Hydration, sporoderm breaking and germination of Cupressus arizonica pollen

I n vitro and in vivo rehydration and germination in Cupressus arizonica pollen were examined using light and scanning electron microscopy. Shed pollen has 12.6% water content, which reduced to 8.2% after dispersal, and this latter pollen survived for some months at room temperature and for years at −10 °C. Rehydration requires breaking of the sporoderm walls and depends on the composition and pH of the rehydration medium. Acidity restrains the breakage, while alkalinity promotes it. Pollen division follows exine shedding and requires the persistence of the mucilaginous layer; hence, pH values countering these outcomes prevent division. Division results in a large and a small cell separated by a callosic wall. A pollen tube develops from the innermost intine of the large cell, which is callosic, and extends into the mucilaginous middle intine. The percentage germination never exceeded 17% in all tested media. In vivo , pollen rehydrates and casts off the exine in the micropylar drop. Drop withdrawal brings pollen to the apical nucellar cells that degenerate in the meantime, and it leaves a deposit on the surface of the micropylar canal. After contaction of the nucellar cells, the pollen flattens and its mucilaginous layer shrinks and disappears. This occurs simultaneously with sealing of the micropylar canal. During this time, pollen divides asymmetrically without the callosic wall, and the larger cell develops a tube in the interface with the nucellus. Only some pollen grains accomplish adhesion to the nucellus and germinate. The in vitro and in vivo developmental stages are discussed.     

Chemical Composition and Biological Activities of Tunisian Cupressus arizonica Greene Essential Oils

The chemical composition of the essential oils obtained by hydrodistillation of leaves, stems, and female cones of Cupressus arizonica Greene , grown in Tunisia, was studied by GC‐FID and GC/MS analyses. Altogether, 62 compounds were identified, 62 in the leaf oil, 19 in the cone oil, and 24 in the stem oil. The cone and stem oils were mainly composed by monoterpene hydrocarbons (96.6 and 85.2%, resp.). In the leaf oil, the total sesquiterpene fraction constituted 36.1% and that of the monoterpene hydrocarbons 33.8% of the total oil composition. The three oils were evaluated for their in vitro herbicidal activity by determining their influence on the germination and the shoot and root growth of the four weed species Sinapis arvensis L., Lolium rigidum Gaudin , Trifolium campestre Schreb ., and Phalaris canariensis L. At the highest doses tested (0.8 and 1.0 mg/ml), the leaf essential oil inhibited either totally or almost completely the seed germination and the shoot and root growth of S. arvensis and T. campestre. The oils were also tested for their antifungal activity; however, their effects on the fungal growth were statistically not significant.     

Pollen production of the genus Cupressus

The study of potential pollen emission in the genus Cupressus was carried out in the city of Córdoba (Spain). Pollen production of 227 trees of the represented species of Cupressus in the area (Cupressus sempervirens, C. macrocarpa and C. arizonica) was studied by counting the number of pollen grains per flower, the number of flowers per branch, and the number of branches per m² of surface area. Total surface area was estimated by considering the shape of the tree as a cone and estimating the average height and radius of each species. Results indicated that there was little variation in the number of pollen grains produced by a flower within each species. Equally, there were few differences in the number of grains per flower among the species, although C. arizonica had slightly fewer than the other species because it possessed fewer stamens per flower. However, there were clear differences in the number of flowers per branch, with C. macrocarpa having the highest floral density, considerably lower in C. arizonica and even lower in C. sempervirens. All of this indicates that the pollen contribution of C. macrocarpa to the atmosphere is far greater than that of C. arizonica and C. sempervirens. The results indicate a different potential emission in each species, and this must be taken into account when determining the quantity of pollen present in the atmosphere. The maximum amount of pollen grains that one individual can spread is provided in this study and would provide useful data in the development of an airborne forecasting system.     

