The signal transducer NPH3 integrates the phototropin1 photosensor with PIN2-based polar auxin transport in Arabidopsis root phototropism.

Under blue light (BL) illumination, Arabidopsis thaliana roots grow away from the light source, showing a negative phototropic response. However, the mechanism of root phototropism is still unclear. Using a noninvasive microelectrode system, we showed that the BL sensor phototropin1 (phot1), the signal transducer NONPHOTOTROPIC HYPOCOTYL3 (NPH3), and the auxin efflux transporter PIN2 were essential for BL-induced auxin flux in the root apex transition zone. We also found that PIN2-green fluorescent protein (GFP) localized to vacuole-like compartments (VLCs) in dark-grown root epidermal and cortical cells, and phot1/NPH3 mediated a BL-initiated pathway that caused PIN2 redistribution to the plasma membrane. When dark-grown roots were exposed to brefeldin A (BFA), PIN2-GFP remained in VLCs in darkness, and BL caused PIN2-GFP disappearance from VLCs and induced PIN2-GFP-FM4-64 colocalization within enlarged compartments. In the nph3 mutant, both dark and BL BFA treatments caused the disappearance of PIN2-GFP from VLCs. However, in the phot1 mutant, PIN2-GFP remained within VLCs under both dark and BL BFA treatments, suggesting that phot1 and NPH3 play different roles in PIN2 localization. In conclusion, BL-induced root phototropism is based on the phot1/NPH3 signaling pathway, which stimulates the shootward auxin flux by modifying the subcellular targeting of PIN2 in the root apex transition zone.

Chloroplast incorporation and long-term photosynthetic performance through the life cycle in laboratory cultures of Elysia timida (Sacoglossa, Heterobranchia).

INTRODUCTION: The Mediterranean sacoglossan Elysia timida is one of the few sea slug species with the ability to sequester chloroplasts from its food algae and to subsequently store them in a functional state in the digestive gland cells for more than a month, during which time the plastids retain high photosynthetic activity (= long-term retention). Adult E. timida have been described to feed on the unicellular alga Acetabularia acetabulum in their natural environment. The suitability of E. timida as a laboratory model culture system including its food source was studied. RESULTS: In contrast to the literature reporting that juvenile E. timida feed on Cladophora dalmatica first, and later on switch to the adult diet A. acetabulum, the juveniles in this study fed directly on A. acetabulum (young, non-calcified stalks); they did not feed on the various Cladophora spp. (collected from the sea or laboratory culture) offered. This could possibly hint to cryptic speciation with no clear morphological differences, but incipient ecological differentiation. Transmission electron microscopy of chloroplasts from A. acetabulum after initial intake by juvenile E. timida showed different states of degradation - in conglomerations or singularly - and fragments of phagosome membranes, but differed from kleptoplast images of C. dalmatica in juvenile E. timida from the literature. Based on the finding that the whole life cycle of E. timida can be completed with A. acetabulum as the sole food source, a laboratory culture system was established. An experiment with PAM-fluorometry showed that cultured E. timida are also able to store chloroplasts in long-term retention from Acetabularia peniculus, which stems from the Indo-Pacific and is not abundant in the natural environment of E. timida. Variations between three experiment groups indicated potential influences of temperature on photosynthetic capacities. CONCLUSIONS: E. timida is a viable laboratory model system to study photosynthesis in incorporated chloroplasts (kleptoplasts). Capacities of chloroplast incorporation in E. timida were investigated in a closed laboratory culture system with two different chloroplast donors and over extended time periods about threefold longer than previously reported.

Arabidopsis homologs of nucleus- and phragmoplast-localized kinase 2 and 3 and mitogen-activated protein kinase 4 are essential for microtubule organization.

