<p>Wheat (<i>Triticum aestivum</i>) and barley (<i>Hordeum vulgare</i>) are the two most important crops in the Triticeae tribe that diverged approximately 11.6 million years ago. Both crops are severely threatened by notorious fungal diseases such as powdery mildew. The pathogens causing powdery mildew disease on cereal crops are <i>Blumeria graminis</i> (<i>syn. Erysiphe graminis</i>), which have evolved and existed as distinct sublineages (called <i>formae speciales</i>, f.sp.). Intriguingly, mildew isolates from each <i>formae speciales</i> subfamily can only cause disease on a cereal host species, but normally not on the others, thus referred to as non-host species. Thus far, powdery mildew resistance genes isolated from wheat, barley and relatives encode mostly nucleotide-binding oligomerization domain-like immune receptors (NLRs) and a few non-NLR proteins (Sánchez-Martín and Keller, <span>2021</span>; Zou <i>et al</i>., <span>2023</span>). While most of the NLR-type <i>Pm</i> genes mediate isolate-specific resistance, the <i>Pm21</i> gene, originally introgressed from wheat wild relative <i>Dasypyrum villosum</i> (<i>Dv</i>), confers broad-spectrum resistance (BSR) to all tested isolates of the wheat powdery mildew, <i>B. graminis</i> f.sp. <i>tritici</i> (<i>Bgt</i>) (Zhang <i>et al</i>., <span>2023</span>). All cloned <i>Pm21</i> homologous genes encode typical coiled-coil (CC)-subtype NLRs (Han <i>et al</i>., <span>2024</span>; He <i>et al</i>., <span>2018</span>, <span>2020</span>; Huang <i>et al</i>., <span>2023</span>; Xing <i>et al</i>., <span>2018</span>; Zhu <i>et al</i>., <span>2023</span>). However, whether the <i>Pm21</i> genes confer BSR to a diversified <i>B. graminis</i> subfamily that colonizes a diverged Triticeae crop, for example, <i>B. graminis</i> f.sp. <i>hordei</i> (<i>Bgh</i>) infecting only barley, remains uninvestigated.</p><p>Our previous analysis reveals that the <i>Pm21</i> locus in <i>Dv</i> accessions harbours at least 38 non-redundant <i>Pm21</i> alleles and these alleles were classified into seven clades, representing a valuable NLR gene pool (He <i>et al</i>., <span>2020</span>). To further explore the <i>Pm21</i> gene resources, the <i>Pm21-B1</i> allele, as a member from the largest clade B and isolated from a resistant <i>Dv</i> accession W619414, was selected for further comparative analysis with the <i>Pm21</i> allele (i.e. <i>Pm21-A1</i> allele from clade A). We aimed to broaden the utilization of <i>Pm21</i> allelic gene pool in diverged Triticeae crops.</p><p>The <i>Pm21-B1</i> allele differs from the <i>Pm21</i> allele by several InDels and many SNPs (He <i>et al</i>., <span>2020</span>), with most polymorphisms resided in the LRR domains (Figure 1a, Figure S1). To assess the function of <i>Pm21</i> and <i>Pm21-B1</i> in diverged crops, we performed single-cell transient gene expression assays in both wheat and barley by particle bombardment. Overexpression of either <i>Pm21</i> or <i>Pm21-B1</
{"title":"An NLR receptor confers broad-spectrum resistance to diversified powdery mildew sublineages in wheat and barley","authors":"Renchun Fan, Lei Feng, Yaling Liu, Qiulian Tang, Yitong Zhao, Yanan Li, Shuangjun Gong, Ruiming Lin, Shuo Huang, Ting Qi, Alexander Förderer, Lijun Yang, Yajun Wang, Jijie Chai, Paul Schulze-Lefert, Huagang He, Qian-Hua Shen","doi":"10.1111/pbi.70038","DOIUrl":"https://doi.org/10.1111/pbi.70038","url":null,"abstract":"<p>Wheat (<i>Triticum aestivum</i>) and barley (<i>Hordeum vulgare</i>) are the two most important crops in the Triticeae tribe that diverged approximately 11.6 million years ago. Both crops are severely threatened by notorious fungal diseases such as powdery mildew. The pathogens causing powdery mildew disease on cereal crops are <i>Blumeria graminis</i> (<i>syn. Erysiphe graminis</i>), which have evolved and existed as distinct sublineages (called <i>formae speciales</i>, f.sp.). Intriguingly, mildew isolates from each <i>formae speciales</i> subfamily can only cause disease on a cereal host species, but normally not on the others, thus referred to as non-host species. Thus far, powdery mildew resistance genes isolated from wheat, barley and relatives encode mostly nucleotide-binding oligomerization domain-like immune receptors (NLRs) and a few non-NLR proteins (Sánchez-Martín and Keller, <span>2021</span>; Zou <i>et al</i>., <span>2023</span>). While most of the NLR-type <i>Pm</i> genes mediate isolate-specific resistance, the <i>Pm21</i> gene, originally introgressed from wheat wild relative <i>Dasypyrum villosum</i> (<i>Dv</i>), confers broad-spectrum resistance (BSR) to all tested isolates of the wheat powdery mildew, <i>B. graminis</i> f.sp. <i>tritici</i> (<i>Bgt</i>) (Zhang <i>et al</i>., <span>2023</span>). All cloned <i>Pm21</i> homologous genes encode typical coiled-coil (CC)-subtype NLRs (Han <i>et al</i>., <span>2024</span>; He <i>et al</i>., <span>2018</span>, <span>2020</span>; Huang <i>et al</i>., <span>2023</span>; Xing <i>et al</i>., <span>2018</span>; Zhu <i>et al</i>., <span>2023</span>). However, whether the <i>Pm21</i> genes confer BSR to a diversified <i>B. graminis</i> subfamily that colonizes a diverged Triticeae crop, for example, <i>B. graminis</i> f.sp. <i>hordei</i> (<i>Bgh</i>) infecting only barley, remains uninvestigated.