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A double-stranded RNA from a Phytophthora species is related to the plant endornaviruses and contains a putative UDP glycosyltransferase gene.

A new dsRNA was isolated from a Phytophthora isolate from Douglas fir. Sequence analysis showed the dsRNA to consist of 13 883 bp and to contain a single open reading frame with the potential to encode a polyprotein of 4548 aa. This polyprotein contained amino acid sequence motifs characteristic of virus RNA-dependent RNA polymerases (RdRps) in its C-terminal region and motifs characteristic of RNA helicases in its N-terminal region. These sequence motifs were related to corresponding motifs in plant viruses in the genus Endornavirus. In phylogenetic trees constructed from the RdRp and helicase motifs of a range of ssRNA and dsRNA viruses, the Phytophthora RdRp and helicase sequences clustered with those of the plant endornaviruses with good bootstrap support. The properties of the Phytophthora dsRNA are consistent with its being classified as the first non-plant member of the genus Endornavirus, for which we propose the name phytophthora endornavirus 1 (PEV1). A region between the RdRp and helicase domains of the PEV1 protein had significant amino acid sequence similarity to UDP glycosyltransferases (UGTs). Two sequence motifs were identified, one characteristic of all UGTs and the other characteristic of sterol UGTs. The PEV1 UGT would be the first for an RNA virus, although ecdysteroid UGT genes have been found in many baculoviruses. The PEV1 UGT was only distantly related to baculovirus ecdysteroid UGTs, which belong to a family distinct from the sterol UGTs.

Amino Acid Motifs↗

Wound-induced terpene synthase gene expression in Sitka spruce that exhibit resistance or susceptibility to attack by the white pine weevil.

We analyzed the expression pattern of various terpene synthase (TPS) genes in response to a wounding injury applied to the apical leader of Sitka spruce (Picea sitchensis Bong. Carr.) genotypes known to be resistant (R) or susceptible (S) to white pine weevil (Pissodes strobi Peck.) attack. The purpose was to test if differences in constitutive or wound-induced TPS expression can be associated with established weevil resistance. All wounding treatments were conducted on 9-year-old R and S trees growing under natural field conditions within the range of variation for weevil R and S genotypes. Representative cDNAs of the monoterpene synthase (mono-TPS), sesquiterpene synthase (sesqui-TPS), and diterpene synthase (di-TPS) classes were isolated from Sitka spruce to assess TPS transcript levels. Based on amino acid sequence similarity, the cDNAs resemble Norway spruce (Picea abies) (-)-linalool synthase (mono-TPS; PsTPS-Linl) and levopimaradiene/abietadiene synthase (di-TPS; PsTPS-LASl), and grand fir (Abies grandis) delta-selinene synthase (sesqui-TPS; PsTPS-Sell). One other mono-TPS was functionally identified as (-)-limonene synthase (PsTPS-Lim). No significant difference in constitutive expression levels for these TPSs was detected between R and S trees. However, over a postwounding period of 16 d, only R trees exhibited significant transcript accumulation for the mono- and sesqui-TPS tested. Both R and S trees exhibited a significant accumulation of PsTPS-LASl transcripts. An assessment of traumatic resin duct formation in wounded leaders showed that both R and S trees responded by forming traumatic resin ducts; however, the magnitude of this response was significantly greater in R trees. Collectively, our data imply that the induced resinosis response is an important aspect of defense in weevil R Sitka spruce trees growing under natural conditions.

Alkyl and Aryl Transferases↗

Developmental and light-regulated expression of individual members of the light-harvesting complex b gene family in Pinus palustris.

