Multiple Isoforms of Phosphoenolpyruvate Carboxylase in the Orchidaceae (Subtribe Oncidiinae): Implications for the Evolution of Crassulacean Acid Metabolism
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Journal of Experimental Botany, Vol. 65, No. 13, pp. 3623–3636, 2014 doi:10.1093/jxb/eru234 This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) RESEARCH PAPER Multiple isoforms of phosphoenolpyruvate carboxylase in the Orchidaceae (subtribe Oncidiinae): implications for the evolution of crassulacean acid metabolism Katia Silvera1,2,*, Klaus Winter1, B. Leticia Rodriguez2, Rebecca L. Albion2 and John C. Cushman2 1 Smithsonian Tropical Research Institute, PO Box 0843-03092, Balboa, Ancon, Republic of Panama 2 Department of Biochemistry & Molecular Biology, MS330, University of Nevada, Reno, NV 89557-0330, USA * To whom correspondence should be addressed. E-mail: [email protected] Received 10 December 2013; Revised 28 April 2014; Accepted 1 May 2014 Abstract Phosphoenolpyruvate carboxylase (PEPC) catalyses the initial fixation of atmospheric CO2 into oxaloacetate and sub- sequently malate. Nocturnal accumulation of malic acid within the vacuole of photosynthetic cells is a typical feature of plants that perform crassulacean acid metabolism (CAM). PEPC is a ubiquitous plant enzyme encoded by a small gene family, and each member encodes an isoform with specialized function. CAM-specific PEPC isoforms probably evolved from ancestral non-photosynthetic isoforms by gene duplication events and subsequent acquisition of tran- scriptional control elements that mediate increased leaf-specific or photosynthetic-tissue-specific mRNA expression. To understand the patterns of functional diversification related to the expression of CAM, ppc gene families and pho- tosynthetic patterns were characterized in 11 closely related orchid species from the subtribe Oncidiinae with a range of photosynthetic pathways from C3 photosynthesis (Oncidium cheirophorum, Oncidium maduroi, Rossioglossum krameri, and Oncidium sotoanum) to weak CAM (Oncidium panamense, Oncidium sphacelatum, Gomesa flexuosa and Rossioglossum insleayi) and strong CAM (Rossioglossum ampliatum, Trichocentrum nanum, and Trichocentrum carthagenense). Phylogenetic analysis revealed the existence of two main ppc lineages in flowering plants, two main ppc lineages within the eudicots, and three ppc lineages within the Orchidaceae. Our results indicate that ppc gene family expansion within the Orchidaceae is likely to be the result of gene duplication events followed by adaptive sequence divergence. CAM-associated PEPC isoforms in the Orchidaceae probably evolved from several independ- ent origins. Keywords: Crassulacean acid metabolism, gene duplication, Orchidaceae, Oncidiinae, phosphoenolpyruvate carboxylase, photosynthesis. Introduction Crassulacean acid metabolism (CAM) is one of three modes families comprising more than 6% of flowering plant spe- of photosynthesis found in vascular plants for the assimi- cies (Griffiths, 1989; Smith and Winter, 1996; Holtum et al., lation of atmospheric CO2. CAM differs from C3 and C4 2007; Silvera et al., 2010a). The multiple independent origins photosynthesis in that CAM plants take up CO2 with low of CAM and the functional convergence of CAM traits in water expenditure at night when evaporative demand is low the many lineages in which it occurs suggest that the start- (Winter and Smith, 1996b; Cushman, 2001). CAM is phy- ing point for CAM evolution might have required relatively logenetically widespread across 343 genera and 35 plant few genetic changes. However, this notion may be simplistic, Abbreviations: ∆H+, titratable acidity; CAM, crassulacean acid metabolism; GSP, gene-specific primer; PEPC, phosphoenolpyruvate carboxylase; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcription-PCR. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 3624 | Silvera et al. given the complexity of the CAM pathway and the fact that Westhoff, 2011), suggesting that these ubiquitous and func- several biochemical and anatomical requirements, and regu- tionally diverse isoforms served as starting points for the latory changes associated with gene expression patterns, are evolution of the C4 and CAM genes (Cushman and Bohnert, all tightly coordinated in CAM plants (Cushman et al., 2008; 1999; Monson, 1999). Furthermore, evidence from PEPC Silvera et al., 2010a). To gain insight into the evolutionary and comparative analysis of the C4-cycle enzymes in C3, history of genes recruited for CAM function, the study of C3–C4 intermediates, and C4 species in the genus Flaveria taxa containing many closely related species with contrast- suggests