</p>\u0000<p>Our previous analysis reveals that the <i>Pm21</i> locus in <i>Dv</i> accessions harbours at least 38 non-redundant <i>Pm21</i> alleles and these alleles were classified into seven clades, representing a valuable NLR gene pool (He <i>et al</i>., <span>2020</span>). To further explore the <i>Pm21</i> gene resources, the <i>Pm21-B1</i> allele, as a member from the largest clade B and isolated from a resistant <i>Dv</i> accession W619414, was selected for further comparative analysis with the <i>Pm21</i> allele (i.e. <i>Pm21-A1</i> allele from clade A). We aimed to broaden the utilization of <i>Pm21</i> allelic gene pool in diverged Triticeae crops.</p>\u0000<p>The <i>Pm21-B1</i> allele differs from the <i>Pm21</i> allele by several InDels and many SNPs (He <i>et al</i>., <span>2020</span>), with most polymorphisms resided in the LRR domains (Figure 1a, Figure S1). To assess the function of <i>Pm21</i> and <i>Pm21-B1</i> in diverged crops, we performed single-cell transient gene expression assays in both wheat and barley by particle bombardment. Overexpression of either <i>Pm21</i> or <i>Pm21-B1</","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"102 4 Pt 1 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-03-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143737210","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}
Jiming Liu, Guochun Zhao, Yulin Zheng, Yuanyuan Xu, Mianzhi Wang, Lu Li, Caowen Sun, Qiuyang He, Rami-Petteri Apuli, Joan Jing Yi Jong, Jia Jun Ngiam, Andrey Vaulin, Roy Jun Kai Tham, Liming Jia, Zhong Chen, Jarkko Salojärvi
Sapindus is an important forest tree genus with utility in biodiesel, biomedicine, biochemistry and forestry. Similar to many perennial crop plants, its breeding is hampered by long generation times and lack of genetic resources. To understand the genome evolution underlying the important bioeconomic traits, we carried out a common garden experiment with 100 Sapindus core germplasm individuals representing three endemic species and 60 populations sampled throughout China. Whole genome sequencing identified a split into six populations according to species and geography. The previously uncharacterized S. delavayi and S. rarak are diploid species, and here we propose hypotheses for their speciation. Selective sweeps suggested stress responses as well as alleles of the genes CYP716A, CAMTA and HD-ZIP involved in triterpenoid saponin biosynthesis to have been under selection in natural populations, while genome-wide association analysis revealed several homologues of fatty acid biosynthesis genes to be associated with kernel fatty acid quality. Our findings elucidate the genetic structure of Sapindus in China, provide target loci for selection and suggest cultivar materials for genetic improvement.
{"title":"Genetic diversity and adaptive evolutionary history of Sapindus in China: insights from whole-genome resequencing of 100 representative individuals","authors":"Jiming Liu, Guochun Zhao, Yulin Zheng, Yuanyuan Xu, Mianzhi Wang, Lu Li, Caowen Sun, Qiuyang He, Rami-Petteri Apuli, Joan Jing Yi Jong, Jia Jun Ngiam, Andrey Vaulin, Roy Jun Kai Tham, Liming Jia, Zhong Chen, Jarkko Salojärvi","doi":"10.1111/pbi.70058","DOIUrl":"https://doi.org/10.1111/pbi.70058","url":null,"abstract":"<i>Sapindus</i> is an important forest tree genus with utility in biodiesel, biomedicine, biochemistry and forestry. Similar to many perennial crop plants, its breeding is hampered by long generation times and lack of genetic resources. To understand the genome evolution underlying the important bioeconomic traits, we carried out a common garden experiment with 100 <i>Sapindus</i> core germplasm individuals representing three endemic species and 60 populations sampled throughout China. Whole genome sequencing identified a split into six populations according to species and geography. The previously uncharacterized <i>S. delavayi</i> and <i>S. rarak</i> are diploid species, and here we propose hypotheses for their speciation. Selective sweeps suggested stress responses as well as alleles of the genes <i>CYP716A</i>, <i>CAMTA</i> and <i>HD</i>-<i>ZIP</i> involved in triterpenoid saponin biosynthesis to have been under selection in natural populations, while genome-wide association analysis revealed several homologues of fatty acid biosynthesis genes to be associated with kernel fatty acid quality. Our findings elucidate the genetic structure of <i>Sapindus</i> in China, provide target loci for selection and suggest cultivar materials for genetic improvement.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"131 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-03-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143737211","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}
Ziyang Zhou, Huichun Zhang, Liming Bian, Lei Zhou, Yufeng Ge
