| In order to explore the physiological and molecular responses of red raspberry seedlings to high temperature stress and the applicable measures to alleviate the heat damage of red raspberry in production,red raspberry cultivars ’Polka’ and ’Heritage’ cultivated in large areas were used as experimental materials,and PGX and shade net were applied to control temperature and shade to study the effects of high temperature stress and shading on cell membrane lipid peroxidation,osmoregulation substances,antioxidant system,transcriptome sequencing and other physiological and biochemical indexes and transcriptional products of red raspberry were studied.The main results are as follows:1.The chlorophyll content of red raspberry ’Polka’ seedlings decreased first and then increased under 35℃,and decreased gradually under 40℃.After 2 days of stress,the chlorophyll content of leaves under 35℃ stress reached the minimum value,which was 3.39 mg.g-1,which was 18.47%lower than that of CK.After 4 days of stress,the chlorophyll content of leaves under 40℃ stress reached the minimum value of 2.65 mg.g-1,which was 34.83%lower than that of CK.2.With the extension of stress time,cell membrane peroxidation increased gradually.The relative electrical conductivity and malondialdehyde content of the red raspberry cultivar’Polka’ under stress at 35℃ and 40℃ showed an upward trend,and reached the maximum at 4 days of stress.The maximum relative conductivity of leaves under 35 ℃ and 40℃ stress were 25.52%and 29.33%,respectively,which were 28.54%and 43.36%higher than those of CK.and the maximum contents of MDA in these leaves were 1.83 and 1.99μmol.g-1.respectively,which were 26.92%and 38.01%higher than those of CK.3.The content of soluble sugar in leaves of red raspberry ’Polka’ seedlings changed little under 35℃ stress,but decreased first and then increased under 40℃ stress,reaching the minimum value of 6.19 mg.g-1 at 2d,which was 6.8%lower than that of CK.The soluble protein content in the leaves of the red raspberry cultivar ’Polka’ under 35℃ stress and 40℃stress showed a gradually increasing trend with the extension of stress time,and the maximum values were 923.93 and 1191.91 mg.g-1,respectively,at 4 days of stress,which were 22.12%and 57.55%higher than those of CK.4.With the extension of stress time,CAT activity of ’Polka’ leaves increased gradually.After 4 days of treatment,the CAT activity of ’Polka’ leaves under 35℃ and 40℃ stress reached the maximum value,which were 137.83/U.g-1.min-1 and 109.25/U.g-1.min-1 respectively,79.14%and 41.99%higher than those of CK.The activity of POD decreased gradually.On the 4th day of treatment,the POD activity of ’Polka’ leaves under 35℃ and 40℃ stress reached the minimum values,58.28 and 66.38/U.g-1.min-1,respectively,which were 28.33%and 18.37%lower than that of CK.The SOD activity of leaves increased first and then decreased.After 2 days of stress,the leaf SOD activities reached the maximum value at 35℃ and 40℃,5.73/U.g-1.min-1 and 4.73/U.g-1.min-1,respectively,which were 28.93%and 6.5%higher than CK.5.With the continuation of the stress time,the net photosynthetic rate of the red raspberry ’Polka’ under the stress of 35℃ and 40℃ showed a gradual decrease trend,and reached the minimum value at 3.36 and 2.38μmol.m-2s-1 respectively after 4 days of stress,which decreased by 41.84%and 58.8%,respectively,compared with CK.Under 35℃and 40℃ stress,the Ci,Gs and Tr increased gradually.After 4 days of stress,under 35℃ and 40℃ stress,the Ci concentration of leaves reached the maximum,which were 222.54 and 247.76 μmol.m-2s-1,respectively,which were 32.29%and 47.27%higher than those of CK.The stomatal conductance of leaves reached the maximum values,which were 0.0853 and 0.102,respectively,which were 94.75%and 132.87%higher than those of CK.The transpiration rate of leaves reached the maximum value,which were 2.15 and 2.55μmol.m-2s-1,respectively,increased by 92.93%and 89.37%,respectively,compared with CK.6.With the extension of stress time,the leaf Fm of red raspberry ’Polka’under 35℃and 40℃ stress decreased gradually,and reached the minimum value after 4 days of stress,which were 21676 and 20623,respectively,which were 6.64%and 4.86%lower than those of CK.The Fv/Fm and Fv/Fo of leaves under 35℃ stress had no significant change,while those of leaves under 40℃ stress showed a continuous downward trend,reaching the minimum values of 0.71 and 2.5 at 4 days of stress,which were 7.79%and 31.71%lower than those of CK.Under 35℃ stress,the leaf Fo decreased gradually,and reached the minimum value of 4783.67 at 4 d stress,which was 6.3%lower than that of CK,while the leaf Fo under 40℃stress showed a gradual increase trend,and reached the maximum value of 5868.33 at 4d of stress,which was 14.95%higher than that of CK.7.The differentially expressed genes were identified under high temperature stress.920 genes were up-regulated and 882 genes were down-regulated in HT35_vs_CK.2872 genes were up-regulated and 2672 genes down regulated in HT40_vs_CK.1823 genes were up-regulated and 2169 genes were down-regulated in HT40_vs_HT35.The Go functions of differentially expressed genes were mainly concentrated in photosynthesis,oxidative stress and other pathways,while KEGG functions were mainly concentrated in the biosynthesis of polyketose units,photosynthesis-antenna proteins and phenylpropanoid biosynthesis.8.Under high temperature stress,42 differentially expressed genes of HSP family were identified.23 genes were up-regulated and 3 genes were down-regulated under 35℃,13 genes were up-regulated and 10 genes were down-regulated under 40℃.As members of HSP family,TRINITY_DN26431_C1_g1、TRINITY_DN22151_C4_g10、TRINITY_DN25473_C0_G1 might play an important role in high temperature stress of red raspberry.9.Two differentially expressed genes related to SOD were identified under high temperature stress,which were all up-regulated under 40℃.25 differentially expressed genes related to POD were up-regulated and down-regulated under high temperature stress.Three differentially expressed genes related to CAT were identified,in which one gene was down-regulated and one was up-regulated under 35℃,and two genes were up-regulated under 40℃.10.A total of 112 differentially expressed genes involved in plant hormone signal transduction were obtained under high temperature stress.11.A total of 38 differentially expressed genes involved in photosynthesis and antenna protein pathway were identified under high temperature stress.Most of the genes encoding PC,Fd,FNR,cytb6f,PSI,PSII and LHC were down regulated.12.With the extension of shading time,the contents of chlorophyll and soluble sugar in leaves of red raspberry ’Heritage’ under shading and CK were all increasing.The content of soluble protein in leaves of all shading treatments decreased,while that of CK increased.The chlorophyll content in leaves of CK was significantly lower than that of the shading,and the soluble sugar and soluble protein content in CK leaves were significantly higher than those of the shading.13.With the extension of shading time,the POD activities in leaves of red raspberry’Heritage’ seedlings increased gradually under full light and shading treatments.After 4 days of stress,the activities of POD in leaves of CK and all shading treatments reached the maximum,and the activities of POD in leaves of all shading treatments were significantly higher than those of CK.The MDA content of CK leaves increased sharply,but the change of MDA content in leaves of different shading treatments was small.The activities of CAT decreased gradually in leaves of CK and shading treatments while the SOD activities changed little. |
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