Identification of a new gene family expressed during the onset of sexual reproduction in the centric diatom Thalassiosira weissflogii.

An intriguing feature of the diatom life cycle is that sexual reproduction and the generation of genetic diversity are coupled to the control of cell size. A PCR-based cDNA subtraction technique was used to identify genes that are expressed as small cells of the centric diatom Thalassiosira weissflogii initiate gametogenesis. Ten genes that are up-regulated during the early stages of sexual reproduction have been identified thus far. Three of the sexually induced genes, Sig1, Sig2, and Sig3, were sequenced to completion and are members of a novel gene family. The three polypeptides encoded by these genes possess different molecular masses and charges but display many features in common: they share five highly conserved domains; they each contain three or more cysteine-rich epithelial growth factor (EGF)-like repeats; and they each display homology to the EGF-like region of the vertebrate extracellular matrix glycoprotein tenascin X. Interestingly, the five conserved domains appear in the same order in each polypeptide but are separated by variable numbers of nonconserved amino acids. SIG1 and SIG2 display putative regulatory domains within the nonconserved regions. A calcium-binding, EF-hand motif is found in SIG1, and an ATP/GTP binding motif is present in SIG2. The striking similarity between the SIG polypeptides and extracellular matrix components commonly involved in cell-cell interactions suggests that the SIG polypeptides may play a role in sperm-egg recognition. The SIG polypeptides are thus important molecular targets for determining when and where sexual reproduction occurs in the field.     

Rapid Evolution of a Sexual Reproduction Gene in Centric Diatoms of the Genus Thalassiosira

Sexual reproduction is commonly assumed to occur in the vast majority of diatoms due to the intimate association of this process with cell size control. Surprisingly, however, little is known about the impact of sexual events on diatom population dynamics. The Sig1 gene is strongly upregulated during sexual reproduction in the centric diatom Thalassiosira weissflogii and has been hypothesized to encode a protein involved in gamete recognition. In the present study, degenerate PCR primers were designed and used to amplify a portion of Sig1 from three closely related species in the cosmopolitan genus Thalassiosira, Thalassiosira oceanica, Thalassiosira guillardii, and Thalassiosira pseudonana. Identification of Sig1 in these three additional species facilitated development of this gene as a molecular marker for diatom sexual events. Examination of the new sequences indicated that multiple copies of Sig1 are probably present in the genome. Moreover, compared to the housekeeping gene β-tubulin, the Sig1 genes of isolates of T. weissflogii collected from different regions of the Atlantic and Pacific oceans displayed high levels of divergence. The Sig1 genes of the four closely related Thalassiosira species also displayed high levels of sequence divergence compared to the levels observed with a second gene, Fcp, probably explaining why Sig1 could not be amplified from more distantly related species. The high levels of sequence divergence both within and between species suggest that Sig1 is rapidly evolving in a manner reminiscent of the manner observed in other genes that encode gamete recognition proteins. A simple model is presented for Sig1 evolution and the implications of such a rapidly evolving sexual reproduction gene for diatom speciation and population dynamics.     

Rapid evolution of a sexual reproduction gene in centric diatoms of the genus Thalassiosira

Sexual reproduction is commonly assumed to occur in the vast majority of diatoms due to the intimate association of this process with cell size control. Surprisingly, however, little is known about the impact of sexual events on diatom population dynamics. The Sig1 gene is strongly upregulated during sexual reproduction in the centric diatom Thalassiosira weissflogii and has been hypothesized to encode a protein involved in gamete recognition. In the present study, degenerate PCR primers were designed and used to amplify a portion of Sig1 from three closely related species in the cosmopolitan genus Thalassiosira, Thalassiosira oceanica, Thalassiosira guillardii, and Thalassiosira pseudonana. Identification of Sig1 in these three additional species facilitated development of this gene as a molecular marker for diatom sexual events. Examination of the new sequences indicated that multiple copies of Sig1 are probably present in the genome. Moreover, compared to the housekeeping gene beta-tubulin, the Sig1 genes of isolates of T. weissflogii collected from different regions of the Atlantic and Pacific oceans displayed high levels of divergence. The Sig1 genes of the four closely related Thalassiosira species also displayed high levels of sequence divergence compared to the levels observed with a second gene, Fcp, probably explaining why Sig1 could not be amplified from more distantly related species. The high levels of sequence divergence both within and between species suggest that Sig1 is rapidly evolving in a manner reminiscent of the manner observed in other genes that encode gamete recognition proteins. A simple model is presented for Sig1 evolution and the implications of such a rapidly evolving sexual reproduction gene for diatom speciation and population dynamics.     

