Freezing induces an increase in leaf spectral transmittance of forest understorey and alpine forbs

Vol.:(0123456789) 1 3 Photochemical & Photobiological Sciences (2022) 21:997–1009 https://doi.org/10.1007/s43630-022-00189-0 ORIGINAL PAPERS Freezing induces anincrease inleaf spectral transmittance offorest understorey andalpine forbs TwinkleSolanki1· JoséIgnacioGarcíaPlazaola2· T.MatthewRobson1 · BeatrizFernándezMarín3 Received: 11 October 2021 / Accepted: 10 February 2022 / Published online: 28 February 2022 © The Author(s) 2022 Abstract Evergreen plants growing at high latitudes or high elevations may experience freezing events in their photosynthetic tissues. Freezing events can have physical and physiological effects on the leaves which alter leaf optical properties affecting remote and proximal sensing parameters. We froze leaves of six alpine plant species (Soldanella alpina, Ranunculus kuepferi, Luzula nutans, Gentiana acaulis, Geum montanum, and Centaurea uniflora) and three evergreen forest understorey species (Hepatica nobilis, Fragaria vesca and Oxalis acetosella), and assessed their spectral transmittance and optically measured pigments, as well as photochemical efficiency of photosystem II (PSII) as an indicator of freezing damage. Upon freezing, leaves of all the species transmitted more photosynthetically active radiation (PAR) and some species had increased ultraviolet-A (UV-A) transmittance. These differences were less pronounced in alpine than in understorey species, which may be related to higher chlorophyll degradation, visible as reduced leaf chlorophyll content upon freezing in the latter species. Among these understorey forbs, the thin leaves of O. acetosella displayed the largest reduction in chlorophyll (−79%). This study provides insights into how freezing changes the leaf optical properties of wild plants which could be used to set a baseline for upscaling optical reflectance data from remote sensing. Changes in leaf transmittance may also serve toindicate photosynthetic sufficiency and physiological tolerance of freezing events, but experimental researchis required to establish this functional association. T. Matthew Robson and Beatriz FernándezMarín shared senior authorship. * T. Matthew Robson matthew[email protected] 1 Organismal andEvolutionary Biology (OEB), Viikki Plant Science Centre (ViPS), Faculty ofBiological andEnvironmental Sciences, University ofHelsinki, 00014Helsinki, Finland 2 Department ofPlant Biology andEcology, University oftheBasque Country (UPV/EHU), Barrio Sarriena s/n, 48940Leioa, Spain 3 Department ofBotany, Ecology andPlant Physiology, University ofLa Laguna (ULL), 38200Tenerife, Spain 998 Photochemical & Photobiological Sciences (2022) 21:997–1009 1 3 Graphical abstract Keywords Frozen leaves· Extreme climatic events· Leaf optical properties· Spectral reflectance· Photoprotection· Leaf pigments 1 Introduction Perennial plants in alpine environments and in temperate understory forests are at natural risk of freezing damage. This risk may be highest in early spring, when photosynthetic tissues emerge from under insulating snow cover, before attaining full frost hardiness [1, 2]. Exposure to such freezing events can damage buds and leaves, leading to reduced flowering or fruiting, and ultimately mortality in both alpine plants [3–6] and forest understorey species [4, 7, 8]. Plants have evolved various adaptations against freezing but nevertheless freezing resistance varies greatly across species, ranging from those that are very temperature sensitive to some alpine and boreal species that even survive extreme freezing temperatures [2, 9, 10]. Canopy cover partially buffers understorey plants from the sharp drops in temperature experienced in open environments of boreal and alpine regions [11], but alternately canopy interception and heterogeneity can reduce snow cover leaving ground vegetation exposed to freezing temperatures. Alpine plants are typically better adapted to freezing conditions than forest understorey species; however, even they can also suffer significant loss of biomass, fecundity and ultimately competitive ability following freezing events [12]. Freezing resistance can also vary through the seasons, among populations within a single species, and even among plant organs and developmental stages [2, 13–15]. Freezing definitely triggers physiological, biochemical and physical alterations in the leaves, regardless the capability of tissues to tolerate sub-zero