UV-B inhibition of hypocotyl growth in etiolated Arabidopsis thaliana seedlings is a consequence of cell cycle arrest initiated by photodimer accumulation.

Ultraviolet (UV) radiation is an important constituent of sunlight that determines plant morphology and growth. It induces photomorphogenic responses but also causes damage to DNA. Arabidopsis mutants of the endonucleases that function in nucleotide excision repair, xpf-3 and uvr1-1, showed hypersensitivity to UV-B (280-320nm) in terms of inhibition of hypocotyl growth. SOG1 is a transcription factor that functions in the DNA damage signalling response after γ-irradiation. xpf mutants that carry the sog1-1 mutation showed hypocotyl growth inhibition after UV-B irradiation similar to the wild type. A DNA replication inhibitor, hydroxyurea (HU), also inhibited hypocotyl growth in etiolated seedlings, but xpf-3 was not hypersensitive to HU. UV-B irradiation induced accumulation of the G2/M-specific cell cycle reporter construct CYCB1;1-GUS in wild-type Arabidopsis seedlings that was consistent with the expected accumulation of photodimers and coincided with the time course of hypocotyl growth inhibition after UV-B treatment. Etiolated mutants of UVR8, a recently described UV-B photoreceptor gene, irradiated with UV-B showed inhibition of hypocotyl growth that was not different from that of the wild type, but they lacked UV-B-specific expression of chalcone synthase (CHS), as expected from previous reports. CHS expression after UV-B irradiation was not different in xpf-3 compared with the wild type, nor was it altered after HU treatment. These results suggest that hypocotyl growth inhibition by UV-B light in etiolated Arabidopsis seedlings, a photomorphogenic response, is dictated by signals originating from UV-B absorption by DNA that lead to cell cycle arrest. This process occurs distinct from UVR8 and its signalling pathway responsible for CHS induction.

Transport of indole-3-butyric acid and indole-3-acetic acid in Arabidopsis hypocotyls using stable isotope labeling.

The polar transport of the natural auxins indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) has been described in Arabidopsis (Arabidopsis thaliana) hypocotyls using radioactive tracers. Because radioactive assays alone cannot distinguish IBA from its metabolites, the detected transport from applied [3H]IBA may have resulted from the transport of IBA metabolites, including IAA. To test this hypothesis, we used a mass spectrometry-based method to quantify the transport of IBA in Arabidopsis hypocotyls by following the movement of [13C1]IBA and the [13C1]IAA derived from [13C1]IBA. We also assayed [13C6]IAA transport in a parallel control experiment. We found that the amount of transported [13C1]IBA was dramatically lower than [13C6]IAA, and the IBA transport was not reduced by the auxin transport inhibitor N-1-naphthylphthalamic acid. Significant amounts of the applied [13C1]IBA were converted to [13C1]IAA during transport, but [13C1]IBA transport was independent of IBA-to-IAA conversion. We also found that most of the [13C1]IBA was converted to ester-linked [13C1]IBA at the apical end of hypocotyls, and ester-linked [13C1]IBA was also found in the basal end at a level higher than free [13C1]IBA. In contrast, most of the [13C6]IAA was converted to amide-linked [13C6]IAA at the apical end of hypocotyls, but very little conjugated [13C6]IAA was found in the basal end. Our results demonstrate that the polar transport of IBA is much lower than IAA in Arabidopsis hypocotyls, and the transport mechanism is distinct from IAA transport. These experiments also establish a method for quantifying the movement of small molecules in plants using stable isotope labeling.

Role for apyrases in polar auxin transport in Arabidopsis.

