Rapid Elimination of the Persistent Synergid through a Cell Fusion Mechanism.

In 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.

ERdj3B-Mediated Quality Control Maintains Anther Development at High Temperatures.

Pollen development is highly sensitive to heat stress, which impairs cellular proteostasis by causing misfolded proteins to accumulate. Therefore, each cellular compartment possesses a dedicated protein quality control system. An elaborate quality control system involving molecular chaperones, including immunoglobulin-binding protein (BiP), heat shock protein70, and regulatory J domain-containing cochaperones (J proteins), allows the endoplasmic reticulum (ER) to withstand a large influx of proteins. Here, we found that Arabidopsis (Arabidopsis thaliana) mutants of ER-localized DnaJ family 3B (ERdj3B), one of three ER-resident J proteins involved in ER quality control, produced few seeds at high temperatures (29°C) due to defects in anther development. This temperature-sensitive fertility defect is specific to the defective interactions of BiP with ERdj3B but not with the other two J proteins, indicating functional differences between ERdj3B and the other J proteins. RNA sequencing analysis revealed that heat stress affects pollen development in both wild-type and mutant buds, but the erdj3b mutant is more susceptible, possibly due to defects in ER quality control. Our results highlight the importance of a specific ER quality control factor, ERdj3B, for plant reproduction, particularly anther development, at high temperatures.

Fertilization-Coupled Sperm Nuclear Fusion Is Required for Normal Endosperm Nuclear Proliferation.

Angiosperms exhibit double fertilization, a process in which one of the sperm cells released from the pollen tube fertilizes the egg, while the other sperm cell fertilizes the central cell, giving rise to the embryo and endosperm, respectively. We have previously reported two polar nuclear fusion-defective double knockout mutants of Arabidopsis thaliana immunoglobulin binding protein (BiP), a molecular chaperone of the heat shock protein 70 (Hsp70) localized in the endoplasmic reticulum (ER), (bip1 bip2) and its partner ER-resident J-proteins, ERdj3A and P58IPK (erdj3a p58ipk). These mutants are defective in the fusion of outer nuclear membrane and exhibit characteristic seed developmental defects after fertilization with wild-type pollen, which are accompanied by aberrant endosperm nuclear proliferation. In this study, we used time-lapse live-cell imaging analysis to determine the cause of aberrant endosperm nuclear division in these mutant seeds. We found that the central cell of bip1 bip2 or erdj3a p58ipk double mutant female gametophytes was also defective in sperm nuclear fusion at fertilization. Sperm nuclear fusion was achieved after the onset of the first endosperm nuclear division. However, division of the condensed sperm nucleus resulted in aberrant endosperm nuclear divisions and delayed expression of paternally derived genes. By contrast, the other double knockout mutant, erdj3b p58ipk, which is defective in the fusion of inner membrane of polar nuclei but does not show aberrant endosperm nuclear proliferation, was not defective in sperm nuclear fusion at fertilization. We thus propose that premitotic sperm nuclear fusion in the central cell is critical for normal endosperm nuclear proliferation.

The ribosomal stalk protein is crucial for the action of the conserved ATPase ABCE1.

The ATP-binding cassette (ABC) protein ABCE1 is an essential factor in ribosome recycling during translation. However, the detailed mechanochemistry of its recruitment to the ribosome, ATPase activation and subunit dissociation remain to be elucidated. Here, we show that the ribosomal stalk protein, which is known to participate in the actions of translational GTPase factors, plays an important role in these events. Biochemical and crystal structural data indicate that the conserved hydrophobic amino acid residues at the C-terminus of the archaeal stalk protein aP1 binds to the nucleotide-binding domain 1 (NBD1) of aABCE1, and that this binding is crucial for ATPase activation of aABCE1 on the ribosome. The functional role of the stalk•ABCE1 interaction in ATPase activation and the subunit dissociation is also investigated using mutagenesis in a yeast system. The data demonstrate that the ribosomal stalk protein likely participates in efficient actions of both archaeal and eukaryotic ABCE1 in ribosome recycling. The results also show that the stalk protein has a role in the function of ATPase as well as GTPase factors in translation.

