ACTA BIOLOGICA SLOVENICA LJUBLJANA 2007 Vol. 50, [t. 1: 31–39 Sprejeto (accepted): 2007-11-26 The life of plants under extreme CO2 Življenje rastlin pri ekstremni koncentraciji CO2 Dominik VODNIK*, Irena MAČEK, Urška VIDEMŠEK & Jože HLADNIK Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia; Phone: +386 14 23 11 61, Fax: +386 14 23 10 88; Email (* corresponding author): dominik.vodnik@bf.uni-lj.si Abstract. Higly elevated and fl uctuating CO2 concentrations at the sites with geogenic CO2 enrichment create unique and often extreme gaseous environment for plant life. In our paper we review the knowledge on plant performance at mofette Stavešinci, which is known for very pure exhalations of CO2. The responses of root processes and those occurring in aboveground parts of the plants are presented and discussed. The primary target of elevated CO2 at NCDS are root- and other belowground processes, while the direct effects on shoots are expected to be minor and only periodical. The successful- lness of plants to cope with adverse conditions can be largely dependent on inherent adaptive mechanisms, which can, however, not be regarded specifi c for the response to elevated CO2. Some species, for example cockspur grass (Echinochloa crus-galli), posess various mechanisms that make them fairly tolerant to extreme mofette environment. Keywords: natural CO2 springs, mofettes, soil CO2 concentration, photosynthesis, respi- ration. Introduction In relation to the problem of global climatic change the effects of elevated CO2 concentrations ([CO2]) on plants have been intensively studied in the last years. The responses of plants have been mainly predicted on the basis of experiments where the effects of a doubled present ambient CO2 concentration, a concentration that can be expected to be reached in the atmosphere in the mid of the 21st century, were compared to a reference state under actual atmospheric concentration. The latter is most commonly considered to be at 350-370 μmol CO2 mol-1. Besides its long-term increase, however, a certain variability of present [CO2] can be measured in space and time (short-term variability). Even recent plants can be confronted with carbon dioxide levels well above the average ambient [CO2]. Such exposure has been reported for forest fl oor plants on humus rich soils and plants in dense stands during the night period (LARCHER 2003, BLANKE 1997, RASCHI unpublished). In both cases high [CO2] can be related to a high rate of autotrophic and heterotrophic respiration. A special environment where [CO2] can reach very high values are natural CO2 springs (NCDSs; mofettes). At these sites geogenic CO2 enriches both soil air and the atmosphere. Especially in the soils, extremely high concentrations can be found (ten-percentage values). Under calm conditions, high [CO2] can be built up in the atmosphere too. Atmospheric increase strongly depends on topography and meteorological conditions that infl uence the mixing of air masses. Frequently enrichment can be limited only to a layer close to the soil surface. Overall, unique and sometimes extreme conditions 32 Acta Biologica Slovenica, 50 (1), 2007 are created at mofettes infl uencing different processes in these ecosystems (Fig. 1). In this paper we review the knowledge on plant performance under natural CO2 enrichment that has been obtained within the research at mofette Stavešinci. Root growth and functioning It is clear that natural geogenic CO2 enrichment primarily infl uences gaseous regime of the soil. Surprisingly, however, a vast majority of studies of NCDSs plants have neglected the possible below- ground effects of elevated [CO2]. Our measurements at the mofette Stavešinci (NE, Slovenia), which is known for very pure CO2 exhalations, showed that mofette soils can be exposed to very high or even extreme CO2 concentrations, that have fairly stable temporal and spatial patterns (VODNIK & al. 2006). High CO2 concentrations infl uence the properties of the soil; they decrease soil pH and redox potential, displace oxygen from the soil profi le, affect the availability of mineral nutrients and soil microbial life (MAČEK 2004, VIDEMŠEK & al. submitted). In the work by JAMNIK (2005) a high corre- lation between spatial pattern soil CO2 concentration and spatial pattern of soil pH was found when both parameters were measured on a small scale (sampling grid with 0.5 m resolution). Similarly, concomitant measurements of soil [CO2] and [O2] revealed a high negative correlation between both gases (VODNIK & PFANZ unpublished). At least for strongly enriched soils it can therefore be expected that direct effects of CO2 on plants are combined with hypoxia or anoxia. Although severely inhibited in growth, the plants of different species are able to sustain these se- vere conditions. A high [CO2] could directly inhibit root growth and functions like aerobic respiration. MAČEK & al. (2005) studied the sensitivity of root respiration in seven NCDSs grass species. By using liquid phase measurements (Clark-type oxygen electrodes) and potassium hydrogencarbonate addition Figure 1: Unique and frequently extreme gaseous conditions are created at mofettes by geogenic CO2 enrichment. They infl uence different processes in plants. ↑ – elevated CO2 concentration, ↑↑ – strongly elevated CO2 concentration. Slika 1: Geogeni CO2 ustvarja na mofetah edinstvene in pogostokrat ekstremne plinske razmere. Te vplivajo na različne procese v rastlinah. ↑ – povečana koncentracija CO2, ↑↑ – močno povečana koncentracija CO2. 33D. Vodnik, I. Maček, U. Videmšek & J. Hladnik: The life of plants under extreme CO2 a high CO2 environment was simulated. Under elevated CO2 (aq) a clear species specifi c decrease in root respiration was found (Fig. 2). At 8.3 mM CO2 (aq), which would correspond to roughly 21% (v/v) of CO2 in the air phase, the respiration was inhibited for 26%, 31% and 41% in E. crus-galli, S. pumila and D. glomerata, res- pectively. In the latter two species, a signifi cant inhibition was found even at 4.2 mM CO2 (aq). It can be generally concluded that signifi cant negative effects of CO2 on root respiration can be expected only at high concentrations that rarely occur in the ‘normal’ soils but can be easily detected at the sites with the geogenic CO2 enrichment. At these sites, species having higher respiratory tolerance to elevated [CO2] could be in advantage (e.g. E. crus-galli). The mechanisms of higher CO2 tolerance have not been elucidated, but might be related to ones underlying plant tolerance to the insuffi cient supply of oxygen (hypoxia). Since elevated [CO2] soil concentrations can be combined with hypoxia it is namely clear, that adaptive and aclimative responses known for plants living at oxygen defi ciency (e.g. in fl ooded soil) would be of benefi t in mofettes. Indeed, many plant species that grow at NCDSs are the ones well known for high tolerance to hypoxia (e. g. Phragmites australis most known in Italian mofette Figure 2: Root respiration of chamber grown Echinochloa crus-galli (L.) PB., Setaria pumila (Pior.) Roem & Schult. and Dactylis glomerata L. seedlings under different CO2 (aq) concentrations during the measure- ments. Values are given as percentages of the control respiration (16.5 ± 3.4, 28.3 ± 2.3 and 29.6 ± 2.2 nmol O2 g-1DW s-1 for E. crus-galli, S. pumila and D. glomerata, respectively), measured at ambient CO2 concentration in the measuring cuvette. Means ± SE are presented, n = 5 (see MAČEK & al. 2005 for details). Slika 2: Dihanje korenin sejank vrst Echinochloa crus-galli (L.) PB., Setaria pumila (Pior.) Roem & Schult. in Dactylis glomerata L. pri povečani koncentraciji CO2 (aq) med meritvijo. Vrednosti so podane kot odstotne vrednosti dihanja korenin v primerjavi s kontrolo, merjeno pri ambientalni koncentraciji CO2 v merilni kiveti (16,5 ± 3,4, 28,3 ± 2,3 in 29,6 ± 2,2 nmol O2 g-1DW s-1, ločeno za vrste E. crus-galli, S. pumila in D. glomerata). Podana so povprečja ± SN, n = 5 (podrobneje opisano v MAČEK & sod. 2005). 