Transcriptomic analysis of early responses of apple flower buds to low temperature before tree dormancy

Abstract：【Objective】Understanding the early responses of apple flower buds to low temperature during dormancy is very important for cold resistance breeding. Various studies have focused on cold responses of multiple plants, such as Arabidopsis, rice, and tomato. Woody plants need to undergo dormancy and have a distinct cold response. Apple is an important fruit tree and can be considered as a model woody plant in laboratory. Previous investigations regarding the transcriptome of the cold responses in apple mainly focused on the leaf tissue in the growing season, little attention has been paid on the flower buds regarding the analysis of differentially expressed genes under the early response to low temperature. Investigation of the early response to cold in apple flower buds before tree dormancy (after fruit harvesting) is helpful for exploring the tolerant mechanism of woody plants during dormancy.【Methods】Flower buds were collected and treated at low temperature (4 ℃) for 45 min (T1), 90 min (T2) or 240 min (T3) on September 25, 2017 and immediately frozen in liquid nitrogen and stored at -80 ℃. Normal temperature treatment (28 ℃) was regarded as the control (C). Total RNA was isolat ed by using the Plant RNAout Kit (160906-50, Tiandz Inc., Beijing, China). Qualified RNA sampleswere used for mRNA purification and cDNA synthesize. After that, cDNA library was constructed and sequenced using the Illumina HiSeq™ 2000. The differentially expressed genes (DEGs) were screened with the Noiseq software. We used a |log2Fold Change| ≥ 2 and p- value ≤ 0.05 as the thresholds of DEGs. The Gene Ontology (GO) and‘Kyoto’Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were conducted using the WEGO software and KEGG online database, respectively. The 8 most enriched GO terms and 8 KEGG pathways in“Biological Process”were extracted and were further validated by quantitative Real-time PCR (qRT-PCR). Transcriptomic data of each sample were obtained and analyzed by high-throughput sequencing. The expression pattern of 9 DEGs involved in enriched GO terms or KEGG pathways were further analyzed by qRT-PCR.【Results】Compared with the control (T0), 237, 508 and 990 differentially expressed genes (DEGs) were detected from T1, T2 and T3, respectively. GO enrichment analysis revealed that the DEGs mainly associated with carbohydraterelated metabolism and single- organism carbohydrate metabolism. For the above enriched GO terms, most of DEGs were up-regulated in the early stage of treatment. In addition, DEGs mainly involved in stimulus response, stress response and DNA transcription in the later stage, and most of these were upregulated. These results indicated that stimulation and stress related DEGs were involved in the early response of apple flower buds to low temperature. According to the results from KEGG enrichment, pathways involved in“plant-pathogen interaction”,“plant hormone signal transduction”,“flavone and flavonol biosynthesis”, and“flavonoid biosynthesis”were significantly enriched (p ≤ 0.05). The above results exhibited that Ca2+ signaling pathway and hormonal signal transduction were involved in Calmodulin/calmodulin proteins (Ca2 + - CaM/CML) metabolism and a variety of hormonal signal transduction pathways. For Ca2 + associated pathway, many DEGs encoding CaM/CML (such as MDP0000808334, MDP0000842520) and WRKY transcription factor (such as MDP0000263349) were significantly upregulated. Furthermore, we detected a large number of up-regulated DEGs involved in Ca2+ signal pathway, such as the DEGs encoding FLS2 (MDP0000728753) and BAK1/BKK1 membrane proteins (MDP0000122792) were significantly up-regulated. In addition, plant hormone signaling pathway also played an indispensable role in the cold response process in apple flower buds. The DEG (MDP0000287039) encoding DELLA protein of gibberellin (GA) metabolism and generating response to GA signal transduction pathway was significantly up- regulated. Furthermore, the key gene MDP0000189486 of ABA metabolism was up- regulated significantly in response to low temperature stress. The key gene MDP0000122792 that encodes a BAK1 protein of brassinosteroid (BR) metabolism and generates response to BR synthesis signal transduction pathway was up-regulated significantly. In addition, in jasmonic acid (JA) synthesis signal transduction pathway, 6 DEGs were up-regulated significantly in the early stage of treatment, which may be related to the expression of stress-related genes, whereas 3 DEGs among them were not obvious. The similar expression patterns of 9 DEGs between RNA- seq and qRT-PCR assays indicated the reliability of the RNA- seq data.【Conclusion】In conclusion, Ca2 + signaling pathway was mainly involved in the cold response of apple flower buds. In addition, ABA, BR and other hormones may also play an important role in regulating the response of apple flower buds to low temperature signals.