• JOU• • ALOF. Plan. PI'~. .., © 1998 by Gustav Fischer Verlag. Jena Interactions between Drought and Elevated CO2 on Alfalfa Plants CRISTINA L. M. SGHERRIl, M. F. NAVARI-IzZOI F. 2 QUARTACCIl, M. MENCONI , I !scituto di Chimica Agraria, Universid degli Studi, Pisa, 56124 Pisa, Italy 2 C.N.R, IA.TA., 50144 Firenze, Italy A. 2 RASCHI , and Received February 24, 1997 . Accepted June 9, 1997 Summary Alfalfa (Medicago sativa L.) plants were grown in open top chambers at ambient (340 ppm) and high (600 ppm) CO2 concentrations. Twenty-five days after the first ,cutting one set of both plants was subjected to water deficit conditions by withholding water for 5 days. A chamber effect on proteolytic activity, monogalactosyl diacylglycerol to digalactosyl diacylglycerol molar ratio, total non-structural carbohydrates and soluble protein contents occurred. In contrast, no change in leaf water potential was observed between plants grown outdoors and inside the chambers. Plants grown at high CO 2 concentration showed a lower decrease in leaf water potential in comparison with plants grown at atmospheric CO 2 when subjected to water stress. Under high CO 2 concentration leaf nitrogen content decreased whereas starch accumulation and a higher proteolytic activity were recorded. Following water depletion, COrenriched plants showed a decrease in total non-structural carbohydrates and soluble proteins. In thylakoid membranes high CO 2 caused an increase in chlorophyll and lipid contents and a degradation of monogalactosyl diacylglycerol. A higher degree of unsaturation in the main thylakoid lipids was also observed. COrenriched plants were less affected by water stress as shown by reduced chlorophyll degradation and a higher membrane stability. Key words: Medicago sativa L., CO2 enrichment, lipids, protein metabolism, thylakoids, total non-structural carbohydrates, water stress. . Abbreviations: DGDG = digalactosyl diacylglycerol; MGDG = monogalactosyl diacylglycerol; OTC open top chamber; TNC = total non-structural carbohydrates; '¥w = water potential. Introduction Atmospheric levels of CO2 are rising at the present and it is estimated that the concentration of CO 2 will reach double the preindustrial levels within the next century (Bazzaz, 1991). Over the years, many short-term physiological studies on the responses of crops to elevated levels of CO2 were conducted. In general, for agricultural crops it was found that elevated levels of CO 2 increase net photosynthetic rates, dry matter production, yields, root/shoot ratio and tuberization (Clifford et al., 1993; MackowIak and Wheeler, 1996). J Plant PhysioL WlL 152. pp. 118-124 (1998) = A consistent response of C 3 plants to high CO 2 is to increase the carbon/nitrogen ratios in their tissues, while the mineral content has been reported to decrease, although the absolute amount per plant may increase (Overdieck, 1993). In several CO 2 enrichment experiments, in particular under reduced supply of nutrients, the stimulation of growth due to elevated CO2 was very limited, while a significant stimulation of photosynthesis was observed (Williams et al., 1988; Grulke et al., 1990). This often results in a disbalance between source (carbon fixation) and sink (structural growth), leading to changes in leaf tissue composition due to the accumulation of excess non-structural carbon. Reductions in the future nutri- Effects of CO2 and Drought on Alfalfa tional quality (C/N ratio) of plants grown in high CO 2 may reduce the populations of herbivorous insects, thereby potentially destabilizing food webs and pollination associations that depend upon them (Bazzaz, 1991). High levels of CO 2 also cause partial closure of leaf stomata and reduce transpiration (Bazzaz, 1991). By increasing the amount--of osmoticum and decreasing transpiration, elevated CO 2 usually allows plants to cope better with water stress (Tyree and Alexander, 1993; Schwanz et al., 1996). Recent data concerning the global climate predict increasing CO 2 levels in conjunction with environmental stress factors, including increased temperature, UV-B radiation and drought (Houghton et al., 1990). One of the first consequences of these kinds of stress is an alteration in the structure and function of cell membranes. Thus, the ability of an organism to maintain its membrane function and composition might indicate its resistance to environmental stresses (Navari-Izzo et al., 1995). The effects of CO2 or drought on plants have been determined separately for a large number of species but very little