- Email: [email protected]
Peeking into pit fields — A new model of secondary plasmodesmata formation
S140
Abstracts / Comparative Biochemistry and Physiology, Part A 150 (2008) S139–S147
proteins). These include ADF, profilin, and CAP (cyclase associated protein). Despite exhibiting some unique biochemistry, these proteins broadly fulfil similar roles to their animal and fungal counterparts. However, genomic sequencing data suggests that several key proteins that signal to ABPs in other eukaryotes (such as WASP and VASP) are missing from plant genomes. This supports the observation that plant ABPs can be regulated by plant-specific signaling systems. Surprisingly, the small number of ABP signalling proteins that are shared between plants and other eukaryotes consist of those utilized predominantly for cell motility in metazoans and protists. Moreover, genetic approaches have shown that these proteins play an essential role in the development of cells in both higher and lower plants. Here we will discuss our recent work in this field.
initiation of the COPII coat at the ER export sites. Using an inducible version of Sar1p-GTP in tobacco leaves we have shown that unexpectedly the first event of Golgi membrane transport to the ER is loss of a trans-Golgi associated matrix protein followed by trans-Golgi located transferases. Subsequently medial and cis-located proteins are trafficked back to the ER. This indicates the presence of a direct pathway from the trans-Golgi to the ER. On rebuilding of the Golgi after either Sar1-GTP or BFA induced dissolution, cis-located proteins assemble on Golgi membranes prior to medial and trans-proteins.
doi:10.1016/j.cbpa.2008.04.343
C1/P4.7 Small GTPases in post-Golgi and endocytic membrane traffic in Arabidopsis
C1/P4.5 Plant microtubules, MAPs and the cytokinetic phragmoplast
I. Moore, C. Chow, O. Teh, H. Neto, C. Foucart, J. Perez-Gomez (University of Oxford)
P. Hussey (University of Durham)
The endomembrane organelles and their associated trafficking pathways synthesise some of the most biologically and commercially important structures in plants. Circumstantial evidence suggests that intracellular membrane trafficking pathways diversified independently in the plant kingdom but documented examples are rare. Rab GTPases are essential regulators of membrane identity and membrane targeting specificity in eukaryotic cells. Rab GTpase families have diversified independently in the animal and plant lineages. We show that in Arabidopsis root tips, the Rab-A2 and Rab-A3 subclasses define a novel post-Golgi membrane domain that communicates with the plasma membrane and early endosomal system and contributes substantially to the cell plate during cytokinesis. In contrast to the Rab-A2 and -A3 subclasses, Rab-A5 protein define a distinct and apparently unique distribution at the periphery of root meristematic cells. We have also employed a screen based on accumulation of secreted GFP to identify mutations that affect biosynthetic membrane traffic in Arabidopsis. Using this screen we identified mutants in the GBF-family ArfGEFGNOM-LIKE1 (GNL1). We show that GNL1 is a BFAresistant GBF protein that functions together with a BFA-sensitive ArfGEF both at the Golgi and in selective endocytosis of PIN2 but not in recycling from endosomes. The evolution of endocytic trafficking in plants was apparently accompanied by neofunctionalisation within the GBF family while in other kingdoms it occurred independently by elaboration of additional ArfGEF families.
Cell division in plant cells is orchestrated by a unique plant microtubule array, the cytokinetic phragmoplast. Plant cells cannot move like animal cells, so differentiation and development is dependent on the direction of cell expansion and the positioning of internal crosswalls. The phragmoplast is responsible for the assembly of the internal wall and the microtubules will guide wall forming vesicles to the site where they coalesce to form the new cell plate. So far we know very little about the regulation of this array, but biochemical and genetic studies are beginning to identify some of the key components. Three of these components are microtubule-associated proteins (MAPs): 1, MOR1/GEM1 a member of the XMAP215 family MAPs; 2, MAP65 suggested to be responsible for the stabilisation of the antiparallel microtubules and a member of a diverse group of midzone MAPs; 3. MAP190 a novel plant microtubule and actin binding protein. Here, using biochemical, cell biological and genetic studies, we present data demonstrating that these MAPs are essential for the integrity of the cytokinetic phragmoplast and for cell division. doi:10.1016/j.cbpa.2008.04.344
C1/P4.6 Dynamics of plant Golgi membranes C. Hawes (Oxford Brookes University); J. Schoberer (BOKU Vienna); E. Hummel, (Oxford Brookes University); A. Osterrieder(Oxford Brookes University) We have previously demonstrated that the plant Golgi apparatus can be highly motile and in many tissues is closely associated with the endoplasmic reticulum. Golgi bodies move in concert with endoplasmic reticulum (ER), export site proteins and with the motile surface of the ER. This secretory unit may be tethered to the ER by a number of Golgi matrix proteins associated with cisternal membranes. Anterograde transport of membrane between the two organelles can be demonstrated by fluorescence recovery after photobleaching techniques, whilst retrograde transport can be studied by the application of the secretory inhibitor BFA. A more controlled reabsorption of Golgi membranes into the ER can be achieved by expression of a GTP-locked mutant version of the small GTPase Sar1p which is responsible for the
