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Cloning and molecular characterization of a glycerol-3-phosphate O-acyltransferase (GPAT) gene from Echium (Boraginaceae) involved in the biosynthesis of cutin polyesters

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Abstract

The glycerol-based lipid polyester called cutin is a main component of cuticle, the protective interface of aerial plant organs also controlling compound exchange with the environment. Though recent progress towards understanding of cutin biosynthesis has been made in Arabidopsis thaliana, little is known in other plants. One key step in this process is the acyl transfer reaction to the glycerol backbone. Here we report the cloning and molecular characterization of EpGPAT1, a gene encoding a glycerol-3-phosphate O-acyltransferase (GPAT) from Echium pitardii (Boraginaceae) with high similarity to the AtGPAT4/AtGPAT8 of Arabidopsis. Quantitative analysis by qRT-PCR showed highest expression of EpGPAT1 in seeds, roots, young leaves and flowers. Acyltransferase activity of EpGPAT1 was evidenced by heterologous expression in yeast. Ectopic expression in leaves of tobacco plants lead to an increase of C16 and C18 hydroxyacids and α,ω-diacids in the cell wall fraction, indicating a role in the biosynthesis of polyesters. Analysis of the genomic organization in Echium revealed the presence of EpGPAT2, a closely related gene which was found to be mostly expressed in developing leaves and flowers. The presence of a conserved HAD-like domain at the N-terminal moiety of GPATs from Echium, Arabidopsis and other plant species suggests a possible phosphohydrolase activity in addition to the reported acyltransferase activity. Evolutive implications of this finding are discussed.

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Abbreviations

AT:

Acyltransferase

BSTFA:

N,O-bis(trimethylsilyl)trifluoroacetamide

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 trimethyl ammonium bromide

DIG:

Digoxigenin

DTNB:

5, 5′-Dithio-bis(2-nitrobenzoic acid)

EtBr:

Ethidium bromide

GC:

Gas-liquid chromatography

G3P:

Glycerol-3-phosphate

GPAT:

Glycerol-3-phosphate O-acyltransferase

HAD:

Haloacid dehalogenase

qPCR:

Quantitative PCR

RACE:

Rapid amplification of cDNA ends

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Acknowledgments

This work was supported by grants from the Ministerio de Ciencia y Tecnología (MCYT, AGL2005-01498/AGR) and Junta de Andalucía (P05-189-AGR). A. Mañas-Fernández was recipient of a postgraduate fellowship from the Junta de Andalucía.

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Author notes

  1. Aurora Mañas-Fernández

    Present address: Department of Botany, University of British Columbia, 6270 University Blvd, Vancouver, BC, V6T 1Z4, Canada

Authors and Affiliations

  1. Universidad de Almería, Grupo de Biotecnología de Productos Naturales (BIO-279), 04120, Almería, Spain

    Aurora Mañas-Fernández, Diego López Alonso & Federico García-Maroto

  2. Department of Plant Biology and Environmental Microbiology, CEA-CNRS-Université Aix Marseille, CEA Cadarache, 13108, Saint Paul lez Durance, France

    Yonghua Li-Beisson

Authors
  1. Aurora Mañas-Fernández
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  2. Yonghua Li-Beisson
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  3. Diego López Alonso
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  4. Federico García-Maroto
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Correspondence to Federico García-Maroto.

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Mañas-Fernández, A., Li-Beisson, Y., Alonso, D.L. et al. Cloning and molecular characterization of a glycerol-3-phosphate O-acyltransferase (GPAT) gene from Echium (Boraginaceae) involved in the biosynthesis of cutin polyesters. Planta 232, 987–997 (2010). https://doi.org/10.1007/s00425-010-1232-8

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