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1.
Physiol Plant ; 176(3): e14378, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38887925

RESUMO

D-2-hydroxyglutarate dehydrogenase (D2HGDH) is a mitochondrial enzyme containing flavin adenine dinucleotide FAD, existing as a dimer, and it facilitates the specific oxidation of D-2HG to 2-oxoglutarate (2-OG), which is a key intermediate in the tricarboxylic acid (TCA) cycle. A Genome-wide expression analysis (GWEA) has indicated an association between GhD2HGDH and flowering time. To further explore the role of GhD2HGDH, we performed a comprehensive investigation encompassing phenotyping, physiology, metabolomics, and transcriptomics in Arabidopsis thaliana plants overexpressing GhD2HGDH. Transcriptomic and qRT-PCR data exhibited heightened expression of GhD2HGDH in upland cotton flowers. Additionally, early-maturing cotton exhibited higher expression of GhD2HGDH across all tissues than delayed-maturing cotton. Subcellular localization confirmed its presence in the mitochondria. Overexpression of GhD2HGDH in Arabidopsis resulted in early flowering. Using virus-induced gene silencing (VIGS), we investigated the impact of GhD2HGDH on flowering in both early- and delayed-maturing cotton plants. Manipulation of GhD2HGDH expression levels led to changes in photosynthetic pigment and gas exchange attributes. GhD2HGDH responded to gibberellin (GA3) hormone treatment, influencing the expression of GA biosynthesis genes and repressing DELLA genes. Protein interaction studies, including yeast two-hybrid, luciferase complementation (LUC), and GST pull-down assays, confirmed the interaction between GhD2HGDH and GhSOX (Sulfite oxidase). The metabolomics analysis demonstrated GhD2HGDH's modulation of the TCA cycle through alterations in various metabolite levels. Transcriptome data revealed that GhD2HGDH overexpression triggers early flowering by modulating the GA3 and photoperiodic pathways of the flowering core factor genes. Taken together, GhD2HGDH positively regulates the network of genes associated with early flowering pathways.


Assuntos
Arabidopsis , Flores , Regulação da Expressão Gênica de Plantas , Giberelinas , Gossypium , Fotoperíodo , Proteínas de Plantas , Gossypium/genética , Gossypium/fisiologia , Gossypium/metabolismo , Flores/genética , Flores/fisiologia , Arabidopsis/genética , Arabidopsis/fisiologia , Arabidopsis/metabolismo , Giberelinas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/genética , Plantas Geneticamente Modificadas , Transporte de Elétrons
2.
Nat Commun ; 15(1): 5224, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38890293

RESUMO

Continued climate change impose multiple stressors on crops, including pathogens, salt, and drought, severely impacting agricultural productivity. Innovative solutions are necessary to develop resilient crops. Here, using quantitative potato proteomics, we identify Parakletos, a thylakoid protein that contributes to disease susceptibility. We show that knockout or silencing of Parakletos enhances resistance to oomycete, fungi, bacteria, salt, and drought, whereas its overexpression reduces resistance. In response to biotic stimuli, Parakletos-overexpressing plants exhibit reduced amplitude of reactive oxygen species and Ca2+ signalling, and silencing Parakletos does the opposite. Parakletos homologues have been identified in all major crops. Consecutive years of field trials demonstrate that Parakletos deletion enhances resistance to Phytophthora infestans and increases yield. These findings demark a susceptibility gene, which can be exploited to enhance crop resilience towards abiotic and biotic stresses in a low-input agriculture.


Assuntos
Doenças das Plantas , Proteínas de Plantas , Solanum tuberosum , Estresse Fisiológico , Solanum tuberosum/genética , Solanum tuberosum/microbiologia , Solanum tuberosum/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estresse Fisiológico/genética , Doenças das Plantas/microbiologia , Doenças das Plantas/genética , Espécies Reativas de Oxigênio/metabolismo , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Secas , Phytophthora infestans , Plantas Geneticamente Modificadas , Produtos Agrícolas/genética , Produtos Agrícolas/microbiologia , Deleção de Genes , Proteômica
3.
Cells ; 13(11)2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38891020

RESUMO

Improving the drought resistance of rice is of great significance for expanding the planting area and improving the stable yield of rice. In our previous work, we found that ROLLED AND ERECT LEAF1 (REL1) protein promoted enhanced tolerance to drought stress by eliminating reactive oxygen species (ROS) levels and triggering the abscisic acid (ABA) response. However, the mechanism through which REL1 regulates drought tolerance by removing ROS is unclear. In this study, we identified REL1 interacting protein 5 (RIP5) and found that it directly combines with REL1 in the chloroplast. We found that RIP5 was strongly expressed in ZH11 under drought-stress conditions, and that the rip5-ko mutants significantly improved the tolerance of rice plants to drought, whereas overexpression of RIP5 resulted in greater susceptibility to drought. Further investigation suggested that RIP5 negatively regulated drought tolerance in rice by decreasing the content of ascorbic acid (AsA), thereby reducing ROS clearance. RNA sequencing showed that the knockout of RIP5 caused differential gene expression that is chiefly associated with ascorbate and aldarate metabolism. Furthermore, multiple experimental results suggest that REL1 is involved in regulating drought tolerance by inhibiting RIP5. Collectively, our findings reveal the importance of the inhibition of RIP5 by REL1 in affecting the rice's response to drought stress. This work not only explains the drought tolerance mechanism of rice, but will also help to improve the drought tolerance of rice.


