Hygromycin B-induced cell death is partly mediated by reactive oxygen species in rice (Oryza sativa L.)

Oung, Hui Min Olivia; Lin, Ke; Wu, Tzong‐Ming; Chandrika, Nulu Naga Prafulla; Hong, Chuang‐Ye

doi:10.1007/s11103-015-0380-4

Cited by 6 publications

(9 citation statements)

References 62 publications

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“…The chemical structure of hygromycin B is given in Scheme 1. [17] The hygromycin X (HygX) isozyme was characterized using crystallography methods and identified as a nonheme iron enzyme that utilizes dioxygen and α-KG. Moreover, it is involved in the ortho-δ-lactone ring formation between two pyran groups (Scheme 1) that acts as a precursor to the hygromycin B synthesis.…”

Section: Introductionmentioning

confidence: 99%

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Mechanism of Oxidative Ring‐Closure as Part of the Hygromycin Biosynthesis Step by a Nonheme Iron Dioxygenase

2021

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Nonheme iron dioxygenases catalyze vital reactions for biosystems including the biosynthesis of antibiotics. One such enzyme, namely the hygromycin biosynthesis enzyme (HygX), performs an oxidative ring-closure reaction to form an orthoÀ ester product, which is a relevant reaction step for drug synthesis and biotechnology. To understand the selective reaction mechanism of oxidative ring-closure to form orthoÀ ester products in HygX, we investigated its catalytic reaction mechanism leading to various products. Large active site cluster models were set-up and various pathways for substrate activation have been calculated. The work identifies a high-valent iron(IV)-oxo species in the quintet spin state as the active oxidant that selectively abstracts a proton of an alcohol group of the substrate, which is followed by a hydrogen atom abstraction from a tertiary CÀ H group and rapid electron transfer. The latter-formed biradical intermediate rearranges to form the desaturated ring-closed product. The calculations show that an active site Lys residue donates positive charge to the metalÀ oxo group and guides the reaction to a chemoselective desaturation pathway.

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Section: Introductionmentioning

confidence: 99%

“…The latter antibiotic was found to be toxic for plants and led to oxidative stress in rice. The chemical structure of hygromycin B is given in Scheme 1 [17] …”

Section: Introductionmentioning

confidence: 99%

Mechanism of Oxidative Ring‐Closure as Part of the Hygromycin Biosynthesis Step by a Nonheme Iron Dioxygenase

2021

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“…Therefore, developing a high-throughput screening platform for rapidly distinguishing transgenic or non-transgenic plants is required to efficiently generate transgene-free genome-edited plants. Our previous study showed that rice seedlings treated with HyB for 12 h markedly increased H 2 O 2 level in non-transformants but not in transgenic rice containing the HPT gene [33]. In this study, we further verified that genome-edited rice progenies containing HPT can also be faithfully reflected by visual detection of the DAB–H 2 O 2 complex in leaves and validated by RT-PCR, which indicates that the H 2 O 2 -based leaf painting assay is a rapid and reliable method.…”

Section: Discussionmentioning

confidence: 99%

“…After being taken up by plants, DAB reacts with H 2 O 2 to form a dark-brown reaction product in the presence of peroxidase [32]. Our recent research indicated that HyB significantly and rapidly enhanced the accumulation of H 2 O 2 in rice leaves [33]. Taking advantage of the high production of H 2 O 2 in plants induced by HyB, we aimed to develop a simple and quick, selection-independent, H 2 O 2 -based assay system for identifying transgenic rice.…”

Section: Introductionmentioning

confidence: 99%

H2O2-Based Method for Rapid Detection of Transgene-Free Rice Plants from Segregating CRISPR/Cas9 Genome-Edited Progenies

