Needle Maturation Research Articles

Effects of elevated ozone and warming on terpenoid emissions and concentrations of Norway spruce depend on needle phenology and age.

Norway spruce (Picea abies (L.) Karst) trees are affected by ongoing climate change, including warming and exposure to phytotoxic levels of ozone. Non-volatile terpenoids and volatile terpenoids (biogenic organic volatile compounds, BVOCs) protect spruce against biotic and abiotic stresses. BVOCs also affect the atmosphere’s oxidative capacity. Four-year-old Norway spruce were exposed to elevated ozone (EO) (1.4 × ambient) and warming (1.1 °C + ambient air) alone and in combination on an open-field exposure site in Central Finland. Net photosynthesis, needle terpenoid concentrations and BVOC emissions were measured four times during the experiment’s second growing season: after bud opening in May, during the mid-growing season in June, and after needle maturation in August and September. Warming increased terpene concentrations in May due to advanced phenology and decreased them at the end of the growing season in matured current-year needles. Ozone enhanced these effects of warming on several compounds. Warming decreased concentrations of oxygenated sesquiterpenes in previous-year needles. Decreased emissions of oxygenated monoterpenes by warming and ozone alone in May were less prominent when ozone and warming were combined. A similar interactive treatment response in isoprene, camphene, tricyclene and α-pinene was observed in August when the temperature and ozone concentration was high. The results suggest long-term warming may reduce the terpenoid-based defence capacity of young spruce, but the defence capacity can be increased during the most sensitive growth phase (after bud break), and when high temperatures or ozone concentrations co-occur. Reduced BVOC emissions from young spruce may decrease the atmosphere’s oxidative capacity in the warmer future, but the effect of EO may be marginal because less reactive minor compounds are affected.

Permafrost Regime Affects the Nutritional Status and Productivity of Larches in Central Siberia

Permafrost exerts strong controls on forest development through nutrient availability. The key question of this study was to assess the effect of site conditions on macroelement concentration and stable isotope (δ13C and δ15N) dynamics during the growing season, and nutrient stoichiometry and resorption efficiency in the foliage of two common larch species in Siberia. Foliar nutrient (N, P and K) concentrations of larches grown on permafrost soils were exceptionally high in juvenile needles compared to those from a permafrost-free region (+50% and 130% for P and K), but were two-fold lower at needle maturation. Within permafrost terrain trees, sites with a warmer and deeper soil active layer had 15–60% greater nutrient concentrations and higher δ15N in their needles compared to shallower, colder soils. Larch of permafrost-free sites demonstrated an enrichment of foliage in 15N (+1.4% to +2.4‰) in comparison to permafrost terrain (−2.0% to −6.9‰). At all sites, foliar δ13C decreased from June to August, which very likely results from an increasing contribution of current photoassimilates to build foliar biomass. With senescence, nutrient concentrations in larch needles decreased significantly by 60–90%. This strong ability of larch to retain nutrients through resorption is the essential mechanism that maintains tree growth early in the growing season when soil remains frozen. The high resorptive efficiency found for K and P for larches established on permafrost suggests nutrient limitation of tree growth within the Central Siberian Plateau not only by N, as previously reported, but also by P and K. The increasing nutrient concentrations and a 15N enrichment of foliage towards warmer sites was paralleled by an up to 50-fold increase in biomass production, strongly suggesting that accelerated nutrient cycling with permafrost degradation contributes to an increased productivity of Siberian larch forests.

Conserved Epigenetic Mechanisms Could Play a Key Role in Regulation of Photosynthesis and Development-Related Genes during Needle Development of Pinus radiata.

