Non-coding RNA Regulation of Secondary Metabolism in Plants

Cover image for research topic "Non-coding RNA Regulation of Secondary Metabolism in Plants"
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Feng Xu
College of Horticulture and Gardening, Yangtze University
Shaohua Zeng
South China Botanical Garden, Chinese Academy of Sciences (CAS)
Su-Sheng Gan
Cornell University
Yongliang Liu
Kentucky Tobacco Research & Development Center, College of Agriculture, Food and Environment, University of Kentucky
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iang","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/2113203/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/2113203/overview","affiliation":{"name":"Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow \u0026 Huai River Valley, College of Agronomy, Anhui Agricultural University, Ministry of Agriculture and Rural Affairs","address":null},"affiliations":[{"name":"Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow \u0026 Huai River Valley, College of Agronomy, Anhui Agricultural University, Ministry of Agriculture and Rural Affairs","address":null}],"nessieId":null},{"fullName":"Wei Gao","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/2126062/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/2126062/overview","affiliation":{"name":"Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow \u0026 Huai River Valley, College of Agronomy, Anhui Agricultural University, Ministry of Agriculture and Rural Affairs","address":null},"affiliations":[{"name":"Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow \u0026 Huai River Valley, College of Agronomy, Anhui Agricultural University, Ministry of Agriculture and Rural Affairs","address":null}],"nessieId":"317828225252"},{"fullName":"Bing-li Jiang","firstName":null,"middleName":null,"lastName":null,"image":null,"loopProfileUrl":null,"affiliation":{"name":"Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow \u0026 Huai River Valley, College of Agronomy, Anhui Agricultural University, Ministry of Agriculture and Rural Affairs","address":null},"affiliations":[{"name":"Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow \u0026 Huai River Valley, College of Agronomy, Anhui Agricultural University, Ministry of Agriculture and Rural Affairs","address":null}],"nessieId":null},{"fullName":"Xue Liu","firstName":null,"middleName":null,"lastName":null,"image":null,"loopProfileUrl":null,"affiliation":{"name":"Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow \u0026 Huai River Valley, College of Agronomy, Anhui Agricultural University, Ministry of Agriculture and Rural Affairs","address":null},"affiliations":[{"name":"Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow \u0026 Huai River Valley, College of Agronomy, Anhui Agricultural University, Ministry of Agriculture and Rural Affairs","address":null}],"nessieId":null},{"fullName":"Ya-ting Jiang","firstName":null,"middleName":null,"lastName":null,"image":null,"loopProfileUrl":null,"affiliation":{"name":"Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow \u0026 Huai River Valley, College of Agronomy, Anhui Agricultural University, Ministry of Agriculture and Rural Affairs","address":null},"affiliations":[{"name":"Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow \u0026 Huai River Valley, College of Agronomy, Anhui Agricultural University, Ministry of Agriculture and Rural Affairs","address":null}],"nessieId":null},{"fullName":"Li-tian Zhang","firstName":null,"middleName":null,"lastName":null,"image":null,"loopProfileUrl":null,"affiliation":{"name":"Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow \u0026 Huai River Valley, College of Agronomy, Anhui Agricultural University, Ministry of Agriculture and Rural Affairs","address":null},"affiliations":[{"name":"Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow \u0026 Huai River Valley, College of Agronomy, Anhui Agricultural University, Ministry of Agriculture and Rural Affairs","address":null}],"nessieId":null},{"fullName":"Yue Zhang","firstName":null,"middleName":null,"lastName":null,"image":null,"loopProfileUrl":null,"affiliation":{"name":"Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow \u0026 Huai River Valley, College of Agronomy, Anhui Agricultural University, Ministry of Agriculture and Rural Affairs","address":null},"affiliations":[{"name":"Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow \u0026 Huai River Valley, College of Agronomy, Anhui Agricultural University, Ministry of Agriculture and Rural Affairs","address":null}],"nessieId":null},{"fullName":"Sheng-nan Yan","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/1943490/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/1943490/overview","affiliation":{"name":"Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow \u0026 Huai River Valley, College of Agronomy, Anhui Agricultural University, Ministry of Agriculture and Rural Affairs","address":null},"affiliations":[{"name":"Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow \u0026 Huai River Valley, College of Agronomy, Anhui Agricultural University, Ministry of Agriculture and Rural Affairs","address":null}],"nessieId":"335008108365"},{"fullName":"Jia-Jia Cao","firstName":null,"middleName":null,"lastName":null,"image":null,"loopProfileUrl":null,"affiliation":{"name":"Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow \u0026 Huai River Valley, College of Agronomy, Anhui Agricultural University, Ministry of Agriculture and Rural Affairs","address":null},"affiliations":[{"name":"Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow \u0026 Huai River Valley, College of Agronomy, Anhui Agricultural University, Ministry of Agriculture and Rural Affairs","address":null}],"nessieId":null},{"fullName":"Jie Lu","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/383454/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/383454/overview","affiliation":{"name":"Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow \u0026 Huai River Valley, College of Agronomy, Anhui Agricultural University, Ministry of Agriculture and Rural Affairs","address":null},"affiliations":[{"name":"Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow \u0026 Huai River Valley, College of Agronomy, Anhui Agricultural University, Ministry of Agriculture and Rural Affairs","address":null}],"nessieId":"120259734923"},{"fullName":"Chuan-xi Ma","firstName":null,"middleName":null,"lastName":null,"image":null,"loopProfileUrl":null,"affiliation":{"name":"Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow \u0026 Huai River Valley, College of Agronomy, Anhui Agricultural University, Ministry of Agriculture and Rural Affairs","address":null},"affiliations":[{"name":"Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow \u0026 Huai River Valley, College of Agronomy, Anhui Agricultural University, Ministry of Agriculture and Rural Affairs","address":null}],"nessieId":null},{"fullName":"Cheng Chang","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/383330/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/383330/overview","affiliation":{"name":"Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow \u0026 Huai River Valley, College of Agronomy, Anhui Agricultural University, Ministry of Agriculture and Rural Affairs","address":null},"affiliations":[{"name":"Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow \u0026 Huai River Valley, College of Agronomy, Anhui Agricultural University, Ministry of Agriculture and Rural Affairs","address":null}],"nessieId":"223338944799"},{"fullName":"Hai-ping Zhang","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/2133964/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/2133964/overview","affiliation":{"name":"Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow \u0026 Huai River Valley, College of Agronomy, Anhui Agricultural University, Ministry of Agriculture and Rural Affairs","address":null},"affiliations":[{"name":"Key Laboratory of Wheat Biology and Genetic Improvement on Southern Yellow \u0026 Huai River Valley, College of Agronomy, Anhui Agricultural University, Ministry of Agriculture and Rural Affairs","address":null}],"nessieId":"180389246019"}],"dates":{"acceptedDate":"2023-01-16","recentDate":"2023-02-01"},"doi":"10.3389/fpls.2023.1107277","frontiersExtra":{"articleType":"Original Research","impact":{"citations":9,"crossrefCitations":0,"downloads":105,"frontiersViews":0,"pmcDownloads":0,"pmcViews":0,"scopusCitations":0,"views":3551},"isPartOfResearchTopic":true,"isPublished":true,"section":"Functional and Applied Plant Genomics"},"guid":1107277,"images":[{"height":309,"url":"https://www.frontiersin.org/files/myhome article library/1107277/1107277_Thumb_400.jpg","width":400,"caption":null},{"height":1429,"url":"https://www.frontiersin.org/files/Articles/1107277/fpls-14-1107277-HTML/image_m/fpls-14-1107277-g001.jpg","width":1851,"caption":"(A) Venn diagram of differentially expressed mRNAs. (B) The KEGG analysis of differentially expressed mRNAs identified in 28DPA-NTvsHT. (C) The KEGG analysis of differentially expressed mRNAs identified in 35DPA-NTvsHT."},{"height":1444,"url":"https://www.frontiersin.org/files/Articles/1107277/fpls-14-1107277-HTML/image_m/fpls-14-1107277-g002.jpg","width":1885,"caption":"(A) Mutation site of ga20ox1. (B) Relative expression levels of GA20ox1 in mutant lines and JM22. Significant differences were determined using Student’ s t-test: **P \u003c 0.01. (C) GA contents of Jimai 22 (JM22) and ga20ox1 seeds at 96 h after imbibition. (D) The seed images of Jimai 22 (JM22) and the EMS mutant ga20ox1 after 96 h imbibition conducted under normal-temperature (25°C/20°C) and high-temperature (35°C/25°C) environments. (E) Germination percentages of JM22 and the EMS mutant ga20ox1 on the 7th day of germination tests conducted under normal-temperature (25°C/20°C) and high-temperature (35°C/25°C) environments. Data represent the mean ± standard error (SE), n = 10–15."},{"height":1828,"url":"https://www.frontiersin.org/files/Articles/1107277/fpls-14-1107277-HTML/image_m/fpls-14-1107277-g003.jpg","width":1295,"caption":"(A) Germination percentages of Col-0, the T-DNA mutant atcdpk24, overexpression plants (35S:TaCDPK21), and complementation of TaCDPK21 (35S:TaCDPK21/atcdpk24) in Arabidopsis. Data are the mean ± standard error (SE), n = 10–15. Significant differences were determined using Student’ s t-test: **P \u003c 0.01. ns represents not significant (B) Relative expression of TaCDPK21 in Col-0, overexpression Arabidopsis plants (35S:TaCDPK21), and complementation of TaCDPK21 (35S:TaCDPK21/atcdpk24). RNA was extracted from seeds imbibed for 24 h. ND represents not detected. (C) Mutation site of cdpk21. * represents terminator (D) The seed images of Jimai 22 (JM22) and the EMS mutant (cdpk21) after 96-h imbibition conducted at normal temperature (25°C/20°C). (E) Germination percentages of JM22 and the EMS mutant (cdpk21) on the 7th day of germination tests conducted at normal temperature (25°C/20°C) and high-temperature (35°C/25°C) environments. Significant differences were determined using Student’ s t-test: **P \u003c 0.01. Data are the mean ± standard error (SE), n = 10–15."},{"height":986,"url":"https://www.frontiersin.org/files/Articles/1107277/fpls-14-1107277-HTML/image_m/fpls-14-1107277-g004.jpg","width":1924,"caption":"(A) The sequence and secondary structure of miR27319. (B) Relative expression of miR27319 in Col-0, 35S:pre-miR27319, and STTM-AtmiR27319. Significant differences were determined using Student’s t-test: **P \u003c 0.01. (C) Seed germination percentages of Col-0, overexpression plants (35S:pre-miR27319), and STTM plants of miR27319 (STTM-AtmiR27319) in Arabidopsis. (D) The seed images of Col-0, overexpression plants (35S:pre-miR27319), and STTM plants of miR27319 (STTM-AtmiR27319) in Arabidopsis."},{"height":1597,"url":"https://www.frontiersin.org/files/Articles/1107277/fpls-14-1107277-HTML/image_m/fpls-14-1107277-g005.jpg","width":1219,"caption":"(A) The seed images of Nipponbare (Nip, WT), overexpression plants (35S:pre-miR27319), and STTM plants of miR27319 (STTM-OsmiR27319) at 7 days after imbibition in rice. (B) Relative expression of Nip and 35S:pre-miR27319. RNA was extracted from seeds imbibed for 24 h. Data are the mean ± standard error (SE). Significant differences were determined using Student’s t-test: **P \u003c 0.01, *P \u003c 0.05. (C) Relative expressions of Nip and STTM-OsmiR27319. RNA was extracted from seeds imbibed for 24 h. Data are the mean ± standard error (SE). Significant differences were determined using Student’s t-test: ** P \u003c 0.01, * P \u003c 0.05. (D) Germination percentages of Nip and 35S:pre-miR27319. Data are the mean ± standard error (SE), n = 10–15. Significant differences were determined using Student’ s t-test: ** P \u003c 0.01, * P \u003c 0.05. (E) Germination percentages of Nip and STTM-OsmiR27319. Data are the mean ± standard error (SE), n = 10–15. Significant differences were determined using Student’ s t-test: **P \u003c 0.01, *P \u003c 0.05."},{"height":1072,"url":"https://www.frontiersin.org/files/Articles/1107277/fpls-14-1107277-HTML/image_m/fpls-14-1107277-g006.jpg","width":1927,"caption":"(A) ABA contents in Nip, 35S:pre-miR27319, and STTM-OsmiR27319. Significant differences were determined using Student’s t-test: **P \u003c 0.01. (B) GA contents in Nip, 35S:pre-miR27319, and STTM-OsmiR27319. Significant differences were determined using Student’s t-test: **P \u003c 0.01. (C) Alpha-amylase activities in Nip, 35S:pre-miR27319, and STTM-OsmiR27319. Significant differences were determined using Student’s t-test: **P \u003c 0.01. (D) Relative expression levels of the key genes involved in ABA and GA biosynthesis, catabolism, and signaling pathways. Gene ID: Os03g0645900 (OsNCED1), Os01g0859300 (OsABI5), Os03g0856700 (OsGA20ox1), Os05g0158600 (OsGA2ox1), and Os01g0357400 (OsAmy1B). Significant differences were determined using Student’s t-test: **P \u003c 0.01."}],"journal":{"guid":373,"name":"Frontiers in Plant Science","link":null,"nessieId":null,"palette":null,"publisher":"Frontiers Media","images":null,"isOnline":null,"isDeleted":null,"isDisabled":null,"issn":null},"link":"https://www.frontiersin.org/articles/10.3389/fpls.2023.1107277","pubDate":"2023-02-01","score":19.47928994082846,"title":"Identification and validation of coding and non-coding RNAs involved in high-temperature-mediated seed dormancy in common wheat","topics":["Seed Dormancy","wheat","Transcriptome sequencing","High-Temperature","Pre-harvest sprouting"],"pdfUrl":"https://www.frontiersin.org/articles/10.3389/fpls.2023.1107277/pdf"},{"__typename":"Feed_Article","_id":"6859a85efd1016fa1b12eaef","abstract":"Acer pictum subsp. mono is a colorful tree species with considerable ornamental and economic value. However, little is known about the metabolism and regulatory mechanism of leaf color change in A. p. subsp. mono. To reveal the molecular mechanism of leaf color change in A. p. subsp. mono, the present study examined the bud mutation branches and compared the metabolites of the red leaves (AR) of the bud mutation branches of A. p. subsp. mono with those of the green leaves (AG) of the wild-type branches. It was found that the chlorophyll and carotenoids content of the red leaves decreased significantly, while anthocyanins, and various antioxidant enzymes increased significantly compared with the green leaves. The glycosides cyanidin, pelargonidin, malvidin, petunidin, delphinidin, and peonidin were detected in AR by liquid chromatography-mass