Regeneration represents a fundamental biological process wherein an organism's tissues or organs repair and replace themselves following damage or environmental stress. In plant systems, injured tree branches can regenerate adventitious buds and develop new crowns through propagation techniques like cuttings and canopy pruning, while transgenic plants emerge via tissue culture in genetic engineering processes intimately connected to plant regeneration mechanisms. The advancement of plant regeneration technology is critical for addressing complex and dynamic climate challenges, ultimately ensuring global agricultural sustainability. This review comprehensively synthesizes the latest genetic transformation technologies, including transformation systems across woody, herbaceous and algal species, organellar genetic modifications, crucial regeneration factors facilitating Agrobacterium-mediated transformations, the intricate hormonal networks regulating plant regeneration, comparative analyses of transient transformation approaches and marker gene dynamics throughout transformation processes. Ultimately, the review offers novel perspectives on current transformation bottlenecks and proposes future research trajectories.
{"title":"Plant genetic transformation: achievements, current status and future prospects","authors":"Peilin Wang, Huan Si, Chenhui Li, Zhongping Xu, Huiming Guo, Shuangxia Jin, Hongmei Cheng","doi":"10.1111/pbi.70028","DOIUrl":"https://doi.org/10.1111/pbi.70028","url":null,"abstract":"Regeneration represents a fundamental biological process wherein an organism's tissues or organs repair and replace themselves following damage or environmental stress. In plant systems, injured tree branches can regenerate adventitious buds and develop new crowns through propagation techniques like cuttings and canopy pruning, while transgenic plants emerge via tissue culture in genetic engineering processes intimately connected to plant regeneration mechanisms. The advancement of plant regeneration technology is critical for addressing complex and dynamic climate challenges, ultimately ensuring global agricultural sustainability. This review comprehensively synthesizes the latest genetic transformation technologies, including transformation systems across woody, herbaceous and algal species, organellar genetic modifications, crucial regeneration factors facilitating Agrobacterium-mediated transformations, the intricate hormonal networks regulating plant regeneration, comparative analyses of transient transformation approaches and marker gene dynamics throughout transformation processes. Ultimately, the review offers novel perspectives on current transformation bottlenecks and proposes future research trajectories.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"29 6 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143569725","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}
H. X. Liu, T. Li, J. Hou, X. T. Yin, Y. Q. Wang, X. M. Si, Shoaib Ur Rehman, L. Zhuang, W. L. Guo, C. Y. Hao, X. Y. Zhang
Plant-specific WUSCHEL-related homeobox (Wox) transcription factors (TFs) are crucial for plant growth and development. However, the molecular mechanism of Wox-mediated regulation of thousand kernel weight (TKW) in crops remains elusive. In this research, we identified a major TKW-associated quantitative trait locus (QTL) on wheat chromosome 5DS by performing a genome-wide association study (GWAS) of a Chinese wheat mini-core collection (MCC) in four environments combined by bulked segregant analysis (BSA) and bulked segregant RNA-sequencing (BSR-seq) of wheat grains exhibiting a wide range of TKWs. The candidate TaWUS-like-5D was highly expressed in developing grains and was found to strongly negative influence grain TKW and wheat yield. Meanwhile, the RNAi lines, CRISPR/Cas9-edited single and double knockout mutants (AABBdd and AAbbdd), as well as the stop-gained aaBB Kronos mutants, exhibited a significant increase in grain size and TKW (P < 0.05 or P < 0.01) and a 10.0% increase in yield (P < 0.01). Further analyses indicated that TaWUS-like-5D regulates TKW by inhibiting the transcription of sucrose, hormone and trehalose metabolism-related genes, subsequently sharply decreasing starch synthesis in wheat grains. The results of this study provide a fundamental molecular basis for further elucidating the mechanism of Wox-mediated regulation of grain development in crops.
{"title":"TaWUS-like-5D affects grain weight and filling by inhibiting the expression of sucrose and trehalose metabolism-related genes in wheat grain endosperm","authors":"H. X. Liu, T. Li, J. Hou, X. T. Yin, Y. Q. Wang, X. M. Si, Shoaib Ur Rehman, L. Zhuang, W. L. Guo, C. Y. Hao, X. Y. Zhang","doi":"10.1111/pbi.70015","DOIUrl":"https://doi.org/10.1111/pbi.70015","url":null,"abstract":"Plant-specific <i>WUSCHEL-related homeobox</i> (<i>Wox</i>) transcription factors (TFs) are crucial for plant growth and development. However, the molecular mechanism of <i>Wox</i>-mediated regulation of thousand kernel weight (TKW) in crops remains elusive. In this research, we identified a major TKW-associated quantitative trait locus (QTL) on wheat chromosome 5DS by performing a genome-wide association study (GWAS) of a Chinese wheat mini-core collection (MCC) in four environments combined by bulked segregant analysis (BSA) and bulked segregant RNA-sequencing (BSR-seq) of wheat grains exhibiting a wide range of TKWs. The candidate <i>TaWUS-like-5D</i> was highly expressed in developing grains and was found to strongly negative influence grain TKW and wheat yield. Meanwhile, the RNAi lines, CRISPR/Cas9-edited single and double knockout mutants (AABB<i>dd</i> and AA<i>bbdd</i>), as well as the stop-gained <i>aa</i>BB Kronos mutants, exhibited a significant increase in grain size and TKW (<i>P</i> < 0.05 or <i>P</i> < 0.01) and a 10.0% increase in yield (<i>P</i> < 0.01). Further analyses indicated that <i>TaWUS-like-5D</i> regulates TKW by inhibiting the transcription of sucrose, hormone and trehalose metabolism-related genes, subsequently sharply decreasing starch synthesis in wheat grains. The results of this study provide a fundamental molecular basis for further elucidating the mechanism of <i>Wox</i>-mediated regulation of grain development in crops.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"91 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143560751","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}
Genome editing based on the homology-directed repair (HDR) pathway enables scar-free and precise genetic manipulations. However, the low frequency of HDR hinders its application in plant genome editing. In this study, we engineered the fusion of Cas9 and a viral replication protein (Rep) as a molecular bridge to tether donor DNA in vivo, which enhances the efficiency of targeted gene insertion via the HDR pathway. This Rep-bridged knock-in (RBKI) method combines the advantages of rolling cycle replication of viral replicons and in vivo enrichment of donor DNA at the target site for HDR. Chromatin immunoprecipitation indicated that the Cas9-Rep fusion protein bound up to 66-fold more donor DNA than Cas9 did. We exemplified the RBKI method by inserting small- to middle-sized tags (33–519 bp) into 3 rice genes. Compared to Cas9, Cas9-Rep fusion increased the KI frequencies by 4–7.6-fold, and up to 72.2% of stable rice transformants carried in-frame knock-in events in the T0 generation. Whole-genome sequencing of 6 plants segregated from heterozygous KI lines indicated that the knock-in events were faithfully inherited by the progenies with neither off-target editing nor random insertions of the donor DNA fragment. Further analysis suggested that the RBKI method reduced the number of byproducts from nonhomologous end joining; however, HDR-mediated knock-in tended to accompany microhomology-mediated end joining events. Together, these findings show that the in vivo tethering of donor DNAs with Cas9-Rep is an effective strategy to increase the frequency of HDR-mediated genome editing.