Needle maturation is a complex process that involves cell growth, differentiation and tissue remodelling towards the acquisition of full physiological competence. Leaf induction mechanisms are well known; however, those underlying the acquisition of physiological competence are still poorly understood, especially in conifers. We studied the specific epigenetic regulation of genes defining organ function (PrRBCS and PrRBCA) and competence and stress response (PrCSDP2 and PrSHMT4) during three stages of needle development and one de-differentiated control. Gene-specific changes in DNA methylation and histone were analysed by bisulfite sequencing and chromatin immunoprecipitation (ChIP). The expression of PrRBCA and PrRBCS increased during needle maturation and was associated with the progressive loss of H3K9me3, H3K27me3 and the increase in AcH4. The maturation-related silencing of PrSHMT4 was correlated with increased H3K9me3 levels, and the repression of PrCSDP2, to the interplay between AcH4, H3K27me3, H3K9me3 and specific DNA methylation. The employ of HAT and HDAC inhibitors led to a further determination of the role of histone acetylation in the regulation of our target genes. The integration of these results with high-throughput analyses in Arabidopsis thaliana and Populus trichocarpa suggests that the specific epigenetic mechanisms that regulate photosynthetic genes are conserved between the analysed species.

Variation in gene expression profile with aging of Pinus radiataD. Don

Variation in gene expression profile with aging of Pinus radiataD. Don

Combined Proteomic and Transcriptomic Analysis Identifies Differentially Expressed Pathways Associated to Pinus radiata Needle Maturation

Needle differentiation is a very complex process that leads to the formation of a mature photosynthetic organ from pluripotent needle primordia. The proteome and transcriptome of immature and fully developed needles of Pinus radiata D. Don were compared to described changes in mRNA and protein species that characterize the needle maturation developmental process. A total of 856 protein spots were analyzed, defining a total of 280 spots as differential between developmental stages, from which 127 were confidently identified. A suppressive subtractive library (2048 clones, 274 non redundant contigs) was built, and 176 genes showed to be differentially expressed. The Joint data analysis of proteomic and transcriptomic results provided a broad overview of differentially expressed pathways associated with needle maturation and stress-related pathways. Proteins and genes related to energy metabolism pathways, photosynthesis, and oxidative phosphorylation were overexpressed in mature needles. Amino acid metabolism, transcription, and translation pathways were overexpressed in immature needles. Interestingly, stress related proteins were characteristic of immature tissues, a fact that may be linked to defense mechanisms and the higher growth rate and morphogenetic competence exhibited by these needles. Thus, this work provides an overview of the molecular changes affecting proteomes and transcriptomes during P. radiata needle maturation, having an integrative vision of the functioning and physiology of this process.

Variations in DNA methylation, acetylated histone H4, and methylated histone H3 during Pinus radiata needle maturation in relation to the loss of in vitro organogenic capability

Needle differentiation is a very complex process associated with the formation of a mature photosynthetic organ. From meristem differentiation to leaf maturation, gene control must play an important role switching required genes on and off to define tissue functions, with the epigenetic code being one of the main regulation mechanisms. In this work, we examined the connections between the variation in the levels of some epigenetic players (DNA methylation, acetylated histone H4 and histone H3 methylation at Lys 4 and Lys 9) at work during needle maturation. Our results indicate that needle maturation, which is associated with a decrease in organogenic capability, is related to an increase in heterochromatin-related epigenetic markers (high DNA methylation and low acetylated histone H4 levels, and the presence of histone H3 methylated at lys 9). Immunohistochemical analyses also showed that the DNA methylation of palisade parenchyma cell layers during the transition from immature to mature scions is associated with the loss of the capacity to induce adventitious organs.

Isotopic studies of leaf water. Part 2: Between-age isotopic variations in pine needles

We studied between-age oxygen and hydrogen isotopic variations of two pine species, red pine ( Pinus resinosa Ait) and white pine ( Pinus strobus L.), with particular attention paid to variations associated with needle maturity. The 2D model by Shu et al. (this volume) was applied to simulate oxygen isotopic ratios of the pine needles; calculated results were compared with data collected from trees in the Hanover, NH area. The calculations indicate that the mechanism most responsible for age-dependent isotopic variations is the variation in transpiration rate along the needle. Because stomata mature progressively from needle tip to needle base, young needles transpire most of their water near the tip. Mature needles, on the other hand, transpire water more evenly along their lengths. We cannot successfully simulate hydrogen isotopic variations, and it remains to be investigated what mechanism or physical process is not included in the model that affects hydrogen isotopic compositions in leaf water.

