Changes of endogenous hormone concentrations and transcriptome analysis during the healing process of greenwood grafting in grape

【Objective】To study the changes of hormone concentrations in the graft union during the healing process of greenwood grafting, the experiment was carried out, combined with transcriptomic data analysis, so as to identify the key hormone changes in the healing process of greenwood grafting in grape, and provide references for revealing the molecular mechanism of the healing process.【Methods】The stion combinations of Tiangong Liren/Beta, Tiangong Moyu/Beta and Shine Muscat/Beta were used as materials and were collected at 1, 4, 7, 10 and 13 days after grafting (DAG), respectively. The concentrations of auxin (IAA), isopentenyladenine (IP), abscisic acid (ABA) and jasmonates (JAs) in the graft union were determined by high performance liquid chromatograph-triple quadrupole tandem mass spectrometry (HPLC-QqQ-MS/MS). Simultaniously, transcriptome sequencing was performed on the graft union of Tiangong Liren/Beta.【Results】From the perspective of morphology, Tiangong Moyu/Beta buds burst first, and the bud swelling was observed at 4 DAG. Tiangong Liren/Beta and Shine Muscat/Beta were observed to have swollen buds at 7 DAG. Different stion combinations com-pleted the healing process within 13 DAG. From the analysis of plant hormone levels, the concentration of IAA increased, while the concentrations of IP, ABA and JAs decreased during healing. From transcriptome level analysis, the transcriptome data of Tiangong Liren/Beta at different stages were pairwise compared, and 6319 differentially expressed genes were screened. 1 DAG vs 13 DAG had the largest number of differentially expressed genes, with 3846, the second was 1 DAG vs 10 DAG, with 3301 and the third was 1 DAG vs 7 DAG, with 2968. The results showed that the gene transcriptional regulation of 7 DAG, 10 DAG and 13 DAG were significantly changed compared with 1 DAG. The 10 DAG vs 13 DAG comparison combination had the smallest number of differentially expressed genes, indicating that there was little difference in the gene expression pattern between 10 DAG and 13 DAG. 6319 differentially expressed genes were divided into 6 clusters by time series analysis. Cluster 5 contained the most differentially expressed genes, which were significantly enriched in xyloglucan, including xyloglucosyl transferase activity, xyloglucan metabolic process and other pathways, its expression showed a downward trend in the whole healing process, and the trend was obvious at 1-4 DAG. The differentially expressed genes contained in Cluster 3 were enriched in auxin-activated signaling pathway, phloem or xylem histogenesis and other pathways, and their expression levels increased at first week after grafting and were higher at 4-10 DAG. In the process of grapevine greenwood grafting healing, 27 differentially expressed genes were screened to participate in the synthesis and metabolism of plant hormones, and 3, 2, 12 and 10 differentially expressed genes were involved in the synthesis and metabolism of auxin, cytokinin, abscisic acid and jasmonic acid, respectively. In the tryptophan-dependent auxin synthesis pathway, L- tryptophan decarboxylase TDC (Vitvi07g00696) and aldehyde dehydrogenase ALDH (Vitvi04g01402) had the highest expression levels at 7 DAG. Isopentenyltransferase IPT (Vitvi07g00154) was highly expressed at first week after grafting, and then showed a downward trend. The expression of cytokinin dehydrogenase CKX (Vitvi04g00161) decreased significantly at 1-4 DAG and 7-10 DAG by 0.66 and 0.60 times, respectively. The expression level of 9- cis- epoxycarotenoid dioxygenase NCED (Vitvi19g01356) was higher at 1-4 DAG and decreased at 4-10 DAG, which was similar to the changing trend of abscisic acid concentration. The expression levels of LOX (Vitvi06g00158), AOS (Vitvi18g00886), OPR (Vitvi18g03162 and Vitvi18g04622) and OPCL1 (Vitvi18g00124) generally decreased at 1-10 DAG, which were consistent with the changing trend of jasmonic acid concentration. 49 differentially expressed genes were selected to participate in plant hormone signal transduction pathway. There were 17 differentially expressed genes involved in auxin signal transduction. They included auxin influx carrier (AUX1), transport inhibitor response protein 1 (TIR1), auxin/indole-3-acetic acid (Aux/IAA), auxin response factor (ARF), acetic acid- amido synthetase (GH3), small auxin- up RNA (SAUR), auxin binding protein 1 (ABP1), plasma membrane H+ -transporting ATPase (AHA) and mitogen activated protein kinase 3 (TMK3). Two-component response regulators (A-ARR) (Vitvi13g01433 and Vitvi17g00732) involved in cytokinin signal transduction were highly expressed at first week after grafting. There were 8 differentially expressed genes were involved in abscisic acid signal transduction, including abscisic acid receptor PYR/PYL, protein phosphatase 2C (PP2C), sucrose non-fermenting 1- related protein kinase 2 (SnRK2) and ABA-responsive element binding factor (ABF). There were 5 differentially expressed genes were involved in jasmonic acid signal transduction, including jasmonic acidamino acid synthetase (JAR1), jasmonate ZIM domain-containing protein (JAZ) and transcription factor MYC2. There were 8 differentially expressed genes were involved in brassinosteroid signal transduction, including brassinosteroid resistant 1/2 (BZR1/2), cyclin D3 (CYCD3) and xyloglucan endotransglucosylase protein (TCH4). There were 3 differentially expressed genes were involved in gibberellinsignal transduction, including gibberellin receptor 1 (GID1) and DELLA protein. There were 3 differentially expressed genes were involved in ethylene signal transduction, including ethylene receptor (ETR), EIN3-binding F-box protein (EBF) and ethylene-responsive transcription factor (ERF). These three differentially expressed genes involved in salicylic acid signal transduction were pathogenesis-related protein 1 (PR1). By WGCNA analysis of transcriptome data, 13 co-expression modules were obtained. The correlation analysis between the module and plant hormone showed that GH3 (Vitvi03g00586), LOX (Vitvi06g00158), AOS (Vitvi18g00886), OPCL1 (Vitvi18g00124), ABF (Vitvi12g01667) and TCH4 (Vitvi11g01682) were positively correlated with the concentrations of JA and IP. AHA (Vitvi14g01888) and TIR1 (Vitvi07g00248) were negatively correlated with the concentrations of ABA and IP.【Conclusion】The changes of plant hormone concentrations in the process of greenwood grafting healing were stage-specific. IAA played a key role in the process of grafting healing. IP, ABA and JAs regulated the healing process by interacting with IAA signal transduction.