EFFECTS OF ULTRASONIC TREATMENT ON FEMALE GAMETOPHYTES OF LAMINARIA JAPONICA (PHAEOPHYTA)

This study on the effects of ultrasonic treatment on female gametophytes of Laminariajaponica showed that:1. Ultrasonic treatment had shortening effect on filaments of female gametophytes. Within certainperiod of time, the average length of filamentous female gametophytes was shortened.2. Ultrasonic treatment had emptying effect on cells. The number of empty cells increased with timeof treatment. Ultrasonic treatment had harmful effect on cells.3. Ultrasonic treatment could break down cell walls. The combination of frequency of 20 kHz, out-put of 15 W, 40 s and 60 s of treatment was best for this purpose. After ultrasonic treatment, the regner-ation of female gametophytes into sporophytes was effected. Female gametophytes could not recover aftertoo long period of treatment.

Laminaria gametophyte clone culture and its application in sporeling cultivation

In China, the total annual production of cultured Laminaria japonica reached half million tons in dry weight there few years. The routine sporeling culture technique conducted in the greenhouse took at least three and half months. In such a case, sometimes the sporelings died within a few days due to destructive diseases. In Order to overcome the mentioned problems, a new sporeling culture technique, the clone technique, is developed. The method includes three stead: (1) Gametophyte clone culture. The spores and the gametophytes are cloned in flasks under favorable environments. (2) Sporeling cul ture. Male and female clones are crushed and spread onto a frame to allow the gametophytes to attach to the substrata. The frames are cultured in tanks, and the sporophytes reach 1 cm in length within one and a half months. (3) Outgrowing of the plant. The frames are put in the open sea when seawater temperature decreased to 20℃. After one month, the sporelings are large enough to be transplanted. It is concluded that the clone technique has the following advantages: (1)Large amount of clones can be produced in a short period of time. (2) Clone seeding method makes it free from the biological rhythm, one can seed the plant anytime all the year round. (3) It takes only one and a half months to complete the process of sporeling cultivation in the greenhouse. At present, this technique is used in the breeding of new strains of Laminaria.

Microsporogenesis and Male Gametophyte Development of Euphorbar pulcherrima

The microsporogenesis and male gametophyte development of Euphorbia pulcherrima were studied with paraffin section method. The results showed that the anther of Euphorbia pulcherrima had four chambers. The development of anther wall was dicotyledonous type; its wall consisted of epidermis, endothecium, middle layer and tapetum. The mature male gametophyte development experienced seven major stages as the followings: archesporial cells, aporgenous cells, microspore mother cells, tetrads of microspores (tetrahedral), uninucleate microspores, 2-uncleate pollens and 2-celled pollen stage. The anther wall varied with the pollen development: the epidermis of anther ruptures at the anther mature stage; the middle layer disappears at tetrad stage; the endothelium became fibrous layer at the anther mature stage; the tapetum (glandular) was the most development at the microspore mother cells stage and disappeared at anther mature stage. The abnormal anther development was observed simultaneously.

Physiological differences in the growth and maturation of Eisenia bicyclis and Ecklonia cava gametophytes in Korea

The objective of this study was to examine the eff ects of temperature and light intensity on growth of female gametophytes of Eisenia bicyclis and Ecklonia cava and responses of these female gametophytes to Fe addition and daylength. Female gametophytes of each species were cultured at four temperatures (10, 15, 20, and 25°C) and under a combination of four light intensities (10, 20, 40, and 80 μmol photons/(m^2 ·s)) with two temperatures (15 and 20°C for E i . bicyclis;20 and 25°C for E c . cava ) to clarify their optimal growth conditions. Growth and maturation of female gametophytes of these two species under a combination of five Fe-EDTA concentrations (0, 1, 2, 4, and 8 μmol/L) and three daylengths (10, 12, and 14 h) were also examined. The growth of E i . bicyclis gametophyte was maximal at approximately 15–20°C, 20 μmol photons/(m^2 ·s), Fe-EDTA concentration of 8 μmol/L and daylengths of 12–14 h. While E c . c ava gametophytes showed optimal growth at approximately 20–25°C, 20 μmol photons/(m^2 ·s), Fe- EDTA concentration of 8 μmol/L and daylength of 14 h. Maturation of gametophytes was enhanced at Fe-EDTA concentration of 4 μmol/L for E i . bicyclis and at 2 μmol/L for E c . c ava . In conclusion, optimal growth temperatures and Fe-EDTA concentrations for maturation of E i . bicyclis and E c . c ava gametophytes were diff erent. Higher optimal growth temperature for E c . c ava gametophytes may contribute to its wider geographical distribution compared to E i . bicyclis which has restricted habitats in Korea. This suggests that addition of Fe ion could be used to recover beds of these two species in barren grounds of Korea.

