Strboh A homologue of NADPH oxidase regulates wound-induced oxidative burst and facilitates wound-healing in potato tubers.

During 30-months of storage at 4 degrees C, potato (Solanum tuberosum L.) tubers progressively lose the ability to produce superoxide in response to wounding, resist microbial infection, and develop a suberized wound periderm. Using differentially aged tubers, we demonstrate that Strboh A is responsible for the wound-induced oxidative burst in potato and aging attenuates its expression. In vivo superoxide production and NADPH oxidase (NOX) activity from 1-month-old tubers increased to a maximum 18-24 h after wounding and then decreased to barely detectable levels by 72 h. Wounding also induced a 68% increase in microsomal protein within 18 h. These wound-induced responses were lost over a 25- to 30-month storage period. Superoxide production and NOX activity were inhibited by diphenylene iodonium chloride, a specific inhibitor of NOX, which in turn effectively inhibited wound-healing and increased susceptibility to microbial infection and decay in 1-month-old tubers. Wound-induced superoxide production was also inhibited by EGTA-mediated destabilization of membranes. The ability to restore superoxide production to EGTA-treated tissue with Ca(+2) declined with advancing tuber age, likely a consequence of age-related changes in membrane architecture. Of the five homologues of NOX (Strboh A-D and F), wounding induced the expression of Strboh A in 6-month-old tubers but this response was absent in tubers stored for 25-30 months. Strboh A thus mediates the initial burst of superoxide in response to wounding of potato tubers; loss of its expression increases the susceptibility to microbial infection and contributes to the age-induced loss of wound-healing ability.

Extraction of RNA from fresh, frozen, and lyophilized tuber and root tissues.

A method for isolating transcriptionally competent RNA from fresh, frozen, and lyophilized plant storage tissues containing high levels of starch and phenolics is described. The protocol avoids the use of guanidium salts, which often lead to the formation of a viscous gel during extraction of high starch-containing tissues, and instead uses a borate-Tris buffer in combination with high concentrations of NaCl, Na2SO3, and sodium dodecyl sulfate in the extraction medium. RNA was extracted from fresh, frozen, and lyophilized tissues of potato tubers, storage roots of sweet potato, radish, and turnip, and rhizomes of ginger. The yield of RNA from potato tubers averaged 281 microg g fresh weight(-1) and 1584 microg g dry weight(-1) from frozen and lyophilized samples, respectively. A260/A230 ratios of potato RNA extracts were 2.2 or greater, indicating minimal contamination by polyphenols and carbohydrates. Similarly, A260/A280 ratios exceeded 1.9, demonstrating minimal contamination of the RNA by tuber protein. While A260/A280 ratios of extracts from the other plant species were somewhat lower than those for potato (average = 1.56 and 1.80 for fresh and lyophilized samples, respectively), A260/A230 ratios averaged more than 2.0, and the RNA extracted from fresh and lyophilized samples of all species was intact, as demonstrated by denaturing agarose-formaldehyde gel electrophoresis. The protocol yielded RNA suitable for downstream molecular applications involving reverse transcription-polymerase chain reaction from all five species. Transcriptionally competent RNA was also recovered from lyophilized potato tuber tissue stored for 6 years (ambient temperature) by a simple modification to the protocol involving extraction in cold acetone. Lyophilization can thus be used to preserve RNA in high starch- and phenolic-containing plant tissues for studies on gene expression.

Catalase activity is necessary for heat-shock recovery in Aspergillus nidulans germlings.

To understand the molecular mechanisms induced by stress that contribute to the development of tolerance in eukaryotic cells, the filamentous fungus Aspergillus nidulans has been chosen as a model system. Here, the response of A. nidulans germlings to heat shock is reported. The heat treatment dramatically increased the concentration of trehalose and induced the accumulation of mannitol and mRNA from the catalase gene catA. Both mannitol and catalase function to protect cells from different reactive oxygen species. Treatment with hydrogen peroxide increased A. nidulans germling viability after heat shock whilst mutants deficient in catalase were more sensitive to a 50 degrees C heat exposure. It is concluded that the defence against the lethal effects of heat exposure can be correlated with the activity of the defence system against oxidative stress.