The mechanism underlying asymmetric bending of lateral petals in Delphinium (Ranunculaceae).

During the process of flower opening, most petals move downward in the direction of the pedicel (i.e., epinastic movement). In most Delphinium flowers, however, their two lateral petals display a very peculiar movement, the mirrored helical rotation, which requires the twist of the petal stalk. However, in some lineages, their lateral petals also exhibit asymmetric bending that increases the degree of mirrored helical rotation, facilitating the formation of a 3D final shape. Notably, petal asymmetric bending is a novel trait that has not been noticed yet, so its morphological nature, developmental process, and molecular mechanisms remain largely unknown. Here, by using D. anthriscifolium as a model, we determined that petal asymmetric bending was caused by the localized expansion of cell width, accompanied by the specialized array of cell wall nano-structure, on the adaxial epidermis. Digital gene analyses, gene expression, and functional studies revealed that a class I homeodomain-leucine zipper family transcription factor gene, DeanLATE MERISTEM IDENTITY1 (DeanLMI1), contributes to petal asymmetric bending; knockdown of it led to the formation of explanate 2D petals. Specifically, DeanLMI1 promotes cell expansion in width and influences the arrangement of cell wall nano-structure on the localized adaxial epidermis. These results not only provide a comprehensive portrait of petal asymmetric bending for the first time but also shed some new insights into the mechanisms of flower opening and helical movement in plants.

Diterpenoid alkaloids isolated from Delphinium trichophorum alleviate pulmonary fibrosis via the TGF-ß/Smad pathway in 3T6 and HFL-1 cells.

Delphinium trichophorum Franch (DTF), a species endemic to China, has been widely used for centuries in Tibet as an indigenous medicine for treating cough, pneumonia, and pulmonary fibrosis. Hetisine-type C20-diterpenoid alkaloids have been reported to be characteristic and active ingredients. Herein, five ones with relatively high contents in D. trichophorum, including 2α,11α,13ß-triacetylhetisine (DTF1), trichodelphinine A (DTF2), trichodelphinine D (DTF3), 2α-acetyl-11α,13ß-dihydroxyhetisine (DTF4), and trichodelphinine C (DTF5), were investigated for anti-fibrosis effects using fibroblasts induced by TGF-ß1 or LPS for the first time. The results showed that all five tested compounds decreased hydroxyproline (HYP) levels and inhibited the abnormal proliferation of 3T6 and HFL-1 cells induced by either TGF-ß1 or LPS. Moreover, DTF1 and DTF2 attenuated the production of collagen (Col-1 and Col-3) at relatively low doses, suggesting their higher efficiency among the five alkaloids. Based on large-scale ligand-based pharmacophore modeling, TGFBR1 was screened as a potential target for these tested alkaloids. The molecular docking results also exhibited high-affinity interactions between TGFBR1 and five alkaloids, especially DTF1 and DTF2. Further experiments revealed that DTF1 and DTF2 could inhibit the expression of TGF-ß1 and α-SMA and the phosphorylation of Smad3 and Smad4 while restoring the expression of Smad7 protein. Overall, DTF1 and DTF2 may reduce collagen generation and delay the development of pulmonary fibrosis by inhibiting the activation of the TGF-ß/Smad signaling pathway. Our results provide experimental and theoretical evidence for DTF1 and DTF2 as superior candidates for further development of anti-fibrotic drugs.

Diterpenoid Alkaloids from Delphinium anthriscifolium var. majus.

Extensive phytochemical investigation on the whole herbs of Delphinium anthriscifolium var. majus led to the identification of fourteen diterpenoid alkaloids, including three new C20-diterpenoid alkaloids (anthriscifolsines A-C, 1-3), six new C19-diterpenoid alkaloids (anthriscifolrines A-F, 4-9), and five know compounds (10-14). Among them, anthriscifolsine A represents a novel C20-diterpenoid alkaloid characterized by a seco C-ring. The structures of the isolated compounds were elucidated by extensive spectroscopic methods, including HR-ESI-MS, X-ray, and 1D and 2D NMR experiments. Bioactivity of compounds 3-6 was evaluated for their cytotoxicity against the MCF-7, HepG2 and H460 cancer cell lines.

Accumulation of mannitol in the cytoplasm and vacuole during the expansion of sepal cells associated with flower opening in Delphinium × belladonna cv. Bellamosum.

