SummaryAccurate prediction of flowering time across diverse environments is crucial for effective crop management and breeding. While the accumulated temperature index (ATI) is widely used as an indicator for estimating flowering time, its traditional definition lacks systematic evaluation and genetic basis understanding. Here, using data from 422 rice hybrids across 47 locations, we identified the optimal ATI calculation window as 1 day after sowing to 26 days before flowering. Based on this redefined ATI, we developed a single‐parameter model that outperforms the state‐of‐the‐art reaction norm index model in both accuracy and stability, especially with limited training data. We identified 10 loci significantly associated with ATI variation, including two near known flowering time genes and four linked to ecotype differentiation. To enhance practical utility, we developed an efficient flowering time prediction kit using 28 functionally relevant markers, complemented by a user‐friendly online tool (http://xielab.hzau.edu.cn/ATI). Our approach can be easily applied to other crops, as ATI is commonly used across various agricultural systems.
摘要准确预测不同环境下的开花时间对作物的有效管理和育种至关重要。虽然积温指数（ATI）被广泛用作估计开花时间的指标，但其传统定义缺乏系统的评估和对遗传基础的了解。在此，我们利用 47 个地点 422 个水稻杂交种的数据，确定了最佳 ATI 计算窗口为播种后 1 天至开花前 26 天。根据这一重新定义的 ATI，我们开发了一个单参数模型，该模型在准确性和稳定性方面都优于最先进的反应标准指数模型，尤其是在训练数据有限的情况下。我们发现了 10 个与 ATI 变化明显相关的基因位点，其中包括两个接近已知花期基因的位点和四个与生态型分化相关的位点。为了提高实用性，我们利用 28 个功能相关的标记开发了一个高效的花期预测试剂盒，并辅以一个用户友好的在线工具 (http://xielab.hzau.edu.cn/ATI)。我们的方法可以很容易地应用于其他作物，因为 ATI 通常用于各种农业系统。
{"title":"Redefining the accumulated temperature index for accurate prediction of rice flowering time in diverse environments","authors":"Xingbing Xu, Qiong Jia, Sijia Li, Julong Wei, Luchang Ming, Qi Yu, Jing Jiang, Peng Zhang, Honglin Yao, Shibo Wang, Chunjiao Xia, Kai Wang, Zhenyu Jia, Weibo Xie","doi":"10.1111/pbi.14498","DOIUrl":"https://doi.org/10.1111/pbi.14498","url":null,"abstract":"SummaryAccurate prediction of flowering time across diverse environments is crucial for effective crop management and breeding. While the accumulated temperature index (ATI) is widely used as an indicator for estimating flowering time, its traditional definition lacks systematic evaluation and genetic basis understanding. Here, using data from 422 rice hybrids across 47 locations, we identified the optimal ATI calculation window as 1 day after sowing to 26 days before flowering. Based on this redefined ATI, we developed a single‐parameter model that outperforms the state‐of‐the‐art reaction norm index model in both accuracy and stability, especially with limited training data. We identified 10 loci significantly associated with ATI variation, including two near known flowering time genes and four linked to ecotype differentiation. To enhance practical utility, we developed an efficient flowering time prediction kit using 28 functionally relevant markers, complemented by a user‐friendly online tool (<jats:ext-link xmlns:xlink=\"http://www.w3.org/1999/xlink\" xlink:href=\"http://xielab.hzau.edu.cn/ATI\">http://xielab.hzau.edu.cn/ATI</jats:ext-link>). Our approach can be easily applied to other crops, as ATI is commonly used across various agricultural systems.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"4 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2024-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142541252","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}
Wen Chen, Dingyi Bai, Yuxi Liao, Qin Yu, Lianyang Bai, Lang Pan
