川藏高海拔不同生态区苹果叶肉细胞叶绿体超微结构的比较

Comparison of chloroplast ultrastructure of‘Golden Delicious' apple leaves in different ecological regions of high attitude areas in Sichuan-Tibet

【目的】探讨川藏高海拔不同生态区‘金冠’苹果叶肉叶绿体超微结构对环境变化的响应与适应,以期为高原苹果叶片生理生态功能研究及栽培管理提供一定的理论依据。【方法】本研究采用透射电镜观察了3个不同生态区‘金冠’苹果叶片叶绿体超微结构的差异,并对叶绿体超微结构参数与主要气象因子的相关性进行了分析。【结果】‘金冠’叶肉细胞中叶绿体呈长椭圆形,具有完整的片层结构,淀粉粒和嗜锇颗粒分布于片层之间,叶绿体在细胞中呈片层纬线平行排列。茂县‘金冠’叶肉细胞叶绿体主要排列于细胞的边缘,类囊体膜遍布整个叶绿体,基粒多,平均达42.5个,基粒片层发达,平均为35.25层;盐源和西藏林芝地区‘金冠’的叶绿体则向细胞中央靠拢,且有少量叶绿体扭曲变形;基粒垛叠程度较低且基粒数及基粒片层均较少,分别为40.3个及16.48层;叶绿体密度以盐源地区最大,每μm2达38.2个,茂县次之,叶绿体密度为每μm233.2个,西藏林芝最小,每μm2仅为22.4个;而叶绿体淀粉粒密度则以西藏林芝地区最多,平均每个基粒为6.6个,茂县最少,平均每个基粒含4.3个,盐源地区居中,平均每个基粒含淀粉粒4.6个;相关性分析表明,年均气温、≥10℃年均积温、一月平均气温、年均极端低温、年日照时数及海拔等生态因子显著影响着叶绿体超微结构参数。【结论】高海拔地区植物叶绿体基粒片层及类囊体垛叠程度下降,淀粉粒含量升高,叶绿体有向细胞中央位移的现象,是高海拔植物对高原地区低温和强辐射的一种适应。

[Objective]This study explored the differences in chloroplast uhrastructure in leaves of ＇Gold- en Delicious＇ apple in 3 different ecological regions （Maoxian, Yanyuan and Nyingchi） and its adaptation to the environments in the high attitude areas of Sichuan-Tibet so as to bring an understanding of physio- logical and ecological functions of leaves, and for cultivation and management. [Methods]The study was carried out from 2012 to 2014 in Horticulture Institute, Sichuan Academy of Agricultural Sciences, Chengdu, China. 20-year-old ＇Golden Delicious＇ apple trees were used as experimental materials, which were planted under standard orchard managements in 3 different ecological regions of the high atti-tude Sichuan-Tibet area. Mature healthy leaves were collected from July to August. The leaf slices were cut off from the primary leaf blade （about 1.0 cm in length and 1.0 cm in width）, fixed in 3.5% glutaral pentanedial （V/V） at 4 ℃ for 2 h, washed four times in 0.1% phosphate buffer （pH 6.8）, postfixed in 2% osmium tetraoxide （OsO4） for 2 h, and then washed twice in 0.1 mol·L^-1 PBS （sodium phosphate buffer, pH 6.8） at 4 ℃. They were then sequentially dehydrated in 30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol each for 15 min. The leaf tissues were embedded in Epon 812 at 37 ℃, 45℃, and 60 ℃ for 12 h at each temperature, and ultra-thin sections （100 nm） were sliced and stained with uranyl ace- tate and lead citrate. The sections were then observed with H-600 IV transmission electron microscopy （TEM; JEM- 1200EX） at an accelerating voltage of 60 kV and chloroplast ultrastructures were photo- graphed. All the experiments were repeated three times, and data were statistically analyzed using DPS 7.05 and SPSS 18.0 software. Significance between means was tested by Duncan＇s multiple range test. [Results] The results showed that chloroplasts in the mesophyll ceils were oval with complete lamellar structures （thylakoids） arranged in parallel with the length of the chloroplast. Starch grains and osmiophil- ic particles were distributed between the thylakoids. Chloroplasts of ＇Golden Delicious＇ sampled in Maox- ian were mainly located at the edges of the cells and had more grana with an average of 42.5 per chloro- plast and 35.25 layers ofthylakoid per granum. Chloroplasts of samples from Yanyuan and Nyingchi were distributed closer to the center of the cells with a few chloroplasts that were distorted, and had fewer gra- na （40.3） with fewer layers of thylakoids （16.48）. The chloroplast density in samples from Yanyuan was largest （38.2 per square microns）, followed by those from Maoxian （33.2 per square microns）, and the low- est was found in samples from Nyingchi （22.4 per square microns）. Highest starch grain density （6.6 per granum） was found in samples from Nyingchi, followed by those from Yanyuan with 4.6 per granum, and the lowest, 4.3 per granum, was in sample from Maoxian. Correlation analysis showed that meteorological factors, including the mean annual temperature, annual ≥10 ℃accumulated thermal units, the average temperature in January, the annual minimum temperature, the average annual sunshine hours and alti- tude, could significantly affected chloroplast ultrastructure. [Conclusion] In this study, it was implied adaptive responses to the plateau environment with low temperatures and intense radiation included chlo- roplast movement to the center of the cells, reduction of thylakoid stacking and increase in starch grains.