... HARVEY J. MARCHANT AND LARRY C. FOWKE' Depcrrtmer~t oJ' Dei~eloprner~tcrl Biology, ... more ... HARVEY J. MARCHANT AND LARRY C. FOWKE' Depcrrtmer~t oJ' Dei~eloprner~tcrl Biology, Reseclrcll School of' Biological Scier~ces ... Spheroplasts (protoplasts) of the inarine dino-flagellate, Got~yaulax have been liberated after detergent treatment and hypotonic shock (Ada ...
ABSTRACT The author highlights his 40-year academic career with emphasis on the major contributio... more ABSTRACT The author highlights his 40-year academic career with emphasis on the major contributions of technicians, graduate students, and postdoctoral fellows in his research laboratory. Postdoctoral research in Canberra, Australia, on algal cell division preceded a faculty position in the Biology Department, University of Saskatchewan, that stretched from 1970 until today. Early work in Saskatoon that focused on cultured plant cells and protoplasts developed into an investigation of the structure and function of plant coated vesicles. A short sabbatical in Sweden resulted in a 15-year research program on somatic embryogenesis in conifers. A return to the study of cell division over the last 10 plus years resulted in the discovery and analysis of a family of plant cell division inhibitors. The author’s story emphasizes the importance of having motivated and creative scientists in the laboratory, but also recognizes the impact of serendipity. International collaborations are also featured.
A mutant of Chlamydomonas reinhardii which lacks a cell wall was fused with Daucus carota protopl... more A mutant of Chlamydomonas reinhardii which lacks a cell wall was fused with Daucus carota protoplasts using polyethylene glycol and the resulting fusion products were cultured. Fusion involved integration of Chlamydomonas and carrot plasma membranes and the release of algal organelles into the carrot cytoplasm. Chlamydomonas basal bodies, nuclei and chloroplasts were frequently observed in the fusion products. Cultured fusion products regenerated cell walls and divided; most Chlamydomonas organelles degenerated during culture but chloroplasts were still recognizable in the carrot cytoplasm after 10.
Cell wall regeneration by protoplasts from Vicia hajastana suspension cultures was investigated w... more Cell wall regeneration by protoplasts from Vicia hajastana suspension cultures was investigated with Calcofluor White ST staining and platinum-palladium surface replicas. Microfibril deposition was initiated after 10-20 minutes of culture and within 20 hours protoplasts were covered with a heavy mat of microfibrils. The early stages of microfibril formation could not be detected with Calcofluor staining.
Cultured soybean cells recovered from a marked decrease in cell division 20 hours after removal o... more Cultured soybean cells recovered from a marked decrease in cell division 20 hours after removal of their cell walls with enzymes and exhibited sustained mitotic activity. Mitosis was essentially similar in both cultured cells and protoplasts. At prophase micrombules aggregated in a clear zone surrounding the nucleus prior to forming the spindle. During metaphase and anaphase chromosomal microtubules were attached to diffuse kinetochores and extended to broad spindle poles; few interzonal microtubules were evident. Considerable endoplasmic reticulum was present at the spindle poles throughout division and may contribute to the new nuclear envelope at telophase. A typical phragmoplast consisting of vesicles, overlapping microtubules and associated electron-dense material appeared earlier in the protoplasts and developed into a thicker ceil plate than found in the cultured cells.
Page 1. Protoplasma 89, 305--316 (1976) 9 by Springer-Verlag 1976 Labelling of Concanavalin A Sit... more Page 1. Protoplasma 89, 305--316 (1976) 9 by Springer-Verlag 1976 Labelling of Concanavalin A Sites on the Plasma Membrane of Soybean Protoplasts ~ FRANCIS A. WILLIAMSON, LARRY C. FOWKE 2, FC CONSTABEL, and OL GAMBORG ...
