The Ubituitin-26S Proteasome and Autophagy Systems Relay Proteome Homeostasis Regulation During Seed Development
Peifeng Yu and Zhihua Hua
Interdisciplinary Program in Molecular and Cellular Biology, Ohio University, Athens, OH, 45701, USA
In plants, ubiquitylation regulates numerous biological functions. Functional studies on the ubituitin-26S proteasome system (UPS) have demonstrated that virtually all aspects of the plant’s life involve UPS-mediated turnover of abnormal or short-term proteins. However, developmental characterization of the UPS activities in seeds remains scarce despite the facts that mutants of several core elements of the UPS are embryonically lethal. Unfortunately, early termination of embryogenesis limits the scope for characterizing the UPS activities throughout seed development. Given both economic and societal impact of seed production, such studies are indispensable. Here, we systematically compared expression changes of multiple 26S subunits along with the entire ubiquitylation substrates in five consecutive seed developmental stages in Arabidopsis thaliana. We discovered that the pool of ubiquitylation substrates in immature siliques declined immediately at two days after pollination (DAP) while both protein and transcript levels of six selected 26S subunits remain no decrease until four DAP. Since autophagy plays the second largest role in maintaining proteome stability, we parallelly studied the activities of five key players in Arabidopsis autophagy pathway. In opposite to the overall decline of UPS activities, the autophagy members displayed up or down expression patterns along with seed development. Hence, the UPS and autophagy may play a relay role in regulating seed development. Consistently, loss-of-function in three proteasome subunits and four autophagy members caused defective embryo development but with various levels indicating functional specialization. We further discovered that expression upregulation of a polyubiquitin gene could significantly increase seed yield demonstrating a possibility for improving crop production through manipulating the UPS activity.
Multimerization variants as potential drivers of neofunctionalization
Youngwoo Lee, Daniel B. Szymanski
Purdue University
Whole-genome duplications are common during evolution, creating genetic redundancy that can enable cellular innovations. Novel protein-protein interactions provide a route to diversified gene functions, but, at present, there is limited proteome-scale knowledge on the extent to which variability in protein complex formation drives neofunctionalization. Here, we used protein correlation profiling to test for variability in apparent mass among thousands of orthologous proteins isolated from diverse species and cell types. Variants in protein complex size were unexpectedly common, in some cases appearing after relatively recent whole-genome duplications or an allopolyploidy event. In other instances, variants such as those in the carbonic anhydrase orthologous group reflected the neofunctionalization of ancient paralogs that have been preserved in extant species. Our results demonstrate that homo- and heteromer formation have the potential to drive neofunctionalization in diverse classes of enzymes, signaling, and structural proteins.
The identification of cytoskeletal determinants of PIN-FORMED protein dynamics within the plasma membrane.
Ayoub Stelate, Katerina Malinska2, Martina Lankova2, Karel Muller2, Roberta Filepova2, Zuzana Amlerova2, Katarzyna Retzer2 and Jan Petrasek1,2
1Department of Experimental Plant Biology, Faculty of Science, Charles University, Vinicna° 5, 128 44 Prague 2, Czech Republic; 2Institute of Experimental Botany, the Academy of Sciences of the Czech Republic, Rozvojova 263,165 02 Prague 6, Czech Republic
PIN-FORMED (PIN) auxin efflux carriers are one of the most studied integral plasma membrane (PM) proteins, important for the generation of auxin concentration gradients regulating plant development. PINs undergo trafficking between PM and intracellular compartments, which modulates the process of cell-to-cell auxin transport and its directionality. Although the molecular machinery that regulates plant vesicle trafficking processes and the role of auxin in this process is intensively studied, the mechanisms regulating dynamics of auxin carriers within the PM, where they perform their role, are still poorly understood. Our previously published data using fluorescence recovery after photobleaching and fluorescence correlation techniques indicated that PINs are present in both highly mobile (20%) and largely immobile domains (80%) within PM. Here we address the involvement of cytoskeleton in the PINs PM nanodomain organization using total internal reflection fluorescence microscopy in tobacco cultured cells carrying inducible genes for GFP-tagged PINs. Image analysis-based quantification of these distributions showed that 3 tested tobacco NtPINs (NtPIN2, 3, 11) displayed protein-specific dynamics between individual nanodomains as well as size of individual nanodomains. In agreement, the application of actin and microtubules drugs, i.e., latrunculin B (Lat B) and oryzalin (Ory), resulted in different reaction of NtPINs within the PM, :NtPIN11 appear to be microtubule dependent and NtPIN2 microtubule and actin- dependent, while NtPIN3 shows no effect after cytoskeletal drugs treatment suggesting that cytoskeleton-dependent fraction does not contribute to the overall auxin transport. Our work is pioneering in the identification of auxin transporting PM nanodomains, which composition is now identified with co-IP/MS to propose a model for the interaction between cytoskeleton and auxin efflux carriers.
The Power of Engineering Organelle Movements in Plants
Jinmo Gu, Julianna K. Vick, Madeline Davis, Alexander Overholt, Andreas Nebenfuehr
Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996-1939
"Organelle movements in plants mainly rely on Myosin class XI motor proteins and actin filament cytoskeleton. Other factors, such as organelle-organelle interactions and hydrodynamic flow, are also considered to contribute to organelle movements, but it is largely unknown how these multiple components affect different organelles’ movements. We hypothesized that each organelle group has a different mechanistic signature underpinning their movements and, in turn, play a different role in creating the overall environment for organelle movements.
In order to understand the movements of specific organelle groups, we generated ‘Booster’ and ‘Anchor’ constructs in agrobacterium binary vectors. Each construct was designed to enhance or suppress the targeted organelle’s movements by expressing an organelle specific membrane marker with either the DUF593 domain from MyoB1, an adaptor protein for Myosin XI, or a microtubule binding domain, respectively. We targeted peroxisomes, endoplasmic reticulum (ER), and mitochondria separately to profile their movements in the leaf epidermal cells of Nicotiana benthamiana. In addition, we examined movements of other, non-targeted organelles including Golgi stacks in cells expressing Booster or Anchor constructs. In all three targeted organelles, we observed a significant increase of linear movements, with their corresponding Booster expression, both in terms of linear speeds and run lengths. Interestingly, both ER and mitochondria Boosters also enhanced linear movements of other non-targeted organelles, whereas peroxisome Boosters were selectively effective in increasing ER movements. By contrast, Anchor expression strikingly diminished the linear movements of targeted organelles, accompanied by morphological alterations especially in mitochondria and ER. No significant effects of Anchors on non-targeted organelles were found, except for a small reduction of ER movements by peroxisome Anchors. Collectively, these Booster and Anchor data suggest that organelle movements in plants are coordinated among different types of organelles although the degree of coordination differs between the organelle groups.
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Root illumination status defines PIN-FORMED2 (PIN2) subcellular distribution and intracellular trafficking
Katarzyna Retzer
Laboratory of Hormonal Regulations in Plants, Institute of Experimental Botany, Czech Academy of Sciences, v.v.i., Rozvojova 263, 165 02 Prague 6, Czech Republic
The sessile life style of plants forces them to adapt constantly to changing environmental conditions. Establishment of auxin gradients underpin modulation of plant architecture during development and adaptation processes. PIN-FORMED (PIN) auxin efflux carrier facilitate active transport of auxin between cells and their abundance and subcellular distribution is rearranged depending on environmental conditions. Continuous modulation of cellular mechanisms including PIN endocytosis from the PM, followed by subsequent exocytosis to the PM or vacuolar degradation are crucial to orchestrate auxin distribution. Plant-cell-biological experiments are often performed using seedlings grown in full light, a growth condition not representative of real-life lighting levels. Using a new growth system, D-root, to grow shoots in light and roots in dark, we observed altered PIN2 turnover in a pharmacological screen depending on the root illumination status. This prompted us to further investigate the effects of D-root on root growth and PIN2 turnover. We observed significant growth and auxin level differences between full-light and D-root grown roots.
Nuclear Movement in Growing Root Hairs in Arabidopsis thaliana Depends on Multiple Mechanisms
Justin Brueggeman and Andreas Nebenfuehr
Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville
Nuclear movement is a common phenomenon in many eukaryotes, primarily during growth and developmental processes. Likewise, nuclear positioning is important in plants, mainly during growth and stress responses. Nuclear movement is particularly important in root hairs, which are single-cell outgrowths from the main root shoot. In root hairs, the nucleus maintains a fixed distance from the root hair tip by moving forward at the same rate as the tip grows. Interestingly, when this distance between the nucleus and the root hair tip is disrupted, root hair growth ceases. Nuclear movement in Arabidopsis thaliana root hairs has been described to be dependent on actin filaments, but not microtubules. Movement along actin filaments is driven by myosin motor proteins and movement of the nucleus has been found to depend on myosin XI-I. Our project sought to test the role of myosin XI-I in nuclear movement during root hair growth. Using a myosin xi-i knockout, our experiments showed that the distance from the nucleus to the tip remains constant, suggesting there may be alternative mechanisms of nuclear movement during root hair growth. To determine whether these movements are actin-based or microtubule-based, we specifically disrupted either of these cytoskeletal networks using the actin-depolymerizing drug Latrunculin B (LatB) and the microtubule depolymerizing drug Oryzalin (Oz), respectively, in both wild type and myosin xi-i knockout (kaku1-3) seedlings. Not only did the nucleus move in the absence of myosin XI-I, but nuclear movement still occurred when the actin cytoskeleton was disrupted with LatB, suggesting that nuclear transport can take place along the microtubules. Conversely, myosin xi-i knockouts treated with oryzalin still demonstrated nuclear movement, suggesting that there may be actin-based processes that may involve different myosins, in addition to myosin XI-I. Together, these experiments demonstrate roles for both actin and microtubules cytoskeletons in the process of nuclear movement in growing root hairs. This suggests the presence of independent but redundant mechanisms to ensure proper nuclear positioning during root hair growth.
The TPLATE complex mediates membrane bending during plant clathrin-mediated endocytosis
Alexander Johnson1 , Dana A Dahhan2*, Nataliia Gnyliukh1*, Walter A Kaufmann1, Vanessa Zheden1, Tommaso Costanzo1, Pierre Mahou3, Mónika Hrtyan1, Jie Wang4,5, Juan Aguilera-Servin1, Daniёl van Damme4,5, Emmanuel Beaurepaire3, Martin Loose1, Sebastian Y Bednarek2 and Jiri Friml1
1Institute of Science and Technology (IST Austria), 3400 Klosterneuburg, Austria; 2UW-Madison, Biochemistry, 215C HF DeLuca Laboratories, Madison, WI, 53706; 3Laboratory for Optics and Biosciences, Ecole Polytechnique, CNRS, Inserm, IP Paris, Palaiseau, France; 4Ghent University, Department of Plant Biotechnology and Bioinformatics, Technologiepark 71, 9052 Ghent, Belgium; 5VIB Center for Plant Systems Biology, Technologiepark 71, 9052 Ghent, Belgium
"Clathrin-mediated endocytosis (CME) plays a critical role in many physiological processes in plants. Despite its physiological significance, little is known about the molecular mechanisms driving and mediating CME in plant cells; particularly how plants can bend their membranes to create endocytic vesicles against their extreme levels of turgor pressure. As plant CME is independent of actin, it indicates that plant CME have a mechanistically distinct solution to drive membrane bending during CME compared to other model systems. By using advanced live quantitative high- and super-resolution imaging of single events of CME, and biochemical analysis of purified clathrin vesicles from plant cells, we surprisingly found that the plant-specific TPLATE complex localizes outside of the clathrin-coated endocytic vesicles. This localization led us to predict that the TPLATE complex is involved in driving membrane bending during CME. Therefore, we tested this hypothesis by directly examining clathrin-coated structures in metal replicas of unroofed protoplasts subjected to TPLATE disruption, using the WDXM2 TPLATE inducible loss-of-function mutant and scanning electron microscopy. We found that cells with disrupted TPLATE failed to generate spherical vesicles and we confirmed this by using scanning transmission electron microscopy tomography to visualize the 3D ultrastructure of the clathrin structures in TPLATE and TPLATE disrupted cells. This demonstrated that the TPLATE complex is required to mediate membrane bending during plant CME. As the TPLATE complex contains protein domains which are homologous to known membrane bending domains in other model systems, we assessed their ability to deform membranes using in vitro biophysical assays and found that the TPLATE complex contains the machinery with the capacity to drive membrane bending. Thus, we redefine the role of the TPLATE complex as a key component of the evolutionarily distinct mechanism mediating membrane bending against high turgor pressure to drive CME in plant cells.
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405nm photostimulation of the endoplasmic reticulum-chloroplast contact site in Arabidopsis hypocotyls causes rapid cytoskeletal depolymerization and elevated cytoplasmic calcium
Sara Maynard, Lawrence R. Griffing
Molecular and Environmental Plant Science Interdisciplinary Program, Department of Biology, Texas A&M University
The network of spatially and temporally interacting signal transduction molecular pathways confers specificity of the plant abiotic stress response. The chloroplast-endoplasmic reticulum (ER) contact site is spatially and temporally highly specific. Stressing it with localized high-fluence 405nm blue light, hereinafter referred to as photostimulation, induces multiple, potentially interacting intra- and intercellular responses. Observing Arabidopsis thaliana seedling hypocotyl cells with fluorescent confocal microscopy before and after photostimulation revealed relative changes in cytosolic calcium and the polymerization of microtubules and actin. Photostimulation induced a near-instantaneous cytosolic calcium wave and rapid depolymerization of both cortical actin and microtubules. These responses localize to the photostimulated, target cell, and while the calcium wave occasionally spreads to adjacent cells at reduced magnitude, preliminary results show that depolymerization of the cytoskeleton does not. While recovery of normal cytosolic calcium usually occurs within one to two minutes in the target cell and more quickly in adjacent cells, recovery of microtubule and actin polymerization occurs with a different, slower time course. Drug-induced depolymerization of the microtubules and actin prior to photostimulation has no effect on the calcium wave, and photostimulation causes no further depolymerization. Our model is that the photostimulation-induced calcium wave contributes to the depolymerization of microtubules and actin in the target cell but is of insufficient magnitude and duration in adjacent cells. Supporting this model is the associated cessation of cytoplasmic streaming in the target cell, but not adjacent cells. Furthermore, a fully functional cytoskeleton is not necessary for propagation of the calcium wave. This system provides a way to reliably study the signal transduction of a specific abiotic stress on the cytoskeleton and calcium signaling at the cell level. Further work investigating the linkage between components of the signal transduction network associated with chloroplast-ER contact sites and the cytoskeleton is ongoing.
