- Email: [email protected]
SECuring the perimeter
Research Update
TRENDS in Plant Science Vol.6 No.6 June 2001
235
Research News
SECuring the perimeter Dominique C. Bergmann Exquisitely regulated trafficking and fusion of vesicles is crucial for proper cell function. The molecules that regulate vesicle fusion are highly conserved among eukaryotes, but they have also undergone expansion and specialization within single genomes. With diversity comes the potential for functions in unique cell processes, and recent work in Arabidopsis reveals how a member of the SEC1 family, KEULE, functions in plant cell cytokinesis.
All cells must ensure that proteins reach the proper cellular compartments, and have sets of regulatory molecules to ensure that this works efficiently and accurately. One of the surprises of the Arabidopsis genome sequence was the enormous number of proteins implicated in vesicle trafficking, an expansion greater than that in even the human genome1. The biological role of these molecules is just beginning to be investigated in plants. Data presented in a recent paper by Farhah Assaad et al. add a specialized role in plant cytokinesis to the repertoire of fusion regulatory molecules2. In plant somatic-cell cytokinesis, nascent cells are divided by a wall built from the inside toward the periphery of the cell. A cytokinesis-specific cytoskeletal array, the phragmoplast, forms from the anaphase spindle after cell division. The phragmoplast contains oriented microtubules, microfilaments, and numerous motor and regulatory proteins, many of which have been characterized only recently3. Within the phragmoplast, the new cell plate is assembled. The cell plate grows by the fusion of Golgi-derived vesicles to form an intricate network of membranous tubes. The plate expands outward, eventually contacting the parental cell walls, and matures to form the new cellulose-rich cell wall (reviewed in Refs 3,4). Several loci required for the execution of cytokinesis in the embryo have been identified, but the molecular identity of only two is known. These two loci are essential components of the vesicle fusion apparatus. KNOLLE, which was cloned several years ago, encodes a protein
similar to syntaxin, and is localized to the forming cell plate in dividing cells5,6. KEULE, the subject of new work by Assaad et al., encodes a homologue of syntaxin’s key binding partner and regulator, sec1 (Ref. 2). The building of a cell plate requires the fusion of vesicles and membranes. This fusion process (analogous to general vesicle trafficking through the secretory pathway) requires the assembly of a protein complex to overcome energetic barriers to fusion at the interface between a vesicle and its membrane target. Contributing to this core complex are membrane-anchored proteins, referred to as SNAREs, at the surfaces of the opposing membranes. Although there is some inherent binding specificity that permits only some combinations of SNAREs to form a complex, many SNAREs are promiscuous7. Because of this, the regulation and specificity of vesicle fusion requires the activity of soluble accessory proteins, such as members of the Sec1 and Rab GTPase families. KEULE was first identified in screens for embryonic pattern mutants8. keule plants arrest as stunted seedlings with radial swelling, and the individual cells of the plants are bloated and disorganized. Electron micrographs of keule sections show multinucleate cells and cell wall stubs or remnants, indicating that the seedling-lethal phenotype is the result of defects in cytokinesis9. knolle plants exhibit similar but more severe
phenotypes than keule plants. However, the phenotypes of null mutations show that neither KEULE nor KNOLLE is absolutely required for cytokinesis. Only embryos missing functional copies of both genes show a complete failure to undergo cytokinesis, arresting as single multinucleate cells10. Evidence that the cytokinesis defect is related to vesicle fusion defects is found in electron micrographs of keule and knolle embryos where unfused vesicles appear at the division plane during telophase and then later near the cell wall stubs10. KEULE appears to have a role in cytokinesis in all somatic cells. Plants containing a transgene that ubiquitously expresses KEULE from a strong promoter exhibit cytokinesis-defective sectors2 (Fig. 1). These sectors probably result from the reduction of KEULE activity through co-suppression because levels of the KEULE transgene RNA are reduced in these cytokinesis-defective sectors compared with wild-type sectors. Consistent with a general requirement for KEULE activity, the KEULE RNA and protein are expressed in all embryonic and post-embryonic tissues2. The large SEC1 family has multiple roles in vesicle fusion11. KEULE and two closely related Arabidopsis homologues define a new branch in the SEC1 family. All three Arabidopsis proteins have closest affinity to yeast Sly1 and Sec1 proteins, which are involved in ER–Golgi transport and exocytosis, respectively1. However, KEULE is not a functional
Fig. 1. Comparison of wild type and 35S::KEULE mutant sectors in flowers. (a) Wild-type Arabidopsis flowers. (b) Sectors with altered KEULE activity. The rough surface of the sectors is due to the presence of bloated epidermal cells at the surface. (c) Enlarged view of boxed region from (b), the swollen, rounded cells are characteristic of cytokinesis defects. Figure modified and reproduced, with permission, from Ref. 2.