Assuntos
Secas , Regulação da Expressão Gênica de Plantas , Oryza , Proteínas de Plantas , Espécies Reativas de Oxigênio , Oryza/genética , Oryza/metabolismo , Oryza/fisiologia , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico , Ácido Abscísico/metabolismo , Cloroplastos/metabolismo , Adaptação Fisiológica/genética , Plantas Geneticamente Modificadas , Ácido Ascórbico/metabolismo , Ligação Proteica , Resistência à Seca
4.
PeerJ ; 12: e17402, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38860212

RESUMO

Background: Global food systems in recent years have been impacted by some harsh environmental challenges and excessive anthropogenic activities. The increasing levels of both biotic and abiotic stressors have led to a decline in food production, safety, and quality. This has also contributed to a low crop production rate and difficulty in meeting the requirements of the ever-growing population. Several biotic stresses have developed above natural resistance in crops coupled with alarming contamination rates. In particular, the multiple antibiotic resistance in bacteria and some other plant pathogens has been a hot topic over recent years since the food system is often exposed to contamination at each of the farm-to-fork stages. Therefore, a system that prioritizes the safety, quality, and availability of foods is needed to meet the health and dietary preferences of everyone at every time. Methods: This review collected scattered information on food systems and proposes methods for plant disease management. Multiple databases were searched for relevant specialized literature in the field. Particular attention was placed on the genetic methods with special interest in the potentials of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and Cas (CRISPR associated) proteins technology in food systems and security. Results: The review reveals the approaches that have been developed to salvage the problem of food insecurity in an attempt to achieve sustainable agriculture. On crop plants, some systems tend towards either enhancing the systemic resistance or engineering resistant varieties against known pathogens. The CRISPR-Cas technology has become a popular tool for engineering desired genes in living organisms. This review discusses its impact and why it should be considered in the sustainable management, availability, and quality of food systems. Some important roles of CRISPR-Cas have been established concerning conventional and earlier genome editing methods for simultaneous modification of different agronomic traits in crops. Conclusion: Despite the controversies over the safety of the CRISPR-Cas system, its importance has been evident in the engineering of disease- and drought-resistant crop varieties, the improvement of crop yield, and enhancement of food quality.


Assuntos
Sistemas CRISPR-Cas , Produtos Agrícolas , Edição de Genes , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Produtos Agrícolas/genética , Produção Agrícola/métodos , Plantas Geneticamente Modificadas/genética , Doenças das Plantas/prevenção & controle , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Abastecimento de Alimentos
5.
Plant Cell Rep ; 43(7): 167, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38865016

RESUMO

KEY MESSAGE: 63 L. bicolor WRKY genes were identified and their informatics was analyzed. The results suggested that the LbWRKY genes involved in the development and salt secretion of salt glands in L. bicolor. Salt stress, as a universal abiotic stress, severely inhibits the growth and development of plants. WRKY transcription factors play a vital role in plant growth and development, as well as in response to various stresses. Nevertheless, little is known of systematic genome-wide analysis of the WRKY genes in Limonium bicolor, a model recretohalophyte. In this study, 63 L. bicolor WRKY genes were identified (LbWRKY1-63), which were unevenly distributed across seven chromosomes and one scaffold. Based on the structural and phylogenetic characteristics, 63 LbWRKYs are divided into three main groups. Cis-elements in the LbWRKY promoters were related to growth and development, phytohormone responses, and stress responses. Colinearity analysis showed strong colinearity between LbWRKYs and GmWRKYs from soybean (Glycine max). Therefore, LbWRKY genes maybe have similar functions to GmWRKY genes. Expression analysis showed that 28 LbWRKY genes are highly expressed in roots, 9 in stems, 26 in leaves, and 12 in flowers and most LbWRKY genes responded to NaCl, ABA, and PEG6000. Silencing LbWRKY10 reduced salt gland density and salt secretion ability of leaves, and the salt tolerance of the species. Consistent with this, genes associated with salt gland development were markedly down-regulated in the LbWRKY10-silenced lines. Our findings suggested that the LbWRKY genes involved in the development and salt secretion of salt glands in L. bicolor. Our research provides new insights into the functions of the WRKY family in halophytes.


