Both LlBTPC and AtBTPC expression patterns are similar to the patterns of the appearance of storage organelles during pollen development in lily and em Arabidopsis /em , respectively. red bars above and the blue bars below the alignment indicate the corresponding peptide sequences of AtPTPC and AtBTPC, respectively, which were detected in the lily anther with LC-MS/MS analysis. The amino acid sequences overlain with pale red and pale blue were used to produce the anti-AtPTPC and anti-AtBTPC antibodies, respectively. 1471-2229-10-200-S2.PDF (13M) GUID:?4A62DC65-F889-4AA8-8F73-6D0469DC9B26 Additional file 3 Immunopurified proteins with FK2 from lily anther. Proteins immunoprecipitated with FK2 from lily anther were subjected to SDS-PAGE and stained with Flamingo?(Bio-Rad Laboratories, CA, USA). Clear bands (marked with lowercase letter) were excised and numbered smearing regions cut into 2-mm-long gel pieces were digested with trypsin for LC-MS/MS analysis. 1471-2229-10-200-S3.PDF (1.3M) GUID:?63E29BF2-2EE2-4A08-82CA-4B99A838AA62 Additional file 4 Table of Ub-related proteins identified from lily anther and the putative em Arabidopsis CAPN1 /em orthologous genes. This additional file contains a table of the Ub-related proteins identified in the lily anther and the putative em Arabidopsis /em orthologous proteins. Candidate proteins with high reliability (MASCOT score 40; em P /em 0.05) are listed. The gel position for each identified polypeptide corresponds to that in Additional file 3. The approximate size of each identified protein was estimated by calculation based on the mobilities of marker proteins (indicated around the left of the panel in Additional file 3). Each em Arabidopsis /em orthologous protein was determined with a BLASTP search at the TAIR website http://www.arabidopsis.org/Blast/index.jsp based on the protein sequence indicated in the corresponding column. The expression of the genes marked with an asterisk was checked by RT-PCR (see Figure ?Physique11). 1471-2229-10-200-S4.PDF (61K) GUID:?E5CCABE8-D015-4EEF-BC02-1497EBE2C4C7 Additional file 5 Primers used in this study. Primers used in this study. 1471-2229-10-200-S5.PDF (7.0M) GUID:?722E07B1-0DE8-4122-A287-32FEBD79AC3C Abstract Background Phospho em enol /em pyruvate carboxylase (PEPC) is a Tranilast (SB 252218) critical enzyme catalyzing the -carboxylation of phospho em enol /em pyruvate (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. Results Expression analyses showed that lily BTPC (LlBTPC) and em Arabidopsis /em 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. Conclusion Our results suggest that an Tranilast (SB 252218) 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 em Arabidopsis /em , 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. Background Phospho em enol /em pyruvate carboxylase (PEPC, EC4.1.1.31) catalyzes the irreversible -carboxylation of phospho em enol /em pyruvate (PEP) to yield oxaloacetate and inorganic phosphate (Additional file 1). PEPC exists widely in plants, algae, and bacteria, but not in animals Tranilast (SB 252218) or fungi Tranilast (SB 252218) [1]. In plants, PEPC acts as an allosteric enzyme and is phosphorylated by PEPC protein kinase [1-3]. Active PEPC commonly consists of a plant-type PEPC (PTPC) homotetramer, and is typically inhibited by L-malate and aspartic acid and activated by glucose-6-phosphate (Glc-6-P). PEPC has been extensively studied in C4 and CAM photosynthesis, because it is usually a critical enzyme catalyzing the initial reaction of atmospheric CO2 fixation [1]. It also plays pivotal metabolic roles in nonphotosynthetic and C3 photosynthetic cells, particularly in the anaplerotic replenishment of the TCA cycle intermediates consumed during lipid synthesis [4], biosynthesis, and nitrogen assimilation [5]. The genomic analysis of the PEPC of em Arabidopsis /em and rice first revealed that higher plants contain a small PEPC family made up of two types of PEPC, PTPC and bacterial-type PEPC (BTPC) [6]. BTPC resembles the bacterial PEPC rather than.