Qi Zou, Tiantian Bao, Lei Yu, Haifeng Xu, Wenjun Liu, Zhiqiang Li, Yansong Zhu, Ran Chen, Xukai Hou, Zongying Zhang, Nan Wang, Xuesen Chen
SummaryApple (Malus domestica Borkh.) is one of the most economically valuable fruit crops globally and a key dietary source for various nutrients. However, the levels of ascorbic acid (AsA) and anthocyanin, essential micronutrients for human health, are extremely low in the pulp of commonly cultivated apple varieties. In the present study, the second‐generation hybrid strain of Xinjiang red‐fleshed apple (‘Zihong No. 1’ × ‘Gala’) was used as the test material. The results revealed that AsA content was significantly higher in red‐fleshed apple pulp than in non‐red‐fleshed varieties, and the expression of MdGLDH, a key gene in the D‐mannose/L‐galactose pathway, correlated strongly with AsA levels. Using the promoter of MdGLDH as bait, an R3‐type MYB transcription factor (TF), MdCPC‐like, was identified through yeast one‐hybrid screening. Further analysis revealed that the overexpression of MdCPCL increased the AsA and anthocyanin levels in both callus and fruits, whereas MdCPCL knockdown led to a reduction in their levels. Moreover, the interaction between MdCPCL and the bHLH TF MdILR3‐like was confirmed, forming the MdCPCL‐MdILR3L complex. This complex significantly enhanced the transcription of downstream target genes MdGLDH and MdANS, promoting the synthesis of AsA and anthocyanins. This study contributes to further enrich the anabolic pathways of AsA and anthocyanin in apples and provides a theoretical foundation for the quality breeding of red‐fleshed apple varieties.
{"title":"The regulatory module MdCPCL‐MdILR3L mediates the synthesis of ascorbic acid and anthocyanin in apple","authors":"Qi Zou, Tiantian Bao, Lei Yu, Haifeng Xu, Wenjun Liu, Zhiqiang Li, Yansong Zhu, Ran Chen, Xukai Hou, Zongying Zhang, Nan Wang, Xuesen Chen","doi":"10.1111/pbi.14567","DOIUrl":"https://doi.org/10.1111/pbi.14567","url":null,"abstract":"SummaryApple (<jats:italic>Malus domestica</jats:italic> Borkh.) is one of the most economically valuable fruit crops globally and a key dietary source for various nutrients. However, the levels of ascorbic acid (AsA) and anthocyanin, essential micronutrients for human health, are extremely low in the pulp of commonly cultivated apple varieties. In the present study, the second‐generation hybrid strain of Xinjiang red‐fleshed apple (‘Zihong No. 1’ × ‘Gala’) was used as the test material. The results revealed that AsA content was significantly higher in red‐fleshed apple pulp than in non‐red‐fleshed varieties, and the expression of <jats:italic>MdGLDH</jats:italic>, a key gene in the D‐mannose/L‐galactose pathway, correlated strongly with AsA levels. Using the promoter of <jats:italic>MdGLDH</jats:italic> as bait, an R3‐type MYB transcription factor (TF), MdCPC‐like, was identified through yeast one‐hybrid screening. Further analysis revealed that the overexpression of <jats:italic>MdCPCL</jats:italic> increased the AsA and anthocyanin levels in both callus and fruits, whereas <jats:italic>MdCPCL</jats:italic> knockdown led to a reduction in their levels. Moreover, the interaction between MdCPCL and the bHLH TF MdILR3‐like was confirmed, forming the MdCPCL‐MdILR3L complex. This complex significantly enhanced the transcription of downstream target genes <jats:italic>MdGLDH</jats:italic> and <jats:italic>MdANS</jats:italic>, promoting the synthesis of AsA and anthocyanins. This study contributes to further enrich the anabolic pathways of AsA and anthocyanin in apples and provides a theoretical foundation for the quality breeding of red‐fleshed apple varieties.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"78 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142936233","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}
Qi Wang, Min Wang, Ai‐Ai Xia, Jin‐Yu Wang, Zi Wang, Tao Xu, De‐Tao Jia, Ming Lu, Wei‐Ming Tan, Jin‐Hong Luo, Yan He
SummaryThe husk leaf of maize (Zea mays) encases the ear as a modified leaf and plays pivotal roles in protecting the ear from pathogen infection, translocating nutrition for grains and warranting grain yield. However, the natural genetic basis for variation in husk leaf width remains largely unexplored. Here, we performed a genome‐wide association study for maize husk leaf width and identified a 3‐bp InDel (insertion/deletion) in the coding region of the nitrate transporter gene ZmNRT2.5. This polymorphism altered the interaction strength of ZmNRT2.5 with another transporter, ZmNPF5, thereby contributing to variation in husk leaf width. We also isolated loss‐of‐function mutants in ZmNRT2.5, which exhibited a substantial decrease in husk leaf width relative to their controls. We demonstrate that ZmNRT2.5 facilitates the transport of nitrate from husk leaves to maize kernels in plants grown under low‐nitrogen conditions, contributing to the accumulation of proteins in maize seeds. Together, our findings uncovered a key gene controlling maize husk leaf width and nitrate transport from husk leaves to kernels. Identification of the ZmNRT2.5 loci offers direct targets for improving the protein content of maize seeds via molecular‐assisted maize breeding.