Assessment and Implications of Intraspecific and Phenological Variability in Monoterpenes of Scots Pine (Pinus sylvestris) Foliage

Scots pine populations contain individuals with widely differing amounts and composition of monoterpenes and exist as one of two chemotypes: with or without delta3-carene. We investigated the significance for ecological studies of two types of variation in monoterpenes: (1) the inherent variability in the concentration of monoterpenes or their relative amounts in needles of seedlings, saplings, and mature trees; and (2) phenological variation in developing needles. The relative composition of needle monoterpenes in 5-year-old saplings changed during the needle development period until the final composition was reached upon needle maturity. Changes in composition depended on chemotype. Needles of the "no-delta3-carene" chemotype had higher absolute concentrations of alpha-pinene, beta-pinene, camphene, and total monoterpenes than "delta3-carene" chemotype. For the "delta3-carene" chemotype, the relative concentration of delta3-carene during the needle growing season and immediately after emergence of seedlings was higher compared to that reached at needle maturity. Repeated removal of single needles (at weekly intervals during growth) from 5-year-old saplings did not influence the composition of monoterpenes. Within a natural Scots pine dominated woodland, 18% of mature Scots pines (N=574) belonged to the "no-delta3-carene" chemotype. Chemotypic variation within populations means that the statistical power with which differences in monoterpene concentrations can be detected is lower when sampling from the whole population compared to sampling within chemotypes. Reduction of this background variation and accounting for chiral variation if present, would significantly aid efficiency, interpretation, and understanding of processes in chemical and ecological research. One method for achieving this is the screening of plants for chemotypes before the establishment of experiments or field sampling regimes. We present a summary of suitable analytical methods for needle tissue that facilitates this prior screening.

Changes in needle quality and larch bud moth performance in response to CO2 enrichment and defoliation of treeline larches

Abstract. 1. It is hypothesised that the larch bud moth cycle is controlled by host‐tree foliage quality. In a Free Air CO2 Enrichment (FACE) experiment at the Swiss alpine treeline (2180 m a.s.l.), the effects of elevated CO2 and previous year defoliation on needle quality of larch and the performance of the larch bud moth were investigated.2. Starch and lignin concentrations increased and water content decreased in elevated CO2‐grown needles compared with ambient CO2 concentration. Defoliation resulted in reduced N, water, starch, and sugar concentrations in needles of the next year generation. No interactions between elevated CO2 and defoliation on needle quality were observed.3. Needle quality changes due to needle maturation over the course of the experiment, however, were much larger than the effects of elevated CO2 and defoliation. For example, N concentration was on average 38% lower and lignin concentration 55% higher at the end of the experiment (early July 2003) than at the beginning (mid June 2003).4. On non‐defoliated trees, larch bud moth larvae grew somewhat more slowly under elevated CO2 compared with ambient CO2 concentration. If, however, trees had been defoliated, this response was reversed, with a faster growth of larch bud moth on high CO2‐exposed trees than on control trees. Pupal weight was not affected by either CO2 treatment or defoliation.5. These results suggest that the larch bud moth has to cope with large changes in food quality due to needle maturation during its development, and that additional CO2‐ and defoliation‐induced alterations in needle chemistry have comparatively minor influences on larch bud moth performance at the treeline.

