Chronic exposure to ionizing radiation elicits growth inhibition and a dynamic oxidative stress response in the shoots of scots pine (Pinus sylvestris) seedlings

Anatomical structure and localisation of lignin in needles and shoots of Scots pine (Pinus sylvestris L.) growing in a habitat with varying environmental characteristics We investigated the influence of a habitat with varying environmental characteristics on the anatomical structure and localisation of lignin in needles and shoots of Scots pine (Pinus sylvestris L.). A dune in South-West Estonia was chosen as the study area because it has extreme environmental characteristics: primitive sandy soil, deficiency of water, heavy winds and high light exposure. Analysis showed that the needles of all age and the current-year shoots of pines growing on the foot of the dune had the largest average cross section, mesophyll and parenchyma areas. The degree of the lignification of needles at the foot, on the slope and on the top of the dune differed distinctly. Intensive lignification of the cellular walls of the xylem was observed in needles and shoots of the pines, growing at the foot, but not in the trees growing on the top of the dune. Analysis showed that the anatomical structure and localisation of lignin, both in needles and in shoots, depend on their age, the trees location (foot, slope or top of the dune) and soil mineral composition.

The changing climate will expose boreal forests to rising temperatures, increasing soil nitrogen (N) levels and an increasing risk of herbivory. The single and interaction effects of warming (+2 °C increase), moderate N addition (30 kg ha-1 year-1) and bark herbivory by large pine weevil (Hylobius abietis L.) on growth and emissions of biogenic volatile organic compounds (BVOCs) from shoots of Scots pine (Pinus sylvestris L.) seedlings were studied in growth chambers over 175 days. In addition, warming and N addition effects on shoot net photosynthesis (Pn) were measured. Nitrogen addition increased both shoot and root dry weights, whereas warming, in combination with herbivory, reduced stem height growth. Warming together with N addition increased current-year shoot Pn, whereas N effects on previous-year shoot Pn were variable over time. Warming decreased non-oxygenated monoterpene (MT) emissions in June and increased them in July. Of individual MT compounds, α-pinene, δ-3-carene, γ-terpinene and terpinolene were among the most frequently responsive compounds in warming treatments in the May-July period. Sesquiterpene emissions were observed only from warming treatments in July. Moderate N addition increased oxygenated monoterpenes in May, and MTs in June and September. However, N addition effect on MTs in June was clearer without warming than with warming. Bark herbivory tended to increase MT emissions in combination with warming and N addition 3 weeks after the damage caused by weevils. Of individual compounds in other BVOC blends, herbivory increased the emissions of methyl-benzene, benzene and hexanal in July. Hence, though both warming and N addition have a potential to change BVOC emissions from Scots pines, the N effect may also be partly cancelled by warming. Furthermore, herbivory pressure in combination with climate warming and N addition may, at least periodically, increase BVOC release to the atmosphere from young Scots pine seedlings.

Ethylene evolution, concentrations of 1-aminocyclopropane-1-carboxylic acid (ACC) and ACC conjugates, activities of ACC synthase and ACC oxidase, and cambial growth as measured by tracheid production were monitored from November to July in 1-year-old shoots, and between July and September in current-year shoots, of Scots pine (Pinus sylvestris L.). Needles, buds and four stem parts (cortex, phloem, cambial region and mature xylem) were surveyed. Ethylene evolution was quantified by gas chromatography. Free ACC and bound ACC (after acidic hydrolysis of ACC conjugates) were quantified by combined gas chromatography-mass spectrometry, with [2H]ACC as an internal standard. Activities of ACC synthase and ACC oxidase were measured in crude protein extracts. We detected high activities of ACC synthase in needles and buds, but not in stem fractions except the phloem. In July, during the period of intensive shoot growth, we found ACC only in buds and needles. In contrast, ACC oxidase activity was high in stem tissues, particularly in the cambial region during the period of rapid tracheid production, but no ACC oxidase activity was detected in needles and buds. Nevertheless, needles evolved large amounts of ethylene. Ethylene was produced by all stem fractions, and the peak rate of ethylene evolution in the cambial region coincided with the period of maximal tracheid production. Conjugated ACC was present in every fraction except mature xylem. The concentration of conjugated ACC decreased when rates of tracheid production and ethylene evolution were high, suggesting that conjugated ACC may serve as a source for ACC in the cambial region. The regulation of ethylene biosynthesis in Scots pine shoots is discussed.