Florogenesis and Female Gametophyte Development in <i>Allium cepa</i>L. cv. Krishnapuram

Florogenesis is one of the most complicated and interesting processes in the nature. This process involves developmental, physiological and molecular events leading to transformation from vegetative to reproductive phase for optimal seed production and the continuation of species. The basic knowledge about flowering processes, male and female sexual systems support basic and applied research and breeding programs. Most of the onion varieties from India are short day varieties, more diverse than other exotic germplasm and useful as a source of new alleles for supporting breeding programs. The present investigation was focused to study for the first time florogenesis process by scanning electron study for the first time florogenesis process by scanning electron microscopy (SEM) and development of female gametophytes by light microscopy in order to acquire basic knowledge useful for optimizing in vitro process to produce gynogenic haploid to support and speed breeding program in short-day onion Allium cepa L. cv. Krishnapuram (KP) or Bangalore Rose. This study revealed that shoot primordium differentiated into inflorescence meristem in the month of December, while seeds were planted in the field in the September. The individual florets are preceded by a variying number of floral initials. The female gametophyte developed from chalazal side megaspore. The embryo sac development is a bisporic Allium type showing short-lived antipodals. The histological study suggests that the use of big or preanthesis flower buds with embryo sac for production of gynogenic haploids to support breeding program in onion cv. Krishnapuram (KP). However further studies are needed for confirmation of this observation.

Recombinant expression of rt-PA gene (encoding Reteplase) in gametophytes of the seaweed Laminaria japonica (Laminariales,Phaeophyta)

The life cycle of seaweed Laminaria japonica involves a generation alternation between diploid sporophyte and haploid gametophte. The expression of foreign genes in sporophte has been proved. In this research, the recombinant expression in gametophyte was investigated by particle bombardment with the rt-PA gene encoding the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction (AMI). Transgenic gametophytes were selected by their resistance to herbicide phosphiothricin (PPT), and proliferated in an established bubble column photo-bioreactor. According to the results from quantitative ELISA, Southern blotting, and fibrin agarose plate assay (FAPA) for bioactivity, it was showed that the rt-PA gene had been inte-grated into the genome of gametophytes of L. japonica, and the expression product showed the ex-pected bioactivity, implying the proper post-transcript modification in haploid gametophyte.

Programmed cell death may act as a surveillance mechanism to safeguard male gametophyte development in Arabidopsis

Programmed cell death(PCD)plays an important role in plant growth and development as well as in stress responses.During male gametophyte development,it has been proposed that PCD may act as a cellular surveillance mechanism to ensure successful progression of male gametogenesis,and this suicide protective machinery is repressed under favorable growth conditions.However,the regulatory mechanism of male gametophyte-specific PCD remains unknown.Here,we report the use of a TdT-mediated dUTP nick-end labelingbased strategy for genetic screening of Arabidopsis mutants that present PCD phenotype during male gametophyte development.By using this approach,we identified 12 mutants,designated as pcd in male gametogenesis(pig).pig mutants are defective at various stages of male gametophyte development,among which nine pig mutants show a microspore-specific PCD phenotype occurring mainly around pollen mitosis I or the bicellular stage.The PIG1 gene was identified by map-based cloning,and was found to be identical to ATAXIA TELANGIECTASIA MUTATED(ATM),a highly conserved gene in eukaryotes and a key regulator of the DNA damage response.Our results suggest that PCD may act as a general mechanism to safeguard the entire process of male gametophyte development.

