EVIDENCE FOR TUBULAR MATING STRUCTURES INDUCED IN EACH MATING TYPE OF HETEROTHALLIC GONIUM PECTORALE (VOLVOCALES,CHLOROPHYTA)1

Gametes were induced separately in cultures of each mating type of the heterothallic, isogamous colonial volvocalean Gonium pectorale O. F. Müll. to examine the tubular mating structure (TMS) of both mating types plus and minus (plus and minus), referred to as “bilateral mating papillae.” Addition of dibutyryl cyclic adenosine monophosphate (DcAMP or db‐cAMP) and 3‐isobutyl‐1‐methylxanthine (IBMX) to approximately 3‐week‐old cultures of each mating type induced immediate release of naked gametes from the cell walls. Both plus and minus gametes formed a TMS in the anterior region of the protoplasts. Accumulation of actin was visualized by antibody staining in the TMS of both mating types as occurs in the TMS (fertilization tubule) of the plus gametes of the unicellular volvocalean Chlamydomonas reinhardtii P. A. Dang. Induction of naked gametes with a TMS in each mating type will be useful for future cell biological and evolutionary studies of the isogametes of colonial volvocalean algae.     

MOLECULAR DELINEATION OF SPECIES AND SYNGENS IN VOLVOCACEAN GREEN ALGAE (CHLOROPHYTA)1

Two species of the colonial green flagellate family Volvocaceae are worldwide in distribution yet exhibit contrasting species structure. Geographically disparate isolates of Gonium pectorale Mueller can interbreed while isolates of Pandorina morum Bory behave quite differently. More than 20 sexually isolated subpopulations occur within this species; these have been termed “syngens” (sensu Sonneborn). Because prezygotic barriers to mating cause intersyngen pairings to fail, breeding analyses cannot be used to estimate genetic relatedness among the syngens of P. morum. DNA comparisons provide an alternative method of assessing genetic relatedness. We compared the nucleotide sequence of the internal transcribed spacer (ITS) region of the nuclear ribosomal repeat among clones of P. morum and of G. pectorale. Members of syngens of P. morum with distribution restricted to one small geographical area show great similarity. Likewise, members of any syngen of worldwide distribution show near uniformity, even those from different continents. However, the ITS sequence of each syngen differs from that of other syngens. In contrast, G. pectorale, which has an ITS region that is remarkably uniform throughout the world, appears to consist of a single syngen within North America and Europe by mating tests. The molecular data are in complete conformity with previous syngen assignment. Because the latter is based on mating affinity, with two complementary mating types per syngen, the evolution of new mating type pairs appears to be the basis of microevolution in these algae. We infer that either P. morum is a more ancient species than G. pectorale or that P. morum has a less stable genome. In either case, the biogeographic distribution of certain syngens may reflect climatological changes of the past.     

Zygote germination in <Emphasis Type="Italic">Pleodorina starrii</Emphasis> (Volvocaceae,Chlorophyta)

Zygote germination of the anisogamous/oogamous colonial green flagellate Pleodorina starrii was observed. After the zygotes were transferred to the usual, illuminated conditions from the dark treatment on the agar plate, they began to germinate. The germinating zygotes gave rise to one or two viable biflagellate gone cells. This type of zygote germination is rare in the colonial Volvocales and may characterize a certain lineage within the anisogamous/oogamous members of the colonial Volvocales. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia.     

Mating type‐dependent partner sensing as mediated by VEL1 in Trichoderma reesei

Sexual development in the filamentous model ascomycete Trichoderma reesei (syn. Hypocrea jecorina) was described only a few years ago. In this study, we show a novel role for VELVET in fungi, which links light response, development and secondary metabolism. Vel1 is required for mating in darkness, normal growth and conidiation. In light, vel1 was dispensable for male fertility but essential for female fertility in both mating types. VEL1 impacted regulation of the pheromone system (hpr1, hpr2, hpp1, ppg1) in a mating type‐dependent manner and depending on the mating partner of a given strain. These partner effects only occurred for hpp1 and hpr2, the pheromone precursor and receptor genes associated with the MAT1‐2 mating type and for the mating type gene mat1‐2‐1. Analysis of secondary metabolite patterns secreted by wild type and mutants under asexual and sexual conditions revealed that even in the wild type, the patterns change upon encounter of a mating partner, with again distinct differences for wild type and vel1 mutants. Hence, T. reesei applies a language of pheromones and secondary metabolites to communicate with mating partners and that this communication is at least in part mediated by VEL1.     

