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Ectopic localization of auxin and cytokinin in tobacco seedlings by the plant-oncogenic AK-6b gene of Agrobacterium tumefaciens AKE10

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Abstract

The oncogenic 6b gene of Agrobacterium tumefaciens induces a number of morphological and metabolic alterations in plants. Although molecular functions associated with the 6b genes have been proposed, including auxin transport, sugar transport, transcriptional regulation, and miRNA metabolism, so far an unequivocal conclusion has not been obtained. We investigated the association between auxin accumulation and tumor development of the tobacco seedlings expressing the AK-6b gene under the control of the dexamethasone-inducible promoter. Indole-3-acetic acid (IAA) localization was examined by immunochemical staining with monoclonal antibody against IAA and by histochemical analysis using the IAA-specific induced construct, DR5::GUS (β-glucuronidase). Both procedures indicated that IAA preferentially accumulated in the tumorous protrusions as well as in newly developing vascular bundles in the tumors. Furthermore, true leaves also showed abaxial IAA localization, leading to altered leaves in which the adaxial and abaxial identities were no longer evident. Co-localization of cytokinin and auxin in the abaxial tumors was verified by immunochemical staining with an antibody against cytokinin. Treatment of AK-6b-seedlings with N-1-naphthylphthalamic acid, an inhibitor of polar auxin transport, promoted the morphological severity of phenotypes, whereas 1-naphthoxyacetic acid, a specific auxin influx carrier inhibitor, induced tumor regression on cotyledons and new tumorous proliferations on hypocotyls. Prominent accumulation of both auxin and cytokinin was observed in both regressed and newly developing tumors. We suggest from these results that modulation of auxin/cytokinin localization as a result of AK-6b gene expression is responsible for the tumorous proliferation.

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Abbreviations

DAPI:

4′,6-Diamidino-2-phenylindole, dihydrochloride

Dex:

Dexamethasone

GUS:

β-Glucuronidase

h:

Hour(s)

Hgr:

Hygromycin

IAA:

Indole-3-acetic acid

Kam:

Kanamycin

min:

Minute(s)

NOA:

1-Naphthoxyacetic acid

NPA:

N-1-Naphthylphthalamic acid

t-ZR:

trans-Zeatin riboside

t-Z:

trans-Zeatin

t-ZRMP:

trans-Zeatin riboside 5′-monophosphate

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Acknowledgments

We thank Dr. Youssefian Shohab (Akita Prefectural University, Akita, Japan) for critical reading of the manuscript, Dr. S. Tiwari (University of Missouri, Columbia, MO, USA) for providing us with the reporter construct; DR5::GUS. We are also grateful to Biotechnology Center of APU for experimental support.

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Author notes

  1. Rui Sato

    Present address: Kyokuto Pharmaceutical Industrial Co., Ltd., 7-8 Nihombashi Kobuna Chuo, Tokyo, 103-0024, Japan

  2. Miho Takahashi

    Present address: Iwate Chikusan Ryutsu Center Co., Ltd., 120 Minami-yachi Inubuchi Shiwa, Shiwa, Iwate, 028-3311, Japan

  3. Noriko Hashiba

    Present address: Kokando Co., Ltd., 2-9-1 Umezawa, Toyama, 930-0055, Japan

  4. Taiki Sawata

    Present address: Shinetsu Myojo Co., Ltd., 942 Akiwa Uedai, Nagano, 386-0041, Japan

Authors and Affiliations

  1. Department of Biological Production, Faculty of Bioresource Sciences, Akita Prefectural University, 241-438 Kaidobata Nishi, Nakano-Aza, Shimoshinjo, Akita, 010-0195, Japan

    Sachiko Takahashi, Rui Sato, Miho Takahashi, Noriko Hashiba, Atsushi Ogawa, Kyoko Toyofuku, Taiki Sawata, Yuki Ohsawa, Kenji Ueda & Hiroetsu Wabiko

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  1. Sachiko Takahashi
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Correspondence to Hiroetsu Wabiko.

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Takahashi, S., Sato, R., Takahashi, M. et al. Ectopic localization of auxin and cytokinin in tobacco seedlings by the plant-oncogenic AK-6b gene of Agrobacterium tumefaciens AKE10. Planta 238, 753–770 (2013). https://doi.org/10.1007/s00425-013-1930-0

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  • DOI: https://doi.org/10.1007/s00425-013-1930-0

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