Effects of climatic warming on cold hardiness of some northern woody plants assessed from simulation experiments

Effects of climatic warming on cold hardiness were investigated for some northern woody plants. In the first experiment, seedlings of Norway spruce ( Picea abies [L.] Karst.), Scots pine ( Pinus sylvestris L.) and lodgepole pine ( Pinus contorta Dougl. var. latifolia Engelm.) were exposed to naturally fluctuating temperatures averaging −6°C (ambient) and 0°C (elevated) for 16 weeks in midwinter before they were thawed and re-saturated with water. In lodgepole pine, needle sugar concentrations had decreased by 15%, and the temperature needed to induce 10% injury to needles in terms of electrolyte leakage had increased by 6°C following treatment to elevated as compared with control temperatures. In contrast, Norway spruce and Scots pine showed no effects. The lack of an effect for Scots pine was ascribed to seedlings containing unusually large energy reserves that buffered respiratory expenditure of sugars. A strong, linear relationship between levels of cold hardiness, assessed by the electrolyte leakage method, and sugars was found when combining data from this and previous, similar experiments. In the second experiment, the evergreen dwarf shrub Empetrum hermaphroditum Hagerup was analysed for leaf cold hardiness, using the electrolyte leakage method, and sugar concentrations in late spring and late autumn during the third year of a warming experiment in a subarctic dwarf shrub community. The objective was to test the hypothesis that warming in the growing season alters hardening/dehardening cycles by increasing soil nitrogen mineralization and plant growth. Data found, however, suggested that cold hardening/dehardening cycles were unaffected by warming.     

Does ice crystal formation in buds explain growth disturbances in boron-deficient Norway spruce?

Loss of apical dominance in boron-deficient trees has been suggested to be due to frost damage of terminal buds and leaders. Excessive nitrogen (N) supply can exacerbate boron (B) deficiency by the dilution-effect. N may also have direct effects on winter hardiness. We studied frost hardening of buds of Norway spruce (Picea abies L. Karst.) in healthy-looking trees and in trees with growth disturbances. The effect of B and N on frost hardiness was studied in a factorial fertilisation experiment during cold acclimation. Frost hardiness was determined by differential temperature analysis (DTA) and scoring of visual damage. In a DTA profile of apical buds with a piece of stem, low-temperature exotherm (LTE) predicted bud injury, while two of the observed high-temperature exotherms and two of the observed intermediate-temperature exotherms were non injurious. Appearance of LTE followed changes in air temperature. The risk of frost damage was not affected by fertilisation treatments or previously observed growth disturbances. However, when the bud structure was deformed by severe B deficiency, the supercooling ability disappeared. Such buds are probably killed by freezing in nature and therefore, frost damage may play a secondary role in the development of growth disturbances.     

Comparing image (fractal analysis) and electrochemical (impedance spectroscopy and electrolyte leakage) techniques for the assessment of the freezing tolerance in olive

Olive growth and productivity are limited by low temperatures mainly during winter, but sometimes also in spring and fall. The most effective way to avoid these damages in areas subjected to these climatic conditions is to select least susceptible varieties, but the choice of the right method to determine cold hardiness is extremely difficult. The aims of the work were (1) to assess LT₅₀ (lethal temperature at which 50% of damage in plants subjected to low temperatures occurs) of some olive varieties in two seasons (summer and winter) and (2) to assess the reliability of different methods to evaluate cold hardiness. LT₅₀ was determined on 21 different olive (Olea europaea L.) Italian varieties by leaf and shoot electrolyte leakage, shoot impedance spectroscopy and leaf color determination of fractal spectrum. All the experiments were conducted on non-acclimated and cold-acclimated plants. Our results showed that all the three methods were able to detect damages on olive plants after exposure to low temperatures, with leaves appearing more sensitive to cold stress than shoots. Among these methods, fractal analysis could be very useful in assessing cold hardiness of plants on the basis of visible injury, without sophisticated or expensive instruments and in a reliable and cost-effective way, using only a scanning device, a personal computer and dedicated freeware software.     

Identification and expression analysis of cold-regulated genes from the cold-hardy Citrus relative Poncirus trifoliata (L.) Raf.

