Xiaoqing Sun, Matthew LaVoie, Paul A Lefebvre, Sean D Gallaher, Anne G Glaesener, Daniela Strenkert, Radhika Mehta, Sabeeha S Merchant, Carolyn D Silflow
Oxygen prevents hydrogen production in Chlamydomonas (Chlamydomonas reinhardtii), in part by inhibiting the transcription of hydrogenase genes. We developed a screen for mutants showing constitutive accumulation of iron hydrogenase 1 (HYDA1) transcripts in normoxia. A reporter gene required for ciliary motility placed under the control of the HYDA1 promoter conferred motility only in hypoxia. By selecting for mutants able to swim even in normoxia, we obtained strains that constitutively express the reporter gene. One identified mutant was affected in a gene encoding an F-box protein 3 (FBXO3) that participates in ubiquitylation and proteasomal degradation pathways in other eukaryotes. Transcriptome profiles revealed that the mutation, termed cehc1-1 (constitutive expression of hydrogenases and copper-responsive genes), triggers the upregulation of genes known to be targets of copper response regulator 1 (CRR1), a transcription factor involved in the nutritional copper signaling pathway and in the hypoxia response pathway. CRR1 was required for upregulating the HYDA1 reporter gene expression in response to hypoxia and for the constitutive expression of the reporter gene in cehc1-1 mutant cells. The CRR1 protein, normally degraded in Cu-supplemented cells, was stabilized in cehc1-1 cells, supporting the conclusion that CEHC1 facilitates CRR1 degradation. Our results describe a previously unknown pathway for CRR1 inhibition and possibly other pathways leading to complex metabolic changes.
{"title":"Identification of a gene controlling levels of the copper response regulator 1 transcription factor in Chlamydomonas reinhardtii","authors":"Xiaoqing Sun, Matthew LaVoie, Paul A Lefebvre, Sean D Gallaher, Anne G Glaesener, Daniela Strenkert, Radhika Mehta, Sabeeha S Merchant, Carolyn D Silflow","doi":"10.1093/plcell/koae300","DOIUrl":"https://doi.org/10.1093/plcell/koae300","url":null,"abstract":"Oxygen prevents hydrogen production in Chlamydomonas (Chlamydomonas reinhardtii), in part by inhibiting the transcription of hydrogenase genes. We developed a screen for mutants showing constitutive accumulation of iron hydrogenase 1 (HYDA1) transcripts in normoxia. A reporter gene required for ciliary motility placed under the control of the HYDA1 promoter conferred motility only in hypoxia. By selecting for mutants able to swim even in normoxia, we obtained strains that constitutively express the reporter gene. One identified mutant was affected in a gene encoding an F-box protein 3 (FBXO3) that participates in ubiquitylation and proteasomal degradation pathways in other eukaryotes. Transcriptome profiles revealed that the mutation, termed cehc1-1 (constitutive expression of hydrogenases and copper-responsive genes), triggers the upregulation of genes known to be targets of copper response regulator 1 (CRR1), a transcription factor involved in the nutritional copper signaling pathway and in the hypoxia response pathway. CRR1 was required for upregulating the HYDA1 reporter gene expression in response to hypoxia and for the constitutive expression of the reporter gene in cehc1-1 mutant cells. The CRR1 protein, normally degraded in Cu-supplemented cells, was stabilized in cehc1-1 cells, supporting the conclusion that CEHC1 facilitates CRR1 degradation. Our results describe a previously unknown pathway for CRR1 inhibition and possibly other pathways leading to complex metabolic changes.","PeriodicalId":501012,"journal":{"name":"The Plant Cell","volume":"3 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142937690","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Da-Gang Hu, Mengxia Zhang, Chunlong Li, Ting-Ting Zhao, Lian-Da Du, Quan Sun, Chu-Kun Wang, Dong Meng, Cui-Hui Sun, Zhangjun Fei, Abhaya M Dandekar, Lailiang Cheng
High carbohydrate availability promotes malic acid accumulation in fleshy fruits, but the underlying mechanism is not known. Here, we show that antisense repression of ALDOSE-6-PHOSPHATE REDUCTASE in apple (Malus domestica) decreases the concentrations of sorbitol and malate and the transcript levels of several genes involved in vacuolar malate transport, including the aluminum-activated malate transporter (ALMT) gene MdALMT9 (Ma1), the P-ATPase gene MdPH5, the MYB transcription factor gene MdMYB73, and the cold-induced basic helix-loop-helix transcription factor gene MdCIbHLH1, in fruit and leaves. We identified a linker histone H1 variant, MdH1.1, which complements the Arabidopsis (Arabidopsis thaliana) H1 deficient mutant and functions as a transcription factor. MdH1.1 activates MdMYB73, MdCIbHLH1, and MdPH5 expression by directly binding to their promoters. MdMYB73, in return, binds to the promoter of MdH1.1 to enhance its transcription. This MdH1.1-MdMYB73 feedback loop responds to sorbitol, regulating Ma1 expression. Antisense suppression of either MdH1.1 or MdMYB73 expression significantly decreases whereas overexpression increases Ma1 expression and malate accumulation. These findings demonstrate that MdH1.1, in addition to being an architectural protein for chromatin structure, operates as a transcription factor orchestrating malic acid accumulation in response to sorbitol, revealing how sugar signaling modulates vacuolar malate transport via a linker histone in plants.