Assuntos
Regulação da Expressão Gênica de Plantas , Família Multigênica , Filogenia , Proteínas de Plantas , Plumbaginaceae , Tolerância ao Sal , Plantas Tolerantes a Sal , Fatores de Transcrição , Plumbaginaceae/genética , Plumbaginaceae/fisiologia , Plantas Tolerantes a Sal/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tolerância ao Sal/genética , Estresse Salino/genética , Estresse Fisiológico/genética , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas/genética , Genes de Plantas
6.
Mol Biol Rep ; 51(1): 731, 2024 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-38869677

RESUMO

BACKGROUND: Chitinase (Chi) is a pathogenesis-related protein, also reported to play an important role in plant responses to abiotic stress. However, its role in response to abiotic stress in barley is still unclear. RESULTS: In this study, a total of 61 Chi gene family members were identified from the whole genome of wild barley EC_S1. Phylogenetic analysis suggested that these family genes were divided into five groups. Among these genes, four pairs of collinearity genes were discovered. Besides, abundant cis-regulatory elements, including drought response element and abscisic acid response element were identified in the promoter regions of HvChi gene family members. The expression profiles revealed that most HvChi family members were significantly up-regulated under drought stress, which was also validated by RT-qPCR measurements. To further explore the role of Chi under drought stress, HvChi22 was overexpressed in Arabidopsis. Compared to wild-type plants, overexpression of HvChi22 enhanced drought tolerance by increasing the activity of oxidative protective enzymes, which caused less MDA accumulation. CONCLUSION: Our study improved the understanding of the Chi gene family under drought stress in barley, and provided a theoretical basis for crop improvement strategies to address the challenges posed by changing environmental conditions.


Assuntos
Quitinases , Secas , Regulação da Expressão Gênica de Plantas , Hordeum , Família Multigênica , Filogenia , Proteínas de Plantas , Estresse Fisiológico , Hordeum/genética , Quitinases/genética , Quitinases/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Estresse Fisiológico/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Arabidopsis/genética , Regiões Promotoras Genéticas/genética , Plantas Geneticamente Modificadas/genética , Perfilação da Expressão Gênica/métodos , Resistência à Seca
7.
Methods Mol Biol ; 2832: 281-290, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38869803

RESUMO

CRISPR/Cas9 system is one of the most often utilized engineering tools for genome editing in many organisms including crop plants and presents great value in both basic and applied research. This is a preferred method because of its relative simplicity, cost-effectiveness, and reliability. The Cas9 nuclease guided by a short single guide RNA (gRNA) can generate double-strand DNA breaks (DSB) at the specific sites in chromosomal DNA. The DSB site is repaired by error-prone repair methods. During repair, some nucleotides are deleted or added at the target site. Here, we present a simplified protocol for generating mutants in gene of interest in rice using CRISPR/Cas9.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Oryza , RNA Guia de Sistemas CRISPR-Cas , Oryza/genética , Edição de Genes/métodos , RNA Guia de Sistemas CRISPR-Cas/genética , Mutação , Plantas Geneticamente Modificadas/genética , Quebras de DNA de Cadeia Dupla , Estresse Fisiológico/genética
8.
BMC Plant Biol ; 24(1): 536, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38862890

RESUMO

BACKGROUND: The heavy metal-associated isoprenylated plant protein (HIPP) is an important regulatory element in response to abiotic stresses, especially playing a key role in low-temperature response. RESULTS: This study investigated the potential function of PavHIPP16 up-regulated in sweet cherry under cold stress by heterologous overexpression in tobacco. The results showed that the overexpression (OE) lines' growth state was better than wild type (WT), and the germination rate, root length, and fresh weight of OE lines were significantly higher than those of WT. In addition, the relative conductivity and malondialdehyde (MDA) content of the OE of tobacco under low-temperature treatment were substantially lower than those of WT. In contrast, peroxidase (POD), superoxide dismutase (SOD), catalase (CAT) activities, hydrogen peroxide (H2O2), proline, soluble protein, and soluble sugar contents were significantly higher than those of WT. Yeast two-hybrid assay (Y2H) and luciferase complementation assay verified the interactions between PavbHLH106 and PavHIPP16, suggesting that these two proteins co-regulated the cold tolerance mechanism in plants. The research results indicated that the transgenic lines could perform better under low-temperature stress by increasing the antioxidant enzyme activity and osmoregulatory substance content of the transgenic plants. CONCLUSIONS: This study provides genetic resources for analyzing the biological functions of PavHIPPs, which is important for elucidating the mechanisms of cold resistance in sweet cherry.


