Agriculture Reference
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removed material from the exposed ends of the plasmodesmata, but there was little
evidence of structural alteration in the plasmodesmal core. In contrast to Tilney
et al.
(1991), Turner
et al.
(1994) found that treatment with detergents removed both the
plasma membrane and the desmotubule from within plasmodesmata, leaving the
cell wall collar undisturbed. These authors concluded that the plasmodesmata collar
is not proteinaceous, but proteins may bind it into the wall. Ritzenthaler
et al.
(2000)
found that treatment with Pronase E and SDS could eliminate DiOC
6
staining of
plasmodesmata in cell wall fragments of
Nicotiana clevelandii
;however, spots of
callose were still identifiable. Treatment with chloroform-methanol, which should
solubilise all membranous material, did not eliminate DiOC
6
staining of plasmod-
esmata. These results led Ritzenthaler
et al.
(2000) to conclude that DiOC
6
staining
of plasmodesmata is unlikely to be due to the lipophilic nature of this stain but may
be correlated to the presence of protein within the plasmodesma. Blackman and
Overall (2001) have suggested that such proteins may be essential for maintaining
the structure of the desmotubule.
Biochemical analysis of plasmodesmal proteins has proven to be problematical
because of the difficulty in obtaining pure extracts that are free from wall contam-
inants (Epel, 1994; Epel
et al.
, 1995, 1996). Filtration, nitrogen bomb disruption
and wall-digesting enzymes have been used to improve plasmodesmata extraction
and to identify several putative proteins enriched in these extracts (Kotlizky
et al.
,
1992; Epel
et al.
, 1996). Antibodies against a 41-kDa protein, isolated from a plas-
modesmal extract, have been localised to plasmodesmata in maize mesocotyl cells
(Epel
et al.
, 1996). A putative plasmodesma-associated protein from maize meso-
coytl, PAP26, was found to cross-react with antibodies raised against connexin from
animal gap junctions (Yahalom
et al.
, 1991). Two monoclonal antibodies (JIM76
and JIM64) raised against maize root plasmodesmata-associated proteins (Turner
et al.
, 1994) were found to label plasmodesmata in trichomes and mesophyll of
N. clevelandii
(Waigmann
et al.
, 1997). Blackman
et al.
(1998) have also identified
four putative plasmodesmata-associated proteins from the alga,
Chara corallina.
Antibodies raised against one of these, a 45-kDa protein, showed localisation to
plasmodesmata. However, to date, no genes encoding these proteins have been iso-
lated or characterised.
Immunogold localisation and rhodamine phalloidin labelling have used to show
that actin is a component along the entire length of plasmodesmata in higher plants
(White
et al.
, 1994), and also in the alga
C. corallina
(Blackman & Overall, 1998).
Anitbodies raised against animal myosin have been localised to plasmodesmata
in root tissue of
A. cepa
,
Zea mays
and
Hordeum vulgare
(Radford & White,
1998). Also, a mung bean myosin antibody was found to localise to plasmodes-
mata in
C. corallina
(Blackman & Overall, 1998). Myosin VIII, an unconventional
myosin found only in plants, has also been localised to developing plasmodesmata
in transverse cell walls of both meristematic and post-mitotically growing root cells
(Reichelt
et al.
, 1999). Tubulin has been found in protein extracts of walls containing
plasmodesmata, but it has not been convincingly localised to plasmodesmata using
immunogold procedures (Blackman & Overall, 1998). The carbohydrate callose,
ab-1,3-glucan, is commonly associated with the extracellular neck region of the
