Takeshi Kuroha, Fabien Lombardo, Watal M. Iwasaki, Svetlana Chechetka, Yoshihiro Kawahara, Akiko Yoshida, Takashi Makino, Hitoshi Yoshida
<p>Genome editing has significantly advanced in recent years, with numerous attempts to integrate it into crop breeding (Gao, <span>2021</span>). Many useful agronomic traits result from subtle changes in gene expression patterns conferred by natural variations (Olsen and Wendel, <span>2013</span>). Therefore, the modification of regulatory sequences through genome editing presents a potential strategy to develop practical breeding resources. Promoters and <i>cis</i>-regulatory elements (CREs) of several target genes have been extensively edited to alter their expression patterns in many crop species including tomato (Rodriguez-Leal <i>et al</i>., <span>2017</span>) and rice (Zhou <i>et al</i>., <span>2023</span>). However, such approaches require numerous genome edits across a wide range of promoter regions or rely on molecular genetic evidence for responsible CREs. Identifying optimal genome-editing target sites within large genome regions to improve desirable agronomic traits remains challenging. Here, we describe creation of quantitative trait variations in panicle branching by precise genome editing of a conserved noncoding sequence (CNS) (Freeling and Subramaniam, <span>2009</span>) located downstream of the rice yield-related gene <i>TAWAWA1</i> (<i>TAW1</i>) (Yoshida <i>et al</i>., <span>2013</span>) and demonstrate the potential of CNSs as targets for genome editing to fine-tune agronomic traits.</p><p><i>TAW1</i> is a member of the ALOG (<i><span style="text-decoration:underline">A</span>rabidopsis</i> <span style="text-decoration:underline">L</span>SH1 and <i><span style="text-decoration:underline">O</span>ryza</i> <span style="text-decoration:underline">G</span>1) gene family encoding putative transcriptional regulators. In grass species, ALOG family proteins are essential for specification of floral organ identity and the normal development of spikelet and inflorescence architecture (Jiang <i>et al</i>., <span>2024</span>; Yoshida <i>et al</i>., <span>2013</span>). In a screen of a transposon-mutagenized rice population, Yoshida <i>et al</i>. (<span>2013</span>) isolated two allelic mutants, <i>taw1</i>-<i>D1</i> and <i>taw1</i>-<i>D2</i> exhibiting elevated <i>TAW1</i> expression and increased panicle branching. Both mutants carried <i>nDart1</i>-<i>0</i> transposons inserted approximately 0.9 kb downstream from the stop codon of <i>TAW1</i> (Figure 1a) (Yoshida <i>et al</i>., <span>2013</span>). Given the high conservation of genes governing inflorescence architecture across grass species, we hypothesized that conserved regulatory sequences would be located near the <i>taw1-D1</i>/<i>-D2</i> insertion sites in these species. We first identified <i>TAW1</i> homologues in monocot species (Table S1; Figure S1), and then compared their genomic sequences (Figure 1a). We identified a CNS (hereafter, <i>TAW1</i>-CNS) in grass species, including the BEP clade, within 50 bp downstream of the transposon insertion sites in <i>taw1-D1</i>/-<i
{"title":"Modification of TAWAWA1-mediated panicle architecture by genome editing of a downstream conserved noncoding sequence in rice","authors":"Takeshi Kuroha, Fabien Lombardo, Watal M. Iwasaki, Svetlana Chechetka, Yoshihiro Kawahara, Akiko Yoshida, Takashi Makino, Hitoshi Yoshida","doi":"10.1111/pbi.70043","DOIUrl":"https://doi.org/10.1111/pbi.70043","url":null,"abstract":"<p>Genome editing has significantly advanced in recent years, with numerous attempts to integrate it into crop breeding (Gao, <span>2021</span>). Many useful agronomic traits result from subtle changes in gene expression patterns conferred by natural variations (Olsen and Wendel, <span>2013</span>). Therefore, the modification of regulatory sequences through genome editing presents a potential strategy to develop practical breeding resources. Promoters and <i>cis</i>-regulatory elements (CREs) of several target genes have been extensively edited to alter their expression patterns in many crop species including tomato (Rodriguez-Leal <i>et al</i>., <span>2017</span>) and rice (Zhou <i>et al</i>., <span>2023</span>). However, such approaches require numerous genome edits across a wide range of promoter regions or rely on molecular genetic evidence for responsible CREs. Identifying optimal genome-editing target sites within large genome regions to improve desirable agronomic traits remains challenging. Here, we describe creation of quantitative trait variations in panicle branching by precise genome editing of a conserved noncoding sequence (CNS) (Freeling and Subramaniam, <span>2009</span>) located downstream of the rice yield-related gene <i>TAWAWA1</i> (<i>TAW1</i>) (Yoshida <i>et al</i>., <span>2013</span>) and demonstrate the potential of CNSs as targets for genome editing to fine-tune agronomic traits.