Primary production, light and vertical mixing in Potter Cove, a shallow bay in the maritime Antarctic

Phytoplankton photosynthesis was measured during spring-summer 1991-1992 in the inner and outer part of the shallow Potter Cove, King George Island. Strong winds characterise this area. Wind-induced turbulent mixing was quantified by means of the root-mean square expected vertical displacement depth of cells in the water column, Z_t. The light attenuation coefficient was used as a measure of the influence of the large amount of terrigenous particles usually present in the water column; 1% light penetration ranged between 30 and 9 m, and between 30 and 15 m for the inner and outer cove, respectively. Obvious differences between photosynthetic capacity [P*_max; averages 2.6 and 0.6 µg C (µg chlorophyll-a)^-1 h^-1] and photosynthetic efficiency {!*; 0.073 and 0.0018 µg C (µg chlorophyll-a)^-1 h^-1 [(µmol m^-2 s^-1)^-1]} values were obtained for both sites during low mixing conditions (Z_t from 10 to 20 m), while no differences were found for high mixing situations (Z_t>20 m). This suggests different photoacclimation of phytoplankton responses, induced by modifications of the light field, which in turn are controlled by physical forcing. Our results suggest that although in experimental work P*_max can be high, wind-induced mixing and low irradiance will prevent profuse phytoplankton development in the area.     

Characteristics of a DO-controlled fed-batch culture of Escherichia coli

The DO-controlled glucose limited fed-batch technique was investigated in an E. coli process for production of a recombinant protein. The k_Lac^* value (oxygen transfer rate at zero oxygen concentration) was calculated from on-line gas analysis data during the process. In the investigated processes with induced production of recombinant protein, the k_Lac^* value decreased drastically several hours after induction. The reason for the decrease was found in increasing concentrations of DNA in the medium and increased viscosity due to cell lysis. The consequences of such a dramatic decrease in the volumetric oxygen transfer coefficient on the glucose feed and specific rates are described in computer simulations and experimental data.     

Comparative study of the fermentation of D-glucose/D-xylose mixtures with Pachysolen tannophilus and Candida shehatae

We have performed a comparative analysis of the fermentation of the solutions of the mixtures of D-glucose and D-xylose with the yeasts Pachysolen tannophilus (ATCC 32691) and Candida shehatae (ATCC 34887), with the aim of producing bioethanol. All the experiments were performed in a batch bioreactor, with a constant aeration level, temperature of 30v°C, and a culture medium with an initial pH of 4.5. For both yeasts, the comparison was established on the basis of the following parameters: maximum specific growth rate, biomass productivity, specific rate of substrate consumption (q_s) and of ethanol production (q_E), and overall ethanol and xylitol yields. For the calculation of the specific rates of substrate consumption and ethanol production, differential and integral methods were applied to the kinetic data. From the experimental results, it is deduced that both Candida and Pachysolen sequentially consume the two substrates, first D-glucose and then D-xylose. In both yeasts, the specific substrate-consumption rate diminished over each culture. The values q_s and q_E proved higher in Candida, although the higher ethanol yield was of the same order for both yeasts, close to 0.4 kg kg^у.     

Vibrational assignments and derived potential energy distributions for tri- and difluoromethyl ketene by density functional calculations

The structures of 3,3,3-trifluoromethyl ketene and 3,3-difluoromethyl ketene were studied by utilizing ab initio calculations with the 6-311++G** basis set at the (B3LYP) Density Functional level. Full optimization was performed for both molecules in their ground and transition states. Energy optimization of the systems under investigation shows that trifluoromethyl ketene exists only in the cis conformation (fluorine atom eclipses the ketene group). Difluoromethyl ketene was predicted to have two stable conformations: the cis (hydrogen atom eclipses the ketene group) and the gauche (fluorine atom eclipses the ketene group) form. The conformational stability of the molecules was found to be governed mainly by electrostatic and molecular orbital interactions. The vibrational frequencies were computed and complete assignments were provided on the basis of normal coordinate calculations and comparison with similar molecules. The potential energy distributions (PED) among symmetry coordinates were derived for the stable conformations of the two molecules.     

