Simple, but not branched, plasmodesmata allow the nonspecific trafficking of proteins in developing tobacco leaves.

Leaves undergo a sink-source transition during which a physiological change occurs from carbon import to export. In sink leaves, biolistic bombardment of plasmids encoding GFP-fusion proteins demonstrated that proteins with an Mr up to 50 kDa could move freely through plasmodesmata. During the sink-source transition, the capacity to traffic proteins decreased substantially and was accompanied by a developmental switch from simple to branched forms of plasmodesmata. Inoculation of sink leaves with a movement protein-defective virus showed that virally expressed GFP, but not viral RNA, was capable of trafficking between sink cells during infection. Contrary to dogma that plasmodesmata have a size exclusion limit below 1 kDa, the data demonstrate that nonspecific "macromolecular trafficking" is a general feature of simple plasmodesmata in sink leaves.     

Developmental Regulation of Intercellular Protein Trafficking through Plasmodesmata in Tobacco Leaf Epidermis

Plasmodesmata mediate direct cell-to-cell communication in plants. One of their significant features is that primary plasmodesmata formed at the time of cytokinesis often undergo structural modifications, by the de novo addition of cytoplasmic strands across cell walls, to become complex secondary plasmodesmata during plant development. Whether such modifications allow plasmodesmata to gain special transport functions has been an outstanding issue in plant biology. Here we present data showing that the cucumber mosaic virus 3a movement protein (MP):green fluorescent protein (GFP) fusion was not targeted to primary plasmodesmata in the epidermis of young or mature leaves in transgenic tobacco (Nicotiana tabacum) plants constitutively expressing the 3a:GFP fusion gene. Furthermore, the cucumber mosaic virus 3a MP:GFP fusion protein produced in planta by biolistic bombardment of the 3a:GFP fusion gene did not traffic between cells interconnected by primary plasmodesmata in the epidermis of a young leaf. In contrast, the 3a MP:GFP was targeted to complex secondary plasmodesmata and trafficked from cell to cell when a leaf reached a certain developmental stage. These data provide the first experimental evidence, to our knowledge, that primary and complex secondary plasmodesmata have different protein-trafficking functions and suggest that complex secondary plasmodesmata may be formed to traffic specific macromolecules that are important for certain stages of leaf development.     

An extraction method for tobacco mosaic virus movement protein localizing in plasmodesmata

Summary. The intercellular communication by plasmodesmata (PD) is important for the growth and development of plants, and the transport of macromolecules through PD is likely to be regulated by developmental signals. While PD in the apical meristem transport macromolecules such as mRNAs, the branched PD in the mature leaf do not transport large macromolecules freely. The changes in PD during development might be important for sink-to-source changes in leaves, but the molecular mechanism is still unknown. Movement proteins (MPs) of the tobacco mosaic virus localize in the branched PD and increase the size exclusion limit, allowing transport of viral RNA. We developed a method for differential extraction of MP from isolated cell walls of transgenic tobacco leaves expressing MP or MP tagged with green-fluorescent protein. Lithium chloride at a concentration of 8 M removed filamentous structures in branched PD, the possible attachment site of MP. As some endogenous proteins were coeluted with MP by the treatment, this extraction method might be a powerful tool for investigating MP-interacting proteins in branched PD. Correspondence and reprints: Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan.     

Non-targeted and targeted protein movement through plasmodesmata in leaves in different developmental and physiological states

Plant cells rely on plasmodesmata for intercellular transport of small signaling molecules as well as larger informational macromolecules such as proteins. A green fluorescent protein (GFP) reporter and low-pressure microprojectile bombardment were used to quantify the degree of symplastic continuity between cells of the leaf at different developmental stages and under different growth conditions. Plasmodesmata were observed to be closed to the transport of GFP or dilated to allow the traffic of GFP. In sink leaves, between 34% and 67% of the cells transport GFP (27 kD), and between 30% and 46% of the cells transport double GFP (54 kD). In leaves in transition transport was reduced; between 21% and 46% and between 2% and 9% of cells transport single and double GFP, respectively. Thus, leaf age dramatically affects the ability of cells to exchange proteins nonselectively. Further, the number of cells allowing GFP or double GFP movement was sensitive to growth conditions because greenhouse-grown plants exhibited higher diffusion rates than culture-grown plants. These studies reveal that leaf cell plasmodesmata are dynamic and do not have a set size exclusion limit. We also examined targeted movement of the movement protein of tobacco mosaic virus fused to GFP, P30::GFP. This 58-kD fusion protein localizes to plasmodesmata, consistently transits from up to 78% of transfected cells, and was not sensitive to developmental age or growth conditions. The relative number of cells containing dilated plasmodesmata varies between different species of tobacco, with Nicotiana clevelandii exhibiting greater diffusion of proteins than Nicotiana tabacum.     

