Gel-free/label-free proteomic analysis of developing rice grains under heat stress.

High temperature markedly reduces the yields and quality of rice grains. To identify the mechanisms underlying heat stress-induced responses in rice grains, proteomic technique was used. Developing Khao Dawk Mali 105 rice grains at the milky, dough, and mature stages were treated at 40 °C for 3 days. Aromatic compounds were decreased in rice grains under heat stress. The protein abundance involved in glycolysis and tricarboxylic acid cycle, including glyceraldehyde 3-phosphate dehydrogenase and citrate synthase, was changed in milky and dough grains after heat treatment; however, none changes in mature grains. The abundance involved in amino acid metabolism was increased in dough grains, but decreased in milky grains. In addition, the abundance involved in starch and sucrose metabolism, such as starch synthase, ADP-glucose pyrophosphorylase, granule-bound starch synthase, and alpha amylase, was decreased in milky grains, but increased in dough grains. A number of redox homeostasis-related proteins, such as ascorbate peroxidase and peroxiredoxin, were increased in developing rice grains treated with heat stress. These results suggest that in response to heat stress, the abundance of numerous proteins involved in redox homeostasis and carbohydrate biosynthetic pathways may play a major role in the development of KDML105 rice grains. BIOLOGICAL SIGNIFICANCE: Yield of Khao Dawk Mali 105 rice, which is an economical aromatic rice, was disrupted by environmental stress. Rice grains developed under heat stress caused loss of aroma compound. To identify the mechanism of heat response in rice grain, gel-free/label-free proteomic technique was used. The abundance of proteins involved in glycolysis and tricarboxylic acid cycle was disrupted by heat stress. High temperature limited starch biosynthesis; however, it enhanced sugar biosynthesis in developing rice grains. Redox homeostasis related proteins were disrupted by heat stress. These results suggest that proteins involved in redox homeostasis and carbohydrate pathway might play a major role in developing grains in Khao Dawk Mali 105 rice under heat stress.

A modified ferrous oxidation-xylenol orange assay for lipoxygenase activity in rice grains.

Ferrous oxidation-xylenol orange assay reagent was reformulated by using spectral analysis of ferric-xylenol orange complex to detect low concentrations of lipoxygenase rice grain products. Reducing the levels of ferrous sulphate and xylenol orange in the FOX reagent enabled the detection of low concentrations of hydroperoxy fatty acid derived from lipoxygenase activity in the range of 0.1-1.5 µM. Protein, substrate and time courses of the modified FOX assay were studied to determine lipoxygenase activity in rice grain. The assay was also applicable as a high throughput technique for comparisons of lipoxygenase activity from various rice varieties. This has important implications for rapid screening for low-lipoxygenase containing rice cultivars in rice breeding program and grain quality during storage.