Plant Development :: Publications :: Abstracts

Abstracts

Colour recovery after photoconversion of H2B::mEosFP allows detection of increased nuclear DNA content in developing plant cells. Plant Physiol. 2011 Nov 22. [Epub ahead of print]
Wozny M, Schattat MH, Mathur N, Barton K, Mathur J.

Many higher plants are polysomatic whereby different cells possess variable amounts of nuclear DNA. The conditional triggering of endocycles results in higher nuclear DNA content (C-value) which in some cases has been correlated to increased cell size. While numerous multi-colored fluorescent protein (FP) probes have revealed the general behaviour of the nucleus and intra-nuclear components, direct visualization and estimation of changes in nuclear-DNA content in live cells during their development has not been possible. Recently, monomeric EosFP (mEosFP) has emerged as a useful photoconvertible protein whose colour changes irreversibly from a green to a red fluorescent form upon exposure to violet-blue light. The stability and irreversibility of red fluorescent mEosFP suggests that detection of green colour recovery would be possible as fresh mEosFP is produced after photo-conversion. Thus a ratiometric evaluation of the red and green forms of mEosFP following photoconversion could be used to estimate production of a core histone such as H2B during its concomitant synthesis with DNA in the S phase of the cell cycle. Here we present proof of concept observations on transgenic tobacco BY2 cells and Arabidopsis plants stably expressing H2B::mEosFP. In Arabidopsis seedlings an increase in green fluorescence is observed specifically in cells known to undergo endo-reduplication. The detection of changes in nuclear DNA content by correlating colour recovery of H2B::mEosFP after photoconversion is a novel approach involving a single fluorescent protein. The method has potential for facilitating detailed investigations on conditions that favor increased cell size and the development of polysomaty in plants.

Correlated behaviour implicates stromules in increasing the interactive surface between plastids and ER tubules. Plant Signaling and Behaviour. 2011 6(5).
Martin H. Schattat, Kiah Barton and Jaideep Mathur

Stromules are extended by plastids but the underlying basis for their extension and retraction had not been understood until recently. Our live-imaging aided observations on coincident plastid stromule branching and ER tubule dynamics open out new areas of investigation relating to these rapid subcellular interactions. The addendum provides a testable hypothesis on the formation of stromules, which argues against the need for new membrane incorporation and suggests that stromal extensions might result from a remodeling of the plastid envelope membrane in an ER aided manner.

Plastid stromule branching coincides with contiguous ER dynamics. Plant Physiology. 2011; 155(4):1667-1677.
Martin H. Schattat, Kiah Barton, Bianca Baudisch, Ralf B. Klossgen and Jaideep Mathur

Stromules are stroma-filled tubules extending from plastids whose rapid extension towards or retraction from other plastids has suggested a role in inter-plastidic communication and exchange of metabolites. Several studies point to sporadic dilations, kinks and branches occurring along stromule length but have not elucidated the underlying basis for these occurrences. Similarly, although specific details on interacting partners have been missing a consensus viewpoint suggests that stromules increase the interactive surface of a plastid with its cytoplasmic surroundings. Here, using live imaging we show that the behaviour of dynamic, pleomorphic stromules strongly coincides with that of cortical endoplasmic reticulum tubules. Co-visualization of fluorescent protein-highlighted stromules and the ER in diverse cell types clearly suggests correlative dynamics of the two membrane-bound compartments. The extension and retraction, as well as directional changes in stromule branches occur in tandem with the behaviour of neighbouring ER tubules. 3D and 4D volume rendering reveals that stromules that extend into cortical regions occupy channels between ER tubules possibly through multiple membrane contact sites. Our observations clearly depict coincidental stromule-ER behaviour and suggest that either the neighbouring ER tubules shape stromules directly or the behaviour of both ER and stromules is simultaneously dictated by a shared cytoskeleton-based mechanism. These new observations strongly implicate the ER membrane in interactions with stromules and suggest that their interacting surfaces might serve as major conduits for bidirectional exchange of ions, lipids and metabolites between the two organelles.

mEosFP based green to red photoconvertible subcellular probes for plants. Plant Physiology. 2010;154(4):1573 - 1587.
Jaideep Mathur, Resmi Radhamony, Alison M. Sinclair, Ana Donoso, Natalie Dunn, Elyse Roach, Devon Radford, S. Mohammad P. Mohaghegh, David C. Logan, Ksenija Kokolic and Neeta Mathur

Photoconvertible fluorescent proteins (FPs) are recent additions to the biologists' toolbox for understanding the living cell. Like GFP, monomeric EosFP is bright green in colour but is efficiently photo-converted into a red fluorescent form using a mild violet-blue excitation. Here we report mEosFP-based probes that localize to the cytosol, plasma-membrane invaginations, endosomes, pre-vacuolar vesicles, vacuoles, the endoplasmic reticulum, Golgi bodies, mitochondria, peroxisomes and the two major cytoskeletal elements, F-actin and cortical microtubules. The mEosFP fusion proteins are smaller than GFP / RFP based probes and as demonstrated here provide several significant advantages for imaging of living plant cells. These include an ability to differentially colour label a single cell or a group of cells in a developing organ; selectively highlight a region of a cell or a sub-population of organelles and vesicles within a cell for tracking them and understanding spatiotemporal aspects of interactions between similar as well as different organelles. In addition, mEosFP probes introduce a milder alternative to FRAP whereby instead of photo bleaching, photoconversion followed by recovery of green fluorescence can be used for estimating subcellular dynamics. Most importantly the two fluorescent forms of mEosFP furnish bright internal controls during imaging experiments and are fully compatible with CFP, GFP, YFP and RFP fluorochromes for use in simultaneous, multi-colour labeling schemes. Photoconvertible mEosFP-based subcellular probes promise to usher in a much higher degree of precision into live imaging of plant cells than has been possible so far using single coloured FPs.

Ethylene receptor ETR2 controls trichome branching by regulating microtubule assembly in Arabidopsis thaliana. J Exp Bot. 2009;60(13):3923-3933.
Plett JM, Mathur J, Regan S.