Adventitous bud induction in vitro from juvenile leaves of Cupressus arizonica Green

Juvenile leaves of Cupressus arizonica Green (3–5 mm in length) from eight week old seedlings, were cultured on liquid medium supplemented with isopentenyladenine (2 mgl^-1). Buds formed from the explants after three weeks of culture, but further growth occurred only after transfer to half-strength medium without plant growth regulators. Histological analysis at different times of culture, showed an early mitotic activity within transfusion tissue, followed by dedifferentiation of epidermal and mesophyll parenchyma cells at the basal zone of the leaves. The differentiation of vascular nodules always preceded bud formation. The difficulty of conifers to root and grow beyond plantlet stage is discussed.     

Identification of Castasterone, 6-Deoxocastasterone, Typhasterol and 6-Deoxotyphasterol from the Shoots of Arabidopsis thaliana

Endogenous brassinosteroids in the shoots of Arabidopsis thalianawere investigated. Castasterone, 6-deoxocastasterone, typhasteroland 6-deoxotyphasterol were identified by GC-MS. The co-occurrenceof 6-deoxo-brassinosteroids and 6-oxo-brassinosteroids suggeststhat there are both early and late C6-oxidation pathways ofbrassinosteroids in A. thaliana. (Received September 17, 1996; Accepted October 11, 1996)     

Refolding of the Cupressus arizonica major pollen allergen Cup a1.02 overexpressed in Escherichia coli

The cDNA encoding an isoform of the cypress major pollen allergen, Cup a1.02, has been cloned and expressed in Escherichia coli as a N-terminal 6x His-tagged protein. To increase recovery, Cup a1.02 was expressed at high levels exploiting the T5 strong promoter and led to accumulate as inclusion bodies. The insoluble purified aggregates were solubilized in 6 M guanidine hydrochloride, immobilized using nickel-chelating affinity chromatography, and successfully refolded by controlled removal of the chaotropic reagent. Enhanced protein refolding was observed by reducing the protein concentration at 0.6-0.8 mg/ml. SDS-PAGE and gel filtration chromatography indicated an apparent molecular mass of approximately 40 kDa and the occurrence of the protein as monomers. The reconstituted fusion protein displayed the same immunological properties of the native Cup a1.02 protein as proven by IgE immunoreactivity. Immunoblotting, ELISA, and histamine release test showed that the tag did not preclude the protein functionality hence validating its correct three-dimensional folding. The protein fold was also assessed by CD spectroscopy and deconvolution of the spectrum allowed to estimate the secondary structure as a prevalence of beta structures (higher than 60%) and a small contribution from alpha helices (less than 12%). The reported procedure has proven to be useful for the production of multi-milligrams of recombinant Cup a1.02 allergen suitable for structural biology studies and for the molecular and functional characterization of the IgE binding sites.     

Effects of AMO-1618 and B-995 on Growth and Endogenous Gibberellin Content of Cupressus arizonica seedlings

Soil drenches of 250, 500 or 1000 mg/l of the growth retardants AMO-1618 or B-995 effectively reduced dry matter production and stem elongation in young seedlings of Cupressus arizonica Greene. In seedlings treated with AMO-1618, the acidic, ethyl acetate-soluble gibberellin-like substances (GAs), as detected. by bioassay, were reduced to almost undetectable levels. However, the endogenous GA content in seedlings treated with B-995 were at least 11-fold greater than in control seedlings and differed as well in chromatographic characteristics, being of a more polar nature than the endogenous GAs of control seedlings. It was concluded that while AMO-1618 probably acts through interference with GA biosynthesis, B-995 may act through the interconversion of GAs.     

Envelope Permeability to Possible Precursors of Carotenoid Biosynthesis during Chloroplast-Chromoplast Transformation

During the transformation of chloroplasts to chromoplasts in Capsicum annuum L., the permeability of the envelope membranes to possible precursors of carotenoid biosynthesis (acetate, mevalonate, citrate) was tested. The plastids were isolated by gel filtration, and the uptake of labeled compounds was measured by a filtering centrifugation technique, using silicone oil. The different ripening stages were characterized by the ratio of chlorophyll to carotenoid content. The chloroplast membranes were shown to be impermeable to all of the metabolites tested. During the transformation there was a sharp increase of membrane permeability. In the mature chromoplast, the permeability to mevalonate and acetate again decreased to about 20% of the maximum value and reached zero for citrate. The results give evidence that during the transformation of chloroplasts to chromoplasts, precursors for carotenoid biosynthesis are translocated from extraplastidic sites into the plastids, there being possibly incorporated into carotenoids.     

