Drought tolerance, abscisic acid and electrolyte leakage in fast-and slow-growing black spruce (Picea mariana) progenies

To investigate the role that drought tolerance plays in growth, abscisic acid (ABA) accumulation and electrolyte leakage during water stress were compared in fast- and slow-growing black spruce ( Picea mariana [Mill.] B. S. P.) progenies. Changes in the ABA content of the needles were quantified using an indirect enzyme-linked immuno-sorbent assay validated by gas chromatography electron capture detection. Needle electrolyte leakage was estimated using a conductivity bridge. Seedlings were stressed using (1) osmotic stress, induced by a stepwise increase in concentrations of polyethylene glycol 3 350 (PEG) for ABA study and (2) air drying for electrolyte leakage study. Progenies did not differ in ABA levels under unstressed conditions, but progeny differences were observed under osmotic stress. Needle ABA content increased up to 500% under osmotic stress. Slow-growing black spruce progenies (25 and 46) accumulated more ABA under moderate (18% PEG), but not severe (25% PEG), osmotic stress. The slow-growing progenies also leaked more electrolytes under moderate to severe water stress and lost 50% electrolytes at a higher xylem tension, suggesting they suffered more injury and were less dehydration tolerant. Our previously-published results showed that slow-growing progenies lost their photosynthesis and stomatal conductance more quickly during osmotic stress and recovered more slowly after rehydration. Therefore, tolerance of dehydration leading to a maintenance of physiological integrity during drought stress could explain the fast growth rates of more vigorous black spruce progenies.     

Winter hardening in first-year black spruce (Picea mariana) seedlings

Winter hardening of first-year black spruce [ Picea mariana (Mill.) B.S.P.] seedlings was studied by assessing a number of morphological and physiological changes under three hardening regimes: 1) early removal (ER), in which seedlings were exposed to natural daylengths and low ambient temperatures outside. 2) extended greenhouse culture (EG), in which seedlings were exposed to natural daylengths and warm temperatures, and 3) short day (SD), in which seedlings were exposed to short daylengths and low ambient temperatures outside. Measurements included needle primordia initiation, embryonic shoot volume, terminal bud mitotic index, embryonic shoot average cell volume, and shoot tip frost hardiness. EG seedlings formed buds containing 4 times as many needle primordia as ER stock. Embryonic shoot volume increased with number of needle primordia initiated, until late in the hardening period, when significant reductions in meristem volumes of SD and EG stock were observed. Frost hardiness increased sooner in seedlings which set bud in response to short days, but SD treatment did not result in significantly greater frost hardiness at the end of the trial. Frost hardiness was correlated with mitotic index of the embryonic shoot. Cell size in the embryonic shoot declined in seedlings of all treatments during hardening, however, EG seedlings had significantly lower cell volumes by the end of the trial in comparison to ER and SD seedlings.     

Drought tolerance in faster- and slower-growing black spruce (Picea mariana) progenies: I. Stomatal and gas exchange responses to osmotic stress

Sixteen years growth in height and basal stem diameter of full-sib black spruce [ Picea mariana (Mill.) B. S. P.] progenies varied with soil moisture availability. The responses to water stress of two faster-growing progenies under drought were compared with two slower-growing progenies to determine the physiological basis of drought tolerance. Six-month-old seedlings were stressed using an osmoticum, polyethylene glycol-3350 (PEG). Seedlings were passed through a series of increasing concentration: 10, 18 and 25% PEG (w/v) each for 3 days to provide solution water potentials of -0.4, -1.0, and -2.0 MPa, respectively. The stress was then relieved by returning the seedlings to nutrient solution without PEG for 24 hours. Gas exchange and water relation parameters were similar in the 4 progenies prior to the imposition of the stress but varied during the stress and after stress relief. The two progenies which grew more vigorously on the driest site maintained significantly higher stomatal conductance, leaf transpiration rate, and net photosynthesis rate during mild (10% PEG) and moderate (18% PEG), but not severe (25% PEG) osmotic stress, and also recovered faster after release of the stress than the other two slower-growing progenies. Black spruce progenies did not differ in xylem water potential or water use efficiency. Progenies capable of faster growth under drought stress were thus characterized by a greater dehydration tolerance, rather than postponement, compared with slower-growing progenies.     