temperatures. Sharp drops in temperature can occur during spring in alpine zones and forest understories at high elevation or latitude [11]. These sudden fluctuations in temperature during periods when full physiological function is still returning to overwintering leaves often causes chlorophyll degradation [16] and stimulates accumulation of antioxidant pigments, such as anthocyanins, which ameliorate stress at low temperatures [17, 18], and other flavonoids which have additional roles including UV-screening, frost protection and herbivory defence [17, 19–21]. Freezing, also generates changes in the pigment composition of leaves: for instance, in frost-tolerant leaves freezing induces the de-epoxidation of xanthophyll cycle carotenoids involved in photoprotection [22], even in darkness [23–25]. In addition to the changes in pigments, freezing temperatures may cause structural changes to the leaf and leaf surface affecting their optical properties. On the one hand, physiological and anatomical responses to avoid leaf damage at cold temperatures, including acclimation of the cell membrane composition and cell structure, may modify leaf optical properties at time-scales of days to seasons [26]. On the other hand, ice accumulation in apoplastic spaces, 999Photochemical & Photobiological Sciences (2022) 21:997–1009 1 3 cellular cytorrhysis, or changes to the cuticle have associated effects on the scattering of radiation in the leaf [26–29]. In fact, ice optical properties greatly differ to those from liquid water, and its birefringence has recently been used to locate ice crystals within leaves and buds through reflected light microscopy [30]. Very few approaches are available to detect exotherms, to visualize ice formation and propagation thermally, or to evaluate ice allocation within plant tissues in order to address the mechanisms and consequences of plant freezing [27, 31–35]. However, by examining the transmittance of radiation in frozen leaves and comparing it with that of fresh leaves we can infer its functional significance, as well as providing information on reflectance that can be scaled-up to interpret remote sensing data [36] with broad applications in crop and ecosystem science. All previous points considered, we aimed to determine the changes induced by freezing in the spectral reflectance and transmittance of leaves from alpine and understorey plants, spanning the ultraviolet (UV; 280–400nm), PAR (400–700nm) and far-red (700–900nm wavelengths) regions of the spectrum. In order to provide insights into physiological processes potentially associated with changes in the spectra, we assessed the photosynthetic yield of photosystem II (Fv/Fm) and optically measured leaf pigments, before and upon freezing. To examine the consistency of these responses between alpine meadow species and boreal forest understorey forbs, we tested several plant species from each environment in spring soon after snowmelt. We expected alpine plant species, from a habitat where fluctuating temperatures and high irradiances are common, to be better acclimated to freezing, including having more photoprotective pigments. Because of this, we also expected alpine species to display a smaller increase in leaf spectral transmittance and smaller drop in photosynthetic yield following freezing compared to the forest understorey species. 2 Materials andmethods 2.1 Collection sites, plant material andexperimental design Leaves of six of the most prominent springtime alpine species [37] were sampled from an alpine meadow in the subnival zone of the western French Alps adjacent to the Station Alpin Joseph Fourier (SAJF) botanical garden at the Col du Lautaret (45.0359°N, 6.4052°E) at 2150m a.s.l, on 28th May 2019. These species were: Soldanella alpina (alpine snowbell), Ranunculus kuepferi (Pyrenean buttercup), Luzula nutans (wood rush), Gentiana acaulis (trumpet gentian), Geum montanum (alpine avens) and Centaurea uniflora (alpine knapweed). Likewise, leaves of three common forb species were sampled from the understorey of a mixed boreal forest [38], dominated by Betula pendulaand Betula pubescens with some Picea abies trees, at Lammi Biological Station LBS, central southern Finland (61.05°N, E 25.04°E) at 130–135m a.s.l on 15th June 2020. These understorey forbs were: Hepatica nobilis (liverwort), Fragaria vesca (wild strawberry) and Oxalis acetosella (wood sorrel). In the alpine meadow, only leaves fromthe previous year that had survived under snow