Four-dimensional quantitative analysis of cell plate development in Arabidopsis using lattice light sheet microscopy identifies robust transition points between growth phases.

Cell plate formation during cytokinesis entails multiple stages occurring concurrently and requiring orchestrated vesicle delivery, membrane remodelling, and timely deposition of polysaccharides, such as callose. Understanding such a dynamic process requires dissection in time and space; this has been a major hurdle in studying cytokinesis. Using lattice light sheet microscopy (LLSM), we studied cell plate development in four dimensions, through the behavior of yellow fluorescent protein (YFP)-tagged cytokinesis-specific GTPase RABA2a vesicles. We monitored the entire duration of cell plate development, from its first emergence, with the aid of YFP-RABA2a, in both the presence and absence of cytokinetic callose. By developing a robust cytokinetic vesicle volume analysis pipeline, we identified distinct behavioral patterns, allowing the identification of three easily trackable cell plate developmental phases. Notably, the phase transition between phase I and phase II is striking, indicating a switch from membrane accumulation to the recycling of excess membrane material. We interrogated the role of callose using pharmacological inhibition with LLSM and electron microscopy. Loss of callose inhibited the phase transitions, establishing the critical role and timing of the polysaccharide deposition in cell plate expansion and maturation. This study exemplifies the power of combining LLSM with quantitative analysis to decode and untangle such a complex process.

A biophysical model for plant cell plate maturation based on the contribution of a spreading force.

Plant cytokinesis, a fundamental process of plant life, involves de novo formation of a "cell plate" partitioning the cytoplasm of dividing cells. Cell plate formation is directed by orchestrated delivery, fusion of cytokinetic vesicles, and membrane maturation to form a nascent cell wall by timely deposition of polysaccharides. During cell plate maturation, the fragile membrane network transitions to a fenestrated sheet and finally a young cell wall. Here, we approximated cell plate sub-structures with testable shapes and adopted the Helfrich-free energy model for membranes, including a stabilizing and spreading force, to understand the transition from a vesicular network to a fenestrated sheet and mature cell plate. Regular cell plate development in the model was possible, with suitable bending modulus, for a two-dimensional late stage spreading force of 2-6 pN/nm, an osmotic pressure difference of 2-10 kPa, and spontaneous curvature between 0 and 0.04 nm-1. With these conditions, stable membrane conformation sizes and morphologies emerged in concordance with stages of cell plate development. To reach a mature cell plate, our model required the late-stage onset of a spreading/stabilizing force coupled with a concurrent loss of spontaneous curvature. Absence of a spreading/stabilizing force predicts failure of maturation. The proposed model provides a framework to interrogate different players in late cytokinesis and potentially other membrane networks that undergo such transitions. Callose, is a polysaccharide that accumulates transiently during cell plate maturation. Callose-related observations were consistent with the proposed model's concept, suggesting that it is one of the factors involved in establishing the spreading force.

AtTRAPPC11/ROG2: A Role for TRAPPs in Maintenance of the Plant Trans-Golgi Network/Early Endosome Organization and Function.

The dynamic trans-Golgi network/early endosome (TGN/EE) facilitates cargo sorting and trafficking and plays a vital role in plant development and environmental response. Transport protein particles (TRAPPs) are multi-protein complexes acting as guanine nucleotide exchange factors and possibly as tethers, regulating intracellular trafficking. TRAPPs are essential in all eukaryotic cells and are implicated in a number of human diseases. It has been proposed that they also play crucial roles in plants; however, our current knowledge about the structure and function of plant TRAPPs is very limited. Here, we identified and characterized AtTRAPPC11/RESPONSE TO OLIGOGALACTURONIDE2 (AtTRAPPC11/ROG2), a TGN/EE-associated, evolutionarily conserved TRAPP protein in Arabidopsis (Arabidopsis thaliana). AtTRAPPC11/ROG2 regulates TGN integrity, as evidenced by altered TGN/EE association of several residents, including SYNTAXIN OF PLANTS61, and altered vesicle morphology in attrappc11/rog2 mutants. Furthermore, endocytic traffic and brefeldin A body formation are perturbed in attrappc11/rog2, suggesting a role for AtTRAPPC11/ROG2 in regulation of endosomal function. Proteomic analysis showed that AtTRAPPC11/ROG2 defines a hitherto uncharacterized TRAPPIII complex in plants. In addition, attrappc11/rog2 mutants are hypersensitive to salinity, indicating an undescribed role of TRAPPs in stress responses. Overall, our study illustrates the plasticity of the endomembrane system through TRAPP protein functions and opens new avenues to explore this dynamic network.

