturer’s guidelines (GE Healthcare). Protein spots were excised manually from Coomassie-stained gels and identified using mass spectrometry analysis. In-Gel Digestion and Nano-LC MALDI-Based Protein Identification and Analysis The bands from 1D-SDS PAGE and the spots from 2D-PAGE were subjected to in-gel digestion as described by Shevchenko et al [33]. The gel pieces containing the protein of interest were digested with either 15 ng/µl trypsin (Promega) or 10 ng/ul Glu-C (Promega). Peptide samples obtained after in-gel digestion were subjected to Nano-LC (Proxeon Biosystems) based reverse phase separation [34]. Fractioned samples were subjected to MALDI TOF/TOF-based acquisition using the Ultraflex III MALDI TOF/TOF instrument (Bruker Daltonics, Germany). Post-acquisition processes, including mass annotation, baseline subtraction, and smoothening, were performed using Flex analysis software version 3.0 through WARP-LC. Protein identification was achieved using Biotools version 3.2 through an in-house licensed Mascot server (version 2.3 - March 2010) [33]. The database search parameters were set as described: fragment masses were searched in NCBInr and in-house generated protein databases through the mascot search engine (version 2.3), taxonomy was unrestricted, enzyme was set as either trypsin or V8-DE (Glu-C), fixed modification included carbamido-methylation of cysteine, variable modification included oxidation of methionine, protein mass was unrestricted, missed cleavage was set to 1, MS tolerance of +/-100 ppm and MS/MS tolerance of +/-0.75 da. Only peptides with an individual ion score of > 40 (p < 0.05) were considered for protein identification. Confocal Microscopy Protoplasts were prepared by partially digesting the mesophyll cells using Cellulase Onuzuka R10 and Pectinase R10 (Sigma) enzymes. Protoplasts were viewed using a confocal microscope: Nikon A-1R inverted microscope. Images were acquired using blue diode laser (ex 405 nm) and signals were collected at the Emission range of 470nm to 550nm for GFP. Chlorophyll auto-fluorescence was also acquired simultaneously. Declarations Contributions: Leelavathi Sadhu: Conceptualization , Project administration, Methodology. Amit Bhardwaj: Conceptualization, Writing – review & editing, Formal analysis, Data curation, Investigation. Krishan Kumar: Conceptualization, Methodology, Writing – review & editing, Validation, Investigation . Saravanan Kumar: Investigation, Writing – review & editing. Abhishek Dass: Investigation, Methodology. Ranjana Pathak: Investigation, Visualization, Pankaj Pandey: Investigation. Bhupendra S. Rawat: Investigation. Vanga Siva Reddy: Conceptualization, Writing – original draft, Supervision, Funding acquisition. Declaration of competing interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments: We are grateful for the excellent technical support from Purnima Kumar and Towa Optics technical team for confocal microscopy. We thank the Department of Biotechnology, Govt. of India, ICGEB, New Delhi and ICAR-IIMR for funding. Data availability All data generated or analysed during this study are included in this published article and the Additional file. Ethics declarations Ethics approval and consent to participate Not applicable. References Goksøyr, J. Evolution of Eucaryotic Cells. Nature 1967 , 214 , 1161–1161, doi:https://doi.org/10.1038/2141161a0. Martin, W.; Müller, M. The Hydrogen Hypothesis for the First Eukaryote. Nature 1998 , 392 , 37–41, doi:https://doi.org/10.1038/32096. Martin, W.; Kowallik, K.V. 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Overproduction of an Alkali-and Thermo-Stable Xylanase in Tobacco Chloroplasts and Efficient Recovery of the Enzyme. Molecular Breeding 2003 , 11 , 59–67, doi:https://doi.org/10.1023/A:1022168321380. Zhu, D.; Xiong, H.; Wu, J.; Zheng, C.; Lu, D.; Zhang, L.; Xu, X. Protein Targeting Into the Thylakoid Membrane Through Different Pathways. Front. Physiol. 2022 , 12 , 802057, doi: https://doi.org/ 10.3389/fphys.2021.802057. Vothknecht, U.C.; Westhoff, P. Biogenesis and Origin of Thylakoid Membranes. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 2001 , 1541 , 91–101, doi:https://doi.org/10.1016/S0167-4889(01)00153-7. Klasek, L.; Inoue, K. Dual Protein Localization to the Envelope and Thylakoid Membranes within the Chloroplast. International review of cell and molecular biology 2016 , 323 , 231–263. doi: https://doi.org/ 10.1016/bs.ircmb.2015.12.008 Brixey, P.J.; Guda, C.; Daniell, H. The Chloroplast psbA Promoter Is More Efficient in Escherichia Coli than the T7 Promoter for Hyperexpression of a Foreign Protein. Biotechnology letters 1997 , 19 , 395–400, doi:https://doi.org/10.1023/A:1018371405675. Kwon, K.-C.; Verma, D.; Jin, S.; Singh, N.D.; Daniell, H. Release of Proteins from Intact Chloroplasts Induced by Reactive Oxygen Species during Biotic and Abiotic Stress. PloS one 2013 , 8 , e67106, doi:https://doi.org/10.1371/journal.pone.0067106. Reddy, V. S., Leelavathi, S., & Bhardwaj, A. (2015). U.S. Patent No. 9,175,300. Washington, DC: U.S. Patent and Trademark Office. Sambrook, J.; Fritsch, E.F.; Maniatis, T.; Russell, D.W.; Green, M.R. Molecular Cloning: A Laboratory Manual ; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1989; ISBN 978-0-87969-309-1. Shevchenko, A.; Tomas, H.; Havli, J.; Olsen, J.V.; Mann, M. In-Gel Digestion for Mass Spectrometric Characterization of Proteins and Proteomes. Nat Protoc 2006 , 1 , 2856–2860, doi: https://doi.org/ 10.1038/nprot.2006.468. Kumar, S.; Kumar, K.; Pandey, P.; Rajamani, V.; Padmalatha, K.V.; Dhandapani, G.; Kanakachari, M.; Leelavathi, S.; Kumar, P.A.; Reddy, V.S. Glycoproteome of Elongating Cotton Fiber Cells. Molecular & Cellular Proteomics 2013 , 12 , 3677–3689, doi: https://doi.org/ 10.1074/mcp.M113.030726. BioRender (2021). Cyanobacteria Structure. https://app.biorender.com/t-5ffdfec7a0005e00aa69816e-cyanobacteria-structure Additional Declarations No competing interests reported. Supplementary Files Additionalfile1SupplementaryFigures.doc Additional file 1: Supplementary Figures S1-S11 Additionalfile2SupplementaryTablesS1S11.xlsx Additional file 2; Supplementary Tables S1-S11 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7128036","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":491161792,"identity":"1282327d-5f0b-44f5-ad3a-46a79d19d7b5","order_by":0,"name":"Leelavathi Sadhu","email":"","orcid":"","institution":"International Centre for Genetic Engineering and Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Leelavathi","middleName":"","lastName":"Sadhu","suffix":""},{"id":491161793,"identity":"f057e6fe-f890-4beb-a335-d8ab299147f5","order_by":1,"name":"Amit Bhardwaj","email":"","orcid":"","institution":"International Centre for Genetic Engineering and Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Amit","middleName":"","lastName":"Bhardwaj","suffix":""},{"id":491161794,"identity":"1829c813-e2d1-49f3-bf77-5192b78dcbd9","order_by":2,"name":"Krishan Kumar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYBACCQbGBmYgzcPGwMD4AMTgI0ULswGUQUgLUCmUzSYBJglpkZx9uPlzQc0dGT6xs88qv+bYybAxMD98dAOPFmm+xDbpGcee8bBJp5vdlt2WDHQYm7FxDh4tcjyMbcw8bIeBWtLYbktuA7KB3pEmoKX5M88/iJZiyW31hLVI8zA2SPO2QbQwftx2mLAWyR7GNmnePrAWZmnGbcd52JgJ+EXiDPvjzzzfDtvLz05j/PhzW7U9P3vzw8f4tKAAZh4wSaxyEGD8QYrqUTAKRsEoGDEAAPDYORqDaIAOAAAAAElFTkSuQmCC","orcid":"","institution":"ICAR-Indian Institute of Maize Research, PUSA Campus","correspondingAuthor":true,"prefix":"","firstName":"Krishan","middleName":"","lastName":"Kumar","suffix":""},{"id":491161795,"identity":"f07b5b5f-aff3-44c1-8a61-37c689be6201","order_by":3,"name":"Saravanan Kumar","email":"","orcid":"","institution":"International Centre for Genetic Engineering and Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Saravanan","middleName":"","lastName":"Kumar","suffix":""},{"id":491161796,"identity":"ebf7a686-3ee6-4af3-a98f-db0ae85c4b3e","order_by":4,"name":"Abhishek Dass","email":"","orcid":"","institution":"International Centre for Genetic Engineering and Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Abhishek","middleName":"","lastName":"Dass","suffix":""},{"id":491161797,"identity":"035ca950-c683-4773-a842-e4f2a40bd577","order_by":5,"name":"Ranjana Pathak","email":"","orcid":"","institution":"International Centre for Genetic Engineering and Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Ranjana","middleName":"","lastName":"Pathak","suffix":""},{"id":491161798,"identity":"705319e6-87c8-4a8b-b473-75da65e7746c","order_by":6,"name":"Pankaj Pandey","email":"","orcid":"","institution":"ICAR-Indian Institute of Maize Research, PUSA Campus","correspondingAuthor":false,"prefix":"","firstName":"Pankaj","middleName":"","lastName":"Pandey","suffix":""},{"id":491161799,"identity":"bb84d185-31ad-4395-b45d-9c583fd8d66c","order_by":7,"name":"Bhupendra S. Rawat","email":"","orcid":"","institution":"International Centre for Genetic Engineering and Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Bhupendra","middleName":"S.","lastName":"Rawat","suffix":""},{"id":491161800,"identity":"e4cea90e-9f87-4edb-9ce1-0b3b83cc27fb","order_by":8,"name":"Vanga Siva Reddy","email":"","orcid":"","institution":"International Centre for Genetic Engineering and Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Vanga","middleName":"Siva","lastName":"Reddy","suffix":""}],"badges":[],"createdAt":"2025-07-15 08:23:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7128036/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7128036/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87716729,"identity":"46575699-c6e8-4935-81df-32cceaa778b6","added_by":"auto","created_at":"2025-07-28 09:14:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":356024,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram showing cell membranes, thylakoids, protein synthesis, export, and localization in the Cyanobacterium and plant cell.\u003c/strong\u003e(A). Cyanobacterium, the photosynthetic progenitor of plastids in higher plants. (B) Plant cell. Proteins encoded by the nuclear genome enter into chloroplasts through various protein import mechanisms and are generally facilitated by targeting signal peptides. (C) Expression and localization of recombinant proteins (e.g., Green Fluorescent Protein) when the gene is expressed under a plastid-specific promoter and integrated into the plastome. Recombinant proteins that are not recognized by the protein export mechanism are expected to remain in the stroma and, with signal peptides, get targeted to thylakoids. (D) The major question being asked is what the fate of and localization of any recombinant protein will be when expressed as fusion proteins containing bacterial secretary SP, recognized by the Sec pathway. If the SP is recognized by the export mechanism, then the recombinant protein may get targeted to thylakoids. Alternately, if plastids have retained the prokaryotic Sec pathway in the plastid envelope despite several changes that took place over a billion years of evolution, the SP is expected to get processed in the envelope, and the recombinant protein is expected to get secreted into the cytoplasm, like other bacteria. Experiments are designed in this study to address this question. OM: outer membrane, IM: inner membrane. IMS: intermembrane space. N: Nucleus.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7128036/v1/059cfd519d78fa6ade7233f8.png"},{"id":87716730,"identity":"b2016716-1658-4719-82ac-954dd96b604c","added_by":"auto","created_at":"2025-07-28 09:14:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":171953,"visible":true,"origin":"","legend":"\u003cp\u003e(A). Expression and secretion of, SP:GFP and GFP from a double membrane containing gram-negative \u003cem\u003eE. coli\u003c/em\u003e. GFP was secreted from \u003cem\u003eE. coli\u003c/em\u003e when expressed along with SP. The recombinant proteins remained inside the bacteria when expressed alone without SP. (B) Western blot analysis to check the expression of SP:GFP in \u003cem\u003eE. coli\u003c/em\u003e. 1. Media supernatant 2. Periplasm 3. Cytosol 4. Membrane fraction. (C) SDS page analysis of co-immunoprecipitated proteins using anti-GFP antibodies from the membrane fraction of \u003cem\u003eE. coli\u003c/em\u003e expressing GFP or SP:GFP. Mass spectrometry-based identification of additional proteins present specifically in SP:GFP expressing \u003cem\u003eE. coli\u003c/em\u003e as compared to GFP expressing one, showed the presence of both SP:GFP (band 3) and GFP (band 4). In addition, FtsY (band 2), a signal-recognition particle receptor in bacteria, was found only in immunoprecipitated proteins from \u003cem\u003eE. coli\u003c/em\u003e expressing SP:GFP. As expected, a single band was observed in \u003cem\u003eE. coli\u003c/em\u003e expressing BSX without SP (band 1). (D) Western blot analysis to identify the processing of SP by tobacco plastids. Anti-GFP antibodies have picked up three bands in all three independent transplastomic lines NtVSRSP-GFP1, NtVSRSP-GFP2 and NtVSRSP-GFP3 tested (lanes 1-3). The upper band corresponded to the unprocessed SP:GFP. Middle and lower bands correspond to SP processed after 20 and 50 amino acids, respectively (see also figures S8, S9, and S10). For a better comparison, the protein extract from the transplastomic line (NtVSRGFP1) expressing GFP alone was used (lane 4). Arrows indicate one unprocessed SP:GFP (upper band) and two processed molecules (middle and lower bands). Note that the lower band corresponds to GFP.