{"title":"A Linker Histone Acts as a Transcription Factor to Orchestrate Malic Acid Accumulation in Apple in Response to Sorbitol","authors":"Da-Gang Hu, Mengxia Zhang, Chunlong Li, Ting-Ting Zhao, Lian-Da Du, Quan Sun, Chu-Kun Wang, Dong Meng, Cui-Hui Sun, Zhangjun Fei, Abhaya M Dandekar, Lailiang Cheng","doi":"10.1093/plcell/koae328","DOIUrl":"https://doi.org/10.1093/plcell/koae328","url":null,"abstract":"High carbohydrate availability promotes malic acid accumulation in fleshy fruits, but the underlying mechanism is not known. Here, we show that antisense repression of ALDOSE-6-PHOSPHATE REDUCTASE in apple (Malus domestica) decreases the concentrations of sorbitol and malate and the transcript levels of several genes involved in vacuolar malate transport, including the aluminum-activated malate transporter (ALMT) gene MdALMT9 (Ma1), the P-ATPase gene MdPH5, the MYB transcription factor gene MdMYB73, and the cold-induced basic helix-loop-helix transcription factor gene MdCIbHLH1, in fruit and leaves. We identified a linker histone H1 variant, MdH1.1, which complements the Arabidopsis (Arabidopsis thaliana) H1 deficient mutant and functions as a transcription factor. MdH1.1 activates MdMYB73, MdCIbHLH1, and MdPH5 expression by directly binding to their promoters. MdMYB73, in return, binds to the promoter of MdH1.1 to enhance its transcription. This MdH1.1-MdMYB73 feedback loop responds to sorbitol, regulating Ma1 expression. Antisense suppression of either MdH1.1 or MdMYB73 expression significantly decreases whereas overexpression increases Ma1 expression and malate accumulation. These findings demonstrate that MdH1.1, in addition to being an architectural protein for chromatin structure, operates as a transcription factor orchestrating malic acid accumulation in response to sorbitol, revealing how sugar signaling modulates vacuolar malate transport via a linker histone in plants.","PeriodicalId":501012,"journal":{"name":"The Plant Cell","volume":"14 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142867299","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Souvik Dhar, Soo Youn Kim, Heeji Shin, Jongsung Park, Ji-Young Lee
Elevated stress signaling compromises plant growth by suppressing proliferative and formative division in the meristem. Plant Elicitor Peptide (PEP), an endogenous danger signal triggered by biotic and abiotic stresses in Arabidopsis (Arabidopsis thaliana), suppresses proliferative division, alters xylem vessel organization, and disrupts cell-to-cell symplastic connections in roots. To gain insight into the dynamic molecular framework that modulates root development under elevated danger signals, we performed a time-course RNA-sequencing analysis of the root meristem after synthetic PEP1 treatment. Our analyses revealed that SALT TOLERANCE ZINC FINGER (STZ) and its homologs are a potential nexus between the stress response and proliferative cell cycle regulation. Through functional, phenotypic, and transcriptomic analyses, we observed that STZ differentially controls the cell cycle, cell differentiation, and stress response genes in various tissue layers of the root meristem. Moreover, we determined the STZ expression level critical for enabling the growth–defense tradeoff. These findings provide valuable information about the dynamic gene expression changes that occur upon perceiving danger signals in the root meristem and potential engineering strategies to generate stress-resilient plants.