Increased drought frequency and severity in a warming climate threaten the health and stability of forest ecosystems, influencing the structure and functioning of forests while having far-reaching implications for global carbon storage and climate regulation. To effectively address the challenges posed by drought, it is imperative to monitor and assess the degree of drought stress in trees in a timely and accurate manner. In this study, a gradient drought stress experiment was conducted with poplar as the research object, and multimodal data were collected for subsequent analysis. A machine learning-based poplar drought monitoring model was constructed, thereby enabling the monitoring of drought severity and duration in poplar trees. Four data processing methods, namely data decomposition, data layer fusion, feature layer fusion and decision layer fusion, were employed to comprehensively evaluate poplar drought monitoring. Additionally, the potential of new phenotypic features obtained by different data processing methods for poplar drought monitoring was discussed. The results demonstrate that the optimal machine learning poplar drought monitoring model, constructed under feature layer fusion, exhibits the best performance, with average accuracy, average precision, average recall and average F1 score reaching 0.85, 0.86, 0.85 and 0.85, respectively. Conversely, the novel phenotypic features derived through data decomposition and data layer fusion methods as supplementary features did not further augment the model precision. This indicates that the feature layer fusion approach has clear advantages in drought monitoring. This research offers a robust theoretical foundation and practical guidance for future tree health monitoring and drought response assessment.
{"title":"Integrating sensor fusion with machine learning for comprehensive assessment of phenotypic traits and drought response in poplar species","authors":"Ziyang Zhou, Huichun Zhang, Liming Bian, Lei Zhou, Yufeng Ge","doi":"10.1111/pbi.70039","DOIUrl":"https://doi.org/10.1111/pbi.70039","url":null,"abstract":"Increased drought frequency and severity in a warming climate threaten the health and stability of forest ecosystems, influencing the structure and functioning of forests while having far-reaching implications for global carbon storage and climate regulation. To effectively address the challenges posed by drought, it is imperative to monitor and assess the degree of drought stress in trees in a timely and accurate manner. In this study, a gradient drought stress experiment was conducted with poplar as the research object, and multimodal data were collected for subsequent analysis. A machine learning-based poplar drought monitoring model was constructed, thereby enabling the monitoring of drought severity and duration in poplar trees. Four data processing methods, namely data decomposition, data layer fusion, feature layer fusion and decision layer fusion, were employed to comprehensively evaluate poplar drought monitoring. Additionally, the potential of new phenotypic features obtained by different data processing methods for poplar drought monitoring was discussed. The results demonstrate that the optimal machine learning poplar drought monitoring model, constructed under feature layer fusion, exhibits the best performance, with average accuracy, average precision, average recall and average F1 score reaching 0.85, 0.86, 0.85 and 0.85, respectively. Conversely, the novel phenotypic features derived through data decomposition and data layer fusion methods as supplementary features did not further augment the model precision. This indicates that the feature layer fusion approach has clear advantages in drought monitoring. This research offers a robust theoretical foundation and practical guidance for future tree health monitoring and drought response assessment.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"22 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-03-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143737212","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}
Maize is a key biomass resource with wide agricultural applications. Anthocyanins, potent antioxidants, offer health benefits like reducing oxidative stress. The biosynthesis of anthocyanins competes with that of lignin for shared metabolic precursors, which can lead to trade-offs in plant growth and feed quality. Higher lignin content can decrease silage digestibility, posing challenges for livestock feed. The maize brown midrib 6 (bm6) mutant, known for reduced lignin, has an unclear genetic basis. Here, we identify ZmGCH1 as the candidate gene for bm6 through fine mapping. Mutations in ZmGCH1 shift precursors from lignin to anthocyanin biosynthesis. Furthermore, we show that ZmGCH1 interacts with ZmPEBP15 to modulate chalcone synthase activity, thereby stabilizing the allocation of precursors between lignin and anthocyanin pathways. To evaluate the practical implications of our findings, we introduced the bm6 mutation into Zhengdan958 and Xianyu335. In vitro rumen digestion assays confirmed that the introduction of the bm6 mutation significantly improved silage digestibility. This discovery not only holds great potential for enhancing silage digestibility but also provides a broader strategy for optimizing maize production to better meet the increasing demands of both the food and livestock feed.