Recent evidence indicates that extracellular nucleotides regulate plant growth. Exogenous ATP has been shown to block auxin transport and gravitropic growth in primary roots of Arabidopsis (Arabidopsis thaliana). Cells limit the concentration of extracellular ATP in part through the activity of ectoapyrases (ectonucleoside triphosphate diphosphohydrolases), and two nearly identical Arabidopsis apyrases, APY1 and APY2, appear to share this function. These findings, plus the fact that suppression of APY1 and APY2 blocks growth in Arabidopsis, suggested that the expression of these apyrases could influence auxin transport. This report tests that hypothesis. The polar movement of [(3)H]indole-3-acetic acid in both hypocotyl sections and primary roots of Arabidopsis seedlings was measured. In both tissues, polar auxin transport was significantly reduced in apy2 null mutants when they were induced by estradiol to suppress the expression of APY1 by RNA interference. In the hypocotyl assays, the basal halves of APY-suppressed hypocotyls contained considerably lower free indole-3-acetic acid levels when compared with wild-type plants, and disrupted auxin transport in the APY-suppressed roots was reflected by their significant morphological abnormalities. When a green fluorescent protein fluorescence signal encoded by a DR5:green fluorescent protein construct was measured in primary roots whose apyrase expression was suppressed either genetically or chemically, the roots showed no signal asymmetry following gravistimulation, and both their growth and gravitropic curvature were inhibited. Chemicals that suppress apyrase activity also inhibit gravitropic curvature and, to a lesser extent, growth. Taken together, these results indicate that a critical step connecting apyrase suppression to growth suppression is the inhibition of polar auxin transport.

Reproducibility of bracket positioning in the indirect bonding technique.

INTRODUCTION: Current studies have compared indirect bonding with direct placement of orthodontic brackets; many of these have shown that indirect bonding is generally a more accurate technique. However, the reproducibility of an indirect bonding setup by an orthodontist has yet to be described in the literature. Using cone-beam computed tomography and computer-assisted modeling software, we evaluated the consistency of orthodontists in placing orthodontic brackets at different times. METHODS: Five orthodontists with experience in indirect bonding were selected to place brackets on 10 different casts at 3 time periods (n = 30 per orthodontist). Each participant completed an initial indirect bonding setup on each cast; subsequent bracket placements were completed twice at monthly intervals for comparison with the initial setup. The casts were scanned using an iCAT cone-beam computed tomography scanner (Imaging Sciences International, Hatfield, Pa) and imported into Geomagic Studio software (Geomagic, Research Triangle Park, NC) for superimposition and analysis. The scans for each time period were superimposed on the initial setup in the imaging software, and differences between bracket positions were calculated. For each superimposition, the measurements recorded were the greatest discrepancies between individual brackets as well as the mean discrepancies and standard deviations between all brackets on each cast. RESULTS: Single-factor and repeated-measure analysis of variance showed no statistically significant differences between time points of each orthodontist, or among the orthodontists for the parameters measured. The mean discrepancy was 0.1 mm for each 10-bracket indirect bonding setup. CONCLUSIONS: Orthodontists are consistent in selecting bracket positions for an indirect bonding setup at various time periods.

Low-fluence red light increases the transport and biosynthesis of auxin.

In plants, light is an important environmental signal that induces photomorphogenesis and interacts with endogenous signals, including hormones. We found that light increased polar auxin transport in dark-grown Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) hypocotyls. In tomato, this increase was induced by low-fluence red or blue light followed by 1 d of darkness. It was reduced in phyA, phyB1, and phyB2 tomato mutants and was reversed by far-red light applied immediately after the red or blue light exposure, suggesting that phytochrome is involved in this response. We further found that the free indole-3-acetic acid (IAA) level in hypocotyl regions below the hook was increased by red light, while the level of conjugated IAA was unchanged. Analysis of IAA synthesized from [¹³C]indole or [¹³C]tryptophan (Trp) revealed that both Trp-dependent and Trp-independent IAA biosynthesis were increased by low-fluence red light in the top section (meristem, cotyledons, and hook), and the Trp-independent pathway appears to become the primary route for IAA biosynthesis after red light exposure. IAA biosynthesis in tissues below the top section was not affected by red light, suggesting that the increase of free IAA in this region was due to increased transport of IAA from above. Our study provides a comprehensive view of light effects on the transport and biosynthesis of IAA, showing that red light increases both IAA biosynthesis in the top section and polar auxin transport in hypocotyls, leading to unchanged free IAA levels in the top section and increased free IAA levels in the lower hypocotyl regions.

