EST analysis of hop glandular trichomes identifies an O-methyltransferase that catalyzes the biosynthesis of xanthohumol

The glandular trichomes (lupulin glands) of hop (Humulus lupulus) synthesize essential oils and terpenophenolic resins, including the bioactive prenylflavonoid xanthohumol. To dissect the biosynthetic processes occurring in lupulin glands, we sequenced 10,581 ESTs from four trichome-derived cDNA libraries. ESTs representing enzymes of terpenoid biosynthesis, including all of the steps of the methyl 4-erythritol phosphate pathway, were abundant in the EST data set, as were ESTs for the known type III polyketide synthases of bitter acid and xanthohumol biosynthesis. The xanthohumol biosynthetic pathway involves a key O-methylation step. Four S-adenosyl-l-methionine-dependent O-methyltransferases (OMTs) with similarity to known flavonoid-methylating enzymes were present in the EST data set. OMT1, which was the most highly expressed OMT based on EST abundance and RT-PCR analysis, performs the final reaction in xanthohumol biosynthesis by methylating desmethylxanthohumol to form xanthohumol. OMT2 accepted a broad range of substrates, including desmethylxanthohumol, but did not form xanthohumol. Mass spectrometry and proton nuclear magnetic resonance analysis showed it methylated xanthohumol to 4-O-methylxanthohumol, which is not known from hop. OMT3 was inactive with all substrates tested. The lupulin gland-specific EST data set expands the genomic resources for H. lupulus and provides further insight into the metabolic specialization of glandular trichomes.     

The anaerobic endurance of elite soccer players improved after a high-intensity training intervention in the 8-week conditioning program

The purpose of this study was to evaluate changes in anaerobic endurance in elite First-league soccer players throughout 2 consecutive seasons, in 2 phases, with and without high-intensity situational drills. Eighteen soccer players were tested before and after the 8-week summer conditioning and again in the next season. The measured variables included 300-yard shuttle run test, maximal heart rate, and maximal blood lactate at the end of the test. During the first phase of the study, the traditional sprint training was performed only 2 x weeks and consisted of 15 bouts of straight-line sprinting. In the second year the 4 x 4 min drills at an intensity of 90-95% of HRmax, separated by periods of 3-minute technical drills at 55-65% of HRmax were introduced. Statistical significance was set at P 相似文献   

Calcium efflux as a component of the hypersensitive response of Nicotiana benthamiana to Pseudomonas syringae

Using a model plant Nicotiana benthamiana, we have demonstrated that initial calcium uptake in response to the HR (hypersensitive response)-causing pathogen Pseudomonas syringae pv syringae 61 is followed by net calcium efflux initiated at about 12 h after the bacterial challenge and sustained for at least 48 h. Our data suggest that calcium not only acts as an important second messenger in the activation of resistance responses but may also be a downstream mediator of later cell death acceleration and completion of the defense reaction. Accordingly, we propose that the existing model of HR should be amended to include a PM Ca(2+) ATP pump as an important component of the HR to pathogens in plants.     

Effects of Verapamil and Gadolinium on Caffeine-Induced Contractures and Calcium Fluxes in Frog Slow Skeletal Muscle Fibers

In this work, we tested whether L-type Ca²⁺ channels are involved in the increase of caffeine-evoked tension in frog slow muscle fibers. Simultaneous net Ca²⁺ fluxes and changes in muscle tension were measured in the presence of caffeine. Isometric tension was recorded by a mechanoelectrical transducer, and net fluxes of Ca²⁺ were measured noninvasively using ion-selective vibrating microelectrodes. We show that the timing of changes in net fluxes and muscle tension coincided, suggesting interdependence of the two processes. The effects of Ca²⁺ channel blockers (verapamil and gadolinium) were explored using 6 mm caffeine; both significantly reduced the action of caffeine on tension and on calcium fluxes. Both caffeine-evoked Ca²⁺ leak and muscle tension were reduced by 75% in the presence of 100 μm GdCl₃, which also caused a 92% inhibition of net Ca²⁺ fluxes in the steady-state condition. Application of 10 μm verapamil to the bath led to 30% and 52% reductions in the Ca²⁺ leak caused by the presence of caffeine for the peak and steady-state values of net Ca²⁺ fluxes, respectively. Verapamil (10 μm) caused a 30% reduction in the maximum values of caffeine-evoked muscle tension. Gd³⁺ was a more potent inhibitor than verapamil. In conclusion, L-type Ca²⁺ channels appear to play the initial role of trigger in the rather complex mechanism of slow fiber contraction, the latter process being mediated by both positive Ca²⁺-induced Ca²⁺ release and negative (Ca²⁺ removal from cytosol) feedback loops. Lana Shabala and Xóchitl Trujillo contributed equally to this study.     

