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article Original Distribution and variations of potassium and calcium in different cross sections of Picea abies (L) Karst needles and Fagus sylvatica (L) leaves exposed to ozone and mild water stress D Le Thiec M Dixon JP Garrec centre de recherches forestières de Nancy, unité écophysiologie forestière, INRA, 54280 Champenoux, France équipe pollution atmosphérique, (Received 8 September 1994; accepted 28 November 1994) Summary — Two clones of 8-year-old Norway spruce trees, and beech trees, planted directly into the soil inopen-top chambers, were exposed to elevated ozone concentrations and subjected to a mild soil drought stress. The nutrient partitioning and intertreatment differences in nutrient levels were studied. In elevated ozone, both clones had increased potassium and calcium levels, whereas in beech, ozone- treated trees had decreased potassium levels. Drought caused decreases in these nutrients for all species. The effect of combining the 2 stresses was more complex, however, and the previously observed effects were not obtained in all cell tissues. Furthermore, they showed both interspecific and interclonal differences. The hypothesis that ozone affects the root nutrition and cell membrane per- meability is discussed. / water stress / potassium / calcium / cell tissues ozone Résumé — Répartition et variations du potassium et du calcium dans différentes coupes trans- versales d’aiguilles de Picea abies (L) Karst et de feuilles de Fagus sylvatica (L) soumis à de l’ozone et à une sécheresse modérée. Deux clones d’épicéas âgés de 8 ans et des hêtres ont été plantés directement dans le sol dans des chambres à ciel ouvert ; ils ont été soumis à des concentrations élevées d’ozone et à un stress hydrique modéré. Les variations et la répartition des éléments minéraux ont été étudiées. Les teneurs en potassium et calcium chez les 2 clones augmentent dans les traite- ments de fumigation ; en revanche chez le hêtre les teneurs en potassium diminuent. La sécheresse appliquée fait diminuer ces teneurs pour tous les arbres. L’application combinée de ces 2 stress est plus complexe et les observations faites auparavant ne se retrouvent pas dans tous les compartiments foliaires et sont différentes entre clones et espèces. L’hypothèse que l’ozone affecte la nutrition miné- rale racinaire et la perméabilité membranaire des cellules est discutée. ozone / sécheresse / potassium / calcium / compartiment cellulaire
INTRODUCTION To better study the interactive effects of ozone and drought stress, an experi- ment was conducted on 2 tree species Atmospheric pollution (especially ozone) (Picea abies and Fagus sylvatica) planted has often been cited as a possible contrib- directly into the soil, enclosed in open-top utory factor in forest decline. Furthermore, chambers and exposed to a soil drying. there is evidence to suggest that current Studies on nutrient distribution at the cel- levels of tropospheric ozone can cause lular level are rare and have never been damage to trees (Dobson et al, 1990) and performed on trees exposed to both real- ozone levels in Europe and North America istic ozone concentrations and drought, are predicted to rise as emissions of their although some work has been done on precursors increase in association with declining spruce trees (Stelzer et al, 1990; increased motor traffic. Much work has thus Fink, 1991 a,b; Garrec et al, 1991; Godde et been carried out to assess the biochemi- al, 1991).The aim of our study was thus cal and physiological response of trees to to determine foliar nutrient changes in this pollutant, but the results have often response to ozone and drought stress and been contradictory. This may be due to to then identify at what cell levels these interspecific differences, but is also the changes occurred. The physiological con- result of differences in the experimental sequences of these changes could then conditions used (Darrall, 1989). Ozone con- be assessed. centrations were often unrealistically high and there has been little work done to study the interaction of ozone with environmental MATERIALS AND METHODS constraints, especially away from the labo- ratory. Prinz et al (1987) showed that forest Experimental site and plant material decline was less severe in wetter years, so studies on the effect of both drought and experiment was carried out at Col du Donon The ozone on trees would seem to be a prior- (Vosges, France: 48° 29’ N; 7° 05’ E) in a mixed ity, but they are still rare. spruce beech forest. This region has relatively A demonstration of the complexity of high levels of tropospheric ozone (38 ppb yearly average). It is 727 m above sea level. interactions between ozone and drought was made by Davidson et al (1992), working Five-year-old Norway spruce (Picea abies (L) Karst) and beech (Fagus sylvatica L) trees with Fagus sylvatica. In addition, ozone has were planted in the soil on 19 April 1990. The been shown to affect both growth and mor- Norway spruce were 2 different clones: clone phology