</p>\u0000<p><i>TAW1</i> is a member of the ALOG (<i><span style=\"text-decoration:underline\">A</span>rabidopsis</i> <span style=\"text-decoration:underline\">L</span>SH1 and <i><span style=\"text-decoration:underline\">O</span>ryza</i> <span style=\"text-decoration:underline\">G</span>1) gene family encoding putative transcriptional regulators. In grass species, ALOG family proteins are essential for specification of floral organ identity and the normal development of spikelet and inflorescence architecture (Jiang <i>et al</i>., <span>2024</span>; Yoshida <i>et al</i>., <span>2013</span>). In a screen of a transposon-mutagenized rice population, Yoshida <i>et al</i>. (<span>2013</span>) isolated two allelic mutants, <i>taw1</i>-<i>D1</i> and <i>taw1</i>-<i>D2</i> exhibiting elevated <i>TAW1</i> expression and increased panicle branching. Both mutants carried <i>nDart1</i>-<i>0</i> transposons inserted approximately 0.9 kb downstream from the stop codon of <i>TAW1</i> (Figure 1a) (Yoshida <i>et al</i>., <span>2013</span>). Given the high conservation of genes governing inflorescence architecture across grass species, we hypothesized that conserved regulatory sequences would be located near the <i>taw1-D1</i>/<i>-D2</i> insertion sites in these species. We first identified <i>TAW1</i> homologues in monocot species (Table S1; Figure S1), and then compared their genomic sequences (Figure 1a). We identified a CNS (hereafter, <i>TAW1</i>-CNS) in grass species, including the BEP clade, within 50 bp downstream of the transposon insertion sites in <i>taw1-D1</i>/-<i","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"27 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-04-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143827209","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}
SummaryNatural genetic variation can be used to improve important crop agronomic traits, and understanding the genetic basis of natural variation in fruit shape can help breeders develop pepper varieties that meet market demand. In this study, we identified a QTL controlling fruit length–width ratio by conventional genetic mapping, encoding a previously uncharacterized gene CaIQD1. Reduced CaIQD1 expression resulted in short and wide fruits in pepper, whereas heterologous overexpression of CaIQD1 resulted in narrower fruits in tomato. Further experiments suggested that CaIQD1 regulates fruit shape in pepper by affecting cell proliferation, expansion and morphological changes. CaIQD1 also has a direct protein interaction with CaOFP20 in CaTRM‐like‐CaOFP20. Reduced CaOFP20 expression caused pepper fruits to become elongated and curved, whereas reduced CaTRM‐like expression led to the formation of rounder fruits. These gene expression changes had a significant effect on the expression of genes related to the cell cycle and cell expansion. The CaTRM‐like‐CaOFP20‐CaIQD1 module may thus represent a conserved regulatory pathway for controlling pepper fruit shape. CaIQD1 also showed direct interactions with the pepper calmodulin CaCaM7, the tubulin CaMAP70‐2 and the microtubule motor protein CaKLCR1, suggesting that the regulation of fruit shape by CaIQD1 is related to changes in microtubule dynamics mediated by Ca2+‐CaM. We also found that CaIQD1 interacts with several homologues of genes that typically regulate fruit shape in other plant species. In summary, our results show that CaIQD1 acts as a core hub in regulating pepper fruit shape through interactions with multiple proteins.
{"title":"The IQ67‐domain protein IQD1 regulates fruit shape through complex multiprotein interactions in pepper (Capsicum annuum L.)","authors":"Lianzhen Mao, Yiyu Shen, Qingzhi Cui, Yu Huang, Xiang Zhang, Junheng Lv, Wujun Xing, Dan Zhang, Naying Fang, Daqing Chen, Zhuoxuan Wu, Peiru Li, Minghua Deng, Lijun Ou, Xuexiao Zou, Zhoubin Liu","doi":"10.1111/pbi.70078","DOIUrl":"https://doi.org/10.1111/pbi.70078","url":null,"abstract":"SummaryNatural genetic variation can be used to improve important crop agronomic traits, and understanding the genetic basis of natural variation in fruit shape can help breeders develop pepper varieties that meet market demand. In this study, we identified a QTL controlling fruit length–width ratio by conventional genetic mapping, encoding a previously uncharacterized gene <jats:italic>CaIQD1</jats:italic>. Reduced <jats:italic>CaIQD1 expression</jats:italic> resulted in short and wide fruits in pepper, whereas heterologous overexpression of <jats:italic>CaIQD1</jats:italic> resulted in narrower fruits in tomato. Further experiments suggested that <jats:italic>CaIQD1</jats:italic> regulates fruit shape in pepper by affecting cell proliferation, expansion and morphological changes. CaIQD1 also has a direct protein interaction with CaOFP20 in CaTRM‐like‐CaOFP20. Reduced <jats:italic>CaOFP20 expression</jats:italic> caused pepper fruits to become elongated and curved, whereas reduced <jats:italic>CaTRM‐like</jats:italic> expression led to the formation of rounder fruits. These gene expression changes had a significant effect on the expression of genes related to the cell cycle and cell expansion. The CaTRM‐like‐CaOFP20‐CaIQD1 module may thus represent a conserved regulatory pathway for controlling pepper fruit shape. CaIQD1 also showed direct interactions with the pepper calmodulin CaCaM7, the tubulin CaMAP70‐2 and the microtubule motor protein CaKLCR1, suggesting that the regulation of fruit shape by CaIQD1 is related to changes in microtubule dynamics mediated by Ca<jats:sup>2+</jats:sup>‐CaM. We also found that CaIQD1 interacts with several homologues of genes that typically regulate fruit shape in other plant species. In summary, our results show that CaIQD1 acts as a core hub in regulating pepper fruit shape through interactions with multiple proteins.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"42 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143822719","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}
