The Functional Domain of GCS1-Based Gamete Fusion Resides in the Amino Terminus in Plant and Parasite Species

Fertilization is one of the most important processes in all organisms utilizing sexual reproduction. In a previous study, we succeeded in identifying a novel male gametic transmembrane protein GCS1 (GENERATIVE CELL SPECIFIC 1), also called HAP2 (HAPLESS 2) in the male-sterile Arabidopsis thaliana mutants, as a factor critical to gamete fusion in flowering plants. Interestingly, GCS1 is highly conserved among various eukaryotes covering plants, protists and invertebrates. Of these organisms, Chlamydomonas (green alga) and Plasmodium (malaria parasite) GCS1s similarly show male gametic expression and gamete fusion function. Since it is generally believed that protein factors controlling gamete fusion have rapidly evolved and different organisms utilize species-specific gamete fusion factors, GCS1 may be an ancient fertilization factor derived from the common ancestor of those organisms above. And therefore, its molecular structure and function are important to understanding the common molecular mechanics of eukaryotic fertilization. In this study, we tried to detect the central functional domain(s) of GCS1, using complementation assay of ArabidopsisGCS1 mutant lines expressing modified GCS1. As a result, the positively-charged C-terminal sequence of this protein is dispensable for gamete fusion, while the highly conserved N-terminal domain is critical to GCS1 function. In addition, in vitro fertilization assay of Plasmodium berghei (mouse malaria parasite) knock-in lines expressing partly truncated GCS1 showed similar results. Those findings above indicate that the extracellular N-terminus alone is sufficient for GCS1-based gamete fusion

The YlmG protein has a conserved function related to the distribution of nucleoids in chloroplasts and cyanobacteria

<p>Abstract</p> <p>Background</p> <p>Reminiscent of their free-living cyanobacterial ancestor, chloroplasts proliferate by division coupled with the partition of nucleoids (DNA-protein complexes). Division of the chloroplast envelope membrane is performed by constriction of the ring structures at the division site. During division, nucleoids also change their shape and are distributed essentially equally to the daughter chloroplasts. Although several components of the envelope division machinery have been identified and characterized, little is known about the molecular components/mechanisms underlying the change of the nucleoid structure.</p> <p>Results</p> <p>In order to identify new factors that are involved in the chloroplast division, we isolated <it>Arabidopsis thaliana </it>chloroplast division mutants from a pool of random cDNA-overexpressed lines. We found that the overexpression of a previously uncharacterized gene (<it>AtYLMG1-1</it>) of cyanobacterial origin results in the formation of an irregular network of chloroplast nucleoids, along with a defect in chloroplast division. In contrast, knockdown of <it>AtYLMG1-1 </it>resulted in a concentration of the nucleoids into a few large structures, but did not affect chloroplast division. Immunofluorescence microscopy showed that AtYLMG1-1 localizes in small puncta on thylakoid membranes, to which a subset of nucleoids colocalize. In addition, in the cyanobacterium <it>Synechococcus elongates</it>, overexpression and deletion of <it>ylmG </it>also displayed defects in nucleoid structure and cell division.</p> <p>Conclusions</p> <p>These results suggest that the proper distribution of nucleoids requires the YlmG protein, and the mechanism is conserved between cyanobacteria and chloroplasts. Given that <it>ylmG </it>exists in a cell division gene cluster downstream of <it>ftsZ </it>in gram-positive bacteria and that <it>ylmG </it>overexpression impaired the chloroplast division, the nucleoid partitioning by YlmG might be related to chloroplast and cyanobacterial division processes.</p

Bacterial Genes in the Aphid Genome: Absence of Functional Gene Transfer from Buchnera to Its Host

Genome reduction is typical of obligate symbionts. In cellular organelles, this reduction partly reflects transfer of ancestral bacterial genes to the host genome, but little is known about gene transfer in other obligate symbioses. Aphids harbor anciently acquired obligate mutualists, Buchnera aphidicola (Gammaproteobacteria), which have highly reduced genomes (420–650 kb), raising the possibility of gene transfer from ancestral Buchnera to the aphid genome. In addition, aphids often harbor other bacteria that also are potential sources of transferred genes. Previous limited sampling of genes expressed in bacteriocytes, the specialized cells that harbor Buchnera, revealed that aphids acquired at least two genes from bacteria. The newly sequenced genome of the pea aphid, Acyrthosiphon pisum, presents the first opportunity for a complete inventory of genes transferred from bacteria to the host genome in the context of an ancient obligate symbiosis. Computational screening of the entire A. pisum genome, followed by phylogenetic and experimental analyses, provided strong support for the transfer of 12 genes or gene fragments from bacteria to the aphid genome: three LD–carboxypeptidases (LdcA1, LdcA2,ψLdcA), five rare lipoprotein As (RlpA1-5), N-acetylmuramoyl-L-alanine amidase (AmiD), 1,4-beta-N-acetylmuramidase (bLys), DNA polymerase III alpha chain (ψDnaE), and ATP synthase delta chain (ψAtpH). Buchnera was the apparent source of two highly truncated pseudogenes (ψDnaE and ψAtpH). Most other transferred genes were closely related to genes from relatives of Wolbachia (Alphaproteobacteria). At least eight of the transferred genes (LdcA1, AmiD, RlpA1-5, bLys) appear to be functional, and expression of seven (LdcA1, AmiD, RlpA1-5) are highly upregulated in bacteriocytes. The LdcAs and RlpAs appear to have been duplicated after transfer. Our results excluded the hypothesis that genome reduction in Buchnera has been accompanied by gene transfer to the host nuclear genome, but suggest that aphids utilize a set of duplicated genes acquired from other bacteria in the context of the Buchnera–aphid mutualism

