Interactions among plants,bacteria, and fungi reduce extracellular enzyme activities under long‐term N fertilization

Atmospheric nitrogen (N) deposition has enhanced soil carbon (C) stocks in temperate forests. Most research has posited that these soil C gains are driven primarily by shifts in fungal community composition with elevated N leading to declines in lignin degrading Basidiomycetes. Recent research, however, suggests that plants and soil microbes are dynamically intertwined, whereby plants send C subsidies to rhizosphere microbes to enhance enzyme production and the mobilization of N. Thus, under elevated N, trees may reduce belowground C allocation leading to cascading impacts on the ability of microbes to degrade soil organic matter through a shift in microbial species and/or a change in plant–microbe interactions. The objective of this study was to determine the extent to which couplings among plant, fungal, and bacterial responses to N fertilization alter the activity of enzymes that are the primary agents of soil decomposition. We measured fungal and bacterial community composition, root–microbial interactions, and extracellular enzyme activity in the rhizosphere, bulk, and organic horizon of soils sampled from a long‐term (>25 years), whole‐watershed, N fertilization experiment at the Fernow Experimental Forest in West Virginia, USA. We observed significant declines in plant C investment to fine root biomass (24.7%), root morphology, and arbuscular mycorrhizal (AM) colonization (55.9%). Moreover, we found that declines in extracellular enzyme activity were significantly correlated with a shift in bacterial community composition, but not fungal community composition. This bacterial community shift was also correlated with reduced AM fungal colonization indicating that declines in plant investment belowground drive the response of bacterial community structure and function to N fertilization. Collectively, we find that enzyme activity responses to N fertilization are not solely driven by fungi, but instead reflect a whole ecosystem response, whereby declines in the strength of belowground C investment to gain N cascade through the soil environment.     

Identification of testis‐specific ubiquitin‐conjugating enzyme in the ascidian Ciona intestinalis

The ubiquitin–proteasome system is known to play a key role in fertilization in ascidians, sea urchins, and mammals. To obtain insights into the ubiquitin‐conjugating enzymes (Ube2) involved in reproductive systems, we systematically explored Ube2 enzymes expressed in the testis of the ascidian Ciona intestinalis. Here, we report cDNA cloning and characterization of a novel type of Ube2r (Ci0100152677) that is capable of making a thiolester bond with ubiquitin. Northern analysis, whole‐mount in situ hybridization and immunocytochemistry indicate that this enzyme is exclusively expressed in the testis, mainly in the germ cells during the late stage of spermatogenesis, and is localized in the sperm head and tail, suggesting possible participation in fertilization or spermatogenesis/spermiogenesis. Mol. Reprod. Dev. 77: 640–647, 2010. © 2010 Wiley‐Liss, Inc.     

太湖地区水稻土不同粒级团聚体中酶活性对长期施肥的响应

以太湖地区水稻土长期肥料试验田为研究对象,采用低能量超声波物理分散法分离土壤团聚体,研究长期不同施肥处理下（化肥与秸秆配施、化肥与猪粪配施、单施化肥和不施肥）耕层土壤团聚体酶活性的变化.结果表明：供试土壤中200～2000 μm和2～20 μm粒组的团聚体占总土壤的60%～70%;施肥显著促进了200～2000 μm团聚体的形成.各种酶活性在不同团聚体中的变化存在差异,脲酶和蔗糖酶在<2 μm的粘粒组中活性最高,纤维素酶、多酚氧化酶和荧光素二乙酸酯（FDA）水解酶则以200～2000 μm粗砂粒中活性最高.肥料的施用特别是化肥与有机肥的配合施用显著提高了200～2000 μm粒组中蔗糖酶、脲酶、纤维素酶和FDA水解酶的活性.以5种酶活性的几何平均数（GMea）作为酶的综合活性指标,发现施肥处理下200～2000 μm粒组中GMea显著高于其他粒组,可见大团聚体中土壤的酶活性对施肥措施响应敏感.有机和无机肥长期配合施用可以通过促进土壤团聚体的聚合和提高较大团聚体中酶活性来提高土壤整体的生物活性功能。     

Microbial activity and soil respiration under nitrogen addition in Alaskan boreal forest

