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Brassinolide-improved development of Brassica napus microspore-derived embryos is associated with increased activities of purine and pyrimidine salvage pathways

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Abstract

Cellular brassinolide (BL) levels regulate the development of Brassica napus microspore-derived embryos (MDEs). Synthesis and degradation of nucleotides were measured on developing MDEs treated with BL or brassinazole (BrZ), a biosynthetic inhibitor of BL. Purine metabolism was investigated by following the metabolic fate of 14C-labelled adenine and adenosine, substrates of the salvage pathway, and inosine, an intermediate of both salvage and degradation pathways. For pyrimidine, orotic acid, uridine and uracil were employed as markers for the de novo (orotic acid), salvage (uridine and uracil), and degradation (uracil) pathways. Our results indicate that utilization of adenine, adenosine, and uridine for nucleotides and nucleic acids increased significantly in BL-treated embryos at day 15 and remained high throughout the culture period. These metabolic changes were ascribed to the activities of the respective salvage enzymes: adenine phosphoribosyltransferase (EC 2.4.2.7), adenosine kinase (EC 2.7.1.20), and uridine kinase (EC 2.7.1.48), which were induced by BL applications. The BL promotion of salvage synthesis was accompanied by a reduction in the activities of the degradation pathways, suggesting the presence of competitive anabolic and catabolic mechanisms utilizing the labelled precursors. In BrZ-treated embryos, with depleted BL levels, the salvage activity of both purine and pyrimidine nucleotides was reduced and this was associated to structural abnormalities and poor embryonic performance. In these embryos, the activities of major salvage enzymes were consistently lower to those measured in their control (untreated) counterparts.

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Abbreviations

APRT:

Adenosine phosphoribosyltransferase

ADK:

Adenosine kinase

IK:

Inosine kinase

BL:

Brassinolide

BRs:

Brassinosteroids

BrZ:

Brassinazole

MDEs:

Microspore-derived embryos

UPRT:

Uracil phosphoribosyltransferase

UDK:

Uridine kinase

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Acknowledgments

This work was supported by a NSERC Discovery Grant to CS and by a Ph.D. scholarship from the Egyptian government to ME. The authors thank Dr. Asami for providing brassinazole.

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Authors and Affiliations

  1. Department of Biological Sciences, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada

    Mark Belmonte

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

    Hiroshi Ashihara

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

    Mohamed Elhiti & Claudio Stasolla

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

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Belmonte, M., Elhiti, M., Ashihara, H. et al. Brassinolide-improved development of Brassica napus microspore-derived embryos is associated with increased activities of purine and pyrimidine salvage pathways. Planta 233, 95–107 (2011). https://doi.org/10.1007/s00425-010-1287-6

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