The single-celled trichome of Arabidopsis thaliana is a widely used model system for studying cell development. While the pathways that control the later stages of trichome development are well characterized, the early signalling events that co-ordinate these pathways are less well understood. Hormones such as gibberellic acid, salicylic acid, cytokinins, and ethylene are known to affect trichome initiation and development. To understand the role of the plant hormone ethylene in trichome development, an Arabidopsis loss-of-function ethylene receptor mutant, etr2-3, which has completely unbranched trichomes, is analysed in this study. It was hypothesized that ETR2 might affect the assembly of the microtubule cytoskeleton based on analysis of the cytoskeleton in developing trichomes, and exposures to paclitaxol and oryzalin, which respectively act either to stabilize or depolymerize the cytoskeleton. Through epistatic and gene expression analyses it is shown that ETR2 is positioned upstream of CHROMATIN ASSEMBLY FACTOR1 and TRYPTICHON and is independent of the GLABRA2 and GLABRA3 pathways. These results help extend understanding of the early events that control trichome development and identify a signalling pathway through which ethylene affects trichome branching.

Rapid peroxisomal responses to ROS suggest an alternative mechanistic model for post-biogenesis peroxisomal life cycle in plants. Plant Signaling and Behaviour. Volume 4, Issue 8. August 2009.
J Mathur.

Plants adapt to and survive in some of the harshest environments. Their success can be ascribed to an ability to maintain an optimal subcellular redox environment. Peroxisomes, ubiquitous ROS producing and scavenging organelles in eukaryotes play an important role in cellular homeostasis. Recently the formation of thin membrane extensions called peroxules has provided further evidence for peroxisomal role in rapidly sensing and responding to alterations in subcellular ROS. Within a cell the transient extension and retraction of peroxules is asynchronous but takes place within seconds. Peroxules follow tracks defined by tubules of the endoplasmic reticulum and their formation does not appear to involve an elaborate transcriptional-translational machinery. Rather the rapidity of peroxisomal responses suggests ROS instigated membrane modifications aimed at local ROS scavenging or leading to peroxisome elongation prior to their fission for increasing peroxisome numbers within a cell. A model on post-biogenesis peroxisomal life-cycle taking cognizance of rapid peroxisomal responses is presented.

Peroxule extension over ER defined paths constitutes a rapid subcellular response to hydroxyl stress. Plant J. 2009: 59: 231 -242
Sinclair AM, Trobacher CP, Mathur N, Greenwood JS, Mathur J.

Plants survive against myriad environmental odds while remaining rooted to a spot. The time scale in which plant cells can respond to environmental cues is seldom appreciated. Fluorescent protein aided live-imaging of peroxisomes reveals that they respond within seconds of exposure to hydrogen peroxide and hydroxyl radicals by producing dynamic extensions called peroxules. Observations of the Arabidopsis flu mutant and treatments with xenobiotics eliciting singlet oxygen and superoxide reactive oxygen species suggest that the observed responses are specific for hydroxyl radicals. Prolonged exposure to hydroxyl radicals inhibits peroxule extension and instead causes motile and spherical peroxisomes in a cell to become immotile and elongate several folds. Expression of a photo-convertible EosFP-PTS1 demonstrates that vermiform peroxisomes result from rapid stretching of individual peroxisomes while the subsequent 'beads-on-a-string' morphology results from differential protein distribution within an elongated tubule. Over time the beads in elongated peroxisomes also extend peroxules randomly before undergoing asynchronous, asymmetrical fission. Peroxule extension does not appear to involve cytoskeletal elements directly but is closely aligned with and reflects the dynamics of ER tubules. Peroxisomal responses reveal a rapidly invoked sub-cellular machinery involved in recognition of hydroxyl stress thresholds and its possible remediation locally through extension of peroxules or globally by increasing peroxisome numbers. A matrix protein retro-flow mechanism that supports peroxisome-ER connectivity in plant cells is suggested.

Visualizing the actin cytoskeleton in living plant cells using a photo-convertible mEos::FABD-mTn fluorescent fusion protein. Plant Methods, 4:21.19th September. 2008. doi:10.1186/1746-4811-4-21.
Schenkel, M, Sinclair, A.M., Johnstone, D., Bewley, J.D. and Mathur J.

The actin cytoskeleton responds quickly to diverse stimuli and plays numerous roles in cellular signalling, organelle motility and subcellular compartmentation during plant growth and development. Molecular and cell biological tools that can facilitate visualization of actin organization and dynamics in a minimally invasive manner are essential for understanding this fundamental component of the living cell.
A novel, monomeric (m) Eos-fluorescent protein derived from the coral Lobophyllia hemprichii was assessed for its green to red photo-convertibility in plant cells by creating mEosFP-cytosolic. mEosFP was fused to the F-(filamentous)-Actin Binding Domain of the mammalian Talin gene to create mEosFP::FABDmTalin. Photo-conversion, visualization and colour quantification protocols were developed for EosFP targeted to the F-actin cytoskeleton. Rapid photo-conversion in the entire cell or in a region of interest was easily achieved upon illumination with an approximately 400 nm wavelength light beam using an epi-fluorescent microscope. Dual color imaging after photo-conversion was carried out using a confocal laser-scanning microscope. Time-lapse imaging revealed that although photo-conversion of single mEosFP molecules can be rapid in terms of live-cell imaging it involves a progressive enrichment of red fluorescent molecules over green species. The fluorescence of photo-converted cells thus progresses through intermediate shades ranging from green to red. The time taken for complete conversion to red fluorescence depends on protein expression level within a cell and the quality of the focusing lens used to deliver the illuminating beam. Three easily applicable methods for obtaining information on fluorescent intensity and colour are provided as a means of ensuring experimental repeatability and data quantification, when using mEosFP and similar photo-convertible proteins.
The mEosFP::FABD-mTn probe retains all the imaging qualities associated with the well tested GFP::mTn probe while allowing for non-invasive, regional photo-conversion that allows colour based discrimination within a living cell. Whereas a number of precautions should be exercised in dealing with photo-convertible probes, mEosFP::FABD-mTn is a versatile live imaging tool for dissecting the organization and activity of the actin cytoskeleton in plants..

The illuminated plant cell. Trends Plant Sci. 12(11):506-513.2007.
Mathur J.

The past decade has provided biologists with a palette of genetically encoded, multicolored fluorescent proteins. The living plant cell turned into a 'coloring book' and today, nearly every text-book organelle has been highlighted in scintillating fluorescent colors. This review provides a concise listing of the earliest representative fluorescent-protein probes used to highlight various targets within the plant cell, and introduces the idea of using the numerous multicolor, subcellular probes for the development of an early intracellular response profile of plants.

Illuminating sub-cellular structures and dynamics in plants: a fluorescent protein toolbox. Can. J, Bot. 84:515-522.2006.
Preetinder K. Dhanoa, Alison Sinclair, Robert T. Mullen and Jaideep Mathur.

