Academic Staff - Associate Professor Jan Marc

Key Words

• Plant cell biology
• Plant development
• Environmental stress signalling
• Signal transduction networks
• Phospholipases
• Cytoskeleton
• Microtubules
• Actin microfilaments

Research interests

Plants are continuously exposed to a variety of environmental signals, both above ground and below. Being sessile, plants have evolved complex cellular and molecular mechanisms that allow them to continuously exploit beneficial signals and adjust to adverse signals in order to optimize their growth and development. The signalling mechanisms generally involve series of interacting molecules that operate in downstream cascades, starting with the interception of signals at cell surface, eliciting metabolic adjustments in the cytoplasm, inducing cytoskeletal remodeling, and regulating gene expression. Research in my laboratory aims at elucidating this mechanism.

Discovery of a plasma membrane–phospholipase–microtubule link

Initially we identified, in collaboration with colleagues at Pennsylvania State University, a 90-kDa protein from tobacco BY-2 membrane fraction that interacts with microtubules (Marc et al. 1996). Immunocytochemistry showed that the protein binds and decorates microtubules in vitro and colocalizes with cortical microtubules in situ. By screening an Arabidopsis cDNA expression library with a monoclonal antibody against the tobacco protein, we subsequently identified a clone encoding a unique phospholipase D (PLD) isoform δ (AtPLDδ) (Gardiner et al. 2001). We cloned and sequenced the gene and characterized the protein. An immunoprecipitate of tobacco BY-2 proteins obtained with the same antibody displayed PLD hydrolytic activity, and PLD activity was dramatically enhanced by tubulin-affinity chromatography of detergent-solubilized membrane proteins, confirming that the PLD-tubulin interaction is functional. Because PLD is a key enzyme in signal transduction pathways, the PLDδ connection represents a “smart” link capable of conveying hormonal and environmental signals to the microtubule cytoskeleton in the cell cortex and then further downstream to the cell interior. This discovery was published in the Plant Cell and attracted a special cover article in Science (Munnik and Musgrave 2001, Science 111:42). It has been acknowledged by Thomson Scientific as belonging to “the top 1% within its field internationally”. This is a major conceptual advance that unifies two established research specializations, opening the door to a new, exciting area of plant biology.

Role of PLD-cytoskeleton interactions in signalling

The PLD family of enzymes hydrolyzes structural membrane phospholipids, generating phosphatidic acid (PA) as a second messenger. PA modulates various cellular activities such as alterations in membrane structure, vesicular trafficking, transcriptional regulation, protein folding and phosphorylation. Individual isoforms of the Arabidopsis PLD family, which have been classified into AtPLDα1,2,3, β1,2, γ1,2,3, δ, ε, and ζ1,2, respond to specific signals and produce specific effects. PLD activity has been related to responses to drought, salinity, cold/freezing, oxidative stress, bacterial and fungal elicitors, nodulation and wounding. The cytoskeleton in interphase cells is organized into highly ordered arrays in the cell cortex next to the plasma membrane, a strategic location for the interception of hormonal and environmental signals. Like PLD, cortical microtubules and actin microfilaments typically respond to environmental stress signals by rapid depolymerization and rearrangement, acting as targets as well as transducers of signals. We have shown that the PLD antagonist, n-butanol, does indeed disrupt microtubule organization and inhibits root elongation in Arabidopsis (Gardiner et al. 2003). Exactly how the activities of individual PLD isoforms and cytoskeletal components are linked and orchestrated is unknown.

Role of PLD in developmental reorientation of cortical microtubule arrays

We developed a highly effective cell model for studies of microtubule organization in living cells using the epidermal cell layer of elongating leaves in leek (Allium porrum) and transient transformation with GFP-MBD microtubule-reporter gene (Marc et al. 1998). A former Honours student, Frank Sainsbury, who since graduated with PhD from the prestigious John Innes Institute for plant science in Britain, used this model to elucidate the mechanistic principle of reorganization of transverse microtubules to longitudinal directions in elongating cells (Sainsbury et al. 2008). Apparently the mechanism involves partial detachment of microtubules from the plasma membrane followed by reorientation of their free ends by the forces of longitudinal cytoplasmic streaming, providing an impetus for microtubule dynamics in the new direction. Interestingly, the reorientation is promoted by applying the PLD antagonist n-butanol, consistent with disruptions of microtubule arrays in response to stress signals.

