- Transcripts encoding several key enzymes involved in chlorophyll biosynthesis were also up-regulated, accompanied by significant differences in the contents of 5-aminolevulinic acid (ALA), porphobilinogen (PBG), protoporphyrin IX (Proto IX), Mg-protoporphyrin IX (Mg-proto IX), protochlorophyllide (Pchl), and photosynthetic pigments. - 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - Full list of author information is available at the end of the article Zhao et al. - a crucial species in the Brassica- ceae family [1]. - Temperature had a significant effect and all 4 of the parameters were significantly dif- ferent between LT and Cont. - There were no significant differences in SLA, total soluble sugar content, and MDA between LT + EBR and Cont (Fig. - The largest change was in the total soluble sugar con- tent among the 4 indicators. - 2 Effects of exogenous EBR application on SLA (a), MSI (b), total soluble sugar content (c), and MDA (d) in wucai leaves under LT stress. - Compared to Cont, MDA content significantly increased by 49.81% in LT plants, and significantly decreased by 30.95% in LT + EBR (Fig. - Compared to LT, SLA signifi- cantly increased by 46.80% in LT + EBR plants (Fig. - Com- pared to LT, SLA significantly increased by 3.27% in LT + EBR (Fig. - Two libraries were constructed from the LT and LT + EBR treatment groups for analysis by RNA-Seq. - The proportion of clean reads in the 2 wucai transcriptome libraries that mapped to the B. - All of the RNA- se- quence data in this article have been deposited in the NCBI- SRA database and are accessible in SRP200451 (https://www.. - A total of 2443 differentially expressed genes (DEGs) were identified between LT and LT + EBR (Fig. - Hierarchical clustering of all of the DEGs was conducted to observe the gene expression patterns and was evaluated by the log 10 RPKMs of the 2 groups (Fig. - Compared to LT, LT + EBR contained 1581 up-regulated and 862 down- regulated genes. - GO and KEGG analyses of the EBR-induced DEGs. - In order to explore the function of the EBR-induced DEGs under LT stress, the DEGs were used in the Gene. - Results uncovered the biological process, molecular function, and cellular component categories for the 2443 DEGs of the 2 groups (Fig. - 2 in the 3 classifica- tions, according to the corresponding -log 10 Pvalue of each term. - In the biological process category, cellular response to far red light (GO:0071490) was the most abundant, followed by cellular response to red light (GO:0071491), indicat- ing the positive regulation of reactive oxygen species biosynthetic processes (GO:1903428), such as photosyn- thesis and light harvesting in photosystem I (GO:. - Similarly, in the cellular component category, photosystem I (GO:0009522) was the most abundant, followed by plastoglobule (GO:0010287), chloroplast thylakoid membrane (GO:0009535), and photosystem II (GO:0009523). - In the molecular function category, oxygen-evolving activity (GO:0010242) was the most abundant, followed by chlorophyll binding (GO:. - Besides, the expression patterns of the cold acclimation, cold-induced and BR-responsive genes were listed in Additional file 7: Table S3 and Additional file 8:. - Additional file 9:. - Significant increases in Chl a , Chl b , Total Chl content, ALA, and PBG were detected in LT + EBR plants after 5 d com- pared to LT, with increases of and 52.06%, respectively. - In contrast, the contents of Proto IX, Mg-Proto IX, and Pchl significantly declined by and 25.78%, respectively, when LT + EBR was compared to LT. - Conversely, significant increases by and 48.58% were noted in the Proto IX, Mg-Proto IX, and Pchl contents, respectively, when LT was compared to Cont. - There were no signifi- cant differences detected between LT + EBR and Cont/. - In LT + EBR plants, the genes involved in photosynthesis were primarily up-regulated (Fig. - The chlorophyll a/b-binding protein was also up-regulated, which is well-known as an important component of the light-harvesting chlorophyll protein complexes I and II [20]. - The Chl a fluorescence transient reflects the