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.

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.

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.

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.

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.

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.

Quality control of nonstop membrane proteins at the ER membrane and in the cytosol.

Since messenger RNAs without a stop codon (nonstop mRNAs) for organelle-targeted proteins and their translation products (nonstop proteins) generate clogged translocon channels as well as stalled ribosomes, cells have mechanisms to degrade nonstop mRNAs and nonstop proteins and to clear the translocons (e.g. the Sec61 complex) by release of nonstop proteins into the organellar lumen. Here we followed the fate of nonstop endoplasmic reticulum (ER) membrane proteins with different membrane topologies in yeast to evaluate the importance of the Ltn1-dependent cytosolic degradation and the Dom34-dependent release of the nonstop membrane proteins. Ltn1-dependent degradation differed for membrane proteins with different topologies and its failure did not affect ER protein import or cell growth. On the other hand, failure in the Dom34-dependent release of the nascent polypeptide from the ribosome led to the block of the Sec61 channel and resultant inhibition of other protein import into the ER caused cell growth defects. Therefore, the nascent chain release from the translation apparatus is more instrumental in clearance of the clogged ER translocon channel and thus maintenance of normal cellular functions.

BiP3 supports the early stages of female gametogenesis in the absence of BiP1 and BiP2 in Arabidopsis thaliana.

Immunoglobulin binding protein (BiP) is an essential heat shock protein 70 (Hsp70) in the endoplasmic reticulum (ER) that functions in various processes including protein translocation, protein folding and quality control. Arabidopsis thaliana harbors ubiquitously expressed genes BIP1 and BIP2, as well as BIP3, which is induced only by ER stress. Recently, we reported that these BIP genes are expressed in male gametophytes and cooperate with each other to support male gametogenesis and pollen competitiveness. Here, we report that the BIP genes cooperate to support female gametogenesis. As reported previously, the bip1 bip2 double mutation causes defects in the fusion of polar nuclei during female gametogenesis. By contrast, the bip triple mutant female gametophytes exhibited defects during the early stages of female gametophyte development, which suggests that BIP3 supports the early stages of female gametophyte development, but not polar nuclear fusion, in the absence of BiP1 and BiP2.

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.

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.

Analyses of protein-protein interactions by in vivo photocrosslinking in budding yeast.

Recent development of methods for genetic incorporation of unnatural amino acids into proteins in live cells enables us to analyze protein interactions by site-specific photocrosslinking. Here we describe a method to incorporate p-benzoyl-L-phenylalanine (pBpa), a photoreactive unnatural amino acid, into defined positions of a target protein in living yeast cells. Photocrosslinking using the pBpa-incorporated proteins has been proven to be a powerful method for analyzing protein-protein interactions at the spatial resolution of amino-acid residues. Since photocrosslinking can be performed for pBpa-incorporated proteins that are properly assembled into a protein complex in living cells, this method will allow us to reveal protein-protein interactions of the target proteins at work.

Tam41 is a CDP-diacylglycerol synthase required for cardiolipin biosynthesis in mitochondria.

CDP-diacylglycerol (CDP-DAG) is central to the phospholipid biosynthesis pathways in cells. A prevailing view is that only one CDP-DAG synthase named Cds1 is present in both the endoplasmic reticulum (ER) and mitochondrial inner membrane (IM) and mediates generation of CDP-DAG from phosphatidic acid (PA) and CTP. However, we demonstrate here by using yeast Saccharomyces cerevisiae as a model organism that Cds1 resides in the ER but not in mitochondria, and that Tam41, a highly conserved mitochondrial maintenance protein, directly catalyzes the formation of CDP-DAG from PA in the mitochondrial IM. We also find that inositol depletion by overexpressing an arrestin-related protein Art5 partially restores the defects of cell growth and CL synthesis in the absence of Tam41. The present findings unveil the missing step of the cardiolipin synthesis pathway in mitochondria as well as the flexibile regulation of phospholipid biosynthesis to respond to compromised CDP-DAG synthesis in mitochondria.

Roles of dom34:hbs1 in nonstop protein clearance from translocators for normal organelle protein influx.

Because messenger RNAs without a stop codon (nonstop mRNAs) generate stalled ribosomes, cells have developed a mechanism allowing degradation of nonstop mRNAs and their translation products (nonstop proteins) in the cytosol. Here, we observe the fate of nonstop proteins destined for organelles such as the endoplasmic reticulum (ER) and mitochondria. Nonstop mRNAs for secretory-pathway proteins in yeast generate nonstop proteins that become stuck in the translocator, the Sec61 complex, in the ER membrane. These stuck nonstop secretory proteins avoid proteasomal degradation in the cytosol, but are instead released into the ER lumen through stalled ribosome and translocator channels by Dom34:Hbs1. We also found that nonstop mitochondrial proteins are cleared from the mitochondrial translocator, the TOM40 complex, by Dom34:Hbs1. Clearance of stuck nonstop proteins from organellar translocator channels is crucial for normal protein influx into organelles and for normal cell growth, especially when nonstop mRNA decay does not function efficiently.

Yos9p and Hrd1p mediate ER retention of misfolded proteins for ER-associated degradation.

The endoplasmic reticulum (ER) has an elaborate quality control system, which retains misfolded proteins and targets them to ER-associated protein degradation (ERAD). To analyze sorting between ER retention and ER exit to the secretory pathway, we constructed fusion proteins containing both folded carboxypeptidase Y (CPY) and misfolded mutant CPY (CPY*) units. Although the luminal Hsp70 chaperone BiP interacts with the fusion proteins containing CPY* with similar efficiency, a lectin-like ERAD factor Yos9p binds to them with different efficiency. Correlation between efficiency of Yos9p interactions and ERAD of these fusion proteins indicates that Yos9p but not BiP functions in the retention of misfolded proteins for ERAD. Yos9p targets a CPY*-containing ERAD substrate to Hrd1p E3 ligase, thereby causing ER retention of the misfolded protein. This ER retention is independent of the glycan degradation signal on the misfolded protein and operates even when proteasomal degradation is inhibited. These results collectively indicate that Yos9p and Hrd1p mediate ER retention of misfolded proteins in the early stage of ERAD, which constitutes a process separable from the later degradation step.

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.

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.