# Arabidopsis Plastid-RNA Polymerase RPOTp Is Involved in Abiotic Stress Tolerance
## 2 Results  
### Knock-Down of RPOTp Alters NaCl, Mannitol and ABA Responses  
To determine if plastid RNA polymerase RPOTp could be involved in plants' tolerance to environmental stress, we studied the sensitivity of Arabidopsis wild-type Columbia-0 (Col-0) and T-DNA mutants _sca3-2_ and _sca3-3_[23] to different abiotic stresses. To this end, Col-0 and _sca3_ mutant seeds were sown in a culture media supplemented with 150 mM NaCl or 350 mM mannitol, which respectively cause ionic and osmotic stress, and their ability to germinate and to form fully expanded green cotyledons (seedling establishment) was examined for 14 DAS (days after stratification). In the absence of stress, the _sca3_ mutant seeds yielded similar germination values to those of the wild-type seeds, although the _sca3-2_ mutant showed slightly lower values than Col-0 during the first culture week (Figure 1a). NaCl or mannitol exogenously applied to the growth medium delayed Col-0 and _sca3_ germination, although this effect was much more pronounced in both mutants, particularly in _sca3-2_ (Figure 1c). In response to mannitol, the germination differences between Col-0 and the _sca3_ mutants became even more evident, and the _sca3-2_ mutant was more sensitive than _sca3-3_ (Figure 1e). Seedling establishment was delayed in the _sca3_ mutants compared to Col-0 for the first 7 DAS in the MS control medium (Figure 1b). This is consistent with the defective photoautotrophic growth previously reported for these mutants [23]. The differences between Col-0 and the _sca3_ mutants were substantially marked in the presence of 150 mM NaCl or 350 mM mannitol, and _sca3-2_ was more sensitive than _sca3-3_ (Figure 1d,f). In line with this, at 10 DAS, 95%, 42% and 25% seedling establishment was achieved for the Col-0, _sca3-3_ and _sca3-2_ mutant seeds, respectively, with 150 mM of NaCl (Figure 1d). Furthermore, at 14 DAS in the presence of mannitol, 90% of the Col-0 seeds yielded seedlings with green expanded cotyledons, as opposed to only 15% and 23% of _sca3-2_ and _sca3-3_, respectively (Figure 1f). The ABA hormone is induced in response to abiotic stress, and plays a fundamental role in adapting plants to adverse environmental conditions [28]. Mutants displaying altered sensitivity to abiotic stress frequently exhibit abnormal responses to ABA [25]. The enhanced sensitivity of the _sca3_ mutants to NaCl and mannitol prompted us to investigate their response to ABA. To do so, the _sca3_ and Col-0 seeds were worn in the MS growth media supplemented with 1.5 or 3 \(\upmu\)M ABA. Germination of the _sca3_ mutants was more sensitive to ABA than that of Col-0, especially _sca3_-2 in the presence of 3 \(\upmu\)M ABA (Figure 2a,b), which is similar to the results obtained with salt or mannitol stress (Figure 1). These differences were more marked from 6 to 10 DAS (Figure 2a,c,e). The ABA concentrations led to a noticeable reduction in seedling establishment in the Col-0 and _sca3_ mutants throughout the study period, although the mutants were more sensitive than Col-0. Accordingly, at 14 DAS, 73% of the Col-0 seeds, and 30% and 37% of the _sca3_-2 and _sca3_-3 seeds, respectively, yielded seedlings with green expanded cotyledons, with 1.5 \(\upmu\)M ABA (Figure 2d). These percentages lowered to 48%, 12% and 24% for Col-0, _sca3_-2 and _sca3_-3, respectively, with 3 \(\upmu\)M ABA (Figure 2f). Figure 1: Effects of NaCl and mannitol on germination and seedling establishment in the wild-type Col-0 and mutants _sca3_-2 and _sca3_-3. Each value corresponds to the mean \(\pm\) standard deviation (SD) of the percentage of germination (**a,c,e**) and seedling establishment (**b,d,f**) in growth media, either without supplementation (**a,b**), or supplemented with 150 mM NaCl (**c,d**) or 350 mM mannitol (**e,f**), for four replicates of at least 50 seeds each per genotype. DAS: days after stratification.