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PubMed2004

In situ c(4) phosphoenolpyruvate carboxylase activity and kinetic properties in isolated digitaria sanguinalis mesophyll cells.

Isolated mesophyll cells from darkened leaves of the C(4) plant Digitaria sanguinalis keep functional plasmodesmata that allow the free exchange of low molecular mass compounds with the surrounding medium. This cell suspension system has been used to measure C(4) PEPC activity in situ using a spectrophotometric assay. Compared to the extracted enzyme assayed in vitro, the essentially non-phosphorylated 'in-cell' C(4) PEPC showed altered functional and regulatory properties. While the S (0.5) for PEP at pH 7.3 was only modestly changed (0.4-0.6 mM), the response to pH was shifted towards the acidic range, being close to the maximal value at pH 7.3. Using expected physiological concentrations of the metabolites, at pH 7.3, the IC(50) for malate showed a five-fold increase, from 1.5 to 8 mM, and was increased further to 22 mM in the presence of the allosteric activator glucose-6-phosphate (4 mM). Thiol compounds like DTT, mercaptoethanol and reduced glutathione weakened the in-situ sensitivity of C(4) PEPC to malate. However, none of them had any effect on this process in vitro. This was not due to thioredoxin-mediated or phoshorylation-dependent processes. Since glutathione is a physiological compound that is present mostly in the reduced state in the cell cytosol, a possible contribution of this thiol to the protection of the enzyme against malate in situ is proposed.

Journal Article↗
PubMed2000

Role of P30 in replication and spread of TMV.

The P30 movement protein (MP) of tobacco mosaic virus is essential for distribution of sites of replication within infected cells and for cell-cell spread of infection. MP is an integral membrane protein and in early and mid-stages of infection causes severe disruption of the cortical endoplasmic reticulum (ER). MP also associates with microtubules, and in late stages is targeted for degradation by the 26S proteosome. During these stages, the ER regains its normal pre-infection configuration. Viral RNA is associated with ER and microtubules in the presence of MP. The MP is phosphorylated and mutation of the phosphorylated amino acid reduced association of MP with the ER, plasmodesmata, and microtubules, and altered the stability of the MP. The nature of the association of MP with vRNA and ER and microtubules, and the role of phosphorylation of MP in each of these functions, if any, remains to be determined.

Biological Transport↗
PubMed2000

Function of microtubules in intercellular transport of plant virus RNA.

Cell-to-cell progression of tobacco mosaic virus (TMV) infection in plants depends on virus-encoded movement protein (MP). Here we show that a conserved sequence motif in tobamovirus MPs shares similarity with a region in tubulins that is proposed to mediate lateral contacts between microtubule protofilaments. Point mutations in this motif confer temperature sensitivity to microtubule association and viral-RNA intercellular-transport functions of the protein, indicating that MP-interacting microtubules are functionally involved in the transport of vRNA to plasmodesmata. Moreover, we show that MP interacts with microtubule-nucleation sites. Together, our results indicate that MP may mimic tubulin assembly surfaces to propel vRNA transport by a dynamic process that is driven by microtubule polymerization.

Amino Acid Motifs↗
PubMed2006

Viroids: an Ariadne's thread into the RNA labyrinth.

Viroids are structurally, functionally and evolutionarily different from viruses. Despite their small, non-protein-encoding, single-stranded circular RNA genome, viroids can infect higher plants and cause certain diseases. Members of the two viroid families, Pospiviroidae and Avsunviroidae, have evolved to usurp the transcriptional machinery of their host nuclei and chloroplasts, respectively, in which replication proceeds through a rolling-circle mechanism involving RNA polymerization, cleavage and ligation. Remarkably, viroids subvert certain DNA-dependent RNA polymerases to transcribe RNA templates, and, in the family Avsunviroidae, post-transcriptional cleavage is catalysed by hammerhead ribozymes. Viroids are models for studying RNA evolution and for analysing RNA transport in plants, because they can move intracellularly, intercellularly through plasmodesmata and to distal parts of the plant through the vascular system. Viroids elicit RNA-silencing phenomena, which might mediate some of their biological properties, including pathogenesis. As some viroids behave as catalytic RNAs, they are regarded as remnants of the RNA world.

