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Distinct fluctuations in nucleotide metabolism accompany the enhanced in vitro embryogenic capacity of Brassica cells over-expressing SHOOTMERISTEMLESS

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Abstract

Besides regulating meristem formation and maintenance in vivo, SHOOTMERISTEMLESS (STM) has been shown to affect embryogenesis. While the over-expression of Brassica napus (Bn)STM enhances the number of microspore-derived embryos produced in culture and their ability to regenerate viable plants, a down-regulation of this gene represses the embryogenic process (Elhiti et al., J Exp Bot, 61:4069–4085, 2010). Synthesis and degradation of pyrimidine and purine nucleotides were measured in developing microspore-derived embryos (MDEs) generated from B. napus lines ectopically expressing or down-regulating BnSTM. Pyrimidine metabolism was investigated by following the metabolic fate of exogenously supplied 14C-uridine, uracil and orotic acid, whereas purine metabolism was estimated by using 14C-adenine, adenosine and inosine. The improvement in embryo number and quality affected by the ectopic expression of BnSTM was linked to the increased pyrimidine and purine salvage activity during the early phases of embryogenesis and the enlargement of the adenylate pool (ATP + ADP) required for the active growth of the embryos. This was due to an increase in transcriptional and enzymatic activity of several salvage enzymes, including adenine phosphoribosyltransferase (APRT) and adenosine kinase (ADK). The highly operative salvage pathway induced by the ectopic expression of BnSTM was associated with a slow catabolism of nucleotides, suggesting the presence of an antagonist mechanism controlling the rate of salvage and degradation pathways. During the second half of embryogenesis utilization of uridine for UTP + UDPglucose (UDPG) synthesis increased in the embryos over-expressing BnSTM, and this coincided with a better post-germination performance. All these events were precluded by the down-regulation of BnSTM which repressed the formation of the embryos and their post-embryonic performance. Overall, this work provides evidence that precise metabolic changes are associated with proper embryo development in culture.

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Abbreviations

APRT:

Adenine phosphoribosyltransferase

ADK:

Adenosine kinase

IK:

Inosine kinase

MDEs:

Microspore-derived embryos

PRPP:

Phosphate independent-phosphoribosylpyrophosphate

STM:

SHOOTMERISTEMLESS

UPRT:

Uracil phosphoribosyltransferase

UDK:

Uridine kinase

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Acknowledgments

This work was supported by a NSERC Discovery Grant to CS.

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Author notes

  1. Mohamed Elhiti

    Present address: Department of Botany, Faculty of Science, Tanta University, Tanta, 31527, Egypt

Authors and Affiliations

  1. Department of Biological Sciences, Graduate School of Humanities and Sciences, Ochanomizu University, Bunkyo-ku, Tokyo, 112-8610, Japan

    Hiroshi Ashihara

  2. Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada

    Claudio Stasolla

Authors
  1. Mohamed Elhiti
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  2. Hiroshi Ashihara
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  3. Claudio Stasolla
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Correspondence to Claudio Stasolla.

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Elhiti, M., Ashihara, H. & Stasolla, C. Distinct fluctuations in nucleotide metabolism accompany the enhanced in vitro embryogenic capacity of Brassica cells over-expressing SHOOTMERISTEMLESS . Planta 234, 1251–1265 (2011). https://doi.org/10.1007/s00425-011-1482-0

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