Rational Design Principles for the Transport and Subcellular Distribution of Nanomaterials into Plant Protoplasts.

The ability to control the subcellular localization of nanoparticles within living plants offers unique advantages for targeted biomolecule delivery and enables important applications in plant bioengineering. However, the mechanism of nanoparticle transport past plant biological membranes is poorly understood. Here, a mechanistic study of nanoparticle cellular uptake into plant protoplasts is presented. An experimentally validated mathematical model of lipid exchange envelope penetration mechanism for protoplasts, which predicts that the subcellular distribution of nanoparticles in plant cells is dictated by the particle size and the magnitude of the zeta potential, is advanced. The mechanism is completely generic, describing nanoparticles ranging from quantum dots, gold and silica nanoparticles, nanoceria, and single-walled carbon nanotubes (SWNTs). In addition, the use of imaging flow cytometry to investigate the influence of protoplasts' morphological characteristics on nanoparticle uptake efficiency is demonstrated. Using DNA-wrapped SWNTs as model nanoparticles, it is found that glycerolipids, the predominant lipids in chloroplast membranes, exhibit stronger lipid-nanoparticle interaction than phospholipids, the major constituent in protoplast membrane. This work can guide the rational design of nanoparticles for targeted delivery into specific compartments within plant cells without the use of chemical or mechanical aid, potentially enabling various plant engineering applications.

Nitroaromatic detection and infrared communication from wild-type plants using plant nanobionics.

Plant nanobionics aims to embed non-native functions to plants by interfacing them with specifically designed nanoparticles. Here, we demonstrate that living spinach plants (Spinacia oleracea) can be engineered to serve as self-powered pre-concentrators and autosamplers of analytes in ambient groundwater and as infrared communication platforms that can send information to a smartphone. The plants employ a pair of near-infrared fluorescent nanosensors-single-walled carbon nanotubes (SWCNTs) conjugated to the peptide Bombolitin II to recognize nitroaromatics via infrared fluorescent emission, and polyvinyl-alcohol functionalized SWCNTs that act as an invariant reference signal-embedded within the plant leaf mesophyll. As contaminant nitroaromatics are transported up the roots and stem into leaf tissues, they accumulate in the mesophyll, resulting in relative changes in emission intensity. The real-time monitoring of embedded SWCNT sensors also allows residence times in the roots, stems and leaves to be estimated, calculated to be 8.3 min (combined residence times of root and stem) and 1.9 min mm-1 leaf, respectively. These results demonstrate the ability of living, wild-type plants to function as chemical monitors of groundwater and communication devices to external electronics at standoff distances.

A Nanobionic Light-Emitting Plant.

The engineering of living plants for visible light emission and sustainable illumination is compelling because plants possess independent energy generation and storage mechanisms and autonomous self-repair. Herein, we demonstrate a plant nanobionic approach that enables exceptional luminosity and lifetime utilizing four chemically interacting nanoparticles, including firefly luciferase conjugated silica (SNP-Luc), d-luciferin releasing poly(lactic-co-glycolic acid) (PLGA-LH2), coenzyme A functionalized chitosan (CS-CoA) and semiconductor nanocrystal phosphors for longer wavelength modulation. An in vitro kinetic model incorporating the release rates of the nanoparticles is developed to maximize the chemiluminescent lifetimes to exceed 21.5 h. In watercress (Nasturtium officinale) and other species, the nanoparticles circumvent limitations such as luciferin toxicity above 400 µM and colocalization of enzymatic reactions near high adenosine triphosphate (ATP) production. Pressurized bath infusion of nanoparticles (PBIN) is introduced to deliver a mixture of nanoparticles to the entire living plant, well described using a nanofluidic mathematical model. We rationally design nanoparticle size and charge to control localization within distinct tissues compartments with 10 nm nanoparticles localizing within the leaf mesophyll and stomata guard cells, and those larger than 100 nm segregated in the leaf mesophyll. The results are mature watercress plants that emit greater than 1.44 × 1012 photons/sec or 50% of 1 µW commercial luminescent diodes and modulate "off" and "on" states by chemical addition of dehydroluciferin and coenzyme A, respectively. We show that CdSe nanocrystals can shift the chemiluminescent emission to 760 nm enabling near-infrared (nIR) signaling. These results advance the viability of nanobionic plants as self-powered photonics, direct and indirect light sources.

