Expression analyses in Ginkgo biloba provide new insights into the evolution and development of the seed.

Although the seed is a key morphological innovation, its origin remains unknown and molecular data outside angiosperms is still limited. Ginkgo biloba, with a unique place in plant evolution, being one of the first extant gymnosperms where seeds evolved, can testify to the evolution and development of the seed. Initially, to better understand the development of the ovules in Ginkgo biloba ovules, we performed spatio-temporal expression analyses in seeds at early developing stages, of six candidate gene homologues known in angiosperms: WUSCHEL, AINTEGUMENTA, BELL1, KANADI, UNICORN, and C3HDZip. Surprisingly, the expression patterns of most these ovule homologues indicate that they are not wholly conserved between angiosperms and Ginkgo biloba. Consistent with previous studies on early diverging seedless plant lineages, ferns, lycophytes, and bryophytes, many of these candidate genes are mainly expressed in mega- and micro-sporangia. Through in-depth comparative transcriptome analyses of Ginkgo biloba developing ovules, pollen cones, and megagametophytes we have been able to identify novel genes, likely involved in ovule development. Finally, our expression analyses support the synangial or neo-synangial hypotheses for the origin of the seed, where the sporangium developmental network was likely co-opted and restricted during integument evolution.

Effects of ultrasonic conditions on the interfacial property and emulsifying property of cellulose nanoparticles from ginkgo seed shells.

Cellulose microparticles from ginkgo seed shells were treated by ultrasonic treatments within the selected output powders (150-600 W) and durations (10-60 min) to produce cellulose nanoparticles. The main aim of this study was to investigate effects of ultrasonic conditions on the interfacial property and emulsifying property of those cellulose nanoparticles. Compared to ultrasonic output powers, ultrasonic durations showed the greater influence on morphology and physical properties of cellulose nanoparticles. Atomic force microscopy revealed that noodle-like cellulose particles with 1100 nm in length gradually became the short rod-like nanoparticles with 300 nm in length with increasing of ultrasonic duration from 10 min to 60 min. Moreover, results of contact angles indicated that ultrasound could significantly improve hydrophobicity of cellulose nanoparticles. The interfacial shear rheology showed that although all cellulose nanoparticles exhibited the similar interface adsorption behavior which showed the initial lag-phase of adsorption, followed by the interface saturation, the time of this initial lag-phase was affected by ultrasonic conditions. The increase of ultrasonic duration and ultrasonic power could shorten the time of this initial lag-phase, suggesting the resulting cellulose nanoparticles easier adsorption at the O/W interface. It was probably attributed to its small size and high hydrophobicity induced by intense ultrasonic treatments. Meanwhile, the cellulose nanoparticles with small size and higher hydrophobicity exhibited the better emulsifying ability to stabilize oil-in-water emulsions due to the formation of the viscoelastic interfacial film. This study improved understanding about changes in interfacial and emulsifying properties of cellulose nanoparticles caused by ultrasonic treatments.

Identification and characterization of long non-coding RNAs involved in embryo development of Ginkgo biloba.

Long non-coding RNAs (lncRNAs) are important regulatory factors for plant growth and development. Despite this, little is known about the regulatory interactions of lncRNAs with mRNA during embryo development. Here, we used a bioinformatics genome-wide approach to identify lncRNAs involved in embryo development of Ginkgo biloba, based on RNA sequencing datasets from G. biloba embryos during early, middle, late developmental stages. In total, 2326 lncRNAs were identified in the G. biloba embryos, of which 1307 and 1019 could be classified as long intergenic non-coding RNAs and antisense lncRNAs, respectively. Among them, a total of 657 differentially expressed lncRNAs were identified in the different developmental stages of the G. biloba embryos. Based on the functional annotation of potential target genes of lncRNAs, 50, 33, and 76 lncRNAs were predicted to target genes involved in plant hormone signal transduction, plant hormone biosynthesis, and circadian rhythm regulation, respectively. A lncRNA (17)-miRNA (25)-PCgene (52) network was constructed for the G. biloba embryo. Three lncRNAs (lnc000823, lnc002072, lnc000866) were predicted as target mimics of miR159, which targeted two transcription factors with variety of functions, Gb_11536 (MYB33) and Gb_23921 (MYB101). The data generated in this study provide a better understanding of the roles of lncRNAs in embryo development of G. biloba and plants in general.

Proteinase inhibitor from ginkgo seeds is a member of the plant nonspecific lipid transfer protein gene family.