Presumable role of plasmodesmata in floral signal transduction in shoot apical meristems of rudbeckia and perilla plants

The number of plasmodesmata was calculated per 1 μm of cell wall length in the central and medullar zones of shoot apical meristems (SAM) in the course of floral transition in a long-day (LD) plant Rudbeckia bicolor Nutt. and a short-day plant Perilla nankinensis Lour. Under the day length unfavorable for flowering (control), the numbers of plasmodesmata differed in the central and medullar zones of SAM, which produce the reproductive organs and stems, respectively. Besides, the numbers of plasmodesmata in the central zone of perilla SAM considerably differed between the anticlinal and periclinal cell walls of the first and second cell layers. Following the photoperiodic induction (PI) with eight LD in rudbeckia and twelve SD in perilla favorable for floral transition, the numbers of plasmodesmata considerably increased in the anticlinal and periclinal cell walls of the first and second cell layers of the central zone; meanwhile in the medullar zone, the numbers of plasmodesmata dropped down following PI. These data show that floral transition presumably involves the activation of cell-to-cell interactions and enhances the signal transduction in SAM.     

Secondary plasmodesmata formation in the minor-vein phloem of Cucumis melo L. and Cucurbita pepo L.

Numerous branched plasmodesmata (pd) are present between bundle-sheath cells (BSCs) and specialized companion cells known as intermediary cells (ICs) in the minor-vein phloem of melon (Cucumis melo L.) and squash (Cucurbita pepo L.). These pd were found to be secondary, i.e., they form across existing walls. Sink, sink-source transition, and source tissues were sampled from developing and mature leaves. In sink tissue, IC precursors divide to produce the two to four ICs and associated sieve elements which are present by the time of the sink-source transition. Plasmodesmata along the interface between the IC precursor and adjacent BSCs in sink tissue are unbranched and few in number. Before the leaf tissue undergoes the sink-source transition, the number of pd channels (individual branches of pd) becomes more numerous. This increase in number of pd channels occurs at least in part and perhaps entirely by branching, resulting in more channels on the IC-side than on the BSC-side. In melon there is a 12-fold increase in the number of pd channels within the IC-side of the interface and a corresponding 9-fold increase in pd channels within the BSC-side. Thus, secondary pd form by the time of the sink-source transition and may be involved in phloem loading and photoassimilate export. The system described is well-defined and amenable to experimental manipulation: secondary pd form in large numbers, at a particular interface, over a short period of time, and in a highly predictable manner.Abbreviations BSC bundle-sheath cell - DAP days after planting - IC intermediary cell - LPI leaf plastochron index - pd plasmodesmata - PI plastochron interval We thank Edith Haritatos, Rich Medville, Esther Gowan, and Nancy Dussault for expert technical assistance. This research was supported by an NSF/DOE/USDA Cornell Plant Science Center fellowship (G.M.V.), Natural Sciences and Engineering Research Council Grant GP0138401 and Université de Montréal, Fonds internes de recherche (D.U.B.), and NSF grant IBN-9419703 (R.T.).     