Citrus is a cold-sensitive genus and most commercially important varieties of citrus are susceptible to freezes. On the other hand, Poncirus trifoliata (L.) Raf. is an interfertile Citrus relative that can tolerate temperatures as low as −26°C when fully cold acclimated. Therefore, it has been used for improving cold tolerance in cold-sensitive commercial citrus rootstock varieties and in attempts to improve scion varieties. In this study, cDNA libraries were constructed from both 2-day cold-acclimated and from non-acclimated Poncirus seedlings using a subtractive hybridization method with the objective of identifying cold-regulated genes. A total of 192 randomly picked clones, 136 from the cold-induced library and 56 from the cold-repressed library, were sequenced. The majority of these clones showed sequence homology to previously identified cold-induced and/or environmental stress-regulated genes in Arabidopsis. In addition, some of them shared homology with cold and/or environmental stress-induced genes previously identified in other herbaceous and woody perennial plants and some showed no homology with sequences in GenBank. When these 192 cDNAs were analyzed by reverse northern blot with cold-acclimated and non-acclimated probes, 92 of the cDNAs displayed significantly increased expression, ranging from 2 to 49-fold, during cold acclimation; all 92 were from the cold-induced library. Surprisingly no clones displayed significantly repressed expression in response to cold. Analysis of a number of selected genes individually in northern blots of mRNA from cold-acclimated and non-acclimated plants largely confirmed the reverse northern analysis, verifying induction of expression of selected cDNAs in response to cold. The results showed that subtractive hybridization is an efficient method for identification of cold-induced genes in plants with limited sequence information available. This study also revealed that genes induced during cold acclimation of the cold-hardy citrus relative P. trifoliata are similar to those in Arabidopsis, indicating that similar pathways may be present and activated during cold acclimation in woody perennial plants.     

Cold acclimation was partially impaired in boron deficient Norway spruce seedlings

Susceptibility of trees to freezing injury has been suggested to increase in boron (B) deficiency but there is no experimental evidence to support this proposition. In this study, Norway spruce (Picea abies L. Karst.) seedlings were cultivated for two growing seasons in deficient, intermediate and ‘optimal’ B levels. Cold hardening of the seedlings was measured after the second growing season. Freezing tolerance in tips of shoots, needles, stems and roots was determined by controlled freezing tests and electrolyte leakage method, and that of buds, in addition, by differential thermal analysis (DTA). Electrical impedance was used to monitor changes in the apoplastic space during cold acclimation. Root dry weight and shoot height growth were lower in B deficiency. Cold acclimation of buds and stems was reduced by B deficiency. When hardened seedlings were subjected to subzero temperatures for 3 weeks, extracellular electrical resistance of stems became the highest at the lowest B supply which was probably due to decreased desiccation tolerance. As a conclusion, susceptibility to freezing damage may be increased by B deficiency in Norway spruce trees.     

Regulation of proline and ethylene levels in rape seedlings for freezing tolerance

This study was aimed to investigate the possibility of regulating free proline content and ethylene production in the resistant to abiotic stress cv. ‘Hornet H’ and the tolerant to stress cv. ‘Sunday’ of winter rapeseed seedlings by pretreatment with exogenous L-proline and L-glutamine in non-acclimated and cold-acclimated seedlings in relation to freezing tolerance. The ratio of proline content in acclimated (at 4°C) versus non-acclimated (18°C) ‘Hornet H’ seedlings increased 2.12-fold and in ‘Sunday’ seedlings 1.95-fold. Exogenously applied, proline and glutamine produced a positive effect on free proline content in both cold-acclimated and non-acclimated seedlings. At a temperature of -1°C the proline content significantly increased in non-acclimated and especially in cold-acclimated seedlings. At an intensified freezing temperature (?3°C, ?5°C, ?7°C), the proline content decreased in comparison with that at ?1°C, but glutamine, especially proline, in cold-acclimated seedlings takes part in free proline level increase and in seedlings’ resistance to freezing. Ethylene production increased in cold-acclimated conditions and under the effect of exogenous proline and glutamine. In freezing conditions, ethylene production decreased, but in cold-acclimated seedlings and under pretreatment of proline and glutamine the ethylene synthesis was intensive. Thus, free proline content and ethylene production increase in cold-acclimated winter rapeseed seedlings and under pretreatment with glutamine and especially with proline. Free proline is involved in the response to cold stress, and its level may be an indicator of cold-hardening and freezing tolerance, but the role of ethylene in the regulation of cold tolerance remains not quite clear.     