The Polycomb repressive complex 2 deposits H3K27me3 and represses transposable elements in a broad range of eukaryotes

The mobility of transposable elements (TEs) contributes to evolution of genomes. Their uncontrolled activity causes genomic instability; therefore, expression of TEs is silenced by host genomes. TEs are marked with DNA and H3K9 methylation, which are associated with silencing in flowering plants, animals, and fungi. However, in distantly related groups of eukaryotes, TEs are marked by H3K27me3 deposited by the Polycomb repressive complex 2 (PRC2), an epigenetic mark associated with gene silencing in flowering plants and animals. The direct silencing of TEs by PRC2 has so far only been shown in one species of ciliates. To test if PRC2 silences TEs in a broader range of eukaryotes, we generated mutants with reduced PRC2 activity and analyzed the role of PRC2 in extant species along the lineage of Archaeplastida and in the diatom P. tricornutum. In this diatom and the red alga C. merolae, a greater proportion of TEs than genes were repressed by PRC2, whereas a greater proportion of genes than TEs were repressed by PRC2 in bryophytes. In flowering plants, TEs contained potential cis-elements recognized by transcription factors and associated with neighbor genes as transcriptional units repressed by PRC2. Thus, silencing of TEs by PRC2 is observed not only in Archaeplastida but also in diatoms and ciliates, suggesting that PRC2 deposited H3K27me3 to silence TEs in the last common ancestor of eukaryotes. We hypothesize that during the evolution of Archaeplastida, TE fragments marked with H3K27me3 were selected to shape transcriptional regulation, controlling networks of genes regulated by PRC2

Cultivation of acidophilic algae Galdieria sulphuraria and Pseudochlorella sp. YKT1 in media derived from acidic hot springs

Microalgae possess a high potential for producing pigments, antioxidants, and lipophilic compounds for industrial applications. However, the cultivation of microalgae comes at a high cost. To reduce the cost, changes from a closed bioreactor to open pond system and from a synthetic medium to environmental or wastewater-based medium are being sought. However, the use of open pond systems is currently limited because of contamination by undesirable organisms. To overcome this issue, one strategy is to combine acidophilic algae and acidic drainage in which other organisms are unable to thrive. Here, we tested waters from sulfuric acidic hot springs (Tamagawa, pH 1.15 and Tsukahara, pH 1.14) in Japan for the cultivation of the red alga Galdieria sulphuraria 074G and the green alga Pseudochlorella sp. YKT1. Both of these spring waters are rich in phosphate (0.043 and 0.145 mM, respectively) compared to other environmental freshwater sources. Neither alga grew in the spring water but they grew very well when the waters were supplemented with an inorganic nitrogen source. The algal yields were ~2.73 g dry weight/L for G. sulphuraria and ~2.49 g dry weight/L for P. sp. YKT1, which were comparable to those in an autotrophic synthetic medium. P. sp. YKT1 grew in the spring waters supplemented either of NH4+, NO3- or urea, while G. sulphuraria grew only when NH4+ was supplemented. For P. sp. YKT1, the spring water was adjusted to pH 2.0, while for G. sulphuraria, no pH adjustment was required. In both cases, no additional pH-buffering compound was required. The phycocyanin of the thermophilic G. sulphuraria is known to be more thermostable than that from the Spirulina platensis currently used in phycocyanin production for commercial use. The phycocyanin content in G. sulphuraria in the Tsukahara water supplemented with NH4+ was 107.42±1.81 μg/mg dry weight, which is comparable to the level in S. platensis (148.3 μg/mg dry weight). P. sp. YKT1 cells in the Tamagawa water supplemented with a nitrogen source formed a large amount of lipid droplets while maintaining cellular growth. These results indicate the potential of sulfuric hot spring waters for large-scale algal cultivation at a low cost

Hierarchal order in the formation of chloroplast division machinery in the red alga Cyanidioschyzon merolae

Chloroplasts have evolved from a cyanobacterial endosymbiont and multiply by dividing. Chloroplast division is performed by constriction of the ring-like protein complex (the PD machinery), which forms at the division site. The PD machinery is composed of cyanobacteria-descended components such as FtsZ and eukaryote-derived proteins such as the dynamin-related protein, DRP5B. In the red alga Cyanidioschyzon merolae, FtsZ ring formation on the stromal side precedes PDR1 and DRP5B ring formation on the cytosolic side. In this study, we impaired FtsZ ring formation in C. merolae by overexpressing FtsZ just before FtsZ ring formation. As a result, PDR1 and DRP5B failed to localize at the chloroplast division site, suggesting that FtsZ ring formation is required for the PDR1 and DRP5B rings. We further found, by expressing a dominant negative form of DRP5B, that DRP5B ring formation begins on the nuclear side of the chloroplast division site. These findings provide insight into how the PD machinery forms in red algae