Development of a Heat-Inducible Gene Expression System Using Female Gametophytes of Arabidopsis thaliana.

Female gametophyte (FG) is crucial for reproduction in flowering plants. Arabidopsis thaliana produces Polygonum-type FGs, which consist of an egg cell, two synergid cells, three antipodal cells and a central cell. Egg cell and central cell are the two female gametes that give rise to the embryo and surrounding endosperm, respectively, after fertilization. During the development of a FG, a single megaspore produced by meiosis undergoes three rounds of mitosis to produce an eight-nucleate cell. A seven-celled FG is formed after cellularization. The central cell initially contains two polar nuclei that fuse during female gametogenesis to form the secondary nucleus. In this study, we developed a gene induction system for analyzing the functions of various genes in developing Arabidopsis FGs. This system allows transgene expression in developing FGs using the heat-inducible Cre-loxP recombination system and FG-specific embryo sac 2 (ES2) promoter. Efficient gene induction was achieved in FGs by incubating flower buds and isolated pistils at 35�C for short periods of time (1-5 min). Gene induction was also induced in developing FGs by heat treatment of isolated ovules using the infrared laser-evoked gene operator (IR-LEGO) system. Expression of a dominant-negative mutant of Sad1/UNC84 (SUN) proteins in developing FGs using the gene induction system developed in this study caused defects in polar nuclear fusion, indicating the roles of SUN proteins in this process. This strategy represents a new tool for analyzing the functions of genes in FG development and FG functions.

Different sets of ER-resident J-proteins regulate distinct polar nuclear-membrane fusion events in Arabidopsis thaliana.

Angiosperm female gametophytes contain a central cell with two polar nuclei. In many species, including Arabidopsis thaliana, the polar nuclei fuse during female gametogenesis. We previously showed that BiP, an Hsp70 in the endoplasmic reticulum (ER), was essential for membrane fusion during female gametogenesis. Hsp70 function requires partner proteins for full activity. J-domain containing proteins (J-proteins) are the major Hsp70 functional partners. A. thaliana ER contains three soluble J-proteins, AtERdj3A, AtERdj3B, and AtP58(IPK). Here, we analyzed mutants of these proteins and determined that double-mutant ovules lacking AtP58(IPK) and AtERdj3A or AtERdj3B were defective in polar nuclear fusion. Electron microscopy analysis identified that polar nuclei were in close contact, but no membrane fusion occurred in mutant ovules lacking AtP58(IPK) and AtERdj3A. The polar nuclear outer membrane appeared to be connected via the ER remaining at the inner unfused membrane in mutant ovules lacking AtP58(IPK) and AtERdj3B. These results indicate that ER-resident J-proteins, AtP58(IPK)/AtERdj3A and AtP58(IPK)/AtERdj3B, function at distinct steps of polar nuclear-membrane fusion. Similar to the bip1 bip2 double mutant female gametophytes, the aterdj3a atp58(ipk) double mutant female gametophytes defective in fusion of the outer polar nuclear membrane displayed aberrant endosperm proliferation after fertilization with wild-type pollen. However, endosperm proliferated normally after fertilization of the aterdj3b atp58(ipk) double mutant female gametophytes defective in fusion of the inner membrane. Our results indicate that the polar nuclear fusion defect itself does not cause an endosperm proliferation defect.

Multiple BiP genes of Arabidopsis thaliana are required for male gametogenesis and pollen competitiveness.

Immunoglobulin-binding protein (BiP) is a molecular chaperone of the heat shock protein 70 (Hsp70) family. BiP is localized in the endoplasmic reticulum (ER) and plays key roles in protein translocation, protein folding and quality control in the ER. The genomes of flowering plants contain multiple BiP genes. Arabidopsis thaliana has three BiP genes. BIP1 and BIP2 are ubiquitously expressed. BIP3 encodes a less well conserved BiP paralog, and it is expressed only under ER stress conditions in the majority of organs. Here, we report that all BiP genes are expressed and functional in pollen and pollen tubes. Although the bip1 bip2 double mutation does not affect pollen viability, the bip1 bip2 bip3 triple mutation is lethal in pollen. This result indicates that lethality of the bip1 bip2 double mutation is rescued by BiP3 expression. A decrease in the copy number of the ubiquitously expressed BiP genes correlates well with a decrease in pollen tube growth, which leads to reduced fitness of mutant pollen during fertilization. Because an increased protein secretion activity is expected to increase the protein folding demand in the ER, the multiple BiP genes probably cooperate with each other to ensure ER homeostasis in cells with active secretion such as rapidly growing pollen tubes.