{"title":"Natural variation in ZmNRT2.5 modulates husk leaf width and promotes seed protein content in maize","authors":"Qi Wang, Min Wang, Ai‐Ai Xia, Jin‐Yu Wang, Zi Wang, Tao Xu, De‐Tao Jia, Ming Lu, Wei‐Ming Tan, Jin‐Hong Luo, Yan He","doi":"10.1111/pbi.14559","DOIUrl":"https://doi.org/10.1111/pbi.14559","url":null,"abstract":"SummaryThe husk leaf of maize (<jats:italic>Zea mays</jats:italic>) encases the ear as a modified leaf and plays pivotal roles in protecting the ear from pathogen infection, translocating nutrition for grains and warranting grain yield. However, the natural genetic basis for variation in husk leaf width remains largely unexplored. Here, we performed a genome‐wide association study for maize husk leaf width and identified a 3‐bp InDel (insertion/deletion) in the coding region of the nitrate transporter gene <jats:italic>ZmNRT2.5</jats:italic>. This polymorphism altered the interaction strength of ZmNRT2.5 with another transporter, ZmNPF5, thereby contributing to variation in husk leaf width. We also isolated loss‐of‐function mutants in <jats:italic>ZmNRT2.5</jats:italic>, which exhibited a substantial decrease in husk leaf width relative to their controls. We demonstrate that ZmNRT2.5 facilitates the transport of nitrate from husk leaves to maize kernels in plants grown under low‐nitrogen conditions, contributing to the accumulation of proteins in maize seeds. Together, our findings uncovered a key gene controlling maize husk leaf width and nitrate transport from husk leaves to kernels. Identification of the <jats:italic>ZmNRT2.5</jats:italic> loci offers direct targets for improving the protein content of maize seeds via molecular‐assisted maize breeding.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"42 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142928991","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>Grain proteins constitute the second most storage substance in rice, of which glutelin accounts for 60%–80% of total protein and is easy to be absorbed by humans (Kumamaru <i>et al</i>., <span>1988</span>). However, for patients with kidney disease and diabetes, excessive glutelin intake is not conducive to recovery. The <i>lgc-1</i> mutant is the earliest discovered low-glutelin material, and <i>Lgc-1</i> regulates glutelin content in rice grains (Iida <i>et al</i>., <span>1993</span>; Kusaba <i>et al</i>., <span>2003</span>), which makes this allele have more extensive application prospects in the cultivation of low-glutelin varieties. Rice eating quality (REQ) is influenced by protein content and composition. Studies have shown that with the increase in protein content, the REQ decreases and the palatability becomes worse (Huang <i>et al</i>., <span>2020</span>). Exogenous glutelin and prolamin could affect REQ, whereas knockout of glutelin-related genes could significantly improve the hardness, appearance and REQ. Therefore, the effect of glutelin on REQ may be greater than prolamin and total protein (Furukawa <i>et al</i>., <span>2006</span>; Huang <i>et al</i>., <span>1998</span>; Yang <i>et al</i>., <span>2022</span>). Furthermore, it was found that the expression of genes related to glutelin synthesis had an important effect on protein content and REQ. As the expression of <i>Nhd1</i> increased, the expression of <i>GluA2</i> was inhibited, resulting in the decrease of glutelin content and protein content, thus improving REQ (Zhang <i>et al</i>., <span>2023</span>). These studies indicated that glutelin can significantly affect REQ, however, fewer studies have been done on japonica hybrid rice with high eating quality and low glutelin.</p><p>Combining with molecular marker and phenotypic screening, three low-glutelin restorer lines, HL8005, HL8023 and HL8027, were screened by crossing two varieties L9037 and R228 (Figure 1a). L9037 is a low-glutelin variety with the genotype <i>Lgc-1</i> (without restoration gene), and R228 is a wide compatibility restorer line (without genotype <i>Lgc-1</i>). The amplified bands of HL8005, HL8023 and HL8027 were consistent with L9037 by molecular markers (Figure 1c; Figure S1). Compared with L9037, the number of grains per panicle decreased by 4.0% for HL8005, increased by 11.8% and 32.2% for HL8023 and HL8027, respectively, and the 1000-grain weight increased by 4.1% and 9.2% for HL8005 and HL8023, respectively, while the 1000-grain weight of HL8027 decreased by 4.6%, and the seed setting rate were all above 75% (Figure 1b). The heading time of three restorer lines was significantly shorter than L9037, and the single plant yield and population yield of three restorer lines were significantly higher than L9037 (Figure S2). The glutelin content of HL8005, HL8023 and HL8027 were significantly lower than R228, but higher than L9037 (Figure 1d). SDS-PAGE of storage profiles showed that the protein composi
谷物蛋白质是水稻中第二大储藏物质，其中谷蛋白占总蛋白质的60%-80%，且易被人体吸收（Kumamaru et al., 1988）。但对于肾病和糖尿病患者，摄入过多的谷蛋白不利于康复。lgc-1突变体是最早发现的低谷蛋白物质，lgc-1调节稻谷蛋白含量(Iida et al., 1993；Kusaba et al., 2003)，这使得该等位基因在低谷蛋白品种的栽培中具有更广泛的应用前景。大米食味品质受蛋白质含量和组成的影响。研究表明，随着蛋白质含量的增加，REQ降低，适口性变差（Huang et al., 2020）。外源谷蛋白和蛋白可影响REQ，敲除谷蛋白相关基因可显著改善硬度、外观和REQ。因此，谷蛋白对REQ的影响可能大于原蛋白和总蛋白(Furukawa et al., 2006；Huang et al., 1998；Yang et al., 2022)。此外，研究还发现，谷氨酸合成相关基因的表达对蛋白质含量和REQ有重要影响。随着Nhd1表达的增加，GluA2的表达受到抑制，导致谷蛋白含量和蛋白含量降低，从而改善REQ （Zhang et al., 2023）。这些研究表明，谷蛋白可显著影响REQ，但对高食性低谷蛋白杂交粳稻的研究较少。结合分子标记和表型筛选，通过L9037和R228两个品种杂交，筛选出3个低谷蛋白恢复系HL8005、HL8023和HL8027（图1a）。L9037是低谷蛋白品种，基因型为Lgc-1（不含恢复基因），R228是广相容性恢复系（不含Lgc-1基因）。HL8005、HL8023和HL8027的扩增条带与L9037的分子标记一致(图1c；图S1)。与L9037相比，HL8005的每穗粒数减少4.0%，HL8023和HL8027的每穗粒数分别增加11.8%和32.2%，HL8005和HL8023的千粒重分别增加4.1%和9.2%，而HL8027的千粒重减少4.6%，结实率均在75%以上（图1b）。3个恢复系抽穗时间均显著短于L9037，单株产量和群体产量均显著高于L9037（图S2）。HL8005、HL8023和HL8027的谷蛋白含量显著低于R228，但高于L9037（图1d）。存储谱SDS-PAGE显示，HL8005、HL8023和HL8027的蛋白质组成与L9037一致，但谷蛋白前体、酸性和碱性亚基少于R228（图1e）。HL8005、HL8023和HL8027的稻粒比L9037长，稻粒宽度和厚度均大于R228，二者的长宽比介于两者之间（图S3a-e）。理化性质分析结果表明，R228、HL8005和HL8027淀粉粉在4 M尿素中开始溶解，溶解度无差异，而L9037和HL8023淀粉粉在5 M尿素中开始溶解。HL8023淀粉粉的溶解度在3个恢复系中最高，高于L9037（图S3f）。HL8005、HL8023和HL8027的直链淀粉含量显著高于两个亲本（图S3g）。HL8005、HL8023和HL8027的起始、峰值和末胶化温度均显著高于L9037，低于R228；HL8005和HL8027的焓显著高于L9037，而HL8023的焓与L9037相似（图S3h）。REQ分析显示，HL8027的REQ优于HL8005和HL8023（图3i - k）。结果表明，含有Lgc-1的3个低谷蛋白恢复系HL8005、HL8023和HL8027的农艺性状优于L9037。3个恢复系中，HL8027的REQ最高，谷蛋白含量为3.0%，比非低谷蛋白品种R228低38.8%。