Behavior of filamentous temperature-sensitive Z2 (FtsZ2) in the male gametophyte during sexual reproduction processes of flowering plants.

Filamentous temperature-sensitive Z (FtsZ) is a critical division protein in bacteria that functions in forming a Z-ring structure to constrict the cell. Since the establishment of the plastid by endosymbiosis of a cyanobacterium into a eukaryotic cell, division via Z-ring formation has been conserved in the plastids of flowering plants. The FtsZ gene was transferred from the cyanobacterial ancestor of plastids to the eukaryotic nuclear genome during evolution, and flowering plants evolved two FtsZ homologs, FtsZ1 and FtsZ2, which are involved in chloroplast division through distinct molecular functions. Regarding the behaviors of FtsZ in nonphotosynthetic cells, the plastid localization of FtsZ1 proteins in the cytoplasm of microspores and pollen vegetative cells but not in generative cells or sperm cells has been reported. On the other hand, the significant accumulation of FtsZ2 transcripts in generative cells has been reported. However, the synthesis of FtsZ2 in the male gamete has not been investigated. Additionally, FtsZ2 behavior has not been analyzed in pollen, a nonphotosynthetic male tissue. Here, we report FtsZ2 protein behaviors in the male gamete by analyzing the localization patterns of GFP-fused protein at various pollen developmental stages and in gametes during the fertilization process. Our results showed that FtsZ2 localization coincided with that of plastids. FtsZ2 protein in male gametes was almost absent, despite the presence of the transcripts. Moreover, transmission of paternal FtsZ2 transcripts to the zygote and endosperm was not observed.

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization.

Flowering plants have a unique sexual reproduction system called 'double fertilization', in which each of the sperm cells precisely fuses with an egg cell or a central cell. Thus, two independent fertilization events take place almost simultaneously. The fertilized egg cell and central cell develop into zygote and endosperm, respectively. Therefore, precise control of double fertilization is essential for the ensuing seed development. Double fertilization occurs in the female gametophyte (embryo sac), which is deeply hidden and covered with thick ovule and ovary tissues. This pistil tissue construction makes observation and analysis of double fertilization quite difficult and has created the present situation in which many questions regarding the mechanism of double fertilization remain unanswered. For the functional evaluation of a potential candidate for fertilization regulator, phenotypic analysis of fertilization is important. To judge the completion of fertilization in Arabidopsis thaliana, the shapes of fluorescence signals labeling sperm nuclei are used as indicators. A sperm cell that fails to fertilize is indicated by a condensed fluorescence signal outside of the female gametes, whereas a sperm cell that successfully fertilizes is indicated by a decondensed signal due to karyogamy with the female gametes' nucleus. The method described here provides a tool to determine successful or failed fertilization under in vivo conditions.

The male gamete membrane protein DMP9/DAU2 is required for double fertilization in flowering plants.

All flowering plants exhibit a unique type of sexual reproduction called 'double fertilization' in which each pollen tube-delivered sperm cell fuses with an egg and a central cell. Proteins that localize to the plasma membrane of gametes regulate one-to-one gamete pairing and fusion between male and female gametes for successful double fertilization. Here, we have identified a membrane protein from Lilium longiflorum generative cells using proteomic analysis and have found that the protein is an ortholog of Arabidopsis DUF679 DOMAIN MEMBRANE PROTEIN 9 (DMP9)/DUO1-ACTIVATED UNKNOWN 2 (DAU2). The flowering plant DMP9 proteins analyzed in this study were predicted to have four transmembrane domains and be specifically expressed in both generative and sperm cells. Knockdown of DMP9 resulted in aborted seeds due to single fertilization of the central cell. Detailed imaging of DMP9-knockdown sperm cells during in vivo and semi-in vitro double fertilization revealed that DMP9 is involved in gamete interaction that leads to correct double fertilization.

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

BACKGROUND: Phosphoenolpyruvate carboxylase (PEPC) is a critical enzyme catalyzing the ß-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. RESULTS: Expression 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. CONCLUSION: 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 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.