effect of PSII afterquantitatively analyzing changes in the OJIP curve (Fig. - The radar maps show the changes in the receptor side and reflection center of PSII (Fig. - For all of the parameters, there were no significant differences detected between Cont and Cont+EBR. - There were also no significant dif- ferences detected in RC/CS 0 , φDo , and φPo between LT and LT + EBR. - LT was associated with significantly higher V j , DI 0 /RC, and F 0 compared to Cont, Cont+EBR, and LT + EBR. - There were no significant differences detected between Cont and Cont+EBR for all of the parameters.. - There were also no significant differences detected in RC/CS 0 , φDo, and φPo between LT and LT + EBR. - Con- versely, Fv/F 0 , φEo, ψ o , and PI abs increased significantly by and 34.38%, respectively, in LT + EBR compared to LT (Fig. - 3 Transcriptome analysis of the DEGs in the LT and LT + EBR treatments of wucai leaves. - a Comparisons of DEGs in the LT and LT + EBR treatments. - Red and green dots represent up- and down-regulated genes, respectively, and gray dots indicate transcripts that did not change significantly in the LT + EBR library compared to LT. - c Hierarchical clustering of all of the DEGs was based on the log 10 RPKM values. - 4 GO and KEGG pathway enrichment analyses of the DEGs in the LT and LT + EBR treatments of wucai leaves. - a GO enrichment analysis with the 30 most enriched GO terms in the 3 categories shown. - is the first demonstration of the molecular mechanisms of wucai responses to EBR treatments under LT. - In order to determine the effect of EBR on LT tolerance, the discussion is focused on contrasting the LT and LT + EBR treatment groups. - MSI can be used to assess the integ- rity of the plasma membrane [29]. - In order to explore the underlying molecular mechanisms, transcriptome sequencing was performed on LT and LT + EBR plants.. - In this study, a signifi- cant increase in endogenous ALA was detected in LT + EBR plants compared to LT. - EBR application caused sig- nificant increases in the ALA and PBG contents, and signifi- cant decreases in Proto IX, Mg-Proto IX, and Pchl compared to LT. - Four transcripts encoding 4 of the key enzymes in- volved in catalyzing the synthesis of these products were all up-regulated by exogenous EBR application. - 7 Effects of exogenous EBR application on photosynthetic pigment contents in wucai leaves under LT stress. - (A – C) Quantification of the Chl a content (a), Chl b content (b), Total Chl content (c), and the Chl a/b ratio calculated from A and B (d). - 8 Effects of exogenous EBR application on porphyrin and chlorophyll metabolism in wucai leaves under LT stress. - Chlorophyll fluorescence transients quantitatively ex- plain dynamic changes in the OJIP curve, which in turn quantitatively explains the PSII light energy absorption, conversion, electron transport, PSII action center, activ- ity on the receptor and donor sides, and redox state of the electron transporter. - In this study, GO and KEGG enrichment analyses suggest that EBR-mediated induc- tion of photosynthesis, chloroplast thylakoid membrane, and chlorophyll biosynthesis may be the primary factors contributing to the distinct photosynthetic characteris- tics of the EBR transcriptome under LT conditions.. - decreased in both LT and LT + EBR plants, especially in LT. - However, the increase was smaller in LT + EBR than in LT (Fig. - 7), indicating there was less relative damage to PSII in LT + EBR. - 9 Heat map of the photosynthesis and photosynthesis-antenna proteins-related DEGs in the LT and LT + EBR treatment of wucai leaves. - The health of PSII is often used to determine the photo- chemical efficiency of the light reactions in photosynthesis II [42]. - SLA and MSI were significantly improved in LT + EBR.. - Specifically, a significant increase in chlorophyll content and significant changes in the chlorophyll fluorescence parameters (i.e., V j , Fv/F 0 , and PI abs ) were detected when LT + EBR was compared to LT. - The regulation of the transcriptome in combination with the physiology of chlorophyll