利用Lgc-1等位基因选育低谷蛋白高食性恢复系和三系杂交粳稻。(a)低谷蛋白恢复系的选育过程。(b)低谷蛋白恢复系植株表型。比例尺= 10厘米。(c) LGC-1基因的PCR检测。(d)谷蛋白含量分析。(e)储存蛋白的SDS-PAGE图谱。(f)低谷蛋白杂交组合的植株表型。比例尺= 10厘米。(g)低谷蛋白杂交组合的产量相关农艺性状。（h, i）低谷蛋白杂交组合的谷蛋白含量(h)和直链淀粉含量(i)分析。(j)低谷蛋白恢复系和杂交组合的熟米外观。 (k)低谷蛋白杂交组合鲜熟米和退熟米的质构分析。(1)低谷蛋白杂交组合稻米口感和食味品质综合评价。数值为平均值±SD （n = 3, *P < 0.05, **P < 0.01，双尾Student's t检验，三个独立实验）。3个低谷蛋白恢复系与粳稻不育系春江23a （A1）、81A （A2）和嘉禾212a （A3）杂交得到9个杂交稻组合，以非低谷蛋白粳稻杂交系嘉优5号为对照（CK）（图1f）。3个不育系的理化品质完全不同，糊化温度和A1直链淀粉含量最高，3个不育系的麸质含量均在4.5%以上（图S4）。C1、C2和C3组合的结实率和千粒重均显著低于CK。C2、C4、C5和C6的单株产量和群体产量显著高于CK(图1g；图S5)。所有组合的谷蛋白含量均显著低于CK，其中C3含量最低（图1h）。C2、C6和C9的直链淀粉含量显著高于CK， C4和C8的直链淀粉含量显著低于CK（图1i）。所有组合的起始、峰值和末端糊化温度均显著高于CK，只有C2的焓低于CK，其他组合的焓均显著高于CK或与CK无差异（图S6a）。C7开始溶解在4m尿素中，C9开始溶解在5m尿素中。在所有组合中，C7的溶解度最差，C9的溶解度最好（图S6b）。除C3、C7和C9外，其余组合的每穗粒数均高于对照。与CK相比，C6、C8和C9的鲜米颜色较亮，而C2、C3、C4和C5的颜色较深，光泽度较低（图1j）。对鲜米和退米的质构分析表明，各组合间的质构参数存在显著差异，硬度和黏附性对REQ影响较大，硬度低、黏附性高的大米食性较好（Li et al., 2016）。C3、C4和C5鲜米的硬度均显著大于CK， C3的黏附性显著高于CK， C7和C9的黏附性显著低于CK。各组合的退煮米饭的质地特征均不如鲜煮米饭。通过对鲜米和退米的质构参数对比分析，C6和C8的质构性能更好（图1k）。味觉和外观综合评分显示，C6得分高于CK，而C2、C3、C7和C9得分显著低于CK（图11）。研究表明，直链淀粉含量、糊化温度和粘附性是评价REQ最广泛接受的指标（Wang et al., 2024）。综合质地分析、口感评价等相关指标，认为C6 （81A/HL8027）的REQ值在9个组合中最佳。综上所述，我们选择了3个低谷蛋白恢复系，发现HL8027具有更好的REQ。随后，将3个恢复系与3个不育系杂交，产生9个组合。其中，81A/HL8027不仅具有低谷蛋白特性，而且REQ最佳，单株产量和群体产量优势明显。研究结果为选育优质低谷蛋白高产杂交稻提供了新的种质资源。
{"title":"Production of grains with low glutelin and high eating quality by using dominant allele Lgc-1 in three-line japonica hybrid rice","authors":"Shikai Hu, Lingwei Yang, Jinyang Cai, Guiai Jiao, Hailong Yang, Suozhen Hui, Liang Zhou, Ruijie Cao, Jingxin Wang, Yujuan Chen, Junchao Fang, Zhonghua Sheng, Shaoqing Tang, Peisong Hu","doi":"10.1111/pbi.14502","DOIUrl":"10.1111/pbi.14502","url":null,"abstract":"<p>Grain proteins constitute the second most storage substance in rice, of which glutelin accounts for 60%–80% of total protein and is easy to be absorbed by humans (Kumamaru <i>et al</i>., <span>1988</span>). However, for patients with kidney disease and diabetes, excessive glutelin intake is not conducive to recovery. The <i>lgc-1</i> mutant is the earliest discovered low-glutelin material, and <i>Lgc-1</i> regulates glutelin content in rice grains (Iida <i>et al</i>., <span>1993</span>; Kusaba <i>et al</i>., <span>2003</span>), which makes this allele have more extensive application prospects in the cultivation of low-glutelin varieties. Rice eating quality (REQ) is influenced by protein content and composition. Studies have shown that with the increase in protein content, the REQ decreases and the palatability becomes worse (Huang <i>et al</i>., <span>2020</span>). Exogenous glutelin and prolamin could affect REQ, whereas knockout of glutelin-related genes could significantly improve the hardness, appearance and REQ. Therefore, the effect of glutelin on REQ may be greater than prolamin and total protein (Furukawa <i>et al</i>., <span>2006</span>; Huang <i>et al</i>., <span>1998</span>; Yang <i>et al</i>., <span>2022</span>). Furthermore, it was found that the expression of genes related to glutelin synthesis had an important effect on protein content and REQ. As the expression of <i>Nhd1</i> increased, the expression of <i>GluA2</i> was inhibited, resulting in the decrease of glutelin content and protein content, thus improving REQ (Zhang <i>et al</i>., <span>2023</span>). These studies indicated that glutelin can significantly affect REQ, however, fewer studies have been done on japonica hybrid rice with high eating quality and low glutelin.</p><p>Combining with molecular marker and phenotypic screening, three low-glutelin restorer lines, HL8005, HL8023 and HL8027, were screened by crossing two varieties L9037 and R228 (Figure 1a). L9037 is a low-glutelin variety with the genotype <i>Lgc-1</i> (without restoration gene), and R228 is a wide compatibility restorer line (without genotype <i>Lgc-1</i>). The amplified bands of HL8005, HL8023 and HL8027 were consistent with L9037 by molecular markers (Figure 1c; Figure S1). Compared with L9037, the number of grains per panicle decreased by 4.0% for HL8005, increased by 11.8% and 32.2% for HL8023 and HL8027, respectively, and the 1000-grain weight increased by 4.1% and 9.2% for HL8005 and HL8023, respectively, while the 1000-grain weight of HL8027 decreased by 4.6%, and the seed setting rate were all above 75% (Figure 1b). The heading time of three restorer lines was significantly shorter than L9037, and the single plant yield and population yield of three restorer lines were significantly higher than L9037 (Figure S2). The glutelin content of HL8005, HL8023 and HL8027 were significantly lower than R228, but higher than L9037 (Figure 1d). SDS-PAGE of storage profiles showed that the protein composi","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"23 2","pages":"374-376"},"PeriodicalIF":10.1,"publicationDate":"2025-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/pbi.14502","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142929481","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Eef M. Jonkheer, Dick de Ridder, Theo A. J. van der Lee, Jorn R. de Haan, Lidija Berke, Sandra Smit
With advances in long-read sequencing and assembly techniques, haplotype-resolved (phased) genome assemblies are becoming more common, also in the field of plant genomics. Computational tools to effectively explore these phased genomes, particularly for polyploid genomes, are currently limited. Here we describe a new strategy adopting a pangenome approach. To analyse both intra- and intergenomic variation in phased genome assemblies, we have made the software package PanTools ploidy-aware by updating the pangenome graph representation and adding several novel functionalities to assess synteny and gene retention, profile repeats and calculate synonymous and nonsynonymous mutation rates. Using PanTools, we constructed and analysed a pangenome comprising of one diploid and four tetraploid potato cultivars, and a pangenome of five diploid apple species. Both pangenomes show high intra- and intergenomic allelic diversity in terms of gene absence/presence, SNPs, indels and larger structural variants. Our findings show that the new functionalities and visualizations are useful to discover introgressions and detect likely misassemblies in phased genomes. PanTools is available at https://git.wur.nl/bioinformatics/pantools.