{"title":"The molecular framework balancing growth and defense in response to PEP-induced signals in Arabidopsis","authors":"Souvik Dhar, Soo Youn Kim, Heeji Shin, Jongsung Park, Ji-Young Lee","doi":"10.1093/plcell/koae327","DOIUrl":"https://doi.org/10.1093/plcell/koae327","url":null,"abstract":"Elevated stress signaling compromises plant growth by suppressing proliferative and formative division in the meristem. Plant Elicitor Peptide (PEP), an endogenous danger signal triggered by biotic and abiotic stresses in Arabidopsis (Arabidopsis thaliana), suppresses proliferative division, alters xylem vessel organization, and disrupts cell-to-cell symplastic connections in roots. To gain insight into the dynamic molecular framework that modulates root development under elevated danger signals, we performed a time-course RNA-sequencing analysis of the root meristem after synthetic PEP1 treatment. Our analyses revealed that SALT TOLERANCE ZINC FINGER (STZ) and its homologs are a potential nexus between the stress response and proliferative cell cycle regulation. Through functional, phenotypic, and transcriptomic analyses, we observed that STZ differentially controls the cell cycle, cell differentiation, and stress response genes in various tissue layers of the root meristem. Moreover, we determined the STZ expression level critical for enabling the growth–defense tradeoff. These findings provide valuable information about the dynamic gene expression changes that occur upon perceiving danger signals in the root meristem and potential engineering strategies to generate stress-resilient plants.","PeriodicalId":501012,"journal":{"name":"The Plant Cell","volume":"40 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142858394","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Daniel B Sloan, Amanda K Broz, Shady A Kuster, Viraj Muthye, Alejandro Peñafiel-Ayala, Jennifer R Marron, Dennis V Lavrov, Luis G Brieba
The widely distributed MutS gene family functions in recombination, DNA repair, and protein translation. Multiple evolutionary processes have expanded this gene family in plants relative to other eukaryotes. Here, we investigate the origins and functions of these plant-specific genes. Cyanobacterial-like MutS1 and MutS2 genes were ancestrally gained via plastid endosymbiotic gene transfer. MutS1 was subsequently lost in seed plants, whereas MutS2 was duplicated in Viridiplantae (i.e., land plants and green algae). Viridiplantae also have two anciently duplicated copies of the eukaryotic MSH6 gene and acquired MSH1 via horizontal gene transfer - potentially from a nucleocytovirus. Despite sharing a name, "plant MSH1" is not directly related to the MSH1 gene in some fungi and animals, which may be an ancestral eukaryotic gene acquired via mitochondrial endosymbiosis and subsequently lost in most eukaryotes. There has been substantial progress in understanding the functions of plant MSH1 and MSH6 genes, but the cyanobacterial-like MutS1 and MutS2 genes remain uncharacterized. Known functions of bacterial homologs and predicted protein structures, including fusions to diverse nuclease domains, provide hypotheses about potential molecular mechanisms. Because most plant-specific MutS proteins are mitochondrial and/or plastid-targeted, the expansion of this family has played a large role in shaping plant organelle genetics.
{"title":"Expansion of the MutS gene family in plants","authors":"Daniel B Sloan, Amanda K Broz, Shady A Kuster, Viraj Muthye, Alejandro Peñafiel-Ayala, Jennifer R Marron, Dennis V Lavrov, Luis G Brieba","doi":"10.1093/plcell/koae277","DOIUrl":"https://doi.org/10.1093/plcell/koae277","url":null,"abstract":"The widely distributed MutS gene family functions in recombination, DNA repair, and protein translation. Multiple evolutionary processes have expanded this gene family in plants relative to other eukaryotes. Here, we investigate the origins and functions of these plant-specific genes. Cyanobacterial-like MutS1 and MutS2 genes were ancestrally gained via plastid endosymbiotic gene transfer. MutS1 was subsequently lost in seed plants, whereas MutS2 was duplicated in Viridiplantae (i.e., land plants and green algae). Viridiplantae also have two anciently duplicated copies of the eukaryotic MSH6 gene and acquired MSH1 via horizontal gene transfer - potentially from a nucleocytovirus. Despite sharing a name, \"plant MSH1\" is not directly related to the MSH1 gene in some fungi and animals, which may be an ancestral eukaryotic gene acquired via mitochondrial endosymbiosis and subsequently lost in most eukaryotes. There has been substantial progress in understanding the functions of plant MSH1 and MSH6 genes, but