Regulation of Oleoresinosis in Grand Fir (Abies grandis) (Coordinate Induction of Monoterpene and Diterpene Cyclases and Two Cytochrome P450-Dependent Diterpenoid Hydroxylases by Stem Wounding)

Oleoresin (pitch) is a defensive secretion composed of monoterpene olefins (turpentine) and diterpene resin acids (rosin) that is produced in grand fir (Abies grandis Lindl.) stems in response to wounding. Monoterpene and diterpene biosynthesis are coordinately induced in wounded stems as determined by monitoring the activity of monoterpene and diterpene cyclases, as well as two cytochrome P450-dependent diterpenoid hydroxylases involved in the formation of ([mdash])-abietic acid, the principal resin acid of this species. The activity of these enzymes reaches maximum levels that are 5- to 100-fold higher than those of nowwounded control stems 10 d after wounding and this is followed by a synchronous decline. The increase in biosynthetic activity is consequently followed by the accumulation of a viscous mass of resin acids, with the loss of the volatile monoterpenes, at the site of injury. The observed coordinate induction of monoterpene olefin and abietic acid bio-synthesis and the results of oleoresin analysis are consistent with the role of the volatile monoterpenes as a solvent for the mobilization and deposition of resin acids at the wound site to seal the injury with a rosin barrier after the evaporation of the turpentine. The last step of resin acid biosynthesis is catalyzed by an operationally soluble aldehyde dehydrogenase that is not inducible by wounding but seemingly is expressed constitutively at a high level. In vivo [14C]acetate feeding and resin analysis indicate that this enzyme is not efficiently coupled to the earlier steps of the pathway.     

Water Permeability of the Wood of Grand Fir (Abies grandis (Doug.) Lindl.) in Relation to Infestation by the Balsam Woolly Aphid, Adelges piceae (Ratz.)

The water permeability of sapwood and heartwood of Abies grandis(Doug.) Lindl. was found for normal trees and those infestedby balsam woolly aphid Adelges Piceae (Ratz.). It was determinedas the rate of flow of water through plugs of the wood of standarddimensions and under constant suction. The permeability of normaltrees was less in the inner than the outer sapwood and thisdifference was correlated with a greater void space (gas-filledtracheids) in the inner sapwood. The permeability of the heartwoodwas less than 5 per cent that of the sapwood. Aphid infestationreduced the permeability of the outer sapwood to about the samelevel as normal heartwood. The infested wood had a high percentageof void space and again permeability was negatively correlatedwith void space. But for a given level of void space the infestedwood had a much lower permeability than normal wood. This suggeststhat there was factor additional to the air in the tracheids,which contributed to the low permeability of infested wood.The possible nature of this factor is briefly discussed.     

Effects of Infestation by the Balsam Woolly Aphid, Adelges Piceae (Ratz.) on the Ultrastructure of Bodered-Pit Membranes of Grand Fir, Abies grandis (Doug.) Lindl.

The ultrastructure of bordered-pit membranes in normal grandfir trees, Abies grandis (Doug.) Lindl., was compared with thatin trees infested with the blasam woolly aphid, Adelges piceae(Ratz). In sapwood of non-infested trees the membranes of earlywoodpits were well perforated, whereas those of latewood pits wereeither heavily incrusted or incompletely developed and showedfew perforations. In the heartwood pit membranes from both earlywoodand latewood were heavily incrusted. In aphid-infested trees all the pit membranes from the sapwoodwere incrusted and resembled those from heartwood of non-infestedtrees. These incurstations reduced the number of pores in themargo of pit membranes, and could account for the reduced permeabilityto water reported for sapwood trees attacked by the aphid. Wesuggest that the incrustation of pit membranes in sapwood inAbies grandis infested with Adelges piceae occurs because theseaphids cause heartwood to form prematurely.     