Generic concept in Chlorella‐related coccoid green algae (Chlorophyta,Trebouxiophyceae)

Using a combined set of sequences of SSU and ITS regions of nuclear‐encoded ribosomal DNA, the concept of the experimental algal genus Chlorella was evaluated. Conventionally in the genus Chlorella, only coccoid, solitary algae with spherical morphology that do not possess any mucilaginous envelope were included. All Chlorella species reproduce asexually by autospores. However, phylogenetic analyses showed that within the clade of ‘true’Chlorella species (Chlorella vulgaris, C. lobophora, and C. sorokiniana), taxa with a mucilaginous envelope and colonial lifeform have also evolved. These algae, formerly designated as Dictyosphaerium, are considered as members of the genus Chlorella. In close relationship to Chlorella, five different genera were supported by the phylogenetic analyses: Micractinium (spherical cells, colonial, with bristles), Didymogenes (ellipsoidal cells, two‐celled coenobia, with or without two spines per cell), Actinastrum (ellipsoidal cells within star‐shaped coenobia), Meyerella (spherical cells, solitary, without pyrenoids), and Hegewaldia (spherical cells, colonial, with or without bristles, oogamous propagation). Based on the secondary structures of SSU and ITS rDNA sequences, molecular signatures are provided for each genus of the Chlorella clade.     

Molecular genetics of coat colour variations in White Galloway and White Park cattle

White Galloway cattle exhibit three different white coat colour phenotypes, that is, well marked, strongly marked and mismarked. However, mating of individuals with the preferred well or strongly marked phenotype also results in offspring with the undesired mismarked and/or even fully black coat colour. To elucidate the genetic background of the coat colour variations in White Galloway cattle, we analysed four coat colour relevant genes: mast/stem cell growth factor receptor (KIT), KIT ligand (KITLG), melanocortin 1 receptor (MC1R) and tyrosinase (TYR). Here, we show that the coat colour variations in White Galloway cattle and White Park cattle are caused by a KIT gene (chromosome 6) duplication and aberrant insertion on chromosome 29 (Cs₂₉) as recently described for colour‐sided Belgian Blue. Homozygous (Cs₂₉/Cs₂₉) White Galloway cattle and White Park cattle exhibit the mismarked phenotype, whereas heterozygous (Cs₂₉/wt₂₉) individuals are either well or strongly marked. In contrast, fully black individuals are characterised by the wild‐type chromosome 29. As known for other cattle breeds, mutations in the MC1R gene determine the red colouring. Our data suggest that the white coat colour variations in White Galloway cattle and White Park cattle are caused by a dose‐dependent effect based on the ploidy of aberrant insertions and inheritance of the KIT gene on chromosome 29.     

Biogenesis of photosynthetic complexes in the chloroplast of Chlamydomonas reinhardtii requires ARSA1, a homolog of prokaryotic arsenite transporter and eukaryotic TRC40 for guided entry of tail‐anchored proteins

as1, for antenna size mutant 1, was obtained by insertion mutagenesis of the unicellular green alga Chlamydomonas reinhardtii. This strain has a low chlorophyll content, 8% with respect to the wild type, and displays a general reduction in thylakoid polypeptides. The mutant was found to carry an insertion into a homologous gene, prokaryotic arsenite transporter (ARSA), whose yeast and mammal counterparts were found to be involved in the targeting of tail‐anchored (TA) proteins to cytosol‐exposed membranes, essential for several cellular functions. Here we present the characterization in a photosynthetic organism of an insertion mutant in an ARSA‐homolog gene. The ARSA1 protein was found to be localized in the cytosol, and yet its absence in as1 leads to a small chloroplast and a strongly decreased chlorophyll content per cell. ARSA1 appears to be required for optimal biogenesis of photosynthetic complexes because of its involvement in the accumulation of TOC34, an essential component of the outer chloroplast membrane translocon (TOC) complex, which, in turn, catalyzes the import of nucleus‐encoded precursor polypeptides into the chloroplast. Remarkably, the effect of the mutation appears to be restricted to biogenesis of chlorophyll‐binding polypeptides and is not compensated by the other ARSA homolog encoded by the C. reinhardtii genome, implying a non‐redundant function.     

Patterns of reproductive isolation within and between two Lygaeus species characterized by sexual conflicts over mating

Theory suggests that, under some circumstances, sexual conflict over mating can lead to divergent sexually antagonistic coevolution among populations for traits associated with mating, and that this can promote reproductive isolation and hence speciation. However, sexual conflict over mating may also select for traits (e.g. male willingness to mate) that enhance gene flow between populations, limiting population divergence. In the present study, we compare pre‐ and post‐mating isolation within and between two species characterized by male–female conflict over mating rate. We quantify sexual isolation among five populations of the seed bug Lygaeus equestris collected from Italy and Sweden, and two replicates of a population of the sister‐species Lygaeus simulans, also collected from Italy. We find no evidence of reproductive isolation amongst populations of L. equestris, suggesting that sexual conflict over mating has not led to population divergence in relevant mating traits in L. equestris. However, there was strong asymmetric pre‐mating isolation between L. equestris and L. simulans: male L. simulans were able to mate successfully with female L. equestris, whereas male L. equestris were largely unable to mate with female L. simulans. We found little evidence for strong post‐mating isolation between the two species, however, with hybrid F₂ offspring being produced. Our results suggest that sexual conflict over mating has not led to population divergence, and indeed perhaps supports the contrary theoretical prediction that male willingness to mate may retard speciation by promoting gene flow.     