{"title":"Modulation of lignin and anthocyanin homeostasis by GTP cyclohydrolase1 in maize","authors":"Mingyue Zhang, Xiaohan Li, Xiao Wang, Shuzhen Jiang, Junli Zhang, Mingfei Sun, Zixian Zhou, Jinxiao Zhang, Mengyao Li, Yanxiao Lv, Enlong Qi, Ziang Tian, Hongjie Zhu, Xuebin Zhang, Xiangyu Zhao, Changcheng Xu, Thomas Lübberstedt, Xiansheng Zhang, Xuerong Yang, Chao Zhou, Hongjun Liu","doi":"10.1111/pbi.70061","DOIUrl":"https://doi.org/10.1111/pbi.70061","url":null,"abstract":"Maize is a key biomass resource with wide agricultural applications. Anthocyanins, potent antioxidants, offer health benefits like reducing oxidative stress. The biosynthesis of anthocyanins competes with that of lignin for shared metabolic precursors, which can lead to trade-offs in plant growth and feed quality. Higher lignin content can decrease silage digestibility, posing challenges for livestock feed. The maize <i>brown midrib 6</i> (<i>bm6</i>) mutant, known for reduced lignin, has an unclear genetic basis. Here, we identify <i>ZmGCH1</i> as the candidate gene for <i>bm6</i> through fine mapping. Mutations in <i>ZmGCH1</i> shift precursors from lignin to anthocyanin biosynthesis. Furthermore, we show that ZmGCH1 interacts with ZmPEBP15 to modulate chalcone synthase activity, thereby stabilizing the allocation of precursors between lignin and anthocyanin pathways. To evaluate the practical implications of our findings, we introduced the <i>bm6</i> mutation into Zhengdan958 and Xianyu335. In vitro rumen digestion assays confirmed that the introduction of the <i>bm6</i> mutation significantly improved silage digestibility. This discovery not only holds great potential for enhancing silage digestibility but also provides a broader strategy for optimizing maize production to better meet the increasing demands of both the food and livestock feed.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"36 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143734441","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}
Nurmansyah, Agnelo Furtado, Pauline Okemo, Robert J. Henry
Australian wild rice species (AWS) possess unique starch properties characterized by a slow digestibility rate. However, the genomic and transcriptomic variations of starch-synthesis-related genes (SSRGs) influencing starch physiochemical properties in AWS remain unclear. Here, we report comparative analyses of 72 SSRGs in wild species, including two AWS (O. meridionalis and Australian populations of O. rufipogon) and the domesticated rice gene pool. Our findings reveal that most SSRGs are more actively expressed in the early stages of seed development. Transcriptome analysis identified differential splicing patterns, with the starch synthesis pathways in Nipponbare and O. rufipogon being more similar than those in O. meridionalis. Three essential starch genes, GBSSI, SSIIa and BEIIb, were more active and had higher expression in AWS compared to Nipponbare, explaining the higher amylose content, gelatinization temperature, soft gel consistency and high retrogradation in the wild rice. Comparative genomics indicated that Asian domesticated rice evolved from a single ancestral allele of GBSSI (Wxlv) and SSIIa (ALKc), but two BEIIb alleles originated from O. rufipogon and O. nivara, the two wild rice species that are considered progenitors of Asian domesticated rice. Additionally, higher expressions of GBSSI, BEI and SSIIIa in O. meridionalis contribute to a slower starch digestibility rate, making its haplotypes valuable for breeding to develop slowly digested starch cultivars. These findings not only provide insight into the evolution of starch gene synthesis during domestication but also pave the way for unlocking desirable gene haplotypes of wild rice to improve starch quality in cultivated rice.
{"title":"Unique starch biosynthesis pathways in wild rice revealed by multi-omics analyses","authors":"Nurmansyah, Agnelo Furtado, Pauline Okemo, Robert J. Henry","doi":"10.1111/pbi.70021","DOIUrl":"https://doi.org/10.1111/pbi.70021","url":null,"abstract":"Australian wild rice species (AWS) possess unique starch properties characterized by a slow digestibility rate. However, the genomic and transcriptomic variations of starch-synthesis-related genes (SSRGs) influencing starch physiochemical properties in AWS remain unclear. Here, we report comparative analyses of 72 SSRGs in wild species, including two AWS (<i>O. meridionalis</i> and Australian populations of <i>O. rufipogon</i>) and the domesticated rice gene pool. Our findings reveal that most SSRGs are more actively expressed in the early stages of seed development. Transcriptome analysis identified differential splicing patterns, with the starch synthesis pathways in Nipponbare and <i>O. rufipogon</i> being more similar than those in <i>O. meridionalis</i>. Three essential starch genes, <i>GBSSI, SSIIa</i> and <i>BEIIb</i>, were more active and had higher expression in AWS compared to Nipponbare, explaining the higher amylose content, gelatinization temperature, soft gel consistency and high retrogradation in the wild rice. Comparative genomics indicated that Asian domesticated rice evolved from a single ancestral allele of <i>GBSSI</i> (<i>Wx</i><sup><i>lv</i></sup>) and <i>SSIIa</i> (<i>ALK</i><sup><i>c</i></sup>), but two <i>BEIIb</i> alleles originated from <i>O. rufipogon</i> and <i>O. nivara</i>, the two wild rice species that are considered progenitors of Asian domesticated rice. Additionally, higher expressions of <i>GBSSI</i>, <i>BEI</i> and <i>SSIIIa</i> in <i>O. meridionalis</i> contribute to a slower starch digestibility rate, making its haplotypes valuable for breeding to develop slowly digested starch cultivars. These findings not only provide insight into the evolution of starch gene synthesis during domestication but also pave the way for unlocking desirable gene haplotypes of wild rice to improve starch quality in cultivated rice.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"87 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143723417","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}