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7128036/v1/8672d900825187003d9b6cb5.png"},{"id":87717381,"identity":"8601cb8f-6d81-42a5-8d5b-366e93e848ef","added_by":"auto","created_at":"2025-07-28 09:22:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":657846,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Confocal image showing the expression of GFP in chloroplasts in the mesophyll cells from the NtVSRGFP1 plant. (B) Autofluorescence of chlorophyll showing the chloroplasts. (C-D) Confocal images showing the expression of GFP in chloroplasts in the mesophyll cells from the NtVSRSP-GFP1 plant. (E) Composite picture showing GFP expression, chlorophyll autofluorescence under UV, under normal light, and a merger of all three images from the NtVSRSP-GFP1 plant. Note the presence of green fluorescence outside the plastids. (F) Composite picture showing leaf trichomes from the same plant. Arrows indicate the presence of GFP in the cytoplasm where there are no plastids (see Figure S7 for 3D images).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7128036/v1/fa9b04b3dc1b22699e8253b5.png"},{"id":87719316,"identity":"ad8b5a95-060a-45bb-a952-ecfc37b9911f","added_by":"auto","created_at":"2025-07-28 09:38:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":156153,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBacterial signal peptide processing by plastids and the model to explain the secretion of recombinant GFP into the cytoplasm.\u003c/strong\u003e(A) Sequence of SP used in this study and signal processing sites (Refer to Figure S11 for N-terminal processing sites determined in E.coli and chloroplast in this study). Actual cleavage sites determined in \u003cem\u003eE. coli\u003c/em\u003e, plastids, and \u003cem\u003eB. subtilis\u003c/em\u003e are shown. (B-C). Model explaining the differentiation of plastids into chloroplasts and the secretion of recombinant SP:GFP. (B) Proplastid where the expression of GFP is very low. (C) Proplastid differentiates into a chloroplast where the expression of GFP is high and the secretion is high. (D) Fully matured chloroplast where expression of GFP is high but the secretion is not very high.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7128036/v1/5d8d22f6b43db386de5a08a2.png"},{"id":87716734,"identity":"769096c1-bd37-4eb8-ae5e-b78310eabb8a","added_by":"auto","created_at":"2025-07-28 09:14:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2268354,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSite-specific integration of GFP and aadA genes into plastid genome through two possible homologous recombination. Physical map of the transplastomic chloroplast genome.\u003c/strong\u003e The GFP is placed under a chloroplast-specific promoter (psbA, brown-colored box) and the pVSRGFP construct is transformed into chloroplasts using a Particle Delivery System (PDS 1000, Biorad). The GFP and aadA (selectable marker) transgenes are expected to be integrated into the intergenic spacer region between rbcL and accD genes through two possible homologous recombination (crossed lines) that can lead to site-specific integration of transgenes into the plastid genome. The Hind III restriction sites used for RFLP analysis and the expected size DNA fragments are indicated.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7128036/v1/a3277c641af9f131efb97d05.png"},{"id":100366580,"identity":"041da3b3-27c6-4770-9661-628668b0d54c","added_by":"auto","created_at":"2026-01-16 07:56:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4817295,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7128036/v1/4d9dc74d-3ad4-4b35-8294-37c5fb200377.pdf"},{"id":87717394,"identity":"2fed20de-63da-4d98-8b87-a74022419deb","added_by":"auto","created_at":"2025-07-28 09:22:09","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3304448,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 1: Supplementary Figures S1-S11\u003c/p\u003e","description":"","filename":"Additionalfile1SupplementaryFigures.doc","url":"https://assets-eu.researchsquare.com/files/rs-7128036/v1/3e3e8d43282b59661fac51a7.doc"},{"id":87716736,"identity":"8293bb58-2963-4e05-bf22-5cc22179ee84","added_by":"auto","created_at":"2025-07-28 09:14:08","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":343679,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 2; Supplementary Tables S1-S11\u003c/p\u003e","description":"","filename":"Additionalfile2SupplementaryTablesS1S11.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7128036/v1/4d2c2ed01d6e835472ab266d.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003ePlastids Retained a Functional Prokaryotic-Like Protein Secretion Pathway That Can Export Proteins Synthesized in Chloroplasts into the Cytoplasm\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eProtein translocation in both bacteria and plants has been the subject of extensive research. In bacteria, most secreted proteins contain an N-terminal signal sequence that directs their translocation. In Gram-positive bacteria such as Bacillus spp., these proteins typically cross only the plasma membrane to reach the extracellular space. In contrast, in Gram-negative bacteria like Escherichia coli, secreted proteins must traverse both the inner (plasma) membrane to enter the periplasm and subsequently cross the outer membrane. In plants, protein sorting is significantly more complex due to the presence of multiple membrane-bound organelles, each requiring distinct targeting signals and specialized translocation mechanisms. Chloroplasts are considered to have evolved from a photosynthetic Cyanobacterium-like organism through endosymbiosis [1\u0026ndash;4]. Through evolutionary processes, a large portion of the chloroplast genome has been transferred to the nuclear genome of the host [5]. Chloroplasts with their own genetic machinery exhibit significant similarities to those of prokaryotes in their DNA replication, transcription, and translation systems [6]. However, proteins encoded