Accumulation and Speciation of Arsenic in Pteris vittata Gametophytes and Sporophytes: Effects of Calcium and Phosphorus

Dear Editor, Environmental arsenic (As) contamination of both soil and water systems is a worldwide problem, and As can enter the human food chain through contaminated agricultural products and drinking water and contribute to the development of lung.

Effects of inorganic carbon concentration and pH on carbonic anhydrase activity of gametophytes of Saccharina japonica

Carbonic anhydrase(CA)was considered to be an important component of carbon concentrating mechanism(CCM)of algae.It was an inducible enzyme.Environmental factors,especially dissolved inorganic carbon and pH,were known to affect CA activity.Effects of inorganic carbon(CO_(2)and HCO_(3)^(-))and pH on CA activity of gametophytes of Saccharina japonica were evaluated in this study.Under high-CO_(2)condition(3%CO_(2)),the activity of external CA(C_(Aext))was significantly decreased(P<0.05)from 33.92 REA/g FW to 27.69 REA/g FW.In contrast,the internal CA(CA_(int))and total CA activities were elevated significantly(p<0.01)from 36.83 REA/g FW to 48.80 REA/g FW,and from 70.75 REA/g FW to 76.49 REA/g FW,respectively.Addition of an appropriate concentration of HCO_(3)^(-)to the medium,CA_(int)and total CA activities were promoted significantly,although the activity of C_(Aext)was inhibited significantly(P<0.05).In higher HCO_(3)^(-)concentration(up to 1680 mg/L),the activity of CA_(int)was promoted to 60.81REA/g FW,which was twice that of the control.Higher activities of CA_(int)and total CA were induced at low pH.At pH 6.0,the highest activities of CA_(int)(110.85 REA/g FW)and total CA(128.17 REA/g FW)were induced.However,at pH 9.0,the activities of CA_(int)and total CA were reduced to the lowest of 23.31REA/g FW and 42.19 REA/g FW,respectively.This is the first report about the successful detection of C_(Aext)activity of gametophytes of S.japonica.The results would provide data for the analysis of Ci acquisition and transport mechanism in S.japonica gametophytes.

Arabidopsis BIG1 and BIG5 are crucial for male gametophyte transmission

Arabidopsis contains five Brefeldin Ainhibited guanine nucleotide exchange factors(BIGs),which play a critical role in vesicle biogenesis for protein traffic from the Golgi to the plasma membrane.Biological processes regulated by BIG1-BIG4 are postulated to be distinct from those by BIG5. However, we show that the self-pollinated BIG1+/- big5 silique do not produce homozygous seeds, and some pollen tubes from BIG1+/- big5 anthers grew slowly in vitro and failed to target nearby ovules in vivo. We identified the big1 big5 homozygote from the progeny of BIG1+/- big5 plants transformed with BIG5, whose expression is driven by a pollen-specific promoter p Lat52, indicating that male gametophyte transmission is blocked in the double mutant. Confocal microscopy indicated that BIG1 and BIG5 are co-localized in trans Golgi network. Thus,our data indicate that BIG1 and BIG5 are crucial for male gametophyte transmission.

Identification and Characterization of Long Non-Coding RNAs Involved in Sex-Related Gene Regulation in Kelp Saccharina japonica

Long non-coding RNAs(lncRNAs)regulate a variety of biological processes,including sexual reproduction and differentiation.Saccharina japonica,a commercially important brown alga in China,shows remarkable sexual dimorphism in haploid gametophytes.The sex of Saccharina japonica gametophytes is determined by UV sexual system.However,no results have been reported on the lncRNAs involved in the sex-related gene regulation of S.japonica.This study identified a number of lncRNAs and assessed their expression levels in male and female gametophytes.Among them,a total of 405 lncRNAs and 211 mRNAs showed differential expressions.Furthermore,the functions of target genes of differentially expressed lncRNAs(DELs)differed from those of differentially expressed genes(DEGs),suggesting that lncRNA may interact with other functional proteins,in addition to DEGs,to involve sex regulation in S.japonica.There were 32 and 90 potential cis-regulatory and trans-regulatory interactions between DELsDEGs,respectively.Five of these lncRNAs(LNC_002974,LNC_021059,LNC_038466,LNC_051584,and LNC_027400)interacted with putative male sex determination region(SDR)genes,suggesting that they act as regulators in gametophytes'sex regulation potentially.Findings from this study contribute to our understanding of the roles of lncRNAs in sex differentiation and lay the foundation for functional studies of candidate lncRNAs in the future.

