Cloé Villard, Idil Baser, Arjen C. van de Peppel, Katarina Cankar, M. Eric Schranz, Robin van Velzen
Cannabinoids, such as tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA) and cannabichromenic acid (CBCA), are bioactive and medicinally relevant compounds found in the cannabis plant ( Cannabis sativa L.). These three compounds are synthesised from a single precursor, cannabigerolic acid (CBGA), through regioselective reactions catalysed by different cannabinoid oxidocyclase enzymes. Despite the importance of cannabinoid oxidocyclases for determining cannabis chemotype and properties, the functional evolution and molecular mechanism of this enzyme family remain poorly understood. To address this gap, we combined ancestral sequence reconstruction and heterologous expression to resurrect and functionally characterise three ancestral cannabinoid oxidocyclases. Results showed that the ability to metabolise CBGA originated in a recent ancestor of cannabis and that early cannabinoid oxidocyclases were promiscuous enzymes producing all three THCA, CBDA and CBCA. Gene duplication and diversification later facilitated enzyme subfunctionalisation, leading to extant, highly‐specialised THCA and CBDA synthases. Through rational engineering of these ancestors, we designed hybrid enzymes which allowed identifying key amino acid mutations underlying the functional evolution of cannabinoid oxidocyclases. Ancestral and hybrid enzymes also displayed unique activities and proved to be easier to produce heterologously than their extant counterparts. Overall, this study contributes to understanding the origin, evolution and molecular mechanism of cannabinoid oxidocyclases, which opens new perspectives for breeding, biotechnological and medicinal applications.
{"title":"Resurrected Ancestral Cannabis Enzymes Unveil the Origin and Functional Evolution of Cannabinoid Synthases","authors":"Cloé Villard, Idil Baser, Arjen C. van de Peppel, Katarina Cankar, M. Eric Schranz, Robin van Velzen","doi":"10.1111/pbi.70475","DOIUrl":"https://doi.org/10.1111/pbi.70475","url":null,"abstract":"Cannabinoids, such as tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA) and cannabichromenic acid (CBCA), are bioactive and medicinally relevant compounds found in the cannabis plant ( <jats:styled-content style=\"fixed-case\"> <jats:italic>Cannabis sativa</jats:italic> </jats:styled-content> L.). These three compounds are synthesised from a single precursor, cannabigerolic acid (CBGA), through regioselective reactions catalysed by different cannabinoid oxidocyclase enzymes. Despite the importance of cannabinoid oxidocyclases for determining cannabis chemotype and properties, the functional evolution and molecular mechanism of this enzyme family remain poorly understood. To address this gap, we combined ancestral sequence reconstruction and heterologous expression to resurrect and functionally characterise three ancestral cannabinoid oxidocyclases. Results showed that the ability to metabolise CBGA originated in a recent ancestor of cannabis and that early cannabinoid oxidocyclases were promiscuous enzymes producing all three THCA, CBDA and CBCA. Gene duplication and diversification later facilitated enzyme subfunctionalisation, leading to extant, highly‐specialised THCA and CBDA synthases. Through rational engineering of these ancestors, we designed hybrid enzymes which allowed identifying key amino acid mutations underlying the functional evolution of cannabinoid oxidocyclases. Ancestral and hybrid enzymes also displayed unique activities and proved to be easier to produce heterologously than their extant counterparts. Overall, this study contributes to understanding the origin, evolution and molecular mechanism of cannabinoid oxidocyclases, which opens new perspectives for breeding, biotechnological and medicinal applications.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"7 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-12-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145836253","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
C. Plasson, J. Balieu, P. Lerouge, M. Bardor, E. Mathieu‐Rivet
{"title":"First Step Towards the Humanisation of Protein N ‐Glycosylation in Chlamydomonas reinhardtii Through Heterologous Expression of α(1,2)‐Mannosidases","authors":"C. Plasson, J. Balieu, P. Lerouge, M. Bardor, E. Mathieu‐Rivet","doi":"10.1111/pbi.70482","DOIUrl":"https://doi.org/10.1111/pbi.70482","url":null,"abstract":"","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"37 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-12-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145836230","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Zhengrong Dai, Yating Li, Changsheng Zhai, Jie Li, Ya Zeng, Jinyao Ouyang, Bing He, Wei Wang, Pingyin Guan, Don Grierson, Kunsong Chen, Wensuo Jia
