Feeding potential of adult Systena frontalis (Coleoptera: Chrysomelidae) on leaves of Hydrangea paniculata (Cornales: Hydrangeaceae).

Systena frontalis (F.) (Coleoptera: Chrysomelidae), also known as the red-headed flea beetle, is a defoliating pest of a variety of crop systems, such as ornamentals and food crops. Leaf consumption by this beetle renders ornamental nursery plants, such as hydrangeas (Hydrangea paniculata Siebold, Hydrangeaceae), unsaleable. In Virginia, this insect has become a major pest at commercial nurseries, and their feeding potential on affected crops has not been quantified. In this study, the extent of their damage to individual leaves and host preference between leaf ages were determined. The rate of defoliation on mature and young hydrangea leaves was measured over 24 and 48 h and between different numbers of adults. A single adult caused up to 10% damage to a young leaf or 5% to a whole mature leaf in 24 h. Without choice, there was a higher percent damage to young leaves. When the size of leaves was controlled by cut-out mature leaves, the area damaged was still higher in young leaves when compared with mature leaves. Adult feeding between mature or young leaves was further investigated by choice assays on a caged plant and within a containerized system. In these choice assays, adults inflicted higher percent damage on mature leaves in both caged plant assays and containerized direct choice assays. The choice assays were more similar to field conditions than the nonchoice assays. This demonstrates that S. frontalis showed a preference for mature leaves over young leaves within hydrangeas.

Phylogenomics and historical biogeography of Hydrangeeae (Hydrangeaceae) elucidate the effects of geologic and climatic dynamics on diversification.

Demonstrating the process of transregional biogeography and mechanisms underlying evolutionary radiations is crucial to understanding biological evolution. Here, we use Hydrangeeae (Hydrangeaceae), a tribe with a unique disjunct distribution and complex trait variations, using a solid phylogenetic framework, to investigate how geographical and climatic factors interact with functional traits to trigger plant evolutionary radiations. We constructed the first highly supported and dated phylogenetic framework using 79 protein-coding genes obtained from 81 plastomes, representing 63 species and all major clades, and found that most extant species originated from asynchronous diversification of two lineages undergoing repeated expansion and retraction, at middle and high latitudes of the Northern Hemisphere between East Asia and North America, during the Eocene to Pleistocene (driven by geologic and climatic dynamics). In accordance with these drivers, interactions of flora between central-eastern China and Japan occurred frequently after the Late Tertiary. We found that resource limitation and range fragmentation probably accelerated the diversification of Hydrangeeae, which supports the resource-use hypothesis. Our study sheds light on the evolutionary radiation and assembly of flora within East Asia, and the East Asian-North American disjunction, through integration of phylogenomic and biogeographic data with functional trait and ecological data.

From dark to darkness, negative phototropism influences the support-tree location of the massive woody climber Hydrangea serratifolia (Hydrangeaceae) in a Chilean temperate rainforest.

Climbing plants rely on suitable support to provide the light conditions they require in the canopy. Negative phototropism is a directional search behavior proposed to detect a support-tree, which indicates growth or movement away from light, based on light attenuation. In a Chilean temperate rainforest, we addressed whether the massive woody climber Hydrangea serratifolia (H. et A.) F. Phil (Hydrangeaceae) presents a support-tree location pattern influenced by light availability. We analyzed direction and light received in two groups of juvenile shoots: searching shoots (SS), with plagiotropic (creeping) growth vs. ascending shoots (AS), with orthotropic growth. We found that, in accordance with light attenuation, SS and AS used directional orientation to search and then ascend host trees. The light available to H. serratifolia searching shoots was less than that of the general forest understory; the directional orientation in both groups showed a significant deviation from a random distribution, with no circular statistical difference between them. Circular-linear regression indicated a relationship between directional orientations and light availability. Negative phototropism encodes the light environment's heterogeneous spatial and temporal information, guiding the shoot apex to the most shaded part of the support-tree base, the climbing start point.

Hydrangenol inhibits lipopolysaccharide-induced nitric oxide production in BV2 microglial cells by suppressing the NF-κB pathway and activating the Nrf2-mediated HO-1 pathway.