Base Sequence↗
PubMed2002

Plant aquaporins: multifunctional water and solute channels with expanding roles.

There is strong evidence that aquaporins are central components in plant water relations. Plant species possess more aquaporin genes than species from other kingdoms. According to sequence similarities, four major groups have been identified, which can be further divided into subgroups that may correspond to localization and transport selectivity. They may be involved in compatible solute distribution, gas-transfer (CO2, NH3) as well as in micronutrient uptake (boric acid). Recent advances in determining the structure of some aquaporins gives further details on the mechanism of selectivity. Gating behaviour of aquaporins is poorly understood but evidence is mounting that phosphorylation, pH, pCa and osmotic gradients can affect water channel activity. Aquaporins are enriched in zones of fast cell division and expansion, or in areas where water flow or solute flux density would be expected to be high. This includes biotrophic interfaces between plants and parasites, between plants and symbiotic bacteria or fungi, and between germinating pollen and stigma. On a cellular level aquaporin clusters have been identified in some membranes. There is also a possibility that aquaporins in the endoplasmic reticulum may function in symplasmic transport if water can flow from cell to cell via the desmotubules in plasmodesmata. Functional characterization of aquaporins in the native membrane has raised doubt about the conclusiveness of expression patterns alone and need to be conducted in parallel. The challenge will be to elucidate gating on a molecular level and cellular level and to tie those findings into plant water relations on a macroscopic scale where various flow pathways need to be considered.

Journal Article↗
PubMed1997

The plant ER: a dynamic organelle composed of a large number of discrete functional domains.

The endoplasmic reticulum (ER) of plants is comprised of a three-dimensional network of continuous tubules and sheets that underlies the plasma membrane, courses through the cytoplasm, and links up with the nuclear envelope. Aside from discussing the dynamic properties of this versatile and adaptable organelle, the review highlights the structure and the functional properties of 16 types of morphologically defined ER membrane domains. Owing to their labile or transient nature, several of these domains can only be visualized reliably through the use of ultrarapid freezing techniques. The ER domains discussed are: the lamin receptor domain; the nuclear pores; the nuclear envelope-ER gates, the microtubule nucleation domains; the protein and oil body-forming domains; the vacuole-forming ER; the actin-binding, the plasma membrane-anchoring and the vacuole and mitochondrion-attachment domains; the lipid recycling ER cisternae and the plasmodesmata. Preliminary evidence suggests that this list will have to be expanded in the near future. Understanding the assembly, the functional roles, and the developmental regulation of these domains has implications both for understanding cell structure and function, and for exploiting plants for agricultural and biotechnological purposes.

Endoplasmic Reticulum↗
PubMed1997

Cell-to-cell movement of potato spindle tuber viroid.

Viroids are non-translatable, autonomously replicating circular RNAs that infect only plants. An important component of the viroid infection process is cell-to-cell movement; however, there is virtually no information available about the pathways and mechanisms of this process. In this study, potato spindle tuber viroid (PSTVd) has been used as a model system to investigate the mechanism of viroid cell-to-cell transport. Infectious RNA transcripts were produced from PSTVd cDNA clones in vitro, labeled with the nucleotide-specific fluorescent dye TOTO-1 iodide, and used for micro-injection. When injected into symplasmically isolated guard cells of mature tomato and tobacco leaves, PSTVd remained in the injected cells; in contrast, PSTVd injected into symplasmically connected mesophyll cells moved rapidly from cell to cell. A 1400 nt RNA containing only vector sequences was unable to move out of the injected mesophyll cells, but when PSTVd was fused to this transcript, the fusion RNA moved from cell to cell. At the DNA level, PSTVd cDNA also appears able to mediate cell-to-cell movement of plasmid DNA. These data indicate that (i) PSTVd moves from cell to cell via plasmodesmata, and (ii) this movement may be mediated by a specific sequence or structural motif.