Climate warming could increase rates of soil organic matter turnover and nutrient mineralization, particularly in northern high‐latitude ecosystems. However, the effects of increasing nutrient availability on microbial processes in these ecosystems are poorly understood. To determine how soil microbes respond to nutrient enrichment, we measured microbial biomass, extracellular enzyme activities, soil respiration, and the community composition of active fungi in nitrogen (N) fertilized soils of a boreal forest in central Alaska. We predicted that N addition would suppress fungal activity relative to bacteria, but stimulate carbon (C)‐degrading enzyme activities and soil respiration. Instead, we found no evidence for a suppression of fungal activity, although fungal sporocarp production declined significantly, and the relative abundance of two fungal taxa changed dramatically with N fertilization. Microbial biomass as measured by chloroform fumigation did not respond to fertilization, nor did the ratio of fungi : bacteria as measured by quantitative polymerase chain reaction. However, microbial biomass C : N ratios narrowed significantly from 16.0 ± 1.4 to 5.2 ± 0.3 with fertilization. N fertilization significantly increased the activity of a cellulose‐degrading enzyme and suppressed the activities of protein‐ and chitin‐degrading enzymes but had no effect on soil respiration rates or ¹⁴C signatures. These results indicate that N fertilization alters microbial community composition and allocation to extracellular enzyme production without affecting soil respiration. Thus, our results do not provide evidence for strong microbial feedbacks to the boreal C cycle under climate warming or N addition. However, organic N cycling may decline due to a reduction in the activity of enzymes that target nitrogenous compounds.     

Roles of mouse sperm‐associated alpha‐L‐fucosidases in fertilization

Sperm‐associated α‐L ‐fucosidases have been implicated in fertilization in many species. Previously, we documented the existence of α‐L ‐fucosidase in mouse cauda epididymal contents, and showed that sperm‐associated α‐L ‐fucosidase is cryptically stored within the acrosome and reappears within the sperm equatorial segment after the acrosome reaction. The enrichment of sperm membrane‐associated α‐L ‐fucosidase within the equatorial segment of acrosome‐reacted cells implicates its roles during fertilization. Here, we document the absence of α‐L ‐fucosidase in mouse oocytes and early embryos, and define roles of sperm associated α‐L ‐fucosidase in fertilization using specific inhibitors and competitors. Mouse sperm were pretreated with deoxyfuconojirimycin (DFJ, an inhibitor of α‐L ‐fucosidase) or with anti‐fucosidase antibody; alternatively, mouse oocytes were pretreated with purified human liver α‐L ‐fucosidase. Five‐millimolar DFJ did not inhibit sperm–zona pellucida (ZP) binding, membrane binding, or fusion and penetration, but anti‐fucosidase antibody and purified human liver α‐L ‐fucosidase significantly decreased the frequency of these events. To evaluate sperm‐associated α‐L ‐fucosidase enzyme activity in post‐fusion events, DFJ‐pretreated sperm were microinjected into oocytes, and 2‐pronuclear (2‐PN) embryos were treated with 5 mM DFJ with no significant effects, suggesting that α‐L ‐fucosidase enzyme activity does not play a role in post‐fusion events and/or early embryo development in mice. The recognition and binding of mouse sperm to the ZP and oolemma involves the glycoprotein structure of α‐L ‐fucosidase, but not its catalytic action. These observations suggest that deficits in fucosidase protein and/or the presence of anti‐fucosidase antibody may be responsible for some types of infertility. Mol. Reprod. Dev. 80: 273–285, 2013. © 2013 Wiley Periodicals, Inc.     

Extracellular Enzyme Activities and Carbon Chemistry as Drivers of Tropical Plant Litter Decomposition

Litter quality parameters such as nitrogen and lignin content correlate with decomposition rates at coarse scales, but fine‐scale mechanisms driving litter decomposition have proven more difficult to generalize. One potentially important driver of decomposition is the activity of extracellular enzymes that catalyze the degradation of complex compounds present in litter. To address the importance of this mechanism, we collected 15 Hawaiian plant litter types and decomposed them in fertilized and control plots for up to two years. We measured litter nutrient content and carbon chemistry prior to decomposition, as well as extracellular enzyme activities, mass loss, and litter nutrient content over time. We found that water‐soluble carbon content, cellobiohydrolase activities, and polyphenol oxidase activities were significantly correlated with mass loss. Enzyme activities and decomposition rate constants both varied significantly by litter type, and fertilization increased mass loss rates in five litter types. Some litter types that decayed faster under fertilization also showed time‐dependent increases in carbon‐degrading enzyme activities, but others decayed faster independent of enzyme changes. These results suggest that extracellular enzyme activities partially determine litter decomposition rates, but high soluble carbon content may circumvent the requirement for enzyme‐catalyzed decomposition.     