the cyanobacterial-like MutS1 and MutS2 genes remain uncharacterized. Known functions of bacterial homologs and predicted protein structures, including fusions to diverse nuclease domains, provide hypotheses about potential molecular mechanisms. Because most plant-specific MutS proteins are mitochondrial and/or plastid-targeted, the expansion of this family has played a large role in shaping plant organelle genetics.","PeriodicalId":501012,"journal":{"name":"The Plant Cell","volume":"22 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142841502","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Root hairs are tip-growing cells that anchor plants in the soil and are critical for water uptake, nutrient acquisition, and plant–environment interactions. While the molecular mechanisms that maintain the polar growth of root hairs through the asymmetric distribution of proteins, such as RHO-RELATED PROTEIN FROM PLANTS 2 (ROP2), have been described, it is unclear whether and how the transcripts encoding these tip-localized proteins are polarly localized and locally translated. Here, we demonstrated that ROP2 mRNA exhibits polar localization in Arabidopsis (Arabidopsis thaliana) root hairs. We showed that region VI (250–350 bp downstream of the stop codon) of the ROP2 3′ untranslated region (UTR) is necessary for proper mRNA localization. Moreover, region VI–mediated ROP2 mRNA polar localization was required for local translation of ROP2 transcripts, contributing to the proper subcellular localization of ROP2. Region III (100–200 bp downstream of the stop codon) influenced the local translation of ROP2 mRNA. Phenotypic investigations demonstrated that both regions III and VI of the ROP2 3′ UTR play crucial roles in modulating root hair growth. These findings help explain the local protein biosynthesis of ROP2, advancing our understanding of the regulatory mechanism and genetic basis of mRNA localization and local translation in plants.
根毛是顶端生长的细胞，可将植物固定在土壤中，对植物的水分吸收、养分获取以及植物与环境的相互作用至关重要。虽然通过 RHO-RELATED PROTEIN FROM PLANTS 2（ROP2）等蛋白质的不对称分布维持根毛极性生长的分子机制已被描述，但编码这些顶端定位蛋白质的转录本是否以及如何极性定位和定位翻译尚不清楚。在这里，我们证明了 ROP2 mRNA 在拟南芥（Arabidopsis thaliana）根毛中表现出极性定位。我们发现 ROP2 3′非翻译区（UTR）的第 VI 区（终止密码子下游 250-350 bp）是 mRNA 正常定位所必需的。此外，区域 VI 介导的 ROP2 mRNA 极性定位是 ROP2 转录本本地翻译所必需的，有助于 ROP2 的正确亚细胞定位。区域 III（终止密码子下游 100-200 bp）影响 ROP2 mRNA 的局部翻译。表型研究表明，ROP2 3′ UTR 的区域 III 和 VI 在调节根毛生长中都起着至关重要的作用。这些发现有助于解释 ROP2 的局部蛋白生物合成，加深了我们对植物 mRNA 定位和局部翻译的调控机制和遗传基础的理解。
{"title":"Polar localization and local translation of RHO-RELATED PROTEIN FROM PLANTS2 mRNAs promote root hair growth in Arabidopsis","authors":"Yuanyuan Li, Sirui Zhu","doi":"10.1093/plcell/koae333","DOIUrl":"https://doi.org/10.1093/plcell/koae333","url":null,"abstract":"Root hairs are tip-growing cells that anchor plants in the soil and are critical for water uptake, nutrient acquisition, and plant–environment interactions. While the molecular mechanisms that maintain the polar growth of root hairs through the asymmetric distribution of proteins, such as RHO-RELATED PROTEIN FROM PLANTS 2 (ROP2), have been described, it is unclear whether and how the transcripts encoding these tip-localized proteins are polarly localized and locally translated. Here, we demonstrated that ROP2 mRNA exhibits polar localization in Arabidopsis (Arabidopsis thaliana) root hairs. We showed that region VI (250–350 bp downstream of the stop codon) of the ROP2 3′ untranslated region (UTR) is necessary for proper mRNA localization. Moreover, region VI–mediated ROP2 mRNA polar localization was required for local translation of ROP2 transcripts, contributing to the proper subcellular localization of ROP2. Region III (100–200 bp downstream of the stop codon) influenced the local translation of ROP2 mRNA. Phenotypic investigations demonstrated that both regions III and VI of the ROP2 3′ UTR play crucial roles in modulating root hair growth. These findings help explain the local protein biosynthesis of ROP2, advancing our understanding of the regulatory mechanism and genetic basis of mRNA localization and local translation in plants.","PeriodicalId":501012,"journal":{"name":"The Plant Cell","volume":"47 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142841503","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Nitrogen is essential for plant growth and development. SNF1-related protein kinase 1 (SnRK1) is an evolutionarily conserved protein kinase pivotal for regulating plant responses to nutrient deficiency. Here, we discovered that the expression and activity of the SnRK1 α-catalytic subunit (SnRK1α1) increased in response to low-nitrogen stress. SnRK1α1 overexpression enhanced seedling tolerance, nitrate uptake