Characterization of Top Leader Elongation in Nordmann Fir (Abies nordmanniana)

Our understanding of the developmental changes that occur during top leader elongation in gymnosperms lags behind that in angiosperms. We developed a semiquantitative method for determining epidermal cell size, by measuring the Feret diameter after cell wall staining of stem epidermal peels. This method allowed a large number of cells to be measured at various locations in the top leader of the Christmas tree Abies nordmanniana. Further, we have identified the growth rate of individual sections of the top leader, and the relationship between cell length and needle arrangement throughout the top leader. At bud break, all stem units begin to elongate simultaneously, but growth ceases from the base upwards during top leader elongation. Long top leaders were characterized by having up to three times as long cells at the base compared to short top leaders, whereas the cell lengths were similar in the apical region independent of the given plant growth capacity. In the basal sector, the level of auxin was much higher, whereas the levels of cytokinins were lower than in the apical sector, causing the auxin/cytokinin ratio to change from about 3 in the apical sector to more than 20 in the basal part. The Fibonacci number changed in the apical sector due to an increased cell number in the stem units and therefore longer distance between the needles. We conclude that the general growth pattern during top leader elongation in A. nordmanniana is similar to angiosperms but differs at the cellular level.

     

Characterization of the constitutive and wound-inducible monoterpene cyclases of grand fir (Abies grandis)

Monoterpene cyclase activity is greatly increased in grand fir (Abies grandis) sapling stems in response to wounding and the composition of the cyclic olefin mixture generated differs from that produced constitutively as determined by radio gas-liquid chromatography. Cell-free extracts from wounded stems and from non-wounded controls were systematically compared for monoterpene cyclase activities following partial purification and separation of these enzymes by anion-exchange chromatography (Mono Q FPLC) and native PAGE. The increase in monoterpene cyclase activity following wounding represents both the apparent enhancement of constitutive cyclase activities and the appearance of novel cyclization enzymes that are absent in nonwounded controls. A pinene cyclase was shown to be the major wound-inducible enzyme directly responsible for oleoresin monoterpene formation and was tentatively identified as a 62-kDa protein by SDS-PAGE.     

Altering product outcome in Abies grandis (-)-limonene synthase and (-)-limonene/(-)-alpha-pinene synthase by domain swapping and directed mutagenesis

(-)-(4S)-limonene synthase (LS) and (-)-(4S)-limonene/(-)-(1S, 5S)-alpha-pinene synthase (LPS) from grand fir (Abies grandis) exhibit nearly 91% sequence identity (93% similarity) at the amino acid level, yet produce very different mixtures of monoterpene olefins. To elucidate critical amino acids involved in determining monoterpene product distribution, a combination of domain swapping and reciprocal site-directed mutagenesis was carried out between these two enzymes. Exchange of the predicted helix D through F region in LS gave rise to an LPS-like product outcome, whereas reciprocal substitutions of four amino acids in LPS (two in the predicted helix D and two in the predicted helix F) altered the product distribution to that intermediate between LS and LPS, and resulted in a 5-fold increase in relative velocity. These results, in conjunction with modeling of the two enzymes, suggest that amino acids in the predicted D through F helix regions are critical for product determination.     

Isolated Trees and Grass Removal Improve Performance of Transplanted Trema micrantha (L.) Blume (Ulmaceae) Saplings in Tropical Pastures

Cattle pastures established in areas previously covered by tropical rainforest can be abandoned in unproductive and degraded conditions. Transplanting native tree species to pastures is one common practice among rainforest restoration techniques, but several environmental barriers compromise transplant success. We assessed whether the presence of isolated trees and the removal of pasture grasses affect survival, growth, and allometry of transplanted saplings of the pioneer tree Trema micrantha (L.) Blume (Ulmaceae) into abandoned pastures in southeast Mexico. An isolated tree was selected in the center of each of four pasture sites of one hectare, and grass treatments were applied under the tree's canopy (0–10 m from the trunk) and in open pasture (15–48 m from the trunk). Grass removal treatments were control (grass present), cut with machete, herbicide application, and total grass removal with a gardening hoe. After 1 year, sapling survival was not different between the canopy and pasture areas (53%). Saplings showed higher survival probability (p < 0.05) in the hoe treatment (63 ± 9%) than in the control treatment (38 ± 9%). Height and crown cover growth rates were faster under the canopy of isolated trees compared with the open pasture. Saplings showed significantly greater crown area/height ratios under the canopy of isolated trees. Stressful environmental conditions restricted sapling growth in the open pasture. We conclude that complete removal of grasses and transplanting T. micrantha saplings in the vicinity of isolated trees can improve transplant success.     

精胺(Spm)在植物响应逆境中的特异性作用

就精胺的代谢和生物合成相关基因以及在植物耐逆境胁迫中的特异性作用研究进展作概述.     

