Proximity driven plastid-nucleus relationships are facilitated by tandem plastid-ER dynamics.

Peri-nuclear clustering (PNC) of chloroplasts has largely been described in senescent and pathogen- or ROS- stressed cells. Stromules, tubular plastid extensions are also observed under similar conditions. Coincident observations of PNC and stromules associate the two phenomena in facilitating retrograde signaling between chloroplasts and the nucleus. However, PNC incidence in non-stressed cells under normal growth and developmental conditions, when stromules are usually not observed, remains unclear. Using transgenic Arabidopsis expressing different organelle-targeted fluorescent proteins we show that PNC is a dynamic subcellular phenomenon that continues in the absence of light and is not dependent on stromule formation. PNC is facilitated by tandem plastid-ER dynamics created through membrane contact sites between the two organelles. While PNC increases upon ER-membrane expansion, some plastids may remain in the peri-nuclear region due to their localization in ER-lined nuclear indentions. Moreover, some PNC plastids may sporadically extend stromules into ER-lined nuclear grooves. Our findings strongly suggest that PNC is not an exclusive response to stress caused by pathogens, high light or exogenous-H2O2 treatment and does not require stromule formation. However, morphological and behavioural alterations in ER and concomitant changes in tandem, plastid-ER dynamics play a major role in facilitating the phenomenon.

Organelle extensions in plant cells.

The life strategy of plants includes their ability to respond quickly at the cellular level to changes in their environment. The use of targeted fluorescent protein probes and imaging of living cells has revealed several rapidly induced organelle responses that create the efficient sub-cellular machinery for maintaining homeostasis in the plant cell. Several organelles, including plastids, mitochondria, and peroxisomes, extend and retract thin tubules that have been named stromules, matrixules, and peroxules, respectively. Here, I combine all these thin tubular forms under the common head of organelle extensions. All extensions change shape continuously and in their elongated form considerably increase organelle outreach into the surrounding cytoplasm. Their pleomorphy reflects their interactions with the dynamic endoplasmic reticulum and cytoskeletal elements. Here, using foundational images and time-lapse movies, and providing salient information on some molecular and biochemically characterized mutants with increased organelle extensions, I draw attention to their common role in maintaining homeostasis in plant cells.

Chloroplast behaviour and interactions with other organelles in Arabidopsis thaliana pavement cells.

Chloroplasts are a characteristic feature of green plants. Mesophyll cells possess the majority of chloroplasts and it is widely believed that, with the exception of guard cells, the epidermal layer in most higher plants does not contain chloroplasts. However, recent observations on Arabidopsis thaliana have shown a population of chloroplasts in pavement cells that are smaller than mesophyll chloroplasts and have a high stroma to grana ratio. Here, using stable transgenic lines expressing fluorescent proteins targeted to the plastid stroma, plasma membrane, endoplasmic reticulum, tonoplast, nucleus, mitochondria, peroxisomes, F-actin and microtubules, we characterize the spatiotemporal relationships between the pavement cell chloroplasts (PCCs) and their subcellular environment. Observations on the PCCs suggest a source-sink relationship between the epidermal and the mesophyll layers, and experiments with the Arabidopsis mutants glabra2 (gl2) and immutans (im), which show altered epidermal plastid development, underscored their developmental plasticity. Our findings lay down the foundation for further investigations aimed at understanding the precise role and contributions of PCCs in plant interactions with the environment.

Peroxisome Mitochondria Inter-relations in Plants.

A large amount of ultrastructural, biochemical and molecular analysis indicates that peroxisomes and mitochondria not only share the same subcellular space but also maintain considerable overlap in their proteins, responses and functions. Recent approaches using imaging of fluorescent proteins targeted to both organelles in living plant cells are beginning to show the dynamic nature of their interactivity. Based on the observations of living cells, mitochondria respond rapidly to stress by undergoing fission. Mitochondrial fission is suggested to release key membrane-interacting members of the FISSION1 and DYNAMIN RELATED PROTEIN3 families and appears to be followed by the formation of thin peroxisomal extensions called peroxules. In a model we present the peroxules as an intermediate state prior to the formation of tubular peroxisomes, which, in turn are acted upon by the constriction-related proteins released by mitochondria and undergo rapid constriction and fission to increase the number of peroxisomes in a cell. The fluorescent protein aided imaging of peroxisome-mitochondria interaction provides visual evidence for their cooperation in maintenance of cellular homeostasis in plants.

Large Cellular Inclusions Accumulate in Arabidopsis Roots Exposed to Low-Sulfur Conditions.