The discovery and development of multi-coloured fluorescent proteins has led to the exciting possibility to observe a remarkable array of sub-cellular structures and dynamics in living cells. This mini-review highlights a number of the more common fluorescent protein probes in plants and is a testimonial to the fact that the plant cell has not lagged behind during the live-imaging revolution and is ready for even more in-depth exploration.

Trichome Cell Morphogenesis in Arabidopsis: a continuum of cellular decisions. Can. J, Bot. 84:604-612.2006.
Jaideep Mathur.

In keeping with the myriad functions carried out by plants, their component cells display an amazing diversity of shapes and sizes. How is a precise cell form achieved? In recent years the single celled, branched, aerial epidermal trichome of Arabidopsis thaliana has emerged as a model cell for understanding the cell biological and molecular basis underlying the development of cell shape in plants. Here I critique the recent information gleaned from dissecting trichome cell morphogenesis in Arabidopsis and identify areas and questions that can be further addressed using this unique cell type.

Local interaactions shape plant cells. Curr Opin Cell Biol. 18:40–46 2006
Jaideep Mathur.

Plant cell expansion is usually attributed to the considerable osmotic pressure that develops within and impinges upon the cell boundary. Whereas turgor containment within expandable walls explains global expansion, the scalar nature of turgor does not directly suggest a mechanism for achieving the localized, differential growth that is responsible for the diversity of plant-cell forms. The key to achieving local growth in plant cells appears to lie not in harnessing turgor but in using it to identify weak regions in the cell boundary and thus creating discrete intracellular domains for targeting the growth machinery. Membrane-interacting phospholipases, Rho-like proteins and their interactors, an actin-modulating ARP2/3 complex with its upstream regulators, and actin-microtubule interactions play important roles in the intracellular cooperation to shape plant cells.

Actin-based motility of endosomes is linked to the polar tip-growth of root hairs. EUROPEAN J. CELL BIOLOGY. 84; 609–621 2005.
Boris Voigt, Antonius Timmers, Jozef Samaj, Andrej Hlavacka, Takashi Ueda, Mary Preuss, Erik Nielsen, Jaideep Mathur, Neil Emans, Harald Stenmark, Akihiko Nakano, Frantisek Baluska and Diedrik Menzel.

Plant tip growth has been recognized as an actin-based cellular process requiring targeted exocytosis and compensatory endocytosis to occur at the growth cone. However, the identity of subcellular compartments involved in polarized membrane trafficking pathways remains enigmatic in plants. Here we characterize endosomal compartments in tip-growing root hair cells. We demonstrate their presence at the growing tip and differential distribution upon cessation of tip growth. We also show that both the presence of endosomes as well as their rapid movements within the tip region depends on an intact actin cytoskeleton and involves actin polymerization. In conclusion, actin-propelled endosomal motility is tightly linked to the polar tip growth of root hairs.

Microtubule plus-ends reveal essential links between intracellular polarization and localized modulation of endocytosis during division-plane establishment in plant cells. BMC Biol. 2005 Apr 14;3(1):11
Dhonukshe P, Mathur J, Hulskamp M, Gadella T Jr

BACKGROUND: A key event in plant morphogenesis is the establishment of a division plane. A plant-specific microtubular preprophase band (PPB) accurately predicts the line of cell division, whereas the phragmoplast, another plant-specific array, executes cell division by maintaining this predicted line. Although establishment of these specific arrays apparently involves intracellular repolarization events that focus cellular resources to a division site, it still remains unclear how microtubules position the cell division planes. Here we study GFP-AtEB1 decorated microtubule plus-ends to dissect events at the division plane. RESULTS: Early mitotic events included guided growth of endoplasmic microtubules (EMTs) towards the PPB site and their coincident localization with endocytic vesicles. Consequently, an endosomal belt lay in close proximity to the microtubular PPB at its maturation and was maintained during spindle formation. During cytokinesis, EMTs radiated from the former spindle poles in a geometrical conformation correlating with cell-plate navigation and tilt-correction. Naphthylphtalamic acid (NPA), an inhibitor of polar auxin efflux, caused abnormal PPBs and shifted division planes. CONCLUSIONS: Our observations reveal a spatio-temporal link between microtubules and intracellular polarization essential for localized endocytosis and precise establishment of the division plane in plants. Additionally, they implicate the growth regulator, auxin, in this important cellular event.

Conservation of boundary extension mechanisms between plants and animals. J. CELL BIOLOGY. 2005.
Jaideep Mathur
Department of Plant Agriculture, University of Guelph, Guelph. ON. Canada N1G2W1

Locomotion clearly sets plants and animals apart. However, recent studies in higher plants reveal cell-biological and molecular features similar to those observed at the leading edge of animal cells and suggest conservation of boundary extension mechanisms between motile animal cells and non-motile plant cells.

The ARP2/3 complex: giving plant cells a leading edge. BIOESSAYS. 27(4). April 2005.
Jaideep Mathur
Department of Plant Agriculture, University of Guelph, Guelph. ON. Canada N1G2W1

The seven-subunit ARP2/3 complex is an efficient modulator of the actin cytoskeleton with well-recognized roles in amoeboid locomotion and sub-cellular motility of organelles and microbes. The recent identification of different subunit-homologs suggests the existence of a functional ARP2/3 complex in higher plants. Mutations in some of the subunits have revealed a pivotal role for the complex in determining the shape of walled cells and focused attention on the interlinked processes of cortical-actin organization, growth-site selection, organelle motility and actin-microtubule interactions during plant cell morphogenesis. The findings supporting a global conservation of molecular mechanisms for membrane protrusion have been further strengthened by the identification of plant homologs of upstream regulators of the complex such as PIR121, NAP125 and HSPC300. As discussed here, the recent studies suggest that there might be hitherto unappreciated molecular and cell-biological commonalities between protrusion-mediated motility of animal cells and polarized, expansion-mediated growth of plant cells.

Cell shape development in Plants. TRENDS IN PLANT SCIENCE. December 2004
Mathur J.
Department of Plant Agriculture, University of Guelph, Guelph. ON. Canada N1G2W1.

The shape of a plant cell has long been the corner stone for diverse areas of plant research but it is only recently that molecular–genetic and cell biological tools have been effectively combined for dissecting plant cell morphogenesis. Increased understanding of polar-growth characteristics for model cell types, availability of numerous morphological mutants and significant advances in fluorescent-protein aided live-cell visualization have provided the major impetus for these analyses. The cytoskeleton and its regulators have emerged as essential components of the scaffold involved in fabricating plant cell shape. This review assimilates information from recent discoveries to derive a simple cytoskeleton-based operational framework for plant cell morphogenesis.