<p>Dinoflagellates are a taxonomically diverse and ecologically significant group of phytoplankton. They are also infamous for their involvement in harmful algal blooms, which have significant ecological and economic impacts. In recent years, substantial advances have been made in the analysis of dinoflagellate genomes, including sequencing, assembly and gene annotation, alongside the accumulation of extensive multi-omics data (González-Pech <i>et al</i>., <span>2021</span>). Despite these developments, the large size and complexity of dinoflagellate genomes present ongoing challenges. Current resources, such as SAGER, primarily focus on genomic and transcriptomic data sets for <i>Symbiodiniaceae</i> (Yu <i>et al</i>., <span>2020</span>).</p><p>In this study, we have developed the first high-precision and comprehensive genome resource database for dinoflagellates, DinoSource (http://glab.hzau.edu.cn/dinosource), which provides 21 genome assemblies for all 20 currently sequenced dinoflagellate species (including two strains of <i>Polarella glacialis</i>) (Table S1). Our database integrates 703 omics samples, which have been generated from our experiments as well as collected from public repositories such as GEO (Gene Expression Omnibus) and SRA (Sequence Read Archive) up to the present date (Figure 1a). The sources and species distribution of the data sets are detailed in the ‘Data’ page of DinoSource (Figures 1b and S1a).</p><figure><picture><source media="(min-width: 1650px)" srcset="/cms/asset/eaf169d6-1eef-4e11-8aa4-58413c9db10c/pbi70054-fig-0001-m.jpg"/><img alt="Details are in the caption following the image" data-lg-src="/cms/asset/eaf169d6-1eef-4e11-8aa4-58413c9db10c/pbi70054-fig-0001-m.jpg" loading="lazy" src="/cms/asset/abbf798d-6830-47f6-b49c-ebeb17ddbea9/pbi70054-fig-0001-m.png" title="Details are in the caption following the image"/></picture><figcaption><div><strong>Figure 1<span style="font-weight:normal"></span></strong><div>Open in figure viewer<i aria-hidden="true"></i><span>PowerPoint</span></div></div><div>Architecture and screenshots of the DinoSource database. (a) Data collection and sources. (b) Species distribution of omics data across different species. (c) DinoSource's web implementation includes three core modules: The boxplot displays expression profiles of a subset of genes associated with ko: K02634 across different treatments in <i>Breviolum minutum</i>. (e) Gene differential expression analysis and functional enrichment analysis tools. (f) The stacked bar plot illustrates the proportion of three 5mC contexts at varying methylation levels across <i>B. minutum</i>. (g) HiGlass visualizes the Hi-C interaction matrices for <i>Symbiodinium microadriaticum</i> (GSM5023543) in the region chr19:800 K–10 MB. The blue triangular box highlights the identified TAD. (h) An example of using comparative genomics tools in DinoSource. The left panel shows a syntenic block located between <i>Fugacium kawagutii</i> and <i>S. mic
{"title":"DinoSource: A comprehensive database of dinoflagellate genomic resources","authors":"Fuming Lai, Chongping Li, Yidong Zhang, Ying Li, Yuci Wang, Qiangwei Zhou, Yaping Fang, Hao Chen, Guoliang Li","doi":"10.1111/pbi.70054","DOIUrl":"https://doi.org/10.1111/pbi.70054","url":null,"abstract":"<p>Dinoflagellates are a taxonomically diverse and ecologically significant group of phytoplankton. They are also infamous for their involvement in harmful algal blooms, which have significant ecological and economic impacts. In recent years, substantial advances have been made in the analysis of dinoflagellate genomes, including sequencing, assembly and gene annotation, alongside the accumulation of extensive multi-omics data (González-Pech <i>et al</i>., <span>2021</span>). Despite these developments, the large size and complexity of dinoflagellate genomes present ongoing challenges. Current resources, such as SAGER, primarily focus on genomic and transcriptomic data sets for <i>Symbiodiniaceae</i> (Yu <i>et al</i>., <span>2020</span>).</p>\u0000<p>In this study, we have developed the first high-precision and comprehensive genome resource database for dinoflagellates, DinoSource (http://glab.hzau.edu.cn/dinosource), which provides 21 genome assemblies for all 20 currently sequenced dinoflagellate species (including two strains of <i>Polarella glacialis</i>) (Table S1). Our database integrates 703 omics samples, which have been generated from our experiments as well as collected from public repositories such as GEO (Gene Expression Omnibus) and SRA (Sequence Read Archive) up to the present date (Figure 1a). The sources and species distribution of the data sets are detailed in the ‘Data’ page of DinoSource (Figures 1b and S1a).