Angiosperms requires light for multiple aspects of chloroplast development, including chlorophyll synthesis and induction of expression of the mRNAs encoding the major polypeptides of the light-harvesting complex of photosystem II (Lhcb genes). In contrast, many conifers, including pines, firs, and spruces, can accumulate chlorophyll and the light-harvesting chlorophyll a/b-binding proteins of photosystem II in complete darkness. To understand the factors responsible for the regulation of expression of individual Lhcb mRNAs in the pine Pinus palustris, we have prepared sequence-specific cDNA probes for each of three family members, Lhcb1*Pp1, Lhcb2*Pp1, and Lhcb2*Pp2, and have studied the expression of two of these, Lhcb1*Pp1 and Lhcb2*Pp2, in detail. The levels of expression of each sequence were disparate, and Lhcb1*Pp1-encoded transcripts were the most abundant in the light. Both Lhcb1*Pp1 and Lhcb2*Pp2 mRNAs were expressed in stems and cotyledons, but Lhcb1*Pp1 mRNA was present at about 10-fold lower levels in stems than in cotyledons, in contrast to Lhcb2*Pp2 mRNA, which was expressed at higher levels in stems than in cotyledons. Both Lhcb1*Pp1 and Lhcb2*Pp2 mRNAs were absent in embryos but were expressed during seedling development. The levels increased with age in both the light and the dark and in both cases were about 2-fold higher in the light than in the dark. Despite the expression of Lhcb1*Pp1 and Lhcb2*Pp2 mRNAs during development in darkness, the levels of both mRNAs increased in dark-grown seedlings given red light in the low fluence range within 2 h of treatment.

Amino Acid Sequence↗

Kinetics of the Daily Rate of Photosynthesis at Low Temperatures for two Conifers.

The daily course of photosynthesis at low temperatures in 2 coniferous species, Pinus ponderosa Laws., and Pseudotsuga menziesii (Mirb.) Franco, were studied using controlled environment facilities. After having been grown at a 23 degrees day, and 19 degrees night for a year, seedlings were acclimatized for 4 months to either a 3 degrees , 7 degrees or 11 degrees day all under 1200 ft-c of light and followed by a 16-hour night at 3 degrees . Measurement of photosynthesis at 1200 ft-c revealed 3 separate responses. First, the rapidity at which the plants attained their maximum photosynthesis when the lights were turned on depended upon the species, the current temperature, and the previous temperature condition to which the plants had become acclimatized. The warmer the day temperature the sooner the daily maximum was reached. Second, fluctuations in the rate of photosynthesis during the day varied with the species and the day temperature. Photosynthesis in both fir and pine kept at an 11 degrees day and pines kept at a 7 degrees day attained a daily peak rate followed by a decline. This decline occurred even though temperature and light were kept constant, the CO(2) level was returned to 320 ppm from 290 ppm, and the plants were kept well watered. At a 3 degrees day neither species showed this decline. Third, a plant transferred to another temperature acquired a new stable daily photosynthetic pattern. The number of days required for stabilization depended upon the previous temperature history of the plant. The adjustment rate was faster when the temperature was raised than when it was lowered.

Journal Article↗

The Structure of Plant Cell Walls: VI. A Survey of the Walls of Suspension-cultured Monocots.

The primary cell walls of six suspension-cultured monocots and of a single suspension-cultured gymnosperm have been investigated with the following results: (a) the compositions of all six monocot cell walls are remarkably similar, despite the fact that the cell cultures were derived from diverse tissues; (b) the cell walls of suspension-cultured monocots differ substantially from those of suspension-cultured dicots and from the suspension-cultured gymnosperm; (c) an arabinoxylan is a major component (40% or more by weight) of monocot primary cell walls; (d) mixed beta-1,3; beta-1,4-glucans were found only in the cell wall preparations of rye grass endosperm cells, and not in the cell walls of any of the other five monocot cell cultures nor in the walls of suspension-cultured Douglas fir cells; (e) the monocot primary cell walls studied contain from 9 to 14% cellulose, 7 to 18% uronic acids, and 7 to 17% protein; (f) hydroxyproline accounts for less than 0.2% of the cell walls of monocots. Similar data on the soluble extracellular polysaccharides secreted by these cells are included.

Journal Article↗

Photosynthetic action spectra of trees: I. Comparative photosynthetic action spectra of one deciduous and four coniferous tree species as related to photorespiration and pigment complements.

