Red and blue light treatments of ripening bilberry fruits reveal differences in signalling through abscisic acid-regulated anthocyanin biosynthesis.

The biosynthesis of anthocyanins has been shown to be influenced by light quality. However, the molecular mechanisms underlying the light-mediated regulation of fruit anthocyanin biosynthesis are not well understood. In this study, we analysed the effects of supplemental red and blue light on the anthocyanin biosynthesis in non-climacteric bilberry (Vaccinium myrtillus L.). After 6 days of continuous irradiation during ripening, both red and blue light elevated concentration of anthocyanins, up to 12- and 4-folds, respectively, compared to the control. Transcriptomic analysis of ripening berries showed that both light treatments up-regulated all the major anthocyanin structural genes, the key regulatory MYB transcription factors and abscisic acid (ABA) biosynthetic genes. However, higher induction of specific genes of anthocyanin and delphinidin biosynthesis alongside ABA signal perception and metabolism were found in red light. The difference in red and blue light signalling was found in 9-cis-epoxycarotenoid dioxygenase (NCED), ABA receptor pyrabactin resistance-like (PYL) and catabolic ABA-8'hydroxylase gene expression. Red light also up-regulated expression of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) domain transporters, which may indicate involvement of these proteins in vesicular trafficking of anthocyanins during fruit ripening. Our results suggest differential signal transduction and transport mechanisms between red and blue light in ABA-regulated anthocyanin and delphinidin biosynthesis during bilberry fruit ripening.

Beyond rest and quiescence (endodormancy and ecodormancy): A novel model for quantifying plant-environment interaction in bud dormancy release.

Bud dormancy of plants has traditionally been explained either by physiological growth arresting conditions in the bud or by unfavourable environmental conditions, such as non-growth-promoting low air temperatures. This conceptual dichotomy has provided the framework also for developing process-based plant phenology models. Here, we propose a novel model that in addition to covering the classical dichotomy as a special case also allows the quantification of an interaction of physiological and environmental factors. According to this plant-environment interaction suggested conceptually decades ago, rather than being unambiguous, the concept of "non-growth-promoting low air temperature" depends on the dormancy status of the plant. We parameterized the model with experimental results of growth onset for seven boreal plant species and found that based on the strength of the interaction, the species can be classified into three dormancy types, only one of which represents the traditional dichotomy. We also tested the model with four species in an independent experiment. Our study suggests that interaction of environmental and physiological factors may be involved in many such phenomena that have until now been considered simply as plant traits without any considerations of effects of the environmental factors.

Xylem anatomical and growth responses of the dwarf shrub Vaccinium myrtillus to experimental CO2 enrichment and soil warming at treeline.

Plant growth responses to environmental changes may be linked to xylem anatomical adjustments. The study of such links is essential for improving our understanding of plant functioning under global change. We investigated the xylem anatomy and above-ground growth of the dwarf shrub Vaccinium myrtillus in the understorey of Larix decidua and Pinus uncinata at the Swiss treeline after 9 years of free-air CO2 enrichment (+200 ppm) and 6 years of soil warming (+4 °C). We aimed to determine the responses of xylem anatomical traits and growth to these treatments, and to analyse xylem anatomy-growth relationships. We quantified anatomical characteristics of vessels and ray parenchyma and measured xylem ring width (RW), above-ground biomass and shoot elongation as growth parameters. Our results showed strong positive correlations between theoretical hydraulic conductivity (Kh) and shoot increment length or total biomass across all treatments. However, while soil warming stimulated shoot elongation and RW, it reduced vessel size (Dh) by 14%. Elevated CO2 had smaller effects than soil warming: it increased Dh (5%) in the last experimental years and only influenced growth by increasing basal stem size. The abundance of ray parenchyma, representing storage capacity, did not change under any treatment. Our results demonstrate a link between growth and stem Kh in V. myrtillus, but its growth responses to warming were not explained by the observed xylem anatomical changes. Smaller Dh under warming may increase resistance to freezing events frequently occurring at treeline and suggests that hydraulic efficiency is not limiting for V. myrtillus growing on moist soils at treeline. Our findings suggest that future higher atmospheric CO2 concentrations will have smaller effects on V. myrtillus growth and functioning than rising temperatures at high elevations; further, growth stimulation of this species under future warmer conditions may not be synchronized with xylem adjustments favouring hydraulic efficiency.

