De novo transcriptome sequencing and gene expression pro�ling of Pinus sibirica under different cold stresses

Background: Pinus sibirica is an evergreen conifer tree species with strong cold stress. However, the transcriptional regulation patterns in response to cold stress have not been reported for P. Sibirica. To gain deeper insights into its regulation process of cold tolerance, transcriptome pro�ling analyses and 12 physiological indices measurement were performed under cold stress (-20 ℃ ) over time. Results: More than 54.1 million clean reads were produced, which were assembled into 97,376 unigenes. Among them, 56,994 unigenes had homology with known genes, 36,836 were assigned to 51 GO (gene ontology) categories and 46,972 were assigned to 24 COG (clusters of orthologous group) categories. P. sibirica showed the highest similarity with sequences from Picea sitchensis. In total, 871, 1397 and 872 DEGs (differentially expressed genes) were identi�ed upon exposure to cold for 6 h, 24 h and 48 h at -20 ℃ , respectively. Nine physiological indices increased signi�cantly (P<0.05) under cold stress, including membrane permeability, relative conductivity, reactive oxygen species, malonaldehyde, peroxidase activity, catalase activity, soluble sugar, soluble protein and proline content. With extension of the cold stress time, 9 physiological indices generally showed a trend toward �rst an increase and then a decrease. The net photosynthetic rate, stomatal conductance and transpiration rate in P. sibirica dropped sharply (P<0.05) in response to cold stress, and they were also decreased signi�cantly (P<0.05) with extension of the stress time at -20 ℃ . Conclusions: There were two cold signal transduction pathways in P. sibirica, the Ca 2+ and ABA (abscisic acid) pathways. The AP2 (ethylene-responsive transcription factor) family and some other transcription factors played an important role in transcriptional regulation. P. sibirica underwent antioxidant and osmotic regulation with changes in the of genes related to cold resistance. Photosynthesis was inhibited, and more DEGs associated with photosynthesis were downregulated under cold stress. The DEGs identi�ed cold signal oxygen species (ROS), malonaldehyde (MDA), peroxidase (POD), catalase (CAT), soluble sugar, soluble protein, proline, net photosynthetic rate, stomatal conductance and transpiration rate. Simultaneously, RNA-seq was performed. This study provides insights into the molecular mechanisms of resistance to cold stress in P. sibirica. The identi�cation of cold response genes such as those encoding signal transduction proteins, TFs, relevant antioxidant enzymes and osmotic substances would provide a valuable genetic resource in future conifer breeding. This study provided a comprehensive description of transcriptomic responses to cold stress in the leaves of P. sibirica with different stress times. Based on physiological data and transcriptomic DEGs data, a cold response model in P. sibirica was roughly summarized. Cold signal was �rst perceived by the plant cell membrane, with membrane rigidity at microdomains, and then the cold signal was transmitted downstream through Ca 2+ and ABA signaling pathways to activate relevant TFs. The ICE-CBF pathway played an important role in the transcriptional regulation of cold stress, as well as the CBF-independent pathway. The action of the TFs triggered a cascade of downstream COR gene, synthesizing various antioxidant enzymes and osmotic protective substances to modulate cellular metabolism homeostasis and improve tolerance to cold stress. Photosynthesis is an essential metabolic process in plants, and it was affected by cold stress even in P. sibirica with strong cold tolerance. The rough cold response model and DEGs involved in the model may facilitate studies of molecular breeding improvement of conifer species.