cover through the winter were sampled, while in the understorey new leaves were also sampled. All leaves were collected close to solar noon between 12:00 and 14:00 to control for diurnal variation in leaf traits. A parallel set of leaves were sampled concurrently from which leaf area, fresh weight and dry mass (after drying to a constant weight at 60°C) were recorded. For alpine species, 6 groups of 5 leaf disks were weighed, whereas among understorey species, 20–25 entire leaves were used, to calculate specific leaf area (SLA: leaf or disk area divided by dry mass) and leaf water content (LWC: one minus dry mass over fresh weight) [39] (Table1). Mean daily temperature, precipitation and relative humidity in the alpine meadow during the experiment were 5.0°C, 0mm and 73% (SAJF weather station, source: https:// www. davismeteo. com/ Vanta gePro2. php) and in the understorey they were 20.6°C, 0mm and 80% (LBS, source: https:// en. ilmat ietee nlait os. fi/) (Fig.1). 2.2 Freezing treatments andchlorophyll fluorescence measurements One mature leaf was harvested from three-to-four different plants of each species (and leaf-age class, where applicable) growing naturally in the alpine meadow and the forest understorey., Immediately after sampling leaveswere taken to the lab for optical measurements. During the transfer period from field to lab, of about 10–15min, the fresh leaves were kept in sealed plastic bags in a cool box. All three sets of measurements: optical properties, chlorophyll fluorescence and leaf pigments were made on every leaf. To freeze the leaves, they were placed horizontally flat between two sheets of paper towel, to ensure that they didn’t crinkle on freezing, returned to sealed plastic bags and transferred to the freezer at − 18°C for 24h in darkness, directly following roomtemperature measurements on fresh leaves. This same set of leaves were remeasured when frozen, keeping the leaves insitu in the freezer to ensure that they remained frozen and at the same temperature throughout all the different measurements. Indices of leaf chlorophyll content, and epidermal flavonoid glycosides (flavonols in dicots., flavones in monocots.) and epidermal anthocyanins were measured once from the adaxial side of each leaf with a Dualex Scientific + (ForceA, Paris Sud, France). Maximum quantum yield of PSII 1000 Photochemical & Photobiological Sciences (2022) 21:997–1009 1 3 (Fv/Fm), as indicator of photosynthetic capacity, was measured with a FluorPen (PSI, Drásov, Czechia) following 30min of dark acclimation using leaf clips. We made a set of measurements of leaf spectral transmittance and reflectance of radiation for each of the three leaves per alpine species or four leaves per understorey species and leaf-age class. These measurements were repeated on the same leaves once frozen, except for reflectance of alpine species which was not logistically possible. Following these measurements and 30-min dark-adaptation, Fv/Fm (mini-PAM, Heinz-Walz, Effeltrich, Germany) was recorded as a means of indirectly assessing true freezing within the tissues of the same set of frozen leaves. The frozen leaves of alpine species were too fragile to allow Dualex and reflectance measurements before leaves defrosted. 2.3 Measuring andprocessing ofleaf optical properties Leaf spectral transmittance and reflectance were measured across the spectral region (250–892nm), encompassing all the wavelengths of ambient UV-B and UV-A radiation, and visible and far-red light that plants naturally receive in sunlight. Reflectance of radiation from the adaxial (upper) side and transmittance to the abaxial (lower) side of one leaf from three-to-four replicate plants per species and age class were measured with a dual integrating-sphere system, Jaz Spectro-Clip (Ocean Optics, Dundane, FL, USA). These measurements were all taken from the same part of the fresh and frozen leaves selected for all other optical measurements, avoiding the midrib where possible. The Jaz Spectro-clip comprises several modules including a dual spectrometer, a pulse xenon light source, data processing unit and battery. The two integrating spheres in the system collect light transmitted though the leaf as well as reflected light. The Jaz modular spectrometer was used as a standalone for optical measurements, the trigger rate was set to 10ms with a hold-off time and trigger delay of 1ms, and the spectrometer integration time was set to 1000ms. Each leaf’s transmittance/reflectance