spectrometry. The cyanidin glycosides increased, and cyanidin 3-O-glycoside was significantly upregulated. We analyzed the transcriptome and small RNA of A. p. subsp. mono leaves and detected 4061 differentially expressed mRNAs and 116 differentially expressed miRNAs. Through miRNA-mRNA association analysis, five differentially expressed modules were found; one miRNA targeted three genes, and four miRNAs targeted a single gene. Among them, miR160b, miR6300, and miR396g were found to be the key miRNAs regulating stable anthocyanin accumulation in A. p. subsp. mono leaves. By revealing the physiological response of leaf color change and the molecular regulatory mechanism of the miRNA, this study provides new insight into the molecular regulatory mechanism of leaf color change, thereby offering a foundation for future studies.","htmlAbstract":"\u003cp\u003e\u003ci\u003eAcer pictum subsp. mono\u003c/i\u003e is a colorful tree species with considerable ornamental and economic value. However, little is known about the metabolism and regulatory mechanism of leaf color change in \u003ci\u003eA. p. subsp. mono\u003c/i\u003e. To reveal the molecular mechanism of leaf color change in \u003ci\u003eA. p. subsp. mono\u003c/i\u003e, the present study examined the bud mutation branches and compared the metabolites of the red leaves (AR) of the bud mutation branches of \u003ci\u003eA. p. subsp. mono\u003c/i\u003e with those of the green leaves (AG) of the wild-type branches. It was found that the chlorophyll and carotenoids content of the red leaves decreased significantly, while anthocyanins, and various antioxidant enzymes increased significantly compared with the green leaves. The glycosides cyanidin, pelargonidin, malvidin, petunidin, delphinidin, and peonidin were detected in AR by liquid chromatography-mass spectrometry. The cyanidin glycosides increased, and cyanidin 3-O-glycoside was significantly upregulated. We analyzed the transcriptome and small RNA of \u003ci\u003eA. p. subsp. mono\u003c/i\u003e leaves and detected 4061 differentially expressed mRNAs and 116 differentially expressed miRNAs. Through miRNA-mRNA association analysis, five differentially expressed modules were found; one miRNA targeted three genes, and four miRNAs targeted a single gene. Among them, miR160b, miR6300, and miR396g were found to be the key miRNAs regulating stable anthocyanin accumulation in \u003ci\u003eA. p. subsp. mono\u003c/i\u003e leaves. By revealing the physiological response of leaf color change and the molecular regulatory mechanism of the miRNA, this study provides new insight into the molecular regulatory mechanism of leaf color change, thereby offering a foundation for future studies.\u003c/p\u003e","authors":[{"fullName":"Baoli Lin","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/1946655/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/1946655/overview","affiliation":{"name":"College of Landscape Architecture, Beijing University of Agriculture","address":null},"affiliations":[{"name":"College of Landscape Architecture, Beijing University of Agriculture","address":null}],"nessieId":null},{"fullName":"He Ma","firstName":null,"middleName":null,"lastName":null,"image":null,"loopProfileUrl":null,"affiliation":{"name":"College of Landscape Architecture, Beijing University of Agriculture","address":null},"affiliations":[{"name":"College of Landscape Architecture, Beijing University of Agriculture","address":null}],"nessieId":null},{"fullName":"Kezhong Zhang","firstName":null,"middleName":null,"lastName":null,"image":null,"loopProfileUrl":null,"affiliation":{"name":"College of Landscape Architecture, Beijing University of Agriculture","address":null},"affiliations":[{"name":"College of Landscape Architecture, Beijing University of Agriculture","address":null},{"name":"Laboratory of Urban and Rural Ecological Environment, Beijing University of Agriculture","address":null}],"nessieId":null},{"fullName":"Jinteng Cui","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/2102721/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/2102721/overview","affiliation":{"name":"College of Landscape Architecture, Beijing University of Agriculture","address":null},"affiliations":[{"name":"College of Landscape Architecture, Beijing University of Agriculture","address":null},{"name":"Laboratory of Urban and Rural Ecological Environment, Beijing University of Agriculture","address":null}],"nessieId":"335008109243"}],"dates":{"acceptedDate":"2022-12-22","recentDate":"2023-01-12"},"doi":"10.3389/fpls.2022.1047452","frontiersExtra":{"articleType":"Original Research","impact":{"citations":4,"crossrefCitations":0,"downloads":737,"frontiersViews":0,"pmcDownloads":0,"pmcViews":0,"scopusCitations":0,"views":1783},"isPartOfResearchTopic":true,"isPublished":true,"section":"Plant Metabolism and Chemodiversity"},"guid":1047452,"images":[{"height":329,"url":"https://www.frontiersin.org/files/myhome article library/1047452/1047452_Thumb_400.jpg","width":400,"caption":null},{"height":2592,"url":"https://www.frontiersin.org/files/Articles/1047452/fpls-13-1047452-HTML/image_m/fpls-13-1047452-g001.jpg","width":3152,"caption":"subsp. mono with bud mutation branches found in the wild. (A) Acer p. subsp. mono with bud mutation branches in autumn. (B, C) are wild-type branches (AG) and bud mutation branches (AR) of A. p. subsp. mono in autumn. (D–F) Green leaves of wild-type branches (AG) and red leaves of bud mutation branches (AR) in autumn."},{"height":660,"url":"https://www.frontiersin.org/files/Articles/1047452/fpls-13-1047452-HTML/image_m/fpls-13-1047452-g002.jpg","width":1417,"caption":"p. subsp. mono leaves. (A–J) show the contents of chlorophyll a, chlorophyll b, carotenoid, anthocyanin, PAL, SOD, POD, CAT, PPO, and soluble sugar in the leaves of A. p. subsp. mono at three stages of S, AG, and AR, respectively. S, AG, and AR denote bud mutation branch leaves in summer, wild branch leaves in autumn, and bud mutation branch leaves in autumn, respectively."},{"height":1099,"url":"https://www.frontiersin.org/files/Articles/1047452/fpls-13-1047452-HTML/image_m/fpls-13-1047452-g003.jpg","width":1404,"caption":"subsp. mono leaves based on targeted metabonomics technology. (A) PCA analysis of metabolites in the leaves of A p. subsp. mono. (B) KEGG classification map of metabolites from the leaves of A p. subsp. mono. (C) Volcanic map of differential metabolites in the leaves of A p. subsp. mono (AR vs. AG). (D) Enrichment analysis of KEGG (AR vs. AG); differential metabolites in the leaves of A p. subsp. mono."},{"height":933,"url":"https://www.frontiersin.org/files/Articles/1047452/fpls-13-1047452-HTML/image_m/fpls-13-1047452-g004.jpg","width":1414,"caption":"(A) Anthocyanins. (B) Nucleotides and derivatives. (C) Carbohydrates and derivatives. (D) Phytohormones."},{"height":1630,"url":"https://www.frontiersin.org/files/Articles/1047452/fpls-13-1047452-HTML/image_m/fpls-13-1047452-g005.jpg","width":1408,"caption":"subsp. mono at different developmental stages and miRNA identity. (A) Length distribution of clean reads. (B) Length distribution of unique reads. (C) Venn Diagram. (D) Length distribution of known miRNA and novel miRNA."},{"height":569,"url":"https://www.frontiersin.org/files/Articles/1047452/fpls-13-1047452-HTML/image_m/fpls-13-1047452-g006.jpg","width":1299,"caption":"(A) GO enrichment analysis of differentially expressed miRNAs target genes. (B) GO enrichment analysis of differentially expressed mRNAs."},{"height":2468,"url":"https://www.frontiersin.org/files/Articles/1047452/fpls-13-1047452-HTML/image_m/fpls-13-1047452-g007.jpg","width":1919,"caption":"Note Green indicates downregulated expression; red indicates upregulated expression."