Plastid gene expression (PGE) must adequately respond to changes in both development and environmental cues. The transcriptional machinery of plastids in land plants is far more complex than that of prokaryotes. Two types of DNA-dependent RNA polymerases transcribe the plastid genome: a multimeric plastid-encoded polymerase (PEP), and a monomeric nuclear-encoded polymerase (NEP). A single NEP in monocots (RPOTp, RNA polymerase of the T3/T7 phage-type) and two NEPs in dicots (plastid-targeted RPOTp, and plastid- and mitochondrial-targeted RPOTmp) have been hitherto identified. To unravel the role of PGE in plant responses to abiotic stress, we investigated if Arabidopsis RPOTp could function in plant salt tolerance. To this end, we studied the sensitivity of T-DNA mutants scabra3-2 (sca3-2) and sca3-3, defective in the RPOTp gene, to salinity, osmotic stress and the phytohormone abscisic acid (ABA) required for plants to adapt to abiotic stress. sca3 mutants were hypersensitive to NaCl, mannitol and ABA during germination and seedling establishment. Later in development, sca3 plants displayed reduced sensitivity to salt stress. A gene ontology (GO) analysis of the nuclear genes differentially expressed in the sca3-2 mutant (301) revealed that many significantly enriched GO terms were related to chloroplast function, and also to the response to several abiotic stresses. By quantitative RT-PCR (qRT-PCR), we found that genes LHCB1 (LIGHT-HARVESTING CHLOROPHYLL a/b-BINDING1) and AOX1A (ALTERNATIVE OXIDASE 1A) were respectively down- and up-regulated in the Columbia-0 (Col-0) salt-stressed plants, which suggests the activation of plastid and mitochondria-to-nucleus retrograde signaling. The transcript levels of genes RPOTp, RPOTmp and RPOTm significantly increased in these salt-stressed seedlings, but this enhanced expression did not lead to the up-regulation of the plastid genes solely transcribed by NEP. Similar to salinity, carotenoid inhibitor norflurazon (NF) also enhanced the RPOTp transcript levels in Col-0 seedlings. This shows that besides salinity, inhibition of chloroplast biogenesis also induces RPOTp expression. Unlike salt and NF, the NEP genes were significantly down-regulated in the Col-0 seedlings grown in ABA-supplemented media. Together, our findings demonstrate that RPOTp functions in abiotic stress tolerance, and RPOTp is likely regulated positively by plastid-to-nucleus retrograde signaling, which is triggered when chloroplast functionality is perturbed by environmental stresses, e.g., salinity or NF. This suggests the existence of a compensatory mechanism, elicited by impaired chloroplast function. To our knowledge, this is the first study to suggest the role of a nuclear-encoded plastid-RNA polymerase in salt stress tolerance in plants.
Keywords: Arabidopsis; RPOTp; abiotic stress; abscisic acid; plastid gene expression; salt stress.