MAPK signalling is necessary for aluminium triggered gene
expression
A mitogen-activated protein kinase (MAPK) cascade is known to convey the
defence signal from the plasma membrane to the nucleus, leading to
changes of gene expression. This signal can be interrupted in grapevine
cell cultures by the specific inhibitor PD98059, such that the
activation of defence genes in response to elicitors is suppressed
(Chang & Nick, 2012). We, therefore, used the same approach to
investigate, whether MAPK signalling was involved in the activation of
phytoalexin genes by Al3+.
As shown in Figure 6A , the MAPK inhibitor PD98059 fully
suppressed the induction of PAL , RS , and STStranscripts in response to aluminium. We tested then, whether this
inhibitor possibly interfered with aluminium-triggered actin remodeling.
We used the same experimental design as in the gene-expression
experiments, i.e. the cells were treated for 30 min with PD98059,
followed by additional 2 hours either in absence or presence of
AlCl3. We observed that the inhibitor by itself already
caused actin remodeling: In addition to a mild bundling and formation of
longitudinal cables (Figure 6B ), punctate signals appeared in
the perinuclear region. These perinuclear dots increased significantly
after the Al3+ treatment (Figure 6C ).
Although such dots had already been observed in response to aluminium
alone (Figure 1 ), their abundance and size differed to an
extent that the quality of this type of actin required a different
quantification strategy scoring the coverage of these actin foci
(Figure 6D, E ). We find a significant increase of these foci
after combined treatment with aluminium and PD98059 as compared to
PD98059 alone. While such foci were occasionally seen also in cells not
treated with PD98059 (see Figure 1B for an example), their
incidence was much lower in absence of this inhibitor (Figure
6E ). These experiments are congruent with a model, where MAPK signaling
acts downstream of aluminium-triggered actin remodeling in response to
aluminium (Figure 6F ). However, since actin foci were also
observed in response to the inhibitor alone indicate a feedback of
inhibited MAPK signalling (or the gene expression altered by this
inhibited signalling) either to actin organisation itself or to a step
upstream of actin organisation.