{"title":"Cas9-Rep fusion tethers donor DNA in vivo and boosts the efficiency of HDR-mediated genome editing","authors":"Zhentao Zhou, Jiahui Xiao, Shuai Yin, Yache Chen, Yang Yuan, Jianwei Zhang, Lizhong Xiong, Kabin Xie","doi":"10.1111/pbi.70036","DOIUrl":"https://doi.org/10.1111/pbi.70036","url":null,"abstract":"Genome editing based on the homology-directed repair (HDR) pathway enables scar-free and precise genetic manipulations. However, the low frequency of HDR hinders its application in plant genome editing. In this study, we engineered the fusion of Cas9 and a viral replication protein (Rep) as a molecular bridge to tether donor DNA <i>in vivo</i>, which enhances the efficiency of targeted gene insertion via the HDR pathway. This Rep-bridged knock-in (RBKI) method combines the advantages of rolling cycle replication of viral replicons and <i>in vivo</i> enrichment of donor DNA at the target site for HDR. Chromatin immunoprecipitation indicated that the Cas9-Rep fusion protein bound up to 66-fold more donor DNA than Cas9 did. We exemplified the RBKI method by inserting small- to middle-sized tags (33–519 bp) into 3 rice genes. Compared to Cas9, Cas9-Rep fusion increased the KI frequencies by 4–7.6-fold, and up to 72.2% of stable rice transformants carried in-frame knock-in events in the T<sub>0</sub> generation. Whole-genome sequencing of 6 plants segregated from heterozygous KI lines indicated that the knock-in events were faithfully inherited by the progenies with neither off-target editing nor random insertions of the donor DNA fragment. Further analysis suggested that the RBKI method reduced the number of byproducts from nonhomologous end joining; however, HDR-mediated knock-in tended to accompany microhomology-mediated end joining events. Together, these findings show that the <i>in vivo</i> tethering of donor DNAs with Cas9-Rep is an effective strategy to increase the frequency of HDR-mediated genome editing.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"35 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143545988","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}
Hongyao Zhu, Tiange Zhou, Jiaming Guan, Zhuo Li, Xiurong Yang, Yuejiao Li, Jian Sun, Quan Xu, Yuan Hu Xuan
The primary goals of crop breeding are to enhance yield and improve disease resistance. However, the “trade-off” mechanism, in which signalling pathways for resistance and yield are antagonistically regulated, poses challenges for achieving both simultaneously. Previously, we demonstrated that knock-out mutants of the Dense and Erect Panicle 1 (DEP1) gene can significantly enhance rice resistance to sheath blight (ShB), and we mapped DEP1's association with panicle length. In this study, we discovered that dep1 mutants significantly reduced rice yield. Nonetheless, truncated DEP1 was able to achieve both ShB resistance and yield increase in japonica rice. To further explore the function of truncated DEP1 in promoting yield and ShB resistance, we generated CRISPR/Cas9-mediated genome editing mutants, including a full-length deletion mutant of DEP1, named dep1, and a truncated version, dep1-cys. Upon inoculation with Rhizoctonia solani, the dep1-cys mutant demonstrated stronger ShB resistance than the dep1 mutant. Additionally, dep1-cys increased yield per plant, whereas dep1 reduced it. Compared to the full DEP1 protein, the truncated DEP1 (dep1-cys) demonstrated a decreased interaction affinity with IDD14 and increased affinity with IDD10, which are known to positively and negatively regulate ShB resistance through the activation of PIN1a and ETR2, respectively. The dep1-cys mutant exhibited higher PIN1a and lower ETR2 expression than wild-type plants, suggesting that dep1-cys modulated IDD14 and IDD10 interactions to regulate PIN1a and ETR2, thereby enhancing ShB resistance. Overall, these data indicate that precise genome editing of DEP1 could simultaneously improve both ShB resistance and yield, effectively mitigating trade-off regulation in rice.
