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).