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Identification of 2,4-D-responsive proteins in embryogenic callus of Valencia sweet orange (Citrus sinensis Osbeck) following osmotic stress

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Abstract

The compound 2,4-Dicholorophenoxyacetic acid (2,4-D) is an important growth regulator which is used in the majority of embryogenic cell and tissue culture systems. However, 2,4-D also appears to have a negative effect on growth and development of plant tissues and organs cultured in vitro. For example, 2,4-D exerts inhibition on in vitro somatic embryo initiation and/or development of most citrus species. To understand the molecular mechanism by which 2,4-D inhibits somatic embryogenesis (SE), proteomic changes of Valencia sweet orange (Citrus sinensis) embryogenic callus induced by treatments with a high concentration of 2,4-D (6 mg l−1) was investigated. Nine 2,4-D-responsive proteins were identified, of which eight were up-regulated and one was down-regulated. Interestingly, three of the eight up-regulated proteins were osmotic stress-associated, suggesting that 2,4-D induced osmotic stress in Valencia embryogenic callus. This speculation was supported by results from our physiological studies: 2,4-D treated callus cells exhibited increased cytoplasm concentration with a significant reduction in relative water content (RWC) and an obvious increase in levels of two osmolytes (proline and soluble sugar). Taken together, our results suggested that 2,4-D could inhibit somatic embryo initiation by, at least in part, inducing osmotic stress to citrus callus cells.

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Abbreviations

2,4-D:

2,4-Dichlorophenoxy acetic acid

2-DE:

Two-dimensional gel electrophoresis

ACN:

Acetonitril

CBB:

Coomassie brilliant blue

CHAPS:

3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonic acid

DTT:

DL-Dithiothreitol

EST:

Expressed sequence tag

FW:

Fresh weight

GSH:

Glutathione

MALDI-TOF:

Matrix-assisted laser desorption ionization time-of-flight

MS:

Mass spectrum

RWC:

Relative water content

SE:

Somatic embryogenesis

TFA:

Trifluoroacetic acid

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Acknowledgments

We thank Dr. Xiao Shunyuan (Center for Biosystems Research, University of Maryland Biotechnology Institute, USA) and Dr Xu Qiang for their suggestions on manuscript preparation. The research was financially supported by the National Natural Science Foundation of China (No. 30570973, 30830078, 30921002), and the Ministry of Education of China (IRT0548).

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  1. National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, 430070, Wuhan, People’s Republic of China

    Zhiyong Pan, Shiping Zhu, Rui Guan & Xiuxin Deng

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  1. Zhiyong Pan
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  2. Shiping Zhu
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  4. Xiuxin Deng
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Correspondence to Xiuxin Deng.

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Pan, Z., Zhu, S., Guan, R. et al. Identification of 2,4-D-responsive proteins in embryogenic callus of Valencia sweet orange (Citrus sinensis Osbeck) following osmotic stress. Plant Cell Tiss Organ Cult 103, 145–153 (2010). https://doi.org/10.1007/s11240-010-9762-0

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