by nucleus-migrated genes are synthesized in the cytoplasm and transported into chloroplasts through multiple pathways, most notably via the TOC\u0026ndash;TIC system (translocons at the outer and inner chloroplast membranes, respectively). Within the chloroplast, many of these proteins are subsequently directed to the thylakoid membranes, where they are translocated by pathways that have direct bacterial homologs, including the Sec-dependent, SRP-dependent, and spontaneous insertion pathways [7\u0026ndash;10]. Although the existence of similar protein secretion mechanisms resembling prokaryotes is reported from plants, they were mainly shown to be involved in the import of nuclear-encoded proteins into plastids and further targeting some of them to thylakoids [11,12]. Several thylakoid signal peptides have been reported that can target proteins for secretion across the plasma membrane of bacteria [13,14]. While mechanisms involved in protein secretion are well-documented in prokaryotes, the functional presence of such mechanisms within contemporary land plant chloroplasts remains ambiguous. Nevertheless, it remains uncertain whether the prokaryotic secretion mechanisms are functionally preserved and capable of exporting proteins expressed in the chloroplasts into the cytoplasm. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Given the above, the present study was aimed at understanding the functional existence of prokaryotic-like protein secretion pathway(s) in present-day land plants. Here we present experimental data based on the expression of Green Fluorescent Protein (GFP) fused with a bacterial signal peptide in the chloroplasts that indicates the existence of a functional protein export mechanism, akin to the protein secretion mechanism observed in prokaryotes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo understand the presence of a functional prokaryotic-like protein secretion mechanism in the chloroplasts of higher plants, a chimeric gene (SP:GFP) coding for Green Fluorescent Protein (GFP) fused at 5\u0026rsquo;-end with a coding region of a bacterial signal peptide (SP) of a cellulolytic enzyme (xylanase, BSX) [15] from Bacillus subtilis, under the control of a chloroplast gene (psbA) promoter was expressed following transplastomic approach [16]. The same construct having the chimeric gene (SP:GFP) under psbA promoter was also expressed in a gram-negative bacterium, Escherichia coli, as psbA gene promoter was shown to be functional \u003cem\u003ein E. coli\u003c/em\u003e, having a double membrane envelope, similar to the chloroplasts and Cyanobacte-rium. Our results showed the presence of GFP in the cytoplasm of transplastomic tobacco cells. Similarly, secretion of GFP was observed in the culture media when the \u003cem\u003eE. coli\u003c/em\u003e was transformed with the same construct. These results suggest the presence of an evolutionarily conserved prokaryotic-like protein secretion mechanism in the land plant chloroplasts, which can be exploited for biotechnological applications.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e\u003cb\u003eA 51-Amino Acid long Signal Peptide from an Extracellular GH10 Xylanase (BSX) of Bacillus spp. Enables Protein Secretion in Gram-Negative Escherichia coli\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePreviously, we identified and characterized a cellulolytic enzyme, GH10 xylanase (BSX), from Bacillus subtilis NG-27 [15]. BSX is produced as a pre-protein with an N-terminal signal peptide (SP) (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA\u003c/b\u003e) that directs its translocation across the membrane for extracellular secretion (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB\u003c/b\u003e) [17,18]. The previously reported crystal structure of mature, extracellular BSX (PDB id \u0026ndash; 2f8q) provided the initial indication that the first 51 amino acids comprise a functional signal peptide [17]. Sequence analysis of the BSX signal peptide (51st amino acids) showed the absence of the conserved twin-arginine motif ([S/T]-R-R-x-F-L-K) required for Tat-pathway-mediated secretion [9], suggesting that BSX could be secreted via some other pathway, such as the Sec-dependent pathway.\u003c/p\u003e\u003cp\u003eTo assess the ability of SP to secrete BSX when expressed in \u003cem\u003eE. coli\u003c/em\u003e, a gram-negative bacterium with a double membrane structure, the BSX was expressed with and without the SP. The results revealed that the BSX enzyme was effectively secreted into the culture medium when co-expressed with the SP (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC and S1D\u003c/b\u003e). The SDS-PAGE analysis of the immunoprecipitated BSX from the membrane fraction using anti-BSX antibodies further confirmed the secretion of BSX when expressed along with SP (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE; Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e; \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). These findings imply that the SP derived from \u003cem\u003eB. subtilis\u003c/em\u003e is recognized and processed by the secretory pathway(s) of \u003cem\u003eE. coli\u003c/em\u003e, leading to the secretion of biologically active BSX into the culture medium.\u003c/p\u003e\u003cp\u003eTo further validate BSX secretion results in \u003cem\u003eE. coli\u003c/em\u003e, the DNA sequence en-coding the SP was translationally fused in-frame to the 5\u0026rsquo; end of the GFP coding region [19], generating a SP:GFP fusion construct in the pET vector system and expressed in E, coli (Rosetta (DE3) cells (see Materials and Methods; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The subcellular fractionation of \u003cem\u003eE. coli\u003c/em\u003e cells expressing SP:GFP revealed the presence of GFP in the culture medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, Lane 1). The GFP protein levels were relatively low in cytoplasmic and periplasmic fractions, whereas a substantial amount of SP:GFP was detected in the membrane-bound fraction, indicative of translocation through the membrane during extracellular secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, Lane 4). These results suggested that the SP can direct the secretion of recombinant proteins expressed in \u003cem\u003eE. coli\u003c/em\u003e, similar to its secretory function in \u003cem\u003eB. subtilis\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eTo determine the cleavage site(s) of the