http://plants.trends.com 1360-1385/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(01)01959-8
Research Update
236
(a)
SEC1 binds syntaxin, keeping it inactive
in ax nt Sy
1
c Se
Target membrane
(b) 1
c Se
Release of SEC1 allows interaction with other SNAREs
v-S NA RE
in ax nt Sy
t-SN ARE
Vesicle
Target membrane
(c)
trans-SNARE complex forms; fusion occurs between membranes
TRENDS in Plant Science
Fig. 2. Cycle of vesicle fusion. (a) Peripheral SEC1 binds syntaxins at the membrane surface, sequestering the syntaxins away from potential interactors. (b) Release of SEC1 (which can induce an activating conformational change in syntaxin) allows syntaxin to bind to SNAREs on the same membrane (t-SNAREs) and on opposing membranes (v-SNAREs). (c) Interactions between SNAREs enables membranes to come in to close proximity so that they can fuse with one another. The final step of the cycle (not shown) is the dissolution of the core complex and rebinding of syntaxin by SEC1.
homologue of the sly1 or sec1 genes because it is unable to complement yeast strains containing mutations in either gene2. What other evidence is there that KEULE behaves as a Sec1 protein? The Sec1 family of proteins is characterized by the following features: • The ability to bind to syntaxins. • The presence of soluble and membrane associated forms. • Peripheral association with membranes. KEULE fulfills these three criteria. In vitro binding assays detect KEULE protein bound to immobilized KNOLLE protein2. Because whole plant extracts were used as the source for KEULE, a http://plants.trends.com
TRENDS in Plant Science Vol.6 No.6 June 2001
requirement for additional factors for KEULE–KNOLLE interactions has not been ruled out. There does appear to be some specificity for KNOLLE binding because the KEULE homologue, AtSec1a, is unable to bind KNOLLE in these assays2. Immunofluorescence data for subcellular localization are not yet available for KEULE, but the protein can be monitored in fractionation experiments. In these, KEULE is found in both membrane pellets and in the cytosolic portions. KEULE can be released from membranes in conditions that enable the release of peripheral, but not integral, membrane proteins2. By contrast, KNOLLE is not released under these conditions and requires the addition of detergents – this is consistent with previous results and predictions that it is a membrane-anchored protein6. Interaction of SEC1 and syntaxin at the membrane
The co-crystal structure of a neuronal Sec1 and syntaxin complex shows an intimate connection between these two molecules at the surface of the membrane (Fig. 2a). The surface of syntaxin that normally binds to other SNAREs to promote vesicle fusion is made unavailable by an interaction with SEC1. Release of SEC1, accompanied by a conformational change in syntaxin, enables this SNARE to interact with other SNAREs on opposing membranes (Fig. 2b). Vesicle fusion is driven by the formation of a core fusion complex assembled from α-helicies contributed by the syntaxin and other v- and t-SNAREs (Fig. 2c). The co-crystal structure of Sec1–syntaxin also reveals that a surface of SEC1 is available to bind other proteins, even when complexed with syntaxin12. If SEC1 regulates vesicle fusion by its interaction with syntaxins, then the regulation of Sec1 itself becomes a crucial question. The model emerging from Arabidopsis is that KEULE and KNOLLE interact to promote vesicle fusion at the phragmoplast. This must be regulated with respect to the cell cycle so that a cell plate is formed only after nuclear division is completed. Are there any candidates for regulators? By analogy to other systems in which the SEC1–syntaxin interaction is modulated by phosphorylation of SEC1, homologues of the cyclin-dependant kinase 5 (cdk5) or protein kinase C (PKC)
