Genetic and environmental contributions to the winter hardiness of conifers

193

Genetic and Environmental Contributions to the Winter Hardiness of Conifers

ALAN R. WELLBURN

Institute of Environmental and Biological Sciences, Lancaster University, Lancaster LA1 4YQ

Summary Both genetic and environmental components are involved in the processes of winter hardening and dehardening which permit needles of conifers like Norway spruce to survive very low temperatures (< -30°C) over winter and to recover fully in time for the following and subsequent seasons. One of the major environmental effects of increasing concentrations of atmospheric ozone (03) in recent summers has been to affect detrimentally the ability of conifer needles to harden properly and at the correct rate the following autumn. Part of the mechanism by which this occurs in the cytoplasm of needle cells has been traced to detrimental effects on both the N 2 fatty acid desaturase and the unusual A 5 desaturase which appears to be part of the low temperature survival mechanism of conifers. The genetic component of winter hardening also involves needle lipids. Studies of lipids in the needles of Norway spruce trees of different provenances growing for many years in the UK have shown that they change during winter hardening as though they were still on trees in the original sites throughout Europe from which the seeds of these trees were initially collected.

Introduction In mid-December 1994, similar seminars on the effects on plants of tropospheric 03 and enhanced ultra-violet radiation, arising from stratospheric 03 depletion, were given on behalf of the Botanical Society of Scotland at the Universities of Dundee, St Andrews, Glasgow, Edinburgh and Aberdeen. Further details con- cerning the general effects of atmospheric O3 and enhanced UV-B on plants given at the beginning and end of these presentations have recently appeared in the proceedings of the Royal Society of Edinburgh (Wellbum, Paul & Mehlhorn, 1994). Consequently, this article focuses on the middle section of the talk which addressed winter hardening in conifers and the detrimental effects of episodes of atmospheric 03 in the summer upon this subsequent process.

Processes involved in winter hardening There is a clear physiological distinction between chilling sensitive (i.e. 5-12 °C), chilling resistant but frost sensitive, and frost resistant plants even at the molecular level (Levitt, 1980; Guy, 1990; H~illgren & C)quist, 1990; Katterman, 1990). In the case of woody plants, those from temperate regions usually show frost injury over the range - 6 to - 12 °C but many conifer species, which naturally experience cold winters, may show resistance to frost injury well below - 4 0 °C (Larcher, 1975) if they have been allowed to develop winter hardiness gradually in response to progressive exposures to lower temperatures and shortening photoperiods.

Winter hardening involves changes in membrane lipid composition, and accumulation of phospholipids, anti-freeze proteins, water bound to the surface

Bot. J. Scotl. 47(2), 193-209

194 ALAN R. WELLBURN

of proteins, and several low molecular weight cryoprotectant compounds such as sucrose, raffinose, and stachyose, plus certain amino and organic acids (Senser et al., 1971 ; H~llgren & Oquist, 1990). Freezing injury develops as a result of ice crystal formation both inside and outside the tissues although up to 40% of intra- cellular water is bound to other compounds and cannot be frozen even below - 4 0 °C (Gusta, 1985). However, if ice crystals do form they subsequently cause loss of turgor, active transport and semi-permeability, degradation of phospholipids, dehydration, and protein denaturation and redistribution (Sakai & Larcher, 1987; H~llgren & Oquist, 1990). Consequently, it is the plant's condition after freeze/thaw cycles that determines the extent of freezing injury.

Certain conifers are remarkable in their ability to harden to very low temperatures. For example, Norway spruce (Picea abies (L.) Karst.) may show freezing tolerance down to - 6 5 °C (Tumanov, 1979). However, there is no deep supercooling in Norway spruce needles frozen to - 3 5 °C and subsequent thawing 10 hours later of needles that are not fully winter-hardened causes an efflux of electrolytes such as K + ions which are symptomatic of a loss of semi- permeability and release of lytic enzymes (Pukacki & Pukacka, 1987). This is linked to a decrease in phenol content because phenolic wastes, normally con- fined to the vacuoles in conifers, are released to the cytosol and oxidized.

