Abstract
Threonyl-tRNA synthetase (ThrRS), one of the aminoacyl-tRNA synthetases (AARSs), plays a crucial role in protein synthesis. However, the AARS functions on rice chloroplast development and growth were not fully appraised. In this study, a thermo-sensitive virescent mutant tsv2, which showed albino phenotype and lethal after the 4-leaf stage at 20°C but recovered to normal when the temperatures rose, was identified and characterized. Map-based cloning and complementation tests showed that TSV2 encoded a chloroplast-located ThrRS protein in rice. The Lys-to-Arg mutation in the anticodon-binding domain hampered chloroplast development under cold stress, while the loss of function of the ThrRS core domain in TSV2 fatally led to seedling death regardless of growing temperatures. In addition, TSV2 had a specific expression in early leaves. Its disruption obviously resulted in the downregulation of certain genes associated with chlorophyll biosynthesis, photosynthesis, and chloroplast development at cold conditions. Our observations revealed that rice nuclear-encoded TSV2 plays an important role in chloroplast development at the early leaf stage under cold stress.
Highlights
MethodsPlant Materials and Growth ConditionsThe thermo-sensitive virescent mutant tsv[2] was discovered in rice mutant pool from Jiahua 1 (wild type, WT, japonica variety) treated with Cobalt-60 gamma rays
The chloroplast is a vital photosynthetic organelle for plant growth and development
We identified and characterized rice tsv[2] mutants with imperfect chloroplasts and albino lethal phenotype at low temperatures, resulting from the abnormal expression of genes associated with chlorophyll biosynthesis, photosynthesis, and chloroplast development
Summary
Plant Materials and Growth ConditionsThe thermo-sensitive virescent mutant tsv[2] was discovered in rice mutant pool from Jiahua 1 (wild type, WT, japonica variety) treated with Cobalt-60 gamma rays. WT and tsv[2] plants were cultured in incubators under controlled 12 h of light and 12 h of dark at a constant temperature of 20 °C, 24 °C, 28 °C and 32 °C, respectively, for phenotypic characterization, pigment analysis, and DNA and RNA extraction. 200 mg fresh leaves were taken from the 3-leaf-stage seedlings cultured at 20°C, 24°C, 28°C and 32°C, respectively and incubated with 5 mL of extraction buffer (ethanol: acetone: water = 5:4:1) at 4 °C in the dark for 18 h. Using spectrophotometer (Beckman Coulter, Danvers, MA, USA), chlorophyll a, b and Car contents were measured according to the modified methods of Arnon (1949) and Wellburn (1994) This experiment were performed out with three biological replicates
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