doi:10.1016/j.cbpa.2008.04.345
doi:10.1016/j.cbpa.2008.04.346
C1/P4.8 Peeking into pit fields — A new model of secondary plasmodesmata formation C. Faulkner, O. Akman, K. Bell, C. Jeffree, K. Oparka (University of Edinburgh) In higher plants, plasmodesmata (PD) are major conduits for cell– cell communication. Primary PD are laid down at cytokinesis while secondary PD arise during wall extension. During tobacco leaf development, the basal cell wall of trichomes extends radially without division, providing a convenient system for studying the origin of secondary PD. We devised a simple freeze-fracture protocol for examining large numbers of PD in surface view. In the post-cytokinetic
Abstracts / Comparative Biochemistry and Physiology, Part A 150 (2008) S139–S147
wall, PD were randomly distributed. As the primary wall extended, PD became ‘twinned’ at the cell periphery, aligned with new cellulose microfibrils. Additional secondary pores were inserted into these sites at right angles to these, giving rise to pit fields composed of several paired PD. Our data are consistent with a model in which peripheral primary PD function as templates to clone additional secondary PD, spatially fixing the position of future pit fields. Contrary to published models that depict secondary PD as fusion profiles of adjacent primary PD, we provide new evidence that secondary PD arise from fissions of peripheral primary PD. Our data are supported by a computational model, ‘Plasmodesmap’, which accurately simulates the formation of pit fields during radial cell–wall extension, producing PD motifs with striking resemblance to those on fractured wall faces. doi:10.1016/j.cbpa.2008.04.347
S141
Leaf epidermal cells contain a cortical ER network comprised of tubules, cisternae and three way junctions. Golgi bodies appear intimately associated with this network, which overlies the actin cytoskeleton. Upon depolymerisation of the actin the network remains intact indicating that additional factors are required for the coordinated response(s) required for the morphological changes from tubules to cisternae and vice versa. Recently, members of a class of proteins termed reticulons were shown in yeast and mammals to modulate ER morphology. Arabidopsis contains at least 19 genes encoding for reticulon-like proteins. We have characterised the shortest member of the family, RTNLB13 (Tolley et al., 2008 Traffic 9:94–102). Overexpression of fluorescent fusions indicate that RTNLB13 is located on the ER, and based on FRAP studies constricts the ER lumen. However, this severe phenotype does not appear to affect Golgi body movement, number, shape or secretion of a reporter protein. Future studies are required to understand the cellular requirement for tubules versus cisternal ER, and whether alterations in this ratio through manipulating reticulon expression levels could affect secretion at the macroscopic level.
C1/P4.9 SNAREs at the traffic junction with signalling, transport and nutrition
doi:10.1016/j.cbpa.2008.04.349
M. Blatt (University of Glasgow); I. Johansson (University of Glasgow); M. Paneque (University of Chile); R. Pratelli (Carnegie Institution of Washington); P. Campanoni (Philip Morris Intnl); S. Sokolovski (University of Dundee); A. Honsbein (University of Glasgow)
C1/P4.11 Integrating cell division and plant development J. Murray (University of Cambridge)
SNARE proteins contribute to vesicle targeting and membrane fusion in all eukaryotic cells, and are essential to the mechanics of cell growth and development. Nonetheless, there is clear evidence that SNAREs play additional roles, for example in facilitating cellular signalling and synaptic transmission. I report here that SYP121, an Arabidopsis SNARE found at the plasma membrane, contributes to scaffolds that anchor ion channels within the plane of the membrane, and it is a critical structural element determining the K+ channel gating and K+ mineral nutrition. Work from my laboratory has shown that SYP121 interacts in vitro and in vivo with the regulatory (‘silent’) K+ channel subunit KC1 which assembles with different inwardrectifying K+ channels to affect their activities in the plant.SYP121-KC1 interaction was found to be specific to these two protein partners and to be essential for the activity of K+ channels, channel-mediated K+ uptake at the root epidermis and for growth. Electrophysiological studies have shown changes in channel gating that can only result from an altered conformation of the channel and profound changes in the exposure of the channel voltage sensor to the membrane electric field. Intriguingly, when heterologously expressed on its own AKT1 does yield an inward-rectifying K+ current, but its gating characteristics – and those of AKT1 expressed together with KC1 – differ significantly from the K+ current in vivo unless co-expressed together with SYP121. In short, SYP121 is a missing component of the K+ channel complex and is essential for its gating in the plant. These results demonstrate a wholly unexpected role for a SNARE in eukaryotes as part of protein complex facilitating mineral nutrition and wholly unrelated to signalling or to membrane vesicle traffic.
