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

<p>Abstract</p> <p>Background</p> <p>Phospho<it>enol</it>pyruvate carboxylase (PEPC) is a critical enzyme catalyzing the β-carboxylation of phospho<it>enol</it>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.</p> <p>Results</p> <p>Expression analyses showed that lily BTPC (LlBTPC) and <it>Arabidopsis </it>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.</p> <p>Conclusion</p> <p>Our 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 <it>Arabidopsis</it>, 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.</p

Rapid Elimination of the Persistent Synergid through a Cell Fusion Mechanism

SummaryIn flowering plants, fertilization-dependent degeneration of the persistent synergid cell ensures one-on-one pairings of male and female gametes. Here, we report that the fusion of the persistent synergid cell and the endosperm selectively inactivates the persistent synergid cell in Arabidopsis thaliana. The synergid-endosperm fusion causes rapid dilution of pre-secreted pollen tube attractant in the persistent synergid cell and selective disorganization of the synergid nucleus during the endosperm proliferation, preventing attractions of excess number of pollen tubes (polytubey). The synergid-endosperm fusion is induced by fertilization of the central cell, while the egg cell fertilization predominantly activates ethylene signaling, an inducer of the synergid nuclear disorganization. Therefore, two female gametes (the egg and the central cell) control independent pathways yet coordinately accomplish the elimination of the persistent synergid cell by double fertilization

Isolation and identification of ubiquitin-related proteins from Arabidopsis seedlings

The majority of proteins in eukaryotic cells are modified according to highly regulated mechanisms to fulfill specific functions and to achieve localization, stability, and transport. Protein ubiquitination is one of the major post-translational modifications occurring in eukaryotic cells. To obtain the proteomic dataset related to the ubiquitin (Ub)-dependent regulatory system in Arabidopsis, affinity purification with an anti-Ub antibody under native condition was performed. Using MS/MS analysis, 196 distinct proteins represented by 251 distinct genes were identified. The identified proteins were involved in metabolism (23.0%), stress response (21.4%), translation (16.8%), transport (6.7%), cell morphology (3.6%), and signal transduction (1.5%), in addition to proteolysis (16.8%) to which proteasome subunits (14.3%) is included. On the basis of potential ubiquitination-targeting signal motifs, in-gel mobilities, and previous reports, 78 of the identified proteins were classified as ubiquitinated proteins and the rest were speculated to be associated proteins of ubiquitinated proteins. The degradation of three proteins predicted to be ubiquitinated proteins was inhibited by a proteasome inhibitor, suggesting that the proteins were regulated by Ub/proteasome-dependent proteolysis

Gamete Attachment Requires GEX2 for Successful Fertilization in Arabidopsis

SummaryFertilization requires recognition, attachment, and membrane fusion between gametes. In metazoans, rapidly evolving surface proteins contribute to gamete recognition and adhesion [1]. Flowering plants evolved a double fertilization process wherein two immotile sperm cells are delivered to female gametes by the pollen tube, guided by elaborate communications between male and female reproductive organs [2–7]. Once released, the sperm cells contact female gametes directly prior to gamete fusion. It remains unclear whether active gamete recognition and attachment mechanisms are required for double fertilization. Here, we provide functional characterization of Arabidopsis GAMETE EXPRESSED 2 (GEX2), which encodes a sperm-expressed protein of unknown function [8]. GEX2 is localized to the sperm membrane and contains extracellular immunoglobulin-like domains, similar to gamete interaction factors in algae and mammals [9, 10]. Using a new in vivo assay, we demonstrate that GEX2 is required for gamete attachment, in the absence of which double fertilization is compromised. Ka/Ks analyses indicate relatively rapid evolution of GEX2, like other proteins involved in male and female interactions [1, 3]. We conclude that surface proteins involved in gamete attachment and recognition exist in plants with immotile gametes, similar to algae and metazoans [11, 12]. This conservation broadens the repertoire of research for plant reproduction factors to mechanisms demonstrated in animals