{"title":"Precise genome editing of Dense and Erect Panicle 1 promotes rice sheath blight resistance and yield production in japonica rice","authors":"Hongyao Zhu, Tiange Zhou, Jiaming Guan, Zhuo Li, Xiurong Yang, Yuejiao Li, Jian Sun, Quan Xu, Yuan Hu Xuan","doi":"10.1111/pbi.70010","DOIUrl":"https://doi.org/10.1111/pbi.70010","url":null,"abstract":"The primary goals of crop breeding are to enhance yield and improve disease resistance. However, the “trade-off” mechanism, in which signalling pathways for resistance and yield are antagonistically regulated, poses challenges for achieving both simultaneously. Previously, we demonstrated that knock-out mutants of the <i>Dense and Erect Panicle 1</i> (<i>DEP1</i>) gene can significantly enhance rice resistance to sheath blight (ShB), and we mapped <i>DEP1</i>'s association with panicle length. In this study, we discovered that <i>dep1</i> mutants significantly reduced rice yield. Nonetheless, truncated DEP1 was able to achieve both ShB resistance and yield increase in japonica rice. To further explore the function of truncated <i>DEP1</i> in promoting yield and ShB resistance, we generated CRISPR/Cas9-mediated genome editing mutants, including a full-length deletion mutant of <i>DEP1</i>, named <i>dep1</i>, and a truncated version, <i>dep1-cys</i>. Upon inoculation with <i>Rhizoctonia solani</i>, the <i>dep1-cys</i> mutant demonstrated stronger ShB resistance than the <i>dep1</i> mutant. Additionally, <i>dep1-cys</i> increased yield per plant, whereas <i>dep1</i> reduced it. Compared to the full DEP1 protein, the truncated DEP1 (dep1-cys) demonstrated a decreased interaction affinity with IDD14 and increased affinity with IDD10, which are known to positively and negatively regulate ShB resistance through the activation of <i>PIN1a</i> and <i>ETR2</i>, respectively. The <i>dep1-cys</i> mutant exhibited higher <i>PIN1a</i> and lower <i>ETR2</i> expression than wild-type plants, suggesting that <i>dep1-cys</i> modulated IDD14 and IDD10 interactions to regulate <i>PIN1a</i> and <i>ETR2</i>, thereby enhancing ShB resistance. Overall, these data indicate that precise genome editing of <i>DEP1</i> could simultaneously improve both ShB resistance and yield, effectively mitigating trade-off regulation in rice.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"67 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143539328","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}
Muhammad Azhar Hussain, Yong Huang, Dan Luo, Sundas Saher Mehmood, Ali Raza, Xuekun Zhang, Yong Cheng, Hongtao Cheng, Xiling Zou, Xiaoyu Ding, Liu Zeng, Liu Duan, Bian Wu, Keming Hu, Yan Lv
SummaryBrassica napus L. (B. napus) is a major edible oil crop grown around the southern part of China, which often faces cold stress, posing potential damage to vegetative tissues. To sustain growth and reproduction, a detailed understanding of fundamental regulatory processes in B. napus against long‐term low temperature (LT) stress is necessary for breeders to adjust the level of LT adaption in a given region and is therefore of great economic importance. Till now, studies on microRNAs (miRNAs) in coping with LT adaption in B. napus are limited. Here, we performed an in‐depth analysis on two B. napus varieties with distinct adaptability to LT stress. Through integration of RNA sequencing (RNA‐seq) and small RNA‐sequencing (sRNA‐seq), we identified 106 modules comprising differentially expressed miRNAs and corresponding potential targets based on strong negative correlations between their dynamic expression patterns. Specifically, we demonstrated that Bna‐miR397a post‐transcriptionally regulates a LACCASE (LAC) gene, BnaLAC2, to enhance the adaption to LT stresses in B. napus by reducing the total lignin remodelling and ROS homeostasis. In addition, the miR397–LAC2 module was also proved to improve freezing tolerance of Arabidopsis, indicating a conserved role of miR397–LAC2 in Cruciferae plants. Overall, this work provides the first description of a miRNA‐mediated‐module signature for LT adaption and highlights the prominent role of laccase in future breeding programme of LT tolerant B. napus.
{"title":"Integrative analyses reveal Bna‐miR397a–BnaLAC2 as a potential modulator of low‐temperature adaptability in Brassica napus L.","authors":"Muhammad Azhar Hussain, Yong Huang, Dan Luo, Sundas Saher Mehmood, Ali Raza, Xuekun Zhang, Yong Cheng, Hongtao Cheng, Xiling Zou, Xiaoyu Ding, Liu Zeng, Liu Duan, Bian Wu, Keming Hu, Yan Lv","doi":"10.1111/pbi.70017","DOIUrl":"https://doi.org/10.1111/pbi.70017","url":null,"abstract":"Summary<jats:italic>Brassica napus</jats:italic> L. (<jats:italic>B. napus</jats:italic>) is a major edible oil crop grown around the southern part of China, which often faces cold stress, posing potential damage to vegetative tissues. To sustain growth and reproduction, a detailed understanding of fundamental regulatory processes in <jats:italic>B. napus</jats:italic> against long‐term low temperature (LT) stress is necessary for breeders to adjust the level of LT adaption in a given region and is therefore of great economic importance. Till now, studies on microRNAs (miRNAs) in coping with LT adaption in <jats:italic>B. napus</jats:italic> are limited. Here, we performed an in‐depth analysis on two <jats:italic>B. napus</jats:italic> varieties with distinct adaptability to LT stress. Through integration of RNA sequencing (RNA‐seq) and small RNA‐sequencing (sRNA‐seq), we identified 106 modules comprising differentially expressed miRNAs and corresponding potential targets based on strong negative correlations between their dynamic expression patterns. Specifically, we demonstrated that <jats:italic>Bna‐miR397a</jats:italic> post‐transcriptionally regulates a LACCASE (LAC) gene, <jats:italic>BnaLAC2</jats:italic>, to enhance the adaption to LT stresses in <jats:italic>B. napus</jats:italic> by reducing the total lignin remodelling and ROS homeostasis. In addition, the <jats:italic>miR397</jats:italic>–<jats:italic>LAC2</jats:italic> module was also proved to improve freezing tolerance of <jats:italic>Arabidopsis</jats:italic>, indicating a conserved role of <jats:italic>miR397</jats:italic>–<jats:italic>LAC2</jats:italic> in <jats:italic>Cruciferae</jats:italic> plants. Overall, this work provides the first description of a miRNA‐mediated‐module signature for LT adaption and highlights the prominent role of laccase in future breeding programme of LT tolerant <jats:italic>B. napus</jats:italic>.