New saga in Finland: The rise of Diplodia sapinea in scots pine.

Conidia of Gremmeniella abietina infected and caused disease symptoms in annual shoots of both Scots pine (Pinus sylvestris) and Norway spruce (Picea abies) seedlings. In Norway spruce shoots the infection remained largely latent, with only a few seedlings showing symptoms. Mycelial growth inside the shoots was faster in Scots pine than in Norway spruce and was favoured by low temperature in both hosts. The shoots of Norway spruce seedlings had higher endophyte populations than those of Scots pine, and the populations were decreased by low temperatures. Reductions in the normal epiphytic or endophytic flora by acid mist treatments seemed to favour the development of G. abietina.

Free amino acid and protein levels, and γ‐glutamyltransferase activity in apical buds and shoots of Scots pine during the growing season. The aim of the study was to obtain the basic information about nitrogen mobilization needed in carrying out studies on the optimal nitrate and ammonium ratios in the metabolism of Scots pine (Pinus sylvestris L.). Considerable seasonal changes in the concentrations of free amino acids and other ninhydrin‐positive low molecular‐weight compounds were observed in the buds and shoots of Scots pine. 43 different amino compounds were identified, the concentrations of arginine, glutamine, glutamic acid, γ‐aminobutyric acid, alanine and aspartic acid being highest at the break of dormancy. The amounts of certain amino compounds decreased during the growing season, those of arginine, ethanolamine and various ammonium compounds in particular. The amount of glutamic and aspartic acids, glycine, alanine and γ‐aminobutyric acid, however, remained relatively constant. The protein concentration and the specific activity of γ‐glutamyltransferase increased towards the end of the growing period. Slight differences were found between the fertilized trees and the control trees.

<p>Plants can emit methane (CH<sub>4</sub>) produced by an unknown aerobic, non-enzymatic process, driven by plant stressors like UV-radiation, elevated temperatures and wounding. In ambient spring conditions in Finland, CH<sub>4</sub> emissions from the shoots of Scots Pine (<em>Pinus sylvestris</em>) correlated with solar radiation independently of temperature (Tenhovirta et al., in revision). The spring-time shoot CH4 emissions also had a diurnal pattern with the highest emissions during noon. It remains unknown whether these emissions are driven directly by solar radiation or indirectly via its effect on tree physiological processed such as photosynthesis or stomatal conductance. Characterizing the ecophysiology of the CH<sub>4</sub> fluxes of tree canopies is a crucial step in order to understand the role of forests in the global CH<sub>4</sub> cycle.</p><p>To test whether shoot CH<sub>4</sub> emissions are driven by tree physiological activity (e.g. stomatal conductance), we conducted a measurement campaign in greenhouse conditions during which Scots pine saplings were exposed to drought. During this 3-month-long campaign, CH<sub>4</sub>, carbon dioxide (CO<sub>2</sub>) and water vapour (H<sub>2</sub>O) fluxes from tree shoots were measured with an automated shoot trace gas flux measurements system (<em>ShoTGa-FluMS</em>)(Kohl, Koskinen et al., 2021). This system is capable of replacing the CO<sub>2</sub> assimilated by the shoots, removing transpired water and cooling the chambers to near ambient temperatures. The experimental setup consisted of six 2-3 year old nursery saplings each with a shoot enclosed inside an automated shoot chamber, alternating (a) in closed loop with a Picarro G2301 cavity ring-down spectroscopy (CRDS) greenhouse gas concentration analyser (CH<sub>4</sub> and CO<sub>2</sub> measurements), (b) in a flow-through setup with a Li-cor 850 CO<sub>2</sub>-H<sub>2</sub>O analyser (photosynthesis and transpiration measurements), or (c) flushed with ambient air. The saplings were exposed to a daily 9-hour photoperiod of ~ 600-800 µmol s<sup>-1</sup> m<sup>-2</sup> photosynthetically active radiation (PAR), and irrigated automatically. Drought was induced by stopping the irrigation and continued to the point where net uptake of CO<sub>2</sub> no longer occurred.</p><p>Our experiment produced a unique dataset of continuous measurements of shoot-level CH<sub>4</sub>, CO<sub>2</sub> and H<sub>2</sub>O fluxes over a period of several weeks. Our preliminary results show small but consistent CH<sub>4</sub> emissions from the shoots of Scots Pine during daylight, supporting our earlier findings of the dependency of shoot CH<sub>4</sub> emissions on light. The data furthermore allows to analyse the effects of drought on tree physiological activity and shoot CH<sub>4</sub> fluxes providing much needed process understanding of shoot CH<sub>4</sub> emissions from boreal trees.</p><p><strong>References</strong></p><p>Kohl, Koskinen et al. 2021. An automated system for trace gas flux measurements from plant foliage and other plant compartments. Atmospheric Measurement Techniques 14: 4445–4460.</p>