Complexity of the auxin biosynthetic network in Arabidopsis hypocotyls is revealed by multiple stable-labeled precursors.

Auxin is a key regulator of plant development and in Arabidopsis thaliana can be synthesized through multiple pathways; however, the contributions of various biosynthetic pathways to specific developmental processes are largely unknown. To trace the involvement of various biosynthetic routes to indole-3-acetic acid (IAA) under conditions that induce adventitious root formation in Arabidopsis hypocotyls, we treated seedlings with three different stable isotope-labeled precursors ([13C6]anthranilate, [15N1]indole, and [13C3]serine) and monitored label incorporation into a number of proposed biosynthesis intermediates as well as IAA. We also employed inhibitors targeting tryptophan aminotransferases and flavin monooxygenases of the IPyA pathway, and treatment with these inhibitors differentially altered the labeling patterns from all three precursors into intermediate compounds and IAA. [13C3]Serine was used to trace utilization of tryptophan (Trp) and downstream intermediates by monitoring 13C incorporation into Trp, indole-3-pyruvic acid (IPyA), and IAA; most 13C incorporation into IAA was eliminated with inhibitor treatments, suggesting Trp-dependent IAA biosynthesis through the IPyA pathway is a dominant contributor to the auxin pool in de-etiolating hypocotyls that can be effectively blocked using chemical inhibitors. Labeling treatment with both [13C6]anthranilate and [15N1]indole simultaneously resulted in higher label incorporation into IAA through [15N1]indole than through [13C6]anthranilate; however, this trend was reversed in the proposed precursors that were monitored, with the majority of isotope label originating from [13C6]anthranilate. An even greater proportion of IAA became [15N1]-labeled compared to [13C6]-labeled in seedlings treated with IPyA pathway inhibitors, suggesting that, when the IPyA pathway is blocked, IAA biosynthesis from labeled indole may also come from an origin independent of the measured pool of Trp in these tissues.

UV-B Irradiation Results in Inhibition of Hypocotyl Elongation, Cell Cycle Arrest, and Decreased Endoreduplication Mediated by miR5642.

UV-B as a component of natural solar radiation can induce damage and morphological development in plants. The UV-B response from germination and early development in seedlings is still largely unknown, with most studies focused on older, light-exposed seedlings. We used fluence response curves measuring hypocotyl length after UV-B exposure coupled with RNA-seq and sRNA-seq evaluation of the early seedling response in the model organism Arabidopsis thaliana. We identified miR5642 as a potential novel key regulator of UV-B responses. miR5642 is a noncanonical miRNA predicted to target previously known and unknown components involved in hypocotyl growth inhibition. These include (i) SMAX1, a signal transmitter for seedling germination and growth; (ii) ZAT1, an uncharacterized transcription factor; and (iii) membrane pores and transporters (VHA-E1, VHA-E3, EPSIN-LIKE and PIP1.4) implicated in cell elongation. In addition, HY5 and HYH, two homologous and redundant transcription factors involved in seedling photomorphogenesis, may interact with these newly identified components. Interestingly, UV-B-induced DNA photodimer formation seems to be the direct trigger leading to inhibition of hypocotyl growth through a combination of cellular decisions including cell cycle arrest, reduced endoreduplication and reduced cell elongation, and this inhibition appears to be modulated by miR5642 target genes.

Manipulation of Continuous and End-of-Day Red/Far-Red Light Ratios Affects Glucobrassicin and Gluconasturtiin Accumulation in Cabbage (Brassica oleracea) and Watercress (Nasturtium officinale), Respectively.