Evaluation of Anticipated Performance Index,biochemical, and physiological parameters of Cupressus arizonica Greene and Juniperus excelsa Bieb for greenbelt development and biomonitoring of air pollution

Abstract

Plants can be used naturally to mitigate environmental pollution, such as air pollution; however, it is important to evaluate plant susceptibility to air pollution when considering green space creation in urban areas. In this study, we investigated the biochemical and physiological parameters, including pH, and ascorbic acid, relative water, total chlorophyl, proline, soluble sugar, protein, and free amino acid contents of two gymnosperm tree species, Cupressus arizonica Greene and Juniperus excelsa Bieb. Using these data, we computed the Air Pollution Tolerance Index (APTI) and Anticipated Performance Index (API) of the trees at five sites in Isfahan, Iran, to determine their applicability for greenbelt establishment. Our results indicated that these evergreen species are good indicators of air pollution and can be used as an early warning tool for air pollution level that is harmful to human health.     

Quantitative analyses of the genetic structure of closely related conifers with disparate distributions and demographics: the Cupressus arizonica (Cupressaceae) complex

Seedling populations of seven Cupressus taxa were compared in common gardens to assess relationships among taxa and to elucidate the genetic structure of three taxa with different distributions and demographics. Discriminant analyses of 15 quantitative traits separated C. sargentii and C. forbesii from taxa within the C. arizonica complex (C. arizonica, C. glabra, C. stephensonii, C. nevadensis, and C. montana); aligned a population of C. arizonica from Sierra de Arteaga, Mexico, with families of the same taxon from southwestern United States; and described a close relationship between C. nevadensis and C. glabra. but nevertheless separated taxa within the C. arizonica complex. Analyses of wind-pollinated progenies of single trees showed the narrow endemic, C. glabra, to have a greater degree of population differentiation than the more broadly dispersed C. arizonica. In addition, genetic variances within the numerically small populations of C. arizonica were about the same as in the much larger populations of C. glabra. The results also suggested that genetic variability in C. stephensonii, a taxon occurring in a single population, may occur primarily among inbred lines. While the genetic structures of C. arizonica and C. glabra have counterparts among broadly dispersed conifers, that of C. stephensonii appears to be unique. Together, the results support the view that genetic structure primarily reflects the uncertain, chance events that are interspersed throughout evolutionary history.     

Fomitiporia cupressicola sp. nov., a parasite on Cupressus arizonica, and additional unnamed clades in the southern USA and northern Mexico, determined by multilocus phylogenetic analyses

Fomitiporia cupressicola sp. nov., found in living Cupressus arizonica, is described on the basis of several collections originating from a high altitude forest in the northern Sierra Madre Occidental, Mexico. The species forms a monophyletic clade, basal to a larger lineage comprising species originating mainly from temperate to Mediterranean areas of the northern hemisphere. The phylogenetic approach in Fomitiporia also revealed multiple unnamed clades within the F. robusta complex in the southern USA and northern Mexico, representing potential species. The status of the F. robusta complex in North America is discussed briefly.     

Dihydrophenanthrene Dimers: Why and Where It Is Possible to Isolate Their Precursors

An interesting class of compounds of natural origin is dihydrophenanthrene dimers, which are characterized by a series of remarkable biological properties. Considering the hypothesis that each dimer is obtained through a biosynthetic mechanism that involves the coupling of the corresponding radicals of the single dihydrophenanthrene unit, we identified 29 dihydrophenanthrenes. Of these dihydrophenanthrenes, 11 were new compounds that could be isolated from 10 different plant species; 11 had already been identified, but not yet isolated in the 17 different plant species from which the corresponding dimers had been isolated; and 7 were known and had been isolated in the same plant sources of the corresponding dimers. A targeted analysis of several natural extracts from specific plant sources would allow the identification of known or new molecules with potential and/or specific biological activities and, in a final analysis, would confirm the relative biosynthetic mechanism.     