Biological Transport↗
PubMed1997

Expression of a luteoviral movement protein in transgenic plants leads to carbohydrate accumulation and reduced photosynthetic capacity in source leaves.

Elucidating the role of viral genes in transgenic plants revealed that the movement protein (MP) from tobacco mosaic virus is responsible for altered carbohydrate allocation in tobacco and potato plants. To study whether this is a general feature of viral MPs, the movement protein MP17 of potato leafroll virus (PLRV), a phloem-restricted luteovirus, was constitutively expressed in tobacco plants. Transgenic lines were strongly reduced in height and developed bleached and sometimes even necrotic areas on their source leaves. Levels of soluble sugars and starch were significantly increased in source leaves. Yet, in leaf laminae the hexose-phosphate content was unaltered and ATP reduced to only a small extent, indicating that these leaves were able to maintain homeostatic conditions by compartmentalization of soluble sugars, probably in the vacuole. On the contrary, midribs contained lower levels of soluble sugars, ATP, hexose-phosphates and UDP-glucose supporting the concept of limited uptake and catabolism of sucrose in the phloem. The accumulation of carbohydrates led to a decreased photosynthetic capacity and carboxylation efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) probably owing to decreased expression of photosynthetic proteins. In parallel, levels of pathogenesis-related proteins were elevated which may be the reason for the obtained limited resistance against the unrelated potato virus Y (PVY)N in the transgenic tobacco plants. Ultrathin sections of affected leaves harvested from 2-week-old plants revealed plasmodesmal alterations in the phloem tissue while plasmodesmata between mesophyll cells were indistinguishable from wild-type. These data favour the phloem tissue to be the primary site of PLRV MP17 action in altering carbohydrate metabolism.

Carbohydrate Metabolism↗
PubMed1998

Genetic evidence for an essential role for potyvirus CI protein in cell-to-cell movement.

The potyvirus cylindrical inclusion (CI) protein, an RNA helicase required for genome replication, was analyzed genetically using alanine-scanning mutagenesis. Thirty-one mutations were introduced into the CI protein coding region of modified tobacco etch virus (TEV) genomes expressing either beta-glucuronidase or green fluorescent protein reporters. Twelve of the mutants were replication-defective in protoplast inoculation assays. Among the 19 replication-competent mutants, several possessed cell-to-cell or long-distance movement defects in tobacco plants. Two mutants, AS1 and AS8, were restricted to single cells in inoculated leaves despite genome amplification levels that were equivalent to that of parental virus. Other mutants, such as AS9 and AS14, were able to move cell to cell slowly but were debilitated in long-distance movement. These data provide genetic evidence for a direct role of CI protein in potyvirus intercellular movement, and for distinct roles of the CI protein in genome replication and movement. In combination with high-resolution ultrastructural analyzes and previous genetic data, these results support a model in which CI protein interacts directly with plasmodesmata and capsid protein-containing ribonucleoprotein complexes to facilitate potyvirus cell-to-cell movement.

Alanine↗
PubMed2000

Movement of plant viruses is delayed in a beta-1,3-glucanase-deficient mutant showing a reduced plasmodesmatal size exclusion limit and enhanced callose deposition.

Susceptibility to virus infection is decreased in a class I beta-1,3-glucanase (GLU I)-deficient mutant (TAG4.4) of tobacco generated by antisense transformation. TAG4.4 exhibited delayed intercellular trafficking via plasmodesmata of a tobamovirus (tobacco mosaic virus), of a potexvirus (recombinant potato virus X expressing GFP), and of the movement protein (MP) 3a of a cucumovirus (cucumber mosaic virus). Monitoring the cell-to-cell movement of dextrans and peptides by a novel biolistic method revealed that the plasmodesmatal size exclusion limit (SEL) of TAG4.4 was also reduced from 1.0 to 0.85 nm. Therefore, GLU I-deficiency has a broad effect on plasmodesmatal movement, which is not limited to a particular virus type. Deposition of callose, a substrate for beta-1,3-glucanases, was increased in TAG4.4 in response to 32 degrees C treatment, treatment with the fungal elicitor xylanase, and wounding, suggesting that GLU I has an important function in regulating callose metabolism. Callose turnover is thought to regulate plasmodesmatal SEL. We propose that GLU I induction in response to infection may help promote MP-driven virus spread by degrading callose.