A double homozygous recessive mutant in the Arabidopsis thaliana homologs of nucleus- and phragmoplast-localized kinase 2 (ANP2) and 3 (ANP3) genes and a homozygous recessive mutant in the mitogen-activated protein kinase 4 (MPK4) gene of Arabidopsis exhibit deficiencies in the overall microtubule (MT) organization, which result in abnormal cell growth patterns, such as branching of root hairs and swelling of diffusely growing epidermal cells. Genetic, pharmacological, molecular, cytological, and biochemical analyses show that the major underlying mechanism for these phenotypes is excessive MT stabilization manifested in both mutants as heavy MT bundling, disorientation, and drug stability. The above defects in MAPK signaling result in the adverse regulation of members of the microtubule-associated protein (MAP65) protein family, including strongly diminished phosphorylation of MAP65-1. These data suggest that ANP2/ANP3, MPK4, and the microtubule-associated protein MAP65-1, a putative target of MPK4 signaling, are all essential for the proper organization of cortical microtubules in Arabidopsis epidermal cells.

Arabidopsis MPK6 is involved in cell division plane control during early root development, and localizes to the pre-prophase band, phragmoplast, trans-Golgi network and plasma membrane.

The proper spatial and temporal expression and localization of mitogen-activated protein kinases (MAPKs) is essential for developmental and cellular signalling in all eukaryotes. Here, we analysed expression, subcellular localization and function of MPK6 in roots of Arabidopsis thaliana using wild-type plants and three mpk6 knock-out mutant lines. The MPK6 promoter showed two expression maxima in the most apical part of the root meristem and in the root transition zone. This expression pattern was highly consistent with 'no root' and 'short root' phenotypes, as well as with ectopic cell divisions and aberrant cell division planes, resulting in disordered cell files in the roots of these mpk6 knock-out mutants. In dividing root cells, MPK6 was localized on the subcellular level to distinct fine spots in the pre-prophase band and phragmoplast, representing the two most important cytoskeletal structures controlling the cell division plane. By combining subcellular fractionation and microscopic in situ and in vivo co-localization methods, MPK6 was localized to the plasma membrane (PM) and the trans-Golgi network (TGN). In summary, these data suggest that MPK6 localizing to mitotic microtubules, secretory TGN vesicles and the PM is involved in cell division plane control and root development in Arabidopsis.

Developmental localization and the role of hydroxyproline rich glycoproteins during somatic embryogenesis of banana (Musa spp. AAA).

BACKGROUND: Hydroxyproline rich glycoproteins (HRGPs) are implicated to have a role in many aspects of plant growth and development but there is limited knowledge about their localization and function during somatic embryogenesis of higher plants. In this study, the localization and function of hydroxyproline rich glycoproteins in embryogenic cells (ECs) and somatic embryos of banana were investigated by using immunobloting and immunocytochemistry with monoclonal JIM11 and JIM20 antibodies as well as by treatment with 3,4-dehydro-L-proline (3,4-DHP, an inhibitor of extensin biosynthesis), and by immunomodulation with the JIM11 antibody. RESULTS: Immunofluorescence labelling of JIM11 and JIM20 hydroxyproline rich glycoprotein epitopes was relatively weak in non-embryogenic cells (NECs), mainly on the edge of small cell aggregates. On the other hand, hydroxyproline rich glycoprotein epitopes were found to be enriched in early embryogenic cells as well as in various developmental stages of somatic embryos. Embryogenic cells (ECs), proembryos and globular embryos showed strong labelling of hydroxyproline rich glycoprotein epitopes, especially in their cell walls and outer surface layer, so-called extracellular matrix (ECM). This hydroxyproline rich glycoprotein signal at embryo surfaces decreased and/or fully disappeared during later developmental stages (e.g. pear-shaped and cotyledonary stages) of embryos. In these later developmental embryogenic stages, however, new prominent hydroxyproline rich glycoprotein labelling appeared in tri-cellular junctions among parenchymatic cells inside these embryos. Overall immunofluorescence labelling of late stage embryos with JIM20 antibody was weaker than that of JIM11. Western blot analysis supported the above immunolocalization data. The treatment with 3,4-DHP inhibited the development of embryogenic cells and decreased the rate of embryo germination. Embryo-like structures, which developed after 3,4-DHP treatment showed aberrant non-compact epidermis with discontinuous ECM at the outer surface as well as much less immunolabelling with the JIM11 antibody. This treatment also decreased the plant regeneration capacity in embryogenic banana cultures. Finally, immunomodulation of surface hydroxyproline rich glycoproteins by co-culture of embryos with the JIM11 antibody resulted in a much lower germination capacity of these embryos. CONCLUSIONS: These results suggest that hydroxyproline rich glycoproteins play an important developmental role, especially in the process of regeneration and germination of embryos during plant regeneration via somatic embryogenesis. Proper content and localization of hydroxyproline rich glycoproteins seem to be essential for the formation and regeneration of banana somatic embryos.