Plant growth is characterised by a continuous indeterminate pattern of growth and the repeated initiation of organs. Growth is ultimately driven by the process of cell division coupled with the subsequent expansion and differentiation of the resulting cells. Both these processes depend on cell cycles-mitotic cycles during the phase of cell production and endocycles during the phase of cell expansion. During organ formation or root growth these two aspects are spatially and/or temporally distinct and the transition between them controls the cellular composition of organs. I will review the overall regulation of cell division in plant growth and development, and focus on data that show a key role for D-type (CYCD) cyclin activity in determining cell division rates and the transition from mitotic to endocycles in leaves and roots. doi:10.1016/j.cbpa.2008.04.350
C1/P4.12 Cyclin dependent protein kinase substrates: Insights into growth control by cell cycle regulators J. Doonan (John Innes Centre); O. Pierrat (John Innes Centre); M. Bush (John Innes Centre); K. Alexiou (John Innes Centre); V. Mikitova (John Innes Centre); G. Kitosis (Athens University); C. Pignocchi (John Innes Centre); L. Mayberry (University of Texas); K. Browning (University of Texas)
doi:10.1016/j.cbpa.2008.04.348
C1/P4.10 Role of reticulons on ER morphology and secretion I. Sparkes (Oxford Brookes University); N. Tolley (Warwick University); C. Hawes (Oxford Brookes University); L. Frigerio (Warwick University)
The plant Cyclin Dependent Protein Kinases (CDKs) comprise a family of at least 8 structurally related kinases, CDKA to G, with diverse cellular functions. Although CDKA is particularly well known for its roles in cell cycle progression, the targets of plant CDKs are not so well understood. As a first step towards understanding CDK function we have undertaken a search for CDK substrates. Bio-informatic analyses and yeast 2-H screens indicate that CDKA interacts with a wide variety of potential substrates and regulatory
Abstracts / Comparative Biochemistry and Physiology, Part A 150 (2008) S139–S147
proteins). These include ADF, profilin, and CAP (cyclase associated protein). Despite exhibiting some unique biochemistry, these proteins broadly fulfil similar roles to their animal and fungal counterparts. However, genomic sequencing data suggests that several key proteins that signal to ABPs in other eukaryotes (such as WASP and VASP) are missing from plant genomes. This supports the observation that plant ABPs can be regulated by plant-specific signaling systems. Surprisingly, the small number of ABP signalling proteins that are shared between plants and other eukaryotes consist of those utilized predominantly for cell motility in metazoans and protists. Moreover, genetic approaches have shown that these proteins play an essential role in the development of cells in both higher and lower plants. Here we will discuss our recent work in this field.
initiation of the COPII coat at the ER export sites. Using an inducible version of Sar1p-GTP in tobacco leaves we have shown that unexpectedly the first event of Golgi membrane transport to the ER is loss of a trans-Golgi associated matrix protein followed by trans-Golgi located transferases. Subsequently medial and cis-located proteins are trafficked back to the ER. This indicates the presence of a direct pathway from the trans-Golgi to the ER. On rebuilding of the Golgi after either Sar1-GTP or BFA induced dissolution, cis-located proteins assemble on Golgi membranes prior to medial and trans-proteins.
doi:10.1016/j.cbpa.2008.04.343
C1/P4.7 Small GTPases in post-Golgi and endocytic membrane traffic in Arabidopsis
C1/P4.5 Plant microtubules, MAPs and the cytokinetic phragmoplast
I. Moore, C. Chow, O. Teh, H. Neto, C. Foucart, J. Perez-Gomez (University of Oxford)
P. Hussey (University of Durham)
The endomembrane organelles and their associated trafficking pathways synthesise some of the most biologically and commercially important structures in plants. Circumstantial evidence suggests that intracellular membrane trafficking pathways diversified independently in the plant kingdom but documented examples are rare. Rab GTPases are essential regulators of membrane identity and membrane targeting specificity in eukaryotic cells. Rab GTpase families have diversified independently in the animal and plant lineages. We show that in Arabidopsis root tips, the Rab-A2 and Rab-A3 subclasses define a novel post-Golgi membrane domain that communicates with the plasma membrane and early endosomal system and contributes substantially to the cell plate during cytokinesis. In contrast to the Rab-A2 and -A3 subclasses, Rab-A5 protein define a distinct and apparently unique distribution at the periphery of root meristematic cells. We have also employed a screen based on accumulation of secreted GFP to identify mutations that affect biosynthetic membrane traffic in Arabidopsis. Using this screen we identified mutants in the GBF-family ArfGEFGNOM-LIKE1 (GNL1). We show that GNL1 is a BFAresistant GBF protein that functions together with a BFA-sensitive ArfGEF both at the Golgi and in selective endocytosis of PIN2 but not in recycling from endosomes. The evolution of endocytic trafficking in plants was apparently accompanied by neofunctionalisation within the GBF family while in other kingdoms it occurred independently by elaboration of additional ArfGEF families.
