HORTSCIENCE 25(8):952-953. 1990. into -80C methanol (mixture of solid CO2 and methanol); the acid was then extracted with 0.6 M HPO3 at 0C. After adding 2,4- Changes in Pyruvic Acid Content and dinitrophenylhydrazine to the extraction, the mixture was allowed to stand for 3 hr at room GPT Activity in Chilling-sensitive and temperature in darkness. Finally, the hydra- zone produced was dissolved in 2 ml of ab- Nonsensitive Crops solute methanol. To separate and quantify the pyruvic acid in the keto acids, a 10-µl Hironobu Tsuchida and Cheng Dan-Hong aliquot of the solution was directly subjected Faculty of Agriculture, Kobe University, Rokkodai-cho, Nadaku, Kobe, to high-performance liquid chromatography Japan (HPLC) . A Gaskuro Kogyo (Tokyo) liquid chro- Kazuko Inoue matograph (Model-570B) equipped with a sampling valve with a 20-µl loop (Rheodyne Consumer Economic Research Institute, 6-28, 1-cho, Nakatsu, Model 7120) was used. The chromato- Ooyodo-ku, Osaka, Japan graphic column (4.0 i-d. × 250 mm) was of stainless steel and packed with Unicil Q- Nobuyuki Kozukue 30 (particle diameter 5 µm, Gaskuro Kog- Department of Home Economics, Kenmei Junior College, 68-Honmachi, yo). The UV detector was set-at 254 nm. A Himeji City, Japan mobile phase [50 chloroform : 5 n-butanol : 5 acetic acid : 50 water (by volume)] was Susumu Mizuno pumped through the column at a flow rate of 1 ml·min-1. The chart speed was adjusted Faculty of Agriculture, Kobe University, Rokkodai-cho, Nadaku, Kobe, -1 to 5 mm·min . Analytical-grade sodium Japan pyruvate was purchased from Wako Pure Additional index words. chilling injury, glutamate-pyruvate transaminase, cucumber, Chemicals (Osaka, Japan). Chromatographic eggplant, carrots, Cucumis sativus, Daucus carota, Solanum melongena peak areas were calculated using a Shimazu Model-RIA electronic integrator (Shimazu Abstract. This study investigated the mechanism of alanine accumulation observed as Seisakusho, Kyoto, Japan). a common factor in chilling-sensitive crops [cucumber (Cucumis sativus L.) and eggplant The determination of GPT activity was (Solanum melongena L.)] during cold storage. Pyruvic acid in acids increased performed according to the method of Jen et rapidly during the storage of these crops at 1C. However, in sensitive crops at 20C or al. (4) or Romani (10); each 30-g sample of in a chilling-resistant crop [carrot (Daucus carota L.)] at 1C, the increase was not the edible portion was blended with 30 ml found. No significant changes in glutamate-pyruvate transaminase (GPT) activity closely of 0.1 MTris·HC1 (pH 7.4) at 4C. The slurry related to the biosynthesis of alanine were found in chilling-sensitive or chilling-resistant was centrifuged at 3000× g and stored at crops during storage at 1, 5, or 20C. We suggest that the accumulated alanine found -20C for 20 min, after which the super- in chilling-sensitive crops may be produced by GPT from accumulated pyruvic acid natant was saturated with ammonium sulfate during cold storage. and allowed to stand overnight at 0C. The precipitate was collected by centrifugation at Fruits and vegetables can roughly be di- 5.3 cm) were used as a chilling-resistant crop. 6000 x g and stored at - 20C until required. vided into those that are sensitive to low All of the crops were purchased in 1985 from The precipitate was dissolved with 5 ml of temperatures, causing injuries during storage the eastern market of Kobe. After purchase, TrisHCl buffer and dialyzed with 0.01 M below 10C, but above 0C, and those that are the crops were washed thoroughly and stored Tris-HCl buffer for 40 hr at 4C. The com- resistant. Assuming certain factors in com- immediately at 1C (80% to 83% RH), 5C ponents of the assay system were maintained mon with these two groups in metabolic (80% to 85% RH), or 20C (80% to 85% RH) at pH 7.4. The assay medium consisted of mechanisms, a series of studies was carried in darkness until used. 80 µmol Tris-HCl buffer (pH 7.4), 10 kg out. We noted that in fruits and vegetables Pyruvic acid was extracted by the method pyridoxal-5-phosphate, 0.3 µmol NADH, 10 highly sensitive to and markedly injured by of Ishetwood and Cruickshank (3). For each µmol alanine, and 25 kg lactic acid dehy- low temperatures, the accumulation of ala- sample, 10 g of the edible portion was placed drogenase (Wako Pure. Chemicals). To the nine is more noticeable (5). To investigate the cause of alanine accu- mulation in-chilling-sensitive crops, we at- tempted to determine the content of pyruvic acid and the changes in glutamate-pyruvate transaminase (GPT) that are closely related to the biosynthesis of alanine in biological samples (11). Mature cucumbers (‘Hokyoku No. 2’- average weight, 49.5 g; length, 19.5 cm; width of center, 2.4 