John Runions

Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants

Expression and tracking of fluorescent fusion proteins has revolutionized our understanding of basic concepts in cell biology. The protocol presented here has underpinned much of the in vivo results highlighting the dynamic nature of the plant secretory pathway. Transient transformation of tobacco leaf epidermal cells is a relatively fast technique to assess expression of genes of interest. These cells can be used to generate stable plant lines using a more time-consuming, cell culture technique. Transient expression takes from 2 to 4 days whereas stable lines are generated after approximately 2 to 4 months.

High-Resolution Whole-Mount Imaging of Three-Dimensional Tissue Organization and Gene Expression Enables the Study of Phloem Development and Structure in Arabidopsis

Currently, examination of the cellular structure of plant organs and the gene expression therein largely relies on the production of tissue sections. Here, we present a staining technique that can be used to image entire plant organs using confocal laser scanning microscopy. This technique produces high-resolution images that allow three-dimensional reconstruction of the cellular organization of plant organs. Importantly, three-dimensional domains of gene expression can be analyzed with single-cell precision. We used this technique for a detailed examination of phloem cells in the wild type and mutants. We were also able to recognize phloem sieve elements and their differentiation state in any tissue type and visualize the structure of sieve plates. We show that in the altered phloem development mutant, a hybrid cell type with phloem and xylem characteristics develops from initially normally differentiated protophloem cells. The simplicity of sieve element data collection allows for the statistical analysis of structural parameters of sieve plates, essential for the calculation of phloem conductivity. Taken together, this technique significantly improves the speed and accuracy of the investigation of plant growth and development.

PIN polarity maintenance by the cell wall in Arabidopsis.

A central question in developmental biology concerns the mechanism of generation and maintenance of cell polarity, because these processes are essential for many cellular functions and multicellular development. In plants, cell polarity has an additional role in mediating directional transport of the plant hormone auxin that is crucial for multiple developmental processes. In addition, plant cells have a complex extracellular matrix, the cell wall, whose role in regulating cellular processes, including cell polarity, is unexplored. We have found that polar distribution of PIN auxin transporters in plant cells is maintained by connections between polar domains at the plasma membrane and the cell wall. Genetic and pharmacological interference with cellulose, the major component of the cell wall, or mechanical interference with the cell wall disrupts these connections and leads to increased lateral diffusion and loss of polar distribution of PIN transporters for the phytohormone auxin. Our results reveal a plant-specific mechanism for cell polarity maintenance and provide a conceptual framework for modulating cell polarity and plant development via endogenous and environmental manipulations of the cellulose-based extracellular matrix.

Movement and Remodeling of the Endoplasmic Reticulum in Nondividing Cells of Tobacco Leaves

Using a novel analytical tool, this study investigates the relative roles of actin, microtubules, myosin, and Golgi bodies on form and movement of the endoplasmic reticulum (ER) in tobacco (Nicotiana tabacum) leaf epidermal cells. Expression of a subset of truncated class XI myosins, which interfere with the activity of native class XI myosins, and drug-induced actin depolymerization produce a more persistent network of ER tubules and larger persistent cisternae. The treatments differentially affect two persistent size classes of cortical ER cisternae, those >0.3 μm2 and those smaller, called punctae. The punctae are not Golgi, and ER remodeling occurs in the absence of Golgi bodies. The treatments diminish the mobile fraction of ER membrane proteins but not the diffusive flow of mobile membrane proteins. The results support a model whereby ER network remodeling is coupled to the directionality but not the magnitude of membrane surface flow, and the punctae are network nodes that act as foci of actin polymerization, regulating network remodeling through exploratory tubule growth and myosin-mediated shrinkage.

Recycling, clustering, and endocytosis jointly maintain PIN auxin carrier polarity at the plasma membrane.

Cell polarity reflected by asymmetric distribution of proteins at the plasma membrane is a fundamental feature of unicellular and multicellular organisms. It remains conceptually unclear how cell polarity is kept in cell wall‐encapsulated plant cells. We have used super‐resolution and semi‐quantitative live‐cell imaging in combination with pharmacological, genetic, and computational approaches to reveal insights into the mechanism of cell polarity maintenance in Arabidopsis thaliana. We show that polar‐competent PIN transporters for the phytohormone auxin are delivered to the center of polar domains by super‐polar recycling. Within the plasma membrane, PINs are recruited into non‐mobile membrane clusters and their lateral diffusion is dramatically reduced, which ensures longer polar retention. At the circumventing edges of the polar domain, spatially defined internalization of escaped cargos occurs by clathrin‐dependent endocytosis. Computer simulations confirm that the combination of these processes provides a robust mechanism for polarity maintenance in plant cells. Moreover, our study suggests that the regulation of lateral diffusion and spatially defined endocytosis, but not super‐polar exocytosis have primary importance for PIN polarity maintenance.