Effects of triadimefon and osmotic stress on plasma membrane composition and ATPase activity in white spruce (Picea glauca) needles

White spruce [ Picea glauca (Moench) Voss.] seedlings were grown in solution culture and treated with 20 mg I^-1 triadimefon [1-(chlorophenoxy)-3,3-dimethyl-1-(1,2,4-triazol-1-yl)-2-butanol] for 4 weeks and then subjected to osmotic stress with polyethylene glycol 3350. Water potentials and electrolyte leakage were measured in control and triadimefon-treated seedlings before and after the plants were subjected to osmotic stress. The plasma membranes were isolated from needles to study their lipid composition and the activity of plasma-membrane bound ATPase. Triadimefon treatment reduced water potentials and increased leakage of electrolytes from seedlings. However, when the seedlings were exposed to osmotic stress, triadimefon-treated plants maintained higher water potentials and leaked less electrolytes compared with the control plants. Both triadimefon and osmotic stress treatments inhibited the activity of plasma membrane-bound ATPase and altered the composition of free sterols in the plasma membranes. Triadimefon-treated plants contained traces of campesterol, which was not present in control. Osmotic stress drastically reduced phospholipid:protein and sterol:protein ratios and increased sterol:phospholipid ratios in the plasma membranes     

Drought tolerance in faster- and slower-growing black spruce (Picea mariana) progenies: II. Osmotic adjustment and changes of soluble carbohydrates and amino acids under osmotic stress

The possibility was considered that osmotic adjustment, the ability to accumulate solutes in response to water stress, may contribute to growth rate differences among closely-related genotypes of trees. Progeny variation in osmotic adjustment and turgor regulation was investigated by comparing changes in osmotic and pressure potentials, soluble carbohydrates, and amino acids in osmotically stressed seedlings in 4 full-sib progenies of black spruce [ Picea mariana (Mill.) B. S. P.] that differed in growth rate under drought. Osmotic stress was induced by a stepwise increase in the concentration of polyethylene glycol (PEG)-3350 from 10 (w/v) to 18 and 25%, which provided osmotic potentials in solution culture of -0.4, -1.0 and -2.0 MPa each for 3 days. All 4 progenies maintained a positive cell turgor even at 25% PEG, due to a significant decline in osmotic potential. Although total amino acids, principally proline, increased, ca 60% of the decrease in osmotic potential was attributable to soluble carbohydrates and glucose was the major osmoregulating solute. There was little progeny variation in any of measured parameters in unstressed seedlings. Compared to two slower-growing progenies, the two progenies capable of more vigorous growth under drought in the field accumulated more soluble carbohydrates (mainly glucose and fructose), developed lower osmotic potential and maintained higher turgor pressure when osmotically-stressed in solution culture. The ability to adjust osmotically and maintain turgor under drought stress could thus be a useful criterion for the early selection of faster-growing, drought-tolerant genotypes.     

Phytochrome action and frost hardening in black spruce seedlings

Black spruce [ Picea mariana (Mill.) B.S.P.] development is sensitive to photoperiod. To date the implication of photoperiod, and especially phytochrome, in the frost hardening process of black spruce has not been fully tested. Two light fluence rates, night interruption of darkness, and red vs far-red radiation treatments were applied to black spruce seedlings, followed by freezing at –6°C. Parallel to the freezing test, growth measurements, bud formation and the xylem water potential estimates of the seedlings were done. While dry weight accumulation depends on the irradiation energy level, bud formation and freezing tolerance are photoperiodically sensitive. Furthermore, bud formation and frost hardening are dependent upon whether phytochrome is in the active form or inactive form, as demonstrated by the positive effect of short days, far-red radiation and the reversal of the red effect by far-red radiation. Also, xylem water potential appears to be influenced by short day and far-red conditioning.     

A fungal endophyte of black spruce (Picea mariana) needles is also an aquatic hyphomycete

An aquatic hyphomycete, Dwayaangam sp., was isolated from superficially sterilized black spruce (Picea mariana) needles submerged in aerated water in a small glass chamber (microcosm). The internal transcribed spacer (ITS) sequence of this fungus and of a commonly encountered foliar endophyte isolated from P. mariana showed a high degree of similarity. When sporulation was induced in the microcosm, both the aquatic hyphomycete and the endophyte isolate produced similar aquatic conidia after 30 days, which is longer than previously documented in similar studies. Without the use of molecular tools, the link between the aquatic and endophytic phases of the fungus would have gone unnoticed. This is the first time that a fungal endophyte of conifer needles has been shown to have an aquatic phase. Its presence both as a foliar endophyte and a sporulating aquatic fungus suggests an alternating life cycle between the two environments.     