INTRASPECIFIC CROSSINGS OF UNDARIA PINNATIFIDA (HARV.) SUR.-A POSSIBLE TIME-SAVING WAY OF STRAIN SELECTION

Unicellular gametophytes of Undaria pinnatifida (Harv.) Sur. were isolated in Qingdao, P. R. China in April 1993 and in Tokushima, southem Japan in March 1995. Diferent intraspecific crossings by using unicellular male and female gametophytes were successfully undertaken in Sept. of 1995 in Qingdao.Sporophytes were transplanted to two different locations for open an cultivation. One was at Zhanqiao (ZQ) Bay where the water current was slower than that of another location - Taipingjiao(TPJ). A total of218 adult sporophytes were harvested on January 12 and 18 from TPJ and ZQ repectively. For eacncombination, 10 sporophytes un cultivated. Analysis of the mprpholgical characteristics of adult spprophytes indicated that the longest length between two bases of the serration of pinnate bldes (W2) is a morphological characteristic that can be transferred from the parent plant to the next generations regardless of environmental variations. There was evidence that W2 was apparently determined by sex-linked factors, i.e, by male parental grametophyte.. Sporophytes from certain crossing combinations showed more vigorous growth than those from other crossing combinations. It is therrfore possible to select grametophyte strains which can be used as parental gametophytes for the seeding production of sporophytes with more vigorous growth within shorter cultivation period. The morphology of hybrids from a Qingdao strain and a Tokushima cultivated strain resembled that of both parental plants in frond feaures (wrinkled or smooth) and W2. Sporophyll formation also varied with strains. The fact that adult sporophytes resulting from the same crossing combinations have identical morphological characteristics under the same environmental conditions indicates the possibility of a new way to select strains which are expected to be ideal for commerical production by purposely selecting, propagating, and seeding unicellular gametophytes for sporeing production through freeliving techniques of gametophytes.

Prediction of Suitable Habitat for Lycophytes and Ferns in Northeast China: A Case Study on Athyrium brevifrons

Suitable habitat is vital for the survival and restoration of a species.Understanding the suitable habitat range for lycophytes and ferns is prerequisite for effective species resource conservation and recovery efforts.In this study, we took Athyrium brevifrons as an example, predicted its suitable habitat using a Maxent model with 67 occurrence data and nine environmental variables in Northeast China.The area under the curve(AUC) value of independent test data, as well as the comparison with specimen county areal distribution of A.brevifrons exhibited excellent predictive performance.The type of environmental variables showed that precipitation contributed the most to the distribution prediction, followed by temperature and topography.Percentage contribution and permutation importance both indicated that precipitation of driest quarter(Bio17) was the key factor in determining the natural distribution of A.brevifrons, the reason could be proved by the fern gametophyte biology.The analysis of high habitat suitability areas also showed the habitat preference of A.brevifrons: comparatively more precipitation and less fluctuation in the driest quarter.Changbai Mountains, covering almost all the high and medium habitat suitability areas, provide the best ecological conditions for the survival of A.brevifrons, and should be considered as priority areas for protection and restoration of the wild resource.The potential habitat suitability distribution map could provide a reference for the sustainable development and utilisation of A.brevifrons resource, and Maxent modelling could be valuable for conservation management planning for lycophytes and ferns in Northeast China.

Karyological observations of Ulva linza chromosomes

Abstract Ulva linza is one of the species that causes green tides in the Yellow Sea,China.Due to the diffi culties in chromosomal preparation,the large numbers of chromosomes,and their relatively small sizes,there have been no reported studies on Ulva macroalgae chromosomes.The karyotypes and chromosomes in U.linza were observed after a series of treatments.The chromosomes were pretreated with 0.1%colchicine for 12 h and then mixed with enzymes.The samples were dropped from 30 cm height onto glass slides,which spread out the surface coat.These pretreatments were the optimal chromosomes preparation treatments.The prepared chromosomes were stained with 4',6-diamidino-2-phenylindole(DAPI),which is a fl uorescent probe sensitive and specifi c to DNA.The chromosome number in the haploid male and female gametophytes was n=9,and was 2 n=18 in the diploid sporophytes.The female gametophyte chromosomes were between 0.804 and 2.292μm in size,the male gametophyte chromosomes were between 0.917 and 2.916μm,and the sporophyte chromosomes were between 0.912 and 2.167μm.The relative sizes of the chromosomes were used to analyze the karyotypes of the female and male gametophyte chromosomes.The results provide a solid foundation for the basic technique that can be used to localize molecular markers of Ulva chromosomes.