of tree root systems (Taylor et al, 780371 from a Polish provenance, Istebna (IST) 1989). Changes to the root system or in and clone 781351 from a French provenance, water availability can have serious implica- Gerardmer (GER). The beech trees were not tions for nutrient uptake by plants, and clonal. The trees were supplied by AFOCEL (Association Forêt Cellulose, Charrey-sur-Saone, changes in the total foliar nutrient concen- France). The soil was classified as "typic dis- tration of trees in response to ozone fumi- trochrept". gation have been observed. Care should Shortly after the trees were planted, 8 open-top be taken in interpreting these results, how- chambers (see Impens, 1992 for details) were ever, as potted plants were used and erected enclosing 9 trees in each chamber - 3 McConnaughay et al (1993) have shown beech and 3 individuals from each of the 2 Nor- that nutrient changes in such trees can be way spruce clones. Four plots of 9 trees without artifacts of the experimental conditions. open-top chambers served as controls.
During the sampling period, the following val- Treatments of microclimatic parameters obtained: were ues Air temperature: 17 ± 4°C An electricdischarge generator (Trailigaz "LABO Leaf-air vapour pressure difference 1.3 ± (VPD): 76") supplied with pure oxygen produced the 0.1 kPa ozone. Twenty-five ppb ozone was continuously added to 2 of the chambers and 50 ppb to humidity of the chamber air: 55 ± 12% Relative another 2 from 17 July 1992 to 29 October 1992. Photon flux density of photosynthetically active Two chambers were ventilated with nonfiltered radiation: 897 ± 254 μmol m s -2 -1 . air, while the remaining 2 were fitted with acti- The differences between chambers with roofs vated charcoal filters which considerably reduced and unroofed chambers were only statistically the ozone levels (by 75% ± 12%). From 30 Octo- significant for light and temperature. Even so, the ber 1992 to 30 March 1993, the added concen- temperature difference was less than 1°C for the trations were reduced by 25 ppb. From 31 March 1993, the experimental treatments were 24-hour average. repeated and continued until 31 October 1993. The ozone concentration in any 1 chamber was recorded for 2 5-minute periods every hour, Pre-dawn leaf water potential using an "Environment SA" analyser. Photo- synthetically active radiation, air temperature and air and soil humidity were also recorded The pre-dawn leaf water potential (Ψ was mea- ) wp (unpublished data). sured using a Skye pressure chamber according to the method in Scholander et al (1965). Mea- In the spring of 1993, work was begun to pre- surements were made throughout the drought pare the drought stress. Wooden structures of stress period. between 4.3 and 4.5 m high were built to sup- port sloping roofs of clear plastic (89% light trans- mission) that prevented rain from entering. The 4 chambers with roofs then had 70 cm deep X-ray microanalysis trenches dug around them to prevent the incursion of soil water into the chambers. The drought stress was begun 1 July 1993 and then the fol- The material to be analysed was prepared lowing treatments were performed: i) filtered air: according to the method used in Le Thiec et al FA; ii) nonfiltered air: NFA; iii) ambient air, out- (1994). Sections of plant material were exam- side chambers: AA; iv) nonfiltered air with 25 ppb ined under a scanning electron microscope ozone added: NFA + 25; v) nonfiltered air with (Stereoscan 90B, Cambridge) at 15 kV, equipped 50 ppb ozone added: NFA + 50. with a dispersive energy microanalysis system (EDX, diode Si-Li; Analyser AN10000 10/25). To Each of these treatments, excepting AA, had ensure that the beam of primary electrons did a drought stressed chamber (DS) and an not penetrate other cells, 1 layer of cells on a unstressed (well-watered) chamber (NS). sheet of aluminium were analysed. All measure- ments were made using an X-ray take-off angle of 45°, a measuring time of 100 s, a magnification Sampling of 6 000 for all examined tissues (stomata, epi- dermis, mesophyll, parenchyma and endoder- mis) and of 400 for the vascular bundle. A ZAF4 The material for microanalysis was sampled on 8 program, FLS, connected to the microanalyser July and 21 August 1993 for beech leaves and gave apparent concentrations of the different ele- 8 July, 21 August and 21 September 1993 for ments analysed. This program takes into account Norway spruce needles, always between 08.00 any variations in volume. In order to convert the and 10.00 GMT. Each sample was composed of microanalysis data into real concentrations (% 2 beech leaves and 5 current-year spruce nee- dry mass), powdered spruce needles and beech dles. The beech leaves were taken from 1 st flush leaves were used as standards (CRM 100 and growth and the needles came from the 1st 3 CRM 101 given by Community Bureau of Refer- whorls. The plant material was immediately ence of the Commission of the European Com- plunged into liquid nitrogen to prevent movement of nutrients after sampling had occurred. munities).
