| Apple fruit sunburn, a common physiological disorder caused by high temperature, drought and strong sunlight, happens widely in main apple-producing areas around the world, especially in the areas with high temperature and drought stress, which results in a huge loss annually. At present, the study on effect of strong light on sunburn is mainly focused on its enhancement of fruit surface temperature or on UV damage. In fact, the threshold temperature was required for sunburn appears to be 3-7℃lower than that of thermal damage in the dark. Studies on plant leaves have shown that both high temperature and strong light can cause photoinhibition, which results in over production of Reactive Oxygen Species (ROS) in photosynthetic electronic chain. Compared with leaf, the photosynthetic capacity of apple green peel is lower, energy absorbed by green peel under high temperature and strong light conditions should be much more than its utilization. However, due to that nearly all of the instruments on study of photosynthesis are designed according to plant leaves and due to the special shape, structure and growth condition of apple fruits, studies on photoinhibition and photoprotection mechanisms in apple fruit peel is very limited up to now. The effect of high temperature and strong light on photosynthesis of apple green fruit as well as the relationship between photoinhibition and oxidation stress has not been clear yet Therefore, it is theoretically and practically important to elucidate the response of photosynthesis and antioxidation system in green peel to high temperature and strong light and to explore the physiological and biochemical mechanism of sunburn, which will help us to improve cultivation practice against sunburn in apple production.In this dissertation, Starkrimson cultivar (Malus domestica Borkh.), a broadly cultivated apple cultivar, were selected⑴to establish a series of approaches to study photosynthetic physiology in apple fruits;⑵to investigate the dynamic variations of relative pigment contents, photosynthetic capacity and respiration rate in apple fruit peel during development of the fruits;⑶to investigate the variations of primary photochemical reactions of apple green peel in response to diurnal changes of incident PFD and air temperature. Furthermore, mechanism of photoinhibition in apple green peel under the cross stress of strong light and high temperature, and the effect of photoinhibition on oxidative stress on apple green peel were also investigated. The main results are as follows:1. Approaches to study photosynthetic physiology in apple peel were established.⑴Approaches to study photosynthesis in apple fruit was improved.By the new approach, the difference of photosynthetic O2 evolution in sun peel, shade peel and longitudinally different parts were compared. It was proved that the central part of sun peel was the desirable representative one for measuring photosynthetic O2 evolution. 500μmol m-2 s-1 PFD and 30℃are optimal measurement condition for photosynthesis in apple peel. In addition, special probes for measuring spectral reflectance and chlorophyll a fluorescence of apple fruit and apparatus for the control of temperature and light for treatment of apple peel or fruit were designed and improved.⑵Nondestructive measurement of pigment content in apple fruits using spectral reflectance was investigated.The results showed that the spectral indices chose in present study were better correlated to the pigment concentrations in fruit peel, the spectral indices were significantly correlated with contents of chlorophyll (a+b) or anthocyanin. It was demonstrated that the relationships of spectral indices to pigment concentrations can be well used for non-destructive and quickly estimating pigment concentrations in apple fruits. The result also showed that photochemical reflectance indices (PRI) was significantly negative correlated with de-epoxidation status of total xanthophylls pigment pool ((0.5A+Z)/( A+Z+V)).2. Dynamic variations of relative pigment contents, photosynthetic capacity and respiration rate in peel during apple green fruit development were investigated.Immature apple fruits had a high content of chlorophyll that gives them the characteristic green color. From the 25th to 83rd day after bloom, the content of both chlorophyll and carotenoid decreased, but the ratio of carotenoid to chlorophyll increased, which indicated that the degradation of chlorophyll was faster than that of carotenoid in green peel. The content of anthocyanin changed in an opposite way to chlorophyll and carotenoid, it was maintained unchanged during the period of 25-61 days after bloom, then increased gradually from the 61st day after bloom.Photosynthesis was higher in immature apple fruit peel. It declined with the progress of fruit development, changing in a similar to chlorophyll content. The contribution of fruit photosynthesis to the total carbon requirement of a fruit was the highest during early development.Before color up, especially in the interval of 33-53 days after bloom, the