Mitogen-activated protein kinase 4 is involved in the regulation of mitotic and cytokinetic microtubule transitions in Arabidopsis thaliana.

• A mitogen-activated protein kinase kinase kinase (MAPKKK) double mutant, Arabidopsis homologue of nucleus and phragmoplast associated kinase (anp) anp2anp3, and the mitogen-activated protein kinase (MAPK) 4 mutant mpk4 of Arabidopsis thaliana show prominent cytokinetic defects. This prompted the analysis of mitotic and cytokinetic progression as a function of MAPK signalling. Mutants were compared with wild types untreated or treated with the specific MAPKK inhibitor PD98059. • This study included phenotype analysis, expression analysis of the MPK4 promoter, immunofluorescent localization of MPK4, tubulin and MAP65-1, and time-lapse microscopic visualization of the mitotic microtubule (MT) transitions in control, mutant and inhibitor-treated cells. • Mutant and inhibitor-treated cells showed defects in mitosis and cytokinesis, including aberrant spindle and phragmoplast formation and drastically delayed or abortive mitosis and cytokinesis. As a result, bi- and multinucleate cells were formed, ultimately disturbing the vegetative tissue patterning. MPK4 was localized to all stages of the expanding phragmoplast, in a pattern similar to that of its putative substrate MAP65-1. • In this study, MPK4 is shown to be involved in the regulation of mitosis/cytokinesis through modulation of the cell division plane and cytokinetic progression.

The endocytic network in plants.

Endocytosis and vesicle recycling via secretory endosomes are essential for many processes in multicellular organisms. Recently, higher plants have provided useful experimental model systems to study these processes. Endocytosis and secretory endosomes in plants play crucial roles in polar tip growth, a process in which secretory and endocytic pathways are integrated closely. Plant endocytosis and endosomes are important for auxin-mediated cell-cell communication, gravitropic responses, stomatal movements, cytokinesis and cell wall morphogenesis. There is also evidence that F-actin is essential for endocytosis and that plant-specific myosin VIII is an endocytic motor in plants. Last, recent results indicate that the trans Golgi network in plants should be considered an integral part of the endocytic network.

Microtubule-dependent motility and orientation of the cortical endoplasmic reticulum in elongating characean internodal cells.

Motility of the endoplasmic reticulum (ER) is predominantly microtubule- dependent in animal cells but thought to be entirely actomyosin-dependent in plant cells. Using live cell imaging and transmission electron microscopy to examine ER motility and structural organization in giant internodal cells of characean algae, we discovered that at the onset of cell elongation, the cortical ER situated near the plasma membrane formed a tight meshwork of predominantly transverse ER tubules that frequently coaligned with microtubules. Microtubule depolymerization increased mesh size and decreased the dynamics of the cortical ER. In contrast, perturbing the cortical actin array with cytochalasins did not affect the transverse orientation but decreased mesh size and increased ER dynamics. Our data suggest that myosin-dependent ER motility is confined to the ER strands in the streaming endoplasm, while the more sedate cortical ER uses microtubule-based mechanisms for organization and motility during early stages of cell elongation. We show further that the ER has an inherent, NEM-sensitive dynamics which can be altered via interaction with the cytoskeleton and that tubule formation and fusion events are cytoskeleton-independent.

D'orenone blocks polarized tip growth of root hairs by interfering with the PIN2-mediated auxin transport network in the root apex.