spectrum was an average of six consecutive individual recordings of spectra without both boxcar-smoothing and non-linearity detection. Measurements of each leaf took c4min., during which time there was no noticeable trend to suggest any short-term time-dependent variation in optical properties. The flash rate was set at 200Hz (or one flash every 5ms) with an intensity of 400 Volts. Each leaf transmittance and reflectance spectrum measured on a sample (S), was matched with a dark (D) and reference (R) measurement, using black and white Spectralon diffusereflectance reference targets (WS-1-SL, Ocean Optics). Table 1 Leaf morphological traits from the alpine and understorey species used in the experiment Leaves of plants growing in the experimental sites at the time of measurement were used to calculate specific leaf area (SLA), leaf area (LA) and leaf water content (LWC), as the mean (± 1 standard deviation) of 20–25 leaves of understorey plants, and of six groups of five leaf disks from alpine species Leaf production Leaf anatomy Leaf shape Trichomes Specific leaf area (mm2 mg−1) ¥ Leaf area (cm2) ¥Leaf water content (%) Alpine species Centaurea uniflora Perennial Dorsiventral Narrow lanceolate Dense short rough hair 15.647 ± 1.532 2.69 ± 0.75 76.78 ± 1.01 Gentiana acualis Annual/perennial Dorsiventral Elliptical to lanceolate Hairless 11.914 ± 1.405 10.16 ± 3.52 70.05 ± 2.10 Geum montanum Perennial Dorsiventral Pinnate Densely Hairy 11.305 ± 0.805 5.87 ± 0.56 64.54 ± 0.64 Luzula nutans Perennial Isobilateral Narrow lanceolate Hairy 18.752 ± 1.375 1.76 ± 0.17 81.07 ± 1.36 Ranunculus kuepferi Perennial Dorsiventral Narrow lanceolate 13.291 ± 1.372 3.82 ± 0.29 79.32 ± 0.06 Soldanella alpina Perennial Dorsiventral Kidney Hairless 12.407 ± 0.77 3.59 ± 1.01 70.57 ± 1.14 Understorey species Hepatica nobilis Once annually Dorsiventral Lobate 3 leaflets Few hairs 25.64 ± 4.33 9.28 ± 3.23 71.49 ± 0.25 Oxalis acetosella Sequentially growing season Isobilateral Palmate trifoliate Few hairs 81.97 ± 13.16 5.53 ± 1.44 82.86 ± 2.59 Fragaria vesca Dimorphic spring/autumn Dorsiventral Palmate 3 leaflets Hairy 48.09 ± 4.54 14.82 ± 2.16 67.08 ± 2.52 1001Photochemical & Photobiological Sciences (2022) 21:997–1009 1 3 The instrument calculates the spectral transmittance (Tλ) and reflectance(Rλ) according to Eq.1. Post-processing of the raw spectra was done using the Photobiology suite of packages in R [40], to detect out-ofrange values. A “lowess” function was selected to smooth the spectrum, after testing and comparing those smooth functions available, reducing the effect of bad pixels without overfitting the data. 2.4 Statistical analyses Differences in the transmittance, reflectance and absorptance of leaves were compared in two ways, treated either as continuous spectra or as discrete spectral regions calculated from these spectra. The mean of spectra (from 3 to 4 leaves) for (1) T 𝜆∕R𝜆= S−D R−D × 100, a species, or freezing treatment, were considered to differ significantly when their 95% confidence bands were nonoverlapping, plotted in the same figure. Whereas, species or freezing-related differences for discrete spectral regions were tested through ANOVA. Two-way ANOVA was used to test whether the spectral transmittance in blue, green, red and far-red (and likewise Fv/Fm) differed among species, before vs upon freezing. Differences in leaf epidermal pigments (flavonols, anthocyanin and chlorophyll content) due to leaf age, among understorey species, and before vs upon freezing, were tested by a threeway ANOVA, and among alpine species leaf epidermal pigments were test using a single factor ANOVA. Function glht from ‘multicomp’ package was used to estimate within-species pair-wise comparisons and multiple comparisons between fresh and frozen leaves. Fig. 1 Time series of mean air temperature measured at 10min interval at both sampling locations; Lammi Biological Station, Finland (understorey species) and the Col du Lautaret, France (alpine species). Inset plots in blue boxes zoom in on the temperature on the day when plant leaves were sampled from both the locations 1002 Photochemical & Photobiological Sciences (2022) 21:997–1009 1 3 3 Results anddiscussion Freezing caused changes in the optical properties (reflectance and transmittance across the UV, PAR and far-red spectrum), as well as pigments (optically measured flavonols, anthocyanins and chlorophylls) of frozen leaves compared with fresh leaves from species growing in alpine and forest