Combined effect of elevated UVB,elevated temperature and fertilization on growth,needle structure and phytochemistry of young Norway spruce (Picea abies) seedlings

Simultaneously with warming climate, other climatic and environmental factors are also changing. Here, we investigated for the first time the effects of elevated temperature, increased ultraviolet‐B (UVB) radiation, fertilization and all combinations of these on the growth, secondary chemistry and needle structure of 1‐year‐old Norway spruce (Picea abies (L.) Karst.) seedlings in an outdoor experiment. After one growing season, elevated temperature increased root : shoot ratio and concentrations of needle piperidine alkaloids, while concentrations of needle catechins and acetophenones and bark flavonoids decreased compared with ambient temperature seedlings. UVB‐radiation increased concentrations of bark condensed tannins, while fertilization increased total biomass and concentrations of needle catechins. In addition to the main effects, concentrations of some individual phenolic compounds showed UV × temperature or UV × temperature × fertilization interactions, and fertilization modified temperature response on root : shoot ratio. All the treatments described here affected the defence chemistry profiles of the seedlings, which may imply some changes in plant‐herbivore interactions in connection with changing climate. The interactions between treatments indicate a need for further experiments involving several simultaneously affecting environmental changes.     

Relationship between respiratory depletion of sugars and loss of cold hardiness in coniferous seedlings over-wintering at raised temperatures: indications of different sensitivities of spruce and pine

Long-term effects of elevated winter temperatures on cold hardiness were investigated for Norway spruce (Picea abies L. Karst.), lodgepole pine (Pinus contorta Dougl.) and Scots pine (Pinus sylvestris L.). Two-year-old seedlings with the same pre-history of growth and cold hardening in the field were maintained from early December to late March at two field sites in northern Sweden and in a cold room. The temperatures at these locations averaged –13·5, –8·9 and 5·5°C, respectively. Following treatments, carbohydrate contents and cold tolerances were assessed. Needle respiration was also analysed during the 5·5°C treatment. Cold tolerance of lodgepole pine and Scots pine was much reduced following the 5·5°C treatment. Cold tolerance was somewhat reduced in lodgepole pine following the –8·9 °C treatment, but was essentially maintained in spruce throughout all treatments. The cold tolerance of needles was strongly correlated with their soluble sugar contents. Spruce maintained cold hardiness by having larger reserves of sugars and lower rates of respiration which decreased more rapidly as sugars were depleted. Tolerance of lodgepole pine to frost desiccation was also much reduced following the 5·5°C treatment.     

Frost hardiness in bark and needles of Norway spruce in southern Sweden

Bark necrosis and resin flows in Norway spruce have increased in southern Sweden over the last few decades. Frost damage late in spring has been suggested as a possible cause, but other factors besides the climate may have contributed to the damage. The nutrient status influences the hardening processes and plants with poor nutritional conditions have an increased sensitivity to frost. In this study the sensitivity to frost of bark and the hardiness status of needles of Norway spruce were compared with the nutrient status at two sites with different soil fertility. The trees were 30-40 years old. The hardiness status of the bark and needles was negatively affected by low concentrations of P and Mg.     

Changes in chemical components in the freshwater apple snail, Pomacea canaliculata (Gastropoda: Ampullariidae), in relation to the development of its cold hardiness

The apple snail, Pomacea canaliculata, is an invasive freshwater snail. It increases its cold hardiness before winter. However, the physiological mechanism of cold hardiness in molluscs is poorly understood, especially in freshwater molluscs. In this study, we examined the changes in low molecular weight compounds, glycogen and lipids, in the body of P. canaliculata in association with the development of cold hardiness. When snails without cold hardiness were experimentally cold-acclimated, the amount of glycerol, glutamine, and carnosine increased, while glycogen and phenylalanine decreased. Overwintering cold-tolerant snails collected from a drained paddy field in November also showed increased glycerol in their bodies with decreasing glycogen concentration, compared to summer snails collected from a submerged field. Water content also decreased during the cold acclimation, although the water loss was minimal. These results indicate that the freshwater snail, P. canaliculata enhances cold hardiness by accumulation of some kinds of low molecular weight compounds in its body as some insects do. However, the actual function of each low molecular compound is still unknown.     