Chemical analysis Sampling was carried out on 20 August 1993 for beech leaves and 21 September 1993 for cur- rent-year spruce needles. The samples were cleaned (by rapid submersion in demineralised, distilled water) and then dried. Phosphorus, sul- phur and cations (Mg, K, Ca, Mn) levels were mineralised (H and HClO Clément, 1977) O 2 ; 4 and measured by ICP (Jobin Yvon JY438 Plus). The levels of soil nutrients were also determined using these techniques (drying at 450°C and min- eralisation with a HF/HClO mixture and then 4 placed in 2% HCl). Statistical analyses The statistical treatment employed was the anal- ysis of variance (a 0.05) by the GLM proce- = dure (SAS Institute Inc, 1985). Test of equality of averages using Student-Newman and Keuls was applied equally (the same letters indicate that averages are not significantly different, and the alphabetic order corresponds to decreasing values). Significance was as follows: ns not = * ** significant; = significant (P < 0.05); highly = significant (P< 0.01); *** extremely significant = (P< 0.0001); α= 0.05. RESULTS relatively constant (-0.82 ± -0.11 MPa; n = 104). There were no significant differences between species, or between ozone treat- Pollution climate and environmental ments. Because the trees were planted in factors the soil, there must have been incursion of soil water from outside the chambers - 1 shows rainfall totals during the veg- Figure despite the trenches that were dug to reduce etation period and the ozone levels (monthly this - or the rooting system of trees was averages) since the trees were planted. deep enough to obtain water. The relatively Ozone showed the often reported fluctua- high rainfall during this period probably kept tions during the year with the maximum lev- the water table at high levels. The Ψ of wp els occurring in spring and summer. The the nonstressed trees remained between Col du Donon is a site where summer ozone - 0.05 and -0.2 MPa. levels often exceed the maximum exposure recommended by the World Health Organ- isation and the EEC. Chamber effects The pre-dawn leaf water potentials showed an initial decrease at the beginning The control trees outside chambers and of the drought period and then remained the trees in the NFA NS chambers were
ing ozone. The potassium levels in Gerard- significantly different for any measured not mer are again increased by exposure to parameter. There 3% difference in was a ozone, but in beech there is observable humidity between the chamber and outside no effect Ca K levels. controls and the increased temperature was on or never more than 2°C. Furthermore, before the drought stress was imposed, there was Microanalysis in effect a replication of 4 treatments (FA, NFA, NFA + 25, NFA + 50); we ensured that there were no a priori differences The microanalysis results for K and Ca are between the same chambers of any 1 treat- shown in table II for the 2 spruce clones and ment. in table III for beech. After analysing the dif- ferent cell tissues, it was found that calcium was most abundant in the epidermis. Potas- Chemical analyses sium was most abundant in the epidermis of beech and the endodermis of Norway The soilanalysis gave the following results: spruce. horizon 0-10 cm: P (&permil;): 0.195 ± 0.05; 5 O 2 - Ca (cmol kg 1.16± 0.39; Mg (cmol + -1 ): + Ozone effects kg0.54 ± 0.21; K (cmol kg0.34 ± + -1 ): ): -1 0.10; Mn (&permil;): 14.25 ± 5.93; The Gerardmer clone showed increasing concentrations of potassium in all tissues, as horizon 20-30 cm: P (&permil;): 0.055 ± 5 O 2 - ozone increased. Such an increase was 0.02; Ca (cmol kg 0.23 ± 0.11; Mg + -1 ): also observed in the epidermis and guard + -1 (cmol kg 0.13 ± 0.03; K (cmol+ kg ): ): -1 cells and to a lesser extent the mesophyll 0.34 ± 0.10; Mn (&permil;): 8.01 ± 4.81. and endodermis of Istebna. However, the The foliar analyses results are shown in vascular bundle of ozone-treated Istebna table I. Ozone had a significant effect on needles had a decreased K level. In ozone- calcium and potassium levels in both treated beech, potassium was decreased species. Drought stress significantly affected in all tissues except the lower epidermis. phosphorus, manganese, calcium and Calcium increases in all tissues, except potassium in both spruce clones and cal- the epidermis, in both clones raised in ele- cium and potassium in beech only. The vated ozone. Beech shows a similar interaction of drought and ozone had a sig- increase apart from in the vascular bundle nificant