is known about their interaction (Ferris and Taylor, 1995; Tschaplinsky et al., 1995). Thus, it is unclear whether the stimulation of growth and photosynthesis provided by high CO2 will persist despite increasing water deficit. There is agreement about the sensitivity of the Mediterranean environment to global change, including changes in water availability, temperature and atmospheric CO 2 , Alfalfa (Medicago sativa L.) is the main forage crop in many areas of the Mediterranean basin, even though its response to elevated CO 2 and drought has never been investigated in detail so far. The aim of the present research was to investigate the changes in leaf protein metabolism and carbohydrate contents as well as thylakoid composition of alfalfa grown in open top chambers (OTCs) under both 600 ppm CO 2 concentration and/or water deficit stress. Materials and Methods Plant material Potted plants of alfalfa (Medicago sativa L.) were grown both in open top chambers (1 m diameter, 1.5 m high), either at ambient (340 ppm) or at high CO2 concentration (600 ppm), and outdoors to check the influence of the OTCs themselves on plant growth. The OTCs were set up in Rapolano Terme (Siena, Italy). The farm belongs to Geogas, a mining company that extracts, purifies and bottles CO2 from natural vents. The OTC air inlets (3 L m -1) are directly connected to the bulk container of the company, avoiding any risk of CO2 shonage during the experiment. The design of the OTCs and the CO2 concentration control system were essentially similar to those described by Ashenden et al. (1992). CO2 concentration inside the chambers was continuously monitored, as were air and humidity. Inside the chambers, daytime air and soil temperatures were on the average 2.5 'C higher and 1.4·C lower than outside during the entire experiment. No significant temperature differences were observed among CO2 and water stress treattnents. The potted plants were grown on a medium fertile soil characterized by neutral reaction. Each pot (9 L) was fertilized according to Raschi et al. (1997). Both inside and outside the chambers the pots were placed in tubes so that the upper rim of the pots was at ground level. A single tube contained 12 pots, each containing 5 plants. After 25 119 days from the first cutting, alfalfa plants were subjected to water deficit stress by withholding water for 5 days. Control plants were maintained at field capacity by regular watering, at the same time, every day. Youngest fully expanded leaves from control and treated plants were harvested at the same time of the day, fresh weight was recorded, and samples were taken for dry weight measurements. Leaf water potential ('Pw) was determined using a pressure-chamber (Navari-Izzo et al., 1990). Isolation ofthylakoid membranes Thylakoid membranes were isolated by homogenizing the leaves in an ice-cold isolation medium (1 : 3 w/v) containing 0.5 moUL sucrose, 75 mmoUL Hepes-KOH, pH 7.5, 10 mmol/L diethyldithiocarbamic acid, 5 mmoUL ascorbic acid, 3.6 mmoUL cysteine and 5 mmollL EDTA following the procedure of Navari-Izzo et al. (1995). Lipid analysis The isolated thylakoids were first boiled in isopropanol for 5 min. Lipids were extracted at 4 ·C with chloroform: methanol (2: 1 v/v), containing butylhydroxytoluol as an antioxidant. Individual polar lipids were fractionated on silica gel plates and quantified by their phosphorus and sugar contents, respectively (Navari-Izzo et al., 1991). The fatty acid methyl ester derivatives (Douce et al., 1990) were analyzed by GLC using a Dani 86.10 HT gaschromatograph equipped with a flame ionization detector. A capillary column (Ld. = 0.32 mm, length = 60 m), with SP 2340 as stationary phase, was used. The operating conditions were: column, 170 ·C; injector and detector, 250 ·C; split ratio 1: 70. Nitrogen was used as carrier gas with a flux of 0.9 mUmin. Determination oftotal nitrogen and soluble and membrane proteins The total nitrogen content was determined by the micro-Kjeldhal method. Soluble protein extraction was carried out at 4 ·C by grinding leaf tissue (0.5 g) in a cold mortar using 100 mmoUL Tris-HCI, pH 7.5, containing 4 % (w/v) Polyclar AT. The homogenate was squeezed through four layers of muslin and centrifuged at 12,000 Kn for 20 min. The supernatant was used for protease and soluble protein determinations. The pelletted thylakoid fraction was first delipidized and membrane proteins were solubilized with 0.1 N NaOH. Protein determinations were performed according to Bensadoun and Weinstein (1976). Protease