Enhanced tolerance to oxidative stress in transgenic tobacco plants expressing three antioxidant enzymes in chloroplasts

The effect of simultaneous expression of genes encoding three antioxidant enzymes, copper zinc superoxide dismutase (CuZnSOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), and dehydroascorbate (DHA) reductase (DHAR, EC 1.8.5.1), in the chloroplasts of tobacco plants was investigated under oxidative stress conditions. In previous studies, transgenic tobacco plants expressing both CuZnSOD and APX in chloroplast (CA plants), or DHAR in chloroplast showed enhanced tolerance to oxidative stresses, such as paraquat and salt. In this study, in order to develop transgenic plants that were more resistant to oxidative stress, we introduced the gene encoding DHAR into CA transgenic plants. Mature leaves of transgenic plants expressing all three antioxidant genes (CAD plants) had approximately 1.6–2.1 times higher DHAR activity, and higher ratios of reduced ascorbate (AsA) to DHA, and oxidized glutathione (GSSG) to reduced glutathione (GSH) compared to CA plants. CAD plants were more resistant to paraquat-induced stress, exhibiting only 18.1% reduction in membrane damage relative to CA plants. In addition, seedlings of CAD plants had enhanced tolerance to NaCI (100 mM) compared to CA plants. These results indicate that the simultaneous expression of multiple antioxidant enzymes, such as CuZnSOD, APX, and DHAR, in chloroplasts is more effective than single or double expression for developing transgenic plants with enhanced tolerance to multiple environmental stresses.     

Distribution of Cd and Zn in annual xylem rings of young spruce trees [Picea abies (L.) Karst.] grown in contaminated soil

Concentrations and total amounts of Cd and Zn in individual annual xylem increments of 5-year-old spruce trees (Picea abies) were investigated after one growing season in contaminated soils. The plants had been potted in soils amended with different concentrations of Cd and Zn and kept in open air conditions. In the outer xylem rings formed during the treatment period lowest concentrations of Cd and Zn were found. In inner rings concentrations of both elements increased and were highest at the pith. Total amounts of both elements showed reversed distribution patterns with highest values in the outermost rings. The observed increase of total dry matter of the rings from pith towards the outer rings was stronger than the parallel increase in total element amounts in the rings. Thus, concentrations dropped in younger rings. The time of the soil contamination event cannot be inferred from element concentrations in annual rings. The results challenge the concept of monitoring historical trends in trace element pollution using analytical data of tree rings of conifers.     

Nitrate reductase in needles,roots and trunk wood of spruce trees [Picea abies (L.) Karst.]

Summary Nitrate reductase (EC 1.6.6.2) activity (NRA), as measured by an in vivo assay, is present in needle leaves and mycorrhizal fine root tips of adult Norway spruce [Picea abies (L.) Karst.] in at least equal amounts on a fresh weight basis, in both adult and 5-year-old trees. NRA could also be demonstrated in trunk wood of deroted trees after fertilization with 5 mM , exhibiting a longitudinal profile in the trunk. Inducibility in needles can more efficiently be achieved by NO₂ (100 g·m^-3) than by 5 mM nitrate, which is effective only in root-amputated trees. A remarkably high level of needle-NRA in unfertilized trees, which are characterized by a very low level of nitrate in the xylem sap, suggests that NRA in spruce needles may in part be constitutive. Organic-N is a major nitrogen source for the needles even in root-amputated trees, indicating pronounced exchange processes between ray parenchyma and trunk xylem, which in turn are modified by the nitrogen source fed to the trunk stump. Intact trees exhibit a very similar amino acid composition of the xylem sap, regardless of whether or has been fed. The amino acid pattern of the needles is not thrown out of balance by flooding with and , which occurs in fertilized derooted trees. This indicates a distinct potential for homoeostasis of nitrogen entrance-metabolism (i.e. NRA and glutamine synthetase activity) in the needles. In the ectomycorrhiza/fine root-system (EMC), marked differences in NRA were observed depending on root-tip diameter and along the longitudinal profile of the fine roots. EMC-nitrate reductase is strongly enhanced by . Needle-NRA exhibits a circannual rhythm. An early summer maximum is followed by a December minimum. This activity pattern matches well the transitory increase of soluble nitrogen in spring and the total protein maximum in winter. In an indirect way assimilatory NRA may well contribute to nitrogen overfertilization (by consumption of NO_X) as one possible cause of the contemporary decline of spruce populations.     

