- splicing landscape during cold acclimation in tea plant. - AS has been implicated in cold acclimation by affecting gene expression in various ways, yet little information is known about how AS influences cold responses in tea plant ( Camellia sinensis. - Results: In this study, the AS transcriptional landscape was characterized in the tea plant genome using high- throughput RNA-seq during cold acclimation. - We summarize the possible existence of 11 types of AS events, including the four common types of intron retention (IR), exon skipping (ES), alternative 5 ′ splice site (A5SS), and alternative 3 ′ splice site (A3SS). - The number of AS events increased rapidly during cold treatment, but decreased significantly following de-acclimation (DA). - Remarkably, the AS isoforms of bHLH transcription factors showed higher expression levels than their full-length ones during cold acclimation. - Conclusion: Our findings demonstrated that changes in AS numbers and transcript expression may contribute to rapid changes in gene expression and metabolite profile during cold acclimation, suggesting that AS events play an important regulatory role in response to cold acclimation in tea plant.. - Cold acclimation (CA) is an im- portant mechanism that has been widely reported to im- prove cold resistance of plants by modulating numerous physiological and biochemical processes [1–3]. - For instance, cold acclimation improves the tolerance of North American Rhododendron from − 7 °C to − 53 °C [4]. - In addition to increases of these substances, many transcription factors and oxidoreductase regulatory mechanisms are also present in plants during cold acclimation. - Plants can also defend against active oxygen through the protection system of oxidoreductases during cold acclimation [9]. - Cold acclimation grants the ability to withstand low temperature and plays an important role in the growth and development of plants.. - The targets of AS in rice were found to include three Serine/. - In tea plant, the contents of sucrose, glucose and fructose were found to be constantly ele- vated during cold acclimation [27]. - But these studies have not focused on the ef- fect of AS on cold stress in tea plants. - In this study, we investigated AS events during cold acclimation with genome-wide analysis in tea plant, which detected a large number of AS occurrences.. - Meanwhile, variations of AS and the related biological functions were analyzed during cold acclimation. - Our results indicated that AS may regulate gene expression and contents of metabolites during cold acclimation. - A number of different AS patterns were found being in- volved in transcriptional regulation during the process of cold acclimation in tea plants, especially the change and function of AS at de-acclimation. - This provides a better understanding of the functions of AS in tea plants responding to cold acclimation.. - Global identification and classification of AS events The RNA-seq data was used to investigate AS events at different periods of cold acclimation (Additional file 4:. - During the sharp cooling treatment of tea plants, the numbers of AS events under low-temperatures [cold acclimation of 6 h at 10 °C, day/night temperature (CS), cold accli- mation of 7 days at 10/4 °C, day/night temperature (CA1) and cold acclimation of 7 days at 4/0 °C, day/. - It is noteworthy that the number of AS events (24,616) tremendously decreased under temperature recovery condition compared with both CA treatment and NA groups, implying that many AS genes had adapted to low-temperature environment and resulted in reduced occurrence of AS events during DA (de-acclimation) of tea plant.. - Among 14,103 identified AS genes at five different time points, 3779 AS genes were conserved during cold acclimation (Fig. - 1 Statistics of all AS events at different time points of cold acclimation treatments. - CA1: cold acclimation of 7 days at 10/4 °C, day/night. - CA2: cold acclimation of 7 days at 4/0 °C, day/night. - The tendency represents the changes in the number of AS events at cold acclimation time points. - Dynamic characterization and analysis of AS events during cold acclimation. - To investigate variation in types of AS events during cold acclimation, we calculated the proportion of the four main AS events (IR, A5SS, A3SS and ES) among the five periods. - It was demonstrated that the number of DAGs gradually increased during cold ac- climation (Fig. - Interest- ingly, pathways relating to sugar, serine and oxidoreduc- tase activity were significantly enriched after cold acclimation compared with non-acclimation.. - Characterization and expression pattern of AS genes during cold acclimation. - To better understand the putative impact of AS during cold acclimation, AS genes associated with cold stress were in- vestigated. - 3 Analysis of AS events at different periods of cold acclimation.a Venn diagram showing the common and unique AS genes at different periods of cold acclimation (NA, CS, CA1, CA2, DA). - CA1: cold acclimation for 7 days at 10/4 °C, day/night. - CA2: cold acclimation for 7 days at 4/0 °C, day/night. - The expression levels of AS transcripts vary significantly among different time periods (Fig. - a Numbers of DAGs and overlap of AS genes among all time points compared with non-acclimation. - b Gene Ontology (GO) enrichment of DAGs of NA vs CS and NA vs DA in cold acclimation ( p <. - CA2: cold acclimation of 7 days at 4/. - The relationship between AS transcripts and metabolites during cold acclimation. - We analyzed the changes of these three metabolite categories together with glucose, fructose, sucrose and raffinose measured in the previous study [28] during cold acclimation (Fig. - We found that a large number of AS variants were in- volved in sugar metabolism (Fig. - Alternative splicing (AS) plays an important role in plant responses to biotic and abiotic stresses such as cold stress and pathogen defense [13, 33]. - severe abiotic stresses in tea plant, and a series of studies have been focusing on tea plant gene regulation during cold