34 Acta Biologica Slovenica, 50 (1), 2007 Il’Bossoleto; Agrostis stolonifera, Juncus effusus and Echinochloa crus-galli in Stavešinci). These species possess adaptive metabolic and morpho-anatomical mechanisms that enable them to cope with adverse gaseous conditions. One of the most evident responses to hypoxia is an increased porosity of the roots. Oxygen defi ciency induces the formation of gas transducing channels in the root cortex (aerenchyma), which deliver atmospheric oxygen from the surface, i. e. via the shoot, to the root and rhizosphere. By this way the negative effects of hypoxia on root aerobic respiration and soil mineral nutrients availability can be mitigated and the action of toxic ions can be limited. It is indicated that similar responses can be found in plants exposed to natural CO2 enrichment, as revealed from the anatomical studies of the roots of maize grown under low and high soil CO2 concentration (Fig.3; VIDEMŠEK 2004, VIDEMŠEK & al. 2006). Figure 3: Aerenchyma in the roots of maize (Zea mays L.) growing in the soil with different geogenic CO2 enri- chment, natural CO2 spring Stavešinci (NE Sloveina); low [CO2] < 1%; high [CO2] > 15 %) (adapted from VIDEMŠEK & al. 2006, with permission). For a general response of maize to naturally elevated CO2 concentrations see also VODNIK & al. 2005. Slika 3: Aerenhimi v koreninah koruze (Zea mays L.), ki je rasla v tleh z različno talno koncentracijo CO2, (low- mala [CO2] < 1%; high-velika [CO2] > 15 %), na območju naravnega izvira CO2 Stavešinci (SV Slovenija) (prirejeno po VIDEMŠEK & al. 2006, z dovoljenjem). Splošni odziv koruze na naravno povečanje CO2 je opisan v članku VODNIK & al. 2005. Shoot growth and functioning A direct effect of elevated CO2 concentrations on the above ground parts of the plants can be expected in natural CO2 springs where an accumulation of CO2 in lower layers of the atmosphere is enabled by topography. A typical example is a karstic dolina Il’Bossoleto which is known for extreme nocturnal [CO2] enrichment (VAN GARDINGEN & al. 1995). Even at fl at area mofettes, however, the CO2 concentrations at canopy height may reach extreme values under calm, non-windy conditions (up to 3000 -10,000 µmol mol-1 in Stavešinci mofette; PFANZ & al. 2004). 35D. Vodnik, I. Maček, U. Videmšek & J. Hladnik: The life of plants under extreme CO2 Table 1: Photosynthetic characteristics of the plants, growing at the Stavešinci mofette. Parameters were derived from A-Ci curves, measured by Li-6400 (Licor, Lincoln, USA) gas exchange system. The differences between high CO2 (20-50% of CO2 in the soil, depth 20 cm) grown plants and control plants of the same species, growing at non-enriched sites within the mofette area, are shown. Preglednica 1: Značilnosti fotosinteze pri rastlinah z območja mofete Stavešinci. Fotosintezni parametri so bili odčitani iz A-Ci krivulij, ki smo jih izmerili z merilnim sistemom Li-6400 (Licor, Lincoln, ZDA). Prika- zane so razlike v fotosnteznih lastnostih rastlin, ki na mofeti rastejo pri veliki koncentraciji CO2, napram rastlinam z lokacij znotraj mofete, kjer obogatitve tal oz. atmosfere z geogenim CO2 ni. Photosynthetic response of plants under elevated [CO2] Plant species Reference Lower rate of CO2 saturated photosynthesis /Phleum pratense/ PFANZ & al. 2007 /Setaria pumila/ VODNIK & al. 2002 /Zea mays/ VODNIK & al. 2005 /Juncus effusus, Alopecurus pratensis, Plantago major/ PFANZ unpublished /Dactylis glomerata, Solidago gigantea/ HLADNIK unpublished Decrease in carboxylation effi - ciency /Phleum pratense/ PFANZ & al. 2007 /Setaria pumila/ VODNIK & al. 2002 /Zea mays/ VODNIK & al. 2005 Increase in CO2 compensation point /Phleum pratense/ PFANZ & al. 2007 /Echinochloa crus-galli/ VODNIK & al. 2002 Photosynthesis The photosynthetic carbon assimilation of plants, growing at NCDSs, has been subjected to quite intensive research. The main interest was to obtain information on the long term effects of elevated [CO2] that could help when predicting future carbon balance of terrestrial ecosystems. Gas exchange measurements that had been performed in nineties revealed inconsistent photosynthetic response of mofette plants to elevated [CO2]. There has been also no adaptive photosynthetic strategy found for autotrophic plants growing under extreme CO2 conditions in natural CO2 spring areas (BADIANI & al. 1999). Latter measurements at Stavešinci mofette clearly showed some common features of the photo- synthetic