Seawater irrigation: antioxidant defence responses in leaves and roots of a sunflower (Helianthus annuus L.) ecotype

Salinity is a widespread environmental stress for crop plants. It is common in arid, semiarid, and coast regions. In those environments, seawater infiltrations can occur or the sea provides the only source of water for irrigation. The effects of 10% and 20% seawater in nutrient solutions were studied in 30 day-old plants of sunflower (Helianthus annuus L.) ecotype Katharina Piacenza. Growth parameters, ascorbate and glutathione contents, and the activities of ascorbate peroxidase and glutathione reductase were determined in shoots and roots. The results showed antioxidative responses of the ecotype to both salt treatments. The different activity patterns of antioxidant molecules and enzymes in the leaves and roots suggested a different kind of reaction to the two seawater concentrations.     

Scavenger Enzyme Activities in Subcellular Fractions of White Clover (Trifolium repens L.) under PEG-induced Water Stress

Scavenger enzyme activities in subcellular fractions under polyethylene glycol (PEG)-induced water stress in white clover (Trifolium repens L.) were studied. Water stress decreased ascorbic acid (AA) content and catalase (CAT) activity and increased the contents of hydrogen peroxide (H₂O₂), thiobarbituric acid reactive substances (TBARS) (measure of lipid peroxidation), and activities of superoxide dismutase (SOD), its various isozymes, ascorbate peroxidase (APOX), and glutathione reductase (GR) in cellular cytosol, chloroplasts, mitochondria, and peroxisomes of Trifolium repens leaves. In both the PEG-treated plants and the control, chloroplastic fractions showed the highest total SOD, APOX, and GR activities, followed by mitochondrial fractions in the case of total SOD and GR activities, whereas cytosolic fractions had the second greatest APOX activity. However, CAT activity was the highest in peroxisomes, followed by the cytosol, mitochondria, and chloroplasts in decreasing order. Although Mn-SOD activity was highest in mitochondrial fractions, residual activity was also observed in cytosolic fractions. Cu/Zn-SOD and Fe-SOD were observed in all subcellular fractions; however, the activities were the highest in chloroplastic fractions for both isoforms. Total Cu/Zn-SOD activity, the sum of activities observed in all fractions, was higher than other SOD isoforms. These results suggest that cytosolic and chloroplastic APOX, chloroplastic and mitochondrial GR, mitochondrial Mn-SOD, cytosolic and chloroplastic Cu/Zn-SOD, and chloroplastic Fe-SOD are the major scavenger enzymes, whereas cellular CAT may play a minor role in scavenging of O₂⁻ and H₂O₂ produced under PEG-induced water stress in Trifolium repens.     

Nutrient content and photosynthesis of young yellowing Norway spruce trees (Picea abies L. Karst.) following calcium and magnesium fertilisation

Severely yellowed ten-year-old spruce trees growing in the Vosges Mountains on an acidic soil were fertilised with Magnesium lime during the spring of 1990. The effects of this treatment were assessed 18 months later. A very significant improvement of the mineral status of the trees was detected, with increasing Mg contents in the needles, and as a consequence, reduced yellowing and improved chlorophyll content. Only slight differences with control trees were observed for height increase. Effects of this improved nutrition on photosynthesis were tested measuring net CO₂ assimilation rates and chlorophyll a fluorescence. Light-saturated net assimilation rates of current-year needles were high, reaching 5.3 mol m^–2 s^–1 on a total needle area basis. The improvement in chlorophyll and Mg content had no significant effect on net assimilation rates or on any parameter describing photochemical functions of both current-and previous-year needles. Despite the strong inter-individual variability in needle chlorophyll and Mg contents (ranging from 0.2 to 0.8 mg g^–1 fresh weight, and 0.05 to 0.5 mg g^-1 dry weight respectively), photochemical efficiency of PS II under limiting irradiance only decreased significantly on older needles displaying Mg contents below 0.1 mg g^–1. It is concluded from these results that spruce trees exhibit a high degree of plasticity with regard to Mg deficiency on acidic soils, and that improved Mg nutrition and increased chlorophyll content do not necessarily improve photosynthesis and height growth.Abbreviations A light-saturated net CO₂ assimilation rate (mol m^–2 s^–1) - g_w light-saturated needle conductance to water vapour (mmol m^–2 s^–1) - _wp and _wm pre-dawn and mid-day needle water potential (MPa) - osmotic potential of sap expressed from needles (MPa) - PFD photosynthetic photon flux density (mol m^–2 s^–1) - F_v/F_m photochemical efficiency of PS II after 20 min dark adaptation - F/F_m ^' photochemical efficiency of PS II reaction centres after 10 min at a PFD of 220 mol m^–2 s^–1     