A Protocol for the Fluorometric Quantification of mGFP5-ER and sGFP(S65T) in Transgenic Plants

The Green Fluorescent Protein (GFP) from Aequorea victoria has begun to be used as a reporter protein in plants. It is particularly useful as GFP fluorescence can be detected in a non-destructive manner, whereas detection of enzyme-based reporters often requires destruction of the plant tissue. The use of GFP as a reporter enables transgenic plant tissues to be screened in vivo at any growth stage. Quantification of GFP in transgenic plant extracts will increase the utility of GFP as a reporter protein. We report herein the quantification of a mGFP5-ER variant in tobacco leaf extracts by UV excitation and a sGFP(S65T) variant in sugarcane leaf and callus extracts by blue light excitation using the BioRad VersaFluor^TM Fluorometer System or the Labsystems Fluoroskan Ascent FL equipped with a narrow band emission filter (510 ± 5 nm). The GFP concentration in transgenic plant extracts was determined from a GFP-standard series prepared in untransformed plant extract with concentrations ranging from 0.1 to 4 g/ml of purified rGFP. Levels of sgfp(S65T) expression, driven by the maize ubiquitin promoter, in sugarcane calli and leaves ranged up to 0.525 g and 2.11 g sGFP(S65T) per mg of extractable protein respectively. In tobacco leaves the expression of mgfp5-ER, driven by the cauliflower mosaic virus (CaMV) 35S promoter, ranged up to 7.05 g mGFP5-ER per mg extractable protein.     

Structural and functional vein maturation in developing tobacco leaves in relation to AtSUC2 promoter activity

Transgenic tobacco (Nicotiana tabacum) plants expressing green fluorescent protein (GFP) from the AtSUC2 promoter were used to study the function of different vein classes in developing leaves. In sink leaves, unloading capacity occurred acropetally, with the class I (midrib) and class II veins becoming functional in phloem unloading before the maturation of the class III veinal network. In contrast, in developing cotyledons and source leaves, loading capacity occurred in a basipetal direction. There was a strong correlation between loading capacity, as assessed by (14)C Suc uptake and companion cell expression of AtSUC2-GFP. Developing cotyledons were shown to utilize all available vein classes for loading. A second line of transgenic plants was produced in which GFP, expressed from the AtSUC2 promoter, was targeted to the endoplasmic reticulum instead of the cytoplasm. In these AtSUC2-GFP-ER plants, GFP was unable to traffic into the sieve element and was restricted solely to the companion cells of source leaf tissues. Partial shading of leaves undergoing the sink-source transition demonstrated that the activation of the AtSUC2 promoter in tobacco was influenced by light. Functional and structural maturation of the minor veins required light or a product of light. The activation of the AtSUC2 promoter within major veins appears to be regulated differently from that in the minor veins. The relationship between AtSUC2 activation and the activity of endogenous tobacco Suc transporters is discussed.     

Cell-to-cell and long-distance trafficking of the green fluorescent protein in the phloem and symplastic unloading of the protein into sink tissues

Macromolecular trafficking within the sieve element-companion cell complex, phloem unloading, and post-phloem transport were studied using the jellyfish green fluorescent protein (GFP). The GFP gene was expressed in Arabidopsis and tobacco under the control of the AtSUC2 promoter. In wild-type Arabidopsis plants, this promoter regulates expression of the companion cell-specific AtSUC2 sucrose-H+ symporter gene. Analyses of the AtSUC2 promoter-GFP plants demonstrated that the 27-kD GFP protein can traffic through plasmodesmata from companion cells into sieve elements and migrate within the phloem. With the stream of assimilates, the GFP is partitioned between different sinks, such as petals, root tips, anthers, funiculi, or young rosette leaves. Eventually, the GFP can be unloaded symplastically from the phloem into sink tissues, such as the seed coat, the anther connective tissue, cells of the root tip, and sink leaf mesophyll cells. In all of these tissues, the GFP can traffic cell to cell by symplastic post-phloem transport. The presented data show that plasmodesmata of the sieve element-companion cell complex, as well as plasmodesmata into and within the analyzed sinks, allow trafficking of the 27-kD nonphloem GFP protein. The data also show that the size exclusion limit of plasmodesmata can change during organ development. The results are also discussed in terms of the phloem mobility of assimilates and of small, low molecular weight companion cell proteins.     

