Jianping Liu, Xin Li, Ke Wang, Tao Wang, Yang Meng, Zhi Peng, Jinli Huang, Jiaohan Huo, Xiaoqi Zhu, Jinyong Yang, Yongxi Fan, Feiyun Xu, Qian Zhang, Zhengrui Wang, Ya Wang, Hao Chen, Weifeng Xu
Heat stress significantly impacts global rice production, highlighting the critical need to understand the genetic basis of heat resistance in rice. U2AF (U2 snRNP auxiliary factor) is an essential splicing complex with critical roles in recognizing the 3′-splice site of precursor messenger RNAs (pre-mRNAs). The U2AF small subunit (U2AF35) can bind to the 3′-AG intron border and promote U2 snRNP binding to the branch-point sequences of introns through interaction with the U2AF large subunit (U2AF65). However, the functions of U2AF35 in plants are poorly understood. In this study, we discovered that the OsU2AF35a gene was vigorously induced by heat stress and could positively regulate rice thermotolerance during both the seedling and reproductive growth stages. OsU2AF35a interacts with OsU2AF65a within the nucleus, and both of them can form condensates through liquid–liquid phase separation (LLPS) following heat stress. The intrinsically disordered regions (IDR) are accountable for their LLPS. OsU2AF35a condensation is indispensable for thermotolerance. RNA-seq analysis disclosed that, subsequent to heat treatment, the expression levels of several genes associated with water deficiency and oxidative stress in osu2af35a-1 were markedly lower than those in ZH11. In accordance with this, OsU2AF35a is capable of positively regulating the oxidative stress resistance of rice. The pre-mRNAs of a considerable number of genes in the osu2af35a-1 mutant exhibited defective splicing, among which was the OsHSA32 gene. Knocking out OsHSA32 significantly reduced the thermotolerance of rice, while overexpressing OsHSA32 could partially rescue the heat sensitivity of osu2af35a-1. Together, our findings uncovered the essential role of OsU2AF35a in rice heat stress response through protein separation and regulating alternative pre-mRNA splicing.
{"title":"The splicing auxiliary factor OsU2AF35a enhances thermotolerance via protein separation and promoting proper splicing of OsHSA32 pre-mRNA in rice","authors":"Jianping Liu, Xin Li, Ke Wang, Tao Wang, Yang Meng, Zhi Peng, Jinli Huang, Jiaohan Huo, Xiaoqi Zhu, Jinyong Yang, Yongxi Fan, Feiyun Xu, Qian Zhang, Zhengrui Wang, Ya Wang, Hao Chen, Weifeng Xu","doi":"10.1111/pbi.14587","DOIUrl":"https://doi.org/10.1111/pbi.14587","url":null,"abstract":"Heat stress significantly impacts global rice production, highlighting the critical need to understand the genetic basis of heat resistance in rice. U2AF (<span style=\"text-decoration:underline\">U2</span> snRNP <span style=\"text-decoration:underline\">a</span>uxiliary <span style=\"text-decoration:underline\">f</span>actor) is an essential splicing complex with critical roles in recognizing the 3′-splice site of precursor messenger RNAs (pre-mRNAs). The U2AF small subunit (U2AF35) can bind to the 3′-AG intron border and promote U2 snRNP binding to the branch-point sequences of introns through interaction with the U2AF large subunit (U2AF65). However, the functions of U2AF35 in plants are poorly understood. In this study, we discovered that the <i>OsU2AF35a</i> gene was vigorously induced by heat stress and could positively regulate rice thermotolerance during both the seedling and reproductive growth stages. OsU2AF35a interacts with OsU2AF65a within the nucleus, and both of them can form condensates through liquid–liquid phase separation (LLPS) following heat stress. The intrinsically disordered regions (IDR) are accountable for their LLPS. OsU2AF35a condensation is indispensable for thermotolerance. RNA-seq analysis disclosed that, subsequent to heat treatment, the expression levels of several genes associated with water deficiency and oxidative stress in <i>osu2af35a-1</i> were markedly lower than those in ZH11. In accordance with this, OsU2AF35a is capable of positively regulating the oxidative stress resistance of rice. The pre-mRNAs of a considerable number of genes in the <i>osu2af35a-1</i> mutant exhibited defective splicing, among which was the <i>OsHSA32</i> gene. Knocking out <i>OsHSA32</i> significantly reduced the thermotolerance of rice, while overexpressing <i>OsHSA32</i> could partially rescue the heat sensitivity of <i>osu2af35a-1</i>. Together, our findings uncovered the essential role of OsU2AF35a in rice heat stress response through protein separation and regulating alternative pre-mRNA splicing.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"12 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143020865","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}
Guanghao Guo, Kaihong Bai, Yikun Hou, Zhen Gong, Huaizhi Zhang, Qiuhong Wu, Ping Lu, Miaomiao Li, Lingli Dong, Jingzhong Xie, Yongxing Chen, Panpan Zhang, Keyu Zhu, Beibei Li, Wenling Li, Lei Dong, Yijun Yang, Dan Qiu, Gaojie Wang, Hee-Kyung Ahn, He Zhao, Chengguo Yuan, Wenqi Shi, Minfeng Xue, Lijun Yang, Dazao Yu, Yusheng Zhao, Yuhang Chen, Hongjie Li, Tiezhu Hu, Guan-Zhu Han, Jonathan D G Jones, Zhiyong Liu
Powdery mildew poses a significant threat to global wheat production and most cloned and deployed resistance genes for wheat breeding encode nucleotide-binding and leucine-rich repeat (NLR) immune receptors. Although two genetically linked NLRs function together as an NLR pair have been reported in other species, this phenomenon has been relatively less studied in wheat. Here, we demonstrate that two tightly linked NLR genes, RXL and Pm5e, arranged in a head-to-head orientation, function together as an NLR pair to mediate powdery mildew resistance in wheat. The resistance function of the RXL/Pm5e pair is validated by mutagenesis, gene silencing, and gene-editing assays. Interestingly, both RXL and Pm5e encode atypical NLRs, with RXL possessing a truncated NB-ARC (nucleotide binding adaptor shared by APAF-1, plant R proteins and CED-4) domain and Pm5e featuring an atypical coiled-coil (CC) domain. Notably, RXL and Pm5e lack an integrated domain associated with effector recognition found in all previously reported NLR pairs. Additionally, RXL and Pm5e exhibit a preference for forming hetero-complexes rather than homo-complexes, highlighting their cooperative role in disease resistance. We further show that the CC domain of Pm5e specifically suppresses the hypersensitive response induced by the CC domain of RXL through competitive interaction, revealing regulatory mechanisms within this NLR pair. Our study sheds light on the molecular mechanism underlying RXL/Pm5e-mediated powdery mildew resistance and provides a new example of an NLR pair in wheat disease resistance.