Resource allocation in different parts of juvenile mountain birch plants: effect of nitrogen supply on seedling phenolics and growth

The composition and concentrations of phenolic compounds were studied in the first true leaves, cotyledons, stems and roots of 2.5-week-old seedlings of mountain birch ( Betula pubescens ssp. czerepanovii ). The differences in secondary compounds among these plant parts were both qualitative and quantitative. In all parts, condensed tannins accounted for more than 50% of the phenolics. In the first true leaves and cotyledons, chlorogenic acid was the most abundant of the HPLC phenolics. The main components in stems were (+)-catechins and rhododendrins whereas in roots, the main components were ellagitannins. The seedlings were grown at three levels of nitrogen supply (very low-N, low-N, moderate-N), and the effect of nitrogen on concentrations of phenolic compounds was studied in all plant parts. The dry weight of all plant parts, except the roots, increased with increased nitrogen. In all parts, the concentration of condensed tannins was higher at lower levels of nitrogen than at moderate-N. The concentrations of total HPLC phenolics and also those of the compound groups of HPLC phenolics were, however, affected only in the first true leaves and roots. The concentrations in the first true leaves were generally higher in seedlings grown at very low-N and low-N than in seedlings grown at moderate-N. The concentrations in roots were highest at low-N. Not all compounds responded to nitrogen supply in the same manner. The changes in concentrations cannot be exclusively interpreted as changes in the accumulation of phenolic compounds, due to dilution caused by the increase in biomass in better nitrogen availability. There were differences in carbon allocation between condensed tannins and HPLC phenolics in seedlings grown at different nitrogen levels.     

The relative hardening of roots and shoots and the influence of day-length during hardening in perennial ryegrass

The influence of long and short days during the hardening period on the cold hardiness of perennial ryegrass seedlings was studied as was the relative hardiness of roots and shoots. Hardiness was assessed by the electrolyte release method which was a measure of the amount of damage subsequent to low temperature treatment. Long days promoted hardiness in shoots of Pax 0tofte plants and in one case under short days the roots were found to be hardy. Generally roots were less hardy than shoots in Pax 0tofte and S23 plants hardened under long days for 2 wk. When hardened at the fourth leaf stage for 2 wk at + 5 ^oC under long day conditions, Pax 0tofte plants were more hardy than those of S23. The long day effect on hardiness was arrived at more rapidly, there being no difference in hardiness after 3 wk in Pax 0tofte hardened under long or short days, whereas a significant degree of hardening was observed after 1 wk of hardening under long days at – 4 ^oC. The results obtained are discussed in relation to winter kill of grasses in the West of Scotland, and it is considered that root damage is not an important factor in causing winter kill. The promotive effect of long days on hardiness when hardening commences in late autumn is considered an advantage in temperate regions as it may also allow early frosts to be withstood.     

Inositol 1,4,5-trisphosphate formation in leaves of winter oilseed rape plants in response to freezing, tissue water potential aed abscisic acid

Time courses of formation of inositol 1,4,5-trisphosphate (IP₃) were followed in the leaves of non-acclimated and cold (2°C)-acclimated winter oilseed rape ( Brassica napus L. var. oleifera ) plants, subjected to different freezing temperatures or to polyethylene glycol 8000 (PEG) and abscisic acid (ABA) treatments. Changes in water potential (Ψ_w) and in ABA level in the frost- and PEG-treated tissues were also determined. Results obtained indicate that temperatures sligthly higher than LT₅₀ induced a transient and substantial increase in IP₃ level, both in non-acclimated and cold-acclimated tissues. At comparable freezing temperature (–5°C) the response of cold-acclimated leaves was lower than that of non-acclimated ones. The PEG-depedent decrease in Ψ_w to –0.9 MPa or ABA (0.1 m M ) treatment gave rise to a transient increase in IP₃ content in non-acclimated tissues only. Collectively, the data indicate that cold acclimation of plants may lead to lower cell responsiveness to the factors studied in terms of induction of IP₃ formation. Changes in the IP₃ content, observed in the present experiments, support our previous suggestion that non-killing freezing temperatures may induce the phosphoinositide pathway, both in non-acclimated and cold-acclimated tissues. Lowering of tissue water potential to some threshold value or a high exogenous ABA supply may mimic the freezing-dependent reaction in the non-acclimated leaves.     