Bioaccumulation of heavy metals and ecophysiological responses to heavy metal stress in selected populations of Vaccinium myrtillus L. and Vaccinium vitis-idaea L.

The aim of this study was to determine the concentrations of heavy metals (Cd, Pb, Zn, Fe, and Mn) in soil, and their bioavailability and bioaccumulation in Vaccinium myrtillus L. and Vaccinium vitis-idaea L. organs. Analysis also concerned the physiological responses of these plants from three polluted sites (immediate vicinity of a zinc smelter in Miasteczko Slaskie, ArcelorMittal Poland S.A. iron smelter in Dabrowa Górnicza-Losien, and Jaworzno III power plant in Jaworzno) and one pseudo-control site (Pazurek nature reserve in Jaroszowiec Olkuski). All of the sites are situated in the southern parts of Poland in the Slaskie or Malopolskie provinces. The contents of proline, non-protein thiols, glutathione, ascorbic acid, and the activity of superoxide dismutase and guaiacol peroxidase in the leaves of Vaccinium myrtillus L. and Vaccinium vitis-idaea L. were measured. In soil, the highest levels of Cd, Pb, and Zn (HNO3 extracted and CaCl2 extracted) were detected at the Miasteczko Slaskie site. At all sites a several times lower concentration of the examined metals was determined in the fraction of soil extracted with CaCl2. Much higher Cd, Pb, Zn and Fe concentrations were found in V. myrtillus and V. vitis-idaea grown at the most polluted site (located near the zinc smelter) in comparison with cleaner areas; definitely higher bioaccumulation of these metals was found in lingonberry organs. Additionally, we observed a large capability of bilberry to accumulate Mn. Antioxidant response to heavy metal stress also differed between V. myrtillus and V. vitis-idaea. In V. myrtillus we found a positive correlation between the level of non-protein thiols and Cd and Zn concentrations, and also between proline and these metals. In V. vitis-idaea leaves an upward trend in ascorbic acid content and superoxide dismutase activity accompanied an increase in Cd, Pb, and Zn concentrations. At the same time, the increased levels of all tested metals in the leaves of V. vitis-idaea were accompanied by a decreased activity of guaiacol peroxidase. In both species increased Mn accumulation caused a decrease in antioxidant response.

Effects of Insect Herbivory on Bilberry Production and Removal of Berries by Frugivores.

The evolutionary purpose of a fleshy fruit is to attract seed dispersers and get the seeds dispersed by frugivorous animals. For this reason, fruits should be highly rewarding to these mutualists. However, insect herbivory can alter plant reproductive success e.g. by decreasing fruit yield or affecting the attractiveness of the fruits to mutualistic seed dispersers. Under natural conditions, we tested the effects of experimental larval-defoliation on berry ripening and consumption of a non-cultivated dwarf shrub, the bilberry (Vaccinium myrtillus L.), which produces animal-dispersed berries with high sugar and anthocyanin concentration. Bilberry ramets with high fruit yield were most likely to have their berries foraged, indicating that frugivores made foraging choices based on the abundance of berries. Moreover, the probability for berries being foraged was the lowest for non-defoliated ramets that grew adjacent to larval-defoliated ramets, even though larval-defoliation did not affect the biochemical composition (total concentrations of anthocyanins, sugars and organic acids) or the probability of ripening of berries. We hypothesise that the lower probability for berries being foraged in these ramets may be a consequence of rhizome- or volatile-mediated communication between ramets, resulting in a priming effect of the herbivore defence and lower attractiveness of the non-defoliated ramets.

Impacts of winter icing events on the growth, phenology and physiology of sub-arctic dwarf shrubs.

