Flavonoids and phenylethanoids from the flowers and leaves of Aeschynanthus species and cultivars (Gesneriaceae).

Forty-one flavones, each one of flavonol, chalcone and dihydroflavonol, two flavanones, and four phenylethanoids were isolated from corollas, calyces and leaves of two Aeschynanthus species, A. fulgens and A. pulcher, and six cultivars, 'Mahligai', 'Mona Lisa', SoeKa', 'Redona', 'Freshya' and 'Bravera'. Flavonoids were mainly the glucuronides and/or methylglucuronides based on hispidulin, nepetin, pectolinarigenin, 6-hydroxyluteolin, scutellarein, apigenin and luteolin, and identified by UV spectra, HR-MS, LC-MS, acid hydrolysis, NMR, and/or HPLC and TLC comparisons with authentic samples. Of these flavonoids, twelve, i.e. hispidulin 7,4'-di-O-glucuronide, 7,4'-di-O-methylglucuronide, 7-O-methylglucuronide-4'-O-glucuronide, 7-O-glucuronide-4'-O-methylglucuronide, 7-O-glucosyl-(1 â†’ 2)-glucuronide and 8-C-glucoside, nepetin 7,4'-di-O-glucuronide, 7-O-glucuronide-4'-O-methylglucuronide and 7-O-methylglucuronide-4'-O-glucuronide, pectolinarigenin 7-O-glucosyl-(1 â†’ 2)-glucuronide and 7-O-xylosyl-(1 â†’ 2)-(6″-malonylglucoside), and 6-hydroxyluteolin 7,4'-di-O-glucuronide, were previously undescribed.

Acylated pelargonidin and cyanidin 3-sambubiosides from the flowers of Aeschynanthus species and cultivars.

Thirteen anthocyanins were isolated from the flowers of two Aeschynanthus species, A. fulgens and A. pulcher, and six cultivars, 'Mahligai', 'Mona Lisa', 'SoeKa', 'Redona', 'Freshya' and 'Bravera', and identified as pelargonidin and cyanidin 3-O-sambubiosides and their malonates, succinates, p-coumarates and caffeates, and pelargonidin 3-O-glucoside by acid hydrolysis, HR-MS and NMR. Of their anthocyanins, pelargonidin 3-O-[xylosyl-(1 â†’ 2)-(6''-malonylglucoside)] (2), pelargonidin 3-O-[xylosyl-(1 â†’ 2)-(6''-succinylglucoside)] (3), pelargonidin 3-O-[xylosyl-(1 â†’ 2)-(6''-E-p-coumaroylglucoside)] (4), pelargonidin 3-O-[xylosyl-(1 â†’ 2)-(6''-Z-p-coumaroylglucoside)] (5), pelargonidin 3-O-[xylosyl-(1 â†’ 2)-(6''-E-caffeoylglucoside)] (6) and cyanidin 3-O-[xylosyl-(1 â†’ 2)-(6''-succinylglucoside)] (9) were reported in nature for the first time.

Identification of anthocyanin and other flavonoids from the green-blue petals of Puya alpestris (Bromeliaceae) and a clarification of their coloration mechanism.

To understand the unique green-blue color of Puya alpestris (Bromeliaceae) flowers, we investigated their constituent anthocyanin and related compounds. An anthocyanin, two undescribed flavonols, and two flavones were isolated and identified as delphinidin 3,3',5'-tri-O-ß-glucopyranoside, myricetin 3-O-[α-rhamnopyranosyl-(1 â†’ 6)-ß-glucopyranoside]-3',5'-di-O-ß-glucopyranoside, myricetin 3,3',5'-tri-O-ß-glucopyranoside, luteolin 4'-O-glucoside, and apigenin 4'-O-glucoside. Furthermore, the presence of chlorophyll has also been revealed. P. alpestris petals show a gradient color appearance: Green-blue at the tip and blue at the base. This color difference between the tip and base was used to analyze the pigment components underlying the green-blue color expression. It was found that the petal tip contains the anthocyanin, flavonols, flavones, and chlorophyll in high quantities. Furthermore, the pH of petal juice was 6.2 and 5.6 at the tip and base, respectively. In vitro reconstruction revealed the blue color expression occurred via an intermolecular copigmentation between the anthocyanin and flavones, as well as yellow color expression, which was due to an increase in the absorption at 400-450 nm of the flavonols under the higher pH conditions. Furthermore, we found that the petal extract obtaining using 50% acetone containing chlorophyll showed the same absorption spectrum as that observed for the raw petal. These results indicate that the green-blue color of P. alpestris flowers is developed via an intermolecular co-pigmentation of the anthocyanin (delphinidin 3,3',5'-tri-O-ß-glucopyranoside) with flavones, such as luteolin 4'-O-glucoside, the yellow color expression of flavonols, such as myricetin 3,3',5'-tri-O-glucoside under relatively high pH conditions in the cell sap, and the presence of chlorophyll.