The application of gibberellin A4/7 (GA4/7) to the stem of previous‐year (1‐year‐old) terminal shoots of Scots pine (Pinus sylvestris) seedlings has been observed to stimulate cambial growth locally, as well as at a distance in the distal current‐year terminal shoot, but the distribution and metabolic fate of the applied GA4/7, as well as the pathway of endogenous GA biosynthesis in this species, has not been investigated. As a first step, we analysed for endogenous GAs and monitored the transport and metabolism of labelled GAs 4, 9 and 20. Endogenous GAs from the elongating current‐year terminal shoot of 2‐year‐old seedlings were purified by column chromatography and high‐performance liquid chromatography and analysed by combined gas chromatography‐mass spectrometry (GC‐MS). GAs 1, 3, 4, 9, 12 and 20 were identified in the stem, and GAs 1, 3 and 4 in the needles, by full‐scan mass spectrometry (GAs 1, 3, 4, 9 and 12) or selected‐ion monitoring (GA20) and Kovats retention index. Tritiated and deuterated GA4, GA9 or GA20 were applied around the circumference at the midpoint of the previous‐year terminal shoot, and metabolites were extracted from the elongating current‐year terminal shoot, the application point, and the 1‐year‐old needles and the cambial region above and below the application point. After purification, detection by liquid scintillation spectrometry and analysis by GC‐MS, it was evident that, for each applied GA, unmetabolised [2H2]GA and [3H]radioactivity were present in every seedling part analysed. Most of the radioactivity was retained at the application point when [3H]GA9 and [3H]GA20 were applied, whereas the largest percentage of radioactivity derived from [3H]GA4 was recovered in the current‐year terminal shoot. It was also found that [2H2]GA9 was converted to [2H2]GA20 and to both [2H2]GA4 and [2H2]GA1, [2H2]GA4 was metabolised to [2H2]GA1, and [2H2]GA20 was converted to [2H2]GA29. The data indicate that for Pinus sylvestris shoots (1) GAs applied laterally to the outside of the vascular system of previous‐year shoots not only are absorbed and translocated extensively throughout the previous‐year and current‐year shoots, but also are readily metabolised, (2) the GA metabolic pathways found are closely related to the endogenous GAs identified, and (3) GA9 metabolism follows two distinctly different routes: in one, GA9 is converted to GA1 through GA4, and in the other it is converted to GA20, which is then metabolised to GA29. The results suggest that the late 13‐hydroxylation pathway is an important route for GA biosynthesis in shoots of Pinus sylvestris, and that the stimulation of cambial growth in Scots pine by exogenous GA4/7 may be due to its conversion to GA1, rather than to it being active per se.

It is essential to have a thorough understanding of the sources and sinks of oxidized nitrogen (NOy) in the atmosphere, since it has a strong influence on the tropospheric chemistry and the eutrophication of ecosystems. One unknown component in the balance of gaseous oxidized nitrogen is vegetation. Plants absorb nitrogenous species from the air via the stomata, but it is not clear whether plants can also emit them at low ambient concentrations. The possible emissions are small and difficult to measure. The aim of this thesis was to analyse an observation made in southern Finland at the SMEAR II station: solar ultraviolet radiation (UV) induced NOy emissions in chambers measuring the gas exchange of Scots pine (Pinus sylvestris L.) shoots. Both measuring and modelling approaches were used in the study. The measurements were performed under noncontrolled field conditions at low ambient NOy concentrations. The chamber blank i.e. artefact NOy emissions from the chamber walls, was dependent on the UV irradiance and increased with time after renewing the Teflon film on chamber surfaces. The contribution of each pine shoot to the total NOy emissions in the chambers was determined by testing whether the emissions decrease when the shoots are removed from their chambers. Emissions did decrease, but only when the chamber interior was exposed to UV radiation. It was concluded that also the pine shoots emit NOy. The possible effects of transpiration on the chamber blank are discussed in the summary part of the thesis, based on previously unpublished data. The possible processes underlying the UV-induced NOy emissions were reviewed. Surface reactions were more likely than metabolic processes. Photolysis of nitrate deposited on the needles may have generated the NOy emissions; the measurements supported this hypothesis. In that case, the emissions apparently would consist mainly of nitrogen dioxide (NO2), nitric oxide (NO) and nitrous acid (HONO). Within studies on NOy exchange of plants, the gases most frequently studied are NO2 and NO (=NOx). In the present work, the implications of the emissions for the NOx exchange of pine were analysed with a model including both NOy emissions and NOy absorption. The model suggested that if the emissions exist, pines can act as an NOx source rather than a sink, even under relatively high ambient concentrations.