},{"height":2308,"url":"https://www.frontiersin.org/files/Articles/1047452/fpls-13-1047452-HTML/image_m/fpls-13-1047452-g008.jpg","width":1332,"caption":"(A) Phylogenetic tree of SUS. (B) Relative expression of ApSUS-mi160b during leaf color transition. (C) Phylogenetic tree of UGP2.(D) Relative expression of ApUPG2-miR396g during leaf color transition. (E) Phylogenetic Tree of UFGT. (F) Relative expression of ApUFGT-miR6300during leaf color transition."},{"height":2472,"url":"https://www.frontiersin.org/files/Articles/1047452/fpls-13-1047452-HTML/image_m/fpls-13-1047452-g009.jpg","width":3071,"caption":"p. subsp. mono leaves. Note Red represents upregulated expression and green represents downregulated expression."}],"journal":{"guid":373,"name":"Frontiers in Plant Science","link":null,"nessieId":null,"palette":null,"publisher":"Frontiers Media","images":null,"isOnline":null,"isDeleted":null,"isDisabled":null,"issn":null},"link":"https://www.frontiersin.org/articles/10.3389/fpls.2022.1047452","pubDate":"2023-01-12","score":9.263313609467483,"title":"Regulatory mechanisms and metabolic changes of miRNA during leaf color change in the bud mutation branches of Acer pictum subsp. mono","topics":["microRNA","regulatory network","Leaf color","Differential metabolite","Acer pictum subsp.mono"],"pdfUrl":"https://www.frontiersin.org/articles/10.3389/fpls.2022.1047452/pdf"},{"__typename":"Feed_Article","_id":"6859a85efd1016fa1b12eaee","abstract":"Crop losses caused by climate change and various (a)biotic stressors negatively affect agriculture and crop production. Therefore, it is vital to develop a proper understanding of the complex response(s) to (a)biotic stresses and delineate them for each crop plant as a means to enable translational research. In plants, the improvement of crop quality by m6A editing is believed to be a promising strategy. As a reaction to environmental changes, m6A modification showed a high degree of sensitivity and complexity. We investigated differences in gene medleys between dark-induced leaf senescence (DILS) and developmental leaf senescence in barley, including inter alia RNA modifications active in DILS. The identified upregulated genes in DILS include RNA methyltransferases of different RNA types, embracing enzymes modifying mRNA, tRNA, and rRNA. We have defined a decisive moment in the DILS model which determines the point of no return, but the mechanism of its control is yet to be uncovered. This indicates the possibility of an unknown additional switch between cell survival and cell death. Discoveries of m6A RNA modification changes in certain RNA species in different stages of leaf senescence may uncover the role of such modifications in metabolic reprogramming. Nonetheless, there is no such data about the process of leaf senescence in plants. In this scope, the prospect of finding connections between the process of senescence and m6A modification of RNA in plants seems to be compelling.","htmlAbstract":"\u003cp\u003eCrop losses caused by climate change and various (a)biotic stressors negatively affect agriculture and crop production. Therefore, it is vital to develop a proper understanding of the complex response(s) to (a)biotic stresses and delineate them for each crop plant as a means to enable translational research. In plants, the improvement of crop quality by m\u003csup\u003e6\u003c/sup\u003eA editing is believed to be a promising strategy. As a reaction to environmental changes, m\u003csup\u003e6\u003c/sup\u003eA modification showed a high degree of sensitivity and complexity. We investigated differences in gene medleys between dark-induced leaf senescence (DILS) and developmental leaf senescence in barley, including \u003ci\u003einter alia\u003c/i\u003e RNA modifications active in DILS. The identified upregulated genes in DILS include RNA methyltransferases of different RNA types, embracing enzymes modifying mRNA, tRNA, and rRNA. We have defined a decisive moment in the DILS model which determines the point of no return, but the mechanism of its control is yet to be uncovered. This indicates the possibility of an unknown additional switch between cell survival and cell death. Discoveries of m\u003csup\u003e6\u003c/sup\u003eA RNA modification changes in certain RNA species in different stages of leaf senescence may uncover the role of such modifications in metabolic reprogramming. Nonetheless, there is no such data about the process of leaf senescence in plants. In this scope, the prospect of finding connections between the process of senescence and m\u003csup\u003e6\u003c/sup\u003eA modification of RNA in plants seems to be compelling.\u003c/p\u003e","authors":[{"fullName":"Elżbieta Rudy","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/1487567/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/1487567/overview","affiliation":{"name":"Department of Plant Physiology, Faculty of Biology, Adam Mickiewicz University in Poznań, Uniwersytetu Poznańskiego 6","address":null},"affiliations":[{"name":"Department of Plant Physiology, Faculty of Biology, Adam Mickiewicz University in Poznań, Uniwersytetu Poznańskiego 6","address":null}],"nessieId":"412317517667"},{"fullName":"Magda Grabsztunowicz","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/2088159/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/2088159/overview","affiliation":{"name":"Department of Plant Physiology, Faculty of Biology, Adam Mickiewicz University in Poznań, Uniwersytetu Poznańskiego 6","address":null},"affiliations":[{"name":"Department of Plant Physiology, Faculty of Biology, Adam Mickiewicz University in Poznań, Uniwersytetu Poznańskiego 6","address":null}],"nessieId":"77310058696"},{"fullName":"Magdalena Arasimowicz-Jelonek","firstName":null,"middleName":null,"lastName":null,"image":null,"loopProfileUrl":null,"affiliation":{"name":"Department of Plant Ecophysiology, Faculty of Biology, Adam Mickiewicz University in Poznań, Uniwersytetu Poznańskiego 6","address":null},"affiliations":[{"name":"Department of Plant Ecophysiology, Faculty of Biology, Adam Mickiewicz University in Poznań, Uniwersytetu Poznańskiego 6","address":null}],"nessieId":null},{"fullName":"Umesh Kumar Tanwar","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/380382/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/380382/overview","affiliation":{"name":"Department of Plant Physiology, Faculty of Biology, Adam Mickiewicz University in Poznań, Uniwersytetu Poznańskiego 6","address":null},"affiliations":[{"name":"Department of Plant Physiology, Faculty of Biology, Adam Mickiewicz University in Poznań, Uniwersytetu Poznańskiego 6","address":null}],"nessieId":"343597915176"},{"fullName":"Julia Maciorowska","firstName":null,"middleName":null,"lastName":null,"image":null,"loopProfileUrl":null,"affiliation":{"name":"Department of Plant Physiology, Faculty of Biology, Adam Mickiewicz University in Poznań, Uniwersytetu Poznańskiego 6","address":null},"affiliations":[{"name":"Department of Plant Physiology, Faculty of Biology, Adam Mickiewicz University in Poznań, Uniwersytetu Poznańskiego 6","address":null}],"nessieId":null},{"fullName":"Ewa Sobieszczuk-Nowicka","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/239334/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/239334/overview","affiliation":{"name":"Department of Plant Physiology, Faculty of Biology, Adam Mickiewicz University in Poznań, Uniwersytetu Poznańskiego 6","address":null},"affiliations":[{"name":"Department of Plant Physiology, Faculty of Biology, Adam Mickiewicz University in Poznań, Uniwersytetu Poznańskiego 6","address":null}],"nessieId":"584116202818"}],"dates":{"acceptedDate":"2022-12-09","recentDate":"2023-01-04"},"doi":"10.3389/fpls.2022.1064131","frontiersExtra":{"articleType":"Perspective","impact":{"citations":13,"crossrefCitations":0,"downloads":2,"frontiersViews":0,"pmcDownloads":0,"pmcViews":0,"scopusCitations":0,"views":3400},"isPartOfResearchTopic":true,"isPublished":true,"section":"Plant