Huang

Oung

et al. 2019

IJMS

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Genome-editing techniques such as CRISPR/Cas9 have been widely used in crop functional genomics and improvement. To efficiently deliver the guide RNA and Cas9, most studies still rely on Agrobacterium-mediated transformation, which involves a selection marker gene. However, several limiting factors may impede the efficiency of screening transgene-free genome-edited plants, including the time needed to produce each life cycle, the response to selection reagents, and the labor costs of PCR-based genotyping. To overcome these disadvantages, we developed a simple and high-throughput method based on visual detection of antibiotics-derived H2O2 to verify transgene-free genome-edited plants. In transgenic rice containing hygromycin phosphotransferase (HPT), H2O2 content did not change in the presence of hygromycin B (HyB). In contrast, in transgenic-free rice plants with 10-h HyB treatment, levels of H2O2 and malondialdehyde, indicators of oxidative stress, were elevated. Detection of H2O2 by 3,3′-diaminobenzidine (DAB) staining suggested that H2O2 could be a marker to efficiently distinguish transgenic and non-transgenic plants. Analysis of 24 segregating progenies of an HPT-containing rice plant by RT-PCR and DAB staining verified that DAB staining is a feasible method for detecting transformants and non-transformants. Transgene-free genome-edited plants were faithfully validated by both PCR and the H2O2-based method. Moreover, HyB induced overproduction of H2O2 in leaves of Arabidopsis, maize, tobacco, and tomato, which suggests the potential application of the DAB method for detecting transgenic events containing HPT in a wide range of plant species. Thus, visual detection of DAB provides a simple, cheap, and reliable way to efficiently identify transgene-free genome-edited and HPT-containing transgenic rice.

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“…Leaf sections from the T 1 progeny of genome-edited rice were incubated with hygromycin B, an antibiotic commonly used for the positive selection of transgenic plants. In the presence of hygromycin B, the genome-edited rice plants that did not retain CRISPR/Cas vectors in their genome could produce reactive oxygen species (e.g., H 2 O 2 ) in their mitochondria and chloroplasts (Oung et al 2015). These elevated H 2 O 2 levels can be visualized directly using 3, 3'diaminobenzidine (DAB) staining (Wu et al 2019).…”

Section: Screening For Marker-free Crispr/cas9-induced Mutantsmentioning

confidence: 99%

How to start your monocot CRISPR/Cas project: plasmid design, efficiency detection, and offspring analysis

Yue

Hong

Wei

et al. 2020

Rice

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The breakthrough CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated genome-editing technology has led to great progress in monocot research; however, several factors need to be considered for the efficient implementation of this technology. To generate genome-edited crops, single guide (sg)RNA and Cas9 DNA are delivered into plant cells and expressed, and the predicted position is targeted. Analyses of successful targeted mutations have revealed that the expression levels, expression timing, and variants of both sgRNA and Cas9 need to be sophisticatedly regulated; therefore, the promoters of these genes and the target site positions are the key factors for genome-editing efficiency. Currently, various vectors and online tools are available to aid sgRNA design. Furthermore, to reduce the sequence limitation of the protospacer adjacent motif (PAM) and for other purposes, many Cas protein variants and base editors can be used in plants. Before the stable transformation of a plant, the evaluation of vectors and target sites is therefore very important. Moreover, the delivery of Cas9-sgRNA ribonucleoproteins (RNPs) is one strategy that can be used to prevent transgene issues with the expression of sgRNA and Cas proteins. RNPs can be used to efficiently generate transgene-free genome-edited crops that can reduce transgene issues related to the generation of genetically modified organisms. In this review, we introduce new techniques for genome editing and identifying marker-free genome-edited mutants in monocot crops. Four topics are covered: the design and construction of plasmids for genome editing in monocots; alternatives to SpCas9; protoplasts and CRISPR; and screening for marker-free CRISPR/Cas9-induced mutants. We have aimed to encompass a full spectrum of information for genome editing in monocot crops.

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Hygromycin B-induced cell death is partly mediated by reactive oxygen species in rice (Oryza sativa L.)

Cited by 6 publications

References 62 publications

Mechanism of Oxidative Ring‐Closure as Part of the Hygromycin Biosynthesis Step by a Nonheme Iron Dioxygenase

Mechanism of Oxidative Ring‐Closure as Part of the Hygromycin Biosynthesis Step by a Nonheme Iron Dioxygenase

H2O2-Based Method for Rapid Detection of Transgene-Free Rice Plants from Segregating CRISPR/Cas9 Genome-Edited Progenies

How to start your monocot CRISPR/Cas project: plasmid design, efficiency detection, and offspring analysis

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