Populations of Polypogon fugax have developed resistance to many acetyl-CoA carboxylase (ACCase)-inhibiting herbicides. This resistance threats the effectiveness and sustainability of herbicide use. In our previous research, a field P. fugax population exhibited GST-based metabolic resistance to the widely used ACCase-inhibiting herbicide quizalofop-p-ethyl. Here, in this current study, we identified and characterized two GST genes (named as PfGSTF2 and PfGSTF58) that showed higher expression levels in the resistant than the susceptible population. Transgenic rice calli overexpressing PfGSTF2, but not PfGSTF58, became resistant to quizalofop-p-ethyl and haloxyfop-R-methyl. This reflects similar cross-resistance pattern to what was observed in the resistant P. fugax population. Transgenic rice seedlings overexpressing PfGSTF2 also exhibited resistance to quizalofop-p-ethyl. In contrast, CRISPR/Cas9 knockout of the orthologue gene in rice seedlings increased their sensitivity to quizalofop-p-ethyl. LC–MS analysis of in vitro herbicide metabolism by Escherichia coli-expressed recombinant PfGSTF2 revealed that quizalofop (but not haloxyfop) was detoxified at the ether bond, generating the GSH-quizalofop conjugate and a propanoic acid derivative with greatly reduced herbicidal activity. Equally, these two metabolites accumulated at higher levels in the resistant population than the susceptible population. In addition, both recombinant PfGSTF2 and PfGSTF58 can attenuate cytotoxicity by reactive oxygen species (ROS), suggesting a role in plant defence against ROS generated by herbicides. Furthermore, the GST inhibitor (NBD-Cl) reversed resistance in the resistant population, and PfGSTF2 (but not PfGSTF58) responded to NBD-Cl inhibition. All these suggest that PfGSTF2 plays a significant role in the evolution of quizalofop resistance through enhanced herbicide metabolism in P. fugax.
{"title":"PfGSTF2 endows resistance to quizalofop-p-ethyl in Polypogon fugax by GSH conjugation","authors":"Wen Chen, Dingyi Bai, Yuxi Liao, Qin Yu, Lianyang Bai, Lang Pan","doi":"10.1111/pbi.14491","DOIUrl":"https://doi.org/10.1111/pbi.14491","url":null,"abstract":"Populations of <i>Polypogon fugax</i> have developed resistance to many acetyl-CoA carboxylase (ACCase)-inhibiting herbicides. This resistance threats the effectiveness and sustainability of herbicide use. In our previous research, a field <i>P. fugax</i> population exhibited GST-based metabolic resistance to the widely used ACCase-inhibiting herbicide quizalofop-p-ethyl. Here, in this current study, we identified and characterized two GST genes (named as <i>PfGSTF2</i> and <i>PfGSTF58</i>) that showed higher expression levels in the resistant than the susceptible population. Transgenic rice calli overexpressing <i>PfGSTF2</i>, but not <i>PfGSTF58</i>, became resistant to quizalofop-p-ethyl and haloxyfop-R-methyl. This reflects similar cross-resistance pattern to what was observed in the resistant <i>P. fugax</i> population. Transgenic rice seedlings overexpressing <i>PfGSTF2</i> also exhibited resistance to quizalofop-p-ethyl. In contrast, CRISPR/Cas9 knockout of the orthologue gene in rice seedlings increased their sensitivity to quizalofop-p-ethyl. LC–MS analysis of <i>in vitro</i> herbicide metabolism by <i>Escherichia coli</i>-expressed recombinant PfGSTF2 revealed that quizalofop (but not haloxyfop) was detoxified at the ether bond, generating the GSH-quizalofop conjugate and a propanoic acid derivative with greatly reduced herbicidal activity. Equally, these two metabolites accumulated at higher levels in the resistant population than the susceptible population. In addition, both recombinant PfGSTF2 and PfGSTF58 can attenuate cytotoxicity by reactive oxygen species (ROS), suggesting a role in plant defence against ROS generated by herbicides. Furthermore, the GST inhibitor (NBD-Cl) reversed resistance in the resistant population, and PfGSTF2 (but not PfGSTF58) responded to NBD-Cl inhibition. All these suggest that PfGSTF2 plays a significant role in the evolution of quizalofop resistance through enhanced herbicide metabolism in <i>P. fugax</i>.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"121 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142519523","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}
Ruben Casatejada‐Anchel, Alejandro Torres‐Moncho, Armand D. Anoman, Nagaveni Budhagatapalli, Ester Pérez‐Lorences, Andrea Alcántara‐Enguídanos, Sara Rosa‐Téllez, Leonardo Perez de Souza, Jochen Kumlehn, Alisdair R. Fernie, Jesús Muñoz‐Bertomeu, Roc Ros