Control of fruit ripening, quality and yield is of major scientific, nutritional and commercial importance. The burst of ethylene (ET) production at the initiation of ripening is the most critical event controlling climacteric (CL) fruit ripening, yet little is known about how it is initiated. ABA is known to be capable of inducing ET production in many biological processes. However, the mechanism for this ABA induced ET production (AEP) and its potential importance in the burst of ET production that initiates ripening are unclear. Here, we report a branched signalling network involving ABA‐activation of multiple SlSnRK2 (SNF1‐related protein kinase 2) kinases, which, when overexpressed in tomato, stimulated ABA‐induced ET production. Two key components, SlSnRK2.1 and SlSnRK2.4, phosphorylate an HD‐Zip homeobox transcription factor, SlHB1, which transcriptionally activates ACC oxidase ( SlACO1 ), required for ethylene synthesis. Concurrently, SlSnRK2.1 and SlSnRK2.4 phosphorylate two mitogen‐activated protein kinases, SlMPK1/2, resulting in the post‐translational stabilisation of ACC synthase (SlACS2), which generates the precursor 1‐aminocyclopropane‐1‐carboxylic acid (ACC) that is converted to ET by ACO1. Removal of SlSnRK2.1 by CRISPR/Cas9 mutation was sufficient to alter the progress of fruit ripening. These results indicate that ABA is a primary hormonal signal modulating CL fruit ripening that stimulates ethylene production by targeting different steps in the ethylene biosynthesis pathway by both transcriptional and post‐translational mechanisms. Further analysis revealed that removal of SlSnRK2.1 signalling also affected other aspects of the life cycle by prolonging the flowering period and suppressing seed development, indicating the potential for modifying fruit cropping and seedlessness.
{"title":"A Branched SNF1 ‐Related Protein Kinase 2 Signalling Cascade Controls ABA ‐Induced Ethylene Production and Regulates Both Fruit Ripening and Reproductive Growth","authors":"Zhengrong Dai, Yating Li, Changsheng Zhai, Jie Li, Ya Zeng, Jinyao Ouyang, Bing He, Wei Wang, Pingyin Guan, Don Grierson, Kunsong Chen, Wensuo Jia","doi":"10.1111/pbi.70501","DOIUrl":"https://doi.org/10.1111/pbi.70501","url":null,"abstract":"Control of fruit ripening, quality and yield is of major scientific, nutritional and commercial importance. The burst of ethylene (ET) production at the initiation of ripening is the most critical event controlling climacteric (CL) fruit ripening, yet little is known about how it is initiated. ABA is known to be capable of inducing ET production in many biological processes. However, the mechanism for this ABA induced ET production (AEP) and its potential importance in the burst of ET production that initiates ripening are unclear. Here, we report a branched signalling network involving ABA‐activation of multiple SlSnRK2 (SNF1‐related protein kinase 2) kinases, which, when overexpressed in tomato, stimulated ABA‐induced ET production. Two key components, SlSnRK2.1 and SlSnRK2.4, phosphorylate an HD‐Zip homeobox transcription factor, SlHB1, which transcriptionally activates ACC oxidase ( <jats:italic>SlACO1</jats:italic> ), required for ethylene synthesis. Concurrently, SlSnRK2.1 and SlSnRK2.4 phosphorylate two mitogen‐activated protein kinases, SlMPK1/2, resulting in the post‐translational stabilisation of ACC synthase (SlACS2), which generates the precursor 1‐aminocyclopropane‐1‐carboxylic acid (ACC) that is converted to ET by ACO1. Removal of <jats:italic>SlSnRK2.1</jats:italic> by CRISPR/Cas9 mutation was sufficient to alter the progress of fruit ripening. These results indicate that ABA is a primary hormonal signal modulating CL fruit ripening that stimulates ethylene production by targeting different steps in the ethylene biosynthesis pathway by both transcriptional and post‐translational mechanisms. Further analysis revealed that removal of <jats:italic>SlSnRK2.1</jats:italic> signalling also affected other aspects of the life cycle by prolonging the flowering period and suppressing seed development, indicating the potential for modifying fruit cropping and seedlessness.