Diterpene composition of Cupressus dupreziana and Cupressus sempervirens

The heartwood of Cupressus dupreziana and C. sempervirens were shown to contain similar mixtures of diterpenes.     

Deciphering the Possible Mechanism of GABA in Tobacco Pollen Tube Growth and Guidance

γ-Aminobutyric acid (GABA) is an inhibitory transmitter in animal central and peripheral nervous systems, and also plays an important role in pollen tube growth and guidance. However, the mechanisms underlying these effects in plants are poorly understood, mainly because the GABA receptor in plants has not been elucidated. To address this issue, we recently created quantum dot probes to identify possible GABA receptors on the membrane surfaces of pollen protoplasts. We found that GABA bound to cell membranes and regulated downstream Ca²⁺ oscillation in the cells. These results provide important clues to further specifying the nature of the binding sites and deciphering the role of GABA as a signal molecule in pollen tube growth and orientation.Key WordS: γ-aminobutyric acid, fertilization, GABA receptor, signal transduction and tobacco     

Photochemistry of NH3, CH4 and PH3. Possible applications to the Jovian planets

Photolysis of NH₃ at 185 nm in the presence of a two-fold excess of CH₄ results in the loss of about 0.25 mole of CH₄ per mole of NH₃ decomposed (ΔCH₄/ΔNH₃). The loss arises from the abstraction of hydrogen atoms from CH₄ by photolytically generated hot hydrogen atoms, the presence of which is established by the constancy of ΔCH₄/ΔNH₃ between 298 and 156 K and by the quenching of the abstraction reaction when either H₂ or SF₆ is added. From the latter result, it can be concluded that NH₃ photolysis in the H₂-abundant atmosphere of Jupiter is not responsible for the presence of the carbon compounds observed there such as ethane, acetylene, and hydrogen cyanide, but may have had a role in the early atmosphere of Titan. Photolysis of PH₃ with a 206 nm light source gives P₂H₄, which in turn is converted to a red-brown solid (P₄?). The course of the photolysis is not changed appreciably when the temperature is lowered to 157 K except that the concentration of P₂H₄ increases. The presence of H₂ has no effect on the P₂H₄ yield. Photolysis of 9∶1 NH₃∶PH₃ gives a rate of decomposition of PH₃ that is comparable with that observed by the direct photolysis of PH₃. Comparable amounts of P₂H₄ and the red-brown solid are also observed. The mechanisms of these photochemical reactions together with their implications to the atmospheric chemistry of Jupiter are discussed. The structures of the compounds responsible for the wide array of colorse.g., brown, red and white, observed in the atmosphere of Jupiter have been the subject of extensive speculation. One theory suggests that these colors are due to organic materials formed by the action of either solar ultraviolet light or electric discharges on mixtures of CH₄, NH₃ and NH₄HS in the Jovian atmosphere (Ponnamperuma, 1976; Khareet al., 1978). An alternative hypothesis is that the colors are due to inorganic compounds resulting from the photolysis of NH₄HS and PH₃ (Lewis and Prinn, 1970; Prinn and Lewis, 1975). In this paper we will summarize our experiments which were designed to test some of these hypotheses.     

5-Dehydro-3-deoxy-D-arabino-heptulosonic acid 7-phosphate. An intermediate in the 3-dehydroquinate synthase reaction.