Applying RNA interference to explore the role of PLD isoforms in microtubule organization and responses to stress signals

A PhD student, Zoe Andreeva, has been working with synthetic siRNAs designed to specifically target individual Arabidopsis PLD isoforms. We evaluated the efficiency and specificity of individual siRNAs using co-transformation of Allium epidermal cells together with GFP-PLD isoform-specific reporter genes, and then used co-transformation with the GFP-MBD microtubule-reporter gene to examine the effect on microtubule organization. The individual knockdowns affected microtubule organization in distinct ways that apparently correspond to the phylogeny of the isoforms according to Allium sequencing data prepared by our colleagues at the Czech Academy of Science in Prague. Strikingly, silencing the isoform AtPLDδ was unique in promoting re-orientation of microtubules to longitudinal directions, suggesting this isoform normally supports anchoring of microtubules in the cell cortex (Andreeva et al. 2009). Subsequently Zoe prepared short hairpin RNA mutants of individual PLD isoforms in Arabidopsis to study their specific roles in cytoskeletal organization and in responses to osmotic stress, abscisic acid (ABA), and the fungal elicitor xylanase.

Proteomic analysis of protein complexes interacting with AtPLDδ

The isoform AtPLDδ is of particular interest since it associates almost exclusively with the plasma membrane, its expression is rapidly upregulated in response to drought, and its hydrolytic product, PA, has been implicated in ABA-mediated responses. Our aim is to elucidate how environmental stress signals initiate protective responses that establish tolerance to drought, salinity, and pathogen attack. Progress in this area requires the use of innovative approaches and technologies. A former PhD student, Angela Ho, used affinity purification and Arabidopsis cell culture transformed with GFP-AtPLDδ chimeric gene as a bait to pull-down interacting proteins, followed by their identification using mass spectrometry. This strategy allowed us to identify a set of proteins that bind either directly or indirectly to AtPLDδ, with implications for fundamental cellular processes such as cytoskeletal rearrangements, vesicular traffic, and cell division (Ho et al. 2009). Ongoing research involves biochemical analysis of predicted actin- and tubulin-binding domains of AtPLDδ, and construction of deletion mutants followed by transformation into Arabidopsis cell suspension to define plasma membrane-binding domains.

Cytoskeletal interactions with phospholipases A (PLA) and C (PLC)

In addition to PLD, we have also discovered cytoskeletal interactions with other members of the phospholipase superfamily. A former PhD student and ARC post-doctoral fellow, John Gardiner, and colleagues have discovered that another phospholipase, PLA2, also interacts with the microtubule cytoskeleton (Gardiner et al. 2008). Similarly, a group of third-year students in Plant Growth and Development performed initial experiments that led to the discovery that phospholipase C likewise interacts with the microtubule and actin microfilaments and also plays a role in shoot and root gravitropism (Andreeva et al. manuscript in revision).

Our research in phospholipase-cytoskeletal interactions is at the cutting edge of contemporary plant biology, as documented by high citation rates of our publications in leading international journals. A student working on Talented Student Program in my lab, David Seung, has won a prestigious award from the American Society of Plant biologist for his research project on the role of ABA in PLD-cytoskeleton interactions. The award includes travel support for presenting his work at the international Plant Biology meeting in Montreál, Canada, 2010. Honours projects on various aspects of our research in this emerging research area are available (see separate listing).

