Quantitative Imaging Reveals Distinct Contributions of SnRK2 and ABI3 in Plasmodesmatal Permeability in Physcomitrella patens.

Cell-to-cell communication is tightly regulated in response to environmental stimuli in plants. We previously used a photoconvertible fluorescent protein Dendra2 as a model reporter to study this process. This experiment revealed that macromolecular trafficking between protonemal cells in Physcomitrella patens is suppressed in response to abscisic acid (ABA). However, it remains unknown which ABA signaling components contribute to this suppression and how. Here, we show that ABA signaling components SUCROSE NON-FERMENTING 1-RELATED PROTEIN KINASE 2 (PpSnRK2) and ABA INSENSITIVE 3 (PpABI3) play roles as an essential and promotive factor, respectively, in regulating ABA-induced suppression of Dendra2 diffusion between cells (ASD). Our quantitative imaging analysis revealed that disruption of PpSnRK2 resulted in defective ASD onset itself, whereas disruption of PpABI3 caused an 81-min delay in the initiation of ASD. Live-cell imaging of callose deposition using aniline blue staining showed that, despite this onset delay, callose deposition on cross walls remained constant in the PpABI3 disruptant, suggesting that PpABI3 facilitates ASD in a callose-independent manner. Given that ABA is an important phytohormone to cope with abiotic stresses, we further explored cellular physiological responses. We found that the acquisition of salt stress tolerance is promoted by PpABI3 in a quantitative manner similar to ASD. Our results suggest that PpABI3-mediated ABA signaling may effectively coordinate cell-to-cell communication during the acquisition of salt stress tolerance. This study will accelerate the quantitative study for ABA signaling mechanism and function in response to various abiotic stresses.

Exposure to heavy metal stress triggers changes in plasmodesmatal permeability via deposition and breakdown of callose.

Both plants and animals must contend with changes in their environment. The ability to respond appropriately to these changes often underlies the ability of the individual to survive. In plants, an early response to environmental stress is an alteration in plasmodesmatal permeability with accompanying changes in cell to cell signaling. However, the ways in which plasmodesmata are modified, the molecular players involved in this regulation, and the biological significance of these responses are not well understood. Here, we examine the effects of nutrient scarcity and excess on plasmodesmata-mediated transport in the Arabidopsis thaliana root and identify two CALLOSE SYNTHASES and two ß-1,3-GLUCANASES as key regulators of these processes. Our results suggest that modification of plasmodesmata-mediated signaling underlies the ability of the plant to maintain root growth and properly partition nutrients when grown under conditions of excess nutrients.

Ectopic Expression of WINDING 1 Leads to Asymmetrical Distribution of Auxin and a Spiral Phenotype in Rice.

Ectopic expression of the rice WINDING 1 (WIN1) gene leads to a spiral phenotype only in shoots but not in roots. Rice WIN1 belongs to a specific class of proteins in cereal plants containing a Bric-a-Brac/Tramtrack/Broad (BTB) complex, a non-phototropic hypocotyl 3 (NPH3) domain and a coiled-coil motif. The WIN1 protein is predominantly localized to the plasma membrane, but is also co-localized to plasmodesmata, where it exhibits a punctate pattern. It is observed that WIN1 is normally expressed in roots and the shoot-root junction, but not in the rest of shoots. In roots, WIN1 is largely localized to the apical and basal sides of cells. However, upon ectopic expression, WIN1 appears on the longitudinal sides of leaf sheath cells, correlated with the appearance of a spiral phenotype in shoots. Despite the spiral phenotype, WIN1-overexpressing plants exhibit a normal phototropic response. Although treatments with exogenous auxins or a polar auxin transport inhibitor do not alter the spiral phenotype, the excurvature side has a higher auxin concentration than the incurvature side. Furthermore, actin filaments are more prominent in the excurvature side than in the incurvature side, which correlates with cell size differences between these two sides. Interestingly, ectopic expression of WIN1 does not cause either unequal auxin distribution or actin filament differences in roots, so a spiral phenotype is not observed in roots. The action of WIN1 appears to be different from that of other proteins causing a spiral phenotype, and it is likely that WIN1 is involved in 1-N-naphthylphthalamic acid-insensitive plasmodesmata-mediated auxin transport.

Probing plasmodesmata function with biochemical inhibitors.

To investigate plasmodesmata (PD) function, a useful technique is to monitor the effect on cell-to-cell transport of applying an inhibitor of a physiological process, protein, or other cell component of interest. Changes in PD transport can then be monitored in one of several ways, most commonly by measuring the cell-to-cell movement of fluorescent tracer dyes or of free fluorescent proteins. Effects on PD structure can be detected in thin sections of embedded tissue observed using an electron microscope, most commonly a Transmission Electron Microscope (TEM). This chapter outlines commonly used inhibitors, methods for treating different tissues, how to detect altered cell-to-cell transport and PD structure, and important caveats.