cm) and eggplants (‘Senryo No. 2’-weight, 64.5 g; length, 9.7 cm; width, 4.1. cm) were used as chilling- sensitive-crops, Chile carrots (‘Hokoku No. 2’-weight, 49.5 g; length, 36.8 cm; width, 952 H ORTS CIENCE, VOL. 25(8), AUGUST 1990 cinic reported that the pathway from oxaloacetic acid to citric acid in a ba- nana peel was inhibited by cold storage. Also, it was reported that keto acids -oxoglu- taric acid and oxaloacetic acid), intermedi- ates in the tricarboxylic acid (TCA) cycle, rapidly accumulated in the tissues of cucum- b&s (1), apples (2), bananas (7), and pep- uers (8). which correlated with the progression of chilling injury. These facts suggest that disruption of the TCA cycle occurs during cold storage. Therefore, by measuring changes in TCA cycle and glycolysis intermediates and enzyme activity in chilling-sensitive crops, which are related to the biosynthesis of metabolites, the cause of pyruvic acid and alanine accumulation produced during cold storage may be elucidated. Literature Cited pyruvic acid and alanine in biological sam- 1. Hirose, T. 1987. Effect of intermittent complete mixture, 0.1 ml of enzyme was warming on chilling injury and contents of added, the changes in optical density at 340 ples: keto acid of cucumber fruits during cold nm were recorded, and, unless otherwise in- storage. Sci. Rpt. Faculty Agr., Kobe Univ., dicated, all assays were run at 25C. The spe- Japan. 17:171-174. cific activity of GPT was expressed as units 2. Hulme, A.C., W.H. Smith, and L.S.C. of activity per gram of protein. In cucumbers, GPT activity increased slightly Wooltoron, 1964. Biochemical changes as- Protein content was determined by the after 4 to 7 days of storage at all tempera- sociated with the development of low tem- method of Lowry et al. (6) using a standard tures and then decreased, but there were no perature breakdown in apples. J. Sci. Food bovine serum albumin (Wako Pure Chemi- significant differences with respect to GPT Agr. 15:303-307. 3. Isherwood, F.A. and D.H. Cruickshank. cals). activity between temperatures (Fig. 2). In 19.54. Chromatographic separation and Pyruvic acid. -Keto acids (i.e., pyruvic eggplants, significant differences in GPT ac- analysis of mixtures of pyruvic, oxaloacetic, acid, -oxoglutaric acid, and oxaloacetic acid) tivity between storage at 1, 5, and 20C did and alpha-ketoglutaric acid. Nature (Lon- play an important role in carbohydrate, lipid, not appear until the 4th day; subsequently, don) 173:121-122. and amino acid metabolism in biological higher GPT activity than initial levels was 4. Jen, J.J. and C.S. Graham. 1975. Glutamic samples. Pyruvic acid in cucumbers (chilling observed even at 1C throughout the storage oxaloacetic transaminase activities during sensitive) increased during storage at 1C, and, periods. GPT activity in carrots stored at 1, maturation. J. Food Sci. 40:934-936. after 14 days, it had increased to more than 5, or 20C decreased until 21 days of storage 5. Kozukue, N., E. Kozukue, T. Hirose, and S. Mizuno. 1984. Accumulation of alanine 7 times the level at 20C (Fig. 1). At 5C, and then increased slightly by 42 days of pyruvic acid content was similar to that at in chilling-sensitive crops. HortScience storage at 1 or 5C. 19:498-501. 20C; however, a slight increase was found The reason why alanine accumulates in 6. Lowry, O.H., N.H. Rosebrough, A.L. Farr, at the end of storage. In eggplants (chilling chilling-sensitive crops during cold storage and R.J. Randall. 1951. Protein measure- sensitive), pyruvic acid increased during is unclear, but the present study has dem- ment with the Folin phenol reagent. J. Biol. storage at 1C and, after 14 days, reached a onstrated that pyruvic acid is rapidly accu- Chem. 193:265-275. level 9 times that measured oil the initial day mulated in chilling-sensitive crops during cold 7. Murata, T. 1969. Physiological and bio- of storage. However, when stored at SC, storage (Fig. 1). Patterson et al. (9) sug- chemical studies of chilling injury in ba- which usually causes chilling injury on cu- gested an increase of GPT activity because nanas. Physiol. Plant, 22:401-411. cumbers and eggplants, the increase in py- 8. Ogata, K.,N. Kozukue, and T. Murata. 1968. passion fruit leaves stored at 0C accumulated Quality changes and the mechanism of chill- ruvic acid was very low compared to the alanine, while glutamic acid was reduced. In ing injury in pepper fruits stored at low-tem- level in eggplants stored at 1C. The reasons this study, GPT activity was examined using perature. J. Jpn. Soc. Hort. Sci. 37:249- are unclear, but it might be that the materials cucumbers and eggplants, which are sensi- 254. used in this study were a little advanced in tive, and carrots, which are not sensitive to 9.