Actin control over microtubules suggested by DISTORTED2 encoding the Arabidopsis ARPC2 subunit homolog. Plant Cell Physiol. 2004 Jul;45(7):813-22.
Saedler R, Mathur N, Srinivas BP, Kernebeck B, Hulskamp M, Mathur J.
Botanical Institute III, University of Koln, Gyrhofstrasse 13, D-50931 Koln, Germany.

In Arabidopsis, based on the randomly misshapen phenotype of leaf epidermal trichomes, eight genes have been grouped into a 'DISTORTED' class. Three of the DIS genes, WURM, DISTORTED1 and CROOKED have been cloned recently and encode the ARP2, ARP3 and ARPC5 subunits respectively, of a conserved actin modulating ARP2/3 complex. Here we identify a fourth gene, DISTORTED2 as the Arabidopsis homolog of the ARPC2 subunit of the ARP2/3 complex. Like other mutants in the complex dis2 trichomes also display supernumerary, randomly localized cortical actin patches. In addition dis2 trichomes possess abnormally clustered endoplasmic microtubules near sites of actin aggregation. Since microtubules are strongly implicated in the establishment and maintenance of growth directionality in higher plants our observations of aberrant microtubule clustering in dis2 trichomes suggests a convincing explanation for the randomly distorted trichome phenotype in dis mutants. In addition, the close proximity of microtubule clusters to the arbitrarily dispersed cortical actin patches in the dis mutants provides fresh insights into cytoskeletal interactions leading us to suggest that in higher plants microtubule arrangements directed towards the establishment and maintenance of polar growth-directionality are guided by cortical actin behavior and organization.

Plant cytoskeleton: reinforcing lines of division in plant cells. Curr Biol. 2004 Apr 6;14(7):R287-9.
Mathur J.
Department of Botany, University of Toronto, 25 Willcocks Street, Toronto, Ontario, M5S 3B2, Canada.

Cytokinesis in plants has unique features concerned with defining and maintaining the line of cell division. Recent studies have identified key cytoskeletal components and events that help to ensure the fidelity of cytokinesis in higher plants.

A novel localization pattern for an EB1-like protein links microtubule dynamics to endomembrane organization. Curr Biol. 2003 Nov 11;13(22):1991-7.
Mathur J, Mathur N, Kernebeck B, Srinivas BP, Hulskamp M.
Botanical Institute III, University of Koln, Gyrhofstrasse 15, D-50931 Koln, Germany. jaideep.mathur@utoronto.ca

A group of microtubule-associated proteins called +TIPs (plus end tracking proteins), including EB1 family proteins, label growing microtubule ends specifically in diverse organisms and are implicated in spindle dynamics, chromosome segregation, and directing microtubules toward cortical sites. Here, we report three new EB1-like proteins from Arabidopsis and provide the intracellular localization for AtEB1, which differs from all known EB1 proteins in having a very long acidic C-terminal tail. In marked contrast to other EB1 proteins, the GFP-AtEB1 fusion protein localizes not only to microtubule plus ends but also to motile, pleiomorphic tubulovesicular membrane networks that surround other organelles and frequently merge with the endoplasmic reticulum. AtEB1 behavior thus resembles that of +TIPs, such as the cytoplasmic linker protein CLIP-170, that are known to associate with and pull along membrane tubules in animal systems but for which homologs have not been identified in plants. In addition, though EB1 proteins are believed to stabilize microtubules, a different behavior is observed for AtEB1 where instead of stabilizing a microtubule it localizes to already stabilized regions on a microtubule. The dual localization pattern of AtEB1 suggests links between microtubule plus end dynamics and endomembrane organization during polarized growth of plant cells.

Arabidopsis CROOKED encodes for the smallest subunit of the ARP2/3 complex and controls cell shape by region specific fine F-actin formation. Development. 2003 Jul;130(14):3137-46.
Mathur J, Mathur N, Kirik V, Kernebeck B, Srinivas BP, Hulskamp M.
Botanical Institute III, University of Koln, Gyrhofstrasse 15, Koln, D-50931, Germany. jaideep.mathur@uni-koeln.de

The generation of a specific cell shape requires differential growth, whereby specific regions of the cell expand more relative to others. The Arabidopsis crooked mutant exhibits aberrant cell shapes that develop because of mis-directed expansion, especially during a rapid growth phase. GFP-aided visualization of the F-actin cytoskeleton and the behavior of subcellular organelles in different cell-types in crooked and wild-type Arabidopsis revealed that localized expansion is promoted in cellular regions with fine F-actin arrays but is restricted in areas that maintain dense F-actin. This suggested that a spatiotemporal distinction between fine versus dense F-actin in a growing cell could determine the final shape of the cell. CROOKED was molecularly identified as the plant homolog of ARPC5, the smallest sub-unit of the ARP2/3 complex that in other organisms is renowned for its role in creating dendritic arrays of fine F-actin. Rescue of crooked phenotype by the human ortholog provides the first molecular evidence for the presence and functional conservation of the complex in higher plants. Our cell-biological and molecular characterization of CROOKED suggests a general actin-based mechanism for regulating differential growth and generating cell shape diversity.

Mutations in actin-related proteins 2 and 3 affect cell shape development in Arabidopsis. Plant Cell. 2003 Jul;15(7):1632-45.
Mathur J, Mathur N, Kernebeck B, Hulskamp M.
Botanical Institute III, University of Koln, D 50931 Koln, Germany. jaideep.mathur@uni-koeln.de

ACTIN-RELATED PROTEINS 2 and 3 form the major subunits of the ARP2/3 complex, which is known as an important regulator of actin organization in diverse organisms. Here, we report that two genes, WURM and DISTORTED1, which are important for cell shape control in Arabidopsis, encode the plant ARP2 and ARP3 orthologs, respectively. Mutations in these genes result in misdirected expansion of various cell types: trichome expansion is randomized, pavement cells fail to produce lobes, hypocotyl cells curl out of the normal epidermal plane, and root hairs are sinuous. At the subcellular level, cell shape changes are linked to severe filamentous actin aggregation and compromised vacuole fusion. Because all seven subunits of the ARP2/3 complex are present in plants, our data indicate that this complex may play a pivotal role during plant cell morphogenesis.

Regulation of cell expansion by the DISTORTED genes in Arabidopsis thaliana: actin controls the spatial organization of microtubules. Mol Genet Genomics. 2003 Jun;269(3):350-60.
Schwab B, Mathur J, Saedler R, Schwarz H, Frey B, Scheidegger C, Hulskamp M.
Zentrum fur Molekularbiologie der Pflanzen, Institut fur Entwicklungsgenetik, Universitat Tubingen, 72070 Tubingen, Germany.

