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

Abstract: 【Objective】This study explored the differences in chloroplast ultrastructure in leaves of‘Gold-en Delicious' apple in 3 different ecological regions(Maoxian,Yanyuan and Nyingchi) and its adaptationto 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 wascarried 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 attitude Sichuan-Tibet area. Mature healthy leaves were collected from July to August. The leaf slices werecut off from the primary leaf blade(about 1.0 cm in length and 1.0 cm in width),fixed in 3.5% glutaralpentanedial(V/V) at 4 ℃ for 2 h,washed four times in 0.1% phosphate buffer(p H 6.8),postfixed in 2%osmium tetraoxide(Os O4) for 2 h,and then washed twice in 0.1 mol·L-1PBS(sodium phosphate buffer,p H 6.8) at 4 ℃. They were then sequentially dehydrated in 30%,50%,70%,80%,90%,95%,and100% 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 k V and chloroplast ultrastructures were photo-graphed. All the experiments were repeated three times,and data were statistically analyzed using DPS7.05 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 cells were oval with complete lamellarstructures(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 weredistributed 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 waslargest(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 pergranum) was found in samples from Nyingchi,followed by those from Yanyuan with 4.6 per granum,andthe lowest,4.3 per granum,was in sample from Maoxian. Correlation analysis showed that meteorologicalfactors,including the mean annual temperature,annual ≥10 ℃accumulated thermal units,the averagetemperature 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 impliedadaptive 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.