Callose deposition at plasmodesmata. J. E. Radford 1, M. Vesk 2, and R. L. Overall 1,*. 1School of Biological Sciences and 2Electron Microscope Unit, ...
Protoplasma (1998) 201: 30-37
PROTOPLASMA 9 Springer-Verlag 1998 Printed in Austria
Callose deposition at plasmodesmata J. E. Radford 1, M. Vesk 2, and R. L. Overall 1,* 1School of Biological Sciences and 2Electron Microscope Unit, University of Sydney, Sydney, New South Wales Received August 8, 1997 Accepted October 16, 1997
Summary. The transport of ions and metabolites through plasmodesmata has been thought to be controlled at the neck region where the cytoplasmic annulus is constricted and where callose has also been localised. In order to determine the possible structural and functional effects of callose, its deposition was inhibited through incubation of the plant tissue with 2-deoxy-D-glucose (DDG) for 1 h prior to fixation in 2.5% glutaraldehyde. The inhibition of callose formation was monitored through aniline blue-induced fluorescence of callose. The neck region of the plasmodesmata from Allium cepa L. roots treated with DDG exhibited a funnel-shaped configuration. This is in contrast to the plasmodesmata from tissue not incubated with DDG, which exhibited constricted necks similar to those previously reported. Both initial dissection and glutaraldehyde fixation induced neck constriction in plasmodesmata, however, dissection of tissue increased the frequency of constrictions. The inhibition of cap lose formation by chemical means showed that the neck constrictions and raised collars in this area are artefacts due to physical wounding and glutaraldehyde fixation. The external electron-dense material observed when tannic acid is included in the primary fixative appears to be unrelated to the deposition of callose at the neck region. Keywords: Cell-to-cell communication; Plasmodesmata; Ultrastructure; Wounding; 2-Deoxy-D-glucose.
Abbreviations: DDG 2-deoxy-D-glucose.
Introduction
Plasmodesmata are the intercellular connections between plant cells that allow cell-to-cell transport of sugars, amino acids, inorganic ions, proteins, and nucleic acids (reviewed by Lucas et al. 1993). Transport is thought to take place via a cytoplasmic annulus
*Correspondence and reprints: School of Biological Sciences, The University of Sydney, Sydney, NSW 2006, Australia.
between the plasma membrane delimiting the plasmodesma and a central axial structure known as the desmotubule (Overall et al. 1982). It has been generally accepted that in most species this cytoplasmic annulus is constricted at the neck regions of the plasmodesma (Robards and Lucas 1990), whilst in some species it is constricted along the length of the plasmodesma (Overall et al. 1982). The constricted neck region is characterised by a raised electron-lucent collar around the opening of the plasmodesmata where the cytoplasmic annulus is narrowed. The repeated observation of this neck constriction has led to the speculation that the neck region is a basic standardised structure, perhaps even an assembly of defined macromolecular construction (Olesen and Robards 1990). However, specimen preparation procedures employed to visualise plasmodesmata, such as dissection and glutaraldehyde fixation, are likely to stimulate the plant material to exhibit wounding responses (Hughes and Gunning 1980, Galway and McCully 1987, Northcote etal. 1989). Callose is a ~-l,3-glucan which is deposited between the plasma membrane and the wall upon wounding of the plant and appears electron-lucent in transmission electron micrographs (reviewed by Stone and Clarke 1992). Stimuli also known to induce callose formation include gentle mechanical perturbation (Jaffe et al. 1985), ultrasound stress (Currier and Webster 1964), chilling (Majumder and Leopold 1967), heating (McNairn 1972), fungal infection (Aist 1976), viral attack (Shimoura and Dijkstra 1975), and cell plasmolysis (Drake et al. 1978). Callose deposition occurs rapidly,
J. E. Radford et al.: Callose deposition at plasmodesmata