Activities of enzymes of starch metabolism in developing Norway spruce [Picea abies (L.) Karst.] needles

Development of spruce needles starts with high levels of starch. These are derived from imported sucrose, and, with some fluctuation, largely vanish during sink/source transition (Hampp et al. 1994, Physiol Plant 90: 299 – 306). In order to get more information about starch metabolism during this period, we collected current year needles of approximately 25-year-old Norway spruce trees [Picea abies (L.) Karst.] for up to 100 days starting from bud break. Levels of extractable activities of α-amylase (EC 3.2.1.1), ADP-glucose pyrophosphorylase (AGP, EC 2.7.7.27), D-enzyme (4-α-D-glucotransferase; EC 2.4.1.25), and of starch phosphorylase (STP, EC 2.4.1.1.) exhibited specific development-related responses. Insoluble starch dissolving α-amylase was close to the limit of detection for up to 70 days after bud break. At this stage, which marked the start of sink/source transition, α-amylase showed a rise in activity which could be related to the activity of sucrose phosphate synthase, a key enzyme of sucrose formation (correlation coefficient r = + 0.93). Similarly, the activity of AGP, a key enzyme of starch synthesis, was low during the initial phase of needle development and started to increase from about 60 days onwards. STP and D-enzyme, both involved in starch cycling, differed from each other. While STP activity changed in parallel to that of AGP, it was only the D-enzyme which showed appreciable rates shortly after bud break. We thus assume that in spruce needles D-enzyme is mainly responsible for starch turnover during the early period of development, whereas needle maturation, i. e. the acquisition of the ability to export photoassimilates, is characterized by an increased turnover of transitory starch – both synthesis (AGP) and degradation (α-amylase, STP) – and this is closely connected to the emergence of activity of the key enzyme of sucrose synthesis, sucrose phosphate synthase.

Terpene changes due to maturation and canopy level in Douglas-fir ( Pseudotsuga menziesii) flush needle oil

Monoterpene accumulation during maturation of the current year's growth of Douglas-fir, Rocky Mountain variety, is characterized by a two-phase response. Beginning at or near budbreak, α-pinene and a tricyclene-(1)-camphene-bornyl acetate biogenetic group accumulate 3–10-fold. This is followed by another lesser increase later in the season of the minor components (E-) ocimene, citronellal, and borneol. In contrast, levels of β-pinene, myrcene, and terpinolene, and the sesquiterpenes γ-cadinene, α-bisabolene, and an oxygenated sesquiterpene, did not vary significantly during needle maturation. These increases occur first in the lower canopy and proceed upward toward the mid- and upper-canopy levels shortly thereafter. Implications of these observations with respect to metabolism, host-insect interactions, and silviculture management are discussed.

Carbon allocation in developing spruce needles. Enzymes and intermediates of sucrose metabolism

In lyophilized needles of Norway spruce (Picea abies [L.] Karsten) and starting from bud break, we determined enzyme activities (sucrose phosphate synthase [SPS; EC 2.4,1.14]. sucrose synthase [SS; EC 2.4,1.13]. acid invertase [AI; EC 3.2,1.26]) and intermediates (starch, sucrose, glucose, fructose; fructose 6‐phosphate, fructose 2.6‐bisphosphate [F26BP]) of carbohydrate metabolism together with needle weight, shoot length, chlorophyll and protein. For up to 110 days after bud break, samples were taken twice a week from about 25‐year‐old trees under field conditions. At least three periods can be distinguished during needle maturation. During the first period (up to 45 days after bud break) Al showed the highest extractable activity. This coincided with very high levels of F26BP (up to 11 pmol [mg dry weight]−1) and a transient increase of starch in parallel to a decrease of sucrose. The interval between 45 and 70 days after bud break was characterized by high SS activity (ratio of fructose/glucose >1), much decreased levels of F26BP (down to below 1 pmol [mg dry weight]−1), and a pronounced increase in the dry weight/fresh weight ratio. In parallel, starch declined and soluble carbohydrates increased. Finally, needle maturation was characterized by decreasing SS and continuously increasing SPS activities, so that the ratio of SPS/SS increased more than 6‐fold. AI. however, did not decline with maturation. Changes in pool sizes of metabolites and enzyme activities (AI. SPS) are consistent with current concepts on sink/source transition. SS is obviously important with regard to the synthesis of structural polysaccharides.

