Abstract:【Objective】To study the transcriptome differences and hormone content changes of Malus sieversii seeds at different storage stages, we screened the plant hormones genes related to dormancy release, and to provide a basis for subsequent studies on the hormonal regulation mechanism of seed dormancy release in Malus sieversii seeds.【Methods】Malus sieversii seeds were used as materials, transcriptome sequencing was performed on control unstratified seeds and seeds after 4 ℃ low-temperature sand storage stratification treatments (30, 60, 90, and 120 d). The content of plant hormones abscisic acid(ABA), gibberellins(GA), auxin(IAA), cytokinin(CTK), the activities of ethylene 1-aminocyclopropane-1-carboxylate oxidase(ACO)and 1-aminocyclopropane-1-carboxylate oxidase synthase(ACS) were measured by using an enzyme-linked immunosorbent assay.【Results】The ABA content of Malus sieversii seeds showed a decreasing trend with the increase of storage time, the maximum content was 80.22 ng·g-1 at 0 d of storage, the content was significantly lower than that of the three periods of 0, 30, and 90 d at 60 d of storage (P ＜0.05), the content reached a minimum of 43.67 ng·g-1 at 120 d. The GA content showed an increasing trend with the increase of storage time, the content was the lowest at 78.42 pmol·g-1 at 0 d, and significantly higher than the other periods at 120 d (P＜0.05), and reached a maximum of 170.67 pmol·g-1 at 120 d. The IAA content showed an increasing trend with storage time, and the content was significantly higher at 90 d than other periods, reaching a maximum value of 41.36 nmol·g-1. The CTK content was significantly higher at 90 d than other periods, reaching a minimum value of 43.67 ng·g-1, it showed a decreasing trend in storage time 0-30 d, 30 d was significantly lower than other periods (P＜0.05), reaching a minimum value of 31.34 ng·g-1, and showed an increasing trend in storage time 30-60 d, suggesting that CTK promotes the accumulation of seed assimilates in this period. The activities of ACC oxidase and ACC synthase, two key enzymes showed inconsistent trends during storage in the ethylene biosynthesis pathway. The activity of ACO was 262.52 ng·g-1 at 90 d of storage, which was significantly higher than that of other periods (P＜0.05), and the activity of ACO was the lowest at 30 d of storage, which was 157.38 ng·g-1. The activity of ACS reached the maximum value of 418.92 ng·g-1 at 120 d of storage, which was significantly higher than that of the three periods of 0, 30 and 60 d (P＜0.05). It indicated that low-temperature sand storage stratification promoted the synthesis of GA, IAA, CTK, ACC oxidase and ACC synthase, and two enzymes of the ethylene synthesis pathway might be more sensitive to low temperature. There were more significant DEGs in M120d_CK120d relative to M0d_CK0d and M30d_CK30d, and in M60d_CK60d and M90d_CK90d, suggesting that the regulation of seed germination and physiological changes were more DEGs in M120 d. There were a total of 7384, 4875 and 3236 significantly differentially expressed genes, significantly differentially up-regulated genes and significantly differentially down-regulated genes in the M30d_CK30d and M60d_CK60d periods, respectively. Gene ontology enrichment of DEGs was performed, and the biological processes were mainly involved in the response to osmotic stress and water deprivation, abscisic acid response, response to salt stress, transcriptional regulation, regulation of seed germination, and gibberellin response. The cellular components are mainly chloroplast stroma, chloroplast envelope and thylakoid. Molecular functions are mainly related to DNA-binding transcription factor activity, phosphatase activity and protein homodimer activation. Multiple plant hormone biological processes remained active and changed during low-temperature sand storage stratification of Malus sieversii seeds, suggesting that it play a role in seed dormancy release activities. KEGG pathway enrichment analysis showed that the main pathways enriched in seeds during low-temperature sand storage stratification at different periods were plant hormone signaling, MAPK signaling pathway-plant, protein processing in endoplasmic reticulum, starch and sucrose metabolism, and glycolysis/glycogenesis. Among them, plant hormones play a key role in regulating seed dormancy and germination in Malus sieversii, and hormone signal transduction pathway-related genes such as ABA, GA, and ETH are involved in processes such as seed dormancy release. Starch and sucrose metabolic pathways are involved in the process of carbon metabolism in the seed embryo, providing carbon source for the seed embryo. Glycogen production metabolic pathway is involved in the synthesis and metabolism of cellular proteins, providing nitrogen source and energy for seed embryo germination. ABA receptor PYR/PYL had 12 genes up-regulated and 3 genes down-regulated, ABA signal transducer protein phosphatases 2C(PP2C) and positive regulator GA receptor gibberellin insensitive dwarf 1(GID1) genes showed up-regulated Three genes were up-regulated and one gene was down-regulated for sucrose non-fermenting-1-related protein kinase 2(SnRK2), and two genes were up-regulated for ABA catabolic hydroxylases 8'-hydroxylases. GA signaling the negatively regulated growth inhibitor DELLA protein was up-regulated in 6 genes and down-regulated in 1 gene, indicating that low-temperature stratification treatment enhanced GA signaling. ETH and IAA were significantly differentially expressed in different stratification stages of Malus sieversii embryos. The ETH receptor ETR, ETHYLENE INSENSITIVE 3/EIN3-LIKE, 1-aminocyclopropane-1-carboxylic acid, a direct precursor of ETH, and ACO were up-regulated expression. 5 IAA-related genes were up-regulated and one was down-regulated, and small auxin up-regulated RNA was up-regulated, indicating that low-temperature stratification promotes IAA synthesis. Cell cycle protein genes were up-regulated to meet the nutritional growth of seed dormancy release. In addition, genes related to sucrose metabolism were screened, sucrose synthase and endoglucanase were up-regulated during low-temperature sand storage stratification. sucrose transport protein STP13 and STP14 were up-regulated, while STP5 and STP10 were down-regulated.【Conclusion】The expression of ERF2-like showed an opposite trend to the changes of ACO and ACS activities, and the expression of PYR1-like and WRKY33 genes decreased in relation to the changes of ABA content, suggesting that the above genes may be involved in the ABA signaling pathway to regulate the process of dormancy release process of Malus sieversii seeds.
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