Arabidopsis SH3P2 is an ubiquitin-binding protein that functions together with ESCRT-I and the deubiquitylating enzyme AMSH3.

Clathrin-mediated endocytosis of plasma membrane proteins is an essential regulatory process that controls plasma membrane protein abundance and is therefore important for many signaling pathways, such as hormone signaling and biotic and abiotic stress responses. On endosomal sorting, plasma membrane proteins maybe recycled or targeted for vacuolar degradation, which is dependent on ubiquitin modification of the cargos and is driven by the endosomal sorting complexes required for transport (ESCRTs). Components of the ESCRT machinery are highly conserved among eukaryotes, but homologs of ESCRT-0 that are responsible for recognition and concentration of ubiquitylated proteins are absent in plants. Recently several ubiquitin-binding proteins have been identified that serve in place of ESCRT-0; however, their function in ubiquitin recognition and endosomal trafficking is not well understood yet. In this study, we identified Src homology-3 (SH3) domain-containing protein 2 (SH3P2) as a ubiquitin- and ESCRT-I-binding protein that functions in intracellular trafficking. SH3P2 colocalized with clathrin light chain-labeled punctate structures and interacted with clathrin heavy chain in planta, indicating a role for SH3P2 in clathrin-mediated endocytosis. Furthermore, SH3P2 cofractionates with clathrin-coated vesicles (CCVs), suggesting that it associates with CCVs in planta Mutants of SH3P2 and VPS23 genetically interact, suggesting that they could function in the same pathway. Based on these results, we suggest a role of SH3P2 as an ubiquitin-binding protein that binds and transfers ubiquitylated proteins to the ESCRT machinery.

First flowering hybrid between autotrophic and mycoheterotrophic plant species: breakthrough in molecular biology of mycoheterotrophy.

Among land plants, which generally exhibit autotrophy through photosynthesis, about 880 species are mycoheterotrophs, dependent on mycorrhizal fungi for their carbon supply. Shifts in nutritional mode from autotrophy to mycoheterotrophy are usually accompanied by evolution of various combinations of characters related to structure and physiology, e.g., loss of foliage leaves and roots, reduction in seed size, degradation of plastid genome, and changes in mycorrhizal association and pollination strategy. However, the patterns and processes involved in such alterations are generally unknown. Hybrids between autotrophic and mycoheterotrophic plants may provide a breakthrough in molecular studies on the evolution of mycoheterotrophy. We have produced the first hybrid between autotrophic and mycoheterotrophic plant species using the orchid group Cymbidium. The autotrophic Cymbidium ensifolium subsp. haematodes and mycoheterotrophic C. macrorhizon were artificially pollinated, and aseptic germination of the hybrid seeds obtained was promoted by sonication. In vitro flowering was observed five years after seed sowing. Development of foliage leaves, an important character for photosynthesis, segregated in the first generation; that is, some individuals only developed scale leaves on the rhizome and flowering stems. However, all of the flowering plants formed roots, which is identical to the maternal parent.

The deubiquitinating enzyme AMSH1 and the ESCRT-III subunit VPS2.1 are required for autophagic degradation in Arabidopsis.

In eukaryotes, posttranslational modification by ubiquitin regulates the activity and stability of many proteins and thus influences a variety of developmental processes as well as environmental responses. Ubiquitination also plays a critical role in intracellular trafficking by serving as a signal for endocytosis. We have previously shown that the Arabidopsis thaliana associated molecule with the SH3 domain of STAM3 (AMSH3) is a deubiquitinating enzyme (DUB) that interacts with endosomal complex required for transport-III (ESCRT-III) and is essential for intracellular transport and vacuole biogenesis. However, physiological functions of AMSH3 in the context of its ESCRT-III interaction are not well understood due to the severe seedling lethal phenotype of its null mutant. In this article, we show that Arabidopsis AMSH1, an AMSH3-related DUB, interacts with the ESCRT-III subunit vacuolar protein sorting2.1 (VPS2.1) and that impairment of both AMSH1 and VPS2.1 causes early senescence and hypersensitivity to artificial carbon starvation in the dark similar to previously reported autophagy mutants. Consistent with this, both mutants accumulate autophagosome markers and accumulate less autophagic bodies in the vacuole. Taken together, our results demonstrate that AMSH1 and the ESCRT-III-subunit VPS2.1 are important for autophagic degradation and autophagy-mediated physiological processes.