Invertase, sucrose synthase and sucrose phosphate synthase in lyophilized spruce needles; microplate reader assays

Data on changes of apparent activities of enzymes involved in sucrose metabolism of developing spruce needles are presented. Extractable activities of sucrose phosphate synthase (SPS, sucrose synthesis), and sucrose synthase (SS) and acid invertase (both sucrolysis) were determined in small volumes using a novel microplate reader system which combined high rates of activity with good reproducibility and high sample throughput. During a developmental period of up to 18 months after bud break characteristic changes in SPS and SS occurred. During the first 4 months of needle development SS declined while SPS increased which is indicative of a transition from net import to net export of photoassimilates (sink/source transition). After needle maturation both enzymes exhibited parallel annual changes with increasing rates towards autumn, which was mirrored by the pool sizes of sucrose (possibly due to the acquisition of frost hardiness). Acid invertase activity was comparable to that of SS but showed only marginal seasonal changes. Approximately 70% of its total activity was found to be soluble.

Ultraviolet‐B and visible light penetration into needles of two species of subalpine conifers during foliar development

ABSTRACTThe depth of penetration of Ultraviolet‐B (UV‐B, 300 and 320 nm) and visible (680 nm) light was measured in foliage of Abies lasiocarpa and Picea engelmannii using a fibre‐optic microprobe. Measurements were made on foliage at four times during development: needles were sampled from within expanding buds (in bud); within 72 h of emergence from the bud scales (emergent); from elongating branches (elongating); and from foliage that emerged the previous summer (mature). Light attenuation in pre‐emergent needles of both species was steep and showed strong wavelength dependence. Short wavelength 300‐nm light was attenuated strongly in the developing epidermal layer, but a significant proportion of this potentially damaging UV‐B radiation penetrated into the mesophyll. For A. lasiocarpa and P. engelmannii, 99% attenuation of 300‐nm light occurred at 51 and 96 μm, respectively, well within the mesophyll. At this stage, however, the bud scales were opaque to light below 400nm. As the epidermal cell walls and cuticle continued to develop and chlorophyll accumulated following emergence from the bud scales, light attenuation, particularly of UV‐B radiation, increased. Although no UV‐B is transmitted through the epidermis‐hypodermis of mature needles, small but measurable quantities of 300‐ and 320‐nm light were measured in the photosynthetic mesophyll of post‐emergent and elongating needles. Thus, shortly after emergence from the bud scales in mid‐June to mid‐July, when incident UV doses are highest, absorption of UV‐B radiation by potentially sensitive chromophores in the mesophyll may disrupt physiological and developmental processes in these species. Soluble UV‐absorbing pigments accumulated during needle maturation for P. engelmannii but not A. lasiocarpa, suggesting that, for A. lasiocarpa at least, the development of effective UV screening properties in the epidermis may not be related to the induction of soluble flavonoids.

Characterization and chemodynamics of plant constituents during maturation, senescence and humus genesis in spruce ecosystems

