Yumiko Iwahashi

Effect of heat stress on tomato fruit protein expression

We delayed the ripening of tomato fruit for several days (average 5 days) by a 1‐day heat treatment at 37—42°C. We analyzed the tomato fruit pericarp proteins, which were altered by the heat stress, using two‐dimensional electrophoresis. Heat stress caused about 23.7% of the proteins in the pericarp to disappear and about 1.1% of new proteins to appear. We determined their apparent molecular mass, isoelectric point, and N‐terminal amino acid sequence. Identified proteins included antioxidant enzymes, heat shock proteins, cell‐wall‐related proteins, etc..

Gene expression profiles of yeast Saccharomyces cerevisiae sod1 caused by patulin toxicity and evaluation of recovery potential of ascorbic acid.

Patulin (PAT) is a fungal secondary metabolite and exhibits various toxicities including DNA damage and oxidative stress. These toxicities are eased by ascorbic acid (AsA). Although a number of studies regarding the mitigating effect of AsA against PAT toxicity have been reported, a comprehensive study about gene expressions is currently underway. Here, we carried out a detailed evaluation of PAT toxicity by co-incubation with AsA using the superoxide dismutase (SOD) mutant. DNA microarray results extracted the alterations in iron transporter and Fe/S cluster assembly genes; some of the genes that constitute the cellular iron transporter systems remained dysfunctional even in the presence of AsA. Meanwhile, AsA treatment reduced the alterations of G1/S phase cell cycle regulation genes. These results suggest that oxidative stress-derived DNA damage still exists, although AsA treatment effectively reduces PAT toxicity. This implies that a combined condition is required for complete blockade of PAT toxicity.

The study of heat stress in tomato fruits by NMR microimaging

The ripening of the tomato fruit was delayed for several days (average 5 days) by a 1-day heat treatment at 42 degrees C. Ethylene production increased during the first 3 h, but, after 6 h inhibition was almost total in tomato fruit incubated at 42 degrees C. However, recovery of ethylene production was rapid if fruits were returned to a temperature of 25 degrees C after heating. In NMR microimaging, three imaging pulse sequences with different repetition and echo times at 42 degrees C were used to obtain the proton density (TR = 6000 ms, TE = 15 ms), the T1 weighted image (TR = 1000 ms, TE = 15 ms) and the T2-weighted image (TR = 6000 ms, TE = 120 ms). After 12 h heating, the water in locular tissues began to show shorter T1 and T2 values. Though the tomatos were returned to 25 degrees C and preserved one more day, the water having a shorter T2 value in locular tissues, did not change. These results show that tomato fruit do not fully recover from heating even after one day, although ethylene production is recovered almost immediately. For this reason, we suggest that some denaturation event inside the tomato, which goes on after the end of heating, is the cause of the delay in tomato ripening.

Regulation of metabolic products and gene expression in Fusarium asiaticum by agmatine addition

The metabolic products resulting from the cultivation of F. asiaticum in agmatine were identified using capillary electrophoresis–time of flight mass spectrometry. Glyoxylic acid was detected from fungal cultures grown in agmatine, while it was absent in control cells. The abundance of other metabolic products of the glycolytic pathway also increased because of agmatine; however, there was no increase in the amounts of pyruvic acid or metabolites from the tricarboxylic acid cycle. Moreover, gene expression levels within Fusarium asiaticum exposed to agmatine were analyzed by DNA microarray. Changes in gene expression levels directed the changes in metabolic products. Our results suggest that acetyl coenzyme A, which is a starting substrate for the biosynthesis of deoxynivalenol (DON), was simultaneously produced by activated β-oxidation. Furthermore, the content of 4-aminobutyrate (GABA) was increased in the agmatine addition culture medium. GABA can be synthesized from agmatine through putrescine and might influence the regulation of DON-related genes.

Addition of Carbon to the Culture Medium Improves the Detection Efficiency of Aflatoxin Synthetic Fungi

Aflatoxin (AF) is a harmful secondary metabolite that is synthesized by the Aspergillus species. Although AF detection techniques have been developed, techniques for detection of AF synthetic fungi are still required. Techniques such as plate culture methods are continually being modified for this purpose. However, plate culture methods require refinement because they suffer from several issues. In this study, activated charcoal powder (carbon) was added to a culture medium containing cyclodextrin (CD) to enhance the contrast of fluorescence and improve the detection efficiency for AF synthetic fungi. Two culture media, potato dextrose agar and yeast extract sucrose agar, were investigated using both plate and liquid cultures. The final concentrations of CD and carbon in the media were 3 mg/mL and 0.3 mg/mL, respectively. Addition of carbon improved the visibility of fluorescence by attenuating approximately 30% of light scattering. Several fungi that could not be detected with only CD in the medium were detected with carbon addition. The carbon also facilitated fungal growth in the potato dextrose liquid medium. The results suggest that addition of carbon to media can enhance the observation of AF-derived fluorescence.

RNA Preparation of Saccharomyces cerevisiae Using the Digestion Method may Give Misleading Results

Zymolyase (lyticase) is used for cell wall digestion in yeast experiments and is needed for incubation processes under moderate experimental conditions. This has been thought to cause unfavorable effects, and many researchers are aware that the enzyme method is unsuitable for RNA preparation following several reports of stress responses to the enzyme process. However, RNA preparation with enzyme digestion continues to be used. This may be because there have been insufficient data directly comparing RNA preparation conditions with previous studies. We investigated the influence of enzyme processes in RNA preparation using a DNA microarray, and compared superoxide dismutase (SOD) activities with a non-treated control and the results of previous research. Gene expressions were commonly changed by enzyme processes, and SOD activities increased only during short-term incubation. Meanwhile, both SOD gene expressions and SOD activity during RNA preparation indicated different results than gained under conditions of long-term incubation. These results suggest that zymolyase treatment surely influences gene expressions and enzyme activity, although the effect assumed by previous studies is not necessarily in agreement with that of RNA preparation.