The organelles of soybean (Glycine max (L.) Merr.) protoplasts were separated using a recently de... more The organelles of soybean (Glycine max (L.) Merr.) protoplasts were separated using a recently developed procedure which allows rapid (3-h) recovery of a fraction enriched for coated vesicles (CVs). As determined by marker-enzyme enrichment and ultrastructural analysis of isolated membrane fractions, endoplasmic reticulum, Golgi membranes, glucan-synthase-II (EC 2.4.1.34)-containing membranes (putative plasma membrane), mitochondria, and CVs were enriched in separate fractions in a sucrose density gradient. Glucan synthase I (EC 2.4.1.12) had the highest specific activity in the Golgi-enriched and CV-enriched fractions and was found to comigrate with CVs upon rate-zonal centrifugation of a CV-enriched fraction. For further elucidation of the role of these latter organelles in cell-wall regeneration, freshly isolated protoplasts were pulsed with [3H]glucose for 20 min, and the disappearance of label from the organelles was followed for the ensuing 1 h. Although a CV-enriched fraction contained glucan synthase I, it contained very small amounts of labeled polysaccharide during the period of study. Pulse-chase experiments with [3H]glucose helped to confirm the role of the Golgi apparatus in secretion of matrix polysaccharides by protoplasts.
A mutant of Chlamydomonas reinhardii which lacks a cell wall was fused with Daucus carota protopl... more A mutant of Chlamydomonas reinhardii which lacks a cell wall was fused with Daucus carota protoplasts using polyethylene glycol and the resulting fusion products were cultured. Fusion involved integration of Chlamydomonas and carrot plasma membranes and the release of algal organelles into the carrot cytoplasm. Chlamydomonas basal bodies, nuclei and chloroplasts were frequently observed in the fusion products. Cultured fusion products regenerated cell walls and divided; most Chlamydomonas organelles degenerated during culture but chloroplasts were still recognizable in the carrot cytoplasm after 10.
In vitro binding experiments were carried out using 32P-labeled cells of the virulent Agrobactenu... more In vitro binding experiments were carried out using 32P-labeled cells of the virulent Agrobactenum tumefaciens strain B6 and Datura imoxia cells from suspension culture. Binding kinetics showed that adherence of bacteria to Datura cells increased gradually during the first 60 minutes and attained a maximum level within 120 minutes of incubation. Maximum binding occurred at pH 6.0. The presence of Ca2+ and Mg2e reduced binding slightly and EDTA had little effect at concentrations of 0.1 to 10 millimolar. The binding of bacteria to Datura cells was temperaturedependent. Escherichis cofi, Salmoneia typhimurium, Rhizobium japonicun, and Micrococcus lysodeikticus did not compete with virulent A. tumefaciens strain B6 for binding to Datura cells. The admixture of avirulent A. tumefiaciens strain IIBNV6 enhanced adherence of virulent A. tumefaciens strain B6 to Daturs cells. Octopine had no effect on the binding of virulent A. tumefaciens strain B6 to Datura cells, but 10 millimolar canavanine was inhibitory. Arginine enhanced the adherence of the bacteria at concentrations higher than 0.1 millimlar. Incubation with DNase, RNase, and lipase did not affect the binding, but protease stimulated the adherence of bacteria to Datura cells. Concanavaline A and soybean lectin had little effect whereas lecithin and lysolecithin enhanced binding slightly. Poly-L-lysine markedly stimulated the bacteria-plant cell adherence. Cells from suspension cultures of pea, vetch, and soybean had a 2to 3-fold higher binding capacity than Datura cells, whereas cells from wheat, corn, rice, and sorghum had a considerably lower affinity for binding with virulent A. tumefaciens strain B6. Bacterial adherence to plant cells was confirmed by autoradiography and electron microscopy. Autoradiwww.plant.org on April 4, 2016 -Published by www.plantphysiol.org Downloaded from
The plasma membrane has a number of important functions in plant cells including the regulation o... more The plasma membrane has a number of important functions in plant cells including the regulation of ion and metabolite transport, hormone transport and action, host pathogen interactions and cell wall biosynthesis and deposition.
... of a coated vesicle-enriched fraction from plant cells BRENT G. MERSEY,13 LARRY C. FOWKE,2 FR... more ... of a coated vesicle-enriched fraction from plant cells BRENT G. MERSEY,13 LARRY C. FOWKE,2 FRED CONSTABEL3 and ELDON H. NEW ... of negatively-stained coated vesicles isolated from suspension-cultured to-bacco cells by a modification of the method of Keen et al. ...