Influence of climate-driven shifts in biomass allocation on water transport and storage in ponderosa pine

Conifers decrease the amount of biomass apportioned to leaves relative to sapwood in response to increasing atmospheric evaporative demand. We determined how these climate-driven shifts in allocation affect the aboveground water relations of ponderosa pine growing in contrasting arid (desert) and humid (montane) climates. To support higher transpiration rates, a low leaf:sapwood area ratio (A_L/A_S) in desert versus montane trees could increase leaf-specific hydraulic conductance (K_L). Alternatively, a high sapwood volume:leaf area ratio in the desert environment may increase the contribution of stored water to transpiration. Transpiration and hydraulic conductance were determined by measuring sap flow (J_S) and shoot water potential during the summer (June-July) and fall (August-September). The daily contribution of stored water to transpiration was determined using the lag between the beginning of transpiration from the crown at sunrise and J_S. In the summer, mean maximum J_S was 31.80LJ.74 and 24.34Dž.05 g m^-2 s^-1 for desert and montane trees (a 30.6% difference), respectively. In the fall, J_S was 25.33NJ.52 and 16.36dž.64 g m^-2 s^-1 in desert and montane trees (a 54.8% difference), respectively. J_S was significantly higher in desert relative to montane trees during summer and fall (P<0.05). Predawn and midday shoot water potential and sapwood relative water content did not differ between environments. Desert trees had a 129% higher K_L than montane trees in the summer (2.41᎒^-5 versus 1.05᎒^-5 kg m^-2 s^-1 MPa^-1, P<0.001) and a 162% higher K_L in the fall (1.97᎒^-5 versus 0.75᎒^-5 kg m^-2 s^-1 MPa^-1, P<0.001). Canopy conductance decreased with D in all trees at all measurement periods (P<0.05). Maximum g_C was 3.91 times higher in desert relative to montane trees averaged over the summer and fall. Water storage capacity accounted for 11 kg (11%) and 10.6 kg (17%) of daily transpiration in the summer and fall, respectively, and did not differ between desert and montane trees. By preventing xylem tensions from reaching levels that cause xylem cavitation, high K_L in desert ponderosa pine may facilitate its avoidance. Thus, the primary benefit of low leaf:sapwood allocation in progressively arid environments is to increase K_L and not to increase the contribution of stored water to transpiration.     

Microbial community composition and function beneath temperate trees exposed to elevated atmospheric carbon dioxide and ozone

We hypothesized that changes in plant growth resulting from atmospheric CO₂ and O₃ enrichment would alter the flow of C through soil food webs and that this effect would vary with tree species. To test this idea, we traced the course of C through the soil microbial community using soils from the free-air CO₂ and O₃ enrichment site in Rhinelander, Wisconsin. We added either ¹³C-labeled cellobiose or ¹³C-labeled N-acetylglucosamine to soils collected beneath ecologically distinct temperate trees exposed for 3 years to factorial CO₂ (ambient and 200 µl l^-1 above ambient) and O₃ (ambient and 20 µl l^-1 above ambient) treatments. For both labeled substrates, recovery of ¹³C in microbial respiration increased beneath plants grown under elevated CO₂ by 29% compared to ambient; elevated O₃ eliminated this effect. Production of ¹³C-CO₂ from soils beneath aspen (Populus tremuloides Michx.) and aspen-birch (Betula papyrifera Marsh.) was greater than that beneath aspen-maple (Acer saccharum Marsh.). Phospholipid fatty acid analyses (¹³C-PLFAs) indicated that the microbial community beneath plants exposed to elevated CO₂ metabolized more ¹³C-cellobiose, compared to the microbial community beneath plants exposed to the ambient condition. Recovery of ¹³C in PLFAs was an order of magnitude greater for N-acetylglucosamine-amended soil compared to cellobiose-amended soil, indicating that substrate type influenced microbial metabolism and soil C cycling. We found that elevated CO₂ increased fungal activity and microbial metabolism of cellobiose, and that microbial processes under early-successional aspen and birch species were more strongly affected by CO₂ and O₃ enrichment than those under late-successional maple.     