within a minute of stimulation (Currier and Webster 1964, Takahashi and Jaffe 1984), and has been localised particularly to plasmodesmata (Northcote et al. 1989, Benhamou 1992, Delmer et al. 1993). Also observed in the neck region outside the plasma membrane in the cell wall are structures which have been termed external sphincters (Olesen 1979, Mollenhauer and Morr6 1987, Badelt et al. 1994). They are differentially stained when tannic acid is added to the primary fixative during preparation for transmission electron microscopic studies. It has been proposed that these sphincter complexes contain callose synthase molecules (Olesen and Robards 1990). Callose can bind large quantities of water and calcium and it has been suggested that after the deposition of callose at the neck region, swelling could occur around the synthase molecules to constrict the plasmodesmata (Olesen and Robards 1990). Concern over the effect of this wound-mediated deposition of callose on the interpretation of plasmodesmata structure and function has been raised in the past but has not been investigated further (Hughes and Gunning 1980, Northcote et al. 1989). 2-Deoxy-Dglucose (DDG), whose action is thought to involve the inhibition of glycosylation of lipid intermediates (Stone and Clarke 1992), has been used previously to inhibit the production of callose in wounded plant tissue (Gale et al. 1984, Jaffe and Leopold 1984, Bayles et al. 1990). In the present study DDG has been used to inhibit the deposition of callose to determine the extent to which callose deposition due to the wounding effects of specimen preparation procedures alters the ultrastructure of plasmodesmata. The findings do not support the suggestion of Olesen and Robards (1990) that the external tannic acid-staining material consists of callose synthase molecules.
Material and methods Plant material Seeds of Allium cepa L. were germinated vertically on moist filter paper at 25 ~ in the dark for four days to obtain straight roots of length 1 to 1.5 cm.
31
Aniline blue-induced fluorescence of callose Following primary fixation of unwounded roots, roots were dehydrated in a progressive ethanol series and infiltrated with paraffin wax. 2-~um longitudinal sections of the first 3 - 4 m m of the root tip were treated with histosol and ethanol to remove the wax, washed in distilled water and mounted in 0.1% aniline blue (Chroma, Medos Co., Mr. Waverley, Australia) in 0.1 M phosphate buffer pH 7.3. After 10 min the specimens were viewed with a Zeiss Axiophot fluorescence microscope under UV excitation. Images of cortical cells were compared with images of controls mounted in phosphate buffer alone to monitor autofluorescence. Aniline blue-induced fluorescence of callose at tire pit fields of plasmodcsmata appears punctate (Currier 1957).
Electron microscopy Following primary fixation, wounded or unwounded roots were postfixed in 1% osmium tetroxide for 1 h, dehydrated in a progressive acetone series, infiltrated and embedded in Spurr's resin in Beem capsules at room temperature, and polymerised overnight at 65 ~ Ultrathin sections of the first 3 m m of the root tip were cut to a thickness of approximately 80-85 n m (silver to pale gold interference co/ours) on a Reichert-Jung Uttracut E ukramicrotome with a Diatome diamond knife. The sections were collected on copper grids coated with 0.2% Pioloform in chloroform and carbon, and subsequently stained with saturated uranyl acetate in 50% ethanol and lead citrate. The sections were then viewed with a Philips 400 electron microscope at I00 kV.
Tannic acid staining Onion roots were treated as above with the addition of 1% tannic acid (Ajax, Seven Hills, Australia) to the primary fixative. Five roots from each treatment were examined for the presence or absence of sphincters throughout the length of the root.
Measurements and statistical analysis 10 roots from each treatment in the chemical inhibition of callose experiments were observed and 100 plasmodesmata from cortical and epidermal cells in the elongation zone of each root (2-3 m m from the tip) were scored as to the structure of their neck regions. The proportion of each structure type from each treatment was compared by Student's t-test. Measurements in the same area were taken of the outer dimensions of the plasmodesmata from longitudinal images of plasmodesmata from both the non-DDG- and DDG-treated tissues. Unwounded and wounded treatments were included together as there was no significant difference found between the treatments (data not shown). Only images in which the trilaminar structure of the plasma membrane could be clearly seen were included.