Haploid, diploid and tetraploid lines of Brassica rapa L. (syn. campestris), and allotetraploid B... more Haploid, diploid and tetraploid lines of Brassica rapa L. (syn. campestris), and allotetraploid B. napus L., were examined to determine the influence of ploidy on floral features, particularly nectary morphology and anatomy, and to relate nectary structure to nectar production capacity. Except for haploids, all lines were rapid-cycling. Average flower dry weight, and petal length and width, were in the descending order B. napus B. rapa (4n) 2n n. Pollen grains of 4n plants were larger than those of 2n plants ; haploids lacked pollen.
ABSTRACT Protoplasts from will carrot (Daucus carota) cell cultures were fused with protoplasts o... more ABSTRACT Protoplasts from will carrot (Daucus carota) cell cultures were fused with protoplasts of the filamentous green alga Stigeoclonium sp. using polyethylene glycol. Following fusion of the algal and higher plant plasma membranes, intact Stigeoclonium chloroplasts, nuclei, and centrioles were observed within the carrot cytoplasm. Other algal organelles were not distinguishable in the fusion products.
The plant CDK inhibitor ICK1 was identi®ed previously from Arabidopis thaliana with its inhibitor... more The plant CDK inhibitor ICK1 was identi®ed previously from Arabidopis thaliana with its inhibitory activity characterized in vitro. ICK1 displayed several structural and functional features that are distinct from known animal CDK inhibitors. Despite the initial characterization, there is no information on the functions of any plant CDK inhibitor in plants. To gain insight into ICK1 functions in vivo and the role of cell division during plant growth and development, transgenic plants were generated expressing ICK1 driven by the cauli¯ower mosaic virus 35S promoter. In comparison to control plants, growth was signi®cantly inhibited in transgenic 35S-ICK1 plants, with some plants weighing <10% of wild-type plants at the 3 week stage. Most organs of 35S-ICK1 plants were smaller. There were also modi®cations in plant morphology such as shape and serration of leaves and petals. The changes were so drastic that 35S-ICK1 plants with strong phenotype no longer resembled wild-type plants morphologically. Analyses showed that increased ICK1 expression resulted in reduced CDK activity and reduced the number of cells in these plants. Cells in 35S-ICK1 plants were larger than corresponding cells in control plants. These results demonstrate that ICK1 acts as a CDK inhibitor in the plant, and the inhibition of cell division by ICK1 expression has profound effects on plant growth and development. They also suggest that alterations of plant organ shape can be achieved by restriction of cell division.
Variation in seasonal sprouting pattern from roots and rhizomes of perennial herbaceous plants in... more Variation in seasonal sprouting pattern from roots and rhizomes of perennial herbaceous plants influence the success of plant proliferation ability, invasiveness and escape from weed control measures. The latter often rely on methods, which repeatedly fragment the underground system, thereby trigger adventitious and axillary buds to sprout, and consequently reduce the amount of stored energy. If carried out at times when no re-growth occurs, treatments will have little effect on weed populations, but cost much in terms of labour and energy.
The cell division cycle in plants as in other eukaryotes is controlled by the cyclin-dependent ki... more The cell division cycle in plants as in other eukaryotes is controlled by the cyclin-dependent kinase (CDK). This CDK paradigm determines that developmental cues and environmental signals need to impinge on the CDK complex to affect the cell cycle. An important part of understanding cell cycle regulation is to understand how CDK is regulated by various factors. In addition, there are features that set the cell cycle regulation in plants apart from that in other eukaryotes such as animals. Our knowledge of the molecular mechanisms that underlie the differences is poor. A family of plant CDK inhibitor proteins has been identified. The plant CDK inhibitors share similarity with a family of animal CDK inhibitors in a small region, while most of the sequence and the structural layout of the plant CDK inhibitors are different from the animal counterparts. Studies of plant CDK inhibitors have been performed mostly with the CDK inhibitors from Arabidopsis called ICKs (also referred to as KRPs). ICKs interact with D-type cyclins and A-type CDK. Overexpression of ICKs has been shown to affect cell division, plant growth, and morphogenesis. Studies of ICKs have also provided insightful information on the control of endoreduplication in plants. These aspects as well as cellular localization and protein regulation of ICKs are reviewed.