SP by the \u003cem\u003eE. coli\u003c/em\u003e secretion machinery, whole-cell lysates from cells overexpressing GFP or SP:GFP were subjected to immunoprecipitation using anti-GFP antibodies, and the resulting bands were analysed via peptide mass fingerprinting. Cells expressing GFP alone exhibited a single band (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, band 1), while cells expressing SP:GFP displayed multiple bands corresponding to full-length SP:GFP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Band 3) and SP-cleaved GFP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Band 4). This analysis revealed that the SP was cleaved at both the 20th and 51st amino acid positions in \u003cem\u003eE. coli\u003c/em\u003e (\u003cb\u003eFigure S3\u003c/b\u003e; \u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e), suggesting that the bacterial secretion machinery recognizes and processes the SP in a manner analogous to \u003cem\u003eB. subtilis\u003c/em\u003e. The cleavage site at the 51st amino acid correlated well with the mature form observed in the BSX crystal structure. Peptide mass fingerprinting also revealed the same cleavage sites (20th and 51st) for SP:BSX, again suggesting that the secretion profile of SP remained similar when fused with two different proteins. These findings indicate that the SP from \u003cem\u003eB. subtilis\u003c/em\u003e is recognized and processed by the secretory machinery of \u003cem\u003eE. coli\u003c/em\u003e, ultimately leading to the extracellular localization of the fused partner.\u003c/p\u003e\u003cp\u003eTo identify potential interaction partners of SP:GFP during secretion, we performed proteomic analysis on protein bands uniquely present in the SP:GFP immunoprecipitation sample. One of the unique bands (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Band 2) in SP:GFP immunoprecipitation sample was characterized as FtsY protein, a signal-recognition receptor in the bacteria (\u003cb\u003eFigure S4 A-B\u003c/b\u003e, and \u003cb\u003eTable S3\u003c/b\u003e). The FtsY is known to interact with the SecYEG translocon channel that connects the cytoplasm to the periplasm in gram-negative bacteria and facilitates the translocation of proteins across the cytoplasmic membrane [20], indicating that the secretion of GFP in \u003cem\u003eE. coli\u003c/em\u003e might be mediated through the Sec pathway. The mechanisms underlying extracellular protein secretion in Gram-negative bacteria are not fully understood, and multiple pathways may be involved in this process. Once the protein localizes to the periplasmic space, it can cross the outer membrane either via passive diffusion or in a type 2 and type 5 secretion system-dependent manner. It is to be noted here that the presence of homologs of the Sec pathway was reported from plants [21], and the Sec pathway has been implicated in the export of proteins having bacterial secretion signals to thylakoids [22]. A maize mutant defective in the expression of FtsY was found to exhibit pleiotropic defects in the thylakoids [23], suggesting that the Sec pathway is essential for the normal development of plastids. However, export of chloroplast-synthesized proteins to the cytoplasm remains poorly characterized.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eBacterial Signal Peptide Facilitates Secretion of Chloroplast-Expressed Recombinant Protein into the Cytoplasm\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the presence of a prokaryotic-like secretion mechanism in pre-sent-day land plant chloroplasts, the gene encoding the SP:GFP fusion protein was cloned into the plastid transformation vector pVSR326 [24], driven by the psbA promoter and terminator. The construct was then site-specifically integrated into the tobacco plastid genome via standard chloroplast transformation protocols [25] (see Materials and Methods). As a control, a construct expressing GFP alone was also cloned into pVSR326 under the same regulatory elements, and stable, homotransplastomic lines were generated.\u003c/p\u003e\u003cp\u003eMolecular analyses confirmed the transplastomic nature of the transformed plants and verified the efficient transcription and translation of both SP:GFP and GFP genes (\u003cb\u003eFigures S5\u0026ndash;S6\u003c/b\u003e). Southern blot analysis demonstrated the site-specific integration of the GFP or SP:GFP transgenes, along with the aadA selectable marker, into the tobacco plastome (\u003cb\u003eFigures S5A\u0026ndash;B\u003c/b\u003e). Northern blot analysis confirmed robust transcription of the respective genes (\u003cb\u003eFigures S5C\u0026ndash;D\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eWestern blot analysis of total leaf protein extracts revealed three distinct bands in all three independent SP:GFP-expressing transplastomic lines (NtVSRSP-GFP1, NtVSRSP-GFP2, and NtVSRSP-GFP3; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, lanes 1\u0026ndash;3). In contrast, the GFP-only transplastomic line (NtVSRGFP1) displayed a single band corresponding in size to the lowest band observed in the SP:GFP-expressing lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, lane 4). These findings suggest that the SP:GFP fusion undergoes processing in chloroplasts.\u003c/p\u003e\u003cp\u003eTo further validate these results, two-dimensional (2D) gel electrophoresis followed by mass spectrometry was performed to identify GFP isoforms (\u003cb\u003eFigure S6; Table S4\u003c/b\u003e). In GFP-only transplastomic plants, a single protein spot was observed within the expected molecular weight and isoelectric point range (\u003cb\u003eFigure S6A\u003c/b\u003e, inset). In contrast, plants expressing SP:GFP exhibited at least two distinct GFP-containing protein spots (\u003cb\u003eFigure S6B\u003c/b\u003e, inset), supporting the hypothesis that the bacterial signal peptide is processed within chloroplasts. These results collectively suggest that the bacterial SP not only remains functional in the chloroplast environment but also facilitates the secretion of recombinant proteins from the chloroplast into the cytoplasm.