respiration rate decreased and was minimized 53 days after bloom, and then it leveled off. The total respiratory rate and the ratio of alternative pathway to the total respirations were higher during the early stage of fruit development. The increase of cyanide-resistant respiration could be in favor of reducing the production of ROS to alleviate oxidative stress in whole cell level.3. The response of PSII primary photochemical reactions and xanthophyll cycle in apple peel to diurnal changes of incident photon flux density (PFD) and air temperature were studied.In a sunny day, with increase of incident PFD, severe photoinhibition occurred from 12:00 to 14:00 in apple peel. Relative activity of oxygen evolving complex (OEC) indicated by a fluorescence index Wk did not change significantly through the day, efficiency that a trapped exciton can move an electron into the electron transport chain beyond QA- (ΨO) reduced from 12:00 to 14:00, indicating that acceptor side of PSⅡwas damaged in apple peel. High irradiance caused a decrease in the density of PSⅡreaction centers per excited cross-section(RC/CS) and resulted in an increase in absorption per active reaction centers(ABS/RC). Since the photochemical reaction in apple peel (TRO/RC) was not able to use excited energy completely at noon, the dissipation per active reaction center (DIO/RC) was increase during that time. Along with appearance of photoinhibition, the de-epoxidation level of xanthophyll pigment pool indicated by PRI markedly increased, indicating that xanthophyll cycle was initiated to prevent PSII reaction centers and electron transport chain against photodamage in apple peel.4. Activity of PSⅡreaction center, antioxidative system in response to strong light and high temperature and the effect of light on oxidative stress under high temperature were explored in apple fruit.To avoid effects of other factors except light and temperature, activity of PSⅡreaction center, antioxidative system in response to strong light and high temperature and the effect of light on oxidative stress under high temperature in apple fruit were explored under controlled light and temperature condition. The results showed that photosynthetic O2 evolution rates in apple peel did not change significantly between 30℃to 44℃, but distinctly declined at 48℃, and every combination of different light intensity with 48℃aggravated the decrease. Analysis of changes in Fv/Fm, H2O2 and MDA indicated that high temperature not only directly damaged photosynthetic apparatus to cause photoinhibition but also aggravated photoinhibition by affecting other biochemical metabolic processes, so high temperature caused photoinhibition under moderate light even under weak light.Under strong light, temperature over 44℃resulted in chlorophyll degradation and deactivation of PSⅡreaction centers. Over 600μmol m-2 s-1 light combined with high temperature accelerated the deactivation of PSⅡreaction centers. Treatments with 48℃in the dark or 30℃combined with 1000μmol m-2 s-1 light did not affect the accept side of PSII. While the combination of 44℃with various light intensity would inhibit the activity of the accept side of PSII.Along with appearance of photoinhibition caused by high temperature combined with light, xanthophyll cycle was initiated to prevent PSII reaction centers and electron transport chain against photodamage in apple peel.The treatment of 44℃combined with 1000μmol m-2 s-1 light greatly reduced the activities of antioxidant enzymes such as SOD, APX and CAT, resulting in over accumulation of ROS in apple peel.The activity of POD and PPO manifested stage heterogeneity, which correlated with the development of apple green fruit. The activity of POD and PPO minimized 56 and 84 days after bloom respectively.The high concentrations of H2O2 and MDA in apple peel treated with high temperature combined with strong light indicated that the antioxidant system was unable to cope with the photooxidation triggered by high temperature coupled with strong light. Under the stress of high temperature combined with light, although down- regulation of photochemistry and xanthophyll cycle were initiated, the ROS was over accumulated due to the broken equilibrium between the scavenging and production of ROS, which enhanced oxidative stress to damage membranes of peel cells including the membranes of thylakoids in apple peel.In conclusion, high temperature brought out oxidative stress in apple green peel; strong light coupled with high temperature caused severe photoinhibition, resulting in over production of ROS in photosynthetic electron chain, which increased the accumulation of ROS in whole cell to accelerate photooxidative damage of apple green peel at high temperature. Severe photoinhibition caused by strong light coupled with high temperature was one of important reasons to cause sunburn under light as well as a key reason to cause the decrease in threshold temperature required for sunburn by 3-7℃lower than that of thermal damage in the dark.
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