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"90 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143538484","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}
Basanta Lamichhane, Sarah-Eve Gélinas, Natacha Merindol, Manoj Koirala, Karen Cristine Gonçalves dos Santos, Hugo Germain, Isabel Desgagné-Penix
Amaryllidaceae alkaloids (AAs) are diverse bioactive metabolites with significant pharmaceutical potential, derived from 4′-O-methylnorbelladine (4′OM). The biosynthesis of these compounds involves the condensation of tyramine and 3,4-dihydroxybenzaldehyde by norbelladine synthase (NBS) and/or noroxomaritidine/norcraugsodine reductase (NR), followed by O-methylation. Cytochrome P450 enzymes, particularly the CYP96T family, introduce further structural diversity through C–C couplings, resulting in lycorine, galanthamine and crinine cores. Despite their importance, the exact biosynthetic pathways remain poorly defined. In this study, we describe key enzymes from Leucojum aestivum (La), providing crucial insight into AA biosynthesis. Transient expression in Nicotiana benthamiana demonstrated that LaNBS and LaNRII catalyse the conversion of tyramine and 3,4-dihydroxybenzaldehyde to norbelladine, which is subsequently O-methylated by a norbelladine-4′-O-methyltransferase (LaN4′OMT) in planta. Co-agroinfiltration of LaNBS, LaNRII, LaN4′OMT and LaCYP96T1 resulted in the production of various phenol-coupled products, with lycorine as the predominant compound, alongside haemanthamine, crinine/vittatine and norgalanthamine. This study identifies LaCYP96T1 and LaCYP96T2 as the first monocot enzymes capable of catalysing all three regioselective C-C phenol couplings and also highlights the substrate promiscuity of LaNRII. The findings not only elucidate critical steps in AA biosynthesis but also open new avenues for biotechnological application in producing valuable alkaloids, offering potential for novel drug development.
{"title":"Elucidating the enzyme network driving Amaryllidaceae alkaloids biosynthesis in Leucojum aestivum","authors":"Basanta Lamichhane, Sarah-Eve Gélinas, Natacha Merindol, Manoj Koirala, Karen Cristine Gonçalves dos Santos, Hugo Germain, Isabel Desgagné-Penix","doi":"10.1111/pbi.70026","DOIUrl":"https://doi.org/10.1111/pbi.70026","url":null,"abstract":"<i>Amaryllidaceae</i> alkaloids (AAs) are diverse bioactive metabolites with significant pharmaceutical potential, derived from 4′-O-methylnorbelladine (4′<i>O</i>M). The biosynthesis of these compounds involves the condensation of tyramine and 3,4-dihydroxybenzaldehyde by norbelladine synthase (NBS) and/or noroxomaritidine/norcraugsodine reductase (NR), followed by <i>O</i>-methylation. Cytochrome P450 enzymes, particularly the CYP96T family, introduce further structural diversity through C–C couplings, resulting in lycorine, galanthamine and crinine cores. Despite their importance, the exact biosynthetic pathways remain poorly defined. In this study, we describe key enzymes from <i>Leucojum aestivum</i> (<i>La</i>), providing crucial insight into AA biosynthesis. Transient expression in <i>Nicotiana benthamiana</i> demonstrated that <i>La</i>NBS and <i>La</i>NRII catalyse the conversion of tyramine and 3,4-dihydroxybenzaldehyde to norbelladine, which is subsequently <i>O</i>-methylated by a norbelladine-4′-<i>O</i>-methyltransferase (<i>La</i>N4′<i>O</i>MT) <i>in planta</i>. Co-agroinfiltration of <i>La</i>NBS, <i>La</i>NRII, <i>La</i>N4′<i>O</i>MT and <i>La</i>CYP96T1 resulted in the production of various phenol-coupled products, with lycorine as the predominant compound, alongside haemanthamine, crinine/vittatine and norgalanthamine. This study identifies <i>La</i>CYP96T1 and <i>La</i>CYP96T2 as the first monocot enzymes capable of catalysing all three regioselective C-C phenol couplings and also highlights the substrate promiscuity of <i>La</i>NRII. The findings not only elucidate critical steps in AA biosynthesis but also open new avenues for biotechnological application in producing valuable alkaloids, offering potential for novel drug development.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"11 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143539332","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>Foxtail millet (<i>Setaria italica</i>), one of the oldest crops originating in China, has increasingly been recognized as a promising C<sub>4</sub> model plant due to its compact diploid genome, short growth cycle and self-pollinating nature (Li and Brutnell, <span>2011</span>). In the past 5 years, significant breakthroughs have been achieved in its basic research and breeding, including high efficient transformation system establishment, telomere-to-telomere (T2T) genome assembly, pan-genome analysis and functional studies (He <i>et al</i>., <span>2023</span>; Tang <i>et al</i>., <span>2023</span>; Yang <i>et al</i>., <span>2020</span>). However, the limited genetic diversity of breeding materials and inefficiencies in identifying target mutants have continued to pose significant challenges in breeding for improved complex agronomic traits and in functional genomics research of this crop.