SummaryIn plants, L‐serine (Ser) biosynthesis occurs through various pathways and is highly dependent on the atmospheric CO2 concentration, especially in C3 species, due to the association of the Glycolate Pathway of Ser Biosynthesis (GPSB) with photorespiration. Characterization of a second plant Ser pathway, the Phosphorylated Pathway of Ser Biosynthesis (PPSB), revealed that it is at the crossroads of carbon, nitrogen, and sulphur metabolism. The PPSB comprises three sequential reactions catalysed by 3‐phosphoglycerate dehydrogenase (PGDH), 3‐phosphoSer aminotransferase (PSAT) and 3‐phosphoSer phosphatase (PSP). PPSB was overexpressed in plants exhibiting two different modes of photosynthesis: Arabidopsis (C3 metabolism), and maize (C4 metabolism), under ambient (aCO2) and elevated (eCO2) CO2 growth conditions. Overexpression in Arabidopsis of the PGDH1 gene alone or PGDH1, PSAT1 and PSP1 in combination increased the Ser levels but also the essential amino acids threonine (aCO2), isoleucine, leucine, lysine, phenylalanine, threonine and methionine (eCO2) compared to the wild‐type. These increases translated into higher protein levels. Likewise, starch levels were also increased in the PPSB‐overexpressing lines. In maize, PPSB‐deficient lines were obtained by targeting PSP1 using Cas9 endonuclease. We concluded that the expression of PPSB in maize male gametophyte is required for viable pollen development. Maize lines overexpressing the AtPGDH1 gene only displayed higher protein levels but not starch at both aCO2 and eCO2 conditions, this translated into a significant rise in the nitrogen/carbon ratio. These results suggest that metabolic engineering of PPSB in crops could enhance nitrogen content, particularly under upcoming eCO2 conditions where the activity of GPSB is limited.
{"title":"Metabolic engineering of the serine/glycine network as a means to improve the nitrogen content of crops","authors":"Ruben Casatejada‐Anchel, Alejandro Torres‐Moncho, Armand D. Anoman, Nagaveni Budhagatapalli, Ester Pérez‐Lorences, Andrea Alcántara‐Enguídanos, Sara Rosa‐Téllez, Leonardo Perez de Souza, Jochen Kumlehn, Alisdair R. Fernie, Jesús Muñoz‐Bertomeu, Roc Ros","doi":"10.1111/pbi.14495","DOIUrl":"https://doi.org/10.1111/pbi.14495","url":null,"abstract":"SummaryIn plants, L‐serine (Ser) biosynthesis occurs through various pathways and is highly dependent on the atmospheric CO<jats:sub>2</jats:sub> concentration, especially in C<jats:sub>3</jats:sub> species, due to the association of the Glycolate Pathway of Ser Biosynthesis (GPSB) with photorespiration. Characterization of a second plant Ser pathway, the Phosphorylated Pathway of Ser Biosynthesis (PPSB), revealed that it is at the crossroads of carbon, nitrogen, and sulphur metabolism. The PPSB comprises three sequential reactions catalysed by 3‐phosphoglycerate dehydrogenase (PGDH), 3‐phosphoSer aminotransferase (PSAT) and 3‐phosphoSer phosphatase (PSP). PPSB was overexpressed in plants exhibiting two different modes of photosynthesis: <jats:italic>Arabidopsis</jats:italic> (C<jats:sub>3</jats:sub> metabolism), and maize (C<jats:sub>4</jats:sub> metabolism), under ambient (aCO<jats:sub>2</jats:sub>) and elevated (eCO<jats:sub>2</jats:sub>) CO<jats:sub>2</jats:sub> growth conditions. Overexpression in <jats:italic>Arabidopsis</jats:italic> of the <jats:italic>PGDH1</jats:italic> gene alone or <jats:italic>PGDH1</jats:italic>, <jats:italic>PSAT1</jats:italic> and <jats:italic>PSP1</jats:italic> in combination increased the Ser levels but also the essential amino acids threonine (aCO<jats:sub>2</jats:sub>), isoleucine, leucine, lysine, phenylalanine, threonine and methionine (eCO<jats:sub>2</jats:sub>) compared to the wild‐type. These increases translated into higher protein levels. Likewise, starch levels were also increased in the PPSB‐overexpressing lines. In maize, PPSB‐deficient