Techniques for assessing the effects of pharmacological inhibitors on intercellular protein movement.

Intercellular protein movement is an important mechanism in plant development. Here we present an integrated protocol that utilizes an inducible system to block plasmodesmata-dependent movement and assessment of fluorescent recovery after photobleaching (FRAP) to identify compounds that influence intercellular protein movement.

Probing protein targeting to plasmodesmata using fluorescence recovery after photo-bleaching.

Fluorescence recovery after photo-bleaching (FRAP) involves the irreversible bleaching of a fluorescent protein within a specific area of the cell using a high-intensity laser. The recovery of fluorescence represents the movement of new protein into this area and can therefore be used to investigate factors involved in this movement. Here we describe a FRAP method to investigate the effect of a range of pharmacological agents on the targeting of Tobacco mosaic virus movement protein to plasmodesmata.

DEVELOPMENTALLY REGULATED PLASMA MEMBRANE PROTEIN of Nicotiana benthamiana contributes to potyvirus movement and transports to plasmodesmata via the early secretory pathway and the actomyosin system.

The intercellular movement of plant viruses requires both viral and host proteins. Previous studies have demonstrated that the frame-shift protein P3N-PIPO (for the protein encoded by the open reading frame [ORF] containing 5'-terminus of P3 and a +2 frame-shift ORF called Pretty Interesting Potyviridae ORF and embedded in the P3) and CYLINDRICAL INCLUSION (CI) proteins were required for potyvirus cell-to-cell movement. Here, we provide genetic evidence showing that a Tobacco vein banding mosaic virus (TVBMV; genus Potyvirus) mutant carrying a truncated PIPO domain of 58 amino acid residues could move between cells and induce systemic infection in Nicotiana benthamiana plants; mutants carrying a PIPO domain of seven, 20, or 43 amino acid residues failed to move between cells and cause systemic infection in this host plant. Interestingly, the movement-defective mutants produced progeny that eliminated the previously introduced stop codons and thus restored their systemic movement ability. We also present evidence showing that a developmentally regulated plasma membrane protein of N. benthamiana (referred to as NbDREPP) interacted with both P3N-PIPO and CI of the movement-competent TVBMV. The knockdown of NbDREPP gene expression in N. benthamiana impeded the cell-to-cell movement of TVBMV. NbDREPP was shown to colocalize with TVBMV P3N-PIPO and CI at plasmodesmata (PD) and traffic to PD via the early secretory pathway and the actomyosin motility system. We also show that myosin XI-2 is specially required for transporting NbDREPP to PD. In conclusion, NbDREPP is a key host protein within the early secretory pathway and the actomyosin motility system that interacts with two movement proteins and influences virus movement.

Salicylic acid regulates Plasmodesmata closure during innate immune responses in Arabidopsis.

In plants, mounting an effective innate immune strategy against microbial pathogens involves triggering local cell death within infected cells as well as boosting the immunity of the uninfected neighboring and systemically located cells. Although not much is known about this, it is evident that well-coordinated cell-cell signaling is critical in this process to confine infection to local tissue while allowing for the spread of systemic immune signals throughout the whole plant. In support of this notion, direct cell-to-cell communication was recently found to play a crucial role in plant defense. Here, we provide experimental evidence that salicylic acid (SA) is a critical hormonal signal that regulates cell-to-cell permeability during innate immune responses elicited by virulent bacterial infection in Arabidopsis thaliana. We show that direct exogenous application of SA or bacterial infection suppresses cell-cell coupling and that SA pathway mutants are impaired in this response. The SA- or infection-induced suppression of cell-cell coupling requires an enhanced desease resistance1- and nonexpressor of pathogenesis-related genes1-dependent SA pathway in conjunction with the regulator of plasmodesmal gating Plasmodesmata-located protein5. We discuss a model wherein the SA signaling pathway and plasmodesmata-mediated cell-to-cell communication converge under an intricate regulatory loop.

A developmental framework for complex plasmodesmata formation revealed by large-scale imaging of the Arabidopsis leaf epidermis.

Plasmodesmata (PD) form tubular connections that function as intercellular communication channels. They are essential for transporting nutrients and for coordinating development. During cytokinesis, simple PDs are inserted into the developing cell plate, while during wall extension, more complex (branched) forms of PD are laid down. We show that complex PDs are derived from existing simple PDs in a pattern that is accelerated when leaves undergo the sink-source transition. Complex PDs are inserted initially at the three-way junctions between epidermal cells but develop most rapidly in the anisocytic complexes around stomata. For a quantitative analysis of complex PD formation, we established a high-throughput imaging platform and constructed PDQUANT, a custom algorithm that detected cell boundaries and PD numbers in different wall faces. For anticlinal walls, the number of complex PDs increased with increasing cell size, while for periclinal walls, the number of PDs decreased. Complex PD insertion was accelerated by up to threefold in response to salicylic acid treatment and challenges with mannitol. In a single 30-min run, we could derive data for up to 11k PDs from 3k epidermal cells. This facile approach opens the door to a large-scale analysis of the endogenous and exogenous factors that influence PD formation.