{"title":"Exploring intra- and intergenomic variation in haplotype-resolved pangenomes","authors":"Eef M. Jonkheer, Dick de Ridder, Theo A. J. van der Lee, Jorn R. de Haan, Lidija Berke, Sandra Smit","doi":"10.1111/pbi.14545","DOIUrl":"https://doi.org/10.1111/pbi.14545","url":null,"abstract":"With advances in long-read sequencing and assembly techniques, haplotype-resolved (phased) genome assemblies are becoming more common, also in the field of plant genomics. Computational tools to effectively explore these phased genomes, particularly for polyploid genomes, are currently limited. Here we describe a new strategy adopting a pangenome approach. To analyse both intra- and intergenomic variation in phased genome assemblies, we have made the software package PanTools ploidy-aware by updating the pangenome graph representation and adding several novel functionalities to assess synteny and gene retention, profile repeats and calculate synonymous and nonsynonymous mutation rates. Using PanTools, we constructed and analysed a pangenome comprising of one diploid and four tetraploid potato cultivars, and a pangenome of five diploid apple species. Both pangenomes show high intra- and intergenomic allelic diversity in terms of gene absence/presence, SNPs, indels and larger structural variants. Our findings show that the new functionalities and visualizations are useful to discover introgressions and detect likely misassemblies in phased genomes. PanTools is available at https://git.wur.nl/bioinformatics/pantools.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"3 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142929482","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}
Kristen Van Gelder, Anuran K. Gayen, Andrew D. Hanson
<p>Continuous directed evolution (CDE) improves the characteristics of a target enzyme by hypermutating the enzyme gene <i>in vivo</i>, coupling enzyme activity to growth of a microbial platform, and selecting for growth rate (Molina <i>et al</i>., <span>2022</span>). Directed evolution can be interfaced with genome editing to expand the gene pool available for plant breeding; this powerful combination (DE–GE) has been neatly termed ‘a Green (r)Evolution’ (Gionfriddo <i>et al</i>., <span>2019</span>). THI4 enzymes, which make the thiazole moiety of thiamin, are good testbed targets for plant CDE technology. Plant THI4s are energy-inefficient suicide enzymes that could potentially be replaced by efficient, non-suicide bacterial THI4s to increase biomass yield by as much as 4% (Joshi <i>et al</i>., <span>2021</span>). However, bacterial THI4s are O<sub>2</sub>-sensitive and otherwise ill-adapted to plants (Joshi <i>et al</i>., <span>2021</span>). We therefore previously ran CDE campaigns in the yeast OrthoRep system to ‘plantize’ bacterial THI4s, that is, to improve function in an aerobic, plant-like milieu (Figure 1a) (García-García <i>et al</i>., <span>2022</span>). Two notably successful campaigns were for the THI4 from <i>Mucinivorans hirudinis</i> (MhTHI4); these campaigns culminated when populations acquired single V124A or Y122C mutations that improved growth to near the wild-type rate (Van Gelder <i>et al</i>., <span>2023</span>). Such culmination can be overcome by increasing the selection pressure (Molina <i>et al</i>., <span>2022</span>).</p> <figure><picture> <source media="(min-width: 1650px)" srcset="/cms/asset/5d1a35f3-b3df-4cfa-bd4e-d6fe7c98b2d5/pbi14563-fig-0001-m.jpg"/><img alt="Details are in the caption following the image" data-lg-src="/cms/asset/5d1a35f3-b3df-4cfa-bd4e-d6fe7c98b2d5/pbi14563-fig-0001-m.jpg" loading="lazy" src="/cms/asset/bd12af5d-8049-40c0-b660-06ab303db600/pbi14563-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>OrthoRep campaigns to plantize a bacterial THI4 and their outcomes. (a) The OrthoRep system. The target enzyme (MhTHI4 V124A), plus or minus a poly(A) tail, is encoded on the cytoplasmic p1 plasmid that also carries a LEU2 marker. p1 is hypermutated by a p1-specific, error-prone DNA polymerase (TP-DNAP1_611 or TP-DNAP1_633) encoded on a nuclear plasmid. The BY4741 platform strain carries a <i>thi4</i>Δ deletion to couple growth to the activity of the THI4 on p1. (b) The combinations of expression-reduction regimes with cold turkey (CT) or gradual (G) selection. (c) The non-synonymous (blue) and synonymous (green) mutations that had swept populations by the end of campaigns. When populations failed to grow early in campaigns, surviving populations were split into subpopulations (A, B, C) and prop