In Scots pine (Pinus sylvestris L.), the total sulphydryl content doubles as the needles harden between - 7 to - 1 0 °C (Levitt et al., 1961) which partly led Levitt (1980) to conclude that freezing causes sulphydryl groups to form disulphide bridges which are not broken upon thawing. This is supported by increases in both levels of glutathione and glutathione reductase activities in Norway spruce needles in the winter (Esterbauer & Grill, 1978) although only 7% of total glutathione occurs in the cytosol (Rennenberg & Lamoureux, 1990).

Many plants accumulate reserves of carbohydrate during hardening, including raffinose and stachyose in conifers (Senser et al., 1971; Senser, 1990), but Levitt (1980) has questioned the overall importance of sugars in the development of freezing tolerance of conifers because cytosolic sucrose concentrations are much lower than vacuolar concentrations before hardening. This is supported by Cannell et al. (1990) who found no increase in total sugars in needles of Sitka spruce (Picea sitchensis (Bong.) Cart.) or Douglas fir (Pseudotsuga menziesii (Mirb.) Franco) between September and March. Similarly, Alscber et al. (1989) found reduced starch contents in red spruce (Picea rubens Sarg.) needles from June to December but maximal levels of starch in the roots in November and minimal levels in July. Meanwhile, soluble sugar contents both in the needles and roots rose from September to December. However, de Hayes (1992) found a strong relationship between total sugar content in red spruce needles and the critical winter temperature for freezing injury, but one-year-old needles, which contained more sugars, were less damaged than current year needles with less sugar. Indeed, differences in frost hardiness between different trees during hardening often reflect earlier differences in assimilation (Sakai & Larcher, 1987). Furthermore, the amount of sugar available to form cryoprotectants is a function of the difference between photosynthesis and respiration (Bytnerowicz & Grulke, 1992).

Carbon dioxide assimilation is much reduced by freezing temperatures in

CONTRIBUTIONS TO THE WINTER HARDINESS OF CONIFERS 195

Scots pine and other conifers and is due to a reduced flux of CO: to the chloroplasts (Larcher & Bauer, 1981), decreased rates of ribulose-l,5-bis-phosphate carboxylation (Strand & Oquist, 1985) and decreases in rates of electron transport (Senser & Beck, 1979) due to a block in electron flow from QA (the primary electron acceptor of PSII) to plastoquinone (Strand & Oquist, 1988). Moreover, there is substantial photoinhibition in Scots pine and Norway spruce during autumn and winter (Oquist & Ogren, 1985; Bolhar-Nordenkampf & Lechner, 1988; Lundmark, H~illgren & Hed~n, 1988) especially when light levels and temperatures rise for a few hours in mid-winter. Part of the cold acclimation mechanism, which counteracts the problems associated with excessive excita- tion, includes higher [3-carotene levels in winter-hardened Scots pine (Oquist, 1986) as well as elevated levels of ascorbate, glutathione, and their associated enzymic acitivites in Norway spruce (Esterbauer & Grill, 1978; Esterbauer, Grill, & Welt, 1980). This down-regulation of photosynthesis by photoinhibition prevents photodamage to the photosystems when prevailing low temperatures limit the rate of photosynthesis (H~illgren & Oquist, 1990).

Increases in rates of dark respiration occur in shoots of white fir (Abies alba Mill.) after freeze-thaw treatments (Bauer, Larcher & Walker, 1975) and these occur partly because repair processes take place (Larcher & Bauer, 1981). The reallocation of carbon reserves to permit this is very evident in timberline trees which are at greater risk from freezing injury (Tranquillini, 1979; Ktirner & Larcher, 1988). Apparent increases in the proportion of dark respiration also occur because there is an inhibition of CO2 assimilation after freezing and thawing of hardened trees such as Scots pine (Strand & Oquist, 1985). If, however, a killing low temperature has not been achieved beforehand, rates of photosynthesis in Scots pine quickly recover after thawing (Strand & Lundmark 1987).