Water relations of coastal and estuarine Rhizophora mangle: xylem pressure potential and dynamics of embolism formation and repair

Physiological traits related to water transport were studied in Rhizophora mangle (red mangrove) growing in coastal and estuarine sites in Hawaii. The magnitude of xylem pressure potential (P_x), the vulnerability of xylem to cavitation, the frequency of embolized vessels in situ, and the capacity of R. mangle to repair embolized vessels were evaluated with conventional and recently developed techniques. The osmotic potential of the interstitial soil water (?_sw) surrounding the roots of R. mangle was c. -2.6LJ.52᎒^-3 and -0.4Lj.13᎒^-3 MPa in the coastal and estuarine sites, respectively. Midday covered (non-transpiring) leaf water potentials (O_L) determined with a pressure chamber were 0.6-0.8 MPa more positive than those of exposed, freely-transpiring leaves, and osmotic potential of the xylem sap (?_x) ranged from -0.1 to -0.3 MPa. Consequently, estimated midday values of P_x (calculated by subtracting ?_x from covered O_L) were about 1 MPa more positive than O_L determined on freely transpiring leaves. The differences in O_L between covered and transpiring leaves were linearly related to the transpiration rates. The slope of this relationship was steeper for the coastal site, suggesting that the hydraulic resistance was larger in leaves of coastal R. mangle plants. This was confirmed by both hydraulic conductivity measurements on stem segments and high-pressure flowmeter studies made on excised leafy twigs. Based on two independent criteria, loss of hydraulic conductivity and proportions of gas- and liquid-filled vessels in cryo-scanning electron microscope (cryo-SEM) images, the xylem of R. mangle plants growing at the estuarine site was found to be more vulnerable to cavitation than that of plants growing at the coastal site. However, the cryo-SEM analyses suggested that cavitation occurred more readily in intact plants than in excised branches that were air-dried in the laboratory. Cryo-SEM analyses also revealed that, in both sites, the proportion of gas-filled vessels was 20-30% greater at midday than at dawn or during the late afternoon. Refilling of cavitated vessels thus occurred during the late afternoon when considerable tension was present in neighboring vessels. These results and results from pressure-volume relationships suggest that R. mangle adjusts hydraulic properties of the water-transport system, as well as the leaf osmotic potential, in concert with the environmental growing conditions.     

Influence of modified oxygen and carbon dioxide atmospheres on mint and thyme plant growth, morphogenesis and secondary metabolism in vitro

. Growth (fresh weight) and morphogenesis (production of leaves, roots and shoots) of mint (Mentha sp. L.) and thyme (Thymus vulgaris L.) shoots were determined under atmospheres of 5%, 10%, 21%, 32%, or 43% O₂ with either 350 or 10,000 µmol mol^-1 CO₂. Plants were grown in vitro on Murashige and Skoog salts, 3% sucrose and 0.8% agar under a 16/8-h (day/night) photoperiod with a light intensity of 180 µmol s^-1 m^-2. Growth and morphogenesis responses varied considerably for the two plant species tested depending on the level of O₂ administered. Growth was considerably enhanced for both species under all O₂ levels tested when 10,000 µmol mol^-1 CO₂ was added as compared to growth responses obtained at the same O₂ levels tested with 350 µmol mol^-1 CO₂. Mint shoots exhibited high growth and morphogenesis responses for all O₂ levels tested with 10,000 µmol mol^-1 CO₂. In contrast, thyme shoots exhibited enhanced growth and morphogenesis when cultured in ₁% O₂ with 10,000 µmol mol^-1 CO₂ included compared to shoots cultured under lower O₂ levels. Essential oil compositions (i.e. monoterpene, piperitenone oxide from mint and aromatic phenol, thymol from thyme) were analyzed from CH₂Cl₂ extracts via gas chromatography from the shoot portion of plants grown at all O₂ levels. The highest levels of thymol were produced from thyme shoots cultured under 10% and 21% O₂ with 10,000 µmol mol^-1 CO_2,and levels were considerably lower in shoots grown under either lower or higher O₂ levels. Higher levels of piperitenone oxide were obtained from mint cultures grown under ₁% O₂ with 10,000 µmol mol^-1 CO₂ compared to that obtained with lower O₂ levels.     