Uneven Distribution of Mating‐Type Alleles Among Togninia minima Isolates,One of the Causal Agents of Leaf Stripe Disease on Grapevines in Northwest Iran

Togninia minima is the main fungal species associated with grapevine leaf stripe disease worldwide. This species is mainly known from its asexual state in nature; nevertheless, a biallelic heterothallic mating strategy has been confirmed for this species based on in vitro crossing studies. There are no data available on the incidence of an active sexual cycle within the populations of this species in many grapevine‐producing countries as well as Iran. The possibility of a clandestine sexual cycle within the Iranian isolates of T. minima was evaluated by analysing the distribution and frequency of the mating‐type alleles on a microspatial and a macrogeographical scales. Towards this aim, a total of 90 T. minima isolates were recovered from grapevines with esca disease from the vineyards in north and north‐western Iran. A multiplex PCR method previously designed by authors was applied for simultaneous identification and determination of the mating‐type alleles in T. minima populations. The results on the screening of mating‐type alleles using multiplex PCR method revealed the mating‐type identity of 77 isolates as Mat1‐2 and 23 isolates as Mat1‐1. Our results showed that both Mat1‐1 and Mat1‐2 isolates are present in a single vineyard and even on single vines. The distribution of mating‐type alleles in the sampled area skewed from the 1 : 1 ratio (77 : 23); however, co‐occurrence of both mating types in a single vineyard and even on single vines is suggestive for the presence of an active sexual cycle for T. minima in north‐western Iran.     

A missense mutation in CHS1, a TIR‐NB protein,induces chilling sensitivity in Arabidopsis

Low temperature is an environmental factor that affects plant growth and development and plant–pathogen interactions. How temperature regulates plant defense responses is not well understood. In this study, we characterized chilling‐sensitive mutant 1 (chs1), and functionally analyzed the role of the CHS1 gene in plant responses to chilling stress. The chs1 mutant displayed a chilling‐sensitive phenotype, and also displayed defense‐associated phenotypes, including extensive cell death, the accumulation of hydrogen peroxide and salicylic acid, and an increased expression of PR genes: these phenotypes indicated that the mutation in chs1 activates the defense responses under chilling stress. A map‐based cloning analysis revealed that CHS1 encodes a TIR‐NB‐type protein. The chilling sensitivity of chs1 was fully rescued by pad4 and eds1, but not by ndr1. The overexpression of the TIR and NB domains can suppress the chs1–conferred phenotypes. Interestingly, the stability of the CHS1 protein was positively regulated by low temperatures independently of the 26S proteasome pathway. This study revealed the role of a TIR‐NB‐type gene in plant growth and cell death under chilling stress, and suggests that temperature modulates the stability of the TIR‐NB protein in Arabidopsis.     

SNPs in SNCA,MCCC1, DLG2, GBF1 and MBNL2 are associated with Parkinson's disease in southern Chinese population

Numerous single nucleotide polymorphisms (SNPs), which have been identified as susceptibility factors for Parkinson's disease (PD) as per genome‐wide association studies, have not been fully characterized for PD patients in China. This study aimed to replicate the relationship between 12 novel SNPs of 12 genes and PD risk in southern Chinese population. Twelve SNPs of 12 genes were detected in 231 PD patients and 249 controls, using the SNaPshot technique. Meta‐analysis was used to assess heterogeneity of effect sizes between this study and published data. The impact of SNPs on gene expression was investigated by analysing the SNP‐gene association in the expression quantitative trait loci (eQTL) data sets. rs8180209 of SNCA (allele model: P = .047, OR = 0.77; additive model: P = .047, OR = 0.77), rs2270968 of MCCC1 (dominant model: P = .024, OR = 1.52), rs7479949 of DLG2 (recessive model; P = .019, OR = 1.52), rs10748818 of GBF1 (additive model: P < .001, OR = 0.37), and rs4771268 of MBNL2 (recessive model: P = .003, OR = 0.48) were replicated to be significantly associated with the increased risk of PD. Noteworthy, a meta‐analysis of previous studies suggested rs8180209, rs2270968, rs7479949 and rs4771268 were in line with those of our cohort. Our study replicated five novel functional SNPs in SNCA, MCCC1, DLG2, GBF1 and MBNL2 could be associated with increased risk of PD in southern Chinese population.     