Changes in lipid composition during winter hardening differ between pines, spruces and other conifers. There is no change in total galactolipid content between summer and winter in Scots pine (Selstam & Oquist, 1985) although the relative incorporation of UDP "C-galactose into monogalactosyl diglyceride (MGDG) compared with that into digalactosyl diglyceride (DGDG) fails from 5.6 in summer to 3.1 in winter (Selstam & Oquist, 1990). By contrast, total amounts of galactolipids and phospholipids increase sharply in winter hardened Norway spruce (Senser & Beck, 1984) and Nothofagus dombeyi BI. (Alberdi et al., 1990). However, in all conifers there is a change in the molar ratio between the MGDG and DGDG of plastids from summer to winter (1.4-2 to 0.6-0.9) with evidence for increased galactosylation during transfer of Scots pines to short days during hardening and decreased galactosylation upon increasing temperatures and lengthening photoperiods during dehardening (Bervaes, Kuiper & Kylin, 1972; Bervaes, Ketchie & Kuiper, 1987). This increase in DGDG during hardening is important for winter hardening in conifers because pure DGDG changes from a crystalline to a gel phase at - 50°C while MGDG changes phase at - 3 0 °C (Shipley, Green & Nichols, 1973).

There are also significant changes in the fatty acid distribution in lipids during winter hardening towards more unsaturated fatty acids, especially lino- lenate (Ag.~2.~q8:3) in the galactolipids (de Yoe & Brown, 1979; Senser, 1982;


Senser & Beck, 1984; Wolfenden, Pearson & Francis, 1991), and in Norway spruce there is an inverse seasonal relationship between short chain (Cl2 and C14) saturated and longer unsaturated C~g fatty acids in certain lipid classes (Puchinger & Stachelberger, 1989). The increase in unsaturation of the galactolipids of Norway spruce chloroplasts is accompanied by an increase in the number of plastids and mitochondria during hardening, but there is no increase in the phospholipid to galactolipid ratio in spruce unlike that in winter-hardened ivy (Senser & Beck, 1984). The lack of apparent change in fatty acid patterns of phospholipids during winter hardening of spruce is surprising because Steponkus (1990) has shown that freeze-induced cell plasmolysis is linked to lipid phase changes (i.e. reduced lamellar-to-hexagonal~ transitions) and fracture-jump lesions in the plasma membranes of winter rye protoplasts which, in turn, are dependent upon changes in lipid composition (Steponkus et al., 1988; Uemura & Steponkas, 1989). Loss of osmotic responsiveness and ultrastructural changes in the plasma membrane, however, are a consequence of severe dehydration, sometimes called winter desiccation, rather than temperature p e r se (Steponkus, 1990). Winter hardening also increases the stability of the plasma membrane to severe dehydration by inducing a lamellar-to-hexagonal, phase transition (Cudd & Steponkus, 1988) which is preceded by dehydration-induced separation or demixing of membrane components. This demixing, in turn, may either be due to crystalline-to-gel (Lo-L~) transitions of phosphatidyl choline (PC; Crowe & Crowe, 1986), which occurs in the endoplasmic reticulum and plasma membrane, or to decreases in hydration repulsion forces around the charged head groups of PC at high osmotic pressures which permit bilayers to come close to each other (Rand & Parsegian, 1986).

In conifers, frost injury is sometimes confused with desiccation damage (Lucas & Penuelas, 1990). Rapid changes in air temperatures and cold drying winds can cause stomatal opening (Tranquillini, 1982) and desiccation may occur despite the fact that rates of winter transpiration are low. Consequently, resistance to winter desiccation is brought about by effective stomatal closure if the cuticle and the epicuticular waxes which fill and surround the stomatal antechamber remain undamaged.