The Plant Organelles Database 3 (PODB3) update 2014: integrating electron micrographs and new options for plant organelle research.

The Plant Organelles Database 2 (PODB2), which was first launched in 2006 as PODB, provides static image and movie data of plant organelles, protocols for plant organelle research and external links to relevant websites. PODB2 has facilitated plant organellar research and the understanding of plant organelle dynamics. To provide comprehensive information on plant organelles in more detail, PODB2 was updated to PODB3 (http://podb.nibb.ac.jp/Organellome/). PODB3 contains two additional components: the electron micrograph database and the perceptive organelles database. Through the electron micrograph database, users can examine the subcellular and/or suborganellar structures in various organs of wild-type and mutant plants. The perceptive organelles database provides information on organelle dynamics in response to external stimuli. In addition to the extra components, the user interface for access has been enhanced in PODB3. The data in PODB3 are directly submitted by plant researchers and can be freely downloaded for use in further analysis. PODB3 contains all the information included in PODB2, and the volume of data and protocols deposited in PODB3 continue to grow steadily. We welcome contributions of data from all plant researchers to enhance the utility and comprehensiveness of PODB3.

BiP-mediated polar nuclei fusion is essential for the regulation of endosperm nuclei proliferation in Arabidopsis thaliana.

Nuclear fusion is an essential process in the sexual reproduction of animals and plants. In flowering plants, nuclear fusion occurs three times: once during female gametogenesis, when the two polar nuclei fuse to produce the diploid central cell nucleus, and twice during double fertilization. The yeast Ig binding protein (BiP) is a molecular chaperone Hsp70 in the endoplasmic reticulum that regulates nuclear membrane fusion during mating. Here we report that in Arabidopsis thaliana, BiP is involved in the fusion of polar nuclei during female gametophyte development. BiP-deficient mature female gametophytes contain two unfused polar nuclei, in spite of their close contact. This indicates a surprising conservation of BiP function in nuclear fusion between plants and yeasts. We also found that endosperm nuclear division becomes aberrant after fertilization of the BiP-deficient female gametophytes with wild-type pollen. This is experimental evidence for the importance of fusion of the polar nuclei in the proliferation of endosperm nuclei.

Nuclear Fusion in Yeast and Plant Reproduction.

Nuclear fusion is essential for the sexual reproduction of various organisms, including plants, animals, and fungi. During the life cycle of flowering plants, nuclear fusion occurs three times: once during female gametogenesis and twice during double fertilization, when two sperm cells fertilize the egg and the central cell. Haploid nuclei migrate in an actin filament-dependent manner to become in close contact and, then, two nuclei fuse. The nuclear fusion process in plant reproduction is achieved through sequential nuclear membrane fusion events. Recent molecular genetic analyses using Arabidopsis thaliana showed the conservation of nuclear membrane fusion machinery between plants and the budding yeast Saccharomyces cerevisiae. These include the heat-shock protein 70 in the endoplasmic reticulum and the conserved nuclear membrane proteins. Analyses of the A. thaliana mutants of these components show that the completion of the sperm nuclear fusion at fertilization is essential for proper embryo and endosperm development.

Structural basis of yeast Tim40/Mia40 as an oxidative translocator in the mitochondrial intermembrane space.