The Effects of Sugars and Ethylene on Apospory and Regeneration in <i>Ceratopteris richardii</i>

In land plants, two distinct generations, gametophyte and sporophyte, alternate to complete the life cycle. Sporophytes undergo meiosis to produce spores, from which gametophytes develop. Gametophytes produce gametes, which participate in fertilization to produce the zygote, the first cell of the sporophyte generation. In addition to this sexual reproduction pathway, some fern species can undergo apospory or apogamy, processes that bypass meiosis or fertilization, respectively, to alternate between the two generations without changing the chromosome number. Apospory is inducible in the laboratory in various fern species simply by altering the sugar level in the media. In sporophytes induced to undergo apospory, sporophyte regeneration is also observed. The ratio of aposporous gametophytes to regenerated sporophytes varies, in a manner consistent with being dependent on sugar level. Whereas the sugar signaling pathway is yet to be elucidated in lower plants, in angiosperms it has been shown to play a regulatory role in controlling essential processes including flowering and embryo development, which give rise to the gametophyte and the next sporophyte generation, respectively. Here, we present evidence for the role of different sugar levels on the balance of apospory and regeneration in the fern Ceratopteris richardii. The demonstration of crosstalk between sugar signaling and the hormone ethylene signaling in angiosperms prompted us to test the effects of this hormone in combination with sugar on apospory vs. regeneration. These results provide insight into how a group of redifferentiating cells determines which generation to become and lay the groundwork for further analysis of this asexual pathway.

Cloning and Bioinformatics Analysis of <i>Rosa rugose S</i>Locus F-Box Gene (<i>RrSLF</i>)

In order to reveal the phenomenon of R. rugose pollination incompatibility, the full-length cDNA sequence of S Locus F-box Gene was cloned for the first time from the pollen of Rosa rugose “Zilong wochi” with RT-PCR and RACE methods and named as RrSLF. The full-length cDNA is 1236 bp with an open reading frame of 1122 bp, encoding 343 amino acids. The derived protein has a molecular weight of 43.7 kD, a calculated pI of 6.24, an F-box conserved domain at position 343 - 741, and belongs to F-box family. The derived protein is a Hydrophobicity protein secreted into the cytoplasm. There is no transmembrane domain and no signal peptide cleavage site, twenty-one Ser phosphorylation sites, seven Thr phosphorylation sites, seven Tyr phosphorylation sites, two N-glycosylation sites, and no O-glycosylation sites. There are 22.25% α-helixes, 31.37% random coil, 32.17% extended peptide chain, and 14.21% β-corner structure. This protein and the SFB/SLF protein from Rosaceae Prunus fruit, including Prunus speciose, share a sequence homology of 59% - 61%;all of the proteins contain an F-box conserved domain, two hypervariable regions HVa, HVb, and two variable regions V1, V2. Furthermore, their phylogenetic relationships are consistent with their traditional classifications. These results were meaningful to reveal the molecular mechanism of Rosa rugose pollination incompatibility and improve the theory and techniques of breeding ornamental Rosa rugose.