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSubcellular Localization of SP:GFP by Confocal Microscopy Reveals Cytoplasmic Distribution of GFP\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo determine the subcellular localization of the GFP signal, protoplasts were isolated from the mesophyll tissue of transplastomic tobacco plants and analysed using confocal laser scanning microscopy. In plants expressing GFP alone, green fluorescence was exclusively localized within the chloroplasts, consistent with plastid-targeted expression and retention (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;B). In contrast, transplastomic plants expressing the SP:GFP fusion protein exhibited green fluorescence in both chloroplasts and the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u0026ndash;F), suggesting that the bacterial signal peptide facilitated the export of GFP from the chloroplasts into the cytoplasm.\u003c/p\u003e\u003cp\u003eThe cytoplasmic distribution of GFP fluorescence was particularly pronounced in cells containing differentiating plastids (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u0026ndash;D), while mesophyll cells with fully developed and mature chloroplasts showed relatively weaker cytoplasmic fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). This differential localization pattern indicates that the efficiency of GFP export may be influenced by the plastid developmental stage. Additionally, GFP fluorescence was observed in the cytoplasm of epidermal trichome cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF), where it appeared more prominent, likely due to the lower abundance of plastids in these cells, thereby reducing signal overlap and masking effects (\u003cb\u003eFigure S7\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMolecular and Proteomic Analyses of Chloroplast-Expressed SP:GFP Reveal Conserved Protein Secretion Mechanisms Shared Between Bacteria and Chloroplasts\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the conserved nature of prokaryotic-type protein secretion mechanisms in plant chloroplasts, mass spectrometry-based analysis was performed. Given the observed cleavage of the BSX signal peptide (SP) after the 20th and 51st amino acids in both Bacillus subtilis and Escherichia coli, it was hypothesized that the SP would undergo similar processing in chloroplasts if analogous mechanisms were present. Mass spectrometry-based identification of the N-terminal region of SP:GFP expressed in chloroplasts revealed cleavage after the 20th and 50th amino acids (corresponding to the lower bands in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, lanes 1\u0026ndash;3; \u003cb\u003eFigure S8\u003c/b\u003e), closely resembling the cleavage pattern observed in \u003cem\u003eB. subtilis\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e (\u003cb\u003eTable S5; Figures S8 - S11\u003c/b\u003e). The cytoplasmic localization of GFP fluorescence further supports the existence of a functionally conserved, prokaryotic-like protein secretion pathway in modern land plant chloroplasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe secretion of BSX and GFP into the extracellular medium when expressed as fusion proteins with the BSX SP in \u003cem\u003eE. coli\u003c/em\u003e, along with the identification of FtsY among co-immunoprecipitated proteins using anti-GFP antibodies, suggests that the Sec pathway is likely involved in this export process in \u003cem\u003eE. coli\u003c/em\u003e. The Sec pathway is also known to facilitate the targeting of a subset of nuclear-encoded proteins, imported into chloroplasts, to the thylakoid membrane [26]. Interestingly, the inner envelope membrane (IEM) and the thylakoid membrane share similar lipid compositions and protein components involved in protein translocation [27, 28].\u003c/p\u003e\u003cp\u003eFor a chloroplast-expressed SP:GFP fusion protein to reach the cytoplasm, as observed in this study, it must first cross the IEM to enter the intermembrane space, followed by translocation across the outer envelope membrane via an unidentified mechanism. The fact that the SP is cleaved at the same sites in both \u003cem\u003eE. coli\u003c/em\u003e and chloroplasts raises the possibility that the chloroplast Sec pathway, which usually mediates thylakoid protein targeting, may also facilitate SP:GFP targeting to the IEM, allowing subsequent export into the intermembrane space. Following this, GFP may either be actively exported into the cytoplasm through a novel pathway or passively diffuse. Alternatively, a thylakoid-independent export mechanism might exist, though its identity remains to be elucidated. The discovery of a functional protein export mechanism in chloroplasts, as suggested by our findings, combined with the well-established capacity of chloroplasts to produce foreign proteins at high levels, opens up exciting possibilities for crop improvement and various biotechnological applications [29, 30]. However, further work is needed to understand the exact mechanism(s) involved in the secretion of proteins by chloroplasts.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn the present study, we have provided evidence that a signal peptide derived from a single membrane containing gram-positive bacteria, \u003cem\u003eBacillus subtilis\u003c/em\u003e, can secrete recombinant proteins by a double membrane containing gram-negative \u003cem\u003eE. coli\u003c/em\u003e. Molecular and proteomic analysis suggested that the secretion of the recombinant proteins might be taking place through the SecY pathway. Similar secretion of recombinant proteins was observed when the SP-GFP fusion protein was expressed in the chloroplasts. Proteomic analysis revealed that the processing of signal peptides in \u003cem\u003eE. coli\u003c/em\u003e and chloroplasts is very similar, suggesting an evolutionarily conserved prokaryotic-like protein secretion mechanism operating in the opposite direction to the well-known protein import mechanisms in chloroplasts. Our results indicate the presence of a unique bidirectional protein trafficking across the double membrane envelope of the plastids. Nonetheless, additional studies are needed to fully understand the molecular components and exact mechanisms underlying protein secretion by chloroplasts.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eCloning and Expression of SP:BSX and SP:GFP in\u003c/strong\u003e \u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFull-length BSX gene along with signal peptide (SP:BSX) was polymerase Chain Reaction (PCR) amplified using genomic DNA of Bacillus sp. NG-27 [15] as a template using xylaFL5 and xyla3-10 primers containing NcoI and BamHI sites, respectively, and cloned into pET14b plasmid, creating the pET14b SP:BSX vector. The BSX signal peptide was also fused with GFP using a PCR-based method and SP:GFP gene was cloned at the same NcoI and BamHI sites of the pET14b plasmid, creating the pET14b SP:GFP vector. To compare the effect of the signal peptide on protein secretion, the GFP gene was cloned into the pET14b vector at the NcoI and BamHI sites, creating a pET14bGFP vector. The cloning of pET14bxyla5-14, carrying the mature part of BSX gene (excluding the Signal peptide) has already been described previously [17].