Effects of atmospheric ozone on winter hardiness There is considerable evidence that summer exposure to elevated concentrations of atmospheric O3 may reduce the winter hardiness of conifers. The first report of an interaction between summer O3 and winter hardiness was made by UK researchers working for the former Central Electricity Generating Board research laboratories at Leatherhead (now closed). They noted that some clonal trees of Norway spruce, left over after a summer-long 03 fumigation in Solardomes similar to those described by Lucas, Cottam & Mansfield (1987), showed signs of visible winter injury in the spring of 1986 following exposure to an early out- of-season frost. The damage, however, was restricted to the older needles (i.e. the 1984 flush) and only occurred on those trees that had previously been exposed to 03 in 1985 when three years old (Brown, Roberts & Blank, 1987). Unfortunately, at the same time, these researchers found that their 03 chambers may have been


contaminated with small amounts of N_,O5 because their 03 generator used a stream of air rather than 02 (Brown & Roberts, 1988).

The following year, Barnes & Davison (1988) at Newcastle University exposed the same eight clones of three-year-old Norway spruce from Bavaria to 120 nl 1 ' 03 for 70 days using controlled environment cabinets, starting in November 1986. Excised shoots were frozen to temperatures between - 6 °C and - 1 9 ° C in January 1987. In four clones, severe freezing damage, scored as % visible injury, subsequently developed on the older needles of plants (i.e. 1985 flush) that had been previously exposed to 03 . Initially, this damage was suggested to provide evidence that 03 predisposed plants to freezing injury (Barnes & Davison, 1988). However, although the visible injury observed was similar to that reported by Brown et at. (1987), it was different from frost damage observed in the field. Moreover, this experiment was conducted late in the year when needles would have been expected already to have acquired considerable frost resistance. In this case, it is more likely that this type of injury was due to O3-induced desiccation (see Barnes, Hull & Davison, 1995).

Using two-year-old Sitka spruce seedlings exposed to either charcoal filtered air (<5 nl 1 -~ 03) or different O3 regimes (70-170 nl 1 -~) for 7 h d -~ for 3 months (June-August 1986) in Solardomes, Lucas et at. (1988) at Lancaster University were able to demonstrate reduced winter hardiness on the first of two occasions (November 1986) when shoots were scored for visible damage after an artificial freezing treatment. The majority of their plants were frost hardy below - 2 0 ° C at the second analysis in December 1986, but at this stage variation between individuals obscured any evidence that O3-treated shoots were less hardy than those grown in clean air. Consequently, they concluded that Sitka spruce exposed to 03 in summer would be more sensitive to early autumn frosts.

Fincher et at. (1989) at the Boyce Thomson Institute in New York State exposed young red spuce trees to 03 in open-top chambers and followed subsequent winter injury. If only those trees showing visible injury were considered, there was a significant effect of summer O3 on numbers of injured shoots observed in spring 1988 following winter frosts. However, summer O3 exposures had no effect on winter injury if all the experimental trees (damaged and undamaged) were ranked according to visible injury. This was the same situation when this experiment was continued for a further year with a similar summer 03 exposure regime (Fincher & Alscher, 1992). This contrasts with the observations of Waite et al. (1994) who found no effect of summer 03 on winter hardening in red spruce at the University of Vermont, Burlington. Indeed, some of their seedlings which had received charcoal-filtered air were significantly less cold tolerant than those which had received elevated 03.

In the Gesellschaft fiir Strahlen- and Umweltforschung (GSF) facility near Munich, six different clones of Norway spruce planted in two different soils were exposed to controls of 03 (25 nl 1-') and acid mists (pH 5.6) or treatments with O 3 (50 nl 1-' with episodic excursions up to 170 nl 1 ~) and acid mist (pH 3). Following an artificial freezing regime, representative of an alpine site showing forest decline, the shoots were scored for visible injury (Senser & Payer, 1989; Senser, 1990). Although the severity of the injury was related to both soil type


and the clone of spruce tested, in this case, no significant effects of exposure to high 03 plus acid mist were detected.