{"title":"The wheat NLR pair RXL/Pm5e confers resistance to powdery mildew","authors":"Guanghao Guo, Kaihong Bai, Yikun Hou, Zhen Gong, Huaizhi Zhang, Qiuhong Wu, Ping Lu, Miaomiao Li, Lingli Dong, Jingzhong Xie, Yongxing Chen, Panpan Zhang, Keyu Zhu, Beibei Li, Wenling Li, Lei Dong, Yijun Yang, Dan Qiu, Gaojie Wang, Hee-Kyung Ahn, He Zhao, Chengguo Yuan, Wenqi Shi, Minfeng Xue, Lijun Yang, Dazao Yu, Yusheng Zhao, Yuhang Chen, Hongjie Li, Tiezhu Hu, Guan-Zhu Han, Jonathan D G Jones, Zhiyong Liu","doi":"10.1111/pbi.14584","DOIUrl":"https://doi.org/10.1111/pbi.14584","url":null,"abstract":"Powdery mildew poses a significant threat to global wheat production and most cloned and deployed resistance genes for wheat breeding encode nucleotide-binding and leucine-rich repeat (NLR) immune receptors. Although two genetically linked NLRs function together as an NLR pair have been reported in other species, this phenomenon has been relatively less studied in wheat. Here, we demonstrate that two tightly linked NLR genes, <i>RXL</i> and <i>Pm5e</i>, arranged in a head-to-head orientation, function together as an <i>NLR</i> pair to mediate powdery mildew resistance in wheat. The resistance function of the <i>RXL</i>/<i>Pm5e</i> pair is validated by mutagenesis, gene silencing, and gene-editing assays. Interestingly, both <i>RXL</i> and <i>Pm5e</i> encode atypical NLRs, with RXL possessing a truncated NB-ARC (nucleotide binding adaptor shared by APAF-1, plant R proteins and CED-4) domain and Pm5e featuring an atypical coiled-coil (CC) domain. Notably, RXL and Pm5e lack an integrated domain associated with effector recognition found in all previously reported NLR pairs. Additionally, RXL and Pm5e exhibit a preference for forming hetero-complexes rather than homo-complexes, highlighting their cooperative role in disease resistance. We further show that the CC domain of Pm5e specifically suppresses the hypersensitive response induced by the CC domain of RXL through competitive interaction, revealing regulatory mechanisms within this NLR pair. Our study sheds light on the molecular mechanism underlying <i>RXL</i>/<i>Pm5e-</i>mediated powdery mildew resistance and provides a new example of an <i>NLR</i> pair in wheat disease resistance.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"10 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142992146","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}
Bei Gao, Jichen Zhao, Xiaoshuang Li, Jianhua Zhang, Melvin J. Oliver, Daoyuan Zhang
<p>The extremophile desert moss <i>Syntrichia caninervis</i>, from the Gurbantunggut Desert in China, was capable of surviving simulated Mars conditions (Li <i>et al</i>., <span>2024</span>). <i>Syntrichia caninervis</i> has become a research model for plant desiccation tolerance (Oliver <i>et al</i>., <span>2020</span>). The chromosome-level genome of <i>S. caninervis</i>, from gametophytes originating from the Mojave Desert, was sequenced and assembled (Silva <i>et al</i>., <span>2021</span>), facilitating research on gene function (Li <i>et al</i>., <span>2023</span>) and comparative and evolutionary genomics (Zhang <i>et al</i>., <span>2024</span>). This <i>S. caninervis</i> genome was considered an initial version (ScMoj v1). Because the ScMoj v1 genome relies on assembly of short reads, it has issues with continuity, gaps and assembly errors related to repetitive sequences. Here we generated a high-quality genome for the <i>S. caninervis</i> isolated from the Gurbantunggut Desert (designated ScGur).</p> <p>Cultured gametophytes propagated from a single female gametophyte (Figure S1) were used for DNA isolation. The genome was assembled from PacBio High Fidelity (HiFi) and Oxford Nanopore Technologies (ONT) ultra-long reads (Table S1) using hifiasm and NextDenovo softwares. The complete circular genomes of the <i>S. caninervis</i> chloroplast (123 124 bp, Figure S2) and mitochondria (108 309 bp, Figure S3) were obtained using GetOrganelle. A single circular bacterial genome of 6 933 718 bp (Figure 1a) was discovered during assembly with high genomic synteny (Figure 1b) to three genomic contigs of <i>Paenibacillus cellulosilyticus</i> (NCBI accession: GCF_013347265.1), indicating an internally symbiotic bacteria <i>Paenibacillus</i> sp. within <i>S. caninervis</i> gametophytes.</p> <figure><picture> <source media="(min-width: 1650px)" srcset="/cms/asset/96930a0f-6765-4442-bb29-f03b2f725bae/pbi14549-fig-0001-m.jpg"/><img alt="Details are in the caption following the image" data-lg-src="/cms/asset/96930a0f-6765-4442-bb29-f03b2f725bae/pbi14549-fig-0001-m.jpg" loading="lazy" src="/cms/asset/eed8b8bf-93e4-4eb0-8fb4-76fbe6812297/pbi14549-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>Complete genome assembly of the desert moss <i>Syntrichia caninervis</i> and its symbiotic bacteria. (a) Complete circular genome of the symbiotic bacteria <i>Paenibacillus</i> sp., illustrated tracks included the GC skew (purple and green), GC content (grey) and the 6-frame protein coding sequences (blue). (b) Collinearity analyses of the symbiotic bacterial genomic sequence with the three contigs of <i>Paenibacillus cellulosilyticus</i> (strain KACC 14175) genome. (c) Overview and comparison of the ScMoj v1 and ScGur T2T genomes. (d) Overview of the genomic s
{"title":"Telomere-to-telomere genome of the desiccation-tolerant desert moss Syntrichia caninervis illuminates Copia-dominant centromeric architecture","authors":"Bei Gao, Jichen Zhao, Xiaoshuang Li, Jianhua Zhang, Melvin J. Oliver, Daoyuan Zhang","doi":"10.1111/pbi.14549","DOIUrl":"https://doi.org/10.1111/pbi.14549","url":null,"abstract":"<p>The extremophile desert moss <i>Syntrichia caninervis</i>, from the Gurbantunggut Desert in China, was capable of surviving simulated Mars conditions (Li <i>et al</i>., <span>2024</span>). <i>Syntrichia caninervis</i> has become a research model for plant desiccation tolerance (Oliver <i>et al</i>., <span>2020</span>). The chromosome-level genome of <i>S. caninervis</i>, from gametophytes originating from the Mojave Desert, was sequenced and assembled (Silva <i>et al</i>., <span>2021</span>), facilitating research on gene function (Li <i>et al</i>., <span>2023</span>) and comparative and evolutionary genomics (Zhang <i>et al</i>., <span>2024</span>). This <i>S. caninervis</i> genome was considered an initial version (ScMoj v1). Because the ScMoj v1 genome relies on assembly of short reads, it has issues with continuity, gaps and assembly errors related to repetitive sequences. Here we generated a high-quality genome for the <i>S. caninervis</i> isolated from the Gurbantunggut Desert (designated ScGur).</p>\u0000<p>Cultured gametophytes propagated from a single female gametophyte (Figure S1) were used for DNA isolation. The genome was assembled from PacBio High Fidelity (HiFi) and Oxford Nanopore Technologies (ONT) ultra-long reads (Table S1) using hifiasm and NextDenovo softwares. The complete circular genomes of the <i>S. caninervis</i> chloroplast (123 124 bp, Figure S2) and mitochondria (108 309 bp, Figure S3) were obtained using GetOrganelle. A single circular bacterial genome of 6 933 718 bp (Figure 1a) was discovered during assembly with high genomic synteny (Figure 1b) to three genomic contigs of <i>Paenibacillus cellulosilyticus</i> (NCBI accession: GCF_013347265.1), indicating an internally symbiotic bacteria <i>Paenibacillus</i> sp. within <i>S. caninervis</i> gametophytes.</p>\u0000<figure><picture>\u0000<source media=\"(min-width: 1650px)\" srcset=\"/cms/asset/96930a0f-6765-4442-bb29-f03b2f725bae/pbi14549-fig-0001-m.jpg\"/><img alt=\"Details are in the caption following the image\" data-lg-src=\"/cms/asset/96930a0f-6765-4442-bb29-f03b2f725bae/pbi14549-fig-0001-m.jpg\" loading=\"lazy\" src=\"/cms/asset/eed8b8bf-93e4-4eb0-8fb4-76fbe6812297/pbi14549-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>Complete genome assembly of the desert moss <i>Syntrichia caninervis</i> and its symbiotic bacteria. (a) Complete circular genome of the symbiotic bacteria <i>Paenibacillus</i> sp., illustrated tracks included the GC skew (purple and green), GC content (grey) and the 6-frame protein coding sequences (blue). (b) Collinearity analyses of the symbiotic bacterial genomic sequence with the three contigs of <i>Paenibacillus cellulosilyticus</i> (strain KACC 14175) genome. (c) Overview and comparison of the ScMoj v1 and ScGur T2T genomes. (d) Overview of the genomic s","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"41 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143021108","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}
Soybean cyst nematode (SCN, Heterodera glycines) is a major pathogen harmful to soybean all over the world, causing huge yield loss every year. Soybean resistance to SCN is a complex quantitative trait controlled by a small number of major genes (rhg1 and Rhg4) and multiple micro-effect genes. Therefore, the continuous identification of new resistant lines and genes is needed for the sustainable development of global soybean production. Here, a novel disease-resistance quantitative trait locus Rscn-16 was identified and fine mapped to an 8.4-kb interval on chromosome 16 using an F2 population. According to transcriptome and metabolome analysis, a UDP-glucosyltransferase encoding gene, GmUGT88A1, was identified as the most likely gene of Rscn-16. Soybean lines overexpressing GmUGT88A1 exhibited increased resistance to SCN, higher isoflavone glycosides and larger seed size while the phenotype of RNA-interference and knockout soybean lines showed sensitivity to SCN and decreased in seed size compared to wild-type plants. GmMYB29 gene could bind to the promoter of GmUGT88A1 and coordinate with GmUGT88A1 to regulate soybean resistance to SCN and isoflavone accumulation. Under SCN infection, GmUGT88A1 participated in the reorientation of isoflavone biosynthetic metabolic flow and the accumulation of isoflavone glycosides, thus protecting soybean from SCN stress. GmUGT88A1 was found to control soybean seed size by affecting transcription abundance of GmSWEET10b and GmFAD3C, which are known to control soybean seed weight. Our findings provide insights into the regulation of SCN resistance, isoflavone content and seed size through metabolic flux redirection, and offer a potential means for soybean improvement.
