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.     

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.     

Effects of preconditioning on electrolyte leakasge and lipid composition in black spruce (Picea mariand) stressed with polyethylene giycol

Black spruce ( Picea mariana Mill. B. S. P.) rooted cuttings were grown in solution culture and preconditioned by osmotically stressing plants with polyethylene glycol. After relief from preconditioning stress, water relations, membrane leakiness, and the composition of lipids and fatty acids were compared in preconditioned and control, unconditioned plants. Both groups of plants were subsequently subjected to a severe osmotic stress with polyethylene glycol and examined again. Osmotic stress decreased shoot water potentials and increased the leakage of electrolytes from shoots of stressed, compared with unstressed, plants. However, both unstressed and stressed preconditioned plants leaked less electrolytes compared with unconditioned plants. Changes in sterol, phospholipid and glycolipid composition were observed in preconditioned unstressed and stressed plants. Sterol and phospholipid levels de clined as a result of stress, and preconditioning resulted in a decline in sterol: phospholipid ratios in plants.     

The effect of freezing nights on photosynthesis, stomatal conductance, and internal CO2 concentration in seedlings of Engelmann spruce (Picea engelmannii Parry)

Abstract The effect of freezing night temperatures on net photosynthesis, stomatal conductance, and internal CO₂ concentration was investigated in unhardened seedlings of Engelmann spruce. Exposure to – 2.5°C in the dark for 10 h caused a slight and reversible reduction in gas-exchange parameters on the following days. Substantial and irreversible inhibition of photosynthesis occurred after exposure to -4°C or –5°C. Despite a parallel decline in stomatal conductance and net photosynthesis, exposure to a hard freeze caused a decrease in the stomatal limitation to gas exchange. Hard-freeze conditions (less than – 4°C) also caused a decrease in carboxylation efficiency and apparent quantum yield, indicating a freeze-induced failure of the dark reactions and electron transport. There was no significant difference in the photosynthetic response to freezing temperatures in different elevational populations of spruce, although acclimatory adjustments were observed. Gas exchange in seedlings grown under cool conditions (14°C day/9°C night) was less affected and recovered more rapidly after exposure to a hard freeze than in seedlings grown under warm conditions (24°C day/19°C night).     

Hygromycin resistance is an effective selectable marker for biolistic transformation of black spruce (Picea mariana)

 Using particle bombardment of mature somatic embryos followed by the induction of secondary embryogenesis in the presence of hygromycin, we produced over 90 lines of transgenic embryonal masses expressing β-glucuronidase from two genotypes of black spruce. Transformation efficiencies of up to 7% (1 transgenic line per 14 embryos bombarded) were achieved by extending the period of selection from 8 to 12 weeks. Proliferation of transformed embryonal masses in the presence of hygromycin had no effect on either embryogenicity or embryo maturation. Southern blot hybridization and PCR amplification confirmed the presence of the hygromycin phosphotransferase gene in genomic DNA. The expression of the β-glucuronidase gene in the needles of regenerated seedlings support the potential for long-term transgene expression in spruce. Received: 1 December 1997 / Revision received: 2 January 1999 / Accepted: 15 June 1999     

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.     

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.     

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.     

A long-term record of carbon exchange in a boreal black spruce forest: means, responses to interannual variability, and decadal trends

We present a decadal (1994–2004) record of carbon dioxide flux in a 160‐year‐old black spruce forest/veneer bog complex in central Manitoba, Canada. The ecosystem shifted from a source (+41 g C m⁻², 1995) to a sink (−21 g C m⁻², 2004) of CO₂ over the decade, with an average net carbon balance near zero. Annual mean temperatures increased 1–2° during the period, consistent with the decadal trend across the North American boreal biome. We found that ecosystem carbon exchange responded strongly to air temperature, moisture status, potential evapotranspiration, and summertime solar radiation. The seasonal cycle of ecosystem respiration significantly lagged that of photosynthesis, limited by the rate of soil thaw and the slow drainage of the soil column. Factors acting over long time scales, especially water table depth, strongly influenced the carbon budget on annual time scales. Net uptake was enhanced and respiration inhibited by multiple years of rainfall in excess of evaporative demand. Contrary to expectations, we observed no correlation between longer growing seasons and net uptake, possibly because of offsetting increases in ecosystem respiration. The results indicate that the interactions between soil thaw and water table depth provide critical controls on carbon exchange in boreal forests underlain by peat, on seasonal to decadal time scales, and these factors must be simulated in terrestrial biosphere models to predict response of these regions to future climate.     

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.     

Leaf responses to drought stress in Mediterranean accessions of Solanum lycopersicum: anatomical adaptations in relation to gas exchange parameters

In a previous study, important acclimation to water stress was observed in the Ramellet tomato cultivar (TR) from the Balearic Islands, related to an increase in the water‐use efficiency through modifications in both stomatal (g_s) and mesophyll conductances (g_m). In the present work, the comparison of physiological and morphological traits between TR accessions grown with and without water stress confirmed that variability in the photosynthetic capacity was mostly explained by differences in the diffusion of CO₂ through stomata and leaf mesophyll. Maximization of g_m under both treatments was mainly achieved through adjustments in the mesophyll thickness and porosity and the surface area of chloroplasts exposed to intercellular airspace (S_c). In addition, the lower g_m/S_c ratio for a given porosity in drought‐acclimated plants suggests that the decrease in g_m was due to an increased cell wall thickness. Stomatal conductance was also affected by drought‐associated changes in the morphological properties of stomata, in an accession and treatment‐dependent manner. The results confirm the presence of advantageous physiological traits in the response to drought stress in Mediterranean accessions of tomato, and relate them to particular changes in the leaf anatomical properties, suggesting specific adaptive processes operating at the leaf anatomical level.     