Comparative isoenergetic action spectra of net photosynthesis for intact, current year foliage of five tree species were determined from 400 to 710 nm by CO(2) exchange analysis. The blue (400 to 500 nm) peak of net photosynthetic activity for the green broadleaves of red alder (Alnus rubra Bong.) was reduced to a plateau for the green needle-leaves of Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) and Sitka spruce (Picea sitchensis [Bong.] Carr.), a shoulder for the blue-green needles of Colorado spruce (Picea pungens Engelm.), and a reduced shoulder for the blue-white needles of Blue spruce (Picea pungens var. hoospii). These differences were attributable neither to a differential blue light stimulation of photorespiration nor to a differential presence of a nonplastid screening pigment. The conifers all had similar carotenoid-chlorophyll ratios, with approximately 50% more carotenoid relative to chlorophyll as compared to red alder. Blue light absorption and low efficiency of energy transfer by the carotenoids probably accounts for the low net photosynthetic activity of the green conifers in blue light as compared to red alder. Leaf form per se (broad versus needle) had no distinguishable influence on these results.

Journal Article↗

Deep undercooling of tissue water and winter hardiness limitations in timberline flora.

Deep undercooled tissue water, which froze near -40 C, was found in winter collected stem and leaf tissue of the dominant timberline tree species of the Colorado Rocky Mountains, Engelmann spruce (Picea engelmannii (Parry) Engelm.) and subalpine fir (Abies lasiocarpa (Hook.) Nutt.), and in numerous other woody species in and below the subalpine vegetation zone. Previous work on numerous woody plants indicates that deep undercooling in xylem makes probable a -40 C winter hardiness limit in stem tissue. Visual injury determinations and electrolyte loss measurements on stem tissue revealed injury near -40 C associated with the freezing of the deep undercooled stem tissue water. These results suggest that the winter hardiness limit of this woody flora is near -40 C. The relevance of deep undercooling in relation to timberline, the upper elevational limit of the subalpine forest, is discussed.

Journal Article↗

Leaf conductance as a function of photosynthetic photon flux density and absolute humidity difference from leaf to air.

FOR AN ENTIRE SEASON OF STOMATAL ACTIVITY, LEAF OR NEEDLE CONDUCTANCE WAS OBSERVED ON FOUR SPECIES, EACH IN A DIFFERENT GENUS: Engelmann spruce (Picea engelmannii Parry ex Engelm.), subalpine fir (Abies lasiocarpa [Hook.] Nutt.), lodgepole pine (Pinus contorta var. latifolia Engelm.), and aspen (Populus tremuloides Michx.). Conductance in the natural environment was described for all species by photosynthetic photon flux density (PPFD) and absolute humidity difference from leaf to air (DAH), as follows: Conductance = b(1) ( radicalPPFD/ radicalDAH) + b(2) ( radicalPPFD/DAH) + b(3) ( radicalPPFD/DAH(2)). The only data not fitting this relationship were conifer data collected after freezing nights or aspen data collected during a short period in August when water stress occurred. In both cases, leaf conductance was reduced. It is proposed that PPFD and DAH are primary factors controlling stomatal function for plants growing in their native range; secondary factors, such as temperature and water stress, affect conductance intermittently, except when plants are growing outside their normal environmental conditions.

Journal Article↗

Enzymic and nonenzymic reduction of (+)-dihydroquercetin to its 3,4,-diol.

A NADPH-dependent reductase activity, capable of converting (+)-dihydroquercetin (2,3-trans) to its 3,4-diol (a leucocyanidin), has been demonstrated in crude, soluble protein extracts derived from cell suspension cultures of Douglas fir (Pseudotsuga menziessi). Neither NADH nor ascorbate substituted as the H-donor. Quantitative analyses were based on the production of cyanidin, the formation of an adduct with vanillin, and on absorbance at 280 nanometers. Nonenzymic reduction of (+)-dihydroquercetin with NaBH(4) produced two presumably isomeric flavan-3,4,-diols. One of these was similar to the enzymically produced diol, based on products isolated by chromatography on paper, on thin-layer cellulose and on C(18) reversed-phase columns (high performance liquid chromatography), and on the conversion of the diol to the all-trans dimer of (+)-catechin upon the addition of (+)-catechin.

Journal Article↗

A comparison of methods for determining compartmental analysis parameters.