Transmission ratio distortion of molecular markers in a doubled haploid population originated from a natural hybrid between Osmunda japonica and O. lancea.

In ferns, intra-gametophytic selfing occurs as a mode of reproduction where two gametes from the same gametophyte form a completely homozygous sporophyte. Intra-gametophytic selfing is considered to be prevented by lethal or deleterious recessive genes in several diploid species. In order to investigate the modes and tempo of selection acting different developmental stages, doubled haploids obtained from intra-gametophytic selfing within isolated gametophytes of a putative F1 hybrid between Osmunda japonica and O. lancea were analyzed with EST_derived molecular markers, and the distribution pattern of transmission ratio distortion (TRD) along linkage map was clarified. As the results, the markers with skewness were clustered in two linkage groups. For the two highly distorted regions, gametophytes and F2 population were also examined. The markers skewed towards O. japonica on a linkage group (LG_2) showed skewness also in gametophytes, and the TRD was generated in the process of spore formation or growth of gametophytes. Also, selection appeared to be operating in the gametophytic stage. The markers on other linkage group (LG_11) showed highest skewness towards O. lancea in doubled haploids, and it was suggested that the segregation of LG_11 were influenced by zygotic lethality or genotypic evaluation and that some deleterious recessive genes exist in LG_11 and reduce the viability of homozygotes with O. japonica alleles. It is very likely that a region of LG_11were responsible for the low frequencies of intra-gametophytic selfing in O. japonica.

Fertility and precocity of Osmunda x intermedia offspring in culture.

The feasibility of later-generation hybrid production in ferns has not been previously studied, although it is a significant factor in relation to reproductive isolation. Osmunda x intermedia, a hybrid between O. japonica and O. lancea, is semifertile and has moderate spore germination rates. Under the artificial conditions of this study, F2 and F3 offspring were formed. Some of the F2 offspring showed precocity, and some of the F3 offspring also showed precocity. This fertility suggests that introgressive hybridization might be ongoing in nature. This also indicates a currently unknown genetic control over the timing of fertile frond production in Osmunda.

Genetic population structure of Osmunda japonica, rheophilous Osmunda lancea and their hybrids.

Rheophilous Osmunda lancea often hybridizes with a dryland ally, Osmunda japonica, to produce O. x intermedia, forming zonation in riverbanks and the adjacent dryland along flooding frequency clines. This study examined the genetic structure of populations consisting of O. x intermedia and the two parental species by analyzing ten nuclear DNA markers [six cleaved amplified polymorphic sequence (CAPS) markers and three simple sequence repeat (SSR) markers developed from an expressed sequence tag (EST) library, and the sequence of the glyceraldehyde-3-phosphate dehydrogenase gene GapCp] and chloroplast DNA sequences. The results suggest that the nuclear genes of O. japonica and O. lancea are genetically differentiated despite shared polymorphism in their chloroplast DNA sequences. This discrepancy may be attributable to natural selection and recent introgression, although it is not evident if introgression occurs between O. japonica and O. lancea in the examined populations. Our findings of putative F2 hybrids in O. x intermedia support its partial reproducibility, and also suggest that formation of later-generation hybrids generates morphological variation in O. x intermedia. O. lancea plants collected from geographically distant localities were genetically very similar, and it is suggested that O. lancea originated monotopically.

Phylogeny and comparative seed morphology of epiphytic and terrestrial species of Liparis (Orchidaceae) in Japan.

To elucidate the evolution of epiphytes in Liparis section Liparis, we examined the phylogenetic relationships of 16 species by using internal transcribed spacer regions of 18S-26S nuclear ribosomal DNA (ITS) and three chloroplast DNA regions (trnS-trnG spacer, trnL with trnL-trnF spacer, and partial matK). Results showed that the epiphytic L. fujisanensis is sister to the terrestrial L. koreana and L. kumokiri, while another epiphyte, L. truncata, is sister to the terrestrial L. krameri. Therefore, the two epiphytic species evolved from terrestrial species independently in section Liparis. Comparative seed morphology revealed that the epiphytes have larger embryos than their closely related terrestrial counterparts. A similar trend toward the increase of embryo size in the two epiphytic species belonging to closely related, but distinct clades suggests that the large embryo may have an advantage in the epiphytic lifestyle. The two epiphytic species share another character state, smaller air spaces in the seed than that of closely related terrestrial species, suggesting possible low dispersibility of the epiphytes.