A high-throughput method for the quantitative analysis of indole-3-acetic acid and other auxins from plant tissue

To investigate novel pathways involved in auxin biosynthesis, transport, metabolism, and response, we have developed a high-throughput screen for indole-3-acetic acid (IAA) levels. Historically, the quantitative analysis of IAA has been a cumbersome and time-consuming process that does not lend itself to the screening of large numbers of samples. The method described here can be performed with or without an automated liquid handler and involves purification solely by solid-phase extraction in a 96-well format, allowing the analysis of up to 96 samples per day. In preparation for quantitative analysis by selected ion monitoring-gas chromatography-mass spectrometry, the carboxylic acid moiety of IAA is derivatized by methylation. The derivatization of the IAA described here was also done in a 96-well format in which up to 96 samples can be methylated at once, minimizing the handling of the toxic reagent, diazomethane. To this end, we have designed a custom diazomethane generator that can safely withstand high flow and accommodate larger volumes. The method for IAA analysis is robust and accurate over a range of plant tissue weights and can be used to screen for and quantify other indolic auxins and compounds including indole-3-butyric acid, 4-chloro-indole-3-acetic acid, and indole-3-propionic acid.     

The RING domain and first zinc finger of TRAF6 coordinate signaling by interleukin-1, lipopolysaccharide, and RANKL

TRAF6, a crucial adaptor molecule in innate and adaptive immunity, contains three distinct functional domains. The C-terminal TRAF domain facilitates oligomerization and sequence-specific interaction with receptors or other adaptor proteins. In conjunction with the dimeric E2 enzyme Ubc13-Uev1A, the N-terminal RING domain of TRAF6 functions as an E3 ubiquitin (Ub) ligase that facilitates its own site-specific ubiquitination through the generation of a Lys-63-linked poly-Ub chain. This modification does not cause its proteasomal degradation but rather serves as a scaffold to activate both the IKK and stress kinase pathways. Connecting the N-and C-terminal regions, the four internal zinc finger (ZF) motifs have yet to be functionally defined. In this study, we examined the role of the ZF domains in interleukin-1, lipopolysaccharide, and RANKL signaling by reconstitution of TRAF6-deficient cells with point mutations or deletions of these ZF motifs. Although ZF domains 2-4 are dispensable for activating IKK, p38, and JNK by interleukin-1 and lipopolysaccharide, the first ZF domain together with an intact RING domain of TRAF6 is essential for activating these pathways. Furthermore, TRAF6 autoubiquitination and its interaction with Ubc13 are dependent on ZF1 and an intact RING domain. Additionally, expression of TRAF6 lacking ZF2-4 in TRAF6-deficient monocytes rescues RANKL-mediated osteoclast differentiation and LPS-stimulated interleukin-6 production. These data provide evidence for the critical role of the Ub ligase activity of TRAF6, which is coordinated via the RING domain and ZF1 to supply the necessary elements in signaling by cytokines dependent upon TRAF6.     

Effect of divalent cations on ion fluxes and leaf photochemistry in salinized barley leaves