Assuntos
Nicotiana , Proteínas de Plantas , Plantas Geneticamente Modificadas , Prunus avium , Nicotiana/genética , Nicotiana/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Prunus avium/genética , Prunus avium/fisiologia , Prunus avium/metabolismo , Resposta ao Choque Frio/genética , Temperatura Baixa , Regulação da Expressão Gênica de Plantas
9.
Funct Plant Biol ; 512024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38870342

RESUMO

Genetic transformation is helpful in enhancing crops, utilising promoters that can be constitutive, inducible, or tissue-specific. However, the use of constitutive promoters may hinder plant growth due to energy consumption during cellular processes. To optimise transgene effects, tissue-specific promoters like root-specific ones prove valuable in addressing root-related issues and enhancing productivity. Yet, identified root-specific promoters in crop are limited. To address this gap, the expression pattern of the root-specific SlREO promoter was examined across various crops. Sequencing confirmed its identity and high homology (99%) with the NCBI database, distinct from other plants tested. Using the PLACE database, six motifs associated with root expression were identified, along with several other important elements. The 2.4kb SlREO promoter was linked to a ß-glucuronidase (GUS) reporter gene alongside the CaMV35S promoter in pRI 201-AN-GUS vectors to study its expression. Histochemistry revealed strong root-specific expression in tomato (Solanum lycopersicum ) root tissues and limited expression in stems. However, the SlREO promoter did not consistently maintain its root-specific expression in other plants. Conversely, the CaMV35S promoter exhibited constitutive expression across all tissues in various plants. This study underscores the potential of the SlREO promoter as a root-specific regulatory element, offering avenues for improving crops, particularly against environmental stresses.


Assuntos
Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Raízes de Plantas , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Solanum lycopersicum , Solanum lycopersicum/genética , Raízes de Plantas/genética , Plantas Geneticamente Modificadas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Glucuronidase/genética , Glucuronidase/metabolismo , Sequência de Bases
10.
Physiol Plant ; 176(3): e14379, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38853306

RESUMO

Drought stress threatens the productivity of numerous crops, including chilli pepper (Capsicum annuum). DnaJ proteins are known to play a protective role against a wide range of abiotic stresses. This study investigates the regulatory mechanism of the chloroplast-targeted chaperone protein AdDjSKI, derived from wild peanut (Arachis diogoi), in enhancing drought tolerance in chilli peppers. Overexpressing AdDjSKI in chilli plants increased chlorophyll content, reflected in the maximal photochemical efficiency of photosystem II (PSII) (Fv/Fm) compared with untransformed control (UC) plants. This enhancement coincided with the upregulated expression of PSII-related genes. Our subsequent investigations revealed that transgenic chilli pepper plants expressing AdDjSKI showed reduced accumulation of superoxide and hydrogen peroxide and, consequently, lower malondialdehyde levels and decreased relative electrolyte leakage percentage compared with UC plants. The mitigation of ROS-mediated oxidative damage was facilitated by heightened activities of antioxidant enzymes, including superoxide dismutase, catalase, ascorbate peroxidase, and peroxidase, coinciding with the upregulation of the expression of associated antioxidant genes. Additionally, our observations revealed that the ectopic expression of the AdDjSKI protein in chilli pepper plants resulted in diminished ABA sensitivity, consequently promoting seed germination in comparison with UC plants under different concentrations of ABA. All of these collectively contributed to enhancing drought tolerance in transgenic chilli plants with improved root systems when compared with UC plants. Overall, our study highlights AdDjSKI as a promising biotechnological solution for enhancing drought tolerance in chilli peppers, addressing the growing global demand for this economically valuable crop.


Assuntos
Ácido Abscísico , Capsicum , Secas , Fotossíntese , Plantas Geneticamente Modificadas , Espécies Reativas de Oxigênio , Capsicum/fisiologia , Capsicum/genética , Capsicum/metabolismo , Fotossíntese/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Ácido Abscísico/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Arachis/genética , Arachis/fisiologia , Arachis/metabolismo , Regulação da Expressão Gênica de Plantas , Complexo de Proteína do Fotossistema II/metabolismo , Clorofila/metabolismo , Antioxidantes/metabolismo , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Resistência à Seca
11.
Theor Appl Genet ; 137(7): 154, 2024 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-38856926