We previously demonstrated the anti-inflammatory effect of water extract of Hydrangea macrophylla in lipopolysaccharide (LPS)-stimulated macrophage cells. Here, we investigated whether hydrangenol, a bioactive component of H. macrophylla, attenuates the expression of nitric oxide (NO) and its associated gene, inducible NO synthase (iNOS), in LPS-stimulated BV2 microglial cells. Our data showed that low dosages of hydrangenol inhibited LPS-stimulated NO release and iNOS expression without any accompanying cytotoxicity. Hydrangenol also suppressed LPS-induced nuclear translocation of nuclear factor-κB (NF-κB) subunits, consequently inhibiting DNA-binding activity of NF-κB. Additionally, the NF-κB inhibitors, pyrrolidine dithiocarbamate (PDTC) and PS-1145, significantly attenuated LPS-induced iNOS expression, indicating that hydrangenol-induced NF-κB inhibition might be a key regulator of iNOS expression. Furthermore, our data showed that hydrangenol suppresses NO production by inducing heme oxygenase-1 (HO-1). The presence of cobalt protoporphyrin, a specific HO-1 inducer, potently suppressed LPS-induced NO production. Hydrangenol also promoted nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and subsequently increased its binding activity at the specific antioxidant response element sites. Additionally, transient knockdown of Nrf2 significantly downregulated hydrangenol-induced HO-1 expression, indicating that hydrangenol-induced Nrf2 is an upstream regulator of HO-1. Taken together, these data suggest that hydrangenol attenuates NO production and iNOS expression in LPS-stimulated BV2 microglial cells by inhibiting NF-κB activation and by stimulating the Nrf2-mediated HO-1 signaling pathway. Therefore, hydrangenol is a promising therapeutic agent for treatment of LPS-mediated inflammatory diseases.

Systematics, biogeography, and character evolution of Deutzia (Hydrangeaceae) inferred from nuclear and chloroplast DNA sequences.

The genus Deutzia (Hydrangeaceae), containing ca. 60 species circumscribed in three sections, is disjunctly distributed in eastern Asia and Central America (Mexico). Although the genus is well delimited, its subdivisions into sections and series have not been the subject of an explicit test of monophyly based on molecular data. A comprehensive examination of the evolutionary relationships within the genus is thus still lacking. We present a fossil-calibrated, molecular phylogeny of Deutzia based on two nuclear ribosomal DNA (ITS and 26S) and three chloroplast DNA regions (matK, rbcL, and trnL-F intergenic spacer). Within this framework, we examine character evolution in petal arrangement, filament shape, and the number of stamens, and infer the ancestral area and biogeographic history of the genus. Our molecular phylogeny suggests that Deutzia is monophyletic. Two major clades are recovered: one composed of the species of sect. Neodeutzia from Mexico, and the other containing all remaining Deutzia species of sections Mesodeutzia and Deutzia from SW China and Northeast Asia. The latter two Asian sections were each revealed to be polyphyletic. The induplicate petals, 2-dentate filaments, and polystemonous androecia are inferred to be ancestral character states. Biogeographic reconstructions suggest a Northeast Asian origin for the genus and subsequent spread to Mexico during the Oligocene and to SW China during the Miocene. Based on our results, a new infrageneric classification of Deutzia inferred from molecular phylogeny is required. We propose to merge sections Mesodeutzia and Deutzia to ensure the monophyly at the sectional level. Cooling trends during the Oligocene resulted in isolation, separating eastern Asian and Mexican taxa, while the warm period during the middle Miocene stimulated the diversification from Northeast Asia to SW China. The uplift in the Qinghai-Tibetan Plateau and monsoon regimes are important in promoting high species diversification of Deutzia in SW China.

A strong 'filter' effect of the East China Sea land bridge for East Asia's temperate plant species: inferences from molecular phylogeography and ecological niche modelling of Platycrater arguta (Hydrangeaceae).

BACKGROUND: In East Asia, an increasing number of studies on temperate forest tree species find evidence for migration and gene exchange across the East China Sea (ECS) land bridge up until the last glacial maximum (LGM). However, it is less clear when and how lineages diverged in this region, whether in full isolation or in the face of post-divergence gene flow. Here, we investigate the effects of Quaternary changes in climate and sea level on the evolutionary and demographic history of Platycrater arguta, a rare temperate understorey shrub with disjunct distributions in East China (var. sinensis) and South Japan (var. arguta). Molecular data were obtained from 14 P. arguta populations to infer current patterns of molecular structure and diversity in relation to past (Last Interglacial and Last Glacial Maximum) and present distributions based on ecological niche modelling (ENM). A coalescent-based isolation-with-migration (IM) model was used to estimate lineage divergence times and population demographic parameters. RESULTS: Combining information from nuclear/chloroplast sequence data with nuclear microsatellites, our IM analyses identify the two varieties as genetically distinct units that evolved in strict allopatry since the mid-Pleistocene, c. 0.89 (0.51-1.2) Ma. Together with Bayesian Skyeline Plots, our data further suggest that both lineages experienced post-divergence demographic growth, followed by refugial isolation, divergence, and in the case of var. arguta post-glacial admixture. However, past species distribution modelling indicates that the species' overall distribution has not greatly changed over the last glacial cycles. CONCLUSIONS: Our findings highlight the important influence of ancient sea-level changes on the diversification of East Asia's temperate flora. Implicitly, they challenge the notion of general temperate forest expansion across the ECS land bridge, demonstrating instead its 'filter' effect owing to an unsuitable environment for certain species and their biological (e.g., recruitment) properties.