activity assay Protease activity assays were performed following Felicioli et al. (1988) using rabbit muscle aldolase as the substrate. Leaf crude extract (0.48 mL) was incubated at 37·C for 30 min in 100 mmoUL Tris-HCI, pH 7.5, with 4.64 units of aldolase. The proteolytic digestion was stopped by a SO-fold dilution with ice-cold 100 mmoUL Tris-HCI, pH 7.5. Another sample, in which the reaction was stopped before digestion, was prepared and used as reference sample. The proreolytic activity was determined by the residual aldolase units. One unit of proteolytic activity is defined as the aldolase units lost per minute of digestion per mg of extract soluble protein. The aldolase activity was assayed at 37'C by monitoring the decrease in absorbance at 340 nm for 3 min. The assay mixture contained 100 mmol/L Tris-HCI, pH 7.5, 1 mmoUL fructose 1,6-bisphosphate, 0.15 mmol/L NADH and 3 units of a a-glycerophosphate dehydrogenase!triosephosphate isomerase mixture (10: 1 w/w). The reaction was initiated by adding aldolase preparation and the amount of sub- 120 CRISTINA L M. SGHERRI, M. F. QuARrACCI, M. MENCONI, A. RAsCHI, and F. NAVARI-Izzo strate transformed per minute was calculated using a molar extinction coefficent of 12,400 for fructose 1,6-bisphosphate. Corrections for any endogenous aldolase activity were not necessary. Daermination offoe amino adJs Free amino acids were extracted for 30 min from 250 mg lyophilized sample with 25 mL ethanol/water (85: 15, v/v) according to Navari-Izzo et aI. (1990). The extracts were filtered, vacuum dried, and dissolved in a known volume of water; amino acids were determined by the ninhydrin method. The amino acids were quantified by comparison with a Calbiochem Behring standard. Ddn'mination oftotal non-structural carbohydrates The youngest fully expanded leaves were excised, immediatdy frozen in liquid nitrogen and stored at -80·C. Tissues were ground with a mortar and pesde and the soluble sugars were extracted three times with distilled water by immersing the homogenized sample in 3r---------------------------------, 2.~ a boiling water bath for 10 min (Wong, 1990). After centrifugation at 12,000 g,. for 15 min, the pellet containing starch was suspended in 0.2 mollL KOH and incubated at l00·C for 30 min. After cooling. the pH of the mixture was adjusted to 4.5 with acetic acid and its digestion was carried out in 0.1 mol/L acetate buffer, pH 4.5, containing 3.2 units mL -I of amylogIucosidase (from Rhizopus; Sigma). Digestion of sucrose in the supernatant of the above centrifugation was carried out in 0.1 mol/L acetate buffer, pH 4.5, by adding 1.5 units mL-I of invertaSe (from Bakers Yeast; Sigma). After incubation at 55 'C for 15 min, digestions were stopped with 24 % (w/v) trichloroacetic acid and samples were centrifuged at 12,000 g,. for 15 min. After washing of the pellets with water, the total extract was brought to volume. InvertaSe and amylogIucosidase treated samples were then assayed for reducing sugars (as glucose equivalents) using the phenol-sulfuric acid method (Dubois et aI., 1956). A glucose was used. standard curve covering the range 8-40 ~olL Total non-structural carbohydrates (TNC) were estimated on the basis of the sum, as glucose equivalents, of soluble sugars and starch. Owing to the presence of background carbohydrate in the enzyme preparation, an enzyme blank was included with the samples. 18r---------------------------------------, 15 W~, 2 o.~ ** C°zXWS ,** 12 3r----------------------------------, 21 18 2.5 CO2 , ** WS, ns c~, 2 1~ i ** A 24 ~ CO2 , ** WS, ** COiXWS, CO, ** ** WS, ** CD2xWS, ** 1.5 12 9 II 3 c o Fig. h Effect of CO2 enrichment (600 ppm) and water stress upon leaf water potential (A) and chlorophyll (B), lipids (C) and lipid to protein ratio (D) of thylakoids of alfalfa (Medicago sativa L). Values represent the mean (n = 6) ± SE. A multifactorial analysis of variance was used to evaluate CO2 enrichment effect (C02), water stress effect (WS) and interaction between CO2 and water stress (C02 xWS). Significance was as follows: ns = not significant; ** = significant at P S 0.01 level. 