Subcellular positioning during cell division and cell plate formation in maize.

Introduction: During proliferative plant cell division, the new cell wall, called the cell plate, is first built in the middle of the cell and then expands outward to complete cytokinesis. This dynamic process requires coordinated movement and arrangement of the cytoskeleton and organelles. Methods: Here we use live-cell markers to track the dynamic reorganization of microtubules, nuclei, endoplasmic reticulum, and endomembrane compartments during division and the formation of the cell plate in maize leaf epidermal cells. Results: The microtubule plus-end localized protein END BINDING1 (EB1) highlighted increasing microtubule dynamicity during mitosis to support rapid changes in microtubule structures. The localization of the cell-plate specific syntaxin KNOLLE, several RAB-GTPases, as well as two plasma membrane localized proteins was assessed after treatment with the cytokinesis-specific callose-deposition inhibitor Endosidin7 (ES7) and the microtubule-disrupting herbicide chlorpropham (CIPC). While ES7 caused cell plate defects in Arabidopsis thaliana, it did not alter callose accumulation, or disrupt cell plate formation in maize. In contrast, CIPC treatment of maize epidermal cells occasionally produced irregular cell plates that split or fragmented, but did not otherwise disrupt the accumulation of cell-plate localized proteins. Discussion: Together, these markers provide a robust suite of tools to examine subcellular trafficking and organellar organization during mitosis and cell plate formation in maize.

Plant cytokinesis and the construction of new cell wall.

Cytokinesis in plants is fundamentally different from that in animals and fungi. In plant cells, a cell plate forms through the fusion of cytokinetic vesicles and then develops into the new cell wall, partitioning the cytoplasm of the dividing cell. The formation of the cell plate entails multiple stages that involve highly orchestrated vesicle accumulation, fusion and membrane maturation, which occur concurrently with the timely deposition of polysaccharides such as callose, cellulose and cross-linking glycans. This review summarizes the major stages in cytokinesis, endomembrane components involved in cell plate assembly and its transition to a new cell wall. An animation that can be widely used for educational purposes further summarizes the process.

Post-Golgi Trafficking and Transport of Cell Wall Components.

The cell wall, a complex macromolecular composite structure surrounding and protecting plant cells, is essential for development, signal transduction, and disease resistance. This structure is also integral to cell expansion, as its tensile resistance is the primary balancing mechanism against internal turgor pressure. Throughout these processes, the biosynthesis, transport, deposition, and assembly of cell wall polymers are tightly regulated. The plant endomembrane system facilitates transport of polysaccharides, polysaccharide biosynthetic and modifying enzymes and glycoproteins through vesicle trafficking pathways. Although a number of enzymes involved in cell wall biosynthesis have been identified, comparatively little is known about the transport of cell wall polysaccharides and glycoproteins by the endomembrane system. This review summarizes our current understanding of trafficking of cell wall components during cell growth and cell division. Emerging technologies, such as vesicle glycomics, are also discussed as promising avenues to gain insights into the trafficking of structural polysaccharides to the apoplast.

Nanosensor Technology Applied to Living Plant Systems.

An understanding of plant biology is essential to solving many long-standing global challenges, including sustainable and secure food production and the generation of renewable fuel sources. Nanosensor platforms, sensors with a characteristic dimension that is nanometer in scale, have emerged as important tools for monitoring plant signaling pathways and metabolism that are nondestructive, minimally invasive, and capable of real-time analysis. This review outlines the recent advances in nanotechnology that enable these platforms, including the measurement of chemical fluxes even at the single-molecule level. Applications of nanosensors to plant biology are discussed in the context of nutrient management, disease assessment, food production, detection of DNA proteins, and the regulation of plant hormones. Current trends and future needs are discussed with respect to the emerging trends of precision agriculture, urban farming, and plant nanobionics.