effect on calcium levels in the where no trend was observed. Istebna clone and potassium levels in Ger- ardmer and beech. Drought effects As the dose increased, there ozone was tendency for a corresponding increase in a The direct effects of drought can be seen calcium. This trend was also observed for from the FA treatments. In spruce, potas- potassium in both spruce clones, but the sium showed decreases in the vascular bun- opposite effect occurred in beech. In filtered dle, endodermis and guard cells, but was air treatments, the drought provoked a con- increased in the mesophyll. Beech had siderable decrease in both calcium and reduced levels in the vascular potassium. The effect of ozone changes potassium bundle, parenchyma, guard cells and lower upon imposition of the drought stress. In epidermis. Calcium levels showed large Istebna there is no longer an increase in decreases in the epidermis of both clones. Ca, but a decrease in response to increas-
DISCUSSION In the mesophyll, however, Istebna had increased levels, whereas Gerardmer’s cal- cium levels were reduced. Calcium The soil analysis showed that there was a decreased in all tissues of beech in good nutrient supply at the site, with no defi- response to drought. ciencies according to the recommended lev- els of Bonneau (1988). Although not defi- cient, magnesium was rather low in the Ozone-drought interactions 20-30 cm horizon. The combined effects of the 2 stresses are Changes in guard cell K concentrations quite complicated. Ozone alone provoked important in regulating stomatal aper- are an increase in potassium in the epidermis of ture (Le Thiec et al, 1994), which in turn can the Gerardmer clone, but in conjunction with limit ozone uptake. This experiment has drought there is no longer such an increase shown increased K concentrations in spruce in NFA + 50. This is also seen in the endo- needles exposed to the ozone treatments, dermis where the ozone-associated compared to FA needles. Associated with increase is restricted to the NFA and NFA + this K increase is an increased stomatal 25 treatments. Potassium levels in Istebna opening. In beech, the reverse pattern was decrease in the mesophyll of NFA + 50, but observed, so a stomatal closing in associa- increase in the endodermis of NFA + 25 and tion with ozone would be expected in beech. in the vascular bundle of all 3 ozone-drought These expectations were confirmed from treatments. stomatal conductance measurements per- formed on the trees on measurement days. Calcium, which had previously shown a Freer-Smith (1993) reports that other work decrease in the epidermis in response to on beech showed a similar reduction in ozone, was increased when the 2 stresses stomatal conductance, as did Taylor and were applied in conjunction in Gerardmer. Dobson (1989) on beech exposed to ambi- Istebna, on the other hand, had a similar ent ozone levels. trend between ozone treatments, but the decrease in FA was less severe. The other The drought stress provoked significant tissues also showed less pronounced decreases in guard cell K levels of Norway changes when ozone was applied in con- spruce in all treatments (this decrease was junction with a drought stress, but the direc- associated with a reduced stomatal con- tion of the effects was nevertheless the ductance). In beech, however, there was same, that is, increasing ozone concentra- no intertreatment difference in K levels and tion was associated with increasing calcium. the levels even showed a relative increase compared to the well-watered treatments. In beech, the previous ozone-associated These results can be explained by refer- potassium decrease in the lower epidermis ence to the diurnal time course of conduc- was no longer apparent in the drought tance measurements. It appears that stressed trees. The guard cells had drought stressed ozone-treated plants close increased K for all 3 ozone treatments, but their stomata earlier in the day and thus effects were no longer apparent in ozone receive a lower dose over a period of time as the parenchyma and even reversed in the ozone levels are greater in the afternoon. vascular bundle. The effects of ozone on In this way the trees are relatively protected calcium were no longer apparent in the epi- from ozone damage and photosynthesis dermis and were diminished in the other tis- more than the well-watered trees in the sues apart from the vascular bundle where morning, when vapour pressure deficits are there is now a decrease.