lines were obtained by targeting <jats:italic>PSP1</jats:italic> using Cas9 endonuclease. We concluded that the expression of PPSB in maize male gametophyte is required for viable pollen development. Maize lines overexpressing the <jats:italic>AtPGDH1</jats:italic> gene only displayed higher protein levels but not starch at both aCO<jats:sub>2</jats:sub> and eCO<jats:sub>2</jats:sub> conditions, this translated into a significant rise in the nitrogen/carbon ratio. These results suggest that metabolic engineering of PPSB in crops could enhance nitrogen content, particularly under upcoming eCO<jats:sub>2</jats:sub> conditions where the activity of GPSB is limited.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"96 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2024-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142490506","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>Transcription factors (TFs), representing 5%–8% of eukaryotic nuclear genome, bind specific DNA sequences like promoters to regulate transcription (Lambert <i>et al</i>., <span>2018</span>). Identifying these sequences is vital for understanding TF functions. Techniques such as chromatin immunoprecipitation sequencing (ChIP-Seq), electrophoretic mobility shift assay (EMSA), yeast one-hybrid (Y1H) assay, dual-luciferase reporter LUC/REN assay, and β-glucuronidase (GUS) reporter are used to validate TF–promoter interactions but require extensive instrumentation and chemicals (Abid <i>et al</i>., <span>2022</span>; Park, <span>2009</span>). An alternative, the RUBY/eYGFPuv assay, uses modified plant leaf colour as a visible, cost-effective method for studying DNA–protein interaction (Sun <i>et al</i>., <span>2023</span>). Advances in genomics, including RNA sequencing and ChIP-Seq, underscore the need for efficient, reliable visual detection systems to map TF binding sites, crucial for elucidating their regulatory roles and broader biological impacts.</p> <p>To develop a visual reporter for TF–DNA interactions, we targeted genes influencing leaf colour by modulating chlorophyll (Chl) degradation. The <i>Stay-Green1</i> (<i>SGR1</i>) gene, crucial for Chl breakdown during senescence, encodes magnesium dechelatase. Mutations in <i>SGR1</i> result in a stay-green phenotype, while overexpression leads to yellowing (Shimoda <i>et al</i>., <span>2016</span>). We chose <i>SGR1</i> from 32 candidates, divided into three subgroups, and cloned <i>SGR1</i> genes from <i>Arabidopsis thaliana</i> (<i>AtSGR1</i>), <i>Oryza sativa</i> (<i>OsSGR1</i>), and two from <i>Ginkgo biloba</i> (<i>GbSGR1</i> and <i>GbSGR1L</i>) (Figure S1). Using the <i>Cauliflower Mosaic Virus 35S</i> promoter, we expressed these genes in <i>Nicotiana benthamiana</i> leaves via <i>Agrobacterium tumefaciens</i>-mediated transformation (Figure S2). All transformed areas exhibited accelerated yellowing, with <i>AtSGR1</i> exhibiting the most rapid and significant Chl degradation, demonstrating its potential as a TF–DNA interaction reporter (Figure 1a–c). Additionally, enhancements including a Kozak consensus sequence for improved translation, darkness to stimulate yellowing, and maintaining temperatures between 22 and 25°C significantly boosted Chl degradation (Figure S3).</p> <figure><picture> <source media="(min-width: 1650px)" srcset="/cms/asset/94290b66-4196-4cb7-b4d0-59dbec6d6b71/pbi14488-fig-0001-m.jpg"/><img alt="Details are in the caption following the image" data-lg-src="/cms/asset/94290b66-4196-4cb7-b4d0-59dbec6d6b71/pbi14488-fig-0001-m.jpg" loading="lazy" src="/cms/asset/8acd5d0a-ed44-420e-8d33-63b02f8f6973/pbi14488-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>Develop