Developmental changes in plasmodesmata in transgenic tobacco expressing the movement protein of tobacco mosaic virus

Summary Cell-to-cell communication in plants occurs through plasmodesmata, cytoplasmic channels that traverse the cell wall between neighboring cells. Plasmodesmata are also exploited by many viruses as an avenue for spread of viral progeny. In the case of tobacco mosaic virus (TMV), a virally-encoded movement protein (MP) enables the virus to move through plasmodesmata during infection. We have used thin section electron microscopy and immunocytochemistry to examine the structure of plasmodesmata in transgenic tobacco plants expressing the TMV MP. We observed a change in structure of the plasmodesmata as the leaves age, both in control and MP expressing [MP(+)] plants. In addition, the plasmodesmata of older cells of MP(+) plants accumulate a fibrous material in the central cavity. The presence of the fibers is correlated with the ability to label plasmodesmata with anti-MP antibodies. The developmental stage of leaf tissue at which this material is observed is the stage at which an increase in the size exclusion limit of the plasmodesmata can be measured in MP(+) plants. Using cell fractionation and aqueous phase partitioning studies, we identified the plasma membrane and cell wall as the compartments with which the MP stably associates. The nature of the interaction between the MP and the plasma membrane was studied using sodium carbonate and Triton X-100 washes. The MP behaves as an integral membrane protein. Identifying the mechanism by which the MP associates with plasma membrane and plasmodesmata will lead to a better understanding of how the MP alters the function of the plasmodesmata.Abbreviations MP movement protein - TMV tobacco mosaic virus     

Secondary plasmodesmata are specific sites of localization of the tobacco mosaic virus movement protein in transgenic tobacco plants.

Expression of the tobacco mosaic virus 30-kD movement protein (TMV MP) gene in tobacco plants increases the plasmodesmatal size exclusion limit (SEL) 10-fold between mesophyll cells in mature leaves. In the present study, we examined the structure of plasmodesmata as a function of leaf development. In young leaves of 30-kD TMV MP transgenic (line 274) and vector control (line 306) plants, almost all plasmodesmata were primary in nature. In both plant lines, secondary plasmodesmata were formed, in a basipetal pattern, as the leaves underwent expansion growth. Ultrastructural and immunolabeling studies demonstrated that in line 274 the TMV MP accumulated predominantly in secondary plasmodesmata of nonvascular tissues and was associated with a filamentous material. A developmental progression was detected in terms of the presence of TMV MP; all secondary plasmodesmata in the tip of the fourth leaf contained TMV MP in association with the filamentous material. Dye-coupling experiments demonstrated that the TMV MP-induced increase in plasmodesmatal SEL could be routinely detected in the tip of the fourth leaf, but was restricted to mesophyll and bundle sheath cells. These findings are discussed with respect to the structure and function of plasmodesmata, particularly those aspects related to virus movement.     

Morphological features of the transgenic tobacco plant shoot expressing the 3-hydroxy-3-methylglutagyl-CoA reductase (<Emphasis Type="Italic">HMG1</Emphasis>) gene in the direct and reverse orientations towards the promoter

3-Hydroxy-3-methylglutaryl-CoA reductase (HMG1) catalyzes the formation of mevalonic acid, the key intermediate of the cytosolic isoprenoid synthesis pathway. The parameters of stem and leaf growth were studied in the transgenic tobacco plants that express the HMG1 gene in both sense and antisense orientations towards the constitutive promoter. The transgenic plant height did not significantly differ from that of the control plants, though the plants carrying the sense copy of the HMG1 gene were considerably taller than plants that carried the antisense gene copy. Plants carrying an extra copy of the HMG1 gene were also characterized by increased leaf area. The number of mesophyll cells calculated per square unit of transgenic plants leaves was smaller than in the control plant leaves, though their volume was not considerably changed in any of the variants, suggesting changes in the cell packing density in leaves.     

Transgenic tobacco plants regenerated from leaf disks can be periclinal chimeras

Amongst rolC transgenic tobacco plants regenerated from leaf disks 6.5% are periclinal chimeras, i.e. plants with genetically different cell populations in different cell layers. The expression of the rolC gene of Agrobacterium rhizogenes causes a reduction in pigment content in leaves. The chimeric composition of the regenerated plants becomes thus apparent as light green leaf tissue in the transgenic region, tissue flanked by dark green wild-type sectors. Southern and northern blot analysis confirmed the chimeric nature of such plants. Investigation of selfed progeny of chimeric plants on selective media indicates that layer invasion in reproductive tissues can occur in tobacco early during the formation of the flower buds. The results show (1) that tobacco plants regenerated from leaf disks and grown on selective media have not necessarily the same clonal origin and (2) that they can give rise to non-transgenic offspring. The chimeric plants provide insight on the effect of rolC gene expression on microsporogenesis.     

