NMR spectroscopy analysis reveals differential metabolic responses in arabidopsis roots and leaves treated with a cytokinesis inhibitor.

In plant cytokinesis, de novo formation of a cell plate evolving into the new cell wall partitions the cytoplasm of the dividing cell. In our earlier chemical genomics studies, we identified and characterized the small molecule endosidin-7, that specifically inhibits callose deposition at the cell plate, arresting late-stage cytokinesis in arabidopsis. Endosidin-7 has emerged as a very valuable tool for dissecting this essential plant process. To gain insights regarding its mode of action and the effects of cytokinesis inhibition on the overall plant response, we investigated the effect of endosidin-7 through a nuclear magnetic resonance spectroscopy (NMR) metabolomics approach. In this case study, metabolomics profiles of arabidopsis leaf and root tissues were analyzed at different growth stages and endosidin-7 exposure levels. The results show leaf and root-specific metabolic profile changes and the effects of endosidin-7 treatment on these metabolomes. Statistical analyses indicated that the effect of endosidin-7 treatment was more significant than the developmental impact. The endosidin-7 induced metabolic profiles suggest compensations for cytokinesis inhibition in central metabolism pathways. This study further shows that long-term treatment of endosidin-7 profoundly changes, likely via alteration of hormonal regulation, the primary metabolism of arabidopsis seedlings. Hormonal pathway-changes are likely reflecting the plant's responses, compensating for the arrested cell division, which in turn are leading to global metabolite modulation. The presented NMR spectral data are made available through the Metabolomics Workbench, providing a reference resource for the scientific community.

Unconventional Protein Secretion in Plants.

Unconventional protein secretion (UPS) describes secretion pathways that bypass one or several of the canonical secretion pit-stops on the way to the plasma membrane, and/or involve the secretion of leaderless proteins. So far, alternatives to conventional secretion were primarily observed and studied in yeast and animal cells. The sessile lifestyle of plants brings with it unique restraints on how they adapt to adverse conditions and environmental challenges. Recently, attention towards unconventional secretion pathways in plant cells has substantially increased, with the large number of leaderless proteins identified through proteomic studies. While UPS pathways in plants are certainly not yet exhaustively researched, an emerging notion is that induction of UPS pathways is correlated with pathogenesis and stress responses. Given the multitude UPS events observed, comprehensively organizing the routes proteins take to the apoplast in defined UPS categories is challenging. With the establishment of a larger collection of studied plant proteins taking these UPS pathways, a clearer picture of endomembrane trafficking as a whole will emerge. There are several novel enabling technologies, such as vesicle proteomics and chemical genomics, with great potential for dissecting secretion pathways, providing information about the cargo that travels along them and the conditions that induce them.

The RAB GTPase RABA1e localizes to the cell plate and shows distinct subcellular behavior from RABA2a under Endosidin 7 treatment.

Cytokinesis in plants requires the activity of RAB GTPases to regulate vesicle-mediated contribution of material to the developing cell plate. While some plant RAB GTPases have been shown to be involved in cell plate formation, many still await functional assignment. Here, we report cell plate localization for YFP-RABA1e in Arabidopsis thaliana and use the cytokinesis inhibitor Endosidin 7 to provide a detailed description of its localization compared to YFP-RABA2a. Differences between YFP-RABA2a and YFP-RABA1e were observed in late-stage cell plates under DMSO control treatment, and became more apparent under Endosidin 7 treatment. Taken together, our results suggest that individual RAB GTPases might make different contributions to cell plate formation and further demonstrates the utility of ES7 probe to dissect them.

On-Demand Formation of Supported Lipid Membrane Arrays by Trehalose-Assisted Vesicle Delivery for SPR Imaging.

The fabrication of large-scale, solid-supported lipid bilayer (SLB) arrays has traditionally been an arduous and complex task, primarily due to the need to maintain SLBs within an aqueous environment. In this work, we demonstrate the use of trehalose vitrified phospholipid vesicles that facilitate on-demand generation of microarrays, allowing each element a unique composition, for the label-free and high-throughput analysis of biomolecular interactions by SPR imaging (SPRi). Small, unilamellar vesicles (SUVs) are suspended in trehalose, deposited in a spatially defined manner, with the trehalose vitrifying on either hydrophilic or hydrophobic SPR substrates. SLBs are subsequently spontaneously formed on-demand simply by in situ hydration of the array in the SPR instrument flow cell. The resulting SLBs exhibit high lateral mobility, characteristic of fluidic cellular lipid membranes, and preserve the biological function of embedded cell membrane receptors, as indicated by SPR affinity measurements. Independent fluorescence and SPR imaging studies show that the individual SLBs stay localized at the area of deposition, without any encapsulating matrix, confining coral, or boundaries. The introduced methodology allows individually addressable SLB arrays to be analyzed with excellent label-free sensitivity in a real-time, high-throughput manner. Various protein-ganglioside interactions have been selected as a model system to illustrate discrimination of strong and weak binding responses in SPRi sensorgrams. This methodology has been applied toward generating hybrid bilayer membranes on hydrophobic SPR substrates, demonstrating its versatility toward a range of surfaces and membrane geometries. The stability of the fabricated arrays, over medium to long storage periods, was evaluated and found to be good. The highly efficient and easily scalable nature of the method has the potential to be applied to a variety of label-free sensing platforms requiring lipid membranes for high-throughput analysis of their properties and constituents.