{"title":"SRS: An intelligent and robust approach for confirmation of plant transcription factor–DNA interactions","authors":"Qi Zhou, Yulu Ye, Haiyan He, Zhigang Meng, Tao Zhou, Jingtao Zhang, Yameng Li, Jilong Zhang, Zhaoyi Liao, Yuan Wang, Sandui Guo, Chengzhen Liang","doi":"10.1111/pbi.14488","DOIUrl":"https://doi.org/10.1111/pbi.14488","url":null,"abstract":"<p>Transcription factors (TFs), representing 5%–8% of eukaryotic nuclear genome, bind specific DNA sequences like promoters to regulate transcription (Lambert <i>et al</i>., <span>2018</span>). Identifying these sequences is vital for understanding TF functions. Techniques such as chromatin immunoprecipitation sequencing (ChIP-Seq), electrophoretic mobility shift assay (EMSA), yeast one-hybrid (Y1H) assay, dual-luciferase reporter LUC/REN assay, and β-glucuronidase (GUS) reporter are used to validate TF–promoter interactions but require extensive instrumentation and chemicals (Abid <i>et al</i>., <span>2022</span>; Park, <span>2009</span>). An alternative, the RUBY/eYGFPuv assay, uses modified plant leaf colour as a visible, cost-effective method for studying DNA–protein interaction (Sun <i>et al</i>., <span>2023</span>). Advances in genomics, including RNA sequencing and ChIP-Seq, underscore the need for efficient, reliable visual detection systems to map TF binding sites, crucial for elucidating their regulatory roles and broader biological impacts.</p>\u0000<p>To develop a visual reporter for TF–DNA interactions, we targeted genes influencing leaf colour by modulating chlorophyll (Chl) degradation. The <i>Stay-Green1</i> (<i>SGR1</i>) gene, crucial for Chl breakdown during senescence, encodes magnesium dechelatase. Mutations in <i>SGR1</i> result in a stay-green phenotype, while overexpression leads to yellowing (Shimoda <i>et al</i>., <span>2016</span>). We chose <i>SGR1</i> from 32 candidates, divided into three subgroups, and cloned <i>SGR1</i> genes from <i>Arabidopsis thaliana</i> (<i>AtSGR1</i>), <i>Oryza sativa</i> (<i>OsSGR1</i>), and two from <i>Ginkgo biloba</i> (<i>GbSGR1</i> and <i>GbSGR1L</i>) (Figure S1). Using the <i>Cauliflower Mosaic Virus 35S</i> promoter, we expressed these genes in <i>Nicotiana benthamiana</i> leaves via <i>Agrobacterium tumefaciens</i>-mediated transformation (Figure S2). All transformed areas exhibited accelerated yellowing, with <i>AtSGR1</i> exhibiting the most rapid and significant Chl degradation, demonstrating its potential as a TF–DNA interaction reporter (Figure 1a–c). Additionally, enhancements including a Kozak consensus sequence for improved translation, darkness to stimulate yellowing, and maintaining temperatures between 22 and 25°C significantly boosted Chl degradation (Figure S3).</p>\u0000<figure><picture>\u0000<source media=\"(min-width: 1650px)\" srcset=\"/cms/asset/94290b66-4196-4cb7-b4d0-59dbec6d6b71/pbi14488-fig-0001-m.jpg\"/><img alt=\"Details are in the caption following the image\" data-lg-src=\"/cms/asset/94290b66-4196-4cb7-b4d0-59dbec6d6b71/pbi14488-fig-0001-m.jpg\" loading=\"lazy\" src=\"/cms/asset/8acd5d0a-ed44-420e-8d33-63b02f8f6973/pbi14488-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>Develop","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"31 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142486539","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}
Elena Garcia-Perez, Marta Vazquez-Vilar, Rosa Lozano-Duran, Diego Orzaez
The growing demand for sustainable platforms for biomolecule manufacturing has fuelled the development of plant-based production systems. Agroinfiltration, the current industry standard, offers several advantages but faces limitations for large-scale production due to high operational costs and batch-to-batch variability. Alternatively, here, we describe the CuBe system, a novel bean yellow dwarf virus (BeYDV)-derived conditional replicative expression platform stably transformed in Nicotiana benthamiana and activated by copper sulphate (CuSO4), an inexpensive and widely used agricultural input. The CuBe system utilizes a synthetic circuit of four genetic modules integrated into the plant genome: (i) a replicative vector harbouring the gene of interest (GOI) flanked by cis-acting elements for geminiviral replication and novelly arranged to enable transgene