Magnesium deficiency in young Norway spruce (Picea abies [L.] Karst.) trees induced by NH4NO3 application

Young Norway spruce trees were grown in 94 pots (2 per pot) on soil substrate derived from granite with low Mg saturation and were fertilized with different amounts of NH₄NO₃ (in total 25, 61, and 97 kmol N ha^-1) over a period of four years, partly at an experimental station, partly at a high-elevation site in the Bavarian Forest. A fourth set of trees received 9.4 kmol Mg ha^-1 in addition to 25 kmol N. Depending on the treatment, needle chlorosis developed in the course of the experiment. Improved light conditions after three years accelerated the yellowing process. The chlorotic Norway spruce trees showed a severe Mg deficiency and an imbalanced N:Mg ratio. The shoot length increment, the stem diameter, and the needle weights however were not influenced by the fertilization. Excessive applications of NH₄NO₃caused the substrate to become depleted of Mg. The successful experimental induction of the characteristic tip yellowing of older needles of Norway spruce growing on acidic soils at higher altitudes allowed hypotheses on the causes and processes of this type of forest decline to be tested.     

Effect of nitrogen on drought strain and nutrient uptake in Norway spruce Picea abies (L.) Karst.) trees

A field lysimeter study was established with the aim of investigating the effect of nitrogen availability upon drought strain in Norway spruce trees. Forest soil (Typic Udipsamment) was filled in lysimeters 1 m in diameter and 1 m deep. Small trees of Norway spruce from five different clones were planted in the lysimeters. Roofs under the canopy of the trees ensured full control of water and nutrient input. Three levels of nitrogen were given to the trees during five years; ambient rainwater, and five and fifteen times this N concentration, respectively. Additional N was given as NH₄NO₃ in irrigation water. Mean annual N-addition during the five years corresponded to 5, 27 and 82 kg per ha and year for the three treatments, respectively. During the third and fifth growth season drought was artificially induced. In addition to a watered control, two levels of drought were applied, representing water deprivation for 2 and 3 months, respectively, in 1990 and 3 and 4 months, respectively in 1992. A higher water consumption in the nitrogen fertilized trees during the droughts resulted in a significantly lower pre-dawn shoot water potential compared to the trees receiving ambient rain N. The interaction between drought and nitrogen fertilization was clear also for photosynthesis and transpiration. A decrease in height- and diameter increment caused by drought was most pronounced in the 82 kg N ha^–1 yr^–1 treatment. A water strain integral showed a strong positive correlation to the needle biomass of the trees. Foliar concentrations of several nutrients decreased significantly with increasing drought strain in the trees. Concentration of potassium and boron were especially low and visual symptoms of deficiency occurred.     

Calcium oxalate crystals in the mature seeds of Norway spruce, Picea abies (L.) Karst.

 The morphology and location of crystals encountered in the mature seeds of Norway spruce, Picea abies (L.) Karst., were examined using light and field emission scanning electron microscopy (FESEM). Crystals of various forms and sizes were discovered in different regions and tissues of seeds, particularly in the testa and the nucellus. Both solitary crystals and druses were occasionally enveloped by protrusions of the megaspore membranes or the cuticle of the megagametophyte. Histological studies and acid solubility tests coupled with analysis using energy dispersive X-ray microanalysis and X-ray diffraction evinced the crystals as calcium oxalate, but were unable to identify different hydration forms. Calcium oxalate crystals were most abundant in the damaged and infected tissues, and in the structures that desiccate during the development of the seed. Based on these observations we concluded that the accumulation of calcium oxalate is a regular process belonging to maturation and defense mechanism in spruce seeds. Received: 1 July 1998 / Accepted: 20 September 1998     

Respiration and photosynthesis in cones of Norway spruce (Picea abies (L.) Karst.)

