In bacteria, the hyper-phosphorylated nucleotide, guanosine 3′,5′-bis(pyrophosphate) (ppGpp), functions as a secondary messenger under stringent conditions. ppGpp levels are controlled by two distinct enzymes, namely RelA and SpoT, in Escherichia coli. RelA–SpoT homologs (RSHs) are also conserved in plants where they function in the plastids. The model plant Arabidopsis thaliana contains four RSHs: RSH1, RSH2, RSH3 and Ca2+-dependent RSH (CRSH). Genetic characterizations of RSH1, RSH2 and RSH3 were undertaken, which showed that the ppGpp-dependent plastidial stringent response significantly influences plant growth and stress acclimation. However, the physiological significance of CRSH-dependent ppGpp synthesis remains unclear, as no crsh-null mutant has been available. Here, to investigate the function of CRSH, a crsh-knockout mutant of Arabidopsis was constructed using a site-specific gene-editing technique, and its phenotype was characterized. A transient increase in ppGpp was observed for 30 min in the wild type (WT) after the light-to-dark transition, but this increase was not observed in the crsh mutant. Similar analyses were performed with the rsh2-rsh3 double and rsh1-rsh2-rsh3 triple mutants and showed that the transient increments of ppGpp in the mutants were higher than those in the WT. The increase in ppGpp in the WT and rsh2 rsh3 accompanied decrements in the mRNA levels of some plastidial genes transcribed by the plastid-encoded plastid RNA polymerase. These results indicate that the transient increase in ppGpp at night is due to CRSH-dependent ppGpp synthesis and that the ppGpp level is maintained by the hydrolytic activities of RSH1, RSH2 and RSH3 to accustom plastidial gene expression to darkness.

The regulatory nucleotides, guanosine 5'-triphosphate 3'-diphosphate (pppGpp) and guanosine 5'-diphosphate 3'-diphosphate (ppGpp), were originally identified in Escherichia coli, and control a large set of gene expression and enzyme activities. The (p)ppGpp-dependent control of cell activities is referred to as the stringent response. A growing number of (p)ppGpp synthase/hydrolase homologs have been identified in plants, which are localized in plastids in Arabidopsis thaliana. We recently reported that the Arabidopsis mutant overproducing ppGpp in plastids showed dwarf chloroplasts, and transcript levels in the mutant plastids were significantly suppressed. Furthermore, the mutant showed more robust growth than the wild type (WT), especially under nutrient-deficient conditions, although the mechanisms are unclear. To better understand the impact of the ppGpp accumulation on plant responses to nutrient deficiency, photosynthetic activities and metabolic changes in the ppGpp-overproducing mutant were characterized here. Upon transition to the nitrogen-deficient conditions, the mutant showed reduction of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) contents, and effective and maximum quantum yield of photosystem II compared with WT. The mutant also showed more obvious changes in key metabolite levels including some amino acid contents than WT; similar metabolic change is known to be critical for plants to maintain carbon-nitrogen balance in their cells. These results suggest that artificially overproducing ppGpp modulates the organelle functions that play an important role in controlling photosynthetic performance and metabolite balance during nitrogen starvation.

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Endometriosis-associated ovarian cancers demonstrate substantial morphological and genetic diversity. The transcription factor, hepatocyte nuclear factor (HNF)-1β, may be one of several key genes involved in the identity of ovarian clear cell carcinoma (CCC). The present study reviews a considerably expanded set of HNF-1β-associated genes and proteins that determine the pathophysiology of CCC. The current literature was reviewed by searching MEDLINE/PubMed. Functional interpretations of gene expression profiling in CCC are provided. Several important CCC-related genes overlap with those known to be regulated by the upregulation of HNF-1β expression, along with a lack of estrogen receptor (ER) expression. Furthermore, the genetic expression pattern in CCC resembles that of the Arias-Stella reaction, decidualization and placentation. HNF-1β regulates a subset of progesterone target genes. HNF-1β may also act as a modulator of female reproduction, playing a role in endometrial regeneration, differentiation, decidualization, glycogen synthesis, detoxification, cell cycle regulation, implantation, uterine receptivity and a successful pregnancy. In conclusion, the present study focused on reviewing the aberrant expression of CCC-specific genes and provided an update on the pathological implications and molecular functions of well-characterized CCC-specific genes.

Abbreviations: CRSH, Ca 2+ -activated RSH; flg22, flagellin 22; (p)ppGpp, 5'-di(tri)phosphate 3'-diphosphate; PAMPs; pathogen-associated molecular patterns; RSH, RelA-SpoT homolog; qRT-PCR, quantitative real-time PCR Abstract In bacteria, the hyper-phosphorylated nucleotides, guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp), function as secondary messengers in the regulation of various metabolic processes of the cell, including transcription, translation, and enzymatic activities, especially under nutrient deficiency. The activity carried out by these nucleotide messengers is known as the stringent response. (p)ppGpp levels are controlled by two distinct enzymes, namely, RelA and SpoT, in Escherichia coli. RelA-SpoT homologs (RSHs) are also conserved in plants and algae where they function in the plastids. The model plant Arabidopsis thaliana contains four RSHs: RSH1, RSH2, RSH3, and Ca 2+ -dependent RSH (CRSH).Genetic characterizations of RSH1, RSH2, and RSH3 were undertaken, which showed that the (p)ppGpp-dependent plastidial stringent response significantly influences plant growth and stress acclimation. However, the physiological significance of CRSH-dependent (p)ppGpp synthesis remains unclear, as no crsh-null mutant has been available. Here to investigate the function of CRSH, a crsh-knockout mutant of Arabidopsis was constructed using a site-specific gene-editing technique, and its phenotype was characterized. A transient increase of ppGpp was observed for 30 min in the wild type (WT) after light-to-dark transition, but this increase was not observed in the crsh mutant. Similar analyzes were performed with the rsh2rsh3 double and rsh1rsh2rsh3 triple mutants of Arabidopsis and showed that the transient increments of ppGpp in the mutants were higher than those in the WT. The increase of (p)ppGpp in the WT and rsh2rsh3 accompanied decrements in the mRNA levels of psbD transcribed by the plastid-encoded plastid RNA polymerase. These results indicated that the transient increase of intracellular ppGpp at night is due to CRSH-dependent ppGpp synthesis and the (p)ppGpp level is maintained by the hydrolytic activities of RSH1, RSH2, and RSH3 to accustom plastidial gene expression to darkness. expression Plants were grown on 0.8% agar-solidified 1/2 MS medium under continuous light (40 µmol photons m −2 s −1 ). Fourteen-day-old plants were transferred to the dark and ~0.1 g shoots were harvested at each of the time points indicated (Fig. 3). Extraction and quantification of ppGpp were as described previously . Western blot analysisProtein content was measured using the Bradford assay Kit (Bio Lab). Total proteins (6 µg each) were applied to an SDS-PAGE gel and electroblotted onto a polyvinylidene difluoride membrane (GE-Healthcare). The membrane was incubated with the CRSH-specific antibody . Immunoreactive proteins were detected using the Alkaline Phosphatase Substrate Kit II (Vector Laboratories).