</p>\u0000<figure><picture>\u0000<source media=\"(min-width: 1650px)\" srcset=\"/cms/asset/eaf169d6-1eef-4e11-8aa4-58413c9db10c/pbi70054-fig-0001-m.jpg\"/><img alt=\"Details are in the caption following the image\" data-lg-src=\"/cms/asset/eaf169d6-1eef-4e11-8aa4-58413c9db10c/pbi70054-fig-0001-m.jpg\" loading=\"lazy\" src=\"/cms/asset/abbf798d-6830-47f6-b49c-ebeb17ddbea9/pbi70054-fig-0001-m.png\" title=\"Details are in the caption following the image\"/></picture><figcaption>\u0000<div><strong>Figure 1<span style=\"font-weight:normal\"></span></strong><div>Open in figure viewer<i aria-hidden=\"true\"></i><span>PowerPoint</span></div>\u0000</div>\u0000<div>Architecture and screenshots of the DinoSource database. (a) Data collection and sources. (b) Species distribution of omics data across different species. (c) DinoSource's web implementation includes three core modules: The boxplot displays expression profiles of a subset of genes associated with ko: K02634 across different treatments in <i>Breviolum minutum</i>. (e) Gene differential expression analysis and functional enrichment analysis tools. (f) The stacked bar plot illustrates the proportion of three 5mC contexts at varying methylation levels across <i>B. minutum</i>. (g) HiGlass visualizes the Hi-C interaction matrices for <i>Symbiodinium microadriaticum</i> (GSM5023543) in the region chr19:800 K–10 MB. The blue triangular box highlights the identified TAD. (h) An example of using comparative genomics tools in DinoSource. The left panel shows a syntenic block located between <i>Fugacium kawagutii</i> and <i>S. mic","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"39 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143819968","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}
Zihao Wei, Meiqi Shang, Zhicheng Jiang, Hong Zhai, Shihan Xing, Zhen Wang, Shaozhen He, Shaopei Gao, Ning Zhao, Huan Zhang, Qingchang Liu
Carotenoid-rich orange-fleshed sweet potato (OFSP) is an important staple diet and source of nutrition in developing countries, including Africa and Asia. However, the regulation of carotenoid biosynthesis remains to be better understood. A natural allelic variation closely linked to carotenoid biosynthesis was identified in the promoter region of the IbbHLH25 gene that encodes a basic helix–loop–helix (bHLH) transcription factor, by transcriptome and haplotype analyses of different flesh colour sweet potato accessions. An 86-bp deletion reduced the transcription of the IbbHLH25 promoter in white- and yellow-fleshed sweet potatoes; however, the deletion was absent in OFSP. IbbHLH25 was highly expressed in the storage roots of carotenoid-rich sweet potato. The overexpression of IbbHLH25 significantly increased the carotenoid contents (by 2.5-fold–6.0-fold) and proportions, especially β-carotene and β-cryptoxanthin; their contents increased by 21.2-fold–55.7-fold and 4.6-fold–9.5-fold, respectively, and their proportions increased by 48.5% and 13.0%, respectively, and the silencing of IbbHLH25 had opposite effects. IbbHLH25 formed heterodimers with IbbHLH66 to directly and synergistically activate the transcription of carotenoid biosynthesis key genes IbGGPPS, IbLCYB and IbBCH. The overexpression of IbbHLH66 significantly increased the carotenoid contents (by 2.3-fold–3.8-fold) and proportions, especially β-carotene and β-cryptoxanthin; their contents increased by 15.2-fold–25.6-fold and 3.1-fold–5.1-fold, respectively, and their proportions increased by 31.1% and 9.6%, respectively. These findings expand our understanding of bHLHs in regulating carotenoid biosynthesis and suggest additional roles in affecting carotenoid component proportions, providing candidate genes for nutritional biofortification of agricultural products.
{"title":"Natural allelic variation of basic helix–loop–helix transcription factor 25 regulates carotenoid biosynthesis in sweet potato","authors":"Zihao Wei, Meiqi Shang, Zhicheng Jiang, Hong Zhai, Shihan Xing, Zhen Wang, Shaozhen He, Shaopei Gao, Ning Zhao, Huan Zhang, Qingchang Liu","doi":"10.1111/pbi.70086","DOIUrl":"https://doi.org/10.1111/pbi.70086","url":null,"abstract":"Carotenoid-rich orange-fleshed sweet potato (OFSP) is an important staple diet and source of nutrition in developing countries, including Africa and Asia. However, the regulation of carotenoid biosynthesis remains to be better understood. A natural allelic variation closely linked to carotenoid biosynthesis was identified in the promoter region of the <i>IbbHLH25</i> gene that encodes a basic helix–loop–helix (bHLH) transcription factor, by transcriptome and haplotype analyses of different flesh colour sweet potato accessions. An 86-bp deletion reduced the transcription of the <i>IbbHLH25</i> promoter in white- and yellow-fleshed sweet potatoes; however, the deletion was absent in OFSP. <i>IbbHLH25</i> was highly expressed in the storage roots of carotenoid-rich sweet potato. The overexpression of <i>IbbHLH25</i> significantly increased the carotenoid contents (by 2.5-fold–6.0-fold) and proportions, especially β-carotene and β-cryptoxanthin; their contents increased by 21.2-fold–55.7-fold and 4.6-fold–9.5-fold, respectively, and their proportions increased by 48.5% and 13.0%, respectively, and the silencing of <i>IbbHLH25</i> had opposite effects. IbbHLH25 formed heterodimers with IbbHLH66 to directly and synergistically activate the transcription of carotenoid biosynthesis key genes <i>IbGGPPS</i>, <i>IbLCYB</i> and <i>IbBCH</i>. The overexpression of <i>IbbHLH66</i> significantly increased the carotenoid contents (by 2.3-fold–3.8-fold) and proportions, especially β-carotene and β-cryptoxanthin; their contents increased by 15.2-fold–25.6-fold and 3.1-fold–5.1-fold, respectively, and their proportions increased by 31.1% and 9.6%, respectively. These findings expand our understanding of bHLHs in regulating carotenoid biosynthesis and suggest additional roles in affecting carotenoid component proportions, providing candidate genes for nutritional biofortification of agricultural products.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"14 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143814132","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}