Sulfur is vital for primary and secondary metabolism in plant roots. To understand the molecular and morphogenetic changes associated with loss of this key macronutrient, we grew Arabidopsis (Arabidopsis thaliana) seedlings in low-sulfur conditions. These conditions induced a cascade of cellular events that converged to produce a profound intracellular phenotype defined by large cytoplasmic inclusions. The inclusions, termed low-sulfur Pox, show cell type- and developmental zone-specific localization. Transcriptome analysis suggested that low sulfur causes dysfunction of the glutathione/ascorbate cycle, which reduces flavonoids. Genetic and biochemical evidence indicated that low-sulfur Pox are the result of peroxidase-catalyzed oxidation of quercetin in roots grown under sulfur-depleted conditions.

A stable JAZ protein from peach mediates the transition from outcrossing to self-pollination.

BACKGROUND: Variations in floral display represent one of the core features associated with the transition from allogamy to autogamy in angiosperms. The promotion of autogamy under stress conditions suggests the potential involvement of a signaling pathway with a dual role in both flower development and stress response. The jasmonic acid (JA) pathway is a plausible candidate to play such a role because of its involvement in many plant responses to environmental and developmental cues. In the present study, we used peach (Prunus persica L.) varieties with showy and non-showy flowers to investigate the role of JA (and JA signaling suppressors) in floral display. RESULTS: Our results show that PpJAZ1, a component of the JA signaling pathway in peach, regulates petal expansion during anthesis and promotes self-pollination. PpJAZ1 transcript levels were higher in petals of the non-showy flowers than those of showy flowers at anthesis. Moreover, the ectopic expression of PpJAZ1 in tobacco (Nicotiana tabacum L.) converted the showy, chasmogamous tobacco flowers into non-showy, cleistogamous flowers. Stability of PpJAZ1 was confirmed in vivo using PpJAZ1-GFP chimeric protein. PpJAZ1 inhibited JA-dependent processes in roots and leaves of transgenic plants, including induction of JA-response genes to mechanical wounding. However, the inhibitory effect of PpJAZ1 on JA-dependent fertility functions was weaker, indicating that PpJAZ1 regulates the spatial localization of JA signaling in different plant organs. Indeed, JA-related genes showed differential expression patterns in leaves and flowers of transgenic plants. CONCLUSIONS: Our results reveal that under stress conditions ­ for example, herbivore attacks ­ stable JAZ proteins such as PpJAZ1 may alter JA signaling in different plant organs, resulting in autogamy as a reproductive assurance mechanism. This represents an additional mechanism by which plant hormone signaling can modulate a vital developmental process in response to stress.

On the relationship between endoreduplication and collet hair initiation and tip growth, as determined using six Arabidopsis thaliana root-hair mutants.

A positive correlation between nuclear DNA content and cell size, as postulated by the karyoplasmic theory, has been confirmed in many plant tissues. However, there is also evidence suggesting that there are exceptions. While in previous reports the cell size:ploidy relationship was studied in intact tissues containing cells of different sizes, here simultaneously developing single cells of collet hairs were used to study endoreduplication in Arabidopsis thaliana mutants that produce hairs of variable size and morphology. Endoreduplication in the root and collet zones of six different root-hair mutants was analysed before and after collet hair development using flow cytometry and confocal microscopy. Additionally, the changes in nuclear size (ploidy), shape, and movement in developing collet hairs of a hybrid between Arabidopsis transgenic line NLS-GFP-GUS and the rhd3 (root hair defective3) mutant were followed using time-lapse confocal microscopy. In this hybrid endoreduplication in the collet hairs was disturbed. However, based on the analyses of all mutants, no correlation was found between hair length and the ploidy of the cells in the collet and root regions. The results indicate that the karyoplasmic ratio is maintained at the beginning of collet-hair development, but tip growth proceeds in a DNA-amount-independent manner. The final size of a collet hair appears to be dependent more on genetic modifiers governing general cell physiology than on its DNA content.

Secretory pathway research: the more experimental systems the better.

Transient gene expression, in plant protoplasts or specific plant tissues, is a key technique in plant molecular cell biology, aimed at exploring gene products and their modifications to examine functional subdomains, their interactions with other biomolecules, and their subcellular localization. Here, we highlight some of the major advantages and potential pitfalls of the most commonly used transient gene expression models and illustrate how ectopic expression and the use of dominant mutants can provide insights into protein function.

Differential coloring reveals that plastids do not form networks for exchanging macromolecules.