MAIN CONCLUSION: The role of mannitol differs from that of glucose, fructose and sucrose in sepal cell expansion associated with flower opening in Delphinium × belladonna. Sepals of Delphinium × belladonna are colored and much larger than the petals. To determine whether the role of mannitol in sepal growth associated with flower opening differs from those of ubiquitous metabolic sugars including glucose, fructose and sucrose, we investigated changes in cell number, subcellular concentrations of soluble carbohydrates, and osmotic potential in sepals during flower opening in Delphinium × belladonna cv. Bellamosum. The number of epidermal cells in the sepals did not increase from the stage when sepal pigmentation started, whereas the cell area increased during flower opening, indicating that petal growth during flower opening depends on cell expansion. Mannitol concentrations in the vacuole at three different stages were approximately 100 mM, which were much higher than the other carbohydrate concentrations, but they decreased slightly at open stage. In contrast, mannitol concentration in the cytoplasm was 56 mM at bud stage, but it increased to 104 mM at open stage. Glucose and fructose concentrations in the vacuole at open stage increased to 45 and 56 mM, respectively. Total osmotic potential in apoplast and symplast, which was partially due to soluble carbohydrates, was almost constant during flower opening. Therefore, mannitol may be acting constitutively as the main osmoticum in the vacuole where it may contribute to the maintenance of the osmotic balance between the cytoplasm and vacuole in open flowers. The role of mannitol differs from those of glucose, fructose, and sucrose in sepal cell expansion in Delphinium × belladonna.

Achievements and perspectives in biochemistry concerning anthocyanin modification for blue flower coloration.

Genetic engineering of roses and other plants of floricultural importance to give them a truly blue petal color is arguably one of the holy grails of plant biotechnology. Toward this goal, bluish carnations and roses were previously engineered by establishing an exclusive accumulation of delphinidin (Dp)-type anthocyanins in their petals via the heterologous expression of a flavonoid 3',5'-hydroxylase gene. Very recently, purple-blue varieties of chrysanthemums were also genetically engineered via a similar biochemical strategy. Although the floral colors of these transgenic plants still lack a true blue color, the basis for the future molecular breeding of truly blue flowers is via the engineering of anthocyanin pathways. Anthocyanins with multiple aromatic acyl groups (often referred to as polyacylated anthocyanins) in the 3'- or 7-position tend to display a more stable blue color than non-acylated anthocyanins. The 7-polyacylation process during the biosynthesis of purple-blue anthocyanins in delphinium (Delphinium grandiflorum) was found to occur in vacuoles using acyl-glucose as both the glucosyl and acyl donor. Glucosyltransferases and acyltransferases involved in anthocyanin 7-polyacylation in delphinium are vacuolar acyl-glucose-dependent enzymes belonging to the glycoside hydrolase family 1 and serine carboxypeptidae-like protein family, respectively. The 7-polyacylation proceeds through the alternate glucosylation and p-hydroxybenzoylation catalyzed by these enzymes. p-Hydroxybenzoyl-glucose serves as the p-hydroxybenzoyl and glucosyl donor to produce anthocyanins modified with a p-hydroxybenzoyl-glucose concatemer at the 7-position. This novel finding has provided a potential breakthrough for the genetic engineering of truly blue flowers, where polyacylated Dp-type anthocyanins are accumulated exclusively in the petals.

The role of acyl-glucose in anthocyanin modifications.

Higher plants can produce a wide variety of anthocyanin molecules through modification of the six common anthocyanin aglycons that they present. Thus, hydrophilic anthocyanin molecules can be formed and stabilized by glycosylation and acylation. Two types of glycosyltransferase (GT) and acyltransferase (AT) have been identified, namely cytoplasmic GT and AT and vacuolar GT and AT. Cytoplasmic GT and AT utilize UDP-sugar and acyl-CoA as donor molecules, respectively, whereas both vacuolar GT and AT use acyl-glucoses as donor molecules. In carnation plants, vacuolar GT uses aromatic acyl-glucoses as the glucose donor in vivo; independently, vacuolar AT uses malylglucose, an aliphatic acyl-glucose, as the acyl-donor. In delphinium and Arabidopsis, p-hydroxybenzoylglucose and sinapoylglucose are used in vivo as bi-functional donor molecules by vacuolar GT and AT, respectively. The evolution of these enzymes has allowed delphinium and Arabidopsis to utilize unique donor molecules for production of highly modified anthocyanins.

Effects of warming on chlorophyll degradation and carbohydrate accumulation of Alpine herbaceous species during plant senescence on the Tibetan Plateau.