<p>Targeted insertion of large DNA fragments holds great promise in crop breeding but is extremely challenging in plants. Prime editing (PE) can efficiently install small genomic insertion through replacement but faces challenges in mediating insertion of >100 bp. To enable larger insertion, a paired PE strategy termed GRAND editing was developed in human cells. In plants, genomic insertions of up to 135 bp were achieved through GRAND editing (Xu <i>et al</i>., <span>2024</span>), but no plants with larger targeted insertion were generated through PE-mediated replacement.</p><p>Here, we attempted to insert three truncated promoters, including 207-bp <i>pGluB4</i>, 204-bp <i>p10kDa</i>, and 206-bp <i>p16kDa</i>, to the 5′ UTRs of <i>OsC1</i>, <i>OsB2</i>, and <i>OsB1</i>, respectively, through PE. An improved prime editor termed ePPEplus (Ni <i>et al</i>., <span>2023</span>) and epegRNA (pegRNA with a tevepreQ1 motif at the 3′ terminus) were used in the PE assays of this study. Expressions of the ePPEplus and epegRNAs were driven by <i>ZmUbi</i> and eCmYLCV promoters (eCmYLCV, 35S enhancer-CmYLCV promoter), respectively. All PE assays in this study were conducted in a <i>japonica</i> cultivar termed Heixiangnuo (HXN). Firstly, we conducted the insertion editing using GRAND editing but detected no targeted insertions in the GRAND editing transgenic plants (Table S1; see Supplemental Methods and Figures S1 and S2 for the design).</p><p>Recently, a PE technology termed template-jumping PE (TJ-PE) was developed for large insertion in human cells (see Figure S3 for the mechanism) (Zheng <i>et al</i>., <span>2023</span>). TJ-PE pegRNA (TJ-pegRNA) contains one reverse transcriptase template (RTT) and two primer binding sites (PBSs), with one PBS matching the pegRNA target and another matching the nicking gRNA target (Figure S3). TJ-PE could mediate 200–800-bp insertion in replacement of the fragment between the two TJ-PE nicks in human cells. Thus, we also conducted the insertion editing using TJ-PE with the same target sites as the above GRAND editing (Figure 1a and Figure S4). In the TJ-PE assays, the TJ-epegRNAs were expressed with pre-tRNA and hepatitis delta virus ribozyme (HDV) processing systems to generate mature epegRNAs (Figure S5). Each TJ-epegRNA expression cassette and the corresponding nicking gRNA cassette were constructed in a binary vector with an ePPEplus cassette to generate one vector for each insertion editing (Figure S5). With each of the TJ-PE vectors, 97–137 transgenic plants were generated through <i>Agrobacterium</i>-mediated transformation. Among these TJ-PE transgenic plants, only two edited plants with truncated <i>pGluB4</i> insertions at <i>OsC1</i> (one with 149-bp <i>pGluB4</i> insertion and another with 139-bp <i>pGluB4</i> insertion) were identified, and no edits were detected in other transgenic plants (Table S2; Figure S6a–c). The 149- and 139-bp insertions occurred at <i>OsC1</i> TJ-epegRNA nicking site with a p
{"title":"Targeted insertion of large DNA fragments through template-jumping prime editing in rice","authors":"Fei Li, Haonan Hou, Minglei Song, Zhen Chen, Ting Peng, Yanxiu Du, Yafan Zhao, Junzhou Li, Chunbo Miao","doi":"10.1111/pbi.70087","DOIUrl":"https://doi.org/10.1111/pbi.70087","url":null,"abstract":"<p>Targeted insertion of large DNA fragments holds great promise in crop breeding but is extremely challenging in plants. Prime editing (PE) can efficiently install small genomic insertion through replacement but faces challenges in mediating insertion of >100 bp. To enable larger insertion, a paired PE strategy termed GRAND editing was developed in human cells. In plants, genomic insertions of up to 135 bp were achieved through GRAND editing (Xu <i>et al</i>., <span>2024</span>), but no plants with larger targeted insertion were generated through PE-mediated replacement.</p>\u0000<p>Here, we attempted to insert three truncated promoters, including 207-bp <i>pGluB4</i>, 204-bp <i>p10kDa</i>, and 206-bp <i>p16kDa</i>, to the 5′ UTRs of <i>OsC1</i>, <i>OsB2</i>, and <i>OsB1</i>, respectively, through PE. An improved prime editor termed ePPEplus (Ni <i>et al</i>., <span>2023</span>) and epegRNA (pegRNA with a tevepreQ1 motif at the 3′ terminus) were used in the PE assays of this study. Expressions of the ePPEplus and epegRNAs were driven by <i>ZmUbi</i> and eCmYLCV promoters (eCmYLCV, 35S enhancer-CmYLCV promoter), respectively. All PE assays in this study were conducted in a <i>japonica</i> cultivar termed Heixiangnuo (HXN). Firstly, we conducted the insertion editing using GRAND editing but detected no targeted insertions in the GRAND editing transgenic plants (Table S1; see Supplemental Methods and Figures S1 and S2 for the design).