</p> <p>To broaden novel sources of genetic diversity and enhance mutant identification efficiency for foxtail millet improvement, we constructed a large EMS-mutagenized library and a data-sharing platform based on the model variety Ci846 (www.setariadb.com/millet/mutation, Figure S3), which is a domesticated Setaria variety with well-established transformation system and efficient indoor research platform (Wang <i>et al</i>., <span>2022</span>). We obtained 9243 M<sub>2</sub> lines, of which 775 (8.38%) displayed various morphological variations under field conditions (Figure 1a,b, Table S14 and Figure S4). We sampled 4109 M<sub>2</sub> plants and re-sequenced them using a mixed pool strategy, and obtained 1950.76 Gb of pair-end reads from 205 mixed pools, with each pool ranging from 8.6 to 12.2 Gb and an average sequencing depth of 22x (Figure 1c). This represents the largest-scale precise genotype data for mutant libraries in crop species to date. The cleaned short-read sequences were then mapped to the Yugu1-T2T reference genome. The mapping rate and coverage rate of sequencing reads were 98.21 ± 0.37% and 94.24 ± 0.37%, respectively (Table S3). A total of 2,899,449 variations were identified across the 205 mixed pools, with an average mutation density of 1/29.70 Kb across all nine chromosomes (Figure 1d). Each mixed pool contained 1,658,170 ± 11,422 SNPs and 252,784 ± 2402 indels. Among the pools, A173 had the smallest number of variations (1,875,480), while A9 had the largest number (1,989,562) (Table S4). We found that C/G to T/A nucleotide transitions were the most prevalent type (928,081, 38.7%) (Table S7) and the Thr/Val to Ala amino acid substitutions occurred at higher frequency (1.42% and 1.46%) than the average amino acid changed rate (0.16%) (Figure 1e, Table S9). Among all samples, indel size ranged from 1 to 31 bp, with an average of 12 bp, and the most frequent indel type was 1 bp deletions (104,581) (Table S5). A total of 2,368,808 mutations occurred in intergenic regions, followed by 1,850,547 upstream variants, 1,770,535 downstream vari
{"title":"An efficient target-mutant screening platform of model variety Ci846 facilitates genetic studies of Setaria","authors":"Hui Zhang, Hongkai Liang, Hui Zhi, Di Yuan, Qi Yao, Renliang Zhang, Lihe Xing, Baolan Yang, Luman Sang, Lirong Zhao, Sha Tang, Liwei Wang, Hailong Wang, Yushuang Ren, Hui Zhang, Yanyan Zhang, Enbo Wang, Xinyu Man, Gongjin Xu, Linlin Zhang, Qiang He, Xianmin Diao, Guanqing Jia","doi":"10.1111/pbi.14594","DOIUrl":"https://doi.org/10.1111/pbi.14594","url":null,"abstract":"<p>Foxtail millet (<i>Setaria italica</i>), one of the oldest crops originating in China, has increasingly been recognized as a promising C<sub>4</sub> model plant due to its compact diploid genome, short growth cycle and self-pollinating nature (Li and Brutnell, <span>2011</span>). In the past 5 years, significant breakthroughs have been achieved in its basic research and breeding, including high efficient transformation system establishment, telomere-to-telomere (T2T) genome assembly, pan-genome analysis and functional studies (He <i>et al</i>., <span>2023</span>; Tang <i>et al</i>., <span>2023</span>; Yang <i>et al</i>., <span>2020</span>). However, the limited genetic diversity of breeding materials and inefficiencies in identifying target mutants have continued to pose significant challenges in breeding for improved complex agronomic traits and in functional genomics research of this crop.</p>\u0000<p>To broaden novel sources of genetic diversity and enhance mutant identification efficiency for foxtail millet improvement, we constructed a large EMS-mutagenized library and a data-sharing platform based on the model variety Ci846 (www.setariadb.com/millet/mutation, Figure S3), which is a domesticated Setaria variety with well-established transformation system and efficient indoor research platform (Wang <i>et al</i>., <span>2022</span>). We obtained 9243 M<sub>2</sub> lines, of which 775 (8.38%) displayed various morphological variations under field conditions (Figure 1a,b, Table S14 and Figure S4). We sampled 4109 M<sub>2</sub> plants and re-sequenced them using a mixed pool strategy, and obtained 1950.76 Gb of pair-end reads from 205 mixed pools, with each pool ranging from 8.6 to 12.2 Gb and an average sequencing depth of 22x (Figure 1c). This represents the largest-scale precise genotype data for mutant libraries in crop species to date. The cleaned short-read sequences were then mapped to the Yugu1-T2T reference genome. The mapping rate and coverage rate of sequencing reads were 98.21 ± 0.37% and 94.24 ± 0.37%, respectively (Table S3). A total of 2,899,449 variations were identified across the 205 mixed pools, with an average mutation density of 1/29.70 Kb across all nine chromosomes (Figure 1d). Each mixed pool contained 1,658,170 ± 11,422 SNPs and 252,784 ± 2402 indels. Among the pools, A173 had the smallest number of variations (1,875,480), while A9 had the largest number (1,989,562) (Table S4). We found that C/G to T/A nucleotide transitions were the most prevalent type (928,081, 38.7%) (Table S7) and the Thr/Val to Ala amino acid substitutions occurred at higher frequency (1.42% and 1.46%) than the average amino acid changed rate (0.16%) (Figure 1e, Table S9). Among all samples, indel size ranged from 1 to 31 bp, with an average of 12 bp, and the most frequent indel type was 1 bp deletions (104,581) (Table S5). A total of 2,368,808 mutations occurred in intergenic regions, followed by 1,850,547 upstream variants, 1,770,535 downstream vari","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"8 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143532514","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}