understorey environments. While a few studies have addressed spectral characterization of leaf-injury induced after freezing in crop plants [36, 41] and in trees [42], to the best of our knowledge, this is the first study examining the spectral transmittance and reflectance of frozen leaves in wild species. The results of this series of measurements of leaf optical properties, and associated leaf pigments in the same leaves, could provide insights into variation in the amount of light reaching the mesophyll upon freezing and set a baseline for remote sensing where such data on leaf transmittance and reflectance of frozen leaves in winter are required as inputs for modeling radiative transfer. 3.1 Freezing increased transmittance ofleaves ofbothalpine andunderstorey species The transmittance spectra of fresh leaves from our studied species followed characteristic patterns similar to those previously reported for leaves from a variety of species [43, 44] (Fig.2). A typical leaf appears green to the human eye, due to the strong absorption by photosynthetic pigments Fig. 2 Measured spectral reflectance and transmittance and calculated absorptance (280–892 nm) for fresh leaves prior to freezing (black line) and for frozen leaves (gray line). The blue band indicates the 95% confidence interval around each spectrum, for mature leaves (solid line) and young leaves (dashed line). Where the bands around spectra are non-overlapping, the difference between them is considered statistically significant. a Alpine species: Each spectrum is the mean of three leaves, with each leaf’s spectrum composed of six consecutive spectra from a single position on the leaf. Note that reflectance spectra were not recorded from frozen leaves except for G. montanum. b Understorey species: Each spectrum is the mean spectra from four leaves collected from different locations in the understorey of the stand. Each leaf’s spectrum is composed of six consecutive spectra from a single position on the leaf 1003Photochemical & Photobiological Sciences (2022) 21:997–1009 1 3 of lightat other wavelengths in the visible spectrum (Photosynthetically Active Radiation—PAR, 400–700nm). At longer wavelengths, in the near and short-wave infrared (700–2500nm) green leaves typically have prominently higher reflectance and transmittance, with very little absorptance; properties linked to their water content and structural features [45–49]. In our experiment, leaf spectral transmittance increased in response to freezing but the size of this effect differed across the spectrum (Fig.3). Overall, the increase in the percentage of radiation transmitted was largest in the far-red (increasing by 4.1% in alpine sp and 7.6% in understorey sp) and in the PAR (1.4% alpine sp and 4.5% understorey sp); where specifically the difference in transmittance between fresh and frozen leaves was greater in the green (500–600nm; 1.84% alpine sp and 7.74% understorey sp) than the red (600–700nm; 0.14% alpine sp and 5.45% understorey sp) and blue (400–500nm; 0.40% alpine sp and 1.85% understorey sp) spectral regions (Figs.2 and 4, TableS5). Overall, frozen leaves also transmitted 0.60% (alpine sp) and 0.24% (understorey sp) more UV-A radiation (315–400nm) than fresh leaves of both the understorey and alpine species, but this effect was minimal in the UV-B (280–315nm) because all leaves transmitted negligible radiation (< 0.1%) in this spectral region, decreasing at shorter wavelengths (Fig.2, TableS5). The enhancement of transmittance induced by freezing was generally greater in the leaves of understorey than alpine species (Fig.2a, b). However, in both environments there were some species which deviated from this general pattern of response. Even once frozen, the leaves of most alpine species (five of the six species) were opaque to UV-A radiation and blue light (Figs.2a, and 4), and L. nutans only transmitted less than 0.02% UV-A radiation (Figs.2a and 4). Luzula nutans was the only monocot among the species that we tested, and had high SLA and LWC compared with the other alpine species (Table1), as well as a very different leaf structure; which may explain this difference in transmittance [50, 51]. A typical monocot leaf has a compact palisade mesophyll often referred to as chlorenchyma and lacks spongy mesophyll; this causes light interception, scattering and transmittance to be very different from species with both spongy and palisade mesophyll cells [52]. Among alpine species, only leaves of