The traditional method for determining compartmental analysis parameters relies on a visual selection of data points to be used for regression of data from each cellular compartment. This method is appropriate when the compartments are kinetically discrete and are easily discernible. However, where treatment effects on compartment parameters are being evaluated, a more objective method for determining initial parameters is desirable.Three methods were examined for determining initial isotopic contents and half-times of (86)Rb elution from cellular compartments using theoretical data with known parameters. Experimental data from roots of Douglas fir (Pseudotsuga menziesii [Mirb.] Franco) and barley (Hordeum vulgare L.) intact seedlings were also used. The three methods were a visually assisted, linear regression on data of semilog plot of isotope elution versus time, a microcomputer-assisted, linear regression on semilog plot where maximization of the square of the correlation coefficient (r(2)) was the criterion to determine data points needed for each regression and a mainframe computer-assisted, direct nonlinear regression on elution data using a model of the sum of three exponential decay functions. The visual method resulted in the least accurate estimates of compartmental analysis parameters. The microcomputer-assisted and nonlinear regression methods calculated the parameters equally well.

Journal Article↗

Flavan-3-ol Biosynthesis : The Conversion of (+)-Dihydromyricetin to Its Flavan-3,4-Diol (Leucodelphinidin) and to (+)-Gallocatechin by Reductases Extracted from Tissue Cultures of Ginkgo biloba and Pseudotsuga menziesii.

Extracts of callus or cell suspension cultures from petioles of Ginkgo biloba catalyzed the production of (+)-gallocatechin (2,3-trans-3,5,7,3',4',5'-hexahydroxy-flavan) from (+)-dihydromyricetin (5'-hydroxy-dihydroquercetin) along with the expected 3,4-cis-diol intermediate, leucodelphinidin, in a NADPH-dependent double-step reductase reaction at pH 7.4. The latter diol, isolated from the above incubation mixture, produced (+)-gallocatechin in a NADPH-dependent reaction. Extracts from tissue cultures derived from needles of Pseudotsuga menziesii (Douglas fir) also produced significant amounts of the 3,4-diol from dihydromyricetin. (+)-Dihydromyricetin, purified via paper chromatography from leaves of Leptarrhena pyrolifolia, was reduced by NaBH(4) to the presumed 3,4-trans-diol and acid epimerized to the 3,4-cis-diol.

Journal Article↗

Buffer capacities of leaves, leaf cells, and leaf cell organelles in relation to fluxes of potentially acidic gases.

Since environmental pollution by potentially acidic gases such as SO(2) causes proton release inside leaf tissues, homogenates of needles of spruce (Picea abies) and fir (Abies alba) and of leaves of spinach (Spinacia oleracea) and barley (Hordeum vulgare) were titrated and buffer capacities were determined as a function of pH. Titration curves of barley leaves were compared with titration curves of barley mesophyll protoplasts. From the protoplasts, chloroplasts and vacuoles were isolated and subjected to titration experiments. From the titration curves, the intracellular distribution of buffering capacities could be deduced. Buffering was strongly pH-dependent. It was high at the extremes of pH but still significant close to neutrality. Owing to its large size, the vacuole was mainly responsible for cellular buffering. However, on a unit volume basis, the cytoplasm was much more strongly buffered than the vacuole. Potentially acidic gases are trapped in the anionic form. They release protons when trapped. The magnitude of diffusion gradients from the atmosphere into the cells, which determines flux, depends on intracellular pH. In the light, the chloroplast stroma, as the most alkaline leaf compartment, has the highest trapping potential. Acidification of the chloroplast stroma inhibits photosynthesis. The trapping potential of the chloroplast is followed by that of the cytosol. Compared with the cytoplasm, the vacuole possesses little trapping potential in spite of its large size. It is particularly small in the acidic vacuoles of conifer needles. In the physiological pH range (slightly above neutrality), chloroplast buffering was about 1 microequivalents H(+) per milligram chlorophyll per pH unit or 35 microequivalents H(+) per milliliter per pH unit in barley or spinach chloroplasts. This compares with SO(2)-generated H(+) production of somewhat more than 1 microequivalent H(+) per milligram chlorophyll per hour, which results from observed SO(2) uptake of leaves when stomata were open and the atmospheric SO(2) concentration was 0.4 microliters per liter (GE Taylor Jr, DT Tingey 1983 Plant Physiol 72: 237-244). At lower SO(2) concentrations, similar H(+) generation inside the cells requires correspondingly longer exposure times.