families might interact with KEULE. The recently completed Arabidopsis genome predicts many proteins in these classes, and, in addition, cell-cycle regulated kinases localized to the phragmoplast are prime candidates for ensuring that the building of the cell plate commences at the appropriate time in the cell cycle3,13. The Arabidopsis genome contains six predicted sec1 homologues and 24 syntaxin-like proteins1; therefore it is likely that each Sec1 will have multiple binding partners. Some Sec1–syntaxin pairs will be used for general cell metabolism, whereas others might also have roles in generating specialized tissues such as pollen tubes or root hairs, or might be needed for germ cell cytokinesis. One tantalizing observation in keule mutants is that root hairs, which expand by tip growth and thus demand polarized secretion, are absent. knolle mutants do not share this defect, suggesting that KEULE has a different partner in this process. Vesicle trafficking has been of interest to many groups working in a variety of systems. The trafficking field has already seen the value of studying specialized cell types (most notably neurons) to identify new variations on the theme of vesicle fusion. Studies of Arabidopsis can contribute by identifying new regulators of vesicle fusion used when building unique cell structures such as the cell plate, root hairs and pollen tubes. KEULE and KNOLLE provide a foothold for understanding the regulation of trafficking during cytokinesis. From this position, the cytokinesis complex can be extended by finding additional interactors through standard biochemical approaches, or by looking for the genetic interactions between knolle or keule and known phragmoplast components or cellcycle regulators. Acknowledgements
I thank Wolfgang Lukowitz, Jane McConnell and Stephanie Mohr for comments on the manuscript and Farhah Assaad for discussion and the images used in Fig. 1. References 1 Sanderfoot, A.A. et al. (2000) The Arabidopsis genome. An abundance of soluble Nethylmaleimide-sensitive factor adaptor protein receptors. Plant Physiol 124, 1558–1569 2 Assaad, F.F. et al. (2001) The cytokinesis gene KEULE encodes a sec1 protein which binds the syntaxin KNOLLE. J. Cell. Biol. 152, 531–544
Research Update
3 Otegui, M. and Staehelin, L.A. (2000) Cytokinesis in flowering plants: more than one way to divide a cell. Curr. Opin. Plant Biol. 3, 493–502 4 Nacry, P. et al. (2000) Genetic dissection of cytokinesis. Plant Mol. Biol. 43, 719–733 5 Lukowitz, W. et al. (1996) Cytokinesis in the Arabidopsis embryo involves the syntaxin-related KNOLLE gene product. Cell 84, 61–71 6 Lauber, M.H. et al. (1997) The Arabidopsis KNOLLE protein is a cytokinesis-specific syntaxin. J. Cell Biol. 139, 1485–1493 7 Yang, B. et al. (1999) SNARE interactions are not selective. Implications for membrane fusion specificity. J. Biol. Chem. 274, 5649–5653
TRENDS in Plant Science Vol.6 No.6 June 2001
8 Mayer, U. et al. (1991) Mutations affecting body organization in the Arabidopsis embryo. Nature 353, 402–407 9 Assaad, F.F. et al. (1996) The KEULE gene is involved in cytokinesis in Arabidopsis. Mol. Gen. Genet. 253, 267–277 10 Waizenegger, I. et al. (2000) The Arabidopsis KNOLLE and KEULE genes interact to promote vesicle fusion during cytokinesis. Curr. Biol. 10, 1371–1374 11 Halachmi, N. and Lev, Z. (1996) The Sec1 family: a novel family of proteins involved in synaptic transmission and general secretion. J. Neurochem. 66, 889–897
237
12 Misura, K.M. et al. (2000) Three-dimensional structure of the neuronal–Sec1–syntaxin 1a complex. Nature 404, 355–362 13 The Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796–815
Dominique C. Bergmann Carnegie Institution of Washington, Dept of Plant Biology, Stanford, CA 94305, USA. e-mail: [email protected]
Antibiotic-free chloroplast genetic engineering – an environmentally friendly approach Henry Daniell, Peter O. Wiebe and Alicia Fernandez-San Millan Chloroplast genetic engineering offers several advantages over nuclear genetic engineering, including gene containment and hyperexpression. However, introducing thousands of copies of transgenes into the chloroplast genome amplifies the antibiotic resistance genes. Two recent articles report different and novel strategies to either remove antibiotic resistance genes or select chloroplast transformants without using these genes. This should eliminate their potential transfer to microorganisms or plants and ease public concerns about genetically modified crops.
