The effects of methionine and cobalt ions on sex expression and development of <Emphasis Type="Italic">Anemia phyllitidis</Emphasis> gametophytes

One of the prime precursor for ethylene synthesis — L-methionine and the inhibitor of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) — Co²⁺-were tested for their effects on sex expression and development of Anemia phyllitidis fern gametophytes. Five concentrations of both chemicals (0, 10, 25, 50, 100 μM) were analysed with reference to antheridia and archegonia formation, number and size of cells as well as thalli length using the three-zone model of gametophyte structure. Both substances, however at different concentrations, enhanced the number of GA₃-induced antheridia and similarly stimulated the cell number and inhibited thalli length. Both of them at 100 μM concentrations without GA₃ induced meristematic area formation while methionine also induced archegonia in the apical parts of gametophytes. These findings correspond with the previous observations concerning the important role of ethylene synthesis precursor (ACC) in controlling gibberellic acid-induced male sex expression in ferns and broaden the knowledge about the mechanisms of fern gametophyte development.     

Elemental composition of the sub-aquatic liverwort Pellia endiviifolia (Dicks.) Dum. in relation to heavy metal contamination

The concentrations of fifteen elements (B, Na, Mg, Al, P, K, Ca, Ti, Mn, Fe, Cu, Zn, Sr, Cd, Pb) in thalli of the sub-aquatic liverwort Pellia endiviifolia (Dicks.) Dum. collected from seven sites in Japan, were analyzed by inductively coupled plasma atomic emission spectrometer (ICP). The average concentration of major essential elements in the thalli was: 49 600 µg g^–1 K, 9 140 µg g^–1 Na, 6740 µg g^–1 Ca, 3 840 µg g^–1 P, and 3 060 µg g^–1 Mg. Thalli of P. endiviifolia from sites polluted by heavy metals (Cu, Zn, Pb) contained high concentrations of these elements (maxima 0.88% Cu, 0.55% Zn and 0.36% Pb in the older thallus), thus demonstrating the potentiality of this liverwort as an indicator for heavy metal contamination.     

麦秆蹄盖蕨（Athyrium fallaciosum）配子体的发育规律及播种密度对配子体性别分化的影响

在组织培养条件下,对麦秆蹄盖蕨（Athyrium fallaciosum）配子体发育的连续过程进行了详细观察。结果表明:麦秆蹄盖蕨孢子为四面体型; 孢子萌发为书带蕨型（Vittaria-type）;原叶体发育为铁线蕨型（Adiantum-type）,成熟原叶体为对称的心形;精子器近圆球形,成熟颈卵器细长,常向原叶体基部倾斜或弯曲。常规播种条件下,发现麦秆蹄盖蕨配子体有雌配子体、雄配子体、雌雄同体配子体和无性配子体类型。配子体的性别随密度不同而呈现一定的变化趋势,雄配子体随密度增大呈上升趋势;雌配子体随密度增大先上升后下降;雌雄同体配子体和无性配子体随密度变化不大。雌配子体和雌雄同体配子体具颈卵器数目一般为10～15个;精子器数目随密度的增大逐渐减少,雄配子体中具有约50个精子器,雌雄同体配子体具有约20个精子器。     

Molecular characterization and expression analysis of a GTP-binding protein (MiRab5) in Mangifera indica

The Rab family, the largest branch of Ras small GTPases, plays a crucial role in the vesicular transport in plants. The members of Rab family act as molecular switches that regulate the fusion of vesicles with target membranes through conformational changes. However, little is known about the Rab5 gene involved in fruit ripening and stress response. In this study, the MiRab5 gene was isolated from stress-induced Mangifera indica. The full-length cDNA sequence was 984 bp and contained an open reading frame of 600 bp, which encoded a 200 amino acid protein with a molecular weight of 21.83 kDa and a theoretical isoelectric point of 6.99. The deduced amino acid sequence exhibited high homology with tomato (91% similarity) and contains all five characteristic Rab motifs. Real-time quantitative RT-PCR analysis demonstrated that MiRab5 was ubiquitously expressed in various mango tree tissues at different levels. The expression of MiRab5 was up-regulated during later stages of fruit ripening. Moreover, MiRab5 was generally up-regulated in response to various abiotic stresses (cold, salinity, and PEG treatments). Recombinant MiRab5 protein was successfully expressed and purified. SDS-PAGE and western blot analysis indicated that the expressed protein was recognized by the anti-6-His antibody. These results provide insights into the role of the MiRab5 gene family in fruit ripening and stress responses in the mango plant.     