The mitochondrial intermembrane space (IMS) contains many small cysteine-bearing proteins, and their passage across the outer membrane and subsequent folding require recognition and disulfide bond transfer by an oxidative translocator Tim40/Mia40 in the inner membrane facing the IMS. Here we determined the crystal structure of the core domain of yeast Mia40 (Mia40C4) as a fusion protein with maltose-binding protein at a resolution of 3 A. The overall structure of Mia40C4 is a fruit-dish-like shape with a hydrophobic concave region, which accommodates a linker segment of the fusion protein in a helical conformation, likely mimicking a bound substrate. Replacement of the hydrophobic residues in this region resulted in growth defects and impaired assembly of a substrate protein. The Cys296-Cys298 disulfide bond is close to the hydrophobic concave region or possible substrate-binding site, so that it can mediate disulfide bond transfer to substrate proteins. These results are consistent with the growth phenotypes of Mia40 mutant cells containing Ser replacement of the conserved cysteine residues.

Expression of GEX1 Orthologs of Brassica rapa and Oryza sativa Rescued the Nuclear Fusion Defect of the Arabidopsis GEX1 Mutant.

Nuclear fusion is required for the sexual reproduction of various organisms, including angiosperms. During the life cycle of angiosperms, nuclear fusion occurs three times: once during female gametogenesis, when the two polar nuclei fuse in the central cell, and twice during double fertilization. Nuclear fusion in plant reproduction is achieved by sequential nuclear fusion events: outer and inner nuclear membrane fusion. Arabidopsis gamete expressed 1 (GEX1) is a nuclear membrane protein of gametes that is required for nuclear fusion during reproduction. Although orthologs of GEX1 have been identified in various land plants, sequence identities are not high, even between angiosperm GEX1 orthologs; the sequence identity between Arabidopsis GEX1 and Oryza sativa GEX1 ortholog is lower than 50%. Here, we found that the expression of GEX1 orthologs of O. sativa, as well as of Brassica rapa from the Arabidopsis GEX1 promoter, rescued the polar nuclear fusion defect of the gex1 mutant. We also found that the expression of these GEX1 orthologs rescued the lethality of the gex1 homozygous mutant, which is proposed to be caused by the sperm nuclear fusion defects upon fertilization. Our results indicate a functional conservation between Arabidopsis and O. sativa GEX1 orthologs, despite their relatively low sequence identities.

The Plant Organelles Database 2 (PODB2): an updated resource containing movie data of plant organelle dynamics.

The Plant Organelles Database (PODB) was launched in 2006 and provides imaging data of plant organelles, protocols for plant organelle research and external links to relevant websites. To provide comprehensive information on plant organelle dynamics and accommodate movie files that contain time-lapse images and 3D structure rotations, PODB was updated to the next version, PODB2 (http://podb.nibb.ac.jp/Organellome). PODB2 contains movie data submitted directly by plant researchers and can be freely downloaded. Through this organelle movie database, users can examine the dynamics of organelles of interest, including their movement, division, subcellular positioning and behavior, in response to external stimuli. In addition, the user interface for access and submission has been enhanced. PODB2 contains all of the information included in PODB, and the volume of data and protocols deposited in the PODB2 continues to grow steadily. Moreover, a new website, Plant Organelles World (http://podb.nibb.ac.jp/Organellome/PODBworld/en/index.html), which is based on PODB2, was recently launched as an educational tool to engage members of the non-scientific community such as students and school teachers. Plant Organelles World is written in layman's terms, and technical terms were avoided where possible. We would appreciate contributions of data from all plant researchers to enhance the usefulness of PODB2 and Plant Organelles World.

LAP5 and LAP6 encode anther-specific proteins with similarity to chalcone synthase essential for pollen exine development in Arabidopsis.