capacity, apoplastic reactive oxygen species (ROS) accumulation, and NADPH oxidase activity in tomato (Solanum lycopersicum L.) under low-nitrogen stress compared to wild type plants, while snrk1α1 mutants exhibited the opposite phenotypes. Mutation of the NADPH oxidase gene Respiratory burst oxidase homolog 1 (RBOH1) suppressed numerous nitrate uptake and metabolism genes during low-nitrogen stress. rboh1 mutants displayed lower NADPH oxidase activity, apoplastic ROS production, and seedling tolerance to low nitrogen. Silencing RBOH1 expression also compromised SnRK1α1-mediated seedling tolerance to low-nitrogen stress. SnRK1α1 interacts with and activates RBOH1 through phosphorylation of three N-terminal serine residues, leading to increased apoplastic ROS production and enhanced tolerance to low nitrogen conditions. Furthermore, RBOH1-dependent ROS oxidatively modified the transcription factor TGA4 at residue Cys-334, which increased NRT1.1 and NRT2.1 expression under low-nitrogen stress. These findings reveal a SnRK1α1-mediated signaling pathway and highlight the essential role of RBOH1-dependent ROS production in enhancing plant tolerance to low nitrogen.
{"title":"SnRK1α1-mediated RBOH1 phosphorylation regulates reactive oxygen species to enhance tolerance to low nitrogen in tomato","authors":"Xuelian Zheng, Hongfei Yang, Jinping Zou, Weiduo Jin, Zhenyu Qi, Ping Yang, Jingquan Yu, Jie Zhou","doi":"10.1093/plcell/koae321","DOIUrl":"https://doi.org/10.1093/plcell/koae321","url":null,"abstract":"Nitrogen is essential for plant growth and development. SNF1-related protein kinase 1 (SnRK1) is an evolutionarily conserved protein kinase pivotal for regulating plant responses to nutrient deficiency. Here, we discovered that the expression and activity of the SnRK1 α-catalytic subunit (SnRK1α1) increased in response to low-nitrogen stress. SnRK1α1 overexpression enhanced seedling tolerance, nitrate uptake capacity, apoplastic reactive oxygen species (ROS) accumulation, and NADPH oxidase activity in tomato (Solanum lycopersicum L.) under low-nitrogen stress compared to wild type plants, while snrk1α1 mutants exhibited the opposite phenotypes. Mutation of the NADPH oxidase gene Respiratory burst oxidase homolog 1 (RBOH1) suppressed numerous nitrate uptake and metabolism genes during low-nitrogen stress. rboh1 mutants displayed lower NADPH oxidase activity, apoplastic ROS production, and seedling tolerance to low nitrogen. Silencing RBOH1 expression also compromised SnRK1α1-mediated seedling tolerance to low-nitrogen stress. SnRK1α1 interacts with and activates RBOH1 through phosphorylation of three N-terminal serine residues, leading to increased apoplastic ROS production and enhanced tolerance to low nitrogen conditions. Furthermore, RBOH1-dependent ROS oxidatively modified the transcription factor TGA4 at residue Cys-334, which increased NRT1.1 and NRT2.1 expression under low-nitrogen stress. These findings reveal a SnRK1α1-mediated signaling pathway and highlight the essential role of RBOH1-dependent ROS production in enhancing plant tolerance to low nitrogen.","PeriodicalId":501012,"journal":{"name":"The Plant Cell","volume":"42 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142815889","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Xueqi Li, Sujie Zhang, Chenyang Wang, Bin Ren, Fang Yan, Shaofang Li, Carl Spetz, Jinguang Huang, Xueping Zhou, Huanbin Zhou
In situ epitope tagging is crucial for probing gene expression, protein localization, and the dynamics of protein interactions within their natural cellular context. However, the practical application of this technique in plants presents considerable hurdles. Here, we comprehensively explored the potential of the CRISPR/Cas nuclease-mediated prime editing and different DNA repair pathways in epitope tagging of endogenous rice (Oryza sativa) genes. We found that a SpCas9 nuclease/microhomology-mediated end joining (MMEJ)-based prime editing (PE) strategy (termed NM-PE) facilitates more straightforward and efficient gene tagging compared to the conventional and other derivative PE methods. Furthermore, the PAM-flexible SpRY and ScCas9 nucleases-based prime editors have been engineered and implemented for the tagging of endogenous genes with diverse epitopes, significantly broadening the applicability of NM-PE in rice. Moreover, NM-PE has been successfully adopted in simultaneous tagging of the MAP kinase (MPK) genes OsMPK1 and OsMPK13 in rice plants with c-Myc and HA tags, respectively. Taken together, our results indicate great potential of the NM-PE toolkit in the targeted gene tagging for Rice Protein Tagging Project, gene function study and genetic improvement.