Do motile spermatozoids limit the effectiveness of sexual reproduction in bryophytes? Not in the liverwort Marchantia polymorpha

To test the premise that fertilization distances in bryophytes are limited by spermatozoid motility, we measured the distances between over 80 000 male and female thalli of Marchantia polymorpha on heathland over 2 successive years after a major fire and recorded the incidence of sporophytes. The first comparative data on spermatozoid numbers per antheridium across bryophytes and pteridophytes were calculated from antheridial sizes and spermatid diameters and cross‐checked with the likely numbers produced by successive antheridial mitoses. Individual antheridia of Marchantia produce over 200 000 spermatozoids. Extrapolating from individual antheridia to the numbers of mature antheridia per antheridiophore and then to individual male plants, we calculated that a single flooding event on a male thallus with 10–12 antheridiophores will result in the release of over 50 million spermatozoids. Assuming radial dispersal in surface water films by lipids released from the dehiscing antheridia, spermatozoids can reach distances exceeding 20 m from the parent plants, in line with our finding of 100% fertilization in female plants of Marchantia up to 19 m from the nearest males, far beyond published fertilization distances in dioicous bryophytes. We attribute this to (i) much greater spermatozoid production and numbers in Marchantia than in other bryophytes and (ii) highly effective sperm transport both within the antheridiophores and archegoniophores via their overlapping scales and grooves in the stalks and between male and female thalli via surface water films. These features, coupled with the massive production of small spores, explain the success of Marchantia as the primary colonist of open habitats.     

Disruption of Ttll5/Stamp Gene (Tubulin Tyrosine Ligase-like Protein 5/SRC-1 and TIF2-associated Modulatory Protein Gene) in Male Mice Causes Sperm Malformation and Infertility

TTLL5/STAMP (tubulin tyrosine ligase-like family member 5) has multiple activities in cells. TTLL5 is one of 13 TTLLs, has polyglutamylation activity, augments the activity of p160 coactivators (SRC-1 and TIF2) in glucocorticoid receptor-regulated gene induction and repression, and displays steroid-independent growth activity with several cell types. To examine TTLL5/STAMP functions in whole animals, mice were prepared with an internal deletion that eliminated several activities of the Stamp gene. This mutation causes both reduced levels of STAMP mRNA and C-terminal truncation of STAMP protein. Homozygous targeted mutant (Stamp^tm/tm) mice appear normal except for marked decreases in male fertility associated with defects in progressive sperm motility. Abnormal axonemal structures with loss of tubulin doublets occur in most Stamp^tm/tm sperm tails in conjunction with substantial reduction in α-tubulin polyglutamylation, which closely correlates with the reduction in mutant STAMP mRNA. The axonemes in other structures appear unaffected. There is no obvious change in the organs for sperm development of WT versus Stamp^tm/tm males despite the levels of WT STAMP mRNA in testes being 20-fold higher than in any other organ examined. This defect in male fertility is unrelated to other Ttll genes or 24 genes previously identified as important for sperm function. Thus, STAMP appears to participate in a unique, tissue-selective TTLL-mediated pathway for α-tubulin polyglutamylation that is required for sperm maturation and motility and may be relevant for male fertility.     

