SummaryGamma-aminobutyric acid (GABA) is a non-protein amino acid commonly present in all organisms. Because cellular levels of GABA in plants are mainly regulated by synthesis (glutamate decarboxylase, GAD) and catabolism (GABA-transaminase, GABA-T), we attempted seed-specific manipulation of the GABA shunt to achieve stable GABA accumulation in rice. A truncated GAD2 sequence, one of five GAD genes, controlled by the glutelin (GluB-1) or rice embryo globulin promoters (REG) and GABA-T-based trigger sequences in RNA interference (RNAi) cassettes controlled by one of these promoters as well, was introduced into rice (cv. Koshihikari) to establish stable transgenic lines under herbicide selection using pyriminobac. T 1 and T 2 generations of rice lines displayed high GABA concentrations (2-100 mg/100 g grain). In analyses of two selected lines from the T 3 generation, there was a strong correlation between GABA level and the expression of truncated GAD2, whereas the inhibitory effect of GABA-T expression was relatively weak. In these two lines both with two T-DNA copies, their starch, amylose, and protein levels were slightly lower than non-transformed cv. Koshihikari. Free amino acid analysis of mature kernels of these lines demonstrated elevated levels of GABA (75-350 mg/ 100 g polished rice) and also high levels of several amino acids, such as Ala, Ser, and Val. Because these lines of seeds could sustain their GABA content after harvest (up to 6 months), the strategy in this study could lead to the accumulation GABA and for these to be sustained in the edible parts.

Intronic regions of eukaryotic genomes accumulate many Transposable Elements (TEs). Intronic TEs often trigger the formation of transcriptionally repressive heterochromatin, even within transcription-permissive chromatin environments. Although TE-bearing introns are widely observed in eukaryotic genomes, their epigenetic states, impacts on gene regulation and function, and their contributions to genetic diversity and evolution, remain poorly understood. In this study, we investigated the genome-wide distribution of intronic TEs and their epigenetic states in the Oryza sativa genome, where TEs comprise 35% of the genome. We found that over 10% of rice genes contain intronic heterochromatin, most of which are associated with TEs and repetitive sequences. These heterochromatic introns are longer and highly enriched in promoter-proximal positions. On the other hand, introns also accumulate hypomethylated short TEs. Genes with heterochromatic introns are implicated in various biological functions. Transcription of genes bearing intronic heterochromatin is regulated by an epigenetic mechanism involving the conserved factor OsIBM2, mutation of which results in severe developmental and reproductive defects. Furthermore, we found that heterochromatic introns evolve rapidly compared to non-heterochromatic introns. Our study demonstrates that heterochromatin is a common epigenetic feature associated with actively transcribed genes in the rice genome.

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Programmable RNA editing offers significant therapeutic potential for a wide range of genetic diseases. Currently, several deaminase enzymes, including ADAR and APOBEC, can perform programmable adenosine-to-inosine or cytidine-to-uridine RNA correction. However, enzymes to perform guanosine-to-adenosine and uridine-to-cytidine (U-to-C) editing are still lacking to complete the set of transition reactions. It is believed that the DYW:KP proteins, specific to seedless plants, catalyze the U-to-C reactions in mitochondria and chloroplasts. In this study, we designed seven DYW:KP domains based on consensus sequences and fused them to a designer RNA-binding pentatricopeptide repeat (PPR) domain. We show that three of these PPR-DYW:KP proteins edit targeted uridine to cytidine in bacteria and human cells. In addition, we show that these proteins have a 5′ but not apparent 3′ preference for neighboring nucleotides. Our results establish the DYW:KP aminase domain as a potential candidate for the development of a U-to-C editing tool in human cells.

Despite lacking Na + -ATPase as a sodium pump in vascular plants, a gene encoding KPA (K + P-type ATPase), a putative animal type-Na + /K + -ATPase, has been isolated from the marine red alga Porphyra yezoensis and designated PyKPA1. To characterize the properties of PyKPA1 and also to confirm its ability to confer salinity tolerance in land plants, transgenic rice plants were produced that expressed the full-length PyKPA1 cDNA under the control of cauliflower mosaic virus 35S RNA promoter. We observed transcriptional activation of the transgene, plasma membrane-localization of the gene product fused with green fluorescent protein in onion epidermal cells, and Na + -ATPase activity in the plasma membrane fraction from transgenic rice plants, indicating that PyKPA1 was functionally expressed in rice plants. Transgenic lines were examined in terms of growth in salinity stress conditions, resulting in protection from a decrease in biomass, although growth of control rice plants was repressed. These results demonstrate the utility of a red algal animal type-sodium pump for conferring salinity tolerance to land plants.