Human DCXR – another ‘moonlighting protein’ involved in sugar metabolism,carbonyl detoxification,cell adhesion and male fertility?

Dicarbonyl/l ‐xylulose reductase (DCXR; SDR20C1), a member of the short‐chain dehydrogenase/reductase (SDR) superfamily catalyzes the reduction of α‐dicarbonyl compounds and monosaccharides. Its role in the metabolism of l ‐xylulose has been known since 1970, when essential pentosuria was found to be associated with DCXR deficiency. Despite its early discovery, our knowledge about the role of human DCXR in normal physiology and pathophysiology is still incomplete. Sporadic studies have demonstrated aberrant expression in several cancers, but their physiological significance is unknown. In reproductive medicine, where DCXR is commonly referred to as ‘sperm surface protein P34H’, it serves as marker for epididymal sperm maturation and is essential for gamete interaction and successful fertilization. DCXR exhibits a multifunctional nature, both acting as a carbonyl reductase and also performing non‐catalytic functions, possibly resulting from interactions with other proteins. Recent observations associate DCXR with a role in cell adhesion, pointing to a novel function involving tumour progression and possibly metastasis. This review summarizes the current knowledge about human DCXR and its orthologs from mouse and Caenorhabditis elegans (DHS‐21) with an emphasis on its multifunctional characteristics. Due to its close structural relationship with DCXR, carbonyl reductase 2 (Cbr2), a tetrameric enzyme found in several non‐primate species is also discussed. Similar to human DCXR, Cbr2 from golden hamster (P26h) and cow (P25b) is essential for sperm–zona pellucida interaction and fertilization. Because of the apparent similarity of these two proteins and the inconsistent use of alternative names previously, we provide an overview of the systematic classification of DCXR and Cbr2 and a phylogenetic analysis to illustrate their ancestry.     

Sperm penetration through oocyte investments in mammals

Literature on the interactions between eutherian gametes is reviewed. The oocyte cumulus complex of the female is surrounded by a zona pellucida, corona radiata, and cumulus layer. Sperm undergo an acrosome reaction before penetrating the zona pellucida. The morphological consequences of this reaction and its possible role(s) in penetration of the oocyte cumulus complex are considered. The acrosomal enzyme, hyaluronidase, has been thought to aid sperm in penetrating the cumulus layer and corona radiata. Several recent investigations, including one that shows that motile cells lacking hyaluronidase can penetrate to the zona surface, do not support this idea. Other possible roles of this enzyme in fertilization are discussed. The development of in vitro fertilization systems that employ physiological numbers (1-100) of sperm will be valuable in studying the mechanisms used by sperm to penetrate the oocyte cumulus complex.     

Haprin‐deficient spermatozoa are incapable of in vitro fertilization

Haprin (TRIM36) is a ubiquitin‐protein ligase that mediates ubiquitination and subsequent proteasomal degradation of target proteins. It is expressed in the testes in both mice and humans and is thought to be involved in spermiogenesis, the acrosome reaction, and fertilization. However, the functional role of Haprin is poorly understood. The aim of this study was to investigate the physiological role of Haprin in fertility. Homozygous haprin‐deficient mice were generated and these mice, and their spermatozoa, were analyzed to detect morphological and fertility‐related abnormalities. In these models, normal spermatogenesis was observed but sperm quality was reduced with haprin‐deficient mice having poorer sperm morphology and motility than wild‐type mice. Interestingly, haprin‐deficient mice showed normal in vivo fertility but could not fertilize oocytes under standard in vitro fertilization conditions. In conclusion, this study demonstrated that Haprin deficiency causes morphological abnormalities in spermatozoa, indicating that Haprin is involved in spermiogenesis.     