Biological Transport↗
PubMed2000

Cell-to-cell movement of TMV RNA is temperature-dependent and corresponds to the association of movement protein with microtubules.

The movement protein (MP) of tobacco mosaic virus (TMV) is essential for spread of the viral RNA genome from cell to cell. During infection, the MP associates with microtubules, and it has been proposed that the cytoskeleton transports the viral ribonucleoprotein complex from ER sites of synthesis to plasmodesmata through which infection spreads into adjacent cells. However, microtubule association of MP was observed in cells undergoing late infection rather than in cells undergoing early infection at the leading edge of expanding infection sites where virus RNA cell-to-cell spread occurs. Therefore, alternative roles for microtubules in virus infection have been proposed, including a role in MP degradation. To further investigate the role of microtubules in virus pathogenesis, we tested the efficiency of cell-to-cell spread of infection and microtubule association of the MP in response to changes in temperature. We show that the subcellular distribution of MP is temperature-dependent and that a higher efficiency of intercellular transport of virus RNA at elevated temperatures corresponds to an increased association of MP with microtubules early in infection.

Green Fluorescent Proteins↗
PubMed2000

Proteolytic processing of CmPP36, a protein from the cytochrome b(5) reductase family, is required for entry into the phloem translocation pathway.

Cucurbita maxima (pumpkin) phloem sap contains a 31 kDa protein that cross-reacts with antibodies directed against the red clover necrotic mosaic virus movement protein (RCNMV MP). Microsequence data from phloem-purified 31 kDa protein were used to isolate a complementary DNA: the open reading frame encodes a 36 kDa protein belonging to the cytochrome b(5) reductase (Cb5R) family; the gene was termed CmPP36. Western analyses established that CmPP36, RCNMV MP and CmPP16 (Xoconostle-Cázares et al., 1999, Science 283, 94-98) are immunologically related, probably due to a common epitope, represented by the NADH(+)-binding domain of CmPP36. An N-terminal 5 kDa membrane-targeting domain is cleaved to produce the 31 kDa Delta N-CmPP36 detected in the phloem sap. Microinjection experiments established that Delta N-CmPP36, but not CmPP36, is able to interact with plasmodesmata to mediate its cell-to-cell transport. Thus, intercellular movement of CmPP36 requires proteolytic processing in the companion cell to produce a soluble, movement-competent, protein. In contrast to RCNMV and CmPP16, Delta N-CmPP36 interacts with but does not mediate the trafficking of RNA. Northern and in situ RT-PCR studies established that CmPP36 mRNA is present in all plant organs, being highly abundant within vascular tissues. In roots of hydroponically grown pumpkin plants, CmPP36 mRNA levels respond to changes in available iron in the culture solution. Finally, enzymatic assays established that both CmPP36 and Delta N-CmPP36 could reduce Fe(3+)-citrate and Fe(3+)-EDTA in the presence of NADH(+). These findings are discussed in terms of the possible roles played by CmPP36 in phloem function.

Amino Acid Sequence↗
PubMed2001

Evidence for expression level-dependent modulation of carbohydrate status and viral resistance by the potato leafroll virus movement protein in transgenic tobacco plants.