Results
Ultrastructure of plasmodesmata DDG treatment and primary fixation Intact onion seedlings (unwounded) were fixed in 2.5% glutaraldehyde in 0.025 M phosphate buffer (pH 7.3) for 2 h at room temperature with or without prior exposure to 104 M DDG (Sigma, Castle Hill, Australia) in distilled water for 1 h. Wounded roots were treated as above except that they were dissected from the seed 1 cm from the root tip with a sharp razor blade in the primary fixative.
The majority of the plasmodesmata of unwounded or wounded tissue had constricted necks and raised collars of electron-lucent material (Figs. 1 a-c and 2 a). These images are similar to the commonly accepted models of plasmodesmata. Also present were plasmodesmata that exhibited straight sides and
32
J.E. Radford et al.: Callose deposition at plasmodesmata
33
J. E. Radford et al.: Callose deposition at plasmodesmata
u l t r a s t r u c t u r e o f the c e l l s a p p e a r e d u n c h a n g e d a p a r t
DT PM A
f r o m m i l d r u f f l i n g o f the p l a s m a m e m b r a n e .
A
Inhibition of caIlose deposition O n i o n r o o t s n o t e x p o s e d to D D G
p r i o r to f i x a t i o n
showed aniline blue-induced fluorescence of callose at pit f i e l d s (Fig. 3 a). O n i o n r o o t s e x p o s e d to D D G f o r 1 h p r i o r to f i x a t i o n n o l o n g e r e x h i b i t e d or e x h i b ited g r e a t l y r e d u c e d p u n c t a t e f l u o r e s c e n c e (Fig. 3 b). In b o t h w o u n d e d and u n w o u n d e d
roots treated with
D D G p r i o r to d i s s e c t i o n and g l u t a r a l d e h y d e f i x a t i o n , DT
the m o s t c o m m o n
A
PM
n e c k c o n f i g u r a t i o n w a s that o f a
f u n n e l - s h a p e d n e c k (Figs. 1 e - h a n d 2 b). T h e d i a m e -
B
~mnimmu~ b Fig. 2 a, b. Simplified diagrams of longitudinal images of plasmodesmata drawn approximately to scale, a Tissue not treated with DDG (as in Fig. 1 a-c) and b tissue treated with DDG prior to fixation (as in Fig. 1 e-h). Labelled dimensions are given in Table 2. DT Desmotubule and associated material, PM plasma membrane, W wall
Table 1. The neck configurations as percentages observed following the various treatments imposed on the tissues during specimen preparation Treatment
Neck configuration (%)
Unwounded Wounded DDG + unwounded DDG + wounded
eonstricted neck
straight
funnelshaped
67.1 _+2.7 86.1 + 2.0 0 0
32.9 _+2.7 13.9 + 2.0 24.9 + 5.0 31.1 + 1.9
0 0 75.2 +_5.0 68.9 _+ 1.9
u n c o n s t r i c t e d n e c k r e g i o n s (Fig. 1 d). W o u n d e d t i s s u e had significantly more closed plasmodesmata unwounded
t i s s u e (t-test, P < 0 . 0 1 ) ( T a b l e
than
1). T h e
Fig. 3 a, b. Aniline blue-induced fluorescence of callose in cortical cells in onion roots, a Roots fixed prior to rinsing and mounting in aniline blue show obvious yellow punctate callose deposits located at the pit fields, as well as blue/green autofluorescence of the walls and nuclei (arrow) due to glutaraldehyde fixation, b The punctate deposits are absent or greatly reduced when the tissue is initially treated with DDG, however, the autofluorescence is still present as is fluorescence of callose in newly formed cell walls (arrow). Bar: 25 ~tm
Fig. 1. a-e Common image of plasmodesmata after conventional fixation procedures: raised collar and constricted neck region (arrowheads). a and b were from the wounded non-DDG treatment; c from the unwounded non-DDG treatment, d Straight configuration, with no neck constriction (arrowheads), that was noted to be present in a small proportion of plasmodesmata in all treatments. This particular image was from the wounded non-DDG treatment, e-h Appearance of the majority of plasmodesmata from tissue treated with DDG prior to aldehyde fixation. Plasmodesmata from this treatment exhibited a funnel-shaped neck configuration (arrowheads). e and f were from the wounded DDG treatment, g and h from the unwounded DDG treatment. Bar: 100 nm
J. E. Radfordet al.: Callosedepositionat plasmodesmata
34 Table 2. Dimensionsof plasmodesmatalcomponentsas viewedin longitudinalsectionsas illustratedin Fig. 2 Treatment
Externaldiameters(nm) A-A B-B