{"title":"Efficient in situ epitope tagging of rice genes by nuclease-mediated prime editing","authors":"Xueqi Li, Sujie Zhang, Chenyang Wang, Bin Ren, Fang Yan, Shaofang Li, Carl Spetz, Jinguang Huang, Xueping Zhou, Huanbin Zhou","doi":"10.1093/plcell/koae316","DOIUrl":"https://doi.org/10.1093/plcell/koae316","url":null,"abstract":"In situ epitope tagging is crucial for probing gene expression, protein localization, and the dynamics of protein interactions within their natural cellular context. However, the practical application of this technique in plants presents considerable hurdles. Here, we comprehensively explored the potential of the CRISPR/Cas nuclease-mediated prime editing and different DNA repair pathways in epitope tagging of endogenous rice (Oryza sativa) genes. We found that a SpCas9 nuclease/microhomology-mediated end joining (MMEJ)-based prime editing (PE) strategy (termed NM-PE) facilitates more straightforward and efficient gene tagging compared to the conventional and other derivative PE methods. Furthermore, the PAM-flexible SpRY and ScCas9 nucleases-based prime editors have been engineered and implemented for the tagging of endogenous genes with diverse epitopes, significantly broadening the applicability of NM-PE in rice. Moreover, NM-PE has been successfully adopted in simultaneous tagging of the MAP kinase (MPK) genes OsMPK1 and OsMPK13 in rice plants with c-Myc and HA tags, respectively. Taken together, our results indicate great potential of the NM-PE toolkit in the targeted gene tagging for Rice Protein Tagging Project, gene function study and genetic improvement.","PeriodicalId":501012,"journal":{"name":"The Plant Cell","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142805437","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Chaochao He, Yue Liang, Runzhou Chen, Yuxiao Shen, Runhui Li, Tingting Sun, Xing Du, Xiaomei Ni, Junzhong Shang, Yanhong He, Manzhu Bao, Hong Luo, Jihua Wang, Pan Liao, Chunying Kang, Yao-Wu Yuan, Guogui Ning
Enhancing the transcriptional activation activity of transcription factors (TFs) has multiple applications in organism improvement, metabolic engineering, and other aspects of plant science, but the approaches remain unclear. Here, we used gene activation assays and genetic transformation to investigate the transcriptional activities of two MYB TFs, PRODUCTION OF ANTHOCYANIN PIGMENT 1 (AtPAP1) from Arabidopsis (Arabidopsis thaliana) and EsMYBA1 from Epimedium (Epimedium sagittatum), and their synthetic variants in a range of plant species from several families. Using anthocyanin biosynthesis as a convenient readout, we discovered that homologous naturally occurring TFs showed differences in the transcriptional activation ability and that similar TFs induced large changes in the genetic program when heterologously expressed in different species. In some cases, shuffling the DNA binding domains and transcriptional activation domains (ADs) between homologous TFs led to synthetic TFs that had stronger activation potency than the original TFs. More importantly, synthetic TFs derived from MYB, NAC, bHLH, and Ethylene-insensitive3-like (EIL) family members containing tandemly repeated ADs had greatly enhanced activity compared to their natural counterparts. These findings enhance our understanding of TF activity and demonstrate that employing tandemly repeated ADs from natural TFs is a simple and widely applicable strategy to enhance the activation potency of synthetic TFs.