High-level constitutive expression of the cell-to-cell movement protein from the phloem-restricted potato leafroll virus (PLRV-MP17) in transgenic tobacco plants leads to growth retardation and severe phenotypic changes of source leaves paralleled by a drastic accumulation of soluble sugars and starch (Herbers et al., 1997). To investigate whether the MP17-induced alteration in carbon metabolism is related to the targeting and modification of specific plasmodesmata (Pd) or is rather due to pleiotropic effects caused by high MP17 protein amounts, non-phenotypic tobacco plants expressing a MP17:GFP fusion protein were obtained and compared with previously described MP17 transgenic lines. Confocal laser scanning microscopy and immunogold labelling studies revealed an overall affinity of MP17 to Pd in vascular and non-vascular tissue of source leaves, whereas in sink leaves GFP fluorescence was restricted to Pd of trichomes. In source leaves, plasmodesmal size exclusion limits of mesophyll cells were likewise increased by MP17 and MP17:GFP independent from steady-state levels of the protein amount and phenotypic alteration. Conversely, carbohydrate contents in source leaves strictly correlated with quantified MP17 protein levels. Low expression of MP17 and MP17:GFP decreased soluble sugars and starch contents in leaves possibly due to changes in plasmodesmal permeability while increasing MP17 protein levels led to carbohydrate accumulation and a stunted growth. Infection of transgenic lines with the unrelated potato virus Y (PVY)N revealed an expression level-dependent mode of MP17-mediated resistance. Phenotypic changes and carbohydrate-mediated defence responses as indicated by elevated levels of PR-protein transcripts were crucial for increased viral resistance, whereas plasmodesmal targeting and modification by MP17 per se had either no effect or even increased susceptibility to PVY. Thus, our results implicate that the absolute level of expression needs to be critically considered when elucidating the effect of MPs on carbon metabolism, biomass allocation and virus resistance.

Base Sequence↗
PubMed2001

Local expression of enzymatically active class I beta-1, 3-glucanase enhances symptoms of TMV infection in tobacco.

Mutant tobacco plants deficient for class I beta-1,3-glucanase (GLU I) are decreased in their susceptibility to virus infection. This is correlated with delayed virus spread, a reduction in the size exclusion limit of plasmodesmata and increased cell-wall deposition of the beta-1,3-glucan callose. To further investigate a role of GLU I during cell-to-cell movement of virus infection, we inserted the GLU I coding sequence into TMV for overexpression in infected cells. Compared with the size of local lesions produced on plants infected with virus expressing either an enzymatically inactive GLU I or a frameshift mutant of the gene, the size of local lesions caused by infection with virus expressing active GLU I was consistently increased. Viruses expressing antisense GLU I constructs led to lesions of decreased size. Similar effects were obtained for virus spread using plants grown at 32 degrees C to block the hypersensitive response. Together, these results indicate that enzymatically active GLU I expressed in cells containing replicating virus can increase cell-to-cell movement of virus. This supports the view that GLU I induced locally during infection helps to promote cell-to-cell movement of virus by hydrolyzing callose. Moreover, our results provide the first direct evidence that a biological function of a plant beta-1,3-glucanase depends on its catalytic activity.

Biological Transport↗
PubMed2003

Engineering photoassimilate partitioning in tobacco plants improves growth and productivity and provides pathogen resistance.

Expression of pathogenesis-related (PR) genes is part of the plant's natural defense response against pathogen attack. To study the in vivo role and function of the maize PRms protein, tobacco plants were transformed with the PRms cDNA under the control of the CaMV35S promoter. Transgenic tobacco plants grow faster and yield more leaf and seed biomass. By using immunoelectron microscopy, we found that PRms is associated with plasmodesmata in leaves of transgenic tobacco plants. Furthermore, we found that activation of sucrose efflux from photosynthetically active leaves and accumulation of higher levels of sucrose in leaf tissues are characteristic features of PRms tobacco plants. This, in turn, results in the constitutive expression of endogenous tobacco PR genes and resistance to phytopathogens. The expression of multiple plant defense genes can then be achieved by using a single transgene. These data provide a new approach for engineering disease-resistant plants while simultaneously improving plant yield and productivity through the modification of photoassimilate partitioning.

Carbohydrate Metabolism↗
PubMed2005

A coiled-coil interaction mediates cauliflower mosaic virus cell-to-cell movement.