Stroma-filled tubules named stromules are sporadic extensions of plastids. Earlier, photobleaching was used to demonstrate fluorescent protein diffusion between already interconnected plastids and formed the basis for suggesting that all plastids are able to form networks for exchanging macromolecules. However, a critical appraisal of literature shows that this conjecture is not supported by unequivocal experimental evidence. Here, using photoconvertible mEosFP, we created color differences between similar organelles that enabled us to distinguish clearly between organelle fusion and nonfusion events. Individual plastids, despite conveying a strong impression of interactivity and fusion, maintained well-defined boundaries and did not exchange fluorescent proteins. Moreover, the high pleomorphy of etioplasts from dark-grown seedlings, leucoplasts from roots, and assorted plastids in the accumulation and replication of chloroplasts5 (arc5), arc6, and phosphoglucomutase1 mutants of Arabidopsis thaliana suggested that a single plastid unit might be easily mistaken for interconnected plastids. Our observations provide succinct evidence to refute the long-standing dogma of interplastid connectivity. The ability to create and maintain a large number of unique biochemical factories in the form of singular plastids might be a key feature underlying the versatility of green plants as it provides increased internal diversity for them to combat a wide range of environmental fluctuations and stresses.

Agrobacterium-derived cytokinin influences plastid morphology and starch accumulation in Nicotiana benthamiana during transient assays.

BACKGROUND: Agrobacterium tumefaciens-based transient assays have become a common tool for answering questions related to protein localization and gene expression in a cellular context. The use of these assays assumes that the transiently transformed cells are observed under relatively authentic physiological conditions and maintain 'normal' sub-cellular behaviour. Although this premise is widely accepted, the question of whether cellular organization and organelle morphology is altered in Agrobacterium-infiltrated cells has not been examined in detail. The first indications of an altered sub-cellular environment came from our observation that a common laboratory strain, GV3101(pMP90), caused a drastic increase in stromule frequency. Stromules, or 'stroma-filled-tubules' emanate from the surface of plastids and are sensitive to a variety of biotic and abiotic stresses. Starting from this observation, the goal of our experiments was to further characterize the changes to the cell resulting from short-term bacterial infestation, and to identify the factor responsible for eliciting these changes. RESULTS: Using a protocol typical of transient assays we evaluated the impact of GV3101(pMP90) infiltration on chloroplast behaviour and morphology in Nicotiana benthamiana. Our experiments confirmed that GV3101(pMP90) consistently induces stromules and alters plastid position relative to the nucleus. These effects were found to be the result of strain-dependant secretion of cytokinin and its accumulation in the plant tissue. Bacterial production of the hormone was found to be dependant on the presence of a trans-zeatin synthase gene (tzs) located on the Ti plasmid of GV3101(pMP90). Bacteria-derived cytokinins were also correlated with changes to both soluble sugar level and starch accumulation. CONCLUSION: Although we have chosen to focus on how transient Agrobacterium infestation alters plastid based parameters, these changes to the morphology and position of a single organelle, combined with the measured increases in sugar and starch content, suggest global changes to cell physiology. This indicates that cells visualized during transient assays may not be as 'normal' as was previously assumed. Our results suggest that the impact of the bacteria can be minimized by choosing Agrobacterium strains devoid of the tzs gene, but that the alterations to sub-cellular organization and cell carbohydrate status cannot be completely avoided using this strategy.

Using ER-Targeted Photoconvertible Fluorescent Proteins in Living Plant Cells.

Photoconvertible fluorescent proteins (pcFPs) enable differential coloring of a single organelle. Several pcFP-based probes have been targeted to the endoplasmic reticulum (ER) and can serve as useful tools to study ER dynamics and interactions with other organelles. Here, we describe the procedure to conduct live-cell imaging experiments using ER-targeted pcFP-based probes. Potential problems that might occur during the experiments, their solutions, and several ways to improve the experiments are discussed.

Color recovery after photoconversion of H2B::mEosFP allows detection of increased nuclear DNA content in developing plant cells.