Plants possess regulatory mechanisms that enhance nitrogen (N) uptake under conditions of spatial and temporal variation in N availability. Study of regulatory mechanisms has focused almost exclusively on the uptake of inorganic N sources (i.e., ammonium (NH4+), nitrate (NO3-). Several lines of evidence, however, suggest that amino acids may constitute a potential source of N for a number of plant species, including conifers. In the present study, we investigated the uptake of amino acids and inorganic N in Scots pine (Pinus sylvestris L.) seedlings grown at different N concentrations. We compared the uptake rate of the individual N sources using U-[13C2], [15N]-glycine, U-[13C6], [15N4]-arginine, 15NH4, or 15NO3, and tested the short-term effect of N supply on the uptake rate of glycine, arginine and in field-grown Scots pine seedlings. Our data indicate that Scots pine seedlings can absorb substantial amounts of N in the form of intact arginine and glycine molecules. The data also suggest that Scots pine seedlings down-regulate their uptake of NH4+-N and arginine-N, but not of glycine-N in response to increased endogenous N concentrations.

Climate change alters both water andCO2availability for plants, but it is largely unknown how they interact with light to affect tree seedling establishment and early growth. Light availability is often regulated by forest management, thus understanding how these resources co‐limit the regeneration success of tree species and populations with contrasting drought tolerances is essential for adaptive forest management and particularly for assisted migration.We studied biomass partitioning of 3‐year‐old Scots pine (Pinus sylvestris) and European black pine (Pinus nigra) seedlings in response to combined effects of light (22% and 40% shade), soil water availability (moist and dry conditions) andCO2(ambient and elevated), and examined the responses of seedlings from Central Alpine and Mediterranean origin. Seedlings of nine populations with varying drought tolerances were grown in a common garden in the European Central Alps. Shoot height, vertical root length, shoot and root biomass of the plants were assessed at the end of the third growing season.Under 40% shade and dry conditions,P. sylvestrisseedlings severely reduced shoot biomass, resulting in an increased specific shoot height (SSH) compared to seedlings under 22% shade and moist conditions. In contrast,P. nigraseedlings retained a constant shoot biomass under all treatment combinations. Seedlings from drier origin were generally larger, heavier and had longer vertical roots than those from wetter locations. In order to keep up shoot height, seedlings from wetter origins disproportionately increasedSSHunder shaded conditions compared to populations from drier origin.Synthesis and applications. Under high light availability, Scots pine (Pinus sylvestris) and European black pine (Pinus nigra) seedlings were well adapted to dry conditions. Moderate shading, however, substantially reduced Scots pine but not black pine growth, and potentially amplified the vulnerability of Scots pine seedlings to drought. Optimising light conditions in forests, for example by thinning, may thus enhance early Scots pine regeneration in a drier future climate.

Food selection by large herbivores occurs at a hierarchy of scales, for example landscape, patch or plant. Several hypotheses regarding food plant selection on patch or plant level have been developed. In this cafeteria-type design field experiment, conducted during one winter immediately after planting, we tested the effect of species mixture on browsing by large herbivores (mainly roe deer) on Scots pine seedlings in mixture with seedlings of ash (highly preferred) or silver birch (less preferred). Browsing on Scots pine was not affected by species mixture, neither in terms of the number of browsed pines nor browsing intensity. Instead, browsed biomass was positively and significantly correlated to the total biomass available for browsing. Also, there were differences due to species, with ash being most browsed (44.6%), followed by Scots pine (18.9%) and silver birch (11.6%). Browsed biomass per browsed seedling, however, was largest for Scots pine. In addition, browsed seedlings were initially taller compared to unbrowsed seedlings for all species. The main management implication in this study is that the species mixture did not influence large herbivore browsing on Scots pine seedlings. Hence, removing or discouraging more (or less) attractive browse species in early stages of pine regeneration activities seems unnecessary from the point of large herbivore browsing.