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"9 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-12-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145829939","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Grey mould, caused by Botrytis cinerea , is a significant postharvest disease leading to substantial economic losses in the grape industry. This study investigated the regulatory mechanism of 3‐methyl‐1‐butanol (3M1B), a yeast volatile substance, on B. cinerea resistance in red grapes ( Vitis vinifera L.). Our findings demonstrate that 3M1B induces resistance by modulating phenylpropane and reactive oxygen species (ROS) metabolism. Specifically, treatment with 50 μL mL −1 3M1B effectively inhibited B. cinerea lesion development on red grape surfaces and delayed fruit quality deterioration. Further analysis revealed that 3M1B enhanced the activity and gene expression of key enzymes in the phenylpropane pathway, including phenylalanine aminase, cinnamic acid‐4‐hydroxylase and 4‐coumaric acid coenzyme A ligase, promoting the accumulation of related secondary metabolites. Furthermore, ROS metabolic analysis showed that 3M1B treatment reduced hydrogen peroxide and superoxide anion accumulation. This treatment also stimulated the activity and transcriptional levels of antioxidant enzymes involved in ROS clearance, and synergistically improved oxidative stress through the glutathione‐ascorbic acid cycle. These results indicate that 3M1B maintains postharvest quality of red grapes by enhancing phenylpropane and ROS metabolism.
{"title":"3‐Methyl‐1‐Butanol Enhances Postharvest Resistance of Red Grapes to Botrytis cinerea by Activating Phenylpropanoid Metabolism and Antioxidant Defences","authors":"Shunjie Zhai, Huijing Guo, Tongrui Sun, Minrui Guo, Jiluan Chen, Jiankang Cao, Wanting Yang, Guogang Chen","doi":"10.1111/pbi.70517","DOIUrl":"https://doi.org/10.1111/pbi.70517","url":null,"abstract":"Grey mould, caused by <jats:italic>Botrytis cinerea</jats:italic> , is a significant postharvest disease leading to substantial economic losses in the grape industry. This study investigated the regulatory mechanism of 3‐methyl‐1‐butanol (3M1B), a yeast volatile substance, on <jats:styled-content style=\"fixed-case\"> <jats:italic>B. cinerea</jats:italic> </jats:styled-content> resistance in red grapes ( <jats:styled-content style=\"fixed-case\"> <jats:italic>Vitis vinifera</jats:italic> </jats:styled-content> L.). Our findings demonstrate that 3M1B induces resistance by modulating phenylpropane and reactive oxygen species (ROS) metabolism. Specifically, treatment with 50 μL mL <jats:sup>−1</jats:sup> 3M1B effectively inhibited <jats:styled-content style=\"fixed-case\"> <jats:italic>B. cinerea</jats:italic> </jats:styled-content> lesion development on red grape surfaces and delayed fruit quality deterioration. Further analysis revealed that 3M1B enhanced the activity and gene expression of key enzymes in the phenylpropane pathway, including phenylalanine aminase, cinnamic acid‐4‐hydroxylase and 4‐coumaric acid coenzyme A ligase, promoting the accumulation of related secondary metabolites. Furthermore, ROS metabolic analysis showed that 3M1B treatment reduced hydrogen peroxide and superoxide anion accumulation. This treatment also stimulated the activity and transcriptional levels of antioxidant enzymes involved in ROS clearance, and synergistically improved oxidative stress through the glutathione‐ascorbic acid cycle. These results indicate that 3M1B maintains postharvest quality of red grapes by enhancing phenylpropane and ROS metabolism.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"25 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-12-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145829940","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pangenome can reveal a large number of variations, providing a more comprehensive view of the genetic diversity of species that a single reference genome cannot surpass. Here, we assembled the haplotype telomere‐to‐telomere genome and 10 chromosome‐level genomes, integrated with two previously reported genomes, and constructed a graph pangenome for P. mume with ‘PmNH_Hap1’ T2T genome as the reference. Core gene families accounted for 64.47%, while non‐essential gene families comprised 35.53%. The total length of the graph pangenome reaches 412.41 Mb, which is a significant increase of 179.60 Mb compared with the ‘PmNH_Hap1’ reference genome, and these unique insertion sequences contain 5918 genes. Additionally, by combining with five Prunus armeniaca genomes for pangenome variation detection, we identified a total of 51 461 non‐redundant SVs, including 15 217 insertions, 21 683 deletions, 13 509 translocations and 1025 inversions. Among these, 60.50% of presence/absence variations were formed by transposons. Pangenome analysis revealed that a 376 bp SV insertion was universally present in the promoter of this gene in P. mume , PmAGL30 can bind to this SV to enhance PmPH4 gene expression and promoted citric acid accumulation in P. mume fruits, leading to extremely significant differences in citric acid content compared with other stone fruits. Additionally, we developed SV molecular markers for the early screening of germplasm with high citric acid content in P. mume fruits. In summary, we constructed a high‐quality graph pangenome that reveals abundant genetic variations, providing valuable insights for variety improvement and molecular breeding in P. mume .