Photosynthesis, photoinhibition, and nitrogen use efficiency in native and invasive tree ferns in Hawaii

Photosynthetic gas exchange, chlorophyll fluorescence, nitrogen use efficiency, and related leaf traits of native Hawaiian tree ferns in the genus Cibotium were compared with those of the invasive Australian tree fern Sphaeropteris cooperi in an attempt to explain the higher growth rates of S. cooperi in Hawaii. Comparisons were made between mature sporophytes growing in the sun (gap or forest edge) and in shady understories at four sites at three different elevations. The invasive tree fern had 12-13 cm greater height increase per year and approximately 5 times larger total leaf surface area per plant compared to the native tree ferns. The maximum rates of photosynthesis of S. cooperi in the sun and shade were significantly higher than those of the native Cibotium spp (for example, 11.2 and 7.1 µmol m^-2 s^-1, and 5.8 and 3.6 µmol m^-2 s^-1 respectively for the invasive and natives at low elevation). The instantaneous photosynthetic nitrogen use efficiency of the invasive tree fern was significantly higher than that of the native tree ferns, but when integrated over the life span of the frond the differences were not significant. The fronds of the invasive tree fern species had a significantly shorter life span than the native tree ferns (approximately 6 months and 12 months, respectively), and significantly higher nitrogen content per unit leaf mass. The native tree ferns growing in both sun and shade exhibited greater photoinhibition than the invasive tree fern after being experimentally subjected to high light levels. The native tree ferns recovered only 78% of their dark-acclimated quantum yield (F_v/F_m), while the invasive tree fern recovered 90% and 86% of its dark-acclimated F_v/F_m when growing in sun and shade, respectively. Overall, the invasive tree fern appears to be more efficient at capturing and utilizing light than the native Cibotium species, particularly in high-light environments such as those associated with high levels of disturbance.     

Elevated CO2 influences nutrient availability in young beech-spruce communities on two soil types

The objectives of this study were to investigate how different soil types and elevated N deposition (0.7 vs 7 g N m^-2a^-1) influence the effects of elevated CO₂ (370 vs 570 µmol CO₂ mol^-1) on soil nutrients and net accumulation of N, P, K, S, Ca, Mg, Fe, Mn, and Zn in spruce (Picea abies) and beech (Fagus sylvatica). Model ecosystems were established in large open-top chambers on two different forest soils: a nutrient-poor acidic loam and a nutrient-rich calcareous sand. The response of net nutrient accumulation to elevated atmospheric CO₂ depended upon soil type (interaction soil 2 CO₂, P<0.05 for N, P, K, S, Ca, Mg, Zn) and differed between spruce and beech. On the acidic loam, CO₂ enrichment suppressed net accumulation of all nutrients in beech (P<0.05 for P, S, Zn), but stimulated it for spruce (P<0.05 for Fe, Zn) On the nutrient-rich calcareous sand, increased atmospheric CO₂ enhanced nutrient accumulation in both species significantly. Increasing the N deposition did not influence the CO₂ effects on net nutrient accumulation with either soil. Under elevated atmospheric CO₂, the accumulation of N declined relative to other nutrients, as indicated by decreasing ratios of N to other nutrients in tree biomass (all ratios: P<0.001, except the N to S ratio). In both the soil and soil solution, elevated CO₂ did not influence concentrations of base cations and available P. Under CO₂ enrichment, concentrations of exchangeable NH₄⁺ decreased by 22% in the acidic loam and increased by 50% in the calcareous sand (soil 2 CO₂, P<0.001). NO₃^- concentrations decreased by 10-70% at elevated CO₂ in both soils (P<0.01).     