3-Dehydroquinate synthase was purified to homogeneity from Escherichia coli. It was found to be a single polypeptide chain of Mr = approximately 57,000. Reaction mixtures of pure enzyme and the substrate, 3-deoxy-D-arabino-heptulosonic acid 7-phosphate, were incubated for short times and treated with NaB3H4. The resulting 3-deoxyheptonic acid 7-phosphate was degraded with sodium periodate, and formic acid representing C-5 of the substrate was isolated. The presence of 3H in the formate corresponding to 15% of the enzyme was interpreted as indicating a 5-dehydro derivative of the substrate as an intermediate of the reaction. Quinic acid, resulting from reduction of 3-dehydroquinate with NaB3H4, was also isolated and degraded with periodate. The formate from C-4 of the quinate was unlabeled, indicating that 3,4-bisdehydroquinate is not an intermediate.     

Two Mitogen-Activated Protein Kinases,MPK3 and MPK6, Are Required for Funicular Guidance of Pollen Tubes in Arabidopsis

Double fertilization in flowering plants requires the delivery of two immotile sperm cells to the female gametes by a pollen tube, which perceives guidance cues, modifies its tip growth direction, and eventually enters the micropyle of the ovule. In spite of the recent progress, so far, little is known about the signaling events in pollen tubes in response to the guidance cues. Here, we show that MPK3 and MPK6, two Arabidopsis (Arabidopsis thaliana) mitogen-activated protein kinases, mediate the guidance response in pollen tubes. Genetic analysis revealed that mpk3 mpk6 double mutant pollen has reduced transmission. However, direct observation of mpk3 mpk6 mutant pollen phenotype was hampered by the embryo lethality of double homozygous mpk3^–/– mpk6^–/– plants. Utilizing a fluorescent reporter-tagged complementation method, we showed that the mpk3 mpk6 mutant pollen had normal pollen tube growth but impaired pollen tube guidance. In vivo pollination assays revealed that the mpk3 mpk6 mutant pollen tubes were defective in the funicular guidance phase. By contrast, semi-in vitro guidance assay showed that the micropylar guidance of the double mutant pollen tube was normal. Our results provide direct evidence to support that the funicular guidance phase of the pollen tube requires an in vivo signaling mechanism distinct from the micropyle guidance. Moreover, our finding opened up the possibility that the MPK3/MPK6 signaling pathway may link common signaling networks in plant stress response and pollen-pistil interaction.In flowering plants, successful fertilization is dependent on extensive cell-cell communication between male and female gametophytes. After landing on a compatible stigma surface, a mature pollen grain germinates to form a pollen tube, which penetrates the stigma, perceives guidance cues along the growth path, and modifies its tip growth direction toward the ovule (Hülskamp et al., 1995). In Arabidopsis (Arabidopsis thaliana), the pollen tube guidance can be divided into two phases: funicular guidance, in which the pollen tube emerges from the septum and proceeds to a funiculus, and micropylar guidance, in which the pollen tube grows toward and enters the micropyle of an ovule (Hülskamp et al., 1995).In pollen tube, it is believed that receptors on the tube tip perceive various guidance cues and regulate downstream signaling pathways to modify tip reorientation toward the ovule (Higashiyama, 2010; Takeuchi and Higashiyama, 2011). Two receptor-like kinase genes, Lost In Pollen tube guidance1 (LIP1) and LIP2, are involved in guidance control of pollen tubes. LIP1 and LIP2 were anchored to the membrane in the pollen tube tip region via palmitoylation, which was essential for their guidance control (Liu et al., 2013). Therefore, LIP1 and LIP2 are the essential components of the receptor complex in micropylar guidance. The Glu receptor-like channels facilitate Ca²⁺ influx across the plasma membrane and regulate pollen tube growth and morphogenesis (Michard et al., 2011). This interesting work revealed that there is a signaling mechanism between the male gametophyte and pistil tissue that is similar to the amino acid-mediated communication in animal nervous systems (Michard et al., 2011). Recent findings also highlight the importance of the endoplasmic reticulum (ER), ion homeostasis, and protein processing in pollen tube guidance (Li et al., 2011; Lu et al., 2011; Li and Yang, 2012). Two pollen-expressed cation proton exchangers (CHXs), CHX21 and CHX23, were reported to mediate K⁺ transport in ER and are essential for the pollen tube to respond to directional signals from the ovule in Arabidopsis (Lu et al., 2011). POLLEN DEFECTIVE IN GUIDANCE1 plays an important role in micropylar guidance in pollen tube (Li et al., 2011). It is an ER luminal protein involved in ER protein retention and interacts with a luminal chaperone involved in Ca²⁺ homeostasis and ER quality control (Li et al., 2011). Therefore, the ER quality control is likely an important mechanism in surveillance of signaling factors in pollen tube guidance (Li and Yang, 2012).In spite of the recent progresses, so far, little is known about the cytoplasmic signaling events in pollen tubes in response to the guidance cues. Mitogen-activated protein kinase (MAPK, or MPK) cascades are conserved signaling pathways that respond to extracellular stimuli and regulate various cellular activities. In Arabidopsis, MPK3 and MPK6 are induced by various biotic and abiotic stresses and collaboratively play important roles in defense response and plant development (Zhang, 2008). Here, we show that MPK3 and MPK6 are also critical to pollen tube guidance. Utilizing a fluorescent reporter-tagged complementation method, we demonstrated that mpk3 mpk6 pollen was defective in pollen tube guidance at the funicular guidance phase. Intriguingly, the micropylar guidance of mpk3 mpk6 pollen tube is not affected.     

