# Arabidopsis Plastid-RNA Polymerase RPOTp Is Involved in Abiotic Stress Tolerance
## 2 Results
### RPOTp Expression is Controlled by Plastid Retrograde Signaling in Response to Salt Stress
\begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Gene**} & \multicolumn{2}{c}{**Normalized Transcript Levels in Col-0 Plants Exposed to 100 mM NaCl Relative to Control Plants**} \\ \cline{2-3}  & **Protein Product** & **Fold Change** & \(p\)**-Value** \\ \hline _Nuclear genes_ & & & \\ _RPOTpSCA3_ & NEP 1 RROTPp & 2.27 \(\pm\) 0.84 & 4.113 \(\times\) 10\({}^{-5}\) ** \\ _RPOTmp_ & NEP 1 RROTPmp & 3.50 \(\pm\) 1.70 & 3.996 \(\times\) 10\({}^{-4}\) ** \\ _RPOTm_ & NEP 1 RPOTm & 1.34 \(\pm\) 0.33 & 0.036 \({}^{*}\) \\ _AOX1A_ & Alternative oxidase 1A & 3.90 \(\pm\) 1.00 & 4.113 \(\times\) 10\({}^{-5}\) ** \\ _LHCB1_ & Light-harvesting complex protein B1 & 0.70 \(\pm\) 0.44 & 0.036 \({}^{*}\) \\ _mTERF5_ & mTERF5 & 0.77 \(\pm\) 0.31 & 0.008 \({}^{**}\) \\ _mTERF9_ & mTERF9 & 1.10 \(\pm\) 0.30 & 0.015 \({}^{*}\) \\ _COR15B_ & Cold-regulated 15B & 4.76 \(\pm\) 2.11 & 2.20 \(\times\) 10\({}^{-10}\) ** \\ _RD29A_ & Responsive to desiccation 29A & 2.74 \(\pm\) 0.76 & 5.83 \(\times\) 10\({}^{-4}\) ** \\ \hline _Chloroplast genes_ & & & \\ _psaA_ & Photosystem I reaction center protein & 1.12 \(\pm\) 0.49 & 0.709 \\ _psaB_ & Photosystem I reaction center protein & 1.04 \(\pm\) 0.28 & 0.709 \\ _psbA_ & Chlorophyll binding protein D1 & 0.60 \(\pm\) 0.22 & 4.113 \(\times\) 10\({}^{-5}\) ** \\ _clcp_ & ATP-dependent protease & 1.18 \(\pm\) 0.27 & 0.036 \({}^{*}\) \\ _rps1B_ & Ribosomal protein S18 & 0.83 \(\pm\) 0.23 & 0.709 \\ _rpaA_ & PEP 2 \({}^{*}\) subunit & 0.63 \(\pm\) 0.27 & 0.001 \({}^{**}\) \\ _rpaB_ & PEP 2 \({}^{*}\) subunit & 0.61 \(\pm\) 0.09 & 0.002 \({}^{**}\) \\ _rpaC1_ & PEP 2 \({}^{*}\) subunit & 0.86 \(\pm\) 0.20 & 0.220 \\ _acC_ & Carboxtransferase 5 subunit of the & 0.88 \(\pm\) 0.35 & 0.709 \\ \cline{2-3}  & Acetyl-CoA carboxylase & 0.82 \(\pm\) 0.35 & 0.709 \\ \hline \hline \end{tabular} \({}^{1}\) NEP: nuclear-encoded plastid RNA polymerase. \({}^{2}\) PEP: plastid-encoded RNA polymerase. Relative expression values were determined as 2\({}^{-2 ACT}\) for each studied gene in 10 DAS Col-0 seedlings exposed to 100 mM after normalization with those of the _ACTIN2_ gene, and compared with those of the Col-0 seedlings in control medium, to which value of 1 was given (see Materials and Methods). Each value corresponds to the mean \(\pm\) standard deviation of the 2\({}^{-2 ACT}\) values obtained using three different biological replicates and triplicate reactions. Values were significantly different from the corresponding wild type at \(p<0.05\) (\({}^{*}\)) or \(p<0.01\) (\({}^{**}\)) using Wilcoxon–Mann–Whitney test.  
\end{table} 
Table 1: Quantitative RT-PCR analysis of the expression of chloroplast and nuclear genes in Col-0 seedlings exposed to 100 mM NaCl. (1.4-fold down-regulated), whereas _AOX1A_ mRNAs markedly increased (3.9-fold up-regulated), in the seedlings exposed to 100 mM NaCl (Table 1). Therefore, these results indicated that our salt stress conditions could trigger organelle retrograde signaling, which would, in turn, modulate the expression of NEP genes. To study the regulation of NEP function by impaired chloroplast biogenesis in-depth, we analyzed the expression of the _RPOTp_ gene in Col-0 seedlings grown in the presence or absence of NF, a bleaching herbicide that inhibits carotenoid biosynthesis and, consequently, chloroplast functionality [36]. When seedlings are treated with NF, the expression of photosynthesis-associated nuclear genes (such as _LHCBI_) is repressed, which has been interpreted as a result of plastid-to-nucleus retrograde signaling [37; 38; 39]. In our study, we included the _gun1-1_ mutant defective in the GUN1 protein, a central integrator of plastid retrograde signals [40]. Compared to the Col-0 seedlings grown on the control media, the presence of NF significantly increased _RPOTp_ transcript abundance (2.4-fold up-regulated) (Figure 4), which was similar to 100 mM NaCl. In the absence of NF, _RPOTp_ expression in the _gun1-1_ mutant was significantly higher than in Col-0 (1.5-fold up-regulated) (Figure 4).