RESUMO

KEY MESSAGE: Our findings highlight a valuable breeding resource, demonstrating the potential to concurrently enhance grain shape, thermotolerance, and alkaline tolerance by manipulating Gγ protein in rice. Temperate Geng/Japonica (GJ) rice yields have improved significantly, bolstering global food security. However, GJ rice breeding faces challenges, including enhancing grain quality, ensuring stable yields at warmer temperatures, and utilizing alkaline land. In this study, we employed CRISPR/Cas9 gene-editing technology to knock out the GS3 locus in seven elite GJ varieties with superior yield performance. Yield component measurements revealed that GS3 knockout mutants consistently enhanced grain length and reduced plant height in diverse genetic backgrounds. The impact of GS3 on the grain number per panicle and setting rate depended on the genetic background. GS3 knockout did not affect milling quality and minimally altered protein and amylose content but notably influenced chalkiness-related traits. GS3 knockout indiscriminately improved heat and alkali stress tolerance in the GJ varieties studied. Transcriptome analysis indicated differential gene expression between the GS3 mutants and their wild-type counterparts, enriched in biological processes related to photosynthesis, photosystem II stabilization, and pathways associated with photosynthesis and cutin, suberine, and wax biosynthesis. Our findings highlight GS3 as a breeding resource for concurrently improving grain shape, thermotolerance, and alkaline tolerance through Gγ protein manipulation in rice.


Assuntos
Grão Comestível , Oryza , Melhoramento Vegetal , Proteínas de Plantas , Termotolerância , Oryza/genética , Oryza/fisiologia , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Termotolerância/genética , Grão Comestível/genética , Grão Comestível/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Fenótipo , Edição de Genes , Álcalis , Sistemas CRISPR-Cas , Plantas Geneticamente Modificadas/genética
12.
Plant Mol Biol ; 114(3): 69, 2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38842584

RESUMO

Petunias are renowned ornamental species widely cultivated as pot plants for their aesthetic appeal both indoors and outdoors. The preference for pot plants depends on their compact growth habit and abundant flowering. While genome editing has gained significant popularity in many crop plants in addressing growth and development and abiotic and biotic stress factors, relatively less emphasis has been placed on its application in ornamental plant species. Genome editing in ornamental plants opens up possibilities for enhancing their aesthetic qualities, offering innovative opportunities for manipulating plant architecture and visual appeal through precise genetic modifications. In this study, we aimed to optimize the procedure for an efficient genome editing system in petunia plants using the highly efficient multiplexed CRISPR/Cas9 system. Specifically, we targeted a total of six genes in Petunia which are associated with plant architecture traits, two paralogous of FLOWERING LOCUS T (PhFT) and four TERMINAL FLOWER-LIKE1 (PhTFL1) paralogous genes separately in two constructs. We successfully induced homogeneous and heterogeneous indels in the targeted genes through precise genome editing, resulting in significant phenotypic alterations in petunia. Notably, the plants harboring edited PhTFL1 and PhFT exhibited a conspicuously early flowering time in comparison to the wild-type counterparts. Furthermore, mutants with alterations in the PhTFL1 demonstrated shorter internodes than wild-type, likely by downregulating the gibberellic acid pathway genes PhGAI, creating a more compact and aesthetically appealing phenotype. This study represents the first successful endeavor to produce compact petunia plants with increased flower abundance through genome editing. Our approach holds immense promise to improve economically important potting plants like petunia and serve as a potential foundation for further improvements in similar ornamental plant species.


Assuntos
Sistemas CRISPR-Cas , Flores , Edição de Genes , Petunia , Proteínas de Plantas , Plantas Geneticamente Modificadas , Petunia/genética , Petunia/crescimento & desenvolvimento , Flores/genética , Flores/crescimento & desenvolvimento , Edição de Genes/métodos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Mutagênese , Regulação da Expressão Gênica de Plantas , Fenótipo
13.
Sci Adv ; 10(23): eadm7452, 2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38848363

RESUMO

Understanding CRISPR-Cas9's capacity to produce native overexpression (OX) alleles would accelerate agronomic gains achievable by gene editing. To generate OX alleles with increased RNA and protein abundance, we leveraged multiplexed CRISPR-Cas9 mutagenesis of noncoding sequences upstream of the rice PSBS1 gene. We isolated 120 gene-edited alleles with varying non-photochemical quenching (NPQ) capacity in vivo-from knockout to overexpression-using a high-throughput screening pipeline. Overexpression increased OsPsbS1 protein abundance two- to threefold, matching fold changes obtained by transgenesis. Increased PsbS protein abundance enhanced NPQ capacity and water-use efficiency. Across our resolved genetic variation, we identify the role of 5'UTR indels and inversions in driving knockout/knockdown and overexpression phenotypes, respectively. Complex structural variants, such as the 252-kb duplication/inversion generated here, evidence the potential of CRISPR-Cas9 to facilitate significant genomic changes with negligible off-target transcriptomic perturbations. Our results may inform future gene-editing strategies for hypermorphic alleles and have advanced the pursuit of gene-edited, non-transgenic rice plants with accelerated relaxation of photoprotection.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Mutagênese , Oryza , Oryza/genética , Edição de Genes/métodos , Alelos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Transgenes , Regulação da Expressão Gênica de Plantas
14.
Plant Mol Biol ; 114(4): 73, 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38874648