The influence of cold acclimation on antioxidative enzymes and antioxidants in sensitive and tolerant barley cultivars

In order to better understand the role of cold acclimation in alleviating freezing injury, two barley cultivars with different cold tolerance, i.e. a sensitive cv. Chumai 1 and a tolerant cv. Mo 103, were used. The freezing treatment increased leaf soluble protein content more in the tolerant cultivar than in the sensitive one. Cold acclimation increased H₂O₂ content of the two cultivars during freezing treatment, especially in Mo 103. Glutathione and ascorbate contents during freezing and recovery were significantly higher in cold-acclimated plants than in non-acclimated ones. Activities of peroxidase, ascorbate peroxidase and glutathione reductase were also higher in cold-acclimated plants than non-acclimated plants during freezing treatment. However, there was no significant difference between cold-acclimated plants and the control plants in catalase activity. It may be assumed that cold acclimation induced H₂O₂ production, which in turn enhanced activities of antioxidative enzymes and synthesis of antioxidants, resulting in alleviation of oxidative stress caused by freezing.     

Changes in wheat leaf phenolome in response to cold acclimation

A study of wheat (Triticum aestivum L.) leaves phenolome was carried out during cold acclimation of the winter (Claire) and spring (Bounty) varieties using a combination of HPLC–ESI–MS techniques. A total of 40 phenolic and flavonoid compounds were identified, and consisted mainly of two coumarin derivatives, eight simple phenolic derivatives, 10 hydroxycinnamoyl amides and 20 flavonoid derivatives. Identification and quantification of individual compounds were performed using an HPLC system coupled with a photodiode array detector and two different ESI–MS systems, in combination with a multiple reaction monitoring (MRM) technique. The analyses indicated that, although there were no qualitative differences in their profiles, the winter variety exhibited a higher phenolic content compared to the spring variety when both were grown under non-acclimated (control) conditions. Cold acclimation, on the other hand, resulted in a significant differential accumulation of phenolic compounds in both varieties: mostly as luteolin C-glycosides and their O-methyl derivatives in the winter variety (Claire) and a derivative of hydroxycinnamoyl amide in the spring variety (Bounty). These compounds accumulated in relatively large amounts in the apoplastic compartment. The accumulation of the O-methylated derivatives was associated with a marked increase in O-methyltransferase (OMT) activity. In addition, the trimethylated flavone, 3′,4′,5′-trimethyltricetin was identified for the first time in the native extracts of both control and cold-acclimated wheat leaves. The accumulation of a mixture of beneficial flavonoids, such as iso-orientin, vitexin and tricin in cold acclimated wheat leaves, attests for its potential as an inexpensive source of a health-promoting supplement to the human diet.     

Decomposition and element concentrations of Norway spruce needle litter with differing B, N, or P status

Adequate boron (B) nutrition may decrease concentrations of phenolic compounds and enhance structural integrity and lignification in plants, compared with suboptimal B. This could affect decomposition in areas where B deficiencies are common. The mass loss and changes in element concentrations in Norway spruce needle litter were studied with combinations of litter from high-B and low-B trees, incubated for 29 months, in either B fertilised or control plots without B addition. The litter originated from the same Norway spruce field experiments. Additionally, the field experiments included long-term N and P treatments. Initially, lowest lignin concentrations were found in Norway spruce litter from the treatment P and particularly in the combination B?+?P, and highest in the B?+?N fertilised plots. The mass loss of Norway spruce litter was not affected by the treatments. However, B_litter increased Cu accumulation. The litter from the B?+?P fertilised plots accumulated considerably more Al, Ca, S and Zn than the other treatments, whereas B together with N reduced the remaining amounts of these elements. Reduced nutrient release from litter may have far-reaching consequences on nutrient cycles in forests.     

Cold hardening of raspberry plants in vitro is enhanced by increasing sucrose in the culture medium

The influence of exogenously applied sucrose on cold hardening of raspberry ( Rubus idaeus L.) in vitro was examined. Raspberry plants (cv. Preussen) were cultured on Murashige-Skoog (MS) media with different levels (1, 3, 5 and 7%) of sucrose and subjected to low-temperature acclimation (3/−3°C day/night temperature, 8-h photoperiod) for 14 days. Cold hardiness (LT₅₀ in controlled freezing), shoot moisture content, osmolality and the amounts of sucrose, glucose and fructose were determined. Exogenously applied sucrose was taken up by plants, but the uptake corresponded to less than 10% of total sugar reserves in the culture. Cold hardiness was primarily affected by acclimation treatment, but sucrose increased cold hardiness of nonacclimated plants and significantly enhanced the effect of acclimation treatment, 5% sucrose in the culture medium being optimal for cold hardening. LT₅₀ values ranged between −4.1 and −7.1°C for nonacclimated, and between −14.2 and −20.7°C for cold-acclimated shoots. Shoot moisture content was inversely related to medium sucrose level and declined only slightly during cold acclimation. After cold acclimation, plant osmolality predicted hardiness better than shoot moisture content. Plant osmolality and sugar content were increased by increasing the medium sucrose level and, to a greater extent, by cold acclimation. Sucrose, glucose and fructose accumulated during hardening. Sucrose was the predominant sugar, and the rate of sucrose accumulation during cold acclimation was independent of the medium sucrose level or the initial plant sucrose content. A close correlation between cold hardiness and total sugars, sucrose, glucose and fructose was established. These results suggest that sugars have more than a purely osmotic effect in protecting acclimated raspberry plants from cold.     