Spruce needles of different age, litter materials and soil samples from the L-, O-and A-horizons of a mor profile were analysed by temperature-programmed pyrolysis (Py) in combination with field ionization mass spectrometry (FIMS). The integrated Py-FI mass spectra give characteristic fingerprints of the biomaterials investigated. The application of principal component analysis to the mass spectral data results in a clear discrimination and classification of the samples reflecting the chemical modifications and transformations of organic matter by biochemical and biogeochemical processes. The chemical compositions are determined by processes such as enrichment and/or translocation of plant constituents (e.g. carbohydrates, lignin, lipids, suberin, and aliphatic polymers) during maturation and senescence of needles; amendment of new components; decomposition; selective preservation and humification processes in the soil environment. During needle maturation, major chemical changes include the decrease of carbohydrate content, condensation of lignin, and crosslinking of waxes. Senescent needles are characterized by lower contents of carbohydrates and lower yields of monomeric pyrolysis products from lignin. The contribution of different litter materials to the humus layer can be estimated by differences in chemical composition. During litter decomposition and humification on the forest floor, carbohydrate content decreases rapidly. The lignin content remains almost constant but some subunits are continuously oxidized. Wax material accumulates until the mechnical disintegration of the needle occurs. In the O-horizons polymeric aliphatic materials are enriched in humified plant remains. A constant increase of aryl-alkyl esters from suberin in the O-horizons is due to both root input and selective preservation. In general, mainly aliphatic polymers and aryl-alkyl esters accumulate during the genesis of mor profiles under conifers.

A comparison of seasonal patterns of photosynthate production and use in branches of red spruce saplings at two elevations

Seasonal patterns of carbon transport from 14C-labeled foliage were examined in red spruce (Picearubens Sarg.) branches at 1935 and 1720 m elevations in the Great Smoky Mountains National Park to characterize possible physiological mechanisms underlying the observed reductions in growth at high elevations. Current and 1-year-old foliage was labeled on 5- to 7-year-old branches of sapling-sized trees in June, August, and October. Retention of 14C was measured after 24 h, and allocation to other branch segments was characterized after 7 days. Total 14C-labeled carbon assimilation was greater in current than in 1-year-old foliage throughout the growing season. This suggests that photosynthetic capacity is high in developing foliage prior to needle maturity and remains high during the first growing season. In August, 14C retention in labeled foliage was less at the higher than at the lower elevation site, possibly reflecting increased respiratory losses that had been previously reported at the higher site. Retention of 14C in current-year shoots as a percent of 14C remaining after 7 days, a measure of sink strength, was high at both sites in June and remained high throughout the season in current-year shoots at the higher elevation site. The relatively high sink activity coupled with the large apportionment of biomass to current-year shoots (19.9 and 26.7% of total branch biomass at the lower and higher elevation sites, respectively) may make red spruce particularly susceptible to stresses such as winter injury that affect young foliage. In addition, the relatively greater sink activity of current-year shoots at the higher elevation site in August and October suggests that the contribution of current-year foliage to overall tree growth may be less at the higher than at the lower elevation site.

Seasonal and age-related variation in the needle quality of five conifer species.

Age changes of foliage resource quality (water, nitrogen, fibre, phenolics and toughness) were studied in five species of conifer (Pinus sylvestris L.), Picea abies (L.) Karsten, Tsuga heterophylla (Rafinesque) Sargent (all Pinaceae), Chamaecyparis lawsonian (Murray) Parlatore and Thuja plicata D. Don (both Cupressaceae) over a 2-year period in an English forest.Mature foliage of Pinus sylvestris was characterized by higher levels of nitrogen, fibre and toughness, and lower phenolics, and that of Tsuga heterophylla by higher levels of phenolics, and lower fibre and toughness levels, than the mature needles of the other species studied. Immature needles had higher levels of water and nitrogen, and lower levels of fibre and toughness, than older needles. Immature needles of Picea abies and Tsuga heterophylla had a high concentration of phenolics, which decreased with needle maturity. By mid-August, the levels of most of the foliar constituents in current-year needles had stabilized at levels maintained for the next year. Sampling revealed a fall in the concentration of phenolics, fibre and water in mature needles between March and June. Possible reasons for this seasonal trend are discussed. The levels of conifer foliar constituents were compared with levels recorded in broadleaf trees. Conifers had greater concentrations of all measured foliar constituents, but, with the exception of the six fold greater toughness of conifer needles, the differences between broadleaves and conifers were less than those between the immature and mature conifer needles. Previous studies have related the abundance of Lepidoptera on conifers to hostplant taxonomic relationships. However, the foliar constituents measured in this study were poor predictors of taxonomic relationships between the conifers. It is suggested that the abundance of Lepidoptera on conifers is not determined by levels of general foliar constituents and the role of other hostplant factors in shaping lepidopteran utilization of conifers is discussed.