lower. Thus, because sampling occurred in This potassium and calcium increase the morning, a relatively greater stomatal could also result from a perturbation of the conductance is found in drought stressed mineral nutrition at root level. Barnes and beech trees. Pfirrmann (1992) and Lucas et al (1993) explained the increase of K and Ca levels in The foliar nutrient content is often used ozone-treated trees by an increasing ion to determine the nutritional status of plants uptake at root level. Furthermore, the and/or to assess if latent damage has increase in K, which is a mobile cation and already occurred (Cape et al, 1990). In gen- which is used as a regulator of numerous eral, the absolute values were not same physiological processes, could be the result found when these analyses were compared of an increased demand from the tree which to the microanalysis results, because the is using it to counteract oxidative stress. An global chemical analysis encompasses sev- increasing metabolism needed to repair eral regions of the foliage (cuticle, cell wall, ozone damage could also cause increas- cellular contents, hypodermis and the resin ing K concentrations (Schier, 1990) and canals), whereas the X-ray microanalysis Cape et al (1990) found that increasing K only includes the cellular contents (vacuole was due to a mechanism to maintain and cytoplasm) and a portion of the cell cationic balance. wall. In contrast to Norway spruce, beech There are several possibilities that could showed decreasing K concentrations as rise to a change in foliar nutrient lev- give ozone exposure increased and Ca levels els. These include soil nutrient availability, were only slightly increased in NFA + 50. root development, foliar leaching, or redis- This could result from more K being leached tribution of nutrients to other parts of the by acid rain; ozone is thought to predispose plant. The good nutritional quality of the soil foliage to such an effect (McLaughlin, 1985; allows us to discount the 1st of these Westmann and Temple, 1989; Pfirrmann et hypotheses. Several other studies, however al, 1990). It could also just result from an (reviewed by Kasana and Mansfield, 1986), ozone-associated efflux of K. Murphy and have shown that ozone can affect the dis- Huerta (1990) found an increase in H tribution of dry matter between the shoots O 2 production in UV-treated plants which led to and roots, leading to a relative reduction in a K efflux. This could be significant as H root O 2 growth. is a product of ozone reactions which occur The potassium and calcium contents inside the plant (Runeckles and Chevone, were increased in the 2 spruce clones and 1992). According to Barnes et al (1990) and these changes could well lead to changes in Taylor et al (1989), ozone can decrease the cell membrane permeability (Heath and beech root biomass and thus nutrient uptake, Castillo, 1988; Chevone et al, 1990; Fink, and this could also produce such a result. 1991 b). It seems that the intercellular deriva- tives of ozone are capable of inhibiting the As previously discussed, ozone is capa- function of membrane pumps and mem- ble of altering cell membrane permeability brane transport mechanisms. Ca ions,2+ and this can cause perturbations in nutrient which are usually expelled from the cell by compartimentalization (Heath and Castillo, an ATP-dependent membrane transport 1988). These become apparent from the mechanism, are now accumulated inside microanalysis results. Fink (1991 b) con- the cell. This can in turn interfere with the cluded that there was an increase in Ca functioning of the numerous enzymes which influx into the cytoplasm of mesophyll cells use this ion as an activator or inhibitor where normally conifers are able to export (Heath and Castillo, 1988). this ion out of the cell. Because, in Norway