Most transformation techniques introduce a gene that confers antibiotic resistance, along with the gene of interest to impart the desired trait. Regenerating transformed cells in growth media containing antibiotic permits the selection of only those cells that have incorporated the foreign genes. Once transgenic plants are regenerated, antibiotic resistance genes serve no useful purpose, but they continue to produce their gene products. One concern is that the antibiotic resistance genes could be transferred to weeds or pathogenic microorganisms in the gastrointestinal tract or soil, making them resistant to treatment with such antibiotics. Antibiotic resistant bacteria are a major problem for modern medicine. The presence of antibiotic resistance gene products in transgenic plants could inactivate oral doses of a clinically important antibiotic1,2. In Germany, genetically modified (GM) crops containing antibiotic resistance genes
have been banned from release3. However, several approaches are available to eliminate antibiotic resistance genes from nuclear transgenic crops1. Advantages of chloroplast genetic engineering
Chloroplast genetic engineering is emerging as an alternative new technology that overcomes many of the concerns of nuclear genetic engineering4. One common environmental concern is the escape of foreign genes through pollen or seed dispersal from transgenic crops to their weedy relatives and thus creating super weeds, or to other crops resulting in genetic pollution2. Several major food corporations require the segregation of native crops from those ‘polluted’ with transgenes. Maternal inheritance of foreign genes through chloroplast genetic engineering is highly desirable where there is the potential for out-cross among crops or between crops and weeds5–7. Yet another concern is the use of nuclear transgenic crops expressing the Bacillus thuringiensis (Bt) toxins at suboptimal levels, resulting in pests developing Bt resistance. However, a study has shown that the insects (which were up to 40 000-fold resistant to other Bt proteins) died when fed on plants hyperexpressing several thousand copies of a novel Bt gene via chloroplast genetic engineering8. Another hotly debated environmental concern is the toxicity of transgenic pollen to non-target insects, such as the Monarch butterflies. Although pollen from a few plants contains
metabolically active plastids, the plastid DNA itself is lost during pollen maturation and hence is not transmitted to the next generation. A recent investigation showed that even though chloroplasts in leaves contained as much as 47% CRY protein of the total soluble protein, the insecticidal protein was absent in transgenic pollen9. In the post-genomic era, emphasis is placed on gene function. Clearly, most desired traits would require engineering of multiple genes or pathways. Therefore, it is essential to establish tools for genetic engineering that would exploit the outcomes of functional genomics. The ability to provide coordinated expression of multiple genes is considered to be the Holy Grail of plant biotechnology to produce valuable agronomic traits. Yet this continues to be challenging in nuclear genetic engineering. For example, in a seven-year-long effort, Xudong Ye et al.10 introduced three genes into rice for a short biosynthetic pathway to express β-carotene, resulting in ‘golden rice’. Although we can now transform several plant species more efficiently, position effect and gene silencing continue to hamper the efficient expression of foreign genes via the nuclear genome. The introduction of multiple genes requires the generation of individual transgenic plants and subsequent back crosses to reconstitute the entire pathway or multisubunit proteins. The expression of polycistrons via the chloroplast genome is an extraordinary opportunity to express foreign pathways in a single
http://plants.trends.com 1360-1385/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(01)01949-5
TRENDS in Plant Science Vol.6 No.6 June 2001
235
Research News
SECuring the perimeter Dominique C. Bergmann Exquisitely regulated trafficking and fusion of vesicles is crucial for proper cell function. The molecules that regulate vesicle fusion are highly conserved among eukaryotes, but they have also undergone expansion and specialization within single genomes. With diversity comes the potential for functions in unique cell processes, and recent work in Arabidopsis reveals how a member of the SEC1 family, KEULE, functions in plant cell cytokinesis.
