| In this study,Malus sieversii f.luteolus D.F.cuet,a subspecies of Xinjiang wild apple(Malus sieversii(Ledeb.)Roem.)with relatively strong cold resistance and Malus sieversii f.arromaticus D.F.Cui et L.Wang is a subspecies with relatively weak cold resistance,these were selected for the study.By observing different periods of early dormancy,dormancy and release dormancy during natural overwintering,the differences in anatomical structure of flower buds,physiological response of branches and transcriptome gene expression levels between the original and introduced habitats were discussed,the mechanism of strong cold resistance of Xinjiang wild apple was preliminarily elucidated from morphology,cytology and molecular.The main results are as follows:(1)Morphological anatomy was used to observe the number and size of starch grains,separation of plasma wall,content and distribution of ice crystals in three different habitats during the three natural overwintering stages of Malus sieversii f.luteolus and Malus sieversii f.aromaticus.The results showed that the number and size of starch grains were negatively correlated with cold resistance.The starch grains of flower bud cells of Malus sieversii f.luteolus with strong cold resistance were small in volume and closely arranged.The starch grains were hydrolyzed completely in dormancy stage,and the starch grains accumulated in dormancy stage was significantly lower than that of the Malus sieversii f.aromaticus with strong cold resistance.In the three natural overwintering stages of the original habitat,the axial diameters of the starch grains were 9.36 μm,1.16 μm and 1.91 μm,which were significantly smaller than the Malus sieversii f.aromaticus’ s Malus sieversii f.aromaticus were10.95 μm,2.88 μm and5.42μm respectively.The axial diameters of starch grains(6.03,1.37,2.92 μm)were significantly lower than those of Malus sieversii f.aromaticus starch grains(7.52,2.51,4.67 μm)in the introduced resource nursery.The results showed that the degree of cytoplasmic wall separation of flower bud was related to the habitat.The degree of cytoplasmic wall separation of Malus sieversii f.luteolus in the original habitat was significantly lower than that of Malus sieversii f.aromaticus,and the degree of cytoplasmic wall separation of Malus sieversii f.luteolus in the resource nursery was significantly higher than that of Malus sieversii f.aromaticus.The results of ice crystal content and distribution indicated that the barrier tissue of Malus sieversii f.luteolus between the base of the main flower bud and the flower axis was a special barrier to prevent ice crystal from transferring from the flower axis to the flower primordium.(2)The electrical conductivity,soluble protein,soluble sugar,free proline,peroxidase(POD),malondialdehyde(MDA),superoxide dismutase(SOD)and other physiological indexes of 1-year-old branches of wild apple with Malus sieversii f.luteolus and Malus sieversii f.aromaticus in three different natural overwintering stages and three different habitats were measured,and the aboriginality and principal component analysis of each physiological index were carried out.The results showed that the content of free proline and SOD activity reached the minimum values in the resting stage,and the SOD activity of the Malus sieversii f.luteolus was 47.10 U/(g·h),which was significantly higher than that of Malus sieversii f.aromaticus 44.98 U/(g·h).The minimum content of free proline was 55.74 μg/g,which was significantly higher than that of Malus sieversii f.aromaticus(33.89 μg/g).The results showed that proline,POD and soluble sugar played important roles in cold resistance of Malus sieversii f.luteolus during early dormancy period,and proline,SOD,POD,MDA played important parts in cold resistance of Malus sieversii f.luteolus during dormancy period,proline,soluble protein,POD and MDA played significant roles in cold resistance of Malus sieversii f.luteolus during release dormancy period.(3)Transcriptomic sequencing technology was used to conduct high-throughput transcriptomic sequencing on the flower buds of Malus sieversii f.luteolus and Malus sieversii f.aromaticus in three different natural overwintering stages and three different habitats.The results showed that the biosynthesis pathway of secondary metabolites,Transferase activity-function of transferable glycosylated molecules plays a fundamental role in cold resistance.The number of differently expressed genes and unique metabolic pathways in the three natural overwintering stages of Malus sieversii f.luteolus with strong cold resistance were the highest in the original habitat(12 770,289)and introduced resource(6 779,255),respectively.Specific metabolic pathway in dormancy: longevity regulation pathway,ABC transporter play specific roles in cold resistance of flower buds of Malus sieversii.Significantly up-regulated genes in metabolic pathways,such as ATG5、SOD3、ABCB1and ABCG2 can be used as potential cold resistance genes.In order to clarify the intrinsic mechanism of cold resistance of Malus sieversii flower buds,this study systematically studied the anatomical structure,cell level and molecular level,so as to obtain reliable conclusions and provide theoretical guidance for the study of cold resistance of Malus sieversii. |
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