{"title":"Multi-omics analysis identified the GmUGT88A1 gene, which coordinately regulates soybean resistance to cyst nematode and isoflavone content","authors":"Haipeng Jiang, Shuo Qu, Fang Liu, Haowen Sun, Haiyan Li, Weili Teng, Yuhang Zhan, Yongguang Li, Yingpeng Han, Xue Zhao","doi":"10.1111/pbi.14586","DOIUrl":"https://doi.org/10.1111/pbi.14586","url":null,"abstract":"Soybean cyst nematode (SCN, <i>Heterodera glycines</i>) is a major pathogen harmful to soybean all over the world, causing huge yield loss every year. Soybean resistance to SCN is a complex quantitative trait controlled by a small number of major genes (<i>rhg1</i> and <i>Rhg4</i>) and multiple micro-effect genes. Therefore, the continuous identification of new resistant lines and genes is needed for the sustainable development of global soybean production. Here, a novel disease-resistance quantitative trait locus <i>Rscn-16</i> was identified and fine mapped to an 8.4-kb interval on chromosome 16 using an F<sub>2</sub> population. According to transcriptome and metabolome analysis, a UDP-glucosyltransferase encoding gene, <i>GmUGT88A1</i>, was identified as the most likely gene of <i>Rscn-16</i>. Soybean lines overexpressing <i>GmUGT88A1</i> exhibited increased resistance to SCN, higher isoflavone glycosides and larger seed size while the phenotype of RNA-interference and knockout soybean lines showed sensitivity to SCN and decreased in seed size compared to wild-type plants. <i>GmMYB29</i> gene could bind to the promoter of <i>GmUGT88A1</i> and coordinate with <i>GmUGT88A1</i> to regulate soybean resistance to SCN and isoflavone accumulation. Under SCN infection, <i>GmUGT88A1</i> participated in the reorientation of isoflavone biosynthetic metabolic flow and the accumulation of isoflavone glycosides, thus protecting soybean from SCN stress. <i>GmUGT88A1</i> was found to control soybean seed size by affecting transcription abundance of <i>GmSWEET10b</i> and <i>GmFAD3C</i>, which are known to control soybean seed weight. Our findings provide insights into the regulation of SCN resistance, isoflavone content and seed size through metabolic flux redirection, and offer a potential means for soybean improvement.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"46 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142990591","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}
Binbin Zhao, Zhanchao Xia, Changhe Sun, Di Yang, Yuelei Zhao, Jiyuan Cao, Yaoyao Li, Haiyang Wang, Baobao Wang
<p>Maize, now ranking #1 in world cereal production (accounting for ~42% of total cereal production worldwide), plays a pivotal role in securing food and feed supply globally (FAO, <span>2023</span>). Historically, increasing planting density has been adopted as a key measurement to increasing maize grain yield per unit land area (Mansfield and Mumm, <span>2014</span>). Plant height (PH) and ear height (EH) are key agronomic traits that determine lodging resistance and thus high-density planting tolerance of maize (Wang <i>et al</i>., <span>2020</span>). Recently, it has been proposed that “Short corn” may represent a future avenue for maize breeding, as it stands up better to windstorms, boost yields and benefit the environment (Stokstad, <span>2023</span>). Nevertheless, a major technical thwart in breeding “Short Corn” is the lack of deployable genes and elite germplasm.</p> <p><i>Brachytic2</i> (<i>Br2</i>) encodes a protein belonging to the multidrug resistant (MDR) class of P-glycoproteins harbouring two transmembrane domains (TMD1 and TMD2), and two nucleotide-binding domains (NBD1 and NBD2), and plays a role in regulating PH via mediating polar auxin transport (Multani <i>et al</i>., <span>2003</span>). Despite its loss-of-function mutants exhibit an extremely dwarf stature, several recent studies reported that mild mutations in the last (fifth) exon of <i>Br2</i> result in milder variation in PH without notable unfavourable effects on other agronomic traits (Wei <i>et al</i>., <span>2018</span>; Xing <i>et al</i>., <span>2015</span>).</p> <p>As PH and EH are complex traits regulated by a large number of quantitative loci and easily influenced by genetic backgrounds and environmental conditions, we wondered if it is possible to generate a series of <i>br2</i> mutant alleles, so as to expand the portfolios of semi-dwarf maize germplasm for tailored breeding of semi-dwarf maize cultivars in different genetic backgrounds.</p> <p>As a proof-of-concept study, we designed a CRISPR/Cas9 vector targeting the last exon of <i>Br2</i>. To increase the targeting efficiency and universality in different genetic backgrounds, we first examined the genetic variation of <i>Br2</i> in 45 representative inbred lines with reported high-quality genome assembly (https://www.maizegdb.org/), and designed 4 conserved targets, with three of them are completely conserved in all the 45 inbred lines, while Target3 is conserved in 38 of the 45 inbred lines (Figure 1a).</p> <figure><picture> <source media="(min-width: 1650px)" srcset="/cms/asset/23d4544b-6d99-49c0-8be3-fb6f4646dd11/pbi14571-fig-0001-m.jpg"/><img alt="Details are in the caption following the image" data-lg-src="/cms/asset/23d4544b-6d99-49c0-8be3-fb6f4646dd11/pbi14571-fig-0001-m.jpg" loading="lazy" src="/cms/asset/774de94f-ada2-4ace-92a4-46a5a22514de/pbi14571-fig-0001-m.png" title="Details are in the caption following the image"/></picture><figcaption> <div><strong>Figure 1<span style="font-weight:normal
{"title":"CRISPR/Cas9-mediated genomic editing of Brachytic2 creates semi-dwarf mutant alleles for tailored maize breeding","authors":"Binbin Zhao, Zhanchao Xia, Changhe Sun, Di Yang, Yuelei Zhao, Jiyuan Cao, Yaoyao Li, Haiyang Wang, Baobao Wang","doi":"10.1111/pbi.14571","DOIUrl":"https://doi.org/10.1111/pbi.14571","url":null,"abstract":"<p>Maize, now ranking #1 in world cereal production (accounting for ~42% of total cereal production worldwide), plays a pivotal role in securing food and feed supply globally (FAO, <span>2023</span>). Historically, increasing planting density has been adopted as a key measurement to increasing maize grain yield per unit land area (Mansfield and Mumm, <span>2014</span>). Plant height (PH) and ear height (EH) are key agronomic traits that determine lodging resistance and thus high-density planting tolerance of maize (Wang <i>et al</i>., <span>2020</span>). Recently, it has been proposed that “Short corn” may represent a future avenue for maize breeding, as it stands up better to windstorms, boost yields and benefit the environment (Stokstad, <span>2023</span>). Nevertheless, a major technical thwart in breeding “Short Corn” is the lack of deployable genes and elite germplasm.</p>\u0000<p><i>Brachytic2</i> (<i>Br2</i>) encodes a protein belonging to the multidrug resistant (MDR) class of P-glycoproteins harbouring two transmembrane domains (TMD1 and TMD2), and two nucleotide-binding domains (NBD1 and NBD2), and plays a role in regulating PH via mediating polar auxin transport (Multani <i>et al</i>., <span>2003</span>). Despite its loss-of-function mutants exhibit an extremely dwarf stature, several recent studies reported that mild mutations in the last (fifth) exon of <i>Br2</i> result in milder variation in PH without notable unfavourable effects on other agronomic traits (Wei <i>et al</i>., <span>2018</span>; Xing <i>et al</i>., <span>2015</span>).</p>\u0000<p>As PH and EH are complex traits regulated by a large number of quantitative loci and easily influenced by genetic backgrounds and environmental conditions, we wondered if it is possible to generate a series of <i>br2</i> mutant alleles, so as to expand the portfolios of semi-dwarf maize germplasm for tailored breeding of semi-dwarf maize cultivars in different genetic backgrounds.</p>\u0000<p>As a proof-of-concept study, we designed a CRISPR/Cas9 vector targeting the last exon of <i>Br2</i>. To increase the targeting efficiency and universality in different genetic backgrounds, we first examined the genetic variation of <i>Br2</i> in 45 representative inbred lines with reported high-quality genome assembly (https://www.maizegdb.org/), and designed 4 conserved targets, with three of them are completely conserved in all the 45 inbred lines, while Target3 is conserved in 38 of the 45 inbred lines (Figure 1a).</p>\u0000<figure><picture>\u0000<source media=\"(min-width: 1650px)\" srcset=\"/cms/asset/23d4544b-6d99-49c0-8be3-fb6f4646dd11/pbi14571-fig-0001-m.jpg\"/><img alt=\"Details are in the caption following the image\" data-lg-src=\"/cms/asset/23d4544b-6d99-49c0-8be3-fb6f4646dd11/pbi14571-fig-0001-m.jpg\" loading=\"lazy\" src=\"/cms/asset/774de94f-ada2-4ace-92a4-46a5a22514de/pbi14571-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","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"10 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142990589","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}
Phosphorus (P) is an essential yet frequently deficient plant nutrient. Optimizing P distribution and recycling between tissues is vital for improving P utilization efficiency (PUE). Yet, the mechanisms underlying the transport and re-translocation of P within plants remain unclear. Here, wide-ranging natural diversity in seed P allocation and positive correlations among yield traits were found using 190 soybean accessions in field trials. Among them, the P-efficient genotype BX10 outperformed BD2 in assessments of PUE that were largely explained through differences in P redistribution from pods to seeds under low P stress. Pods of BX10 were therefore subjected to transcriptome analysis, and GmVPE1 was identified as a vacuolar Pi transporter to investigate further. Importantly, significant DNA polymorphism in GmVPE1 promoter regions was remarkably associated with seed weight among soybean accessions grown on P-deficient soils. Further analyses suggested that mRNA abundance of GmVPE1 in haplotype 2 (Hap) is significantly higher than that GmVPE1Hap1. GmVPE1 was highly upregulated by P deficiency and preferentially expressed in pods, seeds, and seed coats, which was consistent with GUS staining using transgenic soybean plants carrying pGmVPE1Hap2::GUS. Near-isogenic lines carrying the GmVPE1Hap2 allele, along with stable transgenic soybeans overexpressing GmVPE1 in a GmVPE1Hap1 background, had increases in PUE, more seed setting, and greater yields in both greenhouse and field trials than control plants. In summary, natural variation among GmVPE1 alleles determines genetic expression and subsequent P re-translocation phenotypes, which impacts PUE and yield, and thereby makes this an important genetic resource for soybean molecular breeding.