All cells must ensure that proteins reach the proper cellular compartments, and have sets of regulatory molecules to ensure that this works efficiently and accurately. One of the surprises of the Arabidopsis genome sequence was the enormous number of proteins implicated in vesicle trafficking, an expansion greater than that in even the human genome1. The biological role of these molecules is just beginning to be investigated in plants. Data presented in a recent paper by Farhah Assaad et al. add a specialized role in plant cytokinesis to the repertoire of fusion regulatory molecules2. In plant somatic-cell cytokinesis, nascent cells are divided by a wall built from the inside toward the periphery of the cell. A cytokinesis-specific cytoskeletal array, the phragmoplast, forms from the anaphase spindle after cell division. The phragmoplast contains oriented microtubules, microfilaments, and numerous motor and regulatory proteins, many of which have been characterized only recently3. Within the phragmoplast, the new cell plate is assembled. The cell plate grows by the fusion of Golgi-derived vesicles to form an intricate network of membranous tubes. The plate expands outward, eventually contacting the parental cell walls, and matures to form the new cellulose-rich cell wall (reviewed in Refs 3,4). Several loci required for the execution of cytokinesis in the embryo have been identified, but the molecular identity of only two is known. These two loci are essential components of the vesicle fusion apparatus. KNOLLE, which was cloned several years ago, encodes a protein
similar to syntaxin, and is localized to the forming cell plate in dividing cells5,6. KEULE, the subject of new work by Assaad et al., encodes a homologue of syntaxin’s key binding partner and regulator, sec1 (Ref. 2). The building of a cell plate requires the fusion of vesicles and membranes. This fusion process (analogous to general vesicle trafficking through the secretory pathway) requires the assembly of a protein complex to overcome energetic barriers to fusion at the interface between a vesicle and its membrane target. Contributing to this core complex are membrane-anchored proteins, referred to as SNAREs, at the surfaces of the opposing membranes. Although there is some inherent binding specificity that permits only some combinations of SNAREs to form a complex, many SNAREs are promiscuous7. Because of this, the regulation and specificity of vesicle fusion requires the activity of soluble accessory proteins, such as members of the Sec1 and Rab GTPase families. KEULE was first identified in screens for embryonic pattern mutants8. keule plants arrest as stunted seedlings with radial swelling, and the individual cells of the plants are bloated and disorganized. Electron micrographs of keule sections show multinucleate cells and cell wall stubs or remnants, indicating that the seedling-lethal phenotype is the result of defects in cytokinesis9. knolle plants exhibit similar but more severe
phenotypes than keule plants. However, the phenotypes of null mutations show that neither KEULE nor KNOLLE is absolutely required for cytokinesis. Only embryos missing functional copies of both genes show a complete failure to undergo cytokinesis, arresting as single multinucleate cells10. Evidence that the cytokinesis defect is related to vesicle fusion defects is found in electron micrographs of keule and knolle embryos where unfused vesicles appear at the division plane during telophase and then later near the cell wall stubs10. KEULE appears to have a role in cytokinesis in all somatic cells. Plants containing a transgene that ubiquitously expresses KEULE from a strong promoter exhibit cytokinesis-defective sectors2 (Fig. 1). These sectors probably result from the reduction of KEULE activity through co-suppression because levels of the KEULE transgene RNA are reduced in these cytokinesis-defective sectors compared with wild-type sectors. Consistent with a general requirement for KEULE activity, the KEULE RNA and protein are expressed in all embryonic and post-embryonic tissues2. The large SEC1 family has multiple roles in vesicle fusion11. KEULE and two closely related Arabidopsis homologues define a new branch in the SEC1 family. All three Arabidopsis proteins have closest affinity to yeast Sly1 and Sec1 proteins, which are involved in ER–Golgi transport and exocytosis, respectively1. However, KEULE is not a functional
Fig. 1. Comparison of wild type and 35S::KEULE mutant sectors in flowers. (a) Wild-type Arabidopsis flowers. (b) Sectors with altered KEULE activity. The rough surface of the sectors is due to the presence of bloated epidermal cells at the surface. (c) Enlarged view of boxed region from (b), the swollen, rounded cells are characteristic of cytokinesis defects. Figure modified and reproduced, with permission, from Ref. 2.