13C content of ecosystem respiration is linked to precipitation and vapor pressure deficit

Variation in the carbon isotopic composition of ecosystem respiration ('¹³C_R) was studied for 3 years along a precipitation gradient in western Oregon, USA, using the Keeling plot approach. Study sites included six coniferous forests, dominated by Picea sitchensis, Tsuga heterophylla, Pseudotsuga menziesii, Pinus ponderosa, and Juniperus occidentalis, and ranged in location from the Pacific coast to the eastern side of the Cascade Mountains (a 250-km transect). Mean annual precipitation across these sites ranged from 227 to 2,760 mm. Overall '¹³C_R varied from -23.1 to -33.1‰, and within a single forest, it varied in magnitude by 3.5-8.5‰. Mean annual '¹³C_R differed significantly in the forests and was strongly correlated with mean annual precipitation. The carbon isotope ratio of carbon stocks (leaves, fine roots, litter, and soil organic matter) varied similarly with mean precipitation (more positive at the drier sites). There was a strong link between '¹³C_R and the vapor saturation deficit of air (vpd) 5-10 days earlier, both across and within sites. This relationship is consistent with stomatal regulation of gas exchange and associated changes in photosynthetic carbon isotope discrimination. Recent freeze events caused significant deviation from the '¹³C_R versus vpd relationship, resulting in higher than expected '¹³C_R values.     

Physiological basis of spacing effects on tree growth and form in Eucalyptus globulus

We examine the effects of spacing and layout on the growth and form of 3- to 4-year-old Eucalyptus globulus in a farm forestry context. Four planting layouts were chosen. These represented the range commonly in use in farm forestry: block plantings (2Ǹ m), triple rows (2Ǹ m) at 10-m intervals, single rows (2᎒ m) and isolated trees (10᎒ m). The physiological significance of key results is interpreted in terms of changes in the parameters of a simple plantation growth model. Under conditions where levels of direct light are high, for example during summer, block-planted trees intercepted only 38% of the light intercepted by isolated trees. On a stand basis, however, the combination of incident radiation and ground coverage declined with lower stand densities. While stand leaf area index declined from around 6 to 1 with increased spacing, individual tree leaf areas rose from around 50 m² in block plantings to 150 m² in isolated trees. The proportion of above-ground biomass found in stems declined with increasing spacing as the mass in foliage and branches increased. Stems accounted for 65% of above-ground biomass in block-planted trees but only 35% in isolated trees. The contributions of leaves and branches correspondingly rose from 19% to 35% and from 16% to 29%, respectively. Changes in biomass distribution were accompanied by increasing branch number, branch thickness, flatter branch angles and the longer retention of lower branches with greater spacing. These changes have implications for the merchantability of the timber. The efficiency of above-ground radiation conversion was constant at 0.67 g MJ^-1 irrespective of spacing. We estimated that foliar maintenance respiration (R_m) accounted for about 90% of above-ground R_m. On a stand basis R_m costs block plantings 23.90 t DM ha^-1 year^-1 (50% annual above-ground photosynthetic production) compared with 6.22 t DM ha^-1 year^-1 (40% annual above-ground photosynthetic production) in stands of isolated trees.     