Understanding population structure and historical demography in a conservation context: population genetics of the endangered Kirengeshoma palmata (Hydrangeaceae).

PREMISE OF THE STUDY: Both historical and contemporary microevolutionary processes greatly influence the genetic patterns of East Asian plant endemics, but the spatial and temporal contexts of these processes remain poorly understood. Here, we investigate the relative influences of historical and contemporary gene flow and drift on the population genetic structure of Kirengeshoma palmata, a perennial herb from East China and South Japan. METHODS: We used data from nine polymorphic microsatellite loci to assess the levels of genetic diversity, effective population size, and contemporary and historical gene flow for six of the seven known populations. KEY RESULTS: We found high levels of inbreeding and allelic diversity within populations. Both contemporary and historical migration rates among populations were low, and a test of alternate models of population history strongly favored a model of long-term drift-migration equilibrium. We inferred declines in population size ca. 10,000-100,000 yr ago, but failed to detect recent declines. Bayesian clustering divided K. palmata populations into three genetic clusters, two of which were consistent with a glacial refugium hypothesis for two mountain ranges in East China. CONCLUSIONS: These results suggest that anthropogenic fragmentation has had little effect on the genetic characteristics of Chinese K. palmata. Rather, past decline in population size due to Late Pleistocene climate change as well as restricted pollen and seed dispersal may have contributed to low levels of both historical and contemporary gene flow, resulting in high genetic differentiation between adjacent mountain ranges due to genetic drift and inbreeding.

Application of the phylogenetic informativeness method to chloroplast markers: a test case of closely related species in tribe Hydrangeeae (Hydrangeaceae).

In evolutionary biology appropriate marker selection for the reconstruction of solid phylogenetic hypotheses is fundamental. One of the most challenging tasks addresses the appropriate choice of genomic regions in studies of closely related species. Robust phylogenetic frameworks are central to studies dealing with questions ranging from evolutionary and conservation biology, biogeography to plant breeding. Phylogenetic informativeness profiles provide a quantitative measure of the phylogenetic signal in markers and therefore a method for locus prioritization. The present work profiles phylogenetic informativeness of mostly non-coding chloroplast regions in an angiosperm lineage of closely related species: the popular ornamental tribe Hydrangeeae (Hydrangeaceae, Cornales, Asterids). A recent phylogenetic study denoted a case of resolution contrast between the two strongly supported clades within tribe Hydrangeeae. We evaluate the phylogenetic signal of 13 highly variable plastid markers for estimating relationships within and among the currently recognized monophyletic groups of this tribe. A selection of combined loci based on their phylogenetic informativeness retrieved more robust phylogenetic hypotheses than simply combining individual markers performing best with respect to resolution, nodal support and accuracy or those presenting the highest number of parsimony informative characters. We propose the rpl32-ndhF intergenic spacer (IGS), trnV-ndhC IGS, trnL-rpl32 IGS, psbT-petB region and ndhA intron as the best candidates for future phylogenetic studies in Hydrangeeae and potentially in other Asterids. We also contrasted the phylogenetic informativeness of coded indels against substitutions concluding that, despite their low phylogenetic informativeness, coded indels provide additional phylogenetic signal that is nearly free of noise. Phylogenetic relationships obtained from our total combined analyses showed improved resolution and nodal support with respect to recently published results.

Anticoccidial activity of traditional Chinese herbal Dichroa febrifuga Lour. extract against Eimeria tenella infection in chickens.