NOVEL PROTEINS COMPRISING THE STRAMENOPILE TRIPARTITE MASTIGONEME IN OCHROMONAS DANICA (CHRYSOPHYCEAE)1

Two‐dimensional (2‐D) protein analysis of the mastigoneme fraction of the chromophyte alga Ochromonas danica E. G. Pringsh. showed the presence of several component proteins of the tubular mastigoneme. Adding to the reported gene Ocm1, three new genes (Ocm2, Ocm3, and Ocm4) belonging to the Ocm gene family were isolated using degenerate primers designed from predicted Ocm1 amino acid sequences. The predicted polypeptides encoded by Ocm2, Ocm3, and Ocm4 were smaller in size than Ocm1. However, they shared four highly conserved, cysteine‐rich, epithelial growth factor (EGF)‐like motifs, potentially involved in protein–protein interaction. In addition, Ocm2, Ocm3, and Ocm4 showed homology to the SIG protein family in the centric diatom Thalassiosira weissflogii (Grunow) Fryxell et Hasle, which is up‐regulated during early stages of sexual reproduction. Immunofluorescence analysis with a polyclonal antibody against the partial amino acid sequences of Ocm2, Ocm3, and Ocm4 showed that Ocm2 and Ocm3 were located in the basal segment region of mastigonemes attached on the surface of the anterior flagellum, and that Ocm4 was located within the tubular shaft portion similar to Ocm1.     

Recombination between Clonal Lineages of the Asexual Fungus Verticillium dahliae Detected by Genotyping by Sequencing

Most asexual species of fungi have either lost sexuality recently, or they experience recombination by cryptic sexual reproduction. Verticillium dahliae is a plant-pathogenic, ascomycete fungus with no known sexual stage, even though related genera have well-described sexual reproduction. V. dahliae reproduces mitotically and its population structure is highly clonal. However, previously described discrepancies in phylogenetic relationships among clonal lineages may be explained more parsimoniously by recombination than mutation; therefore, we looked for evidence of recombination within and between clonal lineages. Genotyping by sequencing was performed on 141 V. dahliae isolates from diverse geographic and host origins, resulting in 26,748 single-nucleotide polymorphisms (SNPs). We found a strongly clonal population structure with the same lineages as described previously by vegetative compatibility groups (VCGs) and molecular markers. We detected 443 recombination events, evenly distributed throughout the genome. Most recombination events detected were between clonal lineages, with relatively few recombinant haplotypes detected within lineages. The only three isolates with mating type MAT1-1 had recombinant SNP haplotypes; all other isolates had mating type MAT1-2. We found homologs of eight meiosis-specific genes in the V. dahliae genome, all with conserved or partially conserved protein domains. The extent of recombination and molecular signs of sex in (mating-type and meiosis-specific genes) suggest that V. dahliae clonal lineages arose by recombination, even though the current population structure is markedly clonal. Moreover, the detection of new lineages may be evidence that sexual reproduction has occurred recently and may potentially occur under some circumstances. We speculate that the current clonal population structure, despite the sexual origin of lineages, has arisen, in part, as a consequence of agriculture and selection for adaptation to agricultural cropping systems.     

Wound-Released Chemical Signals May Elicit Multiple Responses from an Agrobacterium tumefaciens Strain Containing an Octopine-Type Ti Plasmid

The vir regions of octopine-type and nopaline-type Ti plasmids direct the transfer of oncogenic T-DNA from Agrobacterium tumefaciens to the nuclei of host plant cells. Previous studies indicate that at least two genetic loci at the left ends of these two vir regions are sufficiently conserved to form heteroduplexes visible in the electron microscope. To initiate an investigation of these genetic loci, we determined the DNA sequences of these regions of both Ti plasmids and identified both conserved loci. One of these is the 2.5-kb virH locus, which was previously identified on the octopine-type Ti plasmid but thought to be absent from the nopaline-type Ti plasmid. The virH operon contains two genes that resemble P-450-type monooxygenases. The other locus encodes a 0.5-kb gene designated virK. In addition, we identified other potential genes in this region that are not conserved between these two plasmids. To determine (i) whether these genes are members of the vir regulon and, (ii) whether they are required for tumorigenesis, we used a genetic technique to disrupt each gene and simultaneously fuse its promoter to lacZ. Expression of these genes was also measured by nuclease S1 protection assays. virK and two nonconserved genes, designated virL and virM, were strongly induced by the vir gene inducer acetosyringone. Disruptions of virH, virK, virL, or virM did not affect tumorigenesis of Kalanchöe diagramontiana leaves or carrot disks, suggesting that they may play an entirely different role during pathogenesis.     