Identifying motor proteins that function in male germ unit movement in Arabidopsis thaliana pollen tubes
Tyler Mendes1,2, Norman R. Groves2,3, and Iris Meier2,3,4
1Molecular, Cellular, and Developmental Biology Graduate Program, The Ohio State University, Columbus, OH, USA; 2Department of Molecular Genetics, The Ohio State University, Columbus, OH, USA; 3Center for Applied Plant Sciences, The Ohio State University, Columbus, OH, USA; 4Center for RNA Biology, The Ohio State University, Columbus, OH, USA
Fertilization is a key component of plant reproduction that is necessary for agriculture. While the basic process of fertilization has been understood for centuries, the mechanism underlying how plant sperm is trafficked to the egg remains unknown. To achieve fertilization, the growing pollen tube transports the sperm cell (SCs) to ovules. The SCs are physically connected via a cytoplasmic projection from the lead sperm cell to the vegetative nucleus (VN); this combined complex is referred to as the Male Germ Unit (MGU). Previous research has shown that Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes at the VN nuclear envelope facilitate MGU movement. The LINC complex spans the inner and outer nuclear membranes and connects the nucleoplasm to the cytoskeleton. Null mutants in genes for two plant LINC complex subunits, WIP and WIT, result in a VN movement defect, wherein the SCs lead while the VN trails behind. This defect correlates with a defect in pollen tube burst and, in turn, a loss of seed set. We hypothesize that WIT and WIP act as adapter proteins between the VN envelope and unknown cytoskeletal motor proteins. In plants, there are two kinds of cytoskeletal motor proteins: Kinesins and Myosins. While Myosins have been well-studied in pollen-tube tip growth, the role of Kinesins in pollen tubes has not been established. To determine which motors are involved in VN movement, we have screened insertional mutants in 17 pollen-expressed Kinesins (PEK1-17) for fertility defects. Kinesin-14s represent a significant portion of the PEKs, with 8 of the 17 PEKs being Kinesin-14s. Two of these Kinesin-14s, PEK3 and PEK9, present mild fertility defects in insertional mutants. PEK3 and PEK9 are likely the result of a gene duplication event, leading to the possibility that a more severe fertility defect would be observed in a double mutant. Insertional mutants in a Kinesin-4, PEK14, displayed significant reductions in seed set, on the order of the reduction observed in wit mutants, indicating it may play a role in MGU movement. Future experiments will establish if these genes are associated with the VN envelope, physically interact with WIP and/or WIT, and are required for pollen tube burst.
Arabidopsis myosin XIK interacts with the exocyst complex to facilitate vesicle tethering during exocytosis
Weiwei Zhang, Lei Huang, Chunhua Zhang and Christopher J. Staiger
Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907
Myosin motors are essential players in secretory vesicle trafficking and exocytosis in yeast and mammalian cells; however, similar roles in plants remain a matter for debate, at least for diffusely-growing cells. Here, we demonstrate that Arabidopsis myosin XIK, via its globular tail domain (GTD), participates in the vesicle tethering step of exocytosis through direct interactions with the exocyst complex. Specifically, myosin XIK GTD bound directly to several exocyst subunits in vitro and functional fluorescently-tagged XIK colocalized with multiple exocyst subunits at plasma membrane (PM)-associated stationary foci. Moreover, genetic and pharmacological inhibition of myosin XI activity reduced the rate of appearance and lifetime of stationary exocyst complexes at the PM. By tracking single exocytosis events of cellulose synthase (CESA) complexes (CSCs) with high spatiotemporal resolution imaging and pair-wise colocalization of myosin XIK, exocyst subunits and CESA6, we demonstrated that XIK associates with secretory vesicles earlier than exocyst and is required for the efficient localization and normal dynamic behavior of exocyst complex at the PM tethering site. This study reveals an important functional role for myosin XI in secretion and provides insights about the dynamic regulation of exocytosis in plants.
A machine learning approach to prioritizing functionally active F-box genes in plants
Yang Li, Madhura Mihiranga Yapa, Zhihua Hua
Ohio University
Protein degradation through the Ubiquitin (Ub)-26S Proteasome System (UPS) is a major gene expression regulatory pathway in plants. In this pathway, the 76-amino acid Ub proteins are covalently linked onto a large array of UPS substrates with the help of three enzymes (E1 activating, E2 conjugating and E3 ligating enzymes) and direct them for turnover in the 26S proteasome complex. The S-phase Kinase-associated Protein 1 (Skp1), CUL1, F-box (FBX) protein (SCF) complexes have been identified as the largest E3 ligase group in plants due to the dramatic number increase of the FBX genes in plant genomes. Since it is the FBX proteins that recognize and determine the specificity of SCF substrates, much effort has been done to characterize their genomic, physiological, and biochemical roles in the past over two decades of functional genomic studies. However, the sheer size and high sequence diversity of the FBX gene family demands new approaches to uncover unknown functions. In this work, we first identified 82 known FBX members that have been functionally characterized up to date in Arabidopsis thaliana. Through comparing the genomic structure, evolutionary selection, expression patterns, domain compositions, and functional activities between known and unknown FBX gene members, we developed a neural network machine learning approach to predict whether an unknown FBX member is likely functionally active in Arabidopsis, thereby facilitating its future functional characterization.
Strigolactones and abiotic stress in plants: modulation of abscisic acid transport
Giulia Russo1, Paolo Korwin Krukowski1, Serena Capitanio1, Daniela Minerdi1, Christian Constan Aguilar1, Lorenzo Borghi2, Ivan Visentin1, Andrea Genre3, Francesca Cardinale1, Andrea Schubert1.
1DISAFA - Plant Stress Laboratory, University of Turin, 10095 Grugliasco (TO), Italy; 2Institute of Plant and Microbial Biology, University of Zurich, 8008 Zurich, Switzerland; 3DBIOS - University of Turin, Viale Mattioli 25, 10125 Turin, Italy
Given their role in regulating leaf transpiration and stomata conductance, strigolactones (SL) have emerged as new class of hormones acting in concert with abscisic acid (ABA) to modulate plant tolerance in response to water deprivation.
In order to dissect the molecular mechanisms underlying the cross talk between the latter phytohormones, we evaluated the effect of SL treatment on the localization and abundance of ATP BINDING CASETTE G25 (ABCG25), a well-studied ABA exporter in Arabidopsis thaliana (At). At seedlings expressing a sGFP:ABCG25 construct were subjected to several hormonal and drug treatments, the localization and the accumulation of GFP signal was then monitored in different subcellular compartments in root tips by means of confocal microscopy. The same construct was also inserted in SL-insensitive (d14) At and in SL-deficient (max3) mutants to investigate the dependency of ABCG25 spatial regulation by SL and abiotic stress treatments from known genetic components of the SL pathway. We show that fine tuning the localization and recycling of this transporter might provide a possible mechanism of regulation in ABA homeostasis by SL.
Xyloglucan synthesizing complex: protein-protein interactions, localization, trafficking, and compositional dynamics.
Ning Zhang, Olga Zabotina
Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University
Xyloglucan is abundant and major hemicellulose in Arabidopsis thaliana cell walls and the composition of polysaccharide directly affects their mechanical strength, cell expansion, and plant ability to response to environment stresses. Seven glycosyltransferases are involved in the Xyloglucan biosynthesis in the Golgi apparatus: glucan synthase (CSLC4) synthesizes the xyloglucan glucan backbone; xylosyltransferases XXT1, XXT2 and XXT5 add the xylose residues to the glucan backbone; galactosyltransferases XLT2 and MUR3 and fucosyltransferase (FUT1) further branch glucan backbone and form the mature XLFG type Xyloglucan. Earlier, our lab has confirmed the protein-protein interactions among all seven xyloglucan synthesizing glycosyltransferases using BiFC, IP and pull-down experiments. We hypothesize that the multiprotein complex is required to synthesize complete structure of xyloglucan in Golgi with high efficiency and reproducibility. The open question is how this complex gets assembled, how it is organized and what is relation between its composition and synthesized product. The first question we investigated is whether the protein-protein interactions are required for the Golgi localization of glycosyltransferases involved in the synthesis. To answer this question, we investigated localization of glycosyltransferases fused with CFP/YFP transiently expressed in the protoplasts isolated from different mutants lacking xyloglucan synthesizing proteins. Another study was to investigate how N-terminal tail of these glycosyltrasferases impact their proper localization to Golgi. We truncated the cytosolic tail of XXT2 and XXT5 and found deletion of particular amino acid sequences on cytosolic tail block the Golgi localization of XXTs. We demonstrated that the mutation of the Arg-motif in cytosolic tail of XXT2 and XXT5 resulted in their mis-localization. When stably expressed, the wild type XXT2 can rescue the short root hair phenotype in xxt1xxt2; but the expression of mutated XXT2 is unable to complement root phenotype. Thus, the mutated Arg-motif leads to the mis-localization of XXTs and hence results in termination of xyloglucan formation which was observed before in xxt1xxt2 mutant. Our goal is to understand the dynamics of the complex. First, we decided to evaluate the half-lives of all glycosyltransferases involved in the synthesis. If the glycosyltransferases have the similar half-lives, it is more likely that they function in context of a compositionally steady complex, whereas is the protein have significantly distinct half-lives, the complex composition is dynamic and more transient. Thus, the study of turnover and half-lives of glycosyltransferases can offer some indirect evidence about assembly of XyG synthesizing complexes in Golgi and their co-existence in the complex. We utilized treated with CHI inhibitor the Arabidopsis seedlings expressing CFP-fused proteins and quantified the rate of reduction in fluorescence. Obtained results demonstrated most glycosyltransferases have short and distinct half-lives suggesting the dynamic composition of the complex is most likely. Next, western blotting will be used to reconfirm our observation from fluorescence.
Trafficking SNARE SYP132 contributes to stomatal defenses through regulation of plasma membrane H+-ATPases
Guillermo Baena, Lingfeng Xia, Sakharam Waghmare, Rucha Karnikbr
Plant Science Group, Laboratory of Plant Physiology and Biophysics, Institute of Molecular, Cell and Systems Biology, University of Glasgow, Glasgow G12 8QQ, United Kingdom
In plants stomata are portals for entry of microbial pathogens into the plant. The closing of the stomatal pore is the first line of defence against pathogen infection. However, pathogens such as the Pseudomonas syringae pv. tomato DC3000 can overcome the stomatal defense machinery and re-open the stomata to gain entry into the plant. These mechanisms for plant-pathogen interactions in response to stomatal defenses are known to target the activity of the plasma membrane (PM) H+-ATPases. In guard cells, the PM H+-ATPases energise stomatal movements and their activity is crucial for stomatal opening. Knowledge of mechanisms for PM H+-ATPase regulation in guard cells is therefore essential to understand stomatal defenses of plants.
Recently, the Syntaxin of Plants 132 (SYP132), a plasma membrane trafficking SNARE protein, was found to unusually associate with endocytic traffic which down-regulates PM H+-ATPase density and function at the plasma membrane. This SYP132-associated PM H+-ATPase trafficking is hormone-regulated and it affects plant growth and stomatal responses. The SYP132 is a low abundant SNARE, otherwise noted to be crucial for plant viability, and for defense-related secretory traffic at the plasma membrane. I have uncovered that the SYP132 and its regulation of the PM H+-ATPase also contributes to stomatal defenses in plants. These findings present a new role for the trafficking SNARE SYP132 in plant immunity, through the control of solute transport in guard cells.
Evidence for GTPase rate constant regulation in cortical microtubule dynamics.
Sidney L. Shaw, Timothy Cioffi
Dept of Biology, Indiana University, Bloomington
The cortical microtubule cytoskeleton plays a distinct role in the construction of the primary cell wall. The microtubule array patterns formed at the cell cortex influence subsequent patterning of cellulose into the nascent primary wall, with effects on the wall material properties governing cell expansion. The molecular mechanisms determining how cortical microtubules create a specific array pattern are still mostly unknown. A key factor in creating and maintaining the cortical microtubule array is the persistent addition of tubulin subunits to the microtubule plus end, affecting a form of polymer treadmilling that is critical to array patterning. We have developed high temporal/spatial in vivo tracking methods for examining the dynamic properties of cortical microtubules in super-resolved detail. We observe that transitions from states of microtubule polymer growth to shortening are preceded by a pause state. Using high temporal resolution data, we find that the decision to resume growth or to catastrophe into depolymerization is temporary consistent with the loss of GTP-tubulin at the plus end through hydrolysis to GDP-tubulin. In mutants lacking CLASP expression, we find evidence that the rate of GTP hydrolysis for tubulin subunits binding to the microtubule plus end differs significantly from wild type. Using computational modeling approaches, we provide evidence that plant cells modulate the tubulin GTPase rate constant as a means of increasing the persistence of plus end growth and avoiding microtubule catastrophe in this treadmilling system.
Photostimulation at the ER-chloroplast contact sites induces mitochondrial and peroxisomal clustering.
Megan Paredes, Sara Maynard, and Lawrence Griffing
Biology Department, Texas A&M University, 3258 TAMU College Station, TX USA 77843
Photostimulation of the chloroplast-endoplasmic reticulum (ER) membrane contact site (CERCS) with 405 nm laser light produces a wave of ER stress, visible as luminal protein aggregation, which travels throughout the cellular ER network, but does not move to adjacent cells. The aggregation of the ER luminal proteins is more severe near the photostimulated CERCS, resulting in fewer visible ER tubules labeled with ER-targeted fluorescent protein, but which over several minutes is the site of clustered ER. Mitochondria and peroxisomes also cluster, but their clustering is not restricted to the site of photostimulation, as determined by K-means clustering analysis and visualized with 3D active contouring. Streaming of mitochondria and peroxisomes stops briefly for several seconds near the site of photostimulation and the organelles cluster upon resumption of streaming. The accumulation of peroxisomes might relate to autophagy of the chloroplast when photostimulation results in damage, a hypothesis supported by the accumulation of ATG8-GFP in the area. We propose that the signal produced by the photostimulation, probably calcium release, travels along the ER, which acts as an organelle-crosstalk hub.