Conventionalfixation DDG incubation
30.6 + 1.0 (18) 61.0 _+2.3 (28)
48.2 + 2.8 (20) 37.2 + 1.1 (30)
ter of the plasmodesmata was uniform except for a gradual expansion at both ends. There was no statistical difference in the proportion of straight and funnelshaped plasmodesmata in unwounded and wounded treatments (t-test, P > 0.05). No neck constrictions or raised collars in the plasmodesmata were observed in tissues treated with DDG. Likewise no funnel-shaped plasmodesmata were observed in untreated tissue (Table 1). The outer dimensions of the plasmodesmata were found to vary greatly with the treatments. The outer diameter of the neck regions of the plasmodesmata in DDG-treated tissue was nearly twice that in conventionally treated tissues (Table 2 and Fig. 2).
Putative sphincters Plasmodesmata in all cell types in the roots of onion, when treated with tannic acid, exhibited putative sphincters of external electron-dense material around the neck (Fig. 4 a, b) with the exception of the root cap cells which exhibited no such structures (Fig. 4 c-e). The plasmodesmata from the cells of the root cap appeared identical to those surrounding cell types in all other respects, including the callose-related features (Fig 4 d, e). Discussion The distortion of the neck region observed in conventionally treated tissue appears to be the result of a wounding reaction in the plant. Dissection of onion roots with a sharp razor blade in primary fixative increased the proportion of plasmodesmata with constricted necks above that for unwounded glutaraldehyde fixed tissue. This agrees with the results of Hughes and Gunning (1980) who reported that both glutaraldehyde fixation and dissection produces the same callose deposition responses as a deliberate wound in susceptible species. The deposition of callose is extremely rapid and its effects have no doubt been underestimated in the past.
The ultrastructural study by Ding et al. (1992), in which rapid-freeze fixation techniques were used instead of aldehyde fixation illustrates this point. The brief incubation of dissected tissue in a sucrose buffer, to the extent that mild plasmolysis occurred, would be sufficient to stimulate the deposition of callose at the neck regions of the plasmodesmata. The incubation of tissue in cryoprotectants was also reported to increase the tissue's susceptibility to a wound reaction (Hughes and Gunning 1980). Notwithstanding these observations researchers into the structure and function of plasmodesmata have continued to use both dissection and incubation in cryoprotectants prior to ultrastructural and transport studies. Determination of molecular-exclusion limit and electrical conductivity of plasmodesmata have involved experimental methods likely to induce the deposition of callose. Thus the high resistance noted for plasmodesmata (Spanswick and Costerton 1967, Overall and Gunning 1982) could be attributed to wound-induced constriction of the neck region, due to electrode insertion. Cut tissues exhibit the same plasmodesmatal permeability as intact tissue (Erwee et al. 1985), which implies that the tissue does not distinguish between dissection and microinjection. In Chara and Nitella, thought to have a normal size exclusion limit of around 1 kDa, macromolecules with molecular mass equal to 45 kDa moved from the injected cell to neighbouring cells within 24h after injection (Kikuyama et al. 1992). As in higher plants (Spanswick and Costerton 1967) no transport between cells was noticed immediately after injection. It is interesting to speculate that large molecules may also pass through plasmodesmata of higher plants after the callose deposited through specimen handling is reabsorbed or initially prevented from forming. Thus the in vivo size exclusion limit and diffusion rates through plasmodesmata might be larger than currently estimated. The increased diameter of the plasmodesmata would presumably lead to an increase in the space available for transport through plasmodesmata. Previous estimations of the size exclusion limit and transport pore size may have been based on experimental procedures likely to wound the plant. Indeed, in recent work analysing transport in an unwounded system it was concluded that the current measurements of 1.5-3 nm 2 for plasmodesmatal-pore size might be flawed due to the fact that the flow of sucrose through the plasmodesmata could not meet the carbon demands of root tips (Bret-Harte and Silk