Conserved Gene Order and Expanded Inverted Repeats Characterize Plastid Genomes of Thalassiosirales

Diatoms are mostly photosynthetic eukaryotes within the heterokont lineage. Variable plastid genome sizes and extensive genome rearrangements have been observed across the diatom phylogeny, but little is known about plastid genome evolution within order- or family-level clades. The Thalassiosirales is one of the more comprehensively studied orders in terms of both genetics and morphology. Seven complete diatom plastid genomes are reported here including four Thalassiosirales: Thalassiosira weissflogii, Roundia cardiophora, Cyclotella sp. WC03_2, Cyclotella sp. L04_2, and three additional non-Thalassiosirales species Chaetoceros simplex, Cerataulina daemon, and Rhizosolenia imbricata. The sizes of the seven genomes vary from 116,459 to 129,498 bp, and their genomes are compact and lack introns. The larger size of the plastid genomes of Thalassiosirales compared to other diatoms is due primarily to expansion of the inverted repeat. Gene content within Thalassiosirales is more conserved compared to other diatom lineages. Gene order within Thalassiosirales is highly conserved except for the extensive genome rearrangement in Thalassiosira oceanica. Cyclotella nana, Thalassiosira weissflogii and Roundia cardiophora share an identical gene order, which is inferred to be the ancestral order for the Thalassiosirales, differing from that of the other two Cyclotella species by a single inversion. The genes ilvB and ilvH are missing in all six diatom plastid genomes except for Cerataulina daemon, suggesting an independent gain of these genes in this species. The acpP1 gene is missing in all Thalassiosirales, suggesting that its loss may be a synapomorphy for the order and this gene may have been functionally transferred to the nucleus. Three genes involved in photosynthesis, psaE, psaI, psaM, are missing in Rhizosolenia imbricata, which represents the first documented instance of the loss of photosynthetic genes in diatom plastid genomes.     

The sexual phase of the diatom <Emphasis Type="Italic">Pseudo-nitzschia multistriata</Emphasis>: cytological and time-lapse cinematography characterization

Pseudo-nitzschia is a thoroughly studied pennate diatom genus for ecological and biological reasons. Many species in this genus, including Pseudo-nitzschia multistriata, can produce domoic acid, a toxin responsible for amnesic shellfish poisoning. Physiological, phylogenetic and biological features of P. multistriata were studied extensively in the past. Life cycle stages, including the sexual phase, fundamental in diatoms to restore the maximum cell size and avoid miniaturization to death, have been well described for this species. P. multistriata is heterothallic; sexual reproduction is induced when strains of opposite mating type are mixed, and proceeds with cells producing two functionally anisogamous gametes each; however, detailed cytological information for this process is missing. By means of confocal laser scanning microscopy and nuclear staining, we followed the nuclear fate during meiosis, and using time-lapse cinematography, we timed every step of the sexual reproduction process from mate pairing to initial cell hatching. The present paper depicts cytological aspects during gametogenesis in P. multistriata, shedding light on the chloroplast behaviour during sexual reproduction, finely describing the timing of the sexual phases and providing reference data for further studies on the molecular control of this fundamental process.     