Pollen grains of land plants have evolved remarkably strong outer walls referred to as exine that protect pollen and interact with female stigma cells. Exine is composed of sporopollenin, and while the composition and synthesis of this biopolymer are not well understood, both fatty acids and phenolics are likely components. Here, we describe mutations in the Arabidopsis (Arabidopsis thaliana) LESS ADHESIVE POLLEN (LAP5) and LAP6 that affect exine development. Mutation of either gene results in abnormal exine patterning, whereas pollen of double mutants lacked exine deposition and subsequently collapsed, causing male sterility. LAP5 and LAP6 encode anther-specific proteins with homology to chalcone synthase, a key flavonoid biosynthesis enzyme. lap5 and lap6 mutations reduced the accumulation of flavonoid precursors and flavonoids in developing anthers, suggesting a role in the synthesis of phenolic constituents of sporopollenin. Our in vitro functional analysis of LAP5 and LAP6 using 4-coumaroyl-coenzyme A yielded bis-noryangonin (a commonly reported derailment product of chalcone synthase), while similar in vitro analyses using fatty acyl-coenzyme A as the substrate yielded medium-chain alkyl pyrones. Thus, in vitro assays indicate that LAP5 and LAP6 are multifunctional enzymes and may play a role in both the synthesis of pollen fatty acids and phenolics found in exine. Finally, the genetic interaction between LAP5 and an anther gene involved in fatty acid hydroxylation (CYP703A2) demonstrated that they act synergistically in exine production.

Identification of Tam41 maintaining integrity of the TIM23 protein translocator complex in mitochondria.

Newly synthesized mitochondrial proteins are imported into mitochondria with the aid of protein translocator complexes in the outer and inner mitochondrial membranes. We report the identification of yeast Tam41, a new member of mitochondrial protein translocator systems. Tam41 is a peripheral inner mitochondrial membrane protein facing the matrix. Disruption of the TAM41 gene led to temperature-sensitive growth of yeast cells and resulted in defects in protein import via the TIM23 translocator complex at elevated temperature both in vivo and in vitro. Although Tam41 is not a constituent of the TIM23 complex, depletion of Tam41 led to a decreased molecular size of the TIM23 complex and partial aggregation of Pam18 and -16. Import of Pam16 into mitochondria without Tam41 was retarded, and the imported Pam16 formed aggregates in vitro. These results suggest that Tam41 facilitates mitochondrial protein import by maintaining the functional integrity of the TIM23 protein translocator complex from the matrix side of the inner membrane.

Roles of Tom70 in import of presequence-containing mitochondrial proteins.

Mitochondrial protein traffic requires precise recognition of the mitochondrial targeting signals by the import receptors on the mitochondrial surface including a general import receptor Tom20 and a receptor for presequence-less proteins, Tom70. Here we took a proteome-wide approach of mitochondrial protein import in vitro to find a set of presequence-containing precursor proteins for recognition by Tom70. The presequences of the Tom70-dependent precursor proteins were recognized by Tom20, whereas their mature parts exhibited Tom70-dependent import when attached to the presequence of Tom70-independent precursor proteins. The mature parts of the Tom70-dependent precursor proteins have the propensity to aggregate, and the presence of the receptor domain of Tom70 prevents their aggregate formation. Therefore Tom70 plays the role of a docking site for not only cytosolic chaperones but also aggregate-prone substrates to maintain their solubility for efficient transfer to downstream components of the mitochondrial import machineries.

A vacuolar carboxypeptidase mutant of Arabidopsis thaliana is degraded by the ERAD pathway independently of its N-glycan.

Misfolded proteins produced in the endoplasmic reticulum (ER) are degraded by a mechanism, the ER-associated degradation (ERAD). Here we report establishment of the experimental system to analyze the ERAD in plant cells. Carboxypeptidase Y (CPY) is a vacuolar enzyme and its mutant CPY* is degraded by the ERAD in yeast. Since Arabidopsis thaliana has AtCPY, an ortholog of yeast CPY, we constructed and expressed fusion proteins consisting of AtCPY and GFP and of AtCPY*, which carries a mutation homologous to yeast CPY*, and GFP in A. thaliana cells. While AtCPY-GFP was efficiently transported to the vacuole, AtCPY*-GFP was retained in the ER to be degraded in proteasome- and Cdc48-dependent manners. We also found that AtCPY*-GFP was degraded by the ERAD in yeast cells, but that its single N-glycan did not function as a degradation signal in yeast or plant cells. Therefore, AtCPY*-GFP can be used as a marker protein to analyze the ERAD pathway, likely for nonglycosylated substrates, in plant cells.