连续定向进化（Continuous directed evolution， CDE）通过在体内对酶基因进行超突变，将酶活性与微生物平台的生长耦合，并选择生长速度来改善目标酶的特性（Molina et al., 2022）。定向进化可以与基因组编辑相结合，以扩大可用于植物育种的基因库；这种强大的组合（DE-GE）被整齐地称为“绿色(r)进化”（Gionfriddo等人，2019）。合成硫胺素中噻唑基团的THI4酶是植物CDE技术的良好靶点。植物THI4s是能源效率低下的自杀酶，可能被高效的非自杀性细菌THI4s所取代，从而将生物质产量提高4% （Joshi等人，2021）。然而，细菌THI4s对o2敏感，在其他方面不适应植物（Joshi等，2021）。因此，我们之前在酵母OrthoRep系统中进行了CDE活动，以“培养”细菌THI4s，即改善有氧植物样环境中的功能（图1a） （García-García等人，2022）。两个显著成功的运动是针对来自水蛭杆菌（Mucinivorans hiudinis）的th4 (MhTHI4)；当种群获得单个V124A或Y122C突变时，这些运动达到高潮，这些突变将生长速度提高到接近野生型的速度（Van Gelder等人，2023）。这种高潮可以通过增加选择压力来克服（Molina et al., 2022）。图1在图形查看器中打开powerpointtorthorep活动来规划细菌THI4及其结果。(a) OrthoRep制度。靶酶（MhTHI4 V124A），加上或减少poly(a)尾巴，编码在细胞质p1质粒上，该质粒也携带一个LEU2标记。p1被编码在核质粒上的p1特异性、易出错的DNA聚合酶（TP-DNAP1_611或TP-DNAP1_633）超突变。BY4741平台菌株携带thi4Δ缺失，将生长与p1上THI4的活性结合起来。(b)表达减少方案与冷火鸡（CT）或渐进(G)选择相结合。(c)非同义（蓝色）和同义（绿色）突变在运动结束时横扫人口。当种群在运动早期未能生长时，幸存的种群被分成亚种群（A、B、C）并独立繁殖。被检测的非同义突变的5个突变序列用橙色表示。(d)当清除同义突变并克隆到新鲜p1和新鲜细胞（具有72A尾和TP-DNAP1_611）时，这些突变序列所产生的五个进化群体的生长情况与这些序列所支持的缺乏生长情况进行了比较。将原生MhTHI4、亲本V124A突变体和酵母THI4 （ScTHI4）作为基准以及空载体（EV）对照。对于进化亚群的最后三次传代和用于非同义突变测试的12个重复培养，数据为平均值±SE。在本研究中，我们通过降低MhTHI4靶点的表达，增加了其选择压力，从而降低了酶活性和细胞生长速度，并更新了改进的范围。在OrthoRep中，可以通过缩短目标mRNA的遗传编码多聚(A)尾或减少携带目标基因的质粒（p1）的拷贝数来减少表达(Ravikumar等人，2018；Zhong等人，2018)（图1a）。这些策略导致了更好的，也就是说，更快的增长，种群中含有新的非同义突变的mhthi4，加上同义突变。令人惊讶的是，测试表明，同义突变可能在很大程度上或完全负责观察到的生长速度的改善。由于这样的“海市蜃楼”似乎很可能出现在其他的OrthoRep CDE项目中，我们在这里记录它们作为一个警示案例研究。我们之前的MhTHI4研究使用的是商业编码基因，其中V124A突变体（Van Gelder et al., 2023）是目前研究的起点（附录S1）。和之前一样，平台菌株是BY4741 thi4Δ，它需要噻唑前体（HET）或硫胺素来生长。聚(A)尾从~72A缩短至24A或0A，分别将表达量减少两倍或十倍（Zhong et al., 2018）。为了减少p1拷贝数，将易出错的DNA聚合酶从TP-DNAP1_611改为TP-DNAP1_633，这也提高了突变率（García-García et al., 2022）。每个表达减少方案都被证实如预期的那样起作用（图S1），然后结合“突然停止”选择（不添加硫胺素或HET的培养）或“逐步”选择（最初补充有限的硫胺素或HET，即逐渐减少到零）（García-García等人，2022）。每4-6天进行一次传代。针对每种表达减少机制，对V124A突变体的9个独立群体进行工程化设计，并进行冷断或逐步选择，得到54个初始亚群体（图1b）。 在没有补充的情况下存活下来的亚群被分成3个（记为A、B、C）并独立繁殖。当增长率趋于稳定时，竞选活动就结束了。对来自亚种群的大量DNA进行测序，确定了感兴趣的突变，即那些完全取代了野生型碱基的突变（“横扫”亚种群）。事实证明，突然停止使用的策略是有效的；33代后得到两个亚种群（无A尾的V124A和带TP-DNAP_633的V124A），每代结束时分裂值均达到OD600 1.5 ~ 5.0。总的来说，在选定的亚群中发现了11个非同义突变和9个同义突变(以及两个10B2启动子突变，很可能是中性的，因为10B2已经优化；Zhong等人，2018)（图1c）。值得注意的非同义突变是V124A的逆转和Y122C的取代；这个开关意味着Y122C在功能上更优越。五个具有非保守性非同义突变的序列（图1c）被推进进一步测试并重新合成以清除同义突变。然后将纯化的序列克隆到新鲜的p1质粒和平台细胞中，并在用于进化突变序列的条件下测试细胞生长情况，其中这些未纯化的序列支持生长，而作为起点的V124A突变体不支持生长。请注意，这些培养条件涉及比用于选择V124A突变体的条件更大的曝气（即更高的O2水平）（Van Gelder et al., 2023），并且这会降低互补活性。与未清洗的序列不同，清洗后的序列不支持生长，而酵母THI4阳性对照则支持生长，如预期的那样（图1d）。非同义突变本身不能改善生长，这表明伴随的同义突变对于观察到的生长表型也是必要的，甚至是充分的。基于获得性有益核突变的另一种解释是先验不太可能的(i)因为基因组突变率比OrthoRep靶基因低约10万倍（Molina et al., 2022）， （ii）因为我们以前没有见过这种情况，更不用说同时在不同的人群中(García-García et al., 2022；Van Gelder et al., 2023)。非同义突变可以提高酵母中商业密码子优化细菌酶的性能，这并不奇怪（Lanza et al., 2014）。密码子优化算法强烈倾向于丰富的酵母密码子，这并不总是最有效的方案（Lanza et al., 2014），事实上，获得的同义突变都导致密码子的丰度较低，例如，Gly GGT→GGC, Asp GAT→GAC。令人惊讶的是，同义突变的影响完全支配了运动，并且没有恢复具有实质性益处的新的非同义突变。因此，非同义突变可能是已经存在于有益同义突变产生的基因中的接近中性的客突变。与这种可能性一致的是，非同义突变F14L和I198T（图1c）之前被归类为中性或轻度有害（Van Gelder et al., 2023）。此外，11个非同义突变中，除1个突变外，其余突变均位于非保守或弱保守位置，其中6个突变自然发生（表S1）。请注意，O
{"title":"Mirages in continuous directed enzyme evolution: a cautionary case study with plantized bacterial THI4 enzymes","authors":"Kristen Van Gelder, Anuran K. Gayen, Andrew D. Hanson","doi":"10.1111/pbi.14563","DOIUrl":"https://doi.org/10.1111/pbi.14563","url":null,"abstract":"<p>Continuous directed evolution (CDE) improves the characteristics of a target enzyme by hypermutating the enzyme gene <i>in vivo</i>, coupling enzyme activity to growth of a microbial platform, and selecting for growth rate (Molina <i>et al</i>., <span>2022</span>). Directed evolution can be interfaced with genome editing to expand the gene pool available for plant breeding; this powerful combination (DE–GE) has been neatly termed ‘a Green (r)Evolution’ (Gionfriddo <i>et al</i>., <span>2019</span>). THI4 enzymes, which make the thiazole moiety of thiamin, are good