Developmental disorders in buds and needles of mature Norway spruce, Picea abies (L.) Karst., in relation to needle boron concentrations

Dieback of the terminal shoot and consequently bushy growth induced by boron deficiency have been reported widely throughout the world in several tree species. Recently, similar growth damage was documented in half of the young spruce stands in eastern Finland. To clarify the role of B deficiency, the light microscopic structure of emerging buds and of developing and previous-year needles of mature Norway spruce (Picea abies L. Karst.) from damaged (D stand), partly damaged (PD stand) and healthy (H stand) stands were analysed. The samples, on which needle nutrient concentrations were also determined, were taken seven times between early spring (April) and early winter (November). Cell death characterized by precipitation of the cell content, possibly due to the release of tannins after membrane rupture, was seen in the apex of emerging buds, and this led to fatal damage in about half of the buds in the trees from the D stand, where the needle B concentration was well below the deficiency level of 4–5 mg kg⁻¹. Furthermore, an increase in living cells that accumulated tannins in the vacuoles, which is a common stress and/or defense reaction, was found in the primordial shoots of buds and in the differentiating needles in the PD and D stands. The increase in the areas of the central cylinder and of the xylem found in the needles indicate structural plasticity during needle differentiation to drought. The time frame for bud emergence from late May up to mid-September means that an adequate B supply is necessary throughout the summer in order to avoid fatal bud damage and thus bushy growth of the trees.     

Effect of frost nights and day and night temperature during dormancy induction on frost hardiness, tolerance to cold storage and bud burst in seedlings of Norway spruce

For trees, the ability to obtain and maintain sufficient levels of frost hardiness in late autumn, winter and spring is crucial. We report that temperatures during dormancy induction influence bud set, frost hardiness, tolerance to cold storage, timing of bud burst and spring frost hardiness in seedlings of Norway spruce (Picea abies (L.) Karst.). Bud set occurred later in 12°C than in 21°C, and later in cool nights (7°C) than in constant temperature. One weekly frost night (−2.5°C) improved frost hardiness. Cool nights reduced frost hardiness early, but improved hardiness later during cold acclimation. Buds and stems were slightly hardier in 21°C than in 12°C, while needles were clearly hardier in 12°C. Cold daytime temperature, cool nights and one weekly frost night improved cold storability (0.7°C). Seedlings receiving high daytime temperatures burst buds later, and were less injured by light frost some days after bud burst.     

Restoration of secondary metabolism in birch seedlings relieved from PAL-inhibitor

Phenylalanine ammonia lyase (PAL) plays a key role in phenylpropanoid metabolism, catalyzing the deamination of phenylalanine (Phe) to form trans-cinnamic acid. Inhibitors of PAL have been used to study the physiological role of the different compounds derived from trans-cinnamic acid, and to test theories about a trade-off between growth and defence in plants. In a previous study with birch (Betula pubescens Ehrh.) seedlings, the PAL inhibitor 2-aminoindane-2-phosphonic acid monohydrate (AIP) caused an accumulation of Phe and a strong decrease in the quantity of simple phenolics, soluble condensed tannins and growth, whereas flavonol glycosides were generally not affected. The present study demonstrates restoration of secondary metabolism in the previously AIP treated birch seedlings. Our results indicate that Phe accumulated during PAL inhibition could be partly used to increase the content of the phenolic acids, flavan-3-ols and to some extent the soluble condensed tannins. Seedling growth also increased when the supply of PAL inhibitor ceased. We thereby show that the inhibition of PAL by AIP in vivo is reversible, at least for moderate AIP concentrations and the rate of restoration is dependent on the inhibitor concentration.