# 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  
To gain further insight into the role of PGE in plant responses to abiotic stress, more specifically to salinity, we decided to investigate the effect that salt stress could have on the steady-state levels of the transcripts of the three Arabidopsis NEP genes: _RPOTp_, _RPOTmp_ and _RPOTm_. We decided to perform this analysis on the Col-0 seedlings grown under similar conditions to those we previously assayed (see Figure 1, and Materials and Methods). To this end, Col-0 seedlings were grown in medium supplemented, or not, with 100 mM NaCl. This condition significantly delayed seedling growth, but did not impair growth as severely as 150 mM NaCl (the concentration we used in the germination and seedling establishment assays) (Figure 1). At 10 DAS, total RNA was extracted and retro-transcribed, and cDNAs were analyzed by quantitative PCR (qPCR). To confirm the effectiveness of the salt stress treatment, we first studied the expression of the _RD29A_ (_RESPONSIVE TO DESICCATION 29A_) and _COR15B_ (_COLD-REGILATED 15B_) genes, which are induced by exposure to salinity, cold or ABA [30]. As expected for salt stress, the transcript levels of _RD29A_ and _COR15B_ significantly increased in the Col-0 seedlings grown on 100 mM NaCl (4.8- and 2.7-fold up-regulated, respectively; Table 1). As regards the expression of the NEP genes, the treatment with 100 mM NaCl led to a significant increase in the steady-state levels of the transcripts of them all, albeit to different extents. Accordingly, _RPOTm_, _RPOTp_ and _RPOTmp_ were 1.3-, 2.3- and 3.5-fold up-regulated, respectively (Table 1). To determine the specificity of the effect of salt stress on the expression of the NEP genes, we studied the transcript levels of two nuclear genes that encode chloroplast-targeted proteins also involved in PGE: the mitochondrial transcription termination factors mTERF5 and mTERF9. These mTERFs are functionally related to RPOTp and are required for chloroplast development and response to abiotic stress [31]; furthermore, mTERF5 regulates transcription of the plastid _psbEFLJ_ polycistron [32]. In stark contrast to the NEP genes, we found that 100 mM of NaCl significantly repressed _mTERF5_ expression (1.3-fold down-regulated) and did not noticeably modify _mTERF9_ transcript abundance (Table 1). Together, our results reveal that salinity differentially affected the expression of nuclear genes involved in chloroplast transcription. To investigate if these changes in gene activity could result from retrograde signaling from chloroplasts to the nucleus, we decided to study, in the salt-stressed and non-salt stressed control Col-0 seedlings, the mRNA abundance of two marker genes for the activation of retrograde signaling from the chloroplast and mitochondria to the nucleus. To this end, the expression of the photosynthesis gene _LHCB1_ (encoding the LIGHT-HARVESTING CHLOROPHYLL a/b-BINDING1 protein) and the _AOX1A_ gene (encoding ALTERNATIVE OXIDASE 1A) were analyzed by qRT-PCR. It is well-known that the _LHCB1_ gene is down-regulated when chloroplast biogenesis is perturbed [33], whereas _AOX1A_ expression increases in response to impaired mitochondria function, in order to protect plants from oxidative damage and prevent reactive oxygen species (ROS) production [34; 35]. Compared to the non-stressed seedlings, accumulation of the _LHCB1_ transcripts significantly decreased