Following these initial experiments, the problems associated with visual assessments of winter injury after programmed cooling and thawing were removed when Burr (1987) and Murray, Cape & Fowler (1990) developed quan- titative assays of frost damage based on changes in conductivity as a measure of the rate of electrolyte leakage. These techniques were rapidly adopted, and a number of research groups using this procedure soon confirmed that atmospheric 03 increased sensitivity to winter injury. For example, Chappelka et al. (1990) found visible injury after a late season frost in 1989 on loblolly pine (Pinus taeda L.) seedlings that had previously been exposed to 03 in summer 1988 in open top chambers at Alabama University. Furthermore, damage was much greater on individual trees from an O3-sensitive family (GAKR ! 5-91), and visible symptoms were confirmed using the electrolyte leakage procedure. Edwards, Pier & Kelly (1990), also using loblolly pine seedlings in open top chambers at Oak Ridge National Laboratory, Tennessee, were able to confirm the observations of Chappelka et al. (1990) and showed that summer exposure to 03 resulted in delayed winter hardening. However, this latter group were unable to find evidence of parallel visible symptoms after thawing. This distinction may be important and may partially explain some of the discrepancies in the 03 and winter hardening literature. Certainly, there are clonal differences in winter hardiness following exposure of Norway spruce to concentrations as low as 40 nl 1-' 03 (Skre & Mortensen, 1990).

Meanwhile, Neighbour, Pearson & Mehlhorn (1990) fumigated red spruce seedlings for two seasons in Solardomes at Lancaster University with either charcoal-filtered air (i.e. plus NO, up to 14 nl 1 ') and O3 in the first season (1987) and either charcoal-filtered air, or charcoal and PurafiV M-filtered air (i.e. 40% less NO) and the equivalent 03 regimes (all 70 nl 1 ' 03) with and without PurafiV M filtration the following season (I 988). Following programmed cooling of needles in the winters of 1987-88 and 1988-89, there was a significant effect of summer 03 plus NO exposures on mid-winter electrolyte leakage, but no effect of 03 with reduced NO was found after the second year of exposure. From this they concluded that NO takes part in the mechanism by which episodes of summer 03 affect winter hardening.

There is also evidence that summer 03 may affect individual components of winter hardening. For example, the increases in raffinose (and stachyose) and other soluble sugars that occur in parallel with increasing cold hardiness are delayed in those conifers that have previously been exposed to summer 03 (Alscher et al., 1989; Barnes, Eamus & Brown, 1990).

In the case of fatty acids, however, the full significance of any effect of summer 03 on winter hardening is obscured if only total fatty acids are analysed and the fatty acids are not first hydrolysed from separated lipid classes. Unfortunately, this has been the case with several initial studies of the effects of summer 03 exposure on the fatty acid composition in conifers (e.g. Fangmeier et al., 1990; Kyburz et al., 1991). When the lipids were separated and the fatty acids individu- ally analysed, the molar percentage of octadecatetraenoate (18:4) in MGDG was found to increase and the molar ratios of two forms of linoleate, A~.918:2 and


A9'1218:2 in MGDG decrease during frost hardening in five-year-old Norway spruce trees growing in charcoal-filtered air at Lancaster University, but not in similar trees exposed to episodes of summer 03 for three consecutive summers (Wolfenden & Wellburn, 1991). These authors suggested that the pollutant interferes with the biosynthesis of 18:4 fatty acids by inhibiting AS-desaturation, although significant changes in the degree of unsaturation of MGDG could not then be detected. More recently, however, Wellburn et al. (1994) have found significant decreases in the degree of unsaturation of both C,o and C18 fatty acids, the molar percentage of A5'9'12'1518:4, and the molar ratio of As.918:2 to A9.1218:2 in MGDG at the time of maximum winter hardiness (December) in Norway spruce of similar age which had been exposed to 03 over four consecutive summers in the open top chambers at the Institute of Terrestrial Ecology, Bush Estate, Penicuik, Scotland. Prior to this and using the same set of plants, Cape et al.