RESUMO

Functional genomics through transgenesis has provided faster and more reliable methods for identifying, characterizing, and utilizing genes or quantitative trait loci linked to agronomic traits to target yield. The present study explored the role of Big Grain1 (BG1) gene of rice (Oryza sativa L.) in yield improvement of crop plants. We aimed to identify the genetic variation of OsBG1 in various indica rice cultivars by studying the allelic polymorphism of the gene, while also investigating the gene's potential to increase crop yield through the transgenic approach. Our study reports the presence of an extra 393 bp sequence having two 6 bp enhancer elements in the 3' regulatory sequence of OsBG1 in the large-grain cultivar IR64 but not in the small-grain cultivar Badshahbhog. A single copy of the OsBG1 gene in both the cultivars and a 4.1-fold higher expression of OsBG1 in IR64 than in Badshahbhog imply that the grain size is positively correlated with the level of OsBG1 expression in rice. The ectopic expression of OsBG1 under the endosperm-specific glutelin C promoter in Badshahbhog enhanced the flag leaf length, panicle weight, and panicle length by an average of 33.2%, 33.7%, and 30.5%, respectively. The length of anthers, spikelet fertility, and grain yield per plant increased in transgenic rice lines by an average of 27.5%, 8.3%, and 54.4%, respectively. Heterologous expression of OsBG1 under the constitutive 2xCaMV35S promoter improved the number of seed pods per plant and seed yield per plant in transgenic tobacco lines by an average of 2.2-fold and 2.6-fold, respectively. Improving crop yield is crucial to ensure food security and socio-economic stability, and identifying suitable genetic factor is the essential step towards this endeavor. Our findings suggest that the OsBG1 gene is a promising candidate for improving the grain yield of monocot and dicot plant systems by molecular breeding and genetic engineering.


Assuntos
Grão Comestível , Regulação da Expressão Gênica de Plantas , Nicotiana , Oryza , Proteínas de Plantas , Plantas Geneticamente Modificadas , Oryza/genética , Oryza/crescimento & desenvolvimento , Nicotiana/genética , Nicotiana/crescimento & desenvolvimento , Grão Comestível/genética , Grão Comestível/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas/genética , Sementes/genética , Sementes/crescimento & desenvolvimento
15.
Plant Cell Rep ; 43(7): 172, 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38874775

RESUMO

KEY MESSAGE: The heat stress transcription factor HSFA2e regulates both temperature and drought response via hormonal and secondary metabolism alterations. High temperature and drought are the primary yield-limiting environmental constraints for staple food crops. Heat shock transcription factors (HSF) terminally regulate the plant abiotic stress responses to maintain growth and development under extreme environmental conditions. HSF genes of subclass A2 predominantly express under heat stress (HS) and activate the transcriptional cascade of defense-related genes. In this study, a highly heat-inducible HSF, HvHSFA2e was constitutively expressed in barley (Hordeum vulgare L.) to investigate its role in abiotic stress response and plant development. Transgenic barley plants displayed enhanced heat and drought tolerance in terms of increased chlorophyll content, improved membrane stability, reduced lipid peroxidation, and less accumulation of ROS in comparison to wild-type (WT) plants. Transcriptome analysis revealed that HvHSFA2e positively regulates the expression of abiotic stress-related genes encoding HSFs, HSPs, and enzymatic antioxidants, contributing to improved stress tolerance in transgenic plants. The major genes of ABA biosynthesis pathway, flavonoid, and terpene metabolism were also upregulated in transgenics. Our findings show that HvHSFA2e-mediated upregulation of heat-responsive genes, modulation in ABA and flavonoid biosynthesis pathways enhance drought and heat stress tolerance.


Assuntos
Secas , Regulação da Expressão Gênica de Plantas , Resposta ao Choque Térmico , Hordeum , Reguladores de Crescimento de Plantas , Proteínas de Plantas , Plantas Geneticamente Modificadas , Hordeum/genética , Hordeum/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Resposta ao Choque Térmico/genética , Reguladores de Crescimento de Plantas/metabolismo , Fatores de Transcrição de Choque Térmico/genética , Fatores de Transcrição de Choque Térmico/metabolismo , Clorofila/metabolismo , Estresse Fisiológico/genética , Metabolismo Secundário/genética , Redes e Vias Metabólicas/genética , Resistência à Seca
16.
BMC Biotechnol ; 24(1): 37, 2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38825715

