The powerful potential of amino acid menthyl esters for anti-inflammatory and anti-obesity therapies.

Our newly developed menthyl esters of valine and isoleucine exhibit anti-inflammatory properties beyond those of the well-known menthol in macrophages stimulated by lipopolysaccharide (LPS) and in a mouse model of colitis induced by sodium dextran sulfate. Unlike menthol, which acts primarily through the cold-sensitive TRPM8 channel, these menthyl esters displayed unique mechanisms that operate independently of this receptor. They readily penetrated target cells and efficiently suppressed LPS-stimulated tumour necrosis factor-alpha (Tnf) expression mediated by liver X receptor (LXR), a key nuclear receptor that regulates intracellular cholesterol and lipid balance. The menthyl esters showed affinity for LXR and enhanced the transcriptional activity through their non-competitive and potentially synergistic agonistic effect. This effect can be attributed to the crucial involvement of SCD1, an enzyme regulated by LXR, which is central to lipid metabolism and plays a key role in the anti-inflammatory response. In addition, we discovered that the menthyl esters showed remarkable efficacy in suppressing adipogenesis in 3T3-L1 adipocytes at the mitotic clonal expansion stage in an LXR-independent manner as well as in mice subjected to diet-induced obesity. These multiple capabilities of our compounds establish them as formidable allies in the fight against inflammation and obesity, paving the way for a range of potential therapeutic applications.

Genome editing in rice mediated by miniature size Cas nuclease SpCas12f.

Cas9 derived from Streptococcus pyogenes (SpCas9) is used widely in genome editing using the CRISPR-Cas system due to its high activity, but is a relatively large molecule (1,368 amino acid (a.a.) residues). Recently, targeted mutagenesis in human cells and maize using Cas12f derived from Syntrophomonas palmitatica (SpCas12f)-a very small Cas of 497 a.a, which is a more suitable size for virus vectors-was reported. However, there are no reports of genome editing using SpCas12f in crops other than maize. In this study, we applied SpCas12f to genome editing in rice-one of the most important staple crops in the world. An expression vector encoding rice codon-optimized SpCas12f and sgRNA for OsTubulin as a target was introduced into rice calli by Agrobacterium-mediated transformation. Molecular analysis of SpCas12f-transformed calli showed that mutations were introduced successfully into the target region. Detailed analysis by amplicon sequencing revealed estimated mutation frequencies (a ratio of the number of mutated calli to that of SpCas12f-transformed calli) of 28.8% and 55.6% in two targets. Most mutation patterns were deletions, but base substitutions and insertions were also confirmed at low frequency. Moreover, off-target mutations by SpCas12f were not found. Furthermore, mutant plants were regenerated successfully from the mutated calli. It was confirmed that the mutations in the regenerated plants were inherited to the next-generation. In the previous report in maize, mutations were introduced by treatment with heat shock at 45°C for 4 h per day for 3 days; no mutations were introduced under normal growth conditions at 28°C. Surprisingly, however, mutations can be introduced without heat-shock treatment in rice. This might be due to the culture conditions, with relatively higher temperature (30°C or higher) and constant light during callus proliferation. Taken together, we demonstrated that SpCas12f can be used to achieve targeted mutagenesis in rice. SpCas12f is thus a useful tool for genome editing in rice and is suitable for virus vector-mediated genome editing due to its very small size.

Genome Editing Technology and Its Application to Metabolic Engineering in Rice.

Genome editing technology can be used for gene engineering in many organisms. A target metabolite can be fortified by the knockout and modification of target genes encoding enzymes involved in catabolic and biosynthesis pathways, respectively, via genome editing technology. Genome editing is also applied to genes encoding proteins other than enzymes, such as chaperones and transporters. There are many reports of such metabolic engineering using genome editing technology in rice. Genome editing is used not only for site-directed mutagenesis such as the substitution of a single base in a target gene but also for random mutagenesis at a targeted region. The latter enables the creation of novel genetic alleles in a target gene. Recently, genome editing technology has been applied to random mutagenesis in a targeted gene and its promoter region in rice, enabling the screening of plants with a desirable trait from these mutants. Moreover, the expression level of a target gene can be artificially regulated by a combination of genome editing tools such as catalytically inactivated Cas protein with transcription activator or repressor. This approach could be useful for metabolic engineering, although expression cassettes for inactivated Cas fused to a transcriptional activator or repressor should be stably transformed into the rice genome. Thus, the rapid development of genome editing technology has been expanding the scope of molecular breeding including metabolic engineering. In this paper, we review the current status of genome editing technology and its application to metabolic engineering in rice.