testbed targets for plant CDE technology. Plant THI4s are energy-inefficient suicide enzymes that could potentially be replaced by efficient, non-suicide bacterial THI4s to increase biomass yield by as much as 4% (Joshi <i>et al</i>., <span>2021</span>). However, bacterial THI4s are O<sub>2</sub>-sensitive and otherwise ill-adapted to plants (Joshi <i>et al</i>., <span>2021</span>). We therefore previously ran CDE campaigns in the yeast OrthoRep system to ‘plantize’ bacterial THI4s, that is, to improve function in an aerobic, plant-like milieu (Figure 1a) (García-García <i>et al</i>., <span>2022</span>). Two notably successful campaigns were for the THI4 from <i>Mucinivorans hirudinis</i> (MhTHI4); these campaigns culminated when populations acquired single V124A or Y122C mutations that improved growth to near the wild-type rate (Van Gelder <i>et al</i>., <span>2023</span>). Such culmination can be overcome by increasing the selection pressure (Molina <i>et al</i>., <span>2022</span>).</p>\u0000<figure><picture>\u0000<source media=\"(min-width: 1650px)\" srcset=\"/cms/asset/5d1a35f3-b3df-4cfa-bd4e-d6fe7c98b2d5/pbi14563-fig-0001-m.jpg\"/><img alt=\"Details are in the caption following the image\" data-lg-src=\"/cms/asset/5d1a35f3-b3df-4cfa-bd4e-d6fe7c98b2d5/pbi14563-fig-0001-m.jpg\" loading=\"lazy\" src=\"/cms/asset/bd12af5d-8049-40c0-b660-06ab303db600/pbi14563-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>OrthoRep campaigns to plantize a bacterial THI4 and their outcomes. (a) The OrthoRep system. The target enzyme (MhTHI4 V124A), plus or minus a poly(A) tail, is encoded on the cytoplasmic p1 plasmid that also carries a LEU2 marker. p1 is hypermutated by a p1-specific, error-prone DNA polymerase (TP-DNAP1_611 or TP-DNAP1_633) encoded on a nuclear plasmid. The BY4741 platform strain carries a <i>thi4</i>Δ deletion to couple growth to the activity of the THI4 on p1. (b) The combinations of expression-reduction regimes with cold turkey (CT) or gradual (G) selection. (c) The non-synonymous (blue) and synonymous (green) mutations that had swept populations by the end of campaigns. When populations failed to grow early in campaigns, surviving populations were split into subpopulations (A, B, C) and prop","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"23 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142917510","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}
Pawel Mateusz Mordaka, Kitty Clouston, Aleix Gorchs‐Rovira, Catherine Sutherland, Daniel Qingyang Zhang, Katrin Geisler, Payam Mehrshahi, Alison Gail Smith
SummaryThe green microalga Chlamydomonas reinhardtii is a promising host organism for the production of valuable compounds. Engineering the Chlamydomonas chloroplast genome offers several advantages over the nuclear genome, including targeted gene insertion, lack of silencing mechanisms, potentially higher protein production due to multiple genome copies and natural substrate abundance for metabolic engineering. Tuneable expression systems can be used to minimize competition between heterologous production and host cell viability. However, complex gene regulation and a lack of tight regulatory elements make this a challenge in the Chlamydomonas chloroplast. In this work, we develop two synthetic tuneable systems to control the expression of genes on the chloroplast genome, taking advantage of the properties of the vitamin B12‐responsive METE promoter and a modified thiamine (vitamin B1) riboswitch, along with nucleus‐encoded chloroplast‐targeted regulatory proteins NAC2 and MRL1. We demonstrate the capacity of these systems for robust, fine‐tuned control of several chloroplast transgenes, by addition of nanomolar levels of vitamins. The two systems have been combined in a single strain engineered to avoid effects on photosynthesis and are orthogonal to each other. They were then used to manipulate the production of an industrially relevant diterpenoid, casbene, by introducing and tuning expression of the coding sequence for casbene synthase, as well as regulating the metabolite flux towards casbene precursors, highlighting the utility of these systems for informing metabolic engineering approaches.
