Mechanism Study On Mutation Of OsNaPRT1 In The NAD Salvage Pathway Leading To Withered Leaf Tips In Rice (Oryza Sativa L.)

Rice is the model plant for the research of monocotyledon, and the related agronomic traits of yield are the most important research objectives. Meanwhile, rice is constituted with the “source” and “sink”. Rice leaf is the crucial source of metabolism for photosynthesis and the production of organic energy substrates, and the development of rice leaf is correlated closely with the grain yield. Premature leaf senescence affects plant growth and yield in rice. Nicotinamide adenine dinucleotide(NAD) plays critical roles in cellular redox reactions and remains at a sufficient level in the cell to prevent cell death. Although numerous factors affecting leaf senescence have been identified, few involving NAD biosynthetic pathways have been described for plants. Here, we report the cloning and characterization of Leaf Tip Senescence 1(LTS1) in rice(Oryza sativa), a recessive mutation in the gene encoding O. sativa nicotinate phosphoribosyltransferase(OsNaPRT1) in the NAD salvage pathway. A point mutation in OsNaPRT1 leads to dwarfism and the withered leaf tip phenotypes, and the lts1 mutant displays early leaf senescence compared to the wild type. For these phenotypes, we confirmed the function of OsNaPRT1 by genetic, cytological, biochemical, molecular biological and transcriptome analyses. The main results were summarized as follow:1. Leaf lesions of lts1 appeared at 4 weeks post-germination under natural field conditions. As the plant grew, the spotted area withered at the tillering stage, and the leaf area of senescence shrunk progressively over time. Transmission electron microscopy analysis revealed that numerous osmiophilic globules in mesophyll cells of lts1.2. Western-blot assay revealed the expression of OsNaPRT1 declined clearly in lts1 and the activity of OsNaPRT1 in lts1 was lower than that in wild type.3. Real-time PCR experiments revealed that OsNaPRT1 is widely expressed in different rice tissues tested. The highest level of OsNaPRT1 transcript was observed in leaves, followed by leaf sheaths and panicles; low levels of OsNaPRT1 were detected in stems and roots. OsNaPRT1 expression in fully expanded leaves was examined at different developmental stages, and OsNaPRT1 transcripts almost remained at the same level in lts1 compared with the wild type regardless of the developmental stages. The OsNaPRT1 protein is localized to the nucleus, cytoplasm, and cell membrane.4. Leaf tissue of lts1 exhibited significant DNA fragmentation and H2O2 accumulation, along with up-regulation of genes associated with senescence.5. Leaf nicotinate and nicotinamide contents are elevated in lts1, while NAD levels are reduced. The redundant nicotinate can negatively regulate the height of plants but can’t make a clear effect in senescence of leaf in rice. The accumulation of nicotinamide inhibits the expression of OsSRTs and leads to increased acetylation of histone H3K9, causing the wither leaf tips indirectly.6. Down-regulation of OsSRTs induced histone H3K9 acetylation of senescence-related genes suggesting that deficiency in the NAD salvage pathway can trigger premature leaf senescence due to transcriptional activation of senescence-related genes.