Botanical Studies (2010) 51: 491-510.
Molecular evolution and positive Darwinian selection of the gymnosperm photosynthetic Rubisco enzyme
Da Cheng HAO1*, Jun MU1, and Pei Gen XIAO2
1Biotechnology Institute, College of Environment, Dalian Jiaotong University, Dalian 116028, P.R. China
22Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Beijing 100193, P.R. China
22Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Beijing 100193, P.R. China
(Received August 27, 2009; Accepted February 9, 2010)
ABSTRACT. Although it was found that gymnosperm rbcL of three orders evolves under Darwinian positive selection, it is not clear whether rbcL in other gymnosperm lineages is also subject to positive selection. In this study, eleven gymnosperm groups, representing 393 species at various evolutionary levels, were used to illustrate the molecular adaptation and evolutionary dynamics of gene divergence in rbcLs. rbcL sequences were amplified from 21 Taxaceae and 10 Cephalotaxaceae species. rbcL sequences of other species were retrieved from GenBank. Selective influences were investigated using standard dN/dS ratio methods and more sensitive techniques investigating the amino acid property changes resulting from nonsynonymous replacements in a phylogenetic context. Analyses revealed the presence of positive selection in rbcLs of all gymnosperm groups. Twenty most often positively selected amino acid sites were characterized. In Taxaceae and Cephalotaxaceae, seven amino acid properties, equilibrium constant of ionization -COOH as the most significant, were found to be influenced by destabilizing positive selection. Some amino acid sites relating to these properties were found to be involved in active site, intradimer interaction, dimer-dimer interaction, and interactions with Rubisco small subunits. Moreover, removing amino acid sites that are under positive selection has significant effect on the bootstrap values of phylogenetic reconstruction. Our results suggest that the conservative rbcL evolves under positive selection in gymnosperm lineages. Several regions of rbcL have experienced molecular adaptation which fine-tunes photosynthetic Rubisco performance.
Keywords: Chloroplast rbcL; Gymnosperm; Maximum likelihood; Physicochemical evolution; Positive selection.
INTRODUCTION
The rbcL gene is located in the large single-copy region of the chloroplast genome and encodes large subunits of ribulose-1, 5-bisphosphate carboxylase (Rubisco). The gene is roughly 1425 bp in length, corresponding to 475 amino acids. rbcL protein is characterized by a set of eight a helices and eight parallel p strands that "roll up" to form a barrel with the p strands on the inside. rbcL has been extensively used in determining evolutionary histories at various taxonomic levels (e.g., Muller et al., 2006; Hao et al., 2008), and recently it is recommended to be one of the most useful DNA barcoding markers in species identification (CBOL Plant Working Group, 2009). Moreover, how rbcL and small subunits form the functional Rubisco has been studied extensively, with the hope of manipulating this photosynthetic enzyme and increasing crop yield (Christin et al., 2008).
rbcL is often chosen for phylogenetic reconstructions and it has been sequenced in thousands of plant species. Surprisingly, despite rbcL's physiological importance and abundance of sequence data, rbcL is generally used
*Corresponding author: E-mail: hao@djtu.edu.cn.
as strings of anonymous nucleotides, without regard to its functional evolution. Kapralov and Filatov (2007) searched for positive selection in rbcL sequences from green plants and other phototrophs. Positive selection was found, for the first time, to be present in rbcL of most analyzed land plants, but not in algae and cyanobacteria. Positively selected residues are located in regions important for dimer-dimer, intradimer, large subunit-small sub-unit and Rubisco-Rubisco activase interactions, and that some positively selected residues are close to the active site. Their results demonstrate that despite its conservative nature, rbcL gene evolves under positive selection in land plants. Christin et al. (2008) used phylogenetic analyses on a large data set of C3 and C4 monocots and found that the rbcL gene evolved under positive selection in independent C4 lineages. This confirms that selective pressures on Rubisco have been switched in C4 plants by the high CO2 environment prevailing in their photosynthetic cells. Eight rbcL codons evolving under positive selection in C4 clades were involved in parallel changes among the 23 independent monocot C4 lineages. The introgression of C4-like high-efficiency Rubisco would strongly enhance C3 crop yields in the future CO2-enriched atmosphere (Christin et al., 2008). Recently, rbcL positive selection was also de-