Summary Dark respiration and photosynthetic carbon dioxide refixation in purple and green Picea abies cones were investigated from budbreak to cone maturity. The rate of dark respiration per unit dry weight and CO₂ refixation capacity decreased during cone maturation. At the beginning of the growing season, photosynthetic CO₂ refixation could reduce the amount of CO₂ released by respiration in green and purple cones by 50% and 40%, respectively. The seasonal performance of the components of the cone carbon balance was calculated using information on the seasonal course of respiration, refixation capacity and the light response curves of cone photosynthesis, as well as the actual light and temperature regime in the field. The daily gain of CO₂ refixation reached 28%–34% of respiration in green and 22%–26% in purple cones during the first month of their growth, but decreased later in the season. Over the entire growth period refixation reduced carbon costs of cone production in both cone colour polymorphs by 16%–17%.     

Effects of climate on vertical resin duct density and radial growth of Norway spruce [Picea abies (L.) Karst.]

 Vertical resin duct density in spruce tree-rings was used as a dendroclimatological variable and compared with radial growth. Resin duct density data were statistically stable and distributed with higher mean sensitivity and standard deviation but lower signal-to-noise rate than the corresponding growth rate. Radial growth and resin duct density relationships with climate were investigated using correlation and response function analysis. It was found that resin duct density has a significant positive response to above-normal temperature especially from June to August and a less significant negative response for above-normal precipitation from May – July during the current growing season. Ring width showed a significant negative response to above-normal precipitation from June to August but no response with temperature. Ring width and resin duct density were not related to each other. Sufficient data indicate that vertical resin duct density is a useful variable for dendroclimatology. Received: 14 March 1996 / Accepted: 24 June 1996     

Dynamics of lead accumulation in mycorrhizal and non-mycorrhizal Norway spruce (Picea abies (L.) Karst.)

Twelve-week-old seedlings of Norway spruce (Picea abies (L.) Karst), non-mycorrhizal or mycorrhizal with Laccaria laccata, Paxillus involutus or Pisolithus tinctorius were exposed to 5 M Pb for either 32 or 42 days in a quartz sand-nutrient solution system. Ultrathin sections of mycorrhizal and non-mycorrhizal short roots were examined by X-ray microanalysis. After 42 days Pb treatment, the Pb content of the cortex cell walls was lower in the non-mycorrhizal short roots and in the P. involutus mycorrhizae than in the mycorrhizae of L. laccata or P. tinctorius. The Pb content of the cell walls of the hyphal mantle was higher in P. involutus than in L. laccata or P. tinctorius. The short term experiment over 32 days showed that the Pb content of the cortex cell walls strongly increased during the first 16 days in the non-mycorrhizal roots and the L. laccata mycorrhizae, whereas it increased more slowly in the P. involutus mycorrhizae. After 32 days Pb treatment, the Pb content in the cortex cell walls in the P. involutus mycorrhizae was similar to that in the non-mycorrhizal roots. P. involutus also decreased Pb translocation from the roots to the stems. Mycorrhizal infection was not affected by Pb but with P. involutus, the amount of extramatrical mycelium was reduced by 50% on day 32 compared to day 16. The extramatrical mycelium of L. laccata was not reduced by Pb. It is concluded that ectomycorrhizal fungi differ in their effect on Pb accumulation in the roots of Norway spruce. The binding capacity of the extramatrical mycelium seems to be an important factor.     