G. montanum and R. keupferi transmitted significantly more green light (increasing by 2.7% and 4.8%) and far-red light (increasing by 6.5% and 11.3%) when frozen than fresh (Fig.2a, TableS4); indicating that freezing had a greater impact on leaf optical properties in these species than in the other alpine forbs. Among the understorey species, fresh leaves of O. acetosella had the highest spectral transmittance among the species we measured; and likewise, once frozen, its leaves transmitted the most spectral irradiance (Figs.2b, 4 and TableS4). Irrespective of damage, the fact that O. acetosella has very thin leaves [53–56] likely explains why their transmittance was highest, this contrasts with H.nobilis which has thick leaves through which very little radiation is transmitted (Figs.2b and 4, Table1) [54]. As well as leaf structure, specific leaf area and leaf anatomy, difference in transmittance among species may change due to acclimation to the physical environment, such as temperature fluctuations and high incident irradiance, e.g., inducing phototropin-mediated chloroplast stacking, whereby chloroplasts align against the anticlinal cell walls increasing light transmittance through the leaf [55, 57]. The processes leading to apoplastic ice allocation, organelle reorganization and other ultrastructural changes upon freezing are as yet poorly understood in plant leaves. However, recent methodological advances have enabled ice accommodation within leaf cross-sections to be visualized [30], and for ultrastructural evaluation of frozen leaves by transmission electron microscopy [58]. These techniques appear to be promising new tools to complement the interpretation of changes in spectral signatures of frozen leaves in the near future. Notably, the reflectance of O. acetosella leaves in the PAR increased when frozen, with the green peak seemingly shifting towards longer wavelengths (Fig.2b). Leaf absorbance in this spectral region is mainly attributable to chlorophyll, hence this change may be the result of cell lysis affecting the pH, degrading chlorophyll and producing pheophytin [42]. It is also possible that disruption of the membrane and cell structure of the leaf during freezing contributed to the changes recorded in transmittance and reflectance, reducing the scattering of radiation by internal structures, Fig. 3 Maximum quantum yield of photosystem II (PSII) photochemistry (Fv/Fm) of six alpine species for fresh leaves prior to freezing and for frozen leaves. Each point is the mean ± 1 SE of four leaves. Significant differences between fresh and frozen leaves for each species are indicated as follow: *p < 0.05, **p < 0.01, ***p < 0.001. S. alpina among alpine species, and O. acetosella among all understorey species, had significantly lower value of Fv/Fm than the rest (p < 0.001) 1004 Photochemical & Photobiological Sciences (2022) 21:997–1009 1 3 which would also result in reduced back-scattered radiation escaping the leaf. In all three understorey species, freezing reduced leaf reflectance in the far-red region. The amount of radiation reflected in the far-red is thought to be controlled by surface properties, air spaces and the internal structure of the leaf [52]. Additionally, the phase transition from water to ice has been found to displace reflectance spectra to longer wavelengths in the green and far-red regions during freezing of oil-seed rape (Brassica napus) leaves [36]. When exposed to freezing temperatures and high irradiance, leaves can suffer severe photo-inhibitory stress and damage to the photosynthetic apparatus. In freezing-tolerant species, this risk is effectively counteracted with photoprotective mechanisms [59–61]. Our experiment, however, was conducted in darkness to avoid photoinhibition. Fresh leaves from the understorey species showed high Fv/Fm values of 0.82, and similarly, fresh leaves of the alpine species had high Fv/Fm at 0.78 on average. Values of Fv/Fm slightly below 0.8, such as these, are common under optimal conditions for alpine species generally [24, 25, 62]. Acclimation of photosynthetic apparatus from freezing-tolerant species to low temperatures is usually accompanied by a downregulation of predawn Fv/Fm values [23, 24]. Interestingly, in our study, the Fv/Fm depression due to freezing was higher in the understorey than the alpine species. The greater susceptibility of understorey species to freezing damage may have been exacerbated because they were sampled later into the growing