{"title":"Natural variation in the GmVPE1 promoter contributes to phosphorus re-translocation to seeds and improves soybean yield","authors":"Jiaxin Chen, Wenting Lian, Zhiang Li, Xin Guo, Yaning Li, Hongyu Zhao, Keke Yi, Xinxin Li, Hong Liao","doi":"10.1111/pbi.14592","DOIUrl":"https://doi.org/10.1111/pbi.14592","url":null,"abstract":"Phosphorus (P) is an essential yet frequently deficient plant nutrient. Optimizing P distribution and recycling between tissues is vital for improving P utilization efficiency (PUE). Yet, the mechanisms underlying the transport and re-translocation of P within plants remain unclear. Here, wide-ranging natural diversity in seed P allocation and positive correlations among yield traits were found using 190 soybean accessions in field trials. Among them, the P-efficient genotype BX10 outperformed BD2 in assessments of PUE that were largely explained through differences in P redistribution from pods to seeds under low P stress. Pods of BX10 were therefore subjected to transcriptome analysis, and GmVPE1 was identified as a vacuolar Pi transporter to investigate further. Importantly, significant DNA polymorphism in <i>GmVPE1</i> promoter regions was remarkably associated with seed weight among soybean accessions grown on P-deficient soils. Further analyses suggested that mRNA abundance of <i>GmVPE1</i> in haplotype 2 (Hap) is significantly higher than that <i>GmVPE1</i><sup><i>Hap1</i></sup>. <i>GmVPE1</i> was highly upregulated by P deficiency and preferentially expressed in pods, seeds, and seed coats, which was consistent with GUS staining using transgenic soybean plants carrying <i>pGmVPE1</i><sup><i>Hap2</i></sup>::GUS. Near-isogenic lines carrying the <i>GmVPE1</i><sup><i>Hap2</i></sup> allele, along with stable transgenic soybeans overexpressing <i>GmVPE1</i> in a <i>GmVPE1</i><sup><i>Hap1</i></sup> background, had increases in PUE, more seed setting, and greater yields in both greenhouse and field trials than control plants. In summary, natural variation among <i>GmVPE1</i> alleles determines genetic expression and subsequent P re-translocation phenotypes, which impacts PUE and yield, and thereby makes this an important genetic resource for soybean molecular breeding.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"30 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-01-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142987141","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}
Yinghuang Wu, Xueying Wang, Haoyu Wang, Ying Han, Yaxuan Wang, Chunyu Zou, Jian-Kang Zhu, Ming Li
<p>Base editors (BEs), a groundbreaking class of genome editing tools, enable precise single-nucleotide alterations at target genomic sites, leading to mutations that either disable or enhance gene functions, thus significantly advancing plant functional genomics research and crop enhancement (Li <i>et al</i>., <span>2023</span>). In plants, significant advancements have been made in DNA base editors that can directly modify adenine (A), cytosine (C) and guanine (G) (Li <i>et al</i>., <span>2018</span>; Zong <i>et al</i>., <span>2017</span>). Nevertheless, a direct base editor for thymine (T) remains elusive. Recently, two innovative deaminase-free glycosylase-based base editors were developed: the gTBE for direct T editing (T-to-S conversion, S = G or C) and the gCBE for direct C editing (C-to-G), enabling orthogonal base modifications in mammalian cells (Figure 1a; Tong <i>et al</i>., <span>2024</span>). These base editors utilized the fusion of Cas9 nickase (nCas9) with engineered variants of human uracil DNA glycosylase (UNG), allowing for the direct excision of T or C to generate apurinic/apyrimidinic (AP) sites. However, such direct T base editor has not been developed in plants to date. In this study, we developed a deaminase-free direct T base editor (pTGBE) and direct C base editor (pCKBE, K = G or T) in rice, marking a substantial step forward in expanding genetic manipulation capabilities in plants.</p> <figure><picture> <source media="(min-width: 1650px)" srcset="/cms/asset/0edf6cda-b5d9-4b60-9d14-b9b58df33379/pbi14583-fig-0001-m.jpg"/><img alt="Details are in the caption following the image" data-lg-src="/cms/asset/0edf6cda-b5d9-4b60-9d14-b9b58df33379/pbi14583-fig-0001-m.jpg" loading="lazy" src="/cms/asset/22aec75e-9993-4b32-bbad-22a05d09436c/pbi14583-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>Development of pTGBE and pCKBE base editors in rice. (a) Schematic diagram showing the editing mechanisms of deaminase-free glycosylase-based thymine base editor (gTBE) and deaminase-free glycosylase-based cytosine base editor (gCBE) in mammalian cells. mhUNG, engineered human uracil DNA glycosylase variants. PAM, Protospacer adjacent motif. AP, apurinic/apyrimidinic sites. Star (*) in magenta indicates the nick generated by nCas9. TLS, translesion synthesis. DSB, double-strand break. InDel, insertion and deletion. (b) Plasmid constructs of pTGBE and pCKBE in this study. Short vertical lines represent mutated amino acids. Δ1-88: 1–88 amino acid residue truncation of human UNG2. (c) Summary of the editing efficiencies of pTGBE and pCKBE at the endogenous genes in stable T<sub>0</sub> transgenic lines. Ho, homozygous mutation; He, heterozyous mutation; Bi, biallelic mutation; Chi, chimeric mutation. The PAM sequence is highlighted in
碱基编辑器（BEs）是一类突破性的基因组编辑工具，可在目标基因组位点实现精确的单核苷酸改变，导致基因功能失效或增强的突变，从而极大地推动植物功能基因组学研究和作物改良（Li 等人，2023 年）。在植物中，DNA碱基编辑器取得了重大进展，可以直接修改腺嘌呤（A）、胞嘧啶（C）和鸟嘌呤（G）（Li 等人，2018；Zong 等人，2017）。然而，胸腺嘧啶（T）的直接碱基编辑器仍未出现。最近，人们开发出了两种创新的不含脱氨酶的基于糖基化酶的碱基编辑器：用于直接 T 编辑（T-to-S 转换，S = G 或 C）的 gTBE 和用于直接 C 编辑（C-to-G）的 gCBE，从而在哺乳动物细胞中实现了正交碱基修饰（图 1a；Tong 等人，2024 年）。这些碱基编辑器利用 Cas9 切分酶（nCas9）与人类尿嘧啶 DNA 糖基化酶（UNG）的工程变体融合，允许直接切除 T 或 C 以产生嘌呤/近嘧啶（AP）位点。然而，迄今为止，这种直接的 T 碱基编辑器尚未在植物中开发出来。在这项研究中，我们在水稻中开发出了不含脱氨酶的直接 T 碱基编辑器（pTGBE）和直接 C 碱基编辑器（pCKBE，K = G 或 T），这标志着我们在拓展植物遗传操作能力方面迈出了实质性的一步。(a）示意图显示了哺乳动物细胞中无脱氨酶糖基化酶胸腺嘧啶碱基编辑器（gTBE）和无脱氨酶糖基化酶胞嘧啶碱基编辑器（gCBE）的编辑机制。 mhUNG，工程化人类尿嘧啶 DNA 糖基化酶变体。PAM：邻接原基。AP，嘌呤/近嘧啶位点。品红色星号（*）表示 nCas9 产生的缺口。TLS，转子合成。DSB，双链断裂。InDel，插入和缺失。(b) 本研究中 pTGBE 和 pCKBE 的质粒构建体。短竖线代表突变的氨基酸。 Δ1-88：人 UNG2 的 1-88 个氨基酸残基截断。(c) pTGBE 和 pCKBE 在稳定的 T0 转基因株中对内源基因的编辑效率汇总。Ho，同源突变；He，异源突变；Bi，双倍突变；Chi，嵌合突变。PAM 序列以粗体标出，被编辑的碱基以红色标出。 d, e）pTGBE（d）和 pCKBE（e）对（c）中被编辑位点的 1-20 位（其中 PAM 位于 21-23 位）原间隔序列进行 T 转换和 C 转换的平均频率。(f）示意图，说明 pTGBE 介导的剪接供体位点破坏（从 WT 中的 GU 到 45 号线中的 GG）导致 OsARF24 内含子 1 的保留。(g）WT 和突变体 #45 中 OsARF24 mRNA 的 RT-PCR 分析。M'，DNA 标记。WT'，野生型。(h）（g）中 RT-PCR 扩增子的序列。(为了在水稻中建立 pTSBE，我们将水稻密码子优化的人类尿嘧啶 DNA 糖基化酶变体 UNG2Δ88-Y156A/A214T/Q259A/Y284D （mhUNGv3）（Tong 等人，2024 年）与 nCas9 用 32 氨基酸连接。为了提高核进入效率，我们将一个双核定位信号肽与 UNG 变体融合，从而得到 pTSBE 构建体（图 1b）。我们选择了针对水稻中五个基因的十个内源位点来测试编辑活性和窗口。