Inhibition of bovine sperm‐oocyte fusion by a monoclonal antibody recognising the TEC‐2 epitope on bovine oocytes

The TEC‐2 antigenic determinant is a carbohydrate epitope located on a glycoprotein carrier molecule. In the mouse, this epitope is expressed on the zona pellucida and plasma membrane of the oocyte and is associated with the ZP2 glycoprotein and involved in the secondary sperm receptor mechanism. On the bovine oocyte expression is confined to the plasma membrane. The aim of this study was to determine the role the TEC‐2 epitope plays during fertilization in the bovine species using the monoclonal antibody TEC‐02. Incubating oocytes with the TEC‐02 antibody prior to fertilization inhibited cleavage in a dose‐dependent manner—the cleavage rate decreased as the concentration of the antibody increased. Significantly more sperm were bound to oocytes exposed to TEC‐02 (12 sperm/oocyte) compared to oocytes that were not incubated with the antibody (4 sperm/oocyte). Oocytes treated with the TEC‐02 antibody had a 7.5 ± 3.2% fusion rate and no cortical granule exocytosis compared with oocytes not exposed to the antibody, with 86.5 ± 5.8% of sperm‐oocyte fusions and release of cortical granules. The block to sperm‐oocyte fertilization observed in the pretreated group was overcome using intracytoplasmic sperm injection as the method of fertilization that bypassed the fusion process. Although sperm were binding to the oolemma these results suggest that fusion was not occurring and this may be due to the antibody occupying TEC‐2 epitope sites involved in the fusion process. In conclusion, the TEC‐2 epitope seems to be involved in sperm‐oocyte interaction in the bovine species and appears to be involved specifically during the fusion events of fertilization. Mol. Reprod. Dev. 54:173–178, 1999. © 1999 Wiley‐Liss, Inc.     

Sperm competition,but not major histocompatibility divergence,drives differential fertilization success between alternative reproductive tactics in Chinook salmon

Post‐copulatory sexual selection processes, including sperm competition and cryptic female choice (CFC), can operate based on major histocompatibility (MH) genes. We investigated sperm competition between male alternative reproductive tactics [jack (sneaker) and hooknose (guard)] of Chinook salmon (Oncorhynchus tshawytscha). Using a full factorial design, we examined in vitro competitive fertilization success of paired jack and hooknose males at three time points after sperm activation (0, 15 and 60 s) to test for male competition, CFC and time effects on male fertilization success. We also examined egg‐mediated CFC at two MH genes by examining both the relationship between competitive fertilization success and MH divergence as well as inheritance patterns of MH alleles in resulting offspring. We found that jacks sired more offspring than hooknose males at 0 s post‐activation; however, jack fertilization success declined over time post‐activation, suggesting a trade‐off between sperm speed and longevity. Enhanced fertilization success of jacks (presumably via higher sperm quality) may serve to increase sneaker tactic competitiveness relative to dominant hooknose males. We also found evidence of egg‐mediated CFC (i.e. female × male interaction) influencing competitive fertilization success; however, CFC was not acting on the MH genes as we found no relationship between fertilization success and MH II β₁ or MH I α₁ divergence and we found no deviations from Mendelian inheritance of MH alleles in the offspring. Our study provides insight into evolutionary mechanisms influencing variation in male mating success within alternative reproductive tactics, thus underscoring different strategies that males can adopt to attain success.     

Cysteine-protease involved in male chromatin remodeling after fertilization co-localizes with alpha-tubulin at mitosis

We postulated an essential role for a cysteine-protease in sea urchins sperm histones degradation which follows fertilization. We now report the purification of this enzyme, the determination of its N-terminal amino acid sequence and the localization of the protein with antibodies generated against this amino-terminal peptide. The immunofluorescence data confirmed the presence of this enzyme in the nucleus of unfertilized eggs. After fertilization labeling is observed both in female and male pronuclei suggesting a rapid recruitment of the enzyme to the male pronuclei. Interestingly, we have found that this cysteine-protease persists in the nucleus of the zygotes during S phase of the cell cycle and co-localizes with alpha-tubulin that organizes the mitotic spindle during the initial embryonic cell division.     

Effect of hyaluronidase, beta-glucuronidase and beta-N-acetylglucosaminidase inhibitors on sperm penetration of the mouse oocyte