Effects of Cd and Zn on the development of annual xylem rings of young Norway spruce (Picea abies) plants

Three-year-old spruce (Picea abies) saplings were planted and cultivated for 2 years in pots with 3 1 substrate, consisting of a homogenized mixture of sand, peat and forest soil with a high organic content (volume ratio 11.52). This substrate was amended with 10–180 mol Cd [kg soil dry weight (DW)]^–1, 50–7500 mol Zn (kg soil DW)^–1 (determined with 1 M ammonium acetate extracts) or combinations of both elements. Annual xylem growth rings in stems of plants treated with 50 mol Cd (kg soil DW)^–1 or 7500 mol Zn (kg soil DW)^–1 were significantly narrower than in control plants. Growth reductions were more pronounced in the second year of the experiment. The contents of Cd and Zn in stem wood and needles were positively correlated with the substrate concentrations. The Mg contents of the spruce needles were inversely correlated with soil concentrations of Cd and Zn. Root development was impeded at moderate concentrations of Cd (50 mol kg^–1) or Zn (1000 mol kg^–1) in the substrate. The adverse effects of potentially toxic trace elements, like Cd or Zn, on xylem growth of spruce plants are discussed with regard to possible growth reductions in forest trees under field conditions.     

Decomposition and nitrogen dynamics of fine roots of Norway spruce (Picea abies (L.) Karst.) at different sites

Long-term decomposition and nitrogen dynamics of Norway spruce finest (<1 mm in diameter) and fine (<2 mm in diameter) roots were estimated using the root litter-bag techniques. The seasonal decomposition of the finest roots was investigated in a 40-year-old high site quality stand grown on brown lessive soil at different depths as part of productivity studies. The fine root decomposition studies were conducted on 8 permanent plots in the Estonia with the aim to describe the site variation. The initial material was collected from one of stands (high quality site) and incubated at the depth of 10 cm in 1989 (at one site 1990). The bags were collected once or twice a year except for one site, where the seasonal dynamics was investigated. In all initial and decomposing root samples oven-dry weight, ash and energy content and nitrogen concentration was determined. After five years the finest roots had lost 40% of their initial dry weight, half of it during the first year. The initial concentration of nitrogen was 1.29%, the mean concentrations varied during the incubation from 1.47 to 1.78%. After the first year fine roots had lost 21.0 to 32.7% of their initial dry weight, after two years the weight loss was 22.5 to 43.2%. The initial N concentration in fine roots was 0.73% and in the first years it varied from 0.97 to 1.40% at different sites.     

Quantification of ozone influx and apoplastic ascorbate content in needles of Norway spruce trees (Picea abies L., Karst) at high altitude

The objective of the present study was to investigate whether peak concentrations of ozone can deplete the apoplastic ascorbate pool of needles from Norway spruce trees (Picea abies L. Karst.) and, thereby, contribute to damage to forest trees. Twigs of forest trees grown at high altitude (1950m above sea level; Mt Patscherkofel, Austria) were enclosed in situ in chambers and fumigated for 5-5 or 17 h with ozone concentrations ranging from 60 to 798 nmol mol^?1. Adjacent branches were fumigated with filtered air. Ozone influx into the foliage ranging from 1-7 to 17nmolm^?2s^?1 had little effect on whole-needle ascorbate or glutathione contents. However, apoplastic ascorbate decreased by about 30% when the needles were exposed to environmentally relevant ozone concentrations and increased about 3-fold at higher ozone concentrations. This response suggests the induction of ascorbate as a protective system and may also be important under field conditions. Needles of spruce trees from high altitude that were exposed to chronically increased ozone concentrations contained significantly higher apoplastic ascorbate concentrations than needles from spruce trees from lower altitudes with lower mean atmospheric ozone concentrations. The results show that peak concentrations of ozone do not act in spruce via a depletion of the apoplastic ascorbate pool.     

A close-up view of wood structure and properties across a growth ring of Norway spruce (Picea abies [L] Karst.)

A growth ring of an adult Norway spruce (Picea abies [L] Karst.) was analyzed to a high resolution at the single cell level with respect to structural and mechanical changes during the growth period. For this purpose structural characterization was performed by means of light microscopy, scanning electron microscopy and wide angle X-ray diffraction for investigating the geometry of cells, their cell wall fractions and cellulose microfibril angles (MFA). The mechanical properties were determined in microtensile tests on individual tracheids which had been taken from sequentially cut tangential slices. The results revealed pronounced differences in tensile stiffness between earlywood and latewood cells but only minor differences in tensile stiffness between the cell walls of both tissue types. These comparatively small changes in cell wall stiffness across the growth ring were caused by slight changes in MFA. The findings suggest that trees mainly vary cell size to optimize water transport and mechanical stability during the growth period and that modification of the cell wall organisation plays a minor role.