that key amino acid residue changes are responsi- ing photosynthetic pathways is experimentally helpful. In ble for their acquisition of distinct kinetic and regulatory this context, we use tropical orchids as a study group, because properties (Blasing et al., 2000, 2002; Westhoff and Gowik, CAM is widespread among epiphytes within this large family 2004). Similarly, phylogenetic analysis indicates that multiple of vascular plants (Winter and Smith, 1996a; Silvera et al., origins of C4 photosynthesis in grasses and sedges are the 2009, 2010a, b), and because species within the Orchidaceae likely result of recurring selection acting on a few amino acid exhibit a gradient of photosynthetic pathways ranging from positions of the PEPC enzyme within and across taxonomic C3 photosynthesis to weak- and strong-CAM modes (Silvera scales (Christin et al., 2007, 2012b; Besnard et al., 2009). et al., 2005, 2010a). Weak-CAM species show low-level Phylogenetic analyses also indicate that there was a single CAM activity and typically obtain 5% or less of their car- PEPC origin before the divergence of bacteria and plant bon through the CAM pathway under well-watered condi- lineages (Chollet et al., 1996; Izui et al., 2004; Westhoff and tions. Weak CAM appears to be common among neotropical Gowik, 2004). CAM-specific PEPC isoforms are thought to orchid species (Silvera et al. 2005). have first evolved in response to water deficit from ancestral In vascular plants, phosphoenolpyruvate carboxylase non-photosynthetic isoforms by gene duplication, followed (PEPC; EC 4.1.1.31) belongs to a multigene family, and by acquisition of transcriptional control sequences that medi- each member encodes an enzyme with specialized functions ate leaf- or photosynthetic-tissue-specific increases in mRNA (Gehrig et al., 1995, 1998, 2001; Chollet et al., 1996; Izui et al., expression (Gehrig et al., 2001, 2005; Taybi et al., 2004). For 2004; O’Leary et al., 2011). In species performing C4 photo- example, seven distinct PEPC isoforms were recovered in the synthesis and CAM, one or more ppc genes encode isoforms CAM species Kalanchoe pinnata (Lam.) Pers.: four isoforms of the enzyme that catalyse the fixation of atmospheric CO2 from leaves and three from roots (Gehrig et al., 2005). In into C4-dicarboxylic acids. In CAM plants, CAM-specific Mesembryanthemum crystallinum L., a CAM-specific isoform isoforms catalyse the nocturnal, irreversible β-carboxylation was expressed during the induction of CAM, in addition to – 2+ of phosphoenolpyruvate in the presence of HCO3 and Mg an uninduced housekeeping isoform (Cushman and Bohnert, yielding oxaloacetate and inorganic phosphate. Oxaloacetate 1999). Based on comparative studies of PEPC in many plant is then converted to malate, which is stored as malic acid taxa including orchids performing CAM, Gehrig et al. (2001) in the vacuole. During the subsequent day, malic acid is predicted the clustering of PEPC isoforms according to their decarboxylated, resulting in CO2 release and refixation by taxonomic position and specific function. ribulose-1,5-bisphosphate carboxylase/oxygenase. This Previously, full-length ppc genes were characterized from CO2-concentrating mechanism, or ‘CO2 pump’, suppresses bacteria, several vascular plant species, cyanobacteria, and photorespiration and improves water-use efficiency relative protozoa (Izui et al., 2004). The purpose of our study was to to that of C3 and C4 plant species (Cushman and Bohnert, reconstruct the evolutionary history of PEPC in Orchidaceae. 1997). Cytosolic and chloroplastic PEPC enzymes are found We have characterized the diversity of ppc genes in a phylo- in photosynthetic organisms from higher plants and green genetic context, using partial-length sequences from a closely algae to cyanobacteria and photosynthetic bacteria, and related group of orchid species in the Oncidiinae that express also in non-photosynthetic bacteria and protozoa (Chollet photosynthetic pathways ranging from C3 photosynthesis et al., 1996; Izui et al., 2004). In all plants, ‘housekeeping’ or to weak CAM and strong CAM. The results highlight the non-photosynthetic isoforms of PEPC catalyse anapleurotic evolutionary diversification ofppc gene families and indicate reactions to replenish biosynthetic precursors for the Krebs the possible role of gene duplication and recruitment of ppc cycle. PEPC has many other physiological roles in plants genes for CAM. that include maintaining cellular pH, supplying carbon to N2-fixing legume root nodules, absorbing and transport- ing cations in roots, control of stomatal movements, fruit maturation, and seed germination (Latzko and Kelly, 1983; Materials