Michael Panting, Inger B. Holme, Giuseppe Dionisio, Henrik Brinch-Pedersen
Anti-nutritional factors in plant seeds diminish the utilization of nutrients in feed and food. Among these, protease inhibitors inhibit protein degradation by exogenous proteases during digestion. Through conventional and selection-gene-free genome editing using ovules as explants, we used simplex and multiplex CRISPR/Cas9 for studying the impact of chymotrypsin inhibitor CI-1A, CI-1B and CI-2, Bowman-Birk trypsin inhibitor, Serpin-Z4, and barley ɑ-amylase/subtilisin inhibitor on barley and soybean storage protein degradation. Mutants were generated in the commercial cultivar Stairway, having a high level of protease inhibition, and the barley model cultivar Golden Promise, having a lower inhibition level. In Golden Promise, all individual knockouts decreased the inhibition of the three proteases α-chymotrypsin, trypsin and the commercial feed protease Ronozyme ProAct significantly. The triple knockout of all chymotrypsin inhibitors further decreased the inhibition of α-chymotrypsin and Ronozyme ProAct proteases. Degradations of recombinant barley storage proteins B- and C-hordeins were significantly improved following mutagenesis. In Stairway, a single knockout of CI-1A almost compares to the effect on the proteases achieved for the triple knockout in Golden promise, uncovering CI-1A as the major protease inhibitor in that cultivar. The Stairway mutant demonstrated significantly improved degradation of recombinant barley hordeins and in the soybean storage proteins glycinin and β-conglycinin. The results of this study provide insights into cereal protease inhibitor genes and their negative effects on the degradation of barley storage protein and the most important plant protein from soybeans. The study suggests a future focus on plant protease inhibitors as a major target for improving feed and food protein digestibility.
{"title":"Simplex and multiplex CRISPR/Cas9-mediated knockout of grain protease inhibitors in model and commercial barley improves hydrolysis of barley and soy storage proteins","authors":"Michael Panting, Inger B. Holme, Giuseppe Dionisio, Henrik Brinch-Pedersen","doi":"10.1111/pbi.70065","DOIUrl":"https://doi.org/10.1111/pbi.70065","url":null,"abstract":"Anti-nutritional factors in plant seeds diminish the utilization of nutrients in feed and food. Among these, protease inhibitors inhibit protein degradation by exogenous proteases during digestion. Through conventional and selection-gene-free genome editing using ovules as explants, we used simplex and multiplex CRISPR/Cas9 for studying the impact of chymotrypsin inhibitor CI-1A, CI-1B and CI-2, Bowman-Birk trypsin inhibitor, Serpin-Z4, and barley ɑ-amylase/subtilisin inhibitor on barley and soybean storage protein degradation. Mutants were generated in the commercial cultivar Stairway, having a high level of protease inhibition, and the barley model cultivar Golden Promise, having a lower inhibition level. In Golden Promise, all individual knockouts decreased the inhibition of the three proteases α-chymotrypsin, trypsin and the commercial feed protease Ronozyme ProAct significantly. The triple knockout of all chymotrypsin inhibitors further decreased the inhibition of α-chymotrypsin and Ronozyme ProAct proteases. Degradations of recombinant barley storage proteins B- and C-hordeins were significantly improved following mutagenesis. In Stairway, a single knockout of CI-1A almost compares to the effect on the proteases achieved for the triple knockout in Golden promise, uncovering CI-1A as the major protease inhibitor in that cultivar. The Stairway mutant demonstrated significantly improved degradation of recombinant barley hordeins and in the soybean storage proteins glycinin and β-conglycinin. The results of this study provide insights into cereal protease inhibitor genes and their negative effects on the degradation of barley storage protein and the most important plant protein from soybeans. The study suggests a future focus on plant protease inhibitors as a major target for improving feed and food protein digestibility.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"7 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143723418","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}
Mei Tian, Linglong Luo, Baolong Jin, Jianing Liu, Tong Chen, Jinfu Tang, Ye Shen, Haiyan Zhang, Juan Guo, Huawei Zhang, Guanghong Cui, Luqi Huang
The CRISPR/Cas9 system is a powerful tool for genomic editing with significant potential for gene function validation and molecular breeding in medicinal plants. Salvia miltiorrhiza, a model medicinal plant, was among the pioneers to utilize CRISPR/Cas9 technology, though achieving high-efficiency homozygous knockout mutants has been challenging. In this study, the analysis of variations at 241 single-guide RNA (sgRNA) across different reference genomes and experimental materials was conducted first, leading to the identification of the six-generation inbred line bh2-7 as the most suitable reference genome and experimental material for gene editing research in S. miltiorrhiza. Next, five Agrobacterium rhizogenes strains were evaluated for hairy root induction, editing efficiency, and mutation patterns, with C58C1 and K599 emerging as the most effective delivery systems. Using the CRISPR/Cas9 vector pZKD672, 53 target sites were successfully edited, with K599 achieving 71.07% editing efficiency and 36.74% homozygous or biallelic (HOM) efficiency and C58C1 showing 62.27% editing efficiency and 23.61% HOM efficiency. We thus constructed a large-scale mutant library targeting 121 genes with 170 sgRNAs, yielding 1664 homozygous or biallelic mutants. Analysis of 65 low-efficiency target sites revealed that sgRNA mismatches and secondary structures were key factors reducing HOM efficiency, offering insights for future target design. This study establishes an efficient CRISPR/Cas9 system, advancing precision breeding and metabolic engineering research in medicinal plants.
