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.