(1990) had already shown that 20% shoot death occurs at -23.5 °C by October in those needles exposed to summer O9 compared with a similar mortality at -28 .5 °C for needles from trees growing in charcoal-filtered air. The changes in plastidic MGDG found by Wellbum et al. (1994) could be traced to a significant effect of summer 03 on the A 5- and Al2-desaturases acting upon phosphatidyl choline (PC) in the endoplasmic reticulum. The replacement of the Ar-subset of C,8 fatty acids by an equivalent AS-series was also confirmed by gas chromatog- raphy and mass spectrometry in these studies. Molecular modelling also showed that the AS-forms, which resembled the AMsomers, are very different in shape to the Ar-series which were not found and this may account, in part, for the ability of Norway spruce needles to recover from exposure to extremely low winter temperatures (Wellburn et al., 1994).

Lipid changes in needles of mature Norway spruces of different provenances growing in the UK Large scale differences in winter hardiness between different families and genera of conifers are very evident and there is a good correlation between degree of winter hardiness and the minimum temperatures of their natural ranges (Sakai & Eiga, 1985; Sakai & Larcher, 1987). This adaptation has a genetic basis but the biochemical and physiological changes involved are regulated by an interaction between genotypic and environmental factors (Junttila, 1989). For example, photoperiodic regulation of bud set in Norway spruce is regulated by genes with additive effects (Eriksson et al., 1978) and there is evidence for polygenic inheritance of winter hardiness in Scots pine (Norell et al., 1986). Indeed, winter hardiness can be transferred from hardy species by interspecific hybridization but considerable back-crossing is required to recover desirable commercial attributes (Stushnoff, Junttila & Kaurin, 1985).

Many years ago (mainly from 1968-1971), the UK Forestry Commission (now the Forestry Authority) germinated samples of Norway spruce seeds collected from across Europe (from the Arctic to Bulgaria and France to the Urals) and planted the seedlings out in experimental sites across Britain (see Table 1). Most of these were properly set out trial plots which involved three blocks (normally each of 7 × 7 trees) so that each provenance was initially repre- sented by 147 trees which are now of considerable size (>20 m). Some early


measurements of growth were made in their first years of growth but since then they have been partially thinned (1980), handed over from the research division to the commercial forestry division, and their identification labels are often difficult to find.

Table 1. Experimental plantings by the Forestry Authority across Britain of provenances of Norway spruce, the seeds of which were gathered from all over Europe.

Expt. Year OS Map Altitude Forest No.* Planted Reference (m)

Bin 2 1942 NJ 494407 175 Dean 105 1968 SO 633099 130 Garscrogo 9 1970 NX 778820 70 Halwill 9 1968 SX 418953 200 Kershope 45 1970 NY 471779 165 Minard 6 1969 NR 972953 45 Wark 13 1971 NY 765791 198

* Given by the Forestry Authority.

In the first week of December 1992 and 1993, at a time when maximum winter hardiness had been achieved, we sampled the current year needles of the same provenances of Norway spruce (i.e. tree provenances grown from the same seed batches) in the experimental sites at the Forest of Dean and at Kershope respect- ively (see Table 2). Replicated lipid analyses were carried out on all needle samples from these and other provenances according to the gas chromatographic (GC) procedures described in Wellburn et al. (1994). The set of data from these combined GC analyses is considerable and we are still reanalysing some of the lipid ratios using newer technology (HPLC and mass detection). Never- theless, our GC lipid analyses have been subjected to a series of correlation analyses with latitude, longitude, and altitude from which the provenances were originally obtained and also with the lowest temperature recorded in the neigh- bourhood of collection over the period 1930-65 (Meteorological Office, 1972).