Cell Biology"},"guid":1064131,"images":[{"height":400,"url":"https://www.frontiersin.org/files/myhome article library/1064131/1064131_Thumb_400.jpg","width":379,"caption":null},{"height":1145,"url":"https://www.frontiersin.org/files/Articles/1064131/fpls-13-1064131-HTML/image_m/fpls-13-1064131-g001.jpg","width":1084,"caption":"(A) Regulation of mRNA m6A modification in plants through the action of a network of m6A writers (methyltransferase), erasers (demethylase), and reader proteins. The m6A writer complex consists of the proteins MTA, MTB, FIP37, VIRILIZER, and HAKAI. The m6A modifications can be removed by ALKBH2, ALKBH9B, and ALKBH10B proteins within the nucleus. The ECT2/3/4 and CPSF30 proteins serve as m6A readers that bind specifically to m6A sites and mediate distinct functions. The expression of subunit MTA (pink) was shown to be upregulated during DILS previously (Sobieszczuk- Nowicka et al., 2018). (B) Typical m6A distribution in regions of an mRNA and its readout affects mRNA fates, including trafficking, stability, decay, translation, and localization. Based on: Zheng et al., 2020; Sokpor et al., 2021; modified."}],"journal":{"guid":373,"name":"Frontiers in Plant Science","link":null,"nessieId":null,"palette":null,"publisher":"Frontiers Media","images":null,"isOnline":null,"isDeleted":null,"isDisabled":null,"issn":null},"link":"https://www.frontiersin.org/articles/10.3389/fpls.2022.1064131","pubDate":"2023-01-04","score":23.02071005917165,"title":"N6-methyladenosine (m6A) RNA modification as a metabolic switch between plant cell survival and death in leaf senescence","topics":["abiotic stress","barley","Crop Improvement","RNA modifications","leaf senescence","M6A","Epitranscriptomics"],"pdfUrl":"https://www.frontiersin.org/articles/10.3389/fpls.2022.1064131/pdf"},{"__typename":"Feed_Article","_id":"6859a85efd1016fa1b12eaf1","abstract":"The effect of exogenous salicylic acid (SA) on folate metabolism and the related gene regulation mechanisms is still unclear. In this study, the panicle of foxtail millet with different SA concentrations treatment showed that the 5-M-THF content with 6 mM SA treatment was 2.01 times that of the control. An untargeted metabolomic analysis revealed 275 metabolites were enriched in amino acid metabolism pathways. A transcriptome analysis revealed that differentially expressed genes were mainly enriched in the folate metabolic and amino acid synthesis pathways. Moreover, the miRNA-seq analysis revealed that 33 miRNAs and 51 miRNAs targeted 11 and 15 genes related to the folate and amino acid metabolic pathways, respectively. The miRNA‒mRNA interactions related to the folate and methionine metabolic pathways were analysed. The qRT‒PCR results verified 6 miRNA‒mRNA interactions related to the folate and Met metabolic pathways and were consistent with the prediction. In summary, this study lays the theoretical foundation for elucidating the regulatory mechanisms of folate metabolism.","htmlAbstract":"\u003cp\u003eThe effect of exogenous salicylic acid (SA) on folate metabolism and the related gene regulatory mechanisms is still unclear. In this study, the panicle of foxtail millet treated with different SA concentrations showed that 6 mM SA doubled the 5-methyltetrahydrofolate content compared to that of the control. An untargeted metabolomic analysis revealed that 275 metabolites were enriched in amino acid metabolic pathways. Significantly, the relative content of methionine (Met) after 6 mM SA treatment was 3.14 times higher than the control. Transcriptome analysis revealed that differentially expressed genes were mainly enriched in the folate and amino acid biosynthesis pathways (including Met, Cys, Pro, Ser et\u0026nbsp;al.). The miRNA−mRNA interactions related to the folate and Met metabolic pathways were analyzed and several likely structural gene targets for miRNAs were identified, miRNA-seq analysis revealed that 33 and 51 miRNAs targeted 11 and 15 genes related to the folate and Met pathways, respectively. Eight key genes in the folate metabolism pathway were likely to be up-regulated by 14 new miRNAs and 20 new miRNAs up-regulated the 9 key genes in the Met metabolism pathway. The 6 miRNA−mRNA interactions related to the folate and Met metabolism pathways were verified by qRT−PCR, and consistent with the prediction. The results showed that \u003ci\u003eDHFR1\u003c/i\u003e gene expression level related to folate synthesis was directly up-regulated by Nov-m0139-3p with 3.8 times, but \u003ci\u003eDHFR2\u003c/i\u003e was down-regulated by Nov-m0731-5p with 0.62 times. The expression level of \u003ci\u003eCYSC1\u003c/i\u003e and \u003ci\u003eAPIP\u003c/i\u003e related to Met synthesis were up-regulated by Nov-m0461-5p and Nov-m0664-3p with 4.27 and 1.32 times, respectively. Our results suggested that exogenous SA could induce the folate and Met accumulated in the panicle of foxtail millet. The higher expression level of \u003ci\u003eDHFR1\u003c/i\u003e, \u003ci\u003eFTHFD\u003c/i\u003e, \u003ci\u003eCYSC1\u003c/i\u003e and \u003ci\u003eAPIP\u003c/i\u003e in the folate and Met metabolism pathway and their regulators, including Nov-m0139-3p, Nov-m0717-5p, Nov-m0461-5p and Nov-m0664-3p, could be responsible for these metabolites accumulation. This study lays the theoretical foundation for elucidating the post-transcription regulatory mechanisms of folate and Met metabolism.\u003c/p\u003e","authors":[{"fullName":"Siyu Hou","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/1913804/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/1913804/overview","affiliation":{"name":"College of Agriculture, Institute of Agricultural Bioengineering, Shanxi Agricultural University, Taigu","address":null},"affiliations":[{"name":"College of Agriculture, Institute of Agricultural Bioengineering, Shanxi Agricultural University, Taigu","address":null},{"name":"Shanxi Key Laboratory of Minor Crops Germplasm Innovation and Molecular Breeding, Shanxi Agricultural University","address":null}],"nessieId":null},{"fullName":"Yihan Men","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/2012716/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/2012716/overview","affiliation":{"name":"College of Agriculture, Institute of Agricultural Bioengineering, Shanxi Agricultural University, Taigu","address":null},"affiliations":[{"name":"College of Agriculture, Institute of Agricultural Bioengineering, Shanxi Agricultural University, Taigu","address":null}],"nessieId":null},{"fullName":"Yijuan Zhang","firstName":null,"middleName":null,"lastName":null,"image":null,"loopProfileUrl":null,"affiliation":{"name":"College of Agriculture, Institute of Agricultural Bioengineering, Shanxi Agricultural University, Taigu","address":null},"affiliations":[{"name":"College of Agriculture, Institute of Agricultural Bioengineering, Shanxi Agricultural University, Taigu","address":null},{"name":"Shanxi Key Laboratory of Minor Crops Germplasm Innovation and Molecular Breeding, Shanxi Agricultural University","address":null}],"nessieId":null},{"fullName":"Kai Zhao","firstName":null,"middleName":null,"lastName":null,"image":null,"loopProfileUrl":null,"affiliation":{"name":"College of Agriculture, Institute of Agricultural Bioengineering, Shanxi Agricultural University, Taigu","address":null},"affiliations":[{"name":"College of Agriculture, Institute of Agricultural Bioengineering, Shanxi Agricultural University, Taigu","address":null},{"name":"Shanxi Key Laboratory of Minor Crops Germplasm Innovation and Molecular Breeding, Shanxi Agricultural University","address":null}],"nessieId":null},{"fullName":"Guifang