RESUMO

BACKGROUND: As part of a publicly funded initiative to develop genetically engineered Brassicas (cabbage, cauliflower, and canola) expressing Bacillus thuringiensis Crystal (Cry)-encoded insecticidal (Bt) toxin for Indian and Australian farmers, we designed several constructs that drive high-level expression of modified Cry1B and Cry1C genes (referred to as Cry1BM and Cry1CM; with M indicating modified). The two main motivations for modifying the DNA sequences of these genes were to minimise any licensing cost associated with the commercial cultivation of transgenic crop plants expressing CryM genes, and to remove or alter sequences that might adversely affect their activity in plants. RESULTS: To assess the insecticidal efficacy of the Cry1BM/Cry1CM genes, constructs were introduced into the model Brassica Arabidopsis thaliana in which Cry1BM/Cry1CM expression was directed from either single (S4/S7) or double (S4S4/S7S7) subterranean clover stunt virus (SCSV) promoters. The resulting transgenic plants displayed a high-level of Cry1BM/Cry1CM expression. Protein accumulation for Cry1CM ranged from 5.18 to 176.88 µg Cry1CM/g dry weight of leaves. Contrary to previous work on stunt promoters, we found no correlation between the use of either single or double stunt promoters and the expression levels of Cry1BM/Cry1CM genes, with a similar range of Cry1CM transcript abundance and protein content observed from both constructs. First instar Diamondback moth (Plutella xylostella) larvae fed on transgenic Arabidopsis leaves expressing the Cry1BM/Cry1CM genes showed 100% mortality, with a mean leaf damage score on a scale of zero to five of 0.125 for transgenic leaves and 4.2 for wild-type leaves. CONCLUSIONS: Our work indicates that the modified Cry1 genes are suitable for the development of insect resistant GM crops. Except for the PAT gene in the USA, our assessment of the intellectual property landscape of components presents within the constructs described here suggest that they can be used without the need for further licensing. This has the capacity to significantly reduce the cost of developing and using these Cry1M genes in GM crop plants in the future.


Assuntos
Arabidopsis , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias , Endotoxinas , Proteínas Hemolisinas , Plantas Geneticamente Modificadas , Plantas Geneticamente Modificadas/genética , Arabidopsis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Hemolisinas/genética , Animais , Endotoxinas/genética , Regiões Promotoras Genéticas/genética , Bacillus thuringiensis/genética , Mariposas/genética , Brassica/genética , Controle Biológico de Vetores/métodos , Inseticidas/farmacologia
17.
Plant Signal Behav ; 19(1): 2359257, 2024 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-38825861

RESUMO

Potassium (K+) plays a role in enzyme activation, membrane transport, and osmotic regulation processes. An increase in potassium content can significantly improve the elasticity and combustibility of tobacco and reduce the content of harmful substances. Here, we report that the expression analysis of Nt GF14e, a 14-3-3 gene, increased markedly after low-potassium treatment (LK). Then, chlorophyll content, POD activity and potassium content, were significantly increased in overexpression of Nt GF14e transgenic tobacco lines compared with those in the wild type plants. The net K+ efflux rates were severely lower in the transgenic plants than in the wild type under LK stress. Furthermore, transcriptome analysis identified 5708 upregulated genes and 2787 downregulated genes between Nt GF14e overexpressing transgenic tobacco plants. The expression levels of some potassium-related genes were increased, such as CBL-interacting protein kinase 2 (CIPK2), Nt CIPK23, Nt CIPK25, H+-ATPase isoform 2 a (AHA2a), Nt AHA4a, Stelar K+ outward rectifier 1(SKOR1), and high affinity K+ transporter 5 (HAK5). The result of yeast two-hybrid and luciferase complementation imaging experiments suggested Nt GF14e could interact with CIPK2. Overall, these findings indicate that NtGF14e plays a vital roles in improving tobacco LK tolerance and enhancing potassium nutrition signaling pathways in tobacco plants.


Assuntos
Proteínas 14-3-3 , Regulação da Expressão Gênica de Plantas , Nicotiana , Proteínas de Plantas , Plantas Geneticamente Modificadas , Potássio , Nicotiana/genética , Nicotiana/metabolismo , Proteínas 14-3-3/metabolismo , Proteínas 14-3-3/genética , Potássio/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/metabolismo , Estresse Fisiológico/genética
18.
Mol Plant Pathol ; 25(6): e13483, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38829344