The study was conducted on broiler birds to evaluate the anticoccidial efficacy of an extract of Chinese traditional herb Dichroa febrifuga Lour. One hundred broiler birds were assigned to five equal groups. All birds in groups 1-4 were orally infected with 1.5 × 10(4) Eimeira tenella sporulated oocysts and birds in groups 1, 2 and 3 were medicated with 20, 40 mg extract/kg feed and 2 mg diclazuril/kg feed, respectively. The bloody diarrhea, oocyst counts, intestinal lesion scores, and the body weight were recorded to evaluate the anticoccidial efficacy. The results showed that D. febrifuga extract was effective against Eimeria infection; especially 20 mg D. febrifuga extract/kg feed can significantly increase body weight gains and reduce bloody diarrhea, lesion score, and oocyst excretion in comparison to infected-unmedicated control group.

Development of 12 microsatellite markers for Platycrater arguta (Hydrangeaceae) endemic to East Asia.

PREMISE OF THE STUDY: Microsatellite markers were characterized in Platycrater arguta, a rare endemic shrub from eastern China and southern Japan, to investigate its population genetic structure, phylogeographic patterns, and breeding system. METHODS AND RESULTS: Using both the Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) protocol and the compound microsatellite marker technique, we developed 12 microsatellite markers. All primers showed polymorphism when assessed in 43 individuals from two populations in eastern China and southern Japan. Overall, the number of alleles ranged from five to 21, with an average of 14.91 alleles per locus. CONCLUSIONS: These markers can be used in further studies on population genetics, phylogeography, and breeding system of P. arguta.

The effects of Aphis fabae infestation on the antioxidant response and heavy metal content in field grown Philadelphus coronarius plants.

The purpose of this study was to explore a possible relationship between the soil availability of metals and their concentrations in various parts of Philadelphuscoronarius plants. Moreover, the possible impact of an aphid infestation on the contamination and antioxidant response of plants from the urban environment of Kraków and the reference rural area of Zagaje Stradowskie (southern Poland) was analyzed. The contents of the glutathione, proline, non-protein -SH groups, antioxidants, and phosphorous and the levels of guaiacol peroxidase and catalase activity in leaves and shoots either infested or not by the aphid Aphis fabae Scop., were measured. The potential bioavailability of metals (Cd; Cu; Ni; Pb; Zn) in the soil and their concentrations in P. coronarius plants originating from both sites were compared. The antioxidant responses were generally elevated in the plants in the polluted area. Such reactions were additionally changed by aphid infestation. Generally, the concentrations of metals in the HNO(3) and CaCl(2) extractants of the soils from two layers at the 0-20 and 20-40 cm depths from the polluted area were higher than in those from the reference area. Such differences were found for nickel and lead (in all examined extractants), zinc (in soil extractants from the layer at 20-40 cm) and cadmium (in HNO(3) extractants). Significant positive relationships between the lead concentrations in the soil and in the plants were found. In the parts of plants from the polluted area, higher concentrations of Pb and Zn (leaves and shoots) and Cd (shoots) were recorded. The shoots and leaves of plants infested with aphids had higher concentrations of Zn but lower Pb. Moreover, their leaves had higher contaminations of Cu and Ni. In conclusion, aphids affected not only the antioxidant response of the plants but also their contamination with metals, especially contamination of the leaves.

Dichroa febrifuga Lour. inhibits the production of IL-1beta and IL-6 through blocking NF-kappaB, MAPK and Akt activation in macrophages.

AIM OF THE STUDY: The roots of Dichroa febrifuga Lour. have been used as a traditional antimalarial drug and also used in the treatment of productive cough and unstable fever caused by infection in China and Korea. In this study, we evaluated the anti-inflammatory effect and underlying molecular mechanism of aqueous extract of Dichroa febrifuga (AEDF) in C57BL/6 mouse peritoneal macrophages. MATERIALS AND METHODS: The effect of AEDF on proinflammatory cytokine (IL-1beta and IL-6) production was analyzed by ELISA and real-time RT-PCR. The effects of AEDF on NF-kappaB/IkappaB-alpha/IKK were measured by reporter assay (in RAW 264.7 cells), EMSA, Western blotting and kinase assay. The effects of AEDF on Akt and MAPKs activity were assayed by Western blotting. RESULTS: AEDF inhibited the production of IL-1beta and IL-6, NF-kappaB activation, IkappaB-alpha degradation, and IKK, Akt, ERK1/2 and JNK activities in LPS-stimulated mouse peritoneal macrophages. CONCLUSIONS: These results suggest that AEDF inhibits proinflammatory cytokine (IL-1beta and IL-6) production in LPS-stimulated mouse peritoneal macrophages, and that these effects are mediated by the inhibition of the activity of IKK/IkappaB/NF-kappaB and the phosphorylation of Akt, ERK1/2, and JNK. Our results provide a molecular basis for understanding the inhibitory effects of Dichroa febrifuga roots on endotoxin-mediated inflammation.