... HARVEY J. MARCHANT AND LARRY C. FOWKE' Depcrrtmer~t oJ' Dei~eloprner~tcrl Biology, ... more ... HARVEY J. MARCHANT AND LARRY C. FOWKE' Depcrrtmer~t oJ' Dei~eloprner~tcrl Biology, Reseclrcll School of' Biological Scier~ces ... Spheroplasts (protoplasts) of the inarine dino-flagellate, Got~yaulax have been liberated after detergent treatment and hypotonic shock (Ada ...
ABSTRACT The author highlights his 40-year academic career with emphasis on the major contributio... more ABSTRACT The author highlights his 40-year academic career with emphasis on the major contributions of technicians, graduate students, and postdoctoral fellows in his research laboratory. Postdoctoral research in Canberra, Australia, on algal cell division preceded a faculty position in the Biology Department, University of Saskatchewan, that stretched from 1970 until today. Early work in Saskatoon that focused on cultured plant cells and protoplasts developed into an investigation of the structure and function of plant coated vesicles. A short sabbatical in Sweden resulted in a 15-year research program on somatic embryogenesis in conifers. A return to the study of cell division over the last 10 plus years resulted in the discovery and analysis of a family of plant cell division inhibitors. The author’s story emphasizes the importance of having motivated and creative scientists in the laboratory, but also recognizes the impact of serendipity. International collaborations are also featured.
A mutant of Chlamydomonas reinhardii which lacks a cell wall was fused with Daucus carota protopl... more A mutant of Chlamydomonas reinhardii which lacks a cell wall was fused with Daucus carota protoplasts using polyethylene glycol and the resulting fusion products were cultured. Fusion involved integration of Chlamydomonas and carrot plasma membranes and the release of algal organelles into the carrot cytoplasm. Chlamydomonas basal bodies, nuclei and chloroplasts were frequently observed in the fusion products. Cultured fusion products regenerated cell walls and divided; most Chlamydomonas organelles degenerated during culture but chloroplasts were still recognizable in the carrot cytoplasm after 10.
Cell wall regeneration by protoplasts from Vicia hajastana suspension cultures was investigated w... more Cell wall regeneration by protoplasts from Vicia hajastana suspension cultures was investigated with Calcofluor White ST staining and platinum-palladium surface replicas. Microfibril deposition was initiated after 10-20 minutes of culture and within 20 hours protoplasts were covered with a heavy mat of microfibrils. The early stages of microfibril formation could not be detected with Calcofluor staining.
Cultured soybean cells recovered from a marked decrease in cell division 20 hours after removal o... more Cultured soybean cells recovered from a marked decrease in cell division 20 hours after removal of their cell walls with enzymes and exhibited sustained mitotic activity. Mitosis was essentially similar in both cultured cells and protoplasts. At prophase micrombules aggregated in a clear zone surrounding the nucleus prior to forming the spindle. During metaphase and anaphase chromosomal microtubules were attached to diffuse kinetochores and extended to broad spindle poles; few interzonal microtubules were evident. Considerable endoplasmic reticulum was present at the spindle poles throughout division and may contribute to the new nuclear envelope at telophase. A typical phragmoplast consisting of vesicles, overlapping microtubules and associated electron-dense material appeared earlier in the protoplasts and developed into a thicker ceil plate than found in the cultured cells.
Page 1. Protoplasma 89, 305--316 (1976) 9 by Springer-Verlag 1976 Labelling of Concanavalin A Sit... more Page 1. Protoplasma 89, 305--316 (1976) 9 by Springer-Verlag 1976 Labelling of Concanavalin A Sites on the Plasma Membrane of Soybean Protoplasts ~ FRANCIS A. WILLIAMSON, LARRY C. FOWKE 2, FC CONSTABEL, and OL GAMBORG ...