SUMMARY: The C(18) ketone (5E,7E)-6-methyl-8-(2,6,6-trimethylcyclohex-1-enyl)octa-5,7-dien-2-one (D'orenone) has been postulated to be an early cleavage product of beta-carotene en route to trisporic acids; these act as morphogenetic factors during the sexual reproduction of zygomycetes. Here we report that D'orenone blocks the highly polarized tip growth of root hairs, causing tip growth to stop completely within a few minutes. Importantly, external auxin reverses the effects of D'orenone on root hairs. Further analysis revealed that D'orenone lowers the auxin concentration in trichoblasts via PIN2-mediated auxin efflux to below the critical levels essential for root hair growth. D'orenone specifically increases PIN2 protein abundance without affecting PIN2 transcripts, and the PIN2 expression domain enlarges and shifts basipetally, resulting in more active auxin transport. The observation that D'orenone does not interfere with the root hair growth in roots of null mutant lines provides additional evidence that PIN2 is its specific target.

Getting connected: actin-based cell-to-cell channels in plants and animals.

It has been known for more than one hundred years that plant cells are interconnected by cytoplasmic channels called plasmodesmata. This supracellularity was generally considered to be an exotic feature of walled plants containing immobile cells that are firmly enclosed within robust walls. Unexpectedly, intercellular channels in mobile animal cells have been discovered recently. These are extremely dynamic and sensitive to mechanical stress, which causes their rapid breakage and retraction. Both plasmodesmata and nanotubular cell-to-cell channels are supported by the actin cytoskeleton and exclude microtubules. In this article, we discuss the relevance of cell-to-cell channels not only for intercellular communication but also for the development and morphogenesis of multicellular organisms. We also suggest possible parallels between the cell-to-cell transport of endosomes and intracellular pathogens.

Polar transport of auxin: carrier-mediated flux across the plasma membrane or neurotransmitter-like secretion?

Auxin (indole-3-acetic acid) has its name derived from the Greek word auxein, meaning 'to increase', and it drives plant growth and development. Auxin is a small molecule derived from the amino acid tryptophan and has both hormone- and morphogen-like properties. Although there is much still to be learned, recent progress has started to unveil how auxin is transported from cell-to-cell in a polar manner. Two recent breakthrough papers from Gerd Jürgens' group indicate that auxin transport is mediated by regulated vesicle trafficking, thus encompassing neurotransmitter-like features.

Vesicular trafficking, cytoskeleton and signalling in root hairs and pollen tubes.

Root hairs and pollen tubes show strictly polar cell expansion called tip growth. Recent studies of tip growth in root hairs and pollen tubes have revealed that small GTPases of the Rab, Arf and Rho/Rac families, along with their regulatory proteins, are essential for spatio-temporal regulation of vesicular trafficking, cytoskeleton organization and signalling. ROP/RAC GTPases are involved in a multiplicity of functions including the regulation of cytoskeleton organization, calcium signalling and endocytosis in pollen tubes and root hairs. One of the most exciting recent discoveries is the preferential localization of vesicles of the trans-Golgi network (TGN), defined by specific RAB GTPases, in the apical "clear zone" and the definition of TGN as a bona fide organelle involved in both polarized secretion and endocytosis. The TGN is thought to serve the function of an early endosome in plants because it is involved in early endocytosis and rapid vesicular recycling of the plasma membrane in root epidermal cells.

Microtubule dynamics in root hairs of Medicago truncatula.

The microtubular cytoskeleton plays an important role in the development of tip-growing plant cells, but knowledge about its dynamics is incomplete. In this study, root hairs of the legume Medicago truncatula have been chosen for a detailed analysis of microtubular cytoskeleton dynamics using GFP-MBD and EB1-YFP as markers and 4D imaging. The microtubular cytoskeleton appears mainly to be composed of bundles which form tracks along which new microtubules polymerise. Polymerisation rates of microtubules are highest in the tip of growing root hairs. Treatment of root hairs with Nod factor and latrunculin B result in a twofold decrease in polymerisation rate. Nonetheless, no direct, physical interaction between the actin filament cytoskeleton and microtubules could be observed. A new picture of how the plant cytoskeleton is organised in apically growing root hairs emerges from these observations, revealing similarities with the organisation in other, non-plant, tip-growing cells.

The actin-interacting protein AIP1 is essential for actin organization and plant development.