Cell division in plant cells is orchestrated by a unique plant microtubule array, the cytokinetic phragmoplast. Plant cells cannot move like animal cells, so differentiation and development is dependent on the direction of cell expansion and the positioning of internal crosswalls. The phragmoplast is responsible for the assembly of the internal wall and the microtubules will guide wall forming vesicles to the site where they coalesce to form the new cell plate. So far we know very little about the regulation of this array, but biochemical and genetic studies are beginning to identify some of the key components. Three of these components are microtubule-associated proteins (MAPs): 1, MOR1/GEM1 a member of the XMAP215 family MAPs; 2, MAP65 suggested to be responsible for the stabilisation of the antiparallel microtubules and a member of a diverse group of midzone MAPs; 3. MAP190 a novel plant microtubule and actin binding protein. Here, using biochemical, cell biological and genetic studies, we present data demonstrating that these MAPs are essential for the integrity of the cytokinetic phragmoplast and for cell division. doi:10.1016/j.cbpa.2008.04.344
C1/P4.6 Dynamics of plant Golgi membranes C. Hawes (Oxford Brookes University); J. Schoberer (BOKU Vienna); E. Hummel, (Oxford Brookes University); A. Osterrieder(Oxford Brookes University) We have previously demonstrated that the plant Golgi apparatus can be highly motile and in many tissues is closely associated with the endoplasmic reticulum. Golgi bodies move in concert with endoplasmic reticulum (ER), export site proteins and with the motile surface of the ER. This secretory unit may be tethered to the ER by a number of Golgi matrix proteins associated with cisternal membranes. Anterograde transport of membrane between the two organelles can be demonstrated by fluorescence recovery after photobleaching techniques, whilst retrograde transport can be studied by the application of the secretory inhibitor BFA. A more controlled reabsorption of Golgi membranes into the ER can be achieved by expression of a GTP-locked mutant version of the small GTPase Sar1p which is responsible for the
doi:10.1016/j.cbpa.2008.04.345
doi:10.1016/j.cbpa.2008.04.346
C1/P4.8 Peeking into pit fields — A new model of secondary plasmodesmata formation C. Faulkner, O. Akman, K. Bell, C. Jeffree, K. Oparka (University of Edinburgh) In higher plants, plasmodesmata (PD) are major conduits for cell– cell communication. Primary PD are laid down at cytokinesis while secondary PD arise during wall extension. During tobacco leaf development, the basal cell wall of trichomes extends radially without division, providing a convenient system for studying the origin of secondary PD. We devised a simple freeze-fracture protocol for examining large numbers of PD in surface view. In the post-cytokinetic
Abstracts / Comparative Biochemistry and Physiology, Part A 150 (2008) S139–S147
wall, PD were randomly distributed. As the primary wall extended, PD became ‘twinned’ at the cell periphery, aligned with new cellulose microfibrils. Additional secondary pores were inserted into these sites at right angles to these, giving rise to pit fields composed of several paired PD. Our data are consistent with a model in which peripheral primary PD function as templates to clone additional secondary PD, spatially fixing the position of future pit fields. Contrary to published models that depict secondary PD as fusion profiles of adjacent primary PD, we provide new evidence that secondary PD arise from fissions of peripheral primary PD. Our data are supported by a computational model, ‘Plasmodesmap’, which accurately simulates the formation of pit fields during radial cell–wall extension, producing PD motifs with striking resemblance to those on fractured wall faces. doi:10.1016/j.cbpa.2008.04.347
S141
Leaf epidermal cells contain a cortical ER network comprised of tubules, cisternae and three way junctions. Golgi bodies appear intimately associated with this network, which overlies the actin cytoskeleton. Upon depolymerisation of the actin the network remains intact indicating that additional factors are required for the coordinated response(s) required for the morphological changes from tubules to cisternae and vice versa. Recently, members of a class of proteins termed reticulons were shown in yeast and mammals to modulate ER morphology. Arabidopsis contains at least 19 genes encoding for reticulon-like proteins. We have characterised the shortest member of the family, RTNLB13 (Tolley et al., 2008 Traffic 9:94–102). Overexpression of fluorescent fusions indicate that RTNLB13 is located on the ER, and based on FRAP studies constricts the ER lumen. However, this severe phenotype does not appear to affect Golgi body movement, number, shape or secretion of a reporter protein. Future studies are required to understand the cellular requirement for tubules versus cisternal ER, and whether alterations in this ratio through manipulating reticulon expression levels could affect secretion at the macroscopic level.