Ma","firstName":null,"middleName":null,"lastName":null,"image":null,"loopProfileUrl":null,"affiliation":{"name":"College of Agriculture, Institute of Agricultural Bioengineering, Shanxi Agricultural University, Taigu","address":null},"affiliations":[{"name":"College of Agriculture, Institute of Agricultural Bioengineering, Shanxi Agricultural University, Taigu","address":null}],"nessieId":null},{"fullName":"Hongying Li","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/356407/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/356407/overview","affiliation":{"name":"College of Agriculture, Institute of Agricultural Bioengineering, Shanxi Agricultural University, Taigu","address":null},"affiliations":[{"name":"College of Agriculture, Institute of Agricultural Bioengineering, Shanxi Agricultural University, Taigu","address":null},{"name":"Shanxi Key Laboratory of Minor Crops Germplasm Innovation and Molecular Breeding, Shanxi Agricultural University","address":null}],"nessieId":"25770384997"},{"fullName":"Yuanhuai Han","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/1892541/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/1892541/overview","affiliation":{"name":"College of Agriculture, Institute of Agricultural Bioengineering, Shanxi Agricultural University, Taigu","address":null},"affiliations":[{"name":"College of Agriculture, Institute of Agricultural Bioengineering, Shanxi Agricultural University, Taigu","address":null},{"name":"Shanxi Key Laboratory of Minor Crops Germplasm Innovation and Molecular Breeding, Shanxi Agricultural University","address":null}],"nessieId":"335007979178"},{"fullName":"Zhaoxia Sun","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/1566213/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/1566213/overview","affiliation":{"name":"College of Agriculture, Institute of Agricultural Bioengineering, Shanxi Agricultural University, Taigu","address":null},"affiliations":[{"name":"College of Agriculture, Institute of Agricultural Bioengineering, Shanxi Agricultural University, Taigu","address":null},{"name":"Shanxi Key Laboratory of Minor Crops Germplasm Innovation and Molecular Breeding, Shanxi Agricultural University","address":null}],"nessieId":"343597972321"}],"dates":{"acceptedDate":"2022-11-21","recentDate":"2022-12-06"},"doi":"10.3389/fpls.2022.1023764","frontiersExtra":{"articleType":"Original Research","impact":{"citations":5,"crossrefCitations":0,"downloads":2,"frontiersViews":0,"pmcDownloads":0,"pmcViews":0,"scopusCitations":0,"views":1865},"isPartOfResearchTopic":true,"isPublished":true,"section":"Plant Cell Biology"},"guid":1023764,"images":[{"height":400,"url":"https://www.frontiersin.org/files/myhome article library/1023764/1023764_Thumb_400.jpg","width":242,"caption":null},{"height":1448,"url":"https://www.frontiersin.org/files/Articles/1023764/fpls-13-1023764-HTML/image_m/fpls-13-1023764-g001.jpg","width":876,"caption":"(B): the FA (Folate), THF (Tetrahydrofolate), and 5-M-THF (5-methyltetrahydrofolate) content of the panicle of foxtail millet with above SA treatment. “*” indicates significant difference (p \u003c 0.05), “**” and “***” indicate extremely significant differences (p\u003c0.01 and p\u003c0.001)."},{"height":1028,"url":"https://www.frontiersin.org/files/Articles/1023764/fpls-13-1023764-HTML/image_m/fpls-13-1023764-g002.jpg","width":885,"caption":null},{"height":967,"url":"https://www.frontiersin.org/files/Articles/1023764/fpls-13-1023764-HTML/image_m/fpls-13-1023764-g003.jpg","width":888,"caption":null},{"height":1548,"url":"https://www.frontiersin.org/files/Articles/1023764/fpls-13-1023764-HTML/image_m/fpls-13-1023764-g004.jpg","width":883,"caption":"(A): PYRP2: 5-amino-6-(5-phospho-D-ribitylamino)uracil phosphatase; RFK: bi-functional riboflavin kinase; RIBA1/RIBA2;bi-functional riboflavin biosynthesis protein; MOCS1A/1B: GTP 3’,8-cyclase; HPPK/DHPS1: folate synthesis bi-functional protein; DHFR1/DHFR2: dihydrofolate reductase; FTHFD: formyltetrahydrofolate deformylase; MTRF: methionyl-tRNA formyltransferase; 5-FCL1/5-FCL2: 5-formyltetrahydrofolate cyclo-ligase. (B):CYSC1/CYSK: cysteine synthase; AK: bi-functional aspartokinase/homoserine dehydrogenase; BHMT2: homocysteine S-methyltransferase; metE: 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase; DNMT1: DNA (cytosine-5)-methyltransferase; ACS: 1-aminocyclopropane-1-carboxylate synthase; ACO1/2: 1-aminocyclopropane-1-carboxylate oxidase; AMD1: S-adenosylmethionine decarboxylase proenzyme; SRM: spermine synthase; APIP: bi-functional methylthioribulose-1-phosphate dehydratase/enolase-phosphatase; ADI1: 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase; TAT1/TAT2: tyrosine aminotransferase."},{"height":1478,"url":"https://www.frontiersin.org/files/Articles/1023764/fpls-13-1023764-HTML/image_m/fpls-13-1023764-g005.jpg","width":880,"caption":null},{"height":1788,"url":"https://www.frontiersin.org/files/Articles/1023764/fpls-13-1023764-HTML/image_m/fpls-13-1023764-g006.jpg","width":886,"caption":null},{"height":1166,"url":"https://www.frontiersin.org/files/Articles/1023764/fpls-13-1023764-HTML/image_m/fpls-13-1023764-g007.jpg","width":884,"caption":"“*” indicates significant difference (p \u003c 0.05), “**” and “***” indicate extremely significant differences (p\u003c0.01 and p\u003c0.001)."},{"height":798,"url":"https://www.frontiersin.org/files/Articles/1023764/fpls-13-1023764-HTML/image_m/fpls-13-1023764-g008.jpg","width":1488,"caption":null}],"journal":{"guid":373,"name":"Frontiers in Plant Science","link":null,"nessieId":null,"palette":null,"publisher":"Frontiers Media","images":null,"isOnline":null,"isDeleted":null,"isDisabled":null,"issn":null},"link":"https://www.frontiersin.org/articles/10.3389/fpls.2022.1023764","pubDate":"2022-12-06","score":10.502958579881685,"title":"Role of miRNAs in regulation of SA-mediated upregulation of genes involved in folate and methionine metabolism in foxtail millet","topics":["Salicylic Acid","miRNA","Folate","foxtail millet","Methionine metabolism"],"pdfUrl":"https://www.frontiersin.org/articles/10.3389/fpls.2022.1023764/pdf"},{"__typename":"Feed_Article","_id":"6859a85efd1016fa1b12eaf2","abstract":"The role of terminators is more commonly associated with the polyadenylation and 3’ end formation of new transcripts. Recent evidence, however, suggests that this regulatory region can have a dramatic impact on gene expression. Nonetheless, little is known about the molecular mechanisms leading to the improvements associated with terminator usage in plants and the different elements in a plant terminator. Here, we identified an element in the Arabidopsis HSP18.2 terminator (tHSP) to be essential for the high level of expression seen for transgenes under the regulation of this terminator. Our molecular analyses suggest that this newly identified sequence acts to improve transcription termination, leading to fewer read-through events and decreased amounts of small RNAs originating from the transgene. Besides protecting against silencing, the tHSP-derived sequence positively impacts splicing efficiency, helping to promote gene expression. Moreover, we show that this sequence can be used to generate chimeric terminators with enhanced efficiency, resulting in stronger transgene expression and significantly expanding the availability of efficient terminators that can be part of good expression systems. Thus, our data makes an important contribution toward a better understanding of plant terminators, with the identification of a new element that has a direct impact on gene expression, and at the same time, creates new possibilities to modulate gene expression via the manipulation of 3’ regulatory regions.","htmlAbstract":"\u003cp\u003eThe role of terminators is more commonly associated with the polyadenylation and 3′ end formation of