Characterization of bacterial-type phosphoenolpyruvate carboxylase expressed in male gametophyte of higher plants

Abstract

BackgroundPhosphoenolpyruvate carboxylase (PEPC) is a critical enzyme catalyzing the β-carboxylation of phosphoenolpyruvate (PEP) to oxaloacetate, a tricarboxylic acid (TCA) cycle intermediate. PEPC typically exists as a Class-1 PEPC homotetramer composed of plant-type PEPC (PTPC) polypeptides, and two of the subunits were reported to be monoubiquitinated in germinating castor oil seeds. By the large-scale purification of ubiquitin (Ub)-related proteins from lily anther, two types of PEPCs, bacterial-type PEPC (BTPC) and plant-type PEPC (PTPC), were identified in our study as candidate Ub-related proteins. Until now, there has been no information about the properties of the PEPCs expressed in male reproductive tissues of higher plants.ResultsExpression analyses showed that lily BTPC (LlBTPC) and Arabidopsis BTPC (AtBTPC) were significantly expressed in pollen. The fusion protein AtBTPC-Venus localized in the cytoplasm of the vegetative cell (VC). Both LlBTPC and AtBTPC expression initiated after the last mitosis before pollen germination. Lily PTPC (LlPTPC) and monoubiquitinated LlPTPC (Ub-LlPTPC) remained at constant levels during pollen development. In late bicellular pollen of lily, LlBTPC forms a hetero-octameric Class-2 PEPC complex with LlPTPC to express PEPC activity.ConclusionOur results suggest that an LlBTPC:Ub-LlPTPC:LlPTPC complex is formed in the VC cytoplasm during late pollen development. Both LlBTPC and AtBTPC expression patterns are similar to the patterns of the appearance of storage organelles during pollen development in lily and Arabidopsis, respectively. Therefore, BTPC is thought to accelerate the metabolic flow for the synthesis of storage substances during pollen maturation. Our study provides the first characterization of BTPC in pollen, the male gametophyte of higher plants.

Reciprocal Control of Anaplerotic Phosphoenolpyruvate Carboxylase by in Vivo Monoubiquitination and Phosphorylation in Developing Proteoid Roots of Phosphate-Deficient Harsh Hakea

“…Arabidopsis seedlings and most tissues of the castor plant (Uhrig et al, 2008;O'Leary et al, 2011b), maize (Zea mays) roots and leaves (Prinsi et al, 2009), and lily (Lilium spp.) pollen (Igawa et al, 2010). Prinsi and coworkers (2009) showed that nitrate availability may influence monoubiquitination and phosphorylation of PTPC in maize roots and leaves, respectively.…”

“…In contrast, BTPC genes encode larger 116-to 118-kD polypeptides owing to a unique intrinsically disordered region that mediates BTPC's tight interaction with coexpressed PTPC subunits. This association results in the formation of unusual Class-2 PEPC heterooctameric complexes that are largely desensitized to allosteric effectors and that dynamically associate with the surface of mitochondria in vivo (O'Leary et al, 2009(O'Leary et al, , 2011aIgawa et al, 2010;Park et al, 2012).…”

Phosphoenolpyruvate Carboxylase in Arabidopsis Leaves Plays a Crucial Role in Carbon and Nitrogen Metabolism

“…PEPC is found in all plants, green algae, and cyanobacteria, and in most archaea and nonphotosynthetic bacteria, but not in animals or fungi (Chollet et al, 1996;O'Leary et al, 2011a). Several isoforms of PEPC are present in plants, including planttype PEPCs and one bacterium-type PEPC (Sánchez and Cejudo, 2003;Sullivan et al, 2004;Mamedov et al, 2005;Gennidakis et al, 2007;Igawa et al, 2010). Arabidopsis (Arabidopsis thaliana) possesses three plant-type PEPC genes, AtPPC1, AtPPC2, and AtPPC3, and one bacteriumtype PEPC gene, AtPPC4.…”

Regulatory Phosphorylation of Bacterial-Type PEP Carboxylase by the Ca2+-Dependent Protein Kinase RcCDPK1 in Developing Castor Oil Seeds

“…S3) are conserved among vascular plant BTPC orthologs, including the Arabidopsis BTPC, AtPPC4 (Hill et al, 2014). Thus, future studies need to determine if AtCPK4 and/or AtCPK11 participate in the in vivo regulatory phosphorylation of AtPPC4, particularly in developing pollen where AtCPK4, AtCPK11, and AtPPC4 are all highly expressed (Harper et al, 2004;Igawa et al, 2010). Extracts prepared from flowers of Col-0 plants but not from atcpk11 mutants showed Ca 2+ -dependent kinase activity against castor BTPC at Ser-451 suggesting that AtCPK11 is functionally as well as structurally homologous to RcCDPK1 (Fig.…”

“…Plant BTPC polypeptides and thus Class-2 PEPC complexes have only been documented in biosynthetically active tissues (i.e. developing seeds and pollen, immature leaves), as opposed to PTPCs that are constitutively expressed in the cytosol as housekeeping Class-1 PEPCs (Igawa et al, 2010;O'Leary et al, 2011aO'Leary et al, , 2011b. Although COS Class-1 PEPC localizes diffusely throughout the cytosol, the Class-2 PEPC associates with the outer mitochondrial envelope, an interaction mediated by its BTPC subunits .…”