Due to its tropical origin and adaptation, rice is significantly impacted by cold stress, and consequently sustains large losses in growth and productivity. Currently, rice is the second most consumed cereal in the world and production losses caused by extreme temperature events in the context of "major climatic changes" can have major impacts on the world economy. We report here an analysis of rice genotypes in response to low-temperature stress, studied through physiological gas-exchange parameters, biochemical changes in photosynthetic pigments and antioxidants, and at the level of gene and protein expression, towards an understanding and identification of multiple low-temperature tolerance mechanisms. The first effects of cold stress were observed on photosynthesis among all genotypes. However, the tropical
japonica
genotypes Secano do Brazil and Cypress had a greater reduction in gas exchange parameters like photosynthesis and water use efficiency in comparison to the temperate
japonica
Nipponbare and M202 genotypes. The analysis of biochemical profiles showed that despite the impacts of low temperature on tolerant plants, they quickly adjusted to maintain their cellular homeostasis by an accumulation of antioxidants and osmolytes like phenolic compounds and proline. The cold tolerant and sensitive genotypes showed a clear difference in gene expression at the transcript level for
OsGH3-2
,
OsSRO1a
,
OsZFP245
, and
OsTPP1
, as well as for expression at the protein level for LRR-RLKs, bHLH, GLYI, and LTP1 proteins. This study exemplifies the cold tolerant features of the temperate
japonica
Nipponbare and M202 genotypes, as observed through the analysis of physiological and biochemical responses and the associated changes in gene and protein expression patterns. The genes and proteins showing differential expression response are notable candidates towards understanding the biological pathways affected in rice and for engineering cold tolerance, to generate cultivars capable of maintaining growth, development, and reproduction under cold stress. We also propose that the mechanisms of action of the genes analyzed are associated with the tolerance response.
Analysis of carcinogen-DNA adducts has been regarded as a useful means of assessing human exposure to chemical carcinogens. We have established a method for quantitation of 4-aminobiphenyl (4-ABP)-DNA adducts by alkaline hydrolysis and gas chromatography with negative ion chemical ionization mass spectrometry (GC-NICI-MS). Aliquots of DNA (typically 100 pg/ml) were spiked with an internal standard,d,-4-ABP, and were hydrolyzed in 0.05 N NaOH at 130°C overnight. The liberated 4-ABP was extracted with hexane and derivatized using pentafluoropropionic anhydride in trimethylamine for 30 min at room temperature prior to GC-NICI-MS. With in vitro [3HIN-hydroxy-4-ABP modified DNA standards, we observed 59 ± 7% (n = 9) recovery of the 4-ABP and a linear correlation between hydrolyzed 4-ABP and the adduct levels ranging from about 1 in 1 08to 1 in 104 nucleotides (r = 0.999, n = 9). The method was further validated by comparison of the results with that obtained by the 3 P-postlabeling method. There was excellent agreement (r = 0.994, p<0.001) between the two methods for quantitation of the adduct in eight samples of Salmonella typhimurium DNA treated with 4-ABP and rat liver S9, although the 32P-postlabeling method gave slightly higher values. The DNA adducts in 11 human lung and 8 urinary bladder mucosa specimens were then determined by our GC-NICI-MS method. The adduct levels were found to be <0.32 to 49.5 adducts per 108 nucleotides in the lungs and <0.32 to 3.94 adducts per 108 nucleotides in the bladder samples. Our results indicate that the alkaline hydrolysis/GC-NICI-MS method is sensitive, structure-selective, and accurate, and will be useful for molecular dosimetry of human exposure to this carcinogen.
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