Journal Article↗

Comparison of Proanthocyanidins and Related Compounds in Leaves and Leaf-Derived Cell Cultures of Ginkgo bioloba L., Pseudotsuga menziesii Franco, and Ribes sanguineum Pursh.

Proanthocyanidins, flavan-3-ols, and their flavanoid precursors in leaves and leaf-derived callus and cell suspension cultures have been isolated and analyzed by high performance liquid chromatography with C(18) columns, paper chromatography, and by chemical and spectrophotometric methods. Cultures of Ginkgo biloba and Pseudotsuga menziesii (Douglas-fir) produced much greater amounts of proanthocyanidins than leaves per milligram dry weight. In cultures, however, the prodelphinidin component relative to that of procyanidins decreased; this was most pronounced in Pseudotsuga. In contrast, callus cultures of Ribes sanguineum accumulated proanthocyanidins in amounts about equal to those in intact leaves per milligram dry weight and the prodelphinidin content remained high. Although Ginkgo and Ribes leaves contained major amounts of flavan-3-ols and dimers with the 2,3-cis-stereochemistry, their cultures tended to synthesize 2,3-trans-isomers instead. Glycosides of flavanone and 3-hydroxyflavanone precursors accumulated in medium to high amounts on a dry weight basis in leaves and cultures of Ribes and Pseudotsuga, and the 3'-glycosidic linkage predominated when the latter species was cultured with 2,4-dichlorophenoxyacetic acid rather than naphthaleneacetic acid.

Journal Article↗

Extended Host Range of Agrobacterium tumefaciens in the Genus Pinus.

Two-to 4-month-old seedlings of nine pine species (Pinus eldarica Medw., Pinus elliottii Engelm., Pinus jeffreyi Grev. & Balf., Pinus lambertiana Dougl., Pinus ponderosa Laws., Pinus radiata D. Don, Pinus sylvestris L., Pinus taeda L., Pinus virginiana Mill), Douglas fir (Pseudotsuaa menziesii (Mirb.) Franco) and incense cedar (Libocedrus decurrens Torr.) were inoculated with five strains of Agrobacterium tumefaciens. Transformation occurred in all conifer species tested as determined by gall formation and opine production. The frequency of gall formation varied by host species, by bacterial strain, and was related to the age of the stem when inoculated. Galls were visible 8 to 12 weeks after inoculation and were small (often less than 2.5 millimeters in diameter). Fewer than half (230 of 502) of the galls originally formed on the trees were present after 1 year, and 26 of these grew to diameters greater than 2 centimeters. The majority of these larger galls (18 of 26) were found in P. radiata. Bacterial strain-specific opines were found in 67 of the 81 gall tissues sampled.

Journal Article↗

Cytokinins in Vegetative and Reproductive Buds of Pseudotsuga menziesii.

Immunoaffinity techniques using columns of immobilized antibodies raised against zeatin riboside and isopentenyladenosine were found to be effective in isolating cytoklnins from vegetative, female, and male buds of Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco). The purified cytokinins were separated by reverse phase high performance liquid chromatography and analyzed by radioimmunoassay. Confirmation of cytokinin identities was by gas chromatography-mass spectrometry. Immediately prior to bud burst, all bud types contained three major cytokinins: isopentenyladenosine, zeatin riboside, and a hexose conjugate of zeatin riboside (not zeatin riboside O-glucoside). Zeatin-type cytokinins were present in relatively high concentration in vegetative and female buds. In male buds, however, relatively high levels of isopentenyladenosine were found together with low levels of zeatin-type cytokinins.

Journal Article↗

A gymnosperm extensin contains the serine-tetrahydroxyproline motif.