Spatial Regulation of ABA-Induced ROS Synthesis During Stomatal Closure
Anthony Postiglione
Wake Forest University
Plant hormones trigger a variety of signaling pathways that are critical regulators of plant growth and stress response. Many of these signaling cascades include a burst of reactive oxygen species, which act as important second messengers to carry out specific physiological responses. In response to drought stress, plants must close their stomata in order to prevent excess water loss. This closure is mediated by increased synthesis of the hormone abscisic acid (ABA). Upon binding to its receptor ABA rapidly induces accumulation of ROS which ultimately results in changes in stomatal aperture. ROS levels must be tightly regulated to ensure rapid ROS synthesis for productive signaling while preventing ROS from reaching damaging levels through upregulation of enzymatic and small molecule antioxidants. Chemical ROS show that mutants with decreased synthesis of flavonol antioxidants have increased ROS accumulation and enhanced rate of ABA-dependent guard cell closure. Contrastingly, Arabidopsis mutants with defects in plasma membrane respiratory burst oxidase homolog (RBOH) enzymes RBOHD and RBOHF have impaired guard cell closure and reduced ABA-induced ROS accumulation compared to wild type. However, we detect ABA-induced ROS production in numerous subcellular locations such as the cytoplasm, chloroplasts, nucleus, peroxisomes, and in unidentified punctate structures, some of which do not coincide with plasma membrane localized RBOHs. In order to better understand what types of ROS are induced by ABA, we monitored accumulation of three different chemical ROS probes in Arabidopsis guard cells before and after ABA treatment. Dichlorofluorescein (DCF), a sensor that measures general redox state, displays increased accumulation in the guard cell nuclei and chloroplasts. Peroxy orange 1 (PO1), a hydrogen peroxide-selective sensor, shows apparent increases in the chloroplast boundary while there is no significant increase observed in the nucleus. Lastly, dihydroethidium (DHE), which reacts with superoxide, exhibits a stark increase solely within the nucleus following ABA treatment. However, this increase in DHE signal is mitigated in a mutant deficient in the superoxide-producing enzymes RBOHD and RBOHF. Together, this suggests that different ROS types exhibit differential spatial regulation during ABA-induced stomatal closure. We are also asking whether ROS is produced by RBOH enzymes in sites other than the plasma membrane or by alternate mechanisms. Monitoring the colocalization of RBOHD-GFP with the plasma membrane dye FM4-64 raises the possibility that these puncta may include RBOH enzymes that are internalized from the plasma membrane. We have begun testing the identity of these punctate structures using mutant and transgenic lines containing organelle-specific reporters with early results showing they do not colocalize with peroxisomes nor the endocytic trafficking proteins ara6 and ara7. Altogether, these results will allow us to determine how different types ROS signals are generated as well as how they are spatially communicated throughout Arabidopsis guard cells during drought stress.
Novel vesicular trafficking component modulates Fe sensing and IRT1 accumulation in Arabidopsis thaliana
Alani Antoine-Mitchell1,2; Gayani Ekanayake1,2, Lee-Ann Niekerk3, Nga Nguyen1,2, Marshall Keyster3, David Mendoza-Cozatl2,4, Antje Heese1,2
1Div. of Biochemistry, 2Interdisciplinary Plant Group (IPG), 221 Schweitzer Hall, University of Missouri, Columbia, MO (USA);3Dept. of Biotechnology, University of the Western Cape, Western Cape, South Africa;4Div. of Plant Sciences, University of Missouri, Columbia, MO, (USA)
Plants serve as a vital food source for humans to prevent Fe deficiency that plagues >1.7 billion people worldwide. It is critical that plants take up the correct amounts of Fe, as excess amounts of Fe are highly toxic; but too little Fe uptake is detrimental to plant growth and development. Overall, the underlying molecular mechanisms and components regulating Fe uptake from the soil into roots and its transport into the central vasculature from root to shoot are not well understood.
As a critical component of Fe uptake from the soil, the Arabidopsis root epidermis specific iron transporter, IRON REGULATED TRANSPORTER 1 (IRT1), needs to be at the plasma membrane (PM) in the right abundance, so that Fe can be transported from the soil into roots when needed. Increasing evidence indicates that vesicular trafficking plays a critical role in modulating the PM accumulation of IRT1 for effective Fe uptake. When sufficient amounts of Fe have been acquired, IRT1 is removed from the PM by endocytosis to prevent excess Fe uptake. Under Fe deficiency, new biosynthesis of IRT1 as well as trafficking of IRT1 from endosomal storage compartments result in increased IRT1 accumulation in the PM. Overall, relatively few vesicular trafficking components have been identified that regulate IRT1 trafficking to and from the PM.
Here, we identified VESICULAR TRAFFICKING PROTEIN 5 (VES5) as a novel component for Fe sensing and PM accumulation of IRT1. Using biochemical fractionation, we have evidence that loss of VES5 resulted in altered PM abundance of IRT1. We also observed defects in the expression of Fe-response genes in two independent ves5 mutant alleles as determined by quantitative real-time PCR. In the longer term, our goal is to gain a better understanding in how VES5 regulates a) the PM accumulation of IRT1 and other Fe response components and b) Fe responses between roots and shoots.
Functional Characterization of CSLD2, CSLD3, and CSLD5 Proteins during Cell Wall Synthesis
Jiyuan Yang, Fangwei Gu, Abira Sahu, Sarah Jaksich, Erik Nielsen.
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109
Plant cell expansion is critical for maintaining cell shapes and it requires coordinated activities of various cellular processes. During tip growth, new cell wall materials are deposited at the plasma membrane domain in a restricted manner resulting in a highly polarized cell expansion. Previous studies demonstrated that members of the Cellulose Synthase-Like D (CSLD) subfamily, particularly CSLD3, plays an important role in this process. CSLD3 displayed β-1,4 glucan synthase activity and is also critical for root hair growth. However, it is not known if CSLD3 is essential for development of non-tip growing cells like leaf epidermis and trichomes. In addition, biochemical activities of the other CSLD proteins are also not well understood. Here, we have conducted a detailed phenotypic investigation of csld2, csld3 and csld5 mutants. We show that apart from its role in tip growth of root hairs, CSLD3 is also essential for overall root growth, trichome branching and lobe formation in leaf epidermal cells. Moreover, CSLD5 displayed a unique and irreplaceable function in the formation of cell plates. We also demonstrate the gene dosage effect of CSLD proteins during root hair elongation. We further show that CSLDs do not require the simultaneous presence of different isoforms to perform catalytic cell wall synthase activities. Further in vitro biochemical activity experiments confirmed that CSLD2, CSLD3, and CSLD5 proteins displayed same conserved β-1,4 glucan synthases activities. Thus, this project provides a holistic view of functional activities of vegetatively expressed CSLD proteins in plant growth and development.
An Interrogation of Kinesin-14 Evolution Reveals Novel Motors in the Spindle Midzone for Establishing the Bipolar Mitotic Apparatus
Xiaojiang Guo
Department of Plant Biology, College of Biological Sciences, University of California
Microtubule motors in the Kinesin-14 subfamily proliferated in photosynthetic organisms and they often incorporated novel structural features. To gain insights into the diversified functions of Kinesin-14 motors from an evolutionary perspective, we performed phylogenetic analysis across different eukaryotic kingdoms. Compared to fungi that have a single class of Kinesin-14, the early divergent protist Giardia possesses two classes and the motile green alga Chlamydomonas produces four classes (Kinesin-14I to Kinesin-14IV). The fifth class Kinesin-14V first appeared among immotile green algae and the sixth Kinesin-14VI showed up in mosses, concomitantly with the display of 3D growth. In Arabidopsis, we found that Kinesin-14IVa exhibited cell cycle-dependent dynamics and decorated MTs in the spindle midzone from late prophase to anaphase and ultimately at the cell division site in the developing phragmoplast but was excluded from kinetochore fibers. The spindle midzone association was dependent on the motor activity of Kinesin-14IVa which harbors chromatin and MT-binding domains downstream to the catalytic core. Mutant cells lacking the motor became hypersensitive to low doses of MT-depolymerizing agent oryzalin that did not noticeably affect control cells but left behind discrete kinetochore fibers attached to randomly positioned chromosomes in the mitotic kinesin-14iva cells. Other mitotic MT arrays like those of the preprophase band and phragmoplast, however, were not visibly impacted by the loss of the motor. Therefore, our results revealed the function of the novel Kinesin-14IVa motor in the organization of the bipolar mitotic apparatus by acting on a subpopulation of spindle MTs. Such a function should be conserved in organisms that also have genes encoding Kinesin-14 motors in this class.
CURT1A is required for the assembly of grana stacks at the prolamellar body surface in the Arabidopsis etioplast
Zizhen Liang, Wai Tsun Yeung, Keith Ka Ki Mai, Juncai Ma, Zhongyuan Liu, Yau-Lun Felix Chong, Byung-Ho Kang
School of Life Sciences, Chinese University of Hong Kong
We have investigated the degradation of semicrystalline prolamellar bodies (PLBs) and the assembly of grana thylakoids in high-pressure frozen Arabidopsis cotyledon cells. We employed scanning transmission electron tomography to acquire clear tomography data from thicker PLB volumes in heavily stained etioplast sections. The crystal structure of the Arabidopsis PLB was zinc blende-type as the carbon atom lattice in diamonds, not Wurtzite. Within 1 hr after illumination (HAI), the regular PLB lattice became disorganized from the surface and the crystalline architecture disappeared by 2 HAI. Membrane tubules in the irregular lattice thickened and merge to become fenestrated sheets at the PLB cortex. These new membrane laminae spread out over stroma thylakoids between PLBs or became additional layers of growing grana stacks. As the newly emerging thylakoids have highly curved membranes in their margin, we tested whether CURT1 family proteins are involved in the PLB-to-grana stack conversion. Among CURT1 isotypes, CURT1A was most highly expressed in de-etiolating Arabidopsis cotyledon samples. CURT1A was associated with the PLB surface at the onset of illumination, and they concentrated to puncta at 2 HAI, where grana stacks arose. Etioplasts of T-DNA inserted mutant of CURT1A failed to construct grana stacks from PLBs because nascent thylakoids were swollen and failed to pile up. Our study showed that the transformation of PLB tubules into thylakoid lamellae via fenestrated sheets is analogous to the cell plate assembly from tubulovesicular intermediates and that CURT1A stabilizes the thylakoid edges exposed to the stroma.
Distinct COPII populations in nutrient deprivation and stress conditions
Baiying LI
The Chinese University of Hong Kong
Higher plants live as sessile organisms with large-scale gene duplication events and paralog divergence during evolution. Plant paralogs are expressed tissue-specifically and fine-tuned by phytohormones during various developmental processes. The coat protein complex II (COPII) machinery is a highly conserved vesiculating machinery mediating protein transport from the Endoplasmic Reticulum (ER) to Golgi apparatus in eukaryotes, also a membrane source for autophagosome biogenesis to maintain cellular homeostasis by recycling metabolites via autophagy. Arabidopsis COPII paralogs greatly outnumber those in yeast and mammals. Knowledge of the functional diversity and underlying mechanism of distinct COPII paralogs in regulating protein ER export or autophagic flux and coping with various adverse environmental stresses is in its infancy. Here, our study demonstrated novel roles of specific AtSar1 homologs mediating distinct populations of COPII vesicles in autophagosome formation upon nutrient deprivation, or in ER export under stress conditions. We identified AtSar1d in the plant ATG (autophagy-related gene) interactome and showed that its mutants impinged autophagic flux and displayed starvation relevant phenotypes. AtSar1d and ATG8e interacted via a non-canonical motif. The plant unique Rab1/Ypt1 homologue AtRabD2a functioned as the molecular switch regulating AtSar1d and relevant COPII vesicle population to contribute to the autophagy pathway. On the other hand, we have also identified another plant COPII vesicle population produced in response to phytohormone regulating abiotic stress responses. Phytohormone-induced specific COPII vesicles are regulated by specific COPII paralogs and carry stress-related channels/transporters for alleviating stresses. Our study thus provides new mechanisms underlying stress-modulating endomembrane trafficking in Arabidopsis.
The Plant Energy Sensor SnRK1 Phosphorylates FREE1 to Regulate Autophagosome Closure Through Orchestrating the ESCRTIII Complex and Core Autophagy Machinery
Yonglun Zeng1,8, Baiying Li18, Shuxian Huang1,8, Hongbo Li2, Zhenping Li1, Yixuan Chen1, Chao Yang2, Jiayang Gao1, Sze Wan Lo1, Jierui Zhao4,5, Jinbo Shen6, Caiji Gao2, Yasin Dagdas5, and Liwen Jiang1
1School of Life Sciences, Centre for Cell & Developmental Biology and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China; 2Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University (SCNU), Guangzhou, China; 3CUHK Shenzhen Research Institute, Shenzhen 518057, China; 4Vienna BioCenter PhD Program, Doctoral School of the University at Vienna and Medical University of Vienna, Vienna, Austria; 5Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna BioCenter (VBC), Vienna, Austria; 6State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Hangzhou, China; 7Institute of Plant Molecular Biology and Agricultural Biotechnology, The Chinese University of Hong Kong, Shatin, Hong Kong, China; 8 These authors contribute equally; 9 Lead contact
Plants sense environmental nutrients via the evolutionary conserved energy sensor SnRK1, which participates in a plethora of catabolic processes including autophagy. Autophagy is mediated by de novo formed double-membrane organelle called autophagosome. Although the early steps of autophagosome biogenesis is studied in depth, the closure of the autophagosome, especially the degree to which nutrient sensing impinges on the autophagosome closure remains elusive. Here, we provide the mechanism underlying a plant unique protein FREE1, upon autophagy-induced SnRK1-mediated phosphorylation, functions as a linkage between ATG conjugation system and ESCRT machinery to regulate the autophagosome closure upon nutrient deprivation. Using high-resolution microscopy and 3D-electron tomography, we showed that unclosed autophagosomes accumulated in free1 mutants. Proteomic, cellular and biochemical analysis revealed the mechanistic connection between FREE1 and the ATG conjugation system/ESCRTIII complex in regulating autophagosome closure. Mass spectrometry analysis showed that SnRK1 phosphorylates FREE1 and recruits it to the autophagosomes to promote closure.
Understanding the mechanisms of cell shape regulation in jigsaw puzzle-shaped leaf pavement cells
Francois Jobert, Siamsa M. Doyle, Sijia Liu, Zahra Rahneshan and Stephanie Robert
Umea Plant Science Centre, Swedish University of Agricultural Sciences
Pavement cells are the basic epidermal cell type of the leaf and they have been proposed to play a number of important roles. These include protecting the leaf, spacing out other epidermal cells and contributing to epidermal integrity, mechanical strength and resistance to mechanical stresses (Javelle et al., 2011; Sapala et al., 2018). In many species, pavement cells develop lobes and necks that interdigitate with those of their neighbors to form a jigsaw puzzle-like pattern. While some details regarding the coordinated regulation of these intriguing cell shapes are known, such as the involvement of phytohormone signaling, the cytoskeleton, mechanical stresses and ultimately, the cell wall, many details still remain to be determined (Liu et al., 2021). We are taking several parallel approaches to understand pavement cell shape regulation in Arabidopsis, which displays rather extreme interdigitation, and poplar, which displays varying degrees of interdigitation depending on the accession, leaf type and growth conditions (Liu et al., 2021).