The control of the directionality of cell expansion was investigated using a class of eight genes, the so-called DISTORTED (DIS) genes, that are required for proper expansion of leaf trichomes in Arabidopsis thaliana. By tracing the separation of latex beads placed on the trichome surface, we demonstrate that trichomes grow by diffuse rather than tip growth, and that in dis mutants deviations from the normal orientation of growth can occur in all possible directions. We could not detect any differences in intracellular organization between wild-type and dis-group mutants by electron microscopy. The analysis of double mutants showed that although the expression of the dis phenotype is generally independent of branching and endoreduplication, dis mutations act synthetically in combination lesions in the ZWI gene, which encodes a kinesin motor protein. Using a MAP4:GFP marker line, we show that the organization of cortical microtubules is affected in dis-group mutants. The finding that most dis-group mutants have actin defects suggested to us that actin is involved in organizing the orientation of microtubules. By analyzing the microtubule organization in plants treated with drugs that bind to actin, we verified that actin is involved in the positioning of cortical microtubules and thereby in plant cell expansion.

The Arabidopsis STICHEL gene is a regulator of trichome branch number and encodes a novel protein. Plant Physiol. 2003 Feb;131(2):643-55.
Ilgenfritz H, Bouyer D, Schnittger A, Mathur J, Kirik V, Schwab B, Chua NH, Jurgens G, Hulskamp M.
Zentrum fur Molekularbiologie Pflanzen, Entwicklungsgenetik, Universitat Tubingen, Auf der Morgenstelle 1, D-72076 Tubingen, Germany.

Here, we analyze the STICHEL (STI) gene, which plays an important role in the regulation of branch number of the unicellular trichomes in Arabidopsis. We have isolated the STI locus by positional cloning and confirmed the identity by sequencing seven independent sti alleles. The STI gene encodes a protein of 1,218 amino acid residues containing a domain with sequence similarity to the ATP-binding eubacterial DNA-polymerase III gamma-subunits. Because endoreduplication was found to be normal in sti mutants the molecular function of STI in cell morphogenesis is not linked to DNA replication and, therefore, postulated to represent a novel pathway. Northern-blot analysis shows that STI is expressed in all organs suggesting that STI function is not trichome specific. The analysis of sti alleles and transgenic lines overexpressing STI suggests that STI regulates branching in a dosage-dependent manner.

Microtubules and microfilaments in cell morphogenesis in higher plants. Curr Biol. 2002 Oct 1;12(19):R669-76.
Mathur J, Hulskamp M.
Botanical Institute III, University of Koln, Gyrhofstrasse 15, 50931, Koln, Germany.

Microtubules and microfilaments play important roles in cell morphogenesis. The picture emerging from drug studies and molecular-genetic analyses of mutant higher plants defective in cell morphogenesis shows that the roles played by them remain the same in both tip-growing and diffuse-growing cells. Microtubules are important for establishing and maintaining growth polarity whereas actin microfilaments deliver the materials required for growth to specified sites. The recent cloning of several cell morphogenesis genes has revealed that conserved mechanisms as well as novel signal transduction pathways spatially organize the plant cytoskeleton.

Functional analysis of the tubulin-folding cofactor C in Arabidopsis thaliana. Curr Biol. 2002 Sep 3;12(17):1519-23.
Kirik V, Mathur J, Grini PE, Klinkhammer I, Adler K, Bechtold N, Herzog M, Bonneville JM, Hulskamp M.
University of Koln, Botanical Institute III, Gyrhofstr. 15, Germany.

The biogenesis of microtubules comprises several steps, including the correct folding of alpha- and beta-tubulin and heterodimer formation. In vitro studies and the genetic analysis in yeast revealed that, after translation, alpha- and beta-tubulin are processed by several chaperonins and microtubule-folding cofactors (TFCs) to produce assembly-competent alpha-/beta-tubulin heterodimers. One of the TFCs, TFC-C, does not exist in yeast, and a potential function of TFC-C is thus based only on the biochemical analysis. In this study and in a very recently published study by Steinborn and coworkers, the analysis of the Arabidopsis porcino (por) mutant has shown that TFC-C is important for microtubule function in vivo. The predicted POR protein shares weak amino acid similarity with the human TFC-C (hTFC-C). Our finding that hTFC-C under the control of the ubiquitously expressed 35S promoter can rescue the por mutant phenotype shows that the POR gene encodes the Arabidopsis ortholog of hTFC-C. The analysis of plants carrying a GFP:POR fusion construct showed that POR protein is localized in the cytoplasm and is not associated with microtubules. While, in por mutants, microtubule density was indistinguishable from wild-type, their organization was affected.

Signal transduction: Rho-like proteins in plants. Curr Biol. 2002 Aug 6;12(15):R526-8.
Mathur J, Hulskamp M.
University of Koln, Botanical Institute III, Gyrhofstr. 15, D-50931, Koln, Germany.

Plants lack the Rho and Rac/Cdc42 GTPases that are so important in diverse signal transduction processes in animals. A plant-specific group of Rho-like proteins - Rops - shows striking similarities to their animal relatives, but also exciting differences in their regulation and signal transduction.

The Arabidopsis TUBULIN-FOLDING COFACTOR A gene is involved in the control of the alpha/beta-tubulin monomer balance. Plant Cell. 2002 Sep;14(9):2265-76.
Kirik V, Grini PE, Mathur J, Klinkhammer I, Adler K, Bechtold N, Herzog M, Bonneville JM, Hulskamp M.
Botanical Institute III, University of Koln, Gyrhofstrasse 15, 50931 Koln, Germany.

The control of the stoichiometric balance of alpha- and beta-tubulin is important during microtubule biogenesis. This process involves several tubulin-folding cofactors (TFCs), of which only TFC A is not essential in mammalian in vitro systems or in vivo in yeast. Here, we show that the TFC A gene is important in vivo in plants. The Arabidopsis gene KIESEL (KIS) shows sequence similarity to the TFC A gene. Expression of the mouse TFC A gene under the control of the 35S promoter rescues the kis mutation, indicating that KIS is the Arabidopsis ortholog of TFC A. kis plants exhibit a range of defects similar to the phenotypes associated with impaired microtubule function: plants are reduced in size and show meiotic defects, cell division is impaired, and trichomes are bulged and less branched. Microtubule density was indistinguishable from that of the wild type, but microtubule organization was affected in trichomes and hypocotyl cells of dark-grown kis plants. The kis phenotype was rescued by overexpression of an alpha-tubulin, indicating that KIS is involved in the control of the correct balance of alpha- and beta-tubulin monomers.