C1/P4.9 SNAREs at the traffic junction with signalling, transport and nutrition
doi:10.1016/j.cbpa.2008.04.349
M. Blatt (University of Glasgow); I. Johansson (University of Glasgow); M. Paneque (University of Chile); R. Pratelli (Carnegie Institution of Washington); P. Campanoni (Philip Morris Intnl); S. Sokolovski (University of Dundee); A. Honsbein (University of Glasgow)
C1/P4.11 Integrating cell division and plant development J. Murray (University of Cambridge)
SNARE proteins contribute to vesicle targeting and membrane fusion in all eukaryotic cells, and are essential to the mechanics of cell growth and development. Nonetheless, there is clear evidence that SNAREs play additional roles, for example in facilitating cellular signalling and synaptic transmission. I report here that SYP121, an Arabidopsis SNARE found at the plasma membrane, contributes to scaffolds that anchor ion channels within the plane of the membrane, and it is a critical structural element determining the K+ channel gating and K+ mineral nutrition. Work from my laboratory has shown that SYP121 interacts in vitro and in vivo with the regulatory (‘silent’) K+ channel subunit KC1 which assembles with different inwardrectifying K+ channels to affect their activities in the plant.SYP121-KC1 interaction was found to be specific to these two protein partners and to be essential for the activity of K+ channels, channel-mediated K+ uptake at the root epidermis and for growth. Electrophysiological studies have shown changes in channel gating that can only result from an altered conformation of the channel and profound changes in the exposure of the channel voltage sensor to the membrane electric field. Intriguingly, when heterologously expressed on its own AKT1 does yield an inward-rectifying K+ current, but its gating characteristics – and those of AKT1 expressed together with KC1 – differ significantly from the K+ current in vivo unless co-expressed together with SYP121. In short, SYP121 is a missing component of the K+ channel complex and is essential for its gating in the plant. These results demonstrate a wholly unexpected role for a SNARE in eukaryotes as part of protein complex facilitating mineral nutrition and wholly unrelated to signalling or to membrane vesicle traffic.
Plant growth is characterised by a continuous indeterminate pattern of growth and the repeated initiation of organs. Growth is ultimately driven by the process of cell division coupled with the subsequent expansion and differentiation of the resulting cells. Both these processes depend on cell cycles-mitotic cycles during the phase of cell production and endocycles during the phase of cell expansion. During organ formation or root growth these two aspects are spatially and/or temporally distinct and the transition between them controls the cellular composition of organs. I will review the overall regulation of cell division in plant growth and development, and focus on data that show a key role for D-type (CYCD) cyclin activity in determining cell division rates and the transition from mitotic to endocycles in leaves and roots. doi:10.1016/j.cbpa.2008.04.350
C1/P4.12 Cyclin dependent protein kinase substrates: Insights into growth control by cell cycle regulators J. Doonan (John Innes Centre); O. Pierrat (John Innes Centre); M. Bush (John Innes Centre); K. Alexiou (John Innes Centre); V. Mikitova (John Innes Centre); G. Kitosis (Athens University); C. Pignocchi (John Innes Centre); L. Mayberry (University of Texas); K. Browning (University of Texas)
doi:10.1016/j.cbpa.2008.04.348
C1/P4.10 Role of reticulons on ER morphology and secretion I. Sparkes (Oxford Brookes University); N. Tolley (Warwick University); C. Hawes (Oxford Brookes University); L. Frigerio (Warwick University)
The plant Cyclin Dependent Protein Kinases (CDKs) comprise a family of at least 8 structurally related kinases, CDKA to G, with diverse cellular functions. Although CDKA is particularly well known for its roles in cell cycle progression, the targets of plant CDKs are not so well understood. As a first step towards understanding CDK function we have undertaken a search for CDK substrates. Bio-informatic analyses and yeast 2-H screens indicate that CDKA interacts with a wide variety of potential substrates and regulatory