{"title":"Pangenome Analysis Reveals Structural Variations Associated With Citric Acid Accumulation in Prunus mume","authors":"Xiao Huang, Ximeng Lin, Pengyu Zhou, Wei Tan, Feng Gao, Zhaojun Ni, Ting Shi, Yang Bai, Chengdong Ma, Yufan Ma, Minglu Li, Faisal Hayat, Qixiang Zhang, Ryutaro Tao, Jianmin Wan, Zhihong Gao","doi":"10.1111/pbi.70518","DOIUrl":"https://doi.org/10.1111/pbi.70518","url":null,"abstract":"Pangenome can reveal a large number of variations, providing a more comprehensive view of the genetic diversity of species that a single reference genome cannot surpass. Here, we assembled the haplotype telomere‐to‐telomere genome and 10 chromosome‐level genomes, integrated with two previously reported genomes, and constructed a graph pangenome for <jats:styled-content style=\"fixed-case\"> <jats:italic>P. mume</jats:italic> </jats:styled-content> with ‘PmNH_Hap1’ T2T genome as the reference. Core gene families accounted for 64.47%, while non‐essential gene families comprised 35.53%. The total length of the graph pangenome reaches 412.41 Mb, which is a significant increase of 179.60 Mb compared with the ‘PmNH_Hap1’ reference genome, and these unique insertion sequences contain 5918 genes. Additionally, by combining with five <jats:styled-content style=\"fixed-case\"> <jats:italic>Prunus armeniaca</jats:italic> </jats:styled-content> genomes for pangenome variation detection, we identified a total of 51 461 non‐redundant SVs, including 15 217 insertions, 21 683 deletions, 13 509 translocations and 1025 inversions. Among these, 60.50% of presence/absence variations were formed by transposons. Pangenome analysis revealed that a 376 bp SV insertion was universally present in the promoter of this gene in <jats:styled-content style=\"fixed-case\"> <jats:italic>P. mume</jats:italic> </jats:styled-content> , <jats:italic>PmAGL30</jats:italic> can bind to this SV to enhance <jats:italic>PmPH4</jats:italic> gene expression and promoted citric acid accumulation in <jats:styled-content style=\"fixed-case\"> <jats:italic>P. mume</jats:italic> </jats:styled-content> fruits, leading to extremely significant differences in citric acid content compared with other stone fruits. Additionally, we developed SV molecular markers for the early screening of germplasm with high citric acid content in <jats:styled-content style=\"fixed-case\"> <jats:italic>P. mume</jats:italic> </jats:styled-content> fruits. In summary, we constructed a high‐quality graph pangenome that reveals abundant genetic variations, providing valuable insights for variety improvement and molecular breeding in <jats:styled-content style=\"fixed-case\"> <jats:italic>P. mume</jats:italic> </jats:styled-content> .","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"22 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-12-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145813165","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ran Han, Aifeng Liu, Guang Qi, Chunhao Fang, XiaoLu Wang, Wenjing Xu, Kai Wang, Jihu Li, Qingqi Fan, Dungong Cheng, Faji Li, Haosheng Li, Jianjun Liu, Genying Li, Cheng Liu
Male‐sterile genes and mutants are critical for hybrid seed production in monocotyledonous crops. Lipids are essential structural components of male reproductive organs, such as the anther and pollen. Here, we show that the pollen‐preferential gene TaRIP2 is essential for wheat anther development and pollen formation. RT‐qPCR analysis revealed TaRIP2 is specifically expressed during the callose and tetrad stages. Using CRISPR/Cas9, we generated TaRIP2 mutants ( rip2 ), which displayed smaller, wilted anthers with defective cuticles and a low proportion of viable pollen grains (~5.7%). Microscopy revealed that the mutant Rip2 microspores had a smaller size, a smooth exine lacking sculptural elements and fewer organelles. RNA‐seq identified differentially expressed genes (DEGs) enriched in pathways related to pollen wall formation. KEGG analysis showed these DEGs are involved in cutin, suberine and wax biosynthesis and fatty acid degradation. Fatty acids C16:0, C18:0 and C18:2 were significantly elevated in rip2 anthers. These phenotypes coincided with the downregulation of genes involved in lipid metabolism and anther development. Dual‐luciferase and EMSA assays confirmed TaRIP2 is directly regulated by the transcription factor MYB80. Together, our results show TaRIP2 regulates pollen wall formation through the MYB80‐control lipid metabolic pathways.