Many higher plants are polysomatic whereby different cells possess variable amounts of nuclear DNA. The conditional triggering of endocycles results in higher nuclear DNA content (C value) that in some cases has been correlated to increased cell size. While numerous multicolored fluorescent protein (FP) probes have revealed the general behavior of the nucleus and intranuclear components, direct visualization and estimation of changes in nuclear-DNA content in live cells during their development has not been possible. Recently, monomeric Eos fluorescent protein (mEosFP) has emerged as a useful photoconvertible protein whose color changes irreversibly from a green to a red fluorescent form upon exposure to violet-blue light. The stability and irreversibility of red fluorescent mEosFP suggests that detection of green color recovery would be possible as fresh mEosFP is produced after photoconversion. Thus a ratiometric evaluation of the red and green forms of mEosFP following photoconversion could be used to estimate production of a core histone such as H2B during its concomitant synthesis with DNA in the synthesis phase of the cell cycle. Here we present proof of concept observations on transgenic tobacco (Nicotiana tabacum) Bright Yellow 2 cells and Arabidopsis (Arabidopsis thaliana) plants stably expressing H2B::mEosFP. In Arabidopsis seedlings an increase in green fluorescence is observed specifically in cells known to undergo endoreduplication. The detection of changes in nuclear DNA content by correlating color recovery of H2B::mEosFP after photoconversion is a novel approach involving a single FP. The method has potential for facilitating detailed investigations on conditions that favor increased cell size and the development of polysomaty in plants.

Local interactions shape plant cells.

Plant cell expansion is usually attributed to the considerable osmotic pressure that develops within and impinges upon the cell boundary. Whereas turgor containment within expandable walls explains global expansion, the scalar nature of turgor does not directly suggest a mechanism for achieving the localized, differential growth that is responsible for the diversity of plant-cell forms. The key to achieving local growth in plant cells appears to lie not in harnessing turgor but in using it to identify weak regions in the cell boundary and thus creating discrete intracellular domains for targeting the growth machinery. Membrane-interacting phospholipases, Rho-like proteins and their interactors, an actin-modulating ARP2/3 complex with its upstream regulators, and actin-microtubule interactions play important roles in the intracellular cooperation to shape plant cells.

Membrane contacts with the endoplasmic reticulum modulate plastid morphology and behaviour.

Plastid behaviour often occurs in tandem with endoplasmic reticulum (ER) dynamics. In order to understand the underlying basis for such linked behaviour we have used time-lapse imaging-based analysis of plastid movement and pleomorphy, including the extension and retraction of stromules. Stable transgenic plants that simultaneously express fluorescent fusion proteins targeted to the plastid stroma, and the ER along with BnCLIP1-eGFP, an independent plastid envelope localized membrane contact site (MCS) marker were utilized. Our experiments strongly suggest that transient MCS formed between the plastid envelope and the ER are responsible for their concomitant behaviour.

Plastid stromule branching coincides with contiguous endoplasmic reticulum dynamics.

Stromules are stroma-filled tubules extending from plastids whose rapid extension toward or retraction from other plastids has suggested a role in interplastidic communication and exchange of metabolites. Several studies point to sporadic dilations, kinks, and branches occurring along stromule length but have not elucidated the underlying basis for these occurrences. Similarly, although specific details on interacting partners have been missing, a consensus viewpoint suggests that stromules increase the interactive surface of a plastid with its cytoplasmic surroundings. Here, using live imaging, we show that the behavior of dynamic, pleomorphic stromules strongly coincides with that of cortical endoplasmic reticulum (ER) tubules. Covisualization of fluorescent protein-highlighted stromules and the ER in diverse cell types clearly suggests correlative dynamics of the two membrane-bound compartments. The extension and retraction, as well as directional changes in stromule branches occur in tandem with the behavior of neighboring ER tubules. Three-dimensional and four-dimensional volume rendering reveals that stromules that extend into cortical regions occupy channels between ER tubules possibly through multiple membrane contact sites. Our observations clearly depict coincidental stromule-ER behavior and suggest that either the neighboring ER tubules shape stromules directly or the behavior of both ER and stromules is simultaneously dictated by a shared cytoskeleton-based mechanism. These new observations strongly implicate the ER membrane in interactions with stromules and suggest that their interacting surfaces might serve as major conduits for bidirectional exchange of ions, lipids, and metabolites between the two organelles.

Synchronously developing collet hairs in Arabidopsis thaliana provide an easily accessible system for studying nuclear movement and endoreduplication.

Early Arabidopsis thaliana seedling growth includes the highly synchronous development of hairs from every epidermal cell of the collet (the root-hypocotyl transition zone). The dynamics of collet hair growth, and accompanying nuclear movement and endoreduplication, were followed using a combination of different fluorescent probes for time-lapse imaging and flow cytometry. Using laser-scanning confocal microscopy on the double-transgenic Arabidopsis hybrid line NLS-GFP-GUS × YPM, there appeared to be a correlation between nuclear position and the cell tip during growth of the collet hair cells, as occurs in asynchronously developing root hairs. However, disruption of nuclear movement in the growing collet hairs using low concentrations of cytoskeletal inhibitors demonstrated that nuclear positioning close to the tip of the cell is not essential for tip-directed growth of the hair. Nuclear DNA content increases from 4C to 16C during development of the collet hairs. Following cessation of growth, nuclei moved to the base of the hairs and then their movement became asynchronous and limited. Co-visualization of RFP-highlighted prevacuolar vesicles and GFP-labelled nuclei showed that, whereas small vesicles allowed unimpeded nuclear movement within the hair, transient stops and directional reversals coincided with the presence of larger vesicles in close proximity to the nucleus. Arabidopsis collet hairs provide a robust, easily accessible, naturally synchronized population of single tip-growing cells that can be used as a model cell type for studying nuclear movement and endoreduplication.