Phototropism in the Marine Red Macroalga Pyropia yezoensis

Phototropism is a response to the direction of light that guides growth orientation and determines the shape of plants to optimize photosynthetic activity. The phototropic response is present not only in terrestrial plants but also in water-living algae. However, knowledge about phototropism in Bangiophycean seaweeds is limited. Here, we examined the phototropic response of the red alga Pyropia yezoensis to elucidate the regulatory mechanism of phototropism in Bangiophyceae. When leafy gametophytes and filamentous sporophytes of P. yezoensis were cultured under directional light, phototropism was observed in the gametophytes. Conchosporangia on the sporophytes also exhibited phototropism. Phototropism was positive in the majority of gametophytes and conchosporangia but in some cases was negative. In addition, a strong phototropic response occurred under white light, whereas blue and red light elicited minor and no responses, respectively. This observation is in contrast with the phototropic response in terrestrial plants and several algae, in which blue light is responsible for positive phototropism. Surprisingly, the genome of P. yezoensis has no homologues of the photoreceptors for blue and red light, revealing differences in the regulation of phototropism between terrestrial plants and P. yezoensis . Studies on the phototropism in P. yezoensis could shed light on the evolutional divergence of phototropic responses in plants.

A single silk-and multiple pollen-expressed PMEs at the Ga1 locus modulate maize unilateral cross-incompatibility

The Gametophyte factor1(Ga1) locus in maize confers unilateral cross-incompatibility(UCI), and it is controlled by both pollen and silk-specific determinants. Although the Ga1 locus has been reported for more than a century and is widely utilized in maize breeding programs, only the pollen-specific ZmGa1P has been shown to function as a male determinant;thus, the genomic structure of the Ga1 locus and all the determinants that control UCI at this locus have not yet been fully characterized.Here, we used map-based cloning to confirm the determinants of UCI at the Ga1 locus and maize pan-genome sequence data to characterize the genomic structure of the Ga1 locus. The Ga1 locus comprises one silk-expressed pectin methylesterase gene(PME, ZmGa1F) and eight pollenexpressed PMEs(ZmGa1P and ZmGa1PL1-7).Knockout of ZmGa1F in Ga1/Ga1 lines leads to the complete loss of the female barrier function. The expression of individual ZmGa1PL genes in a ga1/ga1 background endows ga1 pollen with the ability to overcome the female barrier of the Ga1 locus. These findings, combined with genomic data and genetic analyses, indicate that the Ga1 locus is modulated by a single female determinant and multiple male determinants, which are tightly linked.The results of this study provide valuable insights into the genomic structure of the Ga2 and Tcb1 loci and will aid applications of these loci in maize breeding programs.

Emerging role of ER quality control in plant cell signal perception

The endoplasmic reticulum quality control(ER-QC)is a conserved mechanism in surveillance of secreted signaling factors during cell-to-cell communication in eukaryotes.Recent data show that the ER-QC plays important roles in diverse cell-to-cell signaling processes during immune response,vegetative and reproductive development in plants.Pollen tube guidance is a precisely guided cell-cell communication process between the male and female gametophytes during plant reproduction.Recently,the female signal has been identified as small secreted peptides,but how the pollen tube responds to this signal is still unclear.In this review,we intend to summarize the role of ER-QC in plants and discuss the recent advances regarding our understanding of the mechanism of pollen tube response to the female signals.

AtTMEM18 plays important roles in pollen tube and vegetative growth in Arabidopsis

In flowering plants, pollen tube growth is essential for delivery of male gametes into the female gametophyte or embryo sac for double fertilization. Although many genes have been identified as being involved in the process, the molecular mechanisms of pollen tube growth remains poorly understood.In this study, we identified that the Arabidopsis Transmembrane Protein 18(AtT MEM18) gene played important roles in pollen tube growth. The AtT MEM18 shares a high similarity with the Transmembrane 18 proteins(TMEM18s) that are conserved in most eukaryotes and may play important roles in obesity in humans. Mutation in the AtT MEM18 by a Ds insertion caused abnormal callose deposition in the pollen grains and had a significant impact on pollen germination and pollen tube growth.AtT MEM18 is expressed in pollen grains, pollen tubes, root tips and other vegetative tissues. The pollen-rescued assays showed that the mutation in AtT MEM18 also caused defects in roots, stems,leaves and transmitting tracts. AtT MEM18-GFP was located around the nuclei. Genetic assays demonstrated that the localization of AtT MEM18 around the nuclei in the generative cells of pollen grains was essential for the male fertility.Furthermore, expression of the rice TMEM18-homologous protein(OsT MEM18) driven by LAT52 promoter could recover the fertility of the Arabidopsis attmem18 mutant. These results suggested that the TMEM18 is important for plant growth in Arabidopsis.