The organelles of soybean (Glycine max (L.) Merr.) protoplasts were separated using a recently de... more The organelles of soybean (Glycine max (L.) Merr.) protoplasts were separated using a recently developed procedure which allows rapid (3-h) recovery of a fraction enriched for coated vesicles (CVs). As determined by marker-enzyme enrichment and ultrastructural analysis of isolated membrane fractions, endoplasmic reticulum, Golgi membranes, glucan-synthase-II (EC 2.4.1.34)-containing membranes (putative plasma membrane), mitochondria, and CVs were enriched in separate fractions in a sucrose density gradient. Glucan synthase I (EC 2.4.1.12) had the highest specific activity in the Golgi-enriched and CV-enriched fractions and was found to comigrate with CVs upon rate-zonal centrifugation of a CV-enriched fraction. For further elucidation of the role of these latter organelles in cell-wall regeneration, freshly isolated protoplasts were pulsed with [3H]glucose for 20 min, and the disappearance of label from the organelles was followed for the ensuing 1 h. Although a CV-enriched fraction contained glucan synthase I, it contained very small amounts of labeled polysaccharide during the period of study. Pulse-chase experiments with [3H]glucose helped to confirm the role of the Golgi apparatus in secretion of matrix polysaccharides by protoplasts.
A mutant of Chlamydomonas reinhardii which lacks a cell wall was fused with Daucus carota protopl... more A mutant of Chlamydomonas reinhardii which lacks a cell wall was fused with Daucus carota protoplasts using polyethylene glycol and the resulting fusion products were cultured. Fusion involved integration of Chlamydomonas and carrot plasma membranes and the release of algal organelles into the carrot cytoplasm. Chlamydomonas basal bodies, nuclei and chloroplasts were frequently observed in the fusion products. Cultured fusion products regenerated cell walls and divided; most Chlamydomonas organelles degenerated during culture but chloroplasts were still recognizable in the carrot cytoplasm after 10.
In vitro binding experiments were carried out using 32P-labeled cells of the virulent Agrobactenu... more In vitro binding experiments were carried out using 32P-labeled cells of the virulent Agrobactenum tumefaciens strain B6 and Datura imoxia cells from suspension culture. Binding kinetics showed that adherence of bacteria to Datura cells increased gradually during the first 60 minutes and attained a maximum level within 120 minutes of incubation. Maximum binding occurred at pH 6.0. The presence of Ca2+ and Mg2e reduced binding slightly and EDTA had little effect at concentrations of 0.1 to 10 millimolar. The binding of bacteria to Datura cells was temperaturedependent. Escherichis cofi, Salmoneia typhimurium, Rhizobium japonicun, and Micrococcus lysodeikticus did not compete with virulent A. tumefaciens strain B6 for binding to Datura cells. The admixture of avirulent A. tumefiaciens strain IIBNV6 enhanced adherence of virulent A. tumefaciens strain B6 to Daturs cells. Octopine had no effect on the binding of virulent A. tumefaciens strain B6 to Datura cells, but 10 millimolar canavanine was inhibitory. Arginine enhanced the adherence of the bacteria at concentrations higher than 0.1 millimlar. Incubation with DNase, RNase, and lipase did not affect the binding, but protease stimulated the adherence of bacteria to Datura cells. Concanavaline A and soybean lectin had little effect whereas lecithin and lysolecithin enhanced binding slightly. Poly-L-lysine markedly stimulated the bacteria-plant cell adherence. Cells from suspension cultures of pea, vetch, and soybean had a 2to 3-fold higher binding capacity than Datura cells, whereas cells from wheat, corn, rice, and sorghum had a considerably lower affinity for binding with virulent A. tumefaciens strain B6. Bacterial adherence to plant cells was confirmed by autoradiography and electron microscopy. Autoradiwww.plant.org on April 4, 2016 -Published by www.plantphysiol.org Downloaded from
The plasma membrane has a number of important functions in plant cells including the regulation o... more The plasma membrane has a number of important functions in plant cells including the regulation of ion and metabolite transport, hormone transport and action, host pathogen interactions and cell wall biosynthesis and deposition.
... of a coated vesicle-enriched fraction from plant cells BRENT G. MERSEY,13 LARRY C. FOWKE,2 FR... more ... of a coated vesicle-enriched fraction from plant cells BRENT G. MERSEY,13 LARRY C. FOWKE,2 FRED CONSTABEL3 and ELDON H. NEW ... of negatively-stained coated vesicles isolated from suspension-cultured to-bacco cells by a modification of the method of Keen et al. ...
