Abstract
The Kelch-like ECH-associated protein 1 (KEAP1)-nuclear factor E2-related factor 2 (NRF2) module is a key component in the detoxification and antioxidant system in animals, which plays crucial roles in cell homeostasis and cytoprotection, and consequently in carcinogenesis and disease development. However, this system seems to have diverged throughout evolution across different organisms, and the question of whether a similar system exists in plants has thus far remained unresolved. In this study, a KEAP1 ortholog was identified in rice (Oryza sativa L., OsKEAP1) and its properties were characterized via in silico and laboratory studies. To reveal OsKEAP1’s function, two knockdown mutants, oskeap1-1 and oskeap1-2, were generated by targeted mutagenesis in the 5′ untranslated region (UTR) using the CRISPR-Cas9 system. In silico analysis showed that OsKEAP1 has a Kelch-repeat domain which is identical to those of animals and a plant-specific development and cell death (DCD) domain in place of the broad-complex, tramtrack, bric-a-brac (BTB) domain found in animals. Orthologs of OsKEAP1 are present across plant species and all have the DCD domain and the Kelch-repeat domain. OsKEAP1 was proven to be localized to both the cytoplasm and nucleus, in contrast to the exclusive cytoplasm localization of animal KEAP1. Single nucleotide insertions in the 5′ UTR significantly reduced the transcription level of OsKEAP1 in the oskeap1-1 and oskeap1-2 mutants. The oskeap1 mutations greatly impaired plant growth and development, resulting in significant declines in a majority of agronomic and yield-related traits, i.e., plant height, panicle length, grain number per plant, and seed-set rate. The downregulation of OsKEAP1 increased the levels of H2O2, malondialdehyde, and proline while significantly decreasing the expression of two catalase genes in seedlings grown under normal and salt-stressed conditions. The changes in the above phenotypes are either positively or negatively correlated with the degree of OsKEAP1 downregulation. Altogether, we identified a probable KEAP1 ortholog in rice, revealed its unique subcellular localization, and demonstrated its important functions in vegetative and reproductive growth via regulation of the antioxidant response in plants.
Highlights
Cells have developed a myriad of antioxidant mechanisms conferring defense against various redox stresses
Our results revealed that the downregulation of OsKEAP1 increased the level of H2 O2, malondialdehyde, and proline and altered the expression of genes involved in the antioxidant response
The results showed that the the OsKEAP1::green fluorescence protein (GFP) vector were observed under a fluorescence microscope
Summary
Cells have developed a myriad of antioxidant mechanisms conferring defense against various redox stresses. Through bioinformatics-based evolutional analyses, Gacesa R. et al [10] concluded that the KEAP1-NRF2 system exists in organisms from arthropods to mammals, but not in early Eukarya, fungi, Nematoda, and basal metazoa. The same group did identify fungal KEAP1 and NRF2 orthologs, they were distant and quite diverged from animal ones [11]. This discrepancy could be explained by the different parameters and methods used for the homolog search. Gacesa R et al [11] did not identify any KEAP1 and NRF2 orthologs in plants
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