Transcriptome analysis showed that tomato-rootstock enhanced salt tolerance of grafted seedlings was accompanied by multiple metabolic processes and gene differences
Purpose: Grafting is a commonly used cultural practice to counteract salt stress and is especially important for vegetable production. However, it is not clear which metabolic processes and genes are involved in the response of tomato rootstocks to salt stress. Our goals is to elucidate the regulatory mechanism through which grafting enhances salt tolerance. Methods: The salt-sensitive tomato variety Zhongza 9 and the strongly salt-tolerant tomato rootstock variety QZ-006 (selected by the research group in the early stage) were used as experimental materials and were purchased from the Vegetable and Flower Institute of the Chinese Academy of Agricultural Sciences (Beijing, CHN) and Beijing Kaixingelin Agricultural Technology Co., Ltd. (Beijing, CHN);The leaves of the seedlings of every sample was added to 5 mL of HNO3 (65%~68%) after they were heated in a microwave digestion system for 2~3 hours;RNA-seq libraries were sequenced on an Illumina HiSeq X Ten platform;qRT?PCR was performed according to the instructions of a Fast Super EvaGreen qPCR Master Mix Kit (US Everbright®, Inc.);The amino acid contents in the tomato leaves were calculated by the external standard method. The chlorophyll of the seedlings was extracted with acetone-ethanol (1:1) ;An indirect enzyme-linked immunosorbent assay (ELISA) was used to determine auxin (IAA), gibberellin (GA), cytokinin (ZR), brassinolide (BR), abscisic acid (ABA), jasmonic acid (JA) and salicylic acid (SA) contents, and kit purchased from ThermoFisher Scientific Co., Ltd. (USA). Endogenous ethylene (ETH) was collected, fixed and determined according to the methods of Ling (Ling et al.,2008) using a Shimadzu 2010 gas chromatograph equipped with a hydrogen flame ion detector. ETH standard gas was purchased from Sigma Company. Results: Compared with the NGS, the GSs were more salt tolerant, and the Na+ content in the leaves decreased significantly. Through transcriptome sequencing data analysis of 36 samples, we found that GSs exhibited more stable gene expression patterns, with a lower number of DEGs. WRKY and PosF21 transcription factors were significantly upregulated in the GSs compared to the NGSs. Moreover, the GSs presented more amino acids, a higher photosynthetic index and a higher content of growth-promoting hormones. The main differences between GSs and NGSs were in the expression levels of genes involved in the BR signalling pathway, with significant upregulation of XTHs. Conclusions: Our study demonstrates that grafting on salt tolerant rootstocks can bring different metabolic processes and transcription levels changes to scion leaves, thereby the scion leaves show stronger salt tolerance. This information provides new insight into the mechanism underlying tolerance to salt stress regulation and provides useful molecular biological basis for improving plant salt resistance. Overall design: We first evaluated the salt damage index, electrolyte permeability and Na+ accumulation in tomato (Solanum lycopersicum L.) leaves of grafted seedlings (GSs) and nongrafted seedlings (NGSs) subjected to 175 mmol·L- 1 NaCl for 0-96 h,covering the front, middle and rear ranges.The experiment was conducted at the scientific research base of Hebei Agricultural University. (38 ° 10 '- 40 ° 00' N, 113 ° 40 '- 116 ° 20' E). When the tomato seedlings developed four leaves and one heart, the split grafting method was used to obtain GSs. We selected GSs and NGS that were uniform and displayed good growth, rinsed the roots, used Hoagland's nutrient solution formula for hydroponic cultivation, and used an oxygen pump to supplement oxygen to maintain the normal growth of the grafted tomato seedlings for 15 days after grafting