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Cloning and molecular characterisation of a Δ8-sphingolipid-desaturase from Nicotiana tabacum closely related to Δ6-acyl-desaturases

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Abstract

Investigation on the absence of Δ6-desaturase activity in Nicotiana tabacum has led to the cloning of a new desaturase gene from this organism (NTDXDES) that exhibited unexpected biochemical activity. Cladistic analysis shows clustering of NTDXDES together with functional Δ6-acyl-desaturases of near Solanales plants, such as Borago and Echium. This group lies apart from that of previously characterised Δ8-sphingolipid-desaturases, which also includes two putative tobacco members identified in this study. Moreover, strong expression of NTDXDES is found in leaves, flowers, fruits and developing seeds of tobacco plants that is highly dependent on the development phase, with transcriptional activity being higher at stages of active tissue growth. This pattern is similar to that showed by Δ6-acyl-desaturases characterised in Boraginaceae species. However, functional assays using a yeast expression system revealed that the protein encoded by NTDXDES lacks Δ6-desaturase activity, but instead it is able to desaturate sphingolipid substrates by introducing a double bond on the Δ8-position. These data indicate that NTDXDES represent a novel desaturase gene placed in a different evolutionary lineage to that of previously characterised Δ8-desaturases.

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Abbreviations

ACP:

Acyl carrier protein

ALA:

α-linolenic acid

CaMV:

Cauliflower mosaic virus

CSPD:

Disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2′-(5′ chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate

CTAB:

Cetyl-trimetyl-amonium bromide

DIG:

Digoxigenine

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

GLA:

γ-linolenic acid

HPLC:

High pressure liquid chromatography

IPCR:

Inverse PCR

LA:

Linoleic acid

LCB:

Long-chain base

OTA:

Octadecatetraenoic acid

PCR:

Polymerase chain reaction

RNAi:

RNA interference

SDS:

Sodium dodecylsulfate

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Acknowledgements

This work was supported by a grant from the Ministerio de Ciencia y Tecnología (MCYT, AGL2002-00390). J. A. Garrido-Cárdenas was a recipient of a postgraduate fellowship from MCYT. Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council (BBSRC), UK.

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Authors and Affiliations

  1. Area de Bioquímica, Facultad de Ciencias Experimentales, Universidad de Almería, 04120, Almeria, Spain

    Federico García-Maroto & José A. Garrido-Cárdenas

  2. Departamento de Biología Aplicada, Facultad de Ciencias, Universidad de Almería, Campus de la Cañada, 04120, Almeria, Spain

    Diego López Alonso

  3. Rothamsted Research, Harpenden, Herts, AL5 2JQ, UK

    Louise V. Michaelson & Johnathan A. Napier

Authors
  1. Federico García-Maroto
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  2. José A. Garrido-Cárdenas
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  3. Louise V. Michaelson
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  4. Johnathan A. Napier
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  5. Diego López Alonso
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Correspondence to Federico García-Maroto.

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García-Maroto, F., Garrido-Cárdenas, J.A., Michaelson, L.V. et al. Cloning and molecular characterisation of a Δ8-sphingolipid-desaturase from Nicotiana tabacum closely related to Δ6-acyl-desaturases. Plant Mol Biol 64, 241–250 (2007). https://doi.org/10.1007/s11103-007-9148-9

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