Loss‐of‐function of ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (APN1) in Lotus japonicus restricts efficient nitrogen‐fixing symbiosis with specific Mesorhizobium loti strains

The nitrogen‐fixing symbiosis of legumes and Rhizobium bacteria is established by complex interactions between the two symbiotic partners. Legume Fix^– mutants form apparently normal nodules with endosymbiotic rhizobia but fail to induce rhizobial nitrogen fixation. These mutants are useful for identifying the legume genes involved in the interactions essential for symbiotic nitrogen fixation. We describe here a Fix^– mutant of Lotus japonicus, apn1, which showed a very specific symbiotic phenotype. It formed ineffective nodules when inoculated with the Mesorhizobium loti strain TONO. In these nodules, infected cells disintegrated and successively became necrotic, indicating premature senescence typical of Fix^– mutants. However, it formed effective nodules when inoculated with the M. loti strain MAFF303099. Among nine different M. loti strains tested, four formed ineffective nodules and five formed effective nodules on apn1 roots. The identified causal gene, ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (LjAPN1), encodes a nepenthesin‐type aspartic peptidase. The well characterized Arabidopsis aspartic peptidase CDR1 could complement the strain‐specific Fix^– phenotype of apn1. LjAPN1 is a typical late nodulin; its gene expression was exclusively induced during nodule development. LjAPN1 was most abundantly expressed in the infected cells in the nodules. Our findings indicate that LjAPN1 is required for the development and persistence of functional (nitrogen‐fixing) symbiosis in a rhizobial strain‐dependent manner, and thus determines compatibility between M. loti and L. japonicus at the level of nitrogen fixation.     

Common genetic variants in GAL,GAP43 and NRSN1 and interaction networks confer susceptibility to Hirschsprung disease

Hirschsprung disease (HSCR) is a severe multifactorial genetic disorder. Microarray studies indicated GAL, GAP43 and NRSN1 might contribute to the altered risk in HSCR. Thus, we focused on genetic variations in GAL, GAP43 and NRSN1, and the gene‐gene interactions involved in HSCR susceptibility. We recruited a strategy combining case‐control study and MassArray system with interaction network analysis. For GAL, GAP43 and NRSN1, a total of 18 polymorphisms were assessed in 104 subjects with sporadic HSCR and 151 controls of Han Chinese origin. We found statistically significant differences between HSCR and control groups at 5 genetic variants. For each gene, the haplotypes combining all polymorphisms were the most significant. Based on SNPsyn, MDR and GeneMANIA analyses, we observed significant gene‐gene interactions among GAL, GAP43, NRSN1 and our previous identified RELN, GABRG2 and PTCH1. Our study for the first time indicates that genetic variants within GAL, GAP43 and NRSN1 and related gene‐gene interaction networks might be involved in the altered susceptibility to HSCR in the Han Chinese population, which might shed more light on HSCR pathogenesis.     

Localization and partial characterization of a sex-specific enzyme in homothallic and heterothallic mucorales

A study was made of some late reactions in the trisporic acid biosynthetic pathway in Mucor mucedo. Trisporic acids induce sexual reproduction in several Mucorales.Two enzymes involved in these reactions, a NADP-dependent dehydrogenase and an esterase, appeared to be highly specific for the minus mating type.The synthesis of these enzymes is stimulated by trisporic acids, indicating a positive control of these hormones upon their own synthesis.The dehydrogenase was histochemically shown to be concentrated in the zygophores of Mucor mucedominus. In the homothallic Zygorhynchus moelleri the copulating main branch (which is known to have a minus character) appeared to be the major site of dehydrogenase activity.     

Phylogenetic Position and Molecular Chronology of a Colonial Green Flagellate,Stephanosphaera pluvialis (Volvocales,Chlorophyceae), among Unicellular Algae

The genus Balticola comprises a group of unicellular green flagellate algae and is composed of four species formerly classified in the genus Haematococcus. Balticola is closely related to a colonial green flagellate, Stephanosphaera pluvialis. Although the phylogeny among these genera was previously investigated based on two nuclear gene sequences, the phylogenetic sister of S. pluvialis has yet to be determined. In the present study, the species diversity of Balticola and Stephanosphaera was investigated using 18S rRNA gene sequences, and phylogenetic analyses of combined nuclear and chloroplast gene sequences were performed to understand the evolutionary origin of coloniality in Stephanosphaera. The divergence times of four colonial volvocalean flagellates from their respective unicellular sisters were also estimated. Six Balticola genotypes and a single Stephanosphaera genotype were recognized, and one Balticola genotype was resolved as the sister of S. pluvialis, showing that Balticola is a nonmonophyletic genus. The divergence time of Stephanosphaera from its nearest Balticola relative was estimated to be 4–63 million years ago, and these genera represent the most recently diverged pair of unicellular and colonial flagellates among the Volvocales.