Molecular phylogeography of East Asian Kirengeshoma (Hydrangeaceae) in relation to quaternary climate change and landbridge configurations.

Kirengeshoma comprises two species inhabiting warm temperate-deciduous forests in East China/South Japan (Kirengeshoma palmata) and South Korea (Kirengeshoma koreana). A survey of chloroplast (cp) DNA and inter-simple sequence repeats (ISSRs) variation in Kirengeshoma was carried out to determine the population history of a plant taxon around the East China Sea (ECS). CpDNA and ISSRs revealed lower genetic divergence between China and Japan relative to the other contrasts, in line with intrageneric classification. Molecular dating suggests that K. koreana diverged at the Plio-Pleistocene boundary from the Japanese populations, whereas the latter migrated into China during the early-to-mid Pleistocene via the ECS basin. Vicariant segregation of Chinese and Japanese populations likely occurred during the mid-Pleistocene. Mismatch distributions and neutrality tests indicated that Chinese populations expanded their range during the Late Pleistocene, probably during a cold period, whereas those from Japan showed no significant population growth. We conclude that the current distribution and differentiation of components of presently isolated warm temperate-deciduous forests in China, Japan and Korea likely resulted from a combination of relatively ancient vicariant and immigration events, and those from Japan were particularly sensitive to range fragmentation and long-term refugial isolation throughout the Late Pleistocene.

Synthesis and biological evaluation of febrifugine analogues as potential antimalarial agents.

Febrifugine is an alkaloid isolated from Dichroa febrifuga Lour as the active component against Plasmodium falciparum. Adverse side effects have precluded febrifugine as a potential clinical drug. In this study novel febrifugine analogues were designed and synthesized. Lower toxicity was achieved by reducing or eliminating the tendency of forming chemically reactive and toxic intermediates and metabolites. Synthesized compounds were evaluated for acute toxicity and in vitro and in vivo antimalarial efficacy. Some compounds are much less toxic than the natural product febrifugine and existing antimalarial drug chloroquine and are expected to possess wide therapeutic windows. These compounds, as well as the underlying design rationale, may find usefulness in the discovery and development of new antimalarial drugs.

[Fluorescence spectra of Dichroa febrifuga aqueous extraction].

Fluorescence spectra of chang shan (Dichroa febrifuga Lour) aqueous extraction were studied. In the three-dimensional fluorescence contour spectrum, three fluorescence peaks of quinazoline alkaloids, which are the active components of chang shan, were observed. The excitation wavelengths of the peaks were 235, 270 and 320 nm, respectively, and the emission wavelength of all the peaks was 430 nm. Three-dimensional fluorescence contour spectrum is the very image of fingerprint, suitable for qualitative identification of traditional Chinese medicine. In the range of pH 3 to pH 6, the fluorescence spectrum of chang shan aqueous extractions changes with the variation in pH value. The reason for this spectral change might be the protonation of N-1 in quinazolone ring of beta-dichroine (febrifugine) molecule. There is an excellent linear relationship between the fluorescence intensity and the concentration of chang shan under nearly neutral conditions, thereby a quantitative method for the determination of quinazoline alkaloids may be established.

Cytotoxicity of water extracts from leaves and branches of Philadelphus coronarius L.

Philadelphus coronarius L. is big, leggy and deciduous old-fashioned shrub known for its fragrant white flowers in the late spring. Some members of genus Philadelphus L. are known for their antibacterial, antiradical and immunomodulatory effects. Therefore, these herbs represent prospective sources for the isolation of active substances with desired effects. We have investigated the cytotoxicity effects of water extracts from leaves and branches of Philadelphus coronarius L. (Hydrangeaceae). A431 cells (human skin carcinoma cell line) and the human breast adenocarcinoma cell line (MCF-7) were treated with various doses of individual extracts (0,1-100 microg dry matter/ml) for 24 h and 72 h. The highest toxic effects of both plant parts extracts were observed on MCF-7 cells regardless the time of treatment. Cells A431 were less sensitive to toxic effects of leaves and branches extracts but the time dependence was present with the tendency of increased toxicity after chronic treatment. There were no differences in the extent of toxic effects between branches and leaves extracts. The results obtained so far will provide the basis for the future studies with isolated active substances from these extracts.