new transcripts. Recent evidence, however, suggests that this regulatory region can have a dramatic impact on gene expression. Nonetheless, little is known about the molecular mechanisms leading to the improvements associated with terminator usage in plants and the different elements in a plant terminator. Here, we identified an element in the Arabidopsis \u003ci\u003eHSP18.2\u003c/i\u003e terminator (tHSP) to be essential for the high level of expression seen for transgenes under the regulation of this terminator. Our molecular analyses suggest that this newly identified sequence acts to improve transcription termination, leading to fewer read-through events and decreased amounts of small RNAs originating from the transgene. Besides protecting against silencing, the tHSP-derived sequence positively impacts splicing efficiency, helping to promote gene expression. Moreover, we show that this sequence can be used to generate chimeric terminators with enhanced efficiency, resulting in stronger transgene expression and significantly expanding the availability of efficient terminators that can be part of good expression systems. Thus, our data make an important contribution toward a better understanding of plant terminators, with the identification of a new element that has a direct impact on gene expression, and at the same time, creates new possibilities to modulate gene expression via the manipulation of 3′ regulatory regions.\u003c/p\u003e","authors":[{"fullName":"Felipe Fenselau de Felippes","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/966837/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/966837/overview","affiliation":{"name":"Centre for Agriculture and the Bioeconomy, Institute for Future Environments, Queensland University of Technology","address":null},"affiliations":[{"name":"Centre for Agriculture and the Bioeconomy, Institute for Future Environments, Queensland University of Technology","address":null},{"name":"Australian Research Council (ARC) Centre of Excellence for Plant Success in Nature and Agriculture, Queensland University of Technology","address":null}],"nessieId":"214749016784"},{"fullName":"Kylie Shand","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/1800501/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/1800501/overview","affiliation":{"name":"Centre for Agriculture and the Bioeconomy, Institute for Future Environments, Queensland University of Technology","address":null},"affiliations":[{"name":"Centre for Agriculture and the Bioeconomy, Institute for Future Environments, Queensland University of Technology","address":null}],"nessieId":"412317519313"},{"fullName":"Peter M. Waterhouse","firstName":null,"middleName":null,"lastName":null,"image":{"height":null,"url":"https://loop.frontiersin.org/images/profile/303695/70","width":null,"caption":null},"loopProfileUrl":"https://loop.frontiersin.org/people/303695/overview","affiliation":{"name":"Centre for Agriculture and the Bioeconomy, Institute for Future Environments, Queensland University of Technology","address":null},"affiliations":[{"name":"Centre for Agriculture and the Bioeconomy, Institute for Future Environments, Queensland University of Technology","address":null},{"name":"Australian Research Council (ARC) Centre of Excellence for Plant Success in Nature and Agriculture, Queensland University of Technology","address":null}],"nessieId":"120259729975"}],"dates":{"acceptedDate":"2022-04-27","recentDate":"2022-05-16"},"doi":"10.3389/fpls.2022.877793","frontiersExtra":{"articleType":"Original Research","impact":{"citations":16,"crossrefCitations":0,"downloads":82,"frontiersViews":0,"pmcDownloads":0,"pmcViews":0,"scopusCitations":0,"views":4727},"isPartOfResearchTopic":true,"isPublished":true,"section":"Plant Cell Biology"},"guid":877793,"images":[{"height":265,"url":"https://www.frontiersin.org/files/myhome article library/877793/877793_Thumb_400.jpg","width":401,"caption":null},{"height":3009,"url":"https://www.frontiersin.org/files/Articles/877793/fpls-13-877793-HTML/image_m/fpls-13-877793-g001.jpg","width":4550,"caption":"Analysis of the tHSP poly(A) signal. (A) The sequence of the HSP18.2 terminator, with putative poly(A) signals represented in bold/underlined characters. The functional and a potential second near upstream element (NUE and NUE*, respectively) are shown. A region with characteristics compatible with a far upstream element (FUE*) is indicated by a dashed line. The black arrow points to the dominant poly(A) cleavage site (CS). Numbers indicate the nucleotide position relative to the start of the sequence, as well as regions deleted to produce the different constructs shown in Figure 2. (B) Scheme representing the pRBCS::GiFiP::tHSP reporter and variations carrying mutations in either one or both NUEs. (C) A fragment of the tHSP sequence containing the mapped poly(A) sites indicated by numbered arrows. The expected dominant site is shown with a red arrow. (D) Graphs representing the percentage that each of the positions depicted in panel (C) was cloned. Poly(A) mapping data are from a single (2nd_NUE_mut and both_NUE_mut) or two independent experiments (tHSP and 1st_NUE_mut)."},{"height":4051,"url":"https://www.frontiersin.org/files/Articles/877793/fpls-13-877793-HTML/image_m/fpls-13-877793-g002.jpg","width":2217,"caption":"Deletion analysis to assess the effect of different regions of the tHSP on the terminator activity. (A) Representation of the pRBCS::GiFiP reporter using the wild-type tHSP or different versions containing deletions of specific regions. (B) Nicotiana benthamiana leaves agroinfiltrated with the different constructs. Leaves were infiltrated with the test sample (left side) and the wild-type terminator (right side) as reference. Infiltrations for 5′ and 3′ end mutants were carried out in independent experiments and hence, shown separately. (C) GFP fluorescence for deletions affecting the 5′ end of the terminator was calculated from five infiltration spots (originating from four leaves, each from a different plant). Values referent to the 3′ end mutations are from 10 infiltration spots coming from five leaves, each leaf from an independent plant. All values are relative to the wild-type tHSP. Standard error bars are given. Statistically significant differences between the wild-type and each of the mutants (Mann-Whitney U test) are indicated by “**” (p ≤ 0.01)."},{"height":1719,"url":"https://www.frontiersin.org/files/Articles/877793/fpls-13-877793-HTML/image_m/fpls-13-877793-g003.jpg","width":4317,"caption":"Investigation of the capacity of the tHSP 5′ end to improve other terminators’ efficiency. (A) Schematic of the strategy used to test the ability of the tHSP 5′ fragment in improving the activity of weaker terminators. (B) Graphic representation of the different reporter constructs generated to test the impact of the 5′ region of the tHSP on the activity of the ACS2 terminator. (C) Agroinfiltrated leaves representing the GFP expression obtained with different terminator constructs (left side) compared to the wild-type reference (tACS2, right side). (D) Relative GFP fluorescence of at least eight infiltration spots originating from five leaves, each leaf from a different plant (the exact number of independent measurements for each construct is indicated by the numbers in the graph columns). The standard error is shown. Statistically significant differences (p ≤ 0.001; Mann-Whitney U test) are indicated by a “***”."