transcription exclusively upon formation of the circular replicon, (ii) copper-inducible Rep/RepA proteins essential for replicon formation, (iii) the yeast-derived CUP2-Gal4 copper-responsive transcriptional activator for Rep/RepA expression, and (iv) a copper-inducible Flp recombinase to minimize basal Rep/RepA expression. CuSO4 application triggers the activation of the system, leading to the formation of extrachromosomal replicons, expression of the GOI, and accumulation of the desired recombinant protein. We demonstrate the functionality of the CuBe system in N. benthamiana plants expressing high levels of eGFP and an anti-SARS-CoV-2 antibody upon copper treatment. Notably, the system is functional in post-harvest applications, a strategy with high potential impact for large-scale biomanufacturing. This work presents the CuBe system as a promising alternative to agroinfiltration for cost-effective and scalable production of recombinant proteins in plants.
{"title":"CuBe: a geminivirus-based copper-regulated expression system suitable for post-harvest activation.","authors":"Elena Garcia-Perez, Marta Vazquez-Vilar, Rosa Lozano-Duran, Diego Orzaez","doi":"10.1111/pbi.14485","DOIUrl":"10.1111/pbi.14485","url":null,"abstract":"<p><p>The growing demand for sustainable platforms for biomolecule manufacturing has fuelled the development of plant-based production systems. Agroinfiltration, the current industry standard, offers several advantages but faces limitations for large-scale production due to high operational costs and batch-to-batch variability. Alternatively, here, we describe the CuBe system, a novel bean yellow dwarf virus (BeYDV)-derived conditional replicative expression platform stably transformed in Nicotiana benthamiana and activated by copper sulphate (CuSO<sub>4</sub>), an inexpensive and widely used agricultural input. The CuBe system utilizes a synthetic circuit of four genetic modules integrated into the plant genome: (i) a replicative vector harbouring the gene of interest (GOI) flanked by cis-acting elements for geminiviral replication and novelly arranged to enable transgene transcription exclusively upon formation of the circular replicon, (ii) copper-inducible Rep/RepA proteins essential for replicon formation, (iii) the yeast-derived CUP2-Gal4 copper-responsive transcriptional activator for Rep/RepA expression, and (iv) a copper-inducible Flp recombinase to minimize basal Rep/RepA expression. CuSO<sub>4</sub> application triggers the activation of the system, leading to the formation of extrachromosomal replicons, expression of the GOI, and accumulation of the desired recombinant protein. We demonstrate the functionality of the CuBe system in N. benthamiana plants expressing high levels of eGFP and an anti-SARS-CoV-2 antibody upon copper treatment. Notably, the system is functional in post-harvest applications, a strategy with high potential impact for large-scale biomanufacturing. This work presents the CuBe system as a promising alternative to agroinfiltration for cost-effective and scalable production of recombinant proteins in plants.</p>","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":" ","pages":""},"PeriodicalIF":10.1,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142454291","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}
Pramod Sivan, János Urbancsok, Evgeniy N. Donev, Marta Derba-Maceluch, Félix R. Barbut, Zakiya Yassin, Madhavi L. Gandla, Madhusree Mitra, Saara E. Heinonen, Jan Šimura, Kateřina Cermanová, Michal Karady, Gerhard Scheepers, Leif J. Jönsson, Emma R. Master, Francisco Vilaplana, Ewa J. Mellerowicz