Raman imaging to investigate ultrastructure and composition of plant cell walls: distribution of lignin and cellulose in black spruce wood (Picea mariana)

A detailed understanding of the structural organization of the cell wall of vascular plants is important from both the perspectives of plant biology and chemistry and of commercial utilization. A state-of-the-art 633-nm laser-based confocal Raman microscope was used to determine the distribution of cell wall components in the cross section of black spruce wood in situ. Chemical information from morphologically distinct cell wall regions was obtained and Raman images of lignin and cellulose spatial distribution were generated. While cell corner (CC) lignin concentration was the highest on average, lignin concentration in compound middle lamella (CmL) was not significantly different from that in secondary wall (S2 and S2–S3). Images generated using the 1,650 cm⁻¹ band showed that coniferaldehyde and coniferyl alcohol distribution followed that of lignin and no particular cell wall layer/region was therefore enriched in the ethylenic residue. In contrast, cellulose distribution showed the opposite pattern—low concentration in CC and CmL and high in S2 regions. Nevertheless, cellulose concentration varied significantly in some areas, and concentrations of both lignin and cellulose were high in other areas. Though intensity maps of lignin and cellulose distributions are currently interpreted solely in terms of concentration differences, the effect of orientation needs to be carefully considered to reveal the organization of the wood cell wall.The Forest Products Laboratory is maintained in cooperation with the University of Wisconsin. This article was written and prepared by U.S. Government employees on official time, and it is therefore in the public domain and not subject to copyright. The use of trade or firm names in this publication is for reader information and does not imply endorsement by the U.S. Department of Agriculture of any product or service.     

Sequestration of soil nitrogen as tannin-protein complexes may improve the competitive ability of sheep laurel (Kalmia angustifolia) relative to black spruce (Picea mariana)

The role of litter tannins in controlling soil nitrogen (N) cycling may explain the competitive ability of Kalmia relative to black spruce (Picea mariana), although this has not been demonstrated experimentally. Here, the protein-precipitation capacities of purified tannins and leaf extracts from Kalmia and black spruce were compared. The resistance to degradation of tannin-protein precipitates from both species were compared by monitoring carbon (C) and N dynamics in humus amended with protein, purified tannins or protein-tannin precipitates. The purity of the precipitates was verified using solid-state (13)C nuclear magnetic resonance (NMR) spectra. The ability of mycorrhizal fungi associated with both species to grow on media amended with tannin-protein complexes as the principal N source was also compared. The protein precipitation capacity of Kalmia tannins was superior to those of black spruce. Humus amended with protein increased both mineral and microbial N, whereas humus amended with tannin-protein precipitates increased dissolved organic N. Mycorrhizal fungi associated with Kalmia showed better growth than those associated with black spruce when N was provided as tannin-protein precipitates. These data suggest that Kalmia litter increases the amount of soil N sequestered as tannin-protein complexes, which may improve the competitive ability of Kalmia relative to black spruce by favouring N uptake by mycorrhizas associated with the former.     

Multivariate interpretation of the foliar chemical composition of Norway spruce (Picea abies)

Twenty-four chemical elements were analysed by INAA, ICP-AES and CN in needles of Norway spruce (Picea abies (L.) Karst.). Branches were sampled from 54 trees on eight sites in Switzerland and South Germany. From each tree, twigs were sorted into the most recent four or five age classes and their needles analysed separately. All measured concentrations could be considered as log-normally distributed and statistical analyses were, therefore, performed on logarithms. Variance components were estimated by maximum likelihood and compared between elements. Non-essential elements varied more than essential nutrients (Mn was an exception). The sites and the age of the needles were the most important sources of variance. The interaction between site and age, the individual tree, the sampled branch and the residual variance were usually much smaller sources of variance. The effects of the most significant factors – site and age – were further described by principal components and cluster analyses. Mineral elements either increased or decreased with the age of the needles according to their mobility in the phloem. Two different components were identified in the effect of the sites: a geochemical component linked to soil pH and a climate component linked to altitude, temperature and precipitation. Multivariate statistics are discussed as a tool for the interpretation of complex interaction patterns between element concentrations in plants.     

Purification of intact chloroplasts from spruce (Picea abies) by free-flow electrophoresis

Highly purified chloroplasts were isolated from spruce ( Picea abies [L.] Karst.) needles by free-flow electrophoresis. The chloroplast crude suspension was separated into two structurally different populations. The two populations could not be distinguished according to their protein/chlorophyll ratios and protein patterns from two-dimensional gel electrophoresis. The O² evolution, however, showed differences: the chloroplast population deflected towards the cathode contained a major part of structurally intact chloroplasts in contrast to the population deflected towards the anode. The two populations were not contaminated by endoplasmatic membranes or mitochondria.     