Plants are recognized sources of methane (CH4) but plant-mediated CH4 emissions have mostly been studied on herbaceous species, although also trees are known to emit CH4. Emissions from tree canopies likely mostly derive from an aerobic, abiotic process. Aerobic CH4 production from trees has not been thoroughly studied, leaving uncertainties to the global source strength estimates, which vary from 0 to 240 Tg yr-1. Even less is known about how aerobic emissions affect the CH4 cycles in boreal forest ecosystems, as the environmental and physiological drivers are not fully understood. In this study, shoot-level CH4 fluxes of boreal conifer trees were measured outdoors and in the greenhouse, to investigate the environmental and physiological drivers and regulators of shoot-level CH4 fluxes. Most of the measurements were done from saplings of Pinus sylvestris L. (Scots pine), one of the most important tree species of the boreal region of the Eurasian continent, by using chamber enclosure methods with spectral, online greenhouse gas analysers. The measurements were conducted either manually, or with an automated measurement system, developed to overcome issues related to manual measurement techniques. The shoots of Scots pine showed small but significant emissions of CH4 in all experimental setups, the emissions were driven by light, enhanced by elevated temperature, and occurred independently from drought and photosynthesis. Solar radiation was a more significant driver of these CH4 emissions than artificial light with UV-A. These results show that Scots pine canopies have the potential to produce CH4 in a similar process that has been described before for the foliage of herbaceous plants, but these emissions are smaller than the initial estimates of the aerobic CH4 source from vegetation. The boreal forest canopies are sources of CH4 and have the capacity to decrease the CH4 sink strength of boreal upland forests by ~ 5 %.

Economic pressures have driven an ever-widening period during which foresters use machines to plant Scots pine (Pinus sylvestris L.) seedlings. In Fennoscandia, this period has recently stretched to the entire growing season. To evaluate the performance of seedlings planted during this extended period, three experiments were carried out in Central and Northern Finland over 2 years. One-year-old and current-year seedlings were planted in mounds or disc-trenched furrows when soil temperatures were >0°C. When 1-year-old seedlings grown for spring planting and overwintered outdoors were planted after mid-June, more needles browned and growth was reduced, possibly because seedlings were oversized with respect to planting density and the volume of growth media. When current-year seedlings sown in spring were planted from July to November, those planted in late September and October grew less in later years than those planted earlier, but survival was unaffected. No large differences in field performance were found with respect to whether seedlings were planted in mounds or disc-trenched furrows. In conclusion, Scots pine seedlings can be machine planted in mounds or furrows during May and early June (later in the North) and then continued from early August until late September, provided climatic conditions in late spring and early autumn are typical and similar to those experienced in Central Finland.

Phenolic metabolites in forest trees play a key role in the defence against biotic and abiotic stressors, yet we lack information about the effects of combined abiotic factors on phenolic compounds in conifers. We studied the effects of combined abiotic factors (ozone × temperature, ozone × nitrogen, temperature × nitrogen, and ozone × temperature × nitrogen) on phenolic concentrations in stems of Scots pine (Pinus sylvestris L.) seedlings in a 3-year-long field experiment in central Finland. In current-year stems, elevated ozone increased the concentrations of total phenolics, soluble proanthocyanidins, and total proanthocyanidins, while warming reduced the concentrations of piceatannol glucoside, pinosylvin, isorhamnetin + kaempferol-3-rhamnoside, and monocoumaroyl isoquercitrin 1. Complex interaction effects on current-year stems showed that nitrogen addition increased the concentrations of some flavonoids in ambient ozone and temperature levels. In the stems from the previous years, ozone decreased the concentrations of total phenolics, total proanthocyanidins, and several flavonoids in ambient temperature, while warming increased their concentrations in combination with elevated ozone. Our results suggest that phenolic defence responses in Scots pine seedlings are affected by all three factors, but that the level of phenolics in the stems from previous years may increase under the combined exposure to elevated temperature and ozone — an expected climate trend in the Northern Hemisphere.