Wood of broad-leaf tree species is a valued source of renewable biomass for biorefinery and a target for genetic improvement efforts to reduce its recalcitrance. Glucuronoxylan (GX) plays a key role in recalcitrance through its interactions with cellulose and lignin. To reduce recalcitrance, we modified wood GX by expressing GH10 and GH11 endoxylanases from Aspergillus nidulans in hybrid aspen (Populus tremula L. × tremuloides Michx.) and targeting the enzymes to cell wall. The xylanases reduced tree height, modified cambial activity by increasing phloem and reducing xylem production, and reduced secondary wall deposition. Xylan molecular weight was decreased, and the spacing between acetyl and MeGlcA side chains was reduced in transgenic lines. The transgenic trees produced hypolignified xylem having thin secondary walls and deformed vessels. Glucose yields of enzymatic saccharification without pretreatment almost doubled indicating decreased recalcitrance. The transcriptomics, hormonomics and metabolomics data provided evidence for activation of cytokinin and ethylene signalling pathways, decrease in ABA levels, transcriptional suppression of lignification and a subset of secondary wall biosynthetic program, including xylan glucuronidation and acetylation machinery. Several candidate genes for perception of impairment in xylan integrity were detected. These candidates could provide a new target for uncoupling negative growth effects from reduced recalcitrance. In conclusion, our study supports the hypothesis that xylan modification generates intrinsic signals and evokes novel pathways regulating tree growth and secondary wall biosynthesis.
阔叶树种的木材是生物精炼的重要可再生生物质来源，也是基因改良以降低其再抗性的目标。葡萄糖醛酸聚糖（GX）通过与纤维素和木质素的相互作用，在再抗性中发挥着关键作用。为了减少再抗性，我们在杂交杨树（Populus tremula L. × tremuloides Michx.）木聚糖酶降低了树高，通过增加韧皮部生产和减少木质部生产改变了韧皮部活动，并减少了次生壁沉积。转基因品系的木聚糖分子量降低，乙酰基和 MeGlcA 侧链之间的间距缩小。转基因树产生的木质部木质化程度低，次生壁薄，血管变形。未经预处理的酶法糖化葡萄糖产量几乎翻了一番，表明抗逆性降低。转录组学、激素组学和代谢组学数据证明了细胞分裂素和乙烯信号通路的激活、ABA 水平的降低、木质化转录抑制和次生壁生物合成程序子集，包括木聚糖葡萄糖醛酸化和乙酰化机制。研究发现了一些感知木聚糖完整性受损的候选基因。这些候选基因可为解除负生长效应与抗逆性降低之间的联系提供新的靶标。总之，我们的研究支持了木聚糖修饰产生内在信号并唤起调节树木生长和次生壁生物合成的新途径的假设。
{"title":"Modification of xylan in secondary walls alters cell wall biosynthesis and wood formation programs and improves saccharification","authors":"Pramod Sivan, János Urbancsok, Evgeniy N. Donev, Marta Derba-Maceluch, Félix R. Barbut, Zakiya Yassin, Madhavi L. Gandla, Madhusree Mitra, Saara E. Heinonen, Jan Šimura, Kateřina Cermanová, Michal Karady, Gerhard Scheepers, Leif J. Jönsson, Emma R. Master, Francisco Vilaplana, Ewa J. Mellerowicz","doi":"10.1111/pbi.14487","DOIUrl":"https://doi.org/10.1111/pbi.14487","url":null,"abstract":"Wood of broad-leaf tree species is a valued source of renewable biomass for biorefinery and a target for genetic improvement efforts to reduce its recalcitrance. Glucuronoxylan (GX) plays a key role in recalcitrance through its interactions with cellulose and lignin. To reduce recalcitrance, we modified wood GX by expressing GH10 and GH11 endoxylanases from <i>Aspergillus nidulans</i> in hybrid aspen (<i>Populus tremula</i> L. × <i>tremuloides</i> Michx.) and targeting the enzymes to cell wall. The xylanases reduced tree height, modified cambial activity by increasing phloem and reducing xylem production, and reduced secondary wall deposition. Xylan molecular weight was decreased, and the spacing between acetyl and MeGlcA side chains was reduced in transgenic lines. The transgenic trees produced hypolignified xylem having thin secondary walls and deformed vessels. Glucose yields of enzymatic saccharification without pretreatment almost doubled indicating decreased recalcitrance. The transcriptomics, hormonomics and metabolomics data provided evidence for activation of cytokinin and ethylene signalling pathways, decrease in ABA levels, transcriptional suppression of lignification and a subset of secondary wall biosynthetic program, including xylan glucuronidation and acetylation machinery. Several candidate genes for perception of impairment in xylan integrity were detected. These candidates could provide a new target for uncoupling negative growth effects from reduced recalcitrance. In conclusion, our study supports the hypothesis that xylan modification generates intrinsic signals and evokes novel pathways regulating tree growth and secondary wall biosynthesis.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"201 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142486510","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}