CESA TRAFFICKING INHIBITOR inhibits cellulose deposition and interferes with the trafficking of cellulose synthase complexes and their associated proteins KORRIGAN1 and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1.

Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. We monitored the distribution and mobility of fluorescently labeled cellulose synthases (CESAs) in live Arabidopsis cells under chemical exposure to characterize their subcellular effects. CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. KOR1 accumulated in bodies similar to CESA; however, POM2/CSI1 dissociated into the cytoplasm. In addition, MT stability was altered without direct inhibition of MT polymerization, suggesting a feedback mechanism caused by cellulose interference. The selectivity of CESTRIN was assessed using a variety of subcellular markers for which no morphological effect was observed. The association of CESAs with vesicles decorated by the trans-Golgi network-localized protein SYNTAXIN OF PLANTS61 (SYP61) was increased under CESTRIN treatment, implicating SYP61 compartments in CESA trafficking. The properties of CESTRIN compared with known CESA inhibitors afford unique avenues to study and understand the mechanism under which PM-associated CSCs are maintained and interact with MTs and to dissect their trafficking routes in etiolated hypocotyls.

Endosidin 7 Specifically Arrests Late Cytokinesis and Inhibits Callose Biosynthesis, Revealing Distinct Trafficking Events during Cell Plate Maturation.

Although cytokinesis is vital for plant growth and development, our mechanistic understanding of the highly regulated membrane and cargo transport mechanisms in relation to polysaccharide deposition during this process is limited. Here, we present an in-depth characterization of the small molecule endosidin 7 (ES7) inhibiting callose synthase activity and arresting late cytokinesis both in vitro and in vivo in Arabidopsis (Arabidopsis thaliana). ES7 is a specific inhibitor for plant callose deposition during cytokinesis that does not affect endomembrane trafficking during interphase or cytoskeletal organization. The specificity of ES7 was demonstrated (1) by comparing its action with that of known inhibitors such as caffeine, flufenacet, and concanamycin A and (2) across kingdoms with a comparison in yeast. The interplay between cell plate-specific post-Golgi vesicle traffic and callose accumulation was analyzed using ES7, and it revealed unique and temporal contributions of secretory and endosomal vesicles in cell plate maturation. While RABA2A-labeled vesicles, which accumulate at the early stage of cell plate formation, were not affected by ES7, KNOLLE was differentially altered by the small molecule. In addition, the presence of clathrin-coated vesicles in cells containing elevated levels of callose and their reduction under ES7 treatment further support the role of endocytic membrane remodeling in the maturing cell plate while the plate is stabilized by callose. Taken together, these data show the essential role of callose during the late stages of cell plate maturation and establish the temporal relationship between vesicles and regulatory proteins at the cell plate assembly matrix during polysaccharide deposition.

On-demand self-assembly of supported membranes using sacrificial, anhydrobiotic sugar coats.

Borrowing principles of anhydrobiosis, we have developed a technique for self-assembling proteolipid-supported membranes on demand--simply by adding water. Intact lipid- and proteolipid vesicles dispersed in aqueous solutions of anhydrobiotic trehalose are vitrified on arbitrary substrates, producing glassy coats encapsulating biomolecules. Previous efforts establish that these carbohydrate coats arrest molecular mobilities and preserve native conformations and aggregative states of the embedded biomolecules, thereby enabling long-term storage. Subsequent rehydration, even after an extended period of time (e.g., weeks), devitrifies sugar--releasing the cargo and unmasking the substrate surface--thus triggering substrate-mediated vesicle fusion in real time, producing supported membranes. Using this method, arrays of membranes, including those functionalized with membrane proteins, can be readily produced in situ by spatially addressing vitrification using common patterning tools--useful for multiplexed or stochastic sensing and assaying of target interactions with the fluid and functional membrane surface.