Cell division, growth, and cytoplasmic organization require a dynamic actin cytoskeleton. The filamentous actin (F-actin) network is regulated by actin binding proteins that modulate actin dynamics. These actin binding proteins often have cooperative interactions. In particular, actin interacting protein 1 (AIP1) is capable of capping F-actin and enhancing the activity of the small actin modulating protein, actin depolymerising factor (ADF) in vitro. Here, we analyze the effect of the inducible expression of AIP1 RNAi in Arabidopsis plants to assess AIP1s role in vivo. In intercalary growing cells, the normal actin organization is disrupted, and thick bundles of actin appear in the cytoplasm. Moreover, in root hairs, there is the unusual appearance of actin cables ramifying the root hair tip. We suggest that the reduction in AIP1 results in a decrease in F-actin turnover and the promotion of actin bundling. This distortion of the actin cytoskeleton causes severe plant developmental abnormalities. After induction of the Arabidopis RNAi lines, the cells in the leaves, roots, and shoots fail to expand normally, and in the severest phenotypes, the plants die. Our data suggest that AIP1 is essential for the normal functioning of the actin cytoskeleton in plant development.

Plant synapses: actin-based domains for cell-to-cell communication.

For many years it has been known that plants perform rapid long-distance signalling using classical action potentials that have impacts on diverse processes in plants. Plants also synthesize numerous neuronal molecules and fulfill some criteria for intelligent behaviour. Analysis of recent breakthrough data from ecophysiology studies has revealed that plant roots can discriminate between 'self' and 'non-self'; in animals, this ability to discriminate is dependent on the activities of neuronal synapses. Here, we propose that plant cells establish modes of information exchange between each other that have properties in common with neuronal synapses. Moreover, plants also assemble adhesive contacts that orchestrate cell-to-cell communication between the host cells when challenged with pathogens, parasites and potential symbionts. We propose that these adhesive contacts resemble the immunological synapses found in animals.

GFP-FABD2 fusion construct allows in vivo visualization of the dynamic actin cytoskeleton in all cells of Arabidopsis seedlings.

In vivo visualization of filamentous actin in all cells of Arabidopsis thaliana seedlings is essential for understanding the numerous roles of the actin cytoskeleton in diverse processes of cell differentiation. A previously introduced reporter construct based on the actin-binding domain of mouse talin proved to be useful for unravelling some of these aspects in cell layers close to the organ surface. However, cells more deeply embedded, especially stelar cells active in polar transport of auxin, show either diffuse or no fluorescence at all due to the lack of expression of the fusion protein. The same problem is encountered in the root meristem. Recently introduced actin reporters based on fusions between A. thaliana fimbrin 1 and GFP gave brilliant results in organs from the root differentiation zone upwards to the leaves, however failed to depict the filamentous actin cytoskeleton in the transition zone of the root, in the apical meristem and the root cap. To overcome these problems, we have prepared new transgenic lines for the visualization of F-actin in vivo. We report here that a construct consisting of GFP fused to the C-terminal half of A. thaliana fimbrin 1 reveals dynamic arrays of F-actin in all cells of stably transformed A. thaliana seedlings.

Actin-based motility of endosomes is linked to the polar tip growth of root hairs.

Plant tip growth has been recognized as an actin-based cellular process requiring targeted exocytosis and compensatory endocytosis to occur at the growth cone. However, the identity of subcellular compartments involved in polarized membrane trafficking pathways remains enigmatic in plants. Here we characterize endosomal compartments in tip-growing root hair cells. We demonstrate their presence at the growing tip and differential distribution upon cessation of tip growth. We also show that both the presence of endosomes as well as their rapid movements within the tip region depends on an intact actin cytoskeleton and involves actin polymerization. In conclusion, actin-propelled endosomal motility is tightly linked to the polar tip growth of root hairs.

Actin, actin-related proteins and profilin in diatoms: a comparative genomic analysis.