Yarin Livneh, Ehud Leor-Librach, Dor Agmon, Tal Makov-Bouaniche, Vivekanand Tiwari, Ekaterina Shor, Yelena Yeselson, Tania Masci, Arthur Schaffer, Dana Charuvi, Joseph Hirschberg, Alexander Vainstein
Lettuce is widely grown and consumed but provides lower nutritional value compared to other leafy greens, particularly in the essential vitamins A and C. To address this, major control points in carotenoid and ascorbic acid (AsA) production were targeted using a viral-based CRISPR/Cas9 system in the commercial lettuce cultivar ‘Noga’. Knockout of lycopene ε-cyclase (LCY-ε), the enzymatic gatekeeper opposing production of β-branch carotenoids, increased β-carotene (provitamin A) levels up to 2.7-fold and facilitated zeaxanthin accumulation up to 4.3 μg/g fresh weight. Chlorophyll fluorescence measurements revealed that photosystem II efficiency was unaffected in LCY-ε mutants, though their non-photochemical quenching (NPQ) capacity decreased at light intensities above 400 μmol m2 s-1. However, the gene-edited plants exhibited normal growth and comparable plant mass, despite the absence of two major lettuce xanthophylls, lutein and lactucaxanthin. Modifications in a regulatory region in the upstream ORF of GDP-L-galactose phosphorylase 1 and 2 (uGGP1 and uGGP2), the rate-limiting enzyme in AsA production, resulted in an average 6.9-fold increase in AsA levels. The mutation in uGGP2 was found to dominantly influence AsA over-accumulation. Knockout lines that combined the mutations in LCY-ε, uGGP1, uGGP2 and in carotenoid cleavage dioxygenase 4a (CCD4a), an isozyme involved in β-carotene degradation in lettuce, exhibited significantly enhanced content of AsA, β-carotene and zeaxanthin. Our results demonstrate the potential of multi-pathway gene editing to ‘supercharge’ economically important crops such as lettuce as a means to address micronutrient deficiencies in modern diets.
{"title":"Combined enhancement of ascorbic acid, β-carotene and zeaxanthin in gene-edited lettuce","authors":"Yarin Livneh, Ehud Leor-Librach, Dor Agmon, Tal Makov-Bouaniche, Vivekanand Tiwari, Ekaterina Shor, Yelena Yeselson, Tania Masci, Arthur Schaffer, Dana Charuvi, Joseph Hirschberg, Alexander Vainstein","doi":"10.1111/pbi.70018","DOIUrl":"https://doi.org/10.1111/pbi.70018","url":null,"abstract":"Lettuce is widely grown and consumed but provides lower nutritional value compared to other leafy greens, particularly in the essential vitamins A and C. To address this, major control points in carotenoid and ascorbic acid (AsA) production were targeted using a viral-based CRISPR/Cas9 system in the commercial lettuce cultivar ‘Noga’. Knockout of <i>lycopene ε-cyclase</i> (<i>LCY-ε</i>), the enzymatic gatekeeper opposing production of β-branch carotenoids, increased β-carotene (provitamin A) levels up to 2.7-fold and facilitated zeaxanthin accumulation up to 4.3 μg/g fresh weight. Chlorophyll fluorescence measurements revealed that photosystem II efficiency was unaffected in <i>LCY-ε</i> mutants, though their non-photochemical quenching (NPQ) capacity decreased at light intensities above 400 μmol m<sup>2</sup> s<sup>-1</sup>. However, the gene-edited plants exhibited normal growth and comparable plant mass, despite the absence of two major lettuce xanthophylls, lutein and lactucaxanthin. Modifications in a regulatory region in the upstream ORF of <i>GDP-L-galactose phosphorylase 1</i> and <i>2</i> (u<i>GGP1</i> and u<i>GGP2</i>), the rate-limiting enzyme in AsA production, resulted in an average 6.9-fold increase in AsA levels. The mutation in u<i>GGP2</i> was found to dominantly influence AsA over-accumulation. Knockout lines that combined the mutations in <i>LCY-ε</i>, u<i>GGP1</i>, u<i>GGP2</i> and in <i>carotenoid cleavage dioxygenase 4a</i> (<i>CCD4a</i>), an isozyme involved in β-carotene degradation in lettuce, exhibited significantly enhanced content of AsA, β-carotene and zeaxanthin. Our results demonstrate the potential of multi-pathway gene editing to ‘supercharge’ economically important crops such as lettuce as a means to address micronutrient deficiencies in modern diets.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"36 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143532515","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>Fluorescein diacetate (FDA), a cell membrane-permeable esterase substrate, is widely used to assess cell viability. The esterase in viable cells catalyses hydrolysis of diacetate to produce green fluorescein (Rotman and Papermaster, <span>1966</span>). As FDA fluorescence is positively correlated with reactive oxygen species (ROS) levels, it also is used to assess ROS biogenesis (Huang <i>et al</i>., <span>2023</span>). Propidium iodide (PI) is a nuclear dye that produces red fluorescence in inactivated cells, and is usually used to detect apoptosis and inactivated cells (Riccardi and Nicoletti, <span>2006</span>). Greissl (<span>1989</span>) first used FDA/PI staining to assess pollen viability by which viable and aborted pollen grains can be simultaneously identified. Ascari <i>et al</i>. (<span>2020</span>) combined FDA/PI staining with a software to automatically assess viability of mature pollen.