http://plants.trends.com 1360-1385/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(01)01959-8
Research Update
236
(a)
SEC1 binds syntaxin, keeping it inactive
in ax nt Sy
1
c Se
Target membrane
(b) 1
c Se
Release of SEC1 allows interaction with other SNAREs
v-S NA RE
in ax nt Sy
t-SN ARE
Vesicle
Target membrane
(c)
trans-SNARE complex forms; fusion occurs between membranes
TRENDS in Plant Science
Fig. 2. Cycle of vesicle fusion. (a) Peripheral SEC1 binds syntaxins at the membrane surface, sequestering the syntaxins away from potential interactors. (b) Release of SEC1 (which can induce an activating conformational change in syntaxin) allows syntaxin to bind to SNAREs on the same membrane (t-SNAREs) and on opposing membranes (v-SNAREs). (c) Interactions between SNAREs enables membranes to come in to close proximity so that they can fuse with one another. The final step of the cycle (not shown) is the dissolution of the core complex and rebinding of syntaxin by SEC1.
homologue of the sly1 or sec1 genes because it is unable to complement yeast strains containing mutations in either gene2. What other evidence is there that KEULE behaves as a Sec1 protein? The Sec1 family of proteins is characterized by the following features: • The ability to bind to syntaxins. • The presence of soluble and membrane associated forms. • Peripheral association with membranes. KEULE fulfills these three criteria. In vitro binding assays detect KEULE protein bound to immobilized KNOLLE protein2. Because whole plant extracts were used as the source for KEULE, a http://plants.trends.com
TRENDS in Plant Science Vol.6 No.6 June 2001
requirement for additional factors for KEULE–KNOLLE interactions has not been ruled out. There does appear to be some specificity for KNOLLE binding because the KEULE homologue, AtSec1a, is unable to bind KNOLLE in these assays2. Immunofluorescence data for subcellular localization are not yet available for KEULE, but the protein can be monitored in fractionation experiments. In these, KEULE is found in both membrane pellets and in the cytosolic portions. KEULE can be released from membranes in conditions that enable the release of peripheral, but not integral, membrane proteins2. By contrast, KNOLLE is not released under these conditions and requires the addition of detergents – this is consistent with previous results and predictions that it is a membrane-anchored protein6. Interaction of SEC1 and syntaxin at the membrane
The co-crystal structure of a neuronal Sec1 and syntaxin complex shows an intimate connection between these two molecules at the surface of the membrane (Fig. 2a). The surface of syntaxin that normally binds to other SNAREs to promote vesicle fusion is made unavailable by an interaction with SEC1. Release of SEC1, accompanied by a conformational change in syntaxin, enables this SNARE to interact with other SNAREs on opposing membranes (Fig. 2b). Vesicle fusion is driven by the formation of a core fusion complex assembled from α-helicies contributed by the syntaxin and other v- and t-SNAREs (Fig. 2c). The co-crystal structure of Sec1–syntaxin also reveals that a surface of SEC1 is available to bind other proteins, even when complexed with syntaxin12. If SEC1 regulates vesicle fusion by its interaction with syntaxins, then the regulation of Sec1 itself becomes a crucial question. The model emerging from Arabidopsis is that KEULE and KNOLLE interact to promote vesicle fusion at the phragmoplast. This must be regulated with respect to the cell cycle so that a cell plate is formed only after nuclear division is completed. Are there any candidates for regulators? By analogy to other systems in which the SEC1–syntaxin interaction is modulated by phosphorylation of SEC1, homologues of the cyclin-dependant kinase 5 (cdk5) or protein kinase C (PKC)