</p>\u0000<p>Recently, a PE technology termed template-jumping PE (TJ-PE) was developed for large insertion in human cells (see Figure S3 for the mechanism) (Zheng <i>et al</i>., <span>2023</span>). TJ-PE pegRNA (TJ-pegRNA) contains one reverse transcriptase template (RTT) and two primer binding sites (PBSs), with one PBS matching the pegRNA target and another matching the nicking gRNA target (Figure S3). TJ-PE could mediate 200–800-bp insertion in replacement of the fragment between the two TJ-PE nicks in human cells. Thus, we also conducted the insertion editing using TJ-PE with the same target sites as the above GRAND editing (Figure 1a and Figure S4). In the TJ-PE assays, the TJ-epegRNAs were expressed with pre-tRNA and hepatitis delta virus ribozyme (HDV) processing systems to generate mature epegRNAs (Figure S5). Each TJ-epegRNA expression cassette and the corresponding nicking gRNA cassette were constructed in a binary vector with an ePPEplus cassette to generate one vector for each insertion editing (Figure S5). With each of the TJ-PE vectors, 97–137 transgenic plants were generated through <i>Agrobacterium</i>-mediated transformation. Among these TJ-PE transgenic plants, only two edited plants with truncated <i>pGluB4</i> insertions at <i>OsC1</i> (one with 149-bp <i>pGluB4</i> insertion and another with 139-bp <i>pGluB4</i> insertion) were identified, and no edits were detected in other transgenic plants (Table S2; Figure S6a–c). The 149- and 139-bp insertions occurred at <i>OsC1</i> TJ-epegRNA nicking site with a p","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"1 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143814272","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}
Saeideh Dianatkhah, Benjamin Kogelmann, Stanislav Melnik, Florian Eminger, Somanath Kallolimath, Lin Sun, Delia Sumesgutner, Michael W. Traxlmayr, Markus Sack, Eva Stoger, Herta Steinkellner
<p>Sialylated N-glycans are widely distributed in vertebrates and represent the dominant glycoform of many human plasma proteins (Miura <i>et al</i>., <span>2015</span>). Although knowledge of the diverse effects of this glycan formation is rapidly increasing, full understanding of its biological significance remains elusive (Lewis <i>et al</i>., <span>2022</span>). A major reason for this is the difficulty in controlling sialylation in production processes.</p><p>Plants are considered as an effective platform for the production of recombinant proteins used in basic research or for various applications (Eidenberger <i>et al</i>., <span>2023</span>). The platform has recently been extended by so-called plant cell packs (PCPs), three-dimensional, porous plant cell aggregates derived from plant suspension cells. The approach enables high-throughput transient expression of foreign genes and upscaling for subsequent protein purification and characterization (Rademacher <i>et al</i>., <span>2019</span>) and (WO2013113504).</p><p>An important advantage of plant-based expression is the synthesis of N-glycans similar to mammalian cells. Usually, secreted plant glycoproteins are decorated with GlcNAc-terminating complex N-glycans carrying a plant-specific core xylose and α1,3-fucose, so-called GnGnXF structures. Extensive engineering in <i>N. benthamiana</i>, that is, the inactivation of genes responsible for the addition of plant-specific core xylose and fucose, in combination with the overexpression of six foreign genes involved in the human sialylation pathway, resulted in the generation of a plant line (ΔXTFT<sup>Sia</sup>) that synthesizes sialylated N-glycans (Eidenberger <i>et al</i>., <span>2022</span>; Kallolimath <i>et al</i>., <span>2016</span>). One shortcoming of the ΔXTFT<sup>Sia</sup> line is the lower seed production, which makes maintenance and widespread use difficult.</p><p>Here we used hypocotyl of ΔXTFT<sup>Sia</sup> plants as starting material for callus induction, applying a similar method as described recently (Sukenik <i>et al</i>., <span>2018</span>) (Figure S1a, ‘Materials and methods’ section). After tissue dedifferentiation, calli were maintained on semi-solid media by monthly subculturing. Portions of independent calli, PCR-screened for the presence of one of the six foreign glycosylation genes for sialylation (Figures S1a and S1b), were used to initiate suspension cultures, which were maintained for several passages to select for rapid growth (Figure S1a). For the generation of PCPs (Rademacher <i>et al</i>., <span>2019</span>), cells were separated from excess cultivation medium by slow centrifugation in ultrafiltration spin-columns The resulting semi-dry porous cell aggregates (called plant cell packs, PCP<sup>Sia</sup>; Figure S1a) were monitored for expression of recombinant fluorescent protein. PCP<sup>Sia</sup> were incubated with <i>Rhizobium radiobacter</i> (formerly <i>Agrobacterium tumefaciens</i>) suspension cul