{"title":"TaRIP2 Positively Regulates Wheat Pollen Wall Formation Through MYB80 ‐Controlled Lipid Metabolism","authors":"Ran Han, Aifeng Liu, Guang Qi, Chunhao Fang, XiaoLu Wang, Wenjing Xu, Kai Wang, Jihu Li, Qingqi Fan, Dungong Cheng, Faji Li, Haosheng Li, Jianjun Liu, Genying Li, Cheng Liu","doi":"10.1111/pbi.70512","DOIUrl":"https://doi.org/10.1111/pbi.70512","url":null,"abstract":"Male‐sterile genes and mutants are critical for hybrid seed production in monocotyledonous crops. Lipids are essential structural components of male reproductive organs, such as the anther and pollen. Here, we show that the pollen‐preferential gene <jats:italic>TaRIP2</jats:italic> is essential for wheat anther development and pollen formation. RT‐qPCR analysis revealed <jats:italic>TaRIP2</jats:italic> is specifically expressed during the callose and tetrad stages. Using CRISPR/Cas9, we generated <jats:italic>TaRIP2</jats:italic> mutants ( <jats:italic>rip2</jats:italic> ), which displayed smaller, wilted anthers with defective cuticles and a low proportion of viable pollen grains (~5.7%). Microscopy revealed <jats:italic>that the mutant Rip2</jats:italic> microspores had a smaller size, a smooth exine lacking sculptural elements and fewer organelles. RNA‐seq identified differentially expressed genes (DEGs) enriched in pathways related to pollen wall formation. KEGG analysis showed these DEGs are involved in cutin, suberine and wax biosynthesis and fatty acid degradation. Fatty acids C16:0, C18:0 and C18:2 were significantly elevated in <jats:italic>rip2</jats:italic> anthers. These phenotypes coincided with the downregulation of genes involved in lipid metabolism and anther development. Dual‐luciferase and EMSA assays confirmed <jats:italic>TaRIP2</jats:italic> is directly regulated by the transcription factor MYB80. Together, our results show <jats:italic>TaRIP2</jats:italic> regulates pollen wall formation through the MYB80‐control lipid metabolic pathways.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"13 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-12-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145813170","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Nicotine is the most abundant alkaloid produced by Nicotiana species against insect herbivores. Although it has been known for decades that auxin can strongly inhibit nicotine biosynthesis, the underlying mechanisms have remained unclear. Here we demonstrated that NaERF1‐like, the orthologue of NtERF199, is a key regulator of nicotine biosynthesis through gene‐editing and stable overexpression. NaERF1‐like expression was strongly inhibited by auxin treatments, suggesting that it is a target gene regulated by auxin. Yeast two‐hybrid screening revealed that NaARF5, an auxin response factor, interacts with NaERF1‐like. This interaction was further confirmed by GST pull‐down, bimolecular fluorescence complementation, and split‐luciferase complementation. NaARF5 acts as a key negative regulator of nicotine production since both the expression of nicotine‐related genes and nicotine production increased significantly in NaARF5 mutants generated by CRISPR/Cas9 but was severely impaired in NaARF5 overexpression lines. Notably, the inhibition of nicotine‐related genes by auxin, including NaERF1‐like , is alleviated in NaARF5 mutants. Furthermore, the interaction of NaARF5 with NaERF1‐like reduced the binding and activating ability of NaERF1‐like to the promoter of NaPMT1.1 , a key enzyme gene involved in nicotine biosynthesis, as well as the transcriptional activity of NaQPT2 . Thus, we uncovered a new auxin‐mediated regulatory pathway for the inhibition of nicotine biosynthesis through NaARF5 by targeting NaERF1‐like. Our findings provide novel insights into the molecular mechanisms underlying the inhibition of defensive metabolites by auxin and establish NaARF5 as a valuable breeding target for manipulating nicotine production.