{"title":"Boosting transcriptional activities by employing repeated activation domains in transcription factors","authors":"Chaochao He, Yue Liang, Runzhou Chen, Yuxiao Shen, Runhui Li, Tingting Sun, Xing Du, Xiaomei Ni, Junzhong Shang, Yanhong He, Manzhu Bao, Hong Luo, Jihua Wang, Pan Liao, Chunying Kang, Yao-Wu Yuan, Guogui Ning","doi":"10.1093/plcell/koae315","DOIUrl":"https://doi.org/10.1093/plcell/koae315","url":null,"abstract":"Enhancing the transcriptional activation activity of transcription factors (TFs) has multiple applications in organism improvement, metabolic engineering, and other aspects of plant science, but the approaches remain unclear. Here, we used gene activation assays and genetic transformation to investigate the transcriptional activities of two MYB TFs, PRODUCTION OF ANTHOCYANIN PIGMENT 1 (AtPAP1) from Arabidopsis (Arabidopsis thaliana) and EsMYBA1 from Epimedium (Epimedium sagittatum), and their synthetic variants in a range of plant species from several families. Using anthocyanin biosynthesis as a convenient readout, we discovered that homologous naturally occurring TFs showed differences in the transcriptional activation ability and that similar TFs induced large changes in the genetic program when heterologously expressed in different species. In some cases, shuffling the DNA binding domains and transcriptional activation domains (ADs) between homologous TFs led to synthetic TFs that had stronger activation potency than the original TFs. More importantly, synthetic TFs derived from MYB, NAC, bHLH, and Ethylene-insensitive3-like (EIL) family members containing tandemly repeated ADs had greatly enhanced activity compared to their natural counterparts. These findings enhance our understanding of TF activity and demonstrate that employing tandemly repeated ADs from natural TFs is a simple and widely applicable strategy to enhance the activation potency of synthetic TFs.","PeriodicalId":501012,"journal":{"name":"The Plant Cell","volume":"25 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142805459","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Wei Xiao, Yang Zhang, Yue Wang, Yike Zeng, Xiangming Shang, Lin Meng, Yu Zhang, Tian Fang, Peng Xiao, Jing Qu, Yilei Wang, Min Wang, Chunlong Li, Ji-Hong Liu
Plants subjected to cold stress have been observed to accumulate proline, but the underlying regulatory mechanism remains to be elucidated. In this study, we identified a pyrroline-5-carboxylate synthetase (P5CS)-encoding gene (CtrP5CS1) from trifoliate orange (Citrus trifoliata L.), a cold-hardy citrus species, as a critical gene for cold-induced proline accumulation. CtrTGA2 bound directly to the TGACG motif of the CtrP5CS1 promoter and activated its expression. Moreover, CtrTGA2 functioned positively in cold tolerance via modulation of proline synthesis by regulating CtrP5CS1 expression. Up-regulation of CtrP5CS1 and CtrTGA2 under cold stress was dependent on salicylic acid (SA) biosynthesis. CtrTGA2 directly regulated the expression of CtrICS1, a gene encoding isochorismate synthase (ICS) involved in SA biosynthesis, forming a positive feedback loop to intensify the CtrTGA2-mediated transcriptional activation of CtrP5CS1. The cold-induced SA receptor NONEXPRESSOR OF PATHOGENESIS-RELATED GENES3 (CtrNPR3) interacted with CtrTGA2 to inhibit its transcriptional activation activity; however, the inhibition was released by SA. Our results uncover the CtrTGA2-CtrP5CS1/CtrICS1 regulatory module that orchestrates the SA signal to regulate proline synthesis, giving important insights into the transcriptional mechanism underlying proline accumulation in plants under cold stress.
{"title":"The transcription factor TGA2 orchestrates salicylic acid signal to regulate cold-induced proline accumulation in Citrus","authors":"Wei Xiao, Yang Zhang, Yue Wang, Yike Zeng, Xiangming Shang, Lin Meng, Yu Zhang, Tian Fang, Peng Xiao, Jing Qu, Yilei Wang, Min Wang, Chunlong Li, Ji-Hong Liu","doi":"10.1093/plcell/koae290","DOIUrl":"https://doi.org/10.1093/plcell/koae290","url":null,"abstract":"Plants subjected to cold stress have been observed to accumulate proline, but the underlying regulatory mechanism remains to be elucidated. In this study, we identified a pyrroline-5-carboxylate synthetase (P5CS)-encoding gene (CtrP5CS1) from trifoliate orange (Citrus trifoliata L.), a cold-hardy citrus species, as a critical gene for cold-induced proline accumulation. CtrTGA2 bound directly to the TGACG motif of the CtrP5CS1 promoter and activated its expression. Moreover, CtrTGA2 functioned positively in cold tolerance via modulation of proline synthesis by regulating CtrP5CS1 expression. Up-regulation of CtrP5CS1 and CtrTGA2 under cold stress was dependent on salicylic acid (SA) biosynthesis. CtrTGA2 directly regulated the expression of CtrICS1, a