performance in several plant species, growing under elevated CO2. In these experiments, plants with different exposure to geogenic CO2 enrichment were selected on the basis of the soil CO2 concentration, measured in their rooting horizon (depth of 20 cm) (PFANZ & al. 2004). Gas exchange measurements were done on individuals that had been exposed to different rates of soil CO2 enrichment during their growth at the mofette (< 1% of CO2 in the soil = low, >20 % of CO2 = high). The results of these measurements are summed in Table 1. Evidently the photosynthesis of mofette plants is adversely affected by CO2 enrichment which could be the result of direct and/or indirect action of high CO2 concentrations. For Stavešinci mofette it is known that a very sharp gradient of [CO2] exists at the soil/atmosphere interface (VODNIK & al. 2006) and that long term enrichment of the atmosphere is much lower than that of the soil air. It is therefore to presume that the direct negative effects of elevated atmospheric [CO2] concentrations on photosyntheis are minor and most probably occur only periodicaly. On the other hand the soil [CO2] are high or even extreme and far more stable (VODNIK & al. 2006, PFANZ & VODNIK unpublished) and could have an important indirect effect on autotrophic carbon assimilation. 36 Acta Biologica Slovenica, 50 (1), 2007 The reductions of photosynthetic rates and decreases in carboxylation effi ciency can be well related to the lower content of mineral nutrients, in particular nitrogen, in the leaves of high CO2 grown plants. Such decrease was found for different species, Phleum pratense, Dactylis glomerata, Solidago gigantea, Zea mays, Juncus effusus (PFANZ & al. 2004). Also COOK & al. (1998) reported on nitrogen deprivation and photosynthetic reduction in Nardus stricta plants growing at a CO2 spring on Iceland. The same effect of soil CO2 enrichment can even be observed in fertilized plants. In maize fi eld experiment, that was performed within the Stavešinci mofette area, the leaf N content signifi cantly decreased at the parts of the fi eld that were enriched by geogenic CO2, despite fertilization (VODNIK & al. 2005). It is to presume that the main factor infl uencing the mineral nutrition was a reduced availa- bility of minerals in the soil with high CO2 and low O2 concentrations. Reductive conditions in the soil strongly affect chemical reactions in the soil, which can decrease the availability of essential elements, for example nitrogen (via denitrifi cation), sulphate etc. and promote toxicity of other elements (e.g. manganese). When the oxygen is depleted from the soil an additional factor would be lower metabolic capacity of the roots for the mineral uptake (see also MAČEK & al. 2005). In non-fertilized mofette soils the nutrient availability would also be strongly affected by the lower input of organic carbon to the soil, due to lower biomass production, and by the lower rate of mineralization. The observed photosynthetic response can be frequently correlated to a lower content of chlo- rophyll in the leaves of the plants exposed to geogenic CO2 enrichment (VODNIK & al. 2002, PFANZ & al. 2004). Highly elevated CO2 concentrations are refl ected also in the increased levels of some stress related compounds in the leaves (VODNIK & al. 2005). Another aspect on photosynthetic carbon assimilation is the regulation of gas diffusivity between the leaf and the atmosphere. Plants growing at the mofettes are confronted with fl uctuating and extreme air CO2 concentrations and have even more diffi cult task, compared to plants from ‘normal’ sites’, when regulating CO2 uptake and water loss. Stomatal conductance and transpiration Plants have sophisticated regulation of stomata that allow them to achieve suitable stomatal con- ductance under given environmental conditions. The [CO2] in surrounding air is one of the key factors that infl uence stomatal conductance, it is sensed in substomatal cavity by undisclosed mechanism. General assumption is that higher [CO2] leads to reduction of stomatal conductance. When [CO2] is suddenly increased, which can be expected for the mofette conditions, stomata