The extensin family is a diverse group of hydroxyproline-rich glycoproteins located in the cell wall and characterized by repetitive peptide motifs glycosylated to various degrees. The origin of this diversity and its relationship to function led us earlier to compare extensins of the two major groups of angiosperms from which we concluded that the highly glycosylated Ser-Hyp(4) motif was characteristic of advanced herbaceous dicots, occurring rarely or not at all in a representative graminaceous monocot (Zea mays) and a chenopod (Beta vulgaris) representative of primitive dicots. Because these results could arise either from loss or acquisition of a characteristic feature, we chose a typical gymnosperm representing seed-bearing plants more primitive than the angiosperms. Thus, salt eluates of Douglas fir (Pseudotsuga menziesii) cell suspension cultures yielded two monomeric extensins differing in size and composition. The larger extensin reported earlier lacked the Ser-Hyp(4) motif, was rich in proline and hydroxyproline, and contained peptide motifs similar to the dicot repetitive proline-rich proteins. The smaller extensin monomer reported here (Superose-6 peak 2 [SP2]) was compositionally similar to typical dicot extensins such as tomato P1, mainly consisting of Hyp, Thr, Ser, Pro, Val, Tyr, Lys, His, abundant arabinose, and a small but significant galactose content. A chymotryptic peptide map (on Hamilton PRP-1) of anhydrous hydrogen fluoride-deglycosylated SP2 yielded eight peptides sequenced after further purification on a high-resolution fast-sizing column (polyhydroxyethyl aspartamide; Poly LC). Significantly, two of the eight peptides contained the Ser-Hyp(4) motif, consistent both with the SP2 amino acid composition as well as the presence of hydroxyproline tetraarabinoside as a small (4% of total Hyp) component of the hydroxyproline arabinoside profile; thus, hydroxyproline tetraarabinoside corroborates the presence of Ser-Hyp(4), in agreement with our earlier observation that Hyp contiguity and Hyp glycosylation are positively correlated. Interestingly, other peptide sequences indicate that SP2 contains motifs such as Ser-Hyp(3)-Thr-Hyp-Tyr, Ser-Hyp(4)-Lys, and (Ala-Hyp)(n) repeats that are related to and typify dicot extensins P1, P3, and arabinogalactan proteins, respectively. Overall, these peptide sequences confirm our previous prediction that Ser-Hyp(4) is indeed an ancient motif and also strongly support our suggestion that the extensins comprise an extraordinarily diverse, but nevertheless phylogenetically related, family of cell wall hydroxyproline-rich glycoproteins.

Journal Article↗

Adaptive digital notch filter design on the unit circle for the removal of powerline noise from biomedical signals.

This paper investigates adaptive digital notch filters for the elimination of powerline noise from biomedical signals. Since the distribution of the frequency variation of the powerline noise may or may not be centered at 60 Hz, three different adaptive digital notch filters are considered. For the first case, an adaptive FIR second-order digital notch filter is designed to track the center frequency variation. For the second case, the zeroes of an adaptive IIR second-order digital notch filter are fixed on the unit circle and the poles are adapted to find an optimum bandwidth to eliminate the noise to a pre-defined attenuation level. In the third case, both the poles and zeroes of the adaptive IIR second-order filter are adapted to track the center frequency variation within an optimum bandwidth. The adaptive process is considerably simplified by designing the notch filters by pole-zero placement on the unit circle using some suggested rules. A constrained least mean-squared (CLMS) algorithm is used for the adaptive process. To evaluate their performance, the three adaptive notch filters are applied to a powerline noise sample and to a noisy EEG as an illustration of a biomedical signal.

Algorithms↗

Latency measurement improvement of P100 complex in visual evoked potentials by FMH filters.

The ensemble average of Pattern Shift Visual Evoked Potentials (PSVEP) signals is seriously affected by random latency variations encountered in each individual sweep which is modeled as a continuous signal with linear segments and well-shaped triangular peaks. This effect is causing the smoothed peaks of the averaged PSVEP waveforms. It is our objective to restore the degraded peaks and provide accurate information about their exact location. The method used is based on nonlinear filtering of the FIR-Median Hybrid (FMH) type and is recommended as a postfiltering process to the well-known averaging methods of recovering PSVEP signals from noise by time-locking to stimuli. The new technique, tested in signals from clinical observations, has proven very promising.

Artifacts↗