Genome-wide identification and expression analysis of peach auxin response factor gene families

Plant auxin response factors (ARFs) are involved in plant growth, development and multiple other processes. In this study, the ARF gene family in the peach genome was identified by bioinformatics software and RT-PCR. In total, 18 PpARF candidate genes were found in the peach genome. The DNA-binding and ARF domains, as well as motif III and IV of the PpARF gene family were highly conserved. The phylogenetic analysis revealed that PpARF gene family was divided into five classes: Class I (three members), Class II (four members), Class III (five members), Class IV (three members) and Class V (three members). The results of an intron-exon structure analysis indicated that PpARF gene family members were composed of 2–15 exons. A chromosome mapping analysis revealed that PpARF genes were distributed with different densities over eight chromosomes, with the largest number of PpARF genes on chromosome 1 (four genes), followed by chromosome 4 and 6 (three genes each). Only one gene was located on each of chromosome 3, 7 and 8. A conserved motif analysis revealed that the DNA-binding and ARF domains were observed in all PpARF proteins (except for PpARF18). Class I contained no motifs III or IV (except for PpARF7). RT-PCR results indicated that all of the PpARF genes, with the exception of PpARF15 and PpARF17, were expressed in at least one of the tissues (roots, stems, leaves, flowers and five stages of fruit development). These results suggested that the PpARF gene family members are highly and structurally conserved, and are involved in various aspects of peach growth and development, especially in fruit development.     

A gene expression fingerprint of C. elegans embryonic motor neurons

Background

ADP-ribosylation is an enzyme-catalyzed posttranslational protein modification in which mono(ADP-ribosyl)transferases (mARTs) and poly(ADP-ribosyl)transferases (pARTs) transfer the ADP-ribose moiety from NAD onto specific amino acid side chains and/or ADP-ribose units on target proteins.

Results

Using a combination of database search tools we identified the genes encoding recognizable pART domains in the public genome databases. In humans, the pART family encompasses 17 members. For 16 of these genes, an orthologue exists also in the mouse, rat, and pufferfish. Based on the degree of amino acid sequence similarity in the catalytic domain, conserved intron positions, and fused protein domains, pARTs can be divided into five major subgroups. All six members of groups 1 and 2 contain the H-Y-E trias of amino acid residues found also in the active sites of Diphtheria toxin and Pseudomonas exotoxin A, while the eleven members of groups 3 - 5 carry variations of this motif. The pART catalytic domain is found associated in Lego-like fashion with a variety of domains, including nucleic acid-binding, protein-protein interaction, and ubiquitylation domains. Some of these domain associations appear to be very ancient since they are observed also in insects, fungi, amoebae, and plants. The recently completed genome of the pufferfish T. nigroviridis contains recognizable orthologues for all pARTs except for pART7. The nearly completed albeit still fragmentary chicken genome contains recognizable orthologues for twelve pARTs. Simpler eucaryotes generally contain fewer pARTs: two in the fly D. melanogaster, three each in the mosquito A. gambiae, the nematode C. elegans, and the ascomycete microfungus G. zeae, six in the amoeba E. histolytica, nine in the slime mold D. discoideum, and ten in the cress plant A. thaliana. GenBank contains two pART homologues from the large double stranded DNA viruses Chilo iridescent virus and Bacteriophage Aeh1 and only a single entry (from V. cholerae) showing recognizable homology to the pART-like catalytic domains of Diphtheria toxin and Pseudomonas exotoxin A.

Conclusion

The pART family, which encompasses 17 members in the human and 16 members in the mouse, can be divided into five subgroups on the basis of sequence similarity, phylogeny, conserved intron positions, and patterns of genetically fused protein domains.     

Genome-Wide Phylogenetic Analysis of Stress-Activated Protein Kinase Genes in Rice (OsSAPKs) and Expression Profiling in Response to Xanthomonas oryzae pv. oryzicola Infection

All members of the SnRK2 protein kinase gene family encoded by the rice (Oryza sativa L.) genome are activated by hyperosmotic stress, and have been designated as stress-activated protein kinases (SAPKs). In this study, gene structures, phylogeny, and conserved motifs for the entire OsSAPK gene family in rice have been analyzed. Moreover, expression patterns of OsSAPK in response to infection with Xanthomonas oryzae pv. oryzicola (Xoc) were investigated. A total of ten OsSAPK genes in the japonica rice cultivar 9804 were identified and classified into four groups. All genes had similar exon–intron structures and organization of putative motifs/domains, and shared the same four motifs (motifs 1–4). Group I (OsSAPK1 and OsSAPK2) shared another two motifs (motif 5 and motif 10), while group III (OsSAPK8, OsSAPK9 and OsSAPK10) had seven motifs in common (motifs 1–7). Moreover, we found that four OsSAPKs, including OsSAPK3, OsSAPK5, OsSAPK7 and OsSAPK9, were significantly upregulated in response to infection by Xoc in rice plants carrying the nonhost resistance gene Rxo1. Four of the OsSAPK genes in which expression was upregulated were localized to both the cytoplasm and nucleus, but clustered in different groups, suggesting that they are involved in different resistance signal transduction pathways. These results will provide useful information for the future functional dissection of this gene family.     