J. E. Radford et al.: Callose deposition at plasmodesmata 1994). This conclusion has been supported by the observation that the cytoplasmic annulus is enlarged during times of phloem unloading (Schulz 1995). The putative sphincters of electron-dense extracellular material around the necks of plasmodesmata following tannic acid treatment often have clear subunits (Badelt et al. 1994). Olesen and Robards (1990) have suggested that it is these subunits that deposit callose thereby constricting the necks of plasmodesmata. However, this suggestion is contradicted by our finding that callose is deposited at the plasmodesmata of the root cap cells even though they have no electrondense material around their necks following tannic
35 acid treatment. Recent evidence suggests that the callose synthase complex is located vectorially at the plasma membrane (Stone and Clarke 1992) also contradicting the suggestion that the electron-dense subunits visible in the wall around the neck are the source of callose. The absence of sphincters at the root cap cells could be due to the short life span of these cells negating the need for highly regulated transport or could be possibly related to the role of root cap cells as signal transducers for gravitropism (Moore and Maiman 1993), chemical toxicity (Ryan et al. 1993), and hydrosensing (Takahasi and Scott 1993).
Fig. 4 a-e. Plasmodesmata from onion roots that had tannic acid included in the primary fixative, a Plasmodesmata from wounded non-DDG treatments, note external tannic acid-staining material and raised collar (arrowheads). b Appearance of plasmodesmata from roots pretreated with DDG (wounded DDG treatment), note the absence of a collar but the presence of externally stained material (arrowheads). c Appearance of plasmodesmata from root cap ceils in tissue treated with DDG (woundedDDG treatment), note the absence of external material and collars. d and e Appearance of plasmodesmata from the root cap cells in tissue not treated with DDG (wounded non-DDGtreatment), the raised collar is present but the external material is not. Bar: 100 nm
36
If the extracellular tannic acid-stained structures are not involved in the deposition of callose, the question is open as to their function. One interesting possibility is that they could be involved in the regulation of transport through plasmodesmata via an as yet unknown mechanism. This follows the discovery that if actin, which is present along the length of the plasmodesmata, is disrupted by cytochalasin, the necks of the plasmodesmata become enlarged and the external tannic acid structures are lost (White et al. 1994). This suggests that the sphincters may play a structural role in maintaining the neck of the plasmodesmata. Plasmodesmata can modulate their permeability within minutes and yet callose is not fully reabsorbed for 4-24 h (Drake and Carr 1978, Galway and McCully 1987) suggesting that there must be a mechanism other than callose deposition involved in regulation. The present results stress the need for more care to be taken when preparing specimens to prevent the deposition of wound-induced callose in susceptible species. To avoid callose deposition and any effects of chemical treatment cryofixation and freeze substitution would be the logical method of specimen preparation. These results suggest that there are two mechanisms of transport regulation. The first is a gross regulation through wound-induced deposition of callose, which due to time required for reabsorption of callose would be a relatively long-term or emergency regulation method. The second regulatory mechanism involves as yet unknown components that could be related to external tannic acid-stained structures.
Acknowledgements This research was supported by a grant to R. L. Overall from the Australian Research Council.
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