In Arabidopsis, around half of leaf pavement cells arise from spiral patterns of cell divisions forming anisocytic complexes. We previously demonstrated that the phytohormone auxin displays a response gradient within these anisocytic spirals, which fluctuates according to the stage of first lobe formation in the youngest pavement cell (Grones et al., 2020). Our work suggests that directional auxin transport controlled by auxin transporters regulates lobe formation in young pavement cells. We have since revealed that many cell wall-defective mutants display altered auxin response gradients in the anisocytic spirals. According to our previous work and the work of others, cell wall properties play major roles in regulating shape formation in pavement cells (Majda et al., 2017) and we are now investigating the specific cell wall defects of these mutants to further link cell wall properties with auxin-regulated lobe formation in pavement cells.
In poplar, we are investigating the high plasticity of leaf pavement cell shape using a collection of accessions from different Swedish latitudes. We performed detailed morphological analyses of cell shape features in these lines and chose those displaying the most and least complicated cell shapes for genome wide association mapping, RNAseq and quantitative proteomic analysis. The aim of this work is to integrate genomic and proteomic data and compare this among poplar accessions displaying highly lobed and more simple pavement cell shapes to understand the regulation and plasticity of cell shape in this species.
In parallel, we aim to discover new molecular regulators of the highly interdigitated pavement cell shape in Arabidopsis by screening GFP enhancer trap lines for genes expressed exclusively or more strongly in the youngest pavement cells of the anisocytic spirals. In this way, we aim to discover regulators of early lobing events and/or the transition from the non-lobed to lobing stage.
Grones et al., PNAS (2020) 117:16027-16034; Javelle et al., New Phytol (2011) 189:17-39; Liu et al., Ann Rev Plant Biol (2021) in press; Majda et al., Dev Cell (2017) 43:290-304; Sapala et al., eLife (2018) 7:e32794
Cytokinin modulation of the microtubules dynamics, a mechanism regulating cell differentiation in plants
Juan Carlos Montesinos, Clara Sanchez-Rodriguez, Eva Benkova
IST Austria, ETH Zurich
The dynamic network of cellular microtubules provides the necessary platform to accommodate processes associated with the transition of cells through the individual phases of cytogenesis. We have observed that the plant hormone cytokinin fine‚Äêtunes the activity of the cortical microtubules during cell differentiation in Arabidopsis thaliana root cells, delineating the microtubules array orientation and modulating their plus-end growth.
The endogenous upward gradient of cytokinin activity along the longitudinal growth axis in Arabidopsis roots correlates with the required rearrangements of the microtubule cytoskeleton in epidermal cells progressing from the proliferative to the differentiation stage. Controlled increases in cytokinin activity result in premature re‚Äêorganization of the microtubule network from transversal to an oblique disposition in cells prior to their differentiation, whereas attenuated hormone perception delays cytoskeleton conversion into a configuration typical for differentiated cells. We have observed that a specific cytokinin receptor, CRE1/AHK4, and one of the cytokinin-related transcription factors, ARR1, are necessary for the cytokinin-mediated effect on microtubules dynamics. Interestingly, cytokinin also counteracts microtubular rearrangements driven by the hormone auxin. How cytokinin signaling transduces the signal to microtubules and what molecular components are participating in this process is largely unknown. In our current research line, we aim to address these so far unanswered questions and to investigate this unexplored area.
Establishing the molecular role of SINE proteins in regulating stomatal dynamics in Arabidopsis thaliana
Morgan Moser, Iris Meier
Department of Molecular Genetics, The Ohio State University, Columbus, OH
Drought is a devastating natural disaster that contributes to agricultural losses and subsequent food insecurity worldwide every year. The effects of drought on plants are worsened by transpiration (water loss) through the opening of epidermal pores known as stomata. Stomata are composed of a pair of specialized plant cells called guard cells (GCs) that form a pore. Under drought conditions, the plant hormone abscisic acid (ABA) initiates a signaling cascade, causing volume changes in GCs that result in stomatal closure, thereby restricting water loss. Previous work identified two outer nuclear envelope transmembrane proteins that are expressed in GCs, SINE1 and SINE2. ABA-induced stomatal closure is impaired in null mutant lines sine1-1, sine2-1, and sine1-1 sine2-1, with addition of exogenous calcium or hydrogen peroxide inducing closure significantly more than ABA alone. SINE1 has also been shown to associate with F-actin.
To further characterize the molecular role of SINE1 and SINE2 in ABA-induced stomatal closure, we are investigating how the lack of either protein affects three key crucial signaling events in the ABA pathway: actin cytoskeleton rearrangement, calcium signaling/oscillations, and reactive oxygen species (ROS) production. We demonstrate that ROS production is not delayed or abolished in sine mutants in response to ABA. Using genetically-encoded calcium sensors in wild-type and sine mutants, we show that (1) cytoplasmic calcium oscillations are impaired in sine2-1 but not sine1-1; (2) addition of exogenous calcium or disruption of actin organization rescues calcium oscillations in sine2-1; (3) nuclear calcium signaling occurs during ABA-induced stomatal closure and is also disrupted in sine2-1. Additionally, we are testing if SINE1 and/or SINE2 genetically interact with the ARP2/3 complex. ARP2/3 complex null mutants have a impaired stomatal closure, similar to sine mutants, as well as aberrant actin organization. Thus far we show that ABA-induced stomatal closure is also impaired in the arpc4-1 sine1-1 and arpc4-1 sine2-1 double mutants, suggesting SINE1, SINE2, and the ARP2/3 complex may function together. This research will help elucidate regulation of stomatal dynamics in response to stress and can provide valuable information used for drought tolerant crop creation.
Investigating the Role of the Class XI Myosin in F-actin Dynamics required for Sperm Nuclear Migration in the Arabidopsis Central Cell
Umma Fatema Mohammad Foteh Ali, Tomokazu Kawashima
Dept of Plant and Soil Sciences University of Kentucky
Fertilization is the process of fusion of haploid male and female gametes to develop a new individual. In most animals, microtubules drive the migration of the female pronucleus toward the male pronucleus for fertilization. By contrast, the fertilization process in flowering plants is dependent on actin filament (F-actin) dynamics; F-actin, not microtubules, is responsible for sperm nuclear migration. The molecular and cellular mechanisms by which flowering plants utilize F-actin for fertilization are largely unknown. Using the pharmacological and genetic approaches with the combination of live-cell confocal imaging, we have identified the involvement of a class XI myosin, XI-G in the active movement of F-actin essential for sperm nuclear migration. The primary function of plant myosins is a cargo transporter along F-actin, and we discover a non-canonical function of the myosin XI-G that can generate forces for the dynamic movement of F-actin for fertilization. I am further investigating the mechanism of how class XI-G plays its role in the unique F-actin dynamics in the female gametophyte. Knowledge from this project will shed light on our profound understanding of fertilization and cytoskeleton usage in flowering plants.
Plant homologs of PIEZO mechanosensitive ion channels localize to the tonoplast and affect vacuolar morphology
Ivan Radin1,2, Ryan A. Richardson1,2, Joshua H. Coomey1,2, Carlisle S. Bascom3#, Ting Li4, Magdalena Bezanilla3, Elizabeth S. Haswell1,2
1Department of Biology, Washington University in St. Louis, MO USA; 2NSF Center for Engineering Mechanobiology 3 Department of Biological Sciences, Dartmouth College, NH USA; 4California Institute of Technology, Pasadena, CA USA; #Current address: Division of Biological Sciences, University of California San Diego, USA.
All cells and organisms have to perceive and respond to both internal (e.g., osmotic pressure) and external (e.g., gravity and touch) mechanical forces and cues. One of the main mechanisms of mechanoperception is the action of mechanosensitive (MS) ion channels, which open in response to membrane tension. Here we focus on one eukaryote-specific MS channel family, PIEZO. The well-studied animal PIEZO homologs are plasma membrane-based MS calcium channels that play essential roles in the perception of touch, proprioception, shear, and compressive forces, etc. In plants, PIEZOs have been implicated in systemic virus propagation and root cap mechanosensing, but their cellular localization and molecular and cellular mechanisms remain open questions. We worked with the moss Physcomitrium (formerly Physcomitrella) patens, a very versatile plant model species. P.patens is not only a representative of an important land plant lineage, the Bryophytes, but is also amenable to cultivation, genetic modification, and imaging. P. patens has two functionally redundant PIEZO homologs (PpPIEZO1 and PpPIEZO2), the likely products of gene duplication in the ancestor of mosses. Using CRISPR/Cas9 gene editing, we deleted both PpPIEZOs, which altered growth, size, shape, and cytoplasmic calcium oscillations of tip-growing caulonemal cells. Surprisingly, both moss PIEZO homologs localized to vacuolar membranes, in contrast to their animal counterparts. Furthermore, PpPIEZO loss-of-function mutants had large and expanded vacuoles in apical caulonemal cells, unlike the tubule-like morphology of the WT. Overexpression of either PpPIEZO1 or 2 suppressed this vacuolar phenotype but also increased the abundance of intravacuolar membrane structures. We also mutated the channel pore-lining helix residue (R2508K/H) in PpPIEZO2, inspired by mutations of homologous residues in mammalian PIEZOs, which cause altered channel behavior (slower inactivation) and disease. The moss lines carrying these gain-of-function mutations showed dramatic changes in vacuolar morphology including the extreme membrane lamination, where multiple membrane layers were packed on top of each other within the vacuolar lumen. We next tested if vacuolar localization and function are conserved among plant PIEZOs. A homolog from Arabidopsis thaliana, AtPIEZO1, also localized to the tonoplast in moss and Arabidopsis cells and could functionally replace PpPIEZOs in apical caulonemal cells. Furthermore, the vacuoles of pollen tubes (another tip-growing cell type) from atpiezo1 mutants were more expanded and closer to the tip than those in the WT, mirroring our observations in moss. Thus, assuming we can use moss and Arabidopsis as proxies for their lineages, vacuolar localization and a role in controlling vacuolar morphology in tip-growing cells are conserved among PIEZO homologs in land plants. Moreover, the divergence of plant and animal PIEZO homologs could reflect the higher freedom of movement of tonoplast compared with the plasma membrane in plant cells, making it a preferred location to sense and respond to mechanical changes within the cell. Plant PIEZOs may still release calcium into the cytosol, just from vacuolar rather than extracellular stores.
Synergistic roles of Clathrin Coated Vesicle (CCV) components in modulating basal plant immune responses.
Kelly Mason1, Tessa Jennings2, Erica LaMontagne1, Maha Hamed1, Alex Clarke1, Nga Nguyen1, Antje Heese1
1Div. of Biochemistry, 2Div of Plant Sciences, Interdisciplinary Plant Group (IPG), University of Missouri, Columbia MO (USA)
A substantial percentage of crops are lost each year due to pathogenic attack leading to significant economic loss and reduced food security. Understanding the molecular mechanisms behind plant immunity can help to inspire novel approaches to engineering more resistant crop species. A crucial point of contact between a plant cell and its environment is the plasma membrane (PM) because it contains various proteins with immune-related functions. One class of PM proteins are immune receptors that recognize extracellular microbial pathogens and initiate defense responses. Other PM-localized proteins contributing to downstream immune signaling include callose synthases, which are critical for callose deposition outside of the plant cell. Callose is a beta-1,3-glucan that is upregulated and deposited in plant cell walls during plant defense to help reinforce a compromised cell wall or to control plasmodesmata permeability. Increasing evidence shows that clathrin-coated vesicle (CCV) components contribute to effective immune responses by modulating the PM abundance of immune-related proteins, such as immune receptors and callose synthases [1, 2].
The goal of this project is to delineate novel role(s) of CCV components in regulating the PM composition during plant immunity in the model system Arabidopsis thaliana. Here, we focused on two CCV components previously implicated in plant immune signaling and cellular trafficking of PM proteins. Because both single ccv null mutants demonstrated defects in immune signaling, we created a double ccv null mutant to investigate their potential genetic interaction. We observed an upregulation of defense responses, including increased expression of immune marker genes and callose deposition, in the absence of any stimulus (basal conditions). In conclusion, we identified novel genetic interactions between CCV components and their requirements in modulating basal defense responses.
1. Ekanayake, G., E.D. LaMontagne, and A. Heese, Never Walk Alone: Clathrin-Coated Vesicle (CCV) Components in Plant Immunity. Annual Review of Phytopathology, 2019. 57(1): p. 387-409.
2. LaMontagne, E.D. and A. Heese, Trans-Golgi network/early endosome: a central sorting station for cargo proteins in plant immunity. Current Opinion in Plant Biology, 2017. 40: p. 114-121.
Transcriptional competition shapes proteotoxic ER stress resolution
Dae Kwan Ko, Federica Brandizzi
Great Lakes Bioenergy Research Center, Michigan State University, East Lansing, Michigan, 48824
Through dynamic activities of conserved master transcription factors (mTFs), the unfolded protein response (UPR) relieves proteostasis imbalance of the endoplasmic reticulum (ER), a condition known as ER stress. Because dysregulated UPR is lethal, the competence for fate changes of the UPR mTFs must be tightly controlled5. However, the molecular mechanisms underlying regulatory dynamics of mTFs remain largely elusive. Using machine learning and gene-regulatory network analyses in Arabidopsis, we identified the abscisic acid (ABA) signaling regulator G-class bZIP TF2 (GBF2) and the cis-regulatory element G-box as regulatory components of the UPR led by the mTFs, bZIP28 and bZIP60. Here we demonstrate that, by competing with the mTFs at G-box, GBF2 represses UPR gene expression. Conversely, a gbf2 null mutation enhances UPR gene expression and suppresses the lethality of a bzip28 bzip60 mutant in unresolved ER stress. By demonstrating that GBF2 functions as a transcriptional repressor of the UPR, we address the long-standing challenge of identifying shared signaling components for a better understanding of the dynamic nature and complexity of stress biology. Furthermore, our results identify a new layer of UPR gene regulation hinged upon an antagonistic mTFs-GFB2 competition for proteostasis and cell fate determination.