我们共获得了 400 株 T0 稳定编辑植株，Hi-TOM 结果显示，转基因水稻植株中 T 到 S 的碱基转换效率高达 78.05%（图 1c），但基本没有 C 或 G 编辑，所有位点的 A 编辑效率为 1.85%（图 S1a-c）。我们发现 pTSBE 还能诱导插入或缺失（InDels），在 10 个编辑位点上的频率从 20.00% 到 75.32% 不等（图 1c）。值得注意的是，T-to-G 编辑（最多 78.05%，平均 39.21%）在产物中的比例是 T-to-C 编辑（最多 3.70%，平均 2.93%）的 13.38 倍。T-to-G是产生的主要编辑类型，纯度超过80%（图S1d），显示出与哺乳动物细胞截然不同的编辑模式。在哺乳动物细胞中，gTBEv3 具有 T 到 S 的碱基编辑活性，T 到 G 和 T 到 C 的平均编辑效率分别为 27.26% 和 18.75%（Tong 等人，2024 年）。因此，我们将这种 BE 命名为 pTGBE，以更好地反映其在植物中的编辑特性。此外，可编辑范围为 T2-T12、T14 和 T18 位，最佳编辑窗口为 T3-T5 位，其中 T3 位的编辑效率最高（PAM 位置为 21-23）（图 1d）。相比之下，哺乳动物细胞中的 gTBEv3 通常在 T2-T11 位置产生 T 到 S 的转换，最佳编辑窗口在 T5 位置（Tong 等人，2024 年）。T0 事件包括同卵、异卵、双偶或嵌合编辑等位基因（表 S3；图 1c）。在 60 个等位基因中观察到了同源碱基转换。 在所有十个 sgRNA 位点中，OsNRT1.1B-SG3 位点的最高效率为 30.77%（4/13），而 OsARF24-SG2 位点的杂合碱基转换率高达 27.78%（15/54）。类胡萝卜素脱饱和酶（PDS）是参与类胡萝卜素生物合成的关键酶，具有一个关键的单结构域（氨基酸 106-556）。T0 植株 #6 通过 sgRNA OsPDS-SG2 进行同源 T 到 G 编辑，导致第 114 位氨基酸的亮氨酸变为缬氨酸，从而观察到叶片上有白色条纹的白化表型（图 S3）。随着前 mRNA 转录本的加工，AS 可导致内含子保留（IR）、替代 5′剪接、替代 3′剪接和外显子跳接，从而提供不同的基因表达模式（Liu 等，2024 年）。为了说明这一应用，我们设计了特异性靶向 OsARF24 基因 SD 或 SA 位点的 sgRNA（图 S4）。我们发现了一个同源突变体 #45，其内含子 1 的 5′剪接位点上的目标 T 存在 T 到 G 的转换，OsARF24-SG1 将其作为靶标。我们使用外显子 1 的正向引物和外显子 3 的反向引物进行了 RT-PCR 扩增。野生型（WT）植株产生了一个 240 bp 的片段，而突变株 #45 则扩增出了一个 319 bp 的片段（图 1f,g）。对该片段的测序显示，内含子 1 被保留，完全阻止了正常剪接异构体的产生（图 1h）。此外，我们利用 OsARF24-SG2 在 T0 植株中产生了 12 个靶向内含子 7 的 SA 位点的杂合突变体，这将在 T1 植株中产生同源品系，用于鉴定 AS 异构体（图 S4）。为了探索 gCBE 在植物中的编辑类型和效率，我们将水稻密码子优化的人类尿嘧啶 DNA 糖基化酶变体 UNG2Δ88-K184A/N213D/A214V（mhUNGv2）（Tong 等人，2024 年）与 nCas9 融合，设计了一个构建体，以评估其在水稻中的编辑特性（图 1b）。我们选择了针对水稻中三个基因的八个内源位点来测试其编辑活性和窗口。对 255 株 T0 转基因植株进行的 Hi-TOM 测序表明，水稻中的 gCBE 能高效编辑 C 碱基，频率范围在 26.09% 到 61.11% 之间，其中主要的 C 到 G 编辑效率高达 58.33%，C 到 T 的转化率也高达 40.91%，但在所有检测位点上基本没有 A、T 或
{"title":"Targeted deaminase-free T-to-G and C-to-K base editing in rice by fused human uracil DNA glycosylase variants","authors":"Yinghuang Wu, Xueying Wang, Haoyu Wang, Ying Han, Yaxuan Wang, Chunyu Zou, Jian-Kang Zhu, Ming Li","doi":"10.1111/pbi.14583","DOIUrl":"https://doi.org/10.1111/pbi.14583","url":null,"abstract":"<p>Base editors (BEs), a groundbreaking class of genome editing tools, enable precise single-nucleotide alterations at target genomic sites, leading to mutations that either disable or enhance gene functions, thus significantly advancing plant functional genomics research and crop enhancement (Li <i>et al</i>., <span>2023</span>). In plants, significant advancements have been made in DNA base editors that can directly modify adenine (A), cytosine (C) and guanine (G) (Li <i>et al</i>., <span>2018</span>; Zong <i>et al</i>., <span>2017</span>). Nevertheless, a direct base editor for thymine (T) remains elusive. Recently, two innovative deaminase-free glycosylase-based base editors were developed: the gTBE for direct T editing (T-to-S conversion, S = G or C) and the gCBE for direct C editing (C-to-G), enabling orthogonal base modifications in mammalian cells (Figure 1a; Tong <i>et al</i>., <span>2024</span>). These base editors utilized the fusion of Cas9 nickase (nCas9) with engineered variants of human uracil DNA glycosylase (UNG), allowing for the direct excision of T or C to generate apurinic/apyrimidinic (AP) sites. However, such direct T base editor has not been developed in plants to date. In this study, we developed a deaminase-free direct T base editor (pTGBE) and direct C base editor (pCKBE, K = G or T) in rice, marking a substantial step forward in expanding genetic manipulation capabilities in plants.</p>\u0000<figure><picture>\u0000<source media=\"(min-width: 1650px)\" srcset=\"/cms/asset/0edf6cda-b5d9-4b60-9d14-b9b58df33379/pbi14583-fig-0001-m.jpg\"/><img alt=\"Details are in the caption following the image\" data-lg-src=\"/cms/asset/0edf6cda-b5d9-4b60-9d14-b9b58df33379/pbi14583-fig-0001-m.jpg\" loading=\"lazy\" src=\"/cms/asset/22aec75e-9993-4b32-bbad-22a05d09436c/pbi14583-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>Development of pTGBE and pCKBE base editors in rice. (a) Schematic diagram showing the editing mechanisms of deaminase-free glycosylase-based thymine base editor (gTBE) and deaminase-free glycosylase-based cytosine base editor (gCBE) in mammalian cells. mhUNG, engineered human uracil DNA glycosylase variants. PAM, Protospacer adjacent motif. AP, apurinic/apyrimidinic sites. Star (*) in magenta indicates the nick generated by nCas9. TLS, translesion synthesis. DSB, double-strand break. InDel, insertion and deletion. (b) Plasmid constructs of pTGBE and pCKBE in this study. Short vertical lines represent mutated amino acids. Δ1-88: 1–88 amino acid residue truncation of human UNG2. (c) Summary of the editing efficiencies of pTGBE and pCKBE at the endogenous genes in stable T<sub>0</sub> transgenic lines. Ho, homozygous mutation; He, heterozyous mutation; Bi, biallelic mutation; Chi, chimeric mutation. The PAM sequence is highlighted in ","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"119 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-01-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142987139","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}
Kathrin Göritzer, Stanislav Melnik, Jennifer Schwestka, Elsa Arcalis, Margit Drapal, Paul Fraser, Julian K-C. Ma, Eva Stoger
The production of complex multimeric secretory immunoglobulins (SIgA) in Nicotiana benthamiana leaves is challenging, with significant reductions in complete protein assembly and consequently yield, being the most important difficulties. Expanding the physical dimensions of the ER to mimic professional antibody-secreting cells can help to increase yields and promote protein folding and assembly. Here, we expanded the ER in N. benthamiana leaves by targeting the enzyme CTP:phosphocholine cytidylyltransferase (CCT), which catalyses the rate-limiting step in the synthesis of the key membrane component phosphatidylcholine (PC). We used CRISPR/Cas to perform site-directed mutagenesis of each of the three endogenous CCT genes in N. benthamiana by introducing frame-shifting indels to remove the auto-inhibitory C-terminal domains. We generated stable homozygous lines of N. benthamiana containing different combinations of the edited genes, including plants where all three isofunctional CCT homologues were modified. Changes in ER morphology in the mutant plants were confirmed by in vivo confocal imaging and substantially increased the yields of two fully assembled SIgAs by prolonging the ER residence time and boosting chaperone accumulation. Through a combination of ER engineering with chaperone overexpression, we increased the yields of fully assembled SIgA by an order of magnitude, reaching almost 1 g/kg fresh leaf weight. This strategy removes a major roadblock to producing SIgA and will likely facilitate the production of other complex multimeric biopharmaceutical proteins in plants.