A 9-kD proteinase inhibitor was isolated from the seeds of ginkgo (Ginkgo biloba) and purified to homogeneity. This protein was revealed to partial-noncompetitively inhibit the aspartic acid proteinase pepsin and the cysteine proteinase papain (inhibition constant = 10(-5)-10(-4) m). The cDNA of the inhibitor was revealed to contain a 357-bp open reading frame encoding a 119-amino acid protein with a potential signal peptide (27 residues), indicating that this protein is synthesized as a preprotein and secreted outside the cells. Semiquantitative reverse transcription-polymerase chain reaction revealed that this gene expresses only in seeds, not in stems, leaves, and roots, suggesting that the protein is involved in seed development and/or germination. The inhibitor showed about 40% sequence homology with type-I nonspecific lipid transfer protein (nsLTP1) from other plant species. Actually, this inhibitor exerted both lipid transfer activity and lipid-binding activity, while the protein did not show any antifungal and antibacterial activities. Furthermore, the site-directed mutagenesis study using a recombinant ginkgo nsLTP1 revealed that proline (Pro)-79 and phenylalanine-80 are important on phospholipid transfer activity and that Pro-79 and isoleucine-82 are essential for the binding activity toward cis-unsaturated fatty acids. On the other hand, the alpha-helical content of P79A and F80A mutants was significantly lower than that of the wild-type protein. It was noteworthy that the papain-inhibitory activity of P79A and F80A mutants was elevated twice as much as that of the wild-type protein. In summary, we concluded that Pro-79 plays a critical role in both the lipid transfer and binding activities of ginkgo nsLTP1.

Effects of storage temperature on viability, germination and antioxidant metabolism in Ginkgo biloba L. seeds.

The behaviour of the Ginkgo biloba L. seeds was studied during storage at 4 and 25 degrees C. When stored at 25 degrees C, all the seeds died in 6 months. Cold temperatures preserved seed tissue viability for 1 year but did not preserve their capability to germinate, since such capability decreased after 6 months. A significant increase in lipid peroxidation occurred in the seed both in the embryo and in the endosperm. During storage a progressive deterioration of the endosperm tissues was evident. The two major water soluble antioxidants, ascorbate (ASC) and glutathione (GSH), showed different behaviour in the two conditions of storage and in the two main structures of the seed, the embryo and the endosperm. The ASC content of embryos and endosperms remained quite unchanged in the first 9 months at 4 degrees C, then increased. At 25 degrees C a significant decrease in the ASC content in the embryos was evident, whereas it remained more stable in the endosperm. The GSH pool decreased at both storage temperatures in the embryos. As far as the ASC-GSH redox enzymes are concerned, their activities decreased with storage, but changes appeared to be time-dependent more than temperature-dependent, with the exception of the endosperm ascorbate free radical (AFR) reductase (EC 1.6.5.4), the activity of which rapidly decreased at 25 degrees C. Therefore overall the antioxidant enzymes were scarcely regulated and unable to counteract oxidative stress occurring during the long-term storage.

Structural and immunocytochemical characterization of the Ginkgo biloba L. sperm motility apparatus.

Ginkgo biloba and the cycads are the only extant seed plants with motile sperm cells. However, there has been no immunocytochemical characterization of these gametes to determine if they share characteristics with the flagellated sperm found in bryophytes and pteridophytes or might give clues as to the relationships to nonflagellated sperm in all other seed plants. To determine characteristics of proteins associated with the motility apparatus in these motile sperm, we probed thin sections of developing spermatogenous cells of Ginkgo biloba with antibodies to acetylated and tyrosinated tubulin and monoclonal antibodies that recognize mammalian centrosomes and centrin. The blepharoplast that occurs as a precursor to the motility apparatus consists of an amorphous core, pitted with cavities containing microtubules and a surface studded with probasal bodies. The probasal bodies and microtubules within the blepharoplast cavities are labeled with antibodies specific to acetylated tubulin. Positive but weak reactions of the blepharoplast core occur with the centrosomereactive antibodies MPM-2 and C-9. Reactions to centrin antibodies are negative at this developmental stage. From this pre-motility apparatus structure, an assemblage of about 1,000 flagella and associated structures arises as the precursor to the motility apparatus for the sperm. The flagellar apparatus consists of a three-layered multilayered structure that subtends a layer of spline microtubules, a zone of amorphous material similar to that in the blepharoplast, and the flagellar band. Centrin antibodies react strongly with the multilayered structure, the transition zone of the flagella, and fibrillar material near the flagellar base at the surface of the amorphous material. Both the spline microtubules and all of the tubules in the flagella react strongly with the antibodies to acetylated tubulin. These localizations are consistent with the localizations of these components in pteridophyte and bryophyte spermatogenous cells, although the blepharoplast material surrounding and connecting flagellar bases does not occur in the seedless (nonseed) land plants. These data indicate that despite the large size of ginkgo gametes and the taxonomic separation between pteridophytes and Ginkgo biloba, similar proteins in gametes of both groups perform similar functions and are therefore homologous among these plants. Moreover, the presence of acetylated tubulin in bands of microtubules may be a characteristic shared with more derived non-flagellated sperm of other conifers and angiosperms.