{"title":"Auxin‐Induced Nicotine Inhibition Is Mediated by NaARF5 Through the Suppression of NaERF1‐Like Expression and Interaction With NaERF1‐Like in Nicotiana attenuata ","authors":"Miyun Yang, Ahui Tong, Lei Wang, Jinsong Wu","doi":"10.1111/pbi.70524","DOIUrl":"https://doi.org/10.1111/pbi.70524","url":null,"abstract":"Nicotine is the most abundant alkaloid produced by <jats:italic>Nicotiana</jats:italic> species against insect herbivores. Although it has been known for decades that auxin can strongly inhibit nicotine biosynthesis, the underlying mechanisms have remained unclear. Here we demonstrated that NaERF1‐like, the orthologue of NtERF199, is a key regulator of nicotine biosynthesis through gene‐editing and stable overexpression. <jats:italic>NaERF1‐like</jats:italic> expression was strongly inhibited by auxin treatments, suggesting that it is a target gene regulated by auxin. Yeast two‐hybrid screening revealed that NaARF5, an auxin response factor, interacts with NaERF1‐like. This interaction was further confirmed by GST pull‐down, bimolecular fluorescence complementation, and split‐luciferase complementation. NaARF5 acts as a key negative regulator of nicotine production since both the expression of nicotine‐related genes and nicotine production increased significantly in <jats:italic>NaARF5</jats:italic> mutants generated by CRISPR/Cas9 but was severely impaired in <jats:italic>NaARF5</jats:italic> overexpression lines. Notably, the inhibition of nicotine‐related genes by auxin, including <jats:italic>NaERF1‐like</jats:italic> , is alleviated in <jats:italic>NaARF5</jats:italic> mutants. Furthermore, the interaction of NaARF5 with NaERF1‐like reduced the binding and activating ability of NaERF1‐like to the promoter of <jats:italic>NaPMT1.1</jats:italic> , a key enzyme gene involved in nicotine biosynthesis, as well as the transcriptional activity of <jats:italic>NaQPT2</jats:italic> . Thus, we uncovered a new auxin‐mediated regulatory pathway for the inhibition of nicotine biosynthesis through NaARF5 by targeting NaERF1‐like. Our findings provide novel insights into the molecular mechanisms underlying the inhibition of defensive metabolites by auxin and establish NaARF5 as a valuable breeding target for manipulating nicotine production.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"24 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-12-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145813169","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Leaf size and shape are crucial traits affecting photosynthesis. Moderate leaf curling is believed to be beneficial to photosynthesis because upright leaves are ideal for building a canopy structure with reduced shaded areas to improve light penetration and ventilation. In this study, we investigated the cytological features of a natural cotton mutant showing cup‐shaped leaves due to upward curling of leaf edge and identified the gene regulating leaf edge curling. Genetic analysis indicates that the cup‐shaped leaf is caused by mutations in an incomplete dominant gene, designated GhCUP . The gene was mapped to a 206‐kb genomic region containing 16 annotated genes in chromosome A11. Based on the annotation and expression difference of the genes in the interval between the mutant and cotton cultivar with normal leaf shape, virus‐induced gene silencing, and gene overexpression, GH_A11G3479 was identified as GhCUP . The promoter sequence of GhCUP contains several cis ‐elements that could potentially interact with transcription factors involved in leaf development, with some of them mutated in the cup‐shaped leaf mutant. One of those mutations was in a binding site of KAN1 , which might contribute to up‐regulation of GhCUP in the mutant, leading to the mutant phenotype, as KAN1 could interact with the promoter of GhCUP from cotton cultivar with normal leaf shape but binding affinity to the mutant promoter was significantly reduced. Identifying GhCUP provides a novel gene for creating ideal canopy structure by manipulating cotton leaf shape.
