Spinach chloroplasts contain two types of RNA polymerases. One is multimeric and Escherichia coB-like. The other one is not E. coli-like and might represent a monomeric enzyme of 110 kD. The quantitative relation of the two polymerases changes during plant development. This raises the question, how are plastid genes transcribed that contain E. coli-like and non-E, coil-like promoter elements during developmental phases when both enzymes are present? Transcription of the spinach plastid rrn operon promoter is initiated at three sites: P1, PC, and P2. P1 and P2 are preceded by E. coli-like promoter elements that are recognized by E. coil RNA polymerase in vitro. However, in vivo, transcription starts exclusively at PC. We analyzed different promoter constructions using in vitro transcription and gel mobility-shift studies to understand why Pl and P2 are not used in vivo. Our results suggest that the sequence-specific DNA-binding factor CDF2 functions as a repressor for transcription initiation of the E. coli-like enzyme at PI and P2. We propose a mechanism of constitutive repression to keep the rrn operon in all developmental phases under the transcriptional control of the non-E, coli-like RNA polymerase.
Cytokinins (CKs) are known to regulate the biogenesis of chloroplasts under changing environmental conditions and at different stages of plant ontogenesis. However, the underlying mechanisms are still poorly understood. Apparently, the mechanisms can be duplicated in several ways, including the influence of nuclear genes that determine the expression of plastome through the two-component CK regulatory circuit. In this study, we evaluated the role of cytokinins and CK signaling pathway on the expression of nuclear genes for plastid RNA polymerase-associated proteins (PAPs). Cytokinin induced the expression of all twelve Arabidopsis thalianaPAP genes irrespective of their functions via canonical CK signaling pathway but this regulation might be indirect taking into consideration their different functions and versatile structure of promoter regions. The disruption of PAP genes contributed to the abolishment of positive CK effect on the accumulation of the chloroplast gene transcripts and transcripts of the nuclear genes for plastid transcription machinery as can be judged from the analysis of pap1 and pap6 mutants. However, the CK regulatory circuit in the mutants remained practically unperturbed. Knock-out of PAP genes resulted in cytokinin overproduction as a consequence of the strong up-regulation of the genes for CK synthesis.
As multifunctional regulators of physiological processes, phytohormones play an important role in the regulation of expression of the plastid genome and chloroplast biogenesis. Hormones can directly regulate the expression of genes localized in the chloroplast genome. However, many components of the plastid transcription apparatus are encoded by nuclear rather than plastid genes. It remains obscure whether these nuclear genes are subject to hormonal regulation. This is the first study to show that phytohormones exert differential effects on the expression of nuclear genes of the transcription machinery of the Arabidopsis thaliana plastome. RT-PCR analysis showed that the level of transcripts of the majority of studied genes was activated by trans-zeatin but decreased under the influence of ABA, methyl jasmonate, and salicylic acid, whereas ethylene had no significant effect, and the effects of brassinolide depended on the illumination conditions. The results of this study indicate that the hormonal regulation of the plastome expression can be mediated by differential regulation of the nuclear genes encoding plastid transcription machinery components.
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