biosyn- thesis and photosynthesis demonstrates the protective mechanism of EBR treatment in response to LT stress.. - 11 Effects of exogenous EBR application on photosynthesis-related genes in wucai leaves under LT stress. - Wucai seeds were provided by the Vegetable Genetics and Breeding Laboratory of the College of Horticulture, Anhui Agricultural University. - Based on the results of the pilot study, 0.1 μM EBR was identified as the optimum EBR concentration, which was subsequently used as the processing condition in the main experiment (Additional file 4: Figure S4).. - 13 Effects of exogenous EBR application on PSII in wucai leaves under LT stress. - and (4) LT + EBR plants grown with the 0.1 μM EBR pretreatment. - Seedlings in both the LT and LT + EBR groups were transferred to a phytotron set at 7 /3 °C (day/. - After 5 d of LT, func- tional leaf samples from 3 biological replicates were col- lected from LT and LT + EBR for sequencing. - All of the samples were collected at the same time, ground into powder in liquid nitrogen, and immediately stored at − 80 °C for future analyses.. - SLA was calcu- lated as the ratio of the leaf area to dry leaf weight.. - Then, all of the supernatants were brought to a set volume of 10 mL with 80% alcohol and decolorized with 0.01 g activated carbon at 80 °C for 30 min. - The MDA level of all of the plant samples was assessed through lipid peroxidation. - 14 Effects of exogenous EBR application on chlorophyll fluorescence parameters in wucai leaves under LT stress. - RNA extraction, library construction and RNA sequencing Total RNA was extracted from the LT and LT + EBR leaf samples using a mirVana miRNA Isolation Kit (Ambion, TX, USA) following the manufacturer’s instructions.. - A differential expression analysis was performed for both LT and LT + EBR treatments based on the DESeq R package [48], which allowed for statistical analysis using a negative binomial (NB) distribution model. - GO enrichment analysis of the DEGs was performed according to the GOseq R package. - Statistical enrichment of the DEGs. - The absorbance of the supernatant was measured at 3 wavelengths and 663 nm. - After 15 min in darkness, the absorbance of the resulting solution was determined spectrophotometrically at 553 nm [52].. - The absorbance of the supernatant was measured at 575, 590 and 628 nm. - The time resolution of digitization was switched to slower acquisition rates as the kinetics of the fluores- cence signal slowed. - The fluorescence intensity at 20 μ s was considered to be F 0 , while the maximal fluorescence intensity was presumed to be equal to F m , as the intensity was high enough to ensure the closure of all of the reaction centers (RCs) of PSII. - All of the data from the 4 treatments were subjected to an analysis of variance (ANOVA). - A-F rep- resent LT-1, LT-2, LT-3, LT + EBR-1, LT + EBR-2, and LT + EBR-3, respectively.. - A-F represent LT-1, LT-2, LT-3, LT + EBR-1, LT + EBR-2, and LT + EBR-3, respectively.. - The expression patterns of the cold acclimation and cold-induced genes.. - The expression patterns of the BR- responsive genes.. - Parameters derived from the OJIP transient for use in the current study.. - LT + EBR: Low temperature with 24-epibrassinolide. - The raw RNA-Seq data used in this study have been deposited in the Nation Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database under the accession number SRP200451 (https://www.ncbi.nlm.nih.. - Components of the Arabidopsis C-repeat/dehydration- responsive element binding factor cold-response pathway are conserved in Brassica napus and other plant species. - Brassinosteroids participate in the control of basal and acquired freezing tolerance of plants.. - The role of 24- epibrassinolide in the regulation of photosynthetic characteristics and nitrogen metabolism of tomato seedlings under a combined low temperature and weak light stress. - Chlorophyll function in the photosynthetic reaction center. - Differential expression of RNA-Seq data at the gene level – the DESeq package
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