\u003c/p\u003e\n\u003cp\u003eAll four constructs were transformed into Rosetta (DE3) cells (Novagen) for recombinant expression. In case of SP:BSX and SP:GFP, cells were grown at 28oC, and overnight induction was performed by adding IPTG (1mM final concentration). In the case of BSX and GFP, cells were grown at 37oC and induced for 4 hours by adding IPTG (1mM).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCloning and Expression of SP:GFP and GFP in Tobacco Chloroplast\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo express SP:GFP fusion protein in chloroplasts, the SP:GFP gene was placed under a chloroplast gene (psbA) promoter and terminator sequences. For a better comparison, transplastomic plants (transgenic plants transformed with foreign genes into the plastid genome) were also generated using a pVSRGFP vector having the GFP gene alone without the SP [31]. The GFP or SP:GFP gene cassettes are expected to get integrated into the tobacco plastome in a site-specific manner at the intergenic spacer region between rbcL and accD genes through two homologous recombination (as shown below in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). Stable and site-specific integration of GFP, SP:GFP, and the selectable aadA gene into tobacco plastome was confirmed by Southern hybridization (\u003cstrong\u003eFigure S5A-B\u003c/strong\u003e). A 14.3 kb and 14.45 band for GFP and SP:GFP, respectively, provided evidence for stable and site-specific integration of transgenes into tobacco plastome (Figure S5A-B). On the other hand, the presence of an 11.45 kb band in the control untransformed plant and the absence of the same band in the transplastomic plants is proof of the homoplasmy for the transplastome in all three transformed plants analyzed (\u003cstrong\u003eFigure S5B and 5\u003c/strong\u003e). Similarly, Southern hybridization analysis also confirmed the stable and site-specific integration of the GFP gene when tobacco plants were transformed with the pVSRGFP vector (data not shown). Northern blot analysis was used to identify efficient transcription of GFP (Figure S5C) and SP:GFP (\u003cstrong\u003eFigure S5D\u003c/strong\u003e). An expected size transcript of 0.85 kb was observed in the transplastomic plants transformed with SP:GFP gene (\u003cstrong\u003eFigure S5D\u003c/strong\u003e, lanes 1 and 2), and no signal was found in the control untransformed plant (Figure S5D, lane 3). Similarly, an expected size transcript (0.7 kb) was observed in GFP-expressing plants (\u003cstrong\u003eFigure S5C\u003c/strong\u003e, lane 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blotting Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInduced cultures of SP:BSX, SP:GFP, BSX, and GFP were sonicated in 1X PBS containing protease inhibitors (Roche). The supernatant fraction was loaded and subjected to SDS-PAGE analysis [32]. Total protein from tobacco untransformed/control and transplastomic plants (such as NtVSRSP-GFP1, NtVSRSP-GFP2, NtVSRSP-GFP3, and NtVSRGFP1) leaf was extracted as described in our earlier study [16]. Western blotting was performed using the wet transfer method, and in-house developed primary polyclonal antibodies raised against BSX and GFP proteins in rabbits were used to hybridize the PVDF membrane to detect the expression of recombinant proteins following standard procedures [16, 32].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunoprecipitation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInduced culture of SP:BSX, SP:GFP, BSX, and GFP were sonicated in equal volumes of ice-cold 1X TNET buffer (50mM Tris-Cl pH 8.0, 400mM NaCl, 5mM EDTA, and 2% Triton X-100) in the presence of protease inhibitors cocktail (Roche Diagnostics). The clarified extract was incubated with 100ul of Protein-A beads (GE Healthcare) coupled with anti-BSX or anti-GFP antibodies overnight at 4oC. Beads were washed with TNET buffer alone and subsequently with high-salt TNET buffer (600 mM NaCl) and low-salt TNET buffer (50 mM NaCl\u0026thinsp;+\u0026thinsp;0.1% Triton X-100). Finally, proteins were extracted from the Protein-A conjugated beads by boiling at 100\u0026deg;C with 2\u0026times; reducing dye for 5 minutes and subjected to 1D-SDS-PAGE analysis. Protein bands were excised from the Coomassie-stained gel and subjected to MALDI-TOF/TOF (Bruker Daltonics, Germany) analysis as mentioned below.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTwo-Dimensional Gel Electrophoresis Analysis (2D-PAGE)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn equal amount of protein extracted from the same-age leaf tissue expressing GFP and SP:GFP was taken and resolved on a 7cm IpG strip (PH range 4\u0026ndash;7) during first-dimensional isoelectric focusing (IEF) using Ettan IPGphor II IEF system, followed by second dimension SDS-PAGE as per manufacturer\u0026rsquo;s guidelines (GE Healthcare). Protein spots were excised manually from Coomassie-stained gels and identified using mass spectrometry analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn-Gel Digestion and Nano-LC MALDI-Based Protein Identification and Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe bands from 1D-SDS PAGE and the spots from 2D-PAGE were subjected to in-gel digestion as described by Shevchenko et al [33]. The gel pieces containing the protein of interest were digested with either 15 ng/\u0026micro;l trypsin (Promega) or 10 ng/ul Glu-C (Promega). Peptide samples obtained after in-gel digestion were subjected to Nano-LC (Proxeon Biosystems) based reverse phase separation [34]. Fractioned samples were subjected to MALDI TOF/TOF-based acquisition using the Ultraflex III MALDI TOF/TOF instrument (Bruker Daltonics, Germany). Post-acquisition processes, including mass annotation, baseline subtraction, and smoothening, were performed using Flex analysis software version 3.0 through WARP-LC. Protein identification was achieved using Biotools version 3.2 through an in-house licensed Mascot server (version 2.3 - March 2010) [33]. The database search parameters were