stress and cold acclimation . - However, little information is known about AS in tea plant during cold acclimation. - In this study, we in- vestigated the total AS events during cold acclimation in tea plant through high-throughput RNA-seq. - The num- bers and expression levels of AS events varied greatly during cold acclimation, and some AS transcripts were strongly correlated with sugar content (correlation coef- ficient >. - This study provides novel understanding of the roles of AS for cold acclimation in tea plant.. - But little is known about the number of AS changes in response to cold acclimation. - During cold acclimation,. - 6 Expression of AS transcripts analyzed by qRT-PCR during acclimation. - we found that the number of AS transcripts increased about 3000 in response to low temperature, but de- creased by about 6000 during DA (Fig. - this im- plies that low temperature can induce new AS event and AS genes may be adapted to low-temperature en- vironment, which would explain the reduced occur- rence of AS events during DA. - 9 (SRA database number: SRP108833), and found that the number of AS was also increased during CA2 in Yinghong 9 and CA3 in Longjing43, but with differ- ent extents, probably because the varieties and treat- ment conditions were different (Additional file 6:. - Low temperature can affect not only the variation of AS number but also the expression level of AS transcripts to generate a large number of DAGs. - 7 Characteristics of metabolites during cold acclimation. - gradually with increasing cold acclimation (Fig. - In A.thaliana, sugar metabolism also significantly increased during cold acclimation [5]. - 8 Correlation analysis of AS transcripts and sugar content. - The occurrence of AS is influenced by low temperature.. - In addition, a large number of AS events were found in the sugar metabolism and antioxidant pathways such as CsSUS, CsRS and CsPOD. - However, the effect of AS on bHLH transcription factors during cold acclima- tion has not been previously explored. - regulation during cold acclimation. - It is well established that metabolites contents changed dramatically during cold acclimation [50, 51]. - For example, most flavonols and anthocyanins accumulated during cold acclimation in A.thaliana [52], and the accumulation of tri- enoic fatty acid during acclimation is one of the necessary conditions for normal development of leaves at low temperature in tobacco [7]. - In addition, sugar has been widely studied as a class of metabolites playing a role in cold acclimation [5, 53]. - However, a large number of AS events in sugar-related genes have not been reported. - In our correlation analysis of AS and sugar content, levels of the full-length transcript of CsRS-1 (TEA013682.1) had a significant positive correlation with raffinose levels, while levels of its AS isoform CsRS-2 (TEA013682.2) were positively correlated with glucose, su- crose and fructose (Fig. - In conclusion, we analyzed the AS events during cold acclimation of tea plant based on the transcriptome. - Based on these results, we propose that changes in abundances and expression levels of AS transcripts may influence sugar metabolism and the antioxidant enzyme system to impart resistance to cold stress. - This study provides an essential insight into AS events involved in tea plant cold acclimation, and highlights the importance of AS regulatory function during low temperature stress.. - The tea plants were subjected to cold acclimation and de-acclimation with the treatments as follows: Ten well-grown plants were collected for treatments of non- acclimation (NA), CS (sampling after 10 °C for 6 h), CA1 (sampling after 10/4 °C, day/night for 7 days), and CA2 (sampling after 4/0 °C, day/night for another 7 days). - 9 Regulation model of alternative splicing (AS) transcripts in cold acclimation. - Characterization of alternative splicing. - To identify the type of AS events, the AStalavista tool [65]. - Major types of AS events including intron retention (IR, AS code: 1^2-,0), exon skipping (ES, AS code al- ternative 3′ splice site (A3SS, AS code: 1-,2. - Validation of alternative splicing. - Total RNA was isolated from tea plants at different time points of cold acclimation as described above. - smart.embl-heidelberg.de/) was used to predict the domain of AS transcripts with the protein sequences.. - Comparison of AS events in Shuchazao, Longjing43 and Yinghong 9.. - Percentages of four main AS types during cold acclimation.. - Expression analysis of AS transcripts during cold acclimation.. - Correlation analysis of AS transcriptions and sugar content.. - AS: Alternative splicing. - CA: Cold acclimation. - CA1: Cold acclimation of 7 days at 10/4 °C, day/night. - CA2: Cold acclimation of 7 days at 4/0 °C, day/night temperature. - Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. - Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. - A global survey of gene regulation during cold acclimation in Arabidopsis thaliana . - Transcript and metabolite profiling during cold acclimation of Arabidopsis reveals an intricate relationship of cold-regulated gene expression with modifications in metabolite content. - Molecular regulation of CBF signaling in cold acclimation. - Protective systems against active oxygen species in spinach: response to cold acclimation in excess light. - Effects of cold acclimation on sugar metabolism and sugar-related gene expression in tea plant during the winter season. - Global transcriptome profiles of Camellia sinensis during cold acclimation. - Changes in sugar content and fatty acid composition of in vitro sugar beet shoots after cold acclimation: influence on survival after cryopreservation.. - Flavonoids are determinants of freezing tolerance and cold acclimation in Arabidopsis thaliana. - Natural variation in flavonol and anthocyanin metabolism during cold acclimation in Arabidopsis thaliana accessions. - Changes in proline and soluble sugars content during cold acclimation of winter rapeseed shoots in vitro
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