Analysis of RNA-seq libraries
For the LT experiments, two cDNA libraries constructed from grapevine buds collected on April 23 at the ED stage [14] and maintained at 14 ºC (control) and 4 ºC (LT) for one week were sequenced. Two replicates for each treatment were evaluated, and the results concerning the total reads, mapped reads and unique reads are shown in (Table. S1). From the two cDNA libraries, differential gene expression (DEG) analysis revealed 6121 significant differential expressed genes (DEGs), of which 4098 were upregulated and 2093 were downregulated in the buds subjected to LT relative to the control buds (Fig. 1). The DEGs for the buds subjected to SD relative to long days ( LDs) were revealed from [13], 1336 DEGs were reported, of which 400 were upregulated and 936 were downregulated (Fig. 1).
Genes Differentially Expressed In Grapevine Buds Subjected To Lt And Sds
To analyze the genes that were differentially expressed in grapevine buds subjected to LT and SDs, a Venn diagram was constructed (http://bioinfogp.cnb.csic.es/tools/venny/index.html). The results showed that 16 upregulated genes were differentially expressed in grapevine buds subjected to LT and SDs, that 36 downregulated genes were differentially expressed in grapevine buds subjected to both stimuli, that 292 DEGs were upregulated in response to LT and downregulated in response to SDs, and that 46 DEGs were downregulated in response to LT and upregulated in response to SDs (Fig. 2).
Cluster Analysis Of Degs
A cluster analysis of a subset of DEGs expressed in response to LT and SDs was performed using log2(ratio) values for the transcript expression analysis (Fig. 3). The cluster analysis grouped upregulated and downregulated transcripts separately. The results showed that, compared with LD, SD downregulated the expression of most DEGs, while compared with the control conditions, LT upregulated the expression of most DEGs. In addition, the expression of a small number of DEGs simultaneously increased or decreased in response to SDs and LT, while a large number of DEGs whose expression decreased in response to SDs but increased in response to LT were detected (Fig. 3).
Enriched GO categories shared by grapevine buds subjected to LT and SDs based on upregulated DEGs
Enriched GO categories shared by grapevine buds subjected to LT and SDs were determined with the list of upregulated DEGs under both treatments by the use of the multiple query function of the g: Profiler software [15]. The results showed that despite the large number of molecular functions (MF:GO), biological processes (BP:GO) and cellular components (CC:GO) specifically enriched in response to each of the treatments, no MF:GO and few BP:GO were simultaneously enriched in response to both treatments (Fig. 4). The enriched MF:GO, BP:GO and CC:GO shared in response to both treatments are indicated by the same number in both graphs shown in Fig. 4. The only enriched BP:GO shared in response to both treatments was related to polysaccharide (GO:0005976) and carbohydrate metabolic processes (GO:0005975) (Table S2).
Enriched GO categories shared by grapevine buds subjected to LT and SDs based on downregulated DEGs
Enriched GO categories in grapevine buds subjected to LT and SDs were determined with the list of downregulated DEGs under both treatments by the use of the multiple query function of the g: Profiler software [15]. The enriched GO categories shared in response to both treatments are indicated by the same number in both plots shown in Fig. 5. The results indicated that 5 (MF:GO), 18 (BP:GO) and 2 (CC:GO) were shared in response to both treatments. All 5 MF:GO shared in response to both treatments were related to DNA-binding transcription factor activity (GO:0003700), transcription regulatory activity (GO:0140110) and DNA-binding (GO:000677) (Table S3).However, the 18 BP:GO shared in response to both treatments were related to biological regulation (GO:006507), regulation of nitrogen compound metabolic processes (GO:0051171); regulation of primary metabolic processes (GO:0080090); regulation of cellular metabolic processes (GO:0031323), regulation of transcription, DNA-templated (GO:006355); regulation of RNA biosynthetic process (GO:2001141); regulation of macromolecule biosynthetic processes and regulation of gene expression (GO:0010468) (Table S3). Last, the 2 CC:GO shared in response to both treatments were related to the nucleus (GO:0005634).
Enriched GO categories shared by grapevine buds subjected to LT and SDs based on upregulated and downregulated DEGs
According to our Venn diagram, there was a large number of DEGs that were upregulated in grapevine buds subjected to LT but that were downregulated in grapevine buds subjected to SDs (Fig. 3). To investigate the enriched GO categories shared by grapevine buds subjected to LT and SDs, we analyzed the list of DEGs upregulated in response to LT, and the list of DEGs downregulated in response to SDs by using the multiple query function of g: Profiler software [15]. The enriched GO categories shared in response to both treatments are indicated by the same number in both plots shown in Fig. 6. The results indicated that a few MF:GO were shared in response to both treatments and were related to DNA-binding transcription factor activity (GO: 0003700); microtubule binding (GO:008017); tubulin binding (GO:0015631); kinase regulatory activity (GO:0019207) and xyloglucanxyloglucosyl transferase activity (GO: 001672) (Table S4). In contrast, many BP:GO terms were shared in response to both treatments (Fig. 6), and most of them were related to the cell cycle (GO:0007049), cell cycle processes (GO:0022402); the mitotic cell cycle (GO:0000278) and mitotic cell cycle processes (GO:1903047) (Table S4). Additionally, many CC:GO subcategories related mainly to the cell wall (GO:0005618); the microtubule cytoskeleton (GO:0015630); the supramolecular complex (GO:0099080); microtubules (GO:0005874); the cytoskeleton (GO:0005856) and polymeric cytoskeleton fibers (GO:0099513) were shared in response to both treatments (Fig. 6).
Validation Of Rna-seq By Rt-qpcr
Six DEGs were chosen for RT-qPCR analysis to test the reproducibility of the RNA-seq analysis results. In each case, the results from the RT-qPCR and the RNA-seq assays exhibited the same trends (Fig. 7).