Characterization of SUN-domain proteins at the higher plant nuclear envelope

Sad1/UNC-84 (SUN)-domain proteins are inner nuclear membrane (INM) proteins that are part of bridging complexes linking cytoskeletal elements with the nucleoskeleton, and have been shown to be conserved in non-plant systems. In this paper, we report the presence of members of this family in the plant kingdom, and investigate the two Arabidopsis SUN-domain proteins, AtSUN1 and AtSUN2. Our results indicate they contain the highly conserved C-terminal SUN domain, and share similar structural features with animal and fungal SUN-domain proteins including a functional coiled-coil domain and nuclear localization signal. Both are expressed in various tissues with AtSUN2 expression levels relatively low but upregulated in proliferating tissues. Further, we found AtSUN1 and AtSUN2 expressed as fluorescent protein fusions, to localize to and show low mobility in the nuclear envelope (NE), particularly in the INM. Deletion of various functional domains including the N terminus and coiled-coil domain affect the localization and increase the mobility of AtSUN1 and AtSUN2. Finally, we present evidence that AtSUN1 and AtSUN2 are present as homomers and heteromers in vivo, and that the coiled-coil domains are required for this. The study provides evidence suggesting the existence of cytoskeletal-nucleoskeletal bridging complexes at the plant NE.

Cell wall constrains lateral diffusion of plant plasma-membrane proteins

A cell membrane can be considered a liquid-phase plane in which lipids and proteins theoretically are free to diffuse. Numerous reports, however, describe retarded diffusion of membrane proteins in animal cells. This anomalous diffusion results from a combination of structuring factors including protein–protein interactions, cytoskeleton corralling, and lipid organization into microdomains. In plant cells, plasma-membrane (PM) proteins have been described as relatively immobile, but the control mechanisms that structure the PM have not been studied. Here, we use fluorescence recovery after photobleaching to estimate mobility of a set of minimal PM proteins. These proteins consist only of a PM-anchoring domain fused to a fluorescent protein, but their mobilities remained limited, as is the case for many full-length proteins. Neither the cytoskeleton nor membrane microdomain structure was involved in constraining the diffusion of these proteins. The cell wall, however, was shown to have a crucial role in immobilizing PM proteins. In addition, by single-molecule fluorescence imaging we confirmed that the pattern of cellulose deposition in the cell wall affects the trajectory and speed of PM protein diffusion. Regulation of PM protein dynamics by the plant cell wall can be interpreted as a mechanism for regulating protein interactions in processes such as trafficking and signal transduction.

Fluorescence recovery after photobleaching reveals high cycling dynamics of plasma membrane aquaporins in Arabidopsis roots under salt stress

The constitutive cycling of plant plasma membrane (PM) proteins is an essential component of their function and regulation under resting or stress conditions. Transgenic Arabidopsis plants that express GFP fusions with AtPIP1;2 and AtPIP2;1, two prototypic PM aquaporins, were used to develop a fluorescence recovery after photobleaching (FRAP) approach. This technique was used to discriminate between PM and endosomal pools of the aquaporin constructs, and to estimate their cycling between intracellular compartments and the cell surface. The membrane trafficking inhibitors tyrphostin A23, naphthalene-1-acetic acid and brefeldin A blocked the latter process. By contrast, a salt treatment (100 mm NaCl for 30 min) markedly enhanced the cycling of the aquaporin constructs and modified their pharmacological inhibition profile. Two distinct models for PM aquaporin cycling in resting or salt-stressed root cells are discussed.

cg12 expression is specifically linked to infection of root hairs and cortical cells during Casuarina glauca and Allocasuarina verticillata actinorhizal nodule development.

cg12 is an early actinorhizal nodulin gene from Casuarina glauca encoding a subtilisin-like serine protease. Using transgenic Casuarinaceae plants carrying cg12-gus and cg12-gfp fusions, we have studied the expression pattern conferred by the cg12 promoter region after inoculation with Frankia. cg12 was found to be expressed in root hairs and in root and nodule cortical cells containing Frankia infection threads. cg12 expression was also monitored after inoculation with ineffective Frankia strains, during mycorrhizae formation, and after diverse hormonal treatments. None of these treatments was able to induce its expression, therefore suggesting that cg12 expression is linked to plant cell infection by Frankia strains. Possible roles of cg12 in actinorhizal symbiosis are discussed.

A novel function for a redox-related LEA protein (SAG21/AtLEA5) in root development and biotic stress responses

SAG21/AtLEA5 belongs to the late embryogenesis-associated (LEA) protein family. Although it has been implicated in growth and redox responses, its precise roles remain obscure. To address this problem, we characterized root and shoot development and response to biotic stress in SAG21/AtLEA5 over-expressor (OEX) and antisense (AS) lines. AS lines exhibited earlier flowering and senescence and reduced shoot biomass. Primary root length was reduced in AS lines, as was the number of laterals relative to the primary root. Root hair number was unchanged but root hair length was proportional to SAG21/AtLEA5 expression level, with longer root hairs in OEX lines and shorter root hairs in AS, relative to wild type. Growth of the fungal nectroph, Botrytis cinerea and of a virulent bacterial pathogen (Pseudomonas syringae pv. tomato) was affected by SAG21/AtLEA5 expression; however, growth of an avirulent P.syringae strain was unaffected. A SAG21/AtLEA5-YFP fusion was localized to mitochondria, raising the intriguing possibility that SAG21 interacts with proteins involved in mitochondrial ROS signalling, which in turn, impacts on root development and pathogen responses.