Water availability and carbon isotope discrimination in conifers

The stable C isotope composition ('¹³C) of leaf and wood tissue has been used as an index of water availability at both the species and landscape level. However, the generality of this relationship across species has received little attention. We compiled literature data for a range of conifers and examined relationships among landscape and environmental variables (altitude, precipitation, evaporation) and '¹³C. A significant component of the variation in '¹³C was related to altitude (discrimination decreased with altitude in stemwood, 2.53‰ km^-1 altitude, r²=0.49, and in foliage, 1.91‰ km^-1, r²=0.42), as has been noted previously. The decrease in discrimination with altitude was such that the gradient in CO₂ partial pressure into the leaf (P_a-P_i) and altitude were generally unrelated. The ratio of precipitation to evaporation (P/E) explained significant variation in P_a-P_i of stemwood (r²=0.45) and foliage (r²=0.27), but only at low (<0.8) P/E. At greater P/E there was little or no relationship, and other influences on '¹³C probably dominated the effect of water availability. We also examined the relationship between plant drought stress (O) and '¹³C within annual rings of stemwood from Pinus radiata and Pinus pinaster in south-western Australia. Differential thinning and fertiliser application produced large differences in the availability of water, nutrients and light to individual trees. At a density of 750 stems ha^-1, O and '¹³C were less (more negative) than at 250 stems ha^-1 indicating greater drought stress and less efficient water use, contrary to what was expected in light of the general relationship between discrimination and P/E. The greater '¹³C of trees from heavily thinned plots may well be related to an increased interception of radiation by individual trees and greater concentrations of nutrients in foliage - attributes that increase rates of photosynthesis, reduce P_i and increase '¹³C. '¹³C was thus modified to a greater extent by interception of radiation and by nutrient concentrations than by water availability and the '¹³C-O relationship varied between thinning treatments. Within treatments, the relationship between '¹³C and O was strong (0.38<r²<0.58). We conclude that '¹³C may well be a useful indicator of water availability or drought stress, but only in seasonally dry climates (P/E<1) and where variation in other environmental factors can be accounted for.     

Feeding rates of the woodlouse Armadillidium vulgare on herb litters produced at two levels of atmospheric CO2

The consumption and assimilation rates of the woodlouse Armadillidium vulgare were measured on leaf litters from five herb species grown and naturally senesced at 350 and 700 µl l^-1 CO₂. Each type of litter was tested separately after 12, 30 and 45 days of decomposition at 18°C. The effects of elevated CO₂ differed depending on the plant species. In Medicago minima (Fabaceae), the CO₂ treatment had no significant effect on consumption and assimilation. In Tyrimnus leucographus (Asteraceae), the CO₂ treatment had no significant effect on consumption, but the elevated CO₂ litter was assimilated at a lower rate than the ambient CO₂ litter after 30 days of decomposition. In the three other species, Galactites tomentosa (Asteraceae), Trifolium angustifolium (Fabaceae) and Lolium rigidum (Poaceae), the elevated CO₂ litter was consumed and/or assimilated at a higher rate than the ambient CO₂ litter. Examination of the nitrogen contents in these three species of litter did not support the hypothesis of compensatory feeding, i.e. an increase in woodlouse consumption to compensate for low nitrogen content of the food. Rather, the results suggest that in herbs that were unpalatable at the start of the experiment (Galactites, Trifolium and Lolium), more of the the litter produced at 700 µl l^-1 CO₂ was consumed than of that produced at 350 µl l^-1 because inhibitory factors were eliminated faster during decomposition.     

Nitrogen mineralization and phenol accumulation along a fire chronosequence in northern Sweden