{"title":"Genetic Mapping and Functional Characterisation of GhCUP Regulating Leaf Curling in Cotton","authors":"Haonan Shi, Yanqing Qiao, Xiaohu Ma, Qian‐Hao Zhu, Haohong Tang, Yanlong Jin, Xuefeng Wang, Feng Liu, Jie Sun, Fei Xue","doi":"10.1111/pbi.70513","DOIUrl":"https://doi.org/10.1111/pbi.70513","url":null,"abstract":"Leaf size and shape are crucial traits affecting photosynthesis. Moderate leaf curling is believed to be beneficial to photosynthesis because upright leaves are ideal for building a canopy structure with reduced shaded areas to improve light penetration and ventilation. In this study, we investigated the cytological features of a natural cotton mutant showing cup‐shaped leaves due to upward curling of leaf edge and identified the gene regulating leaf edge curling. Genetic analysis indicates that the cup‐shaped leaf is caused by mutations in an incomplete dominant gene, designated <jats:italic>GhCUP</jats:italic> . The gene was mapped to a 206‐kb genomic region containing 16 annotated genes in chromosome A11. Based on the annotation and expression difference of the genes in the interval between the mutant and cotton cultivar with normal leaf shape, virus‐induced gene silencing, and gene overexpression, <jats:italic>GH_A11G3479</jats:italic> was identified as <jats:italic>GhCUP</jats:italic> . The promoter sequence of <jats:italic>GhCUP</jats:italic> contains several <jats:italic>cis</jats:italic> ‐elements that could potentially interact with transcription factors involved in leaf development, with some of them mutated in the cup‐shaped leaf mutant. One of those mutations was in a binding site of <jats:italic>KAN1</jats:italic> , which might contribute to up‐regulation of <jats:italic>GhCUP</jats:italic> in the mutant, leading to the mutant phenotype, as <jats:italic>KAN1</jats:italic> could interact with the promoter of <jats:italic>GhCUP</jats:italic> from cotton cultivar with normal leaf shape but binding affinity to the mutant promoter was significantly reduced. Identifying <jats:italic>GhCUP</jats:italic> provides a novel gene for creating ideal canopy structure by manipulating cotton leaf shape.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"3 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-12-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145813166","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Zhen Xu,Zheng-Shuang Zhu,Hai-Zhu Jia,Jian-Qiang Kong
Apiin, an active flavonoid apioside, occurs in phylogenetically distant plants, such as parsley and chilli pepper (Capsicum annuum L.). Its sporadic distribution suggests the existence of convergent apiin biosynthesis; however, the molecular mechanisms underlying this convergence remain unclear. In this study, apiin biosynthesis in chilli pepper was comprehensively clarified. The structural genes involved in the biosynthesis of apigenin 7-O-glucoside, a precursor of apiin, were conserved using a homology-based approach. An apiin-biosynthetic apiosyltransferase (ApiGT), CaApiGT1, was identified from Capsicum annuum using comparative transcriptomic technology and in vitro enzymatic assays. Despite its strict specificity for the UDP-apiose donor, CaApiGT1 recognised multiple acceptors, including flavonoid 7-, 4'-and 3-O-glycosides. The sequence identity between CaApiGT1 and other apiin-biosynthetic ApiGTs was below 30%. CaApiGT1 (UGT79) and other apiin-biosynthetic ApiGTs (UGT94) belong to different UDP-dependent glycosyltransferase (UGT) families. These evidences collectively confirmed that CaApiGT1 is a novel ApiGT. Thus, convergent apiin biosynthesis was essentially caused by the convergent evolution of ApiGTs, rather than by distinct biosynthetic pathways between phylogenetically distant plants. Subsequently, CaApiGT1 was expressed in Escherichia coli to construct an engineered strain (EAA3PF) for apiin synthesis from apigenin. Additionally, an engineered Saccharomyces cerevisiae strain (YA3P) was developed for de novo apigenin synthesis. Co-cultivation of EAA3PF and YA3P reconstitutes the apiin biosynthetic pathway. This study provides new insight into the molecular convergence and de novo reconstitution of apiin biosynthesis.