set as described: fragment masses were searched in NCBInr and in-house generated protein databases through the mascot search engine (version 2.3), taxonomy was unrestricted, enzyme was set as either trypsin or V8-DE (Glu-C), fixed modification included carbamido-methylation of cysteine, variable modification included oxidation of methionine, protein mass was unrestricted, missed cleavage was set to 1, MS tolerance of +/-100 ppm and MS/MS tolerance of +/-0.75 da. Only peptides with an individual ion score of \u0026gt;\u0026thinsp;40 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were considered for protein identification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConfocal Microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtoplasts were prepared by partially digesting the mesophyll cells using Cellulase Onuzuka R10 and Pectinase R10 (Sigma) enzymes. Protoplasts were viewed using a confocal microscope: Nikon A-1R inverted microscope. Images were acquired using blue diode laser (ex 405 nm) and signals were collected at the Emission range of 470nm to 550nm for GFP. Chlorophyll auto-fluorescence was also acquired simultaneously.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eContributions:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLeelavathi Sadhu:\u0026nbsp;\u003c/strong\u003eConceptualization\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003eProject administration, Methodology.\u003cstrong\u003e\u0026nbsp;Amit Bhardwaj:\u0026nbsp;\u003c/strong\u003eConceptualization, Writing \u0026ndash; review \u0026amp; editing, Formal analysis, Data curation, Investigation. \u003cstrong\u003eKrishan Kumar:\u0026nbsp;\u003c/strong\u003eConceptualization, Methodology, Writing \u0026ndash; review \u0026amp; editing, Validation, Investigation\u003cstrong\u003e. \u0026nbsp;Saravanan Kumar:\u0026nbsp;\u003c/strong\u003eInvestigation, Writing \u0026ndash; review \u0026amp; editing.\u003cstrong\u003e\u0026nbsp;Abhishek Dass:\u0026nbsp;\u003c/strong\u003eInvestigation, Methodology. \u003cstrong\u003e\u0026nbsp;Ranjana Pathak:\u0026nbsp;\u003c/strong\u003eInvestigation, Visualization, \u003cstrong\u003ePankaj Pandey:\u0026nbsp;\u003c/strong\u003eInvestigation. \u003cstrong\u003eBhupendra S. Rawat:\u0026nbsp;\u003c/strong\u003eInvestigation. \u003cstrong\u003eVanga Siva Reddy:\u0026nbsp;\u003c/strong\u003eConceptualization, Writing \u0026ndash; original draft, Supervision, Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We are grateful for the excellent technical support from Purnima Kumar and Towa Optics technical team for confocal microscopy. We thank the Department of Biotechnology, Govt. of India, ICGEB, New Delhi and ICAR-IIMR for funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article and the Additional file.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eGoks\u0026oslash;yr, J. Evolution of Eucaryotic Cells. \u003cem\u003eNature\u003c/em\u003e\u003cstrong\u003e1967\u003c/strong\u003e, \u003cem\u003e214\u003c/em\u003e, 1161\u0026ndash;1161, doi:https://doi.org/10.1038/2141161a0.\u003c/li\u003e\n \u003cli\u003eMartin, W.; M\u0026uuml;ller, M. 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Glycoproteome of Elongating Cotton Fiber Cells. \u003cem\u003eMolecular \u0026amp; Cellular Proteomics\u003c/em\u003e\u003cstrong\u003e2013\u003c/strong\u003e, \u003cem\u003e12\u003c/em\u003e, 3677\u0026ndash;3689, doi: \u003cu\u003ehttps://doi.org/\u003c/u\u003e10.1074/mcp.M113.030726.\u003c/li\u003e\n \u003cli\u003eBioRender (2021). Cyanobacteria Structure. https://app.biorender.com/t-5ffdfec7a0005e00aa69816e-cyanobacteria-structure\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Protein export, Bidirectional protein trafficking, Chloroplasts","lastPublishedDoi":"10.21203/rs.3.rs-7128036/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7128036/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChloroplasts are semiautonomous organelles considered to have evolved from prokaryotic organisms like Cyanobacterium through endosymbiosis. During the course of evolution spanning over a billion years, a part of the organellar genome has migrated to the nucleus, and proteins encoded by such genes are synthesized in the cytoplasm and then imported into chloroplasts for their functions. To import nuclear-encoded and cytoplasm-synthesized proteins, chloroplasts have evolved multiple pathways, and some of them resemble prokaryotic protein export pathways operating in opposite directions. However, it is unknown whether any prokaryotic protein export mechanisms are functionally conserved in present-day land plant chloroplasts, allowing proteins synthesized in the chloroplasts to be exported into the cytoplasm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo understand the existence of any functional protein export pathway in chloroplasts, the coding region of a bacterial signal peptide from \u003cem\u003eBacillus subtilis\u003c/em\u003e, involved in the export of a cellulolytic enzyme (BSX), was translationally fused in-frame with GFP at 5’ end to create SP:GFP fusion protein and expressed in chloroplasts. Here we present data providing evidence that shows the existence of a functional protein export mechanism in the chloroplasts, similar to the prokaryotic protein secretion mechanism, which can be exploited for crop improvement and other biotechnological applications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEvidence for protein export and prior knowledge about protein import by chloroplasts indicate the presence of unique bidirectional trafficking across the chloroplast envelope. Nonetheless, additional studies are needed to fully understand the molecular components and exact mechanisms underlying protein secretion by chloroplasts.\u003c/p\u003e","manuscriptTitle":"Plastids Retained a Functional Prokaryotic-Like Protein Secretion Pathway That Can Export Proteins Synthesized in Chloroplasts into the Cytoplasm","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-28 09:14:03","doi":"10.21203/rs.3.rs-7128036/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"80d41cc7-4e74-4307-b195-c307895a1a8a","owner":[],"postedDate":"July 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-13T09:23:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-28 09:14:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7128036","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7128036","identity":"rs-7128036","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}