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.
Highlights
Phosphoenolpyruvate carboxylase (PEPC) is a critical enzyme catalyzing the b-carboxylation of phosphoenolpyruvate (PEP) to oxaloacetate, a tricarboxylic acid (TCA) cycle intermediate
Our results suggest that bacterial-type PEPC (BTPC) forms a complex with plant-type PEPC (PTPC) and monoubiquitinated PTPC (Ub-PTPC) to accelerate the accumulation of storage substances during pollen maturation
BTPC and PTPC are identified as Ub-related proteins in lily anther, and AtBTPC shows stamen-specific expression With the large-scale purification of Ub-related proteins from the lily anther, 13 proteins were identified as candidate Ub-related proteins by liquid chromatographytandem mass spectrometry (LC-MS/MS)
Summary
Phosphoenolpyruvate carboxylase (PEPC) is a critical enzyme catalyzing the b-carboxylation of phosphoenolpyruvate (PEP) to oxaloacetate, a tricarboxylic acid (TCA) cycle intermediate. PEPC has been extensively studied in C4 and CAM photosynthesis, because it is a critical enzyme catalyzing the initial reaction of atmospheric CO2 fixation [1]. It plays pivotal metabolic roles in nonphotosynthetic and C3 photosynthetic cells, in the anaplerotic replenishment of the TCA cycle intermediates consumed during lipid synthesis [4], biosynthesis, and nitrogen assimilation [5]. BTPC resembles the bacterial PEPC rather than the common plant PEPC in terms of its gene structure and the absence of an N-terminal seryl-phosphorylation domain, a hallmark of PTPC (Additional file 2). Recent studies have indicated that BTPC in developing castor oil seeds (COS) interacts with PTPC to form a heterooctameric complex with PEPC activity [7,8]
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