Tale of the nucleus: A protein in the nuclear membrane is required for correct division plane orientation
M. Arif Ashraf, Michelle Facette
Department of Biology, University of Massachusetts Amherst, Amherst, MA, USAbr>
Both plants and animals rely on asymmetric cell division to generate new cell types, which is a core characteristic of multicellular organisms. Prior to asymmetric cell division, cell polarity is established. Cell polarity establishment and asymmetric cell division are universally important, although proteins important for polarity differ in plants and animals. Zea mays stomatal development serves as an excellent plant model system for asymmetric cell division. In this process, the nucleus migrates to the future division site after polarity establishment and before cytokinesis. In this study, we examined a mutant of the outer nuclear membrane protein, which is part of the LINC (linker of nucleuoskeleton and cytoskeleton) complex. Previously, plants harboring mutations in Mlks2 (Maize LINC KASH AtSINE-like) were observed to have abnormal stomata (Gumber et al. 2019 Nucleus). We confirmed stomatal defects such as abnormal subsidiary cell size and shape, aborted guard mother cell, and extra inter-stomatal cells. We used cell markers to pinpoint the precise defects that lead to abnormal asymmetric divisions. Early markers of cell polarization are normal in mlks2 mutants. Nuclear polarization is impaired. Notably, our data indicate that wrong preprophase band forms in the cells with abnormal nuclear positioning (offset and completely unpolarized). Additionally, spindle orientation is more variable in mlks2 mutants. Misoriented phragmoplasts are observed, consistent with abnormal division planes. Future studies using time-lapse imaging will clarify if cells with abnormal nuclear positioning is directly linked wrong preprophase band and variable spindle angles. We will also determine how nuclear migration and spindle angle ultimately relate to division plane maintenance and phragmoplast guidance. Altogether, our study will help to dissect the role of nuclear movements during different steps of asymmetric cell division including polarization, division plane establishment, division plane maintenance, mitosis, and cytokinesis.
The vacuole - cytoskeleton connection: a handle to regulate growth?
Sabrina Kaiser, Sophie Eisele, Fabian Ries, Frederik Sommer, Felix Willmund, Michael Schroda, David Scheuring
University of Kaiserslautern, 67663 Kaiserslautern, Germany
Changing the morphology of the plant vacuole depends on the integrity of the cytoskeleton. So far, only little is known about the nature of this relationship. The plant-specific Networked (NET) family of membrane-associated actin-binding proteins contains two members, NET4A and NET4B, which are of special interest since NET4A has been shown to localize to the vacuolar membrane. Recently, we could show that NET4 modulates the compactness of vacuoles and this in turn affects its space-filling function and eventually inhibits cell elongation and growth. Since the net4a net4b double mutant only has a mild phenotype and other NETs seem not to compensate, we expected a higher molecular complexity and searched for new interactors. Pull-down experiments followed by MS-MS identified several new interactors, potentially participating in the regulation of the vacuole-cytoskeleton interface.
KIPK-mediated termination of hypocotyl negative gravitropism
Yao Xiao, Benjamin Weller, Dorina P. Janacek, Ulrich Z. Hammes, Claus Schwechheimer
Chair of Plant Systems Biology, School of Life Sciences, Technical University of Munich, Emil-Ramann-Strasse 8, 85354 Freising, Germany
Plants use tropic growth responses to flexibly grow toward or away from directional environmental stimuli (gravity, light, water, salt, etc.). During hypocotyl gravity response, starch-filled amyloplasts in the endodermal cells redistribute to react to changes in the gravity vector. The polarity of the auxin exporter PIN3 (PIN-FORMED3) was proposed to be altered to cause the asymmetric distribution of auxin, which results in the differential cell elongation at the upper and lower side of the hypocotyl. During and after bending, antagonistic control mechanisms must adjust and terminate the bending through auxin-mediated feedback signaling. Until now it is unknown how gravity-trigged amyloplast redistribution is sensed in the cell and how bending is negatively controlled.
We identified the Arabidopsis AGC1 kinase KIPK (KCBP-INTERACTING PROTEIN KlNASE) family as negative regulators of hypocotyl bending during gravitropism. The mutant of the KIPK gene family displays hyperbending of hypocotyls after gravistimulation. The auxin reporter DR5::GUS/GFP shows a decreased auxin content in the cotyledon and hypocotyl of the kipk mutant, suggesting that the overbending cannot be explained by enhanced asymmetric auxin distribution. KIPKs activate PIN3-mediated auxin transport through phosphorylation in a frog oocyte-based transport system. KIPK localizes to the basal plasma membrane in endodermis cells. However, neither a polarity shift of PIN3 nor of KIPK was observed in the endodermal cells of wildtype and kipk mutant hypocotyls. Unlike other AGC1 kinases, KIPK localization is insensitive to phospholipid biosynthesis inhibitors, and its plasma membrane association is mainly determined by its N-terminus and its kinase activity. Taken together, we have identified a novel AGC1 kinase family as a negative regulator of gravitropic responses that may act by controlling PIN auxin transporters at the plasma membrane.
Mechanosensitive ion channel MSL10 genetically and physically interacts with ER-plasma membrane contact site proteins
Jenny CodjoeJenny Codjoe, Elizabeth S. Haswell
Jenny Codjoe
It has recently become clear that many parts of a cell are involved in the perception and response to mechanical forces. Cellular membranes can be tethered to each other at locations called membrane contact sites, and these are important locations of signaling, ionic flux, and lipid transfer in both plants and animals. Endoplasmic reticulum- plasma membrane (ER-PM) contact sites in plants have been implicated in mechanical signaling, but how they participate is unknown. Here, we show that a mechanosensitive ion channel from Arabidopsis thaliana, MSL10, genetically and physically interacts with proteins at endoplasmic reticulum-plasma membrane (ER-PM) contact sites. A gain-of-function MSL10 allele (msl10-3G, MSL10S640L) causes growth retardation and ectopic cell death in adult plants. We sought to uncover components of the signaling pathway promoted by MSL10 that causes these phenotypes. I found that MSL10 co-immunoprecipitates with three major constituents of ER-PM contact sites in plants: the vesicle-associated proteins VAP27-1 and VAP27-3, and synaptotagmin (SYT)1. MSL10 directly interacts with VAP27-1 and VAP27-3 in the split-ubiquitin yeast-2-hybrid system and BiFC. Furthermore, a forward genetic screen identified mutants with missense mutations in SYT5 and SYT7 that cause suppression of msl10-3G phenotypes. At ER-PM contact sites, SYT5 and SYT7 are known to interact with SYT1, a protein that helps maintain PM integrity in response to mechanical pressure (Perez-Sancho 2015), and forms ER-PM contact sites adjacent to those mediated by VAP27-1. Taken together, these results suggest that the cell death-promoting activity of MSL10 can be modulated by its association with ER-PM contact site proteins and raises the possibility that MSL10 regulates ER-PM connectivity. The results of experiments to test the effect of MSL10 on ER-PM contact site morphology using fluorescent markers will be presented. br>
Connected function of PRAF and GNOM in membrane trafficking controls intrinsic cell polarity in plants
Lu Wang1,2, Dongmeng Li1,2, Kezhen Yang3, Juan Dong1,2
1Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA; 2Department of Plant Biology, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901, USA; 3Key Laboratory of Plant Molecular Physiology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
Cell polarity is a fundamental feature underlying cell morphogenesis and organismal development. In the Arabidopsis stomatal lineage, the polarity protein BASL controls stomatal asymmetric cell division. However, the cellular machinery by which this intrinsic polarity site is established remains unknown. Here, we identify the PRAF proteins (containing domains of PH, RCC1 and FYVE) as BASL physical partners and mutating four PRAF members leads to defects in BASL polarization. Members of PRAF proteins are polarized in stomatal lineage cells in a BASL-dependent manner. Developmental defects of the praf mutants phenocopy those of the gnom mutants. GNOM is an activator of the conserved Arf GTPases and plays important roles in membrane trafficking. We further find PRAF physically interacts with GNOM in vitro and in vivo. Thus, we propose that the positive feedback of BASL and PRAF at the plasma membrane and the connected function of PRAF and GNOM in endosomal trafficking establish intrinsic cell polarity in the Arabidopsis stomatal lineage.
Cotton fibers as a case study of how cells set their diameter
Benjamin P. Graham, Ethan T. Pierce, Candace H. Haigler
Department of Crop and Soil Sciences and Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC
How the diameter of elongating plant cells is initially set is poorly understood. The control mechanisms for setting diameter may differ from the classical constraint of diameter via the transverse orientation of cellulose microfibrils and other cell wall components. We investigated mechanisms controlling different cell diameters in the context of cotton fibers, which are highly elongated seed epidermal cells in Gossypium species with worldwide importance as a renewable textile material. The diameter of the fiber cells is a critical factor in the fiber quality parameter of fineness (mTex, mg/km), which in turn impacts the diameter/strength relationship of yarn. The fiber of the most commonly grown cotton species, G. hirsutum, has sub-optimal fineness as compared to the G. barbadense (Pima) cotton, which is preferred for making premium fabrics that are thin and silky while also being strong. This difference in fineness correlates with two fiber types in G. hirsutum, one broad and one narrow (called hemisphere and tapered fibers, respectively), whereas G. barbadense has only one narrow fiber type. Treatment of cultured ovules with the microtubule antagonist, colchicine, demonstrated a brief and transient role of the microtubule array in regulating the formation of narrow fibers as a sub-population on each G. hirsutum ovule. The organization of the microtubule array changes at this time, as we characterized by immunofluorescence microscopy over the first few days of fiber development including fiber initiation, early anisotropic elongation prior to tapering, during the process of tip tapering, and after different fiber diameters had become stabilized prior to rapid elongation. Similar to observations of others during tip refinement of trichome branches, we observed a microtubule depleted zone (MDZ) only at the tips of tapering fibers. Measurements established a moderate relationship between the perimeter of the MDZ and the apical diameter of the tapered G. hirsutum fibers (R2 = 0.73) and a weaker relationship in G. barbadense fibers (R2 = 0.33). Treatment with the cellulose synthesis inhibitor isoxaben at the critical time of tip tapering had less effect on fiber diameter as compared to colchicine, which is consistent with the diameter of elongating cells being set by different mechanisms than the later constraint of diameter via transverse reinforcement. For research support, we thank Cotton Incorporated, Cary, NC.
Clathrin adaptor regulates plasma membrane protein abundance in the roots for effective hormone efflux
Nga Nguyen1, Erica LaMontagne1, Meg Vedra1, Lucia Strader2, Antje Heese1
1Biochemistry Division, IPG, University of Missouri, Columbia, MO, USA; 2Department of Biology, Duke University, Durham, NC, USA
The plant hormone auxin drives growth by mediating cell division and expansion. For proper root growth and development, internal levels of auxin (indole-3-acetic acid; IAA) and its storage form, indole-3-butyric acid (IBA), must be tightly regulated. To maintain appropriate intracellular levels of IBA, plants rely on transporters to be at their correct subcellular location, the plasma membrane (PM), to effectively efflux IBA. In Arabidopsis thaliana roots, proper PM localization and abundance of IBA transporters is dependent on a functional vesicular trafficking network; but few vesicle components are known that regulate PM abundance of IBA transporters, which appear to require vesicle components distinct from those trafficking the PIN auxin transporters. Here, we used large-scale quantitative proteomics of enriched PM to identify a novel role for the clathrin adaptor protein EPSIN1 (EPS1) in regulating PM abundance of the IBA transporter PLEIOTROPIC DRUG RESISTANCE9 / ATP BINDING CASSETTE G37 (PDR9/ABCG37), previously shown to be predominantly expressed in the outermost sides of root cap and root epidermal cells. Loss of EPS1 resulted in reduced PM abundance of PDR9 in roots, which correlated with IBA response and growth defects reminiscent of those observed in pdr9-2 null mutants. Thus, our work expands the limited knowledge of vesicle components that help regulate correct accumulation of IBA transporters at the PM to ensure proper plant growth and development.
Identification of new molecular players during plant cytokinesis
Mingqin Chang, Destiny J. Davis, Luca Comai, Georgia Drakakaki
University of California Davis
Plant cytokinesis is fundamentally different from the cytokinesis in animals and fungi, with that the cytoplasm of the dividing cell is separated by the de novo cell plate formation. Cell plate formation requires the coordinated delivery of membrane and secretory products and removal/recycling of excess materials to the division plane. However, it is unclear how these mechanisms are orchestrated. Overcoming genetic lethality of cytokinesis mutants, we took advantage of Endosidin 7 (ES7), as a cytokinesis specific dissecting tool, toward identifying novel proteins and corresponding transport pathways in cytokinesis. Through screening ethyl methanesulfonate (EMS) mutagenized lines expressing the cytokinesis marker YFP-RABA2A, we obtained ES7 resistant mutants namely es7rs. By genetic mapping and the whole genome sequencing of es7r-3, we identified point mutations in candidate genes contributing to ES7 tolerance. Characterization of the candidate genes and their corresponding pathways in callose deposition during cytokinesis will be presented.
A repeated cysteine-containing motif in AGC1 kinases and its role in cell biology
Alina Graf, Franziska Anzenberger, Claus Schwechheimer
Plant Systems Biology, School of Life Sciences, Technical University of Munich, Emil-Ramann-Straße 8, 85354 Freising, Germany
The directed transport of the phytohormone auxin through polarly localized PIN-FORMED (PIN) auxin efflux carriers is essential for the spatio-temporal control of plant development. The Arabidopsis thaliana serine/threonine kinase D6 PROTEIN KINASE (D6PK) is polarly localized at the plasma membrane of many cells where it co-localizes with PINs and activates PIN-mediated auxin efflux. Its fast cycling to and from the basal plasma membrane indicates that this kinase could act as a molecular switch for polar auxin transport. Our goal is to understand the mechanistic basis of D6PK polarity regulation and cycling.
D6PK is a member of the AGC1 subclade in the AGCVIII subfamily of protein kinases, which are characterized by a DFG to DFD motif exchange and an insertion between the kinase subdomains VII and VIII (middle domain). We previously identified the middle domain to be necessary and sufficient for polar plasma membrane association (Barbosa et al. 2016). Furthermore, the interplay of conserved motifs within the middle domain could represent a novel mechanism for phosphoinositide-recognition and cycling to and from the plasma membrane. Interestingly, a five times repeated cysteine-motif as well as a polybasic motif are required for proper membrane association of D6PK and, thus, for its proper function. Mutations in the cysteine-motif do not affect kinase activity in vitro and are thus particularly involved in mediating the cell biological behavior of the kinase. The effect correlates with the proximity to the polybasic motif, suggesting an interplay of the two motifs. Furthermore, D6PK is targeted by post-translational modifications (PTMs) like S-acylations and strongly phosphorylated at the plasma membrane. Both modifications affect kinase localization. Using structural biology and cell biological methods, we are aiming to resolve the mechanism and interdependency of these motifs and PTMs. Since they are evolutionary conserved to differing degree within the AGC1 clade, resolving their mechanistic role for polarity regulation will lead to a deeper understanding of developmental and tropic processes involving polarly targeted AGC1 kinases in planta.