Diatoms are heterokont unicellular algae with a widespread distribution throughout all aquatic habitats. Research on diatoms has advanced significantly over the last decade due to available genetic transformation methods and publicly available genome databases. Yet up to now, proteins involved in the regulation of the cytoskeleton in diatoms are largely unknown. Consequently, this work focuses on actin and actin-related proteins (ARPs) encoded in the diatom genomes of Thalassiosira pseudonana, Thalassiosira oceanica, Phaeodactylum tricornutum, Fragilariopsis cylindrus and Pseudo-nitzschia multiseries. Our comparative genomic study revealed that most diatoms possess only a single conventional actin and a small set of ARPs. Among these are the highly conserved cytoplasmic Arp1 protein and the nuclear Arp4 as well as Arp6. Diatom genomes contain genes coding for two structurally different homologues of Arp4 that might serve specific functions. All diatom species examined here lack ARP2 and ARP3 proteins, suggesting that diatoms are not capable of forming the Arp2/3 complex, which is essential in most eukaryotes for actin filament branching and plus-end dynamics. Interestingly, none of the sequenced representatives of the Bacillariophyta phylum code for profilin. Profilin is an essential actin-binding protein regulating the monomer actin pool and is involved in filament plus-end dynamics. This is the first report of organisms not containing profilin.

Heterologous DNA Uptake in Cultured Symbiodinium spp. Aided by Agrobacterium tumefaciens.

Plant-targeted pCB302 plasmids containing sequences encoding gfp fusions with a microtubule-binding domain; gfp with the fimbrin actin-binding domain 2; and gfp with AtRACK1C from Arabidopsis thaliana, all harbored in Agrobacterium tumefaciens, were used to assay heterologous expression on three different clades of the photosynthetic dinoflagellate, Symbiodinium. Accessibility to the resistant cell wall and through the plasma membrane of these dinoflagellates was gained after brief but vigorous shaking in the presence of glass beads and polyethylene glycol. A resistance gene to the herbicide Basta allowed appropriate selection of the cells expressing the hybrid proteins, which showed a characteristic green fluorescence, although they appeared to lose their photosynthetic pigments and did not further divide. Cell GFP expression frequency measured as green fluorescence emission yielded 839 per every 106 cells for Symbiodinium kawagutii, followed by 640 and 460 per every 106 cells for Symbiodinium microadriaticum and Symbiodinium sp. Mf11, respectively. Genomic PCR with specific primers amplified the AtRACK1C and gfp sequences after selection in all clades, thus revealing their presence in the cells. RT-PCR from RNA of S. kawagutii co-incubated with A. tumefaciens harboring each of the three vectors with their respective constructs, amplified products corresponding to the heterologous gfp sequence while no products were obtained from three distinct negative controls. The reported procedure shows that mild abrasion followed by co-incubation with A. tumefaciens harboring heterologous plasmids with CaMV35S and nos promoters can lead to expression of the encoded proteins into the Symbiodinium cells in culture. Despite the obvious drawbacks of the procedure, this is an important first step towards a stable transformation of Symbiodinium.

Cell differentiation and the cytoskeleton in Acetabularia.

In multicellular organisms, differentiation of individual cells is typically linked to the development of the whole organism. As cells acquire tissue-specific morphologies and become functionally specialized they lose in turn a number of other functions. A free living, single celled organism, however, maintains all such functions. Compartmentalization and intracellular communication are two basic principles by which expression of specialized features is achieved within a unicell. Both in turn depend on the structure and dynamics of the cytoskeleton. Giant algal unicells lend themselves as experimental models for the study of the cytoskeleton, because the cytoskeletal arrays inside these cells become equally enormous in size. Some of these organisms are large enough to be mistaken for multicellular plants, equipped with holdfast, stem and assimilatory organ. The marine green alga Acetabularia is one of these giant cells, which has already been well known to phycologists and cell biologists for several decades. The current review discusses recent progress in the study of the cytoskeleton in Acetabularia and examines classic concepts of cell morphogenesis from the perspective of cytoskeletal function. Contents Summary 369 I. Introduction 369 II. Morphogenetic stages 371 III. Post-transcriptional control of morphogenesis 687 IV. Apparent plasticity of morphogenesis 389 V. Prospects of using molecular approaches 391.