{"title":"A plant cell-based platform for the expression of complex proteins with fucose-reduced sialylated N-glycans","authors":"Saeideh Dianatkhah, Benjamin Kogelmann, Stanislav Melnik, Florian Eminger, Somanath Kallolimath, Lin Sun, Delia Sumesgutner, Michael W. Traxlmayr, Markus Sack, Eva Stoger, Herta Steinkellner","doi":"10.1111/pbi.70044","DOIUrl":"https://doi.org/10.1111/pbi.70044","url":null,"abstract":"<p>Sialylated N-glycans are widely distributed in vertebrates and represent the dominant glycoform of many human plasma proteins (Miura <i>et al</i>., <span>2015</span>). Although knowledge of the diverse effects of this glycan formation is rapidly increasing, full understanding of its biological significance remains elusive (Lewis <i>et al</i>., <span>2022</span>). A major reason for this is the difficulty in controlling sialylation in production processes.</p>\u0000<p>Plants are considered as an effective platform for the production of recombinant proteins used in basic research or for various applications (Eidenberger <i>et al</i>., <span>2023</span>). The platform has recently been extended by so-called plant cell packs (PCPs), three-dimensional, porous plant cell aggregates derived from plant suspension cells. The approach enables high-throughput transient expression of foreign genes and upscaling for subsequent protein purification and characterization (Rademacher <i>et al</i>., <span>2019</span>) and (WO2013113504).</p>\u0000<p>An important advantage of plant-based expression is the synthesis of N-glycans similar to mammalian cells. Usually, secreted plant glycoproteins are decorated with GlcNAc-terminating complex N-glycans carrying a plant-specific core xylose and α1,3-fucose, so-called GnGnXF structures. Extensive engineering in <i>N. benthamiana</i>, that is, the inactivation of genes responsible for the addition of plant-specific core xylose and fucose, in combination with the overexpression of six foreign genes involved in the human sialylation pathway, resulted in the generation of a plant line (ΔXTFT<sup>Sia</sup>) that synthesizes sialylated N-glycans (Eidenberger <i>et al</i>., <span>2022</span>; Kallolimath <i>et al</i>., <span>2016</span>). One shortcoming of the ΔXTFT<sup>Sia</sup> line is the lower seed production, which makes maintenance and widespread use difficult.</p>\u0000<p>Here we used hypocotyl of ΔXTFT<sup>Sia</sup> plants as starting material for callus induction, applying a similar method as described recently (Sukenik <i>et al</i>., <span>2018</span>) (Figure S1a, ‘Materials and methods’ section). After tissue dedifferentiation, calli were maintained on semi-solid media by monthly subculturing. Portions of independent calli, PCR-screened for the presence of one of the six foreign glycosylation genes for sialylation (Figures S1a and S1b), were used to initiate suspension cultures, which were maintained for several passages to select for rapid growth (Figure S1a). For the generation of PCPs (Rademacher <i>et al</i>., <span>2019</span>), cells were separated from excess cultivation medium by slow centrifugation in ultrafiltration spin-columns The resulting semi-dry porous cell aggregates (called plant cell packs, PCP<sup>Sia</sup>; Figure S1a) were monitored for expression of recombinant fluorescent protein. PCP<sup>Sia</sup> were incubated with <i>Rhizobium radiobacter</i> (formerly <i>Agrobacterium tumefaciens</i>) suspension cul","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"99 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143814271","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}
Zhiyi Fan, Yuan Wang, Yanlei Zhai, Xiaojiao Gu, Kairong Sun, Dan Zhao, Jinying Wang, Pinqi Sun, Hantang Huang, Jiajun He, Yining Wang, Moshe A. Flaishman, Huiqin Ma
The mechanism regulating fruit textural changes has not been fully elucidated. Transcription factor FcERF100 showed rapid transcription repression during drastic texture loss in fig (Ficus carica L.) fruit ripening. Transient overexpression of FcERF100 delayed fig fruit softening and significantly decreased the transcript abundance of a key cell wall-modifying pectate lyase gene, FcPL7. Yeast one-hybrid (Y1H) assay, chromatin immunoprecipitation-qPCR, electrophoretic mobility shift assay (EMSA), and dual-luciferase reporter assay revealed that FcERF100 represses FcPL7 transcription by direct promoter binding via GCC-box and DRE/CRT elements. Stable transgenic fig lines further verified FcERF100's inhibitory effect on FcPL7 expression. We detected FcERF28 as an upstream element of FcERF100 by Y1H and EMSA, revealing its binding to, and activation of FcERF100 by dual-luciferase assay. Taken together, the FcERF28–FcERF100 transcriptional cascade serves as a synergistic flow-limiting valve for FcPL7 abundance. We then identified a NAC transcription factor, FcNOR, using FcERF100 as the bait by yeast two-hybrid screening. FcNOR silencing retarded fig fruit softening, with decreased FcPL7 transcript and pectate lyase activity. FcNOR interacted with FcERF100 to form a protein complex, attenuating FcERF100's transcriptional repression of FcPL7. Moreover, FcNOR bound directly to the promoter of FcERF100 and inhibited its transcription. In addition, ethylene treatment upregulated FcNOR and FcPL7 expression and downregulated FcERF28 and FcERF100 expression. Our findings reveal a novel FcERF100-centered regulatory complex and resolve how the complex achieves the necessary cell wall modification during an early stage of fruit growth and implements drastic softening at fruit ripening by modulating component proportions.