Cabbage (Brassica oleracea) and watercress (Nasturtium officinale) produce glucobrassicin (GBS) and gluconasturtiin (GNST), precursors of chemopreventive compounds. Their accumulation is affected by environmental signals. We studied the impact of the red to far-red light (R/FR) ratio on GBS concentration in red ″Ruby Ball″ and green ″Tiara″ cabbage. Foliar shading, via weed surrogates that competed with cabbage plants for specific durations, induced R/FR variation among treatments. ″Ruby Ball″ GBS concentrations were the highest when R/FR within the canopy was the lowest. ″Tiara″ was unaffected by competition. The same trend was observed in a controlled environment using R and FR LEDs without weeds present. ″Ruby Ball″ subjected to an R/FR = 0.3 treatment had 2.5- and 1.4-fold greater GBS concentration compared to R/FR = 1.1 and 5.0 treatments combined. Watercress given end-of-day (EOD) R and/or FR pulses after the main photoperiod had the lowest GNST concentrations after an EOD FR pulse but the highest concentrations after an R followed by FR pulse.

Phytochromes differentially regulate seed germination responses to light quality and temperature cues during seed maturation.

The ratio of red to far-red light (R : FR) experienced by seeds during maturation affects germination, but the genetic regulation of this effect is poorly understood. In Arabidopsis thaliana, responses to R : FR are governed by five phytochrome photoreceptors, PHYA-PHYE. PHYA, PHYB and PHYE mediate germination, but their roles in germination response to the seed maturation environment are largely unknown. Seeds of A. thaliana phytochrome mutants and natural accessions were matured in a factorial combination of cold (16 degrees C) and warm (24 degrees C) temperatures and high (R : FR = 1) and low (R : FR = 0.6) R : FR environments, resembling sunlight and foliar shade, respectively. Germination was observed in resulting seeds. All five phytochromes mediated germination responses to seed maturation temperature and/or R : FR environment. PHYA suppressed germination in seeds matured under cold temperature, and PHYB promoted germination under the same conditions. PHYD and PHYE promoted germination of seeds matured under warm temperature, but this effect diminished when seeds matured under reduced R : FR. The A. thaliana natural accessions exhibited interesting variation in germination responses to the experimental conditions. Our results suggest that the role of individual PHY loci in regulating plant responses to R : FR varies depending on temperature and provide novel insights into the genetic basis of maternal effects.

Photobiological properties of the inhibition of etiolated Arabidopsis seedling growth by ultraviolet-B irradiation.

Alteration of 'normal' levels of ultraviolet-B light (UV-B, 280-320 nm) can affect plant chemical composition as well as growth; however, little is known about how plants perceive UV-B light. We have carried out fluence response curves, and demonstrated that the growth inhibition of etiolated Arabidopsis thaliana seedlings by low fluence UV light is specific to UV-B and not UV-A (320-390 nm). The response shows reciprocity between duration and intensity, at least over a limited range, and thus depends only on photon fluence and not on photon flux. The action spectrum for this response indicates a peak of maximum effectiveness at 290 nm, and response spectra at different fluences indicate that the most effective wavelength at 30,000 micromol m(-2) is 290 nm, whereas 300 nm light was the most effective at 100,000 micromol m(-2). This response occurs in mutant seedlings deficient in cryptochrome, phytochrome or phototropin, suggesting that none of the known photoreceptors is the major UV-B photoreceptor. Some null mutants in DNA repair enzymes show hypersensitivity to UV-B, suggesting that even at low fluence rates, direct damage to DNA may be one component of the response to UV-B.

3-Acyl dihydroflavonols from poplar resins collected by honey bees are active against the bee pathogens Paenibacillus larvae and Ascosphaera apis.