水稻CAT与逆境应答关系及酶活性分析

过氧化氢酶(catalase,CAT)是一种四聚体血红素酶,主要存在于过氧化物酶体与乙醛酸循环体及相关细胞区域内.水稻中含有CAT1、CAT2和CAT3三种主要同工酶.它们在各组织不同的发育阶段,发挥着重要作用.在本研究解析了水稻过氧化氢酶同工酶基因在NaCl、NaHCO3、Na2CO3、PEG6000、H2O2、低温等逆境处理下mRNA表达特性.结果表明在不同的逆境下过氧化氢酶同工酶在表达上存在差异性.同时利用蛋白表达系统,对水稻过氧化氢酶CAT1和CAT3蛋白进行了纯化实验,对纯化的蛋白的酶活性分析的结果表明,CAT1和CAT3的酶活性没有十分显著的差别.     

Abietadiene synthase from grand fir (Abies grandis): characterization and mechanism of action of the "pseudomature" recombinant enzyme

The oleoresin secreted by grand fir (Abies grandis) is composed of resin acids derived largely from the abietane family of diterpene olefins as precursors which undergo subsequent oxidation of the C18-methyl group to a carboxyl function, for example, in the conversion of abieta-7,13-diene to abietic acid. A cDNA encoding abietadiene synthase has been isolated from grand fir and the heterologously expressed bifunctional enzyme shown to catalyze both the protonation-initiated cyclization of geranylgeranyl diphosphate to the intermediate (+)-copalyl diphosphate and the ionization-dependent cyclization of (+)-copalyl diphosphate, via a pimarenyl intermediate, to the olefin end products. Abietadiene synthase is translated as a preprotein bearing an N-terminal plastidial targeting sequence, and this form of the recombinant protein expressed in Escherichia coli proved to be unsuitable for detailed structure-function studies. Since the transit peptide-mature protein cleavage site could not be determined directly, a truncation series was constructed to delete the targeting sequence and prepare a "pseudomature" form of the enzyme that resembled the native abietadiene synthase in kinetic properties. Both the native synthase and the pseudomature synthase having 84 residues deleted from the preprotein converted geranylgeranyl diphosphate and the intermediate (+)-copalyl diphosphate to a nearly equal mixture of abietadiene, levopimaradiene, and neoabietadiene, as well as to three minor products, indicating that this single enzyme accounts for production of all of the resin acid precursors of grand fir. Kinetic evaluation of abietadiene synthase with geranylgeranyl diphosphate and (+)-copalyl diphosphate provided evidence for two functionally distinct active sites, the first for the cyclization of geranylgeranyl diphosphate to (+)-copalyl diphosphate and the second for the cyclization of (+)-copalyl diphosphate to diterpene end products, and demonstrated that the rate-limiting step of the coupled reaction sequence resides in the second cyclization process. The structural implications of these findings are discussed in the context of primary sequence elements considered to be responsible for binding the substrate and intermediate and for initiating the respective cyclization steps.     

The Functional and Regulatory Mechanisms of the Thellungiella salsuginea Ascorbate Peroxidase 6 (TsAPX6) in Response to Salinity and Water Deficit Stresses

Soil salinization is a resource and ecological problem in the world. Thellungiella salsuginea is becoming a new model plant because it resembles its relative species, Arabidopsis thaliana, in small genome and short life cycle. It is highly tolerant to salinity and drought stresses. Ascorbate peroxidase (APX) is an enzyme that clears H₂O₂ in plants. The function and molecular and regulation mechanisms of APX in T. salsuginea have rarely been reported. In this study, an APX gene, TsApx6, was cloned from T. salsuginea and its responses to abiotic stresses in transgenic Arabidopsis were studied. Under high salinity treatment, the expression of TsApx6 was significantly induced. Under drought treatment, overexpression of TsApx6 increased the survival rate and reduced leaf water loss rate in Arabidopsis. Compared to the wild type plants, high salinity treatment reduced the concentrations of MDA, H₂O₂ and proline but elevated the activities of APX, GPX, CAT and SOD in the TsApx6-overexpressing plants. Meanwhile, germination rate, cotyledon greening, and root length were improved in the transgenic plants compared to the wild type plants under salt and water deficit conditions. Based on these findings, TsApx6 has an important function in the resistance of plants to certain abiotic stresses. The TsApx6 promoter sequence was obtained using Genome Walking technology. Bioinformatics analysis indicated that it contains some cis-acting elements related to stress response. The treatments of salt, dehydration, and ABA induced the expression of Gus gene under the regulation of the TsApx6 promoter. Mutation analysis showed that the MBS motif present in the TsApx6 promoter might be a key negative regulatory element which has an important effect on the growth and developmental process of plants.     