0, 600 ppm CO2; III, 600 ppm CO2 + WS; . , 340 ppm CO2 , ~,340pm CO2 + WS. Effects of CO2 and Drought on Alfalfa 121 Table 1: Chamber effect upon monogaIactosyl diacylglycerol Results (MGDG) to digaIactosyl diacylglycerol (DGDG) molar ratio, total non-structural carbohydrates (TNC), soluble proteins (SP) and proteolytic activity (PA) in alfalfa (Medicago sativa L.) plants grown both in open-top chambers at ambient CO 2 concentration (OTCs) and outdoors. For comparisons among means (n = 6), analysis of variance was used. For each parameter, means in rows followed by different letters are significantly different at the P sO.Ollevel. Proteolytic actlVlty, TNC and soluble protein contents were reduced in alfalfa plants grown in OTCs in comparison with plants grown outdoors, with the exception of the monogalactosyl diacylglycerol (MGDG)/digalactosyl diacylglycerol (DGDG) molar ratio (Table 1). Leaf water potential did not show any difference in plants grown in OTCs under either atmospheric or elevated CO 2, whereas a lower decrease in plants grown at high CO2 was observed under water deficit conditions (Fig. lA). Under elevated CO 2 alfalfa plants showed an increase in chlorophyll and lipid contents in comparison with ambient-C02 plants while following water stress, thylakoid components suffered a general decrease with the exception of lipids (Fig. 1). A less marked chlorophyll decrease upon water depletion was also observed in COrenriched plants (Fig. 1 B). Both under ambient and elevated CO 2 a reduction in the chlorophyll alb ratio, from 3.1 to 2.8 was noted in water stress conditions (data not shown). MGDGIDGOG TNC (mglg OW) SP (mglgOW) PA (nKatfmg SP) OTCs Outdoor 1.03 b 513.50 a 133.49 a 0.96 a 0.43 a 636.00 b 156.00 b 1.10 b High CO 2 concentration affected total nitrogen content, which decreased both in control and stressed plants in comparison with plants grown at ambient CO 2 (Fig. 2 D). Either CO 2 enrichment or water stress caused increases in proteo- 60 50 ~ 40 §. Q.> 30 :J ~ Ol E 20 10 0 60 50 40 ~ ~ CO2, ** WS, ns C02xWS, ns Ol 30 20 c 10 D 0 F•• 2: Effect of CO 2 enrichment (600ppm) and water stress upon proteolytic activity (A), free amino acids (B), soluble proteins (C) and nitrogen content (0) in aIfalfa (Medicago sativa L.) leaves. Otherwise as for Fig. 1. 122 CRlmNA L. M. SGHERRI, M. F. QUARTACCI, M. MENCONI, A. RAsCHI, and F. NAVARl-Izzo 80~-, C~, ** WS, ** C02xWS, ** 800 exception of linolenic acid of MGDG and DGDG {data not shown}. Control and dehydrated plants grown at ambient CO 2 showed similar unsaturation levels, whereas high CO 2 increased it in MGDG and DGDG of water-stressed plants {Table 2}. Discussion 14r-----------------------------------12 C~,* 10 WS, ** C~xWS, 8 ** 6 The influence of OTCs on the microclimate of the plants is called «chamber effect». Irradiance in the chamber has been seen to be reduced compared with the outside by a minimum of 20 % on clear days and by as much as 30 % on overcast days {Ashenden et al., 1992}. This may be of particular significance in photosynthesis and, consequendy, in TNC accumulation {Table I}. Inside the OTCs, air and leaf temperatures were nigher than outside and might have increased sucrose synthesis, sucrose transport and utilization {Farrar and Williams, 1991}, thus decreasing carbohydrate accumulation in the leaf {Table I}. These higher temperatures might have also altered the MGDG/DGDG molar ratio, proteolytic activity and soluble protein content {Table I}. Under CO 2 enrichment a decrease in stomatal conductance (Menconi et al., 1997), probably due to a partial closure of stomata, has been seen to ameliorate midday 'I'w in waterstressed plants, while in well-watered conditions no differences were detected between CO 2-treated and untreated plants (Fig. lA). The higher chlorophyll and lipid contents in elevated COz-grown plants could be explained, at least in part, by the larger number and size of chloroplasts present in 4 B 2 OL-__~ ____~L_ __~ FJgo 3: Effect of CO2 enrichment (600 ppm) and water stress upon total non-structural carbohydrates (A) and starch to soluble sugar ratio (B) in alfalfa (M~d;cago sativa L.) leaves. Otherwise as for Fig. 1. lytic activity and free amino acid content while an interaction between CO 2 and water stress reduced the soluble protein content to the level of plants grown at ambient CO 2 {Fig. 2}. Under CO2 and water stress both TNC and the starch/soluble sugar ratio showed a similar pattern. They had the highest level under high CO2 concentration while following water shortage they were reduced to the level of stressed plants grown at ambient CO2 {Fig. 3}. Examination of thylakoid lipid composition {Fig. 4} indicates that DGDG levels increased both in hydrated and stressed plants grown at high CO 2 concentration whereas MGDG decreased. On the contrary, SQDG and PG levels were unaffected either by CO 2 concentration or by water deficit conditions. The main fatty acids consistendy