T-DNA mediated disruption of essential gametophytic genes in Arabidopsis is unexpectedly rare and cannot be inferred from segregation distortion alone

Many genes are thought to be expressed during the haploid phase in plants, however, very few haploid-specific genes have been isolated so far. T-DNA insertion mutagenesis is a powerful tool for generating mutations that affect gametophyte viability and function, as disruption of a gene essential for these processes should lead to a defect in the transmission of the gametes. Mutants can therefore be screened on the basis of segregation distortion for a reporter resistance gene contained in the T-DNA. We have screened the Versailles collection of Arabidopsis transformants for 1:1 Kan^R:Kan^S segregation after selfing, focussing on gametophyte mutations which show normal transmission through one gametophyte and cause lethality or dysfunction of the other. Only 1.3% (207) of the 16,000 lines screened were scored as good candidates. Thorough genetic analysis of 38 putative T-DNA transmission defect lines (Ttd) identified 8 defective gametophyte mutants, which all showed 0 to 1% T-DNA transmission through the pollen. During the screen, we observed a high background of low-penetrance mutations, often affecting the function of both gametophytes, and many lines which were likely to carry chromosomal rearrangements. The reasons for the small number of retained lines (all male gametophytic) are discussed, as well as the finding that, for most of them, residual T-DNA transmission is obtained through the affected gametophyte.     

Arabidopsis mutant of AtABCG26, an ABC transporter gene, is defective in pollen maturation

In plants, pollen is the male gametophyte that is generated from microspores, which are haploid cells produced after meiosis of diploid pollen mother cells in floral anthers. In normal maturation, microspores interact with the tapetum, which consists of one layer of metabolically active cells enclosing the locule in anthers. The tapetum plays several important roles in the maturation of microspores. ATP-binding cassette (ABC) transporters are a highly conserved protein super-family that uses the energy released in ATP hydrolysis to transport substrates. The ABC transporter gene family is more diverse in plants than in animals. Previously, we reported that an Arabidopsis half-size type ABC transporter gene, COF1/AtWBC11/AtABCG11, is involved in lipid transport for the construction of cuticle layers and pollen coats in normal organ formation, as compared to CER5/AtWBC12/AtABCG12. However, physiological functions of most other ABCG members are unknown. Here, we identified another family gene, AtABCG26, which is required for pollen development in Arabidopsis. An AtABCG26 mutant developed very few pollen grains, resulting in a male-sterile phenotype. By investigating microspore and pollen development in this mutant, we observed that there was a slight abnormality in tetrad morphology prior to the formation of haploid microspores. At a later stage, we could not detect exine deposition on the microspore surface. During pollen maturation, many grains in the mutant anthers got aborted, and surviving grains were found to be defective in mitosis. Transmission of the mutant allele through male gametophytes appeared to be normal in genetic transmission analysis, supporting the view that the pollen function was disturbed by sporophytic defects in the AtABCG26 mutant. AtABCG26 can be expected to be involved in the transport of substrates such as sporopollenin monomers from tapetum to microspores, which both are plant-specific structures critical to pollen development.     

Genome-Wide Identification,Phylogeny and Expression Profile of Vesicle Fusion Components in Verticillium dahliae

Vesicular trafficking plays a crucial role in protein localization and movement, signal transduction, and multiple developmental processes in eukaryotic cells. Vesicle fusion is the final and key step in vesicle-mediated trafficking and mainly relies on SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), the regulators including SM (Sec1/Munc18) family proteins, Rab GTPases and exocyst subunits. Verticillium dahliae is a widespread soil fungus that causes disruptive vascular diseases on a wide range of plants. To date, no genes involved in vesicular fusion process have been identified and characterized in V. dahliae. The recent publication of the draft genome sequence of V. dahliae allowed us to conduct a genome-wide identification, phylogeny and expression profile of genes encoding vesicular fusion components. Using compared genomics and phylogenetic methods, we identified 44 genes encoding vesicle fusion components in the V. dahliae genome. According to the structural features of their encoded proteins, the 44 V. dahliae genes were classified into 22 SNAREs (6 Qa-, 4 Qb-, 6 Qc-, 1 Qbc- and 5 R-types), 4 SM family proteins, 10 Rab GTPases and 8 exocyst proteins. Based on phylogeny and motif constitution analysis, orthologs of vesicle fusion component in filamentous fungi were generally clustered together into the same subclasses with well-supported bootstrap values. Analysis of the expression profiles of these genes indicated that many of them are significantly differentially expressed during vegetative growth and microsclerotia formation in V. dahliae. The analysis show that many components of vesicle fusion are well conserved in filamentous fungi and indicate that vesicle fusion plays a critical role in microsclerotia formation of smoke tree wilt fungus V. dahliae. The genome-wide identification and expression analysis of components involved in vesicle fusion should facilitate research in this gene family and give new insights toward elucidating their functions in growth, development and pathogenesis of V. dahliae.     