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.     

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     

Stomatal and nonstomatal limitations of photosynthesis in 19 temperate tree species on contrasting sites during wet and dry years

A unique approach was used to evaluate stomatal and nonstomatal constraints to photosynthesis in 19 naturally occurring, deciduous tree species on xeric, mesic and wetmesic sites in central Pennsylvania, USA, during relatively wet (1990) and dry (1991) growing seasons. All species exhibited significantly decreased stomatal conductance to CO₂ (g_c) in 1991 compared to 1990. The mesic species had drought related decreases in photosynthesis (A) attributed primarily to increased absolute stomatal limitation to A (L_g), whereas in the wet-mesic species, the absolute mesophyll limitation (Lm) was at least as important as L_g in limiting A during drought. The xeric species maintained relatively high A during drought despite decreased g_c. In the xeric and mesic species, L_m decreased and L_g increased during drought due to stomatal closure. From xeric to mesic to wet-mesic, the relative stomatal limitation (I_g) generally decreased faster, and relative mesophyll limitations to A increased faster, with increasing g_c suggesting greater photosynthetic capacity (i.e. greater potential maximum A) with increasing drought tolerance rank of species. Few species exhibited a significant drought-related decrease in photosynthetic capacity. The results of this landscape-based study indicate that the interaction of stomatal and nonstomatal limitations of A vary in a manner consistent with species' drought tolerance and site conditions, and that nonstomatal constraints to A in field plants during a moderate, season-long drought were generally not as severe as reported in controlled studies.     

Contribution of micro-organisms to the carbon dynamics in black spruce (Picea mariana) forest soil in Canada

In order to clarify the role of micro-organisms in the carbon cycle of the boreal forest ecosystem, the vertical distribution of soil carbon, soil microbial biomass and respiratory activity was studied in a black spruce forest near Candle Lake in Saskatchewan, Canada. The total amount of carbon contained in moss and soil layers (to the depth of 50cm beneath the mineral soil surface) was 7.2kgm^–2, about 47% of which was in the L and FH horizons of the soil. Soil microbial biomass per dry weight of soil was largest in the L horizon, while the biomass per ground area was largest in the FH horizon. Soil respiration rate, measured using a portable infrared gas analyzer, was highest in the FH horizon, exceeding 50% of the total soil respiration. Low but significant CO₂ emission was detected even in deeper soil horizon (E horizon). We also examined the respiration rate of cut roots and the effect of root excision on respiration. The contribution of root respiration to total soil respiration, calculated from root biomass and respiration rate of cut roots, was about 54%. The amount of carbon evolved through microbial respiration during the snow-free season (June–October) was estimated as 221gCm^–2. Micro-organisms in the L horizon showed high respiratory activity as compared with those in deeper soil horizons.     

The relative contribution of elastic and osmotic adjustments to turgor maintenance of woody species.

To determine how tissue water relations vary and contribute to turgor maintenance in species from contrasting ecological zones, seedlings of jack pine ( Pinus banksiana Lamb.), black spruce ( Picea mariana [Mill] B.S.P.) and flooded gum ( Eucalyptus grandis W. Hill ex Maiden) were subjected to an 8 day drought stress by water withholding with and without prior mild water stress conditioning. Jack pine, a deep-rooted species from dry, sandy boreal sites, lost turgor at the lowest relative water content (75–65%) and water potential, and had lowest maximum bulk elastic modulus (E_max of 5.2–5.8 MPa). Although this suggests a high inherent dehydration tolerance, jack pine did not further adjust its elasticity when repeatedly stressed. Black spruce, a shallow-rooted species from predominantly moist sites in the boreal region, lost turgor at intermediate relative water content (86–76%) and water potential, but could adjust its elasticity to maintain turgor in repeatedly stressed tissues. Flooded gum, a deep-rooted species from moist, warm temperate-subtropical regions, had a low inherent drought tolerance since it lost turgor at higher relative water content (88–84%) and water potential, but was capable of some adjustment when the stress was repeated. Elastic adjustment (<3.7 MPa) was more important for turgor maintenance than osmotic adjustment (<0.13 MPa), which was statistically nonsignificant. Maximum bulk modulus of elasticity, but not osmotic potentials at full turgor, was significantly correlated with the relative water content and water potential at zero turgor in droughted seedlings. These results highlight the importance of tissue shrinkage for dehydration tolerance. Both the inherent capacity for turgor maintenance of a species under drought and its ability to adjust to repeated drought should be considered in genetic selections for drought tolerance.     

Sheathing the blade: Significant contribution of sheaths to daytime and nighttime gas exchange in a grass crop

Despite representing a sizeable fraction of the canopy, very little is known about leaf sheath gas exchange in grasses. Specifically, estimates of sheath stomatal conductance, transpiration and photosynthesis along with their responses to light, CO₂ and vapour pressure deficit (VPD) are unknown. Furthermore, the anatomical basis of these responses is poorly documented. Here, using barley as a model system, and combining leaf-level gas exchange, whole-plant gravimetric measurements, transpiration inhibitors, anatomical observations, and biophysical modelling, we found that sheath and blade stomatal conductance and transpiration were similar, especially at low light, in addition to being genotypically variable. Thanks to high abaxial stomata densities and surface areas nearly half those of the blades, sheaths accounted for up to 17% of the daily whole-plant water use, which -surprisingly- increased to 45% during the nighttime. Sheath photosynthesis was on average 17–25% that of the blade and was associated with lower water use efficiency. Finally, sheaths responded differently to the environment, exhibiting a lack of response to CO₂ but a strong sensitivity to VPD. Overall, these results suggest a key involvement of sheaths in feedback loops between canopy architecture and gas exchange with potentially significant implications on adaptation to current and future climates in grasses.