Genes related to the stilbene pathway are induced by Al3+ depending on actin
The previous experiment indicated that RboH mediates the actin response to aluminium. From our previous work, we knew already that activation of RboH can activate genes of the stilbene pathway (Chang et al., 2011). Therefore, we asked, whether actin is necessary for this activation. We selected phenylammonium lyase (PAL ) as the first committed step of the phenylpropanoid pathway, and two subpopulations of the stilbene synthase family, resveratrol synthases (RS ), and canonical stilbene synthases (STS ) as markers for the potential activation of phytoalexin genes by Al3+.
As shown in Figure 3A , Al3+ (200 µM, 2 h) significantly induced the transcripts of all three genes, a bit less pronounced for STS (around 5-fold), as compared to PAL(around 6-fold), and RS (around 7-fold). To test, whether actin filaments are necessary for this activation (Figure 3B ), we first eliminated them by Latrunculin B (Suppl. Figure S1 ), before adding aluminium. For RS and STS , this pretreatment completely eliminated any induction by aluminium. Also for PAL , this effect was observed, although, here, still a net induction of around 3-fold over the control remained. To test, whether bundling of actin was sufficient to induce phytoalexins, we probed the transcript levels of PAL in response to 1 µM of phalloidin administered for 2 h in the absence of Al3+ (Suppl. Figure S2 ). We observed a 10-fold induction, which was comparable to that seen for AlCl3 treatment (Figure 3A ). Thus, bundling of actin filaments (here obtained by phalloidin) can fully mimic the effect of AlCl3 on the induction of PAL(Figure 3B ).
Surprisingly, LatB, if administered in the absence of aluminium, induced a conspicuous activation of all three genes as well (Figure 3A ), which for PAL and RS was even exceeding the reponse obtained by aluminium. In other words: two triggers that each activated gene expression, acted antagonistically, if they were combined. This outcome was not only unexpected, but even seemingly paradox.
To understand, to what extent this induction of transcripts was linked with PTI, which is initiated by a calcium influx that can be measured as extracellular alkalinisation, we probed for potential changes of extracellular pH in response to AlCl3 (Suppl. Figure S3 ), however, we did not observe any alkalinisation Instead, pH dropped slightly, which is linked with the acidification caused by aluminium ions. Consistently, there was also no dependence on GdCl3, an inhibitor of calcium channels that in PTI can block extracellular alkalinisation (Chang & Nick, 2012).