RESUMO

As a universal second messenger, cytosolic calcium (Ca2+) functions in multifaceted intracellular processes, including growth, development and responses to biotic/abiotic stresses in plant. The plant-specific Ca2+ sensors, calmodulin and calmodulin-like (CML) proteins, function as members of the second-messenger system to transfer Ca2+ signal into downstream responses. However, the functions of CMLs in the responses of cotton (Gossypium spp.) after Verticillium dahliae infection, which causes the serious vascular disease Verticillium wilt, remain elusive. Here, we discovered that the expression level of GbCML45 was promoted after V. dahliae infection in roots of cotton, suggesting its potential role in Verticillium wilt resistance. We found that knockdown of GbCML45 in cotton plants decreased resistance while overexpression of GbCML45 in Arabidopsis thaliana plants enhanced resistance to V. dahliae infection. Furthermore, there was physiological interaction between GbCML45 and its close homologue GbCML50 by using yeast two-hybrid and bimolecular fluorescence assays, and both proteins enhanced cotton resistance to V. dahliae infection in a Ca2+-dependent way in a knockdown study. Detailed investigations indicated that several defence-related pathways, including salicylic acid, ethylene, reactive oxygen species and nitric oxide signalling pathways, as well as accumulations of lignin and callose, are responsible for GbCML45- and GbCML50-modulated V. dahliae resistance in cotton. These results collectively indicated that GbCML45 and GbCML50 act as positive regulators to improve cotton Verticillium wilt resistance, providing potential targets for exploitation of improved Verticillium wilt-tolerant cotton cultivars by genetic engineering and molecular breeding.


Assuntos
Cálcio , Resistência à Doença , Gossypium , Doenças das Plantas , Proteínas de Plantas , Gossypium/microbiologia , Gossypium/genética , Gossypium/metabolismo , Gossypium/imunologia , Resistência à Doença/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Cálcio/metabolismo , Regulação da Expressão Gênica de Plantas , Calmodulina/metabolismo , Calmodulina/genética , Arabidopsis/microbiologia , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/metabolismo , Ascomicetos/fisiologia , Ascomicetos/patogenicidade , Plantas Geneticamente Modificadas , Verticillium/fisiologia , Verticillium/patogenicidade
19.
Physiol Plant ; 176(3): e14375, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38837224

RESUMO

MicroRNA(miRNA) is a class of non-coding small RNA that plays an important role in plant growth, development, and response to environmental stresses. Unlike most miRNAs, which usually target homologous genes across a variety of species, miR827 targets different types of genes in different species. Research on miR827 mainly focuses on its role in regulating phosphate (Pi) homeostasis of plants, however, little is known about its function in plant response to virus infection. In the present study, miR827 was significantly upregulated in the recovery tissue of virus-infected Nicotiana tabacum. Overexpression of miR827 could improve plants resistance to the infection of chilli veinal mottle virus (ChiVMV) in Nicotiana benthamiana, whereas interference of miR827 increased the susceptibility of the virus-infected plants. Further experiments indicated that the antiviral defence regulated by miR827 was associated with the reactive oxygen species and salicylic acid signalling pathways. Then, fructose-1,6-bisphosphatase (FBPase) was identified to be a target of miR827, and virus infection could affect the expression of FBPase. Finally, transient expression of FBPase increased the susceptibility to ChiVMV-GFP infection in N. benthamiana. By contrast, silencing of FBPase increased plant resistance. Taken together, our results demonstrate that miR827 plays a positive role in tobacco response to virus infection, thus providing new insights into understanding the role of miR827 in plant-virus interaction.


Assuntos
Resistência à Doença , Regulação da Expressão Gênica de Plantas , MicroRNAs , Nicotiana , Doenças das Plantas , Nicotiana/virologia , Nicotiana/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Doenças das Plantas/virologia , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Resistência à Doença/genética , Frutose-Bifosfatase/genética , Frutose-Bifosfatase/metabolismo , Ácido Salicílico/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tobamovirus/fisiologia , Tobamovirus/genética , Plantas Geneticamente Modificadas
20.
Physiol Plant ; 176(3): e14364, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38837226

RESUMO

Phytoremediation is a promising technology for removing the high-toxic explosive 2,4,6-trinitrotoluene (TNT) pollutant from the environment. Mining dominant genes is the key research direction of this technology. Most previous studies have focused on the detoxification of TNT rather than plants' TNT tolerance. Here, we conducted a transcriptomic analysis of wild type Arabidopsis plants under TNT stress and found that the Arabidopsis cytochrome P450 gene CYP81D11 was significantly induced in TNT-treated plants. Under TNT stress, the root length was approximately 1.4 times longer in CYP81D11-overexpressing transgenic plants than in wild type plants. The half-removal time for TNT was much shorter in CYP81D11-overexpressing transgenic plants (1.1 days) than in wild type plants (t1/2 = 2.2 day). In addition, metabolic analysis showed no difference in metabolites in transgenic plants compared to wild type plants. These results suggest that the high TNT uptake rates of CYP81D11-overexpressing transgenic plants were most likely due to increased tolerance and biomass rather than TNT degradation. However, CYP81D11-overexpressing plants were not more tolerant to osmotic stresses, such as salt or drought. Taken together, our results indicate that CYP81D11 is a promising target for producing bioengineered plants with high TNT removing capability.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Biodegradação Ambiental , Sistema Enzimático do Citocromo P-450 , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas , Trinitrotolueno , Arabidopsis/genética , Arabidopsis/metabolismo , Trinitrotolueno/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Estresse Fisiológico/genética
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