families might interact with KEULE. The recently completed Arabidopsis genome predicts many proteins in these classes, and, in addition, cell-cycle regulated kinases localized to the phragmoplast are prime candidates for ensuring that the building of the cell plate commences at the appropriate time in the cell cycle3,13. The Arabidopsis genome contains six predicted sec1 homologues and 24 syntaxin-like proteins1; therefore it is likely that each Sec1 will have multiple binding partners. Some Sec1–syntaxin pairs will be used for general cell metabolism, whereas others might also have roles in generating specialized tissues such as pollen tubes or root hairs, or might be needed for germ cell cytokinesis. One tantalizing observation in keule mutants is that root hairs, which expand by tip growth and thus demand polarized secretion, are absent. knolle mutants do not share this defect, suggesting that KEULE has a different partner in this process. Vesicle trafficking has been of interest to many groups working in a variety of systems. The trafficking field has already seen the value of studying specialized cell types (most notably neurons) to identify new variations on the theme of vesicle fusion. Studies of Arabidopsis can contribute by identifying new regulators of vesicle fusion used when building unique cell structures such as the cell plate, root hairs and pollen tubes. KEULE and KNOLLE provide a foothold for understanding the regulation of trafficking during cytokinesis. From this position, the cytokinesis complex can be extended by finding additional interactors through standard biochemical approaches, or by looking for the genetic interactions between knolle or keule and known phragmoplast components or cellcycle regulators. Acknowledgements
I thank Wolfgang Lukowitz, Jane McConnell and Stephanie Mohr for comments on the manuscript and Farhah Assaad for discussion and the images used in Fig. 1. References 1 Sanderfoot, A.A. et al. (2000) The Arabidopsis genome. An abundance of soluble Nethylmaleimide-sensitive factor adaptor protein receptors. Plant Physiol 124, 1558–1569 2 Assaad, F.F. et al. (2001) The cytokinesis gene KEULE encodes a sec1 protein which binds the syntaxin KNOLLE. J. Cell. Biol. 152, 531–544
Research Update
3 Otegui, M. and Staehelin, L.A. (2000) Cytokinesis in flowering plants: more than one way to divide a cell. Curr. Opin. Plant Biol. 3, 493–502 4 Nacry, P. et al. (2000) Genetic dissection of cytokinesis. Plant Mol. Biol. 43, 719–733 5 Lukowitz, W. et al. (1996) Cytokinesis in the Arabidopsis embryo involves the syntaxin-related KNOLLE gene product. Cell 84, 61–71 6 Lauber, M.H. et al. (1997) The Arabidopsis KNOLLE protein is a cytokinesis-specific syntaxin. J. Cell Biol. 139, 1485–1493 7 Yang, B. et al. (1999) SNARE interactions are not selective. Implications for membrane fusion specificity. J. Biol. Chem. 274, 5649–5653
TRENDS in Plant Science Vol.6 No.6 June 2001
8 Mayer, U. et al. (1991) Mutations affecting body organization in the Arabidopsis embryo. Nature 353, 402–407 9 Assaad, F.F. et al. (1996) The KEULE gene is involved in cytokinesis in Arabidopsis. Mol. Gen. Genet. 253, 267–277 10 Waizenegger, I. et al. (2000) The Arabidopsis KNOLLE and KEULE genes interact to promote vesicle fusion during cytokinesis. Curr. Biol. 10, 1371–1374 11 Halachmi, N. and Lev, Z. (1996) The Sec1 family: a novel family of proteins involved in synaptic transmission and general secretion. J. Neurochem. 66, 889–897
237
12 Misura, K.M. et al. (2000) Three-dimensional structure of the neuronal–Sec1–syntaxin 1a complex. Nature 404, 355–362 13 The Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796–815
Dominique C. Bergmann Carnegie Institution of Washington, Dept of Plant Biology, Stanford, CA 94305, USA. e-mail: [email protected]
Antibiotic-free chloroplast genetic engineering – an environmentally friendly approach Henry Daniell, Peter O. Wiebe and Alicia Fernandez-San Millan Chloroplast genetic engineering offers several advantages over nuclear genetic engineering, including gene containment and hyperexpression. However, introducing thousands of copies of transgenes into the chloroplast genome amplifies the antibiotic resistance genes. Two recent articles report different and novel strategies to either remove antibiotic resistance genes or select chloroplast transformants without using these genes. This should eliminate their potential transfer to microorganisms or plants and ease public concerns about genetically modified crops.