The chloroplast genome has considerable potential to enhance crop productivity, but it remains underutilized in breeding because it is difficult to modify. This study elucidates the potential of recently developed chloroplast-targeted C-to-T base editors in facilitating the use of the chloroplast genome for crop breeding. The herbicide metribuzin interferes with photosynthesis by binding to the D1 protein of photosystem II, encoded by the chloroplast genome. Naturally occurring D1 mutants with V219I or A251V substitutions are known to have resistance to some herbicides including metribuzin. Here, using the base editors, we introduced these substitutions and showed that the A251V single mutation and the V219 & A251V double mutations conferred significant metribuzin resistance to Arabidopsis thaliana. The V219I & A251V double mutants exhibited increased metribuzin resistance and grew better than the A251V single mutants. Furthermore, the double mutants grew as well as wild-type plants in the absence of metribuzin. The single and double mutants, which are a challenge to obtain through traditional mutagenesis and crossbreeding methods, can be relatively easily generated using C-to-T base editors. In view of the conservation of V219 and A251 across numerous species, C-to-T base editing can potentially confer metribuzin resistance to a wide range of crops. Compared to nuclear genes, chloroplast genes are also less likely to spread into wild populations. Our findings suggest that chloroplast-targeting C-to-T base editors will find many roles in future crop breeding efforts.
{"title":"Resistance to the herbicide metribuzin conferred to Arabidopsis thaliana by targeted base editing of the chloroplast genome","authors":"Issei Nakazato, Wataru Yamori, Hiroyoshi Matsumura, Yuchen Qu, Miki Okuno, Nobuhiro Tsutsumi, Shin-ichi Arimura","doi":"10.1111/pbi.14490","DOIUrl":"https://doi.org/10.1111/pbi.14490","url":null,"abstract":"The chloroplast genome has considerable potential to enhance crop productivity, but it remains underutilized in breeding because it is difficult to modify. This study elucidates the potential of recently developed chloroplast-targeted C-to-T base editors in facilitating the use of the chloroplast genome for crop breeding. The herbicide metribuzin interferes with photosynthesis by binding to the D1 protein of photosystem II, encoded by the chloroplast genome. Naturally occurring D1 mutants with V219I or A251V substitutions are known to have resistance to some herbicides including metribuzin. Here, using the base editors, we introduced these substitutions and showed that the A251V single mutation and the V219 & A251V double mutations conferred significant metribuzin resistance to <i>Arabidopsis thaliana</i>. The V219I & A251V double mutants exhibited increased metribuzin resistance and grew better than the A251V single mutants. Furthermore, the double mutants grew as well as wild-type plants in the absence of metribuzin. The single and double mutants, which are a challenge to obtain through traditional mutagenesis and crossbreeding methods, can be relatively easily generated using C-to-T base editors. In view of the conservation of V219 and A251 across numerous species, C-to-T base editing can potentially confer metribuzin resistance to a wide range of crops. Compared to nuclear genes, chloroplast genes are also less likely to spread into wild populations. Our findings suggest that chloroplast-targeting C-to-T base editors will find many roles in future crop breeding efforts.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"20 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2024-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142451927","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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