Organelle extensions in plant cells.

Cell walls lock each cell in a specific position within the supra-organization of a plant. Despite its fixed location, each cell must be able to sense alterations in its immediate environment and respond rapidly to ensure the optimal functioning, continued growth and development, and eventual long-term survival of the plant. The ultra-structural detail that underlies our present understanding of the plant cell has largely been acquired from fixed and processed material that does not allow an appreciation of the dynamic nature of sub-cellular events in the cell. In recent years, fluorescent protein-aided imaging of living plant cells has added to our understanding of the dynamic nature of the plant cell. One of the major outcomes of live imaging of plant cells is the growing appreciation that organelle shapes are not fixed, and many organelles extend their surface transiently in rapid response to environmental stimuli. In many cases, the extensions appear as tubules extending from the main organelle. Specific terms such as stromules from plastids, matrixules from mitochondria, and peroxules from peroxisomes have been coined to describe the extensions. Here, we review our present understanding of organelle extensions and discuss how they may play potential roles in maintaining cellular homeostasis in plant cells.

The ER Is a Common Mediator for the Behavior and Interactions of Other Organelles.

Optimal functioning of a plant cell depends upon the efficient exchange of genetic information, ions, proteins and metabolites between the different organelles. Intuitively, increased proximity between organelles would be expected to play an important role in facilitating exchanges between them. However, it remains to be seen whether under normal, relatively non-stressed conditions organelles maintain close proximity at all. Moreover, does interactivity involve direct and frequent physical contact between the different organelles? Further, many organelles transition between spherical and tubular forms or sporadically produce thin tubular extensions, but it remains unclear whether changes in organelle morphology play a role in increasing their interactivity. Here, using targeted multicolored fluorescent fusion proteins, we report observations on the spatiotemporal relationship between plastids, mitochondria, peroxisomes and the endoplasmic reticulum in living plant cells. Under normal conditions of growth, we observe that the smaller organelles do not establish direct, physical contacts with each other but, irrespective of their individual form they all maintain intimate connectivity with the ER. Proximity between organelles does increase in response to stress through concomitant alterations in ER dynamics. Significantly, even under increased proximity the ER still remains sandwiched between the different organelles. Our observations provide strong live-imaging-based evidence for the ER acting as a common mediator in interactions between other organelles.

mEosFP-based green-to-red photoconvertible subcellular probes for plants.

Photoconvertible fluorescent proteins (FPs) are recent additions to the biologists' toolbox for understanding the living cell. Like green fluorescent protein (GFP), monomeric EosFP is bright green in color but is efficiently photoconverted into a red fluorescent form using a mild violet-blue excitation. Here, we report mEosFP-based probes that localize to the cytosol, plasma membrane invaginations, endosomes, prevacuolar vesicles, vacuoles, the endoplasmic reticulum, Golgi bodies, mitochondria, peroxisomes, and the two major cytoskeletal elements, filamentous actin and cortical microtubules. The mEosFP fusion proteins are smaller than GFP/red fluorescent protein-based probes and, as demonstrated here, provide several significant advantages for imaging of living plant cells. These include an ability to differentially color label a single cell or a group of cells in a developing organ, selectively highlight a region of a cell or a subpopulation of organelles and vesicles within a cell for tracking them, and understanding spatiotemporal aspects of interactions between similar as well as different organelles. In addition, mEosFP probes introduce a milder alternative to fluorescence recovery after photobleaching, whereby instead of photobleaching, photoconversion followed by recovery of green fluorescence can be used for estimating subcellular dynamics. Most importantly, the two fluorescent forms of mEosFP furnish bright internal controls during imaging experiments and are fully compatible with cyan fluorescent protein, GFP, yellow fluorescent protein, and red fluorescent protein fluorochromes for use in simultaneous, multicolor labeling schemes. Photoconvertible mEosFP-based subcellular probes promise to usher in a much higher degree of precision to live imaging of plant cells than has been possible so far using single-colored FPs.