The plant organelles database (PODB): a collection of visualized plant organelles and protocols for plant organelle research.

The plant organelles database (PODB; http://podb.nibb.ac.jp/Organellome) was built to promote a comprehensive understanding of organelle dynamics, including organelle function, biogenesis, differentiation, movement and interactions with other organelles. This database consists of three individual parts, the organellome database, the functional analysis database and external links to other databases and homepages. The organellome database provides images of various plant organelles that were visualized with fluorescent and nonfluorescent probes in various tissues of several plant species at different developmental stages. The functional analysis database is a collection of protocols for plant organelle research. External links give access primarily to other databases and Web pages with information on transcriptomes and proteomes. All the data and protocols in the organellome database and the functional analysis database are populated by direct submission of experimentally determined data from plant researchers and can be freely downloaded. Our database promotes the exchange of information between plant organelle researchers for the comprehensive study of the organelle dynamics that support integrated functions in higher plants. We would also appreciate contributions of data and protocols from all plant researchers to maximize the usefulness of the database.

Arabidopsis GEX1 Is a Nuclear Membrane Protein of Gametes Required for Nuclear Fusion During Reproduction.

During the life cycle of flowering plants, nuclear fusion, or karyogamy, occurs three times: once during female gametogenesis, when the two polar nuclei fuse in the central cell, and twice during double fertilization. In Arabidopsis thaliana, nuclear fusion events during sexual reproduction proceed without the breakdown of the nuclear envelope, indicating that nuclear membrane fusion is essential for the completion of this process. Arabidopsis gamete expressed 1 (GEX1) is a membrane protein that is conserved among plant species. GEX1 shares homology with the yeast karyogamy protein Kar5, which is primarily expressed in the nuclear membrane. The GEX1 family represents a putative karyogamy factor. Herein, we show that GEX1 is required for the nuclear fusion events in Arabidopsis reproduction. GEX1-deficient mature female gametophytes were found to contain two unfused polar nuclei in close proximity within the central cell. Electron microscopy showed that the outer membrane of the polar nuclei was connected via the endoplasmic reticulum, whereas the inner membrane remained unfused. These results indicate that GEX1 is involved in polar nuclear membrane fusion following the fusion of the outer nuclear membrane. Furthermore, sperm nuclear fusion events were defective in the fertilized egg and central cell following plasmogamy in the fertilization of gex1-1 female gametophytes by gex1-1 pollen. An analysis of GEX1 localization in the female gametophyte using a transgenic line expressing GFP-tagged GEX1 driven by the GEX1 promoter showed that GEX1 is a nuclear membrane protein in the egg and central cell. Time-lapse live-cell imaging showed that in developing female gametophytes, the nuclear GFP-GEX1 signal was first detectable in the central cell shortly before the polar nuclei came in close contact, and then in the egg cell. Thus, we suggest that the GEX1-family proteins are nuclear membrane proteins involved in karyogamy in the reproduction of eukaryotes including flowering plants.

Seeing is believing: on the use of image databases for visually exploring plant organelle dynamics.

Organelle dynamics vary dramatically depending on cell type, developmental stage and environmental stimuli, so that various parameters, such as size, number and behavior, are required for the description of the dynamics of each organelle. Imaging techniques are superior to other techniques for describing organelle dynamics because these parameters are visually exhibited. Therefore, as the results can be seen immediately, investigators can more easily grasp organelle dynamics. At present, imaging techniques are emerging as fundamental tools in plant organelle research, and the development of new methodologies to visualize organelles and the improvement of analytical tools and equipment have allowed the large-scale generation of image and movie data. Accordingly, image databases that accumulate information on organelle dynamics are an increasingly indispensable part of modern plant organelle research. In addition, image databases are potentially rich data sources for computational analyses, as image and movie data reposited in the databases contain valuable and significant information, such as size, number, length and velocity. Computational analytical tools support image-based data mining, such as segmentation, quantification and statistical analyses, to extract biologically meaningful information from each database and combine them to construct models. In this review, we outline the image databases that are dedicated to plant organelle research and present their potential as resources for image-based computational analyses.