Influence of Temperature on Sterol Biosynthesis in Triticum aestivum

Abstract

Sitosterol, campesterol, stigmasterol, and cholesterol were isolated from green wheat (Triticium aestivum var. Monon) seedlings. Sitosterol was the predominant sterol extracted from the shoot, root, and crown tissue. Cholesterol accounted for less that 1% of sterol in shoot tissue with only trace amounts in the root. A temperature change from 10 to 1 C resulted in a general decrease in sitosterol, stigmasterol, and campesterol in the shoot tissue. The cholesterol level was not altered significantly by the temperature change. The sterols in the root responded in a manner very different from those in the shoots. With the reduction in temperature, sterols first decreased and then recovered over a period of 7 to 14 days to levels that were equal to or exceeded the original levels. From these experiments , it would appear that root tissue can acclimate to the lower temperatures and continue sterol synthesis at the normal rate. The level and response of sterols in the crown tissue were intermediate between the root and shoot tissue. At 10 C the crown response was similar to that of root tissue, whereas, at 1 C the response more closely resembled that of the shoot. The investigation of influence of environmental effects on phytosterols has been limited to that of light. Goodwin and Mercer (6) reported that sterol synthesis was not stimulated by placing dark-grown maize seedlings in light. An absence of changes in unsaturated free sterol with a 24-hr period of greening of etiolated pea seedlings was reported by Gaunt and Stowe (4). However, a higher level of total sterol was found by Hirayama and Suzuki (10) when etiolated corn seedlings were compared to light-grown seedlings. Bush et al. (2) found that total sterol was higher in etiolated than green barley shoots and the free sterols accounted for most of this difference. Green tissue has about equal amounts of stigmasterol and si-tosterol, whereas sitosterol predominated in etiolated shoots. From these reports, it is apparent that an environmental factor such as light will influence sterol biosynthesis. Our objective was to determine the influence of another factor, namely temperature , on sterol biosynthesis in shoots, roots, and crown tissue of wheat. 'The investigation reported in this paper (No. 73-3-50) is in connection with a project of the Kentucky Agricultural Experiment Station and is published with approval of the Director. MATERIALS AND METHODS Plant Material. An early maturing, short shoot, high yielding winter wheat (Triticum aestivum var. Monon) capable of developing a relatively high degree of cold hardiness was used in this study. Wheat was seeded in sterilized 10-cm clay pots filled to a uniform level with steam sterilized sand. Six uniformly sized seeds were planted in each pot at a depth of 1 cm. The temperature was maintained at 21 C for 28 days with a 14-hr photoperiod. A complete nutrient solution was applied every 4th day for the duration of the experiment. After the 28-day period at 21 C, the plants were exposed to two different temperature regimes. The temperature treatments were 10 and 1 C for 28 days. Each week three replicates, 18 seedlings, were harvested, plant parts were separated and freeze dried. In another experiment, plants were grown under the same conditions for the first 28 days but altered over the subsequent 35 days. For the 1st day of the 35-day treatment period, the temperature was gradually reduced at a rate of 1 C/hr until 10 C was reached. The plants were maintained at this temperature for 6 additional days. At the end of these 7 days, the temperature was gradually lowered to 1 C on one-half the plants with the remaining plants held at 10 C. The experiment was terminated after 28 days at these two temperatures. This is usually considered to be sufficient time to allow development of cold hardiness (1). Samples were collected every 7 days and handled as described for the first experiment. In a second type of experiment, seedlings were grown on cheesecloth over a continuously aerated water bath containing 0.5 mm CaSO4 in controlled environmental chambers with a 12-hr photoperiod for 7 days. Two temperature regimes were selected: 21 C for 7 days and 21 C for 3.5 days, followed by 3.5 days at 4 C. Shoot and root samples were collected after 3.5 days and 7 days for both temperature regimes. The experiment was repeated three times. Sterol Analysis. Five-gram samples of freeze-dried material were analyzed for total sterols by the gravimetric method of Stedman and Rusaniwskyj (14). Individual sterols were determined by gas chromatography (7, 8). The gas chromatograph was equipped with a coiled glass column (1.8 m X 4 mm) packed with 5% OV-101 on 80 to 90 mesh Anakrom ABS, a hydrogen flame ionization detector, and electronic integrator (3). Operating temperatures were 255, 275, and 300 C for the column oven, injection block, and ionization detector oven, respectively. Helium was used as the carrier gas at a flow rate of 100 ml/min. A standard of cholesterol, campesterol, stig-masterol, and sitosterol (Applied Science Laboratories, Inc.. P. 0. Box 440, State College, Pa.) was analyzed twice daily to correct for detector response. 324