Antitrssanspirant action of abscisic acid and ten synthetic analogs in black spruce

A rapid screening procedure was developed to compare the antitranspirant action of abscisic acid (ABA) and ten synthetic analogs under well-watered and droughted conditions. Compounds were applied to black spruce [ Picea mariana (Mill.) B.S.P.] seedlings at 10 μ M using an aerated root drench. The plants were grown aeroponically under a continuous mist in a misting chamber and a drought stress was then applied, 7 days after treatment with the growth regulators, by switching off the misting unit for 2 h. The activity of 2- cis and 2- trans isomers of 4- transepoxy –β-ionylideneacetic acid, their corresponding methyl esters, and 4 acetylenic analogs were compared with ABA and control, untreated seedlings. Stomatal conductance and transpiration rates declined in treated seedlings after 3 h, but returned to pre-treatment levels 2–7 days after treatment. However, transpiration declined significantly as a result of ABA- and analog-treated seedlings when they were drought stressed 7 days after treatment. Since transpiration declined more than net photosynthesis, water use efficiency increased by up to 75% as a result of ABA analog treatment. The 2- cis -isomers of epoxy-β-ionylideneacetic acids and acetylenic alcohol analogs reduced transpiration and improved water use efficiency more than ABA and 3 out of the 4 2- trans isomers.     

Effects of sealed and vented gaseous microenvironments on the maturation of somatic embryos of black spruce with a special emphasis on ethylene

Maturation of black spruce somatic embryos in sealed and vented microenvironments was investigated. The sealed microenvironment induced a larger number of well-formed mature embryos and less precocious germination than the vented microenvironment. Maturation rate of somatic embryos was not changed either by injection of ethylene into the culture vessel or by its removal by potassium permanganate traps. Increased as well as decreased ethylene concentrations, by the addition of either 1-aminocyclopropane-1-carboxylic acid (precursor of ethylene) or cobalt chloride (inhibitor of ethylene biosynthesis), resulted in a decreased number of embryos produced. However, inhibition of ethylene action by the addition of silver nitrate to the maturation medium did not affect either ethylene concentration or somatic embryo production. It was concluded that ethylene accumulation during maturation has no effect on somatic embryo production. Neither the microenvironment nor the modification of the ethylene metabolism affected conversion rate of somatic embryos into plantlets growing in soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cell wall changes in white spruce (Picea glauca) needles subjected to repeated drought stress

White spruce [ Picea glauca (Moench) Voss] seedlings were preconditioned by subjecting them to 3 cycles of a mild drought stress. After 1 week of stress relief their water status, soluble carbohydrate content and cell wall composition in newly formed needles were examined and compared with those in control seedlings. Both preconditioned and control seedlings were subsequently subjected to a severe drought stress and again analyzed. Preconditioning treatment both before and during subsequent stress exposure lowered osmotic potentials at full hydration, and after the loss of turgor, decreased lignin content and increased hemicellulose content of the cell walls. Severe drought had similar but more drastic effects on seedling water relations, sugar accumulation and cell wall hemicellulose content; it also decreased cell wall pectin levels. The decrease in pectin levels was accompanied by a loss of galactose and glucose from pectic substances. Little change in cellulose content was observed as a result of preconditioning and severe drought.     

Extracellular proteins in embryogenic suspension cultures of Norway spruce (Picea abies)

Embryogenic cell lines of Norway spruce ( Picea abies ) varying in growth habit and morphology were compared as regards profiles of extracellular proteins. Similar proteins were detected in the culture medium by SDS PAGE and in vivo labeling experiments, indicating that the proteins were secreted. Approximately 20 protein bands could be detected in the medium of each cell line. Three of the bands represented glycosylated proteins, as revealed by Concanavalin A staining. Some of the secreted proteins were similar for all tested embryogenic lines of Norway spruce, others were either specific for a group of cell lines or for individual cell lines. A correlation was observed between the morphology of the somatic embryos in a cell line and the presence of secreted proteins. The embryogenic cell lines of Norway spruce can be divided into two main groups. A and B, where A is characterized by somatic embryos with dense embryoheads and B by somatic embryos with loosely aggregated cells in their embryoheads. When proteins secreted from a cell line belonging to group A were added to cell lines belonging to group B, the somatic embryos of the B type developed further and became more similar in morphology to A-type embryos. These observations indicate that cell lines belonging to group A secrete certain proteins to the culture medium that are essential for the development of somatic embryos of Norway spruce.     