Haploid, diploid and tetraploid lines of Brassica rapa L. (syn. campestris), and allotetraploid B... more Haploid, diploid and tetraploid lines of Brassica rapa L. (syn. campestris), and allotetraploid B. napus L., were examined to determine the influence of ploidy on floral features, particularly nectary morphology and anatomy, and to relate nectary structure to nectar production capacity. Except for haploids, all lines were rapid-cycling. Average flower dry weight, and petal length and width, were in the descending order B. napus B. rapa (4n) 2n n. Pollen grains of 4n plants were larger than those of 2n plants ; haploids lacked pollen.
ABSTRACT Protoplasts from will carrot (Daucus carota) cell cultures were fused with protoplasts o... more ABSTRACT Protoplasts from will carrot (Daucus carota) cell cultures were fused with protoplasts of the filamentous green alga Stigeoclonium sp. using polyethylene glycol. Following fusion of the algal and higher plant plasma membranes, intact Stigeoclonium chloroplasts, nuclei, and centrioles were observed within the carrot cytoplasm. Other algal organelles were not distinguishable in the fusion products.
The plant CDK inhibitor ICK1 was identi®ed previously from Arabidopis thaliana with its inhibitor... more The plant CDK inhibitor ICK1 was identi®ed previously from Arabidopis thaliana with its inhibitory activity characterized in vitro. ICK1 displayed several structural and functional features that are distinct from known animal CDK inhibitors. Despite the initial characterization, there is no information on the functions of any plant CDK inhibitor in plants. To gain insight into ICK1 functions in vivo and the role of cell division during plant growth and development, transgenic plants were generated expressing ICK1 driven by the cauli¯ower mosaic virus 35S promoter. In comparison to control plants, growth was signi®cantly inhibited in transgenic 35S-ICK1 plants, with some plants weighing <10% of wild-type plants at the 3 week stage. Most organs of 35S-ICK1 plants were smaller. There were also modi®cations in plant morphology such as shape and serration of leaves and petals. The changes were so drastic that 35S-ICK1 plants with strong phenotype no longer resembled wild-type plants morphologically. Analyses showed that increased ICK1 expression resulted in reduced CDK activity and reduced the number of cells in these plants. Cells in 35S-ICK1 plants were larger than corresponding cells in control plants. These results demonstrate that ICK1 acts as a CDK inhibitor in the plant, and the inhibition of cell division by ICK1 expression has profound effects on plant growth and development. They also suggest that alterations of plant organ shape can be achieved by restriction of cell division.
Variation in seasonal sprouting pattern from roots and rhizomes of perennial herbaceous plants in... more Variation in seasonal sprouting pattern from roots and rhizomes of perennial herbaceous plants influence the success of plant proliferation ability, invasiveness and escape from weed control measures. The latter often rely on methods, which repeatedly fragment the underground system, thereby trigger adventitious and axillary buds to sprout, and consequently reduce the amount of stored energy. If carried out at times when no re-growth occurs, treatments will have little effect on weed populations, but cost much in terms of labour and energy.
The cell division cycle in plants as in other eukaryotes is controlled by the cyclin-dependent ki... more The cell division cycle in plants as in other eukaryotes is controlled by the cyclin-dependent kinase (CDK). This CDK paradigm determines that developmental cues and environmental signals need to impinge on the CDK complex to affect the cell cycle. An important part of understanding cell cycle regulation is to understand how CDK is regulated by various factors. In addition, there are features that set the cell cycle regulation in plants apart from that in other eukaryotes such as animals. Our knowledge of the molecular mechanisms that underlie the differences is poor. A family of plant CDK inhibitor proteins has been identified. The plant CDK inhibitors share similarity with a family of animal CDK inhibitors in a small region, while most of the sequence and the structural layout of the plant CDK inhibitors are different from the animal counterparts. Studies of plant CDK inhibitors have been performed mostly with the CDK inhibitors from Arabidopsis called ICKs (also referred to as KRPs). ICKs interact with D-type cyclins and A-type CDK. Overexpression of ICKs has been shown to affect cell division, plant growth, and morphogenesis. Studies of ICKs have also provided insightful information on the control of endoreduplication in plants. These aspects as well as cellular localization and protein regulation of ICKs are reviewed.
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