Phloem unloading follows an extensive apoplasmic pathway in cucumber (Cucumis sativus L.) fruit from anthesis to marketable maturing stage.

The phloem unloading pathway remains unclear in fruits of Cucurbitaceae, a classical stachyose-transporting species with bicollateral phloem. Using a combination of electron microscopy, transport of phloem-mobile symplasmic tracer carboxyfluorescein, assays of acid invertase and sucrose transporter, and [(14)C]sugar uptake, the phloem unloading pathway was studied in cucumber (Cucumis sativus) fruit from anthesis to the marketable maturing stage. Structural investigations showed that the sieve element-companion cell (SE-CC) complex of the vascular bundles feeding fruit flesh is apparently symplasmically restricted. Imaging of carboxyfluorescein unloading showed that the dye remained confined to the phloem strands of the vascular bundles in the whole fruit throughout the stages examined. A 37 kDa acid invertase was located predominantly in the cell walls of SE-CC complexes and parenchyma cells. Studies of [(14)C]sugar uptake suggested that energy-driven transporters may be functional in sugar trans-membrane transport within symplasmically restricted SE-CC complex, which was further confirmed by the existence of a functional plasma membrane sucrose transporter (CsSUT4) in cucumber fruit. These data provide a clear evidence for an apoplasmic phloem unloading pathway in cucumber fruit. A presumption that putative raffinose or stachyose transporters may be involved in soluble sugars unloading was discussed.

Intracellular trafficking of Potato leafroll virus movement protein in transgenic Arabidopsis.

Intracellular trafficking of viral movement proteins (MPs) in plants has mainly been studied using Tobacco mosaic virus MP30 (TMV MP30) as a model system. Because of the limitations of TMV MP30 expression in Arabidopsis thaliana, these studies have mostly been restricted to tobacco plants. Here we present data on the analysis of transgenic Arabidopsis plants expressing Potato leafroll virus 17-kDa movement protein (MP17) fused to green fluorescent protein. MP17 localizes to secondary branched plasmodesmata (PD) in source but not to simple PD in sink tissues, where MP17 is believed to be degraded by proteolysis. To unravel the intracellular transport path of MP17, we analyzed the relevance of the cytoskeleton and of the secretory pathway on MP17 targeting. To this end, a new incubation system for in vivo analysis of immediate and long-term responses of whole Arabidopsis plants to inhibitor treatments was established. Microscopic and histochemical analysis showed that MP17 is targeted to PD in an actin- and endoplasmic reticulum-Golgi-dependent manner. In contrast, degradation of MP17 in sink tissues required intact microtubules and occurred at 26S proteasomes. Interestingly, inhibition of the 26S proteasome led to aggregation of MP17 in aggresome-like structures. Formation of these structures could be inhibited by colchicine, as was shown for aggresomes in mammalian cells.

Sensitivity of cell hydraulic conductivity to mercury is coincident with symplasmic isolation and expression of plasmalemma aquaporin genes in growing maize roots.

Root elongation occurs as individual cells along the growing zone increase in volume. This increase is caused by water entering the cell either by moving across the cell membrane from the apoplast via aquaporins, or entering through plasmodesmata that symplastically connect cells to each other or with the sieve element. In this investigation we used mercury, a known inhibitor of aquaporin water channels, to manipulate the water permeability of growing maize root cells. 20 micro M HgCl(2) was found to reduce root elongation by around 75% and this reduction in growth was greatest in the older growing cells, with little effect on the younger cells near the root tip. Cell hydraulic conductivity (Lp) of cells close to the root tip (at 3 mm) remained unaffected by mercury treatment in contrast to older growing and non-growing cells where Lp was greatly reduced. Using reverse transcription-polymerase chain reaction analysis, younger root regions were shown to express higher levels of two plasmalemma intrinsic protein genes than older root regions further away from the root tip. However, a gene encoding a tonoplast aquaporin was expressed at similar levels in both regions of the growing zone. The fluorescent tracer, carboxyfluorescein, demonstrated symplastic connection between the phloem and root cortical cells at 3 mm but not at 5 or 20 mm. The data are consistent with a decrease in symplastic continuity along the growing zone and highlight a change in the principal pathway of water uptake during the development of the growing root cell.