respond by relatively fast closure (Fig. 4a). In our research dynamics of the stomatal response was compared for different grassland species from the Stavešinci mofette. A species specifi c response was observed. Plants of cockspur (E. crus-galli) responded to CO2 increase by faster stomatal closure than other species (Fig. 4b) (HLADNIK & VODNIK unpublished data). When the response of plants from low- and high- CO2 environments was compared for selected plant species, no differences were found in dynamics of the response (data not shown). Conclusions CO2 emissions at natural CO2 springs create unique and frequently extreme gaseous conditions for plants. The negative effects of higly elevated [CO2] can been observed on different levels, high [CO2] infl uences growth, photosynthesis, respiration, mineral nutrition etc. Our research at Stavešinci revealed that the responses of plants can be very well correlated to the soil CO2 concentration. Since, in addition, the CO2 enrichments of the atmosphere in this particular mofette are minor and only periodical, it is clear that the root- and other belowground processes are the primary target of the geogenic CO2. This is largely refl ected in disturbances of mineral nutrition and leads to secondary effects such as photosynthetic inhibition. 37D. Vodnik, I. Maček, U. Videmšek & J. Hladnik: The life of plants under extreme CO2 F ig ur e 4: a ) S to m at al r es po ns e of m ai ze ( Ze a m ay s L .) t o th e su dd en c ha ng es o f [C O 2] i n su rr ou nd in g ai r. A rr ow s an d ac co m pa ny in g nu m be rs i nd ic at e ti m e an d th e le ve l [ µ m ol m ol –1 ] o f C O 2 c on ce nt ra ti on c ha ng e (H L A D N IK & V O D N IK 2 00 7, w it h pe rm is si on ). b : S to m at al re sp on se o f E ch in oc hl oa cr us -g al li (E .c -g .) , D ac ty lis gl om er at a (D .g .) , P hl eu m p ra te ns e (P .p ) an d So lid ag o gi ga nt ea ( S .g ) to s ud de n ch an ge in [ C O 2] in s ur ro un di ng a ir . C ha ng e fr om o rd in ar y to d ou bl ed [ C O 2] w as a ch ie ve d at m om en t 0. S to m at al r es po ns e in b ot h ex pe ri m en ts w as m ea su re d w it h L i- 64 00 s ys te m ( L IC O R , L in co ln , Z D A ) at 2 6° C , 1 00 0 µ m ol m –2 s –1 li gh t i nt en si ty a nd r el at iv e hu m id it y of 3 5% . D at a w er e lo gg ed in 5 s ec on d in te rv al s. S li ka 4 : a) O dz iv li st ni h re ž ko ru ze ( Ze a m ay s L .) n a sp re m em bo [ C O 2] v o ko li šk em z ra ku . P uš či ce in s pr em lj aj oč e št ev il ke o zn ač uj ej o ča s in o bs eg s pr em em be [ C O 2] v µ m ol m ol –1 ( H L A D N IK & V O D N IK 2 00 7, z d ov ol je nj em ). b : O dz iv l is tn ih r ež E ch in oc hl oa c ru s- ga lli ( E .c -g .) , D ac ty lis g lo m er at a (D .g .) , Ph le um p ra te ns e (P .p ) in S ol id ag o gi ga nt ea ( S .g ) na h it ro s pr em em bo [ C O 2] iz o bi ča jn e na p od vo je no k on ce nt ra ci jo . S pr em em ba k on ce nt ra ci je C O 2 j e bi la d os ež en a v ča su 0 . O dz iv s m o m er il i z m er il ni m s is te m om L i- 64 00 ( L IC O R , L in co ln , Z D A ) pr i 2 6° C , j ak os ti s ve tl ob e 10 00 µ m ol m –2 s –1 in r el at iv ni v la žn os ti 3 5% . P od at ki s o bi li b el ež en i v in te rv al u 5 se ku nd . 38 Acta Biologica Slovenica, 50 (1), 2007 To some extent plant can cope with adverse growing conditions at the mofettes. Their successfull- ness can be largely dependent on inherent adaptive mechanisms, which can, however, not be regarded specifi c for the response to elevated CO2. They most commonly represent a response strategy to other stressors, that are more common than the higly elevated CO2 concentrations. Some species, for example cockspur grass (Echinochloa crus-galli) posessess various mechanisms that make them fairly tolerant to extreme mofette environment. Acknowledgements The work was supported by grants J4-2186-0486, Z4-3196-0486 (DV) and Research programme P4-0085 from the Ministry of higher education, Science and Technology and the Slovenian Resarch Agency. It was carried out within the framework of the COST 627 action. The authors thank prof. 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