The function of the virion-associated protein (VAP) of cauliflower mosaic virus (CaMV) has long been only poorly understood. VAP is associated with the virion but is dispensable for virus morphogenesis and replication. It mediates virus transmission by aphids through simultaneous interaction with both the aphid transmission factor and the virion. However, although insect transmission is not fundamental to CaMV survival, VAP is indispensable for spreading the virus infection within the host plant. We used a GST pull-down technique to demonstrate that VAP interacts with the viral movement protein through coiled-coil domains and surface plasmon resonance to measure the interaction kinetics. We mapped the movement protein coiled-coil to the C terminus of the protein and proved that it self-assembles as a trimer. Immunogold labeling/electron microscopy revealed that the VAP and viral movement protein colocalize on CaMV particles within plasmodesmata. These results highlight the multifunctional potential of the VAP protein conferred by its efficient coiled-coil interaction system and show a plant virus possessing a surface-exposed protein (VAP) mediating viral entry into host cells.

Amino Acid Sequence↗
PubMed2005

Anatomical and photosynthetic acclimation to the light environment in species with differing mechanisms of phloem loading.

Plants load sugars from photosynthesizing leaves into the phloem of exporting veins either "apoplastically" (by using H+/sucrose symporters) or "symplastically" (through plasmodesmata). The ability to regulate photosynthesis in response to the light environment was compared among apoplastic loaders (pea and spinach) and symplastic loaders (pumpkin and Verbascum phoeniceum). Plants were grown under low light (LL) or high light (HL) or transferred from LL to HL. Upon transfer, pea and spinach up-regulated photosynthesis to the level found in HL-acclimated plants, whereas up-regulation in pumpkin and V. phoeniceum was limited. The vein density of pea and spinach was the same in HL and LL. Although spinach did not exhibit anatomical or ultrastructural acclimation to the light environment, in pea, wall invaginations in minor vein companion (transfer) cells were more extensive in HL. Furthermore, upon transfer from LL to HL, these invaginations increased in mature pea leaves. Foliar starch levels in mature leaves of plants transferred from LL to HL were not greater than in HL-acclimated leaves of either apoplastically loading species. In the symplastic loaders, plasmodesmatal frequency per loading cell did not vary with treatment, but vein density and thus total plasmodesmatal frequency were higher in HL. Upon transfer of symplastic loaders, however, vein density remained low, and starch levels were higher than in HL; the incomplete acclimation of photosynthesis upon transfer is thus consistent with a carbon export capacity physically limited by an inability to increase vein and plasmodesmatal density in a mature leaf.

Acclimatization↗
PubMed2002

Leaf-to-shoot apex movement of symplastic tracer is restricted coincident with flowering in Arabidopsis.

Classical experiments in plant physiology showed that leaves are the source of signals that control the development of flowers from shoot meristems. Additional physiological and genetic experiments have indicated some of the molecules (e.g., gibberellins, cytokinins, and sucrose) that promote flowering in mustards including Arabidopsis. These small hydrophilic molecules are likely to move to the shoot apex symplastically via the phloem and/or via cell-to-cell movement through plasmodesmata. To analyze potential changes in the symplastic trafficking of small molecules during the induction of flowering in Arabidopsis, we measured changes in the flow of symplastic tracers from the leaf to the shoot apex. We previously found that the onset of flowering is coincident with an evident decrease in the leaf-to-shoot trafficking of symplastic tracer molecules; this decrease in trafficking is transitory and resumes when floral development is established. Here we provide detailed analyses of symplastic connectivity during floral induction by monitoring tracer movement under different photoperiodic induction conditions and in a number of genetic backgrounds with altered flowering times. In all cases, the correlation between flowering and the reduction of symplastic tracer movement holds true. The lack of tracer movement during the induction of flowering may represent a change in plasmodesmal selectivity at this time or that a period of reduced symplastic communication is associated with floral induction.

Arabidopsis↗