Differential Response in the Water Status of Immature and Mature Fronds of the Ostrich Fern (Matteuccia struthiopteris [L.] Todaro) to a Mild Water Stress

Experiments were conducted in growth chambers to examine the effect of a mild water stress (−200 kilopascals polyethylene glycol) on frond elongation and water status of the ostrich fern (Matteuccia struthiopteris [L.] Todaro). Measurements were taken for two days, starting one day after the application of polyethylene glycol. Total water potential in control (well-watered) plants was always significantly higher in immature fronds than in mature fronds. The osmotic potential in mature fronds was always significantly lower (about 800 kilopascals) than in immature fronds in both control and stressed plants. In immature fronds, the stress decreased elongation and total water and pressure potentials, while in mature fronds it increased total water and pressure potentials. The decreases in total and pressure potentials in immature fronds were approximately equal to the increases in mature fronds.     

Production of Cytokinin-like Substances by Cylindrocarpon destructans (Zins.) Scholt. Isolates Pathogenic and Nonpathogenic to Fir (Abies alba Mill.) seedlings

The production of cytokinin-like substances by different strains of Cylindrocarpon destructans either pathogenic or non pathogenic to fir (Abies alba Mill.) was studied. The cytokinins were extracted from the fungal culture media and separated by means of column chromatography. The biological activities in different fractions were assayed by callus growth of soybean (Glycine max Merill var. Acme). The amount of these substances was calculated on the basis of fresh weight of callus obtained from cultures containing authentic cytokinin. Cytokinin-like substances were detected in 5 of 6 pathogenic isolates tested and in 2 of 5 non-pathogenic isolates. On the basis of their occurrence in particular column fractions, most of the substances seem to be identical or similar to zeatin and zeatin riboside. In addition the fungi studied produced other, not identified cytokinin-like substances. The synthesis of Ri P can not be excluded. However the method applied did not allow to separate Ri P from zeatin and zeatin riboside.     

Temperature and Photoperiod Influence Postharvest Needle Abscission of Selected Balsam Fir (Abies balsamea L. (Mill.)) Genotypes by Modulating ABA Levels

Seasonal changes in the concentration of abscisic acid (ABA) in current-year needles of two different genotypes (AB-NSD-004 and AB-NSD-184) were monitored in branches collected from 20-year-old balsam fir (Abies balsamea L. (Mill.)) trees over a period of 11 months. A significant genotype × harvesting time interaction was observed for endogenous ABA levels and postharvest needle retention duration (NRD). A consistent pattern of seasonal variation in ABA concentration was observed in both genotypes, with the highest amount of ABA (7,887 ng g^?1 DW) accumulating in April and May. The highest levels of ABA coincided with the lowest postharvest NRD regardless of genotype. Nevertheless, genotypes differed in their ABA concentrations. Branches of genotype AB-NSD-184 sampled during August exhibited 170 days of NRD whereas those collected in May and June registered the lowest NRD of around 40 days. There was a significant negative correlation (P < 0.05) between endogenous ABA concentrations and postharvest NRD in genotype AB-NSD-184. Also, an inverse relationship was observed between the average daily photoperiod and the postharvest NRD (R ² = 0.35; P = 0.000) in the same genotype. Together with average daily temperature, the R ² value for this correlation reached the highest (0.75; P ≤ 0.00). Genotypes differed in their physiological responses to environmental cues and thus differed in their postharvest qualities. Average daily photoperiod and maximum daily temperature are strongly linked to the postharvest NRD through modulating endogenous ABA concentration.     

