Luciferases have been widely utilized as sensitive reporters to monitor gene expression and protein-protein interactions. Compared to firefly luciferase (Fluc), a recently developed luciferase, Nanoluciferase (NanoLuc or Nluc), has several superior properties such as a smaller size and stronger luminescence activity. We compared the reporter properties of Nluc and Fluc in rice (Oryza sativa). In both plant-based two-hybrid and split luc complementation (SLC) assays, Nluc activity was detected with higher sensitivity and specificity than that with Fluc. To apply Nluc to research involving the photoperiodic regulation of flowering, we made a knock-in rice plant in which the Nluc coding region was inserted in-frame with the OsMADS15 gene, a target of the rice florigen Hd3a. Strong Nluc activity in response to Hd3a, and in response to change in day length, was detected in rice protoplasts and in a single shoot apical meristem, respectively. Our results indicate that Nluc assay systems will be powerful tools to monitor gene expression and protein-protein interaction in plant research.

The three-dimensional (3D) arrangement of cells in tissues provides an anatomical basis for analyzing physiological and biochemical aspects of plant and animal cellular development and function. In this study, we established a protocol for tissue clearing and 3D imaging in rice. Our protocol is based on three improvements: clearing with iTOMEI (clearing solution suitable for plants), developing microscopic conditions in which the Z step is optimized for 3D reconstruction, and optimizing cell-wall staining. Our protocol successfully 3D imaged rice shoot apical meristems, florets, and root apical meristems at cellular resolution throughout whole tissues. Using fluorescent reporters of auxin signaling in rice root tips, we also revealed the 3D distribution of auxin signaling events that are activated in the columella, quiescent center, and multiple rows of cells in the stele of the root apical meristem. Examination of cells with higher levels of auxin signaling revealed that only the central row of cells was connected to the quiescent center. Our method provides opportunities to observe the 3D arrangement of cells in rice tissues.

Improving crop yield potential through an enhanced response to rising atmospheric CO 2 levels is an effective strategy for sustainable crop production in the face of climate change. Large-sized panicles (containing many spikelets per panicle) have been a recent ideal plant architecture (IPA) for high-yield rice breeding. However, few breeding programs have proposed an IPA under the projected climate change. Here, we demonstrate through the cloning of the rice (Oryza sativa) quantitative trait locus for MORE PANICLES 3 (MP3) that the improvement in panicle number increases grain yield at elevated atmospheric CO 2 levels. MP3 is a natural allele of OsTB1/FC1, previously reported as a negative regulator of tiller bud outgrowth. The temperate japonica allele advanced the developmental process in axillary buds, moderately promoted tillering, and increased the panicle number without negative effects on the panicle size or culm thickness in a highyielding indica cultivar with large-sized panicles. The MP3 allele, containing three exonic polymorphisms, was observed in most accessions in the temperate japonica subgroups but was rarely observed in the indica subgroup. No selective sweep at MP3 in either the temperate japonica or indica subgroups suggested that MP3 has not been involved and utilized in artificial selection during domestication or breeding. A free-air CO 2 enrichment experiment revealed a clear increase of grain yield associated with the temperate japonica allele at elevated atmospheric CO 2 levels. Our findings show that the moderately increased panicle number combined with large-sized panicles using MP3 could be a novel IPA and contribute to an increase in rice production under climate change with rising atmospheric CO 2 levels. Keywords: rice (Oryza sativa), rising CO 2 , ideal plant architecture, tillering, yield potential.