Transcripts of SA response and synthesis are induced by Al3+ depending on actin
The experiments described above showed that the aluminium induced defence-related transcripts dependending on actin. Actin remodeling is often observed as hallmark for ensuing programmed cell death. In the context of grapevine cells, this has been observed, for instance, during the responses to harpin (Qiao et al., 2010), to the stilbene aglycon resveratrol (Chang et al., 2011), or to the oxylipin derivativecis -3-hexenal (Akaberi et al. 2018). Since defence-related programmed cell death is usually deployed to encounter infection by biotrophic pathogens, a defence type commonly regulated by salicylic acid (SA), we investigated the transcription of PR-1(pathogenesis related 1) (Kobayashi & Kobayashi, 2007) as readout for SA-dependent gene expression. As shown in Figure 5A , the steady-state transcript levels for PR1 went up significantly in response to Al3+, and this induction was strongly (by a factor of almost 3) suppressed by Latrunculin B (Figure 5A ). The effect of aluminium could be efficiently mimicked by Phalloidin (Figure S2 ), a compound stabilising filamentous actin (F-actin) by effectively suppressing actin dynamics. Latrunculin B alone yielded only a minor response (less than 25% of the response seen with Al3+). While these data would place PR1downstream of actin, in a similar way as the phytoalexins synthesis genes (PAL , RS , and STS ), and the transcriptional regulator MYB14 , they also lead to the next question:
If PR1 as SA responsive gene is activated by aluminium through actin-dependent signaling, there should be also a response of SA-synthesis genes. Two biosynthetic pathways, leading to SA, are known (reviewed in Chen et al., 2009) – one pathway uses cinnamonic acid as substrate, and, therefore, depends on the induction ofphenylalanine ammonium lyase (PAL ), while the substrate of the other pathway is isochorismate, and therefore depends on the induction of isochorismate synthase (ICS ). The induction pattern for PAL had already been tested (Figure 3A ), but since this induction might also occur in context with phytoalexins synthesis itself, it does not conclusively indicate a response of SA synthesis. We, therefore, analysed the transcript levels for ICSas well to test, whether they follow the same activation pattern as the phytoalexins genes. As shown in Figure 5B , Al3+ could induce ICS transcripts significantly, although the induction was only around one fifth of that seen for PR1 . This aluminium response was completely suppressed by Latrunculin B, and Latrunculin B alone did not establish the ground levels, but even decreased ICS significantly. In contrast to the pattern seen for PR1 and PAL , phalloidin was not able to mimic aluminium with respect to the induction of of ICS(Figure S2 ).