Scots pine (Pinus sylvestris L.) forests of northern Sweden are often considered to be N limited. This limitation may have been exacerbated by the elimination of wildfire as a natural disturbance factor in these boreal forests. Phenolic inhibition of N mineralization and nitrification (due to litter and exudates of ericaceous shrubs) has been proposed as a mechanism for N limitation of these forests, but this hypothesis remains largely untested. N mineralization rates, nitrification rates, and sorption of free phenolic compounds were assessed along a fire-induced chronosequence in northern Sweden. A total of 34 forest stands varying in age since the last fire were identified and characterized. Overstorey and understorey vegetative composition and depth of humus were analysed in replicated plots at all 34 sites. Eight of the forest stands aged 3-352 years since the last fire were selected for intensive investigation in which ten replicate ionic resin capsules (used to assess net N mineralization and nitrification) and non-ionic carbonaceous resin capsules (used to assess free phenolic compounds) were installed at the interface of humus and mineral soil. A highly significant correlation was observed between site age and net sorption of inorganic N to resin capsules. Net accumulation of NH₄⁺ and NO₃^- on resin capsules followed a linear decrease (R²=0.61, P<0.01) with time perhaps as a result of increased N immobilization with successional C loading. NO₃^- sorption to resin capsules followed a logarithmic decrease (R²=0.80, P<0.01) that may be related to a logarithmic increase in dwarf shrub cover and decreased soil charcoal sorption potential along this chronosequence. A replicated field study was conducted at one of the late successional field sites to assess the influence of charcoal and an added labile N source on N turnover. Three rates of charcoal (0, 100, and 1,000 g M^-2) and two rates of glycine (0 and 50 g N as glycine M^-2) were applied in a factorial design to microplots in a randomized complete block pattern. Net ammonification (as assessed by NH₄⁺ sorption to resins) was readily increased by the addition of a labile N source, but this increase in NH₄⁺ did not stimulate nitrification. Nitrification was stimulated slightly by the addition of charcoal resulting in similar levels of resin-sorbed NO₃^- as those found in early successional sites. Resin-sorbed polyphenol concentrations were decreased with charcoal amendments, but were actually increased with N amendments (likely due to decomposition of polyphenols). Net N mineralization appears to be limited by rapid NH₄⁺ immobilization whereas nitrification is limited by the lack of an appropriate environment or by the presence of inhibitory compounds in late successional forests of northern Sweden.     

Essential oils enhanced by ultra-high carbon dioxide levels from Lamiaceae species grown in vitro and in vivo

The growth (fresh weight), morphogenesis (leaves, roots and shoots) and essential oil composition of mint (Mentha sp. L.) and thyme (Thymus vulgaris L.) plants were determined after 8 weeks under 350, 1,500, 3,000, 10,000 and 30,000 µmol mol^-1 CO₂. Plants were grown in vitro on basal medium (BM) consisting of Murashige and Skoog salts and 0.8% agar that contained either 0 or 3% sucrose under a 16-h (day)/8-h (night) photoperiod at a light intensity of 180 µmol s^-1 m^-2 or in soil in a greenhouse under conditions of natural sunlight. Ultra-high CO₂ levels (i.e. ́,000 µmol mol^-1 CO₂) substantially increased fresh weights, leaves, shoots and roots for all plants compared to plants grown under ambient air (350 µmol mol^-1 CO₂) both in vivo and in vitro. For both species, 10,000 µmol mol^-1 CO₂ was the optimum concentration to obtain the largest growth and morphogenesis responses under in vitro conditions, while the 3,000- to 10,000-µmol mol^-1 CO₂ range provided the largest yields for soil-grown plants. Essential oil composition (i.e. monoterpenes, piperitonone oxide and limonene from mint and aromatic phenol and thymol from thyme) from the shoot portion of plants grown at all CO₂ levels was analyzed in CH₂Cl₂ extracts via gas chromatography. Higher levels of secondary compounds occurred in vitro when cultures were grown under ultra-high CO₂ levels than in ambient air. The concentration of thymol, a major secondary compound in thyme plants grown on BM containing sucrose, was 317-fold higher at 10,000 µmol mol^-1 CO₂ than in plants grown under ambient air conditions with the same BM. The levels of secondary compound in in-vitro-grown plantlets exposed to ultra-high CO₂ concentrations exceeded those occurring in plants grown in the greenhouse under the same CO₂ levels. Substantially higher levels of secondary compound occurred in plants under ultra-high CO₂ levels on BM containing sucrose than on BM lacking sucrose or in soil. Thymol levels in thyme plants grown on BM containing sucrose were 3.9-fold higher at 10,000 µmol mol^-1 CO₂ than in shoots grown on BM without sucrose under the same CO₂ levels. High positive correlations occurred between thymol concentrations and CO₂ levels, fresh weights, shoots, roots and leaves when thyme shoots were grown on BM with sucrose. High positive correlations for thyme shoots grown on BM without sucrose only occurred between thymol concentrations and CO₂ levels, fresh weights, shoots and leaves. No positive correlations between thymol concentrations and CO₂ levels or any growth or morphogenesis responses occurred for thyme shoots when grown in soil.     