在烟草叶中生产复杂的多聚物分泌型免疫球蛋白（SIgA）具有挑战性，其中最重要的困难是蛋白质的完全组装和产量大幅降低。扩大ER的物理尺寸以模拟专业的抗体分泌细胞有助于提高产量并促进蛋白质的折叠和组装。在这里，我们通过靶向 CTP:phosphocholine cytidylyltransferase（CCT）酶扩大了 N. benthamiana 叶片中的ER，该酶催化关键膜成分磷脂酰胆碱（PC）合成过程中的限速步骤。我们利用 CRISPR/Cas 对 N. benthamiana 中的三个内源 CCT 基因分别进行了定点诱变，通过引入移帧嵌段来移除自动抑制的 C 端结构域。我们培育出了含有不同编辑基因组合的 N. benthamiana 稳定同源品系，包括对所有三个同功能 CCT 同源物进行修饰的植株。体内共聚焦成像证实了突变体植株ER形态的变化，并通过延长ER停留时间和促进伴侣蛋白积累，大大提高了两种完全组装的SIgAs的产量。通过ER工程与伴侣蛋白过表达的结合，我们将完全组装的SIgA产量提高了一个数量级，几乎达到了1克/千克鲜叶重量。这一策略消除了生产 SIgA 的主要障碍，并有可能促进在植物中生产其他复杂的多聚生物制药蛋白。
{"title":"Enhancing quality and yield of recombinant secretory IgA antibodies in Nicotiana benthamiana by endoplasmic reticulum engineering","authors":"Kathrin Göritzer, Stanislav Melnik, Jennifer Schwestka, Elsa Arcalis, Margit Drapal, Paul Fraser, Julian K-C. Ma, Eva Stoger","doi":"10.1111/pbi.14576","DOIUrl":"https://doi.org/10.1111/pbi.14576","url":null,"abstract":"The production of complex multimeric secretory immunoglobulins (SIgA) in <i>Nicotiana benthamiana</i> leaves is challenging, with significant reductions in complete protein assembly and consequently yield, being the most important difficulties. Expanding the physical dimensions of the ER to mimic professional antibody-secreting cells can help to increase yields and promote protein folding and assembly. Here, we expanded the ER in <i>N. benthamiana</i> leaves by targeting the enzyme CTP:phosphocholine cytidylyltransferase (CCT), which catalyses the rate-limiting step in the synthesis of the key membrane component phosphatidylcholine (PC). We used CRISPR/Cas to perform site-directed mutagenesis of each of the three endogenous CCT genes in <i>N. benthamiana</i> by introducing frame-shifting indels to remove the auto-inhibitory C-terminal domains. We generated stable homozygous lines of <i>N. benthamiana</i> containing different combinations of the edited genes, including plants where all three isofunctional CCT homologues were modified. Changes in ER morphology in the mutant plants were confirmed by <i>in vivo</i> confocal imaging and substantially increased the yields of two fully assembled SIgAs by prolonging the ER residence time and boosting chaperone accumulation. Through a combination of ER engineering with chaperone overexpression, we increased the yields of fully assembled SIgA by an order of magnitude, reaching almost 1 g/kg fresh leaf weight. This strategy removes a major roadblock to producing SIgA and will likely facilitate the production of other complex multimeric biopharmaceutical proteins in plants.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"29 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-01-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142987140","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}
Jian Wang, Ying Ma, Junhao Tang, Huixin Lin, Guanghong Cui, Jinfu Tang, Juan Liu, Ping Su, Yujun Zhao, Juan Guo, Luqi Huang
<p><i>Andrographis paniculata</i> (Burm.f.) Wall. Ex Nees in Wallich (<i>A. paniculata</i>), an annual medicinal herb of the <i>Acanthaceae</i> family, is widely cultivated for its various medicinal utilities in Southeast and South Asia. Its total extract and monomeric components have a broad range of pharmacological effects including anti-inflammatory, anti-microbial, hepatoprotective and anticancer (Subramanian <i>et al</i>., <span>2012</span>). Numerous bioactive secondary metabolites have been isolated from the leaves and roots of <i>A. paniculata</i>, andrographolide, an <i>ent</i>-labdane diterpenoid, is considered the main bioactive compound (Subramanian <i>et al</i>., <span>2012</span>). For example, Xiyanping®, a traditional Chinese medicine injection made of andrographolide sulfonate, is widely used to treat upper respiratory tract infection, viral pneumonia and bronchitis in China. Due to their medicinal properties, andrographolide biosynthesis has been intensively investigated, genomic data and terpene synthase functions have been reported (Sun <i>et al</i>., <span>2019</span>). However, the enzymes responsible for structural modification that form the key pharmacologically active groups in its biosynthetic pathway remain unknown.</p> <p>The modification steps in andrographolide biosynthesis include hydroxylations at C3, C14, C18 and lactone ring formation at C15–C16. This series of oxidation processes were supposed to be mediated by cytochrome P450 enzymes (CYP450s). In order to accurately screen the CYP450s in andrographolide biosynthesis pathway, we constructed the differential bio-accumulation samples of andrographolide seedlings (Figure S1). After 100 μM MeJA treatment, the production of andrographolide demonstrated significant enhancement at 24 h post-inoculation (hpi) and reached 37.8 mg/g DW at 72 hpi in the leaves, which is approximately 10 times greater than that in the control (Figure 1a). We then constructed the expression atlas and investigated the time-series expression changes of <i>A. paniculata</i>. The expression profiles of samples at 12 hpi, 24 hpi and 48 hpi exhibited significantly different patterns compared to the samples collected at 0 hpi (Figure S2). By applying a cutoff of a four-folds difference in FPKM and a false discovery rate of less than 0.05, we identified that the expression levels of 4463 genes were up-regulated at 12 hpi, 24 hpi or 48 hpi in comparison to the control samples (Figure 1b).</p> <figure><picture> <source media="(min-width: 1650px)" srcset="/cms/asset/3b09830f-074d-4137-b9dc-665ca6ab4fee/pbi14572-fig-0001-m.jpg"/><img alt="Details are in the caption following the image" data-lg-src="/cms/asset/3b09830f-074d-4137-b9dc-665ca6ab4fee/pbi14572-fig-0001-m.jpg" loading="lazy" src="/cms/asset/8c424ac0-7049-492b-92fa-d222a99632a4/pbi14572-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></s