{"title":"Highly efficient Agrobacterium rhizogenes-mediated gene editing system in Salvia miltiorrhiza inbred line bh2-7","authors":"Mei Tian, Linglong Luo, Baolong Jin, Jianing Liu, Tong Chen, Jinfu Tang, Ye Shen, Haiyan Zhang, Juan Guo, Huawei Zhang, Guanghong Cui, Luqi Huang","doi":"10.1111/pbi.70029","DOIUrl":"https://doi.org/10.1111/pbi.70029","url":null,"abstract":"The CRISPR/Cas9 system is a powerful tool for genomic editing with significant potential for gene function validation and molecular breeding in medicinal plants. <i>Salvia miltiorrhiza</i>, a model medicinal plant, was among the pioneers to utilize CRISPR/Cas9 technology, though achieving high-efficiency homozygous knockout mutants has been challenging. In this study, the analysis of variations at 241 single-guide RNA (sgRNA) across different reference genomes and experimental materials was conducted first, leading to the identification of the six-generation inbred line bh2-7 as the most suitable reference genome and experimental material for gene editing research in <i>S. miltiorrhiza</i>. Next, five <i>Agrobacterium rhizogenes</i> strains were evaluated for hairy root induction, editing efficiency, and mutation patterns, with C58C1 and K599 emerging as the most effective delivery systems. Using the CRISPR/Cas9 vector pZKD672, 53 target sites were successfully edited, with K599 achieving 71.07% editing efficiency and 36.74% homozygous or biallelic (HOM) efficiency and C58C1 showing 62.27% editing efficiency and 23.61% HOM efficiency. We thus constructed a large-scale mutant library targeting 121 genes with 170 sgRNAs, yielding 1664 homozygous or biallelic mutants. Analysis of 65 low-efficiency target sites revealed that sgRNA mismatches and secondary structures were key factors reducing HOM efficiency, offering insights for future target design. This study establishes an efficient CRISPR/Cas9 system, advancing precision breeding and metabolic engineering research in medicinal plants.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"23 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143713606","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>Hazels (<i>Corylus</i> L. in birch family) are globally celebrated for their delectable nuts (Molnar, <span>2011</span>). <i>Corylus</i> includes approximately 18 species that are widely distributed across the temperate Northern Hemisphere and is classified into four sections (<i>Acanthochlamys</i>, <i>Siphonochlamys</i>, <i>Colurnae</i> and <i>Phyllochlamys</i>) primarily based on variable bract (Figure 1a). <i>Acanthochlamys</i>, including two <i>C. ferox</i> varieties, is characterized by the spiny structure developed by bract lobes, resembling chestnut fruits, while bract lobes in remaining sections lack of this characteristic. At present, hazelnuts from different cultivars are consumed across 34 countries, with an annual yield of 1.1 million tons of in-shell nuts (FAOSTAT, <span>2023</span>). The primary cultivars are domesticated from <i>C. avellana</i>, and hybridization between some local germplasms (e.g. <i>C. heterophylla</i>) and introduced <i>C. avellana</i> also breed some cultivars with strong adaptability to local environment. With advancements in sequencing, more crops are utilizing pangenome to capture crucial variations—especially genomic structural variations (SVs)—responsible for adaptation and agronomic trait enhancement in molecular breeding (Chen <i>et al</i>., <span>2023</span>). However, only four hazel species currently possess high-quality reference genomes, leaving vast wild hazel species unconcerned (Brainard <i>et al</i>., <span>2023</span>; Li <i>et al</i>., <span>2021</span>). These unconcerned hazels also have some valuable traits. For example, <i>C. ferox</i> has extremely abundant nuts in each infructescence, while the spiny bract impedes its utilization for breeding (Figure 1a).</p><p>To demonstrate developmental variations of spiny bracts, we assembled high-quality chromosome-level genomes of eight wild hazels from seven species, including <i>C. chinensis</i>, <i>C. fargesii</i>, <i>C. wulingensis</i>, <i>C. yunnanensis</i>, <i>C. kweichowensis</i>, <i>C. heterophylla</i> and two <i>C. ferox</i> samples (Table S1). These hazels were collected from diverse geographical locations in China and assembled with the long sequencing reads (HiFi or ONT), NGS and Hi-C approaches. Except for one <i>C. ferox</i> sample with a contig N50 of 1.44 Mb, all of them exhibited a contig N50 longer than 9 Mb (Table S3). Eleven chromosomes were successfully anchored in each genome, revealing significant high collinearity and no large inter-chromosome rearrangement (Figure 1b). All the genomes showed a high quality by different assessments (Table S2). With six published genomes from <i>C. avellana</i>, <i>C. americana</i>, <i>C. heterophylla</i> and <i>C. mandshurica</i> (Table S3), 14 genomes cover four sections in <i>Corylus</i>. We predicted a range of 164–222 Mb for repeat elements and 22 137 to 28 267 for protein-coding genes across different genomes (Tables S4 and S5). Based on these genomes, we further revealed a highly su