Mycorrhizal associations between Tuber melanosporum mycelia and transformed roots of Cistus incanus

We set out to establish root cultures of a host plant with the aim of obtaining dual cultures of Tuber melanosporum mycorrhiza on transformed roots. Seedlings of Cistus incanus germinated under sterile conditions from seeds collected in the wild were treated with Agrobacterium rhizogenes. Nine hairy roots collected from different seedlings were cultured individually by repeated subculturing. The hairy root clones differed in growth rates and in morphology (branching frequency and distance between side roots). Root growth in a liquid medium exhibited a lag phase of about 2 weeks and an exponential phase lasting about 12 days before the start of the stationary phase. Hairy roots could be kept alive on medium M, a special solid minimal medium (low in Fe²⁺, BO₄^3-, Ca²⁺, Cu²⁺ and Zn²⁺, very low in PO₄^3- and lacking MoO₄^2-, NH₄⁺ and Co²⁺), for more than 7 months. T. melanosporum could be grown on the same medium for long periods only by subculturing the fungus with the roots. A mycorrhizal association developed between the roots and the T. melanosporum mycelium within 3 months. The association consisted of elongated roots with a mantle and a Hartig net surrounding two to three layers of cortical cells. Swollen, club-like root tips were discernible 5 months after inoculation. The mycorrhized roots could be subcultured and propagated on medium M and maintain the mycorrhizal association.     

Influence of milk whey, nitrogen and phosphorus concentration on oxalic acid production by Aspergillus niger

A factorial design at two levels was used to determine the effect of milk whey concentration and the addition of nitrogen (as NH₄NO₃) and phosphorus (as KH₂PO₄) on the oxalic acid production by Aspergillus niger. The results of the experiments indicated that milk whey contains enough nutrients for fungus growth, therefore medium supplementing with N and P is not necessary. The optimum milk whey concentration was 100 kg/m³ reaching a final oxalic acid concentration of 37 kg/m³ and a maximum production rate of 3.4 kg/m³ · d. The yield of oxalic acid was 0.4, a very high value compared to previous works.     

Quantitative analyses of shade-shoot architecture of conifers native to New Zealand

Shoot architecture was quantified by measuring the "maximum silhouette area ratio" (R_max). R_max was calculated from the maximum silhouette area (or projected area) of the intact shoot, divided by the silhouette area of the leaves or phylloclades (leaf-like flattened stems) when they are removed from the shoot and laid out flat. Like conifers of the Northern Hemisphere (NH) with non-appressed foliage, the R_max of shade-adapted shoots ranged from 0.5 to 1.0 in New Zealand (NZ) conifers with non-appressed foliage. Defining a "leaf" to mean either a true leaf or a phylloclade, the following was found: leaf area/leaf dry weight, leaf area/shoot dry weight, and leaf dry weight/shoot dry weight, were all similar in the shade-shoots of NZ and NH conifers. None of these variables were significantly correlated with R_max in the NZ conifers, unless species with leaves averaging less than 4 mm² in size were excluded from the analyses. Foliage dry weight/shoot projected area was strongly correlated with R_max. NZ conifers had both smaller and larger mean leaf sizes in comparison to NH conifers. The mean projected area per shade-adapted leaf of NZ conifers varied from 2.7 to 436 mm². In NH conifers, the mean projected area per shade leaf varied from 12 to 83 mm². Except for the strikingly larger range in leaf size in NZ conifers, the data support a hypothesis of strong convergent evolution of shade-shoot architecture in NZ and NH conifers. The results are discussed in relation to photosynthesis, stand production, and the ecological distribution of conifers.