Deciphering the role of the AT-rich interaction domain and the HMG-box domain of ARID-HMG proteins of Arabidopsis thaliana

ARID-HMG DNA-binding proteins represent a novel group of HMG-box containing protein family where the AT-rich interaction domain (ARID) is fused with the HMG-box domain in a single polypeptide chain. ARID-HMG proteins are highly plant specific with homologs found both in flowering plants as well as in moss such as Physcomitrella. The expression of these proteins is ubiquitous in plant tissues and primarily localises in the cell nucleus. HMGB proteins are involved in several nuclear processes, but the role of ARID-HMG proteins in plants remains poorly explored. Here, we performed DNA-protein interaction studies with Arabidopsis ARID-HMG protein HMGB11 (At1g55650) to understand the functionality of this protein and its individual domains. DNA binding assays revealed that AtHMGB11 can bind double-stranded DNA with a weaker affinity (K_d?=?475?±?17.9 nM) compared to Arabidopsis HMGB1 protein (K_d?=?39.8?±?2.68 nM). AtHMGB11 also prefers AT-rich DNA as a substrate and shows structural bias for supercoiled DNA. Molecular docking of the DNA-AtHMGB11 complex indicated that the protein interacts with the DNA major groove, mainly through its ARID domain and the junction region connecting the ARID and the HMG-box domain. Also, predicted by the docking model, mutation of Lys⁸⁵ from the ARID domain and Arg¹⁹⁹ & Lys²⁰² from the junction region affects the DNA binding affinity of AtHMGB11. In addition, AtHMGB11 and its truncated form containing the HMG-box domain can not only promote DNA mini-circle formation but are also capable of inducing negative supercoils into relaxed plasmid DNA suggesting the involvement of this protein in several nuclear events. Overall, the study signifies that both the ARID and the HMG-box domain contribute to the optimal functioning of ARID-HMG protein in vivo.     

Direction of illumination controls gametophyte orientation in seedless plants and related algae

The environmental influences that determine dorsiventral or axial gametophyte orientation are unknown for most modern seedless plants. To fill this gap, an experimental laboratory system was employed to evaluate the relative effects of light direction and gravity on body orientation of the dorsiventral green alga Coleochaete orbicularis, and gametophytes of liverworts Blasia pusilla and Marchantia polymorpha, early-diverging moss Sphagnum compactum, and fern Ceratopteris richardii, the latter functioning as experimental control. Replicate clonal cultures were experimentally illuminated only from above, only from below, or from multiple directions, with the same near-saturation PAR level for periods brief enough to minimize nutrient limitation effects, and orientation of new growth was evaluated. For all species tested, direction of illumination exerted stronger control over gametophyte body orientation than gravity. When illuminated only from below: 1) axial Sphagnum gametophores that had initially grown into an overlying air space inverted growth by 180°, burrowing into the substrate; 2) new growth of dorsiventral Blasia, Marchantia, and Ceratopteris gametophytes–whose ventral rhizoids initially penetrated agar substrate and dorsal surfaces initially faced overlying airspace–twisted 180° so that ventral surfaces bearing rhizoids faced overlying air space and rhizoids extended into the air; and 3) Coleochaete lost typical dorsiventral organization and diagnostic dorsal hairs. Direction of illumination also exerted stronger control over orientation of liverwort new growth than surface contact did. These results indicate that early land plants likely inherited light-directed gametophyte body orientation from ancestral streptophyte algae and suggest a mechanism for reorientation of gametophyte-dominant land plants after spatial disturbance.