The frequency of plasmodesmata increases early in the whole shoot apical meristem of Sinapis alba L. during floral transition

 The frequency of plasmodesmata increases in the shoot apical meristem of plants of Sinapis alba L. induced to flower by exposure to a single long day. This increase is observed within all cell layers (L1, L2, L3) as well as at the interfaces between these layers, and it occurs in both the central and peripheral zones of the shoot apical meristem. The extra plasmodesmata are formed only transiently, from 28 to 48 h after the start of the long day, and acropetally since they are detectable in L3 4 h before they are seen in L1 and L2. These observations indicate that (i) in the Sinapis shoot apical meristem at floral transition, there is an unfolding of a single field with increased plasmodesmatal connectivity, and (ii) this event is an early effect of the arrival at this meristem of the floral stimulus of leaf origin. Since (i) the wave of increased frequency of plasmodesmata is 12 h later than the wave of increased mitotic frequency (A. Jacqmard et al. 1998, Plant cell proliferation and its regulation in growth and development, pp. 67–78; Wiley), and (ii) the increase in frequency of plasmodesmata is observed in all cell walls, including in walls not deriving from recent divisions (periclinal walls separating the cell layers), it is concluded that the extra plasmodesmata seen at floral transition do not arise in the forming cell plate during mitosis and are thus of secondary origin. Received: 4 October 1999 / Accepted: 23 December 1999     

Abnormal cell divisions in leaf primordia caused by the expression of the rice homeobox geneOSH1 lead to altered morphology of leaves in transgenic tobacco

Transgenic tobacco plants were generated carrying a rice homeobox gene,OSH1, controlled by the promoter of a gene encoding a tobacco pathogenesis-related protein (PR1a). These lines were morphologically abnormal, with wrinkled and/or lobed leaves. Histological analysis of shoot apex primordia indicated arrest of lateral leaf blade expansion, often resulting in asymmetric and anisotropic growth of leaf blades. Other notable abnormalities included abnormal or arrested development of leaf lateral veins. Interestingly,OSH1 expression was undetectable in mature leaves with the aberrant morphological features. Thus,OSH1 expression in mature leaves is not necessary for abnormal leaf development. Northern blot and in situ hybridization analyses indicate thatPR1a-OSH1 is expressed only in the shoot apical meristem and in very young leaf primordia. Therefore, the aberrant morphological features are an indirect consequence of ectopicOSH1 gene expression. The only abnormality observed in tissues expressing the transgene was periclinal (rather than anticlinal) division in mesophyll cells during leaf blade initiation. This generates thicker leaf blades and disrupts the mesophyll cell layers, from which vascular tissues differentiate. TheOSH1 product appears to affect the mechanism controlling the orientation of the plane of cell division, resulting in abnormal periclinal division of mesophyll cell, which in turn results in the gross morphological abnormalities observed in the transgenic lines.     

Abnormal cell divisions in leaf primordia caused by the expression of the rice homeobox geneOSH1 lead to altered morphology of leaves in transgenic tobacco

Transgenic tobacco plants were generated carrying a rice homeobox gene,OSH1, controlled by the promoter of a gene encoding a tobacco pathogenesis-related protein (PR1a). These lines were morphologically abnormal, with wrinkled and/or lobed leaves. Histological analysis of shoot apex primordia indicated arrest of lateral leaf blade expansion, often resulting in asymmetric and anisotropic growth of leaf blades. Other notable abnormalities included abnormal or arrested development of leaf lateral veins. Interestingly,OSH1 expression was undetectable in mature leaves with the aberrant morphological features. Thus,OSH1 expression in mature leaves is not necessary for abnormal leaf development. Northern blot and in situ hybridization analyses indicate thatPR1a-OSH1 is expressed only in the shoot apical meristem and in very young leaf primordia. Therefore, the aberrant morphological features are an indirect consequence of ectopicOSH1 gene expression. The only abnormality observed in tissues expressing the transgene was periclinal (rather than anticlinal) division in mesophyll cells during leaf blade initiation. This generates thicker leaf blades and disrupts the mesophyll cell layers, from which vascular tissues differentiate. TheOSH1 product appears to affect the mechanism controlling the orientation of the plane of cell division, resulting in abnormal periclinal division of mesophyll cell, which in turn results in the gross morphological abnormalities observed in the transgenic lines.     