Cytochrome and alternative pathway respiration in white spruce (Picea glauca) roots. Effects of growth and measurement temperature

Interactions between growth temperature and measurement temperature were examined for their effects on white spruce [ Picea glauca (Moench) Voss] root respiration. Total dark respiration rates increased with measurement temperature and were unaffected by growth temperature. Partitioning of respiratory electron flow between the cytochrome and alternative pathways was also unaffected by growth temperature. The proportion of respiration mediated by the alternative pathway was constant at measurement temperatures between 4°C and 18°C, but was increased at higher temperatures. Changes in alternative pathway activity were paralleled by changes in capacity, and the alternative pathway was almost fully engaged at all temperatures. Roots grown at low temperature displayed higher carbohydrate levels than roots grown at higher temperatures, but respiration rate was unaffected. Spruce root respiration did not appear to acclimate to growth temperature, and the alternative pathway was not preferentially engaged at low temperature.     

Diagnosis of damage to Norway spruce (Picea abies) through biochemical criteria

Bioindication can be carried out at different hierarchical levels, eg. cell, organism, and ecosystem. While the monitoring of damage by visible criteria (e.g. loss of needles) is connected with the organism as a whole, the monitoring of damage by biochemical indicators is above all connected with cell metabolism.
The degree of vitality of a tree can be ascertained through the integration of a number of biochemical parameters. Furthermore, a differential diagnosis of a particular stress pattern can be carried out because of the feedback pattern of several biochemical indicators. In order to describe and interpret biochemical or physiological changes that have been caused by a number of factors, multivariate statistical methods are being used more frequently. Apart from cluster and discriminant analysis, it is especially factor analysis which provides a helpful tool when dealing with problems in the field of environmental analysis. Factor analysis can be used for an integrating as well as a differentiating assessment.
Within the framework of forest damage research, numerous changes at the level of cell metabolism have been detected to which a bioindicative character can be attached. A number of physiological and biochemical parameters with bioindicative character concerning Norway spruce are presented.     

Biological activity, identification and quantification of gibberellins in seedlings of Norway spruce (Picea abies) grown under different photoperiods

Short photoperiod induces growth cessation in seedlings of Norway spruce ( Picea abies (L.] Karst.). Application of different gibberellins (GAS) to seedlings growing under a short photoperiod show that GA₉ and GA₂₀ can not induce growth. In contrast application of GA, and GA₄ induced shoot elongation. The results indicate that 3β-hydroxylation of GA₉ to GA₄ and of GA₂₀ to GA₁ is under photoperiodic control. To confirm that conclusion, both qualitative and quantitative analyses of endogenous GAs were performed. GA₁, GA₃, GA₄, GA₇, GA₉, GA₁₂, GA₁₅, GA₁₅, GA₂₀, GA₂₉, GA₃₄ and GA₅₁ were identified by combined gas chromatography-mass spectrometry in shoots of Norway spruce seedlings. The effect of photoperiod on GA levels was determined by using deuterated and ¹⁴C-labelled GAs as intermal standards. In short days, the amounts of GA₉, GA₄ and GA₁ are less than in plants grown in continuous light. There is no significant difference in the amounts of GA₃, GA₁₂, and GA₂₀ between the different photoperiods. The lack of accumulation of GA₉ and GA₂₀ under short days is discussed.     

ABA consumption in norway spruce (Picea abies) and white spruce (Picea glauca) somatic embryo cultures

Summary We investigated abscisic acid (ABA) metabolism among Norway and white spruce somatic embryo cultures which exhibited differences in maturation response when placed on racemic abscisic acid [(±)-ABA]. Differences in metabolic rate among the spruce genotypes could affect the ABA pool available for the maturation process, and might therefore be responsible for the differences in maturation response. The production of cotyledonary (stage 3) somatic embryos in cultures (genotypes) of Norway spruce (PA86:26A and PA88:25B) and of white spruce (WS1F cryoD and WS46) was compared. In each species pair one of the two genotypes failed to show stage 3 embryo development (respectively, PA88:25B and WS46). The investigation of ABA metabolism of each species pair showed that no substantial differences in ABA consumption or in the production of metabolites occurred. In each case ABA was metabolized to phaseic acid and dihydrophaseic acid over the 42-day culture period, metabolites were recoverable from the agar-solidified medium, and the sum of residual ABA and metabolites were equivalent to the ABA initially supplied. The results indicate that the process of ABA metabolism occurs essentially independently of somatic embryo maturation. NRCC no. 37345.