</p> <p>The current FDA/PI methods can only be used to analyse mature pollen because the FDA fluorescent signal produced in developing microspores is weak and quenches very quickly. The commonly used potassium iodide-iodine (I<sub>2</sub>-KI) staining method is also suitable only for mature pollen with accumulated starch. Therefore, the current staining methods are inadequate for investigating viability and abortion processes throughout male development. The precise characterisation of pollen abortion processes usually uses cumbersome, expensive and time-consuming paraffin sectioning. Therefore, we aimed to develop an effective method to simply and efficiently detect abortion in microspores and pollen at different development stages.</p> <p>We first used an FDA/PI solution containing higher concentrations of FDA (130 μg/mL) and PI (87 μg/mL) than previous reports, which was diluted in dH<sub>2</sub>O as described (Jones <i>et al</i>., <span>2016</span>), to analyse microspore viability at tetrad stage (S8b stage) in wild-type rice (<i>Oryza sativa</i> L.). Although FDA/PI/dH<sub>2</sub>O-stained tetrad microspores produced green fluorescence imaged by a confocal microscopy, the green signal was quenched quickly within three minutes, followed by red fluorescence generated by PI (Figure 1a), indicating that the microspores became inactivated quickly in this solution.</p> <figure><picture> <source media="(min-width: 1650px)" srcset="/cms/asset/7e45d6ac-d16a-4f9c-bc1f-3f04d2fe6116/pbi14607-fig-0001-m.jpg"/><img alt="Details are in the caption following the image" data-lg-src="/cms/asset/7e45d6ac-d16a-4f9c-bc1f-3f04d2fe6116/pbi14607-fig-0001-m.jpg" loading="lazy" src="/cms/asset/1941bb72-18d7-40ce-9845-12bde1cfc304/pbi14607-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>FDA/PI fluorescent staining of rice microspores and pollen. The green and r
{"title":"An improved fluorescein diacetate–propidium iodide staining system for assessing microspore and pollen viability at different developmental stages","authors":"Jianian Tang, Qiyu Luo, Huali Li, Zichen Wu, Zhansheng Lin, Sensen Zhang, Letian Chen, Yao-Guang Liu","doi":"10.1111/pbi.14607","DOIUrl":"https://doi.org/10.1111/pbi.14607","url":null,"abstract":"<p>Fluorescein diacetate (FDA), a cell membrane-permeable esterase substrate, is widely used to assess cell viability. The esterase in viable cells catalyses hydrolysis of diacetate to produce green fluorescein (Rotman and Papermaster, <span>1966</span>). As FDA fluorescence is positively correlated with reactive oxygen species (ROS) levels, it also is used to assess ROS biogenesis (Huang <i>et al</i>., <span>2023</span>). Propidium iodide (PI) is a nuclear dye that produces red fluorescence in inactivated cells, and is usually used to detect apoptosis and inactivated cells (Riccardi and Nicoletti, <span>2006</span>). Greissl (<span>1989</span>) first used FDA/PI staining to assess pollen viability by which viable and aborted pollen grains can be simultaneously identified. Ascari <i>et al</i>. (<span>2020</span>) combined FDA/PI staining with a software to automatically assess viability of mature pollen.</p>\u0000<p>The current FDA/PI methods can only be used to analyse mature pollen because the FDA fluorescent signal produced in developing microspores is weak and quenches very quickly. The commonly used potassium iodide-iodine (I<sub>2</sub>-KI) staining method is also suitable only for mature pollen with accumulated starch. Therefore, the current staining methods are inadequate for investigating viability and abortion processes throughout male development. The precise characterisation of pollen abortion processes usually uses cumbersome, expensive and time-consuming paraffin sectioning. Therefore, we aimed to develop an effective method to simply and efficiently detect abortion in microspores and pollen at different development stages.</p>\u0000<p>We first used an FDA/PI solution containing higher concentrations of FDA (130 μg/mL) and PI (87 μg/mL) than previous reports, which was diluted in dH<sub>2</sub>O as described (Jones <i>et al</i>., <span>2016</span>), to analyse microspore viability at tetrad stage (S8b stage) in wild-type rice (<i>Oryza sativa</i> L.). Although FDA/PI/dH<sub>2</sub>O-stained tetrad microspores produced green fluorescence imaged by a confocal microscopy, the green signal was quenched quickly within three minutes, followed by red fluorescence generated by PI (Figure 1a), indicating that the microspores became inactivated quickly in this solution.</p>\u0000<figure><picture>\u0000<source media=\"(min-width: 1650px)\" srcset=\"/cms/asset/7e45d6ac-d16a-4f9c-bc1f-3f04d2fe6116/pbi14607-fig-0001-m.jpg\"/><img alt=\"Details are in the caption following the image\" data-lg-src=\"/cms/asset/7e45d6ac-d16a-4f9c-bc1f-3f04d2fe6116/pbi14607-fig-0001-m.jpg\" loading=\"lazy\" src=\"/cms/asset/1941bb72-18d7-40ce-9845-12bde1cfc304/pbi14607-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>FDA/PI fluorescent staining of rice microspores and pollen. The green and r","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"31 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143517826","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>Alfalfa (<i>Medicago sativa</i> L.), a highly valuable perennial forage legume, is extensively cultivated worldwide (Russelle, <span>2001</span>). As global warming exacerbates evaporation rates, severe drought conditions, characterized by mud cracking, have increasingly affected alfalfa cultivation regions. Drought stress can decrease stomatal conductance, impair photosynthesis activity and induce reactive oxygen species (ROS) accumulation in Alfalfa plants. Therefore, it reduces alfalfa growth and accelerates flowering, leading to significant declines in biomass yield and forage quality. Previous studies have shown that the plant-specific NAC (NAM, ATAF1,2 and CUC2) transcription factors play crucial roles in plant response to diverse environmental stresses. For example, NACs are involved in cold response of tomato, salt tolerance of soybean and disease resistance of <i>Arabidopsis</i>. Recent studies have also highlighted that OsNAC120 and OsNAC016 regulated the balance between plant growth and drought tolerance by promoting gibberellin (GA) biosynthesis, brassinosteroid (BR) signalling and repressing abscisic acid (ABA)-mediated drought responses (Wu <i>et al</i>., <span>2022</span>; Xie <i>et al</i>., <span>2024</span>). These insights provide a framework for developing crop varieties with improved biomass yield under drought conditions.