The Arctic is experiencing the greatest climate change in winter, including increases in freeze-thaw cycles that can result in ice encasement of vegetation. Ice encasement can expose plants to hypoxia and greater temperature extremes, but currently the impacts of icing on plants in the field remain little understood. With this in mind, a unique field manipulation experiment was established in heathland in northern Sweden with ice encasement simulated in early March 2008, 2009 and 2010 until natural thaw each spring. In the following summers we assessed the impacts on flowering, bud phenology, shoot growth and mortality and leaf damage (measured by chlorophyll fluorescence and electrolyte leakage) of the three dominant dwarf shrub species Empetrum nigrum, Vaccinium vitis-idaea (both evergreen) and Vaccinium myrtillus (deciduous). Two consecutive winters of icing decreased V. vitis-idaea flowering by 57%, while flowering of V. myrtillus and E. nigrum remained unaffected. Vaccinium myrtillus showed earlier budburst but shoot growth for all species was unchanged. Shoot mortality of V. myrtillus and V. vitis-idaea increased after the first year (by 70 and 165%, respectively) and again for V. myrtillus following the third year (by 67%), while E. nigrum shoot mortality remained unaffected, as were chlorophyll fluorescence and electrolyte leakage in all species. Overall, the sub-arctic heathland was relatively tolerant to icing, but the considerable shoot mortality of V. myrtillus contrasting with the general tolerance of E. nigrum suggests plant community structure in the longer term could change if winters continue to see a greater frequency of icing events.

Phenolic composition and antioxidant capacity of bilberry (Vaccinium myrtillus) leaves in Northern Europe following foliar development and along environmental gradients.

Bilberry is a characteristic field layer species in the boreal forests and is an important forage plant for herbivores of the North European ecosystem. Bilberry leaves contain high levels of phenolic compounds, especially hydroxycinnamic acids, flavonols, catechins, and proanthocyanidins. We investigated the phenolic composition of bilberry leaves in two studies, one following foliar development in forest and open areas, and the other along a wide geographical gradient from south to north boreal forests in Finland. An analysis of bilberry leaves collected in open and forest areas showed that major phenolic changes appeared in the first stages of leaf development, but, most importantly, synthesis and accumulation of flavonoids was delayed in the forest compared to the high light sites. Sampling along a geographical gradient in the boreal zone indicated that leaves from higher latitudes and higher altitudes had greater soluble phenolic and flavonol levels, higher antioxidant capacity, and lower contents of chlorogenic acid derivatives. The ecological significance of the results is discussed.

Investigating the interaction between ungulate grazing and resource effects on Vaccinium myrtillus populations with integral projection models.

Dense ungulate populations in forest accompanied by high grazing intensities have the potential to affect plant population dynamics, and such herbivory effects on populations are hypothesised to differ along environmental gradients. We investigated red deer grazing and resource interaction effects on the performance and dynamics of the functionally important boreal shrub Vaccinium myrtillus using integral projection models (IPMs). We sampled data from 900 V. myrtillus ramets in 30 plots in two consecutive years across the boreo-nemoral pine forest on the island Svanøy, western Norway. The plots spanned two environmental gradients: a red deer grazing intensity gradient (assessed by Cervus elaphus faecal pellets), and a relative resource gradient (DCA-ordination of species composition). The use of IPMs enabled projections of population growth rate (lambda) using continuous plant size instead of forcing stage division upon the demographic data. We used the environmental gradients as continuous variables to explain the dynamics of V. myrtillus populations and found that both increasing grazing intensity and resource levels negatively affected lambda of the V. myrtillus populations. Interestingly, these factors interacted: the negative effects of grazing were strongest in the resource-rich vegetation, and higher resource levels reduced lambda more strongly than at low resource levels when grazing intensities became higher. Populations with lambda > 1 were projected if the grazing intensity was less than or equal to the mean grazing intensity on the island, and indicated that V. myrtillus is relatively tolerant of grazing. Variance decomposing showed that the decrease of lambda along the grazing gradient, both at low and high resource levels, was largely caused by reductions in plant growth. The use of IPMs together with important environmental gradients offered novel possibilities to study the synthesised effect of different factors on plant population dynamics. Here, we show that the population response of an abundant boreal shrub to ungulate grazing depends on resource level.