{"title":"Genetic insights into developmental variations of spiny bracts among hazels through the pangenome construction","authors":"Zeyu Zheng, Jiaojiao Lv, Zhimin Niu, Jin Zhang, Mingjia Zhu, Hongyin Hu, Wanhe Sun, Jianxiang Ma, Ying Li, Ying Wu, Dandan Wang, Wenjie Mu, Renping Xu, Yun Jiang, Zhiqiang Lu, Jianquan Liu, Yongzhi Yang","doi":"10.1111/pbi.14568","DOIUrl":"10.1111/pbi.14568","url":null,"abstract":"<p>Hazels (<i>Corylus</i> L. in birch family) are globally celebrated for their delectable nuts (Molnar, <span>2011</span>). <i>Corylus</i> includes approximately 18 species that are widely distributed across the temperate Northern Hemisphere and is classified into four sections (<i>Acanthochlamys</i>, <i>Siphonochlamys</i>, <i>Colurnae</i> and <i>Phyllochlamys</i>) primarily based on variable bract (Figure 1a). <i>Acanthochlamys</i>, including two <i>C. ferox</i> varieties, is characterized by the spiny structure developed by bract lobes, resembling chestnut fruits, while bract lobes in remaining sections lack of this characteristic. At present, hazelnuts from different cultivars are consumed across 34 countries, with an annual yield of 1.1 million tons of in-shell nuts (FAOSTAT, <span>2023</span>). The primary cultivars are domesticated from <i>C. avellana</i>, and hybridization between some local germplasms (e.g. <i>C. heterophylla</i>) and introduced <i>C. avellana</i> also breed some cultivars with strong adaptability to local environment. With advancements in sequencing, more crops are utilizing pangenome to capture crucial variations—especially genomic structural variations (SVs)—responsible for adaptation and agronomic trait enhancement in molecular breeding (Chen <i>et al</i>., <span>2023</span>). However, only four hazel species currently possess high-quality reference genomes, leaving vast wild hazel species unconcerned (Brainard <i>et al</i>., <span>2023</span>; Li <i>et al</i>., <span>2021</span>). These unconcerned hazels also have some valuable traits. For example, <i>C. ferox</i> has extremely abundant nuts in each infructescence, while the spiny bract impedes its utilization for breeding (Figure 1a).</p><p>To demonstrate developmental variations of spiny bracts, we assembled high-quality chromosome-level genomes of eight wild hazels from seven species, including <i>C. chinensis</i>, <i>C. fargesii</i>, <i>C. wulingensis</i>, <i>C. yunnanensis</i>, <i>C. kweichowensis</i>, <i>C. heterophylla</i> and two <i>C. ferox</i> samples (Table S1). These hazels were collected from diverse geographical locations in China and assembled with the long sequencing reads (HiFi or ONT), NGS and Hi-C approaches. Except for one <i>C. ferox</i> sample with a contig N50 of 1.44 Mb, all of them exhibited a contig N50 longer than 9 Mb (Table S3). Eleven chromosomes were successfully anchored in each genome, revealing significant high collinearity and no large inter-chromosome rearrangement (Figure 1b). All the genomes showed a high quality by different assessments (Table S2). With six published genomes from <i>C. avellana</i>, <i>C. americana</i>, <i>C. heterophylla</i> and <i>C. mandshurica</i> (Table S3), 14 genomes cover four sections in <i>Corylus</i>. We predicted a range of 164–222 Mb for repeat elements and 22 137 to 28 267 for protein-coding genes across different genomes (Tables S4 and S5). Based on these genomes, we further revealed a highly su","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"23 5","pages":"1396-1398"},"PeriodicalIF":10.1,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/pbi.14568","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143712741","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}
<p>Foxtail millet [<i>Setaria italica</i> (L.) Beauv] is an ancient cereal crop that has been cultivated for grain food and forage consumption globally for over 11 500 years. Despite its long history, foxtail millet continues to be extensively cultivated in developing and underdeveloped countries with low productivity conditions. This is primarily due to its exceptional ability to tolerate drought and barren environments, making it well-suited for grain production in challenging grown conditions. In the last century, significant progress has been made in enhancing the plant architecture of major cereal crops, such as maize (<i>Zea mays</i>) and rice (<i>Oryza sativa</i>), resulting in substantial increases in grain yield potential (Tian <i>et al</i>., <span>2024</span>). However, there has been relatively less emphasis on optimizing the plant architecture of foxtail millet, leading to limited improvements in yield for this ancient crop species to date. While the overexpression of <i>Drooping Leaf 1</i> has been shown to promote more upright leaves in foxtail millet (Zhao <i>et al</i>., <span>2020</span>), compact architecture with reduced leaf angle remains a challenge due to the scarcity of identified germplasm suitable for this purpose.