Accessing Multi-Tiered and Cell-Specific Regulation of Responses to Abiotic Stress in Crops

Abiotic stresses events reduce the maximum crop yield of agricultural systems worldwide. The identification of loci that improve yield maintenance under flooding in rice, provides a key example of the use of natural genetic diversity in crop improvement. The SUBMERGENCE1A (SUB1A) gene is induced by submergence in shoot meristematic regions and confers tolerance to complete submergence by limiting growth. We are interested in how key stress-tolerance genes such as SUB1A and stress responses such as flooding affect gene regulation at the cell-type specific levels. In the root system, specific cell types play key roles under flooding. Cortical cells can differentiate in lysigenous aerenchyma to provide large lacunae that serve as gas conduits from aerial tissue to the root. Our goal is to assess different layers of gene regulation of these specific cell-types in response to different abiotic stresses. To achieve this, we have established non-invasive technologies that allow access to chromatin, nuclear mRNAs, and ribosome-loaded mRNAs of target cell-types. The Isolation of Nuclei TAgged in specific Cell Types was transferred to rice to affinity purify nuclei. We used the WPP domain from AtRANGAP1 or OsRANGAP1 to target GFP-biotin ligase recognition peptide fusion to the nuclear envelope. Translating Ribosome Affinity Purification was established to immunopurify ribosomes using a His-FLAG-GFP-tagged version of a rice ribosomal protein L18. As a proof-of-concept we employed both technologies with near-constitutive promoters to drive expression of the fusion proteins. We have characterized a number of root cell-specific and meristematic promoters for our analysis of abiotic stress responses.