Simultaneous visualization of peroxisomes and cytoskeletal elements reveals actin and not microtubule-based peroxisome motility in plants. Plant Physiol. 2002 Mar;128(3):1031-45.
Mathur J, Mathur N, Hulskamp M.
Botanical Institute III, University of Koln, Gyrhofstrase 15, 50931 Cologne, Germany.

Peroxisomes were visualized in living plant cells using a yellow fluorescent protein tagged with a peroxisomal targeting signal consisting of the SKL motif. Simultaneous visualization of peroxisomes and microfilaments/microtubules was accomplished in onion (Allium cepa) epidermal cells transiently expressing the yellow fluorescent protein-peroxi construct, a green fluorescent protein-mTalin construct that labels filamentous-actin filaments, and a green fluorescent protein-microtubule-binding domain construct that labels microtubules. The covisualization of peroxisomes and cytoskeletal elements revealed that, contrary to the reports from animal cells, peroxisomes in plants appear to associate with actin filaments and not microtubules. That peroxisome movement is actin based was shown by pharmacological studies. For this analysis we used onion epidermal cells and various cell types of Arabidopsis including trichomes, root hairs, and root cortex cells exhibiting different modes of growth. In transient onion epidermis assay and in transgenic Arabidopsis plants, an interference with the actin cytoskeleton resulted in progressive loss of saltatory movement followed by the aggregation and a complete cessation of peroxisome motility within 30 min of drug application. Microtubule depolymerization or stabilization had no effect.

Inactivation of AtRac1 by abscisic acid is essential for stomatal closure. Genes Dev. 2001 Jul 15;15(14):1808-16.
Lemichez E, Wu Y, Sanchez JP, Mettouchi A, Mathur J, Chua NH.
Laboratory of Plant Molecular Biology, Rockefeller University, New York, New York 10021-6399, USA.

Plant water homeostasis is maintained by the phytohormone abscisic acid (ABA), which triggers stomatal pore closure in response to drought stress. We identified the Arabidopsis small guanosine triphosphatase (GTPase) protein AtRac1 as a central component in the ABA-mediated stomatal closure process. ABA treatment induced inactivation of AtRac GTPases and disruption of the guard cell actin cytoskeleton. In contrast, in the ABA-insensitive mutant abi1-1, which is impaired in stomatal closure, neither AtRac inactivation nor actin cytoskeleton disruption was observed on ABA treatment. These observations indicate that AtRac1 inactivation is a limiting step in the ABA-signaling cascade leading to stomatal closure. Consistent with these findings, expression of a dominant-positive mutant of AtRac1 blocked the ABA-mediated effects on actin cytoskeleton and stomatal closure in wild-type plants, whereas expression of a dominant-negative AtRac1 mutant recapitulated the ABA effects in the absence of the hormone. Moreover, the dominant-negative form of AtRac1 could also restore stomatal closure in abi1-1. These results define AtRac1 as a central element for plant adaptation to drought.

Cell growth: how to grow and where to grow. Curr Biol. 2001 May 15;11(10):R402-4.
Mathur J, Hulskamp M.
University of Koln, Botanical Institute III, Gyrhofstr. 15, D-50931, Koln, Germany.

Root hairs provide a model system for studying tip growth in plants. The recent cloning of genes required for tip growth has shed new light on the link between ionic regulation, cell wall assembly and the cytoskeleton in cell growth.

Microtubule stabilization leads to growth reorientation in Arabidopsis trichomes. Plant Cell. 2000 Apr;12(4):465-77.
Mathur J, Chua NH.
Laboratory of Plant Cell Biology, Institute of Molecular Agrobiology, 1 Research Link, National University of Singapore, 117 604, Singapore.

The single-cell trichomes in wild-type Arabidopsis are either unbranched or have two to five branches. Using transgenic Arabidopsis plants expressing a green fluorescent protein-microtubule-associated protein4 fusion protein, which decorates the microtubular cytoskeleton, we observed that during trichome branching, microtubules reorient with respect to the longitudinal growth axis. Considering branching to be a localized microtubule-dependent growth reorientation event, we investigated the effects of microtubule-interacting drugs on branch induction in trichomes. In unbranched trichomes of the mutant stichel, a change in growth directionality, closely simulating branch initiation, could be elicited by a short treatment with paclitaxel, a microtubule-stabilizing drug, but not with microtubule-disrupting drugs. The growth reorientation appeared to be linked to increased microtubule stabilization and to aster formation in the treated trichomes. Taxol-induced microtubule stabilization also led to the initiation of new branch points in the zwichel mutant of Arabidopsis, which is defective in a kinesin-like microtubule motor protein and possesses trichomes that are less branched. Our observations suggest that trichome cell branching in Arabidopsis might be mediated by transiently stabilized microtubular structures, which may form a component of a multiprotein complex required to reorient freshly polymerizing microtubules into new growth directions.

Root hair formation: F-actin-dependent tip growth is initiated by local assembly of profilin-supported F-actin meshworks accumulated within expansin-enriched bulges. Dev Biol. 2000 Nov 15;227(2):618-32.
Baluska F, Salaj J, Mathur J, Braun M, Jasper F, Samaj J, Chua NH, Barlow PW, Volkmann D.
Institute of Botany, Department of Plant Cell Biology, Rheinische Friedrich-Wilhelms University Bonn, Kirschallee 1, Bonn, D-53115, Germany.

Plant root hair formation is initiated when specialized elongating root epidermis cells (trichoblasts) assemble distinct domains at the plasma membrane/cell wall cell periphery complexes facing the root surface. These localities show accumulation of expansin and progressively transform into tip-growing root hair apices. Experimentation showed that trichoblasts made devoid of microtubules (MTs) were unaffected in root hair formation, whereas those depleted of F-actin by the G-actin sequestering agent latrunculin B had their root hair formation blocked after the bulge formation stage. In accordance with this, MTs are naturally depleted from early outgrowing bulges in which dense F-actin meshworks accumulate. These F-actin caps remain associated with tips of emerging and growing root hairs. Constitutive expression of the GFP-mouse talin fusion protein in transgenic Arabidopsis, which visualizes all classes of F-actin in a noninvasive mode, allowed in vivo confirmation of the presence of distinct F-actin meshworks within outgrowing bulges and at tips of young root hairs. Profilin accumulates, at both the protein and the mRNA levels, within F-actin-enriched bulges and at tips of emerging hairs. ER-based calreticulin and HDEL proteins also accumulate within outgrowing bulges and remain enriched at tips of emerging hairs. All this suggests that installation of the actin-based tip growth machinery takes place only after expansin-associated bulge formation and requires assembly of profilin-supported dynamic F-actin meshworks.