Apiin是一种活性类黄酮苷，存在于系统发育较远的植物中，如香菜和辣椒（Capsicum annuum L.）。其零星分布表明存在趋同蜂素生物合成；然而，这种趋同的分子机制尚不清楚。本文对辣椒中蜂素的生物合成进行了较为全面的阐述。利用同源性方法保守了蜂素前体——芹菜素7- o -葡萄糖苷生物合成的结构基因。利用比较转录组学技术和体外酶学分析，从辣椒中鉴定出一种生物合成的ApiGT酶CaApiGT1。尽管CaApiGT1对UDP-apiose供体具有严格的特异性，但它可以识别多种受体，包括类黄酮7-，4'和3- o -糖苷。CaApiGT1与其他蜜蜂生物合成的ApiGTs序列同源性低于30%。CaApiGT1 （UGT79）和其他蜜蜂生物合成ApiGTs （UGT94）属于不同的udp依赖性糖基转移酶（UGT）家族。这些证据共同证实了CaApiGT1是一种新型的ApiGT。因此，趋同的蜂素生物合成本质上是由ApiGTs的趋同进化引起的，而不是由系统发育上遥远的植物之间不同的生物合成途径引起的。随后，在大肠杆菌中表达CaApiGT1，构建由芹菜素合成蜂素的工程菌株（EAA3PF）。此外，还开发了一株工程酿酒酵母（YA3P），用于从头合成芹菜素。EAA3PF和YA3P的共培养重建了蜂素的生物合成途径。该研究为蜂素生物合成的分子聚合和从头重构提供了新的见解。
{"title":"Convergent Evolution and De Novo Reconstitution of Apiin Biosynthesis.","authors":"Zhen Xu,Zheng-Shuang Zhu,Hai-Zhu Jia,Jian-Qiang Kong","doi":"10.1111/pbi.70510","DOIUrl":"https://doi.org/10.1111/pbi.70510","url":null,"abstract":"Apiin, an active flavonoid apioside, occurs in phylogenetically distant plants, such as parsley and chilli pepper (Capsicum annuum L.). Its sporadic distribution suggests the existence of convergent apiin biosynthesis; however, the molecular mechanisms underlying this convergence remain unclear. In this study, apiin biosynthesis in chilli pepper was comprehensively clarified. The structural genes involved in the biosynthesis of apigenin 7-O-glucoside, a precursor of apiin, were conserved using a homology-based approach. An apiin-biosynthetic apiosyltransferase (ApiGT), CaApiGT1, was identified from Capsicum annuum using comparative transcriptomic technology and in vitro enzymatic assays. Despite its strict specificity for the UDP-apiose donor, CaApiGT1 recognised multiple acceptors, including flavonoid 7-, 4'-and 3-O-glycosides. The sequence identity between CaApiGT1 and other apiin-biosynthetic ApiGTs was below 30%. CaApiGT1 (UGT79) and other apiin-biosynthetic ApiGTs (UGT94) belong to different UDP-dependent glycosyltransferase (UGT) families. These evidences collectively confirmed that CaApiGT1 is a novel ApiGT. Thus, convergent apiin biosynthesis was essentially caused by the convergent evolution of ApiGTs, rather than by distinct biosynthetic pathways between phylogenetically distant plants. Subsequently, CaApiGT1 was expressed in Escherichia coli to construct an engineered strain (EAA3PF) for apiin synthesis from apigenin. Additionally, an engineered Saccharomyces cerevisiae strain (YA3P) was developed for de novo apigenin synthesis. Co-cultivation of EAA3PF and YA3P reconstitutes the apiin biosynthetic pathway. This study provides new insight into the molecular convergence and de novo reconstitution of apiin biosynthesis.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"32 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145807961","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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