Research Output

• Andreeva Z, Barton D, Armour WJ, Li MY, Liao L-F, McKellar HL, Pethybridge KA, Marc J (2010) Inhibition of phospholipase C disrupts cytoskeletal organization and gravitropic growth in Arabidopsis roots. Planta (in press)
• Gardiner J, Marc J (2010) Arabidopsis thaliana, a model plant for the neuronal microtubule cytoskeleton? Journal of Experimental Botany (in press)
• Gardiner J, Marc J (2010) Multiple roles of phospholipases in the organisation of cortical microtubules and cellulose microfibrils in plant cells. Journal of Experimental Botany (in press)
• Gardiner J, Marc J, Overall R (2010) PROPSEARCH algorithm reveals putative Arabidopsis coiled-coil cytoskeletal protein homologues. Protoplasma (in press)
• Gardiner J, Overall R, Marc J (2010) The fractal nature of the brain: EEG data suggest that the brain functions as a quantum computer in 5-8 dimensions. Neuroquantology 8, 137-141
• Gardiner J, Marc J (2010) Disruption of normal cytoskeletal dynamics may play a key role in the pathogenesis of epilepsis. Neuroscientist 16, 28-39
• Andreeva Z, Ho AYY, Barthet MM, Potocký M, Bezvoda R, Žárský V, Marc J (2009) Phospholipase D family interactions with the cytoskeleton: isoform δ promotes plasma membrane anchoring of cortical microtubules. Functional Plant Biology 36, 600-612
• Barton D, Braet F, Marc J, Overall R, Gardiner J (2009) ELP3 localises to mitochondria and actin-rich domains at edges of HeLa cells. Neuroscience Letters 455, 60-64
• Shen E, Lei Y, Liu Q, Zheng Y, Song C, Marc J, Wang Y, Sun L, Liang Q (2009) Identification and characterization of INMAP, a novel interphase nucleus and mitotic apparatus protein that is involved in spindle formation and cell cycle progression. Experimental Cell Research 315, 1100-1116
• Ho AYY, Day DD, Brown MH, Marc J (2009) Arabidopsis phospholipase Dδ as an initiator of cytoskeleton-mediated signalling to fundamental cellular processes. Functional Plant Biology 36, 190-198
• Gardiner J, Barton D, Overall O, Marc J (2009) Neurotrophic support and oxidative stress: converging effects in the normal and diseased nervous system. Neuroscientist 15, 47-61
• Sainsbury F, Collings DA, Mackun K, Gardiner J, Harper JDI, Marc J (2008) Developmental reorientation of transverse cortical microtubules to longitudinal directions: a role for actomyosin-based streaming and partial microtubule-membrane detachments. Plant Journal 56, 116-131
• Gardiner J, Overall R, Marc J (2008) Do salivary neurotrophic factors provide neurotrophic support to neurons of the central and peripheral nervous systems including nerves innervating papillae on the tongue? Bioscience 1, 251-254
• Gardiner J, Andreeva Z, Barton D, Ritchie R, Overall R, Marc J (2008) Phospholipase A2 inhibitor, aristolochic acid, disrupts cortical microtubule arrays and root growth in Arabidopsis. Plant Biology 10, 725-731
• Gardiner J, McGee P, Overall, Marc J (2008) Are histones, tubulin, and actin derived from a common ancestral protein? Protoplasma 233, 1-5
• Wei Y, Shen E, Zhao N, Liu Q, Fan J, Marc J, Wang Y, Sun L, Liang Q (2008) Identification of a novel centrosomal protein CrpF46 involved in cell cycle progression and mitosis. Experimental Cell Research 314, 1693-1717
• Gardiner, Marc J, Hall D, Overall R (2008) Defects in tongue papillae and taste sensation indicate a problem with neurotrophic support in various neurological diseases. Neuroscientist 14, 240-250
• Gardiner J, Marc J, Overall R (2008) Cytoskeletal thermal ratchets and cytoskeletal tensegrity: determinants of brain asymmetry and symmetry? Frontiers in Bioscience 1, 4649-4656
• Gardiner J, Barton D, Marc J, Overall R (2007) Potential role of tubulin acetylation and microtubule-based protein trafficking in familial dysautonomia. Traffic 8,1145-1149
• Gardiner J, Collings DA, Harper JDI, Marc J (2003) The effect of the phospholipase D-antagonist 1-butanol on seedling development and microtubule organisation in Arabidopsis. Plant and Cell Physiology 44, 687-696
• Gardiner J, Marc J (2003) Putative microtubule-associated proteins from the Arabidopsis genome. Protoplasma 222, 61-74
• Harper JDI, Weerakoon ND, Gardiner JC, Blackman LM, Marc J (2002) A 75-kDa plant protein isolated by tubulin-affinity chromatography is a peroxisomal matrix enzyme. Canadian Journal of Botany 80, 1018-1027
• Collings DA, Harper JDI, Marc J, Overall RL, Mullen RT (2002) Life in the fast lane: actin-based motility of plant peroxisomes. Canadian Journal of Botany 80, 430-441