{"title":"Regulation of nucleus‐encoded trans‐acting factors allows orthogonal fine‐tuning of multiple transgenes in the chloroplast of Chlamydomonas reinhardtii","authors":"Pawel Mateusz Mordaka, Kitty Clouston, Aleix Gorchs‐Rovira, Catherine Sutherland, Daniel Qingyang Zhang, Katrin Geisler, Payam Mehrshahi, Alison Gail Smith","doi":"10.1111/pbi.14557","DOIUrl":"https://doi.org/10.1111/pbi.14557","url":null,"abstract":"SummaryThe green microalga <jats:italic>Chlamydomonas reinhardtii</jats:italic> is a promising host organism for the production of valuable compounds. Engineering the <jats:italic>Chlamydomonas</jats:italic> chloroplast genome offers several advantages over the nuclear genome, including targeted gene insertion, lack of silencing mechanisms, potentially higher protein production due to multiple genome copies and natural substrate abundance for metabolic engineering. Tuneable expression systems can be used to minimize competition between heterologous production and host cell viability. However, complex gene regulation and a lack of tight regulatory elements make this a challenge in the <jats:italic>Chlamydomonas</jats:italic> chloroplast. In this work, we develop two synthetic tuneable systems to control the expression of genes on the chloroplast genome, taking advantage of the properties of the vitamin B<jats:sub>12</jats:sub>‐responsive <jats:italic>METE</jats:italic> promoter and a modified thiamine (vitamin B<jats:sub>1</jats:sub>) riboswitch, along with nucleus‐encoded chloroplast‐targeted regulatory proteins NAC2 and MRL1. We demonstrate the capacity of these systems for robust, fine‐tuned control of several chloroplast transgenes, by addition of nanomolar levels of vitamins. The two systems have been combined in a single strain engineered to avoid effects on photosynthesis and are orthogonal to each other. They were then used to manipulate the production of an industrially relevant diterpenoid, casbene, by introducing and tuning expression of the coding sequence for casbene synthase, as well as regulating the metabolite flux towards casbene precursors, highlighting the utility of these systems for informing metabolic engineering approaches.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"90 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2024-12-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142888069","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}
SummaryEnhanced grain yield and quality traits are everlasting breeding goals. It is therefore of great significance to uncover more genetic resources associated with these two important agronomic traits. Plant MYB family transcription factors play important regulatory roles in diverse biological processes. However, studies on genetic functions of MYB in rice yield and quality are rarely to be reported. Here, we investigated a nucleus‐localized transcription factor OsMYB73 which is preferentially expressed in the early developing pericarp and endosperm. We generated targeted mutagenesis of OsMYB73 in rice, and the mutants had longer grains with obvious white‐belly chalky endosperm appearance phenotype. The mutants displayed various changes in starch physicochemical characteristics and lipid components. Transcriptome sequencing analysis showed that OsMYB73 was chiefly involved in cell wall development and starch metabolism. OsMYB73 mutation affects the expression of genes related to grain size, starch and lipid biosynthesis and auxin biosynthesis. Moreover, inactivation of OsMYB73 triggers broad changes in secondary metabolites. We speculate that rice OsMYB73 and OsNF‐YB1 play synergistic pivotal role in simultaneously as transcription activators to regulate grain filling and storage compounds accumulation to affect endosperm development and grain chalkiness through binding OsISA2, OsLTPL36 and OsYUC11. The study provides important germplasm resources and theoretical basis for genetic improvement of rice yield and quality. In addition, we enriches the potential biological functions of rice MYB family transcription factors.
{"title":"A novel transcription factor OsMYB73 affects grain size and chalkiness by regulating endosperm storage substances' accumulation‐mediated auxin biosynthesis signalling pathway in rice","authors":"Song Liu, Jiamin Wu, Amos Musyoki Mawia, Xiangjin Wei, Ruijie Cao, Guiai Jiao, Yawen Wu, Jian Zhang, Lihong Xie, Zhonghua Sheng, Shikai Hu, Sanfeng Li, Yusong Lv, Feifei Lu, Yujuan Chen, Sajid Fiaz, Javaria Tabassum, Zhimin Du, Fangyuan Gao, Guangjun Ren, Gaoneng Shao, Peisong Hu, Shaoqing Tang","doi":"10.1111/pbi.14558","DOIUrl":"https://doi.org/10.1111/pbi.14558","url":null,"abstract":"SummaryEnhanced grain yield and quality traits are everlasting breeding goals. It is therefore of great significance to uncover more genetic resources associated with these two important agronomic traits. Plant MYB family transcription factors play important regulatory roles in diverse biological processes. However, studies on genetic functions of MYB in rice yield and quality are rarely to be reported. Here, we investigated a nucleus‐localized transcription factor <jats:italic>OsMYB73</jats:italic> which is preferentially expressed in the early developing pericarp and endosperm. We generated targeted mutagenesis of <jats:italic>OsMYB73</jats:italic> in rice, and the mutants had longer grains with obvious white‐belly chalky endosperm appearance phenotype. The mutants displayed various changes in starch physicochemical characteristics and lipid components. Transcriptome sequencing analysis showed that <jats:italic>OsMYB73</jats:italic> was chiefly involved in cell wall development and starch metabolism. <jats:italic>OsMYB73</jats:italic> mutation affects the expression of genes related to grain size, starch and lipid biosynthesis and auxin biosynthesis. Moreover, inactivation of <jats:italic>OsMYB73</jats:italic> triggers broad changes in secondary metabolites. We speculate that rice <jats:italic>OsMYB73</jats:italic> and <jats:italic>OsNF‐YB1</jats:italic> play synergistic pivotal role in simultaneously as transcription activators to regulate grain filling and storage compounds accumulation to affect endosperm development and grain chalkiness through binding <jats:italic>OsISA2</jats:italic>, <jats:italic>OsLTPL36</jats:italic> and <jats:italic>OsYUC11</jats:italic>. The study provides important germplasm resources and theoretical basis for genetic improvement of rice yield and quality. In addition, we enriches the potential biological functions of rice MYB family transcription factors.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"62 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2024-12-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142888137","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}
Sohee Lim, Hyun Joo Chung, Yoo Jin Oh, Peter Hinterdorfer, Soon Chul Myung, Young-Jin Seo, Kisung Ko
Epithelial cell adhesion molecule (EpCAM) fused to IgG, IgA and IgM Fc domains was expressed to create IgG, IgA and IgM-like structures as anti-cancer vaccines in Nicotiana tabacum. High-mannose glycan structures were generated by adding a C-terminal endoplasmic reticulum (ER) retention motif (KDEL) to the Fc domain (FcK) to produce EpCAM-Fc and EpCAM-FcK proteins in transgenic plants via Agrobacterium-mediated transformation. Cross-fertilization of EpCAM-Fc (FcK) transgenic plants with Joining chain (J-chain, J and JK) transgenic plants led to stable expression of large quaternary EpCAM-IgA Fc (EpCAM-A) and IgM-like (EpCAM-M) proteins. Immunoblotting, SDS–PAGE and ELISA analyses demonstrated that proteins with KDEL had higher expression levels and binding activity to anti-EpCAM IgGs. IgM showed the strongest binding among the fusion proteins, followed by IgA and IgG. Sera from BALB/c mice immunized with these vaccines produced anti-EpCAM IgGs. Flow cytometry indicated that the EpCAM-Fc fusion proteins significantly activated CD8+ cytotoxic T cells, CD4+ helper T cells and B cells, particularly with EpCAM-FcKP and EpCAM-FcP (FcKP) × JP (JKP). The induced anti-EpCAM IgGs captured human prostate cancer PC-3 and colorectal cancer SW620 cells. Sera from immunized mice inhibited cancer cell proliferation, migration and invasion; down-regulated proliferation markers (PCNA, Ki-67) and epithelial–mesenchymal transition markers (Vimentin); and up-regulated E-cadherin. These findings suggest that N. tabacum can produce effective vaccine candidates to induce anti-cancer immune responses.