},{"height":4402,"url":"https://www.frontiersin.org/files/Articles/877793/fpls-13-877793-HTML/image_m/fpls-13-877793-g004.jpg","width":4317,"caption":"Impact of the tHSP 5′ fragment on the activity of different terminators. Illustration of the different approaches (A) and constructs (B) utilized to test the impact of the tHSP 5′ end on different terminators. (C) Nicotiana benthamiana agroinfiltrated leaves representing the improvement of GFP expression as a result of the incorporation of one or two copies of the tHSP 5′ region into the sequence of different terminators. (D) The relative fluorescence of the reporter under the control of wild-type or chimeric terminators is shown. Values are from independent infiltration spots collected from four (tH4), five (tACS2 and tRBCS), or six leaves (tNOS), each one from a different plant. The exact number of replicates analyzed is indicated for each line (numbers shown in the graph columns). Statistically significant differences between wild-type and chimeric terminators, and between regulatory elements carrying one or two copies of the tHSP 5′ fragment are indicated with “**” or “***” for a p-value of p ≤ 0.01 and p ≤ 0.001, respectively (Mann-Whitney U test). (E) GFP fluorescence in 21-days old T1 plants representing a population carrying the wild-type tRBCS compared to chimeric versions containing one or two copies of the tHSP 5′ region (tRBCS_tHSP_5′ and tRBCS_2x_tHSP_5′, respectively). (F) GFP fluorescence relative to the wild-type tRBCS. The number of plants analyzed is indicated for each group. Statistically significant differences (Welsh t-test; two-sample unequal variance; two-tailed distribution) between wild type and chimeric terminators (“**”; p ≤ 0.01), and between regulatory elements carrying one or two copies of the tHSP 5′ fragment are given (“*”; p ≤ 0.05)."},{"height":3012,"url":"https://www.frontiersin.org/files/Articles/877793/fpls-13-877793-HTML/image_m/fpls-13-877793-g005.jpg","width":4317,"caption":"Characterization of tHSP 5′ region. (A) Constructs designed to identify sequence elements contributing to the positive effect exhibited by the tHSP 5′ end. Representative GFP phenotypes (B) and the relative levels of fluorescence (C) of 10 infiltration spots are given (replicates collected from five leaves, one leaf per plant). Mutations resulting in statistically significant changes in fluorescence when compared to tH4_tHSP_5′ are marked with a “**” or “***” (p ≤ 0.01 and p ≤ 0.001, respectively; Mann-Whitney U test). (D) The tHSP 5′ region. Sequences contributing to the improvement of gene expression are shown in black, with the degree of impact reflected in the size of the characters. Lines underneath the sequence highlight possible motifs presented in this fragment."},{"height":3053,"url":"https://www.frontiersin.org/files/Articles/877793/fpls-13-877793-HTML/image_m/fpls-13-877793-g006.jpg","width":4317,"caption":"Analysis of the poly(A) tail formation. (A) Mapping of the poly(A) sites in wild-type and mutant terminators. For each regulatory sequence, a graphic representation of the different terminators with the identified cleavage positions (numbered arrows) and the percentage of these sites are given. The dominant poly(A) sites (as expected from previous publications) are indicated by red arrows. The poly(A) signals are shown as a line above the illustration. (B) The poly(A) tail average size with standard error is presented. The number of clones analyzed is the same as indicated in panel A, except for tHSP (14 clones), tHSP_5′Δ32 (13 clones) and tRBCS (17 clones). All data originated from a single experiment."},{"height":4878,"url":"https://www.frontiersin.org/files/Articles/877793/fpls-13-877793-HTML/image_m/fpls-13-877793-g007.jpg","width":4083,"caption":"The role of the tHSP 5′ fragment on transcription termination, protection against sRNA production and splicing. (A) RT-qPCR analysis to detect read-through and GiFiP expression levels. All values are normalized to GAPDH (GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE) and are relative to the wild-type regulatory sequence. Read-through refers to the detection of a region of the T-DNA downstream of the terminator sequence. For the RT-qPCR analyses, three (tRBCS), four (tNOS), or six (tHSP, tACS2 and tH4) biological replicates were used. Each replicate is an infiltration spot originating from an independent plant. Standard error bars were calculated for the biological replicates. Statistically significant differences observed between the original and modified terminators, or between constructs carrying one versus two fragments of the tHSP (Mann-Whitney U test) are indicated by “*” (p ≤ 0.05) or “**” (p ≤ 0.01). (B) Northern blot to detect sRNAs derived from the GiFiP reporter. U6 was used as the RNA loading control. (C) RT-PCR to test splicing efficiency using primers flanking each of the introns in the GiFiP gene. GAPDH was used as the internal control. (D) Proposed model explaining how terminators carrying elements similar to the one found in the tHSP can contribute to a robust gene expression. (i) In a balanced situation, the RNA POLYMERASE II (RNA Pol II) produces only a small fraction of read-through transcripts, which are readily degraded by the RNA decay pathway. (ii) In cases where the terminator efficiency is not sufficient to cope with high rates of transcription (for instance, when strong promoters are used), the occurrence of read-through transcription increases and the resulting molecules overcome the RNA decay pathway, leading to the production of RDR6-dependent sRNAs and gene silencing. (iii) Terminators containing elements like the one found in the tHSP sequence, contribute to improving transcription termination, which results in low levels of aberrant transcripts being produced. As a consequence, sRNA generation is inhibited, allowing for a robust gene expression."}],"journal":{"guid":373,"name":"Frontiers in Plant Science","link":null,"nessieId":null,"palette":null,"publisher":"Frontiers Media","images":null,"isOnline":null,"isDeleted":null,"isDisabled":null,"issn":null},"link":"https://www.frontiersin.org/articles/10.3389/fpls.2022.877793","pubDate":"2022-05-16","score":29.93786982248528,"title":"Identification of a Transferrable Terminator Element That Inhibits Small RNA Production and Improves Transgene Expression Levels","topics":["Gene Expression","silencing","small RNAs","PTGS","Terminator","Transcription termination","HSP terminator"],"pdfUrl":"https://www.frontiersin.org/articles/10.3389/fpls.2022.877793/pdf"}]],"pageParams":[null]},"dataUpdateCount":1,"dataUpdatedAt":1754639863533,"error":null,"errorUpdateCount":0,"errorUpdatedAt":0,"fetchFailureCount":0,"fetchFailureReason":null,"fetchMeta":null,"isInvalidated":false,"status":"success","fetchStatus":"idle"},"queryKey":["research-topic-articles",36673,1],"queryHash":"[\"research-topic-articles\",36673,1]"},{"state":{"data":{"researchTopicId":36673,"articleViews":11539,"articleDownloads":5124,"topicViews":765,"summary":17428},"dataUpdateCount":1,"dataUpdatedAt":1754639863477,"error":null,"errorUpdateCount":0,"errorUpdatedAt":0,"fetchFailureCount":0,"fetchFailureReason":null,"fetchMeta":null,"isInvalidated":false,"status":"success","fetchStatus":"idle"},"queryKey":["research-topic-impact",36673],"queryHash":"[\"research-topic-impact\",36673]"}]}},"__N_SSG":true},"page":"/research-topics/[id]/[slug]/mag","query":{"id":"36673","slug":"non-coding-rna-regulation-of-secondary-metabolism-in-plants"},"buildId":"ZvyQJ1c6REyZAR__c3437","assetPrefix":"/_rtmag","isFallback":false,"gsp":true,"scriptLoader":[{"id":"google-analytics","strategy":"afterInteractive","children":"(function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':\n new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],\n j=d.createElement(s),dl=l!='dataLayer'?'\u0026l='+l:'';j.async=true;j.src=\n 'https://tag-manager.frontiersin.org/gtm.js?id='+i+dl+ '\u0026gtm_auth=PYjuAXuPWCihEq8Nf7ErrA\u0026gtm_preview=env-1\u0026gtm_cookies_win=x';f.parentNode.insertBefore(j,f);\n })(window,document,'script','dataLayer','GTM-PT9D93K');"}]}