Evolution of two PTENs subfamilies to regulate different vacuolar trafficking routes during plant development
Chloe Champeyroux 1, Bojan Gujas1, Anna Hunkeler1, Mark Roosjen2, Weijers Dolf2, Antia Rodriguez-Villalon1
1Group of Plant Vascular Development, Swiss Federal Institute of Technology (ETH) Zurich, CH-8092, Switzerland. 2Laboratory of Biochemistry, Wageningen University, Stippeneng 4, Wageningen, The Netherlands.
PHOSPHATASE AND TENSIN HOMOLOG DELETED ON CHROMOSOME 10 (PTENs) are original phosphatases with the dual ability to dephosphorylate both lipids and proteins in vitro (Gupta et al., 2002, Pribat et al., 2012). PTENs are found in most eukaryotes. While a single PTEN isoform is encoded in genomes of animals and yeasts, up to four PTENs have been identified in plant genomes. Plant PTENs are divided into two subfamilies (PTEN1s and PTEN2s) with different functions during plant development. Whereas AtPTEN1 has been shown to regulate pollen grain development and pollen tube growth (Gupta et al., 2002, Zhang et al., 2011), recent results from our laboratory suggest that PTEN2s but not PTEN1s are important for xylem, endodermis and, root hair differentiation. Furthermore, PTEN2s have recently been shown to control vacuolar trafficking of soluble (Delgadillo et al., 2020) and endocytic cargoes (Gujas B. unpublished data). To better understand the molecular function of plant PTEN2s, we compared PTEN1s and PTEN2s sequences and identified a plant-specific domain conserved among PTEN2s that is critical for PTEN2s’ function in vacuolar trafficking and xylem development. This domain is important to localize PTEN2s to the Trans-Golgi Network probably by interacting with other proteins. Comparison of PTEN1s and PTEN2s also suggested that they may have different substrate specificities. Although they both can dephosphorylate lipids and proteins in vitro (Gupta et al., 2002, Pribat et al., 2012), they seem to have different preferences in vivo. While AtPTEN1 has been suggested to function as a phosphatase towards PI3P phospholipid (Zhang et al., 2011), in silico and experimental data suggest that PTEN2s function during xylem development would mainly rely on their phosphatase activity towards proteins. Several putative protein targets of PTEN2s have been identified by phosphoproteomics and their role in the regulation of distinct aspects of plant development has been under investigation.
Delgadillo MO, Ruano G, Zouhar J, Sauer M, Shen J, Lazarova A, Sanmartín M, Faat Lai LT, Deng C, Wang P, Hussey PJ, Sánchez-Serrano JJ, Jiang L, and Rojo E, MTV proteins unveil ER- and microtubule-associated compartments in the plant vacuolar trafficking pathway, PNAS (2020) May 5;117(18):9884-9895.
Gupta R, Ting J, Sokolov L, Johnson S and Luan S, A tumor suppressor homolog, AtPTEN1, is essential for pollen development in Arabidopsis, The Plant Cell (2002), Vol. 14, 2495–2507
Pribat A, Sormani R, Rousseau-Gueutin M, Julkowska M, Testerink C, Joubes J, Castroviejo M, Laguerre M, Meyer C, Germain V and Rothan C, A novel class of PTEN protein in Arabidopsis displays unusual phosphoinositide phosphatase activity and efficiently binds phosphatidic acid, Biochem. J. (2012) 441, 161–171
Zhang Y, Li S, Zhou LZ, Fox E, Pao J, Sun W, Zhou C and McCormick S, Overexpression of Arabidopsis thaliana PTEN caused accumulation of autophagic bodies in pollen tubes by disrupting phosphatidylinositol 3-phosphate dynamics, The Plant Journal (2011) 68, 1081–1092
Arabidopsis FLIPPASE as new vesicular trafficking regulator in stress conditions
Adria Sans Sanchez, Vendula Pukysova, Tomasz Nodzynski, Marta Zwiewka
Central European Institute of Technology, Masaryk University, Brno CZ-625 00, Czech Republic
Membrane vesicular trafficking is an essential transport mechanism of proteins and other macromolecules between organelles in plant cells, facilitating maintenance of cellular homeostasis in normal as well as stress conditions. The role of endo- and exocytosis in response to osmotic stress and cellular turgor loss in not full uncovered. It is clear that bending the lipid bilayer occurs during vesicle formation and fusion. This on the functioning of lipid flippases P4-ATPases that are known to be involved in creating lipid asymmetry between leaflets of the membrane and generating its curvature.
Consequently, pleiotropic phenotypes were observed in flippase mutants. It has been shown that defective functioning of flippases leads to loss of the desired lipid distribution within the leaflets, which is likely to result in altered membrane properties.
The creation of membrane curvature and vesicular budding were impaired, leading to impaired protein recruitment to the membrane and trafficking defects. Moreover, it was reported that flipassesfilasses can interact and work with the ARF GEF proteins in plants and yeast cells regulating vesicular trafficking. Here we want to explore the roles of the flippase and other elements of trafficking machinery in the context of vesicular trafficking modification as an plant adaptation strategy to stress.
Inferring the distribution of material secretion for tip-growing cells via a mechano-geometric model
Kamryn Spinelli, Min Wu
Worcester Polytechnic Institute
Many plant cells exhibit tip growth in which the cell elongates while preserving a certain tip shape. Among the broader category of tip-growing walled cells, there is a wide variety of tip shapes, but the details of the self-similar growth are not fully understood. Experimental approaches typically measure strain rate, which is a combined effect of material secretion and elastic deformation, but the individual roles of these two components may be difficult to separate. By computationally simulating the geometry and elastic deformation of the cell, and subsequently inferring the secretion rate needed to sustain self-similar growth, we decouple these two phenomena. We demonstrate that our model can indeed simulate self-similar growth and hence also investigate the strong relationship between cell geometry and the spatial distribution of secretion for tip-growing cells.
PDK1 substrate AGC1 PAX kinase controls protophloem differentiation and vein patterning
A. E. Lanassa Bassukas1, Julia Mergner2, Alina Graf1, Yao Xiao1, Bernhard Kuster2, Claus Schwechheimer1,3
1Chair of Plant Systems Biology, Technical University of Munich (TUM), Emil-Ramann-Strasse 8, 85354 Freising, Germany; 2Chair of Proteomics and Bioanalytics, Technical University of Munich (TUM), Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany
"The AGC1 kinase PAX (PROTEIN KINASE ASSOCIATED WITH BRX) in Arabidopsis thaliana interacts with BRX (BREVIS RADIX) as part of a self-reinforcing polarized and membrane associated molecular module required for functional protophloem sieve element (PPSE) differentiation. The molecular interplay of the two polarly localized proteins BRX and PAX has been implicated in the establishment of high steady state subcellular auxin concentrations within the developing protophloem cells, critically required for the differentiation into PPSE. The molecular encounter of both proteins at the plasma membrane of developing protophloem cells is, in turn, dynamically controlled by cellular auxin levels. While BRX, at high intracellular auxin concentrations, dissociates from the plasma membrane through an AGC1 kinase conveyed phosphorylation-dependent mechanism, AGC1 kinase PAX remains plasma membrane-associated. Moreover, aberrant PAX localization disrupts BRX association and polarization at the plasma membrane and leads to differentiation defects in the PPSE strand equivalent to those observed in the pax knock-out mutants. Yet, the mechanisms controlling PAX localization and kinase activity remain poorly understood.
Recent studies have highlighted the functional requirement of the two orthologous PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASES (PDK-1 and PDK-2) in the differentiation process of PPSEs. Apposite to the phenotypical resemblance of pdk-1 pdk-2 double mutants to the AGC1 kinase pax mutant, PDK-1 and PDK-2 kinases phosphorylate the activation loop of PAX as well as the activation loop of the closely related AGC1 D6-PROTEIN KINASEs (D6PKs) at a conserved serine residue in vitro. PDK-1 and PDK-2-dependent phosphorylation of PAX and the D6PKs, as well as an artificial phosphomimetic variant of the respective serine activation loop phosphosite is sufficient to promote AGC1 kinase trans-catalytic competence of both kinase subclasses PAX and D6PK in vitro.
We use a combinatorial approach of phosphoproteomics, immunohistology and genetics, to explore the PAX-dependent signal transduction in Arabidopsis thaliana PPSE differentiation downstream of PDK-1 and PDK-2. In addition to the already described function of PAX in PPSE differentiation, we uncover its role in cotyledon vein pattering. With this work, we believe to further understand PAX-mediated signalling in space and time and its functional implication in protophloem development and cotyledon vein patterning.
References
Marhava and Bassukas et al. (2018): A molecular rheostat adjusts auxin flux to promote root protophloem differentiation. In Nature 558 (7709), pp. 297- 300. DOI: 10.1038/s41586-018-0186-z.
Yao Xiao and Remko Offringa (2020): PDK1 regulates auxin transport and Arabidopsis vascular development through AGC1 kinase PAX. In Nature Plants 6, pp. 544-555. DOI:10.1038/s41477-020-0650-2
"
Traffic Lights of Xylem Cell Differentiation: PTEN2s Regulation of Tonoplast Dynamics
Gujas Bojan, Rodriguez-Villalon Antia
ETH Zurich, Switzerland
Our understanding of cell differentiation into a given cell type is based on morphology analysis and transcriptomic profiling. Shaping the unique morphology and thus physiology also requires general as well as unique cell-type-specific trafficking pathways. However, the information on cell dynamics is somewhat generalized or disconnected from known mechanisms of a given cell type differentiation. Here we aimed at studying the dependency of centrally positioned vacuole biogenesis on Arabidopsis xylem cell type differentiation.
Xylem vascular cell is a unit of plant water transporting pipeline that endures pressure and provides effective conduct. Its complex cell differentiation program was fine-tuned during the evolution of land plants and involves forming a secondary cell wall (SCW), loading programmed cell death (PCD) factors into the vacuole, and precise timing and execution of it. In our previous study, we discovered a positive correlation between vacuolar trafficking speed and xylem cell differentiation. This follow-up study aimed to find a genetic tool that can abolish the formation of a centrally positioned vacuole and to address its importance for xylem development.
Here we show that overexpression of either of the two closely related PHOSPHATASE AND TENSIN HOMOLOG DELETED ON CHROMOSOME TEN 2 (PTEN2) genes: PTEN2a or PTEN2b disturbs the formation of a centrally positioned vacuole in xylem cells but not in root epidermal cells. Moreover, PTEN2s overexpression prevents delivery of known xylem vacuolar cargos to the vacuole (artificial RFP-AFVY, CELLULOSE SYNTHASE 6, and XYLEM CYSTEINE PEPTIDASE 1). Unexpectedly, these phenotypes correlate with a xylem cell failure to form SCW but not to execute PCD.
Although the double mutants of pten2a pten2b do not exhibit any visible xylem phenotype in optimal in vitro conditions, their protein storage vacuole (PSV) formation morphology is affected. In particular, the double mutant fails to remodel the embryonic vacuole into PSVs in cotyledon pavement cells or root epidermal cells during embryogenesis. Consequently, the double mutant mature embryos contain cells with a single large vacuole intersected by multiple membranes, seemingly failing to separate and create multiple small rounded vacuoles typical for wild-type mature embryos.
Our study sheds new light on a xylem cell vacuole type, its dynamics, and its role during xylem differentiation. Moreover, the similarity between a vascular xylem cell and an embryonic cotyledon pavement or root epidermal cell in tonoplast dynamics challenges our classical view of how we group cells into a cell type. Can plant cell dynamics be a neglected criterion when grouping cells into cell types?
Analysis of aberrant, preprophase band-independent TANGLED1 recruitment to the cell cortex in maize
Aimee N. Uyehara, Lindy A. Allsman, Beatrice N. Diep, Sarah Gayer, Janice J. Kim, Carolyn G. Rasmussen
Department of Botany and Plant Sciences, Center for Plant Cell Biology, Institute for Integrative Genome Biology 900 E University Ave. 3126 Genomics, University of California Riverside, CA 92521
The orientation of cell division in plants is critical for proper growth and development. Establishment of the division plane is mediated through the correct placement of the division site. In plants, the formation of a cortical ring of microtubules and actin called the preprophase band (PPB) sets up the division site and predicts the future cell plate insertion site. A small number of proteins are known to colocalize with the PPB and remain at the division site after PPB disassembly in metaphase. One of these proteins is the microtubule binding protein TANGLED1. The PPB has been shown to be required for TAN localization using chemical treatments that disassembled the PPB or PPB-less mutants in BY2 cells and Arabidopsis respectively. However, the mechanism by which TAN is recruited remains unknown. Here we show that TAN1 can be recruited to the cortex through a PPB-independent mechanism in maize.
Imaging of TAN1-YFP in the PPB-less maize mutant discordia1 (dcd1) and alternative discordia1 (add1) revealed that TAN1 is recruited to the cell cortex in late telophase. Because it is difficult to identify the predicted division site in embryo cells, TAN1 localization was observed in the partial loss of function dcd1 single mutant which makes defective PPBs in leaf asymmetric divisions. We observed TAN1 localization in aberrant de novo cell cortex locations, separate from TAN1 colocalization with mutant defective PPBs. Interestingly, TAN1 localization to the cortex in these aberrant locations seemed to follow the phragmoplast. To determine if aberrant TAN1 localization to the cortex was due to defective PPBs or the phragmoplast, wild-type leaves were treated with CIPC, an herbicide that splits phragmoplasts but does not affect PPBs. TAN1 was observed at the cortex in aberrant locations where phragmoplasts ectopically contacted the cell cortex. Further experiments with chemicals that target the cytoskeleton or alter proper new cell wall formation will provide insight into distinct mechanisms by which TAN1 is recruited to the cell cortex independent of the PPB.
Loss-of-function and localization analysis of Sec6 and Sec3 in Physcomitrium patens
Boyuan Liu, Robert Orr, Angela Ai, Kaye Peterman, Mary Munson, and Luis Vidali
Biology and Biotechnology, Worcester Polytechnic Institute. Biochemistry and Molecular Pharmacology, University of Massachusetts, Medical School. Biological Sciences, Wellesley College.
The exocyst is a protein complex that is essential for cell growth and polarization in mammals and fungi. It tethers secretory vesicles at the plasma membrane for fusion which involves direct interactions of the exocyst with PI(4,5)P2 and SNARE proteins. Although the exocyst is essential for plant development, its precise function is not fully understood. We studied the role of exocyst subunit Sec3 and Sec6 in plant development using moss Physcomitrium patens as a model plant. Here, we silenced the target genes by an adenine phosphoribosyltransferase (APT)-based RNAi technology (APTi) assay. We found that the silencing of all Sec3 paralogs, or Sec6 would stop cell growth and possibly cause cell death. We also show that we can rescue the Sec6 loss-of-function phenotype by expressing an RNAi-resistant target gene. We conclude that Sec6 and Sec3 are essential for plant cell growth and survival. Also, we found Sec6-GFP is localized to the cell plate during cytokinesis, and both Sec6 and PIP2 polarize to the growing apical membrane during tip growth.
