To directly test and further confirm the above
events of Al-induced callose on PD, we performed
immuno-electron microscopy and evaluated callose
levels at PD using a monoclonal antibody raised
against 133 b-d-glucan. In agreement with the confocal images (Fig. 2, E and F), here too the Al-induced
callose was localized preferentially at PD regions
(Fig. 3). The increase in number of gold particles,
especially surrounding PD regions, after Al treatments as well as conspicuous reduction to their number in DDG pretreated roots, all this was apparent
with much more enhanced resolution (Fig. 3, B and
C). The specificity of antibody binding target sites
was also confirmed as the control phloem cells exhibited callose only at sieve tube cell plates (Fig. 3D).
The impact of Al on the callose formation at PD and
the ameliorative effect of DDG was further substantiated by quantitative evaluation of the immunogold
image analysis and by quantitative determination of
total callose at 5-mm root apex (Fig. 4). There was
approximately more than a 3-fold increase in the
number of gold particles localized to the individual
PD after Al treatment (Fig. 4A) and approximately a
2-fold reduction to this level if the roots received
DDG prior to Al treatment (Fig. 4A). This suggests
that DDG can effectively block the Al-induced callose
synthesis at PD. The transfer of living roots to fixative for immunogold labeling seemed to induce a
transient level of callose within controls, which was
also localized preferentially at PD regions (Fig. 3A).
A sharp decrease to callose levels in control plants
(Fig. 4B) fixed after DDG pre-treatment compared
with absolute controls supports this notion. In general, in comparison with the immunogold data the
fluorescence spectrophotometric quantification of
callose contents, which yielded comparable trends,
confirmed both immunogold pattern of callose localization and quantities between treatments (Figs. 3
and 4)