Starch accumulation in sweet potato suspension cells induced by osmotic stress

• Main Authors: Heng Long Wang, 王恆隆
• Other Authors: Jong Ching Su
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/11389040327331711406

博士 === 國立臺灣大學 === 農業化學研究所 === 87 === A b s t r a c t There are two parts in this thesis. At first, the effects of osmotic stress induced by 0.6 M sorbitol on the cell growth and quantitative and qualitative changes in carbohydrates and free amino acids in suspended cells of sweet potato (Ipomoea batatas) were analyzed. Cells transferred into medium without (normal treatment) or with (stress-shocked treatment) 0.6 M sorbitol added, and cells consecutively subcultured under the high stress condition (stress-adapted treatment) were compared. Stress-shocked cells showed cell growth retardation and the induction of plasmolysis. Stress-adapted cells had a shorter lag phase in growth than the stress-shocked, and showed a normal morphology, albeit the size appeared slightly smaller than the normal. Under the stress-shocked condition, the size of amino acid pool (mole/g fresh weight) increased 4-fold relative to the control and stress-adapted cells. The levels of alanine, glutamic acid and its derivatives were especially high, indicating that the changes in the intensity of glycolysis have influenced the amino acid pool. Although proline level showed a 5-fold increase when stress-shocked, it consisted only about 1.5% of total amino acids and thus does not seem convincingly to play an osmotic regulatory function. Among the carbohydrates, sucrose content was high in both stress-shocked and stress-adapted cells. However, a copious amount of starch accumulation occurred only in stress-shocked cells, but neither in normal nor stress-adapted cells, although the latter maintained a higher background level of starch. It is tempting to speculate that sucrose serves as a compatible solute, and starch synthesis from sucrose plays a pivotal role in moderating the hyperosmotic condition. The accumulated starch contained less amylose than the ordinary tuberous root starch, indicating that the pathway of starch synthesis was somewhat altered in the stress-shocked cells. The other experiments investigate events involved in stimulating sucrose accumulation for sweet potato suspension cells exposed to osmotic stress induced by high concentration of sorbitol. The contents of intra- and extra-cellular sugars, the changes of activities of the sucrose-metabolizing enzymes, and the difference of [U-14C] glucose metabolism by a pulse-chase experiment were compared between normal (without sorbitol treatment) and sorbitol-stressed cells. In normal cells, the contents of sucrose and the ratio of sucrose/ hexose were significantly lower than that of stressed-cells. With the onset of osmotic stress, the cells promptly uptake carbon sources by cell-wall-bound invertase via the apoplastic cleavage of sucrose. There are three pieces of evidence indicating that sucrose phosphate synthase (SPS) performs a pivotal role in the sucrose de novo synthesis for sorbitol-stressed cells: 1. The pattern of the increase of sucrose level is consistent with the tendency of SPS activity changes. 2. Northern blot data also showed a close correspondence between the transcript levels and activity of SPS. 3. The glucosyl and fructosyl moiety of labeled sucrose derived from a [U-14C] glucose was symmetric. The stressed-cells simultaneously contain high levels of sucrose-hydrolyzing enzymes, such as alkaline invertase, sucrose synthase (SS), together with the related enzymes located in the cytoplasm for sucrose metabolism, such as fructokinase, glucokinase, and uridine 5’-diphosphoglucose pyrophosphorylase. We may speculate that the futile cycle of sucrose, a continual cycling of degradation and synthesis sucrose, is an active process in the cytoplasm of stressed cells. The stressed cells via a rapid cycling is very sensitivity to respond to the metabolic flux, thus to enhance the carbon partitioning in favor of sucrose accumulation for counteracting the osmotic-stressed condition. The labeled fructose rapidly appeared in the normal cells supplied with [U-14C] glucose. This labeling provide direct evidence for the futile cycle of sucrose is also operation in normal cells. However, the ratio of 14C fructose over 14C glucose is significant lower than 1, indicating that SS contributed partly to the sucrose synthesis for normal cells. Hence, we conclude that the osmotic stress markedly altered the sucrose metabolism in sweet potato cells.