The role of hyaluronidase, beta-glucuronidase and beta-N-acetylglucosaminidase in the penetration by mouse spermatozoa through the layers surrounding the oocyte was investigated by in vitro techniques. Myocrisin, fenoprofen, phosphorulated hesperidin and PS53 (a hydroquinone-sulfonic acid-formaldehyde polymer) inhibited fertilization when incubated with capacitated spermatozoa before the treated spermatozoa were mixed with intact oocytes but not when the inhibitor-treated, capacitated spermatozoa were added to oocytes free of follicle cells. The antifertility activity did not appear to be due to an effect on sperm motility or on the oocytes. These 4 compounds are known hyaluronidase inhibitors and, of the acrosomal enzymes tested, only share inhibition of hyaluronidase. Kinetic studies indicated that myocrisin is a reversible inhibitor of mouse sperm hyaluronidase whereas the other three are irreversible inhibitors. Adding saccharolactone, a beta-glucuronidase inhibitor, or N-acetylglucosaminolactone and N-acetylgalactosaminolactone, beta-N-acetylglucosaminidase inhibitors, to capacitated spermatozoa under the same conditions as the hyaluronidase inhibitors did not decrease fertilization. This was the case even though the beta-glucuronidase or beta-N-acetylglucosaminidase activities of the spermatozoa were completely inhibited, at least at the time that the inhibitor-treated, capacitated spermatozoa were mixed with the oocytes. The hyaluronidase activity of mouse spermatozoa remained unaltered during the incubation period required for capacitation; however, prolonged incubation caused a significant decrease in hyaluronidase. Untreated mouse spermatozoa caused hydrolysis of hyaluronic acid more effectively than did sperm extracts obtained by detergent extraction. These results are consistent with the theory of an essential role of hyaluronidase in mouse fertilization. At least in this species, the enzyme appears to be specifically involved in sperm penetration through the follicle cell layer. The data do not support an essential role for beta-glucuronidase and beta-N-acetylglucosaminidase in the penetration by mouse spermatozoa through the oocyte's investments. In contrast to some other species, sperm capacitation in mice does not result in a loss of hyaluronidase although part of the enzyme activity is lost on prolonged incubation. Mouse spermatozoa appear to be able to digest substrate (hyaluronic acid) even though hyaluronidase is not released.     

Individual and combined effects of disturbance and N addition on understorey vegetation in a subarctic mountain birch forest

Questions: What are the effects of repeated disturbance and N‐fertilization on plant community structure in a mountain birch forest? What is the role of enhanced nutrient availability in recovery of understorey vegetation after repeated disturbance? How are responses of soil micro‐organisms to disturbance and N‐fertilization reflected in nutrient allocation patterns and recovery of understorey vegetation after disturbance? Location: Subarctic mountain birch forest, Finland. Methods: We conducted a fully factorial experiment with annual treatments of disturbance (two levels) and N‐fertilization (four levels) during 1998–2002. We monitored treatment effects on above‐ground plant biomass, plant community structure and plant and soil nutrient concentrations. Results: Both disturbance and N‐fertilization increased the relative biomass of graminoids. The increase of relative biomass of graminoids in the disturbance treatment was over twice that of the highest N‐fertilization level, and N‐fertilization further increased their relative biomass after disturbance. As repeated disturbance broke the dominance of evergreen dwarf shrubs, it resulted in a situation where deciduous species, graminoids and herbs dominated the plant community. Although relative biomass of deciduous dwarf shrubs declined with N‐fertilization, it did not cause a shift in plant community structure, as evergreen dwarf shrubs remained dominant. Both disturbance and N‐fertilization increased the N concentration in vascular plants, whereas microbial biomass N and C were not affected by the treatments. Concentrations of NH₄⁺, dissolved organic N (DON) and dissolved organic C (DOC) increased in the soil after N‐fertilization, whereas concentrations of NH₄⁺ and DON decreased after disturbance. Conclusions: Disturbances caused by e.g. humans or herbivores contribute more to changes in the understorey vegetation structure than increased levels of N in subarctic vegetation. Fertilization accelerated the recovery potential after repeated disturbance in graminoids. Microbial activities did not limit plant growth.     

Structure and diversity of a species-rich grassland community, treated with additional illumination, fertilization and mowing