specific differences. Both stresses were spruce, the increase in potassium was found seen to effect nutrition supply from the roots, in all tissues and ozone attacks the meso- especially in Norway spruce. Membrane phyll first, it would seem that this increase is permeability was probably affected which due to changes in nutritional supply at the led to different nutritional levels between root level rather than a modification of the different tissues - an effect that total foliar membrane permeability of all tissues. Fur- nutritional analyses cannot reveal. thermore, the new growth has been sub- jected to ozone only recently, whereas the root system has developed in an ozone envi- ACKNOWLEDGMENTS ronment over several growing seasons giv- ing more time for ozone to exert its effects. In beech, the situation is different. There is This work was funded by Ministère de l’Agricul- perhaps a modification comparable to ture, Ministère de l’Environnement and Region spruce, but as there is no apparent differ- Lorraine. We thank Mr C Rose for his technical assistance. Also the ONF, D Parker and B Sim- ence in Ca levels in the vascular bundle it peringham for help with the drought stress instal- seems that the effects of membrane lation, G Fiegel of ASPA for supplying us with important. changes are more pollutant concentrations, P Loosveldt for help with Drought acted to decrease K and Ca con- the statistical tests and C Brechet for chemical analyses. centrations in the most internal tissues. The vascular bundle, by way of the xylem, sup- plies nutrients in solution to the various tis- REFERENCES sues and this is regulated by the endodermis (Stelzer, 1990). The drought has, by reduc- ing water supply, also reduced nutrient sup- Barnes JD, Pfirrmann Y (1992) The influence of CO 2 and O singly and in combination, on gas exchange, , 3 ply to the plant. There was a difference growth and nutrient status of radish (Raphanus between clones as regards the response of sativus L). New Phytol 121, 403-412 the mesophyll as only Istebna had an Barnes JD, Reiling D, Davison AW, Renner C (1990) increasing Ca concentration. Fink (1991 b) Interactions between pollutants and winter stress. Environ Poll 53, 235-254 proposed that the ion cannot be evacuated Bonneau M Le diagnostic foliaire. Rev For FrXL, from the cell because of pollution damage, (1988) 19-28 but its accumulation could also be a mech- Freer-Smith PH, Paterson IS, Parkinson JA, Cape JN, anism (linked to ABA production) to protect Wolfenden J (1990) The nutritional status of Picea against the effects of drought. abies (L) Karst across Europe, and implications for ’forest decline’. Trees 4, 211-224 The drought-ozone interaction is difficult Chevone BI, Seiler JR, Melkonian J, Amundson RG to interpret as the results are not a simple (1990) Ozone-water stress interactions. In: Stress summation of the 2 stresses in isolation. In responses in plants: adaptation and acclimation Gerardner, the effects of this interaction are mechanisms (RG Alscher, JR Cumming, eds), Plant Biol 12, 311-328 essentially in the epidermis of NFA + 50 and Clément A (1977) Point 77 sur certaines analyses foli- in the endodermis. The main changes in aires. Internal publication, 25 p Istebna occurred in the endodermis, meso- (1989) The effect of air pollutants on physi- Darrall NM phyll and vascular bundle and in beech, they processes in plants. Plant Cell Environ 12, ological were in the parenchyma and vascular bun- 1-30 dle and the epidermis for Ca only. All these Davidson SR, Ashmore MR, Garrety C (1992) Effects interactions were essentially in the NFA + 50 of ozone and water deficit on the growth and gas exchange of Fagus sylvatica. For Ecol Management treatment. 51, 187-193 To conclude, the effects of ozone and Dobson MC, Taylor G, Freer-Smith PH (1990) The con- drought are antagonistic and show inter- trol of ozone uptake by Picea abies (L) Karst and P
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