Studies On The Molecular Basis Of OsUGP8 Involved In The Regulation Of Lesion Mimic In Rice (Oryza Sativa L.)

Plant lesion mimic mutants (LMM), which spontaneously form necrotic lesions that resemble disease symptoms in the absence of damage, stress conditions or other pathogen attack, are called lesion resembling disease. Partial cell necrosis in these mutants is a kind of programmed cell death (PCD). Here we obtained two rice lesion mimic mutants with spreading cell necrosis through all growth stages, named ugp8-1 and ugp8-2. Using map-based cloning, we isolated OsUGP8 which controlled the development of necrosis in ugp8. The molecular basis of OsUGP8 invold in the regulation of lesions was investigated. The results are as follows.1. Both ugp8-1 and ugp8-2 formed brown and small necrotic spots at the seedling stage.Then the necrosis spread almost all of leaves and finally resulted in the senescnece of leaves. Compared to the wild type (WT), pollen viability was reduced and many agronomic traits were inferior in ugp8-2 mutant. We found that the initiation of lesions was induced by light and cell death occurred accompanied with accumulation of reactive oxygen species (ROS) and callose in the mesophyll cells. The lower chlorophyll content, soluble protein content and peroxidase (POD) activity, the higher malondialdehyde (MDA) content were detected in ugp8-2 mutant than WT. Moreover, cell membranes were destroyed, showing not full vacuolewa, loose and irregular stroma lamella in the mutant.2. The mutant trait of ugp8-1 and ugp8-2 was inherited as a monogenic recessive nuclear gene. Then we fine-mapped the mutated gene to 682 kb region on the short arm of chromosome 8 with 1490 "Dular×ugp8-1" and "Dular×ugp8-2" F2 recessive individuals, including 31 open reading frames (ORFs). Sequence analysis revealed that there were allelic mutations in Os08g0206900, named OsUGP8, which encoded UDP-N-acetylghicosamine pyrophosphorylase (UAGPase). In ugp8-1 mutant, G was replaced with A in the 312th nucleotide downstream of ATG, which caused a change of Trp (W) to a termination codon. In ugp8-2 mutant, G was replaced with A in the 839th nucleotide downstream of ATG, leading to a change of Gly (G) to Glu (E). Genomic complementary test confirmed that the overexpression of OsUGP8 could functionally rescued lesion mimic phenotype of ugp8-2 mutant.3. In the transcriptional level, OsUGP8 was higher expressed in the leaves.It was induced by MV, H2O2 and ACC and inhibited by AVG. Subcellular localization experiment showed that OsUGP8 was present in the cytoplasm and nucleus. The one-way degradation of UDPG in vitro was specifically catalyzed by OsUGP8, producing Glc-1-P and UTP. However, OsUGP4, the homologous protein of OsUGP8, could catalyze the reversible synthesis of UDPG, too. We found that point mutation caused the significant decrease of OsUGP8 activity in vitro and in vivo, accompanied the high accumulation of UDPG in ugp8 mutants. In addition, there was less content of UDPG in OsUGP8 overexpressional transgenic lines than that in WT. These results indicated that OsUGP8 was an important regulatory factor of UDPG metabolism. Moreover, exogenous UDPG could accelerate the formation of necrosis, indicating that UDPG was responsible for the necrotic spots.4. UDPG could induce the expression of OsACSs and OsACOs, which encoded key enzymes involved in ethylene biosynthesis. In addition, ethylene accelerated the development of the lesions obviously. However, ethylene synthesis inhibitors such as CoCl2 and AVG could delay the formation of necrotic spots effectively, which indicated ethylene might play an important role in regulating cell death and the formation of necrosis in ugp8 mutants. Moreover, by transcriptome sequencing we found that the expression level of genes involved in jasmonic acid metabolism and salicylic acid metabolism upregulated in ugp8-2 mutant, which might also be responsible for the regulation of cell death process.