Honey bees, Apis mellifera, collect antimicrobial plant resins from the environment and deposit them in their nests as propolis. This behavior is of practical concern to beekeepers since the presence of propolis in the hive has a variety of benefits, including the suppression of disease symptoms. To connect the benefits that bees derive from propolis with particular resinous plants, we determined the identity and botanical origin of propolis compounds active against bee pathogens using bioassay-guided fractionation against the bacterium Paenibacillus larvae, the causative agent of American foulbrood. Eleven dihydroflavonols were isolated from propolis collected in Fallon, NV, including pinobanksin-3-octanoate. This hitherto unknown derivative and five other 3-acyl-dihydroflavonols showed inhibitory activity against both P. larvae (IC50 = 17-68 µM) and Ascosphaera apis (IC50 = 8-23 µM), the fungal agent of chalkbrood. A structure-activity relationship between acyl group size and antimicrobial activity was found, with longer acyl groups increasing activity against P. larvae and shorter acyl groups increasing activity against A. apis. Finally, it was determined that the isolated 3-acyl-dihydroflavonols originated from Populus fremontii, and further analysis showed these compounds can also be found in other North American Populus spp.

The effect of temperature, photoperiod, and light quality on gluconasturtiin concentration in watercress (Nasturtium officinale R. Br.).

The effects of different growth regimes on gluconasturtiin concentration in watercress (Nasturtium officinale R. Br.) were investigated. Watercress plantlets at the 5th mature leaf stage (ca. 2 weeks old) were exposed to different day and night temperatures, to long (16 h) or short (8 h) days, to red (R) or far-red (FR) light given during the main long day photoperiod, and finally to R or FR light given at the end of the main photoperiod. Watercress plants grown under long days contained a 30-40% higher gluconasturtiin concentration and had a higher fresh weight than watercress plants grown under short days. Watercress plants grown under long days and temperatures of 15 or 10 degrees C had at least a 50% higher gluconasturtiin concentration, but a lower fresh weight, than that of plants grown at 20 or 25 degrees C. Watercress plants grown under metal halide light enriched with R light had approximately a 25-40% higher concentration of gluconasturtiin as compared to the FR-enriched plants. Likewise, a brief R light exposure at the end of the main photoperiod resulted in approximately a 25% or higher concentration of gluconasturtiin as compared to a FR end-of-day exposure. These data indicate that the concentration of gluconasturtiin in watercress can be significantly increased by growing plants at lower temperatures, under long days, and by exposure to R light.

Pain control in orthodontics using a micropulse vibration device: A randomized clinical trial.

OBJECTIVE: To investigate the relationship between a micropulse vibration device and pain perception during orthodontic treatment. MATERIALS AND METHODS: This study was a parallel group, randomized clinical trial. A total of 58 patients meeting eligibility criteria were assigned using block allocation to one of two groups: an experimental group using the vibration device or a control group (n  =  29 for each group). Patients used the device for 20 minutes daily. Patients rated pain intensity on a visual analog scale at appropriate intervals during the weeks after the separator or archwire appointment. Data were analyzed using repeated measures analysis of variance at α  =  .05. RESULTS: During the 4-month test period, significant differences between the micropulse vibration device group and the control group for overall pain (P  =  .002) and biting pain (P  =  .003) were identified. The authors observed that perceived pain was highest at the beginning of the month, following archwire adjustment. CONCLUSION: The micropulse vibration device significantly lowered the pain scores for overall pain and biting pain during the 4-month study period.

Protocol: High-throughput and quantitative assays of auxin and auxin precursors from minute tissue samples.