Role of ARP2/3 complex in Arabidopsis thaliana reproduction
Cifrova Petra, Martinek Jan, Garcia-Gonzalez Judith, Schwarzerova Katerina
Department of Experimental Plant Biology, Faculty of Science, Charles University, Prague, Czech Republic
Pollen tube growth is a finely controlled and polarized process, during which vesicles deliver cell wall material into the apical part of the pollen tube. Clear zone is the most apical part of growing pollen tube, from which big organelles such as vacuoles or amyloplast are excluded. Actin cytoskeleton plays a key role in the pollen tube organization, growth polarity and growth itself. Here we show that ARP2/3 complex, an actin nucleator, is involved in pollen tube growth as well. Our analysis showed that mutants lacking functional ARP2/3 complex or ARP2/3-activating complex have shorter and thicker pollen tubes. Mutants have very short pollen tube clear zone in comparison with wt plants, which corresponds to slower growth of mutant pollen tubes. When we evaluated actin cytoskeleton structure in the clear zone, we found that both actin density and bundling was higher in mutant plants. ß-Glucuronidase reporter gene fused to native promoters of several ARP2/3 subunits confirmed expression of ARP2/3 subunits in growing pollen tubes. Analysis of pollen grains revealed a phenotype of ARP2/3 mutants not yet described. Pollen grains were less viable and smaller. Further, we show that mutants have short siliques with bigger and more round seeds.
Our results show that the loss of ARP2/3 complex results in distinct phenotypes in Arabidopsis reproduction.
Callose and Membrane Dynamics During Cytokinesis In Land Plants And Ancestral Cell Walls
Rosalie Sinclair1, Zaki Jawaid2, Daniel Cox2, Thomas Wilkop3, Georgia Drakakaki1
1Department of Plant Sciences University of California Davis; 2Department of Physics University of California Davis; 3University of Kentucky Light Microscope Core
The cell wall is an integral and dynamic part of plant cells involved in regulating many plant processes. Cell plate formation takes place in four simultaneous stages via the homotypic fusion of vesicles that matures with the deposition of polysaccharides. The polysaccharide callose is thought to stabilize the cell plate and contributing to cell plate maturation. However, despite the central role of cell plate formation, many fundamental questions remain unanswered. In addition, the cytokinesis specific callose synthases are highly recalcitrant to study due to mutant lethality. To overcome these challenges, we have identified novel pharmacological inhibitors, such as endosidin 7 (ES7). ES7 targets callose deposition during late cytokinesis and arrests cell plate maturation, allowing the probing of callose’s spatial-temporal role at the cell plate. Through these studies, callose has proven a crucial transient component to cell plate development in both land plants and algae. A similar necessity for callose has been seen in ancestral cell wall construction, such as in Chlamydomonas reinhardtii and other single and multicellular algae, with sensitivity to ES7 and observed callose during division . We have generated a fluorescently tagged callose synthase (GSL8) and begun probing spatial, temporal dynamics during cell plate development. Using a suite of interdisciplinary techniques, we aim to break apart the molecular black box surrounding callose deposition and membrane development during cytokinesis.
The large scutellar node1 gene is required for auxin-mediated root and vascular development in maize
Janlo M. Robil1, Rhea S. Sablani2, Norman B. Best1, Francine M. Carland3, Paula McSteen1
1Department of Biological Sciences, Interdisciplinary Plant Group, and Missouri Maize Center, University of Missouri, Columbia, MO, USA 65211; 2Department of Biochemistry, Biophysics, and Molecular Biology, Whitman College, Walla Walla, WA, USA 99362; 3Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.
The pleiotropic recessive maize mutant, large scutellar node1 (lsn1), exhibits root and vascular developmental defects in the seedling stage. The lsn1 mutant develops a short primary root with fasciated or flattened tip and abnormal vasculature. The leaves show defects in stomatal cytokinesis and severe aberrations in vein patterning that mimic the effect of treatment with polar auxin transport inhibitor, 1-naphthylphthalamic acid (NPA). Double mutant analyses with auxin biosynthesis and transport mutants in maize indicate that the lsn1 gene functions upstream of auxin transport. Whole genome sequence analysis and positional cloning map the lsn1 locus to 3 Mbp region in chromosome 8, bin 4, which includes a strong candidate gene involved in Golgi-plasma membrane vesicle trafficking. This candidate gene, together with phenotypic and genetic evidence, supports the hypothesis that the lsn1 gene is involved in auxin transport and plays a role in auxin-mediated root and vascular development in maize.
A model for microtubule dynamics in the Arabidopsis phragmoplast
Matthew Hickey, Tetyana Smertenko, Bernard M.A.G. Piette, Sharol Schmidt-Marcec, Andrei Smertenko
Institute of Biologica Washington State University, Durham University
Imaging individual microtubules in the phragmoplast remains impossible due to high concentration of cytoplasmic tubulin. Modeling offers a feasible approach to study the impact of different proteins on phragmoplast microtubule dynamics. The existing model is based on microtubule turnover values (t1/2) generated in tobacco BY-2 tissue culture cells using fluorescence recovery after photobleaching (FRAP). These measurements demonstrated asymmetry of microtubule dynamics in the proximal and distal zones of the phragmoplast. However, such asymmetry has not been shown in Arabidopsis. The main obstacle in determining t1/2 is low photostability of commonly used GFP or mCherry fusions with TuB6 or TuA5. Even expressed under the control of the strong 35S promoter, high zoom settings necessary for imaging relatively small Arabidopsis phragmoplasts leads to photobleaching. We overcame this limitation by using a tubulin isotype that is up-regulated in dividing cells, and by using a more photostable fluorochrome, NeonGreen. Analysis of the publically available A. thaliana gene transcription database (EFP Browser) showed transcription of beta tubulin 2 (TuB2; AT5G62690) was upregulated in the root and shoot apical meristems, developing flowers, and embryos. Homozygous A. thaliana (Col-0) transgenic lines expressing NeonGreen-TuB2 fusion under control of native promoter (proTuB2:NeonGreen:TuB2) labeled major microtubule arrays and showed higher fluorescence signal in the root apical meristem than in differentiated tissues. Higher expression levels of NeonGreen:TuB2 together with higher quantum efficiency and photostability of NeonGreen allowed imaging cells at lower laser power (0.25%) relatively to the regular settings (0.5-1%). FRAP assays were conducted without discernible photobleaching even at high zoom settings. These experiments also demonstrated asymmetry of microtubule dynamics between distal and midzones as has been previously shown in BY-2 cells. The experimental values were used to update the previously published model of phragmoplast microtubule dynamics developed using measurements in tobacco BY-2 cells (Smertenko et al., 2011). We introduced two major modifications to the model: (i) the amount of tubulin in the cell was set as finite; and (ii) rates of microtubule polymerization, transition from pause to growth, transition from catastrophe to pause, and transition from catastrophe to growth were made proportional to the available tubulin concentration; whereas rates of catastrophe, and transitions from growth to pause or catastrophe was inversely proportional to the free tubulin concentration. The model accurately reproduces experimentally determined parameters of tubulin turnover and microtubule length.
Functional Characterization of TAN1 Role in Division Plane Orientation Using an Arabidopsis Synthetic Double Mutant
Alison Mills, Victoria Morris, Carolyn G. Rasmussen.
Department of Biochemistry and Molecular Biology, University of California, Riverside
Division plane orientation is important for plant and animal development, growth, and morphogenesis. TANGLED1 (TAN1) and AUXIN INDUCED IN ROOT CULTURES9 (AIR9) are two unrelated plant microtubule-binding proteins that localize to the division site. Single tan1 and air9 mutants have no discernable phenotypes in Arabidopsis thaliana. However, A. thaliana tan1 air9 double mutants have a synergistic phenotype displaying altered cell file rotation, root growth, and cell division orientation. Transformation with constructs encoding TAN1 or AIR9 is sufficient to rescue these double mutant phenotypes. Surprisingly, driving TAN1 expression with a mitosis-specific promoter fully rescues the double mutant phenotypes, including root growth and cell file rotation. This suggests that TAN1 function is critical during mitosis, and that double mutant phenotypes are a consequence of defects that occurred during mitosis. The first 132 amino acids of the TAN1 protein (TAN1-1-132) localizes to the division site only during telophase and interacts with a number of other proteins involved in division plane orientation, including the division site localized protein PHRAGMOPLAST ORIENTING KINESIN1 (POK1). POK1 fails to localize to the division site during telophase in the tan1 air9 double mutant, suggesting that both TAN1 and AIR9 stabilize POK1 there. Transformation of the tan1 air9 double mutant with TAN1-1-132 significantly rescues the double mutant phenotypes despite only being present at the division site during telophase. Systematic mutagenesis of TAN1-1-132 using six alanine substitutions followed by yeast two-hybrid analysis identified motifs required for TAN1-1-132 interaction with POK1. Alanine substitutions at positions 28-33 in TAN1-1-132 and full length TAN1 disrupted interaction with POK1 and reduced both constructs' ability to rescue the double mutant phenotypes and accumulate at the division site during telophase. Together this evidence suggests that POK1-TAN1 interaction may play a crucial role in TAN1 localization to the division site and TAN1 function.
tls4 functions in auxin mediated vegetative and reproductive development in maize
Leo G Koenigsfeld11,2; Janlo M Robil3; Mika Nevo4; Dennis Zhu3; Norman B Best3, Paula McSteen3
1Department of Plant Science, University of Missouri, Columbia, MO, USA 65211;2Department of Biochemistry, University of Missouri, Columbia, MO, USA 65211; 3Department of Biological Sciences, University of Missouri, Columbia, MO, USA 65211; 4Department of Biology, Whitman College, Walla Walla, WA, USA 99362
The tassel-less4 (tls4) mutant in maize is characterized by reduced plant height, narrow leaves and a smaller or absent tassel (the male inflorescence). Scanning electron microscopy of immature tassel meristems shows defects in both the size of the apical meristem and the production of axillary meristems. As the plant growth hormone, auxin, plays an essential role in apical meristem size and initiation of axillary meristems, stem elongation and leaf margin development, we tested the genetic interaction between tls4 and auxin biosynthesis mutant, vanishing tassel2 (vt2); auxin transport mutant, barren inflorescence2 (bif2) and auxin signaling mutant, Barren inflorescence1 (Bif1). All double mutant combinations showed synergistic interactions in either reproductive (severe tassel defects) or vegetative (reduced apical dominance) development. Therefore, the tls4 gene either, acts parallel to and intersecting with the function of auxin (if the mutation causes a null allele) or acts in the same pathway as auxin (if it’s not a null). Through BSA-seq and fine mapping, we determined that tls4 maps to a 200 kb region, on Chromosome 4 bin 10, containing six genes, one of which has a missense mutation. We are currently testing whether the tls4 candidate gene functions in clathrin-mediated-endocytosis.
Analysis of the preprophase band’s role in recruiting TANGLED1 and its role in division plane orientation in Zea mays
Beatrice N. Diep1, Sarah Gayer2, Janice J. Kim1, Aimee N. Uyehara1, Lindy A. Allsman1, Carolyn G. Rasmussen1
1Department of Botany and Plant Sciences, University of California, Riverside, California, USA; 2Undergraduate Program in Biology, Amherst College, Massachusetts, USA
Cell division is a fundamental process across eukaryotes and improper divisions can have detrimental effects on the organism. In plants, this process is especially important because plant cells cannot migrate and their final positions are permanently established after cytokinesis. To facilitate proper placement of the cell wall, plants employ two unique microtubule structures. The first is a cortical ring of microtubules that form at the cell cortex called the preprophase band (PPB). The PPB forms in preprophase and predicts the future division site, but disassembles to form the metaphase spindle. The second structure is the phragmoplast that inserts the cell plate to the location previously occupied by the PPB. Several proteins help maintain the location of the future division site after PPB disassembly, such as TANGLED1 (TAN1), a microtubule binding protein that colocalizes to the PPB and marks the future division site. The current model is that the PPB establishes the division site and recruits TAN1 to maintain that location after PPB disassembly. While the timing of TAN1 localization is understood, the fundamental mechanism of how TAN1 is recruited to the division site and its role in mediating proper cell division are unclear.
DISCORDIA1 (DCD1) and ALTERNATIVE DISCORDIA1 (ADD1) are redundant genes in Zea mays that promote PPB formation. dcd1 add1 double mutants do not form PPBs and are seedling lethal. We analyzed mutant embryos using confocal microscopy and found that TAN1 does indeed localize in the absence of the PPB, but only in telophase. This suggests that even in the absence of the PPB, TAN1 is still recruited to the cell cortex and that there is an unknown mechanism recruiting TAN1 specifically during telophase. To further investigate how the quality of the PPB may affect TAN1 localization and proper cell division, we imaged leaf tissue from the partial loss of function dcd1 single mutants. Single mutants are reported to have varying PPB defects only in asymmetrically dividing cells. We found that TAN1 had varying localization defects with abnormal and uneven fluorescence, which likely coincides with PPB quality. Additionally, phragmoplast guidance to the correct location seems to correlate with TAN1 localization, and by proxy, the PPB’s establishment of the division site. These two experiments suggest that there are two mechanisms for TAN1 recruitment: a PPB dependent and independent mechanism. Future experiments involving immunoprecipitation will identify potential TAN1 interactors that may be recruiting TAN1 to a division site in telophase, which will provide insight into distinct players involved in cell division.
A Biophysical Model for Plant Cell Plate Development Based on the Contribution of Callose to a Spreading Force
Muhammad Zaki Jawaid1, Rosalie Sinclair2, Vincent Bulone3, Daniel Cox1, and Georgia Drakakaki2
1Department of Physics and Astronomy, University of California, Davis; 2Department of Plant Sciences, University of California, Davis; 3University of Adelaide, School of Agriculture, Food and Wine.
Plant cytokinesis, a fundamental process of plant life, involves de novo formation of a ‘cell plate’ partitioning the cytoplasm of the dividing cell. Cell plate formation is directed by orchestrated delivery, fusion of cytokinetic vesicles, and membrane maturation to form the nascent cell wall by timely deposition of polysaccharides such as callose, cellulose, and crosslinking glycans. In contrast to the role of endomembrane protein regulators the role of polysaccharides, in cell plate development is poorly understood. Callose, a β-1-3 glucan polymer, is transiently accumulated during cell plate expansion to be replaced by cellulose in mature stages. Based on the severity of cytokinesis defects in the absence of callose, it has been proposed that it stabilizes this membrane network structure. However, there is currently no theory to understand its role in cytokinesis.