We examined the changes of species‐rich herbaceous community canopy structure and species diversity in a five‐year field experiment. Above‐ and below‐ground resource addition (fertilization and additional illumination with the help of mirrors) and cessation of annual mowing were applied as experimental treatments in 24 permanent plots of 40×40 cm. Canopy structure was studied by point quadrat sampling using laser beam to randomly position point quadrats at two different angles of observation – 60° and 120° from horizontal, north‐south direction. We hypothezised that the studied plant community is not light‐limited under the normal conditions (non‐fertilized and annually mown) but would become light‐limited after nutrient addition and/or cessation of mowing. In this case the effect of fertilization and cessation of mowing could be mitigated by introducing additional light resource (placing south‐facing mirrors).
Species richness in plots was not significantly altered by experimental treatments, most probably because of a strong input of species from surrounding areas by the means of lateral vegetative spread, compensating for the expected decrease of diversity with fertilization and cessation of mowing. Surprisingly, plant total coverage in a plot was the only variable significantly explaining plot richness, independent of experimental treatments and the year of observation. We therefore studied also species richness per unit coverage (standardized richness) which appeared to be significantly reduced by fertilization. Annual change of plant total coverage of a plot was good predictor of annual change of plot richness, except for the exceptionally dry period (1994–1995). There was no indication of light being limiting for the plants in control plots, but light obviously became limiting after nutrient addition – in fertilized plots additional illumination lead to a notably denser canopy. Generally, the average exposition of plant leaves surface was towards the north, independent of experimental treatments. However, in the exceptionally cloudy and cool year 1996 the situation was radically different – leaves were exposed predominantly towards the south, indicating that multi‐species canopy can plastically react to long‐term changes in light availability.     

Trypsin-like enzyme from eggs of the ascidian (protochordate), Halocynthia roretzi. Purification, properties, and physiological role

A trypsin-like enzyme has been purified to apparent homogeneity from eggs of the ascidian, Halocynthia roretzi, by a procedure including column chromatography on diethylaminoethyl-cellulose, phenyl-Sepharose, and soybean trypsin inhibitor-immobilized Sepharose 4B. The molecular weight of the enzyme was estimated to be 31,000 and 33,000 by gel electrophoresis in sodium dodecyl sulfate under the reducing and the nonreducing conditions, respectively. The isoelectric point of the enzyme was 4.8. The pH optimum of the activity was 8.4. The enzyme was stable between pH 6 and 9 in the presence of 0.005% Brij 35 as a stabilizer. Substrate specificity of the purified enzyme was broad toward various peptidyl-arginine (or -lysine) 4-methylcoumaryl-7-amides and was similar to that of a trypsin-like enzyme found in the fertilization product. The purified enzyme was inhibited by diisopropyl fluorophosphate and a variety of trypsin inhibitors including leupeptin, but not, or scarcely, inhibited by p-chloromercuribenzoic acid, pepstatin, chymostatin, bestatin, elastatinal, and tosyl-phenylalanyl-chloromethane. The rankings in the potencies of leupeptin and its six analogs as the inhibitors of the purified enzyme were well correlated with those found in their inhibitory effects on the expansion of perivitelline space. Thus, the trypsin-like enzyme possibly present in the fertilization product participates in the expansion of perivitelline space of the egg during fertilization of the ascidian.     

Influences of fertilization on population abundance, distribution, and control of Frankliniella occidentalis on chrysanthemum

We examined the effects of fertilization on population abundance and within‐plant distribution of western flower thrips, Frankliniella occidentalis Pergande (Thysanoptera: Thripidae), on potted chrysanthemum, Dendranthema grandiflora (Tzvelev). We also investigated the effects of fertilization on the number of insecticide applications needed to control F. occidentalis on potted chrysanthemum. Under greenhouse conditions, rate of change in population abundance of F. occidentalis increased with fertilization levels from 0 to 100% of the standard fertilization level (375 ppm N) and was four times higher on plants fertilized with the standard level (rate of change = 0.14) than on plants fertilized with 0% during the first 4 weeks after thrips inoculation. Within‐plant distribution of F. occidentalis was influenced by the phenology of the plants rather than total nitrogen content of plant tissues. Prior to flower opening, more F. occidentalis were found in the middle region of the plants. When the flowers began to open, more thrips were found feeding inside the flowers than on the leaves. We further showed that production time, the time from transplantation to flower opening, shortened considerably with increased fertilization level. Production time was shortest, 12 weeks, for plants fertilized with 100% of the standard fertilization level. When the fertilization level was reduced to 20%, production time lengthened to 13 weeks. When fertilization level was reduced to 0%, production time lengthened to 14 weeks. Increased fertilization from 0 to 100% of the standard level did not result in higher numbers of insecticide applications. All three insecticides (acephate, bifenthrin, and spinosad) were effective in keeping the thrips infestation below a predetermined level, five thrips per plant, but bifenthrin required the most number of applications to do so. For chrysanthemum, a fast‐growing crop and heavy utilizer of fertilizer, fertilization influenced not only the population growth of pest insects but also plant production time. As a result, optimizing fertilization level to reduce pest population growth may be a useful tactic in an Integrated Pest Management program for managing F. occidentalis on potted chrysanthemum. However, the effect of fertilization on production time and plant quality should also be considered when implementing this tactic.     

