REGULATING CYTOKININ METABOLISM DURING INFLORESCENCE MERISTEM DEVELOPMENT TO PROMOTE HIGHER RICE PRODUCTIVITY THROUGH CHANGES ON PLANT ARCHITECTURE

The growing world population and the effects of climate change are threatening agricultural production and, consequently, human food security. Current agricultural systems require crops with enhanced yields but with minimal impact on water resources or land used for production. Grain yield in rice (Oryza sativa) is a complex trait determined primarily by three components: the number of panicles, the number of grains per panicle, and grain weight. The cytokinin group of phytohormones positively regulates plant yield through the simultaneous regulation of source capacity (leaf senescence) and sink strength (grain number and size). It is hypothesized that directly modulating endogenous cytokinin content or sensitivity —precisely timed and targeted to the appropriate tissue through genetic engineering— could enhance yield attributes. Consequently, manipulating the phytohormone cytokinin offers a promising strategy to achieve these objectives, given its pivotal role in regulating plant growth and development. In plants, cytokinins are degraded by cytokinin oxidase/dehydrogenase (CKX) enzymes, which catalyze their irreversible oxidative degradation, thereby reducing their levels. In rice, a staple crop feeding billions of people worldwide, previous studies have demonstrated that preventing the degradation of cytokinins by inhibiting or reducing the expression of certain OsCKX genes can improve growth and yield. However, their role in stress tolerance has been neglected. Furthermore, generating rice transgenic lines with multiple osckx gene mutations has shown slight increases in yield. Using the CRISPR/Cas9 genome editing system and a backcross approach, we generated transgenic lines osckx3, osckx5, osckx9, osckx3 osckx5, osckx3 osckx9, osckx5 osckx9, and osckx3 osckx5 osckx9, and performed a detailed phenotypic analysis. Here, we demonstrated that the double mutant osckx5 osckx9 and the triple mutant osckx3 osckx5 osckx9 lines showed improved total grain yield compared to the wild type, primarily due to increased panicle production, a higher number of secondary branches in the main panicle, and increased seed size. A transcriptomic analysis of the inflorescence meristem at early developmental stages of the triple mutant osckx3 osckx5 osckx9 revealed a series of uncharacterized differentially expressed genes whose expression patterns might be influenced by cytokinin levels. These genes represent promising candidates for involvement in panicle architecture determination and are potential targets for rice molecular breeding programs. Drought stress is one of the most damaging factors in agriculture due to the unpredictability of its occurrence, intensity, timing, and duration, as well as its interaction with other abiotic stresses. Since CKX genes have also been shown to be involved in plant tolerance to various abiotic stresses, we analyzed the germination rates of our different osckx mutants under water-deficit conditions generated using polyethylene glycol (PEG). Interestingly, only osckx3 osckx5 osckx9 was able to maintain germination rates (+50%) under increasing PEG concentrations. Additionally, an analysis of drought-responsive gene expression revealed that OsWRKY50 exhibited enhanced expression in osckx3 osckx5 osckx9 seedlings grown under both control and drought stress conditions. Concurrently, the expression of OsP5CS1 was significantly increased in the osckx3 osckx5 osckx9 triple mutant under water-deficit conditions. OsWRKY50, a transcription factor, is involved in the regulation of the peroxidase OsPRX114, which manages the excessive production of reactive oxygen species (ROS) typically generated during biotic or abiotic stresses. Similarly, increased expression of OsP5CS1, a proline biosynthesis gene, may lead to greater proline accumulation, a molecule that aids in reducing the osmotic potential of cells during drought stress periods. Further research is needed to determine if the higher germination rates observed under water-deficit conditions in the osckx3 osckx5 osckx9 mutant are due to more efficient ROS scavenging and/or an increased capacity for proline synthesis and accumulation. Based on our data, the osckx3 osckx5 osckx9 triple mutant represents an ideal gene combination for exploration in rice molecular breeding programs aimed at increasing yield, even under suboptimal water conditions. However, more research is required to elucidate how the manipulation of cytokinin catabolism influences various agronomically important traits.