CYP450酶的催化功能通过与六种穿心莲内酯化合物的酶促反应得到验证，这些化合物被预测为穿心莲内酯生物合成途径的中间体（图S4）。通过分析酶促反应产物并与标准品进行比较，发现TR79615以14-脱氧穿心莲内酯(2)为底物催化生成穿心莲内酯(1)（图1e）。根据系统命名法，TR79615被命名为CYP72F1。产物结构表明CYP72F1可以促进14-脱氧穿心莲内酯的C14羟基化，同时将双键重新排列到C12和C13位置（图1h和S5a）。得到了两种与穿心莲内酯分子量相同的副产物（图1e和S5b、c），推测它们是双键位置不同的穿心莲内酯异构体。植物CYP450酶能够催化具有相同骨架结构的多种底物（Ma etal ., 2021）。CYP72F1微粒体也被发现与穿心莲素发生反应，考虑到植物CYP450酶靶向底物氧化位点的特异性，以及底物与产物极性的差异，我们假设CYP72F1可以催化穿心莲素的C14羟基化和C12、C13双键重排生成3-脱氧穿心莲内酯（图S6）。随着基因组数据的不断进步，越来越多参与植物二萜生物合成途径的关键基因被证明聚集在基因组内（Ma et al., 2021）。根据报道的A. paniculata基因组数据（Sun et al., 2019），我们对CYP72F1等候选基因进行了基因组定位研究，如图1d所示。结果表明，属于CYP72家族的7个CYP450基因与2号染色体上的CYP72F1一起聚集，这些基因之间的距离最小（图1f）。观察这些cyp450在MeJA诱导后的表达趋势（图S7）， TR81244在诱导24 h后表达显著上调后又下调，这与MVA通路基因和GGPPS的表达模式相似（图1c）。这四个CYP72基因随后被克隆并在酵母中表达，用于以穿心莲内酯化合物为底物的酶促反应。结果表明，TR81244可以催化穿心莲苷生成新的产物峰(3)，通过与标准化合物的比较，鉴定产物为14-脱氧穿心莲内酯(2)（图1g和S8a）。因此，TR81244被命名为CYP72A399，并证实了其促进穿心莲素C3羟基化的催化活性（图1h）。CYP72A399还可以催化对-邻苯二酚和16,19-二羟基-对-共醇作为底物，根据CYP450催化位置的特异性，我们推测它还可以催化它们的C3位点生成羟基化产物（图S8b-f）。对2号染色体上的这些CYP72基因以及丹参、黄芩、枸杞等富含二萜的植物进行了染色体定位和共线性分析。这些共线基因在其他物种中也聚集在同一染色体或支架上（图1i），这为进一步探索萜类生物合成途径基因的聚类提供了参考。由于L. japonicus中含有丰富的正链萜（Wang et al., 2022），我们将共线性分析得到的3个L. japonicus cyp450在酵母中进行表达，验证它们是否具有相似的功能。Lej2023与CYP72A399具有相同的催化功能，可催化穿心莲素C3羟基化生成14-脱氧穿心莲内酯（图S9）。在植物中，CYP72家族是参与次生代谢的最大的cyp450家族之一，但CYP72家族基因的生化信息有限。目前发现的CYP72家族蛋白促进了复杂的生物催化过程，如赤霉素（He et al., 2019）、三萜（Biazzi et al., 2015）和生态酸（Yang et al., 2019）途径中的氧化。本研究中鉴定的两个CYP72蛋白扩展了我们对与CYP72家族相关的新型催化功能的理解（图1j）。这些CYP72蛋白被分为两个亚家族，聚集在穿心莲的染色体上，它们持续催化穿心莲内酯生物合成的最后步骤。C14羟基化和C3氧化的催化过程对穿心莲内酯衍生物的形成至关重要，这对增强穿心莲内酯的抗肿瘤活性至关重要（Zhang et al., 2021）。综上所述，本研究报道了A. CYP72家族的两个CYP450基因。 通过萜类途径共表达和基因聚类分析，这两个CYP72 cyp450催化了穿心花内酯生物合成途径的关键步骤C3和C14羟基化和C12-C13双键重排。
{"title":"Two CYP72 enzymes function as Ent-labdane hydroxylases in the biosynthesis of andrographolide in Andrographis paniculata","authors":"Jian Wang, Ying Ma, Junhao Tang, Huixin Lin, Guanghong Cui, Jinfu Tang, Juan Liu, Ping Su, Yujun Zhao, Juan Guo, Luqi Huang","doi":"10.1111/pbi.14572","DOIUrl":"https://doi.org/10.1111/pbi.14572","url":null,"abstract":"<p><i>Andrographis paniculata</i> (Burm.f.) Wall. Ex Nees in Wallich (<i>A. paniculata</i>), an annual medicinal herb of the <i>Acanthaceae</i> family, is widely cultivated for its various medicinal utilities in Southeast and South Asia. Its total extract and monomeric components have a broad range of pharmacological effects including anti-inflammatory, anti-microbial, hepatoprotective and anticancer (Subramanian <i>et al</i>., <span>2012</span>). Numerous bioactive secondary metabolites have been isolated from the leaves and roots of <i>A. paniculata</i>, andrographolide, an <i>ent</i>-labdane diterpenoid, is considered the main bioactive compound (Subramanian <i>et al</i>., <span>2012</span>). For example, Xiyanping®, a traditional Chinese medicine injection made of andrographolide sulfonate, is widely used to treat upper respiratory tract infection, viral pneumonia and bronchitis in China. Due to their medicinal properties, andrographolide biosynthesis has been intensively investigated, genomic data and terpene synthase functions have been reported (Sun <i>et al</i>., <span>2019</span>). However, the enzymes responsible for structural modification that form the key pharmacologically active groups in its biosynthetic pathway remain unknown.</p>\u0000<p>The modification steps in andrographolide biosynthesis include hydroxylations at C3, C14, C18 and lactone ring formation at C15–C16. This series of oxidation processes were supposed to be mediated by cytochrome P450 enzymes (CYP450s). In order to accurately screen the CYP450s in andrographolide biosynthesis pathway, we constructed the differential bio-accumulation samples of andrographolide seedlings (Figure S1). After 100 μM MeJA treatment, the production of andrographolide demonstrated significant enhancement at 24 h post-inoculation (hpi) and reached 37.8 mg/g DW at 72 hpi in the leaves, which is approximately 10 times greater than that in the control (Figure 1a). We then constructed the expression atlas and investigated the time-series expression changes of <i>A. paniculata</i>. The expression profiles of samples at 12 hpi, 24 hpi and 48 hpi exhibited significantly different patterns compared to the samples collected at 0 hpi (Figure S2). By applying a cutoff of a four-folds difference in FPKM and a false discovery rate of less than 0.05, we identified that the expression levels of 4463 genes were up-regulated at 12 hpi, 24 hpi or 48 hpi in comparison to the control samples (Figure 1b).</p>\u0000<figure><picture>\u0000<source media=\"(min-width: 1650px)\" srcset=\"/cms/asset/3b09830f-074d-4137-b9dc-665ca6ab4fee/pbi14572-fig-0001-m.jpg\"/><img alt=\"Details are in the caption following the image\" data-lg-src=\"/cms/asset/3b09830f-074d-4137-b9dc-665ca6ab4fee/pbi14572-fig-0001-m.jpg\" loading=\"lazy\" src=\"/cms/asset/8c424ac0-7049-492b-92fa-d222a99632a4/pbi14572-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></s","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"31 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-01-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142981900","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>Exclusion of Na<sup>+</sup> from the above-ground tissues serves as an important salt-tolerant mechanism in most glycophyte plants, such as maize (Munns and Tester, <span>2008</span>). Existing studies have corroborated that different maize varieties exhibit significant diversity in shoot Na<sup>+</sup> content and then salt tolerance (Liang <i>et al</i>., <span>2024</span>). However, the genetic basis underlying this diversity remains largely unknown, necessitating a comprehensive understanding to sustain breeding for salt-tolerant maize cultivars.</p> <p>In recent decades, numerous genes have been identified to regulate Na<sup>+</sup> transport. The well-known include genes from the <i>NHX</i>, <i>HKT</i>, <i>HAK</i>, <i>CBL</i>, and <i>CIPK</i> gene families (Yang and Guo, <span>2018</span>). Maize has 13 <i>NHX</i>, 3 <i>HKT</i>, 28 <i>HAK</i>, 11 <i>CBL</i>, and 45 <i>CIPK</i> family members (Table S1), which exhibit a varied expression pattern and responses to salt treatment (Method S1; Figure 1a; Figure S1). Given that several genes within these families have been shown to underlie the diversity of shoot Na<sup>+</sup> content and then salt tolerance in maize (Liang <i>et al</i>., <span>2024</span>), we hypothesized that functional variation of additional members of these gene families may also do so. To substantiate this speculation, we obtained 14–623 SNP variants for each of these genes from the genotype data of a population comprised of 508 maize inbred lines (Zhang <i>et al</i>., <span>2019</span>) (Table S1), then analyse the association between these SNP variants and Na<sup>+</sup> content in the shoot tissue of salt-grown seedlings (Method S2). The result indicated that the peak SNP in 17 of these genes respectively explained >1% diversity of the shoot Na<sup>+</sup> content (Figure 1b). Notably, 12 out of these 17 cases explained <2% diversity of the shoot Na<sup>+</sup> content, supporting the notion that various minor-effect variants result in the diversity in the shoot Na<sup>+</sup> content and salt tolerance in maize.