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Nitrate reutilization mechanisms in the tonoplast of two Brassica napus genotypes with different nitrogen use efficiency

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Abstract

Nitrate (NO3 −) can accumulate in high concentrations in plant cell vacuoles if it is not reduced, reutilized or transported into the cytoplasm. Such accumulation of NO3 − in the vacuole occurs when mechanisms for NO3 − assimilation in the cytoplasm are saturated. Moreover, other processes such as efflux across the plasma membrane might affect NO3 − accumulation in the vacuole. These are the main reasons limiting nitrogen use efficiency (NUE) in plants. This study elucidates mechanisms for NO3 − transport from the cytoplasm to vacuoles by the V-proton pump (V-ATPase and V-PPase) and their relationship with different NUE in four Brassica napus genotypes. Pot experiments were conducted in a greenhouse under normal (15.0 mmol L−1) and limited N (7.5 mmol L−1) concentrations of nitrate using B. napus genotypes that demonstrated either high (742 and Xiangyou 15) or low (814 and H8) NUE (g g−1). Specific inhibitors of V-ATPase and V-PPase increased nitrate reductase (NR) activity, resulting in greatly decreased NO3 − in plant tissues. Nitrate reductase activity and NO3 − content correlated more highly to V-PPase activity than they did to V-ATPase activity, and correlation between V-PPase activity and NO3 − content was significantly higher than it was to V-ATPase. Genotypes with high NUE had significantly lower activities of V-ATPase and V-PPase than those with low NUE. In the high-NUE plants, lower activities of V-proton pump underlie mechanisms that result in significantly lower NO3 − content in plant tissues of the high-NUE genotypes than those found in plant tissues of the low-NUE genotypes. Our results show that the tonoplast proton pumps V-PPase and V-ATPase strongly negatively affect NR activity and positively affect NO3 − content. V-PPase contributed more to this regulatory mechanism than did V-ATPase.

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Abbreviations

NUE:

Nitrogen use efficiency

V-ATPase:

Vacuolar H+-ATPase

V-PPase:

Vacuolar H+-pyrophosphatase

NR:

Nitrate reductase

CLC:

Chloride channels

DCCD:

N,N′-Dicyclohexylcarbodiimide

EDTA:

Ethylenediaminetetraacetic acid disodium salt

NRAact:

Nitrate reductase activity

NRAmax:

Maximum nitrate reductase

FW:

Fresh weight

Bafilomycin:

Bafilomycin A1

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Acknowledgments

This work was funded by the National Natural Science Foundation of China (grant numbers 31101596, and 31372130), Open novel science foundation of Hunan province (13K062), National Key Laboratory of Plant Molecular Genetics, The “Twelfth Five-Year” National Science and technology support program (2012BAD15BO4). We thank Abdelbagi M.Ismail from the International Rice Research Institute, Philippines, Tanya Streeter and Juanita Johns from the University of West Florida, United States of America for proofreading drafts of this manuscript.

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    Authors and Affiliations

    1. College of Resources and Environment Sciences, Hunan Agricultural University, Changsha, 410128, Hunan, People’s Republic of China

      Yongliang Han, Qiong Liao, Yin Yu, Haixing Song, Qiang Liu, Xiangmin Rong, Jidong Gu & Zhenhua Zhang

    2. School of Biological Sciences, The University of Hong Kong, Hong Kong SAR, People’s Republic of China

      Jidong Gu

    3. Center for Environmental Diagnostics and Bioremediation, University of West Florida, Pensacola, FL, 32514, USA

      Joe Eugene Lepo

    4. National Center of Oilseed Crops Improvement, Hunan Branch, Changsha, 410128, Hunan, People’s Republic of China

      Chunyun Guan

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    1. Yongliang Han
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    2. Qiong Liao
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    Correspondence to Haixing Song.

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    Communicated by W. Zhou.

    Y. Han, Q. Liao and Y. Yu contributed equally.

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    Han, Y., Liao, Q., Yu, Y. et al. Nitrate reutilization mechanisms in the tonoplast of two Brassica napus genotypes with different nitrogen use efficiency. Acta Physiol Plant 37, 42 (2015). https://doi.org/10.1007/s11738-014-1744-0

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    • DOI: https://doi.org/10.1007/s11738-014-1744-0

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