{"title":"Modification of Fc-fusion protein structures to enhance efficacy of cancer vaccine in plant expression system","authors":"Sohee Lim, Hyun Joo Chung, Yoo Jin Oh, Peter Hinterdorfer, Soon Chul Myung, Young-Jin Seo, Kisung Ko","doi":"10.1111/pbi.14552","DOIUrl":"https://doi.org/10.1111/pbi.14552","url":null,"abstract":"Epithelial cell adhesion molecule (EpCAM) fused to IgG, IgA and IgM Fc domains was expressed to create IgG, IgA and IgM-like structures as anti-cancer vaccines in <i>Nicotiana tabacum</i>. High-mannose glycan structures were generated by adding a C-terminal endoplasmic reticulum (ER) retention motif (KDEL) to the Fc domain (FcK) to produce EpCAM-Fc and EpCAM-FcK proteins in transgenic plants via <i>Agrobacterium</i>-mediated transformation. Cross-fertilization of EpCAM-Fc (FcK) transgenic plants with Joining chain (J-chain, J and JK) transgenic plants led to stable expression of large quaternary EpCAM-IgA Fc (EpCAM-A) and IgM-like (EpCAM-M) proteins. Immunoblotting, SDS–PAGE and ELISA analyses demonstrated that proteins with KDEL had higher expression levels and binding activity to anti-EpCAM IgGs. IgM showed the strongest binding among the fusion proteins, followed by IgA and IgG. Sera from BALB/c mice immunized with these vaccines produced anti-EpCAM IgGs. Flow cytometry indicated that the EpCAM-Fc fusion proteins significantly activated CD8<sup>+</sup> cytotoxic T cells, CD4<sup>+</sup> helper T cells and B cells, particularly with EpCAM-FcK<sup>P</sup> and EpCAM-Fc<sup>P</sup> (FcK<sup>P</sup>) × J<sup>P</sup> (JK<sup>P</sup>). The induced anti-EpCAM IgGs captured human prostate cancer PC-3 and colorectal cancer SW620 cells. Sera from immunized mice inhibited cancer cell proliferation, migration and invasion; down-regulated proliferation markers (PCNA, Ki-67) and epithelial–mesenchymal transition markers (Vimentin); and up-regulated E-cadherin. These findings suggest that <i>N. tabacum</i> can produce effective vaccine candidates to induce anti-cancer immune responses.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"127 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2024-12-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142887239","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}
As 2024 draws to a close, it once again provides an opportunity to recognize and thank everyone who has contributed to the continued success of Plant Biotechnology Journal, a combined effort from the Editors, the reviewers and the Wiley team. I deeply appreciate the diligence and hard work everyone puts into helping authors elevate and improve their manuscripts, ultimately providing useful additions to the published literature.
Plant Biotechnology Journal continues to be a top-ranked journal, maintaining its position in the top five titles in the Plant Sciences category. Our Editors have handled over 1300 submissions so far this year, and we are on track to publish ~250 papers in 2024. It is fair to say that the PBJ team and I are very proud of our metrics and rankings (Impact Factor 11.2 and CiteScore 20.5). Perhaps, a more impressive number is for article downloads, which for 2024 was ~2 million—to my mind, that really does demonstrate relevance and utility. In 2024, PBJ also provided sponsorship to a diverse range of scientific meetings, helping to build communities and forge new connections. A personal highlight for me was the recent meeting in Samson, Turkey entitled Agricultural Biotechnology in the Era of Genome Editing which was organized by AAB and supported by PBJ. I was incredibly impressed with the science presented at this congress as well as the enthusiasm of the participants—I felt lucky to be part of it.
Given the global challenges that we face, the importance of discovery and innovation is ever more apparent and necessary. Although the massive disruption of Covid-19 now seems like a distant memory, it is important to recall that it was scientific innovation (in the form of new vaccines) that stemmed the pandemic. Now we face probably even greater, more complex threats in the form of accelerating climate change and the associated disruption to environments and food production. I believe that plant biotechnology (and PBJ as part of that vibrant community) can play a key role in mitigating these perturbations, and I would encourage everyone to consider how their knowledge and expertise might contribute solutions to these global challenges. Plant biotechnology has delivered amazing disruptive agricultural innovations at a massive scale (such as herbicide-tolerant crops), and I believe that it can and will do so again in response to the climate crisis. In that respect, PBJ will continue to play a role, publishing the best and most useful advances in our field.
Best wishes for 2025,
Johnathan
{"title":"From the Editor-in-Chief","authors":"Johnathan Napier","doi":"10.1111/pbi.14560","DOIUrl":"10.1111/pbi.14560","url":null,"abstract":"<p>As 2024 draws to a close, it once again provides an opportunity to recognize and thank everyone who has contributed to the continued success of <i>Plant Biotechnology Journal</i>, a combined effort from the Editors, the reviewers and the Wiley team. I deeply appreciate the diligence and hard work everyone puts into helping authors elevate and improve their manuscripts, ultimately providing useful additions to the published literature.</p><p><i>Plant Biotechnology Journal</i> continues to be a top-ranked journal, maintaining its position in the top five titles in the Plant Sciences category. Our Editors have handled over 1300 submissions so far this year, and we are on track to publish ~250 papers in 2024. It is fair to say that the PBJ team and I are very proud of our metrics and rankings (Impact Factor 11.2 and CiteScore 20.5). Perhaps, a more impressive number is for article downloads, which for 2024 was ~2 million—to my mind, that really does demonstrate relevance and utility. In 2024, PBJ also provided sponsorship to a diverse range of scientific meetings, helping to build communities and forge new connections. A personal highlight for me was the recent meeting in Samson, Turkey entitled <i>Agricultural Biotechnology in the Era of Genome Editing</i> which was organized by AAB and supported by PBJ. I was incredibly impressed with the science presented at this congress as well as the enthusiasm of the participants—I felt lucky to be part of it.</p><p>Given the global challenges that we face, the importance of discovery and innovation is ever more apparent and necessary. Although the massive disruption of Covid-19 now seems like a distant memory, it is important to recall that it was scientific innovation (in the form of new vaccines) that stemmed the pandemic. Now we face probably even greater, more complex threats in the form of accelerating climate change and the associated disruption to environments and food production. I believe that plant biotechnology (and PBJ as part of that vibrant community) can play a key role in mitigating these perturbations, and I would encourage everyone to consider how their knowledge and expertise might contribute solutions to these global challenges. Plant biotechnology has delivered amazing disruptive agricultural innovations at a massive scale (such as herbicide-tolerant crops), and I believe that it can and will do so again in response to the climate crisis. In that respect, PBJ will continue to play a role, publishing the best and most useful advances in our field.</p><p>Best wishes for 2025,</p><p>Johnathan</p>","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"23 1","pages":"3"},"PeriodicalIF":10.1,"publicationDate":"2024-12-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/pbi.14560","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142886774","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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