Extracellular ubiquitin system implicated in fertilization of the ascidian, Halocynthia roretzi: isolation and characterization

The ubiquitin-proteasome system is essential for intracellular protein degradation, but there are few studies of this system in the extracellular milieu. Recently, we reported that a 70-kDa sperm receptor, HrVC70, on the vitelline coat is ubiquitinated and then degraded by the sperm proteasome during fertilization of the ascidian, Halocynthia roretzi. Here, we investigated the mechanism of extracellular ubiquitination. The HrVC70-ubiquitinating enzyme activity was found to be released from the activated sperm during the fertilization process. This enzyme was purified from an activated sperm exudate, by chromatography on DEAE-cellulose and ubiquitin-agarose columns, and by glycerol density gradient centrifugation. The molecular mass of the enzyme was estimated to be 700 kDa. The purified enzyme requires CaCl2 and MgATP for activity, and is active in seawater. The purified enzyme preparation, but not the crude enzyme preparation, showed narrow substrate specificity to HrVC70. Moreover, ATP and ubiquitin are released from the activated sperm to the surrounding seawater during fertilization. These results indicate that ascidian sperm release a novel extracellular ubiquitinating enzyme system together with ATP and ubiquitin during penetration of the vitelline coat of the egg, which catalyzes the ubiquitination of the HrVC70, an essential component of ascidian fertilization.     

Identification and characterization of PlAlix,the Alix homologue from the Mediterranean sea urchin Paracentrotus lividus

The sea urchin provides a relatively simple and tractable system for analyzing the early stages of embryo development. Here, we use the sea urchin species, Paracentrotus lividus, to investigate the role of Alix in key stages of embryogenesis, namely the egg fertilization and the first cleavage division. Alix is a multifunctional protein involved in different cellular processes including endocytic membrane trafficking, filamentous (F)‐actin remodeling, and cytokinesis. Alix homologues have been identified in different metazoans; in these organisms, Alix is involved in oogenesis and in determination/differentiation events during embryo development. Herein, we describe the identification of the sea urchin homologue of Alix, PlAlix. The deduced amino acid sequence shows that Alix is highly conserved in sea urchins. Accordingly, we detect the PlAlix protein cross‐reacting with monoclonal Alix antibodies in extracts from P. lividus, at different developmental stages. Focusing on the role of PlAlix during early embryogenesis we found that PlAlix is a maternal protein that is expressed at increasingly higher levels from fertilization to the 2‐cell stage embryo. In sea urchin eggs, PlAlix localizes throughout the cytoplasm with a punctuated pattern and, soon after fertilization, accumulates in larger puncta in the cytosol, and in microvilli‐like protrusions. Together our data show that PlAlix is structurally conserved from sea urchin to mammals and may open new lines of inquiry into the role of Alix during the early stages of embryo development.     

Genetic variation in eggplant for Nitrogen Use Efficiency under contrasting NO3‐ supply

Eggplant (Solanum melongena L.) yield is highly sensitive to N fertilization, the excessive use of which is responsible for environmental and human health damage. Lowering N input together with the selection of improved Nitrogen‐Use‐Efficiency (NUE) genotypes, more able to uptake, utilize, and remobilize N available in soils, can be challenging to maintain high crop yields in a sustainable agriculture. The aim of this study was to explore the natural variation among eggplant accessions from different origins, in response to Low (LN) and High (HN) Nitrate (NO₃^‐) supply, to identify NUE‐contrasting genotypes and their NUE‐related traits, in hydroponic and greenhouse pot experiments. Two eggplants, AM222 and AM22, were identified as N‐use efficient and inefficient, respectively, in hydroponic, and these results were confirmed in a pot experiment, when crop yield was also evaluated. Overall, our results indicated the key role of N‐utilization component (NUtE) to confer high NUE. The remobilization of N from leaves to fruits may be a strategy to enhance NUtE, suggesting glutamate synthase as a key enzyme. Further, omics technologies will be used for focusing on C‐N metabolism interacting networks. The availability of RILs from two other selected NUE‐contrasting genotypes will allow us to detect major genes/quantitative trait loci related to NUE.