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Regulation of hydrogen peroxide accumulation and death of Agrobacterium-transformed cells in tomato transformation

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Abstract

Our previous studies demonstrate that Agrobacterium causes tissue browning that subsequently reduces transformation efficiency in tomato transformation. In addition application of lipoic acid (LA) can reduce tissue browning and increase transformation efficiency in different crops. A major challenge in Agrobacterium-mediated plant transformation is the death of Agrobacterium-transformed cells (DATC), which restricts transgenic plant production. Hydrogen peroxide (H2O2) plays a critical role in oxidative stress. However, little is known about the biochemical and molecular mechanisms for DATC. Our biological and correlation analyses showed that Agrobacterium mediated H2O2 accumulation (HOA) and HOA elevation led to DATC during Agrobacterium-mediated tomato cv. MicroTom transformation. Agrobacterium significantly (P < 0.05) increased 4.2- and 1.4-fold expression of WRKY75 (a H2O2-responsive transcription factor) and superoxide dismutase (SOD), respectively, while the application of 4.4 M H2O2 significantly increased 19- and 2.7-fold expression of WRKY75 and 2-cys peroxiredoxin (Cys), respectively, and decreased fivefold SOD expression, compared with a control. LA application significantly (P < 0.05) reduced 1.6-fold HOA and DATC while it significantly increased 1.7-fold expression of Cys, and reduced 2.2- and 1.4-fold expression of WRKY75 and SOD, respectively. The reduction of HOA and DATC was accompanied by suppression of WRKY75 and SOD and activation of Cys. Our results indicated that DATC was regulated by H2O2 that was triggered by Agrobacterium and LA application through their gene regulation. In addition, HOA was associated with a biotic generating reactive oxygen species (ROS) mechanism, and HOA and DATC were likely regulated by an enzymatic ROS scavenging mechanism during Agrobacterium-mediated tomato transformation.

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Abbreviations

CaMV 35S:

Cauliflower mosaic virus 35S

Cys:

2-Cys peroxiredoxin

DATC:

Death of Agrobacterium-transformed cells

gus :

β-Glucuronidase gene

H2O2 :

Hydrogen peroxide

HOA:

H2O2 accumulation

LA:

Lipoic acid

nptII :

Neomycin phosphotransferase gene

ROS:

Reactive oxygen species

SOD:

Superoxide dismutase

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Author contribution

YD supervised and designed research. YD, SZ and AM conducted experiments. YD and AM analyzed data. YD wrote the manuscript. All authors read and approved the manuscript.

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Authors and Affiliations

  1. Institute for Advanced Learning and Research, 150 Slayton Avenue, Danville, VA, 24540, USA

    Yinghui Dan, Song Zhang & Amanda Matherly

  2. Department of Horticulture, Virginia Polytechnic Institute and State University, Blacksburg, VA, 24061, USA

    Yinghui Dan

  3. Department of Forest Resources and Environmental Conservation, Virginia Polytechnic Institute and State University, Blacksburg, VA, 24061, USA

    Yinghui Dan

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  1. Yinghui Dan
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  2. Song Zhang
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  3. Amanda Matherly
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Correspondence to Yinghui Dan.

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Dan, Y., Zhang, S. & Matherly, A. Regulation of hydrogen peroxide accumulation and death of Agrobacterium-transformed cells in tomato transformation. Plant Cell Tiss Organ Cult 127, 229–236 (2016). https://doi.org/10.1007/s11240-016-1045-y

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  • DOI: https://doi.org/10.1007/s11240-016-1045-y

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