The actin cytoskeleton is required to elaborate and maintain spatial patterning during trichome cell morphogenesis in Arabidopsis thaliana. Development. 1999 Dec;126(24):5559-68.
Mathur J, Spielhofer P, Kost B, Chua N.
Laboratory of Plant Cell Biology, Institute of Molecular Agrobiology, National University of Singapore, Singapore, 117 604.

Arabidopsis thaliana trichomes provide an attractive model system to dissect molecular processes involved in the generation of shape and form in single cell morphogenesis in plants. We have used transgenic Arabidopsis plants carrying a GFP-talin chimeric gene to analyze the role of the actin cytoskeleton in trichome cell morphogenesis. We found that during trichome cell development the actin microfilaments assumed an increasing degree of complexity from fine filaments to thick, longitudinally stretched cables. Disruption of the F-actin cytoskeleton by actin antagonists produced distorted but branched trichomes which phenocopied trichomes of mutants belonging to the 'distorted' class. Subsequent analysis of the actin cytoskeleton in trichomes of the distorted mutants, alien, crooked, distorted1, gnarled, klunker and wurm uncovered actin organization defects in each case. Treatments of wild-type seedlings with microtubule-interacting drugs elicited a radically different trichome phenotype characterized by isotropic growth and a severe inhibition of branch formation; these trichomes did not show defects in actin cytoskeleton organization. A normal actin cytoskeleton was also observed in trichomes of the zwichel mutant which have reduced branching. ZWICHEL, which was previously shown to encode a kinesin-like protein is thought to be involved in microtubule-linked processes. Based on our results we propose that microtubules establish the spatial patterning of trichome branches whilst actin microfilaments elaborate and maintain the overall trichome pattern during development.

Cytoskeleton in plant development. Curr Opin Plant Biol. 1999 Dec;2(6):462-70.
Kost B, Mathur J, Chua NH.
Laboratory of Plant Cell Biology, Institute of Molecular Agrobiology, National University of Singapore, 117604, Singapore.

The plant cytoskeleton has crucial functions in a number of cellular processes that are essential for cell morphogenesis, organogenesis and development. These functions have been intensively investigated using single cell model systems. With the recent characterization of plant mutants that show aberrant organogenesis resulting from primary defects in cytoskeletal organization, an integrated understanding of the importance of the cytoskeleton for plant development has begun to emerge. Newly established techniques that allow the non-destructive visualization of microtubules or actin filaments in living plant cells and organs will further advance this understanding.

Gene identification with sequenced T-DNA tags generated by transformation of Arabidopsis cell suspension. Plant J. 1998 Mar;13(5):707-16.
Mathur J, Szabados L, Schaefer S, Grunenberg B, Lossow A, Jonas-Straube E, Schell J, Koncz C, Koncz-Kalman Z.
Max-Planck Institut fur Zuchtungsforschung, Koln, Germany.

A protocol for establishment and high-frequency Agrobacterium-mediated transformation of morphogenic Arabidopsis cell suspensions was developed to facilitate saturation mutagenesis and identification of plant genes by sequenced T-DNA tags. Thirty-two self-circularized T-DNA tagged chromosomal loci were isolated from 21 transgenic plants by plasmid rescue and long-range inverse polymerase chain reaction (LR-iPCR). By bidirectional sequencing of the ends of T-DNA-linked plant DNA segments, nine T-DNA inserts were thus localized in genes coding for the Arabidopsis ASK1 kinase, cyclin 3b, J-domain protein, farnesyl diphosphate synthase, ORF02, an unknown EST, and homologues of a copper amine oxidase, a peripheral Golgi protein and a maize pollen-specific transcript. In addition, 16 genes were identified in the vicinity of sequenced T-DNA tags illustrating the efficiency of genome analysis by insertional mutagenesis.

Transcription of the Arabidopsis CPD gene, encoding a steroidogenic cytochrome P450, is negatively controlled by brassinosteroids. Plant J. 1998 Jun;14(5):593-602.
Mathur J, Molnar G, Fujioka S, Takatsuto S, Sakurai A, Yokota T, Adam G, Voigt B, Nagy F, Maas C, Schell J, Koncz C, Szekeres M.
Max Planck-Institut fur Zuchtungsforschung, Koln, Germany.

The Arabidopsis CPD gene encodes a cytochrome P450 steroid side-chain hydroxylase (CYP90) that plays an essential role in the biosynthesis of the plant hormone brassinolide. Expression of the CPD gene is confined to cotyledons and leaf primordia in etiolated seedlings and detectable in the adaxial parenchyma of expanding leaves in light-grown plants. Transcription of the CPD gene is not affected by the plant growth factors auxin, ethylene, gibberellin, cytokinin, jasmonic acid and salicylic acid, but is specifically down-regulated by brassinolide in both dark and light. Steady-state mRNA levels of a CPD promoter-driven uidA reporter gene correlate with the expression of resident CPD gene in transgenic plants. Intermediates of the early and late C-6 oxidation pathways of brassinolide, carrying C-22 and C-23 side-chain hydroxyls, efficiently inhibit the activity of the CPD promoter. Repression of CPD transcription by brassinosteroids is sensitive to the protein synthesis inhibitor cycloheximide, indicating a requirement for de novo synthesis of a regulatory factor.

The ROOT HAIRLESS 1 gene encodes a nuclear protein required for root hair initiation in Arabidopsis.
Schneider K, Mathur J, Boudonck K, Wells B, Dolan L, Roberts K.
Department of Cell Biology, John Innes Centre, Colney, Norwich, NR4 7UH, UK. kschneid@mpiz-koeln.mpg.de

The epidermis of Arabidopsis wild-type primary roots, in which some cells grow hairs and others remain hairless in a position-dependent manner, has become an established model system to study cell differentiation. Here we present a molecular analysis of the RHL1 (ROOT HAIRLESS 1) gene that, if mutated, prevents the formation of hairs on primary roots and causes a seedling lethal phenotype. We have cloned the RHL1 gene by use of a T-DNA-tagged mutant and found that it encodes a protein that appears to be plant specific. The predicted RHL1 gene product is a small hydrophilic protein (38.9 kD) containing putative nuclear localization signals and shows no significant homology to any known amino acid sequence. We demonstrate that a 78-amino-acid sequence at its amino terminus is capable of directing an RHL1-GFP fusion protein to the nucleus. The RHL1 transcript is present throughout the wild-type plant and in suspension culture cells, but in very low amounts, suggesting a regulatory function for the RHL1 protein. Structural evidence suggests a role for the RHL1 gene product in the nucleolus. We have examined the genetic relationship between RHL1 and GL2, an inhibitor of root hair initiation in non-hair cells. Our molecular and genetic data with double mutants, together with the expression analysis of a GL2 promoter-GUS reporter gene construct, indicate that the RHL1 gene acts independently of GL2.