Most transformation techniques introduce a gene that confers antibiotic resistance, along with the gene of interest to impart the desired trait. Regenerating transformed cells in growth media containing antibiotic permits the selection of only those cells that have incorporated the foreign genes. Once transgenic plants are regenerated, antibiotic resistance genes serve no useful purpose, but they continue to produce their gene products. One concern is that the antibiotic resistance genes could be transferred to weeds or pathogenic microorganisms in the gastrointestinal tract or soil, making them resistant to treatment with such antibiotics. Antibiotic resistant bacteria are a major problem for modern medicine. The presence of antibiotic resistance gene products in transgenic plants could inactivate oral doses of a clinically important antibiotic1,2. In Germany, genetically modified (GM) crops containing antibiotic resistance genes
have been banned from release3. However, several approaches are available to eliminate antibiotic resistance genes from nuclear transgenic crops1. Advantages of chloroplast genetic engineering
Chloroplast genetic engineering is emerging as an alternative new technology that overcomes many of the concerns of nuclear genetic engineering4. One common environmental concern is the escape of foreign genes through pollen or seed dispersal from transgenic crops to their weedy relatives and thus creating super weeds, or to other crops resulting in genetic pollution2. Several major food corporations require the segregation of native crops from those ‘polluted’ with transgenes. Maternal inheritance of foreign genes through chloroplast genetic engineering is highly desirable where there is the potential for out-cross among crops or between crops and weeds5–7. Yet another concern is the use of nuclear transgenic crops expressing the Bacillus thuringiensis (Bt) toxins at suboptimal levels, resulting in pests developing Bt resistance. However, a study has shown that the insects (which were up to 40 000-fold resistant to other Bt proteins) died when fed on plants hyperexpressing several thousand copies of a novel Bt gene via chloroplast genetic engineering8. Another hotly debated environmental concern is the toxicity of transgenic pollen to non-target insects, such as the Monarch butterflies. Although pollen from a few plants contains
metabolically active plastids, the plastid DNA itself is lost during pollen maturation and hence is not transmitted to the next generation. A recent investigation showed that even though chloroplasts in leaves contained as much as 47% CRY protein of the total soluble protein, the insecticidal protein was absent in transgenic pollen9. In the post-genomic era, emphasis is placed on gene function. Clearly, most desired traits would require engineering of multiple genes or pathways. Therefore, it is essential to establish tools for genetic engineering that would exploit the outcomes of functional genomics. The ability to provide coordinated expression of multiple genes is considered to be the Holy Grail of plant biotechnology to produce valuable agronomic traits. Yet this continues to be challenging in nuclear genetic engineering. For example, in a seven-year-long effort, Xudong Ye et al.10 introduced three genes into rice for a short biosynthetic pathway to express β-carotene, resulting in ‘golden rice’. Although we can now transform several plant species more efficiently, position effect and gene silencing continue to hamper the efficient expression of foreign genes via the nuclear genome. The introduction of multiple genes requires the generation of individual transgenic plants and subsequent back crosses to reconstitute the entire pathway or multisubunit proteins. The expression of polycistrons via the chloroplast genome is an extraordinary opportunity to express foreign pathways in a single
http://plants.trends.com 1360-1385/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(01)01949-5