</p><p>Leaf angle, defined as the angle between the culm and leaf midrib, is a crucial aspect of plant architecture that is controlled by a limited number of functional genes. Among these genes, <i>Liguleless1</i> (<i>LG1</i>) and <i>Liguleless2</i> (<i>LG2</i>) are key regulators that play essential roles in lamina joint formation, influencing leaf angle in maize (Walsh <i>et al</i>., <span>1998</span>). <i>LG1</i> has been identified as a transcription factor belonging to the Squamosa Promoter-Binding Proteins (SBP) family, while <i>LG2</i> has been characterized as a basic leucine zipper transcription (bZIP) factor. Genetic analysis of variations indicates that <i>LG1</i> and <i>LG2</i> have the potential to significantly enhance the field productivity of maize varieties, and it has been demonstrated that the functions of <i>LG1</i> and <i>LG2</i> are conserved across various Gramineae crops, including maize (Harper and Freeling, <span>1996</span>), rice (Wang <i>et al</i>., <span>2021</span>), wheat (Yu, <span>2019</span>), sorghum (Brant <i>et al</i>., <span>2021</span>) and sugarcane (Brant <i>et al</i>., <span>2024</span>). However, the roles of <i>LG1</i> and <i>LG2</i> in foxtail millet remain unclear, and as of now, no ligule development-defective mutants have been identified in this coarse cereal crop.</p><p>In this study, <i>SiLG1</i> and <i>SiLG2</i> were identified in foxtail millet through phylogenetic analysis (Figure S1). Both of <i>SiLG1</i> and <i>SiLG2</i> were mainly expressed in leaves, pulvinus, nodes and seeds of foxtail millet (Figure S2). A comprehensive screening analysis was conducted on sequencing data of 1844 worldwide <i>Setaria italica</i> germplasm, which included commercial var
{"title":"De novo creation of narrowed plant architecture via CRISPR/Cas9-mediated mutagenesis of SiLGs in foxtail millet","authors":"Renliang Zhang, Ruifeng Guo, Hui Zhi, Sha Tang, Liwei Wang, Yuemei Ren, Guangbing Ren, Shou Zhang, Jing Feng, Xianmin Diao, Guanqing Jia","doi":"10.1111/pbi.70037","DOIUrl":"https://doi.org/10.1111/pbi.70037","url":null,"abstract":"<p>Foxtail millet [<i>Setaria italica</i> (L.) Beauv] is an ancient cereal crop that has been cultivated for grain food and forage consumption globally for over 11 500 years. Despite its long history, foxtail millet continues to be extensively cultivated in developing and underdeveloped countries with low productivity conditions. This is primarily due to its exceptional ability to tolerate drought and barren environments, making it well-suited for grain production in challenging grown conditions. In the last century, significant progress has been made in enhancing the plant architecture of major cereal crops, such as maize (<i>Zea mays</i>) and rice (<i>Oryza sativa</i>), resulting in substantial increases in grain yield potential (Tian <i>et al</i>., <span>2024</span>). However, there has been relatively less emphasis on optimizing the plant architecture of foxtail millet, leading to limited improvements in yield for this ancient crop species to date. While the overexpression of <i>Drooping Leaf 1</i> has been shown to promote more upright leaves in foxtail millet (Zhao <i>et al</i>., <span>2020</span>), compact architecture with reduced leaf angle remains a challenge due to the scarcity of identified germplasm suitable for this purpose.</p>\u0000<p>Leaf angle, defined as the angle between the culm and leaf midrib, is a crucial aspect of plant architecture that is controlled by a limited number of functional genes. Among these genes, <i>Liguleless1</i> (<i>LG1</i>) and <i>Liguleless2</i> (<i>LG2</i>) are key regulators that play essential roles in lamina joint formation, influencing leaf angle in maize (Walsh <i>et al</i>., <span>1998</span>). <i>LG1</i> has been identified as a transcription factor belonging to the Squamosa Promoter-Binding Proteins (SBP) family, while <i>LG2</i> has been characterized as a basic leucine zipper transcription (bZIP) factor. Genetic analysis of variations indicates that <i>LG1</i> and <i>LG2</i> have the potential to significantly enhance the field productivity of maize varieties, and it has been demonstrated that the functions of <i>LG1</i> and <i>LG2</i> are conserved across various Gramineae crops, including maize (Harper and Freeling, <span>1996</span>), rice (Wang <i>et al</i>., <span>2021</span>), wheat (Yu, <span>2019</span>), sorghum (Brant <i>et al</i>., <span>2021</span>) and sugarcane (Brant <i>et al</i>., <span>2024</span>). However, the roles of <i>LG1</i> and <i>LG2</i> in foxtail millet remain unclear, and as of now, no ligule development-defective mutants have been identified in this coarse cereal crop.</p>\u0000<p>In this study, <i>SiLG1</i> and <i>SiLG2</i> were identified in foxtail millet through phylogenetic analysis (Figure S1). Both of <i>SiLG1</i> and <i>SiLG2</i> were mainly expressed in leaves, pulvinus, nodes and seeds of foxtail millet (Figure S2). A comprehensive screening analysis was conducted on sequencing data of 1844 worldwide <i>Setaria italica</i> germplasm, which included commercial var","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"215 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143695574","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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