Impacts of extreme winter warming events on plant physiology in a sub-Arctic heath community.

Insulation provided by snow cover and tolerance of freezing by physiological acclimation allows Arctic plants to survive cold winter temperatures. However, both the protection mechanisms may be lost with winter climate change, especially during extreme winter warming events where loss of snow cover from snow melt results in exposure of plants to warm temperatures and then returning extreme cold in the absence of insulating snow. These events cause considerable damage to Arctic plants, but physiological responses behind such damage remain unknown. Here, we report simulations of extreme winter warming events using infrared heating lamps and soil warming cables in a sub-Arctic heathland. During these events, we measured maximum quantum yield of photosystem II (PSII), photosynthesis, respiration, bud swelling and associated bud carbohydrate changes and lipid peroxidation to identify physiological responses during and after the winter warming events in three dwarf shrub species: Empetrum hermaphroditum, Vaccinium vitis-idaea and Vaccinium myrtillus. Winter warming increased maximum quantum yield of PSII, and photosynthesis was initiated for E. hermaphroditum and V. vitis-idaea. Bud swelling, bud carbohydrate decreases and lipid peroxidation were largest for E. hermaphroditum, whereas V. myrtillus and V. vitis-idaea showed no or less strong responses. Increased physiological activity and bud swelling suggest that sub-Arctic plants can initiate spring-like development in response to a short winter warming event. Lipid peroxidation suggests that plants experience increased winter stress. The observed differences between species in physiological responses are broadly consistent with interspecific differences in damage seen in previous studies, with E. hermaphroditum and V. myrtillus tending to be most sensitive. This suggests that initiation of spring-like development may be a major driver in the damage caused by winter warming events that are predicted to become more frequent in some regions of the Arctic and that may ultimately drive plant community shifts.

Drought tolerance of juvenile and mature leaves of a deciduous dwarf shrub Vaccinium myrtillus L. in a boreal environment.

The difference between drought tolerance of juvenile and mature leaves of the winter-deciduous dwarf shrub bilberry (Vaccinium myrtillus L.) from a northern boreal environment was investigated. It was hypothesised that mature leaves are more drought sensitive than juvenile leaves. Bilberry plants were allowed to dry out by excluding irrigation when leaves were at juvenile and mature stages. Tissue water content decreased at both phenological stages, but the response was more pronounced in the mature leaves. Anthocyanin concentrations increased as the tissue water content decreased, and again this occurred to a greater extent in the mature leaves. Chlorophyll concentrations decreased only marginally at the juvenile stage, while the decrease was significant in the mature leaves. Chlorophyll degradation was enhanced by drought stress. Soluble proteins decreased and protein oxidation increased in the mature leaves, and degradation of oxidised proteins increased in the drought-stressed plants. The results suggest that leaves of bilberry are more sensitive to drought stress at the mature stage, and that drought stress accelerates senescence at the mature stage. The significance of the results is that dry periods during the juvenility of leaves are not as detrimental as they may be later in summer. In addition, the strategy of a winter-deciduous plant is obviously to protect its perennial parts from severe drought by accelerated leaf senescence at the mature stage. Therefore, the deciduous life form may provide an excellent adaptation against drought also in northern ecosystems. The role of anthocyanins in photoprotection under drought stress is also discussed.

Interactions between gray-sided voles (Clethrionomys rufucanus) and bilberry (Vaccinium myrtillus), their main winter food plant.

