Characterization of a mitochondrial PPR protein in Arabidopsis thaliana
Pentatricopeptide repeat (PPR) proteins are nuclear-encoded proteins, assigned to various aspects of RNA metabolism in mitochondria and chloroplasts such as RNA splicing, editing, processing, and translation, thereby affecting important biological processes. The main function of mitochondria is cellular respiration. Also, they act as sensory organelles, where signals triggered by a wide range of mitochondrial perturbation stimuli cause changes in the nuclear gene expression (Woodson and Chory 2008). The significant phenotypes such as embryo lethality, reduced fertility and a dwarf phenotype, which are associated with mutations of PPR genes emphasize the important roles of PPR proteins in the regulation of plant growth and development (Lurin et al. 2004). In this thesis, a PPR gene is characterized, which is implicated in mitochondrial RNA editing, respiratory activities, affects floral transition, ABA sensitivity and abiotic stress responses in Arabidopsis. Arabidopsis plants with a T-DNA insertion in the PPR gene, AT1G15480, exhibited an early-flowering phenotype. The early-flowering phenotype was observed under both long- and short-days. Genetic complementation analysis reversed the observed phenotype, connecting AT1G15480 to flowering. Due to the observed phenotype, the PPR protein was named PRECOCIOUS1 (POCO1). In this study, it is demonstrated that poco1 mutation affects multiple RNA editing sites in 11 mitochondrial transcripts, supporting a broad role for POCO1 in the regulation of RNA editing efficiency. Conceivably, poco1 showed an impaired respiratory activity, as was shown by a lower respiration rate, ATP level and a higher accumulation of superoxide radicals compared with wild-type plants. To gain insight into the involvement of POCO1 in floral transition, the expression level of FLOWERING LOCUS C (FLC), FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) in poco1 and wild-type plants were investigated. Importantly, the quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis showed that in poco1 the expression level of the potent floral repressor, FLC, and an important promoter of flowering, FT, were significantly down- and up-regulated respectively. Likewise, the expression level of the FLC positive regulator, ABSCISIC ACID-INSENSITIVE 5 (ABI5) was reduced in poco1. Consistent with the qRT-PCR results, poco1 plants showed reduced sensitivity to abscisic acid (ABA) compared with wild-type with respect to primary root growth and days to flowering. Furthermore, the poco1 mutation enhanced the sensitivity to drought stress. To provide an overview of the transcriptome changes in poco1, a RNA-seq analysis was done. Based on the analysis of differentially expressed genes in three comparisons, several biological processes were found to be enriched, which indicates that complex regulatory mechanisms underlie poco1 development. RNA-seq analysis identified many transcripts associated with flowering such as FT, which most likely is involved in the early-flowering phenotype of poco1. Numerous ABA-associated genes, including the core components of ABA signaling along with the important genes for stomatal function, were mostly down-regulated in poco1. Drought and oxidative stress-related genes, including ABA-induced stress genes, were differentially regulated. The RNA-seq analysis identified candidate genes that would be interesting in relation to stress regulation, such as those from the stress-induced nuclear genes encoding mitochondrial proteins (NGEMPs) and drought- and oxidative stress-related genes as well as different families of transcription factors such as ERF, WRKY, MYB, and NAC etc. RNA-seq analysis also uncovered differentially regulated genes encoding various classes of transcription factors and genes involved in cellular signaling. Redox-related genes were affected, suggesting that the redox state in poco1 might be altered. Overall, the data generated in this study can be used to facilitate further dissection of the underlying molecular mechanisms connecting mitochondrial influence and ABA signaling as well as floral transition.
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