Phylogeography of the genus Cardiandra based on genetic variation in cpDNA sequences.

We investigated the phylogenetic relationships within the genus Cardiandra based on plastid DNA sequences. The phylogenetic tree showed that Cardiandra populations from the Ryukyu Islands (Japan) and Taiwan were monophyletic (Ryukyu-Taiwan clade), whereas taxa from China and mainland Japan were sisters to this clade. The divergence time between the Ryukyu-Taiwan clade and the other species was estimated to be 0.082 MYA, i.e., the late Pleistocene. The infrageneric and/or infraspecific differentiation of Cardiandra is estimated to have depended largely on allopatric differentiation caused by the presence or division of the past landbridge of the Ryukyu Islands, which connected mainland Japan to the Asian Continent during the Quaternary.

Suppression of ethanol and lipopolysaccharide-induced liver injury by extracts of Hydrangeae Dulcis Folium in rats.

In female SD rats that were injected with 4 g/kg BW ethanol p.o. followed by a 5 mg/kg BW lipopolysaccharide (LPS) i.v. injection, serum glutamic pyruvic transaminases (GPT) activity increased to about eight times that of normal rats. In this model, rats that had been fed a diet containing 1% Hydrangeae Dulcis Folium (HDF) extracts for fifteen days showed significantly lower serum GPT activity (380.0+/-58.2 IU/l) than the control group (3527.0+/-774.1 IU/l). HDF's efficacy was far superior to milk thistle in this model (2950.0+/-915.9 IU/l). When mouse macrophages were treated with HDF extracts at 50 microg/ml, TNF-alpha production induced by LPS was suppressed to about 10% of the control. Rat serum TNF-alpha levels induced by LPS was decreased to 58.7% of the control by administering 1000 mg/kg BW HDF extract p.o. These results indicate that HDF prevents alcohol-induced liver injury through the inhibition of TNF-alpha production.

[Reactive oxygen forms and luminescence of intact microspore cells]. / Aktivnye formy kisloroda i liuminestsentsiia intaktnykh kletok mikrospor.

The participation of reactive oxygen species (ROS) in luminescence (chemiluminescence and autofluorescence induced by ultraviolet light of 360-380 nm) was analyzed. Microspores, the pollen (male gametophyte) of Hippeastrum hybridum, Philadelphus grandiflorus, and Betula verrucosa and vegetative microspores of the spore-breeding plant Equisetum arvense served as models. It was found that the addition of the chemiluminescent probe lucigenin, which luminesces in the presence of superoxide anionradicals, leads to intensive chemiluminescence of microspores. No emission was observed in the absence of lucigenin and in the presence of the dye luminol as a chemiluminescent probe. The emission decreased significantly if superoxide dismutase, an enzyme of the superoxide anionradical dismutation during which this radical disappeared, was added before the dye addition. The autofluorescence intensity of microspores decreased in the presence of both superoxide dismutase and peroxidase, an enzyme destroying hydrogen peroxide and organic peroxides. The most significant effect was noted after the addition of peroxidase, which indicates a greater contribution of peroxides to this type of emission. The fumigation with ozone, which increases the amount of ROS on the cell surface, enhanced the intensity of the chemiluminescence of microspores with lucigenin, but decreased the intensity of the autofluorescence of microspores. Exogenous peroxides (hydrogen peroxide and tert-butylhydroperoxide) stimulated the autofluorescence of pollen and vegetative spores in a concentration-dependent manner. It was shown that the formation of ROS contributes to the luminescence of plant microspores, which reflects their functional state.

Purification and characterization of an ethylene-induced antifungal protein from leaves of guilder rose (Hydrangea macrophylla).

An ethylene-induced, chitin-binding protein (designated as HM30) from leaves of Hydrangea macrophylla was identified and purified to apparent homogeneity by chitin affinity chromatography followed by FPLC on a Superose 12 column. The molecular mass of HM30 was 30,010.0 Da determined by mass spectrometry and its isoelectric point of 8.4 was estimated by isoelectric focusing. The amino acid composition of HM30 was also determined. The initial 15 amino acid residues of the N-terminal were found to be N-S-M-E-R-V-E-E-L-R-K-K-L-Q-D by automatic Edman degradation. This chitin-binding protein showed antifungal activity toward several crop fungal pathogens. Knowledge of properties of HM30 should be useful for its potential application as a plant fungicidal agent.