Screening of genes related to glutathione metabolism responding to freezing stress in Malus sieversii seedlings in vitro based on transcriptome sequencing

Abstract:【Objective】To explore the response of glutathione metabolism and screen the glutathione metabolity-related genes responding to freezing stress, the possible pathway of freezing resistance was analyzed in Malus sieversii seedlings in vitro.【Methods】The tissue-cultured plantlets of M. sieversiiwere used as the material, the phenotype, maximum quantum yield of photosynthetic system Ⅱ (F_v/F_m), electrolyte leakage, malondialdehyde (MDA), hydrogen peroxide (H2O2) and glutathione (GSH) contents were determined in M. sieversii seedlings in vitro, with the treatments of the control (CK), T6h, T12h, T36h and HF24h at -3 ℃. Transcriptome sequencing analysis was performed on leaves of M. sieversii seedlings treated with CK, T6h and T12h.【Results】Compared with CK, there were no significant changes in leaf shape and color after treatment at -3 ℃ for 6 h, the edge of top leaf on M. sieversii seedlings showed reverse winding, the leaf color became dark, and the apical tip tender leaves were wilting after treatment at -3 ℃ for 12 h, the leaves on M. sieversii seedlings treated were wilting and drooping, and the leaf color was further dark after treatment at -3 ℃ for 36 h. After recovery at 25 ℃ for 24 h, the leaf of wilting curl stretched, the leaf color also became bright, and the brown small spots appeared on the leaf. Compared with CK, Fv/Fm decreased by 20%, 30% and 53% at T6h, T12h and T36h in leaves on M. sieversii seedlings under -3 ℃ low temperature stress, and there was no significant difference between HF24h and CK. Compared with CK, the electrolyte leakage did not increase significantly at T6h, but increased significantly by 50% and 175%, respectively at T12h and T36h, and the electrolyte leakage at HF24h was 48% lower than T36h in leaves on M. sieversii seedlings under -3 ℃ low temperature stress. Compared with CK, the content of MDA did not change significantly at T6h, but increased by 57% and 86% at T12h and T36h, and the MDA content at HF24h was 23% lower than T36h in leaves on M. sieversii seedlings under -3 ℃ low temperature stress. Compared with CK, the hydrogen peroxide content did not change significantly at T6h, but increased by 171% and 291% at T12h and T36h, and the hydrogen peroxide content at HF24 h was 30% lower than T36h in the leaves on M. sieversii seedlings under -3 ℃ low temperature stress. Compared with CK, the glutathione content significantly increased by 40%, 89% and 110% at T6h, T12h and T36h, and no differences were observed between T36h and HF24 h. Transcriptome sequencing was performed on M. sieversii seedlings with the treatment of CK, T6h and T12h at -3 ℃ under simulated freezing conditions. At the transcriptome level, 18 different genes of glutathione metabolism pathway (map00480) were screened, and compared with CK, BGI_novel_G000969, Msi_11B024060, Msi_11B002610, Msi_15B018350 and Msi_11A024060 were down- regulated at T6h and T12h, Msi_08B010370, Msi_11B013970, Msi_ 17B006200, Msi_12A020080, Msi_14A011470, Msi_14B010870, Msi_09B008290, Msi_06A008160, Msi_03A025150, Msi_10A018630, BGI_novel_G001892, Msi_09A009610 and Msi_07A023660 were up-regulated at T6h and T12h. The transcriptome data were analysized by KEGG database, and 18 different genes were annotated as GGCT, GSS, GGT1_5, CARP, speE and IDH1. DPX, PDG and IDH1were respectively involved in the GSH and GSSG homeostatic pathway, so as to reduce the damage by hydrogen peroxide, superoxide anion and other oxides on the tissue-cultured plantlet cells. GST was involved in the reaction, in which GSH reduced substances with high oxidation potential. GGT1_5, CARP and GSS were involved in the process of GSH degradation and synthesis. q- PCR showed that three of the five genes BGI_novel_G001892 (GST), Msi_03A025150 (GST) and Msi_10A018630 (GST) were down-regulated compared with CK at T36h. Two genes including Msi_06A008160 (GPX) and Msi_11B013970 (GST) were up-regulated. Analysis of the expression of GPX and GST at HF24 h showed that BGI_novel_G001892 (GST) was down- regulated, and Msi_06A008160 (GPX), Msi_ 03A025150 (GST), Msi_10A018630 (GST) and Msi_11B013970 (GST) were up- regulated.【Conclusion】The damage of M. sieversii seedlings in vitro increased with the extension of treatment time through the phenotype and physiological indexes determination in M. sieversii seedlings under -3 ℃ simulated freezing stress. GPX and GST were the key genes in glutathione metabolism pathway, which were up-regulated in response to freezing stress in M. sieversii seedlings in vitro.【Objective】To explore the response of glutathione metabolism and screen the glutathione metabolity-related genes responding to freezing stress, the possible pathway of freezing resistance was analyzed in Malus sieversii seedlings in vitro.【Methods】The tissue-cultured plantlets of M. sieversii were used as the material, the phenotype, maximum quantum yield of photosynthetic system Ⅱ (Fv/Fm), electrolyte leakage, malondialdehyde (MDA), hydrogen peroxide (H2O2) and glutathione (GSH) contents were determined in M. sieversii seedlings in vitro, with the treatments of the control (CK), T6h, T12h, T36h and HF24h at -3 ℃. Transcriptome sequencing analysis was performed on leaves of M. sieversii seedlings treated with CK, T6h and T12h.【Results】Compared with CK, there were no significant changes in leaf shape and color after treatment at -3 ℃ for 6 h, the edge of top leaf on M. sieversii seedlings showed reverse winding, the leaf color became dark, and the apical tip tender leaves were