Effect of temperature on needle anatomy and transpiration of Piceamariana after bud initiation

Six-month-old black spruce (Piceamariana (Mill.) B.S.P.) container seedlings were placed in either a cool (18 ± 4: 6 ± 4 °C, light:dark) or a warm (26 ± 3: 18 ± 2 °C, light:dark) hardening regime for 12 weeks after bud initiation to assess the effects of temperature on needle maturation and transpiration. Stomatal transpiration was greater in warm-hardened seedlings than in cool-hardened seedlings. Cuticular transpiration was found to be greater in cool-hardened excised shoots than in warm-hardened shoots. Needle cuticle thickness increased in seedlings exposed to both warm and cool temperatures, however, cuticular growth after bud initiation was greater in the warm-hardening regime.

SEASONAL SECONDARY GROWTH IN NEEDLE LEAVES OF PINUS STROBUS AND PINUS BRUTIA

Mature needles and elongating current year's needles of Pinus strobus growing in Massachusetts and P. brutia growing in Israel were collected monthly or bimonthly for seasonal analysis of leaf cambial activity. Mature needles produced secondary phloem but no xylem, and, regardless of the season, had a cambial zone from 2 to 3 cell layers wide. In the current year's needles maturation was basipetal and the procambium differentiated into primary xylem, primary phloem, and the phloem‐producing vascular cambium before needle maturity. One‐ and 2‐year‐old needles of Pinus strobus produced slightly over 4 cell layers of phloem between April 15 and September 1 of 1983, with a peak production rate of about 2 cell layers per month in May and early June. One‐year‐old needles of P. brutia produced about 6 phloem cell layers in 1983, with phloem being produced throughout the year except in midsummer. This was contrasted by fall and winter dormancy in needles of P. strobus.

Wind Effects on Needles of Timberline Conifers: Seasonal Influence on Mortality

In the Rocky Mountains, USA, year—round wind exposure has been correlated with widespread needle dehydration and death in winter for alpine timberline conifers. Needle death may be due solely to the drying effects of winter wind or may also result from predisposition to winter injury associated with suboptimal summer growth conditions. Our experiments showed that in the lower timerbline ecotone in southwest Wyoming, needle mortality was primarily due to winter wind and cuticle abrasion; death was frequent only in wind—exposed needles of flagged trees and surface needles of krummholz mats. At higher elevations where only krummholz mats and a few flagged trees exist, mortality averaged ≥75% for needles unprotected by snow, regardless of wind exposure. Snow covered needles had low mortality throughout the timberline ecotone. Winter death of naturally wind—exposed needles of Picea engelmannii occurred at ≤—4.5 MPa water potential (≤60% relative water content). Cuticular resistance of wind—exposed needles declined from 100—250 ks/m in autumn to ≤30 ks/m by midwinter. Experimentally reversing the windward—leeward orientation of small, flagged trees in early winter resulted in lower xylem pressure potentials and needle viabilities for newly wind—exposed (originally leeward) compared to newly sheltered (originally windward) shoots. Also, sheltering exposed branches from winter wind on flagged trees in the lower timberline ecotone (3200 m) increased mean overwinter needle survival from near 0 to ≥50% in both Picea engelmannii and Abies lasiocarpa. Scanning electron micrographs of dehydrate wind—exposed needles collected in March at this site showed little cuticular surface wax, probably because of windborne ice crystal abrasion. However, winter dehydration and death in both experimentally and naturally wind—sheltered needles at higher elevation may have been due to inadequate needle maturation during summer, which could act to exclude flagged trees from the upper timberline ecotone.