We compared the abundance, population structure and palatability of bilberry ramets on vole-free islands, islands with voles but no predators (predator-free islands) and mainland sites with both voles and predators. As expected, bilberry biomass was strongly correlated with the herbivory pressure exerted by the voles, since it was significantly lower on the mainland, and much (>80%) lower on the predator-free islands, than on the vole-free islands. However, another finding, which conflicts with hypotheses postulating that herbivory generally induces plant defenses, was that voles preferred ramets from predator-free islands. Bilberry plants were fairly tolerant to grazing since they compensated for some of the lost tissue by producing more new ramets. This response should promote stability in the plant-herbivore interaction by reducing the impact of past grazing on current food production and thus minimizing time delays in the interactions that could potentially generate population cycles.

Explaining bank vole cycles in southern Norway 1980-2004 from bilberry reports 1932-1977 and climate.

Correlations between mast fruiting of bilberry Vaccinium myrtillus and peak levels of Clethrionomys-voles have been reported from both Norway and Finland, but there has been a discussion whether this is a bottom-up or a top-down relationship. In a multiple regression model, 65% of the variation in a bilberry production index calculated from game reports from southern Norway 1932-1977 could be explained by the berry index of the two preceding years and climate factors acting during key stages of the flowering cycle. High vole populations in previous years did not contribute to explain the fluctuation in berry production. I used the selected model and climate data to predict bilberry production for the period 1978-2004. Predicted berry indices of the current and previous year explained 38% and the total amount of precipitation in May-June explained 16% of the variation in a log-transformed snap-trapping index of bank vole Clethrionomys glareolus 1980-2004. The vole index was not related to any of the climate variables used to predict berry production. This pattern supports the hypothesis that vole cycles are generated by changes in plant chemistry due to climate-synchronized mast fruiting.

Nitrogen acquisition from inorganic and organic sources by boreal forest plants in the field.

A wide range of recent studies have indicated that organic nitrogen may be of great importance to plant nitrogen (N) nutrition. Most of these studies have, however, been conducted in laboratory settings, excluding important factors for actual plant uptake, such as competition, mycorrhizal associations and soil interactions. In order to accurately evaluate the importance of different N compounds to plant N nutrition, field studies are crucial. In this study, we investigated short- as well as long-term plant nitrogen uptake by Deschampsia flexuosa, Picea abies and Vaccinium myrtillus from 15NO3-, 15NH4+ and (U-13C, 15N) arginine, glycine or peptides. Root N uptake was analysed after 6 h and 64 days following injections. Our results show that all three species, irrespective of their type of associated mycorrhiza (arbuscular, ecto- or ericoid, respectively) rapidly acquired similar amounts of N from the entire range of added N sources. After 64 days, P. abies and V. myrtillus had acquired similar amounts of N from all N sources, while for D. flexuosa, the uptake from all N sources except ammonium was significantly lower than that from nitrate. Furthermore, soil analyses indicate that glycine was rapidly decarboxylated after injections, while other organic compounds exhibited slower turnover. In all, these results suggest that a wide range of N compounds may be of importance for the N nutrition of these boreal forest plants, and that the type of mycorrhiza may be of great importance for N scavenging, but less important to the N uptake capacity of plants.

Comparison of two methods used to analyse lipid peroxidation from Vaccinium myrtillus (L.) during snow removal, reacclimation and cold acclimation.

Malondialdehyde (MDA) concentration is a widely used method to analyse lipid peroxidation in biological material. In plant tissues, however, certain compounds (anthocyanins, carbohydrates) may interfere with measurements which may lead to an overestimation of the MDA levels. Two methods were compared for analysing lipid peroxidation, either uncorrected or corrected for interfering compounds. The comparison was performed in three separate experiments with respect to cold treatments (snow removal in winter, reacclimation in summer and cold acclimation in autumn) in bilberry (Vaccinium myrtillus L.). During winter and autumn the methods seem to measure different compounds, but during active growth in the summer the difference between the methods was less. This is obviously due to carbohydrates which act as cryoprotectants and increase in concentration during cold acclimation as well as due to the anthocyanins. It is thus suggested that the validity of the uncorrected method to measure MDA and thereby lipid peroxidation is best in plant tissue which is in an active growth state.