An amino acid ester of menthol elicits defense responses in plants.

KEY MESSAGE: Valine menthyl ester (ment-Val) acts as a plant defense potentiator for several crop species including soybean. Terpenoids, including menthol, exhibit potent abilities as plant defense potentiators in agriculture and horticulture. In the current study, we developed new terpene derivatives that consisted of menthol and various amino acids and that were expected to act as powerful plant defense potentiators. We used 6 amino acids possessing low-reactive sidechains to synthesize an array of amino acid ester of menthol (ment-aa) compounds. Transcript levels of two defense genes (pathogenesis-related protein 1 [PR1] and trypsin inhibitor [TI]) were evaluated in leaves of soybean plants 24 h after application of aquatic solution of menthol or menthol-aa, and revealed that the valine menthyl ester (ment-Val) alone elevated the transcript level of defense genes, and it did so only at the low dose of 1 µM, not at higher or lower doses tested. Moreover, it appeared that histone acetylation was involved in this effect. Application of ment-Val enabled soybean plants to sustain the increased transcript levels in their leaves for up to 3 days. Moreover, when ment-Val was additionally applied at day 4, at which time the transcript level had declined to the basal level, the transcript level was re-elevated, indicating the possibility that ment-Val could be repeatedly used to sustain pest control. Ment-Val was found to be chemically stable and effective for defense of several crop species. Collectively, these data show that terpenoid conjugates are useful for pest control instead of or in addition to pesticides.

Plant genome editing: ever more precise and wide reaching.

Genome-editing technologies consisting of targeted mutagenesis and gene targeting enable us to modify genes of interest rapidly and precisely. The discovery in 2012 of CRISPR/Cas9 systems and their development as sequence-specific nucleases has brought about a paradigm shift in biology. Initially, CRISPR/Cas9 was applied in targeted mutagenesis to knock out a target gene. Thereafter, advances in genome-editing technologies using CRISPR/Cas9 developed rapidly, with base editing systems for transition substitution using a combination of Cas9 nickase and either cytidine or adenosine deaminase being reported in 2016 and 2017, respectively, and later in 2021 bringing reports of transversion substitution using Cas9 nickase, cytidine deaminase and uracil DNA glycosylase. Moreover, technologies for gene targeting and prime editing systems using DNA or RNA as donors have also been developed in recent years. Besides these precise genome-editing strategies, reports of successful chromosome engineering using CRISPR/Cas9 have been published recently. The application of genome editing to crop breeding has advanced in parallel with the development of these technologies. Genome-editing enzymes can be introduced into plant cells, and there are now many examples of crop breeding using genome-editing technologies. At present, it is no exaggeration to say that we are now in a position to be able to modify a gene precisely and rearrange genomes and chromosomes in a predicted way. In this review, we introduce and discuss recent highlights in the field of precise gene editing, chromosome engineering and genome engineering technology in plants.

Pest management using mint volatiles to elicit resistance in soy: mechanism and application potential.

Plants can eavesdrop on volatile cues emitted from neighboring plants to boost their defense responses. When 10 categories of mints were tested for their effects on Glycine max (soybean) plants cultivated nearby, candy mint (Mentha × piperita cv. Candy) and peppermint (Mentha × piperita L.) induced the strongest enhancement in RNA levels of defense genes in the soybean leaves. The mechanism by which the mint volatiles enhanced these transcript levels was based on histone acetylation within the promoter regions of defense genes. These increases in transcript levels were induced when receiver plants were cultivated near to candy mint, but the priming of the defense responses was instead induced when receiver plants were cultivated at mid-length intervals. Field assays revealed that anti-herbivore ability of soy was strengthened both by co-cultivation and by pre-incubation of receiver plants with candy mint. The same held true for another receiver, Brassica rapa, when the receiver was co-cultivated or pre-incubated with peppermint. Exposure to mint volatiles resulted in lower damage to receiver plants, although ecological effects on the herbivores and predators probably also contributed. Together, our findings indicate that pest management systems relying on mint as companion plants might be commercially useful for reducing herbivore damage in crops.