gene encoding isochorismate synthase (ICS) involved in SA biosynthesis, forming a positive feedback loop to intensify the CtrTGA2-mediated transcriptional activation of CtrP5CS1. The cold-induced SA receptor NONEXPRESSOR OF PATHOGENESIS-RELATED GENES3 (CtrNPR3) interacted with CtrTGA2 to inhibit its transcriptional activation activity; however, the inhibition was released by SA. Our results uncover the CtrTGA2-CtrP5CS1/CtrICS1 regulatory module that orchestrates the SA signal to regulate proline synthesis, giving important insights into the transcriptional mechanism underlying proline accumulation in plants under cold stress.","PeriodicalId":501012,"journal":{"name":"The Plant Cell","volume":"230 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142805503","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Debamalya Chatterjee, Ziru Zhang, Pei-Yu Lin, Po-Hao Wang, Gurpreet K Sidhu, Neela H Yennawar, Jo-Wei Allison Hsieh, Pao-Yang Chen, Rentao Song, Blake C Meyers, Surinder Chopra
The basal endosperm transfer layer (BETL) of the maize (Zea mays L.) kernel is composed of transfer cells for nutrient transport to nourish the developing kernel. To understand the spatiotemporal processes required for BETL development, we characterized 2 unstable factor for orange1 (Zmufo1) mutant alleles. The BETL defects in these mutants were associated with high levels of reactive oxygen species, oxidative DNA damage, and cell death. Interestingly, antioxidant supplementation in in vitro cultured kernels alleviated the cellular defects in mutants. Transcriptome analysis of the loss-of-function Zmufo1 allele showed differential expression of tricarboxylic acid cycle, redox homeostasis, and BETL-related genes. The basal endosperms of the mutant alleles had high levels of acetyl-CoA and elevated histone acetyltransferase activity. The BETL cell nuclei showed reduced electron-dense regions, indicating sparse heterochromatin distribution in the mutants compared with wild-type. Zmufo1 overexpression further reduced histone methylation marks in the enhancer and gene body regions of the pericarp color1 (Zmp1) reporter gene. Zmufo1 encodes an intrinsically disordered nuclear protein with very low sequence similarity to known proteins. Yeast two-hybrid and luciferase complementation assays established that ZmUFO1 interacts with proteins that play a role in chromatin remodeling, nuclear transport, and transcriptional regulation. This study establishes the critical function of Zmufo1 during basal endosperm development in maize kernels.
{"title":"Maize unstable factor for orange1 encodes a nuclear protein that affects redox accumulation during kernel development","authors":"Debamalya Chatterjee, Ziru Zhang, Pei-Yu Lin, Po-Hao Wang, Gurpreet K Sidhu, Neela H Yennawar, Jo-Wei Allison Hsieh, Pao-Yang Chen, Rentao Song, Blake C Meyers, Surinder Chopra","doi":"10.1093/plcell/koae301","DOIUrl":"https://doi.org/10.1093/plcell/koae301","url":null,"abstract":"The basal endosperm transfer layer (BETL) of the maize (Zea mays L.) kernel is composed of transfer cells for nutrient transport to nourish the developing kernel. To understand the spatiotemporal processes required for BETL development, we characterized 2 unstable factor for orange1 (Zmufo1) mutant alleles. The BETL defects in these mutants were associated with high levels of reactive oxygen species, oxidative DNA damage, and cell death. Interestingly, antioxidant supplementation in in vitro cultured kernels alleviated the cellular defects in mutants. Transcriptome analysis of the loss-of-function Zmufo1 allele showed differential expression of tricarboxylic acid cycle, redox homeostasis, and BETL-related genes. The basal endosperms of the mutant alleles had high levels of acetyl-CoA and elevated histone acetyltransferase activity. The BETL cell nuclei showed reduced electron-dense regions, indicating sparse heterochromatin distribution in the mutants compared with wild-type. Zmufo1 overexpression further reduced histone methylation marks in the enhancer and gene body regions of the pericarp color1 (Zmp1) reporter gene. Zmufo1 encodes an intrinsically disordered nuclear protein with very low sequence similarity to known proteins. Yeast two-hybrid and luciferase complementation assays established that ZmUFO1 interacts with proteins that play a role in chromatin remodeling, nuclear transport, and transcriptional regulation. This study establishes the critical function of Zmufo1 during basal endosperm development in maize kernels.","PeriodicalId":501012,"journal":{"name":"The Plant Cell","volume":"34 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-11-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142718415","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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