Actin is bundled in response to Al3+ in grapevine cells
To address the role of actin in defence, we used aluminium as tool to induce actin remodeling based on our previous work demonstrating aluminium-dependent actin bundling in tobacco (Ahad & Nick, 2007). We therefore tested, whether this response can also be evoked in grapevine cells. In control cells, actin was organised as meshwork of fine filaments (Figure 1A ). Between these filaments, few punctate signals were seen as well, for instance, in the perinuclear region. These punctate structures presumably represent nucleation complexes (Maisch et al., 2009). When the cells were incubated with AlCl3 (200 µM), the actin filaments had, after 2 h, reorganised into dense bundles, often aligned with the long axis of the cell and detached from the membrane (Figure 1B ). In addition, the punctate signals had increased in abundance. To statistically validate this phenomenon, actin bundling was quantified using a quantitative image analysis strategy (Figure 1C ). Compared to the control, the aluminium treatment increased the degree of bundling significantly (by more than 60%).
In order to get insight into the base for the observed bundling, we used Latrunculin B (LatB), a drug that irreversibly sequesters G-actin and prevents F-actin assembly, such that actin polymerisation is blocked. In response to 1 µM LatB (2 h), only short rods of actin were observed, accompanied by increased diffuse fluorescence in the cytoplasm and a higher incidence of punctate signals (Suppl. Figure S1A ). The fine filaments prevalent in control cells (Figure 1A ) were completely eliminated, such that the quantification of actin bundling produced a slightly increased value (compare white bars in Suppl. Figure S1C with those inFigure 1C ). When LatB (1 µM) was administered in presence of AlCl3 (200 µM), the actin filaments were significantly longer (Suppl. Figure S1B ), and the degree of actin bundling was also slightly, but significantly (P = 0.05), increased compared to the situation without AlCl3 (Suppl. Figure S1C ). Aluminium could rescue the effect of Latrunculin B, albeit only partially. This would indicate that a part of the actin response to aluminium is due to a reduced turnover of actin filaments.