| Wheat grain was a living organism, in which a series of physiological reactions including decreased enzyme activity, weakened vitality and respiratory depression may occur in the process of long-term storage and resulted in the deterioration of seeding quality and processing quality. The wheat stored under dry, low oxygen and weak light environment would show a greatly different physiological characteristics with the grain grew in the field. In this paper, we took durum wheat species of Aikang58 and Zhengmai9023 and the soft wheat of Zhengmai004 as the test samples, and determined the physiological metabolic activity regularity, respiration, the formation of energy molecule ATP and the change of germination rate in different varieties of wheat during storage periods. The expression of cell wall expansion(Tri-EXPB1) gene was analyzed by real-time PCR to investigate the effect of physiological deterioration on gene transcription and its correlation with germination percentage. We also determined the proteomics of physiological deterioration area of stored wheat by TMT and LC-MS and identified the physiological deterioration related proteins and their functions. In this paper, we obtained the following results:1. Physiological enzyme activity and germination rate of wheat in different storage timeDehydrogenase, CAT and POD were important protective enzyme in plant metabolic processes which could perfectly reflect the vigor of seeds. The three wheat species of different physiological activities showed different changes tendency in the first two weeks of storage. The variation trend of fresh wheat and 12 months stored wheat at room temperature was not obvious, and the enzyme activity of fresh wheat was slightly higher than that of wheat stored for 12 months. The dehydrogenase activity of high temperature aging wheat increased slightly, and then declined rapidly after 1-2weeks; the CAT and POD activity decreased continuously. For the high temperatureaging wheat of Aikang58 and Zhengmai9023,With the extension of storage time,the three kinds of enzyme activity declined rapidly in the first four weeks, and then slow down slowly. But the change trend of Zhengmai004 was more gentle, and decreased with the deepened of deterioration degree. The results showed that the physiological enzymes activity of Aikang58 was higher than that of the other two wheat cultivars, and the Zhengmai004(soft wheat) showed the lowest enzymes activity.2. The respiration and the ATP production in wheat storageThe studies on respiration and the formation of energy molecule ATP in different wheat storage period showed that the CO2 release of fresh wheat and 12 months stored wheat under room temperature increased steadily as stored time extended, which showed that the respiration of wheat stored at room temperature did not change significantly,which kept the high physiological activity. In the initial stage of high temperature aging,the CO2 release increased slightly, and the increasing trend was much higher than the wheat stored at room temperature. After stored at 45℃ for three weeks the upward trend gradually slowed down to stable and no longer rise. This showed that after a period of high temperature storage, the wheat had gradually lost its physiological activity, and the respiration gradually weakened. And it was obviously that the rising trend of Zhengmai004 was gentler than that of the others in the early stage of storage. The variation trend of ATP content in fresh wheat and stored for 12 months wheat were similar to that of the enzyme activity. In the process of artificial aging, the physiological activity of wheat decreased, the content of ATP reduced step down and at the first four weeks the ATP content had decreased. However the changes of ATP content in wheat at room temperature were not obvious.3. Expression of cell wall expansion protein(Tri-EXPB1) in wheat physiological deterioration area.To understand the physiological deterioration mechanism better in wheat, we usedthe germination wheat as the control group. The results of real-time PCR showed that with the extension of high temperature storage, the expression amount of Tri-EXPB1 gene decreased, and the wheat physiological activity was gradually weakened; The Tri-EXPB1 gene expression of wheat stored at room temperature has no significant change, and the physiological activity decreased trend was not obvious too. The expression of this gene in germination wheat was significantly higher than the other wheat varieties, the expression amount of Tri-EXPB1 gene in germination wheat of Zhengmai004 could reached 4.5 times of the new harvest wheat. This showed that the physiological deterioration had an important influence on the expression amount of cell wall expansion protein(Tri-EXPB1), and weaken its transcriptional activity. The change trend of germination rate of new harvested wheat and stored for 12 months wheat at room temperature was not obvious, but the germination rate of high temperature aging wheat decreased rapidly in the first four weeks. Through the correlation analysis, the correlation coefficient of Tri-EXPB1 expression amount and germination rate was above0.9. It showed that the change trend of Tri-EXPB1 expression amount was corresponding related with that of germination rate, which can be used as a characterization index of physiological deterioration in wheat storage. It was worth mentioning that compared with Aikang58 and Zhengmai9023(soft wheat), the data of Zhengmai004(hard wheat) in the above indicators were low, and the change trend of high temperature aging was relatively gentle. Thus we inferred that Zhengmai004 had a good storage quality.4. Study on the proteomics of wheat physiological deterioration areaThe wheat with different germination rate(WH01, WH20, WH50, WH98, WH100)were analyzed by TMT(tandem mass tag) labeling and LC-MS, statistical analysis of the obtained mass spectrometry data, wheat genome and proteomic matching conditions was made. In total, 6281 proteins were identified, among which 3574 proteins withquantitative information were elicited from Triticum aestivum. During artificial ageing,(WH01vsWH98, WH20vsWH98, and WH50vsWH98),162 differentially expressed proteins were identified including 36 up-regulated and 126 down-regulated proteins mainly involved in disease/defense response and storage proteins were identified during artificial ageing compared to the non-aged seeds. GO annotation and KEGG pathway analysis indicated that the up-regulated proteins mainly involved in ribosome and histone participating in stores proteins degradation; the down-regulated proteins mainly involved in energy supply and response to external environment such as TCA cycle,glycolysis, oxidative stress and pathogen penetration. Among the identified proteins, 531 showed differences(DEPs) in abundance including 312 upregulated and 219 down regulated proteins during seed priming compared to unprimed seeds. The up regulated protein mainly participated in energy supply(TCA cycle, glycolysis), protein synthesis,fatty acid oxidation and synthesis and photogenesis, the down–regulated proteins were mainly stored proteins and proteins related with response to pathogen infection. |
PDF Full Text Request