The results are extremely interesting because they show that several of the fatty acids in the various lipids show a significant correlation with some of these provenance components, especially lowest temperature (see composite Fig. 1A-D for the Forest of Dean site showing significant correlations with molar % fatty acids for each of the fatty acids and Fig. 2A-D for the equivalent molar fatty acid ratios). In the case of DGDG from Forest of Dean (Fig. 1B), eight of the fatty acids correlate significantly with the extreme of temperature of original site and, when the equivalent Kershope provenances are treated in the same way, seven of these significances remain independent of current site and environment.

There is a possible explanation for this. Our samples were always collected at the time of maximum winter hardiness in mid-winter and, as described earlier, there is a change in the molar ratio between MGDG and DGDG of plastids from summer to winter (1.4-2 to 0.6-0.9). Accordingly, those provenances which resist the lowest temperatures appear to have more changes in the unsaturated fatty acids of their DGDG in mid-winter.

C O N T R I B U T I O N S T O T H E W I N T E R H A R D I N E S S O F C O N I F E R S 201

Table 2. Sampled provenances growing in the Forest of Dean and at Kershope.

Lowest~ Provenance Elevation Temperature (seed batch) Source Country Latitude Longitude (m) (°C)

63(498)103 Bicaz*

63(498)108 Brasov*

63(498)110 Cimpeni 1

63(498) 111 Cimpeni 2

63(498)104 Comanesti*

63(498)101 Dorna Cindreni 1"

63(498)100 D o m a Cindreni 2 t

63(498)106 Gheorghieni I t

63(498)107 Gheorghieni 2*

63(4362)3 Klaus*

62(4944)5/100 Lausannet

62(4481)101 Livradois Mass i f t

61(4942)4/100 Lbhner t

63(498)102 Marginha*

61(4944)1/101 Pf'~iffikont

61(4972)100 Rhodope 1"

61(4972)101 Rhodope 2*

63(498) 105 Toplita*

63(498)109 Turda*

62(4481)100 Velayt

61(4943)3/100 Zugerberg t

Rumania

Rumanm

Rumania

Rumanm

Rumania

Rumania

Rumania

Rumania

Rumania

Austria

Switzerland

France

Switzerland

Rumania

Switzerland

Bulgaria

Bulgaria

Rumania

Rumania

France

Switzerland

46040 ' 25°55 ' 1150 - 2 3 . 8 0

45039 ' 25036 ' 1050 - 3 0 . 9 5

46020 ' 23000 ' 1375 - 3 0 . 7 0

46020 ' 23000 ' 1375 - 3 0 . 7 0

46017 ' 26°37 ' 780 - 2 3 . 4 9

47021 ' 25°22 ' 975 - 3 0 . 8 7

46021 ' 23000 ' 1375 - 3 0 . 9 4

46040 ' 25040 ' 1280 - 2 3 . 9 0

46040 ' 25°40 ' 1180 - 2 3 . 8 0

47050 ' 14010 ' 800 - 3 3 . 3 6

46032 ' 6035 ' 400 - 18.30

45030 ' 3030 ' 800 - 2 0 . 1 0

46027 ' 7037 ' 500 - 18.92

47045 ' 25045 ' 670 - 3 0 . 6 8

47015 ' 8045 ' 650 - 2 3 . 3 4

42°00 ' 23030 ' 1000 --21.39

42000 ' 23°00 ' 1400 --21.73

42°56 ' 25°25 ' 880 --23.56

46035 ' 23047 ' 1110 --30.48

45000 ' 3030 ' 900 --20.58

47070 ' 8030 ' 650 --23.44

* Sampled both at Kershope and in the Forest of Dean. t Has been planted at both sites but only sampled in the Forest of Dean. $ Lowest recorded temperature near the site of collection over the period 1930-1965 (Meteorological Office, 1972)

corrected for altitude using the equations of Pielke & Mehring (1977).