{"title":"ERF100 regulated by ERF28 and NOR controls pectate lyase 7, modulating fig (Ficus carica L.) fruit softening","authors":"Zhiyi Fan, Yuan Wang, Yanlei Zhai, Xiaojiao Gu, Kairong Sun, Dan Zhao, Jinying Wang, Pinqi Sun, Hantang Huang, Jiajun He, Yining Wang, Moshe A. Flaishman, Huiqin Ma","doi":"10.1111/pbi.70085","DOIUrl":"https://doi.org/10.1111/pbi.70085","url":null,"abstract":"The mechanism regulating fruit textural changes has not been fully elucidated. Transcription factor FcERF100 showed rapid transcription repression during drastic texture loss in fig (<i>Ficus carica</i> L.) fruit ripening. Transient overexpression of <i>FcERF100</i> delayed fig fruit softening and significantly decreased the transcript abundance of a key cell wall-modifying pectate lyase gene, <i>FcPL7</i>. Yeast one-hybrid (Y1H) assay, chromatin immunoprecipitation-qPCR, electrophoretic mobility shift assay (EMSA), and dual-luciferase reporter assay revealed that FcERF100 represses <i>FcPL7</i> transcription by direct promoter binding via GCC-box and DRE/CRT elements. Stable transgenic fig lines further verified FcERF100's inhibitory effect on <i>FcPL7</i> expression. We detected FcERF28 as an upstream element of <i>FcERF100</i> by Y1H and EMSA, revealing its binding to, and activation of <i>FcERF100</i> by dual-luciferase assay. Taken together, the FcERF28–FcERF100 transcriptional cascade serves as a synergistic flow-limiting valve for FcPL7 abundance. We then identified a NAC transcription factor, FcNOR, using FcERF100 as the bait by yeast two-hybrid screening. <i>FcNOR</i> silencing retarded fig fruit softening, with decreased <i>FcPL7</i> transcript and pectate lyase activity. FcNOR interacted with FcERF100 to form a protein complex, attenuating FcERF100's transcriptional repression of <i>FcPL7</i>. Moreover, FcNOR bound directly to the promoter of <i>FcERF100</i> and inhibited its transcription. In addition, ethylene treatment upregulated <i>FcNOR</i> and <i>FcPL7</i> expression and downregulated <i>FcERF28</i> and <i>FcERF100</i> expression. Our findings reveal a novel FcERF100-centered regulatory complex and resolve how the complex achieves the necessary cell wall modification during an early stage of fruit growth and implements drastic softening at fruit ripening by modulating component proportions.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"23 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143814270","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}
Anya L. Lindström Battle, Angus W. Barrett, Mark D. Fricker, Lee J. Sweetlove
SummaryThe establishment of Nicotiana benthamiana as a robust biofactory is complicated by issues such as product toxicity and proteolytic degradation of target proteins/introduced enzymes. Here we investigate whether biomolecular condensates can be used to address these problems. We engineered biomolecular condensates in N. benthamiana leaves using transient expression of synthetic modular scaffolds. The in vivo properties of the condensates that resulted were consistent with them being liquid‐like bodies with thermodynamic features typical of multicomponent phase‐separating systems. We show that recruitment of enzymes to condensates in vivo led to several‐fold yield increases in one‐ and three‐step metabolic pathways (citramalate biosynthesis and poly‐3‐hydroxybutyrate (PHB) biosynthesis, respectively). This enhanced yield could be for several reasons including improved enzyme kinetics, metabolite channelling or avoidance of cytotoxicity by retention of the pathway product within the condensate, which was demonstrated for PHB. However, we also observed a several‐fold increase in the amount of the enzymes that accumulated when they were targeted to the condensates. This suggests that the enzymes were more stable when localised to the condensate than when freely diffusing in the cytosol. We hypothesise that this stability is likely the main driver for increased pathway product production. Our findings provide a foundation for leveraging biomolecular condensates in plant metabolic engineering and advance N. benthamiana as a versatile biofactory for industrial applications.
{"title":"Localising enzymes to biomolecular condensates increase their accumulation and benefits engineered metabolic pathway performance in Nicotiana benthamiana","authors":"Anya L. Lindström Battle, Angus W. Barrett, Mark D. Fricker, Lee J. Sweetlove","doi":"10.1111/pbi.70082","DOIUrl":"https://doi.org/10.1111/pbi.70082","url":null,"abstract":"SummaryThe establishment of <jats:italic>Nicotiana benthamiana</jats:italic> as a robust biofactory is complicated by issues such as product toxicity and proteolytic degradation of target proteins/introduced enzymes. Here we investigate whether biomolecular condensates can be used to address these problems. We engineered biomolecular condensates in <jats:italic>N. benthamiana</jats:italic> leaves using transient expression of synthetic modular scaffolds. The <jats:italic>in vivo</jats:italic> properties of the condensates that resulted were consistent with them being liquid‐like bodies with thermodynamic features typical of multicomponent phase‐separating systems. We show that recruitment of enzymes to condensates <jats:italic>in vivo</jats:italic> led to several‐fold yield increases in one‐ and three‐step metabolic pathways (citramalate biosynthesis and poly‐3‐hydroxybutyrate (PHB) biosynthesis, respectively). This enhanced yield could be for several reasons including improved enzyme kinetics, metabolite channelling or avoidance of cytotoxicity by retention of the pathway product within the condensate, which was demonstrated for PHB. However, we also observed a several‐fold increase in the amount of the enzymes that accumulated when they were targeted to the condensates. This suggests that the enzymes were more stable when localised to the condensate than when freely diffusing in the cytosol. We hypothesise that this stability is likely the main driver for increased pathway product production. Our findings provide a foundation for leveraging biomolecular condensates in plant metabolic engineering and advance <jats:italic>N. benthamiana</jats:italic> as a versatile biofactory for industrial applications.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"66 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143813758","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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