GA_3对含笑花粉萌发及花粉管生长的影响

以含笑（Michelia figo）花粉为试材,采用花粉离体培养法,研究GA3对含笑花粉萌发和花粉管生长的影响。结果表明,GA3可以促进含笑花粉提早萌发,20～200 mg/L GA3对含笑花粉萌发和花粉管生长起促进作用,浓度超过200 mg/L花粉萌发和花粉管生长均受到抑制。以GA3200 mg/L的促进作用最好。     

Differential Effect of w3 PUFA Supplementations on Na,K-ATPase and Mg-ATPase Activities: Possible Role of the Membrane w6/w3 Ratio

Several functional properties of Na,K-ATPase are strongly dependent on membrane fatty acid composition, but the underlying mechanism is still not well defined. We have studied the effects of two types of supplementations enriched in the w3 polyunsaturated fatty acids on the Na,K-ATPase and Mg-ATPase activities in sciatic nerve (SN) and red blood cells (RBC). Eight groups of rats, controls and diabetics, received a standard diet, supplemented or not with 30 or 60 mg/kg/day of docosahexaenoic acid (DHA) or with soybean for eight weeks. Diabetes induced significant decrease of Na,K-ATPase activity in SN (-23%) and RBC (-25%), without affecting Mg-ATPase activity. In RBC, soybean and DHA supplementations caused significant increases in Na,K-ATPase activity (in various range, +13% to +145%) in all groups, and in Mg-ATPase activity in control soybean (+65%), control and diabetic DHA high dose (+39%, +53%) and diabetic DHA low dose (+131%) groups. In SN, the soybean caused a significant decrease in Na,K-ATPase activity (-26%) and still more in the diabetic group (-53%). The DHA diet induced a slight decrease in activity in control groups, whilst during diabetes, at high dose, we noted an aggravation of this decrease (-36%). Mg-ATPase activity was not modified by supplementations except for the low dose of DHA where the activity was slightly decreased in the control group (-16%). The supplementations induced multiple tissue-specific modifications in the membrane fatty acid composition of RBC and of SN homogenates. Several specific correlations have been found between variations in fatty acids amounts and Na,K-ATPase activity in these tissues but only in RBC for Mg-ATPase activity. Indeed, we observed that the variations in Na,K-ATPase activity are positively and significantly correlated with changes in the omega6/omega3 ratio in SN as well as in RBC. These data clearly show, for the first time, that the diet could modulate the Na,K-ATPase activity via the omega6/omega3 ratio in the membranes. A similar correlation was observed with Mg-ATPase activity in RBC, suggesting also a dietary regulation of the enzyme; but for the SN, this activity might be regulated by a different omega6/omega3 ratio or by another pathway.