Activation of Strigolactone Biosynthesis by the DWARF14-LIKE/KARRIKIN-INSENSITIVE2 Pathway in Mycorrhizal Angiosperms, but Not in Arabidopsis, a Non-mycorrhizal Plant.

Strigolactones (SLs) are a class of plant hormones that regulate many aspects of plant growth and development. SLs also improve symbiosis with arbuscular mycorrhizal fungi (AMF) in the rhizosphere. Recent studies have shown that the DWARF14-LIKE (D14L)/KARRIKIN-INSENSITIVE2 (KAI2) family, paralogs of the SL receptor D14, are required for AMF colonization in several flowering plants, including rice. In this study, we found that (-)-GR5, a 2'S-configured enantiomer of a synthetic SL analog (+)-GR5, significantly activated SL biosynthesis in rice roots via D14L. This result is consistent with a recent report, showing that the D14L pathway positively regulates SL biosynthesis in rice. In fact, the SL levels tended to be lower in the roots of the d14l mutant under both inorganic nutrient-deficient and -sufficient conditions. We also show that the increase in SL levels by (-)-GR5 was observed in other mycorrhizal plant species. In contrast, the KAI2 pathway did not upregulate the SL level and the expression of SL biosynthetic genes in Arabidopsis, a non-mycorrhizal plant. We also examined whether the KAI2 pathway enhances SL biosynthesis in the liverwort Marchantia paleacea, where SL functions as a rhizosphere signaling molecule for AMF. However, the SL level and SL biosynthetic genes were not positively regulated by the KAI2 pathway. These results imply that the activation of SL biosynthesis by the D14L/KAI2 pathway has been evolutionarily acquired after the divergence of bryophytes to efficiently promote symbiosis with AMF, although we cannot exclude the possibility that liverworts have specifically lost this regulatory system.

Development of rat duodenal monolayer model with effective barrier function from rat organoids for ADME assay.

The in-depth analysis of the ADME profiles of drug candidates using in vitro models is essential for drug development since a drug's exposure in humans depends on its ADME properties. In contrast to efforts in developing human in vitro absorption models, only a limited number of studies have explored models using rats, the most frequently used species in in vivo DMPK studies. In this study, we developed a monolayer model with an effective barrier function for ADME assays using rat duodenal organoids as a cell source. At first, we developed rat duodenal organoids according to a previous report, but they were not able to generate a confluent monolayer. Therefore, we modified organoid culture protocols and developed cyst-enriched organoids; these strongly promoted the formation of a confluent monolayer. Furthermore, adding valproic acid to the culture accelerated the differentiation of the monolayer, which possessed an effective barrier function and apicobasal cell polarity. Drug transporter P-gp function as well as CYP3A activity and nuclear receptor function were confirmed in the model. We expect our novel monolayer model to be a useful tool for elucidating drug absorption processes in detail, enabling the development of highly absorbable drugs.

Carlactone is converted to carlactonoic acid by MAX1 in Arabidopsis and its methyl ester can directly interact with AtD14 in vitro.

Strigolactones (SLs) stimulate seed germination of root parasitic plants and induce hyphal branching of arbuscular mycorrhizal fungi in the rhizosphere. In addition, they have been classified as a new group of plant hormones essential for shoot branching inhibition. It has been demonstrated thus far that SLs are derived from carotenoid via a biosynthetic precursor carlactone (CL), which is produced by sequential reactions of DWARF27 (D27) enzyme and two carotenoid cleavage dioxygenases CCD7 and CCD8. We previously found an extreme accumulation of CL in the more axillary growth1 (max1) mutant of Arabidopsis, which exhibits increased lateral inflorescences due to SL deficiency, indicating that CL is a probable substrate for MAX1 (CYP711A1), a cytochrome P450 monooxygenase. To elucidate the enzymatic function of MAX1 in SL biosynthesis, we incubated CL with a recombinant MAX1 protein expressed in yeast microsomes. MAX1 catalyzed consecutive oxidations at C-19 of CL to convert the C-19 methyl group into carboxylic acid, 9-desmethyl-9-carboxy-CL [designated as carlactonoic acid (CLA)]. We also identified endogenous CLA and its methyl ester [methyl carlactonoate (MeCLA)] in Arabidopsis plants using LC-MS/MS. Although an exogenous application of either CLA or MeCLA suppressed the growth of lateral inflorescences of the max1 mutant, MeCLA, but not CLA, interacted with Arabidopsis thaliana DWARF14 (AtD14) protein, a putative SL receptor, as shown by differential scanning fluorimetry and hydrolysis activity tests. These results indicate that not only known SLs but also MeCLA are biologically active in inhibiting shoot branching in Arabidopsis.