Fluorescent Labeling and Confocal Microcopy of Plastids and Stromules.

While chlorophyll has served as an excellent label for plastids in green tissue, the development of fluorescent proteins has allowed their ready visualization in all tissues of the plants, revealing new features of their morphology and motility, including the presence of plastid extensions known as stromules. Gene regulatory sequences in nuclear transgenes that target proteins to plastids, as well as in transgenes introduced into plastid genomes, can be assessed or optimized through the use of fluorescent protein reporters. Fluorescent labeling of plastids simultaneously with other subcellular locations reveals dynamic interactions and mutant phenotypes. Transient expression of fluorescent protein fusions is particularly valuable to determine whether or not a protein of unknown function is targeted to the plastid. Fluorescent biosensors can assay molecules such as ATP, calcium, or reactive oxygen species. Particle bombardment and agroinfiltration methods described here are convenient for imaging fluorescent proteins in plant organelles. With proper selection of fluorophores for labeling the components of the plant cell, confocal microscopy and multiphoton microscopy can produce extremely informative images at high resolution at depths not feasible by standard epifluorescence microscopy.

Protein Bodies in Leaves Exchange Contents through the Endoplasmic Reticulum.

Protein bodies (PBs) are organelles found in seeds whose main function is the storage of proteins that are used during germination for sustaining growth. PBs can also be induced to form in leaves when foreign proteins are produced at high levels in the endoplasmic reticulum (ER) and when fused to one of three tags: Zera®, elastin-like polypeptides (ELP), or hydrophobin-I (HFBI). In this study, we investigate the differences between ELP, HFBI and Zera PB formation, packing, and communication. Our results confirm the ER origin of all three fusion-tag-induced PBs. We show that secretory pathway proteins can be sequestered into all types of PBs but with different patterns, and that different fusion tags can target a specific protein to different PBs. Zera PBs are mobile and dependent on actomyosin motility similar to ELP and HFBI PBs. We show in vivo trafficking of proteins between PBs using GFP photoconversion. We also show that protein trafficking between ELP or HFBI PBs is faster and proteins travel further when compared to Zera PBs. Our results indicate that fusion-tag-induced PBs do not represent terminally stored cytosolic organelles, but that they form in, and remain part of the ER, and dynamically communicate with each other via the ER. We hypothesize that the previously documented PB mobility along the actin cytoskeleton is associated with ER movement rather than independent streaming of detached organelles.

Green to red photoconversion of GFP for protein tracking in vivo.

A variety of fluorescent proteins have been identified that undergo shifts in spectral emission properties over time or once they are irradiated by ultraviolet or blue light. Such proteins are finding application in following the dynamics of particular proteins or labelled organelles within the cell. However, before genes encoding these fluorescent proteins were available, many proteins have already been labelled with GFP in transgenic cells; a number of model organisms feature collections of GFP-tagged lines and organisms. Here we describe a fast, localized and non-invasive method for GFP photoconversion from green to red. We demonstrate its use in transgenic plant, Drosophila and mammalian cells in vivo. While genes encoding fluorescent proteins specifically designed for photoconversion will usually be advantageous when creating new transgenic lines, our method for photoconversion of GFP allows the use of existing GFP-tagged transgenic lines for studies of dynamic processes in living cells.

Fluorescent labeling and confocal microscopic imaging of chloroplasts and non-green plastids.

While chlorophyll has served as an excellent label for plastids in green tissue, the development of fluorescent proteins has allowed their ready visualization in all tissues of the plants, revealing new features of their morphology and motility. Gene regulatory sequences in plastid transgenes can be optimized through the use of fluorescent protein reporters. Fluorescent labeling of plastids simultaneously with other subcellular locations reveals dynamic interactions and mutant phenotypes. Transient expression of fluorescent protein fusions is particularly valuable to determine whether or not a protein of unknown function is targeted to the plastid. Particle bombardment and agroinfiltration methods described here are convenient for imaging fluorescent proteins in plant organelles. With proper selection of fluorophores for labeling the components of the plant cell, confocal microscopy can produce extremely informative images at high resolution at depths not feasible by standard epifluorescence microscopy.

Arabidopsis myosin XI sub-domains homologous to the yeast myo2p organelle inheritance sub-domain target subcellular structures in plant cells.

Myosin XI motor proteins transport plant organelles on the actin cytoskeleton. The Arabidopsis gene family that encodes myosin XI has 13 members, 12 of which have sub-domains within the tail region that are homologous to well-characterized cargo-binding domains in the yeast myosin V myo2p. Little is presently known about the cargo-binding domains of plant myosin XIs. Prior experiments in which most or all of the tail regions of myosin XIs have been fused to yellow fluorescent protein (YFP) and transiently expressed have often not resulted in fluorescent labeling of plant organelles. We identified 42 amino-acid regions within 12 Arabidopsis myosin XIs that are homologous to the yeast myo2p tail region known to be essential for vacuole and mitochondrial inheritance. A YFP fusion of the yeast region expressed in plants did not label tonoplasts or mitochondria. We investigated whether the homologous Arabidopsis regions, termed by us the "PAL" sub-domain, could associate with subcellular structures following transient expression of fusions with YFP in Nicotiana benthamiana. Seven YFP::PAL sub-domain fusions decorated Golgi and six were localized to mitochondria. In general, the myosin XI PAL sub-domains labeled organelles whose motility had previously been observed to be affected by mutagenesis or dominant negative assays with the respective myosins. Simultaneous transient expression of the PAL sub-domains of myosin XI-H, XI-I, and XI-K resulted in inhibition of movement of mitochondria and Golgi.