season than the alpine species, meaning that they are likely to have completely dehardened prior to ourfreezingtreatment [14, 63]. Amongst the understorey species, Fv/Fm was lowered most by freezing in O. acetosella (Fig.3) which was consistent with the largest increase in transmittance occurring in this species, along with the greatest loss of chlorophyll and lowest flavonoid indices Fig. 4 Spectral reflectance, absorptance and transmittance plotted for different spectral regions: UV-B, UV-A, blue, green, red and far-red for all understorey and alpine plant species, comparing fresh leaves prior to freezing against frozen leaves. Spectral regions were calculated from the spectra plotted in Fig.2A, B. Only mature leaves were used in this plot (Fig.2A, B) 1005Photochemical & Photobiological Sciences (2022) 21:997–1009 1 3 (Figs.2b and 5). Amongst alpine species, S.alpina had both the lowest Fv/Fm in fresh leaves (0.72 ± 0.01), and the biggest decrease in frozen leaves (to 0.37 ± 0.04), compared with Fv/Fm in other alpine species which only declined to about 0.60 (Fig.3). While we did not find a difference in the extent to which Fv/Fm in mature leaves, that had overwintered, was depressed by freezing compared with current year’s leaves, previous studies [64, 65] have reported young leaves of alpine species to be more freezing sensitive, since ice formation can more easily damage their photosynthetic apparatus than in mature leaves. 3.2 Changes uponfreezing inoptically measured leaf pigments Leaf chlorophyll, epidermal flavonol and anthocyanin contents were measured optically on the leaves of understorey species before and upon freezing, whereas these optical traits were only measured in fresh leaves of alpine species. The fresh leaves of alpine species contained higher epidermal flavonoids as well as higher chlorophyll content than the fresh leaves of understorey species (Fig.5a and b). This presumably is a consequence of the accumulationof these pigments in response to the higher solar radiation, in particular UV-B radiation, received in the alpine environment than in the boreal understorey, and its colder springtime temperatures with large diurnal fluctuations [38, 66], which stimulate increased photoprotection and chlorophyll accumulation [60, 66]. It is conceivable that excision of the fresh leaves prior to measurement could affect their physiology and pigment values prior to freezing, but comparison with other attached leaves of these species did not provide evidence for such an effect (unpublished data). For all the understorey species, the index of epidermal flavonols was higher than that of anthocyanins, but these indices differed in response to freezing (Fig.5b and c). In understorey species, the epidermal anthocyanin index significantly increased upon freezing in darkness (p < 0.001) (Fig.5c). However, there was no parallel increase in the optical index of UV-screening epidermal flavonols upon freezing in O.acetosella or F.vesca, it only increased in H.nobilis (Fig.5b). Without a quantitative comparison of the metabolites of fresh and frozen leaves, we are not able to attribute these increases in absorbance to changes in the concentration or location of these classes of flavonoid. Even well-acclimated alpine species such as Ramonda myconi cease enzymatic activity at −15°C [24], so at most these leaves would have been metabolically active only as their temperature dropped prior to freezing, meaning there would have been little opportunity for anthocyanin synthesis. In contrast to flavonols, the drop in optically measured leaf chlorophyll content upon freezing differed significantly among species. Chlorophyll content was Fig. 5 Leaf chlorophyll (a), and epidermal flavonol (b) and anthocyanin (c) contents of fresh leaves of six alpine species (Lautaret) before freezing, and three understorey forbs (Lammi) for fresh leaves prior to freezing (red points) and frozen leaves (blue points). Significant differences between fresh and frozen leaves for each species are indicated as black asterisks, and differences between mature and young leaves as blue and red asterisks: *p < 0.05, **p < 0.01, ***p < 0.001. Mean values with different letters indicate significant differences among understorey species (upper case) and alpine species (lower case). Each point is the mean ± 1 SE of 3 and 4 leaves for alpine and understorey species, respectively. Where Dualex data are absent, leaves were too delicate for this measurement when frozen