Perhaps the most interesting result of all is to be found in Fig. 2B which shows that there is a significant correlation (p<0.01) in DGDG between the degree of unsaturation and recorded lowest temperature. There are also some significant correlations with lowest temperatures with the ratio of our unusual A ~ fatty acids to more normal ones (Fig. 2B) which confirms our belief in their importance for survival at low temperatures.

To summarize, there is strong evidence for a genetic component in winter hardening which involves lipids because those in needles of trees of different provenance growing for many years in the UK still change during winter hardening as though they were still on trees in the original sites throughout Europe from which the seeds were collected. This offers some interesting possibilities given the recent advances that have been made in techniques of genetic manipulation including those involving lipids. Indeed, if the genes involved with the A~ desaturase, for example, could be isolated this may lead to some exciting possibilities whereby the natural ranges of some species could be extended into regions where winter temperatures are lower.

Nevertheless, the study of environmental influences on winter hardening cannot be neglected. The health of northern forests is vital for efficient and sustainable timber and pulp production. Any major adverse effect on winter hardening, such as episodes of O3 in summer, will affect this balance and yet there is

202

Fig. 1A MGDG

ALAN R. WELLBURN

Fig. 1B DGDG

Fig. 1C PG

%

Fig. 1D PC

Fig. 1.

[ ] n o slgn~flcance

[ ] p<01

[ ] p < 0.05"

[ ] p<O01 ~

Fig. 1A-D show significant correlations between individual fatty acids (expressed on a molar % basis) in monogalactosyl-diglyceride (MGDG, Fig. IA, top left), digalactosyl-diglyceride (DGDG, Fig. 1B, top right), phosphatidyl-glycerol (PG, Fig. 1C, bottom left) and phosphatidyl- choline (PC, Fig. 1D, bottom right), in the needles of different Norway spruce provenances collected from all over Europe and growing in the Forest of Dean, and original provenance latitude (La), longitude (Lo), altitude (A) and lowest recorded temperature (T) near the site of collection over the period 1930-1965 (Meteorological Office, 1972) corrected for altitude using the equations of Pielke & Mehring (1977). Those fatty acids containing crosses do not occur in sufficient amounts in those particular lipids for correlation analyses to be made.


Fig. 2A MGDG Fig. 2B OGDG

is =u~ W m ~,1

Fatty Acid Ratios Fig. 2C PG Fig. 2D PC

.... I .... ! ° ° ' 1

[ ] no significance

[ ] p<01

[ ] p< 005"

[ ] P < 0.01 ~

Fig. 2. Fig. 2 A - D as legend for Fig. 1A-D but expressed as molar ratios instead o f molar percentages (U = unsaturated, 2 U = one or more unsaturations beyond A 9, S = saturated, t = t r a n s , n = c i s , u = unusual Ag-containing fatty acids, n = normal A 9~ fatty acids, a = u n k n o w n 18:4 fatty acid with

a GC retention t ime as a methyl ester earlier than b which is A 5.9.~2.~518:4).


every evidence that concentrations of atmospheric 0 3 are rising in more north- erly areas as time passes. Understanding of how these processes are affected is important, especially if diagnostic signals can be identified.

Acknowledgements I am grateful to the Department of the Environment and the Natural Environment Research Council for financial support and to the Forestry Authority for allowing access to the Norway spruce provenance trial plots at Kershope and in the Forest of Dean. I also wish to thank Mrs Deborah Robinson for gathering most of the data, for preparing the illustrations, and with my wife (Dr Florence Wellburn), for helping with the sampling in the forests.

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changes in cold hardened leaves of Nothofagus dombeyi. Phytochemistry 29, 2467-2471.

Alscher, R.G., Amundson, R.G., Cumming, J.R., Fellows, S., Fincher, J., Rubin, G., Van Leuken, P. & Weinstein, L.H. (1989). Seasonal changes in the pigments, carbohydrates and growth of red spruce as affected by ozone. New Phytologist 113, 211-223.

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Genetic and environmental contributions to the winter hardiness of conifers