Trafficking of proteins through plastid stromules.

Stromules are thin projections from plastids that are generally longer and more abundant on non-green plastids than on chloroplasts. Occasionally stromules can be observed to connect two plastid bodies with one another. However, photobleaching of GFP-labeled plastids and stromules in 2000 demonstrated that plastids do not form a network like the endoplasmic reticulum, resulting in the proposal that stromules have major functions other than transfer of material from one plastid to another. The absence of a network was confirmed in 2012 with the use of a photoconvertible fluorescent protein, but the prior observations of movement of proteins between plastids were challenged. We review published evidence and provide new experiments that demonstrate trafficking of fluorescent protein between plastids as well as movement of proteins within stromules that emanate from a single plastid and discuss the possible function of stromules.

Analysis of Organelle Targeting by DIL Domains of the Arabidopsis Myosin XI Family.

The Arabidopsis thaliana genome encodes 13 myosin XI motor proteins. Previous insertional mutant analysis has implicated substantial redundancy of function of plant myosin XIs in transport of intracellular organelles. Considerable information is available about the interaction of cargo with the myosin XI-homologous yeast myosin V protein myo2p. We identified a region in each of 12 myosin XI sequences that correspond to the yeast myo2p secretory-vesicle binding domain (the "DIL" domain). Structural modeling of the myosin DIL domain region of plant myosin XIs revealed significant similarity to the yeast myo2p and myo4p DIL domains. Transient expression of YFP fusions with the Arabidopsis myosin XI DIL domain resulted in fluorescent labeling of a variety of organelles, including the endoplasmic reticulum, peroxisomes, Golgi, and nuclear envelope. With the exception of the YFP::MYA1 DIL fusion, expression of the DIL-YFP fusions resulted in loss of motility of labeled organelles, consistent with a dominant-negative effect. Certain fusions resulted in localization to the cytoplasm, plasma membrane, or to unidentified vesicles. The same YFP-domain fusion sometimes labeled more than one organelle. Expression of a YFP fusion to a yeast myo2p DIL domain resulted in labeling of plant peroxisomes. Fusions with some of the myosin XI domains resulted in labeling of known cargoes of the particular myosin XI; however, certain myosin XI YFP fusions labeled organelles that had not previously been found to be detectably affected by mutations nor by expression of dominant-negative constructs.

The Arabidopsis class VIII myosin ATM2 is involved in endocytosis.

Members of the class XI of the myosin superfamily comprising higher plant, actin-based molecular motors have been shown to be involved in peroxisome and Golgi vesicle trafficking comparable to yeast and animal class V myosins. The tasks of the second class of myosins of higher plants, class VIII, are unclear. In this study the class VIII myosin ATM2 from the model plant Arabidopsis thaliana was selected for the examination of cargo specificity in vivo. Fluorescent protein-fusion plasmid constructs with fragments of the ATM2 cDNA were generated and used for Agrobacterium tumefaciens-based transient transformation of Nicotiana benthamiana leaves. The resulting subcellular localization patterns were recorded by live imaging with confocal laser scanning microscopy (CLSM) in epidermal leaf cells. Expression of a nearly full-length construct displayed labeling of filaments and vesicles, a head + neck fragment led to decoration of filaments only. However, expression of fluorescent protein-tagged C-terminal tail domain constructs labeled vesicular structures of different appearance. Most importantly, coexpression of different RFP/YFP-ATM2 tail fusion proteins showed colocalization and, hence, binding to the same type of vesicular target. Further coexpression experiments of RFP/YFP-ATM2 tail fusion proteins with the endosomal marker FYVE and the endosomal tracer FM4-64 demonstrated colocalization with endosomes. Colocalization was also detected by expression of the CFP-tagged membrane receptor BRI1 as marker, which is constantly recycled via endosomes. Occasionally the ATM2 tail targeted to sites at the plasma membrane closely resembling the pattern obtained upon expression of the YFP-ATM1 C-terminal tail. ATM1 is known for its localization at the plasma membrane at sites of plasmodesmata.

Dynamic morphology of plastids and stromules in angiosperm plants.

Labelling of plastids with fluorescent proteins has revealed the diversity of their sizes and shapes in different tissues of vascular plants. Stromules, stroma-filled tubules comprising thin extensions of the stroma surrounded by the double envelope membrane, have been observed to emanate from all major types of plastid, though less common on chloroplasts. In some tissue types, stromules are highly dynamic, forming, shrinking, attaching, releasing and fragmenting. Stromule formation is negatively affected by treatment of tissue with cytoskeletal inhibitors. Plastids can be connected by stromules, through which green fluorescent protein (GFP) and fluorescently tagged chloroplast protein complexes have been observed to flow. Within the highly viscous stroma, proteins traffic by diffusion as well as by an active process of directional travel, whose mechanism is unknown. In addition to exchanging materials between plastids, stromules may also serve to increase the surface area of the envelope for import and export, reduce diffusion distance between plastids and other organelles for exchange of materials, and anchor the plastid onto attachment points for proper positioning with the plant cell. Future studies should reveal how these functions may affect plants in adapting to the challenges of a changing environment.