Pleiotropic control of glucose and hormone responses by PRL1, a nuclear WD protein, in Arabidopsis. Genes Dev. 1998 Oct 1;12(19):3059-73.
Nemeth K, Salchert K, Putnoky P, Bhalerao R, Koncz-Kalman Z, Stankovic-Stangeland B, Bako L, Mathur J, Okresz L, Stabel S, Geigenberger P, Stitt M, Redei GP, Schell J, Koncz C.
Abteilung Genetische Grundlagen der Pflanzenzuchtung, Federal Republic of Germany.

The prl1 mutation localized by T-DNA tagging on Arabidopsis chromosome 4-44 confers hypersensitivity to glucose and sucrose. The prl1 mutation results in transcriptional derepression of glucose responsive genes defining a novel suppressor function in glucose signaling. The prl1 mutation also augments the sensitivity of plants to growth hormones including cytokinin, ethylene, abscisic acid, and auxin; stimulates the accumulation of sugars and starch in leaves; and inhibits root elongation. PRL1 encodes a regulatory WD protein that interacts with ATHKAP2, an alpha-importin nuclear import receptor, and is imported into the nucleus in Arabidopsis. Potential functional conservation of PRL1 homologs found in other eukaryotes is indicated by nuclear localization of PRL1 in monkey COS-1 cells and selective interaction of PRL1 with a nuclear protein kinase C-betaII isoenzyme involved in human insulin signaling.

Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and de-etiolation in Arabidopsis.
Szekeres M, Nemeth K, Koncz-Kalman Z, Mathur J, Kauschmann A, Altmann T, Redei GP, Nagy F, Schell J, Koncz C.<>Institute of Plant Biology, Hungarian Academy of Sciences, Szeged, Hungary.

The cpd mutation localized by T-DNA tagging on Arabidopsis chromosome 5-14.3 inhibits cell elongation controlled by the ecdysone-like brassinosteroid hormone brassinolide. The cpd mutant displays de-etiolation and derepression of light-induced genes in the dark, as well as dwarfism, male sterility, and activation of stress-regulated genes in the light. The CPD gene encodes a cytochrome P450 (CYP90) sharing homologous domains with steroid hydroxylases. The phenotype of the cpd mutant is restored to wild type both by feeding with C23-hydroxylated brassinolide precursors and by ectopic overexpression of the CPD cDNA. Brassinosteroids also compensate for different cell elongation defects of Arabidopsis det, cop, fus, and axr2 mutants, indicating that these steroids play an essential role in the regulation of plant development.

Enhanced Green Fluorescence by the Expression of an Aequorea victoria Green Fluorescent Protein Mutant in Mono- and Dicotyledonous Plant Cells. Proc Natl Acad Sci U S A. 1996 June 11; 93 (12): 5888–5893
C Reichel, J Mathur, P Eckes, K Langenkemper, C Koncz, J Schell, B Reiss, and C Maas
Max-Planck-Institut fur Zuchtungsforschung, Abteilung Genetische Grundlagen der Pflanzenzuchtung, Koln, Germany.

The expression of the jellyfish green fluorescent protein (GFP) in plants was analyzed by transient expression in protoplasts from Nicotiana tabacum, Arabidopsis thaliana, Hordeum vulgare, and Zea mays. Expression of GFP was only observed with a mutated cDNA, from which a recently described cryptic splice site had been removed. However, detectable levels of green fluorescence were only emitted from a small number of protoplasts. Therefore, other mutations in the GFP cDNA leading to single-amino acid exchanges in the chromophore region, which had been previously studied in Escherichia coli, were tested in order to improve the sensitivity of this marker protein. Of the mutations tested so far, the exchange of GFP amino acid tyrosine 66 to histidine (Y66H) led to detection of blue fluorescence in plant protoplasts, while the exchange of amino acid serine 65 to cysteine (S65C) and threonine (S65T) increased the intensity of green fluorescence drastically, thereby significantly raising the detection level for GFP. For GFP S65C, the detectable number of green fluorescing tobacco (BY-2) protoplasts was raised up to 19-fold, while the fluorimetricly determined fluorescence was raised by at least 2 orders of magnitude.

A synthetic cry I-C gene, encoding a Bacillus thuringiensis endotoxin, confers Spodotera resistance in Alfalfa and Tobacco. Proc. Natl. Acad. Sci. USA. Vol. 93, pp. 15012-15017, December 1996
Nicolai Strizhov*, Menachem Keller, Jaideep Mathur*, Zsuzsanna Koncz-Kálmán*, Dirk Bosch§, Evgenia Prudovsky, Jeff Schell*, Baruch Sneh, Csaba Koncz*, and Aviah Zilberstein
* Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Köln, Germany; Department of Botany, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel; and § Department of Molecular Biology, DLO-Centre for Plant Breeding and Reproduction Research (CPRO-DLO), P.O. Box 16, Wageningen, 6700 AA, The Netherlands/

Spodoptera species, representing widespread polyphagous insect pests, are resistant to Bacillus thuringiensis -endotoxins used thus far as insecticides in transgenic plants. Here we describe the chemical synthesis of a cryIC gene by a novel template directed ligation-PCR method. This simple and economical method to construct large synthetic genes can be used when routine resynthesis of genes is required. Chemically phosphorylated adjacent oligonucleotides of the gene to be synthesized are assembled and ligated on a single-stranded, partially homologous template derived from a wild-type gene (cryIC in our case) by a thermostable Pfu DNA ligase using repeated cycles of melting, annealing, and ligation. The resulting synthetic DNA strands are selectively amplified by PCR with short specific flanking primers that are complementary only to the new synthetic DNA. Optimized expression of the synthetic cryIC gene in alfalfa and tobacco results in the production of 0.01-0.2% of total soluble proteins as CryIC toxin and provides protection against the Egyptian cotton leafworm (Spodoptera littoralis) and the beet armyworm (Spodoptera exigua). To facilitate selection and breeding of Spodoptera-resistant plants, the cryIC gene was linked to a pat gene, conferring resistance to the herbicide BASTA.