BACKGROUND: The plant hormone auxin, indole-3-acetic acid (IAA), plays important roles in plant growth and development. The signaling response to IAA is largely dependent on the local concentration of IAA, and this concentration is regulated by multiple mechanisms in plants. Therefore, the precise quantification of local IAA concentration provides insights into the regulation of IAA and its biological roles. Meanwhile, pathways and genes involved in IAA biosynthesis are not fully understood, so it is necessary to analyze the production of IAA at the metabolite level for unbiased studies of IAA biosynthesis. RESULTS: We have developed high-throughput methods to quantify plant endogenous IAA and its biosynthetic precursors including indole, tryptophan, indole-3-pyruvic acid (IPyA), and indole-3-butyric acid (IBA). The protocol starts with homogenizing plant tissues with stable-labeled internal standards added, followed by analyte purification using solid phase extraction (SPE) tips and analyte derivatization. The derivatized analytes are finally analyzed by selected reaction monitoring on a gas chromatograph-mass spectrometer (GC-MS/MS) to determine the precise abundance of analytes. The amount of plant tissue required for the assay is small (typically 2-10 mg fresh weight), and the use of SPE tips is simple and convenient, which allows preparation of large sets of samples within reasonable time periods. CONCLUSIONS: The SPE tips and GC-MS/MS based method enables high-throughput and accurate quantification of IAA and its biosynthetic precursors from minute plant tissue samples. The protocol can be used for measurement of these endogenous compounds using isotope dilution, and it can also be applied to analyze IAA biosynthesis and biosynthetic pathways using stable isotope labeling. The method will potentially advance knowledge of the role and regulation of IAA.

Effect of light on seed germination of eight wetland Carex species.

BACKGROUND AND AIMS: In wetland plant communities, species-specific responses to pulses of white light and to red : far-red light ratios can vary widely and influence plant emergence from the seed bank. Carex species are the characteristic plants of sedge meadows of natural prairie wetlands in mid-continental USA but are not returning to restored wetlands. Little is known about how light affects seed germination in these species-information which is necessary to predict seed bank emergence and to develop optimal revegetation practices. The effects of light on germination in eight Carex species from prairie wetlands were investigated. METHODS: Non-dormant seeds of eight Carex species were used to determine the influence of light on germination by examining: (a) the ability of Carex seeds to germinate in the dark; (b) the effect of different lengths of exposures to white light on germination; (c) whether the effect of white light can be replaced by red light; and (d) whether the germination response of Carex seeds to white or red light is photoreversible by far-red light. KEY RESULTS: Seeds of C. brevior and C. stipata germinated >25 % in continuous darkness. Germination responses after exposure to different lengths of white light varied widely across the eight species. Carex brevior required <15 min of white light for > or =50 % germination, while C. hystericina, C. comosa, C. granularis and C. vulpinoidea required > or =8 h. The effect of white light was replaced by red light in all species. The induction of germination after exposure to white or red light was reversed by far-red light in all species, except C. stipata. CONCLUSIONS: The species-specific responses to simulated field light conditions suggest that (a) the light requirements for germination contribute to the formation of persistent seed banks in these species and (b) in revegetation efforts, timing seed sowing to plant community development and avoiding cover crops will improve Carex seed germination.

Wear comparison of thermoplastic materials used for orthodontic retainers.

Clear thermoplastic retainers are an alternative to fixed lingual retainers and removable Hawley appliances. However, thermoplastic retainers have demonstrated poor wear resistance and durability after only a few months of use. In this study, a simulated wear device was used to compare the wear of different thermoplastic materials used for orthodontic retainers. Three thermoplastic products were evaluated: C+ (Raintree Essix, New Orleans, La),.040-in Invisacryl C (Great Lakes Orthodontics, Towanda, NY), and.040-in TR sheet material (Bay Dental Direct, Bay City, Mich). Twenty specimens were fabricated for each group. The specimens were vacuum thermoformed according to the manufacturers' recommendations and subjected to wear for 1000 cycles in a wear apparatus with steatite ceramic abraders. Depth of wear was determined by surface profilometry. The maximum peak-to-valley measurement was recorded for each specimen. Mean wear (SD) in microns was as follows: C+, 5.9 (2.4); Invisacryl C, 6.1 (2.6); and TR, 1.6 (0.9). One-way analysis of variance detected a significant difference between groups (P <.001). TR material, a hard polyethylene terephthalate glycol copolymer (PETG), demonstrated greater resistance to wear than did the other 2 materials, which were softer, polypropylene-based thermoplastics. There was no evidence to suggest a difference in mean wear between the 2 polypropylene-based materials (P >.05).