• Gardiner JC, Harper, JDI, Weerakoon ND, Collings DA, Ritchie S, Gilroy S, Cyr RJ, Marc J (2001) A 90-kD phospholipase D from tobacco binds to microtubules and the plasma membrane. Plant Cell 13, 2143-2158
• Dibbayawan TP, Harper JDI, Marc J (2001) A γ-tubulin antibody against a plant peptide sequence localises to cell division-specific microtubule arrays and organelles in plants. Micron 32, 671-678
• Harper JDI, Overall RL, Fowke L, Marc J (2000) A centrin homologue is localised across the developing cell plate in gymnosperms and angiosperms. Protoplasma 211, 207-216
• Marc J (1999) Dynamic reorganization of endoplasmic reticulum along actin filaments and microtubules. Biologia 54 (S7), 9-10
• Marc J, Granger CL, Brincat J, Fisher D, Kao TH, McCubbin AG, Cyr R.J (1998) A GFP::MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells. Plant Cell 10, 1927-1940
• Marc J (1997) Microtubule-organizing centers in plants. Trends in Plant Sciences 2, 223-230
• Marc J, Sharkey DE, Durso NA, Zhang M, Cyr RJ (1996) Isolation of a 90-kD microtubule-associated protein from tobacco membranes. Plant Cell 8, 2127-2138
• Dibbayawan TP, Harper JDI, Elliott JE, Gunning BES, Marc J (1995) A γ-tubulin that associates specifically with centrioles in HeLa cells and the basal body complex in Chlamydomonas. Cell Biology International 19, 559-567
• Liu B, Marc J, Joshi HC, Palevitz BA (1993) A γ-tubulin related protein associated with the microtubule arrays of higher plants in a cell cycle dependent manner. Journal of Cell Science 104, 1217-1228
• Marc J, Hackett WP (1992) Changes in the pattern of cell arrangement at the surface of the shoot apical meristem in Hedera helix L. following gibberellin treatment. Planta 186, 503-510
• Marc J, Hackett WP (1991) Gibberellin-induced reorganization of spatial relationships of emerging leaf primordia at the shoot apical meristem of Hedera helix. Planta 185, 171-178
• Mineyuki Y, Marc J, Palevitz BA (1991) Relationship between the preprophase band, nucleus, and spindle in dividing Allium cotyledon cells. Journal of Plant Physiology 138, 640-649
• Hasezawa S, Marc J, Palevitz BA (1991) Microtubule reorganization during the cell cycle in synchronized BY-2 tobacco suspensions. Cell Motility and the Cytoskeleton 18, 94-106
• Marc J, Palevitz BA (1990) Regulation of the spatial order of cortical microtubules in developing guard cells of Allium. Planta 182, 626-634
• Marc J, Hackett WP (1989) A new method for immunofluorescent localization of microtubules in surface cell layers: application to the shoot apical meristem of Hedera. Protoplasma 148, 70-79
• Marc J, Mineyuki Y, Palevitz BA (1989a) The generation and consolidation of a radial array of cortical microtubules in developing guard cells of Allium cepa L. Planta 179, 516-529
• Marc J, Mineyuki Y, Palevitz BA (1989b) A planar microtubule-organizing zone in guard cells of Allium: experimental depolymerization and reassembly of microtubules. Planta 179, 530-540
• Mineyuki Y, Marc J, Palevitz BA (1989) Development of the preprophase band from random cytoplasmic microtubules in guard mother cells of Allium cepa L. Planta 178, 291-296
• Mineyuki Y, Marc J, Palevitz BA (1988) Formation of the oblique spindle in dividing guard mother cells of Allium. Protoplasma 147, 200-203
• Marc J, Gunning BES (1988) Monoclonal antibodies to a fern spermatozoid detect novel components of the mitotic and cytokinetic apparatus in higher plant cells. Protoplasma 142, 15-24
• Marc J, Gunning BES, Hardham AR, Perkin JL, Wick SM (1988) Monoclonal antibodies to surface and cytoskeletal components of the spermatozoid of Pteridium aquilinum. Protoplasma 142, 5-14
• Marc J, Gunning BES (1986) Immunofluorescent localization of cytoskeletal tubulin and actin during spermatogenesis in Pteridium aquilinum (L.) Kuhn. Protoplasma 134, 163-177
• Marc J, Gifford RM (1984) Floral initiation in wheat, sunflower, and sorghum under carbon dioxide enrichment. Canadian Journal of Botany 62, 9 14
• Marc J, Palmer JH (1984) Variation in cell cycle time and nuclear DNA content in the apical meristem of Helianthus annuus L. during the transition to flowering. American Journal of Botany 71, 588 595
• Marc J, Palmer JH (1982) Changes in mitotic activity and cell size in the apical meristem of Helianthus annuus L. during the transition to flowering. American Journal of Botany 69, 768 775
• Palmer JH, Marc J (1982) Wound induced initiation of involucral bracts and florets in the developing sunflower inflorescence. Plant and Cell Physiology 23, 1401 1409
• Marc J, Palmer JH (1981) Photoperiodic sensitivity of inflorescence initiation and development in sunflower. Field Crops Research 4, 155 164