Plant senescence is a critical life history process accompanied by chlorophyll degradation and has large implications for nutrient resorption and carbohydrate storage. Although photoperiod governs much of seasonal leaf senescence in many plant species, temperature has also been shown to modulate this process. Therefore, we hypothesized that climate warming would significantly impact the length of the plant growing season and ultimate productivity. To test this assumption, we measured the effects of simulated autumn climate warming paradigms on four native herbaceous species that represent distinct life forms of alpine meadow plants on the Tibetan Plateau. Conditions were simulated in open-top chambers (OTCs) and the effects on the degradation of chlorophyll, nitrogen (N) concentration in leaves and culms, total non-structural carbohydrate (TNC) in roots, growth and phenology were assessed during one year following treatment. The results showed that climate warming in autumn changed the senescence process only for perennials by slowing chlorophyll degradation at the beginning of senescence and accelerating it in the following phases. Warming also increased root TNC storage as a result of higher N concentrations retained in leaves; however, this effect was species dependent and did not alter the growing and flowering phenology in the following seasons. Our results indicated that autumn warming increases carbohydrate accumulation, not only by enhancing activities of photosynthetic enzymes (a mechanism proposed in previous studies), but also by affecting chlorophyll degradation and preferential allocation of resources to different plant compartments. The different responses to warming can be explained by inherently different growth and phenology patterns observed among the studied species. The results implied that warming leads to changes in the competitive balance among life forms, an effect that can subsequently shift vegetation distribution and species composition in communities.

Identification of the glucosyltransferase gene that supplies the p-hydroxybenzoyl-glucose for 7-polyacylation of anthocyanin in delphinium.

In delphiniums (Delphinium grandiflorum), blue flowers are produced by the presence of 7-polyacylated anthocyanins. The polyacyl moiety is composed of glucose and p-hydroxybenzoic acid (pHBA). The 7-polyacylation of anthocyanin has been shown to be catalysed by two different enzymes, a glucosyltransferase and an acyltransferase; both enzymes utilize p-hydroxybenzoyl-glucose (pHBG) as a bi-functional (Zwitter) donor. To date, however, the enzyme that synthesizes pHBG and the gene that encodes it have not been elucidated. Here, five delphinium cultivars were investigated and found to show reduced or undetectable 7-polyacylation activity; these cultivars synthesized delphinidin 3-O-rutinoside (Dp3R) to produce mauve sepals. One cultivar showed a deficiency for the acyl-glucose-dependent anthocyanin 7-O-glucosyltransferase (AA7GT) necessary for mediating the first step of 7-polyacylation. The other four cultivars showed both AA7GT activity and DgAA7GT expression; nevertheless, pHBG accumulation was significantly reduced compared with wild-type cultivars, whereas p-glucosyl-oxybenzoic acid (pGBA) was accumulated. Three candidate cDNAs encoding a UDP-glucose-dependent pHBA glucosyltransferase (pHBAGT) were identified. A phylogenetic analysis of DgpHBAGT amino acid sequences showed a close relationship with UGTs that act in acyl-glucose synthesis in other plant species. Recombinant DgpHBAGT protein synthesized pHBG and had a high preference for pHBA in vitro. Mutant cultivars accumulating pGBA had very low expression of DgpHBAGT, whereas expression during the development of sepals and tissues in a wild cultivar showed a close correlation to the level of accumulation of pHBG. These results support the conclusion that DgpHBAGT is responsible for in vivo synthesis of pHBG in delphiniums.

Plant toxins that affect nicotinic acetylcholine receptors: a review.

Plants produce a wide variety of chemical compounds termed secondary metabolites that are not involved in basic metabolism, photosynthesis, or reproduction. These compounds are used as flavors, fragrances, insecticides, dyes, hallucinogens, nutritional supplements, poisons, and pharmaceutical agents. However, in some cases these secondary metabolites found in poisonous plants perturb biological systems. Ingestion of toxins from poisonous plants by grazing livestock often results in large economic losses to the livestock industry. The chemical structures of these compounds are diverse and range from simple, low molecular weight toxins such as oxalate in halogeton to the highly complex norditerpene alkaloids in larkspurs. While the negative effects of plant toxins on people and the impact of plant toxins on livestock producers have been widely publicized, the diversity of these toxins and their potential as new pharmaceutical agents for the treatment of diseases in people and animals has also received widespread interest. Scientists are actively screening plants from all regions of the world for bioactivity and potential pharmaceuticals for the treatment or prevention of many diseases. In this review, we focus the discussion to those plant toxins extensively studied at the USDA Poisonous Plant Research Laboratory that affect the nicotinic acetylcholine receptors including species of Delphinium (Larkspurs), Lupinus (Lupines), Conium (poison hemlock), and Nicotiana (tobaccos).

Toxicokinetics of norditerpenoid alkaloids from low larkspur (Delphinium andersonii) orally administered to cattle.