recovered in each polar lipid were myristic, palmitic, a3-trans-hexadecenoic (only in PG), stearic, oleic, linoleic and linolenic acids. In all treatments they did not show any significative difference, with the Table 2: Effects of CO 2 enrichment (600 ppm) and water stress on unsaturation (mol %) of thylakoid polar lipids of alfalfa (M~dicago sativa L.). Values represent the mean (n = 6) ± SE. A multifactorial analysis of variance was used to evaluate the effects of CO2 enrichment (C02), water stress (WS) and CO2 enrichment X water stress (C02 X WS). Significance was as follows: ns = not significant; ** = significant at P S 0.01 level. DGDG, digalactosyl diacylglycerol; MGDG, monogalactosyl diacylglycerol; SQDG, sulfoquinovosyl diacylglycerol; PG, phosphatidylglycerol. A, 340 ppm CO2; H, 600 ppm CO2• Lipid Treamtment U nsaturation MGDG A A+WS H H+WS DGDG A A+WS H H+WS SQDG A A+WS H H+WS PG A A+WS H H+WS 72.2±2.1 72.3±2.2 84.2± 1.9 88.4±2.0 59.0±1.8 55.9±1.9 73.0±2.2 85.3±2.5 42.2±1.5 39.4±1.3 44.4±1.5 43.0±1.4 47.9±1.l 42.2± 1.3 41.7±1.5 49.9± 1.7 ** ns ns ** ** ** ns ns ns ns ns ** 123 Effects of CO 2 and Drought on Alfalfa ~o-, 40 ** c~, WS, ns CO~WS, ns 30 10 B 0 25 20 COz, ** WS, ns C~xWS ns 15 ~ 0 E c 10 5 o 0 FJg.41 Effect of CO 2 enrichment (600ppm) and water stress upon digalactosyl diacylglycerol (A), monogalactosyl diacylglycerol (8), sulfoquinovosyl diacylglycerol (C) and phosphatidylglycerol (D) in alfalfa (M~dicago sativa L.) leaves. Otherwise as for Fig. 1. the tissues exposed to high CO2 levels (Robertson and Leech, 1995). Moreover, the better water use efficiency observed at high CO 2 under water deficit conditions (Bazzaz, 1990) could have limited chlorophyll degradation (Fig. 1 B). Our data show that the activity of the lipid-linked desaturation was affected by CO 2 concentration, which, in contrast, did not influence the chain length of fatty acids (Table 2). The higher lipid to protein ratio, together with an increase in the unsaturation level, have been considered as adaptative responses of plants, by which they cope better with altered environmental conditions (Quartacci et al., 1995). Such responses seem to be a feature of alfalfa plants grown under water deficit conditions at high CO2 (Fig. 1 D and Table 2). The lower MGDG/DGDG molar ratio of thylakoids of stressed plants grown at high CO2 and their higher unsaturation levels (Fig. 4 A, 4 B and Table 2) may reduce the tendency of membranes to form non-lamellar configurations and may help to maintain a more fluid environment (Quartacci et al., 1995). The charged lipids sulfoquinovosyl diacylglycerol (SQDG) and phosphatidylglycerol (PG) (Fig. 4 C, 4 D) are known to interact closely with integral proteins and to be involved in the electron transport between photosystems II and I. Their unchanged levels may have maintained mem- brane protein complex configurations even under water deficit conditions. In addition, due to the capacity of charged lipids to swell with water, messed plants may have also maintained the ability to bind water. Leaf mineral nutrients have been reported to decrease under CO 2 enrichment (Raschi et al., 1997) because of the «carbohydrate dilution effect» that changed leaf chemical composition, in particular concerning the C/N ratio (Fig. 3). Despite the decreased TNC content observed in stressed COzenriched plants, nitrogen did not accumulate (Fig. 2 D), probably due to the reallocation of nitrogen from Rubisco to non-photosynthetic tissues (Raschi et al., 1997). At high CO 2 concentration the accelerated protein metabolism and the decrease in soluble proteins following stress may corroborate the above hypothesis (Fig. 2). Reduction in the amount and/or in the activity of Rubisco may in tum reduce carbon fixation and carbohydrate accumulation (Fig. 3). This reduction occurred although adaptative responses to maintain thylakoid membrane functionality were established (Figs. 1 D, 4 A. 4 B and Table 2). In conclusion, elevated atmospheric CO2 induced changes in protein and carbon metabolism and made thylakoid membranes less susceptible to water deficit conditions. The in- 124 CRISTINA L. M. SGHERRI, M. E QUARrACCI, M. MENCONI, A. RAsCHI, and E NAVARI-Izzo creased tolerance of alfalfa plants to water stress under high CO2 was due not only to a lower decrease in water potential but also to a different chemical composition of membranes that may have rendered them more functional in comparison with plants grown at ambient CO 2 concentration. 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