The Role of Osmotic Potential in Spring Sap Flow of Mature Sugar Maple Trees (Acer saccharum Marsh)

Field measurements of xylem sap osmotic and pressure potentialwere made on sugar maple trecs (Acer saccharum Marsh) duringthe winter and spring of 3 years to determine whether the hydrostaticpressure was osmotically generated. Sap osmotic potential waslow enough to account for the hydrostatic pressure but the dynamicsof its diurnal behaviour indicated that osmotic potential wasnot directly responsible for hydrostatic pressure. The diurnalcourse of hydrostatic pressure showed definite peaks but osmoticpotential often did not. The magnitude of the diurnal changesin hydrostatic pressure was approximately 0·15 MPa whereasthe changes in osmotic potential were only 0·05 MPa.Because the sap osmoticum is primarily sucrose, and starch isstored in the xylem throughout the tree, the temperature dependenceof the sucrose-starch interconversion system was investigated.More active amylase was formed in maple twigs after incubationat 0°C and 4°C than at –3, 6 or 15°C. Therate of starch hydrolysis by maple amylase increased with temperature,reaching a maximum at approximately 45°C. There was somestarch hydrolysis at –3°C. The starch hydrolysis systemthus indicated no critical role for temperature fluctuationsabout 0°C. Starch was found to be densely stored in therays of the trunk and twigs and around the central pith in thetwigs. Key words: Acer succhmum Marsh, Osmotic potential, Xylem sap pressure     

Wound-induced Proteinase Inhibitor in Tomato Leaves: Some Effects of Light and Temperature on the Wound Response

Wounding of single leaflets of young tomato (Lycopersicum esculentum var. Bonnie Best) plants causes the release of a proteinase inhibitor inducing factor. This factor is rapidly transported throughout the plant where it causes accumulation of inhibitor I, a potent inhibitor of several serine proteinases from both animals and microorganisms. The wound-induced accumulation of inhibitor I is both light- and temperature-dependent. In total darkness no accumulation results from wounding. The accumulation exhibits a linear dependence upon light up to 300 foot candles. At 600 foot candles and above, the response is maximal. In light the wound response possesses an unusual temperature dependence with an optimum rate of accumulation near 36 C. Below 20 C no accumulation occurs. The over-all process contains two light- and temperature-dependent steps, one involving wounding and transport, the other involving accumulation.     

Partial purification and characterization of the short-chain prenyltransferases, gernayl diphospate synthase and farnesyl diphosphate synthase, from Abies grandis (grand fir)

In the conifer Abies grandis (grand fir), a secreted oleoresin rich in mono-, sesqui-, and diterpenes serves as a constitutive and induced defense against insects and pathogenic fungi. Geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) synthase, two enzymes which form the principal precursors of the oleoresin mono- and sesquiterpenes, were isolated from the stems of 2-year-old grand fir saplings. These enzymes were partially purified by sequential chromatography on DEAE-Sepharose, Mono-Q, and phenyl-Sepharose to remove competing phosphohydrolase and isopentenyl diphosphate (IPP) isomerase activities. GPP and FPP synthase formed GPP and E,E-FPP, respectively, as the sole products of the enzymatic condensation of IPP and dimethylallyl diphosphate (DMAPP). The properties of both enzymes are broadly similar to those of other prenyltransferases. The apparent native molecular masses are 54 +/- 3 kDa for GPP synthase and 110 +/- 6 kDa fo     

Ontogeny in terminal buds of Abies nordmanniana (Pinaceae) characterized by ubiquitin

Meristematic activity in the bud meristem of Abies nordmanniana was visualized by ubiquitin immunohistochemical localization from before bud break and throughout shoot expansion. Ubiquitin was detected in meristematic cells either in the cytosol or nucleus, or both, depending on tissue type and developmental stage. During winter dormancy, ubiquitin was only observed in the protodermal/hypodermal layers, but at bud break in mid May, the signal expanded to the entire shoot tip. At the end of May, a clear zonation in ubiquitin localization appeared that lasted about one month. Throughout this period, ubiquitin was barely detectable in a central group of cells that might indicate an organizing center with stem cells. At the end of June, coinciding with the transition from scale leaf to needle primordia production, ubiquitin again was more prevalent in the peripheral cell layers. During shoot expansion, a strong ubiquitin signal developed in the axil of all needles. Most of these signals later disappeared, except for those few axils where buds actually developed. A strong ubiquitin signal was also observed in cells lining the young resin ducts. Our data showed that ubiquitin may be used as a marker for metabolic activity associated with seasonal development in the apical meristem.