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Characterization of the NADP malic enzyme gene family in the facultative, single-cell C4 monocot Hydrilla verticillata

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Abstract

Hydrilla verticillata has a facultative single-cell system that changes from C3 to C4 photosynthesis. A NADP+-dependent malic enzyme (NADP-ME) provides a high [CO2] for Rubisco fixation in the C4 leaf chloroplasts. Of three NADP-ME genes identified, only hvme1 was up-regulated in the C4 leaf, during the light period, and it possessed a putative transit peptide. Unlike obligate C4 species, H. verticillata exhibited only one plastidic isoform that may perform housekeeping functions, but is up-regulated as the photosynthetic decarboxylase. Of the two cytosolic forms, hvme2 and hvme3, the latter exhibited the greatest expression, but was not light-regulated. The mature isoform of hvme1 had a pI of 6.0 and a molecular mass of 64 kD, as did the recombinant rHVME1m, and it formed a tetramer in the chloroplast. The recombinant photosynthetic isoform showed intermediate characteristics between isoforms in terrestrial C3 and C4 species. The catalytic efficiency of rHVME1m was four-fold higher than the cytosolic rHVME3 and two-fold higher than recombinant cytosolic isoforms of rice, but lower than plastidic forms of maize. The K m (malate) of 0.6 mM for rHVME1 was higher than maize plastid isoforms, but four-fold lower than found with rice. A comprehensive phylogenetic analysis of 25 taxa suggested that chloroplastic NADP-ME isoforms arose from four duplication events, and hvme1 was derived from cytosolic hvme3. The chloroplastic eudicot sequences were a monophyletic group derived from a cytosolic clade after the eudicot and monocot lineages separated, while the monocots formed a polyphyletic group. The findings support the hypothesis that a NADP-ME isoform with specific and unusual regulatory properties facilitates the functioning of the single-cell C4 system in H. verticillata.

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Abbreviations

CAM:

Crassulacean acid metabolism

NADP-ME:

NADP+-dependent malic enzyme

PEPC:

Phosphoenolpyruvate carboxylase

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Acknowledgments

We thank Ms Margaret Glenn (Center for Aquatic and Invasive Plant Research, University of Florida) for assistance in collecting plant material, and Dr Hiroshi Fukayama (Kobe University, Japan) for his generous gift of anti-rice NADP-ME. We appreciate the technical assistance for BN-PAGE analysis provided by members of the Dr Kenneth Cline laboratory. We also thank Drs Pamela S. Soltis (Florida Museum of Natural History, University of Florida) and Pablo Speranza (Facultad de Agronomía, Montevideo, Uruguay) for help with the phylogenetic analysis. The Amborella trichopoda NADP-ME cDNA was retrieved from the Floral Genome Project (NSF grant PGR-0115684). Financial support was provided by the United States Department of Agriculture, National Research Initiatives Competitive Grants Program grant No. 98-35306-6449.

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  1. Gonzalo M. Estavillo

    Present address: ARC Centre of Excellence in Plant Energy Biology, School of Biochemistry and Molecular Biology (BaMBi), The Australian National University, Building 41, Canberra, ACT, 0200, Australia

  2. Srinath K. Rao

    Present address: Biology Department, Queen’s University, Kingston, ON, Canada, K7L 3N6

Authors and Affiliations

  1. Department of Botany, University of Florida, 220 Bartram Hall, PO Box 118526, Gainesville, FL, 32611-8526, USA

    Gonzalo M. Estavillo, Srinath K. Rao, Julia B. Reiskind & George Bowes

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  1. Gonzalo M. Estavillo
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Correspondence to Gonzalo M. Estavillo.

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Estavillo, G.M., Rao, S.K., Reiskind, J.B. et al. Characterization of the NADP malic enzyme gene family in the facultative, single-cell C4 monocot Hydrilla verticillata . Photosynth Res 94, 43–57 (2007). https://doi.org/10.1007/s11120-007-9212-y

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