Stigma development in Nicotiana tabacum. Cell death in transgenic plants as a marker to follow cell fate at high resolution

Pistil development was studied in transgenic tobacco plants in which the stigma is ablated by expression of a stigma-specific cytotoxic gene. These plants offer a tool to investigate the process of differentiation of the secretory zone, in that cell death caused by barnase activity provides a marker to follow cell fate at high resolution. After fusion of the carpel walls in the region most distal from the ovary, the epidermal cells begin to divide in both wild-type and stigmaless plants. Divisions of the L1 layer of the pistil are immediately followed by the morphogenetic events that lead to three different cell types: rounded-angular cells showing an equal number of anti- and periclinal divisions, cells that are more oblong forming the transition zone, and the square cells of the transmitting tissue dividing mostly anticlinally with respect to the original carpel wall. In the stigmaless plants, cell death caused by the expression ofSTIG 1-barnase begins at stage –1 and proceeds gradually, but is always associated with round epidermal cells and with angular-rounded cells underneath them. Studies at the ultrastructural level show that cell death caused by barnase activity occurs first in solitary cells and gradually extends to groups of cells.In situ hybridizations using the STIG 1 RNA probe in wild-type pistils confirm these results. Most likely, the cells in whichSTIG 1 is expressed are those that have just differentiated into the secretory cell type. Our results indicate that the transition zone or neck is autonomously differentiated from the secretory zone and the transmitting tissue. Furthermore, our results indicate that in both wild-type and stigmaless pistils secretion of lipids most likely occurs through the plasmodesmata. This observation suggests that bulk transport can occur via plasmodesmata.     

Pectin methylesterase-generated methanol may be involved in tobacco leaf growth

Plant leaves undergo a sink-source modification of intercellular macromolecular transport during the transition from carbon import to carbon export. After assessing the role of metabolite signaling in gene regulation in Nicotiana tabacum sink and source leaves, we observed increased pectin methylesterase (PME)-mediated methanol generation in immature leaves. Using suppression subtractive hybridization (SSH), we identified a number of genes whose activity changes from sink to source leaves. The most abundant SSH-identified genes appeared to be sensitive to methanol. We hypothesize that tobacco leaf maturation and the sink-source transition are accompanied by a change in mRNA levels of genes that function in methanol-dependent cell signaling.     

Gating of epidermal plasmodesmata is restricted to the leading edge of expanding infection sites of tobacco mosaic virus (TMV)

Plasmodesmatal gating in epidermal cells of Nicotianatabacum was examined in expanding infection sites of tobacco mosaic virus (TMV) expressing a fusion between the viral movement protein and the green fluorescent protein (MP-GFP). The infection sites were circular in profile and within 3 days post-inoculation had developed a brightly fluorescent leading edge, giving them a characteristic ‘halo’ shape. Co-localization of MP-GFP with callose demonstrated that nearly all epidermal cell plasmodesmata were targeted with MP-GFP. The fusion protein was located in the centre of the plasmodesmal pore, between paired callose platelets. Increase in plasmodesmatal size exclusion limit, as determined by the passage of microinjected 10 kDa Texas Red dextran, was restricted predominantly to cells within the fluorescent halo, and was virtually absent from cells in the centre of the expanding infection site. The plasmodesmata of these cells, however, remained fluorescently labelled with MP-GFP. Injections outside the fluorescent infection site failed to show movement of dextran, while dextran injected into cells at the leading edge moved inwards towards the centre of the lesion but not outwards into cells lacking GFP. Leaf incisions through cells ahead of the infection front halted the advance of the virus, indicating that virus replication was absent in non-fluorescent cells outside the infection site. The data provide the first demonstration that within an expanding infection site plasmodesmatal gating is under temporal control.