OBJECTIVE: To determine the toxicokinetics of N-(methylsuccinimido)anthranoyllycoctonine-type low larkspur alkaloids in beef cattle. ANIMALS: 5 Black Angus steers and 35 Swiss Webster mice. PROCEDURES: -Low larkspur (Delphinium andersonii) was collected, dried, ground, and administered to 5 steers via oral gavage to provide a dose of 12 mg of N-(methylsuccinimido)-anthranoyllycoctonine alkaloids/kg. Steers were housed in metabolism crates for 96 hours following larkspur administration; heart rate was monitored continuously, and blood samples were collected periodically for analysis of serum concentrations of 16-deacetylgeyerline, methyllycaconitine, geyerline, and nudicauline and assessment of kinetic parameters. The LD(50) of a total alkaloid extract from D andersonii was determined in Swiss Webster mice. RESULTS: -The alkaloids were quickly absorbed, with a maximum serum concentration achieved within 18 hours after administration. Geyerline and nudicauline coeluted as 1 peak and were considered together for toxicokinetic analysis. Mean ± SD elimination half-life was 18.4 ± 4.4 hours, 15.6 ± 1.5 hours, and 16.5 ± 5.1 hours for 16-deacetylgeyerline, methyllycaconitine, and geyerline and nudicauline, respectively. There were significant differences in maximum serum concentration, amount absorbed, and distribution half-life among the 4 alkaloids. The mouse LD(50) was 9.8 mg/kg. CONCLUSIONS AND CLINICAL RELEVANCE: -Results suggested that clinical poisoning was likely to be most severe approximately 18 hours after exposure. Cattle should be closely monitored for at least 36 hours after initial exposure. Additionally, a withdrawal time of approximately 7 days would be required to clear > 99% of the toxic alkaloids from the serum of cattle that have ingested low larkspur.

Relationship between petal abscission and programmed cell death in Prunus yedoensis and Delphinium belladonna.

Depending on the species, the end of flower life span is characterized by petal wilting or by abscission of petals that are still fully turgid. Wilting at the end of petal life is due to programmed cell death (PCD). It is not known whether the abscission of turgid petals is preceded by PCD. We studied some parameters that indicate PCD: chromatin condensation, a decrease in nuclear diameter, DNA fragmentation, and DNA content per nucleus, using Prunus yedoensis and Delphinium belladonna which both show abscission of turgid petals at the end of floral life. No DNA degradation, no chromatin condensation, and no change in nuclear volume was observed in P. yedoensis petals, prior to abscission. In abscising D. belladonna petals, in contrast, considerable DNA degradation was found, chromatin was condensed and the nuclear volume considerably reduced. Following abscission, the nuclear area in both species drastically increased, and the chromatin became unevenly distributed. Similar chromatin changes were observed after dehydration (24 h at 60 degrees C) of petals severed at the time of flower opening, and in dehydrated petals of Ipomoea nil and Petunia hybrida, severed at the time of flower opening. In these flowers the petal life span is terminated by wilting rather than abscission. It is concluded that the abscission of turgid petals in D. belladonna was preceded by a number of PCD indicators, whereas no such evidence for PCD was found at the time of P. yedoensis petal abscission. Dehydration of the petal cells, after abscission, was associated with a remarkable nuclear morphology which was also found in younger petals subjected to dehydration. This nuclear morphology has apparently not been described previously, for any organism.

Reproductive and physiological responses to simulated climate warming for four subalpine species.

* The carbon costs of reproduction were examined in four subalpine herbaceous plant species for which number and size of flowers respond differently under a long-term infrared warming experiment. * Instantaneous measurements of gas exchange and an integrative model were used to calculate whole-plant carbon budgets and reproductive effort (RE). * Of the two species for which flowering was reduced, only one (Delphinium nuttallianum) exhibited higher RE under warming. The other species (Erythronium grandiflorum) flowers earlier when freezing events under warming treatment could have damaged floral buds. Of the two species for which flowering rates were not reduced, one (Helianthella quinquenervis) had higher RE, while RE was unaffected for the other (Erigeron speciosus). Each of these different responses was the result of a different combination of changes in organ size and physiological rates in each of the species. * Results show that the magnitude and direction of responses to warming differ greatly among species. Such results demonstrate the importance of examining multiple species to understand the complex interactions among physiological and reproductive responses to climate change.

Changes in flower coloration and sepal anthocyanins of Cyanic delphinium cultivars during flowering.

The changes in flower color related to sepal pigmentation of cyanic Delphinium cultivars were investigated during anthesis. The sepal hues of the purple and blue flowered varieties observed on the initial day of unfurling had changed with a decrease in hue angle three days after anthesis. In both the purple and blue cultivars, violdelphin (3) was the major component on day one of anthesis, and the chromaticity improved with increasing sepal concentrations of violdelphin (3) and cyanodelphin (4) after three days of unfurling. The flower hue was dominated by the constitution of acylated anthocyanins, and the chromaticity was ordered by the sepal concentration. The biosynthesis of cyanodelphin (4) from violdelphin (3) was postulated since an increase in the sepal concentration of cyanodelphin (4) was accompanied by a decrease in violdelphin (3). Acylation of the anthocyanins was initiated by an increase in the respective possible precursors, tulipanin (2) and violdelphin (3), to subsequently synthesize violdelphin (3) and cyanodelphin (4) during flowering.