Photosynthetic characteristics, leaf ionic content, and net fluxes of Na(+), K(+), and Cl(-) were studied in barley (Hordeum vulgare L) plants grown hydroponically at various Na/Ca ratios. Five weeks of moderate (50 mM) or high (100 mM) NaCl stress caused a significant decline in chlorophyll content, chlorophyll fluorescence characteristics, and stomatal conductance (g(s)) in plant leaves grown at low calcium level. Supplemental Ca(2+) enabled normal photochemical efficiency of PSII (F(v)/F(m) around 0.83), restored chlorophyll content to 80-90% of control, but had a much smaller (50% of control) effect on g(s). In experiments on excised leaves, not only Ca(2+), but also other divalent cations (in particular, Ba(2+) and Mg(2+)), significantly ameliorated the otherwise toxic effect of NaCl on leaf photochemistry, thus attributing potential targets for such amelioration to leaf tissues. To study the underlying ionic mechanisms of this process, the MIFE technique was used to measure the kinetics of net Na(+), K(+), and Cl(-) fluxes from salinized barley leaf mesophyll in response to physiological concentrations of Ca(2+), Ba(2+), Mg(2+), and Zn(2+). Addition of 20 mM Na(+) as NaCl or Na(2)SO(4) to the bath caused significant uptake of Na(+) and efflux of K(+). These effects were reversed by adding 1 mM divalent cations to the bath solution, with the relative efficiency Ba(2+)>Zn(2+)=Ca(2+)>Mg(2+). Effect of divalent cations on Na(+) efflux was transient, while their application caused a prolonged shift towards K(+) uptake. This suggests that, in addition to their known ability to block non-selective cation channels (NSCC) responsible for Na(+) entry, divalent cations also control the activity or gating properties of K(+) transporters at the mesophyll cell plasma membrane, thereby assisting in maintaining the high K/Na ratio required for optimal leaf photosynthesis.     

Diel distribution of copepods across a channel of an overwash mangrove island

The distribution of copepod species and their nauplii was studied in a narrow, blind channel on an overwash mangrove island offshore of Belize. Copepodids were sampled with a pump at five stations across the channel during a diel cycle. Diel changes of copepodid stages II – VI were marked by horizontal dispersal of Dioithona oculata, the dominant species, from swarms in the prop roots along the shore during the day to the edge of the prop root habitat at night. Migration of copepodids back to the prop roots appeared to be controlled endogenously because change from a night to a daytime age structure began before first light. Mean copepodid stage at subsurface depths in the channel and prop root edge decreased from 4.2 (with 6.0 = all adults) to 2.9 at predawn to 1.1 during day. The oceanic Oithona nana and O. simplex, and the coastal zone O. fonsecae were evenly distributed with depth and distance from shore during day and night, with avoidance of prop root shoreline during day. These species were much less abundant than Dioithona oculata in the prop roots, but of comparable or greater abundance in the channel. Coastal zone Acartia spinata exhibited evidence of swarming. Nauplii, sampled with a 25m plankton net, were dominated by harpacticoid (50%) and cyclopoid (34%) nauplii, which generally were more abundant at 1m than at the surface and more abundant at night than the day. Lagrangian current measurements indicated velocities at ebb tide twice those of flood tide (1.9 vs. 0.8 cm s^–1) and a minimal residence time of 5 days, which could result in advection of D. oculata nauplii out of the Lair Channel before their recruitment into swarms as copepodid stage II. Previously reported maximum swimming speeds of swarming D. oculata copepodid stages (2.0 cm s^–1) and greater densities in prop roots and near the benthos may help copepodids avoid advection. The swarming behavior and diel horizontal migration (or dispersal) reported for D. oculata appears analogous to that of limnetic zooplankton, which may swarm among macrophytes along shorelines during the day to avoid visual predators and disperse or migrate away from the shoreline at night.     

Kinetics of net H+, Ca2+, K+, Na+, NH+4, and Cl- fluxes associated with post-chilling recovery of plasma membrane transporters in Zea mays leaf and root tissues

Although temperature-induced changes in membrane structure and activity seem to be central to chilling stress perception, the specific details of temperature effects on plant nutrient acquisition remain obscure. In this study, we have undertaken a comparative study of low temperature effects on the activity of plasma membrane transporters of different ions in corn ( Zea mays L.) leaf and root tissues by non-invasive measurements of net ion fluxes using ion-selective microelectrode (the MIFE) technique. Kinetics of net H⁺, Ca²⁺, K⁺, Na⁺,     and Cl^– fluxes were measured as plant tissues recovered after short-term (3 h) chilling stress. The major findings can be summarized as follows: (1) The critical temperatures, under which the recovery of the activity of plasma membrane transporters took place, were found to be the same for all ions measured and are likely to be associated with the phase transition of membrane lipids. (2) The most pronounced was the reduction in net uptake of K⁺ and     (3) Chilling treatment caused a significant net influx of Cl^– and Na⁺ in the leaf tissue. (4) For the same species, the critical temperatures for membrane-transport processes in roots were 2–2.5°C lower than in leaves. Possible physiological significance of these findings is discussed.