</p> <figure><picture> <source media="(min-width: 1650px)" srcset="/cms/asset/8e4bbbc2-3ff7-4170-9019-fbe04fae8566/pbi14553-fig-0001-m.jpg"/><img alt="Details are in the caption following the image" data-lg-src="/cms/asset/8e4bbbc2-3ff7-4170-9019-fbe04fae8566/pbi14553-fig-0001-m.jpg" loading="lazy" src="/cms/asset/c93d960c-5a9f-4f6f-90f3-868b9035436f/pbi14553-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>Various minor-effect variants including an SNP located in the promoter of <i>ZmHAK11</i> underlie the variation in shoot Na<sup>+</sup> content in maize. (a) The transcript levels of the indicated <i>ZmHAK</i>, <i>ZmHKT</i>, <i>ZmNHX</i>, <i>ZmCBL</i>, and <i>ZmCIPK</i> genes under control and s
在大多数糖叶植物（如玉米）中，从地上组织中排除Na+是一种重要的耐盐机制（Munns和Tester， 2008）。已有研究证实，不同玉米品种在茎部Na+含量和耐盐性方面存在显著差异（Liang et al., 2024）。然而，这种多样性的遗传基础在很大程度上仍然是未知的，需要一个全面的了解，以维持耐盐玉米品种的育种。近几十年来，已经发现了许多调节Na+转运的基因。众所周知的基因包括NHX、HKT、HAK、CBL和CIPK基因家族（Yang and Guo, 2018）。玉米有13个NHX家族成员、3个HKT家族成员、28个HAK家族成员、11个CBL家族成员和45个CIPK家族成员（表S1），这些家族成员表现出不同的表达模式和对盐处理的响应(方法S1；图1;图S1)。鉴于这些家族中的几个基因已被证明是玉米茎部Na+含量和耐盐性多样性的基础（Liang et al., 2024），我们假设这些基因家族中其他成员的功能变异也可能起作用。为了证实这一推测，我们从一个由508个玉米自交系组成的群体的基因型数据中获得了这些基因中每个基因的14-623个SNP变异（Zhang et al., 2019）（表S1），然后分析了这些SNP变异与盐苗茎部组织中Na+含量之间的关系（方法S2）。结果表明，其中17个基因的SNP峰值分别解释了茎部Na+含量的1%多样性（图1b）。值得注意的是，这17个案例中有12个解释了茎部Na+含量2%的多样性，这支持了各种小效应变异导致玉米茎部Na+含量和耐盐性多样性的观点。包括位于ZmHAK11启动子中的SNP在内的各种次要变异是玉米茎部Na+含量变化的基础。(a)对照和盐条件下ZmHAK、ZmHKT、ZmNHX、ZmCBL和ZmCIPK基因的转录水平。(b)指示基因内SNP峰对茎部Na+含量多样性的贡献。（c - g）不同基因型和处理2周龄植株的外形(c)、茎部生物量(d)、茎部Na+含量(e)、根系Na+含量(f)和木质部汁液Na+含量(g)。（h, i）用指定质粒转化的酵母细胞在提供指定浓度NaCl (h)或KCl (i)的培养基上生长的情况。（j - m）指定酵母细胞对Na+ (j, k)和k + (l, m)的吸收能力。(n) ZmHAK11在HapA和HapG自交系中的转录水平。(o)烟草叶片中含有指示启动子的绿色荧光蛋白的转录水平。(p) 213个玉米自交系ZmHAK11变异与茎部Na+含量的关系。黄点突出了显著变异之间的中度LD。(q)根据三个显著变异体对ZmHAK11的单倍型进行分类。（r, s）单倍型自交系的茎部Na+含量和ZmHAK11转录物水平。(t)携带ZmHAK11Zheng58 （SNP-1781G）和ZmHAK11Yu82 （SNP-1781C）等位基因的F2植株茎部Na+含量。采用双侧t检验或单因素方差分析确定统计学显著性。ZmHAK4、ZmHKT1;1、ZmHKT1；2和ZmHAK11区域内的峰值snp贡献最大，分别解释了茎部Na+含量变化的7.5%、4.4%、4.0%和2.4%（表S1）。考虑到ZmHAK4、ZmHKT1；1和ZmHKT1；2在以往的研究中已经被研究过（Liang et al., 2024），我们在本研究中确定了HAK11的耐盐作用和功能变异（图1b）。首先，我们创建了两个独立的敲除突变体，hak11-1和hak11-2(方法S3；图S2)。虽然野生型和hak11在对照条件下没有表现出表型差异（图1c），但在盐条件下，突变体的茎部生物量比野生型对照小20%左右（图1d）。同时，与野生型相比，盐条件下hak11突变体的茎部和木质部汁液Na+浓度较高，根部Na+浓度较低（图1e-g）。这些结果表明，ZmHAK11通过阻止Na+在根与茎间的转运，促进了植株对Na+的排斥和耐盐性。与这一观点一致，我们观察到HAK11不太可能影响根组织中Na+的摄取或外排（图S3）以及Na+从茎部到根的转运（图S4）。HAK家族转运蛋白被分为4个簇（簇I-IV）， ZmHAK11属于簇III（图S5）。已有报道表明，集群I成员（ZmHAK5和ZmHAK1）和集群IV成员（ZmHAK4和ZmHAK17）是K+和Na+选择性转运体，分别介导对K+缺乏和盐胁迫的响应（Qin等）。
{"title":"The natural variation in shoot Na+ content and salt tolerance in maize is attributed to various minor-effect variants, including an SNP located in the promoter of ZmHAK11","authors":"Xiaoyan Liang, Limin Wang, Yin Pan, Wenqi Jing, Heyang Wang, Fang Liu, Caifu Jiang","doi":"10.1111/pbi.14553","DOIUrl":"https://doi.org/10.1111/pbi.14553","url":null,"abstract":"<p>Exclusion of Na<sup>+</sup> from the above-ground tissues serves as an important salt-tolerant mechanism in most glycophyte plants, such as maize (Munns and Tester, <span>2008</span>). Existing studies have corroborated that different maize varieties exhibit significant diversity in shoot Na<sup>+</sup> content and then salt tolerance (Liang <i>et al</i>., <span>2024</span>). However, the genetic basis underlying this diversity remains largely unknown, necessitating a comprehensive understanding to sustain breeding for salt-tolerant maize cultivars.</p>\u0000<p>In recent decades, numerous genes have been identified to regulate Na<sup>+</sup> transport. The well-known include genes from the <i>NHX</i>, <i>HKT</i>, <i>HAK</i>, <i>CBL</i>, and <i>CIPK</i> gene families (Yang and Guo, <span>2018</span>). Maize has 13 <i>NHX</i>, 3 <i>HKT</i>, 28 <i>HAK</i>, 11 <i>CBL</i>, and 45 <i>CIPK</i> family members (Table S1), which exhibit a varied expression pattern and responses to salt treatment (Method S1; Figure 1a; Figure S1). Given that several genes within these families have been shown to underlie the diversity of shoot Na<sup>+</sup> content and then salt tolerance in maize (Liang <i>et al</i>., <span>2024</span>), we hypothesized that functional variation of additional members of these gene families may also do so. To substantiate this speculation, we obtained 14–623 SNP variants for each of these genes from the genotype data of a population comprised of 508 maize inbred lines (Zhang <i>et al</i>., <span>2019</span>) (Table S1), then analyse the association between these SNP variants and Na<sup>+</sup> content in the shoot tissue of salt-grown seedlings (Method S2). The result indicated that the peak SNP in 17 of these genes respectively explained >1% diversity of the shoot Na<sup>+</sup> content (Figure 1b). Notably, 12 out of these 17 cases explained <2% diversity of the shoot Na<sup>+</sup> content, supporting the notion that various minor-effect variants result in the diversity in the shoot Na<sup>+</sup> content and salt tolerance in maize.</p>\u0000<figure><picture>\u0000<source media=\"(min-width: 1650px)\" srcset=\"/cms/asset/8e4bbbc2-3ff7-4170-9019-fbe04fae8566/pbi14553-fig-0001-m.jpg\"/><img alt=\"Details are in the caption following the image\" data-lg-src=\"/cms/asset/8e4bbbc2-3ff7-4170-9019-fbe04fae8566/pbi14553-fig-0001-m.jpg\" loading=\"lazy\" src=\"/cms/asset/c93d960c-5a9f-4f6f-90f3-868b9035436f/pbi14553-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>Various minor-effect variants including an SNP located in the promoter of <i>ZmHAK11</i> underlie the variation in shoot Na<sup>+</sup> content in maize. (a) The transcript levels of the indicated <i>ZmHAK</i>, <i>ZmHKT</i>, <i>ZmNHX</i>, <i>ZmCBL</i>, and <i>ZmCIPK</i> genes under control and s","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"2 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-01-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142981901","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}
京公网安备 11010802042870号
京ICP备2023020795号-1
扫码关注我们
求助内容：
应助结果提醒方式：