</p> <p>The <i>Medicago truncatula</i> NAC transcription factor MtNAC33 (Medtr3g096140), one member of the NAC2 subfamily, clusters phylogenetically with <i>Arabidopsis</i> NAC082 and NAC103 (Figure S1). Previous studies revealed that <i>MtNAC33</i> is induced by mannitol and NaCl treatments in <i>Medicago</i> seedlings (Ling <i>et al</i>., <span>2017</span>), but its biological functions remain largely unexplored. To elucidate the role of <i>MtNAC33</i> in drought tolerance, its expression was analysed under mannitol-simulated drought and NaCl-induced salt stress. Results confirmed significant induction of <i>MtNAC33</i> expression in these stress conditions (Figure 1a; Figure S2). To assess its functional role, <i>MtNAC33</i> was overexpressed in <i>Arabidopsis thaliana</i>. Two transgenic lines, MtNAC33OE-A and MtNAC33OE-C, with the highest <i>MtNAC33</i> expression levels, were selected for analysis. Under drought stress (10 days without watering), MtNAC33OE plants exhibited enhanced drought resistance (Figure S3) but showed no significant differences with wild-type plants under salt stress (Figure S4).</p> <figure><picture> <source media="(min-width: 1650px)" srcset="/cms/asset/df058da8-84ac-4d32-b519-67950b50e458/pbi14597-fig-0001-m.jpg"/><img alt="Details are in the caption following the image" data-lg-src="/cms/asset/df058da8-84ac-4d32-b519-67950b50e458/pbi14597-fig-0001-m.jpg" loading="lazy" src="/cms/asset/a97debe3-c7b9-48e4-b492-e0ce7b3352ce/pbi14597-fig-0001-m.png" title="Details are in the caption following the image"/></picture><figcaption> <div><strong>Figure 1<span style="font-weight:normal
{"title":"Overexpression of MtNAC33 enhances biomass yield and drought tolerance in alfalfa","authors":"Ruijuan Yang, Ying Sun, Yan Zhao, Chen Bai, Yaling Liu, Jingzhe Sun, Zhaoming Wang, Feng Yuan, Xiaoshan Wang, Wenwen Liu, Chunxiang Fu","doi":"10.1111/pbi.14597","DOIUrl":"https://doi.org/10.1111/pbi.14597","url":null,"abstract":"<p>Alfalfa (<i>Medicago sativa</i> L.), a highly valuable perennial forage legume, is extensively cultivated worldwide (Russelle, <span>2001</span>). As global warming exacerbates evaporation rates, severe drought conditions, characterized by mud cracking, have increasingly affected alfalfa cultivation regions. Drought stress can decrease stomatal conductance, impair photosynthesis activity and induce reactive oxygen species (ROS) accumulation in Alfalfa plants. Therefore, it reduces alfalfa growth and accelerates flowering, leading to significant declines in biomass yield and forage quality. Previous studies have shown that the plant-specific NAC (NAM, ATAF1,2 and CUC2) transcription factors play crucial roles in plant response to diverse environmental stresses. For example, NACs are involved in cold response of tomato, salt tolerance of soybean and disease resistance of <i>Arabidopsis</i>. Recent studies have also highlighted that OsNAC120 and OsNAC016 regulated the balance between plant growth and drought tolerance by promoting gibberellin (GA) biosynthesis, brassinosteroid (BR) signalling and repressing abscisic acid (ABA)-mediated drought responses (Wu <i>et al</i>., <span>2022</span>; Xie <i>et al</i>., <span>2024</span>). These insights provide a framework for developing crop varieties with improved biomass yield under drought conditions.</p>\u0000<p>The <i>Medicago truncatula</i> NAC transcription factor MtNAC33 (Medtr3g096140), one member of the NAC2 subfamily, clusters phylogenetically with <i>Arabidopsis</i> NAC082 and NAC103 (Figure S1). Previous studies revealed that <i>MtNAC33</i> is induced by mannitol and NaCl treatments in <i>Medicago</i> seedlings (Ling <i>et al</i>., <span>2017</span>), but its biological functions remain largely unexplored. To elucidate the role of <i>MtNAC33</i> in drought tolerance, its expression was analysed under mannitol-simulated drought and NaCl-induced salt stress. Results confirmed significant induction of <i>MtNAC33</i> expression in these stress conditions (Figure 1a; Figure S2). To assess its functional role, <i>MtNAC33</i> was overexpressed in <i>Arabidopsis thaliana</i>. Two transgenic lines, MtNAC33OE-A and MtNAC33OE-C, with the highest <i>MtNAC33</i> expression levels, were selected for analysis. Under drought stress (10 days without watering), MtNAC33OE plants exhibited enhanced drought resistance (Figure S3) but showed no significant differences with wild-type plants under salt stress (Figure S4).</p>\u0000<figure><picture>\u0000<source media=\"(min-width: 1650px)\" srcset=\"/cms/asset/df058da8-84ac-4d32-b519-67950b50e458/pbi14597-fig-0001-m.jpg\"/><img alt=\"Details are in the caption following the image\" data-lg-src=\"/cms/asset/df058da8-84ac-4d32-b519-67950b50e458/pbi14597-fig-0001-m.jpg\" loading=\"lazy\" src=\"/cms/asset/a97debe3-c7b9-48e4-b492-e0ce7b3352ce/pbi14597-fig-0001-m.png\" title=\"Details are in the caption following the image\"/></picture><figcaption>\u0000<div><strong>Figure 1<span style=\"font-weight:normal","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"22 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2025-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143517827","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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