Pseudo-Response Regulator (PRR) Homologues of the Moss Physcomitrella patens: Insights into the Evolution of the PRR Family in Land Plants

The pseudo-response regulators (PRRs) are the circadian clock component proteins in the model dicot Arabidopsis thaliana. They contain a receiver-like domain (RLD) similar to the receiver domains of the RRs in the His–Asp phosphorelay system, but the RLDs lack the phosphoacceptor aspartic acid residue invariably conserved in the receiver domains. To study the evolution of PRR genes in plants, here we characterize their homologue genes, PpPRR1, PpPRR2, PpPRR3 and PpPRR4, from the moss Physcomitrella patens. In the phylogenetic analysis, PpPRRs cluster together, sister to an angiosperm PRR gene subfamily, illustrating their close relationships with the angiosperm PRRs. However, distinct from the angiosperm sequences, the RLDs of PpPRR2/3/4 exhibit a potential phosphoacceptor aspartic acid–aspartic acid–lysine (DDK) motif. Consistently, the PpPRR2 RLD had phosphotransfer ability in vitro, suggesting that PpPRR2 functions as an RR. The PpPRR1 RLD, on the other hand, shows a partially diverged DDK motif, and it did not show phosphotransfer ability. All PpPRRs were expressed in a circadian and light-dependent manner, with differential regulation between PpPRR2/4 and PpPRR1/3. Altogether, our results illustrate that PRRs originated from an RR(s) and that there are intraspecific divergences among PpPRRs. Finally, we offer scenarios for the evolution of the PRR family in land plants.     

Identification of Conserved and Novel microRNAs from Liriodendron chinense Floral Tissues

Background

Liriodendron chinense (L. chinense) is an endangered basal angiosperm plant in China because of its low reproductive efficiency. Recently, miRNAs have obtained great attention because they can play important roles. Through high throughput sequencing technique, large amount of miRNAs were identified from different plant species. But there were few studies about the miRNAs in the basal angiosperms especially in the sexual reproduction process.

Results

Deep sequencing technology was applied to discover miRNAs in L. chinense flowers at different stages. After bioinformatic analysis, 496 putative conserved miRNAs representing 97 families and 2 novel miRNAs were found. Among them, one is previously regarded as gymnosperm specific. Their expressions were further validated by Real-time PCR for 13 selected miRNAs. Putative targeting genes were predicted and categorized with gene ontology (GO) analysis. About ten percents of the targets are involved in the reproduction process. Further expressional analysis showed that many of these miRNAs were highly related to the reproductive growth.

Conclusions

This is the first comprehensive identification of conserved and novel miRNAs in L. chinense. The data presented here might not only help to fill the gap of miRNA registered about basal angiosperm plants but also contribute to understanding the evolution of miRNAs. The differential expression of some of the miRNAs and the prediction of their target genes are also helpful in understanding the regulation of L. chinense sexual reproduction.     

Characterization of a silicon transporter gene family in Cylindrotheca fusiformis: sequences, expression analysis, and identification of homologs in other diatoms

The transport of silicon is an integral part of the synthesis of the silicified cell wall of diatoms, yet knowledge of the number, features, and regulation of silicon transporters is lacking. We report the isolation and sequence determination of five silicon transporter (SIT) genes from Cylindrotheca fusiformis, and examine their expression patterns during cell wall synthesis. The encoded SIT amino acid sequences are highly conserved in their putative transmembrane domains. Nine conserved cysteines in this domain may account for the sensitivity of silicon uptake to sulfhydryl blocking agents. A less conserved C-terminal domain is predicted to form coiled-coil structures, suggesting that the SITs interact with other proteins. We show that SIT gene expression is induced just prior to, and during, cell wall synthesis. The genes are expressed at very different levels, and SIT1 is expressed in a different pattern from SIT 2–5. Hybridization experiments show that multiple SIT gene copies are present in all diatom species tested. From the data we infer that individual transporters play specific roles in silicon uptake, and propose that the cell regulates uptake by controlling the amount or location of each. The identification of all SIT genes in C. fusiformis will enhance our understanding of the mechanism and control of silicon transport in diatoms.