Here we extend the Helfrich free energy model for membranes including a phenomenological spreading force as an “areal pressure” (force per unit distance) generated by callose and/or other polysaccharides. Regular cell plate development in the model is possible, with suitable bending modulus, for a two-dimensional late stage spreading force parameter of between 2-6pN/nm, an osmotic pressure difference of 2-10kPa, and spontaneous curvature between 0-0.04nm^(-1). With these conditions, stable membrane conformation sizes and morphologies emerge in concordance with stages of cell plate development. With no spreading force, the cell plate fails to mature properly, corroborating experimental observations of cytokinesis arrest in the absence of callose. To reach a nearly mature cell plate, our model requires the late-stage onset that the spreading force coupled with a concurrent loss of spontaneous curvature. A simple model based upon production of callose as a quasi-two-dimensional self-avoiding polymer produces the correct phenomenological form of the spreading force and requires biologically relevant levels of callose.
FERONIA-mediated polar accumulation of NORTIA at the filiform apparatus facilitates intra- and inter-specific pollen tube reception in Arabidopsis thaliana
Yan Ju, Jing Yuan, Daniel Jones and Sharon Kessler
Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana USA; Purdue Center for Plant Biology, Purdue University, West Lafayette, Indiana USA
Successful pollen tube reception in flowering plants relies on the intercellular communication between the attracted pollen tube and the receptive synergid cell of the female gametophyte. The pollen tube sends an arrival signal to the synergid and the synergid responds with subcellular changes that lead to release of a synergid signal that induces pollen tube rupture and release of the sperm cells for double fertilization. Mutations with impaired signaling between the synergid and pollen tube results in pollen tube overgrowth in the synergid and plant infertility. NORTIA (NTA) is a synergid-expressed MLO gene that participates in synergid-pollen tube communication. We used live imaging to track NTA subcellular localization changes during pollen tube reception and manipulated NTA localization to put it into a broader molecular pathway. We found that pollen tube arrival triggers the FERONIA-dependent selective trafficking of NTA from Golgi to the filiform apparatus, a region of highly invaginated membranes at the pollen tube entry point. Artificial manipulation of NTA trafficking (either constitutively to the filiform apparatus (faNTA) or retained in the Golgi) revealed that NTA accumulation at the filiform apparatus is necessary and sufficient for MLO function in pollen tube reception. Moreover, overexpression of faNTA in synergids bypasses the upstream FERONIA/LORELEI signal pathway by suppressing pollen tube overgrowth in the mutants. We also show that faNTA promotes interspecific pollen tube reception when Arabidopsis lyrata pollen is used to pollinate Arabidopsis thaliana. We propose that NTA acts independently of FERONIA in the filiform apparatus to provide an additional “bursting” signal to pollen tubes and that the response to these signals from the synergids varies in pollen tubes from different species.
WPRs Acting As a Downstream of PAN2 Receptor Is Involved in the Polarity Establishment in Maize Stomata
Qiong Nan, Michelle R. Facette
Department of Biology, University of Massachusetts, Amherst, MA, 01003
Polarization of cells prior to asymmetric cell division is crucial for correctly orienting the cell division plane, and for subsequent cell fate and overall tissue patterning. In maize stomatal development, polarization of subsidiary mother cells prior to asymmetric division is controlled by the BRK-PAN-ROP pathway. Two catalytically inactive receptor-like kinases, PANGLOSS2 (PAN2) and PAN1, are recruited by BRICK proteins. PAN1 recruits the small GTPase ROP, followed by polarized actin accumulation. Each of these proteins is polarized in subsidiary mother cells, with the polarization of each protein dependent on the previous one. Surprisingly, most of these proteins do not physically interact with one another, indicating there are likely unknown molecules in the pathway. Here, we identify that the WEB1-PMI2 RELATED (WPR) family of proteins plays an important role in subsidiary mother cell polarization. WPRs physically interact with PAN2 and PAN1, and localize polarly in subsidiary mother cellsat sites of guard mother cell contact. The polarized localization of WPR proteins depends on PAN2 but not PAN1. We show that a subgroup of the WPR proteins directly interact with F-actin through their N terminal domain. Together, these results implicate the WPRs act downstream of PAN2, potentially regulating actin filaments during the polarization process.
Endosidin20-1 is more potent than Endosidin20 in inhibiting plant cellulose biosynthesis
Lei Huang, Xiaohui Li, Chunhua Zhang
Department of Botany and Plant Pathology, Purdue University
Endosidin20 (ES20) is a recently identified cellulose biosynthesis inhibitor (CBI) that targets the catalytic site of plant cellulose synthase (CESA). Here, we screened over 600 ES20 analogs and identified nine active analogs named ES20-1 to ES20-9. Among these, endosidin20-1 (ES20-1) had stronger inhibitory effects on plant growth and cellulose biosynthesis than ES20. At the biochemical level, we demonstrated that ES20-1, like ES20, directly interacts with CESA6. At the cellular level, this molecule, like ES20, induced the accumulation of cellulose synthase complexes at the Golgi apparatus and inhibited their secretion to the plasma membrane. Like ES20, ES20-1 likely targets the catalytic site of CESA. However, through molecular docking analysis using a modeled structure of full-length CESA6, we found that both ES20 and ES20-1 might have another target site at the transmembrane regions of CESA6. Besides ES20, other CBIs such as isoxaben, C17, and flupoxam are widely used tools to dissect the mechanism of cellulose biosynthesis and are also valuable resources for the development of herbicides. Here, based on mutant genetic analysis and molecular docking analysis, we have identified the potential target sites of these CBIs on a modeled CESA structure. Some bacteria also produce cellulose, and both ES20 and ES20-1 inhibited bacterial cellulose biosynthesis. Therefore, we conclude that ES20-1 is a more potent analog of ES20 that inhibits intrinsic cellulose biosynthesis in plants, and both ES20 and ES20-1 show an inhibitory effect on bacterial growth and cellulose synthesis, making them excellent tools for exploring the mechanisms of cellulose biosynthesis across kingdoms.
Real-time conversion of tissue-scale mechanical forces into an interdigitated growth pattern
Samuel A. Belteton1, Wenlong Li2, Makoto Yanagisawa3, Faezeh A. Hatam2, Madeline I. Quinn1, Margaret K. Szymanski4, Mathew W. Marley1, Joseph A. Turner2, Daniel B. Szymanski1,5
1Department of Botany and Plant Pathology, 5 Biological Sciences, Purdue University, West Lafayette, IN; 2Mechanical and Materials Engineering University of Nebraska–Lincoln Lincoln NE; 3Department of Botany, University of Wisconsin, Madison, WI; 4 Department of Biochemistry, Indiana University Bloomington, Bloomington, IN.
The leaf epidermis is a dynamic biomechanical shell that integrates growth across spatial scales to influence organ morphology. Pavement cells, the fundamental unit of this tissue, morph irreversibly into highly lobed cells that drive planar leaf expansion. Here we define how tissue-scale cell wall tensile forces and the microtubule–cellulose synthase systems dictate the patterns of interdigitated growth in real time. A morphologically potent subset of cortical microtubules span the periclinal and anticlinal cell faces to pattern cellulose fibres that generate a patch of anisotropic wall. The subsequent local polarized growth is mechanically coupled to the adjacent cell via a pectin-rich middle lamella, and this drives lobe formation. Finite element pavement cell models revealed cell wall tensile stress as an upstream patterning element that links cell- and tissue-scale biomechanical parameters to interdigitated growth. Cell lobing in leaves is evolutionarily conserved, occurs in multiple cell types and is associated with important agronomic traits. Our general mechanistic models of lobe formation provide a foundation to analyse the cellular basis of leaf morphology and function.
Inhibition of actin filament nucleators leads enhances de novo filament nucleation in Arabidopsis.
Liyuan Xu, Lingyan Cao, Christopher J. Staiger
Purdue University
Actin nucleators permit controlled deposition of different actin cytoskeleton structures. Actin-Related Protein 2/3 complex (Arp2/3) and formin are, so far, the only two confirmed actin filament nucleators within plant cells. Numerous in vitro experiments have shown that Arp2/3 is responsible for making new side-branched filaments and formin generates long, non-branched filaments. In addition, in plant cells, the Arp2/3 complex is essential for epidermal cell morphology development. However, the exact role of the Arp2/3 complex in regulating actin structures and single actin filament dynamics in vivo is poorly understood, and the regulatory mechanism of Arp2/3 in plant cell morphology is also currently unknown. Here, we demonstrate for the first time that the Arp2/3 complex is the in vivo nucleator for side-branched actin filaments in Arabidopsis thaliana epidermal cells. We found that Arp2/3 mutants, arp2-1 and arpc2, resulted in dramatically reduced abundance of actin filaments, a reduction in filament bundling, and significantly decreased side-branching nucleation events. To test whether these actin-based phenotypes are the consequence of the loss of a functional Arp2/3 complex, we used CK-666, a small molecule inhibitor for Arp2/3, on both wild-type and mutant plants. CK-666 treatment of wild-type plants recapitulated the actin-based phenotypes of arp2/3 mutants, but applying CK-666 onto arp2/3 mutants did not have any additional effect on overall actin structure and filament dynamics. Thus, we established that CK-666 on is an effective tool for inhibiting plant Arp2/3. Combining the genetic and chemical inhibition of Arp2/3 and formins, we found that both proteins were capable of nucleating side-branched actin filaments, but that Arp2/3-nucleated filaments grew slower and were shorter than formin-nucleated ones. Surprisingly, inhibiting both Arp2/3 and formins leaded to an increased actin filament abundance and enhanced de novo nucleation. Collectively, these observations suggest that plant cells have a different actin filament nucleation mechanism compared to yeast or animal cells.
Identify novel regulators of exocytosis in Arabidopsis through chemical genetics screening
Xiaohui Li, Chunhua Zhang
Department of Botany and Plant Pathology, Center for Plant Biology, Purdue University, West Lafayette, IN
Various proteins are delivered to the plasma membrane (PM) through exocytosis, including proteins involved in cell wall formation, signal transduction, auxin transport, nutrients transport, et al. Exocyst is a conserved octameric complex present in fungi, animals and plants to facilitate the delivery of secretory vesicles to the PM. A small molecule Endosidin2 (ES2) is previously identified as an exocyst inhibitor. PIN2, the auxin efflux carrier protein, is a cargo protein of exocytosis and one of the commonly used marker protein for membrane trafficking study. We are carrying out a large scale forward genetics screening in Arabidopsis using ethyl methanesulfonate (EMS) mutagenized lines expressing PIN2-GFP marker, screening for mutants hypersensitive to ES2. Candidate genes are identified by Next-Generation-Sequencing (NGS)-based fine mapping. We have harbored more than 70 mutant lines hypersensitive to ES2, named as es2s mutants. Among the identified mutants, 20 lines have been mapped so far. The characterization of the es2s mutants is underway.
The epidermis drives twisted growth of Arabidopsis roots
Natasha Bilkey, Juliana Ocasio, Lily Murchison, Ram Dixit
Department of Biology and Center for Engineering Mechanobiology, Washington University in St. Louis
Directional growth of plant organs is determined primarily by the axis of cell expansion, which is specified by the net orientation of the cortical microtubule cytoskeleton. Mutations that perturb the organization of cortical microtubules frequently lead to defective cell expansion and abnormal organ growth. In this work, we used the spiral1-3 (spr1-3) mutant, which exhibits right-handed root twisting, to study how microtubule-level defects result in twisting at the organ level. We found that cortical microtubules in the elongation zone of spr1-3 roots show a consistent left-handed skewed alignment prior to the onset of cell file and root twisting, whereas wild-type (Col-0) microtubule alignment is largely transverse in this zone. Morphometry analysis revealed that individual spr1-3 epidermal cells in the root elongation zone have a subtle but consistent right-handed skewed morphology compared to wild-type. Specifically, the end walls of spr1-3 cells adopt a skewed angle from horizontal compared to wild-type. To determine the relative contribution of different cell layers to root skewing, we complemented the spr1-3 mutant with SPR1-GFP driven by tissue-specific promoters. We found that epidermis-specific expression of SPR1-GFP is sufficient to restore the cell file and root morphology to wild-type, whereas plants expressing SPR1-GFP either in the cortex, endodermis or stele resembled the spr1-3 mutant. These findings suggest that the epidermis entrains the growth of the inner cell layers, perhaps through mechanical coupling.
Novel Cytoskeletal Regulators of Trichome Branching in Arabidopsis
Rachappa Balkunde and Ram Dixit
Department of Biology and Center for Engineering Mechanobiology, Washington University in St. Louis, St. Louis, MO 63130, USA
Using a yeast two-hybrid screen, we have identified a novel family of EB1-interacting proteins in Arabidopsis consisting of three members, which we named Microtubule End-binding Protein1 (MEP1), MEP2 and MEP3. We found that the MEP proteins interact directly with all three members of the Arabidopsis EB1 family in directed yeast two-hybrid and/or pull-down experiments. Live imaging showed that MEP proteins localize to growing plus-ends and to the lattice of cortical microtubules in a manner that is distinct from EB1 proteins. Unexpectedly, we observed that full-length MEP1 and MEP2 proteins label actin filaments in addition to strong microtubule localization. Structure-function analysis revealed that the middle region of all three MEP proteins is sufficient for interaction with EB proteins and for microtubule lattice and plus-end localization, whereas the N terminal region of MEP1 and MEP2 mediates actin localization. By contrast, the C-terminal region of all three MEPs showed diffuse cytoplasmic localization. Genetic analysis showed that MEP2 and MEP3 proteins function as suppressors of trichome branching. While mep2 and mep3 single mutants showed modest increase in the percentage of four-branched trichomes, mep2 mep3 double mutants exhibited trichomes with up to seven branches. The trichome branching phenotype in mep2 mep3 double and mep1 mep2 mep3 triple mutants was indistinguishable, suggesting that MEP1 has no role in trichome branching. Consistent with these findings, reporter gene analysis showed that MEP2 and MEP3 are expressed in trichomes whereas MEP1 is not. The DNA content of trichome nuclei was similar between wild type and mep1 mep2 mep3 triple mutant, indicating that increased trichome branching in the triple mutant is not due to excessive endoreduplication. Ongoing studies are examining the localization of MEP proteins during trichome development and assessing whether microtubule and/or actin filaments dynamics and organization are altered in the mep mutants.
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